CHIRALITY 26:784–789 (2014)
Review Article Acute Toxicity, Bioactivity, and Enantioselective Behavior with Tissue Distribution in Rabbits of Myclobutanil Enantiomers MINGJING SUN, DONGHUI LIU, XINXU QIU, QIAN ZHOU, ZHIGANG SHEN, PENG WANG, AND ZHIQIANG ZHOU* Department of Applied Chemistry, China Agricultural University, Beijing, P.R. China
ABSTRACT The enantioselective bioactivity against pathogens (Cercospora arachidicola, Fulvia fulva, and Phytophthora infestans) and acute toxicity to Daphnia magna of the fungicide myclobutanil enantiomers were studied. The (+)-enantiomer in an antimicrobial activity test was about 1.79–1.96 times more active than the (–)-enantiomer. In the toxicity assay, the calculated 24-h LC50 values of the (–)-form, rac-form and (+)-form were 16.88, 13.17, and 11.91 mg/ L, and the 48-h LC50 values were 10.15, 9.24, and 5.48 mg/L, respectively, showing that (+)-myclobutanil was more toxic. Meanwhile, the enantioselective metabolism of myclobutanil enantiomers following a single intravenous (i.v.) administration was investigated in rabbits. Total plasma clearance value (CL) of the (+)-enantiomer was 1.68-fold higher than its antipode. Significant differences in pharmacokinetics parameters between the two enantiomers indicated that the high bioactive (+)-enantiomer was preferentially metabolized and eliminated in plasma. Consistent consequences were found in the tissues (liver, brain, heart, kidney, fat, and muscle), resulting in a relative enrichment of the low-activity (–)-myclobutanil. These systemic assessments of the stereoisomers of myclobutanil cannot be used only to investigate environmental and biological behavior, but also have human health implications because of the long persistence of triazole fungicide and enantiomeric enrichment in mammals and humans. Chirality 26:784–789, 2014. © 2014 Wiley Periodicals, Inc KEY WORDS: myclobutanil enantiomer; degradation; rabbit INTRODUCTION
In recent years, increasing interest of the scientific community has focused on an emerging class of environmental pollutants, i.e., triazoles as well as the structurally related imidazole fungicides that are used as clinical drugs and pesticides.1,2 As the largest class of synthetic antimycotics, triazole antifungals have been detected worldwide in the environment,3 causing ecological concerns due to their persistence and endocrine-disrupting potency.4–6 Many triazoles are chiral and their enantiomers may have different properties and effectiveness. Chemically, chiral enantiomers are very similar, having the same boiling points, melting points, and typically the same solubility, reactivity, and other chemical properties.7 Nevertheless, the stereospecificity of chiral pesticides may be evident in the activity at the desired biological target and/or at undesirable targets, which results in adverse effects as one form is active against the organisms, and the other form is inactive.8–10 Apart from different biological activities to target biological objects, chiral pesticides usually have enantioselective toxicity, and the processes of absorption, distribution, and degradation in organisms and the environment are often enantioselective.11–13 Myclobutanil is a systemic fungicide belonging to the triazole family of chemicals. It displays an outstanding curative and protective efficacy against powdery mildew of cereal and vegetables.14,15 It is a moderately toxic compound in toxicity class II, and has been found to affect the reproductive abilities of test animals and cause varying degrees of hepatic toxicity and to disrupt steroid hormone homeostasis in © 2014 Wiley Periodicals, Inc.
bioactivity;
acute
toxicity;
enantioselective
rodent.16–19 This compound, which has long persistence in the environment, can be accumulated along the food chain and may therefore represent a risk for human health. Myclobutanil has an asymmetrically substituted C atom and consists of a pair of enantiomers. Previous studies showed that the enantiomers of myclobutanil always have different biological and physiological properties in asymmetry systems.20–22 Nonetheless, as with many chiral pesticides, myclobutanil is generally marketed and applied as the racemate, and owing to the current lack of knowledge concerning the individual bioactivities, toxicities, and degradation of myclobutanil enantiomers, the possible risks about this chiral fungicide are still not clear. So it is of great urgency and necessity to investigate the different effect and behavior of the two isomers to improve their effectiveness, decrease the pesticide residue, protect the environment, and evaluate the risks posed to public health. Daphnia magna, a key food source in the food chain of aquatic ecosystems, is a standard organism frequently used Contract grant sponsor: A Foundation for the Author of National Excellent Doctoral Dissertation of PR China, Program for New Century Excellent Talents in University; Contract grant number: NCET09-0738. Contract grant sponsor: National Natural Science Foundation of China; Contract grant number: 21277171, 21307155. *Correspondence to: Zhiqiang Zhou, Department of Applied Chemistry, China Agricultural University, Yuanmingyuan west road 2, Beijing 100193, P.R. China. E-mail: [email protected] Received for publication 28 February 2014; Accepted 28 May 2014 DOI: 10.1002/chir.22353 Published online 21 July 2014 in Wiley Online Library (wileyonlinelibrary.com).
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ENANTIOSELECTIVE BEHAVIOR OF MYCLOBUTANIL
in ecotoxicity tests, due to its sensitivity to changes of the chemical composition in aquatic environments.23 Recent researches showed that the acute toxicity of the chiral pesticide enantiomers in acute toxicity was enantioselectivity.12 In this study the acute toxicity to Daphnia magna for myclobutanil enantiomers was determined. The inhibitory activities of myclobutanil against Cercospora arachidicola, Fulvia fulva, and Phytophthora infestans were also evaluated using the inhibition zone method. Furthermore, we detected concentrations of myclobutanil enantiomers in tissues and plasma of rabbits after intravenous administration. Through comparing the toxicity, activity, and biological fate in animals, we can provide some suggestions for using myclobutanil enantiomers, and reducing ecological risks caused by chiral pesticides in the environment. MATERIALS AND METHODS Chemicals and Reagents Myclobutanil (96.5%) was obtained from the Institute for Control of Agrichemicals Ministry of Agriculture. The two enantiomers of myclobutanil were prepared by high-performance liquid chromatography (HPLC) with purities >98.0%. The solvents n-hexane, 2-propanol, and acetonitrile, used for mobile phase preparation, were all of HPLC-grade from Fisher Scientific (Fair Lawn, NJ). All other chemicals and solvents were of analytical grade (Beijing Yili Fine Chemicals, China)
Instrumentation and Chromatographic Conditions The HPLC system used involved an Agilent (Palo Alto, CA) 1200 series HPLC equipped with a G1322A degasser, a G1311A quat pump, a G1329A automatic liquid sampler, a G1314B variable wavelength ultraviolet detector, and Agilent 1200 Chemstation software. The column temperature was controlled by an AT-930 heater and cooler column attemperator (Tianjin Automatic Science Instrument, China). Chromatographic separations were performed at room temperature on the Chiralpak AD (amylose tris-(3, 5-dimethylphenylcarbamate)) chiral column (250 × 4.6 mm, 10 μm particle size). The best resolution was achieved using n-hexane: 2-propanol (85:15, v/v) as the mobile phase, at a flow rate of 1.0 mL/min, with 225 nm monitoring wavelength at 20 °C. The elution order of myclobutanil enantiomers was measured by a CHIRALYSER–MP optical rotation detector produced by IBZ MESSTECHNIK (Germany). The (–)-enantiomer of myclobutanil was first eluted from the AD column.
Antimicrobial Activity Test The fungicidal activities against Cercospora arachidicola, Fulvia fulva, and Phytophthora infestans were tested according to the reported inhibition zone method.24 In this method, potato dextrose ager (PDA) was decanted into Petri dishes with actively growing mycelium of pathogenic bacteria when it was cold enough but still liquid. Filter paper disks of 6 mm diameter were sterilized by autoclaving for 15 min at 120 °C and impregnated with 10 μL of stock solutions of myclobutanil enantiomers at different concentrations (ranging from 50 to 800 mg/L). After solidification of the agar, the filter disk was placed onto the middle surface of the PDA, incubated at 25 ± 1 °C, and a light-dark cycle of 12/12 h darklight. Two replicates were done for each concentration, and a blank control was also performed using the solvent without compounds. The antimicrobial activity of each extract was measured as a zone of inhibition of the bacterial growth around the disk. The inhibition percent was used to describe the control efficiency of the compounds. Inhibition percent (%) = inhibition zone diameter in the treatment/hyphal diameter in the control.25
Acute Toxicity Assays Enantioselectivity in acute aquatic toxicity was evaluated via 24-h and 48-h toxicity assays using D. magna as the model species, originally obtained from CABET (Center for Agrochemical Biological and Environmental Technology Institute, Beijing, China). The testing procedures followed the OECD guideline “Acute Immobilization Test.”26 The test
animals used in these experiments were juveniles aged between 6 and 24 h. Prior to the test, a sensitive test for Daphnia to potassium dichromate was performed as a positive control. The D. magna were exposed to a series of test solutions in 50-mL beakers containing rac-, (+), and (–)-myclobutanil separately within a given concentration range (1.25–80 mg/L). Each beaker contained ten D. magnas. Three replicates for each treatment were conducted. The test animals were not further fed and were incubated at 20 ± 1 °C under a light-dark cycle of 16/8 h dark/light. The concentration of enantiomer or racemate that caused 50% mortality of the test population (LC50) was determined using a probit equation with SPSS 16.0 (Chicago, IL).
Animal Treatment Male Japanese white rabbits weighing 2.0 ± 0.25 kg (provided by the Experimental Animal Research Institute of China Agriculture University) were housed under a 12-h light/dark cycle at 20 °C. Racemic myclobutanil was dissolved in alcohol and then diluted to final concentrations with normal saline, and then administered at 30 mg/kg body weight by intravenous injection in the ear vein. Blood from the heart arteries was collected into polypropylene tubes with heparin sodium at 1, 15, 30, 60, 90, 120, 180, and 240 min after treatment. The animals were sacrificed at 5, 60, 120, 180, and 300 min after treatment, and the heart, kidney, liver, lung, fat, muscle, and brain were excised and weighed separately. Plasma and tissue samples were stored at –20 °C for later analysis.
Pretreatment of Plasma and Tissues Plasma sample (1 mL) or homogenized tissue (1 g) was transferred to 15 mL plastic centrifuge tubes, with 5 mL of ethyl ether added, stirred for 2 min on a vortex mixer, and centrifuged at 4000 rpm for 5 min. The extraction was repeated with another 5 mL of ethyl acetate. The organic phase was combined and evaporated to dryness under a stream of nitrogen. The residue was dissolved in 4 mL of acetonitrile, and then partitioned twice with 3 mL of n-hexane, and the acetonitrile phase was evaporated to dryness. All the resulting residue was redissolved in 0.2 mL of 2-propanol, filtered (0.45 mm pore size), and injected into the HPLC for analysis.
Pharmacokinetics Analysis The pharmacokinetic parameters were determined with the Drug and Statistics Software (Section of Quantitative Pharmacology, Chinese Pharmacological Society). An open two-compartment model was used to describe the metabolism of both enantiomers after i.v. administration in plasma. Concentration in the central compartment (C) after a single intravenous dose was expressed as: C ¼ Ae-αt þ Be-βt where t was time, A and B were D(α- k21)/[V1(α-β)] and D(k21 -β))/ [V1(α-β)], respectively, D was dose, V1 was the apparent volume of the central compartment, and k21 was the apparent first-order distribution rate from the peripheral to the central compartment; α and β denote the slopes of α phase and β phase, respectively. The area under the concentration–time cure (AUC) was calculated by the trapezoidal rule. Mean residence time (MRT) was calculated by dividing the area under first-moment curve AUMC by AUC. The distribution and elimination phase half-life (t1/2α, t1/2β) were calculated by 0.693/α and 0.693/β. The total apparent volumes of distribution (Vd) were calculated by D/(βAUC0-∞), and the total body clearance (CL) was calculated by D/AUC0-∞.
RESULTS AND DISCUSSION Enantioselection in Bioactivity
The antimicrobial activity of myclobutanil isomers in the inhibition zone test was expressed by EC50 (the concentration inhibiting mycelial growth by 50%) values after 3 d treatment and the results of the bioactivity of the enantiomers to Cercospora arachidicola, Fulvia fulva, and Phytophthora infestans are shown in Table 1. For all the test pathogens, the bioactivity of both stereoisomers was proportional to their Chirality DOI 10.1002/chir
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TABLE 1. Inhibition of myclobutanil isomers in three fungi Inhibition rate at the different concentrations (%) Fungi Cercospora arachidicola Fulvia fulva Phytophthora infestans (tomato)
Myclobutanil
800 mg/L
400 mg/L
200 mg/L
100 mg/L
50 mg/L
R2
EC50 (mg/L)
(-)-isomer (+)-isomer (-)-isomer (+)-isomer (-)-isomer (+)-isomer
37.78 48.89 36.67 38.89 37.78 43.33
32.22 41.11 31.11 35.56 33.33 37.78
28.89 34.44 27.78 31.11 28.89 32.22
22.22 24.44 21.11 24.44 22.22 25.56
____ ____ 20.00 20.00 18.89 21.11
0.977 0.984 0.970 0.979 0.989 0.996
1146.99 628.17 4569.69 2550.16 2992.50 1523.62
concentration, and the (+)-enantiomer was about 1.79–1.96 times more active than the (–)-enantiomer. The similar result was also reported by Deng’s group found the antifungicidal activity of (+)-myclobutanil to be obviously superior to (–)-myclobutanil by using a growth rate method. 20 Chirality has a considerable influence on the biological activity of triazole fungicides. For instance, the R-enantiomers of diniconazole and uniconazole showed stronger fungicidal activity than the S-enantiomers, whereas the S-enantiomers showed higher plant growth regulating activity.27 The isomers of tebuconazole, flutriofol, and hexaconazole also showed different microbial activity.28 One possible reason for the stereoselectivity of triazole fungicides is that one of the enantiomers better fits the active site of cytochrome P 450.29 Enantioselective in Acute Toxicity
The acute toxicity of myclobutanil and its individual enantiomers to D. magna was expressed by LC50 values at 24 and 48 h and the data are presented in Table 2. According to the "Environmental Safety Evaluation Test Guidelines of Chemical Pesticides," rac-myclobutanil and its individual enantiomers were all moderately toxic to D. magna,30 in which the (+)-enantiomer was more toxic than the racemate and (–)-enantiomer. After longer exposure, differences in toxicity between enantiomers became larger. The
calculated 48-h LC50 of (+)-myclobutanil was nearly twice that for the (–)-enantiomer. The results showed a general agreement in the enantioselectivity between toxicity to D. magna and antimicrobial activity, that is, the active (+)-enantiomer was also the more ecotoxic one. It can be assumed that there might be a common mode of action between some of the target fungi and aquatic invertebrates. Assay Validation
Linear calibration curves were obtained for the (–)-isomer (y = 25.94x + 98.07, R2 = 0.973) and (+)-isomer (y = 25.37x + 87.81, R2 = 0.980) over the concentration range of 0.5–50 μg/mL in drug-free plasma. The recoveries of each enantiomer of myclobutanil were determined by analyzing quality control samples at three spiked concentrations (0.5, 5, 10 μg/mL in plasma and 1, 50, 250 μg/g in tissues). and the lowest recovery was over 80%. The limit of quantification was 0.2 μg/mL in plasma and 0.2 μg/g in tissue samples. Pharmacokinetics
Concentration–time curves of (–)- and (+)-myclobutanil in plasma after i.v. administration of 30 mg/kg b.w. of racemate to the rabbits are shown in Fig. 1A. The enantiomer fraction (EF = [(–)]/([(+)] + [(–)]) was used as a measure of the
TABLE 2. Calculated LC50 values for myclobutanil (–)-isomer Time (hours)
1
LC50(mg/L)
R2
16.88 10.15
0.954 0.991
24 48
rac-compound P
2
0.004 0.005
(+)-isomer
LC50(mg/L)
R2
P
LC50(mg/L)
R2
P
13.17 9.24
0.964 0.989
0.003 0.056
11.91 5.48
0.993 0.969
0.004 0.016
1
Correlation coefficient. Probability (associated with the t-test). A P value < 0.05 indicates that the correlation of the linear equation is significant, and it is calculated using the linear regression model of SPSS 16.0. 2
Fig. 1. Plasma concentration–time curves (A) and EF (B) of myclobutanil enantiomers in rabbits following racemic administration at 30 mg/kg bd wt. Chirality DOI 10.1002/chir
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TABLE 3. Pharmacokinetic parameters of myclobutanil enantiomers following i.v. administration at 30 mg/kg Pharmacokinetic parameters t1/2α (min) t1/2β (min) V1 (L/kg) CL (L/min/kg) AUC0-240 (mg/L · min) AUC0-∞ (mg/L · min) MRT0-240 (min) MRT0-∞ (min)
(–)-myclobutanil
(+)-myclobutanil
8.353 69.315 1.535 0.019 701.948 798.299 75.987 96.857
5.721 63.312 1.516 0.032 436.363 467.531 62.04 68.628
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enantioselectivity of the two enantiomers (Fig. 1B). The concentration of the (+)-enantiomer in plasma was lower than its antipode, causing the enrichment factor (EF) value to increase with time. Based on the statistical analysis of a t-test, the enantioselective is significant (P < 0.05). Compartmental pharmacokinetic analysis showed some statistical differences between the enantiomers in the principal pharmacokinetic parameters (Table 3). The (–)/(+) ratio of the distribution half-life (t1/2α) was 1.46, whereas the (–)/(+) ratio of the elimination half-life (t1/2β) was 1.09. The result indicated that (+)-myclobutanil distributed faster than its antipode in rabbit. The chromatogram of the preferential disappearance of the (+)-enantiomer in plasma is shown in Fig. 2C. The total plasma clearance (CL) of (+)-isomer was more than 1.68-fold higher than that of the (–)-isomer. The
Fig. 2. Representative HPLC chromatogram of (A)100 mg/L myclobutanil (B) drug-free rabbit plasma extract; (C) plasma sample extract (30 min); (D) liver sample extract (60 min); (E) lung sample extract (60 min); (F) brain sample extract (60 min); (G) heart sample extract (60 min); (H) kidney sample extract (60 min); (I) fat sample extract (120 min); (J) muscle sample extract (120 min); (n-hexane/isopropanol = 85/15, flow rate: 1 ml/min, detect wave: 220 nm). Chirality DOI 10.1002/chir
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(–)/(+)-myclobutanil ratios of the AUC0-240 min and AUC0-∞ were 1.51 and 1.71, the ratios of the MRT0-240 and MRT0-∞ were 1.22 and 1.41, respectively. The results suggested that the pharmacokinetics of myclobutanil enantiomers were enantioselective in rabbits. One possible explanation for the significant differences in the pharmacokinetics was the stereoselectivity of plasma protein binding and membrane permeation of myclobutanil. Chiral conversion of myclobutanil in plasma might also contribute to these differences. Distribution in Tissues
Typical chromatograms for (–)- and (+)-myclobutanil in rabbit liver, lung, brain, heart, kidney, fat, and muscle tissues after i.v. administration are shown in Fig. 2D–J, and the distribution data and EF values are shown in Fig. 3. The (+)-enantiomer degraded faster than its antipode in tissues except lung, resulting in the (–)-enantiomer being
enriched from 5 min to the last timepoint. The concentrations of myclobutanil at 60 min were in the following order: lung > liver > kidney > brain > fat > heart > muscle. The concentration of myclobutanil was extremely high in the lung, which caused a lung first-pass effect. Enantioselective disposition could also be attributed to excretion, especially in the kidneys. Renal excretion of racemate may have been enantioselective, with (+)-myclobutanil excreted more rapidly than its antipode. The detection of two enantiomers in brain tissue revealed that both enantiomers could penetrate the blood– brain barrier. Higher concentrations of both enantiomers were found in fat, showing the lipophilic nature of myclobutanil, and the (–)-myclobutanil was more prevalent in fat. Much research has reported the enantioselectivity of chiral chemicals in pharmacokinetics in animals.31,32 Several possible factors are involved in the differential pharmacokinetic behaviors of the enantiomers. The fundamental factor is the characterization of drug binding to plasma and tissue
Fig. 3. Distribution and EF of myclobutanil enantiomers in rabbit tissues. Chirality DOI 10.1002/chir
ENANTIOSELECTIVE BEHAVIOR OF MYCLOBUTANIL
proteins. The other is the preferential tissue distribution of one enantiomer from plasma. Enantioselective metabolism and excretion would also result in the enantioselective behavior of chiral chemicals in animals. CONCLUSION
In this study, we evaluated the enantioselectivity bioactivity and acute toxicity of myclobutanil, and found that the active (+)-enantiomer was also the more toxic one. We also investigated the enantioselective metabolism of myclobutanil enantiomers in rabbits; toxicokinetics analysis showed evidence of enantioselective disposition of two enantiomers in rabbit, and the (+)-enantiomer was preferentially eliminated from plasma and tissues compared with its antipode. ACKNOWLEDGMENTS
Supported by A Foundation for the Author of National Excellent Doctoral Dissertation of PR China, Program for New Century Excellent Talents in University (NCET09-0738), the National Natural Science Foundation of China (21277171, 21307155), the New-Star of Science and Technology supported by Beijing Metropolis, Program for New Century Excellent Talents in University and Program for Changjiang Scholars and Innovative Research Team in University. LITERATURE CITED 1. Zhou Y, Li L, Lin K, Zhu X, Liu W. Enantiomer separation of triazole fungicides by high-performance liquid chromatography. Chirality 2009;21:421–427. 2. Verweij PE, Kema GHJ, Zwaanc B, Melchersa WJG. Triazole fungicides and the selection of resistance to medical triazoles in the opportunistic mould Aspergillus fumigatus. Pest Manag Sci 2013;69:165–170. 3. Kahle M, Buerge IJ, Hauser A, Muller MD, Poiger T. Azole fungicides: occurrence and fate in wastewater and surface waters. Environ Sci Technol 2008;42:7193–7200. 4. Peng X, Huang Q, Zhang K, Yu Y, Wang Z, Wang C. Distribution, behavior and fate of azole antifungals during mechanical, biological, and chemical treatments in sewage treatment plants in China. Sci Total Environ 2012;426:311–317 5. Zarn JA, Brüschweiler BJ, Schlatter JR. Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14 alpha-demethylase and aromatase. Environ Health Persp2003;111:255–261. 6. Baudiffier D, Hinfray N, Ravaud C, Creusot N, Chadili E, Porcher JM, Schulz RW, Brion F. Effect of in vivo chronic exposure to clotrimazole on zebrafish testis function. Environ Sci Pollut R 2013;20:2747–2760. 7. Sekhon BS. Chiral pesticides. J Pestic Sci 2009;34:1–12. 8. Tian Q, Lv C, Wang P, Ren L, Qiu J, Li L, Zhou Z. Enantiomeric separation of chiral pesticides by high performance liquid chromatography on cellulose tris-3,5-dimethyl carbamate stationary phase under reversed phase conditions. J Separ Sci 2007;30:310–321. 9. Nomeir A, Dauterman W. Studies on the optical isomers of EPN and EPNO. Pestic Biochem Phys 1979;10:121–127. 10. Leader H, Casida JE. Resolution and biological activity of the chiral isomers of O-(4-bromo-2-chlorophenyl) O-ethyl S-propyl phosphorothioate (profenofos insecticide). J Agric Food Chem 1982;30:546–551. 11. Monkiedje A, Spiteller M, Bester K. Degradation of racemic and enantiopure metalaxyl in tropical and temperate soils. Environ Sci Technol 2003;37:707–712.
789
12. Jackson BP, Bugge D, Ranville JF, Chen CY. Bioavailability, toxicity, and bioaccumulation of quantum dot nanoparticles to the amphipod Leptocheirus plumulosus. Environ Sci Technol 2012;46:5550–5556. 13. Diao J, Xu P, Wang P, Lu D, Lu Y, Zhou Z. Enantioselective degradation in sediment and aquatic toxicity to Daphnia magna of the herbicide lactofen enantiomers. J Agric Food Chem 2010;58:2439–2445. 14. Lv X, Liu C, Li Y, Gao Y, Guo B, Wang H, Li J. Bioaccumulation and excretion of enantiomers of myclobutanil in Tenebrio molitor larvae through dietary exposure. Chirality 2013;25:890–896. 15. Yan J, Zhang P, Wang X, Wang Y, Zhou Z, Zhu W. Enantioselective degradation of chiral fungicide myclobutanil in rat liver microsomes. Chirality 2014;26:51–55. 16. Rockett JC, Narotsky MC, Thompson KE, Thillainadarajah I, Blystone CR, Goetz AK, Ren H, Best DS, Murrel RN, Nichols HP. Effect of conazole fungicides on reproductive development in the female rat. Reprod Toxicol 2006;22:647–658. 17. Goetz AK, Dix DJ. Toxicogenomic effects common to triazole antifungals and conserved between rats and humans. Toxicol Appl Pharm 2009;238:80–89. 18. Goetz AK, Dix DJ. Mode of action for reproductive and hepatic toxicity inferred from a genomic study of triazole antifungals. Toxicol Sci 2009;110:449–462. 19. Barton H, Tang J, Sey Y, Stanko J, Murrell R. Metabolism of myclobutanil and triadimefon by human and rat cytochrome P450 enzymes and liver microsomes. Xenobiotica 2006;36:793–806. 20. Deng Z, Hu J. Bioactivity investigation of fungicide myclobutanil enantiomers. Agrochemicals 2011;50:371–373. 21. Cheng C, Huang L, Diao J, Zhou Z. Enantioselective toxic effects and degradation of myclobutanil. Chirality 2013;25:858–864. 22. Dong F, Cheng L, Liu X, Xu J, Li J, Li Y, Kong Z, Jian Q, Zheng Y. Enantioselective analysis of triazole fungicide myclobutanil in cucumber and soil under different application modes by chiral liquid chromatography/tandem mass spectrometry. J Agric Food Chem 2012;60:1929–1936. 23. Ma Y, Chen L, Lu X, Chu H, Xu C, Liu W. Enantioselectivity in aquatic toxicity of synthetic pyrethroid insecticide fenvalerate. Ecotox Environ Safe 2009;72:1913–1918. 24. Burlingame EM, Reddish GF. Laboratory methods for testing fungicides used in the treatment of epidermophytosis. Jour Lab Clin Med 1939;24:765–772. 25. Zhao J, Zhou C, Gu Z. Toxicity of several fungicides to Fusarium oxysporum f. sp. melonis. J Shanghai Jiaotong Univ (Agr Sci) 2006;24:386–390. 26. OECD guideline for testing of chemicals 202: Daphnia sp. acute immobilisation test and reproduction test. 2004. 27. Furuta R, Doi T. Enantiomeric separation of diniconazole and uniconazole by cyclodextrin-modified micellar electrokinetic chromatography. J Chromatogr A 1994;676:431–436. 28. Yang L, Li S, Li Y, Gao R. Bioactivity investigation of triazole fungicide enantiomers. Chinese J Pestic Sci 2002;4:67–71. 29. Cao X, Hu M, Zhang J, Li F, Yang Y, Liu D, Liu S. Determination of enantioselective interaction between enantiomers of chiral gamma-aryl1H-1,2,4-triazole derivatives and Penicillium digitatum. J Agric Food Chem 2009;57:6914–6919. 30. Cai D, Yang P, Gong R. Environmental safety evaluation test guidelines of chemical pesticides. Beijing: China Environment Science Press; 1989. p 1–25. 31. Zhu W, Dang Z, Qiu J, Lv C, Jia G, Li L, Zhou Z. Enantioselective toxicokinetics and tissue distribution of ethofumesate in rabbits. Chirality 2007;19:632–637. 32. Kania-Korwel I, El-Komy MH, Veng-Pedersen P, Lehmler HJ. Clearance of polychlorinated biphenyl atropisomers is enantioselective in female C57Bl/6 mice. Environ Sci Technol 2009;44:2828–2835.
Chirality DOI 10.1002/chir
Review Article Acute Toxicity, Bioactivity, and Enantioselective Behavior with Tissue Distribution in Rabbits of Myclobutanil Enantiomers MINGJING SUN, DONGHUI LIU, XINXU QIU, QIAN ZHOU, ZHIGANG SHEN, PENG WANG, AND ZHIQIANG ZHOU* Department of Applied Chemistry, China Agricultural University, Beijing, P.R. China
ABSTRACT The enantioselective bioactivity against pathogens (Cercospora arachidicola, Fulvia fulva, and Phytophthora infestans) and acute toxicity to Daphnia magna of the fungicide myclobutanil enantiomers were studied. The (+)-enantiomer in an antimicrobial activity test was about 1.79–1.96 times more active than the (–)-enantiomer. In the toxicity assay, the calculated 24-h LC50 values of the (–)-form, rac-form and (+)-form were 16.88, 13.17, and 11.91 mg/ L, and the 48-h LC50 values were 10.15, 9.24, and 5.48 mg/L, respectively, showing that (+)-myclobutanil was more toxic. Meanwhile, the enantioselective metabolism of myclobutanil enantiomers following a single intravenous (i.v.) administration was investigated in rabbits. Total plasma clearance value (CL) of the (+)-enantiomer was 1.68-fold higher than its antipode. Significant differences in pharmacokinetics parameters between the two enantiomers indicated that the high bioactive (+)-enantiomer was preferentially metabolized and eliminated in plasma. Consistent consequences were found in the tissues (liver, brain, heart, kidney, fat, and muscle), resulting in a relative enrichment of the low-activity (–)-myclobutanil. These systemic assessments of the stereoisomers of myclobutanil cannot be used only to investigate environmental and biological behavior, but also have human health implications because of the long persistence of triazole fungicide and enantiomeric enrichment in mammals and humans. Chirality 26:784–789, 2014. © 2014 Wiley Periodicals, Inc KEY WORDS: myclobutanil enantiomer; degradation; rabbit INTRODUCTION
In recent years, increasing interest of the scientific community has focused on an emerging class of environmental pollutants, i.e., triazoles as well as the structurally related imidazole fungicides that are used as clinical drugs and pesticides.1,2 As the largest class of synthetic antimycotics, triazole antifungals have been detected worldwide in the environment,3 causing ecological concerns due to their persistence and endocrine-disrupting potency.4–6 Many triazoles are chiral and their enantiomers may have different properties and effectiveness. Chemically, chiral enantiomers are very similar, having the same boiling points, melting points, and typically the same solubility, reactivity, and other chemical properties.7 Nevertheless, the stereospecificity of chiral pesticides may be evident in the activity at the desired biological target and/or at undesirable targets, which results in adverse effects as one form is active against the organisms, and the other form is inactive.8–10 Apart from different biological activities to target biological objects, chiral pesticides usually have enantioselective toxicity, and the processes of absorption, distribution, and degradation in organisms and the environment are often enantioselective.11–13 Myclobutanil is a systemic fungicide belonging to the triazole family of chemicals. It displays an outstanding curative and protective efficacy against powdery mildew of cereal and vegetables.14,15 It is a moderately toxic compound in toxicity class II, and has been found to affect the reproductive abilities of test animals and cause varying degrees of hepatic toxicity and to disrupt steroid hormone homeostasis in © 2014 Wiley Periodicals, Inc.
bioactivity;
acute
toxicity;
enantioselective
rodent.16–19 This compound, which has long persistence in the environment, can be accumulated along the food chain and may therefore represent a risk for human health. Myclobutanil has an asymmetrically substituted C atom and consists of a pair of enantiomers. Previous studies showed that the enantiomers of myclobutanil always have different biological and physiological properties in asymmetry systems.20–22 Nonetheless, as with many chiral pesticides, myclobutanil is generally marketed and applied as the racemate, and owing to the current lack of knowledge concerning the individual bioactivities, toxicities, and degradation of myclobutanil enantiomers, the possible risks about this chiral fungicide are still not clear. So it is of great urgency and necessity to investigate the different effect and behavior of the two isomers to improve their effectiveness, decrease the pesticide residue, protect the environment, and evaluate the risks posed to public health. Daphnia magna, a key food source in the food chain of aquatic ecosystems, is a standard organism frequently used Contract grant sponsor: A Foundation for the Author of National Excellent Doctoral Dissertation of PR China, Program for New Century Excellent Talents in University; Contract grant number: NCET09-0738. Contract grant sponsor: National Natural Science Foundation of China; Contract grant number: 21277171, 21307155. *Correspondence to: Zhiqiang Zhou, Department of Applied Chemistry, China Agricultural University, Yuanmingyuan west road 2, Beijing 100193, P.R. China. E-mail: [email protected] Received for publication 28 February 2014; Accepted 28 May 2014 DOI: 10.1002/chir.22353 Published online 21 July 2014 in Wiley Online Library (wileyonlinelibrary.com).
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in ecotoxicity tests, due to its sensitivity to changes of the chemical composition in aquatic environments.23 Recent researches showed that the acute toxicity of the chiral pesticide enantiomers in acute toxicity was enantioselectivity.12 In this study the acute toxicity to Daphnia magna for myclobutanil enantiomers was determined. The inhibitory activities of myclobutanil against Cercospora arachidicola, Fulvia fulva, and Phytophthora infestans were also evaluated using the inhibition zone method. Furthermore, we detected concentrations of myclobutanil enantiomers in tissues and plasma of rabbits after intravenous administration. Through comparing the toxicity, activity, and biological fate in animals, we can provide some suggestions for using myclobutanil enantiomers, and reducing ecological risks caused by chiral pesticides in the environment. MATERIALS AND METHODS Chemicals and Reagents Myclobutanil (96.5%) was obtained from the Institute for Control of Agrichemicals Ministry of Agriculture. The two enantiomers of myclobutanil were prepared by high-performance liquid chromatography (HPLC) with purities >98.0%. The solvents n-hexane, 2-propanol, and acetonitrile, used for mobile phase preparation, were all of HPLC-grade from Fisher Scientific (Fair Lawn, NJ). All other chemicals and solvents were of analytical grade (Beijing Yili Fine Chemicals, China)
Instrumentation and Chromatographic Conditions The HPLC system used involved an Agilent (Palo Alto, CA) 1200 series HPLC equipped with a G1322A degasser, a G1311A quat pump, a G1329A automatic liquid sampler, a G1314B variable wavelength ultraviolet detector, and Agilent 1200 Chemstation software. The column temperature was controlled by an AT-930 heater and cooler column attemperator (Tianjin Automatic Science Instrument, China). Chromatographic separations were performed at room temperature on the Chiralpak AD (amylose tris-(3, 5-dimethylphenylcarbamate)) chiral column (250 × 4.6 mm, 10 μm particle size). The best resolution was achieved using n-hexane: 2-propanol (85:15, v/v) as the mobile phase, at a flow rate of 1.0 mL/min, with 225 nm monitoring wavelength at 20 °C. The elution order of myclobutanil enantiomers was measured by a CHIRALYSER–MP optical rotation detector produced by IBZ MESSTECHNIK (Germany). The (–)-enantiomer of myclobutanil was first eluted from the AD column.
Antimicrobial Activity Test The fungicidal activities against Cercospora arachidicola, Fulvia fulva, and Phytophthora infestans were tested according to the reported inhibition zone method.24 In this method, potato dextrose ager (PDA) was decanted into Petri dishes with actively growing mycelium of pathogenic bacteria when it was cold enough but still liquid. Filter paper disks of 6 mm diameter were sterilized by autoclaving for 15 min at 120 °C and impregnated with 10 μL of stock solutions of myclobutanil enantiomers at different concentrations (ranging from 50 to 800 mg/L). After solidification of the agar, the filter disk was placed onto the middle surface of the PDA, incubated at 25 ± 1 °C, and a light-dark cycle of 12/12 h darklight. Two replicates were done for each concentration, and a blank control was also performed using the solvent without compounds. The antimicrobial activity of each extract was measured as a zone of inhibition of the bacterial growth around the disk. The inhibition percent was used to describe the control efficiency of the compounds. Inhibition percent (%) = inhibition zone diameter in the treatment/hyphal diameter in the control.25
Acute Toxicity Assays Enantioselectivity in acute aquatic toxicity was evaluated via 24-h and 48-h toxicity assays using D. magna as the model species, originally obtained from CABET (Center for Agrochemical Biological and Environmental Technology Institute, Beijing, China). The testing procedures followed the OECD guideline “Acute Immobilization Test.”26 The test
animals used in these experiments were juveniles aged between 6 and 24 h. Prior to the test, a sensitive test for Daphnia to potassium dichromate was performed as a positive control. The D. magna were exposed to a series of test solutions in 50-mL beakers containing rac-, (+), and (–)-myclobutanil separately within a given concentration range (1.25–80 mg/L). Each beaker contained ten D. magnas. Three replicates for each treatment were conducted. The test animals were not further fed and were incubated at 20 ± 1 °C under a light-dark cycle of 16/8 h dark/light. The concentration of enantiomer or racemate that caused 50% mortality of the test population (LC50) was determined using a probit equation with SPSS 16.0 (Chicago, IL).
Animal Treatment Male Japanese white rabbits weighing 2.0 ± 0.25 kg (provided by the Experimental Animal Research Institute of China Agriculture University) were housed under a 12-h light/dark cycle at 20 °C. Racemic myclobutanil was dissolved in alcohol and then diluted to final concentrations with normal saline, and then administered at 30 mg/kg body weight by intravenous injection in the ear vein. Blood from the heart arteries was collected into polypropylene tubes with heparin sodium at 1, 15, 30, 60, 90, 120, 180, and 240 min after treatment. The animals were sacrificed at 5, 60, 120, 180, and 300 min after treatment, and the heart, kidney, liver, lung, fat, muscle, and brain were excised and weighed separately. Plasma and tissue samples were stored at –20 °C for later analysis.
Pretreatment of Plasma and Tissues Plasma sample (1 mL) or homogenized tissue (1 g) was transferred to 15 mL plastic centrifuge tubes, with 5 mL of ethyl ether added, stirred for 2 min on a vortex mixer, and centrifuged at 4000 rpm for 5 min. The extraction was repeated with another 5 mL of ethyl acetate. The organic phase was combined and evaporated to dryness under a stream of nitrogen. The residue was dissolved in 4 mL of acetonitrile, and then partitioned twice with 3 mL of n-hexane, and the acetonitrile phase was evaporated to dryness. All the resulting residue was redissolved in 0.2 mL of 2-propanol, filtered (0.45 mm pore size), and injected into the HPLC for analysis.
Pharmacokinetics Analysis The pharmacokinetic parameters were determined with the Drug and Statistics Software (Section of Quantitative Pharmacology, Chinese Pharmacological Society). An open two-compartment model was used to describe the metabolism of both enantiomers after i.v. administration in plasma. Concentration in the central compartment (C) after a single intravenous dose was expressed as: C ¼ Ae-αt þ Be-βt where t was time, A and B were D(α- k21)/[V1(α-β)] and D(k21 -β))/ [V1(α-β)], respectively, D was dose, V1 was the apparent volume of the central compartment, and k21 was the apparent first-order distribution rate from the peripheral to the central compartment; α and β denote the slopes of α phase and β phase, respectively. The area under the concentration–time cure (AUC) was calculated by the trapezoidal rule. Mean residence time (MRT) was calculated by dividing the area under first-moment curve AUMC by AUC. The distribution and elimination phase half-life (t1/2α, t1/2β) were calculated by 0.693/α and 0.693/β. The total apparent volumes of distribution (Vd) were calculated by D/(βAUC0-∞), and the total body clearance (CL) was calculated by D/AUC0-∞.
RESULTS AND DISCUSSION Enantioselection in Bioactivity
The antimicrobial activity of myclobutanil isomers in the inhibition zone test was expressed by EC50 (the concentration inhibiting mycelial growth by 50%) values after 3 d treatment and the results of the bioactivity of the enantiomers to Cercospora arachidicola, Fulvia fulva, and Phytophthora infestans are shown in Table 1. For all the test pathogens, the bioactivity of both stereoisomers was proportional to their Chirality DOI 10.1002/chir
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TABLE 1. Inhibition of myclobutanil isomers in three fungi Inhibition rate at the different concentrations (%) Fungi Cercospora arachidicola Fulvia fulva Phytophthora infestans (tomato)
Myclobutanil
800 mg/L
400 mg/L
200 mg/L
100 mg/L
50 mg/L
R2
EC50 (mg/L)
(-)-isomer (+)-isomer (-)-isomer (+)-isomer (-)-isomer (+)-isomer
37.78 48.89 36.67 38.89 37.78 43.33
32.22 41.11 31.11 35.56 33.33 37.78
28.89 34.44 27.78 31.11 28.89 32.22
22.22 24.44 21.11 24.44 22.22 25.56
____ ____ 20.00 20.00 18.89 21.11
0.977 0.984 0.970 0.979 0.989 0.996
1146.99 628.17 4569.69 2550.16 2992.50 1523.62
concentration, and the (+)-enantiomer was about 1.79–1.96 times more active than the (–)-enantiomer. The similar result was also reported by Deng’s group found the antifungicidal activity of (+)-myclobutanil to be obviously superior to (–)-myclobutanil by using a growth rate method. 20 Chirality has a considerable influence on the biological activity of triazole fungicides. For instance, the R-enantiomers of diniconazole and uniconazole showed stronger fungicidal activity than the S-enantiomers, whereas the S-enantiomers showed higher plant growth regulating activity.27 The isomers of tebuconazole, flutriofol, and hexaconazole also showed different microbial activity.28 One possible reason for the stereoselectivity of triazole fungicides is that one of the enantiomers better fits the active site of cytochrome P 450.29 Enantioselective in Acute Toxicity
The acute toxicity of myclobutanil and its individual enantiomers to D. magna was expressed by LC50 values at 24 and 48 h and the data are presented in Table 2. According to the "Environmental Safety Evaluation Test Guidelines of Chemical Pesticides," rac-myclobutanil and its individual enantiomers were all moderately toxic to D. magna,30 in which the (+)-enantiomer was more toxic than the racemate and (–)-enantiomer. After longer exposure, differences in toxicity between enantiomers became larger. The
calculated 48-h LC50 of (+)-myclobutanil was nearly twice that for the (–)-enantiomer. The results showed a general agreement in the enantioselectivity between toxicity to D. magna and antimicrobial activity, that is, the active (+)-enantiomer was also the more ecotoxic one. It can be assumed that there might be a common mode of action between some of the target fungi and aquatic invertebrates. Assay Validation
Linear calibration curves were obtained for the (–)-isomer (y = 25.94x + 98.07, R2 = 0.973) and (+)-isomer (y = 25.37x + 87.81, R2 = 0.980) over the concentration range of 0.5–50 μg/mL in drug-free plasma. The recoveries of each enantiomer of myclobutanil were determined by analyzing quality control samples at three spiked concentrations (0.5, 5, 10 μg/mL in plasma and 1, 50, 250 μg/g in tissues). and the lowest recovery was over 80%. The limit of quantification was 0.2 μg/mL in plasma and 0.2 μg/g in tissue samples. Pharmacokinetics
Concentration–time curves of (–)- and (+)-myclobutanil in plasma after i.v. administration of 30 mg/kg b.w. of racemate to the rabbits are shown in Fig. 1A. The enantiomer fraction (EF = [(–)]/([(+)] + [(–)]) was used as a measure of the
TABLE 2. Calculated LC50 values for myclobutanil (–)-isomer Time (hours)
1
LC50(mg/L)
R2
16.88 10.15
0.954 0.991
24 48
rac-compound P
2
0.004 0.005
(+)-isomer
LC50(mg/L)
R2
P
LC50(mg/L)
R2
P
13.17 9.24
0.964 0.989
0.003 0.056
11.91 5.48
0.993 0.969
0.004 0.016
1
Correlation coefficient. Probability (associated with the t-test). A P value < 0.05 indicates that the correlation of the linear equation is significant, and it is calculated using the linear regression model of SPSS 16.0. 2
Fig. 1. Plasma concentration–time curves (A) and EF (B) of myclobutanil enantiomers in rabbits following racemic administration at 30 mg/kg bd wt. Chirality DOI 10.1002/chir
ENANTIOSELECTIVE BEHAVIOR OF MYCLOBUTANIL
TABLE 3. Pharmacokinetic parameters of myclobutanil enantiomers following i.v. administration at 30 mg/kg Pharmacokinetic parameters t1/2α (min) t1/2β (min) V1 (L/kg) CL (L/min/kg) AUC0-240 (mg/L · min) AUC0-∞ (mg/L · min) MRT0-240 (min) MRT0-∞ (min)
(–)-myclobutanil
(+)-myclobutanil
8.353 69.315 1.535 0.019 701.948 798.299 75.987 96.857
5.721 63.312 1.516 0.032 436.363 467.531 62.04 68.628
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enantioselectivity of the two enantiomers (Fig. 1B). The concentration of the (+)-enantiomer in plasma was lower than its antipode, causing the enrichment factor (EF) value to increase with time. Based on the statistical analysis of a t-test, the enantioselective is significant (P < 0.05). Compartmental pharmacokinetic analysis showed some statistical differences between the enantiomers in the principal pharmacokinetic parameters (Table 3). The (–)/(+) ratio of the distribution half-life (t1/2α) was 1.46, whereas the (–)/(+) ratio of the elimination half-life (t1/2β) was 1.09. The result indicated that (+)-myclobutanil distributed faster than its antipode in rabbit. The chromatogram of the preferential disappearance of the (+)-enantiomer in plasma is shown in Fig. 2C. The total plasma clearance (CL) of (+)-isomer was more than 1.68-fold higher than that of the (–)-isomer. The
Fig. 2. Representative HPLC chromatogram of (A)100 mg/L myclobutanil (B) drug-free rabbit plasma extract; (C) plasma sample extract (30 min); (D) liver sample extract (60 min); (E) lung sample extract (60 min); (F) brain sample extract (60 min); (G) heart sample extract (60 min); (H) kidney sample extract (60 min); (I) fat sample extract (120 min); (J) muscle sample extract (120 min); (n-hexane/isopropanol = 85/15, flow rate: 1 ml/min, detect wave: 220 nm). Chirality DOI 10.1002/chir
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(–)/(+)-myclobutanil ratios of the AUC0-240 min and AUC0-∞ were 1.51 and 1.71, the ratios of the MRT0-240 and MRT0-∞ were 1.22 and 1.41, respectively. The results suggested that the pharmacokinetics of myclobutanil enantiomers were enantioselective in rabbits. One possible explanation for the significant differences in the pharmacokinetics was the stereoselectivity of plasma protein binding and membrane permeation of myclobutanil. Chiral conversion of myclobutanil in plasma might also contribute to these differences. Distribution in Tissues
Typical chromatograms for (–)- and (+)-myclobutanil in rabbit liver, lung, brain, heart, kidney, fat, and muscle tissues after i.v. administration are shown in Fig. 2D–J, and the distribution data and EF values are shown in Fig. 3. The (+)-enantiomer degraded faster than its antipode in tissues except lung, resulting in the (–)-enantiomer being
enriched from 5 min to the last timepoint. The concentrations of myclobutanil at 60 min were in the following order: lung > liver > kidney > brain > fat > heart > muscle. The concentration of myclobutanil was extremely high in the lung, which caused a lung first-pass effect. Enantioselective disposition could also be attributed to excretion, especially in the kidneys. Renal excretion of racemate may have been enantioselective, with (+)-myclobutanil excreted more rapidly than its antipode. The detection of two enantiomers in brain tissue revealed that both enantiomers could penetrate the blood– brain barrier. Higher concentrations of both enantiomers were found in fat, showing the lipophilic nature of myclobutanil, and the (–)-myclobutanil was more prevalent in fat. Much research has reported the enantioselectivity of chiral chemicals in pharmacokinetics in animals.31,32 Several possible factors are involved in the differential pharmacokinetic behaviors of the enantiomers. The fundamental factor is the characterization of drug binding to plasma and tissue
Fig. 3. Distribution and EF of myclobutanil enantiomers in rabbit tissues. Chirality DOI 10.1002/chir
ENANTIOSELECTIVE BEHAVIOR OF MYCLOBUTANIL
proteins. The other is the preferential tissue distribution of one enantiomer from plasma. Enantioselective metabolism and excretion would also result in the enantioselective behavior of chiral chemicals in animals. CONCLUSION
In this study, we evaluated the enantioselectivity bioactivity and acute toxicity of myclobutanil, and found that the active (+)-enantiomer was also the more toxic one. We also investigated the enantioselective metabolism of myclobutanil enantiomers in rabbits; toxicokinetics analysis showed evidence of enantioselective disposition of two enantiomers in rabbit, and the (+)-enantiomer was preferentially eliminated from plasma and tissues compared with its antipode. ACKNOWLEDGMENTS
Supported by A Foundation for the Author of National Excellent Doctoral Dissertation of PR China, Program for New Century Excellent Talents in University (NCET09-0738), the National Natural Science Foundation of China (21277171, 21307155), the New-Star of Science and Technology supported by Beijing Metropolis, Program for New Century Excellent Talents in University and Program for Changjiang Scholars and Innovative Research Team in University. LITERATURE CITED 1. Zhou Y, Li L, Lin K, Zhu X, Liu W. Enantiomer separation of triazole fungicides by high-performance liquid chromatography. Chirality 2009;21:421–427. 2. Verweij PE, Kema GHJ, Zwaanc B, Melchersa WJG. Triazole fungicides and the selection of resistance to medical triazoles in the opportunistic mould Aspergillus fumigatus. Pest Manag Sci 2013;69:165–170. 3. Kahle M, Buerge IJ, Hauser A, Muller MD, Poiger T. Azole fungicides: occurrence and fate in wastewater and surface waters. Environ Sci Technol 2008;42:7193–7200. 4. Peng X, Huang Q, Zhang K, Yu Y, Wang Z, Wang C. Distribution, behavior and fate of azole antifungals during mechanical, biological, and chemical treatments in sewage treatment plants in China. Sci Total Environ 2012;426:311–317 5. Zarn JA, Brüschweiler BJ, Schlatter JR. Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14 alpha-demethylase and aromatase. Environ Health Persp2003;111:255–261. 6. Baudiffier D, Hinfray N, Ravaud C, Creusot N, Chadili E, Porcher JM, Schulz RW, Brion F. Effect of in vivo chronic exposure to clotrimazole on zebrafish testis function. Environ Sci Pollut R 2013;20:2747–2760. 7. Sekhon BS. Chiral pesticides. J Pestic Sci 2009;34:1–12. 8. Tian Q, Lv C, Wang P, Ren L, Qiu J, Li L, Zhou Z. Enantiomeric separation of chiral pesticides by high performance liquid chromatography on cellulose tris-3,5-dimethyl carbamate stationary phase under reversed phase conditions. J Separ Sci 2007;30:310–321. 9. Nomeir A, Dauterman W. Studies on the optical isomers of EPN and EPNO. Pestic Biochem Phys 1979;10:121–127. 10. Leader H, Casida JE. Resolution and biological activity of the chiral isomers of O-(4-bromo-2-chlorophenyl) O-ethyl S-propyl phosphorothioate (profenofos insecticide). J Agric Food Chem 1982;30:546–551. 11. Monkiedje A, Spiteller M, Bester K. Degradation of racemic and enantiopure metalaxyl in tropical and temperate soils. Environ Sci Technol 2003;37:707–712.
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12. Jackson BP, Bugge D, Ranville JF, Chen CY. Bioavailability, toxicity, and bioaccumulation of quantum dot nanoparticles to the amphipod Leptocheirus plumulosus. Environ Sci Technol 2012;46:5550–5556. 13. Diao J, Xu P, Wang P, Lu D, Lu Y, Zhou Z. Enantioselective degradation in sediment and aquatic toxicity to Daphnia magna of the herbicide lactofen enantiomers. J Agric Food Chem 2010;58:2439–2445. 14. Lv X, Liu C, Li Y, Gao Y, Guo B, Wang H, Li J. Bioaccumulation and excretion of enantiomers of myclobutanil in Tenebrio molitor larvae through dietary exposure. Chirality 2013;25:890–896. 15. Yan J, Zhang P, Wang X, Wang Y, Zhou Z, Zhu W. Enantioselective degradation of chiral fungicide myclobutanil in rat liver microsomes. Chirality 2014;26:51–55. 16. Rockett JC, Narotsky MC, Thompson KE, Thillainadarajah I, Blystone CR, Goetz AK, Ren H, Best DS, Murrel RN, Nichols HP. Effect of conazole fungicides on reproductive development in the female rat. Reprod Toxicol 2006;22:647–658. 17. Goetz AK, Dix DJ. Toxicogenomic effects common to triazole antifungals and conserved between rats and humans. Toxicol Appl Pharm 2009;238:80–89. 18. Goetz AK, Dix DJ. Mode of action for reproductive and hepatic toxicity inferred from a genomic study of triazole antifungals. Toxicol Sci 2009;110:449–462. 19. Barton H, Tang J, Sey Y, Stanko J, Murrell R. Metabolism of myclobutanil and triadimefon by human and rat cytochrome P450 enzymes and liver microsomes. Xenobiotica 2006;36:793–806. 20. Deng Z, Hu J. Bioactivity investigation of fungicide myclobutanil enantiomers. Agrochemicals 2011;50:371–373. 21. Cheng C, Huang L, Diao J, Zhou Z. Enantioselective toxic effects and degradation of myclobutanil. Chirality 2013;25:858–864. 22. Dong F, Cheng L, Liu X, Xu J, Li J, Li Y, Kong Z, Jian Q, Zheng Y. Enantioselective analysis of triazole fungicide myclobutanil in cucumber and soil under different application modes by chiral liquid chromatography/tandem mass spectrometry. J Agric Food Chem 2012;60:1929–1936. 23. Ma Y, Chen L, Lu X, Chu H, Xu C, Liu W. Enantioselectivity in aquatic toxicity of synthetic pyrethroid insecticide fenvalerate. Ecotox Environ Safe 2009;72:1913–1918. 24. Burlingame EM, Reddish GF. Laboratory methods for testing fungicides used in the treatment of epidermophytosis. Jour Lab Clin Med 1939;24:765–772. 25. Zhao J, Zhou C, Gu Z. Toxicity of several fungicides to Fusarium oxysporum f. sp. melonis. J Shanghai Jiaotong Univ (Agr Sci) 2006;24:386–390. 26. OECD guideline for testing of chemicals 202: Daphnia sp. acute immobilisation test and reproduction test. 2004. 27. Furuta R, Doi T. Enantiomeric separation of diniconazole and uniconazole by cyclodextrin-modified micellar electrokinetic chromatography. J Chromatogr A 1994;676:431–436. 28. Yang L, Li S, Li Y, Gao R. Bioactivity investigation of triazole fungicide enantiomers. Chinese J Pestic Sci 2002;4:67–71. 29. Cao X, Hu M, Zhang J, Li F, Yang Y, Liu D, Liu S. Determination of enantioselective interaction between enantiomers of chiral gamma-aryl1H-1,2,4-triazole derivatives and Penicillium digitatum. J Agric Food Chem 2009;57:6914–6919. 30. Cai D, Yang P, Gong R. Environmental safety evaluation test guidelines of chemical pesticides. Beijing: China Environment Science Press; 1989. p 1–25. 31. Zhu W, Dang Z, Qiu J, Lv C, Jia G, Li L, Zhou Z. Enantioselective toxicokinetics and tissue distribution of ethofumesate in rabbits. Chirality 2007;19:632–637. 32. Kania-Korwel I, El-Komy MH, Veng-Pedersen P, Lehmler HJ. Clearance of polychlorinated biphenyl atropisomers is enantioselective in female C57Bl/6 mice. Environ Sci Technol 2009;44:2828–2835.
Chirality DOI 10.1002/chir
