Effects of bifenthrin exposure in soil on whole-organism endpoints and biomarkers of earthworm Eisenia fetida

Chemosphere 168 (2017) 41e48

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Effects of bifenthrin exposure in soil on whole-organism endpoints and biomarkers of earthworm Eisenia fetida Lingling Li a, b, Da Yang a, b, Yufang Song a, *, Yi Shi a, Bin Huang a, Jun Yan a, Xinxin Dong c a

Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, 110016, China University of Chinese Academy of Sciences, Beijing, 100049, China c Shenyang Agricultural Environment Monitoring Station, Shenyang, 110016, China b

h i g h l i g h t s
The toxicity of bifenthrin to E. fetida in soil was evaluated synchronously by multi-endpoints in different levels.
Bifenthrin was low toxic in soil but its reproduction toxicity was demonstrated.
EROD activity in earthworm was measured by HPLC and used as a biomarker to assess the effects of bifenthrin.
The toxicity response of worms to the reduced bifenthrin in content, demonstrated the formation of toxic metabolites.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 June 2016 Received in revised form 14 October 2016 Accepted 17 October 2016

In this study, toxic effects of bifenthrin in soil on earthworms were evaluated by acute and chronic toxic endpoints combined with a set of biomarkers. Bifenthrin was moderately toxic in 72-h filter paper test and low toxic in 14-d soil test. The exposure of earthworms to bifenthrin-polluted soil for 8 weeks showed that cocoons were inhibited by high dose of bifenthrin, and larvae were stimulated by low dose but inhibited by high dose of bifenthrin. Furthermore, 28-d soil test on the responses of enzymes associated with antioxidation and detoxification in worms showed that peroxidase (POD) was stimulated by bifenthrin, superoxide dismutase (SOD) inhibited in the early period but stimulated in the later period, glutathione S- transferase (GST) inhibited in the later period, and ethoxyresorufin-O-deethylase (EROD) inhibited at day 3 but markedly stimulated at day 28 at high dose. The different responses of these indexes indicated that multi indexes should be jointly taken into account for comprehensive evaluation of the environmental risk of contaminants in soil. © 2016 Elsevier Ltd. All rights reserved.

Handling Editor: David Volz Keywords: Bifenthrin Fecundity Oxidative stress Detoxification Biomarkers

1. Introduction Pyrethroids are used worldwide to control a wide spectrum of insect pests with the characteristics of high potency, low acute toxicity and easy degradation (Casida and Quistad, 1998; Soderlund et al., 2002). The frequent use of pesticides results in accumulation of pesticide residues in the environment. Pyrethroids have been detected in sediments at levels high enough to cause toxicity to Hyalella azteca (Weston et al., 2004). A recent study on pyrethroids encompassing 25 states across the USA shows that bifenthrin is the most frequently detected pyrethroid (58% of samples), followed by

* Corresponding author. 110016, 72 Wenhua Road, Shenhe District, Shenyang City, Liaoning Province, China. E-mail address: [email protected] (Y. Song). http://dx.doi.org/10.1016/j.chemosphere.2016.10.060 0045-6535/© 2016 Elsevier Ltd. All rights reserved.

permethrin (31%) and cyfluthrin (14%) (Hladik and Kuivila, 2012). Also, residual pyrethroids can have secondary toxic effects on the non-target organisms (Vyjayanthi and Subramanyam, 2002). Bifenthrin is a third-generation synthetic pyrethroid insecticide with the reputation of strong environmental persistence and high insecticidal activity (Mokry and Hoagland, 1990) and is applied commonly in agricultural settings against many insects and mite pests (Wang et al., 2014). As an a-cyano-substituted pyrethroid, bifenthrin is generally more toxic than other pyrethroids to both terrestrial and aquatic insects, and its higher toxicity may be related to its stronger influence on detoxification enzymes (Siegfried, 1993). Up to now, the toxicity of bifenthrin to aquatic organisms has been studied frequently (Weston et al., 2005; Velisek et al., 2009a, 2009b; Beggel et al., 2011). Effective concentrations of bifenthrin

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L. Li et al. / Chemosphere 168 (2017) 41e48

for some species (e.g., Hyalella azteca) were near the analytical chemistry detection limit (~1 ng L1) (Weston and Jackson, 2009). 96 h LC50s of bifenthrin were 1.47 mg L1 for Oncorhynchus mykiss and 5.75 mg L1 for common carp juveniles (Velisek et al., 2009a, 2009b). Regarding the toxicity of bifenthrin to mammals, high dose of bifenthrin resulted in whole-body tremors, twitching, staggered gait, uncoordinated movement, splayed hindlimbs, abnormal posture, clonic convulsions, and abdominogenital staining on Sprague-Dawley rats (Freeman, 1998; Watt, 1998). Comparing with aquatic organisms, terrestrial organisms are more directly exposed to pesticides applied to agricultural soils, since hydrophobic pyrethroids pesticides are strongly absorbed to soil particles and predominantly found in upper soil layers and sediments but not in the water (Maund et al., 2002; Wong, 2006). Thus, more attention should be paid to the effects of pyrethroids in soil systems. However, up to present, only several reports have focused on the effects of pyrethroids on soil organisms. Some pyrethroids, such as deltamethrin, cypermethrin, fenvalerate and lcyhalothrin have been observed to induce biochemical toxicity in earthworm, e.g. Eisenia fetida, Metaphire posthuma and Aporrectodea caliginosa nocturna (Shi et al., 2007; Schreck et al., 2008; Saxena et al., 2014; Song et al., 2015). Sensitivity of soil organisms to pyrethroids is usually described by acute toxicity to meet the requirements for authorization (Hartnik and Styrishave, 2008). Data on pyrethroids sub-lethal toxicity are insufficient, although both sub-lethal effects and long-term survival play important roles in population growth and maintenance (Crommentuijn et al., 1997). Several non-lethal developmental endpoints can be synchronously determined to better evaluate the developmental toxicity of exogenous chemicals (Jin et al., 2009). Hence, it is difficult to evaluate the effects of pyrethroids on populations of soil organisms without sub-lethal toxicity evaluation. As early-warning signals of diagnosis and prognostication, molecular markers of the biological effects of pollutants on organisms (i.e. biomarkers) have been used to evaluate the environmental effects of pollutants, particularly at low concentrations (Livingstone, 1993). Biomarkers should not be applied independently, but as part of a well-designed monitoring programme also including other whole-organism endpoints to provide valuable information about the ecological relevance of sub-lethal stressor effects (Livingstone, 1993; Beggel et al., 2011). Recently, studies have reported the use of multi indexes such as mortality, growth, reproduction and phase II enzyme-cytochrome P450 (CYP) 3A4 in Eisenia fetida for evaluating the toxicity of deltamethrin and fenvalerate in soil under laboratory conditions and found that these multi-endpoints as a whole could provide valuable information for better risk assessment of pyrethroids in soil ecosystem (Shi et al., 2007; Saxena et al., 2014; Song et al., 2015). In this study we focused on the investigation of bifenthrin in soil by integrating whole-organism endpoints (the mortality, growth inhibition, reproduction) with biomarkers (CAT, SOD, POD, GST and CYP1A1 activities) using Eisenia fetida, which is the model species used for acute and sub-acute toxicity tests on general influences of pollutants in soil (OECD, 1984; Reinecke and Reinecke, 1996). The aims are to draw a detailed picture of toxic profile to evaluate the toxicity of bifenthrin in soil, not only from the individual level but also from the biochemical level, and to provide more evidence to reflect the toxicity of bifenthrin on soil organisms. 2. Materials and methods 2.1. Soil and earthworms Soil (0e20 cm) was collected in Shenyang Ecological station,

Liaoning Province, China with characteristics of pH 6.2, KeN 0.091%, total P 0.04%, total K 0.18%, organic matter content 1.65%, cation exchange capacity 12.3 cmol kg1, water holding capacity (WHC) 32%, sand (>50 mm) 22%, silt (1e50 mm) 64%, and clay (<1 mm) 14%. Soil was air-dried and sieved through a 2 mm sieve. Earthworms (Eisenia fetida) were bought from a breeder in Shenyang, and adult worms with well-developed clitella and weight of 300e400 mg were chose to adapt to the soil for more than one week. Before experiments, the earthworms were carried out to rinse out soil and cow dung on moist filter paper for 24 h at 20  C. 2.2. Chemicals Bifenthrin (97% purity) was purchased from Shanghai Forever chemical firm. Glucose-6-phosphate, Glucose-6-phosphate dehydrogenase, b-Nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt (NADPH), 7-Ethoxyresorufin, Resorufin, Ethylene Diamine Tetraacetic Acid (EDTA), DL-Dithiothreitol (DTT), methanol, acetonitrile, glutathione, Tris, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), Bovine serum albumin (BSA), Coomassie brilliant blue G-250 and 1-Chloro-2,4dinitrobenzene were purchased from Sigma-Adrich. P-Nitro-Blue tetrazolium chloride (NBT), methionine, riboflavin and guaiacol were purchased from Sinopharm Chemical Reagent, China. Other chemicals were bought from a chemical firm in Shenyang. 2.3. Acute toxicity tests 2.3.1. Filter paper contact test Filter paper contact test was conducted in accordance with OECD guideline (OECD, 1984). Filter paper was cut to fit the inside of test glass vials (5.0  6.5 cm). For each treatment and control there were ten replicates. Each replicate had one worm. Concentrations spanned a wide range from 62.5 to 2500 mg cm2. Worms were considered dead when they had no response under mechanical stimulus at the front end. Mortality was calculated at 48 h and 72 h. 2.3.2. Soil contact test Sampled soil test was carried out based on the OECD guidelines (OECD, 1984) with some modifications. Bifenthrin dissolved in 50 mL of acetone was added to sampled soil to get the concentration of 40, 80, 120, 160, 200, 240 and 280 mg kg1. Each concentration has 3 containers. After balancing for 48 h, ten worms per container were placed in 500 g soil wetted with distilled water with moisture content of 60% WHC and incubated at 20 ± 1  C (12:12 light:dark cycle). Mortality was assessed at day 7 and day 14. Controls were treated in the same way as the bifenthrin treatments in all tests. 2.4. Reproduction toxicity test Earthworms were exposed to bifenthrin at sub-lethal concentrations of 0, 10, 20, 40, 60, 80 mg kg1 for 8 weeks based on the LC50s of soil acute toxicity tests. For each concentration, there were three containers, each containing 10 adult earthworms. Earthworms were fed with 0.5 g of cow dung per worm once a week. The weights of worms, the number of cocoons and larvae in each dose group were counted every two weeks. The growth-inhibition rate of earthworms was calculated as follows:

GIRn ¼

W0  Wj  100% W0

GIRn is the growth-inhibition rate for dose group n; W0 is the

L. Li et al. / Chemosphere 168 (2017) 41e48

weight on week 0; Wj is the weight on week j (Shi et al., 2007). 2.5. Determination of biomarkers activity 2.5.1. Sample preparation Earthworms were exposed to soil with concentrations of 0, 10, 20, 40, 60, 80 mg kg1 of bifenthrin over 28 days, with three replicates for each concentration, and 20 earthworms in 1000 g soil per replicate. At day 3, 7, 14, 21 and 28, earthworms exposed to each concentration were collected in triplicate for analysis, with three earthworms pooled together as one replicate. Worms were kept on wet filter paper for 24 h to defecate, cut into pieces, washed with 0.15 M KCl, and then homogenized for 1 min in 5 mL homogenization buffer (250 mM sucrose, 50 mM pH 7.5 tris, 1 mM DTT and 1 mM EDTA) by Ultra-Turrax IKA T18 at 12,000 rpm. All procedures were carried out at 4  C. The homogenate was centrifuged at 15,000g at 4  C for 30 min. 1.0 mL of the supernatant was taken out for analysis of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and glutathione S-transferase (GST). Then the remaining supernatant was centrifuged at 150,000g for 1.5 h at 4  C to obtain microsomes. The microsomes were then suspended in 1 mL of store buffer (250 mM sucrose, 50 mM pH 7.5 tris, 1 mM DTT, 1 mM EDTA and 20% glycerol) and stored at 80  C before used to determinate ethoxyresorufin-O-deethylase (EROD) activity. 2.5.2. Enzyme assay CAT activity was determined based on the method of Xu et al. (Xu et al., 1997). 100 mL of supernatant was added into a tube containing 3 mL of 50 mM (pH 7.8) phosphate buffer which contained 50 mM H2O2 for CAT activity measurement at 240 nm. One unit of CAT activity was defined as the enzyme quantity required to consume half of H2O2 in 100 s at 25  C. POD activity was determined according to the method of Kochba (Kochba et al., 1977). The reaction was carried out by adding 100 mL of supernatant into 3 mL of reaction buffer which was prepared by adding 19 mL of 30% H2O2 and 28 mL of guaiacol into 50 mL of potassium phosphate buffer (50 mM, pH 7.8). The absorbance at 470 nm was recorded at the beginning and after incubation for 3 min, respectively. One unit of POD activity was defined as the amount of enzyme that caused an increase of 0.01 absorbance units per minute. SOD activity was determined as described by Giannopolitis (Giannopolitis and Ries, 1977). The reaction system contained 50 mL of supernatant, 2.9 mL of 50 mM phosphate buffer (pH 7.8) containing 1 mM EDTA, 130 mM methionine, 750 mM NBT, and 100 mL of 20 mM riboflavin. The tubes were shaken and illuminated with 4000 Lx fluorescent tubes for 20 min, following which the tubes were covered with a black cloth. Absorbance of the mixture was read at 560 nm. One unit was defined as the amount of enzyme required to cause 50% inhibition of the NBT photoreduction rate (U mg1 Protein). GST activity was determined according to the method of Habig et al. (Habig et al., 1974) with the extinction coefficient of 9.6 mM1 cm1. Reaction system contained 150 mL of 100 mM (pH 6.5) potassium phosphate containing 0.1% Triton X-100, 20 mL of 10 mM glutathione, 20 mL of supernatant and 10 mL of 20 mM CDNB. The change rate of absorbance at 340 nm during the initial 4 min was recorded. The GST activity was expressed as nmol min1 mg1 Protein. EROD activity was assessed by the HPLC method with some modifications based on Belaz and Hanioka (Hanioka et al., 2000; Belaz and Oliveira, 2013). The incubation system was composed of 800 mL of incubation buffer (100 mM pH 7.8 HEPES, 5 mM glucose-6-phosphate, 1 mg mL1 BSA, 5 mM MgCl2, 1 unit mL1 glucose-6-phosphate dehydrogenase), 50 mL of 7-Ethoxyresorufin (100 mM), and 300 mL of microsomes, and 50 mL of NADPH (5 mM) was added to start the reaction. After 20 min, 800 mL of ice-

43

cold methanol was added to stop the reaction. Then, the mixtures were put on ice for 30 min and centrifuged at 10,000g for 15 min at 4  C to obtain the supernatant to measure the content of resorufin by HPLC (Thermo Fisher Ultimate 3000). Samples were injected into a reverse-phase C18 column (Thermo Hypersil Gold 150  4.6 mm, 5 m) at 35  C following the eluent consisting of 20 mM phosphate buffer (pH 6.8), methanol and acetonitrile (52: 45: 3, v/v) at a flow rate of 0.8 mL min1, and detected using fluorescence detector at 530 nm (excitation) and 582 nm (emission). The EROD activity was expressed as the production of resorufin as pmol min1 mg1 Protein. The protein concentration was assayed according to the method of Bradford (Bradford, 1976).

2.6. Bifenthrin degradation in soil The determination of bifenthrin in soil at day 3, 7, 14, 21 and 28 was carried out according to the method of Zhang et al. (2009) with some modifications. 5 g soil was soaked with 25 mL petroleum ether: acetone (v:v ¼ 2:1) after 1 min of vortex oscillation for 24 h. The soaked soil was extracted for 30 min by a KQ-250DE ultrasonic cleaner, then after collection of supernatant, the process was repeated again. 2 g NaCl and 2 g Na2SO4 were added to the supernatant after centrifuging at 4000 rpm for 10 min, and the organic layer was dried under N2 blow with 50  C water bath and dissolve with 2 mL hexane for purification by Florisil SPE column (1 g, 6 mL, Thermo). The eluent was dried under N2 and dissolved to 2 mL with hexane for the analysis by GC-ECD with a DB-1 column (Thermo Finnigan TRACE GC).

2.7. Statistical analysis SPSS 21.0 was used for data analysis. Kolmogorov-Smirnov test and Levene's test were used for normality assumption and homogeneity of variances. One-way analysis of variance (ANOVA) with post hoc comparisons (LSD tests) was used to determine significant differences among treatments (p < 0.05). Logarithmic transformation was applied when data could not meet homogeneity. EROD activity at day 14 followed log-normal distribution. The nonparametric Kruskal-Wallis test was used when the variable could not meet the normality and homogeneity after transformation. Cocoons during 6e8 weeks were tested by non-parametric Kruskal-Wallis test, and there were no significant differences among treatments (P ¼ 0.072). LC50 and associated 95% confidence intervals were estimated using a probit equation.

3. Results 3.1. The acute toxicity Positive correlations were found between the concentration of bifenthrin and the mortality of earthworms both in filter paper and in soil test. No earthworms survived at 72 h when exposed to 2500 mg cm2 of bifenthrin (Fig. 1A). The 48 h and 72 h LC50s of bifenthrin were 440.5 mg cm2 and 163.5 mg cm2, respectively (Table S1). In soil test, the mortality reached 93.3% as earthworms exposed to 280 mg kg1 group for 14 d (Fig. 1B). The LC50s of bifenthrin in soil for E. fetida were 226.0 mg kg1 at day 7 and 166.1 mg kg1 at day 14 (Table S1). The change of mortality in filter paper test was drastic during 0e1000 mg cm2 and then steadied. The mortality in soil test increased continuously with the increase of concentration.

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Fig. 2. Growth inhibition of earthworms exposed to sub-lethal doses of bifenthrin in soil. Error bars indicated standard errors (n ¼ 3). Data with different letters means significant difference at P < 0.05.

Fig. 1. The mortality of earthworms exposed to bifenthrin. (A) The mortality of earthworm exposed to bifenthrin at 0, 62.5, 125, 250, 500, 1000, 2000 and 2500 mg cm2 in filter paper test for 48 h and 72 h. (B) The mortality of earthworm exposed to bifenthrin at 0, 40, 80, 120, 160, 200, 240 and 280 mg kg1 in soil contact test for 7 d and 14 d.

3.2. Reproduction toxicity 3.2.1. Growth inhibition Fig. 2 showed the growth inhibition of bifenthrin on earthworms after exposure of 2, 4, 6 and 8 weeks. The concentrationrelated growth inhibition on earthworm was significant during the whole exposure period (Pearson correlation r ¼ 0.931, P < 0.01; r ¼ 0.908, P < 0.01; r ¼ 0.812, P < 0.01 and r ¼ 0.822, P < 0.01). At 10 mg kg1, no significant effect on growth was determined at whole period. The inhibition effect on worm growth increased with prolonging the exposure time. For example, in the 80 mg kg1 groups, the growth inhibition rates were 13.96% at 2 weeks, 16.95% at 4 weeks, 18.62% at 6 weeks and 25.33% at 8 weeks, respectively. 3.2.2. Cocoon reproduction and larva incubation Table 1 illustrated the influence of bifenthrin on the cocoon reproduction of every two weeks. In the 10e20 mg kg1 groups, the cocoons were more or less similar to that of the control during the first six weeks, and slightly more during 6e8 weeks. In the 40 mg kg1 group, the cocoons were stimulated by bifenthrin during 4e6 weeks significantly. As the concentration increased to 60 and 80 mg kg1, significant inhibition effects occurred during the first 4 weeks. There were only 2.3 and 2.7 cocoons at first 2

weeks. The inhibition effects of 60e80 mg kg1 groups weakened with the prolongation of exposure. The effect of bifenthrin on larva incubation was shown in Table 1. Compared to the control, the 10 mg kg1 groups stimulated the larva incubation significantly after 2 weeks. The larvae from 40 mg kg1 group were similar to those of the controls before 6 weeks exposure, but significant higher during 6e8 weeks. As the concentration increased to 60 and 80 mg kg1, the larva incubation was inhibited significantly during the whole exposure and only 1.3 and 2 larvae were observed at the first 4 weeks. 3.3. Biomarkers 3.3.1. Antioxidant enzyme activity In this study, antioxidant enzyme activities (CAT, POD and SOD) were determined to reflect the bifenthrin stress on earthworms in soil. Fig. 3A indicated at day 3, CAT activity was increased by lower dose of bifenthrin and reduced by high dose (40e80 mg kg1). During the exposure time of 7e21 d, the CAT activities of all bifenthrin groups were similar to those of the controls with no significant differences. As the exposure time prolonged to 28 d, 20 and 40 mg kg1 bifenthrin induced the CAT activity significantly, and the CAT activity of 20 mg kg1 bifenthrin group was 2.26 times as big as that of the control. POD activities in worms exposed to the control and bifenthrin treatments were shown in Fig. 3B. The POD activity was significantly stimulated by bifenthrin with a positive doseeresponse correlation from day 3 to day 14. The POD activities of the 80 mg kg1 group at day 3, 7 and 14 were 6.8, 3.45 and 3.25 times as big as that of the control, respectively. The inductive effects of bifenthrin decreased obviously after 14 d of exposure. Changes of SOD activity of earthworms exposed to bifenthrin were showed in Fig. 3C. At day 3 and day 7, bifenthrin inhibited SOD activity in earthworms significantly. The SOD activity at control was 5.1 times as big as that of 80 mg kg1 group at day 3. SOD activities in bifenthrin groups were stimulated significantly at day 14, and the SOD activity at 40 mg kg1 bifenthrin was 2.27 times as big as that of the control. Thereafter, SOD activity was induced by low-

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Table 1 Cocoon reproduction and larva incubation of earthworm influenced by bifenthrin in soil. Treatment mg kg1

Control 10 20 40 60 80

Cocoons

Larvae

0e2 weeks

2e4 weeks

4e6 weeks

6e8 weeks

0e2 weeks

2e4 weeks

4e6 weeks

6e8 weeks

6 ± 2.0ab 6.7 ± 1.2ab 4 ± 2.7bc 7.0 ± 1.7a 2.3 ± 0.6c 2.7 ± 0.6c

1.7 ± 1.3 ± 1.3 ± 2±1a 0.3 ± 0.3 ±

4 ± 1.7b 5±2b 1.7 ± 1.5b 9.7 ± 2.3a 2 ± 1.7b 3 ± 4.4b

3 ± 1.7a 6.3 ± 5.9a 6 ± 1.7a 5±1a 1.3 ± 0.6a 2 ± 1.7a

0±0a 0±0a 0±0a 0±0a 0±0a 0±0a

5 ± 1.7b 8.3 ± 1.5a 4.7 ± 1.2b 4 ± 2.7bc 1.3 ± 1.5c 2±2bc

10.3 ± 1.5a 12.7 ± 2.1a 9±1ab 12 ± 5.3a 4.3 ± 2.5b 8 ± 3.6ab

11.3 ± 2.5c 19 ± 1.7a 9.3 ± 1.5c 15.3 ± 1.2b 2±1d 4.7 ± 2.1d

0.6a 0.6ab 0.6ab 0.6b 0.6b

Data are expressed as mean ± SD (n ¼ 3). Figures followed by different letters in the same column are significantly different at p < 0.05.

Fig. 3. Activities of (A) CAT, (B) POD, (C) SOD, (D) EROD and (E) GST in earthworms E. fetida after exposure to bifenthrin in soil for 3, 7, 14, 21 and 28 days. Error bars indicated standard errors (n ¼ 3). Data with different letters means significant difference at P < 0.05.

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concentration bifenthrin but was close to that of the control at high-dose bifenthrin. 3.3.2. Metabolic detoxification enzymes Fig. 3D showed the responses of EROD activity of earthworms exposed to bifenthrin in soil. At day 3, there was no big difference between the CYP1A1 activity at low dose of bifenthrin (10e20 mg kg1) and that of the control; however, high dose (40e80 mg kg1) inhibited the EROD activity significantly. At the 7th day, the EROD activities were induced by bifenthrin treatments. And at the 14th and 21th day, no prominent differences of EROD activities between the bifenthrin groups and the control were observed. However, when exposed for 28 d, the stimulation effects of bifenthrin were increased, and the EROD activity increased with the increase of concentration. The EROD activity at 80 mg kg1 bifenthrin was 4.08 times as big as that of the control. As shown in Fig. 3E, at day 3, the GST activity in earthworm was increased by low dose of bifenthrin and reduced by high dose. The GST activities at high dose of bifenthrin (40e80 mg kg1) were significantly lower than those at low dose. At the 14th day, 10 mg kg1 bifenthrin significantly induced the GST activity, which was 1.48 times as big as that of the control. With the extension of exposure time, the significant inhibition effect of bifenthrin on GST activity was observed at day 21 and 28. 3.4. The degradation of bifenthrin in soil Results (Table S2) showed a gradual decrease of bifenthrin in soil during the 28 d exposure. The degradation of bifenthrin corresponded to first-order kinetics with a coefficient of correlation of 0.82e0.99. For the 10 mg kg1 group, the degradation rate of bifenthrin was 11.4% and 54.6% at day 3 and day 28, respectively; for the 80 mg kg1 group, the bifenthrin was reduced by 6.39% and 42.40% after 3 and 28 d, respectively. The half-time of bifenthrin (10e80 mg kg1) in our soil was 30.01e59.24 d. 4. Discussion Despite the widespread use of pyrethroid insecticides in the environment, few studies have been conducted to assess the toxic effects of pyrethroids including bifenthrin in soil. In this study, we focused on the toxic effects of bifenthrin on E. fetida in laboratoryspiked soil using the whole-organism endpoints and molecular biomarkers. The acute toxic effect of bifenthrin was time- and concentration-dependent. According to the classification of chemicals, bifenthrin was moderately toxic (LC50 ¼ 100e1000 mg cm2) in filter paper test and low toxic in soil test (LC50 > 10 mg kg1) (Roberts and Wyman Dorough, 1984; Institute for the Control of Agrochemicals (2004)). The low toxicity of bifenthrin in soil might because the bioavailability of chemicals could be modified by soil phy-chemical properties (Lanno et al., 2004). Although pyrethroids are weak toxicants according their LC50s in soil, their obvious sub-lethal toxicity to soil organisms was observed. When E. fetida was exposed to 25 mg kg1 fenvalerate (14d-LC50 40.77 mg kg1), dominant damage of cuticular membrane, and disintegration of circular and longitudinal muscles were observed (Saxena et al., 2014). When E. fetida was exposed to 5e50 mg kg1 deltamethrin (14d-LC50 432.9 mg kg1), dose-dependent toxic effects on growth and cellulose activity were showed (Shi et al., 2007). Thus, sub-lethal toxicity was important to assess the risk of bifenthrin in this study. The change of biomass is a good indicator of chemical stress (Shi et al., 2007). In this study, the weights of earthworms were significantly inhibited by bifenthrin in soil with dose and timedependent relationship (Fig. 2). In the environment, a relative

biomass loss may be correlated with the strategy for survival (i.e., reducing food intake to avoid the toxins), and would have a substantial influence on population structure by reducing fecundity, recruitment and resistance to environmental stress because homeostatic energy demands are increased to dispose environmental contaminants (Burrows and Edwards, 2002; Amweg et al., 2005). The reproductive strategies were related to the ecological categories in earthworms (Barois et al., 1999). In the study, the reproductive strategies of E. fetida were influenced by bifenthrin. The changes of cocoon reproduction and larva incubation were sensitive to the toxicity of bifenthrin which agreed with previous study (Song et al., 2015). Pollutants can induce excessive free radicals, which may result in oxidative damage to biological macromolecules (Liu et al., 2015). So CAT, POD and SOD activities have been applied to determine the
et al., 2005; Fatima et al., oxidative stress in organisms (Holovska 2007; Song et al., 2009; Zhang et al., 2014). Pyrethroids (cypermethrin, fenvalerate and bifenthrin) have potential to cause severe oxidative stress to rats and mices (Kale et al., 1999; Dar et al., 2013; Jin et al., 2014). Eisenia fetida was demonstrated to be wellequipped to deal with electrophilic species and pro-oxidants (Saint-Denis et al., 1998). However, there are hardly any studies showing the oxidative stress of bifenthrin on soil organism. In this study, CAT, POD and SOD activity in E. fetida exposed to bifenthrin at sub-lethal concentrations were measured. The changes of CAT activities were slight and not significant in most of bifenthrin groups comparing with the controls. CAT activities varied in the controls of different exposure times because of variation in both biological factors and non-biological factors. Similar to our observation, no significant response was observed in CAT activity in Eisenia fetida affected by carbaryl and bromadiolone (Ribera et al., 2001; Liu et al., 2015). POD and SOD activity in earthworm were induced more significantly by bifenthrin. POD activity was stimulated at all concentrations during 3e14 d bifenthrin exposure, which might result from the H2O2 accumulation. SOD activity was inhibited in worms in all bifenthrin treatments during the early exposure, which might be due to the saturated anti-oxidant defenses and the highly reactive O 2 (Wu et al., 2011). A decrease in SOD activity has also been reported in rats treated with bifenthrin and deltamethrin (Dar et al., 2013; Yousef et al., 2006). SOD activity in earthworm increased with the prolongation of exposure, possibly because of the production of O 2 , which stimulated the SOD activity (Song et al., 2009). This study demonstrated exposure to bifenthrin resulted in oxidative damage to E. fetida in soil. CYP1A1, a dominant subform of CYP450 which involved in xenobiotic metabolism in living organisms, is widely used as a biomarker in ecotoxicological studies and specifically assessed by EROD (7-ethoxyresorufin O-deethylase) activity (Bonacci et al., 2003; Bozcaarmutlu et al., 2015). Up to present, only few studies reported the induction of EROD in terrestrial invertebrates (e.g earthworm) exposed to pollutants successfully, because of low activity and interfering substances such as respiratory pigments (Lukkari et al., 2004; Cao et al., 2012; Sanchez-Hernandez et al., 2014). In this study, microsomal fraction of earthworm was used to measure EROD activity successfully based on the method we established for earthworm. Results showed EROD activity exhibited a biphasic response defined by an increase at low dose and decrease at high dose during short-time exposure. This inverted U-shaped relationship seems to be hormetic dose-response model in earthworms. Sanchez-Hernandez et al. (2014) observed the same type response in Aporrectodea caliginosa exposed to chlorpyrifos for 3 days. With the prolongation of exposure, the stimulated effect on EROD activity by bifenthrin was strengthened, showing the presence of bifenthrin induced CYP1A1 in E. fetida. GST, a phase-II biotransformation enzyme, facilitates

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conjugation of electrophilic substances with glutathione and the elimination of toxic compounds (Habig et al., 1974; Bernard et al., 2015). GST activity in earthworm was affected by pollutants such as PAHs, heavy metals and pyrethroids (Velki and Hackenberger, 2013a; Feng et al., 2015; Zhang et al., 2015). In the study, there were no significant differences between most bifenthrin groups and controls before 14 d. The data is in line with previous study, in which no significant variations were observed in GST activity of Eisenia andrei exposed to 0.03e30 mg kg1 dimethoate before two weeks (Velki and Hackenberger, 2013b). After long-term exposure, GST activity in earthworms was inhibited by bifenthrin. The similar phenomenon has been observed in Eisenia andrei exposed to pirimiphos-methyl and deltamethrin (Velki and Hackenberger, 2013a). The decrease in GST activity was likely due to the prolonged retention of bifenthrin in cells resulting in enhanced toxicity and excessively accumulation of radicals to damage certain tissues (Velki and Hackenberger, 2013a; Xu et al., 2015). Our results indicated that exposure to bifenthrin in soil resulted in oxidative stress and biotransformation damage, followed by the decrease of growth and fecundity. All of these responses suggested that multiendpoints are essential and important to diagnose the toxicity of bifenthrin in soil. The degradation reaction of bifenthrin in soil followed the firstorder kinetic, and the half-life was between 30.1 and 59.2 d. However, with the degradation of bifenthrin, the toxicity on worms was not decreased. In previous study, the inconsistence between the changes of toxic effect and the degradation of pyrethroids in soil has also been found (Song et al., 2015). This phenomenon might be due to the formation of toxic metabolites from bifenthrin degradation, such as cyclopropanecarboxylic acid and 2-methyl-3biphenylyl methanol, because the toxicity of metabolite might be higher than the parental compound (Romero et al., 2012). Further studies are needed because of the inconsistence between the degradation of bifenthrin and the enhanced toxicity response.

5. Conclusion Sub-lethal exposure of Eisenia fetida to bifenthrin produced evident toxicity responses including the reproduction toxicity, oxidative stress and metabolic detoxification toxicity. By integrating the acute and chronic toxic endpoints at the wholeorganism level with a group of biochemical biomarkers, the information obtained in this study is useful for developing a scientific theoretical database for future risk assessment of pyrethroids in soil. The inconsistence found between the change of toxic effect and the degradation of bifenthrin in soil indicated existence of possible toxic metabolic products, which need further work on the toxicity and transformation of bifenthrin in soil.

Acknowledgments This work was supported by the National Natural Science Foundation of P. R. China (Grant no. 21377139), the Hundred-Person Program from the Chinese Academy of Sciences awarded to Jun Yan, and National Science and Technology Infrastructure Program of the Ministry of Science and Technology of P. R. China (2012BAD14B022).

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2016.10.060.

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