Diethyl Thiophosphate Research Articles

Determination of diffusion coefficients and diffusion characteristics for chlorferon and diethylthiophosphate in Ca‐Alginate gel beads

Diffusion characteristics of chlorferon and diethylthiophosphate (DETP) in Ca-alginate gel beads were studied to assist in designing and operating bioreactor systems. Diffusion coefficients for chlorferon and DETP in Ca-alginate gel beads determined at conditions suitable for biodegradation studies were 2.70 x 10(-11) m(2)/s and 4.28 x 10(-11) m(2)/s, respectively. Diffusivities of chlorferon and DETP were influenced by several factors, including viscosity of the bulk solution, agitation speed, and the concentrations of diffusing substrate and immobilized cells. Diffusion coefficients increased with increasing agitation speed, probably due to poor mixing at low speed and some attrition of beads at high speeds. Diffusion coefficients also increased with decreasing substrate concentration. Increased cell concentration in the gel beads caused lower diffusivity. Theoretical models to predict diffusivities as a function of cell weight fraction overestimated the effective diffusivities for both chlorferon and DETP, but linear relations between effective diffusivity and cell weight fraction were derived from experimental data. Calcium-alginate gel beads with radii of 1.65-1.70 mm used in this study were not subject to diffusional limitations: external mass transfer resistances were negligible based on Biot number calculations and effectiveness factors indicated that internal mass transfer resistance was negligible. Therefore, the degradation rates of chlorferon and DETP inside Ca-alginate gel beads were reaction-limited.

Performance Assessment and Validation of a Paramagnetic Particle-Based Enzyme-Linked Immunosorbent Assay for Chlorpyrifos in Agricultural Runoff Waters

A commercial magnetic particle-based enzyme-linked immunosorbent assay (ELISA) kit for the insecticide chlorpyrifos [O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate] was evaluated for its specificity, precision, and accuracy, its susceptibility to matrix interferences in agricultural and environmental surface waters, and its comparability to a gas chromatographic/flame photometric (GC/FPD) method for the determination of organophosphorus pesticides in natural waters. Repeatability, reproducibility, and accuracy studies show that the kit satisfies current U.S. Environmental Protection Agency criteria for the assessment of analytical methods. Observable matrix effects were found to be present in all of the environmental test waters, with the slopes of calibration curves generated in each of the test matrices deviating from that of the control matrix by as much as 16%. Specificity studies indicate that the chlorpyrifos polyclonal antibody adequately differentiates the target compound from other structurally similar organophosphorus pesticides, with the exception of its methyl analogue. Cross-reactivity with chlorpyrifos-methyl was approximately 37%, while reactivity with diazinon, pyridaphenthion, diclofenthion, bromiphos-ethyl, bromiphos-methyl, pirimiphos-ethyl, and chlorpyrifos oxon ranged from 1.6 to 10.7%. Cross-reactivity with pirimiphos-methyl, 3,5,6-trichloro-2-pyridinol, diethyl phosphate, and diethyl thiophosphate was negligible (<1%). Validation of the paramagnetic particle ELISA format was accomplished using water samples from two monitoring studies that were collected, split, and analyzed directly by ELISA and by GC/FPD. Results of the two analytical methods were then compared using standard t tests, regression analysis, and differences against mean measurement (bias) plots. While the agreement between the two methods was determined to be satisfactory, ELISA exhibits consistent positive bias in environmental matrices. Several preanalysis mitigation steps were suggested that may help moderate bias, but additional study is recommended to explicate the exact factors responsible for its consistent overestimation of results.

Improved analysis of dialkylphosphates in urine using strong anion exchange disk extraction and in‐vial derivatization

Determination of dialkylphosphates (DAPs) in urine is useful for assessing human exposure to organophosphates (OPs). An improved method for the determination of four DAPs based on a strong anion exchange (SAX) disk extraction and in-vial derivatization was presented in this study. The matrix effect of urine components such as chloride ion and phosphate ion by using a SAX disk to extract DAPs in urine analysis was carefully evaluated. It was observed that the chloride ion mainly affected the extraction of diethylphosphate (DEP), dimethylthiophosphate (DMTP), and diethylthiophosphate (DETP) in urine. The addition of silver hydroxide could significantly improve the extraction efficiencies of these three DAPs, but it decreases the extraction efficiencies of dimethyldithiophosphate (DMDTP) and diethyldithiophosphate (DEDTP). The LOD of this method for DMTP, DETP, DMDTP, and DEDTP are 5, 5, 11, and 5 microg/L, respectively. A pretreatment strategy for the determination of DMTP, DMDTP, DETP, and DEDTP in urine was proposed which can provide reliable and prompt determination of routine urine analysis.

Comparative pharmacokinetics of the organophosphorus insecticide chlorpyrifos and its major metabolites diethylphosphate, diethylthiophosphate and 3,5,6-trichloro-2-pyridinol in the rat

Chlorpyrifos (CPF) is a commonly used diethylphosphorothionate organophosphorus (OP) insecticide. Diethylphosphate (DEP), diethylthiophosphate (DETP) and 3,5,6-trichloro-2-pyridinol (TCPy) are products of metabolism and of environmental degradation of CPF and are routinely measured in urine as biomarkers of exposure. However, because these same chemicals can result from metabolism or by biodegradation, monitoring total urinary metabolite levels may be reflective of not only an individual's contact with the parent pesticide, but also exposure with the metabolites, which are present in the environment. The objective of the current study was to compare the pharmacokinetics of orally administered DEP, DETP and TCPy with their kinetics following oral dosing with the parent insecticide CPF in the rat. Groups of rats were orally administered CPF, DEP, TCPy or DETP at doses of 140μmol/kg body weight, and the time-courses of the metabolites were evaluated in blood and urine. Following oral administration, all three metabolites were well absorbed with peak blood concentrations being attained between 1 and 3h post-dosing. In the case of DEP and TCPy virtually all the administered dose was recovered in the urine by 72h post-dosing, suggesting negligible, if any, metabolism; whereas with DETP, ∼50% of the orally administered dose was recovered in the urine. The CPF oral dose was likewise rapidly absorbed and metabolized to DEP, TCPy and DETP, with the distribution of metabolites in the urine followed the order: TCPy (22±3μmol)>DETP (14±2μmol)>DEP (1.4±0.7μmol). Based upon the total amount of TCPy detected in the urine a minimum of 63% of the oral CPF dose was absorbed. These studies support the hypotheses that DEP, DETP and TCPy present in the environment can be readily absorbed and eliminated in the urine of rats and potentially humans.

Toxicity of diazinon and its metabolites increases in diabetic rats

The effect of diazinon (DZN) on the activities of cholinesterase (ChE) in plasma and acetylcholinesterase (AChE) in erythrocyte and brain was investigated in normal and streptozotocin-induced diabetic rats. Hepatic drug-metabolizing enzyme activity was also estimated by measuring the systemic clearance of antipyrine, and the expression of hepatic cytochrome P450 (CYP) 3A2 and CYP1A2, which is closely related to the metabolism from DZN to DZN-oxon, a strong inhibitor of both ChE and AChE. No significant differences in the activities of ChE in plasma and AChE in erythrocyte were observed between normal and diabetic rats. Treatment with DZN significantly decreased these activities in diabetic rats more than in normal rats 6 h after injection (6.5 mg/kg). Treatment with DZN significantly decreased the activity of AChE in brain of diabetic rats than normal rats 3 h after injection (65 mg/kg), although no significant difference in the activity was found between normal and diabetic rats. The urinary recovery of diethylphosphate (DEP), a metabolite of DZN-oxon, was significantly increased in diabetic rats, but that of diethylthiophosphate (DETP), a metabolite of DZN, was unchanged. Significant increases in the systemic clearance of antipyrine and protein levels of hepatic CYP1A2, not CYP3A2, were observed in diabetic rats. These results suggest the possibility that a metabolite of DZN, DZN-oxon, causes higher toxicity in diabetic rats due to the enhancement of hepatic CYP1A2-mediated metabolism of DZN.

Metabolism of chlorpyrifos and chlorpyrifos oxon by human hepatocytes*

The metabolism of chlorpyrifos (CPS) and chlorpyrifos oxon (CPO) by human hepatocytes and human liver S9 fractions was investigated using LC-MS/MS. Cytochrome P450 (CYP)-dependent and phase II-related products were determined following incubation with CPS and CPO. CYP-related products, 3,5,6-trichloro-2-pyridinol (TCP), diethyl thiophosphate, and dealkylated CPS, were found following CPS treatment and dealkylated CPO following CPO treatment. Diethyl phosphate was not identified because of its high polarity and lack of retention with the chromatographic conditions employed. Phase II-related conjugates, including O- and S-glucuronides as well as 11 GSH-derived metabolites, were identified in CPS-treated human hepatocytes, although the O-sulfate of TCP conjugate was found only when human liver S9 fractions were used as the enzyme source. O-Glucuronide of TCP was also identified in CPO-treated hepatocytes. CPS and CPO were identified using HPLC-UV after CPS metabolism by the human liver S9 fraction. However, CPO was not found following treatment of human hepatocytes with either CPS or CPO. These results suggest that human liver plays an important role in detoxification, rather than activation, of CPS.

Biodegradation of chlorferon and diethylthiophosphate by consortia enriched from waste cattle dip solution

Chlorferon and diethylthiophosphate (DETP) are the hydrolysis products of coumaphos, an organophosphate pesticide. In this research, two consortia of bacterial cultures, one responsible for degrading chlorferon and the other for degrading DETP, were selectively enriched from waste cattle dip solution. The enriched cultures were used as inocula to grow biomass for biodegradation studies. For chlorferon degradation, the optimum biomass concentration was found to be 80 g/L, and pH 7.5 was selected as the optimal operating pH. Chlorferon degradation was characterized by substrate inhibition kinetics with parameter values estimated to be V m = 0.062 ± 0.011 mg/(g-biomass) h, K m = 21 ± 7 mg/L, and K Si = 118 ± 45 mg/L. For DETP degradation, the optimum biomass concentration was found to be 60 g/L, and the optimum pH was in the range of 7.5–8. DETP degradation was characterized by Michaelis–Menten kinetics with parameter values estimated to be V m = 1.52 ± 0.10 mg/(g-biomass) h and K m = 610 ± 106 mg/L.

Organophosphorus pesticide urinary metabolite levels of children in farmworker households in eastern North Carolina

Organophosphorus (OP) pesticide urinary metabolite levels in a sample of farmworker children in North Carolina are documented and compared to national reference data. The relative importance of para-occupational, residential, and environment risk factors are delineated. Urine samples were collected from 60 farmworker children 1-6 years of age, and interviews were completed by their mothers. Urine samples were analyzed for the dialkylphosphate (DAP) metabolites of OP pesticides. Summed molar concentrations of the diethyl and dimethyl DAP metabolites provided summary measures. The farmworker children had relatively high levels of OP pesticide urinary metabolites compared to national reference data; for example, participating children had higher geometric means for diethylphosphate (DEP), diethylthiophosphate (DETP), and the summed diethyl metabolites. However, analyses found no pattern of significant associations between predictors and metabolite levels. Future research requires greater precision in sampling and measurement to determine the risk factors for pesticide exposure among farmworker children.

Synthesis of phosphorothioates using thiophosphate salts

Reactions of O,O'-dialkyl thiophosphoric acids with alkyl halides, in the presence of a base, provide a direct synthetic route to phosphorothioates via O,O'-dialkyl thiophosphate anion formation. Studies on the reaction of ambident nucleophile ammonium O,O'-diethyl thiophosphate with benzyl halides and tosylate in different solvents show that only S-alkylation is obtained. Reaction of this ambident nucleophile with benzoyl chloride (a hard electrophile), gave the O-acylation product. A simple, efficient, and general method has been developed for the synthesis of phosphorothioates through a one-pot reaction of alkyl halides with the mixture of diethyl phosphite in the presence of triethylamine/sulfur/and acidic alumina under solvent-free conditions using microwave irradiation.

Simultaneous determination of urinary dialkylphosphate metabolites of organophosphorus pesticides using gas chromatography–mass spectrometry

In this study, we developed a safe and sensitive method for the simultaneous determination of urinary dialkylphosphates (DAPs), metabolites of organophosphorus insecticides (OPs), including dimethylphosphate (DMP), diethylphosphate (DEP), dimethylthiophosphate (DMTP), and diethylthiophosphate (DETP), using a pentafluorobenzylbromide (PFBBr) derivatization and gas chromatography-mass spectrometry (GC-MS). Several parameters were investigated: pH on evaporation, reaction temperature and time for the derivatization, the use of an antioxidant for preventing oxidation, and a clean-up step. The pH was set at 6, adjusted with K2CO3, and the reaction temperature and time of derivatization were 80 degrees C and 30 min, respectively. Sodium disulfite was chosen as an antioxidant. The clean-up step was performed with a Florisil/PSE mini-column to remove the unreacted PFBBr and sample matrix. This established procedure markedly shortened the sample preparation time to only about 3 h, and completely inhibited the unwanted oxidization of dialkylthiophosphates. The limits of determination (LOD) were approximately 0.3 microg/L for DMP, and 0.1 microg/L for DEP, DMTP, and DETP in 5 mL of human urine. Within-series and between-day imprecision for the present method using pooled urine spiked with DAPs was less than 20.6% in the calibration range of 1-300 microg/L, and the mean recovery was 56.7-60.5% for DMP, 78.5-82.7% for DEP, 88.3-103.9% for DMTP, and 84.2-92.4% for DETP. This method detected geometric mean values of the urinary DAPs in Japanese with and without occupational exposure to OPs, 16.6 or 27.4 for DMP, 1.0 or 0.7 for DEP, 1.3 or 2.3 for DMTP, and 1.0 or 1.1 microg/L for DETP, respectively. The present method, which does not require special equipment except for GC-MS, is quick, safe, and sensitive enough to be adopted in routine biological monitoring of non-occupational as well as occupational exposure to OPs.

Simultaneous determination of six dialkylphosphates in urine by liquid chromatography tandem mass spectrometry

Dialkylphosphates (DAP) are urinary markers of the exposure to organophosphates pesticides. The aim of this study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitative determination of the following DAP: dimethylphosphate (DMP), dimethythiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate (DEP), diethylthiophosphate (DETP) and diethyldithiophosphate (DEDTP). Dibutylphosphate (DBP) was used as internal standard. This method was based on a liquid-liquid extraction procedure, a chromatographic separation using an Inertsil ODS3 C18 column and mass spectrometric detection in the negative ion, multiple reaction monitoring (MRM) mode, following two ion transitions per compound. It yielded a limit of quantification of 2 microg/L for the six compounds and intra-assay coefficients of variation (CV%) lower than 20%. This method was applied to the analysis of urines samples from a small cohort of non-exposed volunteers. At least one of the six DAP was detected in each sample. This result confirmed the feasibility of a LC-MS/MS procedure for monitoring the general population exposure to some frequently employed organophosphate pesticides.

Use of liquid chromatography quadrupole time-of-flight mass spectrometry in the elucidation of transformation products and metabolites of pesticides. Diazinon as a case study

Liquid chromatography (LC) coupled to hybrid quadrupole time-of-flight (QTOF) mass spectrometry (MS) is a useful analytical tool in the elucidation and confirmation of transformation products (TPs)/metabolites of pesticides with a wide range of polarity, in both environmental and biological samples. Firstly, the versatility of LC allows the determination of very distinct TPs/metabolites as chromatographic conditions can be easily changed and optimized depending on the analytical problem. Secondly, the mass accuracy provided by the TOF analyser allows the assignment of a highly probable empirical formula for each compound and the differentiation between nominal isobaric compounds. Finally, the possibility of performing MS/MS spectra with accurate mass measurements can been used for the final characterization of the TPs/metabolites detected and for the differentiation of isomeric compounds. In this study, the insecticide diazinon was used as model compound, and its photodegradation and metabolism have been investigated by LC-QTOF-MS. On one hand, environmental spiked water was irradiated with a mercury lamp for 9 days, sampling 3-mL aliquots approximately every 12 h. On the other hand, both in vitro and in vivo metabolism experiments were carried out with different substrate concentrations and incubation times. After centrifugation, and protein precipitation in the in vitro and in vivo studies, 50-microL aliquots of both environmental and biological samples were directly injected into the LC electrospray ionization QTOF system. The most important transformation processes were found to be hydrolysis of the ester moiety, hydroxylation in the aromatic ring or in one of the alkylic groups, oxidation of the sulfur atom on the P=S cleavage or a combination of these processes, with the highest number of compounds being found in the photodegradation study. Very polar compounds, such as diethyl phosphate and diethyl thiophosphate, were detected after direct injection of the aqueous sample, which was feasible owing to the characteristics of the LC. In MS mode, mass errors were below 3 mDa, leading to an empirical formula for each compound. MS/MS spectra with accurate mass were used for the final elucidation of the compounds detected.

Extractive Spectrofluorometric Determination of Quinalphos Using Fluorescein in Environmental Samples

A simple, sensitive, convenient and low cost spectrofluorometric technique for determination of quinalphos is described. Quinalphos is decomposed with sodium ethoxide. Resultant sodium diethyl thiophosphate (Na-DETP) is extracted with a cationic fluorescein as ion pair complex into 1-chloro-2-nitrobenzene. The fluorescence of the ion pair, fluorescein-DETP is measured at 530 nm after excitation at 490 nm. This technique is applicable for the determination of quinalphos in water, food grains and vegetable samples up to ng level with Amberlite XAD-4.

A case study of acute human chlorpyrifos poisoning: Novel aspects on metabolism and toxicokinetics derived from liquid chromatography–tandem mass spectrometry analysis of urine samples

The metabolic fate of the organophosphorothioate-type insecticide chlorpyrifos (CP) in an acutely intoxicated 59 years old female was investigated by liquid chromatography–electrospray ionisation-tandem mass spectrometry (LC–ESI-MS/MS) analysis of urine samples. Fifteen metabolites of CP and its bioactivated intermediate chlorpyrifos-oxon (CPO), respectively, of which only three have been described in man so far, were identified on the basis of characteristic MS/MS transitions, precursor/product ion and/or neutral loss scans, chlorine isotopomer patterns, and partly by synthesis of reference compounds and subsequent structure confirmation. Three distinct biotransformation routes of CP are proposed: (1) cleavage reactions at the aromatic phosphoester bond, (2) cleavage reactions at the alkyl phosphoester bonds, and (3) glutathione (GSH) dependent nucleophilic substitution of the 6-chlorine at the aromatic moiety. Route (2) has not been reported in humans before and (3) is a hitherto completely unknown scheme of CP metabolism. Urinary markers of the latter were chiefly cysteine S-conjugates of mono-dechlorinated CP, CPO, mono- O-deethyl CP, and mono- O-deethyl CPO as well as the 6-mercapturic acid conjugate of 3,5-dichloro-2-pyridinol. The presence of 3,5-dichloro-6-methylthio-2-pyridinol as well as its O-glucuronide suggests further a cysteine S-conjugate β-lyase mediated degradation. In addition to the qualitative LC–MS/MS screening the renal elimination profiles of the primary products of scheme (1), i.e. diethyl thiophosphate (DETP), diethyl phosphate (DEP), and 3,5,6-trichloro-2-pyridinol (TCP), were monitored over 14 days ( n = 21). A biphasic first-order excretion mechanism with half-lives of 21.5 h (initial fast excretion phase) and 119.5 h (terminal phase) for the sum of free DETP and DEP was found. TCP was hardly eliminated in its free form ( O-glucuronide identified as phase II conjugate) and half-lives calculated for the total amount of TCP (acidic hydrolysis of urine samples) were 40.8 and 150.7 h. The present study gives a more detailed view on the biotransformation of CP and together with the obtained kinetic data adds novel aspects to the limited knowledge of human metabolism of this xenobiotic, in particular at high dosage.

Determination of chlorpyrifos metabolites in human urine by reversed-phase/weak anion exchange liquid chromatography–electrospray ionisation–tandem mass spectrometry

A liquid chromatography–electrospray ionisation–tandem mass spectrometry (LC–ESI–MS/MS) method for the quantification of major chlorpyrifos (CP) metabolites, i.e. diethyl thiophosphate (DETP), diethyl phosphate (DEP), and 3,5,6-trichloro-2-pyridinol (TCP), in human urine was developed. Simultaneous separation of the parent compound and its primary biotransformation products was achieved within 20 min in gradient elution mode employing a mixed-mode reversed-phase/weak anion exchange (RP/WAX) separation principle. The analytical method was developed for a toxicokinetic study of an acute poisoning incidence with a CP containing pesticide formulation. An initial mass spectrometric screening performed with unprocessed urine samples revealed that CP is not excreted unchanged by the kidney. Hence, the quantitative assay was validated for DETP (quantifier transition: m/z 169 → 95, qualifier transition: m/z 169 → 141), DEP ( m/z 153 → 79, 153 → 125), and TCP ( m/z 196 → 35, 198 → 35) taking dibutyl phosphate (DBP) ( m/z 209 → 79, 209 → 153) as internal standard. Clean-up of urine samples prior to LC–ESI–MS/MS analysis was carried out by a liquid–liquid extraction step with a mixture of ethylacetate and acetonitrile (70:30; v/v). Linearity was observed between 0.25 and 75 mg L −1, and the signal-to-noise ratio at 0.25 mg L −1 was better than six for the individual analytes. Recoveries, precision, and accuracies were all adequate across the validated range of 1–75 mg L −1 for the present toxicological case study.

Methyl-parathion and organophosphorous pesticide metabolites modify the activation status and interleukin-2 secretion of human peripheral blood mononuclear cells

Organophosphorous (OP) compounds are the most commonly used pesticides. There are reports on susceptibility to the toxic effects of OP pesticides, but no information exists regarding the toxicity of their metabolites. To determine the metabolites’ contribution to the OP pesticide immunotoxic effects, human peripheral blood mononuclear cells (PBMCs) were treated with the parent compound methyl-parathion (MP) and the following OP pesticide alkyl-phosphorous metabolites: diethylphosphate (DEP), diethylthiophosphate (DETP), diethyldithiophosphate (DEDTP), dimethylphosphate (DMP), and dimethyldithiophosphate (DMDTP). Activation and function of the PBMCs were examined by assessment of phytohemagglutinin (PHA)-induced proliferative response, interleukin-2 (IL-2) secretion, and CD25 and CD69 expression. Treatments with DMP, DEP, DETP and DEDTP for 48 h produced significant toxicity in human PBMCs, but did not affect their proliferative response to PHA. Only MP reduced cell proliferation by 30%. DEDTP decreased the proportion of PBMCs expressing CD25. This effect was associated with a reduction of IL-2 secretion, which was also reduced by MP and DMP treatments. In contrast, DETP and DEDTP treatments increased the expression of CD69. DMP, DETP and DEDTP were more consistently involved in modulating the PBMC response to PHA.

Organophosphorous pesticide exposure alters sperm chromatin structure in Mexican agricultural workers

Our objective was to evaluate alterations in sperm chromatin structure in men occupationally exposed to a mixture of organophosphorus pesticides (OP) because these alterations have been proposed to compromise male fertility and offspring development. Chromatin susceptibility to in situ acid-induced denaturation structure was assessed by the sperm chromatin structure assay (SCSA). Urinary levels of alkylphosphates (DAP) were used to assess exposure. Diethylthiophosphate (DETP) was the most frequent OP metabolite found in urine samples indicating that compounds derived from thiophosphoric acid were mainly used. Chromatin structure was altered in most samples. About 75% of semen samples were classified as having poor fertility potential (>30% of Percentage of DNA Fragmentation Index [DFI%]), whereas individuals without OP occupational exposure showed average DFI% values of 9.9%. Most parameters of conventional semen analysis were within normality except for the presence of immature cells (IGC) in which 82% of the samples were above reference values. There were significant direct associations between urinary DETP concentrations and mean DFI and SD-DFI but marginally ( P = 0.079) with DFI%, after adjustment for potential confounders, including IGC. This suggests that OP exposure alters sperm chromatin condensation, which could be reflected in an increased number of cells with greater susceptibility to DNA denaturation. This study showed that human sperm chromatin is a sensitive target to OP exposure and may contribute to adverse reproductive outcomes. Further studies on the relevance of protein phosphorylation as a possible mechanism by which OP alter sperm chromatin are required.

Concentrations of dialkyl phosphate metabolites of organophosphorus pesticides in the U.S. population.

We report population-based concentrations, stratified by age, sex, and racial/ethnic groups, of dialkyl phosphate (DAP) metabolites of multiple organophosphorus pesticides. We measured dimethylphosphate (DMP), dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate (DEP), diethylthiophosphate (DETP), and diethyldithiophosphate (DEDTP) concentrations in 1,949 urine samples collected in U.S. residents 6-59 years of age during 1999 and 2000 as a part of the ongoing National Health and Nutrition Examination Survey (NHANES). We detected each DAP metabolite in more than 50% of the samples, with DEP being detected most frequently (71%) at a limit of detection of 0.2 microg/L. The geometric means for the metabolites detected in more than 60% of the samples were 1.85 microg/L for DMTP and 1.04 microg/L for DEP. The 95th percentiles for each metabolite were DMP, 13 microg/L; DMTP, 46 microg/L; DMDTP, 19 micro g/L; DEP, 13 microg/L; DETP, 2.2 microg/L; and DEDTP, 0.87 microg/L. We determined the molar sums of the dimethyl-containing and diethyl-containing metabolites; their geometric mean concentrations were 49.4 and 10.5 nmol/L, respectively, and their 95th percentiles were 583 and 108 nmol/L, respectively. These data are also presented as creatinine-adjusted concentrations. Multivariate analyses showed concentrations of DAPs in children 6-11 years of age that were consistently significantly higher than in adults and often higher than in adolescents. Although the concentrations between sexes and among racial/ethnic groups varied, no significant differences were observed. These data will be important in evaluating the impact of organophosphorus pesticide exposure in the U.S. population and the effectiveness of regulatory actions.

Current internal exposure to pesticides in children and adolescents in Germany: urinary levels of metabolites of pyrethroid and organophosphorus insecticides.

Pesticides are widely used throughout the world, in agriculture to protect crops and in public health to control diseases transmitted by vectors or intermediate hosts. After the prohibition of organochlorines such as DDT, today, mainly pyrethroids and organophosphorus insecticides are used. Whereas many studies have been published on background exposure of the population to organochlorines, data on internal exposure of the population to pyrethroids and organophosphorus insecticides are scarce. Here, we report on internal exposure of children and young people, in an urban area in Germany, to pyrethroids and organophosphorus acids, assessed by the analysis of urinary levels of their corresponding specific metabolites. Approximately 673 children and adolescents took part in this voluntary investigation, including 331 children <6 years of age. Their parents stated that they and their children had never used pyrethroids or organophosphorus acids in their homes or for medical reasons. We analysed their spot urine samples for six metabolites of organophosphorus insecticides [dimethyl-phosphate (DMP), diethyl-phosphate (DEP), dimethyl-thiophosphate (DMTP), diethyl-thiophosphate (DETP), dimethyl-dithiophosphate (DMDTP) and diethyl-dithiophosphate (DEDTP)] and for four metabolites of pyrethroids [cis-3-(2,2-dibromo-vinyl)-2,2-dimethyl-cyclopropane carboxylic acid (Br2CA), cis-3-(2,2-dichloro-vinyl)-2,2-dimethyl-cyclopropane carboxylic acid (cis-Cl2-CA ), trans-3-(2,2-dichloro-vinyl)-2,2-dimethyl-cyclopropane carboxylic acid (trans-Cl2-CA) and 4-fluoro-3-phenoxy-benzoic acid (F-PBA)] using gas chromatographic methods with mass-selective detection. The limit of detection was 0.1-0.2 microg/l for pyrethroid metabolites and 1 microg/l for metabolites of organophosphorus acids; in DMP it was 5 microg/l. The 95th percentiles of the urinary metabolite concentrations were, in microgrammes per litre, DMP 158, DMTP 180, DMDTP 12, DEP 17, DETP 8, DEDTP <1; Br2CA 0.30, cis-Cl2-CA 0.44, trans-Cl2-CA 1.22, F-PBA 0.30. There were no correlations between urinary metabolite levels and the age of the children. Current background levels of internal exposure to pyrethroids and organophosphorus insecticides in children and adolescents in Germany are shown. Exposure to these substances in the general population is thought to occur mainly via residues in the diet. The level of background internal pyrethroid exposure in the children is orders of magnitude lower than the corresponding acceptable daily intake (ADI) values published, but the level of internal organophosphate exposure may reach and even exceed ADI values. This observation demands further investigation.

Direct determination of alkyl phosphates in human urine by liquid chromatography/electrospray tandem mass spectrometry.

A novel and rapid procedure based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the determination of dialkyl phosphates (metabolites of organophosphorus pesticides) in human urine has been developed. After addition of 40 mM tetrabutylammonium acetate, 10 microL of urine sample were directly injected into the LC/MS/MS system. The method was validated yielding calibration curves with correlation coefficients greater than 0.997 and repeatability coefficient of variation (CV) lower than 9%. The accuracy and precision were evaluated by direct injection of spiked samples at 10 and 100 microg/L obtaining recoveries between 78 and 119% with coefficients of variation below 12%. Limits of detection of 1 microg/L for diethyl phosphate (DEP), diethylthiophosphate (DETP) and diethyldithiophosphate (DEDTP) and 2 microg/L for dimethyldithiophosphate (DMDTP) were achieved, all the analytes being detected in negative ion mode. The fragmentation pathway of dialkyl phosphates allowed us the use of an additional transition for confirmation in order to improve their identification in real-world samples. The applicability of the LC/MS/MS method was demonstrated by applying it to the analysis of urine samples of farmers exposed to the organophosphorus pesticide chlorpyrifos. Good correlation between application of the product in the field (citrus orchards), concentration levels of dialkyl phosphates and levels of the chlorpyrifos-specific metabolite (1,3,5-trichloro-2-pyridinol) was obtained.