INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
ENVIRONMENTAL HEALTH CRITERIA 63
ORGANOPHOSPHORUS INSECTICIDES: A GENERAL INTRODUCTION
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Labour Organisation, or the World Health Organization.
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the International Labour Organisation,
and the World Health Organization
World Health Orgnization
Geneva, 1986
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CONTENTS
ENVIRONMENTAL HEALTH CRITERIA FOR ORGANOPHOSPHOROUS INSECTICIDES -
A GENERAL INTRODUCTION
PREFACE
1. SUMMARY AND RECOMMENDATIONS
1.1. Summary
1.1.1. General
1.1.2. Properties and analytical methods
1.1.3. Sources; environmental transport and distribution
1.1.4. Environmental levels and exposure
1.1.5. Effects on organisms in the environment
1.1.6. Metabolism
1.1.7. Mode of action
1.1.8. Effects on experimental animals and in vitro
test systems
1.1.9. Effects on human beings
1.1.10. Therapy of poisoning
1.2. Recommendations
2. PROPERTIES AND ANALYTICAL METHODS
2.1. Chemical and physical properties
2.1.1. Effects of light
2.1.2. Effects of solutes and solvents
2.2. Analytical methods
3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE, ENVIRONMENTAL
TRANSPORT AND DISTRIBUTION, EXPOSURE LEVELS
3.1. Sources of pollution
3.2. Environmental transport and distribution
3.2.1. Distribution in air and water
3.2.2. Distribution in food
3.3. Bioaccumulation and degradation in the environment
3.4. Exposure levels
3.4.1. Exposure of the general population
3.4.2. Occupational exposure
4. METABOLISM AND MODE OF ACTION
4.1. Uptake
4.1.1. Dermal uptake
4.1.2. Gastrointestinal tract
4.1.3. Inhalation
4.2. Distribution and storage
4.2.1. Experimental animal studies on distribution
and storage
4.3. Biotransformation
4.3.1. Mixed-function oxidases (MFOs)
4.3.1.1 Oxidative desulfuration
4.3.1.2 Oxidative N - dealkylation
4.3.1.3 Oxidative O -dealkylation
4.3.1.4 Oxidative de-arylation
4.3.1.5 Thioether oxidation
4.3.1.6 Side-chain oxidation
4.3.2. Hydrolases
4.3.3. Transferases
4.3.3.1 Transferases handling primary metabolites
4.3.4. Tissue binding
4.4. Elimination
4.5. Mode of action
4.5.1. Inhibition of esterases
4.5.2. Possible alkylation of biological macromolecules
5. EFFECTS ON ORGANISMS IN THE ENVIRONMENT
5.1. Aquatic organisms
6. EFFECTS ON ANIMALS
6.1. Effects on the nervous system
6.1.1. Effects attributed to interaction with esterases
6.1.1.1 Cholinergic effects
6.1.1.2 Delayed neuropathic effects
6.1.2. Behavioural and other effects on the nervous system
6.2. Other effects
6.2.1. Mutagenic and carcinogenic effects
6.2.2. Teratogenic effects
6.2.3. Effects on the immune system
6.2.4. Effects on tissue carboxyesterases
6.2.5. Sundry other effects of organophosphorus
pesticides
6.2.5.1 Effects on hormones
6.2.5.2 Effects on the reproductive system
6.2.5.3 Effects on the retina
6.2.5.4 Porphyric effect
6.2.5.5 Lipid metabolism
6.2.5.6 Effects causing delayed deaths
6.2.5.7 Selective inhibition of thermogenesis
6.3. Factors influencing organophosphorus insecticide toxicity
6.3.1. Dosage-effect
6.3.2. Age and sex
6.3.3. Nutrition
6.3.4. Effects of impurities and of storage
6.3.4.1 Impurities toxic in their own right
6.3.4.2 Impurities potentiating the toxicity
of the major ingredient
6.3.5. Effects of other pesticides and of drugs
6.3.6. Species
6.3.7. Other factors
6.4. Acquisition of tolerance to organophosphorus
insecticides
6.5. Therapy of experimental organophosphorus poisoning
6.5.1. Palliation
6.5.2. Antagonism of effects of ACh
6.5.3. Reactivation of inhibited AChE
6.5.4. Efficacy of therapy
7. EFFECTS ON MAN
7.1. Acute cholinergic poisoning
7.1.1. Methods for assessing absorption and effects of
organophosphorus insecticides
7.1.1.1 Analysis of urine as a means of monitoring
exposed populations
7.1.1.2 Biochemical methods for the measurement of
effects
7.1.1.3 Electrophysiological methods for the study
of effects
7.1.2. Monitoring studies
7.1.3. Retrospective studies of populations exposed to
organophosphorus pesticides: acute and long-term
exposure
7.2. Other effects on the nervous and neuromuscular system due
to acute or long-term exposure
7.2.1. Delayed neuropathic effects
7.2.2. Behavioural effects
7.3. Effects on other organs and systems
7.4. Treatment of organophosphate insecticide poisoning in man
7.4.1. Minimizing the absorption
7.4.2. General supportive treatment
7.4.3. Specific pharmacological treatment
7.4.3.1 Atropine
7.4.3.2 Oxime reactivators
7.4.3.3 Diazepam
7.4.3.4 Notes on the recommended treatment
REFERENCES
ANNEX I: NAMES AND STRUCTURES OF SELECTED ORGANOPHOSPHORUS
PESTICIDES
ANNEX II: ORGANOPHOSPHORUS INSECTICIDES: JMPR REVIEWS, ADIs,
EVALUATION BY IARC, CLASSIFICATION BY HAZARD, FAO/WHO
DATA SHEETS, IRPTC DATA PROFILE, AND LEGAL FILE
ANNEX III: LD50s AND NO-OBSERVED-ADVERSE-EFFECT LEVELS IN ANIMALS
ANNEX IV: ABBREVIATIONS USED IN THE DOCUMENT
WHO TASK GROUP ON ORGANOPHOSPHOROUS INSECTICIDES
Members
Dr D. Ecobichon, Department of Pharmacology and Therapeutics,
McGill University, Montreal, Quebec, Canada
Dr A.H. El-Sebae, Department of Pesticide Chemistry, Faculty
of Agriculture, University of Alexandria, Alexandria, Egypt
Dr L. Ivanova-Chemishanska, Institute of Hygiene and Occupational
Health, Medical Academy, Sofia, Bulgaria (Vice-Chairman)
Dr M.K. Johnson, Toxicology Unit, Medical Research Council
Laboratories, Carshalton, Surrey, United Kingdom (Rapporteur)
Dr S.K. Kashyap, National Institute of Occupational Health,
Ahmedabad, India
Dr M. Lotti, Institute of Occupational Health, Padua, Italy
Dr L. Martson, All-Union Scientific Research Institute of the
Hygiene and Toxicology of Pesticides, Polymers, and
Plastics (VNIIGINTOX), Kiev, USSRa
Dr U.G. Oleru, College of Medicine, University of Lagos,
Lagos, Nigeria
Dr W.O. Phoon, Department of Social Medicine and Public
Health, National University of Singapore, Outram Hill,
Republic of Singapore (Chairman)
Dr E. Reiner, Institute for Medical Research and Occupational
Health, Zagreb, Yugoslavia
Dr A.F. Rahde, Ministry of Public Health, Porto Alegre, Brazil
Dr J. Sekizawa, National Institute of Hygienic Sciences,
Tokyo, Japan
Observers
Mr R.J. Lacoste, International Group of National Associations
of Pesticide Manufacturers (GIFAP), Brussels, Belgium
Dr W.O. Phoon, International Commission on Occupational
Health, Geneva, Switzerland
Secretariat
Mme B. Bender, United Nations Environment Programme,
International Register of Potentially Toxic Chemicals,
Geneva, Switzerland
Secretariat (contd.)
Dr J.R.P. Cabral, Unit of Mechanisms of Carcinogenesis,
International Agency for Research on Cancer, Lyons, France
Dr K.W. Jager, International Programme on Chemical Safety,
World Health Organization, Geneva, Switzerland (Secretary)
Dr G.J. Van Esch, Bilthoven, The Netherlands (Temporary
Adviser)
Dr C. Xintaras, Office of Occupational Health, World Health
Organization, Geneva, Switzerland
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a Invited, but unable to attend.
NOTE TO READERS OF THE CRITERIA DOCUMENTS
Every effort has been made to present information in the
criteria documents as accurately as possible without unduly
delaying their publication. In the interest of all users of the
environmental health criteria documents, readers are kindly
requested to communicate any errors that may have occurred to the
Manager of the International Programme on Chemical Safety, World
Health Organization, Geneva, Switzerland, in order that they may be
included in corrigenda, which will appear in subsequent volumes.
* * *
Detailed data profiles and legal files for most of the
organophosphorus insecticides can be obtained from the
International Register of Potentially Toxic Chemicals, Palais des
Nations, 1211 Geneva 10, Switzerland (Telephone no. 988400 -
985850).
ENVIRONMENTAL HEALTH CRITERIA FOR ORGANOPHOSPHORUS INSECTICIDES
A WHO Task Group on Environmental Health Criteria for
Organophosphorus Insecticides was held in Geneva on 30 September -
4 October 1985. Dr K.W. Jager opened the meeting on behalf of the
Director-General. The Task Group reviewed and finalized the draft
criteria document.
The drafts of this document were prepared by DR M.K. JOHNSON of
the UNITED KINGDOM MEDICAL RESEARCH COUNCIL.
The efforts of all who helped in the preparation and
finalization of the document are gratefully acknowledged.
* * *
Partial financial support for the publication of this criteria
document was kindly provided by the United States Department of
Health and Human Services, through a contract from the National
Institute of Environmental Health Sciences, Research Triangle Park,
North Carolina, USA - a WHO Collaborating Centre for Environmental
Health Effects. The United Kingdom Department of Health and Social
Security generously supported the cost of printing.
PREFACE
Following the Second World War, organochlorine pesticides made
a major contribution to improvements in agricultural output and in
the control of disease vectors. While the persistence of these
compounds after application was of considerable benefit to the
user, it also introduced problems. As these problems became more
widely appreciated, insect pest control began to rely more on the
anticholinesterase organophosphorus and carbamate ester pesticides.
A large number of such esters have been introduced on the market,
and a much greater number have been screened for pesticidal
activity. Unlike many environmental pollutants, pesticides are
deliberately added to the environment and are devised to be lethal
agents.
It would not be possible to review the class of organo-
phosphorus insecticides in one document, because they are so
numerous (more than 100) and cover a wide range of toxicity.
However, because they have many properties in common, it was
decided to prepare Organophosphorus Insecticides - A General
Introduction to provide background information for brief
Environmental Health Criteria documents on specific organo-
phosphorus insectides.
In addition to the literature cited in the text, much useful
information has been obtained from the following works of
reference: CEC (1977), Kagan (1977, 1985), Hayes (1982), Medved &
Kagan (1983), Worthing (1983), Mel'nikov et al. (1985), and Farm
Chemicals Handbook (1985).
For the purposes of this document, the word "insecticide" is
used more broadly than the strict zoological classification of
insects. Many have some selectivity for particular classes of
pests (mites, aphids, etc.) but, with 2 exceptions, all the
compounds covered in the introductory document exert their primary
effect by inhibiting the vital acetylcholinesterase of the nervous
system.
Non-ester organophosphorus compounds, having herbicidal
activity are not considered, but the herbicide amiprophos ( O -
ethyl- O -4-methyl-6-nitrophenyl N- isopropyl phosphoramidothioate)
and defoliants related to DEF ( S,S,S -tri- n -butylphosphoro-
trithioate) are included because they, in common with
organophosphorus insecticides, are esters and possess the ability
to inhibit tissue esterases and can cause cholinergic and/or
delayed neuropathic responses.
1. SUMMARY AND RECOMMENDATIONS
1.1. Summary
1.1.1. General
At least 100 organophosphorus insecticides have been reviewed
by WHO for consideration as agents for the control of disease
vectors. A large number have been reviewed by the FAO/WHO Joint
Meetings on Pesticide Residues. Unlike many compounds scrutinized
by the IPCS, these compounds are designed to be toxic for certain
pests and are added deliberately to the environment. However, they
have a wide range of acute toxicity for experimental animals, and
it is impossible to review the whole class in a single
comprehensive document. Thus, the purpose of this document is to
give a framework of information and understanding with suitable
illustrations that will provide the background to brief
Environmental Health Criteria documents on specific insecticides.
For the purposes of this document, the word "insecticide" is
used in a broad sense and covers miticides, acaricides, etc. A few
organophosphorus insecticide compounds, with a toxicological mode
of action similar to that of the insecticides, are mentioned,
though they are intended for use as herbicides.
1.1.2. Properties and analytical methods
Organophosphorus insecticides are normally esters, amides, or
thiol derivatives of phosphoric, phosphonic, phosphorothioic, or
phosphonothioic acids. Most are only sightly soluble in water and
have a high oil-to-water partition coefficient and low vapour
pressure.
Physical and chemical data are not given in this introduction
but may be obtained from other sources including other WHO
publications, IRPTC profiles, and the handbooks included in the
list of references. A principal source for analytical methods is
provided by the Codex Alimentarius Commission of the Joint FAO/WHO
Food Standards Programme.
1.1.3. Sources; environmental transport and distribution
While there has been a considerable increase in the annual use
of organophosphorus insecticides for crop protection since 1970,
the overall increase has been less since the early 1980s. However,
new uses and formulations have been introduced. In particular,
parathion and malathion are widely used. Only a few of the less
hazardous organophosphorus insecticides have been evaluated for
disease vector control, and these contribute a very small
percentage to total usage.
With the exception of dichlorvos, most organophosphorus
insecticides are of comparatively low volatility. Dispersion of
spray droplets by wind is possible, but, in general, only small
amounts are likely to be distributed in this way.
The principal route of degradation in the environment seems to
be hydrolysis. In soil and the aqueous environment, the survival
time and the possibility of distribution in water may be influenced
by light intensity and pH. Most organophosphorus insecticides are
more stable in the pH range that may be encountered in the
environment (pH: 3 - 6), than at neutral pH. The influence of
microbiological factors in the degradation of these insecticides in
soil and water may be considerable. Different climatic conditions,
especially temperature and humidity, before, during, and after
spraying may influence the survival time markedly.
1.1.4. Environmental levels and exposure
Apart from occupationally exposed workers or populations
exposed as a result of disease-vector control programmes, marked
exposure of the general population is not expected. While exposure
via foodstuffs is sometimes monitored and controlled, there is
little information about exposure via groundwater, which may reach
drinking-water.
1.1.5. Effects on organisms in the environment
Only a little information is available on the toxicity of
organophosphorus insecticides for fish and aquatic insects. The
mechanism of toxicity has not been shown to be necessarily an
anticholinesterase effect. Lethal concentrations derived from 48-h
exposures in clean laboratory water may be artificially low
compared with exposure in environmental waters.
1.1.6. Metabolism
The metabolic fate of organophosphorus insecticides is
basically the same in insects, animals, and plants. Uptake in
animals and insects may occur through the skin, respiratory system,
or gastrointestinal tract. While uptake of active ingredient
through the skin from powdered or granulated formulations may be
relatively inefficient, the presence of aqueous dispersing agents
or organic solvents in a spray concentrate or formulation may
greatly enhance uptake. Although the actual exposure of the
respiratory system may not be as high as the exposure of skin in
unprotected persons, the efficiency of absorption might be high.
Metabolism occurs principally by oxidation, hydrolysis by
esterases, and by transfer of portions of the molecule to
glutathione. Oxidation of organophosphorus insecticides may result
in more or less toxic products. In general, phosphorothioates are
not directly toxic but require oxidative metabolism to the proximal
toxin. Most mammals have more efficient hydrolytic enzymes than
insects and, therefore, are often more efficient in their
detoxification processes. Birds usually have lower esterase
activity than mammals. The glutathione transferase reactions
produce products, that are, in most cases, of low toxicity.
Hydrolytic and transferase reactions affect both the thioates and
their oxons. Numerous conjugation reactions follow the primary
metabolic processes, and elimination of the phosphorus-containing
residue may be via the urine or faeces. Some bound residues remain
in exposed animals. Binding seems to be to proteins, principally,
and the turnover appears to be related to the half-life of these
proteins. There are limited data showing that incorporation of
residues into DNA occurs only in trace amounts and not by direct
alkylation, such as might be believed to be associated with genetic
damage.
1.1.7. Mode of Action
Organophosphorus insecticides exert their acute effects in both
insects and mammals by inhibiting acetylcholinesterase (AChE) in
the nervous system with subsequent accumulation of toxic levels of
acetylcholine (ACh), which is a neurotransmitter. In many cases,
the organophosphorylated enzyme is fairly stable, so that recovery
from intoxication may be slow.
Because of the greater stability of organophosphorylated AChE
compared with carbamylated enzyme, the ratio of the dose of an
organophosphorus insecticide required to produce mortality and that
which produces minimum symptoms of poisoning is substantially less
than the same ratio for carbamate insecticides. Reactivation of
inhibited enzyme may occur spontaneously, rates of reactivation
depending on the species and the tissue, as well as on the chemical
group attached to the enzyme. In particular, in most mammals,
dimethylphosphorylated AChE undergoes substantial spontaneous
reactivation within one day, which facilitates recovery from
intoxication. Reactivation of inhibited AChE may be induced by
some oxime reagents, and this fact provides opportunities for
therapy. Response to reactivating agents declines with time, and
this process is called "aging" of the inhibited enzyme.
Delayed neuropathy is initiated by attack on a nervous tissue
esterase distinct from AChE. The target has esterase activity and
is called neuropathy target esterase (formerly neurotoxic esterase
(NTE)). The disorder develops not because of loss of esterase
activity but because of some overall change brought about in the
protein molecule resulting from the process of aging of inhibited
NTE: catalytic activity of NTE reappears in the nervous tissue,
even during the period of development of neuropathy. Some
organophosphinates, sulfonyl fluorides, and carbamates may inhibit
NTE and act as protective agents, covering the target with
molecules that cannot engage in the aging reaction. The
structure/activity relationships for inhibitors of NTE differ from
those for AChE, so that pesticides designed as inhibitors of AChE
may be less effective as inhibitors of NTE, and may have low
neuropathic potential.
The rate of reaction of one chosen organophosphorus insecticide
with AChE was many orders higher than its rate of alkylation of the
test nucleophile, 4-nitrobenzylpyridine. On this limited data, it
seems unlikely that alkylation of biological macromolecules by
organophosphorus insecticides would occur in mammals.
1.1.8. Effects on experimental animals and in vitro test systems
The acute toxicity of organophosphorus insecticides is due to
their anticholinesterase action. The oral and dermal LD50s for
many compounds are listed in Annex III. It cannot be over-
emphasized that these numbers are not precise, and substantially
different values may be reported from different sources, even when
the factors of species, age, and sex have been standardized. These
LD50 values range from less than 10 mg/kg body weight to more than
3000 mg/kg for the oral route and, for most compounds, are
significantly higher for the dermal route.
For single exposures, a dose-effect relationship exists between
the dose and the severity of symptoms, and, also, the degree of
AchE inhibition in nervous tissue. The inhibition of blood-AChE
may not be similar to that in nervous tissue. Effects on plasma-
pseudocholinesterase (pseudoChE) are dose-related but are not
correlated with intensity of symptoms. For some insecticides,
pseudocholinesterase is more sensitive to inhibition than AChE,
but, for others, the converse is true.
The majority of organophosphorus insecticides do not cause
delayed neuropathy in test animals at doses up to the LD50. When a
dose is above the LD50 but is given in conjunction with therapy
against anticholinesterase effects, more compounds have been shown
to cause clinical neuropathy and, for others, substantial, but sub-
threshold, effects on NTE have been shown. For other compounds,
only slight effects on NTE have been shown, even at doses much
above the normally lethal dose. The results of dose-response
studies have shown that at least 70% inhibition of NTE in the brain
and spinal cord is required for initiation of delayed neuropathy in
adult hens, the usual test species. This threshold is not so
clearly defined for other species, and laboratory rodents do not
display clinical signs of neuropathy after a single dose. No
marked change in the threshold level of inhibition has been shown
between adult hens of different strains, but more information is
required.
Short- and long-term toxicity studies have been carried out.
While typical cholinergic intoxication only occurs when nervous
tissue AChE is substantially inhibited, the converse may not be
true in cases of long-term exposure because of the development of
tolerance, which is believed to be due, in part, to changes in some
cholinergic receptors. NTE appears to be synthesized continuously.
Consequently, continuous administration of an organophosphorus
compound does not necessarily lead to a continuous increase in the
level of inhibited NTE; the level may tend to reach equilibrium
below the threshold required to initiate neuropathy. With
continuous administration of neuropathic organophosphorus compounds
for up to 90 days, a peak level of about 50% inhibition of NTE must
be maintained to initiate neuropathy.
Acceptable daily intakes (ADIs) have been established as a
result of the evaluation of data by the FAO/WHO Joint Meetings on
Pesticide Residues (JMPR) (Annex II). ADIs are derived from
measurements or estimates of the highest dietary level that does
not cause significant changes in any measured variable, the most
sensitive of which is usually the AChE or pseudoChE activity in
blood. For no-observed-adverse-effect levels, see Annex III.
A variety of behavioural changes have been seen in response to
single or long-term dosing, but, in nearly all of the cases
reported, there was concomitant inhibition of AChE, though not
necessarily up to levels associated with typical signs of
poisoning; dose-response relationships have not always been
established. So far, behavioural tests have not proved adequate to
screen for organophosphate intoxication.
Effects on tissue carboxyesterases may be caused by some
organophosphorus insecticides at doses below those affecting AChE
or ChE. Apart from the delayed neuropathic effect arising from the
inhibition and aging of NTE, inhibition of other carboxyesterases
is not known to have any direct toxic effects. However, prior
inhibition of carboxyesterases may potentiate the toxicity for
mammals of pesticides, such as malathion and most pyrethroids,
which are normally detoxified by tissue esterases.
Various organophosphorus pesticides have been reported to show
positive responses in in vitro mutagenicity tests, but full
experimental details of the tests and control conditions have not
always been available. It can be concluded that some agents are
weakly mutagenic in vitro. Six organophosphorous pesticides have
been evaluated for mutagenic and carcinogenic potential by the
International Agency for Research on Cancer (IARC). In several
cases, the conclusion was that acceptable tests had been performed
with no evidence of carcinogenic potential, while, in others, the
conclusion was that there was "limited evidence consisting of very
small effects above the control background levels in lifetime
studies".
Many organophosphorus insecticides are embryotoxic at doses
that are toxic for the mother. Teratogenic effects have been
reported for trichlorphon in pigs, but few teratogenic effects have
been reported for other compounds.
Some deficiency in immune responses has been reported in
animals dosed with quantities of organophosphorus insecticides that
depressed AChE levels, but not at doses that did not affect AChE.
Several other toxic effects have been claimed after single or
repeated doses of individual compounds, but these effects have not
been reported for a range of the insecticides. Tissues and systems
reported to have been affected include the retina, lung, and
reproductive system.
Differences in toxic dose, but not in the mode of toxicity,
have been reported in animals according to species, age, sex, and
nutritional state. All these factors influence the status of a
variety of metabolizing enzymes in the body, but there is no steady
observable general trend towards increased or decreased toxicity in
response to variations in these variables.
Impurities may be found in either technical grade or formulated
organophosphorus insecticides. The impurities arise during the
synthesis or storage of technical or formulated material. The
levels of impurities may differ according to the route of synthesis
chosen, the formulating ingredients added, or the storage
conditions. Impurities may be toxic in their own right, toxic as
potentiators that block the metabolic degradation of the major
toxic ingredient, or not toxic.
1.1.9. Effects on human beings
Signs and symptoms of acute intoxication by organophosphorus
insecticides include muscarinic, nicotinic, and central nervous
system (CNS) manifestations. Symptoms may develop rapidly, or
there may be a delay of several hours after exposure before they
become evident. The delay tends to be longer in the case of more
lipophilic compounds, which also require metabolic activation.
Symptoms may increase in severity for more than one day and may
last for several days. In severe cases, respiratory failure is a
dominant effect.
In mild cases, or where the compound is disposed of rapidly,
symptoms may regress quite quickly, though depressed blood-ChE
levels may take several weeks to return to normal levels. There
appear to be few long-term effects after acute intoxication, though
weakness and fatigue may persist for several months.
Several methods are available for measuring exposure to, and
effects of, organophosphorus insecticides, and the combined use of
all methods is valuable, both in diagnosis of poisoning and in
determination of exposure. Standard methods for measuring dermal
exposure have been described in technical reports of WHO.
Determination of urinary metabolites provides an indication of
exposure, and analysis of serial samples is more valuable than a
single sample. Methods for the determination of residues are
available, and the Report of the Codex Alimentarius Commission of
FAO/WHO provides details and critical assessment of methods. In
general, it is not possible to relate the concentration of urinary
metabolites to the level of intoxication, though some guidelines
may be developed in connection with the controlled use of any
single organophosphorus pesticide.
Levels of erythrocyte- or whole blood-AChE are a satisfactory
guide to the level of acute intoxication. Plasma or serum levels
of pseudoChE are only useful as indicators of exposure. It is
essential that skin is cleansed carefully before taking blood
samples for analysis. Both enzymes are measurable with good
accuracy using a standard kit suitable for field work and
purchaseable from the World Health Organization; paper tests for
screening purposes have been described. Depression of AChE or
pseudoChE below about 75% of pre-exposure levels is generally
accepted as indicating that a hazard exists, and that workers
should be removed from all contact with the specific insecticide
until the levels recover. Signs of poisoning do not usually appear
until blood levels of AChE are below 50%, while severe poisoning is
usually associated with depression to below 30%. While measurement
of AChE is useful in preventive work and in diagnosis, measuring
the levels of blood-AChE as intoxication or therapy progress is of
less value. Electromyographic (EMG) monitoring of occupationally
exposed workers has been reported to be valuable in assessing
hazard, but there is some dispute, and no certain characteristic
change in EMG has been agreed. Further work under controlled
exposure conditions with parallel chemical, biochemical, and
clinical monitoring is desirable.
In all cases of intoxication, labels from containers should be
preserved, but these may be misleading. Whenever possible, a
sample of the incriminating agent should be stored carefully, and
tissue samples should be taken to aid in the identification of the
active agent.
Delayed neuropathies in occupationally exposed workers have
been reported for only a few of the many currently used
organophosphorus insecticides. For one pesticide, methamidophos,
the syndrome has not been reproduced in experimental animals. There
is no specific treatment for neuropathy, though physiotherapy may
limit the degree of muscle wasting that follows denervation. In
mild cases, some slow improvement can occur, but, in more severe
cases, the defects are permanent. The NTE of human tissue appears
to be similar to the NTE of experimental animals, and
extrapolations from results of laboratory tests in animals may be
of value. Samples of blood lymphocytes provide an accessible
source of NTE for monitoring purposes, though there is some
uncertainty about the stability of NTE in stored lymphocytes.
Continuous long-term exposure to high levels of
organophosphorus insecticides may precipitate typical cholinergic
symptoms, though most of the compounds do not accumulate
extensively in the body. Removal from exposure until AChE levels
return to pre-exposure levels appears to be an adequate health
precaution. There is no clear evidence of adverse effects on
health from long-term exposure to organophosphorus insecticides at
levels that do not affect AChE.
There is limited anecdotal evidence of behavioural effects
arising from long-term, or occasionally even a single, exposure to
one or other organophosphorus insecticide. The reports are
difficult to evaluate and are often complicated by the presence of
other factors, such as endogenous disorders and exposure to other
chemicals.
1.1.10. Therapy of poisoning
Therapy of AChE poisoning by organophosphates may be graded
according to the severity of intoxication. Effective therapy for
most compounds appears to consist of co-administration of atropine
with an oxime reactivating agent plus diazepam. Useful physical
measures include the maintenance of clear airways plus artificial
respiration. Efficacy of oximes may decline as the inhibited AChE
ages. Oxime therapy may continue to be effective in reactivating
AChE, freshly inhibited by inhibitor released from storage in body
depots, long after the bulk of the inhibited enzyme has aged.
There is no known therapy for severe delayed neuropathy. Mild
neuropathies tend to regress, presumably due to some regeneration
or adaptation of peripheral nerves.
1.2. Recommendations
Recommendations for further work on individual organophosphorus
insecticides have been made in the Monographs published in the
Technical Report Series of the JMPR and in some reports from IARC.
Apart from these, some general and specific recommendations are:
1. More up-to-date information should be obtained on the world-
wide production and uses of organophosphorus pesticides.
2. Information is needed on environmental pathways,
concentrations, and distribution of organophosphorus pesticides.
3. There is a need for more information on the occurrence and fate
of organophosphorus insecticides in surface water, soil, and
groundwater, and on their impact on plants, invertebrates, and
mammals.
4. Further studies are necessary on the occurrence of
organophosphorus insecticides in the different food chains
(bioaccumulation) and in the food and drinking-water of man (market
basket or total-diet studies), in order to estimate the daily
exposure of the population.
5. More information should be obtained on the acute and long-term
toxicity of certain organophosphorus insecticides for aquatic and
terrestrial organisms.
6. Apart from a number of studies on human volunteers and a number
of accidents, there is little information on the effects of human
exposure to organophosphorus insecticides. More information should
be collected to evaluate the risks of human exposure to these
compounds.
7. Further work should be done to develop more adequate analytical
methods (i.e., faster procedures and simpler equipment) to
determine organophosphorus residues in biological material (urine,
blood), and also in food. In this connection, the work of the
Codex Alimentarius Commission is noted. Also, further work is
required to develop less hazardous reagents for these analyses.
8. Exposure and health variables in workers exposed occupationally
to only one organophosphorus insecticide at a time should be
carefully monitored. This is an essential background to the more
complex problem of assessment of workers exposed to a variety of
pesticides. Procedures should include validation of methodology of
chemical, biochemical, behavioural, and electrophysiological tests
and should demonstrate the variation in results in both pre-
exposure and post-exposure situations. Adequate groups of matched
controls should also be studied.
9. Measurements of NTE responses in toxicity tests on hens should
be evaluated. The validity and variability of such tests should
be established. Further studies are needed to establish whether
NTE in lymphocytes and/or platelets should be measured in people
exposed to certain organophosphorus insecticides.
10. Enzymes that hydrolyse organophosphates play a role in
detoxifying some organophosphorus insecticides. Further studies
are required to establish whether the activity of these enzymes in
plasma is a good guide to the total hydrolytic capacity of the
whole body.
11. Liasion between National Poison Control Centres and experts
studying the effects of organophosphorus insecticides should be
improved. Preservation of blood, urine, and gastric lavage fluids
might assure the identity of an intoxicating agent. Also,
preservation of autopsied nervous tissue from fatal cases may
facilitate laboratory studies on the dose-response of human nervous
tissue NTE. Such studies may indicate the threshold of NTE
inhibition that might be expected to initiate delayed neuropathy in
man.
12. Information should be obtained concerning the changes in
toxicity due to impurities that can arise in pesticides as a
consequence of different manufacturing processes, the use of
formulating ingredients, and improper storage.
13. Consideration should be given to possible conflicts of
therapeutic procedures recommended for the treatment of poisoning
by other classes of pesticide when dealing with severe intoxication
by mixtures of such compounds with organophosphorus insecticides.
14. Users should be encouraged to be aware of the necessity to
establish a safe re-entry period according to local conditions.
2. PROPERTIES AND ANALYTICAL METHODS
2.1 Chemical and Physical Properties
Various structures of organophosphorus insecticides are
illustrated in Table 1. The compounds are normally esters, amides,
or thiol derivatives of phosphoric or phosphonic acid:
R1 O (or S)
\ ||
P -- X
/
R2
where R1 and R2 are usually simple alkyl or aryl groups, both of
which may be bonded directly to phosphorus (in phosphinates), or
linked via -O-, or -S- (in phosphates), or R1 may be bonded
directly and R2 , bonded via one of the above groups (phosphonates).
In phosphoramidates, carbon is linked to phosphorus through an -NH
group. The group X can be any one of a wide variety of substituted
and branched aliphatic, aromatic, or heterocyclic groups linked to
phosphorus via a bond of some lability (usually -O- or -S-) and is
referred to as the leaving group. The double-bonded atom may be
oxygen or sulfur and related compounds would, for example, be
called phosphates or phosphorothioates (the nomenclature
"thiophosphate" or "thionophosphate" is now less used).
The P=O form of a thioate ester may be referred to as the oxon,
and this is often incorporated in the trivial name (e.g., parathion
is the parent P=S compound of paraoxon).
The variations in the phosphorus group for the insecticides
that have been developed, are shown in Table 1 together with the
common or other names for some pesticides falling into this
classification. The complete structure and names for all the
organophosphorus compounds mentioned are listed in Annex I. It can
be seen that, in terms of numbers of commercial compounds, there
are 3 main groups: phosphates (without a sulfur atom),
phosphorothioates (with one sulfur atom), and phosphorodithioate
(with 2 sulfur atoms). Since the P=S form is intrinsically more
stable, many insecticides are manufactured in this form which can
be converted to the biologically active oxon in tissues. The
manner of this conversion is discussed in section 4.
Specific biotransformation of substituent groups in R1 , R2 , and
X may occur, and this is also considered later. Cleavage of the
direct carbon-to-phosphorus bonds of phosphonates and phosphinates
may occur to a small extent in the final stages of biodegradation,
but is probably insignificant as far as biological effects are
concerned.
Table 1. Variations in the chemical structure of organophosphorus insecticides
---------------------------------------------------------------------------------------------------------
Type of phosphorus group Outline of structure Common or other name
---------------------------------------------------------------------------------------------------------
Phosphate O chlorfenvinphos, crotoxyphos, dichlorvos,
|| dicrotophos, heptenphos, mevinphos, monocroto-
(R-O)2-P-O-X phos, naled, phosphamidon, TEPP, tetrachlor-
vinphos, triazophos
O -alkyl phosphorothioate O amiton, demeton-S-methyl, omethoate, oxydemeton-
|| methyl, phoxim, vamidothion
(R-O)2-P-S-X
S azothoate, bromophos, bromophos-ethyl, chlor-
|| pyriphos, chlorpyriphos-methyl, coumaphos, dia-
(R-O)2-P-O-X zinon, dichlofenthion, fenchlorphos, fenitro-
thion, fenthion, iodofenphos, parathion, para-
thion-methyl, pyrazophos, pyrimiphos-ethyl,
pyrimiphos-methyl, sulfotep, temephos, thionazin
Phosphorodithioate S amidithion, azinophos-ethyl, azinophos-methyl,
|| dimethoate, dioxathion, disulfoton, ethion,
(R-O)2-P-S-X formothion, malathion, mecarbam, menazon, meth-
idathion, morphothion, phenthoate, phorate,
phosalone, phosmet, prothoate, thiometon
S- alkyl phosphorothioate profenofos, trifenofos
R O
\ ||
S ||
\||
P-O-X
/
O
/
R
S- alkyl phosphorodithioate S prothiofos, sulprofos
R-S ||
\||
P-O-X
/
R-O
Phosphoramidate O cruformate, fenamiphos, fosthietan
||
(R-O)2-P-NR2
---------------------------------------------------------------------------------------------------------
Table 1. (contd.)
---------------------------------------------------------------------------------------------------------
Type of phosphorus group Outline of structure Common or other name
---------------------------------------------------------------------------------------------------------
Phosphorotriamidate O triamiphos
||
R2N-P-N
|
NR2
Phosphorothioamidate O methamidophos
||
R-O-P-NR2
|
S-alkyl
S isofenphos
||
(R-O)2-P-NR2
Phosphonate O butonate, trichlorfon
RO ||
\||
P-O-X
/
R
Phosphonothioate S EPN, trichlornat, leptophos, cyanofenphos
R-O ||
\||
P-O-X
/
R
---------------------------------------------------------------------------------------------------------
In order to be useful, these compounds must be reasonably
stable at neutral pH, since many are formulated as concentrates in
oil, in water-miscible solvents such as ethylene glycol monomethyl
ether, or are absorbed on to inert granules for application
directly or after dispersion in water. However, nearly all are
rapidly hydrolysed by alkali and many are also unstable at pH
levels below 2. Phosphoramidates are hydrolysed in an acid-
catalysed reaction, even at pH 4 - 5, and, since acid is produced,
decomposition tends to accelerate due to autocatalysis.
Oxidation of phosphorothioates to phosphates (-P=S --> -P=O)
is potentially dangerous, since the phosphates are more volatile
and are directly toxic agents. This can occur by oxidation of
stored products at elevated temperatures. The enzymatic catalysis
of this reaction is considered in section 4.
Various uncatalysed isomerizations are reported to occur under
forcing conditions of heating at over 100 °C for many hours in the
laboratory (Dauterman, 1971). Also, an isomerization associated
with considerable toxic hazard has been observed during the storage
of some formulations of malathion, particularly under warm humid
climatic conditions:
S O
| ||
(CH3O)2-P-SR ---> CH3S-P-SR
|
OCH3
The S- methyl derivatives, formed from malathion by this
isomerization, potentiate the toxicity of malathion markedly
(section 6.3.4). The isomerization reaction is not completely
understood, but it has been shown to be catalysed by dimethyl
formamide under laboratory conditions (Eto & Ohkawa, 1970). It is
not clear whether all alkyl phosphorothioates are subject to this
reaction, but it probably occurs most readily with the methyl
esters and may be influenced by the formulating agents. The hazard
resulting from isomerization will depend, not only on the extent of
the reaction and the intrinsic toxicity of the product, but also on
the manner of metabolic disposal of the parent compound (see
discussion in section 7).
Besides the various effects of heat and air noted above, both
light and solvent may influence the stability of these
organophosphorus compounds.
2.1.1 Effects of light
Parathion was one of the first organophosphorus compounds in
which the anticholinesterase activity, as measured in vitro, was
shown experimentally to increase during exposure to ultraviolet
radiation (UVR) and sunlight. However, the acute toxicity of
parathion decreased under UVR, although the in vitro
anticholinesterase activity increased as the result of the
formation of more polar products; the metabolites were identified
as paraoxon and the S- ethyl and S -phenyl isomers of parathion,
together with unknown products (Dauterman, 1971). This study
showed that UVR is able to oxidize as well as isomerize parathion.
When parathion-methyl was given the same UVR treatment, only the
methyl homologue of paraoxon was found. In a similar study, in
which EPN was exposed to UVR, the oxygen analogue of EPN and
p -nitrophenol were found together with unidentified resins,
also indicating cleavage of the P-O-aryl bond. Studies with 7
organophosphorus pesticides containing sulfur in a thioether group
indicated that exposure to UVR (254 nm) resulted in a variety of
oxidation products. With phorate, disulfoton, and thiometon, the
corresponding sulfoxides and sulfones were identified as products
of UVR. With thiometon, evidence of oxidation of the thiono sulfur
was also obtained. In all 7 cases, the oxidation products were
more acutely toxic than the parent compound. Exposure of a
carbethoxy analogue of mevinphos to UVR results in another type of
photoisomerization. Starting with either the cis- or the trans-
isomer, or a mixture of the isomers, and exposing the compounds to
UVR, results in a mixture of approximately 30% of the cis- and 70%
of the trans-isomer; in all cases, the trans-isomer was
predominant. When chlorpyrifos is exposed to UVR or sunlight, it
undergoes hydrolysis in the presence of water to liberate 3,5,6-
trichlor-2-pyridinol, which then undergoes complete
photodechlorination with the formation of diols, triols, and
tetraols.
2.1.2 Effects of solutes and solvents
The hydrolysis of organophosphorus compounds is influenced by
solutes, e.g., some amino acids, hydroxylammonium derivatives;
metal ions such as Cu++ act as catalysts.
Solvents used in formulating organophosphorus compounds to
obtain properties that will increase the chances of contact between
the insecticide and the target organism, influence their stability.
It has been found that dimethoate in certain hydroxylic solvents,
particularly 2-alkoxyethanols, increased in toxicity on storage
(Casida & Sanderson, 1963). The acute oral LD50 for rats decreased
from 150 - 250 mg/kg body weight to 30 -40 mg/kg, after 7 months
storage at normal temperatures. Studies indicated that many
reaction products were formed in the presence of methylcellosolve.
The degradation involved hydrolysis of the amide bond, hydrolysis
of ester groups, and loss of the thiono group. The most toxic
fraction was identified as dimethoate with probably one, but
possibly both, of the methyl groups replaced by 2-methoxyethyl
groups. No evidence was obtained for the formation of pyro-
phosphates. In the same study, the toxicity of a few other
phosphorothioate compounds was also found to increase in the
presence of 2-methoxyethanol.
Another type of reaction occurs when organophosphorus compounds
containing a sulfide group (R-S-R) are stored undiluted or in an
aqueous solution. Heath & Vandekar (1957) observed that a 1%
solution of demeton- S -methyl increased in toxicity spontaneously
at 35 °C during the course of one day. This increase was found to
be due to the formation of a transalkylated sulfonium derivative,
the toxicity of which was more than 1000 times that of the parent
compound. A similar reaction has also been shown to take place
with demeton-O (phosphorothioic acid, O,O -diethyl O -[2-
(ethylthio)-ethyl] ether:
S
||
(C2H5O)2-P-OC2H4-SC2H5
Samples of demeton-S-methyl that have been stored for a few months
may contain up to 4% of the sulfonium compound. The
transalkylation reaction is extremely rapid with demetonmethyl, but
slower with demeton.
2.2 Analytical Methods
Procedures consist of sampling, extraction, clean-up of
extract, and determination of compounds. Different procedures are
required for the lipophilic alkali-labile parent pesticides and for
residues that may be mainly stable non-lipophilic hydrolysis
products. Procedures for the determination of pesticide residues
(not only organophosphorus insecticides) are discussed in the
Report of a Joint FAO/WHO Course (Ambrus & Greenhalgh, 1984).
Separation and clean-up usually involve partition between solvents
and chromatography. Detection may be by partially specific colour
reagents or by enzyme inhibition tests applied to spots on thin-
layer chromatographic plates (Stefanac et al., 1976) or by
formation of volatile derivatives suitable for detection by gas
chromatography (Shafik et al., 1973). Diazopentane has been
recommended as a reagent that is less toxic and less difficult to
handle than diazomethane which is often used, but not all workers
regard the modification as satisfactory (Drevenkar et al., 1979).
The hazard of using the volatile and highly carcinogenic
diazomethane as a laboratory reagent should be recognized.
Methods for the the determination of residues of many
individual pesticides are given by the Codex Alimentarius
Commission (1984).
3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE, ENVIRONMENTAL
TRANSPORT AND DISTRIBUTION, EXPOSURE LEVELS
3.1 Sources of Pollution
Organophosphorus pesticides are mainly used in crop protection.
The world-wide consumption of these compounds from 1974-83 is
shown in Table 2. Only parathion and malathion can be shown
separately from the other organophosphorus pesticides. The
information is incomplete since, for example, the USA and some
other countries and regions do not report figures for every year.
However, comparison of the figures given on a yearly basis gives an
idea of the magnitude of the consumption and distribution of the
organophosphorus pesticides throughout the world.
All organophosphorus pesticides are subject to degradation by
hydrolysis yielding water-soluble products that are believed to be
non-toxic at all practical concentrations. The toxic hazard is
therefore essentially short-term in contrast to that of the
persistent organochlorine pesticides, though the half-life at
neutral pH may vary from a few hours for dichlorvos to weeks for
parathion. At the pH of slightly acidic soils (pH 4 - 5), these
half-lives will be extended many-fold. However, constituents of
soil and of river water may themselves catalyse degradation.
3.2 Environmental Transport and Distribution
3.2.1 Distribution in air and water
With the exception of dichlorvos, most organophosphorus
pesticides are of comparatively low volatility. Aerial sprays of
dispersions of organophosphates may be spread by wind, but no
evidence of contamination beyond limits of 1 - 2 km from the
spraying source has been noted.
Three sources of entry into water are possible. One is from
industrial waste or effluent discharged directly into water. A
second is by seepage from buried toxic wastes into water supplies.
Neither of these should be tolerated, since prior treatment of the
waste with alkali (or acid in cases such as diazinon) followed by
neutralization can destroy the toxic agent. Contamination of
running water directly or from run-off during spraying operations
can occur. No studies on the degradation of organophosphorus
pesticides in running water have been noted. In static water, in a
simulated aquatic environment, there is evidence of the
contributions of light, suspended particulates, and bacteria to
degradation. Thus, the degradation of fenitrothion in lake water
under illumination occurred with a half-life of about 2 days,
compared with 50 days in the dark (Greenhalgh et al., 1980).
Furthermore, Drevenkar et al. (1976) concluded that, though
temperature and pH were major factors controlling the rate of
hydrolysis of dichlorvos in water, large differences in the half-
life of this pesticide in different river waters must be attributed
to microbiological factors.
Table 2. Consumption of organophosphorus insecticides (in 100 kg)a
-----------------------------------------------------------------------------------------------------------------
Country Parathion Malathion Other organophosphorus
insecticides
1974 1981 1982 1983 1974 1981 1982 1983 1974 1981 1982 1983
-76 -76 -76
-----------------------------------------------------------------------------------------------------------------
Africa
Burundi 3
Egypt 397 3573 2080 54 267 7200
Gambia 120 1000 477 350
Madagascar 2
Mauritania 50 107
Niger 694 263 151 170 45
Rwanda 1 2 3
Sierra Leone 40
South Africa 24 083
Sudan 6787
Swaziland 17
Zimbabwe 215 450 91 10 3018
North/Central America
Bermuda 7 2
Canada 238 2398 11 519
Cuba 22 667
El Salvador 4000 50 357
Guatemala 7704 1010
Honduras 391 414
Mexico 46 000 50 000 48 000 48 000 3452 12 000 18 000 5000 21 812 54 520 46 350 48 300
Mont Serrat 1 1 1 1
USA 115 000 110 000 15 000 15 000 190 000 175 000
South America
Argentina 1650 4750 2280 2350 2993 8340 15 560
Guyana 52 60
Surinam 28 712
Uruguay 10 105 78 179 45 91 47 201 415 344 735
-----------------------------------------------------------------------------------------------------------------
Table 2. (contd.)
-----------------------------------------------------------------------------------------------------------------
Country Parathion Malathion Other organophosphorus
insecticides
1974 1981 1982 1983 1974 1981 1982 1983 1974 1981 1982 1983
-76 -76 -76
-----------------------------------------------------------------------------------------------------------------
Asia
Bahrain 5
Bangladesh 620 649
Brunei 8 6 20
Burma 34 317
Cyprus 222 1782 842 89 255 212 132 534 591
Hong Kong 1000 414 604 536
India 9657 20 920 30 300 15 640 6800 8000 14 927 40 740 53 510
Israel 8557 11 280 7550 5860
Japan 1800 1000 128 880
Jordan 5000 4500 103 736 40 382
Korea Rep. 711 1426 1601 2759 726 337 24 522 28 224 28 615
Kuwait 4 4
Oman 350 240 830 498 108
Pakistan 982 530 324 90 2381 675 7929 8998 7460
Philippines 4800 630 310 80
Saudi Arabia 55
Sri Lanka 590 1690 20
Turkey 6480 1750 1837 1939 550 577 20 764 11 000 11 550
United Arab 34
Emirates
Europe
Austria 129 201 156 150 839 798 992 993
Czecho- 20 130 44 187 3370 4812 4892
slavakia
Denmark 1581 2578 2334 206 110 108 656 847 1123
Finland 55 74 407
Greece 2873 2837 6493
Hungary 30 599 17 325 11 301 10 190 2711 1584 1598 3130 66 624 77 361 66 848 44 606
Iceland 2 2 2 3 3 2
Italy 23 147 24 231 18 591 8997 6068 5524 87 204 149 558 144 956
Malta 350 250
Norway 196 205 202 212
Poland 530 707 1062 1038 6730 5099 12 246 13 549
Portugal 301 509 643 109 144 227 854 818 880
Sweden 3020 74 60 1029 1264
Switzerland 800 850 800
----------------------------------------------------------------------------------------------------------------
a From: FAO (1984).
3.2.2. Distribution in food
Exposure of food materials to organophosphorus pesticides
occurs chiefly at the crop-growing stage. The scale and frequency
of application varies enormously. Thus, one or 2 applications may
be adequate for pest control in temperate climates, while as many
as 50 applications in one peach-growing season have been reported
for a hot and humid region (Wicker et al., 1979). The amount
remaining on the crop at harvest depends chiefly on the interval
between application and harvest and on the effects of rainfall,
which can wash the active agent off and also provide a milieu for
hydrolysis. Thus, under exceedingly hot and dry conditions, very
high residues of paraoxon were found on citrus plants that had been
sprayed with parathion, 28 days previously: these levels accounted
for the poisoning of several orange pickers, who were working in
the grove at this time, after what is normally an acceptable safe
interval from the time of spraying (Spear et al., 1977). It seems
that both excessive photo-oxidative formation of paraoxon and
absence of hydrolysis or wash-off accounted for the toxic level of
paraoxon. Post-harvest levels of these organophosphorus pesticides
in food appear to decline steadily: this loss is thought to be
principally due to hydrolysis. Dichlorvos, malathion, or
pirimiphos-methyl may be applied to stored grain for the control of
some pests.
For each of the organophosphorus pesticides covered by the
Joint FAO/WHO Meetings, considerable detail is available on the
rate of decline of residues on a wide variety of crops, under
different climatic conditions.
3.3 Bioaccumulation and Degradation in the Environment
While storage in the fat of an organism or animal may reduce
the rate of clearance from that individual, it is unlikely that
significant amounts of an organophosphorus pesticide stored in one
organism could survive the hydrolytic processes of consumption and
digestion to be stored successively by higher members of the food-
chain. Direct poisoning of consumers of sprayed food or pest-
contaminated carcasses can occur, of course.
Degradation in the environment involves both hydrolysis and
oxidation to mono- or di-substituted phosphoric or phosphonic acids
or their thio analogues. There is no evidence that these products
are toxic to any significant extent. If aerial oxidation of a
phosphorothioate precedes hydrolysis, then the product will be a
toxic anticholinesterase, so that hazard due to exposure may
increase for a few days in a dry atmosphere after spraying (section
2.1). Occasionally, other reactions that can be regarded as
chemical degradation yield a more toxic product. Thus, leptophos
(phosphonothioic acid, phenyl-, O -(4-bromo-2,5-dichloro-phenyl)
O -methyl ester) is converted to its desbromo analogue in sunlight,
and the product is considerably more active than the parent in
causing delayed neuropathy (Johnson, 1975b; Sanborn et al., 1977).
Further degradation of the acids to inorganic phosphate is not well
documented, but bacterial cleavage of the carbon-phosphorus bond of
a phosphonate has been reported (Daughton et al., 1979). Whatever
the precise means of degradation, it is clear that residues of most
organophosphorus pesticides are rapidly lost from food crops and
are usually barely detectable 4 weeks after application, though the
exact rate of loss depends on the weather conditions. For a few
organophosphorus pesticides, such as leptophos and fenamiphos, the
residual life is longer (El-Sebae, personal communication, 1985).
Fenamiphos is claimed by its manufacturers to have a residual
activity in soil of "several months" (Bayer, 1971).
3.4 Exposure Levels
3.4.1 Exposure of the general population
Exposure of the general population may occur through the
consumption of foodstuffs treated incorrectly with pesticides or
harvested prematurely before residues have declined to acceptable
levels, from contact with treated areas, or from domestic use.
Exposure of limited populations during disease vector control
is considered below. Significant exposure of the general
population should be unlikely, since the use of these compounds
for crop protection under "good agriculture practice" does not
leave residues that are considered harmful in food. At annual
meetings of the FAO Panel of Experts on Pesticide Residues in Food
and the Environment and the WHO Expert Group on Pesticide
Residues, data accumulated on new and older compounds over the
years are reviewed, and maximum residue limits (MRLs) in various
foods and acceptable daily intakes (ADIs) for the individual
compounds established. The ADIs for the compounds discussed in
this review are given in Annex II.
3.4.2 Occupational exposure
Exposure of factory workers during the undisturbed synthesis of
pesticides is probably negligible, since the processes are carried
out in closed vessels. However, the formulation and dispensing of
formulated pesticides may cause considerable contamination of
workers. The whole range of workers associated with pesticide-
treatment of crops or premises is also liable to exposure as are
both workers and segments of the population during disease vector
control procedures.
Exposure may be via the inhalation, dermal, or oral route.
Dermal contact is the most important route of exposure for
pesticide workers. Durham & Wolfe (1962) described and evaluated
procedures for the use of air samples, pads attached to exposed
body surfaces, and washes, in the direct measurement of the dermal
and respiratory exposure of workers to pesticides. Good methods
are not available for measuring oral exposure. The extent of
exposure depends on disciplined hygiene among workers. Provided
smoking, eating, and drinking in the work area are forbidden, and
these activities are only engaged in after workers have washed
thoroughly, oral intake should be negligible. Exposure by other
routes depends on the amount of protective clothing worn, and, on
the physical state of the pesticide. The majority of
organophosphorus pesticides are liquids having different vapour
pressures at room temperature (i.e., dichlorvos is much more
volatile than malathion); thus, hazard due to inhalation of vapour
varies from compound to compound. The vapour pressure of the
active agent is reduced on dilution with solvent, emulsifier, etc.,
so that the inhalation hazard is reduced, but these additives may
facilitate adsorption of spilled material through the skin. The
likelihood of acute poisoning occurring among process workers seems
greatest when dealing with liquid formulations. It was impossible
to judge the comparative contributions via the dermal and
respiratory routes in the case of poisoning with demeton- S -methyl
reported by Vale & Scott (1974), but it was noted that the area
where intoxication occurred was an unventilated cubicle. The
routine use of gas masks or bottled air respirators may be
necessary, when concentrated liquid pesticides are dispensed. In
studies on several powder-formulated pesticides, Wolfe et al.
(1978) showed that potential dermal exposure markedly exceeded
respiratory exposure; thus, the mean exposure to parathion for the
most contaminated group of workers in a formulation plant was 184
mg/h of work activity for dermal contamination and 0.03 mg/h for
respiratory exposure, the highest values being 33.5 and 33.8 mg/h,
respectively, for one individual. The actual uptake as a result of
such exposures is harder to quantify and may vary according to the
mode of formulation of the pesticide as well as to the
lipophilicity and volatility of the compound and the area of
exposed skin. Using the calculations of Durham & Wolfe (1962), the
highest exposure noted above would have represented 25% of the
toxic dose, had it all been absorbed. However, these authors
calculated that even with contamination by liquid parathion
formulations (which presumably enter more easily through contact
with the skin), the amount absorbed by orchard spraymen was only a
mean 1.23% (0.40 - 1.95% range) of the measured potential dermal
exposure (Durham et al., 1972). The mean dermal and respiratory
exposures of the spraymen were 19 and 0.02 mg/h, respectively,
which was markedly lower than those in the formulating plant. In
view of the inefficiency of absorption, it is perhaps less
surprising that ChE changes were negligible, though total urinary
4-nitrophenol excretion was significant, when a volunteer was
totally covered with 2% parathion dust and enclosed in a rubber
suit for 7 h, spent alternatively in the sun and shade (Hayes et
al., 1964).
Similarly, a 2-h exposure to 48% parathion emulsifiable
concentrate swabbed on the right hand and forearm of a volunteer to
the point of run-off did not cause any change in erythrocyte- or
plasma-ChEs and an average of 10 µg 4-nitrophenol/h was excreted
during the following 24 h. Hayes (1971) stated that absorption of
parathion was tolerated without illness and with little or no
reduction in ChE activity, as long as the concentration of
4-nitrophenol in the urine did not rise above 60 - 80 µg/h (2 ppm),
assuming an average urine excretion of 30 - 40 ml/h.
Studies on orchard spray workers (Wolfe et al., 1967) showed
that, as in the formulation plant, the potential exposure of a
worker without special protective clothing was largely dermal, for
instance, 19.4 mg parathion/h by dermal exposure and 0.02 mg/h
respiratory. This is about 3 times less than the mean exposure of
some formulator/baggers (see above). However, the respiratory
exposure was increased 4-fold, when applying dusts compared with
dilute spray, and 10-fold, when using aerosols of concentrated
pesticide (not necessarily organophosphates). Thus, in the last
case, the respiratory route could be highly important when the
efficiency of absorption is allowed for.
The droplet size in pesticide sprays is influenced by the spray
machinery and a recent study compared potential dermal exposure
during mixing and loading with that during spraying with various
machines. Knapsack spraying seemed to cause much greater dermal
exposure in operators than electrostatic spraying (British
Agrochemical Association, 1983).
Significant exposure of workers may occur when they enter a
previously sprayed crop area for the purposes of further
cultivation or hand-harvesting. The re-entry concept was first
discussed by Milby et al. (1964) in relation to the prevention of
illness. The extent of exposure depends on many factors, including
the physical properties of the pesticide and its biodegradability,
the crop, the nature of the proposed worker operation, and, also,
on the local weather; thus, marked regional differences may occur.
Procedures for determining foliar residues and their dissipation
rates were described by Gunther et al. (1973, 1974), and the topic
was further reviewed by Knaak (1980). Kahn (1979) provided an
outline guide to the procedures and factors to be considered when
performing field studies to establish safe re-entry intervals in
relation to organophosphorus pesticides. One such study was
described by Guthrie et al. (1974). Kahn (1979) cited the US EPA
(1975) in its Registration Procedures as requiring "data necessary
to determine required intervals between pesticide application and
safe re-entry". Re-entry periods appropriate to local conditions
for some pesticides and crops have been reported by Knaak (1980)
and by Kaloyanova-Simeonova & Izmirova-Mosheva (1983).
The situation for workers entering fields that have been
sprayed with some organophosphorus esters differs from that of
workers exposed to dieldrin, for example. In the case of the
former, the oxidation products generated by the action of light and
air may be far more toxic for man than the applied pesticide
(section 2.1), so that the residues on the crops may be more
hazardous for a few days after application than at the time of
application. Degradation is fairly rapid, but clearly a balance of
effects between activation and degradation must be taken into
account, initially.
A "Standard Protocol for Field Surveys of Exposure to
Pesticides" has been published by the World Health Organization
(WHO, 1982).
4. METABOLISM AND MODE OF ACTION
4.1 Uptake
Most organophosphorus pesticides are not ionized and are very
lipophilic. Thus, inhaled or swallowed material will be easily
taken up.
4.1.1 Dermal uptake
Many accidental acute poisonings have occurred following
spillage of pesticide on skin and clothing. The extent of uptake
will depend on persistence time (related to volatility, clothing,
coverage, and thoroughness of washing after exposure), and also on
the presence of solvents and emulsifiers that may facilitate
uptake. However, the evidence concerning parathion, quoted in
section 3.4.2, suggests that dermal absorption is not an efficient
process, under normal working conditions. Experimental
determinations of dermal toxicity depend on the conditions
employed, particularly on whether the treated skin is covered or
not, and on how long the application is left before cleansing.
These are frequently not stated in toxicological reports. With
this limitation in mind, the comparison can be made for the
toxicity of omethoate in rats by 2 routes: the dermal LD50 is 860 -
1020 mg/kg body weight and the oral LD50 is 25 - 28 mg/kg body
weight (FAO/WHO, 1979b). In contrast, uptake through the skin can
be very efficient for more lipophilic agents and, since they avoid
the first-pass metabolic disposal in the liver, agents such as DEF
and EPN may be at least as toxic by the dermal route as by the oral
route in laboratory tests.
4.1.2 Gastrointestinal tract
In rats, the uptake of most of the organophosphorus pesticides
reviewed seems to be rapid and efficient under test conditions
usually involving a dose well below the LD50.
However, the question that does not appear to have been
answered is whether this is true with large doses of low-toxicity
compounds. Thus, the LD50 of bromophos, for rats, is > 3 g/kg
body weight (FAO/WHO, 1973b), but it is not clear whether this low
toxicity is in part a reflection of failure to absorb the majority
of the dose above some unknown threshold. In absorption studies,
using radiolabelled bromophos at a dose of 10 mg/kg body weight,
approximately 96% of the radiolabel was absorbed and excreted in
the urine within 24 h of oral dosing. There is evidence of
comparatively inefficient absorption in hens administered large
doses of very insoluble organophosphorus pesticides with a high
relative molecular mass, such as haloxon [phosphoric acid, 3-
chloro-4-methyl-2-oxo-2H-1-benzopyran-7-yl bis-(2-chloroethyl)
ester]:
or leptophos [phosphonothioic acid, phenyl-, O -(4-bromo-2,5-
dichlorophenyl) O -methyl ester]:
Thus, divided doses may exert a greater toxic effect than the same
amount given as a single large dose (section 6.1.2).
The question of the bioavailability of preparations given by
the oral route needs to be considered. It must be taken into
account when discussing the results obtained for LD50s, and it is
certainly important when considering the toxicity of pesticides
residues. From a chemical point of view, these residues can be
described as parent compounds, free metabolites, and their
conjugates (Kaufman, 1976). The bioavailability of these
fractions and, thus, their toxic potential are not the same
(Dorough, 1976; Marshall & Dorough, 1977). In general, bound
residues appear to have a lower bioavailability and lower toxicity.
This was discussed by Rico & Burgat-Sacaze (1984), and demonstrated
for some pesticides residues by Marshall & Dorough (1977).
4.1.3 Inhalation
Total urinary output of 4-nitrophenol was compared in workers
spraying parathion, who either breathed a pure air supply but did
not wear protective clothing, or who wore total protective
clothing, but did not have any respiratory protection (Durham et
al., 1972). Output derived from the respiratory source compared
with that derived from the dermal source was 1.2% in one test and
12% in another. Since the total exposures by the dermal and
respiratory routes were in the proportion of 1000:1, and the
efficiency of dermal absorption was 1 - 2%, it follows that the
efficiency of absorption by the respiratory route was higher than
20% and could well have been complete.
4.2 Distribution and Storage
The intrinsically reactive chemical nature of organophosphorus
pesticides means that any that enter the body are immediately
liable to a number of biotransformations and reactions with tissue
constituents (particularly tissue proteins carrying esterase active
sites), so that the tracing of radiolabelled material alone does
not give any clue to the distribution of the unchanged parent
compound. It is possible to determine the rate of disposal of
metabolites and thereby to estimate an approximate half-life of
pesticide in the body. Such numbers may be helpful in estimating
safe intervals between successive low exposures, under working
conditions. However, although the half-life of organophosphorus
pesticides and their inhibitory metabolites in vivo is
comparatively short, at least one case of poisoning demonstrated
that significant amounts remained in the body for several weeks
after an acute crisis. Ecobichon et al. (1977) reported a case of
poisoning by fenitrothion. After an effective treatment period
with atropine and an oxime reactivator, leading to 2 days without
symptoms or any therapy, symptoms of nausea and diarrhoea recurred
associated with a decline in the previously restored blood-ChE
levels. These symptoms were controlled by further administration
of oxime, which led to a prompt restoration of the enzyme to a
near-normal level. Further recurrence of symptoms was reported, at
intervals, especially associated with periods of mobilization of
adipose tissue. The conclusion is that the treatment was reversing
the recent inhibition of AChE by a compound that had been stored in
the body and was entering the circulation over a period of many
days.
4.2.1 Experimental animal studies on distribution and storage
In view of the inherent instability of organophosphorus
insecticides, storage in human tissue is not anticipated to be
prolonged (unlike the situation for DDT); population studies,
including analyses of cadavers, do not seem to have been carried
out and would be a pointless exercise. Experimental animal studies
have shown that most of a radiolabelled dose is rapidly excreted in
expired air, urine, and faeces. Thus, it was reported that from 67
to 100% of the administered radioactivity was recovered within 1
week in the combined urine and faeces of cows, rats, and a goat,
given various doses of 32 P-dichlorvos; no organosoluble
radioactivity, which might include unchanged dichlorvos, was
detected after the first 2 h (Blair et al., 1975), though 14 C in
alkyl groups may enter the general metabolic pool and be
incorporated into tissues. Also, phosphorylated proteins are
presumably replaced only by resynthesis and this is a comparatively
slow process with enzymes, such as erythrocyte- and brain-AChE,
typically returning to pre-exposure levels over a period of a few
weeks after irreversible phosphorylation.
In a case of human poisoning by dichlorofenthion, steadily
decreasing concentrations of the pesticide were found in serial fat
biopsy samples up to 48 days after intoxication: the decline
matched a return of blood-ChE levels towards normal, and recovery
of health. Indirect evidence for the short-term storage of
significant amounts of lipophilic organophosphorus compounds was
the return of cholinergic poisoning signs a day or two after
discontinuing oxime therapy in a woman who had been accidentally
poisoned with fenitrothion (Ecobichon et al., 1977). Reinstatement
of therapy led to rapid amelioration of signs and the cycle was
repeated at intervals up to the 15th day after intoxication, after
which her health improved slowly.
4.3 Biotransformation
Alternative metabolic pathways, often available in animals and
man, are listed below, with examples. Most general studies of
pathways have been made on phosphates and their thioate analogues.
Although the ultimate fate of phosphonate pesticides has been
determined, the pathways are usually presumed on the basis of
phosphate studies, which indicate that cleavage of the phosphoric-
carbon bond is limited in mammalian systems. A summary is given
below; further details can be found in Dauterman (1971), Eto
(1974), CEC (1977), and in the annual reports of the Joint FAO/WHO
Meetings on Pesticide Residues in Food (Annex II).
Biotransformation reactions can be divided into three distinct
classes. The former are reactions involving (a) mixed-function
oxidases; (b) hydrolases; and (c) transferases. There is also a
miscellaneous group of unrelated reactions. Binding of
organophosphorus insecticide oxons to tissue is also a significant
biotransformation reaction.
4.3.1 Mixed-function oxidases (MFOs)
Many apparently unrelated substrates can be oxidized by mixed-
function oxidase (MFO) systems associated typically with liver
endoplasmic reticulum, but present also in some other tissues such
as intestine, lung, and kidney. Within the liver, there appears to
be a family of MFOs, possibly with some enzymes in common, but
utilizing slightly different cytochromes of which cytochrome P-450
is the best known. The MFO activity in the liver can vary greatly
according to the nutritional and hormonal state of the animal and
also according to stimuli arising from the ingestion of some
foreign compounds (section 6.3).
4.3.1.1 Oxidative desulfuration
The reaction (Fig. 1) is essentially the activation of the
precursor phosphorothioate to the directly inhibitory phosphate
ester, which is responsible for the inhibition of AChE and for
subsequent toxic effects. There is no evidence of inhibition of
AChE by phosphorothioates occurring under normal situations,
without prior conversion to the phosphate; reports of the
inhibitory power of technical grades of phosphorothioates in vitro
are meaningless, since the activity is almost certainly caused by
traces of the oxon, the activity of which is several orders
greater.
4.3.1.2 Oxidative N -dealkylation
This reaction may be associated with the metabolic activation
of a non-inhibitory precursor, such as schradan, or with
transformation of one inhibitor to another (Fig. 2).
4.3.1.3 Oxidative O -dealkylation
The conversion of triesters to diester is a detoxication
process and was once considered to be mediated only by hydrolytic
enzymes (phosphoryl phosphatases or A-esterases). However, a
reaction requiring liver microsomes, NADPH, and oxygen (the typical
MFO system) deethylates chlorofenvinphos with the production of
acetaldehyde, probably as shown in Fig. 3. It seems that various
phosphates, but not phosphorothioates, are metabolized by this
route in mammals.
4.3.1.4 Oxidative de-arylation
Liver MFOs from rat or rabbit can cleave the acid-anhydride
bond coupling phosphorus to the phenolic group in parathion and
analogues (Nakatsugawa et al., 1968), and the same system may also
be responsible for the cleavage of diazinon (Yang et al., 1971).
In contrast to O -dealkylation noted above, this reaction appears
to deal only with phosphorothioates and not phosphates.
4.3.1.5 Thioether oxidation
Oxidation of sulfur in the phosphorus-sulfur-carbon moiety of
demeton-S or or dimethoate and omethoate has not been reported, but
oxidation is known of carbon-sulfur-carbon moieties with the
formation of sulfoxides and sulfones that are more active AChE
inhibitors than the parent compound and remain in circulation for a
comparatively long time (Fig. 4).
4.3.1.6 Side-chain oxidation
Stepwise oxidation of simple alkyl groups to hydroxy-, oxo-, or
carboxy-derivatives is a well-known process in the metabolism of
many compounds, apart from the organophosphorus compounds. The
conversion of fenitrothion to the water-soluble, 3-carboxy
derivative (Fig. 5) can account for the comparatively low mammalian
toxicity of this pesticide compared with that of the homologous
methyl parathion (rat oral LD50s of about 600 - 800 and 10 - 25
mg/kg body weight, respectively).
4.3.2 Hydrolases
Hydrolysis of the acid anhydride type ester bond of the leaving
group in pesticidal triesters is well known. The monobasic
diesters and their derivatives are the major urinary metabolites of
organophosphorus insecticides (Fig. 6). The enzymes commonly known
as A-esterases or phosphoryl phosphatases are widespread in
mammalian tissues, such as liver, plasma, intestine, etc., though
they are less abundant in many birds and may not be present in some
insects (Brealey et al., 1980). These enzymes are sometimes
referred to as DFPase or paraoxonase according to the substrate
used, but it does not mean that the enzymes are specific only for a
given organophosphorus compound. Although plasma contains enzymes
that can distinguish between closely related structures such as
paraoxon and 4-nitrophenyl, ethyl, or propylphosphonate, the
enzymes are not totally specific (Becker & Barbaro, 1964); the same
is probably true of A-esterase in other tissues.
Hydrolysis of carboxylic acid ester bonds and carboxyl-amide
bonds in organophosphorus insecticides may be catalysed by
carboxylesterases (or B-esterases), which again occur widely in
mammalian tissues. Malathion, which contains 2 carboxylic ester
bonds, is the best-known organophosphorus pesticide that is
hydrolysed in this way (Fig. 7). The importance of this metabolic
route is shown by the fact that the rat oral LD50 for pure
malathion can be reduced from 10 000 to 100 mg/kg body weight, when
the tissue carboxyesterases are inhibited: this profound
potentiation is discussed further in section 5.3.
The hydrolysis of carboxylamide bonds such as in dimethoate is
catalysed by a liver enzyme (Chen & Dauterman, 1971) (Fig. 7).
Although apparently distinct from liver carboxyesterase, it too is
inhibited by its oxygen analogue (omethoate) and also by other
amide-containing phosphates such as dicrotophos.
4.3.3 Transferases
The only transferase reaction that is known to deal with the
intact pesticidal organophosphorus triesters involves glutathione,
which is a required substrate for a number of transferase enzymes
present in liver and some other tissues. The enzymes have limited
but overlapping specificity so that the glutathione transferase
responsible for demethylating methyl paraoxon is distinct from that
which conjugates the 4-nitrophenol group in parathion. Activity in
the liver is greatest with methyl esters, but no evidence has been
found of methyl phosphonates undergoing this reaction (Dauterman,
1971).
4.3.3.1 Transferases handling primary metabolites
Reactions involving the conjugation of carboxylic acids,
alcohols, phenols, and amino, imino, and sulfydryl groups are well-
known and applicable to compounds carrying such groups, formed
after the oxidation, hydrolysis, etc. of an organophosphorus
pesticide. Such conjugation reactions aid in the elimination of
primary degradation products, which are usually devoid of
anticholinesterase activity, though they may cause other toxic
effects if they accumulate in the body.
4.3.4 Tissue binding
It is well-known that active metabolites of most
organophosphorus insecticides react covalently to some extent with
tissue esterases other than AChE. Since few of these esterases
appear vital to health (section 6.2.4), the binding reaction may be
considered a detoxification process. Although the catalytic
activity of these esterases is high, the actual quantity of such
sites is comparatively small. Crude measurements using 32 P-
labelled diisopropyl phosphorofluoridate (an agent reacting with
most organophosphorus-sensitive esterases) suggest that 100 - 150
µg bind per kg body weight of an adult hen injected with about the
LD50 dose (Johnson, M.K., personal communication, 1985). Binding
was principally in liver and muscle. The quantity bound would not
be expected to be much greater whatever the LD50 of an administered
organophosphorus insecticide. Thus, it is a significant proportion
of the total dose only for a very toxic compound such as paraoxon
(Lauwerys & Murphy, 1969) but not for compounds with much higher
LD50s. However, the number of binding sites may, in some cases, be
very significant compared with the quantity of circulating
anticholinesterase oxon that has avoided other metabolic disposal
processes. Molecules of oxon bound to these non-vial sites are
prevented from attacking the vital sites such as AChE or NTE
(section 6.1). Binding sites can therefore be considered an
important second line of defence against intoxication.
The specific problem of tissue binding that leads to
potentiation of the toxicity of malathion and other pesticides
containing carboxylester bonds is discussed in section 6.2.4.
4.4 Elimination
There is no evidence of prolonged storage of organophosphorus
compounds in the body, but the process of elimination can be
subdivided roughly according to the speed of the reactions
involved. Most organophosphorus pesticides are degraded quickly by
the metabolic reactions listed in section 4.3, and the elimination
of the products, mostly in the urine with lesser amounts in faeces
and expired air, is not delayed, so that rates of excretion usually
reach a peak within 2 days and decline quite rapidly. That they do
not almost immediately fall to zero is due to storage in fat and
covalent binding. As indicated in section 4.2, the former process
preserves toxic material, which is slowly released into the
circulation and which is active and is metabolized in the same way
as the bulk of the dose received. Covalent binding involves
phosphorylation of proteins, probably esterases having active
sites, including serine, which are mechanistically related to AChE.
The consequences of such phosphorylation depend on the esterase
involved, but many seem to be of only minor importance to the
continuing health of animals, and temporary inhibition may not be
expressed in physiological defects. The special case of neuropathy
target esterase, which is phosphorylated only by agents capable of
causing delayed neuropathy, is considered later (section 6.1.1.2).
4.5 Mode of Action
4.5.1 Inhibition of esterases
The primary biochemical effect associated with toxicity caused
by organophosphorus pesticides is inhibition of AChE. The normal
function of AChE is to terminate neurotransmission due to ACh that
has been liberated at cholinergic nerve endings in response to
nervous stimuli. Loss of AChE activity may lead to a range of
effects resulting from excessive nervous stimulation and
culminating in respiratory failure and death (section 6.1). The
chemistry of inhibition of AChE and of many other esterases (e.g.,
NTE and liver carboxyesterases, which are discussed elsewhere) by
these chemicals is similar and is given in schematic form in Fig.
8. Following the formation of a Michaelis complex (reaction 1), a
specific serine residue in the protein is phosphorylated with loss
of the leaving group X (reaction 2). Two further reactions are
possible: reaction 3 (reactivation) may occur spontaneously at a
rate that is dependent on the nature of the attached group and on
the protein and is also dependent on the influence of pH and of
added nucleophilic reagents, such as oximes, which may catalyse
reactivation. Reaction 4 ("aging") involves cleavage of an R-O-P-
bond with the loss of R and the formation of a charged mono-
substituted phosphoric acid residue still attached to protein. The
reaction is called "aging" because it is time-dependent, and the
product is no longer responsive to nucleophilic reactivating agents
such as some oximes. Since therapy of organophosphorus compound
poisoning is, in part, dependent on the reactivating power of
oximes (sections 6, 7), understanding of the "aging" reaction is
important. PseudoChE, which is present in blood-plasma and nervous
tissue but has no known physiological function, is inhibited by
organophosphorus compounds in a similar way to AChE, but the
specificity of the 2 enzymes is different. Though no toxic effect
arises as a result of inhibition of pseudoChE, measures of its
inhibition can be made for monitoring purposes (section 7.1.1.2).
4.5.2 Possible alkylation of biological macromolecules
It has been shown, under laboratory conditions, that some
organophosphates could react with and alkylate the reagent
4-nitrobenzylpyridine (Preussmann et al., 1969). The study was
interpreted to imply that the in vivo alkylating potential of some
pesticides was similar to that of the known mutagens dimethyl
sulfate and methyl methanesulfonate. Furthermore, Löfroth et al.,
(1969) derived a substrate constant (a logarithmic measure of
alkylating ability) of 0.75 for dichlorvos, which is intermediate
between those known for methyl and ethyl methanesulfonates.
Concern over the possible mutagenic and carcinogenic potential of
organophosphorus compounds on the basis of the above data was
misplaced, since alternative reactions were not considered.
Compared with the carbon atom of the alkyl group, the phosphorus
atom is markedly more electron-deficient and susceptible to attack
by nucleophiles. Analysis by Bedford & Robinson (1972) of the data
of Löfroth et al. (1969) revealed that the proposed rates of
alkylation by hard nucleophiles were probably combined rates of
phosphorylation and alkylation, and that phosphorylation was the
totally dominant reaction in the case of the hydroxide ion. The
comparison with known mutagens was therefore inappropriate. Two
factors detract further from the toxicological significance of the
alkylation studies. The first is that mammalian tissues (plasma,
liver, etc.) contain active enzymes that catalyse the
phosphorylation of water by the organophosphorus esters. Viewed
inversely, these enzymes (often called A-esterases) catalyse the
hydrolysis of the organophosphorus esters, thereby rapidly reducing
circulating levels of hazardous material. Secondly, the
comparative rate of reaction of most of these pesticides with AChE
is many orders greater than their rate of alkylation of the typical
nucleophile 4-nitrobenzylpyridine: for dichlorvos, the ratio of
rates was 1 x 107 in favour of the inhibitory phosphorylation of
AChE (Aldridge & Johnson, 1977). It follows that, at low exposure
levels, in vivo phosphorylation of AChE and other esterases will
be the dominant reaction with negligible uncatalysed alkylation of
genetic material. Indeed, no such alkylation has been detected in
sensitive in vivo studies designed to check this point (Wooder et
al., 1977). Some catalysed alkylations of glutathione by
organophosphorus compounds are known to occur in vivo (section 4),
but these are essentially detoxification reactions. The topic of
alkylation and the possible mutagenic or carcinogenic consequences
is discussed further in section 6.2.
5. EFFECTS ON ORGANISMS IN THE ENVIRONMENT
5.1 Aquatic Organisms
Organophosphorus insecticides are not very stable in aqueous
media. However, accidental leaching may occur from treated areas
into rivers and lakes where they may exert toxic effects on aquatic
organisms before degradation is complete. In clean water in the
laboratory, toxic effects were seen in several aquatic organisms
when they were exposed to concentrations of organophosphorus
insecticides ranging from 0.01 to 1 mg/litre for 48 h (Nishiuchi,
1981). However, lethal concentrations derived from 48-h exposures
in clear laboratory water may be artificially low compared with the
concentrations that would be effective in true environmental
waters.
One pesticide (phenthoate) appeared to be more toxic for
aquatic insects ("median tolerable limit" = 9 - 75 µg/kg)
(Nishiuchi, 1981) than for one species of fresh-water fish exposed
under apparently similar conditions (20% mortality caused by 263
µg/kg) (Jash & Bhattacharya, 1982).
Accidental release of pesticides in lakes, rivers, and bays
sometimes caused massive death of fish and many of the compounds
were strongly toxic for small aquatic organisms such as Daphnia, as
shown in Table 3.
Table 3. Acute toxicity of organophosphorus pesticides for some aquatic organisms
---------------------------------------------------------------------------------------------------------
Pesticide TLMs for organisms at indicated time (mg/litre) Reference
Carp Goldfish Killifish Guppy Water flea
( Cyprinus (Carassius (Fundulus (Lebistes (Daphnia
carpio auratus) sp) reticulatus pulex
Linné) Peters) Leydig)
48 h 48 h 48 h 48 h 3 h
---------------------------------------------------------------------------------------------------------
Acephate > 40 - > 40 - > 40 Nishiuchi (1974)
Calvinphos > 40 - > 40 - 0.0042 Nishiuchi (1974)
Chlorfenvinphos 0.27 0.34 0.23 0.12 0.011 Yoshida &
Nishiuchi (1972);
Nishiuchi (1974)
Chlorpyrifos 0.13 0.20 0.47 0.74 0.0050 Yoshida &
emulsifiable Nishiuchi (1972);
concentrate) Nishiuchi (1974)
Chlorpyrifos 2.1 - 3.4 - 0.017 Yoshida &
-methyl Nishiuchi (1976)
Cyanofenphos 1.2 1.3 6.3 15 0.0085 Yoshida &
Nishiuchi (1972);
Nishiuchi (1974)
Cyanophos 15 10 ~ 40 28 18 0.34 Yoshida &
(wettable Nishiuchi (1972);
powder) Nishiuchi (1974)
Dialifos 1.3 - 0.80 - 0.027 Yoshida &
Nishiuchi (1976)
Dichlofenthion 5.1 10 ~ 40 1.4 10 0.005 Yoshida &
(emulsifiable (dust Nishiuchi (1972);
concentrate) formulation) Nishiuchi (1974)
Diazinon 3.2 5.1 5.3 4.1 0.08 Yoshida &
Nishiuchi (1972);
Nishiuchi (1974)
Dichlorvos > 40 10 ~ 40 18 - 2.8 Yoshida &
(emulsifiable Nishiuchi (1972)
concentrate)
Dimethoate > 40 > 40 > 40 - 10 ~ 40 Yoshida &
Nishiuchi (1972)
---------------------------------------------------------------------------------------------------------
Table 3. (contd.)
---------------------------------------------------------------------------------------------------------
Pesticide TLMs for organisms at indicated time (mg/litre) Reference
Carp Goldfish Killifish Guppy Water flea
( Cyprinus (Carassius (Fundulus (Lebistes (Daphnia
carpio auratus) sp) reticulatus pulex
Linné) Peters) Leydig)
48 h 48 h 48 h 48 h 3 h
---------------------------------------------------------------------------------------------------------
Dimethylvinphos 5.6 - 1.5 - 0.010 Yoshida &
Nishiuchi (1976)
Dioxathion 10 ~ 40 10 ~ 40 1.4 - 0.007 Yoshida &
(emulsifiable Nishiuchi (1972)
concentrate)
Disulfoton 8.7 10 ~ 40 21 0.37 0.07 Yoshida &
Nishiuchi (1972);
Nishiuchi (1974)
Edifenphos 2.5 1.8 1.8 - 0.27 Yoshida &
(emulsifiable (emulsifiable Nishiuchi (1972)
concentrate) concentrate)
EPN 0.20 0.32 0.50 0.085 0.0017 Yoshida &
Nishiuchi (1972);
Nishiuchi (1974)
Ethion 1.2 1.1 5.5 - 0.005 Yoshida &
Nishiuchi (1972)
Fenitrothion 8.2 3.4 7.0 0.75 0.050 Yoshida &
Nishiuchi (1972);
Nishiuchi (1974)
Fenthion 3.3 1.9 2.5 2.3 0.070 Yoshida &
Nishiuchi (1972);
Nishiuchi (1974)
Formothion 15 10 ~ 40 10 ~ 40 - 5.8 Yoshida &
Nishiuchi (1972)
IBP 10 ~ 40 12 7.2 - 2.3 Yoshida &
(emulsifiable (emulsifiable Nishiuchi (1972)
concentrate) concentrate)
Leptophos > 40 10 ~ 40 8.5 - 0.002 Yoshida &
(emulsifiable Nishiuchi (1972)
concentrate)
---------------------------------------------------------------------------------------------------------
Table 3. (contd.)
---------------------------------------------------------------------------------------------------------
Pesticide TLMs for organisms at indicated time (mg/litre) Reference
Carp Goldfish Killifish Guppy Water flea
( Cyprinus (Carassius (Fundulus (Lebistes (Daphnia
carpio auratus) sp) reticulatus pulex
Linné) Peters) Leydig)
48 h 48 h 48 h 48 h 3 h
---------------------------------------------------------------------------------------------------------
Malathion 23 7.8 0.75 1.4 0.030 Yoshida &
Nishiuchi (1972)
Menazon > 40 > 40 > 40 100 10 ~ 40 Yoshida &
(wettable Nishiuchi (1972);
powder) Nishiuchi (1974)
Methidathion 2.5 2.3 0.034 0.22 0.007 Yoshida &
(emulsifiable Nishiuchi (1972);
concentrate) Nishiuchi (1974)
Naled 1.3 1.2 28 2.3 0.005 Yoshida &
(emulsifiable (emulsifiable Nishiuchi (1972);
concentrate) concentrate) Nishiuchi (1974)
Parathion 4.5 1.7 2.9 - 0.0050 Yoshida &
Nishiuchi (1972)
Parathion-methyl 7.5 10 ~ 40 12 - 0.00050 Yoshida &
Nishiuchi (1972)
Phenkapton 2.0 3.8 3.5 - 0.008 Yoshida &
Nishiuchi (1972)
Phenthoate 2.5 2.4 0.17 0.20 0.008 Yoshida &
Nishiuchi (1972);
Nishiuchi (1974)
Phosalone 1.2 1.2 0.35 - 0.05 Yoshida &
(emulsifiable Nishiuchi (1972)
concentrate)
Phosmet 5.3 4.7 1.8 1.0 0.025 Yoshida &
Nishiuchi (1972);
Nishiuchi (1974)
Pirimiphos-methyl 1.8 - 3.0 - 0.018 Yoshida &
Nishiuchi (1976)
Propaphos 4.8 - 4.1 - 0.0063 Yoshida &
Nishiuchi (1976)
---------------------------------------------------------------------------------------------------------
Table 3. (contd.)
---------------------------------------------------------------------------------------------------------
Pesticide TLMs for organisms at indicated time (mg/litre) Reference
Carp Goldfish Killifish Guppy Water flea
( Cyprinus (Carassius (Fundulus (Lebistes (Daphnia
carpio auratus) sp) reticulatus pulex
Linné) Peters) Leydig)
48 h 48 h 48 h 48 h 3 h
---------------------------------------------------------------------------------------------------------
Propoxur 10 ~ 40 10 ~ 40 10 ~ 40 - 0.37 Yoshida &
Nishiuchi (1972)
Prothiophos 9.5 - 10 - 0.13 Yoshida &
Nishiuchi (1976)
Temivinphos 0.58 - 0.48 - 0.0080 Yoshida &
Nishiuchi (1976)
TEPP 5.6 10 ~ 40 4.8 - 10 ~ 40 Yoshida &
(liquid (liquid (liquid (liquid Nishiuchi (1972)
formulation) formulation) formulation) formulation)
Tetrachlorvinphos 4.3 3.9 4.2 - 0.0035 Yoshida &
(wettable Nishiuchi (1972)
powder)
Thiometon 7.5 10 ~ 40 10 ~ 40 - 5.5 Yoshida &
Nishiuchi (1972)
Trichlorphon 28 10 ~ 40 25 12 0.005 Yoshida &
Nishiuchi (1972);
Nishiuchi (1974)
Vamidothion > 40 > 40 > 40 - > 40 Yoshida &
Nishiuchi (1972)
---------------------------------------------------------------------------------------------------------
Note: Test methods are officially recognized methods based on the Notification of the Ministry of
Agriculture, Forestry and Fishery of Japan, as described in the separate papers.
6. EFFECTS ON ANIMALS
Insecticides are designed as lethal agents. Although they may
be designed to be less toxic for animals than for insects, all
organophosphorus insecticides present a toxic hazard to some
extent. Values for the oral and dermal LD50s in rat, shown for
different compounds in Annex III, range from less than 10 to more
than 3000 mg/kg body weight. The dose-response line for
organophosphorus insecticides is usually steeper than that for
carbamates, though both kill by their anticholinesterase action.
The reason for the difference lies in the faster rate of
spontaneous reactivation of carbamylated AChE compared with
phosphorylated AChE.
By the time the 1984 Joint FAO/WHO Meeting on Pesticide
Residues (JMPR) had ended, the toxicology of 57 organophosphorus
pesticides had been reviewed (Vettorazzi, 1984). Usually, the
compounds had been reviewed several times as additional information
became available. The compounds, with the years of the JMPR
review, and the acceptable daily intake (ADI) advised are listed
Annex II. It also lists the year