Stereoselective Bioaccumulation of Water and Soil-Associated Dufulin

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Stereoselective Bioaccumulation of Water and Soil-associated Dufulin Enantiomers in Tubifex Jing Li, Ping Lu, Deyu Hu, Shouyi Wang, Qingtao Zhang, Yurong Yu, and Song Zeng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03186 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 14, 2017

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Journal of Agricultural and Food Chemistry

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Stereoselective Bioaccumulation of Water and Soil-Associated Dufulin

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Enantiomers in Tubifex

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Jing Li, Ping Lu*, Deyu Hu*, Shouyi Wang, Qingtao Zhang, Yurong Yu, Song Zeng

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Center for Research and Development of Fine Chemicals, Key Laboratory of Green

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Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou

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University, Guiyang 550025, P.R. China

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*

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address: [email protected], [email protected].

Corresponding author: Tel.: +86 851 88292090; fax: +86 851 8292090. E-mail

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ACS Paragon Plus Environment


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ABSTRACT: In this study, the stereoselective bioaccumulation of rac-dufulin, pure

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S-(+)-dufulin, and pure R-(−)-dufulin in tubifex (Oligochaeta, Tubificida) were

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analyzed in spiked-water and spiked-soil systems at low and high dose levels,

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respectively. In the bioaccumulation experiments treated with rac-dufulin, the

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enantioselective behaviors of the enantiomers shown that the concentrations of

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R-(−)-dufulin higher than those of S-(+)-dufulin at two dose levels. However, when

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treated with solely pure S-(+)-dufulin and R-(−)-dufulin, no significant difference of

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concentrations was detected in tubifex. Furthermore, the calculated accumulation

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factors in tubifex indicated that dufulin (racemic or the pure enantiomers) in the

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spiked-soil treatments had higher bioaccumulation potential than in the spiked-water

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treatments. The spiked-soil experiments revealed that the dissipation of dufulin in

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soil was not enantioselective at the enantiomer levels as well as tubifex could reduce

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the concentrations of dufulin in the underlying solid matrix and accelerate its repair

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and detoxification process.

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Keywords: dufulin, tubifex, enantioselectivity, bioaccumulation

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1. INTRODUCTION

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In the ecological transportation and transformation of chiral pesticides, each

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pesticide enantiomer will have different properties. Due to various environmental

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factors, each enantiomer will behave differently in terms of absorption,

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transportation, degradation, and transformation.1–3 It is important to indicate that

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stereochemistry strongly influences biological activity, processes such as degradation

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and metabolic behavior in organisms and in environment.4–7 Therefore, it is not

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sufficient to describe the actual environmental fate and ecological risks of only the

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racemate of chiral pesticides. However, most chiral pesticides are as the form of the

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racemic mixture in using process. So stereoselective kinetic or degradation studies of

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pesticides can improve pesticide safety and reduce contamination of the

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environment.8–13

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Dufulin

((2-fluorophenyl)-(((4-methylbenzothiazol-2-yl)amino)methyl)

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phosphonic acid diethyl ester, Figure 1), a novel chiral pesticide which had good

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antiviral activities against the tobacco mosaic virus (TMV), cucumber mosaic virus

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(CMV), and potato virus Y (PVY),14–19 had been registered by the Ministry of

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Agriculture of China in 2007 (LS 20071280 and 20071282) and subsequently

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produced on an industrial scale for field application. In our previous study, the

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toxicological studies of rac-dufulin (> 98%) had been studied and the results

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revealed that dufulin is a low toxicity pesticide (female/male Sprague Dawley rats:

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oral LD50 > 5000 mg/kg, percutaneous LD50 > 2150 mg/kg, inhalation LC50 > 2000

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mg/m3). Also, the acute toxicity tests of 30% dufulin wettable powder (WP) in 3



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several organisms (zebrafish: LC50 (96 h) > 12.4 mg/L, bee: LC50 (48 h) > 5000

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mg/L, quail: LD50 (7 d) > 450 mg/kg, silkworm: LC50 > 5000 mg/kg) shown that

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dufulin meets the criteria for low toxicity pesticides in environment. Most of the

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researches about dufulin have focused on the racemate rather than the individual

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enantiomers, and the select methods for the achiral analysis and residue

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determination of dufulin in various matrices, such as paddy, tomato, tobacco,

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cucumber, soils, and water, have already been reported.13–15,19,20 Meanwhile,

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previous reports, separately considered rac-dufulin and the individual enantiomers,

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have been restricted to the photolyzation, hydrolyzation, and degradation of dufulin

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in water and soil.13,20 However, the enantioselective bioaccumulation and

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degradation of dufulin at the enantiomeric or racemate level in aquatic animals have

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not been investigated.

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Tubifex, one of the most abundant and ubiquitous groups in freshwater

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ecosystems, is often used as a model organism to assess the bioavailability of

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contaminants in soil because of the special lifestyle.3, 21–25 Tubifex, which is usually

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exposed to environmental pollutants, can cause an effective exchange of material at

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the soil-water interface and accelerate the decomposition of organic matter.26,27 More

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importantly, tubifex can help compounds re-enter the environment through

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bioturbation and accumulate these soil-related chemicals, then consume by predator

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organisms and make a risk to higher trophic levels through the food chain, so toxic

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substances may have trophic transfer and/or bioaccumulation and/or harmful effects

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on organisms through food chains by tubifex.28–30 Despite the numerous studies on 4


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pollutant exposure in several aquatic animal species, only a few studies have been

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carried out to investigate the enantioselective bioaccumulation induced by racemate

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as exposure for tubifex. Previous studies had revealed the bioaccumulation behavior

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of racemic mixtures in tubifex, however, research about enantioselective

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bioaccumulation of the individual enantiomers in this organism has not been

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reported.31–33

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The primary objectives of this study were as follows: 1) to develop suitable

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methods for extraction and detection of rac-dufulin and the enantiomers in tubifex,

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water, and soil samples; 2) to investigate the differences in bioaccumulation behavior

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of rac-dufulin and individual enantiomers in tubifex with different ecological

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environment; and 3) to evaluate the effects of tubifex in the diffusion and

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degradation of dufulin in both soil and water. In addition, our study aims to provide a

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reference for discussing the stereoselective behavior of rac-dufulin and individual

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enantiomers in an aquatic environment.

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2. MATERIALS AND METHODS

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2.1 Chemicals and Reagents

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Rac-dufulin and individual enantiomers with a purity of 99% were obtained

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from Guizhou University. Analytical grade ethyl acetate, dichloromethane,

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petroleum ether, acetone, sodium chloride, and anhydrous sodium sulfate were

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obtained from Chengdu Jinshan Chemical Reagent Co. (Chengdu, China).

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High-performance liquid chromatography (HPLC) grade hexane, isopropyl alcohol,

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and ethanol were obtained from Tedia Co Inc. (Phoenix, USA). C18 (50 μm, 60 Å) 5



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and PSA (40–60 μm) sorbents were purchased from Agela Technologies Inc. (Beijing,

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China).

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2.2 Instruments

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Chiral analysis was performed on an Agilent 1200 Series normal phase

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high-performance liquid chromatography (NP-HPLC) (Agilent Technology, USA)

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equipped with a diode-array detector (DAD). The two enantiomers of dufulin were

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separated on a chiral column (CHIRALPAK@ IA 1.7 μm, 2.1 × 50 mm, Daicel

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Investment Co. Ltd, China), and rac-dufulin was ideally baseline separated on this

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chiral column. According to the research of our group, enantiomers with peaks 1 and

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2 in the chromatogram (Figure S1) are assigned to S-(+)-dufulin and R-(−)-dufulin,

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respectively.13

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2.3 Preparation of Tubifex and the Soil Samples

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Tubifex was obtained from Guigang Flower Market (Guiyang, China). All of the

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worms were acclimated to laboratory conditions for at least 2–3 weeks prior to use.

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To simulate the natural environment for tubifex, the worms were maintained in 2 L

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plastic boxes containing uncontaminated soil and deionized water at 20 ± 2 °C under

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12/12 h light and darkness cycles. The water was replaced weekly and continuously

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aerated.

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Soil from the surface (0–15 cm) without detectable dufulin was collected from a

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Huaxi farm approximately 15 km south of Guiyang, China. After the superficial

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layer (1–2 cm) was removed, the soil was collected to a depth of 10 cm; further, it

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was air-dried, ground, and sifted through a 2 mm mesh. Physicochemical properties 6


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of the soil, which measured according to the Agricultural Industry Standard of the

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People's Republic of China (NY/T 1121), were as follows: organic carbon (OC):

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1.89 ± 0.25%; moisture content (MC): 35.12 ± 0.02%; clay: 22.4 ± 0.22%; sand:

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31.47 ±0.02%; silt: 46.13 ±0.25%; and pH: 7.20 ±0.04.

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2.4 Toxicity Test

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According to the Organization of Arab Petroleum Exporting Countries (OECD)

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guideline 225 (2007) and Test guidelines on environmental safety assessment for

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chemical pesticides,34,35 the test conditions of spiked-water and spiked-soil toxicity

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assay, identical with the bioaccumulation test conditions, was used to test the acute

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and subchronic toxicity to tubifex of rac-dufulin and its two enantiomers. Meanwhile,

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we determined the cultivated concentrations and time of rac-dufulin and its two

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enantiomers according to the tolerance of tubifex in different concentrations of toxic

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solution and exposure time in the spiked-water and spiked-soil experiments. The part

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of detailed experimental procedure is in the Supporting Information.

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2.5 Experimental Design and Sampling

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Three experimental conditions were examined, which are as follows: {+ Tubifex

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+ water}, {+ Tubifex + soil + water}, and {− Tubifex + soil + water}. For each

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experimental condition, the samples were treated with rac-dufulin, pure

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S-(+)-dufulin, and pure R-(−)-dufulin, respectively, at two dose levels. All of the

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treatments were cultured with 12 h of light and 12 h of darkness, and temperature

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was controllable to 20 ± 2 °C.

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The first scenario, was designated as {+ Tubifex + water}, in which dufulin was 7



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accumulated from spiked-water. In this study, a semi-static test was conducted for

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the bioaccumulation experiments (14 days). The exposure media in each beaker (500

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mL) was changed every day to maintain the concentrations at a constant level. Test

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solutions (1 and 10 mg/L of rac-dufulin or 0.5 and 5 mg/L of pure enantiomer) were

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prepared via diluting the stock solutions with dechlorinated tap water containing

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0.01% (v/v) acetone. Each of 198 beakers (11 sampling points, triplicates for one

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sampling points in each of two concentrations of rac-dufulin, pure S-(+)-dufulin, and

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pure R-(−)-dufulin, respectively) placed into 10 g acclimated tubifex. After each 11

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exposure periods (0.5, 1, 2, 3, 5, 7, 9, 10, 11, 13, and 14 days), the living worms

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were removed and washed with dechlorinated tap water until there were no residue

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in the surface of tubifex. The tubifex samples were dried using absorbent paper and

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weighed, then stored at −20 °C until further analysis.

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The second scenario, designated as {+ Tubifex + soil + water}, contained

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tubifex, water, and soil that was spiked with dufulin. Tubifex accumulated

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rac-dufulin and enantiomers from three pathways: overlying water, pore water, and

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ingestion of soil particles. Pure dufulin enantiomers or rac-dufulin dissolved in

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acetone was homogenously dispersed into 100 g (dry weight) of soil to obtain the

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nominal standard solutions and form a series of concentrations contaminated soil (5

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and 50 mg/kg of rac-dufulin, 2.5 and 25 mg/kg of single enantiomers). The spiked

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soil was left in a fumehood overnight to evaporate acetone. After complete

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evaporation of the solvent, the contaminated soil (100 g dry weight) was transferred

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to a 500 mL beaker, then 100 mL of dechlorinated tap water was slowly added to the 8


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beaker along the beaker wall and the overlying water was 2–3 cm. The pre-prepared

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tubifex (10 g per sample) was added test beaker (234 beakers, 13 sampling points,

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triplicates for one sampling points in each of two concentrations of rac-dufulin, pure

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S-(+)-dufulin, and pure R-(−)-dufulin, respectively). For the treatment of {+ Tubifex

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+ soil + water}, tubifex, water and soil samples were all collected after each of 13

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exposure periods (1, 2, 3, 5, 7, 9, 10, 12, 14, 16, 18, 20, and 21 days). Frstly, the

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overlying was aspirated gently with a straw and sampled. Then, the beaker without

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water was put in a water bath (30–35 °C) for 30–45 min to cause the alive worms to

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move to the surface of the soil.37 After that, the worms were separated from the

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beaker, rinsed with dechlorinated tap water, and dried using absorbent paper.24,36−38

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The tubifex samples from each beaker were weighed and the soil, water, and tubifex

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samples from each beaker were frozen at −20 °C for the contamination analysis.

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A negative control experiment, which included only water and spiked soil, was

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performed and labeled as {− Tubifex + soil + water}. The experimental design and

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sampling method were same as the second scenario. The experiments of spiked soil

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(treatment and negative control) were designed randomly, and the test beakers were

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weighed and compensated by addition of dechlorinated tap water every day.

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2.6 Sample Abstraction and Purification

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All of the samples were thawed at room temperature. First, the solid matter

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from the overlying water samples (10 mL per sample) were removed via vacuum

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filtration. Then, a total of 10 mL of saturated salt water was added to the filtered

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overlying water samples. The filtered overlying water samples were extracted twice 9



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with dichloromethane (20 + 20 mL) using a glass separatory funnel. After vigorous

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shaking, the organic phase was obtained, then dehydrated by 3 g anhydrous sodium

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sulfate and concentrated using a rotary evaporator and a nitrogen blow-dry

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instrument. The residue was dissolved in 1.0 mL of isopropyl alcohol and filtered

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through a 0.22 μm plastic microfiber filter prior to the NP-HPLC analysis.

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The ethyl acetate was used to extract the soil samples, and the mixed sorbents

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of C18 and PSA were used to remove the interfering substances of soil. Soil samples

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(10 g per sample), ethyl acetate (25 mL), and 3 g of anhydrous sodium sulfate were

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added to a 50 mL polypropylene centrifuge tube. Then, the mixture was

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vortex-mixed, ultrasonically treated, and centrifuged at 6000 rpm for 5 min. The

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supernatant (15 mL) was transferred to a pear-shaped flask and concentrated to

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dryness on a vacuum rotary evaporator at 45 °C. The residue was dissolved in

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isopropyl alcohol (1.0 mL), and impurities were adsorbed by the mixed sorbents

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(C18 (50 mg) and PSA (50 mg)). Then, the extract was passed through a filter

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membrane (pore size = 0.22 μm) for liquid chromatography analysis.

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For the abstraction of dufulin in tubifex, the 25 mL of ethyl acetate and 8 g

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worms were added to a 50 mL polypropylene centrifuge tube. The mixture was

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homogenized with an IKA T25 homogenizer for 30 s, vortex-mixed for 5 min, and

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then separated via centrifugation at 6000 rpm for 5 min. A portion of 15 mL of

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supernatant was transferred and concentrated using a rotary evaporator and a

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nitrogen blow-dry instrument. The purification process was the same as the soil

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samples described above. Similarly, the extract was passed through a filter 10


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membrane for analysis liquid chromatography.

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2.7 Data Analysis

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To illustrate the stereoselective bioaccumulation of dufulin in tubifex, the

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enantiomeric ratio is usually defined as the enantiomeric fraction (EF) using the

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following equation:

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EF = Ai/AT

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Where Ai is the area of the first eluting enantiomer (S-(+)-dufulin) and AT is the

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sum of areas of both enantiomers (EF = Area1/(Area1 + Area2)). The EF values

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ranges from 0 to 1, and 0.5 indicates a racemate. When the EF values significantly

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deviate from 0.5, enantioselectivity is thought to appear.39

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The dissipation kinetics of dufulin in soil were calculated via fitting the data to a first-order kinetic model:

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C(t) = C(t=0) × exp (−kt)

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Where C(t) (mg/kg) is the concentration of dufulin in the soil at sample time, t

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(days) and k are the elimination rate constant. The elimination half-life (t1/2) is

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calculated by t1/2 = ln2/k.

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In order to express the bioaccumulation of dufulin enantiomers in tubifex,

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accumulation factor (AF) is a function of the relative adsorptive capacities of the

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organism versus the surrounding environment. AF is used for any time point of

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uptake when the steady state has not been reached, whereas the bioaccumulation

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factor (BAF) is defined for steady state conditions.3,40 In our experiments, the steady

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state was not reached, we chose AF to express the relative sorptive capacities of the 11



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organism versus the surrounding environment. The equation for AF is as follows:

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AF (kg/kg) = Ctubifex/C(water/soil)

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Where Ctubifex, Cwater, and Csoil are the concentrations of dufulin enantiomers in

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tubifex, water, and soil, respectively.

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3. RESULTS AND DISCUSSION

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3.1 Establishment and Validation of the Analytical Method

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In this study, we first developed the NP-HPLC method for analyzing the

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enantiomers of dufulin in tubifex and optimized the NP-HPLC method for analyzing

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the enantiomers of dufulin in water and soil. The specific NP-HPLC method

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conditions are shown in Table 1.

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The calibration curves were plotted over the concentrations range from 1.0

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mg/L to 500.0 mg/L (n = 5) for each enantiomer. The regression equations and

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respective correlation coefficients were y = 34.096x + 1.1013 (R2 = 1) for the

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S-(+)-enantiomer and y = 34.079x + 1.7169 (R2 = 1) for the R-(−)-enantiomer. Under

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this NP-HPLC method, the precision and accuracy of the method meet the

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requirements, and the specific numerical records are shown in Table 2. As shown in

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Figure S1, the two stereoisomers were baseline separated. No impurity peaks were

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detected at the same retention time of the stereoisomers.

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3.2 Toxicity Test

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Due to the current classification criteria for the toxicity of toxic substance in

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tubifex have not been declared, in this study, the test guidelines on environmental

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safety assessment for chemical pesticides on earthworm toxicity classification 12


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criteria (LC50 > 10 mg/kg for low toxicity) was referenced. In the earthworm acute

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toxicity test, when earthworms have no death phenomenon with the pesticide

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concentration higher than 100 mg/kg, the toxicity of pesticide is very low for

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earthworms. In our research, tubifex was treated in toxic solution (100 mg/L) and

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contaminated soil (100 mg/kg) for 96 h. The results showed that the tubifex had no

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death phenomenon, therefore, the acute toxicity (96 h, by spiked-water and

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spiked-soil tests) indicated that dufulin (racemate and pure enantiomers) was low

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toxicity.

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Meanwhile, the subchronic toxicity of rac-dufulin and its two enantiomers for

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tubifex was measured by spiked-water and spiked-soil tests. The results revealed that

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the LC50 values (both of spiked-water and spiked-soil tests) generally decreased

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along with the exposure time increased. In the spiked-water test, the LC50 values

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were calculated and shown in Table S1. After 14 days of exposure, the LC50 values

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of rac-dufulin, S-(+)-dufulin and R-(−)-dufulin were 104.42, 49.72 and 51.10 mg/L,

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respectively. Simultaneously, the result from the spiked-soil test implied that the

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values of LC50 (21 days) for rac-dufulin, S-(+)-dufulin and R-(−)-dufulin were

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490.81, 250.41 and 249.39 mg/kg, respectively (Table S2). In spiked-water and

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spiked-soil tests, the values of LC50 of S-(+)-dufulin and R-(−)-dufulin were

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substantially close at the same exposure time. The results, shown in Tables S1 and

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S2, demonstrated that dufulin's toxicity to tubifex is low in spiked-water (7 and 14

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days) and spiked-soil (7, 14, and 21 days) exposure ways. Considering the tolerance

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of tubifex in different media and concentrations (rac-dufulin and pure enantiomers) 13



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and pre-experimental results of different exposure time, we selected 14 and 21 days

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as the exposure time of spiked-water and spiked-soil subchronic exposure

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experiments, respectively. Also, the values of 1/10 and 1/100 of LC50 (14 day) in

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spiked-water experiment and 1/10 and 1/100 of LC50 (21 day) in spiked-soil

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experiment were determined as the high and low two dose levels, respectively, to

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carry out the following bioaccumulation experiments.

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3.3 Bioaccumulation of rac-dufulin and Two Enantiomers in Tubifex

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3.3.1 Enantioselective Bioaccumulation Detection in the Spiked-Water

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Treatment

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In {+ Tubifex + water} experiment, the chromatograms in tubifex detected at

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different time are shown in Figure S2. Figure 2A and 2B show the bioaccumulation

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concentrations of each enantiomer in rac-dufulin and two enantiomers in tubifex

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samples at two dose levels. Clearly, the accumulation models meet with “increase–

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decrease”, whether at low dose level or at high dose level. Through a single skin

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exposure, concentrations of rac-dufulin (S-(+)-dufulin and R-(−)-dufulin: 11.23 and

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15.24 mg/kg, respectively) and the two enantiomers (pure S-(+)-dufulin and pure

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R-(−)-dufulin: 13.92 and 14.07 mg/kg, respectively) both reached the highest level

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on the 10th day at high dose level. Whereas, at the low concentration, concentrations

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of rac-dufulin (S-(+)-dufulin and R-(−)-dufulin: 2.14 and 3.68 mg/kg, respectively)

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and the two enantiomers (pure S-(+)-dufulin and pure R-(−)-dufulin: 2.24 and 2.83

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mg/kg, respectively) both reached the highest level on the 11th day. Then,

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concentrations of rac-dufulin and two enantiomers in tubifex decreased from 11th 14


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day to 14th day at low dose level, and decreased from 10th day to 14th day at high

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dose level, respectively. The phenomenon may be due to autotomy and mucus

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secretion in tubifex. Some studies showed that autotomy has been observed in tubifex

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with exposure to metal and pesticide-contaminated sediment.38,39 Tubifex exposed to

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a high concentration of contaminant in soil presents a high contaminant tolerance,

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and it can complete detoxification via autotomy and increasing mucus secretion.34

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Therefore, decrease in concentration was inevitable between days 11−14 and days

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10−14 owing to the self-detoxification of tubifex at two dose levels. Through

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comparison of high and low doses, concentrations of dufulin in tubifex increased

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with concentrations of dufulin in exposure medium.

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When treated with rac-dufulin, concentrations of R-(−)-dufulin were higher

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than those of S-(+)-dufulin at two dose levels (Figure 2A). Meanwhile, a one-sample

300

t-test was used to compare means of EF values in tubifex with EF = 0.5, and a

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significant deviation (P < 0.05) from 0.5 was detected. As shown in Table 3, EF

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values in tubifex were between 0.30 and 0.43 in a higher concentration and between

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0.41 and 0.46 at a low dose level, respectively. At the same time, data presented

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corresponds to means ± standard deviations (SD) of three independent experiments

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(n = 3). Concentrations and AFs of two enantiomers of dufulin were analyzed using

306

one-way analysis of variance (SPSS 19.0, IBM, Chicago, USA), and a pairwise

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multiple comparison procedure (S-N-K test) was performed to compare results at P

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< 0.05. When treated with rac-dufulin, AF values of R-(−)-dufulin were higher than

309

those of S-(+)-dufulin (Figure 2C). These findings indicated that bioaccumulation of 15



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rac-dufulin was enantioselective with preferential accumulation of R-(−)-dufulin.

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However, when treated with two single enantiomers, AF values and

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concentrations of two single enantiomers were roughly close, and were lower than

313

those treated with rac-dufulin at low and high dose (Figures 2B and 2D). The

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competitive relation and preferred selective enrichment between S-(+)-dufulin and

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R-(−)-dufulin may explain this phenomenon. Precisely, when tubifex was in a

316

competitive relation between S-(+)-dufulin and R-(−)-dufulin, tubifex may have

317

prioritized enrichment of R-(−)-dufulin. Conversely, when tubifex was in a system

318

wherein only S-(+)-dufulin or R-(−)-dufulin existed, no selective enrichment was

319

detected.

320

3.3.2 Enantioselective Bioaccumulation Detection in the Spiked-Soil Treatment

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Compared with {+ Tubifex + water} experiment, the bioaccumulation of

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S-(+)-dufulin and R-(−)-dufulin were also enantioselective in {+ Tubifex + soil +

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water} experiment treated with rac-dufulin at two dose levels. Stereoselective

324

orientation of enantiomers in tubifex was consistent with that of {+ Tubifex + water}

325

experiment. Figure S3 shows the chromatograms of tubifex obtained from this

326

experiment at different sampling points. In Figures 3A and 3B, concentrations of

327

rac-dufulin and two enantiomers in tubifex also followed an “increase–decrease”

328

accumulation trend, and did not reach steady-state equilibrium within 21 days at two

329

dose levels. For the three high dose level treatments (rac-dufulin, pure S-(+)-dufulin,

330

and pure R-(−)-dufulin), concentrations (S-(+)-dufulin and R-(−)-dufulin: 14.85 and

331

20.86 mg/kg, respectively; pure S-(+)-dufulin and pure R-(−)-dufulin: 2.12 and 3.98 16


Page 16 of 39

Page 17 of 39


332

mg/kg, respectively) in tubifex reached the highest level on the 10th day and then

333

decreased between days 10−21. However, at a low concentration, concentrations of

334

rac-dufulin (S-(+)-dufulin and R-(−)-dufulin: 2.12 and 3.98 mg/kg, respectively) and

335

the two enantiomers (pure S-(+)-dufulin, and pure R-(−)-dufulin: 1.73 and 2.59

336

mg/kg) both reached the highest level on the 12th day. By comparing the high and

337

low doses, the concentration of dufulin in tubifex was found to be positively

338

correlated with the concentration of dufulin in the exposed medium. Meanwhile, the

339

concentrations of pure S-(+)-dufulin and pure R-(−)-dufulin in tubifex were

340

approximately equal at low and high dose levels (Figure 3B). The AF values of pure

341

S-(+)-dufulin and pure R-(−)-dufulin were roughly equal and lower than those of the

342

two enantiomers of rac-dufulin at two dose levels (Figures 3C and 3D). This

343

phenomenon can also be explained using the competitive relationship and selective

344

enrichment preference between S-(+)-dufulin and R-(−)-dufulin.

345

The different exposure ways led to the following results: the concentrations of

346

rac-dufulin and two enantiomers in spiked-soil experiment were higher than those in

347

spiked-water experiment at the two dose levels. Through {+ Tubifex + soil + water}

348

experiment treated with rac-dufulin, the results revealed that the concentrations of

349

R-(−)-dufulin accumulated in tubifex were significantly higher than those of

350

S-(+)-dufulin at two dose levels. As shown in Table 3, the EF values (at a low level:

351

0.28−0.38, at a high level: 0.26−0.35) in tubifex deviated from 0.5 to a large extent at

352

the two dose levels, and the AF values of R-(−)-dufulin were larger than those of

353

S-(+)-dufulin in the experiment treated with rac-dufulin in two different 17



Page 18 of 39

354

concentrations, indicating that the enantioselective bioaccumulation of rac-dufulin in

355

the spiked-soil treatment was similar to that in the spiked-water treatment,

356

meanwhile, the AFs of the spiked-soil experiment were more large. In this

357

experiment, tubifex can accumulate dufulin via the skin and ingestion exposure

358

routes. In the spiked-soil treatment, there were more pathways for dufulin entry than

359

in the spiked-water treatment, and the different enrichment pathways may

360

correspond to different enzymes or vectors. The increased number of enrichment

361

pathways may indicate an increase in the chiral selection factor; therefore, the

362

selectivity of dufulin in tubifex was high in the spiked-soil treatment.

363

3.4 Influence of Tubifex on the Environmental Behavior of Dufulin

364

Because of the bioturbation and uptake from tubifex, concentrations of the two

365

enantiomers in the soil and water samples changed continuously. The concentrations

366

of two enantiomers in the soil samples for {+ Tubifex + soil + water} and {− Tubifex

367

+ soil + water} treatments are shown in Figure 4. In these two treatments, no

368

significant enantioselective behavior was detected in the dissipation of rac-dufulin

369

and two enantiomers in the soil. The preference selection in enrichment of

370

rac-dufulin was decided by tubifex rather than the selective dissipation of dufulin in

371

the soil. Similar results were also observed in our previous studies of rac-dufulin in

372

field

373

R-(−)-dufulin.13 In {+ Tubifex + soil + water} treatment, the concentrations of

374

rac-dufulin in soil samples appeared to decrease 80% after 21 days exposure at a

375

high dose level. However, in {− Tubifex + soil + water} treatment, the concentrations

soil

with

no

enantioselective

behavior

between

18


S-(+)-dufulin

and

Page 19 of 39


376

of two pure enantiomers in soil samples appeared to decrease 55% after 21 days

377

exposure at a high dose level. Dissipation kinetics of the two enantiomers from

378

rac-dufulin in soil for {+ Tubifex + soil + water} and {− Tubifex + soil + water}

379

treatments followed first-order kinetics, and the specific results were listed in Table 4.

380

It is obvious that the existence of tubifex hastens the degradation rate of soil in {+

381

Tubifex + soil + water} treatment (S-(+)-dufulin: t1/2 = 7.22 d, R-(−)-dufulin: t1/2 =

382

7.53 d) compared with that in {− Tubifex + soil + water} treatment (S-(+)-dufulin:

383

t1/2 = 20.38 d, R-(−)-dufulin: t1/2 = 23.90 d). The concentrations of the two

384

enantiomers of rac-dufulin in soil under worm-present conditions were lower than

385

those under worm-free conditions during the 21 days exposure period. Therefore,

386

tubifex can repair contaminated soil and refine the contaminated sediment to a

387

certain extent.

388

In Figure 5, dufulin concentrations in the overlying water were detected for

389

both {+ Tubifex + soil + water} (worm-present) and {− Tubifex + soil + water}

390

(worm-free) experiments. Dufulin can spontaneously diffuse from the soil to the

391

overlying water. In the worm-free treatments, no significant enantioselective

392

behavior was detected between S-(+)-dufulin and R-(−)-dufulin, and the

393

concentrations of S-(+)-dufulin and R-(−)-dufulin were constant throughout the

394

entire experiment with their average concentrations of 1.305 and 1.371 mg/L,

395

respectively. Zhang et al.20 found that the S-(+)-enantiomer was hydrolyzed faster

396

than its antipode at pH = 7. Owing to the water samples were separated from the

397

mixed samples (the overlying water and underlying soil) under our experimental 19



398

condition, the result is different to the previous studies. In the worm-present

399

treatments, the concentrations of dufulin first increased and then decreased over time,

400

and the concentrations of S-(+)-dufulin and R-(−)-dufulin in the overlying water

401

were higher than those in the worm-free treatment. Meanwhile, the concentrations of

402

R-(−)-dufulin in the overlying water were higher than those of S-(+)-dufulin in the

403

worm-present treatment. The preferential accumulation of R-enantiomer in tubifex

404

led to the difference in dufulin concentrations. In the worm-present experiment, the

405

average concentrations of S-(+)-dufulin and R-(−)-dufulin were 3.542 and 4.316

406

mg/L, respectively. The presence of tubifex contributed to the conversion of dufulin

407

from the adsorbed state to the dissolved state, thereby it can accelerate the transfer of

408

dufulin in the aquatic environment and increase the effect of dufulin on the toxicity

409

of organisms, such as fish and plankton in aquatic ecosystems.

410

In this study, a sensitive and rapid chiral NP-HPLC method was developed to

411

extract and detect of dufulin in tubifex, soil, and water samples from an artificial

412

ecosystem. For the spike water and spike soil experiments treated by rac-dufulin, the

413

enantioselective behaviors of enantiomers were detected in tubifex, with the AF

414

values and concentrations of R-(−)-dufulin higher than those of S-(+)-dufulin at low

415

and high dose levels. However, a significant difference of concentrations and AF

416

values in tubifex were not detected in the two experiments treated with individual

417

enantiomers of dufulin. Because of the difference in exposure routes, the result was

418

AF(soil)> AF(water) treated by rac-dufulin and individual enantiomers. Moreover, the

419

degradation rate of dufulin in the {+ Tubifex + soil + water} treatment was higher 20


Page 20 of 39

Page 21 of 39


420

than that in {− Tubifex + soil + water} treatment, and the function of bioturbation

421

was significant during the diffusion of dufulin from soil to the overlying water.

422

ASSOCIATED CONTENT

423

Supporting Information

424

The Supporting Information is available free of charge on the ACS Publications

425

website.

426

AUTHOR INFORMATION

427

Corresponding Author

428

*

429

address: [email protected], [email protected].

430

Funding

431

This work was financially supported by National Natural Science Foundation of

432

China (grant numbers: 21365007 and 21507016), the Science and Technology

433

Programs of Guizhou Province (grant number: 20136024).

434

Notes

435

The authors declare no competing financial interest.

436

ABBREVIATIONS USED

437

TMV, tobacco mosaic virus; CMV, cucumber mosaic virus; PVY, potato virus Y;

438

WP,

439

chromatography; DAD, diode-array detector; PSA, primary secondary amine; C18, a

440

kind of bonded phase adsorbents; OECD, Organization of Arab Petroleum Exporting

441

Countries;

Corresponding author: Tel.: +86 851 88292090; fax: +86 851 8292090. E-mail

wettable

EF,

powder;

NP-HPLC,

enantiomeric

normal-phase

fraction;

AF,

high-performance

accumulation

21


factor;

liquid

BAF,


442

bioaccumulation factor; SD, standard deviations; OC, organic carbon; MC, moisture

443

content; LC50, the 50% lethal concentration; LD50, the 50% lethal dose.

444

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445

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446

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effects on bioturbation. Ecotox. Environ. Safe. 2005, 60, 237−246.

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elimination of hexachlorocyclohexane isomers in Tubifex tubifex (Oligochaeta,

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Tubificidae). Environ. Sci. Pollut. Res. 2016, 23, 6990–6998.

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oxidative stress in Tubifex tubifex exposed to hexachlorocyclohexane isomers.

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Soil. Sediment. 2008, 8, 154−164.

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Enantioselective bioaccumulation of soil-associated fipronil enantiomers in

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Tubifex tubifex. J. Hazard. Mater. 2012, 219, 50–56.

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and metabolite formation of triadimefon in Tubifex tubifex. Environ. Sci. Technol.

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2014, 48, 6687−6693.

545

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546

bioaccumulation and dissipation of soil-associated metalaxyl enantiomers in

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Tubifex. Chirality 2014, 26, 33−38.

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549

acute toxicity effects and bioaccumulation of furalaxyl in the Earthworm

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(35) OECD. OECD guidelines for the testing of chemicals: sediment-water 26


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553

(36) Paris-Palacios, S.; Mosleh, Y. Y.; Almohamad, M.; Delahaut, L.; Conrad, A.;

554

Arnoult, F.; Biagianti-Risbourg, S. Toxic effects and bioaccumulation of the

555

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significance of autotomy and its utility as a biomarker. Aquat. Toxicol. 2010, 98,

557

8−14.

558

(37) Lagauzère, S.; Terrail, R.; Bonzom, J. M. Ecotoxicity of uranium to Tubifex

559

tubifex worms (Annelida, Cl itellata, Tubificidae) exposed to contaminated

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sediment. Ecotox. Environ. Safe. 2009, 72, 527–537.

561

(38) Lucan-Bouché, L. B.; Biagianti-Risbourg, S.; Arsac, F.; V, G. An original

562

decontamination process developed by the aquatic oligochaete Tubifex tubifex

563

exposed to copper and lead. Aquat. Toxicol. 1999, 45, 9−17.

564

(39) Méndez-Fernández, L.; Jonge, M. D.; Bervoets, L. Influences of sediment

565

geochemistry on metal accumulation rates and toxicity in the aquatic oligochaete

566

Tubifex tubifex. Aquat. Toxicol. 2014, 157, 109–119.

567

(40) Egeler, P.; Meller, M.; Roembke, J.; Spoerlein, P.; Streit, B.; Nagel, R. Tubifex

568

tubifex as a link in food chain transfer of hexachlorobenzene from contaminated

569

sediment to fish. Hydrobiologia 2001, 463, 171−184.

27



570

Figure Captions

571

Figure 1. Chemical structures of the two dufulin enantiomers.

572

Figure 2. (A) Accumulation curves for rac-dufulin, (B) Accumulation curves for

573

pure S-(+)-dufulin, and pure R-(−)-dufulinin spiked-water treatment (bars indicate

574

standard error), (C) Calculated AF values for rac-dufulin, (D) AF values for pure

575

S-(+)-dufulin and pure R-(−)-dufulin in spiked-water treatment (bars indicate

576

standard error). ∗ indicates a significant difference between the two enantiomers at

577

the same time point (P < 0.05, S-N-K test).

578

Figure 3. (A) Accumulation curves for rac-dufulin, (B) Accumulation curves for

579

pure S-(+)-dufulin, and pure R-(−)-dufulin in tubifex in the spiked-soil treatment

580

(bars indicate standard error), (C) Calculated AF values for rac-dufulin, (D) AF

581

values for pure S-(+)-dufulin and pure R-(−)-dufulin in the spiked-soil treatment

582

(bars indicate standard error).* indicates a significant difference between the two

583

enantiomers at the same time point (P < 0.05, S-N-K test).

584

Figure 4. Dissipation curves of dufulin in the rac-dufulin spiked-soil experiment

585

(bars indicate standard error). ∗ indicates a significant difference between the two

586

enantiomers at the same time point (P < 0.05, S-N-K test).

587

Figure 5. Concentrations of dufulin in the overlying water (bars indicate standard

588

error).

589

28


Page 28 of 39

Page 29 of 39


Table 1. NP-HPLC-method used to analyze rac-dufulin and two enantiomers in overlying water, soil, and tubifex samples. NP-HPLC conditions for dufulin Detector

diode-array detector

Column

CHIRALPAK@ IA

Mobile phase

n-hexane: ethanol (v/v = 97 : 3)

Flow rate

1 mL/min

Injection volume

30 μL

UV detection wavelength

270 nm

Column attemperator

25 °C

29



Page 30 of 39

Table 2. Summary recoveries for rac-dufulin and two enantiomers from overlying water, soil, and tubifex samples.

Samples

Incubated compound

S-(+)-dufulin

Spiked

Average

level

recovery

(mg/kg)

(%, n=5)

0.25

101.27

8.77

1.25

103.19

3.22

25

99.99

4.20

0.25

90.55

9.28

1.25

90.13

1.65

25

98.00

4.16

0.25

99.36

3.21

1.25

97.43

1.66

25

101.23

3.01

0.25

98.07

5.31

1.25

96.33

1.07

25

99.11

3.85

0.2

82.96

2.53

1.0

90.76

1.21

20

95.44

2.18

0.2

86.03

2.24

1.0

92.35

2.25

RSD

Racemate

R-(−)-dufulin

Tubifex

S-(+)-dufulin

Enantiomers

R-(−)-dufulin

S-(+)-dufulin Soil

Racemate

R-(−)-dufulin

30


Page 31 of 39


S-(+)-dufulin Overlying

20

95.42

2.09

0.2

95.50

3.41

1.0

82.91

5.13

20

73.01

1.53

0.2

83.10

0.89

1.0

78.00

4.09

20

72.98

1.58

Racemate water R-(−)-dufulin

31



Page 32 of 39

Table 3. EF (Mean ± SD) of dufulin accumulated in tubifex in spiked-water and spiked-soil treatments. Values of EF Exposure time Spiked-water

Spiked-soil

(days)

a

1 mg/L

10 mg/L

5 mg/kg

50 mg/kg

0.5

0.44 ±0.01

0.30 ±0.05

−a

−

1

0.46 ±0.02

0.31 ±0.02

0.28 ±0.05

0.26 ±0.05

2

0.46 ±0.01

0.36 ±0.02

0.32 ±0.02

0.31 ±0.02

3

0.45 ±0.03

0.38 ±0.01

0.33 ±0.03

0.30 ±0.02

5

0.44 ±0.05

0.39 ±0.01

0.30 ±0.02

0.28 ±0.01

7

0.45 ±0.05

0.35 ±0.06

0.33 ±0.06

0.30 ±0.01

9

0.42 ±0.01

0.39 ±0.01

0.38 ±0.01

0.35 ±0.06

10

0.41 ±0.02

0.37 ±0.03

0.30 ±0.05

0.29 ±0.01

11

0.41 ±0.06

0.39 ±0.01

−

−

12

−

−

0.34 ±0.02

0.33 ±0.03

13

0.42 ±0.05

0.43 ±0.04

−

−

14

0.44 ±0.01

0.43 ±0.02

0.31 ±0.01

0.34 ±0.01

16

−

−

0.28 ±0.03

0.33 ±0.04

18

−

−

0.32 ±0.04

0.33 ±0.02

20

−

−

0.33 ±0.05

0.28 ±0.02

21

−

−

0.30 ±0.02

0.32 ±0.01

No sampling point. 32


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Table 4. Degradation rate constant (k), half-life (t1/2), and correlation coefficient (R2) values for the degradation of rac-dufulin in soil. Incubated

dufulin

k

t1/2 R2

Conditions compound

{+ Tubifex + soil + water}

{− Tubifex + soil + water}

enantiomer

(day-1)

(days)

S-(+)-dufulin

0.096

7.22

0.971

R-(−)-dufulin

0.092

7.53

0.969

S-(+)-dufulin

0.034

20.38

0.984

R-(−)-dufulin

0.029

23.90

0.974

rac-dufulin

rac-dufulin

33



Figure 1. Chemical structures of the two dufulin enantiomers. 96x47mm (300 x 300 DPI)


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Figure 2. (A) Accumulation curves for rac-dufulin, (B) Accumulation curves for pure S-(+)-dufulin, and pure R-(−)-dufulinin spiked-water treatment (bars indicate standard error), (C) Calculated AF values for racdufulin, (D) AF values for pure S-(+)-dufulin and pure R-(−)-dufulin in spiked-water treatment (bars indicate standard error). ∗ indicates a significant difference between the two enantiomers at the same time point (P < 0.05, S-N-K test). 170x120mm (300 x 300 DPI)



Figure 3. (A) Accumulation curves for rac-dufulin, (B) Accumulation curves for pure S-(+)-dufulin, and pure R-(−)-dufulin in tubifex in the spiked-soil treatment (bars indicate standard error), (C) Calculated AF values for rac-dufulin, (D) AF values for pure S-(+)-dufulin and pure R-(−)-dufulin in the spiked-soil treatment (bars indicate standard error).* indicates a significant difference between the two enantiomers at the same time point (P < 0.05, S-N-K test). 170x116mm (300 x 300 DPI)


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Figure 4. Dissipation curves of dufulin in the rac-dufulin spiked-soil experiment (bars indicate standard error). ∗ indicates a significant difference between the two enantiomers at the same time point (P < 0.05, SN-K test). 288x200mm (300 x 300 DPI)



Figure 5. Concentrations of dufulin in the overlying water (bars indicate standard error). 287x201mm (300 x 300 DPI)


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TOC 85x47mm (300 x 300 DPI)


Stereoselective Bioaccumulation of Water and Soil-Associated Dufulin

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