Influence of pH on the Stereoselective Degradation of the Fungicides


(Agroscope), CH-8820 Wädenswil, Switzerland. Many pesticides are chiral and consist of two or more enantiomers/stereoisomers, which may differ in biol...
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Environ. Sci. Technol. 2006, 40, 5443-5450

Influence of pH on the Stereoselective Degradation of the Fungicides Epoxiconazole and Cyproconazole in Soils IGNAZ J. BUERGE,* THOMAS POIGER, MARKUS D. MU ¨ LLER, AND HANS-RUDOLF BUSER Plant Protection Chemistry, Swiss Federal Research Station (Agroscope), CH-8820 Wa¨denswil, Switzerland

Many pesticides are chiral and consist of two or more enantiomers/stereoisomers, which may differ in biological activity, toxicity, effects on nontarget organisms, and environmental fate. In the last few years, several racemic compounds have been substituted by enantiomer-enriched or single-isomer compounds (“chiral switch”). In this context, the stereoselective degradation in soils is an important part of a benefit-risk evaluation, but the understanding of the environmental factors affecting the chiral preferences is limited. In this study, the stereoselective degradation of the fungicides epoxiconazole and cyproconazole was investigated in different soils, selected to cover a wide range of soil properties. The fungicides were incubated under laboratory conditions and the degradation and configurational stability of the stereoisomers were followed over time using enantioselective GC-MS with a γ-cyclodextrin derivative as chiral selector. In alkaline and slightly acidic soils, the degradation of epoxiconazole was clearly enantioselective, whereas in more acidic soils, both enantiomers were degraded at similar rates (overall half-lives 78-184 d). The enantioselectivity, expressed as ES ) (ki - kj)/ (ki + kj), ranged from -0.4 in alkaline soils (faster degradation of enantiomer j) to ∼0 in acidic soils (non-enantioselective), and showed a reasonably linear correlation with the soil pH. The four stereoisomers of cyproconazole (overall halflives 5-223 d) were also degraded at different rates in the various soils, but only the stereoselectivities between epimers showed some correlations with pH, whereas enantioselectivities did not. Both fungicides were configurationally stable in soils, i.e., no enantiomerization or epimerization was observed. Correlations between pH and ES have previously been reported for other pesticides (metalaxyl, dichlorprop, mecoprop), but the presence or absence of such correlations is not obviously linked to the pathways of degradation. It can be assumed that different microorganisms and enzymes are involved in the primary degradation of these compounds, but on which level soil pH has an influence on ES remains to be investigated.

Introduction In the past few years, several racemic chiral pesticides have been replaced by enantiomer-enriched or single-isomer * Corresponding author phone: ++41 44 783 63 83; fax: ++41 44 780 63 41; e-mail: [email protected] 10.1021/es060817d CCC: $33.50 Published on Web 08/05/2006

 2006 American Chemical Society

compounds (1-4). Such a “chiral switch” may allow lower application rates at similar field performance, may result in lower residues in crops and in the environment, and may lead to less side effects on nontarget organisms (1, 5). For various pyrethroid and organophosphorous insecticides, phenoxycarboxylic acid and aryloxyphenoxypropionate herbicides, and triazole fungicides, the efficacy was found to differ significantly between enantiomers or stereoisomers (5, 6); e.g., the 1S2R-stereoisomer of the triazole fungicide triadimenol (structure, see Figure 1) was reported up to 1000fold more fungicidally active than the other three stereoisomers (7-9). Furthermore, for triadimenol stereoisomers, differences were observed with respect to metabolism in barley plants (10) and plant growth regulator activity (11, 12), which may or may not be an unwanted side effect for a fungicide. Triazole fungicides may not only show stereoselective efficacy, plant metabolism, or side effects, but also stereoselective biodegradation in the environment. In this study, two triazoles, epoxiconazole and cyproconazole, were selected to investigate possible stereoselectivity of their biodegradation in soils. The two compounds are widely used broadspectrum fungicides that inhibit the sterol 14R-demethylase, an enzyme involved in the biosynthesis of ergosterol (13). Epoxiconazole and cyproconazole are typically applied as foliar sprays to control diseases caused by ascomycetes, basidiomycetes, and deuteromycetes in cereals, sugar beets, peanut, oilseed rape, or coffee (2, 14). The chemical structures of the two fungicides are shown in Figure 1. Both compounds have two asymmetrically substituted carbon atoms and consist of four stereoisomers, two diastereomeric pairs of enantiomers. The common name epoxiconazole refers to the cis-stereoisomers (2), the major products from the synthesis process; the trans-stereoisomers are byproducts. In the following, for clarity we use the terms cis-epoxiconazole and trans-epoxiconazole, although the latter is not strictly correct. Cyproconazole is an approximate 1:1 mixture of both diastereomers (2). Enantio-/stereoselective degradation in soils has been observed for various chiral pesticides (15-26), but only a few studies tried to elucidate the environmental factors which affected the chiral preferences (20, 22, 25). The enantioselectivity of degradation of the fungicide metalaxyl was found to correlate with the soil pH (25). Furthermore, a reevaluation of published kinetic data for dissipation of the herbicides dichlorprop and mecoprop indicated similar correlations (25) raising the question whether the soil pH is a key parameter for the enantioselectivity of degradation of other chiral compounds. In the present study, the stereoselectivity of epoxiconazole and cyproconazole degradation was therefore investigated in soils with widely differing pH, even though most of these soils were not important for agricultural production. The stereoisomer composition of the residues of a chiral compound in soils is determined by the initial composition at the time of application and possible stereoselective biodegradation, but may also be altered by enantiomerization or isomerization. Information on the configurational stability becomes relevant when assessing the impact of the substitution of a racemic pesticide by an intrinsically more active stereoisomer. For example, isomerization in soil was observed for the triazole fungicide triadimenol (27). Therefore, the process of isomerization was also investigated in the present study for the triazoles epoxiconazole and cyproconazole. VOL. 40, NO. 17, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Structures of the stereoisomers of epoxiconazole and cyproconazole and of the internal standards used in the analytical procedure (propiconazole and triadimenol, respectively). The common name epoxiconazole refers to the cis-stereoisomers, which have 2S3R and 2R3S configuration.

Experimental Section Chemicals. Analytical standards of rac-cis-epoxiconazole ((2RS,3SR)-1-[3-(2-chlorophenyl)-2,3-epoxy-2-(4-fluorophenyl)propyl]-1H-1,2,4-triazole, purity, 99.0%) were obtained from Dr. Ehrenstorfer (Augsburg, Germany), rac-cyproconazole ((2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-cyclopropyl1-(1H-1,2,4-triazol-1-yl)butan-2-ol, 99.7%) and rac-propiconazole (cis-trans-1-[2-(2,4-dichlorophenyl)-4-propyl-1,3dioxolan-2-ylmethyl]-1H-1,2,4-triazole, 99.1%) were from Syngenta (Basel, Switzerland), and rac-triadimenol ((1RS,2RS; 1RS,2SR)-1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol, >99.9%) was from Bayer (Leverkusen, Germany). Soil Samples. Soil samples were collected on October 27 and 28, 2003 (Schachen, Wa¨denswil A, Winzlerboden, Table 1) and between July 28 and August 4, 2004 (Abist, Bra¨mabu ¨ el, Stillberg, Wa¨denswil B) in the Swiss Midland region and in the Swiss Alps (locations, see Figure 2 in ref 25). Three soils were from forests (soil units: cambisol, luvisol, and gleysol), two soils were from alpine grassland (podzol and ranker), and one soil was from farmland (cambisol). Geographical coordinates, altitude, soil unit, sampled horizon and depth, and some soil characteristics (texture, organic carbon, and pH) are listed in Tables 1 and 2. Standard equipment was used for sampling. The soils were kept in the dark at ∼4 °C until used within a few days. Soil Incubation under Aerobic Conditions. The soils were sieved (2 mm) and carefully air-dried at room temperature to obtain a water content of 16-38 g per 100 g dry soil, corresponding to ∼38-47% of the maximum water holding capacity (∼66-70% for soil Bra¨mabu ¨el due to the high organic carbon content). Incubation experiments were carried out in 300-mL Erlenmeyer flasks covered with air-permeable, sterile cellulose plugs. Portions of moist soil (corresponding to 100-150 g dry weight) were filled into the flasks and fortified by dropwise addition of ∼1.0-1.5 mL of an aqueous stock solution containing ∼10-15 µg of rac-cis-epoxiconazole or rac-cyproconazole. The spike level of ∼0.1 µg/g dry soil roughly corresponds to the recommended application rates of typically 125 g epoxiconazole/ha and 60-100 g cyproconazole/ha (2). The soils were carefully mixed and then incubated at ∼20-25 °C in the dark for up to 147 days. The water content of the soils was regularly checked by weighing 5444

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and kept constant by addition of distilled water. At appropriate time intervals, aliquots of 10 g soil were removed and transferred into glass vials for extraction and analysis (see below). Duplicate samples were taken immediately after fortification and mixing to determine the homogeneity of application, recovery, and reproducibility of extraction in the respective soils (see below). Semi-Preparative Isolation and Incubation of Cyproconazole Diastereomers. Separate incubations were carried out with the two diastereomers of cyproconazole in the soils Bra¨mabu ¨ el and Wa¨denswil at a spike level of ∼0.1 µg/g (Table 2). The individual diastereomers (each a racemic mix of the two enantiomers) were collected after non-enantioselective HPLC separation of the technical material on a Zorbax Rx-C18 column (25 cm × 4.6 mm i.d., 5 µm particle size, Agilent, Palo Alto, CA). The HPLC system consisted of a Jasco PU-980 pump, a Jasco AS-1555 autosampler, a Jasco MD-1510 detector (Tokyo, Japan), and a Gilson 202 fraction collector (Middleton, WI). The conditions were as follows: 10 injections of ∼230 µg in 100 µL; eluent water/acetonitrile/ methanol 55:35:10, isocratic; flow rate 1.5 mL/min; detection 240 nm. Fraction A (time of collection 11.25-12.00 min) and fraction B (12.75-13.75 min) were extracted three times with 5-mL portions of dichloromethane. The combined dichloromethane phases were evaporated at room temperature with a gentle draft of air and the residues were redissolved in 50 mL of distilled water (final concentrations 10.9 and 7.7 µg/mL, respectively). Extraction and Cleanup. Two other triazoles, propiconazole and triadimenol, were used as internal standards for epoxiconazole and cyproconazole, respectively. Propiconazole and triadimenol are chiral compounds as well due to two asymmetrically substituted carbon atoms (structures, see Figure 1). They consist of four stereoisomers as two diastereomeric pairs of enantiomers. The internal standards were selected because of their structural similarity to the corresponding analytes. The amount of ∼2 µg of internal standard in 100 µL of methanol was added to 10 g of soil prior to extraction. The soils were first extracted with 5 mL of phosphate buffer (0.1 M, pH 7). After vigorous shaking (∼1 min), 5 mL of acetone was added, again followed by 1 min of shaking. The samples were then centrifuged (2000g for 10-15 min)

and the water/acetone supernatants were transferred into glass vials. The extraction was repeated with 10 mL of methanol. The combined extracts (methanol/acetone/water) were partitioned three times with 5 mL of dichloromethane. The combined dichloromethane phases were evaporated to dryness and the residues were redissolved in ∼200 µL of ethyl acetate. For cleanup, the extracts were passed through silica mini columns (Pasteur pipet with ∼60 mm of silica gel 60, Merck, Darmstadt, Germany, dried at 140 °C during 24 h and then deactivated with 5% water, topped with ∼10 mm of sodium sulfate anhydrous). Epoxiconazole was eluted with 10 mL of ethyl acetate; cyproconazole was eluted with 10 mL of ethyl acetate/methanol 95:5. The eluates were carefully concentrated with a gentle flow of N2 and mild heating to final volumes of ∼1.5 mL ethyl acetate (∼200 µL for cyproconazole samples from incubation in soil Stillberg). Quantification by Non-Enantioselective GC-MS. Summed concentrations of epoxiconazole and cyproconazole stereoisomers, respectively, were determined by nonenantioselective gas chromatography-mass spectrometry (GC-MS) using a Finnigan Voyager quadrupole MS under electron impact ionization (EI, 70 eV, 200 °C) and selectedion-monitoring (SIM) conditions. The GC conditions were as follows: column DB-5 (30 m × 0.25 mm i.d., 0.1 µm film); injection split/splitless (250 °C, 48 s splitless); flow 1 mL/ min He; temperature program 70 °C, 2 min isothermal, 20 °C/min to 140 °C, 4 °C/min to 280 °C, 3 min isothermal hold at this temperature. The following ions were monitored: m/z 192.0 (and 194.0 for confirmatory purposes) for epoxiconazole, m/z 222.0 (139.0) for cyproconazole, m/z 259.0 for propiconazole, and m/z 168.1 for triadimenol. The amounts of epoxiconazole and cyproconazole were determined from peak area ratios relative to the internal standard (propiconazole and triadimenol, respectively; note that peak areas of both diastereomers were summed, see later) and in reference to suitable standard solutions. Recoveries of epoxiconazole, determined immediately after fortification, ranged from 81 to 146%. The high recoveries in some soils were likely due to incomplete extraction of the internal standard. Duplicate samples, however, differed by less than 6%. Therefore, no further efforts were made to improve the extraction procedure. Recoveries of cyproconazole were satisfactory (80-117%) and duplicates differed by less than 8%. Limits of detection (LOD, signal-to-noise ratio of 3) were ∼0.005-0.01 µg/g for epoxiconazole and cyproconazole. Epoxiconazole was not detected in soils fortified with cyproconazole and vice versa, i.e., concentrations in blank soil samples were