Environ. Sci. Technol. 2010, 44, 2042–2047
Environmental Behavior of the Chiral Aryloxyphenoxypropionate Herbicide Diclofop-Methyl and Diclofop: Enantiomerization and Enantioselective Degradation in Soil JINLING DIAO, PENG XU, PENG WANG, YUELE LU, DAHAI LU, AND ZHIQIANG ZHOU* Department of Applied Chemistry, China Agricultural University, Beijing 100193, China
Received December 11, 2009. Revised manuscript received January 31, 2010. Accepted February 2, 2010.
In this study, the degradation of diclofop-methyl (DM) and its main metabolite, diclofop (DC), in two soils under aerobic and anaerobic conditions were investigated using enantioselective HPLC. Under aerobic or anaerobic conditions, rapid hydrolysis to the corresponding acid diclofop (DC) was observed. The results from this study revealed that the degradation of DM in the two soils is not enantioselective, and the calculated half-lives (t1/2) for the two soils were both less than 1 day. However, the degradation of DC in the two soils is enantioselective both under aerobic and anaerobic conditions, and the S-(-)-DC was preferentially degraded, resulting in relative enrichment of the R-(+)-form. The calculated t1/2 values of the enantiomers of DC ranged between 8.7 and 43.3 days for aerobic incubation experiments and between 14.7 and 77.0 days for anaerobic incubation experiments, respectively. The enantiopure S-(-)- and R-(+)-DC were incubated under aerobic conditions, and it revealed significant enantiomerization with inversion of the S-(-)-enantiomer into R-(+)-enantiomer, and vice versa, and the S-(-)-DC showed a significantly higher inversion tendency than the R-(+)-DC.
Introduction Diclofop-methyl (DM), 2-[4-(2,4-dichlorophenoxy)phenoxy]propionate (Figure S1), is a postemergence herbicide that is selective for grasses in broadleaf crops and also controls grass weeds in crops such as wheat and barley (1, 2). It is one of the aryloxyphenoxy propanoate (AOPP) class of herbicides. DM and other AOPP analogs cause nearly immediate growth inhibition of meristems (shoot, intercalary, root) (3-6). Although many herbicides are typically applied to the crop and weed canopy, substantial quantities of the active ingredient penetrate to the soil surface. For example, up to 73% in the case of a controlled foliar application of the herbicide DM (7). Therefore, research on DM has concentrated on its occurrence and fate in surficial soils. Up to 15% of DM is hydrolyzed at the time of application and incorporated into the soil, and as much as 85% is hydrolyzed within 24 h of application (8, 9). Under field conditions DM is known to undergo rapid hydrolysis to its corresponding acid, diclofop * Corresponding author fax: 861062733547; e-mail: zqzhou@ cau.edu.cn. 2042
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(DC, Figure S1)), 2-[4-(2,4-dichlorophenoxy)phenoxy]propionic acid (10). DM and DC both have an asymmetrically substituted carbon atom and consist of a pair of enantiomers (Figure S1). Their absolute configurations were confirmed with (+) rotation of the R-enantiomers and (-) rotation of the S-enantiomers (11). In general, the R-(+)-enantiomers of AOPP herbicides are herbicidally active and are approximately twice as active as respective racemic mixtures (12). The commercial forms of AOPP herbicides are the esters of parent acids to enhance their uptake into plants (13, 14). Several AOPP herbicides such as quizalofop-ethyl, fluazifop, and haloxyfop have had their R-(+)-enantiomers registered and applied worldwide. However, DM, the commercial form of DC, is still widely used as the racemate (15, 16), which is known to hydrolyze rapidly to DC. In theory, enantiomers have identical physical and chemical properties and abiotic degradation rates (17), whereas their individual toxicity, biological activity, and environmental fate have been shown to differ (17-20). As a result, microbially mediated degradation processes in soils can be enantioselective and, therefore, change the enantiomeric composition over time. In some cases, only one enantiomer is being decomposed, while the other enantiomer is being accumulated in the environment. For example, studies have shown that enantiomers of R-HCH, metolachlor, metalaxyl, and benalaxyl behave significantly differently during biodegradation and bioaccumulation in the environment (21-27). Moreover, under certain conditions enantiomerization of chiral compounds, such as dichlorprop and mecoprop, was found (28). Enantiomerization is a process in which the individual enantiomers undergo inversion of their respective configurations, which may influence efficacy and side effects. Recently studies indicated that the S-(-)-enantiomers of DM and DC posed a stronger toxicity to three freshwater algae than the R-(+)-enantiomers (29). For assessing the environmental safety of DM and DC, information on their stereochemistry is very important, which cannot be obtained from achiral analysis. Biodegradation of DM and DC in different soils has been extensively studied, and many studies have demonstrated that the degradation of these chemicals in soil is a microbiologically mediated process (7-10). However, enantioselectivity has seldom been studied for the environmental fates of DM and DC. In this study, the enantioselective degradations of DM and DC were investigated in two soils. Furthermore, we evaluated the potential for the enantioselective degradations of DM and DC in aerobic as well as anaerobic soils. At the same time, we investigated the enantiomerization processes by incubation of racemic and enantiopure compounds.
Experimental Section Chemicals and Reagents. Racmic-diclofop-methyl {2[4-(2,4-dichlorophenoxy)phenoxy] propionate, Rac-DM, purity g 98.0% after purification}was obtained from China Ministry of Agriculture’s Institute for Control of Agrochemicals. Racmic-diclofop {2-[4-(2,4-dichlorophenoxy)phenoxy]propionic acid, Rac-DC, purity g 98.0%} was synthesized in our own laboratory. The enantiomers of DM and DC were prepared on an Agilent HPLC with a preparatory chiral column (250 × 10 mm (I.D.), provided by the Department of Applied Chemistry, China Agricultural University, Beijing). Water was purified by a Milli-Q system. Ethyl ether, n-hexane, 2-propanol (analytical grade), and trifluoroacetic acid (TFA) were from Fisher Scientific (Fair Lawn, NJ, U.S.A.). All other 10.1021/es903755n
2010 American Chemical Society
Published on Web 02/15/2010
chemicals and solvents were analytical grade and purchased from commercial sources. Soil Samples. Two soils representing different physicochemical properties and climatic environments were collected from two distinct sites in China. The Wuxi soil was silt loam soil and located in a subtropical region of china. The Chifeng soil was sandy loam soil and located in a temperate region of china. No detectable DM and DC were found at detectable levels in soils. After collection, the soil samples were air-dried at room temperature, homogenized, and passed through a 2 mm sieve and kept in the dark until used within a few days. Physicochemical properties of the Wuxi soil and Chifeng soil, respectively, were as follows: organic matter, 1.7 ( 0.15% and 1.9 ( 0.12%; clay, 1.7 ( 0.11% and 3.7 ( 0.13%; and, 36.8 ( 1.25% and 74.4 ( 1.82%; silt, 61.5 ( 1.75% and 21.9 ( 1.16%; pH, 5.0 ( 0.2 and 8.1 ( 0.1. Incubation in Soils under Aerobic Conditions. Separate incubation experiments were carried out with the racemic and with the pure S-(-)- and R-(+)-compounds, respectively, using 250 mL Erlenmeyer flasks covered with sterile cotton plugs. To avoid potential effects of solvents upon the microbiological activity of the soils, the following fortification methods were adopted (30). A portion of the sediment (20 g) was first spiked with 200 µL of stock solutions in acetone containing approximately 1000 µg rac-, S-(-)-, or R-(+)-DM and stirred for 5 min. The spiked soils were then allowed to air-dry for 10 min, before the remaining soil (180 g) was added and mixed thoroughly for another 10 min, yielding a fortification level of 5 µg/g (experiment SW1, SW2, and SW3, respectively, for Wuxi soil; SC1, SC2, and SC3, respectively, for Chifeng soil). The soil samples were incubated with a water content of 20-36 g per 100 g of dry soil, corresponding to about 60% of field holding capacity (w/w). The soils were incubated at 25 °C in the dark for 120 days. Soils were checked regularly for water content by weighing and were frequently mixed for aeration. Similar experiments were carried out with rac-, S-(-)-, and R-(+)-DC (experiment SW4, SW5, and SW6, respectively, for Wuxi soil; SC4, SC5, and SC6, respectively, for Chifeng soil). During the incubation, aliquots of 5 g of soil (based on dry weight) were removed from each treatment at different time intervals and immediately transferred into a freezer (-20 °C) to stop degradation. Triplicate samples were taken immediately after fortification and mixing (t ) 0) to determine the recovery and reproducibility of extraction in the soils (see below). Three replicate samples were taken periodically thereafter. Blank determinations of the sediment prior to fortification reavealed no DM or DC present. Incubation in Soils under Anaerobic Conditions. Under anaerobic conditions DM degraded rapidly to DC in a few hours (10), so we only investigated the degradation of DC in these two soils under anaerobic conditions. Incubation experiments were carried out in 50 mL glass bottles with a Teflon septum screw cap. Portions of 20 g of air-dry soils were placed into separate bottles (42 bottles, seven sampling points, triplicates of one of the two soils for one sampling point), in a glovebox under high-purity N2 atmosphere. Twenty milliliters of distilled water, previously purged with N2 to remove O2, was added to each bottle. To favor anaerobic conditions, the soils were preincubated during 1 month prior to addition of rac-DC. The soil samples were fortified by adding 20 µL of an acetone solution of rac-DC with a 50 µL of syringe through the septum (spike level, 5 µg/g of dry soil, experiment SW7 for Wuxi soil, and SC7 for Chifeng). The samples were then incubated at 25 °C in the dark for 120 days. To check the redox conditions, a piece of oxygen indicator (made by MGC, and it consists mainly of resazurin) was added to each bottle. The color of the oxygen indicator was red upon anaerobic conditions; on the contrary, it was blue when
there was only a small amount of oxygen in the bottle. The color of the oxygen indicator stayed red during these anaerobic incubations. Extraction of DM and DC from Soils. Samples were first thawed at room temperature. The 5 g samples (dry weight basis) were then removed and placed into 50 mL polypropylene centrifuge tubes. For extraction of DC, ethyl acetate (20 mL) was added to each tube followed by 200 µL of 6 M HCl to maintain DC in the acid form for efficient extraction. The tube was stirred for 3 min on a vortex mixer, exposed to ultrasonic vibration for 10 min, and then centrifuged at 4000 rpm for 5 min. The extraction was repeated twice with 20 mL of ethyl acetate, and the 3 extracts were combined and filtered through 5 g of anhydrous sodium sulfate for dehydration. The combined extract was evaporated to near dryness on a vacuumed rotary evaporator at 45 °C, and reconstituted in 1.0 mL of hexane. An aliquot (20 µL) was injected into the HPLC. Enantioselective HPLC Analysis. Chromatography was performed using an Agilent 1200 Series HPLC (Agilent Technology) equipped with a G1322A Degasser, G1311A Quat Pump, G1329A ALS, and G1314B VWD. The signal was received and processed by Agilent Chemstation Software. A cellulose-tri(3,5-dimethylphenylcarbamate)-based chiral stationary phase (CDMPC-CSP, provided by the Department of Applied chemistry, CAU, Beijing) was used to separate DM and DC in this study. The CSP and column were prepared according to the procedure described in the literature (31). The detailed HPLC methods and the elution order based on the absolute configuration of DM and DC enantiomers are described elsewhere (32). In the present work, this column was employed in the development of a new method for the simultaneous determination of DM and DC in soil samples. Using a 250 × 4.6 mm (I.D.) column, a simultaneous baseline separation of these compounds was obtained with a mobile phase of n-hexane/2-propanol/TFA (96:4:0.1, v/v/v) with a flow rate of 1.0 mL/min. Trifluoroacetic acid (TFA) was added into the mobile phase for the elution of DC. Chromatographic separation was conducted at a temperature of 15 °C and UV detection at 230 nm. S-(-)-DM, S-(-)-DC, R-(+)-DC, and R-(+)-DM was the elution order as established by comparison with the retention time of the enantiopure enantiomers of DM and DC. No enantiomerization was observed for DM or DC under these analytical conditions. Concentrations were determined by using a peak area, assuming the same response factor for the enantiomers and the racemate. The two compounds gave four separated peaks with retention times of 8.9 min (peak I), 11.0 min (peak II), 20.1 min (peak III), and 23.5 min (peak IV) under the described conditions (Figure S2). A series of blank samples fortified with rac-compounds at 0.1, 1.0, and 10.0 µg/g were determined immediately after fortification into the soils. Preliminary experiments showed that the recovery by the above procedure was >92% for DM or DC in soils. The limit of quantification (LOQ) for these compounds in all samples was found to be 0.3 µg/g based on an acceptable RSD of 20%. The limit of detection (LOD), defined as the concentration with a signal-to-noise ratio of 3, was 0.1 µg/g for all compounds.
Results and Discussion Degradation of DM and Formation of DC in Soils under Aerobic Conditions. In experiment SW1, SW2, SW3, SC1, SC2, and SC3, we found that both enantiomers of DM degraded fast in the two soils, with half-life times less than 1 d (Tables 1-2), when the racemic and the enantiopure S-(-)- and R-(+)-compounds were incubated. The residual concentrations of the two enantiomers were used for estimating the enantiomeric ratio (ER) values during these experiments. The ER was defined as the peak area of the first VOL. 44, NO. 6, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. First-Order Rate Constant (k), Half-Life (t1/2), and Correlation Coefficient (R2) for the Degradation of Enantiomers of DM and DC in Wuxi Soil (pH 5.0) experiment (incubated compound) enantiomer k (day-1) SW1a (rac-DM) SW2a (S-(-)-DM) SW3a (R-(+)-DM) SW4a (rac-DC) SW5a (S-(-)-DC) SW6a (R-(+)-DC) SW7b (rac-DC)
t1/2 (days)
R2
S-(-)-DM R-(+)-DM S-(-)-DM
1.001 0.996 1.108
0.69 ( 0.01 0.98 0.70 ( 0.02 0.98 0.62 ( 0.02 0.95
R-(+)-DM
1.077
0.64 ( 0.01 0.93
S-(-)-DC R-(+)-DC
0.080 0.036
8.7c ( 0.81 0.97 19.2c ( 0.96 0.98
S-(-)-DC
0.093
7.4 ( 0.34 0.94
R-(+)-DC S-(-)-DC R-(+)-DC
0.038 0.047 0.016
18.2 ( 0.72 0.99 14.7c ( 1.03 0.97 43.3c ( 1.54 0.95
a Tthe incubation experiment under aerobic condition. The incubation experiment under anaerobic condition. c Significantly different from each other, P < 0.05 (paired t test). b
TABLE 2. First-Order Rate Constant (k), Half-Life (t1/2) and Correlation Coefficient (R2) for the Degradation of Enantiomers of DM and DC in Chifeng Soil (pH 8.1) experiment (incubated compound) enantiomer k (day-1) a
SC1 (rac-DM) SC2a (S-(-)-DM) SC3a (R-(+)-DM) SC4a (rac-DC) SC5a (S-(-)-DC) SC6a (R-(+)-DC) SC7b (rac-DC)
t1/2 (days)
R
2
S-(-)-DM R-(+)-DM S-(-)-DM
1.859 1.908 2.043
0.37 ( 0.02 0.95 0.36 ( 0.01 0.96 0.34 ( 0.02 0.95
R-(+)-DM
1.993
0.35 ( 0.02 0.94
S-(-)-DC R-(+)-DC
0.048 0.016
14.4c ( 1.09 0.97 43.3c ( 1.23 0.99
S-(-)-DC
0.053
13.1 ( 1.02 0.93
R-(+)-DC S-(-)-DC R-(+)-DC
0.017 0.032 0.009
40.8 ( 1.14 0.99 21.6c ( 1.15 0.93 77.0c ( 2.84 0.93
a The incubation experiment under aerobic condition. The incubation experiment under anaerobic condition. c Significantly different from each other, P < 0.05 (paired t test). b
eluting S-(-)-enantiomer divided by the peak area of the later eluting R-(+)-enantiomer (17). The ER values of rac-DM were found to be constant with time in experiments SW1 and SC1 (Figure S3a and b). A t test between the ER values of rac-DM in the two soils and ER ) 1.0 yielded a p value of 0.217 and 0.10, respectively, indicating that the degradation of DM in the two soils was not enantioselective. From Tables 1 and 2, we found that DM persistence decreases with increasing soil pH (pH Waxi ) 5.0; pH Chifeng ) 8.1), and this conclusion is consistent with that of previous studies (9, 33). DM is hydrolyzed rapidly to DC in crops, soil, and water (10), but degradation of the hydrolysis product, DC, occurs primarily by biological transformation (34). In Figure 1a-c, chromatograms show the elution of DM and DC from samples of experiment SW1 (incubation with rac-DM) after 0, 0.5d, and 4d, respectively, and these chromatograms also show the concurrent formation and subsequent degradation of DC. The identities of DC were confirmed by comparing the residue retention time with the authentic standard. In Figures S4 and S5 we plotted the data from experiment SW1 and SC1 2044
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FIGURE 1. Chromatograms showing elution of DM and DC from the incubation of rac-DM in Wuxi soil (exp.SW1) after (a) 0, (b) 0.5, and (c) 4d. Panel d shows the chromatogram of blank sediment [n-hexane:2-propanol:TFA ) 96:4:0.1, flow rate 1.0 mL/ min]. with rac-DM and those from experiments SW2, SW3, SC2, SC3, respectively, as normalized concentrations (100C/C0) versus time (t). First-order kinetics was assumed for the degradation of the two enantiomers of DM with R2 ranging from 0.93 to 0.99 (Tables 1 and 2), and degradation rate constants were obtained by fitting S-(-)- or R-(+)-DM degradation data from each experiment to the first-order kinetic equation ln(C/C0) ) -kt (35), where C0 is the initial concentration of enantiomer (µg/g), C is its concentration (µg/g) at time t (days), and k is the degradation rate constant. The corresponding half-lives are calculated as t1/2 ) ln 2/k ) 0.693/k (35). From Figures S4a-b and S5a-b, we found that the decrease in the parent compound was accompanied by an increase in the concentration of the metabolite. The data plotted in Figures S4c-d and S5c-d show no formation of S-(-)-form from R-(+)-DM throughout the incubation time and vice versa, indicating that DM configuration was stable in the two soils. For Wuxi soil, during the initial 0.5 day of incubation, the amount of S-(-)-DC and R-(+)-DC detected was about 0.25 µg/g and 0.78 µg/g, respectively, in soil samples spiked with 5 µg/g rac-DM; after further incubation, the concentrations of S-(-)-DC and R-(+)-DC reached a maximum of 2.92 µg/g at day 4 and 4.68 µg/g at day 12, respectively, and then disappeared to 0.18 µg/g at day 30 and 0.27 µg/g at day 100, respectively. For Chifeng soil, during the initial 0.5 day of incubation, the amount of S-(-)-DC and R-(+)-DC detected was about 2.15 µg/g and 2.08 µg/g, respectively, in soil samples spiked with rac-DM; after further incubation, the concentrations of S-(-)-DC and R-(+)-DC reached a maximum of 3.28 µg/g at day 3 and 4.82 µg/g at day 12, respectively, and then disappeared to 0.17 µg/g at day 60 and 0.48 µg/g at day 149, respectively. Degradation of the Metabolite of DC in Soils under Aerobic Conditions. In general, the residues of both enantiomers of DC decreased with time elapsed in experiments SW4 and SC4, when the racemic compound was incubated. In Figure 2a and c, we plotted the data from experiments SW4 and SC4 with rac-DC as normalized concentrations (100C/C0) versus time. The data illustrated the more rapid degradation of the first-eluted S-(-)-enantiomer, leading to residues enriched in R-(+)-enantiomer. The ER values of DC
FIGURE 2. Degradation of DC with rac-DC in (a) Wuxi soil (exp. SW4) and (c) Chifeng soil (exp. SC4). Normalized concentrations (100C/C0) are plotted versus incubation time (d). Note the faster degradation of the S-(-)-enantiomer. Plot of In (ER) from (b) exp.SW4 (Wuxi soil, incubation of rac-DC) and (d) exp. SC4 (Chifeng soil, incubation of rac-DC) versus incubation time showing a linear relationship. consistently decreased with time in experiment SW4 and SC4 (Figure S3c and d). This steady decrease of S-(-)enantiomer concentration in comparison to the R-(+)enantiomer indicates a preferential biotransformation of S-(-)-enantiomer by the soil’s enzymatic systems when this isomer is applied as a racemic mixture. A t test between the ER values of DC in the two soils and ER ) 1.0 yielded a p value of 0.001 and 0.0004, respectively. From these results, it can be concluded that the degradation of DC in the two soils was enantioselective. Assuming that degradation of enantiomers followed firstorder kinetics with a rate constant of kS for the S-(-)enantiomer and a rate constant of kR for the R-(+)enantiomer, ER may be expressed as a function of time (t) in the following relationship (24): ERt ) [S]/[R] ) ER0 × e(kR-kS) ) ER0 × e∆K
(1)
where ER0 is the initial ER value (e.g., ER0 ) 1.02 for racemic compound), [S] is the concentration of the S-(-)-enantiomer at time t, [R] is the concentration of the R-(+)-enantiomer at time t, and ∆k is the difference between kR and kS, which reflects the rate at which ER deviates from ER0 over time. The above relationship can be further expressed in a linear form after logarithmic transformation of ERt (see below): ln(ERt) ) ln(ER0) + ∆k
(2)
Equation 2 indicates a linear relationship between ln(ERt) and t. A plot of ln(ERt) versus t can be used to determine the rate difference ∆k and thus enantioselectivity. For Wuxi soil, in Figure 2a, reasonable fits of the data for rac-DC from experiment SW4 were obtained with kS and kR of 0.080 and 0.036 d-1, respectively (Tables 1 and 2); in Figure 3a, reasonable fits were obtained for the data from the incubation of S-(-)-DC using a rate kS of 0.093 d-1 (exp. SW5); in Figure 3b, a reasonable fit was obtained for the data from the incubation of R-(+)-DC using a rate kR of 0.038 d-1 (exp. SW6). For Chifeng soil, in Figure 2c, reasonable fits of the data for rac-DC from experiment SC4 were obtained with kS and kR of 0.048 and 0.016 d-1, respectively; in Figure 3c, reasonable fits were obtained for the data from the incubation of S-(-)-DC using a rate kS of 0.053 d-1 (exp. SC5); in Figure 3d, a reasonable fit was obtained for the data from the incubation of R-(+)-DC using a rate kR of 0.017 d-1 (exp.
FIGURE 3. Degradation of DC in soil experiments SW5, SW6, SC5, and SC6 with S-(-)- and R-(+)-DC, respectively. (a) Degradation of S-(-)-DC showing concurrent formation of R-(+)-DC in Wuxi soil (exp.SW5), (b) degradation of R-(+)-DC showing concurrent formation of S-(-)-DC in Wuxi soil (exp.SW6), (c) degradation of S-(-)-DC showing concurrent formation of R-(+)-DC in Chifeng soil (exp.SC5), and (d) degradation of R-(+)-DC showing concurrent formation of S-(-)-DC in Chifeng soil (exp.SC6). Normalized concentrations (100C/C0) are plotted versus incubation time (d). SC6). The reason for the differences of kS in experiments SW4, SW5 and SC4, SC5 is unknown. Apparently, the conditions in these experiments were not exactly the same, and it is possible that there is some influence of enantiomer (S-(-)-) on the degradation of (R-(+)-), and vice versa, such as when the racemic compound was incubated. The ER values of DC from experiment SW4 and SC4 showed a continuous decrease (Figure S3c and d). Fitting the measured ER values in Figure 2b,d to incubation time t using eq 2 yielded a linear form for DC (incubation of the rac-DC in Wuxi soil and Chifeng soil, respectively). The rate difference ∆k calculated from these plots are -0.045 d-1 and -0.0303, respectively, which are in agreement with above data (kR - kS ) -0.044 d-1 for Wuxi soil and kR - kS ) -0.032 d-1 for Chifeng soil, respectively). ES that is a measure of enantioselectivity was defined as ES ) (kS - kR)/(kS + kR) in a previous study (36). When the (S)-enantiomer elutes first, as in this study, positive values (0 < ES e 1) indicate a more rapid degradation of (S)enantiomer, while negative values (-1 < ES e 0) indicate a more rapid dissipation of (R)-enantiomer. At an ES value of 0, dissipation is not enantioselective, and at an ES value of 1, degradation is fully enantioselective. In experiment SW4 and SC4, the ES values were 0.38 and 0.50, respectively. In other words, the ES of DC in Chifeng sandy loam soil, which has a higher soil pH, is higher than that in Wuxi silt loam soil. These ES values suggest that the degradation of DC in the two soils is enantioselective, and S-(-)-DC degraded faster than R-(+)-DC. Enantiomerization Degradation of DC in Soils under Aerobic Conditions. Enantiomerization, i.e., the transformation of S-(-)-DC to R-(+)-DC or vice versa, was studied by separate incubations of the two enantiomers of DC under aerobic conditions. In Figure 3a,c we plotted the data from experiment SW5 and SC5, respectively (incubation of the S-(-)-DC in Wuxi soil and Chifeng soil, respectively). The data for S-(-)-DC in Wuxi soil and Chifeng soil show a continuous decrease of the concentration to 1.1% after 47 days of incubation and 0.60% after 90 days of incubation, respectively, of the initial concentrations. At the same time, the concentrations of R-(+)-DC in the two soils increased from initial values of 0 to a maxima of 73 and 54% after 14 days of incubation, VOL. 44, NO. 6, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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respectively, and then decreased again. The curves for the R-(+)- and S-(-)-enantiomers intersected after ≈6 and 7 days of incubation, respectively. That is to say that a 50% inversion of S-(-)-DC in Wuxi soil and Chifeng soil was observed in 6 days and 7 days, respectively. The results from the these experiments illustrate conversion of the S-(-)-DC into R-(+)DC. Furthermore, the results indicated that the extent of the inversion may be dependent on the soil type. In Figure 3b,d we plotted the data from experiment SW6 and SC6, respectively (incubation of the R-(+)-DC in Wuxi soil and Chifeng soil, respectively). The data for R-(+)-DC in Wuxi soil and Chifeng soil show a continuous decrease of the concentration to 1.1% and 15.6% after 120 days of incubation, respectively, of the initial concentrations. At the same time, the concentrations of S-(-)-DC in the two soils increased from initial values of 0 to a maxima of 8.5% after 23 days and 7.7% after 14 days of incubation, respectively, and then decreased again. During the experiments, the concentrations of S-(-)-DC were below those of the respective R-(+)-DC with ERs approaching 0.45 and 0.06 at 90 days for Wuxi soil and Chifeng soil, respectively. The results from these experiments illustrate conversion of the R-(+)-DC into S-(-)-DC, but the inversion-rate of the R-(+)-enantiomer was lower than that of S-(-)-enantiomer. In the following discussion, we try to estimate the rate of enantiomerization of S-(-)-DC to R-(+)-DC or vice versa. Take the case of Wuxi soil, in the initial phase of the experiments of SW5 and SW6 with the individual enantiomers, the concentration of the minor enantiomer is small, and the increase in the concentration of the minor enantiomer is determined by the enantiomerization rate of the major one. The rate of formation of the minor enantiomer, neglecting its degradation, thus is equal to the rate of enantiomerization of the major one, which thus can be estimated from the equation ln[(C0-C)/C] ) kt, where C0 is the initial concentration of the major enantiomer, C is the concentration of the minor enantiomer at time t in the initial phase, and k is the enantiomerization rate constant. The rate of enantiomerization thus determined from experiment SW5 (kSR; S-(-)enantiomer is the major; 0-14 d data) was 0.22 d-1. The rate of enantiomerization determined from experiments SW6 (kRS; R-(+)-enantiomer is the major; 0-23 d data) was 0.024 d-1. The values thus determined are probably higher because of a neglect of the concurrent degradation of the minor enantiomer in the initial phase. The value of kSR/kRS is 9.1, favoring an inversion of S into R enantiomer. Degradation of the Metabolite of DC in Soils under Anaerobic Conditions. DM degraded rapidly to DC under anaerobic condition, we thus only investigated the degradation of DC in the two soils under anaerobic conditions (Tables 1 and 2, exp. SW7 and SC7). Tables 1 and 2 show the halflives (t1/2), the rate constant values in the two soils under anaerobic conditions, and the results indicated that both DC enantiomers were degraded more slowly under anaerobic conditions than under aerobic condition. In Figure S6a,c, we plotted the data from experiments SW7 and SC7 with rac-DC as normalized concentrations (100C/C0) versus time. The plots show significant degradation of both enantiomers but with a clearly more rapid degradation of S-(-)- than R-(+)-DC. The ER values of DC consistently decreased with time in experiment SW7 and SC7 (Figure S3e,f). A t test between the ER values of DC in the two soils and ER ) 1.0 yielded a p value of 0.01 and 0.007, respectively. From these results, it can be concluded that the degradation of DC was enantioselective in the anaerobic soils, the same as that in the aerobic soils. For Wuxi soil, in Figure S6a, reasonable fits of the data for rac-DC from experiment SW7 were obtained with kS and kR of 0.047 and 0.016 d-1, respectively. For Chifeng soil, in Figure S6c, reasonable fits 2046
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of the data for rac-DC from experiment SC7 were obtained with kS and kR of 0.032 and 0.009 d-1, respectively. The ER values of DC from experiment SW7 and SC7 showed a continuous decrease (Figure S3e,f). Fitting the measured ER values in Figure S6b,d to incubation time t using eq 2 yielded a linear form for DC (incubation of the rac-DC in Wuxi soil and Chifeng soil, respectively). The rate difference ∆k calculated from these plots are -0.0314 d-1 and -0.0224, respectively, which are in agreement with above data (kR - kS ) -0.031 d-1 for Wuxi soil, and kR - kS ) -0.023 d-1 for Chifeng soil, respectively). In experiment SW7 and SC7, the ES values were 0.49 and 0.56, respectively. This also suggested that enantioselective degradation occurred for DC under anaerobic conditions, and S-(-)-DC degraded faster than R-(+)-DC. Furthermore, as described above, the direction of enantioselectivity of DC did not shift; in other words, anaerobic conditions did not significantly alter the enantiomeric preference of the microbal community.
Acknowledgments This work was supported by fund from the National Natural Science Foundation of China (Contract grant number: 20777093 and 20707038).
Supporting Information Available Six figures. This material is available free of charge via the Internet at http://pubs.acs.org.
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