Organophosphate pesticide metabolite levels in pre-school children in an agricultural community: within- and between-child variability in a longitudinal study.

This study characterized the within- and between-child variability in dimethylthiophosphate (DMTP) levels in the urine of 44 children living in an agricultural community in central Washington State in December 1997 and 1999. The goal of this analysis was to investigate these variability components during periods when organophosphate pesticides were and were not actively applied to orchards in that community. Each child provided between 10 and 26 biweekly urine samples over a 21-month period, and these samples were analyzed for six dialkylphosphate (DAP) metabolites common to organophosphate pesticides, including DMTP. Previous analysis of this dataset found that DAP concentrations were elevated during months when organophosphate pesticides were applied to orchards in this region. The current analysis demonstrates that the within-child component of day-to-day variability was much greater than the between-child component of variability by a factor of 3-7 across the DAP metabolites that were analyzed. Therefore, organophosphate pesticide exposure appeared to vary more than 3 times from day-to-day than from child-to-child. This finding has important implications for epidemiologic and exposure pathways research, since accounting for within-child variability may increase the power of a study and allow for the detection of differences that would not otherwise be possible without an analysis that separates out the within-child variability.

Impact of dermal absorption factors in occupational exposure assessment: comparison of two models for agricultural reentry workers exposed to azinphosmethyl.

This analysis compares two deterministic reentry exposure models that differ in their treatment of the time dependence of dermal absorption. The first model, called the "traditional model," assumes that dermal absorption is a fixed fraction of the cumulative load on skin at the end of the workshift and that absorption is independent of residence time on the skin. The second model, called the "time-integrated model," incorporates the time dependence of both exposure and absorption by assuming that absorption begins at the outset of exposure and continues through the workshift and beyond, until an effective washing event occurs. These two models were evaluated using previously collected biological monitoring data from apple thinners exposed to azinphosmethyl. Daily doses predicted by the models were compared to doses estimated from the biological sampling results assuming pseudo steady-state excretion. The geometric mean dose estimated from the biological sampling data was 20 microg/kg/day. Corresponding geometric mean doses produced by the traditional model and the time-integrated model were 79 microg/kg/day and 24 microg/kg/day, respectively. When the doses predicted by the traditional model were plotted against those estimated from the biological measurements, the slope of the regression line was significantly greater than 1 (beta = 1.37). However, when this same analysis was conducted for the doses predicted by the time-integrated model, the confidence interval around the slope encompassed 1 (beta = 1.01). Thus, time-integrated treatment of absorption appeared to provide more realistic dose estimates than did the traditional approach.

Fluorescent tracer evaluation of chemical protective clothing during pesticide applications in central Florida citrus groves.

Chemical protective clothing (CPC) is often recommended as a method of exposure mitigation among pesticide applicators. This study evaluated four CPC regimens (cotton work shirts and work pants, cotton/polyester coveralls, and two non-woven garments) during 33 airblast applications of the organophosphorus insecticide ethion in central Florida citrus groves. CPC performance was determined by measurement of fluorescent tracer deposition on skin surfaces beneath garments with a video imaging analysis instrument (VITAE system), and by alpha-cellulose patches placed outside and beneath the garments. Non-woven coveralls allowed significantly greater exposure than did traditional woven garments, primarily because of design factors (e.g., large sleeve and neck openings). The greatest exposure occurred on the forearms beneath the non-woven garments. Fabric penetration was detected for all test garments; 5% to 7% of the ethion measured outside the garments was found beneath the garments. The clothing materials tested were not chemically resistant under these field conditions. Exposurepathways that would probably be undetected by the patch technique were characterized effectively with fluorescent tracers and video imaging analysis. However, the patch technique was more sensitive in detecting fabric penetration. CPC garments have been improved since this study was conducted, but performance testing under field conditions is not widespread. Workers conducting airblast applications would be better protected by closed cab systems or any technology that places an effective barrier between the worker and the pesticide spray.

Biological monitoring survey of organophosphorus pesticide exposure among pre-school children in the Seattle metropolitan area.

In this study we assessed organophosphorus (OP) pesticide exposure among children living in two Seattle metropolitan area communities by measuring urinary metabolites, and identified possible exposure risk factors through a parental interview. We recruited children in clinic and outpatient waiting rooms. We obtained spot urine samples in the spring and fall of 1998 from 110 children ages 2-5 years, from 96 households. We analyzed urine samples for six dialkylphosphate (DAP) compounds, the common metabolites of the OP pesticides. Through parental interviews we gathered demographic and residential pesticide use data. At least one of the DAP metabolites was measured in 99% of the children, and the two predominant metabolites (DMTP and DETP) were measured in 70-75% of the children. We found no significant differences in DAP concentrations related to season, community, sex, age, family income, or housing type. Median concentrations of dimethyl and diethyl DAPs were 0.11 and 0.04 micromol/L, respectively (all children). Concentrations were significantly higher in children whose parents reported pesticide use in the garden (0.19 vs. 0.09 micromol/L for dimethyl metabolites, p = 0.05; 0.04 vs. 0.03 micromol/L for diethyl metabolites, p = 0.02), but were not different based on reported pet treatment or indoor residential use. Nearly all children in this study had measurable levels of OP pesticide metabolites. Some of this exposure was likely due to diet. Garden pesticide use was associated with elevated metabolite levels. It is unlikely that these exposure levels would cause acute intoxication, but the long-term health effects of such exposures are unknown. We recommend that OP pesticide use be avoided in areas where children are likely to play.

Pesticide exposure of children in an agricultural community: evidence of household proximity to farmland and take home exposure pathways.

Children's exposure to organophosphorus (OP) pesticides in an agricultural community in central Washington State was determined. Spot urine and hand wipe samples were collected from 109 children 9 months to 6 years of age, as were house dust samples, and wipe samples from various surfaces. Children were categorized based on parental occupation (agricultural vs nonagricultural) and on household proximity to pesticide-treated orchards. Median house dust concentrations of dimethyl OP pesticides in homes of agricultural families were seven times higher than those of reference families (1. 92 vs 0.27 microg/g; P<0.001). Median pesticide metabolite concentrations in agricultural children were five times higher than those in reference children (0.05 vs 0.01 microg/ml; P=0.09). Median pesticide concentrations in housedust (P=0.01) and metabolite concentrations in urine (P=0.01) from agricultural families were significantly higher in the children living near treated orchards (within 200 ft or 60 m) than those living more distant. Ten of 61 agricultural children had detectable OP pesticide levles on their hands, whereas none of the reference children had detectable levels. These findings indicate that children living with parents who work with agricultural pesticides, or who live in proximity to pesticide-treated farmland, have higher exposures than do other children living in the same community

Saliva biomonitoring of atrazine exposure among herbicide applicators.

A field study was conducted in which saliva samples were collected from a cohort of herbicide applicators during the pre-emergent spray season in Ohio in 1996. Atrazine concentrations were detected in human saliva samples using an enzyme-linked immunosorbent assay (ELISA) method. Trend due to atrazine exposure and subsequent elimination in the body were evidenced by the temporal pattern of decreasing atrazine concentrations in saliva over time. Median salivary concentrations of atrazine on non-spray days were significantly lower than on spray days for each sampling time (Mann-Whitney U-Wilcoxon rank sum test, P < 0.01). Within spray days, median salivary atrazine concentrations were significantly higher on days atrazine was sprayed than on days herbicides other than atrazine were sprayed for each sampling time (Mann-Whitney U-Wilcoxon rank sum test, P = 0.02 for 4 6 p.m. samples, P = 0.04 for bedtime samples, P = 0.03 for next-morning samples). Median salivary atrazine concentrations on days atrazine was sprayed were higher than the median concentration for the corresponding sampling time on non-spray days and on days when other herbicides were sprayed. Salivary concentration of atrazine is a plausible indicator of those days in which atrazine spraying was likely to have occurred. Salivary concentrations of atrazine not only reflect exposures resulting from spraying atrazine, but also exposures from other field activities where applicators may come in contact with atrazine. The results of this study confirmed data from animal experiments that atrazine is able to cross the cell membranes of salivary glands, and can be measured in human saliva with high sensitivity. The sampling method itself is convenient and easy to use in the field, with a high compliance rate, and analytical procedures are rapid and inexpensive. It is, therefore, concluded that saliva sampling of atrazine exposure among herbicide applicators is a feasible biomonitoring method.

Biologically based pesticide dose estimates for children in an agricultural community.

Current pesticide health risk assessments in the United States require the characterization of aggregate exposure and cumulative risk in the setting of food tolerances. Biologic monitoring can aggregate exposures from all sources and routes, and can integrate exposures for chemicals with a common mechanism of action. Its value was demonstrated in a recent study of organophosphorus (OP) pesticide exposure among 109 children in an agricultural community in Washington State; 91 of the children had parents working in agriculture. We estimated individual OP pesticide doses from urinary metabolite concentrations with a deterministic steady state model, and compared them to toxicologic reference values. We evaluated doses by assuming that metabolites were attributable entirely to either azinphos-methyl or phosmet, the two OP pesticides used most frequently in the region. Creatinine-adjusted average dose estimates during the 6- to 8-week spraying season ranged from 0 to 36 microg/kg/day. For children whose parents worked in agriculture as either orchard applicators or as fieldworkers, 56% of the doses estimated for the spray season exceeded the U.S. Environmental Protection Agency (EPA) chronic dietary reference dose, and 19% exceeded the World Health Organization acceptable daily intake values for azinphos-methyl. The corresponding values for children whose parents did not work in agriculture were 44 and 22%, respectively. The percentage of children exceeding the relevant reference values for phosmet was substantially lower (< 10%). Single-day dose estimates ranged from 0 to 72 microg/kg/day, and 26% of these exceeded the EPA acute reference dose for azinphos-methyl. We also generated dose estimates by adjustment for total daily urine volume, and these estimates were consistently higher than the creatinine-adjusted estimates. None of the dose estimates exceeded the empirically derived no-observable-adverse-effect levels for these compounds. The study took place in an agricultural region during a period of active spraying, so the dose estimates for this population should not be considered representative of exposures in the general population. The findings indicate that children living in agricultural regions represent an important subpopulation for public health evaluation, and that their exposures fall within a range of regulatory concern. They also demonstrate that biologically based exposure measures can provide data for health risk evaluations in such populations.

Mechanisms underlying Children's susceptibility to environmental toxicants.

An important public health challenge has been the need to protect children's health. To accomplish this goal, the scientific community needs scientifically based child-specific risk assessment methods. Critical to their development is the need to understand mechanisms underlying children's sensitivity to environmental toxicants. Risk is defined as the probability of adverse outcome and when applied to environmental risk assessment is usually defined as a function of both toxicity and exposure. To adequately evaluate the potential for enhanced health risks during development, both child-specific factors affecting toxicity and exposure need to be considered. In the first section of this article, example mechanisms of susceptibility relevant for toxicity assessment are identified and discussed. In the second section, examples of exposure factors that help define children's susceptibility are presented. Examples of pesticide research from the newly funded Child Health Center at the University of Washington will be given for illustration. The final section discusses the importance of putting these considerations of children's susceptibility into an overall framework for ascertaining relevancy for human risk assessment.

Strategies for assessing children's organophosphorus pesticide exposures in agricultural communities.

Children can be exposed to pesticides from multiple sources and through multiple pathways. In addition to the standard pathways of diet, drinking water and residential pesticide use, children in agricultural communities can be exposed to pesticides used in agricultural production. A research program on children and pesticides was established at the University of Washington (UW) in 1991 and has focused on two major exposure pathway issues: residential proximity to pesticide-treated farmland and transfer of pesticides from the workplace to the home (paraoccupational or take-home exposure). The UW program selected preschool children of agricultural producers and farm workers in the tree fruit region of Washington state as a population that was likely to have elevated exposures from these pathways. The organophosphorus (OP) pesticides were selected as a common class of chemicals for analysis so that issues of aggregate exposure and cumulative risk could be addressed. This paper provides an overview of key findings of our research group over the past 8 years and describes current studies in this field. Soil and housedust concentrations of OP pesticides were elevated in homes of agricultural families (household members engaged in agricultural production) when compared to non-agricultural reference homes in the same community. Dialkyl phosphate metabolites of OP pesticides measured in children's urine were also elevated for agricultural children when compared to reference children and when compared to children in the Seattle metropolitan area. Proximity to farmland was associated with increased OP pesticide concentrations in housedust and OP pesticide metabolites in urine. Current studies include a community-based intervention to reduce parental transfer of pesticides from the workplace, and a systematic investigation of the role of agricultural spray drift in children's exposure to pesticides.

Comparison of three methods for assessment of hand exposure to azinphos-methyl (Guthion) during apple thinning.

Hand exposures of apple thinners to the pesticide azinphos-methyl (Guthion) were measured using three methods (glove, handwash, and wipe). Hand exposure sampling for each method was conducted following apple thinning work for a period of two hours for six to eight workers. Foliar residue samples were collected on each day of hand exposure sampling in the same orchard blocks that were thinned; foliar residues are considered to have been constant during the four-day study, which took place, on average, six days after pesticide application. Hand exposure estimates derived from each of the three methods differed significantly (ANOVA: p < 0.001). Mean measured exposure rates for the glove, handwash, and wipe methods were 6.48, 1.83, and 0.28 mg/hr, respectively. A corrected estimate of hand exposure, 2.7 mg/hr, was calculated from the handwash measurements and the handwash removal efficiency factor from a laboratory study. Comparison with this hand exposure estimate suggests that the glove method produced a 2.4-fold overestimate of exposure, whereas the wipe method produced a 10-fold underestimate. Studies that measure hand exposure to pesticides should include a careful description of sampling methods and should recognize the potential for measurement bias. Furthermore, the standardization and validation of dermal exposure assessment methods are critical to developing more comparable and more accurate pesticide exposure estimates.

Improved cleanup and determination of dialkyl phosphates in the urine of children exposed to organophosphorus insecticides.

Analysis of dialkylphosphate urinary metabolites of organophosphorus insecticides has been used to estimate dose in nonoccupationally exposed populations, including children. Analytical methods must continue to be improved in order to accurately and reproducibly measure less than 10 ng/mL of these metabolites. Dialkyl phosphates are commonly determined as their pentafluorobenzyl bromide derivatives via gas chromatography (GC) with flame photometric detection. Presented here is an improved method for precleanup of urine using solid-phase extraction, followed by derivatization and GC analysis. The method includes the quantitative determination of the following dialkyl phosphate metabolites: dimethylphosphate, diethylphosphate, dimethylthiophosphate, diethylthiophosphate, and dimethyldithiophosphate. Additional cleanup of urine samples allows for increasing sample size and improving sensitivity while minimizing interferences and variability associated with derivatization. Sample aliquot size was 5 mL with limits of quantitation of 10 ng/mL of urine for dimethylphosphate and diethylphosphate and 2 ng/mL of urine for dimethylthiophosphate, diethylthiophosphate, and dimethyldithiophosphate. This level of method sensitivity allows for quantitative determination of trace dialkyl phosphates in approximately 75% of individuals in nonoccupationally exposed populations. This streamlined method increases sample throughput, provides a clean extract for analysis, and requires no custom glassware.

Dermal transfer of chlorpyrifos residues from residential surfaces: comparison of hand press, hand drag, wipe, and polyurethane foam roller measurements after broadcast and aerosol pesticide applications.

Indoor residential pesticide applications present the potential for human exposures, particularly for small children. Personal contact with target and nontarget surfaces can result in transfer of pesticides to the skin, but the magnitude of such transfer is uncertain. This research compared surface sampling techniques [wipe and polyurethane foam (PUF) roller] with the removal ability of human skin following broadcast and total aerosol release applications of Dursban (Dow Elanco, Midland, MI), a residential formulation containing the insecticide chlorpyrifos. Hands were washed immediately after surface contact, following a protocol that included a laboratory-generated adjustment factor to account for incomplete removal of chlorpyrifos from skin. Chlorpyrifos transfer was similar for hand press and hand drag techniques, averaging approximately 1-6 ng/cm2 of carpet contacted. These amounts represented < 1% of the amount of chlorpyrifos deposited on the surfaces 3.5 hr earlier. Chlorpyrifos transfer from carpet to skin was 23-24 times lower than for wipe sampling and 33-36 times lower than for PUF roller sampling (p = 0.0007 and p = 0.0006 for broadcast and aerosol applications, respectively). Hand press sampling removed approximately 4.5 times less chlorpyrifos from nontarget furniture surfaces (12 ng/cm2) than did wipe sampling (56 ng/cm2; p = 0.009). Chlorpyrifos residues on carpet were substantially higher after broadcast applications than after aerosol applications, but residues on such nontarget surfaces as furniture were substantially higher for the aerosol application. This study indicates that human skin removes substantially less residue from carpets and furniture than either conventional wipe or PUF roller sampling methods following residential pest control applications of chlorpyrifos. Although this paper focuses on quantifying residue transfer from surface to skin using different surface sampling techniques, no attempt is made to quantify the amount of chlorpyrifos residue that is subsequently absorbed.

Salivary concentrations of atrazine reflect free atrazine plasma levels in rats.

The protein binding of atrazine in plasma and its effect on salivary excretion of atrazine was determined in male Sprague-Dawley rats. The degree of protein binding of atrazine was determined at 3 steady-state plasma concentrations, 50, 150, and 250 microg/L, using an ultrafiltration technique. In total, 48 arterial blood samples were collected from 18 rats; 38 of 48 blood samples had their time-matched whole saliva samples. The average protein binding of atrazine ranged from 18% to 37%; however, it was not significantly different across the 3 steady-state plasma concentrations nor among the individual rats. Overall, 26% of atrazine was bound to plasma proteins and not available for transport from blood into saliva. Protein binding of atrazine in plasma was not correlated with total atrazine plasma concentration nor with free atrazine plasma concentration, which indicates that the protein-bound fraction of atrazine is independent of plasma concentration within the range measured in this study (30-400 microg/L). The average saliva/plasma (S/P) concentration ratio of atrazine increased from 0.7 using total atrazine plasma concentration to 0.94 (S/fP) when free atrazine plasma concentrations calculated as 26% of protein binding was used. Salivary concentration was highly correlated with free atrazine plasma concentration. The results suggest that salivary concentration of atrazine not only reflects its total plasma level but accurately measures the portion of atrazine (free atrazine) in plasma, which may be of toxicological significance.

Correspondence of salivary and plasma concentrations of atrazine in rats under variable salivary flow rate and plasma concentration.

The stability of the saliva/plasma (S/P) concentration ratio of atrazine was determined under varying conditions of salivary flow rate and plasma concentration of atrazine in Sprague-Dawley rats. In the salivary flow study, whole saliva samples were collected at different salivary flow rates while the plasma concentration of atrazine was maintained at a steady-state level of approximately 150 micrograms/L. In the plasma level study, whole saliva samples were collected at two steady-state plasma concentrations of atrazine (50 and 250 micrograms/L), while salivary flow rate was maintained at a relatively constant level. In both studies, atrazine concentrations in whole saliva and arterial plasma demonstrated a consistent relationship, but salivary concentrations were always lower than those of arterial plasma. Linear regression analysis demonstrated that the S/P concentration ratio of atrazine was not significantly different for salivary flow rates ranging from 23 to 92 microL/min/kg body weight, and did not vary for atrazine plasma concentrations between 30 and 433 micrograms/L. The S/P concentration ratio of atrazine was relatively constant throughout each experimental period (0.68 +/- 0.1 and 0.70 +/- 0.11 for salivary flow and plasma level studies, respectively) and did not differ significantly between rats. When data from both studies were pooled, salivary concentrations were highly correlated with plasma concentrations (r2 = .94). It is concluded that under these experimental conditions, the stability of the S/P concentration ratio of atrazine is not affected by variations in salivary flow rate or atrazine plasma concentrations. The results from this study support the conclusion that atrazine salivary concentrations can be used to predict plasma levels of atrazine in rats.

Second generation video imaging technique for assessing dermal exposure (VITAE System).

Development of a second-generation video imaging technique for assessing occupational skin exposure (VITAE) is described, its performance evaluated, and new procedures for exposure quantification are presented. The current VITAE system has higher resolution in regard to both its picture element array and gray scale when compared with the prototype system. System performance was evaluated during extended field deployment: variability was 3-4% during data acquisition for individual worker evaluation session, and 10% over a 22-day study period. Variabilities attributable to subject positioning and image outlining procedures were 2.7 and 1.2%, respectively. Visual observations of fluorescent tracer deposition on skin were used to classify specific body regions as either exposed of unexposed, and two computer-based classification criteria were tested against the visual classification. These criteria were generally better at minimizing false negative and false positive classification; sensitivity and predictive value reached 95 and 99%, respectively, when analysis was preceded by presampling of a subset of images. Variability in skin pigmentation was found to have a substantial effect on fluorescent tracer qualification, leading to development of new calibration procedures. Standard curves were generated by spotting a range of tracer concentrations on volunteer subjects and quantifying fluorescence with the VITAE system. These data were then grouped either by subject or by the magnitude of the background signal of the unexposed skin. The ability to control for the effects of skin pigmentation was found to be comparable for these grouping methods, indicating that calibration curves can be developed without the creation of a unique curve for each subject.

Simulated dermal contamination with capillary samples and field cholinesterase biomonitoring.

The extensive international use of organophosphorus compounds (OP) results in numerous acute intoxications each year. OPs inhibit acetylcholinesterase, the enzyme responsible for breaking down the neurotransmitter acetylcholine. The World Health Organization recognizes cholinesterase (ChE) biomonitoring as a preventive measure against OP overexposure. The aim of this study was to determine if dermal OP contamination could interfere with current field ChE biomonitoring assays, which use a fingerstick blood sample. In this study we also sought to determine if high levels of a plasma enzyme, A-esterase, could protect ChE from inhibition by hydrolyzing environmentally generated oxons potentially present in a fingerstick sample. A heparinized venous blood sample was collected from a volunteer. Erythrocyte acetylcholinesterase (AChE) and plasma butyrylcholinesterase (PChE) activities were measured using a field-based colorimetric cholinesterase kit. ChE dose-response curves were constructed by allowing 10-microliters blood samples to contact environmentally realistic levels of OP thioate and oxon for 10 s. An inhibition threshold could not be established for PChE when exposed to oxon within the time necessary to perform a fingerstick analysis. AChE was also inhibited by trace amounts of oxon consistent with previously reported environmental levels. These findings suggest that the reliability of field-based biomonitoring results is limited if OP residues remain on a skin surface at the time of sample collection. A-esterase's role in protecting ChE activity was investigated using capillary and venous blood from 30 unexposed individuals. Baseline ChE activities were measured, as were individual A-esterase activities using paraoxon, diazoxon, and phenylacetate as substrates. Results were then compared to ChE activities measured after 10 s of contact with an environmentally realistic amount of OP, containing 1% oxon. Both ChE activities were significantly inhibited, with capillary values being significantly more inhibited than their venous counterparts. However, no protective effect could be associated between the degree of A-esterase activity and the subsequent level of ChE inhibition observed in an individual's blood. These results suggest that (1) if there is any uncertainty about OP skin contamination, venous blood would be a more appropriate specimen to employ when using field ChE biomonitoring kits--it is collected in larger volumes and has essentially no direct contact to dermal surfaces; and (2) A-esterase activity demonstrates no protective effect against ChE inhibition upon a blood droplet's brief contact with an OP residue containing traces of oxon.