Grantee Research Project Results

Final Report: Measurements and Models of Longitudinal Dietary Intake of Pyrethroid and Organophosphate Insecticides by Children

EPA Grant Number: R829396
Title: Measurements and Models of Longitudinal Dietary Intake of Pyrethroid and Organophosphate Insecticides by Children
Investigators: Ryan, P. Barry , Kerr, William L. , Hassan, Sayed
Institution: Emory University , University of Georgia
EPA Project Officer: Aja, Hayley
Project Period: February 1, 2002 through March 31, 2010
Project Amount: $659,764
RFA: Children's Vulnerability to Toxic Substances in the Environment (2001) RFA Text | Recipients Lists
Research Category: Human Health , Children's Health

Objective:

The principal objectives of this investigation are to measure longitudinal dietary exposure of selected insecticides in children, to evaluate the accuracy of children’s dietary exposure models, and to reduce model uncertainty. Our focus is on the validation of urinary biomarkers in children. By measuring environmental media- soil, house dust, food- and modeling intake, we will make a direct comparison to urinary metabolite concentrations.

Our work has focused on three different areas. These may be categorized as Laboratory Methods Development, the Pilot Study, and the Children’s Study. In addition, we accomplished several Other Activities.

Summary/Accomplishments (Outputs/Outcomes):

Laboratory Methods Development
1. Summary
  
  As originally outlined in the proposal, the first year of this investigation focused on the development of laboratory methods for the analysis of pesticides in various environmental and food media. The principal aim was to develop a single analytical method that afforded analysis of both organophosphate (OP) and pyrethroid pesticides using a single sampling and analysis scheme. Delays associated with the transfer of the project from University of Georgia to Emory University resulted in this work being completed in early in 2006. Analysis of Pilot Study (See below) samples began during the summer of 2006. Analysis of Children’s Study samples began in 2008 and was completed prior to the end of the project.
2. Environmental Samples
  
  In our investigation, we have made the artificial distinction that environmental samples include only soil and house dust, while food samples, because of differing analytical schemes, were viewed separately. The air medium was eliminated from consideration due to organophosphorus (OP) and pyrethroid insecticide concentrations below the detection limit for all methods and below levels that add significantly to total exposure burden.
  1. Soil
    
    Due to the organic nature of the OP and pyrethroid insecticides under investigation, it is commonly believed that soil may act as a reservoir for these compounds. Contact with soil and subsequent dermal penetration or inadvertent ingestion leads to exposure and potential health outcomes. Hence developing methods for this important medium was of central interest to our investigation. Scientific posters describing the method were also presented at the 2006 annual meetings of the International Society of Exposure Analysis with manuscript (see Papers Published during Reporting Period) reporting final results from our soil method development has been published.
  2. House Dust
    
    Heterogeneity in the house dust matrix offered new challenges for our analytical scheme. While modifications to the soil method were originally applied to dust, we were not satisfied with the results. Sample cleanup was inefficient and resulted in substantially fouling of columns in gas chromatography. We tracked this down to inefficiencies in the clean-up portion of the analysis. We abandoned this method and implemented a slight modification of our general food extraction and cleanup process during the fall of 2006.
3. Food Samples
  Food is a more complex and diverse matrix for pesticide extraction and analysis than soil or house dust due to the presence of lipids, proteins, and other large organic molecules. Simple extraction in organic solvents produces a very “dirty” sample- one with excess organic matter that interferes with later chromatography. Development of effective clean-up methods is critical. The current literature contains a number of extraction and clean-up procedures for specific foods (e.g., spinach, almonds, wine, others) developed by growers and manufacturers of individual commodities. We have developed a single method that, with only minor modifications, is effective on multiple food groups, with adequate sensitivity to detect OP and pyrethroid residues in the low parts per billion (ppb) range in composited diet samples.
  
  During method development, we composited foods into categories with similar properties with respect to lipid fraction, water content, and the probability of detecting OP and pyrethroid residues. The categories include: Non-Dairy Beverages, Dairy, Above-Ground Vegetables, Below-Ground Vegetables, Fruits and Fruit Juices, Grains, Meat-Fish-Eggs, Fats and Oils, and Beans-Nuts-Legumes-Miscellaneous.
  
  A manuscript was prepared detailing methods for dietary pesticide determination in food. It has since been published in the Journal of Agricultural and Food Chemistry.

Pilot Study
1. Summary of Work
  
  In an effort to pilot our protocols for the Children’s Study, we undertook a study of adults working in our research facility. This study involved 11 individuals who kept four-day duplicate diets, which were separated in nine food categories in accordance with our analysis protocol. Detailed dietary logs were kept as well and each food item weighed. The adults were asked to fill out an activity questionnaire indicating any activities likely to result in exposure to pesticides. Finally, each adult was asked to supply first morning and last evening void urine samples, plus a last evening void before the duplicate diet, and a first morning void after the duplicate diet (a total of 10 samples) for pesticide metabolite analysis. Two full cycles of monitoring were completed. A manuscript detailing the relationship between urinary dialkyl phosphate concentration and environmental concentrations of parent pesticides is in preparation but awaits further analysis of biomarker samples; we would prefer to include measurements of pyrethroid metabolites in this paper as well. A paper on the results of the food analyses was accepted to Environmental Science & Technology. A paper of the results of the soil sampling accepted to Archives of Environmental Contamination and Toxicology.
2. Protocol
  
  The participants in the pilot study were all U.S. adults, ranging in age from 23 to 53, with 7 females and 4 males. The adult pilot protocol involved a 4-day monitoring period. During this 4-day period, participants were asked to keep a weighed duplicate sample of everything eaten. Further, they were asked to composite foods into the nine categories described above (i.e., Non Dairy Beverages, Dairy, Above Ground Vegetables, Below Ground Vegetables, Fruits and Fruit Juices, Grains, Meat/Fish/Eggs, Fats and Oils, Beans/Nuts/Legumes/Miscellaneous). In addition, a food diary was kept in which the food items consumed were described and the weights (grams) of each recorded. Participants were asked to provide first-morning and last-evening void urine samples (to be analyzed for pesticide metabolites) on each of the four duplicate diet days. They were asked to supply an additional last-evening void on the day before sampling started, and a first-morning void on the day after the sampling ended. Finally, composite soil samples and composite house dust samples were collected in the residences of the participants on the final day of sampling. All of the environmental and food samples were analyzed for parent pesticide compounds.
  
  This protocol was quite burdensome and likely could not be implemented in a large-scale investigation with ordinary (i.e., non-researcher) subjects. Yet the data obtained from this pilot should supply useful information regarding the relationship between environmental exposures, dietary exposures, and metabolite concentrations.
3. Data Collection
  
  Two cycles of data collection were completed. These occurred in fall 2005 and winter 2006. In April 2006, we received creatinine concentrations for each urine sample, while in August 2006 we received data on OP dialkylphosphate (DAP) metabolite concentrations. Our paper describing the DAP data (Dietary organophosphorus pesticide intake and urinary dialkylphosphate levels in adult volunteers. Anne M. Riederer, Ronald L. Hunter, Jr., Dana B. Barr, Guy Weekasekera, and P.B. Ryan) was presented as a podium presentation at the 2008 joint conference of the International Society of Exposure Analysis and International Society for Environmental Epidemiology in November 2008.
  
  We have prepared a manuscript describing the environmental sampling from this investigation. A manuscript entitled: Current and historically used pesticides in residential soil from 11 homes in Atlanta, Georgia, USA. Anne M. Riederer, Kimberly D. Smith, Dana B. Barr, Steven W. Hayden, Ronald E. Hunter Jr., and P. Barry Ryan appeared in Archives of Environmental Contamination and Toxicology in late 2009.

Children’s Study
1. Summary
  
  Beginning in September of 2008 and continuing through mid-February 2009, we recruited 31 children in the age range of 3-6 years, and carried out detailed monitoring. We monitored their environment including yard soil, house dust, and food using a detailed protocol designed to assess the impact of environmental exposure and dietary exposure for organophosphorus and pyrethroid pesticides. Dietary exposure was examined in detail with multiple-day, duplicate-diet samples being taken and separated in eight different food categories. Separate analysis was done on each of the categorized food samples. In addition, multiple urine samples were collected from the child that were measured for volume then shipped to the Centers for Disease Control and Prevention for analysis of metabolites, both specific and general, for these pesticides. The simultaneous determination of parent pesticide concentrations in environmental and dietary samples with urinary biomarkers for the parent compounds affords validation of the exposure-biomarker link. Unfortunately, due to backlogs at CDC, analysis of the urine samples was continuously put off. We requested and received a no-cost extension of our study in hopes of receiving these results while still under the umbrella of this study. This did not come to pass and, just prior to the end of the no-cost extension, we requested that samples be returned to us for analysis in our own laboratory. These samples were returned and await analysis.
2. Design
  
  The original design for our children’s study (“Children’s Exposure to Environmental Pesticides (CEEP)”) was developed during September 2005 – January 2006 and reflected the conclusions drawn from the pilot investgation. Briefly, the study was designed to evaluate the validity of using urinary OP and pyrethroid concentrations to estimate intake through diet and environmental pathways in pre-school age children. Our target age group was 3-5 years old for three reasons:
  1. this age range falls within the critical birth to age 6 window of neurodevelopment;
  2. children this age are generally toilet-trained, facilitating urine sample collection, and;
  3. children this age generally spend most of their time in the home environment, under the close supervision of parents or other adults (“primary caregiver”) who would have detailed knowledge of the their dietary habits.
  We modified our original design for the Children’s Study in light of IRB-related delays and related funding shortfalls. We only completed what was originally referred to as Phase II of our study. We targeted thirty children monitoring. Recruitment focused on the Atlanta/DeKalb County area of the State of Georgia. Each child was monitored twice times during the course of the investigation with urine samples (for metabolite analysis,) duplicate food samples, soil, and house dust samples (for parent pesticide and metabolite analysis) taken at each monitoring cycle. Questionnaires and other materials necessary for this investigation were developed.
3. Protocol
  
  The participants in the pilot study were all children, ranging in age from 3 to 5. The Children’s protocol involved a 4-day monitoring period. During this 4-day period, participants were asked to keep a duplicate sample of everything eaten for three of the days. Further, they were asked to composite foods into the nine categories described above (i.e., Non Dairy Beverages, Dairy, Above Ground Vegetables, Below Ground Vegetables, Fruits and Fruit Juices, Grains, Meat/Fish/Eggs, Fats and Oils, Beans/Nuts/Legumes/Miscellaneous) and were given instruction on how to do so. In addition, a food diary was kept in which the food items consumed were. Participants were asked to provide first-morning and last-evening void urine samples (to be analyzed for pesticide metabolites) on each of the three duplicate diet days. They were asked to supply an additional last-evening void on the day before sampling started, and a first-morning void on the day after the sampling ended. Finally, composite soil samples and composite house dust samples were collected in the residences of the participants on the final day of sampling.
4. Data Collection
  
  The monitoring program began in April 2008. We successfully recruited 36 volunteers of whom 31 completed both cycles of data collection. All of the environmental and food have been analyzed as have the questionnaire data. Manuscripts are in the internal review stage

Other Activities
1. Summary
  
  Two other activities were pursued under the funding supplied by this grant. Each is associated with the scope of the work, namely understanding the relationship between exposures to parent pesticides and the presence of measurable metabolites in urine.
2. Analysis of OP and Pyrethroid Metabolites in Environmental Media
  
  The urinary metabolites of organophosphate and pyrethroid pesticides are derived through simple hydrolysis or oxidation processes associated with the ester linkages in these compounds. Such processes can also occur in environmental media without benefit of cytochrome P450 enzyme schemes in the body. Early work in our research group indicated the potential problems associated with this when we found that environmental concentrations of chlorpyrifos could only account for 5-20% of the TCPy metabolite excreted by our subjects in the NHEXAS study (See: MacIntosh DL, Needham LL, Hammerstrom KA, and Ryan PB. A longitudinal investigation of selected pesticide metabolites in urine. J Exp Anal Environ Epidem 1999;9(5):494-501.) Several other research groups have noted similar difficulties in trying to reconcile mass balance on metabolites in urine. However, very little work has been done in trying to understanding alternative exposure pathways to these metabolites.
  
  In an effort to address these issues, we have undertaken a method development study designed to improve analytical techniques for pesticide metabolites in environmental media. Our early work focused on soil but more recent work has focused on the dietary pathway. Food represents a more complex matrix and the polar nature of the metabolites and degradation products has required us to re-work many of our analytical methods for these compounds. Early work by doctoral student Samantha Adkins Radford has shown promise in this area.
3. Analyses of External Data Sets
  
  In an effort to understand dietary and other factors that may influence exposure to pesticides as well as impact upon metabolite measurements, we began investigations of existing data sets in order to glean information on the association between dietary intake and metabolite levels in urine that might help us refine future sampling protocols. Two external data sets in particular, NHANES and NHEXAS-Maryland, provide detailed diet and pesticide concentration data. The 1999-2002 NHANES survey years included a “pesticide subsample” in which urine samples from a representative sample of NHANES subjects (n ~ 3,000) were analyzed for metabolites of OP and pyrethroid insecticides. NHANES also collected 24-hour dietary recall data as well as information on activity patterns and other factors potentially related to pesticide exposure. We combined the NHANES dietary data with urinary data on a commonly-detected pyrethroid metabolite (3-PBA) in an effort to determine which foods, if any, might be correlated with increased metabolite concentrations. Our work in this area lead to a publication at the very beginning of the reporting period. The paper is entitled: Diet and non-diet predictors of urinary 3-phenoxybenzoic acid in NHANES 1999-2002 by Anne M. Riederer, Scott M. Bartell, Dana B. Barr, and P. Barry Ryan. It appeared in Environmental Health Perspectives 2008;116:1015–1022 and is available online.
  
  We also analyzed data from the EPA-funded NHEXAS-Maryland study (PI: Ryan) to determine whether or not certain foods could be identified as key drivers of dietary malathion and chlorpyrifos intake among subjects in this study. In NHEXAS-MD, duplicate diet samples and personal dietary checklists were collected from a stratified probability sample of 80 subjects (13 – 85 years old), living in greater Baltimore, during six, four-consecutive-day sampling periods over a calendar year. We are used both Tobit regression and Bayesian statistical approaches to evaluate which foods on the diet checklists explain the greatest amount of variability in malathion and chlorpyrifos concentrations in the duplicate diet samples. We are also applying the EPA 2006 Organophosphorus Cumulative Risk Assessment Guidelines by conducting statistical analysis on the combined malathion and chlorpyrifos concentrations in methamidophos equivalents. This research is a collaboration with statisticians from the University of California Irvine (Dr. Scott Bartell) and the University of West Georgia (Dr. Ayona Chatterjee). A manuscript reporting results is currently in preparation. However, there have been inconsistencies in the results produced by the Tobit approaches and the Bayesian approaches that have yet to be reconciled.

Conclusions:

With an eye toward understanding the potential for exposure misclassification through the use of biological markers of exposure and we expanded our work to look at environmental degradation of pesticides. We have continued development of methods designed to improve understanding of the environmental and metabolic degradation of pesticides focusing on foods. Future work will continue our investigations through development of methods for the degradation products themselves so that we may see both the loss of parent pesticide and the expected increase in degradation/metabolite concentrations.

Several manuscripts are under development with many approaching the submission stage. In general, our target journals for these manuscripts include Environmental Health Perspectives for those manuscripts with a combination of environmental and biological data, and Environmental Science and Technology, Journal of Agricultural and Food Chemistry, or Analytical Chemistry for the methods development papers. We expect to submit several manuscripts over the nest 12-18 months as data become available on the urinary pesticide metabolites for both our Pilot and Children’s Studies.

Journal Articles on this Report : 4 Displayed | Download in RIS Format

Publications Views
Other project views:	All 14 publications	4 publications in selected types	All 4 journal articles

Publications
Type	Citation	Project	Document Sources
Journal Article	Hunter Jr. RE, Riederer AM, Ryan PB. Method for the determination of organophosphorus and pyrethroid pesticides in food via gas chromatography with electron-capture detection. Journal of Agricultural and Food Chemistry 2010;58(3):1396-1402.	R829396 (Final)	Abstract from PubMed Abstract: ACS Publications-Abstract Exit
Journal Article	Riederer AM, Bartell SM, Barr DB, Ryan PB. Diet and nondiet predictors of urinary 3-phenoxybenzoic acid in NHANES 1999-2002. Environmental Health Perspectives 2008;116(8):1015-1022.	R829396 (Final)	Full-text from PubMed Abstract from PubMed Associated PubMed link Full-text: EHP
Journal Article	Riederer AM, Hunter Jr. RE, Hayden SW, Ryan PB. Pyrethroid and organophosphorus pesticides in composite diet samples from Atlanta, USA adults. Environmental Science & Technology 2010;44(1):483-490.	R829396 (Final)	Abstract from PubMed Full-text: ACS - Full Text HTML Exit
Journal Article	Riederer AM, Smith KD, Barr DB, Hayden SW, Hunter Jr. RE, Ryan PB. Current and historically used pesticides in residential soil from 11 homes in Atlanta, Georgia, USA. Archives of Environmental Contamination and Toxicology 2010;58(4):908-917.	R829396 (Final)	Abstract from PubMed Abstract: SpringerLink-Abstract Exit

Supplemental Keywords:

exposure assessment, pesticides, pyrethroids, organophosphates, biomarkers, pesticide analysis, children,, RFA, Scientific Discipline, Health, PHYSICAL ASPECTS, Toxics, Air, Health Risk Assessment, air toxics, pesticides, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Physical Processes, Biochemistry, Children's Health, genetic susceptability, pesticide exposure, rural communities, urban air, sensitive populations, monitoring, adolescents, organophosphates, multi-pathway study, exposure, age-related differences, dermal contact, children, longitudinal study, human exposure, gender-related variability, pesticide residues, insecticides, environmental toxicant, neurotixics, dust , biological markers, dietary exposure, human health risk, agricultural community, organophosphate pesticides, exposure assessment

Progress and Final Reports:

Original Abstract

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.