Final Report: Saliva Bio-monitoring for Organophosphorus Pesticide Exposures in Children

EPA Grant Number: R828606
Title: Saliva Bio-monitoring for Organophosphorus Pesticide Exposures in Children
Investigators: Fenske, Richard , Barr, Dana Boyd , Lu, Chensheng (Alex) , Rodriguez, Teresa
Institution: University of Washington - Seattle , Centers for Disease Control and Prevention , Emory University , Universidad Nacional Autónoma de Nicaragua–León
EPA Project Officer: Hahn, Intaek
Project Period: September 1, 2000 through August 31, 2003 (Extended to August 31, 2005)
Project Amount: $742,597
RFA: Biomarkers for the Assessment of Exposure and Toxicity in Children (2000) RFA Text |  Recipients Lists
Research Category: Children's Health , Health Effects , Human Health , Health


The overall objective of this research project was to evaluate the feasibility of quantifying children’s exposure to organophosphorus (OP) pesticides through saliva biomonitoring. This work has been part of an ongoing effort to improve exposure assessment methods for children. Two OP pesticides - chlorpyrifos and diazinon - were proposed for this study. A third pesticide, permethrin, was later included in animal studies. These compounds are used widely in both agricultural and residential environments, and the resulting aggregate exposures for children are complex and therefore amenable to biological monitoring.

The specific objectives of this research project were to: (1) determine the pharmacokinetics of these pesticides in saliva and plasma in animals following dermal and oral dosing at variable exposure levels and salivary flow rates; (2) characterize the relationship of pesticide concentrations in saliva and plasma in animals, and evaluate the sensitivity and specificity of salivary excretion for these three compounds at low doses; (3) modify and validate Enzyme-Linked Immunosorbent Assay (ELISA) methods for analyzing chlorpyrifos, diazinon, and permethrin in biological samples; (4) measure children’s exposures to chlorpyrifos and diazinon in saliva and urine following agricultural use of these compounds by their parents; (5) measure adult exposures to chlorpyrifos and diazinon in saliva, plasma, and urine following agricultural use of these compounds; and (6) evaluate pharmacokinetic parameters for these pesticides in children and adults using these measurements.

Summary/Accomplishments (Outputs/Outcomes):

Animal Studies

We conducted a series of controlled dosing studies in animals to determine the pharmacokinetics of these compounds. The study of diazinon has been published (Lu, et al., 2003). Male Spraque-Dawley rats were dosed with 1 or 10 mg/kg diazinon by bolus intravenous injection. Time-matched saliva and arterial blood samples were collected from 10 to 250 minutes post administration. Diazinon was distributed and eliminated rapidly in rats following intravenous bolus injection, according to a two-compartmental pharmacokinetic analysis. Salivary concentrations of diazinon showed a strong correlation with plasma concentrations of diazinon. The saliva/plasma (S/P) concentration ratio of diazinon was not affected by administered dose, sampling time, or salivary flow rate, suggesting that salivary excretion of diazinon in rats is fairly constant. Diazinon concentrations in saliva were consistently lower than those in arterial plasma. The mean S/P concentration ratios of diazinon were 0.16 and 0.13 for 1 and 10 mg/kg intravenous bolus doses, respectively. It is most likely that the incomplete transfer of diazinon from plasma to saliva resulted from protein binding of diazinon in plasma. If the protein-unbound fraction of diazinon in plasma is used to calculate the S/P ratio, the S/P concentration ratio of diazinon is close to unity. The results from this study support the conclusion that diazinon salivary concentrations can not only be used to predict the plasma levels of diazinon in rats, but also can reflect the unbound fraction of diazinon in plasma.

The studies of chlorpyrifos and permethrin were conducted with a similar protocol. Data from animals dosed with chlorpyrifos suggest that chlorpyrifos is metabolized rather rapidly (in minutes) in blood and, therefore, is not able to be measured in saliva. Initially, we were skeptical about the results and repeated the study many times. We also consulted with other researchers who are engaging in saliva research and were able to confirm the validity of our animal data. The saliva samples that we collected from Nicaragua farm workers reaffirmed this finding. We plan to publish this set of results.

Permethrin was selected as one of the chemicals to be measured in saliva, mainly because an ELISA assay was developed recently for this compound. We have been collaborating with Dr. Bruce Hammock at the University of California, Davis, in using ELISA for quantifying pesticide concentrations in various specimen samples. ELISA assays for permethrin and other selected pyrethroid insecticides were only available for use in 2002. Unfortunately, the ELISA assay for permethrin, which was originally developed for analyzing surface and groundwater samples, was not optimized for use in specimen samples. We investigated several approaches, including the use of solid phase extraction, to mitigate this problem, but the results have not been satisfactory. Therefore, we plan to continue these investigations, collecting saliva and plasma samples from rats with several different permethrin doses, and collaborating with Dr. Hammock to improve the permethrin ELISA assay for specimen samples.

Nicaragua Exposure Assessment Study

Our field study in Nicaragua was conducted as a partnership with Dr. Teresa Rodriguez and colleagues at the Programa de Salud Ocupacional y Ambiental, Universidad Nacional Autónoma de Nicaragua, León; and with Dr. Dana Barr and colleagues at the Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, USA. Dr. Rodriguez led the field investigations, and Dr. Barr supervised the analysis of biologic samples. This work is described in a manuscript that has been submitted for publication (Rodriguez, et al.)

Exposures were assessed for 7 small-scale farmers using chlorpyrifos on corn and 10 banana plantation employees applying diazinon, and for 1 child of each worker. Metabolites 3,5,6-trichloropyridinol (TCPY) and 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMPY) were measured in urine before and after applications. TCPY concentrations peaked at 27 and 8.5 hours post-application for applicators and children, respectively (geometric means, 26 and 3.0 µg/L). Proximity to spraying and spray mixture preparation in the home were important exposure factors. IMPY concentrations differed across workers at two plantations (1.3 and 168 µg/L). Children of these workers had little or no diazinon exposure. Plantation workers and their children also were exposed to chlorpyrifos, most likely through contact with chlorpyrifos-impregnated bags used in banana production. Several recommendations are offered: (1) monitor children’s activities during applications; (2) do not store or prepare pesticides in the home; (3) institute good occupational hygiene practices for applicators at banana plantations; and (4) dispose of plastic insecticide bags properly at the worksite.

We also measured diazinon and chlorpyrifos in the plasma and saliva of the applicators, and in the saliva of the children. As stated previously in this report, we were not, however, able to measure chlorpyrifos in saliva because of the rapid metabolism and clearance of chlorpyrifos.

Diazinon concentration-time profiles in saliva varied among applicators at the two plantations, reflecting the marked difference in exposure for these two groups documented with the urinary metabolite data. Salivary concentrations of diazinon measured in plantation 1 workers continued to increase 2 days after self-reported diazinon application, suggesting an on-going exposure among these workers. Salivary diazinon concentrations, however, measured in plantation 2 workers peaked 3 hours after the first application, and then rapidly decreased to non-detectable levels 36 hours after the first application. Diazinon concentrations in saliva were significantly correlated with the time-matched plasma samples collected from the same workers, which is in agreement with the previously published data from animal models. Diazinon exposure among the children of plantation workers was not evident from saliva sampling; the majority of saliva samples did not have detectable diazinon. This finding was consistent with the urinary metabolite results for these children. Severe dehydration was observed in several workers and children, resulting in the loss of some saliva samples and, therefore, an incomplete data set. Regardless of this limitation, the study demonstrated that saliva can be used to assess exposures to diazinon in pesticide applicators and children.


The feasibility of measuring pesticide exposure through saliva sampling was studied in animal models for three chemicals: diazinon, chlorpyrifos, and permethrin. A good correspondence was found for diazinon concentrations in blood and saliva samples, demonstrating that diazinon concentrations in saliva are a reliable indicator of internal dose. Chlorpyrifos was not detected in saliva, probably because of its rapid metabolism. Detection of permethrin in saliva was based on a new analytical method, and results to date have been inconsistent. Additional work is planned to improve this assay. A study of pesticide applicators and their children in Nicaragua provided an opportunity to evaluate saliva sampling in an exposed population. Analysis of urine samples indicated exposure to chlorpyrifos for both applicators and children, but saliva samples did not. This finding was consistent with the animal studies. Exposure to diazinon occurred among applicators, but not among their children. A good correspondence was observed between diazinon concentrations in the workers’ blood and saliva, which is consistent with findings from the animal studies. Diazinon concentrations in saliva also corresponded with excretion of the primary urinary metabolite of diazinon in these applicators. Saliva sampling is an attractive biologic monitoring method because it is simple, non-invasive, and measures pesticides rather than pesticide metabolites. This study demonstrates that saliva sampling is suitable for some compounds, but not for others, and that such sampling is feasible in adults and children.

Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other project views: All 8 publications 3 publications in selected types All 3 journal articles
Type Citation Project Document Sources
Journal Article Lu C, Irish R, Fenske R. Biological monitoring of diazinon exposure using saliva in an animal model. Journal of Toxicology and Environmental Health-Part A 2003;66(24):2315-2325. R828606 (2002)
R828606 (2003)
R828606 (Final)
  • Abstract from PubMed
  • Journal Article Lu C, Rodriguez T, Funez A, Irish RS, Fenske RA. The assessment of occupational exposure to diazinon in Nicaraguan plantation workers using saliva biomonitoring. Annals of the New York Academy of Sciences 2006;1076:355-365. R828606 (Final)
  • Abstract from PubMed
  • Journal Article Rodriguez T, Younglove L, Lu C, Funez A, Weppner S, Barr DB, Fenske RA. Biological monitoring of pesticide exposures among applicators and their children in Nicaragua. International Journal of Occupational and Environmental Health 2006;12(4):312-320. R828606 (Final)
  • Abstract from PubMed
  • Other: IJOEH PDF
  • Supplemental Keywords:

    biomarker, biomonitoring, organophosphorus, OP, pesticides, chlorpyrifos, diazinon, Nicaragua, permethrin, saliva, plasma, children, pharmacokinetics,, RFA, Health, Scientific Discipline, Toxics, Health Risk Assessment, Environmental Chemistry, pesticides, Susceptibility/Sensitive Population/Genetic Susceptibility, Biochemistry, Environmental Monitoring, Children's Health, genetic susceptability, pesticide exposure, sensitive populations, infants, dermal contact, chlorpyrifos, exposure, survey, air pollution, children, children's vulnerablity, insecticides, toxicity, pesticide residues, exposure pathways, saliva, biological markers, dietary exposure, exposure assessment, organophosphate pesticides, Diazinon

    Relevant Websites: Exit
    Synthesis Report of Research from EPA’s Science to Achieve Results (STAR) Grant Program: Feasibility of Estimating Pesticide Exposure and Dose in Children Using Biological Measurements (PDF) (42 pp, 3.87 MB)

    Progress and Final Reports:

    Original Abstract
  • 2001 Progress Report
  • 2002 Progress Report
  • 2003 Progress Report
  • 2004