Urinary Glyphosate, 2,4-D and DEET Biomarkers in Relation to Neurobehavioral Performance in Ecuadorian Adolescents in the ESPINA Cohort.

BACKGROUND: Herbicides are the most used class of pesticides worldwide, and insect repellents are widely used globally. Yet, there is a dearth of studies characterizing the associations between these chemical groups and human neurobehavior. Experimental studies suggest that glyphosate and 2,4-dichlorophenoxyacetic acid (2,4-D) herbicides can affect neurobehavior and the cholinergic and glutamatergic pathways in the brain. We aim to assess whether herbicides and insect repellents are associated with neurobehavioral performance in adolescents. METHODS: We assessed 519 participants (11-17 years of age) living in agricultural communities in Ecuador. We quantified urinary concentrations of glyphosate, 2,4-D, and two N,N-diethyl-meta-toluamide (DEET) insect repellent metabolites [3-(diethylcarbamoyl)benzoic acid (DCBA) and 3-(ethylcarbamoyl)benzoic acid (ECBA)] using isotope-dilution mass spectrometry. We assessed neurobehavioral performance using 9 subtests across 5 domains (attention/inhibitory control, memory/learning, language, visuospatial processing, and social perception). We characterized the associations using generalized estimating equations and multiple imputation for metabolites below detection limits. Models were adjusted for demographic and anthropometric characteristics, urinary creatinine, and sexual maturation. Mediation by salivary cortisol, dehydroepiandrosterone, 17ß-estradiol, and testosterone was assessed using structural equation modeling. RESULTS: The mean of each neurobehavioral domain score was between 7.0 and 8.7 [standard deviation (SD) range: 2.0-2.3]. Glyphosate was detected in 98.3% of participants, 2,4-D in 66.2%, DCBA in 63.3%, and ECBA in 33.4%. 2,4-D was negatively associated with all neurobehavioral domains, but statistically significant associations were observed with attention/inhibition [score difference per 50% higher metabolite concentration (ß)=-0.19 95% confidence interval (CI): -0.31, -0.07], language [ß=-0.12 (95% CI: -0.23, -0.01)], and memory/learning [ß=-0.11 (95% CI: -0.22, 0.01)]. Glyphosate had a statistically significant negative association only with social perception [ß=-0.08 (95% CI: -0.14, -0.01)]. DEET metabolites were not associated with neurobehavioral performance. Mediation by gender and adrenal hormones was not observed. CONCLUSION: This study describes worse neurobehavioral performance associated with herbicide exposures in adolescents, particularly with 2,4-D. Replication of these findings among other pediatric and adult populations is needed. https://doi.org/10.1289/EHP11383.

Biomonitoring of DEET and DCBA in Canadian children following typical protective insect repellent use.

N,N-diethyl-m-toluamide (DEET) is an ingredient found in many consumer insect repellents and its use is recommended to Canadians by government agencies, including Health Canada, for protection against insect bites including mosquitos and ticks. The majority of research on DEET exposure and toxicokinetics in humans has focused on adult populations with little information from vulnerable populations, including children. We aimed to fill this knowledge gap by examining real-world exposure data for DEET and its metabolite 3-diethylcarbamoyl benzoic acid (DCBA) in a sample population of Canadian children. We conducted a 24-h observational exposure human biomonitoring study at three overnight summer camps in Ontario, Canada through July and August 2019. Participating children aged 7-13 years provided multiple spot urine samples over a 24-h period and completed a journal to document insect repellent use and factors that could influence absorption of DEET. Children were instructed to use insect repellent as they usually would while attending a summer camp. Exposure was quantified using the information from the participant's journal and the change in the mass of their insect repellent containers over the course of the study. A total of 389 urine samples were collected from 124 children. Among participants using insect repellent, urinary levels of DEET were elevated between 2 and 8 h post-application and decreased thereafter but remained qualitatively higher than concentrations in participants who did not use insect repellent on the study day, even at 18-22 h post-application. DCBA was the predominant metabolite of DEET exposure in urine. DCBA was elevated between 8 and 14 h post-application, and declined thereafter, but not to the level observed among those who did not use insect repellent on the study day. Children who used more insect repellent, or used higher concentration insect repellent (10%-30% DEET) excreted higher levels of DEET and DCBA. Excreted DEET and DCBA accounted for 0.001% (median) and 1.3% (median) of the estimated applied DEET, respectively. Children did not reach an undetectable level of DEET or DCBA in urine, even among those not using insect repellent during the study day, indicating a potentially complex multi-route exposure to insect repellents in a real world scenario. This work provides targeted biomonitoring data for children intentionally using DEET-based insect repellents for normal protective use, and will support the risk re-evaluation of DEET by Health Canada.

Quantification of DEET and neonicotinoid pesticide biomarkers in human urine by online solid-phase extraction high-performance liquid chromatography-tandem mass spectrometry.

Neonicotinoid insecticides are widely used replacements for organophosphate and carbamate insecticides, but the extent of human exposure is largely unknown. On the other hand, based on urinary concentrations of DEET metabolites, human exposure to N,N-diethyl-m-toluamide (DEET) appears to be widespread. We developed a fast online solid-phase extraction high-performance liquid chromatography-isotope dilution tandem mass spectrometry (HPLC-MS/MS) method to measure in 200 µL of human urine the concentrations of six neonicotinoid biomarkers (acetamiprid, N-desmethyl-acetamiprid, clothianidin, imidacloprid, 5-hydroxy-imidacloprid, thiacloprid), and two DEET biomarkers (3-diethyl-carbamoyl benzoic acid, 3-ethyl-carbamoyl benzoic acid). Limits of detection ranged from 0.01 to 0.1 µg/L, depending on the biomarker. Accuracy ranged from 91 to 116% and precision ranged from 3.7 to 10 %RSD. The presented method can be used to increase our understanding of exposure to neonicotinoid insecticides and DEET, and to evaluate the potential health effects from such exposures.

Urinary concentrations of 3-(diethylcarbamoyl)benzoic acid (DCBA), a major metabolite of N,N-diethyl-m-toluamide (DEET) and semen parameters among men attending a fertility center.

STUDY QUESTION: Are specific gravity (SG)-adjusted urinary concentrations of 3-(diethylcarbamoyl)benzoic acid (DCBA) associated with semen parameters among men attending an academic fertility center? SUMMARY ANSWER: Our study did not demonstrate any association between SG-adjusted urinary DCBA concentrations and semen parameters among men attending an academic fertility center. WHAT IS KNOWN ALREADY: N,N-Diethyl-m-toluamide (DEET) is the most common active ingredient in consumer insect repellents. The recent rise in public health concerns regarding mosquito-borne diseases such as Zika, have led to an increased use of DEET insect repellents, especially among couples planning pregnancy. Animal studies have observed reproductive toxicity from DEET exposure. However, the reproductive health effects of DEET and its metabolites on human reproduction are unknown. STUDY DESIGN, SIZE, DURATION: Between 2007 and 2015, 90 men participating in a prospective cohort study at the Massachusetts General Hospital Fertility Center provided 171 urine samples and 250 semen samples for analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The urinary concentrations of DEET, N,N-diethyl-3-hydroxymethylbenzamide (DHMB) and DCBA were quantified by isotope-dilution tandem mass spectrometry and adjusted by SG. We used linear mixed models to evaluate the association between tertiles of SG-adjusted urinary DCBA concentrations and semen parameters (semen volume, sperm concentration, total sperm count, progressive motility, total progressive motility count, normal morphology and total normal morphology count), adjusting for covariates. DEET and DHMB were not considered for analysis because of the low percentage of detectable concentrations (<7%). Effect modification by BMI and smoking status was explored. MAIN RESULTS AND THE ROLE OF CHANCE: Participants had a median age of 36 years and BMI of 27 kg/m2, and 68% had never smoked. The SG-adjusted geometric mean DCBA urinary concentration was 2.20 µg/l, with 85% detection frequency. The majority of semen parameters fell within the normal range with the exception of progressive motility, where 64% of the men had values below the WHO 2010 lower reference limits. SG-adjusted urinary DCBA concentrations were not associated with semen parameters in unadjusted or adjusted models. Men in the highest tertile of SG-adjusted urinary DCBA concentrations had comparable semen parameters to men in the lowest tertile (2.59 vs. 2.88 ml for semen volume, 47.9 vs. 45.8 million/ml for sperm concentration, 116 vs. 118 million for total sperm count, 25 vs. 24% for progressive sperm motility, and 6.1 vs. 5.8% for morphologically normal sperm). In addition, BMI and smoking status did not modify the associations. LIMITATIONS REASONS FOR CAUTION: We had a relatively small sample size with similar socioeconomic backgrounds and with overall relatively low urinary concentrations of DEET biomarkers. However, our sample size was enough to detect moderate differences with at least 80% statistical power, between the first and third tertiles of urinary DCBA concentrations. Limitations also include possible misclassification of DCBA exposure and difficulties in extrapolating the findings to the general population. WIDER IMPLICATIONS OF THE FINDINGS: Our study found no associations between urinary concentrations of DCBA, a major metabolite of the insect repellent DEET, and semen parameters in men presenting for infertility treatment. While these results are reassuring, further studies including larger sample sizes and higher exposures are warranted. STUDY FUNDING/COMPETING INTEREST(S): The project was financed by the National Institute of Health grants R01ES022955 and R01ES009718 and by grant P30ES000002 from the National Institute of Environmental Health Sciences (NIEHS). None of the authors has any conflicts of interest to declare. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. TRIAL REGISTRATION NUMBER: N/A.

Cross-sectional biomonitoring study of pesticide exposures in Queensland, Australia, using pooled urine samples.

A range of pesticides are available in Australia for use in agricultural and domestic settings to control pests, including organophosphate and pyrethroid insecticides, herbicides, and insect repellents, such as N,N-diethyl-meta-toluamide (DEET). The aim of this study was to provide a cost-effective preliminary assessment of background exposure to a range of pesticides among a convenience sample of Australian residents. De-identified urine specimens stratified by age and sex were obtained from a community-based pathology laboratory and pooled (n = 24 pools of 100 specimens). Concentrations of urinary pesticide biomarkers were quantified using solid-phase extraction coupled with isotope dilution high-performance liquid chromatography-tandem mass spectrometry. Geometric mean biomarker concentrations ranged from <0.1 to 36.8 ng/mL for organophosphate insecticides, <0.1 to 5.5 ng/mL for pyrethroid insecticides, and <0.1 to 8.51 ng/mL for all other biomarkers with the exception of the DEET metabolite 3-diethylcarbamoyl benzoic acid (4.23 to 850 ng/mL). We observed no association between age and concentration for most biomarkers measured but noted a "U-shaped" trend for five organophosphate metabolites, with the highest concentrations observed in the youngest and oldest age strata, perhaps related to age-specific differences in behavior or physiology. The fact that concentrations of specific and non-specific metabolites of the organophosphate insecticide chlorpyrifos were higher than reported in USA and Canada may relate to differences in registered applications among countries. Additional biomonitoring programs of the general population and focusing on vulnerable populations would improve the exposure assessment and the monitoring of temporal exposure trends as usage patterns of pesticide products in Australia change over time.

Novel exposure biomarkers of N,N-diethyl-m-toluamide (DEET): Data from the 2007-2010 National Health and Nutrition Examination Survey.

BACKGROUND: N,N-diethyl-m-toluamide (DEET) is a widely used insect repellent in the United States. OBJECTIVES: To assess exposure to DEET in a representative sample of persons 6years and older in the U.S. general population from the 2007-2010 National Health and Nutrition Examination Survey. METHODS: We analyzed 5348 urine samples by using online solid-phase extraction coupled to isotope dilution-high-performance liquid chromatography-tandem mass spectrometry. We used regression models to examine associations of various demographic parameters with urinary concentrations of DEET biomarkers. RESULTS: We detected DEET in ~3% of samples and at concentration ranges (>0.08µg/L-45.1µg/L) much lower than those of 3-(diethylcarbamoyl)benzoic acid (DCBA) (>0.48µg/L-30,400µg/L) and N,N-diethyl-3-hydroxymethylbenzamide (DHMB) (>0.09µg/L-332µg/L). DCBA was the most frequently detected metabolite (~84%). Regardless of survey cycle and the person's race/ethnicity or income, adjusted geometric mean concentrations of DCBA were higher in May-Sep than in Oct-Apr. Furthermore, non-Hispanic whites in the warm season were more likely than in the colder months [adjusted odds ratio (OR)=10.83; 95% confidence interval (CI), 3.28-35.79] and more likely than non-Hispanic blacks (OR=3.45; 95% CI, 1.51-7.87) to have DCBA concentrations above the 95th percentile. CONCLUSIONS: The general U.S. population, including school-age children, is exposed to DEET. However, reliance on DEET as the sole urinary biomarker would likely underestimate the prevalence of exposure. Instead, oxidative metabolites of DEET are the most adequate exposure biomarkers. Differences by season of the year based on demographic variables including race/ethnicity likely reflect different lifestyle uses of DEET-containing products.

Assessment of dermal absorption of DEET-containing insect repellent and oxybenzone-containing sunscreen using human urinary metabolites.

Mutual enhancement of dermal absorption of N,N-diethyl-m-toluamide (DEET) and oxybenzone (OBZ) has been reported recently with DEET and OBZ being active ingredients of insect repellent and sunscreen, respectively. To assess the reported enhancing effect directly, we used human urinary metabolites as biomarkers; besides, we also sought to determine the best way for concurrent use of these two products without extra absorption of either. Four dermal application methods were used: DEET only (S1), OBZ only (S2), DEET on top of OBZ (S3), and OBZ on top of DEET (S4). Among the study methods, there was a significant difference (p = 0.013), which was attributed to the difference between S1 and S4, suggesting that applying OBZ over DEET on the skin lead to significantly higher absorption of DEET. Using both products in reverse order, (S3) did not result in extra DEET absorption significantly. As for OBZ permeation, no significant difference was observed among the methods. In summary, the enhancement of DEET absorption is confirmed for OBZ being applied over DEET on the skin; should concurrent use of both be necessary, applying sunscreen (OBZ) first and then insect repellent (DEET) with a 15-min interval is recommended.

Urinary biomarkers of exposure to insecticides, herbicides, and one insect repellent among pregnant women in Puerto Rico.

BACKGROUND: There are potential adverse health risks to the mother and fetus from exposure to pesticides. Thus, studies of exposure to pesticides among pregnant women are of interest as they will assist with understanding the potential burden of exposure globally, identifying sources of exposure, and designing epidemiology studies. METHODS: We measured urinary concentrations of the insect repellent N-N-diethyl-meta-toluamide (DEET) and two of its metabolites [3-diethyl-carbamoyl benzoic acid (DCBA) and N,N-diethyl-3-hydroxymethylbenzamide (DHMB)], four pyrethroid insecticide metabolites [4-fluoro-3-phenoxybenzoic acid (4-F-3-PBA); 3-phenoxybenzoic acid (3-PBA); trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (trans-DCCA); and cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid (cis-DBCA)], and two chlorophenoxy herbicides [2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)] in 54 pregnant women from Puerto Rico at three separate time points (20 ± 2 weeks, 24 ± 2 weeks, and 28 ± 2 weeks of gestation). We calculated the distributions of the biomarker concentrations and compared them to those of women of reproductive age from the general U.S. population where available, and estimated the within-subject temporal variability of these repeated measurements. We also collected questionnaire data on demographics, consumption of select fruits, vegetables, and legumes in the past 48-hr, and pest-related issues, and associations between these variables and biomarker concentrations were examined. RESULTS: We found that 95th percentile urinary concentrations of DEET, 3-PBA, trans-DCCA, and 2,4-D were lower than women of reproductive age on the U.S. mainland, whereas 95th percentile urinary concentrations of 4-F-3-PBA, cis-DBCA, and 2,4,5-T were similar. DCBA, the only urinary biomarker detected in >50% of the samples, showed fair to good reproducibility across pregnancy (intraclass correlation coefficient: 0.60). Women were more likely (p <0.05) to have greater urinary concentrations of pesticide biomarkers if they were less educated (DCBA and trans-DCCA), unemployed (DHMB), or married (2,4-D), had consumed collards or spinach in past 48-hr (2,4-D) or had been using insect repellent since becoming pregnant (DCBA), or were involved with residential applications of pesticides (trans-DCCA). CONCLUSIONS: We identified concentrations and predictors of several pesticides among pregnant women in Puerto Rico. Further research is needed to understand what aspects of the predictors identified lead to greater exposure, and whether exposure during pregnancy is associated with adverse health.

A lethal case of DEET toxicity due to intentional ingestion.

A 37-year-old male with prior medical history of profound developmental delay experienced seizure and cardiac arrest following ingestion of 6 ounces of a 40% N, N-diethyl-meta-toluamide (DEET) containing solution. The patient was unresponsive, acidemic, tachycardic and hypotensive on presentation. Over three hospital days, the patient's vitals recovered to baseline but he remained unresponsive and areflexic with fixed and dilated pupils. Non-contrast brain magnetic resonance imaging showed cerebral edema, transtentorial and tonsillar herniations. A rapid, simple and sensitive high-performance liquid chromatography (HPLC) method was utilized for the analysis of postmortem plasma blood and urine samples of a lethal case of DEET intentional ingestion. The method combined the use of C18 SepPak cartridges for solid phase extraction and reversed-phase HPLC. One urine and five blood samples from this patient were analyzed for DEET concentration. Mixtures of serum/urine postcentrifuge were eluted and reduced to 1 mL using a solvent evaporator. Blood in ethylenediaminetetraacetic acid (EDTA), whole blood, serum, blood with heparin and urine DEET concentrations were 9.84, 9.21, 10.18, 8.66dl and 0.642 mg/dL, respectively. All samples were collected <1 h postingestion. Although seizures and cardiac toxicity have been described in other case reports, this case is atypical due to the exceptional dose ingested and the timing of the fluid test samples being drawn so soon following exposure. Although a widely used and extremely safe insect repellent, DEET can be highly toxic in large but easily obtainable doses.

Biomarkers for monitoring profluthrin exposure: urinary excretion kinetics of profluthrin metabolites in rats.

Recently, a pyrethroid profluthrin [(2,3,5,6-tetrafluoro-4-methylphenyl)methyl 2,2-dimethyl-3-(prop-1-enyl)cyclopropane-1-carboxylate] is widely used as mothproof repellents in indoors. The urinary excretion kinetics of its metabolites was examined in rats to search for urinary metabolites suitable as biomarkers of profluthrin exposure in the general population. A single dose (26-400 mg/kg body weight) of profluthrin was administered intraperitoneally to the rats, and then their urine was collected periodically. Four major profluthrin metabolites, 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol (CH3-FB-Al), 4-hydroxymethyl-2,3,5,6-tetrafluorobenzyl alcohol, 4-methyl-2,3,5,6-tetrafluorobenzoic acid and 2,2-dimethyl-3-(1-propenyl)-cyclopropanecarboxylic acid (MCA) were determined in the urine samples by gas chromatography/mass spectrometry. The kinetic evaluation for each metabolite was achieved by moment analysis of the urinary excretion rate versus time curve. The urinary excretion amounts of the three metabolites, expect for MCA, were estimated to be proportional to the amounts of absorbed profluthrin over a wide exposure range. Urinary CH3-FB-Al was considered to be an optimal biomarker for monitoring of profluthrin.

Biotransformation and toxicokinetics of the insect repellent IR3535® in male and female human subjects after dermal exposure.

The absorption and excretion of the insect repellent IR3535(®) was studied in human subjects (five males and five females) after dermal application of approx. 3g of a formulation containing 20% IR3535(®), i.e. the amounts of IR3535(®) applied were between 1.94 and 3.4 mmol/person (418-731 mg/person). Blood and urinary concentrations of IR3535(®) and its only metabolite, IR3535(®)-free acid, were determined over time. In plasma, concentrations of the parent compound IR3535(®) were at or below the limit of quantification (0.037 µmol/L). IR3535(®)-free acid peaked in plasma samples 2-6h after dermal application. Cmax mean values were 5.7 µmol/L in males, 3.0 µmol/L in females and 4.2 µmol/L in all volunteers. Mean AUC values were 41.6, 24.5 and 33.9 µmolL(-1)h in males, females and all subjects, respectively. In urine samples from all human subjects, both IR3535(®) and IR3535(®)-free acid were detectable, however, only very small amounts of IR3535(®) were found. Concentrations of IR3535(®)-free acid were several thousand-fold higher than the parent compound and peaked at the first two sampling points (4h and 8h after dermal application). Overall, IR3535(®) and IR3535(®)-free acid excreted with urine over 48 h representing 13.3 ± 3.05% of the dose applied. Since IR3535(®) is rapidly and extensively metabolized, and IR3535(®)-free acid has a low molecular weight and high water solubility, it is expected that urinary excretion of IR3535(®)-free acid and IR3535(®) represents the total extent of absorption of IR3535(®) in humans. Based on the results of this study, the skin penetration rate of IR3535(®) is 13.3% in humans after dermal application.

Analytical method for urinary metabolites of the fluorine-containing pyrethroids metofluthrin, profluthrin and transfluthrin by gas chromatography/mass spectrometry.

An analytical method was developed for measurement of the major urinary metabolites in rats administered fluorine-containing pyrethroids (metofluthrin, profluthrin and transfluthrin) which are widely used recently as mosquito repellents or mothproof repellents. Eight metabolites, 2,3,5,6-tetrafluorobenzoic acid, 4-methyl-2,3,5,6-tetrafluorobenzoic acid, 2,2-dimethyl-3-(1-propenyl)-cyclopropanecarboxylic acid, 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (carboxylic metabolites), 2,3,5,6-tetrafluorobenzyl alcohol, 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol, 4-methoxymethyl-2,3,5,6-tetrafluorobenzyl alcohol and 4-hydroxymethyl-2,3,5,6-tetrafluorobenzyl alcohol (alcoholic metabolites), were extracted from enzymatic hydrolyzed urine using toluene and then concentrated. After transformation to their tert-butyldimethylsilyl derivatives for carboxylic metabolites or their trimethylsilyl derivatives for alcoholic metabolites, analysis was conducted by gas chromatography/mass spectrometry in the electron impact ionization mode. The calibration curves for each metabolite were linear over the concentration range of 0-20µg/ml in urine, and the quantification limits were between 0.009 and 0.03µg/ml. The relative errors and the relative standard deviations on replicate assays were less than 6% and 5%, respectively, for all concentrations studied. The measurements were accurate and precise. The collected urine samples could be stored for up to 1 month at -20°C in a freezer. The proposed method was applied to the analysis of several urine samples collected from rats treated with these pyrethroids.

Metabolic disposition of the insect repellent DEET and the sunscreen oxybenzone following intravenous and skin administration in rats.

Insect repellent N,N-diethyl-m-toluamide (DEET) and sunscreen oxybenzone have shown a synergistic percutaneous enhancement when applied concurrently. Both compounds are extensively metabolized in vivo into a series of potentially toxic metabolites: 2 metabolites of DEET, N,N-diethyl-m-hydroxymethylbenzamide (DHMB) and N-ethyl-m-toluamide (ET), and 3 metabolites of oxybenzone, 2,4-dihydroxybenzophenone (DHB), 2,2-dihydroxy-4-methoxybenzophenone (DMB), and 2,3,4-trihydroxybenzophenone (THB). In this study, the metabolites were extensively distributed following intravenous and topical skin administration of DEET and oxybenzone in rats. Combined application enhanced the disposition of all DEET metabolites in the liver but did not consistently affect the distribution of oxybenzone metabolites. The DHMB appeared to be the major metabolite for DEET, while THB and its precursor DHB were the main metabolites for oxybenzone. Repeated once-daily topical application for 30 days led to higher concentrations of DEET metabolites in the liver. Hepatoma cell studies revealed a decrease in cellular proliferation from all metabolites as single and combined treatments, most notably at 72 hours. Increased accumulation of DHMB and ET in the liver together with an ability to reduce cellular proliferation at achievable plasma concentrations indicated that simultaneous exposure to DEET and oxybenzone might have the potential to precipitate adverse effects in a rat animal model.

Simultaneous analytical method for urinary metabolites of organophosphorus compounds and moth repellents in general population.

An analytical method was developed for simultaneous measurement of urinary metabolites in the general population exposed to organophosphorus compounds (insecticides, flame retardants and plasticizers) and moth repellents used in Japanese households. Fifteen metabolites, dimethylphosphate, dimethylthiophosphate, diethylphosphate, diethylthiophosphate, di-n-butylphosphate, diphenylphosphate, bis(2-ethylhexyl)phosphate, 2-isopropyl-6-methyl-4-pyrimidinol, 3,5,6-trichloro-2-pyridinol, 3-methyl-4-(methylthio)phenol, 3-methyl-4-nitrophenol, 2,4-dichlorophenol, 2,5-dichlorophenol, 1-naphthol and 2-naphthol, were extracted from hydrolyzed urine by using a sorbent (hydroxylated polystyrene-divinylbenzene copolymers), and then desorbed with methylacetate and acetonitrile, concentrated, and after transformation to their tert-butyldimethylsilyl derivatives, analyzed by gas chromatography/mass spectrometry in the electron impact ionization mode. They could be determined accurately and precisely (quantification limits: 0.8-4 µg/l). The collected urine samples could be stored for up to 1 month at -20°C in a freezer.

Pesticide urinary metabolite levels of children in eastern North Carolina farmworker households.

BACKGROUND: In this investigation we documented the pesticide urinary metabolite levels of farmworker children in North Carolina, determined the number of different metabolites detected for each child, and delineated risk factors associated with the number of metabolites. METHODS: Urine samples were collected from 60 Latino farmworker children 1-6 years of age (34 female, 26 male). Interviews were completed by their mothers in Spanish. We analyzed urine samples for 14 pesticide metabolites, including the organophosphate pesticides chlorpyrifos, coumaphos, diazinon, isazaphos, malathion, pirimiphos, and parathion and its methyl counterpart; a common metabolite of at least 18 pyrethroid insecticides; the repellent DEET; and the herbicides 2,4,5-trichlorphenoxyacetic acid, 2,4-dichlorophenoxyacetic acid, acetochlor, atrazine, and metolachlor. Predictors included measures of paraoccupational, residential, and environmental exposure, child characteristics, and mother characteristics. RESULTS: Thirteen metabolites were present in the urine samples. Organophosphate pesticide metabolites were detected in a substantial proportion of children, particularly metabolites of parathion/methyl parathion (90.0%; geometric mean 1.00 microg/L), chlorpyrifos/chlorpyrifos methyl (83.3%; geometric mean 1.92 microg/L), and diazinon (55.0%; geometric mean 10.56 microg/L). The number of metabolites detected ranged from 0 to 7, with a mode of 4 detected (28.3%). Boys, children living in rented housing, and children with mothers working part-time had more metabolites detected. CONCLUSIONS: Children in farmworker homes experience multiple sources of pesticide exposure. Pesticides may remain in their environments for long periods. Environmental and occupational health changes are needed to address these exposures. Research is needed with more precise measures of exposure and on the health effects of concurrent exposure to multiple pesticides.

High performance liquid chromatographic determination of diazinon, permethrin, DEET (N, N-diethyl-m-toluamide), and their metabolites in rat plasma and urine.

A rapid method was developed for the analysis of the insecticide (A) diazinon (O,O-diethyl O-2-isopropyl-6-methylpyridimidinyl) phosphorothioate, its metabolites (B) diazoxon (O,O-diethyl O-2-isopropyl-6-methylpyridimidinyl) phosphate, and (C) 2-isopropyl-6-methyl-4-pyrimidinol, the insecticide (D) permethrin [3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid (3-phenoxyphenyl)methylester], its metabolites (E) m-phenoxybenzyl alcohol, and (F) m-phenoxybenzoic acid, the insect repellent (G) DEET (N,N-diethyl-m-toluamide), and its metabolites (H) m-toluamide and (I) m-toluic acid in rat plasma and urine. The method is based on using C18 Sep-Pak cartridges (Waters Corporation, Milford, Mass., U.S.A.) for solid phase extraction and high performance liquid chromatography with a reversed phase C18 column, and absorbance detection at 230 nm for compounds A, B, and C, and at 210 nm for compounds D-I. The compounds were separated using a gradient from 1% to 99% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.7 mL/min in a period of 17 min. The limits of detection were ranged between 20 and 100 ng/mL, while limits of quantification were 80-200 ng/mL. The relationship between peak areas and concentration was linear over a range of 100-1000 ng/mL. This method was applied to determine the above insecticides and their metabolites following dermal administration in rats.

Simultaneous determination of malathion, permethrin, DEET (N,N-diethyl-m-toluamide), and their metabolites in rat plasma and urine using high performance liquid chromatography.

A method was developed for the separation and quantification of the insecticide malathion (O,O-dimethyl-S-(1,2-carbethoxyethyl) phosphorodithioate), its metabolite malaoxon (O,O-dimethyl-S-(1,2-carbethoxyethyl) phosphorothioate), the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester), two of its metabolites m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, the insect repellent N,N-diethyl-m-toluamide (DEET), and its metabolites m-toluamide and m-toluic acid in rat plasma and urine. The method used high performance liquid chromatography (HPLC) with reversed phase C(18) column, and UV detection at 210 nm. The compounds were separated using gradient of 45--99% acetonitrile in water (pH 3.5) at a flow rate ranging between 0.5 and 2 ml/min in a period of 15 min. The retention times ranged from 7.4 to 12.3 min. The limits of detection ranged between 20 and 100 ng/ml, while limits of quantitation were 50-150 ng/ml. Average percentage recovery of five spiked plasma samples were 80.1+/-4.2, 75.2+/-4.6, 84.5+/-4.0, 84.3+/-3.4, 82.8+/-3.9, 83.9+/-5.5, 82.2+/-6.0, 83.1+/-4.3, and from urine 78.8+/-3.9, 76.4+/-4.9, 82.3+/-4.5, 82.5+/-3.9, 81.4+/-4.0, 83.9+/-4.3, 81.5+/-5.0, and 84.5+/-3.8 for, malathion, malaoxon, DEET, m-toluamide, m-toluic acid, permethrin, m-phenoxybenzyl alcohol, and m-phenoxybenzoic acid, respectively. The method was reproducible and linear over range between 100 and 1000 ng/ml. This method was applied to analyze the above chemicals and metabolites following combined dermal administration in rats.

Development of a high-performance liquid chromatographic method for the quantification of chlorpyrifos, pyridostigmine bromide, N,N-diethyl-m-toluamide and their metabolites in rat plasma and urine.

A method was developed for the separation and quantification of the insecticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphorothioate), its metabolites chlorpyrifos-oxon (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphate) and TCP (3,5,6-trichloro-2-pyridinol), the anti-nerve agent drug pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide), its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), and its metabolites m-toluamide and m-toluic acid in rat plasma and urine. The method is based on using solid-phase extraction and high-performance liquid chromatography (HPLC) with reversed-phase C18 column, and gradient UV detection ranging between 210 and 280 nm. The compounds were separated using a gradient of 1-85% acetonitrile in water (pH 3.20) at a flow-rate ranging between 1 and 1.7 ml/min over a period of 15 min. The retention times ranged from 5.4 to 13.2 min. The limits of detection ranged between 20 and 150 ng/ml, while the limits of quantitation were between 150 and 200 ng/ml. Average percentage recovery of five spiked plasma samples was 80.2+/-7.9, 74.9+/-8.5, 81.7+/-6.9, 73.1+/-7.8, 74.3+/-8.3, 80.8+/-6.6, 81.6+/-7.3 and 81.4+/-6.5, and from urine 79.4+/-6.9, 77.8+/-8.4, 83.3+/-6.6, 72.8+/-9.0, 76.3+/-7.7, 83.4+/-7.9, 81.6+/-7.9 and 81.8+/-6.8 for chlorpyrifos, chlorpyrifos-oxon, TCP, pyridostigmine bromide, N-methyl-3-hydroxypyridinium bromide, DEET, m-toluamide and m-toluic acid, respectively. The relationship between peak areas and concentration was linear over a range between 200 and 2000 ng/ml.

Simultaneous determination of pyridostigmine bromide, N,N-diethyl-m-toluamide, permethrin, and their metabolites in rat plasma and urine by high-performance liquid chromatography.

A rapid and simple method was developed for the separation and quantification of the anti nerve agent drug pyridostignmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), its metabolites m-toluamide and m-toluic acid, the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester), and two of its metabolites m-phenoxybenzyl alcohol, and m-phenoxybenzoic acid in rat plasma and urine. The method is based on using C18 Sep-Pak cartridges for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with reversed-phase C18 column, and gradient UV detection ranging between 208 and 230 nm. The compounds were separated using gradient of 1 to 99% acetonitrile in water (pH 3.20) at a flow-rate ranging between 0.5 and 1.7 ml/min in a period of 17 min. The retention times ranged from 5.7 to 14.5 min. The limits of detection were ranged between 20 and 100 ng/ml, while limits of quantitation were 150-200 ng/ml. Average percentage recovery of five spiked plasma samples were 51.4+/-10.6, 71.1+/-11.0, 82.3+/-6.7, 60.4+/-11.8, 63.6+/-10.1, 69.3+/-8.5, 68.3+/-12.0, 82.6+/-8.1, and from urine 55.9+/-9.8, 60.3+/-7.4, 77.9+/-9.1, 61.7+/-13.5, 68.6+/-8.9, 62.0+/-9.5, 72.9+/-9.1, and 72.1+/-8.0, for pyridostigmine bromide, DEET, permethrin, N-methyl-3-hydroxypyridinium bromide, m-toluamide, m-toluic acid, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 5000 ng/ml. This method was applied to analyze the above chemicals and metabolites following their administration in rats.

Development and evaluation of LIPODEET, a new long-acting formulation of N, N-diethyl-m-toluamide (DEET) for the prevention of schistosomiasis.

N, N-diethyl-m-toluamide (DEET) is a common and fairly safe active ingredient in many insect repellents. Our recent studies showed that when applied to the skin, DEET has a potent anti-parasitic effect against Schistosoma mansoni. However, the beneficial effects of DEET lasted only for a few minutes, presumably due to its rapid absorption through the skin. In this study, we evaluated different carrier formulations that prolong the activity of DEET in the skin. Among the various formulations analyzed, DEET incorporated into liposomes (LIPODEET) appeared to prolong the activity of DEET for more than 48 hr after a single application. Furthermore, LIPODEET was found to be minimally absorbed through the skin and loss due to washing off was limited. These findings thus suggest LIPODEET is a safe and long-acting formulation of DEET that is quite effective against schistosomiasis.