CHIRALITY 26:155–159 (2014)
Chiral Separation and Enantioselective Degradation of Vinclozolin in Soils HUI LIU, DONGHUI LIU, ZHIGANG SHEN, MINGJING SUN, ZHIQIANG ZHOU,* AND PENG WANG Department of Applied Chemistry, China Agricultural University, Beijing, China
ABSTRACT Vinclozolin is a chiral fungicide with potential environmental problems. The chiral separation of the enantiomers and enantioselective degradation in soil were investigated in this work. The enantiomers were separated by high-performance liquid chromatography (HPLC) on Chiralpak IA, IB, and AZ-H chiral columns under normal phase and the influence of the mobile phase composition on the separation was also studied. Complete resolutions were obtained on all three chiral columns under optimized conditions with the same elution order of (+)/(). The residual analysis of the enantiomers in soil was conducted using accelerate solvent extraction followed by HPLC determination. The recoveries of the enantiomers ranged from 85.7–105.7% with relative standard deviation (SD) of 0.12–3.83%, and the limit of detection (LOD) of the method was 0.013 μg/g. The results showed that the degradations of vinclozolin enantiomers in the soils followed first-order kinetics. Preferential degradation of the ()-enantiomer was observed only in one soil with the largest |ES| value of 0.047, and no obvious enantioselective degradation was observed in other soils. It was found that the persistence of vinclozolin in soil was related to pH values based on the half-lives. The two enantiomers disappeared about 8 times faster in basic soils than that in neutral or acidic soils. Chirality 26:155–159, 2014. © 2014 Wiley Periodicals, Inc. KEY WORDS: vinclozolin; enantiomer; chiral separation; soil; selective degradation INTRODUCTION
It is already well known that a large number of organic agrochemicals are chiral and consist of mixtures of stereoisomers or enantiomers which may have different bioactivity, toxicity, metabolism, excretion, and environmental effects. There is growing concern about the side effects of chiral agrochemicals on nontarget organisms and natural resources.1 For these purposes, it is necessary to investigate the enantiomers separately. Separation of chiral compound enantiomers is a challenging topic in many analytical chemistry areas, especially in the biomedical, pharmaceutical, and environmental fields where pure enantiomeric forms are widely required. Separation techniques such as gas chromatography (GC), high-performance liquid chromatography(HPLC), supercritical fluid chromatography (SFC), and capillary electrophoresis (CE) have been widely employed, although, at present, HPLC still dominates chromatographic chiral analysis of chiral compounds.2 Because it is quite rapid and nondestructive, there is little possibility of epimerization during the analysis.3 Chirality is also popular in pesticides and about 30% of the widely used pesticides are chiral. Vinclozolin [3-(3,5dichlorophenyl)-5-methyl-5-vinyloxazolidine-2,4-dione] (Fig. 1) is a dicarboximide fungicide that is effective in the control of diseases caused by Botrytis cinerea, Sclerotinia sclerotiorum, and Moniliniam spp. It has been widely used in Europe to protect fruits, vegetables, ornamental plants, and turf grasses. The World Health Organization (WHO) has previously evaluated the fungicide vinclozolin as a noncarcinogen (FAO/WHO, 1986), but the U.S. Environmental Protection Agency (U.S. EPA) has classified vinclozolin as a Group C chemical, which indicates a possible human carcinogen (U. S. EPA, 1997). Vinclozolin has been shown to cause abnormal male sexual development in rats,4–6 alter morphological sex © 2014 Wiley Periodicals, Inc.
differentiation in male rats following perinatal exposure,7 and lead to the death of zebrafish embryos, developmental delays, and teratogenic effects, suggesting its potential health hazards. Vinclozolin can undergo chemical hydrolysis, photolysis, or metabolism by mammals and bacterial systems. Szeto et al.8 isolated three hydrolysis products from aqueous buffers which were identified as: 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2methyl-3-butenoic acid(M1), 3’5’-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2), and 3,5-dichloroaniline (M3). The metabolites of vinclozolin have been reported to act as antiandrogens and have adverse effects on reproductive physiology in animals. A number of investigations on the residual analysis of vinclozolin in foods and soils have been conducted by gas chromatography (GC), HPLC, and mass spectrometry.9–15 Solid-phase microextraction (SPME) coupled to GC analysis was evaluated for extracting trace amounts of vinclozolin in soil samples.10 Its dissipation during storage of grape juice was analyzed by liquid–liquid extraction (LLE), gas chromatographic separation, and mass spectrometric detection.16 Pesticides in groundwater, surface water, or the food chain were usually from contaminated soils; for example, grape quality was affected by vinclozolin-polluted soil.17 It was found that the extraction efficiency was influenced by the sorption– desorption,18 pH, heavy metal, and organic matter of soils. Arias-Estévez et al. found that an increasing pH could cause the dissolution of some organic matter and reduce atrazine adsorption, and copper slightly increased atrazine adsorption but Zn acted conversely.19 In subsurface soils with much *Correspondence to: Z. Zhou, Department of Applied Chemistry, China Agricultural University, Beijing 100193, China. E-mail: [email protected] Received for publication 25 September 2013; Accepted 12 November 2013 DOI: 10.1002/chir.22284 Published online 4 February 2014 in Wiley Online Library (wileyonlinelibrary.com).
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performed using an Agilent 1200 series HPLC (Agilent Technology, Palo Alto, CA) equipped with a G1311A pump, G1322A degasser, G1328A injector, a 20 μL sample loop, and G1315A DAD. The signal was received and processed by an Agilent chemstation The polarimeter was provided by IBZ Messtechnik (Germany) with mozartstrafic 14–16, D-30173, 1000 mV, Ser.No.:11/272.
Fig. 1. The chemical structure of vinclozolin.
Chiral Separation
lower organic matter content, Fe oxides could play an important role in overall sorption, because of the net electrical charge as well as the electric potential of the soils.20,21 So the properties of soil should be fully considered in the optimization of pesticide extraction.22 The biodegradation and metabolism of vinclozolin in soils and different plants has been studied extensively and the results have demonstrated that the degradation of this chemical in soil is a microbiologically mediated process. Vinclozolin is quite stable in acidic soils, especially under low temperature.23–26 The moisture content in soil also affected the degradation.27 Few studies in the literature are about chiral separation and enantioselectivity. Vinclozolin was first separated by GC on ACTBDMS-g-CD(OV-1701), ACTHDMS-g-CD(PS-086), and ACTHDMS-g-CD(OV-1701) reported in 1999.28 Wang et al. found that vinclozolin can be separated by cellulose triphenylcarbamate column29 and cellulose-tris-(3,5-dimethylphenylcarbamate) column with n-hexane/isopropanol 99:1 (v/v).30 Vinclozolin has been proven to be enantioselectively degraded in the wine-making process, resulting in the enrichment of ()vinclozolin.31 The enantioselective degradation of vinclozolin in the environmental matrix has not been reported. The chiral separation and the enantioselective degradation of vinclozolin enantiomers in several agriculture soils were investigated in this work. The results may improve our understanding of the environmental behavior of chiral pesticides. EXPERIMENTAL Chemicals and Reagents Rac-vinclozolin (>99.2% purity) was provided by the China Ministry of Agriculture Institute for Control of Agrochemicals. A stock solution of racvinclozolin was prepared in isopropanol and stored at 20 °C. Working standard solutions were obtained by dilutions of the stock solution with isopropanol. Water was purified by a Milli-Q system. Isopropanol, hexane, and ethyl acetate (analytical grade) were purchased from commercial sources, distilled, and filtered through a 0.45 μm filter membrane before use. All other chemicals and solvents were analytical grade and purchased from commercial sources.
The mobile phase was n-hexane/isopropanol or ethanol at a flow rate of 0.8 mL/min. Pesticide solutions were prepared by dissolving the samples in isopropanol at a concentration of 20 mg/L and the injection volume was 20 μL. The chromatographic separation was conducted at 20 °C and 210 nm, Chiralpak IA, IB, and AZ-H column (250 × 4.6 mm .ID) were provided by Daicel Chemical Industries, Arai Plant. Separation factor (α), and resolution factor (Rs) were calculated.
Degradation of Vinclozolin in Soils The test soils were from different agricultural regions (0–10 cm) of China that had not been treated with vinclozolin in the last 5 years. The properties are shown in Table 1. Test soils were adjusted to a moisture content of 30% and maintained in the dark at room temperature for 7 days to activate the microorganisms in soils. Soil subsample (390 g, corresponding to 300 g dry weight) was weighed into a 1000-mL Erlenmeyer flask. In order to add the pesticide to the soil evenly, 10 g of soil was first weighed into a 1000-mL Erlenmeyer flask and 0.5 mL of racemic vinclozolin solution (6000 μg/mL, acetone) was added. After that, another 380 g of soil was added gradually and stirred thoroughly to make a spiked concentration of 5 μg/g (each enantiomer). The flasks were sealed with cotton-wool plugs and stored at 15 °C in the dark for 120 days. The moisture content in each flask was checked gravimetrically at each sampling point. Five grams of the treated soil samples (based on the dry weight) was removed for analysis after incubation points of 0, 1, 3, 5, 7, 10, 14, 21, 28, 35, 50, 65, 80, 100, 120 days and stored at 20 °C before analysis. Each incubation was carried out in triplicate.
Soil Sample Treatment Soil samples were extracted by accelerated solvent extraction (ASE), in which factors such as extraction pressure, temperature, static time, and number of cycles were optimized. Wet soil samples were mixed with diatomite in a ratio of 2:1 in a mortar, and 6.0 g of the mixed sample was placed in the extraction cell and extracted with the following conditions: extraction temperature: 80°C; heating time: 5 min; static extraction time: 10 min; cycles: 2; rinse volume: 10%; nitrogen purge: 60s; extraction solvent: ethyl acetate. The eluting solution was evaporated to near dryness under a stream of nitrogen and reconstituted in 0.5 mL of isopropanol. An aliquot (20 μL) was injected on to the HPLC.
Assay Validation Apparatus The ASE-350 accelerated solvent extraction instrument was from Dionex (Sunnyvale, CA), with three solvent bottles, nitrogen bottle, 34-mL extraction pool, and 60-mL receiving flasks. Chromatography was
A series of working standard solutions (1-100 mg/L) of rac-vinclozolin were prepared. Calibration curves were generated by plotting peak area of each enantiomer versus the concentration. Linear regression analysis was performed using Microsoft-Excel. The concentration of each
TABLE 1. Selected physiochemical properties of the soils Particle size No.
Site
Sand (%)
Silt (%)
Clay (%)
Texture
pH (water)b
Corg [%]
1# 2# 3# 4# 5#
Heilongjiang Shenyang Guangxi Yunnan Gansu
42.15 42.86 42.86 71.77 14.98
33.06 40.82 28.57 12.10 36.44
24.79 16.33 28.57 16.13 48.58
Loam Loam Loam Sandy loam Clay loam
6.01 8.13 5.75 6.28 8.40
31.39 32.36 27.20 20.21 20.27
Chirality DOI 10.1002/chir
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CHIRAL SEPARATION ENANTIOSELECTIVE DEGRADATION VINCLOZOLIN SOIL
enantiomer in samples was calculated by the calibration curves of the corresponding enantiomer using the extended standard method. The limit of detection (LOD) of method for each enantiomer was considered to be the concentration that produced a signal-to-noise (S/N) ratio of 3, and the limit of quantification (LOQ) of method was defined as the lowest concentration in the calibration curve with acceptable precision and accuracy for 20% variability. All quantification was based on HPLC response (peak area of each enantiomer).The concentration of each enantiomer in samples was calculated by calibration curves of the corresponding enantiomer using the extended standard method. Absolute recovery of vinclozolin was determined in all the test soils fortified with racvinclozolin at 1, 5, and 10 μg/g. The enantiomer enantioselectivity (ES) was used to measure the selective degradation in soils. ES was defined as: ES ¼ ðk1 k2 Þ=ðk1 þ k2 Þ When the degradation followed a first-order kinetic, k1 and k2 were used as the degradation rate constant, respectively, for two enantiomers. The |ES| values ranged from 0 to 1. There was no enantioselective behavior if ES = 0.
RESULTS AND DISCUSSION Enantiomeric Separation and Elution Order
The two enantiomers could be completely separated on IA, IB, and AZ-H columns (Fig. 2), in which IB was the best. The chiral separations were performed by HPLC IB columns
using a mobile phase of n-hexane/ isopropanol or ethanol at a flow rate of 0.8 mL/min. The impact of mobile phase composition and temperature on the separation was optimized on the IB column and the results showed the two enantiomers could be fully separated using both isopropanol or ethanol as modifier and better separation was obtained under lower temperature and the mobile phase with less isopropanol or ethanol (Table 2). Assay Validation
Based on the successful chiral resolution of the two enantiomers, the chiral residue analysis method in soil samples was set up. An accelerated solvent extraction method was successfully applied to extract the two enantiomers from soil samples, which proved to be satisfactory for the pesticide with strong adsorption. Good linearity was obtained over the concentration range of 0.5–50 mg/L (n = 7) for both (+)-vinclozolin (y = 158.36x + 106.10,R2 = 0.9995) and ()vinclozolin (y = 158.47x +100.00, R2 = 0.9994). The LOQ for both enantiomers of the method in all samples was found to be 0.043 μg/g, and the LOD for both enantiomers of the method, defined as the concentration with an S/N ratio of 3, was 0.013 μg/g in soil samples (Table 3). The Cva➀ was found to be 0.2% to 5.1%, which proved to be satisfactory (Table 4).
Fig. 2. The chromatograms of the chiral separations of vinclozolin on A:IA column, 1 mL/min,20 °C,n-hexane/ isopropanol = 99:1;B:IB column,0.8 mL/min,20 °C, n-hexane/ isopropanol = 95:5;C: AZ-H column,0.8 mL/min,20 °C,n-hexane/ isopropanol = 99:1.
TABLE 2. The chromatographic separation of vinclozolin with n-hexane/modifier as mobile phase Temperature (°C)
Alcohol modifier (%)
10
Isopronanol, 2% Ethanol, 2% Isopronanol, 2% Ethanol, 2% Isopronanol, 2% Isopronanol, 5% Isopronanol, 10% Ethanol, 2% Ethanol, 5% Ethanol, 10% Isopronanol, 2% Ethanol, 2%
15 20
25
α
Rs
1.11 1.18 1.16 1.17 1.15 1.14 1.12 1.16 1.13 1.12 1.14 1.15
3.07 3.31 2.84 2.87 2.90 2.51 2.19 2.67 2.56 2.06 2.22 2.26
TABLE 4. The CVa of the analysis method for the vinclozolin enantiomers (n = 6) Cva➀ (%)
Interday
Day-to-Day
Concentration (μg/g)
(+) –vinclozolin
() –vinclozolin
1.0 5.0 50 1.0 5.0 50
5.1 0.7 0.4 4.6 2.8 0.4
4.6 0.8 0.2 3.2 1.2 1.1
TABLE 3. Validation of vinclozolin enantiomer analysis Enantiomer (+) –vinclozolin () –vinclozolin
Linear equation
R2
Linear range (μg/mL)
LOD (μg/g)
LOQ (μg/g)
y = 158.36x + 106.10 y = 158.47x + 100.00
0.9995 0.9994
0.5-50 0.5-50
0.013 0.013
0.043 0.043 Chirality DOI 10.1002/chir
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LIU ET AL.
The recoveries of vinclozolin in fortified soil samples were determined at three levels: 0.5, 2.5, and 5 μg/g for both enantiomers and are summarized in Table 5. Recoveries of each enantiomer fortified at three levels ranged from 85.72% to 105.70% with SD from 0.12% to 3.83%. TABLE 5. The recoveries (%) (±SD) of each enantiomer (n = 3) in soil Recovery (%) Test materials Soil 1#
Soil 2#
Soil 3#
Soil 4#
Soil 5#
Rac-vinclozolin fortification (μg/g) 1.0 5.0 10 1.0 5.0 10 1.0 5.0 10 1.0 5.0 10 1.0 5.0 10
(+) – vinclozolin 102.57 ± 1.02 88.79 ± 0.12 96.25 ± 0.50 99.63 ± 1.24 87.65 ± 1.47 96.95 ± 0.35 104.21 ± 2.08 95.21 ± 1.79 102.01 ± 0.79 98.06 ± 3.07 86.36 ± 0.48 105.34 ± 0.43 94.52 ± 1.44 86.25 ± 0.87 98.30 ± 0.78
() – vinclozolin 100.10 ± 1.68 89.43 ± 0.82 96.65 ± 0.42 101.14 ± 1.33 89.55 ± 1.50 96.14 ± 0.35 99.59 ± 1.36 96.86 ± 0.45 101.61 ± 0.52 101.53 ± 3.83 85.72 ± 2.09 105.70 ± 2.17 95.50 ± 1.33 88.40 ± 0.82 100.58 ± 1.51
Enantioselective Degradation in Soils
Degradation curves (concentration in soils versus incubation time) were regressed by Excel 2007 (Microsoft). The first-order polynomial regression analysis model was used to describe the dissipation process for vinclozolin enantiomers in the soils, with R2 values ranging from 0.9559 to 0.9943 (Table 6). It was observed that there was a big difference in the degradation of vinclozolin in five kinds of soils (Fig. 3). Both the enantiomers degraded much faster in soils 2# and 5# (with half-lives of less than 1 week) than in soils 1#, 3#, and 4# (with half-lives more than 3 weeks). From the results it could be inferred that pH was a decisive factor. In alkaline soils 2# and 5# with pH values higher than 8, vinclozolin was significantly degraded, while the degradation was much slower in acidic soils, which may also prove that pH can cause the dissolution of some organic matter and reduce vinclozolin adsorption. The difference of the degradation behavior of vinclozolin among the test soils could be explained that the different types of soils might contain different microorganisms, which preferentially degraded vinclozolin. Enantioselective degradation was observed in soil 3# with halflives of 26.45 d and 24.06 d for (+)–vinclozolin and ()– vinclozolin, respectively. But both enantiomers were metabolized at similar rates in other soils in 4 months, which suggested the degradations in those soils were not enantioselective. Microbial decomposition can play an important role in enantioselective metabolism of many chiral chemicals in soils.
TABLE 6. First order rate constants, k, half-life (t1/2), coefficient (R2), and the ES values of vinclozolin in soils under aerobic conditions Soil
Enantiomer
Soil 1#
(+) () (+) () (+) () (+) () (+) ()
Soil 2# Soil 3# Soil 4# Soil 5#
–vinclozolin –vinclozolin –vinclozolin –vinclozolin –vinclozolin –vinclozolin –vinclozolin –vinclozolin –vinclozolin –vinclozolin
k[d-1]
t1/2[d]
R2
ES
0.0136 0.0143 0.1014 0.1033 0.0262 0.0288 0.0211 0.0203 0.1166 0.1233
50.96 48.46 6.83 6.71 26.45 24.06 32.84 34.14 5.94 5.62
0.9763 0.9777 0.9821 0.9816 0.9943 0.9906 0.9722 0.9559 0.9570 0.9668
0.025
Fig. 3. Typical degradations of vinclozolin in the soils. A, B, C, D and E are respectively for the degradation of vinclozolin in soil 1# to soil 5#. Chirality DOI 10.1002/chir
0.009 0.047 0.019 0.028
CHIRAL SEPARATION ENANTIOSELECTIVE DEGRADATION VINCLOZOLIN SOIL
CONCLUSION
In the present work, the racemic vinclozolin was separated on Chiralpak IA, IB, AZ-H column under normal conditions. The results showed that the IB column was the best choice for separating the racemic vinclozolin. The method was successfully applied to analyze the two enantiomers extracted from soil samples. It was difficult to extract vinclozolin from soils by traditional methods because of the adsorption, so an ASE method was developed which proved to be satisfactory. There was a great difference in the half-lives of vinclozolin in the test soils, indicating that its dissipation depended on soil properties. Vinclozolin degraded faster in the soils with a pH higher than 7. Preferential degradation of the ()-enantiomer was just observed in one soil, leading to a enantioselective degradation with an |ES| of 0.047. Microbial decomposition played an important role in stereoselective metabolism of many chiral chemicals in soils. The underlying processes of both vinclozolin enantiomers in different environmental compartments are still unknown and further studies should be carried out, especially on the mechanism of enantioselectivity. 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. Wang P, Jiang SR, Qiu J, Wang QX, Wang P, Zhou ZQ. Stereoselective degradation of ethofumesate in turfgrass and soil. Pestic Biochem Physiol 2005;82:197–204. 2. Pérez-Fernández V, García MA, Marina ML. Chiral separation of agricultural fungicides. J Chromatogr A 2011;1218:6561– 6582. 3. Li ZY, Zhang ZC, Zhou QL, Wang QM, Gao RY, Wang QS. Stereo- and enantioselective determination of pesticides in soil by using achiral and chiral liquid chromatography in combination with matrix solid-phase dispersion. AOAC Int. 2003;86:521. 4. Monosson E, Kelce WR, Lambright C, Ostby J, Gray LE Jr. Peripubertal exposure to the antiandrogenic fungicide, vinclozolin, delays puberty, inhibits the development of androgen-dependent tissues, and alters androgen receptor function in the male rat. Toxicol Ind Health 1999;15:65. 5. Gray LE Jr, Ostby J, Monosson E, Kelce WR. Environmental antiandrogens: low doses of the fungicide vinclozolin alter sexual differentiation of the male rat. Toxicol Ind Health 1999;15:48. 6. Wolf CJ, LeBlanc GA, Ostby JS, Gray LE Jr. Characterization of the period of sensitivity of fetal male sexual development to vinclozolin. Toxicol Sci 2000;55:152–161. 7. Gray LE Jr, Ostby JC, Kelce WR. Developmental effects of an environmental antiandrogen: the fungicide vinclozolin alters sex differentiation of the male rat. Toxicol Appl Pharmacol 1994;129:46–52. 8. Szeto SY, Burlinson NE, Rahe JE, Oloffs PC. Kinetics of hydrolysis of the dicarboximide fungicide vinclozolin. J Agric Food Chem 1989;37:523. 9. Santana dos Santos TF, Aquino A, Silveira H, Navickiene DS. MSPD procedure for determining buprofezin, tetradifon,vinclozolin, and bifenthrin residues in propolis by gas chromatography–mass spectrometry. Anal Bioanal Chem 2008;390:1425–1430. 10. Lambropoulou DA, Albanis TA. Determination of the fungicides vinclozolin and dicloran in soils using ultrasonic extraction coupled with solid-phase microextraction. Anal Chim Acta 2004;514:125–130.
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11. Frías MM, Frenich, AG, Martínez Vidal JL, Sánchez MM, Olea F, Olea N. Analyses of lindane, vinclozolin, aldrin, p, p’-DDE, o, p’-DDT and p, p’-DDT in human serum using gas chromatography with electron capture detection and tandem mass spectrometry. J Chromatogr B 2001;760; 1–15. 12. Oliva J, Navarro S, Barba A, Navarro G. Determination of chlorpyrifos, penconazole, fenarimol, vinclozolin and metalaxyl in grapes, must and wine by on-line microextraction and gas chromatography. J Chromatogr A 1999;833:43–51. 13. Carlucci G, Di Pasquale D, Ruggieri F, Mazzeo P. Determination and validation of a simple high-performance liquid chromatographic method for simultaneous assay of iprodione and vinclozolin in human urine. J Chromatogr B 2005;828:108–112. 14. Cabras P, Tuberoso C, Melis M, Martini MG. Multiresidue method for pesticide determination in wine by high-performance liquid chromatography. J Agric Food Chem 1992;40:817–819. 15. Sierra-Santoyo A, Barton HA, Hughes MF. Liquid chromatography determination of the anti-androgen vinclozolin and its metabolites in rat serum. J Chromatogr B 2004;809:105–110. 16. Pose-Juan E, Cancho-Grande B, Rial-Otero R, Simal-Gándara J. The dissipation rates of cyprodinil, fludioxonil, procymidone and vinclozoline during storage of grape juice. Food Control 2006;17:1012–1017. 17. González-Rodríguez RM, Rial-Otero R, Cancho-Grande B, GonzalezBarreiro C, Simal-Gándara J. A review on the fate of pesticides during the processes within the food-production chain. Crit Rev Food Sci Nutr 2011;51:99–114. 18. Pateiro-Moure M, Arias-Estévez M, Simal-Gándara J. Critical review on the environmental fate of quaternary ammonium herbicides in soils devoted to vineyards. Environ Sci Technol 2013;47:4984–4998. 19. Arias-Estévez M, Soto-González B, López-Periago E, Cancho-Grande B, Simal-Gándara J. Atrazine sorption dynamics in acid-surface soils. Bull Environ Contam Toxicol 2005;75:264–271. 20. Pateiro-Moure M, Pérez-Novo C, Arias-Estévez M, Rial-Otero R, Simal-Gándara J. Effect of organic matter and iron oxides on quaternary herbicide sorption-desorption in vineyard-devoted soils. J Coloid Interf Sci 2009;333:431–438. 21. Arias-Estévez M, López-Periago E, Martínez-Carballo E, Simal-Gándara J. Carbofuran sorption kinetics by corn crop soils. Bull Environ Contam Toxicol 2006;77:267–273. 22. Pateiro-Moure M, Martínez-Carballo E, Arias-Estévez M, Simal-Gándara J. Determination of quaternary ammonium herbicides in soils: Comparison of digestion, shaking and microwave-assisted extractions. J Chromatogr A 2008;1196–1197:110–116. 23. Walker A, Brown PA, Entwistle AR. Enhanced degradation of iprodione and vinclozo lin in soil. Pestic Sci 1986;17:183–193. 24. Walker A Further observations on the enhanced degradation of iprodione and vinclozolin in soil. Pestic Sci 1987;21:219–231. 25. Garcia-Cazorla J, Xirau-Vayreda M. Monitoring degradation of dicarboximidic fungicide residues in soils. J Agric Food Chem 1998;46:2845–2850. 26. Slade EA, Fullerton RA, Stewart A, Young H. Degradation of the dicarboximide fungicides iprodione, vinclozolin and procymidone in patumahoe clay loam soil, New Zealand. Pestic Sci 1992;35:95–100. 27. Mitchell JA, Cain RB. Rapid onset of the accelerated degradation of dicarboximide fungicides in UK soil with a long history of agrochemical exclusion. Pestic Sci 1996;48:1–11. 28. Bicchi C, Cravotto G, Amato AD, Rubiolo P, Galli A, Galli M. J. Cyclodextrin derivatives in gas chromatographic separation of racemates with different volatility. Part XV: 6-O-t-butyldimethylsilyl- versus 6-O-thexyldimethylsilyl-β and -γ derivatives Microcolumn Sep 1999;11:487. 29. Wang P, Jiang S, Liu D, Shan W, Zhang H, Zhou Z. Chiral Separations of Pesticide Enantiomers by High-Performance Liquid Chromatography Using Cellulose Triphenylcarbamate Chiral Stationary Phase. J Chromatogr Sci 2006;44:602. 30. Wang P, Jiang S, Liu D, Zhang H, Zhou Z. Enantiomeric Resolution of Chiral Pesticides by High-Performance Liquid Chromatography. J Agric Food Chem 2006;54:157. 31. Kodama S, Yamamoto A, Saitoh Y, Matsunaga A, Okamura K, Kizu R, Hayakawa K. Enantioseparation of Vinclozolin by γ-Cyclodextrin-Modified Micellar Electrokinetic Chromatography. J Agric Food Chem 2002;50:1312. Chirality DOI 10.1002/chir
Chiral Separation and Enantioselective Degradation of Vinclozolin in Soils HUI LIU, DONGHUI LIU, ZHIGANG SHEN, MINGJING SUN, ZHIQIANG ZHOU,* AND PENG WANG Department of Applied Chemistry, China Agricultural University, Beijing, China
ABSTRACT Vinclozolin is a chiral fungicide with potential environmental problems. The chiral separation of the enantiomers and enantioselective degradation in soil were investigated in this work. The enantiomers were separated by high-performance liquid chromatography (HPLC) on Chiralpak IA, IB, and AZ-H chiral columns under normal phase and the influence of the mobile phase composition on the separation was also studied. Complete resolutions were obtained on all three chiral columns under optimized conditions with the same elution order of (+)/(). The residual analysis of the enantiomers in soil was conducted using accelerate solvent extraction followed by HPLC determination. The recoveries of the enantiomers ranged from 85.7–105.7% with relative standard deviation (SD) of 0.12–3.83%, and the limit of detection (LOD) of the method was 0.013 μg/g. The results showed that the degradations of vinclozolin enantiomers in the soils followed first-order kinetics. Preferential degradation of the ()-enantiomer was observed only in one soil with the largest |ES| value of 0.047, and no obvious enantioselective degradation was observed in other soils. It was found that the persistence of vinclozolin in soil was related to pH values based on the half-lives. The two enantiomers disappeared about 8 times faster in basic soils than that in neutral or acidic soils. Chirality 26:155–159, 2014. © 2014 Wiley Periodicals, Inc. KEY WORDS: vinclozolin; enantiomer; chiral separation; soil; selective degradation INTRODUCTION
It is already well known that a large number of organic agrochemicals are chiral and consist of mixtures of stereoisomers or enantiomers which may have different bioactivity, toxicity, metabolism, excretion, and environmental effects. There is growing concern about the side effects of chiral agrochemicals on nontarget organisms and natural resources.1 For these purposes, it is necessary to investigate the enantiomers separately. Separation of chiral compound enantiomers is a challenging topic in many analytical chemistry areas, especially in the biomedical, pharmaceutical, and environmental fields where pure enantiomeric forms are widely required. Separation techniques such as gas chromatography (GC), high-performance liquid chromatography(HPLC), supercritical fluid chromatography (SFC), and capillary electrophoresis (CE) have been widely employed, although, at present, HPLC still dominates chromatographic chiral analysis of chiral compounds.2 Because it is quite rapid and nondestructive, there is little possibility of epimerization during the analysis.3 Chirality is also popular in pesticides and about 30% of the widely used pesticides are chiral. Vinclozolin [3-(3,5dichlorophenyl)-5-methyl-5-vinyloxazolidine-2,4-dione] (Fig. 1) is a dicarboximide fungicide that is effective in the control of diseases caused by Botrytis cinerea, Sclerotinia sclerotiorum, and Moniliniam spp. It has been widely used in Europe to protect fruits, vegetables, ornamental plants, and turf grasses. The World Health Organization (WHO) has previously evaluated the fungicide vinclozolin as a noncarcinogen (FAO/WHO, 1986), but the U.S. Environmental Protection Agency (U.S. EPA) has classified vinclozolin as a Group C chemical, which indicates a possible human carcinogen (U. S. EPA, 1997). Vinclozolin has been shown to cause abnormal male sexual development in rats,4–6 alter morphological sex © 2014 Wiley Periodicals, Inc.
differentiation in male rats following perinatal exposure,7 and lead to the death of zebrafish embryos, developmental delays, and teratogenic effects, suggesting its potential health hazards. Vinclozolin can undergo chemical hydrolysis, photolysis, or metabolism by mammals and bacterial systems. Szeto et al.8 isolated three hydrolysis products from aqueous buffers which were identified as: 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2methyl-3-butenoic acid(M1), 3’5’-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2), and 3,5-dichloroaniline (M3). The metabolites of vinclozolin have been reported to act as antiandrogens and have adverse effects on reproductive physiology in animals. A number of investigations on the residual analysis of vinclozolin in foods and soils have been conducted by gas chromatography (GC), HPLC, and mass spectrometry.9–15 Solid-phase microextraction (SPME) coupled to GC analysis was evaluated for extracting trace amounts of vinclozolin in soil samples.10 Its dissipation during storage of grape juice was analyzed by liquid–liquid extraction (LLE), gas chromatographic separation, and mass spectrometric detection.16 Pesticides in groundwater, surface water, or the food chain were usually from contaminated soils; for example, grape quality was affected by vinclozolin-polluted soil.17 It was found that the extraction efficiency was influenced by the sorption– desorption,18 pH, heavy metal, and organic matter of soils. Arias-Estévez et al. found that an increasing pH could cause the dissolution of some organic matter and reduce atrazine adsorption, and copper slightly increased atrazine adsorption but Zn acted conversely.19 In subsurface soils with much *Correspondence to: Z. Zhou, Department of Applied Chemistry, China Agricultural University, Beijing 100193, China. E-mail: [email protected] Received for publication 25 September 2013; Accepted 12 November 2013 DOI: 10.1002/chir.22284 Published online 4 February 2014 in Wiley Online Library (wileyonlinelibrary.com).
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performed using an Agilent 1200 series HPLC (Agilent Technology, Palo Alto, CA) equipped with a G1311A pump, G1322A degasser, G1328A injector, a 20 μL sample loop, and G1315A DAD. The signal was received and processed by an Agilent chemstation The polarimeter was provided by IBZ Messtechnik (Germany) with mozartstrafic 14–16, D-30173, 1000 mV, Ser.No.:11/272.
Fig. 1. The chemical structure of vinclozolin.
Chiral Separation
lower organic matter content, Fe oxides could play an important role in overall sorption, because of the net electrical charge as well as the electric potential of the soils.20,21 So the properties of soil should be fully considered in the optimization of pesticide extraction.22 The biodegradation and metabolism of vinclozolin in soils and different plants has been studied extensively and the results have demonstrated that the degradation of this chemical in soil is a microbiologically mediated process. Vinclozolin is quite stable in acidic soils, especially under low temperature.23–26 The moisture content in soil also affected the degradation.27 Few studies in the literature are about chiral separation and enantioselectivity. Vinclozolin was first separated by GC on ACTBDMS-g-CD(OV-1701), ACTHDMS-g-CD(PS-086), and ACTHDMS-g-CD(OV-1701) reported in 1999.28 Wang et al. found that vinclozolin can be separated by cellulose triphenylcarbamate column29 and cellulose-tris-(3,5-dimethylphenylcarbamate) column with n-hexane/isopropanol 99:1 (v/v).30 Vinclozolin has been proven to be enantioselectively degraded in the wine-making process, resulting in the enrichment of ()vinclozolin.31 The enantioselective degradation of vinclozolin in the environmental matrix has not been reported. The chiral separation and the enantioselective degradation of vinclozolin enantiomers in several agriculture soils were investigated in this work. The results may improve our understanding of the environmental behavior of chiral pesticides. EXPERIMENTAL Chemicals and Reagents Rac-vinclozolin (>99.2% purity) was provided by the China Ministry of Agriculture Institute for Control of Agrochemicals. A stock solution of racvinclozolin was prepared in isopropanol and stored at 20 °C. Working standard solutions were obtained by dilutions of the stock solution with isopropanol. Water was purified by a Milli-Q system. Isopropanol, hexane, and ethyl acetate (analytical grade) were purchased from commercial sources, distilled, and filtered through a 0.45 μm filter membrane before use. All other chemicals and solvents were analytical grade and purchased from commercial sources.
The mobile phase was n-hexane/isopropanol or ethanol at a flow rate of 0.8 mL/min. Pesticide solutions were prepared by dissolving the samples in isopropanol at a concentration of 20 mg/L and the injection volume was 20 μL. The chromatographic separation was conducted at 20 °C and 210 nm, Chiralpak IA, IB, and AZ-H column (250 × 4.6 mm .ID) were provided by Daicel Chemical Industries, Arai Plant. Separation factor (α), and resolution factor (Rs) were calculated.
Degradation of Vinclozolin in Soils The test soils were from different agricultural regions (0–10 cm) of China that had not been treated with vinclozolin in the last 5 years. The properties are shown in Table 1. Test soils were adjusted to a moisture content of 30% and maintained in the dark at room temperature for 7 days to activate the microorganisms in soils. Soil subsample (390 g, corresponding to 300 g dry weight) was weighed into a 1000-mL Erlenmeyer flask. In order to add the pesticide to the soil evenly, 10 g of soil was first weighed into a 1000-mL Erlenmeyer flask and 0.5 mL of racemic vinclozolin solution (6000 μg/mL, acetone) was added. After that, another 380 g of soil was added gradually and stirred thoroughly to make a spiked concentration of 5 μg/g (each enantiomer). The flasks were sealed with cotton-wool plugs and stored at 15 °C in the dark for 120 days. The moisture content in each flask was checked gravimetrically at each sampling point. Five grams of the treated soil samples (based on the dry weight) was removed for analysis after incubation points of 0, 1, 3, 5, 7, 10, 14, 21, 28, 35, 50, 65, 80, 100, 120 days and stored at 20 °C before analysis. Each incubation was carried out in triplicate.
Soil Sample Treatment Soil samples were extracted by accelerated solvent extraction (ASE), in which factors such as extraction pressure, temperature, static time, and number of cycles were optimized. Wet soil samples were mixed with diatomite in a ratio of 2:1 in a mortar, and 6.0 g of the mixed sample was placed in the extraction cell and extracted with the following conditions: extraction temperature: 80°C; heating time: 5 min; static extraction time: 10 min; cycles: 2; rinse volume: 10%; nitrogen purge: 60s; extraction solvent: ethyl acetate. The eluting solution was evaporated to near dryness under a stream of nitrogen and reconstituted in 0.5 mL of isopropanol. An aliquot (20 μL) was injected on to the HPLC.
Assay Validation Apparatus The ASE-350 accelerated solvent extraction instrument was from Dionex (Sunnyvale, CA), with three solvent bottles, nitrogen bottle, 34-mL extraction pool, and 60-mL receiving flasks. Chromatography was
A series of working standard solutions (1-100 mg/L) of rac-vinclozolin were prepared. Calibration curves were generated by plotting peak area of each enantiomer versus the concentration. Linear regression analysis was performed using Microsoft-Excel. The concentration of each
TABLE 1. Selected physiochemical properties of the soils Particle size No.
Site
Sand (%)
Silt (%)
Clay (%)
Texture
pH (water)b
Corg [%]
1# 2# 3# 4# 5#
Heilongjiang Shenyang Guangxi Yunnan Gansu
42.15 42.86 42.86 71.77 14.98
33.06 40.82 28.57 12.10 36.44
24.79 16.33 28.57 16.13 48.58
Loam Loam Loam Sandy loam Clay loam
6.01 8.13 5.75 6.28 8.40
31.39 32.36 27.20 20.21 20.27
Chirality DOI 10.1002/chir
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CHIRAL SEPARATION ENANTIOSELECTIVE DEGRADATION VINCLOZOLIN SOIL
enantiomer in samples was calculated by the calibration curves of the corresponding enantiomer using the extended standard method. The limit of detection (LOD) of method for each enantiomer was considered to be the concentration that produced a signal-to-noise (S/N) ratio of 3, and the limit of quantification (LOQ) of method was defined as the lowest concentration in the calibration curve with acceptable precision and accuracy for 20% variability. All quantification was based on HPLC response (peak area of each enantiomer).The concentration of each enantiomer in samples was calculated by calibration curves of the corresponding enantiomer using the extended standard method. Absolute recovery of vinclozolin was determined in all the test soils fortified with racvinclozolin at 1, 5, and 10 μg/g. The enantiomer enantioselectivity (ES) was used to measure the selective degradation in soils. ES was defined as: ES ¼ ðk1 k2 Þ=ðk1 þ k2 Þ When the degradation followed a first-order kinetic, k1 and k2 were used as the degradation rate constant, respectively, for two enantiomers. The |ES| values ranged from 0 to 1. There was no enantioselective behavior if ES = 0.
RESULTS AND DISCUSSION Enantiomeric Separation and Elution Order
The two enantiomers could be completely separated on IA, IB, and AZ-H columns (Fig. 2), in which IB was the best. The chiral separations were performed by HPLC IB columns
using a mobile phase of n-hexane/ isopropanol or ethanol at a flow rate of 0.8 mL/min. The impact of mobile phase composition and temperature on the separation was optimized on the IB column and the results showed the two enantiomers could be fully separated using both isopropanol or ethanol as modifier and better separation was obtained under lower temperature and the mobile phase with less isopropanol or ethanol (Table 2). Assay Validation
Based on the successful chiral resolution of the two enantiomers, the chiral residue analysis method in soil samples was set up. An accelerated solvent extraction method was successfully applied to extract the two enantiomers from soil samples, which proved to be satisfactory for the pesticide with strong adsorption. Good linearity was obtained over the concentration range of 0.5–50 mg/L (n = 7) for both (+)-vinclozolin (y = 158.36x + 106.10,R2 = 0.9995) and ()vinclozolin (y = 158.47x +100.00, R2 = 0.9994). The LOQ for both enantiomers of the method in all samples was found to be 0.043 μg/g, and the LOD for both enantiomers of the method, defined as the concentration with an S/N ratio of 3, was 0.013 μg/g in soil samples (Table 3). The Cva➀ was found to be 0.2% to 5.1%, which proved to be satisfactory (Table 4).
Fig. 2. The chromatograms of the chiral separations of vinclozolin on A:IA column, 1 mL/min,20 °C,n-hexane/ isopropanol = 99:1;B:IB column,0.8 mL/min,20 °C, n-hexane/ isopropanol = 95:5;C: AZ-H column,0.8 mL/min,20 °C,n-hexane/ isopropanol = 99:1.
TABLE 2. The chromatographic separation of vinclozolin with n-hexane/modifier as mobile phase Temperature (°C)
Alcohol modifier (%)
10
Isopronanol, 2% Ethanol, 2% Isopronanol, 2% Ethanol, 2% Isopronanol, 2% Isopronanol, 5% Isopronanol, 10% Ethanol, 2% Ethanol, 5% Ethanol, 10% Isopronanol, 2% Ethanol, 2%
15 20
25
α
Rs
1.11 1.18 1.16 1.17 1.15 1.14 1.12 1.16 1.13 1.12 1.14 1.15
3.07 3.31 2.84 2.87 2.90 2.51 2.19 2.67 2.56 2.06 2.22 2.26
TABLE 4. The CVa of the analysis method for the vinclozolin enantiomers (n = 6) Cva➀ (%)
Interday
Day-to-Day
Concentration (μg/g)
(+) –vinclozolin
() –vinclozolin
1.0 5.0 50 1.0 5.0 50
5.1 0.7 0.4 4.6 2.8 0.4
4.6 0.8 0.2 3.2 1.2 1.1
TABLE 3. Validation of vinclozolin enantiomer analysis Enantiomer (+) –vinclozolin () –vinclozolin
Linear equation
R2
Linear range (μg/mL)
LOD (μg/g)
LOQ (μg/g)
y = 158.36x + 106.10 y = 158.47x + 100.00
0.9995 0.9994
0.5-50 0.5-50
0.013 0.013
0.043 0.043 Chirality DOI 10.1002/chir
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LIU ET AL.
The recoveries of vinclozolin in fortified soil samples were determined at three levels: 0.5, 2.5, and 5 μg/g for both enantiomers and are summarized in Table 5. Recoveries of each enantiomer fortified at three levels ranged from 85.72% to 105.70% with SD from 0.12% to 3.83%. TABLE 5. The recoveries (%) (±SD) of each enantiomer (n = 3) in soil Recovery (%) Test materials Soil 1#
Soil 2#
Soil 3#
Soil 4#
Soil 5#
Rac-vinclozolin fortification (μg/g) 1.0 5.0 10 1.0 5.0 10 1.0 5.0 10 1.0 5.0 10 1.0 5.0 10
(+) – vinclozolin 102.57 ± 1.02 88.79 ± 0.12 96.25 ± 0.50 99.63 ± 1.24 87.65 ± 1.47 96.95 ± 0.35 104.21 ± 2.08 95.21 ± 1.79 102.01 ± 0.79 98.06 ± 3.07 86.36 ± 0.48 105.34 ± 0.43 94.52 ± 1.44 86.25 ± 0.87 98.30 ± 0.78
() – vinclozolin 100.10 ± 1.68 89.43 ± 0.82 96.65 ± 0.42 101.14 ± 1.33 89.55 ± 1.50 96.14 ± 0.35 99.59 ± 1.36 96.86 ± 0.45 101.61 ± 0.52 101.53 ± 3.83 85.72 ± 2.09 105.70 ± 2.17 95.50 ± 1.33 88.40 ± 0.82 100.58 ± 1.51
Enantioselective Degradation in Soils
Degradation curves (concentration in soils versus incubation time) were regressed by Excel 2007 (Microsoft). The first-order polynomial regression analysis model was used to describe the dissipation process for vinclozolin enantiomers in the soils, with R2 values ranging from 0.9559 to 0.9943 (Table 6). It was observed that there was a big difference in the degradation of vinclozolin in five kinds of soils (Fig. 3). Both the enantiomers degraded much faster in soils 2# and 5# (with half-lives of less than 1 week) than in soils 1#, 3#, and 4# (with half-lives more than 3 weeks). From the results it could be inferred that pH was a decisive factor. In alkaline soils 2# and 5# with pH values higher than 8, vinclozolin was significantly degraded, while the degradation was much slower in acidic soils, which may also prove that pH can cause the dissolution of some organic matter and reduce vinclozolin adsorption. The difference of the degradation behavior of vinclozolin among the test soils could be explained that the different types of soils might contain different microorganisms, which preferentially degraded vinclozolin. Enantioselective degradation was observed in soil 3# with halflives of 26.45 d and 24.06 d for (+)–vinclozolin and ()– vinclozolin, respectively. But both enantiomers were metabolized at similar rates in other soils in 4 months, which suggested the degradations in those soils were not enantioselective. Microbial decomposition can play an important role in enantioselective metabolism of many chiral chemicals in soils.
TABLE 6. First order rate constants, k, half-life (t1/2), coefficient (R2), and the ES values of vinclozolin in soils under aerobic conditions Soil
Enantiomer
Soil 1#
(+) () (+) () (+) () (+) () (+) ()
Soil 2# Soil 3# Soil 4# Soil 5#
–vinclozolin –vinclozolin –vinclozolin –vinclozolin –vinclozolin –vinclozolin –vinclozolin –vinclozolin –vinclozolin –vinclozolin
k[d-1]
t1/2[d]
R2
ES
0.0136 0.0143 0.1014 0.1033 0.0262 0.0288 0.0211 0.0203 0.1166 0.1233
50.96 48.46 6.83 6.71 26.45 24.06 32.84 34.14 5.94 5.62
0.9763 0.9777 0.9821 0.9816 0.9943 0.9906 0.9722 0.9559 0.9570 0.9668
0.025
Fig. 3. Typical degradations of vinclozolin in the soils. A, B, C, D and E are respectively for the degradation of vinclozolin in soil 1# to soil 5#. Chirality DOI 10.1002/chir
0.009 0.047 0.019 0.028
CHIRAL SEPARATION ENANTIOSELECTIVE DEGRADATION VINCLOZOLIN SOIL
CONCLUSION
In the present work, the racemic vinclozolin was separated on Chiralpak IA, IB, AZ-H column under normal conditions. The results showed that the IB column was the best choice for separating the racemic vinclozolin. The method was successfully applied to analyze the two enantiomers extracted from soil samples. It was difficult to extract vinclozolin from soils by traditional methods because of the adsorption, so an ASE method was developed which proved to be satisfactory. There was a great difference in the half-lives of vinclozolin in the test soils, indicating that its dissipation depended on soil properties. Vinclozolin degraded faster in the soils with a pH higher than 7. Preferential degradation of the ()-enantiomer was just observed in one soil, leading to a enantioselective degradation with an |ES| of 0.047. Microbial decomposition played an important role in stereoselective metabolism of many chiral chemicals in soils. The underlying processes of both vinclozolin enantiomers in different environmental compartments are still unknown and further studies should be carried out, especially on the mechanism of enantioselectivity. 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. Wang P, Jiang SR, Qiu J, Wang QX, Wang P, Zhou ZQ. Stereoselective degradation of ethofumesate in turfgrass and soil. Pestic Biochem Physiol 2005;82:197–204. 2. Pérez-Fernández V, García MA, Marina ML. Chiral separation of agricultural fungicides. J Chromatogr A 2011;1218:6561– 6582. 3. Li ZY, Zhang ZC, Zhou QL, Wang QM, Gao RY, Wang QS. Stereo- and enantioselective determination of pesticides in soil by using achiral and chiral liquid chromatography in combination with matrix solid-phase dispersion. AOAC Int. 2003;86:521. 4. Monosson E, Kelce WR, Lambright C, Ostby J, Gray LE Jr. Peripubertal exposure to the antiandrogenic fungicide, vinclozolin, delays puberty, inhibits the development of androgen-dependent tissues, and alters androgen receptor function in the male rat. Toxicol Ind Health 1999;15:65. 5. Gray LE Jr, Ostby J, Monosson E, Kelce WR. Environmental antiandrogens: low doses of the fungicide vinclozolin alter sexual differentiation of the male rat. Toxicol Ind Health 1999;15:48. 6. Wolf CJ, LeBlanc GA, Ostby JS, Gray LE Jr. Characterization of the period of sensitivity of fetal male sexual development to vinclozolin. Toxicol Sci 2000;55:152–161. 7. Gray LE Jr, Ostby JC, Kelce WR. Developmental effects of an environmental antiandrogen: the fungicide vinclozolin alters sex differentiation of the male rat. Toxicol Appl Pharmacol 1994;129:46–52. 8. Szeto SY, Burlinson NE, Rahe JE, Oloffs PC. Kinetics of hydrolysis of the dicarboximide fungicide vinclozolin. J Agric Food Chem 1989;37:523. 9. Santana dos Santos TF, Aquino A, Silveira H, Navickiene DS. MSPD procedure for determining buprofezin, tetradifon,vinclozolin, and bifenthrin residues in propolis by gas chromatography–mass spectrometry. Anal Bioanal Chem 2008;390:1425–1430. 10. Lambropoulou DA, Albanis TA. Determination of the fungicides vinclozolin and dicloran in soils using ultrasonic extraction coupled with solid-phase microextraction. Anal Chim Acta 2004;514:125–130.
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