Stereoselective Bioaccumulation and Metabolite Formation of

May 20, 2014 - Figure 1. Chemical structures of triadimefon and triadimenol. ... Several studies have reported that TF could be transformed to TN at a...
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Stereoselective Bioaccumulation and Metabolite Formation of Triadimefon in Tubifex tubifex Tiantian Liu, Jinling Diao, Shanshan Di, and Zhiqiang Zhou* Department of Applied Chemistry, China Agricultural University, Beijing 100193, P. R. China S Supporting Information *

ABSTRACT: Triadimefon, a chiral fungicide, could be metabolized to triadimenol which has two chiral centers. In this work, Tubifex tubifex was exposed to triadimefon through the aqueous and soil phase to explore the relative importance of the routes of uptake. Bioaccumulation of triadimefon in tubifex was detected in both treatments, and the kinetics of the accumulation processes were significantly different in these two experiments. In spiked water treatment, (S)-triadimefon was preferentially accumulated over the (R)-triadimefon, whereas the enantioselective bioaccumulation was not detected in the spiked soil microenvironment. Simultaneously, four stereoisomers of triadimenol were also found in the tubifex tissue. Although the amount of these stereoisomers were different from each other with relatively more accumulation of the most fungi-toxic stereoisomer (1S,2R), the abundance ratios in the two exposure treatments were similar at the same sampling, following the order (1S,2S) > (1R,2S) > (1R,2R) > (1S,2R). The bioaccumulation factor was calculated for parent compound triadimefon and metabolite enrichment factor for metabolite. The results showed that both uptake routes, epidermal contact in the aqueous phase and ingestion of solid particles in soil, were important to the bioaccumulation of the triadimefon and triadimenol in tubifex.

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INTRODUCTION

Triazoles are a class of systemic fungicides that contain the 1,2,4-triazole moiety. Because of their excellent antifungal activity and relatively low resistance risk, triazoles are becoming the most important class of fungicides, and they are widely used in agricultural applications.1,2 In the molecular structure, a large number of the triazole fungicides have stereogenic centers, and moreover, their metabolic products in various environmental compartments may also be chiral.3 However, most of the triazole fungicides are commercialized as racemate products and released into the environment as an equimolar mixture of enantiomers. It has been widely confirmed that enantiomers of the same compound may perform differently in environmental fate and ecological risk.4−6 Thus, chirality is an important feature in evaluating their environmental behavior and toxicity for triazole-type fungicides. One of these typical triazoles is triadimefon (TF) (Figure 1) with one chiral carbon center. Established data showed that in plants, soils, and animals, the carbonyl group in TF could be easily reduced to alcohol to form the more fungi-active metabolite, triadimenol (TN).7−10 Thus, TN possesses two chiral centers and contains four enantiomeric forms, 1R,2S, 1S,2R, 1R,2R, and 1S,2S. Among these stereoisomers, 1R,2S and 1S,2R constitute one diastereoisomer called triadimenol-A, and 1R,2R and 1S,2S form the other diastereoisomer named triadimenol-B. The enantiomers A1 (1R,2S) and B1 (1R,2R) are metabolites from the (R)-TF, and enantiomers A2 (1S, 2R) and B2 (1S, 2S) are from the (S)TF (Figure 1). Previous studies reported that in some instances transformation products may be more toxic, 11−14 and © 2014 American Chemical Society

Figure 1. Chemical structures of triadimefon and triadimenol. *Indicates the chiral center.

Received: Revised: Accepted: Published: 6687

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consequently, these substances may pose a greater risk to the environment than the parent compound. In addition, the biotransformation of TF into TN is stereoselective,15,16 and the fungicidal activities of four optical isomers of TN differ greatly, with the (1S,2R)-TN isomer shows the highest fungitoxicity (up to 1000-fold more active) than the other three. Yuanbo Li et al. also reported that the toxicities of the four stereoisomers were different from Daphnia magna, following the order (1R,2S)-TN > (1S,2R)-TN > (1R,2R)-TN > (1S,2S)-TN.7 Thus, the stereoselective formation of TN from TF and the associated exposure of the two chiral compounds are important issues for ecological risk assessment. Several studies have reported that TF could be transformed to TN at a relatively fast rate in soil;9,17 however, it is relatively stable in water.18 Although a specialty fungicide applied at low rates, there were 149000 pounds of the active ingredient used in 1988.9,19 As a consequence of their common use, substantial amounts of TF and TN could enter into aquatic system via runoff and spray drift. Triadimenol, for example, has been detected in water samples from ditches and streams at 3 μg L−1.20 In addition to evaluating the potential toxic effects of these contaminations to the aquatic organisms, it is important to understand their accumulation and dissipation characteristics for more comprehensive risk assessment in aquatic biota. Furthermore, because some of the pesticides are readily biotransformed with different rates in different species,21 models based on the physical−chemical properties of these chemicals to predict bioaccumulation in different species may be variable. Limited studies on the bioaccumulation of TF and TN in aquatic organisms have focused on the fish.22 Therefore, there is a need to measure accumulation, assess biotransformation, and track the metabolite formation of TF in other aquatic organisms, such as the benthic fauna. The benthic fauna is of great importance because it represents an essential link in the aquatic food web. Among benthic macroinvertebrates, tubifex worms and, more generally, oligochaetes Tubificidae, are one of the most widespread and ubiquitous groups in freshwater ecosystems.23 These organisms have an intimate contraction with the solid phase and the pore water of the sediment, burrowing the anterior part in the sediment and undulating the posterior part in the overlying water. Thus, this worm is particularly exposed to environmental pollutants via sediment, pore water, and water column through ingestion and/or epidermal contact. Because of the wide distribution and typical benthic lifestyle, tubifex was widely used in assessing the toxicities and accumulations of sedimentassociated contaminations.24,25 Unfortunately, relatively little is known concerning the importance of different exposure routes on contaminations uptake and accumulation. Since contaminations can be taken up and accumulated by benthic invertebrates from different sources, determinations that assess the relative importance of the different uptake routes are helpful for understanding bioaccumulations. In the present study, tubifex worms were exposed to TF through either the aqueous or soil phase to compare the effect of different microcosm exposure on accumulation. A 14 d exposure treatment and a 7 d elimination experiment were conducted in order to determine the enantioselective bioaccumulation and elimination of TF and stereoselective formation of TN in the worm tissue. To our knowledge, this is the first experiment to investigate the enantioselective bioaccumulation and biotransformation of triadimefon in tubifex.

Article

MATERIALS AND METHODS

Reagents. Analytical standards of TF and TN were obtained from the Institute for Control of Agrochemicals, Ministry of Agriculture of China (Beijing, China), with purities of 98.5% and 97.9%, respectively. The ratio of diastereoisomer A (1R,2S; 1S,2R) to diastereoisomer B (1R,2R; 1S,2S) that compose TN is approximately 7:3. Ethyl acetate and n-hexane were obtained from Fisher Scientific (Fair Lawn, NJ) and were of HPLC grade. Methanol and acetonitrile (analytical grade) were purchased from commercial sources. Tubifex. Tubifex was obtained from Beijing Da Senlin Flower Market (Beijing, China). Worms were reared in 2 L plastic tanks containing uncontaminated soil (the same soil used in the following experiment) and deionized water at 21 ± 1 °C with 12 h light/12 h darkness. The water was continuously aerated. The organisms were fed with tetraMin Flakes (Tetra Werke, Melle, Germany) and allowed to acclimatize for 1 week prior to the experiments. Accumulation from the Aqueous Phase. The accumulation and elimination characteristics of TF and TN from the aqueous phase by tubifex were determined. In this scenario, water was the sole source of contamination and living environment with no sediment attendance. Acclimated tubifex (5 g) were exposed to a TF solution in 500 mL beakers. In each experimental container, the spiking solutions were made by adding 100 μL of TF stock solution (1000 mg mL−1, dissolved in acetone) to 100 mL of deionized water. The spiked solutions were refreshed daily. During the experiment, temperature and lighting conditions were set as above, but the organisms were not fed and the water was not aerated. After 7 days of exposure, worms in some beakers were transferred to uncontaminated water to follow the elimination process. The duration of elimination experiment was 7 d, and samplings were performed at days 1, 2, 3, 5, and 7. Worms in the other part of the beakers had a continued exposure with the spiked solutions until 14 d, and worms were sampled at days 1, 2, 3, 5, 7, 10, and 14. At each sampling time, tubifex worms were sifted, washed with distilled water, and finally the water on the surface of the worms was dried by absorbent paper cautiously. In addition, 50 mL of spiked water was collected from each beaker at each sampling time to detect the concentrations of TF and TN. Samples were stored at −20 °C for later extractions and analyses. A control treatment containing TF only and no worms were carried out to determine how the amounts of TF changed in water without the existence of tubifex. Because the spiked solutions were refreshed daily in the test treatment, the duration of the control treatment was 1 day, and samplings were performed at 6, 12, and 24 h. Accumulation from the Soil Phase. A terrestrial soil collected from BaiWang Forest National park (Beijing, China) was used as the solid bottom substrate to simulate one type of sediment. Soil was collected and sieved through 500 μm mesh and then kept in dark at room temperature until it was airdried. No detectable TF or TN was found at detectable levels in soil. Physicochemical characteristics of the soil were as follows: organic carbon (OC), 27.93 ± 1.46%; moisture content (MC), 1.66%; clay, 3.35 ± 0.02%; sand, 60.47 ± 0.25%; silt, 36.19 ± 0.22%; pH, 6.6 ± 0.2. The soil was inoculated and prepared following the procedures reported in our previous study.26 Briefly, 5 mL of stock solution containing 2 mg of TF that dissolved in acetone was added dropwise into 100 gdwt (dry weight) soil. The inoculated soil was homogenized manually 6688

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the relative sorptive capacities of the organism versus the surrounding environmental, and for this treatment it was defined as

using a stainless-steel lab spoon every few hours (during the daytime). Then the spiked soil was left in a fume cupboard overnight to volatilize the solvent completely. The spiked soil (100 gdwt, wet weight) was transferred to a 500 mL beaker, and then 100 mL of deionized water was added to the beaker slowly along the wall. After settling for 24 h, approximately 5 g of tubifex was added to the soil/water system to start the exposure experiment. A control team was set without the addition of worms, while other operations were carried out as the same as above. For this experiment scenario, test organisms, soil and overlying water were collected at days 1, 2, 3, 5, 7, 10, and 14 from both test and control beakers. At each sampling time, all of the overlying water was siphoned out first, and then the beakers were put on ice for 2 h. During this period, tubifex climbed to the soil surface and intertwined together slowly, and the worm aggregation was sampled with forceps and rinsed in deionized water. The beakers were weighed every day, and the loss of water by evaporation was compensated by addition of deionized water. To study the elimination kinetics of TF in the worms bodies, 5 g of tubifex were exposed to TF in 100 gdwt of soil and 100 g of water as in the uptake experiment described above. After 7 days of exposure, the worms were transferred to a new flask with 100 gdwt of clean soil and 100 g water for depuration, which lasted for 7 d. During the depuration period, worms were collected on days 1, 2, 3, 5, and 7. All samples were frozen at −20 °C before analysis. All of the incubations were carried out in triplicate at each sample point. Sample Extractions and Chemicals Analyses. For the analysis of TF and TN, water, soil, and tubifex samples were extracted by ethyl acetate, and an Agilent 7890 gas chromatograph (GC) with electron-capture was used to analyze the sample. The detailed information on the sample extraction, cleanup, and chemicals analyses are provided in the Supporting Information. Data Analysis. Tubifex Worms Elimination. Worms elimination data were also fitted to a first-order kinetic elimination model (eq 1), and the elimination half-life (t1/2) was determined by eq 2 Cw(t ) = Cw(0) × e−k wt

(1)

t1/2 = ln 2/k w = 0.693/k w

(2)

BAF = Cworm/Cse

(5)

where Cworm and Cse are concentrations of compound in tubifex and surrounding environment, respectively. Because tubifex can accumulate TN through the uptake from the surrounding and the metabolism of TF in the tissue, we used the metabolite enrichment factor (MEF) to describe the accumulation of TN. MEF was defined as the ratio of the internal concentration of the metabolite stereoisomer Cworm to the external total concentrations of the corresponding enantiomer of TF and metabolite Cse,(parent+metabolite). MEF = Cworm/Cse,(parent + metabolite)

(6)

Statistical Analysis. Data presented corresponds to means ± standard deviations of three independent experiment (n = 3). The concentrations of TF enantiomers were analyzed using one-way analysis of variance (one-way ANOVA), and a pairwise multiple comparison procedure (S−N−K test) was used to compare results at p < 0.05.

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RESULTS AND DISCUSSION Uptake and Elimination Characteristics from the Water Phase. The accumulation of TF in tubifex from the water phase was determined by exposing the tubifex to a 1 μg mL−1 of TF solution for 14 days, and the results are shown in Figure 2a. It showed a rapid accumulation of TF during the first 3 days, followed by a slower accumulation phase. During the

where Cw(t) is the stereoisomer concentration of TF or TN in tubifex at time t (day) during the elimination phase, Cw(0) is the concentration in the worms at the start of the elimination phase, and kw is the elimination rate constant (day−1). Dissipation of TF in Soil. Changes in the concentration of compound in soil over time were modeled using a first-order exponential decay model Csoil(t ) = Csoil(0) × e−kst

(3)

where Csoil(t) is the stereoisomer concentration of TF or TN in soil at time t (day), Csoil(0) is the concentration of stereoisomer at the start of the test, and ks (day−1) is the rate constant for dissipation from soil. The corresponding half-life was calculated as t1/2 = ln 2/ks = 0.693/ks

Figure 2. Accumulation (a) and elimination (b) curves of TF enantiomers and TN stereoisomers in the tubifex tissue in the TFspiked water treatment (bars are standard error). *Indicates significant difference between the two enantiomers of TN at the same time point (P < 0.05, S−N−K test).

(4)

Bioaccumulation and Metabolite Enrichment Factors. In this work, we used BAF (bioaccumulation factor) to express the bioaccumulation of TF in tubifex tissue. BAF is a function of 6689

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Figure 3. (a) Stereoisomer concentrations of TF from TF-spiked water treatment or TN from TN-spiked water treatment in the tubifex tissue (detected on seven accumulation sampling times) relative to their corresponding concentrations in water. (b) Calculated bioaccumulation factors (BAFs) for rac-TF and metabolite enrichment factors (MAEFs) for rac-TN in the TF-spiked water treatment (bars are standard error).

bioaccumulation period, different concentrations of the two enantiomers of TF in tubifex tissue were observed at the same sample point, with concentrations of the S-form higher than that of the R-form. However, the concentrations of the individual enantiomers were almost the same in water (Table S1, Supporting Information), and the previous study has reported that TF could undergo significant racemization in water.27 These results indicated that the enantioselective bioaccumulation of TF in tubifex tissue was decided by the enantioselective uptake, rather than the selective degradation of TF in water. GC analysis of both the water and organism samples resulted in the appearance of four distinct chromatographic peaks that eluted later than the two peaks of TF. Based on comparison of the retention time with authentic standards, the four peaks were identified as TN [A1 (1R,2S), A2 (1S,2R), B1 (1R,2R), and B2 (1S,2S)]. However, we did not detect the TN enantiomers in controls containing TF only and no worms after incubated for 24 h. Moreover, the TF enantiomers showed no degradation in the control water. Thus, tubifex worms played an very important role in the formation of TN in this spiked water experiment. The stereoisomers of TF and TN concentrations in spiked water during the accumulation process are listed in the Supporting Information, Table S1. During the 14 d exposure period, the loss of TF in the spiked water were determined before the solution was refreshed at each sampling time with a decrease of the concentration up to 27.1% in the whole experiment. We also found that the decrease in the parent compound was accompanied by the formation of the metabolite. These results demonstrated that TN metabolites were only found in the exposure medium when tubifex were present. It is possible that tubifex are capable of producing TN metabolites in vivo and excreting some of them to the surrounding solution. On average, the formation of TN accounted for 95% (77−102%) of TF depletion depending upon the initial concentration of TF in the test water. With the increase of incubation time, the formation of (1R,2S)-TN and (1R,2R)-TN in water decreased steadily; however, the relative amount of (1S,2S)-TN showed an increasing trend. Without regard to the enantiomer conversion, the (1R,2S)-TN and (1R,2R)-TN can be assumed to result solely from reduction of (R)-TF and similarly the (1S,2R)-TN and (1S,2S)-TN from (S)-TF. Thus, (S)-TF experienced stereoselectivity degradation and excretion process in the spiked water with a preferential formation of (1S,2S)-TN, whereas this case is not significant for

(R)-TF with almost the same amount of (1R,2S)-TN and (1R,2R)-TN. From Figure 2a, we found that the bioaccumulation of TF in tubifex tissue was also accompanied by the formation of TN. During the initial 1 day of exposure incubation, the amount of (1S,2S), (1R,2S) and (1R,2R)-TN detected was about 0.24, 0.077, and 0.059 mg kg−1, respectively, in worm samples. After further exposure incubation, the concentrations of (1S,2S)-TN increased continuously while the concentrations of (1R,2S) and (1R,2R)-TN increased slowly and showed a tendency toward stabilization. Thus, the proportion of TN-A to TN-B in the tubifex tissue decreased steadily with increase of incubation time. It is obvious that there was a large difference between the amount of the four TN stereoisomers in water or organism tissue, indicating that the formations and bioaccumulations of TN were diastereoselective. Figure 2b illustrates the depuration profile of the accumulated enantiomers of TN and TF. Elimination kinetics followed a first-order kinetic well (R2 = 0.918−0.991). The elimination rate coefficients (kw) were calculated by fitting data from the depuration phase to eq 1 (Table S2, Supporting Information). As illustrated in Figure 2b, approximately 77% and 76% of the (R)-TF and (S)-TF were depleted or excreted after 24 h, respectively. Significant differences between the concentration of (S) and (R)-TF were detected at most sample times except for the third day, indicating that the (R)-TF was preferentially degraded. The metabolite of TF appeared to be more stable, with the depuration half-life approximately 3 times longer than that of TF. In most of the cases, only three (1R,2S; 1R,2R; 1S,2S) of the four possible TN enantiomers can be determined, and the concentration of (1S,2R)-TN was lower than the LOQ. To compare the accumulation of the TN stereoisomers, a separated experiments was carried out with a TN solution (1 μg mL−1), while other operations were carried out the same way as the accumulation experiment of TF. The uptake results are shown in Figure S1 (Supporting Information), and the accumulation model of TN enantiomers was similar to that of TF. A rapid accumulation was detected during the initial 3 day of incubation; after that the accumulation slowed. The accumulation of the four TN stereoisomers in tubifex did not reach a steady state, and the plots also showed that the bioaccumulation of TN-A enantiomers and TN-B enantiomers were both enantioselective. The enantiomeric composition of TN (1R,2S > 1S,2R > 1S,2S > 1R,2R) detected in worm tissue 6690

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Previous studies25,29 have shown that the aquatic phase could be an important pathway for the bentonic organisms in the accumulation of environmental contaminations. Therefore, the TF in the overlying water was also determined in this study (Figure S2b, Supporting Information). For the control treatment, the concentrations of TF in the overlying water were almost constant and had a slight decrease from the seventh day (Figure S2b, Supporting Information). For the worm-present treatment, the concentrations of TF in the overlying water increased rapidly during the first 2 days, reached a relative stable stage from the second to fifth day, and then decreased. Finally, it reached steady state again on 14th day (Figure S2b, Supporting Information). At the same time, the TN was also found in the overlying water during the whole incubation period. The concentrations of TN increased continually, and followed the order (1S, 2S)-TN> (1R, 2S)TN> (1R, 2R)-TN (Figure S3b, Supporting Information). From Figures S2b and S3b (Supporting Information), we also found the concentrations of TF and TN in test treatments were higher than that in control treatment, demonstrating the bioturbation functions of tubifex. TF Uptake Characteristics from Soil Phase. For the spiked soil experiment, tubifex was exposed in an aquatic microenvironment with TF-inoculated soil and clean water phase for 14 d. Figure 4a describes the accumulation curves of

in this scenario was significant different with that in TF-spiked water test (1S,2S > 1R,2S ≥ 1R,2R). In this work, the bioaccumulation of TF and TN was also assessed through calculation of BAF values. The BAF values calculated in different surrounding environments are shown in Figure 3. From Figure 3a, it can be seen that the BAFs of all the four TN stereoisomers detected during the whole experiment were higher than 1, whereas almost of the BAFs of TF were lower than 1. So it seemed that the metabolite of TF were more inclined to be concentrated by tubifex than the compound itself through water phase exposure. Although the BAFs of the four TN enantiomers at the same sampling were also different, following the order 1S,2S > 1R,2R > 1R,2S > 1S,2R, the enantiomeric profile was invariant in TN-spiked water exposure. We also calculated MEFs (eq 6) for the TN metabolite and compared it to the BAFs of the TF parent compound (Figure 3b). As shown in Figure 3b, the MEFs of rac-TN were close to the BAFs of rac-TF with increase of incubation time. Therefore, the field biomonitoring of TF using tubifex may need to look for the metabolite, TN, and the assessment of environmental behavior of TN is also of concern. Dissipation of TF and Formation of TN in Surroundings. To simulate the living environment of tubifex preferably, the spiked soil was employed as the underlying substrate. The concentration of TF in soil is plotted over time in Figure S2a (Supporting Information) for both the worm-present and control treatments. In general, the residues of both enantiomers of TF decreased with time elapsed in soil. The dissipation kinetics of TF enantiomers in soil followed the first-order kinetic equation. Rate constants and the corresponding half-life were calculated using eqs 3 and 4, listed in Table S3 (Supporting Information). No significantly enantioselective behavior was detected in the dissipation of the TF in the soil. Meanwhile, we found that the participation of tubifex affected the dissipation of TF in soil significantly, with the half-life of 8.88 day in worm-present soil and 16.11 day in control soil. In a previous study,9 the half-life of TF was 18 days in a sandy loam soil, which was consistent with the result in control soil in our present work. The average half-life of TF reported in other studies was 26 days.28 Neera Singh also reported that in flooded soil systems, the higher the soil organic carbon content was, the less persistent was the fungicide, with an half-life of 10.2 day in flooded mollisol.17 These results indicated that the existence of tubifex could accelerate the dissipation of TF in soil. In addition, we also found the formation of (1R,2S), (1R,2R), and (1S,2S)-TN in both test and control soil (Figure S3a, Supporting Information). Combined with the previous study,9 it was speculated that the formation of TN in control soil was due to soil microbial metabolism. In general, the amount of (1R,2S)-, (1R,2R)-, and (1S,2S)-TN increased with the increasing of incubation time in either test or control treatment, except the last sampling time. Compared with the spiked water experiment, the similar result that (1S,2S)-TN was the most abundant metabolite was found. Furthermore, the total concentrations of TN in control soil were higher than that in test treatment in the first day, and the concentrations were lower or no longer significantly different in the other sampling points. The explanation was that tubifex in test treatment could uptake TN. Another possible reason was that the existence of tubifex could quicken the diffusion of TN from soil to the overlying water.

Figure 4. Accumulation (a) and elimination (b) curves of TF enantiomers and TN stereoisomers in the tubifex tissue in the TFspiked soil treatment (bars are standard error).

TF enantiomers. Tubifex rapidly accumulated both enantiomers within the first two days, and concentrations reached the highest level on the second day. After further exposure incubation, the amounts of the (R)-TF and (S)-TF decreased to 1.22 and 1.28 mg kg−1 at day 7, respectively, and then showed a tendency toward stabilization. In this microenvironment, the accumulation curve of the (R)-enantiomer is not 6691

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significant correlations were found between the concentrations in worm tissue and in soil (r = 0.819, p = 0.024 for R-form, r = 0.916, p = 0.004 for S-from), but not between the concentrations in worm tissue and in overlying water. These results demonstrate that the bioaccumulation of TF in spiked soil incubation is governed by the TF concentrations in soil, not the concentrations in overlying water. For the stereoisomers of TN, concentrations of (1R,2S)-TN in tubifex were negatively correlated with the concentration of (1R,2S)-TN in the soil (r = −0.816, p = 0.025) and positively correlated with the concentration of TF in the tissue (r = 0.838, p = 0.018 for R-form, r = 0.837, p = 0.019 for S-form). For (1R,2R) and (1S,2S)-TN, no significant correlation was found neither between the concentrations in worm tissue and in surrounding environment nor between the concentrations of diastereoisomer B and TF in worm tissue. Thus, the bioaccumulations of the (1R,2S)-TN may be regulated in different way from that of (1R,2R) and (1S,2S)-TN. Figure 4b illustrated the depuration kinetics of the accumulated compounds, including (R)-TF, (S)-TF, (1R,2S)TN, and (1R,2R)- and (1S, 2S)-TN in the tubifex tissue. The elimination rate coefficients (shown in Table S3, Supporting Information) for stereoisomers of the two compounds determined in clean soil was not statistically different from that in clean water, and the half-lives were 0.35−0.36 and 1.38− 1.67 for TF and TN, respectively. In addition, no enantioselective phenomenon was detected in the depuration as well as uptake period for the TF enantiomers in spiked soil incubation experiment. The BAFs were also calculated for the TF enantiomers in this scenario. Because tubifex can accumulate TN through the uptake from soil and the metabolism of TF in the tissue, we used MEFs (eq 6) to describe the accumulations of TN stereoisomers and compared them to the BAFs of the TF enantiomers. The results are shown in Figure S5a,b (Supporting Information). As illustrated in Figure S5a (Supporting Information), BAFs of (R)-TF were higher than the MEFs of (1R,2S)- and (1R,2R)-TN, but BAFs of (S)-TF were lower than the MEFs of (1S,2S)-TN. It was consistent with the order of concentrations in tubifex tissue. There was a significant difference in the BAFs of TF between Figures S5a and S5b (Supporting Information). The BAFs of TF was lower than 0.475 in soil (Figure S5a, Supporting Information), but the values increased markedly in overlying water (Figure S5b, Supporting Information). Moreover, the difference in the MEFs of TN between the soil (Figure S5a, Supporting Information) and the overlying water (Figure S5b, Supporting Information) was not significant. These results indicated that the values in the overlying water may be overestimated. The possible reason was the concentrations of TF in tubifex tissue were a combination result of the bioaccumulation that happened through various route including epidermal contact and ingestion of soil particles. Therefore, the BAFs of TF in overlying water (Cworm/Coverlying water) was overestimated greatly. And the water-phase exposure (skin exposure) may be an important route for the enrichment of TN. In other words, the amount of TN in tubifex tissue accumulated from the overlying water by skin was more than that from the soil particles by ingestion. Thus, the MEFs of TN in overly water (Cworm/ Coverlying water(parent+metabolite)) was only overestimated slightly.

significantly (p < 0.05) higher than that of the (S)-enantiomer. In other words, the bioaccumulation behavior of TF in tubifex was nonenantioselective. In addition, all of the four stereoisomers were detected in the worm tissue, but the concentration of (1S,2R)-TN was too low to be quantified during the whole exposure period. The concentrations of the other three stereoisomers varied with the incubation times and followed the order 1S,2S > 1R,2S > 1R,2R. With an increase of exposure time, the (1S,2S)-TN became dominated in enantiomeric composition. The presence of TN in the tissue could be due to the transformation of TF in the organism which was accumulated by tubifex or due to soil microbial metabolism and to the absorption of TN into the tissue from the soil. Two different experiments were conducted to determine whether the tubifex could absorb TF and TF and whether the tubifex could metabolize TF to TN. In the first experiment, 1 mL of acetone solution that contained 0.5 mg of TF was pipetted and added to the filter paper (d = 11 cm). After the solvent was dried under a stream of compressed air, the inoculated filter paper was placed in Petri dishes, and then 2 mL of deionized water was added to each Petri dish. The purpose of added water to the filter paper was to provide a moist environment. After 3 g of worms was added to each dish, the Petri dish was sealed with plastic film with several ventilation holes. TF and TN were both detected in the worms which were sampled after 24 h exposure. The result indicated that TF could be metabolized in vivo in the absence of soil. In the second experiment, tubifex was exposed in the TN spiked soil (10 mg kg−1). Soil and other operations were as the same as the TF spiked soil experiment. In this scenario, TN was also detected in the worm tissue, revealing that tubifex has the ability to accumulate TN from soil. Thus, these results indicated that the presence of TN in the tubifex was a combination of the metabolism of TF in tubifex and the uptake of TN from soil. Previous studies have reported10,30,31 that when organisms exposed to the TF, the product proportion of the (1R,2S; 1S,2R) and (1R,2R; 1S,2S)-TN diastereoisomers might not be the same in the different organisms. For example, black fly larvae30 and trout10 both stereoselectively form TN from TF. The difference was that black fly larvae produce a 5-fold excess in the stereoisomer fraction of diastereomer A (1R,2S; 1S,2R). The data in tubifex were different from the results in black fly larvae and in trout (Figure S4, Supporting Information). The different diastereoisomeric composition of the metabolites in a given species is a result of their metabolic capacity and the specific genes (and their expression) they possess. Because different species have their distinct physiology, they may show different responses to the same pollutant. Thus, it is necessary to have a wide testing of these diastereomers across species to study their influence on organisms and fate in environment comprehensively. The uptake kinetics of the TF and TN were significantly different in the tubifex tissue between the spiked water and spiked soil experiment. In the case of accumulation of the compounds through the aqueous phase, the bioaccumulation of TF and TN had the similar kinetics. However, in the spiked soil experiment, they are accumulated with different kinetics. The two compounds both showed a rapid accumulation during the first 2 days, indicating that the gut was filled with spiked-soil particles. On the 3th day, a rapid decrease in TF accumulation was detected, whereas a sustained and steady increase in uptake were found for TN stereoisomers. For the parent compound, 6692

dx.doi.org/10.1021/es5000287 | Environ. Sci. Technol. 2014, 48, 6687−6693

Environmental Science & Technology

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Article

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ASSOCIATED CONTENT

S Supporting Information *

Details of sample extractions and chemicals analyses, four tables, and four figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 13811992249. Fax: +8610-62733547. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by fund from the National Natural Science Foundation of China (Contract Grant Nos. 41201499 and 21337005).

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dx.doi.org/10.1021/es5000287 | Environ. Sci. Technol. 2014, 48, 6687−6693