Mirror Images
Chiral Chemistry: the ultimate in pollutant speciation
(revised October 2007)
What's in a molecule? We ordinarily think that the diagram of a molecule's structure shows everything about the way the atoms connect to make a specific chemical with defined properties. However, with some molecules, a careful look at their three dimensional structure offers a surprise - there are two ways to connect the atoms. These are called chiral molecules, or chiral chemicals, from the Greek cheir (hand), because, like hands, the two forms of the molecule are non-superimposible mirror images of each other. These two species are called enantiomers.
So what?
Enantiomers have identical physical and chemical properties except when they interact with enzymes or with other chiral molecules; then they usually react differently, or selectively. This enantioselectivity often results in different rates of microbial transformation and differences in activity and toxicity of the two enantiomers. Up to 30% of pesticides are chiral molecules, as are some PCBs and many other pollutants. However, almost all chiral pesticides are manufactured and applied as mixtures of equal amounts of the two enantiomers (racemic mixtures). To make more accurate risk assessments for chiral pesticides, it is necessary to understand the relative persistence and effects of their enantiomers, which should be treated as separate compounds.
The agrochemical industry and government regulators are beginning to take enantioselectivity into account. For example, the (R)-(+)-enantiomer of the herbicide dichlorprop (as well as the (R)-(+)-enantiomers of all the phenoxypropionic acid herbicides) is the active enantiomer, killing the weeds, while the (S)-(-)-enantiomer is inactive (see the dichlorprop structures above); so, to reduce the amount of herbicide used and avoid the possibility of the unnecessary enantiomer causing some adverse impact, several European countries have decreed that only the (R)-enantiomers will be used. Metolachlor, the second most used herbicide in the US , is composed of four enantiomers - two R and two S forms. Because the S-enantiomers are about 10 times more herbicidally active than the R-enantiomers, an 85% S-enriched product is now available for use (Syngenta Crop Protection, Inc.). In fact, in 2001, when US farmers used about equal amounts of the older racemic formulation of metolachlor and the new S-enriched product, sole use of the S-enriched product would have avoided pollution of our environment with about 5000 tons of the unnecessary enantiomer.
The objective of our research is to determine the environmental occurrences, fate and effects of the enantiomers of selected chiral pesticides and other chiral organic pollutants. In the case of pesticides, such data can encourage the production and use of safer, greener products.
Our research approach
Separation: develop analytical methods (GC, HPLC or CE) to separate enantiomers for occurrence, fate, and effects studies.
Occurrence: analyze water, soil, sediment, biota and human exposure samples expected to contain chiral pollutants to determine occurrences and ratios of the enantiomers.
Transformation: conduct laboratory experiments in selected environmental matrices to measure enantioselectivity and rates of enantiomer transformation for pesticides, PCBs and other chiral pollutants.
Exposure: exposures of chiral pesticides and their enantiomers to organisms are done in cooperation with other EPA laboratories (e.g., NHEERL), with extramural collaborators, and by ERD's inhouse Computational Toxicology personnel. Resulting exposure data, which help inform exposure assessment, are derived from extamurally-conducted bioaccumulation experiments as well as from in-house applications of state of the art metabolism studies and metabolomic techniques.
Some results
- Methods have been developed for separation/analysis of enantiomers by GC, CE and HPLC (1-3), including in environmental samples (4).
- The (R)-(-)-enantiomer of o,p'-DDT (5) and the (-)-enantiomer of o,p'-dicofol (6) were shown by endocrine disrupter screening tests to have more estrogenic activity than their opposite enantiomers.
- o,p'-DDD, a chiral metabolite remaining in fish tissue after exposure to DDT, remains primarily as the (S)-enantiomer (7).
- The enantioselectivity occurring during microbial transformation of chiral pesticides in soils may be substantially altered by environmental changes imposed on the soils, thus changing the relative persistence of the enantiomers (8,9).
- Fipronil, a broad spectrum insecticide used for crop and veterinary use, is transformed enantioselectively in anoxic sediments, but the direction of selectivity depends upon sediment properties (9).
- Bromochloroacetic acid, formed by chlorination of drinking waters containing naturally occurring bromide, degraded enantioselectively in all six natural waters and a municipal wastewater effluent into which it was spiked.
- Several chiral PCB congeners occur enantioselectively in lake and river sediments (10,11), indicating that biotransformation has occurred; these congeners also occur selectively in associated biota (12).
- The enantiomers of PCB 84 are metabolized selectively in several rat tissues (13).
- Bioaccumulation of fipronil (14) and myclobutanil, a triazole fungicide (15), by rainbow trout is enantioselective, but most of the chiral organochlorine pesticides and triazoles tested bioaccumulated non-selectively.
- NMR metabolomic analysis of liver tissue of rainbow trout exposed to the separate enantiomers of the conazole fungicide triadimefon show different endogenous metabolite profiles.
- Production and use of target-active single- or enriched-enantiomer pesticides provide green chemistry opportunities (16,17).
Useful publications
1. Garrison, A.W. "Analysis of Chiral Pesticides and Polychlorinated Biphenyl Congeners in Environmental Samples" in Encyclopedia of Analytical Chemistry, R.A. Meyers, ed., pp. 6147-6158, John Wiley & Sons, 2000 and Garrison, A.W. "Issues on the Enantioselectivity of Chiral Agrochemicals" in Chimica Oggi/Chemistry Today, pp. 28-32, October 2002 (General references to chiral chemistry in the environment.)
2. Wong,C.S. and A.W.Garrison. 2000. Enantiomer Separation of Polychlorinated Biphenyl Atropisomers and Polychlorinated Biphenyl Retention Behavior on Modified Cyclodextrin Capillary Gas Chromatography Columns. J.Chromatogr. 866:213-220.
3. Ellington,J.J., Evans,J.J., Prickett,K.B. and Champion,W.L.Jr. 2001. High-Performance Liquid Chromatographic Separation of the Enantiomers of Organophosphorus Pesticides on Polysaccharide Chiral Stationary Phases. J.Chromatogr. 928:145-154.
4. Jarman, J.L., Jones, W.J., Howell, L.A. and Garrison, A.W. 2005. Applications of Capillary Electrophoresis to Study the Enantioselective Transformation of Five Chiral Pesticides in Aerobic Soil Slurries. J.Agric.Food Chem. 53:6175-6182.
5. Wiese, T. Presentation at the Society of Environmental Toxicology and Chemistry, 22nd Annual Meeting, Baltimore, MD , Nov.2001. T. Wiese, S. Nehls and A.W. Garrison, "Enantiomer Selective Estrogen and Antiandrogen Activity of Chiral Pesticides".
6. Hoekstra,P.F., B.K.Burnison, A.W.Garrison, T.Neheli and D.C.G.Muir. 2006. Estrogenic Activity of Dicofol with the Human Estrogen Receptor: Isomer- and Enantiomer-Specific Implications. Chemosphere, 64:174-177.
7. Garrison,A.W., V.A.Nzengung, J.K.Avants, J.J.Ellington and N.L.Wolfe. 1997. Determining the Environmental Enantioselectivity of o,p'-DDT and o,p'-DDD. Proceedings of Dioxin '97, the 17th International Symposium on Chlorinated Dioxins and Related Compounds. Vol.31:256-261
8. Lewis,D.L., Garrison,A.W., Wommack,K.E., Whittemore,A., Steudler,P., Melillo,J.1999 "Influence of environmental changes on degradation of chiral pollutants in soils" Nature, 1999, 401, 898-901.
9. Jones,W.J., Mazur,C.S., Kenneke,J.K. and Garrison,A.W. 2007. "Enantioselective Microbial Transformation of the Insecticide Fipronil in Anoxic Sediments". Accepted for publication Oct. 2007 by Environ.Sc.Technol.
10. Wong,C.S., A.W.Garrison and W.T.Foreman. 2001. Enantiomeric Composition of Chiral Polychlorinated Biphenyl Atropisomers in Aquatic Bed Sediment. Environ.Sci.Technol. 35:33-39.
11. Pakdeesusuk,U., W.J.Jones, C.M.Lee, A.W.Garrison, W.L.O'Niell, D.L.Freedman, J.T.Coates and C.S.Wong. 2003. Changes in Enantiomeric Fractions during Microbial Reductive Dechlorination of PCB132, PCB149 and Aroclor 1254 in Lake Hartwell Sediment Microcosms. Environ.Sci.Technol.37:1100-1107.
12. Wong,C.S., A.W.Garrison, P.D.Smith and W.T.Foreman.2001. Enantiomeric Composition of Chiral Polychlorinated Biphenyl Atropisomers in Aquatic and Riparian Biota. Environ.Sci.Technol. 35:2448-2454.
13. Lehmler,H.-J., Price,D.J., Garrison,A.W., Birge,W.J. and Robertson,L.W. 2003 "Distribution of PCB 84 Enantiomers in C57BL/6 Mice" Fresenius Environmental Bulletin, 12:254-260.
14. Konwick,B.J., Garrison,A.W., Black,M.C., Avants,J.K. and Fisk,A.T. 2006. "Bioaccumulation, Biotransformation and Metabolite Formation of Fipronil and Chiral Legacy Pesticides in Rainbow Trout", Environ.Sci.Technol. 40, 2930-2936.
15. Konwick,B.J., Garrison,A.W., Avants,J.K. and Fisk,A.T. 2006. "Bioaccumulation and Biotransformation of Chiral Triazole Fungicides in Rainbow Trout (Oncorhynchus mykiss)". Aquatic Toxicology. 80, 372-381.
16. Garrison, A.W. 2004 "On the Issue of Enantioselectivity of Chiral Pesticides: A Green Chemistry Opportunity" Green Chemistry, 6:G77-G78.
17. Garrison,A.W. 2006. "Probing the Enantioselectivity of Chiral Pesticides". Environ.Sci.Technol. 40, 16-23.
Current research projects/interests
- Separation of enantiomers of selected conazole fungicides and other chiral pesticides for toxicity testing and metabolism studies
- Metabolic transformation of chiral pesticides to observe enantioselectivity
- Occurrence and enantiomeric ratios (ERs) of o,p'-DDT and -DDD in fish tissue
- Occurrence and ERs of chiral PBTs and metabolites in alligator livers from Lake Apopka, FL
- Transformation of organophosphorus, conazole and other less persistent chiral pesticides in laboratory microcosms
- Occurrence and identification of chiral pollutants in drinking water
- ERs of PCB 95 in human exposure samples (soil, dust, dermal and floor wipes, etc.) with NERL/HEASD/RTP
- Bioaccumulation of separated fipronil enantiomers by various aquatic organisms
- Sorption of chiral pesticides by clay mineral surfaces and humic substances in water
- Measurement of endogenous metabolite profiles by NMR and CE metabolomics of fish and other organisms exposed to pesticide enantiomers
Expected benefits
Risk assessment - increased accuracy of environmental and human risk assessment will result from consideration of enantioselectivity in exposure, environmental fate, metabolism and effects of chiral pollutants. In modeling the fate and effects of chiral pollutants, the two enantiomers should be considered as two compounds.
• Pollution prevention - use of only the target-active enantiomer (or formulation enriched in that enantiomer) of pesticides will reduce the pollutant load and avoid any adverse effects of the other enantiomer - a green chemistry measure.
Participants/affiliations
ORD/NERL/ERD, Athens: Wayne Garrison*, Jimmy Avants, John Kenneke, Matt Henderson, Chris Mazur, Drew Ekman, Jack Jones
ORD/NERL/HEASD, RTP: Elin Ulrich
Extramural: Charles Wong, Chemistry Dept., Univ. of Alberta, Canada; Aaron Fisk and Brad Konwick, School of Forest Resources, Univ. of Georgia; Marsha Black, Dept. of Environmental Health Sciences, Univ. of Georgia; Cindy Lee, Dept. Environmental Engineering and Science, Clemson Univ.; Renee Falconer, Chemistry Dept., Chatham College; Hans-J. Lehmler, Dept. Occupational and Environmental Health, Univ. of Iowa; James Lee, Chiral Technologies, Inc., Exton, PA; Thomas Wiese, College of Pharmacy, Xavier Univ. of Louisiana.
*For more information contact Wayne Garrison (garrison.wayne@epa.gov)