Bioaccumulation and biotransformation of chiral triazole fungicides in rainbow trout (Oncorhynchus mykiss)
Introduction
Current-use pesticides (CUPs) (e.g., atrazine, fipronil, diazinon, permethrin, propiconazole) can be defined as those modern pesticides that are currently registered for use, generally developed from chemical synthesis, and are typically used in the agricultural or lawn care sector. They are generally less environmentally persistent having shorter half-lives and lower bioaccumulation than the organochlorine pesticides that they have replaced. There is concern over the wide application of CUPs and their possible detrimental effects on aquatic ecosystem health that may arise from spray drift or surface run-off after rainfall events. Of particular importance are readily formed in vivo metabolites of CUPs, which may cause greater harm in aquatic biota than their parent compound (Sinclair and Boxall, 2003). Thus, for accurate risk assessment, there is a need to characterize the persistence and accumulation of CUPs in aquatic biota, including biotransformation rates to metabolites. However, there have been few studies that have addressed this issue for CUPs (Konwick et al., 2006), likely due to their short environmental persistence in biota (i.e., low log Kow values) and the inherent difficulty in identifying the numerous potential biotransformation products. In addition, models based on physical–chemical properties of contaminants to estimate biotransformation rates in fish are limited (Borgå et al., 2004), especially concerning CUPs.
In 1996, it was estimated that approximately 25% of CUPs were chiral (Williams, 1996), with that number to likely have risen since then because of the increasing complexity of modern pesticides with the greater likelihood of chiral centers. Chiral chemicals exist as two non-superimposable mirror images called enantiomers, often designated as (+) and (−) based on their rotation of plane-polarized light. The manufacture of chiral chemicals results in a racemic (±) mixture containing an equal percentage (50%) of each enantiomer, the form in which they are typically released into the environment. Chemicals can also contain more than one chiral center and thus exist in other stereoisomeric forms. For example, a chemical with two chiral centers would exist as two diastereomers; these are not mirror images of each other, but each would consist of two enantiomers. Enantiomers, unlike diastereomers, have identical physical–chemical (i.e., achiral) properties (Garrison, 2006), and only show differences in selectivity when in a chiral environment. Thus, the relative abundance of enantiomers is subject to change after metabolic processes due to numerous enzymes and receptors having symmetry (i.e., chiral) dependence; this property has been used recently as a tracer for biotransformation (Vetter et al., 2001, Wong et al., 2004). For example, non-racemic enantiomer residues (expressed as enantiomer ratios or fractions) have indicated that fish can biotransform several chiral organochlorines (OCs) as well as CUPs (Wong et al., 2002, Konwick et al., 2006). The use of changes in relative proportions of diastereomers has rarely been used as a biotransformation tracer, but has similar potential to enantiomer ratios. However, this feature may be compromised by the fact that diastereomers can undergo abiotic reactions at different rates.
Another method based on a curve-linear relationship developed between log Kow and t1/2 for a series of recalcitrant PCBs in juvenile rainbow trout (Fisk et al., 1998) can also provide a means of assessing biotransformation, specifically rates, in fish (Fisk et al., 2000, Buckman et al., 2006, Konwick et al., 2006). Based on this method, non-recalcitrant chemicals whose t1/2 (determined experimentally using the same protocol) fall below this curve-linear relationship are suggested to be biotransformed, whereas those chemicals that fall on or near this relationship are assumed to not undergo biotransformation. This method has been used to generate biotransformation rates for several polychlorinated alkanes and PCBs, and fipronil in juvenile rainbow trout (Fisk et al., 2000, Buckman et al., 2006, Konwick et al., 2006), with the potential application to other CUPs.
Triazoles as well as the structurally related imidazole fungicides are used as clinical drugs and as agricultural pesticides, including applications for the treatment and protection of cereals, soybeans, and a variety of fruits (Roberts and Hutson, 1999). Their fungicidal effect is a result of the inhibition of cytochrome (CYP) P-450 dependent C14 demethylation of lanosterol, an intermediate in ergosterol biosynthesis (Roberts and Hutson, 1999). Other studies have shown that the inhibition of CYP forms is not limited to sterol biosynthesis (Rodrigues et al., 1988, Ronis et al., 1998). In fish, CYP mediated steroid metabolism (Monod et al., 1993), in addition to xenobiotic metabolism (Levine et al., 1999a, Hegelund et al., 2004), can be altered. In fish exposed to propiconazole, for example, a mixed pattern response in metabolism occurs, whereby CYP1A mRNA levels increase, but EROD activity decreases (Levine et al., 1999a). This alteration in metabolism can lead to potentially higher bioaccumulation and toxicity of contaminants. For example, gizzard shad (Dorosoma cepedianum) pre-exposed to clotrimazole had greater bioaccumulation of benzo[a]pyrene compared to fish exposed to benzo[a]pyrene only (Levine et al., 1997). In addition, fathead minnows (Pimphales promelas) that were pretreated with propiconazole showed enhanced acute toxicity after exposure to the pesticide parathion (Levine and Oris, 1999b). Therefore, it is important to understand the fate and toxicokinetics of these fungicides in fish, in part because of their ability to increase the accumulation and toxicity to other contaminants.
We investigated the bioaccumulation and biotransformation of a series of triazole fungicides by dietary exposure to juvenile rainbow trout (Oncorhynchus mykiss) to address the lack of such information for CUPs. The ability of this fish to biotransform these chemicals was assessed through chiral analysis and the use of the log Kow–log t1/2 relationship developed for quantifying biotransformation of organic contaminants in rainbow trout (Fisk et al., 1998, Fisk et al., 2000). α-HCH was included in this study to expand the log Kow–log t1/2 relationship to lower log Kow chemicals. In addition, we attempted to explore what role the functional groups attached to the chiral center of the triazoles had on biotransformation. To our knowledge, this is the first experiment to investigate the bioaccumulation and enantioselective biotransformation of triazole fungicides in fish.
Section snippets
Chemicals and food preparation
All triazoles were obtained from the EPA Repository (EPA National Pesticide Standard Repository, Ft. Meade, MD). α-HCH was obtained from ChemService (West Chester, PA). The purities of all chemical standards were ≥97%. All solvents (Ultra Resi-Analyzed®) were obtained from J.T. Baker (Phillipsburg, NJ).
The spiked food containing a mixture of all the chemicals was made by adding a known quantity of each chemical (dissolved in 300 ml dichloromethane (DCM)) to 150 g of commercial fish food (Zeigler
Effects of pesticide exposure on fish health
Exposure to the triazoles and α-HCH did not appear to influence the health of the rainbow trout. There was no significant difference in lipid percentage or liver somatic index (p > 0.05) between treatment and control fish on any sampling day and there were no signs of stress (e.g., coloration change, behavior) or mortality in either treatment. Body weights (two sample t-test, p = 0.14), and whole fish and liver growth rates (p > 0.05) of the exposed fish were not significant over the course of the
Acknowledgements
We would like to thank the Lake Burton Hatchery, Georgia, for supplying fish, and S. Craven and D. Higginbotham for rainbow trout husbandry and sampling. B.J.K. was supported by a National Network for Environmental Management System Fellowship, funded through the U.S. EPA. This manuscript has been reviewed and approved for publication by the U.S. EPA, but does not necessarily reflect U.S EPA policy.
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