AN INVESTIGATION OF THE SAFETY OF

COTTON REENTRY AFTER

ORGANOPHOSPHATE APPLICATION
JAMES E. BURNS, M.D.1
Institute o f Environmental Medicine,
New York University Medical Center,
550 First Avenue, New York, N.Y. 10016

ROY D. PARKER, M.S.

Texas Agricultural Extension Service,
Texas A &M University Research and Extension
Center, Wesiaco, Texas 78596

Twelve cotton insect scouts were monitored during an entire pesticide application
season by weekly determinations of para-nitrophenol excretion and biweekly cholin-
esterase measurements. These data were compared to weekly reentry intervals and
pesticide application rates to study the validity of a 48-hr minimum reentry interval
for methyl parathion-treated cotton. Methyl parathion represented 54.2% of the
total amount of pesticide used with toxaphene 35.3%, ethyl parathion 4.1%, and
other 6.4%. A significant decrease in erythrocyte and plasma cholinesterase was
observed during initial exposure but during peak exposure the chohnesterase levels
had returned to normal. The maximum mean amount of methyl and ethyl para-
thion absorbed by calculation from PNP excretion was 0.95 mg/24hr (n = 12).
Twelve percent of all reentries were within two days and during one week 48%
occurred during the first two days after pesticide application.

The increased use of organophosphate pesticides in agriculture has created a problem

of farm worker safety. One method of controlling the dermal absorption and potential
toxicity of these compounds is to prevent entry into treated areas until the amount of
pesticide available for absorption is insufficient to cause significant effects.

The factors influencing this reentry interval have been discussed by Ware e t al.(1973).
The experimental methods for these determinations have included artificial exposure
situations using nonagricultural workers during one reentry episode (Ware e t al 1973), and
investigation of foliage residue disappearance (Serat 1973).

This report describes a method of confirming reentry intervals by monitoring cotton

insect scouts throughout an entire pesticide application season without modification of
their behavior. The purpose of the study was to report the physiological results of
organophosphate exposure under actual field conditions.

1U. S. Public Health Service assigned to: Epidemiologic Studies Program, Technical Services Division,
Office of Pesticides Program. U. S. Environmental Protection Agency, during the study reported.

Archives of Environmental Contamination 344

and Toxicology Vol. 3,344-351 (1975)
9 1975 by Springer-Verlag New York Inc.
 Safety of Cotton Reentry 345

Materials a n d M e t h o d s

Cotton scouts. Twelve college-aged men employed by a federal, university and

grower-sponsored cotton pest management programs were enlisted for the study. They
were trained and supervised by an extension entomologist and were responsible for
inspecting certain fields of 77 cooperating growers to determine when the level of insect
infestation warranted pesticide application.

The scouts were hired in early May, 1973, and divided into six teams of two scouts
each. Each team was assigned 17 or 18 fields per day, five days a week, for a total of 521
fields. These fields varied from four to 96 acres and averaged 30 acres with a total acreage
of 15,632. This was 5.8% of the 270,500 acres of cotton planted in the study area. Each
team visited assigned fields weekly on a scheduled day and reported their findings to the
grower. By coordinating activities with the application of pesticides, it was anticipated
that few reentries would be made within the first 48 hr. Efforts were made to enter no
earlier than 24 hr.

In every field examined each scout walked 250 to 350 paces and examined 50
squares and 50 plant terminals. As a result each scout averaged five hr per day in cotton
fields, and by June 25, these activities resulted in significant foliar contact. The standard
uniform was denim trousers, short-sleeved shirt, hat, and leather boots when fields were
dry. On rainy days all scouts went barefooted due to the tenacious adhesiveness of the
soil. Work clothes were changed and baths were taken within two hr of completing work.

After cotton harvest each grower was contacted to determine when his fields were
sprayed, which pesticides were used, and at what rate. The combination of these records
and the field reports allowed determination of reentry interval and pesticides involved for
each exposure. Reentry data were calculated on a random subsample of 15 growers with
2621 acres (16.8% of study acres). Essentially all pesticide was applied by fixed-wing
agricultural aircraft.

Medical data. At the beginning of the study a detailed history and physical examina-
tion was performed on eachscout to rule out complicating medical factors. Blood samples
were not collected for one individual due to persistent syncope on venepuncture. After
being individually informed of the purpose and risks of the study, all other participants
gave their consent to repeated venepuncture and all 12 agreed to donation of urine
samples.

Erythrocyte (AChe) and plasma (BuChe) cholinesterase determinations were made

every other week by the pH Stat method of Nabb and Whitfield (1967). This interval was
selected as a compromise between our need'for data and the participants reluctance to
donate blood. These samples were obtained on Thursday morning and analyzed the same
day. The normal mean was determined on 61 firemen with no known exposure to
anticholinesterase agents. Urinary para-nitrophenol (PNP) excretion was determined by the
gas-liquid chromatographic method of Cranmer (1970). Samples were obtained every
Thursday afternoon, preserved until the following day by the addition of several drops of
346 James E, Burns and Ray D. Parker

37% formaldehyde and then frozen until analysis (Davies et al 1966). One sample
(27 July) represents a 24-hr urine collection.

Results

Table I gives the distribution of reentry intervals by days for the random subsample of
1437 reentries (17.5% of all reentries). Due to the extreme difficulty in determining the
hour of pesticide application the intervals are measured in days. A field sprayed at dusk
and reentered early the following day would be a 12-hr day. A field sprayed at dawn and
reentered the next evening would be a 36-hr day. The time units are therefore in days -+ 12
hr.

The 2% of visits resulting in no entry were rescheduled for the following day. There
were only four recognized episodes of reentry within 12 hr during the study. One of these
was associated with the highest PNP excretion of 2.7 ppm. None of these episodes resulted
in symptomatology and unfortunately no additiorial Che determinations were made. At
no time during the summer did any of the participants develope signs or symptoms
characteristic of organophosphate poisoning.

The laboratory data are presented in Figures 1 and 2. Figure 1 shows the AChe and
BuChe of each individual expressed as a percentage of that individual's pre-exposure value.
The horizontal line at 70% corresponds to two SD below pre-exposure mean. There was
no significant difference between the mean determined on 61 firemen and the pre-
exposure mean for the study subjects. Individual PNP excretion patterns are also included.
The sample on 27 July (arrow) was the 24-hr collection and the mean methyl parathion
equivalent (PNP • 1.9) was 0.32 + 0.26 mg/24hr with a range of 0.08 to 0.98 mg/24hr.
The maximum exposure occurred on 13 July and was approximately equivalent to
absorbing 0.95 mg of methyl parathion in 24 hr. The approximation assumes one L of
urinary output in 24 hr and a constant concentration of PNP. This probably overestimates
the urinary PNP but since urinary PNP only accounts for about 50% of an intravenous dose
of ethyl parathion (Feldmann and Maibach 1970) this approximation seems reasonable.

Figure 2 presents the same data for the cotton scouts as a group expressed as absolute
values for the Che ~m/min/ml). Included in this graph are data points obtained during a

Table I. R e e n t r y Intervals a

Aborted b 1 Day c 2 Days 3 Days 4 Days > 4 Days

2% 5% 7% 8% 7% 71%

a 17.5% (1437) of all reentries

bFields visited but not entered because of recent or current spraying.
cOnly 4 known episodes of <12 hour reentry (out of 8211 reentries).
 Safety of Cotton Reentry 347

preliminary survey the preceeding year under similar circumstances. The 1973 cotton
season was delayed two to four weeks because of heavy rains which is reflected in the
displacement of the Che curves for the two years. The arrow indicates the 24-hr urine
collection.

Figure 2 also shows the weekly percentages of one- and two-day reentries and the
pesticide application rates; these data were obtained on the same subsample of fields. The
application rates are expressed as pounds of technical material per acre reentered. Since
the number of acres reentered per week was constant the units on the ordinate could be
expressed as total pounds of technical material. All pesticides except chlorate-type
defoliants and herbicides are included in this figure.

/
120] n = 11

] 11o1]
~= 1001
o ~ 90"1 ,- --

:=t
~e~ 8O

70. :2SD .................... /

e-

~=~" 110 t
.~ 1001

1
.~ 8o
-2 SD
70 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.5- =

2.0-
o,.
zel. 1.0-

9~ 1.5 -

.5-

0 I ! !
May June July / August
Fig. 1. Cotton scouts, Texas, 1973. Cholinesterase as percent of preexposure values and
PNP excretion of individuals.
348 James E. Burns and Ray D. Parker

Q
14- "'",.. Vertical lines = + 2SD
e = 1972
13- Mean ' " ,, ... ".... " . .
- n= 11
AChe
12-

E 11-
e-
"'"'o
E |

: I Mean
BuChe

PNP 0.5 t
(ppm)
0

1 & 2 da3: l
reentries
(%) o ]
1.50-
]
.•1 Met
" 2 day--~-~ n = 12

~, 1.00-
P
--i
O
ca" .50-

May I
June
!
July
I
August
Fig. 2. Cotton scouts, Texas, 1973. Cholinesterase levels, PNP excretion, reentry intervals,
and pesticide application rates.

The broad hump in the PNP and application rate curves reflects the practice of
spraying cotton fields almost automatically every five days towards the end of the
growing season.

For the entire spray season the proportion of the various pesticides by weight were:
methyl parathion (O,O.dJmethyl-O-p-nitrophenyl phosphorothioate) 54.2%, toxaphene
 Safety of Cotton Reentry 349

(mixture of chlorinated camphene compounds) 35.3%, ethyl parathion (O,O-diethyl-O-p-

nitrophenyl phosphorothioate) 4.1%, and other (mostely organophosphates) 6.4%.

Discussion and Conclusions

Exposure to predominantly methyl parathion (see Fig. 2) under the described condi-
tions resulted in detectable biochemical changes. By June 21 the group reached its
lowest Che level and this depression was significant for both AChe (p < .001) and BuChe
(p < .005~ significance by t-test unless otherwise indicated). This depression occurred
during the increase in organophosphate exposure as indicated by the PNP excretion curve
and the number of pounds of technical material applied per acre. However only a small
proportion of reentries were made at one and two days. A similar though earlier depression
was suggested by the 1972 data which corresponds to the earlier pesticide applications
under more favorable weather conditions. It is evident, however, that two weeks later,
when exposure was at its maximum, the cholinesterase values had returned to normal.
This rise was also seen in 1972. The etiology of this phenomenon is unclear and is possibly
mediated through an as yet unidentified feedback enzyme induction mechanism. Another
possibility is that the changes were laboratory artifacts but this is unlikely in view of the
1972 data. None of the participants received exposure to anticholinesterase agents other
than through their scouting activities.

A similar, though smaller, depression in both Che levels in mid-July corresponds to the
end of the peak exposure period but is not statistically significant. At the end of the
study during low exposure conditions the AChe level again decreases (p < .05) with-
out a corresponding decrease in the BuChe level. This phenomenon was apparently
experienced in 1972 also and is possibly related to the use of phosphate defoliants
(mostly S,S,S-tributyl phosphorotrithioate). This product has a dermal LDs 0 in rats of
850 mg/kg, compared to 67 mg/kg for methyl parathion and 21 mg/kg for ethyl para-
thion, which make this association remote (Hayes 1966).

In a discussion of worker safety it is important to consider individual experience

which may be obscured by observing the central tendency. If we assume that a 30% de-
pression, which is approximately two SD below the mean, of either AChe or BuChe levels
is clinically significant then there were two such depressions in mid-June; one AChe and
one BuChe in different people. In mid-August there were three depressions of AChe
levels, but none of these five episodes were associated with symptomatology or remarkable
PNP excretions.

The 24-hr urine collection, which was unfortunately obtained just past the peak ex-
posure period, indicates that the afternoon urine samples supplied reasonable estimates of
the relative exposure status. It would, of course, be perferable to have frequent 24-hr
urine collections.

There were four epidosdes in which the PNP excretion exceeded one ppm (equivalent
to absorbing and metabolizing 1.9 mg of methyl parathion if urinary volume were one
L). The most dramatic of these was a rise to 2.7 ppm which was associated with entering a
350 James E. Burns and Ray D. Parker

field within one or two hr after spraying. No such association could be determined for the
other episodes nor were there Che depressions or symptoms associated with any of them.

There are apparently no studies relating measured methyl parathion ingestion to PNP
excretion, but Moeiler and Rider (1963) showed that ingesting up to nine mg per day of
methyl parathion produces no significant effect on cholinesterase levels. Hayes et al
(1964) demonstrated that dermal exposure to ethyl parathion sufficient to result in up to
2.37 mg of PNP excreted in 24 hr had no effect on Che levels, but at an excretion rate of
5.93 mg[24 hr Che depression did occur. Thus, our 24-hr PNP excretion level of 0.17 +
0.13 mg/24hr is one-fourteenth the excretion rate known to be associated with no
effects for a more toxic homolog. One important difference in these exposure situations
is that our participants were exposed daffy, whereas the experimental dermal exposures
were isolated events. The possibility of significant quantities of PNP being excreted in
sweat must also be considered. It is assumed that our volunteers received most of their
exposure by dermal absorption after the demonstrations of Durham et al (1972).

The reentry intervals presented in Table I apply to the entire scouting period and are
thereby biased towards longer reentry times in the early part of the season when pesticide
applications were infrequent. The graphic presentation of weekly reentry intervals in
Figure 2 shows that the proportion of one- and two-day reentries roughly parallels the
PNP excretion with the exception of the substantial rise in two-day reentries in late July.

Ware et al (1973)estimated that the arms and hands of cotton scouts would accumulate
six mg of methyl and ethyl parathion if fields treated with a total of one pound/acre
were reentered at 48 hr for five hr/day. Maibach et al (1971) have shown that 8.6% of
parathion applied to the forearm is absorbed. Applying this proportion to Ware's esti-
mate results in 0.52 mg of parathion being absorbed during one working day. This is
approximately two times our estimate of 0.95 mg of methyl parathion being absorbed
and excreted as PNP. Given the many uncontrolled variables such as differential ab-
sorption rates, varying application rates, possible different absorption rates for ethyl and
methyl parathion, varying reentry interval, and the unknown effect of toxaphene it is
surprising that this crude comparison agrees as well as it does.

This study shows 12 cotton insect scouts made up to 48% of their weekly reentries two
days or less from the last pesticide application. These reentries were made into fields
treated at 4-7 day intervals with up to 1.7 pounds per acre of methyl parathion combined
with toxaphene and other materials. The level of exposure indicated by PNP excretion
was low but the cholinesterase data indicate a definite biochemical effect from these
field exposures. There were no symptoms or evidence of disease associated with these
exposures.
Acknowledgements
The authors are grateful for the contributions of the members of the Texas Epidemio-
logic Studies Program Laboratory and the Pest Management Program Staff. This work was
supported by contract number 68-02-0751 from the U. S. Environmental Protection
Agency through the Texas State Health Department.
 Safety of Cotton Reentry 351

References
Cranmer, M. F: Determination of paranitrophenol in human urine. Bull. Environ. Contam.
Toxicol. 5,329 (1970).
Davies, J. E., J. H. Davis, D. E. Frazier, J. B. Mann, and J. O. Welke: Urinary para-
nitrophenol concentrations in acute and chronic parathion exposures. Advances in
Chemistry series, Washington, D. C., American Chemical Society 60, 67 (1966).
Durham, W. F., H. R. Wolfe, and J. W. Elliott: Absorption and excretion of parathion by
spraymen. Arch. Environ. Health 24, 381 (1972).
Feldmann, R. J., and H. I. Maibach: Pesticide percutaneous penetration in man,
abstracted. J. Invest. Dem. 54, 435 (1970).
Hayes, G. R., A. J. Funckes, and W. V. Hartwell: Dermal exposure of human volunteers
to parathion. Arch. Environ. Health 8, 829 (1964).
Hayes, W. J.: Clinical Handbook on Economic Poisons. Washington: U S. Government
Prin ring Office (1966).
Maibach, H. I., R. J. Feldmann, T. H. Milby, and W. F. Serat: Regional variation in
percutaneous penetration in mart. Arch. Environ. Health 23, 208 (1971).
MoeUer, H. C. and J. A. Rider: Further studies on the toxicity of Systox and methyl
parathion. Abstract. Fed. Proc. 22, 189 (1963).
Nabb, D. P., and F. Whitfield: Determination of cholinesterase by an automated pH stat
method. Arch. Environ. Health 15,147 (1967).
Serat, W. F.: Calculation of safe reentry time into an orchard treated with a pesticide
chemical which produces a measurable physiological response. Arch. Environ.
Contain. & Toxicol. 1,170 (1973).
Ware, G. W., D. P. Morgan, B. J. Estesen, W. P. Cahill, and D. M. Whitacre: Establishment
of reentry intervals for organophosphate-treated cotton fields based on human
data: I. Ethyl- and methyl parathion. Arch. Environ. Contam. & T0xicol. 1, 48
(1973).

Manuscript received July 8, 1974; accepted Sept. 26, 1974.