and Arsenic

Chronic Kidney Disease of unknown etiology-

CKDu
Confined to certain geographical area in the
country
Irreversible damage to kidneys.
No specific signs and symptoms
Exact cause is not known.
No cure in western system or any other system
of medicine.
Number of patients increase rapidly.
Becoming the biggest health issue in certain
parts of the country.
Chronic Kidney Disease of unknown etiology-
CKDu
CKDu is not associated with known risk factors
for CKD..
eg. diabetes,hypertention,glomrulonephritis
Histopathological appearance of the disease is
tubulointerstitial that can commonly be
observed in toxic nephropathies.
Clinically disease is characterized by beta 2
microglobulinuria.
Prevalence of chronic kidney
disease of unknown etiology
(CKDu)in Sri Lanka
Girandurukotte 2500 (Badulla District)
Medawachchiya 2000 (Anuradhapura Dist.)
Padaviya 1400 (Anuradhapura Dist.)
Medirigiriya 600 (Polonnaruwa Dist.)
Hingurakgoda 500 (Polonnaruwa Dist.)
Nickawewa 500 (Kurunegala District)
Any hypothesis should explain
Why?
1) Unique geographical distribution of
the CKDu
2) There was no CKDu 20-25 years
back
Current hypotheses on etiology of CKDu
Hypothesis Scientist Comments
High fluoride and
possible exposure
to Aluminum
fluoride
Prof.O.A.Illeperuma-
University of Peradeniya-
2005
This hypothesis cannot explain
the geographical distribution of
CKDu
Entry of excessive
Cadmium via food
chain
Prof.S.Bandara-University
of Peradeniya -2007
New study (Prof.R Chandrajith et
al 2010) revealed Cd is not the
cause
Cyanobacterial
toxins
Dr.Dhammika Menike
Dissanayke-University of
Peradeniya-2010
87% of the population in CKDu
affected area use either dug well
or tube well water for drinking.
Presence of cyanobacteria in
drinking water wells of patients
was marginal-
Prof.M.Amarasinghe 2011
Risk factor
identification
Dr.K.Wanigasuriya et al-
University of Sri
Jayawardenapura 2007
Being a pesticide using-farmer
who drinks well water has been
identified to be of highest risk
Fluoride Hypothesis?
Fluoride poisoning
Skeletal fluorosis and hypothyroidism are main
problems associated with chronic fluoride
poisoning.
Usually liver is affected before kidneys.
Tubulointerstitial nephritis and beta 2
microglobulinuria cannot explain by using
fluoride hypothesis.
Fluoride poisoning
Chronic Cadmium poisoning
Causes,
weak and brittle bones due to osteomalacia
waddling gait due to bone deformities
Chronic kidney disease - Proximal renal
tubulopathy
Itai-itai disease was caused by cadmium
poisoning due to mining in Toyama ,Japan
marked prevalence in older, postmenopausal
women has been observed.
Members of the investigation team
Prof.Nalin de Silva- Dean, Science-University of Kelaniya
Prof.Priyani Paranagama-Head-Chemistry-Uni. of Kelaniya
Prof.Mala Amarasinghe- Head-Botany-Univ. of Kelaniya
Dr.Kithsiri Senanayake- Senior Lecturer-Univ.of Kelaniya
Dr.Kumudu Dahanayake-Acting consultant JMO-Monaragala DGH
Dr.channa Jayasumana-Faculty of Medicine,Rajarata University
Dr.Chinthaka Wijewardhane-MOIC-Padavi Sripura Hospital
Dr.P.Mahamithawa-Faculty of Medicine,Rajarata University
Dr.L.Rajakaruna-Faculty of Medicine,Rajarata University
Dr.D.Samarasinghe-Karawanella Base Hospital
Mr.Saranga Fonseka-University of Kelaniya
Clinical assessments
At Padaviya, Sripura, Wahalkada, Mahawilachchiya, Tantirimale
Number of patients observed-125
Feature No of
patients
%
Hyper pigmentation of
palms
94 75.2%
Hyper pigmentation of
soles
75 60.0%
Keratosis of palms 69 55.2%
Keratosis of soles 57 45.6%
Hyper-pigmentation and keratosis
observed in palms and soles
Other symptoms signs of chronic arsenic toxicity
observed during clinical assessments
Sign/symptom
%
Generalized body weakness 95
Headache 91
Burning of eyes 84
Anaemia 74
Nausia 76
Mild to moderate hepatomegaly and
spleenomegaly
55
Epigastric pain 63
Paresthesia 51
Presence of As was determined in
Urine and blood of patients
Hair samples of CKDu patients
Body parts of deceased patients
Water from paddy fields
Soil
Plants Samples
Arsenic fatal dose-0.2g(200mg)-Jayawardena 2004
Atomic Absorption
Spectrometry at
Analytical chemistry laboratory
Faculty of Science
University of Kelaniya.
Arsenic in Hair
Chronic As poisoning was confirmed by high As levels in
hair samples of CKDu patients.
Sample No Arsenic -mg/kg
Mean-Normal person
(Muzumdar 2000)
0.46
Control 0.220.01
H1 4.561.01
H2 2.700.32
H3 1.270.22
H4 3.970.24
H5 1.940.40
H6 1.870.52
H7 4.780.83
H8 7.031.60
1 Kidney of a person
died from CKDu
1 Kidney of a person
died from another kidney
problem
Presence of As in samples collected from CKDu
postmortem study
Body part As (ug/kg)
P1
As(ug/kg)
P2
Normal values(ug/kg)
Muzumdar 2000
Large Intestine 292.350.2 189.525.6 20
Rectum 301.560.3 283.434.3 20
Liver 395.840.1 264.945.8 30
Thyroid 234.135.3 187.244.6 40
Spleen 255.665.5 273.633.9 20
Kidney 213.537.1 275.345.8 30
Gall stone - 325.129.7 -
Previous studies have shown comparatively high
concentration of As has been accumulated in the
kidney tissues.(Kalia et al-2007)
Kidney is the major route for the excretion of As and
its metabolites from the body and a major site for the
biotransformation of arsenic that ultimately makes
more sensitive organ to arsenic exposure.(Tchounwou-
2003)
Cadmium potentiate As nephrotoxicity during the
long-term and co-exposure.(Hong et al-2004)
Alcohol consumption elevates As absorption and have
synergistic effect on DNA damage by arsenic.(Lingzhi -
2010)
CKDu and (beta) 2 microglobulinurea
High levels of beta 2 microglobulin has been
detected in urine samples of CKDu patients.
Beta 2 microglobulinurea is a feature of As
induced nephrotoxicity.(Hong 2003)
Chronic arsenate poisoning leads to proximal
tubular damage.
Beta 2 microglobulinurea is characteristic for
proximal tubular dysfunction as 99.9%of the
filtered beta2 microglobulin is reabsorbed and
degraded in the Proximal Tubule.(Karisson 1980)
Important references
Prasad GVR, Rossi NF (1995) Arsenic intoxication associated
with tubulointerstitial nephritis. Am J Kidney Diseases
26(2):373-376.
Martha M. Brown; Bonnie C. Rhyne; Robert A. Goyer; Bruce A.
Fowler (1976) Intracellular effects of chronic arsenic
administration on renal proximal tubule cells .Journal of
Toxicology and Environmental Health, Part A: Current Issues
.volume 1,Issue 3: 505 514
Dalal PK,Gangopadhyay AK,Roychowdhury
A.(2008)Identification of indicators of arsenic induced
nephrotoxicity in humans.The internet Journal of
Toxicology;5(2)
Hong F,Jin T,Zhang A. (2004) Risk assessment on renal
dysfunction caused by co-exposure to arsenic and cadmium
using benchmark dose calculation in a Chinese population.
Biometals.;17(5):573-80.
Li Z, Piao F, Liu S, Wang Y, Qu .Sub chronic exposure to arsenic
trioxide-induced oxidative DNA damage in kidney tissue of
mice.Exp Toxicol Pathol. 2010 Sep;62(5):543-7.
Jaime B. Vigo and Joanne T. Ellzey.Effects of arsenic toxicity at the
cellular level: A review. 2006 Tex. J. Micros. 37:2,45-49
Important references
Arsenic in plants
Hard water
and pesticides
As detection in body parts, plants samples and rice -
Methodology-(Lin et al,2004,Yamily et al.,2008)
To determine As content , 0.5 g of each sample was refluxed in 10 mL
of conc. nitric-sulfuric-perchloric acid mixture (4/1/1, v/v/v) for 1 hr.
Formic acid (90%) was then added drop by drop until the red-brown
gas disappeared.
Afterwards, deionized water was added to bring the digest to 50 mL.
The analysis of As was carried out in atomic absorption spectrometry
equipped with a hydride generator.
The analytical methodologies were confirmed by using standard
arsenic solutions.
Each sample was triplicated and analyzed.
Same methodology was repeated without samples and it has been
used as controls.
Site of plant collection
Nelumbo (Flower) 1101 10.2
ug/kg
Calotropis gigantean (Wara) 80.5 2.1
ug/kg
Basawakkulama Tank
Asteracantha
longifolia
(Ikiriya)
5.55 0.1
Calotropis
gigantean
(Wara)
157.6 1.4
Eichhornia
crassipes
553.5 2.4
Marsilea
hirsuta
801.0
Ocimum
sanctum
(Maduruthala)
ND
Bulankulama Tank
Syzygium cumini (Dan) Root 17.850.6
Cynodon dectylon (A grass) 84.051.1
Terminalia arjuna Bark 1152.4
Terminalia arjuna Root high
Tamarindus indica (Siyambala) bark 45.051.0
Azadirachta indica (Kohomba) Bark high
Dichrostachys cinerea (Andara) root ND
Mahawilachchiya Tank
Gliricidiasepium (Gliricidia)
Root
38.450.7
Schleichera oleosa(Kon)
Root
160.12.8
Fruits of shrub
(unidentified)
25.150.8
Diospyros ebenum
(Kaluwara) leaves
53.21.0
Bauhinia racemosa(Maila)
leaves
87.01.3
Agada
225.53.1
Memecylon sp. (Kuratiya)
40.51.1
Pephrosia purpurea(Pila)
root
ND
Schleichera oleosa(Kon)
Bark
ND
Bauhinia racemosa(Maila)
bark
ND
Talawa- ela
As in rice
Rice is particularly susceptible to As accumulation
compared to other cereals.(Heikens,2006)
Baseline levels of As are 10 folds higher than other
cereal grains.(Williams et al.,2007)
50%-90% of total As in grain is inorganic
As.remainder is dimethylarsononic acid(DMA)
.(Heikens,2006)
There are no WHO,EU or US limits for either total As
or inorganic As in food(Francesconi,2007)
Arsenic intake mechanisms of rice plant
Arsenic in Sri Lankan rice-previous studies
2004-34-92
-80-160ug/kg
Chandrajith et al 2010 rice collected from CKDu endemic
regions (Giradurukotte and Nickawewa).(100-260ug/kg)
Year Investigator Institute Machine Arsenic
ug/kg
2004 Jayasekara R
Freitas M.C.
University of Kelaniya
ITN-Portugal
Instrumental neutron
activation followed by
high resolution gama ray
spectrometry at ITN-
Portugal
34-92
2008 Yamily J
Duxbury JM
Cornell university USA
,, ,,
ICP-AES.
Inductively coupled
plasma atomic emission
spectroscopy at cornell
University
30-150
2010 Chandrajith R
Diassanayake C.B.
Thilak Abeysekara
et al
Universiry of Peradeniya
,, ,,
General Hospital Kandy
ICP-MS
Inductively coupled
plasma
mass spectrometry at
the Iwate Medical
University, Japan.
90-260
Region No of samples Arsenic lowest
value(ug/kg)
Arsenic highest
value(ug/kg)
Padaviya 16 225 5.5 1097 21.3
Sripura 12 258 4.9 1495 14.5
Mahawilachchiya 14 314 9.9 1074 12.8
Mihinthale 10 241 2.5 1108 27.3
Kurunegala 12 225 12.8 956 5.4
Monaragala 10 195 7.5 435 4.6
Gampaha 10 266 6.8 585 5.5
Arsenic in Sri Lankan rice (ug/kg)
Soil profile for arsenic-Padaviya
Depth from
surface(feet)
Arsenic concentration
(mg/kg)
Surface 22.1 1.6
01 18.3 1.7
02 24.6 0.9
03 17.0 1.1
04 12.5 0.5
05 07.9 0.8
06 02.4 .08
Drinking water analysis for As
High As concentration was detected in almost all the
drinking water samples.
WHO standard for drinking water- Arsenic
10ug/L
USA is currently contemplating a further
decrease to 5ug/L.
In most of the water samples 20-100 ug/L
Arsenic was detected.
No toxin producing cynobacteria were observed
in our water samples.
Difficulty in elicit As in Hard water
As form strong bonds with Calcium.
As is hidden in hard water. Not sensitive
to conventional analytical methods.
There was no standard method to
analyze As in hard water
We have developed a new method at
Kelaniya.
Arsenic and Hard water
Arsenic forms several calcium arsenate compounds at
room temperature.(Bothe and Brown, 1999, Swash and
Monhemius, 1995)
Ca4(OH)2(AsO4)2.4H2O,
Ca5(AsO4)3OH
Ca3(AsO4)2.3/3H2O
CaHAsO4.xH2O
Ca5H2(AsO4)4
Calcium arsenate-chemistry
Calcium arsenate-chemistry
-Density- 3.620 g/cm
3
, solid
-Melting point- 1045C (decomposes)
-Solubility in water: 130mg/L (25C)
-Solubility in Organic solvents:-insoluble
-Solubility in acids- soluble
Calcium arsenate-chemistry
Calcium arsenate-chemistry
Structure dependence on the pH of the solution
pH 3.06
pH 8.5
pH 10.8
Arsenic detection in Hard water-methodology
Determination of arsenic by direct coprecipitation filtration
technique.
Add few drops of con.HNO3 at the time of sampling.
Add appropriate reagent to the water samples.(Matijevic et
al,1958,Pomerantz et al 1936,Vogels et al, 1998,. Zhaohui Li et al, proceedings
of the 3
rd
international conference in Arsenic in Environment 2010, Tainan,
Thaiwan) temp pH
Filter the precipitate and dissolve.
Add KI before introduce to AAS-HG.
Each sample was triplicated and analyzed.
Same methodology was repeated with deionized water and it has
been used as controls.
References
James V. Bothe, Jr., Paul W. Brown, Arsenic Immobilization by
Calcium Arsenate Formation,Environ. Sci. Technol., 1999, 33 (21),
pp 38063811
Y.N.Zhu,X.H.Zhang,Q.L.Xie,D.Q.Wang,G.W.Cheng,Solubility and
stability of calcium arsenates at 25 C Water, Air, and Soil Pollution
(2006) 169: 221238 volume 169,
James V. Bothe Jr. and Paul W. Brown, The stabilities of calcium
arsenates at 231C,Journal of Hazardous Materials,vol 69,Issue
2,1999,197-207
Potential Source of As Contamination
Arsenic detection in Pesticides-Methodology
1) Weigh 1g of pesticide into a 100ml conical flask
2) Add 10ml of C. HNO
3
(analar grade).
3) Heat to 120
o
C on a hot plate ( temperature should be controlled
as arsenic can react with C. nitric acid and form arsenic acid, its
boiling point is 130
o
C, Hence it will evaporate, if temperature is
above 120
o
C). carry out this under a fume hood.
4) Once the nitric acid is evaporated, add 10 ml of C. HNO
3
again
5) Repeat step 4 for two more times by adding 10 ml x 2 C. HNO
3
6) During the step 2 5 brown fumes will evaporate and a pale
yellow color solution will be formed. It would take about 3 hours
for these steps.
7) Then add 10 ml of 1:4 C H
2
SO
4
: C. HNO
3
to the reaction mixture
and heat at 120
o
C until the mixture is concentrated to about 5 ml.
Arsenic detection in Pesticides-Methodology-cont..
8) If not and still the reaction mixture is pale yellow, add 5 ml of
perchloric acid and continue heating.
9) As the reaction mixture evaporates to about to 5 ml, remove the
flask from the heat and allow to cool to room temperature
10) Pour the reaction mixture to a 100ml volumetric flask and
diluted to 100 ml.
11) Use this solution to measure arsenic and mercury content in ppb
levels and multiply the given figure in 100 to get the amount in
1kg of pesticide.
12) Each pesticide should be done in triplicates
13) Repeat steps 1 11 for in a another conical flask without adding
pesticide and use as the control and this should be done in
triplicates too.
14) The analysis of As was carried out in atomic absorption
spectrometry equipped with a hydride generator.
Trade name Active
ingredient
distributo
r
Type No of
sampl
es
Arsenic(
ug/L)
01 Round Up Glyphosate Lankem weedicide 08 24058
02 Bassa Fenobucarb Lankem Insecticide 05 13810
03 Basudine 50
EC
Diazinon Lankem Insecticide 04 1829
04 Evisect Thiocyclam Lankem Insecticide 04 119514
05 Lannate Methomyl Lankem Insecticide 05 16676
06 Nominee Ispyribac Na Lankem Weedicide 08 7208
07 Pyrinex chloropyrifos Baurs Insecticide 05 22017
08 Ekalux Quinalphos Baurs Insecticide 04 25212
09 Lebaxid Fenthion Bayer Insecticide 04 22525
10 Provado Imidacloprid Heylese Insecticide 04 55215
11 Ricestar Fenoxaprp Heylese Weedicide 05 49910
12
Mimiczo Tebufenozide Heylese Insecticide 04 2568
13
Selecron 50
EC
Profenophos Heylese Insecticide 05 28014
14
Folicur EW Tebuconazole Heylese Fungicide 04 45212
15
Mancozeb Mancozeb Heylese Fungicide 04 72045
16
Agromat EC
40
Diamthooate Heylese Insecticide 04 58515
17
Haydol M60 MCPA Heylese weedicide 06 188825
18
Accurator Carbofuran Heylese Insecticide 10 42516
19
Captaf Captan Harrisons Fungicide 04 198135
20
Calcron Profenophos Harrisons Insecticide 04 48214
21
Trebon Etofennoprox Hacros Insecticide 08 74014
22
Powermate Glyphosate Agrochem weedicide 06 35512
23
Solito Pretilachlor+
Pyribenzoxim
CIC weedicide 04 80535
24
Chikara Chlorpyrifos opexagro Insecticide 04 2484
25
Kemsan 50 Phenthoate Sunagro Insecticide 04 135918
26
Marshal 20 SC Carbosulfan Innovative
pesticides
Insecticide 04 129735
27
Tiller Gold Fenoxaprop+
ethoxysulfan
Harrisons Weedicide 06 245029
28
Quick Quinalphos Harrisons Insecticide 06 285433
29
Diaran Divron CIC Insecticide 04 105018
30
Super Dash Glyphosate CIC Weedicide 04 125036
31
Actara Thiamethoxam CIC Insecticide 04 45017
Trade Name Distributor No of
Samples
Mercury- ug/L
Agromat EC40 % -Insecticide Hayleys 04 235085
Haydol M 60%-Weedicide Hayleys 06 84831
Accurator-Insecticide Hayleys 10 4552750
Nominee-weedicide
a) Solution
b) Surfactant
Lankem 08
1028200
2665144
Tiller Gold-Weedicide Harrisons 06 132556
Quick-Insecticide Harrisons 06 90385
Pattaas-Insecticide Harrisons 06 6002542
Marshal 20 sc-Insecticide Innovative
pesticides
04 91269
Arsenic is it an active ingredient?
"active ingredient" means any substance
which gives a formulated product its
pesticide properties-Control of Pesticides Act-
33 of 1980 Sri Lanka
Arsenic compounds are Highly hazardous
(Class Ib) technical grade active ingredient-
The WHO Recommended Classification of
Pesticides by Hazard and Guidelines to
Classification,2009
As in Pesticides
Arsenic have commonly been used in 1900-1930
in the production of insecticides and herbicides.
Due to the adverse health effect to humans and
the introduction of organic pesticides As
containing pesticides have been banned in
many countries.
It has been estimated that more than 500 000
metric tons of As containing pesticides were at
the stocks at the time of banned.
Source-Pesticide Registrar office-Gannaoruwa, Sri Lanka
Heavy metals in fertilizers
Investigations are being carried out
to detect Arsenic and Mercury in
fertilizers.
Comparatively high level of arsenic
has been detected specially in TSP .
Unique geographical distribution of CKDu in Sri Lanka
a) As retention and soil type
Certain type of soils have more retention ability of
Arsenic.
Adsorption ,fixation of As mainly depend on clay
content and organic matter of the soil.(Smith et al
1998,Bansal , 2010)
CKDu distribution map overlaps with reddish brown
earths and low humic gley soils in the dry zone of Sri
Lanka.
Chandrajith et al (2009) show that dry zone rice field
soils of Sri Lanka are characterized by a high content
of calciumcompared to that of the wet zone.
b)Ground water hardness and CKDu
Total hardness of drinking water of CKDu
patients are very high.
Geographical distribution of patients overlaps
with distribution pattern of high ground water
hardness.
Technical support was given by Water
Resources Board,Sri Lanka
CKDu
1. Arsenic
2. Hard water
3. Reddish brown earths and
low humic gley soil
Possible mechanism to explain the CKDu
Na,Pb,Cu, arsenate
Comparatively high retain in reddish brown earth
Formation of calcium arsenate compounds with
the reaction of calcium in hard water
Entry of calcium arsenate to body mainly via
consuming hard water
Pathophysiology
Calcium arsenate crystals are bound to arsenic
transporters at the liver and transport to kidneys
Deposition of the Calcium arsenate crystals in the
kidney tissues.
Release of the arsenate groups
Replace phosphate by arsenate in the nuclear and
mitochondrial DNAs in the proximal tubular
cells,endothelial cells and interstitial cells.
Formation of abnormal proteins
Activation of immune system and
lymphocyte infiltration
disturbing antioxidant
defense system in
renal tissues
Oxidative injury to
proteins
Accumulation of oxidized
proteins in kidney tissues
Arsenate
Formation of abnormal proteins
Loss of proteins
Endothelial damage
and loss of glomeruli
Tubulointerstitial
nephritis
Cortical
thinning
Medical conditions associated with
chronic Arsenic poisoning
Cancers-Skin,Lung,Kidney
Ischemic Heart Diseases
Cerebro Vascular Diseases
Diabetes Mellitus
Chronic kidney disese
Gastritis
Low resistant to viral fever