AGP:1970/M/12/1 WHO/FOOD ADD/71.42 1970 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, 9-16 November, 1970. FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS WORLD HEALTH ORGANIZATION Rome, 1971 DICHLORVOS Explanation This pesticide was evaluated by the 1965, 1966 and 1967 Joint Meetings of the FAO Working Party and WHO Expert Committee on Pesticide Residues (FAO/WHO 1965, 1967, 1968). Since the previous publications, the results of some additional experimental work have been reported. This new work in summarized and discussed in the following monograph addendum. IDENTITY Composition of the technical product Technical dichlorvos contains not loss than 93 percent w/w of o,o-dimethyl-2, 2-dichlorovinyl phosphate and not more than 7 percent w/w of related compounds. EVALUATION FOR ACCEPTABLE DAILY INTAKE BIOCHEMICAL ASPECTS The rate of detoxification of dichlorvos appears to vary among different mammalian species. Based upon studies where dichlorvos was infused into the jugular vein of animals and the time to mortality measured, the detoxification rate for dogs, pigs, sheep and monkeys is 3.0, 5.5, 0.9 and 0.4 mg/kg/hr., respectively (Young, 1969). Studies in vivo and in vitro in the rat, rabbit, goat and cow using 32P-labelled or unlabelled dichlorvos (Casida et al., 1962; Hodgson and Casida, 1962) have previously been described, and the results of these experiments have fairly clearly established the fate of the phosphorous containing portion of the molecule (FAO/WHO, 1967). Rats were administered intraperitoneally with 0.5 mg of dichlorvos or 1.7 mg of its metabolite o-demethyldichlorvos. Both compounds were 32P-labelled. Dimethylphosphate was by far the most predominant urinary metabolite (64.1 percent); methylphosphoric acid and phosphoric acid accounted for a further 9.2 percent of the radioactivity and the remaining 1.7 percent was o-demethyldichlorvos. In the urine from the rats administered o-demethyldichlorvos directly, 21.2 percent was excreted unchanged, but the major metabolites were methylphosphoric acid and phosphoric acid (43.6 percent) and an unknown compound (metabolite E), which was chromatographically similar to an unknown metabolite obtained from rats fed the related insecticide trichlorphon. This compound was not found after administration of dichlorvos itself. The authors speculated that the compound was a conjugate of a non-toxic metabolite with glucuronic acid, or perhaps sulphuric acid, but this fact was not established. Based upon the results of this experiment, the cleavage of the vinyl group appears to greatly exceed o-demethylation (Bull and Ridgeway, 1969). Three male rats were exposed for one hour to the vapour of dichlorvos labelled with 14C in the vinyl group. A separate group of three male rats of the same age were given an oral dose of the same 14C-labelled dichlorvos. The excretion of the radioactivity of both groups in the urine, faeces and respired air was measured for four days, and the identity of the radioactivity in the liver of the inhalation group was also determined. Metabolism was rapid with both groups, and the rates and routes of excretion were very similar. The radioactivity in the livers of the group studied was found to be largely glycine and serine incorporated into the protein (Blair et al., 1970). Further information on the metabolism of dichlorvos is given in the section entitled "Fate of residues - In animals". The hydrolytic degradation of 32P-labelled dichlorvos was investigated using postmitochondrial supernatant fluid from rat liver and reduced glutathione as a methyl group acceptor. It was found that the rate of hydrolysis of dichlorvos was much greater than was found in some phosphorothiono insecticides. The monomethyl derivative was the major hydrolytic product (Palut et al., 1969). Based upon the experiments described above and those reviewed previously, the metabolic scheme for dichlorovos in represented is Fig. 1. Using purified human serum anticholinesterase, the I50 value for dichlorvos is 10-6M. An enzyme considered to have similar esteratic site, namely chymotrypsin, had a value of 10-3M (Arthur and Casida, 1957). More recently, I50 values for dichlorvos using human plasma and erythrocyte cholinesterase have been shown to be the same, 4.1 × 10-6M (Boyer, 1969). TOXICOLOGICAL STUDIES Special studies on reproduction Chicken embryo When 10 mg of dichlorvos or less were injected into the yolk sac of fertile eggs prior to incubation, there were no teratogenic effects (Roger et al., 1964). When 1 mg dichlorvos was injected in eggs on day four of incubation, borderline teratogenic signs (shortened body and legs) occurred (Roger et al., 1969).Rat Reproduction studies in rats fed 0, 0.1, 1,0, 10.0, 100 or 500 ppm in the diet through three successive generations did not show any significant effect on numbers and sizes of litters, survival of young or growth of young being suckled while dichlorvos was still being fed to the dams. Dichlorvos appeared to be without teratogenic effect (Witherup et al., 1965; see FAO/WHO, 1967). Dichlorvos was administered at unspecified levels intraperitoneally to female rats on day eleven after insemination. Doses of 20 mg/kg body-weight killed the animals; 15 mg/kg produced toxic signs and weight loss. There was no adverse effect on litter size or on the placenta at 15 mg/kg, but three foetuses from one of four litters available had omphaloceles, although none were observed in the six litters from control animals (Kimbrough, 1970). Rabbit A total of 168 adult female rabbits were artificially inseminated with 20-30 million viable and mobile rabbit sperm, and on days 6 to 18 of gestation were administered daily oral doses of 0, 6 or 18 mg/kg body-weight of dichlorvos by capsule, or 31 mg/kg on days 6 to 11 of gestation. Foetuses were recovered by Caesarean section. There was increased incidence of in utero and neonatal toxicity in the top dose group only, which was of doubtful significance. There were no teratogenic effects observed, based upon skeletal examination of the foetuses. Maternal mortality was increased in the 31 mg/kg group (Carson, 1969). In a separate experiment, 104 female rabbits were artificially inseminated with rabbit sperm and administered 0, 3 or 12 mg/kg body-weight of dichlorvos by capsule on gestation days 6 to 16 inclusive. Other rabbits were given higher doses, but they could not tolerate levels of 24 mg/kg or higher, and no live litters were produced from such groups. At 12 mg/kg, there was a significant reduction in the number of implant sites and foetuses compared to the control or lower dose group. At 3 mg/kg, the number of implant sites and foetuses were not lower than the controls. However, one deformed stillborn foetus was encountered from this group. Liver sections from parents and foetuses displayed no pathological abnormalities that could be related to dichlorvos treatment (Vogin, 1969). Pig Female pigs were fed dietary levels of 0, 200, 250, 288, 400, 500 or 750 ppm of dichlorvos for up to 37 months. The animals were first mated after they had received the test diet for six months, and the study was followed through two generations. Initially, a total of 22 females and one male were used, but after the first generation, four more males were used in order to prevent in-breeding. The males received 0, 288 or 400 ppm of dichlorvos in their diets. Dichlorvos did not affect either the numbers of litters or their size and the survival in the groups fed dichlorvos. A total of 490 piglets were examined and none showed anatomical abnormalities (Singh and Rainier, 1966). Special studies on mutagenicity Secondary roots of Vicia fabia were treated with varying concentrations of dichlorvos for one hour. Concentrations down to 1 mM were mostly lethal, and root tips surviving this and lower concentrations had a low mitotic index and showed c-mitotic effects, and chromatid breaks and gaps were observed. In another experiment, a streptomycin dependent, Sd-4, mutant of E. coli was treated in the lug phase at cell densities of about 108 cells/ml during one hour. The LD50 concentration for the bacteria of dichlorvos in the medium was estimated to be between 10 and 60 mM (2200 ppm and 1.3 percent). At concentrations of 1-4 mM, which caused no significant killing, an increased mutation rate was observed when compared with controls and measured as revertants to streptomycin independency (Löfroth, 1969 a, 1969 b). Segments of Vapona-strips of various sizes were placed in an inflated 6000 ml plastic bag containing an uncovered petri-dish with germinating onion seeds. There was no retardation of root growth or mitotic frequency, but the number of chromosome aberrations, when scored at anaphase, increased constantly with increasing size of the Vapona-strip segment. The lowest concentration used was about ten times as great as one strip in a 1000 ft3 room (Sax and Sax, 1968). Attention has been drawn to the alkylating property of a number of alkyl phosphates (Preussman, 1967). For this reason, possible mutagenic effects have been looked for. Calf thymus deoxyribonucleic acid (DNA) was incubated with varying concentrations of dichlorvos solution. In a typical experiment with 2 percent dichlorvos incubated for 66 hours, there was a yield of 1 percent of N-7-methylguanine from the available guanine. Similar experiments with deoxyguanosine gave a 3-5 times higher yield of N-7-methylguanine than from DNA (Löfroth, 1970). Eight mice were injected intraperitoneally with 10-20 mg/kg body-weight of dichlorvos. After 23 hours, 100 cells of their bone marrow were examined. The incidences of aberrations were not greater than observed in four control mice (Hunter, 1970). A human subject was exposed to 1 mg/m3 of dichlorvos for six hours. A sample of blood cultured for leucocytes for 72 hours showed no increase in chromosome aberrations in 100 cells compared to the pre-exposure values (Hunter, 1970). No chromosomal aberrations were seen after examination of blood samples from a family of four female and one male human subjects which had been exposed to dichlorvos strips. A control group had two male and three female subjects (Hine, 1970). Special studies on carcinogenicity Rat Rats (24) were injected subcutaneously, once weekly, with trichlorphon (Dipterex), of which dichlorvos is a known metabolite. Despite a high (unspecified) dosage and a latent period of almost 800 days, only two local sarcomas developed at the site of injection. No data on controls are given. (Preussmann, 1968). In a subsequent publication, Preussmann et al. (1969) refer to the above experiment as a "negative result, presumably due to the very rapid metabolism of Dipterex." Acute toxicity After direct infusion of dichlorvos into a peripheral vein at various rates, pigs can survive an LD50 dose each hour for at least nine hours (page, 1970). The acute toxicity of dichlorvos has been compared with the metabolites. The LD50 values in mg/kg body-weight for intraperitoneal administration to mice are: dichlorvos, 28; o-demethyldichlorvos sodium salt, 1500; dichloroacetaldehyde, 440; 2,2-dichloroethanol, 890; dichloroacetic acid, 250; sodium dichloroacetate, >3000; mono- and dimethylphosphoric acid mixture, 1500; sodium monodimethylphosphoric acid mixture, >3000 (Casida, et al., 1962). Short-term studies Comparative studies on inhalation toxicity Guinea pigs have been exposed for five hours per day for five consecutive days to a concentration of 130 mg dichlorvos/m3 of air without visible effects on health. Rats and mice were not visibly affected by similar exposures to 50 mg/m3. At concentrations above 50 mg/m3, the mice became visibly distressed, and prolonged exposure to 80 mg/m3 was frequently lethal. Rats were less severely affected than mice. In all cases, stress reactions were either apparent during the first hours of exposure or they did not occur at all. No cholinesterase determinations were made (Stevenson and Blair, 1969). Three samples of dichlorvos were investigated (82.5 percent pure, Russian produced, 40% pure, Japanese, and 100% pure from Swiss origin); the oral LD50 for Russian and Japanese samples was similar (87 mg/kg body-weight for mice and 65 mg/kg for rats). The Swiss sample was less toxic: oral LD50, mice: 108 mg/kg. Rabbits were found to be the most sensitive species (oral LD50, Russian sample: 22.5 mg/kg). Inhalation toxicity was investigated for exposure periods of four hours, The highest concentration which could be produced (30 mg/m3) was lethal for mice and rats. The LD50 (four hours) for mice was 13.2 mg/m3 and for rats 14.8 mg/m3. At 5 mg/m3, blood cholinesterase activity in rat was 69 percent of pre-exposure values. A level of 0.5 mg/m3 caused no observable effects in blood cholinesterase. By interpolation, those concentrations of dichlorvos in air were calculated which would after four hours exposure cause a 50 percent drop in blood cholinesterase activity in rat (I50 = 2.4 mg/m3) or a 25 percent drop (I25 = 1 mg/m3). Rats and rabbits were exposed for 4 hours/day for a period of four months to an average dichlorvos vapour concentration of 1 mg/m3 of air. No visible signs of intoxication occurred. Cholinesterase activity in the serums erythrocytes, medullary tissue and liver of rats varied throughout the experiment within the limits of the norm or was slightly depressed. After four months, the depression of cholinesterase activity in the medulla averaged 8 percent in the liver, serum and erythrocytes the depression was 30 percent 22 percent and 24 percent, respectively. Blood sugar content, blood sugar curves after loading with galactose, synthesis of hippuric acid after loading with sodium benzoate and duration of Hexenal induced sleep were not affected. Rabbits exposed under the same conditions showed 11-30 percent reduction of serum cholinesterase activity and 28-30 percent reduction of erythrocyte cholinesterase activity. It was concluded that 1 mg/m3 was the threshold for rats and rabbits which caused an insignificant and variable depression of cholinesterase. It was also the threshold for cats. Rats exposed to an average concentration of 5.2 mg/m3 for 4 hours/day over a period of two months remained visibly healthy. After 15 days, the cholinesterase depression in the medulla was on an average 22 percent in the serum 58 percent in the erythrocytes 22 percent. The duration of drug induced sleep was increased from 33 minutes to 57 minutes. The blood sugar level was unchanged, but the rise of the curve after galactose loading was far higher than in the controls. After 60 days, the blood sugar curve was affected, the duration of Hexenal induced sleep was the same as in the controls, and cholinesterase activity in the medulla fell by an average of 31 percent, in the liver by 35.5 percent, in serum by 52 percent and in erythrocytes by 58 percent. At 8.2 mg/m3 for 45 days, rats lost appetite and weight, and in some cases trembling of the head and entire body was noted. Signs of intoxication occurred in some of the rats and some died (Sasinovich, 1968). OBSERVATIONS IN MAN A total of 10 human subjects, in groups of two, ingested pellets of a slow-release formulation of dichlorvos in daily dosages, doubling weekly, up to 8 mg/kg body-weight/day for periods up to three weeks; one group of two received 16 mg/kg/day for 5.5 days. Doses above 1 mg/kg were administered in two equally divided doses per day. The majority of the clinical side effects occurred after the doses had reached the 8 mg/kg/day level. Most of the subjective clinical complaints referred to the gastrointestinal tract, but none of the studies had to be terminated because of side-effects. The study with 16 mg/kg/day was terminated because of the magnitude of the red blood cell cholinesterase activity depression. Doses of 1-8 mg/kg/day for up to 21 days produced an average plasma cholinesterase activity depression of 70 percent irrespective of dose or duration. Doses of 16 mg/kg/day for 5.5 days produced approximately 90 percent depression of the plasma cholinesterase activity. Red cell cholinesterase activity after multiple dosing of 1 mg/kg/day was 11-27 percent depressed. Doses which reached 8 mg/kg/day caused 45-86 percent depression. At 16 mg/kg/day, depressions of 90 percent were observed. Single doses of dichlorvos needed to be more than 4 mg/kg in order to cause measurable red cell cholinesterase depression. At single doses of 8 mg/kg and higher, maximal plasma cholinesterase inhibition was present and did not increase at higher dosages. Significant and consistent erythrocyte cholinesterase inhibition occurred after single doses of 16 mg/kg (36 percent inhibition) and 32 mg/kg (46 percent inhibition). About 50 percent of the dichlorvos was still present in the excreted pellets, Therefore, the doses absorbed were assumed to be about one half of the above levels (Hine et al., 1966). In another study, 12 male subjects ingested 5 mg of dichlorvos daily in three equal portions with their food until the plasma cholinesterase fell to 75 percent of the pre-exposure value. The time required averaged 12 to 20 days. No effect occurred on erythrocyte cholinesterase. One of the subjects continued the exposure for a total of 77 days. A questionable erythrocyte cholinesterase depression of 10-15 percent occurred during part of the exposure period, but in the beginning and at the end of the exposure period, this activity was normal or slightly above (Hunter, 1970). Six human subjects were exposed to concentrations of dichlorvos in the air, the maximum level being 52 mg/m3 for 62 minutes and the maximum period 240 minutes to 13 mg/m3. No clinical abnormalities were observed, subjective complaints were only related to the dryness of the atmosphere, but the plasma cholinesterase activity was decreased; the extent of the depression was proportional to the product of the concentration and time of exposure. During some of these exposures, a questionable depression of erythrocyte cholinesterase occurred, but the extent of depression was not directly related to the product of the concentration and time of exposure (Hunter, 1969). In six homes in Tucson, Arizona, 18 individuals submitted to exposure to dichlorvos strips and six others served as controls. In order to maximize exposure, the study was made during the winter months, and strips were hung in all rooms in the houses at the rate of 1 strip/1000 ft3. Maximum concentrations of 0.1-0.2 mg/m3 occurred within the first week, levelling off to 0.05-0.10 mg/m3 during the remainder of the regular exposure period of 28 days. Samples of meals and beverages were collected at various intervals. During the exposure period, more than 85 percent of the individual meals contained less than 0.3 ppm dichlorvos and more than half the meals 0.1 ppm or less. Physical examination, complete blood counts, blood chemical profiles and plasma and erythrocyte cholinesterase measurements were performed at various intervals. Levels of dichlorvos in the food eaten ranged from 0.1 to 0.3 ppm. There were no clinical abnormalities evident, nor were there any significant differences in plasma and red-blood cell cholinesterase, blood count and blood chemical profiles between the exposure and pre-exposure periods (Shell, 1970a). A study was made in Italy of the effect of continuous exposure to insecticide levels of dichlorvos by patients in hospital wards. Control values for plasma and erythrocyte cholinesterase were established for 250 healthy male and 100 healthy female adults not exposed to dichlorvos. The variation in the activity of the cholinesterases of the individuals when measured at different times, depending on the time-interval, varied on average between 1.4 and 4.4 percent for the erythrocyte cholinesterase and on average between 5.0 and 14.2 percent for plasma cholinesterase. These were average variations measured in ten to 20 individuals. In single individuals, the maximum variation was 7.7 percent for erythrocyte cholinesterase and 23.2 percent for plasma cholinesterase. A total of 44 adult male patients with diseases other than liver diseases were exposed to atmospheric concentrations of dichlorvos ranging from 0.02 to 0.1 mg/m3 for periods of 3 to 29 days. Another similar group of 22 male patients was exposed to dichlorvos concentrations of 0.1 to 0.28 mg/m3 for periods from 3 to 16 days. Only in the case of five patients exposed 24 hours per day at levels above 0.1 mg/m3 was there a depression of plasma cholinesterase (35 to 72 percent) below the pre-exposure level. There was no depression of erythrocyte cholinesterase nor were any symptoms typical of cholinergic stimulation observed, in any patients. With six other patients suffering from liver disease exposed in the same room, all showed a reduction in plasma cholinesterase (25 to 66 percent of pre-exposure values). Reduction was evident even in patients exposed at levels below 0.1 mg/m3. These patients, because of their liver disease, had already before exposure a considerably reduced plasma cholinesterase activity. However, there was no depression of erythrocyte cholinesterase nor any symptoms of poisoning, in spite of the fact that in these patients with liver insufficiency had a 40 percent depression in plasma cholinesterase lasting for one to three weeks after the removal from exposure. In babies, sick children and women in labour or postpartum exposed to dichlorvos, a similar situation to that of sick adults was seen; depression of plasma (but not erythrocyte) cholinesterase was observed only in some cases when the dichlorvos level was above 0.1 mg/m3. Again, no symptoms were seen. Assuming an inhaled volume of 10 m3/24 hours in adults and 1.4 m3/24 hours in children, the authors calculated that a reduction in plasma cholinesterase for a calculated daily inhalation of 1.7 mg of dichlorvos could occur in adults with normal liver function and 0.2 mg for children. They further calculated that the amount of dichlorvos inhaled which might produce a plasma cholinesterase depression is about the same for adults and children, viz. 0.028 to 0.030 mg/kg body-weight/day. In patients with liver malfunction, daily inhalation intake of 0.34 mg (equivalent to 0.005 to 0.006 mg/kg body-weight/day was sufficient to produce plasma cholinesterase depression (Cavagna et al., 1969; Cavagna and Vigliani, 1970). In two nurseries 22 healthy newborn babies were kept from birth for a period of five days for 18 hours/day in an atmosphere in which average dichlorvos concentrations of 0.15 - 0.16 mg/m3 were maintained. Blood cholinesterase levels were determined at birth and after five days. There were no effects on the health of the babies nor on the activity of their plasma or erythrocyte cholinesterase (Cavagna et al., 1970). In a preliminary study with asthmatic patients, there appeared to be no subjective worsening of asthma during exposure to dichlorvos at levels of 0.1 to 0.2 mg/m3 for two consecutive days. In some cases, an increase in airways resistance and sensitivity to acetylcholine occurred during and after exposure, but these manifestations may be related to other factors (e.g. poor ventilation) than to dichlorvos. Further work in this field is reported to be in progress (Vigliani, 1970). COMMENT On the basis of recent and earlier studies the metabolic pattern of dichlorvos when fed to mammals is now fairly clearly elucidated. Evidence is presented that cleavage of the dichlorovinyl group proceeds much more rapidly than o-demethylation. Information on the exposure of man to varying levels of dichlorvos vapour is available, but such studies do not include assessment as to whether the metabolism is the same after inhalation as after oral intake. Data on the toxicology of the metabolites dichloracetaldehyde and dichloroethanol are considered pertinent in evaluating the toxicity of dichlorvos. A long-term study in rats reported in the monograph from the 1967 Joint Meeting is considered adequate. Feeding studies in rats and rabbits did not show any indication of a teratogenic effect. Additional information on feeding dichlorvos to man has become available, and the no-effect level previously determined in man is used as a basis for establishing an acceptable daily intake. TOXICOLOGICAL EVALUATION Level causing no toxicological effect Man : 0.033 mg/kg body-weight/day ESTIMATE OF ACCEPTABLE DAILY INTAKE FOR MAN 0-0.004 mg/kg body-weight RESIDUES IN FOOD AND THEIR EVALUATION USE PATTERN Pre-harvest treatments In addition to its use on beef and dairy cattle and goats, sheep and pigs, and in and around buildings which house these animals, dichlorvos is finding increasing use on fruit and vegetable crops. Fruit and citrus crops account for more than one third of the agricultural use. Rice accounts for just over one quarter and field crops, including vegetables, approximately one quarter of the agricultural uses. There are minor uses on grapes, glasshouse crops, mushrooms, tobacco, tea, coffee, cocoa and miscellaneous crops. Dichlorvos is known to be registered in at least forty eight countries. Recently it has been introduced as an anthelmintic for pigs, poultry and horses, being administered in the form of plastic granules for this purpose. Post-harvest treatments Dichlorvos is being increasingly used for the control of insect infestation in stored grain. For this purpose it is used as an aerosol or as impregnated resin strips, which are applied in the overhead spaces of storage bins. In some countries, dichlorvos preparations have also been registered for incorporation into stored grain either as a dust, as a direct spray or as an aqueous emulsion. Dichlorvos aerosols, automatic dispensers, sprays and resin impregnated strips are used in many countries for the control of insect infestations in stores, in transport and in warehouses holding processed food. Food processing establishments are also treated with dichlorvos in one of the above forms. Other uses As defined in the monograph of the 1967 Joint Meeting (FAO/WHO 1968), dichlorvos is used extensively in the public health field (homes, hospitals, etc.). It is also used in aircraft during flight. Trials are being carried out on its use by aerial application against insect pests of forests, pastures and field crops. RESIDUES RESULTING FROM SUPERVISED TRIALS Crops grown in the open Table I gives typical results of the analysis for dichlorvos residues following controlled application to various fruit and vegetable crops (Ciba 1970). Crops grown under cover In glasshouses, the rate of decay of dichlorvos residues is fast, with a half-life of approximately one day. Table II gives results of residue trials carried out on crops growing under cover (Ciba 1970). Livestock and poultry Trials in the United States demonstrate that the spraying of dichlorvos on livestock (including cattle, sheep, goats and pigs) and poultry is unlikely to produce significant residues in meat, milk or eggs. When dichlorvos was applied as a single pour-on application of 1 percent at the rate of 16.5 ounces per cow, no residues were TABLE I Crops grown in the open Residue Analytical Limit of Country Commodity Rate of Interval ppm Method Detection Application to Harvest ppm Switzerland Apples 0.05% ai 1 day 0.05 ChE1 0.05 2 days <0.05 Peaches 0.05% ai 0 day 0.8 ChE 0.02 1 day 0.55 " 0.02 2 days 0.35 " 0.02 Strawberries 0.05% ai 2 days 0.35 ChE 0.02 3 days 0.06 " 0.02 8 days <0.02 " 0.02 Carrots 0.025% al. 28 days <0.05 ChE 0.05 31 days <0.05 " 0.05 Cauliflower 0.05% ai 0 days 0.37 ChE 0.04 5 days <0.04 " Brussels 0.05% ai 0 days 1.35 ChE 0.04 sprouts 5 days <0.04 Cabbage 0.05% ai 0 days 1.5 ChE 0.05 1 day 0.14 2 days 0.05 3 days <0.05 Spinach 0.05% 0 days ChE 0.05 1 day 1.6 2 days 0.06 3 days <0.05 TABLE I (Cont'd.) Crops grown in the open Residue Analytical Limit of Country Commodity Rate of Interval ppm Method Detection Application to Harvest ppm Switzerland Lettuce 0.05 0 days ChE 0.05 3 days <0.05 " 0.05 French beans 0.05 0 days 0.3 ChE 0.05 <0.05 Cucumber 0.05 9 days <0.05 ChE 0.05 1 ChE = cholinesterase inhibition method TABLE II Crops grown under cover Residue Analytical Limit of Country Commodity Rate of Interval ppm Method Detection Application to Harvest ppm Switzerland Tomato 20 mg/m3 0 days 0.2 ChE1 0.04 1 day 0.06 " 0.04 2 days 0.04 " 0.04 England Lettuce 130 mg/m3 0 days 78 ChE 0.04 1 day 4.9 70 mg/m3 1 day 2.1 2 days 0.4 3 days 0.2 Germany Lettuce 30 mg/m3 0 days 3.4 B.T.2 0.04 1 day 1.8 3 days 0.3 20 mg/m3 0 days 2.4 B.T. 0.04 1 day 0.9 2 days 0.4 3 days 0.16 Switzerland Cucumber 20 mg/m3 0 days 7.3 ChE 0.04 2 days 0.15 U.S.A. Mushroom 130 mg/m3 1 day 0.1 ChE 0.04 TABLE II (Cont'd.) Crops grown under cover Residue Analytical Limit of Country Commodity Rate of Interval ppm Method Detection Application to Harvest ppm England Mushroom 160 mg/m3 0 days 25 D.N.P.3 0.04 1 day 0.9 3 days 0.27 160 mg/m3 1 day 0.9 D.N.P. 0.04 2 days 0.05 100 mg/m3 0 days 0.3 D.N.P. 0.04 1 day 0.1 1 ChE - cholinesterase inhibition method 2 B.T. - bioassay 3 D.N.P. - 2,4-dinitrophonylhydrazine colorimetric method detectable at the level of sensitivity of the method (0.1 ppm) in any of the milk samples collected from 4-5 hours to 5 days, after treatment (Singh, 1965). In another study (Noetzel, 1964), groups of Holstein and Guernsey cattle received dermal applications, twice daily for 28 consecutive days, of 2 ounces of 0.5 percent dichlorvos and 1 percent dichlorvos, respectively. No residues were detected in the milk (<0.02 ppm). In a similar study (Wisconsin, 1968) in which dichlorvos was dermally applied to lactating dairy cows at the rate of 1 ounce/cow or 3 ounces/cow of a 1 percent dichlorvos for thirty days, milk samples from 1 through 7 days and thereafter at 3 day intervals were analysed for residues of dichlorvos and dichloroacetaldehyde (DCA). No residues were detectable in any of the samples (<0.05 ppm dichlorvos, <0.01 ppm DCA). In other extensive trials (Wisconsin, 1968/1969) Brahma cattle, goats and sheep were sprayed 3 times to saturation (1 gallon) at 14 day intervals with 0.025 percent dichlorvos emulsion. No residues of dichlorvos were detectable at the limits of sensitivity in tissues or organs (including renal, omental and subcutaneous fat, muscle, liver and kidney) of cattle (<0.25 ppm), goats (<0.25 ppm) or sheep (<0.05 ppm). Ivey and Claborn (1969a) sprayed lactating dairy cows for 31 consecutive days with 2 ounces of a 1 percent dichlorvos insecticide spray solution. Milk samples were taken at 2 hours, 1, 2, 4, 8, 16, 24 and 31 days. One day after final treatment, the cows were slaughtered and samples of renal, omental and subcutaneous fat, muscle liver, kidney and blood were taken. Using a highly sensitive flame photometric method (Ivey and Claborn, 1969b) with improved clean-up technique, no residues of dichlorvos were detected in milk (<0.003 ppm) or body tissue (<0.002 ppm). Pigs were fed daily at the high dosage of 9 450 ppm dichlorvos for 90 days (Singh, 1964). Dichlorvos residues were not detectable (<0.05 ppm) in tissues and organs including spleen, kidney, small intestine, lung, heart, muscle and liver 0 and 1 day after last treatment. Analysis of meat, fat, liver and eggs derived from poultry sprayed with 2 ounces of diluted 2 lb/gal dichlorvos insecticide E.C. (0.295 ppm grams DDVP/bird) showed residues of equal to or less than 0.03 ppm when samples of tissue were taken 6 hours, 3, 5 and 7 days after application. Eggs were collected 1, 2, 3, 4, 5, 6 and 7 days after treatment (Shell, 1970b). Poultry exposed to dichlorvon in the form of 2 percent granules (2 lb/100 square feet or 4 lb/100 square feet), or to resin strands containing 20 percent dichlorvos (1 foot of strand/1 foot of cage) caused no residues in either tissue or eggs (<0.02 ppm) (Lancaster, 1962; Loomis and Hodel, 1965; Lancaster, 1963; Shaw, 1964; Nelson, 1968). TABLE III Dichlorvos residues following treatment of animals Reference Animal and Route Quantity Frequency Tissue Residue Analysed1 ppm cm3 days Singh, Cows, pour on 480 1 Milk2 <0.1 1965 Noetzel, Cows, dermal 60 28×1 Milk <0.02 1964 Wisconsin, Cows, dermal 90 30×1 Milk <0.05 1968 Wisconsin, Cows, dermal 4540 3×14 Fat, muscle <0.25 1968, 1969 Goats, dermal 4540 3×14 Fat, muscle <0.25 Sheep, dermal 4540 3×14 Fat, muscle <0.05 Ivey & Claborn, Cows, dermal 60 31×1 Milk <0.003 1969a Cows, dermal 60 31×1 Fat, muscle, <0.002 etc. ppm Singh, Pigs, oral 9450 90×1 All tissues <0.05 1964 g Shell, Chickens, dermal 0.3 1 All tissues3 <0.03 1970b Chickens, dermal 0.3 1 Eggs4 <0.03 Lancaster, Chickens, vapour - 30×1 All tissues <0.2 1962 TABLE III (Cont'd.) Dichlorvos residues following treatment of animals Reference Animal and Route Quantity Frequency Tissue Residue Analysed1 ppm cm3 days Loomis, Chickens, vapour - 30×1 Eggs <0.02 1965 Nelson, Chickens, contact - 28×1 All tissues <0.03 1968 Chickens, contact - 28×1 Eggs <0.05 mg Pitts, Chickens, oral 8.5 over 10 Meat <0.01 1961 Chickens, oral 8.5 over 10 Eggs <0.04 1 Waiting period Nil unless otherwise indicated 2 Waiting period 4 hours and 5 days 3 Waiting period 6 hours 4 Waiting period 1 day and 7 days Uses in the protection of stored products Grain, meals and flour Many of the published and unpublished reports available on the rates of loss of dichlorvos from grain, meals and flour are not reliable, because the investigators have used unsatisfactory methods to recover any residues of dichlorvos from the treated grain and cereal products (see comments under Methods of Residue Analysis). Residues of dichlorvos in flour resulting from the use of resin strips in a flour mill have been investigated (Somme, 1967). Strips were hung at the rate of 1/1000 feet3, and samples of flour made from grain stored in the mill were taken 4 days and 2´ weeks after production had rebegun. Analysis showed that residues of 0.04 ppm and 0.06 ppm dichlorvos were present in the flour and that bran samples averaged 0.09 ppm dichlorvos. In another trial (Shell, 1966a) a "Vapona" strip was hung inside a covered flour bin of 600 ft3 total capacity. A sample of flour drawn from a conveyer belt taking flour from the bottom of the bin one week and two weeks after fitting the strip in a nearly full bin was found to have a dichlorvos residue of 0.03 ppm. Samples taken after the third week and after the bin had been refilled with flour, at the end of the fifth week, contained residues no greater than the limits of detection, Wheatings exposed at a rate of one "Vapona" strip per bin of 1150 feet3 for ten hours to six weeks after the strips had been hung also did not contain residues in the surface layer above the limit of detection (ca. 0.02 ppm), (Shell, 1965a). Dichlorvos has been successfully used to control Oryzaephilus surinamensis and Sitophilus granarius (Green and Wilkin, 1968). The insecticide was sprayed into the air stream from a motorized knapsack sprayer through to a perforated lance which was inserted into the grain. Dichlorvos residues were higher on wheat than on barley and greatest in places where dust and frass had accumulated. When dichlorvos was injected at 20 ppm, the highest residue found after one day was 30 ppm in a dusty pocket of wheat, and this fell to 5.3 ppm after six days. The range of residues reported for barley were 5.8-24.0 ppm after one day, falling to 0.9-1.4 ppm after six days. However, the method of analysis may not have extracted all the dichlorvos from the grain (See Methods of Residue Analysis). In a laboratory study Kirkpatrick et al., (1968) demonstrated that dichlorvos residues of 3.8-8.0 ppm decreased 89 percent and 76 percent, respectively, in 28 days. An unpublished study was conducted by USDA Plant Pest Control (Padget, 1968). Wheat grain was treated with 4, 6 and 8 ppm of dichlorvos formulated from a 25% dichlorvos emulsifiable concentrate. Residues on the wheat were reported to be less than 2 ppm by the third day after application, and by 14 days all dichlorvos residues were <0.5 ppm. Details of the analytical method used were not given. Rice Shell Chemical Company (1969) conducted an extensive study on rough rice and rice products treated for rice weevil control. Rough rice, stored as 45 lb lots were each sprayed with 20 ml of a solution containing 1.5 percent, 1 percent and 0.5 percent dichlorvos prepared from a 23.5% E.C. After treatment, the rough rice was mill processed and the products (brown rice, rice bran, milled rice and rice hulls) were collected. Residue samples were taken 6 hours, 1, 5, 10, 20 and 30 days after application. Dichlorvos residues in the rough rice one day after treatment were 7.3, 4.6 and 1.6 ppm for the 1.5 percent, 1 percent and 0.5 percent applications, respectively. After 30 days, residues for all three treatment rates had dissipated to less than 1.0 ppm. Residues in brown rice and milled rice, after 30 days, were <0.05 ppm for all treatment rates. Rice bran treated at the highest rate contained 0.4 ppm and rice hulls 3 ppm dichlorvos after 30 days. Soybeans Soybeans stored in open bins were exposed to resin strips for intervals ranging from 13 to 126 days (Shell, 1970c). Samples were taken through depths of 0-2" and 2-4" from the surface and the residues observed are summarized in Table IV. TABLE IV Average residues on soybeans after exposure to dichlorvos 0-2 inch depth 2-4 inch depth Period of Strips per 1000 ft3 Strips per 1000 ft3 Exposure, 1 1.5 1 1.5 Days Residues, ppm Residues, ppm 13 .20 - .06 - 28 .06 - .03 - 42 .11 .07 .08 .03 57 - .08 - .03 70 .07 .08 .04 .03 84 .04 .04 .04 .03 TABLE IV (cont'd) Average residues on soybeans after exposure to dichlorvos 0-2 inch depth 2-4 inch depth Period of Strips per 1000 ft3 Strips per 1000 ft3 Exposure, 1 1.5 1 1.5 Days Residues, ppm Residues, ppm 97 .04 .075 .04 .06 111 .05 .04 .04 .04 125 .04 .04 .04 .04 Temperature - 65-96°F Rel. Humidity - 48-76 percent A test conducted by USDA, Stored Products Insect Research and Development Laboratory (Padget, 1969), demonstrated that dichlorvos residues on soybeans decrease rapidly. Initial residues of 5 ppm decreased rapidly to 0.5 ppm or less after one week of storage at 80°F. Only trace amounts (0.1 ppm or less) of dichlorvos were found on the soybeans after the second week of storage. Coffee beans Residues in bagged coffee beans were examined (Shell, 1965a) following storage in a warehouse using resin strips at either one per 1000 cubic feet or 1 per 2000 cubic feet. Some natural ventilation was allowed for 9 hours daily. Samples taken after 15 weeks storage from the surface and interior of the bags did not contain detectable residues (limit 0.01 ppm), but one sample exposed at the rate of one strip per 2000 cubic feet did contain residues of 0.02 ppm, just above the detection limit. Cocoa beans In a trial with stacks of bagged cocoa beans carried out in the same warehouse and under similar conditions (Shell, 1965a) as the above coffee, residues in a bulked sample of cocoa from the top of the stack reached 0.04 ppm, although none were detected in comparable samples from the side of the stack. In a further trial (Shell, 1965a) with another stored supply of cocoa beans, detectable residues (0.01 ppm) were not found after 14 weeks exposure. Another trial (Shell, 1965b) was undertaken in which sacks of cocoa beans were exposed for three months. Surface samples taken from the top sacks were found to have a maximum content of 0.04 ppm, whilst sub-surface samples and samples from the middle of the sack did not contain detectable residues. In a further trial in which exposure was 8 weeks, the maximum residue found in surface samples from the top sacks was only 0.02 ppm. This was mainly in the husks (0.03 ppm); the kernels contained only 0.01 ppm. In a series of trials in Switzerland (Ciba), dichlorvos was applied to cocoa beans in silos at rates ranging from 10 ppm through 100 ppm to 500 ppm. After three weeks storage at 15°C, the residues had fallen to <0.1, 13 and 50 ppm, respectively, on the raw beans. When beans were then roasted, the residues fell to <0.1, 0.2 and 0.5 ppm, respectively. In the samples treated at 500 ppm, most of the residue was in the mash cake with a minimum in the cocoa butter. Further trials (Ciba) showed that the residues in cocoa beans treated at the rate of 12 ppm with dichlorvos emulsion fell from 0.8 to 0.09 ppm during 28 days in storage at 20°C. Groundnuts Sacks of bagged groundnuts (Shell, 1966b) were exposed for 82 days in a warehouse at the recommended rate of one strip/1000 cubic feet. One residue sample taken from the surface (2 cm) layer of the top sack was found to contain 0.10 ppm dichlorvos, but two other similar samples were found to contain only 0.01 ppm dichlorvos. One sample from the middle of the top sacks contained 0.03 ppm, but in two further samples no residues were found. Residues arising from the exposure of foods in processing plants Plants producing foods not processed further after exposure In a cheese factory, in Minnesota, strips were placed throughout the milk receiving and processing areas (Shell, 1964). A monitoring experiment showed that the finished Cheddar cheese did not contain detectable dichlorvos and DCA residues (i.e. below 0.03 ppm and 0.01 ppm, respectively). Similar trials in butter and ice cream factories (Shell, 1965c) showed negligible residues of dichlorvos and DCA in both products. In tests carried out in cooperation with the German Public Health Service in a cheese factory, Lindenberg, Edam and Danish Tilsit cheese (each containing 45% fat) were exposed for four hours. Cheese samples were cut into 20 mm thick slices following normal practice, and exposed for a period of 30 to 60 minutes. Residues, which were determined in the outermost 1 mm layers, were not detectable (Anon., 1966). Strips were hung in a cheese aging room at the rate of one strip/1000 cubic feet. Samples of cheese taken after six and ten weeks of exposure showed residues of <0.04 to 0.10 ppm (Shell, 1965a). In the case of milk (Shell, 1965d), whole milk was exposed in open milk churns for periods of 24 and 48 hours in rooms where fresh strips were installed at the rate of one strip/1000 cubic feet. None of the representative samples taken contained measurable amounts of dichlorvos (detection limit 0.02 ppm). In a study in Kentucky (Shell, 1965d), strips were installed in a farm dairy at the rate of one strip/1000 cubic feet. The air was humid and the temperature about 75°F. The milk samples were taken after being passed through an open coil cooler during the period 0-14 days after the strips were hung. Residues of dichlorvos ranged from the limits of detection to 0.04 ppm. Plants handling foods which are processed after exposure Fresh strips were suspended in areas of a plant where grading and filleting of fish was carried out, and after two days exposure, residues in the prepared product were not detected (detection limit 0.05 ppm) (Shell, 1965e). Exposure tests were also carried out in a slaughterhouse (Shell, 1965c). The various products, beef, liver, T-bone steak, hamburger, beef tongue and heart, pork liver, pork sausage, pork chops, pork tongue and heart were exposed to dichlorvos resin strips during the time normally required for processing. None of them showed any dichlorvos or DCA residues; detection limit was 0.01 ppm. In this test, a sample of bacon showed a residue of 1.4 ppm dichlorvos; DCA was not found (detection limit 0.01 ppm). It was subsequently found, however, that this particular sample had been returned to the cooling room and was exposed for several days longer than normal. Further, data cited later (Shell, 1966c) show that residues of dichlorvos are removed upon cooking; specifically, bacon is cited as an example. An experiment on similar lines was also conducted in a sausage factory where strips were installed throughout the processing areas (Anon, 1966). No residues were detected in either meat or finished sausages. FATE OF RESIDUES In animals Page (1970) has reviewed the extensive literature on the metabolism of dichlorvos and in the same paper has reported on extensive trials designed to show the metabolic fate and tissue residues of dichlorvos following oral administration. The results of this work and earlier studies are summarized in the metabolic chart given under "Biochemical aspects" (Fig. 1). A series of experiments have been conducted recently in pigs to provide further information on the metabolic fate of the dichlorvos portion of the dichlorvos molecule. In a preliminary study, 1-14C labelled dichlorvos was infused into the isolated duodenal loop of anaesthetized male pigs for four hours at the rate of 1 mg/kg body-weight/hour. Urine, bile and peripheral and portal blood were collected hourly and analysed for dichlorvos and its previously reported mammalian metabolites. Dichlorvos, dichloroacetaldehyde and dichloroacetic acid could not be detected in the blood. "The concentration of 30 ppm dichlorvos in the intestinal lumen resulted in less than 0.05 ppm dichlorvos in the blood. Neither could dichloroacetaldehyde or dichloroacetic acid be detected in the blood. The level of 2,2 dichloroethanol in the blood rose from 0.2 ppm after one hour to 0.9 ppm after four hours" whereas o-demethyldichlorvos occurred only near the limit of detection (0.03 ppm or less). In all cases, levels of 14-carbon radioactivity in urine, bile and tissue were higher than the level of 2,2-dichloroethanol, thus in urine the respective figures were 17 ppm compared to 3 ppm; in bile 12 and 0.5; in kidney 21 and 1.2; in liver 17 and 0.9 and in muscle 2.2 and 0.6 ppm. The figures indicated that 2,2-dichloroethanol or its conjugates are not the end products of the metabolism. In other experiments, pigs were given sustained release pellets of 14C- or 36Cl-labelled dichlorvos at an oral dose of 40 mg/kg body-weight, and excreta and tissue were analysed after various intervals. A similar situation to that found in the infusion experiment occurred. It was concluded that there was a rapid dechlorination of dichloroacetaldehyde and that the radioactive carbon enters the metabolic pool possibly via glyoxal (which compound has been found in in vitro but not in in vivo studies). No retention in tissue of any chlorine containing organic compound has been found, and the end product of the chlorine fragment appears to be chloride ion (Page, 1970). All the data support the conclusion that when administered orally over a period of weeks, dichlorvos does not result in detectable toxic residues in tissues of the parent compound or product-related metabolites. The results of some of the published trials are summarized in Table III. In plants New data have become available on the chemical degradation of dichlorvos residues on plants. Careful experiments with various plant species (cotton, maize, peas, beans) have demonstrated that dichlorvos is rapidly lost from leaf surfaces by volatilization and by hydrolysis and, disregarding the fraction of insecticide lost by volatilization during the first few minutes after application, the half-life of the compound under laboratory conditions is of the order of a few hours. A small percentage (0.5-5 percent) of the dichlorvos deposited, appears to penetrate into the waxy layers of leaf and fruit cuticle where it persists longer. Dichlorvos taken up by plants from aqueous solutions by the root system and translocated into aerial parts is less rapidly lost than insecticide applied to leaf and fruit surfaces, but even under these conditions the half-life of the compound is no more than 12 hours. Various measurements have confirmed the rapid disappearance of dichlorvos from vegetable and fruit crops under practical conditions (see Table 1). Residues in vegetables were below 0.1 ppm two days after application and were no longer detectable on the third day. Dichlorvos was somewhat more persistent in fruits, but the residues are below 0.1 ppm three days after application. In vegetable crops or mushrooms grown under cover or in greenhouses, losses of dichlorvos from leaf and fruit surfaces by volatilization are less rapid than outdoors. However, on all crops analysed so far, including such broad leaf vegetables as lettuce, dichlorvos residues have been below 0.5 ppm two days after treatment and below 0.3 ppm after three days. Casida et al. (1962) used 32P-labelling to study the fate of dichlorvos residues on maize, cotton and peas; a 0.1 percent aqueous solution of dichlorvos was applied uniformly to the upper leaf surfaces of each plant. It was found that about half of the dose volatilized within five minutes. An additional 45 percent was absorbed within 20 minutes, so that only 5 percent remained on the surface 20 minutes after application. The surface residue was nonhydrolysed dichlorvos while 70-80 percent of the absorbed material was organosoluble and was presumably nonhydrolysed dichlorvos. However, hydrolysis proceeded rapidly. Ignoring the dichlorvos lost by volatilization during the first five minutes after treatment, the half-life of the dichlorvos was 1.2 hours with loss primarily occurring by hydrolysis. In addition to the volatilization of the dichlorvos, which resulted in the total dichlorvos equivalent to dropping from 100 per leaf at zero time to 35 per leaf at 30 minutes, the hydrolysis products were also lost from the treated leaves. Only 14 units remained after 24 hours and 7 units after 72 hours, despite the fact that no dichlorvos or other organosoluble derivatives were detected after eight hours. The same authors carried out experiments with radio-labelled dichlorvos and showed that, when solutions of the insecticide were absorbed by the roots of plants, 77 percent of the absorbed dichlorvos appeared as hydrolysis products in peas and 33 percent in maize and cotton. Once the plants were removed from the insecticide source, the hydrolysis of the absorbed dichlorvos followed first order reaction kinetics, with half-life values for both maize and cotton of 9 hours and for peas for three hours. Following systemic uptake through the roots of plants, dichlorvos is hydrolysed rapidly, but the products of hydrolysis are not lost from the plant during a 40 hour post-absorption period. Bull and Ridgeway (1969) using 32P labelled dichlorvos showed that 54 percent of the amount applied was lost within one hour by volatilization from the treated leaves. The major metabolite of dichlorvos was dimethyl phosphate; only minor amounts of o-desmethyldichlorvos were detected. Casida et al. (1962) carried out studies with mice on the acute toxicity of dichlorvos and potential hydrolysis products and showed relatively high toxicity associated only with the parent compound. In storage and processing Dichlorvos is readily lost by evaporation from stored products, including raw grain and cereal products. Except at very low temperatures, it appears likely that dichlorvos is also hydrolysed to biologically inactive metabolites. A study (Shell, 1970d) using a number of materials with abnormally high dichlorvos residues showed that residues can be almost completely removed by cooking. Rice containing 4.5 ppm dichlorvos has been shown to lose 90% of the residues on boiling from 20 to 30 minutes. When rice containing 19 ppm was similarly treated, 98% of the residue was destroyed. Samples of flour containing dichlorvos residues in the range 4.5 to 14 ppm have been found to lose about 90% of the residue on baking for 10 to 12 minutes at 230°C in the preparation of biscuits. Boiling the flour with water for 2 minutes, as in the preparation of gravy, has been shown to decrease residue levels by 97%. Residues in cocoa have been shown to be lost during the manufacture of cocoa butter. No dichlorvos was found in the fat, the nibs, liquor or mash cake prepared from beans containing 2.9 ppm dichlorvos. Residues in food moving in commerce Two shiploads of wheat were treated with dichlorvos emulsion immediately prior to shipment from Australia. The dichlorvos was applied at a nominal concentration of 6 ppm (found to be 4.6 -5.5 ppm). Upon arrival in the Netherlands, the grain was thoroughly sampled and all samples were found to contain residues below 2 ppm (range 0.68-1.59 ppm - mean 0.96 ppm). A major portion of each consignment was trans-shipped to the United Kingdom,where it was sampled for analysis on arrival. All samples revealed dichlorvos residues, but at levels lower than those detected in the Netherlands, with a range of 0.4-1.3 ppm and a mean of 0.68 ppm. From the results of analysis prior to loading and at the end of a 7-week voyage, it was calculated that the half-life of the dichlorvos residues was between 20 and 23 days. Before the general release of dichlorvos resin strips, many experiments were carried out to simulate the conditions that would be encountered in practical usage. Subsequent data which related to food exposed in shops, restaurants and homes under various practical conditions where the dichlorvos fumigant strips were used according to directions, broadly show that results from the simulated experiments overestimated the residue levels. Surveys have been carried out in the United Kingdom (17 shops) and in France (20 shops) to establish the residue level of dichlorvos in unwrapped foods bought by the public from shops using the strips at the recommended rate under normal practical conditions. The residues found are shown in Tables V and VI. The somewhat higher results obtained in some of the commodities in the French survey may have been due to the common practice there of closing the shops completely during the heat of the afternoon. TABLE V Residues of dichlorvos (ppm) in food purchased from shops in France Sampling time : days after hanging strips 7 42 70 Commodity Range Mean Range Mean Range Mean Bread <0.05-0.10 <0.05 <0.05 <0.05 <0.05-0.06 <0.05 Cakes <0.05-0.24 0.08 <0.05-0.30 0.10 <0.05-0.17 0.07 Apples <0.05-0.14 0.06 <0.05-0.32 0.07 <0.05-0.30 0.06 Lettuce <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 Tomatoes <0.05-0.08 <0.05 <0.05 <0.05 <0.05 0.05 Ham <0.05-0.08 0.06 <0.05-0.28 0.06 <0.05-0.09 0.05 Cheese (cow) <0.05-0.08 0.06 <0.05-0.16 0.06 <0.05-0.15 0.06 Cheese (goat) <0.05-0.35 0.13 <0.05-0.25 0.08 <0.05-0.30 0.08 A number of surveys have been conducted in the U.S.A. on the residue levels in foods which had been exposed to resin strips in restaurants. In the first of two studies conducted in restaurants near Cincinnati, Ohio, typical restaurant meals were subjected to an exaggerated exposure of 24 hours to 5-10 day old strips. In a second study, meals were exposed for 22 hours to strips which were initially 51 days old. Under the conditions of the first survey, residues were only 0.05-0.15 ppm of apparent dichlorvos: under the second survey, no residues (less than 0.04) were found. In another investigation carried out in four different restaurants in New York City under normal operating conditions, ready to eat meals were sampled as composite samples. Whole meals were sampled at various stages in the life of the strips, ranging from 24 hours to 4 weeks. The meals consisted of various mixtures of meat or fish and vegetables and varied widely, in composition. The following residues were found: Restaurant No. of meals sampled Residue range,ppm A 5 0.04 -0.10 B 5 0.02 -0.11 C 9 0.03 -0.16 D 5 0.03 -0.08 To obtain information on food residues arising from the typical practice of hanging strips in kitchens, surveys were carried out in homes in the U.K. and France. Food samples were made up of whole food and beverage intakes for one day. Each home was sampled the day before the strips were hung and one week and six weeks later. In the U.K. study, no residues exceeded 0.09 ppm, and the mean of all samples taken at one week was 0.034 ppm. The samples at six weeks showed no residues in excess of 0.05 ppm, with a mean value of 0.034 ppm. Similar studies in 14 French homes showed no residues in excess of 0.07 ppm, and the mean of all samples taken at one week was 0.024 ppm. The six week samples showed no residues in excess of 0.03 ppm. METHODS OF RESIDUE ANALYSIS Cholinesterase inhibition A common technique which has been used for routine residue determinations in commodities of known treatment history is based on cholinesterase inhibition. Human and animal plasma cholinesterases are considerably more sensitive to dichlorvos than erythrocyte cholinesterases, and are therefore particularly suitable for residue measurements. Out of date stocks of human blood plasma or horse serum are satisfactory sources of enzyme. Extraction and clean-up prior to the cholinesterase assay are normally fairly simple, involving maceration of the material with a solvent of medium polarity (chloroform, methylene chloride), filtration and careful evaporation of the organic phase in the presence of water. More elaborate procedures may be required for plant materials with a high fat content, each as cocoa beans or groundnuts, and for certain animal products such as milk, cheese and butter. Steam distillation of dichlorvos prior to the enzyme assay has been suggested, not only for removing interference,but also for increasing the specificity of the residue method. For the enzyme assay proper, any of the common techniques for measuring cholinesterase activity may be used. The sensitivity of these methods is 0.05 to 0.1 ppm. Cholinesterase residue analysis for dichlorvos has recently been automated by using Technicon Auto-Analyser facilities. TABLE VI Residues of dichlorvos (ppm) in food purchased from shope in the UK1 Sampling time : days after hanging strips No. of No. of 2 28 70 Commodity samples shops Range Mean2 Range Mean Range Mean Cooked meat 35 7 <0.05-0.13 <0.05 <0.05-0.10 <0.05 <0.05-0.09 <0.05 Cheese 35 7 <0.05-0.07 <0.05 <0.05-0.12 0.05 <0.05-0.12 <0.05 Apples 49 10 <0.05-0.26 0.08 <0.05-0.54 0.14 <0.05-0.19 0.06 Tomatoes 33 11 All <0.05 <0.05 All <0.05 <0.05 <0.05-0.05 <0.05 Lettuce 27 9 All <0.05 <0.05 All <0.05 <0.05 All <0.05 <0.05 Broad 27 9 All <0.05 <0.05 All <0.05 <0.05 All <0.05 <0.05 Cakes 19 7 <0.05-0.07 <0.05 <0.05-0.05 <0.05 <0.05-0.05 <0.05 1 No residues of DCA (0.03 ppm) were found in any of the samples 2 Mean values are geometric means and are calculated by assuming that samples containing less than the detectable level (<0.05 ppm) in fact contain <0.025 ppm Such automated methods permit rapid routine measurements of large series of samples. Since other organophosphorus insecticides or carbamates can interfere, the cholinesterase inhibition method is not suitable for detecting and measuring dichlorvos in samples of unknown or ill-defined treatment history. Both thin-layer and paper chromatographic procedures are available for separating dichlorvos from other cholinesterase-inhibiting pesticides. In these methods, the cholinesterase inhibitors are normally revealed by enzymatic spot tests involving colour reactions of the substrate or its breakdown products after hydrolysis. It must be remembered that dichlorvos is a very volatile compound which, upon solvent evaporation, heating, aeration, etc. is easily lost from vessels or chromatographic support materials. These procedures are therefore recommended for qualitative purposes, but great care must be taken when they are used in quantitative analysis. Bioassay The vinegar fly (Drosophila melanogaster) and daphnia (Daphnia magna) are suitable test organisms for the bioassay of dichlorvos in plant materials. A specific test, which involves absorption barrier chromatography and which distinguishes between dichlorvos and other insecticides, has been described by Sun and Johnson (1963). The sensitivity of the bioassay methods is about 0.1 ppm. Colorimetry Although colorimetric techniques have been developed mainly for measuring traces in air samples or aqueous solutions, some of them can be used for the determination of residues in food materials, provided that the sensitivity required is not better than 0.3-1 ppm. Thus, the color reaction of dichlorvos with alkaline resorcinol has given satisfactory results with extracts of animal food commodities. This method demonstrates not only the parent insecticide, but also dichloroacetaldehyde, one of its potential hydrolysis products. Acting on suggestions made by Hodgson and Casida (1962), Hughes (1963) measured residues of dichlorvos and of dichloroacetaldehyde in air, using colorimetry, following reaction with alkali and 2,4-dinitrophenylhydrazine. This method can also be used for determining residues in food commodities. A further colorimetric procedure, based on formation of an orange-red complex between dichlorvos and acetone in the presence of alcoholic potassium hydroxide, has been described by Mitsui. Gas-liquid chromatography (GLC) The most specific residue methods currently available rely on the separation and detection of dichlorvos by gas chromatography. The sensitivity of these procedures is about equal to that obtained with the cholinesterase techniques, i.e. 0.03-0.1 ppm. Methods have been described by El-Refai and Guiffrida who used the sodium thermionic detector selective to phosphorus, and by Boone (1965) who used a microcoulometric detection system. Both methods use fairly high column temperatures, and the possibility of thermal or catalytic decomposition of dichlorvos on certain column materials must not be overlooked. Gas chromatographic residue measurements in industrial laboratories are usually carried out at lower column temperatures (150°C), and such stationary phases an phenyl diethanolamine succinate and Reoplex 400 have given satisfactory retention times and separations from interfering insecticides. Ivey and Claborn (1969) have published a GLC method for the determination of dichlorvos in milk, eggs and various body tissues of cattle and chickens. Using a gas chromatograph equipped with a flame photometric detector, they were able to detect 0.003 ppm of dichlorvos in milk and 0.002 ppm in body tissues and eggs. Methylene dichloridehexane was used to extract milk and hexane was used for fat and chicken skin. To recover dichlorvos from muscle, blood and eggs the authors used a preliminary extraction with acetonitrile. Abbott at al.(1970) report the successful application of the use of the caesium bromide tipped detector of Hartmann (1966) for the determination of dichlorvos on a variety of foods in a total diet survey. Drager (1968) has published a gas chromatographic method for determining dichlorvos residues in plants and milk. In this method, plant material is extracted by macerating it with methanol and water. The dichlorvos in the extract is partitioned into a mixture of ether and petroleum ether. The residue is then transferred into ethanol, and dichlorvos is determined by gas chromatography using the phosphorous detector. Minett and Belcher (1969) have developed a method for the determination of dichlorvos residues in wheat using GLC techniques. For the extraction of the ground wheat the authors used ethanol. Many of the published methods and many residue reports indicate that a variety of non-polar and polar solvents, such as methylene dichloride were used to extract the dichlorvos residue from the food commodity. Minett and Belcher (1969) and Elms at al. (1970) have shown that dichlorvos cannot be recovered quantitatively from plant materials, especially grain, by the use of solvents even as polar as methylene dichloride. Water or water-miscible solvents, such as methanol or ethanol, are absolutely essential for the recovery of dichlorvos residues from plant materials. Collaborative studies are currently being carried out in the United Kingdom on a method of detecting dichlorvos in grain. The procedure uses GLC with thermionic or flame photometric detection following extraction with methanol or acetone. APPRAISAL Dichlorvos is used extensively for the control of insect pests of importance in public health, in homes, warehouses, food stores and transport, stored grain as well as insects attacking domestic animals. It is also finding increasing use on horticultural and field crops and is registered in over forty eight countries. Dichlorvos impregnated resin strips are extensively used to control insect pests, especially flies, in homes, stores and food processing establishments. Extensive data on residues in food commodities were available from many countries in the form of published reports end submissions from three manufacturers. All data indicate that the level of residues occurring in raw agricultural commodities is low and that the residue levels decline rapidly. Due to the comparatively high vapour pressure of dichlorvos, the applied deposit is quickly lost by volatilization, but that portion which in absorbed into plant tissues undergoes hydrolysis to inactive metabolites. Dichlorvos applied to or fed to domestic animals undergoes rapid detoxification and degradation in all species examined and is unlikely to produce significant residues in meat, milk or eggs. The use of dichlorvos impregnated strips for controlling insects in warehouses, stores and shops gives rise to detectable residues in stored or prepared foods. Residues resulting from exposures at rates much higher than the recommended ones have been shown to decrease very rapidly on exposure to the atmosphere. They also are readily destroyed during the preparation of many products for consumption (e.g. by washing or by cooking). The proposal that a tolerance in meat and meat products was required in view of the use of dichlorvos-resin fumigant strips in meat storage and processing places in some countries (CCPR, 1971) was considered by the Meeting. Considerable data were available on the uptake of dichlorvos by many different foods under varying conditions of storage and exaggerated exposure to dichlorvos-resin strips, It was recognized that, under some conditions, residues as high as 0.1 ppm could result but even in fatty meat products the uptake was only at the surface. Residues declined during atmospheric exposure and were completely destroyed by cooking. Meat and meat products were included among the commodities for which a 0.1 ppm tolerance was recommended. These products were specially considered because of the request made at the 1970 meeting of the Codex Committee on Pesticide Residues (CCPR, 1971). RECOMMENDATIONS FOR TOLERANCES The following tolerances are for residues likely to be found in raw commodities at harvest, in stored products shortly after treatment and in miscellaneous prepared foods exposed during preparation and storage. Recommendations have been made on broad categories of fruit and vegetables, because extensive data have indicated that when dichlorvos is applied to these crops the level of residues shortly afterwards does not vary significantly on different varieties. Due to the transient nature of the insecticide, residue levels will continue to decline during shipment and storage, with a probable half life of less than one day on fresh fruit, vegetables and miscellaneous foods. Residues in food at time of consumption have normally been below the limits of detection by current analytical methods. Foodstuff Tolerance Recommended Period on which recommendation based Cocoa beans 5 ppm Raw grain (wheat, rice, rye, oats, barley, maize, sorghum, etc,) 2 ppm Coffee beans, soybeans, lentils, peanuts (groundnuts) 2 ppm Mushrooms 0.5 ppm 2 days Milled products from raw grain 0.5 ppm Fresh vegetables (excluding lettuce) 0.5 ppm 2 days Tomatoes 0.5 ppm 1 day and post harvest Lettuce 1 ppm 2 days Fresh fruit (apples, pears, peaches, strawberries, etc.) 0.1 ppm 2 days Meat of cattle, sheep, goats, pigs and poultry 0.05 ppm Eggs (on shell free basis) 0.05 ppm Milk (whole) 0.02 ppm Miscellaneous food items not otherwise specified 001 ppm As recommended in previous monographs, the content of dichloroacetaldehyde (DCA) should be reported where known. Furthermore, the amounts found should be added to those for dichlorvos when assessing adherence to the tolerances recommended. FURTHER WORK OR INFORMATION DESIRABLE 1. Continued observation of the effects of repeated exposure of man to dichlorvos in order to determine if there is any qualitative or quantitative differences between the metabolic route following oral or inhalation intake. 2. Data from additional countries on residues in commodities moving in international trade. 3. Analytical methods capable of recovering and determining residues of dichlorvos in foods should be established for regulatory purposes. REFERENCES Abbott, D.C., Crisp, S., Tarrant, K.R. and Tatton, J.O'G. (1970) Pesticide residues in the total diet in England and Wales 1966-69. Pesticide Science, 1 (i):10-13 Anon. (1966) Communication from F.I.D. Nordrhein-Westfalen to Deutsche Shell Chemie, Frankfurt, dated 20.1.66 (unpublished) Anon. (1966) Communication from Hamburg Public Health Service to Deutsche Shell Chemie, Frankfurt, dated 19.7.66 (unpublished) Arthur, B.W. and Casida, J.E. 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Unpublished report from Food and Drug Laboratories Inc., Maspeth, N.Y., prepared for Shell Chemical Co. Casida, J.E., McBride, L. and Niedermeier, R.P. (1962) Metabolism of 2,2-dichlorovinyl dimethyl phosphate in relation to residues in milk and mammalian tissues. J. Agr. Fd. Chem., 10 : 370-376 Cavagna, G., Locati, G. and Vigliani, E.C. (1969) Clinical effects of exposure to DDVP (Vapona) insecticide in hospital wards. Arch. Environmn. Hlth., 19 : 112-123 Cavagna, G., Locati, G. and Vigliani, E.C. (1970) Exposure of newborn babies to 'Vapona' insecticide. Europ. J. Toxicol. 3 : 49-57 Cavagna, G. and Vigliani, E.C. (1970) Problèmes d'hygiène et de sécurité dans l'emploi du Vapona insecticide dans les locaux domestiques. Med. d. Lavoro, 61 : 409-423 CCPR. (1971) Report of the Fifth Session of the Codex Committee on Pesticide Residues para. 165. Alinorm 71/24 Ciba, A.G. (1970) Basle - Submission to FAO/WHO Joint Meeting - Dichlorvos Drager, G. 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Shell Research Tunstall Report No. TLGR. 0024.69 Sun, Y.P. and Johnson, E.R. (1963) A new bioassay technique with special reference to the specific bioassay of dichlorvos insecticide. J. Econ. Entomol., 56 : 635-641 Vigliani, E.C. (1970) Results of recent studies on Vapona. Paper presented at the 7th Shell Industrial Doctors' Meeting, Strasbourg, 27-29 May 1970, submitted by Shell International Research Ltd. Vogin, E.E. (1969) Teratological studies with dichlorvos in rabbits. Unpublished report from Food and Drug Laboratories, Inc., Maspeth, N.Y., prepared for Shell Chemical Co. Wisconsin Alumni Research Foundation No. 8030193 (1968) (in cooperation with Shell Chemical Co. Residue project report (unpublished). Wisconsin Alumni Research Foundation (1968/9) (in cooperation with Shell Chemical Co. Residue project reports on sheep, cattle and goats (unpublished) Witherup, S., Caldwell, J.S. Jr. and Hull, L. 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See Also: Toxicological Abbreviations Dichlorvos (EHC 79, 1988) Dichlorvos (HSG 18, 1988) Dichlorvos (ICSC) Dichlorvos (FAO Meeting Report PL/1965/10/1) Dichlorvos (FAO/PL:CP/15) Dichlorvos (FAO/PL:1967/M/11/1) Dichlorvos (FAO/PL:1969/M/17/1) Dichlorvos (WHO Pesticide Residues Series 4) Dichlorvos (Pesticide residues in food: 1977 evaluations) Dichlorvos (Pesticide residues in food: 1993 evaluations Part II Toxicology) Dichlorvos (IARC Summary & Evaluation, Volume 53, 1991)