Isomers/enantiomers of perfluorocarboxylic acids: Method development and detection in environmental samples.

Perfluoroalkyl substances are globally distributed in both urban and remote settings, and routinely are detected in wildlife, humans, and the environment. One of the most prominent and routinely detected perfluoroalkyl substances is perfluorooctanoic acid (PFOA), which has been shown to be toxic to both humans and animals. PFOA exists as both linear and branched isomers; some of the branched isomers are chiral. A novel GC-NCI-MS method was developed to allow for isomer/enantiomer separation, which was achieved using two columns working in tandem; a 30-m DB-5MS column and a 30-m BGB-172 Analytik column. Samples were derivatized with diazomethane to form methyl esters of the PFOA isomers. In standards, at least eight PFOA isomers were detected, of which at least four were enantiomers of chiral isomers; one chiral isomer (P3) was sufficiently separated to allow for enantiomer-fraction calculations. Soil, sediment and plant samples from contaminated locations in Alabama and Georgia were analyzed. P3 was observed in most of these environmental samples, and was non-racemic in at least one sediment, suggesting the possibility of chirally selective generation from precursors or enantioselective sorption. In addition, the ratio of P3/linear PFOA was inversely related to distance from source, which we suggest might reflect a higher sorption affinity for the P3 over the linear isomer. This method focuses on PFOA, but preliminary results suggest that it should be broadly applicable to other chiral and achiral perfluorocarboxylic acids (PFCAs); e.g., we detected several other homologous PFCA isomers in our PFCA standards and some environmental samples.

Loss of propiconazole and its four stereoisomers from the water phase of two soil-water slurries as measured by capillary electrophoresis.

Propiconazole is a chiral fungicide used in agriculture for control of many fungal diseases on a variety of crops. This use provides opportunities for pollution of soil and, subsequently, groundwater. The rate of loss of propiconazole from the water phase of two different soil-water slurries spiked with the fungicide at 50 mg/L was followed under aerobic conditions over five months; the t(1/2) was 45 and 51 days for the two soil slurries. To accurately assess environmental and human risk, it is necessary to analyze the separate stereoisomers of chiral pollutants, because it is known that for most such pollutants, both biotransformation and toxicity are likely to be stereoselective. Micellar electrokinetic chromatography (MEKC), the mode of capillary electrophoresis used for analysis of neutral chemicals, was used for analysis of the four propiconazole stereoisomers with time in the water phase of the slurries. MEKC resulted in baseline separation of all stereoisomers, while GC-MS using a chiral column gave only partial separation. The four stereoisomers of propiconazole were lost from the aqueous phase of the slurries at experimentally equivalent rates, i.e., there was very little, if any, stereoselectivity. No loss of propiconazole was observed from the autoclaved controls of either soil, indicating that the loss from active samples was most likely caused by aerobic biotansformation, with a possible contribution by sorption to the non-autoclaved active soils. MEKC is a powerful tool for separation of stereoisomers and can be used to study the fate and transformation kinetics of chiral pesticides in water and soil.

Microbial transformation of triadimefon to triadimenol in soils: selective production rates of triadimenol stereoisomers affect exposure and risk.

The microbial transformation of triadimefon, an agricultural fungicide of the 1,2,4-triazole class, was followed at a nominal concentration of 50 µg/mL over 4 months under aerobic conditions in three different soil types. Rates and products of transformation were measured, as well as enantiomer fractions of parent and products. The transformation was biotic and enantioselective, and in each soil the S-(+)-enantiomer reacted faster than the R-(-) one. Rates of the first-order reactions were 0.047, 0.057, and 0.107 d(-1) for the three soils. The transformation involves reduction of the prochiral ketone moiety of triadimefon to an alcohol, resulting in triadimenol, which has two chiral centers and four stereoisomers. The abundances of the four product stereoisomers were different from each other, but abundance ratios were similar for all three soil types. Triadimenol is also a fungicide; the commercial product is composed of two diastereomers of unequal amounts (ratio of about 4.3:1), each having two enantiomers of equal amounts. However, the triadimenol formed by soil transformation of triadimefon exhibited no such stereoisomer profile. Instead, different production rates were observed for each of the four triadimenol stereoisomers, resulting in all stereoisomer concentrations being different from each other and very different from concentration/abundance patterns of the commercial standard. This result is important in risk assessment if the toxicity of the environmental transformation product were to be compared to that of the commercial triadimenol. Because triadimenol stereoisomers differ in their toxicities, at least to fungi and rats, the biological activity of the triadimenol formed by microbes or other biota in soils depends on the relative abundances of its four stereoisomers. This is an exposure and risk assessment issue that, in principle, applies to any chiral pesticide and its metabolites.

Integration of metabolomics and in vitro metabolism assays for investigating the stereoselective transformation of triadimefon in rainbow trout.

Triadimefon is a systemic agricultural fungicide of the triazole class whose major metabolite, triadimenol, also a commercial fungicide, provides the majority of the actual fungicidal activity, i.e., inhibition of steroid demethylation. Both chemicals are chiral: triadimefon has one chiral center with two enantiomers while its enzymatic reduction to triadimenol produces a second chiral center and two diastereomers with two enantiomers each. All six stereoisomers of the two fungicides were separated from each other using a chiral BGB-172 column on a GC-MS system so as to follow stereospecificity in metabolism by rainbow trout hepatic microsomes. In these microsomes the S-(+) enantiomer of triadimefon was transformed to triadimenol 27% faster than the R-(-) enantiomer, forming the four triadimenol stereoisomers at rates different from each other. The most fungi-toxic stereoisomer (1S,2R) was produced at the slowest rate; it was detectable after 8 h, but below the level of method quantitation. The triadimenol stereoisomer ratio pattern produced by the trout microsomes was very different from that of the commercial triadimenol standard, in which the most rat-toxic pair of enantiomers (known as "Diastereomer A") is about 85% of the total stereoisomer composition. The trout microsomes produced only about 4% of "Diastereomer A". Complementary metabolomic studies with NMR showed that exposure of the separate triadimefon enantiomers and the racemate to rainbow trout for 48 h resulted in different metabolic profiles in the trout liver extracts, i.e., different endogenous metabolite patterns that indicated differences in effects of the two enantiomers.

Analysis of the enantiomers of chiral pesticides and other pollutants in environmental samples by capillary electrophoresis.

The generic method described here involves typical capillary electrophoresis (CE) techniques, with the addition of cyclodextrin chiral selectors to the electrolyte for enantiomer separation and also, in the case of neutral analytes, the further addition of a micelle-forming compound such as sodium dodecyl sulfate (SDS) for separation by the micellar electrokinetic chromatography (MEKC) mode of CE. This generic method has broad application for the separation and analysis of enantiomers of chiral pesticides and other small molecules in a variety of environmental matrices. Aqueous samples such as surface water are analyzed after simple filtration, but centrifugation is sometimes necessary for soil-water slurry samples. Soils and sediment must be extracted with a polar organic solvent such as methanol, which needs only to be evaporated to near dryness, diluted with water, and filtered before CE analysis. Simple borate or phosphate-based buffers are usually used in the CE electrolyte. The method must be optimized for the electrolyte composition, including the correct chiral selector and its concentration, as well as for column conditions and instrumental variables such as voltage. Specific methodologies for application of this generic CE method to follow the enantioselective microbial transformation of ruelene, a neutral organophosphorus insecticide, dichlorprop, an ionic phenoxyalkanoic acid herbicide, and bromochloroacetic acid, a drinking water disinfection byproduct, are provided.

Enantioselective Chronic Toxicity of Fipronil to Ceriodaphnia dubia.

Fipronil is a phenylpyrazole pesticide that has greatly increased in popularity in recent years. As a chiral molecule, fipronil is released into the environment as a 1:1 mixture (called a racemate) of its two enantiomers. Previous toxicity work has indicated that the enantiomers of fipronil exhibit significantly different levels of acute toxicity to the nontarget organism Ceriodaphnia dubia. In this work we examine the chronic effects of the pure enantiomers and racemate on the survival, development, mobility, and reproduction of C. dubia adults and the survival and mobility of their offspring. Based on 8-day trials, the (+) enantiomer of fipronil showed a significantly greater reduction in the number of offspring (LOEC = 2 microg/L) than either the racemate (LOEC = 15 microg/L) or the (-) enantiomer (LOEC = 30 microg/L). The (+) enantiomer was also shown to be significantly more toxic to neonates born during the course of the experiment (LC50 24 = 18.1 microg/L, LC50 48 = 10.3 microg/L) than the racemate (LC50 24 = 33.3 microg/L, LC50 48 = 30.3 microg/L), but only after 48 h. Both the (+) enantiomer and the racemate were significantly more toxic to C. dubia than the (-) enantiomer (LC50 24 = 65.2 microg/L, LC50 48 = 50.1 microg/L) at both time points. Qualitative mobility data followed a similar trend, with the (+) enantiomer causing greater impairment in mobility at low concentrations. These data imply that the (-) enantiomer has less impact on the reproductive success of C. dubia than either the (+) enantiomer or the racemate. Enantiomerically pure or enriched formulations of (-) fipronil may reduce impacts to the nontarget organism C. dubia.

Toxicity of fipronil and its enantiomers to marine and freshwater non-targets.

Fipronil is a phenylpyrazole insecticide used in agricultural and domestic settings for controlling various insect pests in crops, lawns, and residential structures. Fipronil is chiral; however, it is released into the environment as a racemic mixture of two enantiomers. In this study, the acute toxicity of the (S,+) and (R,-) enantiomers and the racemic mixture of fipronil were assessed using Simulium vittatum IS-7 (black fly), Xenopus laevis (African clawed frog), Procambarus clarkii (crayfish), Palaemonetes pugio (grass shrimp), Mercenaria mercenaria (hardshell clam), and Dunaliella tertiolecta (phytoplankton). Results showed that S. vittatum IS-7 was the most sensitive freshwater species to the racemic mixture of fipronil (LC50 = 0.65 microg/L) while P. pugio was the most sensitive marine species (LC50 = 0.32 microg/L). Procambarus clarkii were significantly more sensitive to the (S,+) enantiomer while larval P. pugio were significantly more sensitive to the (R,-) enantiomer. Enantioselective toxicity was not observed in the other organisms tested. Increased mortality and minimal recovery was observed in all species tested for recovery from fipronil exposure. These results indicate that the most toxic isomer of fipronil is organism-specific and that enantioselective toxicity may be more common in crustaceans than in other aquatic organisms.

Bioaccumulation and biotransformation of chiral triazole fungicides in rainbow trout (Oncorhynchus mykiss).

There are very little data on the bioaccumulation and biotransformation of current-use pesticides (CUPs) despite the fact that such data are critical in assessing their fate and potential toxic effects in aquatic organisms. To help address this issue, juvenile rainbow trout (Oncorhynchus mykiss) were exposed to dietary concentrations of a mixture of chiral triazole fungicides (bromuconazole, cyproconazole, metconazole, myclobutanil, penconazole, propiconazole, tebuconazole, tetraconazole, and triadimefon) and a chiral legacy pesticide [alpha-hexachlorocyclohexane (alpha-HCH)] to study the bioaccumulation and biotransformation of these CUPs. Fish accumulated all triazoles rapidly during the 8 day uptake phase, and was followed by rapid elimination, which was estimated by taking accelerated sampling times during the 16 day depuration period. Half-lives (t1/2s) and times to 95% elimination (t95s) ranged from 1.0 to 2.5 and 4.5 to 11.0 days, respectively. Chiral analysis suggested no significant selectivity in biotransformation for most of the compounds based on statistically unaltered enantiomer fractions (EFs) in the fish compared to food values; exceptions were a change in EF of myclobutanil and changes in diastereomer fractions (DFs) of propiconazole and cyproconazole. No biotransformation was observed for alpha-HCH based on consistent EFs in the fish throughout the experiment and a t1/2 (15.8 days) that fell within the 95% confidence interval of a log K(ow)-log t1/2 relationship developed for assessing biotransformation of organic contaminants. This relationship did show that biotransformation accounted for the majority (ranging from 59.9 to 90.4%) of the elimination for all triazoles, and that triazole compounds with oxygen and hydroxyl functional groups were more easily biotransformed. This research indicated that chiral analysis may potentially miss biotransformation of CUPs and other potential non-persistent organic contaminants and shows the utility of the log K(ow)-log t1/2 relationship as a mechanistic tool for quantifying biotransformation. Based on the rapid biotransformation of the triazoles, future research should focus on formation of metabolites and their fate and possible effects in the environment.

Distribution of chiral PCBs in selected tissues in the laboratory rat.

The enantiomeric enrichment of polychlorinated biphenyl (PCB) atropisomers has been reported in both wildlife and in humans. The biological processes causing this enrichment are only poorly investigated, a fact that limits the use of enantiomeric fractions (EFs) as a tool to study various processes of environmental relevance. To further understand these enantioselective processes, this study investigates the tissue distribution and EFs of some PCB atropisomers after administration of PCB mixtures to immature male Sprague-Dawley rats. The mixtures selected for this study, Aroclor 1254 and an environmental mixture extracted from Chlorofen-contaminated soil, are qualitatively different and are known to induce different groups of hepatic enzymes. Animals were sacrificed 6 days after dosing, PCBs were extracted, and, whenever possible, the EFs of PCBs 84, 91, 95, 149, 174, and 176 were determined by chiral gas chromatography. The EFs of PCB 95 (adipose tissue, liver, and skin) and PCB 149 (adipose tissue, liver, skin, and blood) in tissues from Aroclor 1254-treated animals differed significantly from EFs in the Aroclor standard, while only EFs of PCB 95 (blood) and PCB 174 (adipose tissue) in tissues from soil-extract-treated animals were different from those of the Chlorofen soil extract. PCB 149 in tissues from soil-extract-treated animals underwent no statistically significant enantiomeric enrichment. These differences in the EFs clearly suggest that the enantioselective enrichment of PCB atropisomers may correlate with exposure history, and with the induction of hepatic enzymes, and that EFs may be useful chemical markers of physiologic and biochemical changes following exposure to PCBs.

Bioaccumulation, biotransformation, and metabolite formation of fipronil and chiral legacy pesticides in rainbow trout.

To assess the fate of current-use pesticides, it is important to understand their bioaccumulation and biotransformation by aquatic biota. We examined the dietary accumulation and enantioselective biotransformation of the chiral current-use pesticide fipronil, along with a mixture of selected chiral [alpha-hexachlorocyclohexane (alpha-HCH), heptachlor epoxide (HEPX), polychlorinated biphenyls (PCBs) 84, 132, 174, o,p'-DDT, and o,p'-DDD] and nonchiral (p,p'-DDT, p,p'-DDD) organochlorine compounds in juvenile rainbow trout (Oncorhynchus mykiss). Fish rapidly accumulated all compounds, as measured in the carcass (whole body minus liver and GI tract) during the 32 d uptake phase, which was followed by varying elimination rates of the chemicals (half-lives (t1/2s) ranging from 0.6 d for fipronil to 77.0 d for PCB 174) during the 96 d depuration period. No biotransformation was observed for alpha-HCH, HEPX, PCB 174, o,p'-DDT, or o,p'-DDD based on consistent enantiomeric fractions (EFs) in the fish and their t1/2s falling on a log K(ow)--log t1/2 relationship established for recalcitrant contaminants in fish. p,p'-DDT and PCBs 84 and 132 were biotransformed based on the former's t1/2 position below the log K(ow)--log t1/2 relationship, and the PCBs change in EF. Fipronil was rapidly biotransformed, based on a change in EF, a t1/2 that fell below the log K(ow)--log t1//2 relationship, which accounted for 88% of its elimination, and the rapid formation of fipronil sulfone, a known metabolite. Fipronil sulfone was found to persist longer (t1/2 approximately 2 d) than its parent compound fipronil (t1/2 approximately 0.6 d) and needs to be considered in fate studies of fipronil. This research demonstrates the utilities of the log K(ow)--log t1/2 relationship as a mechanistic tool for quantifying biotransformation and of chiral analysis to measure biotransformation in fish.

Acute enantioselective toxicity of fipronil and its desulfinyl photoproduct to Ceriodaphnia dubia.

Fipronil is a phenylpyrazole insecticide increasingly used in applications such as rice culture, turf grass management, and residential pest control, with a high probability to contaminate aquatic environments. As a chiral pesticide, fipronil is released to the environment as a racemic mixture (equal amounts of optical isomers called enantiomers). Enantiomers can have different toxicological and biological activity; however, information on these differences, which is necessary for accurate risk assessment of chiral pesticides, is limited. Here we examine the acute toxicity of fipronil enantiomers, the racemate, and its photoproduct (desulfinyl fipronil) to Ceriodaphnia dubia. The 48-h median lethal concentration (LC50) values based on measured concentrations of each compound indicate the (+) enantiomer (LC50 = 10.3 +/- 1.1 microg/L, mean +/- standard error [SE]) was significantly more toxic to C. dubia than either the (-) enantiomer (LC50 = 31.9 +/- 2.2 microg/L) or racemate (LC50 = 17.7 +/- 1.3 microg/L). To account for any potential loss of fipronil through photolysis, tests were performed under light (fluorescent) and dark exposure conditions, and no significant differences in toxicity were observed. Desulfinyl fipronil, the major photodegradation product, which is not chiral, was detected at < 1% of each parent compound in test solutions after 48 h. Separate toxicity tests with desulfinyl fipronil found a > 20-fold higher LC50 (355 +/- 9.3 microg/L) compared to the fipronil racemate, suggesting lower adverse effects to C. dubia as a result of fipronil photolysis. The present results suggest selection of the (-) enantiomer in fipronil production for lower impacts to C. dubia; however, the consistency and relevancy of fipronil's enantiomer-specific activity at both acute and chronic levels of concern to additional target and nontarget species needs further consideration.