Grantee Research Project Results
1999 Progress Report: Assessing Levels of Organophosphorus Insecticides Which Could Expose Children From Pets Treated with Flea Control Insecticides
EPA Grant Number: R825170Title: Assessing Levels of Organophosphorus Insecticides Which Could Expose Children From Pets Treated with Flea Control Insecticides
Investigators: Chambers, Janice E. , Boone, J. Scott , Tyler, John W.
Institution: Mississippi State University
EPA Project Officer: Aja, Hayley
Project Period: October 1, 1996 through September 30, 1999
Project Period Covered by this Report: October 1, 1998 through September 30, 1999
Project Amount: $597,804
RFA: Exposure of Children to Pesticides (1996) RFA Text | Recipients Lists
Research Category: Pesticides , Children's Health , Human Health , Safer Chemicals
Objective:
The objective of this project is to determine the levels of dislodgable insecticide residues which could be transferred to humans from dogs treated with flea control dips or collars. The project completed 12 replications of the chlorpyrifos dip-no shampooing protocol and 12 replications of the chlorpyrifos dip-shampooing protocol, and initiated the phosmet dip protocol during the first grant year. During the second year, 24 replications of the phosmet dip-no shampooing protocol were completed, and a protocol using a tetrachlorvinphos collar was initiated. During the third year, the tetrachlorvinphos collar protocol was completed with 24 replications, and a chlorpyrifos collar protocol was initiated with 12 replications. Because of supplemental funding from the industries formulating the chlorpyrifos collars, urinary metabolite quantification of the chlorpyrifos hydrolysis product trichloropyridinol (TCP) was initiated in an adult and a child in the household of each dog in the chlorpyrifos collar study.
Progress Summary:
During the third grant year, the project completed the tetrachlorvinphos collar study and initiated the chlorpyrifos collar study using the procedures and protocols largely as developed previously. All recruiting of student workers and dogs, along with the owners' consent, were done the same as during the first year. Because of the opportunity to obtain urinary metabolites from people in the households of the dogs (because of the supplemental funding), we developed an informed consent form for the parent participating in the study and an assent procedure for the child which were approved by the Mississippi State University Institutional Review Board.
Briefly, the following procedures were performed. Dogs (24 for the tetrachlorvinphos study or 12 for the chlorpyrifos study of diverse size, breed and fur length, and of both sexes) were recruited and initially shampooed and sampled for residues and plasma cholinesterase activity. None of the dogs showed any evidence of contamination with the test insecticide or of cholinesterase inhibition. They then had an over- the-counter collar with the test insecticide placed around the neck according to package directions. Dogs were sampled by rubbing with a white cotton glove (pre-extracted with dichloromethane) for a 5 minute period. Three fur samples were taken on each sampling occasion: posterior part of the back (back sample), neck with the collar removed (neck without collar) and neck with the collar in place (neck with collar). Fur samples for tetrachlorvinphos were taken before the collar was applied (0 time), and 4 hours and 3, 7, 14, 28, 56, 84, and 112 days after placement of the collar. Fur samples for chlorpyrifos were taken before the collar was applied (0 time), and 4 hours, 1, 3, 7, 14, 28, 56, 84, 112, 140 and 168 days following placement of the collar. Blood samples were taken into EDTA by venipuncture, and the samples were centrifuged to separate plasma. Blood samples were taken on the same occasions as the fur samples. Dogs were shampooed monthly during the course of the experiment.
Gloves were stored at 4°C in the dark until extraction with the Accelerated Solvent Extractor (ASE) using acetone/hexane (50:50) for tetrachlorvinphos or petroleum ether for chlorpyrifos, and were quantified by GC/ECD, using appropriate dilutions to insure that the areas were within the standard curve. The recovery rate was 95-100%.
Plasma samples were refrigerated overnight and were assayed for cholinesterase activity spectrophotometrically on the day after collection. The remaining plasma was frozen in case any samples needed to be repeated. Both acetylthiocholine (the most appropriate substrate for acetylcholinesterase, AChE) and butyrylthiocholine (the most appropriate substrate for butyrylcholinesterase, BChE) were assayed using the cholinesterase inhibitor eserine and the butyrylcholinesterase inhibitor iso-OMPA to characterize activity. Plasma was diluted as necessary in order to insure that activities stayed within the linear range of the assay.
Urine samples have been collected from an adult and a child (age 4-12 years) from each household participating in the chlorpyrifos collar study on the same occasions as the dog samples were taken. These urine samples are currently being run.
Preliminary Data Results: As was true of the residue samples obtained during the first and second grant years with the chlorpyrifos and phosmet dips, there was a large amount of variability among animals. This variability is felt to be of particular value to EPA if probabilistic methods of exposure assessment are used in the risk characterization process.
The following residue data were obtained in the tetrachlorvinphos collar experiment
given as average residue in ug on the glove for each sampling time, followed
by the range of values:
Time | Neck with collar | Neck without collar | Posterior back |
4 hr | 14340, 2227-30348 | 3530, 956-12927 | 185, 16-406 |
3 days | 23728, 5269-41324 | 8042, 1378-13184 | 261, 14-1016 |
7 days | 24039, 8055-77066 | 8674, 1163-17309 | 177, 11-677 |
14 days | 19309, 6888-70453 | 6062, 1163-17309 | 152, 11-486 |
28 days | 12568, 2663-54970 | 3844, 716-11511 | 144, 19-358 |
56 days | 12426, 661-37351 | 2802, 102-14062 | 80, 13-387 |
84 days | 4955, 177-20853 | 953, 67-3046 | 36, 3-168 |
112 days | 3266, 208-17937 | 549, 3-3481 | 34, 1-167 |
No significant plasma cholinesterase inhibition was noted at any sampling time.
The chlorpyrifos collar experiment is currently in progress, with 12 of the replications run. The data accumulated thus far indicate considerably lower levels of transferrable residues of chlorpyrifos than were reported above for tetrachlorvinphos, and also indicated similar levels of transferrable residues throughout the sampling period, with no obvious peak. The results indicated, similar to tetrachlorvinphos, that highest residues were obtained in neck with collar samples (averaging about 300 µg), next highest in neck without collar samples (averaging about 170 µg) and lowest in posterior back samples (averaging about 5 µg). Over the initial 3 week period of the study, plasma butyrylcholinesterase inhibition increased to about 60% and remained at that level for the duration of the study; plasma acetylcholinesterase inhibition remained at about 15% throughout the study.
Evaluation: Data on residues resulting from the tetrachlorvinphos collar indicate that mg quantity levels of the insecticide can be dislodged by a 5 minute rubbing over the collar shortly after administration of the collar. Peak residues were observed at 3-7 days after collar administration, with residues dissipating over the 140 days studied. Highest residues were observed in the samples obtained by rubbing over the collar itself, and the next highest residues were observed on the fur of the neck which had been in close proximity or in direct contact with the collar. However, measurable residues were dislodged from the back of the dog, indicating that these residues can migrate from the collar to distant parts of the dog. The lack of plasma cholinesterase inhibition suggests that little, if any, internalization of the insecticide occurred.
In contrast, substantially lower levels of chlorpyrifos occurred in the fur samples, did not show an early peak followed by dissipation but instead showed reasonably constant levels throughout the study, and there was substantial plasma butyrylcholinesterase inhibition thoughout the study.
We are not sure of the reasons for the differences in transferrable residue patterns between the two compounds. These may be related more to the formulation materials than to the properties of the insecticides themselves. We are also unsure about the human health significance of these residues at this point. The urinary metaobolite data currently being obtained will be very useful in assessing levels of the collar insecticide which might be internalized by the humans in contact with the treated dog.
Difficulties Encountered and Remedial Actions Taken: No significant difficulties have been encountered in any of the technical procedures. All procedures are straight-forward, and are being conducted similar to the first and second grant years. The recruitment of people for the chlorpyrifos collar study has been more difficult than for the previous studies because of the requirement that there be a child in the household between the ages of 4 and 12 years, and because of the requirement that urine samples be obtained. Nevertheless, we have sampled the initial 12 households, and have most of the second set of households identified.
Future Activities:
The experiment with the chlorpyrifos collar is on-going at present, so this will be completed. A manuscript reporting the chlorpyrifos dip data is undergoing revisions in response to the peer review, and manuscripts reporting the phosmet dip and the tetrachlorvinphos and chlorpyrifos data will be written.
Percentage of Project Completed: It is estimated that about 80% of the project's work has been completed.
Quality Assurance: Analytical chemistry data are being generated according to set protocols. The data must be within the standard curve. If the data are outside the standard curve, the samples are diluted and rerun until they are within the standard curve. Data are being checked and verified by two scientists. The plasma cholinesterase values must also be within the linear portion of the standard curve. If samples are in excess of the linear portion, they are diluted until they are in the appropriate region. In addition, protein values, used for standardization of AChE activities, must be within the standard curve.
Journal Articles:
No journal articles submitted with this report: View all 5 publications for this projectSupplemental Keywords:
RFA, Health, Scientific Discipline, Toxics, Environmental Chemistry, Health Risk Assessment, pesticides, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Children's Health, genetic susceptability, health effects, pesticide exposure, risk assessment, sensitive populations, dermal exposure, adult reference dose, Phosmet, health risks, infants, age-related differences, chlorpyrifos, dermal contact, dogs, measuring childhood exposure, exposure, pets, air pollution, children, assessment of exposure, children's vulnerablity, susceptibility, toxicity, human exposure, insecticides, pesticide residues, exposure pathways, harmful environmental agents, dietary exposure, flea control insecticides, organophosphate pesticidesRelevant Websites:
Progress and Final Reports:
Original AbstractThe 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.