Article pubs.acs.org/est
Chiral Triazole Fungicide Difenoconazole: Absolute Stereochemistry, Stereoselective Bioactivity, Aquatic Toxicity, and Environmental Behavior in Vegetables and Soil Fengshou Dong,‡,† Jing Li,‡,† Bezhan Chankvetadze,§ Yongpu Cheng,‡,∥ Jun Xu,‡ Xingang Liu,‡ Yuanbo Li,‡ Xiu Chen,‡ Carlo Bertucci,⊥ Daniele Tedesco,⊥ Riccardo Zanasi,# and Yongquan Zheng‡,* ‡
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, P. R. China § Institute of Physical and Analytical Chemistry, Tbilisi State University, Chavchavadze Ave 3, 0179 Tbilisi, Georgia ∥ Tianjin Agricultural University, Tianjin, 300380, China ⊥ Department of Pharmacy and Biotechnology, University of Bologna, via Belmeloro 6, 40126 Bologna, Italy # Department of Chemistry and Biology, University of Salerno, via Ponte Don Melillo, 84084 Fisciano, Italy S Supporting Information *
ABSTRACT: In this study, the systemic assessments of the stereoisomers of triazole fungicide difenoconazole are reported for the first time, including absolute stereochemistry, stereoselective bioactivity toward pathogens (Alternaria sonali, Fulvia f ulva, Botrytis cinerea, and Rhizoctonia solani), and toxicity toward aquatic organisms (Scenedesmus obliquus, Daphnia magna, and Danio rerio). Moreover, the stereoselective degradation of difenoconazole in vegetables (cucumber, Cucumis sativus and tomato, Lycopersicon esculentum) under field conditions and in soil under laboratory-controlled conditions (aerobic and anaerobic) was investigated. There were 1.33−24.2-fold and 1.04−6.78-fold differences in bioactivity and toxicity, respectively. Investigations on the stereoselective degradation of difenoconazole in vegetables showed that the highest-toxic and lowest-bioactive (2S,4S)stereoisomer displays a different enrichment behavior in different plant species. Under aerobic or anaerobic conditions, (2R,4R)and (2R,4S)-difenoconazole were preferentially degraded in the soil. Moreover, difenoconazole was configurationally stable in the test soil matrices. On the basis of biological activity, ecotoxicity, and environmental behavior, it is likely that the use of pure (2R,4S)-difenoconazole instead of the commercial stereoisomer mix may help to increase the bioactivity and reduce environmental pollution.
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INTRODUCTION Difenoconazole (cis,trans-3-chloro-4-[4-methyl-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-2-yl]phenyl-4-chloro phenyl ether, part A of Figure 1), is a broad-spectrum 1,2,4-triazole fungicide used for the control of fungal diseases on fruits, vegetables, cereals, and other field crops.1,2 The compound was synthesized as a novel demethylation inhibitor (DMI) fungicide and was widely applied for its excellent fast-acting and prominent systemic activity. It has been reported that this fungicide was associated with an increase in the incidence of hepatocellular adenomas and carcinomas in the group of male and female mice following long-term dietary exposure.3 Moreover, difenoconazole was identified as the inhibitor of aromatase activity in human adrenocortical carcinoma cell line H295R.4,5 Compared with other triazole fungicides, difenoconazole is reported to possess relatively high acute toxicity to a wide range of aquatic organisms.3 With respect to its wide © 2013 American Chemical Society
application on crops (especially on rice), the contamination of surface aquatic ecosystems by difenoconazole is of a great environmental concern. However, because this fungicide is used worldwide as foliage-spray or seed-treatment to control vegetables (e.g., tomato and cucumber) diseases, it is also essential to understand the fate of difenoconazole in vegetables and soil for accurate assessment of its stress to food and environment. As difenoconazole possesses two chiral centers, it can exist in four stereoisomeric forms (part B of Figure 1). Because of their different molecular configurations, stereoisomers of chiral pesticides may differ in their binding to structure-sensitive Received: Revised: Accepted: Published: 3386
December 5, 2012 February 27, 2013 March 1, 2013 March 1, 2013 dx.doi.org/10.1021/es304982m | Environ. Sci. Technol. 2013, 47, 3386−3394
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Figure 1. (A) Chemical structure and atom numbering of difenoconazole, (B) ,tructures of the four stereoisomers of difenoconazole. (C) enantioselective HPLC analysis on the stereoisomer mix of difenoconazole (Chiralcel OJ, column mobile phase: n-hexane/ethanol 90:10 (v/v), 0.8 mL/min, 20 °C; DAD detection at 220 nm).
obliquus (Chlorophyceae), water fleas Daphnia magna (Branchiopoda), and tropical fish Danio rerio (Actinopterygii) were determined for the parent pesticide and for the single stereoisomers of difenoconazole. Stereoselectivity was further evaluated during biodegradation in vegetables (cucumber, Cucumis sativus and tomato, Lycopersicon esculentum) and soil under greenhouse and laboratory conditions. Meanwhile, the stabilities of the four individual pure stereoisomers of difenoconazole in soil were also assessed. The results of this study will be relevant for the assessment of a possible substitution of the commercial stereoisomer mix with stereoisomerically enriched difenoconazole, which may be more effective and less toxic toward nontarget organisms.
biological receptors and naturally occurring chiral biomolecules. Apart from different biological activities to target organisms, lots of chiral pesticides have stereoselective toxicity, such as cytotoxicity, endocrine disruption, and carcinogenesis, to nontarget biota.6−8 The processes of absorption, distribution, and degradation in organisms and the environment are often stereoselective.9−13 Stereoselectivity in these processes may result in ecotoxicological effects that cannot be predicted from our existing knowledge. The stereospecific toxicities and degradation rates of chiral pesticides indicate that the traditional practice of treating them as single compounds in risk assessment may well be inappropriate. Therefore, the role of stereoselectivity in environmental safety of current chiral pesticides should be taken into account in risk assessment and regulatory decisions. However, until now, stereoselectivity in biological activity, ecotoxicological effects, and environmental fate of difenoconazole is still unknown. Thus, it is essential to get stereospecific information of the chiral difenoconazole to make a more accurate benefit-risk evaluation. The presence of multiple stereoisomers complicates chemical analysis, interpretation of activity, toxicity, degradation, and risk assessment. In this study, the four stereoisomers of difenoconazole were separated and collected, and their stereochemistry was fully characterized in order to assign the correct absolute configuration to each stereoisomer. Subsequently, the bioactivity toward pathogens (Alternaria sonali, Fulvia f ulva, Botrytis cinerea, and Rhizoctonia solani), and the acute toxicity toward freshwater green algae Scenedesmus
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MATERIALS AND METHODS Chromatographic Separation and Preparation. The stereoisomers of difenoconazole were resolved by highperformance liquid chromatography (HPLC), which was carried out on an Agilent 1100 series HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with a G1315B diode array detector (DAD). The signal was accumulated and processed using the Agilent ChemStation software. Baseline resolution of difenoconazole stereoisomers was achieved on a Chiralcel OJ-H column (250 mm × 4.6 mm i.d., Daicel Chemical Industries, Tokyo, Japan) using a mixture of nhexane/ethanol 90:10 (v/v) as the mobile phase with a flow rate of 0.8 mL/min. The column temperature was maintained at 20 °C and the UV wavelength was set at 220 nm. In the 3387
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test was conducted according to the Standard Protocol.15 Seven concentrations (0.03−2 μg/mL) and two controls (spiked with water and acetone, respectively) were tested in five replicates for each sample of difenoconazole (the stereoisomer mix and the four pure stereoisomers), for a total of 45 tests (each contains five active D. magna). The test animals were not further fed and were incubated at 20 ± 2 °C for 48 h. The LC50 values were determined from the survival data using a probit equation with SPSS 16.0. Tests were considered to be valid if control mortality was (2R,4R) > (2R,4S). To investigate the stereoselective toxicity toward other nontarget organisms, the acute aquatic toxicity for the stereoisomer mix and individual stereoisomers to D. magna and Danio rerio were also measured through 48 and 96 h tests, respectively. Statistic results indicated significant differences in the LC50 values for the stereoisomers of difenoconazole (Table 2), the order of toxicity toward both D. magna and Danio rerio is the same as that toward S. obliquus: (2S,4S)-difenoconazole is the most toxic isomer indicating that the stereoisomers interact with all three aquatic species in a similar manner. Furthermore, LC50 values toward D. magna were lower than those toward Danio rerio and S. obliquus suggesting that D. magna is the most sensitive aquatic species in the three test groups. As mentioned above, the fungicidal bioactivity for the stereoisomers of difenoconazole to the pathogens has been found to be (2R,4S) > (2R,4R) > (2S,4R) > (2S,4S). It is interesting to note that the most active stereoisomer, (2R,4S)difenoconazole, was the least toxic to aquatic species, while the highest toxic isomer, (2S,4S)-difenoconazole, shows the lowest bioactivity. On the other hand, (2S,4S)-difenoconazole was 2.1−6.8 times more toxic than (2R,4S)-difenoconazole to all three aquatic species (Table 2). Although the reasons for this reversal of toxicity and activity among the stereoisomers are not clear, this phenomenon often results from a combination of several factors. First, different species of biological enzymes may have different sensitivities to stereoisomers of chiral compounds. Second, lots of biological activities, especially metabolism, transformation, and accumulation that affect the toxicity in vivo, have been found to be stereoselective.26 Moreover, the in vivo metabolism such as oxidation by microsome may change the toxicity of the compound and the process also can exhibit significant stereoselectivity. Therefore, a comparison between stereoselectivity in fungicidal activity and that in aquatic toxicity reveals that the most ecotoxic stereoisomers are the least fungicidally active. This observation may be of great significance, as stereoselectivity in fungicidal activity may not be used to predict stereoselectivity in aquatic toxicity. The disagreement in stereoselectivity suggests that a common mode of action may not be shared between target species and nontarget organisms. Stereoselective Degradation in Vegetables. Under field conditions, the concentrations of difenoconazole stereoisomers in cucumber and tomato fruits were highest at day 0 (2 h) after the foliar spray treatment (Figure S7 of the Supporting Information), and then decreased gradually with time. The corresponding degradation kinetics of stereoisomers is shown in Table 3. The degradation of four stereoisomers of difenoconazole in tomato and cucumber fruits followed firstorder kinetics (R2 = 0.9453−0.9939), and different half-lives among of stereoisomers were found. (2R,4S)- and (2R,4R)-difenoconazole degraded faster than their antipode in tomato, resulting in tomato enriched with stereoisomers of (2S,4R)- and (2S,4S)-difenoconazole (Figure S7 of the Supporting Information). The EF values for the different diastereomers (EFA and EFB) in tomato were nearly 0.5 in the first day after application, then EFA decreased gradually in the following days from 0.499 ± 0.019 (n = 3) to 0.437 ± 0.009 (n = 3) after 14 days and increased to 0.469 ± 0.018 (n = 3) after 28 days (part A of Figure 2). In contrast, the EFB increased gradually with time, rising from 0.500 ± 0.011 (n
isomer: the third- and fourth-eluted fractions have a good correlation with the calculated properties of (2S,4R)- and (2S,4S)-difenoconazole, respectively. The elution order of the stereoisomers of difenoconazole on the Chiralcel OJ-H column under the optimized conditions can therefore be assessed: (2R,4R), (2R,4S), (2S,4R), (2S,4S) (part C of Figure 1). Full details and results of the experimental and computational procedures for the stereochemical characterization of difenoconazole are reported in Tables S1−S10 and Figures S1−S6 of the Supporting Information. Biotests with Difenoconazole Mix and Pure Stereoisomers. To characterize the isolated stereoisomers of difenoconazole regarding their fungicidal activity, several small-scale biotests with 4 pathogens were performed. The EC50 values of four stereoisomers are shown in Table 1. For all the test pathogens, the order of the bioactivity of the difenoconazole stereoisomers was found to be: (2R,4S) > (2R,4R) > (2S,4R) > (2S,4S). The most remarkable result is the great difference in the biological activities of the stereoisomers against Botrytis cinerea, the activity of the (2R,4S)-isomer being about 24.2 times higher than that of the (2S,4S)-isomer. In short, the (2R,4S)-isomer was very clearly the most effective, whereas the (2S,4S)-isomer showed the lowest activity; the (2R,4S)-isomer was about 4.9−24.2 times more active than (2S,4S)-isomer in the biotest. In view of many triazole fungicides, it is not surprising that chirality in these fungicides can have a decisive influence on the nature and degree of biological activity. For example, the R-enantiomer of diniconazole and uniconazole shows stronger fungicidal activity than the S-enantiomer, whereas the latter has higher plant growth regulating activity. 24 The (1S,2R)-stereoisomer of triadimenol shows the highest fungitoxicity among the four stereoisomers (up to 1000-fold more active than the other three) and the activities of four stereoisomers of paclobutrazol also differ greatly. 25 The results of the biotest are in good agreement with the previously established data for this structurally related class of fungicides. Stereoisomer Selectivity in Acute Aquatic Toxicity. The acute toxicity of the stereoisomer mix of difenoconazole and of its four stereoisomers to S. obliquus was evaluated by using EC50 values as a marker. Significant differences were observed in 96 h EC 50 among the stereoisomers of difenoconazole (Table 2). When comparing EC50 values between the enantiomers of a pair, (2S,4R)-difenoconazole is about 1.9-fold more potent than the (2R,4S)-isomer, whereas the (2S,4S)-isomer is about 1.3-fold more toxic than the (2R,4R)-isomer. Difenoconazole mix had an intermediate potency of toxicity to S. obliquus as compared to its respective Table 2. Acute Aquatic Toxicity Data (EC50 and LC50, μg/ mL) towards S. obliquus, D. magna, and Danio rerio for the Stereoisomer Mix of Difenoconazole and Its Stereoisomers difenoconazole sample
mix (2R,4R) (2S,4S) (2S,4R) (2R,4S)
species S. obliquus (96 h) 1.338 ± 0.02 1.587 ± 0.07 1.196 ± 0.11 1.323 ± 0.02 2.476 ± 0.04
D. magna (48 h) 0.298 ± 0.12 0.253 ± 0.03 0.106 ± 0.02 0.243 ± 0.02 0.719 ± 0.04
Danio rerio (96 h) 1.329 ± 0.02 1.406 ± 0.04 0.616 ± 0.01 1.120 ± 0.03 1.641 ± 0.03 3390
dx.doi.org/10.1021/es304982m | Environ. Sci. Technol. 2013, 47, 3386−3394
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Table 3. First-Order Kinetic Constants (k), Half-Lives (t1/2), Correlation Coefficients (R2), and Final EF Values for the Degradation of Difenoconazole in Tomato and Cucumber under Field Conditions plant tomato
cucumber
stereoisomer (2R,4R) (2S,4S) (2R,4S) (2S,4R) (2R,4R) (2S,4S) (2R,4S) (2S,4R)
k × 102 (d−1) 7.87 7.00 6.51 6.29 53.66 56.60 50.12 47.47
t1/2 (d) a
8.81 9.36a 10.65 11.02 1.29 1.22 1.39 1.46
R2
EFb
0.9453 0.9457 0.9491 0.9502 0.9615 0.9707 0.9939 0.9864
0.469 0.505 0.628 0.439
a
Significantly different from each other, P < 0.05 (Student’s paired ttest). bEF values of each enantiomeric pair at the end of incubation (28 d for tomato, 6 d for cucumber).
Figure 3. Typical HPLC chromatograms of (A) extract from tomato fruits at 14 days after treatment with difenoconazole mix, (B) extract from cucumber fruits at 2 days after treatment with difenoconazole mix.
and cucumber plants. Some pioneer research studies reported that enzymatic systems play an important role in the stereoselective degradation and metabolism of many chiral pesticides in plants.27 For instance, (S)-tebuconazole was preferentially degraded in Chinese cabbage, whereas (R)tebuconazole was preferentially degraded in cucumber. 28 Therefore, (2S,4S)- and (2R,4R)-difenoconazole may be differently restrained by enzymatic systems in tomatoes and cucumbers, many plant medium should be investigated to clarify the stereoselectivity of difenoconazole for developing the stereoisomer-enriched commercial products, especially considering for its application effect and safety, the persistence of (2R,4S)-difenoconazole with highest bioactivity and the fate of (2S,4S)-difenoconazole with highest ecotoxicity in vegetables should be specially focused. Stereoselective Degradation in Soil under Aerobic and Anaerobic Conditions. Difenoconazole was found to be persistent in the test soil matrices under both aerobic and anaerobic conditions (Figure S8 of the Supporting Information). Aerobic degradation of the stereoisomers of difenoconazole generally complied with first-order kinetics, with R2 values ranging from 0.95 to 0.98 (Table 4). The calculated t1/2 values of stereoisomers ranged from 169.0 to 238.9 days (Table 4). During incubation, the concentrations of stereoisomers decreased by ≥32% (Figure S8 of the Supporting Information). It was observed that (2R,4R)- and (2R,4S)-difenoconazole degraded faster than their antipodes. The half-lives of degradation were significantly different between enantiomers (P < 0.05, Student’s paired t-test): 169.0 and 238.9 days for (2R,4R)- and (2S,4S)-difenoconazole, respectively; 223.5 and 173.2 days for (2S,4R)- and (2R,4S)-difenoconazole, respectively. At the same time, EFA and EFB values changed gradually, from 0.497 ± 0.011 (n = 3) to 0.455 ± 0.012 (n = 3) and from
Figure 2. Time development of EFA and EFB values for the diastereomers of difenoconazole in tomato (A) and in cucumber (B).
= 3) to the 0.532 ± 0.012 (n = 3) after 21 days then decreasing to 0.504 ± 0.013 n = 3) after 28 days. Preferential dissipation of (2R,4S)- and (2S,4S)-difenoconazole was observed in cucumber fruits after foliar treatment (Figure S7 in the Supporting Information). Both EFA and EFB value in cucumber were nearly 0.5 at first day after treatment, and then EFA value increased sharply with time, and rose to 0.629 ± 0.016 (n = 3) at 6 days after treatment (part B of Figure 2). Whereas, for EFB, it increased gradually and up to 0.560 ± 0.016 (n = 3) at the fifth day. (2R,4R)- and (2S,4S)difenoconazole were preferentially metabolized by cucumber and could not be detected after 6 days. Meanwhile, the EF changes of difenoconazole in vegetables were all significantly different (P < 0.05, Stendent’s paired test). The typical chromatograms of difenoconazole stereoisomers in tomato and cucumber after treatment are shown in Figure 3. Interestingly, a reversed enantioselectivity direction for (2S,4S)- and (2R,4R)-difenoconazole occurred between tomato 3391
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matrices.29−31 Thus, additional HPLC analyses were performed for an autoclaved aerobic soil sample to determine if the dissipation of difenoconazole was microbially mediated. Difenoconazole loss was minimal (
