Project Number: 9278314 Project Title: A Method for Simultaneous Analysis of Multiple Pesticides Division: DART Project Officer (PO): Raymond Biagini PO Degree: Ph.D. PO Area of Expertise (scientific discipline): Toxicology Project Start Date: 10/98 Project End Date: 09/04 Total Budget over the Project Period: $737,820 Goal/Sub-Goal: Chronic Effects of Pesticide Exposure/Exposure Assessment Research Challenge/Issue: Exposure to pesticides has been suggested to increase the rates of some cancers. Professional pesticide applicators may be exposed to multiple pesticides simultaneously, or over a relatively short time frame. Complete exposure assessment is an essential step in developing a program to prevent pesticide poisonings. Exposure assessment methods development is one of the NORA research priorities. For pesticide applicators, where skin absorption is a common route of exposure, urinary biological monitoring is an essential exposure assessment tool. Historically, urinary biomonitoring has been done using conventional chromatographic techniques which can be expensive, time-consuming, and labor intensive. Immunoassay (IA) offers a faster, easier, and less expensive alternative. However, IA methods, like most urinary biological monitoring methods, are restricted to one analyte per analysis. Hence, an accurate estimate of total pesticide exposure requires separate analyses for each individual pesticide, a task which is costly and time consuming. Consequently, a new multi-analyte urinary biological monitoring methodology was developed via this project. Successful application of multi-analyte technology should allow for a more complete and less costly biological monitoring program for pesticide applicators. A more complete and accurate characterization of workplace exposures will allow for enhanced intervention measures to prevent exposures. At the time this project was conceived, multiplexed fluorescence covalent microbead immunosorbent assays (FCMIA) were new technology. We were aware of this technology from attending scientific meetings and saw how it could be used in defining multiple exposures in Ag workers. NIOSH HHEs and collaborations with other NIOSH investigators indicated that multiple exposures were being experienced by pesticide applicators. The establishment of new analytical methodology for simultaneous multi chemical biological monitoring using new instrumentation is not trivial. We had one of the first commercial instruments ever sold which forced us to "alpha test" the technology. After about 18 months, the instrument supplier shipped us a second generation instrument. Activities: As this was new technology, we had to first become comfortable with the instrumentation and methodology. There were numerous subtle methodologic considerations which we had to
carefully control. For example, if the microspheres were vacuumed too strongly during separations, they could be re-suspended. We had to optimize the microsphere to antigen ratios and also develop the methodololgy for a competitive FCMIA (a term which we invented). Initially we were able to perform urinary analyses for two pesticides simultaneously (atrazine and metolachlor mercapturate). Upon extension of the technology, we expanded the assay to measure three pesticides simultaneously (glyphosate [sensitivity enhanced)], atrazine and metolachlor mercapturate in water and urine) as well as Bacillus thuringiensis insecticidal proteins present in sweet corn. An average of approximately 1.4 FTEs and approximately $20,000 per/yr were devoted to the project. Oorganizations that were engaged in the program activities or benefited by the program accomplishments: Abraxis, LLC and EnviroLogix were collaborators/partners for some parts of the project. Outputs: Article published, professional (juried publication): 1. Biagini, RE, Sammons, DL, Smith, JP, Snawder, JE, Striley, CAF, MacKenzie, BA.Robertson, SR. Development of a Sensitivity Enhanced Multiplexed Fluorescence Covalent Microbead Immunosorbent Assay (FCMIA) for the Measurement of Glyphosate, Atrazine and Metolachlor Mercapturate in Water and Urine. Analytical and Bioanalytical Chemistry, 379:368-374, 2004. 2. Biagini, RE, Murphy, DM, Sammons, DL,. Smith, JP, Striley, CAF and MacKenzie, BA..Development of Multiplexed Fluorescence Microbead Immunosorbent Assays (FMIAs) for Pesticide Biomonitoring. Bull. Environ. Contam Toxicol. (2002) 68:470–477. 3. D.L. Sammons, R.E. Biagini1, J.P. Smith, B.A. MacKenzie, C.A.F. Striley, S.A. Robertson, J.E. Snawder, B.S. Ferguson, K.A. Larkin. Simultaneous Measurement of Bacillus thuringiensis Cry1Ab and Cry3B Proteins in Corn Extracts. ACS Symposium Series Book: Rational Methods for the Selection and Use of Agrochemicals. In Press, 2006 Abstracts: 4. D.L. Sammons, R. E. Biagini, J. P. Smith, B.A. MacKenzie, C.A.F. Striley, S.A. Robertson, J.E. Snawder, B.S. Ferguson, K. A. Larkin. Simultaneous Measurement of Bacillus thuringiensis Cry1A(b) and Cry3B proteins in corn extracts. Pacifichem 2005 Conference, The International Chemical Congress of Pacific Basin Societies, Honolulu, HA, December 15-20, 2005. 5. J.Smith, D. Sammons, R. Biagini, ,J. Snawder, C. Striley, B. MacKenzie and L. Hollstegge. Multiplexed (tetra-plex) urinalyses of pesticides using
Fluorescence Microbead Covalent ImmunoAssays (FMCIAs). Poster, 2005 Toxicology And Risk Assessment Conference, April 25-28, 2005, Fairborn, OH., April 25-28, 2005, Fairborn, OH. 6. CJ Hines, JA Deddens, CAF. Striley, KK Brown, C Lu, BA MacKenzie, RE Biagini, D. Shoemaker,RA Fenske. Biological Monitoring for Atrazine and 2,4-D Among Custom Applicators I: Results of Exposure Determinant Modeling. Utrecht and X2004 -Exposure Assessment in a Changing Environment, Abstract No. 34-2, 2004, Utrecht, The Netherlands. 7. CJ Hines, JA Deddens, CAF Striley, RE Biagini, DA Shoemaker, KK Brown, B A MacKenzie, RD Hull. Biological Monitoring for Urinary Herbicide Biomarkers among Custom Applicators: Application of Mixed-Effect Models. American Industrial Hygiene Conference & Exposition, 2003 8. JP. Smith, DL. Sammons, RE. Biagini, JE. Snawder, CA.F. Striley, BA. MacKenzie. The Use of Fluorescence Microbead Immunosorbent Assays (FCMIAs) for Pesticide Biomonitoring. 226th ACS National Meeting Book of Abstracts, #AGRO 33, 2003. 9. J.Smith, D. Sammons, R. Biagini, ,J. Snawder, C. Striley, B. MacKenzie and L. Hollstegge,. Multiplexed (tetra-plex) urinalyses of pesticides using Fluorescence Microbead Immunoasays. NIOSH Salt Fork XI Interdivisional Meeting in Mineral Wells, WV November,2002 10. RE Biagini, DM Murphy, CAF Striley, BA MacKenzie, and SK Robertson. The Use of Fluorescence Microbead Immunosorbent Assays (FMIAs) for Pesticide Biomonitoring. 2000 International Chemical Congress of Pacific Basin Societies, Book of Abstracts, ISBN0-8412-3775-1, Abstract No. 263, 2000. 11. C.A.F. Striley, R.E. Biagini, J.E. Snawder, B.A. MacKenzie, C.J. Hines. Clinical biological monitoring using immunobiochemical methods. ACS National Meeting, Book of Abstracts, Part 1:106, 2000. Intermediate Outcomes: There is now greater public awareness of methods for the simultaneous analysis of multiple pesticides due to our publications and abstracts. In addition the public is now aware of the availability and adoption of new technologies for the simultaneous analysis of multiple pesticides. We have been contacted by the University of California, Davis, Laboratory of Pesticide and Biotechnology to collaborate on multiplexing more pesticides. External Factors: Partner collaborations enhanced the success of the program. Future Directions: The population is aging and the number of small family farms is decreasing. With the reduction in traditional tobacco farming a large segment of small farms in the southeast US will be
changing to different crops or leaving farming altogether. Secondly, more and more farm workers are non-native and/or migratory. Communication with these workers is difficult because of the language barrier and tracking them for any amount of time becomes problematic. NIOSH researchers continue to develop new laboratory methods to assess worker exposures to agricultural chemicals. These methods are proceeding in two primary directions - multiplexed methods i.e., measuring multiple analytes simultaneously in a single sample and field portable methods. These approaches will allow rapid determination of exposures in the field and also provide near instantaneous evaluation of control practices.