Chemosphere 135 (2015) 250–256
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Toxicity and bioaccumulation of bromadiolone to earthworm Eisenia fetida Jing Liu a,b,c,d, Kang Xiong a,b,c, Xiaoqing Ye a,b,c, Jianyun Zhang a,b,c, Ye Yang a,b,c, Li Ji a,b,c,⇑ a
MOE Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China Zhejiang Provincial Key Laboratory of Organic Pollution Process and Control, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China c Institute of Environmental Science, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China d Research Center for Air Pollution and Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China b
h i g h l i g h t s Bromadiolone is toxic to earthworms. Exposure to bromadiolone inhibits earthworm growth. Exposure to bromadiolone induces malondialdehyde content in earthworms. Bromadiolone in soil is bioaccumulative to earthworms. The BSAFs of bromadiolone decreased as its concentration in soil increased.
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Article history: Received 30 October 2014 Received in revised form 16 April 2015 Accepted 22 April 2015
Keywords: Bromadiolone Anticoagulant rodenticide Earthworm Toxicity Bioaccumulation
a b s t r a c t Bromadiolone, a potent second-generation anticoagulant rodenticide, has been extensively used worldwide for the field control of rodents. Invertebrates may be at risk from primary poisoning as a result of bromadiolone bait applications. However, there are few data regarding the toxicity and bioaccumulation of bromadiolone to earthworms. In this study, we reported that bromadiolone was toxic to earthworms at 1 mg/kg soil, which is a likely concentration in the field following application of bromadiolone baits. Exposure to bromadiolone resulted in a significant inhibition of earthworm growth. The antioxidant activities of superoxide dismutase and catalase were slightly increased in earthworms, while malondialdehyde content (as a molecular marker indicative of the damage to lipid peroxidation) was dominantly elevated over the duration of exposure. Bromadiolone in soil is bioaccumulative to earthworms. The biota to soil accumulation factors (BSAFs) of bromadiolone were concentration dependent and BSAFs decreased as the level of bromadiolone in soil increased. These results suggest earthworms are not only the potential subject to primary poisoning but also the source of secondary exposure for insectivores and scavengers following application of bromadiolone. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Rodents pose a threat to public health, critical habitats, native plants and animals, crops, food and water supplies. Annual costs for total damage from invasive rodents are as high as $19 billion in the USA and $15.9 billion in China (Pimentel et al., 2005; Zeng et al., 2014). The most preferred method for rodent population control is the use of anticoagulant rodenticides (ARs), which are chemical products that interfere with normal blood clotting and cause fatal hemorrhages by inhibiting vitamin K metabolism in liver. ⇑ Corresponding author at: 866 Yuhangtang Road, Hangzhou 310058, China. E-mail address: [email protected] (L. Ji). http://dx.doi.org/10.1016/j.chemosphere.2015.04.058 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
Second-generation ARs (SGARs) generally present more physiological persistence in vertebrate livers than their first-generation counterparts (Thomas et al., 2011). In any case, rodents exposed to SGARs survive for several days and will continue feeding on baits (Sage et al., 2008). The consumption of poisoned rodents by predatory animals can cause secondary poisoning in predators and result in the mortality of non-target species. A number of non-target mammal and bird species have been reported contaminated with SGARs, either directly through consuming baits, or indirectly through secondary poisoning (Hernandez-Moreno et al., 2013; Shore et al., 2003; Thomas et al., 2011). Due to difference in the blood-clotting systems between invertebrates and vertebrates, SGARs are thought to lack insecticidal
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properties (Loof et al., 2011). Nevertheless, it has been reported that the consumption of brodifacoum baits, a widely used SGAR, caused 100% mortality of two Seychelles Islands snail species (Gerlach and Florens, 2000). A previous study detected brodifacoum residues in cave weta (Hemideina thoracica White) (Ogilvie et al., 1997). These observations suggest that invertebrates may be at risk from primary poisoning as a result of SGAR bait applications. Earthworms are one of the most prevalent types of invertebrates that live in close contact with soil particles and represent up to 80% of the total soil biomass. Earthworms are consumed by many species of carnivorous animals, both vertebrate and invertebrate, forming the base of many food chains. If earthworms can carry SGAR residues, they may also be potential sources of secondary exposure for insectivores and scavengers. Bromadiolone is a potent SGAR and has been extensively used worldwide for the field control of rodents. For instance in France, as the only rodenticide authorized for controlling outbreaks of the fossorial water vole, 400 tons of bromadiolone baits (50 mg active ingredient/kg bait) were annually used by farmers (Sage et al., 2007). Bromadiolone is also one of the most popularly used SGARs in China, accounting for about 43% shares in the Chinese rodenticide market (Zeng et al., 2014). Bromadiolone baits were usually distributed in soils by burying in artificial galleries or storage cavities (Sage et al., 2007). The half-live of bromadiolone in various types of soils ranges from 1.8 to 53 days (Sage et al., 2007). Therefore, earthworms may be at risk of consuming bromadiolone. However, information regarding the toxicity of bromadiolone to earthworms and the occurrence of bromadiolone residues in earthworms is not available. Eisenia fetida is the widely used earthworm species for assessment of the ecological risks of toxic substances in the terrestrial environment (Diao et al., 2011; Hayashi et al., 2012). In this study, we determined the acute toxicity of bromadiolone to E. fetida using contact filter paper toxicity and soil toxicity bioassays. The changes in bodyweight and antioxidant defense system induced by bromadiolone in E. fetida were examined. The bioaccumulation of this compound from soil by E. fetida was also assessed. 2. Materials and methods 2.1. Chemicals and reagents Bromadiolone (P98.5% purity, Cat#: P-316S) was purchased from Beijing Helishun Technology Co. Ltd. Stock solutions of bromadiolone were dissolved in ethanol and stored at 20 °C. All other reagents were analytical grade and obtained from commercial sources (Liu et al., 2011; Yao et al., 2013a,b). 2.2. Earthworm Mature earthworms (E. fetida) were obtained from the active central earthworm breeding farm of Zhejiang University. under the condition of The earthworms were kept in an incubation chamber (20 ± 1 °C) at 80–85% relative humidity and the moisture content in soil was 35%. After domestication for 7 days, healthy adult earthworms with clitellum and weighing 200–300 mg were selected for toxicity tests. 2.3. Filter paper contact test According to the OECD guideline 207 (OECD, 1984), the filter paper contact test was conducted to determine the acute toxicity of bromadiolone to earthworms. A piece of filter paper was placed in a flat-bottomed glass vial and treated with bromadiolone dissolved in 1 ml of acetone or acetone alone (solvent control). The
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concentrations of bromadiolone range from 0, 0.1, 1, 10, 100 and 1000 lg/cm to determine concentrations causing 0%, 50% and 100% mortality. After an evaporation period of solvent, 1 ml of deionized water was added to each vial. Ten replicates for each treatment and each vial consisting of one mature worm were used in the experiment. All vials containing earthworms were maintained at 20 ± 1 °C under 80–85% relative humidity. The mortality was assessed after incubation period of 24 and 48 h and LC50 was determined. 2.4. Artificial soil test The artificial soil test was performed according to OECD guideline 207 (OECD, 1984). Artificial soil was composed of 10% peat moss, 20% kaolinite clay, 70% silica sand. For each tested concentration, bromadiolone was dissolved in acetone and mixed into 500 g artificial soil. The mixture was kept open for 3 h to evaporate the acetone. The final moisture contents of artificial soil were adjusted to 35% by the addition of deionized water. Ten adult earthworms were placed in a container containing a total of 0.675 kg treated soil that is equivalent to 0.5 kg dry artificial soil. The concentrations of bromadiolone range from 0, 0.1, 1, 10, 100 and 1000 mg/ kg dry soil to determine concentrations that resulted in 0–100% mortality. Three containers, each containing 10 adult earthworms, were used for each concentration. The containers maintained at 20 ± 1 °C in 80–85% relative humidity. The mortality was assessed after incubation period of 2, 7, 14, 28 and 42 days and LC50 was determined. 2.5. Earthworm exposure and sample collection Based on the LC50 of acute toxicity tests and soil toxicity tests, earthworms were exposed to bromadiolone at sublethal concentrations of 0, 0.1, 1, 10, 20 mg/kg in artificial soil for 2, 7, 14, 28 and 42 days as described above. Three containers, each containing 10 adult earthworms, were used for each concentration. Survival status was recorded, and the earthworms were left for 3 h to void their gut contents. After they were weighed without drying, three live earthworms from each container were stored at 80 °C for enzyme assays and the rest were frozen at 20 °C for bromadiolone residue analysis. Ten grams of soil samples from each container after exposure were collected and stored at 20 °C for the bromadiolone residue analysis. 2.6. Enzyme assays The collected earthworms from different treatment groups were homogenized in an amount of Tris–HCl buffer (100 mmol/L, pH 7.5) that was four times that of their bodyweight and centrifuged at 9000g at 4 °C for 30 min (Li et al., 2011; Liu et al., 2012). The supernatant was collected and employed to determine the contents of malondialdehyde (MDA), catalase (CAT) and superoxide dismutase (SOD) using the commercial kits (Jiancheng Bioengineering Institute, Nanjing, China) as described previously (Wang et al., 2013, 2007; Yang et al., 2014). 2.7. Chemical analysis Soil samples were mixed with 10 g of anhydrous sodium sulfate and 20 ml of a mixture of dichloromethane: acetone (70:30) in a 50 ml Teflon-capped-glass tube. After vortexing for 5 min and ultrasonic treatment for 20 min, the tube was centrifuged at 4500 rpm for 10 min (Niu et al., 2013; Yao et al., 2015; Zhang et al., 2011). The extraction was repeated twice and both liquid phases were transferred into a new tube. After filtering through 20 g of anhydrous sodium sulfate for dehydration, the extracts
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were evaporated to dryness on a rotary evaporator at 45 °C (Niu et al., 2014; Wen et al., 2010; Yao et al., 2014). Finally, the residue was reconstituted in 1 ml methanol and filtered through a 0.22 lm filter prior to high-performance liquid chromatography (HPLC) analysis. The extraction procedure of bromadiolone residues in earthworm was modified from Shore et al. (2003). Earthworm samples were homogenized and blended with anhydrous sodium sulfate. The homogenate was mixed with 20 ml of a mixture of dichloromethane: acetone (70:30), sonicated and then centrifuged. After repeated extraction, the upper organic phase was passed through a funnel with anhydrous sodium sulfate. After evaporation and dissolution, the residue was purified by Alumina-N-solid phase extraction (SPE) column (500 mg, 3 mL, Agilent SampliQ Products). The samples were examined by HPLC (Waters 2695 Separations Module with Waters 2475 Multi-wavelength fluorescence detector) using a Phenomenex Luna C-18 chromatographic column (250 4.6 mm; 5 lm). The injection volume of samples was 20 ll. The mobile phase was 76:24 methanol: water (v/v), supplemented with 0.25% (v/v) acetic acid and 40 mM ammonium acetate and the flow rate was 1.0 ml/min. The column temperature was 30 °C. Bromadiolone residues were detected by fluorescence spectrometry using excitation wavelength at 310 nm and emission wavelength at 390 nm. The average recoveries ranged from 70.3% to 93.0% in earthworm tissue and soil (n P 3 for each concentration). The detection limit was 1.39 lg/kg for both earthworm tissue and soil.
3. Results and discussion 3.1. Acute toxicity In the filter paper contact test, the acute toxicity was measured for bromadiolone. The results demonstrated that the percentage of mortality increased with increasing concentration of bromadiolone (Fig. 1A). The LC50 values for bromadiolone at 24 h and 48 h were 145.70 lg/cm2 (95% CI: 111.91–208.40) and 25.03 lg/cm2 (95% CI: 10.25–61.53), respectively. In the artificial soil test, the mortality increased with increasing concentrations of bromadiolone (Fig. 1B). The LC50 values at 14 d and 28 d were 449.958 mg/kg (95% CI: 190.816–783.000) and 105.593 mg/kg (95% CI: 13.180– 271.787), respectively. The data for the acute toxicity of bromadiolone exhibited a concentration and exposure time dependent relationship (Fig. 1). At 28-day, the mortality significantly raised to 20.0% at the lowest tested concentration of bromadiolone, suggesting that earthworms were adversely affected by soil amended with this rodenticide at 1 mg/kg. This concentration is equivalent to the distribution of bromadiolone from 20 g bait (containing 50 mg active integrant/kg bait) into 1 kg of soil. This scenario is likely to occur in the field following the application of bromadiolone baits, because this rodenticide is generally applied as a product formulated at
2.8. BSAF The relative accumulation of bromadiolone was expressed as biota to soil accumulation factor (BSAF). BSAF (kg wet weight/kg dry weight) was calculated from bromadiolone concentrations (wet weight) in the earthworms and the concentrations (dry weight) in the soil using the mean of the initial and final concentrations during exposure. BSAF (kgoc/kglip) was normalized to lipid content of earthworm and organic carbon (OC) of soil (Diao et al., 2011). The equations are as follows:
BSAFðkg wet weight=kg dry weightÞ ¼ CEW =CS
BSAFðkgoc =kglip Þ ¼ ½CEW FOC ðsoilÞ=½CS Flip ðearthwormÞ In the equations, CEW and CS are concentrations of bromadiolone in earthworm and soil, respectively. FOC (soil) is fraction of organic carbon in soil. The total organic carbon (TOC) in soil was measured using Multi N/C 3100 (Analytik Jena, Germany) and it is 11.07 ± 3.19 g/kg dry weight (Yao et al. 2011, 2013c). Flip (earthworm) is fraction of lipid in earthworm and it is assumed as 1% (Diao et al., 2011).
2.9. Statistical analysis Each treatment was conducted in triplicate. SPSS 17.0 statistical software was used to analyze the experimental data. The values were expressed as mean ± standard error of mean (SEM) for all the experiments. An analysis of variance was conducted to analyze the enzyme activities of earthworms in terms of bromadiolone concentration, time of exposure, and their interaction (Liu et al., 2008; Zhang et al., 2012; Zhao et al., 2014). Significant differences (p < 0.05) between the treatment groups and the control were determined using the post hoc LSD test.
Fig. 1. The mortality of earthworms exposed to bromadiolone. (A) The mortality of earthworm exposed to bromadiolone at concentrations of 1, 10, 100 and 1000 lg/ cm2 by the filter paper contact test for 24 h and 48 h. (B) The mortality of earthworm exposed to bromadiolone at concentrations of 0, 1, 10, 100, 1000 mg/kg by artificial soil test for 14 d and 28 d. Data are expressed as mean ± SEM (n P 3, n: the samples of replicate). Statistical significance versus control group: ⁄p < 0.05, ⁄⁄ p < 0.01, ⁄⁄⁄p < 0.001.
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0.005%, at the ratio of 10–20 g/hole and 5–20 kg/Ha (Giraudoux et al., 2006). Therefore, earthworms are potential non-target species that may be subject to primary poisoning following application of bromadiolone. 3.2. Effects of bromadiolone on earthworm weight Based on the LC50 of soil toxicity tests, earthworms were exposed to bromadiolone at sublethal concentrations of 0.1– 20 mg/kg in artificial soil for 2–42 days. As shown in Fig. 2, the bodyweight of earthworms in control groups was increased after 14 days. At 2-day and 7-day, compared with control groups, significant decrease in weight of earthworms was observed in all treatment groups except for 10 mg/kg. At 14-day and 28-day, the weight change rates of earthworms in groups exposed to 10 or 20 mg/kg were lower than that of control. After exposure for 42 days, the growth of earthworms in all treatment groups was suppressed in a dose-dependent manner. These data indicated that exposure to bromadiolone at sublethal concentrations inhibited earthworm growth. Depletions of glycogen and lipid contents, and decrease in protein content, which were followed by a reduction in growth, have been observed in earthworm or other invertebrates exposed to toxins (Ribeiro et al., 2001; Xu et al., 2015). In this study, the adverse influence may also result from a decrease in these energy reserves in earthworm response to bromadiolone exposure.
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doses of 0.1–20 mg/kg in artificial soil for 2–42 days. As shown in Fig. 3A, in general, SOD activity was slightly up-regulated in earthworms treated with the rodenticide. But the SOD activity was only significantly increased in groups exposed to 1 mg/kg for 2 days and 14 days, or 10 mg/kg for 42 days, compared to the control. The similar pattern of changes in CAT activity was also observed. Although a slight increase in CAT activity was generally induced by bromadiolone exposure from 7 to 42 days, the statistically significant induction of CAT activity was only observed in animals exposed
3.3. Effects of bromadiolone on antioxidant enzymes and lipid peroxidation Environmental contaminants are major hazardous factors to the survival of organisms by eliciting excessive free radicals, such as reactive oxygen species (ROS), which may cause oxidative damage to biological macromolecules, like DNA, protein and lipid. To counteract of ROS, organisms have evolved defensive system which contains various antioxidant enzymes. Normally, a dynamic balance exists between ROS levels and the antioxidant enzyme contents, while excessive ROS production that exceeds the antioxidant capacity causes oxidative stress (Ziech et al., 2010). Superoxide dismutase (SOD) and catalase (CAT), are the most important members of the first antioxidative defense line to scavenge ROS, are well-described biomarkers to determine the oxidative stress profile in the organisms (Wang et al., 2013; Yang et al., 2014; Zhang et al., 2012). In this study, we examined SOD and CAT activities in the earthworms exposed to bromadiolone at
Fig. 2. Weight change rate of earthworms exposed to bromadiolone. Data are expressed as mean ± SEM (n P 3, n: the samples of replicate). Statistical significance versus control group: ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.
Fig. 3. The effects of bromadiolone on antioxidant enzymes and lipid peroxidation in earthworm. (A) SOD activity; (B) CAT activity; (C) MDA content. Data are expressed as mean ± SEM (n P 3, n: the samples of replicate). Statistical significance versus control group: ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.
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to bromadiolone at 20 mg/kg for 14 days, or 0.1 mg/kg and 1 mg/kg for 28 days (Fig. 3B). Malondialdehyde (MDA) is also a commonly used molecular marker indicating the damage of lipid peroxidation caused by redundant free radicals (Wang et al., 2013; Zhang et al., 2012). In the present study, we investigated the changes in MDA content of earthworms treated with bromadiolone. As seen in Fig. 3C, MDA content had a rapid response to the rodenticide treatment. After 2 days, MDA content was significantly greater in the rodenticide-treated groups compared to the control, particularly in the presence of 1, 10 and 20 mg/kg bromadiolone. With increasing exposure duration, the induction of MDA content was more significant. At 42-day, MDA contents in all bromadiolone-exposed groups were extremely higher than the control at 0.1 mg/kg (p < 0.01) and 1–20 mg/kg (p < 0.001). As the important enzymes in the antioxidant system of organisms, SOD and CAT eliminate generated O 2 and H2O2, respectively, and protect cells from the damage during the formation of ROS. A number of studies have shown that the alterations in cellular activities of SOD and CAT were considered as the early biomarkers for determining the toxic effects of pollutants on ecosystems (Wang et al., 2013; Xu et al., 2013; Yang et al., 2014; Zhang et al., 2012). However, in this study, the changes in activities of SOD and CAT were slight but not significant in most of bromadiolone-treated groups. The results showed that the induction of MDA content in earthworm was more dominant than that of SOD and CAT activity. Lipid peroxidation appears to be more sensitive parameter in evaluating the level of oxidative stress in the earthworms in response to bromadiolone exposure. In agreement with our observations, it has been reported that exposure to bromadiolone resulted in increasing levels of lipid peroxidation, but no change in antioxidant capacity, in the tissues of Japanese quails (Ondracek et al., 2012). Lipid peroxidation damages the cell membrane and leads to destruction of membrane lipids (Mylonas and Kouretas, 1999). In addition, as the main end-product of lipid peroxidation, MDA can react with deoxyadenosine and deoxyguanosine in DNA, forming DNA adducts that may cause mutagenesis and carcinogenesis (Bartsch and Nair, 2000). Our results indicated that exposure to bromadiolone resulted in serious oxidative damage and impairment of physiological functions, which subsequently led to a decreasing growth rate in earthworms. 3.4. Bioaccumulation of bromadiolone to earthworm The concentrations of bromadiolone in earthworm tissue and soil were determined. In soil samples, the initial concentrations of bromadiolone were 10 mg/kg and 20 mg/kg. Fig. 4A shows the degradation of bromadiolone during the period of 42 days of incubation. After 2 days, the concentrations of this rodenticide at 10 mg/kg and 20 mg/kg rapidly dropped to 52.1% and 55.6%, respectively. In the 20 mg/kg samples, the concentrations of bromadiolone decreased to approximately 35% at 7-day and reached the steady state levels throughout the exposure duration. However, the concentration of bromadiolone at 10 mg/kg steadily declined to 24.4% at the end of exposure. The elimination rate constant of bromadiolone in soil could be translated into a half-life (t1/2) of 5 days. As shown in Fig. 4B, the concentrations of bromadiolone in earthworm tissue increased from 2 to 14 days. The rapid uptake of bromadiolone was observed, and on the first sample point (2-day), the concentrations of bromadiolone at 10 mg/kg and 20 mg/kg were 0.22 mg/kg and 0.36 mg/kg, respectively. At 14-day, the concentrations reached the maximum as 0.44 mg/kg in both 10 mg/kg and 20 mg/kg groups. After 28 days of exposure, the concentrations in earthworm declined. At 42-day, the concentrations of bromadiolone at 10 mg/kg and 20 mg/kg were
Fig. 4. Concentrations of bromadiolone in soil (A) and earthworm (B) samples. Data are expressed as mean ± SEM (n P 3, n: the samples of replicate).
0.19 mg/kg and 0.30 mg/kg, respectively. These data suggest that bromadiolone can be taken up by earthworms from soil quickly and its accumulation in earthworms reached equilibrium in 14 days. In this study, the relative accumulation of bromadiolone was presented as biota to soil accumulation factor (BSAF) (Fig. 5A). In order to compare with bioaccumulation of other chemicals in earthworm, the activities were normalized to lipid content of earthworm that was assumed as 1% (Diao et al., 2011), and organic carbon (OC) of soil that was determined as 11.07 ± 3.19 g/kg dry weight (Fig. 5B). The BSAF values of bromadiolone of 10 mg/kg groups were higher than those of 20 mg/kg groups during the whole uptake period (Fig. 5). This observation that BSAF values of bromadiolone decreased with increasing soil concentrations indicates that the bioaccumulation of bromadiolone is concentration dependent. Similar trend was reported for perfluoroalkyl substances (PFASs) in earthworm (Zhao et al., 2013). In addition, the decreasing BSAF values with increasing concentration of chemicals in sediment were also found in various organisms, such as chlorinated dibenzo-p-dioxin and dibenzofuran congeners (PCDD/Fs) in dungeness crab (Cretney and Yunker, 2000), 3,4,30 ,40 -tetrachlorobiphenyl (TCBP) in benthic invertebrates (Leppanen et al., 2003), as well as polychlorinated biphenyls (PCBs) in oligochaetes (Sormunen et al., 2008). This phenomenon could be explained by the saturation of binding sites in biota available for adsorption and the resistant desorption of hydrophobic organic compounds at high soil/sediment concentrations. The fact that bromadiolone in soil is bioaccumulative to earthworms suggests that contaminated earthworms are a potential source of secondary exposure of non-target birds and invertebrates to this rodenticide.
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Fig. 5. Calculated biota to soil accumulation factors (BSAFs) for bromadiolone. Data are expressed as mean ± SEM (n P 3, n: the samples of replicate). The unit of BSAFs in panel A and panel B is kg wet weight/kg dry weight and kgoc/kglip, respectively.
4. Conclusions In the present study, the data of acute toxicity demonstrated that exposure to bromadiolone at soil concentration of 1 mg/kg is toxic to earthworms. Exposure to bromadiolone resulted in significant inhibition of growth rate and induction of lipid peroxidation in earthworm. Bromadiolone in soil is bioaccumulative to earthworms. The uptake of bromadiolone by earthworms is concentration dependent and the BSAFs of bromadiolone decreased as its concentration in soil increased. This work provided baseline data on the susceptibility of earthworms to bromadiolone, as a basis for risk assessment for secondary exposure. Acknowledgments This work was supported by the National High Technology Research & Development Program of China (2013AA065202), the National Natural Science Foundation of China (Nos. 21377113, 21307107, 21427815) and Zhejiang Provincial Natural Science Foundation of China (LR15B070001). Reference Bartsch, H., Nair, J., 2000. Ultrasensitive and specific detection methods for exocyclic DNA adducts: markers for lipid peroxidation and oxidative stress. Toxicology 153, 105–114. Cretney, W.J., Yunker, M.B., 2000. Concentration dependency of biota-sediment accumulation factors for chlorinated dibenzo-p-dioxins and dibenzofurans in
255
Dungeness crab (Cancer magister) at marine pulp mill sites in British Columbia, Canada. Environ. Toxicol. Chem. 19, 3012–3023. Diao, J.L., Xu, P., Liu, D.H., Lu, Y.L., Zhou, Z.Q., 2011. Enantiomer-specific toxicity and bioaccumulation of alpha-cypermethrin to earthworm Eisenia fetida. J. Hazard. Mater. 192, 1072–1078. Gerlach, J., Florens, V., 2000. Considering molluscs in rodent eradication projects. Tentacle-TheNewsletter of the IUCN/SSC Mollusc Specialist Group 9, pp. 7–8. Giraudoux, P., Tremollieres, C., Barbier, B., Defaut, R., Rieffel, D., Bernard, N., Lucota, E., Berny, P., 2006. Persistence of bromadiolone anticoagulant rodenticide in Arvicola terrestris populations after field control. Environ. Res. 102, 291–298. Hayashi, Y., Engelmann, P., Foldbjerg, R., Szabo, M., Somogyi, I., Pollak, E., Molnar, L., Autrup, H., Sutherland, D.S., Scott-Fordsmand, J., Heckmann, L.H., 2012. Earthworms and humans in vitro: characterizing evolutionarily conserved stress and immune responses to silver nanoparticles. Environ. Sci. Technol. 46, 4166–4173. Hernandez-Moreno, D., de la Casa-Resino, I., Lopez-Beceiro, A., Fidalgo, L.E., Soler, F., Perez-Lopez, M., 2013. Secondary poisoning of non-target animals in an Ornithological Zoo in Galicia (NW Spain) with anticoagulant rodenticides: a case report. Vet. Med. 58, 553–559. Leppanen, M.T., Landrum, P.F., Kukkonen, J.V.K., Greenberg, M.S., Burton, G.A., Robinson, S.D., Gossiaux, D.C., 2003. Investigating the role of desorption on the bioavailability of sediment-associated 3,4,30 ,40 -tetrachlorobiphenyl in benthic invertebrates. Environ. Toxicol. Chem. 22, 2861–2871. Li, F.X., Liu, J., Jo, M., Curry, T.E., 2011. A role for nuclear factor interleukin-3 (NFIL3), a critical transcriptional repressor, in down-regulation of periovulatory gene expression. Mol. Endocrinol. 25, 445–459. Liu, H.G., Zhao, M.R., Zhang, C., Ma, Y., Liu, W.P., 2008. Enantioselective cytotoxicity of the insecticide bifenthrin on a human amnion epithelial (FL) cell line. Toxicology 253, 89–96. Liu, J., Yang, Y., Zhuang, S.L., Yang, Y., Li, F.X., Liu, W.P., 2011. Enantioselective endocrine-disrupting effects of bifenthrin on hormone synthesis in rat ovarian cells. Toxicology 290, 42–49. Liu, J., Zhao, M., Zhuang, S., Yang, Y., Yang, Y., Liu, W., 2012. Low concentrations of o, p’-DDT inhibit gene expression and prostaglandin synthesis by estrogen receptor-independent mechanism in rat ovarian cells. PLoS ONE 7, e49916. Loof, T.G., Schmidt, O., Herwald, H., Theopold, U., 2011. Coagulation systems of invertebrates and vertebrates and their roles in innate immunity: the same side of two coins? J. Innate Immun. 3, 34–40. Mylonas, C., Kouretas, D., 1999. Lipid peroxidation and tissue damage. In Vivo 13, 295–309. Niu, L.L., Xu, C., Yao, Y.J., Liu, K., Yang, F.X., Tang, M.L., Liu, W.P., 2013. Status, influences and risk assessment of hexachlorocyclohexanes in agricultural soils across China. Environ. Sci. Technol. 47, 12140–12147. Niu, L.L., Xu, C., Xu, Y., Zhang, C.L., Liu, W.P., 2014. Hexachlorocyclohexanes in tree bark across Chinese agricultural regions: spatial distribution and enantiomeric signatures. Environ. Sci. Technol. 48, 12031–12038. OECD, 1984. OECD guideline 207. www.oecd.org/chemicalsafety/risk-assessment/ 1948293 pdf. Ogilvie, S.C., Pierce, R.J., Wright, G.R.G., Booth, L.H., Eason, C.T., 1997. Brodifacoum residue analysis in water, soil, invertebrates, and birds after rat eradication on Lady Alice Island. New Zealand J. Ecol. 21, 195–197. Ondracek, K., Bandouchova, H., Damkova, V., Hilscherova, K., Kral, J., Osickova, J., Mlcakova, V., Pohanka, M., Skochova, H., Vitula, F., Treml, F., Pikula, J., 2012. Risk of combined exposure of birds to cyanobacterial biomass containing microcystins, acetylcholinesterase inhibitor and anticoagulant. Neuroendocrinol. Lett. 33, 155–160. Pimentel, D., Zuniga, R., Morrison, D., 2005. Update on the environmental and economic costs associated with alien-invasive species in the United States. Ecol. Econ. 52, 273–288. Ribeiro, S., Sousa, J.P., Nogueira, A.J.A., Soares, A.M.V.M., 2001. Effect of endosulfan and parathion on energy reserves and physiological parameters of the terrestrial isopod Porcellio dilatatus. Ecotoxicol. Environ. Saf. 49, 131–138. Sage, M., Coeurdassier, M., Defaut, R., Lucot, E., Barbier, B., Rieffel, D., Berny, P., Giraudoux, P., 2007. How environment and vole behaviour may impact rodenticide bromadiolone persistence in wheat baits after field controls of Arvicola terrestris? Environ. Pollut. 148, 372–379. Sage, M., Caeurdassier, M., Defaut, R., Gimbert, F., Berny, P., Giraudoux, P., 2008. Kinetics of bromadiolone in rodent populations and implications for predators after field control of the water vole, Arvicola terrestris. Sci. Total Environ. 407, 211–222. Shore, R.F., Birks, J.D.S., Afsar, A., Wienburg, C.L., Kitchener, A.C., 2003. Spatial and temporal analysis of second-generation anticoagulant rodenticide residues in polecats (Mustela putorius) from throughout their range in Britain, 1992–1999. Environ. Pollut. 122, 183–193. Sormunen, A.J., Leppanen, M.T., Kukkonen, J.V.K., 2008. Influence of sediment ingestion and exposure concentration on the bioavailable fraction of sedimentassociated tetrachlorobiphenyl in oligochaetes. Environ. Toxicol. Chem. 27, 854–863. Thomas, P.J., Mineau, P., Shore, R.F., Champoux, L., Martin, P.A., Wilson, L.K., Fitzgerald, G., Elliott, J.E., 2011. Second generation anticoagulant rodenticides in predatory birds: probabilistic characterisation of toxic liver concentrations and implications for predatory bird populations in Canada. Environ. Int. 37, 914– 920. Wang, L.M., Liu, W., Yang, C.X., Pan, Z.Y., Gan, J.Y., Xu, C., Zhao, M.R., Schlenk, D., 2007. Enantioselectivity in estrogenic potential and uptake of bifenthrin. Environ. Sci. Technol. 41, 6124–6128.
256
J. Liu et al. / Chemosphere 135 (2015) 250–256
Wang, C., Li, Z.Y., Zhang, Q., Zhao, M.R., Liu, W.P., 2013. Enantioselective induction of cytotoxicity by o, p0 -DDD in PC12 cells: implications of chirality in risk assessment of POPs metabolites. Environ. Sci. Technol. 47, 3909–3917. Wen, Y.Z., Yuan, Y.L., Chen, H., Xu, D.M., Lin, K.D., Liu, W.P., 2010. Effect of chitosan on the enantioselective bioavailability of the herbicide dichlorprop to Chlorella pyrenoidosa. Environ. Sci. Technol. 44, 4981–4987. Xu, D.M., Li, C.D., Wen, Y.Z., Liu, W.P., 2013. Antioxidant defense system responses and DNA damage of earthworms exposed to perfluorooctane sulfonate (PFOS). Environ. Pollut. 174, 121–127. Xu, X.B., Shi, Y.J., Lu, Y.L., Zheng, X.Q., Ritchie, R.J., 2015. Growth inhibition and altered gene transcript levels in earthworms (Eisenia fetida) exposed to 2,20 ,4,40 tetrabromodiphenyl ether. Arch. Environ. Contam. Toxicol. Yang, Y., Ma, H.H., Zhou, J.H., Liu, J., Liu, W.P., 2014. Joint toxicity of permethrin and cypermethrin at sublethal concentrations to the embryo-larval zebrafish. Chemosphere 96, 146–154. Yao, Y.J., Shen, R., Pennell, K.G., Suuberg, E.M., 2011. Comparison of the Johnson– Ettinger vapor intrusion screening model predictions with full threedimensional model results. Environ. Sci. Technol. 45, 2227–2235. Yao, Y.J., Shen, R., Pennell, K.G., Suuberg, E.M., 2013a. A review of vapor intrusion models. Environ. Sci. Technol. 47, 2457–2470. Yao, Y.J., Shen, R., Pennell, K.G., Suuberg, E.M., 2013b. Examination of the US EPA’s vapor intrusion database based on models. Environ. Sci. Technol. 47, 1425– 1433. Yao, Y.J., Shen, R., Pennell, K.G., Suuberg, E.M., 2013c. Examination of the influence of environmental factors on contaminant vapor concentration attenuation factors using the U.S. EPA’s vapor intrusion database. Environ. Sci. Technol. 47, 906–913.
Yao, Y.J., Yang, F.X., Suuberg, E.M., Provoost, J., Liu, W.P., 2014. Estimation of contaminant subslab concentration in petroleum vapor intrusion. J. Hazard. Mater. 279, 336–347. Yao, Y.J., Su, Y., Wu, Y., Liu, W.P., He, R., 2015. An analytical model for estimating the reduction of methane emission through landfill cover soils by methane oxidation. J. Hazard. Mater. 283, 871–879. Zeng, J.P., Zeng, C., Ouyang, F., Liu, X.P., Ge, F., 2014. Status Quo, loss and trend of forest bio-disasters from 2000 to 2010 in China. Biol. Disaster Sci. 37, 7–12. Zhang, A.P., Liu, W.P., Yuan, H.J., Zhou, S.S., Su, Y.S., Li, Y.F., 2011. Spatial distribution of hexachlorocyclohexanes in agricultural soils in Zhejiang Province, China, and correlations with elevation and temperature. Environ. Sci. Technol. 45, 6303– 6308. Zhang, Q., Zhao, M.R., Qian, H.F., Lu, T., Zhang, Q., Liu, W.P., 2012. Enantioselective damage of diclofop acid mediated by oxidative stress and acetyl-CoA carboxylase in nontarget plant Arabidopsis thaliana. Environ. Sci. Technol. 46, 8405–8412. Zhao, S.Y., Zhu, L.Y., Liu, L., Liu, Z.T., Zhang, Y.H., 2013. Bioaccumulation of perfluoroalkyl carboxylates (PFCAs) and perfluoroalkane sulfonates (PFSAs) by earthworms (Eisenia fetida) in soil. Environ. Pollut. 179, 45–52. Zhao, M., Zhang, Y., Zhuang, S., Zhang, Q., Lu, C., Liu, W., 2014. Disruption of the hormonal network and the enantioselectivity of bifenthrin in trophoblast: maternal–fetal health risk of chiral pesticides. Environ. Sci. Technol. 48, 8109– 8116. Ziech, D., Franco, R., Georgakilas, A.G., Georgakila, S., Malamou-Mitsi, V., Schoneveld, O., Pappa, A., Panayiotidis, M.I., 2010. The role of reactive oxygen species and oxidative stress in environmental carcinogenesis and biomarker development. Chem. Biol. Interact. 188, 334–339.
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Toxicity and bioaccumulation of bromadiolone to earthworm Eisenia fetida Jing Liu a,b,c,d, Kang Xiong a,b,c, Xiaoqing Ye a,b,c, Jianyun Zhang a,b,c, Ye Yang a,b,c, Li Ji a,b,c,⇑ a
MOE Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China Zhejiang Provincial Key Laboratory of Organic Pollution Process and Control, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China c Institute of Environmental Science, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China d Research Center for Air Pollution and Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China b
h i g h l i g h t s Bromadiolone is toxic to earthworms. Exposure to bromadiolone inhibits earthworm growth. Exposure to bromadiolone induces malondialdehyde content in earthworms. Bromadiolone in soil is bioaccumulative to earthworms. The BSAFs of bromadiolone decreased as its concentration in soil increased.
a r t i c l e
i n f o
Article history: Received 30 October 2014 Received in revised form 16 April 2015 Accepted 22 April 2015
Keywords: Bromadiolone Anticoagulant rodenticide Earthworm Toxicity Bioaccumulation
a b s t r a c t Bromadiolone, a potent second-generation anticoagulant rodenticide, has been extensively used worldwide for the field control of rodents. Invertebrates may be at risk from primary poisoning as a result of bromadiolone bait applications. However, there are few data regarding the toxicity and bioaccumulation of bromadiolone to earthworms. In this study, we reported that bromadiolone was toxic to earthworms at 1 mg/kg soil, which is a likely concentration in the field following application of bromadiolone baits. Exposure to bromadiolone resulted in a significant inhibition of earthworm growth. The antioxidant activities of superoxide dismutase and catalase were slightly increased in earthworms, while malondialdehyde content (as a molecular marker indicative of the damage to lipid peroxidation) was dominantly elevated over the duration of exposure. Bromadiolone in soil is bioaccumulative to earthworms. The biota to soil accumulation factors (BSAFs) of bromadiolone were concentration dependent and BSAFs decreased as the level of bromadiolone in soil increased. These results suggest earthworms are not only the potential subject to primary poisoning but also the source of secondary exposure for insectivores and scavengers following application of bromadiolone. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Rodents pose a threat to public health, critical habitats, native plants and animals, crops, food and water supplies. Annual costs for total damage from invasive rodents are as high as $19 billion in the USA and $15.9 billion in China (Pimentel et al., 2005; Zeng et al., 2014). The most preferred method for rodent population control is the use of anticoagulant rodenticides (ARs), which are chemical products that interfere with normal blood clotting and cause fatal hemorrhages by inhibiting vitamin K metabolism in liver. ⇑ Corresponding author at: 866 Yuhangtang Road, Hangzhou 310058, China. E-mail address: [email protected] (L. Ji). http://dx.doi.org/10.1016/j.chemosphere.2015.04.058 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
Second-generation ARs (SGARs) generally present more physiological persistence in vertebrate livers than their first-generation counterparts (Thomas et al., 2011). In any case, rodents exposed to SGARs survive for several days and will continue feeding on baits (Sage et al., 2008). The consumption of poisoned rodents by predatory animals can cause secondary poisoning in predators and result in the mortality of non-target species. A number of non-target mammal and bird species have been reported contaminated with SGARs, either directly through consuming baits, or indirectly through secondary poisoning (Hernandez-Moreno et al., 2013; Shore et al., 2003; Thomas et al., 2011). Due to difference in the blood-clotting systems between invertebrates and vertebrates, SGARs are thought to lack insecticidal
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properties (Loof et al., 2011). Nevertheless, it has been reported that the consumption of brodifacoum baits, a widely used SGAR, caused 100% mortality of two Seychelles Islands snail species (Gerlach and Florens, 2000). A previous study detected brodifacoum residues in cave weta (Hemideina thoracica White) (Ogilvie et al., 1997). These observations suggest that invertebrates may be at risk from primary poisoning as a result of SGAR bait applications. Earthworms are one of the most prevalent types of invertebrates that live in close contact with soil particles and represent up to 80% of the total soil biomass. Earthworms are consumed by many species of carnivorous animals, both vertebrate and invertebrate, forming the base of many food chains. If earthworms can carry SGAR residues, they may also be potential sources of secondary exposure for insectivores and scavengers. Bromadiolone is a potent SGAR and has been extensively used worldwide for the field control of rodents. For instance in France, as the only rodenticide authorized for controlling outbreaks of the fossorial water vole, 400 tons of bromadiolone baits (50 mg active ingredient/kg bait) were annually used by farmers (Sage et al., 2007). Bromadiolone is also one of the most popularly used SGARs in China, accounting for about 43% shares in the Chinese rodenticide market (Zeng et al., 2014). Bromadiolone baits were usually distributed in soils by burying in artificial galleries or storage cavities (Sage et al., 2007). The half-live of bromadiolone in various types of soils ranges from 1.8 to 53 days (Sage et al., 2007). Therefore, earthworms may be at risk of consuming bromadiolone. However, information regarding the toxicity of bromadiolone to earthworms and the occurrence of bromadiolone residues in earthworms is not available. Eisenia fetida is the widely used earthworm species for assessment of the ecological risks of toxic substances in the terrestrial environment (Diao et al., 2011; Hayashi et al., 2012). In this study, we determined the acute toxicity of bromadiolone to E. fetida using contact filter paper toxicity and soil toxicity bioassays. The changes in bodyweight and antioxidant defense system induced by bromadiolone in E. fetida were examined. The bioaccumulation of this compound from soil by E. fetida was also assessed. 2. Materials and methods 2.1. Chemicals and reagents Bromadiolone (P98.5% purity, Cat#: P-316S) was purchased from Beijing Helishun Technology Co. Ltd. Stock solutions of bromadiolone were dissolved in ethanol and stored at 20 °C. All other reagents were analytical grade and obtained from commercial sources (Liu et al., 2011; Yao et al., 2013a,b). 2.2. Earthworm Mature earthworms (E. fetida) were obtained from the active central earthworm breeding farm of Zhejiang University. under the condition of The earthworms were kept in an incubation chamber (20 ± 1 °C) at 80–85% relative humidity and the moisture content in soil was 35%. After domestication for 7 days, healthy adult earthworms with clitellum and weighing 200–300 mg were selected for toxicity tests. 2.3. Filter paper contact test According to the OECD guideline 207 (OECD, 1984), the filter paper contact test was conducted to determine the acute toxicity of bromadiolone to earthworms. A piece of filter paper was placed in a flat-bottomed glass vial and treated with bromadiolone dissolved in 1 ml of acetone or acetone alone (solvent control). The
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concentrations of bromadiolone range from 0, 0.1, 1, 10, 100 and 1000 lg/cm to determine concentrations causing 0%, 50% and 100% mortality. After an evaporation period of solvent, 1 ml of deionized water was added to each vial. Ten replicates for each treatment and each vial consisting of one mature worm were used in the experiment. All vials containing earthworms were maintained at 20 ± 1 °C under 80–85% relative humidity. The mortality was assessed after incubation period of 24 and 48 h and LC50 was determined. 2.4. Artificial soil test The artificial soil test was performed according to OECD guideline 207 (OECD, 1984). Artificial soil was composed of 10% peat moss, 20% kaolinite clay, 70% silica sand. For each tested concentration, bromadiolone was dissolved in acetone and mixed into 500 g artificial soil. The mixture was kept open for 3 h to evaporate the acetone. The final moisture contents of artificial soil were adjusted to 35% by the addition of deionized water. Ten adult earthworms were placed in a container containing a total of 0.675 kg treated soil that is equivalent to 0.5 kg dry artificial soil. The concentrations of bromadiolone range from 0, 0.1, 1, 10, 100 and 1000 mg/ kg dry soil to determine concentrations that resulted in 0–100% mortality. Three containers, each containing 10 adult earthworms, were used for each concentration. The containers maintained at 20 ± 1 °C in 80–85% relative humidity. The mortality was assessed after incubation period of 2, 7, 14, 28 and 42 days and LC50 was determined. 2.5. Earthworm exposure and sample collection Based on the LC50 of acute toxicity tests and soil toxicity tests, earthworms were exposed to bromadiolone at sublethal concentrations of 0, 0.1, 1, 10, 20 mg/kg in artificial soil for 2, 7, 14, 28 and 42 days as described above. Three containers, each containing 10 adult earthworms, were used for each concentration. Survival status was recorded, and the earthworms were left for 3 h to void their gut contents. After they were weighed without drying, three live earthworms from each container were stored at 80 °C for enzyme assays and the rest were frozen at 20 °C for bromadiolone residue analysis. Ten grams of soil samples from each container after exposure were collected and stored at 20 °C for the bromadiolone residue analysis. 2.6. Enzyme assays The collected earthworms from different treatment groups were homogenized in an amount of Tris–HCl buffer (100 mmol/L, pH 7.5) that was four times that of their bodyweight and centrifuged at 9000g at 4 °C for 30 min (Li et al., 2011; Liu et al., 2012). The supernatant was collected and employed to determine the contents of malondialdehyde (MDA), catalase (CAT) and superoxide dismutase (SOD) using the commercial kits (Jiancheng Bioengineering Institute, Nanjing, China) as described previously (Wang et al., 2013, 2007; Yang et al., 2014). 2.7. Chemical analysis Soil samples were mixed with 10 g of anhydrous sodium sulfate and 20 ml of a mixture of dichloromethane: acetone (70:30) in a 50 ml Teflon-capped-glass tube. After vortexing for 5 min and ultrasonic treatment for 20 min, the tube was centrifuged at 4500 rpm for 10 min (Niu et al., 2013; Yao et al., 2015; Zhang et al., 2011). The extraction was repeated twice and both liquid phases were transferred into a new tube. After filtering through 20 g of anhydrous sodium sulfate for dehydration, the extracts
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were evaporated to dryness on a rotary evaporator at 45 °C (Niu et al., 2014; Wen et al., 2010; Yao et al., 2014). Finally, the residue was reconstituted in 1 ml methanol and filtered through a 0.22 lm filter prior to high-performance liquid chromatography (HPLC) analysis. The extraction procedure of bromadiolone residues in earthworm was modified from Shore et al. (2003). Earthworm samples were homogenized and blended with anhydrous sodium sulfate. The homogenate was mixed with 20 ml of a mixture of dichloromethane: acetone (70:30), sonicated and then centrifuged. After repeated extraction, the upper organic phase was passed through a funnel with anhydrous sodium sulfate. After evaporation and dissolution, the residue was purified by Alumina-N-solid phase extraction (SPE) column (500 mg, 3 mL, Agilent SampliQ Products). The samples were examined by HPLC (Waters 2695 Separations Module with Waters 2475 Multi-wavelength fluorescence detector) using a Phenomenex Luna C-18 chromatographic column (250 4.6 mm; 5 lm). The injection volume of samples was 20 ll. The mobile phase was 76:24 methanol: water (v/v), supplemented with 0.25% (v/v) acetic acid and 40 mM ammonium acetate and the flow rate was 1.0 ml/min. The column temperature was 30 °C. Bromadiolone residues were detected by fluorescence spectrometry using excitation wavelength at 310 nm and emission wavelength at 390 nm. The average recoveries ranged from 70.3% to 93.0% in earthworm tissue and soil (n P 3 for each concentration). The detection limit was 1.39 lg/kg for both earthworm tissue and soil.
3. Results and discussion 3.1. Acute toxicity In the filter paper contact test, the acute toxicity was measured for bromadiolone. The results demonstrated that the percentage of mortality increased with increasing concentration of bromadiolone (Fig. 1A). The LC50 values for bromadiolone at 24 h and 48 h were 145.70 lg/cm2 (95% CI: 111.91–208.40) and 25.03 lg/cm2 (95% CI: 10.25–61.53), respectively. In the artificial soil test, the mortality increased with increasing concentrations of bromadiolone (Fig. 1B). The LC50 values at 14 d and 28 d were 449.958 mg/kg (95% CI: 190.816–783.000) and 105.593 mg/kg (95% CI: 13.180– 271.787), respectively. The data for the acute toxicity of bromadiolone exhibited a concentration and exposure time dependent relationship (Fig. 1). At 28-day, the mortality significantly raised to 20.0% at the lowest tested concentration of bromadiolone, suggesting that earthworms were adversely affected by soil amended with this rodenticide at 1 mg/kg. This concentration is equivalent to the distribution of bromadiolone from 20 g bait (containing 50 mg active integrant/kg bait) into 1 kg of soil. This scenario is likely to occur in the field following the application of bromadiolone baits, because this rodenticide is generally applied as a product formulated at
2.8. BSAF The relative accumulation of bromadiolone was expressed as biota to soil accumulation factor (BSAF). BSAF (kg wet weight/kg dry weight) was calculated from bromadiolone concentrations (wet weight) in the earthworms and the concentrations (dry weight) in the soil using the mean of the initial and final concentrations during exposure. BSAF (kgoc/kglip) was normalized to lipid content of earthworm and organic carbon (OC) of soil (Diao et al., 2011). The equations are as follows:
BSAFðkg wet weight=kg dry weightÞ ¼ CEW =CS
BSAFðkgoc =kglip Þ ¼ ½CEW FOC ðsoilÞ=½CS Flip ðearthwormÞ In the equations, CEW and CS are concentrations of bromadiolone in earthworm and soil, respectively. FOC (soil) is fraction of organic carbon in soil. The total organic carbon (TOC) in soil was measured using Multi N/C 3100 (Analytik Jena, Germany) and it is 11.07 ± 3.19 g/kg dry weight (Yao et al. 2011, 2013c). Flip (earthworm) is fraction of lipid in earthworm and it is assumed as 1% (Diao et al., 2011).
2.9. Statistical analysis Each treatment was conducted in triplicate. SPSS 17.0 statistical software was used to analyze the experimental data. The values were expressed as mean ± standard error of mean (SEM) for all the experiments. An analysis of variance was conducted to analyze the enzyme activities of earthworms in terms of bromadiolone concentration, time of exposure, and their interaction (Liu et al., 2008; Zhang et al., 2012; Zhao et al., 2014). Significant differences (p < 0.05) between the treatment groups and the control were determined using the post hoc LSD test.
Fig. 1. The mortality of earthworms exposed to bromadiolone. (A) The mortality of earthworm exposed to bromadiolone at concentrations of 1, 10, 100 and 1000 lg/ cm2 by the filter paper contact test for 24 h and 48 h. (B) The mortality of earthworm exposed to bromadiolone at concentrations of 0, 1, 10, 100, 1000 mg/kg by artificial soil test for 14 d and 28 d. Data are expressed as mean ± SEM (n P 3, n: the samples of replicate). Statistical significance versus control group: ⁄p < 0.05, ⁄⁄ p < 0.01, ⁄⁄⁄p < 0.001.
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0.005%, at the ratio of 10–20 g/hole and 5–20 kg/Ha (Giraudoux et al., 2006). Therefore, earthworms are potential non-target species that may be subject to primary poisoning following application of bromadiolone. 3.2. Effects of bromadiolone on earthworm weight Based on the LC50 of soil toxicity tests, earthworms were exposed to bromadiolone at sublethal concentrations of 0.1– 20 mg/kg in artificial soil for 2–42 days. As shown in Fig. 2, the bodyweight of earthworms in control groups was increased after 14 days. At 2-day and 7-day, compared with control groups, significant decrease in weight of earthworms was observed in all treatment groups except for 10 mg/kg. At 14-day and 28-day, the weight change rates of earthworms in groups exposed to 10 or 20 mg/kg were lower than that of control. After exposure for 42 days, the growth of earthworms in all treatment groups was suppressed in a dose-dependent manner. These data indicated that exposure to bromadiolone at sublethal concentrations inhibited earthworm growth. Depletions of glycogen and lipid contents, and decrease in protein content, which were followed by a reduction in growth, have been observed in earthworm or other invertebrates exposed to toxins (Ribeiro et al., 2001; Xu et al., 2015). In this study, the adverse influence may also result from a decrease in these energy reserves in earthworm response to bromadiolone exposure.
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doses of 0.1–20 mg/kg in artificial soil for 2–42 days. As shown in Fig. 3A, in general, SOD activity was slightly up-regulated in earthworms treated with the rodenticide. But the SOD activity was only significantly increased in groups exposed to 1 mg/kg for 2 days and 14 days, or 10 mg/kg for 42 days, compared to the control. The similar pattern of changes in CAT activity was also observed. Although a slight increase in CAT activity was generally induced by bromadiolone exposure from 7 to 42 days, the statistically significant induction of CAT activity was only observed in animals exposed
3.3. Effects of bromadiolone on antioxidant enzymes and lipid peroxidation Environmental contaminants are major hazardous factors to the survival of organisms by eliciting excessive free radicals, such as reactive oxygen species (ROS), which may cause oxidative damage to biological macromolecules, like DNA, protein and lipid. To counteract of ROS, organisms have evolved defensive system which contains various antioxidant enzymes. Normally, a dynamic balance exists between ROS levels and the antioxidant enzyme contents, while excessive ROS production that exceeds the antioxidant capacity causes oxidative stress (Ziech et al., 2010). Superoxide dismutase (SOD) and catalase (CAT), are the most important members of the first antioxidative defense line to scavenge ROS, are well-described biomarkers to determine the oxidative stress profile in the organisms (Wang et al., 2013; Yang et al., 2014; Zhang et al., 2012). In this study, we examined SOD and CAT activities in the earthworms exposed to bromadiolone at
Fig. 2. Weight change rate of earthworms exposed to bromadiolone. Data are expressed as mean ± SEM (n P 3, n: the samples of replicate). Statistical significance versus control group: ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.
Fig. 3. The effects of bromadiolone on antioxidant enzymes and lipid peroxidation in earthworm. (A) SOD activity; (B) CAT activity; (C) MDA content. Data are expressed as mean ± SEM (n P 3, n: the samples of replicate). Statistical significance versus control group: ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.
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to bromadiolone at 20 mg/kg for 14 days, or 0.1 mg/kg and 1 mg/kg for 28 days (Fig. 3B). Malondialdehyde (MDA) is also a commonly used molecular marker indicating the damage of lipid peroxidation caused by redundant free radicals (Wang et al., 2013; Zhang et al., 2012). In the present study, we investigated the changes in MDA content of earthworms treated with bromadiolone. As seen in Fig. 3C, MDA content had a rapid response to the rodenticide treatment. After 2 days, MDA content was significantly greater in the rodenticide-treated groups compared to the control, particularly in the presence of 1, 10 and 20 mg/kg bromadiolone. With increasing exposure duration, the induction of MDA content was more significant. At 42-day, MDA contents in all bromadiolone-exposed groups were extremely higher than the control at 0.1 mg/kg (p < 0.01) and 1–20 mg/kg (p < 0.001). As the important enzymes in the antioxidant system of organisms, SOD and CAT eliminate generated O 2 and H2O2, respectively, and protect cells from the damage during the formation of ROS. A number of studies have shown that the alterations in cellular activities of SOD and CAT were considered as the early biomarkers for determining the toxic effects of pollutants on ecosystems (Wang et al., 2013; Xu et al., 2013; Yang et al., 2014; Zhang et al., 2012). However, in this study, the changes in activities of SOD and CAT were slight but not significant in most of bromadiolone-treated groups. The results showed that the induction of MDA content in earthworm was more dominant than that of SOD and CAT activity. Lipid peroxidation appears to be more sensitive parameter in evaluating the level of oxidative stress in the earthworms in response to bromadiolone exposure. In agreement with our observations, it has been reported that exposure to bromadiolone resulted in increasing levels of lipid peroxidation, but no change in antioxidant capacity, in the tissues of Japanese quails (Ondracek et al., 2012). Lipid peroxidation damages the cell membrane and leads to destruction of membrane lipids (Mylonas and Kouretas, 1999). In addition, as the main end-product of lipid peroxidation, MDA can react with deoxyadenosine and deoxyguanosine in DNA, forming DNA adducts that may cause mutagenesis and carcinogenesis (Bartsch and Nair, 2000). Our results indicated that exposure to bromadiolone resulted in serious oxidative damage and impairment of physiological functions, which subsequently led to a decreasing growth rate in earthworms. 3.4. Bioaccumulation of bromadiolone to earthworm The concentrations of bromadiolone in earthworm tissue and soil were determined. In soil samples, the initial concentrations of bromadiolone were 10 mg/kg and 20 mg/kg. Fig. 4A shows the degradation of bromadiolone during the period of 42 days of incubation. After 2 days, the concentrations of this rodenticide at 10 mg/kg and 20 mg/kg rapidly dropped to 52.1% and 55.6%, respectively. In the 20 mg/kg samples, the concentrations of bromadiolone decreased to approximately 35% at 7-day and reached the steady state levels throughout the exposure duration. However, the concentration of bromadiolone at 10 mg/kg steadily declined to 24.4% at the end of exposure. The elimination rate constant of bromadiolone in soil could be translated into a half-life (t1/2) of 5 days. As shown in Fig. 4B, the concentrations of bromadiolone in earthworm tissue increased from 2 to 14 days. The rapid uptake of bromadiolone was observed, and on the first sample point (2-day), the concentrations of bromadiolone at 10 mg/kg and 20 mg/kg were 0.22 mg/kg and 0.36 mg/kg, respectively. At 14-day, the concentrations reached the maximum as 0.44 mg/kg in both 10 mg/kg and 20 mg/kg groups. After 28 days of exposure, the concentrations in earthworm declined. At 42-day, the concentrations of bromadiolone at 10 mg/kg and 20 mg/kg were
Fig. 4. Concentrations of bromadiolone in soil (A) and earthworm (B) samples. Data are expressed as mean ± SEM (n P 3, n: the samples of replicate).
0.19 mg/kg and 0.30 mg/kg, respectively. These data suggest that bromadiolone can be taken up by earthworms from soil quickly and its accumulation in earthworms reached equilibrium in 14 days. In this study, the relative accumulation of bromadiolone was presented as biota to soil accumulation factor (BSAF) (Fig. 5A). In order to compare with bioaccumulation of other chemicals in earthworm, the activities were normalized to lipid content of earthworm that was assumed as 1% (Diao et al., 2011), and organic carbon (OC) of soil that was determined as 11.07 ± 3.19 g/kg dry weight (Fig. 5B). The BSAF values of bromadiolone of 10 mg/kg groups were higher than those of 20 mg/kg groups during the whole uptake period (Fig. 5). This observation that BSAF values of bromadiolone decreased with increasing soil concentrations indicates that the bioaccumulation of bromadiolone is concentration dependent. Similar trend was reported for perfluoroalkyl substances (PFASs) in earthworm (Zhao et al., 2013). In addition, the decreasing BSAF values with increasing concentration of chemicals in sediment were also found in various organisms, such as chlorinated dibenzo-p-dioxin and dibenzofuran congeners (PCDD/Fs) in dungeness crab (Cretney and Yunker, 2000), 3,4,30 ,40 -tetrachlorobiphenyl (TCBP) in benthic invertebrates (Leppanen et al., 2003), as well as polychlorinated biphenyls (PCBs) in oligochaetes (Sormunen et al., 2008). This phenomenon could be explained by the saturation of binding sites in biota available for adsorption and the resistant desorption of hydrophobic organic compounds at high soil/sediment concentrations. The fact that bromadiolone in soil is bioaccumulative to earthworms suggests that contaminated earthworms are a potential source of secondary exposure of non-target birds and invertebrates to this rodenticide.
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Fig. 5. Calculated biota to soil accumulation factors (BSAFs) for bromadiolone. Data are expressed as mean ± SEM (n P 3, n: the samples of replicate). The unit of BSAFs in panel A and panel B is kg wet weight/kg dry weight and kgoc/kglip, respectively.
4. Conclusions In the present study, the data of acute toxicity demonstrated that exposure to bromadiolone at soil concentration of 1 mg/kg is toxic to earthworms. Exposure to bromadiolone resulted in significant inhibition of growth rate and induction of lipid peroxidation in earthworm. Bromadiolone in soil is bioaccumulative to earthworms. The uptake of bromadiolone by earthworms is concentration dependent and the BSAFs of bromadiolone decreased as its concentration in soil increased. This work provided baseline data on the susceptibility of earthworms to bromadiolone, as a basis for risk assessment for secondary exposure. Acknowledgments This work was supported by the National High Technology Research & Development Program of China (2013AA065202), the National Natural Science Foundation of China (Nos. 21377113, 21307107, 21427815) and Zhejiang Provincial Natural Science Foundation of China (LR15B070001). Reference Bartsch, H., Nair, J., 2000. Ultrasensitive and specific detection methods for exocyclic DNA adducts: markers for lipid peroxidation and oxidative stress. Toxicology 153, 105–114. Cretney, W.J., Yunker, M.B., 2000. Concentration dependency of biota-sediment accumulation factors for chlorinated dibenzo-p-dioxins and dibenzofurans in
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Dungeness crab (Cancer magister) at marine pulp mill sites in British Columbia, Canada. Environ. Toxicol. Chem. 19, 3012–3023. Diao, J.L., Xu, P., Liu, D.H., Lu, Y.L., Zhou, Z.Q., 2011. Enantiomer-specific toxicity and bioaccumulation of alpha-cypermethrin to earthworm Eisenia fetida. J. Hazard. Mater. 192, 1072–1078. Gerlach, J., Florens, V., 2000. Considering molluscs in rodent eradication projects. Tentacle-TheNewsletter of the IUCN/SSC Mollusc Specialist Group 9, pp. 7–8. Giraudoux, P., Tremollieres, C., Barbier, B., Defaut, R., Rieffel, D., Bernard, N., Lucota, E., Berny, P., 2006. Persistence of bromadiolone anticoagulant rodenticide in Arvicola terrestris populations after field control. Environ. Res. 102, 291–298. Hayashi, Y., Engelmann, P., Foldbjerg, R., Szabo, M., Somogyi, I., Pollak, E., Molnar, L., Autrup, H., Sutherland, D.S., Scott-Fordsmand, J., Heckmann, L.H., 2012. Earthworms and humans in vitro: characterizing evolutionarily conserved stress and immune responses to silver nanoparticles. Environ. Sci. Technol. 46, 4166–4173. Hernandez-Moreno, D., de la Casa-Resino, I., Lopez-Beceiro, A., Fidalgo, L.E., Soler, F., Perez-Lopez, M., 2013. Secondary poisoning of non-target animals in an Ornithological Zoo in Galicia (NW Spain) with anticoagulant rodenticides: a case report. Vet. Med. 58, 553–559. Leppanen, M.T., Landrum, P.F., Kukkonen, J.V.K., Greenberg, M.S., Burton, G.A., Robinson, S.D., Gossiaux, D.C., 2003. Investigating the role of desorption on the bioavailability of sediment-associated 3,4,30 ,40 -tetrachlorobiphenyl in benthic invertebrates. Environ. Toxicol. Chem. 22, 2861–2871. Li, F.X., Liu, J., Jo, M., Curry, T.E., 2011. A role for nuclear factor interleukin-3 (NFIL3), a critical transcriptional repressor, in down-regulation of periovulatory gene expression. Mol. Endocrinol. 25, 445–459. Liu, H.G., Zhao, M.R., Zhang, C., Ma, Y., Liu, W.P., 2008. Enantioselective cytotoxicity of the insecticide bifenthrin on a human amnion epithelial (FL) cell line. Toxicology 253, 89–96. Liu, J., Yang, Y., Zhuang, S.L., Yang, Y., Li, F.X., Liu, W.P., 2011. Enantioselective endocrine-disrupting effects of bifenthrin on hormone synthesis in rat ovarian cells. Toxicology 290, 42–49. Liu, J., Zhao, M., Zhuang, S., Yang, Y., Yang, Y., Liu, W., 2012. Low concentrations of o, p’-DDT inhibit gene expression and prostaglandin synthesis by estrogen receptor-independent mechanism in rat ovarian cells. PLoS ONE 7, e49916. Loof, T.G., Schmidt, O., Herwald, H., Theopold, U., 2011. Coagulation systems of invertebrates and vertebrates and their roles in innate immunity: the same side of two coins? J. Innate Immun. 3, 34–40. Mylonas, C., Kouretas, D., 1999. Lipid peroxidation and tissue damage. In Vivo 13, 295–309. Niu, L.L., Xu, C., Yao, Y.J., Liu, K., Yang, F.X., Tang, M.L., Liu, W.P., 2013. Status, influences and risk assessment of hexachlorocyclohexanes in agricultural soils across China. Environ. Sci. Technol. 47, 12140–12147. Niu, L.L., Xu, C., Xu, Y., Zhang, C.L., Liu, W.P., 2014. Hexachlorocyclohexanes in tree bark across Chinese agricultural regions: spatial distribution and enantiomeric signatures. Environ. Sci. Technol. 48, 12031–12038. OECD, 1984. OECD guideline 207. www.oecd.org/chemicalsafety/risk-assessment/ 1948293 pdf. Ogilvie, S.C., Pierce, R.J., Wright, G.R.G., Booth, L.H., Eason, C.T., 1997. Brodifacoum residue analysis in water, soil, invertebrates, and birds after rat eradication on Lady Alice Island. New Zealand J. Ecol. 21, 195–197. Ondracek, K., Bandouchova, H., Damkova, V., Hilscherova, K., Kral, J., Osickova, J., Mlcakova, V., Pohanka, M., Skochova, H., Vitula, F., Treml, F., Pikula, J., 2012. Risk of combined exposure of birds to cyanobacterial biomass containing microcystins, acetylcholinesterase inhibitor and anticoagulant. Neuroendocrinol. Lett. 33, 155–160. Pimentel, D., Zuniga, R., Morrison, D., 2005. Update on the environmental and economic costs associated with alien-invasive species in the United States. Ecol. Econ. 52, 273–288. Ribeiro, S., Sousa, J.P., Nogueira, A.J.A., Soares, A.M.V.M., 2001. Effect of endosulfan and parathion on energy reserves and physiological parameters of the terrestrial isopod Porcellio dilatatus. Ecotoxicol. Environ. Saf. 49, 131–138. Sage, M., Coeurdassier, M., Defaut, R., Lucot, E., Barbier, B., Rieffel, D., Berny, P., Giraudoux, P., 2007. How environment and vole behaviour may impact rodenticide bromadiolone persistence in wheat baits after field controls of Arvicola terrestris? Environ. Pollut. 148, 372–379. Sage, M., Caeurdassier, M., Defaut, R., Gimbert, F., Berny, P., Giraudoux, P., 2008. Kinetics of bromadiolone in rodent populations and implications for predators after field control of the water vole, Arvicola terrestris. Sci. Total Environ. 407, 211–222. Shore, R.F., Birks, J.D.S., Afsar, A., Wienburg, C.L., Kitchener, A.C., 2003. Spatial and temporal analysis of second-generation anticoagulant rodenticide residues in polecats (Mustela putorius) from throughout their range in Britain, 1992–1999. Environ. Pollut. 122, 183–193. Sormunen, A.J., Leppanen, M.T., Kukkonen, J.V.K., 2008. Influence of sediment ingestion and exposure concentration on the bioavailable fraction of sedimentassociated tetrachlorobiphenyl in oligochaetes. Environ. Toxicol. Chem. 27, 854–863. Thomas, P.J., Mineau, P., Shore, R.F., Champoux, L., Martin, P.A., Wilson, L.K., Fitzgerald, G., Elliott, J.E., 2011. Second generation anticoagulant rodenticides in predatory birds: probabilistic characterisation of toxic liver concentrations and implications for predatory bird populations in Canada. Environ. Int. 37, 914– 920. Wang, L.M., Liu, W., Yang, C.X., Pan, Z.Y., Gan, J.Y., Xu, C., Zhao, M.R., Schlenk, D., 2007. Enantioselectivity in estrogenic potential and uptake of bifenthrin. Environ. Sci. Technol. 41, 6124–6128.
256
J. Liu et al. / Chemosphere 135 (2015) 250–256
Wang, C., Li, Z.Y., Zhang, Q., Zhao, M.R., Liu, W.P., 2013. Enantioselective induction of cytotoxicity by o, p0 -DDD in PC12 cells: implications of chirality in risk assessment of POPs metabolites. Environ. Sci. Technol. 47, 3909–3917. Wen, Y.Z., Yuan, Y.L., Chen, H., Xu, D.M., Lin, K.D., Liu, W.P., 2010. Effect of chitosan on the enantioselective bioavailability of the herbicide dichlorprop to Chlorella pyrenoidosa. Environ. Sci. Technol. 44, 4981–4987. Xu, D.M., Li, C.D., Wen, Y.Z., Liu, W.P., 2013. Antioxidant defense system responses and DNA damage of earthworms exposed to perfluorooctane sulfonate (PFOS). Environ. Pollut. 174, 121–127. Xu, X.B., Shi, Y.J., Lu, Y.L., Zheng, X.Q., Ritchie, R.J., 2015. Growth inhibition and altered gene transcript levels in earthworms (Eisenia fetida) exposed to 2,20 ,4,40 tetrabromodiphenyl ether. Arch. Environ. Contam. Toxicol. Yang, Y., Ma, H.H., Zhou, J.H., Liu, J., Liu, W.P., 2014. Joint toxicity of permethrin and cypermethrin at sublethal concentrations to the embryo-larval zebrafish. Chemosphere 96, 146–154. Yao, Y.J., Shen, R., Pennell, K.G., Suuberg, E.M., 2011. Comparison of the Johnson– Ettinger vapor intrusion screening model predictions with full threedimensional model results. Environ. Sci. Technol. 45, 2227–2235. Yao, Y.J., Shen, R., Pennell, K.G., Suuberg, E.M., 2013a. A review of vapor intrusion models. Environ. Sci. Technol. 47, 2457–2470. Yao, Y.J., Shen, R., Pennell, K.G., Suuberg, E.M., 2013b. Examination of the US EPA’s vapor intrusion database based on models. Environ. Sci. Technol. 47, 1425– 1433. Yao, Y.J., Shen, R., Pennell, K.G., Suuberg, E.M., 2013c. Examination of the influence of environmental factors on contaminant vapor concentration attenuation factors using the U.S. EPA’s vapor intrusion database. Environ. Sci. Technol. 47, 906–913.
Yao, Y.J., Yang, F.X., Suuberg, E.M., Provoost, J., Liu, W.P., 2014. Estimation of contaminant subslab concentration in petroleum vapor intrusion. J. Hazard. Mater. 279, 336–347. Yao, Y.J., Su, Y., Wu, Y., Liu, W.P., He, R., 2015. An analytical model for estimating the reduction of methane emission through landfill cover soils by methane oxidation. J. Hazard. Mater. 283, 871–879. Zeng, J.P., Zeng, C., Ouyang, F., Liu, X.P., Ge, F., 2014. Status Quo, loss and trend of forest bio-disasters from 2000 to 2010 in China. Biol. Disaster Sci. 37, 7–12. Zhang, A.P., Liu, W.P., Yuan, H.J., Zhou, S.S., Su, Y.S., Li, Y.F., 2011. Spatial distribution of hexachlorocyclohexanes in agricultural soils in Zhejiang Province, China, and correlations with elevation and temperature. Environ. Sci. Technol. 45, 6303– 6308. Zhang, Q., Zhao, M.R., Qian, H.F., Lu, T., Zhang, Q., Liu, W.P., 2012. Enantioselective damage of diclofop acid mediated by oxidative stress and acetyl-CoA carboxylase in nontarget plant Arabidopsis thaliana. Environ. Sci. Technol. 46, 8405–8412. Zhao, S.Y., Zhu, L.Y., Liu, L., Liu, Z.T., Zhang, Y.H., 2013. Bioaccumulation of perfluoroalkyl carboxylates (PFCAs) and perfluoroalkane sulfonates (PFSAs) by earthworms (Eisenia fetida) in soil. Environ. Pollut. 179, 45–52. Zhao, M., Zhang, Y., Zhuang, S., Zhang, Q., Lu, C., Liu, W., 2014. Disruption of the hormonal network and the enantioselectivity of bifenthrin in trophoblast: maternal–fetal health risk of chiral pesticides. Environ. Sci. Technol. 48, 8109– 8116. Ziech, D., Franco, R., Georgakilas, A.G., Georgakila, S., Malamou-Mitsi, V., Schoneveld, O., Pappa, A., Panayiotidis, M.I., 2010. The role of reactive oxygen species and oxidative stress in environmental carcinogenesis and biomarker development. Chem. Biol. Interact. 188, 334–339.
