Arch Environ Contam Toxicol DOI 10.1007/s00244-014-0046-2
Oxidative Stress and Genotoxicity of the Ionic Liquid 1-Octyl-3-Methylimidazolium Bromide in Zebrafish (Danio rerio) Zhongkun Du • Lusheng Zhu • Miao Dong Jinhua Wang • Jun Wang • Hui Xie • Tong Liu • Yingying Guo
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Received: 14 November 2013 / Accepted: 9 May 2014 Springer Science+Business Media New York 2014
Abstract Ionic liquids (ILs) have a great reputation due to their negligible volatility, designability, good stability, and ability to be recycled. They are considered to be ‘‘green’’ solvents and have great promise in many fields. In recent years, the toxicities of ILs have garnered increasing attention as reported by a number of studies. However, previous studies have primarily focused on their lethal toxicities, and data were limited on their toxic effects at nonlethal doses. We performed a study on the toxic effects of 1-octyl-3-methylimidazolium bromide ([Omim]Br) on zebrafish. During a 28-day period, male and female zebrafish were separately exposed to sequential concentrations (0, 5, 10, 20, and 40 mg/L) of [Omim]Br. Fishes were sampled after 7, 14, 21, and 28 days of exposure, and reactive oxygen species (ROS) levels, activities of antioxidant enzymes (superoxide dismutase and catalase), lipid peroxidation (LPO), and DNA damage in fish livers were measured. ROS, LPO, and DNA damage were all induced
Electronic supplementary material The online version of this article (doi:10.1007/s00244-014-0046-2) contains supplementary material, which is available to authorized users. Z. Du L. Zhu (&) M. Dong J. Wang (&) J. Wang H. Xie T. Liu Y. Guo National Engineering Laboratory for Efficient Use of Soil and Fertilizer Resources, Key Laboratory of Agricultural Environment in Universities of Shandong, College of Resources and Environment, Shandong Agricultural University, 61 Daizong Road, Taian 271018, People’s Republic of China e-mail: [email protected]; [email protected] J. Wang e-mail: [email protected] Z. Du School of the Environment, Nanjing University, Nanjing 210023, People’s Republic of China
by the ionic liquid, and antioxidant enzyme activities increased at the beginning and then decreased. These phenomena demonstrate that [Omim]Br can induce oxidative stress and DNA damage in zebrafish.
Ionic liquids (ILs) are salts with a melting point lower than 100 C and are composed entirely of ions (Wilkes 2002). Due to their negligible vapor pressure, good stability, and ability to be recycled, ILs are considered to be environmentally safe ‘‘green’’ solvents and will gradually replace traditional organic solvents in many physical and chemical processes (Matsumoto et al. 2004a). The physical properties of ILs can be tailored by changing their ionic compositions, even by changing the length and branching of the alkyl chain on the cations (Huddleston et al.2001). Therefore, ILs can be synthesized specifically for a certain application. Due to these advantages, ILs have attracted increasing interest in many fields. The application of these liquids in reaction media for organic synthesis, catalysis, or biocatalysis has been well documented (Pham et al. 2010) and has produced good results with the potential for future use. However, ILs cannot be regarded as absolutely green based only on their negligible vapor pressure because their water solubilities cannot be ignored. Large-scale use of ILs in the future may lead to their release into aquatic and soil environments, and their ecotoxicological evaluation is highly relevant. Because the use of ILs is likely to increase, their toxicities have received increasing attention. Matsumoto et al. (2004a, b) reported the toxicity of ILs in bacteria and found that IL toxicity was positively correlated with alkyl chain length. An investigation into the acute toxicity of ILs in zebrafish found that the cation of ILs plays an important role in acute toxicity (Pretti et al.
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2006, 2009). According to (Matsumoto et al. 2004a, b), ILs have antibacterial activity that can decrease the growth rate and productivity of microbes. Toxicities of ILs on other organisms, including earthworms, mice, etc., have also been reported. Although these studies have provided (eco) toxicological assessments of ILs, there is still a lack of knowledge about the toxicities of ILs; data on genotoxicity and bioaccumulation are still insufficient. Until now, there have been almost no data on the genotoxicity of ILs with the exception of Docherty et al. (2006), Zhang et al. (2011), who made the first evaluation of IL genotoxicity and both found that imidazolum ILs show mutagenicity. Imidazolium ILs are the most widely used ILs and have been widely investigated. An imidazolium IL, 1-octyl-3methylimidazolium bromide ([Omim]Br), can be obtained easily and has been widely involved in toxicity studies. Acute toxicity of this IL on goldfish has been reported by (Li et al. 2012); an acute toxicity test showed that the 24-hour LC50 of [Omim]Br for goldfish was 244 mg/L. In the present study, we measured the effects of [Omim]Br on catalase (CAT), superoxide dismutase (SOD), reactive oxygen species (ROS), lipid peroxidation (LPO), and DNA damage in zebrafish during a 28-day period.
[70 %, and pH ranging from 7.4 to 8.1. Fish were exposed to different concentrations of [Omim]Br (0, 5, 10, 20, and 40 mg/L). Males and females were then separated for IL exposure. There were 120 fish/tank and three replicate tanks/group. There was no mortality in both stock cultures and the treatments. The exposure system was semistatic, and [Omim]Br was directly dissolved in water. Half of the water was refreshed every 2 days to clear excreta away and to preserve the IL concentration. The stability and low biodegradation of the imidazolium ILs were documented by Docherty et al. (2006). Degradation experiments have already indicated a high compound stability, which was also confirmed in our laboratory by measuring the concentration of the compound in the exposure tanks at the beginning and end of the experiment. Fishes were fed daily with commercially available dry flakes, except on the day before the test, to protect the fish from interference from feces during the assays. Fishes were sampled on days 7, 14, 21, and 28 of exposure. Fish liver was used as the target organ for toxicity tests, and tests were immediately performed after sampling. ROS Determination
[Omim]Br (purity 99 %, CAS no. 61545-99-1) was obtained from the Chengjie Chemical Co. Ltd. (Shanghai, China), and 20 ,70 -dichlorofluorescin-diacetate (DCFH-DA) was obtained from the Beyotime Institute of Biotechnology (Jiangsu, China). All other chemicals were of analytical purity and purchased from either Sigma Chemical (St. Louis, Missouri, USA) or Beijing Chemical (Beijing, China). Adult male (0.27 ± 0.05 g mean body weight and 2.45 ± 0.01 cm length) and female (0.31 ± 0.07 g mean body weight and 2.59 ± 0.03 cm length) zebrafish (Danio rerio) were purchased from an aquarium (Qixin Aquarium, Taian, China).
Quantitative determination of ROS production was measured as described in the DCFH-DA method using a Diagnostic Reagent Kit. Fish livers were homogenized in 100 mM of ice-cold potassium phosphate buffer (pH 7.4). Subsequently the homogenates were centrifuged at 1,000g for 10 min and the supernatants recentrifuged at 20,000g for 20 min. The mitochondrial protein was then prepared after the pellet was resuspended with ice-cold potassium phosphate buffer (pH 7.4). All of the these manipulations were performed at 4 C. The ROS level was then measured according to the methods of Lawler et al. (2003). The mitochondrial protein supernatants were mixed with 2 lM DCFH-DA and then incubated in a water bath at 37 C for 30 min. Fluorescence intensities were detected at a wavelength of 538 nm with a fluorescent spectrophotometer. The results are expressed as fluorescence intensity per milligram of total protein.
Fish Care and Exposure
Determination of Enzyme Activities
The results of the present study followed the Guiding Principles outlined in the Use of Animals in Toxicology adopted by the Society of Toxicology in 1989. The fish were kept in the laboratory for 2 weeks before the experiments to allow them to adapt to the laboratory conditions. The fishes’ living conditions strictly followed that of Diekmann et al. (2004): a 12:12 h light-to-dark regime, 26 ± 1 C temperature, tap water (aerated and basked for 2 days to dechlorinate before use), oxygen saturation
Fish livers were homogenized in 50 mM of ice-cold potassium phosphate buffer (1:8 w/v [pH 7.0]). The homogenate was then centrifuged at 10,600g at 4 C for 10 min. The supernatant was used immediately for the determination of enzyme activity and protein concentration at room temperature (25 C). CAT activity was determined from the rate of change of ultraviolet absorbance (Xu et al. 1997). CAT activity can be measured by the decrease in ultraviolet absorption over
Materials and Methods Chemicals and Animals
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time due to the degradation of H2O2 by CAT in the sample. One unit (U) of CAT activity was defined as the enzyme quantity required to cause half-degradation of the H2O2 in the sample after 100 s at 25 C normalized by protein concentration. The results are expressed as unit per mg total protein. SOD activity was assayed by its ability to inhibit the photochemical decrease of nitroblue tetrazolium chloride (NBT) (Sun et al. 1988). U of SOD activity was defined as the quantity of enzyme that caused 50 % inhibition of the NBT photoreduction rate. The results are expressed as unit per milligram of total protein.
for the data analyses. The data were subjected to one-way analysis of variance followed by least significant difference test at the p \ 0.05 level of significance. All values are presented as mean ± SD (n = 3).
Results Data are shown with statistical analysis in relation to [Omim]Br treatment concentration (Figs. 1, 2, 3, 4, 5). Statistical analysis of sex discrepancies (Supplemental [S] Figs. S1 to S5) and statistical analysis in relation to the treatment time (Figs. S6 to S10) are given.
Determination of LPO ROS The homogenate used for the LPO test was the same as that used in the enzyme test. Because malondialdehyde (MDA) is the final product of LPO, we measured MDA content as an indicator of LPO. The measurement of MDA was made using the Diagnostic Reagent Kit according to the thiobarbituric acid (TBA) method (Zhang et al. 2013). The assay was monitored for the appearance of the conjugated complex of TBA and MDA at 532 nm. The concentration of MDA is reported as nanomoles of MDA per milligram of total protein.
A liver cell suspension from zebrafish was prepared according to Shao et al. (2012). The resulting cell suspensions were processed by single cell gel electrophoresis (SCGE), which was also performed according to the method described by Shao et al. (2012). The Comet Assay Software Project (CASP 1.2.2, Biolaunching Technologies Co., Ltd., Beijing, China) software program was used to analyze the comet photographs collected with a fluorescent microscope (Olympus BX71). Finally, the olive tail moment (OTM), which is the product of the distance between the center of the head, the center of the tail, and the total DNA percentage in the tail, was used to quantify the extent of DNA damage (Olive et al. 1990; Song et al. 2009).
The effects of [Omim]Br on ROS levels in zebrafish are shown in Figure 1. ROS levels exhibited similar patterns in both male and female fish. [Omim]Br exposure resulted in ROS accumulation, and greater concentrations of [Omim]Br caused greater ROS accumulation. The effects in male fish were as follows: (1) 5 mg/L of [Omin]Br had no effect on ROS level at all sampling times; (2) 10 mg/L of [Omin]Br also caused no change in ROS level on days 7 and 14, but it observably induced an ROS increase on days 21 and 28; (3) in the 20 and 40 mg/L groups, ROS level was steadily greater than that in control; (4) in the 10 and 20 mg/L groups, greater induction of ROS appeared on day 21 (143 % and 157 % of the control, respectively) and then slightly decreased on day 28 day but were still greater than the control (123 % and 133 % of control, respectively); and (5) in the 40 mg/L group, a greater induction of ROS occurred on day 28 (171 % of control). The effects in female fish were as follows: (1) 5 mg/L of [Omin]Br induced ROS level on the days 7 and 21; (2) 10 mg/L of [Omin]Br induced ROS level on days 14 and 28; (3) 20 and 40 mg/L [Omin]Br increased ROS level at all sampling times; (4) in 5 the mg/L group, greater ROS promotion occurred on day 21; and (5) in the 10, 20, and 40 mg/L groups, the greater increase of ROS appeared on the day 14. Regarding the discrepancies in ROS variation between male and female fish (Fig. S1) on day 7 day, the ROS level in male fish was lower than that in female fish when exposed to 5 mg/L [Omin]Br; on day 14 in the 10, 20, and 40 mg/L groups, ROS levels in male fish were lower than those in female fish; on day 21 in the 10 and 20 mg/L groups, ROS levels in male fish were greater than those in female fish.
Statistics
Enzyme Activities
The statistical package for social sciences (SPSS) program (standard version 16.0, SPSS, IBM, Chicago, IL) was used
The effects of [Omim]Br on CAT activities in zebrafish are shown in Figure 2. Generally speaking, CAT activity under
Protein Content Determination Protein content was determined by the Sigma Bradford method (Bradford 1976) using bovine serum albumin as the standard. DNA Damage Determination
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Fig. 1 ROS level in livers of zebrafish exposed to 4 concentrations of [Omim]Br for 28 days. Values are presented as mean ± SD (n = 3). Different letters above the columns indicate significant differences at the p \ 0.05 level between treatments. Pr = protein; fluo = fluorescence
Fig. 2 CAT activities in livers of zebrafish exposed to 4 concentrations of [Omim]Br for 28 days. Values are presented as mean ± SD (n = 3). Different letters above the columns indicate significant differences at p \ 0.05 level between treatments. Pr = protein
Fig. 3 SOD activities in livers of zebrafish exposed to 4 concentrations of [Omim]Br for 28 days. Values are presented as mean ± SD (n = 3). Different letters above the columns indicate significant differences at p \ 0.05 level between treatments. Pr = protein
[Omim]Br exposure showed a time-dependent trend, first induced and then inhibited, in both male and female fish; there is also another trend that the greater [Omim]Br concentration, the earlier the CAT activity turned to being inhibited from being induced in both the male and female groups. For male fish, 5 mg/L of [Omim]Br induced CAT
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activity on day 14, inhibited CAT activity on day 28, and showed no significant effect on CAT activity on days 7 and 21. In the 10 and 20 mg/L groups, CAT activity was induced on day 7 and inhibited on days 21 and 28, whereas it was not statistically different from the control on day 14. In the 40 mg/L group, CAT activity was induced only on
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Fig. 4 LPO level in livers of zebrafish exposed to 4 concentrations of [Omim]Br for 28 days. Values are presented as mean ± SD (n = 3). Different letters above the columns indicate significant differences at p \ 0.05 level between treatments. Pr = protein
Fig. 5 DNA damage level in livers of zebrafish exposed to 4 concentrations of [Omim]Br for 28 days. Values are presented as mean ± SD (n = 3). Different letters above the columns indicate significant differences at p \ 0.05 level between treatments
day 7; at the other sampling times it was inhibited. For females, 5 mg/L of [Omim]Br induced CAT activity on days 7 and 14, inhibited CAT activity on days 21 and 28. In the 10 mg/L group, CAT activity was induced on day 7 and inhibited on day 21, whereas it was not statistically different from the control on days 14 and 28. In the 20 mg/L and 40 mg/L groups, CAT activities were induced on day 7; at the other sampling times it was inhibited. No regularity was observed in the discrepancies between male and female groups (Fig. S2). In the 5 mg/L groups, CAT activity in male fish was lower than that in female fish on days 7 and 14, and then turns toward being greater than that in females on days 21 and 28. This maybe indicates that female fish are more sensitive in CAT activity than male fish. The effects of [Omim]Br on SOD activities in zebrafish are shown in Figure 3. In general, high concentrations of [Omim]Br inhibited SOD activities. Low concentrations of [Omim]Br induced SOD activities before day 7 but inhibited them after day 14. In detail, for male fish, no statistical changes on SOD activities were found in the
5 mg/L group, whereas they were slightly induced in the 10 mg/L group compared with the control on day 7; then SOD activities in both these two groups turned toward being inhibited on the other sampling days. SOD activities in groups exposed to 20 and 40 mg/L of [Omim]Br were inhibited from the beginning to the end of the exposure. For female fish, SOD activities in the 5, 10, and 20 mg/L groups were increased on day 7 and turned to be lower than the control level after 14 days. SOD activity in the 40 mg/ L group was the same level as control on the day 7, then it turned toward being lower at the other sampling times. For differences between male and female groups (Fig. S3), except for in the 40 mg/L group on day 21, male SOD activity was greater than female SOD activity. All other statistical discrepancies (20 and 40 mg/L groups on day 7; 5, 10, and 20 mg/L groups on day 14; 10 mg/L groups on day 21; and 10, 20, and 40 mg/L groups on day 28) showed that SOD activities in male fish were lower than those in male fish under [Omim]Br exposure, which indicates that SOD in male fish was more easily inhibited by [Omim]Br.
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Fig. 6 Comet assay. a Structure of a comet. b Control zebrafish liver cell in comet assay. c–f Different DNA damage extents of female zebrafish exposed to 5, 10, 20, and 40 mg/L of [Omim]Br for 28 days, respectively
Lipid Peroxidation LPO was observed in fishes after exposure to [Omim]Br. As presented in Figure 4, MDA content was primarily enhanced by [Omim]Br in both male and female fish with the highest increase of MDA appearing on day 7 (40 mg/L male group = 230.0 % of the control), then the enhancements gradually decreased. In detail, for male fish, MDA content in all [Omim]Br treatment groups was increased on day 7, and enhancements gradually decreased; on day 28, MDA content in the 5, 10, and 20 mg/L groups was still greater than the control, whereas the MDA content in the 40 mg/L groups decreased to control level. For female fish, 5 mg/L of [Omim]Br had no effect on MDA content on days 7 and 14, but it was induced on days 21 and 28; in the 10 mg/L group, MDA content was only induced on day 21; in the 20 and 40 mg/L groups, MDA content was induced on days 7, 14, and 21 and then decreased to control level on day 28. Regarding discrepancies between male and female fish (Fig. S4), except for day 21 in the 10 mg/L male group, the MDA level was lower than the female MDA level. All other statistical discrepancies (all [Omim]Br treatment groups on days 7 and day 14; the 20 and 40 mg/L groups on day 21; and the 10 and 20 mg/L groups on day 28) showed the reverse result, that is, the LPO level in males was greater than that in females. DNA Damage DNA damage was measured with SCGE. OTM values were used to indicate damage intensities. The DNA damage results caused by [Omim]Br in zebrafish are shown in Figure 5. OTM values in all IL-treated groups were greater
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than in the controls from the beginning to the end of the exposure, which indicates that all concentrations of [Omim]Br in the present study can induce DNA damage in liver cells of zebrafish. In general, greater IL concentrations result in greater levels of DNA damage, and damage is aggravated with increasing exposure time. In 5 mg/L group, the level of DNA damage in male fish was greater than that in female on days 14, 21, and 28; no other statistical discrepancy was observed between male and female fish (Fig. S5). Parts of comet assay photos are shown in Fig. 6. A comet has a head and a tail, but normal cell shows no tail in comet assay; with greater DNA damage, the cell will show a larger tail and a smaller head. The greater concentration of [Omim]Br exposure, the greater the DNA damage of zebrafish liver cell observed.
Discussion Due to their low vapor pressure and many other good properties, ILs have been heavily discussed in green chemistry. Industrial use of ILs is imperative, but are ILs always green? Some toxicological studies of ILs reported a negative answer (Bernot et al. 2005a, b; Cho et al. 2008a, b, c; Yu et al. 2008, 2009). Different ILs have different levels of toxicity, and a given IL may produce different acute toxicity intensities in different organisms. Existing data are mainly about toxic levels of different ILs, but the toxic mechanism of ILs is not yet fully understood. According to Modelli et al. (2008), imidazolium-based ILs are not easily degraded under environmentally realistic conditions; thus organisms may be exposed to them for a
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long time when ILs are released into the environment. Therefore, their chronic effects and toxic mechanisms should be studied further. The results of the present study investigated the effects of [Omim]Br on zebrafish during an exposure period of 28 days. Because the liver is responsible for metabolism and detoxification in animals, important processes pertaining to enzyme synthesis and reactions occur therein, and it is very sensitive to many pollutants (Moreno et al. 2005). Li et al. (2012) reported that the liver is one target organ of IL toxicity in goldfish. We used liver as the target organ to assess the toxic effects of ILs in zebrafish. Oxidative Stress In the present study, ROS were induced in fish by • [Omim]Br. ROS (including O•2 , OH , and H2O2) are generated in processes of normal cell metabolism. They play important roles in apoptosis and signal transmission, but excess ROS have negative effects such as DNA damage, LPO, protein oxidation, and aging (Bayr 2005). Previous studies have also indicated that excess ROS may lead to cardiovascular disease and hearing impairment (Michael et al. 2004; Junichi et al. 1998). In the present study, low concentrations (5 mg/L) of the IL [Omim]Br slightly enhanced the ROS content of zebrafish liver. With greater concentrations of this IL, ROS levels increased too much, which may lead to many health problems. In agreement with this result, we found that [Omim]PF6 can induce ROS in zebrafish liver (Du et al. 2012), and a study by Kumar et al. (2011) found that [C12mim]Br significantly increased • concentrations of O•2 , OH , and H2O2 in the green seaweed Ulva lactuca. These data may indicate a consistent effect of imidazolium ILs of inducing ROS and oxidative stress when they enter organisms. To gather more data on the oxidative stress observed, the response of the antioxidant defense system was studied. The antioxidant defense system includes many antioxidant enzymes that are responsible for eliminating excess ROS to avoid further diseases. CAT and SOD are two important components of the antioxidant defense system. They can decrease the concentrations of H2O2 and O•2 , thus effectively eliminating excess ROS and protecting the organism. They are reasonable markers of oxidative stress and have been well documented (Gomi et al. 1998; Pinho et al. 2005). CAT and SOD activities all showed similar responses to [Omim]Br exposure in the present study: an initial increase followed by a decrease. The increase in enzyme activities is due to an activation of the zebrafish self-defense system. The followed decrease in enzyme activities may be related to many factors. One assumption relates to the expression obstacle of their genes, but
research has yet to confirm this. ROS also inactivate antioxidant enzymes (Tabatabaie & Floyd 1994). Other studies have also discussed the effects of ILs on the antioxidant enzyme systems in different organisms. According to Li et al. (2012), 0.9 mg/L of [Omim]Br induced CAT, SOD, and glutathione peroxidase (GPx) activities in D. magna. In addition, the activities of CAT and SOD were induced, whereas GPx activity was enhanced, when adult goldfish were exposed to 122 mg/L of [Omim]Br for 72 h. Kumar et al. (2011) also reported that SOD activity in U. lactuca was enhanced when exposed to 0.04 mM of [C12mim]Br and was inhibited when exposed to 0.07 mM of [C12 mim]Br. According to Du et al. (2012), [Omim]PF6 also affected SOD and CAT activities in zebrafish, e.g., [Omim]PF6 showed more immediate inhibition of antioxidant enzyme activities at day 7, whereas [Omim]Br caused a transitory increase. The probable cause of this difference is that PF6- can be hydrolyzed to hydrogen fluoride, which can inhibit enzymes (Arning et al. 2008). Therefore, ILs that contain PF6- anion may be more harmful to organisms than those contain Br-. It has been well documented that excess ROS may cause LPO (oxidation of polyunsaturated fatty acids in lipids). LPO can damage biomembranes and can then affect normal cell metabolism and function. MDA is commonly used to indicate LPO because it LPO is the final product in the process (Hiroshi et al. 1979). In the present study, MDA in both male and female fish was induced by [Omim]Br. The highest inductive effect appeared on day 7. Unlike ROS, MDA was not persistently accumulated but gradually decreased after day 7 and practically reached the control level at the end of the 28-day exposure. The nonconformity between MDA and ROS variation seems unreasonable, and we did not perform any further research to determine the cause. However, as mentioned previously, [Omim]Br induced GPx activity, whereas it inhibited CAT and SOD in the study by Li et al. (2012), and it is known that GPx can restrain lipid oxidation. Therefore, we speculate that the same mechanism may exist in our study and that the effects of GPx may facilitate MDA removal. The slight distinction between male and female fish indicates that males may be slightly weaker than females in toxicity tests. The results from the MDA test indicate that the tested concentration of [Omim]Br can lead to LPO in zebrafish, but LPO can be repaired, so it is not a pathway of further [Omim]Br toxicity in zebrafish at related concentrations. Genotoxicity Genotoxicity of [Omim]Br was studied with the comet assay. Obvious comet tails were observed in [Omim]Brtreated groups, and the results indicated that [Omim]Br
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caused DNA damage in zebrafish liver. In the present study, the DNA damage content was positively correlated with IL concentrations, and damage levels increased with the extension of time. DNA damage in the 10, 20, and 40 mg/L groups was much greater than in the 5 mg/L groups after 14 days of exposure. DNA damage in the 5 mg/L groups remained at a steady level. To date, few studies have focused on the genotoxicity of ILs, but all have mentioned that ILs exhibited genotoxicity. Docherty and Zhang reported the mutagenicity of imidazolium-based ILs (Docherty et al. 2006; Zhang et al. 2011). Using comet assay, Kumar et al. (2011) found that [C12mim]Br induced DNA damage in U. lactuca. Our previous study also showed the genotoxicity of [Omim]PF6. These data show that imidazolium-based ILs are genotoxic. Because genotoxicity can lead to DNA lesions that trigger a cascade of biological consequences at the cellular, organ, whole organism, population, and community levels, we cannot regard ILs as ‘‘green’’ as they were once claimed to be. It is well known that oxidative stress plays an important role in DNA damage. Excess ROS is a major cause of oxidative DNA damage. In the present study, DNA damage showed a similar trend to ROS, i.e., they had positive correlations with [Omim]Br concentration and exposure time, so oxidative DNA damage was the major DNA lesion in this study. In addition, 5 mg/L of [Omim]Br exposure did not cause significant ROS accumulation in male fish, but obvious DNA damage was observed in this group; this indicates that oxidative damage may not be the only pathway of DNA damage. Other pathways, such as decreased repair capacity, may be involved. In contrast, DNA damage might affect the expression of antioxidant enzymes; ROS also inactivated antioxidant enzymes, and these two decreased the antioxidant capacity of fish liver and led to further ROS accumulation leading to formation of a vicious cycle. By reason of this, DNA damage and oxidative stress became more and more serious with time in the 10, 20, and 40 mg/L groups. Discrepancies Between Males and Females Lower SOD inhibition, LPO, and DNA-damage induction were found in female fish compared with male fish in the same exposure treatments. This phenomenon shows that female fish have greater ability to decrease oxidative damage. Similar results were found in other species such as flounder and even humans (Giorgio et al. 2001; Katja et al. 2001). This sex discrepancy might be regulated by hormones. It has been reported that estrogen can modulate the expression and function of antioxidant enzymes and confer protect effects against oxidative damage (Adam et al. 2000; Kerstin et al. 2003).
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Conclusion In conclusion, although imidazolium-based ILs have been reported to exhibit low toxicity in fish in acute toxicity studies, low-dose ILs can induce many problems in fish with longer exposure time, including oxidative stress and DNA damage, and this oxidative stress may be the major cause of DNA damage. LPO alteration was not serious enough under the stress of the tested concentrations. We also found that oxidative stress and genotoxicity are universal toxic effects of imidazolium-based ILs in organisms. In ILs, anion PF6inhibits enzymes more strongly than Br-. To decrease the potential risk of ILs, more work should be performed to determine their toxicities rather than guiding IL use. Due to the good stability of ILs, further studies should not focus on their acute toxicity: Chronic toxicity and their mechanisms of action require more attention. Considering the large amount of IL categories, more ILs should be involved in toxicity studies, and their structure-activity relationship must be explored. Acknowledgments The results of the present study was supported by Grants from the National Natural Science Foundation of China [Grants No. 21277083, 41071164, 41001152, and 21377075] and the Specialized Research Fund for the Doctoral Program of Higher Education [Grant No. 20113702110007].
References Adam MP, Pattie SG, Leighann S, Cynthia OH, Larissa Z, Gayle AB et al (2000) Protective effect of estrogens against oxidative damage to heart and skeletal muscle in vivo and in vitro. Proc Soc Exp Biol Med 223:59–66 Arning J, Stolte S, Boschen A, Stock F, Pitner WR, Welz-Biermann U et al (2008) Qualitative and quantitative structure activity relationships for the inhibitory effects of cationic head groups, functionalized side chains and anions of ionic liquids on acetylcholinesterase. Green Chem 10:47–58 Bayr H (2005) Reactive oxygen species. Crit Care Med 33:S498– S501 Bernot RJ, Brueseke MA, Evans-White MA, Lamberti GA (2005a) Acute and chronic toxicity of imidazolium-based ionic liquids on Daphnia magna. Environ Toxicol Chem 24:87–92 Bernot RJ, Kennedy EE, Lamberti GA (2005b) Effects of ionic liquids on the survival, movement, and feeding behavior of the freshwater snail, Physa acuta. Environ Toxicol Chem 24:1759–1765 Bradford MM (1976) A rapid sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 Cho CW, Jeon YC, Pham TPT, Vijayaraghavan K, Yun YS (2008a) The ecotoxicity of ionic liquids and traditional organic solvents on microalga Selenastrum capricornutum. Ecotoxicol Environ Saf 71:166–171 Cho CW, Pham TPT, Jeon YC, Min J, Jung HY, Lee DS et al (2008b) Microalgal photosynthetic activity measurement system for rapid toxicity assessment. Ecotoxicology 17:455–463
Arch Environ Contam Toxicol Cho CW, Pham TPT, Jeon YC, Yun YS (2008c) Influence of anions on the toxic effects of ionic liquids to a phytoplankton Selenastrum capricornutum. Green Chem 10:67–72 Diekmann M, Hultsch V, Nagel R (2004) On the relevance of genotoxicity for fish populations I: effects of a model genotoxicant on zebrafish (Danio rerio) in a complete life-cycle test. Aquat Toxicol 68:13–26 Docherty KM, Hebbeler SZ, Kulpa JC (2006) An assessment of ionic liquid mutagenicity using the Ames test. Green Chem 8:560–567 Du ZK, Zhu LS, Dong M, Wang JH, Wang J, Xie H et al (2012) Effects of the ionic liquid [Omim]PF6 on antioxidant enzyme systems, ROS and DNA damage in zebrafish (Danio rerio). Aquat Toxicol 124–125:91–93 Giorgio F, Patrizia M, Jacopo V, Silvia B, Stefania F, Cristina M P et al (2001) Age and sex influence on oxidative damage and functional status in human skeletal muscle. J Muscle Res Cell Motil 22:345–351 Gomi F, Matsuo M (1998) Effects of aging and food restriction on the antioxidant enzymes activity of rat livers. J Gerontol Ser A 53:B161–B167 Hiroshi O, Nobuko O, Kunio Y (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:158–351 Huddleston JG, Visser AE, Reichert WM, Willauer HD, Broker GA, Rogers RD (2001) Characterization and comparison of hydrophilic and hydrophobic room temperature ionic liquids incorporating the imidazolium cation. Green Chem 3:156–164 Jun-ichi A, Bradford CB (1998) Reactive oxygen species as mediators of signal transduction in cardiovascular disease. Trends cardiovasc med 8:59–64 Katja W, Gary WW, Wilhelm B, Cornelis JFVN, Angela K (2001) Sex-related responses to oxidative stress in primary cultured hepatocytes of European flounder (Platichthys flesus L.). Aquat Toxicol 52:143–155 Kerstin S, Simone R, Sven W, Oliver A, Christian G, Kerstin L et al (2003) Modulation of antioxidant enzyme expression and function by estrogen. Circ Res 93:170–177 Kumar M, Trivedi N, Reddy CRK, Jha B (2011) Toxic effects of imidazolium ionic liquids on the green seaweed Ulva lactuca: Oxidative stress and DNA damage. Chem Res Toxicol 24:1882–1890 Lawler JM, Song W, Demaree SR (2003) Hindlimb unloading increases oxidative stress and disrupts antioxidant capacity in skeletal muscle. Free Radic Biol Med 35:1–9 Li XY, Zeng SH, Dong XY, Ma JG, Wang JJ (2012) Acute toxicity and responses of antioxidant systems to 1-methyl-3-octylimidazolium bromide at different developmental stages of goldfish. Ecotoxicology 21:253–259 Matsumoto M, Mochiduki K, Fukunishi K, Kondo K (2004a) Extraction of organic acids using imidazolium-based ionic liquids and their toxicity to Lactobacillus rhamnosus. Sep Purif Technol 40:97–101 Matsumoto M, Mochiduki K, Kondo K (2004b) Toxicity of ionic liquids and organic solvents to lactic acid-producing bacteria. J Biosci Bioeng 98:344–347 Michael DS, Nadir A, Dipa J, Jake S, Sujatha T, Wayne SQ (2004) Age-related hearing loss and its association with reactive oxygen species and mitochondrial DNA damage. Acta Otolaryngol 552(Suppl):16–24
Modelli A, Sali A, Galletti P, Samorı` C (2008) Biodegradation of oxygenated and non-oxygenated imidazolium-based ionic liquids in soil. Chemosphere 73:1322–1327 Moreno I, Pichardo S, Jos A, Go´mez-Amores L, Mate A, Vazquez CM et al (2005) Antioxidant enzyme activity and lipid peroxidation in liver and kidney of rats exposed to microcystin-LR administered intraperitoneally. Toxicon 45:395–402 Olive PL, Banath JP, Durand RE (1990) Heterogeneity in radiationinduced DNA damage and repair in tumor cells measured using the comet assay. Radiat Res 122:86–94 Pham TPT, Cho CW, Yun YS (2010) Environmental fate and toxicity of ionic liquids: a review. Water Res 44:352–372 Pinho GLL, Moura da Rosa C, Maciel FE, Bianchini A, Yunes JS, Proenca LAO et al (2005) Antioxidant responses and oxidative stress after microcystin exposure in the hepatopancreas of an estuarine crab species. Ecotoxicol Environ Saf 61:353–360 Pretti C, Chiappe C, Pieraccini D, Gregori M, Abramo F, Monni G et al (2006) Acute toxicity of ionic liquids to the zebrafish (Danio rerio). Green Chem 8:238–240 Pretti C, Chiappe C, Baldetti I, Brunini S, Monni G, Intorre L (2009) Acute toxicity of ionic liquids for three freshwater organisms: Pseudokirchneriella subcapitata, Daphnia magna and Danio rerio. Ecotoxicol Environ Saf 72:1170–1176 Shao B, Zhu LS, Dong M, Wang J, Wang JH, Xie H et al (2012) DNA damage and oxidative stress induced by endosulfan exposure in zebrafish (Danio rerio). Ecotoxicology 21:1533–1540 Song Y, Zhu LS, Xie H, Wang J, Wang JH, Liu W et al (2009) Effects of atrazine on DNA damage and antioxidative enzymes in Vicia faba. Environ Toxicol Chem 28:1059–1062 Sun Y, Oberley LW, Li Y (1988) A simple method for clinical assay of superoxide dismutase. Clin Chem 34:497–500 Tabatabaie T, Floyd RA (1994) Susceptibility of glutathione peroxidase and glutathione reductase to oxidative damage and the protective effect of spin trapping agent. Arch Biochem Biophys 314:112–119 Wilkes JS (2002) A short history of ionic liquids—from molten salts to neoteric solvents. Green Chem 4:73–80 Xu JB, Yuan XF, Lang PZ (1997) Determination of catalase activity and catalase inhibition by ultraviolet spectrophotometry. Chin J Environ Chem 16:73–76 Yu M, Li SM, Li XY, Zhang BJ, Wang JJ (2008) Acute effects of 1-octyl-3-methylimidazolium bromide ionic liquid on the antioxidant enzyme system of mouse liver. Ecotoxicol Environ Saf 71:903–908 Yu M, Wang SH, Luo YR, Han YW, Li XY, Zhang BJ et al (2009) Effects of the1-alkyl-3-methylimidazolium bromide ionic liquids on the antioxidant defense system of Daphnia magna. Ecotoxicol Environ Saf 72:1798–1804 Zhang Z, Liu JF, Cai XQ, Jiang WW, Luo WR, Jiang GB (2011) Sorption to dissolved humic acid and its impacts on the toxicity of imidazolium based ionic liquids. Environ Sci Technol 45:1688–1694 Zhang QM, Zhu LS, Wang J, Xie H, Wang JH, Han YN et al (2013) Oxidative stress and lipid peroxidation in the earthworm Eisenia fetida induced by low doses of fomesafen. Environ Sci Pollut Res 20:201–208
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Oxidative Stress and Genotoxicity of the Ionic Liquid 1-Octyl-3-Methylimidazolium Bromide in Zebrafish (Danio rerio) Zhongkun Du • Lusheng Zhu • Miao Dong Jinhua Wang • Jun Wang • Hui Xie • Tong Liu • Yingying Guo
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Received: 14 November 2013 / Accepted: 9 May 2014 Springer Science+Business Media New York 2014
Abstract Ionic liquids (ILs) have a great reputation due to their negligible volatility, designability, good stability, and ability to be recycled. They are considered to be ‘‘green’’ solvents and have great promise in many fields. In recent years, the toxicities of ILs have garnered increasing attention as reported by a number of studies. However, previous studies have primarily focused on their lethal toxicities, and data were limited on their toxic effects at nonlethal doses. We performed a study on the toxic effects of 1-octyl-3-methylimidazolium bromide ([Omim]Br) on zebrafish. During a 28-day period, male and female zebrafish were separately exposed to sequential concentrations (0, 5, 10, 20, and 40 mg/L) of [Omim]Br. Fishes were sampled after 7, 14, 21, and 28 days of exposure, and reactive oxygen species (ROS) levels, activities of antioxidant enzymes (superoxide dismutase and catalase), lipid peroxidation (LPO), and DNA damage in fish livers were measured. ROS, LPO, and DNA damage were all induced
Electronic supplementary material The online version of this article (doi:10.1007/s00244-014-0046-2) contains supplementary material, which is available to authorized users. Z. Du L. Zhu (&) M. Dong J. Wang (&) J. Wang H. Xie T. Liu Y. Guo National Engineering Laboratory for Efficient Use of Soil and Fertilizer Resources, Key Laboratory of Agricultural Environment in Universities of Shandong, College of Resources and Environment, Shandong Agricultural University, 61 Daizong Road, Taian 271018, People’s Republic of China e-mail: [email protected]; [email protected] J. Wang e-mail: [email protected] Z. Du School of the Environment, Nanjing University, Nanjing 210023, People’s Republic of China
by the ionic liquid, and antioxidant enzyme activities increased at the beginning and then decreased. These phenomena demonstrate that [Omim]Br can induce oxidative stress and DNA damage in zebrafish.
Ionic liquids (ILs) are salts with a melting point lower than 100 C and are composed entirely of ions (Wilkes 2002). Due to their negligible vapor pressure, good stability, and ability to be recycled, ILs are considered to be environmentally safe ‘‘green’’ solvents and will gradually replace traditional organic solvents in many physical and chemical processes (Matsumoto et al. 2004a). The physical properties of ILs can be tailored by changing their ionic compositions, even by changing the length and branching of the alkyl chain on the cations (Huddleston et al.2001). Therefore, ILs can be synthesized specifically for a certain application. Due to these advantages, ILs have attracted increasing interest in many fields. The application of these liquids in reaction media for organic synthesis, catalysis, or biocatalysis has been well documented (Pham et al. 2010) and has produced good results with the potential for future use. However, ILs cannot be regarded as absolutely green based only on their negligible vapor pressure because their water solubilities cannot be ignored. Large-scale use of ILs in the future may lead to their release into aquatic and soil environments, and their ecotoxicological evaluation is highly relevant. Because the use of ILs is likely to increase, their toxicities have received increasing attention. Matsumoto et al. (2004a, b) reported the toxicity of ILs in bacteria and found that IL toxicity was positively correlated with alkyl chain length. An investigation into the acute toxicity of ILs in zebrafish found that the cation of ILs plays an important role in acute toxicity (Pretti et al.
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2006, 2009). According to (Matsumoto et al. 2004a, b), ILs have antibacterial activity that can decrease the growth rate and productivity of microbes. Toxicities of ILs on other organisms, including earthworms, mice, etc., have also been reported. Although these studies have provided (eco) toxicological assessments of ILs, there is still a lack of knowledge about the toxicities of ILs; data on genotoxicity and bioaccumulation are still insufficient. Until now, there have been almost no data on the genotoxicity of ILs with the exception of Docherty et al. (2006), Zhang et al. (2011), who made the first evaluation of IL genotoxicity and both found that imidazolum ILs show mutagenicity. Imidazolium ILs are the most widely used ILs and have been widely investigated. An imidazolium IL, 1-octyl-3methylimidazolium bromide ([Omim]Br), can be obtained easily and has been widely involved in toxicity studies. Acute toxicity of this IL on goldfish has been reported by (Li et al. 2012); an acute toxicity test showed that the 24-hour LC50 of [Omim]Br for goldfish was 244 mg/L. In the present study, we measured the effects of [Omim]Br on catalase (CAT), superoxide dismutase (SOD), reactive oxygen species (ROS), lipid peroxidation (LPO), and DNA damage in zebrafish during a 28-day period.
[70 %, and pH ranging from 7.4 to 8.1. Fish were exposed to different concentrations of [Omim]Br (0, 5, 10, 20, and 40 mg/L). Males and females were then separated for IL exposure. There were 120 fish/tank and three replicate tanks/group. There was no mortality in both stock cultures and the treatments. The exposure system was semistatic, and [Omim]Br was directly dissolved in water. Half of the water was refreshed every 2 days to clear excreta away and to preserve the IL concentration. The stability and low biodegradation of the imidazolium ILs were documented by Docherty et al. (2006). Degradation experiments have already indicated a high compound stability, which was also confirmed in our laboratory by measuring the concentration of the compound in the exposure tanks at the beginning and end of the experiment. Fishes were fed daily with commercially available dry flakes, except on the day before the test, to protect the fish from interference from feces during the assays. Fishes were sampled on days 7, 14, 21, and 28 of exposure. Fish liver was used as the target organ for toxicity tests, and tests were immediately performed after sampling. ROS Determination
[Omim]Br (purity 99 %, CAS no. 61545-99-1) was obtained from the Chengjie Chemical Co. Ltd. (Shanghai, China), and 20 ,70 -dichlorofluorescin-diacetate (DCFH-DA) was obtained from the Beyotime Institute of Biotechnology (Jiangsu, China). All other chemicals were of analytical purity and purchased from either Sigma Chemical (St. Louis, Missouri, USA) or Beijing Chemical (Beijing, China). Adult male (0.27 ± 0.05 g mean body weight and 2.45 ± 0.01 cm length) and female (0.31 ± 0.07 g mean body weight and 2.59 ± 0.03 cm length) zebrafish (Danio rerio) were purchased from an aquarium (Qixin Aquarium, Taian, China).
Quantitative determination of ROS production was measured as described in the DCFH-DA method using a Diagnostic Reagent Kit. Fish livers were homogenized in 100 mM of ice-cold potassium phosphate buffer (pH 7.4). Subsequently the homogenates were centrifuged at 1,000g for 10 min and the supernatants recentrifuged at 20,000g for 20 min. The mitochondrial protein was then prepared after the pellet was resuspended with ice-cold potassium phosphate buffer (pH 7.4). All of the these manipulations were performed at 4 C. The ROS level was then measured according to the methods of Lawler et al. (2003). The mitochondrial protein supernatants were mixed with 2 lM DCFH-DA and then incubated in a water bath at 37 C for 30 min. Fluorescence intensities were detected at a wavelength of 538 nm with a fluorescent spectrophotometer. The results are expressed as fluorescence intensity per milligram of total protein.
Fish Care and Exposure
Determination of Enzyme Activities
The results of the present study followed the Guiding Principles outlined in the Use of Animals in Toxicology adopted by the Society of Toxicology in 1989. The fish were kept in the laboratory for 2 weeks before the experiments to allow them to adapt to the laboratory conditions. The fishes’ living conditions strictly followed that of Diekmann et al. (2004): a 12:12 h light-to-dark regime, 26 ± 1 C temperature, tap water (aerated and basked for 2 days to dechlorinate before use), oxygen saturation
Fish livers were homogenized in 50 mM of ice-cold potassium phosphate buffer (1:8 w/v [pH 7.0]). The homogenate was then centrifuged at 10,600g at 4 C for 10 min. The supernatant was used immediately for the determination of enzyme activity and protein concentration at room temperature (25 C). CAT activity was determined from the rate of change of ultraviolet absorbance (Xu et al. 1997). CAT activity can be measured by the decrease in ultraviolet absorption over
Materials and Methods Chemicals and Animals
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time due to the degradation of H2O2 by CAT in the sample. One unit (U) of CAT activity was defined as the enzyme quantity required to cause half-degradation of the H2O2 in the sample after 100 s at 25 C normalized by protein concentration. The results are expressed as unit per mg total protein. SOD activity was assayed by its ability to inhibit the photochemical decrease of nitroblue tetrazolium chloride (NBT) (Sun et al. 1988). U of SOD activity was defined as the quantity of enzyme that caused 50 % inhibition of the NBT photoreduction rate. The results are expressed as unit per milligram of total protein.
for the data analyses. The data were subjected to one-way analysis of variance followed by least significant difference test at the p \ 0.05 level of significance. All values are presented as mean ± SD (n = 3).
Results Data are shown with statistical analysis in relation to [Omim]Br treatment concentration (Figs. 1, 2, 3, 4, 5). Statistical analysis of sex discrepancies (Supplemental [S] Figs. S1 to S5) and statistical analysis in relation to the treatment time (Figs. S6 to S10) are given.
Determination of LPO ROS The homogenate used for the LPO test was the same as that used in the enzyme test. Because malondialdehyde (MDA) is the final product of LPO, we measured MDA content as an indicator of LPO. The measurement of MDA was made using the Diagnostic Reagent Kit according to the thiobarbituric acid (TBA) method (Zhang et al. 2013). The assay was monitored for the appearance of the conjugated complex of TBA and MDA at 532 nm. The concentration of MDA is reported as nanomoles of MDA per milligram of total protein.
A liver cell suspension from zebrafish was prepared according to Shao et al. (2012). The resulting cell suspensions were processed by single cell gel electrophoresis (SCGE), which was also performed according to the method described by Shao et al. (2012). The Comet Assay Software Project (CASP 1.2.2, Biolaunching Technologies Co., Ltd., Beijing, China) software program was used to analyze the comet photographs collected with a fluorescent microscope (Olympus BX71). Finally, the olive tail moment (OTM), which is the product of the distance between the center of the head, the center of the tail, and the total DNA percentage in the tail, was used to quantify the extent of DNA damage (Olive et al. 1990; Song et al. 2009).
The effects of [Omim]Br on ROS levels in zebrafish are shown in Figure 1. ROS levels exhibited similar patterns in both male and female fish. [Omim]Br exposure resulted in ROS accumulation, and greater concentrations of [Omim]Br caused greater ROS accumulation. The effects in male fish were as follows: (1) 5 mg/L of [Omin]Br had no effect on ROS level at all sampling times; (2) 10 mg/L of [Omin]Br also caused no change in ROS level on days 7 and 14, but it observably induced an ROS increase on days 21 and 28; (3) in the 20 and 40 mg/L groups, ROS level was steadily greater than that in control; (4) in the 10 and 20 mg/L groups, greater induction of ROS appeared on day 21 (143 % and 157 % of the control, respectively) and then slightly decreased on day 28 day but were still greater than the control (123 % and 133 % of control, respectively); and (5) in the 40 mg/L group, a greater induction of ROS occurred on day 28 (171 % of control). The effects in female fish were as follows: (1) 5 mg/L of [Omin]Br induced ROS level on the days 7 and 21; (2) 10 mg/L of [Omin]Br induced ROS level on days 14 and 28; (3) 20 and 40 mg/L [Omin]Br increased ROS level at all sampling times; (4) in 5 the mg/L group, greater ROS promotion occurred on day 21; and (5) in the 10, 20, and 40 mg/L groups, the greater increase of ROS appeared on the day 14. Regarding the discrepancies in ROS variation between male and female fish (Fig. S1) on day 7 day, the ROS level in male fish was lower than that in female fish when exposed to 5 mg/L [Omin]Br; on day 14 in the 10, 20, and 40 mg/L groups, ROS levels in male fish were lower than those in female fish; on day 21 in the 10 and 20 mg/L groups, ROS levels in male fish were greater than those in female fish.
Statistics
Enzyme Activities
The statistical package for social sciences (SPSS) program (standard version 16.0, SPSS, IBM, Chicago, IL) was used
The effects of [Omim]Br on CAT activities in zebrafish are shown in Figure 2. Generally speaking, CAT activity under
Protein Content Determination Protein content was determined by the Sigma Bradford method (Bradford 1976) using bovine serum albumin as the standard. DNA Damage Determination
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Fig. 1 ROS level in livers of zebrafish exposed to 4 concentrations of [Omim]Br for 28 days. Values are presented as mean ± SD (n = 3). Different letters above the columns indicate significant differences at the p \ 0.05 level between treatments. Pr = protein; fluo = fluorescence
Fig. 2 CAT activities in livers of zebrafish exposed to 4 concentrations of [Omim]Br for 28 days. Values are presented as mean ± SD (n = 3). Different letters above the columns indicate significant differences at p \ 0.05 level between treatments. Pr = protein
Fig. 3 SOD activities in livers of zebrafish exposed to 4 concentrations of [Omim]Br for 28 days. Values are presented as mean ± SD (n = 3). Different letters above the columns indicate significant differences at p \ 0.05 level between treatments. Pr = protein
[Omim]Br exposure showed a time-dependent trend, first induced and then inhibited, in both male and female fish; there is also another trend that the greater [Omim]Br concentration, the earlier the CAT activity turned to being inhibited from being induced in both the male and female groups. For male fish, 5 mg/L of [Omim]Br induced CAT
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activity on day 14, inhibited CAT activity on day 28, and showed no significant effect on CAT activity on days 7 and 21. In the 10 and 20 mg/L groups, CAT activity was induced on day 7 and inhibited on days 21 and 28, whereas it was not statistically different from the control on day 14. In the 40 mg/L group, CAT activity was induced only on
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Fig. 4 LPO level in livers of zebrafish exposed to 4 concentrations of [Omim]Br for 28 days. Values are presented as mean ± SD (n = 3). Different letters above the columns indicate significant differences at p \ 0.05 level between treatments. Pr = protein
Fig. 5 DNA damage level in livers of zebrafish exposed to 4 concentrations of [Omim]Br for 28 days. Values are presented as mean ± SD (n = 3). Different letters above the columns indicate significant differences at p \ 0.05 level between treatments
day 7; at the other sampling times it was inhibited. For females, 5 mg/L of [Omim]Br induced CAT activity on days 7 and 14, inhibited CAT activity on days 21 and 28. In the 10 mg/L group, CAT activity was induced on day 7 and inhibited on day 21, whereas it was not statistically different from the control on days 14 and 28. In the 20 mg/L and 40 mg/L groups, CAT activities were induced on day 7; at the other sampling times it was inhibited. No regularity was observed in the discrepancies between male and female groups (Fig. S2). In the 5 mg/L groups, CAT activity in male fish was lower than that in female fish on days 7 and 14, and then turns toward being greater than that in females on days 21 and 28. This maybe indicates that female fish are more sensitive in CAT activity than male fish. The effects of [Omim]Br on SOD activities in zebrafish are shown in Figure 3. In general, high concentrations of [Omim]Br inhibited SOD activities. Low concentrations of [Omim]Br induced SOD activities before day 7 but inhibited them after day 14. In detail, for male fish, no statistical changes on SOD activities were found in the
5 mg/L group, whereas they were slightly induced in the 10 mg/L group compared with the control on day 7; then SOD activities in both these two groups turned toward being inhibited on the other sampling days. SOD activities in groups exposed to 20 and 40 mg/L of [Omim]Br were inhibited from the beginning to the end of the exposure. For female fish, SOD activities in the 5, 10, and 20 mg/L groups were increased on day 7 and turned to be lower than the control level after 14 days. SOD activity in the 40 mg/ L group was the same level as control on the day 7, then it turned toward being lower at the other sampling times. For differences between male and female groups (Fig. S3), except for in the 40 mg/L group on day 21, male SOD activity was greater than female SOD activity. All other statistical discrepancies (20 and 40 mg/L groups on day 7; 5, 10, and 20 mg/L groups on day 14; 10 mg/L groups on day 21; and 10, 20, and 40 mg/L groups on day 28) showed that SOD activities in male fish were lower than those in male fish under [Omim]Br exposure, which indicates that SOD in male fish was more easily inhibited by [Omim]Br.
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Fig. 6 Comet assay. a Structure of a comet. b Control zebrafish liver cell in comet assay. c–f Different DNA damage extents of female zebrafish exposed to 5, 10, 20, and 40 mg/L of [Omim]Br for 28 days, respectively
Lipid Peroxidation LPO was observed in fishes after exposure to [Omim]Br. As presented in Figure 4, MDA content was primarily enhanced by [Omim]Br in both male and female fish with the highest increase of MDA appearing on day 7 (40 mg/L male group = 230.0 % of the control), then the enhancements gradually decreased. In detail, for male fish, MDA content in all [Omim]Br treatment groups was increased on day 7, and enhancements gradually decreased; on day 28, MDA content in the 5, 10, and 20 mg/L groups was still greater than the control, whereas the MDA content in the 40 mg/L groups decreased to control level. For female fish, 5 mg/L of [Omim]Br had no effect on MDA content on days 7 and 14, but it was induced on days 21 and 28; in the 10 mg/L group, MDA content was only induced on day 21; in the 20 and 40 mg/L groups, MDA content was induced on days 7, 14, and 21 and then decreased to control level on day 28. Regarding discrepancies between male and female fish (Fig. S4), except for day 21 in the 10 mg/L male group, the MDA level was lower than the female MDA level. All other statistical discrepancies (all [Omim]Br treatment groups on days 7 and day 14; the 20 and 40 mg/L groups on day 21; and the 10 and 20 mg/L groups on day 28) showed the reverse result, that is, the LPO level in males was greater than that in females. DNA Damage DNA damage was measured with SCGE. OTM values were used to indicate damage intensities. The DNA damage results caused by [Omim]Br in zebrafish are shown in Figure 5. OTM values in all IL-treated groups were greater
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than in the controls from the beginning to the end of the exposure, which indicates that all concentrations of [Omim]Br in the present study can induce DNA damage in liver cells of zebrafish. In general, greater IL concentrations result in greater levels of DNA damage, and damage is aggravated with increasing exposure time. In 5 mg/L group, the level of DNA damage in male fish was greater than that in female on days 14, 21, and 28; no other statistical discrepancy was observed between male and female fish (Fig. S5). Parts of comet assay photos are shown in Fig. 6. A comet has a head and a tail, but normal cell shows no tail in comet assay; with greater DNA damage, the cell will show a larger tail and a smaller head. The greater concentration of [Omim]Br exposure, the greater the DNA damage of zebrafish liver cell observed.
Discussion Due to their low vapor pressure and many other good properties, ILs have been heavily discussed in green chemistry. Industrial use of ILs is imperative, but are ILs always green? Some toxicological studies of ILs reported a negative answer (Bernot et al. 2005a, b; Cho et al. 2008a, b, c; Yu et al. 2008, 2009). Different ILs have different levels of toxicity, and a given IL may produce different acute toxicity intensities in different organisms. Existing data are mainly about toxic levels of different ILs, but the toxic mechanism of ILs is not yet fully understood. According to Modelli et al. (2008), imidazolium-based ILs are not easily degraded under environmentally realistic conditions; thus organisms may be exposed to them for a
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long time when ILs are released into the environment. Therefore, their chronic effects and toxic mechanisms should be studied further. The results of the present study investigated the effects of [Omim]Br on zebrafish during an exposure period of 28 days. Because the liver is responsible for metabolism and detoxification in animals, important processes pertaining to enzyme synthesis and reactions occur therein, and it is very sensitive to many pollutants (Moreno et al. 2005). Li et al. (2012) reported that the liver is one target organ of IL toxicity in goldfish. We used liver as the target organ to assess the toxic effects of ILs in zebrafish. Oxidative Stress In the present study, ROS were induced in fish by • [Omim]Br. ROS (including O•2 , OH , and H2O2) are generated in processes of normal cell metabolism. They play important roles in apoptosis and signal transmission, but excess ROS have negative effects such as DNA damage, LPO, protein oxidation, and aging (Bayr 2005). Previous studies have also indicated that excess ROS may lead to cardiovascular disease and hearing impairment (Michael et al. 2004; Junichi et al. 1998). In the present study, low concentrations (5 mg/L) of the IL [Omim]Br slightly enhanced the ROS content of zebrafish liver. With greater concentrations of this IL, ROS levels increased too much, which may lead to many health problems. In agreement with this result, we found that [Omim]PF6 can induce ROS in zebrafish liver (Du et al. 2012), and a study by Kumar et al. (2011) found that [C12mim]Br significantly increased • concentrations of O•2 , OH , and H2O2 in the green seaweed Ulva lactuca. These data may indicate a consistent effect of imidazolium ILs of inducing ROS and oxidative stress when they enter organisms. To gather more data on the oxidative stress observed, the response of the antioxidant defense system was studied. The antioxidant defense system includes many antioxidant enzymes that are responsible for eliminating excess ROS to avoid further diseases. CAT and SOD are two important components of the antioxidant defense system. They can decrease the concentrations of H2O2 and O•2 , thus effectively eliminating excess ROS and protecting the organism. They are reasonable markers of oxidative stress and have been well documented (Gomi et al. 1998; Pinho et al. 2005). CAT and SOD activities all showed similar responses to [Omim]Br exposure in the present study: an initial increase followed by a decrease. The increase in enzyme activities is due to an activation of the zebrafish self-defense system. The followed decrease in enzyme activities may be related to many factors. One assumption relates to the expression obstacle of their genes, but
research has yet to confirm this. ROS also inactivate antioxidant enzymes (Tabatabaie & Floyd 1994). Other studies have also discussed the effects of ILs on the antioxidant enzyme systems in different organisms. According to Li et al. (2012), 0.9 mg/L of [Omim]Br induced CAT, SOD, and glutathione peroxidase (GPx) activities in D. magna. In addition, the activities of CAT and SOD were induced, whereas GPx activity was enhanced, when adult goldfish were exposed to 122 mg/L of [Omim]Br for 72 h. Kumar et al. (2011) also reported that SOD activity in U. lactuca was enhanced when exposed to 0.04 mM of [C12mim]Br and was inhibited when exposed to 0.07 mM of [C12 mim]Br. According to Du et al. (2012), [Omim]PF6 also affected SOD and CAT activities in zebrafish, e.g., [Omim]PF6 showed more immediate inhibition of antioxidant enzyme activities at day 7, whereas [Omim]Br caused a transitory increase. The probable cause of this difference is that PF6- can be hydrolyzed to hydrogen fluoride, which can inhibit enzymes (Arning et al. 2008). Therefore, ILs that contain PF6- anion may be more harmful to organisms than those contain Br-. It has been well documented that excess ROS may cause LPO (oxidation of polyunsaturated fatty acids in lipids). LPO can damage biomembranes and can then affect normal cell metabolism and function. MDA is commonly used to indicate LPO because it LPO is the final product in the process (Hiroshi et al. 1979). In the present study, MDA in both male and female fish was induced by [Omim]Br. The highest inductive effect appeared on day 7. Unlike ROS, MDA was not persistently accumulated but gradually decreased after day 7 and practically reached the control level at the end of the 28-day exposure. The nonconformity between MDA and ROS variation seems unreasonable, and we did not perform any further research to determine the cause. However, as mentioned previously, [Omim]Br induced GPx activity, whereas it inhibited CAT and SOD in the study by Li et al. (2012), and it is known that GPx can restrain lipid oxidation. Therefore, we speculate that the same mechanism may exist in our study and that the effects of GPx may facilitate MDA removal. The slight distinction between male and female fish indicates that males may be slightly weaker than females in toxicity tests. The results from the MDA test indicate that the tested concentration of [Omim]Br can lead to LPO in zebrafish, but LPO can be repaired, so it is not a pathway of further [Omim]Br toxicity in zebrafish at related concentrations. Genotoxicity Genotoxicity of [Omim]Br was studied with the comet assay. Obvious comet tails were observed in [Omim]Brtreated groups, and the results indicated that [Omim]Br
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caused DNA damage in zebrafish liver. In the present study, the DNA damage content was positively correlated with IL concentrations, and damage levels increased with the extension of time. DNA damage in the 10, 20, and 40 mg/L groups was much greater than in the 5 mg/L groups after 14 days of exposure. DNA damage in the 5 mg/L groups remained at a steady level. To date, few studies have focused on the genotoxicity of ILs, but all have mentioned that ILs exhibited genotoxicity. Docherty and Zhang reported the mutagenicity of imidazolium-based ILs (Docherty et al. 2006; Zhang et al. 2011). Using comet assay, Kumar et al. (2011) found that [C12mim]Br induced DNA damage in U. lactuca. Our previous study also showed the genotoxicity of [Omim]PF6. These data show that imidazolium-based ILs are genotoxic. Because genotoxicity can lead to DNA lesions that trigger a cascade of biological consequences at the cellular, organ, whole organism, population, and community levels, we cannot regard ILs as ‘‘green’’ as they were once claimed to be. It is well known that oxidative stress plays an important role in DNA damage. Excess ROS is a major cause of oxidative DNA damage. In the present study, DNA damage showed a similar trend to ROS, i.e., they had positive correlations with [Omim]Br concentration and exposure time, so oxidative DNA damage was the major DNA lesion in this study. In addition, 5 mg/L of [Omim]Br exposure did not cause significant ROS accumulation in male fish, but obvious DNA damage was observed in this group; this indicates that oxidative damage may not be the only pathway of DNA damage. Other pathways, such as decreased repair capacity, may be involved. In contrast, DNA damage might affect the expression of antioxidant enzymes; ROS also inactivated antioxidant enzymes, and these two decreased the antioxidant capacity of fish liver and led to further ROS accumulation leading to formation of a vicious cycle. By reason of this, DNA damage and oxidative stress became more and more serious with time in the 10, 20, and 40 mg/L groups. Discrepancies Between Males and Females Lower SOD inhibition, LPO, and DNA-damage induction were found in female fish compared with male fish in the same exposure treatments. This phenomenon shows that female fish have greater ability to decrease oxidative damage. Similar results were found in other species such as flounder and even humans (Giorgio et al. 2001; Katja et al. 2001). This sex discrepancy might be regulated by hormones. It has been reported that estrogen can modulate the expression and function of antioxidant enzymes and confer protect effects against oxidative damage (Adam et al. 2000; Kerstin et al. 2003).
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Conclusion In conclusion, although imidazolium-based ILs have been reported to exhibit low toxicity in fish in acute toxicity studies, low-dose ILs can induce many problems in fish with longer exposure time, including oxidative stress and DNA damage, and this oxidative stress may be the major cause of DNA damage. LPO alteration was not serious enough under the stress of the tested concentrations. We also found that oxidative stress and genotoxicity are universal toxic effects of imidazolium-based ILs in organisms. In ILs, anion PF6inhibits enzymes more strongly than Br-. To decrease the potential risk of ILs, more work should be performed to determine their toxicities rather than guiding IL use. Due to the good stability of ILs, further studies should not focus on their acute toxicity: Chronic toxicity and their mechanisms of action require more attention. Considering the large amount of IL categories, more ILs should be involved in toxicity studies, and their structure-activity relationship must be explored. Acknowledgments The results of the present study was supported by Grants from the National Natural Science Foundation of China [Grants No. 21277083, 41071164, 41001152, and 21377075] and the Specialized Research Fund for the Doctoral Program of Higher Education [Grant No. 20113702110007].
References Adam MP, Pattie SG, Leighann S, Cynthia OH, Larissa Z, Gayle AB et al (2000) Protective effect of estrogens against oxidative damage to heart and skeletal muscle in vivo and in vitro. Proc Soc Exp Biol Med 223:59–66 Arning J, Stolte S, Boschen A, Stock F, Pitner WR, Welz-Biermann U et al (2008) Qualitative and quantitative structure activity relationships for the inhibitory effects of cationic head groups, functionalized side chains and anions of ionic liquids on acetylcholinesterase. Green Chem 10:47–58 Bayr H (2005) Reactive oxygen species. Crit Care Med 33:S498– S501 Bernot RJ, Brueseke MA, Evans-White MA, Lamberti GA (2005a) Acute and chronic toxicity of imidazolium-based ionic liquids on Daphnia magna. Environ Toxicol Chem 24:87–92 Bernot RJ, Kennedy EE, Lamberti GA (2005b) Effects of ionic liquids on the survival, movement, and feeding behavior of the freshwater snail, Physa acuta. Environ Toxicol Chem 24:1759–1765 Bradford MM (1976) A rapid sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 Cho CW, Jeon YC, Pham TPT, Vijayaraghavan K, Yun YS (2008a) The ecotoxicity of ionic liquids and traditional organic solvents on microalga Selenastrum capricornutum. Ecotoxicol Environ Saf 71:166–171 Cho CW, Pham TPT, Jeon YC, Min J, Jung HY, Lee DS et al (2008b) Microalgal photosynthetic activity measurement system for rapid toxicity assessment. Ecotoxicology 17:455–463
Arch Environ Contam Toxicol Cho CW, Pham TPT, Jeon YC, Yun YS (2008c) Influence of anions on the toxic effects of ionic liquids to a phytoplankton Selenastrum capricornutum. Green Chem 10:67–72 Diekmann M, Hultsch V, Nagel R (2004) On the relevance of genotoxicity for fish populations I: effects of a model genotoxicant on zebrafish (Danio rerio) in a complete life-cycle test. Aquat Toxicol 68:13–26 Docherty KM, Hebbeler SZ, Kulpa JC (2006) An assessment of ionic liquid mutagenicity using the Ames test. Green Chem 8:560–567 Du ZK, Zhu LS, Dong M, Wang JH, Wang J, Xie H et al (2012) Effects of the ionic liquid [Omim]PF6 on antioxidant enzyme systems, ROS and DNA damage in zebrafish (Danio rerio). Aquat Toxicol 124–125:91–93 Giorgio F, Patrizia M, Jacopo V, Silvia B, Stefania F, Cristina M P et al (2001) Age and sex influence on oxidative damage and functional status in human skeletal muscle. J Muscle Res Cell Motil 22:345–351 Gomi F, Matsuo M (1998) Effects of aging and food restriction on the antioxidant enzymes activity of rat livers. J Gerontol Ser A 53:B161–B167 Hiroshi O, Nobuko O, Kunio Y (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:158–351 Huddleston JG, Visser AE, Reichert WM, Willauer HD, Broker GA, Rogers RD (2001) Characterization and comparison of hydrophilic and hydrophobic room temperature ionic liquids incorporating the imidazolium cation. Green Chem 3:156–164 Jun-ichi A, Bradford CB (1998) Reactive oxygen species as mediators of signal transduction in cardiovascular disease. Trends cardiovasc med 8:59–64 Katja W, Gary WW, Wilhelm B, Cornelis JFVN, Angela K (2001) Sex-related responses to oxidative stress in primary cultured hepatocytes of European flounder (Platichthys flesus L.). Aquat Toxicol 52:143–155 Kerstin S, Simone R, Sven W, Oliver A, Christian G, Kerstin L et al (2003) Modulation of antioxidant enzyme expression and function by estrogen. Circ Res 93:170–177 Kumar M, Trivedi N, Reddy CRK, Jha B (2011) Toxic effects of imidazolium ionic liquids on the green seaweed Ulva lactuca: Oxidative stress and DNA damage. Chem Res Toxicol 24:1882–1890 Lawler JM, Song W, Demaree SR (2003) Hindlimb unloading increases oxidative stress and disrupts antioxidant capacity in skeletal muscle. Free Radic Biol Med 35:1–9 Li XY, Zeng SH, Dong XY, Ma JG, Wang JJ (2012) Acute toxicity and responses of antioxidant systems to 1-methyl-3-octylimidazolium bromide at different developmental stages of goldfish. Ecotoxicology 21:253–259 Matsumoto M, Mochiduki K, Fukunishi K, Kondo K (2004a) Extraction of organic acids using imidazolium-based ionic liquids and their toxicity to Lactobacillus rhamnosus. Sep Purif Technol 40:97–101 Matsumoto M, Mochiduki K, Kondo K (2004b) Toxicity of ionic liquids and organic solvents to lactic acid-producing bacteria. J Biosci Bioeng 98:344–347 Michael DS, Nadir A, Dipa J, Jake S, Sujatha T, Wayne SQ (2004) Age-related hearing loss and its association with reactive oxygen species and mitochondrial DNA damage. Acta Otolaryngol 552(Suppl):16–24
Modelli A, Sali A, Galletti P, Samorı` C (2008) Biodegradation of oxygenated and non-oxygenated imidazolium-based ionic liquids in soil. Chemosphere 73:1322–1327 Moreno I, Pichardo S, Jos A, Go´mez-Amores L, Mate A, Vazquez CM et al (2005) Antioxidant enzyme activity and lipid peroxidation in liver and kidney of rats exposed to microcystin-LR administered intraperitoneally. Toxicon 45:395–402 Olive PL, Banath JP, Durand RE (1990) Heterogeneity in radiationinduced DNA damage and repair in tumor cells measured using the comet assay. Radiat Res 122:86–94 Pham TPT, Cho CW, Yun YS (2010) Environmental fate and toxicity of ionic liquids: a review. Water Res 44:352–372 Pinho GLL, Moura da Rosa C, Maciel FE, Bianchini A, Yunes JS, Proenca LAO et al (2005) Antioxidant responses and oxidative stress after microcystin exposure in the hepatopancreas of an estuarine crab species. Ecotoxicol Environ Saf 61:353–360 Pretti C, Chiappe C, Pieraccini D, Gregori M, Abramo F, Monni G et al (2006) Acute toxicity of ionic liquids to the zebrafish (Danio rerio). Green Chem 8:238–240 Pretti C, Chiappe C, Baldetti I, Brunini S, Monni G, Intorre L (2009) Acute toxicity of ionic liquids for three freshwater organisms: Pseudokirchneriella subcapitata, Daphnia magna and Danio rerio. Ecotoxicol Environ Saf 72:1170–1176 Shao B, Zhu LS, Dong M, Wang J, Wang JH, Xie H et al (2012) DNA damage and oxidative stress induced by endosulfan exposure in zebrafish (Danio rerio). Ecotoxicology 21:1533–1540 Song Y, Zhu LS, Xie H, Wang J, Wang JH, Liu W et al (2009) Effects of atrazine on DNA damage and antioxidative enzymes in Vicia faba. Environ Toxicol Chem 28:1059–1062 Sun Y, Oberley LW, Li Y (1988) A simple method for clinical assay of superoxide dismutase. Clin Chem 34:497–500 Tabatabaie T, Floyd RA (1994) Susceptibility of glutathione peroxidase and glutathione reductase to oxidative damage and the protective effect of spin trapping agent. Arch Biochem Biophys 314:112–119 Wilkes JS (2002) A short history of ionic liquids—from molten salts to neoteric solvents. Green Chem 4:73–80 Xu JB, Yuan XF, Lang PZ (1997) Determination of catalase activity and catalase inhibition by ultraviolet spectrophotometry. Chin J Environ Chem 16:73–76 Yu M, Li SM, Li XY, Zhang BJ, Wang JJ (2008) Acute effects of 1-octyl-3-methylimidazolium bromide ionic liquid on the antioxidant enzyme system of mouse liver. Ecotoxicol Environ Saf 71:903–908 Yu M, Wang SH, Luo YR, Han YW, Li XY, Zhang BJ et al (2009) Effects of the1-alkyl-3-methylimidazolium bromide ionic liquids on the antioxidant defense system of Daphnia magna. Ecotoxicol Environ Saf 72:1798–1804 Zhang Z, Liu JF, Cai XQ, Jiang WW, Luo WR, Jiang GB (2011) Sorption to dissolved humic acid and its impacts on the toxicity of imidazolium based ionic liquids. Environ Sci Technol 45:1688–1694 Zhang QM, Zhu LS, Wang J, Xie H, Wang JH, Han YN et al (2013) Oxidative stress and lipid peroxidation in the earthworm Eisenia fetida induced by low doses of fomesafen. Environ Sci Pollut Res 20:201–208
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