Jimmy K. Avants

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 μ g/L) while P. pugio was the most sensitive marine species (LC50 = 0.32 μ g/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.

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 fipronils enantiomer-specific activity at both acute and chronic levels of concern to additional target and nontarget species needs further consideration.

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.

Enantioselective toxicity and bioaccumulation of fipronil in fathead minnows (Pimephales promelas) following water and sediment exposures

Fipronil is a widely used, broad-spectrum pesticide that is applied as an equal mixture of two enantiomers. As regulations on older pesticides become more stringent, production and application of fipronil is expected to grow, leading to increased inputs into aquatic environments and complex exposures to biota. To better understand the potential exposures introduced by fipronil contamination, we conducted subchronic toxicity tests with larval fathead minnows (Pimephales promelas) and waterborne fipronil and its enantiomers and exposed juvenile fathead minnows to fipronil-spiked sediment. Enantioselective toxicity was observed in fish after the 7-d subchronic exposure, with increased toxicity of the racemate and (+) enantiomer observed compared with the (-) enantiomer. Curiously, toxicities of the racemate and (+) enantiomer were not significantly different, even though the racemate contains 50% of the (+) enantiomer and 50% of the less toxic (-) enantiomer. During the sediment exposure, racemic fipronil in sediment was transformed primarily to fipronil sulfide, while exposed fish rapidly accumulated fipronil and/or fipronil sulfide and transformed the majority to fipronil sulfone. Using the results of the sediment-exposure experiment, the authors explored a mechanism that may contribute to the interesting trends in enantioselective toxicity observed during the waterborne exposures. In tandem, the aquatic toxicity experiment and the spiked sediment exposure demonstrate the potentially complex behavior of fipronil in sediment and fish.

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 t1/2 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.

Persistent organochlorine pesticides and their metabolites in alligator livers from Lakes Apopka and Woodruff, Florida, USA

Reproductive disorders in American alligators (Alligator mississippiensis) inhabiting Lake Apopka, Florida, have been observed for several years. Such disorders are hypothesised to be caused by endocrine disrupting contaminants occurring in the lake due to pesticide spills and runoff from bordering agricultural lands. Various studies have resulted in identification of several persistent chlorinated organic pollutants, some of them known endocrine disrupters, in various alligator tissues and fluids. In this report, livers from 12 juvenile alligators inhabiting Lake Apopka and 10 from Lake Woodruff, a control lake, were extracted and analysed using gas chromatography–mass spectrometry with chiral GC columns for identification of both chiral and non-chiral organochlorine pesticides (OCPs, including their metabolites); in so doing, the enantiomer fractions of any chiral OCPs identified were also measured. In Lake Apopka, p,p′-DDE was the most prominent OCP identified, being found in all samples at concentrations ranging from 4 to 779 ng g−1, based on wet weight of the liver samples. Trans- and cis-nonachlor were also detected in all samples at a concentration range of 0.3 to 64 ng g−1; p,p′-DDD was also detected in all samples, but at an even lower concentration of 0.2 to 11 ng g−1. Only 5 chiral OCPs were identified; their enantiomer fractions were mostly non-racemic, indicating pre-ingestion enantioselective biotransformation or enantioselective metabolism by the alligators. p,p′-Dichlorobenzophenone (p,p′-DCBP), a known metabolite of p,p′-dicofol, was detected in all but one sample; most concentrations were <1 ng g−1. Dicofol is known to have been used and spilled near Lake Apopka, and is highly toxic to fish and aquatic invertebrates. Experiments showed that the p,p′-DCBP identified in these samples occurred via thermal degradation during GC analysis of p,p′-dicofol that was present in the liver sample extracts. Only 5 OCPs, at levels much below those in Lake Apopka, were found in control Lake Woodruff.

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.

Enantioseparation of Malathion, Cruformate, and Fensulfothion Organophosphorus Pesticides by Mixed-Mode Electrokinetic Capillary Chromatography

Mixed-mode electrokinetic capillary chromatography (mixed-mode ECC) has been used for the enantioseparation of organophosphorus pesticides. In mixed-ECC, a combination of three pseudostationary phases including surfactants, neutral, and charged cyclodextrins, are used to resolve very challenging enantioseparation problems. The conditions mimic a mixture of micellar electrokinetic capillary chromatography (MECC) and dual-cyclodextrin electrokinetic capillary chromatography (dual-CECC) conditions. In this work SDS, carboxymethyl- g -CD, hydroxypropyl-BCD, and organic modifiers were mixed at various concentrations in order to achieve enantioseparation of three organophosphorus pesticides - cruformate (ruelene), malathion, and fensulfothion. The best condition for separation of ruelene enantiomers was by using a mixture of 70 mM SDS/15 mM carboxymethyl-BCD/45 mM hydroxypropyl-BCD/20% (v/v) acetonitrile in 20 mM borate buffer at pH 8.6, with applied voltage of 25 kV at 25°C. Malathion enantiomers were successfully resolved using either 10 mM SDS/50 mM CM-BCD/40 mM hydroxypropyl-BCD or 50 mM CM-BCD/50 mM hydroxypropyl-BCD/20% (v/v) methanol in 20 mM borate buffer. Fensulfothion enantiomers were successfully resolved using a mixture of 75 mM SDS/12.5 mM carboxymethyl-BCD/45 mM hydroxypropyl-BCD in the same 20 mM borate buffer. The results demonstrate the versatility of the mixed-mode ECC technique in handling very difficult separations such as the organophosphoramidate enantiomers. It offers options for selectivity control by combining three or more pseudostationary phases in the background electrolyte (BGE). The approach to optimization in mixed-mode ECC is generally more straightforward than the use of a solid stationary phase(s) in HPLC. In theory, the separation selectivity of such mixed-mode ECC system can be modified to the extremes of MECC and the dual-CECC of the chiral selectors.

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.