Article pubs.acs.org/JAFC
Enantioselective Determination of Triazole Fungicide Epoxiconazole Bioaccumulation in Tubifex Based on HPLC-MS/MS Chunxiao Liu,† Bo Wang,† Peng Xu,§ Tiantian Liu,† Shanshan Di,† and Jinling Diao*,† †
Department of Applied Chemistry, China Agricultural University, Yuanmingyuan West Road 2, Beijing 100193, China Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Shuangqing Road 18, Beijing 100085, China
§
ABSTRACT: In this study, the enantioselective bioaccumulation of epoxiconazole enantiomers in tubifex (Oligochaeta, Tubificida) was investigated in two uptake pathways. A sensitive and rapid chiral method was developed for the determination of epoxiconazole enantiomers in tubifex and soil based on high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (HPLC-MS/MS). In the spiked-water or spiked-soil treatments, enantioselective bioaccumulation of epoxiconazole in tubifex was obersved. For spiked-water treatment, (−)-epoxiconazole accumulated in tubifex to a greater extent than (+)-epoxiconazole, leading to enrichments with a composition (−) > (+). However, for spiked-soil treatment, the enantioselectivity in tubifex was reversed with a preferential accumulation of (+)-epoxiconazole. Calculated accumulation factors (AFs) indicated that epoxiconazole in spiked-water treatment had higher bioaccumulation potential than that in spiked-soil treatment. The results from the spiked-soil treatment also revealed that the dissipation of epoxiconazole in soil was enantioselective, and tubifex has positive effects on epoxiconazole diffusion from soil to overlying water. KEYWORDS: epoxiconazole, tubifex, enantioselectivity, bioaccumulation
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INTRODUCTION Epoxiconazole is a broad-spectrum triazole fungicide and extensively used worldwide to control diseases caused by fungi, such as Ascomycetes, Basidiomycetes, and Deuteromycetes, in cereals, sugar beet, peanuts, oilseed rape, apple, and ornamentals.1 It is also used to regulate plant growth by acting as an inhibitor of C-14 demethylase in sterol biosynthesis. Epoxiconazole also plays an important role in the formation of ergosterol, a component of fungal cell membrane.2 This fungicide, which is widely used on wheat, sugar beet, triticale, barley, and oat,3 persists in soil and aquatic sediment.4,5 Hence, epoxiconazole in sediment may lead to serious noxious effects on benthic organisms that use sediment as food resources and living environments. After prolonged repeated applications, epoxiconazole could enter into fresh-water circulations. Therefore, the influence of epoxiconazole on the aquatic ecosystem should be investigated. E p o x i c o n a z o l e , ci s - 1- { [ 3 -( 2- c h l o r o p h e n y l )- 2 -( 4fluorophenyl)oxiranyl]ethyl}-1H-1,2,4-triazole, was first produced by BASF Corp. in 1993.6 It consists of two chiral centers in its molecular structure and therefore presents two diastereoisomers with four stereoisomers. However, present commercial epoxiconazole pesticide contains only a pair of enantiomers with 2R,3S- and 2S, 3R-configurations.7 The chemical structures and molecular weights (MWs) of four epoxiconazole stereoisomers are shown in Figure 1. Thus, epoxiconazole is a member of the chiral pesticide family. Organic pesticides play a crucial role in agricultural protection, and among these pesticides, >25% are chiral compounds with at least two mirror-image enantiomers.8 However, most of the currently used chiral pesticides are produced and released into the environment as racemates. Theoretically, enantiomers have identical physical and chemical properties, as well as abiotic degradation rates,9 whereas their individual toxicities, biological © 2013 American Chemical Society
Figure 1. Structures of epoxiconazole stereoisomers. The present commercial product refers to the cis-stereoisomers with 2S,3R and 2R,3S configurations.
activities, and microbial degradation rates have been shown to differ.9−13 Thus, the mechanism, exposure, and effect of each enantiomer should be evaluated when the risk of chiral pesticides to human and wildlife populations is assessed.13 This necessity has led to extensive research on the enantioselectivity of chiral compounds. Several studies have Received: Revised: Accepted: Published: 360
September 9, 2013 December 13, 2013 December 23, 2013 December 23, 2013 dx.doi.org/10.1021/jf403996g | J. Agric. Food Chem. 2014, 62, 360−367
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at room temperature, and stored in the dark until use within a few days. Physicochemical properties of the soil were as follows: organic carbon (OC), 4.28 ± 1.05%; moisture content (MC), 1.37%; clay, 4.31 ± 0.02%; sand, 58.42 ± 0.25%; silt, 37.19 ± 0.22%; and pH, 8.2 ± 0.2. Soil Handling and Spiking. To disperse the test substance epoxiconazole homogeneously within the 100 g dry weight of soil, we performed a stepwise dilution spike procedure.24 First, racemic epoxiconazole was dissolved in acetone to make a stock solution at a concentration of 1000 mg/L. Then, 0.1 mL of acetone solution was added dropwise into the dry soil (100 g) while the mixture was continuously mixed manually for approximately 5 min with a stainless steel laboratory spoon, yielding a nominal epoxiconazole concentration of 1 mg/kgdwt. The spiked soil was left in a fume cupboard for >12 h. After complete evaporation of the solvent, the contaminated soil (100 g dry weight) was transferred to a 500 mL beaker and rehydrated with 100 mL of deionized water. The height of the bottom substrate was 2−3 cm, and the overlying water was 2−3 cm. The test vessels were incubated for a 24 h equilibration period. Prior to tubifex addition, four 50 mL samples of wet soil were weighed, dried at room temperature until the weight became stable, and then reweighed to estimate the moisture content. Bioassay Procedure. To examine the influence of different uptake pathways of epoxiconazole on the total bioaccumulation in tubifex, two types of uptake kinetics were examined resulting from the spiked-water and spiked-soil treatments. The first scenario was designated {+Tub +water}, in which epoxiconazole was accumulated from spiked water. For each individual experiment, acclimated tubifex (10 g) were placed into beakers (21 beakers, 7 sampling points, and triplicates for one sampling point), and the spiking solutions were prepared by adding epoxiconazole dissolved in acetone to deionized water to obtain a final concentration of 1 mg/L. After an exposure period (0.5, 1, 3, 5, 7, 10, and 14 days), the living worms were removed from the beaker, gently passed through a 500 μm sieve to a clean pan, and then washed three times with deionized water. Finally, the peripheral water of the worm samples was dried using absorbent paper, and samples were weighed before storage at −20 °C, and the worm sample for extraction was 5 g. In the present study, only the uptake from the aqueous phase was studied for 14 days of exposure, and this treatment was performed in semistatic conditions with daily water renewal. The second scenario, which consisted of tubifex, water, and spiked soil, was designated {+Tub+soil}, in which epoxiconazole was accumulated from overlying water, pore water, and ingestion of soil particles. Acclimated tubifex (10 g) were added to the test beaker containing unspiked water and spiked soil (21 beakers, 7 sampling points, and triplicates for one sampling point). For the {+Tub+soil} treatment, test organisms, overlying water, and soil were also sampled after an exposure period (1, 3, 5, 7, 10, 14, and 20 days). At each sample point, overlying water was gently poured and initially sampled. Then, the beakers were placed on ice for 2 h, during which the tubifex worms climbed to the soil surface and intertwined slowly together. At this moment, worm aggregation was sampled with forceps and rinsed in deionized water. Water on the surface of the worms was carefully dried by absorbent paper. Soil samples, overlying water, and tubifex sampled from each beaker were weighed and frozen at −20 °C. To compare with the second scenario, a separate experiment (negative control), which was designated {−Tub+soil}, that used only water and spiked soil, was performed (21 beakers, 7 sampling points, and triplicates for one sampling point). The sampling times were also 1, 3, 5, 7, 10, 14, and 20 days. At these sampling points, overlying water and aliquots of 3.15 g of soil (based on dry weight) were removed from each beaker and transferred into 50 mL plastic centrifuge tube for extraction and analysis. For the {+Tub+soil} and {−Tub+soil} treatments, the test beakers were weighed daily, and the loss of water resulting from evaporation was compensated for by adding deionized water. All beakers were cultured in a dark environmental chamber, and temperature was controlled to 20 ± 2 °C. The treatment beakers were arranged in a randomized block design.
shown that enantiomers of epoxiconazole behave differently during biodegradation in the environment.14 Liang et al.6 reported that the dissipation of epoxiconazole enantiomers in grape is enantioselective with relative enrichment of (+)-epoxiconazole, and an enantioselective dissipation also occurred in soil with preferential degradation of the (−)-form under field condition. However, other aspects such as enantioselective bioaccumulation of epoxiconazole in tubifex have not been investigated. Tubifex is a cosmopolitan freshwater oligochaete (Tubificidea). It forms an important link in aquatic food chains.15,16 Given their endobenthic lifestyle, this oligochaete is in intimate contact with the solid phase and the pore water of the sediment.16 In addition, this worm ingests the substrates and utilizes adhering micro-organisms and detrital material.17,18 Tubifex is widely cultured as a fish food.19 Therefore, it is prone to transfer sediment-associated contaminants to higher trophic levels. Thus, tubifex plays an important role in the aquatic ecosystem and has been designated as a representative freshwater benthic infauna for aquatic system bioassays. Several studies have chosen tubifex to study bioaccumulation and sediment toxicity.20−23 However, data on enantioselective bioaccumulation of epoxiconazole enantiomers in Tubifex tubifex have not been reported. The differences in the bioaccumulation behavior of the two enantiomers of epoxiconazole in tubifex were investigated in the present study. Effective methods for the extraction, purification, and detection by high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (HPLC-MS/MS) have been developed to study epoxiconazole enantiomers in tubifex tissue under laboratory conditions. We compared the effect of two different contamination sources, spiked water and spiked soil, on the bioaccumulation of epoxiconazole. The results showed that bioaccumulations of epoxiconazole were both enantioselective in the two contamination treatments. Additionally, we evaluated the effects of tubifex on the diffusion and dissipation of epoxiconazole.
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MATERIALS AND METHODS
Chemicals and Reagents. Reference standards of rac-epoxiconazole (98.4%) were purchased from Shanghai Pesticide Research Institute (Shanghai, China). Acetone, formic acid, methanol, n-hexane, and ethyl acetate were of analytical grade and purchased from Beijing Chemical Reagent Co. Ltd. (Beijing, China). Acetonitrile was of HPLC grade and purchased from J. T. Baker (Phillipsburg, NJ, USA). The Florisil solid phase extraction (SPE) cartridges (500 mg/6 mL) were obtained from Agela Technologies (Tianjin, China) and conditioned with 5 mL of acetone/n-hexane (1:3, v/v) ether before use. Worms and Soil Collection. T. tubifex was obtained from Beijing Xiyuan Flower Market (Beijing, China). The worms were maintained in 2 L plastic tanks containing uncontaminated soil and deionized water at 21 ± 1 °C under 12 h light/12 h darkness. The water was continuously aerated, 75% of which was replaced weekly. The worms were fed TetraMin Flakes (Tetra Werke, Melle, Germany) weekly. For the experiments, adult T. tubifex (aged 5−7 weeks) were used. Prior to the introduction of worms into the treatments, tubifex worms were allowed to live in the uncontaminated environment for 1 week to acclimate. The experimental substrate was terrestrial soil collected from Baiwang Forest National Park northwest of Beijing, China. No detectable epoxiconazole was found at detectable levels in this soil. After the superficial layer of 1−2 cm was removed, the soil was collected to a depth of 10 cm, sieved through a 500 μm mesh, air-dried 361
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Figure 2. Chromatograms of (A) standard solution at 1 mg/L, (B) overlying water spiked at 0.1 mg/L, (C) soil spiked at 0.1 mg/L, and (D) tubifex spiked at 0.1 mg/L. Sample Extraction. The water and overlying water samples (5 mL) were extracted by 10 mL of acetone/ethyl acetate (1:3, v/v) in glass separatory funnels after removal of the solid matter by vacuum filtration. This extraction was repeated twice using fresh solvent. The combined solvent phase was filtered through 5 g of anhydrous sodium sulfate for dehydration, transferred to a pear-shaped flask, and then evaporated to dryness at 35 °C by a vacuum rotary vaporator. The dry extract was redissolved to 1 mL with methanol for analysis on HPLCMS/MS. The wet soil samples were extracted by acetone/ethyl (1:3, v/v) acetate, and interfering substances were cleaned up by Florisil SPE (500 mg) on a cartridge (6 mL). Briefly, soil samples (5 g wet weight per sample), 20 mL of acetone/ethyl (1:3, v/v) acetate, and 2 g of anhydrous sodium sulfate were added to a 50 mL polypropylene centrifuge tube. The tube was capped, vortex-mixed for 3 min, and centrifuged at 3500 rpm for 5 min. The extract was filtered through 5 g of anhydrous sodium sulfate for dehydration and transferred to a pearshaped flask. The remaining part was re-extracted following the same extraction step. Then, the combined extract was concentrated to dryness on a vacuum rotary evaporator at 35 °C. The SPE cartridge was preconditioned by elution with 5 mL of acetone followed by 5 mL of n-hexane and equilibrated with 10 mL of acetone/n-hexane (1:4, v/ v). The sample of dry extract was recovered in 3 mL of 20% acetone in n-hexane, and then the solution was passed through the SPE cartridge. The cartridge was eluted with an additional 7 mL of acetone/n-hexane (1:4, v/v). The eluates were combined with the loading eluates. The combined 10 mL of eluates was collected in a glass tube, evaporated to dryness under a stream of nitrogen, and diluted to 1 mL with methanol after passage through a filter membrane (pore size = 0.45 μm). The tubifex samples were thawed for about 15 min at room temperature. Then, 20 mL of acetone/ethyl (1:3, v/v) acetate was added to a 50 mL polypropylene centrifuge tube containing 5 g of worms. The mixture was homogenized with an Ultra-Turrax T18 homogenizer for 30 s, vortex-mixed for 5 min, and then separated by centrifugation at 3500 rpm for 5 min. The upper organic phase was passed through a funnel with about 5 g of anhydrous sodium sulfate to a pear-shaped flask. The extraction was repeated two more times. The combined extracts were evaporated to dryness at 35 °C and
reconstituted in 5 mL of acetonitrile. Then 5 mL of n-hexane was added for liquid−liquid partition to extract most of the lipid, and this procedure was repeated three times. The upper layer of n-hexane was discarded, and the acetonitrile layer was evaporated to dryness by a vacuum rotary evaporator. The purification process was similar to that of the soil samples described above. Chemical Analysis. A chiral liquid chromatograph coupled with a tandem mass spectrometer was used to determine the concentrations of epoxiconazole enantiomers in the samples. The liquid chromatograph was an Agilent 1200 HPLC system equipped with a G1322A degasser, a G1311A quatpump, a G1316B column compartment, a G1329A autosampler, and a 100 μL sample loop. Stereoselective separation was performed on a new highly efficient chiral column [Phenomenex LuxCellulose-1 column, 250 × 4.6 mm (i.d.)] packed with a chiral stationary phase of cellulose tris(3,5-dimethylphenylcarbamate). The mobile phase consisted of a mixture of acetonitrile/ water/formic acid (5:95:0.1, v/v/v) at flow rate of 0.5 mL/min. Chromatographic separation was conducted at a temperature of 30 °C. The injection volume was maintained at 5 μL. According to a previous paper,25 the enantiomeric elution order of epoxiconazole on the Lux Cellulose-1 column was (−)-isomer first and (+)-isomer second with acetonitrile/water as mobile phase at an optical rotation detection wavelength of 426 nm. An Agilent 6460 triple-quadrupole mass spectrometer equipped with an electrospray ionization source was used for MS/MS analysis (Agilent, USA). The analyses were performed in the positive mode with a 3500 V capillary voltage, 110 V fragmentor, and a 350 °C dry gas temperature. An 11 L/h dry gas flow was used. The nebulizer gas was 99.95% nitrogen with a pressure of 40 psi, and the collision gas was 99.999% nitrogen. The transitions of precursor ion (m/z 330.1) to production (m/z 121 and 141) of epoxiconazole were detected with multiple reaction monitoring (MRM) in positive ion (ESI+) mode. The collision energies for m/z 121 and 141 were 10 and 14 V, respectively, ,and the ion m/z 141 was used for quantification. Masshunter (Agilent, USA) software was used to collect and analyze the data obtained. The linear regression equations and the correlation coefficients of each stereoisomer were obtained from the peak area ratio plotted 362
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Table 1. Summary Recoveries for Epoxiconazole Stereoisomers in Overlying Water, Soil and Tubifex overlying water
soil
tubifex
compound
spiked level (mg/kg)
av recovery (%, n = 3)
RSD
av recovery (%, n = 3)
RSD
av recovery (%, n = 3)
RSD
(−)-epoxiconazole
0.05 0.5 2.5
89.2 86.0 88.3
4.8 5.6 8.2
83.6 80.3 78.2
4.2 4.9 6.8
84.6 83.1 88.5
8.3 4.2 6.4
(+)-epoxiconazole
0.05 0.5 2.5
92.4 89.3 95.2
5.2 7.6 9.3
81.4 85.7 88.2
7.8 6.9 5.4
90.2 85.8 88.7
5.9 7.8 9.6
against its respective concentration (0.01−10 mg/L). Calibration curves were generated by plotting peak area versus the concentration of each enantiomer. The relative standard deviation (RSD) was calculated at the standard range. The standard curves for (−)- and (+)-enationmers showed excellent linearity in the range of 0.01−10 mg/L of each enatiomer (n = 3). For (−)-epoxiconazole, y = 114043x + 23890, R2 = 0.9973; for (+)-epoxiconazole, y = 129131x + 24593, R2 = 0.9963. The precisions of peak areas at the six concentration levels were also good with RSDs of 0.4−0.8% for (−)-epoxiconazole and 0.1−0.8% for (+)-epoxiconazole. A series of blank samples fortified rac-epoxiconazole at 0.1, 1, and 5 mg/kg were determined immediately after fortification into the overlying water, soil, and tubifex tissue. In this work, the LC-MS/MS method was successfully developed to analyze epoxiconazole stereoisomers in water, soil, and tubifex samples. As shown in Figure 2, two stereoisomers were baseline separated. There were no interference peaks eluted at the same retention times as those of the stereoisomers. Table 1 shows the fortified results of epoxiconazole stereoisomers in water, soil, and tubifex samples. Thus, recovery rates and their standard error were acceptable. The LOQs and LODs for both stereoisomers in overlying water were found to be 0.005 and 0.001 mg/kg, and those in soil and tubifex tissue were both 0.03 and 0.006 mg/kg. Data Analysis. The enantiomer fraction (EF) was used to measure the enantioselectivity behavior of epoxiconazole in our experiment. The EF values ranged from 0 to 1, with EF = 0.5 representing the racemic mixture. EF was expressed as
concentrations was observed between 7 and 10 days of exposure, which could be the metabolism and excretion of epoxiconazole caused by the self-detoxification of tubifex. At the end of exposure, the concentrations tended toward a steady state. Additionally, Figure 4 also shows a clearly greater bioaccumulation of (−)-epoxiconazole than of (+)-epoxiconazole. The EF values in tubifex tissue were calculated and are shown in Figure 5. A one-sample t test was performed to compare the means of the EF values in tubifex with EF = 0.5, and the result showed that the EF values significantly deviated from 0.5 (P < 0.005). Therefore, the bioaccumulation of epoxiconazole in tubifex tissue under this treatment was enantioselective. In this study, AF was used to express the bioaccumulation of epoxiconazole enantiomers in tubifex tissue. AF is a function of the relative sorptive capacities of the organism versus the surrounding environment. It is often used to compare the body burden of an organism with the degree of contamination in the water. The following definition is used here: AF = Cworm/Cwater
C worm and C water are concentrations of epoxiconazole enantiomers in tubifex and water, respectively. The AF value at each sampling point was plotted against time. The AF values of (−)-enantiomer were larger than those of (+)-enantiomer, indicating that the (−)-enantiomer was preferentially accumulated over the (+)-enantiomer in tubifex tissue, and a significant difference was observed between the two enantiomers (Figure 4). Thus, the bioaccumulation of epoxiconazole in tubifex tissue exposure was enantioselective. Enantioselective Dissipation of Epoxiconazole in Soil. In general, the residues of both enantiomers of epoxiconazole decreased with prolonged time in the {+Tub+soil} and {−Tub +soil} treatments (Figure 6). For worm-free treatment, the dissipation kinetics of (−)-epoxiconazole and (+)-epoxiconazole in soil followed first-order kinetics with R2 = 0.96 and 0.98, respectively. The first-order kinetic equations were C(t) = 0.4615e−0.063t and C(t) = 0.4781e−0.051t, respectively, where C(t) was the concentration at time t (days). The corresponding halflife was calculated as t1/2 = ln 2/k = 0.693/k, where k is the dissipation rate constant. The (−)-epoxiconazole and (+)-epoxiconazole had half-lives of 11.0 and 13.6 days in soil, respectively, resulting in relative enrichment of the (+)-form in soil (Figure 3C,D). For the worm-present treatment, the dissipation kinetics of (−)-epoxiconazole and (+)-epoxiconazole in soil also followed first-order kinetics with R2 = 0.92 and 0.93, respectively, and the first-order kinetic equations were C(t) = 0.3854e−0.105t and C(t) = 0.4217e−0.079t, respectively. The half-lives of (−)- and (+)-epoxiconazole were 6.6 and 9.2 days in soil, respectively, resulting in preferential dissipation of the (−)-form in soil (Figure 3E,F). Therefore, enantioseletive
EF = peak area of (− )/[(− ) + (+ )] where (−) is the first eluted chromatograph peak of (−)-epoxiconazole and (+) is the second eluted peak of (+)-epoxiconazole. Data presented correspond to mean ± standard deviation of three independent experiments (n = 3). Statistical analysis for the enantioselectivity of epoxiconazole enantiomers was performed using SPSS 18.0. A one-sample t test was used to compare the means of the EF values in tubifex and sediment samples with EF = 0.5. The concentrations and accumulation factors (AFs) of the two enantiomers of epoxiconazole were analyzed using 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
Enantioselective Bioaccumulation in Spiked-Water Treatment. For the spiked-water treatment ({+Tub+water}), in which water was the only supply of living environment and the worm can accumulate contamination via a single skin exposure, concentrations of the two enantiomers of epoxiconazole in tubifex tissue were detected. The typical LC-MS/ MS chromatograms in tubifex obtained from this treatment at different times are shown in Figure 3A,B. Figure 4 shows the bioaccumulation concentrations of each enantiomer of epoxiconazole in tubifex samples under this treatment. On the seventh day, the concentrations of the two enantiomers reached the highest levels. Then, a significant decrease in 363
dx.doi.org/10.1021/jf403996g | J. Agric. Food Chem. 2014, 62, 360−367
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Figure 3. Typical chromatograms of extracts from water in {+Tub+water} treatment after 0.5 days (A) and 7 days (B), extracts from soil in {−Tub +soil} treatment after 1 day (C) and 14 days (D), extracts from soil in {+Tub+soil} treatment after 1 day (E) and 14 days (F), and extracts from tubifex in {+Tub+soil} treatment after 1 day (G) and 14 days (H).
epoxiconazole via skin and ingestion exposure routes, concentrations of epoxiconazole enantiomers in tubifex tissue, overlying water, and spiked soil were evaluated. Figure 7 shows the bioaccumulation curves of the two enantiomers of epoxiconazole in tubifex tissue. Both concentrations of the two enantiomers of epoxiconazole in tubifex tissue persistently increased from days 1 to 5. After a 5 day decline, concentrations increased again and reached steady state as the duration of exposure increased. A significant difference between the two enantiomers was observed during the whole uptake period. The
behaviors were detected in the dissipation of the epoxiconazole in soil. During the whole 20-day exposure, the concentrations of the two enantiomers of epoxiconazole in soil under wormpresent conditions were lower than those under worm-free conditions. Hence, epoxiconazole had lower persistence in soil with the existence of tubifex. In other words, tubifex could play an important role in remediation of contaminated soil. Enantioselective Bioaccumulation in Spiked-Soil Treatment. For the {+Tub+soil} treatment, in which soil was the initial contamination source and tubifex can accumulate 364
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Figure 7. Accumulation curves for epoxiconazole enantiomers in tubifex tissue for spiked-soil treatment (bars are standard error). ∗ indicates significant difference between the two enantiomers at the same time point (P < 0.05, S−N−K test).
Figure 4. Accumulation curves for epoxiconazole in tubifex tissue in spiked-water treatment (bars are standard error). ∗ indicates significant difference between the two enantiomers at the same time point (P < 0.05, S−N−K test).
concentrations of (+)-enantiomer in tubifex tissues were higher than those of (−)-enantiomer, resulting in relative enrichment of the (+)-epoxiconazole (Figure 3G,H). The EF values of epoxiconazole changed with time as shown in Figure 8. A one-
Figure 5. Calculated AF and EF values for epoxiconazole enantiomers in spiked-water treatment (bars are standard error). ∗ indicates significant difference between the two enantiomers at the same time point (P < 0.05, S−N−K test). Figure 8. Calculated AF and EF values for epoxiconazole enantiomers in spiked-soil treatment (bars are standard error). ∗ indicates significant difference between the two enantiomers at the same time point (P < 0.05, S−N−K test).
sample t test between the EF values of epoxiconazole in tubifex and EF = 0.5 yielded a P value of 0.001. These results showed that enantioselective bioaccumulation of epoxiconazole in tubifex occurred. For this treatment ({+Tub+soil}), the AF was defined as
AF = Cworm/Csoil where concentrations of epoxiconazole enantiomers in tubifex and soil were expressed as Cworm and Csoil, respectively. The AF values versus the duration of exposure are plotted in Figure 8. On the basis of the S−N−K test, a significant difference between the AF values of the two enantiomers was observed, and the (+)-enantiomer was preferentially accumulated over the (−)-enantiomer in tubifex tissue. According to Figures 5 and 8, we found that the direction of the enantioselectivity
Figure 6. Dissipation curves of epoxiconazole in spiked soil (bars are standard error). ∗ indicates significant difference between the two enantiomers at the same time point (P < 0.05, S−N−K test).
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varied with incubation conditions. Epoxiconazole was found to be enriched in the (−)-enantiomer in spiked-water treatment. However, enantioselectivity reversal in spiked-soil treatment was observed, resulting in concentrations enriched in (+)-enantiomer. Furthermore, the AF values in spiked-soil treatment were lower than those in spiked-water treatment during the whole exposure period. These interesting phenomena may have resulted from the different uptake pathways of the two treatments. For the spiked-water treatment, the accumulation activities of tubifex are absorbed merely through the epidermis. For spiked-soil treatment, bioaccumulation of epoxiconazole in tubifex may have occurred through ingestion of the soil particles and exposure in overlying water or pore water. Hence, the relative bioaccumulation capacities and bioaccumulation enantioselectivity of epoxiconazole in tubifex are related to the different chiral environments that play a role in the uptake of epoxiconazole. Influence of Bioturbation. Bioturbation is caused by the activity of animal species living at the surface and/or within the sediment superficial layers.26,27 Previous studies have investigated the effects of freshwater infaunal invertebrates on sediment transport and solute composition.27−32 Bioturbation was found to induce a significant release of some xenobiotics from the sediment to the overlying waters. In this study, the concentrations of epoxiconazole enantiomers in overlying water for {+Tub+soil} and {−Tub+soil} treatments were detected. Data from Figure 9 illustrate that the concentrations of
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AUTHOR INFORMATION
Corresponding Author
*(J.D.) Phone: +86 13811992249. Fax: +8610-62733547. Email:
[email protected]. Funding
This work was funded by the National Natural Science Foundation of China (Contracts 41201499 and 21177154) and the Special Fund for Agro-scientific Research in the Public Interest (No. 201203022). Notes
The authors declare no competing financial interest.
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REFERENCES
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Figure 9. Concentrations of epoxiconazole in the overlying water (bars are standard error). ∗ indicates significant difference between the two enantiomers at the same time point (P < 0.05, S−N−K test).
(+)-epoxiconazole in overlying water samples in the two treatments were higher than those of (−)-epoxiconazole. Furthermore, the results also indicated that the concentrations of the two enantiomers in worm-present experiments were higher than those in worm-free experiments. This phenomenon could have been explained by the traversal of epoxiconazole to the spiked soil into the overlying water by simple diffusion processes in the worm-free treatment. However, for the wormpresent treatment, epoxiconazole could pass through the spiked soil into the overlying water by two processes, diffusion and bioturbation. Thus, bioturbation has a positive effect on epoxiconazole diffusion from soil to overlying water. 366
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