EPA-600/1-77-031
June 1977
Environmental Health Effects Research Series
TOXICOLOGY OF ANTICHOLINESTERASE
PESTICIDES
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-77-031
June 1977
TOXICOLOGY OF ANTICHOLINESTERASE PESTICIDES
Elsa Reiner
Principal Investigator
Head, Department of Biochemistry
Institute for Medical Research and Occupational Health
Zagreb, Yugoslavia
Special Foreign Currency Program
PR-2-515-2
Project Officer
Ronald L. Baron
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle Park, North Carolina 27711
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF HEALTH AND ECOLOGICAL EFFECTS
OFFICE OF RESEARCH AND DEVELOPMENT
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory-
RTP, U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views and
policies of the U.S. Environmental Protection Agency, nor does mention of
trade names or commercial products constitute endorsement or recommendation
for use.
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FOREWORD
The many benefits of our modern, developing, industrial
society are accompanied by certain hazards. Careful assessment
of the relative risk of existing and new man-made environmental
hazards is necessary for the establishment of sound regulatory
policy. These regulations serve to enhance the quality of our
environment in order to promote the public health and welfare
and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle
Park conducts a coordinated environmental health research program
in toxicology, epidemiology, and clinical studies using human
volunteer subjects. These studies address problems in air pol-
lution, non-ionizing radiation, environmental carcinogensis and
the toxicology of pesticides as well as other chemical pollutants.
The Laboratory develops and revises air quality criteria documents
on pollutants for which national ambient air quality standards
exist or are proposed, provides the data for registration of new
pesticides or proposed suspension of those already in use, con-
ducts research on hazardous and toxic materials, and is preparing
the health basis for non-ionizing radiation standards. Direct
support to the regulartory function of the Agency is provided in
the form of expert testimony and preparation of affidavits as well
as expert advice to the Administrator to assure the adequacy of
health care and surveillance of persons having suffered imminent
and substantial endangerment of their health.
The use of pesticides to protect man and his environment has
increased substantially over the past few decades. Considerable
efforts have been made to assure the safe use of these toxicants
both to the occupationally exposed and to those who are inadvert-
ently exposed either directly or as a result of pesticide residues
in food or the environment. Studies are reported on the health
aspects of pesticides with the aid of volunteers from occupational
exposure situations. Reports on adverse effects on opthalmologi-
cal parameters of certain organophosphate esters used as pesti-
cides was evaluated using people occupationally exposed. The
significance of these effects were evaluated and the continued
surveillance of the population under substantial risk continues
with emphasis on other health Delated problems.
SJcvhn H. Knelson, M.D.
Health Effects Research Laboratory-RTF
iii
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ABSTRACT
The effect of pesticides was studied in humans and animal
models, and on enzymes in order to develop a better understanding
of their mechanism of action, and thus provide guidance for their
safe use.
It was shown that reduction of human blood cholinesterase
activity is a good indicator of exposure to anticholinesterase
pesticides. Regular measurements of blood cholinesterase
activity and removal of individuals from suspected occupational
environments prevented hazardous absorption of pesticides in
occupationally exposed people. Ophthalmological studies
indicated some functional eye impairment in people exposed to
pesticides for many years.
The effect of one organophosphate ester used as a pesticide
(trichlorfon; Metrifonate®) was studied in detail in humans,
animals (rats) and enzymes (cholinesterases). It was shown that
trichlorfon does not inhibit mammalian cholinesterases; inhibition
of the enzyme is caused by dichlorvos formed from trichlorphon.
The mechanism of reaction of acetylcholinesterase and cholin-
esterase with other inhibitors and with substrates was also
studied in order to evaluate to what extent sites other than the
catalytic sites are involved in the binding of ligands.
Gas chromatographic methods for measuring pesticide
residues were used to determine concentrations of chlorinated
hydrocarbon pesticides in the blood of the general population
in Yugoslavia and for initial studies of organophosphate
pesticide residues in the urine of occupationally exposed workers.
Thin layer chromatography (combined with densitometric measure-
ments of the chromatograms) served to determine organophosphates
in experimental animals.
This report was submitted in fulfillment of a Special
Foreign Currency Program (No. PR 2-515-2) by the Institute for
Medical Research and Occupational Health (Zagreb, Yugoslavia)
under the sponsorship of the U. S. Environmental Protection
Agency. The report covers a period from June 1, 1972, to
May 31, 1976, and work was completed on May 31, 1976.
IV
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CONTENTS
Page
Foreword iii
Abstract iv
Figures vii
Tables viii
Acknowledgments xii
1. Introduction 1
2. Conclusions 2
3. Recommendations 3
4. Blood cholinesterase activities in the
general population and in workers exposed
to pesticides 4
5. Effect of pesticides on eye and sight 17
6. Residues of organophosphorus pesticides
in human urine 34
7. Residues of chlorinated hydrocarbons in
human blood 37
8. Toxic effect of Metrifonate (trichlorphon)
and DDVP (dichlorvos) in rats 39
9. Reaction in vitro of Metrifonate
(trichlorphon) and DDVP (dichlorvos) with
acetylcholinesterase and cholinesterase 48
10. Mechanism of ligand binding to acetyl-
cholinesterase and cholinesterase in vitro 53
11. Evaluation of field methods for determining
the activity of human blood cholinesterases 57
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CONTENTS (cont.)
Page
12. Quantitative determination of organo-
phosphorus pesticides by thin layer
densitometry 60
References 127
Appendices
A. International meeting on cholinesterases
and cholinergic receptors 149
B. Summaries of B. Sc. and M. Sc. Theses 152
C. List of Publications since 1972 156
VI
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FIGURES
Number
1.
2.
3.
Distribution of plasma and whole blood cholin-
esterase activity in nonexposed humans . . . .
Distribution of plasma and whole blood cholin-
esterase activity in relation to sex in
nonexposed humans
Whole blood and plasma cholinesterase activity
in workers engaged in anticholinesterase pesti-
cide formulation
Page
64
i
65
>
66
vn
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TABLES
Number Page
1. List of Compounds (ISO Names, Synonyms and
Formulas) Handled by Occupationally Exposed
Workers 67
2. Days of Exposure per Worker (Production of
Emulsions) 70
3. Days of Exposure per Worker (Production of
Sprays) 71
4. Days of Exposure per Worker (Production of
Powdered Insecticides) 72
5. Average Number of Overtime Work Hours per
Worker in the Production of Emulsions 72
6. The Relationship between Blood Cholinesterase
Activity, and Signs and Symptoms of Poisoning ... 73
7. Symptoms of Poisoning and Cholinesterase
Activities of Exposed Workers 74
8. Blood and Plasma Cholinesterase Activity
During a Workday 76
9. The Mean Value of Blood Cholinesterase Activity
in Workers Engaged in the Formulation of
Thiometon and Dichlorvos 77
10. The Mean Value of Whole Blood and Plasma
Cholinesterase Activity in Agricultural Workers . . 78
11. The Mean Value of Whole Blood and Plasma Cholin-
esterase Activity in Orchard Spraymen 79
12. Mean Number of Leukocytes and Lymphocytes in
Workers Exposed to Organophosphorus Compounds and
in Control Group 80
13. Ophthalmological Findings in Humans Exposed to
Pesticides. Summary of an Epidemiological
Survey 81
viii
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TABLES (cont.)
Number Page
14. Some Data on the Ophthalmologically Examined
Workers 86
15. Complaints of Examined Workers Concerning Eye
and Sight 87
16. Results of External Eye Examination in Exposed
and Control Subjects 88
17. Corneal Curvature in Exposed and Control
Subjects 89
18. Visual Acuity in Exposed and Control Subjects
Before and After Correction 90
19. Intraocular Pressure in Exposed and Control
Subjects 91
20. Number of Patients with Abnormalities of the
Eyeground 92
21. The Results of Perimetric Measurement of the
Right Eye 93
22. The Results of Perimetric Measurement of the
Left Eye 94
23. Dark Adaptation in Exposed Workers and Control
Subjects 95
24. List of Compounds Discussed in Section 6 96
25. Operating Conditions for the Gas Chromatographic
Analysis of Trialkyl Phosphates 97
26. Gas Chromatographic Data for Trialkyl Phosphates. . 98
27. Recoveries Obtained for the Critical Stages of
the Procedure 99
28. Amounts of DEADTP Found in Urine Samples of
Unexposed Subjects 100
29. Amounts of DEADTP Found in Urine Samples and
Blood Cholinestere Activities in Occupationally
Exposed Workers 101
IX
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TABLES (cont.)
Number Page
30. Concentrations of Chlorinated Hydrocarbons in
147 Sa-nples of Human Plasma or Serum 102
31. Acute Toxicity of Metrifonate and DDVP in Rats . . -103
32. Mean Time of the Onset of Cholinergic Symptoms
after Oral or Intravenous Administration of
Metrifonate or DDVP 104
33. Tolerance of Metrifonate and DDVP Infused into
Jugular or Portal Vein 105
34. Brain and Plasma Cholinesterase Activity in Rats
Injected Intravenously with a Single Dose of
Metrifonate or DDVP 1°6
35. Brain and Plasma Cholinesterase Activity in Rats
Repeatedly Treated with Metrifonate or DDVP . . . .107
36. Cholinesterase Activity in a Medium of Different
pH (Intestinal Sac) 108
37. Cholinesterase Activity in a Medium of Different
pH (Abdominal Lymph) 109
38. Metrifonate and/or DDVP Determined by TLC in the
Lymph of Rats 110
39- Second Order Rate Constants for Inhibition of
Acetylcholinesterase and Cholinesterase by DDVP . .111
40. Effect of pH on Inhibition of Bovine Erythrocyte
Acetycholinesterase by DDVP and on the Rates of
Decomposition of Metrifonate 112
41. First-Order Rate Constants for Spontaneous
Reactivation and Aging of Dimethylphosphorylated
Acetylcholinesterase and Cholinesterase 113
42. Decomposition of Metrifonate in Buffer Calculated
from the Kinetics of Inhibition of Cholinesterases .114
43. Decomposition of Metrifonate in Buffer Measured
Polarographically 115
44. Michaelis Constants and Substrate Inhibition
Constants for Acetylcholinesterase and
Cholinesterase 116
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TABLES (cont.)
Number Page
45. K(S) and K(I) Intercepts 117
46. Effect of Temperature on Inhibition of Acetyl-
cholinesterase by Phosphostigmine and Haloxon . . . 118
47. Effect of Temperature on Inhibition of Acetyl-
chqlinesterase by Two Carbamates 119
48. Temperature Conversion Table for Activities of
Human Blood Cholinesterases 120
49. Activity of Human Whole Blood Cholinesterases
Measured by the Tintometric and Spectrophotometric
Methods 121
50. Activity of Human Plasma Cholinesterase Measured
by the Acholest and Spectrophotometric Methods . . . 122
51. Storage Conditions of Human Blood Samples for
Measuring Cholinesterase Activity 123
52. List of Organophosphorus Pesticides Determined by
Thin-layer Densitometry 124
53. R- Values of Organophosphorus Pesticides 125
54. Linearity Ranges and Weighted Variances for the
Thin-layer Chromatographic Determination of Some
Organophosphorus Pesticides 126
XI
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ACKNOWLEDGMENTS
The work presented in this report was performed by the
following staff of the Institute for Medical Research and
Occupational Health (Zagreb):
Senior scientists: R. Plestina (M.D., Ph. D.), V. Simeon
(Ph. D.), M. Skrinjaric-Spoljar (Ph. D.), Z. Stefanac (Ph. D.)
and K. Wilhelm (Ph. D.)
Junior scientists: V- Bradamante-Simic (M.D., M.S.),
K. Fink (B. S.), Dj. Kobrehel (M.S.), B. Krauthacker (M.S.),
Dj. Mrvos (B.S.), B. Radic (M.S.), M. Stipcevic (B.S.),
Z. Vasilic (B.S.) and S. Zima (M.S.)
Technicians: A. Buntic, A. Cernik, T. Fajdetic, J. Kapetan,
M. Kraij, M. Matasin, B. Tkalcevic and B. Volaric
Consultants from two other institutions also participated
in the work: V. Turko (M.D.) from the Chemical works "Chromos"
(Zagreb), and S. Horvat (M.D.), I. Ledic (M.D., Ph. D.),
M. Pejic (M.D.) and M. Piukovic-Plestina (M.D.) from the
Department of Ophthalmology, University Hospital (Zagreb).
XII
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SECTION 1
INTRODUCTION
Anticholinesterase agents are being extensively used as
pesticides in agriculture, industry and for human health on a
worldwide basis. The results presented in this report have been
obtained over a period of four years by the team of workers
listed in Acknowledgments. The report is divided into sections,
each of which is written as a self-contained entity except for
the references which follow the last section.
Sections 2 and 3 contain the main conclusions and recommend-
ations for further studies.
Sections 4 through 7 report results of research on the effect of
pesticides on humans. Section 8 describes in vivo studies on
animal models while Sections 9 and 10 summarize the in vitro
studies on enzyme mechanisms. Sections 11 and 12 deal with
analytical chemistry and biochemistry methods for determining
the effect of pesticides. Appendices A and B reflect two
subjects not included in the EPA program, but which are related
to its subject; An international meeting on cholinesterases
and cholinergic receptors, organized in 1975, is discussed in
Appendix A. Appendix B presents summaries of eight B.S. and M.S.
theses related to the present report which were supervised by
the senior members of our group; this activity arises from the
fact that the Institute for Medical Research and Occupational
Health is also a teaching institution of the University of
Zagreb. Finally, the Reference Section gives a list of publica-
tions pertinent to the subject of this report including our own
which have been published since 1972. The list includes all
of our publications up to June 1976, but does not include
manuscripts submitted for publication.
Several studies described in this report are a continuation
of work started before 1972. Those studies described in
Sections 5, 6, and 7 were initiated in cooperation with EPA and
are now included in the Institute's long term research program.
These studies also reflect the interest of the World Health
Organization, of which organization this Institute is a
Collaborating Center for Pesticide Toxicology.
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SECTION 2
CONCLUSIONS
Human blood cholinesterase activity is a good indicator of
the degree of absorption of anticholinesterase compounds,
provided that preexposure or baseline enzyme activities were
determined in the same subject before as well as after exposure.
This was demonstrated in 485 individuals occupationally exposed
to pesticides. The mean preexposure activities of a given
population could not be taken as control values for an individual
or a particular subject because of individual variations in
cholinesterase activities. Signs and/or symptoms of poisoning
by organophosphate anticholinesterase agents were always
accompanied by inhibition of blood cholinesterase activity; the
reverse was not the case.
The presence of metabolites of organophosphate esters in
urine of occupationally exposed individuals is an adequate index
of exposure. In order to obtain direct evidence of pesticide
absorption, gas chromatographic methods were introduced for
determining pesticide residues in humans. From initial studies
of organophosphate residue analysis measurable amounts of a
phosalone metabolite were present in the urine of 12 out of 13
workers occupationally exposed to phosalone. Blood samples
from 147 individuals not exposed to pesticides all contained
p,p- DDE; five other chlorinated hydrocarbons were also found in
some of the blood samples.
Excessive exposure to pesticides over a period of several
years seems to cause some functional eye impairment; narrowing
of the visual field was more frequently observed in a group of
43 exposed workers than in a group of 20 nonexposed subjects.
The organophosphorus compound trichlorfon, which is a
pesticide and also an antiparasitic drug (Metrifonate ), causes
inhibition of cholinesterases when administered to humans or
animals. It was shown by in vitro and in_ vivo studies that
inhibition of the enzyme is not caused by trichlorfon itself,
but that it is due to dichlorvos formed from trichlorfon by a
nonenzymic rearrangement reaction.
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SECTION 3
RECOMMENDATIONS
In subjects occupationally exposed to anticholinesterase
pesticides, blood cholinesterase activities should be measured
at regular time intervals in order to establish whether or not
hazardous absorption had occurred and to monitor exposure. The
colorimetric method developed by Ellman et al. (1961) is
recommended as the most suitable for measuring cholinesterase
activities; the method should be further developed and made
applicable for field uses. In order to obtain direct evidence
of pesticide exposure, residue analysis should be carried out;
the available analytical methods for determining residues of
cholinesterase inhibitors in humans need to be improved.
There is a need for a better understanding of the mechanism
of action of chlorinated hydrocarbons. So far, direct measure-
ment of residues in humans is the only means bytwhich absorption
is determined. Studies on the body burden of chlorinated
hydrocarbons, and its relation to possible lesions, should be
continued.
In individuals chronically exposed to pesticides, ophtha-
mological studies are required to establish whether or not the
observed functional eye impairment is due to pesticides.
Measurements of blood vitamin A levels should also be carried
out.
Neurophysiological and behavioral studies should be initi-
ated in people occupationally exposed to pesticides in order to
establish whether or not pesticides diminish human functional
capabilities.
Studies of the mechanism of action of pesticides on a
molecular level should continue in order to gain a better under-
standing of their effects on humans.
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SECTION 4
BLOOD CHOLINESTERASE ACTIVITIES IN THE GENERAL POPULATION
AND IN WORKERS EXPOSED TO PESTICIDES
The need for effective pest control is well recognized.
Pesticides are therefore necessary, and their economic value and
efficacy in agriculture, industry, and disease prevention are
unquestioned.
The acute toxicity of most organophosphorus esters used as
insecticides is mainly, if not entirely, due to their inhibition
of cholinesterases. As a result of cholinesterase inhibition
in the nervous system, acetylcholine molecules accumulate at the
synapse, initially causing excessive excitation and later leading
to the blockage of synaptic transmission (Holmstedt 1959). At
present, the only practical way to evaluate the hazards of over-
exposure to anticholinesterase pesticides is to measure the
blood cholinesterase activities in exposed people. In order to
determine the condition which precedes clinical poisoning, an
assessment was made of cholinesterase activities in the blood of
workers employed in the production and application of pesticides.
An attempt was made to correlate the activity of this enzyme with
exposure and with the cholinergic signs and symptoms of poisoning.
The knowledge of the normal baseline blood cholinesterase
activity in the human, and its variation is a necessary qualifi-
cation for an interpretation of the results. Many investigations
have been undertaken to survey blood cholinesterase activities
of normal populations, that is, of groups of individuals that
have not been exposed to cholinesterase inhibitors. Data in the
literature were comprehensively reviewed by Galloway et al.
(1951), Kane (1958), Augustinsson (1955) and Gage (1967). Indi-
vidual results were published by Humiston and Wright (1967) and
Voss and Sachsse (1970) . The results can be compared only by
calculation of the coefficient of variation, because the activities
are expressed in different units according to the method of assay.
The assay of blood cholinesterase activity is fairly simple
(Witter, 1963; Augustinsson 1971) and has been used for a
number of years to assess the extent of human exposure (Hayes
1971; Sandifer et al. 1972; Warnick and Carter 1972). Such
measurements provide a valuable confirmation of the diagnosis of
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organophosphorus poisoning, since the early signs of intoxication
with these substances are rather nonspecific. These measurements
also permit the process of recovery to be followed, and support
the decision of whether or not the patient is fit to return to
work. Also, regular determination of blood cholinesterase may
act as an early warning system for the recognition of excessive
exposure.
Material and methods
Occupationally exposed workers were almost simultaneously
exposed to various organophosphorus or carbamate esters as
active substances. The list of compounds is given in Table 1.
The measurements of blood cholinesterase activity were
performed in the laboratory, while the sampling was done either
in the laboratory or in the field in which case the samples
were kept cool and transferred to the laboratory- Cholinesterase
activity was measured by the spectrophotometric method of
Ellman et al. (1961) as modified for blood and plasma (Wilhelm
1968) .
The reaction mixture contained 3.0 ml of phosphate buffer
(0.1 M, pH 7.4), 100 yl of the thiol reagent (0.01 M 5,5-
dithiobis-2-nitrobenzoic acid) and enzyme preparation (20 yl of
plasma or 5 yl of whole blood). When the substrate was added
(50 yl acetylthiocholine, final concentration 1.0 mM), the
absorbance was read in 1.0 cm cuvettes at 412 rim in the first
and third minute after the addition of substrate, against the
blank, which contained no substrate.
Cholinesterase activity was expressed in ymoles of
thiocholine per min per ml, or in arbitrary units as A absorbance
per min per ml of whole blood or plasma.
Under the conditions described, the activity of whole blood
cholinesterase measured by this method is primarily due to
the activity of erythrocyte cholinesterase. If it is assumed
that the hematocrit is 50%, erythrocyte cholinesterase contri-
butes 92% and plasma cholinesterase only 8% to the total whole
blood activity in uninhibited samples (Wilhelm et al. 1973).
Whenever possible measurements of enzyme activity were
performed before exposure and these values were taken as 100%
in relation to activity determined after exposure. If that
was not possible, 100% cholinesterase activity was determined
much later, i.e. 2-3 months after exposure to any anticholin-
esterase compound. The value so obtained was then considered
as the control value.
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Blood samples were taken by finger-prick. Special care
was taken to remove the insecticide from the skin by washing the
hands with soap and water and rubbing the finger tip thoroughly
with cotton-wool soaked in ethanol.
For determining plasma cholinesterase activity samples
were collected in dry, heparinized, glass capillaries as
described by Stubbs and Fales (I960) . Plasma was separated by
centrifuging the capillaries for 10 min. When necessary, the
nondiluted :.Dimples were transferred to glass containers and kept
in a vacuum flask with ice.
For determining whole blood cholinesterase activity 20 yl
of blood was collected in the laboratory or in the field, in
Sahli pipettes which had been heparinized by rinsing with a 2%
heparin solution immediately before the collection of samples.
If necessary, the blood samples were then transferred, without
dilution, into a glass container which was kept in a vacuum
flask with ice. The same was done with the capillaries contain-
ing plasma.
The blood samples were collected from industrial workers
at intervals which depended on the rate of production. When
the production rate was very high, the sampling was done every
second week.
In workers applying anticholinesterase insecticides in
farms or orchards, blood cholinesterase activities were
measured monthly: before, in the middle, and at the end of
the working season.
Clinical Examinations3 Blood Analyses
Safety-at-work regulations in Yugoslavia require that all
workers employed in the production of pesticides submit to a
continuous health control. This is accomplished in the factory
health unit which is headed by a specialist in occupational
medicine. Health control includes physical examinations twice a
year. From the available medical records a picture of the
general health condition of workers was formed.
The workers engaged in pesticide application are not
medically surveyed continuously because of the seasonal
character of their work. The general condition of their health
was estimated from anamnestic data recorded at the beginning
of a working season.
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RESULTS AND DISCUSSION
Variation in Enzyme Activity within a Population
For the study of the distribution of normal cholinesterase
activities, blood samples were taken from 408 healthy individuals
with no previous exposure to anticholinesterase compounds. Many
of them were later employed in the production and/or application
of anticholinesterase insecticides so that their preexposure
values served as control. The testing was done over a
period of two years. In this way the distribution included
possible external factors which might have influenced normal
values.
The distribution of plasma and whole blood cholinesterase
activity is shown in Figure 1. Enzyme activity is expressed
in ymoles of thiocholine per min per ml, and ranged from 0.27
to 2.6 for plasma, and from 2.7 to 7.7 for whole blood cholin-
esterase. The estimated means were 1.3 and 5.1 respectively.
There is a slight overlap between the highest values of
cholinesterase activities in the plasma and the lowest values
in whole blood. Whole blood cholinesterase activities are
distributed over a much wider range than those in the plasma.
In order to compare our results with those of other invest-
igators who used different techniques, we calculated the results
with respect to the coefficients of variation (S.D. x 100/Mean).
These were 18% for erythrocyte and 25% for plasma cholinesterase.
This is in accordance with the results of other authors, who
report coefficients of 10-15% for erythrocyte and 15-25% for
plasma cholinesterase (Augustinsson 1955; Gage 1967; Voss and
Sachsse 1970).
Figure 2 shows that the distribution of values of plasma
and whole blood cholinesterase activity for males differs from
that for females. The difference between the means of the
male and female groups is statistically significant for
t = 4.62. The difference in the activity of the whole blood
cholinesterase is probably caused by the difference in the
hematocrit values which is due to sex (Augustinsson 1955).
To find out whether the activities of the two enzymes in
man are mutually dependent we statistically analysed the values
of the normal distribution.
The whole blood cholinesterase activity (y) was found to
correlate well with the serum cholinesterase activity (x).
The relationship between the two"activities can be described by
the empirical equation y = (13.0 ± 0.7) + (1.5 ± 0.1)x. This
correlation was found valid for 400 samples.
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The precision of the method was tested on a set of samples
by two subsequent measurements of the activity of individual
samples. The correlation between two pairs of measurements
was r = 0.99 for plasma cholinesterase and r = 0.91 for the
whole blood cholinesterase activities, such a high correlation
permits a precise determination of sample activity by only one
measurement.
From the results of other authors as well as from these
reported it is evident that the measurements of preexposure
enzyme values are indispensable before an individual is allowed
to come into contact with anticholinesterase compounds. Because
of a very wide range of normal activities great errors are
possible in evaluating to what extent, if any, the examined
person was exposed to an anticholinesterase compound. A subject
who normally has a high average cholinesterase value might
suffer a reduction which is due to his occupation, and yet this
might not be recognized if the measurement referred to the
population distribution, as it might well fall within the
normal range of values. Conversely, the measurements made on an
individual not occupationally exposed but with a genetically
controlled atypical low plasma cholinesterase activity, Goedde
et al. (1967) might give rise to unnecessary concern.
Variation in Enzyme Activity within an Individual
The degree of individual variation was studied in 34
subjects by simultaneous measurements of cholinesterase
activities in plasma and whole blood. The successive measure-
ments took place over a period of 15 months. They were per-
formed in healthy adults selected among factory, office and
research workers with no previous exposure to anticholinesterase
compounds.
The measurements were carried out under conditions as
identical as possible. All testing (sampling and measuring) was
done by the same person using the same equipment. The factors
of variation were 5.95 for the activity of whole blood chol-
inesterase and 8.77 for the activity of plasma cholinesterase.
Cholinesterase activity measurements were carried out
over the period of 15 months. The temperature of the buffer
ranged from 18 to 25°C. For the temperature correction factors
the data published by Reiner et al. (1974) were used. Coef-
ficients of variation agree with the data of other authors;
these were 7.2 for whole blood cholinesterase and 11.9 for
plasma cholinesterase activities.
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The number of individuals in each of series of measurements
varied between 28-34 except in the last measurement when only
five subjects were tested.
Some of the individual curves show a slow progressive
decline of activity towards the summer months, but in most of
them this is interrupted by more abrupt variations. The data
were analyzed to ascertain whether the differences between the
results in successive measurements were lesser than predicted
from the overall intra-individual standard deviation; a slight
reduction was found in the order of ten percent. The differences
between successive determinations in some cases exceeded 20
percent. The same differences were observed by other authors
(Fryer et al. 1955; Gage 1955). All subjects were in good
health and it was unlikely that they were suffering from un-
suspected disease and disorders which might have affected blood
cholinesterase activity.
Enzyme Activity -in Workers from Pesticide Manufacture
Two groups of exposed workers were used in the study. The
first group were workers from a manufacturer of anticholin-
esterase pesticides, and the second, workers from a pesticide
application service.
A total of 356 industrial workers who worked in three
different production lines in one plant were followed: 157
were engaged in formulating insecticidal dust or wettable
powder, 136 formulated emulsions and 41 persons worked in the
production of household sprays. In addition, 22 persons working
in the laboratory were also studied.
The workers were aged 21-60 years. Only 85 were continuously
employed in the production of pesticides for a number of years
(2-10) . The others were seasonal workers employed only during
the periods of intense production.
The normal workday was eight hours, but periodically,
because of intense production, overtime, up to 12 hours, was
worked. The process of production in the three lines differs,
so that the type of exposure was also different.
The production of emulsions involves a complex technological
process, and there is a constant danger of workers coming in
contact with active substances or final products, particularly
because the air is contaminated with solvent vapors and active
insecticidal substances. Workers have no fixed place, but move
about as they work. In this study, the workers were most
frequently exposed to phosalone, dimethoate, and dichlorvos.
-------
The formulation of insecticidal dust is automated but in
spite of that workers are exposed to pesticides. Active in-
gredients as a dust can settle onto their hands, clothes, and
hair causing increased exposure. The compounds formulated were
mainly monomethyl carbamates: dioxacarb and carbaryl and only
occasionally, and then for very short time intervals, the organo-
phosphorus compound, phosalone.
In the manufacture of household sprays, dichlorvos and
bromophos we^e produced. The workers handled very low con-
centrations of active substances, and exposure was relatively
low.
In another plant, 83 workers were engaged in the production
of pesticides containing thiometon or dichlorvos as the active
material. Fifty-eight workers were employed continuously for
five or more years while the remaining 28 seasonal workers
worked only for about a three month period during peak produc-
tion. In this plant, the same workers were involved in both
the production of emulsions and the manufacture of household
sprays. Work conditions did not differ from those above.
The demands for insecticides imposed by the market,
especially in spring and summer months, have resulted in an
increase in production. Therefore, in the first, six months
of the year workers are more heavily exposed to organophosphorus
and carbamate insecticides.
Tables 2, 3, and 4 show the average number of days of
exposure and a list of active insecticidal substances to which
a particular worker was exposed in the period from 1971 until
1975. Days of exposure mostly relate to the first half of
the year.
Table 5 shows the average number of overtime work hours
for each worker in the production of emulsions in 1971 and 1972.
The results of measurements of cholinesterase activity
in the blood in exposed workers from all the three production
lines are given in Table 6. The symptoms of poisoning are
recorded with relative enzyme activities.
In order to establish a relationship between exposure
and enzyme activity, the data of the group of workers involved
in emulsion production were analyzed over several years.
Figure 3 shows the mean values of cholinesterase activity in
the blood and plasma for the whole group (N = 11), and also
the duration of exposure between measurements for each worker.
10
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The production Of organophosphorus insecticides increased
between 1971 and 1975 as shown in Tables 2, 3, and 4. We
expected that with the increase in exposure, the number of
blood samples with enzyme activities less than 75% would also
increase. However, Table 6 shows that the greatest number of
blood samples with reduced cholinesterase activity was recorded
in 1971. The accompanying symptoms or signs of poisoning
were also most numerous in the same year.
In 1971 the workers were exposed to and handled mainly
dimetl>oate and chlorfenvinphos, both very dangerous insecticides
which are extremely toxic to mammals. Certain technological
procedures and working conditions provided inadequate protection,
and in addition there was a great deal of overtime work during
the peak of the season (Table 5). Twenty-four workers had some
complaints, and after the cases of poisoning were analysed
working conditions were considerably improved before the 1972
season. Therefore, although the extent of work remained the
same (Tables 2, 3, 4, and Figure 3) there were no cases of
occupational poisoning in 1972, except for one accidental in-
toxication at work.
In 1973 the situation, as noted in 1972, did not change.
Only two exposed workers had blood cholinesterase activities
below 50% which is below the limit for occupational exposure.
They were removed from further exposure. Four workers with
moderate complaints underwent a systematic medical examination.
Their blood cholinesterase activity was above 80% of the normal
value and no signs of poisoning were found.
In the past two years (1974-1975) there has been an in-
crease in the production of insecticides. The number of season-
al workers with no previous experience with insecticides also
increased. From the results presented in Table 6, the number
of reduced enzyme activities again seems to have become higher.
If, however, the accompanying symptoms observed in some workers
are expressed in percent, they are 29% in 1971, 0.95% in 1972,
9% in 1973, 17% in 1974, and 12% in 1975.
Table 7 related to the poisoning of a few workers during
the period of observation. The poisonings were recorded
mostly in 1971 (N = 7). Only those workers are listed for
whom it was possible to ascertain that the accompanying symptoms
were due to excessive exposure to anticholinesterase compounds.
After a two week removal from the place of exposure cholinesterase
activity increased in all these workers. The workers were
exposed to various organophosphorus or even carbamate insecti-
cides so that the spontaneous reactivation of the inhibited
enzyme was not identical in all workers.
11
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It is seen in Table 1 that the workers were .exposed to
organophosphorus compounds containing dimethoxy or a diethoxy
radicals. The acetylcholinesterase of human erythrocytes
inhibited by a dimethoxy organophosphorus compound is known
to reactivate quickly (t, = 51 min) while for the plasma
cholinesterase inhibited by the same group of organophosphorus
compounds the half time reactivation is >^ 83 hours (Skrinjarid
et al. 1973); erythrocyte acetylchoiinesterase inhibited by
diethyl organophosphorus compounds also reactivates faster
(t, = 2.4 days) than plasma cholinesterase (t^ = 30 days)
(Reiner 1971). Further, most organophosphorus insecticides
inhibit, to a greater extent, plasma cholinesterase (Simeon and
Reiner 1973). The emulsion-production workers had a lowered
plasma cholinesterase activity in most measurements which is
in accordance with the literature data about the slow spon-
taneous reactivation and higher inhibition of serum cholin-
esterase. Still, we did not consider this as too important, for
plasma cholinesterase can be highly inhibited without any
consequence to health. In workers exposed to dimethoxy organo-
phosphorus compounds such as dichlorvos, dimethoate and others,
the phenomenon of aging of the inhibited human erythrocyte
cholinesterase, should be borne in mind since it occurs
relatively fast (half-time of aging about four hours) in
contrast to diethyl organophosphorus compounds with a half-time
of aging of about 40 hours (Aldridge and Reiner 1972). The
aged cholinesterase is irreversibly inhibited and cannot be
reactivated even by oximes.
In workers exposed to carbamate insecticides, it was
difficult to assess the actual degree of exposure because of
the already well known property of carbamylated cholinesterase
to reactivate spontaneously in a very short time (Wilson et al.
1960; Reiner 1971). Reiner and Aldridge (1967) showed that the
half-time of reactivation for monomethyl carbamates is 30 minutes,
for dimethyl carbamates only 76 minutes if the cholinesterase
of purified bovine erythrocytes was carbamylated. Some of the
workers from the production of insecticidal powder had blood
cholinesterase activities less than 60% at the time of sampling,
but a 24-hour interruption of exposure was sufficient for cholin-
esterase activity to return to normal. Since the duration of
exposure had no lasting effect on cholinesterase activity and on
workers' health, the cholinesterase activities in these workers
were monitored only 2-3 times a year, during the peak of produc-
tion.
In order to determine the degree of exposure in the course
of one working day, we measured blood and plasma cholinesterase
activities in workers before and after the work shift. The
results are presented in Table 8. In ten workers from the group
engaged in the formulation and packing of phosalone, cholin-
esterase activity was measured at 6 a.m. and at 1 p.m. After
12
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cessation of exposure in five workers the measured blood cholines-
terase activity was the same, while in the other five it was
reduced by 6-10%. Plasma cholinesterase activity in these workers
was reduced by 10-15%.
In another pesticide formulating'plant, workers were engaged
in the production of pesticides containing thiometon or dichlor-
vos. Since for most of the workers no preexposure values were
recorded, it was not possible to calculate the actual degree of
cholinesterase inhibition. However, in comparison with the normal
values determined by the same method in nonexposed workers
enzyme activities were sometimes low. Only the tendency of a
further recovery or diminution of enzyme activity could be
registered. (
)
The results of measurements of enzymes activities in the
workers whose preexposure values were taken are presented in
Table 9. The mean activities of a group of workers engaged in
the formulation of thiometon or dichlorvos are given for a
period of six years. Very often enzyme activities were low.
Almost always these low values were accompanied by symptoms of
poisoning such as headache, fatigue, giddiness and sweating.
Workers Employed in the Application of Insecticides in
Agriculture
To assess the extent of exposure in workers who apply
anticholinesterase insecticides, we chose 129 agricultural
workers engaged in orcharding (41) or farming (88) . Their
jobs are seasonal and last usually about two months. The
working conditions were different from those of industrial
workers described earlier. These individuals work in the open
where the possibility of contamination is smaller. Also, they
come into contact with formulated or finished products and not
with the active pesticide ingredient. However, the exposure can
increase due to windy weather or high temperature.
The workers who sprayed farms were exposed mainly to 5%
phorate, and to a lesser extent to diazinon, methidathion
fenthion, and parathion. The exposure in 1973 was about 30
working days over a period of two months. In 1974 and 1975
the average exposure was 16 days.
After recording preexposure values for each work year in
the course of the working season, in 1973, cholinesterase acti-
vity was measured twice, while in the following years enzyme
activity was determined only once during the exposure period.
The results of measurements are given in Table 10. In the first
year of testing, the group as a whole did not show very reduced
13
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mean values of cholinesterase activity. The lowest blood cho-
linesterase activity was 84% of the preexposure value.
In the second year, of 44 tested workers, six had blood
cholinesterase activity between 60% and 70%, six between 70%
and 80% and the rest over 80%. This decrease indicated that
the workers absorbed a certain amount of organophosphorus
insecticides in the course of work.
The group of orchard sprayrren was exposed to a greater
variety of compounds. In one farm in 1973 phorate (5%),
parathion (46.5%), diazinon (20%) and fenthion (10%) were
applied. The results are given in Table 11. Work conditions
were poor - the work was not mechanized and protective devices
were used infrequently. The exposure to anticholinesterase
compounds was about 60 days over four months. The measurements
were done after approximately 20 days of exposure. Cholines-
terase activities showed no significant trend to decrease,
although the lowest activity was obtained in the third measure-
ment (Mean = 86% for blood cholinesterase and 75% for plasma
cholinesterase). In 1975, the exposure of people employed in
another orchard which used Azinphos-ethyl (25%), phosalone (30%)
and carbaryl (50%) as insecticides was followed. The agricul-
tural work was completely mechanized except in places where the
mixing and diluting of the compounds took place. The workers
were exposed to anticholinesterase compounds continuously for
only two to three days with fortnight breaks. Table 11 shows
that their cholinesterase was practically undiminished, par-
ticularly when data are compared with the former group.
The exposure of workers employed in the agricultural
application of insecticides is essentially different from
that of industrial workers. Work is done in the open with
interruptions, and finished products are used.
Four of eight examined workers employed in the orchard
spraying (1973) showed and reported some signs and symptoms of
poisoning, such as sweating, headache, and nausea. The same
symptoms were observed in farm spraymen whose cholinesterase
activity was below 70%. Although the symptoms were not typical
cholinergic symptoms, it was concluded they were due to the
absorption of a certain amount of anticholinesterase compound.
The workers were in good health and the symptoms cannot be
ascribed to any other cause.
Protection at Work
As protective clothing, industrial workers wore only canvas
clothes, rubber gloves and rubber shoes. For face and eye pro-
tection they occasionally used face shields and goggles, but no
respirators or vapor filters.
14
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Agricultural workers wore only protective clothing, and
periodically gloves and respirators. In some orchards the
spraying operation was highly mechanized. However, neither
the drivers nor the mixers observed protective measures such
as wearing gloves or a respirator. The mixing of concentrate
was done in a most inappropriate way.
Blood Picture and Exposure to Organophosphorus Compounds
According to literature data (Davignon et al., 1965;
Paccaganella et al., 1971) it was expected that increased
exposure of workers employed in the production of organo-
phosphorus insecticides would reduce the total number of
leukocytes, and increase the number of lymphocytes in response
to a possible allergic reaction of the worker to the insecti-
cide in question.
In nine workers who worked in a pesticide industry for a
number of years a differential blood analysis was made. In
addition, the total number of leukocytes and the number of
lymphocytes per 100 leukocytes was measured in the course of
their work with anticholinesterase insecticides. During intense
exposure to these compounds (1970-1974) 53 analyses were carried
out. In the same workers, 50 further analyses were done between
the years 1966 and 1969 when they were not in contact with
anticholinesterases. Ninety-seven differential blood analyses
were done in 48 transportation workers who never had been in
contact with pesticides.
The results are shown in Table 12. No significant differences
in the same workers were noted in the period before and after
exposure to organophosphorus compounds. Also, no differences were
observed between the exposed group and the control group. The
results agree with those of Warnick and Carter (1972) , and
Ensberg et al. (1974), but do not agree with those of Davignon
et al. (1965) and Paccaganella et al. (1971).
SUMMARY
Occupationally-exposed individuals who work in the formu-
lation of anticholinesterase insecticides are exposed to high
concentrations of active substances at their place of work.
Working in a confined space, periodically intense production,
and difficult working conditions all contribute to excessive
absorption of these compounds. By the use of prescribed pro-
tective devices at work, and observing the hygienic and sanitary
regulations, workers can achieve a satisfactory degree of
protection and thus reduce the possibility of contact with
anticholinesterase insecticides.
15
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Regular measurement of cholinesterase activity in these
workers in the course of the work season, i.e., once or twice
in a fortnight during the period of intense production, is
presently the only suitable method for the determination of the
degree of absorption of an anticholinesterase insecticide at
work. With this method one can learn in time whether there are
workers at risk whose enzyme activity is lower than their pre-
exposure value, and thus prevent the danger of further absorption,
In all workers under study a significant drop in blood cholines-
terase activities could always be associated with excessive
exposure due to overtime work and/or lack of protective measures.
However, this does not apply vice versa, i.e., excessive contacts
with anticholinesterases did not always lead to a significant
drop in blood cholinesterase activities. Without exception,
workers showing cholinergic symptoms had their blood cholines-
terase inhibited equal to or greater than 50%. However, con -
plaints from workers were not always related to cholinesterase
inhibition.
Working conditions of those who apply insecticides in agri-
culture are different from the conditions in insecticide produc-
tion. Working in the open is an important factor which helped to
reduce the absorption of anticholinesterase insecticides, and
only bad atmospheric conditions, uncontrolled handling of these
final products, and poor protection at work could result in
excessive absorption. Treating the workers as a group, we did
not find more than about 10% cholinesterase inhibition, although
a few individuals showed inhibition up to 40% and these also had
subjective complaints. In workers exposed to the production of
organophosphorus insecticides, the analysis of differential blood
pictures revealed no change in the leukocyte count nor in the
number of lymphocytes. We believe, therefore, that these com-
pounds have no toxic effect on the blood of exposed workers.
Since enzyme activities vary within a population as well as
within an individual, it is important to establish normal values
for a given population. Plasma and whole blood cholinesterase
activity was determined by a spectrophotometric method in a
group of 408 healthy human subjects (257 males and 151 females)
with no previous exposure to anticholinesterase compounds. The
results of enzyme activities (expressed in ymoles of thiocholine/
min/ml) were distributed over a comparatively large range:
0.35 - 2.45 for plasma, and 2.09 - 8.05 for blood cholinesterase,
with a mean of 1.34 and 5.17 respectively.
Variations within an individual were assessed in 34 subjects
(14 males and 20 females) by measuring cholinesterase activity
in plasma and whole blood every 4-5 weeks over a period of 15
months. The cholinesterase activity fluctuated during that
period with the mean coefficient of variation of 11.9 for plasma
and 7-2 for whole blood. In the summer months, the cholines-
terase activities showed a slight tendency to decrease.
16
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SECTION 5
EFFECT OF PESTICIDES ON EYE AND SIGHT
This section contains two parts: a review of the literature
and the results of current investigations.
The literature review, while not a critical review, was
intended to collect all available information pertinent to the
topic. Conclusions were taken from authors' summaries or
abstracts as published largely in tertiary publications. The
source of information is always quoted as the primary reference
with a notation of secondary or tertiary sources as needed.
In order to make the review as comprehensive as possible, some
references are inserted which are not discussed. Those which
had a very general abstract are marked with an asterisk. When
the same results were published in more than one paper, only
one paper was reviewed; references to the other papers are
marked with an asterisk. Papers which were not accessible,
even as a summary or abstract, are marked with two asterisks.
REVIEW OF THE LITERATURE
For the most part, knowledge of the biological effects of
acute exposure to pesticides has been well documented. The
same cannot be said for the effects of chronic exposure. At
one period it was assumed that the only threat from pesticides
was through by their acute toxic effects. Recently, however, a
number of authors suggest different nonspecific effects of vari-
ous pesticides due to chronic exposure including anticholine-
sterases.
There is a vast quantity of literature on the various effects
of pesticides in experimental animals, many of which have not
been confirmed in humans. For this reason, increased emphasis
on research using human subjects has been recommended by many
researchers, organizations, and government agencies (U.S.
Department of Health, Education and Welfare 1969).
Among many untoward effects of pesticides used in public
health programs and in agriculture, visual impairment has
very recently begun to receive more attention. Since the begin-
ning of this decade a number of reports have appeared primarily
in the Japanese literature describing a visual impairment in
17
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people, including occupationally exposed, the general population,
and children in particular.
Records indicate that Japan is one of the most polluted
countries in the world (Ui, 1972) with one of the world's highest
utilization rates of organophosphorus pesticides per unit area
(Kogure and Imai, 1974) and a corresponding large number of
yearly poisonings. A decade ago Dr. Ron Ohto noticed an unusual
eye disease in children and adults in ti.e Saku locale, Negano
Prefecture, in Central Japan. The disease became known as the
Saku disease. The incidence of the disease increased at a high
rate in many other regions in Japan (Ishikawa and Ohto 1972).
The same or a similar disease, however, as a special nosologic
entity, has not been recognized in other parts of the world.
The first systemic investigations were started in 1969 by
Ishikawa and his coworkers (Ishikawa et al. 1970; Ishikawa 1971a).
From ocular and general signs of poisoning or exposure, Ishikawa
concluded that the Saku disease was due to chronic exposure to
organophosphorus pesticides (Ishikawa 1973a). The figures of
eye impairment, as "one of the most consistent symptoms of
chronic poisoning in people living in agricultural areas,"
gradually became very striking (Uono 1972; Kogure and Imai 1974).
On the basis of an official report submitted to the Japanese
Government, the use of parathion, EPN, TEPP and malathion was
banned and their replacement by less dangerous pesticides among
organophosphorus compounds was encouraged. Ishikawa (1974)
extensively summarized the results of investigations in the Saku
region, tabulating diagnostic criteria, ocular findings, blood
cholinesterase activities and residues of organophosphorus com-
pounds, both in exposed people andicontrol subjects.
Standards for ocular anomalies attributed to organophosphorus
pesticides (Kato 1972) set up by the Japanese Ministry of Public
Welfare include: impaired vision (less than 1.0 after correc-
tion) , visual field stenosis (less than 40 degrees nasal and
less than 60 degrees temporal), progressive myopia, serious
astigmatism (difference between horizontal and vertical radius
larger than 2.0 D), impaired sense of balance (standing time on
one leg less than five seconds), effective cholinesterase reacti-
vator (2-PAM or Padrin) therapy, lowered cholinesterase activity,
and edema and atrophy of the optic nerve (pale in color in the
temporal side of papilla). If five out of the above eight
conditions were positive the diagnosis of the eye damage by
organophosphorus compounds was made (Ishikawa 1972a, 1972b).
The suggestion that the etiology of the disease was asso-
ciated with exposure to organophosphorus pesticides was taken
for granted. As such, some clinicians started to diagnose every
obscure eye impairment in violated areas by measuring blood
cholinesterase activity (Saito and Kubo 1972). Moreover, the
therapy was established along these lines, and patients were
-------
treated with oximes and atropine for many months (Ichii and
Kimata 1973; Ohto 1974; Ozawa et al. 1972). The improvement of
the clinical picture following the oxime therapy is sometimes
regarded as confirming a diagnosis of chronic poisoning with
anticholinesterase (Ohto 1971). Ooishi (1974), however, warns
that unless the pollutant is definitely known, ophthalmologists
should not place a diagnosis such as postbulbar neuritis or toxic
amblyopia, after only a few examinations.
Some authors suggest that organophosphorus compunds might
affect other sensory systems apart from vision. Ozawa et al.
(1975) showed that in patients with chronic organophosphorus
intoxication, the sense of balance can also be disturbed.
Besides eye abnormalities Kwalick (1971) found a significant
difference in the number of individuals with hearing problems
comparing pesticide-exposed subjects with controls. Fuji et al.
(1975) reported on distrubed vestibular function in addition to
other nervous symptoms and visual impairment in subjects who
were in contact with pesticides.
Ep-idemi-ological Studies
Epidemiological studies were conducted on occupationally
exposed workers and on the general population, mostly children,
living in the areas where insecticides were used extensively.
An extensive retrospective investigation, conducted in several
areas in Japan, analyzed the relationship between the use of
organophosphorus compounds and incidence of myopia in
approximately 40,000 school children over a period of 16 years
(Tamura and Mitsui, 1975). A positive correlation was found
between the amount of organophosphorus pesticides used in a
specific year and the incidence of myopia in the following year.
A monitoring of the refractive condition of the eyes revealed
that the subjects from the city of Saku showed a tendency to
become myopic (Otsuka et al. 1972). The axial length of the eye
of the Saku subjects was inclined to be shorter than that of the
controls. It was found that the lower the serum cholinesterase
activity, the greater the axial length. However, there was no
significant correlation between the amount of organophosphorus
residues in the sera and the axial length of the eye. Develop-
ment of myopia caused by chronic organophosphate pesticide
intoxication has been reviewed recently (Ishikawa 1976).
A large ophthalmological trial in which visual field
screening included 13,351 school children living in the region
where insecticides were extensively used revealed visual abnor-
malities in 32 children. In only two cases no explanation for
the abnormality was found (Futenma 1973). According to the
investigation of a large number of school boys, the relationship
between the symptoms of ocular anomalies (set in diagnostic
19
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standards for ocular anomalies attributable to organophosphorus
pesticides) and the extent of exposure was so obscure to be
almost irrelevant (Kato 1972). Also in a much larger study on
30,000 pupils, the results did not prove any statistically signi-
ficant difference in ophthalmological findings between the rural
and urban population (Kato 1973).
However, eye abnormalities in school children living in
violated agricultural areas were observed by other authors. In
an ophthalmological survey on 131 nursery school children living
in an agricultural area where organophosphorus insecticides were
repeatedly detected in appreciable amounts from house dust,
Mitsui (1972) found abnormalities in eyesight. This included:
refraction in 19, narrowing Of the visual fields in 26 and
abnormal eyeground in 8 subjects. Tsukahara (1972) examined 58
children, 18 of whom were chronically exposed to pesticides.
In these children visual acuity could not be corrected with
lenses, and in most of them, papillae were slightly congested
or discolored. Electroretinography revealed no abnormalities.
Among one hundred randomly selected school children between
eight and nine years of age, five to seven percent was found to
have ocular symptoms, either ocular neuritis, constricted visual
fields or astigmatism (Ishikawa et al. 1971). In almpst all of
the twenty-two children (six to twelve years of age) with eye
disorders attributed to the exposure to organophosphorus corn-
ponds, ophthalmological examination revealed remarkable constric-
tion of visual fields (Takaku 1972).
In Japan and in other countries there were several retro-
spective studies in agricultural workers handling pesticides
for a various lengths of time. From ophthalmological examination
of 1,995 agricultural workers and 2,272 students of agricultural
technology, Dugelnij (1971) concluded that organophosphorus
insecticides, to which the workers were mostly exposed, produced
an alteration in the intraocular pressure and in some other
parameters. He pointed out several contraindications concerning
the eye and vision for the subjects who intended to work in dif-
ferent agricultural activities including the handling of agricul-
tural chemicals.
Ophthalmological examinations of 164 Japanese agricultural
workers, handling pesticides for over 20 years, revealed a
considerable number of functional eye impairments (Imaizume et
al. 1971). A similar examination was performed earlier in the
Soviet Union (Medvedovskaya 1967) in a total of 776 agricultural
workers divided into six groups according to the type of work.
The incidence of eye impairment was highest in group of 60
workers whose only job was to handle pesticides (44.8% versus
25.1 to 41.1% in other groups). In another study conducted by
the same author (Medvedovskaya and Golenka 1971) in 33 female
workers highly exposed to pesticides, eye disease was found in
20
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36% mainly located in the anterior portion of the eye. Refrac-
tion anomalies and degenerative alterations of the conjunctiva
(pinguecula and pterygium) were the most frequent findings. In
412 farmers handling chemical poisons, a high incidence of
corneal abnormalities and glaucoma was found (Zarya 1973) .
Nuritdinova (1973) also described an extremely high incidence of
the pathological eye findings among 240 occupationally exposed
workers. In another clinical trial on 683 occupationally exposed
subjects, eye abnormalities were found in 90% (Nuritdinova 1975).
The most frequent findings were distrophic changes of the iris
and narrowing of the visual fields. A variety of systemic, neu-
rological and ocular symptoms were reported for 51 adult patients
who complained of blurred vision (Ohto 1971). Ocular symptoms
included swollen disks, spasms of the ciliary muscle, cataract
and disturbance of the smooth pursuit motion of the eye. In the
60 heavily exposed pesticide workers, no biochemical abnormalities
were noted (Pietsch et al. 1972). Ophthalmological examinations
of these workers revealed a few cataracts and a high incidence •
of cortical wedging, as compared with unexposed subjects. Agri-
cultural workers handling chlorothene complained of epiphora and
photophobia and had very pronounced conjunctivitis (Sivitskaya
and Alekseiicuk 1973). On detailed examination, constricted
visual fields and decreased eye pressure were observed. The
workers handling thiram (tetramethylthiuram disulfide, TMTD) also
had, conjunctivitis, as well as slightly decreased eye pressure
and decreased corneal sensitivity (Sivitskaya 1974).
However the results were not positive in all epidemiological
investigations. A health survey of twelve male professional
pesticide applicators who worked with fenitrothion and dichlorvos
for two to nine years revealed no ophthalmological abnormalities
(Misumi et al 1975). In a retrospective study on 114 persons
who were obviously poisoned by organophorphorus insecticides
eight complained of sight impairment, six of them blaming insec-
ticides as the cause of their troubles (Tabershaw and Cooper
1966). However, the physician in charge thought it was of a
different etiology than organophosphate pesticide posioning or
exposure.
Complaints received from workers exposed to pesticides were
predominantly those of visual acuity disturbance, photophobia
and epiphora (Medvedovskaya and Golenko 1971; Imaizumi et al.
1971; Hanyu and Tsui 1974; Ito 1973). Ophthalmological exami-
nation of the frontal segment frequently revealed conjunctival
hypermia (Kalic-Filipovic et al. 1973; Sivitskaya 1974; Sivitskaya
and Alekseicuk 1973; Hanyu and Tsui 1974; Glazko 1970; Medved-
oskaya and Golenko 1971). Corneal ulcerations were also men-
tioned (Imaizumi and Atsumi 1971). In 23% of 200 pesticide
applicators a new formation of corneal vessels was found (Ito
1973). In these patients serum vitamin B2 was significantly lower,
21
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The occurrance of glaucoma was frequent among occupationally
exposed workers (Medvedovskaya 1967; Nuritdinova 1973; Zarya 1973;
Sivitskaya and Alekseicuk 1973). The narrowing of visual fields
the most frequently report abnormality, was first described by
Ishikawa (1971a) who found a concentric narrowing in all ex-
amined Saku children. Increased incidence of visual field
anomalies was reported as in occupationally exposed adults both
in Japan as well as in occupational.^ exposed adults both in
Japan and in some other countries (Ishikawa et al. 1970, 1971;
Imaizumi et al. 1971; Nekamura et al. 1971; Futenma 1973; Mitsui
1972; Takaku 1972; Galzko 1970; Nuridtinova 1973, 1975; Sivitskaya
and Alekseicuk 1973). Visual field examinations were performed
on 19 children presumed to be victims of organophosphorus
poisoning, on ten normal adults, and on five healthy children
(Ohba et al. 1972). In spite of obvious differences in findings
the authors insist on setting up the standards for visual field
parameters in normal children before making a meaningful compari-
son. A study of the visual field of 15 pilots and two other
persons taken before, during and after occupational exposure to
TEPP did not reveal any meaningful change (Upholt et al, 1956) .
Other functional eye tests were only seldom applied. Abnormal
electroretinographic (ERG) tracings were found in known cases of
intoxication by organophosphorus insecticides (Imai et al. 1973a).
Disturbance of dark adaptation time was mentioned by several
authors (Nuridtinova 1973; Kalic-Filipovic et al. 1973; Glazko
1970). Experimental studies of this problem were reported by
Rubin and Goldberg (1957, 1958). They found that sarin, an
anticholinesterase compound, when inhaled by volunteers signifi-
cantly elevated the absolute visual threshold for up to 24 hours
after exposure. When sarin was applied topically to the conjunc-
tival sac of the eye, a similar effect was not seen (Rubin
et al. 1957). It was therefore concluded that sarin brought about
visual threshold through an extraocular action, probably in the
central nervous system.
Disturbed smooth pursuit motion of the eyeballs in more than
half of the examined subjects was reported by Ishikawa (1971a).
Similar findings were also reported by other authors (Fuji et
al. 1975).
Several authors noticed abnormal pupillary reaction, but
only Ishikawa (1971a) used videopupillographic technique in order
to quantify the observed abnormalities. He found that exposed
subjects and controls significantly differ in the rate of re-
action of light stimuli.
Some epidemiological data discussed above are summarized in
Table 13. In some reports, however, chronic exposure is con-
fused with chronic poisoning. The ophthalmological and biochem-
ical methods used in the reports are only seldom described in
sufficient details to reproduce the results. The criteria for
the diagnosis of eye impairment are not always comparable. The
22
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interpretation of the results described in the Table 13 should
therefore be made very carefully. However, it should be emphasized
that the incidence of different eye damage in examined subjects
varies according to these reports between zero and a hundred
percent.
Case Reports
A 67 year old patient who sprayed malathion and fenitrothiori
for many years complained of gradual deterioration of vision
(Ishikawa 1972c). Ophthalmological examination revealed a loss
and narrowing of the vision, anomalous eye movement and irregular
size of the pupil. These changes disappeared after the treatment
with PAM.
In two adult patients with chronic organophosphorus poison-
ing (Oono et al. 1973) corneal astigmatism, myopia and constric-
tion of visual fields were found. The symptoms gradually re-
turned to normal after five months of therapy with PAM. Similarly
the main symptoms in three children and one adult (disturbance
of vision, peripheral structure of visual field, abnormal re-
fraction, disturbance of eyeball motion) were attributed by the
author to the exposure to anticholinesterase insecticides and
were treated as such (Ohto 1972).
Ophthalmological examination of twelve children with chronic
organophosphorus intoxication (Ohto 1974) revealed apart from
abnormalities of the papilla (congestion and paleness) a con-
striction of the visual fields. Electroretinographic examination
revealed low amplitudes of a-and B-waves.
Campbell (1952) described several cases of polyneuritis in
people who had been using insecticides. In three out of seven
cases retrobulbar neuritis was also present. However the author
was reluctant to claim that the findings were associated only
with the proven exposure to DDT. Besides myopathia, which devel-
oped in two agricultural workers exposed to organophosphorus
compunds, a 47 year old mother and 22 year old son, annular
abnormalities were present around macula lutea with pigment de-
posits (Komoto et al. 1973). Bilateral optic neuritis was
diagnosed in a 47 year old subject (Uchida 1974). He had retinal
chorioidal degeneration, and also suffered from neurosyphilis
and diabetes. However, he frequently used malathion and since
visual disturbances improved after a therapy with PAM, the case
was considered to be a poisoning with organophosphates.
A rare transient bilateral internal ophthalmoplegia was
observed in an adult with a history of exposure to a commercial
insecticide containing pyrethrins which suggested a possibility
of a toxic etiology of the disease (Weintraub, 1971). Twelve
hospitalized patients treated for eye impairment claimed by the
23
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author (Ohto 1974) to have been caused by pesticides were founa
to suffer from progressive pigmentary degeneration without any
family connection. Five cases of upward gaze paresis or para-
lysis combined with optico-autonomic neuropathy have been des-
cribed in pesticide applicators who had handled different organo-
phosphorus pesticides for at least five years (Ishikawa et al.
1976) .
Untoward Effects of Anticholinesterases Uses as Drugs in Opthal-
mologioal Treatment
Exactly one century has elapsed since physostigmine was
introduced as the first anticholinesterase drug for the medical
treatment of glaucoma. A variety of different acetylcholinesterase
inhibitors have been used since for the treatment of this disease.
With the use of prescribed concentrations, applied locally to the
eye, adequate control of glaucoma can be maintained generally
without the occurrence of side effects. If these occur, however,
they are obviously of systemic cholinergic stimulation through
cholinesterase inhibition or are most likely associated with
this mechanism (Ellis 1966) .
However, local application of anticholinesterase miotics in
glaucoma treatment caused lenticular changes in a number of
patients (Axelsson and Holmberg 1966; DeRoetth 1966; Axelsson
1968b; Abraham and Teller 1969) but not in guinea pigs (Axelsson
1971). In human subjects occupationally exposed to antichol-
inesterase agents lenticular changes have been described only
seldom (Pietsch et al. 1972; Komoto et al. 1973). The application
of miotics which are of carbamic acid origin, although antichol-
inesterases, have never caused lenticular opacities (Axelsson
1968a).
The mechanism by which these agents produce cataracts is
still unclear. The presence of lenticular cholinesterase as
well as its inhibition by anticholinesterase miotics were
clearly demonstrated (DeRoetth 1966; Laties 1969). However, it
seems that there is no direct relationship between inhibition
of cholinesterases and obstruction of lens clarity (Michon and
Kinoshita 1968). The cataracts initiated by anticholinesterase
agents applied topically are specific in type (Shaffer and
Hetherington 1966) if compared with common cataracts seen in
elderly people. The cataractogenic activity of different chem-
ical agents including anticholinesterase drugs has been ade-
quately reviewed (Paterson 1971; Gehring 1971).
A case report (Hornblass et al. 1974) deals with topical
application of thiotepa which caused a delayed side effect -
periorbital skin depigmentation. The mechanism of depigmentation
is completely unclear.
24
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Studies on Experimental Animals
Different abnormalities in chronic organophosphorus intoxi-
cation of experimental animals observed by a number of authors
point out the predominance of sensory neuropathy as compared
with affection of the motor nerve -system. Other authors however
found no eye and sight abnormalities in animals treated with
certain anticholinesterases which are frequently blamed for
causing eye damage in man. Thus Nishimura and coworkers treated
beagles with fenitrothion 20 mg/kg/day for 10 months and on
ophthalmological examinations found no difference from control
animals in many of the parameters measured (Nishimura et al.,
1974). Neither did Ogata (1972a) find any meaningful clinical
or pathophysiological anomaly in dogs treated with the same or
similar compound. Blindness in cattle in some areas in Japan was
formerly attributed to a direct ;effect of anticholinesterases.
However, Ohishi (1972) regards it to be connected with vitamin A
deficiency, since serum and liver carotene was found to be ex-
tremely low as was serum and liver vitamin A.
Morphological and Biochemical Changes
Severe ocular abnormalities, such as turbidity, protuberance
and projection of cornea were found in rats fed on a diet con-
taining up to 600 ppm of fenitrothion (Ooka et al. 1973). How-
ever, although marked inflammatory changes were seen in all
corneal layers by light and electron microscopy, no significant
changes were observed in the retina or in the optic nerve. The
incidence of corneal opacities produced by this compound or
similar compounds, could be appreciably reduced by subcutaneous
application of glutathion (Hikita and Ishikawa 1972). In
another experiment, besides atropine and PAM, Naito et al (1973)
tested glutathion in the therapy of corneal abnormalities in rats
given organophosphorus compounds and found that glutathion was
not therapeutic. In rabbits sprayed with a high concentration of
fenitrothion a decrease in the radius of corneal curvature was
observed (Kawai and Naito 1974). The same was noticed in beagles
treated with a dose of 5 mg/kg/day fenitrothion during 10 months
(Kawamura et al. 1974). The corneal curvature was considerably ,
reduced until growth stopped. Even smaller doses produced similar
effects (Uede et al. 1972).
Myopia appeared in beagles only four months after beginning
the application of ethylthiometon at a dose of 5 to 15 mg/day/
dog (Tokoro 1973). Astigmatism was also present and the findings
resembled the clinical picture reported in children from the
Saku district. In the experiment carried out by Suzuki an^
Ishikawa (1974) all dogs treated with the same compound showed
myopia after 12 months of administration. Myopia progressed
until cessation of administration. Thiometon given to dogs at
25
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a dose of 0.8 - 1.0 ing/day over a period of 100 days caused
myopia in three out of five animals (Ishikawa 1972d). Other
corneal abnormalities in experimental animals treated with
different anticholinesterases, were described (Maebashi et al.
1972; Naito et al. 1973a, 1973b).
The mechanism of formation of described corneal abnormal-
ities is unknown. Corneal epithelium contains acetylcholine and
the enzymes acetylcholinesterase and cholinacetyltransferase are
very active 'Howard and Wilson 1972) but whether these facts are
in any way connected with the abnormalities reported in experi-
mental animals and humans remains to be explained.
Myopia frequently observed in children and in experimental
animals was correlated with the exposure to organophosphorus
compounds. In thiometon treated beagles a significant inverse
correlation between blood cholinesterase activity and the length
of the optic axis was found (Otsuka 1971).
After a repeated administration of ethylthiometon to a
beagle over a period of two years the basement membrane between
ciliary muscle cells was extremely thickened as revealed by
electron microscopy (Araki et al 1973). In an identical experi-
ment the fine structure of the ciliary muscle of beagle dogs
treated with ethylthiometon revealed unique membraneous struc-
tures occupying the cytoplasm of one third of the muscle fibres
(Suzuki and Ishikawa 1974) . Structural changes of the ciliary
muscle were also found in rabbits and monkeys treated with
dichlorvos (Matsushima et al. 1973). Similarly, morphological
changes in the extraocular muscles of beagles treated with
ethylthiometon were described by Mukuno and Imai (1973). In
monkeys and rabbits treated repeatedly with fenitrothion, motor
and plates changed conspicuously especially in the outer ocular
muscle (Matsushima et al. 1972). In the same animal species
treated with dichlorvos or fenitrothion Yamazaki and coworkers
reported alteration of the junctional fold and synaptic vesicle,
a swelling and a decrease in the number of mitochondria in the
motor and plates of the external eye muscle (Yamazaki et al.
1975). These results suggest a possibility of the chronic pro-
gressive paralysis of extraocular muscles in man.
Microscopical changes in the optic nerve fibers were found
in beagles treated with thiometon for two years (Kano et al.
1975). A dose response relationship was observed with respect
to the ratio of medulated nerve fibres. A similar compound
produced axonal denaturation of the optic nerve in the beagle,
so that the mechanism of action of organophosphorus compounds on
eye might be connected with the inhibition of protein synthesis
and repression of axoplasmic flow (Mukuno et al. 1973; Mukuno and
Ishikawa 1975). A histopathological study of the canine optic
nerve and retina was undertaken to investigate morphologically
and quantitatively the structural changes caused by repeated
26
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administration of ethylthiometon (Uga et al, 1976). The optic
nerve was found to be invariably affected and the number of mye-
linated axons was markedly reduced. Changes in the retina were
mainly seen in the pigment epithelium.
In rabbits fed on a diet containing fenitrothion significant
changes in the composition of fatty acids in the optic nerve were
found (Imaizumi et al. 1973). The same compound produced retinal
changes in puppies (Homma 1973). At the beginning of administra-
tion, the reddening of the papilla and retina and a dilatation
of the central vein of the retina were found. Discoloration of
the papilla and retina was seen after a long time.
Functional Changes.
Retinal function has been extensively studied but little is
known about how it is impaired by chemical agents, pesticides in
particular.
To explain some functional anomalies observed in experi-
mental animals Miyata and coworkers studied localization of
cholinesterases in rat's eye (Miyata et al. 1974). Acetylochol-
inesterase was found to be present in the retina and choline-
sterase in the pigmentary epithelial layer. By histochemical
techniques, cholinesterase was found to be only slightly inhibited
after the administration of 5 mg/kg of fenthion and completely
inhibited after 100 mg/kg.
Electroretinographic studies carried out in mice treated
with parathion showed that the c-wave disappears quickly and
that the latency time of the B-wave increases and its altitude
decreases (Carricaburu and Lacroix 1973). The electroretino-
graphic measurements were also carried out in flies poisoned by
parathion (Carricaburu, 1971). Retinal disburbances in rats
given fenthion were recorded by electroretinography by many
authors (Miyata et al. 1973; Imai 1974a, 1974b, 1975; Imai et al.
1973b). The maximum noneffeet level was found to be very low,
5 yg/kg, i.e. 1/60,000 of the LD5Q dose of the compound (Imai
1974a). According to the same group of authors, the maximum non-
effect of fenthion for the retina was estimated to correspond to
25 mg of the compound for a human subject (Imai et al. 19373b).
The authors therefore conclude that ERG examinations should be
performed in suspected poisonings by organophosphorus compounds.
There are very few reports which describe other functional
tests in experimental animals. In an interesting experiment
Lehner (1973) found no measurable effect on color vision in
dieldrin treated ducks. Rats fed on a diet containing 50 ppm
of disulphoton over a period of two months showed no performance
deficit in tests in which depth perception and discrimination
were required (Clark 1972).
27
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OpUfhalmological Examination of Workers Exposed to Pesticides
The purpose of our present work was to investigate the
existence of morphological changes of the eye and functional
eye impairment in workers exposed to pesticides, who had been
under medical observation for many years and whose exposure was
assessed in several ways. These were predominatly the same
workers who were taken into the study o.c the relationship of
pesticide exposure and its effect on blood cholinesterases
(Section 4). The ophthalmological studies were undertaken
because there is no doubt that among many subjects (about 100,000)
described in the literature at least some showed eye and visual
impairment. The extent to which this is related to pesticides
requires further elucidation.
Exposed Workers and Control Subjects
A total of 63 workers were taken in this study. They were
divided into three groups according to the exposure to pesti-
cides. Some data on the ophthalmologically examined workers
are shown in Table 14.
Group A — Consisting of 31 workers was selected according
to the length of time each worker was engaged in pesticide
production. Only those working longer than five years were
taken in the study. All of them were well trained permanent
staff, mostly from the production of pesticide emulsions.
Initially, the working conditions of those occupationally
exposed to pesticides were rather poor, but they improved gradually.
The active ingredients produced were predominantly, but not
exclusively, anticholinesterase compounds, consisting mainly of
different types of dimethoxy and diethoxy phosphates of thiophos-
phates and carbamates. A few compounds were chlorinated hydro-
carbons or herbicides. Workers had no fixed working place and
an irregular rotating system was introduced to equalize exposure.
Usual working time was eight hours a day, five days a week for
9-10 months a year. However, overtime work was quite common,
particularly when seasonal production had to be increased. A
regular monitoring of cholinesterase activity showed an asso-
ciation between overtime work and decreased blood cholinesterase
activity. The details concerning the extent of exposure, type
of compounds and their effect on blood cholinesterases are
described in Section 4.
Group B — Among twelve workers, seven were engaged for more
than five years in pesticide application, largely in public
health programs, but also in desinfestation of food stores. Two
workers were pilots who applied pesticides in agriculture and
28
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those remaining supervised pesticide production. A common factor
for all the people in this group was irregular exposure, good
protective measures and regime at work. All this made for a
considerably milder exposure than for workers in group A. Again,
the active materials were predominantly anticholinesterase com-
pounds except for pilots who were equally exposed to different
fertilizers and herbicides. Blood cholinesterase activity data,
monitored over a long period of time, did not reveal any toxi-
cologically meaningful pattern of enzyme inhibition among these
workers. Similarly, other routine biochemical and hematological
tests required by clinicians at their periodical medical checkup,
were normal.
Group C -- This group of 20 subjects was formed to serve as
control. The workers were recruited among those of similar
socio-economic status as for the two previous groups. They were
matched by age and habits, but had no greater contact with pesti-
cides than the general population. They worked either in internal
transport or in the storage of end products which were not insec-
ticides. Thus except by sex, the three groups were matched as
closely as possible.
Methods
The ophthalmological examinations took place at the Ophthal-
mology Department of the University Hospital, Zagreb Yugoslavia.
Throughout the study, all examinations were performed by the same
team of ophthalmologists. The examiners were not aware of workers'
exposure. As a rule, in each subject all the examinations were
completed within one or two consecutive days. However, whenever
additional tests were needed, the workers were asked to come as
many times as needed. The prerequiste condition was imposed that
before any ophthalmological examination was made the worker must
have felt healthy and be fit to work.
All the workers were questioned about previous eye or other
relevant diseases, family history and present complaints con-
cerning the eye and vision.
By external eye examination all changes on the eyelids, in
the motility of eye-balls, bulbar and palpebral conjunctiva,
cornea and lacrimal apparatus were recorded. Whenever necessary
an examination by slit lamp microscopy (up to 64 x magnification)
was performed.
The corneal curvature was determined with a keratometer
which was either Javal or Bausch & Lomb. The results were cal-
culated as differences in dioptrics between the two meridians
and as corneal curvature expressed in millimeter values.
29
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Visual acuity was determined with the Snellen chart under
standard illumination. The vision was expressed by a fraction
in which the numerator denotes the distance at which the test
was conducted (5 or 6m) and the denominator the distance at
which the smallest letters read subtend a visual angle of five
minutes. Whenever possible, the refractive errors were cor-
rected to as near normal sight as possible. In patients with
discrepancy in the visual acuity anr1 estimated refraction ability
a sciascopic test was carried out after adequate cycloplegia.
This was, ho./ever, always done after measuring of internal eye
pressure but before perimetry. Testing of accommodation ability
and focusing power was done with Jager reading charts Ji - Js.
Measurement of the intraocular eye pressure was performed
in all the workers with the same Schi^tz' tonometer. The eye
pressure was expressed in millimeters of mercury after the
results were calculated from the instrument's calibration graphs.
In patients who showed even minimal abnormality in the pressure,
the measurements were repeated several times. If again found
abnormal, the patient was thoroughly investigated in order to
exclude or confirm the diagnosis of glaucoma. Applanation tono-
metry and gonioscopy were standard tests carried out in these
patients.
Ocular media and eyeground were examined after the pupil
was widened with a short lasting mydriatic drug. Special atten-
tion was paid to estimate the transparency of ocular media.
Visual fields were examined in detail in all workers with
a Goldmann perimeter 940 (Haag-Streit). This was done after
visual acuity determination and if necessary after its appro-
priate correction by lenses. The basic illumination of the
perimeter's calota was kept constant, 1,000 asb. The size of
the target was also constant, being 0.25 mm2, but its illumi-
nation varied. Relative intensity of the illuminated target
was 1.00 (Ii,), 0.315 (I3), 0.10 (I2) and 0.0315 (ix) of the
basic illumination. The examinations were carried out according
to the principle "nonseen - seen" at 15° intervals. These
values were later computed for each eye, each isopter and each
of the three groups. The means were also calculated.
Dark adaptation was determined in most workers from the
three groups on a Goldmann - Weekers adaptometer. The examined
person was kept in absolute darkness for three minutes and then
exposed to light of 2,000° asb. over five minutes. After that,
the absolute threshold of excitation was determined at one and
two minute intervals over the period of 25 minutes. The exam-
ined area was in the paracentral region 11° remote from the
center of fixation. The target was a 100% contrast black-white
stripped plate with a 56 mm diameter. The method applied was
again "nonseen - seen" i.e. starting with complete darkness,
the illumination of the target was gradually increased until
30
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the object was seen. The individual results obtained as adap-
tation curves were expressed in logarithmic values for each
minute, and means calculated for each group.
Color vision was tested in two ways: with a dichromatic
test according to several pseudoisochromatic tables (Ishikara
tables 1964; Velhagen, 23rd edition; Color Vision Test 1957)
and with an anomaloscopic instrument according to Nagel, Type I.
The test with pseudoisochromatic tables was carried out in
daylight for each eye separately. The examined subject was
not allowed to wear glasses while examined.
The examination with the Nagel, Type I anomaloscope was
conducted by applying the Rayleigh equations. The anomalic
quotient (AQ) was calculated from the results obtained.
Results and Discussion
The complaints received from workers exposed to pesticides
(Groups A and B) were predominantly lacrimation, photophobia
and a subjective feeling of burning and itching of the eyes.
These complaints were not received from control subjects. The
summary of complaints is shown in Table 15. Some complaints
which were obviously not associated with exposure to pesticides,
were disregarded.
The results of external eye examination are summarized in
Table 16. By external eye examination conjunctival injection
of different degrees was found in more than half of the examined
workers from group A. This finding could explain frequent
complaints received from exposed workers. However, in group B
similar changes were not observed, while 8 out of 20 control
subjects showed changes similar to those in group A. Dilated
episcleral blood vessels were seen in many exposed subjects.
Apart from the dilated episcleral blood vessels, which are not
usually so frequent in the general population, the other find-
ings were commonly recorded abnormalities, conjunctival infec-
tion being slightly more frequent in exposed workers.
In three patients from group A an irregular astigmatism was
diagnosed. In one case the abnormality was in a very advanced
stage. Otherwise, corneal curvature measurements revealed no
striking differences in the groups tested. The difference be-
tween the two meridians (placed at 90°) was within the expected
physiological difference (less or around 1 D). These values
were not statistically different from control subjects. The
results are shown in Table 17.
31
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Visual acuity was determined in all workers and if found to
be lower than 1.0 it was corrected by lenses to as near normal
as possible. The results showing visual acuity of both eyes
before and after correction with lenses are;plotted in Table 18.
With very few exceptions the visual acuity which was lower
than normal (1.0) could have been adequately corrected by lenses.
A rather rough method for testing accommodation ability and
focusing power did not reveal any greater impairment.
The measurement of internal eye pressure by means of Schi^tz'
tonometer revealed values which are expected in this population.
However, in workers exposed to pesticides the internal eye pres-
sure was significantly lower (P<0.01) if compared with control
subjects. The results of these measurements are shown in Table
19. Although several workers had a considerably higher pressure
possibly indicating glaucoma, applanation tonometry and gonio-
scopy confirmed this diagnosis in only one worker from Group A.
This was an advanced stage of an open angle glaucoma which had
not been recognized before. The results of this measurement
were omitted from the table.
Ophthalmoscopic examination of the eyeground revealed only
mild degenerative changes of the retina, mostly in elderly pa-
tients. In addition, in several patients retinal blood vessels
were slightly altered, but the findings were not very striking.
The findings are summarized in Table 20.
Opacities of optic media were found only in two workers
from group A and in one from group B. In all others, and in
group C, the optic media were absolutely clear.
The perimetric measurements revealed a significant nar-
rowing of the visual field in the second and third isopeter.
The location of this defect is rather unusual and at present
cannot be explained. The results expressed as mean values for
each isopter and each group are shown separately for each eye
in Tables 21 and 22. At several points the difference between
the two isopters is statistically highly significant (P<0.001).
Since all the measurements were carried out on the same instru-
ment, by the same person and under the same conditions there is
little doubt that the differences were an artefact. However,
it is obvious that the constriction of the visual field was very
mild and therefore its pathophysiological meaning should be
carefully considered.
Color vision testing showed no striking differences between
the three groups tested. The anomaloscopic quotient (AQ) was
found to be close to the normal physiological value in all
workers.
32
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Dark adaptation ability was slower in workers exposed to
pesticides than in control subjects (Table 23). The difference
became significant (P<0.05) only at 15th minute, being consid-
erably larger at 21st minute (P<0.001).
Although some morphological changes and functional eye im-
pairment were found more frequently in exposed workers than in
control subjects, additional tests and a larger number of exam-
ined subjects are required before any firm conclusion on the
effect of pesticides on eye and sight is made.
Summary
The effect of pesticides on eye and sight is comprehen- '
sively reviewed and 147 literature references are provided.
Data were not reviewed .in a critical way. The results of epi-
demiological studies on exposed humans conducted in several
countries are tabulated (Table 13).
The results of our own investigations which were initiated
recently are presented in ten tables. Ophthalmological exami-
nations were carried out mainly in the workers described in
Section 4. Sixty-three wo'rkers were examined of whom 20 served
as controls.
The analysis of the results revealed the narrowing of the
peripheral visual field (particularly in the second and third
isopter of either eye) in exposed workers when compared with
control subjects. The difference was significant (P<0.05 -
0.001) in the lower temporal region. So far this finding has no
explanation.
Internal eye pressure was found to be lower in exposed
workers than in controls (P<0.01).
The dark adaptation ability after 15 minutes was consider-
ably slower than in controls (P<0.05 - 0.001).
In other clinical parameters there were no great differ-
ences between exposed workers and control subjects.
33
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SECTION 6
RESIDUES OF ORGANOPHOSPHORUS PESTICIDES IN HUMAN URINE
For guidelines to control occupational exposure to organo-
phosphorus pesticides, the determination of organophosphorus
residues has been suggested. For this reason, the method of
Shafik et al. (1974) has been introduced. In this study, the
determination in a group of 13 workers of one metabolite of
phosalone is described.
Materials and Methods
Table 24 shows the names, abbreviations and formulas of
compounds used in this work.
N-amyl-N'-nitro-N-nitrosoguanidine produced by Aldrich
Chemical Co., Milwaukee, Wis. was obtained from the EPA
Repository at Research Triangle Park, N. C., as were all
standard substances and mixtures used. All other reagents
were of the highest purity grade available, and the solvents
were further purified.
The gas chromatograph, Varian Aerograph Model 2860, with
Alkali Flame lonization Detector (AFID) with RbaSOi* salt tip
was used. The gas chromatographic column used was: Borosil-
icate glass, 6' x 1/4" o.d. packed with 4% SE-30 and 6%
OV-210 Gas-Chrom Q 80/100 mesh.
i
The operating conditions for the gas chromatographic
analysis, the sensitivity, and retention times are listed in
Tables 25 and 26.
*
The following stages of the original procedure of Shafik
et al. (1974) were tested with standard solutions:
Alkylation—Known amounts of dimethyl phosphoric acid
were dissolved in hexane and the alkylation with diazopentane
performed, omitting the cleanup by chromatography on silica
gel columns.
34
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The effects of various parameters were tested on the yield
of the alkylation (Table 27):
- added amount of freshly prepared diazopentane;
- aging of diazopentane solutions (with 1 ml of diazopentane
added);
- duration of the alkylation (with 1.5 ml of diazopentane
added).
Alkylation and column chromatography—As already noted in
the original procedure, the elution pattern in the chromato-
graphic separation on silica gel columns may vary from one
laboratory to another, indicating a need for establishing a
standard elution pattern.
Using a 3 x 1 cm column of silica gel (2 g silica gel and
2 g NaaSOz.) the impurities were eluted with 20 ml CH2C12 at
the beginning of the separation. An additional volume of
20 ml CH2C12 with 3% of acetone contained the whole amount
of DMAP and DEAP. This volume was evaporated to dryness and
the residue dissolved in 5.0 ml of hexane for gas chromato-
graphic analysis.
DEADTP was partially eluted with 3 ml of a solvent mixture
CH2C12 4- hexane (1 + 1) , and an additional 3 ml contained the
rest. Both fractions were united, evaporated to dryness and the
residue dissolved in 2.0 ml of hexane for gas chromatographic
analysis. Further fractions contained only the impurities. Mean
values of the recovery in these stages are shown in Table 27.
Extraction, alkylation and column chromatography—The
critical stages of the procedure - Extraction, alkylation and
column chromatography - were tested by adding DMP, DEP and
DEDTP to urine samples of unexposed subjects. The results
are shown in Table 27. Recoveries calculated in Table 27
are based on the amount of standard added to the samples.
Results and Discussion
Urine (10 - 20 ml) was collected from workers industrially
exposed in the formulation of phosalone. Samples were taken on
a Tuesday at the end of the workday. From each sample, two
aliquots of 2 ml each were analyzed for DEDTP- To another two
aliquots (2.0 ml each) DEDTPK was added (900 ng per ml urine) in
order to evaluate the recovery. The eluates from the silica gel
columns were evaporated to dryness and dissolved in 2.0 ml hexane
For the gas chromatographic analysis 1.6 to 1.8 yl of the hexane
solution were injected. The identification of the compound was
based on the retention time as compared to known standards. The
smallest amount detectable was 7 ng DEADTP per ml urine. The
35
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obtained results are summarized in Tables 28 and 29; the tables
also contain the results obtained on urine samples of 8 unexposed
individuals.
In urine samples from five subjects not exposed to pesti-
cides, the amount of DEADTP was < 7 ng/ml urine. The exception
was subject V.B.S. who had < 7 ng/ml on one day, and 33 ng/ml
several days later. In urine samples from workers formulating
phosalone the amount of DEADTP was between 12 and 105 ng/ml
urine. The exception was one worker whose urine contained < 7
ng/ml. Consequently, it seems that 12 out of 13 workers absorbed
phosalone.
Blood cholinesterase activities were always measured in
these workers at the beginning of the same workday as sample
urine collection was made. All activities were within 85 to
100% of the preexposure value, except for subject I. S., whose
whole blood cholinesterase activity was 73%, while the plasma
activity was 100%.
The recovery of DEADTP added to urine samples varied from
12 to 32%, with a mean value of 21%. Such recoveries are low
indicating that the method requires improvement. As some reagents
used in the procedure are hazardous (nitrosoguanidine and
diazopentane) it would be advantageous to develop a more suitable
method.
Summary
The gas chromatographic method of Safik et al. was applied
to measure one metabolite of phosalone in urine samples from
13 workers formulating phosalone. In 12 of the 13 workers
measurable amounts of 0,0-diethyl phosphorodithioate (DEDTP)
were found. The recovery of the procedure was tested by adding
known amounts of DEDTP to urine, and it was shown that 21% of
the added compound could be recovered.
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SECTION 7
RESIDUES OF CHLORINATED HYDROCARBONS IN HUMAN BLOOD
There are no readily available data on the content of
chlorinated hydrocarbon residues in blood of the population of
Yugoslavia. The use of chlorinated hydrocarbons has recently
been restricted, and it was of interest to initiate a survey
which would establish the present levels and enable a later
followup of the body burden of chlorinated hydrocarbon pesti-
cides. A small number of individuals (147) in a continental
town in Croatia was selected, and their plasma or serum were
analyzed for those pesticides which are still in use or were
recently used.
Methods
The chlorinated hydrocarbons were determined by gas-liquid
chromatography. The extraction of compounds was done within
24 hours after sampling, and the extraction procedure was ac-
cording to Dale et al. (1970). From each individual two 1.0 ml
samples (acidified with formic acid) were extracted with hexane
(four 3 ml aliquots). The combined extracts were purified on a
Florisil column (1 g Florisil + ca. 1 g anhydrous Na2SoiJ.
The hexane was evaporated from the eluates, and the compounds
were dissolved in 1.0 ml hexane. The concentrations of chlori-
nated hydrocarbons were determined on a Varian 2800 gas-liquid
chromatograph with an H3 - Sc electron capture detector. Three
different column packings were used: Column A - 1.95% QF-1 and
1.5% OV-17 on Gas Chrom Q, 80-100 mesh; Column B - 4% SE-30 and
6% OV-210 on Gas Chrom Q, 80-100 mesh; Column C - 3% QF-1 and
6% DC-200 on Varaport 30. The best separation was obtained on
Column B. On all columns, the compounds were identified by
their retention times as compared with known standards. Solu-
tions of standards in hexane were treated in the same way as
the hexane extracts. The purification of extracts on Florisil
column is not included into the procedure of Dale et al. (1970).
The purification was necessary because fatty acids were found
in the extract. It was shown that except for deildrin, no loss
of chlorinated hydrocarbons occurs by that procedure. The con-
centrations of dieldrin are therefore not quoted.
37
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Results and Discussion
Samples of plasma (92) or serum (55) of 147 individuals
were analyzed for their content of chlorinated hydrocarbons.
The individuals were 65 males and 82 females; their mean age was
42 years, ranging from 8 to 92 years. Blood samples were ob-
tained from clinical laboratories during the year 1975. To our
knowledge none of the individuals had a previous occupational
or accidental exposure to chlorinated hydrocarbons or pesticides
in general. The results are presented in Table 30. All samples
contained p,p"-DDE, but p,p'-DDT was found in only 20 samples.
In five (out of the 20 samples) the concentration of p,p'-DDT
was higher than p,p'-DDE; in the other 15 samples the reverse
was true. The mean ratio of p,p'-DDE/p,p'-DDT in these 20 sam-
ples was 2.3. Similar to p,p'-DDT, the other compounds were
not found in all samples analyzed. The concentration range for
a given compound is large and the concentrations agree with
published data for other populations (Davies 1972; Kolmodin-
Hedman 1974).
Summary
Samples of plasma or serum of 147 individuals of a general
population in a continental town in Croatia were analyzed for
chlorinated hydrocarbons. The compounds were determined by gas-
liquid chromatography and identified by their retention times
as compared with known standards. All samples contained p,p'-
DDE (mean cone. 35.3 ppb), but p,p"-DDT was found in only 20
samples (mean cone. 22.7 ppb). The concentrations of the other
compounds were 3.25, 4.09, 12.2 and 11.6 ppb for a-HCH, lindane,
aldrin and p,p'-DDD respectively.
38
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SECTION 8
TOXIC EFFECT OF METRIFONATE (TRICHLORFON) AND
DDVP (DICHLORVOS) IN RATS
Metrifonate is the international nonproprietary name for
the pharmaceutical substance 0,0-dimethyl 2 ,2,2-trichloro-l-
hydroxyethyl phosphonate. This compound was first prepared by
Lorenz and coworkers (Lorenz et al. 1955) and was introduced
by Bayer, West Germany in 1952. It has been commercially avail-
able since then. As a pesticide, with the common name trichlor-
fon, it has been recommended for use as follows: against flies
and other household pests (WHO Expert Committee on Insecticides
1963), in agriculture (Goeldner 1960; Muller 1961), in veterinary
practice as an antiparasitic drug in the treatment of various
helminths, and for the control of ectoparasites (Boyd and Bullard
1968; Khan et al. 1972; Khan 1973) . It is also known to be
effective in the treatment of human S_. haematobium infection
(Davis and Bailey 1969; Plestina et al. 1972).Its toxicity to
mammals is surprisingly low in comparison with other organophos-
phorus compounds. The rat oral LDso is approximately 600 mg/kg
(DuBois and Cotter 1955; Gaines 1969). Insecticidal activity
of trichlorfon is attributed to its conversion to dichlorvos
(0,0-dimethyl 2,2-dichlorovinyl phosphate, DDVP) (Metcalf et al.
1959). However, DDVP has not been found in any appreciable
amounts after the administration of trichlorfon to experimental
animals (Robbins et al. 1956; Arthur and Casida 1957). According
to some authors (Fukuto and Metcalf 1969), trichlorfon itself is
devoid of anticholinesterase action but others (Arthur and Casida
1957; Hobbiger 1963; Hassan et al 1965) regard it as a direct
inhibitor of cholinesterase. The in vitro studies on that
question are described in Section 9. DDVP is a well known and
widely used insecticide, considerably more toxic for mammals
than trichlorfon (Gaines 1969). It was also used as an anti-
parasitic drug (Chavarria et al. 1969? Hass 1970). However, due
to its high toxicity this practice has been discontinued.
It has been known for a long time that the rearrangement
reaction leading to the conversion of trichlorfon to DDVP occurs
in vitro the rate being faster at higher pH (Barthel et al. 1955).
However, little is known about the extent to which this conversion
takes place in mammals, particularly in humans. Since trichlor-
fon is proposed to be administered therapeutically to patients,
children in particular, in relatively large repeated doses, its
conversion to DDVP might cause untoward effects of the otherwise
39
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beneficial drug. Therefore, its conversion to DDVP and the
combined effects of the two compounds should not remain unknown
and uncontrolled. If DDVP is a step in the breakdown of tri-
chlorfon, the effects of DDVP are inevitably included into data
obtained by in vivo toxicological studies on trichlorfon.
Materials and Methods
I
Only male albino rats bred at the Institute for Medical
Research and Occupational Health in Zagreb and fed on ordinary
rat feed ("Pliva," Zagreb) were used in the experiments. If
not otherwise stated rats were 10 to 12 weeks old and approxi-
mately 300 grams body weight.
Metrifonate (trichlorfon) was a gift from Bayer Farbenf abrik,
Wuppertal , West Germany. It was in a form for human use (the
purity was not stated). DDVP (93% pure) and DDT [1, 1 , 1-trichloro
2 , 2-bis (p-chlorophenyl) ethane] were obtained from the World
Health Organization. TMB-4 (1 ,1 '-trimethylene-bis- (4-formyl-
pyridinium) and PAM-2 (pyridine-2-aldoxime methiodide both as
the chloride salt were gifts of the Research Institute of
"Bosnalijek" - Sarajevo. SKF-525A (2-diethylaminoethyl-3 , 3-
diphenylpropylacetate) was a gift from late Professor Stern of
Sarajevo. All other chemicals were of commercial origin.
Acute toxicity testing was done by determining LDso and
ED 50 values after oral or intravenous administration of metri-
fonate or DDVP to rats of different ages. These values were
calculated on the basis of 24 hour mortality rate by the method
of moving averages (Thompson 1947) using the tables given by
Weil (1952). The animals were carefully observed and the onset,
intensity, and duration of cholinergic symptoms were recorded.
For some experiments , when a compound had to be infused or
blood withdrawn, rats were anesthetized with ether, and either
the jugular or a branch of portal vein were cannulated with
Clay-Adams polythene cannula (PE-50 and PE-10 respectively) .
For slow infusion experiments, after the animals had recovered
(two to three hours) metrifonate or DDVP were infused at dif-
ferent concentrations which were calculated so that the rate of
entrance of the compound corresponded to a multiple of a single
intravenous LDso value of that particular compound. The onset
of cholinergic symptoms and time of death were recorded and
from these values the results were calculated and expressed as
numbers of respective I.V- LD5o dose received until the onset
of symptoms or until death.
Cholinesterase activity in plasma and brain homogenates was
measured spectrophotometrically (Ellman et al . 1961). At given
times animals were killed by carbon monoxide or by ether, and two
40
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to three milliliters of blood was taken from the heart, centri-
fuged and plasma separated. The brain was removed, weighed and
homogenized in cold saline in a concentration of 40 mg/ml. The
results were expressed as percentages of the respective activity
which was determined in 40 healthy rats.
The technique of the intestinal sac (Wilson and Wiseman
1954) was used for assessing metrifonate transfer through the
intestinal wall. The noneverted sac of rat jejunum was filled
with 0.6 ml of 0.1 M phosphate buffer pH 5.0, 7.4 or 9.0 con-
taining 50 or 100 yg of metrifonate. The filled intestinal sac
was then placed into a buffered solution (40 ml) of hemolyzed
human erthrocytes, 5.0, 7.4 or 9.0, The temperature was kept
at 37°C. The cholinesterase activity of the hemolyzed erthro-
cytes was measured instantly, and at given time intervals up
to 180 minutes. The enzyme activity found at "0" time was taken
as 100 percent, and all other activities were expressed as per-
centages of that value.
Abdominal lymph was collected from large abdominal lympha-
tics. These were 'cannulated with polythene cannulae (PE-50)
while rats were under ether anesthesia. After recovery, rats
were dosed orally with either metrifonate or DDVP. When injec-
ted with high doses of DDVP, rats were protected with atropine
(10 mg/kg) and PAM-2 (100 mg/kg) injected intramuscularly just
before dosing. Lymph was collected for 30 minutes. The amount
collected was divided in half and added to 4.0 ml of hemolyzed
human erythrocyte in phosphate buffer of pH 5.0 or 9.0. The
incubation media were kept at 37°C. Cholinesterase activity
was measured after five minutes of incubation and then at inter-
vals until 60 minutes.
The TLC method (Beck and Sherman 1968) was slightly modi-
fied for the quantitative determination of metrifonate and DDVP.
The two compounds were extracted with ethylacetate from blood
and homogenates of brain and liver. The extract was applied
directly to thin layer plates, and the plates were developed
with a mixture of chloroform and acetone (1:1). The detection
was carried out with AgNO3, and the intensity of the spots was
quantified on a Model 111 fluorometer Camag (Switzerland).
Results and Discussion
Acute Toxicity in Relation to Age
The results of LD50 and ED50 (with 95% confidence limits)
determined in rats of different age after oral and intravenous
administration are shown in Table 31.
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While LDso value for metrifonate given intravenously did
not change with age, considerable differences were found after
its oral administration. The youngest rats, two weeks old, were
much more sensitive than adults. Surprisingly, the four weeks
old were the least susceptible. The differences in DDVP treated
rats were hardly noticeable. The ratios of oral and intravenous
LDso values were much smaller for metrifonate than for DDVP.
In rats after the oral administration of metrifonate the
onset of symptoms was much delayed as compared to DDVP. The
results are shown in Table 32. Even after an intravenous injec-
tion of metrifonate there was a considerable delay in the ap-
pearance of cholinergic symptoms, while after an intravenous
injection of DDVP symptoms developed within the first minute
after injection.
The symptoms observed in rats given metrifonate or DDVP
did not differ from those seen in rats injected with other
organophosphorus compounds. They were cholinergic in nature,
tremor, fasciculations and salivation dominated the clinical
picture. The rats dying from high doses of either compound
showed very pronounced convulsions and died of respiratory
failure.
The Role of the Liver in Metrifonate Toxicity
All compounds absorbed from the gastrointestinal tract have
to pass through the liver before being distributed in the body.
It is well known that liver plays an essential role in many
chemical processes which involve absorbed compounds. It was
therefore our aim to find out whether the liver has any influ-
ence on the toxicity of metrifonate.
The effects of metrifonate infusion into the portal circu-
lation of rats are similar to the effects when infused into
the jugular vein. The tolerance of the drug until death and
until the appearance of the first cholinergic symptoms was
similar, regardless of the route of administration. The results
are shown in Table 33. The rats infused with metrifonate at
the rate of one or two single I.V. LD5o doses/hour tolerated
totally only slightly over one I.V. LDso dose. When DDVP was
slowly infused either in the portal or in the jugular vein, the
tolerance was considerably higher. Even at a rate of two I.V.
LDso doses/hour, infused in the jugular vein, rats tolerated up
to 17 LD5o doses, the number being higher when the compound
was infused into the portal vein. Also the ratio between the
dose producing death and the dose producing the first noticeable
symptoms was much higher for DDVP than for metrifonate by either
route of administration.
42
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It is anticipated that if the conversion of metrifonate to
DDVP is assisted by the liver enzymes, their stimulation or
inhibition should either increase or decrease metrifonate tox-
icity. The purpose of the experiments carried out in rats was
to find out whether a sublethal dose of metrifonate would become
lethal in rats pretreated with microsomal enzyme stimulators, or
a lethal dose would become nonlethal in rats pretreated with
microsomal enzyme inhibitors. The stimulating agents used were
DDT or phenobarbital, and inhibitory agents were carbon disulfide
or SKF-525A. The procedure was the same as described by De
Matteis (1971). Altogether 14 groups, including adequate con-
trols, totaling 224 animals were used in these experiments. DDT
and CSz do not seem to have influence on the toxic effects of
DDVP. However, DDT, phendbarbital and SKF-525A showed a certain
protective effect against the highest dose of metrifonate. The
rats pretreated with CSa died after an injection of 150 or 300
mg/kg of metrifonate. It seems likely that microsomal stimula-
ting agents decrease the acute toxicity of metrifonate by stim-
ulating the detoxifying mechanisms. The protective effect of
SKF-525A might be due to its atropiniformic action.
*
Although from the described experiments there was no sup-
porting evidence that the conversion of metrifonate to DDVP is
assisted by the liver, we wanted to find out more directly
whether the concentration of DDVP in the blood which has just
passed through the liver is higher than, equal to or lower than
the concentration of DDVP in the blood coming into the liver.
This was assessed by measuring the inhibitory power of the blood
taken from the jugular and portal veins of rats given metri-
fonate or DDVP orally- Samples of blood taken either from the
protal or jugular vein had a similar inhibitory effect for cho-
linesterase preparations after appropriate incubation in vitro.
These results indicate that the conversion of metrifonate to
another inhibitor in mammals is not influenced by the liver and
therefore the conversion which takes place in vivo is most
likely a nonenzymic process.
Brain and Plasma Cholinesterase Activity
In order to avoid any possible effect of intestinal absorp-
tion of the compounds, in these experiments we used only the
intravenous route of administration. Plasma and brain cholin-
esterase activity was determined in rats at given times after
a single injection of a sublethal dose of metrifonate (300 mg/
kg) or DDVP (2.5 mg/kg).
The results are listed in Table 34. The enzyme inhibition
was very pronounced shortly after* dosing. The recovery of
enzyme activity was also fast. Six hours after the injection
the recovery was nearly complete, and on the following day inhi-
bition was almost nonexistent. No striking differences in the
43
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trend of recovery of either enzyme were found between rats injec-
ted with metrifonate and those injected with DDVP. Plasma
cholinesterase activity regained its normal activity within a
week after dosing. In rats given metrifonate brain choline-
sterase activity reached its normal value only one month after
the treatment.
One problem encountered in the assessment of the toxic pro-
perties of metrifonate in mammals is the aging of cholinesterases.
The use of metrifonate as a drug which ought to be administered
repeatedly to patients suffering from £3. haematobium infection,
bears a danger of cumulation of phosphorylated cholinesterase
which cannot be reactivated/ but only synthesized de novo.
The most pronounced inhibition of both brain and plasma
cholinesterase was found between 15 and 30 minutes after an
intravenous injection of either compound. The spontaneous
enzyme recovery, however, which takes place from that time on,
is more or less completed by 24 hours after dosing. Therefore,
the timing 30 minutes and 24 hours was selected for measuring
the extent of enzyme aging in rats dosed repeatedly with metri-
fonate or DDVP. Groups of six rats were injected intravenously
at daily intervals with 300 mg/kg of metrifonate or 2.5 mg/kg
DDVP. Brain and plasma cholinesterase activity was measured
30 minutes and 24 hours after each of five consecutive doses
were injected. The results expressed as mean values for six
animals are shown in Table 35.
The pattern of the enzyme inhibition was similar for the
two compounds. In either case brain cholinesterase was more
inhibited than plasma. The cumulation of the remaining un-
reacted enzyme was obvious, particularly in the brain of rats
treated with metrifonate.
In a separate set of experiments rats were injected intra-
venously with metrifonate (300 mg/kg) or DDVP (2.5 mg/kg) and
killed at given times. To measure the spontaneous reactivation
of cholinesterase activity in vitro it was monitored in plasma
and homogenates of brains which were taken at different times,
up to 24 hours after dosing, and kept at 37°C up to 24 hrs. In
rats treated with metrifonate no spontaneous reactivation occur-
red in homogenates of brain taken at any time after dosing and
kept at 37°C. In plasma samples taken a few hours after dosing
and kept under the same conditions, the recovery of enzyme acti-
vity was found to be moderate. In brain and plasma samples
taken from rats by the third hour after dosing with DDVP, spon-
taneous reactivation continued in vitro at approximately the
same rate as found in vivo. *
After TMB-4 was added to all samples taken up to six hours
after dosing (at a final concentration of 2 x 10~* M), the
reactivation of cholinesterases was fast and almost complete.
44
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All this indicates that up to approximately six hours after
dosing, the spontaneous reactivation, as well as the aging of
the enzymes, are completed. The lack of spontaneous reactiva-
tion in brain homogenates of rats dosed with metrifonate even
soon after dosing could be understood if one takes into account
the total amount of the compound injected (300 mg/kg as compared
with 2.5 mg/kg of DDVP).
The observed in vivo recovery of the enzymes after admini-
stration of metrifonate agrees well with the results obtained
in vitro on enzymes inhibited by DDVP (Skrinjarid-Spoljar et al.
1973) .
Absorption of Metrifonate from the Gastrointestinal Tract
The pH of the gastrointestinal tract in mammals varies con-
siderably and might have an influence on the nonenzymic rear-
rangement of metrifonate molecule even before its absorption.
This problem has been studied in several ways.
With the intestinal sac technique it was found that regard-
less of the metrifonate concentration and the pH of the buffer
in the sac, no inhibition of the incubation media occurred by
the fifth minute of the incubation. However, after 15 minutes
and at later time intervals, the extent of inhibition was very
much dependent upon the dose and the pH, becoming faster with
increasing concentrations and with increasing pH in the intes-
tinal sac. The results are presented in Table 36.
The rate of inhibition by 50 yg of metrifonate at pH 9.0
was very much like that of 100 yg of metrifonate at pH 7.4. In
the experiments in which the pH in the intestinal sac was 5.0
and in the incubation medium 9.0 the rate of inhibition was
slightly lower than when the pH values of the media were re-
versed. The maximal rate of inhibition was obtained with the
pH of both media was 9.0.
The question of whether or not metrifonate crosses the
intestinal wall unchanged or after conversion to DDVP was also
assessed in separate experiments in which inhibitory power of
abdominal lymph collected from rats immediately after oral
administration of metrifonate or DDVP was tested against cho-
linesterase preparation. As shown in Table 37, the rate of
inhibition was considerably faster if the lymph from rats given
metrifonate was incubated with the enzyme preparation at pH 9.0
than at pH 5.0. For comparison, rats were dosed orally with an
approximately equitoxic dose of DDVP, but the inhibitory power
of the collected lymph was very mild. Therefore, the dose of
DDVP applied was considerably increased (280 mg/kg). The rats
were protected with atropine and oximes, which in our case could
not influence the results. As seen from Table 37, there was no
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difference when the collected lymph was incubated with acetyl-
cholinesterase at pH 5.0 or 9.0. The experiments of Reiner and
Aldridge (1967) demonstrated that the rate of inhibition for
dimethylphosphates is somewhat lower at pH 5.0 than at pH 9.0.
However, a similar rate of inhibition in the case of DDVP-treated
rats strongly suggests that the difference found in the metri-
fonate treated rats was not due to the effect of pH on inhibi-
tion. This therefore indicates thaj; tr.e higher the pH the
faster is the conversion of metrifonate to DDVP which in turn
more rapidly inhibits cholinesterase (cf. Section 9, Table 40).
Metrifonate and/or DDVP were determined by TLC method in
the lymph collected from rats during the 60 minutes after oral
dosing with metrifonate (300 mg/kg) or DDVP (280 mg/kg). When
lymph was collected in an acid medium of pH between 2 and 3
(a drop of formic acid was added to a container before collection
started) no DDVP was found in the lymph. The results are shown
in Table 38. On the contrary, when lymph was not acidified and
its pH was between 7 and 8, a certain amount of DDVP was found
in addition to metrifonate. In the lymph of rats given DDVP
the amount of DDVP recovered was similar regardless of whether
the lymph was acidified or not.
These results indicate a possibility that metrifonate is
transferred through the intestinal wall predominantly or entirely
unchanged and that the conversion to DDVP takes place afterwards.
Although the experiments described have clarified certain points
in connection with the absorption of metrifonate, its pharmaco-
kinetics remains to be elucidated.
Summary
Metrifonate (trichlorfon) is a promising antiparasitic drug
having a relatively low acute mammalian toxicity. This
anticholinesterase compound is also in wide use as an insec-
ticide.
The aim of the study was to study conditions which might
influence its low toxicity to mammals.
In contrast to DDVP, the oral LD50 for metrifonate in rats
varies with age, being higher in the very young.
As with many other direct cholinesterase inhibitors, cho-
linergic symptoms appeared immediately after an intravenous
injection of DDVP but there was a considerable delay (3-4 min.)
in the appearance of symptoms in rats given an intravenous
injection of metrifonate. Given by either route metrifonate
causes inhibition of both brain and plasma cholinesterases.
46
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Although the rate of reactivation in vivo is fast, it is never
complete, since approximately 20% of the enzyme undergoes the
aging reaction. Repeated administration of metrifonate causes
cumulation of the unreactivable cholinesterases.
The conversion of metrifonate to DDVP seems to be an
entirely nonenzymic process, since the liver enzymes, appear
to have no influence on its toxicity.
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SECTION 9
REACTION IN VITRO OF METRIFONATE (TRICHLORPHON) AND DDVP
(DICHLORVOS) WITH ACETYLCHOLIKESTERASE AND CHOLINESTERASE
Metrifonate (trichlorfon, 0,0-dimethyl 2,2,-trichloro-l-
hydroxyethyl phosphonate) and DDVP (dichlorvos, 0,0-dimethyl
2,2-dichlorovinyl phosphate) are used as pesticides, and
metrifonate is also used as an antiparasitic drug. Both com-
pounds are unstable in aqueous solutions; metrifonate rearranges
into DDVP, and DDVP undergoes hydrolysis.
Metrifonate (CH30)2P(O)C - CC13
I
(trichlorphon) OH
DDVP (CH3O)2P(0)OCH = CC12
(dichlorvos)
It is known that DDVP is an inhibitor of cholinesterases,
but there are controversial views on the reaction of metrifonate
with cholinesterases. The present studies were therefore under-
taken to establish whether metrifonate is an inhibitor of
mammalian and parasitic cholinesterases. The approach in these
studies was a kinetic analysis of cholinesterase inhibition by
metrifonate. In order to analyze the kinetic data it was
also necessary to study the inhibition of cholinesterase by
DDVP under the same conditions as those used in metrifonate
studies. Further it was necessary to study the rates of
spontaneous reactivation and aging of an enzyme phosphorylated
by DDVP, since such data did not exist in the literature.
Methods
The activity of cholinesterases at pH 7.4 was measured by
the spectrophotometrie method of Ellman et al. (1961) with
acetylthiocholine as substrate; the thiocholine formed upon
hydrolysis of the substrate reacts with the thiol reagent DTNB
forming 4-nitrothiophenol, the absorbance of which is measured
48
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spec tr ©photometric ally at 412 nm. The activity of cholinesterases
at pH 6.0 was measured by the pH-stat method of Jensen-Holm et al .
(1961) with acetylcholine as substrate; the acetic acid formed
upon hydrolysis of the substrate was titrated with NaOH to main-
tain constant pH. The decomposition of metrifonate in buffer
solutions was measured polarographically by the method of Giang
and Caswell (1957). Under our experimental conditions the half-
wave potential of metrifonate was at - 0.8 v.
Results and Discussion
The rate of inhibition of mammalian acetylcholinesterase
(two species) and mammalian cholinesterase (three species) by
DDVP was determined at different concentrations of DDVP and
various times of inhibition (Skrinjaric-Spoljar et al. 1973;
Reiner et al. 1975). All reactions followed the kinetics of
a bimolecular reaction and the calculated second order rate
constants of inhibition (ka) at 25°C and 37 °C are listed in
Table 39. Of the five enzymes studied, human plasma cholines-
terase was most inhibited by DDVP. At 37 °C the rate of inhibi-
tion is faster than at 25°C, but this effect was only slightly
manifested for the inhibition of horse plasma cholinesterase.
This finding is in agreement with our earlier studies on the
effect of temperature in which it was shown that phosphorylation
and carbamylation of horse serum cholinesterase increases with
temperature between 5° and 30°C, and then decreases from 30°C
to 40°C (Simeon, Reiner and Vernon, 1972) .
The effect of pH on inhibition of bovine erythrocyte
acetylcholinesterase by DDVP is given in Table 40. At both
pH 6.0 and pH 7.4 DDVP is about equally effective as inhibitor.
This pH effect is consistant with our earlier finding that pH
values below 8 have little effect on inhibition of acetylcho-
linesterase by nonionic molecules (Reiner and Aldridge 1967) .
The dimethylphosphorylated enzyme, formed upon inhibition
by DDVP of acetylcholinesterase and cholinesterase, undergoes
two reactions which occur simultaneously: spontaneous reactiva-
tion and aging. It was shown that the rate of formation of the
active enzyme EH can be calculated from the following theo-
retical equation (Skrinjaric-Spoljar et al . 1973):
- rv + k } t
- e r ag >
k + k
r ag
[E ] is the concentration of the inhibited enzyme at zero
time, and k and k are the first order rate constants of
spontaneousrreactivltion and aging, respectively. These con-
stants were determined experimentally and are listed in Table 41
As shown in the table, dimethylphosphorylated human plasma
49
-------
cholinesterase is very stable, and only an approximate k con-
stant could be evaluated. However, human erythrocyte acetylcho-
linesterase reactivates spontaneously with a half-time of 51
min; the half-time of aging is 3.9 hr.
Solutions of metrifonate cause enzyme inhibition when added
to mammalian acetylcholinesterase and cholinesterase. Assuming
that inhibition is due only to DDVP formed from metrifonate, it
is possible to calculate the rate of formation of DDVP from
metrifonate. Such experiments and subsequent calculations were
conducted on acetylcholinesterase from two species and cholines-
terase from three species, and the results are presented in
Table 42. Further, the rate of formation of DDVP from metrifonate
was determined under the same experimental conditions by a direct
method (polarographically), and these results are given in
Table 43. The sets of values in Tables 42 and 43 are in good
agreement, and such agreement is a kinetic proof that metrifonate
itself is not an inhibitor of mammalian cholinesterases; inhibi-
tion by metrifonate solutions is caused by the DDVP formed from:
metrifonate in a nonenzymic rearrangement reaction. It has to
be emphasized that in the experiments in Table 42 only 1% - 2%
of metrifonate decomposed into DDVP, and the calculated k values
are therefore subject to greater variations than those given in
Table 43, which were obtained from decomposition curves where
more than 50% of metrifonate decomposed.
The above conclusions are substantiated by studying the
effect of pH on inhibition of mammalian cholinesterases
(Table 40). The rate of enzyme inhibition by metrifonate solu-
tions is lower at pH 6.0 than at pH 7.4. This difference is not
due to the lower rate of inhibition by DDVP, but is due to the
lower conversion of metrifonate into DDVP at pH 6.0.
Studies of the reaction of parasitic cholinesterases with
metrifonate and DDVP are still in progress. So far only studies
on the parasite Metastrongylus apri (M. apri) have been completed.
The inhibition of the cholinesterase in M. apri was measured
at 25°C in 0.1 M phosphate buffer pH 7.4, and the activity was
determined spectrophotometrically with acetylthiocholine and
butyrylthiocholine as substrates.
DDVP is an inhibitor of the cholinesterases in M. apri.
The degree of inhibition is the same irrespective of which sub-
strate is used to measure the enzyme activity. This result
provides strong evidence that acetylthiocholine and butyryl-
thiocholine are hydrolyzed by the same cholinesterase in M. apri.
The second order rate constant (k ) of inhibition by DDVP~~is
7.2 x 10" M~1min~1; this is of the same order of magnitude as
the k for the inhibition of mammalian cholinesterases.
a
50
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Solutions of metrifonate inhibit the hydrolysis of acetyl-
thiocholine and butyrylthiocholine; the degree of inhibition is
the same for both substrates. From a kinetic analysis of the
inhibition curves it follows that metrifonate itself is no
inhibitor of the cholinesterase; inhibition is entirely due to
DDVP formed from metrifonate. The kinetic analysis was the same
as used in our studies of mammalian cholinesterases.
Summary
Metrifonate is not an inhibitor of mammalian cholinesterases
Inhibition of mammalian cholinesterases in metrifonate solutions
is due to DDVP formed from metrifonate in a nonenzymic reaction.
The same conclusion applies to inhibition of the cholinesterase
from the parasite Metastrongylus apri. 1
A comparison of three groups of results obtained in vitro
(this Section) and in vivo (Section 8) supports such a conclusion,
When DDVP is injected to rats (I.V.), the onset of symptoms
is immediate, while after metrifonate there is a delay of 3-4
min before the symptoms appear. The LD50 for DDVP is 3-5 mg/kg
and for metrifonate 450 mg/kg. If DDVP is the toxic compound,
about 1% of the metrifonate has to decompose into DDVP in order
to produce the toxic dose. From measurements in_ vitro it follows
that about 2 min are required for 1% metrifonate" to decompose
into DDVP at 37°C. This time is in excellent agreement with the
delay observed in the onset of symptoms.
Metrifonate administered to people causes inhibition of
human blood cholinesterases. Plasma cholinesterase is always
more inhibited than the erythrocyte acetylcholinesterase. This
finding is to be expected from measurements in vitro, supposing
that DDVP, and not metrifonate, is the enzyme inhibitor; the
rate constant of inhibition by DDVP (measured in vitro at 37°C)
is 6.6 times higher for human plasma cholinesterase than for
human erythrocyte acetylcholinesterase.
The rates of spontaneous reactivation in vivo following
administration of metrifonate correspond to the rates of
spontaneous reactivation in vitro after inhibition by DDVP.
After a single oral dose of metrifonate, human blood cholines-
terases become inhibited in vivo within about half an hour;
after that time the enzyme activities begin to recover. The
half-time for the recovery iri vivo of human plasma cholines-
terase is about 180 h (calculated from mean values obtained in
blood samples from 60 treated children), and the half-time
for the erythrocyte acetylcholinesterase of the same group is
about 300 h. The rate of spontaneous reactivation of human
blood cholinesterases after inhibition in vitro by DDVP was
measured at 37°C. The rate of spontaneous reactivation of
51
-------
human plasma cholinesterase is slow, and only the minimum
for the rate constant could be obtained; this accounts for a
half-time = 83 h.Human erythrocyte acetylcholinesterase reacti-
vates spontaneously much faster (half-time 51 min), but it also
undergoes aging (half-time 233 min). It can be proved theo-
retically that after 300 h,83% of the inhibited enzyme should
have reactivated spontaneously.
52
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SECTION 10
MECHANISM OF LIGAND BINDING OF ACETYLCHOLINESTERASE AND
CHOLINESTERASE IN VITRO
It is known that some substrates react with cholinesterases
in such way that they cause substrate inhibition (Simeon 1974 a,
1974 b). It is also known that some reversible inhibitors react
with acetylcholinesterase in a way analogous to substrate inhi-
bition (Aldridge and Reiner 1972; Reiner and Simeon 1975). It
has been therefore suggested that both reactions occur at the
same site, and that this site is an allosteric site of acetyl-
cholinesterase (Aldridge and Reiner 1972). To investigate fur-
ther this hypothesis, the kinetics of the enzyme-substrate and
enzyme-inhibitor reaction was studied with particular emphasis
on the mechanism of reversible binding of ligands to acetyl-
cholinesterase and cholinesterase.
Materials and Methods
The enzymes were bovine erythrocyte acetylocholinesterase
(Winthrop Ltd., New York, NY, USA) and horse serum choline-
sterase (Sigma Chemical Comp., St. Louis, MO, USA). The sub-
strates were acetylthiocholine iodide (AChS), phenylacetate
(PhAc), benzoylcholine chloride (BzCh) and acetylcholine per-
chlorate (ACh). The inhibitors were two organophosphorus com-
pounds, and two carbamates. All experiments were done in 100 mM
phosphate buffer pH = 7.4. The activity towards acetylthio-
choline was measured by the spectrophotometric method of Ellman
et al. (1961) and towards phenylacetate by the spectrophotometric
method of Krupka (1966). The activity towards acetylcholine was
measured by the pH-stat method of Jensen-Holm (1961). The
activity of serum cholinesterase towards benzoylcholine was
measured by the spectrophotometric method of Kalow et al. (1956)
and the pH-stat method of Jensen-Holm (1961).
Results and Discussion
Competition between Substrates for Acetylcholinesterase and
Cholinesterase
The kinetic constants for the reaction of acetylcholine-
sterase and cholinesterase (at 25°C) with a single substrate are
53
-------
listed in Table 44. The Michaelis constant for acetylcholine
and acetylcholinesterase was calculated from a plot of [S]/vo
vs. [S], where vo is the enzyme activity -
For the reaction of benzoylcholine and cholinesterase the
above plot is nonlinear. The Hill equation was therefore
applied, and the Michaelis constant (Km) and the Hill coefficient
(nn) were obtained from the X2 - analysis which was performed
in order to derive the best set of Km and nH values which fits
the experimental results. The eubstrate inhibition constant,
Kss, for benzoylcholine and cholinesterase was calculated from
a plot of I/VQ vs. [S]; this plot is linear indicating that the
enzyme inhibited by the substrate itself does not hydrolyse at
a measurable rate, i.e. 3 = 0 (Simeon 1974 b). The above plot
for the reaction of acetylcholine and acetylcholinesterase is
nonlinear indicating that 6 is different from zero. The KSS and
B values given in Table 44 were obtained from the experimental
data in a way described earlier (Simeon 1974 b) by the least-
squares estimation.
The competition between two substrates for their reaction
with the enzyme was studied (at 25°C) so that the hydrolysis of
only one substrate was measured at a time. For instance, when
acetylthiocholine was the substrate and acetylcholine the inhi-
bitor, the hydrolysis of acetylthiocholine was measured by the
Ellman spectrophotometric method (1961); by this method only the
formation of thiocholine, and not choline, is determined. All
results were presented in such way that the substrate concentra-
tion was plotted on the abscissa, and the ratio v [I]/(vo-v)
plotted on the ordinate; vo is the enzyme activity when no inhibi-
tor is present, and v is the activity at the same substrate con-
centration but with the inhibitor present. [I] is the inhibitor
concentration; the range of the inhibitor concentrations used in
the experiments is given in Table 45. At each substrate concen-
tration at least two inhibitor concentrations were tested, and
each experiment was repeated 2 or 3 times. It was possible to
calculate mean values because the ratio v [I]/(vo-v) was constant
at any given substrate concentration irrespective of the concen-
tration of the inhibitor.
In the kinetic equation derived earlier (Aldridge and
Reiner 1972; Reiner and Simeon 1975) the mechanism of compe-
tition between substrates and inhibitors can be identified from
the numerical values of the intercepts on the abscissa and
ordinate, if the results are plotted as described above. When
the intercept of the line or curve with the abscissa, K(S) ,
equals the Michaelis constant, substrate and inhibitor compete
at the catalytic site. When K(S) equals Kss, the competition
takes place at the substrate inhibition site. The intercept
with the ordinate, K(I), corresponds to the dissociation con-
stant between enzyme and inhibitor. In Table 45 are listed the
intercepts calculated for the reactions studied. In all reactions
54
-------
but one the plot of v[I]/(v -v) vs. [S] is linear. The exception
is the hydrolysis of acetylthiocholine by cholinesterase with
phenylacetate as inhibitor; the intercepts in that case were
assessed from the initial slope of the curve. For both enzymes,
the calculated K(S) and K(I) constants are all of the same order
of magnitude as the corresponding Michaelis constants for the
same compound when used as a single substrate. This is true
irrespective of whether the enzyme is or is not inhibited by
the single substrate. (i.e. K does or does not reveal itself
within the studied concentration range) . It also applies irres-
pective of whether or not the reaction of the single substrate
follows the Michaelis or Hill equation.
The standard errors of the calculated constants are large
(Table 45). Therefore, the differences which exist between K
on one side, and K(S) and K(I) on the other side cannot be tamen
as meaningful in terms of reaction mechanisms. However, the
values of K(S) and K(I) for acetylcholine, acetylthiocholine
and benzoylcholine are all on the average two orders of magnitude
lower than the corresponding substrate inhibition constants K
This implies that K(S) and K(I) do not correspond to K . Itss
also implies that the competition between two substratll is
basically a competition at the catalytic site of the enzyme.
The substrate inhibition site does not show up in the kinetics
of competition between substrates. This is different from the
competition between the same substrates and coumarin derivatives
where the substrate inhibition site was kinetically prevalent.
Effect of Temperature on Phosphorylation and Carbamylation of
Acetylcholinesterase
Studies of the effect of temperature on acylation reactions
have proved to be effective tools in evaluating reversible
intermediates in the reaction, as shown for the acylation of
human and horse serum cholinesterase (Simeon, Reiner and Vernon
1972). Following up that approach, studies were expanded to
bovine erythrocyte acetylcholinesterase as enzyme. The inhi-
bitors were: haloxon [ (0,0-bis (g-chloroethyl)0-(3-chloro-4-methyl-
7-coumarinyl)], phosphate, phosphostigmine methylsulfate(0,0-
diethyl-3-trimethyl-ammoniumphenyl phosphate), neostigmine methyl-
sulfate (3-trimethyl-ammonium-phenyl-N,N-dimethylcarbamate) and
3-isopropylphenyl-N-methylcarbamate.
The effect of temperature was studied at 7 different tem-
peratures, between 5°C and 40°C, in 1.0 mM or 0.1 M phosphate
buffer pH 7.4. The enzyme activity was measured with acetyl-
thiocholine, acetylcholine or phenylacetate as substrates.
At a constant concentration of haloxon and phosphostigmine
the plot log enzyme activity vs. time of inhibition was linear.
55
-------
However, the sldpes of lines, i.e., the first order rate con-
stants, were not a linear function of the inhibitor concentra-
tion. This is a kinetic proof of a reversible complex between
enzyme and inhibitor. The exception was the inhibition by phos-
phostigmine at 40°C for which a linear correlation was obtained.
From a Wilkinson plot, the dissociation constant K(I) of the
reversible complex was calculated. For a given inhibitor, this
constant is the same, irrespective of which method or substrate
was used to determine the enzyme accivi-y. The values of K(I)
and of the second order rate constants of phosphorylation (ka)
are given in Table 46. The increase of the rate constants of
phosphorylation with temperature follows the Arrhenius equation,
with an activation energy of 20 kcal mol"1 for inhibition by
phosphostigmine.
The inhibition of acetylcholinesterase by the two carbamate
inhibitors follows the kinetics of a second order reaction with
one component (the inhibitor) in excess. No reversible complex
between enzyme and inhibitor is detectable from the kinetic ana-
lysis. The values of the ka constants are given in Table 47.
The rates of carbamylation increase with increasing temperature
and follow the Arrhenius equation. The activation energies for
inhibition by neostigmine and 3-isopropylphenyl-N-methylcar-
bamate are 13 and 10 kcal mol"1 respectively.
Summary
The kinetics of competition of pairs of two substrates for
bovine erythrocyte acetylcholinesterase (acetylcholine hydrolase,
EC 3.1.1.7) and horse serum cholinesterase (acylcholine acyl-
hydrolase, EC 3.1.1.8) was studied (at 25°C) so that the hydro-
lysis of only one substrate was measured at a time. The sub-
strates were acetylthiocholine, phenylacetate and benzoylcholine;
the same compounds, and also acetylcholine, were used as com-
peting substrates i.e. inhibitors. The substrate inhibition
constants (Kss) and Michaelis constants for the reaction of a
single substrate were also determined. It was concluded that
the substrate inhibition site in the enzyme does not show up in
the competition between two substrates.
t
The effect of temperature (5° - 40°C) was studied on rever-
sible and irreversible inhibition of bovine erythrocyte acetyl-
cholinesterase by two organophosphorus compounds and two car-
bamates. The second order rate constants of inhibition increased
with increasing temperature, and the increase followed the
Arrhenius equation. From the kinetics of phosphorylation by the
two organophosphorus compounds (haloxon and phosphostigmine) it
was concluded that both compounds form a reversible complex with
the enzyme. No reversible complex could be detected from the
kinetics of carbamylation by neostigmine and 3-isopropylphenyl-
N-methyl-carbamate.
56
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SECTION 11
EVALUATION OF FIELD METHODS FOR DETERMINING THE ACTIVITY
OF HUMAN BLOOD CHOLINESTERASES
Absorption of organophosphorus compounds and carbamates
are so far best determined by measuring the activity of human
blood cholinesterases. Sometimes it is not possible to measure
the activity in a laboratory, and suitable field methods are
therefore required. In this report, three methods are compared
and temperature conversion tables are provided for one of the
methods.
Methods
The following methods were used: spectrophotometric method
of Ellman et al. (1961) and two field methods, the Acholest
(Herzfeld and Stumpf 1955; Plestina 1966) and tintometric
(Edson 1958; Vandekar and Svetlicic 1966).
Results and Discussion
The spectrophotometric method of Ellman et al. (1961) is
widely used for measuring the activity of acetylcholinesterase
and cholinesterase. The procedure is simple and the results
well reproducible. The method is used as a laboratory method
and more recently attempts have been underway to apply it in
the field. When the enzyme activity is measured under field
conditions, the temperature of assay is that of its surrounding.
Temperature conversion tables are therefore required to enable
the conversion of activities determined at different temperatures.
As no temperature conversion tables have been published for the
Ellman method, the aim of our work was to provide such tables
(Reiner et al. 1974) showing the effect of temperature on human
blood cholinesterases.
The following experimental conditions were chosen: tempera-
ture range 10°C to 40°C, substrate concentration 1.0 mM acetyl-
thiocholine, pH of assay 7.4, phosphate buffer 0.1 M. The above
temperature range seems to cover field conditions in different
countries of the world. The substrate concentration of 1.0 mM
was chosen for both human whole blood and plasma cholinesterase
57
-------
because it is convenient to have only one substrate concentration
in routine measurements, and this concentration in routine
measurements, and this concentration gives suitable activities
for both preparations. At a pH of 7.4, the nonenzymic hydrolysis
of 1.0 mM acetylthiocholine is slow, and no corrections for
spontaneous hydrolysis are required. The phosphate buffer was
chosen because the effect of temperature on the pH of that
buffer is small; and the buffer can therefore be prepared at any
temperature between 10°C and 40°C. The buffer concentration of
0.1 M has a good buffering capacity; the addition of whole blood
or plasma will not alter the pH, also the pH will remain constant
during assay.
The results are presented in Table 48. When the temperature
increases from 10°C to 40°C, the activity of whole blood cholines-
terase will increase 2.60 times and that of plasma cholinesterase
3.48 times.
Besides the Ellman method there are two other methods which
are also widely used for determining the activity of human blood
cholinesterases. These are the Acholest (Herzfeld and Stumpf
1955; Plestina 1966) and the tintometric (Edson 1958; Vandekar
and Svetlicic 1966) methods. Both are field methods and they
were developed to evaluate the absorption of organophosphorus
compounds. When these methods are used for measuring enzyme
activities in persons exposed to dimethylphosphates or carbamates
their accuracy requires verification. This is because of the
spontaneous reactivation of the inhibited enzyme which might
give rise to inaccurate results.
The two field methods were tested under laboratory condi-
tions and the Ellman spectrophotometric method was used as a
reference method (Wilhelm et al. 1973). The inhibitors were
four N-methylcarbamates. The results are presented in Tables 49
and 50. When plasma cholinesterase activity was measured by the
Acholest and Ellman method, no difference was found. However,
the enzyme activity in whole blood determined by the tintometri6
method was <_ 11% higher than when the same sample was measured
by the Ellman method.
It is not always possible to measure human blood cholines-
terases immediately after the sampling. When blood samples of
people exposed to cholinesterase inhibitors are stored, the
effect of storage upon enzyme activity should be evaluated.
Human blood cholinesterases were inhibited by N-methylcarbamates
and stored at different pH values, temperatures and sample dilu-
tions. Storage conditions as shown in Table 51 were found
satisfactory (Wilhelm and Reiner 1973) .
58
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The same conditions are likely to apply to other cholines-
terase inhibitors because massive dilution slows down cholines-
terase inhibition during storage, the low pH slows down
spontaneous reactivation of the enzyme (Reiner and Aldridge
1967) and the low temperature slows down both the inhibition
and spontaneous reactivation. Uninhibited human blood samples
treated in the same way retained their cholinesterase activities
unaltered.
Summary
Temperature conversion tables for the activities of human
whole blood and plasma cholinesterases were developed for the
temerature range 10°C to 40°C for the spectrophotometric method
of Ellman.
Storage conditions of human blood were established for
measuring the cholinesterase activities after inhibition by
compounds which produce an easily reactivatable enzyme.
Two field methods, the Acholest and tintometric, were
shown to compare favorably with the Ellman spectrophotometric
method, which was used as the reference method.
59
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SECTION 12
QUANTITATIVE DETERMINATION OF ORGANOPHOSPHORUS PESTICIDES
BY THIN-LAYER DENSITOMETRY
For the determination of organophosphorus pesticides thin-
layer chromatograms were evaluated quantitatively by direct den-
sitometric measurements of the difference in the coloration or
fluorescence of spots and layer after visualization with silver
nitrate or by acetylcholinesterase inhibition (Stefanac et al.
1976).
Materials and Methods
The origin of organophosphorus standards used (Table 52) is
as follows: dichlorvos 93%, WHO, Geneva, Switzerland; trichlorfon,
Bayer AG, Elberfeld, West Germany; Malathion 96%, the reference
standard, Cat. No. 2576, EPA,. (USA); Parathion 99%, Bayer AG,
Leverkusen, West Germany.
Stock solutions were prepared by weighing pure substances
and dissolving them in ethanol. The same solvent was used for
further dilutions.
Layer: 0.5 mm of Kieselgel G nach Stahl, Type 60, Merck,
Darmstadt, West Germany. TL chromatograms were developed with
the solvent system chloroform : acetone (1:1) (Beck and Sherman
1968). For the separation of malathion and parathion, whenever
necessary, the solvent system acetone : benzene : n-hexane
(25:10:65) was used (Greve 1972).
The solvents were purified in the usual way and freshly
distilled fractions were taken daily for the detection by enzyme
inhibition.
Silver nitrate (AgN03) reagent for detection (Beck and Sher-
man 1968): a solution of 2.0 g AgNOs in 25 ml of bidistilled water
was filled up to 100 ml with acetone and left to stand 10-14 days
in the refrigerator before use.
Enzyme solution (Ortloff and Franz 1965) : a 10% aqueous
solution of rat plasma diluted with Sorensen's buffer pH 8.0 to
volume ratio 1:1.
60
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Substrate solution A (Ortloff and Franz 1965) :.a 0.5%
solution of N-methylindoxylacetate in the solvent system acetone
: water (2:3).
Substrate solution B (Mendoza and Shielde 1971) : 1 mg of
indophenylacetate was present in each milliliter of absolute
ethanolic solution.
For densitometric measurements the Camag T-Scanner (Camag,
Miittenz, Switzerland) was used. This procedure was the following:
the volume applied to the TL was 10 yl throughout. After devel-
opment and evaporation of solvents, the chromatograms were
sprayed with the reagent for detection. Uniformly colored spots,
suitable for quantitative evaluation, appeared after the plates
were warmed for 30 minutes at 135°C.
The spots were brown for dichlorvos, yellow-brown for tri-
chlorfon, dark yellow for malathion and pale yellow for parathion.
Densitometric measurements were performed with a VIS light source
and primary filter (A = 460-470 run) only.
Detection by enzyme inhibition: the developed and dried
chromatograms with dichlorvos and/or trichlorfon spots were
uniformly sprayed with the enzyme solution and placed for 30
minutes in a moisture saturated atmosphere. Wet plates were
then cautiously sprayed with the substrate solution A. After a
few minutes dark violet spots became visible in UV light (366 nm)
against the intensively fluorescent background. After spraying
with the substrate solution the chromatograms were placed in a
dark place and measured after 30 minutes.
Densitometric measurements were performed with a VIS light
source and a primary filter (A = 360 nm) and a secondary filter
(A = 415 nm) .
Bromination (Mendoza and others 1968) : parathion and mala-
thion were oxidized by exposing the chromatograms to bromine
vapors for 5 minutes. After this time the adhering bromine was
expelled by flushing the chromatograms with air for 20 minutes.
The plates with a 0.25 nm thick layer were sprayed with the
enzyme solution and placed for 30 minutes in a tank saturated
with water vapor.
The wet chromatograms were sprayed with substrate solution
B and dried at 37°C during 20 minutes. Light yellow spots on a
pink background were measured with a VIS light source using the
secondary filter (A = 510 nm) only-
61
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Results and Discussion
The calibration curves for the quantitative determination
of organophosphorus pesticides were constructed by taking the
ratio of sample peak area to standard peak area as the ordinate
to eliminate possible variations in experimental conditions. The
peak areas were determined by the Monte Carlo method (Turina
et al. 1974). The ranges of linearity and reproducibilities
obtained for the studied pesticides witii the procedures described
are shown in Table 54.
As the conversion of trichlorfon into dichlorvos is a spon-
taneous process the assessment of its actual quantity at a given
moment has been the objective of numerous studies. Considering
the different toxicities of trichlorfon and dichlorvos on one
hand, and the readiness for conversion under biological condi-
tions on the other, the usefulness of the procedure which enables
simultaneous determination of both pesticides becomes evident.
In the experiments using silver nitrate as the reagent for
spot detection, no evidence was found that the conversion of
trichlorfon to dichlorvos takes place in the course of analysis.
The reproducibility of results, for the two compounds present
simultaneously, was acceptable for the amounts of 0.1-1 yg and
1-4 yg for dichlorvos and trichlorfon respectively. However,
the much higher sensitivity obtained by spot detection based on
cholinesterase inhibition by organophosphorus pesticides proves
that the conversion goes on continuously.
Thin-layer chromatographic assay of trichlorfon using spot
detection based on enzyme inhibition yielded an intense spot
with an Rf value characteristic of dichlorvos and a slightly
visible spot with the Rf value of trichlorfon (Table 53). The
determinations of trichlorfon performed at fixed time intervals
from the start of the experiment and extrapolation to zero time
make it possible to assess directly, but only approximately, the
trichlorfon initially present.
The measurement of fluorescence-quenching intensity as a
function of the time elapsed between the spraying with the sub-
strate and the densitometric measurement showed reproducible
values within the interval of 5-65 min. As the maximum value
is reached after about 30 minutes the spots of the standard were
measured in the 28th and 34th minute and those of the sample in
the 30th and 32nd minute.
The bromination procedure was used for the conversion of
parathion and malathion to the corresponding oxygen derivatives
suitable for the detection by enzyme inhibition. It was shown
by a series of experiments that the sensitivity of detection
does not depend on the bromination time for malathion. However,
the best results for parathion were obtained with the bromination
time of 5 minutes.
62
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The described analytical procedures under carefully defined
experimental conditions were successfully applied for the deter-
mination of organophosphorus pesticides in the aqueous environ-
ment and also in biological samples for toxicological research.
Summary
A method for quantitative thin-layer chromatographic analysis
of organophosphorus pesticides is described. The spots visualized
with silver nitrate or by enzyme inhibition were quantitatively
evaluated by direct densitometric measurements of chromatograms.
The detection with silver nitrate, although neither selec-
tive nor sensitive, proved to be useful for the determination
of dichlorvos and trichlorfon present simultaneously in concen-
trations of 0.1-1 yg/10 yl and 1-4 yg/10 yl respectively.
63
-------
180
160
UO
120
>100
•jjr
HI
a 80
LLJ
or
"- 60
40
20
-
-
•
•
r
---
^M^
PLASMA
N = 400
X = 1.34
SD = 0.33
WHOLE BLOOD
N = 408
X = 5.10
SD = 0.94
0.35 1.05 1.75 2.45 3.15 3.85 4.55 525 5.95 6.65 7.35
ACTIVITY (p moles of thiocholine/min/ ml)
Figure 1.-Distribution of plasma and whole blood cholinesterase
activity in nonexposed humans
aos
-------
PLASMA
WHOLE BLOOD
50
40
^ 30
0 20
FREQUEN
*- -»
0 0
30
20
10
m
•
-
-
•
•
•
•
FEMALES
-| N = 148
"r-T
—
X = 1.24
SD = 0.31
•••
-,
MALES
N = 252
^^m
X = 1.39
SD = 0.32
, — h- 1
•
•
rvT
•
•
•
H
^^m
H
—
n
FEMALES
N =151
X = 4.54
SD = 0.84
^M>
Tru
MALES
N=257
X = 5.37
SD = 1.01
___ ___
r
— ~~|
0.35 1.40 2.45 2.45 3.85 525 6.65 8.05
ACTIVITY (jj mole of thiochoLine /min/ml )
Figure 2.- Distribution of plasma and whole blood
cholinesterase activity in relation to sex
in nonexposed humans
65
-------
100
90
60
40
20
100
^o
0 80
60
lU
20
100
a:
LU 80
i-
tn
UJ 60
z
-J 40
O
X
O 20
100
80
60
40
20
o—
U _n.
1972
n n R
1973
1974
n
n n n
n
n
n
1975
n
I II III IV
V VI VII VIII IX X XI XII
MONTHS
30
20
10
30
20
10
a:
o
30
20
10
30
20
10
Figure 3.-Whole blood (•) and plasma (o) cholinesterase
activity in exposed workers. Number of work
days is shown by means of bars (D).
66
-------
TABLE 1. LIST OF COMPOUNDS HANDLED BY OCCUPATIONALLY EXPOSED WORKERS
ISO name
Synonym Formula
AZINPHOS-ETHYL Gusathion A (C2H50)2 P(S)-S-CH2—N'
BROMOPHOS
Nexion
Cl
(CH30)2P(S)-0- « VBr
\^=
Cl
CARBARYL
Sevin
0-C(0)-NHCH,
CHLORFENVINPHOS
Birlane
Sapecron
(C2H50)2P1Q)-OC=CHC1
Cl
Cl
DIAZINON
Bausudin
Neocidol/
Nucidol
CH-
N^S:H
7-rC^ J-0-P(S)(C2H50)2
continued
67
-------
TABLE 1. (continued)
ISO name
DICHLORVOS
DIMETHOATE
DIOXACARB
FENTHIQN
METHIDATHION
MONOCROTOPHOS
Synonym
DDVP
Nuvon
Nogos
Vapona
DDVF
Dederap
Rogor
Roxion
Cygon
Dimetate
Fostion MM
Perfekthion
Elocron
Famid
Lebaycid
Tiguvon
Baytex
Baycid
Entex
Mercaptophos
Qualeton
Ultracide-
Geigy
Supracide
Nuvacron
Azodrin
Formula
(CH30)2P(0)-0-CH=CC12
(CH30)2P(S)-S-CH2-CO-NHCH3
Q-C(0)-NHCH3
\ / V0 — CH2
(CH30)2P(S)-0-/ VsCH3
I ......
CH3
ffH 0^ Pf^} ^..TH N-.I — - -.. . .r-n
d i L\ \
V
OCH3
(CH30)2P(0)-0-C(CH3)=CH-C(0)-NHCH3
continued
68
-------
TABLE 1. (continued)
ISO name
NALED
PARATHION
Synonym Formula
Dibram (CH30)2P(0)-0-CHBr-CBrCl2
Folidol (C?Ht.O)?P(S)-0-/ \-NO?
Thiophos ' D \ /
Bladan
Niran
Fosferno
SNP
PHORATE
Thimate
PHOSALONE
Zolone
(C2H50)2P(S)-SCH2-N
THIOMETON
Ekatin
(CH30)2P(S)-S-(CH2)2-S-C2H5
ZINEB
Dithane Z-78
Parzatezineb
H2CNH-C(S)S
Zn
H2CNH-C(S)S
69
-------
TABLE 2. DAYS OF EXPOSURE PER WORKER (PRODUCTION OF EMULSIONS)
Year
1971
1972
1973
1974
1975
Phosalone
(35*)
10.3
17.6
72.8
95.0
88.0
Chlor-
fenvinphos
(24*)
37.5
56.4
2.3
0
0
Dichlorvos
(50%)
0.4
10.5
7.3
10.0
5.0
Dimethoate
(40%)
26.6
3.5
1,1
3.0
2.0
Naled
(64.5%)
-
4.1
0.3
0
0
Mono-
crotophos
(20%)
-
-
8.9
1.2
1.0
Total
(days)
74.8
92.3
92.7
109.2
96.0
The concentration of the active matter in the final product is given in parentheses.
-------
TABLE 3. DAYS OF EXPOSURE PER WORKER (PRODUCTION OF SPRAYS)
Year
197;
1972
1973
1974
1975
Dichlorvos
(0.8%)
60
48
77
72
85
Dichlorvos
(0.3%)
Bromophos
(0.8%)
-
-
26
48
57
Bromophos
(0.1%)
120
48
27
30
22
Total
(days)
180
96
130
150
164
The concentration of the active matter in the final product is given in
parentheses.
71
-------
TABLE 4. DAYS OF EXPOSURE PER WORKER (PRODUCTION OF POWDERED INSECTICIDES)
Year
1973
1974
1975
Z1neb
(65%)
9.
-
-
Dioxacarb
(2.5%)
186
117
96
Carbaryl
(50%)
85
48
-
Phosalone-
suspension
(30%)
8
-
188
Total
(days)
288
165
284
The concentration of the active matter in the final product is given in
parentheses.
TABLE 5. AVERAGE NUMBER OF OVERTIME WORK HOURS PER WORKER IN THE PRODUCTION
OF EMULSIONS
Year Phosalone Chlorfenvinphos Dimethoate (ho )
1971 13.6 35.6 28.9 78.1
1972 5.0 49.8 6.1 60.9
72
-------
TABLE 6. THE RELATIONSHIP BETWEEN BLOOD CHOLINESTERASE ACTIVITITY AND SIGNS
AND SYMPTOMS OF POISONING IN WORKERS MANUFACTURING ANTICHOLINESTER-
ASE PESTICIDES IN ONE PLANT
Year
1971
1972
1973
1974
1975
Inhibition of cholin-
esterase activity in
whole blood (%)
0 -
25 -
50 -
75 -.
0 -
25 -
50 -
75 -
0 -
25 -
50 -
75 -
0 -
25 -
50 -
75 -
0 -
25 -
50 -
75 -
25
50
75
100
25
50
75
100
25
50
75
100
25
50
75
100
25
50
75
100
Number of individuals
without
complaints
59
18
5
82
99
6
105
47
19
1
67
56
37
9
102
83
71
6
1
with complaints
and/or symptoms
1
14
6
3
24
1
1
4
1
1
6
14
2
1
17
1
9
8
1
161
19
Total
517
67
73
-------
TABLE 7. SYMPTOMS OF POISONING AND CHOLINESTERASE ACTIVITIES OF EXPOSED
WORKERS
Subjects Exposure Duration
to of
Compound Exposure
M.B,
S.DJ.
S.V.
T.Z.
J.Z.
G.R.
K.J.
J.B.
K.M.
R.Z.
Dimethoate 2 months
Phosalone
" 40 days
2 months
15 days
7 days
33 days
30 days
21 days
15 days
12 days
Signs and symptoms
recorded
loss of appetite,
weakness
tiredness, exhaustion,
tightness in chest,
cough
stomach
discomfort
hand-sweating,
headache
vomiting, weakness,
salivation,
tiredness, abdominal
cramps
vomiting, slower
pupillary reaction
weakness, exhaustion
weakness, headache,
exhaustion
tiredness, sleepiness,
myosis
abdominal cramps,
Whole blood ChE
activity (%)
while
having
symptoms
55
59
49
56
24
37
44
40
9
21
two
weeks
later
70
65
69
76
48
66
62
53
22
38
C.J. Dimethoate 34 days
Phosalone,
Dichlorvos
vomiting
tiredness, sleepiness,
headache
continued
54
72
74
-------
TABLE 7. (continued)
Subjects Exposure
to
Compound
D.B.
N.Z.
M.K.
K.M.
K.S.
V.J.
B.V.
Dimethoate
Phosalone
Dichl orvos
Dimethoate
Phosalone
n
Dioxacarb
Carbaryl
ii
Phosalone
OP and car-
bamate in-
secticides
Duration
of
Exposure
7 days
3 months
3 months
4 months
Signs and symptoms
recorded
weakness,
vomiting
weakness,
headache,
sickness
headache,
dizziness
tightness
headache,
sweati ng ,
sickness,
in chest,
Whole blood ChE
activity (%)
while two
having weeks
symptoms later
24 42
36.5 68
52.7 62.7
37.5 83.1
stiff extremities
4 months
20 days
Continuous
laboratory
work
dizziness
vomiting
headache,
sweating,
vomiting
, sickness,
sickness
tiredness,
41.5 87.2
51.4 81.4
47.8 70.7
B.J.
weakness, vomiting,
suffocation
44.4
71.1
75
-------
TABLE 8. BLOOD AND PLASMA CHOLINESTERASE ACTIVITY DURING A WORKDAY*
Worker
1
2
3
4
5
6
7
8
9
10
Enzyme
preparation
blood
plasma
blood
plasma
blood
plasma
blood
pi asma
blood
pi asma
blood
plasma
blood
plasma
blood
pi asma
blood
plasma
blood
plasma
Blood and pi
measured at
90
72
90
100
98
90
87
100
78
82
93
83
96
92
88
90
95
99
93
100
asma cholinesterase activity
(%)
6 a.m. measured at 1 p.m.
81
65
83
100
97
90
82
100
71
74
93
83
90
78
87
90
94
97
94
98
* Measured at the beginning and at the end of a work shift in workers from
the production of emulsion (exposure to phosalone).
76
-------
TABLE 9. THE MEAN VALUE OF BLOOD CHOLINESTERASE ACTIVITY IN WORKERS ENGAGED
IN THE FORMULATION OF THIOMETON AND DICHLORVOS*
Year
1970
1971
1972
1973
1974
1975
Date
4
20
8
20
1
30
19
10
17
13
19
11
16
23
9
20
3
10
21
10
February
March
April
May
September
October
April
June
June
April
April
July
July
July
April
February
March
July
July
November
Mean
85
74
72
62
79
92
72
67
63
91
80
47
63
65
91
72
75
68
66
92
± SEM
2.41
3.89
2.21
2.67
3.81
2.67
5.76
6.58
3.20
3.27
7.51
6.47
4.77
6.87
2.44
3.34
7.74
2.98
3.18
1.60
N
20
22
19
14
18
14
9
12
12
10
9
12
18
7
19
22
8
17
19
24
Expressed in percent of their preexposure values.
77
-------
TABLE 10. THE MEAN VALUE OF WHOLE BLOOD AND PLASMA CHOLINESTERASE ACTIVITY
IN AGRICULTURAL WORKERS*
1973
April
May
After 10 days of exposure
After 20 days of exposure
Whole blood Plasma
Whole blood Plasma
Mean
SEM
100
± 2.57
99
± 3.12
98
± 3.25
98
± 4.20
N
10
8
8
1974
1975
April
After 16 days of exposure
Mean
SEM
Whole blood
87
± 1.49
Plasma
89
± 1.47
44
43
Apri 1
After 16 days of exposure
Mean
SEM
Whole blood
95
± 1.08
Plasma
93
± 1.65
34
33
Expressed in percent of their preexposure values,
78
-------
TABLE 11. THE MEAN VALUE OF WHOLE BLOOD AND PLASMA CHOLINESTERASE ACTIVITY
IN ORCHARD SPRAYMEN*
1973
Mean
SEM
N
Whole
100
± 1.
8
27 April
blood Plasma
100
13 ± 3.44
8
26 May
Whole blood
93
± 5.35
8
Plasma
89
± 3.36
8
2 July
Whole blood
86
± 3.82
6
Plasma
75
± 7.24
6
1975
Mean
SEM
N
26 May
(after 2-3 days of
Whole blood
94
± 1.35
33
exposure)
Plasma
95
± 1.47
32
30 May
(after 2-3 days of
Whole blood
97
± 1.26
20
exposure)
Plasma
94
± 2.35
20
* Expressed in percent of their preexposure values.
79
-------
TABLE 12. MEAN NUMBER OF LEUKOCYTES AND LYMPHOCYTES IN WORKERS EXPOSED TO
ORGANOPHOSPHORUS COMPOUNDS AND IN CONTROL GROUP
Subjects
(number)
Exposed
workers
(9)
Control
group
(48)
Mean values
1966 - 1969
1970 - 1973
leukocytes lymphocytes leukocytes lymphocytes
per 100 leuk. per 100 leuk.
7270
35
6964
6164
32
35
80
-------
TABLE 13. OPHTHALMOLOGICAL FINDINGS IN HUMANS EXPOSED TO PESTICIDES,
SUMMARY OF AN EPIDEMIOLOGICAL SURVEY
SUBJECTS (NUMBER)
EXPOSURE
FINDINGS
REFERENCE
00.
Children (324) and
adults (255)
Occupat. exposed
(200) and control
subjects (50)
Adult patients (51)
Patients "with OP
poisoning" (262)
School children
(40,000)
Children (4-16 yrs)
(71) and students
(20)
School children
(13,351)
General exposure in
agricultural area
Not specified
Not specified
Not specified
General exposure to
pesticides applied in
agriculture
Diminished visual acuity,
narrowing of the peripheral
visual field, myopia and
occasionally astigamatism
Hearing and eye problems
(unspecified) significantly
higher in exposed subjects
Swollen disc, atropic disc,
cataract, disturbance of smooth
pursuit motion of the eye
Unspecified eye damage
Myopia
General exposure in two Reduced vision, narrowing of
different areas the vision, refraction anomaly,
affected eye movement
General exposure in
agricultural area
Only in 2 cases unexplained
constriction of visual
fields
Ishikawa, 1973
Kwalick, 1971
Ohto, 1971
Uomo, 1972
Tamura and
Mitsui, 1975
Ishikawa, 1971a
Futenma, 1973
Continued
-------
TABLE 13. (continued)
SUBJECTS (NUMBER)
EXPOSURE
FINDINGS
REFERENCE
School children by
random (100)
Children (5-15 vrs)
din. cases (53)
Children (58)
Children from nursery
« school (131)
Children (9-11 yrs)
(30,000)
Children with OP
related eye disorders
(22)
Adults with OP related
eye disorders (215)
Agricultural workers
(1955) and students
(2272)
General exposure in
agricultural area
(SAKU)
General exposure in
agricultural area
(SAKU)
17 exposed (?) to
pesticides
General exposure - in
agric. area (Tokushima)
General exposure -
town population versus
agric. population
Not specified
Not specified
Organophosphates
5-7% ocular symptoms, visual
field constriction, poor vision:
astigmatism
Disturbed visual acuity, nar-
rowing of visual field, astig-
matism, disturbed smooth pursuit
motion
Ishikawa, 1972a
Ishikawa, et al
1970
Disturbed visual acuity, congested Tsukahara, 1972
and discolored papillae
Narrowing of visual field (in 26) Mitsui, 1972
disturbed sight (in 19), abnormal
eyeground (8)
No great difference of town
versus agricultural population
Damaged retina, narrowed
vision
Padrin proved effective for
recovery of eyesight
Aberration in intraocular
pressure
Kato, 1973
Takaku, 1972
Takaku, 1972
Dugelnij, 1971
Continued
-------
TABLE 13. (continued)
SUBJECTS (NUMBER)
EXPOSURE
FINDINGS
REFERENCE
CO
10
Farmers handling
pesticides (164)
Farmers handling
pesticides (60) and
other agricultural
workers (716)
Agricultural workers
handling pesticides
(33)
Agricultural workers,
formerly poisoned
(114)
Pesticide applicators
(12)
Outpatients with
signs of pesticide
injury (253)
Occupational ly
poisoned workers
(240)
Organic mercury,
chlorinated hydro-
carbons, organo-
phosphates for more
than 20 yrs
Constricted visual field in
25%, disturbed accommodation
in 29%
Different pesticides Pterygium, conjunctivitis
Malathion, DDT,
dithiocarbamates
Organophosphorus
compounds
Fenitrothion and
dichlorvos 2 to 9 yrs
Not specified
Different pesticides
7-20 yrs
Lacrimation, photophobia,
refraction anomalies,
pterygium, increased intra-
ocular pressure
Subjective loss of eyesight
in 8 workers
No abnormalities
Myopia, astigmatism, narrowing
of visual field
Increased intraocular pressure,
glaucoma in 4.9%, constricted
visual field, retinal
angiopathy
Imaizumi, et al.
1971
Medvedovskaya,
1967
Medvedovskaya
and Golenko,
1971
Tabershaw and
Cooper, 1966
Misumi, et al.,
1975
Nakamura, et al.,
1971
Nuridtinova,
1973
Continued
-------
TABLE 13. (continued)
SUBJECTS (NUMBER)
EXPOSURE
FINDINGS
REFERENCE
00
Workers (683)
Workers and
farmers (60)
Agricultural
workers (412)
Agricultural
workers (?)
Agricultural
workers (77)
Farmers (25)
Pesticide appli-
cators (200)
Occupational exposure
up to 18 yrs
Occupational exposure
(application,
formulation)
Handling DDT and
trichlorphon, 2 to
10 yrs
Chlorothene
Tetramethylthiuram
disulphide
Chlorthalonil (mostly)
and OP compounds
Not specified
Eye abnormality in 90%, narrowing Nuritdinova,
of visual field, distrophic
changes of iris increased dark
adaptation time
Cortical wedging
Glaucoma in 6.5%, corneal
abnormalities
Epyphora, photophobia,
conjunctivitis, constricted
visual field, increased
intraocular pressure
Conjunctivitis, decreased corneal
sensitivity, increased intra-
ocular pressure
Dimness of vision, con-
junctival hyperemia
Corneal irritation, photophobia
lacrimation, formation of
corneal vessels
1975
Pietsch, 1972
Zarya, 1973
Sivitskaya and
Alekseicuk,
1973
Sivitskaya, 1974
Hanyu and Tsuji,
1974
Ito, 1973
Continued
-------
TABLE 13. (continued)
SUBJECTS (NUMBER)
EXPOSURE
FINDINGS
REFERENCE
00
(Jl
Pesticide plant
workers (90)
Agricultural workers
(260)
Children (after OP
poisoning) and
adults (34)
Pilots (17)
Adults (5)
Organochlorine, mercury, Conjunctiyal hyperemia, tearful
OP and carbamates, eyes, diminution of adaption
6-10 years to dark, color vision
disturbance, visual acuity
troubles
Parathion and other
OP comp., granosan,
aldrin, DNOC
19 children presumed
to be victims of OP
poisoning, others
normal children and
adults
TEPP
OP compounds
Blepharoconjunctivitis,
pherygium, dystrophy of iris,
color vision impaired, narrowing
of visual field, reduced dark
adaptation
Greater threshold intensity
among children exposed
to pesticides
No abnormalities
Disturbed visual acuity,
visual field tenosis,
abnormal motion of
eyeballs
Kalid-Filipovi
et al., 1973
Glazko, 1970
Ohba, et al.,
1972
Upholt et al.,
1956
Fuji, et al.,
1975
-------
TABLE 14. SOME DATA ON THE OPHTHALMOLOGICALLY EXAMINED WORKERS
00
o\
Number of
Group workers examined Age
Male Female Total mean
A 14 17 31 22 -
42.2
B 10 2 12 30 -
42.3
Range
± S.E.
60
(±1.6)
51
(±1.9)
Type of work
Pesticide pro-
duction and
formulation
Pesticide applica-
tion and super-
vision of production
20 0 20 28 - 50 Internal
transport and
work in stores
-------
TABLE 15. COMPLAINTS OF EXAMINED WORKERS CONCERNING EYE AND SIGHT
Complaint
Lacrimation
Photophobia
Burning and
itching
Frequent
conjunctivitis
Other complaints
No complaints
A
11
6
4
1
5
10
Group
B
2
1
4
2
1
4
C
0
0
1
1
5
13
87
-------
TABLE 16. RESULTS OF EXTERNAL EYE EXAMINATION IN EXPOSED AND CONTROL SUBJECTS
Findings
Conjunctiva!
infection
Dilated episcleral
vessels
Abnormal pupils
Other abnormalities
Normal
A
16
12
2
2
4
Group
B
0
5
1
2
5
C
8
3
1
1
9
88
-------
TABLE 17. CORNEAL CURVATURE IN EXPOSED AND CONTROL SUBJECTS
CO
Clinical parameter Eye
Difference between
horizontal and vertical
meridian (expressed in Right
diopters)
Left
Corneal radius
(horizontal and
vertically expressed ' Right
in mm)
Left
Statistical
parameter
n
T
S.E.
n
T
S.E.
n
T
S.E.
n
J
S.E.
A
29
0.59
0.08
28
0.57
0.07
17
7.91-7.80
0.07-0.08
16
7.89-7.81
0.08-0.08
Group
B C
12 20
1.03 0.57
0.242 0.10
12 20
1.05 0.69
0.246 0.13
20
7.95-7.84
0.07-0.06
20
7.92-7.83
0.07-0.06
-------
TABLE 18. VISUAL ACUITY IN EXPOSED AND CONTROL SUBJECTS BEFORE AND AFTER CORRECTION
ID
o
Eye
Right
Left
Visual
acuity
> 1.0
= 1.0
< 1.0
> 1.0
= 1.0
< 1.0
A
Before
5
18
8
5
17
8
After
9
19
3
9
20
1
Group
B
Before
0
8
4
1
7
4
After
0
11
1
1
8
3
C
Before
13
2
5
13
3
4
After
13
6
1
14
5
1
-------
TABLE 19. INTRAOCULAR PRESSURE IN EXPOSED AND CONTROL SUBJECTS MEASURED
BY SCHIOTZ' TONOMETER AND CONVERTED TO MILLIMETERS MERCURY
Group
Eye Statistical Significance
parameter ABC
n 28 12 17 (A:C) p<0.01
Right I 16.24 16.15 18.75 (B:C) p<0.01
S.E. 0.57 0.97 0.61
n 27 12 17 (A:C) p<0.01
Left I 15.87 14.92 18.49 (B:C) p<0.01
S.E. 0.59 1.05 0.54
91
-------
TABLE 20. NUMBER OF PATIENTS WITH ABNORMALITIES OF THE EYEGROUND
Clinical findings
Abnormal retinal
arteries or veins
Degenerative or
chorioretinitic
changes
Changes of the
papilla
Other findings
Normal
A
3
5
3
1
20
Group
B
3
0
0
3
6
C
3
1
4
0
12
92
-------
TABLE 21. THE RESULTS OF PERIMETRIC MEASUREMENT OF THE RIGHT EYE IN EXPOSED WORKERS
Degree
Isop- Group
ter 0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345
T7T A 21 20 18 17 16 15 15 15 15 15 16 16 16 16 17 17 17 17 18 18 19 20 20 20
* ** ** ** **
C 22 21 19 18 17 16 15 16 16 16 16 17 19 19 19 19 19 19 19 20 21 22 22 23
B 21 21 19 18 16 16 16 16 17 17 17 18 17 18 18 18 18 18 18 18 20 21 22 23
A 38 36 34 31 28 27 26 26 27 28 28 29 29 29 30 30 31 31 32 34 36 37 38 38
* * ** ** ** ** ******* * *
******
1/2 C 41 39 36 33 30 28 27 28 29 30 32 32 33 33 33 33 34 34 35 36 38 40 41 42
*****
B 39 38 35 32 30 28 27 28 28 29 29 30 30 30 30 30 31 31 33 34 36 38 39 39
A 55 52 48 44 40 38 36 36 37 39 40 41 41 42 42 43 43 44 46 48 51 54 56 56
* * ** ** ** ** ** ** ** -**
*******
1/3 C 58 55 51 46 42 3? 37 37 38 40 42 43 44 45 45 46 47 48 48 51 53 56 58 59
** ** **
* *
B 58 53 49 46 43 41 39 39 40 43 43 43 44 43 43 43 43 45 45 49 53 56 58 59
A 75 70 63 58 53 49 47 48 49 50 52 54"54 54 55 55 56"58 61 65"70 74 76 77
* ** *
1/4 C 75 70 65 57 52 48 47 46 47 49 52 53 55 56 57 57 59 60 62 65 70 74 76 78
B 77 70 64 58 53 51 49 49 50 52 55 55 56 56 56 56 57 59 62 67 73 77 80 80
*=p<0.05; **=p<0.02; **.=p<0.01; **.=p<0.001
In group A, n=24 and B, n=10 and in control subjects Group C, n=23. Each figure is a mean value of the
group expressed in degrees.
-------
TABLE 22. THE RESULTS OF PERIMETRIC MEASUREMENT OF THE LEFT EYE IN EXPOSED WORKERS
Degree .-- —
Isop- Group
ter 0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345
A 16 16 15 15 15 15 15 16IT19 21 22 23 22 22 21201918 1717171616
I/I C 18 18 17 16 16 15 15 16 17 18 20 22 23 23 23 22 21 20 19 18 18 18 18 18
B 17 17 17 16 16 16 16 16 16 17 18 19 19 20 20 19 18 17 16 16 16 16 17 17
A 29 29 28 28 27 27 26 28 30 33 36 38 39 39 39 3735 33 3131312929 29
* * * ****** ** ** *
* * * *
1/2 C 30 31 30 29 28 27 27 28 30 33 37 40 43 42 42 40 38 36 34 34 33 32 32 31
* ** ** ** ** ** ** ** **
** ** ** ** *
B 28 28 28 28 28 27 28 30 30 32 36 38 39 39 38 36 34 31 30 29 29 29 29 28
A 41 41 40 39 38 38 38 39 42 46 52 56 58 57 57 54 50 46 44 43 42 42 41 41
vo ** * ** ** ** ** ** ** ** *
4^ * ** ** ** * *
1/3 C 43 43 42 41 39 38 37 39 42 46 53 57 59 60 60 57 54 50 47 46 45 44 44 43
* ** ** ** ** ** **
*****
B 41 41 40 39 39 38 39 41 44 48 54 57 58 58 56 53 49 46 44 43 42 42 41 41
A 55 54 53 51 50 48 49 50 54 60 63 74 77 77 77 73 68 64 60 58 57 56 55 55
1/4 C 55 55 54 51 48 47 47 49 54 60 68 74 77 78 78 75 70 65 62 59 57 55 55 55
*
B 55 55 54 52 50 49 49 52 57 64 72 77 79 79 78 74 68 64 61 57 77 56 55 55
* = p<0.05; ** = p<0.02; ** = p<0.01; JJ = p<0.001
In Group A, n=23 and B, n=10 and in control subjects Group C, n=23. Each figure is a mean value of the
group expressed in degrees.
-------
TABLE 23. DARK ADAPTATION IN EXPOSED WORKERS AND CONTROL SUBJECTS
Minutes
Group 1 2 3 4 5 6 7 8 9 10 11 13 15 17 19 21 23 25
(n-?n\ 5-28 4-89 4-67 4-54 4-46 4-30 4.12 3.93 3.73 3.58 3.43 3.20 3.05 2.94 2.84 2.78 2.71 2.68
\i\-z\j) + +# ++ #* ** **
* ***** *
(n=20) 5<26 4'86 4'64 4>5° 4'41 4'28 4'09 3'89 3<7° 3'53 3'36 3'06 2'84 2^68 2'56 2'48 2'41 2'37
/85j 5.21 4.86 4.70 4.42 4.31 4.33 4.04 3.87 3.70 3.61 3.45 3.20 3.06 2.92 2.82 2.73 2.70 2.61
^ * = p
-------
TABLE 24. LIST OF COMPOUNDS DISCUSSED IN SECTION SIX
Name (abbreviation)
Formula
0,0-Dimethyl phosphate (DMP)
0,0-Diethyl phosphate (DEP)
0,0-Dimethyl-0-amyl phosphate (DMAP)
0,0-Diethyl-0-amyl phosphate (DEAP)
0,0-Dimethyl-S-amyl phosphorodithioate (DMADTP)
0,0-Diethyl-S-amyl phosphorodithioate (DEADTP)
0,0-Diethyl phosphorodithioate (DEDTP)
0,0-Diethyl phosphorodithioate potassium salt (DEDTPK)
Phosalone
(CH30)2P(0)OH
(C2H50)2P(0)OH
(CH30)2P(0)OC5HU
(C2H50)2P(0)OC5Hn
(CH30)2P(S)SC5Hn
(C2H50)2P(S)SC5Hn
(C2H50)2P(S)SH
(C2H50)2P(S)SK
-CH2-S-P(S)(OC2H5)2
= 0
-------
TABLE 25. OPERATING CONDITIONS FOR THE GAS CHROMATOGRAPHIC ANALYSIS OF
TRIALKYL PHOSPHATES
Column temperature 190-195°C
Injection block temperature 225-230°C
Detector block temperature 235-245°C
Nitrogen (carrier) flow rate 30-45 ml/min
Hydrogen flow rate 35± 3 ml/min
Airflow rate 235±10 ml/min
97
-------
TABLE 26. GAS CHROMATOGRAPHIC DATA FOR TRIALKYL PHOSPHATES, ALKALI
FLAME DETECTION SYSTEMS*
Compound
DMAP
DEAP
DMADTP
DEADTP
Retention
time (min)
1.1
1.4
1.8
2.3
Sensitivity (pg)
4:1 signal/noise
9
12
7
7
* Column 4% SE-30 and 6% OV-210. Temp. 195°C; carrier flow 40 ml
N2/min.
98
-------
TABLE 27. RECOVERIES OBTAINED FOR THE CRITICAL STAGES OF THE PROCEDURE
vo
vo
Alkylating reagent
prepared freshly
ALKYLATION Alkylating reagent
Prepared earlier
Alkylation time
ALKYLATION
AND COLUMN
CHROMATOGRAPHY
EXTRACTION,
ALKYLATION
AND COLUMN
CHROMATOGRAPHY
0.5 ml added
1.5 ml added
2.0 ml added
2 days
3 days
1 week
10 min
20 min
30 min
Mean values
DMAP
54 (4)
62 (4)
64 (6)
48 (4)
41 (6)
48 (6)
61 (4)
49 (4)
47 (4)
45 (8)
RECOVERY (%)
of (n) determinations
DEAP DEADTP
21(10) 23 (9)
-------
TABLE 28. AMOUNTS OF DEADTP FOUND IN URINE SAMPLES OF UNEXPOSED SUBJECTS
DEADTP (rig/ml urine)
Subject
Found Recovery (%)
M.S. < 7 19
M.S. < 7 20
V.B.S. < 7 32
J.B. < 7 21
J.S. < 7 28
B.T. < 7 24
V.B.S. 33 14
M.S. < 7 21
B.T. < 7 25
100
-------
TABLE 29. AMOUNTS OF DEADTP FOUND IN URINE SAMPLES AND BLOOD CHOLINESTERASE
ACTIVITIES IN OCCUPATIONALLY EXPOSED WORKERS
Subject
S.B.
R.V.
J.K.
M.V.
I. P.
I.H.
B.J.
I.S.
S.B.
J.S.
S.V.
J.H.
S.D.
DEADTP
Found
105
34
22
43
28
25
30
35
22
16
41
<7
12
(ng/ml urine)
Recovery (%)
13
19
20
16
12
13
19
24
19
22
29
25
25
Cholinesterase activity
(% of preexposure value)
Whole blood
100
93
94
100
100
98
100
73
86
93
92
100
100
Plasma
100
96
100
97
99
90
98
100
85
99
100
86
100
101
-------
TABLE 30. CONCENTRATIONS OF CHLORINATED HYDROCARBONS IN 147 SAMPLES OF
HUMAN PLASMA OR SERUM
Concentration
(ppb)
^uuijjuuim
a-HCH
Lindane
Aldrin
p.p'-DDE
p,p'-DDD
p,p'-DDT
3
4
12
35
11
22
Mean
.25
.09
.2
.3
.6
.7
±
± 0
± 0
± 3
± 1
± 2
± 3
SEM
.50
.62
.0
.5
.7
.3
(n)*
(57)
(23)
(22)
(147)
(7)
(20)
Range
0
0
0
8
3
2
.11
.45
.51
.4
.0
.2
- 15
- 15
- 50
-118
- 23
- 81
* (n) is the number of samples which contain the particular compound.
102
-------
TABLE 31. ACUTE TOXICITY OF METRIFONATE AND DDVP IN RATS
o
CO
Compound
Metri-
fonate
DDVP
Route of
Admini-
stration
Oral
Intra-
venous
Oral
Intra-
venous
2 4
LD50 LD50 Oral LD50
(mg/kg) (mg/kg) I.V. LD50
179 1237
(147-218) (-)
2.74
450
(401-
505)
85.7
(65-
113)
..... . „ , i z o
— -'-• 13. 3
5.4
(-)
Age (weeks)
8
LD50 Oral LD50
(mg/kg) I.V. LD50
1070
(953-1201)
2.38
450
(401- 505)
79.3
(60-105)
. . in Q
10. y
4.2
(-)
LD50 Oral LD50
(mg/kg) I.V. LD50
505
(-)
1.12
450
(401-
505)
70.7
(59-86)
OO p.
£J. V
3.0
(2.6-3.4)
12
ED50 LDso
(mg/kg) ED50
63.0 8.0
(55-72)
56.2 8.0
(51-62)
12.6 5.6
(10-16)
0.71 4.2
(0.6-0.8)
-------
TABLE 32. MEAN TIME OF THE ONSET OF CHOLINERGIC SYMPTOMS*
Age (weeks)
Route of
Compound
Metn'fpnate
DDVP
Administration
Oral
Intravenous
Oral
Intravenous
2 4
7.6 10,3
(8) (12)
5.0
(16)
2.8
(16)
<1
8
9.6
(12)
5.6
(16)
4.0
(16)
<1
12
11.5
(16)
4.7
(22)
4.9
(16)
<1
* (In minutes) after oral or intravenous administration of metrifonate or
DDVP. The number of rats is given in parentheses
104
-------
TABLE 33. TOLERANCE OF METRIFONATE AND DDVP*
o
en
Compound
Rate of
infusion
JUGULAR VEIN
Until
onset
Until
(I.V. LDso/hr) of symptoms
I S.E.X X
Metn'fonate
DDVP
0.25
0.50
1.0
2.0
4.0
8.0
2.0
4.0
8.0
0.37
0.33
0.44
0.74
0.93
0.65
0.72
0.94
0.07
0.01
0.05
0.08
0.11
0.01
0.05
0.08
3.21
2.43
1.13
1.25
1.40
2.00
17.68
5.24
2.27
death
S.E.JT
0.11
0.21
0.12
0.08
0.19
0.11
0.55
0.13
0.06
Death
Until
PORTAL VEIN
onset
Until
Symptoms of symptoms
X S.E.X X
6.6
3.4
2.8
1.9
2.2
27.2
7.3
2.4
0.39
0.38
0.51
0.77
1.07
~2.6
1.47
1.47
0.04
0.04
0.05
0.04
0.11
0.37
0.22
3.19
2.47
1.25
1.50
1.67
2.27
28.20
11.44
7.13
death
S.E.X"
0.06
0.23
0.12
0.10
0.08
0.11
1.33
0.20
0.56
Death
Symptoms
6.3
3.3
2.9
2.2
2.1
-11
7.8
4.9
Infused into jugular or portal vein and expressed as number of intravenous LD50 doses received
until the onset of cholinergic symptoms or until death.
-------
TABLE 34. BRAIN AND PLASMA CHOLINESTERASE ACTIVITY*
Time after
dosing
30 min
60 "
90 "
120 "
180 "
6 hrs
12 "
24 "
2 days
6 "
8 "
16 "
32 "
Metrifonate (300 mg/kg)
Brain ChE
Plasma ChE
1.7 (0.8) 7.0 (1.7)
6.0 (2.0) 7.0 (0.7)
12.7 (2.2) 14.3 (2.1)
13.8 (2.11
34.6 (3.21
1 19.4 (3.4)
) 33.3 (2.4)
67.5 (2.9) 73.4 (3.3)
67.9 (3.5) 73.8 (6.7)
83.8 (2.6]
94.8 (8.3)
77.5 (3.3) 93.0 (3.2)
82.6 (2.9
77.4 (3.3
87.5 (3.1
95.0 (4.0)
101.9 (2.4)
_
DDVP
Brain ChE
14.9 (1.3)
29.8 (1.1)
36.0 (2.6)
49.8 (1.2)
56.2 (2.1)
85.1 (1.7)
82.6 (3.5)
87.4 (1.8)
89.5 (1.8)
81.9 2.4)
81.9 (3.0)
83.7 (1.3)
(2.5 mg/kg)
Plasma ChE
40.6 (2.1)
44.3 (3.6)
59.2 (4.4
73.7 (5.3
92.7 (8.2
94.2 (5.0)
99.6 (3.5)
102.6 (6.7)
106.2 (7.0)
101.1 (5.1)
89.5 (2.1)
-
96.2 (3.3) -
1
Percent in rats injected intravenously with a single dose of
metrifonate or DDVP. Each figure is a mean value (±S.E.X) of five
to twelve animals.
106
-------
TABLE 35. BRAIN AND PLASMA CHOLINESTERASE ACTIVITY*
Compound
injected
Metrifonate
300 mg/kg
I.V.
DDVP
2.5 mg/kg
I.V.
Sampling after
dosing
Enzyme Time (h)
Brain 0.5
24
Plasma 0.5
24
Brain 0.5
24
Plasma 0.5
24
Number of applications
1
1.7
83.8
7.0
94.8
14.9
87.4
40.6
102.6
2
3.0
78.7
6.7
86.9
22.4
92.7
47.3
120.5
3
4.4
61.8
9.2
59.9
24.2
78.2
49.5
118.7
4
2.1
45.5
3.1
87.0
18.9
64.1
47.3
109.0
5
2.5
42.6
4.7
72.0
13.9
60.5
25.4
107.9
* Percent in rats repeatedly treated with metrifonate or DDVP. Each figure represents a mean
value obtained in four rats. x
-------
TABLE 36. PERCENT CHOLINESTERASE ACTIVITY AND pH:
Quantity of pH
metrifonate
in the sac In the
sac
0 5.0
(control) 9.0
5.0
5.0
_, 5.0
00 50 yg 7.4
9.0
9.0
9.0
5.0
100 yg 7.4
9.0
Time of
In the
medium
9.0
5.0
5.0
7.4
9.0
7.4
5.0
7.4
9.0
7.4
7.4
7.4
5
101
100
98
97
92
94
98
97
97
98
102
15
99
100
96
94
90
99
81
83
73
93
88
83
incubation (min)
20
102
98
99
91
80
76
74
53
86
77
59
30
99
99
97
90
43
86
58
54
26
80
51
28
45
101
98
95
77
15
37
28
11
53
26
14
60
101
101
87
64
12
47
16
17
10
36
17
11
90
104
97
88
36
11
6
17
6
8
12
4
120
99
99
78
24
7
18
5
12
7
7
5
9
180
99
99
67
16
5
11
3
8
6
4
3
8
at different pH values. Each figure in a buffered medium at pH 5, 7.4 or 9 is a mean of four
determinations. ~ •
-------
TABLE 37. PERCENT CHOLINESTERASE ACTIVITY OF ABDOMINAL LYMPH AND:pH*
o
10
Incubation time
Rats dosed
with
Metrifonate
(300 mg/kg
P.O.)
DDVP (45
mg/kg p.o.)
DDVP (280
mg/kg p.o.)
pH of the
medium
5.0
9.0
5.0
9.0
5.0
9.0
1
87
83
101
95
95
98
5
77
47
98
96
86
84
10
66
29
97
94
83
80
(min)
15
54
21
94
90
73
72
30
44
12
91
93
66
62
45
31
8
89
84
56
51
60
27
6
82
89
48
52
* Measured in the course of incubation in a medium at different pH values of abdominal lymph .collected
from rats during 30 minutes after oral dosing with metrifonate or DDVP.
-------
TABLE 38. METRIFONATE OR DDVP IN LYMPH OF RATS GIVEN METRIFONATE OR DDVP
ORALLY*
Rats treated pH of the
with lymph
Metrifopate 7-8
(300 mg/kg p.o.)
2-3
DDVP 7 - 8
(2.5 mg/kg p.o.)
2-3
Compound recovered from
the lymph (yg/ml )
Metrifonate
14.4 (±6.3)
22.3 (±2.6)
0
0
DDVP
3.8
0
3.4
2.6
(±1.9)
(0)
(±1.3)
(±1.8)
* Determined by the TLC method of Beck and Sherman (1968).
110
-------
TABLE 39. SECOND ORDER RATE CONSTANTS* FOR INHIBITION (ka) OF
ACETYLCHOLINESTERASE AND CHOLINESTERASE BY DDVP
Enzyme
Acetylchol inesterase
Bovine erythrocytes
Rat brain
Choi inesterase
Human plasma
Horse plasma
Rat plasma
10"4 x ka (M"1 min"1)
a
25° 37°
2.62 - 1
6.85 15.4
50.1 87.0
7.77 8.78
6.90 18.3
* in 0.1 M buffer pH 7.4 (from Reiner et al. 1975)
111
-------
TABLE 40. EFFECT OF pH ON (a) INHIBITION OF BOVINE ERYTHROCYTE ACETYL-
CHOLINESTERASE BY DDVP AND (b) ON THE RATES OF DECOMPOSITION
OF METRIFONATE*
pH 6.0 pH 7.4
(a)
k_ (second order rate
a
constant of inhibition
by DDVP (M"1 min'1) 4.2 x 104 4.7 x 104
(b)
k (first order rate
constant of decomposition
of metrifonate)
(min"1) (0.73±0.12) x 10"4 (22.2±1.7) x 10"4
* Values calculated from the kinetics of inhibition of bovine erythrocyte
acetylcholinesterase in metrifonate solutions. Both effects were measured
in 1.0 mM buffer at 37°C (from Reiner et al. 1975).
112
-------
TABLE 41. FIRST-ORDER RATE CONSTANTS FOR SPONTANEOUS REACTIVATION (kr) AND
AGING (kag) OF DIMETHYLPHOSPHORYLATED ACETYLCHOLINESTERASE AND
CHOLINESTERASE*
Enzyme
102 (kr±S.E.)
(min"1)
102 (kag±S.E.)
(min"1)
Acetylcholinesterase
Human erythrocytes 1.36 ±00.14 (12) 0.297 ±0.018 (20)
Bovine erythrocytes 0.92 ± 0.05 (7)
Rat brain 0.606±0.057 (12) 0.1720±0.0070 (19)
Cholinesterase
Human plasma >^ 0.014
Rat plasma 0.504±0.028 (24) approx. 0.058
* At 37°C and pH=7.4 in 0.1 M buffer. The numbers in parentheses represent
the number of points from which the rate constants were calculated (from
Skrinjaric-Spoljar et al. 1973)
113
-------
TABLE 42. DECOMPOSITION OF METRIFONATE*
Enzyme Metriforiate
104 x k (miiT1)
(mM) 25° 37<
Acetylcholi nesterase
Bovine erythrocytes 0.15 3.69
0.50 2.80
Rat brain 0.15 5.08 28.2
0.50 3.48 36.4
Choiinesterase
Human plasma 0.01 7.08 41.6
0.05 2.39 46.2
0.10 4.17
Horse plasma 0.10 3.97 39.4
0.50 2.68 30.8
1.00 1.46
Rat plasma 0.15 3.13 20.0
0.50 2.47 22.2
In 0.1 M buffer pH 7.4. Values calculated from the kinetics of
inhibition of acetylcholinesterase and cholinesterase. First order
rate constants for decomposition (k) are given (from Reiner et al.
1975)
114
-------
TABLE 43. DECOMPOSITION OF METRIFONATE*
Metrifonate
(mM)
0.150
15.0
25°
7.27 ± 0.39 (33)
6.05 ± 0.52 (18)
104 x k (mirf1)
37°
53.1 ± 1.2
37.1 ± 1.2
(20)
(40)
* In 0.1 M buffer pH=7.4. The rate of decomposition was measured polaro-
graphically. First order rate constants (k) ± standard errors are
given. The numbers in parentheses are the number of individual results
from which k was calculated (from Reiner et al. 1975)
115
-------
TABLE 44. MICHAELIS CONSTANTS (Kj AND SUBSTRATE INHIBITION CONSTANTS
(Kss) FOR ACETYLCHOLINESTERASE AND CHOLINESTERASE*
Substrate (mM) Km (mM) Kss(mM)
Acetylcholinesterase
Acetylcholine (0.01-1.0) 0.15 ± 0.01
Acetylcholine (1.0-100) - 9 (e-0.2)
Acetylthiocholine 0.11 14
Phenylacetate 2.6
Choiinesterase
Benzoylcholine (0.01-10) 0.4 (nH=0.7)
Benzoylcholine (10-100) - 56±5 (g=0)
Acetylcholine 1.2 to 3.2
Acetylthiocholine 0.6 (nu=0.8)
n
Phenylacetate 3.8
* n^ is the Hill coefficient. 3 is defined in the text. The range of
substrate concentrations used in the experiments is given in brackets.
116
-------
TABLE 45. INTERCEPTS (± STANDARD ERRORS) ON THE ABSCISSA K(S) AND ORDINATE K(I) OF THE HUNTER AND
DOWNS PLAT*
Substrate
K(S) (mM)
Inhibitor (mM)
(mM)
Acetylcholinesterase
Acetylthiocholine
Acetylthiocholine
Phenyl acetate
Phenyl acetate
0.17 ± 0.17
0.22 ± 0.14
3.8 ± 0.8
6.2 ± 1.2
Acetylcholine (0.5-10)
Phenylacetate (2.6-15)
Acetylthiocholine (0.2-5)
Acetylcholine (0.5-5)
0.22 ± 0.22
4.9 ± 6.1
0.25 ± 0.03
0.54 ± 0.05
-. Cholinesterase
~>j
Acetylthiocholine
Acetylthiocholine
Acetylthiocholine
Phenylacetate
Benzoylcholine
0.53 ± 0.17
•v* 1.0
0.37 ± 0.55
± 0.4
± 0.45
2.5
0.17
Acetylcholine (5-20)
Phenylacetate (1-10)
Benzoylcholine (0.05-1)
Acetylcholine (5-15)
Acetylthiocholine (10-50)
2.6
± 0.77
4.0
0.03 ± 0.04
±0.7
± 3.3
3.1
1.3
The range of inhibitor concentrations used in the experiments is given in brackets.
-------
TABLE 46. EFFECT OF TEMPERATURE ON INHIBITION OF ACETYLCHOLINESTERASE BY
PHOSPHOSTIGMINE AND HALOXON*
Temp. Phosphostogmine
°C 10~V K(I)
a
fM^min"1) (pM)
5.0
10 0.557 70
11
16 0.944 73
18
22 1.59 235
25
30 3.73 124
32 3.89 168
40 8.46
(n)
(5)
-
(4)
-
(3)
-
(3)
(3)
(7)
Haloxon
10"5ka K(I) (n)
(M^mirT1) (yM)
0.099 3.64 (28)
-
0.226 4.21 (12)
-
0.394 5.45 (3)
-
1.25 6.42 (87)
-
2.38 9.12 (3)
5.45 6.54 (7)
* k is the second order rate constant of phosphorylation and K(I) is the
dissociation constant for the enzyme-inhibitor complex (n) is the number
of experiments.
118
-------
TABLE 47. EFFECT OF TEMPERATURE ON INHIBITION OF ACETYL-
CHOLINESTERASE BY TWO CARBAMATES*
Inhibitor
3-isopropyl phenyl -
-N-methyl carbamate
Neostigmine
t°C
11
25
40
5.6
25.6
39.5
10"5 (k+S.E.)
a
(M"1 min"1)
2.27 ± 0.06
5.35 ± 0.05
10.3 ± 0.1
1.59 ± 0.07
6.71 ± 0.64
25.1 ± 0.1
(n)
(5)
(5)
(6)
(4)
(5)
(3)
*ka (± standard error) is the second order rate constant of
a
inhibition; (n) is thfe number of experiments.
119
-------
TABLE 48. TEMPERATURE CONVERSION TABLE FOR ACTIVITIES OF
HUMAN WHOLE BLOOD AND PLASMA CHOLINESTERASES*
Temperature
(0°C)
10
15
20
25
30
35
40
Whole blood
cholinesterase
1.00
1.25
1.51
1.85
2.19
2.40
2.60
Plasma
cholinesterase
1.00
1.35
1.64
2.10
2.52
3.00
3.48
* Measured by the method of Ellman et al. (1961). Activities
at 10°C were taken as 1.00 (from Reiner et al. 1974).
120
-------
TABLE 49. ACTIVITY OF HUMAN WHOLE BLOOD CHOLINESTERASES AFTER INHIBITION
BY DIFFERENT CARBAMATES*
Carbamate
Carbaryl
Propoxur
Landrin
Promecarb
Con-
centra-
tion
(vM)
2.0
4.0
8.0
16.0
1.0
3.0
9.0
7.0
14.0
28.0
0.1
0.4
1.0
4.0
Whole blood chol
activity
Tintometric
method
92
80
67
54
68
48
35
65
49
35
93
77
55
30
inesterase
(«)
Spectrophoto-
metric method
89
76
60
49
65
37
26
58
44
29
91
72
51
28
* Measured by tintometric and spectrophotometric methods and expressed as
a percentage of the control value (from Wilhelm et al. 1973).
121
-------
TABLE 50. ACTIVITY OF HUMAN PLASMA CHOLINESTERASE AFTER INHIBITION BY
DIFFERENT CARBAMATES*
Carbamate
Propoxur
Landrin
Promecarb
Con-
centra-
tion-
(yM)
1.0
3.0
9.0
7.0
14.0
28.0
0.1
0.4
1.0
4.0
Plasma chol
Acholest method
96
87
74
67
58
46
87
71
53
26
inesterase activity (%)
Spectrophotometric
method
92
86
71
72
58
45
88
74
50
21
* Measured by Acholest and Spectrophotometric methods and expressed as a
percentage of the control value (from Wilhelm et al. 1973).
122
-------
TABLE 51. STORAGE CONDITIONS OF HUMAN BLOOD SAMPLES FOR MEASURING
CHOLINESTERASE ACTIVITY*
20 yl blood from finger prick
Diluted into 6.0 ml 20 mM buffer
pH 5.0
Stored up to 4 hours at 4°C
6.0 ml 0.1 M buffer pH 7.4
(containing substrate and DTNB)
added and cholinesterase acti-
vity measured by the Ellman
method
* From Mil helm and Reiner 1973.
123
-------
TABLE 52. LIST OF ORGANOPHOSPHORUS PESTICIDES DETERMINED
BY THIN-LAYER DENSITOMETRY
Name
Formula
dichlorvos (DDVP)
trichlorron (metrifonate)
malathion
parathion
,,
f-
(CH,0)9P(0)CH=CC1
H (•
(CH30)2P(0)CH(OH)CC13
(CH30)2P(S)S-CH-CH2C(0)-OCH2CH3
C(0)-CH2CH3
(CH3CH20)2P(S)0-C6H4(p-N02)
124
-------
TABLE 53. Rf VALUES OF ORGANOPHOSPHORUS PESTICIDES IN-
VESTIGATED IN TWO SOLVENT SYSTEMS
Compound
dichlorvos
trichlorfon
malathion
parathion
Rf values
Solvent system I
0.52
0.36
0.62
0.62
Solvent system II
0.38
0.53
0.64
Solvent system I, chloroform: acetone (1:1); solvent system II,
benzene: acetone: n-hexane (10:25:65).
125
-------
TABLE 54. LINEARITY RANGES AND WEIGHTED VARIANCES FOR THE THIN-LAYER CHROMATOGRAPHIC DETERMINATION
OF SOME ORGANOPHOSPHORUS PESTICIDES
M
Detection of s_p_ots
Organophosphorus
pesticide
Dichlorvos
trichlorfon
malathion
parathion
dichlorvos*
trichlorfon*
With AgN03 reagent
Range
0.1
1.0
2.0
2.0
0.1
1.0
of linearity
- 0.7 yg
- 5.0 yg
- 10.0 yg
- 18.0 yg
- 1.0 yg
- 4.0 yg
Weighted variance
0.0128
0.0074
0.0095
0.0381
0.0200
0.0193
By acetylcholinesterase inhibition
Range of linearity Weighted variance
0.1 - 3.0 ng 0.0632
100 - 300 ng
0.25- 0.7 yg 0.0262
0.01- 4.0 ng 0.0450
-
-
* Simultaneous determination.
-------
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*Tokoro, T. , Suzuki, K. , Otsuka, J. , and Suzuki, H. 1973. The
changes or refraction and ophthalmological pressure of
experimental chronically intoxicated beagles by an organo-
phosphorus pesticide. Nippon Ganka Gakkai Zasshi (Acta Soc.
Ophthalmol. Jpn. ) 77:40. From: Health Aspects Pestic.
str. Bull.~6Tl973) no. 73-2498.
Op
Ab
*Tsuchiya, T. 1973. A report on the state of Organophosphorus
pesticide application and the cases considered to be intoxi-
cation by the pesticide in Miyazaki district. Rinsho Ganka
(Clin. Ophthalmol . ) 27:1166. From Pestic. Abstr. 7(1974)
no. 74-1611.
Tsukahara, I. 1972. Examination for chronic pesticide poisoning
based on examinations of visual acuity, eyegrounds and EPG.
Ganka Rinsho Iho (Jpn. Rev. Clin. Ophthalmol . ) 66:838-848.
From: Health Aspects Pestic. Abstr. Bull. 6(1973)
no. 73-0624.
Turina, S., Klasinc, L., and Jamnicki, V. 1974. Area determi-
nation under chroma tographic curves using the Monte Carlo
method. Chroma tographia 7;(4)203-204.
Uchida, T. 1974. A case of retinochoroid pathosis due to
chronic intoxication by Organophosphorus pesticides. Ganka
Rinsho Iho (Jpn. Rev. Clin. Ophthalmol.) 68:191-192.
From: Pestic. Abstr. 771974) no. 74-1106.
145
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Ueda, K., Nishimura, M., Maebashi, H., Miyazaki, I., Ogita, S.,
and Dawamura, A. 1972. Experimental chronic intoxication
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Noson Igakkai Zusshi (J. Jpn. Assoc. Rural med.) 21:98-99.
From: Pestic. Abstr. 6(1973) no. 73-0390.
Uga, S., Ishikawa, S., and Mukuno, K. 1976. Histopathological
study of the canine optic nerve and retina induced by
organophosphate pesticides. Nippon Ganka Gakki Zasshi
(Acta Soc. Ophthalmol. Jpn.) From: author's manuscript.
Ui, J. 1972. Pollution disasters in Japan. Lakartidningen
69:2789-2796. From: Health Aspects Pestic. Abstr. Bull.
5(1972) no. 72-2512.
Uono, M. 1972. Organophosphorus poisoning. Shinkei Kenkyu No
Shimpo (Adv. Neurol. Sci.) 16:892-901. From: Health
Aspects Pestic. Abstr. Bull. 6(1973) no. 73-2427.
Upholt, W. M., Quinby, G. E., Batchelor, G. S., and Thompson,
J. P- 1956. Visual effects accompanying induced miosis.
AMA. Arch. Ophthalmol. 56:128-134.
U.S. Department of Health, Education and Welfare 1969. Report
of the Secretary's Commission on Pesticides and Their
Relationship to Environmental Health, Parts I and II.
Vandekar, M., and Svetlicid, B. 1966. Observations on the
toxicity of three anticholinesterase insecticides in a
village. Scale trial and comparison of methods used for
determining cholinesterase activity. Arh. Hig. Rada Toksikol.
17:135-150.
Voss, G., and Sachsse, K. 1970. Red cell and plasma choline-
sterase activities in microsamples of human and animal blood
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Warnick, S. I., and Carter, J. E. 1972. Some Findings in a
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146
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Wilhelm, K. 1968. Determination of human plasma cholinesterase
activity by adapted Ellman's method. Arch. Hig. Rada
Toksikol. 19:199-207.
Wilhelm, K.: Variations in blood cholinesterase activities in
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Health, Brighton 1975, Abstr. p. 160.
Wilhelm, K., Plestina, R. : The influence of previous carba-
moylation on cholinesterase activity in rats poisoned with
some organophosphorus compounds (in Croatian), 1st
Yugoslav Congress of Toxicologists, Herceg-Novi 1974, Abstr.
no. 83.
Wilhelm K., Plestina, R., Svetlicic, B.: The toxicity of
Ekatin and its action on occupationally exposed workers
(in Croatian) , Meeting ojf Chemists of_ Croatia, Zagreb 1973,
Abstr. p. 465.
Wilhelm, K., and Reiner, E. 1973. Effect of sample storage on
human blood cholinesterase activity after inhibition by
carbamates. Bull. WHO 48:235-238.
Wilhelm, K. , Vandekar, M. , and Reiner,- E. 1973. Comparison of
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mates. Bull. WHO 48:41-44.
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235:2312-2315.
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everted small intestine for the study of the transference
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Witter, R. R. 1963. Measurement of blood cholinesterase.
Arch. Environ. Health. 6:537-563.
WHO Expert Committee on Insecticides 1963. Recommended method
for vector control. Tech. Rept. ser. no. 265.
Yamazaki, H., Shimazaki, K., Kurosawa, K., and Suzuki, A. 1975.
Electron microscopic study on the subacute toxicity of
organophosphorus pesticides. Nippon Noson Igakkai Zasshi
(J. Jpn. Assoc. Rural Med.) 24:1-7. From: Pestic. Abstr.
8(19757" no.
147
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Zarya, K. I. 1973. Izmenenija organa zrenija u kolhoznikov,
rabotajuscin s pesticidami. Vestn. Oftalmol. 3:80-82.
Note:
* Very general abstract - Some results were published in more
than on^ paper; only one paper reviewed;
**
Article not accessible, even as summary or abstract.
148
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APPENDIX A
INTERNATIONAL MEETING ON CHOLINESTERASES AND CHOLINERGIC RECEPTORS
The Institute for Medical Research and Occupational Health,
Zagreb, Yugoslavia, organized an International Meeting on
Cholinesterases and Cholinergic Receptors, which was held in
Split from the 1st to the 5th of April 1975. The meeting was
sponsored by the Yugoslav Academy of Sciences and Arts, and
received substantial support from the Scientific Research
Council of the Republic of Croatia.
The aim of the meeting was to discuss the molecular proper-
ties of Cholinesterases and cholinoreceptors. It was felt that
an informal exchange of views would be extremely desirable for
future developments in that field, as no such meeting was held
since the two macromolecules had been purified and isolated.
In this aim, the meeting was fully successful and excelled in
a stimulating working atmosphere to which everyone contributed.
During the meeting it became obvious that great interest exists
in models for the binding sites of Cholinesterases, and a gen-
eral discussion was organized on the last day, summarizing old
and new concepts on that particular aspect of research.
The participation in the meeting was by invitation through
the Organizing Committee which consisted of the following mem-
bers: W. N. Aldridge (Great Britain), E. DeRobertis (Argentina),
Edith Heilbronn (Sweden), G. Kato (Canada), I. A. R. Main (USA),
J. Massoulie (France), D. B. Millar (USA), R. D. O'Brien (USA),
Elsa Reiner (Yugoslavia) and A. 0. Zupancic Yugoslavia).
Twenty-nine papers were presented. The total number of partici-
pants was 45.
The Proceedings of the meeting are published as No. 3,
(XII + 344 pp), Vol 47_ of Croatica Chemica Acta and also in the
form of a book as an offprint of the same issue of the journal.
The editor of the Proceedings was E. Reiner, and the subeditors
were M. Skrinjari6-Spoljar and V. Simeon. The Proceedings com-
prise 28 full-length papers and the discussion following each
paper. Two participants did not send their manuscripts and one
paper was included which was not read. The Proceedings also
included a general discussion on models for the ligand binding
sites of Cholinesterases.
149
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The following are the titles and authors of the papers:
Molecular Forms of Acetylcholinesterase (J. Massoulie, S. Bon,
F. Rieger, and M. Vigny), Molecular Structure and Catalytic
Activity of Membrane-bound Acetylcholinesterase from Electric
Organ Tissue of the Electric Eel (I. Silman and Y. Dudai), Com-
parative Studies on the Molecular Properties of Purified Acetyl-
cholinesterpse from Human Erythrocytes and from the Electric
Organ of Electrophorus Electricus (U. Brodbeck, P. Ott, and
Therese Wiedmer), Properties of the Bobble and Membrane-bound
Forms of Acetylcholinesterase Present ii. Pig Brain (D. T.
Plummer, C. A. Reavill and C. H, S. Mclntosh), Survey of Major
Points of Interest about Reactions of Cholinesterases (W. N.
Aldridge), Catalysis by Acetylcholinesterase. The Rate-limiting
Steps Involved in the Acylation of Acetylcholinesterase by
Acetic Acid Esters and Phosphorylating Agents (T. L. Rosenberry),
Spectroscopic Studies of Ligand Interactions with Acetylcholine-
sterase (P. Taylor, J. Lwebuga-Mukasa, H. Berman, and S. Lap:),
Interaction of Ligand Binding Sites of Acetylcholinesterase (G.
Mooser and D. S. Sigman), The Reaction Mechanism of Butyryl-
cholinesterase (K.-B. Augustinsson and H. Erikson), Allosteric
Reactions of Horse Serum Cholinesterase (Ljubica Kramaric),
Methanesulphonyl Fluoride: A Probe of Substrate Interactions in
the Catalytic Site of Acetylcholinesterase (R. M. Krupka),
Characterization of the Active Site of Acetylcholinesterases
by Application of Sterically Modified Acetylcholine Homologues
(W. H. Hopff, G. Riggio, A. Hofmann and P- G. Waser) , Kinetic
Study of the Effect of Substrates on Reversible Inhibition of
Cholinesterase and Acetylcholine'sterase by two Coumarin Deri-.
vatives (Elsa Reiner and Vera Simeon) , Non-competitive Inhibi*-
tipn of Flyhead Acetylcholinesterase by Oxime Carbamates.
Kinetic Evidence for Non-productive Binding to the Catalytic
Site (P- J. Jewess and N. R. McFarlane), The Comparative Bio-
chemistry of Mammalian and Insect Acetylcholinesterase with
Reference to the Selective Inhibition by Organophosphates and
Carbamates (K. Hellenbrand and R. M. Krupka), Structural Changes
in Acetylcholinesterase under the Influence of Some Ligands
(M. R. Pavlic), The Use of Affinity Gels for the Study of the
Ligand Binding Properties of Mammalian Acetylcholinesterase
(G. Hollunger and B. Niklasson), Regulatory Properties of Mem-
brane Bound Acetylcholinesterase from Red Cell Ghosts (H. Kuhnen),
Inhibition of Acetylcholinesterase by N-Alkylpyridinium and N-
Alkylpyridinium-2-aldoxime Salts (L. P. A. de Jong and G. Z.
Wolring), Effects of Natural Polyamines on Membrane-bound and
Solubilized Acetylcholinesterase of Human Red Cells (A. Kossorotow
and H. U. Wolf), Current Research on the Nature of Cholinergic
Receptors (Edith Heilbronn), Heterogeneities in Acetylcholine
Receptor from Torpedo Species (R. D. O'Brien and R. E. Gibson),
Structure and Function of the Acetylcholine Receptor (M. E.
Eldefrawi and Amira T. Eldefrawi), Actions of Histrionicotoxin
on Acetylcholine Receptors (G. Kato, M. Glavinovic, J. Henry,
150
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K. Krnjevic, E. Puil and B. Tattrie), Purification of Acetyl-
choline Receptor Enriched Membranes by Use of Affinity Parti-
tioning (S. D. Flanagan, P. Taylor, and S. H. Barondes),
Isolation of a Housefly Head Protein Fraction that Exhibits High
Affinity Binding of Cholinergic Ligands (P. J. Jewess, B. S.
Clarke, and J. F. Donnellan), Regulatory Properties and Cooper-
ativity of Membrane Bound Muscarinic Receptors of Intestinal
Smooth Muscle Cells (Dida Kuhnen-Clausen), Cholinergic Muscarinic
Receptor: Biochemical and Light Autoradiographic Localization
in the Brain (H. I. Yamamura, K.-J. Chang, M. J. Kuhar, and S.
H. Snyder), Immunization of Rabbits with Purified Nicotinic
Acetylcholine Receptor (Ch. Mattsson and Edith Heilbronn), The
Acetylcholine Receptor of the Mammalian Neuromuscular Junction
(E. A. Barnard).
151
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APPENDIX B
SUMMARIES OF
B.S. AND M.S. THESES
V. Bradamante-Simid; The Effect of Anticholinesterase Pesti-
cides in Occupationally Exposed Workers, M.S. Thesis, University
of Zagreb (1975).
The effects of anticholinesterase insecticides were studied
in workers employed in the production and formulation of insec-
ticides as well as in workers who apply them in agriculture.
Acetylcholinesterase and cholinesterase activity was fol-
lowed in workers during the working season and the onset of
symptoms and signs of poisoning following exposure to anticho-
linesterase insecticides were observed.
In addition to these parameters the mode and duration of
exposure and the types of insecticides to which workers were
exposed in the course of work were determined. As both indus-
trial and agricultural workers were exposed to a combination of
insecticides it was not possible to ascertain which insecticide
was predominant in producing a biochemical lesion and/or cho-
linergic symptoms.
The studies in workers formulating the insecticides were
carried out from 1971 to 1974, while studies in agricultural
workers were done in 1973 and 1974.
The usefulness of protective devices during the production
and formulation of insecticides has been confirmed. It was
also found in the same workers that a long-term exposure to
organophosphorus insecticides does not induce changes in the
differential blood picture.
K. Fink; The Persistence of some Organophosphorus Pesticides
in Aqueous Media, B.S. Thesis, University of Zagreb (1975).
The aim of this work was to find a suitable procedure for
the accumulation of organophosphorus pesticides present as
micropollutants in aqueous media. The efficiency of extraction
with different solvents and solvent systems was compared with
the efficiency of adsorption on the activated carbon microcolumn.
The latter procedure was utilized in the studies of the persis-
tence of some organophosphorus pesticides added to river water
152
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samples. Some possibilities for simultaneous determination of
dichlorvos and trichlorphon by reaction gas chromatographic
analysis were investigated.
Dj. Kobrehel; Kinetics of Acetylcholinesterase Inhibition by
Two Coumarin Derivatives, M.S. Thesis, University of Zagreb
(1975). y
The kinetics of inhibition of acetylcholinesterase by
[0,0,-bis(3-chloroethyl)0-(3-chloro-4-methyl-7-coumarinyl)
phosphate] (haloxon) and 3-chloro-7-hydroxy-4-methylcoumarin
was studied at 5°C in phosphate buffer pH 7.4. The substrate
was acetylthiocholine.
Haloxon reacts with acetylcholinesterase irreversibly
(rate of phosphorylation k = 9.90 x 103 NT1 min~M the enzyme-
haloxon complex is 3.64 yM (calculated from the rate of phos-
phorylation) and 3.49 yM (calculated from the kinetics of compe-
tition between haloxon and acetylthiocholine).
3-Chloro-7-hydroxy-4-methyl coumarin inhibits the enzyme
reversibly, with dissociation constant K(I) = 1.1 yM.
The substrate-inhibition constant K for acetylthiocholine
is 3.0 mM (obtained from the activity-pSs§urve). From the
kinetics of reversible inhibition by haloxon and 3-chloro-7-
hydroxy-4-methyl coumarin the dissociation constants for the
enzyme-substrate reaction are K(S) = 2.50 mM and 1.26 mM, respec-
tively. Comparing the K and K(S) constants it appears that
the inhibitors react at €ne same site of acetylcholinesterase
like the substrate, when it inhibits the enzyme. This site is
assumed to be an allosteric site of acetylcholinesterase.
B. Krauthacker; Stability of 0,0-Dimethyl 2,2,2-trichloro-l-
hydroxyethyl phosphonate (Metrifonate) in Aqueous Solutions
and the Mechanism of Inhibition of Cholinesterases in Metrifonate
Solutions, M.S. Thesis, University of Zagreb (1974).
The mechanism of inhibition of Cholinesterases in solutions
of 0,0-dimethyl 2,2,2-trichloro-l-hydroxyethyl phosphonate
(metrifonate) was studied on 0.1 M phosphate buffer pH 7.4 at
25°C and 37°C. It was shown that the inhibition of Choline-
sterases is not caused by metrifonate, but by 0,0-dimethyl 2,2-
dichlorovinyl phosphate (DDVP) which was formed by nonenzymic
rearrangement from metrifonate. This result was obtained from
a comparison between the kinetics of enzyme inhibition and the
kinetics of the nonenzymic rearrangement from metrifonate which
was measured polarographically. The enzyme preparations, used
in this work were: acetylcholinesterase (E.G. 3.1.1.7) of rat
brain and bovine erythrocytes and cholinesterase (E.G. 3.1.1.8.)
of human horse and rat serum.
153
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Dj. Mrvos; Decomposition of 0,0-Dimethyl 2,2-dichlorovinyl-phos-
phate (DDVP) in Rabbit Serum, B.S. Thesis, University of Zagreb
(1975).
The rate of DDVP decomposition in rabbit serum was measured
in 0.1 M phosphate buffer pH 7.4, at 25°C and 37°C by thin-layer
densitometry at four different DDVP concentrations.
The reaction did not follow the Michaelis kinetics at
either temperature. At 25°C the rate oi decomposition was 0.10
ymol min'V'ml""1 serum (DDVP = 20..0 mM) , At 37°C the rate was
about five times higher.
The rate of spontaneous decomposition of DDVP was also
measured by thin-layer densitometry, and the results agreed with
those obtained by other methods.
Only one product containing chlorine was observed either
in the enzymic or in spontaneous degradation of DDVP indicating
that decomposition consisted in the hydrolysis of the ester bond
of the P-0-CH=CHC12 moiety.
B. Radic; Determination of Organophosphorus Pesticides in Bio-
logical Material by Means of Thin-Layer Chromatography. M.S.
Thesis, University of Zagreb (1975).
A method for simultaneous determination of metrifonate and
dichlorvos in biological material has been developed.
Due to the complex structures of compounds, not only in
the original samples, but also in the extracts, thin-layer chro-
matography was chosen because it enables separation and quanti-
tative determination of these compounds.
Samples of blood, brain and liver homogenates of experi-
mental animals were used as biological material.
Calibration lines for the quantitative determination by
direct measurement on the thin-layer had been made, and the
method was applied to animals treated with metrifonate and
dichlorvos. Absorption, distribution and persistence of exam-
ined compounds were followed after intravenous administration.
It has been established that metrifonate decomposes into
dichlorvos, because after intravenous injection of metrifonate
it was found that dichlorvos was also present in the blood.
The results for dichlorvos obtained by thin-layer chroma-
tography were compared with those using gas chromatography.
154
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Z. Vasilic; Quantitative Determination of Organophosphorus
Pesticides by Thin Layer Densitometry, B.S. Thesis, University
of Zagreb (1976).
A method for a quantitative thin-layer chromatographic
analysis of organophosphorus pesticides is described. The spots
visualized with silver nitrate or by enzyme inhibition were
quantitatively evaluated by direct densitometric measurements of
chromatograms.
The detection with silver nitrate, although neither spe-
cially selective nor sensitive, proved to be useful for the
determination of dichlorvos and trichlorfon present simultan-
eously in concentrations of 0.1 - 1 yg and 1-4 yg/per 10 yl
respectively.
S. Zima: Total Organic Carbon Determination in Water, M.S.
Thesis, University of Zagreb (1975).
A method has been developed for the determination of total
organic carbon present as a pollutant in water. An apparatus
for microanalysis of carbon and hydrogen in organic compounds
has been modified to enable a continuous flow of water sample
into the combustion tube, the latter being packed with copper
oxide and heated to 700°C.
The amount of carbon dioxide obtained by combustion of the
polluted water sample in a stream of oxygen has been determined
gravimetrically. The water of the sample and the water produced
by the pyrolysis of the organic matter was eliminated by con-
densation in a trap connected to the end of the combusion tube.
The results of carbon determinations (total carbon, organic
and inorganic carbon), of the following are discussed: aqueous
solutions of pure organic compounds of known composition;
organic and inorganic compounds present simultaneously in aqueous
solution, and in samples of waste waters and surface waters.
The amount of sample is 3 - 5 ml. The analysis can be
completed in roughly one hour. The reproducibility of the
results is shown to be ± 2 ppm of carbon. The lower limit of
carbon content is not critical, as it can be widened by dilution
of the sample.
155
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APPENDIX C
LIST OF PUBLICATIONS SINCE 1972
Papers
1. Drevenkar, V., Fink K., Stipcevic, M., Stengl, B.: The fate
of pesticides in aquatic environment; I. The persistence of
some organophosphorus pesticides in river water,.Arhiv- hig.
rada 2j6 (1975) 257-266.
2. PleStina, R., Davis, A., Bailey, D. R.: Effect of metrifo-
nate on blood cholinesterases in children during the treat-
ment of schistosomiasis, Bull, Wld Hlth Org., 46 (1972)
747-759.
3. Plestina, R., Svetlicic, B.: Toxic effects on two carbamate
insecticides in dogs, Arh. hig. rada, 2_4_ (1973) 217-225.
4. Plestina, R.: Toxicology of some anticholinesterases used
in disinsection (in Croatian), Pomorska biblioteka, 26^ (1975)
689-695).
5. Reiner, E., Buntic, A., Trdak, M., Simeon, V.: Effect of
temperature on the activity of human blood cholinesterases,
Arch. Toxicol., 32^ (1974) 347-350.
6. Reiner, E., Krauthacker, B., Simeon, V., Skrinjaric-Spoljar,
M.: Mechanism of inhibition in vitro of mammalian acetylcho-
linesterase and cholinesterase in solutions of 0,0-dimethyl
2,2,2-trichloro-l-hydroxyethyl phosphonate (trichlorphon),
Biochem. Pharmacol., 24 (1975) 717-722.
7. Reiner, E., Simeon, V.: Kinetic study of the effects of sub-
s strates on reversible inhibition of cholinesterase and ace-
tylcholinesterase by two coumarin derivatives, Croat. Chem.
Acta 4J7_ (1975) 321-331.
8. Simeon, V.: Effect of acetylcholine on inhibition of cho-
linesterases by acylating inhibitors, Arh. hig. rada, 25
(1974) 51-56.
156
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9. Simeon, V.: Michaelis constants and substrate inhibition
constants for the reaction of acetylthiocholine with
acetylcholinesterase and cholinesterase, Croat. Chem. Acta,
4£ (1974) 137-144.
10. Simeon, V., Reiner, E.: Comparison between inhibition of
acetylcholinesterase and cholinesterase by some N-methyl
and NN-dimethyl-carbamates, Arh. hig. rada, 24 (1973)
199-206.
11. Simeon, V., Reiner, E. , Vernon, C. A.: Effect of tempera-
ture and pH on carbaraoylation and phosphorylation of serum
cholinesterases; Theoretical interpretation of activation
energies in complex reactions, Biochem. J., 130 (1972)
515-524.
12. Simeon, V., Skrinjaric-Spoljar, M. , Wilhelm, K.: Reactiva-
tion of phosphorylated cholinesterases in vitro and pro-
tecting effects in vivo of some pyridinium and quinolinium
oximes, Arh. hig. rada, 24_ (1973) 11-18.
13. Svetlicic, B., Wilhelm, K.: Methods for measuring exposure
to anticholinesterase insecticides, Arh. hig. rada, 24
(1973) 357-365.
14. Skrinjarid-Spoljar, M., Simeon, V., Reiner, E.: Spontaneous
reactivation and aging of dimethylphosphorylated acetylcho-
linesterase and cholinesterase, Biochim. Biophys. Acta, 315
(1973) 363-369.
15. Wilhelm, K., Plestina, R., Svetlicic, B.: Blood cholineste-
rase activity in workers exposed to organophosphorus insec-
ticide "Ekatin" (in Croatian), Arh. hig. rada, 2_4 (1973)
107-116.
16. Wilhelm, K., Reiner, E.: Effect of sample storage on human
blood cholinesterase activity after inhibition by carbamates,
Bull. Wld Hlth Org., 48^ (1973) 235-238.
17. Wilhelm, K., Vandekar, M., Reiner, E.: Comparison of methods
for measuring cholinesterase inhibition by carbamates, Bull.
Wild Hlth Org., 4_8_ (1973) 41-44.
18. Zima, S., Sliepcevic, Z., Stefanac, Z.: A simple assembly
for carbon determination in water, Croat. Chem. Acta 47
(1975) 611-616.
157
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Communications
19. Drevenkar, V., Fink, K., Stipcevic, M., Tkalcevid, B.: The
fate of some organophosphorus pesticides in river water,
Scientific Session on Environmental Analysis, Szombathely
1975, (unpublished).
20. Drevenkar, V., Stengl, B., Tkalcevid, B., Besic", J.:
Determination of organophosphorus insecticides present as
micropollutants in surface waters, Scientific Session on
Environmental Analysis, Szombathely 1974, (unpublished).
21. Drevenkar, V., Stengl, B., Stipcevic, M: A method for the
determination of organophosphorus pesticides in water,
Meeting of Chemists of Croatia, Zagreb 1975, Abstracts,
p. 200.
22. Kiseljak, B., Wilhelm, K.: Determination of insecticidal
carbamates in biological material (in Croatian), 3rd Yugo-
slav Congress of Pure and Applied Chemistry, Ljubljana
1972., Abstract No. IX-10.
23. Krauthacker, B., Reiner, E., Stipcevid, M., Stefanac, Z.:
Chlorinated hydrocarbons in human blood (in Croatian),
9th Congress of the Ugoslav Physiological Society, Porto-
roz 1975, Abstract. 125.
24. Pelicari6, B., Reiner, E., Becajac, S., Krvavica, S.: Spe-
cificity of cholinesterases in Paramphystomum cervic (Tre-
matoda) and Ascaris suum (Nematoda) (in Croatian), 9th
Congress of the Ugoslav Physiological Society, Portoroz
1975, Abstract No. 180.
25. Plestina, R.: Practicability of blood cholinesterase
activity measurements for assessment of overexposure to
anticholinesterases, 3rd International Congress of Pesticide
Chemistry, Helsinki 1974, Abstract Book, No. 333.
26. Plestina, R.: The toxicology of some anticholinesterases
used in disinsection (in Croatin), 1st Yugoslav Symposium
of Naval Medicine, Trogir 1974, Abstract Book, No. 74.
27. Plestina, R., Fajdetid, T., Matasin, M.: The effect of pH
on transport of 0,0-dimethyl 2,2,3-trichloro-l-hydroxyethyl
phosphonate (metrifonate) through the rat intestinal sac
(in Croatin), 9th Congress of the Yugoslav Physiological
Society, Portoroz 1975, Abstracts No. 186.
28. Plestina, R., Piukovic-Plestina, M., Horvat, S.: Ophthal-
mological examination of workers exposed to pesticides, 18th
International Congress on Occupational Health, Brighton
1975, Abstracts, p. 472.
158
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29. Plestina, R., Rodrigues, F. P., Wilhelm, K.: Influence of
inhibition and stimulation of microsomal enzymes on toxic
effects of some organophosphorus compounds (in Croatian),
8th Congress of the Yugoslav Physiological Society, Opatija
1973, Abstracts No. 151.
30. Plestina, R., Svetlicid, B.: The toxicity of two monomethyl
carbamates after different routes of application to dogs
(in Croatian), 8th Congress of the Yugoslav Physiological
Society, Opatija 1973, Abstracts No. 152.
31, Plestina, R., Wilhelm, K.: The effect of metrifonate (tri-
chlorfon) on rat cholinesterase (in Croatian), 1st Yugoslav
Congress of Toxicologists, Herceg-Novi 1974, Abstracts No.
48.
32. Radic, B., Stefanac, Z., Wilhelm, K.: Determination of
metrifonate and dichlorvos in biological material by thin
layer chromatography, Meeting of Chemists of Croatia,
Zagreb 1975, Abstracts, p. 261.
33. Reiner, E., Krauthacker, B., Rodrigues, P. F.: Stability of
metrifonate and DDVP in aqueous solutions, and inhibition of
acetylcholinesterase in metrifonate solution, Meeting of
Chemists of Croatia, Zagreb 1973, Abstracts, p. 307.
34. Reiner, E., Krauthacker, B., Simeon, V., Spoljar, M.:
Mechanism of inhibition of mammalian cholinesterases in
trichlorphon solutions, 3rd International Congress of
Pesticide Chemistry, Helsinki 1974, Abstract Book, No.
256.
35. Reiner, E., Simeon, V-: Effect of substrates on reversible
inhibition of acetylcholinesterase and cholinesterase by two
coumarin derivatives, 9th FEES Meeting, Budapest 1974,
Abstracts of Communications, No. s2i7.
36. Reiner, E., Simeon, V., Kobrehel, Dj.: Comparison between
substrate inhibition of acetylcholinesterase and reversible
inhibition by some organophosphorus compounds, 8th FEES
Meeting, Amsterdam 1972, Abstracts of Communications, No. 428.
37. Reiner, E., Skrinjaric-Spoljar, M., Kraij, M.: Cholinesterase
in Metastrongylus apri (M. apri): Substrate specificity and
inhibition by trichlorphon solutions, 10th FEES Meeting,
Paris 1975, Abstracts No. 771.
38. Simeon, V.: Michaelis constants for cholinesterases and the
influence of substrate on inhibition by anticholinesterases
(in Croatian), 8th Congress of the Yugoslav Physiological
Society, Opatija 1973, Abstract No. 94.
159
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39. Simeon, V., Kobrehel, Dj.: Effect of ionic strength and
temperature on reversible and progressive inhibition of
acetylcholinesterase by some inhibitors, Meeting of
chemists of Croatia, Zagreb 1973, Abstracts, p. 305.
40. Simeon, V., Reiner, E.: Effect of temperature on interaction
of serum cholinesterases with acylating inhibitors and sub-
strates (in Croatian), 3rd Yugoslav Congress of Pure and
Applied Chemistry, Ljubljana 1972, Abstract No. VI-20.
41. Simeon, V., Reiner, E.: Competition of two substrates in
their reactions with acetylcholinesterase and cholineste-
rase, 2. Jahrestagung der Osterreichischen biochemischen
Gesellschaft, Graz 1975, Programme, p. 3 (unpublished).
42. Siroki, M., Stefanac, Z. : Nitrate determination in water by
an extraction-visible spectrophotometric method, Scientific
Session on Environmental Analysis, Szombathely 1974 (unpub-
lished) .
43. Skrinjarid-Spoljar, M., Reiner, E.: Spontaneous reactiva-
tion and aging of dimethylphosphorylated cholinesterases
(in Croatian), 3rd Yugoslav Congress of Pure and Applied
Chemistry, Ljubljana 1972, Abstract No. VI-22.
44. Wilhelm, K.: Monitoring of the effects of anticholineste-
rase pesticides in exposed workers, 3rd International
Congress of Pesticides Chemistry, Helsinki 1974, Abstract
Book, No. 301.
45. Wilhelm, K.: Variations in blood cholinesterase activities
in nonexposed humans, 18th International Congress on Occu-
pational Health, Brighton 1975, Abstracts, p. 160.
46. Wilhelm, K., Plestina, R.: The influence of previous carba-
moylation on cholinesterase activity in rats poisoned with
some organophosphorus compounds (in Croatian), 1st Yugoslav
Congress of Toxicologists, Herceg-Novi 1974, Abstract No.
83.
47. Wilhelm, K., Plestina, R., Svtlicic, B.: The toxicity of
Ekatin and its action on occupationally exposed workers
(in Croatian), Meeting of Chemists of Croatia, Zagreb 1973,
Abstracts, p. 465.
Books
48. Aldridge, W. N., Reiner, E.: "Enzyme inhibitors as sub-
strates. Interaction of esterases with esters of organo-
phosphorus and carbamic acids", North Holland Publ. Co.,
Amsterdam (1972) XVI + 328 pp.; First reprint, 1975.
160
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49. Reiner, E. (Editor): "Cholinesterases and cholinergic re-
ceptors", Proceedings of the International Meeting, Split
1975, Croatica Chemica Acta, Zagreb (1975), XII + 344 pp.
Reviews
50. Krauthacker, B.: Methods for determination of 0,0-dimethyl
2,2,2-trichloro-l-hydroxyethyl phosphonate (DMTHF) (in
Croatian), Arh. hig. rada, 2£ (1975) 157-169.
51. Simeon, V.: Comparison of biochemical properties of cholin-
esterases (in Croatian), Arh. hig. rada, £3_ (1972) 29-39.
52. Wilhelm, K.: Toxicology and prevention of poisoning with
some pesticides used for protection of stored food stuff
(in Croatian) , Biljna zastita, !L9 (1975) 87-90 (part I)
and 126-129 (part II).
Theses
53. Bradamante-Simic, V.: The effect of anticholinesterase
pesticides in occupationally exposed workers (in Croatian),
M. Sc. Thesis, University of Zagreb, 1975.
54. Fink, K.: The persistence of some organophosphorus pesti-
cides in aqueous media (in Croatian), B. Sc. Thesis, Uni-
versity of Zagreb, 1975.
55. Kobrehel, Dj.: Kinetics of acetylcholinesterase inhibition
by two coumarin derivatives (in Croatian), M. Sc. Thesis,
University of Zagreb, 1975.
56. Krauthacker, B.: Stability of 0,0-dimethyl 2,2,2-trichloro-
1-tiydroxyethyl posphonate (metrifonate) in aqueous solutions
and the mechanism of inhibition of cholinesterases in metri-
fonate solutions (in Croatian), M. Sc. Thesis, University
of Zagreb, 1974.
57. Mrvos, Dj.: Decomposition of 0,0-dimethyl 2,2-dichloro-
vinylphosphate (DDVP) in rabbit serum (in Croatian), B. Sc.
Thesis, University of Zagreb, 1975.
58. Radic, B.: Determination of organophosphorus pesticides in
biological material by means of thin layer chromatography
(in Croatian), M. Sc. Thesis, University of Zagreb, 1975.
161
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59. Vasilic, Z.: Quantitative determination of organophospho-
rus pesticides by thin layer densitometry (in Croatian),
B. Sc. Thesis, University of Zagreb, 1976.
60. Zima, S.: Total organic carbon determination in water (in
Croatian), M. Sc. Thesis, University of Zagreb, 1975.
162
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
EPA-600/1-77-031
3. RECIPIENT'S ACCE
.TITLE AND SUBTITLE
Toxicology of Anticholinesterase Pesticides
5. REPORT DATE
June 1977
6. PERFORMING ORGANIZATION CODE
. AUTHOR(S)
Elsa Reiner
' PERFORMING ORGANIZATION NAME AND ADDRESS
Institute for Medical Research and Occupational Health
Zagreb
Yugoslavia
10. PROGRA
1EA615
I. CONTRACT/GRANT NO.
SFCP-PR-2-515-2
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
13. TYPE OF REPORT AND PERIOD
RTP, NC
14. SPONSORING AGENCY CODE
EPA 600/11
fif
Iff Park
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The effect of pesticides was studied in humans and animal models, and on enzymes
in order to develop a better understanding of their mechanism of action, and thus
provide guidance for their safe use. It was shown that reduction of human blood
cholinesterase activity is a good indicator of exposure to anticholinesterase pestici-
des. Regular measurements of blood cholinesterase activity and removal of individuals
from suspected occupational environments prevented hazardous absorption of pesticides
in occupationally exposed people. Ophthalmological studies indicated some functional
eye impairment in people exposed to pesticides for many years.
The effect of one organophosphate ester used as a pesticide (trichlorfon; Metri-
fonate(R)) was studied in detail in humans, animals (rats) and enzymes (cholinesterase
It was shown that trichlorfon does not inhibit mammalian cholinesterases; inhibition
of the enzyme is caused by dichlorvos formed from trichlorfon . The mechanism of
reaction of acetylcholinesterase and cholinesterase with other inhibitors and with
substrates was also studied in order to evaluate to what extent sites other than the
catalytic sites are involved in the binding of ligands. Gas chromatographic methods
for measuring pesticide residues were used to determine the concentrations of chlorina
ted hydrocarbon pesticides in the blood of the general population in Yugoslavia and
for studies of organophosphate residues in urine.
17.
KEY WORDS AND DOCUMENT ANALYSIS
a.
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COS AT I Field/Group
toxicity
cholinesterase inhibitors
pesticides
trichlorfon
Metrifonate
06 F
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
175
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
163
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