ARTICLE pubs.acs.org/est
Microbial Transformation of Triadimefon to Triadimenol in Soils: Selective Production Rates of Triadimenol Stereoisomers Affect Exposure and Risk Arthur W. Garrison,*,† Jimmy K. Avants,‡ and W. Jack Jones† †
National Exposure Research Laboratory, Ecosystems Research Division, U.S. Environmental Protection Agency, Athens, Georgia, United States ‡ Senior Service America, U.S. Environmental Protection Agency, NERL/ERD, Athens, Georgia, United States ABSTRACT: 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-(þ)-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.
’ INTRODUCTION Triadimefon (Figure 1) [1-(4-chlorophenoxy)-3,3-dimethyl1-(1H-1,2,4-triazol-1-yl)-2-butanone, CAS 43121-43-3] is a 1,2,4-triazole fungicide used for the control of powdery mildews and fungi on fruits, vegetables, turf grasses, and other agricultural crops. Although a specialty fungicide applied at low rates, there were 149 000 pounds of the active ingredient used in 1988. 1 Triadimefon is a systemic foliar fungicide that acts by inhibiting steroid demethylation.2 It is enzymatically reduced in plants, soils, and fungi to the more fungi-active metabolite, triadimenol (Figure 1) [β-(4-chlorophenoxy)-R-(1,1-dimethylethyl)-1H1,2,4-triazole-1-ethanol, CAS 55219-65-3], which is also used as an agricultural fungicide. Triadimefon has a single chiral center and thus exists as two enantiomers. Its metabolic transformation to triadimenol involves the reduction of a carbonyl group to an alcohol, resulting in formation of a second chiral center3 (Figure 1). Thus, triadimenol consists of two diastereomers: A [enantiomers A1 (1R,2S) and A2 (1S,2R)] and B [enantiomers B1 (1R,2R) and B2 (1S,2S)], for a total of four stereoisomers. Diastereomers A and B of triadimenol, and the four stereoisomers that make up these diastereomers, are each produced from triadimefon in different relative amounts by plants and r 2011 American Chemical Society
fungi; the proportions of these stereoisomers may differ depending upon the species of fungi or plant.4,5 In addition, the relative sensitivity of various fungi to the stereoisomers of triadimenol is dependent on the fungal species.6 In most species of fungi, triadimenol is a more active fungicide than triadimefon. The enzymatic reduction of triadimefon may therefore be regarded as an activation process.7 Since triadimefon and triadimenol are both used as agricultural fungicides, there is concern regarding potential human exposure, as well as wildlife exposure, from their residues in the environment. The technical formulation of triadimefon (Bayleton, 92.6% triadimefon) has an acute oral LD50 in rats of 569 mg/kg and in rabbits and dogs of about 500 mg/kg. The LC50s in rainbow trout, bluegill sunfish, and goldfish are 14, 11, and 10 mg/L, respectively.8 The EPA classifies triadimenol as moderately toxic, similar in toxicity to triadimefon for rainbow trout and bluegill: LC50s are 19 and 15 mg/L, respectively.9 Received: October 12, 2010 Accepted: January 31, 2011 Revised: January 25, 2011 Published: February 22, 2011 2186
dx.doi.org/10.1021/es103430s | Environ. Sci. Technol. 2011, 45, 2186–2193
Environmental Science & Technology
ARTICLE
Figure 1. Metabolic transformation of triadimefon to triadimenol. The reduction of a carbonyl group to an alcohol yields a second chiral center and four stereoisomers.27
Detection of triadimefon in natural streams or sediments has not been reported, possibly because of its relatively fast biotransformation to triadimenol in soils.10 If it does reach water, however, triadimefon is very stable; in water with a pH of 3.0, 6.0, or 9.0, almost 95% of the compound remained after 28 weeks.8 In soil systems, triadimefon generally degrades relatively fast while triadimenol degrades slowly. In a silty clay loam soil, triadimefon had a DT50 (the time by which 50% of the pesticide has disappeared) of 15 days and yielded an almost equimolar concentration of triadimenol (about 38 μg/g) after about 30 days. This product was “rather stable” and showed no degradation after 50 days.11 In another study using a field lysimeter, triadimefon was applied to sandy soils (pH 6.7) at 11.05 kg/ha in September and October; triadimenol residues were still detectable in samples collected in early June the following year and could still control fungi.12 Triadimenol has been found in streamwater at 3 μg/L.13 In still other work, the DT50 of triadimefon was 18 days in a sandy loam soil, but 6 days in loamy soil, indicating that degradation varies with soil type. Other reported soil DT50s for triadimefon are 14-60 days with an average of 26 days.14 In addition to possible exposure of triazole fungicides and their metabolites to humans and wildlife through soil and water residues, the stereoselective transformation of these chemicals is also of concern. Nearly all of the conazole fungicides are chiral, which is an important feature in evaluating their environmental behavior and toxicity. It is well-known that many pesticides are chiral and can be metabolized enantioselectively by microbes in various environmental media,15-17 becoming depleted in one enantiomer while enriched in the other. In addition, their metabolites may be chiral, as is often the case with conazoles, and can themselves be metabolized enantioselectively. Established data show that a wide variety of chiral pesticide residues occur nonracemically.18-20
A frequently occurring complication with chiral pesticides, common to almost all conazoles and pyrethroids, for example, is the occurrence of more than one chiral center (see triadimenol, Figure 1); this results in four or more stereoisomers, each possibly having different biological properties. For example, it is known that triadimenol diastereomer A is 10 times more acutely toxic to rats (oral LD50) than is diastereomer B.21 This presence of multiple stereoisomers complicates chemical analysis, interpretation of fate and toxicity data, and risk assessment. Several reports have appeared in the literature over the past 5 years on the stereoselective biotransformation of pyrethroid pesticides, which have more than one chiral center. For example, Qin et al.22 reported observations of the enantioselective biodegradation of cis-bifenthrin, cis-permethrin, cypermethrin, and cyfluthrin in soil and sediment. This work was followed by a study of permethrin degradation in soil and sediment using 14C-labeled permethrin.23 A more recent study24 investigated the stereoselective biotransformation of permethrin to more estrogenic metabolites in fish. However, aside from pyrethroids, in-depth investigations in environmental matrices of the stereoselective biotransformation of pollutants with one chiral center to create metabolites with more than one such center are very rare.25 Thus, the stereoselective formation of triadimenol from triadimefon and the associated exposure of both chiral fungicides are important issues for both human health and ecological risk assessment. In this research, the biotic transformations of triadimefon to triadimenol in three different aerobic soils were followed over time to quantify degradation rates and product formation and determine enantiomeric fractions (EF) of parent triadimefon and relative abundances and EFs of the four triadimenol stereoisomeric products. It was observed that relative abundances of the triadimenol stereoisomers produced in soils do not match those of commercial triadimenol standards. Results of this novel research suggest that stereoselectivity in 2187
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Environmental Science & Technology transformations of conazole fungicides need to be considered in assessing their risk.
’ MATERIALS AND METHODS Chemicals and Soils. Triadimefon and triadimenol standards were obtained from the U.S. Environmental Protection Agency National Pesticide Standard Repository (Ft. Meade, MD) with purities of 99.4 and 96.4%, respectively. Solutions of each fungicide were analyzed by GC-MS using selective ion monitoring (SIM) (see GC-MS Analysis below) to check for purity, which nominally matched the given purity. Methanol, MTBE, and other organic solvents were of analytical grade from Fisher Chemicals (Fair Lawn, NJ). Reagent water for all experiments was produced by a Barnstead Nanopure Infinity water purification system (Thermo Scientific). The four triadimenol stereoisomers were separated by Regis Technologies, Inc. (Morton Grove, IL). In brief, the process involved preparative separation from racemic triadimenol using supercritical fluid chromatography (SFC) with a RegisPack (Regis Technologies) chiral preparative column followed by further separation into purer components by an AS-H chiral analytical HPLC column. The purity of each separated stereoisomer ranged from 98 to 100%. Similar methods were used by Chiral Technologies, Inc. (Exton, PA) to separate the two triadimefon enantiomers; i.e., separation by SFC using a ChiralpakAD-H (Chiral Technologies, Inc.) chiral preparative column to provide enantiomers of >99% purity. Soil sources and collection data are as follows: Soil 1: J. Phil Campbell Natural Resource Conservation Center, USDA, Watkinsville, GA. This was a composite agricultural soil collected from the A horizon of a cropped field. After collection, the soil was air-dried, sieved to
