Ecotoxicology and Environmental Safety 117 (2015) 1–6
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Study on the stereoselective degradation of three triazole fungicides in sediment Qing Zhang, Liangliang Zhou, Yu Yang, Xiude Hua, Haiyan Shi, Minghua Wang n Department of Pesticide Science, College of Plant Protection, Nanjing Agricultural University, State & Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing 210095, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 4 January 2015 Received in revised form 12 March 2015 Accepted 13 March 2015
The stereoselective degradation behaviors of chiral triazole fungicides (hexaconazole, flutriafol and tebuconazole) in sediment were investigated under laboratory conditions. The enantiomers were completely separated by high-performance liquid chromatography on a cellulose tris(3-chloro-4-methylphenylcarbamate) (Lux Cellulose-2) column. The mean recoveries of hexaconazole, flutriafol and tebuconazole in sediment ranged from 86.7% to 105.9%. The methods were successfully applied for the enantioselective degradation analysis of fungicides in sediment. The results showed that the dissipation of hexaconazole, flutriafol and tebuconazole stereoisomers in sediment followed first-order kinetics (R2 40.95). The degradation rate of the enantiomers was different in sediment, and the ( )-enantiomer (t1/2 was 86 days for hexaconazole, 139 for flutriafol and 136 for tebuconazole) degraded faster than the ( þ)-enantiomer (t1/2 was 94 days for hexaconazole, 144 for flutriafol and 151 for tebuconazole) in native condition. The fungicides were degraded slowly, and no significant enantioselective degradation were observed under sterilized conditions. The results may hold promising implications for the environmental and ecological risk assessment of three important chiral triazole fungicides. & 2015 Elsevier Inc. All rights reserved.
Keywords: Stereoselective degradation Sediment Hexaconazole Flutriafol Tebuconazole
1. Introduction Triazole fungicides are widely used broad-spectrum fungicides that inhibit the sterol 14α-demethylase, an enzyme involved in the biosynthesis of ergosterol (Buerge et al., 2006). Almost all triazole fungicides are chiral due to the presence of the asymmetrically substituted carbon atoms in the triazol alkyl moiety. These chiral molecule chemicals consist of two or four stereoisomers. Although enantiomers have identical physico-chemical properties, their interactions with biological macromolecules are often chiral-selective, leading to enantiomer selectivity in biodegradation, ecotoxicity and human health effects. These interactions may also contribute to differences in biological activity, toxicity on beneficial or nontarget organisms and environmental fate (Ribeiro et al., 2012; Dong et al., 2013; Han et al., 2013; Sun et al., 2012;). However, chiral pesticides are generally produced and used as racemates or mixtures of stereoisomers. The introduction of inactive isomers into the environment may result in unwanted side effects. Therefore, there is an increasing interest in evaluating the enantioselective behavior of chiral pesticides in the environment. Hexaconazole, flutriafol and tebuconazole possess an n
Corresponding author. Fax: þ 86 25 84395479. E-mail address: [email protected] (M. Wang).
http://dx.doi.org/10.1016/j.ecoenv.2015.03.014 0147-6513/& 2015 Elsevier Inc. All rights reserved.
asymmetrically substituted carbon atom and consist of a pair of enantiomers (Fig. 1), which are registered as racemic products in many countries. They are typically applied as foliar sprays to control diseases caused by ascomycetes, basidiomycetes and deuteromycetes of cereals, vegetables and fruits (Wang et al., 2012a; Zhang et al., 2014). Although the triazole fungicides are extremely persistent in soil, they may appear in aquatic systems and enriched river sediments via runoff or soil erosion (Belluck et al., 1991; Yan et al., 2014). As a result, sediments are usually regarded as important sinks or receptors of pesticides. As is well known, in addition to stereoselective degradation and transformation in the environment (Dong et al., 2013; Han et al., 2013; Wang et al., 2014), triazole fungicides enantiomers also have different toxic effects on aquatic organisms (Obliquus, 2012; Zhang et al., 2012a; Zhu et al., 2007; Cao et al., 2009). When racemic compounds are employed, approximately 50% or more of ineffective or less active products will be incorporated into the environment (Pérez-Fernandez et al., 2011; Armstrong et al., 1993; Lewis et al., 1999; Zhang et al., 2012b). These ineffective enantiomers are widely used in agricultural systems and enriched river sediments, and they may lead to more side effects on nontarget aquatic organisms (such as algae and zebrafish). To the best of our knowledge, no study has been reported about the enantioselective degradation behavior of hexaconazole, flutriafol and tebuconazole in sediment. Sediment is an important ecosystem for aquatic
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Fig. 1. Molecular structures (nindicates chiral center) and typical chromatograms of the chiral separations of fungicides (hexaconazole, flutriafol and tebuconazole).
2. Materials and methods
working solutions with concentrations of 0.5, 1, 2, 5, 10 and 20 mg L 1 were prepared by serial dilution. Purified water was obtained using a MUL-9000 water purification system (Nanjing Zongxin Water Equipment Co. Ltd., China). HPLC-grade acetonitrile was purchased from TEDIA (Fairfield, USA), and Cleanert florisil (500 mg, 6 mL) were obtained from Agela Technologies (Tianjin, China). The mobile phase was filtered through a 0.22 mm filter membrane (Tengda, Tianjin, China). Sodium chloride, anhydrous sodium sulfate, ethyl acetate, acetonitrile, acetone and n-hexane of analytical grade were purchased from commercial sources. The chromatographic separation of the enantiomers was performed on an Agilent 1200 HPLC system (Agilent, USA) with UV detection on chiral Lux Cellulose-2 [cellulose tris(3-chloro-4-methylphenylcarbamate)] column supplied by Phenomenex (Torrance, USA); The column was packed with 3-μm particles with 150 mm 2.0 mm i.d. Sediment samples were collected from Taihu Lake (0–10 cm surface layer) located in Suzhou, China. Taihu Lake is the third largest freshwater body downstream of the Yangtze River, China. The sediment samples were air-dried at room temperature, passed through a 2-mm sieve and stored in the dark. The sediments showed no hexaconazole, flutriafol and tebuconazole before fortification. The physicochemical characteristics of the sediment were as follows: pH 7.007 0.10; organic matter, 1.41 70.10%; clay, 18.70 70.81%; sand, 6.55 70.13; and silt, 74.75 71.23%.
2.1. Chemicals and materials
2.2. Incubation experiments
Standard of rac-hexaconazole (499.9% purity), rac-flutriafol (4 97.3% purity) and rac-tebuconazole ( 498.2% purity) were obtained from the China Shanghai Pesticide Research Institute (Shanghai, China). Enantiomers of hexaconazole, flutriafol, tebuconazole (purity Z 97.0%) were obtained from the Shanghai Chiralway Biotech Co., Ltd. (Shanghai, China). Stock solutions of hexaconazole, flutriafol and tebuconazole standard (1000 mg L 1) were prepared in acetonitrile and stored at 20 °C. Standard
Incubation experiments on hexaconazole, flutriafol and tebuconazole in native and sterile sediment under laboratory conditions were conducted to evaluate the contribution of abiotic degradation and biodegradation to the dissipation of triazole fungicides in sediment. Tests on the degradation of hexaconazole, flutriafol and tebuconazole were conducted with the racemic compounds using 100-mL polypropylene centrifuge tubes sealed with sterile cotton plugs (Xu et al., 2012). To avoid the potential effects
organisms. It is necessary to establish the appropriate method for the separation and study of the enantioselective dissipation kinetics of hexaconazole, flutriafol and tebuconazole residues. Such a study would provide more information relevant to environmental and ecological risk assessment. The objective of this work was to study the enantioselective degradation and persistence of the racemic forms of hexaconazole, flutriafol and tebuconazole in Taihu Lake sediment using reversephase and chiral high-performance liquid chromatography (HPLC). The enantiomers of three triazole fungicides were completely separated on Lux Cellulose-2 columns by Phenomenex (Torrance, USA) packed with cellulose tris(3-chloro-4-methylphenylcarbamate) combined with acetonitrile and water as the mobile phase. In addition, the stereoselective degradation of hexaconazole, flutriafol and tebuconazole was investigated in the enantiomers’ native as well as sterilized conditions. The current report is the first to investigate the enantioselective dissipation of three triazole fungicides in sediment. The results of this research, which are relevant to stereoselectivity and degradation in sediment, may hold implications for improving the environmental and human risk assessment of these three widely used chiral triazole fungicides (Li et al., 2012).
Q. Zhang et al. / Ecotoxicology and Environmental Safety 117 (2015) 1–6
of solvents on the microbiological activity of the soils, the following fortification methods were adopted (Brinch et al., 2002). A portion of the sediment (20 g) was first spiked with 100 μL of stock solution in acetone containing approximately 1000 μg of fungicide (hexaconazole, flutriafol or tebuconazole) and stirred for 5 min. The soil (180 g) was added and mixed thoroughly for 15 min, yielding a fortification level of 2.5 μg g 1 for each enantiomer. After the solvent was completely evaporated, the sediment (10 g) was transferred to a 100 mL polypropylene centrifuge tube and rehydrated with 10 g of deionized water, and the overlying water height was approximately 2–3 cm (Di et al., 2013). The sediment was incubated at 25 71 °C in the dark and weighted regularly for water content. Three replicate samples were removed from each treatment at different time intervals (0, 1, 3, 7, 10, 14, 21, 30, 45, 60, 90, 120 and 150 days). Approximately 10 g of sediment weighted in 100 mL polypropylene centrifuge tubes was sterilized by autoclaving twice at 121 °C for 30 min in 24 h intervals. The sterilized samples were fortified with 50 μg of racemic fungicide (hexaconazole, flutriafol or tebuconazole), and 10 g sterile water was added to the samples under 15 min of ultraviolet irradiation on clean bench. The samples were incubated at 25 71 °C in the dark and removed from each treatment at different time intervals. 2.3. Sample preparation and extraction Ethyl acetate (50 mL) was added to each polypropylene centrifuge tube (10 g of sediment). The tubes were stirred for 3 min on a vortex mixer and extracted by sonication for 10 min, and 2 g sodium chloride was added for complete extraction. The tube was then stirred for 1 min on a vortex mixer and centrifuged for 5 min at 4000 rpm. The supernatant (25 mL) was evaporated to neardryness on an evacuated rotary evaporator at 45 °C and redissolved in a 5-ml mixture of hexane and acetone (98:2, v/v) for cleanup. 2.4. Sample purification procedure The concentrated extracts were transferred to a florisil cartridge. The Cleanert florisil-SPE was rinsed with 6 mL hexane– acetone (95:5, v/v) and then eluted with 15 mL hexane–acetone (85:15, v/v). All eluates were collected and evaporated to dryness under vacuum at 40 °C. The sample was dissolved in 1 mL acetonitrile and filtered through a 0.22-μm filter for HPLC analysis. 2.5. Enantiomer HPLC determination The chromatographic separation of the enantiomers of hexaconazole, flutriafol and tebuconazole were performed on an Agilent 1200 HPLC (Agilent, USA) with chiral Lux Cellulose-2 columns and an injection volume of 20 μL. The mobile phase consisted of acetonitrile and water (40:60 for flutriafol, 67:33 for hexaconazole, 68:32 for tebuconazole, v/v) and delivered at a different rate (0.8 mL min 1 for flutriafol, 0.65 mL min 1 for hexaconazole, 0.7 mL min 1 for tebuconazole). The column was kept at 30 °C, and UV detection was performed at 223 nm for tebuconazole, 210 nm for flutriafol and 220 nm for hexaconazole. The chromatograms of raceme and enantiomers were shown in Fig. 1. The retention times between the two enantiomers were distinguished with complete separation (17.98, 19.50 min for flutriafol; 12.15, 16.43 min for hexaconazole; 11.04, 12.44 min for tebuconazole). The (þ )-enantiomer was eluted earlier than the ( )-enantiomer for tebuconazole and hexaconazole, whereas the ( )-flutriafol was confirmed as the first eluted enantiomer and the (þ )-flutriafol as the second eluted enantiomer.
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2.6. Calibration curves and assay validation The linearities of the fungicides were established using a series of standard solvent solutions and standard matrix-matched enantiomer solutions by plotting peak areas against analyte concentrations from 0.5 to 20 mg L 1. The linear regression results obtained from the matrix-matched calibration curves were evaluated for the matrix effect. Method accuracy and precision were evaluated by recovery studies using spiked samples at three concentration levels (0.05, 0.25 and 2.5 mg kg 1 for each enantiomer based on five replicates) over three different days. The spiked samples were equilibrated for 2 h and then processed according to the above-described procedures. Blank analyses were performed to check for interference from the matrix. The precision of the method was determined by repeatability and reproducibility studies and expressed as relative standard deviation (RSD). The limit of detection (LOD) and limit of quantification (LOQ) for the enantiomers of hexaconazole, flutriafol and tebuconazole were considered concentrations in a sample matrix, resulting in peak areas with signal-to-noise (S/N) ratios of 3 and 10. The capacity factor for the (þ)- and ( )-enantiomers (k1 and k2, respectively), the separation factor (α), and the resolution (Rs) were calculated. A kinetic study of the enantiomers in sediment was performed by plotting residue concentration against time; the degradation rate constant k of the enantiomers were calculated from Eq. (1), and the half-life (t1/2, day) was estimated from Eq. (2).
C = C0e−kt
(1)
t1/2 = ln2/k = 0.693/k
(2)
Residual concentration data of the two enantiomers were used to estimate the enantiomer ratio (ER) during experiments. The ER was defined as the concentration of the ( )-enantiomer divided by the concentration of the ( þ)-enantiomer, where an ER of 1 represents the racemate (Wang et al., 2007). A more rapid dissipation of the (þ)-enantiomer with ER more than 1, whereas ( )-enantiomer degraded faster than the (þ)-form. The enantiomeric selectivity (ES) was also used to confirm the overall trend of the enantioselective dissipation process of hexaconazole, flutriafol and tebuconazole in sediment (Buerge et al., 2003). Enantiomeric selectivity (ES) was defined by Eq. (3).
ES = (k (+) − k (−))/(k (−) + k (+))
(3)
Positive values (0 o ESr1) indicate a more rapid dissipation of the (þ )-enantiomer, and negative values ( 1 rES o0) indicate a more rapid dissipation of the ( )-enantiomer. An ES value of 0 indicates a dissipation that is not enantioselective (Wang et al., 2012b).
3. Results and discussion 3.1. Matrix effects and linearity Sample extraction and cleanup procedures based on the modified QuEChERS method facilitated ultrasonic extraction. Methods for analyzing hexaconazole, flutriafol and tebuconazole in sediment were successfully developed and validated by HPLC. The two enantiomers could be completely separated with a resolution (Rs) of over 1.5 (1.82 for flutriafol, 3.11 for tebuconazole and 9.1 for hexaconazole), and there were no endogenous interference peaks at the same retention time of the three fungicides. The linear regression results for the standard solution and matrix-matched calibration curves for each enantiomer are summarized in Table 1. Good linear calibration curves for each
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Table 1 Matrix effects and linearity for tebuconazole, flutriafol and hexaconazole enantiomers. Compound
Concentration (mg L 1)
Standard solution linear equation, R2
Matrix-matched linear equation, R2
P-value
( þ )-Tebuconazole ( )-Tebuconazole ( þ )-Flutriafol ( )-Flutriafol ( þ )-Hexaconazole ( )-Hexaconazole
0.5–20 0.5–20 0.5–20 0.5–20 0.5–20 0.5–20
y¼ 37.57x 1.512, 0.9999 y¼ 37.39x 2.555, 0.9999 y¼ 42.10xþ 0.951, 0.9999 y¼ 41.95x 1.860, 0.9999 y¼ 33.05xþ 2.133, 0.9999 y¼ 33.17x þ3.189, 0.9999
y¼ 37.31xþ 0.245, 0.9993 y¼ 37.22x 1.280, 0.9994 y¼ 43.10x 3.112, 0.9992 y¼ 43.22x 3.279, 0.9996 y ¼33.86x 1.389, 0.9995 y ¼33.33x 1.066, 0.9994
0.085 0.094 0.486 0.197 0.296 0.058
enantiomer were obtained over the range of 0.5–20 μg mL 1 with R2 Z0.9992. The matrix effect was calculated by comparing standard solution curves with matrix-matched calibration curves using a two-tailed paired t-test with confidence interval of 95%. Table 1 showed that no significant matrix-induced enhancement or suppression effect was observed for each enantiomer and that the matrix effect could be negligible (P4 0.05). 3.2. Validation of the method The recoveries and RSD values are shown in Table 2. The mean recoveries of hexaconazole, flutriafol and tebuconazole at three spiked levels in sediment ranged from 86.7% to 105.9%. The relative standard deviation (RSD) values ranged from 2.1% to 8.7% for intra- and inter-day studies. The recovery and precision results indicate that the method was efficient and reliable. The LOD and LOQ were estimated to be 0.013 and 0.030 mg kg 1 for each enantiomer of hexaconazole, flutriafol and tebuconazole at low concentrations. 3.3. Degradation of hexaconazole, flutriafol and tebuconazole in sediment in native condition The degradation kinetics of hexaconazole, flutriafol and tebuconazole enantiomers in sediment followed first-order kinetics, as shown Table 3, and the typical chromatograms are shown in Fig. 2. The concentrations of the enantiomers were different in the chiral HPLC chromatograms for the three fungicides at 120 days. The
residues of both enantiomers of hexaconazole, flutriafol and tebuconazole decreased with time in sediment, and the data show that the ( )-enantiomer (t1/2 ¼ 86 days for hexaconazole, 139 for flutriafol and 136 for tebuconazole) degrades faster than the (þ)-enantiomer (t1/2 ¼94 days for hexaconazole, 144 for flutriafol and 151 for tebuconazole) with a significant difference (Po 0.05, Student’s paired t-test). The ER values of the fungicides consistently decreased over time (Fig. 2). A t-test between the ER values of hexaconazole, flutriafol and tebuconazole in sediment and ER ¼ 1.0 yielded P values of 0.00021, 0.00017, and 0.01028, respectively. These results indicate that enantioselectivity existed during the dissipation of the three fungicides in sediment. The data show that the ( )-enantiomer degraded more rapidly, leading to residues enriched in ( þ)-enantiomer during incubation. The degradation of pesticides in soil or sediment has been attributed to microorganisms (Buerge et al., 2006; Wu et al., 2013; Yuan et al., 2014; Zhi et al., 2014). The ES values observed in the present study also suggest that the compounds examined are, at least to some extent, degraded by biological processes, whereas abiotic degradation would generally not be enantioselective (ES¼ 0). In this study, the ES value was 0.045 for hexaconazole, 0.020 for flutriafol and 0.052 for tebuconazole (Table 3). These ES values suggest that the dissipation of hexaconazole, flutriafol and tebuconazole in sediment is enantioselective, and ( )-enantiomer degraded faster than the (þ)-form. According to a previous report, the enantiomers may be preferentially degraded or transformed by micro-organisms in the environment (Lewis et al., 1999).
Table 2 Recovery and RSD for tebuconazole, flutriafol and hexaconazole enantiomers in sediment. Compound
Spiked level (mg kg 1)
Intra-day (n ¼5)
Inter-day (n¼ 15) RSD (%)
Day 1
( þ )-Tebuconazole
0.05 0.25 2.5 ( )-Tebuconazole 0.05 0.25 2.5 ( þ )-Flutriafol 0.05 0.25 2.5 ( )-Flutriafol 0.05 0.25 2.5 ( þ )-Hexaconazole 0.05 0.25 2.5 ( )-Hexaconazole 0.05 0.25 2.5
Day 2
Day 3
Average Recoveries (%)
RSD (%) Average Recoveries (%)
RSD (%) Average Recoveries (%)
RSD (%)
92.0 92.7 89.2 87.6 89.4 87.9 104.3 86.9 90.2 105.9 86.7 88.9 98.6 90.3 91.7 95.6 90.0 91.1
3.8 4.2 5.3 2.4 2.1 3.2 4.1 4.1 2.3 5.7 4.3 5.3 3.6 3.6 4.2 3.8 4.8 3.8
5.1 3.9 6.7 3.1 3.7 4.3 7.9 5.9 3.7 8.7 5.2 4.9 2.5 2.9 3.8 2.1 3.9 5.9
5.9 5.1 7.5 4.1 4.3 5.9 8.5 3.9 3.9 7.3 2.7 4.2 3.2 4.1 3.3 4.8 5.7 4.9
88.7 91.2 87.5 87.3 88.7 88.3 96.7 94.6 91.7 88.7 93.7 97.3 98.7 89.3 90.9 91.7 94.3 90.7
95.6 94.9 97.8 92.0 95.6 91.1 93.8 87.9 95.8 95.4 90.4 93.9 94.6 88.9 89.5 94.3 89.3 88.5
4.3 4.7 6.6 3.2 3.9 4.4 6.1 4.5 3.3 7.4 4.5 4.6 2.2 3.1 2.9 3.3 4.6 5.1
Q. Zhang et al. / Ecotoxicology and Environmental Safety 117 (2015) 1–6
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Table 3 Regression functions, half-life (t1/2), correlation coefficient (R2), and ES values for the degradation of tebuconazole, flutriafol and hexaconazole enantiomers in native and sterile sediment.
Native sediment
Sterile sediment
a b
Enantiomer
Regression functionsa
R2
Half-life ( t1/2, day)b
ES
( þ )-Tebuconazole ( )-Tebuconazole ( þ )-Flutriafol ( )-Flutriafol ( þ )-Hexaconazole ( )-Hexaconazole ( þ )-Tebuconazole ( )-Tebuconazole ( þ )-Flutriafol ( )-Flutriafol ( þ )-Hexaconazole ( )-Hexaconazole
Ct ¼2.3854e 0.0046x Ct ¼2.3339e 0.0051x Ct ¼2.2240e 0.0048x Ct ¼2.2259e 0.0050x Ct ¼2.3149e 0.0074x Ct ¼2.2250e 0.0081x Ct ¼2.3744e 0.0026x Ct ¼2.3638e 0.0026x Ct ¼2.3035e 0.0025x Ct ¼2.3098e 0.0025x Ct ¼2.4109e 0.0050x Ct ¼2.3901e 0.0050x
0.9775 0.9677 0.9557 0.9682 0.9703 0.9712 0.9651 0.9644 0.981 0.9595 0.9845 0.9774
150.65 71.65 135.8872.07 144.38 71.07 138.60 70.98 93.65 71.18 85.56 71.38 266.54 73.58 266.54 73.23 277.2073.76 277.2073.37 138.60 72.14 138.60 72.01
0.052 0.020 0.045 0 0 0
The regressive functions are based on the mean value of three replicates. Values represent the means (n¼ 3).
Fig. 2. Typical chromatograms and time development of enantiomeric ratio (ER) on native condition. Chromatograms of hexaconazole (a), flutriafol (b) and tebuconazole (c) in sediment after 120 days. ER of hexaconazole (d), flutriafol (e) and tebuconazole (f) in sediment.
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3.4. Degradation of hexaconazole, flutriafol and tebuconazole in sediment under sterilized condition The degradation kinetics of the three fungicides showed firstorder behavior under the sterilized condition, with the correlation coefficient values (R2) ranging from 0.9595 to 0.9845 (Table 3). The results of the kinetics study of fungicide residues in sediment are shown in Table 3. The enantiomers have the same half-life and the ES values was 0 during the period of incubation, indicating three triazole fungicides (hexaconazole, flutriafol and tebuconazole) degradation was not enantioselective in sediment in the sterilized condition. The dissipation of the three fungicides was rather slow, with half-life values ranging from 139 to 277 days (Table 3). The degradation order of the three fungicides was observed to be hexaconazole 4tebuconazole4 flutriafol. The dissipation of flutriafol was slowest in the sterilized condition, with a half-life of 277 days, and tebuconazole was dissipated at a rate similar to that observed for flutriafol (t1/2 ¼267 days). Most importantly, the halflife value of hexaconazole in sediment was 139 days less than two times that of the other fungicides. The observed differences in the persistence of the three fungicides in sediment may be determined by their branch chain structure because the fungicides have the same core skeleton structure. It is clear that the persistence of the fungicides in the sterilized condition was two times longer than that in the native condition. The degradation of hexaconazole, flutriafol and tebuconazole involved both abiotic degradation and biodegradation in native soil, whereas only abiotic degradation occurred in sterile soil. The difference in the degradation behavior of the fungicides in sediment could be greatly attributed to microbial populations equipped with a variety of metabolic enzymes, which are preferential degraders of different enantiomers (Zhi and Ji, 2014). This could be one reason for the different ranking order of the two enantiomers of fungicides with respect to their degradation rate constants.
4. Conclusions In this study, an effective method for enantioselective determination of hexaconazole, flutriafol and tebuconazole enantiomers in sediment was established and validated. The methods were successfully used to study the degradation of the stereoisomers in sediment. The result shows that the degradation of the hexaconazole, flutriafol and tebuconazole enantiomers in sediment occurs with some chiral preference in the native condition. The ( )-enantiomer degraded faster than the ( þ)-form, leading to residues enriched in the (þ)-form. The different degradation rates of the enantiomers and their chiral preference may depend on the variety of microbial populations with genes harboring different metabolic enzymes that occur in sediment. These findings may hold implications for improving environmental and ecological risk assessment and are also relevant to pesticide registration and approval.
Acknowledgments This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (201203022) and the project of Graduates’ Research Innovative Programs from Colleges and Universities of Jiangsu Province (KYLX-0581). References Armstrong, W.D., Reid, G.L., Hilton, M.L., Chang, C., 1993. Relevance of enantiomeric separations in environmental science. Environ. Pollut. 79, 51–58.
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Enantioselective degradation of metalaxyl in cucumber, cabbage, spinach and pakchoi. Chemosphere 95, 241–246. Wang, X.Q., Jia, G.F., Qiu, J., Diao, J., Zhu, W., Lv, C., Zhou, Z.Q., 2007. Stereoselective degradation of fungicide benalaxyl in soils and cucumber plants. Chirality 19, 300–306. Wang, X.Q., Wang, X.S., Zhang, H., Wu, C., Wang, X.Y., Xu, H., Li, Z., 2012a. Enantioselective degradation of tebuconazole in cabbage, cucumber, and soils. Chirality 24, 104. Wang, X.Q., Zhang, H., Xu, H., Wang, X.Y., Wu, C., Yang, H., Wang, Q., 2012b. Enantioselective residue dissipation of hexaconazole in cucumber (Cucumis sativus L.), head cabbage (Brassica oleracea L. var. caulorapa DC.), and soils. J. Agr. Food Chem. 60, 2212–2218. Wu, C.W., Sun, J.Q., Zhang, A.P., Liu, W.P., 2013. Dissipation and enantioselective degradation of plant growth retardants paclobutrazol and uniconazole in open field, greenhouse, and laboratory soils. Environ. Sci. Technol. 47, 843–849. Xu, Y.X., Zhang, H., Zhuang, S.L., Yu, M., Xiao, H., Qian, M., 2012. Different enantioselective degradation of pyraclofos in soils. J. Agr. Food Chem. 60, 4173–4178. Yan, Y.F., Zhang, Z.E., Zhang, L., Chen, Y.R., Tang, Q., 2014. Dynamic modeling of biogas upgrading in hollow fiber membrane contactors. Energy Fuels 28, 5745–5755. Yuan, L., Zhi, W., Liu, Y., Karyala, S., Vikesland, P.J., Chen, X., Zhang, H., 2014. Lead toxicity to the performance, viability, and community composition of activated sludge microorganisms. Environ. Sci. Technol. 49, 824–830. Zhang, H., Qian, M.R., Wang, X.Q., Wang, X.Y., Xu, H., Qi, P., Wang, M.H., 2012a. Analysis of tebuconazole and tetraconazole enantiomers by chiral HPLC-MS/MS and application to measure enantioselective degradation in strawberries. Food Anal. Method 5, 1342–1348. Zhang, H., Qian, M.R., Wang, X.Q., Wang, X.Y., Xu, H., Wang, Q., Wang, M.H., 2012b. HPLC–MS/MS enantioseparation of triazole fungicides using polysaccharidebased stationary phases. J. Sep. Sci. 35, 773–777. Zhang, Q., Tian, M.M., Wang, M.Y., Shi, H.Y., Wang, M.H., 2014. Simultaneous enantioselective determination of triazole fungicide flutriafol in vegetables, fruits, wheat, soil, and water by reversed-phase high-performance liquid chromatography. J. Agr. Food Chem. 62, 2809–2815. Zhi, W., Ge, Z., He, Z., Zhang, H., 2014. Methods for understanding microbial community structures and functions in microbial fuel cells: a review. Bioresour. Technol. 171, 461–468. Zhi, W., Ji, G., 2014. Quantitative response relationships between nitrogen transformation rates and nitrogen functional genes in a tidal flow constructed wetland under C/N ratio constraints. Water Res. 64, 32–41. Zhu, W.T., Qiu, J., Dang, Z.H., Lv, C., Jia, G., Li, L., Zhou, Z.Q., 2007. Stereoselective degradation kinetics of tebuconazole in rabbits. Chirality 19, 141–147.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Study on the stereoselective degradation of three triazole fungicides in sediment Qing Zhang, Liangliang Zhou, Yu Yang, Xiude Hua, Haiyan Shi, Minghua Wang n Department of Pesticide Science, College of Plant Protection, Nanjing Agricultural University, State & Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing 210095, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 4 January 2015 Received in revised form 12 March 2015 Accepted 13 March 2015
The stereoselective degradation behaviors of chiral triazole fungicides (hexaconazole, flutriafol and tebuconazole) in sediment were investigated under laboratory conditions. The enantiomers were completely separated by high-performance liquid chromatography on a cellulose tris(3-chloro-4-methylphenylcarbamate) (Lux Cellulose-2) column. The mean recoveries of hexaconazole, flutriafol and tebuconazole in sediment ranged from 86.7% to 105.9%. The methods were successfully applied for the enantioselective degradation analysis of fungicides in sediment. The results showed that the dissipation of hexaconazole, flutriafol and tebuconazole stereoisomers in sediment followed first-order kinetics (R2 40.95). The degradation rate of the enantiomers was different in sediment, and the ( )-enantiomer (t1/2 was 86 days for hexaconazole, 139 for flutriafol and 136 for tebuconazole) degraded faster than the ( þ)-enantiomer (t1/2 was 94 days for hexaconazole, 144 for flutriafol and 151 for tebuconazole) in native condition. The fungicides were degraded slowly, and no significant enantioselective degradation were observed under sterilized conditions. The results may hold promising implications for the environmental and ecological risk assessment of three important chiral triazole fungicides. & 2015 Elsevier Inc. All rights reserved.
Keywords: Stereoselective degradation Sediment Hexaconazole Flutriafol Tebuconazole
1. Introduction Triazole fungicides are widely used broad-spectrum fungicides that inhibit the sterol 14α-demethylase, an enzyme involved in the biosynthesis of ergosterol (Buerge et al., 2006). Almost all triazole fungicides are chiral due to the presence of the asymmetrically substituted carbon atoms in the triazol alkyl moiety. These chiral molecule chemicals consist of two or four stereoisomers. Although enantiomers have identical physico-chemical properties, their interactions with biological macromolecules are often chiral-selective, leading to enantiomer selectivity in biodegradation, ecotoxicity and human health effects. These interactions may also contribute to differences in biological activity, toxicity on beneficial or nontarget organisms and environmental fate (Ribeiro et al., 2012; Dong et al., 2013; Han et al., 2013; Sun et al., 2012;). However, chiral pesticides are generally produced and used as racemates or mixtures of stereoisomers. The introduction of inactive isomers into the environment may result in unwanted side effects. Therefore, there is an increasing interest in evaluating the enantioselective behavior of chiral pesticides in the environment. Hexaconazole, flutriafol and tebuconazole possess an n
Corresponding author. Fax: þ 86 25 84395479. E-mail address: [email protected] (M. Wang).
http://dx.doi.org/10.1016/j.ecoenv.2015.03.014 0147-6513/& 2015 Elsevier Inc. All rights reserved.
asymmetrically substituted carbon atom and consist of a pair of enantiomers (Fig. 1), which are registered as racemic products in many countries. They are typically applied as foliar sprays to control diseases caused by ascomycetes, basidiomycetes and deuteromycetes of cereals, vegetables and fruits (Wang et al., 2012a; Zhang et al., 2014). Although the triazole fungicides are extremely persistent in soil, they may appear in aquatic systems and enriched river sediments via runoff or soil erosion (Belluck et al., 1991; Yan et al., 2014). As a result, sediments are usually regarded as important sinks or receptors of pesticides. As is well known, in addition to stereoselective degradation and transformation in the environment (Dong et al., 2013; Han et al., 2013; Wang et al., 2014), triazole fungicides enantiomers also have different toxic effects on aquatic organisms (Obliquus, 2012; Zhang et al., 2012a; Zhu et al., 2007; Cao et al., 2009). When racemic compounds are employed, approximately 50% or more of ineffective or less active products will be incorporated into the environment (Pérez-Fernandez et al., 2011; Armstrong et al., 1993; Lewis et al., 1999; Zhang et al., 2012b). These ineffective enantiomers are widely used in agricultural systems and enriched river sediments, and they may lead to more side effects on nontarget aquatic organisms (such as algae and zebrafish). To the best of our knowledge, no study has been reported about the enantioselective degradation behavior of hexaconazole, flutriafol and tebuconazole in sediment. Sediment is an important ecosystem for aquatic
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Fig. 1. Molecular structures (nindicates chiral center) and typical chromatograms of the chiral separations of fungicides (hexaconazole, flutriafol and tebuconazole).
2. Materials and methods
working solutions with concentrations of 0.5, 1, 2, 5, 10 and 20 mg L 1 were prepared by serial dilution. Purified water was obtained using a MUL-9000 water purification system (Nanjing Zongxin Water Equipment Co. Ltd., China). HPLC-grade acetonitrile was purchased from TEDIA (Fairfield, USA), and Cleanert florisil (500 mg, 6 mL) were obtained from Agela Technologies (Tianjin, China). The mobile phase was filtered through a 0.22 mm filter membrane (Tengda, Tianjin, China). Sodium chloride, anhydrous sodium sulfate, ethyl acetate, acetonitrile, acetone and n-hexane of analytical grade were purchased from commercial sources. The chromatographic separation of the enantiomers was performed on an Agilent 1200 HPLC system (Agilent, USA) with UV detection on chiral Lux Cellulose-2 [cellulose tris(3-chloro-4-methylphenylcarbamate)] column supplied by Phenomenex (Torrance, USA); The column was packed with 3-μm particles with 150 mm 2.0 mm i.d. Sediment samples were collected from Taihu Lake (0–10 cm surface layer) located in Suzhou, China. Taihu Lake is the third largest freshwater body downstream of the Yangtze River, China. The sediment samples were air-dried at room temperature, passed through a 2-mm sieve and stored in the dark. The sediments showed no hexaconazole, flutriafol and tebuconazole before fortification. The physicochemical characteristics of the sediment were as follows: pH 7.007 0.10; organic matter, 1.41 70.10%; clay, 18.70 70.81%; sand, 6.55 70.13; and silt, 74.75 71.23%.
2.1. Chemicals and materials
2.2. Incubation experiments
Standard of rac-hexaconazole (499.9% purity), rac-flutriafol (4 97.3% purity) and rac-tebuconazole ( 498.2% purity) were obtained from the China Shanghai Pesticide Research Institute (Shanghai, China). Enantiomers of hexaconazole, flutriafol, tebuconazole (purity Z 97.0%) were obtained from the Shanghai Chiralway Biotech Co., Ltd. (Shanghai, China). Stock solutions of hexaconazole, flutriafol and tebuconazole standard (1000 mg L 1) were prepared in acetonitrile and stored at 20 °C. Standard
Incubation experiments on hexaconazole, flutriafol and tebuconazole in native and sterile sediment under laboratory conditions were conducted to evaluate the contribution of abiotic degradation and biodegradation to the dissipation of triazole fungicides in sediment. Tests on the degradation of hexaconazole, flutriafol and tebuconazole were conducted with the racemic compounds using 100-mL polypropylene centrifuge tubes sealed with sterile cotton plugs (Xu et al., 2012). To avoid the potential effects
organisms. It is necessary to establish the appropriate method for the separation and study of the enantioselective dissipation kinetics of hexaconazole, flutriafol and tebuconazole residues. Such a study would provide more information relevant to environmental and ecological risk assessment. The objective of this work was to study the enantioselective degradation and persistence of the racemic forms of hexaconazole, flutriafol and tebuconazole in Taihu Lake sediment using reversephase and chiral high-performance liquid chromatography (HPLC). The enantiomers of three triazole fungicides were completely separated on Lux Cellulose-2 columns by Phenomenex (Torrance, USA) packed with cellulose tris(3-chloro-4-methylphenylcarbamate) combined with acetonitrile and water as the mobile phase. In addition, the stereoselective degradation of hexaconazole, flutriafol and tebuconazole was investigated in the enantiomers’ native as well as sterilized conditions. The current report is the first to investigate the enantioselective dissipation of three triazole fungicides in sediment. The results of this research, which are relevant to stereoselectivity and degradation in sediment, may hold implications for improving the environmental and human risk assessment of these three widely used chiral triazole fungicides (Li et al., 2012).
Q. Zhang et al. / Ecotoxicology and Environmental Safety 117 (2015) 1–6
of solvents on the microbiological activity of the soils, the following fortification methods were adopted (Brinch et al., 2002). A portion of the sediment (20 g) was first spiked with 100 μL of stock solution in acetone containing approximately 1000 μg of fungicide (hexaconazole, flutriafol or tebuconazole) and stirred for 5 min. The soil (180 g) was added and mixed thoroughly for 15 min, yielding a fortification level of 2.5 μg g 1 for each enantiomer. After the solvent was completely evaporated, the sediment (10 g) was transferred to a 100 mL polypropylene centrifuge tube and rehydrated with 10 g of deionized water, and the overlying water height was approximately 2–3 cm (Di et al., 2013). The sediment was incubated at 25 71 °C in the dark and weighted regularly for water content. Three replicate samples were removed from each treatment at different time intervals (0, 1, 3, 7, 10, 14, 21, 30, 45, 60, 90, 120 and 150 days). Approximately 10 g of sediment weighted in 100 mL polypropylene centrifuge tubes was sterilized by autoclaving twice at 121 °C for 30 min in 24 h intervals. The sterilized samples were fortified with 50 μg of racemic fungicide (hexaconazole, flutriafol or tebuconazole), and 10 g sterile water was added to the samples under 15 min of ultraviolet irradiation on clean bench. The samples were incubated at 25 71 °C in the dark and removed from each treatment at different time intervals. 2.3. Sample preparation and extraction Ethyl acetate (50 mL) was added to each polypropylene centrifuge tube (10 g of sediment). The tubes were stirred for 3 min on a vortex mixer and extracted by sonication for 10 min, and 2 g sodium chloride was added for complete extraction. The tube was then stirred for 1 min on a vortex mixer and centrifuged for 5 min at 4000 rpm. The supernatant (25 mL) was evaporated to neardryness on an evacuated rotary evaporator at 45 °C and redissolved in a 5-ml mixture of hexane and acetone (98:2, v/v) for cleanup. 2.4. Sample purification procedure The concentrated extracts were transferred to a florisil cartridge. The Cleanert florisil-SPE was rinsed with 6 mL hexane– acetone (95:5, v/v) and then eluted with 15 mL hexane–acetone (85:15, v/v). All eluates were collected and evaporated to dryness under vacuum at 40 °C. The sample was dissolved in 1 mL acetonitrile and filtered through a 0.22-μm filter for HPLC analysis. 2.5. Enantiomer HPLC determination The chromatographic separation of the enantiomers of hexaconazole, flutriafol and tebuconazole were performed on an Agilent 1200 HPLC (Agilent, USA) with chiral Lux Cellulose-2 columns and an injection volume of 20 μL. The mobile phase consisted of acetonitrile and water (40:60 for flutriafol, 67:33 for hexaconazole, 68:32 for tebuconazole, v/v) and delivered at a different rate (0.8 mL min 1 for flutriafol, 0.65 mL min 1 for hexaconazole, 0.7 mL min 1 for tebuconazole). The column was kept at 30 °C, and UV detection was performed at 223 nm for tebuconazole, 210 nm for flutriafol and 220 nm for hexaconazole. The chromatograms of raceme and enantiomers were shown in Fig. 1. The retention times between the two enantiomers were distinguished with complete separation (17.98, 19.50 min for flutriafol; 12.15, 16.43 min for hexaconazole; 11.04, 12.44 min for tebuconazole). The (þ )-enantiomer was eluted earlier than the ( )-enantiomer for tebuconazole and hexaconazole, whereas the ( )-flutriafol was confirmed as the first eluted enantiomer and the (þ )-flutriafol as the second eluted enantiomer.
3
2.6. Calibration curves and assay validation The linearities of the fungicides were established using a series of standard solvent solutions and standard matrix-matched enantiomer solutions by plotting peak areas against analyte concentrations from 0.5 to 20 mg L 1. The linear regression results obtained from the matrix-matched calibration curves were evaluated for the matrix effect. Method accuracy and precision were evaluated by recovery studies using spiked samples at three concentration levels (0.05, 0.25 and 2.5 mg kg 1 for each enantiomer based on five replicates) over three different days. The spiked samples were equilibrated for 2 h and then processed according to the above-described procedures. Blank analyses were performed to check for interference from the matrix. The precision of the method was determined by repeatability and reproducibility studies and expressed as relative standard deviation (RSD). The limit of detection (LOD) and limit of quantification (LOQ) for the enantiomers of hexaconazole, flutriafol and tebuconazole were considered concentrations in a sample matrix, resulting in peak areas with signal-to-noise (S/N) ratios of 3 and 10. The capacity factor for the (þ)- and ( )-enantiomers (k1 and k2, respectively), the separation factor (α), and the resolution (Rs) were calculated. A kinetic study of the enantiomers in sediment was performed by plotting residue concentration against time; the degradation rate constant k of the enantiomers were calculated from Eq. (1), and the half-life (t1/2, day) was estimated from Eq. (2).
C = C0e−kt
(1)
t1/2 = ln2/k = 0.693/k
(2)
Residual concentration data of the two enantiomers were used to estimate the enantiomer ratio (ER) during experiments. The ER was defined as the concentration of the ( )-enantiomer divided by the concentration of the ( þ)-enantiomer, where an ER of 1 represents the racemate (Wang et al., 2007). A more rapid dissipation of the (þ)-enantiomer with ER more than 1, whereas ( )-enantiomer degraded faster than the (þ)-form. The enantiomeric selectivity (ES) was also used to confirm the overall trend of the enantioselective dissipation process of hexaconazole, flutriafol and tebuconazole in sediment (Buerge et al., 2003). Enantiomeric selectivity (ES) was defined by Eq. (3).
ES = (k (+) − k (−))/(k (−) + k (+))
(3)
Positive values (0 o ESr1) indicate a more rapid dissipation of the (þ )-enantiomer, and negative values ( 1 rES o0) indicate a more rapid dissipation of the ( )-enantiomer. An ES value of 0 indicates a dissipation that is not enantioselective (Wang et al., 2012b).
3. Results and discussion 3.1. Matrix effects and linearity Sample extraction and cleanup procedures based on the modified QuEChERS method facilitated ultrasonic extraction. Methods for analyzing hexaconazole, flutriafol and tebuconazole in sediment were successfully developed and validated by HPLC. The two enantiomers could be completely separated with a resolution (Rs) of over 1.5 (1.82 for flutriafol, 3.11 for tebuconazole and 9.1 for hexaconazole), and there were no endogenous interference peaks at the same retention time of the three fungicides. The linear regression results for the standard solution and matrix-matched calibration curves for each enantiomer are summarized in Table 1. Good linear calibration curves for each
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Table 1 Matrix effects and linearity for tebuconazole, flutriafol and hexaconazole enantiomers. Compound
Concentration (mg L 1)
Standard solution linear equation, R2
Matrix-matched linear equation, R2
P-value
( þ )-Tebuconazole ( )-Tebuconazole ( þ )-Flutriafol ( )-Flutriafol ( þ )-Hexaconazole ( )-Hexaconazole
0.5–20 0.5–20 0.5–20 0.5–20 0.5–20 0.5–20
y¼ 37.57x 1.512, 0.9999 y¼ 37.39x 2.555, 0.9999 y¼ 42.10xþ 0.951, 0.9999 y¼ 41.95x 1.860, 0.9999 y¼ 33.05xþ 2.133, 0.9999 y¼ 33.17x þ3.189, 0.9999
y¼ 37.31xþ 0.245, 0.9993 y¼ 37.22x 1.280, 0.9994 y¼ 43.10x 3.112, 0.9992 y¼ 43.22x 3.279, 0.9996 y ¼33.86x 1.389, 0.9995 y ¼33.33x 1.066, 0.9994
0.085 0.094 0.486 0.197 0.296 0.058
enantiomer were obtained over the range of 0.5–20 μg mL 1 with R2 Z0.9992. The matrix effect was calculated by comparing standard solution curves with matrix-matched calibration curves using a two-tailed paired t-test with confidence interval of 95%. Table 1 showed that no significant matrix-induced enhancement or suppression effect was observed for each enantiomer and that the matrix effect could be negligible (P4 0.05). 3.2. Validation of the method The recoveries and RSD values are shown in Table 2. The mean recoveries of hexaconazole, flutriafol and tebuconazole at three spiked levels in sediment ranged from 86.7% to 105.9%. The relative standard deviation (RSD) values ranged from 2.1% to 8.7% for intra- and inter-day studies. The recovery and precision results indicate that the method was efficient and reliable. The LOD and LOQ were estimated to be 0.013 and 0.030 mg kg 1 for each enantiomer of hexaconazole, flutriafol and tebuconazole at low concentrations. 3.3. Degradation of hexaconazole, flutriafol and tebuconazole in sediment in native condition The degradation kinetics of hexaconazole, flutriafol and tebuconazole enantiomers in sediment followed first-order kinetics, as shown Table 3, and the typical chromatograms are shown in Fig. 2. The concentrations of the enantiomers were different in the chiral HPLC chromatograms for the three fungicides at 120 days. The
residues of both enantiomers of hexaconazole, flutriafol and tebuconazole decreased with time in sediment, and the data show that the ( )-enantiomer (t1/2 ¼ 86 days for hexaconazole, 139 for flutriafol and 136 for tebuconazole) degrades faster than the (þ)-enantiomer (t1/2 ¼94 days for hexaconazole, 144 for flutriafol and 151 for tebuconazole) with a significant difference (Po 0.05, Student’s paired t-test). The ER values of the fungicides consistently decreased over time (Fig. 2). A t-test between the ER values of hexaconazole, flutriafol and tebuconazole in sediment and ER ¼ 1.0 yielded P values of 0.00021, 0.00017, and 0.01028, respectively. These results indicate that enantioselectivity existed during the dissipation of the three fungicides in sediment. The data show that the ( )-enantiomer degraded more rapidly, leading to residues enriched in ( þ)-enantiomer during incubation. The degradation of pesticides in soil or sediment has been attributed to microorganisms (Buerge et al., 2006; Wu et al., 2013; Yuan et al., 2014; Zhi et al., 2014). The ES values observed in the present study also suggest that the compounds examined are, at least to some extent, degraded by biological processes, whereas abiotic degradation would generally not be enantioselective (ES¼ 0). In this study, the ES value was 0.045 for hexaconazole, 0.020 for flutriafol and 0.052 for tebuconazole (Table 3). These ES values suggest that the dissipation of hexaconazole, flutriafol and tebuconazole in sediment is enantioselective, and ( )-enantiomer degraded faster than the (þ)-form. According to a previous report, the enantiomers may be preferentially degraded or transformed by micro-organisms in the environment (Lewis et al., 1999).
Table 2 Recovery and RSD for tebuconazole, flutriafol and hexaconazole enantiomers in sediment. Compound
Spiked level (mg kg 1)
Intra-day (n ¼5)
Inter-day (n¼ 15) RSD (%)
Day 1
( þ )-Tebuconazole
0.05 0.25 2.5 ( )-Tebuconazole 0.05 0.25 2.5 ( þ )-Flutriafol 0.05 0.25 2.5 ( )-Flutriafol 0.05 0.25 2.5 ( þ )-Hexaconazole 0.05 0.25 2.5 ( )-Hexaconazole 0.05 0.25 2.5
Day 2
Day 3
Average Recoveries (%)
RSD (%) Average Recoveries (%)
RSD (%) Average Recoveries (%)
RSD (%)
92.0 92.7 89.2 87.6 89.4 87.9 104.3 86.9 90.2 105.9 86.7 88.9 98.6 90.3 91.7 95.6 90.0 91.1
3.8 4.2 5.3 2.4 2.1 3.2 4.1 4.1 2.3 5.7 4.3 5.3 3.6 3.6 4.2 3.8 4.8 3.8
5.1 3.9 6.7 3.1 3.7 4.3 7.9 5.9 3.7 8.7 5.2 4.9 2.5 2.9 3.8 2.1 3.9 5.9
5.9 5.1 7.5 4.1 4.3 5.9 8.5 3.9 3.9 7.3 2.7 4.2 3.2 4.1 3.3 4.8 5.7 4.9
88.7 91.2 87.5 87.3 88.7 88.3 96.7 94.6 91.7 88.7 93.7 97.3 98.7 89.3 90.9 91.7 94.3 90.7
95.6 94.9 97.8 92.0 95.6 91.1 93.8 87.9 95.8 95.4 90.4 93.9 94.6 88.9 89.5 94.3 89.3 88.5
4.3 4.7 6.6 3.2 3.9 4.4 6.1 4.5 3.3 7.4 4.5 4.6 2.2 3.1 2.9 3.3 4.6 5.1
Q. Zhang et al. / Ecotoxicology and Environmental Safety 117 (2015) 1–6
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Table 3 Regression functions, half-life (t1/2), correlation coefficient (R2), and ES values for the degradation of tebuconazole, flutriafol and hexaconazole enantiomers in native and sterile sediment.
Native sediment
Sterile sediment
a b
Enantiomer
Regression functionsa
R2
Half-life ( t1/2, day)b
ES
( þ )-Tebuconazole ( )-Tebuconazole ( þ )-Flutriafol ( )-Flutriafol ( þ )-Hexaconazole ( )-Hexaconazole ( þ )-Tebuconazole ( )-Tebuconazole ( þ )-Flutriafol ( )-Flutriafol ( þ )-Hexaconazole ( )-Hexaconazole
Ct ¼2.3854e 0.0046x Ct ¼2.3339e 0.0051x Ct ¼2.2240e 0.0048x Ct ¼2.2259e 0.0050x Ct ¼2.3149e 0.0074x Ct ¼2.2250e 0.0081x Ct ¼2.3744e 0.0026x Ct ¼2.3638e 0.0026x Ct ¼2.3035e 0.0025x Ct ¼2.3098e 0.0025x Ct ¼2.4109e 0.0050x Ct ¼2.3901e 0.0050x
0.9775 0.9677 0.9557 0.9682 0.9703 0.9712 0.9651 0.9644 0.981 0.9595 0.9845 0.9774
150.65 71.65 135.8872.07 144.38 71.07 138.60 70.98 93.65 71.18 85.56 71.38 266.54 73.58 266.54 73.23 277.2073.76 277.2073.37 138.60 72.14 138.60 72.01
0.052 0.020 0.045 0 0 0
The regressive functions are based on the mean value of three replicates. Values represent the means (n¼ 3).
Fig. 2. Typical chromatograms and time development of enantiomeric ratio (ER) on native condition. Chromatograms of hexaconazole (a), flutriafol (b) and tebuconazole (c) in sediment after 120 days. ER of hexaconazole (d), flutriafol (e) and tebuconazole (f) in sediment.
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3.4. Degradation of hexaconazole, flutriafol and tebuconazole in sediment under sterilized condition The degradation kinetics of the three fungicides showed firstorder behavior under the sterilized condition, with the correlation coefficient values (R2) ranging from 0.9595 to 0.9845 (Table 3). The results of the kinetics study of fungicide residues in sediment are shown in Table 3. The enantiomers have the same half-life and the ES values was 0 during the period of incubation, indicating three triazole fungicides (hexaconazole, flutriafol and tebuconazole) degradation was not enantioselective in sediment in the sterilized condition. The dissipation of the three fungicides was rather slow, with half-life values ranging from 139 to 277 days (Table 3). The degradation order of the three fungicides was observed to be hexaconazole 4tebuconazole4 flutriafol. The dissipation of flutriafol was slowest in the sterilized condition, with a half-life of 277 days, and tebuconazole was dissipated at a rate similar to that observed for flutriafol (t1/2 ¼267 days). Most importantly, the halflife value of hexaconazole in sediment was 139 days less than two times that of the other fungicides. The observed differences in the persistence of the three fungicides in sediment may be determined by their branch chain structure because the fungicides have the same core skeleton structure. It is clear that the persistence of the fungicides in the sterilized condition was two times longer than that in the native condition. The degradation of hexaconazole, flutriafol and tebuconazole involved both abiotic degradation and biodegradation in native soil, whereas only abiotic degradation occurred in sterile soil. The difference in the degradation behavior of the fungicides in sediment could be greatly attributed to microbial populations equipped with a variety of metabolic enzymes, which are preferential degraders of different enantiomers (Zhi and Ji, 2014). This could be one reason for the different ranking order of the two enantiomers of fungicides with respect to their degradation rate constants.
4. Conclusions In this study, an effective method for enantioselective determination of hexaconazole, flutriafol and tebuconazole enantiomers in sediment was established and validated. The methods were successfully used to study the degradation of the stereoisomers in sediment. The result shows that the degradation of the hexaconazole, flutriafol and tebuconazole enantiomers in sediment occurs with some chiral preference in the native condition. The ( )-enantiomer degraded faster than the ( þ)-form, leading to residues enriched in the (þ)-form. The different degradation rates of the enantiomers and their chiral preference may depend on the variety of microbial populations with genes harboring different metabolic enzymes that occur in sediment. These findings may hold implications for improving environmental and ecological risk assessment and are also relevant to pesticide registration and approval.
Acknowledgments This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (201203022) and the project of Graduates’ Research Innovative Programs from Colleges and Universities of Jiangsu Province (KYLX-0581). References Armstrong, W.D., Reid, G.L., Hilton, M.L., Chang, C., 1993. Relevance of enantiomeric separations in environmental science. Environ. Pollut. 79, 51–58.
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