vvEPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-82-001 July 1982
Project Summary
Toxicology of Pesticides
Elsa Reiner
Documented in this report are the
results of five toxicological studies of
pesticide compounds conducted by the
Institute for Medical Research and
Occupational Health, Zagreb, Yugosla-
via, for the U.S. Environmental Protection
Agency.
In the first study, the reactions of two
groups of esterases (cholinesterases
and arylesterases) with substrates and
inhibitors were investigated. Procedures
for monitoring the absorption of phosa-
lone and malathion in occupationally
exposed workers by determination of
pesticide residues in the urine were
developed in the second study. This
detection technique was compared to
the traditional blood cholinesterase in-
hibition method to determine which
was a more rapid detector of organo-
phosphorus poisoning.
The third study surveyed the residues
of chlorinated hydrocarbons in human
milk and blood samples taken from the
general population, and compared the
observed levels with those found in the
serum of workers exposed to pesticides.
In the fourth study, cholinesterase ac-
tivity was used to assess the effects of
recent changes made in the protective
procedures for occupationally exposed
workers. Finally, in the last study, the
alleged effect of pesticides on the eye
and on vision was studied and the results
discussed.
In addition, the report includes sum-
maries of seven students' theses related
to the work conducted by the Institute.
This Project Summary was developed
by EPA '5 Health Effects Research Lab-
oratory, Research Triangle Park, NC, to
announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
Of all chemical compounds commer-
cially used, pesticides pose one of the
greatest and most direct threats to
human health. To assure that all pesti-
cides intended for widespread use are
nonhazardous to humans, the U.S. En-
vironmental Protection Agency's (EPA's)
Health Effects Research Laboratory
screens proposed compounds through
extensive toxicity testing. The labora-
tory tests and analyzes compounds to
provide the data necessary for EPA's
regulatory activities, including the regis-
tration of new pesticides and suspen-
sion of pesticides proven hazardous
after initial approval. The laboratory
also develops and tests procedures for
monitoring workers occupationally ex-
posed to pesticides. Data gathered
through such monitoring efforts are also
used to support regulatory actions.
In support of the Health Effects
Research Laboratory's testing and mon-
itoring efforts, the Institute for Medical
Research and Occupational Health
(IMORH), Zagreb, Yugoslavia, has con-
ducted five pesticide studies over a
four-year period. This project summary
presents and discusses the results of
each of these studies. Only the major
details of experimental procedures,
however, are provided.
Esterases and
Organophosphorus Compounds
The focus of the first of the five stu-
dies conducted by IMORH was to deter-
mine the reactions of two groups of
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esterases—cholinesterases and aryles-
terases—with their substrates and or-
ganophosphorus (OP) inhibitors.
Procedure
Pyridinium oximes have long been
recognized as effective reactivators of
cholinesterases (ChE's) inhibited by OP
compounds. It is their reactivating ability
that makes them useful in the preven-
tion and therapy of OP poisoning. In this
study, 25 pyridinium oximes were syn-
thetized in the laboratory and tested in
vitro for the reactivation of phosphory-
lated and phosphonylated acetylcholin-
esterases (AChE's). The oximes were
also tested in vivo as protective com-
pounds against OP poisoning.
After initial testing, the binding sites
for substrates and inhibitors of AChE
and ChE were studied in detail. The
kinetics of competition between pairs
of substrates for AChE and ChE were
investigated to determine whether or
not the substrate-inhibition site takes
part in the reaction.
In addition, ChE's and arylesterases
were tested comparatively in several
species using 0,O-dimethyl-2,2-dichlo-
rovinylphosphate (DDVP) as ChE inhibi-
tor and arylesterase substrate.
Results
Sixteen of the 25 newly synthetized
compounds were tested for their ability
to reactivate human erythrocyte methyl-
ethoxyphosphonylated AChE. Of these
16 oximes, three proved to be fairly
good reactivators. The reaction of these
three compounds was initially rapid, but
they slowly tapered down to an equili-
brium. Although all three compounds
had a similar reactivation efficiency,
none had better reactivation properties
than TMB-4, a well-known reactivator
of OP-inhibited AChE which was tested
in the study as a reference compound.
The same 1 6 compounds discussed
above were also investigated to deter-
mine their protective characteristics
against AChE inhibition by 0-1,2,2-tri-
methylpropylphosphonofluoridate (So-
man). Protective efficiency was evalu-
ated by comparing AChE inhibition by
Soman, with and without the tested
compound. Benzolcarbonyl, cyclohexyl-
carbonyl, and amidocarbonyl exerted a
good protective effect against AChE in-
hibition by Soman; TMB-4 offered no
protection from Soman inhibition.
The nine other oximes (bispyridinium)
were tested for reactivation potency
and therapeutic effect on two OP com-
pounds, DDVP and O-ethyl-S-2-diiso-
propylaminoethyl methylphosphonothi-
oate (VX). Enzyme reactivation was
measured on human erythrocyte AChE,
and therapeutic effect was evaluated
on male albino rats. The oximes with a
hydroxyamino group in position 4 in the
pyridinium ring were good reactivators
of both phosphorylated and phospho-
nylated AChE. The same oximes were
also effective (given with atropine)
against VX and DDVP poisoning.
To determine whether or not the sub-
strate-inhibitor site in an enzyme takes
part in the reaction, the binding sites in
bovine erythrocyte AChE were studied
in the presence of several different
inhibitors. After a series of kinetic experi-
ments investigating paired substrate
competition for erythrocyte AChE and
horse serum ChE, it was concluded that
the substrate-inhibition site was not in-
volved in the competition between two
substrates. However, when the OP
compound haloxon was the inhibitor,
the reaction did occur near the site.
To determine whether regeneration in
vivo was spontaneous or was due to
enzyme synthesis, comparative studies
of the reaction of ChE's and arylester-
ases with DDVP were conducted in var-
ious species. Pure DDVP and DDVP de-
rived from metrifonate were used as the
ChE inhibitor and arylesterase sub-
strate. Kinetic analysis of in vivo rat
brain and plasma date showed that re-
generation of enzyme activities after
DDVP treatment could be attributed en-
tirely to spontaneous activation of the
inhibited enzymes; no difference was
seen in the kinetics of the reaction in
different species, and the activity of the
arylesterases was of the same order in
mammalian and nonvertebrate tissues.
Regeneration of ChE activity in human
plasma and erythrocytes in vivo was at-
tributed to enzyme synthesis, but since
6 h elapsed before ChE determination,
the enzyme had reached an aged, non-
reactable form.
Residues of OP Pesticides
in Human Urine
In the second study, IMORH re-
searchers compared two methods—the
blood ChE inhibition method and the
urine pesticide residues method —to
establish which of the two was a more
reliable indicator of the amount of OP
pesticides absorbed by occupationally
exposed workers. Urinary metabolite
analysis was hypothesized to detect the
absorption of OP pesticides prior to any
depression of ChE activity, thus permit-
ting protective measures to be taken
sooner than allowed by the standard
ChE inhibition method.
Procedure
To evaluate this hypothesis, the ef-
fects of phosalone, malathion, and qui-
nalphos on occupationally exposed
workers were investigated by analyzing
urine samples obtained after exposure
and by measuring blood ChE inhibition
levels.
Urine samples from 1 2 workers ex-
posed to phosalone and 14 workers ex-
posed to malathion were analyzed for
their alkali metal salts concentration.
These salts result from the hydrolysis
and metabolism of the dialkyl esters of
the acids produced during OP pesticide
degradation. Determination of the
amounts of these salts in urine and
blood samples can be used to detect OP
poisoning. Simulated samples were pre-
pared for purposes of comparison by
adding a definite amount of standard
salt to the urine of nonexposed persons.
In addition, a volunteer subject with
no previous exposure to phosalone was
experimentally exposed for the duration
of one working day, and then again on
the three following days. Urine samples
were taken at the beginning and the end
of work, and 4 to 5 h after termination
of exposure. Using the data obtained
from this experiment, the period neces-
sary for the complete excretion of pesti-
cide residues and the appropriate time
for urine sampling were determined.
Analysis of samples obtained from
workers exposed to phosalone and
malathion consisted of measuring the
concentration of 0,0-diethyl phospho-
rodithoate (DEDTPK) and 0,0-dimethyl
phosphorodithoate (DMDTPK) alkali
metal salts in the urine. Various diazoal-
kanes were applied to convert the salts
into more volatile trialkyl derivatives for
gas chromatography (GO analysis.
Urine samples from malathion-exposed
workers were collected at the end of
work hours. Urine samples from phosa-
lone-exposed workers were collected
both prior to the beginning of work and
at the end of work.
The absorption of quinalphos by 1 1
exposed workers from a pesticide pro-
duction plant was studied by determin-
ing the amount of 0,0-diethyl phosphate
(DEP) and 0,0-diethyl phosphorothionate
potassium salt (DETPK) in the workers'
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urine. Urine samples were collected at
the start and end of work at intervals of
30 days during three months and imme-
diately after one month's vacation. Blood
samples were taken at the beginning of
work on the same days as urine sam-
ples were taken. Eleven urine samples
were also collected from non-exposed
workers and analyzed for comparison.
Results
DEDTPK was found in the urine of all
1 2 production workers exposed to pho-
salone. Only the two highest values
measured in the urine samples were
also detected by the blood ChE inhibi-
tion method, supporting the hypothesis
set forth at the beginning of the study.
DMDTPK concentrations found in 10
of 14 urine samples obtained from
workers exposed to malathion fell within
the range of 449 to 1072 ng/ml of
urine. In remaining urine samples, and in
nine samples from non-exposed persons,
no malathion residues were detected.
The highest concentration of malathion
residue in urine was accompanied by
the lowest ChE activity.
Results from the experimental expo-
sure of a volunteer to phosalone showed
that the amount of residues excreted in-
creased gradually, and the highest value
was reached 4 to 5 h after exposure.
Subsequently, the amount of residue
decreased abruptly, but was still meas-
urable at the beginning of the following
work day. Residues increased system-
atically when exposure was prolonged
for several days. The volunteer's blood
and plasma ChE was only slightly
reduced during exposure.
The total amount of all metabolites
excreted during one day was determined
a better indicator of exposure than me-
tabolite concentration in a single urine
fraction. However, because of the latter
technique's inconvenience, the concen-
trations of DEDTPK taken for the same
period of time after exposure can be
used instead.
Results from the study conducted to
compare the effect of quinalphos on
blood ChE inhibition and urine residues
concentration showed that the highest
concentration of residues in urine was
followed by the greatest decrease of
ChE activity. However, since the
workers tested alternated three to five
days between work with OP compounds
and other compounds such as carba-
mates, no determination of one method's
desirability over the other could be made.
Residues of Chlorinated
Hydrocarbons in Human Milk
and Blood
The third IMORH study was conducted
to determine the amount of chlorinated
hydrocarbon (HO residues in serum
samples taken from two groups of occu-
pationally exposed workers and from the
general population of four different parts
of Yugoslavia. In addition, mother's
serum, mother's milk, and umbilical
cord serum samples taken between
1977 and 1979 in the Yugoslavian
town of Croatia were analyzed for DDT
content.
Procedures
Pesticide residue levels in 262 blood
serum samples taken from members of
the general population and in 78 serum
samples taken from exposed workers
were determined by GC. Samples were
analyzed for p,p1-DDT, p^-DDE, and
p,p1-DDD, Lindane, and a-HCH.
Residue levels in mother's serum,
mother's milk, and cord blood serum
were also determined by GC. Concen-
trations of p,p1-DDE, p,pi-DDD, and
p,p1-DDT were measured in 34 human
milk samples collected three to five
days after delivery and in 37 samples
collected 1 Vi to 55 weeks after delivery.
Two extraction methods for milk were
used and compared.
In the first method, 1 ml milk was
extracted with acetronitrile and the ex-
tracts combined with sodium sulfate.
This mixture was then extracted with
hexane, and the hexane extracts puri-
fied on a florisil column. Organochlorine
compounds retained on the column were
eluted with hexane, and the eluates
evaporated to dryness in a nitrogen
stream. The compounds were redis-
solved for GC analysis.
In the second method, 0.5 ml milk
was partitioned with methanol and po-
tassium carbonate. The mixture was
extracted with hexane, and the hexane
extracts evaporated to dryness. The
compounds were redissolved in hexane
for GC analysis.
For extracting residues from the
mother's and cord serum samples,
serum was partitioned with formic acid
and the resulting mixture extracted with
hexane. The hexane extracts were
washed and purified on a florisil column.
Compounds retained on the column
were eluted with hexane, and the elu-
ates evaporated to dryness in a nitrogen
stream. The compounds were redis-
solved in hexane and analyzed.
Results and Discussion
Concentrations of DDT and hexachlo-
rocyclohexane residues found in the
blood serum of the general population
fell within the range reported for other
countries. Few samples contained Lin-
dane or a-HCH, but the mean concen-
trations for these compounds were
much lower for the general population
than for occupationally exposed workers.
Exposed workers also had a higher inci-
dence of residues in the serum.
Differences in the mean concentration
values for p.pT-DDE measured in milk
samples obtained at the beginning of
lactation and samples obtained during
the subsequent 55 weeks were not
found to be significant. The two extrac-
tion methods for milk compared favor-
ably for p,p1-DDE. The content of DDT
derivatives in milk fell within the middle
of the range for European and non-
European countries.
Concentrations in mothers' milk were
2.1 times higher than in mothers' serum.
Cord blood serum contained lower con-
centrations of p,p1-DDE than the
mother's serum. Serum samples from
non-pregnant women had the same
DDT content as those from mothers at
delivery.
Biochemical and Clinical Effects
of Pesticides in Humans
The IMORH's fourth study attempted
to relate clinical symptoms in workers
to the degree of their exposure to a
wide variety of pesticides. Vitamin A
levels and DDT concentrations in ex-
posed workers were measured and
compared to values obtained for non-
exposed persons.
Procedures
To evaluate the absorption of pesti-
cides by 567 industrial workers during
the period from 1970 to 1979, meas-
urements of blood ChE were taken
regularly. Only 1 70 of the 567 workers
studied worked at any of three produc-
tion lines (dust or wettable powder,
emulsion, or household sprays) in one
plant for 2 to 1 4 years. The others were
seasonal workers hired for short periods
of intense production.
In the nine-year study period, workers
were exposed to OP compounds, carba-
mate insecticides, herbicides, fungi-
cides, and other compounds. Plasma
and erythrocyte ChE activity were de-
termined by spectrophotometry.
Vitamin A levels in the serum of 65
exposed workers were measured in
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1976, and again in 1977 for some.
Vitamin A levels for the control group
were measured in both 1976 and
1977. Vitamin A was determined by
spectrophotometry with anitmonium
trichloride.
Total DDT in the serum and ChE ac-
tivity in the blood and plasma of ex-
posed workers were also measured to
verify that pesticides were absorbed.
Since exposure to certain pesticides
was not continuous, ChE activity was
monitored both before the beginning of
work with AChE pesticides and during
the course of work with these pesticides.
Results
Since the production of OP insecticides
increased during the 10-year observa-
tion period, the number of blood samples
with decreased enzyme activity was also
expected to increase. However, the
greatest depression of enzyme activity
below 50% of normal occurred in the
first two years, when workers were ex-
posed to the extremely toxic insecti-
cides dimethoate and chlorfenvinphos.
Once this was noted working condi-
tions were improved, resulting in no
cases of poisoning in 1972 and only
two in 1 973. Enzyme depression below
50% again rose in the following years
when production of insecticides in-
creased and more inexperienced workers
were hired.
Results of measurements of Vitamin
A showed no effect of pesticides on
Vitamin A levels. All control and ex-
posed group Vitamin A levels fell within
the same range along with levels meas-
ured for standards.
Measurements of ChE activity made
before the initiation of work with AChE
pesticides showed that ChE activity
was not reduced more than 20% in any
of the groups. During the course of
work with pesticides the same workers
showed greatly reduced ChE activity
levels, indicating that the workers had
absorbed quantities of the pesticides.
Workers exposed to chlorinated HC's
had higher serum DDT levels than con-
trols. DDT levels found in workers not
exposed to chlorinated HC's were com-
parable to those found in the general
population earlier.
Conclusions
When protective devices and sanita-
tion regulations were enforced after the
first two years, the absorption of AChE
insecticides was satisfactorily reduced.
In addition, weekly measurement of ChE
activity was shown to be a practical
method for determining worker risk and
thus allowing the prevention of further
pesticide absorption.
Effect of Pesticides on the
Eye and Vision
The last study reported by IMORH
was conducted to investigate the ef-
fects of pesticides on the human eye
and vision.
Procedures
Fifty-seven of the permanent workers
exposed to AChE pesticides in the pre-
vious study (47 production and 10 pes-
ticide application workers) were selected
for this investigation. Eighteen of the
workers were exposed from one to five
years, and the other 39 were exposed
for over five years. Only 1 1 were older
than 45. A detailed history of illnesses
possibly connected with eyesight was
gathered for each worker, and all work-
ers underwent opthamological exami-
nations, including tonometry, opthal-
moscopy, slit-lamp biomicroscopy,
keratometry, and visual acuity tests.
Peripheral vision was measured by a
Goldmann perimeter, and dark adapta-
tion ability was assessed on a Goldmann-
Weekers adaptometer. Workers with
visual abnormalities were treated by
standard procedures.
Results
Twenty-three of the production
workers complained of lacrimation, pho-
tophobia, itching and burning, and other
maladies. Only three of the application
(agricultural and public health) workers
complained of these eye problems.
The most frequently observed abnor-
mality of the frontal eye segment was
dilated or tortous episcleral blood vessels.
The incidence of these abnormalities
was much higher in the exposed workers
than in the same-age members of the
general population. Limited conjunctival
injection, abnormal pupils, and lens
opacities were also noted in some
workers. Four production and one appli-
cation worker had pronounced astigma-
tisms. Keratometric measurements
showed little abnormality in the majority
of the workers, however.
Increased intraocular pressure was
noted in six workers; open angle glau-
coma was subsequently diagnosed in
one of the six, but the phenomenon re-
mains unexplained for the other five.
Also unexplained is the constriction of
the visual field observed in 11 produc-
tion and 2 application workers. This was
the most common of all abnormalities
observed in the workers tested.
Dark adaptability was measured as
remarkably slow in 2 of 1 6 production
workers tested, but no or little differ-
ence was noticed in the other 14
workers.
Appended Students' Theses
The seven students' theses sum-
marized in an appendix to the report
include the following titles:
• Preparation of Silyl Derivatives for
the Gas Chromatographic Analysis
of Organophosphorus Pesticide
Residues
• Simultaneous Gas Chromatographic
Determination of Alkali Metal Salts
of 0,0-Diethyl-, 0,0-Dimethyldithio-,
and 0,0-Diethylthio-phosphoric Acid.
• Toxic Effects of Metrifonate in
Mammals
• Mechanism of Inhibition of Acetyl-
cholinesterase by Some Oximes
• Determination of Organophosphorus
Pesticide Residues by Gas Chroma-
tography
• Organophosphorus Pesticides in
Surface Waters
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Elsa Reiner is with the Institute for Medical Research and Occupational
Health, Zagreb, Yugoslavia.
William F. Durham is the EPA Project Officer (see below).
The complete report, entitled "Toxicology of Pesticides, " (Order No. PB 82-226
077; Cost: $9.00, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use $300
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