Studies in Environmental Science 27

FLUORIDE RESEARCH 1985

Selected Papers from the 14th Conference of the International Society for Fluoride Research, Morioka, Japan, 12-15 June 1985

Edited by

Humio Tsunoda
Department of Hygiene & Public Health, School of Medicine, I wate Medical University, 19-1 lchimaru, Morioka, 020 Japan

Ming-Ho Yu
Huxley College of Environmental Studies, Western Washington University, Bellingham, WA 98225, U.S.A.

ELSEVIE R

1986

Amsterdam - New York - Oxford - Tokyo

E L S E V I E R SCIENCE PUBLISHERS B.V. Sara Burgerhartstraat 25 P.O.B o x 21 1, 1000 A E Amsterdam, T h e Netherlands

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0 Elsevier Science Publishers B.V., 1986

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In Memory of

NORYKO TSUNODA

XI11

PREFACE T h i s volume c o n t a i n s f o r t y - n i n e p a p e r s . They c o n s i s t o f a s e l e c t i o n o f t h e research papers, and some r e v i e w p a p e r s , i n Morioka, t h a t were presented a t t h e The p a p e r s a r e c o n c e r n e d p o l l u t i o n caused b y animals, and 1 4 t h C o n f e r e n c e o f t h e I n t e r n a t i o n a l S o c i e t y f o r F l u o r i d e R e s e a r c h (ISFR) h e l d f r o m 1 2 - 1 5 June 1985, inorganic fluorides; humans. F l u o r i d e i s a common e l e m e n t and i t i s known t o be w i d e l y d i s t r i b u t e d i n nature. With regard t o t h e p u b l i c h e a l t h i m p l i c a t i o n s o f f l u o r i d e , appears toxic that a t t e n t i o n has been f o c u s e d of this substance l a r g e l y on t h e b e n e f i c i a l and its effect effects i n d r i n k i n g water
it

Japan.

w i t h a n a l y t i c a l methods f o r f l u o r i d e :

environmental

and t h e e f f e c t s o f f l u o r i d e on p l a n t s ,

and on

t e e t h . Because o f i t s w i d e s p r e a d and g r o w i n g use i n i n d u s t r i e s , i n d u s t r i a l areas i n t h e world.

however,

f l u o r i d e i s now f o u n d i n i n c r e a s i n q amounts i n t h e s u r r o u n d i n g s o f many

I n s e v e r e l y p o l l u t e d a r e a s , f l u o r i d e has
i t c o u l d a l s o con-

been shown t o cause i n j u r i e s t o p l a n t s and a n i m a l s ; c e i v a b l y cause a d v e r s e e f f e c t s on humans. I n t h e past,

as a consequence o f i n d u s t r i a l d i s c h a r g e , Japan has u n f o r e p i s o d e s as Minamata d i s e a s e l i k e m e t h y l mer-

t u n a t e l y e x p e r i e n c e d such p o l l u t i o n - r e l a t e d

and I t a i - I t a i d i s e a s e . I t i s w i d e l y known t h a t f l u o r i d e , organisms, mental We

c u r y and cadmium w h i c h p r o d u c e d t h e s e e p i s o d e s , c a n a c c u m u l a t e i n l i v i n q c a u s i n g i n j u r i e s . To u n d e r s t a n d f u l l y t h e e f f e c t s o f e n v i r o n i n c l u d i n g humans, for an i n t e r d i s c i p p o l l u t a n t s on l i v i n g systems, feel strongly that the basis

l i n a r y approach t o r e s e a r c h i s needed. understanding f l u o r i d e - r e l a t e d i n a i r and w a t e r , b u t also p r o b l e m s l i e s i n h a v i n g sound knowledge c o n c e r n i n g a l l phases o f f l u o r i d e occurrence, i n c l u d i n g not o n l y t h e f l u o r i d e t h a t i n o r g a n s and t i s s u e s o f l i v i n g o r g a n i s m s . The s e c t i o n " A n a l y t i c a l Methods f o r F l u o r i d e " was i n c l u d e d as one o f t h e m a i n themes i n t h e conference widely based o n t h i s employed in consideration. reported

I t was hoped t h a t ,
research in such

w h i l e newly fields as

developed a n a l y t i c a l medicine, endeavor. Thanks to the

methods

i n t h e c o n f e r e n c e m i g h t be m r e a n a l y t i c a l c h e m i s t s m i g h t also use to the ISFR

furthering l e a r n ways

fluoride

d e n t i s t r y and b i o c h e m i s t r y ,

t h i s opportunity t o

i n which t h e y c o u l d c o n t r i b u t e by many scientists, the

interest

shown

14th

C o n f e r e n c e was w e l l dent i s t r y ,
b

attended: science,

n e a r l y 200 p a r t i c i p a n t s came f r o m e l e v e n

d i f f e r e n t c o u n t r i e s . They r e p r e s e n t e d such d i v e r s e f i e l d s veterinary pharmacology, chemistry, environmental science. Altogether,

as m e d i c i n e ,
biology, and

n e a r l y 100 p a p e r s were p r e s e n t e d i n

XIV
the oral tations sessions and i n t h e p o s t e r s e s s i o n s . Both forms o f presenSeveral of

g e n e r a t e d i n t e r e s t i n q and w i d e - r a n g i n g d i s c u s s i o n s . These methods can no doubt are thus fluoride in be utilized i n this

t h e s e p a p e r s were c o n c e r n e d w i t h n e w l y - d e v e l o p e d analysis. research, addition, and t h e p a p e r s of are airborne not included pollution, this volume

methods f o r f l u o r i n e i n many areas o f publication. fluorosis, be published In and in

we a r e p l e a s e d t o i n c l u d e s e v e r a l

o t h e r papers d e a l i n q w i t h skeletal The m r e t h a n t h i r t y

case s t u d i e s papers that

r e p o r t s on t h e b i o c h e m i c a l e f f e c t s o f f l u o r i d e . included "Fluoride", t h e o f f i c i a l j o u r n a l o f t h e ISFR.

will

The success of t o the s k i l l greatly

t h e m e e t i n g was due u l t i m a t e l y t o t h e p a r t i c i p a n t s and We a r e

and a s s i s t a n c e o f t h e c h a i r m e n o f t h e s e s s i o n s .

i n d e b t e d t o each o f t h e c o n t r i b u t o r s o f t h e s e p r o c e e d i n g s who we g r a t e f u l l y acknowledge t h e c o n t r i b u t i o n made b y t h e D i r e c t o r o f N i s h i Matsuzono C l i n i c , M o r i o k a , who She passed Dr and a s p e c i a l held in

c o o p e r a t e d i n many ways c o n c e r n i n g t h e p u b l i c a t i o n .
I n conclusion,

l a t e D r N o r i k o Tsunoda,

n o t o n l y gave s u b s t a n t i a l f i n a n c i a l a s s i s t a n c e t o t h e c o n f e r e n c e , b u t was a l s o i t s h o s t e s s and o r g a n i z e d a l l o f t h e s o c i a l programs. away o n 18 J u l y 1985, f r i e n d o f M.H. Conference Petersburg, Noriko and N o r i k o Tsunoda was t h e d e a r w i f e o f one o f us, H. Tsunoda, Yu and h i s w i f e , the was Ervena. Utah, International and i n Logan, acquainted with Fluoride Symposium investigators a p p r o x i m a t e l y one month a f t e r t h e m e e t i n g .

S i n c e a t t e n d i n q t h e 1 2 t h ISFR

St.
in

Florida,

USA,

respectively,

i n 1982, D r involved

Tsunoda

many

f l u o r i d e research.

I t was h e r s i n c e r e hope t h a t t h e ISFR C o n f e r e n c e h e l d She made e v e r y e f f o r t

i n M o r i o k a , where she l i v e d , w o u l d be s u c c e s s f u l . t o ensure i t s success. F o r t h e s e r e a s o n s we d e d i c a t e t h i s volume, Tsunoda.

t o a s s i s t h e r husband and f r i e n d s p r i o r t o and t h r o u g h o u t t h e c o n f e r e n c e

i n memoriam, t o D r Noriko

Humi o Tsunoda Ming-Ho Yu

Tokyo January 1986

xv
ACKNOWLEDGMENTS On b e h a l f o f t h e O r g a n i z i n g C o m m i t t e e o f t h e 1 4 t h C o n f e r e n c e o f t h e I S F R , we w i s h t o e x p r e s s o u r deep g r a t i t u d e t o t h e many i n s t i t u t i o n s , organizations. cooperation particular, and i n d i v i d u a l s f o r t h e i r s u p p o r t , f i n a n c i a l assistance. President. and In which c o n t r i b u t e d t o t h e success o f t h e Conference. we w o u l d l i k e t o t h a n k D r . K i j u r o O b a r a ,

Iwate

Medical University, Public Health, University, Ohbori,

Dr.

Yoshito Tsuji, S a n s h i Abe,

Chief Director, President,

Japanese S o c i e t y o f N i p p o n Gakuen

and Dr.

Higashi

who s e r v e d a s C o n f e r e n c e H o n o r a r y A d v i s o r s ; and D r . T s u t o m u Iwate University,

I w a t e M e d i c a l U n i v e r s i t y , Dr. S e i k o I s h i k a w a . Iwate Medical University,

Dr.

S h u n i c h i Sato,

and D r . Y a s u h i r o S u z u k i ,

Yamagata

U n i v e r s i t y , who s e r v e d as C o n f e r e n c e A d v i s o r s . W e g r e a t l y a c k n o w l e d g e t h e f o l l o w i n g i n s t i t u t i o n s and o r g a n i z a t i o n s w h i c h sponsored t h e Conference; I n t e r n a t i o n a l S o c i e t y f o r F l u o r i d e Research: I w a t e M e d i c a l U n i v e r s i t y : Japanese S o c i e t y f o r Hygiene: Japanese S o c i e t y o f P u b l i c H e a l t h : A s s o c i a t i o n o f I n d u s t r i a l Health: S o c i e t y f o r A n a l y t i c a l Chemistry. Our s i n c e r e a p p r e c i a t i o n i s e x t e n d e d t o members o f t h e C o n f e r e n c e E x e c u t i v e C o m m i t t e e and S e c r e t a r i a l S t a f f f o r o r g a n i z i n g and h a n d l i n g t h e many d e t a i l s i n v o l v e d i n t h e C o n f e r e n c e . t h a n k Dr. Masanobu T a t s u m i , I n particular, we w o u l d l i k e t o Japan S o c i e t y o f A i r P o l l u t i o n ; Japan and Japan

Dr. S h i r o Sakurai,

D r . S h i g e n a o Nakaya, D r . Mr.

M i t s u o M i t a , D r . K a z u y o s h i I t a i , D r . K e i k o Nagayama, M r . T o r u Sato. M a t s ud a.
We

T o s h i t a k a H o r i u c h i , D r . Y o i c h i I i j i m a , D r . K o s u k e Okada, and Mrs. M i c h i k o

are deeply grateful

f o r the financial Morioka,

assistance given t o t h i s and i n d i v i d u a l s : Iwate Medical Morioka Medical and Sakata,

Conference by t h e f o l l o w i n g i n s t i t u t i o n s , Iwate Prefecture, Association, Co-op,

City o f

organizations.
City o f

Iwate Dental Association,

I w a t e Hea1,th S e r v i c e A s s o c i a t i o n ,

Department o f P u b l i c H e a l t h a t Fukushima Medical College,

Keiryokai (Alumni Association o f Iwate Medical University):

Professor Emeritus Tadashi I g a r i ( I w a t e Medical U n i v e r s i t y ) , Prof. Teru Takanohashi ( I w a t e U n i v e r s i t y ) . (Oshuku S p r i n g H o s p i t a l ) , Osamu T a k a h a s h i (Hanamaki ( K a w a s a k i S t e e l Co.).

M r . A k i o Yamaraki and Dr. Yasuo Kuboya Dr.

and Dr. Yoshinori Shoji

Dr. Sadayoshi Ichinohe ( I c h i n o h e C l i n i c ) ,

Mental Hospital),

XVI
Lastly, we w o u l d l i k e a l s o t o t h a n k t h e f o l l o w i n g members o f t h e A l u m n i I w a t e Medical f o r t h e i r c o o p e r a t i o n and d e v o t i o n : M r s . T o m o k o Endo.

A s s o c i a t i o n o f t h e D e p a r t m e n t o f H y g i e n e and P u b l i c H e a l t h , University,

Dr.

Susumi Atsumu,

Dr.

Shoko F u j i m o r i ,

Dr.

Yutaka

Fujino,

Dr.

S h o i c h i r o Fusa,

Dr.

S e t s u H a s h i m o t o , Dr. C h i s a t o Hamajima.

Dr.

T o k u z o I k e n o , D r . S a d a t o I c h i n o h e , Dr. K a z u k o I c h i n o w a t a r i , Ichinowatari, Matsuda, T a k a t s u g u Kudo, Sadakatsu Naito, Kyoko Odashima, Orikasa, Saka, Sekiya, Dr. Y a s u h i r o Kudo, Dr. M a t s u o Kumagai,

Dr. Yoshim1

D r . Y u t a k a I s h i z a w a , D r . T a k a s h i I t o , D r . S a i c h i Kon, D r .
Mr.
Kazuhiro

D r . Susumu Motoyama.

Dr.

H i r o s h i Mural,

Dr. S h i g e r u Nagasawa.

Dr.

Mrs. Yoko Nakaya, Dr. H i d e o N i h e i , Dr. K e n i c h i Ohsawa.

M r . YUJI

Noru,

Miss

D r . S h u n p e i O i k a w a , Dr. T a d a s h i O g i w a r a , D r . K e i k o Onodera, D r . T a k e o Ohsawa, D r . Choshun

Mrs. M l h o k o S a i t o , Dr. M a s a t a k a Mrs. A t s u k o

Dr. K e n i c h i Onodera, Dr.

Mrs.

Fumihiko Saito,

Mr.

Ken S a i t o ,

D r . H i n a Sato, Dr. N o b u h i s a Sato,

Re1 k o S h i r u k u i s h i , K e n i c h i Takeuchi,

Mr. Shigeki Sasaki,

Mr.

D r . T s u n e a r i Sugawara,
Dr. Ryozo Tanaka,

Mamoru S u z u k i . Dr.

D r . Y o s h i o S h i m a z a k i , D r . H i d e k o Sonoda, D r . I k u r o T a k a h a s h i , D r . Susumu
Takahashi. Dr.

Mr.

S e n j i r o Tanaka,

Mitsugu Tobari,

Dr. Kazuo T o d o r i ,

Dr. M i n o r u Uematsu.

Dr. Masao Yamada, Dr.

K e n j i Y a z a k i , Dr. Y u t a k a Y a s u i , D r . Masao Y o s h i d a . D r . H i d e k o Y o s h i m u r a , Mrs. Y o s h i k o Y o s h i o k a . and Dr. T a k e h i s a Yoshiyama.

H. Tsunoda

M.

H. Yu
h e l d i n Morioka,

O r g a n i z i n g C o m m i t t e e o f t h e 1 4 t h C o n f e r e n c e o f t h e ISFR. Japan, Dr. J u n e 12-15,

1985.
Professor, Dept. o f Hygiene

Humio Tsunoda ( C o n f e r e n c e Chairman), Public Health, Iwate Medical U n i v e r s i t y Professor,

Dr. K e i i c h i r o Fuwa,

Dept. o f C h e m i s t r y ,

U n i v e r s i t y o f Tokyo Iwate

Dr. T s u y o s h i Katayama,
Medical U n i v e r s i t y

Professor,

Dept. o f P r e v e n t i v e D e n t i s t r y ,

D r . J i r o Matsushima, P r o f e s s o r , Dr.
Kan-ichi University Ohshima,

Faculty o f Agriculture, Mie University Dept. o f Veterinary Pathology, Iwate

Professor,

D r . S h i g e r u Ono, P r o f e s s o r , D e p t . o f B i o c h e m i s t r y ,

Iwate Medical U n i v e r s i t y Tokyo

Dr. Y o s h i n o r i Takaesu,
Dental College

Professor,

Dept. o f P r e v e n t i v e D e n t i s t r y ,

Dr.

F u m i y o s h i Yanagisawa, Un iv e r s it y

Professor Emeritus,

Tokyo Medical - Dental

XVII
Dr. Yasuhisa Yoshida,
Medical College P r o f e s s o r , Dept. o f H y g i e n e

- Public

H e a l t h , Osaka Western

D r . Ming-Ho Yu.

Professor,

Huxley College o f Environmental Studies,

Washington U n i v e r s i t y

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 3-14 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in T h e Netherlands

ANALYTICAL C H E M I S T R Y AND BIOGEOCHEMISTRY OF FLUORINE: AN HISTORICAL V I E W

K E I I C H I R O FUWA
Department o f Chemistry, F a c u l t y o f Science, Bunkyo-ku, Tokyo 11 3, J a p a n ABSTRACT The a n a l y s i s o f f l u o r i n e i s one o f t h e m o s t d i f f i c u l t e l e m e n t a l a n a l y s e s i n t h e h i s t o r y o f a n a l y t i c a l chemistry. c o l o r i m e t r y and s p e c t r o c h e m i s t r y . Each o f t h e r e p r e s e n t a t i v e i i l e t h o d s i n t h e f i e l d o f f l u o r i d e a n a l y s i s was d e s c r i b e d w i t h p a r t i c u l a r e m p h a s i s on Some b i o g e o c h e m i c a l a n d e n v i r o n m e n t a l aspects o f f l u o r i n e were a l s o mentioned. INTRODUCTION F l u o r i n e . t h e f i r s t member o f t h e h a l o g e n s , i s an e l e m e n t w i t h u n i q u e p h y s i c a l and c h e m i c a l p r o p e r t i e s . t h e most n o n - m e t a l l i c nature, I t has t h e h i g h e s t e l e c t r o n a f f i n i t y o r i t s compounds o f t e n d i s p l a y For instance, fluorides in and t h e r e f o r e , The U n i v e r s i t y o f Tokyo,

d i f f e r e n t p r o p e r t i e s f r o m t h o s e o f o t h e r halogens.

o f a l k a l i n e e a r t h s s u c h as CaF2 and S r F 2 h a v e l o w s o l u b i l i t y i n w a t e r ,

c o n t r a s t t o c h l o r i d e s o r b r o m i d e s s u c h a s CaC12 a n d S r C 1 2 o r C a B r 2 a n d

SrBr2.
scopic.

Whereas s i l v e r f l u o r i d e (AgF) i s e a s i l y s o l u b l e i n w a t e r o r h y g r o s i l v e r c h l o r i d e (AgC1) o r s i l v e r b r o m i d e ( A g B r ) a r e t h e l e a s t F l u o r i d e g i v e s complex anions w i t h s i l i c o n . itself. The g e o c h e m i s t r y , aluminum forming SiFi2,

s o l u b l e compounds. and i r o n ,

A l F g 3 and FeFg3, w h i c h a r e s p e c i e s o f t e n f o u n d i n
biogeochem-

n a t u r a l w a t e r s b e s i d e s f l u o r i d e (F-)

i s t r y and b i o c h e m i s t r y o f f l u o r i d e a r e s p e c i a l and a r e o f p a r t i c u l a r i n t e r e s t because o f t h e s e fundamental s p e c i a l L h a r a c t e r i s t i c s i n b o t h p h y s i c s and c h e m i s t r y . The a n a l y t i c a l c h e m i s t r y o f f l u o r i n e i s a l s o u n i q u e i n t h a t i t i s one o f t h e m o s t d i f f i c u l t e l e m e n t s t o h a n d l e i n t e r m s o f b o t h c h e m i c a l and i n s t r u m e n t a l t e c h n i q u e s u s e d i n i t s a n a l y s i s . A n a l y t i c a l Chemistry o f Fluorine A l m o s t a l l c l a s s i c a l and i n s t r u m e n t a l summary of each method with special a n a l y t i c a l methods have been The f o l l o w i n g i s a b r i e f e m p h a s i s o n c o l o r i m e t r y and a p p l i e d t o t h e a n a l y s i s o f f l u o r i n e (Table I). s p e c t r o c h e m i c a l methods, Gravimetry.

w h i c h h a v e been s t u d i e d i n t h i s l a b o r a t o r y and a r e CaFZ, lead chlorofluoride, PbClF, and

considered most u s e f u l f o r f l u o r i n e analysis. Calcium fluoride, LaF3, lanthanum f l u o r i d e , a r e common i n s o l u b l e s p e c i e s f o r t h e g r a v i m e t r i c

TABLE I
METHODS OF FLUORINE ANALYSIS Method Gra v i met ry Species etc. CaF2, PbClF, LaF3 i zari ne S; AgN03/PbCl F Ion Selective Electrode, LaF, SiFg2, AlFi3 19F(n,p)190

Ti tration
Electrochemical Ion Chromatography Radiochemical Colorimetry Spectrochemical

Th( N03)4/A1
F-,

"F(r1,2n)~*F,

Zr-Alizarin, Al-Hematoxylin La-ALC(TRLCC) Emission; CaF, SrF, BaF, F Absorption; AIF, InF

analysis of fluoride (1).

A well-known classical Berzelius-Hillebrand

method of analyzing fluoride in rocks is based on dissolving fluoride (CaF2) after alkaline fusion o f the sample powder followed by the addition of calcium chloride in excess to the solution. Titration. Fluoride i n aqueous solution can be titrated by thorium nitrate, Th(N03)4, using alizarin S as an indicator, or by argentimetric titration of chloride after separating the fluoride as PbClF (1.2). Electrochemical method. Among several electrochemical techniques, the one using an ion selective electrode for fluoride with LaF3 membrane is the most useful ( 3 ) . This method i s simple, and can distinguish fluoride ions from other complexed species, and the sensitivity is high enough, 20 ppb of
F-,

so that it can be applied to most determinations. Ion chromatography. Ion chromatography has been developed recently and

has become one o f the most convenient methods for anion determinations in natural water ( 4 ) . Fluoride can be determined by ion chromatography, with proper caution against interfering elements. Radiochemical method. Activation analysis of fluorine can be achieved by nGutron bombardment of samples in an atomic pile and counting the resulting r a d i o a c t i v e species. T h e reaction m a y be either 19F(n,2n)18F or ' ' F ( n , ~ ) ~ ' 0 . The half-lives of each atom are 1.87h for 1 8 F and 29.5s for "0, respectively (5.6)
_ Colorimetry. ____

The color bleaching effect by fluoride on yellowish

titanium color, developed with hydrogen peroxide in an acidic solution was one of the early colorimetric methods used for fluoride determination ( 7 ) . As a more sensitive fading c o l o r i m e t r y f o r fluoride, z i r c o n i u m -

5
a l i z a r i n a t e (8) o r a l u m i n u m h a s e m a t o x y l i n e ( 9 ) w e r e u t i l i z e d i n o r d e r t o determine f l u o r i d e c o n t e n t o f n a t u r a l water i n c l u d i n g sea water. a b s o r p t i o n s p e c t r a o f t h e s e compounds w e r e i n v e s t i g a t e d (Fig. The 1) (10).

F i g u r e 1.

Colorimetry o f f l u o r i d e

Although use o f t h e c o l o r f a d i n g e f f e c t s o f f l u o r i d e f o r i t s d e t e r m i n a t i o n was s o m e w h a t s u c c e s s f u l , and West ( I I ) , i.e.. (ALC.1.2-dihydroxy a c o l o r d e v e l o p i n g compound was f o u n d b y B e l c h e r t h e r e d d i s h lanthanum complex w i t h a l i z a r i n complexon

anthraqhinonyl-3-methylamine-N, N - d i a c e t i c a c i d ) changes
r e s u l t i n g 7n a c o m p l e x w i t h an a b s o r p I n order t o increase t h e sensi-

t o blue by reacting w i t h fluoride,

t i o n maximum a t 620 nm. T h i s La-ALC m e t h o d has been e m p l o y e d i n m o s t o f t h e c o l o r i m e t r y o f f l u o r i d e i n r e c e n t years. t i v i t y o f t h e method, absorption cell, quently, a technique using t o t a l a r e f l e c t i o n long c a p i l l a r y

was a p p l i e d (12). T h i s t e c h n i q u e u s e d a " l i q u i d c o r e o p t i c a l f i b e r " as a n t h e core l i q u i d being t h e s o l u t i o n i n which t h e m a t e r i a l The s o u r c e l i g h t t r a v e l s t h r o u g h n1, n2, o r i s d i s s o l v e d . so t h a t t h e a b s o r p t i o n c e l l c o u l d be v e r y l o n g , and conse-

o f high analytical sensitivity.

and t h e r e f o r e ,

the solution,

the r e f r a c t i v e index o f the solution,

o u g h t t o be g r e a t e r t h a n t h a t o f t h e c e l l w a l l , n1

' n2

(1)

Then t h e c r i t i c a l a n g l e i i s e x p r e s s e d b y S i n i = n2/nl and t h e i n c i d e n t s o u r c e l i g h t , can t r a v e l intensity, i n Figure through as S n e l l ' s l a w holds.

(2)
w h i c h h a s a l a r g e r i n c i d e n t a n g l e t h a n i, reflection without losing i t s

the c e l l by t o t a l

A t y p i c a l i n s t r u m e n t a l d i a g r a m i s shown

2,

i n w h i c h an o p t i c a l f i b e r t y p e c a p i l l a r y c e l l i s seen a t t h e

6 center. S i n c e a g l a s s o r p y r e x c a p i l l a r y c e l l i s used, an o r g a n i c s o l v e n t has t o be used.

w i t h a r e f r a c t i v e index higher than t h a t of pyrex, C a r b o n d i s u l f i d e , w h o s e i n d e x i s 1.66, and c a r b o n d i s u l f i d e ,

1.48,

o r a m i x t u r e of a n o r g a n i c s o l v e n t

i s t h e b e s t f o r t h i s purpose.

A diethylaniline
With t h i s

d e r i v a t i v e o f ALC was f o u n d t o be more s u i t a b l e f o r t h e s o l u b i l i z a t i o n o f i t s l a n t h a n u m and f l u o r i d e d e r i v a t i v e s measured. tion, paper. i n t o carbon d i s u l f i d e . t o t a l r e f l e c t i o n long c a p i l l a r y technique. 0.01 p p b F i n w a t e r c a n b e

A d e t a i l e d d e s c r i p t i o n o f t h e a n a l y t i c a l procedure, instrumenta-

a s w e l l as some e x p e r i m e n t a l r e s u l t s a r e r e p o r t e d e l s e w h e r e i n t h i s

LENS F I LTER "OPT I CAL FIBER"

SAMPLE

RECORDER

F i g u r e 2.

D e t e c t i o n system w i t h " h o l l o w f i b e r "

long c a p i l l a r y c e l l

Spectrochemical analysis.

Emission spectrochemical analysis o f f l u o r i n e

b y D C a r c u s i n g a CaF b a n d a t 5 2 9 nm w a s f i r s t e m p l o y e d b y P a p i s h ( 1 4 ) . T h i s p a r t i c u l a r b a n d a s w e l l a s o t h e r CaF b a n d s a n d t h e m o n o f l u o r i d e s o f strontium author (15). and b a r i u m i n a DC a r c were further investigated by the which give T a b l e I 1 shows v a r i o u s m e t a l l i c m o n o f l u o r l d e s ,

e i t h e r e m i s s i o n o r a b s o r p t i o n bands i n a h i g h t e m p e r a t u r e medium (16).

All

e m i s s i o n b a n d s o f a l k a l i n e e a r t h m o n o f l u o r i d e s i n v e s t i g a t e d i n a DC a r c b y e i t h e r naked e y e o b s e r v a t i o n o r s p e c t r o p h o t o g r a p h i c t e c h n i q u e s w e r e f o u n d t o be enriched i n t h e negative flame region. n e g a t i v e f l a m e e n r i c h m e n t o f CaF bands. G o l d s c h m i d t a n d S t r o c k (17). cathode, i n w h i c h 20-30 Figure 3 illustrates the T h i s phenomenon c a n be c o n s i d e r e d

e q u i v a l e n t t o t h e cathode l a y e r enrichment f o r m e t a l l i c species s t u d i e d by By o b s e r v i n g w i t h t h e n a k e d e y e t h e g r e e n i s h O.OOl%F was y e l l o w CaF b a n d a t 5 2 9 nm e n r i c h e d i n t h e n e g a t i v e f l a m e n e a r t h e l o w e r mg o f CaC03 d i l u t e d s a m p l e i s l o a d e d ,

TABLE I 1 BAND HEADS OF METAL-MONOFLUORIDES Wavelength (nm) M9F CaF Abs.

-

F.E.

D.E.
(ev)

539.42V 606.47V 529.10R 324.54v

10 10 10
-

4.5 5.4

8 10 10 10 7 10 10 4 10 10 10
10 10

SrF

663.27V 577.95R 352.98V

10 8 8 10
-

5.45

BaF

711.60R 495.08V 380.99V

5.8 7.85 6.85 6.1 5.35 4.5 3.5 5.2

BF
A1 F GaF InF T1F

195.74 227.47v 211.02R 233.73v 219.80R 493.20V 351.78V 242.41V

10
10

10
-

CuF
MnF

10 10

F r o m : R. Spectra"

W.

6. P e a r s e a n d A. G. G a y d o n : " T h e I d e n t i f i c a t i o n o f M o l e c u l a r

d e t e c t e d (15). available, e.g.,

S r F a t 577 nm a n d BaF a t 6 9 4 nm g i v e s i m i l a r o r s l i g h t l y
b u t t h e y a r e u s e f u l w h e n t h e CaF b a n d i s n o t F i g u r e 4 shows a r e p r o d u c t i o n o f BaF bands. fluorine atomic w h e n t h e r e g i o n a t 5 2 9 nm i s i n t e r f e r e d w i t h b y o t h e r

l o w e r s e n s i t i v i t i e s (18). e l e m e n t s such as barium.

As a t o m i c a b s o r p t i o n s p e c t r o s c o p y b e c a m e p o p u l a r , a b s o r p t i o n was i n v e s t i g a t e d . fluorine a t

I t was f o u n d t h a t t h e r e s o n a n c e l i n e o f
Diatomic and aluminum sharp

95 nm

was t o o s h o r t f o r r e g u l a r a p p l i c a t i o n s (19).

f l u o r i d e species were i n v e s t i g a t e d i n t h i s laboratory, p r o m i s i n g (20).

m o n o f l u o r i d e ( A l F ) w i t h a b a n d h e a d a t 2 2 7 nm w a s f o u n d t o b e t h e m o s t

A t t h i s wavelength, 5). Thus,

A1F g i v e s a " l i n e - l i k e ' '

a b s o r p t i o n peak ( F i g . apparatus

h i g h temperature absorption spectrometry o f Any r e g u l a r a t o m i c a b s o r p t i o n light source should provide The g r a p h i t e f u r n a c e was f o u n d that the

f l u o r i n e has become a u s e f u l method. can be used e x c e p t c o n t i n u o u s s p e c t r a f r o m a h y d r o g e n lamp.

I -

negative flame center of arc column

in
B

CaF

(bh529 1 A )

F i g u r e 3. F l u o r i n e d e t e r m i n a t i o n b y DC a r c (CaF s p e c t r a ) A p p r o x . 30 mg s a m p l e i s d i l u t e d w i t h CaC03. DC 1 0 0 V, 8 - 1 0 A i s u s e d . CaF ( b h 529.1 nm) i s e n r i c h e d m o s t i n t h e n e g a t i v e f l a m e n e a r t h e l o w e r e l e c t R e p r o d u c e d f r o m : Fuwa K ( 1 9 5 1 ) J J p n Chem SOC 71:341 r o d e i n B.

_ _ _ _ B a F bh 5000 6A ---- B a F bh 4992 1A

---- B a F bh 4950 8 A

'

F i g u r e 4. F l u o r i n e d e t e r m i n a t i o n b y DC a r c ( 5 a F ) . A i s t a k e n on t h e photo p l a t e and 5 i s t h e s p e c t r u m observed BaS04/5aF2. R e p r o d u c e d f r o m : Fuwa b y t h e n a k e d eye. A p p r o x i m a t e l y O.l%F i s d e t e c t e d . K ( 1 9 5 5 J J J p n Chem SOC 76:14

W a v e l e n g t h (nm) F i g u r e 5.

Absorption s p e c t r u m o f A1F

p a r t i c u l a r l y s e n s i t i v e and t h e d e t e c t i o n l i m i t has r e a c h e d a s s m a l l a q u a n t i t y a s 0.021 n g

F.

For most p r a c t i c a l purposes,

a platinum atomic

l i n e f r o m a P t h o l l o w c a t h o d e l a m p t o g e t h e r w i t h a D2 l a m p f o r b a c k g r o u n d c o r r e c t i o n seems t o b e t h e b e s t , a l t h o u g h t h e f o r m a t i o n o f t h e A1F m o l e c u l e i n s i d e t h e medium i s o f t e n d i s t u r b e d b y e x i s t i n g f o r e i g n m a t t e r (21.22). g i v e n e l s e w h e r e i n t h i s paper. I n t h e l a s t t e n years, e m i s s i o n s p e c t r o m e t r y has regained a t t e n t i o n i n s p e c i a l l y designed i n d u c t i v e l y the f i e l d o f fluorine analysis since s e n s i t i v i t y (23).

d e t a i l e d d e s c r i p t i o n and i t s a p p l i c a t i o n t o some b i o l o g i c a l s a m p l e s h a v e

c o u p l e d a r g o n p l a s m a was f o u n d e x c e l l e n t i n t e r m s o f b o t h p r e c i s i o n and

I t g i v e s s i g n a l s as s e n s i t i v e as a t o m i c a b s o r p t i o n does
I n addition, a simultaneous determination system.

f o r most m e t a l l i c elements.

s y s t e m has been c o n v e n i e n t l y d e s i g n e d w i t h a p o l y c h r o m a t o r d e t e c t o r combined w i t h a computerized read-out atomic absorption. However,

I t has s i n c e r e p l a c e d a
elements i n c l u d i n g apparently the

large p a r t o f the f i e l d o f elementary analysis previously c a r r i e d o u t by most o f t h e non-metallic f l u o r i n e a r e excluded f r o m t h i s t e c h n i q u e again, h i g h e x c i t a t i o n energies. most energetic region, However, atom, been m a i n l y because o f t h e i r

Only between t h e i n d u c t i o n c o i l s ,

was some a t o m i c f l u o r i n e e m i s s i o n o b s e r v e d (24). t h e m e t a s t a b l e He has c o u l d e x c i t e f l u o r i n e as method for fluoride

when h e l i u m g a s i s e m p l o y e d a s t h e p l a s m a gas,

w h i c h has a s u f f i c i e n t l y h i g h energy l e v e l , investigated and established TMCS, as a

i s i l l u s t r a t e d i n F i g u r e 6. determination.

Use o f m i c r o w a v e i n d u c e d p l a s m a ( M I P ) , novel

By s i m p l y s h a k i n g an aqueous s a m p l e c o n t a i n i n g f l u o r i d e f l u o r i d e i s converted t o tetramethyl The l a t t e r i s e x t r a c t e d i n t o an o r g a n i c s o l v e n t and

w i t h tetramethyl chlorosilane, f l u o r o s i l a n e , TMFS.

10

F i g u r e 6.

E n e r g y d i a g r a m o f f l u o r i n e and z i n c w i t h M I P as an e l e m e n t In

t h e e x t r a c t i s t h e n analyzed b y gas chromatography, s e l e c t i v e d e t e c t o r (25). H i s t o r y o f spectroscopy.

It i s said t h a t h i s t o r y repeats r t s e l f .

s c i e n t i f i c h i s t o r y man c a n a l s o f i n d r e p e a t e d d e v e l o p m e n t s i n some f i e l d s . S p e c t r o s c o p y i s p e r h a p s one o f t h e b e s t examples. described i n t h i s section, As h a s a l r e a d y b e e n I n T a b l e 111, It i s t h e r e c u r r e n c e o f e m i s s i o n and a b s o r p t i o n

spectrometry i n h i s t o r i c a l terms i s indeed most s t r i k i n g .

t h e e s s e n c e o f t h i s r e p e a t e d h i s t o r y o f s p e c t r o s c o p y i s shown.

i n t e r e s t i n g t h a t b o t h a t o m i c a b s o r p t i o n and p l a s m a e m i s s i o n h a v e r e g a i n e d t h e i r r e p u t a t i o n and p r o v e d i n v a l u a b l e i n t h e a r e a o f f l u o r i d e a n a l y s i s . Fluorine i n Hokutolite H o k u t o l i t e i s a n i s o m o r p h o u s m i x t u r e o f BaS04 and PbS04, m i n e r a l s produced i n b o t h Hokuto h o t spring, h o t s p r i n g , A k i t a , J a p a n ( F i g . 7). studied i n detail. t e m p e r a t u r e w a s 97C. pH 1.2, Taipei, one of t h e r a r e T a i w a n and Tamagawa where t h e

H o k u t o l i t e f r o m T a m a g a w a h o t s p r i n g was and w h i c h c o n t a i n e d

It i s t h e d e p o s i t from a unique h o t spring, d u e m a i n l y t o HC1.

r a d i o a c t i v i t y d u e t o ThX, Ra ( F i g . 8). f l u o r i d e as a m i n o r c o n s t i t u e n t , F i g u r e 8, source, 0.01-0.05%

Since the spring water contained As s h o w n i n

the deposition o f fluoride i n Hokutolite

w a s i n v e s t i g a t e d b y t h e e m i s s i o n s p e c t r o m e t r y o f BaF (18). near t h e j u n c t i o n o f Shibukuro River,

F was f o u n d i n H o k u t o l i t e d e p o s i t e d f a r f r o m t h e
where b o t h t h e t e m p e r a t u r e

and t h e a c i d c o n c e n t r a t i o n o f t h e s p r i n g w a t e r w e r e s u b s t a n t i a l l y l o w e r e d . The H o k u t o l i t e f r o m Tamagawa h o t s p r i n g h a s been named one o f t h e n a t i o n a l

11
TABLE I 1 1 HISTORY OF SPECTROSCOPY Year Name Fact Emission o r absorption
-

1666
1817 1859 1955

S i r I . Newton

D i s c o v e r y o f c o l o r and v i s i b l e spectrum Fraunhofer Lines K i r c h h o f f ' s Law D i s c o v e r y o f Rb, Cs, T1 Atomic Absorption P l a s m a emission

emission absorption emission absorption erni s s i o n

J. F r a u n h o f e r
G.R.
Kirchhoff

R. Bunsen
S i r A. Walsh
V.A.
Fassel

1974

F i g u r e 7.

H o k u t o l i t e producing areas

Hokuiollte 2

D i a g r a m s h o w i n g t h e g e o g r a p h y of Tamagawa H o t S p r i n g a r e a ( A k i t a F i g u r e 8. Pref., Japan)

12
s p e c i a l p r o d u c t s f o r c o n s e r v a t i o n i n Japan and m o s t o f i t i s b e i n g k e p t a t t h e Geological Department of A k i t a U n i v e r s i t y , Biogeochemistry o f F l u o r i n e Some c r u c i a l bioelement, f a c t s about t h e biogeochemistry o f f l u o r i n e a r e reproduced Fluorine i s not c o n s i d e r e d an e s s e n t i a l e l e m e n t o r as i t has i n h i b i t o r y e f f e c t s
i t i s needed f o r h e a l t h y

Japan.

i n T a b l e I V (26). l i v i n g matter.

w h i c h i s needed f o r t h e n o r m a l b i o l o g i c a l f u n c t i o n o f any

I t i s o f t e n considered toxic,

on many enzymes.

But, as i s shown i n T a b l e I V ,

g r o w t h o f mammalian teeth,

b e s i d e s b e i n g a c o n s t i t u e n t o f bones and t e e t h . Many s u b j e c t s r e l a t e d t o t h e s e a r e

O D t i m a l u p t a k e and o p t i m a l c o n c e n t r a t i o n s i n aqueous m e d i a , f o o d and b o d i e s a r e obviously t h e most important. described elsewhere i n t h i s paper, t h e r e f o r e o n l y a few r e c e n t environm e n t a l problems o f f l u o r i n e a r e mentioned here: F l u o r i d a t i o n o f d r i n k i n q water. s t i l l a social, A l t h o u g h t h i s p r o b l e m i s n o t new,
it i s

e n v i r o n m e n t a l a n d h e a l t h p r o b l e m f o r humans.

Fluoride a t

0.5-1.0

ppm i n d r i n k i n g w a t e r i s c o n s i d e r e d o p t i m a l .

Recommended concen-

t r a t i o n s may depend on f a c t o r s s u c h a s t h e c o n s t i t u e n t s o f w a t e r and t o t a l c o n t e n t o f f l u o r i n e i n foods. Ozone d e p l e t i o n i n t h e s t r a t o s p h e r e . O r g a n o f l u o r i n e g a s e s s u c h a s Freon, CC12F2, a r e f o u n d t o d e c o m p o s e o z o n e i n t h e o z o n e l a y e r o f t h e s t r a t o -

TABLE I V EIOGEOCHEMISTRY OF FLUORINE Abundance i n t h e U n i v e r s e : l g n e o u s Rocks: 625ppm; Sandstones: F r e s h Water: 270ppm; 0.09ppm; 3D0-900OF/1O6Si Shales: Seawater: 740ppm 1.3ppm L i m e s t o n e s : 330ppm

Air:

0.01pgm-3

S o i l s : 200ppm: f i x e d i n many c l a y m i n e r a l s and i n a p a t i t e . The v e g e t a t i o n f r o m F - r i c h s o i l s i s t o x i c t o g r a z i n g mammals. F - d e f i c i e n c y i s a s s o c i a t e d w i t h d e n t a l decay. M a r i n e P l a n t s : 4.5ppm; Land P l a n t s : 0.5-40ppm 150-500ppm i n mammalian M a r i n e A n i m a l s : 2ppm; Land A n i m a l s : s o f t t i s s u e s , 1500ppm i n bones.

Function: n o t proved e s s e n t i a l , moderately t o x i c t o a l l organisms, b u t a i d s p r o d u c t i o n o f sound t e e t h i n mammals. From Bowen, H.J.M.: T r a c e E l e m e n t s i n B i o c h e m i s t r y , Academic P r e s s , London,

1966

13
sphere. As a r e s u l t ,

UV r a d i a t i o n f r o m t h e s u n r e a c h i n g t h e e a r t h w i l l be
This i s The m a n u f a c t u r e o f F r e o n h a s been

more i n t e n s e and w i l l i n c r e a s e t h e i n c i d e n c e o f human s k i n c a n c e r . deemed a g l o b a l e n v i r o n m e n t a l p r o b l e m . s t o p p e d i n t h e U.S.A. F l u o r i n a t i o n o f chemicals. result,

R e c e n t l y , c h l o r i n a t e d h y d r o c a r b o n s s u c h as As a

PCB, DDT, BHC. a n d D i o x i n , h a v e c a u s e d m u c h e n v i r o n m e n t a l c o n c e r n . t i o n t o some e x t e n t . Consequently,

f l u o r i n a t i o n o f i n d u s t r i a l o r g a n i c c h e m i c a l s has r e p l a c e d c h l o r i n a there i s a trend t o increasing the

f l u o r i n e b u r d e n i n t h e e n v i r o n m e n t , w h i c h i s c o n s i d e r e d t o be one o f t h e f u t u r e environmental problems.

REFERENCES
1 . H i l l e b r a n d WF,
New Y o r k L u n d e l l GEF (1953) EB ( 1 9 6 9 ) Applied Inorganic Analysis. Wiley,

2. K o l t h o f f
3. 4.

IM, Sandell M a c M i l l a n , New Y o r k

Q u a n t i t a t i v e Chemical Analysis. Selective Ion-sensitive

R o s s J r JW, R i s e m a n JH, K r u e g e r J A ( 1 9 7 3 ) Electrodes. Butterworths S m a l l H, S t e v e n s TS,

Bauman WC ( 1 9 7 5 ) A n a l Chem 47:1801-1809

5. L e o n h a r d t W ( 1 9 6 3 ) K e r n e n e r g i e 6:45-46

6. B l a c k b u r n R (1964) A n a l Chem 36:669-671

7. S a n d e l l EB ( 1 9 5 9 ) C o l o r i m e t r i c D e t e r m i n a t i o n o f T r a c e s o f M e t a l s . I n t e r s c i e n c e , New Y o r k 8. 9. B o l t z DF ( 1 9 5 8 ) C o l o r i m e t r i c D e t e r m i n a t i o n o f N o n m e t a l s . New Y o r k Fuwa K (1954) J a p a n A n a l y s t 3:98-104 U n i v e r s i t y o f Tokyo A n a l Chem 56:1640-1644 Addison-Wesley. L e o n a r d MA, West TS ( 1 9 5 8 ) J Chem Soc:2390-2393 U n i v e r s i t y o f Tokyo Spectrochemical Analysis. Interscience.

10. Fuwa K ( 1 9 5 4 ) PhD t h e s i s , 11. B e l c h e r R.

12. Fuwa K.

We1 L.

F u j i w a r a K (1984)

13. We1 L ( 1 9 8 2 ) MS t h e s i s , 14. A b r e n s LH, R e a d i ng

T a y l o r SR (1961)

15. Fuwa K (1950) J J a p a n Chem SOC 71:341-343 16. P e a r s e RWB, Gaydon AG (1950) Chapman and H a l l , London The I d e n t i f i c a t i o n o f M o l e c u l a r S p e c t r a .

17. S t r o c k L W ( 1 9 3 6 ) S p e c t r u m A n a l y s i s w i t h t h e C a r b o n A r c C a t h o d e Laser. Adam H i l g e r , London 18. Fuwa K (1955) J J a p a n Chem SOC 76:14-17 19. S l a v i n W ( 1 9 6 8 ) A t o m i c A b s o r p t i o n S p e c t r o s c o p y . I n t e r s c i e n c e . 20. Tsunoda K. 21. Tsunoda K. F u j i w a r a K. C h i b a K, I t a i K, Fuwa K (1977) A n a l Chem 49:2035-2039 Fuwa K (1979) A n a l Chem 51:2059-2061 F l u o r i d e 17:27-35 H a r a g u c h i H, New Y o r k

22. F u j i m o r i S,

Tsunoda H ( 1 9 8 4 )

14

23. 24.

F a s s e l VA,

K n i s e l e y RN

(1974)

A n a l Chem Hughes

F r y RC, N o r t h w a y SJ. B r o w n RM,

46:lllOA-l120A SK (1980) A n a l Chem 52:1716-1722

W T n e f o r d n e r JD Academic Press,

25. C h i b a K, Y o s h i d a K, T a n a b e K, O z a k i M, H a r a g u c h l H, (1982) Anal Chem 54:761-765

26. B o w e n H J M (1966) T r a c e E l e m e n t s i n B i o c h e m i s t r y .
London

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 15-23 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

15

SPECTROCHEMICAL ANALYSIS

OF

FLUORINE BY ALUMINUM MONOFLUORIDE ABSORPTION I N

H I G H TEMPERATURE

KIN-ICHI

TSUNODA, H I R O K I HARAGUCHI,

AND K E I I C H I R O FUWA
7-3-1 Hongo, Bunkyo-ku, Tokyo

Department o f Chemistry, 113, J a p a n

U n i v e r s i t y o f Tokyo,

ABSTRACT Spectrochemical a n a l y s i s of f l u o r i n e by aluminum monofluoride absorption i s described i n detail. method a r e reviewed. Firstly, the analytical characteristics o f this Then, t h e e f f e c t s o f m a t r i x m o d i f i e r s such as

a l k a l i n e e a r t h m e t a l s o n A1F m o l e c u l a r a b s o r p t i o n a r e d i s c u s s e d o n t h e b a s i s o f t i m e - r e s o l v e d measurements o f t h e m o l e c u l a r a b s o r p t i o n s o f v a r i o u s monofluorides. I n addition, t h e determination o f f l u o r i n e i n urine, serum, and m i l k s a m p l e s b y A1F m o l e c u l a r a b s o r p t i o n i s shown a s an a p p l i c a t i o n o f t h i s m e t h o d t o b i o l o g i c a l samples. INTRODUCTION S e v e r a l y e a r s ago, t h e a u t h o r s d e v e l o p e d a new s p e c t r o c h e m i c a l m e t h o d f o r where m o l e c u l a r a b s o r p t i o n o f aluminum monofluof l u o r i n e d e t e r m i n a t i o n , t h a t i s , a1 u m i n u m m o n o f l u o r i d e (A1 F) m o l e c u l a r absorption spectrometry, r i d e p r o d u c e d i n a h i g h t e m p e r a t u r e g r a p h i t e f u r n a c e was m e a s u r e d a t 227.45 nm u s i n g a d e u t e r i u m l a m p o r a p l a t i n u m h o l l o w c a t h o d e l a m p as t h e l i g h t s o u r c e (1,Z). T h i s m e t h o d has some s u p e r i o r a n a l y t i c a l f e a t u r e s s u c h a s ng o f 1% a b s o r p t i o n ) , a p p l i c a b i l i t y t o s m a l l volume h i g h s e n s i t i v i t y (0.021

(5-20 VL) o f s a m p l e s , and s i m p l i c i t y o f s a m p l e p r e t r e a t m e n t . The m e t h o d has s i n c c been s t u d i e d f u r t h e r and u t i l i z e d f o r t h e d e t e r m i n a t i o n o f t r a c e amounts o f f l u o r i n e i n d i f f e r e n t samples n o t o n l y i n t h i s l a b o r a t o r y b u t a l s o i n s o m e o t h e r s (3-8). s a m p l e s (6). More r e c e n t l y , Tsunoda

a J . ,

i n p a r t i c u l a r , have success-

f u l l y u t i l i z e d t h i s method f o r t h e d e t e r m i n a t i o n o f f l u o r i n e i n b l o o d serum a s u b s t a n t i a l improvement of t h i s method has These s t u d i e s h a v e made i t p o s s i b l e t o been r e p o r t e d b y I t a i I n t h i s paper,

gtd. ( 7 ) .

e v a l u a t e t h e p o t e n t i a l i t y of t h i s method as w e l l as i t s l i m i t a t i o n . r e c e n t s t u d i e s on b o t h a f r a m e w o r k and an a p p l i c a t i o n o f A1F m o l e c u l a r a b s o r p t i o n s p e c t r o m e t r y w i t h an e l e c t r o t h e r m a l g r a p h i t e f u r nace a r e i n t r o d u c e d a f t e r a b r i e f r e v i e w o f t h e a n a l y t i c a l c h a r a c t e r i s t i c s o f t h i s method.

16
Analytical Features o f Aluminum Monofluoride S p e c t r o m e t r y w i t h an E l e c t r o t h e r m a l G r a p h i t e F u r n a c e T h e m o l e c u l a r a b s o r p t i o n s p e c t r u m o f A1F. Molecular Absorption

which i s produced i n a h i g h g r a p h i t e furnace,

t e m p e r a t u r e m e d i u m s u c h a s f l a m e s o r an e l e c t r o t h e r m a l

p r o v i d e s a s h a r p a b s o r p t i o n b a n d n e a r 227.45 nm a s shown i n F i g u r e 1. When an excess amount o f aluminum compared t o f l u o r i n e i s i n t r o d u c e d i n t o t h e medium. t h e i n t e n s i t y o f m o l e c u l a r a b s o r p t i o n o f A1F t h u s f o r m e d i s p r o p o r Thus, f l u o r i n e can Because t h e t i o n a l t o t h e a m o u n t o f f l u o r i n e p r e s e n t i n t h e sample.

b e d e t e r m i n e d b y m e a s u r i n g t h e A1F m o l e c u l a r a b s o r p t i o n . f u r n a c e i s so e f f i c i e n t , m i n e d (1). 227.438 Moreover, and 227.485

f o r m a t i o n o f a l u m i n u m m o n o f l u o r i d e m o l e c u l e s i n an e l e c t r o t h e r m a l g r a p h i t e subnanogram q u a n t i t i e s o f f l u o r i n e c a n be d e t e r -

i t h a s been shown t h a t t h e p l a t i n u m a t o m i c l i n e s a t
nm a r e g o o d a b s o r b e r s o f t h e A1F m o l e c u l a r band. simultaneous d e u t e r i u m lamp, w h i c h i s t h e m o s t commonly

Consequently,

when a p l a t i n u m l a m p i s u s e d a s a l i g h t s o u r c e ,

background c o r r e c t i o n u s i n g a

u s e d c o n v e n t i o n a l a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t e r , c a n be a p p l i e d w i t h o u t a n y i n s t r u m e n t a l m o d i f i c a t i o n (2). I n t h i s study, a commercial a t o m i c a b s o r p t i o n spectrometer w i t h a s i m u l taneous background c o r r e c t i o n system (Model 170-50 f r o m H i t a c h i Co., Ltd..

0.3

: A1 I 2 2 6 3

0.2
P)

.
'

m C

f
n
4:
u)

0.1

ol

226

227
Wavelength. nm

228

F i g u r e 1. M o l e c u l a r a b s o r p t i o n s p e c t r u m o f A1F i n an e l e c t r o t h e r m a l g r a p h i t e furnace. -A-: B a c k g r o u n d s p e c t r u m o b t a i n e d w i t h 5 111 o f 0.01 M a l u m i n u m n i t r a t e s o l u t i o n ; -0-: s p e c t r u m o b t a i n e d w i t h 4.5 n g F a d d e d t o t h e d r i e d b a c k ground solution.

17
Japan) e q u i p p e d w i t h an e l e c t r o t h e r m a l g r a p h i t e f u r n a c e a t o m i z e r (FLA 1 0 0 f r o m N i p p o n J a r r e l l A s h Co.. molecular absorption. L t d . ) w a s u s e d f o r t h e m e a s u r e m e n t o f A1F The a n a l y t i c a l p r o c e d u r e and t h e i n s t r u m e n t a l c o n d i -

. The a n a l y t i c a l p r o c e d u r e i s b a s i c a l l y t h e t i o n s a r e summarized i n Table I

same a s t h a t o f g r a p h i t e f u r n a c e a t o m i c a b s o r p t i o n s p e c t r o m e t r y , e x c e p t t h a t a d d i t i o n o f a l u m i n u m and m a t r i x m o d i f i e r s s u c h a s F e ( I I 1 ) and S r ( I 1 ) were r e q u i r e d (see below). v o l u m e r e q u i r e d was 5-20 uL. The a n a l y t i c a l w o r k i n g r a n g e f o r A1F m o l e c u l a r ng and t h e s a m p l e This s e n s i t i v i t y is s l i g h t l y b e t t e r o r almost and t h e l a n t h a n u m / a l i z a r i n a b s o r p t i o n s p e c t r o m e t r y i n t h i s s y s t e m w a s ca. 0.1-1.0

t h e same a s a n y o t h e r m e t h o d f o r f l u o r i n e d e t e r m i n a t i o n s u c h as t h e f l u o r i d e i o n s e l e c t i v e e l e c t r o d e ( I S E method), f l u o r i d e b l u e method. I t s h o u l d be n o t e d , h o w e v e r , t h a t t h e s e n s i t i v i t y o f B a c k g r o u n d a b s o r p t i o n due t o NaCl up t o Even when t h e b a c k g r o u n d m e a s u r e m e n t o f A1F a b s o r p t i o n o f t e n s u f Recently,

i t was f o u n d t h a t t h i s b l a n k

t h i s method v a r i e s w i t h t h e t y p e o f f u r n a c e , a p p a r e n t l y more so t h a n t h e measurement o f a t o m i c a b s o r p t i o n . 0.05

c o u l d be c o r r e c t e d b y t h e p r e s e n t s y s t e m .

c o r r e c t i o n s y s t e m was u t i l i z e d , fered from a high blank signal.

s i g n a l was m a i n l y due t o t h e m o l e c u l a r a b s o r p t i o n o f a l u m i n u m o x i d e enhanced b y t h e p r e s e n c e o f o x y g e n a s an i m p u r i t y i n a r g o n s h e a t h gas. b l a n k s i g n a l can, therefore, t h e a r g o n g a s u s i n g an o x y g e n t r a p p i n g column. The be decreased c o n s i d e r a b l y b y p u r i f i c a t i o n o f

TABLE I EXPERIMENTAL PROCEDURES AND CONDITIONS FOR FLUORINE DETERMINATION BY A j F MOLECULAR ABSORPTION SPECTROMETRY WITH AN ELECTROTHERMAL GRAPHITE FURNACE A p p l i c a t i o n o f aluminum s o l u t i o n ' (0.01

M, 2 0 u l )
D r y i n g I (150"C, A s h i n g I (600"C, 20 s ) 15 s )

Cooling o f furnace A p p l i c a t i o n o f sample s o l u t i o n ( 5 u l ) D r y i n g I 1 (150"C, A s h i n g 11 (600"C, 20 s )

30 s )

A t o m i z a t i o n and measurement (2500C.

7 s)

' E x p e r i m e n t a l c o n d i t i o n s a t e a c h s t a g e a r e shown i n p a r e n t h e s e s . 'Aluminum n i t r a t e s o l u t i o n c o n t a i n i n g f e r r i c n i t r a t e (0.01 n i t r a t e ( 0 . 0 1 M)

1

M) and s t r o n t i u m

18

This method can detect not only inorganic fluoride but also organic fluorine. that is, covalently bound fluorine ( 8 ) . However, the application of this method is limited to nonvolatile fluorine compounds as pointed out by Venkateswarlu ( 8 ) . Volatile fluorine compounds may escape from the furnace before the formation of AlF molecules and predecomposition is required for such samples. Effects of Cations on the Formation of A1F Molecules in an Graphite Furnace Electrothermal

As described previously, the nature of A1F molecular absorption spectrometry is similar t o t h a t o f graphite furnace atomic absorption spectrometry. However, the former method also has other aspects because of the complexity of the molecular formation process of A1F in a graphite furnace. In particular, the effects of different cations on this method are noteworthy. It has been shown that some transition metals such as iron, nickel, This situation can be seen i n Figure 2. and cobalt reduced the background absorption due to aluminum oxide (1). In this figure, background absorption due to aluminum solution was considerably reduced by the addition of Co(I1). This is interpreted as being due to Co(I1) cutting the chemical bond of aluminum and oxygen ( 9 ) . Moreover, alkaline earth metals, especially strontium, were found to act as a spectral buffer for enhancing the signals and eliminating the influence of concomitants in the A1F molecular absorption method (1). Thus, i n practice, Sr(I1) and Fe(II1) have been added to the aluminum solution as matrix modifiers for the determination of fluorine
in

actual samples (1-4).

Figure 2. Background absorption spectra i n an electrothermal graphite furnace. -0-: Aluminum nitrate solution (0.01 M); -A-: Mixed solution of aluminum nitrate (0.01 M) and strontium (0.01 M); -0-: Mixed solution of aluminum nitrate (0.01 M). and cobalt nitrate (0.01 M).

19
TABLE I 1 S E N S I T I V I T I E S O F FLUORINE DETERMINATION BY DIATOMIC MOLECULAR ABSORPTION SPECTROMETRY WITH AN ELECTROTHERMAL GRAPHITE FURNACE Species Wavelength (nmf 227.45 211.23 233.75 358.8 606.44 662.94 500.5 Sensitivity C o m p o s i t i o n of A d d i t i v e s

(1% Abs.,ng)
0.085 0.021 0.16 0.11 1.5 0.50 0.38 1.5

A1 F GaF InF M9F CaF SrF BaF

none Fe(II1). Fe(III), Sr(I1) Sr(I1)

Fe( 1 1 1 ) . K ( I )

To u n d e r s t a n d t h e e f f e c t s o f c o e x i s t i n g c a t i o n s . m e t a l s and Group I I I B e l e m e n t s . m o l e c u l e s w e r e o b t a i n e d (10.11).

these s t u d i e s were

extended t o t h e molecular absorption o f o t h e r monohalides o f a l k a l i n e e a r t h F i r s t , the absorption spectra o f those Table I 1 summarizes t h e s e n s i t i v i t i e s o f Although aluminum other

the molecular absorptions o f various monofluorides.

monofluoride molecular absorption provides the highest sensitivity,

m o n o f l u o r i d e m o l e c u l e s a l s o h a v e r e l a t i v e l y s h a r p and s t r o n g band s p e c t r a . These s p e c t r a c a n a l s o b e u t i l i z e d f o r f l u o r i n e d e t e r m i n a t i o n i n t h e same way a s t h e a l u m i n u m m o n o f l u o r i d e spectrum. Furthermore, time-resolved signals o f the molecular absorption o f those monofluorides w i t h various c o e x i s t i n g c a t i o n s were o b t a i n e d u s i n g a r a p i d response measurement system i n o r d e r t o i n v e s t i g a t e t h e i n f l u e n c e of t h e s e c a t i o n s . example. ug/ml), tion, The t i m e - r e s o l v e d s i g n a l p r o f i l e s o f GaF m o l e c u l a r a b s o r p t i o n a r e s h o w n i n F i g u r e 3 a s a n P r o f i l e a) shows GaF m o l e c u l a r a b s o r p t i o n w i t h t h e s o l u t i o n o f p r o f i l e b ) w i t h Ga ( 1 0 m M ) . Na ( 1 0 m M ) a n d F ( 2 I n addip r o f i l e c ) w i t h Ga ( 1 0 m M ) , G a ( l 0 mM) and F ( 2 u g / m l ) ,

S r (10 mM) and F ( 2 ug/ml).

1 0 m M Fe was added t o e a c h o f t h e s a m p l e s t o m i n i m i z e t h e m o l e c u l a r I n p r o f i l e c, t h e a b s o r p t i o n s i g n a l was but The t e m p e r a t u r e The f i r s t peak c o r r e s p o n d s t o t h a t o f p r o f i l e a,

absorption o f g a l l i u m oxides. s p l i t i n t o t w o peaks.

t h e s e c o n d peak a p p e a r e d i n t h e h i g h e r t e m p e r a t u r e r e g i o n . a b s o r p t i o n ( p r o f i l e d).

r e g i o n o f t h e second peak i n p r o f i l e c was c l o s e t o t h a t o f S r F m o l e c u l a r S i m i l a r r e s u l t s w e r e o b t a i n e d f o r a l l o t h e r monoThese o b s e r v a t i o n s s u g g e s t t h e c o m p l e x f l u o r i d e s o f Group I I I B e l e m e n t s .

t

f o r m a t i o n o f f l u o r i d e w i t h c o e x i s t i n g c a t i o n s on t h e g r a p h i t e s u r f a c e and

20

Time, s

F i g u r e 3. T i m e - r e s o l v e d s i g n a l p r o f i l e s o f GaF m o l e c u l a r a b s o r p t i o n . a, Ga and Fe ( e a c h 0.01 M) and 2 u g / m l o f F; b, Ga, Fe and Na ( e a c h 0.01 M ) and 2 u g / m l o f F; c , Ga, Fe and S r ( e a c h 0.01 M ) and 2 u g / m l o f F: d, S r (0.01 M ) and 2 u g / m l o f F ( S r F m o l e c u l a r a b s o r p t i o n p l o t t e d ) . C a t i o n s w e r e added as n i t r a t e . GaF a n d S r F m o l e c u l a r a b s o r p t i o n s w e r e o b t a i n e d a t 211.45 nm and 663.1 nm, r e s p e c t i v e l y .

t h e e f f e c t o f t h e c a t i o n s on c o n t r o l l i n g t h e v a p o r i z a t i o n o f f l u o r i d e . Moreover, furnace.

i t has been observed t h a t t h e a p p a r e n t t e m p e r a t u r e o f a S r F

Thus, t h e e n h a n c i n g e f f e c t o f S r ( I 1 ) i s i n t e r p r e t e d as a r e s u l t

m o l e c u l e i s a l m o s t t h e s a m e a s t h a t o f a n A1F m o l e c u l e i n t h e g r a p h i t e

of p r o t e c t i o n o f f l u o r i d e b y S r ( I 1 ) u n t i l t h e t e m p e r a t u r e o f A1F f o r m a t i o n
on t h e g r a p h i t e s u r f a c e i s r e a c h e d . D e t e r m i n a t i o n o f F l u o r i n e i n B i o l o g i c a l S a m p l e s b y A1F M o l e c u l a r A b s o r p t i o n Spectrometry F l u o r i n e i n u r i n e , serum, t i o n o f th!s tion of fluorine and m i l k s a m p l e s w e r e d e t e r m i n e d a s an a p p l i c a T a b l e I 1 1 shows t h e d e t e r m i n a u r i n e sample (Standard Reference The u r i n e s a m p l e s w e r e d i l u t e d w i t h The a n a l y t i c a l p r o c e d u r e was t h e same m e t h o d t o b i o l o g i c a l samples. i n a freeze-dried

M a t e r i a l 2671 f r o m NBS i n U.S.A.)

(3).

d i s t i l l e d w a t e r b y v a r i o u s d i l u t i o n f a c t o r s and a p p l i e d d i r e c t l y t o t h e A 1 F m o l e c u l a r a b s o r p t i o n measurements. a s d e s c r i b e d i n S e c t i o n 2.

A s c a n be seen f r o m t h e t a b l e , f l u o r i d e concen-

t r a t i o n s o b t a i n e d b y t h i s m e t h o d w e r e i n good a g r e e m e n t w i t h b o t h c e r t i f i e d

21

TABLE 111 DETERMINATION OF FLUORINE I N FREEZE-DRIED U R I N E FROM NBS (SRM 2671) Samples Dilution factor Found, r.cg/mL Certified values w/ml 7.14 f 0.48

AI ~1
elevatedlevel sample low-level sample

I SE*
7.6 k 0.3 7.00 f 0.24 7.00 f 0.24 0.80 f 0.03 0.80 f 0.05

100 40 20
10

7.1 f 0.1 6.90 f 0.18 7.10 f 0.18 0.89 f 0.03 0.81 f 0.05

0.84 5 0.08

' A l F molecular absorption spectrometry 2 F l u o r i d e i o n s e l e c t i v e e l e c t r o d e method

NBS v a l u e s and t h o s e o b t a i n e d by t h e I S E method.

I n addition,

the f l u o r i n e and t h e In

c o n t e n t i n b l o o d serum samples was a l s o d e t e r m i n e d by t h i s method,

r e s u l t s were compared w i t h t h o s e o b t a i n e d by t h e I S E method ( T a b l e I V ) . water,

t h i s e x p e r i m e n t , t h e serum samples were d i l u t e d 10 t i m e s w i t h d i s t i l l e d and A 1 F m o l e c u l a r a b s o r p t i o n s were measured i n t h e same manner as w i t h u r i n e s a m p l e s . R e c o v e r y v a l u e s o f ca. 100% i n T a b l e I V i n d i c a t e t h a t A 1 F m o l e c u l a r a b s o r p t i o n method i s a r e l i a b l e method f o r t h e d e t e r m i n a t i o n
o f f l u o r i n e i n b l o o d serum.

On t h e o t h e r hand,

t h e v a l u e s o b t a i n e d by t h i s As aluminum mono-

method were much h i g h e r t h a n t h o s e by t h e I S E method.

f l u o r i d e m o l e c u l a r a b s o r p t i o n s p e c t r o m e t r y can d e t e c t n o t o n l y i o n i c f l u o r i d e , b u t a l s o c o v a l e n t l y bound f l u o r i n e ( s e e S e c t i o n 2). t h e d i f f e r e n c e

TABLE I V

D E T E R M I N A T I O N OF FLUORINE I N BLOOD SERUM

A1F method' Added Recovery3 (r.cg/mL) (Z) 0.34 f 0.04 0.31 f 0.04 105 95 95 95

Sample

Found (w/mL) 0.13 f 0.02 0.12 f 0.02 0.21 f 0.03 0.16 ? 0.02

I S E method2
Found (ug/mL)

I
I1 111

0.076 f 0.002 0.025 f. 0.001 0.020 f 0.001 0.024 f 0.001

0.40

f 0.04

IV

0.35 f 0.04

' A l F molecular absorption spectrometry 2 F l u o r i d e i o n s e l e c t i v e e l e c t r o d e method 3 F l u o r i d e was added a t 0.2 vg/ml

I

22
between t h e t w o methods may be a t t r i b u t a b l e t o t h e e x i s t e n c e o f s o - c a l l e d "nonionic fluorine" i n serum samples (6,12). however, r e m a i n unclear. t h i s method The a c t u a l c h e m i c a l f o r m s o f "nonionic fluorine,"

A l t h o u g h t o t a l f l u o r i n e i n b o t h u r i n e and serum samples can be d e t e r m i n e d b y d i r e c t m e a s u r e m e n t u s i n g t h e A1F m o l e c u l a r a b s o r p t i o n , samples. As shown i n T a b l e V, cannot be a p p l i e d d i r e c t l y t o t h e d e t e r m i n a t i o n o f f l u o r i n e i n bovine m i l k l o w e r a n a l y t i c a l v a l u e s were o b t a i n e d w i t h F u r t h e r i n v e s t i g a t i o n suggested t h a t t h e d i r e c t a n a l y s i s o f t w i c e d i l u t e d m i l k samples by t h i s method compared t o t h o s e f o r t h e d i s t i l l e d samples. c o n t a i n e d i n m i l k samples. t h i s d i f f e r e n c e was m a i n l y due t o t h e i n t e r f e r e n c e o f o r g a n i c m a t t e r s A l t h o u g h i n t e r f e r e n c e by o r g a n i c m a t t e r s w i t h p r e t r e a t m e n t w i l l be necessary f o r samples t h i s m e t h o d was p a r t i a l l y r e m o v e d b y t h e a d d i t i o n o f a l a r g e amount o f m a t r i x m o d i f i e r such as S r ( I I ) , containing high concentrations o f organic matters.

TABLE V

D E T E R M I N A T I O N OF FLUORIDE I N B O V I N E MILK BY A1F MOLECULAR ABSORPTION

SPECTROMETRY

Method Direct-' D r y ashing'

F l u o r i d e Content (ng/ml)

33.6 ?r 15.9

105.3 f 19.8

'The samples were d i l u t e d t w i c e w i t h d i s t i 1 l e d water. 'The samples were dry-ashed w i t h t h e a d d i t i o n o f sodium carbonate and t h e n d i s t i l l e d w i t h a steam d i s t i l l a t i o n s ys tern.

CONCLUSION
AluminiAm m o n o f l u o r i d e m o l e c u l a r a b s o r p t i o n s p e c t r o m e t r y has e x c e l l e n t a n a l y t i c a l f e a t u r e s as w e l l as some l i m i t a t i o n s . samples, T h i s method i s a p p l i c a b l e t o t h e d e t e r m i n a t i o n o f f l u o r i n e i n v a r i o u s b i o l o g i c a l and e n v i r o n m e n t a l a l t h o u g h i t may need o c c a s i o n a l sample p r e t r e a t m e n t such as d r y a s h i n g f o l l o w i n g d i s t i l l a t i o n o r d i f f u s i o n techniques. t r i b u t e t o d i f f e r e n t areas o f f l u o r i d e research.

It i s hoped t h a t

t h e p o t e n t i a l i t y o f t h e method becomes w i d e l y known and t h a t i t can con-

23

REFERENCES 1. Tsunoda K, 2. Tsunoda K, F u j i w a r a K, Fuwa K (1977) A n a l Chem 49:2035-2039 Fuwa K (1979) A n a l Chem 51:2059-2061 Fuwa K (1980) A n a l Chem 52:1582-1585 C h i b a K, H a r a g u c h i H, H a r a g u c h i H,

3. C h i b a K, Tsunoda K.

4. T a k a t s u A. C h i b a K, O z a k i M, F u w a K, H a r a g u c h i H ( 1 9 8 4 ) S p e c t r o c h i m A c t a 39B:365-370

5. D i t t r i c h K (1979)

A n a l Chim A c t a 111:123-135 Tsunoda H (1984) W i n t e r LD, F l u o r i d e 17:27-35 H a g e n D F ( 1 9 8 3 ) A n a l Chem

6. F u j i m o r i S, I t a i K,
7. I t a i K. 8. V e n k a t e s w a r l u P, 55: 2232-2236 9. Tsunoda K. 10. Tsunoda K.

Tsunoda H (1985) A n a l Chim A c t a 171:293-301 P r o k o p RA,

F u j i W a r a K, H a r a g u c h i H.

Fuwa K (1978) A n a l Chem 50:861-865 Fuwa K (1984) S p e c t r o c h i m A c t a 35B:715-729

11. Tsunoda K, C h i b a K. H a r a g u c h i H, C h a k r a b a r t i CL, Fuwa K (1982) Canadian J S p e c t r o s c 27:94-97

12. Taves DR (1968)

N a t u r e 220:582-583

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 25-29 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

25

DETERMINATION OF SUBMICROGRAM QUANTITIES OF FLUORIDE BY A R A P I D AND HIGHLY SENSITIVE METHOD

K A Z U Y O S H I I T A I AND HUM10 TSUNODA

D e p a r t m e n t o f H y g i e n e and P u b l i c H e a l t h , U n i v e r s i t y , M o r i o k a , I w a t e 020, J a p a n School o f Medicine, Iwate Medical

ABSTRACT

A new m e t h o d h a s b e e n d e v e l o p e d f o r t h e d e t e r m i n a t i o n o f s u b m i c r o g r a m
q u a n t i t i e s o f f l u o r i d e . It c o n s i s t s o f s e p a r a t i n g f l u o r i d e f r o m t h e t e s t sample b y t h e p y r o h y d r o l y t i c s e p a r a t i o n method and d e t e r m i n a t i o n o f t h e r e s u l t i n g aqueous HF s o l u t i o n b y e l e c t r o t h e r m a l m o l e c u l a r a b s o r p t i o n spect r o m e t r y as a l u m i n u m m o n o f l u o r i d e ( A l F ) . sensitive, T h i s method i s rapid, highly and c a n be used f o r t h e d e t e r m i n a t i o n o f t o t a l ( o r g a n i c and

i n o r g a n i c ) f l u o r i d e i n v a r i o u s o r g a n i c compounds and b i o l o g i c a l m a t e r i a l s .

INTRODUCTION

A number o f a n a l y t i c a l m e t h o d s h a v e been d e s c r i b e d f o r t h e d e t e r m i n a t i o n
o f f l u o r i n e i n o r g a n i c f l u o r i d e compounds

and b i o l o g i c a l m a t e r i a l s (1-3). I n addition,

Many o f t h e s e m e t h o d s a r e r a t h e r c o m p l e x and t i m e - c o n s u m i n g . a c c u r a t e l y determined.

when t h e f l u o r i n e c o n c e n t r a t i o n s o f t h e s a m p l e s a r e l o w , t h e y c a n n o t b e

A r a p i d and h i g h l y s e n s i t i v e m e t h o d f o r f1uo:ide
developed.

d e t e r m i n a t i o n h a s been electro-

I t combines t h e p y r o h y d r o l y t i c s e p a r a t i o n method w i t h

t h e r m a l a l u m i n u m m o n o f l u o r i d e (A1F) m o l e c u l a r a b s o r p t i o n s p e c t r o m e t r y . The p y r o h y d r o l y t i c s e p a r a t i o n method i s v a l u a b l e f o r r a p i d i s o l a t i o n o f f l u o r i n e f r o m i n o r g a n i c r e f r a c t o r y compounds, many o r g a n i c m a t e r i a l s (4-7). m i n a t i o n by extraneous f l u o r i d e s . s u c h as c a l c i u m f l u o r i d e , o r from I n a d d i t i o n , t h i s method i s f r e e f r o m contaBy u s e o f an a u t o s a m p l e r , f l u o r i n e can

be a c c u r a t e l y and p r e c i s e l y d e t e r m i n e d a t t h e p p b l e v e l b y e l e c t r o t h e r m a l A1F m o l e c u l a r a b s o r p t i o n s p e c t r o m e t r y . MATERIAL AND METHODS Apparatus F i g u r e 1 shows t h e a p p a r a t u s u s e d i n t h e p y r o h y d r o l y s i s . t i o n tube,

A quartz reac-

550 m m l o n g w i t h an i n t e r n a l d i a m e t e r o f 1 3 m m i s m a i n t a i n e d b y

26
a main-heater a t a p p r o x i m a t e l y 1100C. The t u b e i s g r a d u a l l y heated t o

500C w i t h a s u b - h e a t e r . O x y g e n g a s i s s c r u b b e d w i t h 0.2N KOH s o l u t i o n , s i l i c a g e l , a n d a c t i v e charcoal, and p a s s e d t h r o u g h t h e t u b e w i t h w a t e r v a p o r a t a r a t e o f a p p r o x P l a t i n u m gauze, used a s a c a t a l y s t f o r t h e o x i d a t i o n . i m a t e l y 1 l/min.

is

p l a c e d a t t h e end o f t h e t u b e .

A p i e c e o f q u a r t z wool i s placed behind t h e A suit-

p l a t i n u m gauze f o r t r a p p i n g v o l a t i l e m e t a l s and m e t a l o x i d e s (6). r e n e b o t t l e w i t h cover. The a p p a r a t u s as f o l l o w s :

a b l e a m o u n t o f c o n d e n s a t e i s w e i g h e d w i t h an e l e c t r i c b a l a n c e i n a p o l y s t y used f o r d e t e r m i n a t i o n o f f l u o r i n e i n t h e condensate a r e equipped w i t h a and a u t o Ltd.).

a t o m i c a b s o r p t i o n s p e c t r o m e t e r m o d e l AA-855.

d e u t e r i u m s i m u l t a n e o u s b a c k g r o u n d c o r r e c t i o n s y s t e m ; g r a p h i t e f u r n a c e atomi z e r , m o d e l FLA-100; sampler, m i c r o p r o c e s s o r r e a d o u t s y s t e m , m o d e l MC-100; ( a 1 1 m a n u f a c t u r e d b y N i p p o n J a r r e l l - A s h Co., model AS-301

F i g u r e 1. A d i a g r a m s h o w i n g a p p a r a t u s u s e d f o r p y r o h y d r o l y s i s 1: 0.2" KOH s o l u t i o n : 2: S i l i c a g e l ; 3: A c t i v e c h a r c o a l : 4: F l o w m e t e r : 5: W a t e r f l a s k ( 9 0 C ) ; 6: R e a c t i o n t u b e ( Q u a r t z ) ; 7: S u b h e a t e r (0-500C); 8: M a i n h e a t e r ( 1 1 0 0 C ) : 9: S a m p l e b o a t ; 1 0 : P l a t i n u m g a u z e ; 1 1 : Q u a r t z w o o l : 12: Condenser; 13: S t y r e n e b o t t l e : 14: E l e c t r i c b a l a n c e .

Reagents

A s t a n d a r d f l u o r i d e s o l u t i o n was p r e p a r e d b y d i s s o l v i n g a n a l y t i c a l - g r a d e
s o d i u m f l u o r i d e (Wako P u r e C h e m i c a l s ) i n d i s t i l l e d w a t e r .

A 1% a l u m i n u m

s o l u t i o n w a s p r e p a r e d b y d i s s o l v i n g 5 g o f a l u m i n u m m e t a l p o w d e r (99.5%. Wako) i n 1 0 m l o f c o n c e n t r a t e d n i t r i c a c i d a n d d i l u t i n g t o 5 0 0 m l w i t h d i s t i l l e d water. t o 1000 m l . Procedure S a m p l e s r a n g i n g f r o m s e v e r a l mg t o g w e r e w e i g h e d i n t o t h e s a m p l e b o a t , and w e r e i n t r o d u c e d i n t o t h e s u b - h e a t e r a t u r e o f t h e sub-heater

4

A 2% b a r i u m s o l u t i o n was p r e p a r e d b y d i s s o l v i n g 38.06 g
i n d i s t i l l e d w a t e r and d i l u t i n g

o f a n a l y t i c a l - g r a d e b a r i u m n i t r a t e (Wako)

p a r t o f t h e tube.

When t h e t e m p e r -

was g r a d u a l l y r a i s e d t o 500C,

t h e s a m p l e b o a t was

i n t r o d u c e d i n t o t h e c e n t e r o f t h e main-heater.

F l u o r i n e i n t h e s a m p l e was

27
i s o l a t e d a s HF a n d w a s c o n d e n s e d w i t h w a t e r v a p o r b y t h e c o n d e n s e r . w i t h cover. lected.
AP

a p p r o p r i a t e a m o u n t o f t h e c o n d e n s a t e was w e i g h e d i n t o a p o l y s t y r e n e b o t t l e C o n d e n s a t e s r a n g i n g f r o m 5 t o 20 g ( m l ) w e r e u s u a l l y c o l s u c h a s CaF2, t h e a d d i t i o n o f some w h i c h a c t e d a s an a c i d f l u x , was r e q u i r e d (7). For very resistant materials,

tungsten t r i o x i d e ,

Measurement o f F l u o r i n e i n C o n d e n s a t e The p r o c e d u r e f o r t h e d e t e r m i n a t i o n o f f l u o r i n e i n t h e c o n d e n s a t e b y e l e c t r o t h e r m a l A1F m o l e c u l a r a b s o r p t i o n s p e c t r o m e t r y i s shown i n F i g u r e 2

(5).

To 800 p 1 o f t h e c o n d e n s a t e o r a s t a n d a r d s o l u t i o n i n a p o l y e t h y l e n e The p o l y e t h y l e n e c u p was s e t i n t h e Conditions f o r

c u p was added 200 p l o f t h e a l u m i n u m s o l u t i o n c o n t a i n i n g 0.1% a l u m i n u m and

1% b a r i u m a s n i t r a t e , r e s p e c t i v e l y .
autosampler.

The m i x t u r e c o n t a i n i n g t h e c o n d e n s a t e and t h e a l u m i n u m s o l u T h e a b s o r b a n c e o f A1F g e n e r a t e d w a s

t i o n was a u t o m a t i c a l l y i n J e c t e d i n t o t h e g r a p h i t e f u r n a c e . m e a s u r e m e n t a r e s h o w n i n F i g u r e 2.

a u t o m a t i c a l l y m e a s u r e d and t h e f l u o r i n e c o n c e n t r a t i o n o f t h e c o n d e n s a t e was determined by a microprocessor readout system from a c a l i b r a t i o n curve obtained by using standard f l u o r i d e solutions.

Condensate

; At
U
I

1 -

800,~ l soIution'200p

P o l y e t h y l e n e cup

Autosampler (20,u Q ) D r y i n g (,l50V 30sec) I C h a r r i n g ( 8 3 O C 15sec) A t o m i z i n g (2800C 7 s e e ) Measurement

F i g u r e 2. P r o c e d u r e f o r d e t e r m i n a t i o n o f f l u o r i d e b y A l F m o l e c u l a r a b s o r p t i o n spectrometry. A1 s o l u t i o n c o n s i s t s o f 0.1% A1 and 1% BA a s a n i t r a t e .

I I

RESULTS AND D I S C U S S I O N Recoveries o f f l u o r i n e f r o m several standard m a t e r i a l s by use o f t h e p r e s e n t m e t h o d a r e shown i n T a b l e I. I n a l l t h e f l u o r i d e compounds t e s t e d , t h e f l u o r i n e contents found were i n agreement w i t h t h e c a l c u l a t e d values, a n d t h e r e c o v e r y w a s n e a r l y 100%.

As observed by Warf gt

aj.

(4).

fluo-

r i d e s c a n be g r o u p e d i n t o t w o c a t e g o r i e s : and a s l o w l y p y r o h y d r o l y z a b l e group. alkaline-earth

1

a r a p i d l y p y r o h y d r o l y z a b l e group, F l u o r i n e compounds

The f l u o r i d e s o f t h e a l k a l i n e and

m e t a l s b e l o n g t o t h e l a t t e r g r o u p (6).

28 TABLE I

RECOVERIES

OF

FLUORINE FROM STANDARD MATERIALS

% F calculated
NaF CaF2 45.2 48.7 76.4 11.0

% F found
45.5 48.2' 76.1 10.7

Recovery (%) 107 99.0 99.6 97.3

N ( CF2CF2CF2CF3)3
Cg IH 5 C6 H4 F

'W03 was added

o f the alkaline-earth resistant. an a c i d f l u x .

metals,

such as c a l c i u m f l u o r i d e .

are very heat

F l u o r i n e i n t h e s e m a t e r i a l s was s e p a r a t e d b y t h e u s e o f W03 as A c c o r d i n g t o Leuven Q t d l (7). i n t h e absence o f t h e f l u x I n t h i s method, the

t h e r e c o v e r y o f f l u o r i n e i n t h e s e m a t e r i a l s was p o o r , b e l o w 50%, b u t i n t h e p r e s e n c e o f t h e f l u x t h e r e c o v e r y w a s n e a r l y 100%. r e c o v e r y o f f l u o r i n e i n CaF2 w i t h t h e u s e o f W03 was n e a r l y 100%. T a b l e I 1 shows t h e f l u o r i n e c o n t e n t s o f v a r i o u s o r g a n i c m a t e r i a l s d e t e r m i n e d b y t h i s method. I n order f o r rapid pyrohydrolytic separation o f f l u o r i n e i n t h e s e m a t e r i a l s a s HF, f l u o r i n e c o n t e n t o f orchard leaves, w a s r e p o r t e d t o b e 4 ppm. m e t h o d ( 1 ) was 5.320.61

W03 w a s a d d e d a s a n a c i d f l u x .

The

NBS S t a n d a r d R e f e r e n c e M a t e r i a l 1571,

W i t h t h i s m e t h o d , i t w a s f o u n d t o b e 4.6 ppm. and t h e r e l a t i v e s t a n d a r d d e v i a t i o n was

T h e a v e r a g e f l u o r i n e c o n t e n t i n t h e same s a m p l e d e t e r m i n e d b y A.O.A.C. ppm (n=7),

11.5%.

The a v e r a g e v a l u e o f u n p o l i s h e d r i c e o b t a i n e d b y t h i s m e t h o d was The a v e r a g e f l u o r i d e v a l u e i n t h e same s a m p l e a s d e t e r m i n e d b y m e t h o d ( 1 ) was 0.7020.13 ppm ( n = 5 ) , and t h e r e l a t i v e s t a n d a r d

0.57 ppm. t h e A.O.A.C.

TABLE I 1 DETERMINATION Sample

OF

FLUORIDE I N V A R I O U S MATERIALS n x f S.D. (uLl/g) 4.6 0.57 2.4

c.v

(%)

O r c h a r d 1e a v e s Unpolished r i c e Human h a i r Human serum

0.21

4.6 3.4

6
5 4

2 0.02

f 0.24

10.0
14.0

0.043 f 0.006

29 d e v i a t i o n was 18.6%. Sakurai

& &.

(9) r e p o r t e d t h a t t h e average f l u o r i n e f o r which l i t t l e information i s The a v e r a g e was 2.4 ppm and t h e The l a r g e v a r i a t i o n , c o m p a r e d t o

c o n t e n t i n u n p o l i s h e d r i c e i n J a p a n was 0.89 ppm. The f l u o r i n e c o n t e n t o f human h a i r , available, r e l a t i v e s t a n d a r d d e v i a t i o n w a s 10%. t h o s e f o r t h e p l a n t s a m p l e s shown, r e a c t i o n tube. was d e t e r m i n e d b y t h i s method.

i s p r e s u m a b l y due t o t h e h a i r b e i n g

s c a t t e r e d b y a n e l e c t r o s t a t i c f o r c e w h i l e t h e s a m p l e was p l a c e d i n t h e The f l u o r i n e c o n c e n t r a t i o n o f human s e r u n i s s o l o w t h a t a c c u r a t e d e t e r m i n a t i o n h a s been d i f f i c u l t . i s needed f o r t h e d e t e r m i n a t i o n . done w i t h o n l y 1 m l o f sample. f l u o r i n e i n t h e sample.

A l a r g e volume o f serum sample

t h e d e t e r m i n a t i o n was

W i t h t h i s method,

The r e l a t i v e l y h i g h r e l a t i v e s t a n d a r d the extremely low concentration o f

d e v i a t i o n seen i n T a b l e I 1 i s due t o

CONCLUSION T h e c o m b i n e d u s e o f p y r o h y d r o l y s i s a n d e l e c t r o t h e r m a l A1F m o l e c u l a r a b s o r p t i o n s p e c t r o m e t r y h a s made i t p o s s i b l e t o d e t e r m i n e f l u o r i n e i n many m a t e r i a l s r a p i d l y and a c c u r a t e l y . (e.g. CaF2) c a n b e a c c o m p l i s h e d . I n t h e presence o f a c a t a l y s t such as Aluminum m o n o f l u o r i d e (AlF) m o l e c u l a r When s a m p l e s a r e o b t a i n e d f r o m pyro-

W03, a r a p i d s e p a r a t i o n o f f l u o r i n e f r o m i n o r g a n i c r e f r a c t o r y c o m p o u n d s
a b s o r p t i o n s p e c t r o m e t r y c a n be u s e d t o d e t e r m i n e t h e l e v e l o f f l u o r i n e i n a s a m p l e o f 20 g l w i t h h i g h a c c u r a c y . hydrolysis, c o n t a m i n a t i o n b y extraneous f l u o r i d e i s avoided.

ACKNOWLEDGEMENT T h i s research i s supported by Grant-in-Aid R e s e a r c h (No. 60480193) f o r 1985-1987 S c i e n c e and C u l t u r e o f t h e G o v e r n m e n t o f Japan f o r Co-operative S c i e n t i f i c from t h e M i n i s t r y o f Education,

REFERENCES
1. O f f i c i a l Methods o f A n a l y s i s (1970) 6.023 B a i l e y JJ, G e h r i n g DG ( 1 9 6 1 ) 1 1 t h Ed AOAC. W a s h i n g t o n , USA,

2.
3. 4.

A n a l Chem 33:1760

V e n k a t e s w a r l u P ( 1 9 7 7 ) M e t h B i o c h e m A n a l 24:93 W a r f JC, C l i n e WD, Tevebaugh RD (1954) A n a l Chem 26:342 K a k a b a d s e GJ, M a n o h i n N a t u r e 229:626 B e r n s EG, Van L e u v e n HCE, 296: 36 I t a i K, Tsunoda H. I t a i K, S a k u r a i S.

5.

B,

B a t h e r JM, W e l l e r EC.

Woodbridge

(1971)

6.
7. 8. 9.

Van D e r Zwaan PW (1972) A n a l C h i m A c t a 59:293 R o t s c h e i d GJ, B u i s W J ( 1 9 7 9 ) F r e s e n i u s Z A n a l Chem I k e d a M (1985) A n a l C h i m A c t a 171:293 Tsurfoda H (1983) F l u o r i d e 16:175

H. Tsunoda and M.-H. Y u (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 31-42 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

31

PLASMA E M I S S I O N SPECTROMETRY OF FLUORINE AND I T S ENVIRONMENTAL APPLICATION

H I R O K I HARAGUCHI
Department o f Chemistry, F a c u l t y o f Science, Bunkyo-ku, T o k y o 11 3, J a p a n The U n i v e r s i t y o f Tokyo,

ABSTRACT Spectrochemical a n a l y s i s o f f l u o r i n e by plasma emission spectrometry i s described. First, p l a s m a e m i s s i o n s p e c t r o m e t r y and i t s p r e s e n t s t a t u s a r e reviewed e s p e c i a l l y f o r t h e d e t e r m i n a t i o n o f n o n m e t a l l i c elements. Secondly atomic emission spectrometry u s i n g h e l i u m microwave-induced plasma ( M I P ) a t atmospheric pressure, w h i c h i s an e f f i c i e n t e x c i t a t i o n s o u r c e f o r n o n m e t a l i s d i s c u s s e d a s an e l e m e n t Finally, the determination l i c elements as w e l l as m e t a l l i c elements, s e l e c t i v e d e t e c t o r i n gas c h r o m a t o g r a p h y (GC).

o f f l u o r i n e i n w a t e r s a m p l e s b y t h e GC/helium M I P s y s t e m i s shown as an e x p e r i m e n t a l a p p l i c a t i o n o f h e 1 i u m M I P t o e n v i r o n m e n t a l and g e o c h e m i c a l sampl es.

INTRODUCTION S p e c t r o c h e m i c a l a n a l y s i s u s i n g h i g h t e m p e r a t u r e media has e v o l v e d s i n c e t h e w o r k o f B u n s e n a n d K i r c h h o f f (1). g r a p h i t e furnaces, emission, They used a c h e m i c a l f l a m e as an e x c i t a t i o n s o u r c e i n f l a m e e m i s s i o n s p e c t r o m e t r y . Flames, a r c s , sparits, d i s c h a r g e t u b e s and s o f o r t h h a v e b e e n u s e d as a t o m i z a and f l u o r e s c e n c e s p e c t r o m e t r y . t i o n / e x c i t a t i o n s o u r c e s i n a n a l y t i c a l a t o m i c s p e c t r o s c o p y such as a t o m i c absorption, A t o m i c a b s o r p t i o n s p e c t r o m e t r y (AAS) w a s d e v e l o p e d a b o u t 30 y e a r s ago, and i t h a s b e e n u s e d a s a p o w e r f u l a n a l y t i c a l t o o l i n t r a c e e l e m e n t a n a l y s i s (2). I n AAS, f l a m e s and e l e c t r o t h e r m a l g r a p h i t e f u r n a c e s a r e c o m m o n l y As i s w e l l k n o w n , employed as e f f i c i e n t a t o m i z e r s f o r most elements. n o n m e t a l l i c elements.

however, AAS i s used f o r t h e d e t e r m i n a t i o n o f m e t a l l i c e l e m e n t s and n o t T h e r e a s o n why a t o m i c a b s o r p t i o n o r e v e n a t o m i c e m i s s i o n s p e c t r o m e t r y cannot be a p p l i e d t o t h e d e t e r m i n a t i o n o f non-metall i c e l e m e n t s i s t h a t t h e i r a t o m i c r e s o n a n c e l i n e s e x i s t i n t h e vacuum u l t r a v i o l e t region, nation. W i t h f l u o r i n e , t h e w a v e l e n g t h o f t h e a t o m i c r e s o n a n c e l i n e i s 95.5 nm. w h i C h r e q u i r e s an e x c i t a t i o n e n e r g y o f 12.92 eV. The e x p e r i m e n t a l i n c o n v e a s shown i n T a b l e I. T h e r e f o r e , molecular absorption o r e m i s s i o n s p e c t r o m e t r y h a s been e x a m i n e d f o r n o n m e t a l l i c e l e m e n t d e t e r m i -

32

TABLE I ATOMIC RESONANCE LINES OF NONMETALLIC ELEMENTS Element Wavelength (nm) 121.6 165.7 113.5 130.2 95.5 177.5 180.7 134.7 148.8 183.0 E x c i t a t io n E n e r g y (eV) 10.21 7.45 70.87 9.48 12.92 6.95 6.83 9.16 8.29 6.78 Ionization E n e r g y (eV) 13.60 11.26 14.53 13.62 17.42 10.49 10.36 12.97 11.81 10.45

C
N

F
P

S
c1 Br

nience

of

vacuum UV s p e c t r o s c o p y and l a c k o f e f f i c i e n t e x c i t a t i o n s o u r c e s However, i f an e x c i t a t i o n s o u r c e w h i c h a l l o w s

have hampered t h e d e v e l o p m e n t o f a n a l y t i c a l a t o m i c s p e c t r o s c o p i c t e c h n i q u e s f o r f l u o r i n e determination. invented, sible. t h e e x c i t a t i o n o f f l u o r i n e a t o m s much h i g h e r t h a n t h e l e v e l a t 12.91 eV i s t h e n a t o m i c e m i s s i o n s p e c t r o m e t r y o f f l u o r i n e w i 11 become posR e c e n t d e v e l o p m e n t o f r a r e gas p l a s m a s u s i n g h e l i u m and a r g o n Hence p l a s m a e m i s s i o n s p e c t r o m e t r y w i l l be

p r o v i d e s such a p o t e n t i a l i t y .

b r i e f l y discussed i n t h e n e x t section. Plasma e m i s s i o n s p e c t r o m e t r y f o r s p e c t r o c h e m i c a l a n a l y s i s P l a s m a e x c i t a t i o n s o u r c e s u s i n g r a r e gases s u c h as a r g o n and h e l i u m have been d e v e l o p e d f o r e m i s s i o n s p e c t r o c h e m i c a l a n a l y s i s . c a l l e d plasma emission spectrometry. a commonly u s e d s p e c t r o m e t r i c m e t h o d f o r t r a c e a n a l y s i s . because i t has many a n a l y t i c a l advantages, (3). The p l a s m a e x c i t a t i o n s o u r c e s w h i c h a r e now commonly used and commerc i a l l y a v a i l a b l e a r e as f o l l o w s : a r g o n gas; and, rarely, ( 1 ) d i r e c t c u r r e n t p l a s m a (DCP). u s i n g .commonly, p l a s m a (MIP), using (2) i n d u c t i v e l y c o u p l e d p l a s m a ( I C P ) , n i t r o g e n gas; (3) m i c r o w a v e - i n d u c e d a r g o n gas u s i n g argon The method i s now However, plasma As d e s c r i b e d p r e v i o u s l y , AAS has been

e m i s s i o n s p e c t r o m e t r y has s u b s t a n t i a l l y r e p l a c e d AAS i n t r a c e a n a l y s i s i n c l u d i n g h i g h s e n s i t i v i t y and p r e c i s i o n , w i d e d y n a m i c ranges, a n d s i m u l t a n e o u s m u l t i - e l e m e n t a n a l y s i s

o r h e l i u m gas.

33

ICP is excited by a radio frequency of 27.12 MHz. OCP and ICP using argon gas have been established as excitation sources for atomic emission spectrometry, and are extensively used for the determination of elements 4 ) . Despite their excellent excitation capability, including P, S. and I (
they cannot be applied to the determination of most nonmetallic elements, especially halogens, for reasons mentioned previously. In 1976, Beenakker designed a new microwave cavity, making it possible to sustain helium plasma at atmospheric pressure ( 5 ) . A schematic drawing of the Beenakker-type microwave cavity is shown i n Figure 1. The cavity is

T-@
c

observe

Figure 1. A simple drawing o f TM type cavity. (I), cylindrical wall; fixed bottom; (3), removajO\id; (4) quartz discharge tube: (5). holder; (6) coupling loop; (7) connector: (8) Teflon insulator: (9, 10) tuning screw.

(2).

made of copper metal with a diameter of about 10 cm. At the center, the cavity has a hole of 8 mm. i n which a quartz discharge tube of 6 mm in 0.d. is set. Helium plasma is sustained inside a discharge tube of 1-3 m m i n 1.d. by applying microwave power of 75-100 W with the helium gas flow rate of 80-500 ml/min. The helium M I P at atmospheric pressure is operated at the lower power with a relatively small gas flow rate, and thus it is a small string-like plasma. Therefore, only gaseous volatile compounds can be directly introducsd into the plasma. Some electrothermal vaporization devices are required when nonvolatile or metallic compounds are analyzed ( 6 ) . Despite this limitation in sample introduction, the large excitation energy of helium plasma offers an advantage over argon plasma. That is, the excitation erlergy o f helium plasma is about 19.5 eV, while that of

34

a r g o n p l a s m a i s a b o u t 11.7 eV. a t o m s (Hem o r A r m ) . f o r example,

The e x c i t a t i o n p o w e r i n h e l i u m o r a r g o n

plasma i s determined by t h e e x c i t a t i o n energy o f h e l i u m o r argon metastable

I n T a b l e 11, e m i s s i o n w a v e l e n g t h s i n t h e r e g u l a r U V
Fluorine,

and v i s i b l e r e g i o n s f o r s e v e r a l n o n m e t a l l i c e l e m e n t s a r e shown. a t 690.3 nm as the

when e x c i t e d up t o 14.53 eV p r o v i d e s a n a t o m i c e m i s s i o n l i n e t r a n s i t i o n t o t h e e n e r g y l e v e l a t 12.92 eV i n s t e a d o f Such an e x c i t a t i o n can o n l y be emission l i n e s i n the regular Generally i f excitation a t a level higher

t h e v a c u u m e m i s s i o n l i n e a t 95.5 nm. r e a l i z e d w i t h h e l i u m plasma.

t h a n t h e f i r s t e x c i t a t i o n l e v e l i s performed, level t o the lower level. TABLE I 1

UV a n d v i s i b l e r e g i o n s a r e o b s e r v e d a s t h e t r a n s i t i o n s f r o m t h e h i g h e r

WAVELENGTHS AND EXCITATION ENERGIES OF NONMETALLIC ELEMENTS I N THE REGULAR UV AND VISIBLE REGIONS Element Wavelength (nm) 656.3 486.1 247.9 746.8 777.2 690.3 253.6 190.0 479.5 I 1 470.5 I 1 Excitation E n e r g y (eV) 12.09 2.75 7.68 1.99 0.74 4.53 7.18 6.50 5.89 4.28 6.92 12.45

H
C
N
0
F P

S c1
Br

206.2
516.1 I 1

_I<

I 1 i n d i c a t e s i o n i c l i n e s , and o t h e r l i n e s a r e a t o m i c l i n e s .
e l e m s

____ P l a s m a e m i s s i o n s p e c t r o m e t r y o f f l u o r i n e and i t s a p p l i c a t i o n t o an s e l e c t i v e d e t e c t o r i n gas c h r o m a t o g r a p h y

I n T a b l e 111, i n t e n s i t i e s (7). plasma. The

t h e wavelength t a b l e s o f f l u o r i n e emission l i n e s e x c i t e d These e m i s s i o n l i n e s w e r e i d e n t i f i e d f r o m t h e e m i s s i o n C,

w i t h h e l i u m MIP a r e shown a l o n g w i t h t r a n s i t i o n a s s i g n m e n t s and r e l a t i v e s p e c t r u m w h i c h was o b s e r v e d b y i n t r o d u c i n g gaseous c6F6 i n t o t h e h e l i u m w a v e l e n g t h t a b l e s o f n o n m e t a l l i c e l e m e n t s (H,

N, 0, F, P,

35

S.

C1,

Br,

I ) e x c i t e d w i t h h e l i u m M I P were proposed i n t h i s l a b o r a t o r y (7).

many a t o m i c e m i s s i o n l i n e s a r e o b s e r v e d i n I n t h e t a b l e , I.P.. o r eV). atomic lines,

As can be seen i n T a b l e 111, the v i s i b l e region. t i o n p o t e n t i a l (cm-

I, IR. and

IMIT

indicate ionizarespectively.

o b s e r v e d r e l a t i v e i n t e n s i t y , and

r e p o r t e d r e l a t i v e i n t e n s i t y i n t h e MIT Wavelength T a b l e s (8). f l u o r i n e atomic lines.

The i n t e r f e r i n g band means t h a t some m o l e c u l a r e m i s s i o n bands o v e r l a p w i t h Such a t o m i c e m i s s i o n l i n e s a r e n o t a v a i l a b l e f o r a n a l y s i s because o f band i n t e r f e r e n c e s .

I t c a n be n o t e d f r o m T a b l e I 1 1

t h a t t h e e m i s s i o n l i n e a t 685.602 nm i s t h e b e s t one f o r f l u o r i n e d e t e r m i n a t i o n because i t i s m o s t i n t e n s e and has no i n t e r f e r i n g band. TABLE 111

OBSERVED E M I S S I O N LINES

OF FLUORINE I N ATMOSPHERIC PRESSURE HELIUM-MIP

140,554 cm- (17.42 eV) Energy (cm- ) Interfering Band
CN 2nd

F l u o r i n e I.P. Wavelength (nm)

Intensity Transition IR IMIT 4 3 4

I
I I I I I I

775.470 757.341 755.224 748.272 742.564 739.868 733.195 731.102 720.237 712.799 703.745 690.982 690.246 687.022 685.602 683.426 677.397 641.366 634.850 623.964

60
40 40 80 150 400

104,732-1 17,624 102.841-1 16,042 102.681 -1 1 5.91 9 102,681-1 16,042 102,681-1 16.144 102,407-1 1 5.91 9 102.407-116.042 104,732-118,406 105,057-1 18.938 105.057-119.083 104,732-1 18.938 102,841-1 17.309 102,681-1 17.165 102,841-1 17.393 102,407-1 16,988 102,681-1 17,309 132,407-117.165 102.841-1 18.429 102,681-118,429 102,407-118.429

2
8 32 15

200
125 125 150

I
I

5
3 11 17 15 44 15 100 21 15 17 32 32

I I
I

N$,

N2 2nd

200
150 500 150 1000 300 100 150

I I I 1
I

NH 2nd

I I
I

200
300

OH 2nd(w)
OH 2nd(w)

36
As m e n t i o n e d p r e v i o u s l y , a r g o n I C P i s n o t a s u i t a b l e e x c i t a t i o n s o u r c e f o r n o n m e t a l l i c e l e m e n t s because o f i t s l i m i t a t i o n i n e x c i t a t i o n e n e r g y . Recently, however, f l u o r i n e e m i s s i o n l i n e s e x c i t e d w i t h a r g o n ICP w e r e report, f l u o r i n e emission l i n e s were and t h e y r e p o r t e d (9). According t o t h i s

o b s e r v e d a t a r e l a t i v e l y l o w e r p o s i t i o n o f t h e p l a s m a o n l y when v o l a t i l e o r g a n i c compounds w i t h o u t h u m i d i t y w e r e i n t r o d u c e d i n t o t h e I C P . w e r e m o s t l y weak o r i n s e n s i t i v e l i n e s . available f o r the determination o f fluorine i n trace quantities. As a G C d e t e c t o r f o r h a l o g e n s , t h e E C D ( e l e c t r o n c a p t u r e d e t e c t o r ) i s commonly used because o f i t s h i g h s e n s i t i v i t y . t o a l l halogens, i.e., t o f l u o r i n e w h i l e i t i s t o o t h e r halogens. used as a However, t h e ECD r e s p o n d s
i t has n o e l e m e n t s e l e c t i v i t y and i s n o t s e n s i t i v e

A r g o n ICP, t h e r e f o r e , i s n o t y e t

On t h e o t h e r h a n d , h e l i u m M I P Thus, i f h e l i u m M I P i s The has

can d e t e c t each element by wavelength s e l e c t i o n . GC/helium M I P system.

GC d e t e c t o r , i t can be s e n s i t i v e and e l e m e n t - s e l e c t i v e .
i n w h i c h h e l i u m M I P i s u s e d as t h e GC d e t e c t o r .

been d e v e l o p e d and s u c c e s s f u l l y a p p l i e d t o a n a l y s i s o f v a r i o u s samples e s p e c i a l l y f o r c h e m i c a l s p e c i a t i o n (10-16).

A s c h e m a t i c d i a g r a m o f t h e G C / M I P s y s t e m u s e d i n t h i s l a b o r a t o r y i s shown
i n F i g u r e 2. The i n s t r u m e n t a l c o m p o n e n t s a n d some o p e r a t i n g c o n d i t i o n s a r e summarized i n Table IV.

A S h i m a d z u GC-6A d u a l c o l u m n g a s c h r o m a t o g r a p h

r---------

GC

F i g u r e 2. S c h e m a t i c d i a g r a m o f t h e G C - M I P s y s t e m . ( S ) helium tank, (T) t h e r m a l c o n d u c t i v i t y d e t e c t o r , (H) h e a t e d t r a n s f e r t u b e , (C) m i c r o w a v e c a v i t y , ( G ) m i c r o w a v e g e n e r a t o r , (L) l e n s , ( M ) m o n o c h r o m a t o r . ( P ) p h o t o m u l t i p l i e r , ( V ) h i g h v o l t a g e s u p p l y , (A) p i c o a m m e t e r , ( R ) c h a r t r e c o r d e r .

37
e q u i p p e d w i t h a t h e r m a l c o n d u c t i v i t y d e t e c t o r (TCD) i s employed. i s used f o r m o n i t o r i n g t h e s o l v e n t s i g n a l s . The TCD

The i n t e r f a c e b e t w e e n t h e g a s The

chromatograph and t h e d i s c h a r g e t u b e o f t h e M I P i s c o n s t r u c t e d f r o m a h i g h t e m p e r a t u r e e l e c t r o m a g n e t i c f o u r - w a y v a l v e and a h e a t e d t r a n s f e r tube. four-way l o c a t e d on t h e f r o n t panel o f t h e gas chromatograph. t h e GC column. i n g g l a s s tape, TABLE IV INSTRUMENTAL COMPONENTS AND OPERATING CONDITIONS v a l v e i s a t t a c h e d t o t h e TCD o v e n and c a n be o p e r a t e d b y a s w i t c h The v a l v e i s u s e d f o r

s o l v e n t v e n t i l a t i o n when a l a r g e a m o u n t o f s o l v e n t v a p o r i s e l u t e d o u t f r o m The t r a n s f e r t u b e ( 5 0 cm l o n g ) i s c o n s t r u c t e d f r o m an i n n e r a copper p r o t e c t i o n tube, a thermocoupler, shieldnichrome wire, and o u t e r g l a s s t a p e i n s u l a t i o n . n i c k e l t u b e ( 2 mm i.d.),

I tern
Gas c h r o m a t o g r a p h Detector C a r r i e r gas ( f l o w r a t e ) Microwave generator Microwave c a v i t y Discharge tube Plasma gas ( f l o w r a t e ) Monochromator Grating Photomult i p 1i e r tube Amp1 i f i e r

Model o r C o n d i t i o n S h i m a d z u GC-6A T h e r m a l c o n d u c t i v i t y (TCD) H e l i u m (80 m l / m i n ) 2.45 GHz, 20-200 W ( I t o h Chotampa, Ltd.) B e e n a k k e r ' s TMol0 Quartz tube H e l i u m (80 m l / m i n ) 50-cm f o c a l l e n g t h ( N i p p o n J a r r e l l - A s h Co.) cavity

1200 g r o o v e s / m m
R955 (Hamamatsu P h o t o n i c s , Ltd.)

DC a m p l i f i e r ( p a r t o f ICAP-500) ( N i p p o n J a r r e l l - A s h Co.)

D e t e r m i n a t i o n o f u l t r a t r a c e l e v e l s o f f l u o r i d e i n w a t e r and u r i n e s a m p l e s I n r e c e n t years, t h e d e t e r m i n a t i o n o f f l u o r i n e i n v a r i o u s samples has been e x t e n s i v e l y i n v e s t i g a t e d because o f c l i n i c a l interest. and e n v i r o n m e n t a l has been An i o n - s e l e c t i v e e l e c t r o d e f o r f l u o r i d e i o n ( F - I S E ) As i s w e l l known, t h e F-ISE

w i d e l y used as a c o n v e n t i o n a l a n a l y t i c a l method because o f i t s convenience a n d e c o n o m y (17-20). f l u o r i d e ions only,

i s sensitive t o free

and i t i s i n f l u e n c e d b y c o e x i s t i n g c a t i o n s .

Other

a n a l y t i c a l m e t h o d s f o r f l u o r i n e d e t e r m i n a t i o n a r e c o l o r i m e t r y (21) and g a s

38

chromatography (22).

Recently, a molecular absorption s p e c t r o m e t r i c method

f o r f l u o r i n e d e t e r m i n a t i o n was d e v e l o p e d , i n which t o t a l f l u o r i n e was d i r e c t l y d e t e r m i n e d by m e a s u r i n g aluminum m o n o f l u o r i d e (AlF) m o l e c u l a r absorption a t 227.45 nm (23-25).

I n t h i s s e c t i o n , t h e f e a s i b i l i t y of t h e GC-MIP

system i s evaluated a s a and applied t o The

conventional a n a l y t i c a l method f o r f l u o r i n e determination, r e l i a b i l i t y of t h e a n a l y t i c a l values obtained by t h a t obtained by t h e F-ISE.

t h e a n a l y s i s o f s e a w a t e r , t a p w a t e r , pond w a t e r , and u r i n e (26).

GC-MIP

i s compared with

MATERIALS AND METHODS

The GC-MIP system used i s s i m i l a r t o t h e instrument shown in Figure 2.

Pyrex g l a s s chromatographic column packed w i t h 15% DC-200 on 80/100 mesh U n i p o r t 8 a n d a n o t h e r Pyrex g l a s s column packed w i t h 3% OV-17 on 80/100 mesh Uniport HP were used. The operating c o n d i t i o n s of t h e gas chromatoThe plasma o p e r a t i n g c o n d i t i o n s a n d g r a p h a r e summarized i n T a b l e V.

d e t e c t i o n systems a r e t h e same a s those shown i n Table IV. The f l u o r i d e ions in water and u r i n e samples were e x t r a c t e d by t h e f o l lowing procedure: Four ml of s a m p l e s o l u t i o n were t a k e n i n a 15 ml t e s t and 1 ml of 9.5 M H C 1 and 1 ml of t u b e w i t h a ground g l a s s s t o p p e r , of t o l u e n e ) were t h e n added.

e x t r a c t a n t s o l u t i o n (0.5 g of t r i r n e t h y l c h l o r o s i l a n e (TMCS) dissolved i n 1 1 The t e s t t u b e was shaken v i g o r o u s l y f o r 30

TABLE V

OPERATING CONDITIONS OF GAS CHROMATOGRAPH

Experimental Conditions Column Column packing Column temp. ("C) I n j e c t i o n temp. ("C) Detector oven temp. ("C) Transfer tube temp. ("C) C a r r i e r gas C a r r i e r g a s flow r a t e (ml/min) Pyrex g l a s s 1 m x 3 mm i.d. 15% OC-200 on 80/100 mesh Uniport B Pyrex g l a s s 3 m x 3 mm i . d . 3% OV-17 on 80/100 mesh Uniport HP 80 150

40
160 160 170 Helium 80

150
160 Helium 80

39
min.

A p a r t o f TMCS i n t h e o r g a n i c p h a s e r e a c t e d w i t h f l u o r i d e i o n s i n t h e
p r o d u c i n g TMFS (trimethylfluorosilane). Therefore, fluo-

sample s o l u t i o n ,

r i d e i o n s i n t h e aqueous phase w e r e e x t r a c t e d I n t o t h e o r g a n i c phase as

TMFS.
phase,

F o l l o w i n g t h e s e p a r a t i o n o f t h e o r g a n i c phase f r o m t h e aqueous t h e o r g a n i c p h a s e was t r a n s f e r r e d i n t o a s a m p l i n g t u b e . the analytical nm was used. One u l o f s a m p l e e x t r a c t a n t

I n t h e measurements o f plasma e m i s s i o n spectrometry, a t o m i c l i n e o f f l u o r i n e a t 685.6 s o l u t i o n was i n j e c t e d i n t o t h e GC column. t i m e (24 s).

The TMFS f l u o r i n e p e a k a p p e a r e d t h e four-way valve

f i r s t and was d e t e c t e d b y t h e M I P d e t e c t o r a t i t s c h a r a c t e r i s t i c r e t e n t i o n I m m e d i a t e l y a f t e r t h e TMFS peak appeared, was s w i t c h e d t o v e n t t h e s o l v e n t w i t h m o n i t o r i n g of t h e s o l v e n t p e a k s b y t h e TCD. T h i s p r o c e d u r e was n e c e s s a r y t o a v o i d e x t i n g u i s h i n g t h e p l a s m a and t o m a i n t a i n good p l a s m a s t a b i l i t y . t o 1 vg/ml

A t y p i c a l chromatogram o f f l u o r i n e w i t h
Only a TMFS s i g n a l c o r r e s p o n d i n g

t h e GC-MIP s y s t e m i s shown i n F i g u r e 3. d e t e c t i o n (lower curve), ( u p p e r curve).

f l u o r i n e was s e l e c t i v e l y o b s e r v e d i n t h e c h r o m a t o g r a m u s i n g M I P w h i l e i t w a s n o t d e t e c t e d u s i n g TCD d e t e c t i o n

S 1 2 3 4 5 6 7 8 9

RetentionTime (min)
F i g u r e 3. T y p i c a l c h r o m a t o g r a m s o f f l u o r i n e w i t h t h e GC-MIP system. (a) TMFS, ( b ) b e n z e n e , ( c ) TMCS. ( d ) t o l u e n e . U p p e r : TCD d e t e c t i o n . Lower: M I P detection.

40 RESULTS The d e t e c t i o n l i m i t s , e x t r a c t i o n e f f i c i e n c i e s , r e p r o d u c i b i l i t i e s . and l i n e a r d y n a m i c r a n g e s o b t a i n e d f o r f l u o r i n e ( p r e s e n t a s TMFS) w i t h t h e DC-

200

column are summarized i n Table VI.

Preconcentration o f the extract a n d 95% e x t r a c t i o n e f f i c i e n c y u s e o f 4 u1 s a m p l e s t h i s procedure allowed

4 t i m e s g a v e 3% r e l a t i v e s t a n d a r d d e v i a t i o n ,
w i t h a 1 v1 sample. A l t h o u g h p r e c o n c e n t r a t i n g t h e e x t r a c t 10

T h i s i s a v e r y r e l i a b l e method o f f l u o r i n e analysis. t i m e s and

r e s u l t e d i n a somewhat p o o r e r e x t r a c t i o n e f f i c i e n c y ,

f l u o r i n e d e t e c t i o n a t a v e r y l o w c o n c e n t r a t i o n w i t h good r e p r o d u c i b i l i t y . A l u m i n u m i o n s i n t e r f e r e d w i t h t h e d e t e r m i n a t i o n o n l y when t h e i r c o n c e n t r a t i o n was 100 t i m e s g r e a t e r t h a n t h a t o f f l u o r i d e i o n s . The G C - M I P s y s t e m was a p p l i e d t o t h e d e t e r m i n a t i o n o f f l u o r i n e i n seaand u r i n e . The c o n v e n t i o n a l F - I S E m e t h o d was The The r e s u l t s a r e p r e s e n t e d i n T a b l e V I I . water, t a p water, pond water, a l s o used f o r analysis. other.

a n a l y t i c a l d a t a o b t a i n e d f r o m t h e s e t w o methods a r e c o n s i s t e n t w i t h each The r e c o v e r i e s o f t h e G C - M I P s y s t e m w e r e i n t h e r a n g e o f 95% t o

110%. and t h e r e p r o d u c i b i l i t y o f a n a l y s i s i n s e a w a t e r was a b o u t 6%.

CONCLUSION Element-selective characterization. detection a t the ultratrace level u t i l i z i n g atomic useful technique f o r trace spectroscopic methods i s a n e w l y developed,

Among t h o s e a n a l y t i c a l a t o m i c s p e c t r o m e t r i c methods, t h e

G C / h e l i u m M I P m e t h o d f o r f l u o r i n e i s a s a c c u r a t e and s e n s i t i v e a s t h e F-ISE m e t h o d w i t h t h e a d v a n t a g e s o f a g r e a t e r s e l e c t i v i t y and w i d e r a p p l i c a b i l ity. We hope t h a t i n t h e n e a r f u t u r e p l a s m a e m i s s i o n s p e c t r o m e t r y u s i n g h e l i u m M I P and a r g o n I C P c o m b i n e d w i t h g a s o r l i q u i d c h r o m a t o g r a p h y w i l l be f u r t h e r d e v e l o p e d a s a u s e f u l a n a l y t i c a l method. TABLE V I

ANALYTICAL

FIGURES OF MERIT

FOR THE GC-MIP

Sample Size, vl DL vg/ml

SYSTEM WITH A DC-ZOO EE %

RSD

COLUMN^
LDR Decades

Preconcentrated i n extraction, 4 times Preconcentrated i n e x t r a c t i o n , 10 t i m e s

1 4

0.02
0.004

95

3.0

3.3

75

6.5

4.1

'DL=detection l i m i t ; EE=extrattion e f f i c i e n c y : RSD=relative standard d e v i a t i o n : LDR=linear dynamic range.

41
TABLE V I I APPLICATION OF THE G C - M I P SYSTEM TO DETERMINATION OF FLUORINE I N V A R I O U S SAMPLES ( c o n c . o f F; u g / m l ) GC-MIP i n sample' added t o sample 1.25 0.10 0.10 1.00 total Recovery added

F-ISE i n sample'

Seawater Tap w a t e r Pond w a t e r Urine

1.30f0.06 0.11~0.01 0.13tO. 01 0.98t0.03

1.30f0.03 0.12+0.01 0.11*0.01 0.93+0.03

2.48

1.18 0.11 0.10 0.95

94 110 100 95

0.23
0.21 1.66

'The v a l u e s are estimated concentration d e v i a t i o n s corresponding t o the e t e r r e a d i n g s (mV). 'The v a l u e s f a r e e s t i m a t e d s t a n d a r d d e v i a t i o n s f o r t h r e e s e p a r a t e measurements.

REFERENCES
1. K i r c h h o f f G, dam. PP Bunsen R (1860) Ann Phys Chem 110:161-165 E l s e v i e r Amster-

2. C a n t l e JE(ed)(1982) A t o m i c A b s o r p t i o n S p e c t r o m e t r y .
3. F a s s e l V A (1979) Anal Chem 51:1290A-l308A

4. Fuwa K, H a r a g u c h i H ( e d s ) ( 1 9 8 0 ) I n d u c t i v e l y C o u p l e d P l a s m a A t o m i c E m i s s i o n S p e c t r o m e t r y ( i n Japanese). Nanko-do. Tokyo, pp 5. Beenakker C I M (1980) S p e c t r o c h i m A c t a 31B:483-486

6. Chiba K,
7. Tanabe K, 9. F r y RC,

Kurosawa M. Haraguchi H,

Tanabe K,

Haraguchi H (1984) Chem L e t t 1984:75-78 MIT Press, Cambridge

Fuwa K (1981) S p e c t r o c h i m A c t a 36B:119-127 Wavelength Tables.

8. H a r r i s o n GH (1979) M.I.T. 10. Tanabe K, Haraguchi H,

N o r t h w a y SJ, Brown RM, Hughes SK (1980) Anal Chem 52:1716-1722 Fuwa K (1981) S p e c t r o c h i m A c t a 36B:633-639

11. C h i b a K, Y o s h i d a K. T a n a b e K, H a r a g u c h i H, Fuwa K ( 1 9 8 3 ) A n a l Chem 1 2 55:450-453 12. Chiba K, Haraguchi H (1983) A n a l Chem 55:1504-1508

13. Fuwa K. H a r a g u c h i H, M o r i t a M, Van L o o n JC ( 1 9 8 2 ) J S p e c t r o s c S O C J p n 31:289-305

14. E t t r e LS (1978) J Chromatog S c i 16:396-417 15. Carnahan JW, M u l l i g a n KJ, Caruso JA (1981) Anal Chim A c t a 130:227-241 16. Haraguchi H, T a k a t s u A (1984) Bunseki:108-114 17. Taves DR (1968) T a l a n t a 15:1015-1023 18. V e n k a t e s w a r l u P (1974) Anal Chem 46:878-882 19. V e n k a t e s w a r l u P (1975) C l i n Chim A c t a 59:277-282

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20. C h i b a K, T s u n o d a K, Umezawa Y, H a r a g u c h i H. F u j i w a r a A n a l Chem 52:596-598

21. V e n k a t e s w a r l u P.

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K (1980)

S i t a P (1971)

A n a l Chem 43:758-760

22. B e l i s l e J, Hagen DF (1978) A n a l Biochem 87:545-555 23. Tsunoda K, F u j i w a r a K. Fuwa K (1977) A n a l Chem 49:2035-2039 Fuwa K (1980) A n a l Chem 52:1582-1585 Fuwa K, H a r a g u c h i H (1984) S p e c t r o c h i m O z a k i M. 24. C h i b a K, Tsunoda K, 25. T a k a t s u A. C h i b a K, A c t a 39B:365-370 H a r a g u c h i H,

26. C h i b a K, Y o s h i d a K, Tanabe K. O z a k i M, H a r a g u c h i H, W i n e f o r d n e r , JD, Fuwa K (1982) A n a l Chem 54:761-764

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 43-46 0 1986 Elsevier Science Publishers B.V.. Amsterdam - Printed in The Netherlands

43

SEPARATION A N D DETERMINATION

OF

FLUORIDE I O N B Y HIGHLY SELECTIVE I O N -

EXCHANGE R E S I N S H I S A S H I TANAKA,
YOSHITO OKABAYASHI, TERUMICHI NAKAGAWA, A I K O INOUE,

R I E OH,

AND MASAHIKO C H I K U M A
F a c u l t y o f Pharmaceutical Sciences. K y o t o 606, J a p a n ABSTRACT New f u n c t i o n a l r e s i n s f o r t h e s e l e c t i v e c o l l e c t i o n o f f l u o r i d e i o n s w e r e p r e p a r e d b y t h e m o d i f i c a t i o n o f an a n i o n - e x c h a n g e r e s i n w i t h l a n t h a n u m c o m p l e x e s o f a1 i r a r i n - c o m p l e x o n e ALC-La b o t h ion-exchange and p h y s i c a l (ALC) o r i t s s u l f o n a t e d compound (ALCS). w h e r e a s ALCS-La i s adsorbed by resin. and o r ALCS-La a d s o r p t i o n o n t o anion-exchange i s adsorbed by p h y s i c a l adsorption, K y o t o U n i v e r s i t y , Y o s h i d a , Sakyo-ku,

A d s o r p t i o n o f f l u o r i d e i o n s b y t h e r e s i n l o a d e d w i t h ALC-La complete.

t h e d e s o r p t i o n o f f l u o r i d e i o n s w i t h sodium hydroxide s o l u t i o n were a l m o s t These r e s i n s m a i n t a i n e d t h e c a p a b i l i t y o f f l u o r i d e i o n a d s o r p Interferences by ions. acid. c a t i o n s and a n i o n s I n t e r f e r e n c e by these ions Satisfactory results t i o n even a f t e r b e i n g used t e n times.

w e r e n e g l i g i b l e e x c e p t f o r Fe3+ a n d AT3+

c o u l d be masked by t h e a d d i t i o n o f t a r t a r i c r i d e i o n s i n t a p water. INTRODUCTION

were o b t a i n e d by t h e use o f t h e s e r e s i n s i n t h e p r e c o n c e n t r a t i o n o f f l u o -

F l u o r i n e i s an e l e m e n t t h a t commands a t t e n t i o n b o t h as a h a z a r d o u s e l e ment and a n u t r i e n t . The s e n s i t i v i t y o f t h e ion-selective electrode method i s n o t always s a t i s f a c t o r i l y h i g h f o r t h e d e t e r m i n a t i o n o f f l u o r i d e i o n s i n b i o l o g i c a l o r e n v i r o n m e n t a l samples. i s o f t e n necessary. However, Preconcentration o f the ions developed. This paper a h i g h l y s e l e c t i v e and e f f e c t i v e s o r b e n t f o r resins f o r the selective collec-

t h e c o l l e c t i o n o f t h e f l u o r i d e i o n has n o t been deals w i t h t h e development o f f u n c t i o n a l t i o n o f f l u o r i d e ion. react with fluoride

A t t e m p t s w e r e made t o d e v e l o p f u n c t i o n a l r e s i n s ion specifically. For t h i s purpose, i.e., alizarin-

t h r o u g h m o d i f i c a t i o n o f a n i o n - e x c h a n g e r e s i n w i t h some r e a g e n t s w h i c h c o m p l e x o n e - l a n t h a n u m c o m p l e x (ALC-La) and i t s s u l f o n a t e d compound (ALCS-La) i t s high selecdue t o t h e

(1) w e r e examined.

ALCS-La

has t w o c h a r a c t e r i s t i c s , On t h e o t h e r h a n d ,

t i v i t y and s e n s i t i v i t y t o w a r d s f l u o r i d e i o n s . and i t s a b i l i t y t o a d s o r b onto t h e ion-exchange resin. ALCS-La,

44
presence o f a s u l f o n a t e group, anion-exchange r e s i n , above. g e n t (2). has an i o n - e x c h a n g e c a p a b i l i t y w i t h an

i n a d d i t i o n t o t h e t w o c h a r a c t e r i s t i c s mentioned

A r e a g e n t s u c h a s ALCS-La i s r e f e r r e d t o a s a t e r f u n c t i o n a l r e a F i g u r e l s h o w s t h e s t r u c t u r e o f ALC a n d ALCS s y n t h e s i z e d f r o m a c i d and i m i n o d i a c e t i c acid. Lanthanum complexes o f

alizarine-5-sulfonic r i d e ion. MATERIALS AND METHODS Preparation o f resin

t h e s e c h e l a t i n g a g e n t s f o r m deep c o l o r e d t e r n a r y c o m p l e x e s w i t h t h e f l u o -

An aqueous s o l u t i o n o f ALC o r ALCS ( 4 0 u m o l e ) was m i x e d w i t h 0.1 M n i t r i c a c i d s o l u t i o n o f La(N03)3 (40 umole), d i l u t e d t o 100 m l , and t h e r e s u l t i n g s o l u t i o n was
-

f o l l o w i n g a d j u s t m e n t o f t h e pH t o 4.5

5.0.

An a n i o n 1 9) w a s

e x c h a n g e r e s i n , A m b e r l i t e - C G 4 0 0 ( 1 0 0 - 2 0 0 mesh; n i t r a t e f o r m ; hours. The r e s i n (40 u m o l e / g anion-exchange and a i r d r i e d .

a d d e d t o t h e s o l u t i o n o f ALC o r ALCS, a n d t h e m i x t u r e w a s s t i r r e d f o r 2 4 r e s i n ) was c o l l e c t e d , washed w i t h water,

A d s o r p t i o n and d e s o r p t i o n o f f l u o r i d e i o n T h e r e a c t i o n c o n d i t i o n s a r e d e s c r i b e d i n t h e l e g e n d o f F i g u r e 2. f l u o r i d e i o n was d e t e r m i n e d b y i o n s e l e c t i v e e l e c t r o d e method. RESULTS AND D I S C U S S I O N The a d s o r p t i o n o f f l u o r i d e i o n s d e c r e a s e d w i t h an i n c r e a s e i n t h e pH o f the solution. d u e t o c o m p e t i t i o n b e t w e e n f l u o r i d e i o n s and h y d r o x i d e i o n s . The a d s o r p t i o n and t h e d e s o r p t i o n o f f l u o r i d e i o n s I n addition, the capability o f the resin resin T h i s suggests t h e p o s s i b l e d e s o r p t i o n o f f l u o r i d e i o n s b y t h e sodium hydroxide solution. w e r e f o u n d t o be a l m o s t c o m p l e t e . (Fig. The

f o r f l u o r i d e i o n a d s o r p t i o n was m a i n t a i n e d e v e n a f t e r b e i n g u s e d t e n t i i n e s

2).

The a d s o r p t i o n o f f l u o r i d e i o n s b y t h e a n i o n - e x c h a n g e

shown i n F i g u r e 2 i n d i c a t e s t h a t u n s e l e c t i v e a d s o r p t i o n o f t h e f l u o r i d e i o n a s an a n i o n t o t h e a n i o n - e x c h a n g e extent. o f ALC-La, group. ALC-La, The a m o u n t o f ALCS-La r e s i n m a y h a v e t a k e n p l a c e t o some S i m i l a r s a t i s f a c t o r y r e s u l t s w e r e o b t a i n e d b y t h e u s e o f ALCS-La. l o a d e d on t h e r e s i n was f o u n d t o be a l m o s t t w i c e t h a t i s a more advantageous reagent than because o f t h e ion-exchange c a p a c i t y e x h i b i t e d by t h e s u l f o n a t e loaded r e s i n can adsorb g r e a t e r amounts o f f l u o r i d e The p r e s e n c e o f common a n i o n s s l i g h t l y T h i s may be c o n s i d e r e d a d i s t u r b a n c e , However, the selective

T h i s i n d i c a t e s t h a t ALCS-La b e c a u s e ALCS-La loaded resin.

i o n s t h a n ALC-La s i n c e an

decreases f l u o r i d e i o n adsorption.

u n s e l e c t i v e a d s o r p t i o n o f i o n s o n t h e a n i o n e x c h a n g e r e s i n may

take place together w i t h a s e l e c t i v e adsorption.

H *\

'
ALC

Hz-N(

CHzCOOH CHZCOOH

K+

miz
o;
/

45

-N

c ~ C O O H HzCOOH

ALCS

F i g u r e 1.

S t r u c t u r e o f ALC and ALCS

i /
p
100 0

I I 8
I

T i m e s of use

F i g u r e 2.

--

'

'

910

Reuse o f t h e r e s i n ALC r e s i n , ----- CG-400

ALC R e s i n :

40 u m o l e / g - r e s i n 10 cm x 10 mm

R e s i n column:

Adsorption Conditions: f l o w r a t e : 0.23 ml/rnin F-: 38 ppm i n 0.1 M NaC1, pH 4.2 ( a c e t a t e b u f f e r ) Desorption Conditions: eluent: 10 m l o f 1N f l o w r a t e : 1 ml/min

NaOH

46
adsorption o f fluoride ions by the functional s i t e o f the resin i s not i n t e r f e r e d w i t h i n t h e presence o f o t h e r anions. With the exception o f The Satisfactory A13+ a n d Fe3+, m o s t o f t h e m e t a l i o n s d i d n o t s h o w a n y i n t e r f e r e n c e . a d d i t i o n o f t a r t a r i c a c i d c o u l d mask t h e s e m e t a l i o n s . r e s u l t s w e r e o b t a i n e d when ALCS-La c e n t r a t i o n o f f l u o r i d e i o n s i n t a p water.

l o a d e d r e s i n was a p p l i e d t o t h e p r e c o n The r e s i n s p r e s e n t e d h e r e a r e

u s e f u l sorbents f o r t h e s e l e c t i v e c o l l e c t i o n o f f l u o r i d e ions. REFERENCES

1.
2.

L e o n a l d MA, N a k a y a m a M, 31 :269-274

M u r r a y GT ( 1 9 7 4 ) A n a l y s t 99:645-651 I t o h K. C h i k u m a M, S a k u r a i H. Tanaka H (1984) T a l a n t a

Fluoride Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 47-58 0 1986 Elsevier Science Publishers B.V.. Amsterdam - Printed in The Netherlands

H. Tsunoda a n d M.-H. Y u (Editors)

47

G A S CHROMATOGRAPHICAL MICRO-ANALYSIS O F FLUORIDE U S I N G TRIMETHYLCHLOROS I LANE Y O S H I H I R O ZAIMA AND S H I G E R U GOT0

D e p a r t m e n t o f E n v i r o n m e n t a l H e a l t h and Hygiene, M e d i c a l School, University ABSTRACT An a c c u r a t e and r a p i d m e t h o d f o r f l u o r i d e d e t e r m i n a t i o n b y g a s c h r o m a t o g r a p h y combined w i t h s o l v e n t e x t r a c t i o n has been e s t a b l i s h e d . extracted w i t h trimethylchlorosilane Fluoride i s solution into T r a c e amounts T h i s method Osaka

fTMCSl f r o m

acidified

n-hexane as t r i m e t h y l f l u o r o s i l a n e (TMFS) i n a s e a l e d v e s s e l . o f s h a k i n g and g l a s s v e s s e l s a r e a v a i l a b l e f o r t h e e x t r a c t i o n .

o f f l u o r i d e i n t h e r a n g e o f 1 t o 50 n g c a n b e e x t r a c t e d w i t h i n o n e m i n u t e has been s u c c e s s f u l l y a p p l i e d t o t h e d e t e r m i n a t i o n o f f l u o r i d e d i s t r i b u t i o n i n t h e u p p e r i n c i s o r enamel o f an a c u t e l y f l u o r o s e d e x p e r i m e n t a l rat. INTRODUCTION For t h e d e t e r m i n a t i o n o f s m a l l amounts o f f l u o r i d e , t h e f l u o r i d e ions p e c i f i c e l e c t r o d e h a s b e e n w i d e l y used. r a n g e o f 5 t o 8, T h i s method i s based on t h e d e t e r m i n a t i o n o f t h e i o n i c a c t i v i t y o f f l u o r i n e i n s o l u t i o n s w i t h a pH and t h u s r e q u i r e s d i s s o l v i n g s a m p l e s b e f o r e t h e a n a l y s i s . a s h i n g and I n t h e a n a l y s i s o f f l u o r i d e i n b i o l o g i c a l and a i r samples, d i f f u s i o n m e t h o d (3,4), are, therefore,

f l u o r i d e s e p a r a t i o n p r o c e d u r e s s u c h a s t h e a1 k a l i n e f u s i o n m e t h o d ( l , Z ) , and s t e a m d i s t i l l a t i o n method ( 5 ) a r e a v a i l a b l e . and These d i s s o l u t i o n and s e p a r a t i o n t e c h n i q u e s c a u s e e r r o n e o u s r e s u l t s , fluoride. Gas c h r o m a t o g r a p h y c o m b i n e d w i t h e x t r a c t i o n o f f l u o r i d e f r o m an a c i d i f i e d s o l u t i o n i n t o an o r g a n i c s o l v e n t i s c o n s i d e r e d u s e f u l f o r t h e a n a l y s i s o f t r a c e amounts o f f l u o r i d e . fluoride determination. the organic solvents, o f 3.8 t o 3 8 u g p e r m l . and Bock and Semmler ( 6 ) a p p l i e d t h i s method t o They used t r i e t h y l s i l a n o l cyclohexane and triethylThey

n o t s u i t a b l e f o r t h e d e t e r m i n a t i o n o f t r a c e amounts o f

c h l o r o s i l a n e a s t h e s i l i l a t i o n r e a g e n t s , t e t r a c h l o r e t h y l e n e and m - x y l e n e a s as an i n t e r n a l s t a n d a r d . obtained a l i n e a r c a l i b r a t i o n curve f o r f l u o r i d e i n t h e c o n c e n t r a t i o n range The s h a k i n g t i m e f o r f l u o r i d e e x t r a c t i o n was 40 t o t r i r n e t h y l c h l o r o s i l a n e (TMCS) a s t h e

50 m i n u t e s .
Fresen, Cox a n d W i t t e r ( 7 ) u s e d

48 sililation reagent, 2-methylbutane as t h e i n n e r standard, and benzene as

t h e o r g a n i c s o l v e n t . They

d e t e r m i n e d t h e f l u o r i d e c o n t e n t s i n u r i n e and M u n k s g a a r d and B r u n n a p p l i e d t h i s method t o d e t e r m i n e t h e f l u o r i d e Although t h i s method is a p p l i c a b l e f o r v a r i o u s long extraction time

s e r u m s a m p l e s a f t e r a 30 m i n u t e e x t r a c t i o n p e r i o d .

(8), a n d G i b b s e t a l . ( 9 )
c o n t e n t i n t o o t h enamel. samples, involved. U s i n g TMCS,

r e s u l t s a r e erroneous because o f t h e

Yamaya and Y o s h i d a ( 1 0 ) e x t r a c t e d f l u o r i d e b y d i f f u They e x a m i n e d a n a l y t i c a l

s i o n and d e t e r m i n e d i t b y gas chromatography. TMCS. solvent, and s a m p l e s o l u t i o n s .

c o n d i t i o n s s u c h as s t a n d i n g t i m e o r d i f f u s i o n p e r i o d and t h e q u a n t i t i e s o f But they d i d not consider t h e volat i l i t y o f t r i m e t h y l f l u o r o s i l a n e (TMFS) f o r m e d f r o m TMCS and f l u o r i d e .

I n o r d e r t o i m p r o v e t h e gas c h r o m a t o g r a p h i c method combined w i t h s o l v e n t extraction, the authors studied the extracting conditions t h a t could a f f e c t i n c l u d i n g t h e a c i d i t y o f t h e aqueous l a y e r , and t h e v e s s e l s u s e d f o r f l u o r i d e e x t r a c t i o n . and t h e i n t e r n a l and g l a s s v i a l s standard, time In s t a n d i n g t i m e f r o m e x t r a c t i o n t o gas f l u o r i d e determination, chromatographic analysis,

and t e m p e r a t u r e f o r t h e e x t r a c t i o n ,

t h i s s t u d y TMCS w a s u s e d a s t h e f l u o r i d e e x t r a c t a n t , a n d n - h e x a n e a n d 2 m e t h y l b u t a n e were used as t h e s o l v e n t , tively. respecI t becomes c l e a r f r o m t h e r e s u l t s t h a t ,

t o extract fluoride,

s h a k i n g f o r one m i n u t e i s s u f f i c i e n t

are available.

E x t r a c t i o n w i t h a s e a l e d v e s s e l h a v i n g a s c r e w cap and a s i l i c o n e r u b b e r septum l a m i n a t e d w i t h a Teflonm sheet i s e f f e c t i v e i n p r e v e n t i n g t h e evaporation o f v o l a t i l e TMFS (b.p.. 16.4"C). The s t a n d a r d d e v i a t i o n o f fluoride i n t h e d e t e r m i n a t i o n o f 1 u g o f f l u o r i d e c a n be r e d u c e d t o b e l o w 1%. When t h e c o n c e n t r a t i o n o f 2 - m e t h y l b u t a n e extractant. i s s e t a t 0.7 u g / m l , t h e r a n g e o f 1 t o 50 n g c a n b e d e t e r m i n e d u s i n g t h e V - v i a l

w i t h 50 u l

By u s e o f t h i s method, t h e f l u o r i d e d i s t r i b u t i o n i n t h e enamel

o f t h e u p p e r i n c i s o r o f an a c u t e l y f l u o r o s e d e x p e r i m e n t a l r a t was s u c c e s s f u l l y determined. MATERIALS AND METHODS Apparatus Gas c h r o m a t o g r a p h : Y a n a g i m o t o , model G-180 w i t h F I D 1 0 % s i l i c o n e o i l DC Column: S t a i n l e s s s t e e l ( 4 6 X 4m), ABS 6 9 0 / 1 0 0 Oven t e m p e r a t u r e : C a r r i e r gas:

200 o n

Anakrom

70C
150C f l o w r a t e 30 m l / m i n H i t a c h i , model 708

I n j e c t o r temperature:

nitrogen,

Atomic absorption spectroscopy:

49
Reagents n-Hexane: Water: Wako p u r e chem.. f o r d e t e r m i n a t i o n o f p e s t i c i d e r e s i d u e 300 d e m i n e r a l i z e d and d o u b l e d i s t i l l e d

A s o l u t i o n c o n t a i n i n g 0.9 mg t r i m e t h y l c h l o r o s i l a n e (TMCS) Extractant: and 1.7 v g 2 - m e t h y l b u t a n e p e r m l o f n-hexane was used. The s o l u t i o n was s t o r e d i n a sealed g l a s s b o t t l e i n a deep-freezer.
S t o c k s o l u t i o n o f f l u o r i d e : An a q u e o u s s o l u t i o n c o n t a i n i n g 2 0 0 0 ppm F ( 2 . 2 1 0 0 g o f d r i e d NaF p e r 5 0 0 m l ) w a s p r e p a r e d . F r o m t h i s s o l u t i o n , t h e r e q u i r e d standard s o l u t i o n s were prepared by d i l u t i o n w i t h p i p e t s and b e a k e r s o f p o l y e t h y l e n e . Procedure

A s a m p l e s o l u t i o n was t a k e n i n a v e s s e l and p l a c e d i n a c o l d r o o m (5C)

f o r one hour. sheet. V-vial F o l l o w i n g a d d i t i o n o f t h e e x t r a c t a n t t h e v e s s e l was s e a l e d m i n i v i a l and w i t h a screw cap and a s i l i c o n e r u b b e r septum l a m i n a t e d w i t h a T e f l o n @ The a m o u n t o f aqueous l a y e r i n a p o l y c a r b o n a t e t u b e , were 5 m l . 2.5 m l , a n d 0.25 m l , respectively, w h i l e those o f the The v e s s e l was the

e x t r a c t a n t were 1 ml,

0.5 m l , a n d 50 v l , r e s p e c t i v e l y .

shaken v i g o r o u s l y b y hand a t a r a t e

of a b o u t 200 r e c i p r o c a t i o n s p e r m i n u t e .

I n c a s e s w h e r e t h e e x t r a c t i o n was done a t d i f f e r e n t t e m p e r a t u r e s , a d j u s t e d t o t h e d e s i r e d t e m p e r a t u r e b e f o r e shaking.

v e s s e l s p r e p a r e d were a l l o w e d t o s t a n d more t h a n one h o u r i n t h e room Two v l o f t h e e x t r a c t w e r e p i p e t t e d w i t h a m i c r o s y r i n g e p i e r c i n g t h r o u g h t h e s e p t u m and i n j e c t e d i n t o t h e g a s c h r o m a t o g r a p h column. butane. RESULTS AND D I S C U S S I O N Blank c o r r e c t i o n The TMFS and 2 - m e t h y l b u t a n e (Fig. 1). p e a k s w e r e s y m m e t r i c a l and s e p a r a t e d n i c e l y Extraction R e l a t i v e peak h e i g h t s o f t h e b l a n k TMFS w i t h s t a n d a r d d e v i a t i o n s The f l u o r i d e c o n t e n t w a s d e t e r m i n e d f r o m t h e p e a k h e i g h t r a t i o o f TMFS t o 2 - m e t h y l -

a t a c o n c e n t r a t i o n l e v e l o f 0.2 ppm F - a r e l i s t e d i n T a b l e I. acidified with perchloric a n c e (C.V.) w a s 0.67%. then, a c i d (0.47 mol/l).

o f f l u o r i d e was c a r r i e d o u t i n a p o l y c a r b o n a t e t u b e w i t h t h e aqueous l a y e r
R e s u l t s o f s i x t e e n measurem e n t s w e r e i n g o o d a g r e e m e n t w i t h e a c h o t h e r and t h e c o e f f i c i e n t o f v a r i T h e r e l a t i v e p e a k h e i g h t o f t h e b l a n k w a s 0.6% o f t h a t o f t h e s t a n d a r d s o l u t i o n and was w i t h i n e x p e r i m e n t a l m e a s u r i n g e r r o r . I n t h i s study, b l a n k c o r r e c t i o n was n o t made. was A c i d i t y o f aqueous l a y e r H y d r o c h l o r i c a c i d and p e r c h l o r i c a c i d w e r e used and 1 vg o f Ff o r m e d a f t e r one m i n u t e s h a k i n g f o l l o w e d b y one m i n u t e s t a n d i n g . e x t r a c t e d i n t h e p o l y c a r b o n a t e t u b e . Gas c h r o m a t o g r a p h i c a n a l y s i s was p e r Figure 2

50

F i g u r e 1. D e t e r m i n a t i o n o f f l u o r i d e b y gas c h r o m a t o g r a p h y . t r i m e t h y l f l u o r o s i l a n e ; B = 2 - m e t h y l b u t a n e ; C = n-hexane.

= start;

A =

a
c
U

0 .c
c

A c i d i t y of aqueous layer

( rnol I1 )

F i g u r e 2. R e l a t i o n s h i p b e t w e e n e x t r a c t i o n r a t e and m o l a r c o n c e n t r a t i o n o f a c i d i n t h e a q u e o u s l a y e r . 1 v g F (0.2 ppm F ) w a s e x t r a c t e d i n a p o l y c a r b o n a t e c e n t r i f u g e t u b e following 1 m i n s h a k i n g . 0. HC1; e. HC104.

51
TABLE I R E L A T I V E PEAK HEIGHT (TMFS/2-METHYL-BUTANE) STANDARD SOLUTION AND BLANK

OF

F- Conc.
(ppm) 0.2

Mean k S.D.

C.V. (%)
0.67

16 5

0.8300 f 0.0056 0.0050 f 0 . 0 0 1 4 polycarbonate

27.00

e x t r a c t i o n vessel: aqueous l a y e r : organic layer: s h a k i ng t i me:

5 ml

1 ml 1 min

shows t h e r e l a t i o n s h i p b e t w e e n e x t r a c t i o n r a t e and m o l a r c o n c e n t r a t i o n o f each a c i d i n t h e aqueous l a y e r . higher than 5 mol/l, E x t r a c t l o n r a t e was e x p r e s s e d a s a p e r c e n t a g e o f t h e r e l a t i v e peak h e i g h t o f TMFS t o t h a t i n t h e r a n g e o f a c i d i t y where t h e r e l a t i v e peak h e i g h t became c o n s t a n t and H y d r o c h l o r i c a c i d and p e r c h l o r i c a c i d showed t h e The e x t r a c t i o n r a t e was

C.V.

w a s l e s s t h a n 1%.

same e x t r a c t i o n r a t e f o r t h e same m o l a r i t y .

r e d u c e d b y 2% w h e n t h e a c i d i t y w a s r e d u c e d f r o m 5 m o l / l o r m o r e t o 0.5

mol/l.

I n t h i s study,

p e r c h l o r i c a c i d was used.

S ha k inq t ime
F i g u r e 3 shows t h e r e l a t i o n s h i p b e t w e e n s h a k i n g t i m e and t h e e x t r a c t i o n r a t e a t t h r e e c o n c e n t r a t i o n l e v e l s o f p e r c h l o r i c a c i d (0.13, mol/l). One u g o f F-

0.47,

and 2.35

was e x t r a c t e d i n a p o l y c a r b o n a t e t u b e and d e t e r m i n e d

b y g a s c h r o m a t o g r a p h y f o l l o w i n g s h a k i n g a n d o n e m i n u t e s t a n d i n g a t 5C. The s h a k i n g t i m e i s e x h i b i t e d i n t h e l o g a r i t h m i c s c a l e o n t h e t r a n s v e r s a l axis. The r a t e o f f l u o r i d e e x t r a c t i o n r o s e s t e e p l y as s h a k i n g t i m e M i n i m u m t i m e r e q u i r e d t o a c c o m p l i s h maximum e x t r a c t i o n The t i m e b u t was 2 m i n i n c r e a s e d and r e a c h e d a p l a t e a u w h i c h corresponded t o t h e a c i d i t y o f t h e aqueous l a y e r . became l o n g e r as t h e a c i d i t y o f t h e aqueous l a y e r was l o w e r e d . r e q u i r e d was o n l y 5 s e c o n d s when t h e a c i d i t y was 2.35 m o l / l . when t h e a c i d i t y was 0.19 mol/l. I n a p r e v i o u s s t u d y on t h e d e t e r m i n a t i o n o f f l u o r i d e b y gas chromatography, min. leak. s h a k i n g was c a r r i e d o u t w i t h a m e c h a n i c a l s h a k e r f o r m o r e t h a n 30 a s e a l e d v e s s e l a n d TMCS w e r e u s e d a n d a r a p i d Such a l o n g s h a k i n g i s d i s a d v a n t a g e o u s when t h e e x t r a c t i o n v e s s e l s I n t h i s study,

e x t r a c t i o n o f f l u o r i d e was a c c o m p l i s h e d . Standing time a f t e r shaking F i g u r e 4 shows t h e r e l a t i o n s h i p b e t w e e n s h a k i n g t i m e and r a t e o f f l u o r i d e

52

( %)
c

100

I
? ! 50
C

I c

"

x W

0 1 oo

lo'
Shaking

1 o2
time

o3
( sec

10

Figure 3 . Effect of shaking time on the extraction rate of fluoride (1 pg F). The aqueous layer was acidified with HC104. 0 , 2.35 mol/l; a, 0.47 rnol/l; 0, 0.19 mol/l.

5haklng

time

(sec)

Figure 4 . Effect o f 2 4 hours standing on the extraction rate of fluoride (1 p g F). Concentrations of HC104: 0 . 2.35 rnol/l; a, 0.47 mol/l; 0 ,0.19 mol/l. extraction after standing 24 hours following shaking. The experimental conditions were the same as described previously. The extraction vessels were allowed to stand in the cold (5C) for 24 hours after shaking. It i s clear from Figures 3 and 4 that fluoride unextracted after shaking was extracted at the rate of 90% a day by diffusion. Maximum or ceiling value
o f the extraction rate rose only 0.6% after 24 hours for each acidity and the increase was not significant statistically.

53
When t h e a c i d i t y o f t h e aqueous l a y e r was 0.47 i n g t i m e w a s m o r e t h a n 20 s e c ( F i g . 3). t h e e x t r a c t i o n vessel, ppm, respectively. standing I t i s c l e a r t h a t t h e m a t e r i a l o f t h e vessel used f o r f l u o r i d e mol/l, t h e r e q u i r e d shakFor The

A s h a k i n g t i m e o f one m i n u t e was

c h o s e n and t h e e f f e c t o f t h e s t a n d i n g t i m e a f t e r s h a k i n g was s t u d i e d . p o l y c a r b o n a t e t u b e s and m i n i v i a l s w e r e used. c o n c e n t r a t i o n s o f p e r c h l o r i c a c i d a n d f l u o r i d e w e r e 0.47 m o l / l , F i g u r e 5 shows t h e change i n e x t r a c t i o n r a t e f o r a 50-hour period. Also,

a n d 0.2

e x t r a c t i o n . p o l y c a r b o n a t e and g l a s s , d i d n o t a f f e c t t h e e x t r a c t i o n r a t e . t h e standing t i m e f o l l o w i n g shaking increased t h e e x t r a c t i o n r a t e a t Assuming t h a t t h e e x t r a c t i o n r a t e i s c o n s t a n t t h e c o e f f i c i e n t o f v a r i a n c e o f a l l measures Combined w i t h t h e c o e f f i c i e n t f r o m T a b l e I t h e r a t e o f 0.02% a n h o u r . i n F i g u r e 5 i s o n l y 1.09%. (0.67%). g r a p h i c method. Temperature The c o n c e n t r a t i o n s o f f l u o r i d e and p e r c h l o r i c a c i d u s e d w e r e 0.2 ppm and 0.47 m o l / l . respectively. The a q u e o u s s o l u t i o n a n d t h e e x t r a c t i o n w e r e p o u r e d i n t o a p o l y c a r b o n a t e c e n t r i f u g e t u b e a t 5C and s e a l e d i m m e d i a t e l y w i t h a screw cap and a s i l i c o n e r u b b e r septum l a m i n a t e d w i t h a T e f l o n @ sheet. The t u b e was p l a c e d i n t h e r o o m a d j u s t e d t o t h e p r e s c r i b e d t e m p e r a temperature. t u r e ( 0 . 1 3 and 20C) f o r one h o u r f o l l o w e d b y one m i n u t e ' s s h a k i n g . F i g u r e 6 shows t h e r e l a t i o n s h i p b e t w e e n e x t r a c t i o n r a t e and The e x t r a c t i o n r a t e i s e x p r e s s e d a s t h e p e r c e n t a g e o f t h e r e l a t i v e p e a k

f o r 50 h o u r s f o l l o w i n g s h a k i n g ,

t h e e r r o r o f d e t e r m i n a t i o n w i l l be w i t h i n 2% b y t h e gas c h r o m a t o -

10 20 30 LO 50 Standing time after extraction ( hr)

F i g u r e 5. E f f e c t o f s t a n d i n g t i m e on t h e e x t r a c t i o n r a t e o f f l u o r i d e . F l u o r i d e (0.2 ppm F) was e x t r a c t e d i n a p o l y c a r b o n a t e c e n t r i f u g e t u b e and glass minivial. The c o n c e n t r a t i o n o f HC104 was 0.47 m o l / l .

54

10 15 Temperature

20

( ' C

F i g u r e 6. E f f e c t o f t e m p e r a t u r e on t h e e x t r a c t i o n r a t e o f f l u o r i d e . o f f l u o r i d e was e x t r a c t e d i n a p o l y c a r b o n a t e c e n t r i f u g e t u b e . h e i g h t o f TMFS t o t h a t a t t u r e rose,

1 ug

5C.

A t t e m p e r a t u r e s above

5C.

a s t h e ternpera-

t h e e x t r a c t i o n r a t e d e c r e a s e d and t h e s t a n d a r d d e v i a t i o n o f t h e T h e c o e f f i c i e n t s o f v a r i a n c e a t 0, 5, 1 3 respectively. T h i s means t h a t Accordingly,

d e t e r m i n e d v a l u e became l a r g e . and 20C w e r e 0.67%.

0.68%,

1.36% and 1.71%.

t h e v o l a t i l e TMFS e v a p o r a t e d m o r e as t h e t e m p e r a t u r e r o s e . t e m p e r a t u r e l o w e r t h a n 5C. Vessels used f o r f l u o r i d e e x t r a c t i o n

f o r an a c c u r a t e d e t e r m i n a t i o n , t h e e x t r a c t i o n s h o u l d be c a r r i e d o u t a t a

As s h o w n i n F i g u r e 5,
fluoride extraction,

p o l y c a r b o n a t e c e n t r i f u g e t u b e s and g l a s s v i a l s To s t u d y t h e e f f e c t o f v e s s e l s o n The c o n c e n t r a t i o n o f p e r c h l o r i c mol/l w h i l e those of f l u o S h a k i n g t i m e was one m i n u t e and t h e They show a v e r y good

e x h i b i t e d t h e same e x t r a c t i o n r a t e s . m i n i v i a l s , and V - v i a l s w e r e compared. r i d e w e r e i n t h e r a n g e o f 0 t o 50 ppm. t e m p e r a t u r e was 5C.

t h e c a l i b r a t i o n c u r v e s made w i t h p o l y c a r b o n a t e t u b e s ,

a c i d i n t h e aqueous l a y e r was a d j u s t e d t o 0.47

The c a l i b r a t i o n c u r v e s a r e shown i n F i g u r e s 7-9. linearity. b o t h g l a s s v i a l s w e r e 0.9989 0.9 and 0.9999,

The c o r r e l a t i o n c o e f f i c i e n t s f o r p o l y c a r b o n a t e t u b e s a n d f o r respectively. The a m o u n t o f f l u o r i d e corresponding t o

I r r e s p e c t i v e o f t h e k i n d o f v e s s e l used, 1 m l o f e x t r a c t a n t c o n t a i n i n g mg TMCS c o u l d e x t r a c t u p t o 50 u g o f f l u o r i d e . e x t r a c t e d w i t h a p o l y c a r b o n a t e tube, ug, 1.186 ug, and 1.175 ug, m i n i v i a l and V - v i a l

t h e r e l a t i v e peak h e i g h t o f TMFS t o t h e i n t e r n a l s t a n d a r d o f 1.0 w e r e 1.786 respectively. The d i f f e r e n c e i n r e l a t i v e peak (Table h e i g h t s among d i f f e r e n t k i n d s o f v e s s e l s was o n l y 1% and was a l m o s t e q u i v a lent t o the dispersion o f the determinations

I).

I t can be

55

/
50

Fluorine content

pg )

F i g u r e 7. C a l i b r a t i o n c u r v e o f f l u o r i d e u s i n g p o l y c a r b o n a t e c e n t r i f u g e tubes. The a m o u n t s o f aqueous l a y e r and e x t r a c t a n t a r e 5 m l and 1 m l . r e s p e c t i v e l y . 0 . d e n o t e s t h e r a n g e o f f l u o r i d e 0-5 ppm; 0.d e n o t e s t h e r a n g e o f f l u o r i d e 10-50 ppm.

3130

/ I
Fluorlne content

a
(yg)

t!

10

F i g u r e 8. Calibration curve o f f l u o r i d e using glass minivials. The a m o u n t s o f a q u e o u s l a y e r a n d e x t r a c t a n t a r e 2.5 m l a n d 0.5 m l , r e s p e c 0 , f l u o r i d e 0-5 ppm; 0 , f l u o r i d e 10-50 ppm. tively. concluded, therefore, t h a t t h e r a t e o f f l u o r i d e e x t r a c t i o n was n o t a f f e c t e d

by t h e k i n d o f v e s s e l s used f o r e x t r a c t i o n . A n a l y s i s o f nanogram f l u o r i d e To d e t e r m i n e n a n o g r a m q u a n t i t i e s o f f l u o r i d e , i n g o f 0.9 mg TMCS and 0.7 p g 2 - m e t h y l b u t a n e prepared. was e x t r a c t e d and d e t e r m i n e d . c u r v e was o b t a i n e d . another e x t r a c t a n t consistwas p e r m i l l i l i t e r o f n-hexane

By u s i n g t h i s e x t r a c t a n t and a V - v i a l ,

0 t o 50 ng o f f l u o r i d e
a linear calibration The p r o even i n a

As shown i n F i g u r e 10,

The c o e f f i c i e n t o f c o r r e l a t i o n was 0.9990.

posed method makes t h e t r a c e a n y a l y s i s o f f l u o r i d e p o s s i b l e , v o l u m e o f s a m p l e s o l u t i o n a s s m a l l as 250 ul.

56

Fluorine content

( pg)

o f aqueous layer and extractant are 250 111 and

Figure 9. Calibration curve of fluoride using glass V-vials. The amounts 50 111. respectively. 0 , fluoride 0-5 ppm; 0, fluoride 10-50 ppm.

10

20
Fluorine

30

40

50
( ng )

content

Figure 1 0 . Calibration curve of fluoride using glass V-vials. The amount of 2-methylbutane, internal standard, in n-hexane was reduced to 0.7 ug/ml. . The other conditions are the same as shown i n Figure 9 Fluoride in the tooth enamel of rats By using the proposed method, the fluoride distribution in the enamel of the upper incisor of an acutely fluorosed experimental rat could be determined. In experiment, a 2.5% NaF solution was injected subcutaneously into weight. a male rat (SD strain, 160 f 59) at a dose of 0.5 mg per 100 g body Four days after the injection, the rat was sacrificed and the upper incisors were extracted. They were fixed with 10% formaldehyde

57

solution, etching.

d e h y d r a t e d and embedded i n a p o l y e s t e r r e s i n .

They w e r e s l i c e d .

200 urn t h i c k t r a n s v e r s a l l y , a n d c o a t e d w i t h

n a i l enamel t o a v o i d a c i d The c a l c i u m

The e n a m e l o f t h e s u r f a c e was e t c h e d w i t h p e r c h l o r i c a c i d . and

t h e f l u o r i d e c o n t e n t was d e t e r m i n e d b y gas c h r o m a t o g r a p h y . troscopy.

c o n t e n t ? n t h e aqueous l a y e r was d e t e r m i n e d b y a t o m i c a b s o r p t i o n spec-

As shown i n F i g u r e 11, b o t h f l u o r i d e and c a l c i u m w e r e r e d u c e d a t

The These r e s u l t s

t h e h y p o m i n e r a l i z e d zone and i n c r e a s e d a t t h e h y p e r m i n e r a l i z e d zone. r a t i o s o f f l u o r i d e t o c a l c i u m a l s o showed a s i m i l a r t r e n d . o f not only calcium but also fluoride. d e p o s i t e d t o g e t h e r w i t h enamel m a t r i x .

indicate that, through f l u o r i d e t o x i c i t y , ameloblasts a r r e s t the deposition And i t may s u g g e s t t h a t f l u o r i d e i s

The d i s t r i b u t i o n p a t t e r n o f c a l c i u m a g r e e s w i t h t h e r e s u l t o b t a i n e d f r o m e l e c t r o n m i c r o p r o b e a n a l y s i s b y Suga (11).

CONCLUSION
The a u t h o r s h a v e e s t a b l i s h e d an i m p r o v e d gas c h r o m a t o g r a p h i c m e t h o d combined w i t h e x t r a c t i o n o f f l u o r i d e as t r i m e t h y l f l u o r o s i l a n e . Various

S
FlGu

HPOl DEJ HPR

F I Cd ( x ~ O )- ~

2i
0
10 20 3 0 40 50 Depth

(prn)

F i g u r e 11. D i s t r i b u t i o n o f f l u o r i d e and c a l c i u m i n t h e e n a m e l o f t h e u p p e r i n c i s o r o f an a c u t e l y f l u o r o s e d e x p e r i m e n t a l r a t . U p p e r p i c t u r e shows t h e contact microradiograph: S, s u r f a c e o f t h e e n a m e l : HPO, h y p o m i n e r a l i z e d zone: HPR, h y p e r m i n e r a l i z e d zone: DEJ, d e n t i n e - e n a m e l j u n c t i o n .

58

f a c t o r s a f f e c t i n g t h e e x t r a c t i o n have been s t u d i e d i n d e t a i l and s u i t a b l e c o n d i t i o n s have been e s t a b l i s h e d . The i m p r o v e d m e t h o d p r o v e d e f f e c t i v e f o r t h e d e t e r m i n a t i o n o f f l u o r i d e d i s t r i b u t i o n i n t h e enamel o f u p p e r i n c i s o r o f an a c u t e l y f l u o r o s e d e x p e r i m e n t a l r a t .

REFERENCES
1. Remmert LF, P a r k s AM, McBurney EH (1953) A n a l Chew 25:450-453

2. N i o s h Manual o f A n a l y t i c a l M e t h o d s (1977) I n : c a l Methods, 2nd ed. v o l 1:212-1--212-4 4. W i l l a r d HH,

N I O S H Manual o f A n a l y t i -

3. S i n g e r L, A r m s t r o n g WD (1954) A n a l Chem 26:904-046 W i n t e r OB (1933) I n d Eng Chem A n a l Ed 5:7-10

5. I n t e r s o c i e t y C o m m i t t e e o n M e t h o d f o r A m b i e n t A i r S a m p l i n g and A n a l y s i s (1969) H l t h Lab S c i 6:84-101

6. Bock R. Semmler HJ (1967) F r e s e n i u s ' Z A n a l Chem 230:161-184 B r u n n C (1973) A r c h s O r a l B i o l 18:735-744 7. F r e s e n JA, Cox FH, W i t t e r MJ (1968) Pharm Weekblad 103:909-914

8. Munksgaard EC,
60:770-775

9. G i b b s M, R e t i e f DH. B r a d l e y EL, T a y l o r RE, W a l k e r A R ( 1 9 8 1 ) J D e n t Res

10. Yamaya K, Y o s h i d a M (1984) J Chem SOC, J p n 4:563-568

11. S u g a S ( 1 9 8 3 ) I n : S u g a S ( e d ) M e c h a n i s m s o f T o o t h E n a m e l F o r m a t i o n . Q u i n t e s s e n c e P u b l i s h i n g Co, I n c . Tokyo, pp 157-303

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 59-71 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

59

DETERMINATION

OF

FLUORIDE IN DRINKING WATER BY COLORIMETRY WITH A TOTAL-

REFLECTION LONG CAPILLARY CELL WE1 L E I , KITAO FUJIWARA, AND K E I I C H I R O FUWA Department of Chemistry, F a c u l t y o f Science. Bunkyo-ku, T o k y o 113, J a p a n The U n i v e r s l t y o f Tokyo,

ABSTRACT C o l o r i m e t r i c d e t e r m i n a t i o n o f f l u o r i d e i q n s i n d r i n k i n g w a t e r was c a r r i e d o u t i n a l o n g c a p i l l a r y c e l l (LCC). C o l o r d e v e l o p m e n t was b a s e d o n t h e La-ALC w a s a d d e d t o t h e and was m i x e d w i t h a c o m p l e x o n e f o r m e d was e x t r a c t e d Lanthanum-a1 i z a r i n complexone (La-ALL) method: s a m p l e and t h e fluoro-lanthanum-alizarin

w i t h isoamyl alcohol containing N,N-diethylaniline solution 1.57).

m i x t u r e o f c a r b o n d i s u l f i d e and a c e t o n e ( f i n a l r e f r a c t i v e i n d e x o f a n a l y t e
=

Extension o f c e l l length longer than 1 m provided h i g h l y Enhancement o f a b s o r p t i o n was 0.5-2.5 respectively. x lo3 times D e t e c t i o n l i m i t and r e c o v e r y of t h i s m e t h o d w e r e The p r e s e n t m e t h o d was

sensitive colorimetry. g r e a t e r u s i n g a 4 m LCC.

1 0 n g / L a n d 93-104% ( f o r 50 u g / 1 ) ,

a p p l i e d t o t h e d e t e r m i n a t i o n o f f l u o r i d e i n d r i n k i n g w a t e r samples c o l l e c t e d f r o m v a r i o u s s i t e s i n Tokyo c i t y .

INTRODUCTION Long c a p i l l a r y c e l l I t i s known t h a t t h e c o n c e n t r a t i o n o f f l u o r i d e i n d r i n k i n g w a t e r c o r r e l a t e s d i r e c t l y w i t h t h e o c c u r r e n c e o f d e n t a l d i s e a s e (1,Z). n a t i o n o f f l u o r i d e i n d r i n k i n g water, t h e environmental f i e l d . c o m p l e x o n e (La-ALC) t h a n 20 ng/ml, Recently, cells. determination o f fluoride, therefore, The d e t e r m i i s an i m p o r t a n t s u b j e c t i n

Although c o l o r i m e t r y using lanthanum a l i z a r i n i s one o f t h e most s e n s i t i v e methods f o r t h e i t cannot d e t e c t f l u o r i d e concentrations of l e s s

(3-6)

w h e n a n o r d i n a r y c o l o r i m e t r i c c e l l ( 1 cm c e l l ) i s u s e d . the

l o n g c a p i l l a r i e s ( l o n g e r t h a n 1 m) w e r e used as c o l o r i m e t r i c

Because o f t h e e x t e n s i o n o f o p t i c a l p a t h l e n g t h i n s i d e t h e c e l l ,

u s e o f a l o n g c a p i l l a r y c e l l (LCC) p r o v i d e s e n h a n c e d d e t e c t i o n p o w e r i n o r d i n a r y c o l o r i m e t r y (7). cells, F i g u r e 1 s h o w s t h e o r d i n a r y c e l l s ( 1 cm a n d 5 I n ordinary w h i c h obeys t h e Lambertcm) and v a r i o u s t y p e s o f LCCs e m p l o y e d i n t h e p r e s e n t s t u d y . t h e s e n s i t i v i t y o f absorption spectrometry,

60

Ordinary Cells

Capillary Cells

(5)
1 . -

--I

L = 2 , 4 , 1 0 m,....

L=25,50m,

1 .

F i g u r e 1. O r d i n a r y and l o n g c a p i l l a r y c e l l s f o r s p e c t r o p h o t o m e t r y . (l'), 1 cm c e l l ; (2'), . 5 cm c e l l ; ( 1 ) . l i n e a r t y p e ; (2), c u r v e d t y p e ; ( 3 ) , l o o p e d t y p e ; (4), s p i r a l t y p e , ( 5 ) , " o p t i c a l f i b e r " t y p e .

Beer law, i s determined by t h e l i g h t pathlength w i t h t h e source l i g h t p a r a l l e l w i t h t h e c e l l wall.

of

the

sample

cell.

I n t h i s system,

t h e d i a m e t e r o f t h e a p e r t u r e o f t h e LCC was s m a l l e r t h a n r e s u l t i n g i n m u l t i r e f l e c t i o n o f source l i g h t Thus, when u s i n g a l i n e a r l y shaped 1 However, i t T h i s c a u s e d t h e o p t i c a l p a t h l e n g t h t o become l o n g e r

t h a t o f t h e s o u r c e l i g h t beam, inside the capillary.

m LCC,

t h a n t h e a p p a r e n t l e n g t h o f t h e LCC. hundred t i m e s t h a t o f o r d i n a r y an o r d i n a r y l a b o r a t o r y . looped (Fig. 1(3)),

t h e a b s o r b a n c e o f t h e s o l u t i o n was enhanced b y m o r e t h a n a f e w spectrometry (Fig. l(1)). more t h a n 1 m long,

1s

r a t h e r i n c o n v e n i e n t t o h a n d l e a l i n e a r LCC whose l e n g t h i s m o r e t h a n 1 m i n For c e l l s curved (fig. 1(2)), o r s p i r a l (Fig. l ( 4 ) ) forms a r e preferable. However,

when t h e aqueous s o l u t i o n i s a p p l i e d t o t h e LCC, shape f r o m l i n e a r i t y c a u s e s a s u b s t a n t i a l c a p a c i t y o f t h e LCC.

the transformation o f c e l l

loss i n the l i g h t transmission

I t was f o u n d t h a t o n l y 0.001% o f t h e i n c i d e n t l i g h t

c o u l d reach t h e e x i t o f t h e s i n g l e looped c e l l . Condition f o r t o t a l r e f l e c t i o n For m a i n t a i n i n g source l i g h t t r a n s m i s s i o n i n s i d e t h e long c a p i l l a r y c e l l , successive t o t a l r e f l e c t i o n s must continue a t t h e i n t e r n a l w a l l o f t h e capillary. law. Namely, This i s o n l y possible by applying a solvent, w h i c h has a r e f r a c t i v e index higher than t h a t o f t h e c e l l material, w h i c h obeys S n e l l ' s

the required condition f o r a t o t a l r e f l e c t i o n c e l l is:

61

"2 sin (90"-0) 2 n1

where 0 is the angle of the incident light, and n1 and n2 are the refractive indexes of the solvent and cell material (Pyrex), respectively. Figure 2 shows the patterns of source light transmission i n an ordinary 1 crn cell, L C C with specular reflection, and L C C with total reflection, where 10 and

I o ' are the intensities of source light before and after passing through the cell containing sample solutions, respectively. When n1 is smaller
than n2, a s is the case when water is used as the solvent: n 1 = 1.33 (water), n2 = 1.474 (Pyrex), the light transmits through the internal cell wall with specular reflection (middle of Fig. 2 ) . In this case the internal cell wall must be coated with a light reflective material. Since light reflectivities of most coating materials are smaller than loo%, the source light suffers strong attenuation during transmission i n this type of cell due to multiple reflection. On the other hand, when n1 is greater than n2, i.e., when refractive index of the solution exceeds that of the cell rnateria1,light transmission efficiency of the L C C is greatly improved. In other words, the source light is propagated through the inside cell wall via total reflections without the loss of intensity.

Io-c

I0

I0

--

(D

-I0f

ordinary cell specular reflection LCC ( "1<"2 )

-1

I o = I 0 ' I o f I. >>> I

t o t a l reflection LCC ( n1>n2 1

'

I.

Iof

nl: refractive index of solvent n2: refractive index of c e l l wall

Figure 2. Patterns of source light transmissions ( 1 0 ) i n the ordinary cell, L C C with specular reflection, and L C C with total reflection.

For total reflection long capillary cell spectrometry, carbon disulfide much higher than that of the Pyrex cell. is mainly used as the solvent, as its refractive index is 1.62, which is It is noted that the structure of

optical fiber is in principle the same as that of a long capillary cell

62

(8,9). F i g u r e l ( 5 ) s h o w s a l i q u i d c o r e o p t i c a l f i b e r c e l l w h o s e i n t e r n a l
d i a m e t e r i s 250 pm. The s a m p l e v o l u m e r e q u i r e d i s o n l y a f e w m l f o r a 50 m long cell. When a 100 m o p t i c a l f i b e r c e l l i s used, a b s o r b a n c e i s enhanced m o r e t h a n 6x104 t i m e s t h a t w i t h o r d i n a r y s p e c t r o m e t r y . I n t h i s paper, t h e a p p l i c a t i o n o f t o t a l r e f l e c t i o n LCC t o t h e d e t e r m i n a t i o n o f f l u o r i d e i n t h e d r i n k i n g w a t e r samples o f Tokyo c i t y i s reported. MATERIALS AND METHODS Reagents S u b b o i l e d w a t e r d i s t i l l e d b y a q u a r t z d i s t i l l e r was u s e d t h r o u g h o u t t h e experiment. opment f o r (La-ALC) A l l t h e r e a g e n t s u s e d w e r e o f a n a l y t i c a l grade. Color deuelcomplexone the Figure 3 f l u o r i d e i o n s was b a s e d o n t h e l a n t h a n u m - a l i z a r i n e m u s t be d i s s o l v e d i n c a r b o n d i s u l f i d e . a l i z a r i n c o m p l e x o n e (La-ALC-F)

m e t h o d (3-6).

F o r a t t a i n i n g t o t a l r e f l e c t i o n i n t h e LCC,

d e v e l o p e d c o l o r (La-ALC-F) (La-ALC),

shows t h e m o l e c u l a r s t r u c t u r e s o f t h e l a n t h a n u m - a l i z a r i n fluoro-lanthanum La-ALC

comoplexone The c o l o r

and fluoro-N,N-

diethylanilino-lanthanum a l i z a r i n c o m p l e x o n e (La-ALC-DA-F).
developing reagent, amine-N,N-diacetic lanthanum (Fig. ALC-F ( F i g . 3 ( B ) ) . tion o f fluoride. ALC-F

(Lanthanum-1.2-dihydroxanthraquinonyl-3-methyland f l u o r i d e , a

acid) contains two molecules o f water attached t o I n t h e p r e s e n c e o f b o t h La-ALC f o r m i n g La-

3(A)).

f l u o r i d e i o n s u b s t i t u t e s one o f t h e w a t e r m o l e c u l e s i n La-ALC. However, La-ALC-F

T h i s p r i n c i p l e i s w i d e l y used f o r c o l o r i m e t r i c d e t e c -

is insoluble i n organic solvents

Thus, La-

because one m o l e c u l e o f w a t e r i s s t i l l a t t a c h e d t o lanthanum. o t h e r ligand. I n t h e presence experiment, N.N-diethylaniline

i s n o t s o l u b l e i n c a r b o n d i s u l f i d e a n d m u s t b e s u b s t i t u t e d b y some was used as a

A ( La-ALC )

C
( La-ALC-DA-F )

( La-ALC-F)

(red color)

(purple color)

(blue c o l o r )

F i g u r e 3. M o l e c u l a r s t r u c t u r e s o f l a n t h a n u m a l i z a r i n c o m p l e x o n e and f l u o (A), La-ALC. m a j o r c o n s t i t u e n t o f " D o t i t e A l f u s o n e " ; r i d e derivatives. soluble i n ( B ) . La-ALC-F. i n s o l u b l e i n o r g a n i c s o l v e n t ; (C). La-ALC-DA-F, organic solvent.

63
s u b s t i t u t e f o r water, C o l o r development. To a 25 m l s a m p l e s o l u t i o n , ALC), r e s u l t i n g i n LaALC-DA-F (Fig. 3(C)), which i s soluble

i n o r g a n i c s o l v e n t s (10).

5 m l o f 5% l a n t h a n u m - a l i z a r i n c o m p l e x o n e (LaAcetone improves

10 m l a c e t o n e ,

and 2 m l a c e t y l a c e t o n e w e r e added.

t h e c o l o r development, and i r o n .

and a c e t y l a c e t o n e was t h e m a s k i n g a g e n t f o r a l u m i n u m F o u r m l o f 5% N , N - d i e t h y l a n i l i n e The La-ALC-DA-F in

A p o r t i o n o f t h i s s o l u t i o n was t a k e n and i t s a b s o r p t i o n was

f o r m e d was e x t r a c t e d This mixture

m e a s u r e d b y an o r d i n a r y c o l o r i m e t e r . i s o a m y l a l c o h o l s o l u t i o n was added. i n t o t h e isoamyl alcohol.

One m l o f t h e e x t r a c t a n t was m i x e d w i t h 99 m l o f

a m i x t u r e c o n s i s t i n g o f 80% c a r b o n d i s u l f i d e and 20% a c e t o n e . reflection cell. La-ALC-DA-F

h a s a r e f r a c t i v e i n d e x o f 1.57 a n d i s s u i t a b l e a s a s o l v e n t f o r t h e t o t a l i s n o t s o l u b l e i n pure carbon d i s u l f i d e . La-ALCThe t w o e x t r a c t e d i n t o isoarnyl alcohol. F i g u r e 4 i s a p h o t o g r a p h w h i c h s h o w s t h e a c t u a l c o l o r s o f La-ALC,

F i n aqueous p h a s e and La-ALC-DA-F

When La-ALC-F tion. color.

t e s t t u b e s o n t h e l e f t s i d e s h o w t h e c o l o r o f La-ALC w h i c h i s r e d a t pH 7. forms, t h e c o l o r changes t o p u r p l e . The t w o t e s t t u b e s o n shows t h e r i g h t s i d e show t h e o r g a n i c and aqueous phases a f t e r s o l v e n t e x t r a c The u p p e r l a y e r i s i s o a m y l a l c o h o l . On t h e o t h e r hand, The e x t r a c t e d La-ALC-DA-F a blue color. o f La-ALC. t h e b l a n k (La-ALC-DA) gives a pale yellow

The aqueous p h a s e i s i n t h e l o w e r l a y e r and m a i n t a i n s t h e r e d c o l o r

F i g u r e 4. C o l o r d e v e l o p m e n t o f l a n t h a n u m a l i z a r i n c o m p l e x o n e a n d f l u o r i d e ( A ) ( l ) . a q u e o u s s o l u t i o n (La-ALC): ( A ) ( 2 ) , a q u e o u s s o l u t i o n derivatives. 1o w e r (La-ALC-F): ( B ) ( 3 ) , u p p e r 1 a y e r : is o a m y l a 1 c o h o 1 p h a s e (La-ALC-DA); l a y e r : a q u e o u s p h a s e (La-ALC): ( B ) ( 4 ) , u p p e r l a y e r : i s o a m y l a l c o h o l p h a s e (La-ALC-DA-F): l o w e r l a y e r : aqueous phase (La-ALC).

64

Apparatus F i g u r e 5 shows t h e LCC a b s o r p t i o n s p e c t r o m e t r y s y s t e m .

A tungsten

i n c a n d e s c e n t l a m p (Kondo S i l v a n i a , n e u t r a l d e n s i t y (N.D) filter.

300 W ) was u s e d as t h e l i g h t source.

The i r r a d i a t i o n i n t e n s i t y o f t h e l i g h t s o u r c e was a d j u s t e d b y u s i n g a The m o n o c h r o m a t o r was u n d e r t h e c o n t r o l o f a and t h e o u t p u t s i g n a l f r o m t h e p h o t o m u l t i a m p l i f i e r ( Y o k o k a w a E l e c t r i c W o r k : t y p e YEW a n d / o r was d i g i t i z e d w i t h a 1 2 - b i t p e r s o n a l c o m p u t e r (NEC, 3121). PC8001),

p l i e r was a m p l i f i e d b y a chart recorder (Hitachi;

DC

The a m p l i f i e d o u t p u t f r o m t h e DC a m p l i f i e r was r e c o r d e d b y a s t r i p t y p e 056).

A/D

c o n v e r t e r ( D a t e 1 t y p e ADC HX 12BGC) w h i c h was s e t i n t o t h e same u n i v e r s a l b o a r d i n t h e 1/0 u n i t (NEC PC 8 0 1 2 ) f r o m w h i c h t h e d a t a w e r e b e i n g a c q u i r e d b y t h e same p e r s o n a l c o m p u t e r . c e l l s were employed. I n t h i s experiment, two types o f c a p i l l a r y In One w a s l i n e a r l y s h a p e d a n d t h e o t h e r s p i r a l .

o r d e r t o keep t h e s a m p l e v o l u m e down,

a c e l l equipped w i t h a c a p i l l a r y w i t h The v o l u m e o f s a m p l e

a n i n s i d e d i a m e t e r o f l e s s t h a n 1 m m was used. used f o r c o n v e n t i o n a l c o l o r i m e t r y .

r e q u i r e d w a s a p p r o x i m a t e l y 1 m l f o r a 1 m LCC ( 1 m m i.d.).

A UV-210A w a s

ILCC I
I 1
SPAPLE

MONOCHROMATOR

Computer-control l e d absorption spectrophotometer w i t h t o t a l F i g u r e 5. r e f l e c t i o n long c a p i l l a r y cell.

65
RESULTS AND DISCUSSION T r a n s m i s s i o n and a b s o r p t i o n s p e c t r a o f a n a l y t e F i g u r e 6 shows t h e t r a n s m i s s i o n s p e c t r a o f La-ALC-DA carbon d i s u l f i d e i n a 2 m l o n g c a p i l l a r y c e l l . a n a q u e o u s s o l u t i o n o f La-ALC-F i n F i g u r e 7. B o t h La-ALC-F a n d a n La-ALC-DA-F and La-ALC-DA-F in The a b s o r p t i o n s p e c t r a o f s o l u t i o n i n carbon i n carbon

d i s u l f i d e o b t a i n e d b y a n o r d i n a r y s p e c t r o p h o t o m e t e r ( 1 cm c e l l ) a r e shown i n a q u e o u s p h a s e a n d La-ALC-DA-F d i s u l f i d e p r o v i d e a n a b s o r p t i o n m a x i m u m a t a b o u t 6 2 0 nm. l e n g t h a t 620 nm f o r b o t h w a t e r and c a r b o n d i s u l f i d e was used.

So, t h e w a v e -

:. 2 3 0 0 400 500 600 700 800

J . J

:.ar c ar

v)

2 3 0 0 400 500 600 700 800

1 k
# J ,

i_

400

500

600 700 Wavelength (nm)

800

300 400 500 600 700 800 Wavelength (nrn)

(A),
in

F i g u r e 6. La-ALC-DA F i g u r e 7. H20; (B).

T r a n s m i s s i o n s p e c t r a w i t h a 2 m l o n g t o t a l r e f l e c t i o n LCC. i n CS2; (B), La-ALC-DA-F i n CS2. A b s o r p t i o n s p e c t r a w i t h a n o r d i n a r y 1 cm c e l l . ( A ) La-ALC-F La-ALC-DA-F i n CS2.

P a t h w a y o f l i g h t w i t h s p e c u l a r and t o t a l r e f l e c t i o n i n LCC F i g u r e 8 shows t h e c a l i b r a t i o n c u r v e s o f f l u o r i d e when a p p l y i n g aqueous a n d c a r b o n d i s u l f i d e s o l u t i o n s t o 70 cm LCC. u s e d a s t h e medium. reflection. The p a t h w a y s o f l i g h t i n t h e c e l l c a n be c l a s s i f i e d i n t o t w o p a t t e r n s (11). One i s t h e m e r i d i o n a l r a y w h i c h i s t h e m a j o r l i g h t t r a n s m i s s i o n and t h e o t h e r i s h e l i c a l ray, which p a t t e r n i n a specular r e f l e c t i o n c e l l , When c a r b o n d i s u l f i d e w a s pathlength o f t h e source u s e d a s t h e s o l v e n t , a b s o r b a n c e was t w i c e a s h i g h as t h a t when w a t e r was T h i s means t h a t t h e o p t i c a l l i g h t i n t h e c e l l f o r specular r e f l e c t i o n i s d i f f e r e n t from t h a t f o r t o t a l

i s t h e m a j o r o p t i c a l p a t h w a y i n t h e t o t a l r e f l e c t i o n LCC ( F i g .

9). H e l i c a l
However

p r o g r e s s i o n c a n b e c o n s i d e r e d t h e c a s e i n s p e c u l a r r e f l e c t i o n LCC.

i t i s much a t t e n u a t e d due t o a number o f r e f l e c t i o n s whose c o e f f i c i e n t i s

66 below

100%.

Since t h e frequencies o f source l i g h t r e f l e c t i o n i n m e r i d i o n a l h e l i c a l progression, the attenuation therefore, the

progression are lower than those i n o f source v i a h e l i c a l progression.

l i g h t suffered v i a meridional progression i s s m a l l e r than t h a t I n t h e specular r e f l e c t i o n c e l l , mainly, contributes t o the l i g h t

t r a n s m i t t e d l i g h t v i a m e r i d i o n a l pathways, x sec 8 (Fig. 9(1)). On t h e o t h e r h a n d ,

d e t e c t o r a t t h e e x i t o f t h e LCC, w h e r e t h e o p t i c a l p a t h l e n g t h i s g i v e n b y b the effective pathlength of h e l i c a l r a y i s g i v e n b y b, x c o s e c @ ( F i g . 9(2)).

1.5V V

total reflection

rl
1u

1.0-

specular

10

1s

20

Concentration of Fluoride ( 11;: in1

F i g u r e 8. cm).

C a l i b r a t i o n c u r v e s w i t h s p e c u l a r and t o t a l r e f l e c t i o n c e l l s

(70

meridional ray

helical ray

--

b specular

F i g u r e 9. P a t h w a y s o f l i g h t s o u r c e p r o g r e s s i o n i n LCC. (I), r e f l e c t i o n : m e r i d i o n a l ray; ( Z ) , t o t a l r e f l e c t i o n : h e l i c a l ray.

67
T h e o r e t i c a l e q u a t i o n s o f a b s o r b a n c e f o r LCC Based on t h e a b o v e c o n s i d e r a t i o n o n o p t i c a l p a t h l e n g t h , the theoretical s e n s i t i v i t y o f s p e c u l a r r e f l e c t i o n LCC ( m e r i d i o n a l r a y ) c a n be shown a s :

A(absorbance) = l o g

I 0
I

'

P(X) exp[-a(h)b

sec 8 c k ( c ) ] dXd0

And t h e t h e o r e t i c a l s e n s i t i v i t y o f t o t a l r e f l e c t i o n LCC c a n be g i v e n as:

A(absorbance)

log

2
I

w h e r e 10 i s t h e i n t e n s i t y o f i n c i d e n t l i g h t . I i s t h e i n t e n s i t y o f t r a n s mitted light,

h i s t h e w a v e l e n g t h o f t h e l i g h t source,
b i s t h e c e l l length,

P(A)

i s the spectral

d i s t r i b u t i o n o f t h e l i g h t source, f u n c t i o n o f w a v e l e n g t h (A), absorbent, t h e experiment,

a(X) i s t h e a b s o r p t i o n c o e f f i c i e n t as a
c i s the concentration o f

k ( c ) i s a l m o s t e q u a l t o one i n t h e a b s o r p t i o n r a n g e o b s e r v e d i n

8 i s t h e a n g l e of i n c i d e n c e o f s o u r c e m e r i d i o n a l l i g h t , and

@ i s t h e angle o f incidence o f source h e l i c a l l i g h t .

Dependence o f s e n s i t i v i t y o f LCC on 8 and @ F i g u r e 1 0 s h o w s t h e d e p e n d e n c e s o f c a l c u l a t e d s e n s i t i v i t y o f LCC o n a n g l e s o f s o u r c e l i g h t i n c i d e n c e ( 0 , @(go"-@)) f o r m e r i d i o n a l and rays. These a r e o b t a i n e d a c c o r d i n g t o E q u a t i o n s (1) and t h e i n t e n s i t y o f I. helical

(2)

shown above.

I n t h e case o f t o t a l r e f l e c t i o n ,

d o e s n o t depend o n t h e Therefore, when

a n g l e s o f 0 and I$ b u t t h e i n t e n s i t y o f I i s much d e p e n d e n t o n t h e s e a n g l e s . When t h e a n g l e s 8 a n d (go"-@) i n c r e a s e , and (90"-I$) a r e c l o s e t o go", infinite. However,

1 decreases.

these angles increase, t h e absorbance a l s o increases.

When t h e a n g l e s 8

t h e a b s o r b a n c e i n c r e a s e s and f i n a l l y becomes and c a n n o t con-

the i n t e n s i t y o f I ( l i g h t transmitted through the

s a m p l e s o l u t i o n ) a l s o d e c r e a s e s w i t h i n c r e a s i n g 0 t o go", t r i b u t e t o t h e d e t e c t i o n signal. Cell lenqth

The c a l i b r a t i o n c u r v e s o b t a i n e d f r o m t h e t o t a l r e f l e c t i o n c e l l s o f v a r y i n g l e n g t h s a r e s h o w n i n F i g u r e 11. Clearly, the sensitivity i n the

68

90r

Figure 1 0 . The dependence of calculated sensitivity of LCC on angles 8 and of incidence of source light. 8, angle of incidence of meridional ray: @, angle of incidence of helical ray.

Figure 11. cell s.

CalibratTon curves obtained from various total reflection

total reflection LCC was dependent on cell length. It i s noted that the calibration curves showed strong curvature. This is due to the changes i n optical pathlength with variation in absorption of the solution: the optical path becomes shorter for solutions of higher concentrations. Thus the enhancement of smaller absorbance is more obvious than that of higher

A n absorbance o f 0.0002 and 0.002 given by an ordinary 1 cm cell can be amplified 2000 and 700 times, respectively, with a 4 m cell. The amplification o f absorbance by LCC is t w o to six times
absorbances for the LCC. greater than that of cell length.

69

C a l i b r a t i o n s i g n a l s and d e t e c t i o n l i m i t The a c t u a l c a l i b r a t i o n s i g n a l s o b t a i n e d b y a 4 m l o n g c e l l a r e shown i n F i g u r e 12. Aqueous s o l u t i o n s o f d i f f e r e n t f l u o r i d e c o n c e n t r a t i o n s w e r e t r e a t e d and e x t r a c t e d i n t o a carbon d i s u l f i d e - a c e t o n e m i x t u r e d e s c r i b e d previously. I n t h e p r e s e n t system, t h e d e t e c t i o n l i m i t was found t o be 10 n g / l w i t h a 4 m t o t a l r e f l e c t i o n LCC.

F i g u r e 12. A c t u a l c a l i b r a t i o n s i g n a l s ( t r a n s m i s s i o n s i g n a l s ) f o r f l u o r i d e o b t a i n e d f r o m t o t a l r e f l e c t i o n LCC (4 m) w i t h LaALC-DA-F.

TABLE I

R E C O V E R I E S OF FLUORIDE ADDED TO D R I N K I N G WATER ( n g / m l )

Fluoride Concentration Found Added Result (ng/ml) (ng/ml) (ng/ml)

Sampl e l

Recovery ( % )

A B
C

107
92.8 120 153 90.0

50

151 149 173 196 130

96.2

50
50
50

104
102
96.6 92.9

D E

50

'Samples were d i l u t e d 50 t i m e s

70
Recoveries T a b l e I shows t h e r e c o v e r i e s o f t h e f l u o r i d e added t o v a r i o u s k i n d s o f d r i n k i n g water, 50 ng/ml c o l l e c t e d o n t h e U n i v e r s i t y o f T o k y o campus. i o n s w e r e 1 n t h e r a n g e o f 93-104%. Recoveries o f lndicatlng the fluoride

v a l i d i t y o f t h i s method i n t h e a n a l y s i s o f f l u o r i d e i n d r i n k i n g water. Application t o drinking water analysis T a b l e I 1 shows t h e a n a l y t i c a l r e s u l t s o f t h e f l u o r i d e i n d r i n k i n g w a t e r c o l l e c t e d f r o m v a r i o u s areas o f Tokyo c i t y , c o l o r i m e t r y w i t h a 1 cm c e l l . ordinary colorimetry. ng/ml, Japan, using the present ordinary c o l o r i m e t r i c t e c h n i q u e w i t h a t o t a l r e f l e c t i o n LCC (0.7 m ) a n d

It i s c l e a r t h a t t h e values obtained from t h e p r e s e n t method i s more e f f e c t i v e i n e s p e c i a l l y those l o w e r than 10

t h e t o t a l r e f l e c t i o n LCC m e t h o d a g r e e d w e l l w i t h t h o s e r e s u l t i n g f r o m Furthermore, d e t e r m i n i n g t r a c e amounts o f f l u o r i d e ,

which cannot be d e t e c t e d by o r d i n a r y c o l o r i m e t r y .

TABLE I 1 ANALYTICAL RESULTS

OF

FLUORIDE I N DRINKING WATER

OF

TOKYO CITY
ng/ml Presenf method (LCC. 0 . 7 m ) 107 124 175 4.352

Conc. o f F, Sampling s t a t i o n Ordinary colorimetry ( 1 cm c e l l ) 110 120 170

1 ) U n i v . o f Tokyo, 3) I b i d .

D e p t . o f Chem.

2 ) I b i d , D e p t . o f Chem. f o r L a b o r a t o r y Use 4 ) I b i d , T r e a t e d b y I o n Exchange R e s i n 5) I b i d , Student Union

ND3
95 80

93.8
83.1 10.7 166 126 10.5 88.4

6) I b i d , Student Union
7) I b i d . Student Union

ND
165 125

8) I b i d . S t u d e n t U n i o n
9 ) I b i d , Dept. o f P h y s i c s 10) I b i d . Dept. o f P h y s i c s 11) R e s i d e n t i a l Area, Bunkyo-ku

ND
90

' S a m p l e was d i l u t e d 20 t i m e s 'After 3ND, deionization n o t detected

71
REFERENCES 1. Fuwa K (1978) D e n t a l R e v i e w 4 2 7 : l l l - 1 1 6 Underwood EJ (1975) T r a c e E l e m e n t s i n Human and A n i m a l N u t r i t i o n . Maruzen Co. Academic P r e s s , New York, p p 369-400 F r e r e FJ (1961) A n a l Chem 33:644-645 Yamamura SS, Wade MA, S i k e s JH (1962) A n a l Chem 34:1308-1312 H a s h i t a n i H. H a s h i t a n i H, Fuwa K, Wei Y o s h i d a H, Y o s h i d a H,

2.
3. 4. 5. 6. 7.

Muto H (1967) B u n s e k i Kagaku 16:44-46

A d a c h i T (1979) B u n s e k i Kagaku 28:680-684

We1 L, F u j i w a r a K.

Fuwa K (1983) A n a l Chem 55:951-955

8.
9.

L,

F u j i w a r a K (1984) A n a l Chem 56:1640-1644

F u j i w a r a K,

Fuwa K (1985) A n a l Chem 57:1012-1016

10. I n s t i t u t e o f E n v i r o n m e n t a l Science, B e i j i n g (1976) P r i n c i p a l Knowledge a b o u t P h y s i c a l and C h e m i c a l A n a l y s i s o f Water. C o n s t r u c t i o n I n d u s t r i a l Press. B e i j i n g , pp 194-197 11. We1 L. F u j i w a r a K, Fuwa K (1985) J S p e c t r o s c SOC Japan 29:173-176

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 73-80 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

73

DETERMINATION OF TOTAL FLUORINE AND IONIZABLE FLUORINE LEVELS I N BLOOD SERA

-

A COMPARATIVE STUDY

OF V A R I O U S

ANALYTICAL METHODS

YOKO BESSHO,

M I S A K O TOMITA. Y O S H I H I R O KANEKO
Showa

Department o f Hygiene and O r a l H e a l t h , School o f D e n t i s t r y , U n i v e r s i t y , 1-5-8, H a t a n o d a i , Shinagawa-ku. T o k y o 142, J a p a n

ABSTRACT

A c o m p a r a t i v e s t u d y was c a r r i e d o u t i n w h i c h f o u r a n a l y t i c a l m e t h o t i s w e r e
t e s t e d f o r t h e d e t e r m i n a t i o n o f i o n i z a b l e and t o t a l n e w b o r n c a l f serum: f l u o r i n e contents i n i n the (1) d i r e c t measurement o f i o n i z a b l e f l u o r i n e

serum u s i n g t h e f l u o r i d e i o n - s e l e c t i v e e l e c t r o d e ; ( 2 ) p r e t r e a t m e n t w i t h l o w t e m p e r a t u r e oxygen p l a s m a a s h i n g f o l l o w e d by t h e f l u o r i d e i o n - s e l e c t i v e e l e c t r o d e method o r b y gas c h r o m a t o g r a p h y ; ( 3 ) s e p a r a t i o n o f f l u o r i n e by m i c r o d i f f u s i o n f o l l o w e d by i t s d e t e r m i n a t i o n by gas chromatography; and ( 4 ) d i r e c t e x t r a c t i o n w i t h 4% TMCS n-hexane f o l l o w e d b y gas c h r o m a t o g r a n h y . The f l u o r i d e e l e c t r o d e m e t h o d w i t h o u t a n y p r e t r e a t m e n t p r o v e d e f f i c i e n t i n determining the ionizable fluorine. t o t a l f l u o r i n e i n t h e serum. The b e s t r e s u l t s w e r e o b t a i n e d f r o m t h e l o w t e m p e r a t u r e o x y g e n o l a s m a a s h i n g method f o l l o w e d by gas chromatography. sample. The v a l u e s r e s u l t i n g f r o m
111 t h e

B o t h t h e m i c r o d i f f u s i o n m e t i o d and

t h e d i r e c t e x t r a c t i o n method proved d e f i c i e n t f o r t h e d e t e r m i n a t i o n o f

t h i s p r o c e d u r e p r e s u m a b l y i n d i c a t e d an a c t u a l t o t a l f l u o r i n e c o n t e n t

INTRODUCTION Numerous analytical methods and p r e t r e a t m e n t p r o c e d u r e s havc been Findings have however, r e p o r t e d f o r t h e d e t e r m i n a t i o n o f t h e f l u o r i d e i n serum samples. on t h e l e v e l o f f l u o r i d e r e p o r t e d b y d i f f e r e n t r e s e a r c h e r s , been c o n t r a d i c t o r y . Ashed s a m p l e s have been used t o s e p a r a t e f l u o r i n e b y m i c r o d i f f u s i o n . R e c e n t l y , however,

i t has been r e p o r t e d t h a t s a t i s f a c t o r y r e s u l t s w e r e

o b t a i n e d b y u s i n g s t r o n g p e r c h l o r i c a c i d t o s i m u l t a n e o u s l y decompose and d i f f u s e samples r i c h i n o r g a n i c m a t t e r ( 1 , Z ) . I n t h i s study, we t e s t e d v a r i o u s a n a l y t i c a l methods, e i t h e r alone o r i n c o m b i n a t i o n w i t h some p r e t r e a t m e n t p r o c e d u r e s , i n an a t t e m p t t o f i n d an

74 adequate method f o r t h e d e t e r m i n a t i o n of c o n t e n t s i n c a l f serum. t h e i o n i z a b l e and t o t a l f l u o r i n e

MATERIALS AND METHODS The f o l l o w i n g a p p a r a t u s was u s e d t o c o n d u c t t h e s e e x p e r i m e n t s : t e m p e r a t u r e a s h e r ( B r a n s o n / I P C 1005-248 AN); GC-7AG,

(1) l o w

(2) gas chromatograph (Shimazu

96-09); (5)

FID);

(3) M i c r o p r o c e s s o r i o n a n a l y z e r ( O r i o n R e s e a r c h M o d e l 901);
e l e c t r o d e ( O r i o n Research Model

(4) combination f l u o r i d e

s h a k i n g a p p a r a t u s (Thomas S c i e n t i f i c T - 2 2 s ) . I r v i n e S c i e n t i f i c ' s n e w b o r n c a l f s e r u m was u s e d a s t h e t e s t s a m p l e s e r u m t h r o u g h o u t t h i s work. The i o n i z a b l e f l u o r i d e i n t h e s e r u m was d e t e r m i n e d including d i r e c t l y by a f l u o r i d e i o n - s e l e c t i v e electrode. Three p r o c e d u r e s were used t o d e t e r m i n e t h e t o t a l f l u o r i n e , b o t h i o n i z a b l e a n d combined, o f one a n a l y t i c a l methods. i n t h e serum. Each o f t h e s e was a c o m b i n a t i o n

of t h e t h r e e p r e t r e a t m e n t m e t h o d s w i t h o n e o r b o t h o f t h e t w o
The p r e t r e a t m e n t m e t h o d s u s e d w e r e l o w t e m p e r a t u r e microdiffusion. and d i r e c t e x t r a c t i o n . The e l e c t r o d e method,

oxygen plasma ashing (3)' and g a s c h r o m a t o g r a p h y .

a n a l y t i c a l methods used were t h e f l u o r i d e i o n - s e l e c t i v e The f o l l o w i n g t h r e e p r o c e d u r e s w e r e used:

( 1 ) I n t h i s p r o c e d u r e t h e s e r u m f l u o r i n e c o n t e n t was d e t e r m i n e d f i r s t b y
t h e f l u o r i d e i o n - s e l e c t i v e e l e c t r o d e method f o l l o w i n g p r e t r e a t m e n t w i t h l o w t e m p e r a t u r e oxygen plasma ashing. a g a i n b y gas chromatography (Fig. F l u o r i d e i n t h e s a m p l e was d e t e r m i n e d 1).

( 2 ) T h i i was a c o m b i n a t i o n o f m i c r o d i f f u s i o n and g a s c h r o m a t o g r a p h y ( F i g .
2).
A T u p p e r w a r e m i n i c u p was u s e d as a d i f f u s i o n b o t t l e .
The s a m p l e was p l a c e d i n t o t h e m i n i c u p ,

A polyethylene

t u b e c o n t a i n i n g a p i e c e o f f i l t e r p a p e r w i t h s o d i u m h y d r o x i d e was p l a c e d i n t h e minicup. f o l l o w e d by t h e addit i o n o f 60% p e r c h l o r i c a c i d s o l u t i o n s a t u r a t e d w i t h h e x a m e t h y l d i s i l o x a n e

(HMDS) as a r e l e a s i n g s o l u t i o n .
chromatography. attempted. high value. However,

The f l u o r i d e r e l e a s e d was a n a l y z e d b y gas

The e l e c t r o d e m e t h o d f o r a n a l y z i n g t h e f l u o r i d e was t h e sodium h y d r o x i d e p r e s e n t i n t h e sample s o l u t i o n

i n t e r f e r e d w i t h f l u o r i n e m e a s u r e m e n t b y t h e e l e c t r o d e and g a v e a f a l s e l y

(3) I n t h i s p r o c e d u r e t h e s e r u m s a m p l e was e x t r a c t e d d i r e c t l y w i t h o u t
p r i o r a s h i n g o r d e c o m p o s i t i o n t r e a t m e n t ( F i g . 3). A 60% p e r c h l o r i c a c i d (TMCS) s o l u t i o n and a hexane s o l u t i o n c o n t a i n i n g 4% t r i m e t h y l c h l o r o s i l a n e

w e r e u s e d f o r t h e e x t r a c t i o n . The f l u o r i n e c o n t a i n e d i n t r i m e t h y l f l u o r o s i l a n e (TMFS) i n t h e h e x a n e l a y e r was t h e n d e t e r m i n e d b y g a s c h r o m a t o g r a p h y .

75
F i g u r e 1. S c h e m a t i c d i a gram showing d e t e r m i n a t i o n o f t o t a l F i n serum by F i o n e l e c t r o d e and b y g a s chromatography.

Sercim I .5Illl
IJW
11111

Ash

I---

I)W

1.5IIII

II.0 l a y e r

I lexanc layer
GC

(1-11))

1 1 ? 0 layer

Icxnllc?

GC

laycr
(I 11 1)

76

Serum 2mi
4% TMCS-hexane Irnl
609; HClOs 2-4ml

H20

layer

Hexane layer
GC (FID)

F i g u r e 3. Schematic diagram showing d e t e r m i n a t i o n o f t o t a l F i n serum by d i r e c t e x t r a c t i o n f o l l o w e d by gas chromatography.

RESULTS

AND DISCUSSION
4 ) . a 100% r e c o v e r y o f t h e f l u o r i n e
the experimental value f o r the

W i t h t h e m i c r o d i f f u s i o n method (Fig. was

added t o t h e serum as sodium f l u o r i d e was observed when t h e d i f f u s i o n t i m e

3 hours o r longer.
Moreover,

I n contrast,

f l u o r i n e i n t h e serum i t s e l f was i n c r e a s e d o n l y g r a d u a l l y w i t h t h e d i f f u sion time. even a f t e r a d i f f u s i o n t i m e o f

74

hours, t h e f l u o r i n e

l e v e l observed never exceeded t h e v a l u e o f i o n i z a b l e f l u o r i n e measured by t h e d i r e c t e l e c t r o d e method.

O ' i

Diffusion time, h r

12

2 1

F i g u r e 4. E f f e c t o f d i f f u s i o n t i m e on t h e F v a l u e d e t e r m i n e d and t h e A 2 m l s e r u m s a m p l e w i t h o r w i t h o u t added r e c o v e r y r a t e o f added F. f l u o r i d e was t r e a t e d w i t h t h e m i c r o d i f f u s i o n method u s i n g 4 m l 60% HC104 s a t u r a t e d w i t h HMDS f o r v a r i o u s t i m e p e r i o d s . The r e l e a s e d F was t h e n d e t e r m i n e d b y gas chromatography.

77

The r e s u l t s o b t a i n e d w i t h t h e d i r e c t e x t r a c t i o n m e t h o d w e r e a l r i l o s t t h e same as w i t h m i c r o d i f f u s i o n . longed t o 8 hours (Fig. The f l u o r i n e l e v e l o b t a i n e d n e v e r e x c e e d e d t h e i o n i z a b l e one i n t h e serum, e v e n t h o u g h t h e e x t r a c t i o n t i m e was p r o -

5).

O i 2 3 1 6 Extraction t i m e s hP

F i g u r e 5. E f f e c t o f e x t r a c t i o n t i m e o n t h e F v a l u e d e t e r m i n e d i n serum. A 2 m l s e r u m s a m p l e was e x t r a c t e d w i t h 2 m l 60% HC104 and 1 m l 4% TMCS i n nhexane. The e x t r a c t e d

F was t h e n d e t e r m i n e d b y g a s c h r o m a t o g r a p h y .

F i g u r e s 5 and 7 show t h e r e l a t i o n s h i p b e t w e e n t h e s a m p l e v o l u m e and t h e a m o u n t o f f l u o r i d e o b t a i n e d b y m i c r o d i f f u s i o n and t h e a m o u n t o b t a i n e d b y d i r e c t extraction, gas c h r o m a t o g r a p h y . a b l e s i n b o t h cases. i o n i z a b l e f l u o r i n e only, respectively. I n b o t h cases, f l u o r i n e was d e t e r m i n e d b y A l i n e a r r e l a t i o n s h i p e x i s t e d between t h e t w o v a r i The v a l u e s shown i n t h e t w o f i g u r e s r e p r e s e n t t h e as mentioned p r e v i o u s l y . subse-

F i g u r e 8 shows t h e r e l a t i o n s h i p b e t w e e n t h e f l u o r i n e c o n t e n t s d e t e r m i n e d b y t h e e l e c t r o d e m e t h o d and t h o s e d e t e r m i n e d b y g a s c h r o m a t o g r a p h y , method. q u e n t t o p r e t r e a t m e n t o f t h e serum sample b y t h e l o w t e m p e r a t u r e a s h i n g An e x c e l l e n t a g r e e m e n t w a s f o u n d b e t w e e n t h e d e t e r m i n e d v a l u e s even though t h e v a l u e s o b t a i n e d f r o m gas Chromatography f r o m b o t h methods, samp 1es. The f l u o r i d e c o n t e n t s i n t h e c a l f s e r u m s a m p l e d e t e r m i n e d b y d i f f e r e n t a n a l y t i c a l m e t h o d s a r e s u m m a r i z e d i n T a b l e I. ug/ml. The mean v a l u e o f t h e i o n i z a b l e f l u o r i d e l e v e l d e t e r m i n e d b y t h e d i r e c t e l e c t r o d e m e t h o d w a s 0.169 Samples t h a t were p r e t r e a t e d w i t h t h e l o w t e m p e r a t u r e a s h i n g method and t h e n d e t e r m i n e d f o r f l u o r i d e b y t h e e l e c t r o d e method o r b y gas c h r o -

were s l i g h t l y h i g h e r t h a n t h o s e f r o m t h e e l e c t r o d e method i n most o f t h e

78

F i g u r e 6. The r e l a t i o n s h i p b e t w e e n s a m p l e v o l u m e a n d t h e F v a l u e s d e t e r m i n e d b y t h e m i c r o d i f f u s i o n method. The v o l u m e o f 60% HC104 u s e d was t h r e e t i m e s t h a t o f t h e s e r u m sample. The r e l a t i o n s h i p b e t w e e n s a m p l e v o l u m e and t h e F v a l u e s d e t e r F i g u r e 7. m i n e d b y d i r e c t e x t r a c t i o n method. The v o l u m e o f 60% HC104 u s e d was t h r e e t i m e s t h e v o l u m e o f t h e s e r u m sample.

2 E !

E .& 1.51.2.

0.9.
0.6.

u m *
L

c ._

0.304 0

0.3

U.6

0.9

1.2

1.5

T o t a l F in s e r u m ( G C ) ( r J q / m l )

F i g u r e 8. The r e l a t i o n s h i p b e t w e e n t o t a l F v a l u e s o b t a i n e d b y g a s chromat o g r a p h y (GC) and b y F i o n e l e c t r o d e m e t h o d ( I E ) .

I9
matography gave h i g h e r v a l u e s : 0.199 p g / m l by t h e e l e c t r o d e method, and

0.232 p g / m l b y g a s c h r o m a t o g r a p h y . i.e.,

M o r e o v e r , when a s t a n d a r d a d d i t i o n even h i g h e r v a l u e s were o b t a i n e d ,

method was a p p l i e d t o t h e above methods, tography.

0.257 p g / m l b y t h e e l e c t r o d e m e t h o d , and 0.264 p g / m l b y g a s c h r o m a An e x a m p l e o f t h e c a l i b r a t i o n c u r v e f o r t h e s t a n d a r d a d d i t i o n w i t h t h e gas c h r o m a t o g r a p h i c method f o l l o w i n g t h e This

method i s shown i n F i g u r e 9. Contrary t o this, pretreatment, e i t h e r w i t h m i c r o d i f f u s i o n o r w i t h d i r e c t e x t r a c t i o n , t h e v a l u e s o b t a i n e d n e v e r exceeded t h e l e v e l of t h e i o n i z a b l e f l u o r i n e . indicates w i t h t h e s e procedures. C o n c e r n i n g m i c r o d i f f u s i o n , V e n k a t e s w a r l u (4) and Paez f l u o r i d e i n b l o o d serum. t h a t t h e c o m b i n e d f l u o r i n e i n b l o o d serum c a n n o t be d e t e c t e r '

fi g .

(5) p o i n t e d

o u t a s i m i l a r f a c t i n t h e i r c o m p a r a t i v e s t u d i e s on methods f o r e v a l u a t i n g They f o u n d m i c r o d i f f u s i o n t o be i n e f f e c t i v e as a p r e t r e a t m e n t p r o c e d u r e f o r d e t e r m i n i n g t h e t o t a l f l u o r i n e c o n t e n t when i t was d i r e c t l y a p p l i e d t o unashed samples.

TABLE I FLUORIDE CONTENTS I N CALF SERUM DETERMINED BY DIFFERENT ANALYTICAL METHODS

Method
~~~

F i n Serum (w/m1) Mean ? SD

0.169 0.199 0.257 0.232 0.264 0.165 0.003

Ratio t o Ionizable F

IE' (ionizable)
IE-sL2 Ashing IE-st. GC-st. Ashing GC-st. add3 add3

15
9 36 8 31 4

1.00
1.18 1.52 1.37 1.56 0.98 0.92

0.019

----0.011

----0.008

D if f 4 E x t r . 5-

GC

GC

0.1 55

_____

'F ion electrode 'Standard c u r v e method 3 S t a n d a r d a d d i t i o n method

4 M i c r o d i f f us i o n method 5 D i r e c t e x t r a c t i o n method

80

-c,5

c.5

1.3

1.5
(ug)

2.0

F added

F i g u r e 9. C a l i b r a t i o n c u r v e o f t h e s t a n d a r d a d d i t i o n m e t h o d f o r F d e t e r m i n a t i o n b y gas chromatography.

CONCLUSION The u s e o f t h e f l u o r i d e i o n - s e l e c t i v e e l e c t r o d e w i t h o u t a p r i o r s e p a r a t i o n p r o c e d u r e was f o u n d t o b e e f f i c i e n t f o r t h e d e t e r m i n a t i o n o f t h e i o n i z a b l e f l u o r i n e i n b l o o d serum. f l u o r i n e i n b l o o d serum, t i o n proved deficient. For the determination o f the t o t a l pretreatment w i t h microdiffusion o r d i r e c t extracThe b e s t r e s u l t s w e r e o b t a i n e d b y u s e o f t h e l o w

t e m p e r a t u r e ashing method f o l l o w e d b y gas c h r o m a t o g r a p h y o r b y f l u o r i d e e l e c t r o d e measurement, a l t h o u g h t h e l a t t e r method gave a s l i g h t l y l o w e r value. The v a l u e s o b t a i n e d f r o m t h e s e p r o c e d u r e s p r e s u m a b l y i n d i c a t e an a c t u a l t o t a l f l u o r i n e c o n t e n t i n t h e sample.

REFERENCES
1. H a r a K, Hayashi

H,

H i r o k a w a K.

I i z u k a K (1983) J D e n t H l t h 33:296-297

2.
3. 4. 5.

D a b e k a RD, M c K e n z i e AD, 62: 1065-1069 T o m i t a M, Paez DM, 13: 65-70 S u z u k i S,

C o n a c h e r HBS ( 1 9 7 9 ) J A s s o c O f f A n a l Chem

K u r o i w a S, Kaneko Y (1983) J D e n t H l t h 33:183-196 Dapas J r 0, C o r o n a t o

V e n k a t e s w a r l u P (1975) Biochem Med 14:368-371 deB1anchi LP, G i l BA,

RG (1980) F l u o r i d e

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 81-88 0 1986 Elsevier Science Publishers B.V.. Amsterdam - Printed in The Netherlands

81

SAMPLING OF HYDROGEN FLUO2IDE BY DIFFUSIONAL TRANSPORT I N A CIRCULAR GLASS TUBE COATED WITH SODIUM CARBONATE TAKAAKI YANAKA

N i i g a t a P r e f e c t u r a l Research L a b o r a t o r y Sowa, N i i g a t a C i t y , J a p a n 950-21

f G r

H e a l t h and E n v i r o n m e n t ,

314-1

ABSTRACT Hydrogen f l u o r i d e o f t h r o u g h a t u b e whose l o w c o n c e n t r a t i o n s (0.004 inside wall was c o a t e d

-

2 ppm) was p a s s e d
carbonate. For laminar

w i t h sodium

R e y n o l d s n u m b e r s o f t h e f l o w w e r e b e t w e e n 2 5 0 a n d 10,000. with the theoretical f o r m u l a o f G o r m l e y and Kennedy.

flow, t h e d i s t r i b u t i o n o f f l u o r i d e on t h e i n s i d e w a l l corresponded w e l l For t u r b u l e n t flow, t o the distance From t l i e s e t h e d i f f u s i o n c o e f f i c i e n t was e x p r e s s e d a s a f u n c t i o n of R e y n o l d s number. S a m p l i n g e f f i c i e n c i e s o f t h e t u b e a r e shown i n r e l a t i o n from t h e tube entrance, w i t h t h i s t y p e o f tube, o f 0.4 t o 1 cm. t h e f l o w r a t e and t h e R e y n o l d s number.

r e s u l t s , i t a p p e a r s t h a t h y d r o g e n f l u o r i d e c a n be e f f e c t i v e l y c o l l e c t e d s e v e r a l d e c a c e n t i m e t e r s l o n g and w i t h a d i a m e t e r

INTRODUCTION I n the f i e l d o f aerosol science, a method o f d i f f u s i o n a l t r a n s p o r t o f The m a i n p a r t o f p a r t i c l e s b y B r o w n i a n movement o n t o t h e i n s i d e w a l l o f a t u b e h a s been used f o r t h e m e a s u r e m e n t o f p a r t i c l e s i z e and c o n c e n t r a t i o n . t h e t u b e f o r t h i s m e t h o d i s c a l l e d a d i f f u s i o n tube. the tube is usually c i r c u l a r o r f l a t l y rectangular. For P o i s e u i l l e f l o w through a c i r c u l a r tube, t h e d i s t r i b u t i o n of d i f f u s i o n a l t r a n s p o r t o n t o t h e i n s i d e w a l l was s o l v e d t h e o r e t i c a l l y b y G o r m l e y and Kennedy ( 1 ) u n d e r t h e c o n d i t i o n o f n e g l e c t i n g a x i a l d i f f u s i o n . The d i f f u s i o n e q u a t i o n i n a s t e a d y s t a t e i s The c r o s s s e c t i o n o f

and b o u n d a r y c o n d i t i o n s a r e x =

Co ( c o n s t )

r = R , C = O

82 I n these equations, x i s t h e distance from the tube entrance, r i s r a d i a l coordinate, particles,

i s t h e c o n c e n t r a t i o n o f a e r o s o l a t (X,r),

i s t h e mean v e l o -

c i t y t h r o u g h t h e tube,

D i s t h e Brownian d i f f u s i o n c o e f f i c i e n t o f aerosol The s o l u t i o n i s

and R i s t h e r a d i u s o f t h e t u b e .

c(x) =
LO

0.8191 exp(-14.63A)

0.0976 exp(-89.2A)

W h e r e C ( x ) i s t h e m e a n c o n c e n t r a t i o n o f a e r o s o l a t a d i s t a n c e x. d i f f u s i o n parameter f o r a c i r c u l a r tube

0 . 0 3 2 5 exp(-228A)

+ ...

(3)

A i s the

D x 4VR

= X / P e , d = 2 R d

and Pe i s t h e d i f f u s i v e P e c l e t number ( v d / D ) . F o r t u r b u l e n t f l o w t h r o u g h a t u b e , M o n t g o m e r y and C o r n (2) s u m m a r i z e d t h e theories o f deposition o f p a r t i c l e s claimed by Friedlander were f a r h i g h e r t h a n t h e t h e o r e t i c a l values. o f Friedlander gt c o m p l e t e l y solved. The d i f f u s i o n c o e f f i c i e n t o f t h e m o l e c u l a r m o t i o n o f g a s i s b y f a r h i g h e r t h a n t h a t o f p a r t i c l e s b y Brownian motion. F o r t h i s reason,

&

2 1 . (3).

D a v i e s (4). and B e a l (5). and p o i n t e d o u t t h a t t h e i r e x p e r i m e n t a l r e s u l t s B u t L i u and Agarwal (6) c l a i m e d t h a t t h e i r e x p e r i m e n t a l values were i n agreement w i t h t h e t h e o r i e s

2 1 . and

Beal.

It i s presumed t h a t t h e t h e o r y o f d i f -

f u s i o n a l deposition o f p a r t i c l e s i n a t u r b u l e n t f l o w i n a tube i s s t i l l not

i f the inside

w a l l o f a tube i s coated w i t h a m a t e r i a l which reacts r a p i d l y w i t h a s p e c i f i c gas, and t h e n a g a s c o n t a i n i n g t h a t s p e c i f i c g a s f l o w s t h r o u g h t h e tube,

i t i s e x p e c t e d f r o m Boundary c o n d i t i o n s 2 and S o l u t i o n 3 t h a t t h e
O k i t a e t al.

s p e c i f i c gas can be e f f e c t i v e l y c o l l e c t e d on t h e i n s i d e w a l l i n a s h o r t l e n g t h o f tube.

(7) showed t h a t a t u b e c o a t e d w i t h sodium

B u t s a m p l i n g e f f i c i e n c y has n o t

carbonate c o u l d c a p t u r e hydrogen f l u o r i d e .

been i n v e s t i g a t e d u n d e r d i f f e r e n t c o n d i t i o n s . The p u r p o s e o f t h i s p a p e r i s t o show t h e d i s t r i b u t i o n o f d i f f u s i o n a l t r a n s p o r t o f h y d r o g e n f l u o r i d e i n a c i r c u l a r g l a s s t u b e whose i n s i d e w a l l i s c o a t e d w i t h sodium c a r b o n a t e f o r b o t h l a m i n a r and t u r b u l e n t f l o w s . MATERIALS AND METHODS

A i r c o n t a i n i n g 0.004 - 2 ppm h y d r o g e n f l u o r i d e g e n e r a t e d b y a P e r m e a c a l
Permeator (PD
-

10, G a s t e c Co.)

was

passed through Pyrex g l a s s tubes

83
( d i f f u s i o n t u b e ) o f 0.4.

0.6,

and 1.0 cin i.d.,

whose i n s i d e w a l l s w e r e The appara-

c o a t e d w i t h s o d i u m c a r b o n a t e ( d r i e d a f t e r b e i n g c o a t e d w i t h 1M Na2C03). F i g u r e 1 i s a s c h e m a t i c d i a g r a m o f t h e e x p e r i m e n t a l apparatus. d i l u t e d hydrogen f l u o r i d e . o f t h e d i f f u s i o n tube, t h e d i f f u s i o n tube, t u s c o n s i s t s o f a g a s g e n e r a t i o n s y s t e m and a c o l l e c t i n g s y s t e m f o r t h e

A T e f l o n t u b e E,

whose r a d i u s was equal t o t h a t

was u s e d f o r t h e f u l l d e v e l o p m e n t o f t h e f l o w

p r o f i l e . The l e n g t h o f t h e t u b e was 1 6 0 cm (8). A t t h e d o w n s t r e a m end o f an a l k a l i impregnated f i l t e r was s e t i n a T e f l o n h o l d e r The t o c o l l e c t hydrogen f l u o r i d e t h a t passed t h r o u g h t h e d i f f u s i o n tube. 51-A, Toyo R o s h i Co.,

a l k a l i impregnated f i l t e r was prepared by s o a k i n g a c e l l u l o s e f i l t e r (No.

4 cm i n e f f e c t i v e d i a m e t e r ) i n a s o d i u m c a r b o n a t e
f o l l o w e d by a i r - d r y i n g a t 60C. I n t h i s experiment, and

s o l u t i o n o f ca.

O.lM,

t h e t e m p e r a t u r e o f t h e gas f l o w i n g t h r o u g h t h e t u b e was m a i n t a i n e d a t

201rZ"C,

and t h e p r e s s u r e was k e p t a t 760 - 773 T o r r f o r l a m i n a r f l o w ,

760 -860 T o r r f o r t u r b u l e n t f l o w .
A f t e r sampling, t h e d i f f u s i o n t u b e was c u t i n t o s e v e r a l s e c t i o n s and each The f l u o r i d e i n t h e a l k a l i impreg, T h e amount o f p i e c e was washed w i t h 25 m l o f water.

n a t e d f i l t e r was e x t r a c t e d w i t h 2 5 m l o f warm w a t e r . trode.

f l u o r i d e i n t h e s e samples was d e t e r m i n e d b y a f l u o r i d e i o n s e l e c t i v e e l e c -

-v

F i g u r e 1. Schematic diagram o f t h e e x p e r i m e n t a l apparatus. ( C ) c o m p r e s s o r , ( V ) v a l v e , ( G ) r e g u l a t o r , ( A ) a i r f i l t e r , (P) P e r m e a c a l P e r m e a t o r , ( R ) f l o w m e t e r , ( M ) gas m i x e r , ( E ) T e f l o n Tube, (D) d i f f u s i o n tube, ( F ) a l k a l i impregnated f i l t e r , ( T ) thermometer.

RESULTS AND

DISCUSSION

F u l l y developed f l o w The d i s t r i b u t i o n o f d i f f u s i o n a l d e p o s i t i o n o f hydrogen f l u o r i d e i n t h e d i f f u s i o n t u b e was measured by s e t t i n g t h e e n t r a n c e r e g i o n a t i t s upstream. The e x p e r i m e n t a l r e s u l t s f o r P o i s e u i l l e f l o w and f u l l y developed t u r b u l e n t f l o w a r e i l l u s t r a t e d i n F i g u r e s 2 and 3, r e s p e c t i v e l y . The c o - o r d i n a t e s

0.1

>
0
0

1 0

0.01

R e = 707 R e = 283

0.001

100

x /d

200

3oa

F i g u r E 2. D i f f u s i o n a l t r a n s p o r t o f h y d r o g e n f l u o r i d e i n a d i f f u s i o n t u b e f o r P o i s e u i l l e f l o w . Re, R e y n o l d s n u m b e r ; C(x), mean c o n c . o f a e r o s o l a t a d i s t a n c e x; Co, c o n c . a t e n t r a n c e ; x 1 d i s t a n c e f r o m t h e t u b e e n t r a n c e ; d, i n s i d e : d = 1.0 cm. d i a m e t e r o f tube. o : d = 0.4 cm; + : d = 0.6 cm; D i f f u s j o n a l t r a n s p o r t o f hydrogen f l u o r i d e i n a d i f f u s i o n tube i n f u l l y developed t u r b u l e n t flow. F i g u r e 3. Re, R e y n o l d s number; C(x), mean conc. o f a e r o s o l a t a d i s t a n c e x; Co. conc. a t e n t r a n c e ; x, d i s t a n c e f r o m t u b e e n t r a n c e ; d, i n s i d e d i a m e t e r o f t u b e . o : d = 0.4 cm.

85

a r e i n d i c a t e d i n d i m e n s i o n l e s s v a l u e s as t h e r a t i o o f t h e d i s t a n c e f r o m t h e t u b e i n l e t t o t h e d i a m e t e r o f t h e t u b e and t h e f r a c t i o n o f h y d r o g e n f l u o r i d e a r r i v i n g a t t h e cross-section. Each p l o t i s t h e mean v a l u e f o r 2-

4 r e p l i c a t e s o f t h e experiment,
1.d.

a n d t h e r e p r o d u c i b i l i t y w a s v e r y good.

When t h e R e y n o l d s number was l e s s t h a n e v e r y R e y n o l d s number, e n t r a n c e o f t h e tube.

2,000, t u b e s w i t h 0.4.

0.6 o r 1.0 cm
For

w e r e u s e d , w h i l e b e y o n d 2,000. t h o s e w i t h 0.4 cm 1.d.

w e r e used.

t h e a r r i v i n g f r a c t i o n o f hydrogen f l u o r i d e is l i n e a r

on a s e m i - l o g a r i t h m i c g r a p h w i t h t h e e x c e p t i o n o f a s h o r t r e g i o n n e a r t h e

I f D i s a s s u m e d t o b e 0.26 c m 2 / s , a n d t h e d i s t a n c e

f r o m t h e t u b e e n t r a n c e i s expressed b y t h e d i m e n s i o n l e s s v a l u e (x/d)/Pe. a l l e x p e r i m e n t a l r e s u l t s i n P o i s e u i l l e f l o w (Re<2.300) a r e i n a g r e e m e n t w i t h t h e t h e o r e t i c a l v a l u e s o f G o r m l e y and Kennedy ( 1 ) as i l l u s t r a t e d i n F i g u r e 4. S i n c e t h e v a l u e o f 0.26 c m 2 / s i s n e a r l y e q u a l t o t h e m o l e c u l a r d i f f u s i o n c o e f f i c i e n t o f w a t e r i n a i r (0.25 c r n 2 / s a t 2O"C, 7 6 0 T o r r ) . a n d t h e m o l e c u l a r w e i g h t of hydrogen f l u o r i d e i s n e a r l y equal t o t h a t o f water,
i t seems r e a s o n a b l e t o c o n s i d e r t h a t t h e v a l u e o f

0.26 c m 2 / s i s t h e

m o l e c u l a r d i f f u s i o n c o e f f i c i e n t o f hydrogen f l u o r i d e i n a i r .

Comparison o f e x p e r i m e n t a l r e s u l t s i n P o i s e u i l l e f l o w w i t h theoF i g u r e 4. r e t i c a l v a l u e s s o l e d by-Gormley and Kennedy. D, B r o w n i a n d i f f u s i o n c o e f f i c i e n t (=0.26 cm /s); C(x), m e a n c o n c . o f a e r o s o l a t a d i s t a n c e X ; Co, c o n c . a t e n t r a n c e ; X, d i s t a n c e from t u b e e n t r a n c e : d, i n s i d e d i a m e t e r o f t u b e : Pe, d i f f u s i v e P e c l e t number (=Vd/D)

86

F i g u r e 5. Diffusion coefficient o f hydrogen f l u o r i d e o f f u l l y developed f l o w i n d i f f u s i o n tube. Re, R e y n o l d s numb r; D, B r o w n i a n d i f f u s i o n c o e f f i c i e n t o f a e r s o l p a r t i c l e s . D = 0.26 cm / s ( 2 8 3 < R e < 2 , 3 0 0 ) ; D = 2.04 l o g ( R e 1 1 5 0 0 ) cm / s (2.300iRe<10,000)

I f t h e f l o w i n t h e t u b e e x c e e d s t h e c r i t i c a l R e y n o l d s number (ca. 2,300).

diffusional transport onto the inside wall becomes m o r e r a p i d t h a n u n d e r The r e s u l t s o f t h e c a l c u l a t i o n laminar conditions. T h i s phenomenon c a n b e e x p l a i n e d b y a d d i n g t h e e f f e c t s

o f eddy d i f f u s i o n t o m o l e c u l a r d i f f u s i o n . numbers.

o f D f o r t u r b u l e n t f l o w a r e shown i n F i g u r e 5 a c c o r d i n g t o R e y n o l d s The e x p e r i m e n t a l f o r m u l a f o r D w h i c h c a n be a p p l i e d t o R e y n o l d s n u m b e r s f r o m 2,300 t o 10,000 i s Re

2.04 l o g __ 1500

cm2/s

(4)

Therefore,

s u b s t i t u t i n g F o r m u l a ( 4 ) i n t o t h e f i r s t t e r m o f S o l u t i o n (3). transport for turbulent flow can be

the distribution o f diffusional e x p r e s s e d as f o l l o w s :

-

c(x) =
CO

0.819[

-29.8

vd

log Re 1500

(5)

(2,300~Re~10,000) Developing f l o w T h e t u b e E. i l l u s t r a t e d i n F i g u r e 1, w a s r e p l a c e d w i t h t h e a p p a r a t u s The c y l i n d e r was made o f a c r y l i c r e s i n , and a T o y o R o s h i Co.) w a s u s e d f o r a r r a n g i n g s t r e a m l i n e s . and t h e d i s t a n c e from t h e f i l t e r t o t h e F i g u r e 7 shows t h e d i f f u s i o n i l l u s t r a t e d i n F i g u r e 6. T e f l o n f i l t e r (PF-1,

T h e i n s i d e d i a m e t e r w a s 4 cm,

e n t r a n c e o f t h e d i f f u s i o n t u b e was a b o u t 5 cm. l a m i n a r and t u r b u l e n t f l o w .

c o e f f i c i e n t s o f hydrogen f l u o r i d e c o r r e s p o n d i n g t o Reynolds numbers i n There were no s i g n i f i c a n t d i f f e r e n c e s between

t h e s e e x p e r i m e n t a l r e s u l t s and t h o s e o f e x p e r i m e n t s i n v o l v i n g f u l l y d e v e l oped f l o w .

I
a

-+
out

F i g u r e 6. Apparatus f o r developing f l o w i n t h e entrance r e g i o n o f d i f f u ( F ) T e f l o n f i l t e r , (D) d i f f u s i o n tube, s c a l e u n i t i n m i l l i s i o n tube. meters.

1.0 0

0.5 -

OL 100

too0

. . . .I
loo00

Re

F i g u r e 7. D i f f u s i o n c o e f f i c i e n t o f hydrogen f l u o r i d e o f d e v e l o p i n g f l o w i n tube. D i s t a n c e f r o m T e f l o n f i l t e r and e n t r a n c e o f d i f f u s i o n t u b e i s a p p r o x i m a t e l y 5 cm. Re, R e y n o l d s n u y e r ; D, B r o w n i a n d i f f u s i o n c o e f f i c i e n t o f aerosol particles. D = 0.26 cm / s ( 2 5 0 < R e < 2 , 3 0 0 ) , D = 2.04 l o g ( R e / 1 5 0 0 ) c m / s ( 2,300 < Re< 10.000)

88 R e l a t i o n b e t w e e n s a m p l i n g e f f i c i e n c y and p a r a m e t e r s ( x . 99%) a n d d i s t a n c e x,

Q and Re).

F i g u r e 8 shows t h e r e l a t i o n b e t w e e n s a m p l i n g e f f i c i e n c i e s (90, 95 and f l o w r a t e Q a n d R e y n o l d s n u m b e r Re.

If t h e f l o w r a t e
If the flow rate

i s constant i n the laminar condition, i s constant i n turbulent flow, From t h e s e r e s u l t s ,

t h e s a m p l i n g e f f i c i e n c y i s dependent

on t h e t u b e l e n g t h r e g a r d l e s s o f t h e r a d i u s o f t h e t u b e . t h e tube diameter) t h e higher t h e sampling e f f i c i e n c y .

t h e h i g h e r t h e Reynolds number ( t h e s m a l l e r

i t a p p e a r s t h a t h y d r o g e n f l u o r i d e c a n be e f f e c t i v e l y

c o l l e c t e d b y a d i f f u s i o n t u b e s e v e r a l deca c e n t i m e t e r s l o n g and w i t h a d i a m e t e r f r o m 0.4 t o 1 cm.

0 =90%

95%

99%

150r

100 100 -

150 -

50

50 -

OC

10

20
Q

30
CLhinI

F i g u r e 8. R e l a t i o n between s a m p l i n g e f f i c i e n c y o f d i f f u s i o n tube, d i s t a n c e , f l o w r a t e , a n d R e y n o l d s n u m b e r . Re, R e y n o l d s n u m b e r ; Q. f l o w r a t e ; x, d i s t a n c e f r o m tube entrance.

REFERENCES

1.

G o r m l e y PG,

Kennedy M (1949) P r o c Roy I r i s h Acad 52A:163-169 C o r n M (1970) J A e r o s o l S c i 1:185-213 J o h n s t o n e HF (1957) I n d Engng Chem 49:1151 New York, p 436 W e s t i n g h o u s e E l e c t r i c C o r p o r a t i o n P u b l i c a t i o n WAPD-TM-

2.
3. 4.

M o n t g o m e r y TL, F r i e d l a n d e r SK, B e a l SK (1968) 765 L i u BYH.

D a v i e s CN (1966) A e r o s o l S c i e n c e . A c a d e m i c P r e s s ,

5.
6.

A g a r w a l JK (1974) J A e r o s o l S c i 5:145-155 Theory. McGraw-Hill. pp

7.
8.

O k i t a T, S u g a i R, K a n e d a K ( 1 9 7 0 ) J J a p a n SOC A i r P o l l 5 : 8 8 S c h l i c h t i n g H (1968) Boundary-Layer

282 -

561

H. Tsunoda and M.-H. Yu (Editors)

Fluoride Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 91-97 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

91

FLUORIDE TRANSPORT I N TERRESTRIAL ECOSYSTEMS AROUND INDUSTRIAL AREAS

FRANK M U R R A Y ~
Department of B i o l o g i c a l Sciences, Wales, A u s t r a l i a U n i v e r s i t y o f Newcastle, New S o u t h

School o f E n v i r o n m e n t a l and L i f e Sciences, Murdoch 'Present address: U n i v e r s i t y , Murdoch. W e s t e r n A u s t r a l i a 6150, A u s t r a l i a ABSTRACT

A E u c a l y p t u s - d o m i n a t e d f o r e s t e c o s y s t e m and an A v i c e n n i a m a r i n a - d o m i n a t e d
m a n g r o v e f o r e s t e c o s y s t e m a r o u n d f l u o r i d e e m i s s i o n s o u r c e s i n New S o u t h Wales. A u s t r a l i a , have been s u b j e c t t o c o n t i n u o u s f l u o r i d e i n p u t s i n c e S t u d i e s o f t h e e f f e c t s o f f l u o r i d e e m i s s i o n s on t h e e c o l o q y o f and f l u o r i d e - s e n s i t i v e t r e e s and s h r u b s a b o u t 1970.

t h e s e e c o s y s t e m s h a v e shown t h a t t r e e s b e 1 i e v e d t o be f l u o r i d e - s e n s i t i v e demonstrated changes i n metabolism, i n areas w i t h h i g h f l u o r i d e i n p u t died. i n a number o f ecosystem components. F l u o r i d e concentrations increased Important t r a n s f e r processes o f

t h r o u g h f a l l and s t e m f l o w , w e r e r e s p o n s i b l e f o r s i g n i f i c a n t f l u o r i d e f l u x e s . T h r e e zones c o u l d be d e f i n e d i n a r e a s s u b j e c t t o h i g h f l u o r i d e i n p u t and are characterized by the following:

1.
2.

M a j o r e f f e c t s r e s u l t i n g i n v i s i b l e i n j u r y and l o s s o f s e n s i t i v e e x p o s e d s p e c i e s f r o m t h e ecosystem. Subtle e f f e c t s d e m o n s t r a t e d b y changes i n p l a n t m e t a b o l i s m w i t h p o t e n r e p r o d u c t i o n and d i s e a s e r e s i s t a n c e o f s e n s i b i o m a s s and n u t r i e n t c y c l i n g . t i a l e f f e c t s on g r o w t h , t i v e species. species composition,

A t an e c o s y s t e m l e v e l , t h e r e may be l o n g - t e r m changes i n
diversity,

3.

Innocuous

e f f e c t s c h a r a c t e r i z e d by t h e ecosystem a c t i n g as a s i n k f o r

f 1u o r i d e .
INTRODUCTION F l u o r i d e i s w i d e l y d i s t r i b u t e d i n t h e n a t u r a l environment, i n rocks, s o i l s , groundwater, surface waters, air, ranking 13th It occurs among t h e e l e m e n t s i n o r d e r o f a b u n d a n c e i n t h e e a r t h ' s c r u s t .

p l a n t s and a n i m a l s . The m a i n aluminum

F l u o r i d e i s a l s o an i m p o r t a n t i n d u s t r i a l e m i s s i o n w h i c h i s r e l e a s e d when fluoride-containing smelters, works, m a t e r i a l s a r e heated t o h i g h temperatures. phosphate f e r t i l i z e r works, brick. i n d u s t r i a l processes involved are c o a l - f i r e d power stations, steel plants, o i l r e f i n e r i e s and some o r e - r e f i n i n g and c h e m i c a l p l a n t s .

t i l e and g l a s s

92
E f f e c t s on P l a n t s F l u o r i d e i s one o f t h e m o s t p h y t o t o x i c o f t h e common a i r p o l l u t a n t s . a i r p o l l u t a n t i n E u r o p e ( 1 ) a n d t h e f o u r t h i n t h e U S A (2). d i o x i d e o r n i t r o g e n oxides. In t e r m s o f i t s e f f e c t on p l a n t s i t i s r e g a r d e d a s t h e s e c o n d m o s t i m p o r t a n t I t may c a u s e sulphur For i n j u r y a t c o n c e n t r a t i o n s i n a i r a b o u t 100 t i m e s l o w e r t h a n ozone, p a s s e d on t o a n i m a l s w h i c h consume f l u o r i d e - c o n t a m i n a t e d systems. The e f f e c t s o f f l u o r i d e o n f o r e s t e c o s y s t e m s a r e d e t e r m i n e d by: form o f the fluoride, t h e concentration, d u r a t i o n o f exoosure. (a) f a c t o r s r e l a t i n g t o f l u o r i d e e x p o s u r e such as t h e c h e m i c a l and p h y s i c a l windspeed and o t h e r c l i m a t i c c o n d i t i o n s : cesses, and ( b ) f a c t o r s r e l a t i n g t o t h e r e s p o n s e o f

I t i s a l s o a c c u m u l a t i v e i n p l a n t s and may be
vegetation.

t h e s e r e a s o n s f l u o r i d e h a s t h e c a p a c i t y t o h a v e l o n g - t e r m e f f e c t s o n eco-

t h e e c o s y s t e m s u c h as t h e r e s i s t a n c e o f t h e e c o s y s t e m c o m p o n e n t s o r p r o d u r i n g t h e p e r i o d o f exposure. agricultural plants t o fluoride, r e l a t i v e l y l i t t l e i s known I n a d d i t i o n some k n o w l e d g e o f The accuA l t h o u g h t h e r e i s a s u b s t a n t i a l amount o f i n f o r m a t i o n r e l a t i n g t o t h e responses of a b o u t t h e responses o f f o r e s t e d ecosystems. the r a t e o f uptake of

f l u o r i d e by ecosystems i s r e q u i r e d i n order t o

i m p r o v e models o f f l u o r i d e d i s p e r s i o n around e m i s s i o n sources.

r a c y o f p r e d i c t i o n s r e s u l t i n g f r o m t h e s e models depends i n p a r t on t h e a s s u m p t i o n s t h e y make a b o u t r a t e s o f r e m o v a l o f a i r b o r n e f l u o r i d e b y veget a t i o n and s o i l , b u t l i t t l e f i e l d d a t a i s a v a i l a b l e f o r most ecosystems. I n t h i s p a p e r some c h a r a c t e r i s t i c s o f t h e i n p u t o f f l u o r i d e t o e c o s y s t e m s a r e examined w i t h emphasis on t h e t r a n s p o r t o f f l u o r i d e w i t h i n forested ecosystems. C h a r a c t e r i s t i c s o f F l u o r i d e E n t r y t o F o r e s t e d Ecosystems F l u o r i d e e n t e r s f o r e s t e d e c o s y s t e m s a r o u n d e m i s s i o n s o u r c e s b y means o f a e r i a l d e p o s i t i o n o n t o v e g e t a t i o n and s o i l s u r f a c e s , t r a t i o n s due t o n a t u r a l o r a n t h r o p o g e n i c c o n t a m i n a t i o n . F l u o r i d e d e p o s i t e d o n l e a v e s may be i n a g a s e o u s o r p a r t i c u l a t e f o r m , i n solution associated w i t h precipitation. adhere t o t h e l e a f surface b u t they o n l y s l o w l y cross t h e c u t i c l e . r a t e o f t r a n s c u t i c u l a r m i g r a t i o n i n c r e a s e s w i t h h u m i d i t y (3). Gaseous f l u o r i d e s may be a d s o r b e d o n t h e s u r f a c e o f t h e l e a f o r may e n t e r t h e l e a f through t h e stomata where t h e m a j o r i t y remains i n a f r e e i o n i c f o r m and i s t r a n s l o c a t e d t o p e r i p h e r a l a r e a s o f t h e l e a f i n t h e t r a n s p i r a t i o n s t r e a m (4). Solutions o f fluoride i n rain, mist, f o g o r snow p r o b a b l y but c r o s s t h e c u t i c l e more q u i c k l y t h a n p a r t i c u l a t e s under most c o n d i t i o n s , or The P a r t i c u l a t e f l u o r i d e s may o r by transport i n s u r f a c e w a t e r o r g r o u n d w a t e r s f r o m o t h e r a r e a s w i t h h i g h f l u o r i d e concen-

93
appear u n l i k e l y t o g a i n e n t r y t o t h e l e a f t h r o u g h t h e stomata. Fluorides

deposited on o t h e r p l a n t organs such as branches a r e l e s s l i k e l y t o g a i n e n t r y t o t h e p l a n t a l t h o u g h some u p t a k e o f g a s e o u s f l u o r i d e s b y l e n t i c e l s p r o b a b l y o c c u r s (4). ratios. F l u o r i d e I n p u t Processes I n o r d e r t o q u a n t i f y some o f t h e p r o c e s s e s b y w h i c h f l u o r i d e e n t e r s and i s t r a n s p o r t e d w i t h i n f o r e s t e d ecosystems, were studied. F l u o r i d e e n t r y i n t o a E u c a l y p t u s f o r e s t n e a r an a l u m i n u m s m e l t e r a t K u r r i Kurri. A u s t r a l i a was e s t i m a t e d u s i n g r a i n f a l l c o l l e c t o r s . The s o u r c e was e m i t t i n g a b o u t 200 t o n n e s o f g a s e o u s and p a r t i c u l a t e f l u o r i d e s p e r y e a r , l a r g e l y f r o m r o o f vents. e a r t h ' s surface), canopy), The r a i n f a l l c o l l e c t o r s w e r e n o t e v e n t - a c t u a t e d . ( p r e c i p i t a t i o n t h a t has p a s s e d t h r o u g h a l e a f F i v e c o l l e c t o r s were They m e a s u r e d c l e a r f a l l ( p r e c i p i t a t i o n g r a v i t a t i o n a l l y t r a n s f e r r e d t o t h e throughfall a n d s t e m f l o w ( p r e c i p i t a t i o n d i v e r t e d down t h e sterns o f p l a n t s ) , a t d i s t a n c e s o f 0.5 a n d 9.9 k m f r o m t h e t w o n a t u r a l f o r e s t e d ecosystems Organs w i t h l a r g e s u r f a c e area:volume r a t i o s have g r e a t e r f l u o r i d e c o n c e n t r a t i o n s t h a n those w i t h s m a l l s u r f a c e area:volume

b u t were a l s o c o n t i n u o u s l y exposed t o d r y d e p o s i t i o n . used t o measure each parameter, source, f o r a p e r i o d o f 1037 d a y s (5).

The r e s u l t s show t h a t f l u o r i d e c o n c e n t r a t i o n s i n s t e m f l o w w e r e m o r e t h a n one and a h a l f t i m e s t h o s e i n t h r o u g h f a l l and a l m o s t f i v e t i m e s t h o s e i n c l e a r f a l l i n t h e p o l l u t e d a r e a s ( F i g u r e 1). The f l u o r i d e c o n c e n t r a t i o n s i n 15 The f l u o r i d e c l e a r f a l l , t h r o u g h f a l l and s t e m f l o w i n t h e p o l l u t e d a r e a w e r e a l m o s t 10, and 27 t i m e s t h e r e s p e c t i v e v a l u e s f o r t h e c o n t r o l area. i n p u t t o t h e ecosystem, sites, areas.

a l l o w i n g f o r t h e percentage t r e e cover a t t h e study was 0.39 g / m 2 / y r In

was c o n s i d e r a b l y g r e a t e r as t h r o u g h f a l l t h a n c l e a r f a l l i n b o t h The n e t t h r o u g h f a l l ( t h r o u g h f a l l m i n u s c l e a r f a l l ) b u t o n l y 0.016 g / m 2 / y r i n t h e c o n t r o l area.

i n t h e p o l l u t e d ecosystem, other studies (6,7),

estimates indicate t h a t stemflow f l u x represents less However, t h i s n u t r i e n t i n p u t

t h a n 5% o f n u t r i e n t i n p u t i n o p e n f o r e s t s . tree. Fluoride i n Vegetation

i s i m p o r t a n t because i t i s d e p o s i t e d i n a s m a l l a r e a around t h e base o f t h e

The a m o u n t o f f l u o r i d e i n v e g e t a t i o n c a n be e x p r e s s e d i n t e r m s o f concent r a t i o n s i n one o r g a n such as p l a n t leaves, o r i n a number o f organs, b u t

i n r e l a t i o n t o e c o s y s t e m s i t i s b e s t e x p r e s s e d a s t h e mass o f f l u o r i d e p e r
u n i t area, t h a t i s b y summing t h e s t a n d i n g c r o p o f f l u o r i d e ( t h e p r o d u c t o f The f l u o r i d e s t a n d i n g c r o p i n u n d e r s t o r y and g r o u n d d r y mass p e r u n i t a r e a and f l u o r i d e c o n c e n t r a t i o n ) f o r a l l o f t h e l a r g e s t e c o s y s t e m components.

94

0.5km

DISTANCE FROM A SOURCE

--

O.Okm

FLUORIDE FLUX ( 0 avi2yr-1

F i g u r e 1: forest.

Some f l u o r i d e t r a n s p o r t p r o c e s s e s a n d f l u x e s i n a E u c a l y p t u s

c o v e r s p e c i e s i n t h e E u c a l y p t u s f o r e s t s was e s t i m a t e d b y c l e a r i n g a l l biomass f r o m f o u r r e p l i c a t e d p l o t s f o r each o f t w o years, p l a n t species p r e s e n t i n each p l o t . a n d 1.4 mg F / m 2 a t 9.9 k m ( T a b l e I ) . The s t a n d i n g c r o p o f f l u o r i d e i n p l a n t l i t t e r was m e a s u r e d q u a r t e r l y f o r t w o y e a r s a t each o f f i v e s i t e s . nents: leaves, b r a n c h e s ( > 5 mm), components such as f l o w e r s , was d r i e d , L i t t e r was d i v i d e d i n t o d i f f e r e n t compot w i g s (< 5 mm), bark, and m i s c e l l a n e o u s Each The r e s u l t s show and m e a s u r i n g d r y w e i g h t s and f l u o r i d e c o n c e n t r a t i o n f o r each c o m p o n e n t f o r e a c h o f a b o u t 30 The t o t a l f l u o r i d e s t a n d i n g c r o p i n u n d e r s t o r y a n d g r o u n d c o v e r s p e c i e s w a s f o u n d t o b e 10.2 mg F / m 2 a t 0.5 k m

f r u i t and p a r t l y decomposed p l a n t l i t t e r .

w e i g h e d and a n a l y z e d f o r f l u o r i d e s e p a r a t e l y .

t h a t t h e t o t a l f l u o r i d e s t a n d i n g c r o p i n p l a n t l i t t e r w a s 1 5 7 mg F / m 2 a t

0.5 km a n d 1 3 mg F / m 2 a t 9.9 kni.

As a c o m p a r i s o n , a s t u d y o f a mangrove f o r e s t g r o w i n g n e a r a hydrogen f l u o r i d e m a n u f a c t u r i n g f a c i l i t y on Kooragang I s l a n d ,

N.S.W.,

Australia,

and

a s i m i l a r m a n g r o v e f o r e s t i n a r e l a t i v e l y u n p o l l u t e d a r e a ( G o s f o r d ) showed t h a t f l u o r i d e i n p u t t o t h e Kooragang I s l a n d ecosystem as b u l k d e p o s i t i o n o f

95 TABLE I FLUORIDE FLUX AND STANDING CROP I N FORESTED ECOSYSTEMS Eucalyptus f o r e s t ecosystem 1. 2. 3. D i s t a n c e f r o m s o u r c e (km) Bulk deposition o f f l u o r i d e (g/m2/yr) Fluori e standing crop (mgF/m ) i n a ) u n d e r s t o r y and groundcover species b) p l a n t l i t t e r c ) t o t a l above g r o u n d standing crop 4. F l u o r i d e t r a n s f e r (mgF/m2/yr) a ) 1 it t e r f a l l NA
-

Mangrove f o r e s t ecosystem 0.5 0.31 50 0.04

0.5
0.93

9.9 0.21

10.2 157 ND

1.4 13 ND

NA NA 270

NA

NA 4.0

54

2.0

176

1.4

samples n o t a v a i l a b l e

ND - n o t d e t e r m i n e d
and t h i s v a r i e d w i t h t h e d i s t a n c e and d i r e c t i o n Most o f t h e

f l u o r i d e was 0.31 g / m 2 / y r

o f e a c h s i t e i n r e l a t i o n t o f l u o r i d e e m i s s i o n s o u r c e s (8). f l u o r i d e i n p u t occurred. a l l gaseous f l u o r i d e . The a b o v e - g r o u n d and 21.7 d r y t / h a ,

i n p u t a t Kooragang I s l a n d was gaseous f l u o r i d e , b u t a s m a l l p a r t i c u l a t e Bulk deposition o f f l u o r i d e a t a r e l a t i v e l y virtually u n c o n t a m i n a t e d s i t e a t F u l l e r t o n Cove was a b o u t 0.04 g/m2/year.

b i o m a s s a t b o t h s i t e s w a s p r a c t i c a l l y i d e n t i c a l (21.8 respectively) b u t t h e f l u o r i d e concentrations i n leaves Consequently t h e above-ground fluoride i n the

and branches were a l m o s t t w o o r d e r s o f m a g n i t u d e g r e a t e r a t Kooragang I s l a n d than a t Gosford. G o s f o r d (0.04 k g F / h a ) . I n t h e mangrove f o r e s t , a comparison o f p l a n t l i t t e r f a l l . f l u o r i d e conc e n t r a t i o n and f l u o r i d e d e p o s i t i o n i n l i t t e r f a l l a t t h e K o o r a g a n g I s l a n d s i t e and a t F u l l e r t o n Cove showed t h a t a l t h o u g h l i t t e r f a l l r a t e s w e r e s i m i l a r (562 and 514 g/m2/yr, 1.42 mg F/m2/yr, r e s p e c t i v e l y ) t h e r a t e s o f f l u o r i d e deposit i o n i n l i t t e r f a l l w e r e a l s o t w o o r d e r s o f m a g n i t u d e d i f f e r e n t (176 and r e s p e c t i v e l y ) due t o t h e d i f f e r e n c e s i n f l u o r i d e concenThese l i t t e r t r a t i o n i n a l l components o f p l a n t l i t t e r a t t h e t w o s i t e s . t i o n a t t h e same l a t i t u d e (8). s t a n d i n g c r o p w a s m u c h g r e a t e r a t K o o r a g a n g I s l a n d (2.7 k g F / h a ) t h a n a t

f a l l r a t e s a r e s i m i l a r t o o t h e r d a t a r e p o r t e d f o r mangrove l i t t e r p r o d u c -

96
As a consequence o f f l u o r i d e i n p u t by l i t t e r f a l l ,

w e t d e p o s i t i o n and d r y i n s o i l s a t Koora-

deposition,

buffer-extractable

f l u o r i d e concentrations

gang I s l a n d were v a r i a b l e , b u t were g e n e r a l l y w i t h i n t h e range 4-15 u g F/g.

A t F u l l e r t o n Cove and G o s f o r d t h e b u f f e r - e x t r a c t a b l e f l u o r i d e concent r a t i o n s i n s o i l were r e l a t i v e l y c o n s t a n t w i t h mean f l u o r i d e c o n c e n t r a t i o n s o f 2.0 and 1.9 u g F / g . r e s p e c t i v e l y .

S o i 1s

F l u o r i d e i s r e l e a s e d f r o m p l a n t l i t t e r by f i r e and i t has been e s t i m a t e d t h a t about 68% i s e x p o r t e d f r o m t h e ecosystem as gaseous and p a r t i c u l a t e fluoride, w i t h 32% r e m a i n i n g as ash. High s o l u b l e f l u o r i d e c o n c e n t r a t i o n s These c o n c e n t r a t i o n s c o u l d a l s o Most s o i l s o c c u r i n t h e ash and have been found t o measure up t o about 1600 u g / g a f t e r a b u s h f i r e around an aluminum s m e l t e r ( 9 )
w i l l u l t i m a t e l y leach t h e water-soluble

impede g e r m i n a t i o n and s e e d l i n g g r o w t h f o r a p e r i o d o f t i m e b u t r a i n f a l l fluoride i n t o the soil. a c t as a s i n k f o r f l u o r i d e . They have a h i g h c a p a c i t y t o b i n d f l u o r i d e i n Some s a n d y and s a l i n e and consequently

an i n s o l u b l e and u n a v a i l a b l e form, and t h e f a c t o r s r e s p o n s i b l e f o r t h i s a r e i r o n , a l u m i n u m , c a l c i u m and o t h e r a g e n t s ( 1 0 , l l ) . s o i l s l a c k t h e s e f a c t o r s and may n o t have t h i s c a p a c i t y , r i d e t o be t a k e n up by r o o t s , o r leached t o groundwaters.

t h e r e may be some p o t e n t i a l i n s o i l s w i t h t h e s e c h a r a c t e r i s t i c s f o r f l u o -

DISCUSSION
I n t h e a s s e s s m e n t o f t h e e f f e c t s o f f l u o r i d e s on f o r e s t e d a r e a s a r o u n d e m i s s i o n sources i t i s n e c e s s a r y t o c o n s i d e r t h a t ecosystems c o n s i s t o f o r g a n i s m s w h i c h a r e o r g a n i z e d i n s t r u c t u r a l and f u n c t i o n a l c o m p o n e n t s u n i t e d by t r a n s f e r p r o c e s s e s (12). Consequently, evaluations o f the as e f f e c t s o f f l u o r i d e on ecosystem processes d i f f e r c o n s i d e r a b l y f r o m evaluat i o n s o f t h e e f f e c t s on i n d i v i d u a l species. even t h e dominant species, t h e s e n s i t i v e p o i n t s w i t h i n t h e e c o s y s t e m s may be f u n c t i o n a l p r o c e s s e s . N a t u r a l f o r e s t ecosystems a r e u s u a l l y complex and i n o r d e r t o understand t h e e f f e c t s o f f l u o r i d e on t h e s e ecosystems, t h e i r r e a c t i o n s need t o be u n d e r s t o o d . decomposers (12). t h e most s e n s i t i v e organisms and ecosystem processes need t o be i d e n t i f i e d and t h e c h a r a c t e r i s t i c s o f I n f o r e s t e d ecosystems t h e most s e n s i t i v e organisms t o t h e e f f e c t s o f a i r p o l l u t a n t s a r e t h e producers and w h i c h a r e a l s o t h e t w o most i m p o r t a n t groups o f organisms w i t h i n f o r e s t e d e c o s y s t e m s , as t h e y c o n t r o l t h e p r o c e s s e s o f u p t a k e and t r a n s f e r o f energy and m i n e r a l s which may be r e a d i l y d i s t u r b e d (13). a d v e r s e l y a f f e c t ecosystems i n a s u b t l e manner i n i t i a l l y , more obvious i m p a c t s w i t h t i m e i f t h e s t r e s s continues.

It i s

b y means o f i m p a c t s a t t h e s e l e v e l s t h a t p o l l u t a n t s s u c h a s f l u o r i d e may developing i n t o

97
E c o s y s t e m s a r o u n d m a j o r e m i s s i o n s o u r c e s may b e r e c o g n i z e d a s c a p a b l e of b e i n g c l a s s i f i e d i n t o d i s t i n c t zones. The a r e a n e a r e s t t o t h e e m i s s i o n S e n s i t i v e species Further s o u r c e may be s u b j e c t t o m a j o r i m p a c t s a s s o c i a t e d w i t h s i g n i f i c a n t e c o l o g i c a l d e g r a d a t i o n o f f u n c t i o n a l and a e s t h e t i c a t t r i b u t e s . a r e r e m o v e d and v i s i b l e i n j u r y t o f o l i a g e i s o b v i o u s i n t h i s zone. w h i c h s u b t l e e f f e c t s o f f l u o r i d e may be r e c o g n i z e d . w i t h p o t e n t i a l e f f e c t s on growth, s e n s i t i v e species.

f r o m t h e e m i s s i o n s o u r c e o r i n l e s s exposed l o c a t i o n s i s f o u n d a zone i n I n t h i s zone t h e r e a r e changes o r s u b t l e e f f e c t s d e m o n s t r a t e d by a l t e r a t i o n s i n p l a n t m e t a b o l i s m r e p r o d u c t i o n and d i s e a s e r e s i s t a n c e o f t h e r e may b e l o n g - t e r m changes

A t an ecosystem l e v e l ,

i n s p e c i e s c o m p o s i t i o n , d i v e r s i t y , b i o m a s s and n u t r i e n t c y c l i n g . Beyond t h i s zone i s a zone o f innocuous e f f e c t s c h a r a c t e r i z e d b y t h e ecosystem a c t i n g as a s i n k f o r f l u o r i d e . t h i s zone. ACKNOWLEDGEMENTS The a s s i s t a n c e o f A l c a n A u s t r a l i a Ltd, A u s t r a l i a n F l u o r i n e C h e m i c a l s , R o s l y n Avery, John Clancy. Rosemary H u t c h i s o n , M a r i n a McGlinn, Elizabeth R a t c l i f f e and D a v i d R o s h i e r i s g r a t e f u l l y a c k n o w l e d g e d . REFERENCES 1. K n a b e W ( 1 9 7 8 ) I n : O l s c h o w y G ( e d ) U n w e l t s c h u t z i n d. B u n d e s r e p u b l i k D e u t s c h l a n d . P a u l Parey, Hamburg, p p 697-709 The f l u o r i d e c o n c e n t r a t i o n s i n some e c o s y s tem components, and f l u o r i d e f l u x i n c y c l i n g processes a r e i n c r e a s e d i n

2. Heck W W (1982)

I n : U n s w o r t h MH, O r m r o d DP ( e d s ) E f f e c t s o f Gaseous A i r P o l l u t i o n i n A g r i c u l t u r e and H o r t i c u l t u r e . B u t t e r w o r t h S c i e n t i f i c , London, pp 411-435

3.

In: Proc I n t Clean A i r M c C u n e DC. S i l b e r m a n DH, W e i n s t e i n LH ( 1 9 7 7 ) C o n g r 4 t h . Tokyo, J a p a n e s e U n i o n o f A i r P o l l u t i o n P r e v e n t i o n A s s o c i a t i o n s , Tokyo, p p 116-118 W e i n s t e i n LH (1977) J Occup Med 19:49-78 M u r r a y F (1985a) P e r s p e c t i v e s E n v i r o n B o t 1 ( i n p r e s s )
A t t i w i l l PM.

4. 5.

6. O v i n g t o n JD (1954) F o r e s t r y 27:41-53
7. G u t h r i e HB, 9. L e u n i g R (1978) A u s t J B o t 26:189-201

8. M u r r a y F (1985b) J A p p l E c o l 22:277-285
M u r r a y F (1981) I n : Webb KA. S m i t h AJ ( e d s ) S e v e n t h I n t e r n a t i o n a l C l e a n A i r C o n f e r e n c e : Ann A r b o r S c i e n c e . Ann A r b o r , Mich.. p p 451-462

10. M u r r a y F ( 1 9 8 3 ) Water,

Air,

S o i l P o l l u t 20:361-367 B) 7:83-95

11. M u r r a y F ( 1 9 8 4 ) E n v i r o n P o l l u t (Ser. 13. E h r l i c h PR.

12. S m i t h WH ( 1 9 8 4 ) F o r e s t E c o l Manage 9:193-219 Mooney HA ( 1 9 8 3 ) B i o S c i e n c e 33:248-254

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 99-106 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

99

PROSPECTIVE SURVEY ON FLUORIDE CONTENTS OF R I C E

S H I R O SAKURAI, KAZUYOSHI I T A I , AND HUM10 TSUNODA

Department o f Hygiene and P u b l i c Health, U n i v e r s i t y , Morioka, I w a t e 020 Japan School o f Medicine, I w a t e Medical

ABSTRACT

A p r o s p e c t i v e 17-year s u r v e y was conducted on changes i n f l u o r i d e c o n t e n t

i n u n p o l i s h e d r i c e f r o m an a r e a n e a r an a l u m i n u m r e f i n e r y p l a n t . Rice samples were a l s o taken f o r a n a l y s i s from areas around v a r i o u s types o f i n d u s t r i e s known t o e m i t f l u o r i d e , and t h e r e s u l t s were compared w i t h t h o s e o b t a i n e d 10 y e a r s before. It was shown t h a t u n p o l i s h e d r i c e r e a d i l y accuThe f l u o r i d e c o n t e n t s m u l a t e d a i r b o r n e f l u o r i d e and t h a t i t c o u l d s e r v e as an i n d i c a t o r f o r longt e r m and l o w l e v e l exposure t o a t m o s p h e r i c f l u o r i d e . o f unpolished r i c e grown i n areas around ceramics works were g e n e r a l l y h i g h e r t h a n t h o s e found i n samples c o l l e c t e d f r o m o t h e r i n d u s t r i a l areas. A l t h o u g h areas p o l l u t e d by a i r b o r n e f l u o r i d e e m i t t e d f r o m c e r a m i c s works are l i m i t e d t o those surrounding t h e plants, t e r e d a c r o s s Japan. problem.
It i s possible,

many c e r a m i c s works l i e s c a t t h a t i n g e s t i o n o f excessive

therefore,

amounts o f f l u o r i d e - c o n t a m i n a t e d

r i c e c o u l d become a s e r i o u s p u b l i c h e a l t h

I t i s s t r e s s e d t h a t c o n t r o l m e a s u r e s s h o u l d be t a k e n t o c u r b

f l u o r i d e e m i s s i o n a t c e r a m i c s works.

INTRODUCTION A c c u m u l a t i o n o f h i g h l e v e l s o f f l u o r i d e i n v e g e t a t i o n i s commonly observed i n areas p o l l u t e d by a i r b o r n e f l u o r i d e . The most i m p o r t a n t probSince r i c e is rice fluolem i n v o l v e d i n human h e a l t h i s t h e i n t a k e o f e x c e s s i v e amounts o f f l u o r i d e through i n g e s t i o n o f foods contaminated by t h e p o l l u t a n t . t h e main s t a p l e f o r t h e Japanese, ingestion o f fluoride-contaminated

c o u l d a c c o u n t f o r a s i g n i f i c a n t p a r t o f t o t a l f l u o r i d e i n t a k e s i n Japan

(1).
ties.

The p r e s e n t s t u d y was c a r r i e d o u t i n an a t t e m p t t o d e t e r m i n e

r i d e c o n t a m i n a t i o n i n r i c e caused by s e v e r a l t y p e s o f i n d u s t r i a l a c t i v i C u r r e n t c o n c e r n w i t h a i r b o r n e f l u o r i d e p o l l u t i o n was a l s o considered.

100
MATERIALS AND METHODS A p r o s p e c t i v e 17-year s u r v e y was c o n d u c t e d t o s t u d y t h e f l u o r i d e c o n t e n t i n u n p o l i s h e d r i c e t a k e n f r o m an a r e a n e a r an aluminum r e f i n e r y p l a n t . T o t a l p r o d u c t i o n o f a l u m i n u m b y t h e p l a n t was 40.000 t o n s p e r y e a r . R i c e s a m p l e s w e r e c o l l e c t e d a n n u a l l y b e g i n n i n g i n 1967. R i c e s a m p l e s w e r e a l s o t a k e n f r o m a r e a s a r o u n d 1 8 i n d u s t r i a l p l a n t s known t o e m i t f l u o r i d e i n t o t h e atmosphere. r e f i n e r y p l a n t s (A-F), smelters, They c o n s i s t e d o f s i x aluminum three steel tile, f o u r p h o s p h a t e f e r t i l i z e r p l a n t s (A-D),

and f i v e c e r a m i c s w o r k s i n c l u d i n g t h o s e p r o d u c i n g c h i n a ,

r o o f t i l e and g l a s s f i b e r . D e t e r m i n a t i o n o f f l u o r i d e f o r t h e r i c e s a m p l e s t a k e n p r i o r t o 1984 was p e r f o r m e d a c c o r d i n g t o t h e A.O.A.C. m e t h o d (2). F l u o r i d e ions i n s o l u t i o n Samples t a k e n w e r e t h e n d e t e r m i n e d b y t h e i o n - s e l e c t i v e e l e c t r o d e method. d u r i n g 1 9 8 4 w e r e d e t e r m i n e d b y I t a i ' s m e t h o d (3). RESULTS F i g u r e 1 shows t h e l o n g - t e r m I n 1968, changes i n f l u o r i d e c o n t e n t s o f u n p o l i s h e d r i c e f r o m a n a r e a n e a r a n a l u m i n u m r e f i n e r y p l a n t d e s i g n a t e d a s P l a n t B. when v i s i b l e i n j u r y o f v e g e t a t i o n was v e r y s e v e r e t h e e i g h t - h o u r with w i t h i n a 0.5 km r a d i u s o f t h e p l a n t . Even w i t h i n a respecmean f l u o r i d e c o n c e n t r a t i o n i n t h e a t m o s p h e r e was f o u n d t o be 3.3 ppb, a maximum o f 14.8 ppb, 1.0 km r a d i u s o f t h e p l a n t , t h e v a l u e s w e r e 1.6 p p b a n d 4.8 ppb,

. ! ? I0.0.0
Q

12.0-

2
0

::I
8.0.. 2.0

1st Electrolvsas closed

-+ --+-

Y i t h i n 0 . 5 km

- Beyond 0.5 km

md E l e c t r o l y s i s closed
P l s n f ctosed

F i g u r e 1. Changes i n f l u o r i d e c o n t e n t s o f u n p o l i s h e d r i c e f r o m a r e a s n e a r an a l u m i n u m r e f i n e r y p l a n t ( B ) .

101
tively. T h e m e a n f l u o r i d e c o n t e n t o f u n p o l i s h e d r i c e w a s 1 2 ppm ( 4 ) . Subsequently, In

1972 t h e f i r s t e l e c t r o l y s i s p l a n t , was c l o s e d . r i c e s a m p l e s was o b s e r v e d .

t h e main source o f f l u o r i d e emission,

a marked r e d u c t i o n o f f l u o r i d e c o n t e n t s i n t h e F l u o r i d e l e v e l s i n t h e samples have f u r t h e r

d e c r e a s e d t o 1.5 pprn s i n c e 1 9 8 1 when t h e p l a n t was c l o s e d c o m p l e t e l y .

a
Q

I c

K 0 0

LL

0 3

cunl"

CERnlCs

PH(1BcRTE

87EEL

F i g u r e 2-1. F l u o r i d e c o n t e n t s (mean) i n u n p o l i s h e d r i c e i n t h e s u r r o u n d i n g s o f 1 8 i n d u s t r i a l p l a n t s (1975).

i i

0 3

A B C D E F A P C D E A I C D A B C

F i g u r e 2-2. F l u o r i d e c o n t e n t s (mean) i n u n p o l i s h e d r i c e i n t h e s u r r o u n d i n g s o f 1 8 i n d u s t r i a l p l a n t s (1984).

102
The d a t a on t h e f l u o r i d e c o n t e n t s o f u n p o l i s h e d r i c e t a k e n f r o m a r e a s around i n d u s t r i a l f l u o r i d e sources a r e shown i n F i g u r e 2 w h i l e t h e i r maximum v a l u e s a r e shown i n F i g u r e 3.
I t i s o b v i o u s t h a t t h e maximum v a l u e s

appear t o be independent o f t h e d i s t a n c e f r o m e m i s s i o n s o u r c e (Fig.

3).

E n n
6
I

0
L

I? .c
.v)

0
C

n
3

. c

0
v)

+.
C

C 0)

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3 LI

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F i g u r e 3-1. F l u o r i d e c o n t e n t s (max) i n u n p o l i s h e d r i c e i n t h e s u r r o u n d i n g s o f 18 i n d u s t r i a l p l a n t s (1975).

Ea

v) *

C 0) c C

0 0

F l u o r i d e c o n t e n t s (max) i n u n p o l i s h e d r i c e i n t h e s u r r o u n d i n g s F i g u r e 3-2. o f 18 i n d u s t r i a l p l a n t s (1984).

103
The d a t a o b t a i n e d f r o m t h i s s t u d y were compared w i t h t h o s e o b t a i n e d f r o m a s i m i l a r s u r v e y p e r f o r m e d 10 y e a r s before. p l a n t s had been r e d u c e d t o a n o r m a l l e v e l . aluminum p l a n t s showed a s i m i l a r r e d u c t i o n .
I t was o b s e r v e d t h a t t h e

f l u o r i d e c o n t e n t s o f r i c e f r o m t h e v i c i n i t y o f many phosphate f e r t i l i z e r R i c e samples f r o m a r e a s near On t h e o t h e r hand, r i c e sam-

p l e s o b t a i n e d f r o m areas around s e v e r a l c e r a m i c s works w h i c h showed a h i g h f l u o r i d e l e v e l o f 19 ppm i n 1975 s t i l l had f l u o r i d e l e v e l s i n excess o f 10 ppm i n 1984, a l t h o u g h t h e p o l l u t e d areas were l i m i t e d t o t h o s e a d j a c e n t t o A f l u o r i d e c o n t e n t as h i g h as 37.8 ppm was found i n t h e e m i s s i o n sources.

r i c e samples o b t a i n e d f r o m an a r e a near a r o o f t i l e m a n u f a c t u r i n g p l a n t .

DISCUSSION
R e l a t i o n s h i p between a i r b o r n e f l u o r i d e and f l u o r i d e c o n t e n t o f r i c e The r e s u l t s o f t h e p r o s p e c t i v e 17-year r i c e samples decreased, as e x p e c t e d , s u r v e y conducted i n an area near aluminum r e f i n e r y p l a n t B have c l e a r l y shown t h a t f l u o r i d e c o n t e n t s o f t h e w i t h decreasing concentration o f t h e f l u o r i d e c o n t e n t s i n unpolf l u o r i d e i n t h e atmosphere. f l u o r i d e e m i s s i o n source. The f l u o r i d e c o n t e n t o f u n p o l i s h e d r i c e i s a good i n d i c a t o r o f long-term and s u c c e s s i v e e x p o s u r e t o a t m o s p h e r i c f l u o r i d e s i n c e t h e e a r o f a r i c e p l a n t r a r e l y a b s c i s e s as l e a v e s do, u n l e s s t h e c o n c e n t r a t i o n o f a i r b o r n e f l u o r i d e i s so h i g h as t o cause n e c r o s i s and d e a t h o f t h e p l a n t . Concerning t h e d o s e - r e l a t i o n s h i p between t h e l e v e l o f a t m o s p h e r i c f l u o r i d e and t h e f l u o r i d e c o n t e n t i n u n p o l i s h e d r i c e , more t h a n 4.0 However, I n actuality,

i s h e d r i c e had been reduced t o a

normal l e v e l f o l l o w i n g t h e removal o f t h e

i t was f o u n d i n t h i s

f i e l d study (1) t h a t the f l u o r i d e content i n unpolished r i c e increased t o ppm when t h e a t m o s p h e r i c f l u o r i d e c o n c e n t r a t i o n exceeded 1 ppb hydrogen f l u o r i d e ppb d u r i n g t h e r i c e g r o w i n g period. T o m a t s u r i (5) exposed r i c e p l a n t s t o 2-5 f o r 40 days and concluded t h a t t h e c o r r e l a t i o n between t h e c o n c e n t r a t i o n o f a t m o s p h e r i c f l u o r i d e and t h e f l u o r i d e c o n t e n t i n t h e l e a v e s o f r i c e p l a n t s was s i g n i f i c a n t , not. b u t t h a t t h e c o r r e l a t i o n between t h e c o n c e n t r a t i o n o f a t m o s p h e r i c f l u o r i d e and t h e f l u o r i d e c o n t e n t i n t h e u n p o l i s h e d r i c e was I n t h i s e x p e r i m e n t , t h e c o n t r o l p l a n t s w e r e a l s o e x p o s e d t o 0.1 ppb The c o n c e n t r a t i o n u s e d may h a v e been t o o h i g h f o r a This R i c e can a c c u m u l a t e f l u o r i d e when t h e exposure p e r i o d i s prohydrogen f l u o r i d e . control.

l o n g e d , e v e n when t h e l e v e l o f a i r b o r n e f l u o r i d e may b e v e r y l o w . t h e e x p o s u r e g r o u p and t h e c o n t r o l g r o u p becomes i n s i g n i f i c a n t . h i s conclusion,

c o u l d account f o r t h e f a c t t h a t t h e d i f f e r e n c e i n f l u o r i d e l e v e l s between Despite Tomatsuris study (5) i m p l i e s t h a t t h e f l u o r i d e content i n

104
u n p o l i s h e d r i c e s e r v e s as a good i n d i c a t o r f o r l o w l e v e l e x p o s u r e t o a i r borne f l u o r i d e . Nakao

a.( 6 )

measured t h e c o n c e n t r a t i o n s o f gaseous f l u o r i d e i n t h e They r e p o r t e d t h a t t h e eight-hour

s u r r o u n d i n g s o f c e r a m i c s w o r k s (D).

m a x i m u m f l u o r i d e c o n c e n t r a t i o n i n t h e a t m o s p h e r e w a s 3 - 4 ppb, w h i l e t h e f l u o r i d e c o n t e n t o f t h e u n p o l i s h e d r i c e was 8.5 ppm. R e l a t i o n s h i p between y i e l d o f r i c e and f l u o r i d e c o n t e n t i n u n p o l i s h e d r i c e T o m a t s u r i ( 5 ) f u m i g a t e d r i c e w i t h 5 p p b HF a n d c o n c l u d e d t h a t HF h a d n o e f f e c t on t h e y i e l d o f r i c e , even though i t s f l u o r i d e c o n t e n t exceeded 8 ppm.

A s i m i l a r f i n d i n g has been r e p o r t e d b y Katsumi

t d.(7).

On t h e

o t h e r hand, Nakao @, d .(6), based on t h e i r f i e l d o b s e r v a t i o n s , s u g g e s t e d t h a t an i n c r e a s e d f l u o r i d e c o n c e n t r a t i o n i n t h e atmosphere a f f e c t e d t h e g r o w t h and y i e l d o f r i c e . Tsunoda

eC, d.(8) a l s o r e p o r t e d i n t h e i r f i e l d
T h i s i s e q u i v a l e n t t o a p p r o x i m a t e l y 20 Cho g t

s t u d y t h a t up t o 20% r e d u c t i o n i n y i e l d o c c u r r e d when t h e f l u o r i d e c o n t e n t o f p o l i s h e d r i c e exceeded 10 ppm. ppm f l u o r i d e i n u n p o l i s h e d r i c e (4,9).

2 1 . (10)

a l s o presented

evidence i n d i c a t i n g a s i g n i f i c a n t y i e l d r e d u c t i o n i n r i c e fumigated w i t h

HF.

I n t h e p r e s e n t s t u d y , a 90% r e d u c t i o n i n y i e l d was o b s e r v e d i n r i c e The f l u o r i d e

c o l l e c t e d f r o m a n a r e a 1 5 0 m f r o m t h e c e r a m i c s w o r k s (C). c o n t e n t i n t h e u n p o l i s h e d r i c e was f o u n d t o be 37.8 ppm. K a t s u m i @,

d.(11)

exposed r i c e p l a n t s t o 5 ppb HF w h i l e h a v i n g t h e husk Judging f r o m t h e observation unpolished r i c e accumulated

wrapped o r t h e l e a v e s o f t h e p l a n t s c u t o f f . e i t h e r treatment, t h e y showed c l e a r l y t h a t

t h a t t h e r e was no i n c r e a s e i n f l u o r i d e c o n t e n t i n t h e u n p o l i s h e d r i c e i n a t m o s p h e r i c f l u o r i d e o n l y t h r o u g h t h e husk and t h a t t h e r e was no t r a n s l o c a t i o n o f f l u o r i d e from t h e leaves t o t h e unpolished rice. The f o r e g o i n g d e s c r i p t i o n l e a d s t o t h e c o n c l u s i o n t h a t f l u o r i d e c o n t e n t i n u n p o l i s h e d r i c e i s a good i n d i c a t o r o f l o n g - t e r m t o atmospheric fluoride. R e l a t i o n between f l u o r i d e c o n t e n t o f r i c e and d i s t a n c e f r o m e m i s s i o n s o u r c e The f l u o r i d e c o n t e n t i n r i c e a p p e a r s t o be i n d e p e n d e n t o f t h e d i s t a n c e f r o m e m i s s i o n source. Concerning t h i s , Nakao and l o w l e v e l e x p o s u r e

&a.(6)

concluded t h a t t h e as a r i c e p l a n t

r i c e p l a n t absorbed atmospheric f l u o r i d e t h r o u g h stomata, c o u l d b e c l a s s i f i e d as " s t o m a t a type", r i d e content o f unpolished rice. b e made,

and t h e y f e l t t h a t t h e r e was a c l o s e

r e l a t i o n s h i p b e t w e e n t h e c o n c e n t r a t i o n o f a i r b o r n e f l u o r i d e and t h e f l u o As t h e c o n c e n t r a t i o n s o f a t m o s p h e r i c

f l u o r i d e w e r e n o t measured i n t h e p r e s e n t s t u d y ,

no d e t a i l e d comment c o u l d

but nevertheless i t i s possible t h a t the f a c t o r a f f e c t i n g the w h i c h i n t u r n i s dependent on w i n d d i r e c t i o n , r a t h e r than

f l u o r i d e c o n t e n t o f u n p o l i s h e d r i c e may be t h e c o n c e n t r a t i o n o f f l u o r i d e i n t h e atmosphere, t h e d i s t a n c e f r o m t h e e m i s s i o n source.

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R e c e n t p r o b l e m s a s s o c i a t e d w i t h a i r b o r n e f l u o r i d e p o l l u t i o n i n Japan I n Japan, from
i t was r e c o g n i z e d t h a t f l u o r i d e c o n t e n t s i n r i c e c r o p s o b t a i n e d

a r e a s a r o u n d many p h o s p h a t e f e r t i l i z e r p l a n t s and a l u m i n u m r e f i n e r y Presumably, t h i s was due e i t h e r o r t o reduction i n operation Several unpolished r i c e however, still

p l a n t s had b e e n r e d u c e d t o n o r m a l l e v e l s .

t o i n s t a l l a t i o n o f e m i s s i o n c o n t r o l systems, samples obtained from areas

o r c l o s u r e o f p l a n t s as a r e s u l t o f depression.

a r o u n d some c e r a m i c s w o r k s ,

showed h i g h l e v e l s o f f l u o r i d e . Japanese c e r a m i c s w o r k s a r e o f s m a l l s c a l e and t h e t o t a l volume o f e x h a u s t g a s f r o m t h e m i s a b o u t 1-3 N k m 3 / h r . The o b s e r v a t i o n t h a t t h e f l u o r i d e c o n t e n t o f unpolished r i c e obtained i n t h e v i c i n i t y o f these works was s t i l l h i g h may b e due t o t h e c h e m i c a l f o r m o f f l u o r i d e i n t h e e f f l u e n t . Itai

d.(12)

r e p o r t e d t h a t 70 t o 80% o f t o t a l a t m o s p h e r i c f l u o r i d e came Nakao

from ceramics manufacturing plants. emissions from tunnel

g d. (13)

studied fluoride

k i l n s o f t i l e w o r k s and c o n c l u d e d t h a t t h e f l u o r i d e The e x p o s e d r i c e p l a n t s may

i n t h e e f f l u e n t was l a r g e l y i n g a s e o u s f o r m . I n c r e a s e i n body burden o f f l u o r i d e

a c c u m u l a t e a h i g h l e v e l o f f l u o r i d e r e l e a s e d f r o m c e r a m i c s works. I n c r e a s e i n t h e body burden o f f l u o r i d e a s s o c i a t e d w i t h i n c r e a s e i n t h e f l u o r i d e c o n t e n t o f r i c e may come i n t o q u e s t i o n as r e p o r t e d p r e v i o u s l y (1). F o r example, quently, market, r i c e has been g r o w n d e s p i t e q u a l i t a t i v e and q u a n t i t a t i v e Conser e d u c t i o n s i n r i c e c r o p s o r a r e d u c t i o n i n t h e v a l u e o f goods.
i t on t h e market.

t h e f a r m e r s may be f o r c e d t o consume p o l l u t e d r i c e w i t h o u t p u t t i n g When s u c h c o n t a m i n a t e d r i c e i s s o l d a n d f o u n d i n t h e t h e p u b l i c w i l l be e x p o s e d t o it, r e s u l t i n g i n e x c e s s i v e i n t a k e o f Since ceramics works l i e scattered across t h e country, such a

fluoride.

p r o b l e m as t h i s c o u l d become a m a t t e r o f concern. CONCLUSION

A p r o s p e c t i v e 1 7 - y e a r s u r v e y was p e r f o r m e d t o t r a c e t h e changes i n
f l u o r i d e c o n t e n t o f u n p o l i s h e d r i c e f r o m an a r e a n e a r an a l u m i n u m r e f i n e r y plant. Additionally, f l u o r i d e l e v e l s o f unpolished r i c e from areas a d j a c e n t t o v a r i o u s t y p e s o f i n d u s t r i e s c o n s i d e r e d t o be f l u o r i d e e m i s s i o n s o u r c e s w e r e s t u d i e d and c o m p a r e d w i t h t h e r e s u l t s o b t a i n e d f r o m a s i m i l a r i n v e s t i g a t i o n c o n d u c t e d 10 y e a r s b e f o r e . t h e f o l l o w i n g c o n c l u s i o n s were reached: 1. P o l l u t i o n b y a i r b o r n e f l u o r i d e d i s a p p e a r e d when t h e e m i s s i o n s o u r c e was removed. F l u o r i d e c o n t e n t i n u n p o l i s h e d r i c e was f o u n d t o s e r v e as a good i n d i c a t o r o f l o n g - t e r m and l o w l e v e l e x p o s u r e t o a t m o s p h e r i c f l u o r i d e . Based o n r e s u l t i n g o b s e r v a t i o n s ,

2.

106
3. The f l u o r i d e c o n t e n t o f u n p o l i s h e d r i c e grown i n a r e a s around cerami c s w o r k s was g e n e r a l l y h i g h and a l e v e l as h i g h as 37.8 ppm was found i n samples o b t a i n e d f r o m t h e v i c i n i t y o f one o f t h e s e p l a n t s . 4. A l t h o u g h areas p o l l u t e d by a i r b o r n e f l u o r i d e e m i t t e d f r o m ceramics works a r e l i m i t e d t o those areas surrounding t h e p l a n t s , t h e r e are many c e r a m i c s works i n Japan. The p o s s i b i l i t y e x i s t s t h a t i n g e s t i o n o f f l u o r i d e - c o n t a m i n a t e d r i c e i n l a r g e q u a n t i t i e s c o u l d become a s e r i o u s p u b l i c h e a l t h p r o b l e m . I t i s s t r e s s e d t h a t t h e r e i s a need f o r c o n t r o l l i n g t h e f l u o r i d e e m i s s i o n a t c e r a m i c s works.

REFERENCES 1. Sakurai S, I t a i K. Tsunoda H (1983) F l u o r i d e 16:175-180 (ed)(1970) O f f i c i a l Methods o f A n a l y s i s o f t h e AOAC, p p 405-411 Washing-

2. H o r w i t z t o n , D.C..

3. I t a i K, Tsunoda H ( 1 9 8 5 ) I n : Tsunoda H and Yu MH ( e d s ) P r o c e e d i n g s o f t h e 1 4 t h Conference o f t h e I n t e r n a t i o n a l S o c i e t y f o r F l u o r i d e Research. E l s e v i e r , Tokyo 4. Tsunoda H, K u n i t a H (1973) Kougai t o T a i s a k u 9:69-75 5. T o m a t s u r i M (1971) J Japan

SOC of A i r P o l l u t i o n 6:336-342
Fukuda K, H a y a s h i K. S a i t o K (1980)

6. Nakao M, Tanaka F, T a c h i b a n a C. Zenkokukougaikenkaishi 2:45-55 8. Tsunoda H, Haneda

7. Katsumi H (1984) B u l l Fukui P r e f A g r i Exp S t a 8:12-14

(1968) Sangyou Kankyou Kougaku 59:2-11 Res Rept ORD26-1(SPMU), pp 29-38

9. Hara S (1983) B u l l J o s a i Den U n i v 12:71-82

10. Cho JK, K i m BY,
12. I t a i K, Jeh SY (1984)

11. Katsumi H (1978) B u l l Fukui P r e f A g r i Exp S t a 9:34-37

Sakurai S , Tsunoda H (1983) Tanaka F, F l u o r i d e 16:229-234 Zenkokukougaikenkaishi 13. Nakao M, Tachibana C. 8: 81 -86 S a i t o K (1983)

H. Tsunoda a n d M.-H. Y u (Editors)

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Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 107-112 0 1 9 8 6 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

FLUORIDE ABSORPTION AND EXCRETION I N HUMAN SUBJECTS FOLLOWING INGESTION OF F-CONTAMINATED AGRICULTURAL PRODUCTS

H U M I O TSUNODA AND N O R I K O TSUNODA

D e p a r t m e n t o f H y g i e n e and P u b l i c H e a l t h , U n i v e r s i t y , M o r i o k a 020, J a p a n School o f Medicine, Iwate Medical

ABSTRACT F i v e v o l u n t e e r s p a r t i c i p a t e d i n a s t u d y i n w h i c h f l u o r i d e u p t a k e and e x c r e t i o n were i n v e s t i g a t e d f o l l o w i n g t h e i n g e s t i o n o f r i c e and g r e e n l e a f y v e g e t a b l e s p r o d u c e d i n an a r e a known t o be p o l l u t e d b y a i r b o r n e f l u o r i d e . The r e s u l t s w e r e c o m p a r e d w i t h t h o s e o b t a i n e d f r o m e x p e r i m e n t s i n w h i c h t h e s u b j e c t s were f e d d i e t s c o n s i s t i n g o f s i m i l a r b u t non-contaminated foods. The a v e r a g e d a i l y i n t a k e o f f l u o r i d e b y e a c h p a r t i c i p a n t was 7.84 mg when t h e s u b j e c t s w e r e on t h e e x p e r i m e n t a l menu, w h i l e i t was 1.89 mg when t h e y The r a t e a p p r o x w e r e o n t h e c o n t r o l menu. A h i g h r a t e ( 9 2 % ) o f f l u o r i d e a b s o r p t i o n w a s o b s e r v e d when t h e s u b j e c t s w e r e on t h e e x p e r i m e n t a l menu. i m a t e d t h a t o f NaF. I n addition, t h e r e t e n t i o n t i m e o f f l u o r i d e s contained

i n t h e e x p e r i m e n t a l m e a l s was f o u n d t o b e l o n g e r t h a n t h a t o f NaF. The i m p o r t a n c e o f f l u o r i d e a c c u m u l a t i o n i n a g r i c u l t u r a l p r o d u c t s i n r e l a t i o n t o p u b l i c h e a l t h i n J a p a n was d i s c u s s e d .

INTRODUCTION One o f t h e c h a r a c t e r i s t i c s o f f l u o r i d e - i n d u c e d a i r pollution i s the a c c u m u l a t i o n o f f l u o r i d e s i n v e g e t a t i o n g r o w n i n t h e p o l l u t e d a r e a (1.2). F e e d i n g d o m e s t i c a n i m a l s a g r i c u l t u r a l products contaminated by f l u o r i d e s has been shown t o r e s u l t i n f l u o r o s i s , l i v e s t o c k i n d u s t r y (3,4).
I n Japan,

posing a serious problem i n the

a c c u m u l a t i o n o f f l u o r i d e i n r i c e and l e a f y v e g e t a b l e s grown i n and h a s been h e a l t h c o n c e r n (7.8). When h u m a n s i n g e s t such f l u o r i d e -

a r e a s p o l l u t e d b y a i r b o r n e f l u o r i d e h a s been r e c o g n i z e d (5,6), of public

contaminated a g r i c u l t u r a l products, t h e r e s u l t i n g t o x i c i t y may be d e t e r mined by t h e e x t e n t t o w h i c h t h e f l u o r i d e i s absorbed t h r o u g h t h e gastrointestinal tract.

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T h i s p a p e r d e a l s w i t h a b a l a n c e s t u d y i n human s u b j e c t s i n w h i c h t h e i n t a k e and e x c r e t i o n o f f l u o r i d e t h r o u g h i n g e s t i o n o f f l u o r i d e - c o n t a m i n a t e d a g r i c u l t u r a l p r o d u c t s w e r e examined. MATERIALS AND METHODS F i v e h e a l t h y male a d u l t s p a r t i c i p a t e d i n t h i s study. They w e r e a s k e d t o with b o a r d t o g e t h e r and p e r f o r m d e s k w o r k i n a d e s i g n a t e d h o u s e f o r f i v e days. The s u b j e c t s w e r e f e d d i e t s c o n s i d e r e d a d e q u a t e f o r e a c h i n d i v i d u a l r e s p e c t t o t h e i r c a l o r i c and p r o t e i n a l l o w a n c e s . days, On t h e s e c o n d and t h i r d while during the

t h e s u b j e c t s w e r e f e d m e a l s c o n t a i n i n g r i c e and g r e e n l e a f y veget h e m e a l s i n c l u d e d r i c e and v e g e t a b l e s g r o w n i n an a r e a t h e f o o d and d r i n k i n g e s t e d b y each To d e t e r m i n e 24-hour u r i n a r y and f e c a l s a m p l e s

t a b l e s p r o d u c e d i n an a r e a p o l l u t e d b y a i r b o r n e f l u o r i d e , o t h e r t h r e e days, w i t h no known f l u o r i d e p o l l u t i o n . Throughout t h e e x p e r i m e n t a l period, the total d a i l y excretion o f fluoride,

p a r t i c i p a n t was w e i g h e d and t h e f l u o r i d e c o n t e n t d e t e r m i n e d . w e r e t a k e n , b e g i n n i n g a t 8 a.m., was d e t e r m i n e d . b a s e d o n t h e A.O.A.C.

and t h e f l u o r i d e c o n t e n t i n each sample S a m p l e s w e r e a s h e d a n d t h e a s h was w h i l e u r i n a r y f l u o r i d e was

s o a s t o p r e v e n t an

D e t e r m i n a t i o n o f f l u o r i d e c o n t e n t s i n f o o d and f e c e s was m e t h o d (11). The r e l e a s e d f l u o r i d e was t h e n d e t e r m i n e d by

subjected t o d i s t i l l a t i o n .

t h e f l u o r i d e i o n - s e l e c t i v e e l e c t r o d e (12,13), d e t e r m i n e d d i r e c t l y b y t h e same m e t h o d (14).

The e x p e r i m e n t s w e r e c o n d u c t e d d u r i n g t h e w i n t e r , e x t e n s i v e l o s s o f f l u o r i d e t h r o u g h p e r s p i r a t i o n (9,lO). RESULTS

The f l u o r i d e c o n t e n t s o f t h e a g r i c u l t u r a l p r o d u c t s f r o m b o t h p o l l u t e d and c o n t r o l areas d i f f e r e d f r o m each o t h e r . ppm, respectively, w h i l e t h e y w e r e 0.57 Average f l u o r i d e c o n t e n t s o f r i c e ppm a n d 0.44-0.94 ppm, respecand g r e e n l e a f y v e g e t a b l e s f r o m t h e p o l l u t e d a r e a w e r e 5.2 ppm and 7.4-10.2 tively, i n t h e c o n t r o l samples. Because t h e

The d a i l y i n t a k e o f f l u o r i d e f r o m f o o d and t h e f l u o r i d e e x c r e t i o n i n u r i n e a n d f e c e s f o r e a c h i n d i v i d u a l a r e s h o w n i n T a b l e I. quantities o f f o o d i n g e s t e d and c o m p o s i t i o n o f t h e m e a l s v a r i e d s l i g h t l y The a v e r a g e d a i l y i n t a k e o f f l u o r i d e b y each

a m o n g t h e p a r t i c i p a n t s , i n t a k e s o f f l u o r i d e d i f f e r e d f r o m d a y t o d a y and from individual t o individual. c o n t r o l meal. T a b l e I 1 shows t h e r a t e o f d a i l y u r i n a r y and f e c a l f l u o r i d e e x c r e t i o n i n r e l a t i o n t o corresponding f l u o r i d e i n t a k e . The a v e r a g e r a t e o f f l u o r i d e while e x c r e t i o n was 69% when t h e s u b j e c t s w e r e f e d n o n - c o n t a m i n a t e d food, p a r t i c i p a n t w a s 7.84 mg f r o m t h e e x p e r i m e n t a l m e a l , a n d 1.89 mg f r o m t h e

109
TABLE I FIVE-DAY INTAKE AND EXCRETION OF FLUORIDE BY VOLUNTEERS Subject D a i l y F l u o r i d e I n t a k e o r O u t p u t , mg 2 3 4 7.58 3.45 0.57 8.37 3.61 0.48 8.12 2.35 0.25 8.50 2.04 0.99 8.33 2.73 0.81 8.18k0.36 2.84+0.68 0.62+0.29 7.24 2.69 0.36 7.26 2.55 0.39 7.33 3.15 0.09 8.36 3.37 0.74 7.35 1.88 1.27 7.51k0.48 2.73+0. 58 0.57k0.45 2.12 1.72 0.11 2.12 1.99 0.16 1.98 0.77 0.12 2.24 0.93 0.27 2.04 1.19 0.17 2.1 OkO. 10 1.32k0.52 0.17+0.06

Day 1 Intake' Output Urine Feces Intake Output Intake Output Intake Output Intake Output Urine Feces Urine Feces Urine Feces Urine Feces 1.53 0.99 0.54 1.66 1.15 0.33 1.72 0.81 0.52 1.60 0.26 0.91 1.52 0.59 0.16

5 1.93 1.30 0.38 1.93 1.39 0.27 1.22 0.09 2.09 0.77 0.17 1.85 0.94 0.13

S.S.

H.T.

M.K.

2.02

Y.K.

K.S.

Average' Intake Output

Urine Feces

1.61 +O. 09 0.76kO.35 0.49+0.28

1.96+0.09 1.12k0.26 0.21 +o. 12

' I n t a k e i n c l u d e s f l u o r i d e s i n f o o d and beverage 'Values a r e means and s t a n d a r d d e v i a t i o n

i t was 43% when t h e y were f e d e x p e r i m e n t a l food.

These r e s u l t s e x c l u d e t h e t h e average e x c r e t i o n o f

f i r s t dzy's d a t a as t h e y were a f f e c t e d by t h e f o o d i n g e s t e d p r i o r t o t h e i n i t i a t i o n o f t h e experiment.

On t h e o t h e r hand,

f e c a l f l u o r i d e was 8% w i t h l i t t l e v a r i a t i o n between days, w h i l e t h e u r i n a r y e x c r e t i o n r a t e was l o w e r on t h o s e days when t h e s u b j e c t s were f e d c o n t a m i n a t e d f o o d t h a n when t h e y were f e d t h e non-contaminated food. Since t h e The r a t e o f f l u o r i d e a b s o r p t i o n t h r o u g h i n g e s t i o n o f t h e e x p e r i m e n t a l f o o d can be e s t i m a t e d f r o m t h e f l u o r i d e e x c r e t e d i n t h e feces. a b s o r p t i o n f o r t h e a g r i c u l t u r a l p r o d u c t s i s 92%. I n a d d i t i o n , e x c r e t i o n , i.e.. u r i n a r y and f e c a l f l u o r i d e e x c r e t i o n . p e r c e n t a g e o f f e c a l e x c r e t i o n o f f l u o r i d e i s a b o u t 8%. t h e r a t e o f f l u o r i d e the fluoride r e t e n t i o n r a t e i n t h e body can a l s o be e s t i m a t e d f r o m t o t a l t h e f l u o r i d e
I t was o b s e r v e d

t h a t f l u o r i d e r e t e n t i o n r e s u l t i n g from i n g e s t i o n o f the contaminated a g r i c u l t u r a l p r o d u c t s was g r e a t e r t h a n t h a t o f t h e c o n t r o l s .

110

TRBLE 11
RATE OF FLUORIDE EXCRETION Siibject Item

Day 1

Rate o f D a i l y Excretion, %

5 87.1 67.4 19. *' 86.0 72.0 14.0 64.9 60.4 4.5 44.9 36.8 8.1 57.8 50.8 7.0 68.1k16.3 57.5+12.6 10.7k 5.5

s. s.
H. T.

Total Urine Feces Total Urine Feces Total Urine Feces Total Urine Feces Total Urine Feces Total Urine Feces

100.0 64.7 35.3 89.2 69.3 19.9 77.3 47.1 30.2 73.2 16.3 56.9 49.3 38.8 10.5 77.8217.1 47.2k19.1 30.6k15.7

53.0 45.5 7.5 48.8 43.1 5.7 32.0 28.9 3.1 35.6 24.0 11.6 42.5 32.8 9.7 42.427.8 34.9k8.2 7.5k3.0

42.2 37.2 5.0 40.5 35.1 5.4 44.2 43.0 1.2 49.2 40.3 8.9 42.9 25.6 17.3 43.8t3.0 36.226.0 7.625.4

86.3 81.1 5.2 101.4 93.9 7.5 45.0 38.9 6.1 53.6 41.5 12.1 66.6 58.3 8.3 70.6220.8 62.7k21.7 7.8k 2.4

M. K.
Y.K.

K. S.
Average1

'Values

a r e means and s t a n d a r d d e v i a t i o n

DISCUSSION
As m e n t i o n e d p r e v i o u s l y , problems associated w i t h fluoride-induced air p o l l u t i o n l i e i n t h a t i t n o t o n l y caused i n j u r y t o p l a n t s b u t a l s o r e s u l t s i n f l u o r i d e a c c u m u l a t i o n i n v e g e t a t i o n exposed t o t h e p o l l u t a n t . c o n t a r n i n a t e d v e g e t a t i o n (3.4.8). 'Several types o f agricultural p r o d u c t s c o m m o n l y consumed b y humans a r e known t o a c c u m u l a t e f l u o r i d e . green l e a f y vegetables, I n Japan, Numerous r e p o r t s have been p u b l i s h e d d e s c r i b i n g f l u o r o s i s i n a n i m a l s f e d f l u o r i d e -

i t h a s been o b s e r v e d t h a t r i c e and
produced i n p o l l u t e d Problems associated

main s t a p l e s i n t h i s country,

areas c o u l d c o n t a i n f l u o r i d e t h a t i s s e v e r a l t o more t h a n t e n t i m e s h i g h e r t h a n t h a t f o u n d i n n o n - c o n t a m i n a t e d p r o d u c t s (5,6). w i t h t h e c o n s u m p t i o n o f such c o n t a m i n a t e d p r o d u c t s have been r e c o g n i z e d

(8.15).

The t o x i c i t y o f f l u o r i d e i s d e p e n d e n t o n t h e s o l u b i l i t y o f v a r i o u s Results obtained from t h i s a r e a s was g r e a t e r t h a n

t y p e s o f f l u o r i d e and t h e a m o u n t a b s o r b e d (16).

s t u d y show t h a t t h e a b s o r p t i o n r a t e o f f l u o r i d e s c o n t a i n e d i n r i c e and green l e a f y vegetables produced i n f l u o r i d e - p o l l u t e d

90%, a p p r o x i m a t i n g t h a t of NaF (9.17).

111
I n a d d i t i o n , t h e r a t e o f u r i n a r y e x c r e t i o n o f f l u o r i d e as a r e s u l t o f i n g e s t i n g t h e c o n t a m i n a t e d f o o d was f o u n d t o be l o w e r t h a n t h a t o f i n g e s t i n g MaF ( 9 , 1 4 ) , s u g g e s t i n g a l o n g e r r e t e n t i o n t i m e i n t h e body. For t h i s r e a s o n , t h e f l u o r i d e s c o n t a i n e d i n r i c e and l e a f y v e g e t a b l e s a r e o f (nuch c o n c e r n i n t e r m s o f t c l e i r i n f l u e n c e on p u b l i c h e a l t h . T h e r e i s a need f o r c o n t r o l l i n g f l u o r i d e e m i s s i o n b y i n d u s t r i e s so t h a t r i c e and g r e e n l e a f y v e g e t a b l e s g r o w n i n t h e v i c i n i t y o f i n d u s t r i a l p l a n t s may b e f r e e f r o m accumulation o f the pollutant. CONCLUSION T h e a b s o p t i o n o f f l u o r i d e t h r o u g h t h e g a s t r o i n t e s t i n a l t r a c t h a s been s t u d i e d i n f i v e human v o l u n t e e r s . and g r e e n They w e r e f e d d i e t s c o n s i s t i n g o f r i c e e a f y v e g e t a b l e s p r o d u c e d i n an a r e a known t o be p o l l u t e d b y I n addition,
i t was o b s e r v e d t h a t t h e

a i r b o r n e f u o r i d e . The a v e r a g e r a t e o f f l u o r i d e a b s o r p t i o n b y t h e s e p a r t i c i p a n t s was f o u n d t o be a b o u t 92%. body t h a n t h a t o f NaF. The i m p o r t a n c e o f p r e v e n t i n g f l u o r i d e a c c u m u l a t i o n b y a g r i c u l t u r a l p r o d u c t s g r o w n i n f l u o r i d e p o l l u t e d a r e a s i n r e l a t i o n t o human h e a l t h i s stressed. f l u o r i d e present i n these foods e x h i b i t e d a longer r e t e n t i o n t i m e i n the

REFERENCES 1. Tsunoda H,
Haneda M (1968) I n d u s t r E n v i r o n Eng 59:2-11

2. C o m m i t t e e o n B i o l o g i c E f f e c t s o f A t m o s p h e r i c P o l l u t a n t s (1971) F l u o r i d e s . NAS. Washington, DC. p p 77-162

3. P h i l l i p s PH, Greenwood DA, Hobbs CS, H u f f m a n C F (1955) The F l u o r o s i s Problem i n L i v e s t o c k Production. A Report o f t h e Committee on Animal N u t r i t i o n . NAS-NCR P u b l i c a t i o n 381, Washington, DC, p p 1-20 4. S h u p e J L ( 1 9 6 9 ) F l u o r o s i s o f L i v e s t o c k . A i r Q u a l i t y M o n o g r a p h 69-4, A m e r i c a n P e t r o l e u m I n s t i t u t e , New York, p p 1-29 5. Tsunoda H, 6. S a k u r a i S, K u n i t a H (1973) J E n v i r o n P o l l u t C o n t r o l 9:69-75 I t a i K, Tsunoda H (1983) F l u o r i d e 16:175-180

7. Tsunoda H (1971) J Japan SOC S a f e t y Eng 10:75-85

8. Tsunoda H (1978) N i p p o n D e n t Rev 427:137-150

9. H o d g e HC, S m i t h FA ( 1 9 6 5 ) I n : S i m o n s JH ( e d ) F l u o r i n e C h e m i s t r y , V o l 4. Academic Press, New York, p p 137-176 10. C r e m e r HD. B i t t n e r W ( 1 9 7 0 ) Health. WHO, Geneva, p p 75-91 I n : E r i c s s o n Y ( e d ) F l u o r i d e a n d Human
o f A n a l y s i s o f AOAC 1 1 t h ed.

11. H o r o w i t z W (ed) (1970) O f f i c i a l M e t h o d s Washington, DC, p p 405-411

12. F r a n t MS, 13. N e e f u s JD. C h o l a k J,

Ross J W (1966) S c i e n c e 154:1553-1554 S a l t z m a n BE (1970) Am I n d Hyg Assoc J 1:168

14. I s h i k a w a S (1976) I w a t e I g a k u Z a s s h i 28:207-222

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15. T s u n o d a H. S a k u r a i S , I t a i K. (1984) F l u o r i d e 17:159-167

S a t 0 T. Nakaya S . M i t a M,

Tatsumi M

16. E a g e r s R Y ( 1 9 6 9 ) T o x i c P r o p e r t i e s E l s e v i e r , Essex, p 33

o f I n o r g a n i c F l u o r i n e Compounds.

17. Largent EJ (1961) Fluorosis-The Health Aspects of F l u o r i n e Compounds. Ohio S t a t e U n i v e r s i t y P r e s s , Columbus, p p 34-39

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 113-125 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

113

EFFECTS O F EFFLUENTS FROM A F O S S I L FUEL POWERED GENERATING PLANT O N THE HEALTH OF BEEF CATTLE

J A M E S L. SHUPE, ARLAND E. OLSON, JAY W.

BAGLEY. RAGHUBIR P. SHARMA, AND GENE W.

CALL,

JOHN E. BUTCHER, CLELL V.

MILLER

D e p a r t m e n t o f V e t e r i n a r y S c i e n c e , UMC 56. U t a h S t a t e U n i v e r s i t y , L o g a n , U t a h 84322, U.S.A. ABSTRACT E f f l u e n t waters from a coal-fueled e l e c t r i c i t y generating p l a n t were s p r i n k l e - i r r i g a t e d on forage crops which were grazed o r harvested f o r w i n t e r feed f o r c a t t l e . f o r drinking. ogy, The w a t e r s w e r e h i g h e r t h a n n o r m a l i n t o t a l d i s The c a t t l e s o m e t i m e s had a c c e s s t o t h i s w a t e r o r heavy m e t a l c o n c e n t r a t i o n s i n blood, selected s o l v e d s a l t s and f l u o r i d e s . c l i n i c a l chemistries.

A n a l y s e s h a v e shown no c o n s i s t e n t a b n o r m a l i t i e s i n IhematolF l u o r i n e c o n t e n t o f bone and some u r i n e s a m p l e s T e e t h t h a t h a v e f o r m e d and m i n e r a l i z e d w h i l e t h e n e i t h e r the e f f l u e n t water, nor forage sprinkle-

s o f t t i s s u e s o r bones. were s l i g h t l y elevated. sis. Independently,

c a t t l e w e r e on t h e f a r m h a v e m o d e r a t e t o m a r k e d l e s i o n s o f d e n t a l f l u o r o i r r i g a t e d w i t h t h a t w a t e r h a v e a h i g h enough f l u o r i n e c o n t e n t t o i n d u c e t h e s e v e r i t y o f d e n t a l l e s i o n s observed, b u t t h e combined sources have induccd chronic fluoride toxicosis i n the cattle. INTRODUCTION F o r s e v e r a l decades, types o f industry. s c i e n t i s t s a t U t a h S t a t e U n i v e r s i t y (USU) h a v e Seen f i n d i n g s are from a planned t e n year w i t h e f f l u e n t waters from a coalThe e f f l u e n t w a t e r i s o r i g i n a l l y s t u d y i n g t h e e f f e c t s on t h e e n v i r o n m e n t o f e m i s s i o n s f r o m many d i f f e r e n t The c u r r e n t 5 - y e a r s t u d y t o m o n i t o r h e a l t h r e l a t e d e f f e c t s on c a t t l e a f t e r i n g e s t i o n o f f o r a g e c r o p s t h a t h a v e been s p r i n k l e - i r r i g a t e d fueled e l e c t r i c i t y generating plant.

i n t r o d u c e d i n t o t h e g e n e r a t i o n p l a n t f r o m r e l a t i v e l y p r i s t i n e sources. Regulations f o r b i d t h e discharge o f any w a t e r from t h e p l a n t back i n t o n a t u r a l s t r e a m channels. chemicals. The w a t e r i s r e c i r c u l a t e d t e n t i m e s f o r c o o l i n g Smoke is also and p o l l u t i o n c o n t r o l p u r p o s e s u n t i l t h e w a t e r i s h e a v i l y l a d e n e d w i t h Most o f t h e p l a n t e f f l u e n t s a r e i n t h e d i s c h a r g e water. Some o f t h e w a t e r e v a p o r a t e s , b u t much stack emissions are negligible. i n t o a storage reservoir. used t o s p r i n k l e i r r i g a t e crops. The h e a v i l y p o l l u t e d w a t e r i s d i s c h a r g e d The c r o p s a r e d i v e r s i f i e d b u t m o s t a r e

114
f o r a g e c r o p s f o r g r a z i n g by c a t t l e o r h a r v e s t f o r w i n t e r feed. MATERIALS AND METHODS V e g e t a t i o n g r o w t h and d e v e l o p m e n t i s e v a l u a t e d throughout t h e growing s e a s o n and c o m p a r e d t o c o n t r o l v e g e t a t i o n o n t h e USU f a r m . fluorine. D e t a i l e d p h y s i c a l and c l i n i c a l e x a m i n a t i o n s a r e c o n d u c t e d on t h e c a t t l e periodically t o evaluate t h e i r h e a l t h status. s p e c i f i c elemental contents. selected tissues. measured. o f i n o r g a n i c elements. Analytical on t h e r e s e a r c h f a r m . U t a h s e r v e as c o n t r o l s . Heavy m e t a l s and o t h e r e l e m e n t s w e r e a n a l y z e d f o r b y u s i n g a p p r o p r i a t e flame/flameless atomic absorption spectrometry. and u r i n e w e r e c o n d u c t e d w i t h a s p e c i f i c F l u o r i d e analyses o f blood Routine d i g e s t i o n Values a r e i o n electrode. A n i m a l s o f d i f f e r e n t ages a r e a n n u a l l y s e l e c t e d a n d s a c r i f i c e d and t i s s u e s c o l l e c t e d and a n a l y z e d f o r P a t h o l o g i c e v a l u a t i o n s a r e a l s o made o f B l o o d s a m p l e s f r o m a l l a n i m a l s a r e a n a l y z e d f o r a number B i o c h e m i c a l and p h y s i o l o g i c a l p a r a m e t e r s a r e a l s o r e s u l t s a r e compared w i t h e a r l i e r r e s u l t s o f a n i m a l s C o m p a r a b l e n o r m a l c a t t l e a t t h e USU f a r m n e a r Logan, Analyses are b o r o n , and made o n v e g e t a t i o n s a m p l e s f o r l e a d , c o p p e r , z i n c , cadmium,

o r o t h e r p r e p a r a t i o n o f a l l s a m p l e s was done p r i o r t o a n a l y s e s . an ima1 s. RESULTS AND D I S C U S S I O N To d a t e , a l l analyses o f blood, l i v e r ,

g r o u p e d a c c o r d i n g t o t h e t i m e o f y e a r o f s a m p l i n g and t h e age g r o u p o f t h e

kidney. s k e l e t a l muscle, lung, zinc. magnelead but are

s p l e e n a n d bone h a v e b e e n w i t h i n t h e n o r m a l r a n g e s f o r i r o n , s i u m , c o p p e r , cadmium, chromium, m e r c u r y and lead.

O f these elements,

v a l u e s h a v e shown t h e g r e a t e s t f l u c t u a t i o n s t o d a t e i n t h e s t u d y , s t i l l b e l o w l e v e l s w h e r e c l i n i c a l e f f e c t s w o u l d be a p p a r e n t .

A l l serum e l e c t r o l y t e ,
normal ranges f o r c a t t l e .

enzyme o r o t h e r b i o c h e m i c a l v a l u e s a r e w i t h i n t h e The t e s t s i n d i c a t e n o a l t e r a t i o n o f t h e p h y s i o There were time-toseasonal, or b u t t h e s e a r e e i t h e r o f no

l o g i c a l s t a t e o r organ d y s f u n c t i o n i n these animals. t i m e v a r i a t i o n s observed i n s e l e c t e d parameters,

adverse p h y s i o l o g i c a l consequence o r r e f l e c t t h e b i o l o g i c a l , Similarly,

age v a r i a t i o n i n t h e a n i m a l s f r o m whom t h e s a m p l e s w e r e o b t a i n e d . t h e h e m a t o l o g i c a l parameters o f a l l a n i m a l s were n o t i n f l u factors. enced by t h e e n v i r o n m e n t a l reported f o r cattle. The e s s e n t i a l m e t a l s i n b l o o d o r s e r u m o f t h e s e a n i m a l s h a v e been w i t h i n

A l l c u r r e n t values a r e comparable t o

t h o s e o b t a i n e d f r o m e a r l i e r s a m p l i n g s and a r e w i t h i n t h e n o r m a l r a n g e s

115

the normal ranges. Slight variations may be due to biological variation or environmental factors, but did not significantly alter the levels of these elements and none of the values posed any health risk. The fluoride content o f the urine o f normal cattle ingesting a normal diet fluctuates, but it is usually less than 6 ppm F. Few of the samples collected to date were above 6 ppm F (Table 1). The average urine fluoride content o f all animals has been below the level normally associated with detrimental lesions. The fluoride levels i n urine and bone do, however, reflect an elevated fluoride intake by these animals. Urinary fluoride is an index of the exposure to this element although wide variations can be expected. An increase in the bone and urine fluoride is also consistent with increased amounts of fluoride in the water and vegetation.

TABLE I FLUORIDE CONTENT

OF

URINE FROM RESEARCH FARM CATTLE

PPM F

Age (years)
3.7 3.7 7.6 3.7 8.7 1.7 1.6 2.7

8.9 3.3 2.3 1.2 5.3 2.6 2.7 7.6 0.8 4.6

1.6
5.7 1.7 1.6 3.7 3.6 6.7 1.6 0.5 2.7 2.7 7.5

1.2
0.8 5.4 6.3 2.8 1.7 0.4 0.8 2.7 9.0

116
Other evidence o f increased l e v e l s o f f l u o r i d e i n t a k e are present. Once t h e t e e t h

F l u o r i d e d e n t a l l e s i o n s a r e i n d u c e d w h i l e t e e t h a r e f o r m i n g and m i n e r a l i z i n g p r i o r t o e r u p t i n g i n t o t h e o r a l c a v i t y ( F i g . 1). have formed, mineralized, and e r u p t e d , c h a r a c t e r i s t i c enamel m o t t l i n g . e x c e s s i v e f l u o r i d e does n o t induce

Dental l e s i o n s c h a r a c t e r i s t i c o f f l u o r i d e Teeth t h a t were formed

t o x i c o s i s a r e p r e s e n t i n t h e c a t t l e t h a t had permanent i n c i s o r t e e t h form, m i n e r a l i z e and e r u p t w h i l e on t h e R e s e a r c h Farm. l e s i o n s ( F i g . 2). b e f o r e t h e c a t t l e w e r e p l a c e d on t h e f a r m showed n o f l u o r i d e i n d u c e d The f l u o r i d e i n d u c e d d e n t a l l e s i o n s a r e m o r e s e v e r e i n indicat h e second p a i r o f permanent i n c i s o r t e e t h t h a n i n t h e f i r s t p a i r , t h e i r v e g e t a t i o n and w a t e r i n t a k e ( F i g s .

t i n g an i n c r e a s e d l e v e l o f f l u o r i d e i n t a k e a s t h e c a t t l e g r e w and i n c r e a s e d

3-4).

The c l a s s i f i c a t i o n o f s l i g h t moderate e f f e c t s

d e n t a l f l u o r o s i s i n t h e f i r s t p a i r o f permanent i n c i s o r s ,

i n t h e second p a i r , and marked t o s e v e r e e f f e c t s i n t h e t h i r d and f o u r t h p a i r s i n d i c a t e t h a t t h e a n i m a l s r e c e i v e d more than t h e t o l e r a n c e t h r e s h o l d l e v e l o f f l u o r i d e when t h e t e e t h w e r e f o r m i n g ( T a b l e 11).

F i g u r e 1. Photograph o f dissected a n t e r i o r mandible of a 16 month o l d h e i f e r . The e r u p t e d t e e t h a r e a l l d e c i d u o u s . The d i s s e c t i o n shows t h e f o r m i n g and m i n e r a l i z i n g f i r s t ( 1 ) a n d s e c o n d (2) p a i r s o f p e r m a n e n t i n c i sors. Number 1 shows t y p i c a l m o d e r a t e f l u o r i d e i n d u c e d l e s i o n s .

117

F i g u r e 2. P e r m a n e n t i n c i s o r t e 3 t h f r o m a 4-1/2 y e a r o l d H e r e f o r d . T h e s e t e e t h were formed p r i o r t o exposure t o h i g h f l u o r i d e i n t a k e and a r e essent i a l l y normal.

Photograph o f dissected a n t e r i o r mandible of a 2 year 8 month F i g u r e 3. old H e r e f o r d female. The f i r s t p a i r o f p e r m a n e n t i n c i s o r s (1) has erupted. The s e c o n d p a i r (2) a r e f o r m e d , m i n e r a l i z e d and show soiro f l u o r i d e induced mottling. The c r o w n o f t h e L h i r d p a i r o f i n c i s o r s ( 3 ) i s m o s t l y f o r m e d and p a r t i a l l y m i n e r a l i z e d .

118

P e r m a n e n t i n c i s o r t e e t h o f a 3 y e a r 8 m o n t h o l d cow t h a t was F i g u r e 4. r a i s e d o n t h e Research Farm and exposed t o e x c e s s i v e f l u o r i d e . Note t h e a b n o r m a l d i s c o l o r a t i o n and c h a l k y appearance o f t h e second, t h i r d and f o u r t h p a i r s o f teeth. TABLE I 1 PERMANENT I N C I S O R TOOTH CLASSIFICATIONS OF CATTLE R A I S E D ON RESEARCH FARM Age ( y e a r s ) Incisor Classifications

2-3

4-7-7-7 3-3-7-7
i f

3-7-7-7 2-6-7-7

3-4

2-3-3-7 1-3-7-7 1-3-4-7

+++

2-3-4-7 2-3-4-6 2-3-4-7

4-5

2-3-4-6

5 +

2-3-4-4 2-3-3-3

++

3-3-4-4

F o r t h e purpose o f c l i n i c a l l y c l a s s i f y i n g t h e v a r i o u s degrees o f d e n t a l fluorosis, s t a n d a r d s have been developed. Normal: t o o t h h a s n o r m a l shape and s i z e . a d o p t e d a n d a r e u s e d (1-4). w h i t e appearance o f enamel;

0.

smooth, t r a n s l u c e n t , glossy

1.

Questionable effect: determine; may have

a b e r r a t i o n s w i t h e x a c t cause d i f f i c u l t t o enamel opaque, flecks; dull, cavities,

i f present,

may b e u n i l a t e r a l o r b i l a t e r a l ; n o m o t t l i n g .

2.

Slight

effect:

slight,

chalky-white cross s t r i a -

t i o n s ( m o t t l e d enamel); a b n o r m a l wear;

may h a v e s l i g h t d i s c o l o r a t i o n b u t n o

t o o t h w i l l h a v e n o r m a l shape and s i z e .

119 3.
Moderate effect: diffuse, dull, chalky-white enamel

(generalized mottling);

t o o t h may h a v e a s l i g h t l y i n c r e a s e d enamel may b e c r e a m

r a t e o f wear and may be s t a i n e d .

4.

Marked e f f e c t :

definite mottling:

c o l o r e d and evidence

h y p o p l a s i a and hypomineralization: d e f i n i t e abrasion o f tooth:

e n a m e l may b e p i t t e d and e r o d e d ; s t a i n i n g may o r may n o t be present.

5.

Severe e f f e c t : classification.

e f f e c t s more severe t h a n d e s c r i b e d i n number f o u r

T e e t h c l a s s i f i e d a s n u m b e r 2 ( s l i g h t ) o r 3 ( m o d e r a t e ) d o n o t w e a r away any f a s t e r t h a n normal t e e t h and have cosmetic, animals. e f f e c t s a r e i m p o r t a n t t o people, teeth. however. non-detrimental effects i n The e c o n o m i c i m p a c t i s n e g l i g i b l e i n a n i m a l s . Such c o s m e t i c

D e n t a l f l u o r o s i s i n a n i m a l s i s u s u a l l y diagnosed by e x a m i n i n g t h e i n c i s o r The use o f b o t h d i r e c t and back l i g h t i n g o f t h e t e e t h enhances t h e Cheek ( p r e m o l a r and m o l a r ) t e e t h a r e more d i f f i i l l u m i n a t i o n o f t h e t e e t h i s o f t e n poor, the a c c u r a c y o f examination.

c u l t t o examine i n t h e l i v e a n i m a l because i t i s sometimes d i f f i c u l t t o r e s t r a i n the animal properly, tongue and u n s w a l l o w e d f o o d may i n t e r f e r e , and d i s c o l o r a t i o n r e s u l t i n g f r o m f a c t o r s o t h e r t h a n f l u o r i d e c o m p l i c a t e t h e examination. The c r i t e r i a used i n d i a g n o s i n g and e v a l u a t i n g whether d e n t a l f l u o r o s i s i s a f f e c t i n g premol a r s and m o l a r s a r e b a s e d o n t h e d e g r e e o f s e l e c t i v e a b r a s i o n a n d a r e c o r r e l a t e d w i t h t h e degree o f i n c i s o r f l u o r o s i s . P r e m o l a r and m o l a r abraAbraded cheek t e e t h Damaged t e e t h s i o n i s d e l a y e d somewhat beyond t h a t o f i n c i s o r t e e t h .

can cause i m p r o p e r m a s t i c a t i o n and u t i l i z a t i o n o f roughage.

may be s o m e w h a t p r o t e c t e d b y a d j a c e n t sound t e e t h t h a t a r e n o t abraded. A b r a s i v e f e e d s s u c h as c o a r s e , f i b r o u s , t o u g h r o u g h a g e w i l l i n c r e a s e t h e r a t e o f d e n t a l abrasion. t o x i c o s i s i n animals, I n d i a g n o s i n g and e v a l u a t i n g c h r o n i c f l u o r i d e symptoms and l e s i o n s (5). t h e cheek t e e t h s h o u l d be examined and t h e f i n d i n g s

c o r r e l a t e d w i t h o t h e r fluoride-induced

The degree o f d e n t a l f l u o r o s i s can u s u a l l y be c o r r e l a t e d w i t h t h e amount o f f l u o r i d e i n t h e bones, t h e d e g r e e o f o s t e o f l u o r o s i s . t h e d u r a t i o n o f exposure, ingestion, age o f the animal during the time of therefore, excessive fluoride t h e amount o f f l u o r i d e ingested, The d e n t a l l e s i o n s , toxicosis. and o t h e r p h y s i o l o g i c processes are useful i n c l i n i c a l l y however, the

o f t h e body.

diagnosing fluoride

To b e m o s t m e a n i n g f u l ,

f l u o r o t i c d e n t a l changes must be c o r r e l a t e d w i t h o t h e r symptoms and l e s i o n s indicative o f fluoride toxicosis. n o t be used as t h e I n o t h e r words, d e n t a l l e s i o n s s h o u l d s o l e c r i t e r i o n when e v a l u a t i n g f l u o r i d e t o x i c o s i s .

I n g e s t e d f l u o r i d e s have g r e a t a f f i n i t y f o r bone as w e l l a s f o r d e v e l o p i n g

120
and m i n e r a l i z i n g t e e t h . d u r i n g an animal's l i f e ; s i v e t o exce.;sive E x c e s s i v e f l u o r i d e s may a f f e c t bones a t a n y t i m e however, bones i n y o u n g a n i m a l s a r e m o r e r e s p o n There

f l u o r i d e l e v e l s t h a n a r e those o f mature animals. To d a t e ,

has been an i n c r e a s e i n t h e f l u o r i d e c o n t e n t o f bone f r o m t h e s a c r i f i c e d a n i m a l s t h a t w a s a g e r e l a t e d ( T a b l e 111). no c h a r a c t e r i s t i c radiographfor a f l u o r i d e induced l e s i o n s o f bone h a v e b e e n o b s e r v e d g r o s s l y , R e s e a r c h Farm. However, an increased ingestion o f

i c a l l y o r m i c r o s c o p i c a l l y i n any o f t h e necropsied animals f r o m t h e fluorides p r o l o n g e d p e r i o d o f t i m e c a n c a u s e d e f i n i t e c h a r a c t e r i s t i c bone changes. TABLE 111 FLUORINE CONTENT

OF BONES FROM

SACRIFICED CATTLE

Age ( m o n t h s ) 18

Metacarpal 343 595 954 1080

PPM

F -

dried fat-free basis Metatarsal 352

Rib 455 1 1 50 1925 1940

32
91 106

670
1020 1110

I f t h e amounts o f f l u o r i d e i n g e s t e d a r e s u f f i c i e n t l y h i g h e r t h a n normal
over an appreciable l e n g t h o f time, evident. Grossly, s t r u c t u r a l bone changes w i l l become They a r e bones t h a t a r e s e v e r e l y a f f e c t e d b y f l u o r i d e appear

c h a l k y w h i t e w i t h a roughened i r r e g u l a r p e r i o s t e a l surface. l a r g e r i n d i a m e t e r and h e a v i e r t h a n n o r m a l . The t y p e o f bone c h a n g e s seen depend o n ' f a c t o r s toxicosis.

that influence fluoride Osteo-

One o r m o r e o f t h e f o l l o w i n g c o n d i t i o n s m a y o c c u r :

porosis, osteosclerosis. hyperostosis. osteophytosis. o r osteomalacia. C h a r a c t e r i s t i c h i s t o l o g i c a l changes a r e a s s o c i a t e d w i t h t h e v a r i o u s degrees o f osteofluorosis. (1,2,4,6,7). These h a v e b e e n e l u c i d a t e d , d e s c r i b e d and i l l u s t r a t e d

A s l i g h t i n c r e a s e i n t h e f l u o r i d e c o n t e n t o f bone above t h e normal

c o n c e n t r a t i o n c a n o c c u r w i t h o u t d e t e c t a b l e chnnges i n bone s t r u c t u r e o r function. T h i s a p p e a r s t o be t h e s t a t u s o f R e s e a r c h Farm a n i m a l s c u r r e n t l y A s l i g h t t o moderate increase i n t h e f l u o r i d e content induces As a m o u n t s o f b o n e f l u o r i d e i n c r e a s e , p r o g r e s s i v e s t r u c t u r a l It i s important to ( T a b l e 111). function.

o n l y s l i g h t m i c r o s c o p i c bone changes w i t h no d e t e c t a b l e a l t e r a t i o n o f changes o c c u r t h a t r e s u l t i n d e f i n i t e bone l e s i o n s .

d i s c e r n t h e d i f f e r e n c e b e t w e e n n o r m a l g r o w t h and r e m o d e l i n g o f bone, fluoride-induced lesions.

and

The d e g r e e o f p r o g r e s s i v e s t r u c t u r a l bone c h a n g e s d u e t o e x c e s s i v e f l u o r i d e i n t a k e and t h e i r p a t t e r n s o f c o m b i n a t i o n s a r e g o v e r n e d b y t h e v a r i o u s factors t h a t influence the manifestations o f fluoride toxicosis. Some o f t h e g r o s s and h i s t o l o g i c a l c h a n g e s i n d u c e d b y f l u o r i d e t o x i c o s i s may resemb l e bone l e s i o n s and a l t e r a t i o n s t h a t a r e a s s o c i a t e d w i t h o t h e r bone diseases. T h e r e f o r e , t h e l e s i o n s observed must be c o r r e l a t e d c a r e f u l l y w i t h o t h e r l e s i o n s and s y m p t o m s i n m a k i n g a d e f i n i t i v e d i a g n o s i s o f f l u o r i d e t o x i c o s i s (5.8). Bone c h a n g e s i n t h e r e s e a r c h f a r m a n i m a l s h a v e n o t y e t a p p e a r e d and t h u s t h e r e has been none o f t h e n o n - s p e c i f i c lameness o r s t i f f n e s s t h a t i s s o m e t i m e s seen i n a d v a n c e d c a s e s o f o s t e o f l u o r o s i s . E x c e s s i v e f l u o r i d e s f o r c a t t l e may a r i s e f r o m s e v e r a l s o u r c e s w h i c h may include: f o r a g e s c o n t a m i n a t e d e i t h e r d i r e c t l y o r i n d i r e c t l y w i t h indusw a t e r w h i c h has a h i g h f l u o r i d e c o n t e n t , e i t h e r n a t u r a l l y , or polluted with industrial t r i a l effluents; effluents.

as i n t h e c a s e o f m o s t g e o t h e r m a l w a t e r s ,

M i n e r a l supplements may e x c e e d recommended f l u o r i d e c o n t e n t F l u o r i d e s a r e c u m u l a t i v e and, t h e r e -

(9).
fore,

H i g h f l u o r i d e c o n t e n t s o i l may b e c a r r i e d b y w i n d o r b e r a i n s p l a s h e d c o m b i n a t i o n s o f moderate amounts o f f l u o r i d e s f r o m t h e above sources t o date, had a c c e s s a t l e a s t p a r t o f

o n t o f o r a g e s w h i c h a r e t h e n consumed.

may add u p t o an e x c e s s i v e t o t a l i n t a k e . The b e e f c a t t l e i n t h i s s t u d y have, t h e t i m e t o t w o f l u o r i d e sources, n a m e l y c o n t a m i n a t e d f o r a g e s and w a t e r .

N e i t h e r o f these sources i s e x t r e m e l y h i g h i n f l u o r i n e content (Tables I V -

V I ) b u t t h e combined s o u r c e s can i n d u c e t h e t y p e s and s e v e r i t y o f d e n t a l

l e s i o n s we h a v e seen t o d a t e . harvested, i n g water, water. power generating plant.

A l l o f t h e f o r a g e s consumed,

b o t h g r a z e d and

h a v e been s p r i n k l e - i r r i g a t e d

w i t h the e f f l u e n t water from the

T h i s w a t e r i s n o t i n t e n t i o n a l l y provided as d r i n k -

b u t c a t t l e have had a c c e s s t o it, and have d r u n k i t a t t i m e s .

E f f o r t s a r e now b e i n g made t o p r e v e n t c a t t l e a c c e s s t o t h e h i g h F c o n t e n t This should lessen t h e s e v e r i t y o f t h e f l u o r i d e toxicosis. There a r e a p p a r e n t l y few f l u o r i d e c o n t a i n i n g emissions f r o m t h e stacks o f t h e g e n e r a t i n g p l a n t because f l u o r i d e c o n t e n t o f n a t i v e u n i r r i g a t e d p l a n t s i n t h e v i c i n i t y o f t h e R e s e a r c h Farm i s w e l l w i t h i n e x p e c t e d n o r m a l l i m i t s , e v e n a t t h e e n d o f t h e g r o w i n g season when F c o n t e n t w o u l d u s u a l l y be e x p e c t e d t o be a t i t s h i g h e s t l e v e l . We h a v e s e e n t h e e f f e c t o r a p p l i c a t i o n o f s e v e r a l o f t h e f a c t o r s t h a t influence the expression o f chronic f l u o r i d e toxicosis. Those f a c t o r s are:

(1) amount o f f l u o r i d e i n g e s t e d ; (2) d u r a t i o n o f i n g e s t i o n : (3) b i o a v a i l -

122 TABLE I V FLUORIDE CONTENT OF ALFALFA AT RESEARCH FARM A n a l y s e s PPM F1 July August 10.8

June Area

October 36.9 23.3

4.0 13.0

8.6
59.2

Area I I B

7.7

' A n a l y s e s a r e o n d r y w t . b a s i s and a r e an a v e r a g e o f 3 samples.

TABLE V FLUORIDE CONTENT OF MIXED PASTURE AT RESEARCH FARM A n a l y s e s PPM F1 July August 28.

June Area I A A r e a 111 Area I I I B 40.4 40.1 7.0

October 17.5

a
54.5 51.8

19.9
18.2

41.3

'Analyses a r e on d r y w t .

b a s i s and a r e an a v e r a g e o f 3 samples.

TABLE V I FLUORIDE ANALYSES (PPM) OF IRRIGATION SYSTEM WATER Time August

~ ~~

July Area I A r e a I1 Area I11 5.6

October 4.7

5.5 5.8 5.8

a b i l i t y o f f l u o r i d e i n g e s t e d ; (4) s p e c i e s o f a n i m a l i n v o l v e d ; (5) age a t t i m e of excessive f l u o r i d e ingestion:

(6) l e v e l o f n u t r i t i o n ;

(7) f l u c t u a -

t i o n s i n ingestion levels o f f l u o r i d e ( i n t e r m i t t e n t ingestion);

(8) g e n e r a l

s t a t e o f h e a l t h o f t h e animal; (9) a d d i t i v e o r s y n e r g i s t i c e f f e c t s f r o m

123
exposure t o o t h e r substances: c a u s e d b y p o o r management:

(10) o t h e r s t r e s s f a c t o r s such as t h o s e

and ( 1 1 ) i n d i v i d u a l b i o l o g i c a l r e s p o n s e .
i t i s apparent

T h i s r e p o r t i n c l u d e s work conducted through t h e f i f t h y e a r o f a planned

10 y e a r p r o j e c t .
coal-fired

On t h e b a s i s o f r e s u l t s o b t a i n e d t h u s f a r ,

t h a t e f f e c t s on t h e h e a l t h s t a t u s o f a n i m a l s r a i s e d i n t h e v i c i n i t y o f t h e power-generation p l a n t and f e d o n v e g e t a t i o n i r r i g a t e d w i t h t h e e f f l u e n t s f r o m t h e p o w e r p l a n t has been l i m i t e d t o d e n t a l f l u o r o s i s and s l i g h t l y i n c r e a s e d f l u o r i d e c o n t e n t o f t h e bone and u r i n e . Dental l e s i o n s c h a r a c t e r i s t i c o f f l u o r i d e t o x i c o s i s were present i n t h e c a t t l e t h a t had p e r m a n e n t i n c i s o r t e e t h f o r m , t h e R e s e a r c h Farm. i n the f i r s t pair, m i n e r a l i z e and e r u p t w h i l e on The f l u o r i d e i n d u c e d d e n t a l l e s i o n s a r e u s u a l l y m o r e i n d i c a t i n g an i n c r e a s e d l e v e l o f f l u o r i d e i n t a k e a s t h e The c l a s -

s e v e r e i n t h e s e c o n d and s u b s e q u e n t p a i r s o f p e r m a n e n t i n c i s o r t e e t h t h a n c a t t l e g r e w and i n c r e a s e d t h e i r v e g e t a t i o n and w a t e r i n t a k e . sors, moderate e f f e c t s i n t h e second p a i r ,

s i f i c a t i o n o f s l i g h t d e n t a l f l u o r o s i s i n t h e f i r s t p a i r o f permanent i n c i and m a r k e d t o s e v e r e e f f e c t s i n For reference, t h e t h i r d and f o u r t h p a i r s i n d i c a t e t h a t t h e a n i m a l s r e c e i v e d m o r e t h a n t h e t h r e s h o l d l e v e l o f f l u o r i d e when t h e t e e t h w e r e f o r m i n g . animal species are given i n Table V I I . c a n be e x t r a p o l a t e d . When i n g e s t i o n o f e l e v a t e d l e v e l s o f f l u o r i d e i s s u s p e c t e d a n d s u p p o r t i v e c l i n i c a l evidence i s lacking, w i t h a d i a g n o s i s (10). urinalysis, e s t a b l i s h e d f l u o r i d e t o l e r a n c e l e v e l s i n b o t h f e e d and w a t e r f o r s e v e r a l Tolerance l e v e l s f o r o t h e r species

i f p r o p e r l y evaluated,

can a s s i s t older

S e v e r a l v a r i a b l e s m u s t be k e p t i n m i n d when e v a l u -

a t i n g u r i n e analyses f o r fluoride.

A t a given f l u o r i d e intake level,

animals w i l l v o i d more f l u o r i d e i n t h e u r i n e than w i l l younger animals. The f l u o r i d e c o n t e n t o f t h e u r i n e o f n o r m a l c a t t l e i n g e s t i n g a n o r m a l d i e t fluctuates, b u t i t i s u s u a l l y l e s s t h a n 6 ppm F. The f l u o r i d e l e v e l s i n u r i n e The a v e r a g e u r i n e f l u o r i d e c o n t e n t o f m o s t o f t h e a n i m a l s o n t h e R e s e a r c h F a r m was b e l o w t h e l e v e l o f h e a l t h c o n c e r n . and bone do, however, r e f l e c t an e l e v a t e d f l u o r i d e i n t a k e b y these animals. Urinary fluoride i s

T h i s has been c o n f i r m e d b y t h e d e n t a l c l a s s i f i c a t i o n o f c a t t l e r a i s e d o n t h e f a r m and u r i n a r y f l u o r i d e l e v e l s i n some a n i m a l s . expected. an i n d e x o f t h e e x p o s u r e t o t h i s e l e m e n t a l t h o u g h w i d e v a r i a t i o n s c a n be An i n c r e a s e i n t h e b o n e a n d u r i n e f l u o r i d e i s a l s o c o n s i s t e n t Samples

w i t h t h e i n c r e a s e d a m o u n t o f f l u o r i d e i n t h e w a t e r and v e g e t a t i o n .
M o n i t o r i n g o f a n i m a l h e a l t h w i l l c o n t i n u e o n t h i s R e s e a r c h Farm. t i c t o b i o l o g i c a l e f f e c t s o f i n g e s t e d f l u o r i d e s on c a t t l e .
w i l l be a n a l y z e d f o r s e v e r a l e l e m e n t s t h a t may b e s y n e r g i s t i c o r a n t a g o n i s -

124
TABLE V I I A G U I D E TO FLUORIDE TOLERANCE' LEVELS I N FEED AN0 WATER FOR DOMESTIC

ANIMALS~

BASED ON CLINICAL

SIGNS

AND LESIONS

Species

Cattle, Cattle, Cattle, Cattle, Sheep, Lambs, Horses

D a i r y and B e e f H e i f e r s Dairy, Mature Beef, Mature Finishing Breeding Feeder

30 40 50 100

2.5

3 4 12 12
-

a
15 8 15

60
150

5 -

60 70
100 150 50 45

45 10
-

Swine, G r o w i n g Turkeys, Growing Chickens, Growing Dogs, G r o w i n g Mink, Growing

a a
13

10 - 12 3 3 -

1 8 i o l o g i c a l e f f e c t s depend on t h e f a c t o r s m e n t i o n e d i n t h e t e x t . 'The v a l u e s s h o u l d b e r e d u c e d p r o p o r t i o n a l l y w h e n b o t h w a t e r a n d f e e d c o n t a i n a p p r e c i a b l e amounts o f f l u o r i d e s . 3 T h i s i s a s u g g e s t e d g u i d e when F i n t h e f e e d i s e s s e n t i a l l y t h e s o u r c e o f fluoride. T o l e r a n c e s b a s e d on s o d i u m f l u o r i d e o r o r o t h e r f l u o r i d e s o f s im i1a r t o x ic it y. 4The a v e r a g e a m b i e n t a i r t e m p e r a t u r e and t h e p h y s i c a l and b i o l o g i c a l a c t i v i t y o f t h e a n i m a l s i n f l u e n c e t h e a m o u n t o f w a t e r consumed and hence t h e w i d e range o f t o l e r a n c e l e v e l s suggested. For a c t i v e animals i n a warm c l i m a t e , t h e l o w e r v a l u e s s h o u l d be u s e d as c r i t i c a l l e v e l i n d i c a tors. REFERENCES

1. S h u p e JL ( 1 9 6 7 ) I n : I V t h I n t e r n M e e t i n g o f t h e W o r l d A s s o c f o r B u i atrics, P u b l No 4. Zurich, S w i t z e r l a n d , p p 15-30 H a r r i s LE,

2.

Shupe JL, M i n e r ML. V e t Res 24:964-984

Greenwood DA,

S t o d d a r d GE (1953) Amer J

3. S h u p e JL, A l t h e r EW ( 1 9 6 6 ) I n : E i c h l e r 0, F o r a n A, H e r k e n H, W e l c h AD, S m i t h FA ( e d s ) Handbook o f E x p e r i m e n t a l P h a r m a c o l o g y . S p r i n g e r - V e r l a g , New York, 20:307-354 4. S h u p e J L , O l s o n AE ( 1 9 8 4 ) I n : S h u p e J L . L e o n e NE, P e t e r s o n HB ( e d s ) F l u o r i d e s - E f f e c t s o n V e g e t a t i o n , A n i m a l s and Humans. P a r a g o n P r e s s , p p 319-338

5. Shupe J L (1970) Amer V e t P u b l , Wheaton,

Ill, pp 288-301

125 6. J o h n s o n LC ( 1 9 6 5 ) I n : S i m o n s JH ( e d ) F l u o r i n e C h e m i s t r y , V o l 4. Acad e m i c Press, New York, pp 424-441 7. M i l l e r GW, Shupe JL (1962) Amer J V e t Res 23(92):24-31 8. Shupe JL (1961) Can V e t J 2 3 6 9 - 3 7 6 9. A A F C O ( 1 9 8 2 ) O f f i c i a l P u b l i c a t i o n . Assoc Amer Feed C o n t r o l O f f i c i a l s , I n c , C h a r l e s t o n , W Va 10. Shupe JL. H a r r i s LE, Greenwood DA, B u t c h e r JE, N i e l s o n HM (1963) Amer J V e t Res 24:300-30

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 127-134 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

127

DENTAL LESIONS I N CATTLE AND SHEEP DUE TO INDUSTRIAL POLLUTION CAUSED BY COAL COMBUSTION

FRANKLIN

RIET-CORREA~,

MARIA

c.

M ~ N D E Z ~ ANA , L. SCHILD~.

JOXO A.

OLIVEIRA~,
Ufpel,

AND O D A I R ZENEBON'
' L a b o r a t b r i o R e g i o n a l De D i a g n 6 s t i c o . 96100 P e l o t a s , RS., 5 r a z i l F a c u l d a d e De V e t e r i n s r i a . Brazil

'Institute
ABSTRACT

A d o l f o Lutz,

Szo P a u l o , SP.,

D e n t a l l e s i o n s i n c a t t l e w e r e s t u d i e d i n 11 f a r m s l o c a t e d w i t h i n 1.2 t o 9.6 km f r o m a c o a l c o m b u s t i o n t h e r m o e l e c t r i c p l a n t . c a t t l e showed e v i d e n c e o f c h r o n i c f l u o r i d e p o i s o n i n g . a l t e r a t i o n was d e n t a l a t t r i t i o n . complete a t t r i t i o n o f i n c i s o r s a t 6 t o 7 years. f u n c t i o n y = 4.11 ppm. I n c i s o r t e e t h o f some The m o s t i m p o r t a n t

C a t t l e f r o m f a r m s n e a r t h e p l a n t had The d e g r e e o f wear was Sheep had s i m i l a r t h e abra-

r e l a t e d t o t h e d i s t a n c e between t h e f a r m s and t h e p l a n t as a l i n e a r

(-0.42~);

r 2 = 0.75

(P<0.05).

lesions t o those o f cattle.

Bone f l u o r i d e l e v e l s w e r e f r o m 265 t o 5673 emitted into the

Two m e c h a n i s m s seem t o b e r e s p o n s i b l e f o r d e n t a l l e s i o n s :

sive ei'fect o f p a r t i c u l a t e matter, especially s i l i c a , atmosphere,

and t h e e f f e c t o f f l u o r i d e d e c r e a s i n c t h e e n a m e l r e s i s t a n c e .

INTRODUCTION F l u o r i d e i n t o x i c a t i o n has been r e c e n t l y d e s c r i b 2 d i n B r a z i l a s a conseq u e n c e o f i n d u s t r i a l p o l l u t i o n c a u s e d b y p h o s p h a t e F r o c e s s i n g p l a n t s (1). I n a n a r e a w i t h i n t h e m u n i c i p a l i t y o f Bag&. s t a t e o f R i o G r a n d e d o S u l , where a c o a l combustion e l e c t r i c power p l a n t i s located, animals. many f a r m e r s c l a i m e d t h a t a t m o s p h e r i c e m i s s i o n o f t h i s p l a n t caused d e n t a l a t t r i t i o n i n The o b j e c t i v e o f t h i s w o r k was t o s t u d y d e n t a l l e s i o n s i n c a t t l e and sheep f r o m t h e area.

MATERIALS AND METHODS D e n t a l l e s i o n s were s t u d i e d i n 11 f a r m s l o c a t e d a t d i f f e r e n t d i s t a n c e s and d i r e c t i o n s f r o m t h e t h e r m o e l e c t r i c p l a n t . making b l a c k and w h i t e p r i n t s .

A t each farm,

incisor teeth

o f a v a r i a b l e n u m b e r o f c a t t l e ( T a b l e I) were observed and photographed, The d e g r e e o f l e s i o n o f e a c h i n c i s o r was

TABLE I EFFECT OF INDUSTRIAL POLLUTION ON I N C I S O R LESION AND ATTRITION Farma Number o f cattle observed Distance from p l a n t (krn) min. max. X

OF

CATTLE Mean d e g r e e o f lesion Mean d e g r e e of a t t r i t i o n ( X ? SX)

Number o f i n c i s o r t e e t h a f f e c t e d w i t h each d e g r e e o f l e s i o n 0 1 2 3 4

EF RM TA

34

1.2 1.4 0.8 3.3 3.3 4.5 4.5 5.5 6.4 7.6

3.7 4.8 5.4 8.0 6.8 6.8 9.0 9.6 9.5 9.4

2.45 3.1 3.1 5.65 5.05 5.65 6.75 7.55 7.95 8.5

80 171 45 120 85 216 200 140 147 179

12 59 14 47 16 34 70
18

11 13 1 3
1

1 1
0

37
26 30 30 38 37 21 35 28

6 0 0

0.55 0.36 0.27 0.31 0.18 0.17 0.26 0.13 0.23 0.25

3 . 4 8 f 0.27 2.24
f

0.21

_--

DX EC
GL

0 0 0
0
1

0
0

141 k 0.22

---

5
0

0
0

224 f 0 . 1 8 170 k 0.18

ES

PF
JM
EL

38 32

0 3
9

0 2

0 0 0

56

0.26

The d e g r e e o f l e s i o n o f i n c i s o r t e e t h f r o m f a r m JE t h e Dhotos o b t a i n e d

was n o t d e t e r m i n e d due t o t h e low q u a l i t y

Of

c l a s s i f i e d i n t o t h e f o l l o w i n g s c o r e s : 0) w i t h o u t l e s i o n s ; 1 ) s l i g h t m o t t l i n g o f e n a m e l ; 2) d e f i n i t e m o t t l i n g ; 3) d e f i n i t e m o t t l i n g . hypocalcification: and i n c r e a s e d w e a r when c o m p a r e d w i t h a d j a c e n t i n c i s o r s . p l e t e wear were n o t considered. h y p o p l a s i a and T e e t h w i t h comThe

4) d e f i n i t e m o t t l i n g , hypoplasia o r hypocalcification
The mean s c o r e o f a l l p e r m a n e n t i n c i s o r

t e e t h o b s e r v e d i n e a c h f a r m was d e s i g n e d as mean d e g r e e o f l e s i o n . studied by regression analysis. Dental

mean d e g r e e o f l e s i o n i n r e l a t i o n t o t h e d i s t a n c e f r o m t h e p l a n t was a t t r i t i o n o f i n c i s o r t e e t h was s t u d i e d i n 6 f a r m s w i t h r e c o r d s of The age c a l c u l a t e d b y i n c i s o r s w e a r was d e t e r m i n e d b y The d i f f e r e n c e ( i n y e a r s ) b e t w e e n t h e age

a n i m a l ages.

c o m p a r i s o n w i t h b l a c k and w h i t e p r i n t s f r o m n o r m a l c a t t l e b e l o n g i n g t o t h e Pelotas U n i v e r s i t y farm. c a l c u l a t e d b y t h e wear, tion. a n d t h e r e a l age was d e s i g n a t e d a s d e g r e e o f a t t r i i n relation t o the

The mean d e g r e e o f a t t r i t i o n i n e a c h f a r m ,

d i s t a n c e f r o m t h e p l a n t , was s t u d i e d b y r e g r e s s i o n a n a l y s i s . L e s i o n s i n m o l a r and p r e m o l a r t e e t h w e r e o b s e r v e d i n 20 s l a u g h t e r e d cows. I n c i s o r t e e t h f r o m t h e s e a n i m a l s were f i x e d i n lox f o r m a l i n e , w i t h f o r m i c a c i d and s o d i u m c i t r a t e and embedded i n p a r a f f i n . w e r e s t a i n e d w i t h h e m a t o x y l i n and e o s i n . Humerus and m a n d i b l e s w e r e c o l l e c t e d f r o m s l a u g h t e r h o u s e s o r f r o m a n i m a l s w h i c h d i e d f r o m d i f f e r e n t causes. F l u o r i d e l e v e l s i n bone ashes w e r e determined w i t h t h e f l u o r i d e i o n electrode. D e n t a l l e s i o n s i n sheep w e r e s t u d i e d i n 20 a n i m a l s o f d i f f e r e n t a g e s f r o m f a r m s RM, decalcified Sections

E F a n d JE.

The f a r m s w e r e c o d e d u s i n g t h e i n i t i a l s o f t h e

o w n e r ' s names. RESULTS I n c i s o r t e e t h o f some c a t t l e showed c h a l k y w h i t e , c o l o r a t i o n and h y p o p l a s i a o f enamel. y e l l o w o r brown d i s The number of Such l e s i o n s w e r e c o n s i d e r a b l e i n t h e degree o f l e s i o n

t h o s e f a r m s n e a r t h e p l a n t and d i s c r e t e i n t h e o t h e r s . c a t t l e and p e r m a n e n t i n c i s o r s o b s e r v e d i n e a c h f a r m , o f individual i n c i s o r teeth, s h o w n i n F i g u r e 1. f r o m t h e p l a n t a r e s h o w n i n T a b l e I.

t h e mean d e g r e e o f l e s i o n a n d t h e d i s t a n c e Farms and p l a n t l o c a l i z a t i o n a r e

R e g r e s s i o n a n a l y s i s o f t h e d e g r e e o f l e s i o n as a Regression a n a l y s i s

f u n c t i o n o f t h e d i s t a n c e f r o m t h e p l a n t i s p r e s e n t e d i n F i g u r e 2. D e n t a l a t t r i t i o n was t h e m o s t i m p o r t a n t a l t e r a t i o n . p r e s e n t e d i n F i g u r e 3. incisors, o f t h e d e g r e e o f wear as a f u n c t i o n o f t h e d i s t a n c e f r o m t h e p l a n t i s T e e t h o f many a n i m a l s w i t h 8 p e r m a n e n t i n c i s o r s I n some a n i m a l s w i t h 4 o r 6 p e r m a n e n t w e r e w o r n t o gum l e v e l ( F i g . 4).

t h e 1, was s h o r t e r t h a n o t h e r s a s a c o n s e q u e n c e o f w e a r ( F i g .

5).

130

F i g u r e 1. D r a w i n g o f t h e a r e a s t u d i e d s h o w i n g t h e f a r m s and p l a n t (UTPM) l o c a l i z a t i o n . T h e t o t a l s u r f a c e o f t h e 1 1 f a r m s i s a p p r o x i m a t e l y 11.500 h e c t a r e s and t h e w h o l e a r e a 30.000 h e c t a r e s .

Mean d e g r e e o f t o o t h l e s i o n i n e a c h f a r m ( y a s a f u n c t i o n o f F i g u r e 2. y = 0 . 4 7 1 7 + ( - 0 . 0 3 6 ~ ) ; r 4 . 4 2 (PcO.05). t h e d i s t a n c e f r o m t h e p l a n t (x).

131

DISTANCE

, m m

f i g u r e 4. S e v e n - y e a r - o l d cow f r o m f a r m TA. I n c i s o r s a r e w o r n t o gum l e v e l showing c o n s i d e r a b l e amounts o f secondary d e n t i n e . Severe g i n g i v a l hyperplasia.

I n many a n i m a l s p e r m a n e n t i n c i s o r s s h o w e d v a r i a b l e a m o u n t s o f s e c o n d a r y d e n t i n w h i c h a p p e a r e d d a r k b r o w n o r b l a c k ( F i g . 4); c a v i t y was exposed. h y p e r p l a s i a ( F i g . 4-5). teeth. H i s t o l o g i c a l l e s i o n s o f p e r m a n e n t i n c i s o r t e e t h showed h y p e r p l a s i a o f t h e c e m e n t and p r o l i f e r a t i o n o f s e c o n d a r y d e n t i n . I n many t e e t h , t h i s secondi n others the pulp The m a j o r i t y o f a n i m a l s showed c o n s i d e r a b l e g i n g i v a l D e c i d u o u s i n c i s o r s a l s o showed e x c e s s i v e wear.

E x c e s s i v e and uneven a t t r i t i o n was a l s o o b s e r v e d i n m o l a r and p r e m o l a r

132
a r y d e n t i n had m o r e w e a r t h a n p r i m a r y d e n t i n and e n a m e l , c a v i t y impacted w i t h d e t r i t u s . l e a v i n g an e m p t y The

I n o t h e r s t h e p u l p c a v i t y was exposed.

g i n g i v a showed m a r k e d h y p e r p l a s i a o f t h e s t r a t i f i e d squamous e p i t h e l i u m .

T h r e e - y e a r - o l d cow f r o m f a r m RM. Central incisors are shorter F i g u r e 5. Gingival hyperplasia. t h a n I 2 a s a c o n s e q u e n c e o f wear.

TABLE I 1 FLUORIDE LEVELS I N BOVINE BONE ASHES FROM DIFFERENT FARMS Farm Age F l u o r i d e (ppm) Humerus Mandible

DX
PF

5.65 7.55 6.75 3.10 5.65 4.30 2.45

adults

559-491-475 41 3-21 Oa 265 594 1937 681 832 5673-3966 3680-31 56 1526-1 502 11 72-1 091 1081 702-533-508 490-461 954-772 1317 2931 -2580-2554 2244-2087-1 990 1852-1 770-1 476 1281-975-849 81 0-747-733 714-667-528 499-435-389 386

3 years 4 years 3 years

adults adults adult

ES
RM
GL

JE
EF

1-7 y e a r s

aEach v a l u e i s f r o m a d i f f e r e n t a n i m a l .

133
T e e t h o f n e a r l y a l l s h e e p w i t h 8 p e r m a n e n t i n c i s o r s w e r e w o r n t o gum level. Wear was a l s o e x c e s s i v e i n a n i m a l s w i t h 2 p e r m a n e n t i n c i s o r s : in sheep w i t h 4 o r 6 p e r m a n e n t i n c i s o r s t h e I 1 was s h o r t e r t h a n t h e o t h e r s . R e s u l t s o f f l u o r i d e d e t e r m i n a t i o n s i n bone a s h e s a r e shown i n T a b l e 11.

DISCUSSION
The r e s u l t s o f t h i s w o r k e s t a b l i s h e d t h e d i a g n o s i s o f f l u o r i d e p o i s o n i n g as a c o n s e q u e n c e o f c o a l c o m b u s t i o n ; otherwise it i s clear t h a t fluoride i s Dental lesions o f t h e most severe T o x i c l e v e l s o f f l u o r i d e were M o s t i n c i s o r s showed which can not the only f a c t o r responsible f o r the occurrence o f dental lesions i n c a t t l e and sheep i n t h e a r e a s t u d i e d . degree, s u c h as t h o s e s c o r i n g 3 o r 4 i n enamel h y p o p l a s i a , w e r e o b s e r v e d m a i n l y i n t h e farms l o c a t e d near t h e plant. d e t e c t e d i n bone a s h e s i n a n i m a l s f r o m t h e s e f a r m s . be o b s e r v e d i n a n i m a l s n o t e x p o s e d t o f l u o r i d e . distance from the plant. F l u o r i d e i n t o x i c a t i o n was d e s c r i b e d i n a p r e v i o u s p a p e r i n t h e m u n i c i p a l i t y o f R i o G r a n d e (1).

l e s i o n s w i t h s c o r e 1 , r e p r e s e n t e d b y s l i g h t m o t t l i n g o f enamel,

T h i s i s confirmed by t h e

l o w c o e f f i c i e n t o f i n d e t e r m i n a t i o n b e t w e e n t h e d e g r e e o f l e s i o n s and t h e

Comparing t h e a l t e r a t i o n s observed i n b o t h munici-

palities,

we c a n c o n c l u d e t h a t d e n t a l l e s i o n s c h a r a c t e r i s t i c o f f l u o r o s i s t h a n i n R i o Grande where t h e d i s e a s e o c c u r r e d as a I n R i o Grande, i n a farm t h e m e a n d e g r e e o f l e s i o n w a s 1.99,

w e r e l e s s m a r k e d a n d f l u o r i d e l e v e l s w e r e l o w e r i n Bag6 a s a c o n s e q u e n c e o f coal combustion, consequence o f rock phosphate processing.

s i t u a t e d 5.5 km f r o m t h e f a c t o r i e s ,

w h i l e i n B a g e i n a f a r m l o c a t e d 2.4 km f r o m t h e p l a n t t h e mean d e g r e e was

0.55.
Grande.

O t h e r w i s e d e n t a l a t t r i t i o n was m o r e m a r k e d i n Bag& t h a n i n R i o i n d i c a t i n g t h a t f l u o r i d e p o i s o n i n g i s n o t t h e u n i q u e cause o f t e e t h t h e amounts o f p a r t i c u as t h e p l a n t w i t h a precipi-

l e s i o n s due t o i n d u s t r i a l p o l l u t i o n caused by c o a l combustion. U n t i l O c t o b e r 1984 when t h i s w o r k was f i n i s h e d , l a t e s e m i t t e d i n t o t h e atmosphere were very high, t a t o r s o f v e r y l o w e f f i c i e n c y (0% t o 50%).

c a p a c i t y o f 126 m e g a v o l t s was e q u i p p e d w i t h o n l y 2 e l e c t r o s t a t i c

Taking i n t o account a d a i l y

c o n s u m p t i o n o f a p p r o x i m a t e l y 1 8 6 7 t o n s o f c o a l , c o n t a i n i n g 52% t o 5 9 % o f a s h e s , t h e p r o d u c t i o n o f p a r t i c u l a t e s w a s a b o u t 855 t o n s d a i l y , o f w h i c h

40% t o 80% w e r e e l i m i n a t e d w i t h t h e f l u e g a s ( S e c r e t a r y o f H e a l t h , E n v i r o n mental Department, R i o Grande do Sul.

1985.

Unpublished data).

Such Unpub-

p a r t i c u l a t e s w e r e composed m a i n l y o f S i O 2 (70%), A1203 (21.4%) and Fez03

(4.1%) ( A n d r a d e A.
lished data).

1985. U n i v e r s i d a d e F e d e r a l d o R i o G r a n d e d o Sul.

Considering t h e abrasive e f f e c t o f these elements deposited i n considera b l e amounts on pastures,

it i s evident that a t least two factors are

134
r e s p o n s i b l e f o r t o o t h wear: a b r a s i o n caused b y t h e f l y a s h d u r i n g g r a z i n g mainly

and c h e w i n g and t h e e f f e c t o f f l u o r i d e d e c r e a s i n g t h e enamel r e s i s t a n c e . A b r a s i o n b y p h y s i c a l a g e n t s i s a w e l l known mechanism o f t o o t h wear, i n sheep i n w h i c h t h e i n g e s t i o n o f s o i l , i n feces, i s r e l a t e d t o t o o t h a t t r i t i o n (2.3). e s t i m a t e d b y t h e amount o f s i l i c a The p o s s i b i l i t y o f o t h e r

elements contained i n t h e p a r t i c u l a t e c o n t r i b u t i n g t o t h e e t i o l o g y o f t e e t h l e s i o n s c a n n o t be e x c l u d e d . D e n t a l l e s i o n s o f c a t t l e and sheep d e s c r i b e d i n t h i s p a p e r w e r e c e r t a i n l y an i m p o r t a n t c a u s e o f e c o n o m i c l o s s e s t o t h e b e e f and w o o l i n d u s t r y o f t h e area studied. formed. fortunately, i n O c t o b e r 1 9 8 4 t h e p l a n t i n s t a l l e d t w o new t h u s l e a v i n g o n l y 7.6% i n e l e c t r o s t a t i c p r e c i p i t a t o r s a b l e t o r e m o v e 99.4% o f t h e p a r t i c u l a t e s They a l s o remove 91.3% o f t h e f l u o r i d e , t h e f l u e g a s (4). For these reasons i t i s expected t h a t t h e seriousness o f

d e n t a l l e s i o n s w i l l decrease. ACKNOWLEDGEMENTS T h i s w o r k was s u p p o r t e d b y EMBRAPA/UEPAE/Pelotas. REFERENCES 1. R i e t - C o r r e a F, O l i v e i r a JA. M6ndez MC, S c h i l d AL ( 1 9 8 3 ) P e s q V e t B r a s 3: 107-114 A r n o l d GW, McManus WR, 107 H e a l y WB, Page AL, E l s e e w i AA. Bush I G (1966) A u s t J Exp A g r i c Anim Husb 6 : l O l -

2.
3. 4.

L u d w i g TG (1965) N Z J A g r i c Res 8:737-752 S t r a u g h a n I R (1979) R e s i d u e R e v i e w s 71:83-120

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985,Studies in Environmental Science, Volume 27, pp. 135-141 0 1986 Elsevier Science Publishers B.V.. Amsterdam - Printed in The Netherlands

135

HEALTH EFFECTS NETHERLANDS

OF A H Y D R O G E N

FLUORIDE E M I T T I N G GLASS F I B R E PLANT I N T H E

KLAAS BIERSTEKER AND JAN S. M.

BOLEIJ
University, Gen.

Department o f Environmental Health, Agricultural F o u l k e s w e g 43. 6703 BM Wageningen, The N e t h e r l a n d s

ABSTRACT The h e a l t h e f f e c t s o f a g l a s s f i b r e p l a n t , studied i n a v i l l a g e close t o the plant. d i s t a n c e served as c o n t r o l i n t h e study. e m i t t i n g 12-15 k g HF/hr. were

A v i l l a g e u p w i n d a t 1 5 km

The s o c i o e c o n o m i c d a t a o f b o t h

p o p u l a t i o n s i n d i c a t e d good c o m p a r a b i l i t y e x c e p t f o r a h i g h e r l e v e l o f environmental concern i n t h e p o l l u t e d v i l l a g e . The c o l l e c t e d d a t a showed t h a t l o c a l l y g r o w n v e g e t a b l e s had a h i g h e r f l u o r i d e c o n t e n t i n t h e exposed v i l l a g e . only slightly, however, l o c a l vegetables. Lung f u n c t i o n s f o r 40 d a y s , o f 20 s c h o o l c h i l d r e n i n b o t h v i l l a g e s w e r e t e s t e d d a i l y t o study There were a few days w i t h b u t no e f f e c t on lung T h i s d i f f e r e n c e may be a f t e r t h e c h i l d r e n had been i n t h e o u t d o o r a i r , Urine concentrations differed p r o b a b l y as a r e s u l t o f l i m i t e d consumption o f t h e

p o t e n t i a l acute e f f e c t s o f a i r pollution. f u n c t i o n was observed.

increased p o l l u t i o n l e v e l i n t h e exposed v i l l a g e , o f pulmonary symptoms t h a n t h a t o f t h e c o n t r o l s . e x p o s e d v i 1 1 age.

The e x p o s e d v i l l a g e h a d m o r e p e o p l e w i t h a h i s t o r y

due t o t h e h i g h e r degree o f environmental concern o f t h e people i n t h e The s t u d e n t s who p a r t i c i p a t e d i n t h e s t u d y consumed a m e a l o f l o c a l l y g r o w n k a l e and c o l l e c t e d t h e i r u r i n e f o l l o w i n g t h e meal. l e v e l s o f t h e k a l e r e a c h e d 100 ppm. rapid resorption, b u t stayed below danger l e v e l . The f l u o r i d e Urinary content d i d not contradict the

INTRODUCTION T h e r e was c o n c e r n a b o u t r e s p i r a t o r y e f f e c t s and q u a l i t y o f l o c a l l y g r o w n vegetables i n a v i l l a g e located close t o a glass f i b r e p l a n t i n the north of t h e Netherlands. The g l a s s f i b r e p l a n t ( 8 0 0 employees, 28000 t o n s p r o d u c t i o n a n n u a l l y ) u s e d CaF2 i n t h e g l a s s o v e n s t o i m p r o v e t h e q u a l i t y o f the fibres.

It was assumed t h a t ,

due t o t h e h i g h t e m p e r a t u r e ,

HF w a s NO,).

e m i t t e d as gas t o g e t h e r w i t h t h e e m i s s i o n s o f t h e oven b u r n e r s (SO2,

136
T h e r e w e r e s i x c h i m n e y s , 30 m h i g h t o d i s p e r s e t h e e m i s s i o n s . p l a n t d i r e c t o r , b e t w e e n 12-15 k g p e r h o u r . The p l a n t

w a s i n c o n t i n u o u s o p e r a t i o n a n d t h e HF e m i s s i o n v a r i e d , a c c o r d i n g t o t h e Concern about h e a l t h e f f e c t s who a d v i s e d p e o p l e n o t This advice was s t i m u l a t e d b y t h e p r o v i n c i a l h e a l t h i n s p e c t o r ,

t o consume m o r e t h a n t w i c e a week l o c a l l y g r o w n v e g e t a b l e s . f l u o r i d e content. I n an e a r l i e r s t u d y i n t h e s o u t h o f t h e N e t h e r l a n d s (1,Z) l i v i n g c l o s e t o an a l u m i n u m s m e l t e r .

was b a s e d o n a s m a l l s a m p l e o f v e g e t a b l e s t h a t had been a n a l y z e d f o r u r i n e had been

used f o r b i o l o g i c a l m o n i t o r i n g o f t h e f l u o r i d e exposure o f school c h i l d r e n The f l u o r i d e e m i s s i o n s t h e r e w e r e i n d u s t f o r m and i t was d e m o n s t r a t e d t h a t p r o p e r w a s h i n g o f t h e v e g e t a b l e s b e f o r e c o o k i n g removed most o f t h e f l u o r i d e s . Compared w i t h o t h e r f l u o r i d e compounds, t o t h e s k i n and mucosa. i n t h e e x p o s e d v i 1 1age.

HF i s known t o be v e r y a b r a s i v e

A s e c o n d p o i n t o f i n t e r e s t was t h e r e f o r e w h e t h e r

an e f f e c t on t h e r e s p i r a t o r y t r a c t o f s c h o o l c h i l d r e n c o u l d be d e m o n s t r a t e d

A group o f environmental
c a r r i e d o u t t h e f i e l d work.

h y g i e n e s t u d e n t s d e v e l o p e d a s t u d y d e s i g n and T h i s paper i s a condensed v e r s i o n o f t h e f i n a l

r e p o r t t h a t was s u b m i t t e d t o t h e l o c a l and p r o v i n c i a l h e a l t h a u t h o r i t i e s , t h e p l a n t d i r e c t o r and t h e p o p u l a t i o n o f t h e v i l l a g e W e s t e r b r o e k i n 1978. MATERIALS AND METHODS Design o f t h e study The s t u d y a t t e m p t e d t o answer t w o q u e s t i o n s : a. b.

Is t h e r e a demonstrable e f f e c t on pulmonary h e a l t h i n t h e v i l l a g e ?
Is t h e r e a d e n t a l h e a l t h r i s k i n c o n s u m i n g l o c a l v e g e t a b l e s ?
l u n g f u n c t i o n t e s t s o f 20 c h i l d r e n w e r e

To a n s w e r t h e f i r s t q u e s t i o n ,

s t u d i e d d a i l y on s c h o o l g o i n g d a y s i n t h e m o r n i n g a f t e r p l a y t i m e o u t d o o r s i n W e s t e r b r o e k a n d i n a c o n t r o l v i l l a g e l o c a t e d 1 5 km f r o m t h e p l a n t a n d s o c i o e c o n o m i c a l l y comparable. schools, I n addition the parents o f schoolchildren d i s t r i b u t e d weekly by the two were asked t o r e g i s t e r on s p e c i a l forms, p a s t week. To a n s w e r t h e s e c o n d q u e s t i o n v e g e t a b l e s w e r e c o l l e c t e d t h r e e t i m e s and analyzed f o r f l u o r i d e content.

w h e t h e r t h e r e had b e e n r e s p i r a t o r y s y m p t o m s i n t h e f a m i l y i n t h e

A l s o t h e u r i n e o f s c h o o l c h i l d r e n was anat h e students organized two s i m i l a r t o register

lyzed f o r f l u o r i d e content. l o o k i n g meals, The i m m i s s i o n

Finally,

o n e w i t h p o l l u t e d k a l e and one w i t h c l e a n k a l e ,

t h e e f f e c t on e x c r e t i o n o f f l u o r i d e s w i t h urine. F l u o r i d e concentrations c l o s e t o t h e p l a n t were determined several t i m e s and i t was d e m o n s t r a t e d by sampling separately f o r particulate fluorides t h a t p r a c t i c a l l y a l l o f t h e f l u o r i d e was p r e s e n t a s g a s ( T a b l e I).

137 TABLE I
PRESENCE OF FLUORIDES I N DOUBLE FILTER SAMPLES I N EXPOSED VILLAGE (4 HOUR AVERAGES )

time

particJes

w/mJ

21 21-22 25 31 36 37 38 38

13.00-1 7.00 22.00- 2.00 5.00- 9.00 5.00- 9.00 13.00-1 7.00 5.00- 9.00 5.00- 9.00 13.00-17.00

16.3 3.5 3.4 3.0 4.3 16.7 6.0 13.6

0 0.5 0 0 0 0 0 0.1

TABLE I 1 FREQUENCY OF FLUORIDE AN0 SO2 CONCENTRATIONS AT THE PLAYGROUNDS OF THE SCHOOLS
F Iuorjde ( i d m )

Exposed village A 37 0 1 (+) 1 () 39 Exposed village A 26 6 4 1 0 3 40

Control village B 37 1 0 0 average A: average B: (+)A-day 21: (+)A-day 24: 0.40 ug/m3 0.12ug/rn3 8.50 ugF-/m3 2.30 vgF-/m3

<1 1-2 2-3 >3 Total SO2 (w/m3)

38
Control village B 22 9 8 1 1 0 41 average A: 18.35 ug/m3 3 vg/m pgS02/m3 vgSO2/m; ugS02/m

<I0 10-30 30-50 50-70 70-90 >90 Total

(1

average B: 16.13 ()A-day 7: 107.00 day 21: 96.00 day 29: 232.00

138

SO2

and HF w e r e m e a s u r e d a s 1 h o u r a v e r a g e s a t t h e p l a y g r o u n d o f t h e As s h o w n i n T a b l e I 1 t h e r e w e r e o n l y t w o d a y s w i t h

s c h o o l e a c h m o r n i n g when t h e c h i l d r e n p e r f o r m e d l u n g f u n c t i o n t e s t s a f t e r p l a y t i m e outdoors. e l e v a t e d HF c o n c e n t r a t i o n s and t h r e e d a y s w i t h e l e v a t e d a t t h e exposed school. RESULTS AND D I S C U S S I O N E f f e c t s on l u n q f u n c t i o n Both schools provided

SO2

concentrations

20

c h i l d r e n who w e r e w i l l i n g t o p a r t i c i p a t e i n t h e The

l u n g f u n c t i o n t e s t i n g t h a t t o o k p l a c e e a c h d a y a t a b o u t 1O:OO a.m.

t e s t s w e r e c o n d u c t e d w i t h a w e t Lode s p i r o m e t e r and a pneumotachograph. The d a t a w e r e f e d i n t o a c o m p u t e r and p r i n t o u t s f o r e a c h c h i l d and each g r o u p o f c h i l d r e n w e r e a v a i l a b l e a t t h e end o f t h e s t u d y . F o r a n a l y s i s o f an e f f e c t on l u n g f u n c t i o n s , d a y s 21 and 24 w e r e c o m p a r e d w i t h s u r r o u n d i n g d a y s and a l s o w i t h d a y s w i t h s i m i l a r w e a t h e r c o n d i t i o n s , b u t w i t h o u t HF i m m i s s i o n s . B o t h t y p e s o f s t a t i s t i c a l a n a l y s i s f a i l e d t o d e m o n s t r a t e a l o w e r i n g o f l u n g f u n c t i o n s o n t h e p o l l u t e d days. d a y w i t h an i n c r e a s e d SO2 c o n c e n t r a t i o n o f ZOO+ p g S02/m3 A m u l t i p l e r e g r e s s i o n a n a l y s i s f o r a l l days, temperature, villages, r e l a t i v e humidity, v a r i a b l e s demonstrated e f f e c t s o f time, R e s p i r a t o r y symptoms The p a r e n t s of a l l s c h o o l c h i l d r e n w e r e a s k e d w e e k l y t o l i s t r e s p i r a t o r y s y m p t o m s t h a t h a d o c c u r r e d i n t h e f a m i l y o n a f o r m w i t h q u e s t i o n s s u c h as: d u r i n g t h e l a s t week, w h i c h members o f t h e f a m i l y had a f e v e r , a r u n n i n g nose, a sore throat, etc. T a b l e 111 shows t h e p e r c e n t a g e o f p a r t i c i p a n t s p e r i o d . T h e r e w e r e 316 Westerbroek Whether t h i s i n b o t h v i l l a g e s who had s y m p t o m s d u r i n g a 12-week The s i n g l e also failed t o

d e m o n s t r a t e an e f f e c t o n l u n g f u n c t i o n p a r a m e t e r s o f t h e s c h o o l c h i l d r e n . u s i n g number o f t h e day, r u n n i n g nose, SO2 and HF as i n d e p e n d e n t t e m p e r a t u r e and common c o l d i n b o t h

b u t no e f f e c t o f t h e t w o a i r p o l l u t a n t s i n t h e e x p o s e d v i l l a g e .

r e s p o n d e n t s i n W e s t e r b r o e k and 2 2 6 i n t h e c o n t r o l v i l l a g e . showed a h i g h e r p e r c e n t a g e w i t h symptoms and d o c t o r v i s i t s . population o r t o a i r p o l l u t i o n i s hard t o prove.

i s due t o g r e a t e r c o n c e r n a b o u t h e a l t h i n W e s t e r b r o e k t h a n i n t h e c o n t r o l An a t t e m p t t o c o r r e l a t e w e e k l y a v e r a g e s o f HF p o l l u t i o n l e v e l s w i t h w e e k l y p r e v a l e n c e s o f symptoms d i d n o t s h o w s i g n i f i c a n t r e l a t i o n s . When t h e s c h o o l c h i l d r e n u n d e r w e n t a p h y s i c a l examination a t t h e beginning o f t h e study, The r e a s o n f o r t h e d i f f e r e n c e s i s n o t c l e a r . r e p o r t i n g o f symptoms.

i t was e v i d e n t t h a t

W e s t e r b r o e k a l s o had m o r e c h i l d r e n w i t h a h i s t o r y o f r e s p i r a t o r y d i s e a s e . T h e r e was m o r e e n v i r o n m e n t a l c o n c e r n i n t h e e x p o s e d v i l l a g e and t h i s may h a v e i n f l u e n c e d t h e

139
TABLE 111 REPORTED RESPIRATORY SYMPTOMS I N THE TWO VILLAGES (AVERAGE RESPONDENTS) Exposed Running nose Fever Cough Sore t h r o a t Doctors v i s i t Absent from school o r from work PERCENTAGE

OF

Control

a. 3
1.2 8.6
4.5

9.1 7.9 2.9 1.2 0.8

0.5

1.3 1.5

Fluoride i n urine o f schoolchildren The p e r c e n t a g e o f s c h o o l c h i l d r e n c o n s u m i n g v e g e t a b l e s f r o m t h e i r own g a r d e n was l o w e r i n W e s t e r b r o e k t h a n i n t h e c o n t r o l v i l l a g e ( T a b l e I V ) . The u s e o f f l u o r i d e t a b l e t s b y t h e c h i l d r e n was l e s s t h a n 1 0 p e r c e n t . Compared w i t h t h e c h i l d r e n u s i n g f l u o r i d a t e d a r i n k i n g w a t e r i n t h e N e t h e r lands,

the l e v e l s w e r e low, and t h e r e were no s i g n i f i c a n t d i f f e r e n c e s

The absence o f d i f f e r e n c e s i s p r o b a b l y due t o l o c a l vegetables containing elevated f l u o r i d e

between t h e t w o v i l l a g e s . t h e i r r e g u l a r consumption of levels.

V e g e t a b l e s w e r e c o l l e c t e d t h r e e t i m e s i n t h e e x p o s e d v i l l a g e and i n t h e control village. K a l e as a w i n t e r v e g e t a b l e was c h o s e n a s a good c o l l e c t o r

TABLE I V FLUORIDE CONCENTRATION ( P P M )

I N URINE

OF

THE SCHOOLCHILDREN

Use o f t o o t h p a s t e o r t a b l e t s w i t h FYes Period Exposed: Nov. March June Oct. Number Average Number No Average

1976 1977 1977 1977

za

23 20 34

0.41 0.40 0.41

0.45

37 39 36 39

0.38 0.37 0.31 0.31

Control : Nov. 1976 M a r c h 1977 J u n e 1977 Oct. 1977

17 20
34

15

0.45 0.53

0.44 0.32

19 18 19 15

0.31

0.30 0.28
0.31

140
TABLE V FLUORIDE CONTENT

SOURCE
Rank O r d e r distance

OF

KALE I N WESTERBROEK WITH INCREASING DISTANCE FROM THE

November unwashed washed 81 20 21 20 17 81

-

March unwashed washed 83

-

1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 Control v i l l a g e 1 2 3 4

58

14 14 10 54

16

66

39 37 53 56 27 40 12

39 30 43 32 21 34 8

28 53 32 93 61 58 86 60
-

17 43 23 71 48 42 49
-

50

44 52 38 31

41 31 34 28

6 6 7 6 7

6 4 4 7

5 6 4 4 8

6
6 4 4

o f f l u o r i d e s i n e a r l i e r studies. g i v e n i n T a b l e V. s m e l t e r (2), The k a l e e x p e r i m e n t During t h e i r stay, kale,

The d a t a f o r t w o o f t h e k a l e s a m p l e s a r e

I n c o n t r a s t w i t h e a r l i e r s t u d i e s around an aluminum

washing d i d n o t remove most o f t h e f l u o r i d e . t h e s t u d e n t s consumed a k a l e meal t w i c e ( a m i x o f once p r e p a r e d w i t h u n p o l l u t e d k a l e and once T h e i r u r i n e was c o l l e c t e d a t Table V I gives t h e f l u o The k a l e

p o t a t o e s and meat),

prepared w i t h l o c a l l y grown p o l l u t e d kale. r i d e c o n c e n t r a t i o n s f o r b o t h occasions.

r e g u l a r i n t e r v a l s f o r 24 h o u r s a f t e r t h e meals.

It i s e v i d e n t t h a t t h e k a l e i n t h e
the nonpolluted kale

exposed v i l l a g e s contained b i o l o g i c a l l y a v a i l a b l e f l u o r i d e s . c o n t e n t was 1 6 ppm i n f r e s h k a l e ( 9 4 ppm i n d r y r e s t ) , c o n t a i n e d 0.6 p p m ( 4 pm i n d r y r e s t ) . t y p i c a l f o r k a l e grown c l o s e t o t h e factory.

The l e v e l o f t h e p o l l u t e d k a l e was

D a i l y consumption o f l a r g e q u a n t i t i e s o f such k a l e m i g h t l e a d t o u n d e s i r a b l e u p t a k e o f f l u o r i d e s (3), b u t i t i s i m p r o b a b l e t h a t t h i s occurs.

141
TABLE V I FLUORIDE CONTENT O F U R I N E I N P P M AFTER CONSUMPTION OF NONPOLLUTED A N D POLLUTED KALE H o u r s A f t e r N o n p o l l u t e d and P o l l u t e d M e a l Student

0-3 0.45 0.37 0.46 0.28 0.32 0.35 0.34 0.55

3-6 0.35 0.32 0.56 0.36 0.21 0.24 0.15 0.39

Day 1 6-10

10-24
0.41 0.57 0.54 0.36 0.46 0.64 0.23 0.47

0-3 1.43 1.19 1.72 1.16 1.28 1.23 1.70 1.67

3-6 2.66 2.14 3.61 3.33

----

Day 2

6-10

10-24 0.89 0.98 0.89 0.90 0.92 1.12 0.92 0.88

1 2 3 4 5 6 7 8

0.64
0.61

0.50 0.59 0.32 0.88 0.21 0.55

3.13 3.10 3.09

0.86 1.56 1.12 2.55 0.63 1.82 2.68 2.03

CONCLUSIONS An a c u t e e f f e c t o f h y d r o g e n f l u o r i d e i m m i s s i o n on c h i l d r e n ' s l u n g f u n c t i o n s c o u l d n o t be demonstrated. T h e r e was a s o m e w h a t h i g h e r r a t e o f s e l f W h e t h e r t h i s was due r e p o r t e d r e s p i r a t o r y symptoms i n t h e exposed v i l l a g e . q u a l i t y c o u l d n o t be e s t a b l i s h e d . As f o r t h e u p t a k e o f f l u o r i d e s w i t h l o c a l l y g r o w n v e g e t a b l e s , l e v e l t h a t c a u s e s e n a m e l damage. even an u n u s u a l l y h i g h e x p e r i m e n t a l r e s u l t among t h e s t u d e n t s r e m a i n e d b e l o w t h e

more t o environmental concern than t o environmental d i f f e r e n c e s i n a i r

REFERENCES

1. 2. 3.

236

B i e r s t e k e r K. Z i e l h u i s RL. Van L e e u w e n P ( 1 9 7 6 ) T S o c G e n e e s k 54:230B i e r s t e k e r K. Z i e l h u i s RL, B a c k e r D i r k s 0, Van L e e u w e n P. Van R a a y A D r i n k i n g Water

(1977) Env Res 13:129-134

E n v i r o n m e n t a l P r o t e c t i o n Agency (1985) N a t i o n a l P r i m a r y R e g u l a t i o n s . F l u o r i d e . Fed Reg 50(93):20164-20175

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 143-154 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in T h e Netherlands

143

FLUORIDE

EMISSION FROM TUNNEL KILNS OF TILE WORKS

MAKOTO NAKAO AND F U M I O TANAKA S h i m a n e P r e f e c t u r a l I n s t i t u t e o f P u b l i c H e a l t h and E n v i r o n m e n t a l S c i e n c e , Nishi-Hamasada, Matsue, Shimane, 690-01, J a p a n ABSTRACT F l u o r i d e e m i s s i o n f r o m t u n n e l k i l n s has been assessed based on d a t a o b t a i n e d a t s e v e r a l t i l e works i n t h e Iwarni D i s t r i c t , P r e f e c t u r e , Japan. t h e f l u e gas. w e s t e r n Shimane The r e s u l t s showed an a v e r a g e e m i s s i o n o f 24 mgF/m3N i n The b u l k o f t h e f l u o r i d e i n t h e f l u e gas was i n

F o l l o w i n g an a l k a l i c l e a n i n g p r o c e s s t h e c o n c e n t r a t i o n

d e c r e a s e d t o 1 mgF/m3N. m a t e r i a l clay. general.

g a s e o u s f o r m , a n d m o s t o f t h e f l u o r i d e e m i t t e d may h a v e o r i g i n a t e d f r o m F l u o r i d e r e l e a s e r a t e i n t h e f i r i n g p r o c e s s was 40-60% i n

The q u a n t i t y o f f l u o r i d e e m i t t e d f r o m a t u n n e l k i l n was a p p r o x i m a t e l y i n p r o p o r t i o n t o t h e w e i g h t o f t h e product. used. Based on t h e s e d a t a , The w e i g h t o f t h e p r o d u c t c a n be e s t i m a t e d f r o m t h e number o f p r o d u c t s o r f r o m t h e q u a n t i t i e s o f m a t e r i a l the t o t a l fluoride emission from tunnel k i l n s estimated. The f l u o r i d e e m i s s i o n f a c t o r f o r t h r o u g h o u t t h e P r e f e c t u r e was

t u n n e l k i l n s was 1 2 0 g F / t o n o f m a t e r i a l used. INTRODUCTION Sekishugawara, a t i l e w i t h a 360-year h i s t o r y , i s manufactured i n t h e I w a m i D i s t r i c t . w h i c h i s l o c a t e d i n t h e w e s t e r n p a r t o f Shimane P r e f e c t u r e . Because o f t h e i n t r o d u c t i o n o f t u n n e l k i l n s and t h e housing.boom. as t h e h i g h q u a l i t y o f t h e t i l e , 1965, r e a c h i n g 6 0 m i l l i o n p i e c e s a n n u a l l y 1 0 y e a r s ago, and now 1 9 0 m i l l i o n p i e c e s . 20-100C as w e l l p r o d u c t i o n has r a p i d l y grown s i n c e about 138 m i l l i o n p i e c e s
i s fired

5 y e a r s ago,

Since t h e Sekishu-tile

a t a t e m p e r a t u r e o f 1200-13OO0C,

h i g h e r than t h a t employed i n

o t h e r t i l e p r o d u c t i o n a r e a s i n Japan, t i o n areas.

the r a t e a t which f l u o r i d e is

r e l e a s e d f r o m r a w m a t e r i a l i s c o n s i d e r e d t o be h i g h e r t h a n i n o t h e r p r o d u c F u r t h e r m o r e , due t o t h e i n c r e a s e i n t h e number o f k i l n s . t h e and r e g i o n a l c o n c e n t r a t i o n o f k i l n s i n r e c e n t y e a r s , the larger kiln-size. I n fact,

p o s s i b i l i t y e x i s t s t h a t s e r i o u s l o c a l a i r p o l l u t i o n m i g h t r e a d i l y occur. damages t o v e g e t a t i o n and t h e s i l k w o r m i n d u s t r y h a v e been r e c o g The n i z e d s i n c e around 1972 i n areas n e a r t i l e m a n u f a c t u r i n g works.

a u t h o r s h a v e been i n v o l v e d i n i n v e s t i g a t i n g t h e s t a t e o f a i r p o l l u t i o n b y

144
f l u o r i d e s (1-7). I n t h i s paper, t h e s t a t e o f f l u o r i d e e m i s s i o n from tunnel

k i l n s p r e s e n t i n most o f t h e t i l e works i s r e p o r t e d . MATERIALS AND METHODS Fifty-seven tunnel k i l n s f o r Sekishu-tile manufacture were c l a s s i f i e d by and t o p - f i r i n g ) , producf i r i n g methods (8) ( s i d e - f i r i n g , etc.) o f t h e i n d i v i d u a l k i l n . f o r the investigation. f l u e gas m o n i t o r i n g , on. Gaseous and p a r t i c u l a t e f o r m s o f f l u o r i d e s w e r e c o l l e c t e d s e p a r a t e l y w i t h sandwich-firing,

t i o n , and o p e r a t i n g c o n d i t i o n s ( f i r i n g t e m p e r a t u r e , c a r r i a g e f e e d r a t e , T h i r t e e n t y p i c a l k i l n s were then selected and so Factors investigated included operating conditions,

f l u o r i n e c o n t e n t i n r a w m a t e r i a l s and p r o d u c t s ,

M i 1 1 i p o r e F i 1t e r s ( T y p e AAWP).
M a t e r i a l c l a y and g l a z e w e r e t a k e n f r o m t h e c l a y y a r d and g l a z e t a n k , respectively. A b o u t t e n p i e c e s o f u n f i r e d t i l e s o f Type I o r Type I V p e r carriage. Three t o t e n k i l n were t a k e n b e f o r e t h e y were loaded on t h e f i r i n g process.

p i e c e s f r o m each t y p e o f f i r e d t i l e s were t a k e n i m m e d i a t e l y a f t e r t h e Samples w e r e p u l v e r i z e d i n t o 100 mesh o r s m a l l e r f o r a n a l y s i s b y t h e t h e r m a l d e c o m p o s i t i o n m e t h o d - i o n e l e c t r o d e m e t h o d (5). RESULTS AND D I S C U S S I O N C o n c e n t r a t i o n and c o m p o s i t i o n o f f l u o r i d e s i n f l u e qas The c o n c e n t r a t i o n o f g a s e o u s f l u o r i d e s e m i t t e d f r o m t u n n e l k i l n s was 9.23 0 mgF/m3N ( m e a n o f 1 3 k i l n s : mgF/m3N ( m e a n o f 6 k i l n s : mgF/m3N (mean o f 4 k i l n s : 2 8 mgF/m3N), 2 3 mgF/m3N). Based o n f i r i n g methods, k i I n s w a s 9.2-27 gaseous f l u o r i d e c o n c e n t r a t i o n o f t h e s i d e - f i r i n g - t y p e 2 0 mgF/m3N). 24 mgF/m3N) and 25-30

The c o n c e n t r a t i o n s w e r e 21-27 mgF/m3N (mean o f 3 k i l n s : r e s p e c t i v e l y . There k i l n s seemed t o h a v e as seen i n F i g u r e 1, methods,

f o r t h e s a n d w i c h - and t o p - f i r i n g

seemed t o be l i t t l e d i f f e r e n c e i n g a s e o u s f l u o r i d e c o n c e n t r a t i o n s b e t w e e n d i f f e r e n t f i r i n g methods, though t h e t o p - f i r i n g - t y p e Also, somewhat h i g h e r c o n c e n t r a t i o n t h a n t h e o t h e r s . On t h e o t h e r h a n d , k i l n s : 1.0 mgF/m3N).

gaseous f l u o r i d e c o n c e n t r a t i o n i n t h e f l u e gas d i d n o t f l u c t u a t e g r e a t l y . f o l l o w i n g the a l k a l i cleaning process the f l u o r i d e mgF/m3N ( m e a n o f 3 T h e p e r c e n t a g e o f f l u o r i d e r e m o v a l was. t h e r e f o r e , c o n c e n t r a t i o n i n t h e f l u e g a s d e c r e a s e d t o 0.78-1.4 e s t i m a t e d t o be m o r e t h a n 90%. The e f f e c t s o f f l u o r i d e s o n a n i m a l s and p l a n t s depend o n t h e p h y s i c a l and chemical p r o p e r t i e s o f f l u o r i d e s , and i n s o l u b l e p a r t i c u l a t e form, and t h e i r c o n c e n t r a t i o n s . i n t h a t order. I n general, t h e t o x i c i t y o f f l u o r i d e i s h i g h e r i n gaseous form, s o l u b l e p a r t i c u l a t e , F o r example, i t has been

145

11 I-

Fluorine concentration ( P P ~
F i g u r e 1. E x a m p l e o f c o n t i n u o u s a u t o m a t i c m e a s u r e m e n t r e c o r d o f g a s e o u s f l u o r i n e c o n c e n t r a t i o n i n f l u e gas. r e p o r t e d t h a t gaseous f l u o r i d e s a r e e i g h t t o t e n t i m e s more t o x i c t h a n p a r t i c u l a t e f l u o r i d e s (9). fore, t h e atmosphere. T a b l e I shows t h e c o n c e n t r a t i o n s o f v a r i o u s f o r m s o f f l u o r i d e s i n f l u e gas. form. S i n c e a p a r t i c u l a t e f o r m o f f l u o r i d e was d e t e c t e d i n o n l y one sample, Gaseous f l u o r i d e s a r e h i g h l y p o i s o n o u s t o a n i m a l s and p l a n t s . and m o s t p a r t i c u l a t e - f o r m f l u o r i d e s a r e s o l u b l e , Furi t a p p e a r s t h a t f l u o r i d e s e m i t t e d f r o m t u n n e l k i l n s w e r e m o s t l y i n gaseous thermore, s i n c e 70-80% o f t h e a t m o s p h e r i c f l u o r i d e s a r o u n d t i l e w o r k s a r e emphasis To e v a l u a t e f l u o r i d e t o x i c i t y .
i t is, there-

i m p o r t a n t t o know t h e p r o p e r t i e s o f f l u o r i d e s i n t h e f l u e gas and i n

i n gaseous form, works.

s h o u l d be focused on measures t o c o n t r o l f l u o r i d e p o l l u t i o n f r o m t i l e F l u o r i n e c o n t e n t s i n m a t e r i a l c l a y and g l a z e F l u o r i d e s c o n t a i n e d i n t h e f l u e gas o r i g i n a t e f r o m m a t e r i a l c l a y and glaze. material To s e c u r e t h e r e f r a c t o r i n e s s t o w i t h s t a n d h i g h - t e m p e r a t u r e c l a y s a r e g e n e r a l l y m i x e d i n t o t h e compound m a t e r i a l . firing and t o s e c u r e t h e q u a l i t y o f t h e f i n i s h e d S e k i s h u - t i l e , several kinds o f Material The f l u o r i n e mean: 311 ppm) 18-21 ppm

c l a y s c a n b e c a t e g o r i z e d i n t o c l a y , sand, a n d s i l i c a sand. c o n t e n t i n t h e m a t e r i a l s was 195-438 ppm ( s a m p l e number: 40; f o r clay, 45-217 ( s a m p l e number: 2; mean: 20 ppm) f o r s i l i c a sand,

ppm ( s a m p l e number: 11; mean: 1 3 8 ppm) f o r sand,

and 227-330 ppm ( s a m p l e On t h e o t h e r h a n d , t h e

n u m b e r 1 1 ; mean: 2 7 5 p p m ) f o r c o m p o u n d m a t e r i a l . f l u o r i n e c o n t e n t i n g l a z e was 80-268

ppm ( s a m p l e n u m b e r : 2 2 ; mean: 1 7 4

146
TABLE I

GASEOUS AND PARTICULATE FORMS OF FLUORIDE I N THE FLUE GAS

Kiln Gaseous Form P a r t i c u l a t e Form Soluble Insoluble ND Total

9.2 23

ND

9.2 23 19 27 21 23.4

ND
ND

ND
ND

19
27 21 23 ND < 0.1 mgF m3N ( s o l u b l e ) ND < 1 mgF/m N ( i n s o l u b l e ) ppm). Thus,

ND
ND
0.4

ND

ND
ND

No m a t e r i a l w i t h an e x t r e m e l y h i g h c o n c e n t r a t i o n was o b s e r v e d ,

and

t h e r e were no marked d i f f e r e n c e s between d i f f e r e n t k i n d s o f m a t e r i a l s . since t h e f l u c r i d e content i n glaze i s considerably lower than t h a t furthermore, since the quantity f o r Type I o f m a t e r i a l c l a y o r compound m a t e r i a l , and, tile, a few percent i n weight),

o f g l a z e u s e d i s m u c h s m a l l e r t h a n t h a t o f m a t e r i a l c l a y (e.g. k i l n s may o r i g i n a t e f r o m m a t e r i a l c l a y , r e l e a s e d i n t h e f i r i n g process. Release r a t e o f f l u o r i d e i n f i r i n g process

most o f t h e f l u o r i d e s e m i t t e d from tunnel although t h e r e i s a tendency t h a t

t h e f l u o r i n e c o n t a i n e d c l o s e r t o t h e s u r f a c e o f a t i l e i s more e a s i l y

F l u o r i n e e m i s s i o n f r o m t u n n e l k i l n s i s c o n s i d e r e d dependent on t h e f l u o r i n e c o n t e n t o f t h e r a w m a t e r i a l and t h e f l u o r i n e r e l e a s e r a t e f r o m t i l e s i n t h e f i r i n g process. fluorides, And, t h e r e l e a s e r a t e i s supposed t o be the form o f ten and so f o r t h . i n f l u e n c e d b y t h e c o m p o s i t i o n o f m a t e r i a l c l a y and g l a z e , f i r i n g c o n d i t i o n , t i l e shape,

To s t u d y t h e d i f f e r e n c e s b e t w e e n t h e r e l e a s e r a t e and t i l e shape, were c o l l e c t e d f o r measurement o f f l u o r i n e content.

samples f r o m each o f t h e f o u r k i n d s o f t i l e s f i r e d s i m u l t a n e o u s l y i n a k i l n

As shown i n T a b l e 11, t h e r e l e a s e r a t e was 48-62%, d e p e n d e n t o n t h e shape

of the tile.

Though t h e shape, s i z e , and t h i c k n e s s v a r i e d w i t h t h e k i n d s

of tiles, weight. examined. rates.

l a r g e r and t h i c k e r t i l e s a r e c o n s i d e r e d t o h a v e a h e a v i e r p r o d u c t The r e l a t i o n s h i p b e t w e e n r e l e a s e r a t e and p r o d u c t w e i g h t was t h u s The r e s u l t s showed t h a t t h e h e a v i e r t i l e s had l o w e r r e l e a s e

147 TABLE I 1 FLUORINE RELEASE RATE ACCORDING TO TYPES

OF

TILE

Release Rate (%) Product Weight (kg)

47.4 2.90

48.8 3.55

58.7 2.84

61.6 2.06

II

TABLE I 1 1 FLUORINE RELEASE RATE OF TYPE I T I L E

Kiln

Firing Method

F l u o r i n e C o n t e n t (ppm) Fired U n f ir e d

Release Rate (%)

X A
S i d e - f i r i ng S i d e - f i r ing S i d e - f ir i n g Side-f i r ing Sandwich-firing Sandwich-firing Sandwich-firing Sandwich-firing Top-firing Top-firing

21 52.3 47.0 59.2 81.6 58.5 45.9 61.9 56.7 43.4 44.0

Product Weight (kg)

264 300 255 228 248 255 268 231 320 284

126 159 104 42 103 138 102 100 181 159

2.95 3.07 2.84 3.31 3.06 3.07 2.96 2.96 3.01 3.19

C
D

E
F H

I J
K

z=-

x - y

x100

148
S i n c e l i t t l e d i f f e r e n c e was f o u n d i n s i z e a n d w e i g h t b e t w e e n d i f f e r e n t t i l e w o r k s , and s i n c e t h e f o r m o f f l u o r i d e s and t h e c o m p o s i t i o n o f compound m a t e r i a l and g l a z e w e r e c o n s i d e r e d t o b e s i m i l a r among t i l e w o r k s , w o r k s depended o n d i f f e r e n t f i r i n g c o n d i t i o n s . f r o m Type I t i l e s o f d i f f e r e n t t i l e works. r e l e a s e r a t e r a n g e d f r o m 43.4-81.6% E x c e p t f o r k i l n G,
i t was

supposed t h a t t h e d i f f e r e n c e i n r e l e a s e r a t e o f Type I t i l e s between t i l e

So,

the influence o f f i r i n g

c o n d i t i o n s o n t h e r e l e a s e r a t e was s t u d i e d b y c o m p a r i n g t h e r e l e a s e r a t e

A s s h o w n i n T a b l e 111, t h e

w i t h a mean o f 55.1%.

w h i c h had a h i g h e r f i r i n g temperature than t h a t o f

m o s t S e k i s h u - t i l e k i l n s b y a b o u t 50C b e c a u s e o f t h e u s e o f a h i g h m e l t i n g g l a z e a n d a s l o w e r f e e d r a t e o f t h e c a r r i a g e , t h e r e l e a s e r a t e w a s 43.461.9% w i t h a mean o f 52.1%. Concerning t h e f i r i n g method, perhaps because o f the f a s t e r carriage feed-rate, sidered small. I n conclusion, t h e r e l e a s e r a t e i n c r e a s e d as t h e t i l e s i z e o r t h e f i r i n g temperature decreased, t o p - f i r i n g k i l n s had a s l i g h t l y l o w e r B u t , t h e i r d i f f e r e n c e was con-

r e l e a s e r a t e t h a n o t h e r f i r i n g methods.

o r as t h e c a r r i a g e f e e d - r a t e
firing,

increased.

for a

regular tunnel k i l n f o r the Sekishu-tile

t h e r e l e a s e r a t e was

40-

60%.
E s t i m a t e d and measured f l u o r i n e e m i s s i o n F l u o r i n e e m i s s i o n f r o m a t u n n e l k i l n c a n be e s t i m a t e d b y u s i n g E q u a t i o n

( 1 ) f r o m t h e m a t e r i a l balance.
Ec
where:
=

Ci(FIi

WI,

x (1-~1,/100)

- Fol x Wgl]

(1)

Ec

= F l u o r i n e emission (gF/day)
i

F I ~ = F l u o r i n e c o n t e n t i n u n f i r e d t i l e s o f Type Fo1 = F l u o r i n e c o n t e n t i n f i r e d t i l e s o f Type

i

(ppm)

(ppm)

W I ~ = W e i g h t o f u n f i r e d t i l e s o f Type

(ton/day)

Woi
mi

= W e i g h t o f f i r e d t i l e s o f Type i ( t o n / d a y ) = Q u a n t i t y o f w a t e r a t t a c h e d t o u n f i r e d t i l e s o f Type
i

(%)

I n t h e c a l c u l a t i o n o f f l u o r i n e emission from tunnel k i l n s using Equation t h e v a l u e s c a l c u l a t e d f r o m t h e p r o d u c t i o n and t h e m e a s u r e d u n i t w e i g h t (I), o f e a c h t i l e t y p e w e r e used f o r W I ~and

Wol,

and t h e measured v a l u e s f o r

FI,

a n d Fol,

and a l s o ,

measured v a l u e s o f Type

t i l e w e r e u s e d f o r mi On t h e

w h i c h was c o n s i d e r e d t o show l i t t l e d i f f e r e n c e w i t h t i l e t y p e . c o n c e n t r a t i o n i n t h e f l u e gas and t h e d r y - g a s volume.

o t h e r hand, m e a s u r e d f l u o r i n e e m i s s i o n was c a l c u l a t e d f r o m t h e f l u o r i n e T h e e s t i m a t e d a n d m e a s u r e d v a l u e s a r e p l o t t e d i n F i g u r e 2. While the

149

0
c1

o Side-f i r i n g
A w i ch f 0 Sand Top-firin;/
0

- i r ing Top-firing o
A
0

m 3
09'

ia

Measured v a l u e (KgF/day) F i g u r e 2. R e l a t i o n b e t w e e n e s t i m a t e d and measured f l u o r i n e e m i s s i o n .

e s t i m a t e d values tended t o be l a r g e r t h a n t!ie measured values f o r k i l n s w i t h h i g h f l u o r i n e emission, s l o p e o f 45". Consequently, p l o t t e d p o i n t s gathered near t h e l i n e w i t h t h e since t h e estimated values almost coincided a m a t e r i a l balance f o r f l u o r i n e The m e a s u r e d v a l u e s o f f l u o r i n e e m i s s i o n

w i t h t h e measured v a l u e s f o r t u n n e l k i l n s , seemed t o e x i s t i n t u n n e l k i l n s .
w i l l

be u s e d i n t h e d i s c u s s i o n h e r e a f t e r .

R e l a t i o n s h i p b e t w e e n f l u o r i n e e m i s s i o n and w e i q h t o f f i r e d p r o d u c t s Since f l u o r i d e s e m i t t e d from tunnel k i l n s o r i g i n a t e from raw materials,

i t i s supposed t h a t t h e e m i s s i o n i s r e l a t e d t o t h e q u a n t i t y o f m a t e r i a l s o r

t h e weight o f products. b y E q u a t i o n (2).

The r e l a t i o n b e t w e e n f l u o r i n e e m i s s i o n and t h e The r e l a t i o n s h i p i s e x p r e s s e d

w e i g h t o f p r o d u c t s i s p l o t t e d i n F i g u r e 3.

Em

0.13 x W
and W,

(2)
weight o f products a high correla-

w h e r e Em r e p r e s e n t s f l u o r i n e e m i s s i o n (kgF/day). (ton/day). t i o n i s recognized.

S i n c e t h e c o e f f i c i e n t o f c o r r e l a t i o n i s 0.97,

The v i r t u a l l y l i n e a r r e l a t i o n s h i p b e t w e e n f l u o r i n e e m i s s i o n and w e i g h t o f t h e product suggests t h a t f l u o r i n e content i n raw m a t e r i a l s , f i r i n g method, k i Ins, etc. are not significant factors, t i l e shape, though they s l i g h t l y r a t h e r than f i r i n g

i n f l u e n c e t h e e m i s s i o n . Thus, t o e s t i m a t e f l u o r i n e e m i s s i o n f r o m t u n n e l k i I n p r o d u c t i o n c a p a c i t y s h o u l d be i d e n t i f i e d .

150

t
0

Side-firing

a 4 0

60

Product w e i g h t (ton/day) F i g u r e 3. R e l a t i o n b e t w e e n f l u o r i n e e m i s s i o n and p r o d u c t w e i g h t . Therefore, k i l n s using sandwich-firing should

method o r o t h e r f i r i n g c o n d i t i o n s . or top-firing methods,

which are being b u i l t i n c r e a s i n g l y recently,

be examined s i n c e t h e y have a c a p a c i t y t w o t o t h r e e Method o f e s t i m a t i o n o f f l u o r i n e e m i s s i o n

times larger than

c o n v e n t i o n a l k i l n s and n a t u r a l l y h a v e h i g h e r f l u o r i d e e m i s s i o n s . E s t i m a t i o n o f f l u o r i d e emission f r o m tunnel k i l n s i s e s s e n t i a l f o r promoting environmental p r o t e c t i o n against f l u o r i d e p o l l u t i o n . as e n v i r o n m e n t a l i m p a c t assessment, e v a l u a t i o n o f data, a r e g i o n a l p o l l u t i o n c o n t r o l program a r e a l l important. To e s t i m a t e t h e f l u o r i n e e m i s s i o n , E q u a t i o n ( 2 ) c a n be used. Although t h e method f o r e v a l u a t i n g p r o d u c t w e i g h t f r o m t h e u n i t w e i g h t o f a p r o d u c t and t h e number o f p r o d u c t s i s h i g h l y a c c u r a t e , cated. Therefore, every kiln. I n practice, t h e measurement i s c o m p l i Such m e a s u r e s and f o r m u l a t i o n o f

i t i s d i f f i c u l t t o obtain a real product weight f o r

s u c h m e t h o d s as u s i n g t h e p r o d u c t i o n o r t h e quani n which t h e product w e i g h t (product-

t i t i e s o f r a w m a t e r i a l s may be adopted. The m e t h o d o f u s i n g t h e p r o d u c t i o n , n u m b e r b a s i s ) i s a p r o d u c t o f t h e p r o d u c t i o n c a l c u l a t e d i n t e r m s o f Type I t i l e ( t h e q u o t i e n t o f t h e t o t a l o u t p u t d i v i d e d b y t h e u n i t p r i c e o f Type I t i l e i s t r a d i t i o n a l l y c a l l e d t h e p r o d u c t i o n a t t i l e w o r k s ) and t h e u n i t w e i g h t o f t h e ' T y p e I t i l e ( a s s h o w n i n T a b l e 111. u n i t w e i g h t o f T y p e I t i l e i s a b o u t 3 Kg w i t h l i t t l e d i f f e r e n c e among t i l e w o r k s : o f 3.0 t h e e s t i m a t i o n ) i s expressed a t f o l l o w s : therefore, use Kg a s a u n i t w e i g h t m a y h a v e l i t t l e i n f l u e n c e o n t h e a c c u r a c y o f

151
W ,
=

3 x P/30

(3)
and P

where U p i s p r o d u c t w e i g h t c a l c u l a t e d from p r o d u c t number (ton/day), The method o f u s i n g t h e q u a n t i t i e s o f raw m a t e r i a l s , used,

i s p r o d u c t number c a l c u l a t e d i n t e r m s o f Type I t i l e ( l o 3 pieces/month). i n which the product weight ( m a t e r i a l basis) i s evaluated from t h e q u a n t i t y of m a t e r i a l c l a y w i t h c o r r e c t i o n s f o r t h e q u a n t i t y o f water attached t o t h e m a t e r i a l w h i c h was t h e mean o f m e a s u r e d v a l u e s , 16-20%) and 446%). w h i c h was t h e mean of measured values, c l a y ( 1 8 % was used,

i g n i t i o n l o s s (5% was used, i s expressed i n E q u a t i o n (4).

wr
where Wr

R x (1

--

100

100

(4)

i s t h e p r o d u c t w e i g h t c a l c u l a t e d from m a t e r i a l q u a n t i t y (ton/day) Here, the quantity of

and R t h e q u a n t i t y o f m a t e r i a l c l a y used (ton/day).

t h e g l a z e used i s n e g l e c t e d f o r t h e reason d e s c r i b e d p r e v i o u s l y . The c a l c u l a t e d v a l u e s f r o m E q u a t i o n ( 3 ) and E q u a t i o n ( 4 ) a r e p l o t t e d t o t h e measured v a l u e s i n F i g u r e 4. w i t h a s l o p e o f 45". racy. I n b o t h cases, c a l c u l a t e d on t h e p r o d u c t number b a s i s and on t h e m a t e r i a l basis, p l o t t e d p o i n t s g a t h e r e d near a l i n e

Thus, i t was f o u n d t h a t t h e p r o d u c t w e i g h t c o u l d be

e s t i m a t e d f r o m e i t h e r E q u a t i o n (3) o r E q u a t i o n (4) w i t h c o n s i d e r a b l e accu-

t
0

Product-number bas i s

o Material basis

20

40

60

Reasured value ( t o d d a y )

F i g u r e 4.

R e l a t i o n between c a l c u l a t e d and measured p r o d u c t w e i g h t .

152
Emission f a c t o r s tunnel k i l n s Emission f a c t o r i s g e n e r a l l y used t o o b t a i n t h e amount o f discharged environmental pollutant. i n Table IV. o f the side-firing Emission f a c t o r s f o r t u n n e l k i l n s a r e summarized i n s u f f i c i e n t m e a s u r e s f o r f u e l economy, based on f u e l consumption, kilns Because o f t h e

t y p e have h i g h e r r a t e s o f f u e l consumption and t h e r e f o r e compared w i t h t h e number The mean However, b a s e d on t h e w e i g h t o f p r o d u c t s ,

lower fluorine emission factors, t h e o t h e r kiln-types, o f products,

o r the quantities o f materials,

l i t t l e difference i n fluorine basis.

e m i s s i o n f a c t o r i s r e c o g n i z e d b e t w e e n d i f f e r e n t f i r i n g methods. emission f a c t o r s a r e 157 gF/ton on a product-weight p i e c e s on a product-number basis, Method o f r e d u c t i o n o f f l u o r i n e e m i s s i o n To r e d u c e t h e f l u o r i n e e m i s s i o n f r o m t u n n e l k i l n s , considered. content, These i n c l u d e selection o f

443 gF/103

and 1 2 0 g F / t o n on a m a t e r i a l b a s i s . s e v e r a l m e t h o d s may b e and

raw m a t e r i a l s w i t h l o w e r f l u o r i n e

c o n t r o l o f t h e f l u o r i n e release r a t e from raw materials, industry, however,

t r e a t m e n t o f t h e f l u e gas. I n the Sekishu-tile clay, and a l s o , since selection o f the material c l a y i s d i f f i c u l t i n view o f t h e possible exhaustion o f e x c e l l e n t m a t e r i a l s i n c e f l u o r i d e compounds s u c h a s f l u o r i t e o r c r y o l i t e a r e reduction o f f l u o r i n e emission by improvement o f s u c h as s l a k e d n o t u s e d as t h e s o l v e n t o f t h e g l a z e , On t h e o t h e r hand,

s e l e c t i o n o f r a w m a t e r i a l s c a n n o t be e x p e c t e d . control o f the f l u o r i n e release rate, t h e o p e r a t i o n o f k i l n s or a d d i t i o n o f a r e l e a s e i n h i b i t o r . l i m e , t o t h e m a t e r i a l c l a y may b e c o n s i d e r e d . I n the Sekishu-tile i n d u s t r y , however, s i n c e improvement i n t h e o p e r a t i o n by increaso f k i l n s h a s been a c h i e v e d f r o m t h e v i e w p o i n t o f f u e l economy,

i n g the feed r a t e o f the carriage o r by lowering the f i r i n g temperature w i t h i n t h e r a n g e so t h a t t h e q u a l i t y o f p r o d u c t s m i g h t s t i l l be m a i n t a i n e d , f u r t h e r d e c r e a s e o f t h e f l u o r i n e r e l e a s e r a t e c a n n o t be e x p e c t e d f o r m o s t o f t h e t i l e works. Fluorine release inhibitor i s said t o exhibit the Therefore, development o f inexpenc o n t r o l e f f e c t o n l y when i t i s added i n s u c h abundance a s t o a d v e r s e l y a f f e c t t h e q u a l i t y o f t h e p r o d u c t (10). Thus, s i v e and m o r e e f f e c t i v e i n h i b i t o r s i s needed. w h i l e keeping t h e q u a l i t y o f t h e Sekishu-tile a t i t s present level, t h e r e seems t o be n o o t h e r way t o e f f e c t i v e l y r e d u c e f l u o r i n e e m i s s i o n t h a n t o t r e a t f l u e gas.

A f l u o r i n e r e m o v a l r a t e o f m o r e t h a n 90% c a n b e

a c h i e v e d b y t h e a 1 k a l i c l e a n i n g method.

CONCLUSIONS
The a c t u a l s t a t e o f f l u o r i n e e m i s s i o n f r o m t u n n e l k i l n s was i n v e s t i g a t e d

TABLE I V

E M I S S I O N FACTORS OF TUNNEL KILNS

Kiln Fuel Consumpti on (l/ton-product) (lo3, F l u e gas Volume
pry)

/ton-product)

Fuel Basis (kgF/kl)

F l u o r i n e Emission Product-weight Product-number M a t e r i a1 Basis Basis Basis (gF/ton-product) (gF/103pieces) ( g F / t o n - m a t e r i a l )

Total

61-237 ( n = l l . 123) 122-237 (n=6. 167) 61-113 (n=4, 8 4 ) 83-89 (n=3, 8 6 )

3.9-1 4.3 ( n = l l , 7.4) 6.1-14.3 (n=6, 9.1) 4.8-7.6 (n=3, 5.8) 3.9-5.1 4.5)

0.78-0.88 ( n = l l , 1.30) 0.78-1.61 (n=6, 1.08) 1.07-1.88 (n=3, 1.55) 1.39-1.73 (n=2, 1.56)

11 7-256 ( n = l l , 157) 11 7-256

281 -658 ( n = l l , 443) 281 -658 (n=6, 471) 323-51 1 (n=3, d02) 380-463 (n=2, 422)

91 -1 44 (n=9, 120) 91-1 41 (n=4, 117) 105-1 56 (n=3, 123) 96-1 44 (n=2, 120)

S id e - f ir ing

( n = 6 , 174)
121-167

S a n d w i c h - f i r i ng

(n=3, 138)
119-154 (n=2. 137)

Top-firing

(n=2.

154

among t h e S e k i s h u - t i l e m a n u f a c t u r i n g i n d u s t r i e s s p e c i f i c t o t h e Iwami D i s t r i c t , Shi mane P r e f e c t u r e . T h e mean c o n c e n t r a t i o n o f g a s e o u s f l u o r i d e s i n t h e f l u e g a s was 2 4 mgF/m3N. nificant.

T h e bulk of t h e f l u o r i d e s i n t h e f l u e g a s was i n g a s e o u s form,

f l u o r i d e s was found t o be i n s i g Fluorine release r a t e F l u o r i d e o r i g i n a t i n g from t h e g l a z e i s s c a r c e i n t h e f l u e gas.

and t h e c o n t r i b u t i o n of p a r t i c u l a t e - f o r m

a n d m o s t o f i t may o r i g i n a t e f r o m m a t e r i a l c l a y .

from raw m a t e r i a l s i n t h e f i r i n g p r o c e s s was 40-60% i n g e n e r a l . T h e q u a n t i t y o f f l u o r i d e s e m i t t e d from a t u n n e l k i l n i s a p p r o x i m a t e l y i n p r o p o r t i o n t o t h e w e i g h t o f t h e p r o d u c t , w h i c h i n turn c a n be e s t i m a t e d e i t h e r from t h e number of p r o d u c t s o r from t h e q u a n t i t i e s of m a t e r i a l c l a y used. By t h i s m e t h o d , t h e t o t a l f l u o r i n e e m i s s i o n f r o m t u n n e l k i l n s The f l u o r i n e e m i s s i o n f a c t o r f o r t h r o u g h o u t t h e p r e f e c t u r e was e s t i m a t e d .

t u n n e l k i l n s was 120 gF/ton o f m a t e r i a l used. T h e r e s e e m s t o b e no o t h e r way f o r an e f f e c t i v e r e d u c t i o n o f f l u o r i n e e m i s s i o n from t u n n e l k i l n s t h a n t h e t r e a t m e n t of f l u e gas. method. REFERENCES 1. Nakao M, Kimura T, S a i t o K (1973) Report o f Shimane P r e f I n s t Pub Hlth Environ S c i e n c e 15:52-53 Fluorine r e m o v a l r a t e o f m o r e t h a n 90% c a n be a c h i e v e d by t h e a l k a l i c l e a n i n g

2. Nakao M, Tanaka F (1977) Shimane PIOPHAES Report 19:96-97

3. T a n a k a F, Fukada K , Nakao M, Kimura T ( 1 9 7 7 ) S h i m a n e PIOPHAES R e p o r t 19 :65-72 4. Shimane P r e f e c t u r e (1976) Report of Survey f o r A d d i t i o n a l D e s i g n a t i o n of Toxic S u b s t a n c e s (on commission of t h e Environ Agency) 5. Shimane P r e f e c t u r e (1979) Report on F l u o r i d e E m i t t i n g Source Survey (on commission of t h e Environ Agency)

6. Nakao M, Tanaka F, Tachibana C, S a i t o K (1979) Shimane PIOPHAES Report 21 :75-76

7. Nakao M, T a n a k a F, T a c h i b a n a C. Fukada K , H a y a s h i K , S a i t o K ( 1 9 8 0 ) J Environ Lab Assoc 5:105-115

8. Tanaka M (1980) Handbook of Clay-Tile.

Gihodo, Tokyo

9. Tsunoda F, S a k u r a i S, I t a i K and Nakaya S (1978) C r i t e r i a on F l u o r i d e s (on commission of t h e Environ Agency) 10. Gunma P r e f e c t u r e (1975) Report of Gunma P r e f I n s t I n d u s t S c i Tech 28

Fluoride Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 155-163 0 1 9 8 6 Elsevier Science Publishers B.V., Amsterdam - Printed in T h e Netherlands

H. Tsunoda and M.-H. Yu (Editors)

155

D I S T R I B U l I O N OF FLUORIDE I N H U M A N B O N E S COLLECTED FOR THE EVALUATION O F INDUSTRIAL AIR POLLUTION

M A R C A. V A N D E N H E E D E ~ , J A C Q U E S 1.1. TIMPERMAN*, JAN A.

AUBIN

M. H E Y N D R I C K X ~ .

AND

CORDONNIER~
S t a t e U n i v e r s i t y o f Ghent, H o s p i t a a l s t r a a t 13, Ghent. P r o f . J.

'Department o f Toxicology, B-9000 Ghent. B e l g i u m

'Department o f Legal Medicine, State U n i v e r s i t y o f K l u y s k e n s s t r a a t 29, B-9000 Ghent. B e l g i u m

ABSTRACT F l u o r i d e c o n c e n t r a t i o n s w e r e d e t e r m i n e d i n human bones i n o r d e r t o e s t a b l i s h w h e t h e r bone f l u o r i d e l e v e l s c o u l d be i n d i c a t i v e o f a c h r o n i c e x p o s u r e t o a f l u o r i d e contaminated environment. cases, The bone s a m p l e s s t u d i e d w e r e I n ten o f these the individuals f r e e o f any f l u o r i d e m a i n l y o b t a i n e d f r o m t h i r t e e n p o s t mortem examinations. t h e p e r s o n s i n v o l v e d had l i v e d i n r u r a l a r e a s , p o l l u t i o n ( r e f e r e n c e cases). t i n g plant. I n t h e o t h e r t h r e e cases,

i n v o l v e d had l i v e d f o r many ,;ears p r o c u r e d by a 71-year-old t i o n o f t h e hip. f l u o r i d e intoxication.

i n t h e neighborhood o f a f l u o r i d e e m i t -

I n a d d i t i o n , an a n a l y s i s was a l s o p e r f o r m e d o n a bone f r a g m e n t woman who u n d e r w e n t a n a r t h r o p l a s t i c r e c o n s t r u c T h i s p a t i e n t a l s o had a p r e v i o u s h i s t o r y o f c h r o n i c

The f l u o r i d e c o n c e n t r a t i o n s measured i n t h e f o u r s u s p e c t e d cases w e r e w i t h i n t h e l i m i t s i n d i c a t e d b y t h e r e f e r e n c e cases. As s u c h t h e y w e r e n o t d i r e c t l y i n d i c a t i v e o f a c h r o n i c exposure t o i n d u s t r i a l f l u o r i d e p o l l u t i o n .

INTRODUCTION With t h e c u r r e n t increased a t t e n t i o n t o environmental protection.

i t has

been n o t e d t h a t t h e r i s i n g i n d u s t r i a l f l u o r i d e e m i s s i o n s a s an e l e m e n t of a i r p o l l u t i o n s h o u l d i n d e e d t r i g g e r c o n c e r n among t h o s e i n v o l v e d w i t h public health protection. However,

i t i s known t h a t t h e d i a g n o s i s of
Apart from a

c h r o n i c f l u o r i d e i n t o x i c a t i o n i n man i s v e r y d i f f i c u l t . s e r i e s o f n o n s p e c i f i c symptoms, b l o o d and u r i n e samples.

the only practical c r i t e r i a available f o r

e s t a b l i s h i n g a d i a g n o s i s a r e X - r a y e v i d e n c e and f l u o r i d e d e t e r m i n a t i o n s i n The p u r p o s e o f t h i s s t u d y was t o e s t a b l i s h w h e t h e r a g i v e n f l u o r i d e c o n c e n t r a t i o n f o u n d i n t h e bone t i s s u e o f an i n d i v i d u a l s u s p e c t e d o f h a v i n g been e x p o s e d t o t o x i c l e v e l s o f f l u o r i d e i n

156
t h e a i r c o u l d be i n d i c a t i v e o f a c h r o n i c f l u o r i d e i n t o x i c a t i o n . ma-lerial obtained from t h i r t e e n r o u t i n e f o r e n s i c autopsies. cases, The accumI n three

u l a t i o n o f f l u o r i d e i n human bone t i s s u e was s t u d i e d b y a n a l y z i n g bone t h e p o s t m o r t e m bone s a m p l e s o r i g i n a t e d f r o m p e r s o n s who had been The p o s t m o r t e m bone s a m p l e s c o l l e c t e d i n t h e o t h e r t e n cases The f l u o r i d e c o n c e n t r a t i o n s f o u n d i n t h e l a t t e r cases c o u l d I n view o f u n i f o r m i t y bone s a m p l e s w e r e t a k e n f r o m t h e same a n a t o m i c s i t e s T h e s e s i t e s , a s s h o w n i n F i g u r e 1. t h e "Manubrium" o f t h e sternum,

l i v i n g f o r a number o f years i n t h e neighborhood o f a f l u o r i d e - e m i t t i n g factory. pollution. b e l o n g e d t o p e r s o n s who h a d b e e n l i v i n g i n a r e a s f r e e o f a n y known f l u o r i d e t h u s be c o n s i d e r e d as r e f e r e n c e v a l u e s o r blanks. and r e p r o d u c i b i l i t y , i n a l l t h e p o s t mortem cases studied.

a r e t h e " P o s t e r i o r Calvarium" o f t h e s k u l l ,

t h e "Caput" o f t h e femur, and a p a r t o f t h e r i b . F r o m t w o i n d i v i d u a l s who h a d b e e n l i v i n g i n a n a r e a o f h e a v y f l u o r i d e c o n t a m i n a t i o n ( p o s t m o r t e m c a s e s 1 2 and 13), femur was a l s o a n a l y z e d . bone s a m p l e s , t h e "Trochanter t h e "Caput" Major" o f t h e o f t h e femur was a l s o A p a r t f r o m t h e d e t e r m i n a t i o n s p e r f o r m e d on t h e woman d u r i n g an a r t h r o p l a s t i c r e c o n : ! ? z . > i t s l z i n J a n u a r y 1977.

w h i c h were c o l l e c t e d p o s t mortem,

s u r g i c a l l y removed f r o m a 71-year-old struction of the hi?

iii

t h e '!i?jv?rsi':;

F i g u r e 1.

N a t u r e o f t h e bone samples.

157
analyzed. T h i s c a s e was c h o s e n a f t e r i t was shown d u r i n g a p r e v i o u s s t a y w h e r e t h e woman was u n d e r g o i n g t r e a t t h a t t h e p a t i e n t had i n c r e a s e d f l u o as compared w i t h t h e

i n t h e U n i v e r s i t y H o s p i t a l s i n 1975, m e n t f o r a r t h r i t i s and p o l y n e u r i t i s .

r i d e c o n c e n t r a t i o n s i n t h e u r i n e ( u p t o 3.88 ppm).

t o l e r a n c e v a l u e s d e s c r i b e d b y V i g l i a n i (1) and Hodge

gt G.

(2).

A t this

t i m e t h e woman d r a n k b o t t l e d V i c h y m i n e r a l w a t e r a t an a m o u n t o f a b o u t 1.5

L a day.

D u r i n g h e r s t a y i n t h e h o s p i t a l t h e u r i n a r y f l u o r i d e concenThe a n a l y s i s r e v e a l e d t h a t t h e V i c h y w a t e r c o n t a i n e d u p t o 9 L a t e r i t was c o n f i r m e d b y V e r g o t e gt

t r a t i o n r e m a i n e d h i g h so an a n a l y s i s o f s e v e r a l b o t t l e s o f V i c h y w a t e r was performed. ppm o f f l u o r i d e .

~ 1 (. 3)

that the

f l u o r i d e c o n c e n t r a t i o n s i n b o t t l e d V i c h y w a t e r s were h i g h as opoosed t o those found i n o t h e r m i n e r a l waters. o f Vichy water, A f t e r d i s c o n t i n u i n g t h e consumption t h e u r i n a r y f l u o r i d e c o n c e n t r a t i o n decreased.

few

m o n t h s a f t e r t h e p a t i e n t had l e f t t h e h o s p i t a l . f l u o r i d e c o n c e n t r a t i o n was n o t e d again. plant,

an i n c r e a s e o f t h e u r i n a r y

Further investigation revealed of the wind,

t h a t t h i s woman a l s o l i v e d i n t h e n e a r v i c i n i t y o f a f l u o r i d e e m i t t i n g and t h a t a c c o r d i n g t o t h e p r e v a i l i n g d i r e c t i o n a t m o s p h e r i c f l u o r i d e l e v e l s o f u p t o 57 pg/m3 w e r e m e a s u r e d i n h e r g a r d e n a t a d i s t a n c e o f a b o u t 700 m f r o m t h e p l a n t . Measurements performed a f t e r w a r d s on p l a n t m a t e r i a l g r o w i n g i n h e r g a r d e n r e v e a l e d t h a t t h e l e a v e s o f t h e b r u s h w o o d and some v e g e t a b l e s c o n t a i n e d f l u o r i d e i n e x c e s s i v e l y h i g h c o n c e n t r a t i o n s (bush rose, a d r y w e i g h t basis). pollution. MATERIALS AND METHODS Reaqents A l l c h e m i c a l s used w e r e r e a g e n t g r a d e and w e r e u s e d w i t h o u t f u r t h e r purification, desiccator. F l u o r i d e s t o c k s o l u t i o n ( 1 , 9 0 0 p p m ) w a s p r e p a r e d b y d i s s o l v i n g 4.199 g s o d i u m f l u o r i d e i n d o u b l e d i s t i l l e d w a t e r a n d d i l u t i n g t o 1 L. double d i s t i l l e d water. T o t a l i o n i c s t r e n g t h a d j u s t m e n t b u f f e r (TISAB) was p r e p a r e d by a d d i n g 57 mL g l a c i a l a c e t i c a c i d , 5 8 g s o d i u m c h l o r i d e a n d 0.3 g s o d i u m c i t r a t e t o 500 mL o f d o u b l e d i s t i l l e d w a t e r . t o pH 5.0
-

37.2

ppm;

parsley,

29.1 ppm;

b o t h c a l c u l a t e d on

The r i s e i n u r i n a r y f l u o r i d e c o n c e n t r a t i o n c o u l d t h u s

be b r o u g h t i n r e l a t i o n w i t h a r e n e w e d e x p o s u r e t o e n v i r o n m e n t a l f l u o r i d e

except f o r sodium f l u o r i d e ,

a Merck suprapure product,

which

was p r e v i o u s l y d r i e d f o r 6 h o u r s a t 110C and s u b s e q u e n t l y s t o r e d i n a

Fluoride

standard s o l u t i o n s were prepared from t h e stock s o l u t i o n by d i l u t i n g w i t h

The s o l u t i o n was t i t r a t e d w i t h 5 M NaOtl

5.5 and f u r t h e r d i l u t e d t o 1 L w i t h d o u b l e d i s t i l l e d w a t e r .

158

Instrumentation The f o l l o w i n g i n s t r u m e n t a t i o n was u t i l i z e d : t y p e K R 1 7 0 (W.C. H e r a e u s G.M.B.H., Hanau); e l e c t r o d e (Beckman Model 39600): t r o d e (Beckman M o d e l 39402). Sample P r e p a r a t i o n Each bone s a m p l e was chopped i n s m a l l p i e c e s , w h i c h w e r e b o i l e d t w i c e i n d o u b l e d i s t i l l e d w a t e r f o r 10 m i n u t e s and d r i e d . d u r e s o f S i n g e r and A r m s t r o n g (4) and Zober and According t o t h e proceS c h e l l m a n n (5) a w e l l (1) m u f f l e f u r n a c e , Heraeus

(2) f l u o r i d e i o n s e l e c t i v e

s i n g l e j u n c t i o n reference calomel elec-

R e a d i n g s w e r e made on a Beckman E x p a n d o m a t i c

SS-2 pH m e t e r w i t h expanded s c a l e c a p a b i l i t y .

d e f i n e d a m o u n t o f d r y bone t i s s u e ( 1 t o 1 0 g r a m s ) was w e i g h e d i n a t a r e d q u a r t z c r u c i b l e w i t h c o v e r ( c a p a c i t y : 5 0 mL) a n d a s h e d f o r 1 5 h o u r s i n a m u f f l e f u r n a c e a t 550C. Fluoride Determination The a p p l i e d m e t h o d was a s l i g h t m o d i f i c a t i o n o f a t e c h n i q u e f o r t h e d e t e r m i n a t i o n o f f l u o r i d e i n v e g e t a t i o n p r e v i o u s l y d e s c r i b e d b y Van d e n tieede After cooling i n
2

desiccator,

t h e corresponding

amount o f a s h was d e t e r m i n e d and homogenized.

2 d.(6).

An a c c u r a t e l y w e i g h e d a m o u n t o f a s h ( 1 5 t o 2 0 mg) w a s The o b t a i n e d s o l u t i o n w a s p a r t i a l l y Finally the solution a d d i t i o n o f 2 0 mL a f l u o r i d e i o n selec-

d i s s o l v e d i n 3.5 mL o f 0.25 M HC1. b y t i t r a t i n g w i t h a 0.05

n e u t r a l i z e d b y a d d i n g 4 mL o f 0.125 M NaOH a n d f u r t h e r a d j u s t e d t o pH 4.7 M sodium a c e t a t e s o l u t i o n . was d i l u t e d t o 20 mL w i t h d o u b l e d i s t i l l e d w a t e r . After

o f a t o t a l i o n i c s t r e n g t h a d j u s t m e n t b u f f e r (TISAB),

t i v e e l e c t r o d e and a s i n g l e j u n c t i o n r e f e r e n c e c a l o m e l e l e c t r o d e were i m m e r s e d i n t o t h e s o l u t i o n w h i c h was s t i r r e d u n t i l a c o n s t a n t m i l l i v o l t a g e r e a d i n g was o b t a i n e d . standard stock solution. o f 0.25 D i f f e r e n t f l u o r i d e standard solutions, containing
To 5 mL o f each f l u o r i d e s t a n d a r d s o l u t i o n 3.5

f l u o r i d e i o n c o n c e n t r a t i o n s b e t w e e n 0.38 and 38 ppm, w e r e p r e p a r e d f r o m t h e mL The After M HC1 a n d 6 mL o f 0.125 M NaOH w e r e s u b s e q u e n t l y added.

s o l u t i o n w a s a d j u s t e d t o pH 4.7 b y t i t r a t i n g w i t h a 0.05 M s o d i u m a c e t a t e s o l u t i o n and f u r t h e r d i l u t e d t o 20 m l w i t h d o u b l e d i s t i l l e d w a t e r . f o r m e d as a l r e a d y d e s c r i b e d . a d d i t i o n o f 2 0 mL o f TISAB, a f l u o r i d e i o n a c t i v i t y m e a s u r e m e n t w a s p e r The f l u o r i d e c o n t e n t s o f t h e bone a s h s a m p l e s were c a l c u l a t e d from a c a l i b r a t i o n curve, drawn by p l o t t i n g t h e m i l l i voltage readings w i t h t h e standard solutions on the rectangular a x i s against t h e i r fluoride o f 58 m V p e r decade. i o n a c t i v i t i e s ( c o n c e n t r a t i o n s ) on t h e l o g a r i t h m i c T h i s g r a p h was a s t r a i g h t l i n e w i t h a s l o p e a x i s o f s e m i l o g a r i t h m i c paper.

159

RESULTS AND DISCUSSION

D a t a e x h i b i t i n g t h e r e p r o d u c i b i l i t y o f t h e proposed method f o r t h e d e t e r m i n a t i o n o f f l u o r i d e i n bone t i s s u e , "Trochanter Major" o f t h e femur, obtained by determining f l u o r i d e i n f i v e f r a g m e n t s o f b o t h t h e " P o s t e r i o r C a l v a r i u m " o f t h e s k u l l and t h e a r e s u m m a r i z e d i n T a b l e I. A l l b o n e o f w h i c h no s p e c i f i c a t i o n s were f r a g m e n t s o r i g i n a t e d f r o m t h e same body, before the actual study started.

a v a i l a b l e , s i n c e t h e y w e r e c o l l e c t e d t o p e r f o r m some p r e l i m i n a r y t e s t s Some i m p o r t a n t s p e c i f i c a t i o n s r e g a r d i n g t h e t h i r t e e n p o s t m o r t e r n c a s e s s t u d i e d a r e s h o w n i n T a b l e 11. TABLE

A l l data

REPRODUCIBILITY OF THE PROPOSED METHOO FOR THE DETERMINATION OF FLUORIDE I N BONE TISSUE ( F I V E FRAGMENTS O F BOTH THE POSTERIOR CALVARIUM OF THE SKULL AN0 THE TROCHANTER MAJOR OF THE FEMUR ARE TAKEN FROM THE SAME BODY) Nature o f the bone sample Skull 1 2 3
4

Weight (9) d r y bone ash 1.502 0.854 1.272 1.153 0.884 1.012 0.567 0.793 0.707 0.666

ratio' 1.5 1.5 1.6 1.6 1.5

F l u o r i d e C o n c e n t r a t i o n (ppm) d r y bone ash 1,112 863 71 1 705 795 837.20 166.86 74.62 19.93 844 931 836 700 852 832.6 83.34 37.27 10.01 1.650 1,300 1.140 1.150 1,175 1,283 214.93 96.12 16.75 1,300 1,475 1,300 1.125 1,400 1.320 131.58 58.84 9.97

x
SD

1.54 0.05 0.02 3.56 1.5 1.6 1.6 1.6 1.6

sx
CV% Femur 1 2 3 4 5 1.527 0.382 0.990 0.992 0.241 0.637 0.503 0.319
jT

0.808
0.524

1.50 0.04 0.02 2.83

SD

sx
CV%

' d r y bone/ash

160
TABLE I 1 SPECIFICATIONS ABOUT THE CASES STUDIED Case Sex Age Cause o f Death Profession a b

M
M F F

74 53
20

Drowning Drowning T r a f f i c accident F a l l from a b u i l d i n g Strangulation Drowning Drowning B a r b i t u r a t e overdose E x h a u s t i o n and f a l l T r a f f i c accident B r o n c h i a l carcinoma Cerebrovascular accident Cardiovascular s c l e r o s i s

Retired Dealer i n c a t t l e fodder Housekeeper Factory-worker Old servant Baker Reti red Metal-worker Housekeeper Housekeeper
So 1d ie r

4 days 1 day 3 days 3 days

1 day

2 3 4 5
6

50 70
63

M
M

3 days 3 days 3 days 1 day

1 day 70 days

7 8 9 10 11 12 13

M
M

73 49 79 74 53 76 72

F
F M F

Housekeeper Farmer

18 months

2 months

a: t i m e i n t e r v a l between d e a t h and autopsy o r exhumation b: l i v i n g o r w o r k i n g i n an a r e a suspected o f c o n t a m i n a t i o n

TABLE 111 DATA CONCERNING THE ASHING AND THE FLUORIDE DETERMINATIONS AS PERFORMED ON THE BONE T I S S U E S COLLECTED I N THIRTEEN POST MORTEM CASES AND A SURGICALLY REMOVED BONE FRAGMENT Case N Nature o f t h e bone sample Sternum (Manubrium) Femur (Caput) Rib Skull (Posterior Calvarium) Sternum (Manubrium) Femur (Caput) Rib Skull (Posterior C a l v a r i urn) Sternum (Manubrium) Femur (Caput) Rib Skull (Posterior Calvarium) Weight ( 9 ) d r y bone ash ratio F l u o r i d e Conc. (ppm) ash d r y bone

1.953 3.187 1.273 5.213 1.806 2.978 2.722 2.330 1.730 2.570 2.566 4.156

0.633 0.819 0.708 3.126 0.125 1.040 1.449 1.543 0.235 0.684 1.275 2.631

3.1 3.9 1.8 1.6 14.4 2.9 1.9 1.5 7.3 3.7 2.0 1.6

3.653 3,523 3.132 3,266 833 1,247 91 1 642 2,525 3.083 1.822 1,913

1,184 905 1,742 1,958 58 435 485 425 343 821 905 1,211

161

Sternum (Manubrium) Femur ( C a p u t ) Rib Skull (Posterior Calvarium) Sternum (Manubrium) Femur (Caput) Rib Skull (Posterior Calvarium) Sternum (Manu b r ium) Femur ( C a p u t ) Rib Skull (Posterior Calvarium) Sternum (Manubrium) Femur ( C a p u t ) Rib Skull (Posterior Calvarium) Sternum (Manubrium) Femur ( C a p u t ) Rib Skull (Posterior Cal v a r i urn)

2.632 3.305 2.185 4.141 4.094 12.130 3.361 5.991 3.792 6.197 3.533 7.442 3.132 7.110 2.584 5.314 5.447 6.242 2.489 7.810

0.081 1.648 1.319 2.685 0.819 3.806 1.848 3.61 5 0.637 2.335 1.355 4.585 0.558 2.527 1.231 3.306 1.526 2.045 1.006 4.778

32.5 2.0 1.7 1.5 5.0 3.2 1.8 1.6 6.0 2.7 2.6 1.6 5.6 2.8 2.1 1.6 3.6 3.0 2.5 1.6

223 452 369 31 1 1,636 1,655 1,288 1,532 1.199 1,380 1.131 869 1,529 2.134 1,636 1,665 1,016 883 908 767

7 225 223 201 327 51 9 708 925 201 520 434 535 21 2 7 58 779 1,036 285 289 367 469

Sternum (Manubrium) Femur ( C a p u t ) Rib Skull (Posterior Calvarium) Sternum (Manubrium) Femur (Caput) Rib Skull (Posterior Calvarium) Sternum (Manubrium) Femur ( C a p u t ) Rib Skull (Posterior Calvarium) Sternum (Manubrium) Femur ( C a p u t ) Femur ( T r o c h a n t e r ) Rib Skull (Posterior Calvarium) Sternum (Manubrium) Femur ( C a p u t ) Femur ( T r o c h a n t e r ) Rib Skull (Posterior Calvarium) Femur ( C a p u t )

3.574 4.463 2.646 3.895 5.426 5.132 3.471 4.077 3.017 6.041 1.860 4.606 1.139 2.295 1.276 0.746 1.955 2.818 2.497 2.289 2.320 3.803 5.392

0.858 1.053 0.816 2.378 1.392 1.724 0.997 2.667 0.582 1.402 0.987 2.899

4.2 4.2 3.2 1.6 3.9 3.0 3.5 1.5 5.2 4.3 1.9 1.6
-

1,162 1.475 1.023 955 798 850 787 749 877 825 71 1 700 1,300 1,403 1,061 1,157 1.033 1,414 1,598 1,483 1,357 1,109 2,177

266 348 31 5 583 205 286 226 490 169 191 377 441 433 528 467 432 668 576 644 508 581 709 963

10

11

12

13

14

' d r y bone/ash

162
c o n c e r n i n g t h e a s h i n g and t h e f l u o r i d e d e t e r m i n a t i o n s as p e r f o r m e d on t h e bone t i s s u e s c o l l e c t e d i n t h o s e t h i r t e e n cases, ("Caput" woman, w e r e l i s t e d i n T a b l e 111. and t h e bone f r a g m e n t o f t h e f e m u r ) w h i c h was s u r g i c a l l y removed f r o m t h e 71-year-old As c o u l d b e e x p e c t e d , t h e r a t i o o f t h e How-

w e i g h t o f t h e d r y bone t o t h e w e i g h t o f t h e c o r r e s p o n d i n g amount o f ash r e m a i n e d v e r y c o n s t a n t f o r c o m p a c t b o n e t i s s u e s s u c h as t h e s k u l l . range. ever, f o r h i g h l y v a s c u l a r i z e d bone t i s s u e s t h i s v a l u e v a r i e d o v e r a w i d e As i l l u s t r a t e d i n T a b l e s I V and V t h e f l u o r i d e l e v e l s f o u n d i n t h e 58.90

-

bone samples o r i g i n a t i n g f r o m t h e t e n r e f e r e n c e cases showed h i g h l y i n d i v i d u a l d i f f e r e n c e s (CV%: range. 68.21). However, t h o s e f o u n d i n t h e bone

=

s a m p l e s w h i c h o r i g i n a t e d f r o m a s i n g l e p e r s o n u s u a l l y w e r e i n t h e same T h i s i n d i c a t e d a good c o r r e l a t i o n (r 0.94 - 0.99) without a sign i f i c a n t d i f f e r e n c e (pO.05).

TABLE I V

MEAN VALUES OF THE FLUORIDE CONCENTRATIONS FOUND I N THE TEN REFERENCE CASES
Sternum Femur 1,668.20 984.44 311.31 59.01 Rib 1,300.70 766.08 242.86 58.90 Sku1 1 1,266.90 864.14 273.26 68.21

X
SD

1,457.10 982.87 310.81 67.45

SX
CVZ

X: mean; SD: s t a n d a r d d e v i a t i o n : SX: s t a n d a r d e r r o r : CV%: c o e f f i c i e n t o f variation %

TABLE V CORRELATION DATA CALCULATED FOR THE TEN REFERENCE CASES DeterminatioQ Intercept constant, r a 0.89 0.89 0.93 0.97 0.96 0.96 -114.20 72.97 -1 48.40 192.54 46.94 -1 75.38 Slope b 0.33 0.74 0.96 0.87 1.11 1.26 Correlation coefficient, r 0.94 0.95 0.96 0.99

femur femur femur skul 1 skul 1 rib (y=a+bx; iJ=lO;

skull rib sternum rib sternum sternum

p=NS)

0.98
0.98

163 It was a l s o i n t e r e s t i n g t o n o t e t h a t t h e maximum ( c a s e 1) a s w e l l a s t h e

m i n i m u m f l u o r i d e l e v e l s ( c a s e 4) w e r e f o u n d i n t h e bone s a m p l e s t a k e n f r o r r p e r s o n s who h a d b e e n l i v i n g i n r u r a l a r e a s , fluoride pollution. supposedly f r e e f r o m i n d u s t r i a l found T h i s suggests t h a t t h e f l u o r i d e concentrations

i n t h e f o u r suspected cases were w i t h i n t h e l i m i t s o f t h e s o - c a l l e d r e f e r ence v a l u e s o r blanks. population.

I t c o u l d be c o n c l u d e d t h a t u p t a k e and m i n e r a l i z a -

t i o n o f f l u o r i d e i n t h e bones o f t h e s e persons were t h o s e o f t h e random F r o m t h e s e f i g u r e s h o w e v e r i t was i m p o s s i b l e t o d e t e r m i n e persons. which were t h e fluoride-exposed CONCLUSION Cur i n v e s t i g a t i o n s
i.e.,

p e r f o r m e d o n a s e r i e s o f human bones i n d i c a t e d t h a t found indeed v a r i e d f r o m l o w t o h i g h levels,

the f l u o r i d e concentrations

f r o m a b o u t 300 t o 3 , 0 0 0 p p m ( i n b o n e ash). whose

H o w e v e r , i t w a s n o t e d t h a t f l u o r i d e c o n c e n t r a t i o n s u p t o 2,000 ppm ( i n b o n e a s h ) w e r e f o u n d i n a b o n e f r a g m e n t p r o c u r e d b y a n o l d woman, e x p o s u r e t o i n d u s t r i a l f l u o r i d e e m i s s i o n s was u n d e n i a b l e . p e r c e n t a g e o f t h e r e f e r e n c e c a s e s l o w e r l e v e l s w e r e found. h i g h amounts, however, were a l s o detected. I n a large Exceptionally

The r e a s o n f o r t h e s e e m e r g i n g

concentrations remained obscure since the available information did not s p e c i f y t h e source o f p o s s i b l e contamination. REFERENCES 1. V i g l i a n i EC (1959) Med d L a v o r o 50:323-327 F l u o r i d e s and iluman H e a l t h . H o d g e HC, S m i t h FA. G e d a l i a 1 ( 1 9 7 0 ) I n : W o r l d H e a l t h O r g a n i z a t i o n , Monograph S e r i e s No.59, Geneva, p p 141-161 V e r g o t e G, Van Z e l e W, B e l g 56: 130-1 34 Z o b e r A,

2.
3. 4. 5. 6.

De C l e r c k F,

H e y n d r i c k x A (1979) F a r m T i j d s c h r

S i n g e r L, A r m s t r o n g WD (1968) A n a l Chem 40:613-614 S c h e l l m a n n B (1975) Z K l i n Chem K l i n B i o c h e m 13:197-201 H e y n d r i c k x AM, Van Peteghem CH, Van Z e l e W A (1975) Van d e n Heede MA,

J A s s o c O f f A n a l Chem 58:1135-1137

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 165-172 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

165

DENTAL FLUOROSIS I N D U C E D BY WATER-BORNE FLUORIDE I N THE EFFLUENT O F A N ALUMINUM PLANT

TORU YAMAUCHI~,

YASUO H O N M A ~ , AND HUMIO

TSUNODA~

' D e p a r t m e n t o f P u b l i c H e a l t h , F u k u s h i m a M e d i c a l C o l l e g e , F u k u s h i m a 960, Japan 'Department o f H y g i e n e and P u b l i c H e a l t h , I w a t e M e d i c a l U n i v e r s i t y , Japan

ABSTRACT Twenty-six c a s e s o f d e n t a l f l u o r o s i s (DF) i n c l u d i n g p r e s u m a b l e c a s e s w e r e o b s e r v e d among y o u n g p e o p l e ( l e s s t h a n 24 y e a r s o l d ) i n a v i l l a g e w h e r e t h e a i r was s l i g h t l y p o l l u t e d b y a i r - b o r n e f l u o r i d e ( F ) f r o m an a l u m i n u m p l a n t .

I t was shown b y an e p i d e m i o l o g i c a l s t u d y t h a t s t r e a m s i n t h e v i l l a g e w e r e
c o n t a m i n a t e d b y h i g h l e v e l s o f F f r o m an e f f l u e n t o f t h e a l u m i n u m p l a n t and t h e r e s i d e n t s u s u a l l y consumed t h e s e s t r e a m - w a t e r s purposes, w i t h o u t any knowledge o f F p o l l u t i o n . daily, even f o r d r i n k i n g The h i g h l e v e l o f F i n t h e

e f f l u e n t r e s u l t e d f r o m a wet scrubber system i n t h e plant.

INTRODUCTION T h e r e h a v e been many r e p o r t s on e n v i r o n m e n t a l p o l l u t i o n b y a i r - b o r n e f l u o r i d e f r o m i n d u s t r i a l s o u r c e s (1-4). i n g water-borne o f water-borne fluoride, However, few papers e x i s t concernAs f o r t h e h e a l t h e f f e c t f l u o r i d e f r o m i n d u s t r i a l sources.

a l m o s t a l l t h e cases r e p o r t e d e a r l i e r were caused

by consumption o f water containing high concentrations o f f l u o r i d e from s u c h n a t u r a l s o u r c e s as s p r i n g s o r w e l l - w a t e r s f l u o r i d e f r o m an i n d u s t r i a l s o u r c e i s r e p o r t e d .

(5,6).
Although i t occurred about

I n t h i s paper, t h e o u t b r e a k o f d e n t a l f l u o r o s i s i n d u c e d b y w a t e r - b o r n e

15 y e a r s ago,

t h e r e s u l t s o f t h e study are s t i l l considered t o be valuable

today i n terms o f c o n t r o l l i n g environmental p o l l u t i o n . MATERIALS AND METHODS The s u b j e c t a r e a o f t h e e p i d e m i o l o g i c a l s u r v e y was a s m a l l v i l l a g e named Nagao o f K i t a k a t a C i t y , Japan (Fig. 1). i n t h e n o r t h w e s t e r n p a r t o f Fukushima Prefecture, determined p r i o r t o t h e epiT h e r e was an a l u m i n u m p l a n t l o c a t e d a b o u t 2 km f r o m Nagao. survey, a r e a l s o shown i n F i g u r e 1

F l u o r i d e l e v e l s i n t h e a i r around t h e p l a n t , demiological surrounded m a i n l y by r i c e f i e l d s .

(2,4).

The p l a n t was

166

F i g u r e 1. S u r v e y e d a r e a a n d f l u o r i d e l e v e l s Tn t h e a i r (2). s e n t a t m o s p h e r i c f l u o r i d e l e v e l s (ugF/dm2/mon.).

Figures repre-

The s u b j e c t s o f t h e e p i d e m i o l o g i c a l s u r v e y w e r e a l l r e s i d e n t s o f Nagao and w e r e 7 t o 6 9 y e a r s o f age. graphs. The s u r v e y i n c l u d e d t h e f o l l o w i n g : 1. D e n t a l e x a m i n a t i o n o f a l l s u b j e c t s b y d i r e c t e x a m i n a t i o n a n d p h o t o -

2. G e n e r a l h e a l t h e x a m i n a t i o n o f t h e
i n c l u d i n g p h y s i c a l examination, (protein, g l u c o s e and pH).

s u b j e c t s t e n y e a r s o l d and above, and u r i n a l y s i s

blood analysis (hematocrit),

3.

M e a s u r e m e n t o f f l u o r i d e c o n t e n t i n u r i n e ( s p o t and 24 h o u r u r i n e ) . and t h e d e t e r m i n a t i o n s

4. E n v i r o n m e n t a l s u r v e y i n c l u d i n g a q u e s t i o n n a i r e a b o u t t h e s o u r c e s o f
w a t e r f o r d a i l y c o n s u m p t i o n i n t h e p a s t and p r e s e n t , o f f l u o r i d e l e v e l s i n w e l l - w a t e r and s t r e a m - w a t e r RESULTS The s u r v e y was c o n d u c t e d i n 1971.
i n t h e e x a m i n a t i o n (85% c o v e r a g e ) ,

i n the village.

O u t o f 220 s u b j e c t s , and i n p a r t i c u l a r ,

185 p a r t i c i p a t e d

a l l c h i l d r e n 7 t o 15

years o l d received t h e examination. Dental examination The r e s u l t s o f t h e d e n t a l e x a m i n a t i o n a r e shown i n F i g u r e 2. the prevalence o f dental f l u o r o s i s (mottled teeth)

A s t u d y on

indicated that the

167

DMF Rate 20

- 24 (9)

1 p

?,S

0.0

16 18 (1 0 13 15 (1 7) 10 12 I 2 1)
1 - $

(1 5)

SO\

30 20 10 0 PREVALENCE OF MOTTLED TEETH

48

Ap. (n)

DMFTlperson

F i g u r e 2.

Results o f dental examination

highest occurrence t h e s u b j e c t s were, 24,

was i n t h e 10 t o 15 y e a r age g r o u p , t h e lower t h e prevalence.

and t h a t t h e o l d e r

No m o t t l e d t e e t h w e r e
and

o b s e r v e d i n t h e g r o u p aged 25 y e a r s o r o l d e r . a n o t h e r 1 3 c a s e s a s h a v i n g m o t t l e d t e e t h (MI t h e s e c a s e s i s i l l u s t r a t e d i n F i g u r e 3.

O u t o f 78 SubJeCtS aged 7 t o

1 3 c a s e s (17%) w e r e d i a g n o s e d a s p r e s u m a b l e d e n t a l f l u o r o s i s (MQ),

M2).

The d i s t r i b u t i o n o f

Wet scrubber in Aluminum Plant

F i g u r e 3.

D i s t r i b u t i o n o f d e n t a l f l u o r o s i s and F - l e v e l s (ppm). 0 ,c a s e o f d e n t a l f l u o r o s i s ; Q , case o f presumable d.f.

i n stream-waters

168
I n contrast, t h e DMF (decayed, missing, and f i l l e d ) r a t e i n d i c a t e d t h a t who a l s o h a d t h e h i g h e s t

t h e l o w e s t l e v e l was i n t h e g r o u p aged 1 0 t o 1 5 prevalence o f dental fluorosis. Fluoride content i n urine

DMFT/person was t h e l o w e s t i n t h e g r o u p

aged 7 t o 9 y e a r s and i n c r e a s e d w i t h age o f t h e s u b j e c t s . F l u o r i d e c o n c e n t r a t i o n s f o u n d i n t h e 24 h o u r u r i n e a r e shown i n F i g u r e 4. The mean l e v e l o f f l u o r i d e i n u r i n e i n c r e a s e d w i t h i n c r e a s e i n age u n t i l I n n e i t h e r spot t h e age g r o u p o f 50 y e a r s i n m a l e and 4 0 y e a r s i n f e m a l e .

n o r 24 h o u r u r i n e s a m p l e s w e r e a b n o r m a l l y h i g h l e v e l s o f f l u o r i d e found.

1.5 -Male

i O T 10
r

2Ll
29

30 3 9
49

19

5:0 59

6,0 AGE GROUP

F i g u r e 4.

F-contents

n 24 hour u r i n e samples o f t h e r e s i d e n t s .

Other h e a l t h examination F i g u r e 5 shows t h e mean l e v e l o f h e m a t o c r i t ( H t ) b y d i f f e r e n t age groups. No a b n o r m a l H t l e v e l s n o r p a t h o l o g i c a l c o n d i t i o n s w e r e o b s e r v e d i n t h e examination. F l u o r i d e l e v e l i n w a t e r samples The f l u o r i d e c o n c e n t r a t i o n s i n t h e w e l l - w a t e r s a m p l e s a r e shown i n T a b l e

I. E x c e p t f o r o n e s a m p l e
was 0.057 ppm.

w h i c h s h o w e d m o r e t h a n 0.1 ppm.

37 o u t o f 46

s a m p l e s (80%) g a v e l e v e l s w i t h i n 0.050 t o 0 . 0 9 9 ppm. a n d t h e mean F l e v e l The f l u o r i d e l e v e l s i n 20 w a t e r s a m p l e s f r o m t h e s t r e a m s i n Nagao v i l l a g e a r e shown i n F i g u r e 3. i n t o t h e s e streams. 8.20 ppm. The e f f l u e n t f r o m t h e a l u m i n u m p l a n t was d i s c h a r g e d The mean F l e v e l was 3.34 ppm w i t h a r e r g e o f 2.10 t o

l e v e l s o f t h e samples taken f r o m s i t e s near t h e p l a n t were

h i g h e r than those o f samples taken f r o m t h e streams d i s t a n t f r o m t h e plant.

169

I Male

V'

A 0g e Group

12 1519 2 9

39

49

59

69

F i g u r e 5.

D i s t r i b u t i o n o f h e m a t o c r i t b y age g r o u p s .

TABLE I FLUORIDE LEVEL I N WELL-WATERS Fluoride level (PPm) L e s s t h a n 0.049 Number o f w e l l s

8 37

(17.4%) (80.4%)

0.050

0.099

0.100 o r more
Total

l a ( 2.2%)
46
~~

aMean l e v e l was 0.68 ppm (0.057 ppm i f t h e h i g h e s t one was e x c l u d e d ) .

On e x a m i n a t i o n o f t h e d i s t r i b u t i o n o f d e n t a l f l u o r o s i s c a s e s , m a n y w e r e d i s t r i b u t e d i n t h e west p a r t o f t h e v i l l a g e where t h e f l u o r i d e l e v e l s i n t h e stream-water w e r e h i g h e r t h a n 3.0 ppm. Results o f questionnaire F i g u r e 6 shows t h e a n n u a l t r e n d o f t h e s o u r c e s o f w a t e r f o r d a i l y cons u m p t i o n i n Nagao, b a s e d o n r e s u l t s o f t h e q u e s t i o n n a i r e . daily, and h a l f I n t h e 1950s, n e a r l y 80% o f t h e r e s i d e n t s c o n s u m e d t h e s t r e a m - w a t e r n e a r t h e i r h o u s e s
3f

them used t h e w a t e r f o r d r i n k i n g purposes. These propor-

t i o n s b e g a n t o d e c r e a s e i n t h e b e g i n n i n g o f t h e 1960s w h e n t h e a l u m i n u m p l a n t began t o t r e a t i t s e m i s s i o n w i t h wet-scrubbers.

170

F i g u r e 6.

H i s t o r y o f w a t e r s u p p l y and consumption 1 Aluminum p l a n t began t o t r e a t i t s e f f l u e n t . 2 Many c a r p i n t h e streams d i e d f r o m an unknown cause. 3 Aluminum p l a n t began t o o p e r a t e i t s wet-scrubber system.

DISCUSSION
S e v e r a l c a s e s o f p r e s u m e d d e n t a l f l u o r o s i s w e r e o b s e r v e d among s c h o o l c h i l d r e n i n Nagao v i l l a g e , Fukushima P r e f e c t u r e , Japan, fluoride were i n 1968 and 1969. There was an aluminum p l a n t near Nagao and i t was known t h a t t h e a i r i n t h e s u r r o u n d i n g a r e a was p o l l u t e d b y a i r - b o r n e Although the f l u o r i d e l e v e l s i n the those i n non-polluted several air s e r i o u s enough t o a f f e c t cases o f d e n t a l (Figs. 1 , 2 ) , t h e y fluorosis from the plant. a i r i n 1968 were s l i g h t l y h i g h e r t h a n n o t c o n s i d e r e d t o be I n fact, villages only

the health o f the residents. were found i n o t h e r

(3)

( T a r o m a r u v. and T a k a y o s h i v., t h a t i n Nagao as w e l l .

F i g . 1 ) w h i c h w e r e c l o s e r t h a n Nagao t o t h e therefore, was planned

a l u m i n u m p l a n t , and w h e r e t h e f l u o r i d e l e v e l i n t h e a i r was h i g h e r t h a n An e p i d e m i o l o g i c a l survey, t o i n v e s t i g a t e t h e cause o f t h e o u t b r e a k o f d e n t a l f l u o r o s i s i n Nagao. The aluminum p l a n t s t a r t e d i t s o p e r a t i o n i n 1943, p r o d u c t i o n was mo.re t h a n 50,000 tons. The p r e v a l e n c e o f d e n t a l f l u o r o s i s among y o u n g p e o p l e l i v i n g i n l o c a l areas where f l u o r i d e c o n t a m i n a t e d w a t e r was consumed had been e s t i m a t e d t o b e 1 0 t o 1 3 % (7). hand, T h i s s u r v e y showed a p r e v a l e n c e o f 33.3%. On t h e o t h e r t h e p r e v a l e n c e of d e n t a l c a r i e s i n Nagao was compared w i t h t h o s e i n and i n 1965 t h e annual

Y a m a s h i n a c i t y and S h u g a k u i n t o w n ( 6 ) , b o t h i n K y o t o . I n Y a m a s h i n a c i t y ,

171
TABLE I 1

COMPARISON OF DMF RATE AND DMFT/PERSON I N NAGAO V. OTHER AREAS

Area

WITH

Nagao V.

7- 9 10-1 2 13-1 5

6.9 5.1 5.8 4.9-1 1.3 10.6-1 1 .4 12.7-14.1 1 1 .8-20.0 13.5-17.3 16.6-23.2

0.73 1 .oo 1.59 0.32-1.48 1.73-3.81 3.34-3.81 0.84-2.28 2.90-4.17 4.43-6.46

Yamashina C. ( 1 964)

(6)

7- 9 10-12 13-1 5

Shugakuin T. ( 1 964)

(6)

7- 9 10-1 2 13-1 5

f l u o r i d a t e d w a t e r was s u p p l i e d .

B o t h D M F r a t e and DMFT/person i n Nagao

w e r e much l o w e r t h a n i n t h o s e t w o a r e a s ( T a b l e 11). young people aged 10 t o 15. r e s i d e n t s i n Nagao. However,

A high prevalence o f

d e n t a l f l u o r o s i s and a l o w p r e v a l e n c e o f d e n t a l c a r i e s were observed among R e s u l t s f r o m t h e d e n t a l e x a m i n a t i o n suggested the low content o f f l u o r i d e i n the u r i n e o f consumed a t t h e t h e p o s s i b i l i t y o f exposure t o r e l a t i v e l y h i g h l e v e l s o f f l u o r i d e by t h e t h e r e s i d e n t s and t h e l o w l e v e l o f f l u o r i d e i n w e l l - w a t e r s t h e p a s t and n o t a t t h e t i m e o f t h e survey. A n a l y s i s o f t h e q u e s t i o n n a i r e showed t h a t n e a r l y 8 0 % o f t h e r e s i d e n t s consumed t h e s t r e a m - w a t e r n e a r t h e i r h o u s e s d a i l y , ( F i g . 6). high. The f i n d i n g s o f t h e e p i d e m i o l o g i c a l survey d e s c r i b e d above have l e a d t o t h e f o l l o w i n g conclusion: The e f f l u e n t f r o m t h e a l u m i n u m p l a n t c a u s e d T h i s had o c c u r r e d s i n c e to f l u o r i d e p o l l u t i o n o f t h e streams i n t h e v i l l a g e . and,
i n particular,

t i m e o f survey suggested t h a t t h e f l u o r i d e p o l l u t i o n m i g h t have o c c u r r e d i n

a b o u t h a l f o f t h e m consumed t h e w a t e r f o r d r i n k i n g p u r p o s e s i n t h e 1 9 5 0 s T h i s c o u l d e x p l a i n why c a s e s o f d e n t a l f l u o r o s i s w e r e d i s t r i b u t e d a l o n g t h e streams where t h e f l u o r i d e c o n c e n t r a t i o n s o f t h e w a t e r were

t h e b e g i n n i n g o f t h e 1960s, when t h e p l a n t i n s t a l l e d a w e t - s c r u b b e r i n t o t h e streams.

c o l l e c t gas c o n t a i n i n g h i g h l e v e l s o f f l u o r i d e and d i s c h a r g e d t h e e f f l u e n t The r e s i d e n t s a l o n g t h e s t r e a m s had consumed t h e stream-

172
w a t e r w i t h o u t any k n o w l e d g e o f t h e p o l l u t i o n . were j u s t growing a t t h a t time. I n t h e 1960s, h a r d l y anybody, n a t u r a l l y n o t t h e residents o r t h e workers o f t h e p l a n t , o r even t h e s c i e n t i s t s s p e c i a l i z i n g i n f l u o r i d e p o l l u t i o n , c o n s i d e r e d t h e hazardous e f f e c t o f water-borne f l u o r i d e discharged from i n d u s t r i a l s o u r c e s (5.6). fluoride, While air-borne f l u o r i d e i s known t o a f f e c t water-borne v e g e t a t i o n and a n i m a l s s u c h a s l i v e s t o c k g r a z i n g o n g r a s s , i r r e s p e c t i v e o f i t s source, The h i g h l e v e l s o f f l u o r i d e

i n t h e i r d r i n k i n g w a t e r c a u s e d d e n t a l f l u o r o s i s i n y o u n g p e o p l e whose t e e t h

may d i r e c t l y a f f e c t human h e a l t h .

The e p i s o d e r e p o r t e d h e r e s u g g e s t s t h a t a m e a s u r e t a k e n a g a i n s t one t y p e o f e n v i r o n m e n t a l p o l l u t i o n m i g h t have caused a n o t h e r t y p e o f p o l l u t i o n w h i c h a d v e r s e l y had a f f e c t e d t h e h e a l t h o f t h e r e s i d e n t s . REFERENCES

1.

A g a t e JN, B e l l GH, B o d d i e GF, B o w l e r RG. B u c k e l l M, C h e e s e m a n EA, D o u g l a s THJ. D r u e t t HA, G a r r a d J , H u n t e r 0, P e r r y K M A . R i c h a r d s o n JD, d e V.Weir JB ( 1 9 6 8 ) I n d u s t r i a l f l u o r o s i s : a s t u d y o f t h e h a z a r d t o man and a n i m a l n e a r F o r t W i l l i a m , S c o t l a n d . Med Res C o u n c i l Mem No 22, 47. HM S t a t i o n a r y O f f i c e , London T s u n o d a H, 59: 2-1 1 Haneda M (1968) Sangyou-Kougaku ( I n d u s t r i a l Engineering)

2.
3. 4. 5. 6.

Tsunoda H (1968) J J p n SOC A i r P o l l 2:135-137 Tsunoda H (1970) J E n v i r o n P o l l C o n t r o l 6(7):16-20 and 6(8):3-8

Cornmi t t e e o n B i o l o g i c a l E f f e c t o f A t m o s p h e r i c P o l l u t i o n : F l u o r i d e s ( 1 9 7 1 ) N a t Acad S c i e n c e s , W a s h i n g t o n , DC, pp 5-11 L e o n e NC, M a r t i n AE. M i n o g u c h i G, S c h l e s i n g e r ER, S i d d i q u i A H ( 1 9 7 0 ) WHO (Geneva): 2 7 3 - 3 2 1 T s u j i Y (1973) J E n v i r o n P o l l C o n t r o l 9(4):4-10

7.

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 173-18C 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

173

COMMUNITY DENTAL FLUOROSIS AND THE FLUORIDE CONTENT OF SALIVA AND U R I N E I N THAI CHILDREN PRATHIP PHANTUMVANIT172, Y U P I N SONGPAISAN2,

AND R U D I G. SCHAMSCHULA3
Chulalongkorn

'Department o f Biochemistry, F a c u l t y o f D e n t i s t r y , U n i v e r s i t y , H e n r i - D u n a n t Road, Bangkok 10500, T h a i l a n d 'Intercountry Thai land Centre f o r Oral Health, Sydney,

M i n i s t r y o f Pub1 i c H e a l t h , C h i a n g m a i , Australia

3 1 n s t i t u t e o f D e n t a l Research,

ABSTRACT C o m m u n i t y i n d i c e s o f f l u o r o s i s ( F c i ) and t h e f l u o r i d e ( F ) c o n c e n t r a t i o n s i n u r i n e and s a l i v a w e r e assessed i n 184 c h i l d r e n , f r o m Chiangmai Province, Thailand, aged 11 t o 13 y e a r s , The who h a d been c o n s u m i n g w a t e r c o n t a i n i n g

0.05 ppm, 0 . 3 8 ppm, 0.76 ppm, 1.76 ppm. a n d 3.09 ppm F, r e s p e c t i v e l y .

t e n t l y w i t h increase i n F c o n t e n t o f t h e water supply, r e l a t i o n s h i p i n each instance. t h e w a t e r was 0.44 ppm; An F c i l e v e l o f 0.4, considered i n s i g n i f i c a n t i n populations, t r a t i o n s w e r e 0.84 ppm and 33.2 ppb.

mean F c i v a l u e s a n d t h e u r i n a r y a n d s a l i v a r y F l e v e l s i n c r e a s e d c o n s i s exhibiting a sigmoid a t which fluorosis i s

was r e a c h e d when t h e F c o n t e n t i n These r e s u l t s c o n f i r m e d indicators o f F

c o r r e s p o n d i n g mean u r i n a r y and s a l i v a r y F concenrespectively.

t h e usefulness o f u r i n a r y and s a l i v a r y F as short-term exposure i n small groups o f children.

The f i n d i n g s a l s o c a u t i o n a g a i n s t

applying e x i s t i n g formulas f o r determining optimal F l e v e l s o f water supply i n d e v e l o p i n g and t r o p i c a l c o u n t r i e s w i t h o u t e x p l o r i n g t h e d i e t a r y and n u t r i t i o n a l c h a r a c t e r i s t i c s o f t h e community concerned.

INTR3DUCTION T h a i l a n d i s one o f t h e m a i n f l u o r s p a r p r o d u c i n g c o u n t r i e s i n t h e w o r l d . Much o f t h e m i n e r a l i s m i n e d f r o m numerous d e p o s i t s s i t u a t e d i n t h e n o r t h e r n p a r t o f t h e c o u n t r y w h e r e L e a t h e r w o o d and a s s o c i a t e s f o u n d h i g h p r e v a l e n c e o f d e n t a l f l u o r o s i s (1). E n d e m i c f l u o r o s i s h a s been c o n f i r m e d where f l u o r i d e ( F ) to r a n g i n g f r o m 0.01 i n t h e n o r t h e r n p r o v i n c e o f Chiangmai i n a r e c e n t study, mapping o f d r i n k i n g w e l l w a t e r s d i s c l o s e d v a r i a t i o n s 4.38 ppm ( 2 ) . highly contrasting levels o f F n;Ltilr I
Y ,G-

The e x i s t e n c e o f s u b s t a n t i a l r u r a l p o p u l a t i o n s exposed t o
17

r a t e r p r o v i d e d an o p p o r t u n i t y f o r

f u r t h e r i n v e s t i g a t i o n s of f l u c r ? ; ' e

7nd u t i l i z a t i o n .

174
The a s s o c i a t i o n b e t w e e n F c o n c e n t r a t i o n s i n d r i n k i n g w a t e r and t h e p r e v a l e n c e and s e v e r i t y o f d e n t a l f l u o r o s i s h a s b e e n w e l l e s t a b l i s h e d u n d e r t e m p e r a t e c l i m a t i c c o n d i t i o n s (3,4). q u a n t i t a t i n g c u r r e n t F i n t a k e (5). Under t h e s e c o n d i t i o n s , I n contrast, the F content o f u r i n e has been w i d e l y used as an i n d i c a t o r o f F u t i l i z a t i o n and f o r s a l i v a r y F h a s r a r e l y been u t i l i z e d f o r corresponding purposes i n f i e l d studies. The p u r p o s e o f t h e p r e s e n t s t u d y was t o e x a m i n e t h e i n t e r r e l a t i o n s between a wide range o f n a t u r a l l y o c c u r r i n g F i n t h e water, indices o f dental fluorosis (Fci)(6) and t h e F levels of community u r i n e and

a c t i v a t e d whole saliva, under t h e t r o p i c a l c o n d i t i o n s p r e v a i l i n g i n t h e C h i a n g m a i area. MATERIALS AND METHODS P r i o r t o f i n a l d e f i n i t i o n o f t h e sample, t o 13 years, 1,687 s c h o o l c h i l d r e n , aged 11 who w e r e c o n s t a n t r e s i d e n t s o f o n e o f t h e 71 r u r a l c o m m u n i t i e s were asked t o c o l l e c t w a t e r f e l l within the f i v e F divia n d 2.4-4.8 ppm. were The Those c h i l d r e n whose combined w a t e r 1.5-2.0,

o f t h r e e n e i g h b o u r i n g d i s t r i c t s o f Chiangmai, samples f r o m t h e i r d r i n k i n g w e l l s . supply, sions, i.e., i n c l u d i n g h o u s e h o l d and s c h o o l w a t e r ,

<0.1:

0.3-0.5:

0.7-0.9;

r e g a r d e d as e l i g i b l e and were asked t o p a r t i c i p a t e i n t h e study. sample comprised 184 o f t h e consenting c h i l d r e n , f u l f i l l q u o t a s o f 30 t o 40 p e r F d i v i s i o n , t i o n s o f b o t h sexes.

randomly selected t o

w i t h approximately equal proporF l u o r o s i s was r e c o r d e d

Dental examinations were c a r r i e d o u t a t t h e schools

i n n a t u r a l d a y l i g h t u s i n g v i s u a l and t a c t i l e means. tooth, and averaged f o r each s u b j e c t .

a c c o r d i n g t o Dean's i n d e x (6) as t h e i n d i v i d u a l s c o r e f o r e a c h p e r m a n e n t The F c i means w e r e c a l c u l a t e d separ a t e l y f o r each o f t h e f i v e F d i v i s i o n s . Two s a m p l e s e a c h o f u r i n e and s t i m u l a t e d w h o l e s a l i v a w e r e c o l l e c t e d f r o m e a c h s u b j e c t i n t h e m o r n i n g w i t h an i n t e r v a l o f t w o weeks b e t w e e n t h e duplicates. buffer, F i e l d b l a n k s w e r e made u p w i t h e a c h b a t c h o f s a m p l e s . The s a m p l e s and b l a n k s w e r e a n a l y z e d f o r F i n a 1O:l d u r e s d e s c r i b e d p r e v i o u s l y (7). The r e l a t i o n s h i p s between t h e F c o n c e n t r a t i o n s i n t h e w a t e r s u p p l y and t h o s e i n t h e b i o l o g i c a l samples, and t h e F c i o b t a i n e d f r o m e a c h F d i v i s i o n s u s p e n s i o n o f TISAB

u s i n g a s p e c i f i c i o n e l e c t r o d e ( O r i o n 940911) and s t a n d a r d p r o c e -

were examined u s i n g l i n e a r r e g r e s s i o n analyses. RESULTS T a b l e I shows t h e m a i n c h a r a c t e r i s t i c s o f t h e s a m p l e s s t u d i e d , t i o n o f children according t o F divisions. including t h e F c o n c e n t r a t i o n s i n t h e h o u s e h o l d and s c h o o l w a t e r s and t h e d i s t r i b u -

175
TABLE I SAMPLE CHARACTERISTICS No. o f Children M F Total 15 16 24 24 15 24 39 38 39 40 28

F division
1

Range ( PPm)

Household 0.05k0.04a 0.39k0.14 0.74k0.15 1.82+0. 34 3.50k1.20

F i n w a t e r (ppm)
School 0.04k0.02 0.38k0.06 0.78+0.20 1.69kO. 11 2.67k0.84

Combined 0.05k0.03 0.38k0.09 0.76k0.08 1.76kO. 17 3.09kO. 57

<o. 1
0.3-0.5 0.7-0.9 1.5-2.0 2.4-4.8

2
3 4 5

22
15 16 13

aMeans k S.D.

The mean u r i n a r y and s a l i v a r y F c o n c e n t r a t i o n s and t h e F c i v a l u e s i n e a c h d i v i s i o n a r e shown i n T a b l e 11. V a r i a t i o n s o f t h e u r i n a r y and s a l i v a r y F l e v e l s i n each o f t h e F d i v i s i o n s a r e i n d i c a t e d by standard deviations. Several o f these a r e high, b u t a r e n o t excessive i n t h e c o n t e x t o f i n d i v i d ual physiological differences. v a r i a t i o n (CV), 30-502, The g e n e r a l r a n g e o f t h e c o e f f i c i e n t s o f I n contrast, the coefficients f o r Fci 1.e.. 2672, i n d i c a t e s moderate o v e r l a p o f values between neigh-

b o u r i n g d i v i s i o n s f o r m o s t samples. 129%, and 952,

values are h i g h l y variable i n the f i r s t three F divisions,

respectively, r e f l e c t i n g t h e occasional incidence o f s i g n i f t h e mean u r i n a r y F c o n c e n t r a t i o n a l w a y s exceeds t h a t T h i s e f f e c t i s most

i c a n t degree o f f l u o r o s i s a t r e l a t i v e l y l o w F l e v e l s i n t h e water. I n absolute terms, o f water, i n d i c a t i n g c o n t r i b u t i o n f r o m o t h e r sources.

TABLE I 1 FLUORIDE CONCENTRATIONS I N D R I N K I N G WELL WATER, U R I N A R Y A N D SALIVARY SAMPLES, AND COMMUNITY FLUOROSIS I N D E X ( F c i ) ACCORDING TO F D I V I S I O N

F Division

Water-F (PPm) 0.05+0. 04a 0.39kO. 1 4 0.74kO. 1 5 1.82kO. 34 3.50k1.20

Urinary F ( PPm) 0.33kO. 1 4 0.86k0.32 1.03k0.49 2.57k1.08 3.79k1.84

Salivary F (PPb) 28.2k12.5 32.4k14.0 34.4k14.1 58.0k25.6 77.1k36.7

Fci

0.06k0.16 0.31 k0. 40 0.64k0.61 1.52kO. 72

3
4 5 aMeans k S.D.

2.30kO. 73

176

.C

q
1

T I

T 3
Well-water fluoride, ppn1
The relationship b e t w e e n f l u o r i d e c o n c e n t r a t i o n i n t h e d r i n k i n g F i g u r e 1. w e l l w a t e r i n C h i a n g m a i and community f l u o r o s i s i n d e x ( F c i ) .

1 I I

Well-water

fluoride, ppm

F i g u r e 2. The r e l a t i o n s h i p b e t w e e n w e l l - w a t e r r i d e i n c h i l d r e n o f Chiangmai.

f l u o r i d e and u r i n a r y f l u o -

177
a p p a r e n t i n t h e l o w F d i v i s i o n s and i s i n c r e a s i n g l y m a r k e d w i t h i n c r e a s e i n

F c o n c e n t r a t i o n i n t h e water.
t o F i n u r i n e i s 1:6.6,

Thus,

i n Division 1 the r a t i o o f F i n water

w h e r e a s i n D i v i s i o n 5 i t i s 1:1.08.

With s a l i v a r y

F,

t h e a b s o l u t e c o n c e n t r a t i o n i s a l w a y s much l o w e r t h a n t h a t i n w a t e r and The a s s o c i a t i o n b e t w e e n t h e F c o n t e n t o f w e l l w a t e r and t h e F c i ( F i g . 1).

u r i n e , b u t s t i l l shows a c o n s i s t e n t i n c r e a s e w i t h b o t h p a r a m e t e r s . u r i n a r y F (Fig. (r=0.989-0.990).

Z ) , and s a l i v a r y F (Fig.
However,

3) shows a r i s i n g t r e n d i n t h e
i n part, to

lneans w i t h i n c r e a s e d f l u o r i d e e x p o s u r e . The c o r r e l a t i o n i s n e a r l y a b s o l u t e i t s h o u l d be n o t e d t h a t t h i s i s due, variability. t h e u s e o f means, which eliminates within-group The r e g r e s -

s i o n e q u a t i o n s and t h e s l o p e s o f t h e l i n e s o f b e s t f i t i n d i c a t e t h a t t h e u n i t change i n each dependent v a r i a b l e i s s u b s t a n t i a l w i t h change i n F concentration i n t h e water.

A closer examination o f Figures 1 - 3 dis-

c l o s e s a d e f i n i t e t r e n d o f s i g m o i d shape between each p a i r o f v a r i a b l e s , indicating t h a t the cause-effect r e l a t i o n s h i p i s c o n s i d e r a b l y reduced a t t h e l o w and h i g h e n d s o f t h e s c a l e s .

1
I

Well-water

fluoride, ppm

F i g u r e 3. The r e l a t i o n s h i p b e t w e e n w e l l - w a t e r r i d e i n c h i l d r e n o f Chiangmai.

f l u o r i d e and s a l i v a r y f l u o -

178
DISCUSSION
The h i g h c o r r e l a t i o n b e t w e e n F c o n t e n t o f w a t e r and F c i f o u n d i n t h i s s t u d y i s i n a c c o r d w i t h p r e v i o u s o b s e r v a t i o n s i n t e m p e r a t e c l i m a t e s (3,4). However, t h e 0.4 l i m i t o f F c i , w h e r e Dean (8) c o n s i d e r e d t h e p r e v a l e n c e and was r e a c h e d i n t h e p r e s e n t s t u d y a t a ppm ( F i g . severity o f fluorosis insignificant, c o n t r a s t w i t h t h e 0.8-1.2 t e m p e r a t e areas. 0.44-0.74

v e r y l o w w a t e r F c o n c e n t r a t i o n o f 0.44 Similarly,

1).

This i s i n sharp w h i c h was r e p o r t e d corresponded t o ppm)(4). One

ppm F r a n g e u s u a l l y c o n s i d e r e d a p p r o p r i a t e f o r t h e F c i r a n g e o f 0.4-0.6,

b y Dean ( 8 ) t o b e o f b o r d e r l i n e p u b l i c h e a l t h s i g n i f i c a n c e , t o r s i n t h e U.S.A. (1.0-1.5 p p m ) ( 3 ) a n d i n D e n m a r k (1.5-2.0

ppm F i n t h e w a t e r , a s o p p o s e d t o r a n g e s r e p o r t e d b y i n v e s t i g a -

obvious reason f o r t h e d i f f e r e n c e s between these f i n d i n g s i s t h e higher w a t e r i n t a k e under t r o p i c a l c o n d i t i o n s . ( T a b l e 11) s u g g e s t s t h a t o t h e r f a c t o r s , However, a c o n s i s t e n t and s u b s t a n such as F i n foods, may c o n t r i b u t e t i a l e l e v a t i o n o f u r i n a r y F o v e r t h e F c o n t e n t o f w a t e r shown i n t h i s s t u d y s i g n i f i c a n t l y t o F exposure and t h e h i g h Fci. j e c t s t o F u t i l i z a t i o n and d e n t a l f l u o r o s i s , c h i l d r e n (10). I n t h e same c o n t e x t , A n o t h e r a s p e c t t o be con-

s i d e r e d i s t h e l i k e l y c o n t r i b u t i o n o f p o o r n u t r i t i o n a l s t a t u s o f t h e subas s u g g e s t e d b y an e a r l y s t u d y i n a parallel i n I t a l y (9) and b y more r e c e n t o b s e r v a t i o n s i n A u s t r a l i a n A b o r i g i n a l

it i s o f interest that,

s t u d y i n Hungary, t i o n s o f 1.6-3.1

w h e r e n o m a l n u t r i t i o n was found,

f l u o r o s i s posed no

c l i n i c a l o r a e s t h e t i c problems a t t h e community l e v e l where F concentrappm w e r e f o u n d i n t h e w a t e r (7). and i n samThe f o r e g o i n g a s p e c t s d e s e r v e f u r t h e r i n v e s t i g a t i o n s i n y o u n g e r age groups where permanent t o o t h f o r m a t i o n i s s t i l l i n progress, p l e s s e l e c t e d b a s e d on c o n t r a s t i n g n u t r i t i o n a l s t a t u s . I n children, u r i n a r y F excretion i s n o t always expected t o p a r a l l e l

i n t a k e f r o m d r i n k i n g w a t e r t o t h e same e x t e n t a s i n a d u l t s , because o f d e p o s i t i o n o f F i n d e v e l o p i n g and m i n e r a l i z i n g h a r d t i s s u e s . i n 7 t o 12 and 1 4 t o 1 8 - y e a r - o l d children. However, relaC o l l i n s and S e g r e t o ( 1 1 ) f o u n d a s t r o n g d i r e c t a s s o c i a t i o n i n t h i s r e s p e c t

A s i m i l a r dose-response

t i o n s h i p was d e m o n s t r a t e d i n t h i s s t u d y . a l s o f o u n d i n H u n g a r i a n c h i l d r e n (7). As i n t h e H u n g a r i a n s t u d y (7). that,

A c o r r e s p o n d i n g a s s o c i a t i o n was

although the urinary

concentration

was t w i c e a s h i g h i n t h e T h a i s a m p l e a t a l l l e v e l s o f F i n t h e w a t e r . a p o s i t i v e a s s o c i a t i o n b e t w e e n w a t e r and s a l i v a r y F l e v e l s was c o n f i r m e d i n t h e p r e s e n t s t u d y .

It s h o u l d be n o t e d

e v e n t h o u g h i d e n t i c a l c o l l e c t i o n and a n a l y t i c a l m e t h o d s w e r e used, Urinary F l e v e l s were a l s o t w i c e as I t may be assumed t h a t

t h e s a l i v a r y F c o n t e n t s o f t h e Thai samples were t h r e e t o f o u r t i m e s h i g h e r than those i n t h e Hungarian study. h i g h i n t h i s s t u d y as i n t h e Hungarian study.

179
differences i n climatic conditions, d i e t a r y patterns, and n u t r i t i o n a l

s t a t u s c o u l d have c o n t r i b u t e d t o t h e s e v a r i a t i o n s . D e s p i t e t h e d i f f e r e n c e s i n u r i n a r y and s a l i v a r y F c o n c e n t r a t i o n s f o u n d i n t h i s s t u d y and i n H u n g a r y (7). both instances. Thus, i n c r e a s e i n t h e mean F v a l u e s o f t h e b i o l o g i c a l s a m p l e s w i t h i n c r e a s e d F c o n c e n t r a t i o n i n t h e w a t e r was c o n s i s t e n t i n t h e present f i n d i n g s c o n f i r m t h a t both parameters are useful indicators o f short-term systemic F turnover i n r e l a t i v e l y small g r o u p s o f s u b j e c t s c o n s i s t i n g o f 20 t o 40. The s i g m o i d r e l a t i o n s h i p b e t w e e n t h e F c o n c e n t r a t i o n i n t h e w a t e r and a l l t h r e e dependent v a r i a b l e s examined supports t h e a s s e r t i o n t h a t t h e f o r m o f a s s o c i a t i o n b e t w e e n d e n t a l c o n d i t i o n s and t h e i r d e t e r m i n a n t s i s o f i m p o r t a n c e (12). T a k i n g F i g u r e 1 as an example, the i n f l e x i o n s o f the curve s u g g e s t t h a t i n t h e s e samples, t h e preva:ence and s e v e r i t y o f f l u o r o s i s i n c r e a s e d m o s t s t e e p l y w h e n F l e v e l i n t h e w a t e r w a s b e t w e e n 0.4 ppm a n d 2.0 ppm. I n t h e p r e s e n c e o f a h i g h i n f l e x i o n t h e i m p a c t i s so s t r o n g t h a t as t h e g r e a t m a j o r i t y o f t e e t h have Whereas f l u o r o s i s was o f no consequence i n t h e l i t t l e a d d i t i o n a l damage i s p o s s i b l e , a l r e a d y been a f f e c t e d . H u n g a r i a n s t u d y (7).

i t i s o f i n t e r e s t i n t e r m s o f F e x p o s u r e and u t i l i z a n a i l s and h a i r ) was a l s o

t i o n t h a t t h e a s s o c i a t i o n b e t w e e n t h e F c o n c e n t r a t i o n i n w a t e r and t h o s e i n u r i n e and s a l i v a (as w e l l as t h o s e i n p l a q u e , curvilinear, r e s e m b l i n g t h e s i g m o i d r e l a t i o n s h i p found i n t h i s study.

m a j o r i t y o f t h e samples used i n t h i s study were w e l l s u p p l i e d o r

oversupplied w i t h water-borne i n p r o g r e s s w i t h WHO s u p p o r t . areas i n Thailand, justified. where,

F and s t u d i e s o n d e f l u o r i d a t i o n a r e a l r e a d y
However, t h e r e a r e numerous F - d e f i c i e n t and

i n v i e w o f i n c r e a s i n g c a r i e s i n c i d e n c e (13),

w a t e r f l u o r i d a t i o n o r o t h e r f l u o r i d e s u p p l e m e n t s w o u l d be b e n e f i c i a l

Such m e a s u r e s r e q u i r e p r i o r d e t e r m i n a t i o n o f o p t i m a l F concenApplication o f the and

t r a t i o n i n t h e w a t e r , f o r w h i c h f o r m u l a s based on temperature-dependent w a t e r c o n s u m p t i o n h a v e been d e v e l o p e d (3.14.15). G a l a g a n - V e r m i l l i o n f o r m u l a (14), intended f o r unusual c l i m a t i c c o n d i t i o n s . ppm a p p e a r s t o

w o u l d i n d i c a t e an o p t i m a l l e v e l o f 0.7 ppm f o r m o s t a r e a s o f T h a i l a n d , e v e n t h e m o r e c o n s e r v a t i v e M i n o g u s h i (15) a p p r o a c h o f 0.55 be e x c e s s i v e . These f i n d i n g s i n d i c a t e t h a t , pre-fluoridation developing countries,

p a r t i c u l a r l y i n t r o p i c a l and exploration o f the nutritional and t h e u s e o f r e l i a b l e

t r i a l s should include determination

o f F i n t a k e f r o m sources o t h e r t h a n water,

s t a t u s o f t h e community w i t h emphasis on c h i l d r e n ,

b i o l o g i c a l i n d i c a t o r s o f a c t u a l F e x p o s u r e and u t i l i z a t i o n . ACKNOWLEDGEMENT T h i s s t u d y was s u p p o r t e d t h r o u g h WHO A g r e e m e n t No D2/181/52. t h a n k Ms. J. S a r d o - I n f i r r i The a u t h o r s and D r . T h a w o r n A n u m a r n r a j a d h o n f o r l o g i s t i c s

180
arrangements, schools, M i s s P. S. H. Un f o r a d v i c e , and t h e p r i n c i p a l s , M i s s M. T a n t a n a d i l o k f o r t e c h n i and c h i l d r e n a t v a r i o u s

c a l assistance,

teachers,

f o r t h e i r cooperation.

REFERENCES 1. L e a t h e r w o o d EC. B u r n e t t GW. Pub H1t h 55: 1792-1 799 C h a n d r a v e j j s m a r n R. S i r i k a y a P (1965) Am J

2. P h a n t u m v a n i t P, S o n g p a i s a n Y. V i s a r u r a t h S . P a t i m a n u k a s e m P ( 1 9 8 2 ) O s t e r r Z S t o m a t o l 79:303-304
3. G a l a g a n DJ.
4. Lamson GG ( 1 9 5 3 ) P u b l H l t h Rep 68:497-508 Rhodes I n t e r n a t i o n a l D e n t a l F l u o r o s i s and C a r i e s . M d l l e r IJ (1965) S c i e n c e P u b l i s h e r s , Copenhagen, p p 146-147 Dean HT ( 1 9 3 4 ) J Am D e n t Assoc 21:1321-1426 S c h a m s c h u l a RG. S u g a r E, Un PSH, T o t h K. C o m m u n i t y D e n t O r a l E p i d e m i o l 13:104-107 B a r m e s DE. A d k i n s BL ( 1 9 8 5 )

5. E r i c s o n Y (1971) A c t a O d o n t Scand 29:43-51 6. 7.

F l u o r i n e and D e n t a l Health. Am 8. D e a n HT ( 1 9 4 2 ) I n : M o u l t o n F R ( e d ) Assoc Adv S c i , W a s h i n g t o n DC, p p 23-31 9. M a s s l e r M, S c h o u r I (1952) J Am D e n t Assoc 44:156-169 1980-82, Insti10. S c h a m s c h u l a RG. C o o p e r MH (1982) I n : B i e n n i a l Report, t u t e o f D e n t a l R e s e a r c h , Sydney, p p 29-31 11. C o l l i n s EM, S e g r e t o VA ( 1 9 8 4 ) J D e n t c h i l d 51:352-355

12. S c h a m s c h u l a R G , A d k l n s BL, Agus HM. B a r m e s DE, C h a r l t o n G ( 1 9 7 8 ) I n : B i b b y BG, S h e a r n RJ ( e d s ) P r o c e e d i n g s , M e t h o d s o f C a r i e s P r e d i c t i o n .

Spec S u p p l t o M i c r o b i o l A b s t r a c t s , p 289-297 13. P a r n - y a - g h a r m Bangkok 2002. 14. G a l a g a n DJ, P h a n t u m v a n i t P. S o n g p a i s a n Y ( 1 9 8 2 ) I n : S y m p o s i u m C h u l a l o n g k o r n U n i v e r s i t y . Bangkok, p p 15.1-15.21

Y,

V e r m i l l i o n JR ( 1 9 5 7 ) Pub H l t h Rep 72:191-193 World Health Organi-

15. M i n o g u c h i G ( 1 9 7 0 ) I n : F l u o r i d e s and Human H e a l t h . z a t i o n , Geneva, p p 294-304

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 181-186 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

181

SKELETAL FLUOROSIS A S SEEN I N A H I G H FLUORIDE ZONE

SHIV

CHANDRA~

D e p a r t m e n t o f P r e v e n t i v e and S o c i a l M e d i c i n e , J a i p u r : 302004, I n d i a

S.M.S.

Medical College, J.L.N.

P r e s e n t Address: D e p a r t m e n t o f P r e v e n t i v e and S o c i a l M e d i c i n e , M e d i c a l C o l l e g e , A j r n e r : 305001, I n d i a .

ABSTRACT An e p i d e m i o l o g i c a l s t u d y o f e n d e m i c s k e l e t a l f l u o r o s i s h a s b e e n c a r r i e d o u t among p e r s o n s 1 5 y e a r s o l d and a b o v e i n a n a r e a w h e r e t h e d r i n k i n g w a t e r c o n t a i n e d 5 ppm F and above. lower limbs, 17.46%; I n a s a m p l e o f 1 2 6 p e r s o n s t h e sequence 26.98%; pain i n 12.69%. Examination o f persons w i t h k y p h o s i s and When t h r e e 3.46%. o f c o m p l a i n t s n o t e d w a s : b a c k a c h e , 31.74%; j o i n t p a i n s , and p a r e s t h e s i a , 17.96% e a c h ; a n y s u g g e s t i v e symptoms r e v e a l e d : r i g i d i t y o f spine,

b r i s k r e f l e x e s , 46.51%:

and bony e x o s t o s i s ,

symptoms and t h r e e s i g n s were p r e s e n t t o g e t h e r , case o f c l i n i c a l s k e l e t a l f l u o r o s i s . t h e s t u d y area.

a p e r s o n was d i a g n o s e d a s a

Thus 1 5 p e r s o n s w e r e f o u n d t o be

s u f f e r i n g f r o m s k e l e t a l f l u o r o s i s g i v i n g an o v e r a l l p r e v a l e n c e o f 1.13% i n U r i n a r y f l u o r i d e e x c r e t i o n i n t h e s e i n d i v i d u a l s was h i g h e r T h i s d i f f e r e n c e was f o u n d Measuret h a n t h e f l u o r i d e l e v e l i n t h e i r d r i n k i n g water.

s t a t i s t i c a l l y s i g n i f i c a n t when c o m p a r e d a g a i n s t a c o n t r o l group.

m e n t o f t h e I n d e x o f I n d e p e n d e n c e i n t h e A c t i v i t i e s o f D a i l y L i v i n g among t h e s e i n d i v i d u a l s d i s c l o s e d some l i m i t a t i o n i n p e r f o r m i n g d a i l y a c t i v i t y .

INTRODUCTION I n t h e f l u o r o t i c b e l t s s p r e a d t h r o u g h o u t w e s t and s o u t h I n d i a , f l u o r o s i s c o n t i n u e s t o be a p r o b l e m o f p u b l i c h e a l t h magnitude. skeletal Epidemio-

l o g i c a l s t u d i e s o f endemic f l u o r o s i s c a r r i e d o u t i n v a r i o u s areas i n d i c a t e t h a t p u b l i c h e a l t h e x p e r t s m u s t d e v e l o p a s i m p l e and f e a s i b l e method o f i d e n t i f y i n g t h e m e d i c a l g e o g r a p h y o f f l u o r o s i s i n s u c h zones. t i v e s o f t h e p r e s e n t work are: (1) measuring t h e prevalence o f s k e l e t a l f l u o r o s i s i n a f l u o r o t i c b e l t , (2) developing c l i n i c a l diagnosis o f skeletal fluorosis i n the t h e absence of t e c h n i c a l i n v e s t i g a t i v e aids, and ( 3 ) m e a s u r i n g t h e i n t e n s i t y o f d i s e a s e a t an i n d i v i d u a l l e v e l . field in The o b j e c -

182
MATERIALS AND METHODS T h i s s t u d y was c a r r i e d o u t i n a r u r a l a r e a i n w e s t e r n I n d i a w h e r e p u b l i s h e d (1,Z). I n t h e present paper observations r e l a t e d t o s k e l e t a l The a r e a was d i v i d e d i n t o t w o p a r t s b a s e d on A r e a I, 5.1 t o 1 0 ppm; A r e a 11. involvement are presented. 10.1 ppm a n d a b o v e . RESULTS S i n c e s k e l e t a l f l u o r o s i s i s a d i s e a s e i n a d u l t s i n a n e n d e m i c zone, p e r s o n s 1 5 y e a r s o l d and above w e r e i n t e r v i e w e d and e x a m i n e d f o r t h e p r e s ence o f s k e l e t a l f l u o r o s i s . S i n c e n o symptoms r e l a t e d t o s k e l e t a l i n v o l v e p r e v a l e n c e was i n v e s t i g a t e d The m o s t common symptom was backache t h e increase m e n t w e r e seen i n t h e 1 5 t o 19 y e a r s o l d group, i n p e r s o n s 20 y e a r s o l d and above.

the F concentrations i n d r i n k i n g water:

s e e n i n 28.57% o f t h e s a m p l e i n A r e a I a n d 35.71% i n A r e a 11, r e s p e c t i v e l y ( T a b l e I). W i t h t h e i n c r e a s e i n F c o n t e n t o f d r i n k i n g w a t e r , i n t h e number o f s y m p t o m a t i c s (p<0.005). I n symptomatics found d u r i n g i n t e r v i e w s . c l i n i c a l e x a m i n a t i o n was made f o r t h e r i g i d i t y o f s p i n e , k y p h o s i s , b r i s k r e f l e x e s and bony e x o s t o s i s . B r i s k r e f l e x e s was t h e m o s t f r e q u e n t l y e n c o u n t e r e d s i g n w h i l e bony e x o s t o s i s was seen i n o n l y t h r e e p e r s o n s ( T a b l e 11). The s y m p t o m a t i c s h a v i n g one o r m o r e s i g n s w e r e 72.72% i n A r e a 11, c o m p a r e d t o 28.57% i n A r e a I. W i t h t h e increase i n F concentrations i n t h e d r i n k i n g water, t h i s increase i n was found statistically significant

TABLE I OBSERVED SYMPTOMS OF SKELETAL FLUOROSIS SAMPLE A R E A ' Percent 14.28 28.57 24.28 14.28 60.00 N=56 12 20 17

SYMPTOMS

N=70
10 20 17 10 42

I1

Percent 21.42 35.71 30.35 10.71 78.57

P a i n i n Lower Limbs Backache Pain i n J o i n t s Number o f P a r a e s t h e s i a T o t a l Symptomatics X2=4.94; d.f.=l; ~ ~ 0 . 0 5

6
44

' F l u o r i d e i n d r i n k i n g w a t e r : A r e a I, 5.1-10.0

ppm; A r e a 11. 210.1 ppm

183 TABLE I 1 OBSERVED SIGNS OF SKELETAL FLUOROSIS

~ ~

SIGNS

N=42 3 7 10

SAMPLE
Percent 7.15 16.66 23.80

AREA^
N=44 14 10 30 3

11

Percent 31.81 22.72 68.18 6.81

R i g i d i t y o f Spine Kyphosis B r i s k Reflexes Bony E x o s t o s e s S y m p t o m a t i c s h a v i n g one o r more s i g n s Xz16.75: d.f.=l; p<O.OOl

0.00

12

28.57

32

72.72

F l u o r i d e i n d r i n k i n g w a t e r : A r e a I . 5.1-10.0

ppm: A r e a 11.

10.1 ppm

t h e number o f s y m p t o m a t i c s h a v i n g one o r m o r e p o s i t i v e s i g n s was s t a t i s t i c a l l y h i g h l y s i g n i f i c a n t (p<O.OOl). P e r s o n s h a v i n g any t h r e e symptoms and s i g n s s u c h as t h o s e shown i n T a b l e

I and I 1 w e r e c l a s s i f i e d as c a s e s o f s k e l e t a l f l u o r o s i s .
c a s e was a 2 8 - y e a r - o l d

Thus,

a total of female,

1 5 c a s e s o f s k e l e t a l f l u o r o s i s w e r e d e t e c t e d ( T a b l e 111). b o t h o f whom consumed w a t e r c o n t a i n i n g 10.1 ppm F and above. cases, 12 were bona f i d e r e s i d e n t s and t h r e e were m i g r a n t s . m i g r a n t s were female. U r i n a r y F e x c r e t i o n o f t h e s e c a s e s was

The y o u n g e s t

m a l e w h i l e t h e o l d e s t c a s e was a 7 0 - y e a r - o l d

O f t h e s e 15
A l l of the

compared t o t h a t o f a c o n t r o l

g r o u p w h o w e r e o f t h e s a m e s e x , h a d d r i n k i n g w a t e r f r o m t h e same s o u r c e , had s i m i l a r r e s i d e n t i a l s t a t u s , and w e r e w i t h i n t e n y e a r s o f t h e age c l a s s . O n l y 12 c o n t r o l cases c o u l d be obtained. fluorosis, except f o r a 48-year-old Among t h e c a s e s o f s k e l e t a l u r i n a r y F was a l w a y s h i g h e r t h a n t h e F c o n t e n t i n d r i n k i n g w a t e r m a l e who e x c r e t e d 11.2 ppm F i n u r i n e whereas h i s The c o n t r o l g r o u p r e v e a l e d However, the relationship

d r i n k i n g w a t e r c o n t a i n e d 11.8 ppm F ( T a b l e I V ) . an e r r a t i c p a t t e r n o f u r i n a r y F e x c r e t i o n .

b e t w e e n u r i n a r y F e x c r e t i o n and t h e F c o n t e n t i n d r i n k i n g w a t e r was f o u n d t o b e s t a t i s t i c a l l y s i g n i f i c a n t ( ~ ~ 0 . 0 5 i )n c a s e s c o m p a r e d t o c o n t r o l s . T h e p a t t e r n o f u r i n a r y F e x c r e t i o n s e e n among c h i l d r e n i n t h e s t u d y a r e a has been p u b l i s h e d e l s e w h e r e (3).

184

TABLE I 1 1 DISTRIBUTION OF SKELETAL FLUOROSIS ACCORDING TO FLUORIDE CONTENT IN DRINKING WATERa Cases of Skeletal Fluorosis Age Group (Years) Area I (N=38)
0

Male

Area I 1 (N=30)
1

Female Area I Area I 1 (N=32) (N=26)

0
0 0 0 0 0

Total
(N=126) 1 1 6 2 5 15 (11.9%)

20 - 29 30 - 39 40 50 60
-

0
0 0

49 59

1 5 1 1

lb lb 1

- above

Ib 1 (3.12%)

Total Grand Total

(5.26%)

(30.0%)

3 (11.54%)
4 (6.8%)

11 (16.17%)

'Fluoride in drinking water: Area I , 5.1-10.0 pprn; Area 11. 2 10.1 ppm bMi grants

TABLE i V URINARY FLUORIDE EXCRETION AMONG CASES OF SKELETAL FLUOROSIS AND CONTROLS Fluoride i n Drinking Water
(ppm)

Cases of Skeletal Fluorosis Age Sex Urinary F

( PPm)

Age

Controls Sex Urinary F

( PPm)

6.8 10.7

70 60

M
F M

a. a
38.0 38.8 28.0 18.8 15.6 19.2 11.2 14.2 14.8
70.8

7.8

64 52 48 56 40 25 27 45 45 40 50 55

M F

4.2
4.

48 48 56 45 48 28 50 48 45 60

16.4 9.8 30.0 15.6 9.2 7.6 5.3 5. a 6.9 15.2

M M M M F
M
F

M M F M
F

M
M

11.8

t=2.16; d.f.=2; ~ ~ 0 . 0 5

185
The I n d e x o f I n d e p e n d e n c e i n t h e A c t i v i t i e s o f D a i l y L i v i n g ( 4 ) was measured i n t h e 15 cases o f s k e l e t a l f l u o r o s i s and was compared w i t h t h e r e s t o f t h e p o p u l a t i o n ( 6 2 7 p e r s o n s ) 20 y e a r s o l d and a b o v e l i v i n g i n t h e same a r e a . O n l y 40% o f t h e s k e l e t a l f l u o r o s i s c a s e s w e r e i n d e p e n d e n t i n c a r r y i n g o u t a l l t h e s i x f u n c t i o n s c o n s i d e r e d i n measuring t h e ADL index, w h i l e i n t h e g e n e r a l p o p u l a t i o n 96.4% c o u l d p e r f o r m t h e same f u n c t i o n i n d e p e n d e n t l y (Table V).

TABLE V

I N D E X OF INDEPENDENCE I N A C T I V I T I E S OF DAILY LIVING I N PERSONS 20 YEARS OLD AND ABOVE

Skeletal Fluorosis Index Number (N=l5) Percent Number (N=612) 590 17 Others Percent

A B

40.00

96.40 2.77

33.33
6.66

0 0 0

0.00
0.00

D
E F
G
Others

0.00

0.00
6.66

0.00
0.50

0.00 13.33

0 2

0.00 0.33

DISCUSSION
D u r i n g an e p i d e m i o l o g i c a l s t u d y o f endemic f l u o r o s i s i n a remote r u r a l area i n I n d i a , t h e need t o c l i n i c a l l y diagnose t h e cases o f s k e l e t a l f l u o On t h e b a s i s o f t h r e e s y m p t o m s and t h r e e s i g n s , c a s e s r o s i s was f e l t . fluorosis.

w h i c h met i n v e s t i g a t i v e c r i t e r i a were l a b e l e d as s u f f e r i n g f r o m s k e l e t a l S k e l e t a l f l u o r o s i s was observed more i n males t h a n i n f e m a l e s as reported e a r l i e r by J o l l y

d.(5).

T h i s o c c u r s l a r g e l y because males

p e r f o r m more s t r e n u o u s work w h i c h r e s u l t s i n a h i g h e r w a t e r i n t a k e . Fewer cases a r e observed i n f e m a l e s i n t h e s t u d y area a l s o due t o t h e i r m i g r a t i o n f r o m n o n - f l u o r o t i c areas when t h e y marry. Mathur e t al. Singh and J o l l y (6) n o t e d backache i n persons t h e y examined f o r s k e l e t a l i n v o l v e m e n t i n a f l u o r o t i c b e l t .

( 7 ) a l s o r e p o r t e d b a c k a c h e i n 78.2% o f t h e p e r s o n s i n a
H i g h e r amounts o f c a l c i u m i n t h e d r i n k -

f l u o r o t i c b e l t i n t h e same region. t a l f l u o r o s i s i n t h e s t u d y area.

i n g w a t e r i n t h e s t u d y a r e a was r e s p o n s i b l e f o r l o w e r p r e v a l e n c e o f s k e l e -

186
REFERENCES 1. C h a n d r a S, S h a r m a R, T h e r g a o n k a r VP, C h a t u r v e d i O r a l E p i d e m i o l 8:92-96

SK ( 1 9 8 0 ) Comm D e n t -

2.

Chandra S (1983) E p i d e m i o l o g i c a l s t u d i e s o f endemic f l u o r o s i s i n n o r t h e r n I n d i a . P a p e r p r e s e n t e d a t 1 3 t h C o n f e r e n c e o f I.S.F.R., New D e l h i . Nov. 13-17 Chandra S, T h e r g a o n k a r VP (1984) F l u o r i d e 17:155-159 M a l j o r d C (1963) K a t z S, F o r d AB. M a s k o w i t z RW, J a c k s o n BA, M a l j o r d WJ. JAMA 185: 91 4-91 5 S i n g h A. J o l l y SS (1961) Q u a r t J Med 30:357-360

3.
4.

5.
6. 7.

J o l l y SS, S i n g h BM, M a t h u r OC. M a l h o t r a KC (1968) BMJ 4:427-429 M a t h u r GM. T a m b o l i BL, M a t h u r RM, Ray AK, M a t h u r GL, G o y a l OP ( 1 9 7 6 ) I n d J PSM 7:90-96

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 187-191 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

187

LONG-TERM RETENTION OF FLUORIDE I N THE BONES OF FORMER ALUMINUM WORKERS CHARLES A. JEAN M. BAUD, SEONG BANG, CHRISTIANE DEMEURISSE, H E N R I J. TOCHON-DANGUY, 1, r u e M i c h e l -

VERY

I n s t i t u t e o f Morphology, Centre M i d i c a l U n i v e r s i t a i r e , S e r v e t , 121 1 Gengve 4, S w i t z e r l a n d ABSTRACT

F l u o r i d e d e p o s i t e d i n t h e s k e l e t o n i s p r o g r e s s i v e l y removed b y osteoclast i c r e s o r p t i o n process. i n n e w l y f o r m i n g bone.

A small p a r t o f the released f l u o r i d e i s recycled A t e v e r y step,

the crystallographic properties o f c r y s t a l s i z e and/or p e r f e c t i o n , unit

t h e bone m i n e r a l ( c r y s t a l l i n i t y index, tion.

c e l l p a r a m e t e r s ) a r e c l o s e l y c o r r e l a t e d t o t h e l o c a l f l u o r i d e concentra-

E l i m i n a t i o n o f f l u o r i d e f r o m t h e skeleton,

and a c c o m p a n y i n g c h a n g e s i n

t h e bone m i n e r a l s u b s t a n c e , w e r e s t u d i e d o n 1 0 2 i l i a c b i o p s i e s f r o m a l u m i n u m w o r k e r s 0-32 y e a r s a f t e r e x p o s u r e t o f l u o r i d e ceased.

68 m a l e

F l u o r i d e i n m i n e r a l i z e d t i s s u e was d e t e r m i n e d w i t h a s p e c i f i c e l e c t r o d e

(1).

The f l u o r i d e c o n t e n t o f c o m p a c t bone was f o u n d t o d e c r e a s e p r o confirming earlier indirect b u t i n o p p o s i t i o n t o t h e o p i n i o n o f Dominok gt (Fig. 1).

g r e s s i v e l y w i t h t i m e a f t e r e x p o s u r e h a s ended, e s t i m a t i o n s (2), The e l i m i n a t i o n r a t e was a b o u t 6.5%/year

2 1 . (3).

I n comparison, t h e

t u r n o v e r r a t e e s t i m a t e d by h i s t o m o r p h o m e t r i c methods amounts t o 8-l0%/year

(4).

The d i f f e r e n c e i n d i c a t e s t h a t f l u o r i d e r e l e a s e d d u r i n g bone r e s o r p a process e x p e r i m e n t a l l y

t i o n i s i n p a r t r e c y c l e d d u r i n g bone f o r m a t i o n , p r o v e d b y Guo g t

d.(5).
as d i s p l a y e d b y e l e c t r o n p r o b e Xi s characterized by an i r r e g u l a r r e p a r t i t i o n o f

Topographic d i s t r i b u t i o n o f f l u o r i d e , r a y microanalysis (6.7),

t h e f i e l d s w i t h a h i g h f l u o r i d e l e v e l d u r i n g t h e p e r i o d o f exposure (Fig. 2) and b y an e v o l u t i o n t o w a r d s l o w l e v e l s and homogeneity a f t e r t h e end o f c o n t a m i n a t i o n ( F i g . 3). The c r y s t a l l i n i t y o f t h e bone m i n e r a l s u b s t a n c e was e v a l u a t e d b y i n f r a r e d s p e c t r o m e t r y (8). The c r y s t a l l i n i t y i n d e x d e c r e a s e s as a f u n c t i o n o f d e c r e a s i n g f l u o r i d e c o n t e n t (Fig. w i d t h o f X-ray d i f f r a c t i o n

4).
the i n t e n s i t y ( 9 ) . The B v a l u e s

C r y s t a l s i z e and/or p e r f e c t i o n i s i n v e r s e l y c o r r e l a t e d w i t h B values, lines a t half i n c r e a s e when t h e f l u o r i d e c o n c e n t r a t i o n i s f a l l i n g ( F i g .

5).

The d i m e n s i o n s o f t h e u n i t c e l l o f t h e a p a t i t e w e r e d e t e r m i n e d b y means

188

*I* F
.8
.6

Duration of Exposure to F= 30'- 3 years = 5 5 % 6 years Age at withdrawal

.4
.2

10 15 20 Years after withdrawal

25

F i g u r e 1. F l u o r i d e c o n t e n t o f t h e i l i a c c o m p a c t bone t i s s u e m i n e r a l o f a l u m i n u m w o r k e r s , 0 t o 2 5 y e a r s a f t e r e x p o s u r e t o f l u o r i d e ceased.

.2

it

:I
E
4o

Duration of Exposure l o F - 4 3 years Duration of Post-exposure = 0

1700 3400 5100 6800 8500 10200 11900 13600 15300 p p rn F

Control

40

20

1
I

1700 3400 5100 6800 8500 10200119001360015300 p p m F

F i g u r e 2. H i s t o g r a m s showing t h e d i s t r i b u t i o n o f f l u o r i d e i n t h e i l i u m o f an aluminum w o r k e r exposed t o f l u o r i d e f o r 43 y e a r s ( t o p ) and o f a c o n t r o l s u b j e c t o f t h e same age group (bottom).

:i
#
20 60

189
Duration of Expowre l o F = I 4 years Duration o f P-I-exposun

= 20 years

1tW 2200 Ooo 4400 3500 6640 vpm F

Control

10

it

20

11002100uoO44005500~ ppmF

F i g u r e 3. H i s t o g r a m s showing t h e d i s t r i b u t i o n o f f l u o r i d e c o n c e n t r a t i o n s , i n p e r c e n t a g e volume o f compact bone t i s s u e , i n t h e i l i u m o f an aluminum w o r k e r 20 y e a r s a f t e r e x p o s u r e t o f l u o r i d e c e a s e d ( t o p ) and o f a c o n t r o l s u b j e c t o f t h e same age group (bottom).

75

0 Controls
0 @ Exposed 0 After

withdrawal

70

E .-

65

2.

60
0

.5

1.0

*I. F
F i g u r e 4. V a r i a t i o n o f t h e c r y s t a l l i n i t y i n d e x o f t h e i l i a c compact bone t i s s u e mineral asa f u n c t i o n o f f l u o r i d e concentration.

190

' ( J I 0 )

re')=?
600
0

0 Controls @Exposed

o After withdrawal r = -. 78

500

400

300

.5
O/o

1.0

F i g u r e 5. V a r i a t i o n o f t h e c r y s t a l s i z e a n d / o r p e r f e c t i o n ( B (31.0) v a l u e ) o f t h e i l i a c c o m p a c t bone t i s s u e m i n e r a l a s a f u n c t i o n o f f l u o r i d e concentration.

(All

0 Controls

Exposed
0After withdrawal

9.44 -

r = -. 94

I
0

.5

'lo F

1.0

F i g u r e 6. V a r i a t i o n o f the a p a t i t e u n i t c e l l a parameter o f the i l i a c c o m p a c t bone t i s s u e m i n e r a l as a f u n c t i o n o f f l u o r i d e c o n c e n t r a t i o n .

191
o f h i g h r e s o l u t i o n X-ray d i f f r a c t i o n (9). decreasing f l u o r i d e c o n c e n t r a t i o n (Fig. The a p a r a m e t e r o f t h e u n i t c e l l

o f t h e bone a p a t i t e c r y s t a l s l e n g t h e n s p r o g r e s s i v e l y as a f u n c t i o n o f

6).

The c o r r e l a t i o n b e t w e e n f l u o r i d e c o n t e n t and t h e o t h e r p a r a m e t e r s demons t r a t e s t h a t t h e f l u o r i d e i s t h e most i m p o r t a n t f a c t o r f o r t h e progressive a l t e r a t i o n o f t h e bone m i n e r a l d u r i n g t h e p o s t - c o n t a m i n a t i o n p e r i o d as w e l l as d u r i n g t h e p e r i o d o f e x p o s u r e t o f l u o r i d e . REFERENCES 1. 2. McCann HG (1968) A r c h O r a l B i o l 13:475-477 L a r g e n t EJ (1952) A r c h I n d u s t r H y g i e n e Dominok G, Siefert

- Occup

Med 6:37-42 Techniques

3.
4. 5.

K,

F r e g e J, Dominok B (1984) F l u o r i d e 17:23-26

P a r f i t t A M (1983) I n : R e c k e r RR (ed) Bone H i s t o m o r p h o m e t r y : and I n t e r p r e t a t i o n . CRC Press, I n c , Boca Raton, p p 143-223

Guo MK,

M e s s e r HH, Ophaug R,

S i n g e r L (1985) J D e n t Res 64:357

6.

Baud CA, Bang S (1972) I n : S h i n o d a G, K o h r a K, I c h i n o k a w a T (eds) Proc e e d i n g s o f t h e S i x t h I n t e r n a t i o n a l C o n f e r e n c e o n X-ray O p t i c s and M i c r o a n a l y s i s . U n i v e r s i t y o f Tokyo Press, p p 835-840 B a n g S , B a u d CA, B o i v i n G, D e m e u r i s s e C, G o s s i M, T o c h o n - D a n g u y HJ. Very J M (1978) I n : F l u o r i d e and Bone, P r o c 2nd Symp CEMO. Huber, Bern, p p 168-175 B a u d CA, P o u e z a t JA, s u p p l : 452-456 Tochon-Danguy

7.

8.
9.

HJ ( 1 9 7 6 )

C a l c i f T i s s u e Res 2 1

V e r y JM, Baud CA (1984) I n : D i c k s o n GR (ed) Methods o f C a l c i f i e d T i s s u e P r e p a r a t i o n . E l s e v i e r , Amsterdam, pp 369-387

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 193-199 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

193

SYMPTOMATOLOGY OF WORKERS I N THE FLUORIDE INDUSTRY AND FLUORSPAR PROCESSING PLANTS

V I K A S K.

DESAI.

BHARAT S.

BHAVSAR,

NATVARLAL R.

MEHTA,

DINESH KUMAR SAXENA Surat. 395 001,

AND SHANTILAL L. KANTHARIA

Community M e d i c i n e Department, Government M e d i c a l College, Gujarat, India

ABSTRACT R a p i d i n d u s t r i a l d e v e l o p m e n t w i t h i n c r e a s i n g u s e o f f l u o r i d e compounds i n I n d i a n e c e s s i t a t e s b a s e l i n e s t u d i e s on i n d u s t r i a l were undertaken i n G u j a r a t State. HF, f r e e z i n g gas, cryolite, etc., fluorosis. Two s t u d i e s Another One on w o r k e r s i n i n d u s t r i e s p r o d u c i n g was c o n d u c t e d f r o m 1976 t o 1979.

o n w o r k e r s i n f l u o r s p a r p r o c e s s i n g p l a n t s was p e r f o r m e d f r o m 1981 t o 1984.

O f 4 3 8 i n d u s t r i a l w o r k e r s c o n s u m i n g d r i n k i n g w a t e r c o n t a i n i n g <0.8 ppm F.
34% h a d c o m p l a i n t s . M a j o r c o m p l a i n t s w e r e p a i n and s t i f f n e s s o f t h e l u m b a r T h e i r mean u r i n a r y s p i n e (14.1%) a n d p a i n and s t i f f n e s s o f j o i n t s (12.1%).

F l e v e l w a s 1.96 ppm.

O f 3 6 6 p l a n t w o r k e r s w h e r e d r i n k i n g w a t e r c o n t a i n e d 0.8
61.48% h a d c o m p l a i n t s . l u m b a r s p i n e (42.6%). p a i n (34.9%).

3.0 ppm F,

M a j o r c o m p l a i n t s w e r e p a i n and s t i f f n e s s o f t h e p a i n a n d s t i f f n e s s o f j o i n t s (23.2%) and e p i g a s t r i c ppm. Symptoms

T h e i r mean u r i n a r y F l e v e l was 3.17

I n b o t h groups o f w o r k e r s symptoms were more severe i n employees o f h i g h r i s k departments. The s y m p t o m s w e r e r e l a t e d t o mode o f exposure. w e r e u n r e l a t e d t o d e n t a l f l u o r o t i c c h a n g e s o r r a d i o l o g i c a l changes.

INTRODUCTION R a p i d i n d u s t r i a l d e v e l o p m e n t w i t h i n c r e a s i n g u s e o f F compounds i n I n d i a n e c e s s i t a t e s b a s e l i n e s t u d i e s on t h e i n d u s t r i a l f l u o r o s i s problem. studies, plants, f r e e z i n g gas, cryolite, etc., and on w o r k e r s i n f l u o r s p a r Two t h e f i r s t o f t h e i r k i n d i n I n d i a o n i n d u s t r i e s p r o d u c i n g HF, processing were undertaken i n G u j a r a t State.

D e n t a l and s k e l e t a l f l u o r o s i s a r e c o n s p i c u o u s and t h o r o u g h l y s t u d i e d m a n i f e s t a t i o n s o f t h e i n d u s t r i a l and e n d e m i c f l u o r o s i s p r o b l e m . been a c c e p t e d t h a t t h e y a r e n o t t h e o n l y f e a t u r e s o f t h e d i s e a s e .

I t h a s now

194
Roholm ( 1 ) i n h i s c l a s s i c book h a s d e s c r i b e d a w i d e s p e c t r u m o f m a n i f e s t a t i o n s o t h e r t h a n i n v o l v e m e n t o f bones and t e e t h . were a l s o d e s c r i b e d b y M u r r a y and W i l s o n (4). Such m a n i f e s t a t i o n s

(2). W a l d b o t t ( 3 ) . F r a d a g t

21.

C z e r w i n s k i and L a n k o s z ( 5 ) i n t h e i r s t u d i e s on human f l u o r o s i s .

T h i s study a t t e m p t s t o analyze b a s e l i n e i n f o r m a t i o n o f symptomatology i n i n d u s t r i a l and p l a n t w o r k e r s who h a v e d i f f e r e n t b a c k g r o u n d s and d i f f e r e n t environments. MATERIALS AND METHODS

A t o t a l o f 4 3 8 i n d u s t r i a l w o r k e r s , i n c l u d i n g 286 m a n u a l l a b o r e r s , w h o
c o n s u m e d d r i n k i n g w a t e r c o n t a i n i n g 0.8 r e s i d e n t s o f nearby c i t i e s o r v i l l a g e s .
In addition,

ppm F w e r e s t u d i e d .

They w e r e who con-

366 p l a n t w o r k e r s ,
-

i n c l u d i n g 247 t r i b a l l a b o r e r s , ppm F w e r e s t u d i e d .

sumed w a t e r c o n t a i n i n g 0.8

3.0

They w e r e r e s i d e n t s Spot u r i n e samples view)

o f a workers' colony o r nearby t r i b a l v i l l a g e s . W o r k e r s w e r e i n t e r v l e w e d a n d c l i n i c a l l y examined. were c o l l e c t e d from selected. 319 p l a n t w o r k e r s . X-rays w e r e c o l l e c t e d f r o m 210 i n d u s t r i a l w o r k e r s and f i r s t m o r n i n g u r i n e s a m p l e s

( R t . f o r e a r m , A.P.

w e r e t a k e n o f 183 i n d u s t r i a l w o r k e r s and 82 p l a n t w o r k e r s who w e r e r a n d o m l y Water samples were c o l l e c t e d f r o m t h e source.

F levels o f water

and u r i n e s a m p l e s w e r e d e t e r m i n e d u s i n g an O r i o n i o n s p e c i f i c e l e c t r o d e . RESULTS AND D I S C U S S I O N E n d e m i c f l u o r o s i s h a s been e x t e n s i v e l y s t u d i e d and r e p o r t e d f r o m I n d i a , w h i l e i n d u s t r i a l f l u o r o s i s i s a new f i e l d . p r e v i o u s and c u r r e n t F i n t a k e s , individual susceptibility, pany c l a s s i c a l and teeth. changes extent, Inter-individual variations are known i n v a r i o u s t y p e s o f m a n i f e s t a t i o n s o f f l u o r o s i s . and n u t r i t i o n a l s t a t u s , etc. Roholm (1) has They a r e based on

mode and d u r a t i o n o f F i n t a k e ,

M a n i f e s t a t i o n s o t h e r t h a n s k e l e t a l and d e n t a l f l u o r o s i s p r e c e d e o r accomi n c h r o n i c f l u o r i d e exposure. d e s c r i b e d a w i d e s p e c t r u m o f m a n i f e s t a t i o n s o t h e r t h a n i n v o l v e m e n t o f bones s u c h as n e u r o m u s c u l a r symptoms, t a c h y c a r d i a , p o l y d i p s i a , and W a l d b o t t and C e c i l i o n i ( 6 ) have a l s o d e s c r i b e d gastro-intestinal, respiratory a l l e r g i c s k i n lesions.

symptomatology r e l a t e d t o musculoskeletal,

system and c h i z z o l a macula i n neighborhood f l u o r o s i s . c o u n c i l s t u d y ( 7 ) r e v e a l e d m i l d d y s p e p t i c symptoms.

A medical research
The

S y m p t o m a t o l o g y o f i n d u s t r i a l and p l a n t w o r k e r s i s shown i n T a b l e I. over-all respectively. P a i n and s t i f f n e s s o f t h e spine,

p r e v a l e n c e r a t e was 34% and 61.5% i n i n d u s t r i a l a n d p l a n t w o r k e r s , predominantly t h e lumbar

s p i n e , and p a i n and s t i f f n e s s o f j o i n t s , p r e d o m i n a n t l y knee j o i n t s , w e r e

195

TABLE I SYMPTOMATOLOGY

OF WORKERS
Percent

Symptoms P a i n and s t i f f n e s s o f s p i n e P a i n and s t i f f n e s s o f j o i n t Nausea, Headache Muscle s t i f f n e s s , Paresthesia

L o s s o f w e i g h t and weakness

Industry 14.1 12.1 2.7 1.8 twitching 3.3 3.8 2.5 3.4 1.3 4.7 1.5 34.0 18.0

Plant 42.6 20.8

vomiting,

epigastric pain

34.9
9.6 5.2 0.3 5.5 3.3 3.3 3.3 3.3 61.5 48.4

Emphysema, asthma, c h e s t p a i n Hearing disturbances Nasal c o n j u n c t i v a l i r r i t a t i o n U r i n a r y complaints Overall prevalence Specific

t h e m a i n symptoms i n b o t h groups. 34.9% o f t h e p l a n t w o r k e r s ,

B u r n i n g e p i g a s t r i c p a i n was r e p o r t e d b y t h e symptoms can b e t h i s study can

compared t o 2.7% b y t h e i n d u s t r i a l w o r k e r s .

T o i d e n t i f y symptoms r e l a t e d t o i n d u s t r i a l exposure,
fluoride-related industry.

compared w i t h those found i n i d e n t i c a l p o p u l a t i o n s n o t employed i n

I n t h e absence o f s u c h r e c o r d s ,

s e r v e as a b a s e l i n e . S p e c i f i c symptoms r e l a t e d t o bones and j o i n t s w h i c h can be s t r o n g l y a t t r i b u t e d t o F e x p o s u r e w e r e h i g h e r (48.4%) i n p l a n t w o r k e r s t h a n i n i n d u s t r i a l w o r k e r s (18.0%) I n d u s t r i a l w o r k e r s who consumed w a t e r w i t h n o r m a l F l e v e l s w e r e exposed t o t h e same e n v i r o n m e n t t h r o u g h o u t t h e y e a r s o f t h e i r e m p l o y m e n t i n t h e absence o f i n t e r d e p a r t m e n t a l r o t a t i o n a t t h e t i m e o f study. w o r k e r s w e r e a t r i s k o f e x p o s u r e t o F - c o n t a i n i n g gas and d u s t . The m a j o r i t y o f t h e p l a n t w o r k e r s ( l a b o r e r s ) w e r e r e s i d e n t s o f s u r r o u n d i n g t r i b a l v i l l a g e s w i t h d i f f e r e n t w a t e r F l e v e l s b e t w e e n 0.8 Compared t o o t h e r d e p a r t m e n t s ,
-

Industrial

3.0 ppm.

h i g h r i s k d e p a r t m e n t s had a s i g n i f i c a n t l y

h i g h p r o p o r t i o n o f w o r k e r s who w e r e e m p l o y e d f o r l e s s t h a n t e n y e a r s , w e r e f r o m a r e a s w h e r e t h e r e was m o r e t h a n 1 ppm F i n t h e w a t e r , and w e r e a t r i s k o f b e i n g exposed t o F - c o n t a i n i n g dust.

196
Symptomatology i n t h i s s t u d y appears t o r e l a t e s t r o n g l y w i t h t h e mode o f exposure. workers. (7). exposure. H y d r o f l u o r o s i s and i n g e s t i o n o f F - c o n t a i n i n g d u s t r e s u l t e d i n
I n t h e s t u d i e s b y R o h o l m ( 1 ) and t h e M e d i c a l R e s e a r c h C o u n c i l

h i g h e r p r e v a l e n c e o f s p e c i f i c symptoms and b u r n i n g e p i g a s t r i c p a i n i n p l a n t r a d i o l o g i c a l c h a n g e s w e r e r e l a t e d t o d u s t i n h a l a t i o n and d e g r e e o f

I n t h e M e d i c a l Research C o u n c i l s t u d y ( 7 ) u r i n a r y F e x c r e t i o n
s p e c i f i c symptoms i n t h i s s t u d y show a s i g n i f i c a n t S p e c i f i c symptoms were s i g n i f i c a n t l y

was r e l a t e d t o s e v e r i t y o f exposure. As shown i n Table 11, a s s o c i a t i o n t o r i s k o f exposure.

h i g h e r i n w o r k e r s i n h i g h r i s k d e p a r t m e n t s i n i n d u s t r y (29.2%) and i n p l a n t s (70.9%) t h a n i n w o r k e r s o f l o w r i s k d e p a r t m e n t s i n i n d u s t r y (9.9%) and i n p l a n t s (26.9%). respectively. S p e c i f i c symptoms w e r e h i g h e r i n i n d u s t r i a l w o r k e r s w i t h m o r e t h a n 10 y e a r s employment (19.8%) t h a n i n t h o s e w i t h l e s s t h a n 10 y e a r s employment (12.0%). health. T h i s suggests a c u m u l a t i v e e f f e c t I n p l a n t workers, o f t h e e n v i r o n m e n t on workers This i s s p e c i f i c symptoms were s i g n i f i c a n t l y h i g h e r i n

t h o s e employed f o r l e s s t h a n 10 y e a r s (52.3%) t h a n o t h e r s (33.3%).

because a m a j o r i t y o f h i g h r i s k department w o r k e r s a r e employed f o r l e s s t h a n f i v e y e a r s a f t e r w h i c h t h e y e i t h e r s h i f t t o s a f e r departments o r l e a v e t h e job. S p e c i f i c symptoms w e r e s i g n i f i c a n t l y a s s o c i a t e d w i t h d r i n k i n g w a t e r F level. 7.63, These s y m p t o m s w e r e p r e s e n t i n d.f.= 2, ~~0.05). 40.5%, 58.3% and 61.5% o f t h e w o r k e r s e x p o s e d t o <1, 1-2 and 2-3 ppm F i n t h e w a t e r , r e s p e c t i v e l y (X:

TABLE I 1

WORKERS

SPECIFIC SYMPTOMS I N RELATION TO R I S K OF EXPOSURE TO INDUSTRY AND PLANT

R i s k o f Exposure High Moderate

S p e c i f i c Symptoms I n d u s t r y workers P l a n t workers Present Absent Present Absent

(29.2%) 14 (12.2%) (9.9%) X2 d.f. P 25.48 2

50

121 (70.8%) 101 (87.8%) 137 (89.1%)

61 (70.9%) 84 (52.2%) 32 (26.9%) 40.43 2

25 (29.1%) 17 (47.8%) a7 (73.1%)

Low

15

<0.05

<O. 05

197
F l u o r o t i c d e n t a l changes a r e a r e s u l t o f c h i l d h o o d exposure t o h i g h F levels. I t s s i g n i f i c a n t a s s o c i a t i o n w i t h s p e c i f i c symptoms i n p l a n t worke r s i n d i c a t e s c o n t r i b u t i o n o f water F l e v e l t o which workers are continuo u s l y exposed t o f r o m b i r t h i n a d d i t i o n t o F - c o n t a i n i n g d u s t exposure a t t h e plants. 111). I n t h e absence o f h y d r o f l u o r o s i s i n i n d u s t r i a l w o r k e r s t h e r e was n o a s s o c i a t i o n b e t w e e n d e n t a l c h a n g e s and s p e c i f i c s y m p t o m s ( T a b l e

TABLE I 1 1

S P E C I F I C SYMPTOMS I N RELATION TO FLUOROTIC DENTAL CHANGES I N INDUSTRY AND PLANT WORKERS

F l u o r o t i c D e n t a l Changes I n d u s t r y workers P l a n t workers Present Absent Present Absent

S p e c i f i c Symptoms Present Absent

7 (8.9%) 72 (91.1%)
79

35 (9.7%) 324 (90.3%) 359

102 (57.6%) 75 (42.4%)

177

80 (42.3%)
109 (57.7%) 189

Total X2 d.f. P

0.05 1 >O. 05

8.56 1 <O. 05

U r i n a r y F e x c r e t i o n i n t h e p r e s e n c e o f h y d r o f l u o r o s i s and F - c o n t a i n i n g d u s t exposure a t p l a n t s showed a s i g n i f i c a n t r e l a t i o n t o s p e c i f i c symptoms, w h e r e t h e mean u r i n a r y F l e v e l was s i g n i f i c a n t l y h i g h e r i n w o r k e r s w i t h s p e c i f i c symptoms t h a n t h o s e n o t e x h i b i t i n g any symptoms. s h i p was n o t observed i n i n d u s t r i a l w o r k e r s (Table I V ) . Such a r e l a t i o n -

O f t h e t o t a l w o r k e r s X-rayed, 30.5% o f p l a n t w o r k e r s
l o g i c a l changes.

o n l y 11.4% o f t h e i n d u s t r i a l w o r k e r s and

had b o t h s p e c i f i c symptoms and q u a l i t a t i v e r a d i o -

D y s p e p t i c symptoms a r e d e s c r i b e d i n a M e d i c a l Research C o u n c i l Study (7). I n t h e aluminum i n d u s t r y s t u d y a t F o r t W i l l i a m , t h i s study. a s i g n i f i c a n t l y h i g h preval e n c e o f b u r n i n g e p i g a s t r i c p a i n (34.9%) i s n o t e w o r t h y i n p l a n t w o r k e r s i n P r e v a l e n c e o f b u r n i n g e p i g a s t r i c p a i n was s i g n i f i c a n t l y h i g h e r

(52.3%) i n h i g h r i s k departments t h a n i n l o w r i s k departments (30.3%; Table V).

The M e d i c a l R e s e a r c h C o u n c i l S t u d y ( 7 ) a l s o r e p o r t e d an a s s o c i a t i o n between d y s p e p t i c symptoms and s e v e r i t y o f exposure.

198
TABLE I V SPECIFIC SYMPTOMS I N RELATION TO URINARY F EXCRETION I N INDUSTRY AND PLANT

WORKERS

S p e c i f i c Symptoms Present Absent

Mean U r i n a r y Industry

L e v e l (ppm) Plant

% o f Workers w i t h >4 ppm L e v e l

Industry Plant

1.95 t1.90 2.10

t1.76

3.24 Q.59 2.78

G ' . 41

18.4 20.2

28.9 17.3

X*
P

0.5

2.29

2.26
>O. 05

2.49
<0.05

>O. 05

<O. 05

H y d r o f l u o r o s i s i s a l s o p r e s e n t i n h i g h r i s k departments.

However,

the That

f a c t that burning epigastric pain i s not related t o water F levels indicates t h a t d u s t i n g e s t i o n p l a y s a r o l e i n h i g h r i s k departments. t h e same c o n c l u s i o n . T h e m e a n u r i n a r y F l e v e l (3.55 ppm) a n d w o r k e r s w i t h m o r e t h a n 4 ppm ppm u r i n a r y F l e v e l s (30.3%) w e r e s i g n i f i c a n t l y h i g h e r i n w o r k e r s w i t h b u r n i n g e p i g a s t r i c p a i n t h a n i n w o r k e r s w i t h o u t b u r n i n g e p i g a s t r i c p a i n (2.85 b u r n i n g e p i g a s t r i c p a i n i s n o t r e l a t e d t o f l u o r o t i c d e n t a l changes suggests

TABLE V BURNING EPIGASTRIC P A I N I N RELATION EXPOSURE I N PLANT WORKERS Burning Epigastric Pain Present Absent R i s k o f Exposure Moderate

TO R I S K OF E X P O S U R E AND

DURATION O F

High

Low

D u r a t i o n o f Exposure (years) L e s s t h a n 10 M o r e t h a n 10

45 (52.3%) 41 (47.7%) 86 10.25 2 <O. 05

62 (38.5%) 99 (61.5%) 161

36 (30.3%) 83 (69.7%) 119

123 (41.0%) 177 (59.0%) 300 3.39 1 >0.05

19 (28.8%) 47 (71.2%) 66

Total

d.f. P

X2

199 and

19.3%. r e s p e c t i v e l y ) .

T h i s suggests absorption o f F from r e l a t i v e l y

non-water-soluble CONCLUSION

f l u o r s p a r d u s t i n g e s t e d by workers.

T h i s b a s e l i n e s t u d y o f i n d u s t r i a l and p l a n t w o r k e r s u n d e r p r e v a i l i n g c o n d i t i o n s r e v e a l s t h a t s y m p t o m s r e l a t e d t o bones and j o i n t s w e r e t h e m a j o r ones. S p e c i f i c s y m p t o m s w e r e r e l a t e d t o mode o f F i n j e s t i o n , s e v e r i t y o f exposure, d u r a t i o n o f e x p o s u r e and w a t e r F l e v e l . Only i n t h e presence o f h y d r o f l u o r o s i s were s p e c i f i c symptoms r e l a t e d t o f l u o r o t i c d e n t a l changes and u r i n a r y F e x c r e t i o n . Ingestion o f fluorspar dust resulting i n burning Such a s t u d i e s o f workers t o undere p i g a s t r i c p a i n was r e l a t e d t o s e v e r i t y and d u r a t i o n o f e x p o s u r e . baseline study w i l l help i n further follow-up p l a n n i n g o f p r e v e n t i v e measures. REFERENCES

s t a n d t h e e f f e c t o f e x p o s u r e i n t h e w o r k i n g e n v i r o n m e n t on t h e i r h e a l t h and

1.
2.

Roholrn K (1937) F l u o r i n e I n t o x i c a t i o n , L e w i s and Co. Ltd., London M u r r a y MM. W i l s o n DC W a l d b o t t GL

A C l i n i c a l H y g e n i c Study. H. K.

3. 4. 5. 6. 7.

(1946) L a n c e t 2:821-824 (1962) I n t A r c h A v e r g 20(Suppl):1-60 F r a d a G, M e n t e s a n a G, N a l b o n e G (1963) M i n e r v a Med 54:451-459 C z e r w i n s k i E, L a n k o s z W (1977) F l u o r i d e 10:125-136 W a l d b o t t GL, C e c i l i o n i VA (1969) C l i n T o x i c o l 2:387-396 M e d i c a l R e s e a r c h C o u n c i l Memorandum (1949) No 22 I n d u s t r i a l F l u o r o s i s
HMSO. London

H. Tsunoda and M.-H. Y u (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 203-210 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

203

INFLUENCE OF PROTEIN AND ASCORBIC A C I D ON FLUORIDE-INDUCED CHANGES I N BLOOD COMPOSITION AND SKELETAL FLUORIDE DEPOSITION I N M I C E

MING-HO

YU AND HUEY-LIY S. HWANG I d s s t e r n Washington U n i v e r s i t y .

Huxley College o f Environmental Studies, B e l l i n g h a m , W a s h i n g t o n 98225, U.S.A.

ABSTRACT F e m a l e m i c e ( N u s m u s c u l u s . S w i s s W e b s t e r s ) f e d a d i e t c o n t a i n i n g low (4%) p r o t e i n s were t e s t e d f o r t h e e f f e c t s o f supplemental a c i d on body weight, tion. f l u o r i d e and a s c o r b i c and b l o o d composithe fluoride deposition i n the tibia.

The r e s u l t s w e r e c o m p a r e d w i t h t h o s e o b t a i n e d f r o m c o n t r o l m i c e f e d A t t h e end o f 12 weeks,

a d i e t c o n t a i n i n g n o r m a l (27%) p r o t e i n . weight,

a n i m a l s m a i n t a i n e d o n t h e l o w p r o t e i n d i e t showed a 40% r e d u c t i o n i n b o d y w h i c h was u n a f f e c t e d b y s u p p l e m e n t a l f l u o r i d e a t 1 0 ppm. Fluoride a d m i n i s t r a t i o n e n h a n c e d t h e t i b i a f l u o r i d e l e v e l b y 550% i n t h e l o w p r o t e i n g r o u p a n d b y 160% i n t h e n o r m a l p r o t e i n group. These i n c r e a s e s were marThe e f f e c t k e d l y d i m i n i s h e d i n t h e presence o f supplemental ascorbic acid. s i m i l a r t o t h a t on b o n e f l u o r i d e d e p o s i t i o n .

o f f l u o r i d e a n d a s c o r b i c a c i d o n s e r u m a l k a l i n e p h o s p h a t a s e a c t i v i t y was

A pronounced i n c r e a s e i n t h e

enzyme a c t i v i t y was o b s e r v e d e s p e c i a l l y i n t h e l o w p r o t e i n group.

INTRODUCTION M a n y e p i d e m i o l o g i c a l a n d e x p e r i m e n t a l s t u d i e s h a v e shown t h a t d i e t a r y f a c t o r s s u c h a s p r o t e i n s , c a l c i u m , v i t a m i n C, effects o f fluoride. i n d u c e d t o x i c i t y (1,Z). etc.. can m o d i f y t h e t o x i c G e n e r a l u n d e r n u t r i t i o n a p p e a r s t o enhance f l u o r i d z The a s s o c i a t i o n o f a s c o r b i c a c i d w i t h f l u o r i d e

t o x i c i t y a p p e a r s t o be f i r s t r e p o r t e d b y P h i l l i p s i n 1933 (3). t h e ensuing s t u d i e s have demonstrated mitigation of fluorosis m e n t a l a n i m a l s b y t h e a d m i n i s t r a t i o n o f v i t a m i n C (4-7).

A number O F
i n experiwhile others

s h o w e d t h a t t h e a d d i t i o n o f t h e v i t a m i n h a d n o b e n e f i c i a l e f f e c t s (8). Many s t u d i e s h a v e shown d i s t u r b a n c e s i n s k e l e t a l d e v e l o p m e n t i n humans and e x p e r i m e n t a l a n i m a l s f e d a d i e t d e f i c i e n t i n p r o t e i n s (9-11). study w i t h guinea pigs, d i e t a r y v i t a m i n C, Parker In their

fi d.( 1 2 )

investigated the interaction o f and showed i n c r e e s e d

p r o t e i n , and c a l c i u m w i t h f l u o r i d e ,

a c c u m u l a t i o n o f f l u o r i d e i n t h e g r o u p n a i n t a i n e d on a l o w p r o t e i n d i e t .

204

Reports on the interaction of dietary factors with fluoride, however, have been largely concerned with epidemiological studies on humans o r experimental studies on domestic animals. For studies dealing with the effect o f vitamin C, guinea pigs or monkeys have been widely used. Little information is available on the effects of fluoride on wildlife or the effect of ascorbic acid on animals that do not require this vitamin because of their ability to synthesize it. Newman and Yu (13) observed marked dental disfigurement and abnormal tooth wear patterns in black-tailed deer taken from an area near an industrial fluoride source. More recently, Yu and Driver (14) reported that supplementation o f fluoride in the diet of growing cockerels resulted in significantly altered tissue ascorbic acid and dehydroascorbic acid levels. Thrs suggests that in animals that do not require vitamin C, supplemental fluoride and ascorbic acid may induce a response similar to that in those animals that require the vitamin. The present work was initiated to further test this possibility. In particular, the interaction of protein and supplemental ascorbic acid with fluoride in laboratory mice was studied.

MATERIALS AND METHODS

One-week-old female mice (Mbl musculus. Swiss Websters) were fed diets containing either normal (27%) protein o r low (4%) protein for three weeks. The diets were obtained from Nutritional Biochemicals, Cleveland, Ohio,

U.S.A.

The

normal protein diet consisted of vitamin free casein, 27%;

starch, 32%; sucrose, 27%; vegetable oil, 10%: and salt mixture and vita% . The low protein diet differed from the normal protein diet only mins, 4 in the casein and starch contents, which were 4% and 55%, respectively. At the end of the three-week period, the mice in both diet groups were divided into four sub-groups varying only in drinking water: the control group continued to receive water with no supplements.while the others received 0 ppm F as NaF, 20 ppm ascorbic acid, and 10 pprn F water supplemented with 1 plus 20 ppm ascorbic acid, respectlvely. In all cases the animals received the food and drinking water ad libitum. These regimens were continued for 12 weeks. At the end o f the experimental period, the mice were sacrificed and the blood samples and the tibias were collected. The tibias were dried, defatted. and ashed. The fluoride content of the bone ash was then determined by a fluoride specific ion electrode by the method of Singer and Armstrong (15). respectively. The serum samples were analyzed for albumin, glucose, The analyses were carried out in an ACCU-STAT blood chemiscalcium, and cholesterol contents, and the alkaline phosphatase activity, try analyzer (Clay-Adams, Inc.. New Jersey, U . S . A . ) .

205
RESULTS The e x p e r i m e n t a l m i c e m a i n t a i n e d on t h e l o w p r o t e i n d i e t e x h i b i t e d a general poor growth, (Table I). a n d t h e r e was a m o r e t h a n 40% (p<0.05) reduction i n While body w e i g h t i n comparison w i t h t h e c o n t r o l a n i m a l s f e d t h e normal p r o t e i n F l u o r i d e o r a s c o r b i c a c i d a l o n e c a u s e d l i t t l e change. b o t h NaF and a s c o r b i c a c i d were supplemented a t t h e same t i m e , t h e r e was a s l i g h t r e d u c t i o n (6%) i n t h e body w e i g h t o f t h e normal p r o t e i n group, w h i l e a 19% i n c r e a s e i n t h e l o w p r o t e i n g r o u p was o b s e r v e d . dence, however. A d m i n i s t r a t i o n o f 10 However, t h e r e was a These d i f f e r e n c e s were n o t found t o be s t a t i s t i c a l l y s i g n i f i c a n t a t t h e 95% l e v e l o f c o n f i As shown i n Table 11. an average o f 123 ppm F was observed 1n t h e t i b i a s o f t h e c o n t r o l m i c e r e c e i v i n g n o n f l u o r i d a t e d water. ppm

i n the drinking water resulted i n

s i g n i f i c a n t i n c r e a s e i n bone

f l u o r i d e i n b o t h n o r m a l and l o w p r o t e i n g r o u p s . groups.

s t r i k i n g d i f f e r e n c e i n bone f l u o r i d e r e t e n t i o n b e t w e e n t h e t w o d i e t a r y The i n c r e a s e i n f l u o r i d e d e p o s i t i o n i n t h e n o r m a l p r o t e i n g r o u p whereas i n t h e l o w proThe d i m i n u t i o n was espeThese i n c r e a s e s w e r e d i m i n i s h e d when a t t h e concentrations o f f l u o r i d e was 160% (p<O.Ol) compared t o t h e w a t e r c o n t r o l s , t e i n g r o u p i t was 550% (p<O.OI). a s c o r b i c a c i d was supplemented s i m u i t a n e o u s l y . c i a l l y marked f o r t h e l o w p r o t e i n group: and a s c o r b i c a c i d used, For t h e mice fed normal protein,

a 47% decrease i n bone d e p o s i t i o n was observed. s u p p l e m e n t a l f l u o r i d e caused l i t t l e

change i n serum a l k a l i n e phosphatase a c t i v i t y w h i l e i n t h e presence o f

TABLE I

EFFECTS OF FLUORIDE AND ASCORBIC A C I D ON BODY WEIGHT OF M I C E FED NORMAL AND L O W PROTEIN DIETS
Treatment Normal P r o t e i n Body Weight (9) Control NaF2 32.5k5.4' 32.7k5.8 33.1k9.6 30.4k3.2 Percent o f Control Low P r o t e i n Body Weight (9) 20.0k2.8 20.5k3.3 20.7k1.7 23.8k0.8 Percent o f Control

(4) (3) (3) (4)

(3) (3) (3)

102
103 119

100 101 94

A A ~
NaF

AA

(2)

' V a l u e s a r e means f S.D.

' F l u o r i d e and a s c o r b i c a c i d (AA) were supplemented i n t h e d r i n k i n g w a t e r a t 10 ppm and 20 ppm. r e s p e c t i v e l y .

206 a s c o r b i c a c i d a l o n e , a 14% r e d u c t i o n was o b s e r v e d ( T a b l e 111). a c t i v i t y w e r e shown i n a l l s u b - g r o u p s . Far the

a n i m a l s f e d a l o w - p r o t e i n d i e t , h o w e v e r , m a r k e d i n c r e a s e s i n t h e enzyme The s u b - g r o u p t r e a t e d w i t h NaF I n t h e presence o f showed a 54% e l e v a t i o n o v e r t h e w a t e r c o n t r o l s . a s c o r b i c a c i d t h i s i n c r e a s e was d i m i n i s h e d t o 21%.

TABLE I 1

EFFECTS OF FLUORIDE AND ASCORBIC A C I D Old FLUORIDE DEPOSITION I N T I B I A S M I C E FED NORMAL AND L O W PROTEIN D I E T S
Treatment Normal P r o t e i n Low P r o t e i n

OF

F conc.
P Pm

N
(4) (3) (3) (4)

Percent o f Control

F conc.
PPm

N (3) (3) (3)

Percent o f Control

Con t r o 1 NaF2 A A ~ NaFtAA 'Values a r e

123+46' 320k147 105+29 280k137 means

260 85 227

227k122 1810k743 289283 964+348

653 104 348

(2)

_C

S.D.
water a t

' F l u o r i d e and a s c o r b i c a c i d (AA) were supplemented i n t h e drinl:ir,g 10 ppm and 20 ppm, r e s p e c t i v e l y .

TABLE I 1 1 EFFECTS OF FLUORIDE AND A S C O R B I C A C I D ON SERUM ALKALINE PHOSPHATASE ACTIVITY OF M I C E FED NORMAL AND L O W PROTEIN DIETS Treatment Normal P r o t e i n Enzyme activity Control NaF2 32k4.9l 33k3.5 2721.4 32k0.6 N (4) Percent Enzyme activity 82k8.5 125kl. 1 7625.6 99+2.1 Low P r o t e i n Percent

of C o n t r o l 103 86 100

o f Control

(3)
(3) (3)

153 93

(3)
(3)
(4)

A A ~
Na F+AA

(2)

121

'Values a r e means k S.D.

Babson u n i t .

' F l u o r i d e and a s c o r b i c a c i d (AA) were supplemented i n t h e d r i n k i n g w a t e r a t 10 ppm and 20 ppm, r e s p e c t i v e l y .

TABLE IV EFFECTS OF FLUORIDE AND ASCORBIC ACID ON SERUM COMPOSITION OF MICE FED NORMAL AND LOW PROTEIN DIETS
Treatment Albumin g/dl Control NaF2 Normal Protein Glucose mg/dl Cholesterol mg/dl Ca mg/dl

A1 bumi n
g/dl

G1 ucose
mg/dl

Cholesterol mg/dl
~~

Ca mg/dl

A A ~
Na F+AA

2.341 (0.05) 2.30 (0.14) 2.53 (0.25) 2.49 (0.20)

2 12 (31.7) 205 (5.0) 225 (21.8) 207 (24.7)

147 (16.9) 139 (12.7) 126 (15.3) 134 (7.9)

(0.6)

9.6

10.0 (2.3) 9.4 (0.6) 9.1 (0.3)

1.93 (0.27) 1.86 (0.08) 1.96 (0.29) 2.73 (0.53)

153 (28.4) 176 (17.6) 218 (46) 175 (20.5)

124 (19.1) 135 (35.3) 158 (26.3) 168 (24.7)

8.7 (0.3) 9.9 (1.5) 8.4 (0.5) 9.3 (0.5)

'Values are means and S.D.. in parenthesis. 'Fluoride and ascorbic acid (AA) w e r e supplemented in the respectively.

drinking

water a t 10 ppm and 20 ppm,

208 Whereas b o t h s e r u m g l u c o s e and a l b u m i n l e v e l s i n m i c e m a i n t a i n e d o n t h e low-protein d l e t were g e n e r a l l y l o w e r than those i n t h e normal p r o t e i n group, f l u o r i d e o r a s c o r b i c a c i d induced no s i g n i f i c a n t changes ( T a b l e I V ) . For t h e Serum c a l c i u m and c h o l e s t e r o l l e v e l s w e r e no-t shown t o be s i g n i f i c a n t l y a l t e r e d b y t h e a d m i n i s t r a t i o n o f e i t h e r f l u o r i d e o r a s c o r b i c acid. some i n c r e a s e s i n s e r u m c h o l e s t e r o l c o n t e n t , 95% l e v e l o f c o n f i d e n c e . m i c e f e d a l o w - p r o t e i n d i e t , b o t h f l u o r i d e and a s c o r b i c a c i d r e s u l t e d i n b u t t h e d i f f e r e n c e s between t h e e x p e r i m e n t a l and t h e w a t e r c o n t r o l s w e r e f o u n d n o t s i g n i f i c a n t a t t h e

DISCUSSION
The e x p e r i m e n t a l d a t a p r e s e n t e d h e r e c l e a r l y d e m o n s t r a t e t h e i m p o r t a n c e o f d i e t a r y p r o t e i n i n t h e r e t e n t i o n o f f l u c r i d e i n t h e bone o f m i c e , low dietary protein resulted i n higher fluoride e x p e r i m e n t a l c o n d i t i o n s used, accumulation. o b s e r v a t i o n w i t h g u i n e a p i g s h a s b e e n r e p o r t e d (12). i.e.,

A similar
Under t h e

s u p p l e m e n t a t i o n o f f l u o r i d e a t 10 ppm i n t h e

d r i n k i n g w a t e r d e p o s i t e d n e a r l y f i v e t i m e s more f l u o r i d e i n t h e t i b i a of mice maintained on a low p r o t e i n d i e t than i n t h e normal p r o t e i n group r e c e i v i n g t h e s a m e a m o u n t o f f l u o r i d e i n t h e d r i n k i n g w a t e r ( T a b l e 11). Even w i t h o u t f l u o r i d e s u p p l e m e n t a t i o n , t h e l o w p r o t e i n group accumulated The f l u o r i d e i n t h e bone 125% m o r e f l u o r i d e t h a n t h e n o r m a l p r o t e i n group.

o f t h e c o n t r o l a n i m a l s p r e s u m a b l y was d e r i v e d f r o m t h e d i e t i t s e l f . d e p o s i t i o n i n t h e bone o f m i c e .

It

appears t h a t d i e t a r y p r o t e i n i s an independent f a c t o r a f f e c t i n g f l u o r i d e T h a t s u p p l e m e n t a l a s c o r b i c a c i d m a r k e d l y reduced f l u o r i d e r e t e n t i o n i n t h e bones o f t h e e x p e r i m e n t a l a n i m a l s i s c l e a r l y shown i n T a b l e 11. It i s i m p o r t a n t t o n o t e t h a t t h e d i m i n i s h i n g e f f e c t o f a s c o r b i c a c i d i s more p r o n o u n c e d i n t h e l o w p r o t e i n g r o u p (46%) t h a n i n t h e n o r m a l p r o t e i n g r o u p (12%). W h i l e t h e r e a s o n f o r s u c h d i f f e r e n c e i s n o t known, i t s implication i n t e r m s o f h e a l t h e f f e c t may be i m p o r t a n t . The e a r l i e r o b s e r v a t i o n s t h a t

v i t a m i n C a d m i n i s t r a t i o n p r e v e n t e d t h e d e v e l o p m e n t o f s e v e r e bone f l u o r o s i s

(7).

o r d i m i n i s h e d t h e t o x i c e f f e c t s o f f l u o r i d e i n bones o f monkeys (ll),

0-:

may be e x p l a i n e d i n t e r m s o f t h e r e d u c t i o n i n f l u e n c e d by t h e vitamin.

bone f l u o r i d e a c c u m u l a t i o n

As shown i n T a b l e 111, s e r u m a l k a l i n e p h o s p h a t a s e a c t i v i t y was m a r k e d l y

e n h a n c e d i n a l l t r e a t m e n t g r o u p s m a i n t a i n e d on a l o w p r o t e i r ! d i e t . fluoride retention

s t r i k i n g s i m i l a r i t y e x i s t s b e t w e e n s e r u m a l k a l i n e p h o s p h a t a s e a c t i v i t y and i n t h e bone w i t h r e s p e c t t o t h e i n f l u e n c e o f f l u o r i d e exposure t o supplemental f l u o r i d e and a s c o r b i c a c i d s u p p l e m e n t a t i o n : activity,

enhanced f l u o r i d e d e p o s i t i o n i n t h e bone and serum a1 k a l i n e p h o s p h a t a s e and s u p p l e m e n t a t i o n o f a s c o r b i c a c i d d i m i n i s h e d t h e i n c r e a s e

209 ( T a b l e s I 1 and 111). The d i f f e r e n c e s i n enzyme a c t i v i t y e x h i b i t e d b y t h e Since t h e p r e c i s e metabolic the implication because t h e However,

t w o d i e t a r y p r o t e i n groups a r e considerable.

f u n c t i o n s o f t h e enzyme a r e n o t y e t f u l l y understood, a s s o c i a t e d w i t h t h e s e d i f f e r e n c e s i s n o t known. associated w i t h l i p i d transport, p r o c e s s o f b o n e s y n t h e s i s (16), t h e s e f u n c t i o n s m y be a l t e r e d .

i n any o f t h e v a r i o u s organs.

enzyme may f a c i l i t a t e t r a n s f e r o f m e t a b o l i t e s a c r o s s c e l l m e m b r a n e s , b e and be i n v o l v e d i n t h e c a l c i f i c a t i o n t h e p o s s i b i l i t y e x i s t s t h a t one o r m o r e o f

A p a t h o l o g i c a l l e g i o n may a l s o be p r e s e n t

I t i s n o t i c e a b l e t h a t r e p o r t s on c h a n g e s i n s e r u m a l k a l i n e p h o s p h a t a s e
a c t i v i t y as a r e s u l t o f exposure t o f l u o r i d e have been c o n t r a d i c t o r y . W h i l e o u r o b s e r v a t i o n s a r e c o n s i s t e n t w i t h t h o s e r e p o r t e d b y Reddy and S r i k a n t i a (11). 21). and tlough and Freeman (1~/), t h e y c o n t r a s t w i t h o t h e r s s h o w i n g a l o w e r e d enzyme a c t i v i t y a s s o c i a t e d w i t h p r o t e i n m a l n u t r i t i o n (18These d i s c r e p a n c i e s p r e s u m a b l y a r e caused b y such f a c t o r s as t h e s p e c i e s o f a n i m a l s and e x p e r i m e n t a l c o n d i t i o n s used, i n a d d i t i o n t o t h e c o m p l e x n a t u r e o f t h e enzyme i t s e l f . ACKNOWLEDGEMENT We w i s h t o t h a n k t h e B u r e a u o f F a c u l t y R e s e a r c h , University, f o r s u p p o r t i n t h e c o u r s e o f t h i s work. Western Washington

REFERENCES
1. K r i s h n a m a c h a r i KAVR, K r i s h n a s w a m y K (1974) I n d J Med Res 62:1415-1423

2.

Suttie J W (1983) I n : Shupe JL, P e t e r s o n HB. Leone NC ( e d s ) F l u o r i d e s E f f e c t s o n V e g e t a t i o n , A n i m a l s , a n d Humans. P a r a g o n P r e s s , S a l t L a k e C i t y , p p 291-304 P h i l l i p s PH (1933) J B i o l Chem 100:79 Evans RJ, P h i l l i p s PH (1939) J N u t r 18:353-360 Wadhwani TK (1952) I n d Med Gaz 87:5-7 Wadhwani TK ( 1 9 5 5 ) I n d J Med Res 45:377-386 V e n k a t e s w a r l u P, J h a GJ, Deo MG, J o n e s PRM, 9eddy GS, P a r k e r CM, Newman JR.
Yu MH,

3. 4. 5. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

6. Wadhwani TK ( 1 9 5 4 ) J I n d I n s t S c i 36:64-69
Rao ND ( 1 9 5 7 ) I n d J Med Res 45:377-386 R a m a l i n g a s w a m y V ( 1 9 6 8 ) Amer J P a t h 53:1111-1123

Dean RFA (1959) J P e d i a t 54:176 S r i k a n t i a SG ( 1 9 7 1 ) M e t a b o l i s m 20:642-656 S h a r n a RP, Shupe J L ( 1 9 7 9 ) C l i n T o x i c o l 15:301-311

Yu MH ( 1 9 7 6 ) J W i l d l i f e D i s 12:39-41

D r i v e r CJ (1982) F l u o r i d e 15:97-104

S i n g e r LD, A r m s t r o n g WD (1968) A n a l Chem 40:613-614 T i e t z NW ( 1 9 7 9 ) I n : G r i f f i t h s JC ( e d ) C l i n i c a l E n z y m o l o g y . M a s s o n P u b l i s h i n g , New York, p p 69-79

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17.

Hough VH,

Freeman S (1942) Amer J P h y s i o l 138:184-189 Hughes RE (1978) S p e c i a l i a 15:429

18.
19.

D a v i e s JEW, E l l e r y PM, H e y w o r t h PG, S c h w a r t z R (1956) J C l i n P a t h 9:33

20.
21.

W a t e r l o w JC, S t e p h e n JML (1969) P r o c N u t r SOC 28:234-242 E d o z i e n JC (1961) P e d i a t r i c s 27:325-333

H.Tsunoda and M.-H. Y u (Editors)

Fluoride Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 211-224 0 1 9 8 6 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

211

CHANGES I N THE METABOLISM

OF

GLUCOSE AND CALCIUM FOLLOWING A SINGLE LARGE

DOSE OF FLUORIDE TO RATS

YASUNOBU SUKETA, Y U K A R I KANAMOTO, YUKO SATOH, AND SHOJI OKADA S h i z u o k a C o l l e g e o f P h a r m a c y , D e p t o f E n v i r o n m e n t a l B i o c h e m i s t r y , 2-2-1 O s h i k a , S h i z u o k a , S h i z u o k a 422, J a p a n

ABSTRACT Serum g l u c o s e l e v e l was e l e v a t e d i m m e d i a t e l y a f t e r i p a d m i n i s t r a t i o n o f a s i n g l e l a r g e d o s e o f f l u o r i d e (NaF 3 5 m g / k g ) t o r a t s . propranolol, glucose was b u t n o t b y thyroid-parathyroidectomy. found t o be a s s o c i a t e d The e l e v a t i o n o f d i benamine o r glucose-6rats. E l e v a t i o n o f seruni serum g l u c o s e was m a r k e d l y s u p p r e s s e d b y a d r e n a l e c t o m y ,

w i t h enhancement o f

phosphatase a c t i v i t i e s i n t h e l i v e r and k i d n e y o f f l u o r i d e - t r e a t e d On t h e o t h e r hand, tion.

r e n a l c a l c i u m was e l e v a t e d g r a d u a l l y as c o m p a r e d t o t h e

a u g m e n t o f s e r u m g l u c o s e and c y c l i c AMP f o l l o w i n g t h e f l u o r i d e a d m i n i s t r a The e l e v a t i o n o f r e n a l c a l c i u m was m a r k e d l y s u p p r e s s e d b y t h r y r o i d parathyroidectomy.

INTRODUCTION Taylor

gt

4. ( 1 )

r e p o r t e d t h a t a d m i n i s t r a t i o n o f n e a r - l e t h a l doses o f mg/kg, i v ) markedly increased u r i n a r y sugar excretion.

f l u o r i d e (NaF 20-30

T h e i r f i n d i n g s w e r e c o n f i r m e d and i t was shown t h a t a s i n g l e l a r g e d o s e o f f l u o r i d e (NaF 3 5 m g / k g , i p ) c a u s e d t h e e l e v a t i o n o f n o t o n l y u r i n a r y g l u c o s e e x c r e t i o n b u t a l s o serum g l u c o s e c o n c e n t r a t i o n (2a). r i d e administration. cium i n f l u x . Suketa gt

. a !

( 2 b ) f o u n d t h a t t h e c a l c i u m c o n t e n t i n t h e k i d n e y s was a u g m e n t e d b y f l u o G l u c o s e p r o d u c t i o n i s known t o he r e g u l a t e d b y c a l I n t h e p r e s e n t s t u d y , a p o s s i b l e m e c h a n i s m f o r changes i n t h e

m e t a b o l i s m o f g l u c o s e and c a l c i u m i n r a t s f o l l o w i n g a s i n g l e l a r g e d o s e o f f l u o r i d e was i n v e s t i g a t e d .

MATERIALS AND METHODS Materials Glucose-6-phosphate (sodium s a l t ) , (disodiurn salt),

D-fructose-1.6-diphosphate ( s o d i u m
6-nicotinamide adenine dinucleo-

salt), phosphoenolpyruvate (nonopotassium s a l t ) , i n o s i n e 5'-diphosphate g l u t a t h i o n e ( r e d u c e d form),

212
t i d e , r e d u c e d f o r m (NADH). ma1 i c d e h y d r o g e n a s e a n d ( - ) - e p i n e p h r i n e Louis. Mo); (hydrochloride), were

o b t a i n e d f r o m S i g m a C h e m i c a l Company ( S t . c h l o r i d e ) and D L - p r o p r a n o l o l (Kyoto, Japan); i c a l Industries, (Boston, Treatment Mass).

dibenamine (hydro-

from Nakarai Chemical Ltd.

17-hydroxycorticosterone and g l y c o g e n , f r o m Wako P u r e ChemL t d . (Osaka, Japan); and s o d i u m b i ~ a r b o n a t e - [ C ' ~ ] ( O . 2 5 f r o m New E n g l a n d N u c l e a r

mCi/mg) and c a l c i u m ~ h l o r i d e - [ C a ~ ~ ] ( 2 3 . 1uCi/mg), 8

M a l e W i s t a r a l b i n o r a t s w e i g h i n g 95-105 g w e r e u s e d i n t h i s s t u d y . r a t s w e r e m a i n t a i n e d i n an a i r - c o n d i t i o n e d r o o m a t d i e t M F ( O r i e n t a l Y e a s t Co. L t d . , s e t of experiments t o

The

22"C,

and w e r e f e d b a s a l

Tokyo, J a p a n ) and w a t e r ad l l b i t u m .

At

t h e end o f s i x days, t h e r a t s w e r e f a s t e d 24 h o u r s b e f o r e and a f t e r each m i n i m i z e g l u c o s e 3 b s o r p t i o n f r o m t h e g u t and Adrenalectomy and t h y r o i d s t a b i l i z e u r i n a r y e x c r e t i o n o f glucose. w a t e r f o r 5 days.

p a r a t h y r o i d e c t o m y w e r e c a r r i e d o u t on r a t s m a i n t a i n e d o n b a s a l d i e t M F and F o l l o w i n g a s i n g l e l p a d m i n i s t r a t i o n o f f l u o r i d e (NaF. used as c o n t r o l s , t h e r a t s were

3 5 m g / k g ) o r c h l o r i d e (NaC1 48.1 r n g / k g ) ,
I n j e c t i o n o f epinephrine, 0101 Epinephrine and/or

a n e s t h e t i z e d w i t h e t h e r and k i l l e d b y c a r d i a c p u n c t u r e .

17-hydroxycorticosterone, d i b e n a m i n e and pro=

17-hydroxycorticosterone ( 3 ) w e r e i n j e c t e d i p 1 0 n i i n
D i benami ne (4) a n d / o r p r o p r a n o l o l (5)

a f t e r the fluoride administration.

w e r e i n j e c t e d s c 10 m i n a f t e r t h e f l u o r i d e a d m i n i s t r a t i o n . D e t e r m i n a t i o n o f g l u c o s e and 17-hydroxycorticosterone. Serum and u r i n a r y g l u c o s e and 17-hydroxycorticosterone w e r e d e t e r m i n e d b y t h e m e t h o d s of H u l t m a n Enzyme a s s a y The a c t i v i t i e s o f g l u c o s e - 6 - p h o s p h a t a s e , phosphoenolpyruvate carboxykinase, m i n e d b y t h e m e t h o d s o f S w a n s o n (8), Assay o f c a l c i u m f l u x D e t e r m i n a t i o n o f r e n a l and h e p a t i c c a l c i u m - i n f l u x and - e f f l u x was c a r r i e d o u t w i t h k i d n e y and l i v e r s l i c e s a c c o r d i n g t o t h e m e t h o d o f W a l l a c e (12) u s i n g 4 5 ~ a . D e t e r m i n a t i o n o f magnesium and c a l c i u m C a l c i u m and magnesium w e r e m e a s u r e d b y t h e m e t h o d o f W i l l i s ( 1 3 ) . Determination o f protein P r o t e i n was d e t e r m i n e d b y t h e method o f L o w r y aJ.(l4).

( 6 ) . and P o r t e r and S i l v e r (7).

respectively.

fructose-1.6-diphosphatase.
B a l l a r d and Hanson

and g l y c o g e n p h o s p h o r y l a s e w e r e d e t e r M c G i l v e r y (9).

( 1 0 ) . a n d M e y e r g t g l . (ll), respectively.

g G.

213
P r e p a r a t i o n s o f m i c r o s o m e s , c y t o s o l and g l y c o g e n g r a n u l e s M i c r o s o m e s and c y t o s o l w e r e p r e p a r e d a c c o r d i n g t o t h e m e t h o d o f J d r g e n s e n (15). At the time o f sacrifice, t h e r a t s w e r e a n e s t h e t i z e d w i t h e t h e r and k i l l e d by cardiac puncture. The k i d n e y s and l i v e r s w e r e r e m o v e d and t h e sucrose-0.03M histidine

t i s s u e s (1 g) were i m m e d i a t e l y homogenized i n a P o t t e r - E l v e h j e m t e f l o n g l a s s h o m o g e n i z e r w i t h 5 m l o f i c e - c o l d 0.25M b u f f e r (pH 7.2). w e r e o b t a i n e d b y c e n t r i f u g a t i o n ( 2 5 3 0 0 x g. Reproducible p r e p a r a t i o n s o f t h e heavy microsomal f r a c t i o n 30 m i n ) o f t h e s u p e r n a t a n t Cytosol Glycogen at

a f t e r s e d i m e n t a t i o n o f t h e m i t o c h o n d r i a a t 1 0 8 0 0 x g f o r 3 0 min. ( 1 0 5 0 0 0 x g. 6 0 m i n ) f r o m t h e s u p e r n a t a n t ( 2 5 3 0 0 x g, 3 0 m i n ) . g r a n u l e s were p r e p a r e d a c c o r d i n g t o t h e method o f Meyer p r e p a r a t i o n s ( 1 mg o f p r o t e i n / m l o f t h e r e s p e c t i v e b u f f e r ) -20C.

f r a c t i o n s were o b t a i n e d by s e d i m e n t a t i o n o f t h e l i g h t m i c r o s o m a l f r a c t i o n

9d.(ll).These
were s t o r e d

RESULTS AND DISCUSSION

E f f e c t o f f l u o r i d e a d m i n i s t r a t i o n on serum g l u c o s e Serum g l u c o s e was e l e v a t e d t o a maximum 3 h o u r s f o l l o w i n g a s i n g l e i p a d m i n i s t r a t i o n o f f l u o r i d e (NaF 3 5 m g / k g ) ( F i g . la). E l e v a t i o n o f serum

SERUM

U 3

0
U w

5
z
0
0

12

24

m
W

URINE

l n

.
z

2 M

-6

12

24

HOURS AFTER ADMINISTRATION

F i g u r e 1. Changes i n s e r u m and u r i n a r y g l u c o s e c o n t e n t s o f r a t s . The r a t s were k i l l e d a t v a r i o u s t i m e s f o l l o w i n g a s i n g l e i p a d m i n i s t r a t i o n o f f l u o r i d e (NaF, 3 5 rng/kg). T h e v a l u e s a r e a v e r a g e a n d S.E. (n=8). Fluoride0 i n ) Figure t r e a t e d (.)in F i g u r e l a . Control(=) and f l u o r i d e - t r e a t e d ( lb. " S i g n i f i c a n t d i f f e r e n c e f r o m t h e c o n t r o l . p<O.O2.

214
g l u c o s e b y f l u o r i d e was s u g g e s t e d t o b e p o s s i b l y d u e t o d e c r e a s e d u r i n a r y glucose excretion, o r t o enhancement o f g l y c o l y s i s o r gluconeogenesis. To t e s t these p o s s i b i l i t i e s , v a l u e s 6-12 changes i n u r i n a r y g l u c o s e e x c r e t i o n induced by U r i n a r y g l u c o s e e x c r e t i o n r e a c h e d maximum lb). These

f l u o r i d e w e r e f i r s t examined.

hours f o l l o w i n g t h e f l u o r i d e a d m i n i s t r a t i o n (Fig.

r e s u l t s suggest t h a t t h e e l e v a t i o n o f serum glucose i n f l u o r i d e - t r e a t e d r a t s i s n o t l i k e l y due t o d e c r e a s e d u r i n a r y g l u c o s e e x c r e t i o n . To t e s t w h e t h e r o r n o t e l e v a t i o n o f s e r u m g l u c o s e was a s s o c i a t e d w i t h a c t i v a t i o n o f g l y c o l y s i s o r gluconeogenesis-related fluoride enzymes, t h e e f f e c t s o f a d m i n i s t r a t i o n on t h e a c t i v i t i e s o f glucose-6-phosphatase. p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e and g l y c o g e n As shown i n T a b l e I ,

fructose-1.6-diphosphatase.

p h o s p h o r y l a s e ( a a n d b) i n t h e l i v e r w e r e e x a m i n e d .

t h e a c t i v i t y o f g l u c o s e - ~ 6 - p h o s p h a t a s e was m a r k e d l y enhanced b y f l u o r i d e .

TABLE I FLUORIDE-INDUCED C H A N G E S I N THE A C T I V I T I E S OF HEPATIC ENZYMES RELATED TO GLUCONEOGENESIS AND GLYCOLYSIS. Enzyme Hour a f t e r adrninistration Enzyme a c t i v i t y nmol/min/mg protein R e l a t i v e value 1 1.75a 1 1.02 1. 16b 1

Glucose-6-phosphatase

0
3

168.0 f 9 . 8 294.5 f 12.5 1320 f 47 1345 f 41 1530

Fructose-l.6d i phosphatase

0
1

3
Phosphoenol p y r u v a t e c a r b o x y k i nase

71

D
3

60.7 f 3.3 30.7 f 4.5 540 f 9 270 f 8 1 6 0 0 t 24 600 f 180

0. 50a
1

a Glycogen phosphorylase b

0
3

0. 50a
1

0
3

0. 38a

T h e r a t s w e r e k i l l e d 1 o r 3 h o u r s a f t e r f l u o r i d e a d m i n i s t r a t i o n (NaF 3 5 m g / k g . i p ) o r c h l o r i d e a d m i n i s t r a t i o n (NaC1 48.7 m g / k g . i p ) a s c o n t r o l t o rats. V a l u e s a r e a v e r a g e s o b t a i n e d f r o m f o u r t o s i x r a t s f SE. a v b S i g n i f i c a n t c h a n g e f r o m z e r o t i m e : ap<O.Ol; bp<0.05.

215
E f f e c t o f a d r e n a l e c t o m y on e l e v a t i o n of s e r u m g l u c o s e c a u s e d administration a d r e n a l g l u c o c o r t i c o i d s (3). n a l f u n c t i o n by f l u o r i d e , by fluoride

The p r o d u c t i o n o f g l u c o s e i s known t o be s t i m u l a t e d b y e p i n e p h r i n e a n d / o r I n o r d e r t o examine t h e enhancement o f adreAs shown a d r e n a l e c t o m y was c a r r i e d o u t o n r a t s . I n addition,

i n T a b l e 11. a d r e n a l e c t o m y m a r k e d l y s u p p r e s s e d t h e e l e v a t i o n o f s e r u m glucose induced by f l u o r i d e . r e n a l glucose-6-phosphatase by adrenalectomy. t h e e n h a n c e m e n t o f h e p a t i c and a c t i v i t y was f o u n d t o be c o m p l e t e l y s u p p r e s s e d s u g g e s t t h a t e l e v a t i o n o f serum g l u c o s e a c t i v i t y i n the rats.

These r e s u l t s ,

may b e due t o t h e e n h a n c e m e n t o f g l u c o s e - 6 - p h o s p h a t a s e l i v e r and k i d n e y s i n f l u o r i d e - t r e a t e d

TABLE I 1 EFFECT OF ADRENALECTOMY ON ELEVATIONS OF SERUM GLUCOSE, AND OF HEPATIC AND RENAL GLUCOSE-6-PHOSPHATASE A C T I V I T I E S BY A SINGLE LARGE DOSE OF FLUORIDE Treatment Intact Serum g l u c o s e c o n c e n t r a t i o n ( m g / d l ) Adrenalectomized

(AX)

Suppression by AX (%)

Control

104.8 f 8.2 187.3

f

56.6 f 10.5
55.8 f 4.1

46.0 100.6

F 1u o r i d e
Treatment

11.8

H e p a t i c gl ucose-6-phosphatasea Intact Adrenalectomized

(AX)

Suppression by AX (%)

Control F1 u o r i d e Treatment

168.6 f 9.8 294.4 f 12.5

138.9 f 0.9 139.7 f 2.7

Renal glucose-6-phosphatasea

17.6
99.5

Intact

Adrenalectomized

(AX)

Suppression b y AX ( X )

Control

70.0 f 1.9
195.9
?

54.1 i 1.4

22.7 102.6

F 1u o r i d e

2.8

50.4

4.8

The r a t s w e r e k i l l e d 3 h o u r s a f t e r f l u o r i d e a d m i n i s t r a t i o n (NaF 35 mg/kg. i p ) o r c h l o r i d e a d m i n i s t r a t i o n (NaC1 48.7 m g / k g . i p ) a s c o n t r o l t o r a t s . V a l u e s a r e a v e r a g e s o b t a i n e d f r o m f o u r t o s i x r a t s i SE. a E n z y m e a c t i v i t y i s e x p r e s s e d a s n m o l e s P i l i b e r a t e d p e r mg p r o t e i n p e r min.

TABLE 111
SUPPRESSIVE EFFECTS OF PROPRANOLOL AND/OR DIBENAMINE ON ELEVATIONS OF HEPATIC AND RENAL GLUCOSE-6PHOSPHATASE ACTIVITIES, AND OF SERUM GLUCOSE BY A SINGLE LARGE DOSE OF FLUORIDE Glucose-6-phosphatase activity Treatment Enzyme acti v i tya Liver Suppression by antagonist ( % ) 0 64.7b 76.4b 104b
-

Serum glucose Concentration (mg/dl) Suppression by antagonist (Z)

Kidney Enzyme Suppression acti vi tya by antagonist ( % ) 300.4 f 171.3 f 189.9 ?I 121.2 f 18.2 9.2 5.9 9.9

Fluoride None Pr

Di Pr+Di
Control None

475.5 f 42.3 257.6 k 4.0 218.4 rf 15.5 125.4 f 6.8 139.0

k

0 69.3b 59.3b 96.1b

-

155.0 104.3 112.2 95.1

15.5

k
k k

6.2 6.7 4.3 7.3 4.1

D
63.qb 53.5b 73.Zb

17.4

114.0 k 8.0

Propranolol (Pr) (1.0 mg/kg) or dibenamine (Di) (2.0 mg/kg) was subcutaneously two times immediately and 30 min after fluoride administration (NaF 35 mg/kg, ip) or chloride administration (NaC1 48.7 mg/kg, ip) as control to rats. The rats were killed 3 hours after the fluoride dose. Values are averages obtained from four rats k SE. aEnzyme activity is expressed as nmoles Pi liberated per mg protein per min. bSuppression by antagonist: p<O.O1

217

Suppressive effects of propranolol and/or dibenamine on elevation of qlucose-6-phosphatase activity and serum qlucose after a single larqe dose of fluoride The contribution of adrenomedullary function to elevation o f serum glucose i n fluoride-treated rats was examined by use of dibenamine (4) and/or propranolol (5) as an 01- or &blocker, respectively. The elevation of

0101.

serum glucose was significantly suppressed by dibenamine and/or propranHowever, the suppression of serum glucose level by concomitant

treatment with dibenamine and propranolol was not significantly higher than that by dibenamine or propranolol. alone (Table 111). Increase in glucose-6-phosphatase activities in the liver and kidneys was suppressed by dibenamine and/or propranolol (Table 111). These results suggest that elevation of serum glucose in fluoride-treated rats may be due to the stimulation of adrenomedullary function by fluoride. Changes in adrenocortical function and serum glucose caused by fluoride Production of glucose i s known to be stimulated by glucocorticoids secreted by the adrenal cortex ( 3 ) . Changes i n 1 7 - h y d r o x y c o r t i c o s t e r o n e in

u l

z _ d
0
CC
I - P

p :

9-

URINE

2
*

6 -

Figure 2 . Changes in serum and urinary 1 7 - h y d r o x y c o r t i c o s t e r o n e contents o f rats. The rats were killed at various times following a single ip administration of fluoride (NaF, 35 mg/kg). Values are average and S.E. (n=8). Fluoride-treated ( 0 ) . Figure 2a. Control ( I and ) fluoride-treated (a), Figure 2b. Significant difference from the control, +piO.O2, +%p<o. 05.

N W

TABLE I V EFFECTS OF E P I N E P H R I N E AND 17-HYDROXYCORTICOSTERONE ON HEPATIC AND RENAL GLUCOSE-6-PHOSPHATASE AND ON SERUM GLUCOSE OF RATS TREATED WITH FLUORIDE
~~

ACTIVITIES,

Glucose-6-phosphatase Treatment Enzyme activitya Liver Stimulation b y Ep o r Hc

activity Enzyme activitya Kidney Stimulation b y Ep o r Hc

Serum g l u c o s e Concentration (mg/dl) Stimulation b y Ep o r Hc

(Z)
0

(Z)
0

(Z)

Fluoride

None
Hc

294.5 f 1 2 . 5 349.1 f 10.2 350.2 ?r 13.5 346.5 f 14.1 168.6 f 9.8

195.9 216.1 210.5

*
f f

2.8 5.4 5.7

187.3 f 11.8

0
40.1 103.7b 170.4b
-

32. qb 33. Ob 30. gb

-

28.gb 26.8b 24.8b

-

229.3

6.4

EP

214.6 f 6.1

296.0 f 35.8 365.9 f 45.2 104.8 f 8.2

HctEp Control None

70.0 f 1.9

E p i n e p h r i n e (56.6 v g / k g ) a n d / o r 17-hydroxycorticosterone ( 1 0 0 u g / k g ) w e r e a d m i n i s t e r e d i p 1 0 m i n a f t e r a s i n g l e d o s e o f f l u o r i d e (NaF 3 5 m g / k g , i p ) t o r a t s . T h e r a t s w e r e k i l l e d 3 h o u r s a f t e r a s i n g l e d o s e o f f l u o r i d e o r c h l o r i d e (NaC1 48.7 rng/kg, i p ) as c o n t r o l t o r a t s . V a l u e s a r e a v e r a g e s o b t a i n e d f r o m f o u r r a t s f SE. aEnzyrne a c t i v i t y i s e x p r e s s e d as n m o l e s P i l i b e r a t e d p e r rng p r o t e i n p e r min. b S t i r n u l a t i o n b y Ep a n d / o r Hc:p<O.Ol

219
t h e s e r u m and u r i n e w e r e e x a m i n e d 0-24 h o u r s f o l l o w i n g a s i n g l e i p a d m i n i s tration o f fluoride. i s t r a t i o n (Fig. 2a). S e r u m 17-hydroxycorticosterone w a s f o u n d t o b e Moreover, u r i n a r y 17-hydroxycorticosterone 2b). excretion i n c r e a s e d p r i o r t o an e l e v a t i o n o f s e r u m g l u c o s e f o l l o w i n g f l u o r i d e a d m i n was i n c r e a s e d b y f l u o r i d e ( F i g . Moreover, These d a t a i n d i c a t e t h a t a d r e n o c o r -

t i c a l f u n c t i o n was s t i m u l a t e d b y f l u o r i d e . r e g u l a t i o n o f serum g l u c o s e l e v e l b y g l u c o c o r t i c o i d s has been Thus, t h e e f f e c t s o f 17serum glucose level in and/or epinephrine on r e p o r t e d t o b e a s s o c i a t e d w i t h e p i n e p h r i n e (4). hydroxycorticosterone fluoride-treated kidney.

r a t s w e r e e x a m i n e d a s a c o m p a r i s o n w i t h t h e e f f e c t s on t h e a c t i v i t i e s i n t h e l i v e r and

c o r r e s p o n d i n g changes i n g l u c o s e - 6 - p h o s p h a t a s e

E p i n e p h r i n e and 17-hydroxycorticosterone c o u l d be r e c o n f i r m e d t o a c t i v i t i e s i n t h e l i v e r and k i d n e y were m a r k e d l y i n c r e a s e i n enzyme a c t i v i t y b y c o n c o m i t a n t injection

i n c r e a s e s e r u m g l u c o s e l e v e l a s shown i n T a b l e I V . Glucose-6-phosphatase (Table IV). alone. On t h e o t h e r h a n d , Goiter-like a n i n c r e a s e i n p l a s m a PTH l e v e l s i s k n o w n t o b e a c h a r a c t e r i s t i c symptom o f human e n d e m i c f l u o r o s i s t h y r o x i n e a n d PTH ( 1 8 ) . Thus, However, e n h a n c e d b y t h e i n j e c t i o n o f e p i n e p h r i n e a n d 17-hydroxycorticosterone was n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t b y i n j e c t i o n o f e i t h e r d r u g

w i t h Genu Valgum (16).

s w e l l i n g o f t h e t h y r o i d o f a d o g was r e p o r t e d t o be i n d u c e d b y i n order t o understand t h e r e l a t i o n s h i p f u n c t i o n and e l e v a t i o n o f serum E l e v a t i o n o f serum

between changes i n t h y r o i d - p a r a t h y r o i d glucose i n f l u o r i d e - t r e a t e d on t h e animals. rats,

thyroid-parathyroidectomy was p e r f o r m e d

T h e r e s u l t s a r e s h o w n i n T a b l e V. livers and

g l u c o s e b y f l u o r i d e was n o t s u p p r e s s e d b y thyroid-parathyroidectomy. Changes i n c a l c i u m and cAMP i n t h e administration kidneys a f t e r f l u o r i d e

The mode o f t r a n s m i s s i o n o f t h e membrane-bound a d e n y l a t e c y c l a s e a c t i v i t y i s n o t y e t c l e a r a n d i n some t a r g e t c e l l s t h e a c t i v i t y i s k n o w n t o be m a r k e d l y i n f l u e n c e d b y La2+ i o n s (19). On t h e o t h e r h a n d , Thus, fluoride i s known t o s t i m u l a t e a d e n y l a t e c y c l a s e i n v i t r o (20). f l u o r i d e were examined. t h e changes i n

c a l c i u m and cAMP i n t h e l i v e r and k i d n e y f o l l o w i n g a s i n g l e l a r g e d o s e o f

A s s h o w n i n F i g u r e s 3 a a n d b, r e n a l a n d h e p a t i c
The e l e v a t i o n o f c a l c i u m c o n c e n t r a t i o n s

cAMP c o n c e n t r a t i o n s w e r e i n c r e a s e d a p p a r e n t l y p r i o r t o e l e v a t i o n o f t h e c a l c i u m c o n t e n t s i n t h e s e organs. i n t h e k i d n e y and l i v e r i n d u c e d b y f l u o r i d e was s i g n i f i c a n t l y s u p p r e s s e d b y

thyroid-parathyroidectomy ( 2 1 )( Ta b l e V I ) .

220

TABLE V EFFECT OF THYROID-PARATHYROIOECTOMY O N ELEVATIONS O F S E R U M AN0 U R I N A R Y GLUCOSE BY A SINGLE LARGE DOSE OF FLUORIDE Serum g l u c o s e c o n c e n t r a t i o n ( m g / d l ) a Intact TPTX Suppression b y TPTX ( % )

Treatment

Control F1 u o r i d e

71.5 f 13.1 133.6 f 1 1 . 6

85.8 f 9.0 131.4

?

-10.7 3.9

14.9

Treatment

U r i n a r y g l u c o s e e x c r e t i o n (mg/day/lOOg)b Intact TPTX

Suppression b y TPTX ( % )

Control

0.334 k 0.042 5.359 f 1.711

0.423 f 0.040 7.048 f 2.715

-26.7 -33.6

F1u o r i d e

The r a t s w e r e k i l l e d 3a o r 24b h o u r s a f t e r f l u o r i d e a d m i n i s t r a t i o n (NaF 35 mg/kg, i p ) o r c h l o r i d e a d m i n i s t r a t i o n (NaC1 48.7 mg/kg. i p ) as c o n t r o l t o rats. V a l u e s a r e a v e r a g e s o b t a i n e d f r o m e i g h t r a t s t SE. S u p p r e s s i o n b y TPTX: n o s i g n i f i c a n c e (p>O.O5)

lL==++dw
0 3
6 12

Ca

2.

HOURS AFTER ADMINISTRATION

F i g u r e 3. E f f e c t o f f l u o r i d e a d m i n i s t r a t i o n o n c a l c i u m and cAMP c o n t e n t s i n t h e k i d n e y a n d l i v e r . C h a n g e s i n t h e c a l c i u m a n d cAMP c o n t e n t s w e r e d e t e r m i n e d a t v a r i o u s t i m e s a f t e r a s i n g l e f l u o r i d e a d m i n i s t r a t i o n (NaF, 35 T h e v a l u e s a r e a v e r a g e a n d S.E. (n=6). 0 . c a l c i u m ; 0,CAMP. mg/kg).

221

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222 In vitro 1 iv e r effects o f f l u o r i d e and PTH o n c a l c i u m - f l u x i n t h e kidney and

C o n t r a r y t o e x p e c t a t i o n , t h e h e p a t i c and r e n a l c a l c i u m e f f l u x was n o t s u p p r e s s e d b y e i t h e r f l u o r i d e o r PTH ( T a b l e V I I ) . d e c r e a s e d b y b o t h f l u o r i d e and PTH, I n t h i s experiment, Moreover, t h e c a l c i u m i n f l u x i n r e n a l and h e p a t i c m i t o c h o n d r i a and c y t o s o l s was s i g n i f i c a n t l y as shown i n T a b l e V I I I . a c t i v i t i e s (22-24) in
i t w a s f o u n d t h a t e l e v a t i o n o f s e r u m g l u c o s e was

a s s o c i a t e d w i t h enhancement o f g l u c o s e - 6 - p h o s p h a t a s e t h e l i v e r and k i d n e y o f f l u o r i d e - t r e a t e d t i o n o f serum glucose.

r a t s . R e n a l a n d h e p a t i c CAMP

concentrations were increased markedly p r i o r t o t h e fluoride-induced elevaHowever, e l e v a t i o n o f r e n a l and h e p a t i c c a l c i u m c o n t e n t s c o u l d n o t b e d e m o n s t r a t e d i n a s s o c i a t i o n w i t h t h e augment o f serum glucose by t h e f l u o r i d e administration.

TABLE V I I EFFECTS OF FLUORIDE AND PARATHORMONE ON CALCIUM-WASHOUT I N RAT KIDNEY 45Ca E f f l u x ( n m o l / g / m i n ) Kidney Control 18.75 f 0.53 ( 4 ) 10-3 M 20.25 k 1.28 ( 4 ) 30.50 f 2 . 7 0 a ( 4 ) 18.75 f 1.35 ( 4 ) 16.50 f 1.50b(4) Liver 16.43 f 0.60 ( 4 ) 17.40 f 1.28 ( 4 ) 20.70 f 1 . 5 0 a ( 4 ) 19.28 f 0.75 ( 4 ) 22.35 f 1 . 0 5 a ( 4 )

F-

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2TH

1 pg m l - I 10 pg m l - I

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223

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224

REFERENCES 1. T a y l o r JM, S c o t t JK. M a y n a r d EA. S m i t h FA, Hodge H D ( 1 9 6 1 ) T o x i c o l A p p l Pharmac 3:278-289 S a k a s h i t a T, Okada S (1985) T o x i c o l A p p l

2a. S u k e t a Y, Asao Y, Kanamoto Y, Pharmac 80:199-205 2b. S u k e t a Y, 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

M i k a m i E, H a y a s h i M (1977) T o x i c o l A p p l Pharmac 39:313-319

E x t o n JH, FrTedmann N, Wong EH, B r i n e a u JP. C o r b i n J B i o l Chem 247:3579-3588 T o l b e r t MEM, S a i t o h Y, U i M (1976) Biochem P h a r m a c o l 25:841-845

JD,

P a r k CR (1972)

B u t c h e r FR, F a i t JN (1973) J B i o l Chem 248:5686-5692

H u l t m a n E (1959) N a t u r e 183:108-109 P o r t e r CC, S i l b e r RC ( 1 9 5 0 ) J B i o l Chem 1 8 4 : 2 0 1 - 2 0 7 Swanson M A (1955) I n : C o l o w i c k SP, l o g y . Acad Press, New York, V o l 2, K a p l a n NO (eds) Methods i n EnzymoFP 541-543 Methods i n

M c G i l b e r t RW ( 1 9 5 5 ) I n : C o l o w i c k SP, K a p l a n NO ( e d s ) , Enzymology. Acad Press, New York. V o l 2, p p 543-546 B a l l a r d FJ, Hanson RW (1969) J B i o l Chem 244:5625-5630 M e y e r F, H e i l m e y e r J r LMG, H a s c h k e RJ, 245: 6642-6648 W a l l a c e J. P i n t a d o E,

F i s c h e r E H ( 1 9 7 0 ) J B l o l Chem

S c a r p a A (1983) F E B S L e t t e r s 151:83-88 F a r r AF. R a n d a l l RJ ( 1 9 5 1 ) J B i o l Chem

W i l l i s JB (1960) S p e c t r o c h i m A c t a 16:259-272 L o w r y OH, R o s e b r o u g h NJ, 193: 265-275 K r i s h n a m a c h a r i KAVR,

J d r g e n s e n PL (1968) B i o c h i m B l o p h y s A c t a 151:212-224 S i v k u m a r B (1976) F l u o r i d e 9:185-200 McLaren JR (1976) F l u o r i d e 9:105-116 Ashmore J, Weber G (1959) V a t a m i n e s Hormones 17:91-132 Bradshaw RA, S u k e t a Y, S u k e t a Y, S u k e t a Y, S u k e t a Y, F r a z i e r WA (1977) C u r r Top C e l l Regul 1 2 : l - 3 7 R a l l TW. S u t h e r l a n d EM (1958) J B i o l Chem 232:1065-1076 Kanamoto Y (1983) T o x ~ c o l o g y26:335-345 S a t o M (1980) T o x i c o l A p p l Pharmac 52:386-390 S a t o M. K u r a M (1980) E x p e r i e n t i a 36:438-439 Yamada M, Hasegawa J, Asao Y (1982) M o l Pharmac 22:116-120

20.
21. 22. 23. 24.

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 225-229 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

225

FLUORIDE AND CALCIUM INTERRELATIONSHIP I N MATERNAL AND CORD BLOOD SERA MOT00 NIWA, JUN TAKIMOTO, AND TATSUHIKO TSUJI D e p a r t m e n t o f P r e v e n t i v e and C o m m u n i t y D e n t i s t r y , N i p p o n D e n t a l U n i v e r s i t y , F u j i m i , Chiyoda-ku, T o k y o 102, J a p a n

ABSTRACT M a t e r n a l and c o r d s e r u m p a i r s w e r e c o l l e c t e d f r o m 121 d e l i v e r i e s . m a t e r n a l t o t a l s e r u m F was f o u n d t o b e 0.072+0.051 t o t a l s e r u m F was 0.055+0.028 ug/ml. ug/ml. The while the fetal but the differ-

T o t a l F l e v e l s i n m a t e r n a l serum were

f o u n d t o be s i g n i f i c a n t l y h i g h e r t h a n t h o s e i n c o r d serum, significant. On t h e o t h e r hand,

e n c e i n i o n i c F l e v e l s b e t w e e n m a t e r n a l and c o r d s e r a was n o t s t a t i s t i c a l l y t h e t o t a l and i o n i c Ca l e v e l s i n m a t e r n a l serum w e r e f o u n d t o be s i g n i f i c a n t l y l o w e r t h a n t h o s e i n c o r d serum. c o r d sera.

p o s i t i v e c o r r e l a t i o n was f o u n d b e t w e e n t h e i o n i c F l e v e l s i n m a t e r n a l and M a t e r n a l i o n i c Ca l e v e l s c o r r e l a t e d s i g n i f i c a n t l y a n d p o s i No s i g n i f i c a n t c o r r e l a t i o n w a s f o u n d These f i n d i n g s d o n o t s u p p o r t I o n i c F found i n t h e serum i n d i c a t e s t i v e l y w i t h t h o s e i n c o r d serum. t h e concept o f a placental barrier. tions.

b e t w e e n Ca and F i n m a t e r n a l and c o r d sera.

t h a t t h e r e i s no d i s e q u i l i b r a t i o n o f i o n i c F i n m a t e r n a l a n d f e t a l c i r c u l a -

INTRODUCTION I n o r d e r t o i n c r e a s e t h e knowledge o f t h e pharmacokinetics o f f l u o r i d e

( F ) i n t h e body,
f e t u s (1). determined unknown

i t i s i m p o r t a n t t o s t u d y t h e t r a n s f e r o f F from mother t o

However, because o f l a c k o f samples, o n l y a few a u t h o r s have

F l e v e l s i n human m a t e r n a l and c o r d b l o o d s e r a .

The e x a c t

q u a n t i t a t i v e r e l a t i o n s h i p b e t w e e n m a t e r n a l and c o r d b l o o d F l e v e l s r e m a i n s

(2. 3).

S i n c e il number m a t e r n a l and c o r d b l o o d s a m p l e s w e r e s i m u l t h e i r i o n i c and t o t a l F l e v e l s were studied. I n addiw h i c h h a s a h i g h a f f i n i t y f o r F, w e r e

taneously obtained. a l s o determined.

t i o n , t h e l e v e l s o f c a l c i u m (Ca),

MATERIALS AND METHODS I n c l u d e d i n t h e p r e s e n t s t u d y w e r e p r e g n a n t wcmon who d e l i v e r e d a t an OB-

G Y N h o s p i t a l i n Tokyo, Japan, b e t w e e n 1 9 8 2 a n d 1984.

lected durlng parturition;

C o r d b l o o d was c o l -

m a t e r n a l b l o o d was w i t h d r a w n a s soon a s p o s s i b l e

226
after parturition. B o t h b l o o d s a m p l e s w e r e o b t a i n e d f r o m 92 women. respectively. Ionic

and t o t a l F l e v e l s i n b l o o d s e r u m w e r e d e t e r m i n e d b y t h e F e l e c t r o d e m e t h o d and t h e A1F m o l e c u l a r a b s o r p t i o n m e t h o d (4), molecular absorption analysis, RESULTS AND D I S C U S S I O N I o n i c and t o t a l F l e v e l s i n b o t h m a t e r n a l and c o r d b l o o d p r o d u c e d a l m o s t r e g u l a r frequency d i s t r i b u t i o n patterns. Mean i o n i c F l e v e l s w e r e comparCa a b l e i n m a t e r n a l and c o r d b l o o d s e r a , w h i l e t o t a l F l e v e l s i n c o r d b l o o d s e r u m w e r e s i g n i f i c a n t l y l o w e r t h a n t h o s e i n m a t e r n a l b l o o d serum. respectively. I o n i c and t o t a l Ca l e v e l s i n b l o o d s e r a w e r e d e t e r m i n e d b y t h e Ca e l e c t r o d e m e t h o d and b y

TABLE I

FLUORIDE

AND CALCIUM

LEVELS IN MATERNAL AND CORD SERA (n=92)'

M a t e r n a l Serum C o r d Serum

P
NS
~0.01

I o n i c f l u o r i d e (ppm) T o t a l f l u o r i d e (ppm) I o n i c c a l c i u m (mM) T o t a l c a l c i u m (mM) ' V a l u e s a r e means f

0.036
0.070

*
k

0.004 0.040 0.062

0.035 0.055 0.965 2.939

0.004 0.028 0.088 0.148

0.944

*
f

NS

2.585 f 0.177

<0.001

S.D.

NS: ~ 0 . 0 5

TABLE I 1 CONCENTRATIONS Feto-maternal

OF I O N I C

FLUORIDE I N 1,lATERNAL AND CORD SERA n Frequency (%) 7.6

rate'

85 90 95

7
9 15 14 47 92

85 90
100

9.8 16.3 15.2 51.0

99.9

48.9

95 - 1 0 0
-

Total

I o n i c F i n c o r d serum I o n i c F i n m a t e r n a l serum

100 ( % )

227
l e v e l s i n c o r d b l o o d serum were s i g n i f i c a n t l y h i g h e r t h a n t h o s e i n m a t e r n a l blood serum (Table I). I o n i c F l e v e l s i n c o r d b l o o d serum were l o w e r than t h o s e i n m a t e r n a l b l o o d s e r u m i n 48.9% o f t h e s a m p l e s , and t h e y w e r e e q u a l t o o r h i g h e r t h a n t h o s e i n m a t e r n a l b l o o d s e r u m i n t h e r e m a i n i n g 51.1%. T o t a l F l e v e l s were l o w e r i n c o r d b l o o d serum t h a n i n m a t e r n a l b l o o d serum i n 64.2% o f t h e s a m p l e s a n d h i g h e r i n t h e r e m a i n i n g 35.8%. the rates o f transfer, therefore, f e t u s more e a s i l y than t o t a l Judging from i o n i c F seems t o b e t r a n s f e r r e d t o t h e

( T a b l e s I 1 a n d 111). I n o r d e r t o e v a l u a t e

t h e c o r r e l a t i o n b e t w e e n F a n d Ca l e v e l s i n m a t e r n a l a n d c o r d b l o o d s e r a , t h e c o r r e l a t i o n c o e f f i c i e n t w a s d e t e r m i n e d f o r i o n i c a n d t o t a l F a n d Ca levels. (Figs.

A s i g n i f i c a n t c o r r e l a t i o n was f o u n d b e t w e e n i o n i c F and Ca l e v e l s
1 a n d 2).

-t
g 0011 a
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. ..

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M a t e r n a l serum (ppm)
F i g u r e 1. S c a t t e r d i a g r a m and r e g r e s s i o n l i n e o f i o n i c f l u o r i d e c o n c e n t r a t i o n s i n c o r d and m a t e r n a l s e r a .

Newborns were grouped by sex i n o r d e r t o d e t e r m i n e t h e c o r r e l a t i o n c o e f f i c i e n t b e t w e e n s e r u m F l e v e l s and b i r t h w e i g h t . t i o n was found. p4I.01) serum, However,
No s i g n i f i c a n t c o r r e l a -

a s i g n i f i c a n t n e g a t i v e c o r r e l a t i o n ( r = -0.3,

was p r e s e n t b e t w e e n m a t e r n a l age and t o t a l F l e v e l s i n c o r d b l o o d suggesting a v e r y c l o s e c o r r e l a t i o n between t h e t w o parameters.

228
TABLE 111 CONCENTRATIONS OF TOTAL FLUORIDE I N MATERNAL AND CORD SERA Feto-maternal r a t e1 n 5 Frequency (%) 5.4 19.6 27.2 12.0 35. 64.2

25 50 100
-

25 50 75

ia
25 11 33 92

75 - 1 0 0
-

Total 1

100.0

T o t a l F i n c o r d serum T o t a l F i n m a t e r n a l serum

100

(z)

..

. .

. . : ::: :/
..

..

Y=0.64X+0.36

Maternal serum (ri

'

F i g u r e 2. S c a t t e r d i a g r a m and r e g r e s s i o n l i n e o f i o n i c c a l c i u m c o n c e n t r a t i o n s i n c o r d and m a t e r n a l sera.

CONCLUSION R e s u l t s f r o m t h e p r e s e n t s t u d y s u g g e s t t h a t w h i l e Ca, w h i c h i s needed f o r f e t a l g r o w t h , was t r a n s f e r r e d t o t h e f e t u s t o a c o n s i d e r a b l e e x t e n t , t h e t r a n s f e r o f F was r a t h e r r e s t r i c t e d . Furthermore, organic F levels (which This also are probably equal t o t o t a l F l e v e l s minus i o n i c F l e v e l s ) decreased i n c o r d b l o o d s e r u m as c o m p a r e d t o t h o s e i n m a t e r n a l b l o o d serum.

229
suggests a p a r t i a l b l o c k i n g a t t h e p l a c e n t a l b a r r i e r . I n order t o deterhowever, studies

mine t h e e x t e n t t o which F i s t r a n s f e r r e d i n t o t h e fetus,

i n v o l v i n g a g r e a t e r number o f s a m p l e s ( i n c l u d i n g d e t e r m i n a t i o n o f p l a c e n t a l

F l e v e l s ) a s w e l l a s a n i m a l s t u d i e s a r e needed.
REFERENCES 1. 2. G e d a l i a I (1979) I n : A r m s t r o n g WD. 434 Shen YW. N i s h i d a A. F l u o r i d e s and Human H e a l t h .

WHO,

Geneva,

p 103

S i n g e r L,

M a k o w s k i E ( 1 9 7 0 ) Am J O b s t e t G y n e c o l 107:432-

3.
4.

T a v e s DR (1974) Am J O b s t e t G y n e c o l 119:205-207 N i w a M (1984) O d o n t o l o g y 72:871-881

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 231-239 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

231

FLUORIDE T O X I C I T Y : FORMATI O N A.

ERYTHROCYTE MEMBRANE ABNORMALITY AND "ECHINOCYTE"

K.

SUSHEELA AND SURESH K.

JAIN A1 1

F l u o r o s i s and F l u o r i d e Research L a b o r a t o r y , D e p a r t m e n t o f Anatomy, I n d i a I n s t i t u t e o f M e d i c a l S c i e n c e s , New D e l h i - 1 1 0 029, I n d i a ABSTRACT

I n g e s t i o n o f f l u o r i d e i n excess leads t o i t s accumulation i n t h e e r y t h r o c y t e membrane. The b i o c h e m i c a l and s t r u c t u r a l a b n o r m a l i t i e s d e t e c t e d a r e r e p o r t e d i n t h i s communication. B l o o d s a m p l e s o f r a b b i t s a d m i n i s t e r e d w i t h 1 0 mg NaF/kg b o d y w e i g h t a t 24 hour i n t e r v a l s f o r v a r y i n g d u r a t i o n s were investigated. a f f l i c t e d w i t h h y d r o f l u o r o s i s were a l s o investigated. F l u o r i d e and c a l c i u m c o n t e n t s o f serum and e r y t h r o c y t e membrane w e r e e s t i m a t e d u s i n g an i o n s e l e c t i v e e l e c t r o d e and a n a t o m i c a b s o r p t i o n spectrometer respectively. The e r y t h r o c y t e m o r p h o l o g y was s t u d i e d u s i n g a scanning e l e c t r o n microscope. The r e s u l t s i n d i c a t e t h a t f l u o r i d e a c c u m u l a t i o n i n t h e e r y t h r o c y t e memb r a n e l e d t o d e p l e t i o n o f c a l c i u m i n t h e membrane. Erythrocytes w i t h However, s p i n o u s p r o j e c t i o n s w e r e a t a maximum o f 7% i n human s u b j e c t s . f l u o r i d e was i n c r e a s e d f r o m 1-20 months. Human s u b j e c t s

t h e number i n t h e r a b b i t r o s e f r o m 1% t o 9% a s t h e d u r a t i o n o f e x p o s u r e t o

I t was a l s o e v i d e n t t h a t o l d e r

e r y t h r o c y t e s were more s e r i o u s l y a f f e c t e d t h a n t h e n e w l y formed e r y t h r o c y t e s as f l u o r i d e a c c u m u l a t i o n increased. T h i s s t u d y has c o n f i r m e d t h a t f l u o r i d e i s one o f t h e c h e m i c a l a g e n t s responsible f o r inducing echinocyte formation. INTRODUCTION Susheela e t al. (1) have r e p o r t e d an a c c u m u l a t i o n o f f l u o r i d e i n t h e Changes e r y t h r o c y t e membrane i n r a b b i t s t h a t had i n g e s t e d e x c e s s f l u o r i d e . excess f l u o r i d e ingestion. ingestion.

h a v e been r e p o r t e d i n t h e h e m a t o l o g i c a l p r o f i l e o f r a b b i t s a s a r e s u l t o f Anemia a l s o occurred as a r e s u l t o f f l u o r i d e The c o n c l u s i o n s d r a w n w e r e b a s e d o n s t u d i e s c o n d u c t e d u s i n g 10,

30 and 50 mg d o s e s o f f l u o r i d e a d m i n i s t e r e d t o r a b b i t s f o r f i v e m o n t h s (2).
I n f o r m a t i o n o n b l o o d c e l l p r o f i l e was f u r t h e r augmented b y s t u d i e s cond u c t e d b y S u s h e e l a a n d J a i n (3). According t o these i n v e s t i g a t o r s t h e t h o s e a n i m a l s t h a t w e r e on e r y t h r o c y t e and l e u c o c y t e p o p u l a t i o n r e v e a l e d a s i g n i f i c a n t r e d u c t i o n a f t e r

6 and 1 2 m o n t h s o f f l u o r i d e i n g e s t i o n . However,

232
NaF f o r 7 , m o n t h s p r o d u c e d o f f s p r i n g w h i c h d i d n o t r e v e a l a n y d e v i a t i o n e i t h e r i n e r y t h r o c y t e o r leucocyte population. and t h e i r o f f s p r i n g . Basophils. lymphocytes and hemoglobin c o n t e n t were a l s o s i g n i f i c a n t l y reduced i n b o t h t r e a t e d a n i m a l s The r e d u c t i o n i n b l o o d c e l l c o u n t and h e m o g l o b i n Rao c o n t e n t was c o n s i d e r e d as a r e s u l t o f a d r e n a l c o r t i c a l m a l f u n c t i o n . g l a n d and r e d u c t i o n i n d e l t a 5,3

and S u s h e e l a ( 4 ) h a v e r e p o r t e d e a r l i e r t h a t h y p e r t r o p h y o f t h e a d r e n a l b e t a s t e r o i d dehydrogenase a c t i v i t y was due t o e x c e s s i v e f l u o r i d e i n g e s t i o n . The p r e s e n t communication r e p o r t s t h a t e r y t h r o c y t e membrane a b n o r m a l i t y o c c u r s i n human s u b j e c t s a f f l i c t e d w i t h s k e l e t a l f l u o r o s i s and i n r a b b i t s t h a t i n g e s t e x c e s s i v e amounts o f f l u o r i d e . MATERIALS AN0 METHODS Human S u b j e c t s A f f l i c t e d w i t h S k e l e t a l F l u o r o s i s B l o o d a n d u r i n a r y s a m p l e s w e r e c o l l e c t e d f r o m t w o p a t i e n t s ( A and B) a f f l i c t e d w i t h s k e l e t a l f l u o r o s i s diagnosed r a d i o l o g i c a l l y and t h e b l o o d samples were used f o r s t u d i e s on e r y t h r o c y t e morphology u s i n g SEM. R a b b i t Model Male a l b i n o r a b b i t s w e i g h i n g 750-1000 g each were k e p t under l a b o r a t o r y conditions, segregated a t random i n t o t w o groups, and f e d a balanced d i e t The a n i m a l s o b t a i n e d f r o m H i n d u s t a n Lever (Bombay) and w a t e r ad l i b i t u m . f o r a p e r i o d o f 20 months. sex-matched c o n t r o l s .

i n one group were g i v e n o r a l l y 10 mg o f NaF/kg body w e i g h t one dose d a i l y A n i m a l s i n t h e s e c o n d g r o u p s e r v e d as age and B l o o d was d r a w n a t i n t e r v a l s o f 1,

3,

9 and 20

months a f t e r f l u o r i d e t r e a t m e n t .

Blood samples f r o m c o n t r o l a n i m a l s were The samples were used f o r scanning

a l s o c o l l e c t e d a t t h e same i n t e r v a l s . plasma c a l c i u m c o n t e n t d e t e r m i n a t i o n s .

e l e c t r o n m i c r o s c o p i c s t u d i e s o f e r y t h r o c y t e s , serum f l u o r i d e c o n t e n t and Scanning E l e c t r o n M i c r o s c o p i c S t u d i e s o f E r y t h r o c y t e s Blood samples f r o m humans and r a b b i t s were t r a n s f e r r e d i n t o c o l d b u f f e r e d (pH 7.4) g l u t e r a l d e h y d e (2%) f o r 45 minutes. After fixation, samples were a i r d r i e d and washed t h r e e t i m e s i n c o l d p h o s p h a t e s p u t t e r c o a t e d w i t h gold. b u f f e r (pH 7.4) (5). A t h i n f i l m o f

t h e m a t e r i a l t h u s o b t a i n e d was p l a c e d o n a s m a l l s t u b , Scanning E l e c t r o n Microscope 501. C o u n t i n g o f Abnormal E r y t h r o c y t e s

The c e l l s were scanned a t 30 K V u s i n g a P h i l i p s

A m i n i m u m o f 1 1 and a maximum o f 300 c e l l s f r o m a n u m b e r o f s p e c i m e n s

o b t a i n e d f r o m t h e 2 p a t i e n t s i n v e s t i g a t e d were counted on t h e t e l e v i s i o n s c r e e n o f t h e s c a n n i n g e l e c t r o n m i c r o s c o p e a t a m a g n i f i c a t i o n o f 640. F u r t h e r , a minimum o f 78 and a maximum o f 310 c e l l s f r o m a number o f s t u b s

233
obtained from t h e experimental animals ( f o r d e t a i l s r e f e r Table I ) were a l s o counted. A b n o r m a l c e l l s among t h e t o t a l p o p u l a t i o n o f c e l l s w e r e s c o r e d and t h e p e r c e n t a g e o f a b n o r m a l c e l l s was c a l c u l a t e d s i m u l t a n e o u s l y .

A s e r i e s o f p h o t o g r a p h s w e r e t a k e n f r o m t h e s a m e f i e l d s a t t h e s a m e magn i f i c a t i o n ( 6 4 0 X), and c e l l s w e r e c o u n t e d f r o m t h e p h o t o g r a p h s t o c o n f i r m t h e o b s e r v a t i o n s made on t h e s c r e e n . Serum F l u o r i d e D e t e r m i n a t i o n (Human and R a b b i t ) Serum f l u o r i d e was d e t e r m i n e d b y u s i n g a pH m e t e r m o d e l PHM 8 4 ( R a d i o meter, Copenhagen) w i t h F- s p e c i f i c e l e c t r o d e s a s d e s c r i b e d b y H a l l

&

d.

(6).
Plasma C a l c i u m E s t i m a t i o n ( R a b b i t ) T o t a l c a l c i u m f r o m t h e p l a s m a o f t h e r a b b i t was d e t e r m i n e d w i t h a Pye Unicam A t o m i c A b s o r p t i o n S p e c t r o p h o t o m e t e r B a r k e r gt meter M o d e l SP-900 as described by The

d. (7).

The p r e c i s i o n was c h e c k e d b y i n t e r n a l s t a n d a r d s . (Radiometer, Copenhagen) with

p l a s m a c a l c i u m i o n c o n c e n t r a t i o n o f r a b b i t w a s e s t i m a t e d b y u s i n g a pH model PHM 8 4 calcium-specific The e l e c t r o d e s b y a p p l y i n g t h e m e t h o d o f H u l a n i c k i a n d T r o j a n o w i c z (8). a l l possible contamination. RESULTS E r y t h r o c y t e Morphology E r y t h r o c y t e s f r o m human s u b j e c t s a f f l i c t e d w i t h f l u o r o s i s and s o d i u m f l u o r i d e t r e a t e d r a b b i t s revealed c e r t a i n s p e c i f i c m o r p h o l o g i c a l changes w h i c h a r e d e p i c t e d i n F i g u r e s la,b,c and 2a,b,c, la, respectively. 2a).

g l a s s w a r e and s a m p l i n g b o t t l e s w e r e c l e a n e d i n h o t n i t r i c a c i d t o r e m o v e

It i s c l e a r

f r o m t h e s e e l e c t r o n m i c r o g r a p h s t h a t t h e e a r l i e s t change o b s e r v e d was t h a t t h e o u t e r membrane h a s a w a v y o u t l i n e ( F i g s . The c r e n a t i o n o f t h e c e l l s enhanced t o t h e e x t e n t t h a t p r o j e c t i o n s f r o m t h e s u r f a c e o f t h e e r y t h r o c y t e i n a l l d i r e c t i o n s appeared as l a r g e spinous processes (Figs. 1 b . c a n d 2b,c). B l o o d s a m p l e s o b t a i n e d f r o m n o r m a l human s u b j e c t s and c o n t r o l r a b b i t s d i d n o t r e v e a l any such a b n o r m a l i t y . The human s u b j e c t s i n v e s t i g a t e d had s e r u m f l u o r i d e c o n t e n t s r a n g i n g f r o m 0.12 t o 0.2 ppm ( n o r m a l v a l u e : 0.02 ppm) and u r i n a r y f l u o r i d e r a n g i n g f r o m 1.7 t o 2.8 ppm a n d t h e n u m b e r o f a b n o r m a l e r y t h r o c y t e s w e r e 4.4% a n d 6.7% i n the two subjects investigated. I n rabbits, t h e p e r c e n t a g e o f e r y t h r o c y t e s w h i c h had undergone such In m o r p h o l o g i c a l changes i n c r e a s e d w i t h d u r a t i o n o f exposure t o f l u o r i d e . 1.28% i n r a b b i t s t r e a t e d f o r one month,

T a b l e I i t i s r e v e a l e d t h a t t h e p e r c e n t a g e o f a b n o r m a l e r y t h r o c y t e s was whereas i n a n i m a l s t r e a t e d f o r 9 a n d 2 0 m o n t h s t h e a b n o r m a l a p p e a r i n g e r y t h r o c y t e s i n c r e a s e d f r o m 6.25% t o

234

F i g u r e la. Scanning e l e c t r o n micrograph o f e r y t h r o c y t e of human p a t i e n t A showing normal e r y t h r o c y t e s ( a ) and t h o s e w i t h c r e n a t e d margin ( X 6300). F i g u r e Ib. Scanning e l e c t r o n micrograph o f e r y t h r o c y t e o f human p a t i e n t B s h o w i n g a n o r m a l e r y t h r o c y t e and a n o t h e r w i t h wavy o u t l i n e and s p i n o u s membrane o u t g r o w t h s ( X 13125). F i g u r e Ic. Scanning e l e c t r o n micrograph o f e r y t h r o c y t e o f human p a t i e n t B showing a l a r g e number o f s p i n o u s membrane o u t g r o w t h s ( X 9800).

235

F i g u r e 2a. S c a n n i n g e l e c t r o n m i c r o g r a p h o f e r y t h r o c y t e o f r a b b i t t r e a t e d w i t h NaF f o r 9 m o n t h s ( X 15750). N o t e t h e wavy m a r g i n o f e r y t h r o c y t e . F i g u r e s 2b,c. Scanning e l e c t r o n micrographs o f e r y t h r o c y t e s o f r a b b i t t r e a t e d w i t h NaF f o r 1 9 m o n t h s ( X 17500). N o t e t h e m e m b r a n e a b n o r m a l i t y l e a d i n g t o spinous p r o j e c t i o n .

236

9.0%. I t i s a l s o e v i d e n t t h a t t h e s e r u m f l u o r i d e c o n t e n t i n c r e a s e d f r o m
0.09 ppm i n n o r m a l a n i m a l s t o 0.33 ppm and 0.78 ppm o v e r a p e r i o d o f 1-20
months o f NaF t r e a t m e n t . The number o f abnormal e r y t h r o c y t e s i n c r e a s e d as c i r c u l a t i n g l e v e l s o f f l u o r i d e increased. The t o t a l and i o n i c c a l c i u m c o n t e n t s o f t h e plasma i n r a b b i t s i n c r e a s e d s i g n i f i c a n t l y (P<0.05) ( T a b l e 11) a f t e r f l u o r i d e i n g e s t i o n f o r 9 months.

TABLE I

SERUM FLUORIDE CONTENT AND PERCENTAGE OF ABNORMAL ERYTHROCYTE POPULATION I N HUMAN SUBJECT AFFLICTED WITH FLUOROSIS AND I N R A B B I T S EXPOSED TO NaF FOR VARIOUS TIME INTERVALS
Duration o f exposure t o NaF Serum Flevels ( PPm) Percentage o f erythrocytes with altered morphology Number o f s t u b s examined w i t h r e f e r e n c e t o number o f human/animals s t u d i e d

Human S u b j e c t s P a t i e n t A (35 y r ) Patient 6 (51 y r ) Rabbits Normal

0.2
0.12

6.7%
4.4%

3 stubs/patient A 3 stubs/patient B
4 stubs/2 animals 4 stubs/2 animals

0.09

-_-_-

1 month
9 months

0.33
0.63

I . 28%
6.25%
9.0%

6 stubs/3 animals
6 stubs/2 animals

20 months

0.78

TABLE I 1

TOTAL

AND I O N I C CALCIUM CONCENTRATIONS (PPM) I N PLASMA

T o t a l Calcium PPm (n=5)

OF

RABBITS

I o n i c Calcium PPm (n=4)

Normal R a b b i t s Rabbits t r e a t e d w i t h NaF f o r 9 months 'Values a r e means

61.6 76.3
S.D.

6.7' 5.3

16.35 18.52

? ?

1.48

1.22 (p<0.05)

237
DISCUSSION The m o r p h o l o g i c a l changes o f e r y t h r o c y t e s i n human s u b j e c t s a f f l i c t e d w i t h s k e l e t a l f l u o r o s i s and t h e e x p e r i m e n t a l f l u o r o s i s i n r a b b i t s r e p o r t e d i n t h e p r e s e n t s t u d y b e a r c o n s i d e r a b l e s i m i l a r i t y t o r e d c e l l membrane pathological stages I

I 1 r e f e r r e d t o as e c h i n o c y t e I

I 1 (9).

I t has

been r e p o r t e d t h a t e c h i n o c y t e f o r m a t i o n can be i n d u c e d by (a) washing o f r e d c e l l s f r e e o f p l a s m a and e x a m i n i n g b e t w e e n g l a s s s l i d e s , factors, and ( c ) i n t r i n s i c f a c t o r s . I n t h e p r e s e n t study, (b) e x t r i n s i c washing erythro-

c y t e s t o remove p l a s m a and e x a m i n i n g them between g l a s s s l i d e s d i d n o t occur s i n c e t h e experimental procedure i n v o l v e d an e n t i r e l y d i f f e r e n t method. The e f f e c t o f g l a s s c a n a l s o b e r u l e d o u t a s s u c h c h a n g e s w e r e n o t o b s e r v e d i n b l o o d s a m p l e s o b t a i n e d f r o m n o r m a l m a t e r i a l and p r o c e s s e d s i m u l t a n e o u s l y i n i d e n t i c a l manner. B e c a u s e o f t h e s c a r c i t y o f human m a t e r i a l , only the erythrocytes from f l u o r o s e d r a b b i t s were f u r t h e r i n v e s t i g a t e d f o r t h e p o s s i b l e involvement o f e x t r i n s i c and i n t r i n s i c f a c t o r s r e s p o n s i b l e f o r p r o d u c i n g e r y t h r o c y t e membrane a b n o r m a l i t y s i m i l a r t o echinocyte I lecithin, b i l e acids, barbiturates,

11.
lysophenylbutazone,

I t i s known t h a t c e r t a i n c h e m i c a l s u b s t a n c e s s u c h a s f a t t y a c i d s ,
dipyridamole,

s a l i c y l a t e a n d r o s e b e n g a l c a n i n d u c e e c h i n o c y t e t r a n s f o r m a t i o n (9.10). According t o Hoffman (lo), a l t e r a t i o n s i n t h e morphology o f t h e e r y t h r o c y t e depend upon a t r a n s m e m b r a n e c o n c e n t r a t i o n g r a d i e n t o f r e s p e c t i v e c h e m i c a l agents. R e c e n t s t u d i e s h a v e shown t h a t f l u o r i d e p e n e t r a t e s b i o l o g i c a l hydrogen f l u o r i d e , whose a b s o r p t i o n i n b o t h humans and r a t s , as i s t h e c a s e w i t h submembranes a s t h e u n d i s s o c i a t e d a c i d , r a t e i s pH-dependent stances such as s a l i c y l i c acid,

a c e t y l s a l i c y l i c a c i d and some b a r b i t u r a t e s Hence, f l u o r i d e c a n

(11).

T h i s pH d e p e n d e n t a b s o r p t i o n o f f l u o r i d e t h r o u g h t h e membranes l e a d s

t o t h e f o r m a t i o n o f a t r a n s m e m b r a n e g r a d i e n t (12). formation.

a l s o b e g r o u p e d w i t h t h o s e a g e n t s w h i c h a r e known t o c a u s e e c h i n o c y t e

I t may a l s o be p o s s i b l e t h a t c e r t a i n i n t r i n s i c f a c t o r s a r e r e s p o n s i b l e
f o r t h e o b s e r v e d t r a n s f o r m a t i o n o f e r y t h r o c y t e s r e p o r t e d i n t h i s communication. calcium. Weed a n d C h a i l l e y (13) h a v e s u g g e s t e d t h a t m o r p h o l o g i c a l t r a n s f o r The i n c r e a s e i n p e r m e a b i l i t y t o c a l c i u m i s a t t r i b u t e d t o e n e r g y Susheela and J a i n (14) have r e p o r t e d a dehydrogenase mation i s associated with a generalized increase i n permeability t o depletion o f the erythrocytes.

s i g n i f i c a n t decrease i n t h e a c t i v i t i e s o f glucose-6-phosphate i n t a k e s o f sodium f l u o r i d e .

and p y r u v a t e k i n a s e i n e r y t h r o c y t e s f r o m r a b b i t s f o l l o w i n g e x c e s s i v e Reduced e r y t h r o c y t e membrane c a l c i u m c o n t e n t , and enhanced plasma and h e m o l y s a t e c a l c i u m l e v e l s have been r e p o r t e d by

238
J a i n a n d S u s h e e l a (15), fluoride toxicity. s u g g e s t i n g enhanced p e r m e a b i l i t y o f c a l c i u m i o n s i n

The o b s e r v e d e r y t h r o c y t e membrane a b n o r m a l i t y i n human fluoride

s u b j e c t s a f f l i c t e d w i t h s k e l e t a l f l u o r o s i s and i n e x p e r i m e n t a l r a b b i t s e x p o s e d t o f l u o r i d e may be a s s o c i a t e d w i t h an i n d i r e c t e f f e c t o f energy d e p l e t i o n o f erythrocytes. Therefore, o n i n t r i n s i c f a c t o r s s u c h a s i n c r e a s e d membrane p e r m e a b i l i t y t o c a l c i u m and the present study points out However, t h e p o s s i b i l i t y t h a t t h e o b s e r v e d e r y t h r o c y t e membrane a b n o r m a l i t y may h a v e been c a u s e d b y f l u o r i d e and o t h e r c a u s a t i v e f a c t o r s .

i t s h o u l d be

n o t e d t h a t t h e e n t i r e e r y t h r o c y t e p o p u l a t i o n d o e s n o t show t h e a b n o r m a l i t y .
I n t h e t w o human s u b j e c t s s t u d i e d t h e p e r c e n t a g e o f a b n o r m a l e r y t h r o c y t e

r a n g e d f r o m 4.4

t o 6.7% w h i l e i n t h e r a b b i t s t h e number r a n g e d f r o m 1.28 t o T h i s i s p o s s i b l y due t o In

9.0% when e x p o s e d t o f l u o r i d e f r o m 1 t o 20 months.

t h e f a c t t h a t e r y t h r o c y t e s a r e known t o h a v e a d e f i n i t e l i f e span. erythrocytes

humans e r y t h r o c y t e s , t h i s r a n g e s f r o m 120 t o 130 days, w h e r e a s i n r a b b i t

i t i s 6 8 days.

It t h e r e f o r e emerges t h a t i n o l d e r
This explains the

e r y t h r o c y t e s t h e f l u o r i d e c o n t e n t i n t h e membrane may b e h i g h e r and m o r e severely affected than t h a t i n newly formed cells. f o r m a t ion. I n conclusion, r e a s o n why o n l y a c e r t a i n p e r c e n t a g e o f c e l l s h a v e s h o w n " e c h i n o c y t e "

i t may be s t a t e d t h a t f l u o r i d e i s one o f t h e c h e m i c a l
formation.

substances r e s p o n s i b l e f o r "echinocyte" ACKNOWLLDGEMENTS We a r e g r a t e f u l t o Dr. T.

C.

Anand Kumar,

Director,

Institute for

Research i n Reproduction (IRR), and Dr.

Bombay,

f o r a l l o w i n g us t o use t h e s c a n n i n g and t o Dr. K a m l a G o p a l k r i s h n a W e also

e l e c t r o n microscope f o r p r e l i m i n a r y studies, thank Mr. assistance.

Ashok Kumar o f I R R f o r t h e i r h e l p d u r i n g t h e SEM s t u d i e s .

S.

C.

P.

Sharma o f t h e e l e c t r o n m i c r o s c o p e l a b o r a t o r y o f t h e A l l New D e l h i . f o r h i s technical

I n d i a I n s t i t u t e o f Medical Sciences (AIIMS), mentation Centre (RSIC) f a c i l i t y ,

W e acknowledge t h e use o f t h e Regional S o p h i s t i c a t e d I n s t r u -

AIIMS,

w h e r e t h i s s t u d y was c o m p l e t e d .

T h i s i n v e s t i g a t i o n was s u p p o r t e d b y g r a n t s made a v a i l a b l e t o one o f u s

( A K S ) b y t h e D e p a r t m e n t o f E n v i r o n m e n t , Government o f I n d i a , New D e l h i .
REFERENCES 1. S u s h e e l a AK. Sharma YD, F l u o r i d e 15(4):173-177 3. S u s h e e l a AK, Mohan J, S i n g h M, Jagannath

B,

J a i n SK

(1982)

2. H i r a o M (1972) F l u o r i d e 5(1):33-37 J a i n SK (1983a) B u l l E n v i r o n Contam T o x i c o l 30:388-393

4. Rao K,

S u s h e e l a A K (1979) F l u o r i d e 12:67-71

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5. C l a r k e JA, S a l s b u r y AJ (1967) N a t u r e 215:402-404

6. H a l l LL, S m i t h PA, De L o p e z OH, 1458 G a r d n e r DE ( 1 9 7 2 ) C l i n Chem 1 8 : 1 4 5 5 -

7. B a r k e r DH, R e n d e r AC. M i t t a l BM, S h a n b h a g SU, S h a r m a VN, S h a r m a L S (1976) I n : H e m p h i l DD ( e d ) T r a c e S u b s t a n c e s i n E n v i r o n m e n t a l H e a l t h . V o l X, U n i v o f M i s s o u r i , C o l u m b i a 8. H u l a n i c k i A. T r o j a n o w i c z M (1974) A n a l y t i c a C h i m i c a A c t a 68:155-160 9. M o h a n d a s N. Weed R I , B e s s i s M ( 1 9 8 0 ) I n : T r u m p BF, A r s t i l a A U ( e d s ) P a t h o b i o l o g y o f C e l l Membranes. Academic Press, p p 41-91 10. H o f f m a n

JF

(1972) c f : P a t h o b i o l o g y o f C e l l Membranes,

1980

11. W h i t f o r d CM.

P a s h l e y DH (1984) C a l c i f T i s s u e I n t 36(3):302-307

12. M i l l e r GW. P u s h n i k JC, G i a n n i n i J, B r i s k i n OD (1985) 1 4 t h C o n f e r e n c e o f I n t e r n a t i o n a l S o c i e t y f o r F l u o r i d e Research, M o r i o k a 13. Weed R I , C h a i l l e y B ( 1 9 7 3 ) I n : B e s s i s M. Week R I . L e p l o n g PF ( e d s ) Red C e l l Shape, P h y s i o l o g y P a t h o l o g y , U l t r a s t r u c t u r e . S p r i n g e r V e r l a g , p 55 14. S u s h e e l a AK, 15. J a i n SK, J a i n SK (1983b) I R C S 11:14-15 S u s h e e l a AK B u l l E n v i r o n Contam T o x i c o l ( i n p r e s s )

H. Tsunoda and M.-H. Y u (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 241-251 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

241

A MODEL FOR SUBCELLULAR

DISTRIBUTION OF

FLUORIDE

I N H I G H E R PLANTS:

RELATION TO I N VITRO CONCENTRATIONS R E Q U I R E D FOR ENZYME I N H I B I T I O N

GENE W.

MILLER, JAMES C.

PUSHNIK, AND JOHN G I A N N I N I

Logan, UT 84322, U.S.A.

B i o l o g y Department, ABSTRACT

Utah State University,

P h y s i o l o g i c d a t a o f whole p l a n t response t o e l e v a t e d ambient a i r l e v e l s o f f l u o r i d e w o u l d seem i n c o n s i s t e n t w i t h c o n c e n t r a t i o n s r e q u i r e d t o a f f e c t b i o c h e m i c a l mechanisms i n v i t r o . been o b t a i n e d by s t e a d y - s t a t e Reconciliation o f the apparently higher

-~ i n v i t r o c o n c e n t r a t i o n s and t h e a n t i c i p a t e d l o w e r i n v i v o o b s e r v a t i o n s has
modeling o f t h e s u b c e l l u l a r p a r t i t i o n i n g o f d i f f u s i o n gradient. f l u o r i d e s i n t o c e l l u l a r f r a c t i o n s d r i v e n by a non-ionic

A p p l i c a t i o n o f t h i s p r e d i c t i v e model t o r e p o r t e d l i t e r a t u r e v a l u e s and p h y s i o l o g i c o b s e r v a t i o n s h a s r e s u l t e d i n an u n d e r s t a n d i n g o f t h e sequence o f biochemical a l t e r a t i o n s o f metabolism associated w i t h elevated ambient atmospheric concentrations o f f l u o r i d e . T h i s m o d e l c a n be a p p l i e d t o o t h e r e n v i r o n m e n t a l p o l l u t a n t s w h i c h a c t a s weak a c i d - b a s e c o u p l e s and may be u s e d t o p r e d i c t t h e c o n c e n t r a t i o n s o f t h e s e t o x i c compounds a t s u b c e l l u l a r sites. Such a m o d e l w o u l d p r o v i d e u s e f u l i n f o r m a t i o n i n s t u d y i n g c o n t e m I n f o r m a t i o n on t h e s u b c e l l u l a r p a r t i levels of p o r a r y problems such as a c i d r a i n .

tioning o f pollutants w i l l help elucidate the i n i t i a l s i t e s o f t o x i c i t y i n t h e o r g a n i s m and a i d i n t h e d e v e l o p m e n t o f r e a l i s t i c a l l o w a b l e t h e s e compounds i n t h e e n v i r o n m e n t . FLUORIDE I N THE ENVIRONMENT The p h y t o t o x i c e f f e c t s o f f l u o r i d e on p l a n t s and t h e i r c h e m i s t r y h a s been w e l l documented. generating plants, w o r k s and g l a s s , l o n g e d exposure. tissue, V e g e t a t i o n damage has been a t t r i b u t e d t o i n d u s t r i a l coal-fired electric bricksuperphosphate p l a n t s , and c e r a m i c f a c t o r i e s . aluminum r e f i n i n g plants, atmospheric f l u o r i d e e m i s s i o n s f r o m copper smelters,

F o l i a r damage i s c h a r a c t e r i z e d b y

i n i t i a l g e n e r a l l e a f c h l o r o s i s and e v e n t u a l m a r g i n a l n e c r o s i s w i t h p r o The a v a i l a b l e d a t a s u g g e s t t h a t f o l i a r n e c r o s i s i n s u s c e p t i b l e p l a n t s o c c u r s a t a t h r e s h o l d r e s p o n s e o f 40 ppm f l u o r i d e i n t h e w h i l e l e s s s e n s i t i v e s p e c i e s do n o t e x h i b i t v i s i b l e symptoms u n t i l h i g h e r l e v e l s o f a c c u m u l a t i o n o c c u r . A c c o r d i n g t o a N a t i o n a l Academy o f S c i e n c e R e p o r t (NAS),

40 pprn w o u l d r e s u l t f r o m e x p o s u r e t o a v e r a g e a m b i e n t

a i r c o n c e n t r a t i o n s o f g a s e o u s f l u o r i d e s o f 0.5 u g / m 3 f o r a p e r i o d o f 3 0

242
days: m o r e r a p i d a c c u m u l a t i o n h a s been d e m o n s t r a t e d w i t h h i g h e r e x p o s u r e

l e v e l s (1). Environmental Factors A f f e c t i n q P l a n t Uptake The p r i n c i p a l mode o f f l u o r i d e e n t r y i n t o p l a n t s i s t h r o u g h t h e s t o m a t e s d u r i n g a c t i v e g a s exchange. enter the leaf, opening. tal factors: S o l u b l e p a r t i c u l a t e forms o f f l u o r i d e can a l s o b o t h b y d i f f u s i o n t h r o u g h t h e c u t i c l e and v i a t h e s t o m a t a 1 1i g h t , temperature, w a t e r balance, nutritional status, age

These p a s s i v e a c c u m u l a t i o n s a r e r e s p o n s i v e t o v a r i o u s e n v i r o n m e n A l l t h e s e i n t e r a c t i n g f a c t o r s m o d i f y f o l i a r response. The d a r k

and p l a n t s p e c i e s .

Dark exposure o f Jerusalem c h e r r y l e a v e s t o gaseous f l u o r i d e r e s u l t e d i n l o w e r a c c u m u l a t i o n l e v e l s t h a n l i g h t t r e a t e d c o n t r o l s (2). t r e a t e d p l a n t s d i s p l a y e d no v i s i b l e f o l i a r damage: t r a n s f e r r e d t o t h e l i g h t s e v e r e damage r e s u l t e d . Sunflowers accumulated h i g h e r l e v e l s o f f l u o r i d e a t elevated temperature, w h i l e g l a d i o l u s d i s p l a y e d t h e opposite response b u t w i t h g r e a t e r v i s i b l e damage (3). P o o v a i a h and W i e b e ( 4 ) d e m o n s t r a t e d t h a t s o y b e a n s f u m i g a t e d w i t h f l u o r i d e d i s p l a y e d i n c r e a s e d v i s i b l e damage a t e l e v a t e d t e m p e r a t u r e even a f t e r t h e t e r m i n a t i o n o f t h e f u m i g a t i o n . P l a n t s g r o w i n g u n d e r w a t e r s t r e s s c o n d i t i o n s a p p e a r t o be m o r e r e s i s t a n t t o f l u o r i d e d a m a g e (5). b u t t h i s does n o t appear t o be t h e case i f t h e i m p o s i t i o n o f w a t e r s t r e s s o c c u r s a f t e r f u m i g a t i o n i s t e r m i n a t e d (4). Accumulation o f F l u o r i d e by t h e F o l i a q e o f Higher P l a n t s Once f l u o r i d e h a s p e n e t r a t e d l e a f e n t r y r e s i s t a n c e s , t h e a p o p l a s t t o w a r d t h e l e a f t i p s and margins, accumulation.
it dissolves i n the

however,

when t h e y w e r e

s u b s t o m a t a l l i q u i d p h a s e s and moves w i t h t h e t r a n s p i r a t i o n a l s t r e a m t h r o u g h the principal sites o f F l u o r i d e does n o t d i s t r i b u t e e v e n l y a c r o s s t h e l e a f b u t G a r r e c gJ

a p p e a r s t o a f f e c t t h e c e l l s c l o s e s t t o t h e s i t e e n t r y and a l o n g t h e t r a n s p i r a t i o n a l p a t h w a y (6).

d.( 7 )

employed e l e c t r o n microprobe

a n a l y s i s of f l u o r i d e exposed f i r n e e d l e s and d e m o n s t r a t e d t h a t f l u o r i d e d i s t r i b u t e d i n a steep g r a d i e n t from t h e l e a f t i p d i m i n i s h i n g toward t h e l e a f base. These i n v e s t i g a t o r s a l s o e s t a b l i s h e d t h a t t h e g r a d i e n t d i s t h e f i r s t i n t h e l e a f t i p and a s e c o n d a t t h e zone w h i c h p l a y e d t w o peaks,

separated t h e n e c r o t i c region from t h e uninjured tissue. C e l l u l a r Location o f F l u o r i d e by F r a c t i o n a t i o n Studies F r a c t i o n a t i o n s t u d i e s on f l u o r i d e exposed t o m a t o p l a n t s r e v e a l e d t h a t f l u o r i d e was f o u n d i n d e c r e a s i n g c o n c e n t r a t i o n s i n t h e c e l l w a l l s , plasts, soluble proteins, mitochondria, and m i c r o s o m e s (8). chloroThe h i g h f l u o -

r i d e c o n c e n t r a t i o n a s s o c i a t e d w i t h t h e c e l l w a l l i s i n accordance w i t h t h e known h i g h c a l c i u m c o n c e n t r a t i o n s o f t h a t f r a c t i o n . The a c c u m u l a t i o n i n t h e c h l o r o p l a s t was s u b s t a n t i a t e d b y Chang and Thompson (9) and i s c o n s i s t e n t

243
w i t h known u l t r a s t r u c t u r a l (10.11) and p h y s i o l o g i c r e s p o n s e s (12).

P h y s i o l o g i c Response t o E l e v a t e d F l u o r i d e L e v e l s The e a r l i e s t m a n i f e s t a t i o n s o f f l u o r i d e e x p o s u r e a r e a l t e r a t i o n s i n t h e r a t e s o f p h o t o s y n t h e s i s and r e s p i r a t i o n . I n v i v o s t u d i e s h a v e shown t h a t These p h o t o s y n t h e t i c and r e s p i r a t o r y a c t i v i t i e s r e s p o n d m o s t r a p i d l y t o f l u o r i d e e x p o s u r e a s q u i c k l y a s 2 h o u r s a f t e r f u m i g a t i o n w i t h 4 0 p p b HF. p r o c e s s e s a r e s i g n i f i c a n t l y a l t e r e d (13). R e c e n t s t u d i e s on t h e e f f e c t s o f

f l u o r i d e on t h e b i o c h e m i s t r y o f c h l o r o p l a s t s h a v e c l e a r l y d e m o n s t r a t e d t h a t t h e i n h i b i t i o n i s a s s o c i a t e d w i t h t h e a c t i o n o f t h e F i o n on t h e photosynthetic c o u p l i n g f a c t o r (ATPase) (14; Table

I).

I n contrast t o the

observed f l u o r i d e i n h i b i t i o n o f photosynthetic activities, mitochondrial r e s p i r a t i o n i s i n i t i a l l y enhanced b u t w i t h c o n t i n u e d e x p o s u r e i n h i b i t i o n o f o x y g e n c o n s u m p t i o n i s s e e n (15,16). While decreased t i s s u e . r e s p i r a t i o n i s t h e reason f o r g e n e r a l l y a t t r i b u t e d t o i n h i b i t i o n o f r e s p i r a t o r y enzymes, t h e i n i t i a l s t i m u l a t i o n i s somewhat obscure. M e t a b o l i c s t u d i e s show v a r y i n g d e g r e e s o f f l u o r i d e s u s c e p t i b i l i t y o f enzymes. Cytoplasmic enzymes, such as enolase (17) and phosphog l u c o m u t a s e (18) d i s p l a y s e n s i t i v i t i e s t o l o w l e v e l s o f s o l u b l e f l u o r i d e ( 5

mM).

B i o e n e r g e t i c w o r k h a s shown t h a t t h e m i t o c h o n d r i a l ( 1 5 ) and c h l o r o Some o f t h e m i t o c h o n d r i a l b o u n d

p l a s t l o c a l i z e d ATPases ( 1 9 ) a r e i n h i b i t e d a t a p p a r e n t l y h i g h e r c o n c e n t r a t i o n s o f f l u o r i d e (30 mM; T a b l e I ) . enzymes (dehydrogenases), however, a r e i n h i b i t e d s i g n i f i c a n t l y a t l o w e r

f l u o r i d e c o n c e n t r a t i o n s ( T a b l e I). T h e t o n o p l a s t a s s o c i a t e d ATPase d i s p l a y s a s e n s i t i v i t y t o f l u o r i d e s i m i l a r t o t h e m i t o c h o n d r i a l ATPase (20). b u t t h e m e m b r a n e pH g r a d i e n t f o r m a t i o n i s i n h i b i t e d a t 1 0 m M (Table I). The p l a s m a l e m m a l ATPase a p p e a r s t o be s e n s i t i v e t o f l u o r i d e i n h i b i t i o n a t 5

mM ( T a b l e I ) and i s i n h i b i t e d a t t h e l o w e s t c o n c e n t r a t i o n o f a l l membrane
bound A T P a s e s ( 2 1 ) . M c N u l t y and L o r d s (22) r e p o r t e d t h a t h i g h e r c o n c e n t r a t i o n s o f p h o s p h o r y l a t e d n u c l e o t i d e s were detected i n h i g h e r p l a n t s t r e a t e d w i t h f l u o r i d e than i n comparable c o n t r o l plants. T h e s e a u t h o r s s u g g e s t e d t h a t f l u o r i d e may t h u s i n c r e a s i n g t h e oxygen consumption d i s r u p t basic c e l l u l a r energetics,

b y i n c r e a s i n g t h e a m o u n t s o f p h o s p h a t e a c c e p t o r s ( A D P ) o r d o n o r s (ATP). R e v i e w o f t h e r e l e v a n t l i t e r a t u r e shows t h a t b o t h s i t u a t i o n s a r e o b s e r v e d . Yang a n d M i l l e r ( 1 8 ) p r o p o s e d t h a t i n c r e a s e d ADP c o n c e n t r a t i o n s w e r e responsible for f l u o r i d e s t i m u l a t i o n o f r e s p i r a t i o n i n n e c r o t i c tissues. i n v e s t i g a t e d t h e r e s p i r a t i o n o f f l u o r i d e fumigated mitochonM i l l e r and M i l l e r (16)

t i s s u e and t h e ATPase a c t i v i t y o f m i t o c h o n d r i a i s o l a t e d f r o m t h i s t i s s u e . T h e s e a u t h o r s f o u n d a good c o r r e l a t i o n among t i s s u e r e s p i r a t i o n , d r i a l respiration, a n d ATPase a c t i v i t y . These o b s e r v a t i o n s l e d t o t h e

244 p o s t u l a t i o n t h a t r e s p i r a t o r y s t i m u l a t i o n was c o n t r o l l e d b y t h e r e l a t i v e r a t i o o f c e l l u l a r ADP/ATP. A l t e r n a t e l y i t was s u g g e s t e d t h a t HF was a c t i n g These s t u d i e s a s a c l a s s i c weak u n c o u p l e r o f t h e r e s p i r a t o r y c h a i n (15). r e s p i r a t o r y a c t i v i t y (23).

Model

w e r e c o n s i s t e n t w i t h e a r l i e r r e s u l t s o f t h e c o m p a r i s o n o f DNP a n d H F o n Ballentyne

(24).

on t h e o t h e r h a n d , r e p o r t e d

i n c r e a s e s i n t h e l e v e l s o f ATP e x t r a c t e d f r o m f l u o r i d e f u m i g a t e d t i s s u e s .

o f Fluoride Subcellular Distribution:

Physical Factors

I n an a t t e m p t t o e q u a t e c o n c e n t r a t i o n s o f t i s s u e f l u o r i d e i n v o l v e d i n t h e
-~ i n vivo

o b s e r v a t i o n s o f a t m o s p h e r i c f l u o r i d e damage w i t h i n v i t r o e x p e r a steady-state The m o d e l i s b a s e d o n t h e weak a c i d (pKa 3.45) R e l a t i v e concen-

imental levels associated w i t h biochemical alterations, m o d e l o f s u b c e l l u l a r d i s t r i b u t i o n i s proposed. physical properties o f fluoride i n solution: and e x i s t s i n t w o f o r m s a t p h y s i o l o g i c a l pH: Henderson-Hasselbalch e a u a t i o n as f o l l o w s : pH=pKa

HF i s a

F- and HF.

t r a t i o n s - o f these i o n i c species are mathematically predictable by the

log [F-]/[HF]

The t w o i o n i c f o r m s e x h i b i t e x t r e m e l y d i f f e r e n t p e r m e a b i l i t y c o e f f i c i e n t s t h r o u g h l i p i d b i l a y e r membranes (25). IO-cm/sec, The c h a r g e d i o n c a n b e d e m o n s t r a t e d t o c r o s s a m o d e l membrane ( l e c i t h i n and c h o l e s t e r o l ) b i l a y e r a t 4.9 f 2.3 x w h i l e t h e u n d i s s o c i a t e d m o l e c u l e moves a p p r o x i m a t e l y 6 o r d e r s o f m a g n i t u d e m o r e r a p i d l y (1.4

0.3 x 1 0 - 4 c m / s e c ) .

A p r e d i c t a b l e con-

sequence o f t h e s e p r o p e r t i e s i s t h a t a d i f f e r e n t i a l s u b c e l l u l a r d i s t r i b u t i o n w i l l o c c u r b a s e d p r i m a r i l y o n l o c a l i z e d pH g r a d i e n t s w i t h i n t h e c e l l . T h i s d i s t r i b u t i o n w i l l occur as t h e f l u o r i d e t r a n s p i r a t i o n a l route, s u b c e l l u l a r compartments. moves f r o m t h e a p o p l a s t i c a c r o s s t h e plasmalemma, and e v e n t u a l l y i n t o t h e The movement i s d e p e n d e n t on t h e a t m o s p h e r i c and

t i s s u e f l u o r i d e c o n c e n t r a t i o n s and t h e m a g n i t u d e and d i r e c t i o n o f t h e pH d i f f e r e n c e on t w o s i d e s o f a membrane.

Mechanics

o f Fluoride Partitioninq Within the Cell

R e g r e s s i o n m o d e l s have been p r e s e n t e d w h i c h r e l a t e t h e a m b i e n t atmospheric f l u o r i d e concentrations w i t h the r a t e o f f o l i a r accumulation

(1.26).

The d o s e - r a t e

response f o r accumulation o f atmospheric f l u o r i d e i s

described by t h e equation:

AF

= KCT

where K i s t h e accumulation c o e f f i c i e n t ,

C represents t h e concentration of

h y d r o g e n f l u o r i d e i n ug/m3 and T i s t h e t i m e o f e x p o s u r e i n days. The v a l u e

245
o f K h a s b e e n d i f f i c u l t t o e s t a b l i s h f o r i n d i v i d u a l s p e c i e s and w i t h v a r y ing environmental conditions. R e g r e s s i o n m o d e l v a l u e s h a v e been p r i m a r i l y d e t e r m i n e d i n e n c l o s e d e n v i r o n m e n t a l chambers i n w h i c h t h e r e d u c t i o n o f a i r movement e s t a b l i s h e s a l a r g e r e s i s t a n c e b o u n d a r y l a y e r and r e s u l t s i n l o w e r a c c u m u l a t i o n t h a n w o u l d b e e x p e c t e d u n d e r f i e l d c o n d i t i o n s (26). result, lower, t h a n m i g h t be e x p e c t e d , under f i e l d conditions.

As a

t h e K v a l u e s f o r m o s t p l a n t s d e t e r m i n e d i n t h e s e c h a m b e r s w o u l d be These l i m i t a t i o n s

i n p r e d i c t i n g a c c u m u l a t i o n r a t e s o f a m b i e n t gaseous hydrogen f l u o r i d e a r e recognized, b u t a c c u m u l a t i o n s o f f l u o r i d e i n t h e v a r i o u s s u b c e l l u l a r comp a r t m e n t s r e s u l t f r o m t h e r e s i s t a n c e s e n c o u n t e r e d d u r i n g t h e p a s s i v e movement o f f l u o r i d e i n t h e t r a n s p i r a t i o n a l f l o w t o w a r d t h e l e a f margin. The f i r s t m a j o r r e s i s t a n c e b a r r i e r t o c e l l u l a r f l u o r i d e a c c u m u l a t i o n imposed by t h e l e a f i s t h e c e l l wall. g u i s h a b l e spaces; I n t h e WFS, sion. (27). Cell walls consist o f two distinand t h e Donnan f r e e s p a c e (DFS). while a w a t e r f r e e s p a c e (WFS)

c a t i o n s and a n i o n s a r e i n r o u g h l y e q u a l c o n c e n t r a t i o n s ,

t h e DFS c o n t a i n s f i x e d n e g a t i v e c h a r g e s and t e n d s t o p r o m o t e a n i o n e x c l u C e l l w a l l s a r e v i e w e d as c o n s i s t i n g o f m a c r o p o r e and m i c r o p o r e s p a c e s The f o r m e r i s u n c h a r g e d a n d c o r r e s p o n d s t o t h e WFS. while the latter

r e p r e s e n t s t h e DFS w h i c h i s h i g h l y c h a r g e d due t o i o n i z a b l e u r o n i c a c i d and p e c t i c substances. monovalent ions. The DFS d i s p l a y s p r e f e r e n t i a l b i n d i n g o f d i v a l e n t o v e r Sequestration o f ions by t h e c e l l w a l l i s p r i m a r i l y C a l c i u m i s o r d i n a r i l y t h e m a j o r c a t i o n bound i n Concentration o f t h i s ion i n the relative

e l e c t r o s t a t i c i n nature.

the c e l l w a l l i n t h e form o f calcium-pectate. degree o f s u s c e p t i b i l i t y t o f l u o r i d e t o x i c i t y .

v a r i e s w i t h p l a n t s p e c i e s and may be a c o n t r i b u t i n g f a c t o r

As a t m o s p h e r i c f l u o r i d e f o l l o w s t h e t r a n s p i r a t i o n a l

movement t o w a r d t h e
i t may b e g i n t o

l e a f m a r g i n t h r o u g h t h e macropore space i n t h e c e l l w a l l , due t o accumulated c a l c i u m (Fig. wall. This displacement 1).

p a r t i t i o n i n t o t h e m i c r o p o r e s p a c e s w h i c h h a v e become c h a r g e c o m p e n s a t e d

As t h e f l u o r i d e

concentration from the c e l l

i n c r e a s e s i n t h e m i c r o p o r e space, f l u o r i d e w i l l d i s p l a c e Ca i s due t o t h e s t r e n g t h s o f t h e CaF2 vs. s o l u b i l i t y products. bound. Ca p e c t a t e as i l l u s t r a t e d

large difference i n binding by t h e i r respective is

The c e l l w a l l w i l l a c c u m u l a t e F u n t i l a l l t h e Ca

T h i s phenomenon i s a n a l o g o u s t o a c a t i o n e x c h a n g e c o l u m n b i n d i n g T h i s phenomenon

a n i o n s u n t i l t h e a c t i v e s i t e s a r e c o m p l e t e l y exhausted.

might account f o r the d i f f e r e n c e i n the tolerance o f d i f f e r e n t p l a n t s p e c i e s t o t h r e s h o l d l e v e l s of a c c u m u l a t e d f l u o r i d e a s t h e c e l l w a l l c a l c i u m w o u l d be a c t i n g as a b u f f e r a g a i n s t c e l l u l a r f l u o r i d e a c c u m u l a t i o n . Once t h e b u f f e r i n g c a p a c i t y o f t h e c e l l w a l l i s d e p l e t e d b y p r e c i p i t a t i o n a s f l u o r o c o m p l e x e s o f t h e p o l y a n i o n b o u n d d i v a l e n t c a t i o n s ( F i g . 1). the

246

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Ca

F i g u r e 1. T h e i n t e r a c t i o n o f f l u o r i d e w i t h c a l c i u m p e c t a t e . A m o d e l d e p i c t i n g t h e b u f f e r i n g a c t i o n o f t h e c e l l w a l l on f l u o r i d e , t h e d i s p l a c e m e n t o f c a l c i u m f r o m t h e p e c t a t e f r a c t i o n and p r e c i p i t a t i o n o f CaF2. f l u o r i d e i o n encounters t h e second b a r r i e r t o t h e i r accumulation, plasmalemma. The p l a s m a l e m m a i s t h e l i m i t i n g membrane o f t h e c e l l i n s o m u c h a s i t d i s p l a y s s e l e c t i v i t y i n p e r m e a b i l i t y t o substances d i f f u s i n g from t h e exterior. The p l a s m a l e m m a i s a p r o t e i n and l i p i d c o m p l e x i n w h i c h many o f The d r i v i n g f o r c e f o r t h e t r a n s p o r t o f t h e s e m o l e The movement o f H+ t o t h e e x t e r i o r r e s u l t s i n t h e pH t h e p r o t e i n s f u n c t i o n a s t r a n s p o r t m o l e c u l e s f o r t r a n s m e m b r a n e i o n and m e t a b o l i t e movement. h i g h e r H+ c o n c e n t r a t i o n . a p p r o x i m a t e l y 5.8. c u l e s i s a n a s y m m e t r i c p r o t o n p u m p i n g , ApH, i n w h i c h t h e e x t e r i o r h a s a l o w e r i n g o f t h e pH o f t h e a p o p l a s t i c s p a c e a d j a c e n t t o t h e plasmalemma. the

R e d u c t i o n i n pH i n t h i s r e g i o n o f t h e c e l l w o u l d r e s u l t

i n a m a j o r s h i f t i n t h e f l u o r i d e Henderson-Hasselbalch e q u i l i b r i u m d i s t r i b u t i o n i n f a v o r o f t h e protonated form,

HF.

S i n c e t h e HF f o r m h a s b e e n

demonstrated t o be a p p r o x i m a t e l y 6 o r d e r s o f magnitude more permeable than

241

t h e n o n p r o t o n a t e d c o u n t e r p a r t (28).

a h i g h e r r a t e o f d i f f u s i o n w o u l d be D i f f u s i o n t o w a r d s an a r e a o f HowSince

e x p e c t e d a c r o s s t h e p l a s m a l e m m a ( F i g . 2).

l o w e r c o n c e n t r a t i o n would continue u n t i l e q u i l i b r i u m i s established. H e n d e r s o n - H a s s e l b a l c h e q u i l i b r i u m t o w a r d t h e n o n p r o t o n a t e d f o r m , F-. m e a b l e t o t h e F- f o r m ,

e v e r , t h e pH o f t h e c y t o p l a s m i s m a i n t a i n e d a t 7.2 w h i c h a g a i n s h i f t s t h e t h e HF f o r m i s t h e d i f f u s i n g s p e c i e s and t h e membrane i s r e l a t i v e l y i m p e r a n e t a c c u m u l a t i o n i n t h e c e l l w i l l o c c u r d r i v e n by t h e m a g n i t u d e o f t h e n o n i o n i c d i f f u s i o n g r a d i e n t o f t h e HF f o r m . The m a g n i t u d e o f t h i s g r a d i e n t i s d i r e c t l y d e p e n d e n t o n t h e t r a n s m e m b r a n e pH gradient.

EXTERIOR Space Membrane FHF FHi F HF H HF F FHF HF HF HF HF HF HF

INTERIOR

H HF

HF

<:+
High pH

Space

L o w PH

F i g u r e 2. H y p o t h e t i c a l model o f f l u o r i d e a c c u m u l a t i o n b y a n o n i o n i c d i f f u s i o n gradient. I n c r e a s e o f p r o t o n s a t t h e e x t e r i o r o f t h e membrane i n d u c e s HF f o r m a t i o n e n h a n c i n g f l u o r i d e t r a n s p o r t a c r o s s t h e membrane. I n t h e cytoplasm t h e accumulated f l u o r i d e would randomly d i s t r i b u t e by free diffusion. This free diffusion within the c e l l w i l l result i n the Based o n t h e Hendersonmovement o f t h e f l u o r i d e i n t o t h e v a r i o u s s u b c e l l u l a r c o m p a r t m e n t s v i a t h e same p r i n c i p l e s as t h e t r a n s p l a s m a l e m m a movement. Hasselbalch c a l c u l a t i o n s t h e two p r i n c i p a l s i t e s of accumulation would appear t o be t h e c h l o r o p l a s t and t h e m i t o c h o n d r i a : e f f e c t s w o u l d a l s o be e x p e c t e d i n t h e t o n o p l a s t and m i c r o s o m a l v e s i c l e s ( F i g .

3).
I n t h i s organ-

A c c u m u l a t i o n w i t h i n t h e m i t o c h o n d r i a w o u l d r e s u l t f r o m pH d e p e n d e n t m e c h a n i s m s s i m i l a r t o t h o s e o p e r a t i n g i n t h e plasmalemma. elle, d i e n t v i a a c t i v e extrusion o f proton, a c t i v e r e s p i r a t i o n r e s u l t s i n g e n e r a t i o n o f a t r a n s m e m b r a n e pH g r a -

H+ i o n s , w h i c h a r e u s e d f o r t h e

t r a n s p o r t o f m e t a b o l i t e s a c r o s s t h e membrane and a l s o d r i v e r e s p i r a t o r y

248

cell wall

5.8

cytoplasm

PH

7 2

F i g u r e 3. T h e d i f f u s i o n o f f l u o r i d e ( H F ) a c r o s s t h e p l a s m a accumulation i n various organelles o f t h e c e l l . phosphorylation.

Ir
l e a l margan

membrane and

C y t o p l a s m i c f l u o r i d e ( l i p i d s o l u b l e HF) n e a r t h e o r g a n e l l e Since t h e m a t r i x of

membrane s u r f a c e w o u l d r e a d i l y move i n t o t h e membrane.

t h e m i t o c h o n d r i a h a s a pH o f a b o u t 7.8 w h e r e F- w o u l d a c c u m u l a t e , a n o n i o n i c d i f f u s i o n g r a d i e n t w o u l d r e s u l t i n t h e one-way movement o f c y t o p l a s m i c HF i n t o t h e m i t o c h o n d r i a 1 m a t r i x r e s u l t i n g i n a c c u m u l a t i o n . The c h l o r o p l a s t r e s p o n s e w o u l d be s i m i l a r i n p r i n c i p l e b u t d i f f e r e n t i n d i r e c t i o n o f t h e p r o t o n movement. Under l i g h t c o n d i t i o n s photosynthetic e l e c t r o n t r a n s p o r t moves p r o t o n s f r o m t h e s t r o m a i n t o t h e l u m e n o f t h e t h y l a k o i d r e s u l t i n g i n t h e r e d u c t i o n o f t h e s t r o m a l pH t o a v a l u e g r e a t e r t h a n 8.0. form. T h i s r e d u c t i o n i n t h e pH o f t h e c h l o r o p l a s t s t r o m a c r e a t e s a s i n k f o r t h e p o r t i o n o f t h e c y t o p l a s m i c f l u o r i d e w h i c h i s p r e s e n t i n t h e HF The s t e e p n e s s o f t h i s g r a d i e n t makes t h e c h l o r o p l a s t t h e p r i m a r y b u t more s i t e o f fluoride accumulation w i t h i n the cell. O t h e r c e l l u l a r c o m p o n e n t s w o u l d a l s o be a f f e c t e d b y f l u o r i d e , functions, t h a n f r o m a c c u m u l a t i o n (Table I). l i k e l y f r o m i n h i b i t i o n o f e n z y m e s a s s o c i a t e d w i t h membrane o r m e t a b o l i c The t o n o p l a s t i s a p r i m e example o f t h i s response. T o n o p l a s t ATPases a r e s u s p e c t e d t o be l o c a l i z e d ATPases or the membrane pH gradient

on t h e c y t o p l a s m i c s i d e o f t h e membrane and a r e s e n s i t i v e t o f l u o r i d e . Inhibition o f tonoplast transport b r e a k d o w n o f t h e membrane. The f o r e g o i n g d i s c u s s i o n p r o v i d e s a m o d e l f o r t h e s u b c e l l u l a r p a r t i t i o n o f f l u o r i d e s i n t o t h e v a r i o u s o r g a n e l l e s based o n s t e a d y s t a t e m a t h e m a t i c a l f o r m a t i o n w o u l d r e s u l t i n d i s f u n c t i o n ( c h a n g e i n p e r m e a b i l i t y ) and e v e n t u a l

249

TABLE I
FLUORIDE EFFECTS ON ENZYMATIC REACTIONS Inhibition % Concentration mM

Enzymes o r System Glycolysis Phosphoglucomutase UOP glucose-fructose transglucosylase Enolase

Reference

29 29 94
(Mg

5 1 1 mM: Poi3: 5 mM)

30

Respiration Succinic dehydrogenase Malate dehydrogenase Peroxidase Ascorbic acid oxidase Polyphenol oxidase Photosynthesis PGA-Dependent 0 evolution (chloroplasts? Photosynthetic Phosphorylation, Membrane-Associated ATPases Mitochondria Chloroplast Tonoplast Plasma membrane

29 8 60 86 16

10 10

10 10

10

31 31 31 32 32

40

10
10

14 14

30

30 54 42 40

30 30 30 5 (Mg/ATP. 1 : 1 )

15 19 20 21

Other Membrane (Tonoplast) pH gradient formations

24

10

20

equation. This is summarized in Table I 1 and Figure 3, assuming a pKa of 3.45. The calculated values (Table 11) are based on the equilibrium distribution of fluoride in adjacent cellular compartments induced by pH gradients and calculated using the Henderson-Hasselbalch, equation (e.g., a
25 fold accumulation could be expected between the apoplast and the cyto-

plasm, a 6.3 and 5.0 fold increase i s expected in the chloroplast and the mitochondria, respectively, relative to the cytoplasmic fluoride level).

25 0

TABLE I 1 HENDERSON-HASSELBALCH DISTRIBUTION OF SUBCELLULAR FLUORIDE Fluoride Concentration (mM) (PPm) 0.1 2.5 1.9 47.5 298.3 190.0

Cellular Fraction Apopl a s t Cytoplasm Chloroplast Mitochondria

PH 5.8 7.2 8.0 7.8

15.7
10.0

C a l c u l a t i o n s determined u s i n g t h e Henderson-Hasselbalch e q u a t i o n assuming a pKa o f 3.45. F l u o r i d e accumulation process. earlier, i n t h e l i v i n g t i s s u e i s a d y n a m i c and c o n t i n u o u s as d i s c u s s e d

Accumulation o f atmospheric f l u o r i d e i n the f o l i a g e o f higher a l l o f which p r i m a r i l y a f f e c t the concentration o f f l u o r i d e The s u b c e l l u l a r p a r t i t i o n i n g o f The h y p o t h e t i c a l

plants i s subjected t o various environmental conditions, p r e s e n t w i t h i n t h e t r a n s p i r a t i o n a l stream.

f l u o r i d e would be determined by t h e f l u o r i d e c o n c e n t r a t i o n p r e s e n t i n t h e n o n i o n i c f o r m a t t h e e x t e r i o r s u r f a c e o f t h e plasmalemma. model i l l u s t r a t e s t h a t even s l i g h t e l e v a t i o n i n t h e l e v e l o f f l u o r i d e s above t h e b u f f e r i n g c a p a c i t y o f t h e c e l l w a l l w i l l v e r y r a p i d l y r e s u l t i n high l e v e l o f organelle accumulation. These s l i g h t i n c r e a s e s r e s u l t i n Such a model h i g h a c c u m u l a t i o n l e v e l s i n t h e c h l o r o p l a s t and m i t o c h o n d r i a r e s u l t i n g f r o m a h i g h pH g r a d i e n t b e t w e e n t h e o r g a n e l l e a n d c y t o p l a s m . i n d i c a t e s t h a t t h e use o f h i g h e r f l u o r i d e l e v e l s i n i n v i t r o o r g a n e l l e o r enzyme s t u d i e s i s v a l i d and c o m p a r a b l e t o u s i n g much l o w e r c o n c e n t r a t i o n s i n i n v i v o whole organism studies. REFERENCES

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3. 4.

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DC. p p 295

M a c l e a n DC, S c h n e i d e r RE, W e i n s t e i n LH (1978) I n : 7 1 s t Annual M e e t i n g o f t h e A i r P o l l u t i o n C o n t r o l A s s o c i a t i o n , Houston, Texas M a c l e a n DC, P o o v a i a h BW. A p p l e g a t e HG, S c h n e i d e r RE (1973) J E n v i r o n Q u a l i t y 2:501-503 Wiebe HH (1973) P l a n t P h y s i o l 51:396-399 Adams DF (1960) B o t Gar 121:223-227

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6.
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G a r r e c JP, M o r l e v a t JP, J o l i v e t J, L h o s t e A, F o u r c y A ( 1 9 7 4 ) C R A c a d S c i / P a r i s 279:61-64 G a r r e c JP, B l a n c h a r d B, B r u n JC, B i s c h A. B l i g n y R, F o u r c y A ( 1 9 7 3 ) C R Acad S c i / P a r i s 277:805-808

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L e d b e t t e r MC, Mavrodineanu R, I n s t 20:331-341 Chang CW, G a r r e c JP.

Weiss AJ (1960) C o n t r i b Boyce Thompson

Thompson CR (1966) P l a n t P h y s i o l 41:211-213 166 pp L h o s t e A M (1976) F l u o r i d e 9:63-70

Wei LL (1972) Ph D Thesis. U t a h S t a t e Univ.

W e i n s t e i n LH, Alscher-Herman (1980) I n : U n s w o r t h MH, Osmrod DP (eds) E f f e c t s o f Gaseous P o l l u t a n t s i n A g r i c u l t u r e and H o r t i c u l t u r e . P r o c 32nd School i n Agr Sci, U n i v o f N o t t i n g h a m School o f Agr B u t t e r w o r t h s , London B e n n e t t JH, G i a n n i n i JL,
H i l l AC (1973) J E n v i r o n Qua1 2:526-530

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M i l l e r GW,

P u s h n i k JC (1985) F l u o r i d e 18:72-79

P u s h n i k JC. M i l l e r GW ( 1 9 8 3 ) I n : S u s h e e l a A K ( e d ) F l u o r i d e T o x i c i t y . I n t SOC F l u o r i d e Res, Kalpana P r i n t i n g House, New D e l h i M i l l e r JE, M i l l e r GW (1974) P h y s i o l P l a n t 32:115-121 M i l l e r GW (1958) P l a n t P h y s i o l 33:199-206 Yang SF, M i l l e r GW (1963) Biochem J 88:505-509 M i l l e r GW, P u s h n i k JC (1985) P l a n t and C e l l Physiology G i a n n i n i JL. (submitted) (submitted)

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18. 19.

20. G i a n n i n i JL, M i l l e r GW. P u s h n i k JC, B r i s k i n . D (1985) P l a n t P h y s i o l o g y

21. G i a n n i n i J, P u s h n i k J, B r i s k i n D, M i l l e r G ( 1 9 8 5 ) P l a n t S c i e n c e ( i n
press)

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McNulty IB,

L o r d s JL (1960) S c i 122:1553-1554

Yu MH, M i l l e r GW (1967) P l a n t and C e l l P h y s i o l 8:483-493 Bal l e n t y n e OJ (1983) P l a n t P h y s i o l 7 2 : 2 2 ( a b s t r a c t ) G u t k n e c h t J, W a l t e r A (1980) B i o c h i m B i o p h y s A c t a 644:153-156 W e i n s t e i n LH (1977) J Occup Med 19:49-78 P i t n a m MG (1977) Ann Rev P1 P h y s i o l 28:71-88 W h i t f o r d G M ( 1 9 8 3 ) I n : Shupe JL. P e t e r s o n HB, L e o n e NC ( e d s ) Fluor i d e s : E f f e c t s o n V e g e t a t i o n , A n i m a l s and Humans. P a r a g o n P r e s s , p p 167-182 Yang SF, M i l l e r GW (1963) Biochem J 88:509-517 M i l l e r GW (1958) P l a n t P h y s i o l 33:199-206 L o v e l a c e CJ, M i l l e r GW (1967) J H i s t o c h e m Cytochem 15:195-201 Lee CJ, 181 M i l l e r GW, W e l k i e GW (1965) A i r Water P o l l u t I n t e r n J 10:169-

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H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 253-256 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

253

THE EFFECT
PROTOPLAST

OF

FLUORIDE

ON DNA

AND RNA SYNTHESIS BY BARLEY MESOPHYLL

YUMIN L I AND H U I X I A N MA
Department o f I n d u s t r i a l Hygiene, Hunan I n s t i t u t e o f M e t a l l u r g i c a l Protect i o n , Shu Mu Ling, Changsha, Hunan, China

ABSTRACT T r e a t m e n t w i t h f l u o r i d e d e c r e a s e d t h e s y n t h e s i s r a t i o o f DNA and i n c r e a s e d t h a t o f RNA i n b a r l e y ( H o r d i u m d i s t i c h o n v a r nudum) m e s o p h y l l protoplasts. E x p e r i m e n t a l r e s u l t s showed t h a t NaF promoted 3H-UdR i n c o r p o r a t i o n i n t o

RNA.

The a v e r a g e p r o m o t l o n r a t i o s w e r e 18.3%. 39.5%, and 49.9% when NaF 10.0, and 100 ppm, r e s p e c t i v e l y . However NaF 1.0, 10.0,

c o n c e n t r a t i o n s w e r e 1.0,

i n h i b i t e d 3H-TdR i n c o r p o r a t i o n i n t o DNA. A v e r a g e i n h i b i t i o n r a t i o s w e r e 11.2%. 11.1%. 22.8%, and 64.3% when NaF c o n c e n t r a t i o n s w e r e 0.1, and 100 ppm. r e s p e c t i v e l y

INTRODUCTION The e f f e c t s o f f l u o r i d e on p l a n t s c a n b e shown a t v a r i o u s l e v e l s o f b i o l o g i c a l organization. Mohamed 0.01 Those e f f e c t s , as o c c u r r i n g i n t h e t i s s u e , cellul a r , o r g a n e l l e , and m o l e c u l a r l e v e l s , w e r e r e v i e w e d b y M i l l e r

fit. (1).

e G . demonstrated

t h a t g r o w i n g young o n i o n r o o t s t r e a t e d w i t h H y d r o g e n f l u o r i d e was

M NaF showed c h r o m o s o m a l a b e r r a t i o n s (2).

t h o u g h t t o b l o c k t h e r e p l i c a t i o n o f DNA i n t o m a t o p l a n t s (3). I n o r d e r t o s t u d y t h e e f f e c t s o f f l u o r i d e on DNA and RNA s y n t h e s i s , b a r l e y (Hordium d i s t i c h o n v a r nudum) m e s o p h y l l p r o t o p l a s t s w e r e t r e a t e d w i t h d i f f e r e n t c o n c e n t r a t i o n s o f sodium f l u o r i d e and t h e i n c o r p o r a t i o n o f 3H-TdR ( t h y m i d i n e ) o r 3H-UdR ( u r i d i n e ) i n t o DNA o r R N A was d e t e r m i n e d . P r e l i m i n a r y r e s u l t s i n d i c a t e d t h a t f l u o r i d e caused t h e s y n t h e s i s r a t i o s o f

DNA t o be decreased and RNA t o be increased.

MATERIALS AND METHODS L a b e l e d 3H-TdR

(4.5

C/mM)

and 3H-UdR (20 C / m M )

were produced by t h e

Shanghai Atomic I n s t i t u t e . B a r l e y (Hordium d i s t i c h o n v a r &) seeds were g e r m i n a t e d on n y l o n net, A f t e r g r o w i n g f o r 6 days, and t h e s e e d l i n g s were grown by w a t e r c u l t u r e .

254
t h e l e a v e s w e r e c u t o f f and t h e f i b e r l a y e r was r e m o v e d f r o m t h e b o t t o m surface. 1.5 T h e l e a v e s w e r e i n c u b a t e d i n 4% p e c t i n a s e a n d 4 % c e l l u l a s e f o r The i n c u b a t i o n The m e s o p h y l l h o u r s a t 28C t o c a u s e t h e c e l l w a l l t o p e e l o f f .

m i x t u r e was t h e n f i l t e r e d t h r o u g h a n y l o n s i e v e o f 300-400 mesh and c e n t r i f u g e d a t 1000 rpm t o o b t a i n t h e m e s o p h y l l p r o t o p l a s t s . c o u n t was 1 x 106/m1. v i t a l it y . S a m p l e s w e r e d i v i d e d i n t o f i v e g r o u p s based on t h e amount o f NaF used: 0 (control), p r o t o p l a s t s w e r e s u s p e n d e d i n M r a s h i g e and Skoog c u l t u r e m e d i a and t h e c e l l M o r e t h a n 95% o f t h e p r o t o p l a s t s w e r e shown t o h a v e

0.1.

1.0,

10.0,

a n d 1 0 0 ppm NaF. Each o f t h e t u b e s p r o t o p l a s t preparaThe c o n t r o l

The r e a c t i o n was a l l o w e d t o p r o c e e d i n t e s t t u b e s . c o n t a i n e d a r e a c t i o n m i x t u r e c o n s i s t i n g o f M S media, t i o n . NaF, a n d 3H-TdR o r 3H-UdR, d i d n o t c o n t a i n added NaF. i n c u b a t e d f o r 12 hours.

i n a t o t a l v o l u m e o f 1.0 m l .

The t u b e s c o n t a i n i n g t h e r e a c t i o n m i x t u r e w e r e A t t h e end o f t h e i n c u b a t i o n p e r i o d . t h e tubes

w e r e s u b m e r g e d i n i c e - w a t e r t o s t o p t h e r e a c t i o n . T h e m i x t u r e was t h e n f i l t e r e d t h r o u g h g l a s s f i b e r a n d washed w i t h 10 m l e a c h o f 0.7 M m a n n i t o l , 100% e t h a n o l , and 10% t r i c h l o r o a c e t a t e . The d r i e d g l a s s f i b e r was p l a c e d i n t o l i q u i d s c i n t i l l a t i o n v i a l s c o n t a i n i n g 1 0 m l s c i n t i l l a t i o n liquid. and t h e r a d i o a c t i v i t y was d e t e r m i n e d i n a s c i n t i l l a t i o n s p e c t r o m e t e r . Promotion o r i n h i b i t i o n r a t i o s o f i n c o r p o r a t i o n were c a l c u l a t e d according t o t h e f o l l o w i n g formula:

Ratio =

CPMNaF - C P M c o n t r o l

x 100 (CPM, c o u n t s p e r m i n u t e )

RESULTS R e s u l t s f r o m p r e l i m i n a r y e x p e r i m e n t s showed t h a t b o t h 3H-TdR and 3H-UdR a t t a i n e d t h e i r peak o f i n c o r p o r a t i o n i n 1 0 h o u r s a n d r e m a i n e d t h e r e f o r 1 8 hours. I n c u b a t i o n t i m e i n t h i s e x p e r i m e n t was. therefore, s e t a t 1 2 hours. T h e i n c o r p o r a t i o n o f 3H-TdR w a s i n h i b i t e d b y NaF. r a t i o s w e r e 11.1%, 19.9%, 22.7%. w e r e 0.1. Average i n h i b i t i o n

a n d 64.3% w h e n t h e c o n c e n t r a t i o n s o f NaF The

1.0,

10.0, a n d 100 ppm. r e s p e c t i v e l y ( T a b l e I ) .

on t h e o t h e r hand, was p r o m o t e d b y NaF. 39.5%, and 1 0 0 ppm, r e s p e c t i v e l y ( T a b l e 11). and 49.9% when t h e c o n c e n t r a -

The i n c o r p o r a t i o n o f 3H-UdR, t i o n s o f NaF w e r e 1.0.

a v e r a g e p r o m o t i o n r a t i o s w e r e 18.3%.

10.0,

255

TABLE I EFFECT OF NaF ON THE INCORPORATION OF 3H-TdR INTO DNA NaF ( PPm) CPM o f each e x p e r i m e n t 1 2.836 2.592 2,388 2,286 1,202 2 5,062 4.586 4,070 4,019 1,853 3 5,431 4,899 4,535 4,182 2,194 4 4,563 4,034 3,541 3,445 1,556 5 3,560 3.104 2,681 2,631 1,050 6 3,748 3.216 2,976 2.906 1,181 Range o f Average i n h i b i t i o n in h i b i t i on r a t i o (%) r a t i o (%)

8.6-14.2 15.8-24.7 19.4-26.1 57.6-70.5

11.2k2.17 19.9k3.41 22.7k2.46 64.3t5.03

0.1 1.0 10.0 100.0

TABLE I 1 EFFECT NaF

OF NaF ON THE INCORPORATION OF 3H-UdR INTO RNA

CPM o f each e x p e r i m e n t

( PPm)

1 5,869 5,822 7,072 8,498 8.622

2 7,602 7,564 9,282 10.920 11,190

3 6,973 6,966 8,096 9,469 10,083

4 2,831 2,814 3,261 3,751 4,258

5 2,674 2,663 3,142 3,655 4,109

6 4,556 4,553 5,394 6,542 7,135

Range o f promotion r a t i o (%)

-

Average promotion r a t i o (%)

0.1 1.0 10.0 100.0

-0.8-0.6 15.2-22.1 32.5-44.8 44.6-56.7

18.3k2.62 39.5k5.14 49.9f4.55

DISCUSSION

Mohamed

& d.r e p o r t e d t h a t v i g o r o u s young o n i o n r o o t s t r e a t e d w i t h 0.01

Mohamed c o n c l u d e d t h a t HF w o u l d B a l e and H a r t ( 4 )

M NaF showed chromosomal a b e r r a t i o n (2).

b l o c k DNA r e p l i c a t i o n d i r e c t l y o r i n d i r e c t l y (3).

t r e a t e d b a r l e y (Hordium v u l g a r e ) s e e d l i n g r o o t t i p s w i t h e i t h e r 0.01

M NaF
It

o r 0.01 M HF and n o t e d a m a r k e d l y s l o w e d r a t e o f s e e d l i n g g r o w t h . a f f e c t e d by fluoride. 3H-TdR

a p p e a r s , t h e r e f o r e , t h a t DNA, t h e b a s i c m a t e r i a l o f c e l l n u c l e i . m u s t b e S i n c e t h y m i d i n e i s a component o f DNA. t h e r a t i o s o f With barley The r e s u l t s o b t a i n e d i n i n c o r p o r a t i o n i n t o DNA can r e p r e s e n t i t s r e p l i c a t i o n .

m e s o p h y l l p r o t o p l a s t s F- i n h i b i t e d DNA s y n t h e s i s .

t h i s e x p e r i m e n t appear t o be c o n s i s t e n t w i t h t h o s e o f o t h e r e x p e r i m e n t s .

256

F l u o r i d e has been r e p o r t e d t o cause a decrease i n polysome l e v e l s and an i n c r e a s e i n t h e number and c o n c e n t r a t i o n o f ribosomes and s u b u n i t s combined (5). Chang (6) compared t h e b i o c h e m i c a l mechanisms o f g r o w t h r e t a r d a t i o n The f l u o r i d e - i n d u c e d biochemical a l t e r a t i o n caused by f l u o r i d e and ozone. particles. promoted.
I t s h o u l d be s t r e s s e d t h a t t h e s e r e s u l t s a r e p r e l i m i n a r y .

was t h o u g h t t o c o n s i s t o f t h e d i s i n t e g r a t i o n o f t h e polysome i n t o s m a l l e r
I t i s p o s s i b l e t h a t 3H-UdR may come i n t o c o n t a c t w i t h ribosomes

more r e a d i l y .

T h i s c o u l d e x p l a i n why 3H-UdR i n c o r p o r a t i o n r a t i o s w e r e Further

s t u d i e s on t h e mechanisms i n v o l v e d i n t h e observed changes a r e necessary.

REFERENCES
1. M i l l e r GW, Yu MH, Pushnik JC (1983) In: Shupe JL, P e t e r s o n HB, Leone NC ( e d s ) F l u o r i d e s - E f f e c t s on V e g e t a t i o n , A n i m a l s and Humans. P a r a g o n Press, Inc., S a l t Lake City, pp 83-104 Mohamed AH, B a l e SS, Applegate HG, S m i t h

2.
3. 4.

J D (1966) Can J Genet C y t o l 8:241-244

Mohamed AH (1968) J A i r P o l l u t C o n t r o l Assoc 18:395-398 H a r t GE (1973) Can J G e n e t i c C y t o l 15:695-702 Chang CW (1970) Can J Biochem 48:450-454 Chang CW (7978) F l u o r i d e 11:55-59

5.
6.

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 257-261 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

257

THE EFFECTS

OF

FLUORIDE ON THE A C T I V I T I E S

OF

SEVERAL ENZYMES A!!D

ON CYCLIC

AMP LEVELS I N HUMAN LYMPHOCYTES I N VITRO TSUTOMU SATO, MOT00 NIWA, YOSHIKAZU NISHIDA. AND TATSUHIKO TSUJI D e p a r t m e n t o f P r e v e n t i v e and C o m m u n i t y D e n t i s t r y , 1-19-20, F U j i m i . Chiyoda-ku. T o k y o 102. J a p a n Nippon Dental U n i v e r s i t y .

ABSTRACT T h e a c t i v i t y o f g l y c o l y t i c e n z y m e s (ALD a n d LDH) a n d p h o s p h a t a s e s ( A l p and AcP). and cAMP l e v e l s i n human l y m p h o c y t e s c u l t u r e d i n t h e p r e s e n c e o f T h e i r m o r p h o l o g y was Loss o f lymphocyte v i a b i l i t y the controls. The g l y c o l y t i c

F (0.53 m M o r 1.05 mM) f o r 24 h o u r s w e r e d e t e r m i n e d .

examined by t r a n s m i s s i o n e l e c t r o n microscopy. was n o t shown i n f l u o r i d e - t r e a t e d c e l l s . observed between f l u o r i d e - t r e a t e d c e l l s and

No m o r p h o l o g i c a l d i f f e r e n c e s w e r e

enzymes and p h o s p h a t a s e s w e r e n o t a f f e c t e d b u t cAMP l e v e l s w e r e enhanced b y

1.05 m M F.

INTRODUCTION A l t h o u g h t h e e f f e c t s o f f l u o r i d e ( F ) on m a m m a l i a n c e l l s h a v e b e e n s t u d i e d under v a r i o u s c o n d i t i o n s , f e w s t u d i e s h a v e been c a r r i e d o u t on i m m u n o c y t c s .

It i s w e l l k n o w n t h a t a l a r g e n u m b e r o f e n z y m a t i c r e a c t i o n s ( i n c l u d i n g
g l y c o l y t i c e n z y m e s ) a r e i n h i b i t e d b y F.

On

t h e o t h e r hand,

i t has been
Phosphatases

s h o w n t h a t F e n h a n c e s t h e p r o d u c t i o n o f a d e n o s i n e 3', 5 ' - m o n o p h o s p h a t e

(CAMP)

b y s t i m u l a t i n g t h e a c t i v i t y o f a d e n y l a t e c y c l a s e (1).

h a v e a l s o b e e n shown t o b e a c t i v a t e d b y F (2,3). a c t i v i t y o f g l y c o l y t i c enzymes and p h o s p h a t a s e s , observed under a t r a n s m i s s i o n e l e c t r o n microscope.

Thus, i n t h i s s t u d y , t h e and cAMP l e v e l s i n human

l y m p h o c y t e s c u l t u r e d i n t h e p r e s e n c e o f F w e r e examined. The m o r p h o l o g y was

MATERIALS AND METHODS Preparation o f lymphocytes L y m p h o c y t e s w e r e s e p a r a t e d f r o m f r e s h venous b l o o d s a m p l e s f r o m t e n human v o l u n t e e r s (age: Sweden). L y m p h o c y t e c u l t u r e s and F t r e a t m e n t . S e p a r a t e d l y m p h o c y t e s w e r e washed w i t h Ca2+/Mg2+s o l u t i o n (PBS-) r e e phosphate b u f f e r fetal and s e e d e d i n RPMI 164.0 s u p p l e m e n t e d w i t h 20% ( v / v ) 24-31) b y c e n t r i f u g a t i o n o n F i c o 1-Paque (Pharmacia.

258
calf serum at a density o f 2-5x106 cells per plastic tube. After

p r e i n c u b a t i o n f o r 2 4 h o u r s a t 37C ( 0 t i m e ) , nM.

F s o l u t i o n (NaF) was added t o

t h e p r e i n c u b a t e d l y m p h o c y t e s a t t h e f i n a l c o n c e n t r a t i o n o f 0.53 m M o r 1.05 T h e s e s a m p l e s w e r e f u r t h e r i n c u b a t e d a t 37C f o r 2 4 h o u r s , and b i o c h e m i c a l and m o r p h o l o g i c a l s t u d i e s w e r e c a r r i e d o u t . Enzyme a s s a y F o u r c e l l u l a r enzymes, (LDH), i n c u l t u r e d lymphocytes. tured using a Teflon-glass An a u t o a n a l y z e r , C y c l i c AMP a s s a y L y m p h o c y t e s w e r e washed a n d d i s r u p t e d b y t h e same p r o c e d u r e d e s c r i b e d a b o v e . A f t e r t h e a d d i t i o n o f c o l d TCA t o t h e h o m o g e n a t e . centrifuged. t h e m i x t u r e was The s u p e r n a t a n t was c o m b i n e d w i t h e t h e r s a t u r a t e d w i t h w a t e r . JCA-N6R i n c l u d i n g a l d o l a s e (ALD), l a c t i c dehydrogenase were measured a c i d p h o s p h a t a s e (AcP) and a1 k a l i n e p h o s p h a t a s e ( A l p ) .

L y m p h o c y t e s w e r e washed w i t h c o l d PBS- a n d susCells, maintained on ice, were rupEnzyme a c t i v i t y h o m o g e n i z e r and t h e n u s e d f o r t h e enzyme assay. was u s e d f o r t h e assay.

p e n d e d i n t h e same b u f f e r s o l u t i o n .

(JEOL).

was e x p r e s s e d a s s p e c i f i c a c t i v i t y .

A f t e r m i x i n g , CAMP i n t h e aqueous l a y e r was q u a n t i f i e d w i t h a r a d i o i m m u n o a s s a y k i t (YAMASA). T h e r e s u l t s w e r e e x p r e s s e d a s f e m t o m o l s p e r mg c e l l protein. Preparation f o r transmission e l e c t r o n microscopy L y m p h o c y t e s w e r e washed a s d e s c r i b e d above. P r i m a r y f i x a t i o n was done i n 2.5% g l u t a r a l d e h y d e i n p h o s p h a t e b u f f e r s o l u t i o n c o n t a i n i n g 0.05 A f t e r t w o h o u r s p o s t f i x a t i o n i n 2% o s m i u m t e t r o x i d e , embedded i n EPON 812.

M sucrose.

t h e c e l l s were stained

f o r o n e h o u r w i t h 2% u r a n y l a c e t a t e a n d l e a d , d e h y d r a t e d i n e t h a n o l , and T h i n s e c t i o n s w e r e c u t o n LKB U l t o r o t o m e I V ( m o d e l 8800) and e x a m i n e d w i t h a JEOL l O O C e l e c t r o n m i c r o s c o p e a t 8 0 kV. RESULTS The a d d i t i o n o f F (0.53 rnM o r 1.05

mM) t o t h e c u l t u r e media d i d n o t
determined using t h e trypan The e f f e c t s o f f l u o r i d e on Figures

a f f e c t t h e v i a b i l i t y o f l y m p h o c y t e s w h i c h was b l u e d y e e x c l u s i o n m e t h o d ( d a t a n o t shown). enzyme a c t i v i t i e s i n l y m p h o c y t e s a r e shown i n d i v i d e d i n t o t w o groups: However.

1-4.

The b a s a l ALD

a c t i v i t y m e a s u r e d i n t e n c o n t r o l s a m p l e s ( n o t t r e a t e d w i t h F) c o u l d be a h i g h a c t i v i t y group, and a l o w a c t i v i t y group. t h e i r a c t i v i t i e s w e r e n o t c o n s i s t e n t l y a f f e c t e d b y F ( F i g . 1).

T h e LDH a c t i v i t y w a s a l s o n o t a f f e c t e d b y F ( F i g . 24 h o u r p e r i o d ( F i g .

2).

The,AcP a c t i v i t i e s

i n l y m p h o c y t e s c u l t u r e d w i t h F d i d n o t change f r o m c o n t r o l v a l u e s w i t h i n a

3).

S i n c e t h e A1P a c t i v i t y i n l y m p h o c y t e s was t o o l o w d a t a c o u l d be o b t a i n e d o n l y f r o m f i v e p e r t h e e f f e c t o f F o n A1P a c t i v i t y w a s Therefore,

f o r an a c c u r a t e d e t e r m i n a t i o n , s o n a l s a m p l e s ( F i g . 4).

259

2
0 L4

1000

a
\

z
> 4

E -4

500

u u
irJ
I : 4
ci

F
F i g u r e 1. hours.

0.53 ( nM

1.05
)

0.53
F ( mM

1.05

ALD a c t i v i t i e s o f l y m p h o c y t e s c u l t u r e d w i t h o r w i t h o u t F f o r 24

F i g u r e 2. LDH a c t i v i t i e s o f l y m p h o c y t e s c u l t u r e d w i t h o r w i t h o u t F f o r 24 hours. A c t i v i t i e s o f t w o samples were n o t detected.

0.53
F ( W )

1.05

A l p a c t i v i t i e s o f l y m p h o c y t e s c u l t u r e d w i t h o r w i t h o u t F f o r 24 F i g u r e 3. hours. A c t i v i t i e s o f f i v e samples were n o t detected. F i g u r e 4. AcP a c t i v i t i e s o f l y m p h o c y t e s c u l t u r e d w i t h o r w i t h o u t hours. A c t i v i t i e s o f t w o samples were n o t detected.

f o r 24

260

o b s c u r e i n t h i s study.

C y c l i c AMP l e v e l s were enhanced by 1.05 m M F (Fig.

5).

U l t r a s t r u c t u r a l d i f f e r e n c e s c o u l d n o t be found between c o n t r o l c e l l s

and f l u o r i d e - t r e a t e d c e l l s ( F i g . 6A-C).

0
F i g u r e 5. 2 4 Hours.

0.53
F ( d 1 )

1 .05

C y c l i c AMP l e v e l s i n lymphocytes c u l t u r e d w i t h o r w i t h o u t F f o r

261

Figure 6. Electron micrographs of lymphocytes c u l t u r e d i n t h e absence (A: c o n t r o l ) and p r e s e n c e (B: 0.53 mM, C: 1.05 m M ) o f f l u o r i d e f o r 2 4 hours. The s p a r s e cytoplasm and few o r g a n e l l e s were shown i n both c o n t r o l and Ft r e a t e d c e l l s . N: nucleus, M: mitochondrion, Ps: pseudopod, Bar=l.O urn.

REFERENCES 1 . Rall TW. Sutherland EW (1958) J Biol Chem 2321065-1076

2.
3. Lai CC, Singer L, Armstrong WD (1974) Clin Orthop Re1 Res 100:387-391 Farley JR, Wergedal JE, Baylink DJ (1983) Science 222:330-332

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 263-266 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

263

INDUCTION OF PROTEIN I N HELA CELLS BY SODIUM FLUORIDE TOSHIO I M A I , K U M I GOJO, TATSUHIKO TSUJI D e p a r t m e n t o f P r e v e n t i v e and C o m m u n i t y D e n t i s t r y N i p p o n D e n t a l U n i v e r s i t y , 1-9-20, F u j i m i Chiyoda-Ku. Tokyo, 102. J a p a n ABSTRACT When HeLa c e l l s w e r e t r e a t e d w i t h s o d i u m f l u o r i d e , synthesis inhibited. cells, of a specific protein with a 77000 d a l t o n s , t h e r e was an i n c r e a s e d weight of about molecular TSUTOMU SATO, R Y O I C H I NISHIKAWA, MOT00 NIWA AND

w h i l e t h e s y n t h e s i s o f m o s t o t h e r c e l l u l a r p r o t e i n s was C l o n e 1-5c-4 When HeLa c e l l s the not

T h i s p h e n o m e n o n w a s c o m m o n t o HeLa-S3 c e l l s ,

human d i p l o i d f i b r o b l a s t c e l l s and HT-1080 c e l l s .

were t r e a t e d w i t h bromide o r iodide, sodium f l u o r i d e d i d n o t occur. observed. INTRODUCTION

h o w e v e r , t h e phenomenon o b s e r v e d w i t h was

M o r e o v e r , when s t r e s s e d b y t e m p e r a t u r e ,

i n d u c t i o n o f s p e c i f i c 77KD p r o t e i n s y n t h e s i s i n H e L a c e l l s

I t i s w e l l known t h a t sodium f l u o r i d e i s c y t o t o x i c a t h i g h c o n c e n t r a t i o n s , c a u s i n g g r o w t h i n h i b i t i o n a n d c e l l d e a t h (1-4). The m a i n c a u s e o f t h i s e f f e c t i s p r o b a b l y i n h i b i t i o n o f c e l l u l a r p r o t e i n s y n t h e s i s (4,5). P r e v i o u s s t u d y h a s shown t h a t when HeLa c e l l s w e r e e x p o s e d t o s o d i u m f l u o ride, the synthesis of a particular protein w i t h a molecular weight of w h i l e t h e synthesis o f most o t h e r The p u r p o s e o f t h e p r e s e n t s t u d y was a b o u t 77.000 d a l t o n s (77KD) was i n d u c e d , c e l l u l a r p r o t e i n s was i n h i b i t e d (6). cells. MATERIALS AND METHODS Cell culture HeLa c e l l s , HeLa S3 c e l l s , C l o n e 1-5c-4 cells, human d i p l o i d f i b r o b l a s t c e l l s and HT-1080 c e l l s w e r e c u l t i v a t e d i n m o n o l a y e r c u l t u r e s w i t h E a g l e ' s m i n i m a l e s s e n t i a l medium s u p p l e m e n t e d w i t h 20 p g / m l k a n a m y c i n e and 10% h e a t i n a c t i v a t e d f e t a l c a l f s e r u m a t 37C i n a 5% C02 a t m o s p h e r e . Labeling condition Twenty-four hours a f t e r seeding c e l l s , s o d i u m f l u o r i d e was added t o t h e The m e d i u m was c u l t u r e m e d i u m a t a f i n a l c o n c e n t r a t i o n o f 1.5 mM. a n d t h e c u l t u r e s w e r e t h e n i n c u b a t e d f o r 2.5.

t o i n v e s t i g a t e t h e m e c h a n i s m i n v o l v e d i n t h e s y n t h e s i s o f 77KD p r o t e i n i n

5.0 a n d 7.5 h o u r s , r e s p e c t i v e l y .

264
r e m o v e d f r o m t h e c u l t u r e s and r e p l a c e d w i t h P B S ( - ) and I 4 C - p r o t e i n A f t e r labeling, h y d r o l y s a t e (0.5 vCi/dish) c o n t a i n i n g 1.5 m M NaF

and l a b e l e d f o r 1 hour a t 37C.

t h e c e l l s were washed w i t h PBS(-) and were scraped o f f t h e

p l a t e s and homogenized u s i n g a t e f l o n homogenizer. Gel e l e c t r o p h o r e s i s Samples o f 1 4 C - p r o t e i n h y d r o l y s a t e - l a b e l e d p r o t e i n were analyzed i n 11and t h e n exposed t o X13% g r a d i e n t p o l y a c r y l a m i d e g e l s a c c o r d i n g t o t h e procedure o f Laemmli (7). Gels were t r e a t e d w i t h sodium s a l i c y l a t e ( 8 ) , d r i e d . ray f i l m .

RESULTS AND D I S C U S S I O N
P r e v i o u s s t u d i e s have r e v e a l e d a t r e n d o f i n c r e a s e d s y n t h e s i s o f t h e 77KD p r o t e i n i n c e l l s i n t h e presence o f sodium f l u o r i d e . general.

A q u e s t i o n arose as
human

t o whether t h i s 77KD p r o t e i n was unique t o HeLa c e l l s o r t o human c e l l s i n

To i n v e s t i g a t e o t h e r human c e l l s , H e l a S3, C l o n e 1-5c-4,

and

diploid fibroblasts and l a b e l e d .

HT-1080

c e l l s were

t r e a t e d w i t h sodium f l u o r i d e gels. The

The l a b e l e d p r o t e i n s were f r a c t i o n a t e d on SDS-polyacrylamide s o d i u m f l u o r i d e ( F i g . 1). other cells. t o human c e l l s .

s y n t h e s i s o f 77KD p r o t e i n i n HeLa S3 c e l l s was i n d u c e d b y t r e a t m e n t w i t h I n d u c t i o n o f 77KD p r o t e i n was a l s o o b s e r v e d i n T h i s suggests t h a t i n d u c t i o n o f 77KD p r o t e i n m i g h t be common

Two t y p e s o f e x p e r i m e n t s w e r e done t o i n v e s t i g a t e t h e s p e c i f i c i t y o f sodium f l u o r i d e i n r e l a t i o n t o t h e s y n t h e s i s o f 77KD p r o t e i n . c e l l s were t r e a t e d w i t h bromide o r i o d i d e f o r 5 hours, amide g e l a n a l y s i s . o b s e r v e d ( F i g . 2). Second, c e l l s w e r e e x p o s e d t o a t e m p e r a t u r e o f 29C f o r 5 h o u r s i n t h e presence o r absence o f sodium f l u o r i d e . period, o f sodium f l u o r i d e , First, HeLa l a b e l e d f o r 1 hour,

and t h e p a t t e r n o f t h e s y n t h e s i z e d p r o t e i n was examined by SDS-polyacrylThe c o n c e n t r a t i o n o f t h e h a l i d e s used i n t h e s e s t u d i e s The i n d u c t i o n o f 77KD p r o t e i n s y n t h e s i s was n o t

(80 m M ) was c y t o t o x i c .

A t t h e end o f t h e s h i f t e d - d o w n
I n t h e absence the synthesis However, the These r e s u l t s

c e l l s were r e t u r n e d t o 37C and l a b e l e d f o r 1 hour. I n t h e presence o f sodium f l u o r i d e ,

t h e i n d u c t i o n o f 77KD p r o t e i n s y n t h e s i s i n s h i f t e d - d o w n

c e l l s was n o t observed.

o f 77KD p r o t e i n was d i m i n i s h e d w i t h most c e l l u l a r p r o t e i n . a b i l i t y t o i n d u c e 77KD p r o t e i n s y n t h e s i s was n o t l o s t . non t o sodium f l u o r i d e t r e a t m e n t .

suggest t h a t t h e i n d u c t i o n o f 77KD p r o t e i n s y n t h e s i s i s a s p e c i f i c phenome-

Figure 1. Induction o f 77KD protein in HeLa S3 cells by sodium fluoride. Cells were treated with 1.5 m M NaF for 0 (1). 2.5 hours (2) and 7.5 hours ( 3 ) . respectively. After treatment, the cellular proteins w e r e labeled w i t h protein hydrolysate for 1 hour. The labeled proteins were analyzed in SDS-polyacrylamide gel and visualized by fluorography. Black arrow indicates the 77KD protein. Figure 2 . Fluorogram o f SDS-polyacrylamide slab gel of 14C-protein hydrolysate-labeled proteins from HeLa cells treated with fluoride, bromide and iodide. Cells were treated with drug at 37C for 5 hours and then labeled for 1 hour. (1) control: (2) 1.5 m M NaF; (3) 80 m M bromide; (4) 80 m M iodide. Black arrow indicates the 77KD protein.

0)

01

266
ACKNOWLEDGEMENT We t h a n k Dr. M. U e d a f o r h e l p f u l d i s c u s s i o n a n d f o r r e v i e w i n g t h e manuscript. REFERENCES 1. B e r r y RJ, T r i l l w o o d W (1963) B r i t Med J 23:1964 A r m s t r o n g WC, B l o m q u i s t CH, S i n g e r B r i t Med J 1:486-488 B e r r y RJ (1969) F l u o r i d e 2:157-167 I m a i T, N i w a M, Ueda M (1983) A c t a p h a r m a c o l e t t o x i c o l 52:8-11 H o l l a n d R I (1979) A c t a p h a r m a c o l e t t o x i c o l 49:96-101 I m a i T, N i s h i k a w a R. N i w a M (1983) J D e n t Res 6 2 : S p e c i a l L a e m m l i UK (1970) N a t u r e (London) 227-680-685 C h a m b e r l a i n JP (1979) A n a l Biochem 98:132-135 I s s u e 692

2.
3. 4. 5.

L,

P o l l o c k ME, M c L a r e n LC ( 1 9 6 5 )

6.
7. 8.

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 267-276 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

267

EFFECTS OF FLUORIDES ON ORAL MICROORGANISMS MASATO I I D A , TSUGUO KAWASAKI, MASAHIRO FURUTANI, AND TAKUMI SAT0 Shiga U n i v e r s i t y o f Medical

D e p a r t m e n t o f O r a l and M a x i l l o f a c i a l S u r g e r y , S c i e n c e , S e t a O h t s u 520-21 Japan ABSTRACT

The p u r p o s e o f t h i s s t u d y was t o e x a m i n e t h e e f f e c t s o f f l u o r i d e s on v a r i o u s o r a l microorganisms i n c l u d i n g Streptococcus, Pseudomonus, B a c t e r o i d e s and Fusobacterium, mutans), Streptococcus mutans ( S .
S.

Actinomyces,

Candida.

on t h e a c i d production o f

and o n t h e s y n t h e s i s o f w a t e r i n s o l u b l e

g l u c a n c a t a l y z e d b y e x t r a c e l l u l a r enzyme ( g l u c o s y l t r a n s f e r a s e ) d e r i v e d f r o m mutans. C a n d i d a and Pseudomonus w e r e h i g h e r t h a n t h o s e f o r t h e o t h e r NaF a r r e s t e d b a c t e r i a l g r o w t h b a c t e r i o s t a t i c a l l y b u t d i d glucan. G l u c a n s y n t h e s i s was T h e m i n i m u m i n h i b i t o r y c o n c e n t r a t i o n s o f f l u o r i d e p r o v i d e d a s NaF f o r Actinomyces, microorganisms.

not a f f e c t the synthesis o f water-insoluble

i n h i b i t e d o n l y b y CoF2 a t c o n c e n t r a t i o n s o f 600-1000 ppm F. L a c t i c a c i d p r o d u c t i o n was s u p p r e s s e d i n b o t h t h e c e l l r e s t i n g and c e l l g r o w i n g systems. F l u o r i d e s o f b i v a l e n t c a t i o n s s u c h a s SnF2 a n d CoF2 i n h i b i t e d t h e g r o w t h o f t h e m i c r o o r g a n i s m s m o r e m a r k e d l y t h a n NaF. INTRODUCTION F l u o r i d e s a r e w i d e l y used i n p r e v e n t i v e d e n t i s t r y s u c h a s t o p i c a l a p p l i c a t i o n and f l u o r i d a t i o n b e c a u s e o f t h e i r c a r i o s t a t i c e f f e c t s . m a t e r i a l through formation o f f l u o r a p a t i t e t h e t o o t h (1-4). Fluorides, however, and CaF2. Such e f f e c t s a r e p r o d u c e d a s a r e s u l t o f e n h a n c e d s t r e n g t h and a c i d t o l e r a n c e o f t o o t h S t u d i e s on t h e e f f e c t s o f f l u o r i d e s have focused l a r g e l y on improvement of a c i d t o l e r a n c e o f a r e a l s o known t o h a v e a n t i b a c t e r i a l B i b b y a n d Van K e s t e r n (6) e f f e c t s on d e n t a l p l a q u e - f o r m i n g o r a l microbes i n culture, b a c t e r i a (5).

r e p o r t e d i n 1940 t h a t f l u o r i d e s r e d u c e d t h e p r o d u c t i o n o f o r g a n i c a c i d s b y and s u g g e s t e d t h a t t h e y a f f e c t e d b a c t e r i a l g r o w t h They a l s o d e t e r m i n e d t h e o f NaF f o r S t r e p t o c o c c u s t o be 250 and s a c c h a r o m e t a b o l i s m a t l o w c o n c e n t r a t i o n s . minimum i n h i b i t o r y c o n c e n t r a t i o n ( M I C ) f l u o r i d e concentrations. However,

ppm F, and r e p o r t e d s u p p r e s s i o n o f t h e p r o d u c t i o n o f o r g a n i c a c i d s a t l o w e r much o f t h e e f f e c t o f f l u o r i d e on i n d i g enous o r a l m i c r o o r g a n i s m s r e m a i n s o b s c u r e . The p u r p o s e o f t h i s s t u d y was t o e x a m i n e t h e e f f e c t s o f f l u o r i d e s o n

268

various oral microorganisms including Streptococcus, Actinomyces, Candida. Pseudomonus, Bacteroides and Fusobacterium. on acid production of Strepto-

~coccus mutans (S. mutans).

and on the synthesis of water-insoluble glucan catalyzed by extracellular enzyme (glucosyltransferase, EC. 2.4.1.5)

derived from S_ mutans. In addition, the effects of NaF and other fluorides on cariogenic S . mutans at various concentrations were compared
in_ vitro. _ _

MATERIALS AND METHODS

Bacterial strains Oral microorganisms used in this study were provided by the Department of Bacteriology, Osaka Dental College and Shiga University of Medical Science. Bacteria were repeatedly stored once a month i n 10 ml brain-heart infusion agar media (BHI, Difco) at the Department of Oral Surgery, Shiga University of Medical Science. For preincubation, the culture ( 0 . 1 ml) was inoculated culture ( 0 . 1 ml) was reinoculated to the same media and incubated aerobically and anaerobically at 37C for 1 8 hours. Table I shows the number and kind o f strains of microorganisms used in the present study. Chemi ca 1 s Sodium fluoride was obtained from the Hashimoto Chemical Co. while 1 1 other fluorides were obtained from the Nakarai Chemical Co., Japan. to a 10 ml broth and incubated at 37C for 24 hours. The incubated

TABLE I BACTERIAL STRAINS

Streptococcus ( I 3a) S. mutans (13) Bacteroides (19) B. melaninogenicus (8)
B. intermedius (3)

Fusobacteri um (9) F. nucleatum (5) F. mortiferum (2) F. vorium (2) Actinomyces (10) A. viscosus (5)

B. asaccharolyticus (3) B. gingivalis (2)

B. fragilis (3) Candida (10)

C. albicans (5)
C. tropicalis (4) C. krusei (1) anumber of strain

naeslandi (1) Pseudomonus (10) P. aeruginosa (10)

A. A. A. A.

islaelli (2) odontolyticus (1) meyeri (1)

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C u l t u r e media

A B H I b r o t h was used as t h e l i q u i d medium, and agar p l a t e s were prepared

b y a d d i n g B a c t o a g a r ( D i f c o ) t o BHI f o r a e r o b i c microorganisms. a n a e r o b i c medium (GAN, N i s s u i ) was used f o r a n a e r o b i c microorganisms. Measurement o f M I C and b a c t e r i a l growth The M I C v a l u e s w e r e d e t e r m i n e d w i t h t h e t h e a g a r d i l u t i o n t e c h n i q u e a c c o r d i n g t o t h e method o f t h e Japanese S o c i e t y f o r Chemotherapy (7).
l y 6x106 c e l l s / m l

A Gifu

One

p l a t i n u m l o o p o f t h e p r e - i n c u b a t e d sample s o l u t i o n ( c o n t a i n i n g approximatef o r aerobs and 8x108 c e l l s / m l f o r anaerobs) was added t o ppm F, verified with a agar media w i t h f l u o r i n e c o n c e n t r a t i o n s o f 0-5960 f l u o r i d e i o n e l e c t r o d e ( O r i o n Research). 37"C, observed was d e t e r m i n e d t o be t h e M I C . W h i l e i n v e s t i g a t i n g t h e b a c t e r i a l growth, Colony-forming a f t e r 0, 4, u n i t s (CFU) t h e t u r b i d i t y absorbance was 100-10) a t 500 nm. NaCl was and measured e v e r y hour w i t h a s p e c t r o p h o t o m e t e r ( H i t a c h i ,

A f t e r 24 hours o f i n c u b a t i o n a t

t h e m i n i m a l c o n c e n t r a t i o n a t w h i c h no b a c t e r i a l c o l o n y was g r o s s l y

were s i m u l t a n e o u s l y measured on B H I agar p l a t e s As a c o n t r o l , a t t h e same c o n c e n t r a t i o n s as NaF,

8,

12. and 24 h o u r s o f i n c u b a t i o n .

added t o t h e l i q u i d and agar media m a n n e r as above. c u l t u r e over time. Measurement o f pH and l a c t i c a c i d

m e a s u r e m e n t o f t u r b i d i t y and C F U was made d u r i n g i n c u b a t i o n i n t h e same G r o w t h c u r v e s o f C F U w e r e p l o t t e d as l o g l o

CFU/ml o f

The s a m p l e s o l u t i o n s w e r e c e n t r i f u g e d a t 1500 x g f o r 1 5 m i n and t h e pH o f t h e s u p e r n a t a n t was measured w i t h a pH m e t e r ( H i t a c h i - H o r i b a GC-7A) by an o r d i n a r y m e t h y l - e s t e r i f i c a t i o n method. F7). A n a l y s i s o f l a c t i c a c i d was conducted w i t h a gas chromatograph (Shimazu P r i o r t o t h e analysis. l a c t i c a c i d was i d e n t i f i e d i n t h e s t a n d a r d and sample s o l u t i o n s by i t s mass number (105). i n a mass s p e c t r o g r a p h (Shimazu AUTO GCMS 6020). mutans AHT ( s e r o type, a), Preparation o f dextransucrase Dextransucrase was d e r i v e d f r o m S. (c). OMZ-176 (d),

NCTC 10449

OMZ-175 ( f ) and N I D R 6715 (9).

A f t e r preculturing i n BHI a 0.1 m l s o l u t i o n was

b r o t h f o r 24 and 18 hours t w i c e a t 37C a e r o b i c a l l y , as d e s c r i b e d below.

i n o c u l a t e d i n 30 m l B H I b r o t h f o r t h e purpose o f p r e p a r i n g d e x t r a n s u c r a s e The p r e c u l t u r a l f l u i d s o f S. m u t a n s w e r e i n o c u l a t e d i n t o 3 1 B H I b r o t h and i n c u b a t e d a t 37C f o r 18 hours. The c u l t u r e s were t h e n c e n t r i f u g e d a t Following centrifugation t h e preand t h e 7,200 x g f o r 20 m i n and t h e s u p e r n a t a n t was t r e a t e d w i t h 60% s a t u r a t e d ammonium s u l f a t e a t 4C f o r 18 hours. c i p i t a t e was d i s s o l v e d i n 75 m l 0.05 M p h o s p h a t e b u f f e r (pH 6.8). m i x t u r e was d i a l y z e d a g a i n s t t h e same b u f f e r o v e r n i g h t .

The d i a l y z a t e was

270
c e n t r i f u g e d t o r e m o v e i n s o l u b l e m a t e r i a l s and t h e s u p e r n a t a n t was used a s a crude p r e p a r a t i o n o f dextransucrase. The c r u d e enzyme p r e p a r a t i o n f r o m c o l u m n c h r o m a t o g r a p h y o n B i o G e l A-15m d e s c r i b e d p r e v i o u s l y (8). Enzyme a s s a y Enzyme a c t i v i t y was d e t e r m i n e d b y t h e b l u e d e x t r a n m e t h o d ( 9 ) a n d / o r I 4 C s u c r o s e m e t h o d (10). NaCl was u s e d a s t h e c o n t r o l . R e l a t i v e enzyme a c t i v i t y i n t h e r e a c t i o n m i x t u r e a t e a c h f l u o r i d e concent r a t i o n was e x p r e s s e d i n t e r m s o f t h e p e r c e n t a g e o f a c t i v i t y o b t a i n e d w i t h t h e s t a n d a r d m i x t u r e , as shown b e l o w : T h i s p r e p a r a t i o n was a d j u s t e d t o convert sucrose t o w a t e r i n s o l u b l e glucan a t a r a t e o f

0 . 6

umol/ml/min.

S.

m u t a n s OMZ-176 w a s p u r i f i e d b y as

and s u b s e q u e n t l y o n h y d r o x y l a p a t i t e .

T h i s enzyme p r e p a r a t i o n g a v e t h r e e bands o n a n a l y s i s b y SDS-PAGE

Relative

enryme activity

A c t i v i t y a t each f l u o r i d e c o n c e n t r a t i o n A c t i v i t y w i t h t h e standard m i x t u r e

A l l t e s t s were performed i n t r i p l i c a t e .
RESULTS T a b l e 11 shows t h e M I C d i s t r i b u t i o n of NaF f o r o r a l m i c r o o r g a n i s m s . The

M I C f o r S. m u t a n s w e r e l o w e r t h a n 1 8 0 ppm

F.

The M I C f o r B a c t e r o i d e s and Pseudomonus and

F u s o b a c t e r i u m w e r e w i d e l y d i s t r i b u t e d w i t h i n t h e r a n g e o f 22.5 t o 1440 ppm

F.

On t h e o t h e r h a n d , t h e M I C v a l u e s f o r A c t i n o m y c e s ,

Candida were h i g h e r t h a n those f o r o t h e r microorganisms. TABLE I 1 SENSITIVITY DISTRIBUTION OF ORAL MICROORGANISMS O r g a n i sms 22.5 Bacteroides Fusobacterium A c t i nomyces 2 1 45 2 1 1

M I C o f NaF

90
4 2 4

180
1

360 8 3

720
1 2

1440

2880

5760 (ppm

F)

1
2 7 6 6 4 4

1
8

S. m u t a n s
Pseudomonus Candida

271
S. -~ mutans

g r o w t h a f t e r 24 h o u r s o f i n c u b a t i o n i n t h e p r e s e n c e o f NaF i s B a c t e r i a l growth, expressed i n t e r m s o f absorbance t o 360 ppm. Fluo-

p r e s e n t e d i n F i g u r e 1.

d e c r e a s e d w i t h i n c r e a s e i n NaF c o n c e n t r a t i o n f r o m 22.5

r i d e l e v e l s b e l o w 22.5 ppm had n o e f f e c t and t h e p r e s e n c e o f added NaCl had no i n h i b i t o r y e f f e c t w i t h i n t h e c o n c e n t r a t i o n r a n g e t e s t e d . T h e t i m e c o u r s e f o r t h e g r o w t h o f S. mutans as a f f e c t e d b y d i f f e r e n t F l u o r i d e a t 22.5 ppm c o n c e n t r a t i o n s o f f l u o r i d e i s s h o w n i n F i g u r e 2. g r o w t h o f t h e t e s t o r g a n i s m was o b s e r v e d . b a c t e r i a l growth.

showed n o i n h i b i t o r y e f f e c t , w h i l e a t 4 5 ppm F a s l i g h t r e d u c t i o n i n t h e A d d i t i o n o f 90 ppm F was c l e a r l y i n h i b i t o r y t o g r o w t h . F l u o r i d e l e v e l s b e t w e e n 180 a n d 3 6 0 ppm a r r e s t e d

A t t h e s e f l u o r i d e l e v e l s t h e g r o w t h was r e d u c e d b y
u n i t s compared w i t h t h e c o n t r o l . L a c t i c a c i d c o n t e n t s a n d pH

a p p r o x i m a t e l y 1.5 l o g l o

f i g u r e 3 shows t h e e f f e c t o f f l u o r i d e on pH v a l u e s and f i n a l l a c t i c a c i d c o n t e n t d u r i n g t h e g r o w t h o f S. m u t a n s Gs-5. values were i n v e r s e l y related.

A s i m i l a r o b s e r v a t i o n was a l s o made f r o m
dextran a t low The d i f f e r e n c e while

e x p e r i m e n t s u s i n g r e s t i n g c e l l s ( d a t a n o t shown). T h e e f f e c t o f NaF o n t h e s y n t h e s i s o f w a t e r - i n s o l u b l e f l u o r i d e c o n c e n t r a t i o n s (0-1000 ppm) i s shown i n F i g u r e 4a. distinct, tions, was n o t s i g n i f i c a n t .

i n a c t i v i t i e s a t d i f f e r e n t c o n c e n t r a t i o n s o f f l u o r i n e and c h l o r i n e ,

The e f f e c t o f f l u o r i d e a t h i g h e r c o n c e n t r a F o r t h i s purpose, f l u o r i n e and c h l o r i n e

c o m p a r a b l e t o t h o s e i n a 2% NaF s o l u t i o n u s e d f o r d i r e c t t o p i c a l

a p p l i c a t i o n , was t h e n s t u d i e d .

N.n

=;

46

90

180

360

Concentration of F. pprn F i g u r e 1. E f f e c t s o f NaF o n t h e g r o w t h o f S. m u t a n s s t r a i n s . T u r b i d i t y a b s o r b a n c e was measured a f t e r i n c u b a t i o n f o r 24 h o u r s w i t h a s p e c t r o p h o t o m e t e r ( H i t a c h i , 100-10) a t 5 5 0 nrn. A s t h e c o n t r o l , N a C l w a s added t o t h e l i q u i d .

212

22.5 45 0

90

110

300 (pprnF)

, I

I2

'

24

Incubation Time. hr

F i g u r e 2. E f f e c t s o f NaF o n t h e g r o w t h o f S. m u t a n s s t r a i n s . C o l o n y - f o r m i n g u n i t s (CFU) w e r e m e a s u r e d on B H I a g a r p l a t e s a f t e r i n c u b a t i o n f o r 0, 4. 8, 12, a n d 2 4 h o u r s . G r o w t h c u r v e s o f C F U w e r e p l o t t e d a s l o g l o CFU/ml i n c u l t u r e o v e r t i m e .

(Iacric acid mmoltml)

4
Of

; 1

12

'

1 -

Incubation Time. hr

F i g u r e 3. E f f e c t o f f l u o r i d e on l a c t i c a c i d f o r m a t i o n d u r i n g g r o w t h o f S, m u t a n s Gs-5. c o n c e n t r a t i o n s o f 0, tested.

1000, 2000, 4000,

6000, 8000 a n d 10000 p p m w e r e

As shown i n F i g u r e 4b,

no c h a n g e s i n d e x t r a n s y n t h e s i s w e r e n o t e d The enzyme u s e d was p u r i f i e d d e x t r a n Crude dextransucrase p r e p a r a t i o n s from

despite these h i g h concentrations. s u c r a s e f r o m S. m u t a n s 0142-176. different strains of results.

&

mutans were subsequently t e s t e d w i t h s i m i l a r

273

2'%

I
250
500
IOU0
wniF

NaF

1000

2000

4000

6000

8000

10000

Concentration of F. ppm
F i g u r e 4. The e f f e c t o f NaF on t h e s y n t h e s i s o f w a t e r - i n s o l u b l e l o w ( a ) and h i g h ( b ) c o n c e n t r a t i o n s . dextran a t

Experiments were c a r r i e d out t o t e s t the e f f e c t o f d i f f e r e n t f l u o r i d e compounds on t h e g r o w t h o f S. 111. mutans. The r e s u l t s a r e s u m m a r i z e d i n T a b l e T h e M I C o f CoF2 a n d SnF2 w e r e l o w e r t h a n t h o s e o f o t h e r f l u o r i d e s . The e f f e c t o f f l u o r i d e s o n t h e s y n t h e s i s o f w a t e r - i n s o l u b l e G l u c a n s y n t h e s i s was i n h i b -

B a c t e r i a l g r o w t h was i n h i b i t e d b a c t e r i c i d a l l y a t 2MIC and 3MIC o f b o t h CoF2 a n d SnF2. g l u c a n was e x a m i n e d as i s shown i n F i g u r e 4.

i t e d a p p r o x i m a t e l y 20% b y CoF2 o n l y a t c o n c e n t r a t i o n s o f 600-1000 ppm F.

DISCUSSION
F l u o r i d e s a r e w i d e l y u s e d i n t h e f i e l d o f p r e v e n t i v e d e n t i s t r y b e c a u s e of t h e i r anticarious effects. i n s e n s i t i v i t y t o NaF. Candida, results. The p r e s e n t s t u d y shows t h a t NaF h a s d i f f e r e n t These

M I C a g a i n s t o r a l m i c r o o r g a n i s r s and t h a t b a c t e r i a l s p e c i e s and s t r a i n s v a r y
The M I C o f S. m u t a n s w e r e l e s s t h a n 180 ppm. observations are consistent w i t h those o f M a l t z s e n s i t i v e than Streptococcus, On t h e o t h e r hand, mutans.

d . &

(5).

Actinomyces,

and Pseudomonus s t r a i n s w e r e shown i n t h e p r e s e n t s t u d y t o be l e s s B a c t e r o i d e s and F u s o b a c t e r i u m t o t h e M I C Maltz

fi fl

( 5 ) r e p o r t e d t h a t A c t i n o m y c e s was

m o r e s e n s i t i v e t o t h e b a c t e r i c i d a l a c t i v i t y o f NaF t h a n w e r e s a l i v a r i u s a n d S. known.

S,

sanguis.

S,

The r e a s o n f o r t h e s e c o n t r a s t i n g r e s u l t s i s n o t The a n t i m i c r o -

I t may i n d i c a t e t h a t d i f f e r e n t mechanisms a r e i n v o l v e d i n t h e

b a c t e r i c i d a l (MBC) and b a c t e r i o s t a t i c ( M I C ) a c t i o n s o f NaF.

214 TABLE I11 EFFECTS OF DIFFERENT FLUORIDE COMPOUNDS ON S. m u t a n s

F 1u o r i d e s
NaF

MIC
1 8 0 ppm

Inhibitory effect bacteriostatic

GTA a c t i v i t y not effective

KF
CsF NH4F NaHF2 NH4F HF AlF3
b

1 8 0 ppm

bacteriostatic

not effective

CrF3
K 2 T i F6 L iF COF~ SnF2 360 ppm 45 PPm bacteriostatic bactericidal bactericidal not effective effective n o t determined

45 PPm

b i a l e f f e c t o f f l u o r i d e s o n B a c t e r o i d e s m e l a n i n o g e n i c u s s u b s p e c i e s and B a c t e r o i d e s a s a c c h a r o l y t i c u s was r e p o r t e d b y Yoon g&

d. (11).

When t h e y

e x a m i n e d t h e v i a b i l i t y o f e a c h o r g a n i s m a f t e r e x p o s u r e t o NaF f o r 8 hours. t h e r e s u l t s showed t h a t NaF was t h e l e a s t e f f e c t i v e a s c o n c e n t r a t i o n s o f 500 a n d 7 0 0 ppm F w e r e r e q u i r e d t o a f f e c t t h e v i a b i l i t y . s t r a i n s was shown t o b e h i g h . I n the MIC e v a l u a t i o n i n t h e present study, t h e s u s c e p t i b i l i t y o f most Bacteroides F u s o b a c t e r i u m and B a c t e r o i d e s s t r a i n s showed Thus, the results growth a s i m i l a r s e n s i t i v i t y t o NaF. T h e M I C v a l u e s f o r P s e u d o m o n u s a n d C a n d i d a were h i g h e r than t h o s e o f o t h e r s except f o r Actinomyces. T h e a d d i t i o n o f 9 0 ppmF p r o v i d e d a s NaF slightly. reduced s u g g e s t t h a t NaF h a s s e l e c t i v e e f f e c t s o n o r a l m i c r o o r g a n i s m s . bacterial Changes i n CFU o f t h e b a c t e r i a l c u l t u r e i n d i c a t e d m a r k e d i n h i b i and s u g g e s t e d mutans i n g r o w t h and b a c t e r i o s t a t i c e f f e c t a t 1 8 0 and r e p o r t e d a s i g n i f i c a n t a r r e s t o f S. T h i s s t u d y a l s o showed d i s t i n c t

t i o n o f b a c t e r i a l g r o w t h i n t h e p r e s e n c e o f 1 8 0 ppm F a s NaF, complete a r r e s t o f b a c t e r i a l 3 6 0 ppm F. Brown

&

d.( 1 2 )

t h e p r e s e n c e o f 3 0 0 ppm a n d 600 ppm F. f o r m o u t h w a s h (100-500 ppm F). Bibby

g r o w t h i n h i b i t i o n o f S. m u t a n s a t f l u o r i n e i o n c o n c e n t r a t i o n s c o m m o n l y used

gt . a !

r e p o r t e d i n h i b i t o r y e f f e c t s o f NaF o n t h e g r o w t h a n d a c i d L a c t o b a c i l l u s a n d o t h e r o r a l m i c r o b e s (5).

production o f Streptococcus,

275 According t o these authors, t h e a c i d p r o d u c t i o n o f S t r e p t o c o c c u s and L a c t o -

b a c i l l u s d e c r e a s e d b y a b o u t 50% i n i n c u b a t i o n m e d i a c o n t a i n i n g 1 0 0 ppm NaF. Their r e s u l t s are consistent w i t h these findings t h a t acid production d e c r e a s e d a b o u t 40% i n t h e p r e s e n c e o f 90 ppm F. I n addition, B u n i c k and K a s h k e t (12), s t u d y i n g t h e e f f e c t s o f f l u o r i n e on p u r i f i e d e n o l a s e d e r i v e d f r o m S, s a l i v a r i e . enzymic a c t i o n on enolase. water-insoluble while Shklair

S. m u t a n s a n d S. s a n g u i s ,

suggested t h a t f l u o r i n e exerted a n t i b a c t e r i a l e f f e c t s through i t s a n t i The e f f e c t s o f f l u o r i n e on t h e s y n t h e s i s o f S h i m u r a and O n i s h i d e x t r a n h a v e b e e n e x t e n s i v e l y examined.

(14) r e p o r t e d t h a t p r o d u c t i o n o f d e x t r a n d e c r e a s e d i n t h e p r e s e n c e o f NaF,

9 ~ 1(. 15)

o b s e r v e d no change i n p r o d u c t i o n a t a f l u o r i n e

c o n c e n t r a t i o n o f 100 ppm d e s p i t e a n o b v i o u s i n h i b i t i o n o f b a c t e r i a l g r o w t h . I n t h e w o r k o f P a t r i c k (16) w i t h v a r i o u s s t r a i n s o f S. m u t a n s , f l u o r i n e was shown t o e n h a n c e t h e s y n t h e s i s o f w a t e r - i n s o l u b l e d e x t r a n b y a c c e l e r a t i n g t h e dextransucrase a c t i v i t y . o f water-insoluble enhanced. These d i s c r e p a n c i e s i n d i c a t e d t h a t p r o d u c t i o n d e x t r a n was i n h i b i t e d b y t h e p r e s e n c e o f NaF i n t h e c e l l s y s t e m was e x a m i n e d no

g r o w i n g s y s t e m b u t t h a t t h e p r o d u c t i o n p e r u n i t o f b a c t e r i a l p r o t e i n was A l t h o u g h t h e e f f e c t o f NaF i n t h e c e l l - f r e e

i n t h i s s t u d y a t h i g h e r c o n c e n t r a t i o n s t h a n t h o s e employed b y S h k l a i r ,
i n h i b i t i o n o f d e x t r a n s y n t h e s i s was o b s e r v e d . T h e f i n d i n g s t h a t Li', Na', e n t w i t h t h o s e o f S h k l a i r ( 1 5 ) a n d C a r l s s o n (17).

These f i n d i n g s a r e c o n s i s t a n d Cs3+ h a d n o e f f e c t o n

K+,

NH4+,

Cr',

d e x t r a n s y n t h e s i s a r e c o n s i s t e n t w i t h t h o s e o f C a r l s s o n ( 1 7 ) and W i l l i a m

(18).
Co2+,

T h e s e a u t h o r s , h o w e v e r , f o u n d t h a t b i v a l e n t c a t i o n s s u c h a s Zn2+, Mg2+,

Ni2+,

and Cu2+ i n h i b i t e d g l u c a n s y n t h e s i s . b u t AlF3. buffer. ZnF2,

I n t h i s study, CaF2,

Co2+

was shown t o i n h i b i t g l u c a n s y n t h e s i s , phate,

MgF2 and SnF?

w e r e n o t o b s e r v e d t o f o r m p r e c i p i t a t e s i n t h e p r e s e n c e o f p o t a s s i u m phosa c e t i c a c i d b u f f e r and t r i s - m a l a t e S n F 2 a n d CoF2 g a v e l o w e r M I C f o r S. m u t a n s t h a n o t h e r f l u o r i d e s . seems t o h a v e a s p e c i f i c s y n e r g i s m w i t h f l u o r i n e and v a r i o u s c a t i o n s , t h e c a s e o f SnF2. a c t i o n o f CoF2. B e c a u s e o f t h e s p e c i f i c c y t o t o x i c i t y o f Co2+, CoF2 as i n

F u r t h e r s t u d i e s a r e needed t o c l a r i f y t h e a n t i b a c t e r i a l caution

s h o u l d be e x e r c i s e d i n t h e c l i n i c a l a p p l i c a t i o n o f t h i s agent. I n t h e present study, antibacterial effects. NaF a n d o t h e r f l u o r i d e s w e r e s c r e e n e d f o r t h e i r The g r e a t e r a n t i b a c t e r i a l e f f e c t o f CoF2 t h a n t h a t

o f NaF, s u g g e s t e d b y t h e c o m p a r i s o n o f M I C and i n s o l u b l e d e x t r a n f o r m a t i o n ,
w i l l s e r v e as a u s e f u l g u i d e l i n e i n t h e development o f t h i s study.

CONCLUSION The f o l l o w i n g r e s u l t s w e r e o b t a i n e d .

276

T h e M I C o f f l u o r i d e p r o v i d e d a s NaF f o r A c t i n o m y c e s .

Candida and

_____ Pseudomonus
S. _ m u t_ ans) _

were higher than those f o r t h e o t h e r microorganisms.

=

(Candida > A c t i n o m y c e s > Pseudomonus > > B a c t e r o i d e s

Fusobacterium >

The i n h i b i t o r y e f f e c t o f NaF was r e l a t e d t o f l u o r i n e c o n c e n t r a t i o n and observed as a b a c t e r i o s t a t i c action. CoF2 and SnF2 i n h i b i t e d b a c t e r i a l g r o w t h a t c o n c e n t r a t i o n s o f 2MIC and 3MIC b a c t e r i c i d a l 1y. E x c e p t f o r CoF2, n o e f f e c t s o f f l u o r i d e (0-10000 ppm F ) o n t h e s y n t h e s i s o f w a t e r i n s o l u b l e g l u c a n w e r e observed. REFERENCES

1. Huysen GV.

M u h l e r JC (1948) J D e n t Res 27:46-51 M u h l e r JC (1955) J D e n t Res 34:350-357

2. H a t t o n WE, H e b e r g a l l WH,

3. C u r r e s s TM (1966) NZ D e n t J 62:26-31 4. Shannon I L (1970) J O r a l Med 25:12-17 5. M a l t z M, E m i l s o n CG (1982) J D e n t Res 61:786-790

6. B i b b y BG, Van K e s t e r e n M (1940) J D e n t Res 19:391-402

7. Japanese C o m m i t t e e f o r Judgment o f M I C ( i n J a p a n e s e ) ( l 9 8 1 ) Chemotherapy 29: 76-81 8. F u r u t a n i M, 56: 793 Y a g i T, I w a k i M, I w a k i M, I i d a M. S a t 0 T, N o z a k i K (1984) S e i k a g a k u

9. F u r u t a n i M, Y a g i T, H e a l t h 35:173-179 10. R o b y t e JF,

I i d a M, N o z a k i K, S a t 0 T ( 1 9 8 5 ) J D e n t

C o r r i n g a n AL (1977) A r c h Biochem B i o p h y s 83:726-781 H o r t o n I M , S t r e c k f u s s JL, Dreizen

11. Yoon A, Newman MG (1980) J C l i n P e r i o d o n t o l 7: 489-494 12. Brown LR, H a n d l e r SH, Res 50: 159-1 67 14. S h i m u r a N, 15. S h k l a i r IL,

(1980) J D e n t

13. B u n i c k FT, K a s h k e t SK (1981) I n f e c t Immun 34:856-863 O n i s h i M (1978) J D e n t Res 57:928-931 W a l t e r RG, L a m b e r t s BL (1977) AADR 56:147 C a r i e s Res 4:97-113

16. P a t r i c k T (1981) J D e n t Res 51:1601-1610 17. C a r l s s o n J (1970) A L e v a n s u c r a s e f r o m S, mutans. 18. W i l l i a m R (197) C a r b o Res 42:325-331

H.Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science,Volume 27, pp. 277-284 0 1986 Elsevier Science PublishersB.V., Amsterdam - Printed in The Netherlands

277

EXCITATION

OF

SKELETAL MUSCLE

BY FLUORIDE

TOSHIMI HATTORI AND HIROSHI MAEHASHI Department of Dental Pharmacology, Matsumoto Dental College, Shiojiri 39907. Japan

ABSTRACT The effects of NaF on the mechanical and electrical responses of sciatic nerve-sartorius muscle preparations from the bullfrog were investigated i n an attempt to elucidate the mechanism of excitation of skeletal muscle . 1 mM augmented the induced by fluoride. NaF at concentrations above 0 twitch and tetanus evoked by indirect stimulation of the partially fatigued muscle, and above 5.0 m M it induced fibrillation. Sodium oxalate at 2.5
mM.

sodium citrate at 1.7 m M , and calcium-deficient Ringer's solution neither induced fibrillation nor augmented the twitch. NaF increased the amp1 itude of the miniature endplate potential (m.e.p.p.) even under pre-

treatment with 16 u M neostigmine. but the extent of the increment was only . 1 m M adrenaline, 1.0 m M one half as much as that by NaF alone. NaF, 0 theophylline, and 4.0 m M dibutyryl cyclic A M P , raised the m.e.p.p. frequency and increased the quanta1 content of the endplate potential. These findings suggest that fluoride may induce excitation of skeletal muscle in a manner unrelated to decalcification and that such excitation may be due to the activation of adenylate cyclase in the nerve ending, the inhibition of cholinesterase, and to increased sensitivity of the ZndPlate to acetylcholine.

INTRODUCTION Fluoride affects the l i v i n g cell i n various ways, for example, decalcification, inhibition o f enzymes, and activation of adenylate cyclase, etc. It also excites skeletal muscles. Fibrillation is observed in acute fluoride poisoning (1). and fluoride decurarizes muscles blocked by 4tubocurarine ( 2 ) . Many investigators have ascribed acute fluoride poisoning to decalcification (3). However, there are some reports indicating that it is due rather to other properties of fluoride, e.g., its inhibitory effect on cholinesterase ( 4 ) . Most studies on fluoride toxicity have been performed in vivo. The experimental data are complex, and the mechanism of

278
t h e poisoning i s s t i l l unclear. skeletal muscle o f t h e frog, An i n v i t r o s t u d y h a s b e e n m a d e o n t h e

e f f e c t s o f s o d i u m f l u o r i d e on m e c h a n i c a l a n d e l e c t r i c a l r e s p o n s e s o f t h e t o d e t e r m i n e w h e t h e r o r n o t f l u o r i d e causes muscle e x c i t a t i o n t h r o u g h a mechanism independent o f d e c a l c i f i c a t i o n . MATERIALS AND METHODS Sciatic nerve-sartorius m u s c l e p r e p a r a t i o n s f r o m t h e b u l l f r o g Rana c a t e s The b e i a n a ( w e i g h i n g b e t w e e n 1 0 0 and 2 0 0 g) w e r e u s e d a s t h e m a t e r i a l . p r e p a r a t i o n s h a v e been d e s c r i b e d p r e v i o u s l y (5). The p r e p a r a t i o n was (volume: v e r t i c a l l y f i x e d o n t h e b o t t o m o f a Magnus t u b e T w i t c h and t e t a n u s The ressupramaximum; 10 m l ) c o n t a i n i n g a e r a t e d R i n g e r ' s s o l u t i o n .

were evoked b y e l e c t r i c a l s t i m u l a t i o n o f t h e n e r v e o r t h e muscle. n e r v e was s t i m u l a t e d w i t h a s u c t i o n e l e c t r o d e ( v o l t a g e : pectively). d u r a t i o n , 0.1 m s e c ; f r e q u e n c y , 0.1 a n d 50 Hz f o r t w i t c h a n d t e t a n u s , f i e l d s t i m u l a t i o n w i t h p l a t i n u m p l a t e s ( 2 0 0 V, 0.1-1.0 l o w i n g t r e a t m e n t w i t h d - t u b o c u r a r i n e c h l o r i d e (6.0-30 (SB-IT. N i h o n Kohden).

D i r e c t s t i m u l a t i o n o f t h e m u s c l e was done t r a n s v e r s e l y b y m s e c , 0.1 H z ) f o l -

ugiml).

Tensions i n transducer

t h e muscle were measured i s o m e t r i c a l l y w i t h a f o r c e - d i s p l a c e m e n t t i o n was d e t e r m i n e d b y a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y .

The c o n c e n t r a t i o n o f c a l c i u m i n t h e R i n g e r ' s s o l u -

E l e c t r i c a l r e s p o n s e s o f t h e m u s c l e f i b e r were i n t r a c e l l u l a r l y recorded w i t h a n o s c i l l o s c o p e (VC-10. f i l l e d w i t h 3M KC1. extracellularly. p o t e n t i a l (e.p.p.). For N i h o n Kohden) a glass microelectrode The compound a c t i o n p o t e n t i a l o f t h e n e r v e was d e r i v e d d e t e r m i n a t i o n o f t h e quanta1 content o f t h e endplate and t h e m u s c l e was t r e a t e d w i t h a p e r f u s a t e c o n t a i n i n g 0.5 w e r e r e c o r d e d (6). log, Quanta1 c o n t e n t (number o f nerve

m M CaC12 a n d 6.0 m M MgC12 t h r o u g h o u t t h e e x p e r i m e n t a n d t h e e.p.p.

m i n i a t u r e e n d p l a t e p o t e n t i a l (m.e.p.p.) a m p l i t u d e o f m.e.p.p.)

w a s o b t a i n e d b y b o t h t h e d i r e c t m e t h o d ( m e a n a m p l i t u d e o f e.p.p./mean and t h e f a i l u r e s method, i m p u l s e s / n u m b e r o f f a i l u r e s o f e.p.p.)

(6).

The v a l u e s o b t a i n e d b y t h e

d i r e c t m e t h o d w e r e c o r r e c t e d f o r n o n l i n e a r s u m m a t i o n (7). The c o m p o s i t i o n o f n o r m a l R i n g e r ' s s o l u t i o n ( i n m M ) was a s f o l l o w s : NaC1, 5.6. 1 1 0 ; KC1, 1.9; CaC12, 1.1; NaH2P04,

0.4;

NaHC03.

2.4:

and g l u c o s e ,

T h e pH w a s a d j u s t e d t o 7.3.

The f o l l o w i n g c h e m i c a l s w e r e used:

s o d i u m f l u o r i d e (NaF), (d-Tc. (Sigma). Yoshitomi);

sodium o x a l a t e , sodium c i t r a t e . and t h e o p h y l l i n e and d i b u t y r y l c y c l i c AMP (dbcAMP)

(Na k a r a i ); n e o s t i g m i n e m e t h y l s u 1f a t e ( S h io n o g i); d - t u b o c u r a r in e c h 1 o r id e adrenaline (Daiichi);

A l l e x p e r i m e n t s w e r e c a r r i e d o u t a t r o o m t e m p e r a t u r e (20-25C).

219 RESULTS NaF (0.1-10 mM) dose-dependently a u g m e n t e d t h e t w i t c h and t e t a n u s e v o k e d b y i n d i r e c t s t i m u l a t i o n o f t h e p a r t i a l l y f a t i g u e d m u s c l e whose t w l t c h t e n s i o n had d e c r e a s e d t o 60-70% o f t h e m a x i m a l v a l u e . t h e t w i t c h i s i l l u s t r a t e d i n F i g u r e 1. t i o n a s h i g h a s 1 0 mM, induced by d i r e c t stimulation. t h e u n s t i m u l a t e d muscle. T h e Ca c o n c e n t r a t i o n i n t h e R i n g e r ' s s o l u t i o n w a s l o w e r e d f r o m 1.1 t o However, NaF. The e f f e c t o f NaF on even a t a concentra-

augmented n e i t h e r t h e maximal t w i t c h n o r t h e t w i t c h NaF a b o v e 5.0 m M i n d u c e d f i b r i l l a t i o n o f

0.94 mM b y 5 mM NaF.
Ca-reducing e f f e c t , s o d i u m o x a l a t e (2.5 c o n t a i n i n g 0.94 twitch.

To c o m p a r e NaF w i t h v a r i o u s c h e m i c a l a g e n t s f o r t h e i r we i n v e s t i g a t e d t h e e f f e c t s o f t h e d e c a l c i f y i n g d r u g s , m M ) and s o d i u m c i t r a t e (1.7 mM), which are equivalent and C a - d e f i c i e n t R i n g e r

t o 5.0 m M NaF i n t e r m s o f C a - b i n d i n g c a p a c i t y ,

m M Ca.

None o f t h e s e i n d u c e d f i b r i l l a t i o n o r a u g m e n t e d t h e

180,

F i g u r e 1. L o g d o s e - r e s p o n s e c u r v e o f NaF o n t h e t w i t c h t e n s i o n o f t h e z a r t o r i u s muscle evoked b y s c i a t i c n e r v e s t i m u l a t i o n o f a p r e p a r a t i o n f r o m the bullfrog. NaF a u g m e n t e d t h e t w i t c h d o s e - d e p e n d e n t l y . Each p o i n t r e p r e s e n t s t h e mean o f r e l a t i v e v a l u e s (8-9 e x p e r i m e n t s ) o f t h e t w i t c h t e n s i o n s o b s e r v e d 3 m i n u t e s a f t e r a p p l i c a t i o n o f NaF c o m p a r e d w i t h t h a t before the application. The v e r t i c a l b a r s i n d i c a t e t h e s t a n d a r d d e v i a t i o n . +$, ?yric: S i g n i f i c a n t l y d i f f e r e n t f r o m t h e v a l u e b e f o r e t h e a p p l i c a t i o n a t p<0.05 and p<O.Ol, r e s p e c t i v e l y .

280

200 r

2
u

180

$ e
ti d

140120-

looL0:5 1:O 2 1 0 5:O NgF concentration (mM)

Figure 2. Rise of m.e.p.p. frequency produced by NaF. Each polnt represents the mean o f flve relatlve values compared with those before the application. )ep~0.05,i'"p<O.Ol.

The effects of NaF on the nerve and muscle were studied to ascertain that 0 mM) they were not the sites of NaF action. As expected, NaF (5.0 and 1 had no of the (r.p.), muscle effect on their electrical responses, i.e., the alpha-wave amplitude compound action potential of the nerve and the resting potential membrane resistance, and wave form of the action potential of fiber remained unchanged by NaF treatment.

The influence of NaF on the electrical phenomena in the endplate was also NaF at 0.5-5.0 m M raised the m.e.p.p. frequency (i.e. the rate of spontaneous release of the transmitter) and increased the amplitude investigated. of not only the m.e.p.p. but also the e.p.p. The increase of the m.e.p.p. frequency produced by NaF is shown in Figure 2. Since the elevation of m.e.p.p. frequency and e.p.p. amplitude seemed to be related to the improvement of transmitter release from the nerve ending, the effect of NaF on the quantal content of e.p.p. (i.e., the amount of transmitter released in response to a single nerve impulse) was studied. NaF increased the quantal contents calculated by both methods, as illustrated in Figure 3. The quantal content obtained by the direct method was corrected by Martin's method (7); namely, the value was multiplied by (r.p.-15)/(r.p.-15-e.p.p.) after r.p. was substituted by 89.4 or 92.0, obtained from measurement during the control or test period. In addition, the effects of adrenaline. an activator of adenylate cyclase. theophylline, an inhibitor of phosphodiesterase, and dbcAMP, a derivative o f cyclic AMP, on the transmitter release were studied to see how NaF affected the nerve ending. The quantal content and m.e.p.p. frequency were increased by 0 . 1 mM adrenaline, 1 . 0 mM theophylline, and 4 . 0 mM dbcAMP (Table I ) .

TABLE I

E F F E C T S O F ADRENALINE, THEOPHYLLINE. A N D dbcAMP

m.e. p. p. F R E Q U E N C Y . Treatment Mean A m p l i t u d e o f e.p.p. ( m V )

ON T H E

QUANTAL CONTENT O F e.p.p.

A N D O N THE

Mean A m p l i t u d e o f m.e.p.p. (mV)

Corrected Quanta1 Content

Frequency o f m.e.p.p. (/min)

Control A d r e n a l i n e , 0.1 mM Control Theophylline, Control DbcAMP, "p<O. 05.

0.70

0.85
1.37

0.30 0.30 0.31 0.36 0.26 0.23

2.52 3.31 4.53 5.15 3.06 4.35

23.7 30.0 29.3 51.0 49.3 61.3

1.0 mM

1.50 0.13 0 . 8 7 ' '

4.0 mM

282

140r t
c

130 -

5 120.
-

10010:5 1:O 2:O 5:O NaF concentration (mM)

Figure 3. Increment o f quanta1 content of e.p.p. produced by NaF. Each point represents the same as in Figure 2. 0 : direct method, A : failures . 0 5, 3*"p<0.0 1. m e t h od. 3c p < 0

The effect of neostigmine, an inhibitor of cholinesterase (ChE), was investigated to find out whether the increase in the m.e.p.p. amplitude induced by NaF was related to C h E inhibition. Figure 4 shows that 5.0 m M NaF increased the m.e.p.p. amplitude about twice as much as neostigmine (16 uM). a concentration 8 times higher than that which inhibited ChE up to

80%. NaF increased the amplitude even after neostigmine application. but the increase was only one half as much as that by NaF alone (Fig. 4 ) .

180r

I d 120

"i
100

NaF

Figure 4. Effects of NaF and neostigmine on m.e.p.p. amplitude. NaF increased the m.e.p.p. amplitude more markedly than neostigmine. The extent of the increment o f m.e.p.p. amplitude by N a f under pretreatment with neostigmine was only half as much as that by NaF alone. Each column represents the same as in Figure 2.

283

DISCUSSION
NaF a t c o n c e n t r a t i o n s above 0.1 stimulation, l a t e o r sodium c i t r a t e , twitch. Moreover, mM augmented t h e t w i t c h evoked by n e r v e However, s o d i u m oxaa n d a b o v e 5.0 mM i n d u c e d f i b r i l l a t i o n .

b o t h a t a c o n c e n t r a t i o n e q u i v a l e n t t o 5.0 m M NaF,

or C a - d e f i c i e n t R i n g e r caused n e i t h e r f i b r i l l a t i o n n o r augmentation o f
These r e s u l t s i n d i c a t e t h a t NaF a t c o n c e n t r a t i o n s l o w e r t h a n 5.0 s i n c e NaF h a d no e f f e c t o n t h e t w i t c h e v o k e d b y d i r e c t s t i m u l a S i n c e d e c r e a s i n g t h e e x t e r n a l Ca c o n c e n t r a mM induces e x c i t a t i o n t h r o u g h a m e c h a n i s m u n r e l a t e d t o d e c a l c i f i c a t i o n . t i o n n o r on t h e e l e c t r i c a l responses o f t h e muscle, i t i s u n l i k e l y t o a c t on t h e muscle f i b e r d i r e c t l y . t i o n raises the e x c i t a b i l i t y o f the nerve wave f o r m r e m a i n e d unchanged. Therefore,

( 8 ) . t h e e f f e c t o f NaF o n t h e
I t s a m p l i t u d e and

compound a c t i o n p o t e n t i a l o f t h e n e r v e was i n v e s t i g a t e d .

t h e s i t e o f a c t i o n o f NaF seems

t o be n e i t h e r t h e n e r v e n o r m u s c l e b u t t h e n e u r o m u s c u l a r j u n c t i o n .

P.s t h e m.e.p.p.

frequency i s e n t i r e l y c o n t r o l l e d by t h e conditions o f t h e i n c r e a s e i n f r e q u e n c y b y NaF s h o w e d t h a t NaF

p r e s y n a p t i c membrane (9), release. mitter. I n addition, Thus,

a c t e d on t h e n e r v e e n d i n g and i n c r e a s e d t h e s p o n t a n e o u s r a t e o f t r a n s m i t t e r

i t a l s o s u g g e s t e d t h a t t h e i n c r e a s e i n t h e e.p.p.
was measured. NaF a t

a m p l i t u d e may b e d u e t o i m p r o v e m e n t o f t h e e v o k e d r e l e a s e o f t h e t r a n s t h e q u a n t a l c o n t e n t o f t h e e.p.p.

0.5-

5.0 m M i n c r e a s e d t h e q u a n t a l c o n t e n t , i n d i c a t i n g t h a t
amount o f t r a n s m i t t e r r e l e a s e d b y a n e r v e impulse. S i n c e NaF a c t i v a t e s a d e n y l a t e c y c l a s e , various t i s s u e s (lo), increased the a l l o f w h l c h show a s i m i l a r a c t i o n ,

NaF i n c r e a s e d t h e

t h u s r a i s i n g t h e cAMP l e v e l i n theophylline, and dbcAMP, This Each o f t h e t e s t e d d r u g s frequency. This idea i s

t h e e f f e c t s o f adrenaline, were studied.

q u a n t a l c o n t e n t a n d r a i s e d t h e m.e.p.p.

i m p l i e s t h a t cAMP may be i n v o l v e d i n t h e t r a n s m i t t e r r e l e a s e . t r a n s m i s s i o n (11). dbcAMP, theophylline,

s u p p o r t e d b y m a n y r e p o r t s d e s c r i b i n g t h e r o l e o f cAMP i n n e u r o m u s c u l a r F o r example, W i l s o n (12) i n v e s t i g a t e d t h e e f f e c t s o f and a m i n o p h y l l i n e ( a n i n h i b i t o r o f p h o s p h o d i e s t e r a s e ) and r e p o r t e d t h a t a l l o f t h e t e s t e d releasable store, t h a t NaF i m p r o v e s

o n t h e t r a i n s o f r e p e t i t i v e e.p.p.s and m o b i l i z a t i o n r a t e ,

d r u g s i n c r e a s e d t h e q u a n t a l c o n t e n t o f t h e f i r s t e.p.p.. m o b i l i z a t i o n , and s t o r a g e o f Ach. We b e l i e v e , t h e r e f o r e ,

s u g g e s t i n g t h a t cAMP was i n v o l v e d i n t h e s y n t h e s i s ,

Ach r e l e a s e t h r o u g h e l e v a t i o n o f cAMP c o n c e n t r a t i o n s i n t h e n e r v e e n d i n g . B e c a u s e NaF r e d u c e s t h e a c t i v i t y o f ChE, m.e.p.p. a m p l i t u d e was i n v e s t i g a t e d . therefore, t h e i n f l u e n c e o f NaF o n t h e amp1 iT h e i n c r e a s e i n t h e m.e.p.p.

t u d e b y NaF u n d e r p r e t r e a t m e n t w i t h n e o s t i g m i n e was o n l y o n e h a l f a s much as t h a t b y NaF a l o n e ; t h e p o t e n t i a t i o n o f t h e m.e.p.p. a c t i o n o f NaF. amplitude may be p a r t i a l l y due t o t h e a n t i - C h E

284
NaF i n c r e a s e d t h e m.e.p.p. stigmine. t o Ach, a m p l i t u d e e v e n u n d e r p r e t r e a t m e n t w i t h neo-

T h i s i n d i c a t e s t h a t NaF r a i s e d t h e s e n s i t i v i t y o f t h e e n d p l a t e

f o r t h e a m p l i t u d e o f t h e m.e.p.p.

i s c o n t r o l l e d by p r o p e r t i e s o f

t h e p o s t s y n a p t i c membrane (9). t h e i n c r e a s e i n t h e m.e.p.p.

Koketsu and Gerard ( 2 ) have d e s c r i b e d t h a t

a m p l i t u d e induced by NaF i s due t o t h e r i s e o f t h e mechanism o f e x c i t a t i o n o f t h e NaF a c t i v a t e s adenylcAMP improves frequency i s

Ach s e n s i t i v i t y r a t h e r t h a n t o ChE i n h i b i t i o n . F r o m t h e r e s u l t s d e s c r i b e d above, a t e cyclase, raised, s k e l e t a l muscle by NaF i s t h o u g h t t o be as f o l l o w s : spontaneous o r evoked r e l e a s e o f t h e t r a n s m i t t e r ; ChE i s i n h i b i t e d , a n d t h e Ach increased;

t h u s r a i s i n g t h e cAMP l e v e l i n t h e n e r v e ending: t h e m.e.p.p.

s e n s i t i v i t y o f the endplate i s f i n a l l y f i b r i l l a t i o n occurs o r

d e p o l a r i z a t i o n o f t h e e n d p l a t e r e g i o n by Ach i s made so i n t e n -

s i v e t h a t t h e a c t i o n p o t e n t i a l i s generated; It

t h e t w i t c h i s a u g m e n t e d as a r e s u l t o f r e c r u i t m e n t o f t h e m u s c l e f i b e r s .
IS

concluded t h a t f l u o r i d e may induce e x c i t a t i o n o f s k e l e t a l muscles i n t h a t i s , e x c i t a t i o n may be due t o the i n h i b i t i o n o f

a fashion unrelated t o d e c a l c i f i c a t i o n ; ChE,

t h e a c t i v a t i o n o f a d e n y l a t e c y c l a s e i n t h e nerve ending,

and t o t h e i n c r e a s e d s e n s i t i v i t y o f t h e e n d p l a t e t o Ach.

REFERENCES
1. Tappeiner H. (1889) Arch Exper P a t h o l Pharmakol 25:203-224

2. Koketsu K, Gerard RW (1956) Am J P h y s i o l 186:278-282

3. Haynes RC. M u r a d F ( 1 9 8 0 ) I n : Goodman
c o l o g i c a l B a s i s o f Therapeutics, 1550 6 t h ed.

LS.

G i l m a n A ( e d s ) The PharmaM a c M i l l a n . New York, pp 1525-

4.

Roholm K ( 1 9 3 8 ) I n : Handbuch d e r E x p e r i m e n t e l l e n P h a r m a k o l o g i e , Erganzungswerk, 7. J u l i u s S p r i n g e r , B e r l i n , pp 1-62

5. H a t t o r i T (1984) Matsumoto Shigaku 10:17-23

6. Del C a s t i l l o J, K a t z B (1954) J P h y s i o l 124:560-573

7. M a r t i n AR (1955) J P h y s i o l 130:114-122
8. Frankenheuser B, 9. K a t z B (1962) 10. S u t h e r l a n d EW, 11. Goldberg AL, Hodgkin AL (1957) J P h y s i o l 137:218-244 Menon T ( 1 9 6 2 ) J B i o l Chen 237:1220-1227 Proc Roy SOC B i o l 155:455-477 R a l l TW, S i n g e r J J (1969) Proc Nat Acad S c i 64: 134-141

12. Wilson DF (1974) J Pharmacol Exp Ther 188:447-452

H.Tsunoda and M.-H. Y u (Editors) Fiuoride Research 1985, Studies in Environmental Science,Volume 27, pp. 285-297 0 1986 Elsevier Science PublishersB.V., Amsterdam - Printed in The Netherlands

285

BIOLOGICAL SIGNIFICANCE OF FLUORIDE I N FISH TEETH SHOICHI SUGA'. YASUHIKO TAK12, AND KOJI WADA3

'Address all correspondence to Dr. S. Suga, Department of Pathology, Nippon Dental University. 1-9-20, Fujimi, Chiyoda-ku, Tokyo, Japan 'Tokyo University o f Fisheries, Tokyo, Japan 3National Research Institute of Aquaculture, Mie. Japan ABSTRACT terms of the fluoride concentration in the enameloid of teeth: one group containing fluoride of more than about 1.4% i n the enameloid, and the other containing
in

The teleost fishes may be classified into t w o groups

less than about 0.3%. There is no correlation between the fluoride concentration i n the enameloid or that i n the environmental water, the shape and size of teeth which show diverse morphological adaptation, and the feeding habits. The fluoride concentration in the enameloid i s related to the phylogeny of fishes. The enameloid is considered to provide a site where excess fluoride i n the body fluids is excreted i n order to detoxify the fishes. INTRODUCTION The fluoride concentration in the enamel of mammals is generally considered closely related to that i n their drinking water and diet. However, preliminary investigations of fluoride concentration in the teeth o f various teleosts and elasmobranchs from both marine and freshwater indicated that fish may be divided into t w o groups i n terms of fluoride concentrations i n the enameloid: one group containing more than about 2.5% fluoride in the enameloid, and the other containing less than about 0.17%. I n addition, it can be shown that the fluoride concentration in the enameloid might be related to fish phylogeny rather than to the environmental waters. (Marine water over all the oceans contains about 1.3 ppm fluoride (1) whereas freshwater contains less than 0 . 1 ppm) (2-4). I n order to study more precisely the relationship between the fluoride concentration in the enameloid, the fish phylogeny and the environmental water, quantitative fluoride analyses were performed on the teeth of many fishes collected systematically. Efforts were focused particularly on bony fishes (Osteichthyes) because there are more species of bony fishes in the world than cartilaginous fishes (Chondrichthyes) and they are distributed
in

various geographical and environmental conditions.

286 The e n a m e l o i d o f f i s h h a s been c o n s i d e r e d a s a p r i m i t i v e f o r m o f enamel i n amphibians. r e p t i l e s and mammals.

It i s h i g h l y m i n e r a l i z e d ,

although

i t s o r g a n i c m a t r i x c o m p r i s e s b o t h e c t o d e r m a l and m e s o d e r m a l p r o d u c t s w h i l e t h e enamel o f t e t r a p o d s c o n t a i n s o n l y e c t o d e r m a l products. T h i s paper i s a r e v i e w o f t h e r e s u l t s o f o u r r e c e n t i n v e s t i g a t i o n s i n t o t h e t e e t h o f f i s h e s b e l o n g i n g t o some i m p o r t a n t g r o u p s (5-7). MATERIALS AND METHODS The g r o u n d s e c t i o n s o f t e e t h and bone embedded i n p o l y e s t e r r e s i n w e r e f i r s t microradiographed u s i n g a s o f t X-ray mineralization pattern, b l o c k w i t h epoxy resin. apparatus t o examine t h e and t h e n m o u n t e d on a p o l i s h e d s u r f a c e o f a l u m i n u m A f t e r applying a t h i n coating o f carbon t o the quantitative and l i n e scan e l e c t r o n The e l e c t r o n m i c r o p r o b e a p p a r a t u s used equipped w i t h a wavelength d i s p e r s i v e

p o l i s h e d s u r f a c e o f t h e specimens, microprobe analyses were performed. was a Shimadzu-ARL, m o n o c h r o m e t e r (WDX). t y p e EMX-SM,

Q u a n t i t a t i v e f l u o r i d e a n a l y s i s was c a r r i e d o u t b y m e a s u r i n g t h e i n t e n s i t y of

FKa e m i s s i o n o b t a i n e d b y t h e m o d e o f

point

analysis,

using the Point the middle

c o n v e r g e n t m e t h o d w i t h f l u o r a p a t i t e ( D u r a n g o ) a s a s t a n d a r d sample. a n a l y s i s was made m a i n l y i n t h e s u r f a c e l a y e r o f e n a m e l o i d . l a y e r o f dentin, and t h e t o o t h s u p p o r t i n g bone.

RESULTS AND DISCUSSION Distribution o f fluoride i n the teeth A l i n e s c a n a n a l y s i s was c a r r i e d o u t a l o n g a l i n e c r o s s i n g b o t h e n a m e l o i d and d e n t i n l a y e r s o f t h e t e e t h . i n t h e dentin.
I n t h e t e e t h o f some f i s h e s ,

the results

i n d i c a t e d t h a t f l u o r i d e c o n c e n t r a t i o n was much h i g h e r i n t h e e n a m e l o i d t h a n The c o n c e n t r a t i o n was t h e h i g h e s t i n t h e s u r f a c e l a y e r o f Although the f l u o r i d e t h e e n a m e l o i d and d e c r e a s e d g r a d u a l l y t o w a r d s t h e e n a m e l o i d - d e n t i n j u n c t i o n and t h e n showed a marked d e c r e a s e i n t h e d e n t i n . l e v e l was v e r y l o w i n t h e d e n t i n , pulpal side towards the enameloid-dentin
i t tended t o increase s l i g h t l y from t h e

j u n c t i o n i n the narrow dentin

F i g u r e 1. A c a n t h o p a g r u s s c h l e g e l i ( m a r i n e f i s h o f t h e P e r c i f o r m e s ) . a - M i c r o r a d i o g r a m o f g r o u n d s e c t i o n o f d e v e l o p i n g and e r u p t e d t e e t h . E l e c t r o n m i c r o p r o b e l i n e s c a n a n a l y s i s was p e r f o r m e d a l o n g t h e l i n e s d r a w n o n t h e m i c r o r a d i o g r a m ( x 80). b - L i n e s c a n a n a l y s i s o f CaKa, MgKa and FKa p e r f o r m e d a l o n g l i n e b o n t h e d e v e l o p i n g t o o t h germ a t t h e l a t e stage. c-Line scan a n a l y s i s performed a l o n g l i n e c on t h e e r u p t e d t o o t h . F l u o r i d e c o n c e n t r a t i o n i s much h i g h e r i n t h e e n a m e l o i d t h a n i n t h e Magnesium c o n c e n t r a t i o n i s l o w e r i n t h e e n a m e l o i d t h a n i n t h e dentin. d e n t i n . ES, e n a m e l o i d s u r f a c e ; EDJ, e n a m e l o i d - d e n t i n j u n c t i o n .

287

1b

Enamcsloidl

Ic

Enamsloid

Dbntin

288

l a y e r adjacent t o the enameloid-dentin

j u n c t i o n ( F i g s . 1 a n d 5).

A high
but

f l u o r i d e c o n c e n t r a t i o n was o b s e r v e d n o t o n l y i n t h e e r u p t e d e n a m e l o i d , ( F i g . 1).

a l s o i n t h e d e v e l o p i n g e n a m e l o i d a t a l a t e s t a g e o f m i n e r a l i z a t i o n (7.8) On t h e o t h e r h a n d , i n t h e t e e t h o f o t h e r f i s h e s , b o t h e n a m e l o i d 6). and d e n t i n a l s o showed a v e r y l o w f l u o r i d e l e v e l ( F i g . The P e r c i f o r m e s , fishes,

Fluoride concentration i n the teeth o f Perciform fishes (5) one o f t h e l a r g e s t o r d e r s o f t e l e o s t f i s h e s o f bony T h e s e f r e s h w a t e r s p e c i e s c a n be c l a s s i f i e d i n c l u d e a c o n s i d e r a b l e number o f f r e s h w a t e r s p e c i e s i n a d d i t i o n t o

t h e predominant marine forms. freshwater forms, e.g.,

i n t o s e v e r a l g r o u p s w h i c h d i f f e r i n t h e p r o c e s s o f e s t a b l i s h m e n t as primary freshwater fishes which are s t r i c t l y secondary f r e s h w a t e r f i s h e s which are i n t o l e r a n t o f s a l i n e environments, a c e r t a i n extent,

r a t h e r s t r i c t l y c o n f i n e d t o f r e s h w a t e r b u t may be t o l e r a n t o f s e a w a t e r t o and v i c a r i o u s f r e s h w a t e r f i s h e s t h a t a r e s t r i c t l y f r e s h w a t e r r e p r e s e n t a t i v e s o f p r i m a r i l y m a r i n e g r o u p s (9). The s p e c i m e n s used r e p r e s e n t 78 s p e c i e s f a l l i n g i n t o 40 f a m i l i e s i n 10 suborders. The t e e t h o f f i s h e s o f t h e s e o r d e r s showed v a r i o u s shapes and Some f i s h e s h a d s e p a r a t e a n d w e 1 1 - d e v e l o p e d , m o l a r - 1 ik e teeth arrangements.

(Fig. l a ) o r c h i s e l - l i k e (Fig. 2) t e e t h and o t h e r f i s h e s had sharp-edged, b e a k - l i k e t o o t h p l a t e s composed o f s u c c e s s i v e r o w s o f a r r o w h e a d - l i k e w h i c h w e r e s u r r o u n d e d b y a l v e o l a r bone ( F i g . 3). The d i s t r i b u t i o n o f f l u o r i d e c o n c e n t r a t i o n s d e t e r m i n e d b y p o i n t a n a l y s i s i s p l o t t e d i n F i g u r e 4. t o 5.45%, The e n a m e l o i d o f a l l t h e P e r c i f o r m s p e c i e s r a n g i n g f r o m 1.44% w h e r e a s t h e d e n t i n and bone c o n t a i n e d l o w e r respectively. The f l u o r i d e c o n c e n t r a t i o n o f examined c o n t a i n e d r a t h e r h i g h f l u o r i d e concentrations, i n the surface layer, t h a n 0.75% and 0.63% f l u o r i d e ,

t h i s o r d e r w a s m u c h h i g h e r t h a n t h a t i n h u m a n e n a m e l ( a b o u t 0.2% i n t h e s u r f a c e l a y e r ) and i n t h e e n a m e l o i d o f C y p r i n u s c a r p i o (common c a r p : 0.17%)(4) and Fugu r u b r i p e s r u b r i p e s ( p u f f e r : 0.17%)(6). It was r e c o g n i z e d t h a t t h e r e was n o s i g n i f i c a n t d i f f e r e n c e i n f l u o r i d e c o n c e n t r a t i o n among

m a r i n e and f r e s h w a t e r s p e c i e s o f d i f f e r e n t d i v i s i o n s . Fluoride concentration i n the teeth o f Tetraodontiform fishes (6) The T e t r a o d o n t i f o r m s a r e c o n s i d e r e d t o h a v e e v o l v e d f r o m P e r c i f o r m s d u r i n g t h e Eocene p e r i o d o f t h e C e n o z o i c Era. f i e d i n t o t w o suborders, l i e s and t h e m o r e a d v a n c e d T e t r a o d o n t o i d e i l o n g r o o t s (Fig. teeth, 5a). On t h e o t h e r hand, L i v i n g examples a r e c l a s s i f a m i l i e s (10). t h e p r i m i t i v e B a l i s t o i d e i which contains s i x famicontaining four chisel-like teeth with

The B a l i s t o i d e i h a v e s e p a r a t e and well-developed,

t h e T e t r a o d o n t o i d e i have a k n i f e 6a).

shaped p l a t e f o r m e d b y f u s i o n o f s u c c e s s i v e r o w s o f d e v e l o p i n g p l a t e - l i k e w h i c h a r e s u r r o u n d e d b y a l v e o l a r bone ( F i g .

289

F i g u r e 2. M i c r o r a d i o g r a m o f c h i s e l - l i k e t o o t h o f E p i n e p h e l u s l a t i f a s c i a t u s ( m a r i n e f i s h o f t h e P e r c i f o r m e s ) ( x 100)(5) F i g u r e 3. M i c r o r a d i o g r a m o f p l a t e - l i k e t o o t h o f S c a r u s g i b b u s ( m a r i n e f i s h o f t h e P e r c i f o r m e s ) ( x 80)(5)

290

A q u a n t i t a t i v e p o i n t analysis o f f l u o r i d e (Fig. 7 ) i n d i c a t e d t h a t t h e
enameloid o f all the Balistoidel specimens contained a very hlgh concentration o f fluoride. r a n g i n g f r o m 2.31% t o 5.16%. whereas i n t h e

T e t r a o d o n t o i d e i t h e c o n c e n t r a t i o n ranged o n l y f r o m 0.14% t o 0.28%. It s h o u l d a l s o be n o t e d t h a t t h e e n a m e l o i d o f t h e f a m i l y f r o m w h i c h t h i s o r d e r may have d e r i v e d ( t h e A c a n t h u r i d a e o f t h e P e r c i f o r m e s ) c o n t a i n e d v e r y h i g h amounts o f f l u o r i d e .

It i s speculated t h a t the f l u o r i d e concentrating

mechanism may have been l o s t d u r i n g t h e e v o l u t i o n o f t h e T e t r a o d o n t o i d e i . F l u o r i d e c o n c e n t r a t i o n s i n t h e d e n t i n r a n g e d f r o m 0.14% t o 0.82% i n a l l t h e f i s h e s o f t h i s o r d e r w i t h a tendency t o be s l i g h t l y h i g h e r i n t h e B a l i s t o i d e i than i n the Tetraodontoidei. s i m i l a r pattern. Role and s i g n i f i c a n c e o f h i g h d e p o s i t i o n o f f l u o r i d e i n t h e f i s h enameloid The f i n d i n g s d e s c r i b e d above s u p p o r t t h e p r e v i o u s s p e c u l a t i o n t h a t t h e f l u o r i d e concentration mental water. i n the enameloid o f f i s h t e e t h i s r e l a t e d t o phylogeny o f f i s h e s r a t h e r t h a n t h e f l u o r i d e c o n c e n t r a t i o n i n t h e e n v i r o n R e s u l t s o f o t h e r r e c e n t i n v e s t i g a t i o n s p e r f o r m e d on t h e s u c h as t h e O s t a r i o p h y s a n f i s h e s o f a l s o support t h i s speculation. t e e t h o f f i s h e s o f o t h e r formes, F l u o r i d e i n t h e bone showed a

t e l e o s t s (11) and t h e elasmobranchs (12), size,

The t e e t h o f f i s h e s show a r e m a r k a b l e r a n g e o f a d a p t a t i o n i n f o r m and a s s o c i a t e d w i t h f e e d i n g h a b i t s and p h y s i c a l p r o p e r t i e s o f d i e t . i n these studies

However,

(5,6.8).

t h e r e was no e v i d e n c e t h a t a I n o t h e r words,

particular

c o r r e l a t i o n e x i s t e d between t h e f l u o r i d e c o n c e n t r a t i o n i n t h e enameloid and f o r m and s i z e o f t h e e n a m e l o i d . morphological adaptation o f teeth. Crystallographic investigation indicated that, enameloid, fluoride i s apparently present i n the high fluoride i n the apatite as the evolution o f the c h e m i c a l c o m p o s i t i o n o f f i s h e n a m e l o i d i s c o m p l e t e l y independent o f t h e

(13). C ~ ~ O ( P ~ ~ ) ~ ( Fc. rOCal0(P04)6F2 H)

The f a c t t h a t t h e f l u o r i d e c o n c e n t r a t i o n i n t h e d e n t i n and b o n e o f a l l t h e f i s h e s e x a m i n e d was v e r y l o w , w h e r e a s t h e e n a m e l o i d o f some s p e c i e s contained high fluoride, indicates t h a t the enameloid-forming cells, e s p e c i a l l y t h e ameloblasts. have a p e c u l i a r a b i l i t y t o c o n c e n t r a t e t h e

f l u o r i d e i n t o d e v e l o p i n g enameloid. R e c e n t l y , i t has b e e n f o u n d t h a t i r o n i n t h e e n a m e l o i d shows a s i m i l a r phenomenon, a l t h o u g h t h e r e l a t i o n s h i p b e t w e e n i t s c o n c e n t r a t i o n and f i s h group i s n o t i d e n t i c a l w i t h t h a t o f f l u o r i d e (11, mineralization,

14).
one c o n t a i n i n g h i g h

I n o r d e r t o u n d e r s t a n d t h e r o l e o f f l u o r i d e and i r o n i n e n a m e l o i d d e v e l o p i n g enameloids o f t w o f i s h e s , f l u o r i d e and i r o n (Pagrus m a j o r o f t h e P e r c i f o r m s ) and t h e o t h e r c o n t a i n i n g

Enamel o i d

Dentin

6
5

4
3

1
3
2

291

Bone

0 Marine f i s h

0 Primary freshwater f i s h

Vicarious freshwater f i s h Secondary freshwater f i s h

1
0.9 0.8 0.7 0.6

5
W

1 0.9
0,8

1
0.9

@,8
0.7

0.

0. 0.
0.

@,6

d 0.5
I

0.5
0,4

0 3

0.4
(J,

0.

0.3

0.2

Human enamel \.(surface layer) rubripes. r u b r i pes

i-w
1

0.

0,2

Cyprinus

carpio

0,l

n,iJ

0,l

F i g u r e 4. D i s t r i b u t i o n o f 78 p e r c i f o r m s p e c i e s e x a m i n e d i s p l o t t e d a c c o r d i n g t o t h e f l u o r i d e c o n c e n t r a t i o n s i n t h e e n a m e l o i d , d e n t i n a n d b o n e (5). The f l u o r i d e c o n c e n t r a t i o n s i n human m a t u r e d enamel ( s u r f a c e l a y e r ) ( 4 ) . i n t h e e n a m e l o i d o f C y p r i n u s c a r p i o ( 4 ) and Fugu r u b r i p e s r u b r i p e s (6). and i n fluorapatite are also plotted.

292

v e r y l o w f l u o r i d e and i r o n ( C y p r i n u s c a r p i o o f t h e C y p r i n i f o r m e s ) were e x a m i n e d u s i n g m i c r o r a d i o g r a p h y and e l e c t r o n m i c r o p r o b e a n a l y s i s (7). The p r o g r e s s i v e m i n e r a l i z a t i o n p a t t e r n r e v e a l e d b y m i c r o r a d i o g r a p h y was almost the same i n these two species. The process o f progressive The m i n e r a l i z a t i o n i s c o n s i d e r e d t o be d i v i d e d i n t o a t l e a s t t h r e e s t e p s . formation,

t h i r d step, w h i c h seems t o c o r r e s p o n d t o t h e m a t u r a t i o n s t a g e o f enamel i s composed o f t w o d i f f e r e n t p r o c e s s e s : one w h i c h p r o g r e s s e s and f r o m t h e i n n e r m o s t l a y e r towards t h e m i d d l e l a y e r a t t h e e a r l y stage,

t h e other which progresses from t h e surface l a y e r towards the inner l a y e r

b 5 F :3.89
ES

Dentin

.>
/
7 I .

-.

, . * *

F : OI 2 4
,

F i g u r e 5. B a l i s t o i d e s c o n s p i c i l l u m ( m a r i n e f i s h o f t h e B a l i s t o i d e i o f Tetraodontiformes)(6). a - M i c r o r a d i o g r a m o f g r o u n d s e c t i o n ( x 100) b - L i n e s c a n a n a l y s i s o f CaKcr, MgKa a n d FKcr b y t h e e l e c t r o n m i c r o p r o b e , p e r f o r m e d a l o n g t h e l i n e c r o s s i n g t h e e n a m e l o i d and d e n t i n . Fluoride c o n c e n t r a t i o n i s much h i g h e r i n t h e e n a m e l o i d t h a n i n t h e d e n t i n . Magnesium c o n c e n t r a t i o n i s l o w e r i n t h e e n a m e l o i d t h a n i n t h e d e n t i n .

293

smeloid

Dentin

ES
x

**

F i g u r e 6. T r i o d o n m a c r o p t e r u s ( m a r i n e f i s h o f t h e T e t r a o d o n t o i d e i o f T e t r a o d o n t i f o r m e s ) ( x 120)(6)-a - M i c r o r a d i o g r a m o f g r o u n d s e c t i o n ( x 100) b - L i n e s c a n a n a l y s i s o f CaKcc, MgKa a n d FKa b y t h e e l e c t r o n m i c r o p r o b e , p e r f o r m e d a l o n g t h e l i n e c r o s s i n g t h e m a t u r e d e n a m e l o i d and d e n t i n l a y e r s . F l u o r i d e c o n c e n t r a t i o n i s v e r y l o w i n b o t h t h e e n a m e l o i d and t h e d e n t i n . Magnesium i s l o w e r i n t h e e n a m e l o i d t h a n i n t h e d e n t i n .

294
a t t h e l a t e r stage, iron. and i s a c c o m p a n i e d b y h e a v y d e p o s i t i o n o f f l u o r i d e and i n t h e developing enameloid. are independent.
i t i s considered

S i n c e t h e t i m i n g o f d e p o s i t i o n and d i s t r i b u t i o n p a t t e r n s o f t h e s e

elements are n o t i d e n t i c a l

t h a t t h e i r c o n c e n t r a t i n g mechanisms these two fishes.

No p a r t i c u l a r

d i f f e r e n c e i n t h e p a t t e r n o f p r o g r e s s i v e m i n e r a l i z a t i o n was f o u n d b e t w e e n

I t i s t h u s c o n c l u d e d t h a t f l u o r i d e and i r o n d o n o t p l a y

any s p e c i a l r o l e i n t h e p r o c e s s o f e n a m e l o i d m i n e r a l i z a t i o n . The s i g n i f i c a n c e o f t h e d u a l d e p o s i t i o n o f f l u o r i d e and i r o n i n t h e developing e n a m e l o i d o f some f i s h e s i s n o t u n d e r s t o o d .

A very similar

phenomenon has been p o i n t e d o u t i n t h e m i n e r a l i z e d t i s s u e s o f o t h e r b i o l o g i c a l systems, s u c h a s t h e t e e t h o f r a d u l a o f some m o l l u s k s (15). t h o u g h no r e a s o n a b l e i n t e r p r e t a t i o n has been g i v e n f o r such p e c u l i a r c o m b i n a t i o n s o f f l u o r i d e and i r o n d e p o s i t i o n i n t h e h e a v i l y m i n e r a l i z e d tissues. I n certain epithelial tissues (kidney, digestive glands and hepatopancreas) o f m o l l u s k s , h i g h l y i n s o l u b l e i n t r a c e l l u l a r m i n e r a l s appear
i n a form o f e i t h e r crystallographically pure deposits containing w e l l

o r i e n t e d c r y s t a l s o r o f t e n amorphous m a t e r i a l s . c o n s i s t mainly o f calcium, contain also small aluminum. cadmium, lead, magnesium. iron, zinc,

They h a v e been shown t o such as s i l v e r , There i s no

phosphate and c a r b o n a t e i o n s and copper, and s i l i c o n .

amounts o f v a r i o u s heavy metals,

evidence f o r t h e i r recycling.

Such a b i o m i n e r a l i z a t i o n h a s been s u g g e s t e d

as a c e l l u l a r d e t o x i c a t i o n mechanism f o r c a l c i u m w h i c h i s one o f t h e p h a r m a c o l o g i c a l l y m o s t a c t i v e c a t i o n s and h e a v y m e t a l i o n s (16).

It i s speculated t h a t t h e h i g h l y m i n e r a l i z e d enameloid p r o v i d e s a s i t e
w h e r e e x c e s s a m o u n t s o f f l u o r i d e and i r o n i n t h e b o d y f l u i d s a r e e x c r e t e d i n order t o d e t o x i f y t h e fishes. gland. The a m e l o b l a s t s a c t a l s o a s an e x c r e t i n g The e n a m e l o i d i s c o n s i d e r e d t o be a s u i t a b l e t i s s u e t o f i r m l y f i x d o e s n o t h a v e conFurther-

t h e e l e m e n t s e x c r e t e d , a s c o m p a r e d w i t h t h e d e n t i n a n d bone, b e c a u s e t h e f u l l y mineralized enameloid i s chemically very stable, t a c t with l i v i n g cells, the fish. and i s n o t so p e r m e a b l e t o b o d y f l u i d s . therefore,

more, f i s h t e e t h a r e c o n t i n u o u s l y b e i n g r e p l a c e d t h r o u g h o u t t h e l i f e s p a n o f

It i s considered,

that t h i s peculiar property o f

e n a m e l o i d e n a b l e s t h e f i s h e s t o r e m o v e e x c e s s f l u o r i d e and i r o n c o n t i n u o u s l y i n t o t h e i r s u r r o u n d i n g w a t e r s a s s t a b l e c h e m i c a l compounds w h i c h do F i g u r e 7. H i s t o g r a m s h o w i n g t h e f l u o r i d e c o n c e n t r a t i o n s i n t h e e n a m e l o i d . The f l u o r i d e cond e n t i n and bone o f t h e f i s h e s o f T e t r a o d o n t i f o r m s (6). c e n t r a t i o n o f a l l t h e p r i m i t i v e B a l i s t o i d e i e x a m i n e d was h i g h e r t h a n 2.31%, w h e r e a s t h a t o f t h e a d v a n c e d J e t r a o d o n t o i d e i w a s l e s s t h a n 0.28%. The f l u o r i d e c o n c e n t r a t i o n s i n m a t u r e d and u n e r u p t e d human enamel and i t s t h e o r e t i c a l v a l u e i n t h e f l u o r a p a t i t e a r e a l s o shown.

295
Fluoride concentration (%) Triacanthodidae Triacanthodes nnamalus Triacanthidae Triacanthus breuirost r i s Ballst idae Canthidermis maculatus Balistoides conspicrllum Xanthichthys mento Monacanthdae S tephanolepi s cirrhifer S t ephanoleprs japonicus Rudarius ercodes Nauodon modes f us Alutera monoceros Pseudalutarrus nasicornis Aracandae Kenirocapros aculealus Ostracidae Ost r aci on cubicus Triodontdae Triodon macroplerus Tetraodontdae Fugu rubripes rubripes
Fugu pardale Fugu nrphobles Fugu vermiculare porphyreum Fugu vermrculare uermiculare Lagocephalus lunaris spadiceus Tetraodon hrspidus Canthigaster rivulata Diodontdae Chilomycterus affznrs
0

0.25

Om

"

LI

I I1

w o . 1 9

0.49

a
I

= r"cTJ

Enameloid

Dentin

0 Bone

D i odon ho lac ant hu s

Diodon lrturosus
Diodon h y s t r i r
~ ~

Molidae Mola mola Fluorapatite Human tooth

Very thin enarneloid. could not be analysed

. : ;: ; .

~~

- ....._. _.;_ . * :_. .: . .. : .....-_ : . : . .:_..._._ .......... ..,..,.: .. ..:. ...._ . .....: 11.71
'

296
n o t p o l l u t e t h e i r environment (7)(Table

I).

Thus, t h e c o n c e n t r a t i o n s o f T h i s s p e c u l a t i o n may be sup-

f l u o r i d e and i r o n i n t h e e n a m e l o i d seem t o b e r e l a t e d t o t h e e v o l u t i o n o f t h e s y s t e m i c system t o c o n t r o l body f l u i d s . p o r t e d b y t h e f a c t t h a t t h e f l u o r i d e c o n c e n t r a t i o n s i n t h e d e n t i n a n d bone o f t h e f i s h e s o f B a l i s t o i d e i and P e r c i f o r m e s whose e n a m e l o i d c o n t a i n s h i g h f l u o r i d e a r e s l i g h t l y h i g h e r t h a n t h o s e o f f i s h e s o f T e t r a o d o n t o i d e i whose e n a m e l o i d c o n t a i n s l o w f l u o r i d e (5,6). TABLE

HISTOLOGICAL AND CHEMICAL CHARACTERISTICS OF THE ENAMELOID, DENTIN AND BONE O F F I S H A S A S I T E WHERE E X C E S S FLUORIDE AND I R O N I N BODY F L U I D S A R E EXCRETED. Enameloid M i n e r a l i z a t i o n degree Amount o f o r g a n i c substances Chemical s t a b i l i t y Permeability o f body f l u i d s Resorption by c l a s t i c cells F l u o r i d e and i r o n deposited very high D e n t i n and bone

1ow

+
high

+++
+++
t

+
-

1ow

a r e removed i n t o environment as s t a b l e c h e m i c a l compounds b y a t t r i t i o n and s h e d d i n g of teeth

are released p a r t l y t o body f l u i d s by r e s o r p t i o n and chemical exchange

REFERENCES 1. G r e e n h a l g h R, R i l e y JP ( 1 9 6 3 ) N a t u r e 197:371-372 Wada K. Ogawa M (1976) J D e n t Res 55D:117 Ogawa M (1978) J D e n t Res 57A:28D

2. Suga S, Suga S.
3. Suga S, 4. Wada K,

S u g a S, Wada K. O g a w a M ( 1 9 8 0 ) I n : O m o r i M. W a t a b e N ( e d s ) T h e M e c h a n i s m s o f B i o m i n e r a l i z a t i o n i n A n i m a l s and P l a n t s . T o k a i U n i v P r e s s p p 229-240 T a k i Y, Wada K. Wada K ( 1 9 8 3 ) J a p a n J I c h t h y o l 30:81-93 Ogawa M (1981) J a p a n J I c h t h y o l 28:304-312 Elsevier

5. Suga S, 6. Suga S,

7. Suga S (1984) I n :

F e a r n h e a d RW. Suga S ( e d s ) T o o t h Enamel I V . S c i e n c e P u b l i s h e r s BV Amsterdam, pp 472-477 r i d e M e t a b o l i s m . V e r l a g W i l h e l m M a u d r i c h , Wien, M y e r s GS ( 1 9 5 1 ) S t a n f o r d I c h t h o l B u l l 4 : l l - 2 1 pp 79-88

8. S u g a S, Wada K, O g a w a M ( 1 9 8 1 ) I n : B i n d e r K. H o h e n e g g e r M ( e d s ) F l u o 9.

297

10. T y l e r JC (1980) O s t e o l o g y , Phylogeny, and H i g h e r C l a s s i f i c a t i o n o f t h e F i s h e s o f t h e O r d e r P l e c t o g n a t h i ( T e t r a o d o n t i f o r r n e s ) . US D e p t Commerce N a t O c e a n A t m o s A d m i n N a t M a r F i s h S e r v N O A A T e c h Rep NMFS C i r c 434: 422 11. S u g a S , T a k i Y ( 1 9 8 4 ) J D e n t Res 6 3 : 1 8 5 12. S u g a S ( 1 9 8 4 ) J D e n t Res 6 3 : 5 6 0 13. LeGeros RZ. Suga S (1980) C a l c i f T i s s u e I n t 32:169-174 14. Suga S, Ogawa M (1980) J D e n t Res 59D31891 15. Lowenstam HA (1967) S c i e n c e 156:1373

16. S i r n k i s s K (1977) C a l c i f T i s s Res 24:199-200

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 299-305 0 1986 Elsevier Science Publishers B.V., Amsterdam -Printed in The Netherlands

299

EFFECTS OF FLUORIDE ON DEVELOPING ENAMEL AND DENTIN OF FAT I N C I S O R S TOSHIO ABE,

M I C H I O MASUOKA,

MASAJI

NOMURA, AND

HIROAKI

MIYAJIMA

D r u g S a f e t y E v a l u a t i o n L a b o r a t o r i e s , C e n t r a l Research D i v i s i o n , Takeda C h e m i c a l I n d u s t r i e s , Ltd., 569 6-10-1 H i m u r o - c h o T a k a t s u k i , Osaka, Japan

ABSTRACT The e f f e c t s o f f l u o r i d e on t h e d e v e l o p i n g e n a m e l and d e n t i n o f r a t i n c i sors were investigated. agents, dine-r-5-carboxylate These a n t i - n e o p l a s t i c enamel Rats were t r e a t e d o r a l l y w i t h t h e a n t i - n e o p l a s t i c 4-dioxopyrimiand NaF a t d i f f e r e n t d o s e s f o r e i g h t weeks. and a p p e a r e d t o r e l e a s e induced mottled Histoshowed I n the calcioT e g a f u r a n d TAC-278 T e g a f u r a n d e t h y l t-6-butoxy-5-fluorohexahydro-2, (TAC-278), agents c o n t a i n f l u o r i n e (F)

5-fluorouraci 1 a f t e r t h e treatment. pathologically, ameloblasts,

c h a r a c t e r i z e d by pigment-free

and c h a l k y w h i t e areas.

a t t h e s e c r e t o r y t o m a t u r a t i o n stage, and f o r m e d a c y s t i c i r r e g u l a r a r r a y . Microradiographically,

d e g e n e r a t i o n and n e c r o s i s ,

dentin, t h e r e were f o c a l o r d i f f u s e hypoplastic d e f e c t s r e s u l t i n g from d e g e n e r a t i o n and a t r o p h y o f o d o n t o b l a s t s . t r a u m a t i c l i n e s r e s u l t i n g f r o m damage t o s e c r e t o r y a m e l o b l a s t s , s t a g e w e r e o b s e r v e d i n t h e enamel. and c a l c i o -

t r a u m a t i c zones r e s u l t i n g f r o m damage t o a m e l o b l a s t s a t t h e m a t u r a t i o n T h e s e c h a n g e s w e r e e s s e n t i a T l y t h e same The

as t h e a b n o r m a l i t y r e s u l t i n g f r o m NaF t r e a t m e n t .

concentration i n the

i n c i s o r s w a s h i g h e s t i n t h e NaF t r e a t e d r a t s , treated rats.

a n d l o w e s t i n t h e TAC-278 From t h e s e r e s u l t s , i t

The s e v e r i t y o f g r o s s l e s i o n o f t h e i n c i s o r s and t h e F l i k e NaF, a n e x c e s s i v e d o s e o f T e g a f u r o r TAC-278 a f -

c o n c e n t r a t i o n i n t h e i n c i s o r s were w e l l correlated. i s suggested that,

f e c t s t h e d e v e l o p i n g e n a m e l and d e n t i n o f r a t i n c i s o r s .

INTRODUCTION The e f f e c t s o f f l u o r i n e ( F ) on d e v e l o p i n g enamel and d e n t i n has been r e p o r t e d (1-7). The t o x i c e f f e c t s o f e x c e s s i v e f l u o r i d e e x p o s u r e on t o o t h The s e v e r i t y o f f o r m a t i o n i s c a l l e d f l u o r o s i s o r m o t t l e d enamel (8-10). ( 1 1,12). M o t t l e d enamel, c i f i e d areas, c h a r a c t e r i z e d by pigment-free, c h a l k y w h i t e and h y p o c a l was o b s e r v e d i n t h e i n c i s o r s o f r a t s t r e a t e d w i t h t h e a n t i -

f o r m a t i v e d e f e c t s i s d i r e c t l y r e l a t e d t o t h e amount o f f l u o r i d e i n g e s t e d

300

n e o p l a s t i c agents,

T e g a f u r a n d e t h y l t-6-butoxy-5-fluorohexahydro-2. a f t e r t h e treatment.

4-

dioxopyrimidine-r-5-carboxylate (TAC-278).
appeared t o r e l e a s e 5 - f l u o r o u r a c i l I n t h i s study, concentration. series.

These d r u g s c o n t a i n F and

these [mottled t e e t h were examined u s i n g h i s t o l o g i c

methods, c o n t a c t m i c r o r a d i o g r a p h y , and an a n a l y t i c method f o r i n c i s a l F The s t u d y was c a r r i e d o u t as t w o i n d e p e n d e n t e x p e r i m e n t a l I n t h e second e x p e r i m e n t , I n t h e f i r s t e x p e r i m e n t , t h e h i s t o p a t h o l o g i c a l and m i c r o r a d i o -

g r a p h i c changes o f t h e i n c i s o r s were s t u d i e d . was s t u d i e d . MATERIALS AND METHODS

t h e r e l a t i o n o f t h e F c o n c e n t r a t i o n t o t h e s e v e r i t y o f t h e i n c i s o r damage

To s t u d y t h e h i s t o p a t h o l o g i c a l and m i c r o r a d i o g r a p h i c changes o f t h e incisors. four-week-old 30 f e m a l e W i s t a r r a t s w e r e t r e a t e d o r a l l y w i t h 200 When t h e m g / k g / d a y o f T e g a f u r o r 500 m g / k g / d a y o f TAC-278 f o r e i g h t weeks. 10% n e u t r a l b u f f e r e d f o r m a l i n t h r o u g h t h e a s c e n d i n g a o r t a . o e r i o d o n t a l t i s s u e , d e m i n e r a l i z e d w i t h 2 Na-EDTA, B e r l i n b l u e , P A S a n d azan.

d o s i n g p e r i o d was c o m p l e t e d , e a c h a n i m a l was a n e s t h e t i z e d and p e r f u s e d w i t h One i n c i s o r f r o m e a c h a n i m a l was r e m o v e d w i t h t h e s h e a t h o f a l v e o l a r bone and s o f t embedded i n p a r a f f i n , s e c t i o n e d s a g i t t a l l y a t 5 Dm, and s t a i n e d w i t h h e m a t o x y l i n and e o s i n (H-E),

A s e c o n d i n c i s o r was r e m o v e d and embedded i n

Four microradiographs o f

o o l y e s t e r r e s i n ( R i g o l a c B ) a f t e r e t h a n o l d e h y d r a t i o n and s a g i t t a l s e c t i o n s , 5 0 t o 7 5 um t h i c k , w e r e p r e p a r e d b y g r i n d i n g . c o n t a c t microradiographic apparatus. ences i n m i n e r a l content. undemineralized s e c t i o n were prepared a t d i f f e r e n t exposure t i m e s i n a The e x p o s u r e f a c t o r s w e r e s e l e c t e d e m p i r i c a l l y t o p r o d u c e maximum c o n t r a s t b e t w e e n a r e a s w i t h s m a l l d i f f e r The m i c r o r a d i o g r a p h s w e r e p r e p a r e d o n Kodak Developer. S p e c t r o s c o p i c F i l m 649-0 and d e v e l o p e d i n a Kodak X - r a y i n c i s o r damage, 20 t h r e e - w e e k - o l d

To s t u d y t h e r e l a t i o n s h i p b e t w e e n F c o n c e n t r a t i o n s and s e v e r i t y o f t h e female W i s t a r r a t s were d i v i d e d e q u a l l y Anii n t o f o u r groups: NaF, t h e c o n t r o l g r o u p and t h r e e e x p e r i m e n t a l g r o u p s .

m a l s o f t h e e x p e r i m e n t a l g r o u p s w e r e t r e a t e d o r a l l y w i t h 21 m g / k g / d a y 100 mg/kg/day o f Tegafur, T h e s e d o s e s w e r e e q u i v a l e n t t o 9.5 v g F / k g / d a y . added f l u o r i d e . (13). Student's t - t e s t

of

o r 1 4 0 m g / k g / d a y o f TAC-278 f o r e i g h t weeks.
The c o n t r o l s r e c e i v e d no

When t h e d o s i n g p e r i o d was c o m p l e t e d ,

F concentration i n

t h e l o w e r i n c i s o r s was d e t e r m i n e d b y t h e L a n t h a n - A l i z a r i n C o m p l e x o n m e t h o d was a p p l i e d t o e x a m i ne t h e s i g n i f i c a n c e o f d i f f e r e n c e

b e t w e e n t h e means o f measurements.

301
I?tSU LTS

A t t h e end o f t h e f i r s t e x p e r i m e n t a l p e r i o d ,
r a t s t r e a t e d w i t h T e g a f u r o r TAC-278. f r e e and c h a l k y w h i t e c o l o r . s o r s and i n T e g a f u r - t r e a t e d rats.

u p p e r and l o w e r i n c i s o r s o f

t h e c o n t r o l r a t s showed a d i s t i n c t , o r a n g e p i g m e n t a t i o n . The i n c i s o r s o f on t h e o t h e r hand, showed p i g m e n t r a t s showed These c h a n g e s w e r e m a r k e d i n t h e l o w e r i n c i Some o f t h e T e g a f u r - t r e a t e d

a l m o s t no i n c i s o r p i g m e n t a t i o n . B e f o r e e x p l a i n i n g t h e h i s t o p a t h o l o g i c a l f i n d i n g s o f t h e i n c i s o r s , we w i l l b r i e f l y describe t h e morphogenesis o f r a t incisors. calcify, Rat i n c i s o r s grow, and and e r u p t c o n t i n u o u s l y t h r o u g h o u t t h e l i f e o f t h e a n i m a l ,

t h e r e f o r e each t o o t h shows t h e c o m p l e t e l i f e c y c l e o f t o o t h development f r o m i n c e p t i o n t o m a t u r i t y (10). o f r a t i n c i s o r s (2,4,10,14), sors i n t o f o u r stages: maturation. I n t h e enamel o r g a n o f r a t s t r e a t e d w i t h T e g a f u r o r TAC-278, a t t h e t h e s e c r e t o r y t o m a t u r a t i o n s t a g e w e r e damaged. e r a t i o n and n e c r o s i s , t h e a r e a o f c y s t i c d e g e n e r a t i o n o f t h e enamel organ, ameloblasts 1-3). In They showed degend e f e c t i v e enamel AmeloBased o n t h e m o r p h o l o g i c a l c l a s s i f i c a t i o n m a t r i x formation, transition, and we d i v i d e d t h e d e v e l o p i n g e n a m e l i n r a t i n c i proliferation,

and f o r m e d a c y s t i c i r r e g u l a r a r r a y ( F i g s .

m a t r i x a n d d e p o s i t i o n o f amorphous o r g a n i c m a t e r i a l s w e r e o b s e r v e d . a l i t t l e i r o n p i g m e n t (Fig. g r a n u l a r shape. ameloblasts.

b l a s t s a t t h e l a t e m a t u r a t i o n s t a g e w e r e i r r e g u l a r l y a r r a y e d and c o n t a i n e d

4).

T h i s pigment e x h i b i t e d a g l o b u l a r o r round
: : 1 7 s

No a b n o r m a l i t y

o/>serv?:I

i , 1 .;;A,.

' . , - " l i f e r a t i o n stage o f

A section from the posterior t h i r d o f a lower i n c i s o r o f a r a t F i g u r e 1. A c y s t i c degeneration t r e a t e d w i t h 500 m g / k g o f TAC-278 f o r e i g h t weeks. o f t h e e n a m e l o r g a n ( 0 ) a n d a f o c a l h y p o p l a s t i c d e f e c t o f t h e d e n t i n (v) a r e observed. An a r r o w i n d i c a t e s t h e d i r e c t i o n o f i n c i s o r e r u p t i o n . H-E s t a i n , x 85.
F i g u r e 2. A section from the posterior t h i r d o f a lower incisor o f a r a t t r e a t e d w i t h 200 mg/kg o f T e g a f u r f o r e i g h t weeks. Cystic degeneration o f t h e e n a m e l o r g a n and a b n o r m a l e n a m e l m a t r i x a r e o b s e r v e d . An a r r o w i n d i H--E s t a i n , x 170. cates t h e d i r e c t i o n o f i n c i s o r eruption.

ZOE:

303
I n t h e m i c r o r a d i o g r a p h s o f i n c i s o r s o f r a t s t r e a t e d w i t h T e g a f u r o r TAC278, calcio-traumatic l i n e s c o n s i s t i n g o f b o t h h y p e r - and h y p o m i n e r a l i z e d T h i s p a t t e r n was e v i d e n c e d b y a r a d i o p a q u e C a l c i o - t r a u m a t i c Tones 6). The s u r f a c e l a y e r c o m p o n e n t s c r o s s e d t h e enamel f r o m t h e d e n t i n o e n a m e l j u n c t i o n t o t h e s u r f a c e o f t h e e n a m e l ( F i g . 5). zone f o l l o w e d b y a zone o f d i s t i n c t r a d i o l u c e n c y . were a l s o observed i n t h e enamel o f t h e s e r a t s (Fig. f o c a l l y h y p o m i n e r a l i z e d e n a m e l b e n e a t h it. (Fig. 7). Fluorine and

o f t h e enamel was h y p e r m i n e r a l i z e d o n l y w i t h r e s p e c t t o t h e r e l a t i v e l y I n a d d i t i o n t o these lesions, t h e p r e s e n c e o f c a l c i f i e d b o d i e s p e r i p h e r a l t o t h e d e f e c t s was o b s e r v e d The r e s u l t s o f t h e second e x p e r i m e n t a r e g i v e n i n T a b l e I. t h e l o w e s t i;; c .': i n t h e iilci;c.:"s
-[.!. $:,-!/?
-'-

c o n c e n t r a t i o n i n t h e i n c i s o r s was t h e h i g h e s t i n t h e NaF t r e a t e d r a t s ,

ti-ezL::l

rr?'is.

The s e v z r i t y o f t h e d i s c o l o r a t i o n

'i7s

c2rr:ilzt>d

t i i t ?

t ! iF ~ 1 ~ 0 ~ i i l -c72 n c e n t r a t i o n .

F i g u r e 7. A m i c r o r a d i o g r a p h o f a l o w e r i n c i s o r f r o m a r a t t r e a t e d w i t h 500 mg/kg o f TAC-278 f o r e i g h t weeks. C a l c i f i e d bodies are located outside o f t h e d e f e c t enamel (E). x 85. TABLE I

F CONCENTRATION I N THE I N C I S O R S AND DISCOLORATION OF THE I N C I S O R S

Mol e c u 1a r Weight
--

F concentration
(ppm) 60.0 f 1 3 . 3 a 604.6 f 37.gb 453.2
f 57.gb

Compound

D iscolorationc

Control NaF Tegafur TAC-278

42 200 276

146.6 f 1 7 . Z b

++ +++ ++ +

a V a l u e s a r e mean f SD. b S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l , p<O.OI. no change; +, s l i g h t ; 'Gradings o f d i s c o l o r a t i o n : ++, m o d e r a t e ; +++, s e v e r e

-.

304
DISCUSSION
The e f f e c t s o f F on r a t i n c i s o r s h a v e been r e p o r t e d b y many w o r k e r s u s i n g d i f f e r e n t t e c h n i q u e s (1-7). follows. T h e i r f i n d i n g s c o u l d be s u m m a r i z e d as areas severe demonChronic A c u t e f l u o r o s i s p r o d u c e s h o r i z o n t a l bands o f p i g m e n t - f r e e

i n r a t i n c i s o r enamel c o r r e s p o n d l n g t o t h e t i m i n g o f i n j e c t i o n s . f l u o r o s i s i n t h e r a t i n c i s o r enamel v a r i e s f r o m l o s s o f p i g m e n t t o enamel hypoplasia. The a m e l o b l a s t s a r e v e r y s e n s i t i v e t o f l u o r i d e , s t r a t e d by e a r l y reduction i n height. b o d i e s a r e found.

Then f o l l o w s p r o l i f e r a t i o n o f t h e

enamel o r g a n l e a d i n g t o f o r m a t i o n o f c y s t s i n w h i c h amorphous c a l c i f i e d Microradiographic studies o f fluorosed r a t incisor As a r e s u l t o f h i g h d o s a g e s o f f l u o r i d e , striation, four enairiel h a v e shown a h y p e r m i n e r a l i z e d s u r f a c e l a y e r s i m i l a r t o t h a t f o u n d i n human f l u o r o s e d t e e t h . a b n o r m a l i t i e s may be seen i n t h e d e n t i n : h y p o i n i n e r a l i z e d i n t e r g l o b u l a r spaces, o u t l i n e o f t h e dentin. The h i s t o p a t h o l o g i c a l and m i c r o r a d i o g r a p h i c f i n d i n g s i n t h e p r e s e n t s t u d y o f r a t s . t r e a t e d w i t h T e g a f u r o r TAC-278 w e r e e s s e n t i a l l y t h e same as t h o s e observed i n f l u o r o s i s o f r a t i n c i s o r s . C y s t i c d e g e n e r a t i o n o f e n a m e l o r g a n and a b n o r m a l e n a m e l m a t r i x f o r m a t i o n w e r e o b s e r v e d i n t h e i n c i s o r s o f r a t s t r e a t e d w i t h T e g a f u r o r TAC-278. T h e s e c h a n g e s e x h i b i t e d t h e damage and p r o l i f e r a t i o n o f a m e l o b l a s t . surrounding ameloblasts continued t o form normal matrix. b l a s t s a t t h e l a t e m a t u r a t i o n stage were i r r e g u l a r l y arrayed, a l i t t l e i r o n pigment. o f pigment-free enamel. c a l c i o - t r a u m a t i c l e s i o n s were observed i n the These c a l c i o - t r a u m a t i c zone was a l s o These and l e s i o n s d e v e l o p e d when l i m i t e d r e g i o n s o f a m e l o b l a s t a t r o p h i e d o r d i e d . a r e a s o f a m e l o b l a s t e p i t h e l i u m became s u r r o u n d e d b y e n a m e l m a t r i x . hypoplastic defects,

and g r o s s d e f o r m a t i o n o f t h e e x t e r n a l

I n t h i s way, Ameloand c o n t a i n e d

These c h a n g e s w e r e c o n s i d e r e d t o be r e l a t e d t o t h a t

Microradiographically,

e n a m e l o f r a t s t r e a t e d w i t h T e g a f u r o r TAC-278. t h e m a t r i x f o r m a t i o n s t a g e (4.5).

l i n e s i n t h e e n a m e l w e r e c o n s i d e r e d t o r e s u l t f r o m damage t o a m e l o b l a s t s a t The c a l c i o - t r a u m a t i c studies, c o n s i d e r e d t o r e s u l t f r o m damage t o a m e l o b l a s t s a t t h e m a t u r a t i o n s t a g e

(4.5).

Based on l i g h t and e l e c t r o n m i c r o s c o p i c a l

i t was assumed

t h a t t h e h y p o m i n e r a l i z e d zone i n d i c a t e d a d i s t u r b e d f u n c t i o n o f t h e s e c r e t o r y a m e l o b l a s t s d u e t o f l u o r i d e (11,15). Concerning t h e pathogenesis o f t h e enamel l e s i o n s i n t h e p r e s e n t study, h i s t o p a t h o l o g i c a l f i n d i n g s and m i c r o r a d i o g r a p h i c f i n d i n g s w e r e w e l l c o r r e lated.

305
Hypoplastic d e f e c t s i n t h e d e n t i n were observed i n r a t s t r e a t e d w i t h T e g a f u r o r TAC-278.
excessive
o f t h e e n a m e l (8.10).

These changes w e r e c o n s i d e r e d t o be t h e r e s u l t o f an

dosage o f F because t h e d e n t i n w a s n o t a s s e n s i t i v e t o F a s t h a t Tegafur, o r TAC-278, each con-

A l t h o u g h t h e r a t s w e r e t r e a t e d w i t h NaF. taining equivalent molecular weight o f F, TAC-278 t r e a t m e n t .

the F concentration i n the inci-

s o r s was f o u n d t o be t h e h i g h e s t a f t e r NaF t r e a t m e n t and t h e l o w e s t a f t e r These d i f f e r e n c e s w e r e c o n s i d e r e d t o be r e l a t e d t o t h e S e v e r i t y o f g r o s s l e s i o n of t h e i n c i The r a t e o f m e t a b o l i s m o f t h e compound.

s o r s and t h e F c o n c e n t r a t i o n i n t h e i n c i s o r s w e r e w e l l c o r r e l a t e d . (11,12). From t h e s e r e s u l t s , sors. REFERENCES 1. Schour I , S m i t h MC (1934) P r o c SOC Exp B i o l Med 3 2 : l - 2
i t i s suggested t h a t ,

e x t e n t o f damage i s d i r e c t l y r e l a t e d t o t h e amount o f f l u o r i d e i n g e s t e d l i k e NaF. an e x c e s s i v e dose o f

T e g a f u r o r TAC-278 a f f e c t s t h e d e v e l o p i n g e n a m e l a n d d e n t i n o f r a t i n c i -

2. O t a k e T (1960) O d o n t o l o g y 48:l-54
3. Yaeger JA (1966) Am J A n a t 118:665-684
4. I s h i d a T (1970) J a p J O r a l B i o l 12:88-107

5. Hasegawa K (1971) J a p J O r a l B i o l 13:239-254

6.
I j u i n N (1971) J a p J O r a l B i o l 13:39-61
7. S h i n o d a H (1975) C a l c i f T i s s Res 18:91-100

8. P i n d b o r g J J ( 1 9 7 0 ) P a t h o l o g y o f t h e D e n t a l H a r d T i s s u e s . M u n k s g a a r d , Copenhagen. p 162 9. N i k i f o r u k G. G r a i n g e r RM (1964) I n : B e a t o n GH. McHenry EW (eds) N u t r i t i o n - A Comprehensive Treatise. Academic Press, New York, pp 417-461 10. S c h o u r I , M a s s l e r M ( 1 9 4 9 ) I n : F a r r i s EJ, G r i f f i t h JQ ( e d s ) The R a t i n L a b o r a t o r y I n v e s t i g a t i o n . J B L i p p i n c o t t Company, London, pp 104-165 11. K r u g e r BJ (1970) A r c h O r a l B i o l 15:109-114 12. Angmar-Mansson

B,

E r i c s s o n Y,

Ekberg, 0 (1976) C a l c i f T i s s Res 22:77-84

13. P h a r m a c e u t i c a l S o c i e t y o f J a p a n (1980). K i n b a r a P u b l i c a t i o n , Tokyo, P 60 14. Warshawsky H, S m i t h CE (1974) A n a t Rec 179:423-446 15. W a l t o n RE, Eisenmann.

DR (1974) A r c h O r a l B i o l 19:171-182

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 307-312 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

307

EFFECTS OF LONG TERM,

LOW DOSE INGESTION

OF

FLUORIDE ON THE THYROID GLAND

I N RATS

MITSURU
TANAKA,

TSUCHIDA~,

ISAO

O K A Y A S U ~YUHKO K O H Y A M A ~ . HIROKO

KURIHARA~,

HEIZO

FUMIYOSHI Y A N A G I S A W A ,

C H I G U S A DATE3, MASAYUKI HAYASH13. K A Y O

MU13, AND MASAHIRO ASADA3

D e p a r t m e n t o f E p i d e m i o l o g y , M e d i c a l R e s e a r c h I n s t i t u t e . T o k y o M e d i c a l and D e n t a l U n i v e r s i t y , No. 3-10, 2-Chome, K a n d a - S u r u g a d a i C h i y o d a - k u , T o k y o 101, J a p a n Pathology Center, U n i v e r s i t y H o s p i t a l . and D e n t a l U n i v e r s i t y 3Department o f P u b l i c Health, ABSTRACT Four-week-old c o n t a i n i n g 0, 3. W i s t a r m a l e r a t s w e r e g i v e n d i s t i l l e d w a t e r ad l i b i t u m School o f Medicine. Tokyo M e d i c a l

Osaka C i t y U n i v e r s i t y M e d i c a l S c h o o l

6,

12,

a n d 2 5 ppm F- a s NaF.

A t t h e end o f 20 months,

m o r p h o l o g i c a l changes and e n d o c r i n o l o g i c a l d a t a on t h e t h y r o i d g l a n d i n these animals were studied. H y p e r p l a s t i c nodules c o n s i s t i n g o f t h y r o i d p a r a f o l l i c u l a r c e l l s (C c e l l s ) w e r e o b s e r v e d i n t h e e x p e r i m e n t a l a n i m a l s r e c e i v i n g F-. these hyperplastic cells. m o r p h o l o g i c a l changes. highly significant. INTRODUCTION Many e p i d e m i o l o g i c a l s t u d i e s (1,2) and e x p e r i m e n t a l s t u d i e s (3.4) on t h e e f f e c t s o f f l u o r i d e on t h e t h y r o i d h a v e been r e p o r t e d . consensus o f o p i n i o n on t h e i r r e l a t i o n s h i p . However t h e r e i s n o Both c a l c i t o n i n and c h a r a c t e r i s t i c s e c r e t o r y g r a n u l e s w e r e d e t e c t e d i n t h e c y t o p l a s m s o f The s t r i k i n g i n c r e a s e o f s e r u m c a l c i t o n i n l e v e l The t h y r o i d f o l l i c l e s o f t h e e x p e r i m e n t a l a n i m a l s although t h e d i f f e r e n c e s were n o t seen i n r a t s w i t h h y p e r p l a s i a o f p a r a f o l l i c u l a r c e l l s s u b s t a n t i a t e d t h e were l a r g e r than those o f t h e controls,

Although there i s a r e p o r t

i n d i c a t i n g t h a t g o i t e r w o u l d b e i n d u c e d b y f l u o r i d e and a c c o m p a n y i n g i n s u f f i c i e n t i o d i n e i n t a k e ( 5 ) , i t h a s b e e n c o n c l u d e d b y WHO t h a t n o s p e c i f i c t o x i c i t i e s o f f l u o r i d e on t h e t h y r o i d e x i s t (6). T h i s e x p e r i m e n t a l w o r k was done t o s t u d y t h e e f f e c t o f a l o n g - t e r m , dose i n g e s t i o n o f sodium f l u o r i d e on t h e t h y r o i d g l a n d i n r a t s . s e r u m and e n d o c r i n o l o g i c a l o b s e r v a t i o n s .

low

Emphasis

was p l a c e d on t h e m o r p h o l o g i c a l changes o f t h e t h y r o i d g l a n d as w e l l as

308
MATERIALS AND METHODS Four-week-old group) and g i v e n 12, were s a c r i f i c e d . W i s t a r male r a t s were d i v i d e d i n t o f i v e groups respectively. (8-11 rats/ d i s t i l l e d d r i n k i n g w a t e r ad l i b i t u m c o n t a i n i n g 0. 3,

6,

and 2 5 ppm F- (NaF),

A t t h e end o f 20 m o n t h s t h e a n i m a l s
Hematoxylin-eosin F o r measurement of were U s i n g a MOP

H i s t o l o g i c s e c t i o n s were obtained l o n g i t u d i n a l l y from t h e

middle portions o f b i l a t e r a l lobes o f the thyroid. s t a i n i n g was done f o r r o u t i n e h i s t o l o g i c o b s e r v a t i o n s . t h e s i z e s o f t h y r o i d f o l l i c l e s on t h e c u t surface, Videoplan. section,

microphotographs

taken f r o m t h e m i d d l e p o r t i o n o f each l o b e o f t h e t h y r o i d . a t random i n each group. m e t h o d (7). Serum c a l c i t o n i n , t h y r o i d s t i m u l a t i n g h o r m o n e (TSH),

t h e a r e a s and d i a m e t e r s o f 232 t h y r o i d f o l l i c l e s w e r e measured I n order t o detect calcitonin i n the tissue d e p a r a f f i n i r e d sections were stained by t h e immunoperoxidase t h y r o x i n e (T4). and

t r i i o d o t h y r o n i n e (T3) w e r e t e s t e d b y t h e s t a n d a r d r a d i o i m m u n o a s s a y method. RESULTS The t h y r o i d f o l l i c l e s o f t h e e x p e r i m e n t a l a n i m a l s w e r e l a r g e r t h a n t h o s e i n t h e c o n t r o l group, i z e d i n T a b l e I. a s s h o w n i n F i g u r e s 1 a n d 2. Their follicular e p i t h e l i a were a l s o flattened. The d a t a on t h y r o i d f o l l i c l e s a r e summar-

The mean v a l u e s o f b o t h t h e a r e a s a n d d i a m e t e r s o f

t h y r o i d f o l l i c l e s o f t h e e x p e r i m e n t a l g r o u p s w e r e much g r e a t e r t h a n t h o s e o f t h e c o n t r o l group. High incidence o f h y p e r p l a s t i c nodules c o n s i s t i n g o f p a r a f o l l i c u l a r c e l l s

( C c e l l s ) was o b s e r v e d i n t h e e x p e r i m e n t a l a n i m a l s ,

w h i l e no p r o l i f e r a t i o n

o f p a r a f o l l i c u l a r c e l l s as a mass was seen i n t h e c o n t r o l g r o u p ( T a b l e 11). Where m e d u l l a r y p r o l i f e r a t i o n o f p a r a f o l l i c u l a r c e l l s f o r m e d a mass i t . was d i a g n o s e d as a h y p e r p l a s t i c nodule. M u l t i p l e h y p e r p l a s t i c nodules were o c c a s i o n a l l y seen i n a s i n g l e t h y r o i d . normal p a r a f o l l i c u l a r c e l l s i n t h e t h y r o i d , dase method (Fig. 3). i s t i c s e c r e t o r y granules,

I n addition,

c a l c i t o n i n was

d e t e c t e d a s a d a r k d e p o s i t i o n i n t h e c y t o p l a s m o f p r o l i f e r a t i n g as w e l l as a s shown b y t h e i m m u n o p e r o x i and p o l y Electron microscopic observation revealed characterabundant rough endoplasmic r e t i c u l u m s ,

ribosomes i n t h e cytoplasms o f h y p e r p l a s t i c p a r a f o l l i c u l a r c e l l s . As s h o w n i n T a b l e 1 1 1 , s e r u m c a l c i t o n i n l e v e l s w e r e e n h a n c e d i n t h e F t r e a t e d groups. I n p a r t i c u l a r , t h e g r o u p r e c e i v i n g 2 5 ppm F- s h o w e d a Furthermore, i n most cases t h e s e r u m c a l c i t o n i n l e v e l was v a l u e s i x t i m e s h i g h e r t h a n t h e c o n t r o l group. where h y p e r p l a s t i c nodules were observed, a l s o enhanced. The serum T j v a l u e s o f t h e e x p e r i m e n t a l g r o u p s t e n d e d t o be h i g h e r t h a n

309

F i g u r e 1. H i s t o l o g y o f t h e t h y r o i d i n t h e c o n t r o l group. The f o l l i c l e s o f t h e t h y r o i d a r e u n i f o r m l y s m a l l i n s i z e . The p a r e n chymatous p a r a t h y r o i d g l a n d can be seen ir! t h 2 u p p e r l e f t p o r t i o n . 'Iir>stGxylin-eosin s'iiin. T'2 x.

H i s t o l o g y o f t h e t h y r o i d i n t h e g r o u p r e c e i v i n g 2 5 ppm F-. F i g u r e 2. The f o l l i c u l a r The f o l l i c l e s o f t h e t h y r o i d a r e l a r g e and v a r y i n s i z e . iIc!.i;.toxylin-eosin stain. 200 x. e p i t h e l i a (2t-2 r a t i i c r i'1at:;c;icd.

D e t e c t i o n o f c a l c i t o n i n i n h y p e r p l a s t i c p a r a f o l l i c u l a r c e l l s by F i g u r e 3. the immunoperoxidase technique. N o r m a l p a r a f o l l i c u l a r c e l l s a l s o show p o s i t i v e r e a c t i o n i n t h e surrounding t h y r o i d tissue. 400 x.

310 TABLE I S I Z E OF FOLLICLES I N THE T H Y R O I D GLAND Treatmen t Control F-, Area (urn2) Diameter (wn)

1999.1 5132.6 4545.0'' 4810.0 4737.8

1828.3l 4850.8 4070.8

46.7 73.1 70.25e 69.7 70.4

t
f

19.0 34.6 29.5 35.6 32.8

3 ppm

t
f

6 ppm
12 ppm 25 ppm
1L

f
f

5297.1 i 4432.4
i

" S i g n i f i c a n t l y d i f f e r e n t (piO.05) ' v a l u e s a r e means S.D.

f r o m t h e c o n t r o l group

TABLE I 1

I N C I D E N C E OF HYPERPLASTIC NODULE OF PARAFOLLICULAR CELLS I N THE THYROID GLAND

F l u o r i n e (F-), ppm

6
3/9

12 7/10

25 7/10

H y p e r p l a s t i c nodule' 'No.

0/8

3/11

o f case t h a t had h y p e r p l a s t i c nodules i n t h e t h y r o i d / t o t a l

TABLE 111 HORMONES I N SERUM

(F1

f SD)

Control

50.8 k 41.6 120.6 182.5

f

34.1 47.6 50.5

f f

10.8 24.8 17.8"

2.2
1.6

f
f

0.2

15.5 29.8 18.7 24.0 30.0

4.9 f 0 . 8 6 . 0 f 1.0" 6. o. 7Qit 6.9 k 1.2'"' 4.4 f 0.6

F-,

3 ppm
6ppm

58.9* 102.8"

0.7

80.3f30.2
f

50.4f12.3
f

2.7f0.6" 2.1 f 0 . 7 2.3

f

1 2 ppm 25 ppm

319.1 f 209.2'ii:

62.0 f 25.2"

0.6

J ,.t z - S i g n i f i c a n t l y d i f f e r e n t (~~0.05) from t h e c o n t r o l s S i g n i f i c a n t l y d i f f e r e n t (p<O.Ol) f r o m t h e c o n t r o l s

311
t h e c o n t r o l s a l t h o u g h no a p p r e c i a b l e d i f f e r e n c e s seen. i n s e r u m T4 v a l u e s w e r e

S e r u m TSH v a l u e s w e r e h i g h e r i n a n i m a l s r e c e i v i n g 3.

6. a n d 1 2 ppm

F- t h a n i n t h e c o n t r o l s .
No h i s t o l o g i c a l d i f f e r e n c e s w e r e f o u n d b e t w e e n t h e e x p e r i m e n t a l a n d c o n t r o l groups. a n i ma1 s s t u d i e d . Parathyroid glands were parenchymatous i n a l l o f t h e

DISCUSSION
Histological changes o f t h y r o i d glands, h y p e r p l a s t i c nodules c o n s i s t i n g were

o f p a r a f o l l i c u l a r c e l l s , c o n f i r m e d b y t h e i m m u n o p e r o x i d a s e method.
f o u n d i n a n i m a l s r e c e i v i n g F-. i n t h e cytoplasm, (8,9).

E l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s showed

c h a r a c t e r i s t i c s e c r e t o r y g r a n u l e s and a b u n d a n t r o u g h e n d o p l a s m i c r e t i c u l u m s s u g g e s t i n g enhanced a c t i v i t y o f t h e s e h y p e r p l a s t i c c e l l s observations The r e s u l t s o f t h e h i s t o I n c r e a s e d serum c a l c i t o n i n v a l u e s a l s o s u b s t a n t i a t e d

on h y p e r p l a s t i c nodules o f p a r a f o l l i c u l a r c e l l s . studies. These r e s u l t s r e v e a l e d t h a t l o n g - t e r m

l o g i c a l i n v e s t i g a t i o n agree w e l l w i t h r e s u l t s o b t a i n e d f r o m b i o c h e m i c a l i n g e s t i o n o f F- b y t h e r a t i n d u c e d

h y p e r p l a s t i c nodules i n t h e t h y r o i d gland. a l t e r a t i o n s o f c a l c i u m i n bones ( 1 0 , l l ) m i g h t have on t h y r o i d function.

It i s n o t known w h e t h e r t h e

changes were caused d i r e c t l y by f l u o r i d e o r i n d i r e c t l y t h r o u g h m e t a b o l i c

o r i n t h e k i d n e y s (12,13).

I t i s s t i l l c o n t r o v e r s i a l a s t o w h a t e f f e c t s l o n g - t e r m i n g e s t i o n o f FThe h i s t o l o g i c a l changes o f t h e t h y r o i d T4 a n d TSH v a l u e s d o n o t i n c l u d i n g t h e appearance o f l a r g e t h y r o i d f o l l i c l e s l i n e d w i t h r a t h e r f l a t t e n e d e p i t h e l i a , a n d h i g h o r l o w s e r u m T3, suggest any u n i f o r m , Nevertheless, T3 v a l u e s , f u n c t i o n a l s t a t e o f t h e t h y r o i d gland. The s e r u m T 4 and

t h e h i s t o l o g i c c h a n g e s d e s c r i b e d h e r e may s u g g e s t a s l i g h t do n o t s u p p o r t h y p o f u n c t i o n o f t h e t h y r o i d g l a n d sug-

h y p o f u n c t i o n o f t h e t h y r o i d g l a n d i n r a t s r e c e i v i n g F-. however, gested by t h e h i s t o l o g i c a l f i n d i n g s .

detailed examination o f thyroid

f u n c t i o n i n c o n j u n c t i o n w i t h f l u o r i d e a d m i n i s t r a t i o n i s t h u s necessary. REFERENCES 1. S i d d i q u i AH (1955) B r i t Med J 11:1408-1413

2. S i n g h

A,

J o l l y SS,

B a n s a l BC,

M a t h o u r CC (1963) M e d i c i n e 42:229-246

3. H a r r i s NO, 4. G a l l e t t i PM, 5. Day TK, Geneva

Hayes RL (1955) J D e n t R e s e a r c h 34:470-477 J o y e t G (1958) J C l i n E n d o c l i n o l 18:1102-1110

Powell-Jackson

PR (1972) L a n c e t (May 27):1135-1138

6. WHO ( 1 9 7 0 )

F l u o r i d e a n d Human H e a l t h .

WHO M o n o g r a p h S e r i e s NO. 59,

312
7. Hsu SM, R a i n e L, F a n g e r H (1981) J. H i s t o c h e m , Cytochem 29:577-580 Nunnemacher G. W o l f e HJ (1977) Lab I n v e s t 36:237-248 Endocrine Pathology, W i l l i a m s - W i l k i n s Co.

8. De L e l l i s RA.

9. B l o o d w o r t h JMB (ed) (1982) B a l t i m o r e and London

10. Sundstbrm B (1972) A c t a P a t h M i c r o b i o l Scand Sec A 80:17-20

11. I s h i d a K (1981) O r a l H y g i e n e 31:2-16 12. K o u r K,

S i n g h J (1980) F l u o r i d e 13:163-167 H e a t h H (1981) N E n g l J Med 304:269-278 13. A u s t i n LA,

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 313-324 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

313

EXPERIMENTAL

STUDIES

ON

THE

EFFECTS O F

FLUORIDATION O N

RATS

WITH

EXPERIMENTALLY INDUCED GLOi~lERULONEPHRITIS TAKUMI SATO. KAZUSATA YOSHITAKE, MASATO I I D A . AND TSUGUO KAWASAKI Shiga U n i v e r s i t y o f Medical

D e p a r t m e n t o f O r a l and M a x i l l o f a c i a l S u r g e r y . Science, Japan ABSTRACT

The i n f l u e n c e o f f l u o r i d e i n g e s t i o n on r a t s w i t h g l o m e r u l o n e p h r i t i s h a s been i n v e s t i g a t e d . Thirty-day-old W i s t e r r a t s were given nephrotoxin, body w e i g h t , and f i v e d a y s l a t e r , w e r e p r o v i d e d 1 0 ppm F ( a s NaF s o l u t i o n ) During t h e experimental period, i n the incisors, the experimental period, mandibles, tibias. and m a i n t a i n e d f o r 100 days. o c c u r r e n c e o f m o t t l e d enamel

and u r i n e and b l o o d c h e m i s t r y w e r e s t u d i e d . t h e animals were s a c r i f i c e d ,

A t t h e end o f

and s t u d i e d f o r t h e His-

presence o f d e n t a l c a r i e s i n molars, molars,

and t h e f l u o r i d e c o n t e n t s o f i n c i s o r s ,

and s e v e r a l s o f t t i s s u e s w e r e d e t e r m i n e d .

t o l o g i c a l e x a m i n a t i o n s w e r e p e r f o r m e d o n some o r g a n s . No s i g n i f i c a n t d i f f e r e n c e s i n b l o o d c h e m i s t r y o r i n h i s t o p a t h o l o g y o f s o f t tissues, except t h e kidney, were observed between t h e r a t s w i t h r e n a l d i s e a s e and t h e c o n t r o l s . stage o f t h e experiment. i n t h e hard tissues. INTRODUCTION E v e r s i n c e t h e p u b l i c a t i o n o f r e p o r t s i n t h e 1930's on e p i d e m i o l o g i c a l s t u d i e s o f t h e r e l a t i o n b e t w e e n f l u o r i d e i n g e s t i o n and d e n t a l d e n t a l c a r r i e s p r e v e n t i o n programs. conditions, w a t e r f l u o r i d a t i o n o r t o p i c a l a p p l i c a t i o n o f f l u o r i d e h a v e become p a r t o f

A d e c r e a s e i n b o d y w e i g h t was n o t e d i n t h e e a r l y
Furthermore, n e p h r i t i c animals given d r i n k i n g

w a t e r s u p p l e m e n t e d w i t h 1 0 ppm F showed a m a r k e d l y e l e v a t e d f l u o r i d e l e v e l

I t i s now w i d e l y a c c e p t e d t h a t adeFluoride as w e l l as perform anti-bacte-

q u a t e use o f f l u o r i d e c o u l d r e d u c e d e n t a l c a r i e s considerably. may a c t t o s t r e n g t h e n d e n t a l h a r d t i s s u e s , r i a l and a n t i - e n z y m a t i c a c t i o n s on t o o t h s u r f a c e s .

It i s suggested t h a t

f l u o r i d e i s an e s s e n t i a l e l e m e n t as it i s an e f f e c t i v e f a c t o r i n t h e m i n e r a l i z a t i o n o f h a r d t i s s u e (1). n o t c l e a r l y understood. f l u o r i d e absorption, On t h e o t h e r h a n d , i t s m e t a b o l i s m i s Concerning absorption, There i s no agreement on t h e mechanism i n v o l v e d i n d i s t r i b u t i o n , and e x c r e t i o n .

t h e e x i s t e n c e o f b o t h a c t i v e t r a n s p o r t and p a s s i v e d i f f u s i o n h a s been shown

314
i n t h e r a t (2.3). The a c t i o n and e f f e c t o f f l u o r i d e on a n i m a l s d e p e n d o n s e v e r a l f a c t o r s such as dose and t y p e o f f l u o r i d e compounds, cation, organ as w e l l as age and sex. healthy adults, e x c r e t e d i n t h e u r i n e (4). t r o i n t e s t i n a l secretion, disturbances. However, m-thod and f r e q u e n c y o f a p p l i In and p h y s i o l o g i c a l c o n d i t i o n s o r s p e c i f i c i t y i n r e a c t i o n s o f each E x c r e t i o n o f f l u o r i d e i s m a r k e d l y high. more t h a n 90% o f t h e i n g e s t e d f l u o r i d e i s c o n s i d e r e d t o be O t h e r r o u t e s w i l l be t h r o u g h s a l i v a and gaslacrimal, and mammary e x c r e t i o n s . Adminissweat,

t r a t i o n o f a l a r g e d o s e o f f l u o r i d e has been r e p o r t e d t o c a u s e k i d n e y r e p o r t s on t h e e x c r e t i o n and a c c u m u l a t i o n o f f l u o r i d e under t h e c o n d i t i o n o f k i d n e y d i s f u n c t i o n a r e r a r e i n t h e l i t e r a t u r e

(5).
These e x p e r i m e n t s were designed t o i n v e s t i g a t e t h e i n f l u e n c e o f f l u o r i d e a d m i n i s t r a t i o n on a n i m a l s w i t h e x p e r i m e n t a l l y induced k i d n e y disease. Rats w i t h e x p e r i m e n t a l g l o m e r u l o n e p h r i t i s (Masugi's n e p h r i t i s ) were g i v e n f l u o r i d a t e d d r i n k i n g w a t e r and t h e i r t e e t h and b i o l o g i c a l c o n d i t i o n s w e r e studied. MATERIALS AND METHODS F o r t y - f o u r W i s t e r s t r a i n p u r e m a l e r a t s w e r e used. For preparing nephrotoxin (anti-rat The r a t s w e r e b r e d Japan. ten and s u p p l i e d by t h e Pure S t r a i n Animal Center o f Kyoto U n i v e r s i t y , mature r a t s and t h r e e h e a l t h y m a t u r e r a b b i t s were used.

n e p h r o c o r t e x r a b b i t serum),

An e m u l s i o n cononce e v e r y

t a i n i n g 20% r a t k i d n e y c o r t e x was homogenized and 5 m l o f t h e homogenate was i n j e c t e d i n t o t h e abdominal c a v i t y o f t h e r a b b i t 16 t i m e s , t h r e e t o f o u r days. test. I m m u n o l o g i c a l v a l u e was checked by b l o o d s e d i m e n t a t i o n f o r 30 min. Anti-rat-nephrocortex antigen

Two weeks a f t e r t h e f i n a l i n j e c t i o n , t h e s e r u m was c o l l e c t e d and

i n a c t i v a t e d by h e a t i n g a t 65C

was o b t a i n e d by t h e a d d i t i o n o f 0.5% c a r b o l i c acid.

To produce Masugi's n e p h r i t i s ,

a volume o f t h e a n t i g e n was i n j e c t e d i n t o 30 days o f age. Dose o f s e n s i t i z a -

t h e t a i l v e i n o f t h e experimental rats, T h i s induced marked g l o m e r u l o n e p h r i t i s .

t i o n was 0.5 m l o r 0.25 m l o f t h e a n t i g e n p e r 100 g body w e i g h t o f t h e r a t .

To s t u d y t h e e f f e c t o f f l u o r i d e on r a t s w i t h induced g l o m e r u l o n e p h r i t i s .
t h e a n i m a l s were d i v i d e d i n t o 5 groups: water only (controls); nephrotoxin. Group A a n i m a l s r e c e i v e d d i s t i l l e d G r o u p nF r e c e i v e d G r o u p N A r e c e i v e d i n t r a v e n o u s i n j e c t i o n o f 0.5ml

p l u s d i s t i l l e d w a t e r as d r i n k i n g w a t e r :

i n t r a v e n o u s i n j e c t i o n o f 0.25 c o n t a i n i n g 10 ppm F- (as NaF):

m l o f t h e nephrotoxin,

plus d i s t i l l e d water

Group FIF r e c e i v e d 0.5 m l o f t h e n e p h r o t o x i n , and Group F r e c e i v e d d i s t i l l e d

p l u s d i s t i l l e d w a t e r c o n t a i n i n g 10 ppm F-;

315
w a t e r c o n t a i n i n g 1 0 ppm F- as d r i n k i n g w a t e r and n o i n j e c t i o n . F l u o r i d e c o n t a i n i n g d r i n k i n g w a t e r was g i v e n s i n c e t h e f i f t h day a f t e r t h e i n j e c t i o n o f the nephrotoxin. pellet. The a n i m a l s were m a i n t a i n e d f o r a p e r i o d o f 100 days f o l l o w i n g a d m i n i s t r a t i o n o f t h e n e p h r o t o x i n . A t ten-day i n t h e i n c i s o r s were examined, intervals, body w e i g h t and changes and b l o o d and u r i n e samples were analyzed. The a n i m a l s w e r e a l l f e d a b a s a l d i e t o f O r i e n t a l

A t t h e end o f t h e e x p e r i m e n t a l p e r i o d , t h e a n i m a l s were s a c r i f i c e d . C a r i o u s
l e s i o n s o f t h e m o l a r s were examined and t h e f l u o r i d e c o n t e n t s o f s e v e r a l h a r d and s o f t t i s s u e s determined. on d i f f e r e n t t i s s u e s , pancreas, RESULTS I n c r e a s e o f body w e i g h t and g e n e r a l f i n d i n g s The c h a n g e s i n t h e b o d y w e i g h t o f t h e a n i m a l s d u r i n g t h e e x p e r i m e n t a l p e r i o d a r e shown i n T a b l e I. I n c r e a s e s i n b o d y w e i g h t i n g r o u p s NA. nF. Rats t r e a t e d w i t h b o t h and NF w e r e l o w e r t h a n t h o s e i n g r o u p s A and F. w i t h F alone. On t h e o t h e r hand, s a l i v a r y glands, H i s t o p a t h o l o g i c a l s t u d i e s were a l s o made liver, heart, lungs, spleen, i n c l u d i n g t h e kidneys, and t e s t e s .

n e p h r o t o x i n and f l u o r i d e showed l e s s i n c r e a s e i n w e i g h t t h a n t h o s e t r e a t e d n o s i g n i f i c a n t d i f f e r e n c e was f o u n d between t h e c o n t r o l s and t h o s e a n i m a l s r e c e i v i n g f l u o r i d e alone. TABLE I

CHANGES I N THE BODY WEIGHT OF EXPERIMENTAL ANIMALS

Group N Average body w e i g h t ( 4 ) initial final i n c r e a s e d f SDa 92.3 85.5 81.4 91.4 94.6 341.5 274.4 279.2 280.9 320.9 249.2 f 24.7 191.2 f 30.2 197.8 f 14.0 198.5 k 58.4 226.3 f 21.7 P (by t - t e s t )

10
5 7 gb 15

NA
nF

<o. 01 <o. 001

~0.05

NF
F

aSD s t a n d a r d d e v i a t i o n b e x c e p t one animal d i e d d u r i n g t h e e x p e r i m e n t a l p e r i o d

As f o r abnormal c o n d i t i o n s ,

t w o o f t h e r a t s i n groups NF showed abdominal and a n o t h e r i n t h e same group d i e d on One o f t h e r a t s

s w e l l i n g w i t h i n one t o t h r e e weeks,

t h e 5 3 r d day o f t h e e x p e r i m e n t a f t e r s u f f e r i n g d i a r r h e a .

316
i n g r o u p nF showed d y s p n e a and r h i n o r r h e a . M a c r o s c o p i c o b s e r v a t i o n o f i n c i s o r enamel M o s t mai-ked c h a n g e s o c c u r r e d i n f o u r o u t o f t h e s i x a n i m a l s i n g r o u p NF,
i.e., l i n e a r w h i t e c o l o r c h a n g e s w e r e o b s e r v e d i n i n c i s o r enamel.

I n the

r e s t o f t h e groups, observed.

w h i t e d i s c o l o r a t i o n o r i r r e g u l a r w h i t e spots were o f t e n

Occurrence o f d e n t a l c a r r i e s The number and d e g r e e o f c a r r i e s o c c u r r i n g i n t h e m o l a r a r e shown i n F i g u r e 1. number of The f l u o r i d e - t r e a t e d carious g r o u p s a l l showed a s i g n i f i c a n t l y s m a l l e r groups. The nont e e t h compared t o t h e c o n t r o l carious incidence,

f l u o r i d a t e d n e p h r i t i s g r o u p ( g r o u p NA) a l s o s h o w e d a s i g n i f i c a n t l y l o w e r number o f c a r i o u s m o l a r s , controls. and c a r i o u s e x t e n t t h a n t h e The number o f c a r i o u s m o l a r s and c a r i e s e x t e n t a r e s i g n i f i c a n t l y

l o w e r i n g r o u p NF o r i n g r o u p nF t h a n i n g r o u p F. F l u o r i d e c o n t e n t s o f h a r d t i s s u e s and some s o f t t i s s u e s The f l u o r i d e c o n t e n t s o f m o l a r s w e r e h i g h e r t h a n i n c i s o r s i n n o n - f l u o r i d a t e d g r o u p s (A

- MA),

w h i l e those o f i n c i s o r s were h i g h e r than molars i n

fluoride-treated

g r o u p s (nF,

NF - F ) ( F i g .

2).

The f l u o r i d e c o n t e n t o f The f l u o r i d e c o n t e n t In

f e m u r s was h i g h e r t h a n m a n d i b l e s i n g r o u p s A and NA. each o f t h e hard t i s s u e s , mandibles),

o f m a n d i b l e s was h i g h e r t h a n f e m u r s i n g r o u p s NF and F t h a n i n o t h e r s . a n d g r o u p n F w a s h i g h e r t h a n g r o u p F.

g r o u p NA was h i g h e r t h a n g r o u p A ( e x c e p t f o r This indicates that

3.0

2.0
I

T II
1.0

carious molars

carious incidence =group

carious extent

carious grade NA

0 group A I g r o u p nF
F i g u r e 1.

! I l group l

group NF

The number and e x t e n t o f c a r i e s i n m o l a r s .

317

200

400

600

800

1000

F-ppm

Femurs Mandibles Molars Incisors Kidneys Liver Heart Lungs Testicles Spleen Pancreas Submandibular salivary glands Ogroup =group -group group group
A
NA

nF
NF F

F i g u r e 2.

F l u o r i d e c o n t e n t o f s e v e r a l t i s s u e s and organs.

a n i m a l s w i t h e x p e r i m e n t a l n e p h r i t i s t e n d t o have h i g h e r l e v e l s o f f l u o r i d e i n hard tissues. I n the f l u o r i d e content o f s o f t tissues, observed. l e v e l s t h a n n o n - f l u o r i d a t e d groups. H i s t o l o g i c a l observation o f s o f t t i s s u e s Kidney. F i g u r e 4. H i s t o p a t h o l o g i c a l changes o f t h e k i d n e y i n t h e r a t w i t h induced The d a t a a r e summarized i n P r e v a l e n t c h a n g e s a r e a t r o p h y and d e g e n e r a t i o n o f g l o m e r u l i , These changes a r e marked i n groups n e p h r i t i s a r e shown i n F i g u r e s 3-1 t h r o u g h 4. no marked d i f f e r e n c e s were G e n e r a l l y speaking, f l u o r i d a t e d g r o u p s showed h i g h e r f l u o r i d e F l u o r i d e l e v e l i t s e l f , however, was

n o t r e l a t e d t o t h e presence o f n e p h r i t i s .

e x u d a t i o n and r o u n d c e l l i n f i l t r a t i o n i n i n t e r s t i t i u m o r c a p s u l e , and d e g e n e r a t i o n and n e c r o s i s o f tubules.

318

F i g u r e 3-1. H i s t o p a t h o l o g i c a l changes o f t h e r a t k i d n e y o f g r o u p NA. I n f i l t r a t i o n o f r o u n d c e l l s i n i n t 2 r s t i t i u n a:>:: ? i e o t \ ? i ? . c r \ s t s i : i t h e t u b u l e s ( l e f t , r 2 - t :ic. 3), an:: f i ' 2 r i n o i : l <qen:rr\tioi? 0 - F cj1o;:zrcli ( r i ? h t , r a t no. 6).

F i g u r e 3-2. Kidney o f group NF, r a t no. 4. F i b r o u s l e s i o n o f g l o m e r u l i , h y p e r p l a s t i c changes o f m a l p i g h i a n corpuscles, r e g e n e r a t i o n o f t u b u l a r e p i t h e l i u m and i n f l a m m a t o r y round c e l l i n f i l t r a t i o n on i n t e r s t i t i u m .

319

F i g u r e 3-3. K i d n e y o f g r o u p nF, r a t no. 4. Marked i n f i l t r a t i o n o f r o u n d c e l l s and f i b r o s i s i n t h e i n t e r s t i t i u m , p r o t e i n c a s t s i n t h e tubules, hyperplasia o f rnalpighian corpuscles, atrophy and f i b r o s i s o f PI----

,.

F i g u r e 3-4. K i d n e y o f g r o u p nF, r a t no. 2. I n f i l t r a t i o n o f r o u n d c e l l s i n i n t e r s t i t i u m , marked d e g e n e r a t i o n and n e c r o s i s o f t u b u l e s , f i b r o p l a s t i c h y p e r p l a s i a o f m a l p i g h i a n c o r p u s c l e s and These changes a r e d i a g n o s e d as a t r o p h i c s c l e r o t i c atrophy o f glomeruli. kidney.

rnultinuclear Fibrous hypertrophy embo 1 i sm

l-l

inside

capsule

adhesion with glomerulus scar c h a n g e compensatory h y p e r t r o p h y

F i g u r e 4. H i s t o l o g i c a l o b s e r v a t i o n s o f t h e kidney.

321
NF,
nF and NA, b u t t h e d e g r e e and s i t e s o f c h a n g e s d i f f e r e d i n e a c h a n i m a l Some o f t h e s e c h a n g e s w e r e a l s o n o t e d i n

e v e n w i t h i n t h e same group. control and F g r o u p s . Other orgaE. and t e s t e s ,

Microscopic observation o f the tissues from the liver, m a i n l y i n blood vessels from high t o

heart, lungs, spleen, pancreas, t h y r o i d , s a l i v a r y glands, adrenal glands r e v e a l e d s e v e r a l h i s t o l o g i c a l changes, and m e s e n c h y m a l components. l o w , g r o u p s N F > n F > a n d F. significant. The d e g r e e o f t h e s e c h a n g e s w e r e ,

However, t h e r e s u l t s w e r e n o t s t a t i s t i c a l l y

-____ Urine analysis.

as w e l l .

P r o t e i n u r i a was o b s e r v e d i n g r o u p NF r a t s a t an e a r l y

p e r i o d o f t h e e x p e r i m e n t , a n d d u r i n g t h e l a s t 4 0 d a y s i n g r o u p s nF a n d N F Some w e r e a l s o n o t e d i n g r o u p A.

No s i g n i f i c a n t d i f f e r e n c e s were

observed between groups.

-__Blood analysis.
range,

Serum t o t a l p r o t e i n l e v e l s w e r e m o s t l y w i t h i n n o r m a l

except f o r t h e f i n a l days o f t h e experiment. however (Fig.

No marked d i f f e r e n c e s

were noted between groups, urea nitrogen, w i t h i n a n o r m a l range.

5).

Data on serum c r e a t i n i n e . were f o u n d t o be m o s t l y

and n o n - p r o t e i n

n i t r o g e n (NPN)

I n t h e i n d u c e d n e p h r i t i s g r o u p , b l o o d c h l o r i n e t e n d e d t o i n c r e a s e f r o m 30 d a y s t o 50 d a y s , w h e n t h e c o n c e n t r a t i o n s o f g r o u p NA w e r e 1.5 t i m e s , a n d t h o s e o f g r o u p s nF a n d N F w e r e t w i c e t h o s e o f t h e c o n t r o l s . h a l f o f t h e experimental period, n o r m a l ( F i g . 6). I n the last t h e c h l o r i n e l e v e l s were decreased t o

L Q)

10

30

50

60

70

80

90

100

u )

W group D--W

A,

Experimental period, days group NA, h - - A group

C-. g r o u p F

nF.

g r o u p NF,

F i g u r e 5.

Serum t o t a l p r o t e i n o f e x p e r i m e n t a l r a t s .

322

*ool
W 150

10
O---C

30
qCOUp

Experimental period, days

A.6-dgpuop NA,t--Agroup
NF,-group

50

60

7 0 80 90 1 0 0

nF,

)-*group

F i g u r e 6.

Blood c h l o r i n e c o n t e n t o f experimental r a t s .

DISCUSSION
F l u o r i d e a b s o r p t i o n i n a n i m a l s i s c o n s i d e r e d t o be m a i n l y v i a t h e s m a l l i n t e s t i n e and s t o m a c h t h r o u g h p a s s i v e d i f f u s i o n (2). t h e kidney i n t o urine.
I n h e a l t h y persons,

W h i l e some f l u o r i d e

i s bound t o h a r d t i s s u e s , m o s t o f t h e i n g e s t e d f l u o r i d e i s e x c r e t e d t h r o u g h f l u o r i d e i n t a k e a t t h e u s u a l f l u o r i d a t i o n l e v e l may I n patients s u f f e r i n g renal dysfunction,

n o t a f f e c t renal functions. however,

t h e body f l u o r i d e b u r d e n m i g h t b e i n c r e a s e d , and i t i s supposed

t h a t t h e t h r e s h o l d s a f e t y range m i g h t be narrowed. m e t a b o l i s m and t h i s a f f e c t s s o f t t i s s u e s . i n view o f these considerations.

It i s a l s o considered

t h a t r e n a l d y s f u n c t i o n has c l o s e and i m p o r t a n t i n t e r a c t i o n s w i t h f l u o r i d e The p r e s e n t s t u d y was d e s i g n e d

D r i n k i n g w a t e r c o n t a i n i n g 10 ppm F i n r a t s i s c o n s i d e r e d t o be e q u i v a l e n t t o 1 p p m F i n h u m a n s (6,7). I n 1970. Hodge a n d T a v e s (8) r e p o r t e d t h e p r o b a b i l i t y and d a n g e r o f Yuds a f e t y margin narrowing i n i n d i v i d u a l s s u f f e r i n g renal disturbances. kin l e v e l s between t w o groups o f p a t i e n t s w i t h r e n a l dysfunction,

g t gl. ( 9 ) o b s e r v e d n o s i g n i f i c a n t d i f f e r e n c e s i n u r i n a r y f l u o r i d e
one group

c o n s u m i n g w a t e r f l u o r i d a t e d w i t h 1.0 ppm F and a n o t h e r g r o u p w i t h 0.1 ppm

F.

Smith

g . (10)

a l s o r e p o r t e d t h a t among o l d e r p e o p l e w i t h and w i t h Dialysis

out nephritis,

t h e r e were no s i g n i f i c a n t d i f f e r e n c e s i n t h e l e v e l o f u r i n -

a r y f l u o r i d e c o n c e n t r a t i o n s b e f o r e and a f t e r f l u o r i d a t i o n .

323
patients with renal failure, Dialysis patients w i l l on t h e o t h e r hand, were r e p o r t e d t o have membranous

s e v e r a l t i m e s h i g h e r serum f l u o r i d e

l e v e l s t h a n h e a l t h y i n d i v i d u a l s (11).

h a v e o t h e r p r o b l e m s s u c h as n o n - d e s i r e d

exchange o f f l u o r i d e w i t h o t h e r e l e m e n t s i n c l u d i n g c a l c i u m ions. The r e s u l t s o f t h i s s t u d y d e m o n s t r a t e d t h a t l o w c o n c e n t r a t i o n s o f f l u o r i d e s u c h a5 f o u n d i n m u n i c i p a l f l u o r i d a t i o n m i g h t n o t a f f e c t t h e c o n d i t i o n and s e v e r i t y o f g l o m e r u l o n e p h r i t i s . affect histological features of many s o f t Also, n e p h r i t i s m i g h t n o t tissues. There were no

d i f f e r e n c e s i n body w e i g h t and m o s t o f t h e b l o o d c h e m i s t r y d a t a b e t w e e n g r o u p s nF,NF and NA. T h i s s u g g e s t s t h a t c o n s u m p t i o n o f 10 ppm f l u o r i d e b y n e p h r i t i c r a t s does n o t a d d i t i o n a l l y a f f e c t t h e g r o w t h and p a t h o l o g i c a l c o n d i t i o n s o f t h e kidneys.

I t m u s t be m e n t i o n e d t h a t t h e r a t , a r o d e n t , shows d i f f e r e n t d e n t i t i o n
t h a n humans, a s i t s i n c i s o r s c o n t i n u e t o e r u p t . The f l u o r i d e l e v e l anaSince f l u o r i d e l y z e d i n t h i s s t u d y may n o t be f u l l y a p p l i c a b l e t o humans. f l u o r i d e accumulation i n t h e hard tissue.

c o n t e n t s i n bones and t e e t h w e r e e l e v a t e d i n n e p h r i t i c r a t s , s u g g e s t i n g Decrease o f d e n t a l c a r i e s i n c i d e n c e and o t h e r c a r i e s i n d e x w i l l b e a r e f l e c t i o n o f t h i s phenomenon. CONCLUSION The e f f e c t s o f d r i n k i n g f l u o r i d a t e d w a t e r o n r a t s w i t h i n d u c e d g l o m e r u l o n e p h r i t i s were studied. I n c r e a s e i n b o d y w e i g h t was d e p r e s s e d s 1 i g h t : y compared t o t h e c o n t r o l groups, i n the experimental r a t s b u t i t was n o t i n f l u e n c e d b y d r i n k i n g Both

f l u o r i d a t e d w a t e r . D a t a o n u r i n e and b l o o d a n a l y s i s showed t h a t t h e c h a r a c t e r i s t i c f e a t u r e s o f n e p h r i t i s were n o t enhanced b y f l u o r i d a t i o n . content o f hard tissues. n e p h r i t i c c o n d i t i o n s and f l u o r i d e supplements i n c r e a s e d t h e f l u o r i d e T h i s may h a v e r e s u l t e d i n t h e d e c r e a s e o f d e n t a l c a r i e s observed i n these exoeriments. REFERENCES 1.

WHO:

WHO T e c h Rep, No 582. WHO, Geneva, 1973

2.
4.

S a t o T ( 1 9 7 8 ) B u l l S t o m a t o l o g y K y o t o U n i v 18:16-27 B i t t n e r W (1968) D t s c h Z a h n a r z t l Z 23:123-128 Springer-Verlag,

3. S a t 0 T (1971) J D e n t H l t h 21:339-365 5. S m i t h FA, G a r d n e r DE ( 1 9 6 6 ) P h a r m a c o l o g y o f F l u o r i d e s . N.Y. P a r t I , p 1 2 9 6. 7. M i y a z a k i Y, K y o t o , p 80

I s h i k a w a G, A k i y o s h i M ( 1 9 5 4 ) O r a l P a t h o l o g y I. N a g a s u e , U n i v 3:244-275

S a t 0 T (1963) B u l l S t o m a t o l o g y K y o t o

324

8. Hodge HC, Taves DR (1970) I n : F l u o r i d e s and Human H e a l t h , WHO Monogr Ser No 59, pp 249-255
9. Y u d k i n EP. 10. S m i t h FA, Gardner DE,

WHO, Geneva,

C z e r n i e j e w s k i J, B l a y n e y JR (1954) J Dent Res 33:691-692 Hodge HC (1955) A r c h I n d u s t r H e a l t h 11:2-10 S c r i b n e r BS (1968) Trans Am

11. Taves DR. Freeman RB. Kamm DE, Ramos CP, SOC A r t i f I n t Organs 14:412-414

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 325-332 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

325

MECHANISM OF FLUORIDE ABSORPTION FROM THE GASTROINTESTINAL TRACT IN RATS

T.I\KUMI

S A T O ~ , KAZUSADA YOSHITAKE~

AND GONJIRO

HI TO MI^
Shiga U n i v e r s i t y o f Medical Fukui. Fukui Medical College,

' D e p a r t m e n t o f O r a l and M a x i l l o f a c i a l S u r g e r y , S c i e n c e , O t s u . S h i g a , 520-21 J a p a n ' D e p a r t m e n t o f D e n t i s t r y and O r a l S u r g e r y , 910-11 J a p a n

ABSTRACT The m e c h a n i s m i n v o l v e d i n t h e a b s o r p t i o n o f f l u o r i d e b y t h e g a s t r o i n t e s t i n a l t r a c t has been s t u d i e d b y t h e g a s t r o i n t e s t i n a l r e f l u x method i n s i t u o f r a t . R e f l u x was a p p l i e d t o t h e stomach, mouth o f each animal. t h e stomach small intestine, rectum, and When

A t certain intervals during the reflux. a portion o f

t h e r e f l u x s o l u t i o n was t a k e n f o r d e t e r m T n a t i o n o f f l u o r i d e u p t a k e .

o r t h e s m a l l i n t e s t i n e was r e f l u x e d f o r 1 hour, t h e r a t e o f These r e s u l t s i n d i c a t e

f l u o r i d e a b s o r p t i o n was d e c r e a s e d w i t h i n c r e a s e i n f l u o r i d e c o n c e n t r a t i o n s , and i n t h e p r e s e n c e o f d i n i t r o p h e n o l o r s a l i n e . n o t be l i m i t e d t o p a s s i v e t r a n s p o r t alone. t h a t t h e u p t a k e o f f l u o r i d e f r o m t h e g a s t r o i n t e s t i n a l t r a c t o f t h e r a t may

INTRODUCTION F l u o r i d e (F) i s w i d e l y u s e d f o r t h e p r e v e n t i o n o f d e n t a l c a r i e s . Fluorid a t i o n h a s b e e n a d o p t e d i n many c o u n t r i e s a s a p o s i t i v e means f o r t h e p r e v e n t i o n o f d e n t a l c a r i e s i n p u b l i c h e a l t h p r o g r a m s a n d WHO h a s p r o p o s e d t h a t t h i s method be p r a c t i c e d as w i d e l y as p o s s i b l e . r e s u l t s have n o t been s a t i s f a c t o r y . water. Consequently, Although t h e dynamics experimental o f F i n v i v o h a v e b e e n s t u d i e d .by m a n y i n v e s t i g a t o r s ( 1 - 5 ) .

it i s difficult to

d e t e r m i n e an a d e q u a t e d o s e o f F f o r f l u o r i d a t i o n o f c o m m u n i t y d r i n k i n g

It

i s i m p o r t a n t t o c l a r i f y t h e mechanism o f F a b s o r p t i o n f r o m t h e

g a s t r o in t e s t ina 1 t r a c t . I n t h i s paper, e x p e r i m e n t s were conducted t o s t u d y t h e a b s o r p t i o n o f F f r o m t h e g a s t r o i n t e s t i n a l t r a c t i n s i t u o f r a t s b y means o f a r e f l u x method.

MATERIALS AND METHODS M a l e r a t s w e i g h i n g 120-140 g, a f t e r 24 h o u r s f a s t i n g , were anesthesized A w i t h u r e t h a n a n d p l a c e d i n a r e f l u x a p p a r a t u s a s s h o w n i n F i g u r e 1.

326

Figure 1: Front cutaway diagram of apparatus used f o r r e f l u x experiments of r a t stomach and i n t e s t i n e .

r e f l u x i n g f l u i d ( 7 5 ml) containing NaF s o l u t i o n s of k n o w n was refluxed through cne stomach and small i n t e s t i n e .

concentrations

P r i o r t o t h e experiment on F absorption from t h e stomach, d i s t i l l e d water was used a s t h e r e f l u x f l u i d and t h e c h l o r i d e c o n t e n t of t h e r e f l u x was measured o v e r a 90 min p e r i o d . The amount of c h l o r i d e s e c r e t e d from t h e stomach w a l l i n t o t h e r e f l u x f l u i d d u r i n g t h i s p e r i o d was a p p r o x i m a t e l y

1,500 ug/dl.

T h i s i s n e g l i g i b l e compared w i t h t h e c h l o r i d e c o n t e n t of Sodium f l u o r i d e dissolved in

physiological s a l i n e . Consequently, t h e amount o f c h l o r i d e s e c r e t e d i n t o t h e g a s t r i c j u i c e was not taken i n t o account. concentrations. Dinitrophenol (DNP), d i s t i l l e d w a t e r o r p h y s i o l o g i c a l s a l i n e was used f o r r e f l u x a t v a r y i n g an uncoupler of o x i d a t i v e phosphoryl a t i o n , was used a t Z X I O - ~ M a n d ~ x I O - ~ M . The concentrations of F- and C1ions i n t h e r e f l u x were measured by t h e ion e l e c t r o d e method. Time 0 was defined a s 15 min a f t e r the beginning of infusion.

A t 15,

30

45, a n d 60 min t h e r e a f t e r , two 0.5 ml r e f l u e n t samples were taken, one f o r

t h e d e t e r m i n a t i o n of F c o n c e n t r a t i o n and t h e o t h e r f o r t h e c o r r e c t i o n of concentration f o r t h e f l u c t u a t i o n in water content of t h e r e f l u x during t h e course of t h e experiment. To c o r r e c t t h e f l u c t u a t i o n in F concentration t o the reflux fluid. following equation: absorbance of phenol red a t t h e beginning of r e f l u x measured absorbance of phenol red
i n

t h e r e f l u x , phenol red,

considered t o be s c a r c e l y absorbed by t h e g a s t r o i n t e s t i n a l t r a c t , was added Fluoride concentration was corrected according t o the

corrected concentration

measured x concentration

327
I n t h e e x p e r i m e n t o n a b s o r p t i o n f r o m t h e r e c t u m , a v i n y l t u b e was conn e c t e d t o t h e u p p e r p a r t o f t h e r e c t u m and anus. a b s o r p t i o n f r o m t h e mouth, c o n n e c t e d t o it. The e x p e r i m e n t a l m e t h o d s u s e d f o r r e f l u x i n g t e s t s o l u t i o n s , and f o r and d e t e r m i n i n g t h e l o s s o f F f r o m r e f l u x f l u i d i n c h r o n o l o g i c a l sequence, was f i x e d t o t h e mouth w i t h c y a n o a c r y l a t e , F o r t h e e x p e r i m e n t on a s p e c i a l l y p r e p a r e d a d a p t e r shown i n F i g u r e 2 and t h e r e f l u x a p p a r a t u s was

F absorption from physiological saline containing varying concentrations o f

F w e r e t h e same a s w i t h t h e s t o m a c h and s m a l l i n t e s t i n e .

Rec i r c u -

nm g;,e s e r v o i r

rnm
F i g u r e 2. D i a g r a m o f a p p a r a t u s u s e d i n r e f l u x e x p e r i m e n t o f r a t mouth.

RESULTS
A b s o r p t i o n f r o m t h e stoE& T i m e 0 was d e f i n e d a s 15 m i n a f t e r t h e b e g i n n i n g o f t h e r e f l u x . shows Figure 3 t h e l o g a r i t h m o f t h e c o r r e c t e d F c o n c e n t r a t i o n s as a b s o r b a n c e o f t h e An a p p r o x i m a t e l y r e c t i l i n e a r r e s p o n s e was seen. The r a t e o f F

r e f l u x a t 15, 30, 45, a n d 60 r n i n t h e r e a f t e r f o r s a m p l e s c o n t a i n i n g 50 a n d

100 u g F / m l .

2 0 .-235000 [
LI

-n

$0.150
0

15 3 0 4 5 6 0 Time i n m i n u t e
0 0

5 0 yg/ml o----o1 0 0 yg/ml

F i g u r e 3. R e l a t i o n s h i p b e t w e e n F c o n c e n t r a t i o n s and r e f l u x t i m e o f f l u o r i d e s o l u t i o n f o r f l u o r i d e a b s o r p t i o n f r o m t h e stomach o f t h e rat.

328
a b s o r p t i o n was a f f e c t e d b y t h e F c o n c e n t r a t i o n i n t h e r e f l u x f l u i d . l i q u i d was i n c r e a s e d , c o n s t a n t v a l u e (Fig. t h e r a t e o f a b s o r p t i o n decreased, When

approaching a As

4).

The r a t e o f NaF a b s o r p t i o n f r o m s a l i n e was l o d e r e s p e c i a l l y a t lower F concentrations. however, the difference

t h a n t h a t f r o m d i s t i l l e d water, became s m a l l e r ,

t h e F c o n c e n t r a t i o n o f t h e r e f l u x was increased.

and a l m o s t disappeared a t 900 ugF/ml.

.:Absorption from d i s t i l l e d water 0 : A b s o r p t i o n from 0 . 9 % N a C l s o l u t i o n

F i g u r e 4. Effect of f r o m t h e stomach. f l u o r i d e c o n c e n t r a t i o n on t h e a b s o r p t i o n o f f l u o r i d e

Absorption from t h e small i n t e s t i n e F l u o r i d e i n t h e r e f l u x f l u i d was d e t e r m i n e d e v e r y 1 5 m i n and t h e l o g a r i t h m o f c o n c e n t r a t i o n s p l o t t e d a g a i n s t t i m e showed a r e c t i l i n e a r r e g r e s s i o n as w i t h t h e stomach. shown i n F i g u r e 5. dependent. The r e l a t i o n s h i p between F c o n c e n t r a t i o n i n t h e r e f l u x and t h e r a t e o f a b s o r p t i o n a t t h e end o f t h e 1 hour r e f l u x p e r i o d i s W h i l e t h e a b s o r p t i o n i n t h e s m a l l i n t e s t i n e was generi t was a l s o c o n c e n t r a t i o n -

a l l y more e f f i c i e n t t h a n i n t h e stomach,

W i t h an i n c r e a s e i n F c o n c e n t r a t i o n s i n t h e r e f l u x ,

the rate of

F a b s o r p t i o n decreased.

The presence o f c h l o r i d e g e n e r a l l y decreased t h e The d e c r e a s e i n t h e r a t e o f a b s o r p t i o n was i n d i c a t i n g a diminished i n f l u A d d i t i o n o f DNP t o t h e

r a t e o f a b s o r p t i o n ( F i g . 5).

more pronounced a t l o w e r F c o n c e n t r a t i o n s ,

ence o f c h l o r i d e w i t h i n c r e a s i n g F c o n c e n t r a t i o n s .

r e f l u x s o l u t i o n decreased t h e r a t e o f F a b s o r p t i o n (Fig.

5).

329

c
. P i

20
15.o

E
0

.-l

a
0
4

;10 . o
0 m
LI

5.o
1

200 400 600 800 1000 Fluoride c o n c e n t r a t i o n ( p g / m l )

distilled 0.9% N a C l distilled distilled water solution water c o n t a i n i n g 2 x 1 0 - 4 DNP w a t e r c o n t a i n i n g 4 x 1 0 - 4 DNP

:Fluoride :Fluoride A:Fluoride A :Fluoride

0 0

in in in in

Figure 5. Effect of fluoride concentration in the reflux on the absorption of fluoride from the small intestine.

Absorption from the rectum The rate of F disappearance from the reflux to aqueous solution with varying concentrations of NaF over a period of 60 m i n was plotted against the initial concentration of the reflux. Unlike those of the stomach and

4.0

n C
0
LI

~1

200 1000 1400 Flucride concentration

(

p / m U

do

0 : F l u o r i d e i n d i s t i l l e d water i n 0.9% NaCl s o l u t i o n .:Fluoride

Figure 6. Effect of fluoride concentration on the absorption of fluoride from the rectum.

330

small intestine, the rate remained approximately constant with increases

F concentrations in the reflux.

in

When NaF-containing saline was refluxed, little or no influence of chloride on F absorption was observed (Fig. 6 ) . Absorption from the mouth When the rate o f F absorption was plotted against time, a rectilinear relationship was seen over a 1 hour period, as with the rectum. As shown in Figure 7, the rate of absorption remained approximately constant at 2%. The absorption of F from the oral mucosa was unaffected by changes in Fion or C1- ion concentrations (Fig. 7 ) .

0 200 4 0 0 6 0 0 8 0 0 Flurode concentration ( pg/ml) o : F l u o r i d e i n d i s t i l l e d water .:Fluoride i n 0.9% NaCl s o l u t i o n

Figure 7. Effect of fluoride concentration on the absorption o f fluoride from the mouth.

DISCUSSION
As shown in Figure

3 for the reflux through the stomach, the rectilinear

relationship between the logarithm of F concentrations o f the reflux fluid and time suggests that F was absorbed according to first-order kinetics. And the results shown in Figures 4 and 5 indicate that F absorption from the stomach and small intestine depended on F concentrations, being more efficient at lower concentrations. With increasing F concentrations, however, the rate of absorption approached a constant value. The rate of F absorption from saline was generally lower than that from distilled water, and the influence of C1- ions was especially pronounced at lower F concentrations. The decrease of F absorption in the presence of C1- ions may be explained by their inhibitory effect on F absorption. The data on F absorption were employed in the Lineweaver-Burk equation ( 6 ) . assuming that active transport operated mainly within the concentration range studied. The results are shown in Figure 8. Rectilinear regressions were obtained with and without chloride. The two lines, moreover, were found to intersect at the ordinate. This indicates a competi-

331

0.0020

1 0.0015V
0.0010-

..
0.005

0.010

0.015 o*020

S:Initial concentration (Absorbance) V:Rate of transport (Absorbance/hr.) 0:Fluoride in distilled water :Fluoride in NaCl solution
F i g u r e 8. L i n e w e a v e r - B u r k i n t e s t i n e o f rat. plot: Transport o f f l u o r i d e through the small

tive inhibition a c t i o n o f C1-

i n a n enzyme r e a c t i o n and,

i n t h i s case, an i n h i b i t o r y

ions on F absorption.

Even t h o u g h e n z y m a t i c a c t i o n on F t h e presence o f a rate-1 i m i t i n g Decrease i n t h e r a t e o f F or competitive inhibi-

a b s o r p t i o n c a n n o t be d e f i n i t e l y c o n c l u d e d ,

s t e p and c o m p e t i t i v e i n h i b i t i o n b y c o e x i s t i n g c h l o r i d e i s l i k e l y i n F absorption from the gastrointestinal tract. r e q u i r i n g mechanism. t i o n by, On t h e o t h e r hand, inhibitors, a b s o r p t i o n i n t h e p r e s e n c e o f DNP a l s o s u g g e s t s i n v o l v e m e n t o f an e n e r g y C o n c e n t r a t i o n dependency o f , c h l o r i d e suggests t h a t a c t i v e t r a n s p o r t operates i n F absorption. t h e o b s e r v a t i o n t h a t t h e r a t e o f a b s o r p t i o n approached a o r i n t h e presence o f

constant l e v e l w i t h increasing F concentrations, suggests absorption by s i m p l e d i f f u s i o n . t r a c t (Fig.

It i s p o s s i b l e t h a t

b o t h t h e s e m e c h a n i s m s may be i n v o l v e d i n t h e a b s o r p t i o n s f F f r o m t h e gastrointestinal

8).
The i n f l u e n c e o f c h l o r i d e i o n s was Parkins

F a b s o r p t i o n f r o m t h e m o u t h and r e c t u m showed no c o n c e n t r a t i o n dependency

under t h e experimental conditions. s l i g h t and F was f o u n d t o be a b s o r b e d f r o m t h e s e s i t e s a l m o s t a l o n g t h e c o n c e n t r a t i o n g r a d i e n t b y a s i m p l e mechanism.

&

d.( 7 , 8 )

u s e d an In this

e v e r t e d s a c t e c h n i q u e t o d e m o n s t r a t e t h e a c t i v e t r a n s p o r t o f F, paper,

and s t a t e d

opinions c o n t r a r y t o t h e conventional concept o f s i m p l e d i f f u s i o n .

t h e m e t h o d o f i n t e s t i n a l r e f l u x i n s i t u was used t o e l u c i d a t e t h e and a b s o r p t i o n b y a c t i v e t r a n s p o r t was shown t o

mechanism o f F a b s o r p t i o n f r o m each p a r t o f t h e d i g e s t i v e t r a c t under identical host factors,

332
occur i n a d d i t i o n t o absorption by s i m p l e d i f f u s i o n i n t h e s m a l l i n t e s t i n e and stomach. CONCLUSION The m e c h a n i s m o f F a b s o r p t i o n f r o m t h e g a s t r o i n t e s t i n a l t r a c t was s t u d i e d by u s i n g t h e r e f l u x method i n s i t u o f r a t s . occurred not only through passive diffusion. The e x p e r i m e n t a l r e s u l t s b u t t h r o u g h a mechanism The a n t a g o n i s t i c showed t h a t t h e a b s o r p t i o n o f F f r o m t h e stomach and s m a l l i n t e s t i n e involving active transport or facilitated diffusion. e f f e c t o f C1- i o n s on F a b s o r p t i o n was a l s o d e m o n s t r a t e d .

REFERENCES
1. 2.
3. 4. 5. 6. C a r l s o n CH, 104: 235-23'7 S t o o k e y GK, 301 Armstrong

WD,

S i i l g e r L (1960)

P r o c S o c e x p B i o l Med

C r a n e DB, M u h l e r JC ( 1 9 6 4 ) P r o c S o c e x p B i o l Med 1 1 5 : 2 9 1 -

Wagner MJ ( 1 9 6 2 ) J d e n t Res 41:667-671 Wallace-Durbin Z i p k i n I, N a t h a n s D,

P ( 1 9 5 4 ) J d e n t Res 33:789-800
Ross JE ( 1 9 6 0 ) B i o c h i m B i o p h y s A c t a 41:271 F a u s t RG (1966)

L i k i n s RC ( 1 9 5 7 ) Am J P h y s i o l 191:549-550 T a p l e y DF,

7.

P a r k i n s FM. H o l l i f i e l d JW, M c C a s l i n AJ, L i u Wu S-M, B i o c h e m B i o p h y s A c t a 126:513-524

8. P a r k i n s F M ( 1 9 7 1 ) B i o c h i m B i o p h y s A c t a 241:507-512

Fluoride Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 335-339 0 1 9 8 6 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

H. Tsunoda a n d M.-H. Yu (Editors)

335

OSSIFICATIONS AND CALCIFICATIONS OF MUSCLE AND TENDON INSERTIONS I N HUMAN INDUSTRIAL FLUOROSIS

JURGEN FRANKE
D e p a r t m e n t o f O r t h o p e d i c S u r g e r y , M e d i c a l Academy E r f u r t . R e g i e r u n g s s t r 42. DDR-5010, German D e m o c r a t i c R e p u b l i c F l u o r o s i s i s a c h r o n i c f l u o r i d e i n t o x i c a t i o n i n humans and a n i m a l s . There are f o u r types fluorides: of human f l u o r o s i s a c c o r d i n g to the source o f content i n

I.

Endemic f l u o r o s i s . T h i s i s caused b y a d r i n k i n g water. Africa.

high fluorine

Examples a r e found i n I n d i a .

and i n N o r t h and S o u t h

2.

Wine f l u o r o s i s .

T h i s was r e p o r t e d b y S o r i a n o o f S p a i n i n 1965(1).

Here h i g h amounts o f f l u o r i d e were added i n t o t h e w i n e t o p r e v e n t abnormal f e r m e n t a t i o n .

3.
4.

Neiqhborhood f l u o r o s i s .

T h i s o c c u r s v e r y r a r e l y i n humans and c a t t l e rare o c c u p a t i o n a l d i s e a s e among i.e.. (1) p e r i o s t o muscle

near f l u o r i d e e m i t t i n g industries. Industrial fluorosis. This i s a w o r k e r s of f l u o r i n e p r o c e s s i n g o r m a n u f a c t u r i n g i n d u s t r i e s . Human f l u o r o s i s i s c h a r a c t e r i z e d b y t h r e e phenomena, s i s w i t h f o r m a t i o n o f exophytes, i n s e r t i o n s and j o i n t c a p s u l e s ; spongiosa. Radiologically, i n b e g i n n i n g s t a g e s t h e r e a p p e a r s t o be a c o n d e n s a t i o n o f an e n l a r g e m e n t o f t h e bone t r a b e c u l a e i n t h e l u m b a r t h e x-rays o f t h e s p i n e show an e x t r e m e m a r b l e - l i k e t h e bone s t r u c t u r e , I n advanced stages, w i t h o s s i f i c a t i o n s o f ligaments,

(2) e n d o s t o s i s ; and (3) s c l e r o s i s o f t h e

s p i n e and p e r i o s t e a l a p p o s i t i o n s o n t h e bones o f f o r e a r m s and l o w e r l e g s . s c l e r o s i s o f t h e bones w i t h o s s i f i c a t i o n o f t h e l o n g i t u d i n a l ligaments. The f o r m a t i o n o f o u t g r o w t h s and s p u r s a t t h e m u s c u l a r i n s e r t i o n s and o s s i f i c a t i o n s o f the ligaments o f the p e l v i c f l o o r complete t h i s picture. O u t g r o w t h s l i k e a saw b l a d e a l s o d e v e l o p b e t w e e n t h e r a d i u s and u l n a and t h e t i b i a and f i b u l a . Pathologic-anatomical a r e as f o l l o w s : t u d i n a l ligaments; f i n d i n g s f r o m a severe case o f i n d u s t r i a l f l u o r o s i s o n t h e s p i n e we s e e a c o m p l e t e o s s i f i c a t i o n o f t h e l o n g i t h e s m a l l v e r t e b r a l j o i n t s and c o s t o v e r t e b r a l j o i n t s a r e t h e muscular attachments These changes a r e seen c l e a r l y o n t h e p e l v i s : a

ankylosed by o s s i f i c a t i o n o f t h e capsules, p r o t r u d e as exostoses.

336
projection o f t h e m u s c u l a r a t t a c h m e n t s on t h e i l i a c c r e s t and p u b i c bone, p u b i c symphysis. and l i g a m e n t s The Peripherally, saw-blade-like a p p o s i t i o n s between t h e

and o s s i f i c a t i o n s o f t h e s a c r o i l i a c j o i n t s , o f the pelvic floor.

r a d i u s and u l n a (Fig. 1) o r between t h e t i b i a and f i b u l a a r e found. e n i n g o f t h e w h o l e c o r t i c a l i s b y p e r i o s t o s i s and e n d o s t o s i s .

l o n g bones show an i n c r e a s e o f t h e bone c r o s s - s e c t i o n w i t h d i s t i n c t t h i c k H i s t o l o g i c a l f i n d i n g s i n t h i s s t u d y w e r e b a s e d o n t h e a n a l y s i s o f 41 i l i a c c r e s t b i o p s i e s and 3 a u t o p s i e s a t d i f f e r e n t s t a g e s o f f l u o r o s i s (2-4). These f i n d i n g s i n c l u d e : c o a r s e n i n g and c o n d e n s a t i o n o f t h e s p o n g y b o n e p e r i o s t e a l f o r m a t i o n o f f i b r o u s bone w i t h t r a n s f o r m a t i o n i n t o l a m e l l a r bone l e a d i n g t o t h e f o r m a t i o n o f e x o p h y t e s t h i c k e n i n g and s p o n g i o s a t i o n o f t h e c o r t i c a l i s irregular matrix formation w i t h a high turnover rate f o c i w i t h a coarse f i b r o u s structure, charides, i n some cases, a h i g h c o n t e n t o f mucopolysacand w i t h an i n c l i n a t i o n t o c a l c i f i c a t i o n an i n c r e a s e and t h i c k e n i n g o f t h e o s t e o i d seams sometimes w i t h t r a n s f o r m a t i o n

1.
2.

3.
4.

5.
6.
1. 2.

D i f f e r e n t phenomena w e r e f o u n d e s p e c i a l l y on t h e p e r i o s t e a l s u r f a c e : s u b p e r i o s t e a l f i b r o u s bone f o r m a t i o n , i n t o l a m e l l a r bone new bone f o r m a t i o n i n t o t h e c o n n e c t i v e t i s s u e o f t h e p e r i o s t e u m , m o s t l y i n t o l i g a m e n t s and m u s c u l a r i n s e r t i o n s ( F i g . 2) ossification o f inserting periosteal fibers w i t h transformation i n t o l a m e 1 1 a r bone s o m e t i m e s some f i b r o u s s t r u c t u r e s a r e n o t o s s i f i e d b u t e n c l o s e d by n e w l y f o r m e d bone and t h e n i n c r u s t e d w i t h m i n e r a l s t h e s e c a l c i f i e d f o c i ( i n c r u s t a t i o n f o c i ) a r e t o be f o u n d i n t h e n e w l y formed c o r t i c a l i s j u s t below t h e periosteum; coarse, t h e f o c i a r e PAS p o s i t i v e ( F i g . the fiber structure i s

3.
4.

5.

3) and e x h i b i t a b l u i s h c o l o r

upon s t a i n i n g w i t h a l i z a r i n b l u e and d a r k b l u e w i t h t o l u i d i n e b l u e I n t h e m i c r o r a d i o g r a m t h e s e f o c i show a h i g h m i n e r a l c o n t e n t ( F i g . 4). The s c a n n i n g - e l e c t r o n - m i c r o s c o p i c rosis. f i n d i n g s (SEM)

(5) a r e b a s e d on exam-

i n a t i o n s o f bones o f t h r e e p e r s o n s i n d i f f e r e n t s t a g e s o f i n d u s t r i a l f l u o The s t u d i e s w e r e c a r r i e d o u t o n t h e p e r i o s t e a l and f r a c t u r e s u r t i b i a e and v a u l t s o f cranium. I n t h e case o f beginning a r e a s w i t h s w e l l i n g o r edema o f t h e c o l l a g e n o u s f i b e r s on t h e faces o f ribs, fluorosis,

p e r i o s t e a l s u r f a c e o f t h e r i b w e r e found. Sometimes t h e s e f i b e r s a r e i m p r e g n a t e d w i t h g l o b u l a r and c r y s t a l l i n e material, b u t on t h e s k u l l s u r f a c e areas w i t h d i s t i n c t m i n e r a l i z a t i o n o f c o l l a g e n o u s f i b e r s i n s e r t i n g i n t o t h e bone were a l s o found.

A l l these

337

F i g u r e ?.
p

,:,:e.in 7

New b o n e f o r m a t i o n a t t h e p e r i o s + ? ; l ( + ) I:.!. x 1 2 5

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338

Figure 4. M i c r o r a d i o g r a m o f i l i a c crest, f l u o r o s i s s t a g e 1 1 1 ; typical "calcified focus" ( t ) . x 125

Figure 5. SEM: periosteal surface o f tibia in moderately advanced fluorosis: highly-mineralized, broken-off insertion of a tendon. x 2250

339
s t r u c t u r e s r e p r e s e n t a s t a g e b e t w e e n n o r m a l c o n n e c t i v e t i s s u e and i m m a t u r e , s l i g h t l y m i n e r a l i z e d bone t i s s u e . I n m o d e r a t e f l u o r o s i s i t was o b s e r v e d t h a t t h e p e r i o s t e a l s u r f a c e o f t h e t i b i a had a m o r e p r o n o u n c e d edema o f t h e s t i l l d i r e c t e d c o l l a g e n o u s f i b e r s . The h i g h l y - m i n e r a l i z e d , impressive (Fig. 5). the collagenous f i b e r s are broken-off i n s e r t i o n s o f t e n d o n s i n t o t h e bone w e r e

I n advanced f l u o r o s i s on t h e r i b s u r f a c e , matrix. Under h i g h e r m a g n i f i c a t i o n ,

a b n o r m a l l y t h i n , i r r e g u l a r l y o r i e n t e d and p a r t i a l l y covered b y a t y p i c a l s i n g l e f i b e r s as w e l l as t h e w h o l e bone s u r f a c e h a v e a g r a n u l a r - l i k e c o v e r i n g . I n conclusion, a t f i r s t a s l i g h t edema and i m p r e g n a t i o n w i t h g l o b u l a r and i n severe c r y s t a l l i n e m a t e r i a l i n t h e p e r i o s t e a l c o l l a g e n o u s f i b e r s ' were observed. The edema and i m p r e g n a t i o n s i n c r e a s e a s t h e d i s e a s e a d v a n c e s and, fluorosis, t h i c k c r y s t a l l i n e c o v e r i n g s on t h e bone s u r f a c e o c c u r s . be m i n e r a l ized. Some S E M f i n d i n g s a r e c l o s e l y c o m p a r a b l e w i t h t h e n o r m a l h i s t o l o g y o f fluorosis: The a p p o s i t i o n a l p e r i o s t e a l o s s i f i c a t i o n o b s e r v e d i n b e g i n n i n g f l u o r o s i s corresponds t o t h e i m p r e g n a t i o n o f s o f t t i s s u e i n t h e SEM p i c t u r e , w h i c h i s an i n t e r m e d i a t e s t a g e b e t w e e n c o n n e c t i v e t i s s u e and i m m a t u r e bone. a c o m p l e t e l y i r r e g u l a r o r i e n t a t i o n o f a b n o r m a l l y t h i n f i b e r s and The f i b e r s o f t h e

m u s c u l a r a t t a c h m e n t s and t h e t e n d o n i n s e r t i o n s w e r e o b v i o u s l y t h e f i r s t t o

It c o r r e s p o n d s t o t h e f o r m a t i o n o f i m m a t u r e p e r i o s t e a l f i b r o u s bone
w h i c h a f t e r w a r d s w i l l be t r a n s f o r m e d I n t h i s p r o c e s s t h e t e n d i n o u s and m u s c u l a r i n s e r t i o n s I n t h e normal h i s t o l o g i c a l p i c t u r e , areas w i t h t h e so-called " c a l c i f i e d o r encrustation foci."

i n t h e normal h i s t o l o g i c a l p i c t u r e , i n t o l a m e l l a r bone. a r e a f f e c t e d most severely. t i s s u e were a l s o observed, However, The c o m p l e t e l y a t y p i c a l

p u r e i n c r u s t a t i o n s o f c o n n e c t i v e t i s s u e w i t h o u t t r a n s f o r m a t i o n i n t o bone t h i s phenomenon c a n b e o b s e r v e d m o r e o f t e n i n t h e SEM p i c t u r e . s t r u c t u r e and a r r a n g e m e n t o f c o l l a g e n o u s f i b e r s

on t h e p e r i o s t e a l bone s u r f a c e i n s e v e r e f l u o r o s i s i n t h e SEM p i c t u r e a r e i n accordance w i t h t h e i r r e g u l a r m a t r i x i n t h e normal h i s t o l o g i c a l p i c t u r e . REFERENCES 1. S o r i a n o M ( 1 9 6 5 ) Rev C l i n E s p a n o l a 97:375-388 F r a n k e J (1972) F l u o r i d e 8:61-83 4. 5. F r a n k e J, H o r n V (1976) F l u o r i d e 9:127-137 F r a n k e J. R u n g e H, F e n g l e r F ( 1 9 7 8 ) I n : C o u r v o i s i e r B. D o n a t h A, Baud, CA ( e d s ) Symposium CEMO 11: F l u o r i d e and Bone. Ed M'edecine e t H y g i e n e , Geneva, p p 129-143 F l u o r i d e 5:182-198 R u n g e H, F e n g l e r F, A u e r m a n n E, Lenart G (1975) F r a n k e J , R a t h F.

2.
3.

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 341-346 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

341

TOPOGRAPHICAL LOCALIZATION OF FLUORIDE I N BONE TISSUE

SEONG BANG AND CHARLES A.

BAUD
1. r u e M i c h e l -

I n s t i t u t de M o r p h o l o g i e . C e n t r e M i d i c a l U n i v e r s i t a i r e , Servet, 1211 Genhve 4, S w i t z e r l a n d ABSTRACT

The e l e c t r o n m i c r o p r o b e was used t o demonstrate t h e t o p o g r a p h i c a l d i s t r i b u t i o n o f f l u o r i d e (F) a t t h e m i c r o s c o p i c a l l e v e l i n a n i m a l and human bone tissues. The f e m o r a l bones o f w e a n l i n g m i c e m a i n t a i n e d on f l u o r i d a t e d d r i n k i n g w a t e r f o r p e r i o d s o f up t o 12 months were analyzed. graph o f F
Kcl

The c o m p o s i t e photoModerate

X-ray images showed heavy d e p o s i t s o f F i n t h e p e r i o s t e a l and

e n d o s t e a l a r e a s o f bone t i s s u e f o r m e d d u r i n g t h e e x p e r i m e n t . t h e b e g i n n i n g o f t h e experiment.

q u a n t i t i e s o f F were a l s o found i n t h e remnants o f woven bone formed b e f o r e I n t h e i l i a c bone b i o p s i e s f r o m o s t e o p o r o t i c p a t i e n t s t r e a t e d w i t h 12 months, the treatment,

for

a h i g h l e v e l o f F was observed i n t h e bone l a y e r s formed d u r i n g whereas i n t h e l a m e l l a r bone t i s s u e formed b e f o r e t r e a t m e n t ,

t h e F l e v e l was f o u n d t o b e s i m i l a r t o t h a t i n t h e c o n t r o l s e x c e p t i n a t h i n layer l i n i n g the surface i n the v i c i n i t y o f the c a p i l l a r y blood vessels. Thus, i t c o u l d be p o s t u l a t e d t h a t s y s t e m i c a l l y i n g e s t e d F i s d e p o s i t e d p r i m a r i l y i n t h e s i t e s o f new bone formed d u r i n g t h e p e r i o d s o f F i n g e s t i o n i n b o t h cases, and a l s o s e c o n d a r i l y b y a d i f f u s i o n p r o c e s s i n t h e w h o l e p r e - e x i s t i n g woven bone i n m i c e , and i n a n a r r o w s u b s u r f a c e b a n d o f t h e l a m e l l a r bone formed b e f o r e t r e a t m e n t i n human s u b j e c t s . INTRODUCTION S y s t e m i c a l l y i n g e s t e d f l u o r i d e (F) i s e i t h e r e x c r e t e d i n t h e u r i n e and feces, o r d e p o s i t e d p r i n c i p a l l y i n t h e c a l c i f i e d t i s s u e s o f t h e body. F has been employed i n t h e A l t h o u g h e x c e s s i v e i n t a k e may induce f l u o r o s i s , Thus,

t r e a t m e n t o f o s t e o p o r o s i s and i n t h e p r o p h y l a x i s a g a i n s t d e n t a l c a r i e s . t h e r e i s a c o n s i d e r a b l e i n t e r e s t i n t h e l o c a l i z a t i o n o f absorbed F. This i n t e r e s t arises from both the possible therapeutic o r prophylactic a c t i o n o f t h i s e l e m e n t and a l s o t h e p o t e n t i a l h a z a r d o f an i n c r e a s e d F accumulation i n t h e c a l c i f i e d tissues. T h i s paper i s a r e p o r t on t h e F d i s t r i b u t i o n p a t t e r n o f t h e bones o f m i c e w h i c h had been g i v e n F i n t h e i r drinking water, and o f i l i a c c r e s t

342 b i o p s i e s t a k e n f r o m o s t e o p o r o t i c p a t i e n t s who had been t r e a t e d w i t h sodium fluoride. MATERIALS AND METHODS E x p e r i m e n t a l Animals 21-day-old m i c e were m a i n t a i n e d on f l u o r i d a t e d d r i n k i n g w a t e r (100 ppm F) f o r up t o 12 months.

A t t h e b e g i n n i n g o f t h e experiment,
The femoral

t e t r a c y c l i n e was bones o f m i c e

i n j e c t e d i n t r a p e r i t o n e a l l y f o r bone l a b e l i n g . r a y m i c r o a n a l y s i s (1-4). Human Bone B i o p s i e s I l i a c c r e s t biopsies,

m i croprobe.

s a c r i f i c e d a t d i f f e r e n t p e r i o d s o f t i m e were p r e p a r e d f o r e l e c t r o n probe X-

t a k e n f r o m o s t e o p o r o t i c p a t i e n t s who had been were analyzed by e l e c t r o n

t r e a t e d w i t h NaF ( 3 0 mg F / d a y ) f o r 1 2 months,

RESULTS A c o m p o s i t e X-ray image o f F Ka e m i s s i o n o f t h e f e m o r a l bone o f a mouse g i v e n f l u o r i d a t e d w a t e r f o r 45 days showed d e p o s i t s o f F i n t h e p e r i o s t e a l and e n d o s t e a l l a y e r s , as i n d i c a t e d b y a r r o w s a and b i n F i g u r e 1. These l a y e r s a r e l a b e l e d w i t h t e t r a c y c l i n e as shown i n F i g u r e 2, months) F - i n g e s t e d mice, corresponding t o t h e bone t i s s u e formed d u r i n g t h e e x p e r i m e n t a l period. I n t h e l o n g - t e r m (12 t h e F was d i s t r i b u t e d r a t h e r h o m o g e n e o u s l y t h r o u g h o u t t h e bone s e c t i o n s i n c e t h e woven bone was b e i n g r e p l a c e d by new l a m e l l a r b o n e ( F i g s . 3 a n d 4). A n a l y s i s o f t h e human bone b i o p s i e s showed a h i g h c o n c e n t r a t i o n o f F i n t h e p e r i o s t e a l and e n d o s t e a l l a y e r s , osteons. i n t h e t r a b e c u l a r bone, and i n c e r t a i n However, These were a l l formed d u r i n g t h e p e r i o d o f F-treatment.

i n t h e l a m e l l a r b o n e t i s s u e f o r m e d b e f o r e t h e t r e a t m e n t , t h e F l e v e l was f o u n d t o be s i m i l a r t o t h a t i n t h e c o n t r o l s e x c e p t i n a t h i n l a y e r l i n i n g the surface i n the v i c i n i t y o f t h e c a p i l l a r y blood vessels ( F i g s . 5 and

6).
DISCUSSION
Chemical analyses o f t h e F c o n t e n t o f whole bone as used by Weidmann and Weatherell (5) t o i n v e s t i g a t e t h e uptake o f F i n t h e various s k e l e t a l t i s s u e s o f r a b b i t d o n o t p r o v i d e d e t a i l s on t h e d i s t r i b u t i o n o f F i n t h e bone t i s s u e i t s e l f .

By u t i l i z i n g t h e r a d i o a c t i v e i s o t o p e o f f l u o r i n e (F18) f o r t h e a u t o r a d i o g r a p h i c s t u d i e s i n b o n e s and t e e t h , V o l k e r d.(6).

W a l l a c e - D u r b i n (7). Perkinson

ad. (8)

and E r i c s s o n

gfl.( 9 )

reported

t h a t i n e x p e r i m e n t a l a n i m a l s t h e a r e a o f g r e a t e s t F18 c o n c e n t r a t i o n was i n

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346 t h e most r e c e n t l y c a l c i f i e d bone, and t h e y i n d i c a t e d t h a t t h e a c c u m u l a t i o n

i n b o n e seemed t o d e p e n d u p o n t h e v a s c u l a r i t y o f t h e bone a n d i t s g r o w t h a c t i v i t y . Although t h e autoradiographic technique i s n o t s u i t a b l e f o r a long-term F-experiment because o f t h e s h o r t h a l f - l i f e o f F18, t h e i r findings are q u i t e s i m i l a r t o those o f t h e e l e c t r o n microprobe analysis. I n t h e p r e s e n t study, a t t e m p t s w e r e made t o show some o f t h e X-ray e m i s s i o n images i n mosaic f o r m t o p r o v i d e a maximum o f i n f o r m a t i o n f r o m e x t e n s i v e a r e a s o f t h e bone s e c t i o n . The

distribution pattern o f the The

e x p e r i m e n t a l mouse was s i m i l a r t o t h a t o f t h e human bone t i s s u e . b l o o d v e s s e l s t o t h e bone 1ayers. Thus,

i t may be p o s t u l a t e d t h a t s y s t e m i c a l l y - i n g e s t e d

i n g e s t e d F d e p o s i t e d i n t h e n e w l y f o r m e d bone: F a l s o d i f f u s e d f r o m t h e

i s deposited

p r i m a r i l y i n t h e s i t e s o f new bone formed d u r i n g t h e p e r i o d s o f e x i s t i n g woven bone i n mice,

ingestion

i n b o t h cases, and, s e c o n d a r i l y , b y a d i f f u s i o n p r o c e s s i n t h e w h o l e p r e and i n a n a r r o w s u b s u r f a c e band o f t h e l a m e l l a r bone formed b e f o r e t r e a t m e n t i n human subjects. REFERENCES 1. Bang S (1976) Revue mens s u i s s e Odonto-Stomatol 86:838-863

2.
3. 4.

Bang S ( 1 9 7 8 ) I n : C o u r v o i s i e r B, D o n a t h A, Baud CA ( e d s ) F l u o r i d e and Bone. Huber, Bern. pp 77-81 Baud CA, Bang S (1970) I n : Huber. Bern, pp 27-36 V i s c h e r ThL (ed) F l u o r i d e i n M e d i c i n e .

Baud CA, Bang S (1972) I n : S h i n o d a G. K o h r a K, I c h i n o k a w a T ( e d s ) Proceedings o f t h e S i x t h I n t e r n a t i o n a l Conference on X-ray O p t i c s and Microanalysis. U n i v e r s i t y o f Tokyo Press, pp 841-846. Weidmann SM, W e a t h e r e l l JA (1959) J P a t h o l B a c t e r i o l 78:243-255 V o l k e r JF, Sognnaes RF. B i b b y GB (1941) Amer J P h y s i o l 132707-712 Wallace-Durbin P (1954) J Dent Res 33:789-800 P e r k i n s o n JD, W h i t n e y I B . M o n r o e RA. L o t z WE, Comar CL (1955) Amer J P h y s i o l 182:383-359 E r i c s s o n Y, U l l b e r g S. A p p e l g r e n LE ( 1 9 6 0 ) A c t a O d o n t o l Scand 18:253261

5.
6. 7. 8. 9.

Fluoride Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 347-355 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in T h e Netherlands

H. Tsunoda and M.-H. Y u (Editors)

347

BONE STATIC AND D Y N A M I C HISTOMORPHOMETRY IN E N Q E M I C SKELETAL FLUOROSIS

SURENDRA P.

S. TEOTIA, M O H I N I TEgTIA, AND DHARAM P. S I N G H

Lala Lajpat

P o s t G r a d u a t e D e p a r t m e n t o f Human M e t a b o l i s m and E n d o c r i n o l o g y , R a i M e m o r i a l M e d i c a l C o l l e g e , M e e r u t - 2 5 0 102, I n d i a

ABSTRACT Quantitative tetracycline-based a n a l y s i s o f i l i a c bone b i o p s y was p e r f o r m e d i n 17 p a t i e n t s w i t h endemic s k e l e t a l f l u o r o s i s . endemic f l u o r o s i s areas s i n c e t h e i r b i r t h . d u r a t i o n o f symptoms 7.6 y e a r s . :!ster

A l l had l i v e d i n

The mean age was 25.6 y e a r s and None

The mean f l u o r i d e c o n t e n t o f t h c d r i n k i n g i n t e s t i n a l malabsorption o r renal o f fluoride,

was 17.8 ppm and t h e mean d a i l y i n t a k e o f f l u o r i d e was 26.2 mg.

o f t h e p a t i e n t s had V i t a m i n D d e f i c i e n c y , dysfunction.

P e r t i n e n t b i o c h e m i c a l f i n d i n g s w e r e : r a i s e d p l a s m a le?.ls a l k a l i n e p h o s p h a t a s e and iPTH.

and i n c r e a s e d u r i n a r y f l u o r i d e e x c r e t i o n .

S t a t i c and d y n a m i c h i s t o m o r p h o m e t r i c m e a s u r e m e n t s r e v e a l e d t h e p r o f i l e s o f o s t e o m a l a c i a and s e c o n d a r y h y p e r p a r a t h y r o i d i s m i n v a r y i n g c o m b i n a t i o n s i n all cases. I n c r e a s e s i n o s t e o i d volume, osteoid surfaces, o s t e o i d seams w i d t h , r e s o r p t i o n s u r f a c e s and m i n e r a l i z a t i o n l a g t i m e and d e c r e a s e s i n corrected apposition rate, b o n e f o r m a t i o n r a t e s and t e t r a c y c l i n e l a b e l e d s u r f a c e s w e r e t h e v a r i a b l e f e a t u r e s r e c o r d e d i n each case. D y n a m i c bone h i s t o r n o r p h o m e t r y has f u r t h e r i n c r e a s e d t h e u n d e r s t a n d i n g o f t h e e f f e c t s o f f l u o r i d e on i n t r a s k e l e t a l m e c h a n i s m s a f f e c t i n g t h e s t r u c t u r e and m e t a b o l i s m o f t h e bone b u t i t d o e s n o t a l l o w c l e a r - c u t d i f f e r e n t i a t i o n f r o m m e t a b o l i c bone d i s e a s e . End>mic s k e l e t a l f l u o r o s i s , therefore, may be r e g a r d e d as a p a r a m e t a b o l i c bone d i s e a s e .

INTRODUCTION Endemic s k e l e t a l f l u o r o s i s c o n t i n u e s t o r e m a i n a c h a l l e n g i n g n a t i o n a l h e a l t h p r o b l e m i n m o s t p a r t s o f t h e \ w o r l d (1). s t u d i e s on i t s c l i n i c a l . biochemical, metabolic, a s p e c t s h a v e been r e p o r t e d . s k e l e t a l f l u o r o s i s a r e sparse.

I n e a r l i e r w o r k s (2-8).

e n d o c r i n e and r a d i o l o g i c a l

R e p o r t s o n bone h i s t o m o r p h o m e t r y i n e n d e m i c The o n l y r e p o r t s p r o v i d i n g a d e q u a t e d a t a on

human e n d e m i c s k e l e t a l f l u o r o s i s a r e t h o s e o f T e o t i a and T e o t i a (9-13).

348

The hi stopathogenesi s and hi stomorphometry of bone disease produced by chronic fluoride intoxication still remain incompletely understood. There appear to be no published reports o n human e n d e m i c skeletal fluorosis employing a comprehensive histomorphometric analysis of non-decalcified sections of bone that have been labeled at mineralization zones with double tetracycline labels. This extended work on quantitative analysis of labeled iliac crest biopsies will serve t w o major needs: 1. Is bone hlstomorphometry useful for the diagnosis of skeletal fluoro51 5 7

2 . To achieve a better understanding of bone disease and toxic effects of fluoride on intraskeletal mechanisms, structure and metabolism of the bone.
MATERIALS AND METHODS Seventeen patients of endemic skeletal fluorosis were studied during the period 1972-1984 (Table I). since their hi rth.

All had been living in endemic fluorosis areas

TABLE I CLASSIFICATION OF SEVERITY OF FLUOROSIS C1 inical

Mi

Id:

Generalized bone and joint pains Generalized bone and joint pains, stiffness and rigidity,

Moderate:

restricted movements at spine and joints. Severe: Symptoms of moderate with deformities of spine and limbs,

knock knees, crippl ing/bed-ridden state.

Mi Id:
Mod era te :

Radiological Only osteosclerosis Osteosclerosis. periosteal bone formation, calcifications o f interosseous membrane, ligaments. capsules, muscular attachments, tendons. Findings as in moderate, exostoses, osteophytosis and

Severe:

associated metabol i c bone disease.

349
P e r t i n e n t 1a b o r a t o r y i n v e s t i g a t i o n s i n c l u d e d d e t e r m i n a t i o n s >f p l a s m a fluoride, (IPTH). calcium, phosphate, alkaline phosphatase, calcium, 25creh y d r o x y c h o l e c a l c i f e r o l (25-OHD), Twenty-four a t i n i n e c l e a r a n c e (Ccr), a n d i m m u n o r e a c t i v e p a r a t h y r o i d hornlo:? and t u b u l a r r e a b -

hour u r i n e d e t e r m i n a t i o n s o f f l u o r i d e . n e p h r o g e o u s c y c l i c AMP (Nc'AMP)

s o r p t i o n o f p h o s p h a t e (TRP) w e r e made. accepted p u b l i s h e d procedures. Bone b i o p s y :

A l l t h e e s t i m a t i o n s w e r e made u s i n g

F l u o r i d e c o n c e n t r a t i o n s were measured w i t h

a f l u o r i d e s e l e c t r o d e u s i n g a PHM64 r e s e a r c h r a d i o m e t e r . P r i o r t o b i o p s y e a c h p a t i e n t was g i v e n o x y t e t r a c y c l i n e f o l l o w e d b y an 11 day i n t e r v a l and t h e n d e m e c l o c y c l i n e h y d r o c h l o r i d e 300 mg 8 h o u r l y h y d r o c h l o r i d e 500 mg 8 h o u r l y f o r 3 d a y s , w i t h no m e d i c a t i o n , f o r 3 days.

T h r e e d a y s a f t e r t h e s e c o n d l a b e l a f u l l t h i c k n e s s b i o p s y was T h i s produced a c o r d o f bone w i t h c o r t e x a t each The w h o l e s p e c i m e n was p r e embedding, staining,

o b t a i n e d f r o m t h e i l i a c c r e s t u n d e r l o c a l a n e s t h e s i a u s i n g a 7.5 m m i n t e r nal diameter trephine. end, t r a b e c u l a r bone and m a r r o w i n between.

served i m m e d i a t e l y i n 70 p e r c e n t e t h a n o l . S e c t i o n i n g , u s i n g s t a n d a r d p r o c e d u r e s and p r i n c i p l e s (10). s t u d i e d i n c l u d e d s t r u c t u r a l measurements, and r e s o r D t i o n i n d i c e s . RESULTS

m i c r o s c o p i c e x a m i n a t i o n and q u a n t i t a t i v e h i s t o m o r p h o m e t r y w e r e p e r f o r m e d The v a r i o u s p a r a m e t e r s formation s u r f a c e measurements,

The d i a g n o s i s o f e n d e m i c s k e l e t a l f l u o r o s i s was c o n f i r m e d i n a l l p a t i e n t s b y c h a r a c t e r i s t i c r a d i o l o g i c a l f i n d i n g s and h i g h c o n c e n t r a t i o n s o f f l u o r i d e i n t h e i r p l a s m a and u r i n e and i n d r i n k i n g w a t e r . The c l i n i c a l and r a d i o l o g ical severity o f skeletal f l u o r o s i s was c l a s s i f i e d a s m i l d , m o d e r a t e and

20

40

6 0 00

1 0 0

( % TOTAL s uR FAC E )

OSTEOID SURFACE

CORRECTED

-0.2
0.4

0.6

08 1 . 0

I?

APPOSITION

RATE

(ym/d)

F i g u r e 1. Bone h i s t o m o r p h o m e t r i c i n d i c e s i n e n d e m i c s k e l e t a l f l u o r o s i s a s c o m p a r e d t o t h e p a r a m e t e r s seen i n m e t a b o l i c bone d i s e a s e .

cn

TABLE I 1 CLINICAL DETAILS OF PATIENTS STUDIED Case Age No. (Yrs) Fluoride i n water (PPm) Fluoride intake (rng/d) Duration o f symptoms (Years) Severity o f Skeletal Fluorosis Clinical Severe Severe Severe Severe Severe Severe Severe Moderate Moderate Radiological Severe Severe Severe Severe Severe Severe Severe Moderate Severe Moderate Severe Severe Severe Severe Severe Mild Dental F 1u o r o s i s (Grade)

Sex

20 10 23 21 65 13 30 32 35 42 15

25 25 25 25 25 25 25 25 25 8.5 25 18 8 6.5 8.5 5.5 4.5

36 39 30 28 50 32 40 36 32 12 27 20 16 14 21 11

2 3 4 5 6
7

M
M

M M
M M F

3 3 4
20 10

3
3

3 4

0
4 3 3 3 2 1 2 2 3 1
1

8 9

6 5
6 4

F
F F F

10
11

MiI d
Severe Severe Severe Moderate Severe Moderate

12 13 14 15 16 17

9 18
23 30 30
29

3
6 20 15 5
6

F
M F

F
F

MiId

MiI d

351

TABLE 111 PLASMA COMPOSITION

Case Fluoride
No.

(wM/1)
~

Calcium (mg/dl) 9.5 10.0 9.6 10.2 11.0 10.0 9.0 9.8 10.5 10.0 9.5 9.8 7.6 8.4 1.7 10.4 8.9 3.5 4.0 3.7 4.8 3.0 4.2 4.0 3.2 4.0 3.4 3.2 5.8 5.0 4.2 3.6 3.8
4.9

Alk Ptase (KAU/dl)

35 30 16 31 76 57 46 32 25 28 45 45 53 18 116 27 1" 15.0 16.5 12.0 17.5 23.0 18.0 14.0 25.5 20.0 15.0 12.0 12.0 14.0 20.0 16.0 14.0 l".1400 2275 1300 1150 5050 2700 1225 1350 1050 1200 1625 1500 900 1200 2800 ~600 1lQO 18 20 15 16 22 20 15 30 22 1 8 16 1 8 19 30 16 22 15

1 2 3 4 5 6 7 8 9 10 11

12 13 14 15 16 17

8.5 12.0 1.5 5.8 12.7 10.5 8.0 5.6 6.6 3.2 9.5 4.2 6.5 10.2 7.5 3.2 3.4

Figure 2. Wide osteoid seams and hook resorption of the trabeculae. Findi n g s are suggestive o f osteomalacia a n d secondary hyperparathyroidism (undecalcified villanueva osteochrome section 100 x ) .

352

TABLE I V URINARY COMPOSITION

-

Case

No.

Fluoride (mg/d)

Calcium
(mg/d)

TRP
(%)

Nc'AMP (nm/mg Cr) 1.32 6.75 3.20 2.23 5.56 4.89 1.29 1.35 4.25 1.39 2.85 2.62 1.65 3.85 7.95 2.25 1 . 7 '

Ccr (ml/min) 130 120 128 120 125 123 126 118 120 135 116 122 135 126 122 134
1 q,>

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

11.0 16.8 5.0 13.4 36.0 15.0 13.0 9.4 5.8 5.8 13.0 1.5 11.5 9.2 6.8 6.8 5.4

41 62 32 40 76 56 35 46 50 44 58 57 53 68 85 74 130

78 64 80 70 68 75 82 83 68 85 74 82 78 84 51 82 86

F i g u r e 3. Undecalcified section s a m e a s Figure 2 . Photomicrograph taken under fluorescence microscope revealed only a few areas of tetracycline labels indicating lack of mineralization zones.

353
severe (Table I). rigidity,

A l l w e r e s y m p t o m a t i c and had vague p a i n s , s t i f f n e s s ,

backache, restricted joint The g r o s s l y l i m i t e d movements o f

and l i m i t a t i o n s i n s p i n a l movements,

movements and i n a b i l i t y t o c l o s e f i s t s . f l u o r o s i s was p r e s e n t i n 11 p a t i e n t s .

t h e spine, f l e x i o n d e f o r m i t i e s a t t h e h i p s and knees s u g g e s t t h a t severe C l i n i c a l and b i o c h e m i c a l o b s e r v a t i o n s a r e summarized i n Tables I

-

I V and

r e s u l t s o f h i s t o m o r p h o m e t r y a r e summarized i n T a b l e V and F i g u r e s 1-4. TABLE V

BONE HISTOMORPHOMETRIC MEASUREMENTS

S t a t i c Variables O s t e o i d Volume ( % TT) O s t e o i d S u r f a c e (%TS) O s t e o i d Seam Width (u) R e s o r p t i o n Lacunae w i t h O s t e o c l a s t s ( % TBS) Dynamic V a r i a b l e s C o r r e c t e d a p p o s i t i o n r a t e (urn/d) Mineralization lag time (d) Labeled s u r f a c e ( % 0s) Bone f o r m a t i o n r a t e (pm3/um2)

(MEAN f SD)

E. S. F.
4.64 f 1.97 55.04 18.78 30.29 f 21.53 4.00 f 3.58
_+

Normal 1.8 13.5 11.25 2.9 0.7 t 9.0 f 5.15 f 0.2

f

0.30 0.14 276.50 f 165.62 8.89 21.17 5.20 f 3.01

+_ +_

0.7 0.2 26.25 f 10.15 63.5 f 17.2 21.5 f 16.2

F i g u r e 4. Radiograph showing g e n e r a l i z e d o s t e o s c l e r o s i s and m u l t i p l e c y s t i c r e s o r p t i o n a r e a s . The p r e s e n c e o f h y p e r p a r a t h y r o i d i s m s e c o n d a r y t o t h e f l u o r o s i s i s suggested.

354 DISCUSSION
C l i n i c a l d e t a i l s o f t h e p a t i e n t s s t u d i e d a r e g i v e n i n T a b l e 11. t h e p a t i e n t s had d i e t a r y v i t a m i n - D renal dysfunction. deficiency, None o f i n t e s t i n a l malabsorption o r Twenty-four hour u r i n e

P l a s m a b i o c h e m i c a l f i n d i n g s showed r a i s e d f l u o r i d e , TRP was l o w and c r e a t i n i n e c l e a r a n c e

a l k a l i n e p h o s p h a t a s e and iPTH l e v e l s ( T a b l e 111). f l u o r i d e and Nc'AMP w e r e i n c r e a s e d , was n o r m a l ( T a b l e I V ) .

Q u a n t i t a t i v e m e a s u r e m e n t s w e r e made o f s t a t i c and d y n a m i c v a r i a b l e s on undecalcified sections o f tetracycline-based i l i a c c r e s t biopsies obtained The p a r a m e t e r s f r o m 1 7 p a t i e n t s o f e n d e m i c s k e l e t a l f l u o r o s i s ( T a b l e V). r o i d i s m i n v a r y i n g c o m b i n a t i o n s i n a l l cases. Bone h i s t o m o r p h o m e t r i c m e a s u r e m e n t s showed i n c r e a s e d v a l u e s o f o s t e o i d volume, o s t e o i d surfaces, o s t e o i d seams w i d t h and r e s c r p t i o n l a c u ' n a e cont a i n i n g o s t e o c l a s t s . D y n a m i c m e a s u r e m e n t s r e v e a l e d d e c r e a s e d a p p o s i t i o n and bone f o r m a t i o n r a t e s , l a g t i m e ( F i g s . 1-3). t e t r a c y c l i n e l a b e l i n g and i n c r e a s e d m i n e r a l i z a t i o n I n t h e p r e s e n c e o f d e p r e s s e d a p p o s i t i o n and bone Bone h i s t o m o r p h o m e t r i c intakes,

s t u d i e d r e v e a l e d t h e p r o f i l e s o f o s t e o m a l a c i a and s e c o n d a r y h y p e r p a r a t h y -

formation rates. osteosclerosis i n skeletal fluorosis could r e s u l t from the increased volume o f p a r t i a l l y m i n e r a l i z e d osteoid. plasma l e v e l s o f f l u o r i d e , changes i n a l l t h e cases s t u d i e d c o r r e l a t e d w i t h d a i l y f l u o r i d e f i n d i n g s i n t h e i r s k e l e t a l radiographs (Fig. I n p r e v i o u s r e p o r t s (9-13).

a l k a l i n e p h o s p h a t a s e and iPTH a n d a l s o w i t h t h e

4).

i t was shown t h a t t h e h i s t o p a t h o l o g i c a l

p i c t u r e o f s k e l e t a l f l u o r o s i s w a s c h a r a c t e r i z e d b y i n c r e a s e d o s t e o i d and supernormal p r o p o r t i o n s o f i n c o m p l e t e l y m i n e r a l i z e d bone ( o s t e o m a l a c i a ) . i n c r e a s e d t r a b e c u l a r r e s o r p t i o n o f bone w i t h o s t e o c l a s t s and m a r r o w f i b r o s i s (secondary hyperparathyroidism). d o m i n a n t l y woven bone, l a m e l l a r o r i e n t a t i o n o f t h e bone. appear t h i c k w i t h excess calcium. reported i n patients o f industrial Histomorphometric increased production o f immature preI n u n c o m p l i c a t e d c a s e s t r a b e c u l a e may S i m i l a r h i s t o l o g i c a l f e a t u r e s h a v e been f l u o r o s i s (14). fluorosis did not p o o r l y f o r m e d h a v e r s i o n s y s t e m s and d i s o r d e r e d

a n a l y s i s o f i l i a c bone i n s k e l e t a l

p r o v i d e any p r e c i s e o r c h a r a c t e r i s t i c i n f o r m a t i o n t o p e r m i t p e n e t r a t i n g s e p a r a t i o n f r o m m e t a b o l i c bone disease. profiles, however. H i s t o m o r p h o m e t r i c and dynamic understanding, and i n s i g h t

provided a better viewpoint,

i n t o t h e t o x i c e f f e c t s o f c h r o n i c i n g e s t i o n o f n a t u r a l f l u o r i d e on i n t r a s k e l e t a l mechanisms, s t r u c t u r e and m e t a b o l i s m o f t h e bone.

This informa-

t i o n has a l l o w e d t h e i n s t i t u t i o n o f a p p r o p r i a t e p r e v e n t i v e and t h e r a p e u t i c m e a s u r e s a d a p t e d t o f l u o r i d e i n d u c e d bone d i s e a s e ( o s t e o m a l a c i a and seconda r y h y p e r p a r a t h y r o i d ism).

355
It i s b e l i e v e d t h a t s i m i l a r h i s t o d y n a m i c b e h a v i o r o f bone may o c c u r i n p a t i e n t s o f o s t e o p o r o s i s t r e a t e d w i t h sodium f l u o r i d e , and o s t e o m a l a c i a and s e c o n d a r y h y p e r p a r a t h y r o i d i s m may be t h e p o t e n t i a l r i s k s o f t h i s t h e r a p y . ACKNOWLEDGEMENT We a r e g r a t e f u l t o P r o f e s s o r A. M. P a r f i t t . D i r e c t o r , B o n e a n d M i n e r a l D i v i s i o n , H e n r y F o r d H o s p i t a l . D e t r o i t , USA, a n d t o l a t e P r o f e s s o r C. h e l p i n t h e s t u d y o f t h e bone h i s t o m o r p h o m e t r y and t h e d e t e r m i n a t i o n s 25-OHD and p a r a t h y r o i d hormone. REFERENCES 1. 2. 3. 4. 5. T e o t i a SPS. T e o t i a SPS, T e o t i a M, T e o t i a SPS, T e o t i a M ( 1 9 8 4 ) J Assoc Phys I n d i a 32:347-352 K u n w a r KB, T e o t i a SPS, T e o t i a M (1969) F l u o r i d e 2:142-152 Kunwar K B ( 1 9 7 1 ) A r c h D i s C h i l d 46:686-691 K u n w a r K B ( 1 9 7 3 ) I n : F r a m e 6, P a r f i t t A M a n d

E.
of

D e n t o f U n i v e r s i t y C o l l e g e H o s p i t a l M e d i c a l S c h o o l London f o r t h e i r k i n d

T e o t i a M ( 1 9 7 3 ) B r i t Med J 1:637-640

T e o t i a M, T e o t i a SPS,

Duncan H ( e d s ) C l i n i c a l A s p e c t s o f M e t a b o l i c Bone D i s e a s e . E x c e r 7 t a M e d i c a , Amsterdam, pp 232-238

6. T e o t i a SPS, T e o t i a M, S i n g h RK, T a v e s OR. H e e l s S ( 1 9 7 8 ) J A s s o c P h y s

I n d i a 26:995-1000 7. T e o t i a SPS. T e o t i a M. S i n g h R K , T e o t i a N P S , T a v e s DR, H e e l s S, D ' M e l low,

V P ( 1 9 7 8 ) F l u o r i d e 12:115-119
T e o t i a SPS, S i n g h RK (1979) F l u o r i d e 12:58-64 S i n g h CV. Tomar NPS (1984)

8.
9. 10. 11.

T e o t i a M,

T e o t i a SPS, T e o t i a M, S i n g h DP, A n a n d V. F1 u o r i d e 17: 14-22 F a c c i n i JM, T e o t i a SPS,

T e o t i a SPS (1974) C a l c T i s s Res 16:45-57 T e o t i a M, R o h t a g i VK, T e o t i a N P S ( 1 9 7 4 ) J I n d i a n Med

ASSOC

63:207-211

12. 13.
14.

Teotia

SPS, T e o t i a M.
T e o t i a M.

S i n g h RK, T e o t i a NPS ( 1 9 7 4 ) I n : P r o c e e d i n g s o f Hyderabad, I n d i a , p p 425-434 T e o t i a NPS (1976) F l u o r i d e 9:91-98

t h e s y m p o s i u m on f l u o r o s i s . T e o t i a SPS,

F r a n k e J ( 1 9 7 2 ) F l u o r i d e 5:182-199

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 357-367 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

357

I N V E S T I G A T I O N S ON THE RELEVANCE O F DEFLUORIDATED WATER AND NUTRITIONAL SUPPLEMENTS I N FLUOROSIS ENDEMIC AREAS I N ANDHRA PRADESH, I N D I A I(I3LA RAJYALAKSHMI,

N. V.

RAMAMOHAN RAO, AND NEELAM K R I S H N A

I n s t i t u t e o f P r e v e n t i v e M e d i c i n e , P u b l i c H e a l t h L a b o r a t o r i e s and Food ( , i e a l t h ) A u t h o r i t y . A.P., H y d e r a b a d 500 029 I n d i a

ABSTRACT Nalgonda i n Andhra P r a d e s h i s one o f t h e d i s t r i c t s i n I n d i a w h i c h i s severely a f f e c t e d by f l u o r o s i s . disease. Several hundred pcople a r e c r i p p l e d b y t h e C o n t r o l l e d s t u d i e s w e r e made t o d e t e r m i n e t h e e f f e c t i v e n e s s o f Seventy-two p a t i e n t s i n each o f f o u r

p r o v i d i n g d e f l u o r i d a t e d d r i n k i n g w a t e r and n u t r i t i o n a l supplements b o t h s e p a r a t e l y as w e l l as i n combination. v i l l a g e s who w e r e i n t h e age g r o u p s o f 1-5,

5-10,

10-18.

and above 1 8 y e a r s a n d biochemi-

o l d w e r e p r o v i d e d d e f l u o r i d a t e d w a t e r and n u t r i t i o n a l s u p p l e m e n t s f o l l o w i n g b a s e l i n e s t u d i e s o n d i e t a r y i n t a k e and c l i n i c a l , ical profiles. radiological, The r e s u l t s o f i n v e s t i g a t i o n s c o n d u c t e d o n c e i n t h r e e

m o n t h s e a c h y e a r a f t e r commencement o f t h e i n t e r v e n t i o n p r o g r a m w e r e comp a r e d w i t h i n i t i a l data.

A g r a d u a l r e l i e f i n t h e c l i n i c a l symptonis o f b o d y p a i n s and j o i n t p a i n s
was o b s e r v e d . slow. Radiological p r o f i l e s o f the patients indicated that fluor i l e r e m o b i l i z a t i o n f r o m bones i s r e v e r s i b l e b u t t h e r a t e o f r e m o v a l i s S t a t i s t i c a l evaluation o f biochemical data revealed s i g n i f i c a n t The r e d u c t i o n i n a l k a l i n e p h o s p h a t a s e and s e r u m f l u o r i d e c o n c e n t r a t i o n s .

r e s u l t s o f t h e i n v e s t i g a t i o n h a v e shown t h a t t h e a f f l i c t i o n c a n be s i g w i f i c a n i l y reduced through t h e simultaneous consumption o f d e f l u o r i d a t e d d r i n k i n g w a t e r and n u t r i t i o n a l s u p p l e m e n t s .

INTRODUCTION 5jalgonda d i s t r i c t i n A n d h r a P r a d e s h i s one o f t h e a r e a s i n I n d i a w h i c h i s severely a f f e c t e d by f l u o r o s i s .

A number o f v i l l a g e s i n t h i s d i s t r i c t h a v e
Epidemiological surveys revealed

e x c e s s i v e f l u o r i d e i n t h e i r d r i n k i n g w a t e r and s e v e r a l t h o u s a n d i n h a b i t a n t s are suffering from skeletal fluorosis. t h a t persons o f l o w income g r o u p s w i t h i n a d e q u a t e n u t r i t i o n a r e more a f f e c t e d t h a n t h o s e i n h i g h e r i n c o m e g r o u p s who h a v e a m o r e b a l a n c e d d i e t .
!t i s a l s o o b s e r v e d t h a t a f f e c t e d p e o p l e a s w e l l a s c a t t l e r e c o u p t h e i r

h e a l t h on m i g r a t i o n t o a r e a s w h e r e t h e f l u o r i d e c o n t e n t i n w a t e r i s l o w .

358
As a r e s u l t o f t h e s e o b s e r v a t i o n s and o t h e r r e p o r t s i n t h e l i t e r a t u r e (1-

5). t h e e f f e c t i v e n e s s o f p r o v i d i n g n u t r i t i o n a l s u p p l e m e n t s a n d d e f l u o r i dated water separately, was s t u d i e d . MATERIALS AND METHODS as w e l l as i n c o m b i n a t i o n , t o fluorosis patients who h a d b e e n c o n s u m i n g d r i n k i n g w a t e r c o n t a i n i n g h i g h l e v e l s o f f l u o r i d e

A t o t a l o f 207 p a t i e n t s w e r e s e l e c t e d f r o m t h e v i l l a g e s o f B a t l a p a l l y ,
S i v a n n a g u d a and M a r r i g u d a w h e r e t h e d r i n k i n g w a t e r s o u r c e c o n t a i n e d f l u o r i d e i n t h e r a n g e s o f ( a ) 1.1-4.0 and above. mg/l, ( b ) 4.1-8.0 mg/l, a n d ( c ) 8.1 m g / l P e o p l e i n a non-endemic v i l l a g e w e r e used as a c o n t r o l group.

S a m p l e s of d r i n k i n g w a t e r i n t h e f o u r v i l l a g e s w e r e a n a l y z e d f o r v a r i o u s c h e m i c a l p a r a m e t e r s f o l l o w i n g t h e p r o c e d u r e s recommended b y A.P.H.A. protein, carbohydrates, calcium, p h o s p h o r u s and v i t a m i n s .

(6).

D i e t a r y surveys were conducted t o evaluate d e f i c i e n c i e s i n calories, Dietary intake o f f o o d m a t e r i a l s was d e t e r m i n e d b y w e i g h t metbfod, and n u t r i t i o n a l paramet e r s w e r e c a l c u l a t e d i n a c c o r d a n c e w i t h t h e I C M R m a n u a l (7). C l i n i c a l examination o f p a t i e n t s I n c i d e n c e o f d e n t a l f l u o r o s i s was s t u d i e d u n d e r t h e f o l l o w i n g c l a s s i f i c a tions: Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 criteria:
-

n o r m a l , t r a n s l u c e n t , smooth and g l o s s y t e e t h white opacities, f a i n t yellow l i n e c h a n g e s o f Grade 1 and b r o w n s t a i n b r o w n l i n e , p i t t i n g a n d c h i p p e d o f f edges b r o w n b l a c k and l o s s o f t e e t h skeletal fluorosis was categorized by t h e following

Incidence o f

0 - normal
1 2
-

m i l d s y m p t o m a t i c ; r a d i o g r a p h s w i t h i n c r e a s e d bone d e n s i t y moderate symptomatic; s t i f f n e s s , r i g i d i t y , severe: o f s p i n e and j o i n t s p a i n , aches symptomatic w i t h o s t e o p h y t o s i s e x o s t o s i s marked l i m i t a t i o n

F o r r a d i o l o g i c a l e v a l u a t i o n r a d i o g r a p h s o f f o r e a r m , knee j o i n t s , and s p i n e were taken. Biochemical s t u d i e s B l o o d and u r i n e samples were c o l l e c t e d f r o m b o t h normal persons o f t h e c o n t r o l g r o u p and f r o m f l u o r o s i s o a t i e n t s and a n a l y z e d f o r b i o c h e m i c a l parameters. A f t e r c o l l e c t i n g b a s e l i n e d a t a and e v a l u a t i n g t h e r e s u l t s , t i o n p r o g r a m was i n i t i a t e d b y p r o v i d i n g t h e f o l l o w i n g : an i n t e r v e n -

359 Group 1 - d e f l u o r i d a t e d w a t e r Group 2 Group 3 Group 4

-

n u t r i t i o n a l supplements d e f l u o r i d a t e d w a t e r and n u t r i t i o n a l s u p p l e m e n t s no d e f l u o r i d a t e d water o r n u t r i t i o n a l supplements ( c o n t r o l )

The d e f l u o r i d a t i o n p r o c e s s d e v e l o p e d a t t h e I n s t i t u t e c o n s i s t e d o f a d d i n g c a l c i u m c a r b o n a t e p o w d e r t o t h e r a w w a t e r f o l l o w e d b y t h e a d d i t i o n o f known q u a n t i t i e s o f s a t u r a t e d f i l t e r e d a l u m s o l u t i o n and m i x i n g f o r 1 0 m i n u t e s . N u t r i t i o n a l supplements

A m i x t u r e o f "Hyderabad Mix",
ties:

which i s manufactured l o c a l l y ,

contains the

f o l l o w i n g r a w f o o d s and was s u p p l i e d t o t h e p a t i e n t s i n a p p r o p r i a t e q u a n t i -

2. 3.
4.

1.

wheat b e n g a l gram jaggery groundnut

70 9 35 g 23 9 12 4
140 g c l i n i c a l and r a d i o l o g i c a l

Following the intervention the biochemical, p r o f i l e s o f t h e p a t i e n t s were studied. RESULTS AND DISCUSSION

A t o t a l o f 207 p a t i e n t s r e p r e s e n t i n g b o t h s e x e s i n d i f f e r e n t age g r o u p s
( a g e s 0-5. 5-10, 10-18, and 18+) w e r e s t u d i e d i n t h e t h r e e v i l l a g e s . Some o f t h e p a t i e n t s e x a m i n e d a r e shown i n F i g u r e s 1-4. and s k e l e t a l f l u o r o s i s . degree o f s e v e r i t y , contents o f water, C l i n i c a l and r a d i o l o g i -

c a l p r o f i l e s o f t h e p a t i e n t s revealed t h a t t h e y were s u f f e r i n g from dental Several f a c t o r s appear i m p o r t a n t i n a f f e c t i n g t h e f l u o r i d e and a l k a l i n i t y i n c l u d i n g n u t r i t i o n a l status, age and o c c u p a t i o n .

TABLE I

INCIDENCE

OF FLUOROSIS

IN

BAT LAP ALLY^

Dental Grade I1 I11 -

Age G r o u p (Years)

Sk e l e t a 1
IV MiI d
Moderate
-

I
2

Severe
-

0-5
5-10
10-1 8 18+ Total

2
1

7
3
4 14

2
7
11 20

4 1 5

6 6
12

8
9

' F l u o r i d e c o n t e n t i n d r i n k i n g w a t e r was ~ 8 . 0m g / l .

FIG. I

PATIENTS WITH FIG. 2 PATIENT WITH RIGID SPINAL COLUMN

KNOCK K N E E AND NOW LEGS

FIG. 4 FIG. 3 PATIENT HAVING KEPHOSIS AND BOW LEGS

FEMALE HAVING BOW LEGS

361
T a b l e I shows t h e i n c i d e n c e o f d i s e a s e among s u b j e c t s o f B a t l a p a l l y v i l l a g e w h e r e t h e maximum f l u o r i d e c o n c e n t r a t i o n i n d r i n k i n g w a t e r r a n g e d f r o m 8.0-8.5 mg/l. T a b l e s I 1 and I 1 1 show i n c i d e n c e o f d i s e a s e among a n d 1.1 t o 4.0 m g / l , respectively. The s u b j e c t s o f S i v a n n a g u d a and M a r r i g u d a w h e r e t h e f l u o r i d e c o n t e n t s i n t h e w a t e r w e r e 4.1 t o 8.0 m g / l M o s t o f t h e v i l l a g e r s s u r v e y e d w e r e f o u n d t o h a v e p o o r n u t r i t i o n . The f o o d t h e y consumed was d e f i c i e n t i n s e v e r a l e s s e n t i a l n u t r i e n t s . d i e t a r y s t a t u s o f t h e p e o p l e i n t h e t h r e e v i l l a g e s i s shown i n T a b l e I V .

TABLE I 1

INCIDENCE

OF FLUOROSIS

IN SIVANNAGUDA~
Dental Grade Skel e t a l

Age Group (Years)

I1

111
-

IV
-

MiI d
-

Floderate
-

Severe
-

0-5
5-1 0 10-1 8 18+ Total

2
1
~

4
8 12

4
10 16

9
2
-

6
10 16

8
-

10 17 27

15

24

30

11

' F l u o r i d e c o n t e n t i n d r i n k i n g w a t e r was 4 . 1 t o 8 . 0 m g / l

TABLE I 1 1

INCIDENCE

OF FLUOROSIS

IN MARRIGUDA~
Dental Grade I1 I11

Aqe G r o u p (Years)' 0-5 5-1 0 10-18 18+ Total

Sk e l e t a l
IV
-

MiId
-

Moderate
~

Severe
-

2
-

6 6
4
16

10
17 29

6
10 20

20
26

2 2

' F l u o r i d e c o n t e n t i n d r i n k i n g w a t e r was 1 . 1 t o 4.0 m g / l .

362
TABLE IV DIETARY STATUS OF PATIENTS Village Proteins (9) 9.9-27.7 14.7-29.1 22.1-42.2 Energy Calcium (mg) 127-1 63 71-158 72-222 Phosphorus (m9) 243-675 268-675 657-1203 Iron (mg) 4.1-13.9 5.7-13.2 8.8-20.0

(K calorie)
584-1464 691 -1 4 9 0 998-2007

:larriguda Sivannaguda Batlapally

N i n i,num

requirements

19.2-53.7

1350-2500

400-800

--

1.5-28.0

Biochemical a n a l y s i s The b l o o d p r o f i l e s o f t h e p a t i e n t s showed e l e v a t e d a l k a l i n e p h o s p h a t a s e v a l u e s r a n g i n g f r o m 15-59 i n c r e a s e o f 5-10 c o n t r o l g r o u p (0.01-0.06). The c o n c e n t r a t i o n o f f l u o r i d e i n t h e u r i n e o f t h e s u b j e c t s r a n g e d f r o m 7.5-31 m g / l w h i l e t h a t o f t h e c o n t r o l g r o u p was 0.8-0.9 mg/l. No s i g n i f i c a n t c h a n g e s c o u l d be o b s e r v e d i n u r i n a r y p h o s p h a t e , n i n e c l e a r a n c e values. I n t e r v e n t i o n proqram After intervention, t h e b l o o d and u r i n e samples o f t h e p a t i e n t s were The r e s u l t s w e r e No s i g n i f i c a n t c h a n g e s w e r e o b s e r v e d i n o r i n u r i n a r y magnea n a l y z e d f o r d i f f e r e n t p a r a m e t e r s o n c e i n t h r e e months. subjected t o s t a t i s t i c a l analysis. t h e b l o o d urea, sium, phosphate, creatinine. p h o s p h o r u s and magnesium, creatin~ne, o r creatiunits, and a serum f l u o r i d e c o n c e n t r a t i o n area f o l d (0.15-0.8) c o m p a r e d t o v a l u e s o f t h e non-endemic

c r e a t i n i n e , and c r e a t i n i n e c l e a r a n c e .

A moderate increase
but the

i n p r o t e i n and c a l c i u m p r o f i l e s o f t h e p a t i e n t s w e r e observed, v a r i a t i o n s w e r e n o t f o u n d t o be s t a t i s t i c a l l y s i g n i f i c a n t .

A summary o f s e r u m f l u o r i d e ,

a l k a l i n e p h o s p h a t a s e and c a l c i u m v a l u e s i n The d a t a i n d i c a t e a

p a t i e n t s o f B a t l a p a l l y v i l l a g e i s s h o w n i n T a b l e V. s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n b e t w e e n G r o u p s 11, c o n t r o l g r o u p (Group I).

111 a n d I V a n d t h e

T h e r e d u c t i o n i s s i g n i f i c a n t ( ~ ~ 0 . 0 5f )o r G r o u p

I V ( n u t r i t i o n a l supplement

defluoridation).

A l t h o u g h u r i n a r y c a l c i u m and the

f l u o r i d e c o n t e n t s s h o w a g e n e r a l r e d u c t i o n i n a l l g r o u p s ( T a b l e V), differences are not significant. h i g h compared t o t h o s e o f t h e c o n t r o l group.

The u r i n a r y f l u o r i d e v a l u e s a r e u n i f o r m l y

It i s e v i d e n t t h a t t h e

f l u o r i d e a l r e a d y absorbed i s r e m o b i l i z e d and e x c r e t e d i n t h e u r i n e even a f t e r c e s s a t i o n o f excessive f l u o r i d e i n g e s t i o n t h r o u g h d r i n k i n g water.

363
TABLE V

SUMMARY OF BIOCHEMICAL PROFILE

OF

BATLAPALLY PATIENTS

Initial Mean f S.D.

A t 12 m o n t h f o l l o w - u p Mean f S.D.

SERUM
F l u o r i d e (mg/l) Group I Group I I Group 1 1 1 G r o u p IV A l k Phost (KAU) Group I Group I 1 G r o u p I11 Group I V

0.18 0.19 0.18 0.17

f f f f

0.04

0.14 f 0 . 0 3

0.04 0.03 0.03

0.13 0.12

0.03

0.12 i 0 . 0 2
f

0.02

26.07 25.81

f
?

9.71 8.89

15.66 23.06 18.96

8.32 10.34 8.69

12.88 i 8.72
? f

37.57 f 12.87 32.28 i 10.91

URINE
C a l c i u m (mg % ) Group I Group I 1 Group I11 G r o u p IV F l u o r i d e (mg/l) Group I Group I 1 Group I 1 1 Group IV

9.81

2.68

5.76 5.23 4.10 5.80

f
f

1.83 1.57 1.03

10.89 i 3.37 9.91 f 3.08 9.95 i 3.24

1.46

11.05 11.03 14.18

1.39 1.17 1.88

10.91 9.40 10.95

f f f

2.48 1.62 2.55

12.04 f 1.57

1 0 . 9 3 f 2.16

The c h a n g e s i n t h e b i o c h e m i c a l p r o f i l e s o f c a l c i u m , g r o u p s c o m p a r e d t o t h o s e o f t h e non-endemic a t t h e 12 month f o l l o w - u p area.

f l u o r i d e and a l k a l i n e

p h o s p h a t a s e a r e a l s o d i s c e r n i b l e i n i n d i v i d u a l p a t i e n t s o f d i f f e r e n t age I n i t i a l v a l u e s and t h o s e s e r u m and u r i n a r y o f serum a l k a l i n e phosphatase,

f l u o r i d e and u r i n a r y c a l c i u m i n s u b j e c t s o f B a t l a p a l l y v i l l a g e and t h e N a c h a r a m c o n t r o l g r o u p a r e s h o w n i n T a b l e VI. e n t groups. However,

It i s seen f r o m t h e t a b l e

t h a t t h e r e d u c t i o n i n t h e r e s p e c t i v e parameters a r e s i g n i f i c a n t i n d i f f e r s e r u m a l k a l i n e p h o s p h a t a s e and f l u o r i d e l e v e l s a r e

364 much h i g h e r t h a n t h o s e o f t h e c o n t r o l s even 12 months a f t e r t h e i n t e r v e n t i o n was i n j t i a t e d . The r e s u l t s o f t h e b i o c h e m i c a l s t u d i e s t h u s p o i n t o u t i m p o r t a n t phenomenon: ( 1 ) p r o v i d i n g d e f l u o r i d a t e d w a t e r o r n u t r i t i o n a l supplements b r i n g a b o u t s i g n i f i c a n t changes i n serum f l u o r i d e and a l k a l i n e phosphatase, i.e.. r e d u c t i o n o f these parameters, and (2) s u b j e c t s r e c e i v i n g d e f l u o r i d a t e d w a t e r and n u t r i t i o n a l supplements e x c r e t e more f l u o r i d e i n u r i n e t h a n t h o s e r e c e i v i n g n u t r i t i o n a l supplements alone.

TABLE V I INDIVIDUAL BLOOD AND U R I N E PROFILES OF BATLAPALLY SUBJECTS Parameters Age Group 1 Initial
A t 12 month follow-up

Nacharam c o n t r o l group 1.5

Serum A1 k a l i n e Phosphatase (MU)

0-5

63.6
31.0 25.0 28.0 33.0 48.0 0.2 0.2 0.4 9.8 11.0 17.0 28.6 5.0

34.0 20.0 16.0 14.0 20.0 24.0 0.1 0.1 0.1 4.0 9.4 11.6 17.5 16.0

N+D
5-1 0 10-18

N+D
N

16.0 18.5

N+D
18+ Serum F1u o r i de 18+ N+D

20.5

D
N

0.06
0.04

(mgll)
Urinary Calcium (mg%) 18+ 10-18

N+D N+D N+D

0.06
9.2 10.0 9.8

D
Urinary Fluoride (mgll) 10-18

N
N+D

1.4
1.6

'D

d e f l u o r i d a t e d water

N = n u t r i t i o n a l supplements

365
C l i n i c a l examination

A l l t h e p a t i e n t s e x a m i n e d s h o w e d mod'erate t o good' r e l i e f f r o m t h e c T i n i c a l symptoms. water. F o r t h e s u b j e c t s o f B a t l a p a l l y . t h e r e l i e f was g r e a t e s t among t h o s e r e c e i v i n g n u t r i t i o n a l s u p p l e m e n t s i n a d d i t i o n t o d e f l u o r i d a t e d The r e s u l t s o f c l i n i c a l e x a m i n a t i o n a f t e r i n t e r v e n t i o n a r e shown i n T a b l e s V I I t h r o u g h X.

TABLE V I I SALIENT CLINICAL FEATURES - BATLAPALLY CONTROL GROUP

No.
1.

S.

0-5
-

Age 5-10 10-18

i8+

M
2 2

Sex

F
0
1

C l i n i c a l Features Initial Final vague body p a i n and j o i n t p a i n s b o d y aches, back ache, arthralgia, and s t i f f n e s s generalized body p a i n s , b a c k ache, stiffness, swelling o f joints, limitat i o n o f movements, rigidity o f the s p i n e and numbness complaints o f pains only joint

2
-

2.

c o m p l a i n t s o f back a c h e and s t i f f n e s s complaints o f s t i f f ness. swelling of j o i n t s , n o change i n l i m i t a t i o n o f movements, rigidity of t h e spine, slight i m p r o v e m e n t i n numbness

3.

TABLE

vrrr

S A L I E N T CLINICAL FEATURES - BATLAPALLY DEFLUORIDATION GROUP

S. NO.
1.

0-5 5-10 10-18 18+

Age

Sex

F
0
1

C l i n i c a l Features Initial Final vague body j o i n t pains and complaints o f pains only joint

1
-

1 2

2.

Body aches, back aches, arthralgia, and s t i f f n e s s generalized body pains, backache, s t i f f ness. s w e l l in g joints, r i g i d i t y of t h e s p i n e and numbness

c o m p l a i n t s o f back a c h e and s t i f f n e s s complaints o f s t i f f of ness, swelling j o i n t s , no change i n l i m i t a t i o n o f movements, rigidity of spine, slight imp r o v e m e n t o f numbness

3.

366
TABLE I X SALIENT CLINICAL FEATURES
-

BATLAPALLY NUTRITION GROUP Sex C l i n i c a l Features Initial Final vague pains body joint

S.

NO.

0-5 5-10 10-18 18+

1
-

Age

1.

0
5

1
2

no c o m p l a i n t s of body j o i n t p a i n s
no c o m p l a i n t s

2.
3.

body aches, back aches, arthralgia, and s t i f f n e s s generalized body pains, backaches, stiffness, swelling of joints, limitat i o n o f movements, rigidity of the s p i n e , and numbness

no c o m p l a i n t s

I t was o b s e r v e d t h a t p a t i e n t s i n t h e c o n t r o l g r o u p c o n t i n u e d t o s u f f e r
f r o m a l l t h e i n i t i a l symptoms. T h e r e has b e e n s i g n i f i c a n t i m p r o v e m e n t i n Patients t h e p h y s i c a l d i s a b i l i t i e s experienced by t h e s u b j e c t s r e c e i v i n g n u t r i t i o n a l supplements alone o r i n c o m b i n a t i o n w i t h d e f l u o r i d a t e d w a t e r . r e c e i v i n g t h e s e a l o n e were r e l i e v e d o f vague p a i n s b u t j o i n t p a i n s pers i s t e d . R e s u l t s s u g g e s t t h a t some o f t h e s y m p t o m s e x p e r i e n c e d b y t h e p e o p l e c a n be a t t r i b u t a b l e t o m a l n u t r i t i o n .

TABLE X SALIENT CLINICAL FEATURES

-

BATLAPALLY NUTRITION AND DEFLUORIDATION GROUP Sex C l i n i c a l Features Initial Final malnutrition b o d y p a i n s and j o i n t pa1 n s b o d y ache, b a c k a c h e , artbralgia generalized body pains, backache, stiffness. swelling o f j o i n t s . numbness moderate improvement no c o m p l a i n t s no c o m p l a i n t s no c o m p l a i n t s

NO.

S.

0-5 5-10 10-18 18+

Age

1.

3
-

1
1

2
2
0 0

2 .
3.
4.

3
-

2
-

2 2

367
Radiological examination F i n d i n g s o f r a d i o l o g i c a l p r o f i l e s r e v e a l no change and i n c l u d e l i t t l e regression i n coarse t r a b e c u l a t i o n pattern, formations, decreased o s t e o s c l e r o s i s , o s s e o u s membrane. d e c r e a s e d p e r i o s t e a l new bone and d e c r e a s e d o s s i f i c a t i o n o f i n t e r -

The r e s u l t s i n d i c a t e t h a t t h e p r o c e s s o f m o b i l i z a t i o n o f

skeletal f l u o r i d e i s reversible but it takes a long time f o r substantial r e c t i f i c a t i o n o f skeletal abnormalities. C l i n i c a l d a t a f r o m r a d i o g r a p h s and b i o c h e m i c a l p r o f i l e s o f b l o o d and u r i n e s a m p l e s i n d i c a t e t h a t s i g n i f i c a n t r e l i e f c a n be o b t a i n e d i n p a t i e n t s w i t h s e v e r e f l u o r o s i s s y m p t o m s b y u s i n g d e f l u o r i d a t e d w a t e r and b y e a t i n g a nutritional diet.

REFERENCES
1. 2. Ramamohan Rao NV. R a j y a l a k s h m i K ( 1 9 7 4 ) I n : P r o c S i g n F l u o r o s i s I n d Acad Geo SOC, p p 273-284 Incidence o f Fluorosis i n R a j y a l a k s h m i K, Ramamohan Rao N V ( 1 9 8 3 ) N a l g o n d a D i s t r i c t , A.P. i n r e l a t i o n t o C h e m i c a l C h a r a c t e r i s t i c s o f P o t a b l e W a t e r and S t a p l e Foods. Paper p r e s e n t e d a t 1 3 t h Conference o f I S F R Nov 14-17, New D e l h i R a j y a l a k s h m i K, New D e l h i Ramamohan Rao NV ( 1 9 8 3 ) I b i d - r e p o r t s u b m i t t e d t o DOE

3. 4.

R a j y a l a k s h m i K, V e n k i a h KR, Ramamohan Rao NV, K r i s h n a N ( 1 9 8 4 ) R e l e v a n c e o f d e f l u o r i d a t e d w a t e r and n u t r i t i o n a l s u p p l e m e n t s t o F l u o r o s i s . R e p o r t s u b m i t t e d t o IDRC. Canada Ramamohan Rao NV, R a j y a l a k s h m i K, S u r y a p r a k a s h Rao K, N a v n e e t h a Rao T (1983) S t u d i e s on Geo-chemistry o f f l u o r i d e d i s t r i b u t i o n i n w a t e r s o u r c e s o f N a l g o n d a D i s t r i c t , A.P. Paper p r e s e n t e d a t 1 3 t h Conference o f ISFR Nov 14-17, New D e l h i S t a n d a r d M e t h o d s f o r t h e E x a m i n a t i o n o f W a t e r and Waste Water, APHA, AWWA, WPCF. USA (1981). 1 4 ed

5.

6.
7.

N u t r i t i v e V a l u e s o f Food M a t e r i a l s (1975) I n d i a n C o u n c i l o f M e d i c a l Research. New D e l h i

H. Tsunoda and M.-H. Y u (Editors)

Fluoride Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 369-378 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in T h e Netherlands

369

FLUORIDE UPTAKE BY DECIDUOUS ENAMEL FROM FLUORIDE MOUTHRINSE AND NATURAL FLUORIDE I N TtIE WATER YOUICHI

IIJIMA

AND TSUYOSHI KATAYAMA I w a t e Medical U n i v e r s i t y School o f

D e p a r t m e n t of P r e v e n t i v e D e n t i s t r y , D e n t i s t r y , Morioka, Japan

ABSTRACT The e f f e c t s o f f l u o r i d e m o u t h r i n s e (F:500 ppm. t i o n o f n a t u r a l w a t e r w i t h 0.3-3.2

5 t i m e s / w e e k ) and consump-

ppm F on f l u o r i d e u p t a k e b y d e c i d u o u s

e n a m e l w e r e i n v e s t i g a t e d u s i n g 135 e x f o l i a t e d t e e t h . F l u o r i d e m o u t h r i n s e i n c r e a s e d t h e f l u o r i d e c o n c e n t r a t i o n o f t h e t o p 1 vm l a y e r o f t h e e n a m e l f r o m 4,300 t o 7,300 ppm w h e n t h e r i n s i n g p e r i o d w a s i n c r e a s e d f r o m one t o f o u r years. No s t a t i s t i c a l l y s i g n i f i c a n t i n c r e a s e s

i n f l u o r i d e c o n c e n t r a t i o n were found i n t h e enamel 5

urn

f r o m t h e surface.

These r e s u l t s show t h a t f l u o r i d e u p t a k e b y m o u t h r i n s e was m o s t l y r e s t r i c t e d t o t h e o u t e r m o s t l a y e r s o f t h e enamel. F l u o r i d e c o n c e n t r a t i o n s i n t h e d e c i d u o u s enamel s u r f a c e o f i n h a b i t a n t s consuming n a t u r a l f l u o r i d e i n t h e water were s i g n i f i c a n t l y higher than t h o s e i n t h e enamel o f s u b j e c t s consuming w a t e r w i t h v e r y l o w f l u o r i d e o r those i n enamel t r e a t e d w i t h f l u o r i d e mouthrinse. Fluoride levels o f However, a p p r o x i m a t e l y 10,000 pprn w e r e f r e q u e n t l y f o u n d i n t h e t o p 1 Um l a y e r o f t h e e n a m e l f r o m s u b j e c t s l i v i n g i n h i g h f l u o r i d e a r e a s ( o v e r 1.0 ppm). t h e f l u o r i d e c o n c e n t r a t i o n s a t an e n a m e l d e p t h o f 5 Um f r o m of t h e cont.rol. an area w i t h

0.3 ppm F i n t h e d r i n k i n g w a t e r w e r e n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h o s e
H i g h f l u o r i d e p e n e t r a t i o n was f o u n d i n b o t h t h e o u t e r m o s t and d e e p e r l a y e r s o f t h e d e c i d u o u s enamel.

INTRODUCTION F l u o r i d e i s w i d e l y used i n p r e v e n t i v e d e n t a l programs. r i d e i n c o m m u n i t y p r e v e n t i v e measures, The u s e o f f uoand i n d i v i d u a l l y based procedures,

i n d i v i d u a l s e l f c a r e h a v e been e x t e n s i v e l y r e v i e w e d r e c e n t l y (1,Z).

F uo-

r i d e decreases t h e a c i d s o l u b i l i t y o f enamel by c o n v e r t i n g enamel hydroxya p a t i t e t o f l u o r a p a t i t e ( 3 ) . Because o f t h i s , an i n c r e a s e i n enamel f l u o r i d e c o n c e n t r a t i o n has been s u g g e s t e d t o have c a r i o s t a t i c b e n e f i t s , and t h e r e i s much e v i d e n c e f a v o r i n g a n t i c a r i e s e f f e c t s o f e n a m e l f l u o r i d e

(4,5).

370
It i s general Iv
stage.
aLL~2)Lcii

hat t n e a m o u n t o f f l u o r i d e a c q u i r e d i n t h e
maturation However,

enamel v a r i e s c o n s i d e r a b l y w i t h t h e l e n g t h o f t h e p r e - e r u p t i v e t h e r e f o r e a c q u i r e much l e s s f l u o r i d e t h a n t h e p e r m a n e n t t e e t h (6).

D e c i d u o u s t e e t h have a s h o r t e r p e r i o d o f enamel m a t u r a t i o n and

l i t t l e e v i d e n c e i s a v a i l a b l e a s t o how n e u t r a l f l u o r i d e m o u t h r i n s e and n a t u r a l f l u o r i d e i n t h e water a f f e c t t h e f l u o r i d e uptake i n deciduous enamel. I n a p r e v i o u s r e p o r t , i t was d e m o n s t r a t e d t h a t e x t r e m e l y h i g h f l u o r i d e l e v e l s , a p p r o x i m a t e l y 10,000 ppm, w e r e f r e q u e n t l y f o u n d a t a d e p t h o f 1 p m i n t h e s u r f a c e e n a m e l o f i n d i v i d u a l s f r o m an a r e a w i t h a h i g h l e v e l o f n a t u r a l f l u o r i d e i n t h e w a t e r ( o v e r 1.0 ppm)(7). The p r e s e n t s t u d y was c a r r i e d o u t t o i n v e s t i g a t e f l u o r i d e u p t a k e b y deciduous enamel f r o m m o u t h r i n s e and n a t u r a l f l u o r i d e i n t h e d r i n k i n g water. MATERIALS AND METHODS S p e c i mens Deciduous enamel specimens employed i n t h i s study were d e r i v e d from a t o t a l o f 135 e x f o l i a t e d i n c i s o r s and m o l a r s f r o m s c h o o l c h i l d r e n who had r e s i d e d c o n t i n u o u s l y s i n c e b i r t h f o r 6-11 y e a r s i n one o f t w o a r e a s : one a r e a w i t h 0.3 t o 3.2 ppm n a t u r a l f l u o r i d e i n t h e d r i n k i n g w a t e r , and a n o t h e r a r e a w i t h l e s s t h a n 0.1 ppm i n i t s w a t e r s u p p l y ( T a b l e I). F i f t e e n t e e t h w i t h no h i s t o r y o f t o p i c a l f l u o r i d e a p p l i c a t i o n s f r o m t h e l o w f l u o r i d e community were d e s i g n a t e d as t h e c o n t r o l group, ppm, and a n o t h e r 60 t e e t h o b t a i n e d f r o m t h e same a r e a b u t t r e a t e d w i t h f l u o r i d e m o u t h r i n s e

(F:500

5 times/week)

were d i v i d e d e q u a l l y i n t o 4 groups a c c o r d i n g t o r i n s i n g A n o t h e r 60 t e e t h o b t a i n e d f r o m t h e c o m m u n i t y

p e r i o d s o f one t o f o u r y e a r s .

w i t h n a t u r a l f l u o r i d e i n t h e w a t e r were d i v i d e d i n t o 4 groups according t o

TABLE I

DECIDUOUS ENAMEL SPECIMENS EMPLOYED I N THIS STUDY

Enamel specimens

F content o f d r i n k i n g water (PPm)

< 0.1

Type o f t e e t h (surface)

No. o f teeth

Age (Yr)

Control F-mouthrinse ( 5 0 0 ppm F 5 t imes/week) Natural F

Molars Incisors (labial) Molars (buccal )

15
60

10

< 0.1

6 - 7

0.3

3.2

60

11

371
t h e f l u o r i d e l e v e l s i n t h e d r i n k i n g water. d i s t i l l e d water, The t e e t h w e r e c l e a n e d f o r 60 Speci-

seconds w i t h a r o t a t i n g b r u s h w i t h o u t pumice, p l a c e d i n a beaker c o n t a i n i n g and s t i r r e d f o r 24 h o u r s w i t h a m a g n e t i c s t i r r e r . mens w i t h o u t c a r i e s l e s i o n s o r o t h e r d e f e c t s o n t h e e n a m e l s u r f a c e w e r e employed i n t h i s study. A n a l y t i c a l procedures s i x successive l a y e r s o f enamel (sampling area: a p p r o x i m a t e l y

20

mm2)

w e r e d i s s o l v e d i n a p l a s t i c t u b e c o n t a i n i n g 1.0 m l o f 0.5M p e r c h l o r i c a c i d w i t h g e n t l e s h a k i n g . T h e f i r s t t h r e e l a y e r s w e r e e t c h e d f o r 5, 10, a n d 1 5 seconds, respectively, and t h e l a s t t h r e e l a y e r s were each e t c h e d f o r a n o t h e r 30 seconds. E t c h i n g was t e r m i n a t e d b y w a s h i n g t h e e t c h e d s u r f a c e

w i t h d i s t i l l e d w a t e r f o r 1 5 s e c o n d s . 4.0 m l o f a 0.5M t r i s o d i u m c i t r a t e s o l u t i o n was t h e n added t o t h e e t c h e d s o l u t i o n . Concentrations e l e c t r o d e (96-09, of fluoride were determined Cambridge, by the Mass.) F-specific and t h o s e o f The O r i o n R e s e a r c h Inc..

c a l c i u m b y a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y (508A,

H i t a c h i , Tokyo).

a m o u n t o f e n a m e l r e m o v e d and t h e d e p t h o f e a c h l a y e r w e r e c a l c u l a t e d assumi n g t h e c a l c i u m c o n t e n t o f e n a m e l t o b e 3 7 w e i g h t % a n d t h e d e n s i t y 2.95 gIcm3. T h e r e was a c l o s e a g r e e m e n t b e t w e e n t h e f l u o r i d e d i s t r i b u t i o n c u r v e s e x p e r i m e n t a l l y o b t a i n e d and t h e o r e t i c a l Therefore, Once and exponential curves(8)

[Y

2 X-b,
um].

w h e r e Y i s f l u o r i d e c o n c e n t r a t i o n i n ppm.

and X i s t h e d e p t h i n

t h e e q u a t i o n was o b t a i n e d b y a p p l y i n g t h e l e a s t s q u a r e s method.

t,

a r e e x p e r i m e n t a l l y determined,

i t i s p o s s i b l e t o c a l c u l a t e and
Fluoride concentrations

compare f l u o r i d e c o n c e n t r a t i o n s a t a g i v e n depth. w e r e c a l c u l a t e d f o r 1, surface. Statistical analysis

5,

10,

20. 3 0 ,

a n d 40 Um l a y e r s o f t h e e n a m e l

The mean f l u o r i d e c o n c e n t r a t i o n s and t h e s t a t i s t i c a l s i g n i f i c a n c e o f t h e d i f f e r e n c e s among t h e c o n t r o l , f l u o r i d e mouthrinse, and n a t u r a l f l u o r i d e g r o u p s w e r e c o m p a r e d b y t h e Newman-Keuls RESULTS F l u o r i d e mouthrinse group The mean f l u o r i d e c o n c e n t r a t i o n s and t h e d i s t r i b u t i o n o f t h e e n a m e l l a y e r s t r e a t e d w i t h f l u o r i d e m o u t h r i n s e a r e shown i n T a b l e I 1 and F i g u r e 1, respectively. T r e a t m e n t w i t h f l u o r i d e mouthrinse increased t h e f l u c r i d e ppm when t h e The f l u o r i d e concenF l u o r i d e uptake by concentration o f the 1 u m l a y e r o f enamel f r o m 4,300 t o 7,300 r i n s i n g p e r i o d was i n c r e a s e d f r o m one t o f o u r y e a r s . t r a t i o n was p r o p o r t i o n a l t o t h e l e n g t h o f m o u t h r i n s i n g . t e s t f o r m u l t i p l e c o m p a r i s o n s (9).

372

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t h e enamel i n t h i s g r o u p was t h r e e t o f o u r t i m e s h i g h e r t h a n t h a t o f t h e c o n t r o l s ( T a b l e 11).

While t h e f l u o r i d e c o n c e n t r a t i o n s o f t h e
t w i c e as h i g h as t h o s e o f t h e c o n t r o l , between t h e enamel beyond 5 (Fig.

5 urn l a y e r s o f e n a m e l w e r e o n l y A

the difference i n f l u o r i d e contents

and t h a t o f t h e c o n t r o l was o n l y s l i g h t .

d i m i n i s h e d f l u o r i d e u p t a k e was o b s e r v e d i n e a c h o f t h e s u c c e e d i n g l a y e r s

1).

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F i g u r e 1. Fluoride distribution i n deciduous t e e t h t r e a t e d w i t h f l u o r i d e r n o u t h r i n s e . o : c o n t r o l ; A : m o u t h r i n s e f o r 1 yr; A: r n o u t h r i n s e f o r 2 y r ; A: m o u t h r i n s e f o r 3 yr: A : m o u t h r i n s e f o r 4 yr.

Natural f l u o r i d e group F l u o r i d e c o n c e n t r a t i o n s and d i s t r i b u t i o n o f t h e e n a m e l f r o m a r e a s w i t h d i f f e r e n t l e v e l s o f n a t u r a l f l u o r i d e i n t h e d r i n k i n g w a t e r a r e shown i n T a b l e I11 and F i g u r e 2. The c o n c e n t r a t i o n s i n t h e e n a m e l f r o m t h i s g r o u p An i n c r e a s e i n Furtherwere s i g n i f i c a n t l y higher than those i n t h e controls.

f l u o r i d e c o n t e n t i n t h e d r i n k i n g w a t e r was p o s i t i v e l y a s s o c i a t e d w i t h i n c r e a s e i n f l u o r i d e c o n c e n t r a t i o n o f t h e o u t e r l a y e r s o f enamel. more, h i g h c o n c e n t r a t i o n s o f f l u o r i d e i n t h e o u t e r m o s t l a y e r s o f t h e enamel

were always associated w i t h r e l a t i v e l y h i g h concentrations i n t h e i n t e r i o r r e g i o n , w i t h t h e e x c e p t i o n s o f t h e c o n t r o l s and t h o s e f r o m an a r e a w i t h 0.3 ppm f l u o r i d e i n t h e d r i n k i n g w a t e r . The mean f l u o r i d e c o n c e n t r a t i o n s o f t o f i v e times higher fluoride t h e 40 um l a y e r s o f t h e e n a m e l s h o w e d t w o

u p t a k e t h a n t h o s e o f t h e c o n t r o l s and 0.3 ppm f l u o r i d e g r o u p ( T a b l e 111). S a m p l e s f r o m a r e a s w i t h g r e a t e r t h a n 1.0 p p m n a t u r a l f l u o r i d e i n t h e d r i n k i n g water r e s u l t e d i n t h e h i g h e s t f l u o r i d e uptake: approximately

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1 0 , 0 0 0 ppm f l u o r i d e i n t h e t o p 1 urn l a y e r o f t h e e n a m e l w a s f r e q u e n t l y observed. I n contrast, e n a m e l f l u o r i d e u p t a k e s o f t h e c o n t r o l s and t l i e 0.3 ppm f l u o r i d e g r o u p w e r e n o t s i g n i f i c a n t l y d i f f e r e n t f r o m e a c h o t h e r ( F i g .

10

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Deplhbm)

30

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F i g u r e 2. Fluoride distributions i n deciduous t e e t h f r o m areas w i t h natur a l f l u o r i d e i n t h e w a t e r . N a t u r a l f l u o r i d e i n t h e w a t e r : o : <0.1 p p m 1.0 ppm; m: 1.7 ppm; 3.2 ppm. ( c o n t r o l ) ; 0 :0.3 ppm;

a:

.:

Statistical evaluation The mean f l u o r i d e c o n c e n t r a t i o n s and t h e s i g n i f i c a n c e o f t h e d i f f e r e n c e s between t h e c o n t r o l s , f l u o r i d e mouthrinse, c o m p a r e d ( T a b l e s I V a n d V). and n a t u r a l f l u o r i d e g r o u p s w e r e The d a t a on f l u o r i d e c o n t e n t s i n t h e 1 p m

l a y e r o f t h e samples i n d i c a t e d t h a t a s i g n i f i c a n t l y h i g h e r f l u o r i d e uptake o c c u r r e d i n b o t h f l u o r i d e m o u t h r i n s e and n a t u r a l w a t e r g r o u p s t h a n i n t h e controls. Although f l u o r i d e uptake b y samples f r o m areas w i t h more than they ppm f l u o r i d e and f l u o f l u o r i d e mouth-

1.0 ppm f l u o r i d e w e r e n o t s i g n i f i c a n t l y d i f f e r e n t f r o m one a n o t h e r ,

w e r e s i g n i f i c a n t l y d i f f e r e n t f r o m t h o s e o f t h e 0.3 r i d e mouthrinse groups (Table IV). However, s i g n i f i c a n t d i f f e r e n c e s between t h e c o n t r o l s ,

r i n s e , a n d t h e 0.3 p p m f l u o r i d e g r o u p s w e r e n o t s h o w n f o r t h e 5 t o 40 um l a y e r s ( T a b l e V).

A somewhat s m a l l e r u p t a k e o f f l u o r i d e i n t o deeper l a y e r s

o f e n a m e l was f o u n d w i t h t h e 0.3 ppm f l u o r i d e group. A p a r t f r o m t h i s g r o u p , a c o n s i d e r a b l e u p t a k e o f f l u o r i d e i n t o t h e d e e p e r l a y e r s o f enamel was f o u n d ( T a b l e V).

376

TABLE I V NEWMAN-KEULS TEST ON DIFFERENCES BETWEEN ALL PAIRS TRATIONS I N THE 1 Urn LAYER OF THE ENAMEL
F mouthrinse (year)

OF MEAN FLUORIDE CONCEN-

1 Urn l a y e r

N a t u r a l f l u o r i d e v a l u e (ppm) 4 0.3 1.0 1.7 3.2

Control

aMR-l: M o u t h r i n s e f o r 1 y e a r bFW-0.3: 0.3 ppm F i n d r i n k i n g w a t e r

~~~~~~~

~~

itP<0.O5

i"cp<o~0~

TABLE V NEWMAN-KEULS TEST ON DIFFERENCES BETWEEN ALL PAIRS TRATIONS I N THE 5-40 urn LAYER OF THE ENAMEL F mouthrinse (year) Control

OF

MEAN FLUORIDE CONCEN-

5-40 u r n layer

N a t u r a l f l u o r i d e v a l u e (ppm) 4 0.3 1.0 1.7 3.2

Control MR-1 a MR-2 MR-3 MR-4

F W-0.3b

FW-1.0 FW-1.7 FW-3.2

aMR-l: M o u t h r i n s e f o r 1 y e a r bFW-0.3: 0.3 ppm F i n d r i n k i n g w a t e r

"p<O.O5

+"i'p<O.OI

377
OISCUSSION T h e r e i s s u f f i c i e n t e v i d e n c e t o i n d i c a t e t h a t o l d e r (10). more h i g h l y m i n e r a l i z e d enamel (11) o r enamels c o n t a i n i n g h i g h f l u o r i d e c o n c e n t r a t i o n s (12) can a c q u i r e l e s s f l u o r i d e w h i l e porous enamel (13) o r n e w l y erupted t e e t h ( 1 0 ) a c q u i r e more.

It i s conceivable,

therefore,

t h a t f l u o r i d e uptake

b y m o u t h r i n s i n g may be r e s t r i c t e d t o t h e o u t e r m o s t l a y e r s o f t h e enamel. The e n a m e l s p e c i m e n s e m p l o y e d i n t h i s s t u d y w e r e n o t t r e a t e d w i t h f l u o r i d e mouthrinse immediately a f t e r eruption. ment f o r about 5 years. By t h e t i m e s c h o o l - b a s e d m o u t h r i n s e f l u o r i d e concentrations were was f i r s t a p p l i e d , t h e d e c i d u o u s t e e t h h a d b e e n e x p o s e d t o an o r a l e n v i r o n Despite this, increased i n t h e 1 pm l a y e r w i t h increase i n t h e length o f mouthrinsing. T h i s o b s e r v a t i o n i s i n c o n t r a s t w i t h t h e f i n d i n g o f M e l l b e r g g t ~ l (. 14). They d i d n o t remove t h e o u t e r m o s t enamel l a y e r (1 p m ) and c o n c l u d e d t h a t f l u o r i d e p e n e t r a t i o n d i d n o t e x t e n d b e y o n d a p p r o x i m a t e l y 5 pm. F l u o r i d e u p t a k e b y t h e e n a m e l i n a r e a s w i t h m o r e t h a n 1.0 ppm f l u o r i d e i n t h e d r i n k i n g w a t e r was h i g h e r t h a n t h a t o f t h e c o n t r o l s as w e l l as enamel t r e a t e d w i t h f l u o r i d e mouthrinse. eralized. Consequently, the From b i r t h t o e r u p t i o n o f t h e

d e c i d u o u s t e e t h t h e s u r f a c e o f t h e e n a m e l a p p e a r s t o be i n c o m p l e t e l y m i n t h e o u t e r m o s t and d e e p e r l a y e r s o f e n a m e l w i l l be As t h e m a t u r a t i o n p r o c e s s p r o c e e d s , post-eruptive mineraliacquisiand able t o acquire additional f l u o r i d e through contact w i t h t i s s u e f l u i d s (pre-eruptive acquisition). z a t i o n becomes m o r e c o m p l e t e , tion)(l5). and t h e a m o u n t o f f l u o r i d e a c q u i r e d i n t o t h e

o u t e r m o s t enamel l a y e r s w i l l d i m i n i s h (pre- and/or

T h e r e f o r e t h e h i g h f l u o r i d e u p t a k e f o u n d i n t h e o u t e r m o s t and acquisition o f fluoride. f o o d s t u f f (17), and i n c i p i e n t c a r i e s ( 1 9 ) may p o s s i b l y a f f e c t f l u o r i d e u p t a k e This suggests t h a t

d e e p e r l a y e r s o f e n a m e l i n t h i s s t u d y may b e d u e t o b o t h p r e - e r u p t i v e post-eruptive p l a q u e (18), V a r i o u s f a c t o r s s u c h a s t o p i c a l l y a p p l i e d f l u o r i d e (16). b y d e c i d u o u s enamel as w e l l as b y permanent t e e t h .

e n a m e l may a c q u i r e f l u o r i d e t h r o u g h o u t t h e l i f e o f t h e t e e t h . REFERENCES 1. B u r t BA, H a u g e j o r d e n 0, H e i n W , H o r o w i t z HS, H u n t e r PB. M o r r i s o n EC, O ' B r e i n - M o r a n E, Stamm J (1984) I n t e r D e n t J 34:141-158

2.
3. 4.

E r i c s s o n Y (ed) (1978) C a r i e s Res 1 2 ( s u p p 1):7-64

M i r NA,
Bishoff

H i g u c h i W I (1969) A r c h s o r a l B i o l 14:904-920 B r a d l e y EL, B a r b a k o w FH, F r i e d m a n M, V a n d e r M e r w e EHM. C u t r e s s TW, Katayama B a l l M E (1979) N Z d e n t J 75:94-106 (1985) C a r i e s Res 19:262-265

R e t i e f DH,

J I (1979) J O r a l P a t h o l 8:224-236

5. Speake JD, 7. I i j i m a Y,

6. M e l l b e r g JR ( 1 9 7 7 ) J P r e v e n t D e n t 4:8-20

378
8. A t h a n a s s o u l i TM, 7: 368-373 9. P a p a s t a t h o p o u l o s DS. H a d j i i o a n n o u TP (1973) C a r i e s Res

W i n e r BJ ( 1 9 7 1 ) S t a t i s t i c a l P r i n c i p l e s i n E x p e r i m e n t a l D e s i g n . 2 n d Ed. McGraw-Hill, New York, p p 201 Nicholson C R (1968) J D e n t Res 4/:17b Aasenden R. Brudevold Bakhos Y (1982) C a n e s Res 16:243-248 Mellberg JR (1969) J D e n t Res 48:302-306

10. Mellberg JR, 11. Brudevold

F.

12. Nicholson C R ,
13. Aasenden R. 14. Mellberg JR.

F.

MaCann H G (1968) Archs o r a l B i o l 13:543-552 H a l l s w o r t h A S ( 1 9 7 7 ) C a r i e s Res

Nicholson C R (1974) Caries Res 8:148-154

15. W e a t h e r e l l JA, D e u t s c h D, R o b i n s o n C, 11 ( s ~ p p1 ):85-115

16. K i r k e g a a r d E (1977) C a n e s Res 11:16-23

17. B a u m e LJ, V u l l i e m o z JP ( 1 9 7 0 ) A r c h s o r a l B i o l 1 5 : 4 3 1 - 4 4 3 18. T u r t o l a L O (1977) Scand J D e n t Res 85:373-379 19. T a k a e s u Y , I i j i m a Y ( 1 9 7 6 ) I n : S u g a S. I s h i i T ( e d s ) C a r i e s S u s c e p t i b i l i t y . S t r u c t u r e and Composition o f t h e S u r f a c e Enamel. K o k u h Hoken Kyokai. Tokyo, p p 117-132

H. Tsunoda and M.-H. Yu (Editors)

379

Fluoride Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 379-388 0 1 9 8 6 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

A S T U D Y ON THE APPEARANCE

OF

DENTAL FLUOROSIS I N RELATION

TO

ENAMEL

DEVELOPMENT AND FLUORIDE INTAKE TAKUO I S H I I AND HARUO NAKAGAKI D e p a r t m e n t o f P r e v e n t i v e D e n t i s t r y and D e n t a l P u b l i c H e a l t h , A i c h i - G a k u i n U n i v e r s i t y S c h o o l o f D e n t i s t r y , 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464. Japan

ABSTRACT F l u o r i d e - r i c h (7.8 examination, ppm) w a t e r was i n a d v e r t e n t l y s u p p l i e d t o t h e I k e n o D u r i n g t h e 1973 Limited a r e a o f A i c h i P r e f e c t u r e i n J a p a n f r o m 1 9 6 0 t o 1973.

n o r m a l e n a m e l o n t h e u p p e r i n c i s o r was f o u n d i n c h i l d r e n who

w e r e m o r e t h a n 38 m o n t h s o l d when t h e w a t e r s u p p l y was s t a r t e d . c h i l d r e n who w e r e b o r n d u r i n g a p e r i o d 29 m o n t h s p r i o r t o , a f t e r t h e w a t e r s u p p l y was s t a r t e d .

o p a c i t y o n t h e c e r v i c a l p o r t i o n o f t h e u p p e r i n c i s o r was o b s e r v e d among and 8 months children I n t h e 1984 e x a m i n a t i o n ,

e x p o s e d t o t h e w a t e r u n t i l a b o u t 35 m o n t h s o f age showed s e v e r e o r m o d e r a t e d e n t a l f l u o r o s i s on t h e u p p e r i n c i s o r s ; m o n t h s showed m i l d o r v e r y m i l d f l u o r o s i s : condition. c h i l d r e n exposed f o r 24 t o 33 and c h i l d r e n who consumed t h e

w a t e r f o r 1 1 t o 22 m o n t h s showed q u e s t i o n a b l e f l u o r o s i s o r a n o r m a l T h e s e f i n d i n g s seemed t o p r e s e n t some i n f o r m a t i o n o n t h e c o r r e l a t i o n b e t w e e n f l u o r i d e a c t i o n and t h e s t a g e o f e n a m e l f o r m a t i o n .

INTRODUCTION T h e r e have been more t h a n 300 r e p o r t s on t h e e p i d e m i o l o g i c a l s t u d y o f d e n t a l f l u o r o s i s i n J a p a n s i n c e 1925. D e n t a l f l u o r o s i s h a s been f o u n d i n b u t o n l y f o u r areas have been Moreover, t h e r e are few r e p o r t s a l m o s t a l l o f t h e p r e f e c t u r e s o f Japan, d e c r e a s e d t o t h e i r p r e s e n t l o w l e v e l (1).

s t u d i e d d u r i n g t h e p e r i o d o f t i m e i n which cases o f d e n t a l f l u o r o s i s d e s c r i b i n g t h e changes i n appearance o f d e n t a l f l u o r o s i s caused b y exposure t o w a t e r w i t h a h i g h f l u o r i d e c o n t e n t f o r l i m i t e d b u t known p e r i o d s d u r i n g t o o t h formation. From December 1960 t o F e b r u a r y 1973, d r i n k i n g w a t e r c o n t a i n i n g a h i g h l e v e l o f f l u o r i d e (7.8 p p m ) w a s i n a d v e r t e n t l y s u p p l i e d t o t h e I k e n o a r e a (Pop. 423) o f A i c h i P r e f e c t u r e i n Japan. The h i g h l e v e l o f f l u o r i d e was n o t r e c o g n i z e d u n t i l 1 0 y e a r s l a t e r when d e n t a l f l u o r o s i s was shown among

380
school c h i l d r e n . I n February 1973, t h e w a t e r s u p p l y was r e p l a c e d w i t h one The t e e t h o f t h e a f f e c t e d c h i l d r e n I n 1984. 1 6 c h i l d r e n , aged

c o n t a i n i n g l e s s t h a n 0.2 ppm f l u o r i d e .

h a v e been e x a m i n e d a n n u a l l y s i n c e 1973 (2,3).

1 2 t o 15 years,

were re-examined as a f o l l o w - u p s t u d y (4).

The p u r p o s e o f t h e p r e s e n t s t u d y was t o a s s e s s t h e c o r r e l a t i o n b e t w e e n t h e degree o f d e n t a l f l u o r o s i s and t h e d u r a t i o n o f f l u o r i d e exposure. MATERIALS AN0 METHODS The s t u d y i n c l u d e d e x a m i n a t i o n o f 101 c h i l d r e n f r o m t h e I k e n o area. w e r e b o r n i n t h e a r e a b e t w e e n 1959 and 1972, f l u o r i d e - r i c h w a t e r u n t i l 1973. They and h a d been c o n s u m i n g

They w e r e e x a m i n e d a n n u a l l y w h i l e t h e y

w e r e a t t e n d i n g t h e l o c a l e l e m e n t a r y s c h o o l . V i s u a l and t a c t i l e d e n t a l e x a m i n a t i o n s o f t h e d e n t a l f l u o r o s i s were p e r f o r m e d i n accordance w i t h t h e c r i t e r i a d e s c r i b e d b y Dean (5). The t e e t h w e r e n o t d r i e d b u t w e r e c l e a n e d w i t h gauze as r e q u i r e d . tion. RESULTS The r e s u l t s f r o m t h e surveys o f 1973 and 1980. dental f l u o r o s i s , o r severe dental f l u o r o s i s l e s s t h a n t h r e e y e a r s o f age. i n d i c a t i n g t h e changes i n respectively. Moderate a r e p r e s e n t e d i n Tables I and 11, Intra-oral photographs were t a k e n a t each examina-

was seen i n c h i l d r e n exposed t o f l u o r i d e f r o m No c h i l d r e n who were younger t h a n t w o y e a r s

TABLE I

PERCENTAGE O I S T R I B U T I O N OF DENTAL FLUOROSIS I N SUBJECTS I N 1973

C l a s s i f i c a t i o n (Dean) MiId Moderate

Age'

Very M i l d

Severe

(%)

(%)

(%)

(%I

0-7 0-8

100.0
100.0

100.0 100.0 100.0 100.0 100.0 100.0 100.0

100.0 100.0 66.7 100.0 100.0

100.0

50.0 57.1 33.3 75.0

0-9
0-1 0 0-1 1
0-1 2

100.0
100.0

100.0 100.0
100.0

50.0 28.6
33.3 33.3

1-1 3 2-1 4
3-1 5

66.7
100.0

100.0

100.0

100.0 33.3

33.3

0.0

' B e g i n n i n g and e n d i n g ages o f c h i l d r e n exposed t o F - r i c h water

381
TABLE I 1 PERCENTAGE DISTRIBUTION OF DENTAL FLUOROSIS I N SUBJECTS IN 1980 C l a s s i f i c a t i o n (Dean) MiId Moderate

Age

Very M i l d

(2)

(2)

(70)

Severe

(2)

10
3

0.0

0.0
0.0 20.0 100.0 100.0
100.0

0.0 0.0
0.0 100.0
100.0 100.0

0.0 0.0

0
0-1
0-2
0-3

0.0
80.0

5
8

100.0
100.0 100.0

0.0 0.0
71.4

7
7

0-4

85.7

l B e g i n n i n g and e n d i n g ages o f c h i l d r e n exposed t o F - r i c h w a t e r

o f age when t h e w a t e r was changed showed moderate o r s e v e r e d e n t a l f l u o r o -

sis.
The c h i l d r e n w e r e d i v i d e d i n t o t h r e e g r o u p s a c c o r d i n g t o t h e s t a g e o f t o o t h development d u r i n g t h e p e r i o d of exposure t o f l u o r i d e - r i c h water. G r o u p A. b e f o r e 1961. Group B. Group C. The enamel of t h e permanent t e e t h developed d u r i n g t h e p e r i o d A g r e a t e r p a r t o f t h e enamel o f t h e permanent t e e t h was formed o f exposure t o h i g h f l u o r i d e . a f t e r t h e w a t e r s u p p l y was changed (Table 111). The e n a m e l o f t h e p e r m a n e n t t e e t h had been p a r t i a l l y f o r m e d

A t y p i c a l example o f d e n t a l f l u o r o s i s observed among s u b j e c t s i n group A

i s shown i n F i g u r e 1. The c h i l d was b o r n i n S e p t e m b e r 1958 a n d was 2 7 The p r e m o l a r s months o l d when t h e f l u o r i d e - r i c h w a t e r s u p p l y was s t a r t e d .

showed m o d e r a t e f l u o r o s i s , b u t t h e d i f f e r e n c e b e t w e e n i n c i s a l l y p l a c e d normal enamel and g i n g i v a l l y p l a c e d f l u o r o s e d enamel was c l e a r l y seen on t h e c a n i n e s and m a x i l l a r y i n c i s o r s . s t a r t o f t h e w a t e r supply. fluoride-rich incisors. S i m i l a r o b s e r v a t i o n s w e r e made i n the c h i l d r e n b o r n d u r i n g a p e r i o d 29 m o n t h s b e f o r e , and 8 m o n t h s a f t e r

C h i l d r e n o l d e r t h a n 38 months who r e c e i v e d t h e

w a t e r showed d e n t a l f l u o r o s i s i n some p r e m o l a r s b u t n o t i n

A l l t h e c h i l d r e n i n g r o u p B, b o r n a f t e r t h e w a t e r was s u p p l i e d , showed
severe o r moderate f l u o r o s i s on a l m o s t a l l o f t h e i r teeth. r i c h w a t e r p r e n a t a l l y u n t i l e i g h t y e a r s o f age. F i g u r e 2 shows t h e t e e t h of a boy b o r n i n March 1963 who had been exposed t o t h e f l u o r i d e T h e r e was s e v e r e d e n t a l f l u o r o s i s on t h e i n c i s o r s and even on t h e deciduous molars.

382

TABLE 111

OF THIS STUDY
'59'601

RELATION BETWEEN THE PERIOD OF FLUORIDE-RICH WATER SUPPLY AND THE SUBJECTS calender year 7 8 9 '80 84

1 2 3 4 5 6 7 8 9'701 2
r

13

.year of birth
- 6 aye

11

pa,

F i g u r e 1. Teeth o f a 14-year-old g i r l was 27 months old.

girl.

F l u o r i d e exposure s t a r t e d when t h e

A l m o s t a l l o f t h e i n c i s o r s o f c h i l d r e n i n group C showed m i l d o r moderate fluorosis; t h e p r e m o l a r s showed o n l y q u e s t i o n a b l e changes. However, the f i r s t m o l a r s and c e n t r a l i n c i s o r s showed severe f l u o r o s i s i n c h i l d r e n who were more t h a n t h r e e y e a r s o f age when t h e w a t e r s u p p l y was changed. F i g u r e 3 i l l u s t r a t e s t y p i c a l f l u o r o s i s p a t t e r n s f r o m each group. o f t h e t o o t h crown, t o o t h surface. Incis o r s and m o l a r s i n group A showed f l u o r o s e d enamel on t h e c e r v i c a l p o r t i o n and changed e n a m e l on p r e m o l a r s r e a c h e d a l l o f t h e i n c i s o r s i n g r o u p C showed w h i t e and and normal enamel i n t h e p o r t i o n On t h e o t h e r hand,

s t a i n e d enamel c l o s e t o t h e i n c i s a l t i p , o f t h e g i n g i v a l margin,

b u t t h e d i f f e r e n c e b e t w e e n c h a n g e d e n a m e l and

normal enamel was n o t as c l e a r as i n group A.

383

F i g u r e 2. Teeth o f an 8-year-old u n t i l t h e boy was 8 y e a r s old.

boy.

F l u o r i d e exposure s t a r t e d p r e n a t a l l y

Tooth
Group A

16

14

11

Group B

Group C

a00a a (3 0
........
.:.>:.......

n~or~
filj.

or
0r

MO

..... .... ...... ... .....

........... ..:.:.>>

r.10

........... ........... .....

....... ........ ....... ............. ...

. ..... .. . ...

.... .. ...... ..: ::

, :

F i g u r e 3. Appearance o f s e l e c t e d t e e t h r e l a t i n g t h e p e r i o d o f exposure t o f l u o r i d e - r i c h w a t e r s u p p l y u s i n g Dean's c l a s s i f i c a t i o n .

The s u b j e c t s i n group C were d i v i d e d i n t o t h r e e sub-groups i n a f o l l o w - u p s t u d y c o n d u c t e d i n 1984. t h e i n c i s o r s was d u l l , e x a m i n a t i o n o f 1980, g r e a t (Fig. 4). C h i l d r e n e x p o s e d t o f l u o r i d e f o r m o r e t h a n 35 and compared w i t h t h e r e s u l t s o f t h e months showed t h e most severe e f f e c t s on t h e upper i n c i s o r s . The enamel o f chalky-white l o s s e s o f o u t e r m o s t e n a m e l and s t a i n e d a r e a s w e r e

Loss o f enamel and m a g n i f i c a t i o n o f s t a i n e d areas were n o t t h e c h i l d r e n exposed f o r more t h a n 35 months were The i n c i s o r s o f c h i l d r e n exposed f o r 24 t o They w e r e i n

f o u n d i n y o u n g e r c h i l d r e n e x p o s e d t o t h e w a t e r f o r l e s s t h a n 3 3 months. F o l l o w i n g these findings, separated from o t h e r children. t h e second sub-group o f Group C.

33 m o n t h s w e r e c l a s s i f i e d as m o d e r a t e , m i l d , o r v e r y m i l d .

384

Fig. 4-1

Fig. 4-2

Fig. 4-3

. Teeth o f a girl exposed to fluoride-rich water prenatally until Figure 4 40 months old. Figure 4-1. X-ray photograph of teeth when the subject was 51 months old ( 1 973.11). Figure 4-2. (1980.5) Figure 4-3. (1984.6)

385

F i g . 5-1

F i g . 5-2

F i g . 5-3

F i g u r e 5. T e e t h o f a b o y exposed t o f l u o r i d e - r i c h w a t e r p r e n a t a l l y u n t i l 33 m o n t h s old. F i g u r e 5-1. X-ray p h o t o g r a p h of t e e t h when t h e s u b j e c t was 44 m o n t h s o l d (1973.11). (1980.5) F i g u r e 5-2. F i g u r e 5-3. (1984.6)

386
F i g u r e 5 shows t h e t e e t h o f a boy who r e c e i v e d f l u o r i d e - r i c h water u n t i l X-ray

33 months o f age. H i s upper c e n t r a l i n c l s o r s were c l a s s i f i e d as moderate,

b u t t h e c o n d i t i o n s were n o t a g g r a v a t e d f o r t h e l a s t f o u r years. photographs of t h e t e e t h showed t h a t t h e permanent i n c i s o r s had developed m o r e t h a n h a l f o f t h e i r t o o t h c r o w n s when t h e w a t e r s u p p l y was c h a n g e d . , B u t t h e d i f f e r e n c e i n t h e c o n d i t i o n b e t w e e n i n c i s a l l y p l a c e d e n a m e l and g i n g i v a l l y p l a c e d e n a m e l was n o t c l e a r l y seen. t o 22 months. These c h i l d r e n showed v e r y m i l d , 6). The t h i r d s u b - g r o u p o f w a t e r f o r 11 q u e s t i o n a b l e o r normal Group C c o n s i s t e d o f c h i l d r e n who r e c e i v e d t h e f l u o r i d e - r i c h enamel changes (Fig.

F i g . 6-1

F i g . 6-2

F i g u r e 6. Teeth o f a g i r l exposed t o f l u o r i d e - r i c h 22 months old. F i g u r e 6-1. (1980.5) F i g u r e 6-2. (1984.6)

water p r e n a t a l l y u n t i l

387

DISCUSSION
Schour and S m i t h ( 6 ) f o u n d t h r o u g h a n i m a l e x p e r i m e n t s t h a t f l u o r i d e e x e r t e d a d i r e c t l o c a l a c t i o n on e n a m e l - f o r m i n g h a v e been v o i c e d r e c e n t l y a b o u t t h e s t a g e o r t h a t a r e more s e n s i t i v e t o f l u o r i d e . M a n y e x p e r i m e n t s w i t h r a t s p r o d u c e d m o r p h o l o g i c a l changes 1n i n c i s o r s when f l u o r i d e was a d m i n i s t e r e d a t t h e s e c r e t o r y s t a g e o f enamel f o r m a t i o n cells. Different opinions formation s t a g e s o f enamel

(7-9).

Suga and K a t a g i r i

( 1 0 ) r e p o r t e d t h a t a h y p o m i n e r a l i z e d l e s i o n was S h i n o d a (11) r e p o r t e d t h a t t h e l a t e f o r m a t i v e F e j e r s k o v and h i s co-

i n d u c e d b y a s i n g l e i n j e c t i o n o f NaF i n t h e e a r l y m a t u r a t i o n z o n e o f t h e i n c i s o r enamel of a r a t . a m e l o b l a s t s were p a r t i c u l a r l y s e n s i t i v e t o f l u o r i d e .

w o r k e r s ( 1 2 ) s u g g e s t e d t h a t t h e a m e l o b l a s t s a r e a b l e t o l a y down m a t r i x , b u t i t i s d u r i n g t h e f o l l o w i n g m i n e r a l i z a t i o n p r o c e s s t h a t d i s t u r b a n c e s may occur. The p r e s e n t s t u d y h a s d e m o n s t r a t e d t h a t e f f e c t s o f w a t e r c o n t a i n i n g 7.8 ppm f l u o r i d e d i d n o t a p p e a r on t h e u p p e r i n c i s o r s o f c h i l d r e n c o n s u m i n g t h e w a t e r when t h e y w e r e more t h a n 3 8 m o n t h s o l d . ages when t h e w a t e r s u p p l y was changed, Regarding the childrens' t h o s e who w e r e m o r e t h a n 33 m o n t h s

o l d had s e v e r e d e n t a l f l u o r o s i s on t h e i r u p p e r i n c i s o r s .
It i s i n t e r e s t i n g t h a t , on c o m p a r i n g t h e X-ray photographs o f c h i l d r e n

exposed t o f l u o r i d e - r i c h

w a t e r u n t i l 33 and 4 0 m o n t h s o f age,

the differ-

e n c e i n t o o t h g r o w t h was n o t g r e a t b u t t h e d i f f e r e n c e i n t h e g r a d e o f d e n t a l f l u o r o s i s w a s v e r y c l e a r ( F i g s . 3 a n d 4). p o r t e a r l i e r r e p o r t s b y Dean t e n 13-year These o b s e r v a t i o n s sup-

Gal.

(13).

Approximately ten years a f t e r

t h e change i n t h e communal w a t e r s u p p l y , b a u x i t e was r e s u r v e y e d . o l d c h i l d r e n who had r e c e i v e d t h e f l u o r i d e - r i c h e i g h t were affected. Among t h e s i x 1 2 - y e a r f o r t h r e e years,

O f the

water supply old children

who had been e x p o s e d t o t h e w a t e r f o r t w o y e a r s , m o t t l e d enamel b u t o f t h e seven 11-year-old water f o r o n l y a year, fluorosis.

t h r e e showed e v i d e n c e o f mild" dental

c h i l d r e n who had consumed t h e

o n l y one showed a d e g r e e o f " v e r y

These f i n d i n g s i n d i c a t e t h a t t h e l a t t e r p h a s e o f e n a m e l f o r m a f l u o r i d e i s s t o r e d i n t h e bones

t i o n h a s an i m p o r t a n t i n f l u e n c e on t h e a p p e a r a n c e o f d e n t a l f l u o r o s i s . During a period o f high f l u o r i d e intake, (14). F l u o r i d e f r o m such sources m i g h t produce t h e i n d i s t i n c t d i f f e r e n c e

b e t w e e n f l u o r o s e d e n a m e l p l a c e d on t h e i n c i s o r p o r t i o n and n o r m a l enamel p l a c e d on t h e g i n g i v a l p o r t i o n i n group C o f t h i s study. o f f l u o r i d e ' s i n f l u e n c e occurred, seems t o be m o r e i m p o r t a n t . The o u t e r l a y e r o f enamel b e g i n s t o m i n e r a l i z e v e r y r a p i d l y d u r i n g t h e l a t t e r s t a g e o f m a t u r a t i o n and f i n a l l y shows t h e h i g h e s t d e g r e e o f m i n e r a l i z a t i o n (15). The f l u o r i d e c o n c e n t r a t i o n i n c r e a s e d t o a peak a t t h e e a r l y

I f t h i s extension

t h e l a t t e r s t a g e o f enamel f o r m a t i o n

388
s t a g e o f m a t u r a t i o n o f t h e enamel, m a t u r a t i o n p r o c e e d e d (16,17). and t h e n t e n d e d t o f a l l as enamel The

D u r a t i o n o f t h e s e c r e t o r y and maturation

phases o f enamel d e v e l o p m e n t i n human p e r m a n e n t t e e t h i s n o t known. and p e r m a n e n t enamel d e v e l o p m e n t i n humans.

f i n d i n g s i n t h e p r e s e n t s t u d y suggest a c o r r e l a t i o n between f l u o r i d e a c t i o n I n t h i s o b s e r v a t i o n t h e g r a d e s o f d e n t a l f l u o r o s i s w e r e changed by t h e d u r a t i o n of f l u o r i d e intake, g i v e n t h e same c o n c e n t r a t i o n o f f l u o r i d e .

it

When p a r t o f t h e f l u o r i d e i n d u c e d change o c c u r s d u r i n g t o o t h f o r m a t i o n , cation.

a p p e a r s d i f f i c u l t t o c a t e g o r i z e enamel change a c c o r d i n g t o Dean's c l a s s i f i A s y s t e m f o r c l a s s i f y i n g enamel d e f e c t s based on t h e i r appearance as u s e d b y I s h i i and S u c k l i n g (4). or alternative w o u l d be u s e f u l t o assess (DDE I n d e x F D I ) ( 1 8 )

c l a s s i f i c a t i o n s f o r d e n t a l f l u o r o s i s (19,20)

enamel changes s i m i l a r t o t h o s e o b s e r v e d i n t h i s s t u d y . REFERENCES 1. I s h i i T, I s h i i T, K a t o K, S a k a k i b a r a Y (1982) J D e n t a l H e a l t h 32:78-102 P r o g - A b s t 61:651

2.
3. 4. 5. 6. 7.

S a k a k i b a r a Y (1983) IADR,

I s h i i T, N a k a g a k i H ( 1 9 8 4 ) I n : F e a r n h e a d RW, S u g a S ( e d s ) T o o t h Enamel I V . E l s e v i e r S c i e n c e Pub1 i s h e r s , Amsterdam, p p 338-341 I s h i i T, Schour I , Weber D, Suckling G (submitted) S m i t h MC (1935) JADA 22:796-813 Yaeger JA (1964) JDR 43:50-56 Eisenmann DR (1974) A r c h s o r a l B i o l 19:171-182 Dean HT (1934) JADA 21:1421-1426

8.
9. 10. 11. 12. 13. 14. 15. 16. 17.

Ishida T (1970) J a p J O r a l Biol 12:88-107

W a l t o n RE, Suga S. Katagiri

(1967) JDR 46:134-135

S h i n o d a H (1975) C a l c i f T i s s Res 18:91-100 F e j e r s k o v 0, S i l v e r s t o n e LM, M e l s e r B. M b ' l l e r I J ( 1 9 7 5 ) C a r i e s Res 9~190-219 Dean HT, Suga S, McKay FS. Kamio N, E l v o v e E (1938) Pub H e a l t h r e p 53:1736-1748 W h i t f o r d GM (1985) I A D R A b s t 64:675 Doh A (1977) Jap J O r a l B i o l 19:219-238 Angmar-mansson D e u t s c h D,

B, L i n d h V.
Y o s h i d a S,

G e d a l i a I (1982) C a r i e s Res 16:428-432

D e u t s c h D, S h a p i r a L. A l a y o f f A, L e v i e l D, Y o e l i Z, A r a d A ( 1 9 8 4 ) I n : Fearnhead RW, Suga S (eds) T o o t h Enamel I V . E l s e v i e r Science Publishers, Amsterdam, p p 234-239 T h y l u s t r u p A, F e j e r s k o v 0 (1978) Comm D e n t O r a l E p d e m i o l 6:315-328 K i n g m a n A, M e y e n s RJ ( 1 9 8 4 ) FDI T e c h n i c a l R e p o r t No 1 5 (1982) I n t D e n t J 32:159-167 H o r r o w i t z HS. H e i f e t z SB, D r i s c o l l WS, JADA 109: 37-41

18. 19.

20.

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 389-394 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

389

GASTROINTESTINAL ABSORPTION OF FLUORIDE I N HUMANS - A COMPARATIVE STUDY OF NaF AND CaF2

NORIKO TSUNODA, S H I R O SAKURAI AND HUM10 TSUNODA

D e p a r t m e n t o f H y g i e n e and P u b l i c H e a l t h , U n i v e r s i t y , M o r i o k a 020, J a p a n School o f Medicine, I w a t e Medical

ABSTRACT Twenty-three changes male s u b j e c t s p a r t i c i p a t e d i n a study i n which d i f f e r e n c e s and u r i n a r y fluoride concentrations subsequent t o i n t h e g a s t r o i n t e s t i n a l a b s o r p t i o n o f f l u o r i d e were examined based on i n serum i n g e s t i o n o f NaF o r CaF2. R a p i d f l u o r i d e a b s o r p t i o n was o b s e r v e d w i t h NaF. and t h e s e r u m f l u o r i d e c o n c e n t r a t i o n r e a c h e d a p e a k 30 m i n u t e s a f t e r f l u o r i d e a d m i n i s t r a t i o n .

With CaF2,

however,

f l u o r i d e a b s o r p t i o n was much l o w e r t h a n t h a t o f NaF, and t h e serum

accounting f o r o n l y e i g h t t o t e n percent o f the l a t t e r ,

f l u o r i d e l e v e l d i d n o t peak u n t i l t w o hours f o l l o w i n g i t s a d m i n i s t r a t i o n . A m a r k e d d i f f e r e n c e i n t h e e x t e n t o f f l u o r i d e a b s o r p t i o n was a l s o o b s e r v e d w i t h CaF2, suggesting t h e presence o f i n d i v i d u a l v a r i a t i o n .

INTRODUCTION The t o x i c i t y o f o r a l l y a d m i n i s t e r e d f l u o r i d e i s d e p e n d e n t on i t s c h e m i c a l form. Presumably, t h i s i s r e l a t e d t o t h e v a r y i n g r a t e s o f f l u o r i d e absorpproperties, including solubility, of compounds (1). t i o n due t o d i f f e r e n c e s i n c h e m i c a l various fluorine-containing r e c e n t years,

W i t h t h e advent o f r a p i d advances i n t h e m i c r o a n a l y s i s o f f l u o r i d e s i n
i t i s now p o s s i b l e t o d e t e r m i n e w i t h h i g h a c c u r a c y t r a c e

amounts o f f l u o r i d e ,

such as t h o s e found i n t h e serum

(2-4).

This paper i s concerned w i t h a study i n which t h e r a t e o f f l u o r i d e a b s o r p t i o n t h r o u g h t h e g a s t r o i n t e s t i n a l t r a c t i n humans was i n v e s t i g a t e d f o l l o w i n g t h e i n g e s t i o n o f NaF o r CaF2. t r a s t i n g s o l u b i 1 it i e s . t w o f l u o r i d e compounds w i t h con-

MATERIALS AND METHODS Twenty-three t h i s study. h e a l t h y male adults, a l l i n t h e i r twenties, participated i n Subjxts i n They w e r e d i v i d e d i n t o t w o g r o u p s : NaF and CaF2.

390
t h e NaF g r o u p (n=12) w e r e o r a l l y a d m i n i s t e r e d a 200 m l s o l u t i o n c o n t a i n i n g

1 0 mg F a s NaF. w h i l e t h o s e i n t h e CaF2 ( n = l l ) g r o u p r e c e i v e d 20 mg p o w d e r e d CaF2 and 200 m l w a t e r . t h i s group,

as

A h i g h e r a m o u n t o f F was a d m i n i s t e r e d i n
Fluo-

as a p r e l i m i n a r y s t u d y r e v e a l e d no a p p r e c i a b l e changes i n

f l u o r i d e e x c r e t i o n s u b s e q u e n t t o a d m i n i s t r a t i o n o f 1 0 mg F a s CaF2. state.

r i d e s were a d m i n i s t e r e d i n t h e morning w h i l e t h e s u b j e c t s were i n a f a s t i n g Experiments were c a r r i e d o u t d u r i n g t h e w i n t e r t o avoid excessive t h u s i n f l u e n c i n g t h e e x p e r i m e n t a l d a t a (5.6). p e r s p i r a t i o n by t h e subjects, m i n u t e , 3 0 m i n u t e , 1,

B l o o d s a m p l e s w e r e t a k e n p r i o r t o f l u o r i d e a d m i n i s t r a t i o n , and a t 15

2.

4,

6. 9. 12, a n d 2 4 h o u r i n t e r v a l s , r e s p e c t i v e l y ,
The v o l u m e and c o n c e n t r a t i o n o f e a c h o f t h e F l u o r i d e c o n c e n t r a t i o n was d e t e r m i n e d r e g u l a r meals were p r o v i d e d o n l y a f t e r while drinking water

f o r f l u o r i d e determination.

u r i n e samples were a l s o determined. During t h e experimental period, t h e 12-hour was p r o v i d e d ad l i b i t u m .

by use o f t h e f l u o r i d e i o n - s e l e c t i v e e l e c t r o d e method (2,3,7). b l o o d and u r i n e s a m p l e s had been t a k e n ,

RESULTS
Changes i n serum f l u o r i d e c o n c e n t r a t i o n s a t d i f f e r e n t t i m e i n t e r v a l s f o l l o w i n g t h e o r a l a d m i n i s t r a t i o n o f f l u o r i d e s a r e shown i n F i g u r e 1. immediately following f l u o r i d e administration, a t 30 m i n u t e s . restored t o p e a k i n g a t 0.341fO.076 It ppm i s c l e a r t h a t s e r u m f l u o r i d e l e v e l s o f t h e NaF g r o u p s h o w e d a r a p i d r i s e A l t h o u g h f l u o r i d e l e v e l s d i m i n i s h e d subsequently, t h e y were and d i d n o t

s t i l l h i g h e r t h a n t h o s e shown p r i o r t o f l u o r i d e i n g e s t i o n , n o r m a l v a l u e s u n t i l a f t e r 24 h o u r s .

4 Time, hr

12

24

F i g u r e 1: Changes i n serum f l u o r i d e a d m i n i s t r a t i o n o f NaF and CaF2.

concentrations following oral

391
On t h e o t h e r h a n d , t h e s e r u m f l u o r i d e l e v e l s o f t h e CaF2 g r o u p d i d n o t show a p r o n o u n c e d r i s e f o l l o w i n g i n d i v i d u a l subjects. F i g u r e 1, f l u o r l d e i n t a k e and varied widely w i t h and i t ppm.
As shown i n

An e n h a n c e d l e v e l w a s s e e n a f t e r o n e h o u r ,

p e a k e d a t t w o h o u r s s h o w i n g a l o w e r v a l u e o f 0.073+0.065 t r a t i o n o f NaF and CaF2 v a r i e d m a r k e d l y .

t h e p a t t e r n and peak l e v e l s o f s e r u m f l u o r i d e f o l l o w i n g a d m i n i s -

A t t h e peak l e v e l ,

administration

o f CaF2 a t a n a m o u n t e q u i v a l e n t o f 1 0 mg F. b y t h e NaF g r o u p . much s l o w e r ,

a s w a s t h e c a s e w i t h NaF.

r e s u l t e d i n a s e r u m f l u o r i d e l e v e l w h i c h was o n l y 10.7% o f t h a t e x h i b i t e d I t a p p e a r s t h a t a b s o r p t i o n o f f l u o r i d e f r o m CaF2 w a s and i t s r a t e much l o w e r t h a n t h a t o f NaF. whose peak

Two s u b j e c t s i n t h e CaF2 g r o u p showed a g r e a t e r i n c r e a s e i n s e r u m f l u o r i d e l e v e l s t h a n t h a t d e m o n s t r a t e d b y t h e r e s t o f t h e group, c o n c e n t r a t i o n s a v e r a g e d 0.045+0.021 ppm ( F i g .

2).

Whereas t h e l a t t e r v a l u e Nevertheless,

w a s 6.6% o f t h e c o r r e s p o n d i n g v a l u e o f t h e NaF g r o u p , t h e v a l u e s s h o w n b y t h e t w o s u b j e c t s w e r e as h i g h a s 21% and 31%. r e s p e c t i v e l y . i n a l l cases, administration. p e a k v a l u e s w e r e n o t a t t a i n e d u n t i l t w o h o u r s a f t e r CaF2 m u c h l a t e r t h a n t h a t o b s e r v e d w i t h t h e NaF g r o u p .

It

s h o u l d b e m e n t i o n e d t h a t no o t h e r e f f e c t s w e r e o b s e r v e d ,

s u g g e s t i n g an

i n d i v i d u a l v a r i a t i o n i n t h e a b s o r p t i o n o f f l u o r i d e when CaF2 was used. T a b l e I shows t h e c u m u l a t i v e u r i n a r y f l u o r i d e e x c r e t i o n a s t h e p e r c e n t a g e o f f l u o r i d e o r a l l y ingested. o t h e r hand, f o r t h e CaF2 g r o u p , F o r t h e NaF g r o u p , urinary excretion o f On t h e f l u o r i d e a m o u n t e d t o a b o u t 20% a t t w o h o u r s , e x c r e t e d w i t h i n 24 h o u r s o f a d m i n i s t r a t i o n . I n a d d i t i o n , as shown i n F i g u r e 3, e x c e p t f o r t h e t w o s u b j e c t s s h o w i n g a n enhanced s e r u m f l u o r i d e level, t h e c u m u l a t i v e e x c r e t i o n r a t e was 4.7k1.82. ppm and 0.22 ppm, respectively. F o r t h e t w o s u b j e c t s whose s e r u m f l u o r i d e p e a k e d a t 0.17 t h e c o r r e s p o n d i n g v a l u e s w e r e 19.9% and 31.5%. and 48.1% a t 24 h o u r s .

o n l y 8.5% o f t h e i n g e s t e d f l u o r i d e w a s

5 0.301
Q

--*--

_.

Subject A

Subiect 6

Time, hr
F i g u r e 2: Changes i n s e r u m f l u o r i d e c o n c e n t r a t i o n s f o l l o w i n g o r a l a d m i n i s t r a t i o n o f CaF2.

392
TABLE I CUMULATIVE RATE OF URINARY FLUORINE EXCRETION FOLLOWING ORAL ADMINISTRATION OF NaF AND CaF2 ( M e a n f S.D.) Time a f t e r a d m i n i s t r a t i o n Hr 1 NaF ( n = 1 2 ) CaF2 ( n = l l )

2 4 6 9 12 24

9.9 18.5 _+ 29.4 f 35.3 40.1 f 42 .8 48.1 f

_+

2.9 4.0 4.0 4.1 4.1 4.5

3.5

0.4

_+

4.2 5.6 6.6 7.3 8.5

2.1 f 2.6
f
f

0.3

f
f f

5.4 6.6 7.7 8.3 9.0

8
L

401

--o----0--

Subject A Subject B

Time, hr
F i g u r e 3: Cumulative r a t e o f urinary f l u o r i d e excretion f o l l o w i n g oral a d m i n i s t r a t i o n o f CaF2.

DISCUSSION F l u o r i d e a b s o r p t i o n t h r o u g h t h e g a s t r o i n t e s t i n a l t r a c t i n humans depends on t h e s o l u b i l i t y o f f l u o r i d e s . According t o t h e experimental data from metabolic fluoride and McClure i s 62-69%. b a l a n c e s t u d i e s o n human a d u l t s r e p o r t e d b y L a r g e n t (8) the rate o f fluoride absorption for soluble fluo-

gtt. ( 9 ) ,

r i d e s s u c h a s NaF i s 89-97% w h e r e a s f o r i n s o l u b l e f l u o r i d e s s u c h a s CaF2 i t These d a t a w e r e based on t h e t o t a l f l u o r i d e i n t a k e and t h e As a r e s u l t o f r e c e n t a d v a n c e s i n serum f l u o r i d e l e v e l s can a l s o be d e t e r t h e p r e s e n t s t u d y i n c l u d e d serum f l u o f l u o r i d e e x c r e t i o n i n u r i n e and feces. quantitative analysis o f fluoride, mined accurately. I n view o f this,

393
r i d e c o n c e n t r a t i o n s a s w e l l a s t h e c o m m o n l y u s e d u r i n a r y f l u o r i d e l e v e l s as an i n d i c a t o r o f been examined. The r e s u l t s o b t a i n e d f r o m t h i s s t u d y h a v e c o n f i r m e d e a r l i e r f i n d i n g s t h a t i n a d u l t s t h e a b s o r p t i o n o f f l u o r i d e s u c h a s NaF was r a p i d a n d c o n s i d e r a b l e (8-12). On t h e o t h e r hand, t h e f l u o r i d e a b s o r p t i o n f r o m compounds s u c h as CaF2 w a s l o w e r t h a n t h a t r e p o r t e d b y L a r g e n t ( 8 ) a n d M c C l u r e f l u o r i d e absorption, whereby changes i n t h e r a t e o f f l u o r i d e a b s o r p t i o n caused by d i f f e r e n c e s i n t h e s o l u b i l i t y o f f l u o r i d e s have

gal.

(9). A

m a j o r i t y o f t h e s u b j e c t s i n t h e CaF2 g r o u p e x h i b i t e d a n a b s o r p t i o n r a t e t h a t w a s o n l y 6.6% o f t h e v a l u e s h o w n b y NaF w h e n v i e w e d a t p e a k f l u o r i d e c o n c e n t r a t i o n i n t h e serum. the urine, When v i e w e d a t o f 2 4 - h o u r f l u o r i d e e x c r e t i o n i n t h e f l u o r i d e a b s o r p t i o n r a t e w a s e s t i m a t e d t o b e o n l y 10%. The r e a s o n f o r t h e s e d i f f e r e n c e s i s n o t known. e.g., inflammation o r ulcers,

a l t h o u g h i n some i n s t a n c e s t h e r a t e was h s h i g h as 20-30% o f t h e c o r r e s p o n d i n g v a l u e o f NaF. i s possible, (5,13). however, t h e g a s t r i c mucosa,

It

t h a t t h e pH o f t h e g a s t r i c j u i c e and t h e t r o u b l e s o f may h a v e a n e f f e c t

The i n f l u e n c e o f h o s t f a c t o r s s u c h a s t h e s e a w a i t s f u r t h e r s t u d y .

Recently, a c r i t i c a l problem a s s o c i a t e d w i t h a i r b o r n e f l u o r i d e i n d u c e d p o l l u t i o n i n Japan h a s been t h e a c c u m u l a t i o n o f f l u o r i d e i n a g r i c u l t u r a l p r o d u c t s (14,15). The f i n d i n g s o f t h i s s t u d y s t r e s s e d t h e i m p o r t a n c e o f including solubility, i n evaluating the i d e n t i f y i n g n o t o n l y the f l u o r i d e content o f a g r i c u l t u r a l products, but also the chemical properties, i n f l u e n c e o f p o l l u t e d f o o d p r o d u c t s on humans. ACKNOWLEDGEMENT T h i s w o r k was s u p p o r t e d b y a G r a n t - i n - A i d 60480193) f o r 1985-1987 o f t h e Government o f Japan. REFERENCES 1. E a g e r s R Y ( 1 9 6 9 ) T o x i c P r o p e r t i e s o f I n o r g a n i c F l u o r i n e C o m p o u n d s . E l s e v i e r . Essex, p 33 2. T a v e s DR (1968) T a l a n t a 15:1015-1023 3. F u j i m o r i S, I t a i K, Tsunoda H (1984) F l u o r i d e 17:27-35 4. V e n k a t e s w a r l u P (1983) I n : Shupe JL. P e t e r s o n HB, Leone NC ( e d s ) F l u o r i d e s : E f f e c t s o n V e g e t a t i o n , A n i m a l s a n d Humans. Paragon Press I n c . S a l t L a k e City, Utah, pp 21-52 Simons JH (ed) F l u o r i n e C h e m i s t r y , 5. Hodge HC, S m i t h FA (1965) I n : 4. A c a d e m i c P r e s s , New York. p p 137-176 6. 7. C r e m e r HD, B i t t n e r W ( 1 9 7 0 ) I n : E r i c s s o n Y ( e d ) F l u o r i d e H e a l t h . WHO, Geneva, p p 75-91 F r a n t MS, Ross J W (1966) S c i e n c e 154:1553-1554 Vol. f o r Co-operative R e s e a r c h (No. f r o m t h e M i n i s t r y o f Education, Science and C u l t u r e

a n d Human

394 The H e a l t h A s p e c t s o f F l u o r i n e Compounds. 8. L a r g e n t EJ (1961) F l u o r o s i s : O h i o S t a t e U n i v Press, Columbus, p p 34-39 9. M c C l u r e FJ, M i t c h e l l HH, 27: 159-1 70 H a m i l t o n TS, K i n s e r C A ( 1 9 4 5 ) J i n d u s t r Hyg

10. H e n s c h l e r 0. B i t t n e r W. P a t z J (1975) I n : K u h l e n c o r d t F. K r u s e HP (eds) C a l c i u m M e t a b o l i s m , Bone and M e t a b o l i c Bone Diseases. S p r i n g e r - V e r l a g . B e r l i n , New York, p p 111-121
11. E k s t r a n d J. 12: 31 1-317 12. Spak CJ, 13. W h i t f o r d 14. Tsunoda A l v a n G, Borbs, N o r l i n A (1977) Eur J C l i n Pharrnacol

E k s t r a n d J, Z y l b e r s t e i n (1982) C a r i e s Res 16:249-256

GM,

P a s h l e y DH,

D i r k s e n TR (1982) J D e n t Res 61:291

(1978) N i p p o n D e n t Rev 427:137-150 I t a i K, Tsunoda H (1983) F l u o r i d e 16:175-180

15. S a k u r a i S.

H. Tsunoda and M.-H. Y u (Editors)

Fluoride Research 1 9 8 5 , Studies in Environmental Science, Volume 27, pp. 395-400 0 1 9 8 6 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

395

POWER SPECTRUM ANALYSIS OF U R I N A R Y FLUORIDE EXCRETION I N HUMAN ADULTS

TOSHITAKA H O R I U C H I ,

I K U O NASU, AND MOT01 MORIMOTO

Nihon U n i v e r s i t y School o f D e n t i s t r y a t Matsudo, D e p a r t m e n t o f D e n t a l P u b l i c H e a l t h , 2-870-1 S a k a e c h o - N i s h i , M a t s u d o - s h i , C h i b a 271, J a p a n

ABSTRACT The p o w e r s p e c t r u m a n a l y s i s o f t h e r h y t h m o b t a i n e d f r o m a s t u d y n which eported

6 h e a l t h y a d u l t s were f e d a space f o o d 3 t i m e s a day f o r 6 days i s

here. fluoride.

Urine samples were c o l l e c t e d every 4 hours f o r determin t i o n o f The r e s u l t s w e r e u s e d f o r t h e c a l c u l a t i o n o f t h e a u t o c o r r e l a t i o n The e x p e r i m e n t a l d a t a i n d i c a t e t h a t t h e main and 1 2 h o u r s . The d a t a s u g g e s t p e r i o d i c i -

c o e f f i c i e n t and p o w e r s p e c t r u m . c y c l e w i t h m i n o r p e a k s a t 6,

r h y t h m e x h i b i t e d by u r i n a r y f l u o r i d e e x c r e t i o n c o n s i s t e d o f a 24-hour

8 .

t i e s i n u r i n e v o l u m e and 4 and 6 h o u r f e e d i n g c y c l e s o f a d u l t s u b j e c t s .

INTRODUCTION U r i n e a n a l y s i s i s f r e q u e n t l y used i n s t u d y i n g t h e m e c h a n i s m o f exogenous fluorine excretion. (1,2). duced. Twenty-four hour u r i n e samples a r e g e n e r a l l y analyzed analysis by time

To e v a l u a t e c h a n g e s i n u r i n a r y f l u o r i n e e x c r e t i o n ,

s e r i e s w o u l d seem an e f f e c t i v e m e t h o d b e c a u s e u r i n e i s c o n t i n u o u s l y p r o -

A detailed,

s e r i a l a n a l y s i s u s i n g s p o t u r i n e s a m p l e s h a s many

a d v a n t a g e s i n t e r m s o f r e d u c i n g t h e b u r d e n on t h e p a r t o f t h e s u b j e c t s and i n t h e t i m e and c o s t i n v o l v e d , as w e l l a s w i t h t h e p o s s i b i l i t y t h a t m o r e a c c u r a t e i n f o r m a t i o n may be o b t a i n e d . I n p r e v i o u s work, a power spectrum, d i u m (4,5), However, l i t t l e information i s a v a i l a b l e c o n c e r n i n g d e t a i l e d a n a l y s i s o f s p o t u r i n e samples. s p o t u r i n e s a m p l e s w e r e e x a m i n e d and t h e e x i s t e n c e o f I n t h i s study, p e r i o d i c i t y i n u r i n a r y f l u o r i n e e x c r e t i o n was r e p o r t e d (3).

w h i c h i s a method used i n t h e a n a l y s i s o f e l e c t r o m y o c a r -

was e m p l o y e d i n t h e a n a l y s i s o f t h e c o m p o s i t i o n a n d p e r i o d i c i t y

o f f l u o r i n e excreted i n spot u r i n e i n a d u l t subjects.

MATERIALS AND METHODS Six healthy male adults, o f vegetables, shuttle.

1 9 t o 4 0 y e a r s o f age, w e r e g i v e n s p a c e f o o d
U.S.A. The food, composed 1 2 : 3 0 P.M., and 6 : 3 0 and c r a c k e r s , i s b e i n g used i n t h e space

m a n u f a c t u r e d b y O r e g o n F r e e z e Dry F o o d s , Inc., chicken stew,

I t w a s p r o v i d e d 3 t i m e s d a i l y a t 8:30 A.M.,

396

P.M.

Water was given 3 times d a i l y . T o t a l d a i l y w a t e r i n t a k e was 1400 m l ,

i n c l u d i n g t h e h o t w a t e r used f o r r e s t o r i n g t h e f r e e z e - d r i e d food samples.

T h e s u b j e c t s were a l l o w e d t o l e a d r e g u l a r l i v e s e x c e p t t h a t t h e y w e r e
housed a t n i g h t i n a l o c a t i o n f a c i l i t a t i n g t h e study. Urine s a m p l e s were c o l l e c t e d e v e r y 4 hours f o l l o w i n g i n g e s t i o n of t h e e x p e r i m e n t a l food f o r t h e e n t i r e e x p e r i m e n t a l p e r i o d of 6 days.
To a n a l y z e t h e p e r i o d i c i t y o f u r i n a r y f l u o r i d e e x c r e t i o n ,

Urinary t h e power

f l u o r i d e c o n t e n t was d e t e r m i n e d by t h e f l u o r i d e ion m e t e r method (6,7). s p e c t r u m ( 8 . 9 ) was o b t a i n e d by u s e o f t h e a u t o c o r r e l a t i o n c o e f f i c i e n t s described previously (3).

RESULTS AND

DISCUSSION
day e x p e r i m e n t a l period. W i t h some

F i g u r e 1 shows t h e amount of f l u o r i d e e x c r e t e d i n t h e u r i n e e v e r y 4 hours by t h e 6 participants during t h e G e x c e p t i o n s , u r i n a r y f l u o r i d e l e v e l s p l o t t e d a g a i n s t s a m p l i n g time form an

23

23

23

Clock time

23

23

Figure 1.

Dally u r i n a r y e x c r e t i o n of f l u o r i d e i n s i x a d u l t s

397
M - s h a p e d c u r v e . T h e p e a k s a p p e a r a t t h e 11:OO a n d 19:OO s a m p l i n g t i m e s . The d a t a c o n c e r n i n g t h e a m o u n t o f f l u o r i n e e x c r e t e d i n t h e u r i n e a t e a c h s a m p l i n g t i m e a r e s u m m a r i z e d i n T a b l e I. s a m p l e s w e r e c o l l e c t e d e v e r y 2 h o u r s (3). meal. I n a previous experiment. urine The amount o f f l u o r i n e e x c r e t e d

i n u r i n e showed a d e f i n i t e t e n d e n c y t o i n c r e a s e i m m e d i a t e l y a f t e r each I n t h e p r e s e n t study, u r i n e samples were c o l l e c t e d every 4 hours, When a d i e t c o n t a i n i n g a n e q u a l a m o u n t o f however, t h e d a i l y f l u o r i n e and a p p r o x i m a t e l y 470 a s s t a t e d b y Tsunoda (10). subjects, 1.e.. a 19-yearand t h e a m o u n t f o r e a c h t i m e p e r i o d was averaged. T h e r e f o r e , a c l e a r - c u t d i f f e r e n c e was n o t observed. f l u o r i n e was i n g e s t e d b y a l l t h e SubJects, ug i n t h e 40-year-old age o f t h e SubJect. o l d and 4 0 - y e a r - o l d s u b J e c t ( T a b l e 11).

e x c r e t i o n was a p p r o x i m a t e l y 350 u g i n t h e 1 9 - y e a r - o l d Thus,

t h e amount o f f l u o r i n e e x c r e t e d i n u r i n e t e n d e d t o i n c r e a s e w i t h i n c r e a s i n g B o t h young and m i d d l e - a g e d males, showed a l m o s t t h e same c i r c a d i a n v a r i a t i o n s .

It

TABLE I

U R I N A R Y FLUORIDE LEVEL AT EACH SAMPLING TIME

Time 23: 00-07: 00 07:00-11:00 11 :00-1 5: 00 15: 00-1 9: 00 19: 00-23: 00 Mean 10.0 16.9 20.1 14.4 22.5 Max. 14.0 21.3 24.7 18.5 25.1 Min. 7.4 12.2 17.8 11.7 20.4

S. D.
2.6 3.5 3.2 2.3 3.9

Data i s f o r 6 s u b j e c t s f o r 6 days (ug/hr)

TABLE 11 24-HOUR URINARY EXCRETION OF FLUORIDE I N ADULTS ( p g / d a y ) Subject TH Mean 349 348 335 395 343 472 Max. 418 417 372 452 369 520 M i n. 31 2 292 292 280

S. D.
38 45 34 57 20 45

KO
IG
KH

HK
HT

310
382

398
i s d i f f i c u l t t o determine the d e f i n i t e p e r i o d i c i t y o f circadian variations as d i f f e r e n t f a c t o r s a r e i n v o l v e d . periodicity. The d a t a o n u r i n a r y f l u o r i n e e x c r e t i o n w e r e e x a m i n e d f o r t h e a u t o c o r r e l a t i o n coefficient. As shown i n F i g u r e 2, all 6 s u b j e c t s showed p e r i o d i c i t y The w h e r e t h e a u t o c o r r e l a t i o n c o e f f i c i e n t a p p r o a c h e d 1 e v e r y 24 h o u r s . p e r i o d i c i t i e s showed no s p e c i f i c p a t t e r n s as shown p r e v i o u s l y w i t h Na. and C1 (3.11). o f a W-shape, t h e (-) i n s t e a d o f a V-shape. The d a t a f r o m t h e q u a n t i t a t i v e a n a l y s i s o f f l u o r i n e excreted i n u r i n e were then analyzed by t i m e s e r i e s t o evaluate

K.

Changes i n t h e c o r r e l a t i o n c o e f f i c i e n t s i n t h e (-) a r e a w e r e The c o e f f i c i e n t f o r e v e r y 24 h o u r s i n

a r e a a p p r o a c h e d zero.

To a c c u r a t e l y a s s e s s v a r i a t i o n i n t h e (-) a r e a , f a s t F o u r i e r t r a n s f o r m was u s e d t o d e t e r m i n e t h e p o w e r s p e c t r u m o f t h e a u t o c o r r e l a t i o n c o e f f i c i e n t . A t w o - p e a k p o w e r s p e c t r u m a p p e a r e d w i t h s u b j e c t s T.H. three-peak w i t h H.K. s p e c t r u m w i t h K.O., 3). a n d I.G.. a

K.H.,

a n d H.T.,

and a four-peak a n d H.T..

spectrum

(Fig.

The f i r s t peak o f t h e p o w e r s p e c t r a a p p e a r e d i n a 24

h o u r c y c l e i n s u b j e c t s T.H.,

K.O.,

I.G..

H.K..

The s e c o n d peak

. . . . . . .. ... . . . ..-. - . . ......... . . . .. -.. ..

..

..

..

. . --. ..............
.
~ ~~

.* *~

24

48

72

96

Time

1M

F i g u r e 2.

Autocorrelation coefficient o f urlnary fluoride

399

iii

'

Period(hr) F i g u r e 3. Power s p e c t r u m o f u r i n a r y f l u o r i d e

a p p e a r e d s o m e t i m e d u r i n g t h e t i m e p e r i o d s o f 12, w i t h subject

8. a n d 6 h o u r s .

These

p e r i o d i c i t i e s a l l c o r r e s p o n d e d t o d i v i s o r s o f 24 h o u r s .

The f i r s t peak

K.H.

appeared a t 12 hours,

u n l i k e t h a t o f t h e other subjects.

Changes i n t h e p o w e r s p e c t r a o f Na, other subjects,

and C1 e x c r e t e d i n t h e u r i n e b y t h i s u r i n e e x c r e t i o n p a t t e r n compared t o

s u b j e c t a l s o showed p e r i o d i c i t i e s ( 1 1 ) w h i c h w e r e d i f f e r e n t f r o m t h o s e o f suggesting a different i.e.. e.g.. t h a t o f t h e other 5 subjects. Circadian periodicity, urine, t h e n a t u r a l environment, the main rhythm o f f l u o r i n e excretion i n tide. and w i t h t h e c i r c a d i a n p e r i o d i c i t y o f (12) and Sulzman (13). which are It i s . h i g h l y probable t h a t seems t o b e i n a g r e e m e n t w i t h c h r o n o t r o p i s m ( p e r i o d i c v a r i a t i o n s ) i n

u r i n e volume reported by M i l l s e t al. t h e p e r i o d i c i t i e s o f 6, 8, i n t a k e (131,

g e n e r a t e d b y endogenous o s c i 1l a t o r y systems.

and 12 h o u r s a r e due t o t h e rhythm o f food The r h y t h m o f s l e e p i n g

from the f a c t that the urinary fluorine excretion i s influ-

e n c e d b y t h e f l u o r i n e i n g e s t e d a t meal t i m e (3).

400
and w a k i n g , light-dark cycle, etc.. c a n be c o n s i d e r e d o t h e r f a c t o r s , b u t no

d e f i n i t e c o n c l u s i o n c a n be d r a w n i n t h e p r e s e n t s t u d y .

I t w o u l d seem r e a s o n a b l e t o assume t h a t m e t a b o l i s m a n d e x c r e t i o n o f
v a r i o u s m a t e r i a l s a r e repeated i n a temporal p a t t e r n i n t h e l i f e process i n a s h o r t c y c l e r a t h e r than c i r c a d i a n p e r i o d i c i t y o f constant rhythm. u l t r a d i a n rhythm. F l u o r i n e shows an u l t r a d i a n r h y t h m , w h e r e a s Na, i.e..

K,

and

C1 show a c i r c a d i a n r h y t h m ( 3 , l l ) . ment, and t h r e e - d i m e n s i o n a l l y , CONCLUSION

These f a c t s s u g g e s t a u n i q u e r e a c t i v i t y t h e most negative ele-

or material property o f fluorine (electronically,

t h e s m a l l e s t atom n e x t t o hydrogen).

T h i s e x p e r i m e n t a l d a t a i n d i c a t e s t h a t t h e u l t r a d i a n r h y t h m e x h i b i t e d by u r i n a r y f l u o r i n e e x c r e t i o n c o n s i s t s o f a 24,

1 2 , 8,

and 6 h o u r c y c l e

a c c o r d i n g t o t h e p o w e r s p e c t r u m method. T h e s e p e r i o d i c i t i e s s u g g e s t p e r i o d i c i t i e s o f u r i n e v o l u m e and 4 and 6 h o u r f e e d i n g c y c l e s o f a d u l t S u b j e c t s .

REFERENCES

1.
2.

6: 503-514
Hodge HC,

D e r r y b e r r y OM,

B a r t h o l o m e w MD,

F l e m i n g RBL (1963)

Arch Environ Health

S m i t h FA (1970) J A i r P o l l A s s o c 20:226-232 Nasu I. M o r i m o t o M (1984) F l u o r i d e 17:173-177

3.

H o r i u c h i T,

4. C h a f f i n DB (1969) J Occup Med 11:109-115 5. W a l t o n JH (1952) J N e u r o l N e u r o s u r g P s y c h i a t 15:219-226

6. H o r i u c h i T, K a s a i G, T e r a m o t o K. M o r i m o t o M (1977) J D e n t H e a l t h 27:102

7. O r i o n R e s e a r c h I n c (1971) S p e c i f i c I o n E l e c t r o d e T e c h n o l o g y 3:8
8. A k a i k e K ( 1 9 8 5 )
Power Spectrum A n a l y s i s o f I r r e g u l a r O s c i l l a t i o n . I n s t i t u t e o f S t a t i s t i c a l M a t h e m a t i c s , p p 37-40 A s a k u r a Shuppan. p p 40-51

9. H i n o M (1980) P o w e r S p e c t r u m A n a l y s i s .

10. Tsunoda H, S a k u r a i S, I t a i K, S a t 0 T, Nakaya S, M i t a M, T a t u m i M (1983) F 1u o r i d e 17: 15971 67

11. H o r i u c h i T.
(unpublished) S t u d y f o r t h e movement o f u r i n a r y f l u o r i d e i n human a d u l t s

12. M i l l s JN, S t a n b u r y SW (1952) P h y s i o l 117:22 13. S u l z m a n FM, F u l l e r CA, Moore-Ede MC (1977) P h y s i o l Behav 18:775-779

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 401-406 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

401

NORMAL FLUORIDE CONCENTRATIONS I N SPOT SAMPLES OF U R I N E

KAORU KASAHARA, TAKESHI KONDO. AND TAKASHI NAKANE

D e p a r t m e n t o f Communi t y D e n t i s t r y , Gobara S h i o j i r i C i t y 399-07, J a p a n Matsumoto Dental College, 1780 Hirooka

ABSTRACT I n an a t t e m p t t o e v a l u a t e n o r m a l f l u o r i d e c o n c e n t r a t i o n s i n u r i n e , s a m p l e s o f u r i n e w e r e c o l l e c t e d f r o m 1,213 working environment, Mid-morning spot h e a l t h y Japanese m a l e a d u l t s , were determined.

c o n s i d e r e d t o be f r e e f r o m u n u s u a l f l u o r i d e exposure i n t h e i r l i v i n g o r and t h e i r f l u o r i d e c o n t e n t s u r i n e s a m p l e s w e r e c o l l e c t e d f r o m 1,047 G r o u n d S e l f - D e f e n s e F l u o r i d e c o n c e n t r a t i o n s w e r e d e t e r m i n e d u s i n g a n F-

F o r c e o f f i c i a l s aged 1 8 t o 58, and t h e f i r s t - m o r n i n g u r i n e s a m p l e s f r o m 172 f a r m e r s aged 50 t o 69. e l e c t r o d e a f t e r t h e f l u o r i d e was s e p a r a t e d b y a m i c r o d i f f u s i o n method. S t a t i s t i c a l a n a l y s i s o f t h e d a t a was c a r r i e d o u t f o l l o w i n g l o g a r i t h m i c t r a n s f o r m a t i o n o f t h e values, since t h e d i s t r i b u t i o n o f f l u o r i d e concentrat i o n s was n e a r l y l o g a r i t h m i c a l l y n o r m a l . G e o m e t r i c mean v a l u e s o f t h e u r i n a r y f l u o r i d e c o n c 2 n t r a t i o n s o f t h e S e l f D e f e n s e F o r c e o f f i c i a l s (N=1040) and t h e f a r m e r s (N=164) w e r e e s t i m a t e d t o b e 0.32 m g / l a n d 0.50 m g / l , respectively.

INTRODUCTION U r i n a r y e x c r e t i o n o f f l u o r i d e i s w i d e l y a c c e p t e d as one o f t h e b e s t i n d i c e s o f f l u o r i d e a b s o r p t i o n (1). i n d e x o f h e a l t h c a r e (2.3). tion, 24-hour a r e employed r o u t i n e l y . Normal f l u o r i d e e x c r e t i o n i n t h e 24-hour j e c t s has been r e p o r t e d b y Tsunoda u r i n e s a m p l e s o f J a p a n e s e subb u t r e p o r t s on t h e normal spot F l u o r i d e l e v e l s i n t h e u r i n e samples therefore, b e e n u s e d a s an o f i n d u s t r i a l w o r k e r s e x p o s e d t o f l u o r i d e have, u r i n e s a m p l e s s h o u l d be used.

F o r an a c c u r a t e e v a l u a t i o n o f f l u o r i d e e x c r e However, s p o t samples o f u r i n e

g d.( 3 ) ,

f l u o r i d e c o n c e n t r a t i o n o f s p o t samples o f u r i n e have been c o n t r a d i c t o r y . I n an a t t e m p t t o e v a l u a t e normal f l u o r i d e c o n c e n t r a t i o n s i n urine.' s a m p l e s w e r e c o l l e c t e d f o r m 1,219 h e a l t h y Japanese male a d u l t s c o n s i d e r e d

t o be f r e e f r o m f l u o r i d e exposure i n t h e i r l i v i n g o r w o r k i n g environment, and t h e f l u o r i d e c o n c e n t r a t i o n s o f t h e s e u r i n a r y samples were determined.

402
MATERIALS AND METHODS I n t h i s s t u d y , t w o d i f f e r e n t e x p e r i m e n t a l g r o u p s w e r e examined. m o r n i n g u r i n e s a m p l e s (8:30-11:OO Self-Defense Force a.m.) Midw e r e c o l l e c t e d f r o m 1,047 G r o u n d aged 50 t o 69. residing i n

(GSDF) o f f i c i a l s

aged 18 t o 58, and t h e f i r s t - m o r n i n g

u r i n e samples were c o l l e c t e d f r o m 172 farmers,

d i f f e r e n t areas. F l u o r i d e c o n c e n t r a t i o n s i n t h e d r i n k i n g w a t e r i n b o t h areas were l e s s t h a n 0.1 m g / l and f l u o r i d e i n t h e a i r was n o t d e t e c t a b l e .

To s e p a r a t e t h e f l u o r i n e i n u r i n e ,

a m i c r o d i f f u s i o n method was employed

(4).

A 5 m l u r i n e s a m p l e was d i f f u s e d i n 2.7 M HC104 ( H M D S - s a t u r a t e d ) .

F l u o r i n e was t r a p p e d i n f i l t e r paper t r e a t e d w i t h 0.5 t h e f l u o r i d e was e l u t e d by shaking.

NaOH.

The f i l t e r

paper was t h e n p l a c e d i n a t e s t t u b e c o n t a i n i n g 1 m l o f d i s t i l l e d w a t e r and F l u o r i d e concentrations i n the eluates w e r e m e a s u r e d b y t h e f l u o r i d e i o n - s c l ? c C i v a 31.?c-:r>:.'? . i n l i i ~ o d . F i g u r e 1

shc'~.i; .i:is ~ . : i ; x ~ r o t : ?I iZsY ! f a r ,Tic;po?i f-F:!-io?.

F i g u r e 1.

Apparatus used f o r m i c r o d i f f u s i o n .

RESULTS The minimum and maximum f l u o r i d e c o n c e n t r a t i o n s i n t h e mid-morning u r i n e s a m p l e s w e r e f o u n d t o b e 0.046 m g / l and 4.6 m g / l . m o r n i n g u r i n e were 0.070 mg/l and 10 mg/l. m o r n i n g u r i n e s a m p l e s showed m o r e t h a n 1.2 m g F / l . and t h o s e i n t h e f i r s t Seven o f t h e midThey w e r e exand n i n e o f t h e f i r s t respectively.

m o r n i n g u r i n e s a m p l e s g a v e v a l u e s h i g h e r t h a n 1.4 m g F / l .

c l u d e d f r o m t h e s t a t i s t i c a l a n a l y s i s , as t h e y were c o n s i d e r e d abnormal. F i g u r e s 2 a n d 3 show t h e h i s t o g r a m s o f f l u o r i d e c o n c e n t r a t i o n s i n t h e mid-morning and t h e f i r s t - m o r n i n g urine. These r e s u l t s suggest t h a t t h e d i s t r i b u t i o n o f u r i n a r y f l u o r i d e c o n c e n t r a t i o n s i n s p o t samples i s logar i t h m i c a l l y normal.

400

300

200

100

ik
20 0

403

1
.8
1.2
1.6

0 0
.24 .48

.72

.96

1.2

.4

F conc. i n u r i n e ( m g / l )

F conc. i n u r i n e (mgll)

F i g u r e 2. (N=l040). F i g u r e 3. (N=l64).

Hi,-togram

o f fluoride concentrations i n t h e mid-morning

urine

Histogram o f f l u o r i d e concentrations i n the f i r s t - m o r n i n g

urine

(21 99 r

0.05

0.1

0.5

F concentrotlons In urlne Img/l)

F i g u r e 4. C u m u l a t i v e f r e q u e n c y c u r v e o f f l u o r i d e concentrations i n spot sampling urine. 0 . mid-morning u r i n e ; O , f i r s t - m o r n i n g u r i n e .

404
F i g u r e 4 shows t h e c u m u l a t i v e f r e q u e n c y c u r v e o f u r i n a r y f l u o r i d e concent r a t i o n s on l o g a r i t h m i c p r o b a b i l i t y paper. The d i s t r i b u t i o n of u r i n a r y since Thus s t a t i s t i c a l f l u o r i d e c o n c e n t r a t i o n s was c o n f i r m e d t o b e l o g a r i t h m i c a l l y n o r m a l , t h e c u m u l a t i v e f r e q u e n c y gave e s s e n t i a l l y s t r a i g h t l i n e s . the values. T a b l e s I and I 1 show t h e g e o m e t r i c mean v a l u e s and s t a n d a r d d e v i a t i o n of u r i n a r y f l u o r i d e c o n c e n t r a t i o n s o f s p o t samples. The g e o m e t r i c mean v a l u e s respectively. urinary fluoo f t h e f l u o r i d e c o n c e n t r a t i o n s i n t h e mid-morning and t h e f i r s t - m o r n i n g u r i n e s a m p l e s w e r e c a l c u l a t e d t o be 0.32 m g / l and 0.50 m g / l , F i g u r e 5 i l l u s t r a t e s t h e n o r m a l r a n g e (Meang r i d e c o n c e n t r a t i o n s i n d i f f e r e n t age groups.

a n a l y s i s o f t h e d a t a was p e r f o r m e d a f t e r t h e l o g a r i t h m i c t r a n s f o r m a t i o n of

* 2S.D. 4 ) o f

TABLE I FLUORIDE CONCENTRATIONS ( m g / l ) I N THE M I D - M O R N I N G U R I N E OF MALE ADULTS Age Geometric S. D. Mean 0.25 0.29 0.36 0.35 0.37 0.32 1.57 1.72 1.73 1.60 1.66 1.71

n 97 447 182

18-1 9 20-29 30-39 40-49 50-58 Total

209
105
1040

TABLE I 1 FLUORIDE CONCENTRATIONS (mg/l) I N THE FIRST-MORNING U R I N E OF MALE ADULTS Geometric Mean S. D. 0.49 0.50 1.65 1.57 1.61

n 50-59 60-69 Total a3 81 164

0.50

405

10

20

30 40
Age group

50

ll

60s

F i g u r e 5. N o r m a l r a n g e (Mean + 2S.D. ) o f u r i n a r y f l u o r i d e c o n c e n t r a t i o n s i n d i f f e r e n t age groups. , a i d - m o r n g n g ; 0 ,f i r s t - m o r n i n g u r i n e .

DISCUSSION
F l u o r i d e c o n c e n t r a t l o n s i n u r i n e samples a r e o f t e n d i r e c t l y determined using a fluoride ion-selective i s e a s y t o use, e l e c t r o d e p o t e n t i a l i s long, electrode. The d i r e c t d e t e r n i n a t i o n m e t h o d b u t t h e t i m e r e q u i r e d t o r e a c h an e q u i l i b r i u m o f t h e e s p e c i a l l y a t l o w f l u o r i d e c o n c e n t r a t i o n s (2,

5).

I n t h i s s t u d y . f l u o r i n e was s e p a r a t e d f r o m u r i n e s a m p l e s b y m i c r o and i t s c o n c e n t r a t i o n d e t e r m i n e d as a s a m p l e f i v e t i m e s

d i f f u s i o n method,

i t s o r i g i n a l concentration.

With t h i s method,

t h e e l e c t r o d e p o t e n t i a l can t h e concentWe h a v e t h e geomet-

be s t a b i l i z e d i n a few minutes.

I n s t a t i s t i c a l analysis o f u r i n a r y f l u o r i d e concentrations,
r a t i o n s a r e o f t e n r e g a r d e d a s n o r m a l d i s t r i b u t i o n (2.5-7,9). l o g a r i t h m i c a l l y normal, analysis. as was s u g g e s t e d b y T o y o t a (8). Thus,

confirmed that the distribution o f urinary fluoride concentrations i s r i c mean s h o u l d b e u s e d i n s t e a d o f t h e a r i t h m e t r i c mean f o r t h e s t a t i s t i c a l T h e r e h a v e b e e n many r e p o r t s c o n c e r n i n g t h e n o r m a l f l u o r i d e c o n c e n t r a t i o n s o f s p o t samples o f u r i n e u r i n e , 0.32 m g / l , o t h e r w o r k e r s (5-7).

(2,5-9).

The mean v a l u e o f m i d - m o r n i n g

o b t a i n e d i n t h i s study i s l o w e r than those r e p o r t e d by T h i s d i s c r e p a n c y may b e d u e p a r t l y t o t h e u s e o f

g e o m e t r i c means i n s t a t i s t i c a l a n a l y s i s and t o t h e u n i q u e l i f e - s t y l e o f t h e p a r t i c i p a t i n g GSDF o f f i c i a l s . A k i n i v i a r e p o r t e d t h e mean f l u o r i d e c o n c e n t r a t i o n o f t h e f i r s t - m o r n i n g u r i n e t o be 0.457 m g / l ( 9 ) . O u r d a t a w e r e s i m i l a r t o t h i s value, s t a n d a r d d e v i a t i o n was v e r y l a r g e . h a b i t s and l i f e - s t y l e s may h a v e i n f l u e n c e d f l u o r i d e e x c r e t i o n . . but the A possible explanation i s t h a t eating

406

CONCLUSION S t u d i e s were made on t h e normal f l u o r i d e c o n c e n t r a t i o n s o f s p o t samples o f u r i n e among Japanese male s u b j e c t s . The d i s t r i b u t i o n o f f l u o r i d e concent r a t i o n s i n s p o t samples was n e a r l y l o g a r i t h m i c a l l y normal. 1,040 Ground Self-Defense F o r c e o f f i c i a l s was 0.32 mg/l, The g e o m e t r i c mean f l u o r i d e c o n c e n t r a t i o n o f t h e mid-morning u r i n e samples o b t a i n e d from while the f i r s t m o r n i n g u r i n e samples c o l l e c t e d f r o m 164 f a r m e r s gave a v a l u e o f 0.50 mg/l.

REFERENCES

1.

I s h i k a w a S (1976) J I w a t e Med Ass 28:207-222 Med School 24:56-67

2. Y o s h i d a Y, T o y o t a S , Kono K, Watanabe M, I w a s a k i K ( 1 9 7 8 ) B u l l Osaka

3.
4. 5. 6. Tsunoda H, S a k u r a i S , I t a i K, (1984) F l u o r i d e 17: 159-167 K a m i j o h K, Kondo T (1978) S a t o T, Nakaya S, M i t a M, Tatsumi

Matsumoto Shigaku 4:150-153 Okudera H. M i y a s i t a N, I i z u k a Y (1974) J

Yasaki T, Ohhashi K, Dent H l t h 24:191-206 Seki Y.

Kubota M,

I c h i k a w a Y (1975) Jap J I n d H e a l t h 17:236-237

7.

K o n i s h i M, I w a t a M. Minagawa Y (1975) Jap J I n d H e a l t h 17:238-239 Toyota S (1979) Jap J I n d H e a l t h 21:335-348 A k i n i w a K (1981) F l u o r i d e R 1:75-77

a.
9.

F l u o r i d e Research 1985, Studies in Environmental Science, V o l u m e 27, pp. 407-414 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

H. Tsunoda a n d M.-H. Y u (Editors)

407

FLUORIDE CLEARANCE I N THE AGING KIDNEY

KOI C H I K O N O ~ , Y ASUH ISA Y O S H ID A ~ , HI ROSHI Y O S H I H I R O TAKEDA',

'Department o f Environmental Health,

Y A M A G A T A ~ , YOSHIHISA

TANI M U R A ~ .

AKIRA HARADA3, AND KAZUHIDE DO14 Fukui M e d i c a l School, Japan Takatsuki,

' D e p a r t m e n t o f H y g i e n e and P u b l i c H e a l t h , Osaka M e d i c a l C o l l e g e , Osaka 569, J a p a n ( a d d r e s s c o r r e s p o n d e n c e t o O r . Y. Y o s h i d a ) 3Kansai T e c h n i c a l C e n t r e f o r O c c u p a t i o n a l Health, 4Matsushita E l e c t r o n i c s Corporation. Japan Japan

ABSTRACT Several c h a r a c t e r i s t i c s o f t h e aging process related t o renal function and f l u o r i d e m e t a b o l i s m w e r e i n v e s t i g a t e d . 1088 h e a l t h y s u b j e c t s by 10-year

A cross-sectional

analysis o f There

age g r o u p s showed a p r o g r e s s i v e l i n e a r The a m o u n t o f

d e c l i n e i n c r e a t i n i n e c l e a r a n c e ( C C r ) and f l u o r i d e c l e a r a n c e (CF). w a s a s i g n i f i c a n t r e d u c t i o n i n C C r a n d CF a f t e r a g e 80. f l u o r i d e f i l t e r e d t h r o u g h t h e g l o m e r u l i p e r m i n u t e (FF). a g e 79. The 2 4 - h o u r

t u b u l a r reabsorp-

t i o n o f f l u o r i d e (TRF) a n d i t s r a t e (%TRF), s e e m e d t o r e m a i n s t a b l e u n t i l e x c r e t i o n o f f l u o r i d e a n d CF w e r e w e l l c o r r e l a t e d i n 7 p a t i e n t s w i t h c h r o n i c r e n a l f a i l u r e (CRF) and The r e s u l t s s u g g e s t w i t h e a c h o t h e r (r=0.83)

11 h e a l t h y s u b j e c t s a f t e r f l u o r i d e a d m i n i s t r a t i o n . k i d n e y d i s e a s e b u t a l s o w i t h a d v a n c i n g age.

t h a t r e n a l f l u o r i d e clearance decreased n o t o n l y w i t h the evidence o f For t h e h e a l t h care o f workers e x p o s e d t o f l u o r i d e and o f p o p u l a t i o n s l i v i n g i n f l u o r i d e p o l l u t e d a r e a s , age may b e an i m p o r t a n t f a c t o r t o be c o n s i d e r e d .

INTRODUCTION The m a j o r p a t h w a y o f f l u o r i d e e x c r e t i o n f r o m t h e human b o d y i s v i a t h e k i d n e y (1,2,3). h e a l t h y person, d u r i n g a 24-hour Since f l u o r i d e has a s h o r t b i o l o g i c a l p e r i o d (4.5,6). half l i f e i n a the 40 t o 70% o f t h e i n g e s t e d f l u o r i d e a p p e a r s i n t h e u r i n e When r e n a l f u n c t i o n d e t e r i o r a t e s , possibly resulting i n

a b i l i t y t o e x c r e t e f l u o r i d e markedly decreases, greater retention o f fluoride. u r e m i c p a t i e n t s (7,8.9).

Some i n v e s t i g a t o r s d e m o n s t r a t e d t h a t f l u o -

r i d e c o n c e n t r a t i o n s i n t h e plasma and bone s i g n i f i c a n t l y i n c r e a s e d i n On t h e o t h e r h a n d , i t h a s b e e n s h o w n t h a t m a n y F l u o r i d e metaboa s p e c t s o f k i d n e y f u n c t i o n d e c r e a s e w i t h age ( l D , l l , l Z ) .

408

lism in elderly persons, however, remains incompletely understood. function and fluoride metabolism.
( S F ) and urine ( U F ) ,

This

paper deals with the characteristics of the aging process related to renal Fluoride concentrations in the serum the amount o f tubular reabsorpthe renal clearance of fluoride (CF).

fluoride filtered through the glomeruli per minute (FF). tion of fluoride (TRF) and its rate (%TRF),

and phenolsulfonphthalein

excretion (PSP, 15 min) were investigated i n healthy subjects living in a rural area of Japan. In addition, the relationship between 24-hour urinary excretion and renal clearance of fluoride was studied i n patients with chronic renal failure (CRF) and healthy controls following fluoride administration. MATERIALS AND METHODS In the first part of the study, 2-hour renal clearance determinations were performed on 1088 healthy individuals (aged 35 to 88) living i n a rural area of Japan. Fasting blood samples and urine specimens were taken from the subjects between 6:DO and 8 : O O a.m. Before the sampling, the subjects were given 300 ml water in order to increase the amount of urine. None of the subjects were known to be on drugs and none had been exposed to any unusually high level of fluoride before the investigation. Water fluoridation is not practiced in Japan, and in the community under study the fluoride level in the drinking water was approximately 0 . 1 ppm. In the second part of the study, seven patients with chronic renal failure (aged 40 to 78) and 1 1 healthy controls (aged 20 to 57) were selected. After an overnight fasting, the subjects were given 4 mg of fluoride by a single oral administration with water. Blood samples and 24-hour urine specimens were collected after the fluoride administration. Renal clearance of fluoride (CF) was calculated according to the follow4 ) : ing formula (

CF (ml/min/1.48 m2)

U-F

(ug/ml) x V (ml/min)/S-F (vg/ml) x 1.48 (m2)/A in2

where U-F is the concentration of fluoride in urine at the specific time (ug/ml): S-F is the concentration of fluoride in serum at the same time (vg/ml); V is the urinary flow during 1 minute at the same time (ml/min);

1.48 m2 is the mean body surface area of Japanese: and A m 2 is the body surface area of the subject. The amount of fluoride filtered through the glomeruli per minute (FF) was calculated by the following equation: FF(ug/min/1.48 m2) = S-F (vg/ml) x GFR (ml/min/1.48 m2) where GFR i s the glomerular filtration rate, which is the creatinine clearance (CCr) in this study.

409

The t u b u l a r r e a b s o r p t i o n o f f l u o r i d e (TRF) a n d t h e r a t e o f t u b u l a r r e a b s o r p t i o n o f f l u o r i d e (%TRF) w e r e d e t e r m i n e d f r o m t h e f o l l o w i n g e q u a t i o n : TRF ( u g / m i n / 1 . 4 8

m2)

FF (ug/min/1.48

m2)

[U-F(ug/ml)
m2)]

x V (ml/min)]

%TRF ( % ) = [l-CF ( m l / m i n / 1 . 4 8

m2)/GFR
-

(ml/min/1.48

x 100 period

The e x c r e t i o n r a t i o o f f l u o r i d e i n t h e u r i n e d u r i n g t h e 24-hour was c a l c u l a t e d u s i n g t h e f o r m u l a : ( 2 )

( 1 ) u g / d o s e ( 4 0 0 0 u g ) x 100 w h e r e u r i n e volume, w h i l e (2) i s the

(1) i s t h e base l i n e v a l u e c a l c u l a t e d f r o m t h e u r i n a r y c o n c e n t r a t i o n o f f l u o r i d e b e f o r e t h e e x p e r i m e n t and 24-hour t o t a l e x c r e t i o n v a l u e i n a 24-hour experiment.

F l u o r i d e c o n c e n t r a t i o n s i n t h e serum and u r i n e w e r e measured b y t h e f l u o r i d e e l e c t r o d e m e t h o d u s i n g an O r i o n M o d e l 901 i o n a n a l y z e r (13). C r e a t i n i n e c o n c e n t r a t i o n s i n t h e serum and u r i n e were measured b y t h e F o l i n - W u m e t h o d u s i n g a H i t a c h i M o d e l 706D a u t o a n a l y z e r . D i f f e r e n c e s b e t w e e n means w e r e e v a l u a t e d b y S t u d e n t ' s t - t e s t . RESULTS The r e s u l t s o f t h e c r o s s - s e c t i o n a l and f l u o r i d e c l e a r a n c e ( C C r a n a l y s e s i n 1088 h e a l t h y s u b j e c t s by creatinine t h e amount o f f l u o r i d e f i l t e r e d tubular reabsorption o f fluoride The mean c o n c e n t r a t i o n s o f CF, FF. TRF a n d %TRF w e r e 1.18 u g / m i n / 1 . 4 8
m2,

1 0 - y e a r a g e g r o u p s o n s e r u m a n d u r i n a r y f l u o r i d e ( S F a n d UF). and CF), t h r o u g h t h e g l o m e r u l i p e r m i n u t e (FF),

(TRF) and i t s r a t e (%TRF) a r e shown i n T a b l e I. u g / m l (ppm), 84.57 ug/min/1.48 r e s p e c t i v e l y . The v a l u e s f o r C C r ,

in2,

s e r u m and u r i n a r y f l u o r i d e i n t h e s e s u b j e c t s w e r e 14.42 u g / l ( p p b ) and 0.78 ml/min/1.48

m2,

43.30

ml/min/1.48

m2,

0.71

a n d 38.74%,

respectively.

SF

and UF s l i g h t l y i n c r e a s e d w i t h 1).

a d v a n c i n g age.

A p r o g r e s s i v e l i n e a r d e c l i n e w a s o b s e r v e d i n C C r a n d CF. CF was shown t o

The

When e x p r e s s e d a s m e a n s o f d e c a d e a g e g r o u p s ( F i g .

r e m a i n s t a b l e u n t i l age 50 and t h e r e a f t e r decreased p r o g r e s s i v e l y . T h e r e was a s i g n i f i c a n t r e d u c t i o n i n FF, TRF, and %TRF a f t e r age 80.

l o w e s t v a l u e s i n C C r and CF w e r e o b s e r v e d a t t h e age i n t e r v a l o f 80 t o 89. T a b l e s I 1 and I 1 1 show c r e a t i n i n e and f l u o r i d e c l e a r a n c e s t u d i e s ( C C r , CF, F F . TRF, a n d %TRF) a n d p h e n o l s u l f o n p h t h a l e i n ( P S P ) e x c r e t i o n t e s t i n t h r e e d i f f e r e n t age g r o u p s . y e a r age g r o u p (pi0.01). i n t h e age 60-79 The v a l u e s f o r C C r ,

CF and PSP e x c r e t i o n t e s t

f o r t h e 60-79 y e a r age g r o u p w e r e m a r k e d l y l o w e r t h a n t h o s e f o r t h e 35-59 A b o u t 1 5 % d e c r e a s e i n t h e s e f u n c t i o n s was shown y e a r age group. y e a r age group, c o m p a r e d w i t h t h e 35-39 %TRF i n t h e 6 0 - 7 9

T h e r e w a s a s i g n i f i c a n t r e d u c t i o n a f t e r a g e 80. r e m a i n s t a b l e u n t i l a g e 79. t h a n t h o s e i n o t h e r age g r o u p s .

F F a n d TRF s e e m e d t o

y e a r age g r o u p was h i g h e r

F i g u r e 2 shows t h e r e l a t i o n s h i p b e t w e e n f l u o r i d e c l e a r a n c e (CF) a n d t h e

24-

h o u r u r i n a r y f l u o r i d e e x c r e t i o n (%) f o r 7 p a t i e n t s w i t h c h r o n i c r e n a l

TABLE I CLEARANCE Age

~

STUDIES BY 10-YEAR AGE GROUP I N HEALTHY I N H A B I T A N T S L I V I N G I N

A RURAL AREA OF JAPAN (MEAN k SD) FF ug/mi n / l .48m

~~

S- F ugll

U-F vglml

CCr

m! / m i n / l 48m2

CF

TY

%TRF (%)

35-39 40-49 50-59 60-69 70-79 80-89

78

12.0527.61 13.09k8.18 15.0821 1.92 15.09t9.26 15.1529.63 15.00+7.08 14.42t10.10

0.71k0.48 0.69k0.43 0.84kO. 56 0.78tO. 57 0.79k0.58 0.79t0.28 0.78k0.53

99.54k24.59 96.30k32.27 84.74254.50 81.34t53.07 65.00k31.09 53.68t25.00 84.57k46.74

47.69t38.68 46.63t33.28 39.70k42.56 37.30t31.51 31.71k22.02 30.33k20.78 43.30k35.92

1.01k0.89 1.13k0.90

0.62k0.87 0.66k0.91 0.70t1.19 0.81k1.31 0.65k0.89 0.53t0.64 0.71k1.12

43.02k40.95 41.15k47.98 31.57156.09 45.05t49.61 44.92k40.81 34.97t30.77 38.74k51.33

248
403 249 97 13

1.24t1.22
1.22k1.35 1.04k1.10 0.96k0.87 1.18k1.15

T o t a l 1088 or a v e .

411

TABLE I 1

SERUM AND URINARY FLUORIDE, AND FLUORIDE CLEARANCE STUDIES IN THREE GROUPS

AGE

35-59 60-79 80-89

P<0.01

729 346 13

14.07k10.45 15.10k9.38 15.00k7.08

0.78k0.52 0.78k0.57 0.79k0.28

91.90k45.51]] 77.01k48.76 53.68+25.00

42.45k39.301 35.53k29.41 30.33QO. 78

_L

failure (CRF) and for 1 1 healthy controls following administration o f 4 mg of fluoride. The 24-hour urinary fluoride excretion and fluoride clearance were well correlated with each other (r=0.83) i n these subjects. I n the healthy SubJeCtS, about 50% of the ingested fluoride was excreted into the urine in a 24-hour period. The excretion rate decreased with the diminution of the fluoride clearance and when fluoride clearance decreased 20%. the 24-hour excretion of fluoride also diminished 20%.

Figure I. Cross-sectional differences in fluoride clearance with age. The number o f subjects in each group is indicated above the abscissa. Values plotted are means ?r SEM.

412

TABLE I 1 1 GLOMERULAR FLUORIDE F I L T R A T I O N , TUBULAR FLUORIDE REABSORPTION AND I T S RATE, AND PHENOLSULFONPHTHALEIN I N THREE AGE GROUPS
Age

FF

pg/mi n/l .48m

JRF

%TRF

(%I

% (15 min)

PSP

35-59 60-79 80-89

729 346 13

1.19k1.09 1.17t1.29 0.96k0.87

0.68t1.07 0.77t1.22 0.53k0.64

35.96t52.28 45.02k48.871 34.97k30.77

"

32.04k9.60 29.44t10.6I 21.37t7.51

]I7$

2 X
a ,

50

Y = 0.94X-0.07 r = +0.83

Fluoride clearance ,ml/min/l

.48m2

Figure 2. Correlation between fluoride clearance and rate of urinary fluoride excretion in 24 hours.

DISCUSSION

Once renal function is impaired the excretion of fluoride i n the urine decreases (3). A previous report showed that the 24-hour excretion of fluoride in patients with chronic renal failure ( C R F ) was significantly 4 ) . It is widely recognized lower (p<O.OOI) than that in healthy subjects ( that i n elderly persons there is a gradual failure i n renal functions. including glomerular filtration, renal blood flow, a n d the ability to concentrate urine. The decrease in renal functions is attributed mainly to

413
involution. (10,14,15). Among t h e f a c t o r s c o n t r i b u t i n g t o t h i s d e c r e a s e a r e r e d u c t i o n s Creatinine clearance i s simple. i n e x p e n s i v e and does n o t Thus, the determination

i n t h e r e n a l v a s c u l a r bed w h i c h can be a c c e l e r a t e d b y a r t e r i o s c l e r o s i s r e q u i r e a d m i n i s t r a t i o n o f an exogenous s u b s t a n c e .

o f g l o m e r u l a r f i l t r a t i o n r a t e (GFR) h a s b e e n f o u n d t o b e c l i n i c a l l y u s e f u l . D u r a t i o n o f u r i n e c o l l e c t i o n f o r t h e m e a s u r e m e n t o f C C r v a r i e s f r o m 1 t o 24 hours. A c c o r d i n g t o Wesson ( 1 6 ) a 2 4 - h o u r c o l l e c t i o n seemed m o s t u s e f u l as However, t h e e f f e c t o f d i u r n a l v a r i a t i o n i n GRF c o u l d be a v o i d e d . o r workers i n a plant.

i t may

n o t b e p r a c t i c a b l e i n a s t u d y t h a t i n v o l v e s a l a r g e number o f i n d i v i d u a l s

A two-hour

clearance examination performed i n the

e a r l y morning. as done i n t h i s study, appears t o be s u i t a b l e f o r a r a p i d evaluation o f the renal function. The r e s u l t s o b t a i n e d i n t h i s s t u d y d e m o n s t r a t e a h i g h l y s i g n i f i c a n t ager e l a t e d d e c l i n e i n c r e a t i n i n e c l e a r a n c e and f l u o r i d e c l e a r a n c e (CF) h e a l t h y persons. a b o u t 50% o f C C r , demonstrated T h e f i n d i n g s o n CF, agree w i t h t h e r e p o r t b y Waterhause study. i n which i n these subjects averaged

et

gil.. (6).

A
has

d e c l i n e i n C C r a n d C F among t h e d i f f e r e n t d e c a d e age g r o u p s was w e l l i n this By age 9 0 e a c h o f t h e s e f u n c t i o n s d e c r e a s e d t o a v a l u e a p p r o x i m a t e l y 50 t o 6 0 % o f w h a t i t w a s a t t h e a g e o f

3 5 t o 39.
FF,
TRF and %TRF w e r e a l s o m a r k e d l y d e c r e a s e d a f t e r age 80. The e x p e r i m e n t a l d a t a o f W h i t f o r d (17) s u g g e s t t h a t 35 t o 45% o f t h e f i l t e r e d and t h e r e a b s o r p t i o n was I t seems t h a t

f l u o r i d e was r e a b s o r b e d i n t h e p r o x i m a l t u b u l e ,

i n v e r s e l y r e l a t e d t o t h e pH v a l u e o f t h e t u b u l a r f l u i d . As shown i n t h i s s t u d y , t h e 24-hour 2). fluoride

a b o u t 38% o f t h e f i l t e r e d f l u o r i d e i s r e a b s o r b e d i n h e a l t h y s u b J e c t s . excretion correlated well t h e 24-hour e x c r e t i o n o f

w i t h CF.

When C F f e l l b e l o w 10 m l / m i n / 1 . 4 8

m,

f l u o r i d e markedly decreased (Fig.

T h i s i n d l c a t e s t h a t t h e 24-hour

e x c r e t i o n o f f l u o r i d e i s d e p e n d e n t o n CF. The r e s u l t s o b t a i n e d i n t h i s s t u d y s u g g e s t t h a t r e n a l f l u o r i d e c l e a r a n c e decreases n o t o n l y w i t h t h e evidence o f kidney disease b u t a l s o w i t h a d v a n c i n g age. Therefore, f o r t h e h e a l t h c a r e o f workers exposed t o f l u o r i d e and o f p o p u l a t i o n s l i v i n g i n f l u o r i d e p o l l u t e d a r e a s , age may be an i m p o r t a n t f a c t o r t o be considered. REFERENCES

1.

E k s t r a n d J ( 1 9 7 8 ) C a r i e s Res 1 2 1 2 3 - 1 2 7 S p e n c e r H, L e w i n I , W i a t r o s k i E. S a m o c h s o n J ( 1 9 7 0 ) Am J M e d 4 9 : 8 0 7 813 W h i t f o r d G M , P a s h l e y DH ( 1 9 7 9 ) I n : J o h a n s e n E, T a v e s DR, O l s e n TO ( e d s ) C o n t i n u i n g E v a l u a t i o n o f t h e Use o f F l u o r i d e s . W e s t v i e w P r e s s I n c , B o u l d e r , p p 187-221

2.
3.

414

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Kono K, Yoshida Y, 22: 33-40

Watanabe M, Tanimura Y, H i r o t a T (1984) Ind Health

Macheshwar VS, B r u n e t t i AJ, L e i b i n L, Newbrun E, Hodge HC (1981) A m J C1 i N u t r 34:2679-2684 Waterhause C, Taves D. Munzer A (1980) C l i n S c i 58:145-152 S i d d i q u i JY, S i m p s o n SW, E l l i s H E , K e r r DMS, A p p l e t o n O R , R o b i n s o n BH, Hawkins JB, Robertson PW, Taves OR (1971) Proc E u r D i a l y s i s Transp l a n t Ass 8:149-159 Sreepada Rao JK, Friedman EA (1975) Kidney I n t 7:125-129 T a v e s D R , F r e e m a n RB, Kamm DB, Ramos SP, S c r i b n e r BH ( 1 9 6 8 ) T r a n s A m Soc A r t i f Intern Organs 14:412-414 F r i e d m a n SA, R a i z n e r A E , Rosen H, Solomon N A , Sy W ( 1 9 7 2 ) A n n I n t e r n Med 76:41-45 Lewis WH, Alving AS (1938) A m J P h y s i o l 123:500-515 Rowe JW, A n d r e s R , T o b i n JD. N o r r i s A H , Shock N W ( 1 9 7 6 ) 31 155-163

J Geront

Y o s h i d a Y , T o y o t a S , Kono K, W a t a n a b e M , I w a s a k i K ( 1 9 7 8 ) B u l l Osaka Med School 24:56-67 Sworn MJ, Fox M (1972) Brit 3 Uro 44:377-383 Wardener HE (1985) In: Wardener H E ( e d ) T h e Kidney. C h u r c h i l l L i v i n g s t o n e , Edinburgh, pp 128-131 Wesson CG J r ( 1 9 6 9 ) In: Wesson LG J r ( e d ) Kidney. Grune - S t r a t t o n , New York P h y s i o l o g y o f t h e Human

Whitford GM, P a s h l e y DH, S t r i n g e r GI (1976) Am J P h y s i o l 230:527-532

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 415-422 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

415

KINETICS OF FLUORIDE EXCRETION IN HUMAN SALIVA

YASUHISA Y O S H I D A ~ , K O I C H I K O N O ~ . M I S U Z U W A T A N A B E ~ . S U M I E I N O U E ~ . T O S H I Y U K U
HIROTAl. MICHIZO MURAOl, AND HIDEHIRO NAGAIE3 'Department o f Hygiene and P u b l i c Health, Osaka Medical College, T a k a t s u k i . O s a k a , 569. J a p a n 'Department of Environmental Health, F u k u i Medical School, Japan 3Department of P h y s i c a l Education. Osaka Dental U n i v e r s i t y , Japan

ABSTRACT The f l u o r i d e found i n s a l i v a may depend l a r g e l y on serum f l u o r i d e c o n t e n t w h i c h i s a f f e c t e d m a i n l y by t h e d i e t and by t h e k i d n e y f u n c t i o n o f t h e individual. The p r e s e n t i n v e s t i g a t i o n was concerned w i t h f l u o r i d e c o n t e n t s comof t h e whole and p a r o t i d s a l i v a from r e s i d e n t s of a n o n - f l u o r i d a t e d e x c r e t e f l u o r i d e was markedly decreased. Whole and p a r o t i d s a l i v a and serum s a m p l e s were o b t a i n e d f r o m e i g h t h y d r o f l u o r i c a c i d (HF) p l a n t w o r k e r s , f i v e p a t i e n t s w i t h CRF, and 40 h e a l t h y c o n t r o l s . The s a m p l e s were t a k e n a t 6:OD a.m. and t h i s was f o l l o w e d by t h e r e s p e c t i v e f l u o r i d e dose. in t h e f a s t i n g s t a t e , S a l i v a r y and serum

munity and from p a t i e n t s w i t h c h r o n i c r e n a l f a i l u r e (CRF), whose a b i l i t y t o

s a m p l e s were c o l l e c t e d a t f i x e d i n t e r v a l s o v e r a 48-hour period. I n t h e h e a l t h y s u b j e c t s , a h i g h d e g r e e o f c o r r e l a t i o n ( r = + 0 . 9 9 ) was o b t a i n e d between serum and s a l i v a r y f l u o r i d e l e v e l s . t o t h o s e of t h e s e r a . Changes i n f l u o r i d e c o n t e n t of t h e whole and p a r o t i d s a l i v a a f t e r f l u o r i d e i n t a k e were s i m i l a r The s a l i v a r y f l u o r i d e c o n t e n t of p a t i e n t s w i t h C R F was t h r e e t o s i x t i m e s h i g h e r t h a n t h a t of h e a l t h y s u b j e c t s a t any t i m e of t h e day. In HF p l a n t workers, p o s t - s h i f t s a l i v a r y f l u o r i d e c o n t e n t was f i v e t o 50 t i m e s h i g h e r t h a n t h a t i n s e r a , p o s s i b l y d u e t o o r a l c o n t a m i n a t i o n from t h e work environment. T h e s e r e s u l t s s u g g e s t t h a t f l u o r i d e c o n t e n t s o f t h e w h o l e and p a r o t i d s a l i v a r e f l e c t e d t h e body b u r d e n o f f l u o r i d e subjects.
i n

non-fluoridated healthy

INTRODUCTION S e v e r a l i n v e s t i g a t o r s (1-4) have d e t e r m i n e d human s a l i v a r y f l u o r i d e cont e n t s and observed t h a t t h e v a l u e s w e l l r e f l e c t t h e serum f l u o r i d e concen-

416

trations. On the other hand, a correlation exists between serum fluoride levels and the body burden o f fluoride and its excretion. T h e s e characteristics are utilized in biological monitoring of fluoride among indus5 ) . Determination of fluoride level in the serum makes it trial workers ( 6 ) . possible to diagnose certain diseases, such as chronic renal failure ( In the present investigation, the kinetics o f fluoride excretion in human saliva has been studied.

MATERIALS AND METHODS

Forty healthy male and female subjects, eight workers from a hydrofluoric acid (HF) manufacturing plant, and five patients with chronic renal failure, were employed in this study. The patients with chronic renal failure s h o w e d over 5 mg/dl creatinine and over 50 mg/dl BUN. S o m e o f these patients w e r e using artificial dialysis. T h e H F plant workers' routine work involved washing TV picture tubes at in electronics department. The

HF concentration in the working environment was about 3 ppm.

Salivary specimens were obtained by stimulation of the top of the subject's tongue w i t h l e m o n drops. T h e fluoride concentrations in parotid saliva, obtained by use o f Curby's plastics cap ( 3 ) with negative pressure, and in mixed saliva, naturally obtained in the oral cavity. w e r e determined. O n e ml o f the saliva s a m p l e w a s centrifuged at 1,000 xg f o r 15 min.
A

0.5 ml aliquot of the supernatant was then used for fluoride determination.
The determination was carried out with a microprocessor ion analyzer, Model

901, and total ion strength a d j u s t m e n t buffer T y p e 1 1 1 , o f the U.S. Orion 7 ) . For the determination of fluoride concentrations i n Research Company ( saliva and serum, s p e c i m e n s w e r e taken at 6:OO a.m. f r o m subjects in the
fasting state. After a certain amount of fluoride dosing, specimens were collected at fixed intervals over a period of 48 hours. Portions o f serum and saliva s p e c i m e n s w e r e also used f o r the determination o f calcium, sodium and chloride ions.

RESULTS
The correlation between salivary fluoride and serum fluoride levels of adult subjects is shown in Figure 1. Although the salivary specimens were whole saliva, the correlation coefficient between serum fluoride and salivary fluoride was as high as 0.99. Figure 2 s h o w s a survey o f salivary and s e r u m fluoride levels f o r 24 hours following the intake of 5 mg of fluoride by healthy adult subjects.

417

300
3

c ci

m i

-?-Ze
0
100 200 S a l i v a r y fluoride, n g / m l

300

6 1 0 1 2

Time, hr

Figure 1. Correlation between salivary fluoride and serum fluoride levels in human subjects. Figure 2. 5 mg F. Salivary fluoride and serum fluoride levels following intake of

The serum fluoride level rapidly increased 30 min after fluoride intake and then gradually decreased. It is seen that changes in salivary fluoride level paralleled those of serum fluoride. The correlation between mixed and parotid salivary fluoride levels following the intake of 5 mg fluoride by a healthy subject is shown in Figure 3 . The mixed salivary fluoride level changed i n parallel with the parotid saliva fluoride level. It should be pointed out that the parotid saliva fluoride level was always slightly lower than the mixed salivary fluoride level. Figure 4 shows the response of mixed salivary fluoride levels when the intake of fluoride was increased form 3 to 10 mg. A dose-response is clearly shown. Similar results were obtained when the number of subjects was increased to five. Thus, the salivary fluoride level coincided well with that of the serum. Similar results to those of parotid saliva were obtained with the mixed saliva. Calcium, sodium, and chloride ions i n the serum and saliva showed a nearly constant diurnal variation with time. As shown i n Table I . no correlation was observed between fluoride ions and calcium ions. Table I 1 shows the correlation between the salivary fluoride and serum fluoride levels in two patients with chronic renal failure. Changes in the fluoride levels up to 48 hours after the intake of 5 mg fluoride are shown.

418

Time, h r

Mixed and p a r o t i d s a l i v a r y f l u o r i d e l e v e l s i n a s u b j e c t f o l l o w F i g u r e 3. i n g i n t a k e o f 5 mg F.

T i m e , hr

Response o f mixed s a l i v a r y f l u o r i d e l e v e l s f o l l o w i n g i n t a k e o f F i g u r e 4. v a r i o u s amounts o f f l u o r i d e .

The m i x e d s a l i v a r y f l u o r i d e l e v e l s i n t h e s e p a t i e n t s were t w o t o f o u r t i m e s h i g h e r t h a n t h o s e o f serum f l u o r i d e . T a b l e 111 shows a d i u r n a l v a r i a t i o n o f s a l i v a r y f l u o r i d e l e v e l s i n t h e patients w i t h chronic renal failure. h i g h e r t h a n t h o s e o f t h e c o n t r o l group. T h e i r f l u o r i d e l e v e l s w e r e much Therefore,

i t can be s a i d t h a t t h e

mixed s a l i v a r y f l u o r i d e l e v e l r e f l e c t s t h e s t a t u s o f t h e k i d n e y f u n c t i o n and t h a t i t i s p o s s i b l e t h a t t h e v a l u e s may be u t i l i z e d f o r t h e d i a g n o s i s o f chronic renal failure.

419
TABLE I
CHANGES I N SERUM AND SALIVARY F LEVELS I N NORMAL SUBJECTS (N=5) FOLLOWING INTAKE OF 5 'MG F

Time a f t e r F intake (hr) 0 0.5 1

2

Serum
F ng/ml

Mixed s a l i v a T-Ca m E q / l 4.8420.22 4.78t0.05 4.68tO. 15 4.27tO. 37 3.87k0.66 4.77t0.27

F ng/ml
17.7k2. 52 223.3k4.77 171.7k18.9 106.0k16.4 66.3k7.51 40.723.06

T-Ca mEq/l 2.3821.07 2.85t0.58 2.64t1.20 2.3321.17 2.31k0.82 2.19k1.57

16.0t3.6 236.625.8 184.3k4.0 123.3k10 72.7k11 41.6k5.6

4 12

TABLE I 1 CHANGES I N SERUM AND SALIVARY F LEVELS I N PATIENTS WITH CHRONIC RENAL FAILURE (CRF) FOLLOWING INTAKE OF 5 MG F Time a f t e r F intake (hr) Fluoride level (vg/l) Patient A Serum Saliva 54 210 120 70 244 180 110 80 60 Patient B Serum Saliva 40 230 120 70 44 50 120 440 260 145 160 160

0 0.5 4 12 24 48

90
60 50

TABLE I 1 1 DIURNAL VARIATION O F SALIVARY F LEVELS I N PATIENTS WITH C H R O N I C RENAL FAILURE ( C R F ) F l u o r i d e l e v e l , ng/ml
SubJ e c t
a.m.

p.m.

6 925 86
-

8 17k10 150 180

-

10 16k9

12 18k10 105 82

2 17k9 250

4 13k6 86 120 38

6 15k10 80 132
-

8 2Ot9 50 153 42

10 1326 70

12 12+6
-

C o n t r o l (n=9) Patient

A (dialysis) B (dialysis)
C

82

72 -

420 Among w o r k e r s o f t h e HF m a n u f a c t u r i n g p l a n t , p o s t - s h i f t However, salivary fluoride

c o n c e n t r a t i o n s w e r e a l w a y s 5 t o 50 t i m e s h i g h e r t h a n t h e v a l u e s i n serum.

i t w o u l d b e r e a s o n a b l e t o assume t h a t s u c h h i g h v a l u e s w e r e due t o

o r a l contamination o f f l u o r i d e f r o m t h e i r working environment.

DISCUSSION
A p r o p e r e v a l u a t i o n o f t h e body burden o f f l u o r i d e , s o u r c e o f burden, r e c o g n i z e d t o be i n d i s p e n s a b l e . hours. regardless o f the as a means o f p r e v e n t i n g f l u o r i d e p o i s o n i n g has been The m a j o r i t y o f t h e f l u o r i d e t a k e n i n t o

t h e body o r a l l y o r t h r o u g h t h e a i r passage i s e x c r e t e d i n t h e u r i n e w i t h i n The l e v e l o f u r i n a r y f l u o r i d e has, t h e r e f o r e , been c o n s i d e r e d a m e a s u r e m e n t o f f l u o r i d e c o n c e n t r a t i o n i n t h e b l o o d has as c o m p a r e d t o t h a t i n u r i n e . First, low fluogood i n d i c a t o r o f t h e b o d y b u r d e n o f f l u o r i d e . As a s p e c i a l case, r i d e clearance excretion, been c o n s i d e r e d i m p o r t a n t ,

i s o f t e n o b s e r v e d b e c a u s e o f r e n a l h y p o f u n c t i o n among t h e Due t o d i s t u r b a n c e o f f l u o r i d e Accumulai n t h e b l o o d a r e r e l a t i v e l y h i g h when

o l d o r patients w i t h chronic renal failure. fluoride concentrations

compared w i t h t h e amount o f f l u o r i d e d i s c h a r g e d i n t o t h e u r i n e . t i o n o f f l u o r i d e i n t h e b l o o d can cause f l u o r i d e poisoning.

Second, s t u d i e s c o n d u c t e d i n t h i s l a b o r a t o r y (8) s h o w e d t h a t a l a r g e amount o f f l u o r i d e moved i n t o t h e b l o o d f r o m t h e i n j u r e d s k i n o f victims. death.

I t was p o i n t e d o u t t h a t ,

HF

burn

i n t h i s case,

through reaction with

f l u o r i d e , b l o o d c a l c i u m l e v e l was m a r k e d l y l o w e r e d , p o s s i b l y l e a d i n g t o Clinically.
i t i s n e c e s s a r y t o m o n i t o r t h e b l o o d f l u o r i d e and

c a l c i u m c o n c e n t r a t i o n s and t o a d m i n i s t e r c a l c i u m b y i n t r a v e n o u s i n j e c t i o n i m m e d i a t e l y and r e p e a t e d l y , save t h e l i f e o f t h e p a t i e n t . I n r o u t i n e m e d i c a l e x a m i n a t i o n o f i n d u s t r i a l HF w o r k e r s and i n t h e t r e a t m e n t o f HF b u r n s , a n a l y s i s o f b l o o d f l u o r i d e i s n e c e s s a r y a n d e f f e c t i v e . However, r e p e a t e d s a m p l i n g o f t h e b l o o d i s o f t e n p a i n f u l t o t h e s u b j e c t . t o m a i n t a i n t h e necessary l e v e l o f calcium t o

I f s a l i v a does r e f l e c t t h e f l u o r i d e c o n c e n t r a t i o n i n t h e blood,
S a l i v a i s n o t rnerejy f i l t e r e d blood, s a l i v a r y gland. sodium,
al.

i t may be

possible t o s u b s t i t u t e t h i s f o r blood sampling i n b i o l o g i c a l monitoring. b u t i s an e x t e r n a l s e c r e t i o n b y t h e Consequently, J e n k i n s (3) n o t e d t h a t g e n e r a l l y t h e r e was However, E r i c s s o n ( 1 ) and H e n s c h l e r t h i s i s due t o t h e f a c t

no c o r r e l a t i o n b e t w e e n s e r u m and s a l i v a i n t h e c o n c e n t r a t i o n s o f c a l c i u m , chloride, and o t h e r i o n s .

(2) r e p o r t e d t h a t f l u o r i d e c o n c e n t r a t i o n i n t h e s a l i v a was e x c e p t i o n Presumably, u n l i k e o t h e r ions, f l u o r i d e i o n s a r e n o t re-absorbed b y t h e e x c r e t o r y

a l l y s i m i l a r t o t h a t i n t h e serum. that, d u c t o f t h e s a l i v a r y gland.

421
At first, i n healthy a d u l t subjects, t h e c o r r e l a t i o n between mixed s a l i and r = t0.99.
= +0.98)(10).

v a r y a n d s e r u m f l u o r i d e w a s c a l c u l a t e d a s y = 1 . 0 4 ~+ 1.06, w i t h p a r o t i d s a l i v a (r =

T h i s c o e f f i c i e n t i s a p p r o x i m a t e l y t h e same as t h a t r e p o r t e d by E k s t r a n d ( 9 )

+0.99) a n d t h a t o f P a t z e t a l . ( r

The t e s t was f o l l o w e d b y a 5 mg f l u o r i d e b u r d e n t e s t . l o w e d t h a t i n t h e serum w i t h o u t a t i m e lag.

I n t h i s t e s t i t was

observed t h a t t h e f l u o r i d e c o n c e n t r a t i o n i n t h e mixed s a l i v a c l o s e l y f o l I n t h e i r f l u o r i d e b u r d e n t e s t s , E k s t r a n d g J g l . ( l l ) a n d H e n s c h l e r gJ g l . (2) h a v e shown a d o s e - r e s p o n s e relationship i n the fluoride concentration i n t h e serum. S i m i l a r r e s u l t s were obtained i n t h i s study o f f l u o r i d e

c o n c e n t r a t i o n s i n t h e m i x e d s a l i v a i n r e s p o n s e t o t h e a d m i n i s t r a t i o n o f 3,

5. a n d 1 0 mg o f f l u o r i d e ( F i g . 4).
E k s t r a n d ( 9 ) and P a t z ng/ml

aJ.(lO)

r e p o r t e d f l u o r i d e c o n c e n t r a t i o n s o f 9-15 ng/ml i n t h e m i x e d s a l i v a . They a l s o

i n t h e p a r o t i d s a l i v a and 20-30

s t a t e d t h a t t h e p a r o t i d s a l i v a s e r v e d a s an i n d i c a t o r o f t h e f l u o r i d e c o n c e n t r a t i o n i n t h e serum. Yao and G r d h ( 1 2 ) o b s e r v e d t h a t t h e f l u o r i d e m i x e d s a l i v a was s i m i l a r t o concentration i n the centrifugally-separated

t h a t i n t h e p a r o t i d s a l i v a . The c o r r e l a t i o n b e t w e e n f l u o r i d e c o n c e n t r a t i o n s
i n t h e m i x e d s a l i v a and t h e p a r o t i d s a l i v a shown i n t h i s s t u d y s u p p o r t t h e

above-mentioned observations. The r e s u l t s o f t h i s s t u d y show t h a t t h e m i x e d s a l i v a , obtainable, i n t h e s e r u m w i t h o u t a t i m e lag. which i s e a s i l y s e r v e s a s a good d i r e c t i n d i c a t o r o f t h e f l u o r i d e c o n c e n t r a t i o n

On t h e o t h e r hand,

with patients suffering

Fluoride The

c h r o n i c r e n a l f a i l u r e a n d w o r k e r s i n HF m a n u f a c t u r i n g p l a n t s , d i f f e r e n t r e s u l t s were o b t a i n e d compared w i t h t h o s e f r o m h e a l t h y s u b j e c t s . four concentrations i n t h e mixed s a l i v a o f these p a t i e n t s were always two t o t i m e s h i g h e r t h a n t h e c o r r e s p o n d i n g v a l u e s i n t h e serum. i n c r e a s e d f l u o r i d e c o n c e n t r a t i o n i n t h e m i x e d s a l i v a may b e d u e t o an enhanced s e l e c t i v e s e c r e t i o n o f t h e s a l i v a r y g l a n d i n r e s p o n s e t o t h e h i g h serum f l u o r i d e l e v e l , o r due t o an e x t r e m e l y l o w w a t e r i n t a k e because o f r e n a l insufficiency. The d i u r n a l v a r i a t i o n o f t h e f l u o r i d e c o n c e n t r a t i o n s i n t h e s a l i v a o f t h e h e a l t h y s u b j e c t s was 9 t o 20 n g / m l on t h e average. p a t i e n t s s u f f e r i n g c h r o n i c r e n a l fa1 lure, diets, ten times that o f the controls. W i t h i n d u s t r i a l HF w o r k e r s , T h i s was i n f l u e n c e d t o In some e x t e n t b y t h e i n t a k e o f f o o d s a n d t e a c o n t a i n i n g f l u o r i d e .

i n spite o f s t r i c t l y controlled

f l u o r i d e c o n c e n t r a t i o n i n t h e s a l i v a o c c a s i o n a l l y reached as h i g h as This suggests a high concentration o f

f l u o r i d e i n t h e blood o f these patients. because o f d i r e c t c o n t a m i n a t i o n i n t h e o r a l e s t i m a t i o n o f serum f l u o r i d e However, c a v i t y by f l u o r i d e i n t h e working environment. c o n c e n t r a t i o n u s i n g s a l i v a may be e r r o n e o u s .

w i t h acute f l u o r i d e

422
p o i s o n i n g s u c h a s i n HF b u r n s , s a l i v a r y f l u o r i d e l e v e l s e r v e s a s a good

i n d i c a t o r o f serum f l u o r i d e c o n c e n t r a t i o n . CONCLUSION S a l i v a r y f l u o r i d e c o n c e n t r a t i o n s w e r e c o m p a r e d w i t h s e r u m f l u o r i d e conc e n t r a t i o n s among h e a l t h y s u b j e c t s , w i t h chronic renal failure. i n d u s t r i a l HF w o r k e r s , a n d p a t i e n t s F o r t h e h e a l t h y s u b j e c t s ( N = 40). t h e r e l a and r +0.99, as t h e

t i o n s h i p between t h e f l u o r i d e c o n c e n t r a t i o n i n t h e mixed s a l i v a and t h a t i n t h e s e r u m c a n b e e x p r e s s e d w i t h y = 1 . 0 4 ~ + 1.06 r e g r e s s i o n e q u a t i o n and c o r r e l a t i o n c o e f f i c i e n t , A l s o i n t h e f l u o r i d e burden t e s t , v e r y s i m i l a r t o t h o s e i n t h e serum. e a s i l y sampled, Therefore,

=

respectively. t h e mixed saliva, which i s

s a l i v a r y f l u o r i d e c o n c e n t r a t i o n s were

c a n be u s e d t o e s t i m a t e t h e f l u o r i d e c o n c e n t r a t i o n i n t h e

s e r u m and t o e v a l u a t e t h e f l u o r i d e b u r d e n o f an i n d i v i d u a l . S p e c i a l a t t e n t i o n s h o u l d be p a i d t o t h e f a c t t h a t w i t h p a t i e n t s s u f f e r i n g c h r o n i c r e n a l f a i l u r e , due t o t h e i r c o n c e n t r a t e d s a l i v a , and w i t h indust r i a l HF w o r k e r s i m m e d i a t e l y a f t e r t h e s h i f t , tamination i n the oral cavity. t h a n t h a t i n t h e serum i s o f t e n obtained. d u e t o d i r e c t f l u o r i d e cona higher salivary fluoride concentration

REFERENCES
1. E r i c s s o n Y (1969) C a r i e s Res 3:159-166

2. H e n s c h l e r

D. B U t t n e r W, P a t z J ( 1 9 7 5 ) I n : and M e t a b o l i c Bone D i s e a s e . S p r i n g e r - V e r l a g ,
WM

C a l c i u m M e t a b o l i s m , Bone B e r l i n , pp 112-121 4 t h Ed.

3. J e n k i n s JN (1978) The P h y s i o l o g y and B i o c h e m i s t r y o f t h e Mouth. B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n , London

4. J e n k i n s JN, E d g e r

(1977) C a r i e s Res 1:226-242 K o n o K, Harada A (1980) T a n i m u r a Y. A r c h I n d Hyg T o x i c o l Hirota T (1984) I n d u s t

5. T o y o t a S, Y o s h i d a Y, 30: 957-966

6. K o n o K,

Y o s h i d a Y, H e a l t h 22: 33-40

W a t a n a b e M,

T o y o t a S, Kono K, 7. Y o s h i d a Y, Osaka Med S c h o o l 24:56-67

Watanabe M,

I w a s a k i K, H i r o t a T.

Kato I (1978) B u l l T a n i m u r a Y, Shibuya

8. Kono K,
9.

Y o s h i d a Y, H a r a d a A, Watanabe M, Y (1982) B u l l Osaka Med S c h o o l 28:124-133 H e n s c h l e r D,

E k s t r a n d J ( 1 9 7 7 ) C a l c i f T i s s Res 23:225-228 W u r z b u r g HF (1977) D t s c h Z a h n a r z t l Z 32:482-486 B o r b s LO, N o r l i n A (1977) Europ J C l i n Pharmacol

10. P a t z VS.

11. E k s t r a n d J. A l v s l G, 12: 31 1-317 12. Yao K,

Grdh P (1970) C a r i e s Res 4:321-331

H. Tsunoda and M.-H. Yu (Editors) Fluoride Research 1985, Studies in Environmental Science, Volume 27, pp. 423-429 0 1986 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

423

AN INTERNATIONAL COMPARISON OF TRACE AMOUNTS OF FLUORIDE CONTAINED I N HUMAN

HAIR
YASUHIRO TAKAGI, YOSHITERU OHKUBO, TSUNEO ASADA, AND SUSUMU MATSUDA

Fukui P r e f e c t u r a l I n s t i t u t e o f P u b l i c Health,

39-4,

Harame, F u k u i . J a p a n

ABSTRACT Fluoride concentrations countries, method. Samples f r o m I n d i a n r e s i d e n t s gave h i g h e r f l u o r i d e l e v e l s t h a n t h o s e f r o m other residents. O n l y s a m p l e s f r o m I n d i a showed d i f f e r e n c e s w i t h sex, and m a l e h a i r samples gave h i g h e r v a l u e s t h a n f e m a l e samples. age group. S m o k i n g and f i s h e a t i n g h a b i t s w e r e shown t o h a v e an e f f e c t on f l u o r i d e a c c u m u l a t i o n i n t h e h a i r , b u t t h e h a i r f l u o r i d e l e v e l was u n a f f e c t e d b y t h e p r a c t i c e o f h a i r d y i n g a n d p e r m a n e n t wave. Some c o r r e l a t i o n b e t w e e n f l u o r i d e and o t h e r m i n e r a l e l e m e n t s i n t h e h a i r samples were a l s o observed. The I n d i a n m a l e i n t h e h a i r s a m p l e s o f 306 r e s i d e n t s f r o m f i v e India, i n c l u d i n g Japan,

U.S.A.,

Canada,

and P o l a n d w e r e

d e t e r m i n e d b y means o f t h e o x y g e n b o m b - f l u o r l d e i o n s e l e c t i v e e l e c t r o d e

s a m p l e s a l s o showed an i n c r e a s i n g l e v e l o f f l u o r i d e w i t h i n c r e a s e i n each

INTRODUCTION T r a c e e l e m e n t s i n t h e human b o d y h a v e b e e n r e c o g n i z e d as an i m p o r t a n t i n d e x o f e n v i r o n m e n t a l p o l l u t i o n i n r e c e n t y e a r s (1). Use o f human h a i r has a d v a n t a g e s i n t h a t t h e c o l l e c t i o n and t r a n s p o r t o f s a m p l e s a r e easy, and t h e c o n c e n t r a t i o n s o f v a r i o u s e l e m e n t s a r e h i g h compared t o t h o s e i n b l o o d and u r i n e . However, hair, For t h i s reason, h a i r s a m p l e s have been used t o s t u d y t h e i n f l u e n c e o f l o n g t e r m e x p o s u r e t o l o w c o n c e n t r a t i o n s o f p o l l u t a n t s (6.8). l i t t l e i n f o r m a t i o n i s a v a i l a b l e c o n c e r n i n g f l u o r i d e i n human h a i r . I n v i e w o f t h e i n t e r e s t i n t h e d i s t r i b u t i o n o f t r a c e e l e m e n t s i n human an i n t e r n a t i o n a l s u r v e y was c o n d u c t e d t o s t u d y t h e i r d i s t r i b u t i o n i n H a i r s a m p l e s t a k e n f r o m U.S.A.. Canada, populations o f different origin.

P o l a n d , Japan, and I n d i a w e r e a n a l y z e d f o r f l u o r i d e c o n t e n t b y means o f t h e oxygen b o m b - f l u o r i d e i o n e l e c t r o d e method. study the correlation, An a t t e m p t w a s a l s o m a d e t o age, sex

i f any,

between e l e m e n t a l c o n c e n t r a t i o n ,

424

and l i v i n g s t y l e o f t h e i n d i v i d u a l s i n a p a r t i c u l a r c o u n t r y . f i n d i n g s a r e r e p o r t e d here. MATERIALS AND METHODS Sampling procedure

Some o f t h e

The n u m b e r o f d o n o r s f r o m s e l e c t e d l o c a t i o n s i n N o r t h A m e r i c a , a g e s o f t h e J a p a n e s e p a r t i c i p a n t s r a n g e d f r o m 20 the participants from India. years. Poland, Canada,

Europe, The

a n d A s i a a n d t h e d i s t r i b u t i o n o f t h e s a m p l e s a r e s h o w n i n T a b l e I.
-

70 y e a r s , w h i l e t h o s e o f
f r o m 1 9 t o 80 and i m m e d i general

a n d U.S.A..

The h a i r s a m p l e f r o m e a c h i n d i v i d u a l was p r o c u r e d b y n o r m a l c u t t i n g Each d o n o r was a s k e d t o c o m p l e t e a sex, domicile.

and c o l l e c t e d w i t h c a r e t o a v o i d a n y e x t e r n a l c o n t a m i n a t i o n . a t e l y s e a l e d i n a c l e a n p l a s t i c bag. health, o c c u p a t i o n and l i v i n g s t y l e . p e r s o n a l q u e s t i o n n a i r e d e s c r i b i n g h , s / h e r age,

TABLE

DISTRIBUTION

OF

HAIR SAMPLES TAKEN FROM DIFFERENT POPULATIONS Location Fukui New D e l h i Bombay Mon c t on Scranton Poznan Male 29 40 Fema 1e 44 62 Total

Country Japan India Canada

73
102 53 43 35
~

33
21

20
22 13 161

U. S. A.
P o l and Total

22
145

306

A n a l y t i c a l method H a i r samples f r o m each i n d i v i d u a l were c u t i n t h e l a b o r a t o r y t o about 3

mm l e n g t h s .

They

were washed w i t h acetone and w a t e r , and t h e n acetone A p p r o x i m a t e l y 1.0 g o f t h e d r i e d

again,

and d r i e d a t room t e m p e r a t u r e .

h a i r s a m p l e was p l a c e d i n t h e p l a t i n u m b o a t o f an o x y g e n bomb c o n t a i n i n g 10
m l 0.5N KOH a s a n a b s o r b e r .

T h e s a m p l e w a s b u r n e d i n t h e b o m b b y t h e m e t h o d o f T a k a g i gJ F o l l o w i n g t h e combustion, water. atmosphere,

a1.(7).

t h e bomb was s h a k e n v i g o r o u s l y and c o o l e d i n i c e

When t h e i n t e r n a l p r e s s u r e o f t h e bomb was r e s t o r e d t o t h a t o f t h e t h e c o n t e n t s o f t h e bomb w e r e washed t w i c e w i t h t h e a b s o r b e r

425 and t h e w a s h i n g s w e r e combined. a t 135-145C t h e d i s t i l l a t e w a s 100 m l . The c o m b i n e d w a s h i n g s w e r e s t e a m d i s t i l l e d The i o n i c s t r e n g t h o f t h e d i s t i l l a t e was solution.

i n t h e presence o f p e r c h l o r i c a c i d - s i l v e r p e r c h l o r a t e u n t i l

a d j u s t e d w i t h t h e same v o l u m e o f TISAB ( s o d i u m c i t r a t e b u f f e r ) s e l e c t i v e e l e c t r o d e method.

The f l u o r i d e c o n t e n t o f t h e m i x t u r e was t h e n d e t e r m i n e d b y t h e f l u o r i d e i o n The c o m p o s i t i o n o f d i f f e r e n t m i n e r a l e l e m e n t s o t h e r t h a n f l u o r i d e was determined by emission spectrum a n a l y s i s and a t o m i c a b s o r p t i o n s p e c t r o metry. RESULTS AND DISCUSSION R e s u l t s o f t h e a n a l y s i s showed t h a t t h e h i s t o g r a m o f f l u o r i d e c o n c e n t r a t i o n s i n t h e h a i r was c l o s e t o a n o r m a l d i s t r i b u t i o n .

c o m p a r i s o n o f t h e mean f l u o r i d e c o n c e n t r a t i o n s i n t h e h a i r s a m p l e s f r o m

e a c h o f t h e f i v e c o u n t r i e s a r e s h o w n i n F i g u r e 1. T h e h a i r s a m p l e s f r o m I n d i a n m a l e s w e r e f o u n d t o c o n t a i n a much h i g h e r f l u o r i d e l e v e l t h a n t h a t i n I n d i a n f e m a l e samples. I n addition, t h e f l u o r i d e c o n t e n t was s i g n i f i c a n t l y h i g h e r t h a n t h o s e o f samples f r o m o t h e r n a t i o n a l s r e g a r d l e s s o f sex ( ~ ~ 0 . 0 1 ) On . t h e o t h e r hand, t h e r e w e r e no d i f f e r e n c e s i n f l u o r i d e l e v e l s b e t w e e n m a l e and f e m a l e s a m p l e s f r o m o t h e r f o u r t o d i f f e r e n t age g r o u p s . countries. The f l u o r i d e c o n c e n t r a t i o n s i n t h e h a i r s a m p l e s w e r e c o m p a r e d a c c o r d i n g The r e s u l t s showed t h a t I n d i a n m a l e s a m p l e s f o r On a l l age g r o u p s h a d h i g h e r f l u o r i d e l e v e l s t h a n t h o s e f r o m o t h e r c o u n t r i e s . a n d t h a t t h e d i f f e r e n c e s b e c a m e h i g h e r w i t h i n c r e a s e i n a g e ( F i g . 2). t h e o t h e r hand, f e m a l e h a i r samples f r o m a l l f i v e c o u n t r i e s a n y age e f f e c t o n f l u o r i d e l e v e l s ( F i g . d i d n o t show

LL

15!
C

3).

I male
female

. ,

426

o , \
0

o INDIA rn JAPAN A U.S.A..CANADA POLAND

F i g u r e 2. C o m p a r i s o n o f F c o n c e n t r a t i o n i n human ( m a l e ) h a i r a c c o r d i n g t o age groups.

Cn \
07

female
0

rn J A P A N

INDIA

C 0

U.S.A.,CANADA POLAND

''

21-30

31;LO

L1-SO Age

51-60

F i g u r e 3. C o m p a r i s o n o f F c o n c e n t r a t i o n i n human ( f e m a l e ) h a i r a c c o r d i n g t o age groups.

The e f f e c t o f s m o k i n g on f l u o r i d e c o n c e n t r a t i o n s i n t h e h a i r was s t u d i e d based on t h e r e s u l t s o f t h e q u e s t i o n n a i r e . a n d U.S.A. countries. females), smoking. P 9 I 9 (5). The d a t a o n s a m p l e s f r o m Canada were pooled as no d i f f e r e n c e s were found between these two

A s s h o w n i n F i g u r e 4,

i n Canada,

U.S.A.,

and I n d i a (except

samples f r o m smokers had h i g h e r f l u o r i d e l e v e l s t h a n t h o s e o f But no d i f f e r e n c e s were observed w i t h t h e e x t e n t o f

n o n - s m o k e r s (pi0.01).

I t h a s b e e n r e p o r t e d t h a t t h e f l u o r i d e c o n t e n t i n t o b a c c o i s 100

0 ._ c
c
L

\,in.n
1
C

01

Smoking Hobit L_I1 YES

13

8
0

50

I*
1

427

NO

T
I

n L
INDIA
F i g u r e 4.

CANADA

E f f e c t o f smoking on F c o n c e n t r a t i o n s i n t h e h a i r .

E x a m i n a t i o n o f t h e e f f e c t o f f i s h e a t i n g on f l u o r i d e c o n t e n t s o f h a i r samples r e v e a l e d t h a t t h o s e who e a t l i t t l e f i s h had a h i g h e r f l u o r i d e l e v e l t h a n f i s h e a t e r s ( ~ ~ 0 . 0 5 ) . B u t among t h e f i s h e a t e r s , the frequency o f f i s h e a t i n g was n o t f o u n d t o a f f e c t t h e f l u o r l d e l e v e l i n t h e h a i r ( F i g .

5).

I t i s c o n s i d e r e d t h a t t h e c o n t r i b u t i o n o f f i s h e a t i n g t o f l u o r i d e was The p r a c t i c e o f h a i r d y i n g and permanent wave was n o t found t o i n f l u e n c e

insignificant. f l u o r i d e l e v e l s i n t h e hair.

I t i s c o n s i d e r e d t h a t f l u o r i d e i n human h a i r i s r e l a t e d t o i n g e s t i o n o f
f o o d and w a t e r c o n t a i n i n g i t . zone, I n areas where l a r g e q u a n t i t i e s o f highf l u o r i d e c o n t a i n i n g t e a (2) and g r a i n s (3) a r e consumed, t h e d a i l y f l u o r i d e i n t a k e may b e h i g h , and i n t h e t o r r i d

leading t o accumulation o f

F i g u r e 5.

Ib
NO
YES

1-2

L -4

5-6

E f f e c t o f f i s h e a t i n g on F c o n c e n t r a t i o n s i n t h e h a i r .

428

TABLE I 1 CORRELATION BETWEEN FLUORIDE AND OTHER MINERAL ELEMENTS I N HUMAN H A I R (MALE) Country Japan India Element A1 Cr A1 Contents (vglg) 16.8 0.13
r

0.448 0.277 0.557 0.451 0.408 0.324 0.418 0.629 0.422 0.474 0.393 0.499 0.383 0.379 0.479

Cr Ca

64.7 0.14 0.67 863.5 0.048 1.25 10.01 0.294 0.494 8.34 0.041 15.9 0.45

U.S.A.

As Cd Pb Sn Cd

Canada

co Na P o l and
-:+:
> ,

Pb

Cr

, ,

. p<o. 0 5

p<o.o1

TABLE I 1 1 CORRELATION BETWEEN FLUORIDE AND OTHER MINERAL ELEMENTS I N HUMAN HAIR (FEMALE) Country Japan Element Contents
(11919)
r

P
c

Sb K Na
Mg

0.976 10.8 425.3 318.6 32.4 0.228 12.1 36.0 0.184 0.318 2.38 0.333 176.9

0.519 0.320 0.353 0.308 0.497 0.372 0.338 0.415 0.41 6 0.647 0.580 0.548 0.489

c ;${c

India

A1 Cd cu Fe

U. S.A.
P o l and

v
Cd Pb Ni Zn

-:c

429 fluoride i n the hair. h a i r i s very limited, t i o n s i n human h a i r . T a b l e s I 1 a n d 111 show t h e c o r r e l a t i o n b e t w e e n f l u o r i d e c o n t e n t s and t h o s e o f o t h e r e l e m e n t s i n t h e h a i r samples. common c o r r e l a t i o n was o b s e r v e d ( T a b l e 111), c o r r e l a t i o n s w e r e shown ( T a b l e 11). s a m p l e s f r o m U.S.A. o b s e r v e d ( p < 0.01 ). This p r e l i m i n a r y survey, though n o t conclusive, does s u g g e s t t h a i f l u o r i d e c o n t e n t s i n h a i r c a n be u s e f u l i n s t u d y i n g t h e i m p a c t o f e n v i r o n m e n t a l a n d d i e t a r y f a c t o r s o n humans i n g e n e r a l . ACKNOWLEDGEMENT We d e e p l y t h a n k Prof. P r o f . J. A. Vinson ( U n i v e r s i t y o f Scranton, Canada), Dr. A. Poland) and Dr. W h i l e i n f e m a l e s a m p l e s no i n m a l e s a m p l e s a f e w common I n contrast, i n The l i t e r a t u r e o n t h e e f f e c t o f t o b a c c o o n human b u t t o b a c c o c o n t a i n s a h i g h l e v e l o f f l u o r i d e (5.6).

So i t i s s u g g e s t e d t h a t s m o k i n g h a b i t s may i n f l u e n c e f l u o r i d e c o n c e n t r a -

E x a m p l e s a r e s a m p l e s f r o m I n d i a and

J a p a n w h e r e b o t h A1 a n d C r w e r e f o u n d t o b e c o r r e l a t e d .

a n d C a n a d a , c o r r e l a t i o n b e t w e e n F a n d Cd a n d Pb w a s

U.S.A.),

M. C. M e h r a ( M o n c t o n U n i v e r s i t y ,

Kaniewski ( I n s t i t u t e B. K. P u r i ( I n d i a n

o f S o c i a l M e d i c i n e M e d i c a l Academy, I n s t i t u t e o f Technology, mented on t h e manuscript.

I n d i a ) who h e l p e d c o l l e c t h a i r s a m p l e s and comT h i s e x p e r i m e n t was, i n part, supported by a

g r a n t f r o m Nissan Science Foundation.

REFERENCES
1. F l y n n A ( 1 9 7 7 ) J A p p l N u t r 29:51-57 H a r r i s o n M F (1949) B r i t J N u t r 3:162-167 M c C l u r e F J (1949) Pub H e a l t h Rep 64:1061 Okamoto T, Okamoto T, R e a v e s RD, 14: 579-584 M a t s u h i s a T (1965) J Food Hyg SOC J a p a n 6(4):382-387 M a t s u h i s a T (1967) J p n J Pub H e a l t h 14(3):123-126 J o l l y KW, B u c k l e y PD ( 1 9 7 5 ) B u l l E n v i r o n C o n t a r n T o x i c o l

2.
3. 4.

5.

6.
7.

T a k a g i Y. T a n a k a H, M a s u n a g a S ( 1 9 7 5 ) J Food H y g S O C J a p a n 1 6 ( 2 ) : 1 1 9 123 T a k a g i Y ( 1 9 8 1 ) J p n J Hyg 36:824-829

8.

431

AUTHOR

INDEX Hitomi, G . , 325 Honma, Y . , 1 6 5 H o r i u c h i , T., 395 Hwang, H . L . S . , 203 113 I i d a , M., 2 6 7 , 3 1 3 I i j i m a , Y., 3 6 9 Imai, T., 263 Inoue, Inoue, Ishii, Itai, 43 415 T., 379 K . , 25, 99
A.,

Abe, T . , 299 H s a d a , PI., 3 0 7 A s a d a , T., 4 2 3

Bagley, C . V . , B a n g , S.,

187, 341

Baud, C . A . , 187, 3 4 1 B e s s h o , Y., 73 B h a v s a r , B.S., 1 9 3 B i e r s t e k e r , K . , 135 B o l e i j , J.S.M., 135 Butcher, J.E., 113 C a l l , J.W., 1 1 3 C h a n d r a , S . , 181 C h i k u m a , M., 4 3 Cordonn i e r , J. A . , Uate, C., 307

S.,

J a i n , S.K.,

231

155

Kanamoto, Y., 211 Kaneko, Y., 73 K a n t h a r i a , S.L., 193 Kasahara, K., Katayama, T., 401 369 313

Demeurisse, C . , 187 Desai, V.K., 193 Uoi, K . , 407

K a w a s a k i , T., 2 6 7 , Kohyama, Y . , 3 0 7 Kondo, T . , 4 0 1 Kono, K . , 4 0 7 , 4 1 5 Krishna, N., 357 K u r i h a r a , H., 307

F r a n k e , J . , 335 Fujiwara, K., 59 F u r u t a n i , IY., 2 6 7 Fuwa, K . , 3, 1 5 , 59

L e i , W., G i a n n i n i , J . , 241 Gojo, K , , 263 Goto, S., 47 H a r a d a , A., Hattori, T., Hayashi, M., H i r o t a , T., 407
15, 31
Li,
Y.,

59 253

Ma, H . , 2 5 3 M a e h a s h i , H., Masuoka, M . , M a t s u d a , S.,

277 299 423

H a r a g u c h i , H.,

277 307

Heyndrickx, A.M., 415

155

Mehta, M.R., 193 MGndez, M . C . , 127 M i l l e r , G.W., 113, 241 Miyajima, H., 299

432

Morimoto, Mui,

M., 307 415 91

395

Singh, Suga,

D.P.,
S.,

347
Y.,

K.,
M.,

Songpaisan, Suketa, Susheela, Y.,

173

Murao, Murray, Nagaie,

285 211
A.K.,

F., H.,

231

415

Nakagaki, Nakagawa, Nakane, Nakao, Nasu, T.,

M.,

H.,
T., 143 395 Y.,

379 43 401

Takagi, Takeda, Taki,

Y.,

Y.,
Y.,

423 407 285

J.,

Takimoto, Tanaka,
F.,

225

I.,

143 307 43 Y., 407 347 347 155 J.M.,

Nishida, Niwa, M.,

257

Tanaka, ranaka, 263 Teotia, Teotia,

H.,
H.,

Nishikawa, Nomura, Oh,

K.,

K.,
225, 299

263 257,

Tanimura,

M., 43 Y.,
S.,

M.,

S.P.S.,

Timperman, 423
Y.,

Ohkubo, Okada,

Tochon-Oanguy, 43 Tomita, Tsuji, 127

!I., 73 M., H., K., N.,

H.J.,
307 257, 99, 389

187

Okabayashi, Okayasu, Oliveira, Olson,

211 I., 307 J.A., 113 P., 241 173

Tsuchida, T., Tsunoda, Tsunoda, Tsunoda,

225, 25, 15

263 107, 1 6 5 , 359

A.E.,

107,

Phantumvanit, Pushnik,
J.C.,

Van d e n H e e d e , Very,
J.M.,

M.A.,

155

187 285
M., H.,

Rajyalakshmi, Ramamohan Rao, Riet-Correa. Sakurai, Sato, Sato, Satoh, Saxena, Schild, Sharma, Shupe, T., T.,
Y.,

K.,
F.,

357 357 Wada, K., 127 389 325 Watanabe, Yamagata, Yamauchi, Yanaka,
T., Y.,

N.V.,

415 407 165 F., 81 407, 415 325 307

S.,

99,

267, 257, 211

D.K.,

313, 263 193

T.,

Yanagisawa, Yoshida, 173 Yu,

M.H.,

Schamschula, A.L., R.P.,

R.G.,

Yoshitake, Zaima, Y.,

O.,

K.,
203 47

313,

127 113 113

J.L.,

Zenebon,

127

433

KEYWORD I N D E X (Page number r e f e r s t o t h e f i r s t A b s o r p t i o n mechanism, Accumulation, 277 Active transport, A d r e n a l ec tomy, Aging, 407 products, 91, 99, 107 15, 25 Echinocyte, Ecosystem, 43 277 299 231 91 325 341 25 107 155 Agricultural 211 325 241 325 page o f t h e a r t i c l e concerned.) 369 127 165 81 253 165 intake,

Deciduous enamel, Defluoridation, Dental Dental DNA/RNA attrition, fluorosis, synthesis,

357

Acetylcholine sensitivity,

0 i f f us i o n a1 t r a n s p o r t ,
Drinking water, 379 59,

Duration o f fluoride

Air pollution,

Airborne fluoride, Aluminum p l a n t , Analysis, 73, 165

Aluminum m o n o f l u o r i d e , 155

Effect o f chloride, Electron-probe, Electrothermal, nace, 15 143 99

Anion-exchange resin, Anti-cholinesterase, Anti-neoplastic Apatite, Ascorbic Bacterial Blood, Bone, CaF2, CAMP, 415 73 187, 389 341 187 acid, 203 267

agents,

Electrothermal graphite furEmission factor, Emission sources, Enamel, 47, 379 285 127 3 231

growth,

Biogeochemistry, B l o o d serum,

Enameloid,

Environmental pollution, Environmental problem, E r y t h r o c y t e membrane, Evolution, 285 395 Excretory rhythm,

C a l c iu m - f 1 u x 211, 113 Cattle,

211 F-electrode, F i e l d work, 127 Fish teeth, Fluoride,

257

401
379 285 313

C a t t l e and sheep, 113 Chronic Coal

Chronic f l u o r id e toxicosis, renal failure, 127 81 59 415

Fluoridation,

91,

99,

155, 263, 415 401

187, 285, 341 389

225,

241,

253, 47,

combustion, 3, 225

F1 u o r i d e a b s o r p t i o n ,

325,

Coated d i f f u s i o n tube, Colorimetry, Cord serum,

-, -,

analysis,

-, c l e a r a n c e , 4 0 7
concentration,

434

-, c o n t e n t , 4 2 3

Human b o n e s , 59 Human h a i r ,

155 335 107, 423

-, d e t e r m i n a t i o n , 1 5 , -, e x c r e t i o n , 1 0 7 -, i o n , 4 3 , 3 0 7 -, m e t a b o l i s m , 3 1 3 -, m o u t h r i n s e , 3 6 9 -, p o i s o n i n g , 1 2 7 -, t o x i c i t y , 2 3 1 -, u p t a k e , 3 6 9 -, w o r k e r , 4 0 7 , 4 1 5
F1 u o r i d e s , Fluorine, Fluorosis, Functional 267 73

Human f l u o r o s i s , Human s u b j e c t s ,

389

Hydrogen f l u o r i d e sampl i n g ,

81
H y p e r p a r a t h y r o i d ism, Hyperplasia, 307 99 113 423 347

Indicator plant, Industry, 193

Influencing factors,

Fluorine determination, 181, 347, 43 resin,

31
379

I n t e r n a t i o n a l comparison,

357,

L a m i n a r and t u r b u l e n t f l o w s , 81 Lanthanum complex o f s u l f o n a t e d a1 i z a r i n - c o i n p l e x o n e , 47 43 Litterfall, Logarithmic 91 341 normal d is t r i b u 59 73 Localization, tion, 401 203

Gas c h r o m a t o g r a p h i c d e t e c tion, 31 143 absorption, 135 313 267 257 Gas c h r o m a t o g r a p h y , Gaseous f l u o r i d e , Gastrointestinal 107 Glass f i b r e plant,
G 1 o m e r u 1 o n e p h r it is ,

Long c a p i l l a r y c e l l , 47, Low-protein diet, Lung f u n c t i o n , 135 Low t e m p e r a t u r e a s h i n g , Lymphocyte, 257 313 253

Glucosyl transferase, G l y c o l y t i c enzyme, Hard tissue, Health care, plasma, HF, 135, 31 263 241 313 407, 415

H e 1 i um m i c r o w a v e - i n d u c e d HeLa c e l l s ,

Masugi's nephritis, M a t e r n a l serum, MIC, 267 73, 225

Mesophyll protoplast, 193 Microdiffusion, Mice, 347 203

High r i s k department,

401

H i stodynamic,
Histological,

347 335 and rnicro-

Molecular absorption spectrometry, NaF, 389 fluoride, 369

H i stomorphometry,

15,

25

Histopathologic radiographic Hokutolite, 3

, 299

Natural

435

kutritional

supplements,

357

Smoking a n d f i s h e a t i n g , Sodium c a r b o n a t e , 8 1 S o d i u m f l u o r i d e , 277 Sources, 113 Space food,

395 113 193

423

Oral

administration, 389
267

Oral microorganisms,
Osteomalacia, 347

Species tolerance, Parafoll icular c e l l s , 307 P a r t i c u l a t e f l u o r i d e , 143 Pathologic-anatomical, Phosphatase, 257

335

S p e c i f i c symptoms, Spectroscopy, 3

Spot samples of u r i n e , 401 Subcellular distribution, 241 Symptoms,

THC-278, 193
299

P l a n t , 193 P 1 asnia e m i s s i o n s p e c t r o m e t r y ,
31

Power s p e c t r u m , 3 9 5 P r o s p e c t i v e s u r v e y , 99
77KD P r o t e i n , 2 6 3

Tegafur,

29Y

Tnroughfal 1 , 9 1 T h y r o i d-par at h y r o i d e c t o m y ,
211

PTH,

211

Pyrohydrolysis f l u o r i n e det e r m i n a t i o n , 25 Rat, 47

Thyroid g l a n d , 307 Tibia, 203 T i l e works, Total Tunnel

143

reflection, 59
kiln,
143

Rat incisor, 299

Rats,
307

Transmitter release, 277

Recycling, 187 Heflux method, 3 2 5 Removal,

187

Unpolished r i c e , 9 9 Urinary f l u o r i d e excretion,

181

Renal f u n c t i o n , 4 0 7 Salivary fluoride,

173, 415

Urinary f l u o r i d e , Urine, 135 Vegetables,

135

173,

395

School c h i l d r e n , 3 7 9 S e l e c t i v e c o l l e c t i o n , 43
SEM-f indings,

335 211

Serum g l u c o s e ,
203

Water f l u o r i d e , 1 7 3 Water-Dorne f l u o r i d e , 1 6 5 Water s a m p l e s , 3 1

Serum a1 k a l i n e p h o s p h a t a s e , S k e l e t a l muscle, 277