Pesticide Uptake Across the Amphibian Dermis Through Soil and Overspray Exposures.

For terrestrial amphibians, accumulation of pesticides through dermal contact is a primary route of exposure in agricultural landscapes and may be contributing to widespread amphibian declines. To show pesticide transfer across the amphibian dermis at permitted label application rates, our study was designed to measure pesticide body burdens after two simulated exposure scenarios. We compared direct exposures, where amphibians were present when spraying occurred, to indirect exposures, where amphibians were exposed to soils after pesticide application. During summer 2012, we reared barking (Hyla gratiosa) and green treefrogs (H. cinerea) through 60-90 days post-metamorphosis at a United States Environmental Protection Agency research laboratory. We tested exposure for 8 h to five pesticide active ingredients (imidacloprid, atrazine, triadimefon, fipronil, or pendimethalin) in glass aquaria lined with soil in the laboratory. We quantified total pesticide body burden and soil concentrations using liquid chromatography-mass spectrometry. All individuals in both treatments had measurable body burdens at the end of the study. A randomized block design analysis of variance (n = 18) showed that body burdens (p = 0.03) and bioconcentration factors (BCFs) (p = 0.01) were significantly greater in the direct overspray treatment relative to the indirect soil spray treatment for both species and tested pesticides. BCFs ranged from 0.1 to 1.16 and from 0.013 to 0.78 in the direct and indirect treatments, respectively. Our study shows dermal uptake for multiple pesticides from both direct spray and indirect soil exposures and provides empirical support for the degree to which terrestrial phase amphibians have higher body burdens after overspray pesticide exposure.

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.

Estrogenic activity of dicofol with the human estrogen receptor: Isomer- and enantiomer-specific implications.

Dicofol is a non-systemic acaricide/miticide currently registered in the US and Canada for use on a wide variety of crops. This agrochemical has been identified as a potential candidate substance for the United Nations Economic Commission for Europe (UN-ECE) Persistent Organic Pollutant (POP) Protocol and implicated as a potential "endocrine disrupting compound". The technical product is usually synthesized from technical DDT and consists of approximately 80% and 20% of p,p'- and o,p'-dicofol isomers. The o,p'-substituted isomer of dicofol is chiral and may have enantiomer-specific activity; however, the stereospecific activity of o,p'-dicofol has not been reported. In this study, we examined the isomer- and enantiomer-specific endocrine disruption potential of dicofol using yeast-based steroid hormone receptor gene transcription assay designed with the human estrogen receptor (hER). Estrogenic activity of (+)-17-beta estradiol (positive control), p,p'-dicofol, racemic o,p'-dicofol [(+/-)-o,p'-dicofol] and the individual o,p'-dicofol enantiomers was measured via quantification of beta-galactosidase. The (+/-)-o,p'- and p,p'-dicofol were weak estrogen mimics (EC(50): 4.2 x 10(-6) and 1.6 x 10(-6)M, respectively) relative to estradiol (3.7 x 10(-10)M). For o,p'-dicofol, the beta-galactosidase induction by (-)-o,p'-dicofol (EC(50): 5.1 x 10(-7)M) was greater than the racemic mixture. However, the (+)-o,p'-dicofol enantiomer was found to have negligible estrogenic activity. These data indicate that dicofol is a weak hER agonist due to activity of the achiral p,p'-isomer and (-)-o,p'-substituted enantiomer and emphasizes the influence of chemical structure and configuration on biological responses to exposure from chiral compounds.

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.

Effects of PCB 84 enantiomers on [3H]-phorbol ester binding in rat cerebellar granule cells and 45Ca2+-uptake in rat cerebellum.

There is evidence that polychlorinated biphenyl (PCB) congeners with ortho chlorine substituents have potential to cause neurotoxicity. Many PCB congeners implicated in these neurotoxic effects are chiral. It is currently unknown if the enantiomers of chiral PCB congeners have different neurotoxic effects. We herein report the effect of racemic 2,2',3,3',6-pentachlorobiphenyl (PCB 84) and its enantiomers on two neurochemical measures, protein kinase C (PKC) translocation as determined by [3H]-phorobol ester binding in cerebellar granule cells and Ca2+-sequestration as determined by 45Ca2+-uptake by microsomes isolated from adult rat cerebellum. Both (+)- and (-)-PCB 84 increased [3H]-phorobol ester binding in a concentration-dependent manner with (-)-PCB 84 being slightly more potent. Racemic PCB 84 was significantly more potent and efficacious than the pure enantiomers alone. (-)- and (+)-PCB 84 each inhibited microsomal 45Ca2+-uptake to a similar extent, whereas racemic PCB 84 was more potent and efficacious. These results indicate that PCB 84 enantiomers alone can have different potencies, and these may differ from that of the racemic mixture, observations that may have important implications for understanding the mechanisms of neurotoxicity of chiral PCB congeners.

Application of capillary electrophoresis to study the enantioselective transformation of five chiral pesticides in aerobic soil slurries.

The enantiomers of five chiral pesticides of environmental interest, metalaxyl, imazaquin, fonofos (dyfonate), ruelene (cruformate), and dichlorprop, were separated analytically using capillary electrophoresis (CE) with cyclodextrin chiral selectors. For metalaxyl, imazaquin, and fonofos, aqueous slurries of soil samples from two sites in Georgia and one in Ohio were spiked with the racemate of each pesticide at 50-60 mg/L of aqueous phase of the slurry, and CE analyses were performed at various time intervals to determine enantiomer fractions (EF). Metalaxyl underwent enantioselective transformation; in one soil, the half-life of the target active R-(+)-enantiomer was 17 days while that for the S-(-)-enantiomer was 69 days. Transformation occurred more slowly in the other two soils but was still selective for the R-(+)-enantiomer. Imazaquin and fonofos exhibited nonselective enantiomer loss over their 3 months of incubation time; this could have been due to abiotic or nonselective microbial reactions. Ruelene and dichlorprop were transformed selectively in a variety of soils in a previously reported study (7) that showed the influence of environmental changes on the transformation of chiral pollutants in soils; analytical methods used in that study are reported here to further illustrate the application of CE. CE is shown to be a simple, efficient, and inexpensive way to follow the transformation of chiral pesticides in laboratory microcosms where concentrations can be made high enough (25-50 mg/L initial racemate concentration) for detection of residual parent enantiomers during most of the process.

Liquid chromatographic separation of the enantiomers of trans-chlordane, cis-chlordane, heptachlor, heptachlor epoxide and alpha-hexachlorocyclohexane with application to small-scale preparative separation.

Analytical high-performance liquid chromatographic separations of the individual enantiomers of five polychlorinated compounds were obtained on polysaccharide stereoselective HPLC columns. The enantiomers of the pesticides trans-chlordane, cis-chlordane and heptachlor were separated on CHIRALCEL OD using a hexane mobile phase. The enantiomers of the heptachlor metabolite, heptachlor epoxide, were separated on CHIRALPAK AD using a methanol mobile phase. The enantiomers of alpha-hexachlorocyclohexane (alpha-HCH), were separated on CHIRALCEL OJ using a hexane/2-propanol mobile phase. Similar chromatographic conditions using preparative columns were used to isolate approximately 250 mg of each of the individual enantiomers. The purified individual enantiomers have been submitted for testing of their endocrine disruptor (ED) activity.

Enantioselective microbial transformation of the phenylpyrazole insecticide fipronil in anoxic sediments.

Fipronil, a chiral insecticide, was biotransformed initially to fipronil sulfide in anoxic sediment slurries following a short lag period. Sulfidogenic or methanogenic sediments transformed fipronil with half-lives of approximately 35 and 40 days, respectively. In all microbially active sediment slurries tested, the transformation of fipronil to fipronil sulfide was enantioselective. In the sulfidogenic sediment slurry, the enantiomeric fraction (EF) of fipronil decreased from an initial racemic EF value of 0.46 to a value of 0.22 during the incubation period of active fipronil transformation, indicating preferential transformation of the S-(+)-enantiomer. A previously unidentified product, 5-amino1-[2,6-dichloro-4-(trifluoromethyl)-phenyl]-4-(trifluoromethylthio)-1-H-pyrazole-3-carboxyamide, or fipronil sulfide-amide, was detected in the sulfidogenic slurries and coincided with the loss of fipronil sulfide. Biota from methanogenic freshwater sediment slurries also transformed fipronil enantioselectively but with a preference for the R-(-)-enantiomer. In all microbially inhibited (autoclaved) sediment slurries tested, no changes in the enantiomeric fractions of fipronil were observed and only low levels (< 5% of the added fipronil) of the fipronil sulfide metabolite were detected. In defined (model) chemical experiments, solutions of pyrite (FeS2) and iron sulfide (FeS) non-enantioselectively transformed fipronil primarily to either 2,6-dichloro-4-(trifluoromethyl)-aniline or to fipronil sulfide and fipronil amide, respectively. This report provides the first experimental evidence of enantioselective microbial transformation of fipronil in a natural environment (soil, water, and sediment) as well as identification of a novel fipronil biotransformation product.

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.

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.