Changes in CpG Methylation of the Vitellogenin 1 Promoter in Adult Male Zebrafish after Exposure to 17α-Ethynylestradiol.

Numerous pharmaceutical and industrial chemicals are classified as endocrine-disrupting chemicals (EDCs) that interfere with hormonal homeostasis, leading to developmental disorders and other pathologies. The synthetic estrogen 17α-ethynylestradiol (EE2) is used in oral contraceptives and other hormone therapies. EE2 and other estrogens are inadvertently introduced into aquatic environments through municipal wastewater and agricultural effluents. Exposure of male fish to estrogens increases expression of the egg yolk precursor protein vitellogenin (Vtg), which is used as a molecular marker of exposure to estrogenic EDCs. The mechanisms behind Vtg induction are not fully known, and we hypothesized that it is regulated via DNA methylation. Adult zebrafish were exposed to either dimethyl sulfoxide or 20 ng/L EE2 for 14 days. Messenger RNA (mRNA) expression and DNA methylation were assessed in male zebrafish livers at 0, 0.25, 0.5, 1, 4, 7, and 14 days of exposure; and those of females were assessed at 13 days (n ≥ 4/group/time point). To test the persistence of any changes, we included a recovery group that received EE2 for 7 days and did not receive any for the following 7 days, in the total 14-day study. Methylation of DNA at the vtg1 promoter was assessed with targeted gene bisulfite sequencing in livers of adult male and female zebrafish. A significant increase in vtg1 mRNA was observed in the EE2-exposed male fish as early as 6 h. Interestingly, DNA methylation changes were observed at 4 days. Decreases in the overall methylation of the vtg1 promoter in exposed males resulted in levels comparable to those in female controls, suggesting feminization. Importantly, DNA methylation levels in males remained significantly impacted after 7 days post-EE2 removal, unlike mRNA levels. These data identify an epigenetic mark of feminization that may serve as an indicator of not only estrogenic exposure but also previous exposure to EE2. Environ Toxicol Chem 2024;00:1-10. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Polybrominated Diphenyl Ethers in Human Milk and Serum from the U.S. EPA MAMA Study: Modeled Predictions of Infant Exposure and Considerations for Risk Assessment.

BACKGROUND: Serum concentrations of polybrominated diphenyl ethers (PBDEs) in U.S. women are believed to be among the world's highest; however, little information exists on the partitioning of PBDEs between serum and breast milk and how this may affect infant exposure. OBJECTIVES: Paired milk and serum samples were measured for PBDE concentrations in 34 women who participated in the U.S. EPA MAMA Study. Computational models for predicting milk PBDE concentrations from serum were evaluated. METHODS: Samples were analyzed using gas chromatography isotope-dilution high-resolution mass spectrometry. Observed milk PBDE concentrations were compared with model predictions, and models were applied to NHANES serum data to predict milk PBDE concentrations and infant intakes for the U.S. population. RESULTS: Serum and milk samples had detectable concentrations of most PBDEs. BDE-47 was found in the highest concentrations (median serum: 18.6; milk: 31.5 ng/g lipid) and BDE-28 had the highest milk:serum partitioning ratio (2.1 ± 0.2). No evidence of depuration was found. Models demonstrated high reliability and, as of 2007-2008, predicted U.S. milk concentrations of BDE-47, BDE-99, and BDE-100 appear to be declining but BDE-153 may be rising. Predicted infant intakes (ng/kg/day) were below threshold reference doses (RfDs) for BDE-99 and BDE-153 but above the suggested RfD for BDE-47. CONCLUSIONS: Concentrations and partitioning ratios of PBDEs in milk and serum from women in the U.S. EPA MAMA Study are presented for the first time; modeled predictions of milk PBDE concentrations using serum concentrations appear to be a valid method for estimating PBDE exposure in U.S. infants.

Inhibition of the Human ABC Efflux Transporters P-gp and BCRP by the BDE-47 Hydroxylated Metabolite 6-OH-BDE-47: Considerations for Human Exposure.

High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low µM concentrations (IC50 = 11.7 µM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50 = 45.9 µM (BDE-47) vs. IC50 = 9.4 µM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [(3H)-paclitaxel and (3H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity. These findings suggest that some effects previously attributed to BDE-47 in biological systems may actually be due to 6-OH-BDE-47. Considerations for human exposure are discussed.

An Integrated Experimental and Computational Approach for Characterizing the Kinetics and Mechanism of Triadimefon Racemization.

Enantiomers of chiral molecules commonly exhibit differing pharmacokinetics and toxicities, which can introduce significant uncertainty when evaluating biological and environmental fates and potential risks to humans and the environment. However, racemization (the irreversible transformation of one enantiomer into the racemic mixture) and enantiomerization (the reversible conversion of one enantiomer into the other) are poorly understood. To better understand these processes, we investigated the chiral fungicide, triadimefon, which undergoes racemization in soils, water, and organic solvents. Nuclear magnetic resonance (NMR) and gas chromatography / mass spectrometry (GC/MS) techniques were used to measure the rates of enantiomerization and racemization, deuterium isotope effects, and activation energies for triadimefon in H2 O and D2 O. From these results we were able to determine that: 1) the alpha-carbonyl carbon of triadimefon is the reaction site; 2) cleavage of the C-H (C-D) bond is the rate-determining step; 3) the reaction is base-catalyzed; and 4) the reaction likely involves a symmetrical intermediate. The B3LYP/6-311 + G** level of theory was used to compute optimized geometries, harmonic vibrational frequencies, nature population analysis, and intrinsic reaction coordinates for triadimefon in water and three racemization pathways were hypothesized. This work provides an initial step in developing predictive, structure-based models that are needed to identify compounds of concern that may undergo racemization. Chirality 28:633-641, 2016. © 2016 Wiley Periodicals, Inc.

Improving infant exposure and health risk estimates: using serum data to predict polybrominated diphenyl ether concentrations in breast milk.

Women in the United States have breast milk concentrations of polybrominated diphenyl ethers (PBDEs) that are among the highest in the world, leading to concerns over the potential health implications to breastfeeding infants during critical stages of growth and development. Developing cost-effective and sustainable methods for assessing chemical exposures in infants is a high priority to federal agencies and local communities. PBDE data are available in nationally representative serum samples but not in breast milk. As a method to predict PBDE concentrations in U.S. breast milk, we present the development of congener-specific linear regression partitioning models and their application to U.S. serum data. Models were developed from existing paired milk and serum data and applied to 2003-2004 NHANES serum data for U.S. women. Highest estimated median U.S. breast milk concentrations were for BDE-47 (30.6 ng/g lipid) and BDE-99 (6.1 ng/g lipid) with the median concentration of Σ7PBDEs estimated at 54.2 ng/g lipid. Predictions of breast milk PBDE concentration were consistent with reported concentrations from 11 similarly timed U.S. studies. When applied to NHANES data, these models provide a sustainable method for estimating population-level concentrations of PBDEs in U.S. breast milk and should improve exposure estimates in breastfeeding infants.

Human and rat ABC transporter efflux of bisphenol a and bisphenol a glucuronide: interspecies comparison and implications for pharmacokinetic assessment.

Significant interspecies differences exist between human and rodent with respect to absorption, distribution, and excretion of bisphenol A (BPA) and its primary metabolite, BPA-glucuronide (BPA-G). ATP-Binding Cassette (ABC) transporter enzymes play important roles in these physiological processes, and their enzyme localization (apical vs. basolateral) in the plasma membrane allows for different cellular efflux pathways. In this study, we utilized an ATPase assay to evaluate BPA and BPA-G as potential substrates for the human and rat ABC transporters: P-glycoprotein (MDR1), multidrug resistance-associated proteins (MRPs), and breast cancer-resistant protein (BCRP). Based on high ATPase activity, BPA is likely a substrate for rat mdr1b but not for human MDR1 or rat mdr1a. Results indicate that BPA is a potential substrate for rat mrp2 and human MRP2, BCRP, and MRP3. The metabolite BPA-G demonstrated the highest apparent substrate binding affinity for rat mrp2 and human MRP3 but appeared to be a nonsubstrate or potential inhibitor for human MRP2, MDR1, and BCRP and for rat mdr1a, mdr1b, and bcrp. Analysis of ABC transporter amino acid sequences revealed key differences in putative binding site composition that may explain substrate specificity. Collectively, these results suggest that in both rat and human, apical transporters efflux BPA into the bile and/or intestinal lumen. BPA-G would follow a similar pathway in rat; however, in human, due to the basolateral location of MRP3, BPA-G would likely enter systemic and portal blood supplies. These differences between human and rodent ABC transporters may have significant implications for interspecies extrapolation used in risk assessment.

Development and application of a physiologically based pharmacokinetic model for triadimefon and its metabolite triadimenol in rats and humans.

A physiologically based pharmacokinetic (PBPK) model was developed for the conazole fungicide triadimefon and its primary metabolite, triadimenol. Rat tissue:blood partition coefficients and metabolic constants were measured in vitro for both compounds. Pharmacokinetic data for parent and metabolite were collected from several tissues after intravenous administration of triadimefon to male Sprague-Dawley rats. The model adequately simulated peak blood and tissue concentrations but predicted more rapid clearance of both triadimefon and triadimenol from blood and tissues. Reverse metabolism of triadimenol to triadimefon in the liver was explored as a possible explanation of this slow clearance, with significant improvement in model prediction. The amended model was extrapolated to humans using in vitro metabolic constants measured in human hepatic microsomes. Human equivalent doses (HEDs) were calculated for a rat no observable adverse effect level (NOAEL) dose of 3.4mg/kg/day using area under the concentration curve (AUC) in brain and blood for triadimefon and triadimenol as dosimetrics. All dosimetric-based HEDs were 25-30 fold above the human oral reference dose of 0.03mg triadimefon/kg/day, but did not account for intra-human variability or pharmacodynamic differences. Ultimately, derivations of this model will be able to better predict the exposure profile of these and other conazole fungicides in humans.

Differences between human and rat intestinal and hepatic bisphenol A glucuronidation and the influence of alamethicin on in vitro kinetic measurements.

The extent to which membrane-disrupting agents, such as alamethicin, may alter cofactor transport and influence in vitro kinetic measurements of glucuronidation is a major concern regarding the characterization and extrapolation of inter- and intraspecies pharmacokinetics of bisphenol A (BPA). An additional concern is the omission of a BPA intestinal metabolism component in current pharmacokinetic models used to assess oral exposure. In this study, BPA glucuronidation in native hepatic microsomes from female rat and female human liver displayed higher V(max) values than that in males. In the presence of alamethicin, all hepatic V(max) values increased; however, this increase was disproportionately greater in males and gender differences were no longer observed. Female rats exhibited a much higher K(m) than all other species and genders; the addition of alamethicin had little influence on K(m) values for any of the test systems. The dissimilar K(m) measured for female rat suggests that different UDP-glucuronosyltransferase (UGT) enzyme(s) are involved in BPA glucuronidation. The presence of different UGTs in female rat was confirmed using Hill coefficients measured from diclofenac-mediated chemical inhibition assays within hepatic microsomes and purified human UGT2B7 and UGT2B15. Mixed-gender human intestinal microsomes showed little BPA glucuronidation reactivity compared with those from male rat intestine. Male rat intestinal microsomes in the presence of alamethicin exhibited a V(max) that was nearly 30-fold higher than that for mixed human microsomes. The species and gender metabolic differences we observed between rat and human liver and intestine provide key information for delineating BPA pharmacokinetics needed for human health risk assessment.

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.

Gender and species differences in triadimefon metabolism by rodent hepatic microsomes.

Understanding the potential differences in metabolic capacity and kinetics between various common laboratory species as well as between genders is an important facet of chemical risk assessment that is often overlooked, particularly for chemicals which undergo non-cytochrome P450 mediated metabolism. The use of physiologically based pharmacokinetic (PBPK) models to better describe chemical exposure is made more powerful by incorporation of high quality in vitro kinetic data. To this end, metabolism of the conazole fungicide triadimefon was studied in hepatic microsomes of both genders of SD rats and CD-1 mice. Triadimefon depletion and triadimenol formation were measured in each type of microsomes. Michaelis-Menten regressions were applied to metabolic data and V(MAX) and the Michaelis constant (K(M)) values calculated. Male SD rats metabolized triadimefon more rapidly than female SD rats or either gender of CD-1 mouse. K(M) values were in the micromolar range, indicating the possibility of competitive inhibition with endogenous substrates. Intrinsic clearances derived from kinetic parameters indicate that triadimefon metabolism is blood-flow limited in all organisms studied with the possible exception of female rat. The in vitro half-life method was investigated as a less resource intensive method for the derivation of intrinsic clearance, and was found to be useful as a complement to the traditional Michaelis-Menten approach.

Mechanistic approach to understanding the toxicity of the azole fungicide triadimefon to a nontarget aquatic insect and implications for exposure assessment.

Mechanistic and stereoselective based in vitro metabolism assays were utlilized to gain insight into the toxic mode of action of the 1,2,4-triazole fungicide, triadimefon, with black fly (Diptera: Simuliidae) larvae. Based on results from enzyme inhibitor studies, the metabolism of triadimefon in black fly larvae microsomes was found to occur predominantly via an oxidative P450-mediated pathway; triadimenol was formed via the stereoselective reduction of the prochiral carbonyl group of triadimefon. The relatively minor contribution of carbonyl reduction suggests that triadimefon may inhibit ecdysone 20-monooxygenase and disrupt insect molting hormone biosynthesis. 48-h LC50 tests for triadimefon and triadimenol with black fly larvae yielded median values (with 95% confidence intervals) of 6.1 (5.8-6.4) and 22.3 (20.3-24.1) mg/L respectively. The exposure of black fly larvae to sublethal concentrations of triadimefon resulted in increased microsomal P450 activity and affected the microsomal rates of both triadimefon depletion and triadimenol formation. In contrast to trout, black fly larvae produced a higher fraction of the more toxic triadimenol stereoisomers, which may explain in part why triadimefon exhibited a significantly greater toxicity with black fly larvae than trout. These results illustrate that while LC50 tests conducted with commercial triadimenol would presumably expose each organism to the same relative abundance of the four triadimenol stereoisomers, LC50 tests with triadimefon ultimately expose each organism to a unique set of triadimenol stereoisomers depending upon the organism's stereoselective metabolism.

Contrasting influence of NADPH and a NADPH-regenerating system on the metabolism of carbonyl-containing compounds in hepatic microsomes.

Carbonyl containing xenobiotics may be susceptible to NADPH-dependent cytochrome P450 (P450) and carbonyl-reduction reactions. In vitro hepatic microsome assays are routinely supplied NADPH either by direct addition of NADPH or via an NADPH-regenerating system (NRS). In contrast to oxidative P450 transformations, which occur on the periphery of a microsome vesicle, intraluminal carbonyl reduction depends on transport of cofactors across the endoplasmic reticulum (ER) membrane into the lumen. Glucose 6-phosphate, a natural cofactor and component of the NRS matrix, is readily transported across the ER membrane and facilitates intraluminal NADPH production, whereas direct addition of NADPH has limited access to the lumen. In this study, we compared the effects of direct addition of NADPH and use of an NRS on the P450-mediated transformation of propiconazole and 11 beta-hydroxysteroid dehydrogenase type 1 (HSD1) carbonyl reduction of cortisone and the xenobiotic triadimefon in hepatic microsomes. Our results demonstrate that the use of NADPH rather than NRS can underestimate the kinetic rates of intraluminal carbonyl reduction, whereas P450-mediated transformations were unaffected. Therefore, in vitro depletion rates measured for a carbonyl-containing xenobiotic susceptible to both intraluminal carbonyl reduction and P450 processes may not be properly assessed with direct addition of NADPH. In addition, we used in silico predictions as follows: 1) to show that 11 beta-HSD1 carbonyl reduction was energetically more favorable than oxidative P450 transformation; and 2) to calculate chemical binding score and the distance between the carbonyl group and the hydride to be transferred by NADPH to identify other 11 beta-HSD1 substrates for which reaction kinetics may be underestimated by direct addition of NADPH.

Mechanistic investigation of the noncytochrome P450-mediated metabolism of triadimefon to triadimenol in hepatic microsomes.

Recently, much emphasis has been placed on understanding the toxic mode of action of the 1,2,4-triazole fungicides (i.e., conazoles) in an effort to improve and harmonize risk assessment. Relative to other conazoles, triadimefon is unique with respect to tumorigenesis in rodents, and it has been proposed that triadimefon does not share a common mechanism of toxicity with other conazoles. We postulate that one reason for this difference is that while many conazoles are metabolized via an oxidative P450-mediated pathway, triadimefon is not. In studies conducted with rat hepatic microsomes, triadimenol was identified as the major metabolite (approximately 80%) of triadimefon metabolism, and reduction of the carbonyl group in triadimefon occurred stereoselectively with preferential formation of the less toxic triadimenol B diastereomer. Using chemical inhibitors of P450s (i.e., clotrimazole and 1-aminobenzotriazole) and carbonyl reducing enzymes (i.e., glycyrrhetinic acid, quercitrin, and cortisone), both triadimefon depletion and triadimenol formation were found to be mediated by 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1). Studies examining NADPH production and inhibitor studies for glucose-6-phosphate translocation across the endoplasmic reticulum (ER) membrane implicated hexose-6-phosphate dehydrogenase (H6PDH) in the metabolism of triadimefon as well. These results ultimately associate triadimefon metabolism not only with steroidogenesis (i.e., 11 beta-HSD1) but carbohydrate metabolism (i.e., H6PDH) as well. Considering the impact of triadimefon on these biochemical pathways may help explain some of triadimefon's unique toxicological effects relative to other conazole fungicides.

Cross-species comparison of conazole fungicide metabolites using rat and rainbow trout (Onchorhynchus mykiss) hepatic microsomes and purified human CYP 3A4.

Ecological risk assessment frequently relies on cross-species extrapolation to predict acute toxicity from chemical exposures. A major concern for environmental risk characterization is the degree of uncertainty in assessing xenobiotic biotransformation processes. Although inherently complex, metabolite identification is critical to risk assessment since the product(s) formed may pose a greater toxicological threat than the parent molecule. This issue is further complicated by differences observed in metabolic transformation pathways among species. Conazoles represent an important class of azole fungicides that are widely used in both pharmaceutical and agricultural applications. The antifungal property of conazoles occurs via complexation with the cytochrome P450 monooxygenases (CYP) responsible for mediating fungal cell wall synthesis. This mode of action has cause for concern regarding the potential adverse impact of conazoles on the broad spectrum of CYP-based processes within mammalian and aquatic species. In this study, in vitro metabolic profiles were determined for thirteen conazole fungicides using rat and rainbow trout (Oncorhynchus mykiss) liver microsomes and purified human CYP 3A4. Results showed that 10 out of the 13 conazoles tested demonstrated identical metabolite profiles among rat and trout microsomes, and these transformations were well conserved via both aromatic and aliphatic hydroxylation and carbonyl reduction processes. Furthermore, nearly all metabolites detected in the rat and trout microsomal assays were detected within the human CYP 3A4 assays. These results indicate a high degree of metabolic conservation among species with an equivalent isozyme activity of human CYP 3A4 being present in both the rat and trout, and provides insight into xenobiotic biotransformations needed for accurate risk assessment.

In vitro metabolism of the fungicide and environmental contaminant trans-bromuconazole and implications for risk assessment.

trans-Bromuconazole is a chiral chemical representative of a class of triazole derivatives known to inhibit specific fungal cytochrome P-450 (CYP) reactions. Kinetic measurements and delineation of metabolic pathways for triazole chemicals within in vitro hepatic microsomes are needed for accurate risk assessment and predictive in vivo physiological modeling. The studies described here were conducted with rat liver microsomes to determine Michaelis-Menten saturation kinetic parameters (Vmax and KM) for trans-bromuconazole using both substrate depletion and product formation reaction velocities. Kinetic parameters determined for trans-bromuconazole depletion at varying protein levels incubated at physiological temperature 37 degrees C resulted in a KM value of 1.69 microM and a Vmax value of 1398 pmol/min/mg protein. The concomitant linear formation of two metabolites identified using liquid chromatography/time-of-flight mass spectrometry (LC/MS-TOF) and LC-MS/MS indicated hydroxylation of the trans-bromuconazole dichlorophenyl ring moiety. KM values determined for the hydroxylated metabolites were 0.87 and 1.03 microM, with Vmax values of 449 and 694 pmol/min/mg protein, respectively. Chemical inhibition assays and studies conducted with individual purified human recombinant enzymes indicated the CYP3A subfamily was primarily responsible for biotransformation of the parent substrate. Additionally, trans-bromuconazole was found to undergo stereoselective metabolism as evidenced by a change in the enantiomeric ratio (trans-/trans+) with respect to time.

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.

Use of pretreatment zones and zero-valent iron for the remediation of chloroalkenes in an oxic aquifer.

Pretreatment zones (PTZs) composed of sand, 10% zero-valent iron [Fe(0)]/sand, and 10% pyrite (FeS2)/sand were examined for their ability to prolong Fe(0) reactivity in above ground column reactors and a subsurface permeable reactive barrier (PRB). The test site had an acidic, oxic aquifer contaminated with tetrachloroethylene (PCE) and trichloroethylene (TCE). The 10% FeS2 and 10% Fe(0) PTZs removed dissolved oxygen and affected the pH and E(h) in the PTZ. None of the PTZs had any effect on pH or E(h) in the 100% Fe(0) zone. Nitrate and sulfate were removed more quickly in the Fe(0) zones preceded by either the 10% Fe(0) PTZ or 10% FeS2. PCE first-order degradation rate constants (k(obs)) decreased significantly (> 80%) with increasing column pore volumes regardless of the PTZ material used. k(obs) finally leveled off after approximately 1 yr of operation. The column results predict that the PRB will experience a breakthrough of PCE in 3-5 yr and illustrate the importance of incorporating temporal variations in degradation rate constants when designing PRBs.

Reductive dehalogenation of halomethanes in iron- and sulfate-reducing sediments. 1. Reactivity pattern analysis.

The incorporation of reductive transformations into environmental fate models requires the characterization of natural reductants in sediments and aquifer materials. For this purpose, reactivity patterns (range and relative order of reactivity) for a series of 14 halogenated methanes were measured in iron- and sulfate-reducing sediments and two representative model systems: adsorbed Fe(II)/goethite [Fe(II)ads/alpha-FeOOH] and iron sulfide (FeS). Both Fe(II)ads and FeS are naturally occurring reductants. The strong similarity in reactivity patterns between the iron- and sulfate-reducing sediments suggests that the two share a common reductant despite their different chemical compositions (i.e., the sulfate-reducing sediment contained FeS). An orthogonal regression analysis of the halomethane transformation rate data in the sediment and model systems supports the assumption that a common mechanism for halomethane transformation exists between the sediments and the Fe(II)ads/alpha-FeOOH system and further corroborates the conclusion that Fe(II) adsorbed to Fe(III)-containing minerals is the dominant reductant in both sediment systems. Weak (0.5 N) and strong (6.0 N) acid extraction of the sediments indicated that solid-phase Fe(II) was 67% higher in the sulfate-reducing sediment than in the iron-reducing sediment, which is consistent with the observations that the halomethanes were transformed a factor of 3 times faster in the sulfate-reducing sediment and that Fe(II) was the dominant reductant.