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Environmental Health Criteria 63

ORGANOPHOSPHORUS : A GENERAL INTRODUCTION

Please note that the layout and pagination of this web version are not identical with the printed version. Organophophorus insecticides: a general introduction (EHC 63, 1986)

INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

ENVIRONMENTAL HEALTH CRITERIA 63

ORGANOPHOSPHORUS INSECTICIDES: A GENERAL INTRODUCTION

This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the International Labour Organisation, or the World Health Organization.

Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization

World Health Orgnization Geneva, 1986

The International Programme on Chemical Safety (IPCS) is a joint venture of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization. The main objective of the IPCS is to carry out and disseminate evaluations of the effects of chemicals on human health and the quality of the environment. Supporting activities include the development of epidemiological, experimental laboratory, and risk-assessment methods that could produce internationally comparable results, and the development of manpower in the field of toxicology. Other activities carried out by the IPCS include the development of know-how for coping with chemical accidents, coordination of laboratory testing and epidemiological studies, and promotion of research on the mechanisms of the biological action of chemicals.

ISBN 92 4 154263 2

The World Health Organization welcomes requests for permission to reproduce or translate its publications, in part or in full. Applications and enquiries should be addressed to the Office of Publications, World Health Organization, Geneva, Switzerland, which will be glad to provide the latest information on any changes made to the text, plans for new editions, and reprints and translations already available.

Page 1 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

(c) World Health Organization 1986

Publications of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention. All rights reserved.

The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries.

The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters.

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. 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 2.2. Analytical methods

3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE, ENVIRONMENTAL TRANSPORT AND DISTRIBUTION, EXPOSURE LEVELS

3.1. Sources of 3.2. Environmental transport and distribution 3.2.1. Distribution in air and water 3.2.2. Distribution in 3.3. and degradation in the environment 3.4. Exposure levels 3.4.1. Exposure of the general population 3.4.2. Occupational exposure

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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. 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 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 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 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 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

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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 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 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 7.4.3.2 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 and Therapeutics, McGill University, Montreal, Quebec, Canada

Dr A.H. El-Sebae, Department of 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

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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

------a Invited, but unable to attend.

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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 control began to rely more on the anticholinesterase organophosphorus and 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.

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It would not be possible to review the class of organo- 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, , etc.) but, with 2 exceptions, all the compounds covered in the introductory document exert their primary effect by inhibiting the vital 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 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, , 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 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.

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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, and 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 , 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 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, , 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

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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 . 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 . Most have more efficient hydrolytic than insects and, therefore, are often more efficient in their detoxification processes. Birds usually have lower 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 , 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 (ACh), which is a . In many cases, the organophosphorylated 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 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 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.

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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 . 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

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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 , 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.

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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

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been reported for only a few of the many currently used organophosphorus insecticides. For one pesticide, , 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 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 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.

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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 Control Centres and experts studying the effects of organophosphorus insecticides should be improved. Preservation of blood, urine, and 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

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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 or aryl groups, both of which may be bonded directly to phosphorus (in ), or linked via -O-, or -S- (in ), or R1 may be bonded directly and R2 , bonded via one of the above groups (). In , 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 or and related compounds would, for example, be called phosphates or phosphorothioates (the nomenclature "" or "thionophosphate" is now less used).

The P=O form of a thioate ester may be referred to as the , and this is often incorporated in the trivial name (e.g., parathion is the parent P=S compound of ).

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 ------ O , crotoxyph || , heptenphos, m

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(R-O)2-P-O-X phos, , , vinphos, triazophos

O -alkyl phosphorothioate O amiton, -S-methyl, || methyl, , vamidothio (R-O)2-P-S-X

S azothoate, bromophos, brom || pyriphos, chlorpyriphos-me (R-O)2-P-O-X zinon, dichlofenthion, fen thion, , iodofenph thion-methyl, pyrazophos, pyrimiphos-methyl, sulfote

Phosphorodithioate S amidithion, azinophos-ethy || , , di (R-O)2-P-S-X , malathion, mec idathion, morphothion, phe , , protho

S- alkyl phosphorothioate , 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, , fo || (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

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RO || \|| P-O-X / R

Phosphonothioate S EPN, trichlornat, leptopho 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 o& 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 phosphorothio!tes 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 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

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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 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 , , 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 to UVR results in another type of photoisomerization. Starting with either the cis- or the trans- , 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 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

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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

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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 ) 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 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 O

1974 1981 1982 1983 1974 1981 1982 1983 1 -76 -76 ------Africa

Burundi 3 Egypt 397 3573 2080 5 Gambia 120 Madagascar 2 Mauritania 50 1 Niger 694 263 1 Rwanda 1 2 3 Sierra Leone 40 South Africa 2 Sudan 6 Swaziland 1 Zimbabwe 215 450 91 10

North/Central America

Bermuda 7 2 Canada 238 2398 1 Cuba 2 El Salvador 4000 50 3 Guatemala 7704 1010 Honduras 391 414 Mexico 46 000 50 000 48 000 48 000 3452 12 000 18 000 5000 2 Mont Serrat 1 1 USA 115 000 110 000 15 000 15 000

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South America

Argentina 1650 4750 2280 2350 2 Guyana 52 Surinam 28 Uruguay 10 105 78 179 45 91 47 2 ------

Table 2. (contd.) ------Country Parathion Malathion O

1974 1981 1982 1983 1974 1981 1982 1983 1 -76 -76 ------Asia

Bahrain 5 Bangladesh Brunei Burma 34 317 Cyprus 222 1782 842 89 255 212 1 Hong Kong 1 India 9657 20 920 30 300 15 640 6800 8000 1 Israel 8 Japan 1800 1000 Jordan 5000 4500 Korea Rep. 711 1426 1601 2759 726 337 2 Kuwait 4 4 Oman 350 240 Pakistan 982 530 324 90 2381 675 7 Philippines 4800 630 310 Saudi Arabia 55 Sri Lanka 590 1690 Turkey 6480 1750 1837 1939 550 577 2 United Arab 34 Emirates

Europe

Austria 129 201 156 150 8 Czecho- 20 130 44 187 3 slavakia Denmark 1581 2578 2334 206 110 108 6 Finland 55 74 4 Greece 2873 2837 6 Hungary 30 599 17 325 11 301 10 190 2711 1584 1598 3130 6 Iceland 2 2 2 3 Italy 23 147 24 231 18 591 8997 6068 5524 8 Malta 350 Norway 1 Poland 530 707 1062 1038 6 Portugal 301 509 643 109 144 227 8 Sweden 3020 74 60 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 in temperate climates, while as many as 50 applications in one peach-growing season have been reported

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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, (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 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" (, 1971).

3.4 Exposure Levels

3.4.1 Exposure of the general population

Exposure of the general population may occur through the

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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 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

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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

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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 , 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 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

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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 [, 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 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

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liable to a number of 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

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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 , or with transformation of one inhibitor to another (Fig. 2).

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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).

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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.

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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

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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, , 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

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phosphorylation of proteins, probably esterases having active sites, including , 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 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

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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.

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One pesticide () 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 organism ------Pesticide TLMs for organisms at indicated time (mg/litre) Carp Goldfish Killifish Guppy Water ( Cyprinus (Carassius (Fundulus (Lebistes (Daphn carpio auratus) sp) reticulatus pulex Linné) Peters) Leydig 48 h 48 h 48 h 48 h 3 h ------ > 40 - > 40 - > 40 Calvinphos > 40 - > 40 - 0.0042 Chlorfenvinphos 0.27 0.34 0.23 0.12 0.011

Chlorpyrifos 0.13 0.20 0.47 0.74 0.0050 emulsifiable concentrate)

Chlorpyrifos 2.1 - 3.4 - 0.017 -methyl

Cyanofenphos 1.2 1.3 6.3 15 0.0085

Cyanophos 15 10 ~ 40 28 18 0.34 (wettable powder) Dialifos 1.3 - 0.80 - 0.027

Dichlofenthion 5.1 10 ~ 40 1.4 10 0.005 (emulsifiable (dust concentrate) formulation)

Diazinon 3.2 5.1 5.3 4.1 0.08

Dichlorvos > 40 10 ~ 40 18 - 2.8 (emulsifiable concentrate)

Dimethoate > 40 > 40 > 40 - 10 ~ 4

------

Table 3. (contd.) ------Pesticide TLMs for organisms at indicated time (mg/litre) Carp Goldfish Killifish Guppy Water ( Cyprinus (Carassius (Fundulus (Lebistes (Daphn carpio auratus) sp) reticulatus pulex

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Linné) Peters) Leydig 48 h 48 h 48 h 48 h 3 h ------Dimethylvinphos 5.6 - 1.5 - 0.010

Dioxathion 10 ~ 40 10 ~ 40 1.4 - 0.007 (emulsifiable concentrate)

Disulfoton 8.7 10 ~ 40 21 0.37 0.07

Edifenphos 2.5 1.8 1.8 - 0.27 (emulsifiable (emulsifiable concentrate) concentrate)

EPN 0.20 0.32 0.50 0.085 0.0017

Ethion 1.2 1.1 5.5 - 0.005

Fenitrothion 8.2 3.4 7.0 0.75 0.050

Fenthion 3.3 1.9 2.5 2.3 0.070

Formothion 15 10 ~ 40 10 ~ 40 - 5.8

IBP 10 ~ 40 12 7.2 - 2.3 (emulsifiable (emulsifiable concentrate) concentrate)

Leptophos > 40 10 ~ 40 8.5 - 0.002 (emulsifiable concentrate)

------

Table 3. (contd.) ------Pesticide TLMs for organisms at indicated time (mg/litre) Carp Goldfish Killifish Guppy Water ( Cyprinus (Carassius (Fundulus (Lebistes (Daphn 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

Menazon > 40 > 40 > 40 100 10 ~ 40 (wettable powder) 2.5 2.3 0.034 0.22 0.007 (emulsifiable concentrate)

Naled 1.3 1.2 28 2.3 0.005 (emulsifiable (emulsifiable concentrate) concentrate)

Parathion 4.5 1.7 2.9 - 0.0050

Parathion-methyl 7.5 10 ~ 40 12 - 0.0005

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Phenkapton 2.0 3.8 3.5 - 0.008

Phenthoate 2.5 2.4 0.17 0.20 0.008

Phosalone 1.2 1.2 0.35 - 0.05 (emulsifiable concentrate)

Phosmet 5.3 4.7 1.8 1.0 0.025

Pirimiphos-methyl 1.8 - 3.0 - 0.018

Propaphos 4.8 - 4.1 - 0.0063

------

Table 3. (contd.) ------Pesticide TLMs for organisms at indicated time (mg/litre) Carp Goldfish Killifish Guppy Water ( Cyprinus (Carassius (Fundulus (Lebistes (Daphn carpio auratus) sp) reticulatus pulex Linné) Peters) Leydig 48 h 48 h 48 h 48 h 3 h ------ 10 ~ 40 10 ~ 40 10 ~ 40 - 0.37

Prothiophos 9.5 - 10 - 0.13

Temivinphos 0.58 - 0.48 - 0.0080

TEPP 5.6 10 ~ 40 4.8 - 10 ~ 4 (liquid (liquid (liquid (liqui formulation) formulation) formulation) formul

Tetrachlorvinphos 4.3 3.9 4.2 - 0.0035 (wettable powder) Thiometon 7.5 10 ~ 40 10 ~ 40 - 5.5

Trichlorphon 28 10 ~ 40 25 12 0.005

Vamidothion > 40 > 40 > 40 - > 40

------Note: Test methods are officially recognized methods based on the Notification o Agriculture, Forestry and Fishery of Japan, as described in the separate pa

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

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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 (ADI) advised are listed Annex II. It also lists the year of review by IARC. Moreover, it gives the WHO recommended classification by acute toxic hazard (WHO, 1984a) and an indication on whether WHO/FAO issued a "Data Sheet on Chemical Pesticides" on this substance.

Details of the tests and of the results are recorded in the appropriate published evaluations from the FAO/WHO Joint Committee Meetings, which have been summarized and commented on by Vettorazzi (1979).

It should be realized that, while reports on toxicological tests may give some indication of the purity of the sample as percentage content of the major active ingredient, there is seldom any indication of the nature or quantity of the impurities or of whether the impurities may significantly influence toxicity. It cannot be emphasized too strongly that measurements such as acute LD50s are not absolute values.

The LD50 values for one typical compound, fenitrothion, in various species when administered by various routes are listed in Table 4 (FAO/WHO, 1970b, 1975b). From this Table, it is clear that there are marked differences in the LD50 depending on species and . Variability in LD50 values for the same route and species is often considered to be due to the vehicule in which the pesticide is applied, which may influence its uptake into the body. However, it is also possible that such differences are real and can throw light on mechanisms of toxicity and/or of detoxication, thus making more intelligent assessment of toxicity possible. It can be argued that the presence of the very toxic impurities in some formulations of diazinon (section 6.3.4) could have been deduced much earlier from differences in LD50 data. Likewise, the Pakistani poisonings due to strong potentiators in stored malathion (Baker et al., 1978) (section 6.3.4) might have been prevented, if more questions had been asked about the range of LD50 values given in the literature for malathion. Table 4. Comparison of acute toxicity data for fenitrothion listed in evaluation reports of joint FAO/WHO meetingsa ------Animal Sex Route LD50 (mg/kg body weight) ------1970 Report

Mouse M oral 1336 Mouse F oral 1416 Mouse M ip 115 Mouse F ip 110 Mouse iv 220 Rat M oral 740 Rat F oral 570 Rat M ip 135 Rat F ip 160 Rat iv 33 Guinea-pig M oral 500 Guinea-pig oral 1850

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Guinea-pig M ip 110 Guinea-pig iv 112 Cat oral 142

1975 Report

Mouse M oral 1030 Mouse F oral 1040 Rat M oral 330 Rat F oral 800 Ring-neck pheasant oral 34.5 Mallard duck oral 2550 Dog oral MLDb 681 mg/kg Rat M oral 940 Rat F oral 600 ------a From: FAO/WHO (1970b, 1975b). b MLD = minimum lethal dose.

6.1 Effects on the Nervous System

6.1.1 Effects attributed to interaction with esterases

6.1.1.1 Cholinergic effects

(a) Acute toxicity

If non-ester herbicidal compounds are excluded, then the acute toxicity of all other organophosphorus pesticides shares the basic mechanism outlined in section 4.5.1, involving inhibition of AChE, accumulation of ACh, and over-stimulation of some central cholinergic and of the sympathetic and parasympathetic nervous systems. Signs and symptoms of poisoning are described fully in section 7.1. Death is caused by respiratory failure due to a combination of blocking of the respiratory centre, bronchospasm, and paralysis of the respiratory muscles.

(b) Chronic toxicity

The cholinergic effects brought about by repeated administration of less than a single fatal dose are similar in type to the acute single-dose effects and are discussed in section 6.3.1. For the majority of these compounds, long-term feeding tests have been performed to establish the no-observed-adverse- effect levels. In every case, except bromophos-ethyl, the most sensitive indicator of an effect was depression of ChE activity in plasma or erythrocytes. The phenomenon of tolerance to repeated doses of anticholinesterase compounds is covered in sections 6.3.1 and 6.4. For bromophos-ethyl, it has been reported that the urinary excretion of ascorbic acid and dehydroascorbic acid was slightly increased in beagle dogs given 0.39 mg/kg body weight daily, for 18 weeks, at which dose, depression of serum-ChE was not significant (FAO/WHO, 1973b).

6.1.1.2 Delayed neuropathic effects

Delayed neuropathy has occurred occasionally in man and experimental animals after intoxication with a variety of organophosphorus esters. The subject has been reviewed by Johnson (1975a,b, 1980, 1982a) and by Davis & Richardson (1980). An account suitable for physicians is given by Lotti et al. (1984). Delayed neuropathy is not inevitably associated with intoxication by organophosphorus pesticides (Johnson, 1982a; Soliman et al., 1982). Improvements in the therapy of acute poisoning (section

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7.4) mean that higher doses of some organophosphorus insecticides can now be tolerated without fatal consequences. However, many organophosphorus pesticides that might, theoretically, cause neuropathy, would only do so at a dose far above the lethal dose.

(a) Characteristics

Regardless of the severity of anticholinesterase effects, there is a delay after intoxication before neuropathic signs and symptoms appear. In the adult hen, which is the test species of choice, this delay is 8 - 14 days, while in man, the delay may be up to 4 weeks after acute exposure. The first symptoms are often sensory with tingling and burning sensations in the limb extremities followed by weakness in the lower limbs and ataxia. This progresses to paralysis, which, in severe cases, affects the upper limbs also. Children and young animals are less severely affected than adults, but recovery is slow and seldom complete in adults; with the passage of time, the clinical picture changes from a flaccid to a spastic type of paralysis. Cats, hens, and a number of larger species are affected by a single dose. Repeated dosing does not reduce the delay in onset to less than 8 days from the first dose. Baboons, monkeys, and marmosets do not respond easily to single doses of several typical neuropathic esters, and it is difficult to produce typical delayed neurotoxic effects in rodents, even by repeated dosing. In early histological examinations, methods were used that showed mainly degeneration of the fatty myelin sheath surrounding long nerve axons and names such as "Organophosphate Demyelinating Disease" are still erroneously used, in spite of later work that showed that the nerve axon itself was primarily affected and damage to the myelin sheath was secondary (Cavanagh, 1954; Bouldin & Cavanagh, 1979). The preparation of tissues and identification of lesions are described by Bradley (1976), Bickford & Sprague (1984), and by Prentice & Roberts (1984) in the Workshop Report edited by Cranmer & Hixson (1984). This publication also covers many aspects of mechanism and testing.

(b) Mechanism

The first essential step in the initiation of the delayed neuropathic effect of an organophosphate is phosphorylation of a target protein in the nervous system. The protein has esteratic enzyme activity. The phosphorylation, which was originally studied radiochemically, can be monitored conveniently as progressive inhibition of the activity of this enzyme, which is now referred to as Neuropathy Target Esterase (NTE or Neurotoxic Esterase) (Johnson, 1980, 1982a). The second, and equally essential, step is the transformation of the phosphorylated NTE to a modified form: one of the remaining ester bonds of the inhibitor molecule attached to the NTE active site undergoes a biochemical cleavage leaving an ionized acidic residue bound to the protein: this residue is negatively charged and the reaction is referred to as "aging". Both inhibition and aging of inhibited NTE are essential to initiate neuropathy, but the role of the negative charge in the initiation of axonal degeneration is not known. The process of "aging" of inhibited NTE has some analogy with the better-known "aging" of inhibited AChE. However, the analogy does not last above the level of enzyme inhibition. Acute toxicity arises directly from the loss of catalytic activity of AChE, leading to accumulation of excess physiological substrate. Mere loss of catalytic activity of NTE (without aging) does not initiate neuropathy (see below). There is no evidence of a deleterious accumulation of a physiological substrate for NTE or lack of hydrolysis products after inhibition in vivo, and the effect of

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the negative charge may be focused on some quite separate process.

When NTE has been inhibited by a suitable phosphate, phosphonate, or , aging is always possible, and this process has been shown to occur rapidly with a wide variety of neurotoxic esters and hen NTE. However, after inhibition by phosphinates (which contain 2 phosphorus-carbon bonds) or by sulfonyl fluorides, no hydrolysable bonds remain in the attached inhibitor molecule. Thus, aging is not possible, and these compounds are not neuropathic in vivo. Whenever hen NTE can be phosphinylated or sulphonated in vivo, the birds become resistant to challenge doses of typical neuropathic esters, because the 2-step initiation process has been blocked halfway. Similarly, carbamates do not form aged inhibited enzymes and are either totally without effect (anti- carbamates are poor inhibitors of NTE) or inhibit the enzyme, but do not age. Thus, they protect the hen in the same way as the phosphinates. Now that this mechanism is understood, it is obvious that carbamates need not be subjected to tests that were designed to detect delayed neuropathic potential in organophosphorus anticholinesterase pesticides.

The events that follow inhibition and aging of NTE are not known, but it is clear that, in adult hens, detectable delayed neuropathic events (clinical or histological) are never seen after a single dose/exposure of an organophosphorus ester, unless there is at least 70% (probably 80 - 90%) inhibition of the NTE in the brain and spinal cord soon after dosing (4 - 40 h). Owing to the synthesis of fresh protein, this inhibition declines markedly during the 8 to 14-day delay period, and there is no correlation between neuropathy and NTE inhibition measured at the time that clinical signs reach their peak. NTE has been found in the nervous tissue of all mammals and birds examined and can be inhibited, even in species such as the rat, in which there is no obvious clinical neuropathic response to a single dose. It has been shown recently that, when single doses of certain neuropathic organophosphorus compounds are given to rats, degenerative lesions develop in their peripheral nerves and spinal cord. Although these lesions are similar to those in ataxic hens, they are not accompanied by clinical signs. The dose-response of NTE inhibition and the neurological damage in rats are well correlated, and, as in hens, the lesions are prevented by protectively predosing the rats with sulfonyl fluoride (Padilla & Veronesi, 1985; Veronesi & Padilla, in press). Human NTE has been examined in vitro (Lotti & Johnson, 1978), and its response to inhibitors is similar to that of the hen enzyme (I50s differed by not more than 4-fold). What is not known is the numerical value of the threshold of inhibition of NTE in man that is associated with clinical neuropathy. However, it is known that numerous people treated only with atropine for poisoning by trichlorfon have survived and then developed neuropathy (Johnson, 1981a). In contrast, it is very difficult to produce neuropathy with trichlorfon in the hen, without enormous doses coupled with prophylaxis as well as therapy for severe anticholinesterase effects. It seems, therefore, that in the case of this pesticide, the chances of a severely poisoned man developing neuropathy rather than dying are greater than those of the hen. It might be deduced that the threshold level of NTE inhibition for neuropathic effects in man is somewhat less than the 70% value for hen, and caution should be applied in the extrapolation from hen tests. This argument is merely a comparison of the two hazards, death and neuropathy: it does not give any guide to the relative dose required to intoxicate man and hen.

(c) Delayed testing

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Two distinct procedures are currently practiced by testers, and each depends on the anticipated hazard. In acute tests, it is recommended that hens surviving an LD50 test (preferably a test in which the therapeutic use of atropine raised the LD50) should be observed for three weeks for abnormalities of gait and then be re-dosed and watched for a further three weeks, after which a thorough histological examination of the distal ends of neurons in the spinal cord and peripheral nerve should be performed. Long- term neurotoxicity tests require feeding hens for up to 90 days with doses in the diet ranging up to those causing obvious adverse cholinergic effects; evaluation is by the same criteria as for the acute test. It may be argued that long-term tests should be omitted, in view of the greater speed and simplicity of the acute test in obtaining yes/no answers and the fact that no pesticidal compound has ever been found to give a positive clinical response in feeding trials when negative in the 2-massive-doses test. The practice in the United Kingdom has been to voluntarily exclude from agricultural use any compound shown to be neuropathic in acute tests. Guidelines for the performance of acute and long-term delayed neurotoxicity tests are available (United Kingdom PSPS, 1979; US EPA, 1982; OECD, 1983). The OECD Guidelines point out that acute tests would be improved if they were accompanied by assays of NTE inhibition in the brain and spinal cord, one and two days after dosing. Results of assays would provide graded dose- response relationships instead of the present all-or-none scoring. Some examples are given in Johnson (1975b). Particularly useful are data such as those obtained with malathion (Johnson, 1981b), which showed negligible (< 10%) inhibition of NTE at the LD50 dose in hens. These data indicate a different order of safety compared with that of some other compounds which caused 50 - 60% inhibition, though there was no visible clinical response. A way to integrate the NTE test efficiently into delayed neurotoxicity test protocols has been suggested by Johnson (1984). However, further discussion to improve this method is required.

(d) Delayed neurotoxic response to long-term feeding

For some neuropathic compounds, the potency of a single dose may be matched or even exceeded by the effect of the same amount spread over a few days. In particular, compounds such as TOCP or leptophos, which are very poorly soluble in water and are required in a very large single dose to be effective, may well be absorbed with greater efficiency in a divided lower-dose regime. However, the results of various studies (Johnson, 1982a) have shown that, as the dose is reduced further, there is a clear cut-off point below which there does not appear to be any cumulative effect. Thus, TOCP in the diet of hens at 400 mg/kg produced neuropathy in 21 days, while a level of 100 mg/kg diet did not cause any detectable clinical or histological damage after 140 days (Barnes, 1975). Monitoring of the NTE response to continuous feeding of non- neurotoxic regimes of 2 organophosphates (0.125 mg DFP/kg for 5 days per week, over 4 weeks, or 2.5 mg mono-2-cresyl diphenyl phosphate/kg, daily, for 10 weeks) showed that NTE levels in the brain and spinal cord were depressed within 2 - 3 weeks to about 45 - 55% of normal and remained unchanged thereafter; the equilibrium is presumbably the result of daily synthesis matching daily inhibition. These doses did not cause detectable neuropathy during either the feeding period or the 3 ensuing weeks (Lotti & Johnson, 1980). On the basis of these and other studies, Johnson (1982a) concluded that the hen nervous system might tolerate the biochemical defect of prolonged inhibition of NTE to about 50% normal level brought about by long-term dosing but could not

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tolerate the brief 80 - 90% inhibition that follows a single larger dose. If long-term feeding tests in hens are required, then weekly measurements of the level of NTE inhibition should provide valuable predictive information, early in the test (Johnson, 1984). Since it is necessary to kill birds for tissue sampling, more birds may be necessary, at least in the early stages.

(e) Results of delayed neurotoxicity testing

No pesticidal organophosphorus compounds, giving negative results in massive-dose tests, have caused delayed neuropathy in long-term feeding studies. With the exception of trichlorfon, where 2 doses, 3 days apart, were necessary (Johnson 1970, 1981a), the massive-dose tests have been single doses given with prophylaxis and therapy (eserine + atropine and atropine + oxime - repeated if necessary) to ensure that doses above LD50 could be examined. In Table 5, known pesticidal compounds are listed according to the doses known to produce marked-to-severe neuropathy in the majority of birds tested. Administration by either the oral or dermal route may be effective.

The review by Johnson (1975b) is believed to list all pesticidal and non-pesticidal compounds, or their derived oxons, tested and reported on, up to that time. The JMPR Reports were not included as a literature source. NTE responses are given where these have been measured. Johnson (1982a) lists a number of pesticides for which both clinical and NTE tests had been reported since 1975 and notes that carbophenthion, , diazinon, fenitrothion, malathion, methyl parathion, omethoate, and parathion can positively be declared non-neuropathic on the basis of negligible NTE inhibition responses, while chlorpyrifos, methamidophos, and salithion gave intermediate NTE responses without clinical expression at the doses tested. Isofenphos ( o -ethyl- o -2-iso-propoxycarbonylphenyl isopropylphosphor- amidothioate) has been shown by NTE assays and clinical tests to cause delayed neuropathy in hens at about 20 x LD50 (Wilson et al., 1984), while the insecticidal synergist o-n -propyl- o -(2-propynyl) phenylphosphonate was neuropathic by both criteria at about the LD50 (Soliman, 1982).

Table 5. Organophosphorus pesticides causing delayed neuropathy in hens after a single dose ------Compound Dose (mg/kg) Reference and route ------ 25 im Bidstrup et al. N,N -diisopropylphosphoro- (1953) diamidic fluoride haloxon 1000 oral Malone (1964) 3-chloro-4-methyl-2-oxo- 2H-1-benzopyran-7-yl- bis-(2-chloroethyl) phosphate

EPN 40-80 sc Witter & Gaines (1963) trichlornat 310 oral Johnson (1975b) ethyl 2,4,5-trichlorophenyl 300-400 oral Johnson (1975b) phenylphosphonate ethyl 2,4-dichlorophenyl > 2000 oral Abou-Donia et al. phenylphosphonothioate (1979)

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leptophos 400-500 oral Abou-Donia et al. (1974); Johnson (1975b) desbromoleptophos 60 oral Johnson (1975b)

S,S,S -tributyl phosphoro- 1110 sc Johnson (1970) trithioate (DEF) cyanofenphos > 100 oral Ohkawa et al. (1980) isofenphos 100 oral Wilson et al. O -ethyl O -2-isopropoxy- (1984) carbonylphenyl isopropyl- phosphoramidothioate

O-n- propyl O -(2-propynyl) 400 oral Soliman (1982) phenylphosphonate dichlorvosb 100 scc Caroldi & Lotti (1981) amiprophos 600 oralc,d Huang et al. O -ethyl O -4-methyl-6- (1979) nitrophenyl N- isopropylphos- phoramidothionate

Table 5 (contd.) ------Compound Dose (mg/kg) Reference and route ------ 50 oralc Abou-Donia et al. 500 dermal (1982) chlorpyrifos 150 oral Lotti et al. (1986) salithion 120 oral El-Sebae et al. (1981) ------a Dose needed to cause marked-to-severe neuropathy in the majority of birds tested. b 50% formulation in hydrocarbons: dose calculated as active ingredient. c Mild neuropathy only at maximum tolerated dose. d Test performed in cockerels.

The statement by Namba et al. (1971) that chlorpyrifos produced neuropathy in hens seems to have been without foundation and may arise from the misreading of a report by Gaines (1969), which mentioned that chlorpyrifos caused a rapid onset short-term weakness (sometimes called paralytic effect) similar to that caused by malathion. However, one recent case of human poisoning and laboratory tests with doses well above the unprotected LD50 have shown neuropathic effects from this pesticide (Lotti & Moretto, in press).

(f) Structure/activity relationships

As noted above, not all organophosphorus pesticides cause delayed neuropathy. In vivo tests and target-enzyme studies listed by Johnson (1975b) can be condensed according to a number of

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factors as listed by Johnson (1980, 1982a):

(1) Factors that increase delayed neurotoxicity potential more than acute toxicity are:

(a) choice of phosphonates or phosphoramidates rather than analogous phosphates;

(b) increase in chain-length or hydrophobicity of R1 and R2 ; and

(c) a leaving group X, which does not sterically hinder approach to the active site of NTE;

(2) Factors that decrease the comparative potential are:

(a) the converse of (1) a, b, and c;

(b) choice of R or X groups that are very bulky (naphthyloxy) or non-planar;

(c) choice of a nitrophenyl group at X (a steric effect?);

(d) choice of comparatively more hydrophilic X groups (oximes or heterocyclics); and

(e) choice of thioether linkages at X.

Considering these factors, it can be seen why malathion and diazinon are both far below the neurotoxicity hazard line, why, in its homologous series, only dichlorvos is not neurotoxic at the LD50, why EPN, a phosphonothioate with a hydrophobic phenyl group at R1 is neurotoxic, even with a 4-nitrophenyl leaving group, and why it is not surprising that other phenylphosphonothioates such as desbromoleptophos, or cyanofenphos are also neurotoxic. The apparent non-neurotoxicity of the ethyl analogue of leptophos (Hollingshaus et al., 1979) seems to contradict (1-b) above, but the dominant factor seems to be the problem of absorption of this very poorly soluble compound after oral dosage (Hansen & Hansen, 1985).

6.1.2 Behavioural and other effects on the nervous system

The problems of interpreting behavioural changes in relation to inhibition of AChE and the time-dependent changes in these variables have been discussed by Bignami (1976) and Bignami et al. (1975). It appears that some learned responses may be acquired more quickly by rats recovering from an inhibitory dose of, e.g., DFP (1 mg/kg body weight). This may be a sign of changed inhibitory responses in learning pathways, but it is difficult to say whether it can be classified as a toxic response.

Numerous research workers have reported changes at doses that affect levels of AChE, but without overt signs of intoxication. For example, Kaloyanova-Simeonova (1961) noted that small doses of chlorthion (5 or 10 mg/kg body weight in the rat) intensified conditioned motor reflexes and depressed them at higher doses: ChE levels were depressed in all cases and were restored more slowly than the return to normal reflex activity. Reiter et al. (1975) did not find any effects on performance of learned visual discrimination tasks by monkeys at doses of parathion of 0.5 mg/kg body weight, which depressed blood levels of AChE by about 25% and those of pseudoChE by about 35%. Doses causing 40% or more inhibition of AChE were associated with decreased responses, which

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returned to normal faster than the recovery of AChE levels. A very sensitive response to the (pinacolyl methylphosphonofluoridate) was reported in one out of several behavioural tests by Wolthuis & Vanwersch (1984). In an open-field test, a number of performance variables were affected by a dose of only 3% of the LD50. Effects were also seen with doses of 4 - 6% LD50 of 2 carbamates but not below 30% LD50 of the pesticide, TEPP, or of another nerve agent, (isopropyl methylphosphono- fluoridate). Unfortunately, the effects on ChE levels were not measured. There is no obvious reason for the contrasting effects of TEPP and sarin, on the one hand, and of soman and , on the other. The authors noted that, judged by poisoning signs at near-lethal doses, soman exerted a greater proportion of its effects centrally than the other 2 organophosphorus esters. The fact that changes may be seen in some, but not all behavioural responses at a certain dose emphasizes the statement by Revzin (1983) that no single behavioural or neurophysiological test can give a definite conclusion about organophosphate toxicity.

There appears to be only one other report in the literature of a behavioural change in animals at doses less than those that inhibit ChEs (Desi et al., 1971). Behavioural and EEG changes were noted in rats fed bromophos at 500 or 100 mg/kg diet for 6 weeks, doses that also caused ChE inhibition, but, at 30 and 10 mg/kg, there were no effects on ChE, though some behavioural changes were still observed. It is not clear whether undosed animals were handled in precisely the same fashion as the dosed.

However, in contrast to the above, Desi (1983) reported tests applied after 3 months of daily consumption of diet containing various percentages of the LD50 of bromophos. The lowest concentration that produced changes in a behavioural test (maze running) and in EEG (both the complex EEG and computer-analysed segments of the EEG) was about 0.26% of LD50, daily, but this also caused significant changes in erythrocyte-AChE and plasma-ChE. Among 6 organophosphorus pesticides assessed by these procedures, none produced changes greater than that caused by the vehicle alone without also causing effects on ChE. This conclusion differs from that written by the author.

Analysis of the EEG records of a small group of rhesus monkeys has been carried out both before, and one year after, intoxication with the potent anticholinesterase agent sarin (isopropyl methylphosphonofluoridate) (Duffy & Burchfield, 1980). Three animals received a single "large dose" (5 µg/kg body weight iv), and 3 others received a series of 10 injections of 1 µg sarin/kg body weight im at weekly intervals: there were 10 controls. The "large dose" animals had generalized convulsions and were maintained on Gallamine relaxant with artificial respiration; small-dose animals were considered in pilot studies to be near the threshold of poisoning, but showed few overt signs; they did not receive relaxant or artificial respiration. Twenty-four hours after a "large dose", marked differences were seen in the EEG frequency spectrum, which is not surprising. However, one year after the dose, there was still a small increase in the percentage energy in the beta-2 region of the spectrum and the change was said to be statistically significant. Some changes in the beta region were detectable in all 6 dosed monkeys one year after; no significant changes were seen in the 10 controls. However, apparently the changes were only seen under some lighting conditions and were small compared with differences between the frequency spectra of the only 2 controls for which data were shown. The statistical treatment of the acquired data seems valid, but the

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actual values measured one year after dosing are obviously well within the normal range. No indication was given of how much variation can be caused in the pattern of undosed animals by variations in the conditions of handling and observation, or what the range is for apparently identically treated normal animals. The doubts pertaining to the toxicological significance of these measurements apply also to some human studies using the same technique (section 7.2.2).

Effects of cholinergic agents on the visual system have been monitored by Revzin (1980). In urethane-anaesthetised pigeons with implanted electrodes, various changes in the response of specific neurones of the optic tectum and of the hippocampus were noted after doses both of mevinphos and of atropine. The author claims that the procedure was sensitive in detecting effects in the absence of detectable peripheral parasympathetic signs. However, the lowest effective dose of mevinphos was only one-third of that (0.15 mg/kg body weight) which produced parasympathetic signs that would certainly be associated with substantial inhibition of AChE. Thus, the claim to sensitivity of this complex procedure in surgically modified birds seems excessive.

Some possible effects of anticholinesterases on non- cholinesterase targets were considered by O'Neill (1981). No clear effects seem definable at concentrations lower than those that inhibit AChE and some are probably secondary to stimulation of non- cholinergic nerves with cholinergic innervation. However, an endopeptidase that can hydrolyse putative transmitter peptides in the nervous system is known to be inhibited by di-isopropyl phosphorofluoridate at a concentration similar to that which inhibits AChE (Kato et al., 1980). Thus, some involvement of non- cholinergic pathways in the causing of CNS effects in organophosphate poisoning cannot be excluded. Moreover, some anticholinesterases also exert effects directly at the cholinergic receptor as well as by the inhibition of AChE (Karczmar & Ohta, 1981).

6.2 Other Effects

A variety of histological lesions has been described at autopsy in animals severely poisoned with organophosphate pesticides. However, very few effects are described in the absence of obvious poisoning or at doses that do not markedly inhibit the ChEs. In the following sections, the evidence is reviewed for effects not obviously attributable to inhibtion of either AChE or NTE.

6.2.1 Mutagenic and carcinogenic effects

The proposed theoretical basis for believing that dimethyl or diethyl phosphate pesticides might be mutagenic or carcinogenic has already been discussed (section 4.5.2). This basis was shown to be defective by Bedford & Robinson (1972). No alkylation was detected in N7 of guanine in RNA and DNA of liver of animals that were exposed for 12 h to working concentrations of dichlorvos of 64 g/m3 (0.064 g/litre air) (Wooder et al., 1977); these authors contrasted their data with earlier published work showing modest alkylation of guanine in suspensions of cells exposed to very high solution concentrations of dichlorvos. In vivo, the acute anticholinesterase effects limit the circulating concentration that can be tolerated by any and there is also strong preference for phosphorylation of biological scavenger molecules rather than for alkylation built into the organophosphorus

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pesticide molecules.

It has frequently been suggested that dichlorvos has carcinogenic potential because of observed mutagenic effects in in vitro test systems. The IARC (1979) Working Group accepted extensive data showing no evidence for the mutagenicity of dichlorvos in mammals. FAO/WHO (1978b) accepted animal studies showing no dose-related carcinogenic effects in life-time studies carried out at doses that depressed blood-ChE levels.

In a 78-week feeding test on groups of 50 male and 50 female mice (B6 C3 F1 hybrid), no dose-related effects were seen when the diet contained about 600 or 300 mg dichlorvos/kg, respectively. The IARC Working Group evaluating this study noted that a few oesophageal tumours were seen in treated mice. It appears that this fact influenced their verdict that "the available data do not allow an evaluation of the carcinogenicity of dichlorvos to be made" (IARC, 1979).

An IARC Monograph (1983) included evaluations of 5 widely-used organophosphate pesticides (malathion, methyl parathion, parathion, , and trichlorfon). In several cases, the conclusions were that acceptable tests had been performed with no evidence of carcinogenic effects or of mutagenic effects in mammals. For others, the conclusions were of "limited evidence" consisting of very small effects above the control background levels in life-time studies. None of these compounds was judged to be a strong mammalian mutagen or and the same statement is true for all organosphosphorus pesticides that have been evaluated by FAO/WHO Working Parties or by other authorities. However, recent published results show that controversies do occur when evaluating the outcome of carcinogenicity studies. Huff et al. (1985) reevaluated the pathology of the original studies by the US National Cancer Institute in 2 different strains of rats. Histopathological reexamination confirmed the earlier conclusions that malathion and were not carcinogenic. These conclusions differed from those of Reuber (1985) who evaluated the same studies and rated both compounds as carcinogenic.

6.2.2 Teratogenic effects

Defects in the development of fertilized hen eggs, injected with various organophosphates, are known, but many of these are associated with the inhibition of the enzyme kynurine formamidase and a depression of NAD levels at a critical period of development (Seifert & Casida, 1980). This pathway is not critical in mammals, and no equivalent effects are known. If teratogenicity is taken to mean induction of malformations in live offspring without decrease in number of births (i.e., no embryotoxicity), then for the vast majority of organophosphorus pesticides, no adverse effects of continuous feeding of organophosphates on pre- or postpartum mortality have been reported, nor have embryonic defects been proved, except at doses that significantly retarded growth in the mother (Vergieva, 1983). Single high doses causing significant toxic effects in mothers may be deleterious: a number of these toxicity-linked effects have been summarized by Seifert & Casida (1980). Kimbrough & Gaines (1968) reported deaths, and that resorptions were increased in pregnant rats given a single high dose of parathion or diazinon on the 11th day of gestation. However, these effects were associated with significant toxic effects on the mothers. Similarly, trichlorfon at very high doses (400 mg/kg per day with the dose divided into 3 spaced aliquots) and given on days 6 - 15 of gestation, produced defects in

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offspring: each dose produced cholinergic symptoms; no effects were seen in rats, mice, or hamsters when the daily dose was 200 mg/kg (Staples & Goulding, 1979). However, a specific defect consisting of hypoplasia of the cerebellum in offspring was noted, both in field cases and experimental tests, 8 pregnant pigs were dosed once or twice with neguvon (a veterinary grade of trichlorfon) between days 55 and 70 of pregnancy (Knox et al., 1978). Doses were 50 - 60 mg/kg body weight, which caused maximum inhibition of erythrocyte-ChE levels of 40 -80%, without overt signs of poisoning. The hypoplasia was accompanied by severe ataxia and tremors, while voice and vision appeared unaffected. No defects were seen in the offspring of over 100 control sows housed with the dosed animals, and serological and virological tests did not show anything incriminating. A genuine teratogenic effect of moderate doses of trichlorfon commonly used in veterinary practice has been demonstrated in the pregnant pig. However, it may be that the herds tested were unusually susceptible, since (apparently) congenital tremor had been known in litters borne over many years in the area where the trichlorfon-induced effects were seen.

6.2.3 Effects on the immune system

In a review, Zackov (1983) stated that "most ... organo- phosphorus pesticides elicit autoimmune reactions and suppress the production of antibodies against vaccines". No evidence was given and it is not clear whether the statement was claiming specific effects or referred to doses that are sufficient to produce a range of toxic effects.

Shtenberg et al. (1974) claimed that oral doses of methyl- nitrophos (fenitrothion) or chlorphos (trichlorfon) at 5 or 7 mg/kg body weight per day, for an unspecified period, suppressed haemaglutinin levels in rats immunized against sheep red blood cells; these doses would have significantly inhibited ChEs and were said to be more effective in rats fed a protein-deficient diet. Dandliker et al. (1979) reported a depression in antibody titre in rats in the 6 - 7 weeks of an immunization procedure that commenced either one day before or one day after (both statements are made) an oral dose of half of the LD50 of parathion. The immunization consisted of weekly intramuscular doses of 400 µg fluorescein- labelled ovalbumin with Freunds Complete Adjuvant. The confusion concerning the order of intoxication and primary immunization is very important when such a high dose was given, since cholinergic symptoms, fluid loss, imbalance, and general debility would have been marked. Administration to mice of 0.1 x LD50 of parathion for 8 days led to a 10% loss in body weight. Immunization on the 9th day showed a statistically-significant (34%) reduction in the number of antibody plaque-forming cells derived from the spleen, 4 days after immunization, but the reduction was insignificant (10% only) when immunization was carried out on day 10, though several animals in the group died at this point due to accumulated toxic effects (Wiltrout et al., 1978).

Some decreased antibody titres "proportional" to AChE inhibition were noted in response to prolonged doses of malathion or dichlorvos (0.025 - 0.4 x LD50 given 5 days per week for 6 weeks) (Desi et al., 1978). Clearly, none of the above responses can be dissociated from the general cholinergic intoxicating effects of the pesticides. There do not appear to be any reports of studies on the immunological status of healthy animals receiving doses causing little or no depression in ChE levels.

6.2.4 Effects on tissue carboxyesterases

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A variety of carboxyesterases abound in serum, liver, intestine, and other tissues (section 4.4). Although inhibition of one specific carboxyesterase (NTE) has toxic sequelae (section 6.1.1.2), no direct deleterious effects of inhibiting other carboxyesterases have been demonstrated. However, they may contribute markedly to the metabolic disposal of malathion and certain other organophosphorus pesticides, so that inhibition of tissue carboxyesterases may potentiate the toxicity of such pesticides (section 6.3.5). The structure/activity relationships for inhibition of these enzymes by organophosphorus compounds inevitably differ from those for inhibition of AChE. For EPN and fenchlorphos, both serum- and liver-carboxyesterases of rats were markedly more sensitive than brain- and serum-AChE; in other cases, the liver enzymes, but not those in serum, were more sensitive (Su et al., 1971).

6.2.5 Sundry other effects of organophosphorus pesticides

Very few effects other than those described in the sections above have been noted, except those arising from ill-health due to severe anticholinesterase effects. Thus, impaired growth rate is commonly associated with a rapid depression in AChE levels to less than 50%, but much lower levels can be tolerated without ill- effects, if the depression is brought about over several weeks, and then maintained for up to a year (section 6.3.1).

Various changes in glucose metabolism, in serum enzymes, and in other clinical chemical variables have been reported after single, acute, or repeated doses of various pesticides at from one-tenth to one-quarter of the LD50, daily (Dimov & Kaloyanova, 1967; Enan et al., 1982).

A reversible, mild muscle-necrotising effect could be detected histologically in the diaphragm muscles of rats, 24 h after a dose of paraoxon, parathion, or other anticholinesterases, sufficient to cause marked fasciculations (Fenichel et al., 1972; Dettbarn, 1984). The damage appeared to be a function of the prolonged cholinergic stimulation of the muscle, since it was entirely prevented by doses of atropine, which prevent fasciculations, or by alpha- applied directly to the myoneural junction (Salpeter et al., 1979).

Several adverse effects attributed only to certain organophosphorus esters are listed below. The list may be of value in promoting more careful examination of intoxicated animals or human beings.

6.2.5.1 Effects on hormones

Changes in the diurnal pattern of plasma-ACTH and adrenal levels of some related enzymes have been reported in rats maintained with dichlorvos in the drinking-water at 2 mg/litre: blood-ChE levels were not affected (Civen et al., 1980). However, the weight gain of the treated animals was only half that of the controls, and the fluid consumption was increased by 20%. These deleterious effects could be due to diarrhoea and imbalance of fluids with inevitable repercussions on hormonal levels, etc.; preferential effects of the dichlorvos on intestinal esterases might be the primary effect.

6.2.5.2 Effects on the reproductive system

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Damaged seminiferous tubules were reported in mice given either a single dose of about half of the LD50 of dichlorvos or 18 doses of about one-tenth of the LD50 (Krause & Homola, 1974). However, the doses were high, the percentage increases seem small, and the number of samples taken was very small; thus, no statistical evaluation is possible. Amiprophos has been reported to cause some gonadotrophic effects in adult cockerels (Huang et al., 1979), but details are few.

6.2.5.3 Effects on the retina

Fenthion administered intramuscularly at about one-quarter of the LD50 (50 mg/kg body weight), every 4 days for one year (solvent, if any, not stated), affected the electroretinogram in 2 strains of rats (Wistar and black Long-Evans) within 3 months and abolished it after one year (Imai, 1977). Similar studies involving the administration of either fenitrothion or ethylthiometon to beagle dogs for 5 days per week for 2 years, were reported by Ishikawa & Miyata (1980). Doses that depressed plasma-ChE levels to about 30% of normal, throughout the period, led to changes in optical function after 13 months continuous exposure and to morphological changes in the ciliary muscle at termination.

6.2.5.4 Porphyric effect

Daily application of technical (85%) diazinon (20 or 40 mg/kg body weight) to the skin of Dark Agouti rats just above the tail produced a 4-fold increase in faecal porphyrins, after 8 - 12 weeks (Bleakley et al., 1979). There was no increase in urinary porphyrins and no effect when diazinon was given in food (about 8 mg per day to rats, initially weighing 180 g). The pattern of porphyrins excretion was said to be indistinguishable from porphyria cutanea tarda. However, the increase in excreted porphyrins in classical porphyria cutanea tarda may be as much as 1000-fold, with massive amounts in the urine as well as in the faeces. The authors failed to reproduce even this small effect in rats when they used more pure (97%) diazinon (Nichol et al., 1982). However, they also found that an impurity in stored technical material, isodiazinon (diethyl 2-isopropyl-6-methyl-4- S - pyrimidinyl phosphorothioate), was very effective in causing porphyrin accumulation, when added to cultures of chick hepatocytes; confusingly, however, the accumulated porphyrin was coproporphyrin rather than the expected protoporphyrin. Although human poisoning with diazinon is not uncommon, there has only been one report implicating technical diazinon in a few cases of porphyria cutanea tarda in occupationally-exposed workers (Bopp & Kasminsky, 1975). Further experimental animal studies seem warranted. No reports have been found of porphyria connected with exposure to other organophosphorus pesticides.

6.2.5.5 Lipid metabolism

Organophosphate esters can inhibit the activities of some triglyceridases and lipases in vitro and in vivo. However, no repercussions from such inhibition were found when appropriate parameters were measured in rats fed for one year with either of 2 pesticides that caused marked (60 - 80%) depression of blood-ChE. Thus, male or female rats were fed either a normal diet or a diet enriched sufficiently with fat to increase aortic fatty acids by 170%. No changes occurred in: the hormone-sensitive lipase and lipoprotein lipase in adipose tissue; the free and total fatty acids and total glycerol and total cholesterol in serum; and the total fatty acids, cholesterol, and glycerol in the aorta (Buchet

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et al., l977). The pesticides were chlorpyriphos (100 mg/kg diet) and triamiphos (10 mg/kg) and the findings were contrary to those from a preliminary study by the same workers.

6.2.5.6 Effects causing delayed deaths

Although not pesticidal agents themselves, two of the phosphorothiolate impurities found in technical malathion and some analogues of these impurities caused delayed effects, which were lethal for rats at doses below the cholinergic LD50 (Aldridge et al., 1979; Mallipudi et al., 1979; Verschoyle et al., 1980). The compounds were O,O,S -trimethyl phosphorothioate (I), O,S,S - trimethyl phosphorodithioate (II), and the ethyl analogues (III and IV). After an oral LD50 dose (as low as 26 mg/kg body weight for II), all 4 compounds produced cholinergic responses that lasted less than 24 h and deaths at this time were only seen with IV: doses 2 - 6 x LD50 were needed to cause cholinergic deaths with I - III. Rats dosed at, or near, the LD50 recovered from the initial cholinergic effects, but by day 3, they had lost weight and were panting with laboured respiration; deaths occurred 3 - 6 days after dosing. However, survivors appeared normal, 10 days after dosing. Death was due to pulmonary insufficiency associated with progressive cell proliferation (Dinsdale et al., 1982; Imamura et al., 1983; Aldridge & Nemery, 1984), and combined therapy with atropine and oxime was ineffective. The biochemical mechanism of these effects is not fully known, but it is probable that the proximal toxin is produced in the lung by oxidative attack on the alkylthio moiety of the compounds (Aldridge et al., 1985). The activities of brain-AChE and plasma-ChE and carboxylesterase, which were partially inhibited during the first day after dosing, increased thereafter and were at least 50% of the levels of activity in the controls at the time of death. The margin between delayed death LD50 and cholinergic LD50 was markedly less with the triethyl, than with trimethyl compounds. Such effects were seen with S,S,S -trimethyl phosphorotrithiolate but not with the higher analogue ( O -ethyl S,S -di- n -propyl phosphorodithiolate) or with methamidophos (Verschoyle & Cabral, 1982).

A different form of delayed acute toxicity was reported to occur 4 days after large oral doses of DEF in hens. The effect was not seen after a single dose, sufficient to cause delayed neurotoxicity, was given subcutaneously (Johnson, 1970) or dermally (Abou-Donia et al., 1980). The effect was distinct from both cholinergic and delayed neuropathic effects and is attributed to the acute toxicity of n- butyl mercaptan produced by the degradation of DEF in the gastrointestinal tract.

6.2.5.7 Selective inhibition of thermogenesis

The defoliant DEF ( S,S,S -tri- n -butyl phosphorotrithiolate) acted as an anticholinesterase at high doses, but, at lower doses (60 - 200 mg/kg in rats and mice), it caused a profound fall in body temperature (as much as 10 °C over a few h), without marked sedation; deaths occurred mostly after the depression had persisted for a day (Ray, 1980). The effect was different from the smaller atropine-sensitive changes due to some cholinomimetics and was due to blocking of cold-induced thermogenesis without affecting heat conservation mechanisms. The effect seems unique to the DEF chemical structure. Ray & Cunningham (1985) demonstrated that the effect was a selective action on a central thermogenic control mechanism rather than on peripheral thermogenic processes and that it was probably due to a metabolite of DEF rather than to the

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parent compound.

6.3 Factors Influencing Organophosphorus Insecticide Toxicity

6.3.1 Dosage-effect

The lethal effects of organophosphorus insecticides are due to severe cholinergic effects arising from excessive inhibition of AChE. With few exceptions, the AChE activity of the tissues is inhibited soon after the administration of acutely toxic doses of all anti-AChE agents. This is true, not only for compounds that do not require metabolic conversion to anti-AChE agents, but also for most phosphorothioates, phosphorodithioates, and phosphorodiamidates that are oxidized by the liver to metabolites with anti-AChE activity. In general, the duration of action of most anti-AChE agents is relatively short, as evidenced by considerable reversal of the inhibition within a few days. For this reason, a 10-day observation period is sufficient for acute LD50 measurements on all anti-AChE agents that have been studied, and 2 days suffice for most.

The data in Table 6 give a comparison of the maximal amount of inhibition of the ChE activities in the brain, submaxillary glands, and serum of rats for several compounds, all of which were given at dose levels equivalent to 5/8 of the LD50. The time at which maximal inhibition occurred and the period required for complete reversal of the inhibition are also presented. From these examples, it can be seen that, in general, equivalent fractions of the LD50 of various anti-ChE compounds produce similar levels of inhibition of ChEs, though the LD50 values for the various compounds differ considerably. The time at which maximal inhibition of ChEs occurs varies from 15 min to 3 h after administration. In some cases, the AChE activity of the brain and parasympathetic nervous system, as indicated by the submaxillary gland, and the non-specific ChE of the serum are inhibited to the same extent by a particular compound. However, notable exceptions are OMPA, which is converted in the liver to a very labile inhibitor that never reaches the brain, and Guthion, which does not inhibit non-specific ChE.

Differences between the responsiveness of rat brain- and serum- ChE have been reported also in the case of dietary administration of various organophosphorus insecticides (Su et al., 1971). Thus, a level of only 40 mg EPN/kg fed for 1 week reduced brain-ChE to 50%, while 125 mg/kg was needed to achieve the effect in serum; the sensitivity was reversed for fenchlorphos, while the sensitivities of the 2 tissues were similar for demeton. It is not clear whether these differences reflect differences in access of the compounds to their targets, differences between the tissue AChEs, or the fact that pseudoChE is present as well as AChE in the serum of rats, so that assay of the hydrolysis of ACh using serum measures both enzymes, whereas, in the brain, the activity is about 90% specific.

Marked differences in the rate of reversal of the inhibitory effects on ChEs of different compounds, in vivo, are shown in Table 6. In view of the variable duration of action of various anti-ChE agents, the performance of assays on the tissues of animals at intervals after acutely toxic doses provides a great deal of useful information regarding the toxicity of these compounds. The transition from a tolerable to a lethal dose (either acute or chronic) often occurs within a 2 to 4-fold range. This is not surprising, since the AChE of nervous tissue and effector organs must be inhibited by 50 - 80% before pharmacological effects can be

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seen (Holmstedt, 1959). The increment in dose to raise inhibition to 90% with associated deaths is not very great.

Table 6. Onset and duration of the anticholinesterese action of some organophosphorus compounds in ratsa ------Compound Maximum inhibition of cholinesterase (%) Dose Brain Serum Sub- Time to Time to (mg/kg maxillary maximum complete body gland inhib- reversal weight) tion (h) (h) ------Iso-Systox 1.0 85 80 75 3.0 120 (demeton- S )b Disyston 1.25 75 85 75 3.0 120 (disulfoton) Guthion 3.5 60 0 50 0.5 24 (azinophosmethyl) Dipterex 140.0 85 82 85 0.25 6 (trichlorfon) Octamethyl 5.0 0 85 88 2.0 144 pyrophosphor- tetramide (OMPA)c ------a From: DuBois (1963). b Phosphorothioic acid, O -[2-(ethylthio)ethyl] O,O -diethyl ester. c Octamethylpyrophosphoramide.

The general correlation of inhibition of ChE with symptoms of poisoning is also seen with repeated dosing with organo-phosphates, but details vary greatly. It is always true that a prerequisite of death is profound inhibition of AChE in the central and/or peripheral nerves, but the tolerable maximum inhibition increases when this level is reached, stepwise, over a period of 2 - 4 weeks or more. Thus, when commercial Systox (containing about equal amounts of the O - and S -isomers of demeton) was fed to rats at 20 mg/kg diet, there were no signs of poisoning, though, at the end of 16 weeks, the brain- and whole blood-ChE activities were 26 and 28% of normal, respectively (Barnes & Denz, 1954). At 50 mg/kg, 3 out of 12 rats died, but all others in the group improved after initial marked signs of poisoning during the first month, their food intake increased above normal (and therefore their actual dose increased), and their growth rate became normal. This improvement was not due to any marked increase in ability to detoxify the agent, since, at the end of 16 weeks, these apparently healthy rats had only 7 - 8% of normal AChE activity in the brain and blood. This level would be associated with fatalities if brought about by a single dose; indeed, at the end of the study, the animals were consuming daily a dose equivalent to 96% of the single-dose LD50. In a similar way, Barnes & Denz (1951) found that parathion in the diet at 100 or 75 mg/kg was lethal for the majority of rats in large groups within 3 - 4 weeks, while results with 50 mg/kg were variable (26/72 deaths in one trial and 3/36 in a later trial) and no deaths attributable to poisoning occurred in groups fed 20 or 10 mg/kg. The rats surviving 50 mg/kg showed clinical signs of intoxication (notably fasciculations) and ate less, initially: they ate normally after 3 weeks, but failed to gain weight as rapidly as the controls. However, signs of poisoning decreased in severity and frequency during the third month and seldom reappeared during the remainder of a year's feeding. This pattern of response and of adaptation is typical for all anticholinesterase pesticides. When monitoring of enzymes is carried out in parallel with feeding, the level of

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activity often rises to a steady state after an initial decline. Factors determining the fraction of an LD50 dose that is tolerable include:

(a) Speed of absorption, of subsequent metabolic activation, and of elimination of the compound. Thus, for demeton- S - methyl, much of the sulfoxide metabolite from a single dose will still be circulating on the following day, though the parent compound may not linger. In such a case, lethal concentrations will build up more easily than with, say, trichlorfon, which is converted to the inhibitory dichlorvos, both of which are rapidly eliminated;

(b) The net rate of formation of a stable form of inhibited AChE arising from the 3 reactions of inhibition, reactivation, and aging described in section 4.5. The relationship of the chemical structure of an oxon inhibitor to rates of these reactions is complex and also varies between species. AChEs from the rat have not been purified and subjected to extensive kinetic study in vitro. In most studies, crystallized (not 100% pure) bovine erythrocyte-AChE has been used. Rates of spontaneous reactivation and aging for this enzyme inhibited with dimethoxy, diethoxy, and ethoxy, ethanthio-substituted phosphates are shown in Table 7.

Although data derived in this way cannot be transposed directly to in vivo situations, they are consistent with the well-known fact that, after poisoning by a sub-lethal dose of some dimethyl phosphates, recovery with the disappearance of symptoms is complete within a few h. The value of k+3 for erythrocyte-ChE taken from rats dosed in vivo with dimethyl phosphate, was reported to be 57 x 10-4 (a half-life of inhibited enzyme of 2 h). One day after such a sub-lethal dose, most of the rat AChE will again be in the uninhibited form with a small fraction in the aged inhibited form. In contrast, not more than half of the inhibited enzyme would be expected to be reactivated in one day after poisoning with diethyl phosphate and a substantial proportion of the inhibited enzyme would be aged, so that recovery to 100% activity would be a very slow process, depending on the synthesis of fresh enzyme. It follows that markedly different outcomes would be expected from repeated intoxication with doses of dimethyl phosphate and diethyl phosphate which both caused an initial response of, say, 50% inhibition, the former would be less hazardous than the latter. This interpretation concurs with the fact that rats can survive daily doses of 25% of the LD50 of trichlorfon, but only about 12% of the LD50 of parathion (DuBois, 1963). DuBois (1963) also pointed out that ChE inhibition mounted steadily as a result of daily doses of OMPA and that toxic effects were seen when inhibition reached about 70%. It is believed that no spontaneous reactivation of ChE occurs after inhibition by phosphoramidates, which makes such compounds intrinsically undesirable as pesticides.

Table 7. Rates of spontaneous reactivation (k+3 ) and of aging (k+4 ) of bovine erythrocyte cholinesterase after inhibition in vitro a ------O Rate constants x 104 || R1 (per min) at pH 7.4 || / and 37 °C Enz-O-P substituents \ R2

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R1 R2 k+3 k+4 ------OCH3 -OCH3 115 14

-OCH3 -SCH3 1170 95

-OC2 H5 -OC2 H5 2.0 2.2

-OC2 H5 -SC2 H5 270 32 ------a From: Clothier et al. (1981).

Dose-effect relationships for delayed neurotoxicity have been listed (Johnson, 1975b). It has been noted that, as for cholinergic effects, levels of long-term dosing can be found that are detectable by a biochemical response at the primary target but have no clinically- or histologically-observable correlate. The threshold of the tolerable response is probably a permanent inhibition or 40 - 50% of NTE (Johnson, 1982a).

6.3.2 Age and sex

It is well-known that the microsomal MFOs and other drug- metabolizing enzymes are present at comparatively low levels in neonatal animals, but activity develops to approximately the adult level early in maturation. Since MFOs are involved in both the activation and degradation of many organophosphorus pesticides (section 4.3), the likely net result in terms of LD50 is hard to predict. One-day-old rats were 9 times more susceptible to malathion than 17-day-old animals (Mendoza, 1976). The toxicity of methyl parathion and of parathion for rats decreased from birth through the developmental period: the decrease was best correlated with the increasing capacity of the animals to metabolize the oxygen analogues by both oxidative and hydrolytic pathways (Benke & Murphy, 1975). The LD50 (ip) of trichlorfon in adult male rats was reported to be 250 mg/kg body weight, compared with 190 mg/kg in male weanlings (FAO/WHO, 1972b). Whether this is a significant difference is not clear. Liver MFO activity fluctuates according to the hormonal status of female animals. LD50 values quoted for males and females often differ, but these values generally arise from different laboratory animals subjected to many variable factors (including the purity of the test sample). Among several representative pesticides surveyed for this review, only parathion showed a marked and apparently real difference in LD50s between the sexes, the oral LD50 in male rats being 5 - 30 mg/kg body weight, depending on the solvent, compared with 1.8 - 5 mg/kg in females (FAO/WHO, 1964).

6.3.3 Nutrition

It is well-known that liver MFO activity can be manipulated by administering a diet severely deficient in protein. The consequences can be dramatic in terms of the toxicity of compounds, such as , which undergo a single biotransformation step leading to a directly toxic product. However, as with the age and sex factors discussed above (section 6.3.2), several metabolic steps may be affected. No clear-cut effects seem to have been reported. Thus, the acute toxicity of diazinon is greater (up to 2-fold) in rats maintained on a diet, either very low (4%), or very high (81%) in protein compared with a standard (29%) protein diet (FAO/WHO, 1971b). A similar increase in toxicity is seen with naled ( O,O -dimethyl O -1,2-dibromo-2,2- dichloroethyl phos-phate) (Kaloyanova & Tasheva, 1983). Boyd

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(1969) reported that, while increases in toxicity were 2-3 fold for diazinon, malathion, and demeton, the increase in parathion toxicity was 7.6 fold, in malnourished rats. Whether the effects at such extremes were directly due to changes in the biotransformation of the agent or to the animals becoming generally unhealthy is not clear.

6.3.4 Effects of impurities and of storage

Insecticides are manufactured and formulated in various ways and in many countries. There may be significant differences in these procedures and in the conditions of storage of formulated products. These factors can influence the nature and extent of impurities present in the material that is ultimately applied.

Impurities in a pesticide may be of very low toxicity (the majority), may be toxic in their own right (more or less toxic than the major component), or they may be potentiators of the toxicity of another component.

6.3.4.1 Impurities toxic in their own right

(a) Non-anticholinesterase effects

The most dramatic example of an impurity exerting an effect different from that of the principal component does not come from the realm of organophosphorus pesticides. The potent toxicant 2,3,7,8-tetrachlorodibenzodioxin (TCDD) may be present in the herbicide 2,4,5-trichlorophenoxyacetic acid so that, even at a few mg/kg, the effects of the impurity may dominate the toxicological response. An analogous non-cholinergic response due to impurities is not known in anticholinesterase pesticides. Questions have been asked about the possible mutagenic effects of trimethyl phosphate, which may be present at a few percent in some technical preparations of dimethyl phosphates, but the possible exposure in vivo is limited by the main anticholinesterase response to the pesticide, and there appears to be no evidence for mutagenic effects in mammals of any organophosphorus pesticide (section 6.2.1).

(b) Anticholinesterase effects

LD50 values for technical preparations of diazinon have varied over an unusually wide range. The oral LD50 for the rat was reported to be 76 - 108 mg/kg body weight in 1964 (FAO/WHO, 1964) and 250 - 466 mg/kg in 1971 (FAO/WHO, 1971b). A major contributing factor, according to the latter report, was the presence of highly toxic in earlier samples; it was implied that the impurities had been produced during storage and eliminated by stabilization (detail unspecified) of formulated material. It seems likely that the concerned in this improvement was monothiono-TEPP with an oral LD50 in mice of about 4 mg/kg body weight (Margot & Gysen, 1957). Both the sulfotepp and monothiono- TEPP content of an emulsifiable concentrate of diazinon and its toxicity increased rapidly when it was stored in tinned-steel containers instead of in inert-lined aluminium ones (Soliman et al., 1982). However, in 1979, it was reported that sulfotepp was also present in many standard and formulated preparations of diazinon at concentrations ranging from 0.2 to 0.8% and that the percentage was unrelated to the age of the sample (Meier et al., 1979). It seems likely that this impurity was formed during the synthesis of diazinon using diethyl phosphorothiochloridate. Sulfotepp was 60 - 80 times more toxic than diazinon for the rat,

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so that at least one-third of the toxicity of typical diazinon might be attributed to the impurity. This calculation is probably an underestimate, since it seems that metabolic disposal of an impurity is often slowed markedly by competition from the major component. It can be said that there may be "reverse potentiation" of the toxicity of the impurity by the major component (diazinon).

Formation of pyrophosphates is implicit in the mode of synthesis of the many organophosphorus pesticides with a phosphorochloridate or phosphorothionochloridate as a precursor. TEPP or its methyl analogue would be unlikely to survive much aqueous washing during production, but the mixed mono- or disulfo analogues may well survive, unless deliberately eliminated. It seems likely that sulfotepp is generally present in parathion (Diggory, 1977); however, since both the parent and the impurity have similar LD50s, pure and impure preparations do not differ significantly. It is a curious fact that the lower the true toxicity of a pesticide, the more marked may be the effect of an impurity in changing its toxicity. It might be very rewarding both to analyse more thoroughly and to reexamine the of low-toxicity pesticides such as bromophos which is said to have an LD50 in various mammals of 3 - 8 g/kg body weight (FAO/WHO, 1973b); even this low toxicity might be attributable to the impurities rather than to the pure compound. An analogous situation certainly pertains concerning the potentiation of malathion (see below).

6.3.4.2 Impurities potentiating the toxicity of the major ingredient

There does not appear to be any indication that the MFO status of animals is different after administration of technical grade organophosphorus insecticides compared with pure. Alterations of the MFO status of animals because of diet, sex, drugs, etc., discussed in sections 6.3.2, 6.3.3, and 6.3.5, are unpredictable in their effect on the LD50. In contrast, inhibition of the esteratic capacity of mammals increases the toxicity of pesticides that depend principally on tissue esterases in their metabolism (section 4.3). Malathion is a notable example of an organophosphorus pesticide in which the toxicity is enhanced when tissue esterases are inhibited. Until comparatively recently, such inhibition was only known in situations where an unrelated organophosphorus ester was administered to test animals a short time before the malathion. However, it is now known that several impurities present in most samples of malathion prepared for use as pesticides, are capable of inhibiting tissue carboxylesterases. Some of these impurities act very rapidly and so prevent the normal metabolism of malathion and potentiate its toxicity. This enhanced toxicity has been expressed in man. Several hundred spray workers were intoxicated while spraying certain formulations of malathion in Pakistan, and 5 died (Baker et al., 1978). Isomalathion and several trimethyl phosphorothiolates are found in most commercial preparations of malathion, but the levels depend markedly on the formulation and on storage conditions. The potentiating power of small amounts of these impurities are shown in Table 8. Examination of samples of formulated malathion, known to be unusually toxic, showed a fair correlation of toxicity only with the percentage content of isomalathion (Aldridge et al., 1979; Miles et al., 1979). However, the correlation was imperfect when a large number of samples were examined and the addition of known further amounts of pure isomalathion to the formulated samples caused more than the expected potentiation (Aldridge et al., 1979). The authors concluded that, although isomalathion contributed the main effect, there was also significant potentiation by other agents present in the samples; the chief candidate was O,S,S -phosphorodithioate.

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Most unformulated samples of technical grade malathion seem to have an LD50 for rats in the range of 1500 - 2000 mg/kg body weight. Such material contains some potentiating impurities (Pellegrini & Santi, 1972; Umetsu et al., 1977), but has proved acceptable as a basis for formulated insecticides with little toxic hazard. However, it is now clear that some formulated samples increase markedly in both their impurity content and toxicity, when they are stored at elevated temperatures. Not only are temperature and time important, but also the formulating agents (Table 9), and almost half of the malathion lost from Formulation C is apparently transformed to isomalathion with a massive potentiation, whereas the increase in toxicity is less marked in Formulations A and B, in which a much smaller proportion of lost malathion is converted to isomalathion.

Table 8. Potentiation of acute oral toxicity for rats by impurities added to malathion ------Compound Amount Potentiation Purified Reference added added ratio found malathion (%) used (LD50 mg/kg body weight) ------isomalathion 0.4 3 10 700 Aldridge et al. 0.6 5 (1979) 2 12 8 25 0.05 3 12 500 Umetsu et al. 0.1 4 (1977) 0.5 6 2 10

O,S,S -trimethyl 0.15 3 10 700 Aldridge et al. phosphorodithioate 0.3 4 (1979) 0.5 8 1.0 13 2.0 20 0.05 4 12 500 Umetsu et al. 0.2 6 (1977) 0.5 7 0.035 2 8000 Pellegrini & 0.1 3 Santi (1972) 0.2 4 0.5 7

O,O,S -trimethyl 0.3 2 10 700 Aldridge et al. phosphorothioate 1.3 4 (1979) 0.2 3 12 500 Umetsu et al. 1 4 (1977) 0.2 3 8000 Pellegrini & 0.5 4 Santi (1972)

O,O,S -trimethyl 1.5 2 10 700 Aldridge et al. phosphorodithioate 5 5 (1979) 1 4 12 500 Umetsu et al. 5 5 (1977) 3.5 2 8000 Pellegrini & 4.5 3 Santi (1972) ------

There is much evidence that potentiation of malathion by extraneous compounds is associated with the inhibition of

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carboxylesterases. Using malathion as specific substrate, Talcott et al. (1979b) showed that isomalathion and O,S,S -trimethyl phosphorodithioate were potent inhibitors of rat liver and plasma malathion carboxylesterase, in vitro and in vivo; a partially- purified sample of carboxylesterase from human liver was also sensitive to isomalathion (Talcott et al., 1979a).

The same hazard exists with impure samples of phenthoate as for malathion. Pellegrini & Santi (1972) showed that technical samples containing 61 - 91% of the principal ester had LD50s (rat oral) of 78 - 243 mg/kg body weight, while a purified preparation (98.5%) had an LD50 of 4700 mg/kg, though its toxicity for insects increased approximately in proportion to the purity. The principal potentiating impurities were the S -methyl isomer and the identical trimethyl phosphorothiolates found in malathion. For phenthoate, as for malathion, the vulnerability to potentiation lies in the presence of the hydrolysable ethoxycarbonyl ester bond which, in pure samples, is the key to low mammalian toxicity.

Table 9. Effects of formulating agents and of storage time and temperature on composition and toxicity of malathion (50% wdp)a ------Storage conditions Composition (%) Oral LD50 Temperature Time Malathion Isomalation (rat) (°C) (days) ------Formulation A

0 48.0 0.38 2800 38 60 45.5 0.37 2230 90 44.2 0.49 1740 55 6 47.6 0.30 2520 13 46.4 0.37 1760 90 1 40.9 0.69 950

Formulation B

0 48.8 0.18 2540 38 60 47.2 0.79 1130 90 46.5 0.81 1330 55 6 45.2 0.67 1200 13 43.6 0.55 1170 90 1 39.9 0.32 1900

Formulation C

0 50.6 0.61 2660 38 90 44.9 3.7 590 55 6 46.2 3.4 535 13 43.7 3.5 555 ------a From: Miles et al. (1979).

The presence of the S -methyl isomer (0.32%) in a commercial fenitrothion formulation was shown by Miles et al. (1979). The concentration increased to > 1% during accelerated storage tests, but there was no concomitant increase in the toxicity of the formulation. This observation on a compound not heavily dependent on esterases for the primary step of detoxication, points to the need to consider biochemical mechanisms in assessing possible hazards. There need be no general concern about the presence of small amounts of isomers in organophosphorus pesticides. The

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presence of isoparathion in parathion is well-known, but there is no suggestion of any marked change in toxicity brought about by this impurity.

Some organophosphorus pesticides contain carboxylamide bonds rather than carboxyester. These include dimethoate, dicrotophos, , phosphamidon, and acephate. There appears to be little evidence that impurities in commercial formulations of any of the above pesticides markedly alter the toxicity, apart from a small (1.6x) decrease in the mammalian toxicity of acephate, after storage for 6 months at 40 °C; the insecticidal activity of the compound was unchanged (Umetsu et al., 1977). During the storage period, the concentration of various impurities changed, but no relationship between these changes and altered toxicity was obvious.

6.3.5 Effects of other pesticides and of drugs

All organophosphorus and carbamate insecticides exert their acute toxic action by attack on the AChE. Thus, it follows that exposure to more than one such pesticide will usually produce at least an additive effect. Besides this simple effect, other pesticides or drugs may also influence the toxicity of an individual organophosphorus pesticide by interfering with its metabolism, activation, and disposal.

Not all organophosphorus pesticides and probably no carbamate pesticides inhibit tissue carboxylesterase to potentiate malathion in the manner discussed in section 6.3.4. However, like all other enzymes, the carboxylesterases have their own structure-activity pattern: this has not been worked out in a systematic fashion. It is clear that, with some compounds, profound inhibition of liver carboxylesterase can be achieved without inhibition of AChE sufficient to cause signs of poisoning. This class includes many thioalkyl esters such as the trimethyl esters and also S,S,S -tri- n -butyl phosphorotrithioate (DEF), which are potent potentiators of malathion toxicity via carboxylesterase inhibition. EPN is a phenylphosphorothioate insecticide that also acts in this way. Tables 10 and 11 show examples in which measurements of effects on tissue carboxylesterases and AChE are of value in predicting the potentiation of malathion toxicity (Murphy, 1969).

Table 10. Comparison of enzyme inhibition caused by several pesticidesa ------Insecticide Dietary concentration (mg/kg) resulting in (period) 40 - 60% inhibition of: Red cell- Liver- Plasma- cholinesterase malathionase malathionase ------Parathion 3 5 5 (7 days) Fenchlorphos 500 30 30 (7 days) Malathion 500 100 500 (30 days) ------a From: Murphy (1969).

Table 11. Effect of feeding fenchlorphos on in vivo anticholinesterase activity of malathiona ------Fenchlorphos Malathion Inhibition of brain concentration challenge dose AChE 1 h after

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in diet (mg/kg) (mg/kg ip) challenge (%) ------0 200 13

30 0 1

30 200 61 ------a From: Murphy (1969).

Potentiation of the toxicity of organophosphate compounds for mammals not containing a carboxylester function, does not appear to be a significant hazard, though it is possible that potentiation of some carboxylamide pesticides by an analogous inhibition of tissue amidases may occur; certainly, EPN potentiates the toxicity of dimethoate (El-Sebae, 1980). Potentiation of the toxicity of organophosphorus pesticides for insects by inhibition of MFO activity is well-known, and many potent synergists are used in agriculture for this purpose (Wilkinson, 1971), but much less has been reported concerning similar effects in mammals. This may reflect the greater versatility of mammals compared with insects in disposing of organophosphorus esters (section 4.3). Competition for one metabolic route within the animal often does not greatly alter its total capacity to deal with a foreign compound. Keplinger & Deichmann (1967) combined pairs of various pesticides in proportion to their oral LD50s, determined individually, and then measured the oral toxicity of the mixtures in rats or mice. They calculated a ratio of expected LD50/observed LD50 where "expected LD50" was the sum of half the LD50 value of each of the 2 constituents. Their study included 7 chlorinated hydrocarbons, the carbamate , and 5 organophosphates including diazinon, malathion, and parathion. With a "no-effect" ratio of 1.0, they considered measured ratios greater than 1.75 or less than 0.57 as probably significant of real effects. In a number of cases, less than additive effects were noted for combinations of a chlorinated hydrocarbon and an organophosphate, e.g., + diazinon (0.55), DDT + malathion (0.54), + carbonylfenthion (0.54): this might well be expected if the mode of toxicity were different and metabolic pathways were not markedly altered. The only cases of potentiation involving organophosphates were also not surprising. A triple combination of parathion, malathion, and had a ratio of 1.99. This effect was probably due to simple potentiation of malathion by parathion, since chlordane alone did not potentiate either organophosphorus compound. Mixtures of Aramite (sulfurous acid 2-chloroethyl 2-[4-(1-1-dimethylethyl)-phenoxyl-1-methylethyl ester) with several organophosphates in mice had ratios of 1.86 - 2.14. However, since the LD50 of Aramite in mice is high (2000 mg/kg body weight), it may be that the 500 mg/kg administered in a mixture was absorbed more efficiently and was therefore more effective proportionately than the much higher LD50 dose of Aramite alone.

As noted above, aldrin had little effect on the toxicity of organophosphorus insecticides, when administered at the same time. However, a number of chlorinated hydrocarbons (aldrin, DDT, chlordane, etc.) are well-known as stimulators of MFO activity in (principally) the mammalian liver. This activity increased markedly during a period of a few days after dosage, and pretreatment of mice with aldrin (16 mg/kg body weight), 4 days before a challenge dose of 6 different organophosphates, markedly reduced (up to 5-fold) the toxicity of each (Murphy, 1969). Similar results were obtained with other classes of MFO inducers such as phenobarbital. Murphy points out that the mechanism of these effects probably includes stimulation of liver

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carboxylesterase as well as MFO.

From the observations above, it appears that simple mixing of an organophosphorus insecticide with a chlorinated hydrocarbon is unlikely to adversely influence the acute toxicity for mammals, as expressed by LD50 value. However, administration of DDE at 55 mg/kg diet to adult male Japanese quail led to an increasing susceptibility to challenge doses of parathion (2.5 mg/kg) administered orally; mortality in these birds increased from 0 to 30% after 1 week of feeding and 60% after 3 weeks (Ludke, 1977).

The problem of potentiation by some organophosphates of the toxicity of pesticides containing carboxyl ester bonds may be significant. This has been demonstrated for malathion in animals (section 6.3.4) and in man (section 7.1.3). It may also be a problem for insecticides for most of which degradation by mammalian carboxyesterases is a significant detoxification pathway (Miyamoto, 1976).

6.3.6 Species

No clear ranking of species sensitivity to organophosphorus pesticides as a class can be given. A general impression is that mice, hamsters, and guinea-pigs may be more sensitive than rats, with respect to a number of compounds, but the converse is seldom true. However, most available data have been produced with little reference to conditions of husbandry, diet, hormonal status, etc., so that only very marked differences, which do not usually seem to exist, would emerge. Birds tend to be more sensitive to organophosphorus pesticides (Schafer, 1972) and amphibians less sensitive than mammals. It has been suggested, but not confirmed, that these differences might be due to differences in the activity of enzymes in species that hydrolyse organophosphorus compounds and thereby contribute to detoxification.

The toxicity of several organophosphorus pesticides for different species seems to be inversely related to the activity of the plasma A esterase, which degrades the pesticide oxon. Such activity is considerably lower in birds than in mammals (Machin et al., 1978). When 14 avian species were compared with 5 mammalian, the average plasma activity against pirimiphos-methyl oxon was 170 times less, and that against paraoxon, at least 13 times less (Brealey et al., 1980). Differences in liver microsomal oxidative activity involved in the metabolism of several organophosphorus pesticides by mammals and birds were less profound, though fish liver was less active (Miyamoto & Ohkawa, 1978).

A multitude of factors contribute to the great difference between the oral toxicity of chlorofenvinphos for the rat (10 mg/kg body weight) and that for the dog (> 5000 mg/kg). These include efficiency of absorption, at least 2 metabolic detoxification processes, the rate of uptake by the brain, and a 7-fold difference in the sensitivity of the brain AChE to this compound in the 2 species (Hutson & Hathway, 1967; Donninger, 1971).

Adult hens, cats, dogs, and larger farm animals are all susceptible to organophosphorus delayed neurotoxicants (Davis & Richardson, 1980; Johnson, 1982a). There is no clear ranking of dose-sensitivity, though hens seem to be most uniformly responsive. The full clinical response is not easily seen in laboratory primates and rodents, though morphological damage may be detected (section 6.1.1.2).

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6.3.7 Other factors

The effects of solvents on chemical stability and isomerization reactions were noted in section 4.5. The effects of formulation agents on stability and, therefore, on the toxicity of malathion have also been noted previously (section 6.3.4). It is likely that percutaneous absorption will be greater for liquid formulations than for powders but that powders may adhere longer thereby enhancing an effect, if proper hygiene is not observed. A number of examples are quoted by El-Sebae (1980), in which formulated pesticides were more toxic than the technical preparation (Table 12). In many cases, this is due to the fact that the solvent in the formulation facilitates the uptake of the pesticide into the body. The toxicity of other components of the formulation may play a role (especially in the case of pesticides of very low toxicity, such as tetrachlorvinphos) (Table 12), as well as potentiation. El-Sebae noted that, in some cases, the I50 for inhibition of ChEs in vitro by these compounds differed. This could be relevant in the case of the directly-active oxon-type pesticide tetrachlorvinphos, but in cases where the test was performed with a thioate, the anticholinesterase activity would depend almost entirely on trace oxon impurities, which are often destroyed rapidly in vivo and contribute little to toxicity compared with the bulk of oxon produced metabolically.

Table 12. Comparative toxicity for mice of some technical and formulated organophosphorus pesticidesa ------Pesticide 24-h oral LD50 (mg/kg body weight) Technical Formulated ------phosfolanb 12 11 chlorpyriphos 140 60 leptophos 162 83 tetrachlorvinphos 5000 1800 ------a From: El-Sebae (1980). b (diethoxyphosphinothioyl)dithioimidocarbonic acid, cyclic ethylene ester.

6.4 Acquisition of Tolerance to Organophosphorus Insecticides

This topic has been discussed in section 6.3.1, where it was noted that when AChE levels were reduced progressively over a number of days or weeks, animals showed cholinergic signs of poisoning which, in animals that survived, decreased in severity and sometimes disappeared completely, though ChE inhibition was maintained. This phenomenon is separate from the fact that permanent inhibition of 30 - 50% is ineffective in producing measurable symptoms. The basis for acquired tolerance is not fully known, though a "down " in the muscarinic ACh receptor is thought to be a contributary cause. This involves both reduced sensitivity and reduced numbers of receptors (Costa et al., 1982).

6.5 Therapy of Experimental Organophosphorus Poisoning

The understanding of the mechanism of acute toxicity of organophosphorus pesticides has provided the basis for rational therapy. The effects of inhibition of AChE, as described in

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section 6.1.1, are common to all organophosphorus pesticides intoxications. However, the speed of onset and the rate of unaided recovery from sub-lethal doses vary greatly, depending on the chemical nature of the pesticide, the route of exposure, and on whether this exposure was a sudden overwhelming dose or a drawn- out process.

Factors leading to a slow onset of symptoms include:

(a) Slow absorption or metabolic activation: this is often associated with extremely low and therefore with the presence of large hydrophobic groups in the ester molecule; pesticides such as haloxon, chlorpyrifos, and leptophos are of this type;

(b) Persistence in the system of a comparatively stable inhibitor of ChEs. This could be as a low concentration of an active inhibitor such as demeton- S -methyl sulfoxide or high concentrations of a weak inhibitor such as methamidophos.

Factors leading to rapid clearance of symptoms include:

(a) Rapid clearance of the pesticide and its active agents, as with trichlorfon and dichlorvos; rapid clearance occurs also with nerve agents such as soman and sarin, which have been much used in studies on therapy in experimental animals.

(b) A slow rate of aging of inhibited AChE giving opportunity for reactivation (spontaneous or induced) to occur; diethyl phosphorylated AChE ages more slowly than dimethyl or diisopropyl.

(c) Rapid spontaneous reactivation of inhibited AChE, such as occurs after inhibition by all dimethyl or bis-2- chloroethyl phosphates. In this case, the possibility of reinhibition by a persistent compound will affect the picture.

The factors noted above, which influence speed of onset and remission of effects, influence the prognosis for response to therapy but do not markedly alter the nature of optimal treatment. Maximum benefit comes from combined treatment with an drug (usually atropine) plus a reactivator of inhibited ChE (an oxime) with diazepam, and also artificial respiration. The effects of the components will be discussed individually below.

6.5.1 Palliation

Artificial respiration alone can be very effective in maintaining life, since the primary cause of death in organophosphorus poisoning is respiratory failure (section 6.1.1). Such treatment gains time for the processes, natural or imposed, that lead to the return of sufficient ChE activity to maintain life.

6.5.2 Antagonism of effects of ACh

Atropine antagonizes many of the peripheral muscarinic effects of excess ACh and also some central effects. However, there was no correlation between the peripheral anticholinergic activity of a range of atropine-like drugs and their capacity when used alone (or

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in conjunction with oximes) (section 6.5.3) to protect against the lethal effects of sarin (Coleman et. al., 1962; Brimblecombe et. al., 1970). Moreover, the ranking of the therapeutic efficacy of atropine analogues varied according to the test species (rat, mouse, or guinea-pig), and all effects were small (protective ratio < 1.5) in mice and guinea-pigs, but much larger and more variable (1.2 - 9.3) in rats. The ranking changed yet further when the drug was combined with oxime P2S (see below), though the protective ratio with some compounds rose to 18 - 24 in guinea-pigs, 9 - 80 in rats, but only 1.8 - 6.3 in mice. These confusing effects are now thought to be at least partially due to an anticonvulsant effect contributed variously by the atropine analogues. Anticonvulsants often supplement the effects of atropine or of combined atropine/oxime therapy (see below). In particular, diazepam (valium) is known both to raise the LD50 and speed recovery in some cases (Johnson & Wilcox, 1975). These authors implied that the mechanism might be partly direct antagonism to some central effects of ACh and partly indirect. When diazepam is included in the therapeutic package, there appears to be little evidence that alternative anticholinergic drugs to the well-proved atropine are superior (Green et al., 1977). It has been reported that, in prophylaxis against the toxicity of DFP in mice, the protection factor was 28 when atropine and were used but 180 when Dexetimide (a drug with strong central anticholinergic activity) was substituted for atropine. The particular advantage claimed for this drug was that, in the therapy of rabbits intoxicated with up to 60 x LD50 of paraoxon or 80 x LD50 of DFP, one single intravenous injection of Dexetimide (8 - 16 mg/kg body weight) was effective in conjunction with obidoxime, whereas repeated doses of atropine were necessary (Bertram et al., 1977). Dexetimide is used for the treatment of parkinsonism and is available commercially. However, Dexetimide has not been evaluated in conjunction with diazepam or compared with atropine plus diazepam, and no details were given of the hazards of its use and side-effects in control animals.

6.5.3 Reactivation of inhibited AChE

As indicated in section 4.5.1, inhibited ChEs can be reactivated in vitro by treatment with appropriate nucleophilic agents, of which of the oxime N- methylpyridinium-2-aldoxime are the most commonly used (the is known as and the methanesulfonate as P2S). In some countries, obidoxime (ToxogoninR ), which is a bis-quaternary oxime, is recommended at slightly different doses than pralidoxime, but the mode of action is similar. The scope for further improvement in the design of therapeutic oximes is discussed by Gray (1984).

In vivo, there are 2 limitations to the benefits to be obtained from the use of these agents:

(a) Access

The quaternary oximes are thought not to cross the blood-brain barrier easily (Taylor, 1980). However, some experimental work, summarized by Lotti & Becker (1982b), suggests that there may be limited access, and this may have a significant, albeit small, effect in reversing inhibition of AChE to improve the clinical state. Other more direct beneficial action of the oximes directly at in the medullary respiratory centre cannot be ruled out. The prompt improvement in the level of consciousness observed and in the EEG of an intoxicated child

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when iv infusion of 2-PAM was commenced (Lotti & Becker, 1982b) also seemed to indicate that there was some access to important brain regions. It has been claimed that obidoxime (25 mg/kg body weight) injected intraperitoneally in rats, 5 min after a dose of armin, was effective in reactivating 33% of the inhibited AChE of the ponto-medullary region, which contains the centre for control of respiration (Vasic et al., 1977). However, there were no controls appropriate to disprove the alternative explanation that the oxime had altered the circulating level of armin (by direct destructive interaction or otherwise), which, although interesting, is unlikely to be relevant to therapy instituted later after dosing, nor is such destruction-protection unique to toxogonin.

(b) Aging of inhibited AChE

As noted in section 4.5.1, inhibited AChE is converted by a time-dependent reaction to a form resistant to reactivators. Thus, oxime therapy becomes less effective with time after poisoning. The rate of aging of dialkyl-phosphorylated AChE is Me > IsoPr > Et (O'Brien, 1967), but little has been published on the rate of aging when phosphonyl groups derived from pesticides are attached to the enzyme. When one or both of the residual alkyl groups are attached to phosphorus through sulfur rather than oxygen, the rates of both spontaneous reactivation and of aging of inhibited bovine erythrocyte AChE are markedly increased (Clothier et al., 1981). Ethoprophos is one of the newer pesticides that contain such a residual alkylthio group; no published studies are known of therapy after poisoning with this or related pesticides.

6.5.4 Efficacy of therapy

As reported above, the combination of atropine plus oxime is far more effective in most cases than the mere summed effects. This is because the peripheral neuromuscular junctions (particularly the diaphragm) and the sympathetic ganglia, where oximes reactivate AChE, are nicotinic and are unaffected by atropine, so that separate aspects of intoxication are treated by the two agents. There is speculation that some oximes may exert a protective effect by acting as depolarizing agents at the . This may also account for the slight therapeutic effects of some analogues of toxogonin which do not have any nucleophilic oxime group or reactivating power (Schoene & Oldiges, 1973). Diazepam, also, is ineffective, except in combination with atropine and oxime.

Persistence of the toxic agent may interfere with successful therapy. Thus, single doses of atropine + oxime were of only marginal efficacy in altering the LD50 of isofenphos in rats (FAO/WHO, 1982b). However, when therapeutic doses were given repeatedly at about 12-h intervals, for 2 - 3 days, the LD50 for rats was raised about 4-fold; for hens, the increase was 15-fold (Wilson et al., 1984). It appears that the failure of one-shot therapy in this case was due to persistence of the toxic agent rather than to a different mode of intoxication or to formation of an inhibited form of AChE that resisted reactivation. The same situation may pertain for profenofos intoxication, which appears not to respond well to therapy (El-Sebae, personal communication, 1985).

7. EFFECTS ON MAN

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7.1 Acute Cholinergic Poisoning

The clinical picture of organophosphorus intoxication results from accumulation of ACh at nerve endings. The syndrome is described in detail in several major references (Namba et al., 1971; Kagan, 1977; Taylor, 1980; HMSO, 1983; Plestina, 1984). The symptoms can be summarized in three groups as follows:

(a) Muscarinic manifestations

- increased bronchial secretion, excessive sweating, salivation, and lachrymation;

- pinpoint pupils, bronchoconstriction, abdominal cramps ( and diarrhoea); and

- bradycardia.

(b) Nicotinic manifestations

- fasciculation of fine muscles and, in more severe cases, of diaphragm and respiratory muscles; and

- tachycardia.

(c) Central nervous system manifestations

- headache, dizziness, restlessness, and ;

- mental confusion, convulsions, and coma; and

- depression of the respiratory centre.

All these symptoms can occur in different combinations and can vary in time of onset, sequence, and duration, depending on the chemical, dose, and route of exposure. Mild poisoning might include muscarinic and nicotinic signs only. Severe cases always show central nervous system involvement; the clinical picture is dominated by respiratory failure, sometimes leading to pulmonary oedema, due to the combination of the above-mentioned symptoms.

Clinical diagnosis is relatively easy and is based on:

(a) medical history and circumstances of exposure; and

(b) presence of several of the above-mentioned symptoms, in particular, bronchoconstriction and pinpoint pupils not reactive to the light. Pulse rate is not of diagnostic value, because the AChE effects on the heart reflect the complex innervation of this organ. On the other hand, since changes in the conduction and excitability of the heart might be life- threatening, monitoring should be performed.

Confirmation of diagnosis is made by measurement of AChE in RBC or plasma-pseudoChE, and, also, of the dibucaine number (to rule out genetic deficiencies).

Measurements of blood-ChE during therapy are also useful in assessing the treatment with oximes, though there might not be a correlation between the severity of symptoms and the degree of ChE inhibition: comparison should be made with pre-exposure levels, wherever possible.

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Chemical analysis of body fluids (urine, blood, gastric lavage) should be made in order to identify the compounds that caused poisoning.

7.1.1 Methods for assessing absorption and effects of organophosphorus insecticides

As well as assessments of general health and behaviour, the study of the effects of this class of pesticides is favoured compared with that of some other classes since the basic biochemical mechanisms (inhibition of esterases) are known for the major toxic effects. Biochemical and neuro-physiological techniques, relevant to the principal effects of all the compounds, have been established. Identification of the monobasic acid type of urinary metabolite, which is commonly produced, is an indicator of exposure rather than of an effect, but it seems appropriate to outline the technique in this sub-section. Wherever possible, test findings should be compared with pre-exposure measurements on the same individual.

7.1.1.1 Analysis of urine as a means of monitoring exposed populations

As previously discussed in section 4, organophosphorus pesticides may undergo hydrolysis in vivo to yield substituted phosphoric acids that are subsequently excreted in urine. Advances in gas chromatography and combined gas chromatography/mass spectrometry (CG/MS) have made it possible to analyse the urine of exposed persons for the presence of appropriate metabolites. It is usually necessary to preserve the sample by the addition of chloroform, to concentrate or extract the metabolite(s), and to convert them to suitably-volatile derivatives that can be detected by GC. Obviously, access to a well-equipped analytical laboratory, capable of the quick processing of samples, is a necessary factor if monitoring by urine analysis is proposed. However, in some cases, simpler and sensitive colorimetric tests are available for screening the urine of exposed persons. Thus, 4-nitrophenol can be measured directly in the urine of workers exposed to parathion (Wolfe et al., 1970).

Consideration of the concentration of metabolite(s) in the urine can be helpful in determining patterns of exposure, and these concentrations can be calibrated against the effects on AChE for a particular pesticide. However, the time-course and peak of excretion of metabolites appears to vary according to dose (Bradway et al., 1977), so that serial sampling and analyses of urine are desirable. Levels of metabolite alone cannot be considered a guide to hazard. This is obvious when it is realized that pesticides that have very different toxicities may yield identical acidic metabolites. Thus, the level of metabolites in urine, after exposure to sufficient amounts of the very toxic parathion-methyl to depress blood-AChE to 50%, will be much lower than that of the identical metabolites, following exposure to the related fenitrothion, which is about 40 times less toxic.

7.1.1.2 Biochemical methods for the measurement of effects

AChE is present in human erythrocytes (RBC) and is the same as the enzyme present in the target synapses. Thus, levels of AChE in RBC are assumed to mirror the effects in the target organs. However, it must be borne in mind that this assumption is only correct when the organophosphate has equal access to blood and synapses. In the case of acute poisoning, a high inhibition of

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RBC-AChE is pathognomonic, but, in the follow-up of the intoxication, it might not be correlated with the severity of symptoms. In the case of repeated exposures, additional difficulties in interpretation arise from possible development of tolerance. However, monitoring of pre- and post-exposure levels of AChE in RBC gives a good measure of the effects of an exposure (Kaloyanova, 1975). In cases where the pre-exposure AChE level is not known (as in accidental poisoning), reference can be made to a mean population AChE activity. Blood-plasma contains a related enzyme called ChE or pseudoChE, which contributes to the whole- blood enzymatic activity; the contribution of plasma-ChE in assays of AChE will depend on the type and concentration of the substrate used. PseudoChE has no known physiological function and can be inhibited selectively by some compounds without causing a toxic response. The sensitivities of AChE and ChE to inhibitors differ, so that measurements of the ability of whole-blood samples to hydrolyse the usual analytical substrates give only an approximate estimate of the activity of the erythrocyte-AChE. However, under many kinds of field conditions, procedures using whole blood, are more practical than those using separated erythrocytes. Quite commonly, pseudoChE is more sensitive to inhibitors. Thus, if separation of plasma and erythrocytes is possible, prior to assay, an indication of exposure can be obtained by assay of pseudoChE only. Examples of selected organophosphorus insecticides, arranged according to their ability to inhibit preferentially either plasma or red cell-ChE in man, are given in Table 13 (Hayes, 1982).

Table 13. Selected organophosphorus insecticides arranged according to their ability to inhibit either plasma- or red cell-cholinesterase in mana ------Plasma enzyme more inhibited RBC enzyme more inhibited ------Chlorpyrifos Demeton Mevinphos Diazinon Parathion dichlorvos Parathion-methyl malathion Mipafox Trichlorphon ------a Modified from: Hayes (1982).

ChE assay procedures vary greatly in sophistication, but the most satisfactory is that based on the procedure of Ellman et al. (1961). A field method and kit for whole blood- and plasma-ChE determination have been developed (WHO, 1984b). Quick methods exist for the determination of ChE in serum using paper tests (Izmirova, 1980) and for the colorimetric determination of ChE in whole blood (Tintometer): these may be useful in the differential diagnosis of organophosphate poisoning. Interpretation of the test results is discussed in section 7.1.2.

The potential for delayed neuropathic response to an organophosphorus ester can be predicted by the assay of the esteratic activity of the target protein (NTE), in autopsy brain samples from dosed adult hens (section 6.1.2). It has been shown (Dudek et al., 1979; Richardson & Dudek, 1983) that a low level of similar enzyme activity resides in lymphocytes and that there may be correlations under some circumstances between neurotoxic dose and available lymphocyte enzyme activity. The possibility of monitoring exposed individuals by means of human lymphocyte or platelet NTE activity is being explored (Lotti et al., 1983; Bertocin et al., 1985; Maroni & Bleeker, 1986).

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7.1.1.3 Electrophysiological methods for the study of effects

Electromyographic (EMG) studies using non-invasive surface electrodes have been claimed to give sensitive indications of exposure to organophosphorus pesticides, even in situations where blood-ChE activity has returned to normal levels (Jager et al., 1970; Roberts, 1976). The method requires electro-physiological equipment and a very skilled practitioner. There is still considerable doubt about the validity of some published studies. Reproducibility is known to be very sensitive to local factors such as temperature of the skin, and conflicting results have been published, some of which show small increases and some, small decreases in the ampli-tude of evoked muscle , in response to nerve stimulation. These findings have been reviewed by LeQuesne & Maxwell (1981), who noted that changes that have been reported tended not to be dose-related. In addition, they evaluated the technique under controlled circumstances. In a treatment to eradicate parasitic schistosomes, 55 children were dosed orally with trichlorfon, 3 times, at 2-weekly intervals, at doses that measurably depressed blood-ChE (mean 50%), but were not enough to cause overt toxic effects, apart from mild cramps and diarrhoea in a few cases. Only 3 children showed a significant alteration in electromyographic response. Shortly after the last (and highest) dose of 10 mg/kg body weight, 3 children developed repetitive activity recorded over the thenar muscles following supramaximal stimulation of the median nerve at the wrist. The activity consisted of a small potential at the end of the main muscle response and was characterized by being abolished by a second stimulus 30 or 80 milliseconds after the first, or by maximum voluntary contraction for 10 seconds; the amplitude of the response to the second stimulus was not reduced. These characteristics are necessary criteria that distinguish (these) dose-related responses from pre-existing natural (and idiosyncratic) responses, which can otherwise confuse EMG studies in a population. Changes in amplitude measured on 52 control subjects (mean 13.8 ± 2.5 SD), on 2 occasions (2 weeks apart), ranged from +5 to -3 mV. Thus, EMG does not appear to give a highly sensitive measure of exposure to an ingested organophosphorus compound.

7.1.2 Monitoring studies

Measurement of whole blood-AChE is the most widely adopted method for monitoring the effects of occupational exposure to organophosphorus insecticides. Physiological variations in blood- ChE levels occur in a healthy person and are seen among a population. It has been estimated that the coefficient of variation for AChE activity in samples from an individual is 8 - 11%, and that a decrease of 23% below pre-exposure level may, therefore, be considered significant. If the average of several pre-exposure values were available, then a decrease of 17% would be significant. It has been recommended that, if measured activity is reduced by 30% or more of the pre-exposure value, AChE measurements should be repeated at appropriate intervals to confirm the results. Depressions of AChE or ChE in excess of 20 - 25% are considered diagnostic of exposure but not, necessarily, indicative of hazard. Depressions of 30 - 50% or more are considered indicators for removal of an exposed individual from further contact with pesticides until levels return to normal. Work procedures and hygiene should also be checked (Zielhuis, 1972; WHO, 1975; CEC, 1977; Kaloyanova et al., 1979; Plestina, 1984).

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Urinary metabolites have been monitored as a means of comparing the efficiency of absorption of a pesticide by different routes.

The reports of the annual Joint FAO/WHO Working Parties, mentioned previously, contain summaries of numerous controlled exposure studies. No cases appear to be known of significant clinical effects in man in the absence of depression of plasma- or erythrocyte-ChE levels. No-observed-adverse-effect levels have been calculated on this basis, where the data are available, or have been estimated for man by extrapolation of the available data for exposed animals.

7.1.3 Retrospective studies of populations exposed to organophosphorus pesticides: acute and long-term exposure

Many thousands of cases of acute poisoning by organophosphorus pesticides have been recorded (Namba et al., 1971). The majority have been due to parathion and methyl parathion. Thus, Namba (1974) in a discussion of the relative toxicities of parathion and malathion, quoting Japanese Government statistics for the 7 years 1958-62, 1966, and 1967, stated that there were 3311 accidental or occupational poisonings due to parathion including 188 deaths, while for malathion, the numbers were 63 and 10, respectively. He noted that the difference was not due to the restricted use of malathion.

In the context of this general introduction, no descriptions and breakdown will be given of retrospective studies of populations exposed to organophosphorus pesticides. Such figures are relevant to individual substances and will be given in the appropriate Environmental Health Criteria.

It is generally thought that the only long-term effects attributable to overt or subclinical acute intoxication with organophosphorus compounds, or to prolonged low-level exposure, are behavioural (rather doubtful), and delayed-onset neuropathy in the case of certain compounds; these are dealt with in section 7.2.

>7.2 Other Effects on the Nervous and Neuromuscular System Due to Acute or Long-Term Exposure

7.2.1 Delayed neuropathic effects

The characteristics of this disorder are given in section 6.1.2. The agents most commonly causing delayed neuropathy in man are triaryl phosphate esters used in, e.g., hydraulic fluids; these do not have any AChE activity and are not pesticides. In Table 14, organophosphorus pesticides are listed for which there is reasonable evidence that they have caused delayed neuropathy in man.

Table 14. Organophosphorus pesticides reported to cause delayed neuropathy in man ------Pesticide Number Reference of cases ------mipafox 2 Bidstrup et al. (1953) leptophos 8 Xintaras et al. (1978); FAO/WHO (1979b) methamidophos 9 Senanayake & Johnson

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(1982) trichlorphon many Shiraishi et al. (1977); Hierons & Johnson (1978); Johnson (1981a) trichlornat 2 Jedrzejowska et al. (1980); Willems (1981)

EPN 3a Xintaras & Burg (1980)

Chlorpyrifos 1 Lotti & Moretto (1986) ------a Moderate effects only and possibly other etiological factors.

The cases with mipafox involved a single occupational exposure to a compound that was developed before delayed neuropathy was a recognized hazard. The cases with EPN and leptophos arose through repeated occupational exposure with inadequate precautions. Apparently cholinergic effects were often experienced, but not at the level of severe poisoning. A few cases with methamidophos and trichlorfon involved substantial occupational exposure, which caused severe acute poisoning prior to the development of neuropathy, but the majority of cases involved accidental or deliberate ingestion of quantities that might well have been fatal but for medical intervention. The fact that most of the cases listed are due to exposure to phosphonates or phosphoramidates is in line with the structure-activity relationships listed in section 6.1.2.

The value of measuring the neuropathy target esterase of human lymphocytes as a predictive monitor was proposed by Dudek et al. (1979) and Richardson & Dudek (1983). Lotti et al. (1983) reported occupationally related changes in the lymphocyte NTE in spraymen during seasonal spraying of DEF, but overt neuropathy was absent. In a case of self-poisoning by a mixture of pesticides including chlorpyrifos (about 20 g diluted in petroleum distillates), Osterloh et al. (1983) noted that signs of cholinergic poisoning were limited and they attributed death to the effects of chlorophenoxyacetic acids. Brain esterases were not inhibited at autopsy, but erythrocyte and peripheral nerve AChE levels were about 22% of normal and nerve NTE was about 30%. This substantial inhibition of NTE suggested that treated survivors of severe poisoning by chlorpyrifos might well develop delayed neuropathy. This prediction was confirmed in a recent case of self-intoxication with chlorpyrifos (estimated dose 300 mg/kg body weight) in which very low levels of lymphocyte-NTE were found, 30 days after intoxication and after recovery from very severe cholinergic effects. Typical moderate polyneuropathy developed in the following days (Lotti & Moretto, 1985).

Allegations have been made against a few other organophosphorus insecticides including malathion, omethoate, and parathion, though many accidental and intentional poisonings by these agents have not had any neuropathic sequelae. Experimental evidence against a neuropathic potential in these compounds is strong (section 6.1.2). However, in view of the serious paralytic effects involved, the evidence adducing that these pesticides were the causal agents is reviewed below.

(a) Malathion

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Two alleged cases can be discounted. Petry (1958) reported a case in which a physician contaminated himself frequently with chlorinated hydrocarbon pesticides during day-long gardening activities, about once per week, over a 10-year period. In 1954, he commenced using 6% malathion in a "hose-on" device for garden pest control and, in 1955, he commenced using 50% malathion in a hand spray, both indoors and outside. He soon developed signs of chronic anticholinesterase poisoning (generalized weakness and tremor, irritability, difficulty in focusing). He eventually collapsed and his general condition improved in hospital. However, he continued to experience generalized weakness and particular weakness in the right shoulder girdle, right serratus anterior, and both peroneal muscle groups, and these symptoms persisted to some extent for over a year. The distribution of these symptoms of deficient muscle performance is quite atypical for delayed neuropathy and seems more likely to be due to prolonged moderate cholinergic insult from malathion precipitating weakness, anorexia, and weight loss, which then precipitated further ill-health as previously-stored chlorinated hydrocarbons was mobilized from degraded fat stores. DDT at 23 mg/kg plus a high level of organic chloride were found in a subcutaneous fat biopsy. A muscle- necrotising effect, due to prolonged cholinergic stimulation, as described in section 6.2.6, is also possible.

A separate case report of ascending paralysis following malathion intoxication (Healy, 1959) concerned an 18-month-old child exposed daily for 6 weeks to malathion from a garden spray. Contamination was dermal and also by inhalation and ingestion, leading ultimately to a cholinergic crisis and a prolonged period of weakness including extensive flaccid paralysis for several days. The condition responded to atropine and rest within 4 weeks. In spite of the author's conclusion that this was a "demyelinating" (i.e., delayed neuropathic) disorder, the picture is typical of prolonged cholinergic insult responding to atropine and the comparatively slow clearance of accumulated pesticide with recovery from excessive nerve-muscle stimulation.

(b) Omethoate

A typical delayed neuropathy followed ingestion of an organophosphorus pesticide with suicidal intent (Curtes et al., 1979). Identification of the actual pesticide ingested was doubtful depending on a later hearsay report concerning a bottle in a garden shed, the contents of which were not analysed. Lotti et al. (1981) assayed both the AChE and the NTE activities in autopsied brain after a fatal intoxication with omethoate and found that, even at the fatal dose, the NTE levels were normal while the AChE activity was highly inhibited. Considering this data together with evidence of similar non-inhibition in experimental hens at up to 8 x the unprotected LD50, the authors concluded that, though the neuropathy was typical, it was likely that the toxic agent was some other organophosphorus compound more liable to cause neuropathy, such as trichlorfon or trichlornat (Table 13).

(c) Parathion

Only two cases of permanent incapacity have been attributed to parathion, though thousands of parathion poisonings are known (see earlier). Petry (1951) attributed a case of neuropathy to the aftermath of several occupational incidents of cholinergic poisoning, but the history is entirely atypical in that symptoms developed only 4 months, rather than 2 - 4 weeks, after the last exposure. Causation by parathion is therefore very unlikely. A

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farmer deliberately ingested parathion at a dose estimated to be at least 150 g (perhaps 500 x the estimated human lethal dose) in 600 ml of . Vigorous therapy preserved his life, though he remained in a deep coma for 7 weeks. On recovery from this experience, he was found to be suffering from flaccid paralysis of both legs and weakness of both hands with muscle atrophy (De Jager et al., 1981; 1982); partial recovery occurred during one year.

Lotti & Becker (1982b) have discussed the complicating factors of the potentially supra-lethal dose of methanol and of the long coma. However, the clinical picture is not unlike a true moderate organophosphorus-induced neuropathy. At such a colossal dose, the possibility of ingesting a significant amount of a neuropathic impurity in the parathion must be recognized. This could be ethyl bis-(4-nitrophenyl) phosphorothioate; small but significant amounts of the appropriate oxon are present in some samples of paraoxon made from diethyl phosphorochloridate (Johnson, 1982b), and this oxon is a potent inhibitor of NTE.

(d) Other organophosphorus pesticides

Besides the atypical case attributed to malathion, Petry (1958) described another case in which symptoms persisted after a cholinergic crisis that followed severe intermittent exposures over 3 seasons to a variety of insecticides including parathion, EPN, DDT, dieldrin, and lead arsenate. Some of the persistent symptoms might be compatible with a mild peripheral neuropathy. Among the agents used, lead arsenate and dieldrin would be expected to contribute damage to the nervous system and EPN at about the LD50 level causes neuropathy in hens and man (Tables 4, 13).

A case of slow-onset profound weakness with complete recovery within 3 months following contamination of an agricultural worker with the cotton defoliant, merphos ( S,S,S -tri- n -butyl phosphorothioite), was thought by the author to be of the delayed neuropathy type (Fisher, 1977). However, the clinical picture showed signs that are not seen in acute organophosphate intoxication (influenza-like onset of the syndrome and high level of protein in the spinal fluid). These signs are, however, characteristic of a Guillan-Barré syndrome, which might have been coincidental to the merphos exposure only 4 days previously. It is possible that, later, a mild organophosphorus neuropathic effect was superimposed on the Guillan-Barré effect, since merphos can produce neuropathy in experimental animals (Johnson, 1970, 1975b). Recovery from very mild neuropathies is usually complete.

7.2.2 Behavioural effects

Although many epidemiological studies have been carried out, few controlled studies on man have been reported. It is generally recognized that there are behavioural and psychic changes during overt clinical poisoning by organophosphorus insecticides and that these may take several months to regress (Karczmar, 1984). However, there is no information to suggest that effects occur at exposure levels that do not either alter ChE levels or produce physical symptoms. Levin & Rodnitsky (1976) have reviewed the literature, including their own work, on different aspects of behaviour as affected by organophosphates. Much was based on generalized complaints from workers occupationally exposed to many agricultural chemicals (and probably also to automobile fuels and lubricants and to ). In summary, they found that, in human subjects sufficiently exposed to organophosphates to depress plasma- or erythrocyte-ChEs, some or all of the following

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behavioural variables might be impaired. In cognition: vigilance, information processing and psychomotor speed, and memory; in speech: both performance and perception; in psychic state: increased tendencies to depression, anxiety, and irritability; and in EEG records: a tendency to faster frequencies and higher voltages. They also concluded that the EEG abnormalities were positively related to the level of AChE inhibition during the initial stages of inhibition. Concerning studies on asymptomatic workers at risk from repeated exposure to organophosphorus pesticides, they considered that the evidence was equivocal for the presence of less severe or latent forms of any behavioural abnormalities. However, Duffy & Burchfield (1980) claimed that changes in the EEG of individuals accidentally exposed to the nerve-agent sarin could be detected twelve months after the exposure. This study was a sequel to the study on monkeys described in section 6.1.2, and the analyses of EEGs was performed as noted there. They claimed significant differences (very small differences analysed by complex statistical procedures) between the group of sarin-exposed workers and controls, particularly in the region of beta-rhythm (but the comment on the spread among normal monkeys should be noted). However, the authors were unable to pick out sarin-exposed individuals on the basis of the EEG. They also found (small) increased amounts of REM-sleep in the exposed workers. Without controlled exposure and serial monitoring of effects in individuals, little can be deduced from these apparent marginal changes.

7.3 Effects on Other Organs and Systems

Very few effects, other than those described in sections 7.1 and 7.2, have been noted, except those arising from ill health due to severe anticholinesterase effects.

Several adverse effects attributed to only one organophosphorus ester will be listed under the individual substances.

7.4 Treatment of Organophosphate Insecticide Poisoning in Man

All cases of organophosphorus poisoning should be dealt with as an emergency and the patient sent to hospital as quickly as possible. Although symptoms may develop rapidly, delay in onset or a steady increase in severity may be seen up to 48 h after ingestion of some formulated organophosphorus insecticides.

Extensive descriptions of treatment of poisoning by organophosphorus insecticides are given in several major references (Kagan 1977; Taylor 1980; HMSO, 1983; Plestina 1984) and will also be included in the IPCS Health and Safety Guides to be prepared for selected organophosphorus insecticides.

The treatment is based on:

(a) minimizing the absorption;

(b) general supportive treatment; and

(c) specific pharmacological treatment.

7.4.1 Minimizing the absorption

When dermal exposure occurs, decontamination procedures include removal of contaminated clothes and washing of the skin with alkaline soap or with a solution. Particular care should be taken in cleaning the skin area where venupuncture

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is performed. Blood might be contaminated with direct-acting organophosphorus esters, and, therefore, inaccurate measures of ChE inhibition might result. Extensive eye irrigation with water or saline should also be performed. In the case of ingestion, vomiting might be induced, if the patient is conscious, by the administration of ipecacuanha syrup (10 - 30 ml) followed by 200 ml water. This treatment is, however, contraindicated in the case of pesticides dissolved in hydrocarbon solvents. Gastric lavage (with addition of bicarbonate solution or activated charcoal) can also be performed, particularly in unconscious patients, taking care to prevent aspiration of fluids into the lungs (i.e., only after a tracheal tube has been placed).

The volume of fluid introduced into the stomach should be recorded and samples of gastric lavage frozen and stored for subsequent chemical analysis. If the formulation of the pesticide involved is available, it should also be stored for further analysis (i.e., detection of toxicologically relevant impurities). A purge to remove the ingested compound can be administered.

7.4.2 General supportive treatment

Artificial respiration (via a tracheal tube) should be started at the first sign of respiratory failure and maintained for as long as necessary.

Cautious administration of fluids is advised, as well as general supportive and symptomatic pharmacological treatment and absolute rest.

7.4.3 Specific pharmacological treatment

7.4.3.1 Atropine

Atropine should be given, beginning with 2 mg iv and given at 15 to 30-min intervals. The dose and the frequency of atropine treatment varies from case to case, but should maintain the patient fully atropinized (dilated pupils, dry mouth, skin flushing, etc.). Continuous infusion of atropine may be necessary in extreme cases and total daily doses up to several hundred mg may be necessary during the first few days of treatment.

7.4.3.2 Oxime reactivators

Cholinesterase reactivators (e.g., pralidoxime, obidoxime) specifically restore AChE activity inhibited by organophosphates. This is not the case with enzymes inhibited by carbamates. The treatment should begin as soon as possible, because oximes are not effective on "aged" phosphorylated ChEs (section 6.5.3). However, if absorption, distribution, and metabolism are thought to be delayed for any reasons, oximes can be administered for several days after intoxication. Effective treatment with oximes reduces the required dose of atropine. Pralidoxime is the most widely available oxime. A dose of 1 g pralidoxime can be given either im or iv and repeated 2 - 3 times per day or, in extreme cases, more often. If possible, blood samples should be taken for AChE determinations before and during treatment. Skin should be carefully cleansed before sampling. Results of the assays should influence the decision whether to continue oxime therapy after the first 2 days.

The possible beneficial effects of oxime therapy on CNS-derived symptoms is discussed in section 6.5.3.

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7.4.3.3 Diazepam

Diazepam should be included in the therapy of all but the mildest cases. Besides relieving anxiety, it appears to counteract some aspects of CNS-derived symptoms, which are not affected by atropine. Doses of 10 mg sc or iv are appropriate and may be repeated as required (Vale & Scott, 1974). Other centrally acting drugs and drugs that may depress respiration are not recommended in the absence of artificial respiration procedures.

7.4.3.4 Notes on the recommended treatment

(a) Effects of atropine and oxime

The combined effect far exceeds the benefit of either drug singly.

(b) Response to atropine

The response of the eye pupil may be unreliable in cases of organophosphorus poisoning. A flushed skin and drying of secretions are the best guide to the effectiveness of atropinisation. Although repeated dosing may well be necessary, excessive doses at any one time may cause toxic side-effects. Pulse-rate should not exceed 120/min.

(c) Persistence of treatment

Some organophosphorus pesticides are very lipophilic and may be taken into, and then released from, fat depots over a period of many days. It is therefore quite incorrect to abandon oxime treatment after 1 - 2 days on the supposition that all inhibited enzyme will be aged. Ecobichon et al. (1977) noted prompt improvement in both condition and blood-ChEs in response to pralidoxime given on the 11th - 15th days after major symptoms of poisoning appeared due to extended exposure to fenitrothion (a dimethyl phosphate with a short half-life for aging of inhibited AChE).

(d) Dosage of atropine and oxime

The recommended doses above pertain to exposures, usual for an occupational setting, but, in the case of very severe exposure or massive ingestion (accidental or deliberate), the therapeutic doses may be extended considerably. Warriner et al. (1977) reported the case of a patient who drank a large quantity of dicrotophos, in error, while drunk. Therapeutic dosages were progressively increased up to 6 mg atropine iv every 15 min together with continuous iv infusion of pralidoxime chloride at 0.5 g/h for 72 h, from days 3 to 6 after intoxication. After considerable improvement, the patient relapsed and further aggressive therapy was given at a declining rate from days 10 to 16 (atropine) and to day 23 (oxime), respectively. In total, 92 g of pralidoxime chloride and 3912 mg of atropine were given and the patient was discharged on the thirty-third day with no apparent sequelae.

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Annex I. Names and structures of selected organophosphorus pesticides ------Common name Trade or CAS chemical name Molecular Relative other formula molecular name mass ------acephate Orthene phosphoramidothioic C4 H10 NO3 PS 183.18 Ortran acid, acetyl-, O,S- dimetyl ester

amidithion Thiocron phosphorodithioic C7 H16 NO4 PS2 273.33 acid, O,O -dimethyl S -[2-[(2-methoxy- ethyl)amino]-2-oxo- ethyl/ester

amiton Citram phosphorothioic C10 H24 NO3 PS 269.38 Inferno acid, S -[2-(diethyl- Metramac amino)ethyl] O,O -di- Tetram ethyl ester

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative

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other name formula molecular mass ------azinophos- Cotnion- phosphorodithioic C12 H16 N3 O3 PS2 345.4 ethyl ethyl acid, O,O -diethyl- Gusathion S -[[4-oxo-1,2-benzo- Ethyl, triazin-3(4H)-yl] Guthion methyl] ester ethyl, Trazotion (Russian)

azinophos- Cotnion- phosphorodithiotic C10 H12 N3 O3 PS2 317.34 methyl methyl acid, O,O -dimethyl- gusathion S -[[4-oxo-1,2,3- guthion benzotriazin-3(4H)- metiltr- yl]methyl] ester azotion

azothoate alamos phosphorothiotic C14 H14 ClN2 O3 PS 356.78 acid, O -[4-[(4- chlorophenyl)azo] phenyl] O,O -dimethyl ester

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------bromophos netal phosphorothioic C8 H8 BrCl2 O3 PS 366.0 nexion acid, O -(4-bromo- 2,5-dichlorophenyl) O,O -dimethyl ester

bromophos- nexagan phosphorothioic C10 H12 BrCl2 O3 PS 394.06 ethyl acid, O -(4-bromo- 2,5-dichlorophenyl) O,O -diethyl ester

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butonate tribufon butanoic acid, C8 H14 Cl3 O5 P 327.54 2,2,2-trichloro-1- (dimethoxyphosphi- nyl) ethyl ester

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------chlorfen- birlane phosphoric acid, C12 H14 Cl3 O4 P 359.58 vinphos sapecron 2-chloro-1-(2,4- supona dichlorophenyl) ethenyl dimethyl ester

chlorpyri- dursban phosphorothioic C9 H11 Cl3 NO3 PS 350.59 fos lorsban acid, O,O -diethyl O -(3,5,6-trichloro- 2-pyridinyl) ester

chlorpyri- fospirate phosphorothioic C7 H7 Cl3 NO3 PS 322.53 fos reldan acid, O,O -dimethyl methyl zertell O -(33,5,6-trichloro- 2-pyridinyl) ester

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------coumaphos agridip phosphorothioic C14 H16 Cl05 PS 326.78

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asunthol acid, O -(3-chloro- co-ral 4-methyl-2-oxo-2H- meldane 1-benzopyran-7-yl) muscatox O,O -diethyl ester resistox suntol

crotoxy- ciodrin 2-butenoic acid, C14 H19 O6 P 314.3 phos 3-[(dimethoxyphos- phinyl)oxy]-, 1-phenylethyl ester

crufomate montrel phosphoramidic C12 H19 ClNO3 P 291.74 ruelene acid, methyl-, 2-chloro-4(1,1- dimethylethyl)- phenyl methyl ester

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------demeton- metasystox phosporothioic C6 H15 O3 PS2 230.3 S -methyl methyl acid, S -[2-(ethyl- isosystox thio)ethyl] O,O - dimethyl ester

diazinon basudin phosphorothioc C12 H21 N2 O3 PS 304.38 dazzel acid, O,O -diethyl diazajet O -[6-methyl-2-(1- diazide methylethyl)-4- diazol pyrimidinyl] ester gardentox nucidol

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dichlo- ECP phosphorothioic C10 H13 Cl2 O3 PS 315.16 fenthion hexa-nema acid, O -(2,4-di- mobilawn chlorophenyl) O,O - nemacide diethyl ester

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------dichlorves atgard phosphoric acid, C4 H7 Cl2 O4 P 220.98 canogard 2,2-dichloro- cekusan ethenyl dimethyl DDVP ester dedevap equigard herkal marvex nuvan task vapona

dicroto- bidrin phosphoric acid, C8 H16 NO5 P 237.22 phos carbicron 3-(dimethylamino)- ektafos 1-methyl-3-oxo-1- propenyl dimethyl ester

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------dimethoate cygon phosphorodithioic C5 H12 NO3 PS2 229.27 daphene acid, O,O -dimethyl dimeton S -[2-(methylamine)- ferkethion 2-exoethyl] ester fortion

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fosfamid fosfotox lurgo perfektion rebelate rogor roxion

dioxathion delnav phosphorodithioic C12 H26 O6 P2 S4 456.56 kavadel acid, S-S' -1,4- navadel diexane-2,3-diyl ruphos O,O,O',O' -tetra- ethyl ester

disulfoton dimaz phosphorodithioic C8 H19 O2 PS3 274.42 disyston acid, O,O -diethyl disystox S -[2-(ethylthio) frumin ethyl] ester solvirex

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------

EPN phosphonothioic C14 H14 NO4 PS 323.32 acid, phenyl- O - ethyl O -(4-nitro- phenyl) ester

bladan phosphorodithioic C9 H22 O4 P2 S4 384.49 fosfono 50 acid, S,S' -methy- nialate lene O,O,O',O'- redocid tetramethyl ester seprathion

Page 105 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

fenamiphos nemacur phosphoramidic C13 H22 NO3 PS 272.34 acid, (1-methyl- ethyl)-ethyl 3- methyl-4-(methyl- thio)phenyl ester

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------fenchlor- ectoral phosphorothioic C8 H8 Cl3 O3 PS 321.54 phos etrolene acid, O,O -dimethyl korlane O -(2,4,5-trichloro- nanchlor phenyl) ester nankor trolene viozene

fenitro- accothion phosphorothioic C9 H12 NO5 PS 277.25 thion cyfen acid, O,O -dimethyl cytel O -(3-methyl-4- felithion nitrophenyl) pester metathion nitrophos nevathion sumithion

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------fensulfo- dasanit phosphorothioic C11 H17 O4 PS2 308.37 thion terracur-P acid, O,O -diethyl O -[4-(methyl-

Page 106 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

sulfinyl)phenyl] ester

fenthion baycid phosphorothioic C10 H15 O3 PS2 278.34 baytex acid, O,O -dimethyl entex O -[3-methyl-4- lebaycid (methylthio)phenyl] mercaptophos ester queletox tiguvon

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------ Dyfonate O -ethyl S -phenyl C10 H15 OPS2 246.3 (RS)-ethyl-phospho- nodithioate

formothion aflix phosphorodithioic C6 H12 NO4 PS2 257.28 anthio acid, S -[2-(formyl- methylamino)-2-oxo- ethyl] O,O -dimethyl ester

fosthietan phosphoramidic C6 H12 NO3 PS2 241. acid, 1,3- dithietan-2- ylidene-, diethyl ester

------

Page 107 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------hepteno- hostaquick phosphoric acid, C9 H12 ClO4 P 250.63 phos ragadan 7-chlorobicyclo- [3.2.0]hepta-2,6- dien-6-yl dimethyl ester

idofenphos alfacron phosphorothioic C8 H8 Cl2 IO3 PS 412.99 nuvanol-N acid, O -(2,5- dichloro-4- iodophenyl) O,O - dimethyl ester

isofenphos oftanol , 2- C15 H24 NO4 PS 345.43 [[ethoxy-[(1- methyl-ethyl)amino] phosphinothioyl]- oxy]-, 1-methyl- ethyl ester

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------leptophos Phosvel O -(4-bromo-2,5-di- C13 H10 BrCl2 O2 PS 412.1 Abar chlorophenyl) O - methyl phenylphos- phonothioate

malathion carbetox butanedioic acid, C10 H19 O6 PS2 330.38 carbefos [(dimethoxyphos- chemation phinothioyl)thio]-,

Page 108 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

cythion diethyl ester emmatos fyfanon kypfos sadafos zithiol

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------mecarbam afos carbamic acid, C10 H20 NO5 PS2 329.4 muratox [[[(diethoxyphos- pestan phinothioyl)thio]- acetyl]methyl]-, ethyl ester

menazon azidithion phosphorodithioic C6 H12 N5 O2 PS2 281.32 saphizon acid, S -[(4,6-di- saphos amino-1,3,5- sayfor triazin-2-yl)- syphos methyl] O,O - dimethyl ester

mephosfolan Cytrolane diethyl(4-methyl-1, C8 H16 NO3 PS2 269.3 3-dithiolan-2- ylidene)phosphor amidate

------

Annex I. (contd.) ------

Page 109 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------methamido- hamidop phosphoramidothioic C2 H8 NO2 PS 141.14 phos monitor acid, O,S -dimethyl tamaron ester

methida- supracide phosphorodithioic C6 H11 N2 O4 PS2 302.34 thion ultracide acid, S -[(5- methoxy-2-oxo-1,3, 4-thiadiazol-3(2 H)- yl)-methyl] O,O - dimethyl ester

mevinphos gestid 2-butenoic acid, C7 H13 O6 P 224.17 menite 3-[(dimethoxyphos- phosdrin phinyl)oxy]-, phosfene methyl ester

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------monocro- azodrin phosphoric acid, C7 H14 NO5 P 223.19 tophos monocron dimethyl 1-methyl- nuvacron 3-(methylamino)-3- oxo-1-propenyl ester

morpho- ekatin phosphorodithioic C8 H16 NO4 PS2 285.34 thion morphotox acid, O,O -dimethyl

Page 110 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

S -[2-(4-morpho- linyl)-2-oxoethyl] ester

naled arthodibrom phosphoric acid, C4 H7 Br2 Cl2 O4 P 380.8 bromex 1,2-dibromo-2,2- dibrom dichloroethyl dimethyl ester

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------omethoate dimethoxon phosphorothioic C5 H10 NO4 PS 213.21 folimat acid, O,O -dimethyl S -[2-(methylamino)- 2-oxoethyl] ester

oxydeme- phosphorothioic C6 H15 O4 PS2 246.3 ton- acid, S -[2-(ethyl- methyl sulfinyl)ethyl] O,O -dimethyl ester

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------parathion alkron phosphorothioic C10 H14 NO5 PS 291.28 alleron acid, O,O -diethyl cerothion O -(4-nitrophenyl) danthion ester ekatox folidol fosfex

Page 111 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

kypthion niran stathion sulphos

parathion- amofos phosphorothioic C8 H10 NO5 PS 263.22 methyl dalf acid, O,O -dimethyl metafos O -(4-nitrophenyl) metaphor ester matron nitrox tekwaisa thiophenit vofatox

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------phenthoate pap benzeneacetic acid, C12 H17 O4 PS2 320.38 papthion alpha-[(dimethoxy- tanone phosphinothioyl)- cidial thio]-, ethyl ester

phorate granutox phosphorodithioic C7 H17 O2P S3 260.39 rampart acid, O,O -diethyl thimet S -[(ethylthio)methyl] vegfru ester

phosalone azofene phosphordithioic C12 H15 ClNO4 PS2 367.82 benzphos acid, S -[(6-chloro- rubitox 2-oxo-3(2 H)- benzoxa- zolone zolyl)methyl] O,O - diethyl ester

Page 112 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------phosmet decemthion phosphorodithioic C11 H12 NO2 PS2 317.33 appa acid, S -[(1,3-di- ftalophos hydro-1,3-dioxo-2H- imidan isoindol-2-yl)methyl] prolate O,O -diethyl ester smidan

phospha- dimeron phosphoric acid, C10 H19 ClNO5 P 299.72 midon famfos 2-chloro-3-(diethyl- amino)-1-methyl-3- oxo-1-propenyl di- ethyl ester

Cyolane P,P-diethyl cyclic- C7 H14 NO3 PS2 255.3 Cyolan ethylene ester of Cyalane phosphonodithiomido- Cylan carbonic acid

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------phoxim baythion 3,5-dioxa-6-aza- C12 H15 N2 O3 PS 298.32 valexon 4-phosphaocta-6-ene- volaton S -nitrile, 4-ethoxy- 7-phenyl, 4-sulfide

Page 113 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

profenofos Curacron O -(4-bromo-2-chloro- C11 H15 BrCl03 PS 373.6 Selecron phenyl) O -ethyl S - propyl phosphoro- thioate

prothiofos Tokuthion dichlorophenyl O - C11 H15 Cl2 O2 PS2 345.2 ethyl S- propyl phos- phorodithioate

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------ fal phosphorodithioic C9 H20 NO3 PS2 285.39 fostion acid, O,O -diethyl oleofac S -[2-[1-methylethyl)- telefos amino]-2-oxo-ethyl] ester

pyrimiphos- fernex phosphorothioic C13 H24 N3 O3 PS 302.46 ethyl primieid acid, O -[2-(diethyl- primotec amino)-6-methyl-4- pyrimidinyl] O,O - diethyl ester

pyrimiphos- actellic phosphorothioic C11 H20 N3 O3 PS 274.4 methyl actellifog acid, O -[2-(diethyl- blex amino)-6-methyl-4- silosan pyrimidinyl] O,O - dimethyl ester

Page 114 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------pyrazophos afugan pyrazolo[1,5a]pyri- C14 H20 N3 O5 PS 373.4 curamil midine-6-carboxylic acid, 2-[(diethoxyd- phosphinothioyl)oxy]- 5-methyl-, ethyl ester

bladafume thiodiphosphoric C8 H20 O5 P2 S2 322.34 dithiofos acid, tetraetyl dithione ester dithiotep

sulprofos Bolstar O -ethyl O -[4- C12 H19 O2 PS3 322.4 (methylthio) phenyl] phenyl] S -propyl phosphorodithioate

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------temephos abate phosphorothioic C16 H20 O6 P2 S3 466.48 abathion acid, O,O' -(thio- biothion di-4,1-phenylene) difethos O,O,O',O' -tetra- nimitox methyl ester

Page 115 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

TEPP bladan diphosphoric acid, C8 H20 O7 P2 290.22 bladex tetraethyl ester fosuex grisol hexamite lirohex mortopal nifos

tetrachlor- appex phosphoric acid, C10 H9 Cl4 O4 P 365.96 vinphos gardcide 2-chloro-1-(2,4,5- gardona trichlorophenyl)- rabon ethenyl dimethyl stirofos ester

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------thiometon ekatin phosphorodithioic C6 H15 O2 PS3 246.36 intrathion acid, S -[2-(ethyl- thio)ethyl] O,O - dimethyl ester

thionazin cynem phosphorothioic C6 H13 N2 O3 PS 248.26 nemafos acid, O,O -diethyl zinophos O -pyrazin-2-yl ester

triamiphos wepsin phosphonic diamide, C12 H19 N6 CP 294.34 P -(5-amino-3-phenyl- 1 H- 1,2,4-triazol-1- yl)- N,N,N' -tetra- methyl

Page 116 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------triazophos Hostathion O,O -diethyl O -(1H- C12 H16 N3 O3 PS 313.3 HOE 2960 1.2.4-triazol-3-yl) phosphorothioate

trichlor- anthion phosphonic acid, C4 H8 Cl3 O4 P 257.44 fon bovinox (2,2,2-trichloro- briton 1-hydroxyethyl)-, cehuion dimethyl ester clorofos ciclosom danex dipterex dylox metrifonate proxol

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------trichlor- agrisil phosphonothioic C10 H12 Cl3 O2 PS 333.6 nat agritox acid, ethyl-, O - ethyl O -(2,4,5- trichlorophenyl) ester

trifenofos RH 218 O -ethyl- S -propyl- O- C11 H14 Cl3 O3 PS 363.6 (2,4,6-tri-chloro- phenyl) phosphoro thioate

Page 117 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

------

Annex I. (contd.) ------Common name Trade or CAS chemical name Molecular Relative other name formula molecular mass ------vamidothion kilyal phosphorothioic C8 H18 NO4 PS2 287.36 trucidor acid, O,O -dimethyl vamidoate S -[2-[[1-methyl-2- (methylamino)-2- oxoethyl]thio]ethyl] ester

------

Annex II. Organophosphorus pesticides: JMPR reviews, ADIs, Evaluation by IARC, C Hazard, FAO/WHO Data Sheets, IRPTC Data Profile and Legal Filea ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------Acephate 1984 0-0.0005 1985a (temporary) 1982 0-0.003 1983b + II (temporary) 1983a 1981i 0-0.02 1982b 1982a 1979i 0-0.02 1980b 1980a 1978 0-0.02 1979a 1976 0-0.02 1977b 1977a

Azinphos 1983i no ADI 1984a + + -ethyl 1973 no ADI 1974b IB 1974a

Azinphos 1974i 1975b + + I -methyl 1975a 1973 0-0.0025 1974b 1974a 1972i 0-0.0025 1973b 1973a 1968 0-0.0025 1969b 1969a

Page 118 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

1965 1965b 0-0.0025 1965a 1963 0-0.0025 1964 ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------Bromophos 1984i 0-0.04 1985b 1982i 0-0.04 1983b + + I 1983a 1978i 0-0.04 1979a 1977 0-0.04 1978b 1978a 1975i 0-0.006 1976b (temporary) 1976a 1972 0-0.006 1973b (temporary) 1973a

Bromophos 1978i 0-0.003 1979a + + I -ethyl 1977i 0-0.003 1978b 1978a 1975 0-0.003 1976b 1976a 1972 0-0.003 1973b (temporary) 1973a

Carbophen- 1983i 0-0.0005 1984a + + I othion 1980 0-0.0005 1981b 1981a 1979 0-0.0005 1980b 1980a 1977 0-0.0002 1978b (temporary) 1978a 1976 temporary 1977b ADI withdrawn 1977a 1972 0-0.005 1973b (temporary) 1973a

Chlorfen- 1971 0-0.002 1972b + + IA vinphos 1972a ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------Chlorpy- 1983i 0-0.01 1984a + + I rifos 1982 0-0.01 1983b 1983a 1981i 0-0.001 1982b

Page 119 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

1982a 1977 0-0.001 1978b 1978a 1975i 0-0.0015 1976b 1976a 1974i 0-0.0015 1975b 1975a 1972 0-0.0015 1973b 1973a

Chlorpy- 1979i 0-0.01 1980b + + rifos- 1980a methyl 1975 0-0.01 1976b II 1976a

Chlorthion 1965 no ADI 1965b 1965a - 1963 no ADI 1964

Coumaphos 1983i ADI withdrawn 1984a + + I 1980i ADI Withdrawn 1981b 1981a 1978i 0-0.005 1979b (temporary) 1979a 1975i 0-0.005 1976b (temporary) 1976a 1972i 0-0.0005 1973b (temporary) 1973a ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------1968 0-0.0005 1969b (temporary) 1969a

Crufomate 1972i 0-0.1 1973b I 1973a 1968 0-0.1 1969b 1969a

Cyano- 1983 ADI withdrawn 1984a II fenphos 1982i 0-0.001 1983b (temporary) 1983a 1980 0-0.001 1981b (temporary) 1981a 1978i 0-0.005 1979a (temporary)

Cyano- 1975 0-0.005 1976b fenphos (temporary)

Page 120 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

1976a

Demeton 1983i no ADI 1984a + + (see also 1982 ADI withdrawn 1983a disulfoton) 1975 0-0.005 1976b IA

1976a 1967i 0-0.0025 1968b 1968a 1965 0-0.0025 1965b 1965a 1963 0-0.0025 1964 ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------Demeton- S - 1984 no ADI 1985b methyl and 1983i no ADI 1984a + + I Related 1982 ADI withdrawn 1983a Compounds 1979i 0-0.005 (the 1980b (see also total demeton- oxydemeton- S -methyl, 1980a I methyl for demeton- S - 1963 to 1968 methyl sulfo- evaluations) xide and deme- ton- S -methyl sulfone not to exceed this figure) 1973 0-0.005 (the 1974b total demeton 1974a - S -methyl, demeton- S - methyl sulf- oxide and demeton- S - methyl sulfone not to exceed this figure) ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------Demeton- 1984 no ADI 1985b S -methyl 1983i no ADI 1984a sulfoxide 1982 ADI 1983a IB (see withdrawn oxydemeton- methyl for 1963 to 1968

Page 121 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

evaluation) (see demeton- S -methyl and related compounds after 1968)

Dialifos 1982 ADI 1983a II withdrawn 1978i 0-0.003 1979a 1976 0-0.003 1977b 1977a

Diazinon 1979i 0-0.002 1983a + + I 1980a 1975i 0-0.002 1976b 1976a 1970 0-0.002 1971b 1971a 1968i 0-0.002 1969b 1969a 1967i 0-0.002 1968b 1968a 1966 0-0.002 1969b 1967a 1965 0-0.002 1965b 1965a 1963 no ADI 1964 + ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------Dichlorvos 1977 0-0.004 1978b Vol 20 + + IB p.97 1978a 1974i 0-0.004 1975b 1975a 1970 0-0.004 1971b 1971a 1969i 0-0.004 1970b 1970a 1967 1968b 0-0.004 1968a 1966 1967b 0-0.004 1967a 1965 no ADI 1965b 1965a

Dimethoate 1984 0-0.002 1985b (temporary) 1983i 0-0.02 1984a + + 1978i 0-0.02 1979a 1977i 0-0.02 1978b Vol. 15 I page 177 1970i 0-0.02 1971b 1971a

Page 122 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

1967 0-0.02 1968b 1968a 1966i 0-0.004 1967b 1967a 1965 0-0.004 1965b 1965a 1963 0-0.004 1964 ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------Disulfoton 1984i 0-0.002 1985b (see also 1981i 0-0.002 1982b + + demeton) 1982a 1979i 0-0.002 1980b I 1980a 1978i 0-0.002 1979a 1975 0-0.002 1976b 1976a 1973 0-0.001 1974b (temporary) 1974a

Edifenphos 1981 0-0.003 1982b + IB 1982a 1979 0-0.003 1980b (temporary) 1980a 1976 0-0.003 1977b (temporary) 1977a

Ethion 1985 0-0.0005 1986b (temporary) 1983i 0-0.001 1984a + + I (temporary) 1982 0-0.001 1983b (temporary) 1983a 1975i 0-0.005 1976b 1976a 1972 0-0.005 1973b 1973a ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------1970i 0-0.00125 1971b 1971a

Page 123 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

1969i 0-0.00125 1970b 1970a 1968 0-0.00125 1969b 1969a

Ethoprophos 1983 no ADI 1984a IA

Etrimfos 1982 0-0.003 1983b + + II 1983a 1980 0-0.003 1981b (temporary) 1981a

Fenamiphos 1985 0-0.0003 1986b (temporary) 1980i 0-0.0006 1981b I 1981a 1978i 0-0.0006 1979b R30-17 1977i 0-0.0006 1978b 1978a 1974 0-0.0006 1975b 1975a

Fenclorphos 1983i 0-0.01 1984a + + I 1972i 0-0.01 1973b 1973a 1968 0-0.01 1969b 1969a ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------Fenitro- 1984 0-0.003 1985a thion 1983i 0-0.001 1974a + + (temporary) 1982 0-0.001 1983b (temporary) 1983a II 1979i 0-0.005 1980b 1980a 1977 0-0.005 1978b 1978a 1976i 0-0.005 1977b 1977a 1974 0-0.005 1975b 1975a 1969 0-0.001 1970b (temporary) 1970a

Fensulf- 1983i 0-0.0003 1984a + + othion 1982 0-0.0003 1983b 1983a IA 1972 0-0.0003 1973b

Page 124 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

1973a ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------Fenthion 1983i 0-0.001 1984a + 1980 0-0.001 1981b 1981a IB 1979 0-0.0005 1980b (temporary) 1980a 1978 0-0.0005 1979b (temporary) 1979a 1977i 0-0.0005 1978b (temporary) 1978a 1975 0-0.0005 1976b (temporary) 1976a 1971 0-0.0005 1972b (temporary) 1972a

Formothion 1978i 0-0.02 1979a + + I 1973 0-0.02 1974b 1974a 1972i no ADI 1973b 1973a 1969 no ADI 1970b 1970a

Isophenphos 1984i 0-0.0005 1985a 1982 0-0.0005 1983b IB (temporary) 1983a 1981 0-0.0005 1982b (temporary) 1982a ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------Iodophenphos O

Leptophos 1978i ADI 1979b + + withdrawn 1979a 1976i 0-0.001 1977a I (temporary) 1975 0-0.001 1976b

Page 125 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

(temporary) 1976a 1974 No ADI 1975b 1975a

Malathion 1984 0-0.02 1985b 1977i 0-0.02 1978b Vol. 30 + + page 103 1978a 1975i 0-0.02 1976b I 1976a 1973i 0-0.02 1974b 1974a 1970i 0-0.02 1971b 1971a 1969i 0-0.02 1970b 1970a 1968i 0-0.02 1969b 1969a 1967i 0-0.02 1968b 1968a 1966 0-0.02 1967b 1967a 1965 0-0.02 1965b 1965a 1963 0-0.02 1964 ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------Mecarbam 1985 0-0.0005 1986b (temporary) 1983 0-0.001 1984a IB (temporary) 1980 0-0.001 1981b (temporary) 1981a

Methacrifos 1982 0-0.0003 1983b - (temporary) 1983a 1980 0-0.0003 1981b (temporary) 1981a

Methamido- 1985 0-0.0006 1986b phos 1984i 0-0.0004 1985a (temporary) 1982 0-0.0004 1983b + + IB (temporary) 1983a 1981i 0-0.002 1982b 1982a 1979i 0-0.002 1980b 1980a 1976 0-0.002 1977b

Page 126 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

1977a

Methida- 1979i 0-0.005 1980b + + I thion 1980a 1977i 0-0.005 1978a 1975 0-0.005 1976b 1976a 1972 0-0.005 1973b (temporary) 1973a ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe> : meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------Methyl parathion (see parathionmethyl)

Mevinphos 1972 0-0.0015 1973b + + IA 1973a 1965 no ADI 1965b + 1965a 1963 no ADI 1964

Monocroto- 1975 0-0.0006 1976b + + IB phos 1976a 1972 0-0.0003 1973b 1973a

Omethoate 1985 0-0.0003 1986b 1984i 0-0.0005 1985a (temporary) 1981 0-0.0005 1982b IB (temporary) 1982a 1980i 0-0.0005 1981b (temporary) 1981a 1979 0-0.0005 1980b (temporary) 1980a 1978 0-0.0005 1979b (temporary) 1979a 1975 0-0.0005 1976b (temporary) 1976a 1971 0-0.0005 1972b (temporary) 1972a ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si

Page 127 of 135 Organophophorus insecticides: a general introduction (EHC 63, 1986)

in: FAO/WHO genicity Profile filef o by ------Oxydemeton- 1968i ADI 1969b methyl withdrawn 1969a IB (referred in 1967 0-0.0025 1968b 1963 and 1965 1968a reports as 1965 0-0.0025 1965b demeton- S - 1965a methyl 1963 0-0.0025 1964 sulfoxide) (see demeton- S - methyl and related comp- ounds for evaluations after 1968)

Parathion 1984i 0-0.005 1985a 1970i 0-0.005 1971b Vol. 30 + + I page 153 1971a 1969i 0-0.005 1970b 1970a 1967 0-0.005 1968b 1968a 1965 0-0.005 1965b 1965a 1963 0-0.005 1964 ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by h ------Parathion- 1984 0-0.02 1985b methyl (evaluated 1982 0-0.001 1983b vol. page + + under methyl (temporary) 30-131 parathion 1983a in 1963 and 1980 0-0.001 1981b 1965) (temporary) 1981a 1979 0-0.001 1980b (temporary) 1980a 1978i 0-0.001 1979b (temporary) 1979a 1975 0-0.001 1976b (temporary) 1976a 1972i 0-0.001 1973b (temporary) 1973a 1968 0-0.001 1969b

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(temporary) 1969a 1965 0-0.01 1965b 1965a 1963 0-0.01 1964

Phenthoate 1984 0-0.003 1985b 1981i 0-0.001 1982b + + I (temporary) 1982a 1980 0-0.001 1981b (temporary) 1981a ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------Phorate 1985 0-0.0002 1986b 1984i 0-0.0002 1985a (temporary) 1983 0-0.0002 1984a + + IA (temporary) 1982 0-0.0002 1983b (temporary) 1983a 1977 No ADI 1978b established 1978a

Phosalone 1976i 0-0.006 1977b + + I 1977a 1975i 0-0.006 1976b 1976a 1972 0-0.006 1973b 1973a

Phosmet 1984i 0-0.02 1985b 1981i 0-0.02 1982b + + I 1982a 1979 0-0.02 1980b 1980a 1978 0-0.005 1979b (temporary) 1979a 1977 -corrigenda 1978b to 1976 evaluations - 1977i no ADI 1978a 1976i no ADI 1977b 1977a ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m

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meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------Phosphamidon 1985 0-0.0005 1986b 1982 0-0.001 1983b + + IA (temporary) 1983a 1974i 0-0.01 1975b 1975a 1972i 0-0.001 1973b 1973a 1969i 0-0.001 1970b 1970a 1968 0-0.001 1969b 1969a 1966 0-0.001 1967b 1967a 1965 no ADI 1965b 1965a 1963 no ADI 1964

Phoxim 1984 0-0.001 1985b 1983i 0-0.0005 1984a (temporary) 1982 0-0.0005 1983b (temporary) 1983a II

Pirimiphos- 1983 0-0.01 1984a methyl 1979i 0-0.01 1980b 1980a 1977i 0-0.01 1978b I 1978a 1976 0-0.01 1977b 1977a 1974 0-0.005 1975b (temporary) 1975a ------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------Temephos + + 0

Thiometon 1979 0-0.003 1980b + + IB 1980a 1976i 0-0.005 1977b (temporary) 1977a 1973 0-0.005 1974b (temporary) 1974a 1969 no ADI 1970b 1970a

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Triazophos 1983i 0-0.0002 1984a I (temporary) 1982 0-0.0002 1983b (temporary) 1983a

------

Annex II. (contd.) ------Compound Year of ADIb Evaluation IARCd Availability W JMPR (mg/kg body by JMPRc : Evaluation of IRPTCe : m meeting weight) Published of Carcino- Data Legal si in: FAO/WHO genicity Profile filef o by ------Trichlorfon 1978 0-0.01 1979b + + II 1979a 1975 0-0.005 1976b Vol. 30 (temporary) page 207 1976a 1971 0-0.01 1972b (temporary) 1972a

Trichloronat 1971 no ADI 1972b + + IA 1972a

Vamidothion 1985 0-0.0003 1986b (temporary) 1982 0-0.0003 1983b + + IB (temporary) 1983a 1973 no ADI 1974b 1974a ------a Adapted from: Vettorazzi & van den Hurk (1984). b ADI = acceptable daily intake. c JMPR = Joint Meeting on Pesticide Residues (FAO/WHO). d IARC = International Agency for Research on Cancer (WHO, Lyons, France). e IRPTC = International Register of Potentially Toxic Chemicals (UNEP, Geneva) f From: IRPTC (1983). g From: WHO (1984a). See this reference for classification of organophosphates this annex.

The hazard referred to in this Classification is the acute risk for health ( single or multiple exposures over a relatively short period of time) that mi accidentally by any person handling the product in accordance with the direc by the manufacturer or in accordance with the rules laid down for storage an by competent international bodies.

Classification relates to the technical material, and not to the formulated product: ------Class LD50 for the rat (mg/kg body weight) Oral Dermal Solids Liquids Solids Liquids ------IA Extremely hazardous 5 or less 20 or less 10 or less 40 or l IB Highly hazardous 5 - 50 20 - 200 10 - 100 40 - 40 II Moderately hazardous 50 - 500 200 - 2000 100 - 1000 400 - 4

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III Slightly hazardous over 500 over 2000 over 1000 Over 40 O Unlikely to present acute hazard in normal use ------h WHO/FAO Data Sheets on Pesticides with number and year of appearance. i No toxicological evaluation - residues only.

N.B. References to Annex II are listed in the reference list of the main docume

Annex III. LD50s and no-observed-adverse-effect levels in animals ------Chemical Acute LD50 No-observed-adverse-effect level in (mg/kg body weight)a animals (rats unless otherwise stat Oral Dermal (mg/kg (mg/kg body Duration diet) weight) of test ------Azinophos- 16.4 2.5 0.125 2 years methyl 80 (guinea-pig) 5 (dog) 0.125 (dog) 2 years

Bromophos- 71 - 127 1366 (rabbit) 0.78 2 years ethyl 10 (dog) 0.26 (dog) 2 years 225 - 550 (mice)

Bromophos 3750 - 7700 20 (dog) 0.5 (dog) 1 year 2829 - 5850 0.4 (man) 4 weeks (mice) 720 (rabbit)

Carbopheno- 32.3 1270 (rabbit) 3 0.15 3 generati thion 0.02 (dog) 3 months 0.01 (man) 1 month

Chlorfen- 10 - 39 31 - 108, 1 0.05 3 months vinphos 117 - 200 (mice) 417 - 4700 1 dog 0.05 (dog) 16 weeks (rabbit) 300 - 1000 (rabbit) > 12 000 (dog)

Chlorpyr- 135 - 163 approximately 0.1 2 years ifos 2000 500 (guinea-pig) (rabbit) 0.1 (dog) 90 days 32 (chicken) 0.1 (man) 1 month 1000 - 2000 (rabbit)

Crufomate 770 - 950 40 2 2 year 400 - 600 40 (dog) 1 dog 75 days (rabbit)

Demeton 2.5 - 12 ------

Annex III. (contd.) ------Chemical Acute LD50 No-observed-adverse-effect level in (mg/kg body weight)a animals (rats unless otherwise stat Oral Dermal (mg/kg (mg/kg body Duration diet) weight) of test ------Demeton- S - 57 - 106 302 methyl 110 (guinea-pig)

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Diazinon 300 - 850 > 2150 2 0.1 90 days 0.02 (dog) 31 days 0.05 (monkey) 2 years 0.02 (man) 37 days

Dichlorvos 56 - 108 75 - 210 0.033 (man) 28 days

5 0.25 15 weeks Dimethoate 320 - 380 0.2 (man) 57 days 15 (pheasant) 40 (duck)

Dioxathion 43 235 3 0.15 13 weeks 0.075 (dog) 90 days 0.075 (man) 28 days

Disulfoton 2.6 - 8.6 ca 20 1 0.05 2 years 1 (dog) 0.025 (dog) 12 weeks 0.075 (man) 30 days

Ethion 24.4 - 208 915 3 0.15 13 weeks (rabbit) 0.125 (dog) 90 days

Fenamiphos 15.3 - 19.4 500 3 0.17 2 years 10 (dog) 1 (dog) 0.025 (dog) 2 years 75 - 100 (guinea-pig) 12 (hen)

Fenchlor- 1740 2000 0.5 2 years phos 1 (dog) 2 years ------

Annex III. (contd.) ------Chemical Acute LD50 No-observed-adverse-effect level in (mg/kg body weight)a animals (rats unless otherwise stat Oral Dermal (mg/kg (mg/kg body Duration diet) weight) of test ------Fenitro- 250 - 500 > 3000 5 0.25 34 weeks thion 870 (mice) (mice) 10 (dog) 0.3 (dog) 12 months

Fenthion 190 - 315 330 - 500 3 0.15 2 years 3 (dog) 0.09 2 years 0.07 (monkey) 1 year 0.02 (man) -

Formothion 365 - 500 > 1000 20 1 2 years 40 (dog) 1 (dog) 2 years

Malathion 2800 4100 100 5 2 years (rabbit) 0.2 (man) 88 days

Methida- 25 - 54 1546 - 1663 4 0.2 104 weeks thion 25 - 20 (mice) 0.25 (monkey) 23 months 0.11 (man) 6 weeks

Mevinphos 3 - 12 1 - 90 0.37 0.02 2 years 7 - 18 (mice) 16 - 34 0.025 (dog) 2 years (rabbit) 0.014 (man) 30 days

Monocroto- 14 - 23 336 0.5 0.025 12 weeks phos (rabbit) 0.5 (dog) 0.0125 (dog) 13 weeks

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Omethoate ca 50 700 1 0.05 3 months 0.025 (dog) 12 months

Parathion 3.6 - 13 6.8 - 21 0.05 (man) 3 weeks

Parathion- 2 0.1 2 years methyl 14 - 24 67 0.3 (man) 30 days ------

Annex III. (contd.) ------Chemical Acute LD50 No-observed-adverse-effect level in (mg/kg body weight)a animals (rats unless otherwise stat Oral Dermal (mg/kg (mg/kg body Duration diet) weight) of test ------Phosalone 120 - 170 1500 25 1.25 2 years 180 (mice) > 1000 25 (dog) 0.625 2 years (rabbit) 380 (guinea-pig) 290 (pheasant)

Phospha- 17 - 30 374 - 530 2 0.1 12 weeks midon 0.5 (dog) 90 days

Pirimiphos- 2050 > 2000 10 0.5 2 years methyl (rabbit) 5 (mouse) 0.5 (mouse) 80 weeks 1180 (mice) 0.25 (man) 28 days 1000 - 2000 (guinea-pig) 1150 - 2300 (rabbit) 30 - 60 (hen)

Thiometon 120 - 130 > 1000 2.5 0.12 2 years 6 (dog) 0.5 (dog) 2 years

Trichlorfon 560 - 630 > 2000 50 2.5 2 years 50 (dog) 1.25 (dog) 4 years (d ------a From: Worthing (1983). N.B. This reference is listed in the reference list of the main document.

Annex IV. Abbreviations used in the document

ACh acetylcholine

AChE acetylcholinesterase

ACTH adrenocorticotropic hormone

ADI acceptable daily intake

ChE cholinesterase

CNS central nervous system

DDE dichlorodiphenyldichloroethylene

DDT dichlorodiphenyltrichloroethane

DEF S,S,S -tributyl phosphorotrithioate

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DFP di-isopropyl fluorophosphate

EEG electroencephalogram

EMG electromyography

EPN o -ethyl- O -(4-nitrophenyl)phenylphosphonothioate

FAO Food and Agricultural Organization (United Nations)

IARC International Agency for Research on Cancer im intramuscular

IPCS International Programme on Chemical Safety (World Health Organization)

IRPTC International Register of Potentially Toxic Chemicals (United Nations Environment Programme) iv intravenous

JMPR FAO/WHO Joint Meeting on Pesticide Residues

MFO mixed-function oxidase

MLD minimum lethal dose

MRL maximum residue limit

NAD nicotinamide-adenine-dinucleotide

NADPH nicotinamide-adenine-dinucleotide phosphate (reduced form)

NTE neuropathy target esterase (formerly neurotoxic esterase)

OMPA octamethylpyrophosphorictetramide

2-PAM pyridine-2-aldoxime methyl chloride pseudoChE pseudocholinesterase sc subcutaneous

TCDD 2,3,7,8-tetrachlorodibenzo-1,4-dioxin

TEPP

TOCP tri- o -cresyl phosphate

UVR ultraviolet radiation

See Also: Toxicological Abbreviations

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