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A simple and rapid method is described for the determination of the non-registered pesticides, captafol, quintozene (PCNB), cyhexatin and 1-naphthylacetic acid (NAA), in fruits. These pesticides were extracted with acidified acetone, then captafol and PCNB were purified with a Florisil mini column and analyzed by GC-ECD. Cyhexatin was ethylated with ethylmagnesium bromide, and the ethyl derivative was analyzed by GC-FPD (Sn filter). NAA was purified with liquid-liquid extraction and determined by HPLC equipped with a fluorescence detector. These analytes were identified with GC/MS or LC/MS. The minimum identified concentration of the pesticides was below 0.2 ng per injection, which corresponds to a detection limit of below 0.02 microgram/g in the original samples. Recoveries of the pesticides spiked at 0.1 microgram/g into apple, Japanese pear and melon were greater than 61%.

In studies on the mechanisms of mutagenic and carcinogenic action of captan and captafol-related chloroalkylthiocarboximide fungicides, two effects were tested: (i) the effect of both compounds on the activity of eukaryotic topoisomerases I and II in vitro, and (ii) their mutagenic and recombinagenic activity in the somatic mutation and recombination test (SMART) in wing cells of Drosophila melanogaster. Only captafol inhibited the activity of topoisomerase I (10-20% inhibition of activity in the range of 10-100microM). In contrast, both chemicals decreased the activity of topoisomerase II already at 1microM concentration (50 and 20% inhibition of activity by captafol and captan, respectively).Genotoxicity was tested in vivo by administrating both compounds by acute (3h) and chronic feeding (48h) of 3-day-old larvae. In acute feeding, captan and captafol demonstrated positive results only for small single and total spots in 10-100mM exposure concentration range. Both chemicals were inconclusive for large single spots, as well as for twin spots. In chronic treatment, captan showed positive results only for small single and total spots at 2.5 and 5mM concentrations. Captafol gave inconclusive results over all concentrations tested. The results of the acute treatment experiments which have been performed at very high doses (50% toxicity at higher doses) indicate very weak overall mutagenic activity of both test fungicides.

The simultaneous polarographic determination of the ternary mixture of captan-captafol and folpet is studied. The polarographic signals of these compounds in their mixture show a high overlapping. For this reason different chemometric methods such as PLS, PCR and artificial neuronal network (ANN) have been utilized for the simultaneous determination of these compounds in mixtures. The calibration model is built from solutions containing river water of known pesticide concentrations and the signals obtained by Sampled DC and DPP (differential pulse polarography) have been used. The analysis of both synthetic and real samples (river water) has been carried out by PLS with satisfactory results in most cases. It is possible to determine 0.25 ppm of each pesticide in river water samples after a preconcentration step by extraction into diethyl ether. ANN has also been applied to improve the results obtained by the PLS tool when the sampled DC current is recorded or when liquid-solid extraction with C18 cartridges is performed.

A rapid, reliable method for the multiresidue analysis of eight commonly used fungicides by micellar electrokinetic chromatography (MEKC) was developed. Excellent separation of the eight fungicides (carbendazim, metalaxyl, captan, procymidone, folpet, captafol, vinclozolin and iprodione) is achieved within about 10 min by using optimized electrophoretic conditions that include the addition of a mixture of organic modifiers to the running buffer for improved resolution. The sensitivity of the method is enhanced by using an enrichment step that involves on-column high-salt stacking. Limits of detection in the microgram-per-liter region and relative standard deviations from 2.1 to 5.9% are thus obtained for the fungicides without detracting from peak resolution. These results reveal that the high-salt stacking method provides highly improved sensitivity and enables highly flexible adjustment of the selectivity of the separation method. Also, the method surpasses other stacking alternatives used in MEKC and affords routine analyses of fruit juice containing fungicides at trace levels following a straightforward sample treatment. The robustness of the high-salt stacking method as demonstrated in this work makes MEKC methods involving stacking procedures an attractive choice for routine analyses.

The modifying effects of captafol and protective effects of L-cysteine on the development of glutathione S-transferase placental form-positive (GST-P +) foci of the liver and expression of proliferating cell nuclear antigen (PCNA) in the kidney were investigated in a medium-term bioassay using D-galactosamine (DGA) in rats. Male 6-week-old F344 rats were initially given a single i.p. injection (200 mg/kg) of diethylnitrosamine (DEN) and after 2 weeks on basal diet, received two i.p. injections of DGA (300 mg/kg) at the ends of weeks 2 and 5, and were fed a diet supplemented with test chemicals for weeks 3-8. Animals in group 1 were given 1500 ppm captafol in the diet, while group 2 received 1500 ppm captafol in diet as well as 1500 ppm L-cysteine in drinking water, animals in control group being given basal diet alone. Positive results regarding increased numbers and areas of GST-P + liver cell foci were obtained in rats treated with captafol alone. On the other hand, significant reduction by L-cysteine in the areas of GST-P + liver cell foci initiated by DEN and promoted by captafol was observed. In addition, the PCNA-labelling indices of renal tubule cells were elevated in rats treated with captafol alone and significantly reduced in rats treated simultaneously with L-cysteine. The protocol used in the present study therefore allowed the in vivo determination of promoting effects of captafol and inhibitory influence of L-cysteine by analyzing GST-P + foci in the livers as marker lesions, within a relatively short period of 8 weeks. Thus, this bioassay protocol could have applicability as a new in vivo assay system for the screening of hepatic carcinogenic or anti-carcinogenic agents.

The fungicide, captan, induces a cellular stress response in the soil nematode Caenorhabditis elegans. Transgenic C, elegans, which produce beta-galactosidase as a surrogate stress protein, reveal that captan-induced stress is localized mainly to muscle cells of the pharynx. The stress response is elicited by captan concentrations above 5 ppm and occurs within five hours of the initial exposure to the fungicide. Higher concentrations of captan, up to the solubility limit, increase the intensity of the response. Adult nematodes are significantly more sensitive to captan than are larvae. Captan also inhibits feeding in C. elegans, and nematodes exposed to captan rapidly cease muscular contractions in the pharynx. Stress induction and feeding inhibition are also caused by the related fungicides, captafol and folpet, but not by the parent compounds, phthalimide and tetrahydrophthalimide. The inhibition of feeding caused by compounds which elicit the cellular stress response may be an important survival mechanism for C, elegans.

Cytotoxic and cell-transforming activities of the three fungicides, captan, captafol and folpet, have been studied in an experimental in vitro model by exposing BALB/c 3T3 cells to the chemicals with or without S-9 mix-induced bioactivation. Cytotoxicity of the three compounds was reduced in the presence of the metabolizing system. Each assayed pesticide displayed cell-transforming ability in the presence of the metabolizing system. The relative efficiency was: captafol > captan > folpet. Cell transformation was considered to be due to carcinogenesis-promoting activity. These data, obtained in a medium-term (6-8 weeks) experimental model, contribute to a better understanding of the action of the three pesticides in the multistep carcinogenesis process and provide more information concerning the oncogenic risk of these xenobiotic compounds for humans.

Early life-stage survival, motility, and growth toxicity tests were carried out on common carp (Cyprinus carpio L.) endotrophic embryo, with two reference molecules (captafol and DNOC), from fertilization to the end of the first two-thirds period of mortalities by starvation. Thirteen days duration exposure was performed in daily renewed medium, at 24.5 degrees C, in standard synthetic water, at three pHs (6.9, 7.8, 9.0), in the presence of 10 mM/liter nontoxic pH buffer. Nominal concentrations of toxicants were 0.0, 0.25, 0.5, 1.0, and 2.0 mg/liter. Toxic effects on survival, motility, and growth decreased with increasing pH. From pH 6.9 to 9.0, the "no-observed-effect concentrations" differed by a factor greater than 8. Motility and growth cannot be considered, at population level, as true sublethal embryo-larval toxicity criteria. The toxicological interpretation of these results is discussed. They confirm the need of multifactorial methods for toxic risks and effects assessment on fish early life stages in the environment.

The mutagenic activity of captan and captafol was tested using Ames strains and strains showing an SOS response. Captafol was mutagenic in S. typhimurium strain TA102 (uvr+) and captan in strain TA104 (uvrB). Both captan and captafol elicit damages in DNA recognized by correndonuclease II, as shown by the repair test, and induced the SOS repair system in E. coli PQ37 (uvrA) strain. Only captafol induced the SOS system in PQ35 (uvr+). The lack of induction of beta-galactosidase at nonpermissive temperature in E. coli MD332 (dnaCs uvrA) strain showed that neither chemical was able to produce DNA breaks. In V79 Chinese hamster fibroblasts higher induction of c-mitosis by captafol than by captan (22% and 15% over the control, respectively) was accompanied by a higher decrease in nonprotein sulfhydryl groups, mainly GSH (41% and 77%, respectively). The content of protein sulfhydryl groups was decreased by either fungicide to a similar extent.

The Health Effects Division of the Office of Pesticide Programs (OPP) assessed the carcinogenic potential of three structurally related chloroalkylthiodicarboximide fungicides using a consensus peer review process and EPA's 1986 guidelines for cancer risk assessment. All of the fungicides were categorized as Group B2 (probable human) carcinogens based upon findings of an increased incidence of malignant tumors, or combined malignant and benign tumors, in multiple experiments involving different strains of mice and rats. The primary sites of tumor formation with the chloroalkylthiodicarboximide fungicides in male and/or female mice (CD-1 and B6C3F1) were the gastrointestinal tract (captan, folpet, and captafol), the lymph system (folpet and captafol), and the vascular system (captafol). The main sites of tumor formation in rats of one or both sexes (CR CD, Wistar, or F344 strains) were the kidney (Captan and captafol), uterus (captan), mammary gland and liver (captafol). In addition, positive trends for thyroid, testicular, mammary gland, and lymph node tumors were observed with folpet in the same strains of rats. All three of the compounds exhibited positive mutagenic activity in a variety of in vitro short-term tests for gene mutation, DNA repair, and chromosomal aberrations in prokaryotic and eukaryotic cells, but were not genotoxic in available studies performed under in vivo conditions. The assessment of human cancer risk for captan, folpet, and captafol was made using low-dose extrapolation models.

Tests were conducted to determine the effects of fungicides, captafol and chlorothalonil, on microbial and enzymatic activities in sandy loam. The results indicated that when captafol or chlorothalonil was added to the sandy loam, bacterial and fungicidal populations initially decreased with the treatments but recovered rapidly to levels similar to those in the controls. No inhibition on oxidation of soil ammonia or organic sulfur was observed. The fungicide treatments significantly increased oxygen consumption from the decomposition of organic matter indigenous to the soil. Both fungicides suppressed invertase and amylase for 1 day. However, the inhibitory effect disappeared after 2 days. Captafol depressed dehydrogenase for 4 days and recovered to equal to that of control after 7 days. No inhibitory effect on urease and phosphatase was shown with the fungicidal treatments. Although some stimulatory influences of fungicides on microbial and enzymatic activities were found in the soil, in no instance were the effects dramatic or sufficient enough to be considered important to soil fertility.

The effects of subchronic administration of captafol were studied in B6C3F1 mice given dose levels of 0, 0.3, 0.625, and 1.25% in the diet for 12 wk. There was a dose-related decrease in body weight gain during the 12-wk experiment and a loss of body weight in the 1.25% group of both sexes. Whiles the mice given captafol consumed less diet than the control mice, this was not directly dose-related. The relative weights of liver demonstrated a tendency for dose-dependent increase. Light-microscopic examination revealed cytoplasmic vacuolar degeneration, depending in severity on the dosage, in the livers of both sexes given captafol. In conclusion, the findings obtained from the present subchronic toxicity study indicated the liver to be a primary target organ.

A 34-year-old chemical manufacturing worker had new onset of work-related asthma after several years of exposure to the fungicide, captafol. On specific bronchial challenge testing, he demonstrated a marked and persistent fall in FEV1. Cessation of exposure resulted in improved symptoms and pulmonary function. The delay in symptoms after several years of workplace exposure and the dual reaction demonstrated on specific bronchial challenge testing suggest sensitization to some component of technical-grade captafol, but an IgE response was not detected.

An interlaboratory study of the determination of captan, folpet, and captafol in tomatoes, cucumbers, and apples was conducted by 4 laboratories using wide-bore capillary column gas chromatography with electron capture detection. The 3 fungicides were determined using the Luke et al. multiresidue method modified to include additional solvent elution in the optional Florisil column cleanup step used with this method. The crops were fortified with each fungicide at 3 levels per crop. Mean recoveries ranged from 86.2% for a 25.1 ppm level of captan in apples to 115.4% for a 0.288 ppm level of captafol in apples. Interlaboratory coefficients of variation ranged from 3.4% (24.7 ppm folpet) to 9.7% (0.243 ppm captafol) for tomatoes; from 2.8% (2.0 ppm captafol) to 8.2% (24.8 ppm captan) for cucumbers; and from 1.5% (0.234 ppm folpet) to 22.1% (0.266 ppm captafol) for apples.

A simple and rapid colorimetric method for the microdetermination of captafol (difolatan), based on its reaction with a dithiocarbamate, has been developed. The bright yellow colour which develops instantaneously on mixing the fungicide with the reagent is stable for at least 12 h. The method has been successfully adapted to the determination of captafol in its formulated products and residues on grains and apples.

During two vegetation periods, the behaviour of the triadimefon metabolite, triadimenol, in different plant parts of winter wheat and in soil was investigated. The fungicide Bayleton DF (triadimefon + captafol) was applied at the beginning of earing. Different ratios of triadimenol-A/-B were found in individual plant parts. Triadimenol-A predominated in the two uppermost leaves, and triadimenol-B in the roots and in the soil. No residues of triadimenol were found in grain at harvest time (detection limit 0.01 mg/kg).

Captafol fed at concentrations of 0, 0.075, 0.15, 0.3, and 0.6% to both sexes of F344 rats for 13 weeks produced dose-related decreases in body weight in males and females given 0.15% or higher concentrations. A dose-dependent decrease in urinary pH was observed in males receiving 0.3 or 0.6% and in females given 0.15% or higher concentrations of captafol. The 0.3 and 0.6% doses produced slight increases in leukocyte count and glutamic-pyruvic transaminase activity in females, along with a mild increase in alkaline phosphatase activity in the 0.6% case. The liver- and kidney-to-body weight ratios were increased in both male and female rats. Histopathological changes were observed in the forestomach, liver, and kidney. Squamous cell hyperplasia and edema accompanied by polynuclear leukocyte infiltration and dilation of vessels in the lamina propria were observed in the forestomach of both sexes given 0.15% or higher concentrations. Oval cell proliferation was apparent around Glisson's sheath in the livers of females given 0.3 and 0.6% captafol. Multifocal appearance of karyocytomegaly and tubular cell atypia in the proximal tubules of the kidney was found in the 0.3 and 0.6% groups of both sexes.

The fungicide captafol has been reported as causing irritant and allergic contact dermatitis in humans and in guinea pigs. This study investigated the ability of purified captafol to cause contact sensitization in BALB/c mice. Female mice were pretreated with an intraperitoneal injection of cyclophosphamide or saline. Applications of captafol (18.7 or 37.4 mg/ml), dinitrofluorobenzene (DNFB; 5 mg/ml) or solvent (4:1 acetone:ethanol mixture) were administered to the shaved abdomen on 2 consecutive days, or on days 1, 2, 8, 15, 22 and 29. Following challenge with captafol (37.4 mg/ml) or DNFB (2 mg/ml) on the right ear 6 days after the last induction, ear thickness ratios (right ear/left ear) were significantly larger after challenge in captafol-induced and DNFB-induced mice compared to control mice. A slightly larger response was observed with the smaller induction dose level of captafol and with multiple inductions over the course of a month. The overall maximum response to captafol was not increased by pretreatment with cyclophosphamide. Histologically, ears from captafol-induced and DNFB-induced mice showed edema and cellular infiltration. This study demonstrated the ability of captafol to produce contact hypersensitivity in the BALB/c mouse.

In a 4-year study, the behaviour of the residues of the fungicide captafol in different plant parts of winter wheat and in the soil was investigated. The fungicide Bayleton DF (captafol + triadimefon) was applied at the beginning of earing. Captafol residues clearly showed a high dependence on weather conditions in all examined aerial parts of the plant. After a 9-year application of captafol, there was no evidence of an enrichment of residues in the soil. At harvest, captafol residues in grains always were below the maximum residue limits of 0.5 mg/kg established in West Germany.

A mutagenic evaluation of captan, folpet, captafol, dichlofluanid and related compounds was carried out using the Salmonella/mammalian microsome test using strains TA102 and TA104. These strains contain A-T base pairs at the site of the mutation in contrast to the other Salmonella tester strains that detect mutagens damaging G-C base pairs. In addition, the excision repair system of the TA102 strain is still intact. Captan and folpet were mutagenic in strain TA104, captafol was mutagenic in strain TA102, whereas the remaining test compounds (dichlofluanid, tetrahydrophthalimide and thiozolidine-4-carboxylic acid) were not mutagenic in either strain. In conclusion, we consider it of value to add these two strains to those already used in the Ames test in order to increase confidence in our ability to detect mutagens and to shed further light on their mechanism of action.

The potential carcinogenicity of captafol in B6C3F1 mice was examined. Captafol was given at levels of 0 (control), 0.075, 0.15 or 0.3% in the diet to a total of 203 males and 203 females for 96 weeks, after which time the animals were returned to basal diet for a further 8 weeks. Mice surviving 42 weeks or longer were included in the effective numbers. Males and females given 0.3% captafol showed increased cumulative mortalities in the final quarter period of the experiment. Significant increases in the development of neoplastic lesions were found in the heart, spleen, forestomach, small intestine and liver of mice of both sexes treated with captafol. Tumors induced by captafol were, histologically, hemangioendothelioma in the heart, hemangioma or hemangioendothelioma in the spleen, papilloma and squamous cell carcinoma in the forestomach, adenoma and adenocarcinoma in the small intestine, and hyperplastic nodule and hepatocellular carcinoma in the liver. These results demonstrate a broad-spectrum carcinogenicity of captafol in B6C3F1 mice.

Two multiresidue methods, the Mills method and the Luke et al. method, are widely used for the determination of pesticides in foods. These methods were evaluated for the determination of the fungicides captan, folpet, and captafol in selected fruits and vegetables. The analytical behavior of standards through these methods was investigated first. Recoveries from apples, strawberries, lettuce, and tomatoes fortified with these compounds at levels of 0.2-5.9 ppm were then obtained. The analytes were quantitated by gas chromatography with electron capture detection, using a column of 5% SP-2401 on 100-120 mesh Supelcoport. Recoveries of captan, folpet, and captafol from fortified crops ranged from 69 to 78, 90 to 93, and 67 to 83%, respectively, by the Mills procedure and from 87 to 102, 81 to 106, and 91 to 109%, respectively, by the Luke et al. method modified to include additional solvent elution of the optional Florisil column.

Three groups of Fischer 344 rats were fed Merpafol (Makhteshim, Israel), an agricultural fungicide, in increasing concentrations for a period of 2 years. A control group was maintained under identical conditions, but without the addition of the fungicide in the diet. A range of nonneoplastic and neoplastic lesions were observed in the kidneys of rats fed Merpafol. The histogenesis of the renal tumors is discussed in relation to chemical-induced epithelial hyperplasia and cystic tubular dilation. Comparative aspects of renal cyst development and carcinoma in humans are also discussed.