FAO/PL:1969/M/17/1 WHO/FOOD ADD./70.38 1969 EVALUATIONS OF SOME PESTICIDE RESIDUES IN FOOD THE MONOGRAPHS Issued jointly by FAO and WHO The content of this document is the result of the deliberations of the Joint Meeting of the FAO Working Party of Experts and the WHO Expert Group on Pesticide Residues, which met in Rome, 8 - 15 December 1969. FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS WORLD HEALTH ORGANIZATION Rome, 1970 FORMOTHION IDENTITY Chemical name S-(N-formyl-N-methylcarbamoylmethyl) dimethyl phosphorothiolothionate Synonyms S-(N-formyl-N-methylcarbamoylmethyl) O,O-dimethyl phosphorodithioate, Anthio(R) Structural formulaOther relevant chemical properties Yellow oil with onion-like odour or white crystalline solid; d420 1.361; soluble in alcohols, ether, chloroform, ketones, aromatic solvents; insoluble in paraffin solvents and water; stable in apolar solvents, unstable at alkaline pH. Formothion is available as a 25 percent and 40 percent emulsifiable concentrate (ingredients other than formothion undefined). BIOCHEMICAL ASPECTS Absorption, distribution and excretion Single oral doses of 10 mg/kg body-weight of formothion, labelled with carbon14 in the carbamoyl group, were administered by stomach tube to male rats. Autoradiographs indicated that the compound was readily absorbed from the stomach. Within 30 minutes after giving the dose, a high level of radioactivity was found in the liver and kidney, although the major activity was still in the stomach. Six hours after administration the activity in the stomach area was low; the main activity then being in the kidneys, with less activity in the liver, intestines, pancreas and thymus. After 24 hours from the time of administration of the dose, distinct radioactivity was found only in the thymus. The urine was the major source of excretion; it contained 98-99 percent of the radioactivity compared to the faeces which had only 1-2 percent. A total of 51 per cent of the administered radioactivity had been excreted in the urine after four hours and 96 percent after 24 hours. The presence of considerable radioactivity in the bile indicated that there was also excretion of the compound and its metabolites in the bile. However, because so little radioactivity was encountered in the faeces nearly all of these compounds must be re-absorbed in the intestine (Klotsche, 1969a). Biotransformation Formothion appears to be completely metabolized in male rats. The only metabolite found, which is said to be of known structure is termed "formothion acid", and this compound is eliminated in the urine more rapidly than other polar metabolites of unknown structure. ("Formothion acid" to presumably O,O-dimethyl-S-carboxymethyl phosphorodithioate or a phosphate analogue of that compound). All these metabolites including "formothion acid" contain nearly all the administered radioactivity, which indicates that they still contain the carboxyl-carbon of the labelled formothion (Klotsche 1969a). Although it had been stated that formothion "is converted to dimethoate in the animal" (rat) (Carshalton, 1965), no dimethoate has been demonstrated in the metabolic studies performed with formothion in rats (Klotsche 1969a). However dimethoate has been identified in plants treated with formothion (Klotsche 1969b). Effect on enzymes and other biochemical parameters Experiment in vitro with rat serum cholinesterase have demonstrated that formothion has anti-cholinesterase activity (Klotsche 1961). For human serum anti-cholinesterase the I50 was found to be 1.91 × 10-5 (Zehnder, 1961). Special studies on neurotoxicity Chicken Two groups, each containing three hens were given 100 mg/kg body-weight of formothion by intramuscular injection; two more groups were given 150 mg/kg. In one of each of the groups the hens were protected against the lethal effects of formothion with pralidoxime and atropine. All the unprotected and one of the protected hens in the 150 mg/kg group died from acute poisoning while the other hens survived and did not show any clinical symptoms of neurotoxicity during an observation period of 22 and 29 days after administration of the 100 and 150 mg/kg doses, respectively. A positive control group given an unspecified dose of triorthocresyl phosphate (TOCP) displayed distinct signs of paralysis (Sandoz, 1964a; Klotsche 1966). Special studies on potentiation Rat Single oral doses of formothion were administered jointly to rats with the following organophosphorus compounds: diazinon, dimethoate, malathion, parathion, phosphamidon and thiometon. There were no effects other than additive except for the formothion - dimethoate combination where there was some indication of a potentiating effect (Klotsche 1969a), Special studies on teratogenicity Rabbit Groups of 10 rabbits were given daily doses by stomach tube of 6 and 30 mg/kg bodyweight of formothion, from the sixth to the eighteenth day of pregnancy. Several groups served as controls. Pregnancy rate, number of implantations, live and dead foetuses, embryonic and foetal deaths and resorptions were comparable in the test and control groups. The same was true of the foetal and placental weights and the number of malformations of organs and the skeletal systems in the foetuses (Klotsche 1969a). Acute toxicity LD50 mg/kg Animal Route body-weight References Mouse (M) oral 190 Klotsche 1966 Mouse (F) oral 195 Klotsche 1966 Rat (M) oral 370-400 Klotsche 1966 Rat (F) oral 500-540 Klotsche 1966 Rat (F) oral 424 Carshalton, 1965 Rat (M) i.v. 36 Klotsche 1966 Guinea-pig oral 560 Sandoz, 1968 Rabbit (M) oral 570 Klotsche 1969a Rabbit (M) i.v. 20 Klotsche 1969a Cat (F) oral 213 Klotsche 1966 Chicken i.v. 20 Klotsche 1966 The symptoms of poisoning were the same as for other anti-cholinesterase organophosphorus compounds. In most of the rat experiments the onset of symptoms was delayed until 70-90 minutes after the oral administration (Carshalton, 1965; Sandoz, 1968). Short-term studies Chicken Three groups of young cocks, each group comprising five birds, were fed for three weeks on grain impregnated with approximately 0, 65 and 330 mg/kg of formothion. In the group that received grain having 330 mg/kg, weight loss occurred in the first week, probably due to food aversion, but the original weight was restored by the end of the test period. Haematological examination revealed no abnormalities indicative of toxic effects in any group. Serum cholinesterase was normal in the group receiving the wheat containing 65 mg/kg but in those on the 330 mg/kg level there was a reduction of 30 percent after 10 days and 40 percent after 21 days compared to the controls (Sandoz, 1963; Klotsche, 1966). Dog Groups of three dogs each comprising two males and one female were given doses of 0, 2, 8 mg/kg body-weight of formothion orally in gelatine capsules for six months. Another group was given 16 mg/kg for three months and then 32 mg/kg for the remaining three months, while a further group was given 100 mg/kg for a maximum period of nine weeks. There were no deaths in the animals given doses of 32 mg/kg or less, but two of the three dogs in the 100 mg/kg group died. These two dogs, together with the three controls, one animal in the 2 mg/kg group and the three animals in the 32 mg/kg group were submitted to gross and histopathological examination at autopsy. In the 100 mg/kg group there was a rapid drop in serum cholinesterase, typical symptoms of anti-cholinesterase poisoning and weight loss. Decrease in eosinophile leucocyte counts and great increase in relative adrenal weights were found in the two dogs which died but the surviving dog made rapid recovery of weight and serum cholinesterase upon withdrawal of formothion. Food intake, haematology, urinalysis and liver function tests showed no abnormalities attributable to formothion in the dogs given 32 mg/kg or lower. A slight loss of weight was observed at 32 mg/kg. Weekly determination of serum cholinesterase activity gave fluctuating values both in the controls and in the animals given up to 32 mg/kg (Klotsche, 1966). Pheasant Three groups of pheasants, each containing two male and two females, received wheat grain treated with 0, 0.15 or 0.75 percent of formothion for three weeks. The decrease in food intake in the group on the high level was thought to be largely responsible for a considerable loss of weight. The weights in the other groups remained constant, or increased slightly. No toxic symptoms were noted and haematology showed no pathological changes in any of the animals. The activity of serum cholinesterase in the birds on the high strength compared with that of the controls, was reduced by about 45 percent. The transaminase values were normal, indicating that there was no liver damage (Sandoz, 1964b; Klotsche, 1966). Rat Two groups, initially comprising 15 male rats per group, were given, by stomach tube, doses of 35 mg/kg and 90 mg/kg body-weight of formothion. The doses were administered six-times weekly for one month and then five times weekly for two months. Only one of the 15 animals survived 90 mg/kg while 13 survived 35 mg/kg. Serum cholinesterase after three months was 25 percent and 46 percent of normal in the 90 and 35 mg/kg groups, respectively. No changes due to formothion were found upon gross and histopathological examination of the dead or in the surviving animals after sacrifice (Klotzsche, 1966; Klotsche, 1969a). Groups of rats, initially comprising 25 male and 25 female animals were given by stomach tube 0, 3, 5, 9 and 16 mg/kg body-weight of formothion daily for six months. Levels of 3 and 5 mg/kg were tolerated without any clinical symptoms, while the rats given 9 mg/kg showed slight, and those given 16 mg/kg appreciable symptoms which disappeared after four weeks of receiving the compound, indicating that a tolerance to the compound developed. A decrease in weight-gain for male rats was found in the 9 and 16 mg/kg groups. Haematological and urine examination revealed no dose-dependent changes. One rat of each sex from each group was sacrificed monthly. Some decrease in serum cholinesterase activity was found in the test groups. In the 3 mg/kg group, though, the activity found from the fourth month and onward was comparable to the controls. After six months the cholinesterase activity in the 5 mg/kg group was approximately 50 percent and in the 16 mg/kg group 30 percent of that of the control group. The average weights of liver and spleen (10 animals) were not different in the test and control groups. Gross and histological examinations (two animals from each group) revealed no changes which could be attributed to formothion (Klotsche, 1966; Klotsche, 1969a). Groups of varying numbers of rats were given formothion percutaneously in daily doses of 0, 70 and 140 mg/kg body-weight for three weeks. After this time, the surviving animals were sacrificed and these animals and those which died during the treatment were subjected to autopsy. Gross pathology showed no differences in organ-weights. The animals which received 140 mg/kg displayed slight histological changes in the liver (fine-droplet fatty infiltration, nuclear pyknosis, single cell degeneration) and in the adrenal glands (increased plasmavacuolization in the zone fasciculata) (Klotsche and Rüttiman, 1965). Long-term studies No information available. COMMENT Formothion seems to be rapidly and completely metabolized in the rat, the only animal from which information on metabolism is available. The analogy of this compound with dimethoate, a transformation product in plants, was considered but there is no information that such a transformation occurs in animals, the only metabolite characterized being "formothion acid". Based upon a six-month study in the dog, there is a no-effect level of 16 mg/kg body-weight per day. However, the rat appears to be a more sensitive animal; an effect of 3 mg/kg body-weight produced a depression of serum cholinesterase and a no-effect level was not found. There is some indication of a possible potentiation of formothion and dimethoate. No information is available on long-term studies in animals or observations in man. The experimental studies available are considered insufficient for establishing an acceptable daily intake for man. RESIDUES IN FOOD AND THEIR EVALUATION USE PATTERN Formothion is a phosphorus-containing systemic insecticide with activity as a contact and stomach poison. It has no ovicidal action. It is used on a wide variety of crops (cotton, rice, cereals, sugarcane, oil seeds, sugar-beets, citrus, grapes, fruits, vegetables, potatoes, tomatoes, coffee, tea, and tobacco) against a wide range of sucking pests and a number of biting and chewing insects (aphids, fruit flies, olive flies, Mangold flies, white flies, thrips, Jassids, mites, scale insects, and mealybugs). Waiting periods between last application and harvest are generally one or two weeks, depending on country and crop, but may be as much as six weeks. Formothion is registered in more than 50 countries in Europe, South America, Africa, Asia, and Australia. Pre-harvest treatments Formothion is usually applied as a 0.1-0.2% spray. Post-harvest treatments None are known. Other uses Formothion is applied as a spray on ornamental plants and tobacco. RESIDUES RESULTING FROM SUPERVISED TRIALS Residues of formothion, after normal application (0.1-0.2% spray), generally decrease to less than 1 ppm after 10 days. Persistence of residues depends on type of crop and environmental factors (rainfall, temperature). Residues are usually formothion and dimethoate (O,O-dimethyl S-(N-methylcarbamoylmethyl) phosphorodithioate), the latter being formed by loss of the N-formyl group. (Dimethoate was evaluated by FAO/WHO in 1967). The oxygen analogue of dimethoate, dimethoxon, may also form. In trials on fruits and vegetables, residues often diminish to 0.1 ppm or less in 14 days. In some instances (sugar-beets and potatoes) no residues were found after treatment. In typical trials following normal practice, the maximum residues found are listed in the following table: Typical maximum residues found following normal application Maximum residues when sampled, Spray Day respectively, ppma/ Crop application sampled Formothion Dimethoate Sugar-beets 0.2% 0,7,10 nd,nd,nd nd,nd,nd Cherriesb/ 0.2% 0,14,21 0.4,nd,nd 1.6,0.3,0.1 Tomatoes 0.15% 0,29 0.2,nd 0.4,0.06 Grapes 0.15% 0,56 0.8,nd 0.8,0.05 Plums 0.15% 0,16 0.6,<0.05 0.15,<0.05 Coffee leavesc/ 0.5%(2X) 1/4,14,28 61,nd,nd 53,7,0.6 Strawberries 0.2% 0,14,21 0.2,nd,nd 0.4,0.3,0.1 Potatoes 0.2% 3,7 nd,nd nd,nd Apples 0.2% 1,7 0.25,nd 0.25,0.1 Black currants 0.2% 0,7 0.6,nd 1.2,1.6d/ Snap beans 0.2% 0.5,7 tr,nd 0.75,0.35 Wheat 700 cc/ha 7,14,21 nd,nd,nd 0.12,0.23,0.11 Sugarcane plant ca. 0.3 kg/ha 30 nd nd (4X) Typical maximum residues found following normal application (continued) Maximum residues when sampled, Spray Day respectively, ppma/ Crop application sampled Formothion Dimethoate Olives 0.12% (2X) 10,20 nd,nd nd,nd Olive oil 0.12% 10,20 nd,nd nd,nd Oranges peel 0.12% 15,29 0,17,0.31 1.30,1.70 pulp 0.12% 15,29 nd,nd tr,tr whole fruit 0.12% 15,29 0.12,nd 0.55,0.18 Grapefruit peel 0.12% 15,29,96 0.35,0.14,tr 2.10,1.20,0.22 pulp 0.12% 15,29,96 nd,nd,nd 0.14,tr,nd whole fruit 0.12% 15,29,96 tr,0.08,tr 0.49,0.27,tr leaves 0.12% 96 nd 0.63 Cole crops 0.2% 60-105 nd nd a/ tr - trace; nd - none detected. Fingings of more than 0.1 ppm dimethoxon are given in footnotes. b/ Up to 0.35 ppm dimethoxon found in other trials. Oxon generally less than 0.1 ppm at 21 days. c/ Deliberate overdose to study degradation. Test conducted in greenhouse. d/ 0.2 ppm dimethoxon FATE OF RESIDUES According to Sandoz (Anon., 1969) the active residues after treatment of plants with formothion are formothion, dimethoate, and dimethoxon. The data of Table I indicate that formothion has usually degraded to dimethoate almost completely after seven days with the exception of residues on the peel of oranges and grapefruits, on which 0.17 and 0.35 ppm residues of formothion were found after 15 days. A recent study on the metabolism of 32P-labelled dimethoate is of interest because dimethoate is the principal persistent residue of formothion. It was applied to bean plants four ways, and the only significant residue besides dimethoate was the oxygen analogue (Lucier and Menzer, 1968). Information on the fate of residues in mammals is scanty. However 14C-labelled formothion administered orally to the rat was completely metabolized, and elimination was essentially complete in 24 hours; the only metabolite of well known structure is said to be formothion acid (Anon., 1969); presumably this is the thiocarboxy derivative CH3O S \ " P-S-CH2COOH / CH3O which is also a metabolite of dimethoate in mammals (FAO/WHO, 1968). Evidence of residues in food in commerce or at consumption No definite data available. However, the statement is made that residues are rapidly degraded by high temperature, especially by cooking. METHODS OF RESIDUE ANALYSIS A residue method for formothion should determine the parent insecticide and the two metabolites, dimethoate and dimethoxon. The method utilized to obtain almost all the data of this report requires a thorough cleanup of the plant extract (several column chromatographies and binary solvent partitions) followed by paper chromatography with formamide as the immobile phase (Faderl, 1962). Recoveries are 70-80% for formothion, 85-95% for dimethoate and 70-80% for dimethoxon. Sensitivities are 0.04-0.06 ppm. Determinations are semi-quantitative (± 25%) below 3 ppm. A thin-layer chromatographic procedure is also described but it does not appear to have any significant advantage over the paper procedure (Anon., 1969). The paper chromatographic method, although acceptable, is not the method of choice today. Since formothion is usually converted to dimethoate within a week and the waiting interval after treatment is at least one week, only dimethoate (and possibly dimethoxon) is likely to remain at harvest. Methods for the analysis of dimethoate (and dimethoxon) already cited (FAO/WHO, 1968) should therefore be useful for formothion and perhaps directly applicable. An international interlaboratory study on methods for dimethoate residues in crops has recommended a colorimetric procedure devised by Frehse (Joint Dimethoate Residues Panel, 1968). Use of a gas chromatographic method with flame-photometric or thermionic detection should give improved accuracy, sensitivity, specificity, and reliability in analyzing for residues of formothion and its metabolites. Such a method will be more rapid and less subject to error because little or no cleanup is needed. As part of method development an exhaustive extraction (e.g. Soxhlet) should be used to check on completeness of extraction (Bowman et al., 1968). Gas chromatographic methods for qualitative detection of both formothion and dimethoate have appeared (Askew et al., 1969; Ruzicka et al., 1967; Ebing, 1968). A gas chromatographic procedure for multiresidue detection and analysis of organophosphorus pesticides is likely to prove suitable, and such a procedure should be established. NATIONAL TOLERANCES AND WITHHOLDING PERIODS Tolerance (ppm) Withholding Country (Various fruits and vegetables) period (weeks) Belgium - 2 Denmark - 1 E.E.C. 0.6 (proposed) - France - 1 Germany (Fed. Rep.) 0.6* 2 Great Britain - 1 Netherlands 0.5 2 Sweden - 3 (proposed) Switzerland 0.3 6** * 0.5 ppm dimethoate + 0.1 ppm formothion ** For cherry flies, 3 weeks APPRAISAL Formothion is a phosphorus-containing systemic insecticide used on a wide variety of crops in many countries to control many sucking pests and some biting and chewing insects. It is usually applied as a 0.1-0.2 percent spray prepared from a 25 or 40 percent emulsifiable concentrate (ingredients other than formothion undefined). Residues are generally 0.3-0.5 ppm or less after one or two weeks and at harvest usually consist of dimethoate (to which it degrades) and occasionally traces of dimethoxon. Information on mammalian metabolism is scanty. However, if the residue at harvest is dimethoate and its oxon, the information on dimethoate already on record (FAC/WHO 1968) should suffice. Formothion and dimethoate should be considered together since their significant residues appear to be identical at harvest; that is, the effect of applying dimethoate and formothion will be additive. No data are available on residues in meat and milk, in total diets, in foods in commerce, or in cooked or processed foods. A sensitive and reliable analytical method in needed for regulatory purposes. A gas chromatographic analysis with a flame photometric or thermionic detector in suggested. Specificity is high and sensitivity is usually 0.01 ppm or better. Methods of this kind have been published and should be evaluated. RECOMMENDATIONS FOR TOLERANCES, TEMPORARY TOLERANCES OR PRACTICAL RESIDUE LIMITS TEMPORARY TOLERANCES Pre-harvest Tolerance Crop interval, days ppm Strawberries 14 0.3 Blackcurrants 7 2.0 Insufficient data were available on which to base recommendations for grapes, wheat and citrus fruit. FURTHER WORK OR INFORMATION REQUIRED (before an acceptable daily intake for the parent compound can be established) 1. Short-term studies in a non-rodent mammalian species with cholinesterase determination. 2. Long-term studies in rats. 3. Information on ingredients in technical products produced by several manufacturers. 4. Data on animal metabolism and residues in meat and milk of animals consuming agricultural products treated in accordance with good agricultural practice. 5. Data on disappearance of residues during storage, processing and cooking. 6. Data on residue levels in raw agricultural commodities moving in commerce and in total diet studies. DESIRABLE 1. Metabolic studies in non-rodent mammalian species. 2. Observations in man. 3. Evaluation of a gas chromatographic method for residue analysis and for regulatory purposes. REFERENCES Anon. (1969) Formothion. An organophosphorus systemic insecticide. Submitted by the Swiss delegation to the Codex Committee on Pesticide Residues Askew, J., Ruzicka, J.H. and Wheals, B.B. (1969) A general method for the determination of organophosphorus pesticide residues in river waters and effluents by gas, thin-layer and gel chromatography. Analyst 94, 275-83 Bowman, M.C., Leuck, D.B. and Beroza, M. (1968) Procedures for extracting residues of phosphorus insecticides and metabolites from field-treated crops. J. Agr. Food Chem. 16, 796-802 Carshalton. (1965) WHO insecticide evaluation and testing programme. Stage I. Toxicity report. OMS 968. Unpub. Rept. from the Toxicology Research Unit, Carshalton Ebing, W. (1968) Gaschromatischer Rückstandnachweis von 47 phosphorhaltigen Insektizid-Wirketoffen nach einer Einheitverfahren. Pflanzenschutz-Berichte 38, 1-22 Faderl, N. (1962) Methode zur Bestimmung von Mikromengen organischer Phosphorinsektizide. Mitt. Geb. Lebensmittelunters. Hyg. 53, 154-75 FAO/WHO. (1968) 1967 evaluations of some pesticide residues in food. FAO/PL:1967/M/11/1; WHO/Food Add. 68.30. Joint Dimethoate Residues Panel. (1968) The determination of dimethoate residues in fruits and vegetables; report. Analyst 93, 756-66 Klotsche, C. (1961) Formothion, ein neuer systemicher phosphorsaureester geringerer Giftigkeit. Mitt. Lebensmitt. Hyg., 52; 340-9 Klotsche, C. and Rüttiman, G. (1965) Subacute dermal toxicity of Anthio (containing 24% of formothion as active ingredient). Unpub. Rept. produced and submitted by Sandoz, Ltd., Basle Klotsche, C. (1966) Toxikologische Untersuchungen mit dem systemischen phosphorsaureester Formothion. Int. Arch. Gewerbepath. Gewerbehyg. 22.246-61 Klotsche, C. (1969a) Formothion. An organophosphorus insecticide. Unpub. Rept. on animal toxicology submitted by Sandoz, Ltd., Basle Klotsche, C. (1969b) Formothion. An organophosphorus insecticide. Unpub. Rept. on residues in plants submitted by Sandoz, Ltd., Basle Lucier, G.W. and Menzer, R.E. (1968) Metabolism of dimethoate in bean plants in relation to its mode of application. J. Agr. Food Chem. 16, 936-45 Ruzicka, J., Thomson, J. and Wheals, B.B. (1967) The gas-chromatographic examination of organophosphorus pesticides and their oxidation products. J. Chromatogr. 30, 92-9 Sandoz. (1963) Toxicity of Anthio to birds. Unpub. Rept. produced and submitted by Sandoz, Ltd., Basle Sandoz. (1964a) Neurotoxicity of Anthio. Unpub. Rept. produced and submitted by Sandoz, Ltd., Basle Sandoz. (1964b) Toxicity of Anthio to birds (pheasants) Unpub. Rept. produced and submitted by Sandoz, Ltd., Basle Sandoz. (1968) Formothion. Unpub. summary report produced and submitted by Sandoz, Ltd., Basle Zehnder, K. (1961) Personal communication cited by Klotsche, 1961
See Also: Toxicological Abbreviations Formothion (WHO Pesticide Residues Series 2) Formothion (WHO Pesticide Residues Series 3)