A detailed urinary excretion time course study of captan and folpet biomarkers in workers for the estimation of dose, main route-of-entry and most appropriate sampling and analysis strategies.

Captan and folpet are two fungicides largely used in agriculture, but biomonitoring data are mostly limited to measurements of captan metabolite concentrations in spot urine samples of workers, which complicate interpretation of results in terms of internal dose estimation, daily variations according to tasks performed, and most plausible routes of exposure. This study aimed at performing repeated biological measurements of exposure to captan and folpet in field workers (i) to better assess internal dose along with main routes-of-entry according to tasks and (ii) to establish most appropriate sampling and analysis strategies. The detailed urinary excretion time courses of specific and non-specific biomarkers of exposure to captan and folpet were established in tree farmers (n = 2) and grape growers (n = 3) over a typical workweek (seven consecutive days), including spraying and harvest activities. The impact of the expression of urinary measurements [excretion rate values adjusted or not for creatinine or cumulative amounts over given time periods (8, 12, and 24 h)] was evaluated. Absorbed doses and main routes-of-entry were then estimated from the 24-h cumulative urinary amounts through the use of a kinetic model. The time courses showed that exposure levels were higher during spraying than harvest activities. Model simulations also suggest a limited absorption in the studied workers and an exposure mostly through the dermal route. It further pointed out the advantage of expressing biomarker values in terms of body weight-adjusted amounts in repeated 24-h urine collections as compared to concentrations or excretion rates in spot samples, without the necessity for creatinine corrections.

Toxicokinetics of captan and folpet biomarkers in orally exposed volunteers.

The time courses of key biomarkers of exposure to captan and folpet was assessed in accessible biological matrices of orally exposed volunteers. Ten volunteers ingested 1 mg kg(-1) body weight of captan or folpet. Blood samples were withdrawn at fixed time periods over the 72 h following ingestion and complete urine voids were collected over 96 h post-dosing. The tetrahydrophthalimide (THPI) metabolite of captan along with the phthalimide (PI) and phthalic acid metabolites of folpet were then quantified in these samples. Plasma levels of THPI and PI increased progressively after ingestion, reaching peak values ~10 and 6 h post-dosing, respectively; subsequent elimination phase appeared monophasic with a mean elimination half-life (t(½) ) of 15.7 and 31.5 h, respectively. In urine, elimination rate time courses of PI and phthalic acid evolved in parallel, with respective t(½) of 27.3 and 27.6 h; relatively faster elimination was found for THPI, with mean t(½) of 11.7 h. However, phthalic acid was present in urine in 1000-fold higher amounts than PI. In the 96 h period post-treatment, on average 25% of folpet dose was excreted in urine as phthalic acid as compared with only 0.02% as PI. The corresponding value for THPI was 3.5%. Overall, THPI and PI appear as interesting biomarkers of recent exposure, with relatively short half-lives; their sensitivity to assess exposure in field studies should be further verified. Although not a metabolite specific to folpet, the concomitant use of phthalic acid as a major biomarker of exposure to folpet should also be considered.

Toxicokinetics of captan and folpet biomarkers in dermally exposed volunteers.

To better assess biomonitoring data in workers exposed to captan and folpet, the kinetics of ring metabolites [tetrahydrophthalimide (THPI), phthalimide (PI) and phthalic acid] were determined in urine and plasma of dermally exposed volunteers. A 10 mg kg(-1) dose of each fungicide was applied on 80 cm(2) of the forearm and left without occlusion or washing for 24 h. Blood samples were withdrawn at fixed time periods over the 72 h following application and complete urine voids were collected over 96 h post-dosing, for metabolite analysis. In the hours following treatment, a progressive increase in plasma levels of THPI and PI was observed, with peak levels being reached at 24 h for THPI and 10 h for PI. The ensuing elimination phase appeared monophasic with a mean elimination half-life (t(½) ) of 24.7 and 29.7 h for THPI and PI, respectively. In urine, time courses PI and phthalic acid excretion rate rapidly evolved in parallel, and a mean elimination t(½) of 28.8 and 29.6 h, respectively, was calculated from these curves. THPI was eliminated slightly faster, with a mean t(½) of 18.7 h. Over the 96 h period post-application, metabolites were almost completely excreted, and on average 0.02% of captan dose was recovered in urine as THPI while 1.8% of the folpet dose was excreted as phthalic acid and 0.002% as PI, suggesting a low dermal absorption fraction for both fungicides. This study showed the potential use of THPI, PI and phthalic acid as key biomarkers of exposure to captan and folpet.

Toxicokinetic modeling of captan fungicide and its tetrahydrophthalimide biomarker of exposure in humans.

Measurement of tetrahydrophthalimide (THPI) in urine has been used for the biomonitoring of exposure to the widely used captan fungicide in workers. To allow a better understanding of the toxicokinetics of captan and its key biomarker of exposure, a human multi-compartment model was built to simulate the transformation of captan into THPI and its subsequent excretion while accounting for other non-monitored metabolites. The mathematical parameters of the model were determined from best-fits to the time courses of THPI in blood and urine of five volunteers administered orally 1mg/kg and dermally 10mg/kg of captan. In the case of oral administration, the mean elimination half-life of THPI from the body (either through faeces, urine or metabolism) was found to be 13.43 h. In the case of dermal application, mean THPI elimination half-life was estimated to be 21.27 h and was governed by the dermal absorption rate. The average final fractions of administered dose recovered in urine as THPI were 3.6% and 0.02%, for oral and dermal administration, respectively. Furthermore, according to the model, after oral exposure, only 8.6% of the THPI formed in the GI reaches the bloodstream. As for the dermal absorption fraction of captan, it was estimated to be 0.09%. Finally, the average blood clearance rate of THPI calculated from the oral and dermal data was 0.18 ± 0.03 ml/h and 0.24 ± 0.6 ml/h while the predicted volume of distribution was 3.5 ± 0.6l and 7.5 ± 1.9l, respectively. Our mathematical model is in complete accordance with both independent measurements of THPI levels in blood (R(2)=0.996 for oral and R(2)=0.908 for dermal) and urine (R(2)=0.979 for oral and R(2)=0.982 for dermal) as well as previous experimental data published in the literature.

Gas-chromatography mass-spectrometry determination of phthalic acid in human urine as a biomarker of folpet exposure.

Agricultural workers are exposed to folpet, but biomonitoring data are limited. Phthalimide (PI), phthalamic acid (PAA), and phthalic acid (PA) are the ring metabolites of this fungicide according to animal studies, but they have not yet been measured in human urine as metabolites of folpet, only PA as a metabolite of phthalates. The objective of this study was thus to develop a reliable gas chromatography-tandem mass spectrometry (GC-MS) method to quantify the sum of PI, PAA, and PA ring-metabolites of folpet in human urine. Briefly, the method consisted of adding p-methylhippuric acid as an internal standard, performing an acid hydrolysis at 100 °C to convert ring-metabolites into PA, purifying samples by ethyl acetate extraction, and derivatizing with N,O-bis(trimethylsilyl)trifluoro acetamide prior to GC-MS analysis. The method had a detection limit of 60.2 nmol/L (10 ng/mL); it was found to be accurate (mean recovery, 97%), precise (inter- and intra-day percentage relative standard deviations <13%), and with a good linearity (R (2) > 0.98). Validation was conducted using unexposed peoples urine spiked at concentrations ranging from 4.0 to 16.1 µmol/L, along with urine samples of volunteers dosed with folpet, and of exposed workers. The method proved to be (1) suitable and accurate to determine the kinetic profile of PA equivalents in the urine of volunteers orally and dermally administered folpet and (2) relevant for the biomonitoring of exposure in workers.

Urinary excretion of tetrahydrophtalimide in fruit growers with dermal exposure to captan.

The relation between dermal and respiratory exposure and uptake into the body of captan, measured as 24 hr cumulative tetrahydrophtalimide (THPI) dose, was studied among 14 male fruit growers applying pesticides in orchards in the Netherlands. No contribution of respiratory exposure was observed on THPI in the urine. Dermal exposure, measured with skin pads, showed a clear relation with THPI in urine when exposure was estimated from exposure on skin pads of ankles and neck. No relation was found for total dermal exposure, calculated from measured exposure on skin pads of representative skin areas according to models described in the literature. Determinants of exposure such as use of a cabin on the tractor, use of gloves during mixing and loading, and use of rubber boots also explained THPI in urine very well. This finding corroborated the findings on measured dermal exposure. Results indicate that more attention should be paid to skin areas which are suspected to be most permeable for a chemical under study. It was concluded that dermal exposure data can be linked better to biological monitoring based on empirical findings as gathered in a pilot study on exposure of specific body areas than on estimations of total skin dose.

Metabolism of N-[4-chloro-2-fluoro-5-[(1-methyl-2- propynl)oxy]phenyl]-3,4,5,6-tetrahydrophthalimide (S-23121) in the rat: I. Identification of a new, sulphonic acid type of conjugate.

1. Several metabolites of 14C-labelled N-[4-chloro-2-fluoro-5-[(1-methyl- 2-propynyl)oxy]phenyl]-3,4,5,6-tetra-hydrophthalimide (S-23121) were identified. 2. The major urinary metabolites were found to be 4-chloro-2-fluoro-5-hydroxyaniline, its sulphate and glucuronide by t.l.c. cochromatography with authentic standards. 3. The major faecal metabolites in addition to the parent compound were six sulphonic acid conjugates having a sulphonic acid group incorporated into the double bond of the 3,4,5,6-tetrahydrophthalimide moiety. These sulphonic acid conjugates have never been reported previously for this type of compound. 4. To confirm the mechanism of biosynthesis of the sulphonic acid conjugates, sodium sulphate, cysteine and glutathione labelled with 35S were administered to the male rat together with unlabelled S-23121. The same faecal metabolites as those detected in faeces of the rat dosed with 14C-labelled S-23121 were similarly found after dosing with any of the 35S-labelled chemicals. Their biosynthesis was most pronounced with 35S-labelled sodium sulphate, implying that the sulphonic acid is incorporated into the double bond after reduction of sulphate to sulphite.

Captan metabolism in humans yields two biomarkers, tetrahydrophthalimide (THPI) and thiazolidine-2-thione-4-carboxylic acid (TTCA) in urine.

Captan fungicide (N-(trichloromethylthio)-4-cyclohexene-1,2-dicarboximide) metabolism in two human volunteers rapidly yields THPI (tetrahydrophthalimide) and TTCA (thiazolidine-2-thione-4-carboxylic acid). The work was done to evaluate usefulness of TTCA and THPI as biomarkers of occupational exposure and to compare human and rat dermal absorption and metabolism. THPI in 12h urine ranged from MDL (5 ppb) to 640 ppb and was stable for at least one year. TTCA was also a stable metabolite, but the MDL was 50 ppb. THPI was detectable in urine for 72 hours following oral dosages of 1 mg/kg, but most was eliminated 0-24 h. No THPI was detectable in urine following application of a chloroform solution to hands, forearms, or inguinal region. Dermal absorption and metabolism of captan are substantially different in humans and rats.

Determination of tetrahydrophtalimide and 2-thiothiazolidine-4-carboxylic acid, urinary metabolites of the fungicide captan, in rats and humans.

Capillary gas chromatographic (GC) methods using sulphur and mass selective detection for the qualitative and quantitative determination of tetrahydrophtalimide (THPI) and 2-thiothiazolidine-4-carboxylic acid (TTCA), urinary metabolites of the fungicide captan in rat and humans, were developed. Urinary detection limits were 2.7 micrograms/l for THPI and 110 micrograms/l for TTCA. Intraperitoneal and oral administration of captan to rats resulted in a 48-h cumulative urinary excretion of THPI of 1%-2% and 3%-9% of the dose, respectively. Cumulative urinary excretion of TTCA over 48 h ranged from 2% to 5% of the captan dose for the respective routes of administration. In urine of non-exposed human subjects, neither THPI nor TTCA could be detected. In urine of fruit-growers who were occupationally exposed to captan, both THPI and TTCA could be detected. Based on these results, THPI and TTCA are proposed as promising parameters for the biological monitoring of occupational exposure to captan.

Gas chromatographic determination of the captan metabolite tetrahydrophthalimide in urine.

A gas-liquid chromatographic (GLC) method is described for the determination of the captan metabolite tetrahydrophthalimide in human urine. The sample is extracted with chloroform and cleaned up on a Florisil column. The extract is then analyzed by GLC, using a nitrogen-phosphorus-selective detector. Recoveries from urine samples fortified at 0.03-0.5 ppm ranged from 82 to 87%, and were qualitatively confirmed by GLC-mass spectrometry.