J BIOCHEM MOLECULAR TOXICOLOGY Volume 28, Number 2, 2014
Toxicity of Imidazolium-Based Ionic Liquids on Physa Acuta and the Snail Antioxidant Stress Response Junguo Ma,1 Xiangyi Dong,1 Qian Fang,1 Xiaoyu Li,1 and Jianji Wang2 1 College of Life Science, Henan Normal University, Xinxiang, Henan Province, People’s Republic of China; E-mail: [email protected]; [email protected] 2 Key
Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan Province, People’s Republic of China Received 30 August 2013; revised 12 October 2013; accepted 16 October 2013
ABSTRACT: In the present study, the acute and developmental toxicities of imidazolium ionic liquids (ILs) with different alkyl chain lengths, as well as the antioxidant response and lipid peroxidation levels were evaluated in the snail, Physa acuta. Longer alkyl chains corresponded to increased IL toxicity in snails. Long-term IL exposure at lower concentrations inhibited snail growth and reproduction. We also found that IL inhibited the activities of superoxide dismutase (SOD) and glutathione S-transferase (GST), promoted the activity of catalase (CAT), and increased the glutathione content. However, SOD, GST, and CAT activities returned to control levels after 96 h of recovery. In addition, malondialdehyde levels were increased in treatment groups compared with the control and did not return to control levels even after a recovery period, indicating that ILs induced lipid peroxidation in snail viscera. These results suggest that oxidative stress and lipid peroxidation may be involved in the mechanism of toxC 2013 Wiley Periodicals, Inc. J. Biochem. icity for ILs. Mol. Toxicol. 28:69–75, 2014; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21537
KEYWORDS: Ionic Liquid; Physa Acuta; Toxicity; Antioxidant Enzymes; Lipid Peroxidation
INTRODUCTION Ionic liquids (ILs) are salts with relatively low melting points (below 100◦ C), and they are expected to Correspondence to: Xiaoyu Li. Contract Grant Sponsor: National Science Foundation of China. Contract Grant Number: 31172415, 20573019, and 20573034. Contract Grant Sponsor: Key Subjects of Biology and Ecology in Henan Province, People’s Republic of China. C 2013 Wiley Periodicals, Inc.
replace conventional volatile organic solvents to reduce air pollution [1, 2]. In recent years, ILs have attracted considerable attention due to their unique characteristics and properties, such as negligible vapor pressure, high thermal stability, and excellent solvation ability [3–5]. Therefore, they have been regarded as promising “green” solvents in manufacturing, processing, and cleaning technologies [6–10]. However, IL toxicity to aquatic organisms and possible environmental risks to aquatic ecosystems are a potential concern because most ILs are water soluble, poorly decomposed by microorganisms, and prone to entering the aquatic environment by accidental discharges [11–15]. Hence, the available literature regarding IL toxicity to aquatic organisms has been expanding; for example, IL-toxicity evaluations are available for algae [16, 17], cladocerans [18–20], and fish [21]. In contrast, limited reports regarding the toxicity of ILs to snails are available, including the works of Bernot et al. [22] and Li et al. [23]. The snail Physa acuta is a globally invasive species [24,25]. However, these snails are very sensitive to toxicant exposure and thus, appropriate for toxicity testing [26]. Imidazolium-based ILs were evaluated in the present study based on a two-point rationale. First, these chemicals usually have moderate or relatively decreased toxicity in organisms. Second, and more importantly, the toxic response of organisms to ILs is typically dose dependent, and the results from toxicity testing for common imidazolium ILs can be contextualized with the results of our previous studies [18,21,23,27,28]. The current study aimed to determine the acute toxicities of the imidazolium ILs with different alkyl chain lengths in adult snails, the reproductive and developmental toxicities of ILs in juvenile snails, and ILs’ effects on the snail antioxidant system to elucidate the biochemical mechanism of IL-induced toxicity.
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MATERIALS AND METHODS ILs and Chemicals The imidazolium-based ILs employed in the present study were 1-hexyl-3-methylimidazolium bromide ([C6 mim]Br), 1-octyl-3-methylimidazolium bromide ([C8 mim]Br), 1-decyl-3-methylimidazolium and 1-dodecyl-3bromide ([C10 mim]Br), methylimidazolium bromide ([C12 mim]Br). They were purchased from Hubei Hengshuo Chemical Company, Ltd., Wuhan, Hubei, People’s Republic of China, and their purities were 99%. Analytical grade reagents were purchased from Sigma (St Louis, MO). The IL solutions were prepared by dissolving the ILs in distilled water, followed by dilution to the desired concentrations with aerated tap water when tests were performed.
Snails P. acuta specimens were collected from a fishery pond in China. Specimens were maintained in our laboratory in glass jars (3 L in volume) with aerated tap water (total hardness 340 mg/L, pH 7.6, turbidity 1.5 NTU, total dissolved solid content 660 mg/L) at 20 ± 1◦ C and a photoperiod of 16 h:8 h light/darkness for several generations prior to the experiment. The snails were fed ad libitum with commercial goldfish food (Wannong Fishery Company, Xinxiang, Henan, People’s Republic of China) at a rate of 0.5%–1% of body weight per day, and the water was changed weekly. The adult snails had an average wet weight of 30.74 ± 2.57 mg and shell length of 7.26 ± 0.98 mm, and the juvenile snails (30 days after hatching) had an average wet weight of 8.85 ± 0.93 mg and shell length of 4.25 ± 0.36 mm. The adult and juvenile snails were used for the toxicity tests and antioxidant system assays described below. Generally, the first oviposition of the snail (sexual maturity) occurred at 45 to 60 days after hatching; following this developmental time point, the snails laid eggs (20–40) encapsulated in egg sacks once every 1 to 3 days. Snail longevity was approximately 12–18 months under our laboratory conditions.
Determination of the Acute Toxicity of Imidazolium ILs with Different Alkyl Chain Lengths in Adult Snails The acute toxicities of the imidazolium-based ILs with different alkyl chain lengths, that is, [C6 mim]Br, [C8 mim]Br, [C10 mim]Br, and [C12 mim]Br, were determined in adult snails (average wet body weight 30.74 ± 2.57 mg and average shell length 7.26 ±
FIGURE 1. Death rates in adult snails exposed to a concentration range of imidazolium ILs with different alkyl chain lengths. The acute toxicity tests were carried out according to the Spearman– K¨arber method [29], as described in section 2.3. Data are presented as the mean ± standard deviation (SD). Asterisks denote a response that is significantly different from controls (**P < 0.01). A, [C6 mim]Br; B, [C8 mim]Br; C, [C10 mim]Br; and D, [C12 mim]Br.
0.98 mm) according to the Spearman–K¨arber method [29]. Ten snails were placed in a 500-mL beaker containing 300 mL of a range of concentrations of IL solutions, as shown in Figure 1. The control snails were also bred temporarily in a beaker, but with aerated tap water only. The beakers were covered with cotton J Biochem Molecular Toxicology
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gauze to prevent the snails from escaping. Each test was conducted in triplicate. Any dead snails were removed immediately from the beaker, and the total numbers of dead and surviving snails were recorded in each group during the exposure period. The 96 h 50% of lethal concentration (LC50 ) values and their 95% confidence limits were calculated using SPSS 11.5.
Determination of the Subchronic Toxicity of [C8 mim]Br in Juvenile Snails Subchronic toxicity testing was designed to evaluate the effects of [C8 mim]Br exposure on the growth, development, and reproduction of juvenile snails. The tests included four groups, of which three served as IL-treatment groups (1, 4, 8 mg/L of [C8 mim]Br) and one served as a control. In the treatment groups, a total of 12 juvenile snails (30 days after hatching), with an average wet weight of 8.85 ± 0.93 mg and an average shell length of 4.25 ± 0.36 mm, were exposed to [C8 mim]Br solutions at concentrations of 1, 4, and 8 mg/L in a 500 mL beaker for 45 days. Control-group snails were placed in a beaker with aerated tap water for the same period. Each test was conducted in duplicate. During the testing, the snails were fed with commercial goldfish food, 7 mg/L of dissolved oxygen was maintained by aeration, and the water containing either the [C8 mim]Br solution for the treatment groups or water only for the control group was changed every other day. The numbers of egg sacks in every group were recorded once the snails began laying eggs. No snail deaths occurred during the IL exposure period. After 45 days of exposure, the body weight and body length of every snail in both the treatment and control groups were measured, and the total numbers of egg sacks in every group were tabulated.
Visceral Antioxidant System and Lipid Peroxidation Assays with Adult Snails A total of 20 adult snails (30.74 ± 2.57 mg) were exposed to 12.14 mg/L (approximately 1/10th of the LC50 for [C8 mim]Br in adult snails) or 24.28 mg/L (approximately 1/5th of the LC50 ) of [C8 mim]Br solution in a 500-mL beaker for 96 h. The beaker was covered with cotton gauze to prevent the snails from escaping. The control snails were bred temporarily in a beaker with aerated tap water. Each test was conducted in triplicate. Five snails were collected from each group after 12, 48, and 96 h of exposure for the antioxidant system and lipid peroxidation assays. The remaining five snails were moved to untreated, aerated tap water immediately after 96 h of IL exposure. These snails were allowed to recover in the IL-free water for another 96 h, J Biochem Molecular Toxicology
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and then they were also harvested for the biochemical assays. After sampling at each time point, the snail viscera were quickly isolated, washed with ice-cold 0.9% saline solution, blotted, and weighed. Then, they were homogenized in a 1/10 (w/v) ratio with cold phosphatebuffered saline (pH 7.2). Each homogenate sample was centrifuged at 15,000× g for 10 min at 4◦ C, and the resultant supernatant was stored at –20◦ C for biochemical assays. Visceral antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST), and the contents of glutathione (GSH) and malondialdehyde (MDA) were determined using a Diagnostic Reagent Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, People’s Republic of China), according to the manufacturer’s instructions. The results of antioxidant enzymatic assays are reported in units of enzymatic activity per milligram of protein (U/mg protein). Lipid peroxidation was expressed as nmoles MDA per milligram protein. The total protein content of each sample was determined using the Diagnostic Reagent Kit, with serum albumin as a standard.
Statistical Analyses Data were analyzed using a one-way analysis of variance followed by least significant difference determination using SPSS 11.5. P values less than 0.05 were considered statistically significant.
RESULTS Acute Toxicity in Adult Snails The results of the acute toxicity tests are reported in Figure 1. These results demonstrated that among the tested imidazolium ILs, [C6 mim]Br was the least toxic salt and [C12 mim]Br was the most toxic salt in adult snails (Figure 1). The 96 h LC50 values for [C6 mim]Br, [C8 mim]Br, [C10 mim]Br, and [C12 mim]Br in adult snails were 359.6 ± 11.6, 109.3 ± 4, 8.96 ± 5.66, and 1.35 ± 0.24 mg/L, respectively.
Subchronic Toxicity in Juvenile Snails The subchronic toxicity test results showed that [C8 mim]Br exposure significantly decreased the total number of egg sacks and the body length and weight gain at higher IL exposure concentrations (4 and 8 mg/L) when compared with the control groups (Figure 2A–2C). Further, a low IL exposure concentration (1 mg/L) also decreased body-weight gains
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observed between the treatment and control groups (Figure 3A–3C), suggesting that the IL-treated snails can recover from acute IL toxicity. In addition, GSH content in the IL-treated snails tended to increase following the exposure, even for snails allowed a recovery period (Figure 4A).
Lipid Peroxidation Induced by IL Exposure in Adult Snails MDA levels in the snail viscera in all treatment groups were significantly higher than that of the control groups, including the snails from the recovery test (Figure 4B), indicating that IL exposure induced lipid peroxidation in the snail viscera.
DISCUSSION
FIGURE 2. Total egg sacks, body length gain, and body weight gain of juvenile snails after 45 days of [C8 mim]Br exposure. Subchronic toxicity testing was conducted as described in section 2.4. Data are presented as the mean ± SD. Asterisks denote a response that is significantly different from controls (*P < 0.05, **P < 0.01). A, total egg sacks; B, gained body length; and C, gained body weight.
(Figure 2C). These results suggest that IL exposure may inhibit the growth and reproduction of snails.
Responses of the Visceral Antioxidant System to IL-Induced Toxicity in Snails CAT activities in snail viscera increased at 12 h of IL exposure, decreased at 48 h, and increased again at 96 h in the treatment groups when compared with the control groups (Figure 3A). SOD and GST activities increased after 12 and 48 h of IL exposure, followed by decreased activity at 96 h of exposure (Figure 3B and 3C). However, for snails allowed a 96 h recovery period in the IL-free water, no activity differences were
The results of the acute toxicity tests revealed that all imidazolium ILs with different alkyl chain lengths were toxic to snails. On the basis of the LC50 values obtained in this study, [C8 mim]Br and [C6 mim]Br were moderately toxic, whereas [C10 mim]Br and [C12 mim]Br were highly toxic. These results also indicated that ILs with longer alkyl chains were associated with greater toxicity, suggesting that IL toxicity may be determined mainly by the cationic component of the molecule, consistent with the results obtained previously in bacteria [30], alga [16], and daphnia [27]. Generally, ILs with longer alkyl chains have increased lipophilic properties and enhanced membrane permeability; therefore, they may cause greater toxicity in organisms. Subchronic toxicity testing is a very important determination of the harmful effects of chemicals at low doses on organisms over a long-term exposure. In this study, we found that 45 days of [C8 mim]Br exposure not only inhibited the growth of juvenile snails but also abated their reproductive ability. In addition, we also observed that [C8 mim]Br exposure decreased the hatching rate of P. acuta embryos and promoted the embryonic death rate [23]. These results suggest that the imidazolium ILs may cause reproductive and embryonic toxicity in snails. The antioxidant response system plays a key role in capturing and eliminating the excess toxicant-induced reactive oxygen species (ROS) that may damage intracellular macromolecules and initiate lipid peroxidation of the cellular membrane. The results of the current study showed that acute exposure to [C8 mim]Br induced changes in the activities of SOD, CAT, and GST and also altered the GSH content in snail viscera. This finding is consistent with our previous work regarding IL toxicity in Daphnia magna [27]. In addition, in J Biochem Molecular Toxicology
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FIGURE 3. Activities of antioxidant enzymes in the viscera of adult snails exposed to 12.14 or 24.28 mg/L of [C8 mim]Br, with and without a 96-h recovery period. [C8 mim]Br exposure procedures and the method of determination of CAT, SOD, and GST activities are described in section 2.5. Data are presented as the mean ± SD. Asterisks denote a response that is significantly different from controls (*P < 0.05, **P < 0.01). A, CAT activity; B, SOD; and C, GST.
this study, we also found that the antioxidant enzyme activities recovered to control levels after the animals were rescued from the stress of IL exposure. However, GSH levels failed to return to normal after 96 h of recovery. MDA is the product of the reaction between ROS and unsaturated fatty acids in cellular membranes, and increased concentrations of MDA in tissue indirectly reflect cellular membrane injury. Excess ROS induced by chemicals or a decrease in cellular antioxidant levels may lead to oxidative stress, culminating in cellular damage in organisms. In particular, lipid peroxidation is considered to be a deleterious result of ROS or oxidative stress. In this study, we observed that MDA levels J Biochem Molecular Toxicology
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in snail viscera from all treatment groups, including the recovery group, were significantly higher than that of the snail viscera from control group (Figure 4B), indicating that lipid peroxidation occurred in snail viscera during the IL exposure. The persistent change in the MDA levels in the snails from the recovery group suggests that the excess ROS were not successfully eliminated by the snail antioxidant system. The significantly increased MDA levels provide direct evidence for lipid peroxidation in snail viscera. This result also indicates that ROS and the antioxidant system are likely involved in the toxicity mechanism of ILs in snails. In conclusion, our results demonstrated that imidazolium ILs are toxic to P. acuta, and longer alkyl
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FIGURE 4. The GSH and MDA concentrations in the viscera of adult snails exposed to 12.14 or 24.28 mg/L of [C8 mim]Br, with and without a 96-h recovery period. [C8 mim]Br exposure procedures and the method of determination of GSH and MDA contents are described in section 2.5. Data are presented as the mean ± SD. Asterisks denote a response that is significantly different from controls (*P < 0.05, **P < 0.01). A, GSH contents; and B, MDA.
chains were associated with increased IL toxicity. Moreover, long-term IL exposure at low concentrations inhibited the growth and reproduction of the snails. Finally, IL exposure changed the activities of antioxidant enzymes and induced lipid peroxidation in snail viscera.
REFERENCES 1. Welton T. Room-temperature ionic liquids. Solvents for synthesis and catalysis. Chem Rev 1999;99:2071–2084. ¨ 2. Ranke J, Stolte S, Stormann R, Arning J, Jastorff B. Design of sustainable chemical products—the example of ionic liquids. Chem Rev 2007;107:2183–2206. 3. Wilkes JS. A short history of ionic liquids—from molten salts to neoteric solvents. Green Chem 2002;4:73–80. 4. Yang JZ, Lu XM, Gui JS, Xu WG. A new theory for ionic liquids—the interstice model Part 1. The density and surface tension of ionic liquid. Green Chem 2004;6:541–543. 5. Jungnickel C, Luczak J, Ranke J, Fernandez J, Muller A, Thoming J. Micelle formation of imidazolium ionic liquids in aqueous solution. Colloids Surf, A 2008;316:278– 284. 6. Brennecke JF, Maginn EJ. Ionic liquids: Innovative fluids for chemical processing. AIChE J 2001;47:2384–2389. 7. Gordon CM. New developments in catalysis using ionic liquids. Appl Catal A 2001;222:101–117.
8. Rantwijk F, Madeira Lau R, Sheldon RA. Biocatalytic transformations in ionic liquids. Trends Biotechnol 2003;21:131–138. 9. Weyershausen B, Lehmann K. Industrial application of ionic liquids as performance additives. Green Chem 2004;7:15–19. 10. Hough WL, Smiglak M, Rodriguez H, Swatloski RP, Spear SK, Daly DT, Pernak J, Grisel JE, Carliss RD, Soutullo MD, Davis JH, Rogers RD. The third evolution of ionic liquids: Active pharmaceutical ingredients. New J Chem 2007;31:1429–1436. 11. Gathergood N, Scammells PJ, Garcia MT. Biodegradable ionic liquids: Part III. The first readily biodegradable ionic liquids. Green Chem 2006;8:156–160. 12. Romero A, Santos A, Tojo J, Rodriguez A. Toxicity and biodegradability of imidazolium ionic liquids. J Hazard Mater 2008;151:268–273. ¨ 13. Czerwicka M, Stolte S, Muller A, Siedlecka EM, Gołebiowski M, Kumirska J, Stepnowski P. Identification of ionic liquid breakdown products in an advanced oxidation system. J Hazard Mater 2009;171:478–483. 14. Petkovic M, Ferguson JL, Nimal Gunaratne HQ, Ferreira R, Leit˜ao MC, Seddon KR, Rebelo LPN, Pereira CS. Novel biocompatible cholinium-based ionic liquids—toxicity and biodegradability. Green Chem 2010;12:643–649. 15. Pham TPT, Cho C-W, Yun Y-S. Environmental fate and toxicity of ionic liquids: A review. Water Res 2010;44:352– 372. 16. Cho CW, Jeon YC, Pham TP, Vijayaraghavan K, Yun YS. The ecotoxicity of ionic liquids and traditional organic
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solvents on microalga Selenastrum capricornutum. Ecotoxicol Environ Safe 2008;71:166–171. Latała A, Nedzi M, Stepnowski P. Toxicity of imidazolium and pyridinium based ionic liquids towards algae. Bacillaria paxillifer (a microphytobenthic diatom) and Geitlerinema amphibium (a microphytobenthic blue green alga). Green Chem 2009;11:1371–1376. Luo YR, Li XY, Chen XX, Zhang BJ, Sun ZJ, Wang JJ. The developmental toxicity of 1-methyl-3octylimidazolium bromide on Daphnia magna. Environ Toxicol 2008;23(6):736–744. Pretti C, Chiappe C, Baldetti I, Brunini S, Monni G, Intorre L. Acute toxicity of ionic liquids for three fresh water organisms: Pseudokirchneriella subcapitata, Daphnia magna and Danio rerio. Ecotoxicol Environ Safe 2009;72:1170– 1176. Ventura S P M, Goncalves A M M Goncalves, F, Coutinho J A P. Assessing the toxicity on [C3mim][Tf2N] to aquatic organisms of different trophic levels. Aquatic Toxicol 2010;96:290–297. Li XY, Zeng SH, Dong XY, Ma JG, Wang JJ. Acute toxicity and responses of antioxidant systems to 1methyl-3-octylimidazolium bromide at different developmental stages of goldfish. Ecotoxicology 2012;21:253– 259. Bernot RJ, Kennedy EE, Lamberti GA. Effects of ionic liquids on the survival, movement, and feeding behavior of the freshwater snail, Physa acuta. Environ Toxicol Chem 2005;24:1759–1765.
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23. Li XY, Dong XY, Bai X, Liu Li, Wang JJ. The embryonic and post-embryonic developmental toxicity of imidazolium-based ionic liquids on Physa acuta. Environ Toxicol 2013 (Online available). 24. Albrecht C, Oliver Kroll O, Terrazas EM, Wilke T. Invasion of ancient Lake Titicaca by the globally invasive Physa acuta (Gastropoda: Pulmonata: Hygrophila). Biol Invasions 2009;11:1821–1826. 25. Guo YH, Wang CM, Luo J, He HX. Physa acuta found in Beijing, China. Chinese J Zool 2009;44:127–128. 26. Guo EM, Zhao GF. Reproductive behavior and embryonic development in Conch Physa acuta. Fishery Sci 2009;28:594–597. 27. Yu M, Wang SH, Luo YR, Li XY, Zhang BJ, Wang JJ. Effects of the1-alkyl-3-methylimidazolium bromide ionic liquids on the antioxidant defense system of Daphnia magna. Ecotoxicol Environ Safe 2009;72:1798–1804. 28. Ma JM, Cai LL, Zhang BJ, Hu LW, Li XY, Wang JJ. Acute toxicity and effects of 1-alkyl-3-methylimidazolium bromide ionic liquids on green algae. Ecotoxicol Environ Safe 2010;73:1465–1469. 29. K¨arber G. Beitrag zur kollektiven behandlung pharmakologischer reihenversuche. Arch Exp Pathol Pharmakol 1931;162:480–482. ¨ 30. Ranke J, Molter K, Stock F, Bottin-Weber U, Poczobutt J, Hoffmann J, Ondruschka B, Filser J, Jastorff B. Biological effects of imidazolium ionic liquids with varying chain lengths in acute Vibrio fischeri and WST-1 cell viability assays. Ecotoxicol Environ Safe 2004;58:396–404.
Toxicity of Imidazolium-Based Ionic Liquids on Physa Acuta and the Snail Antioxidant Stress Response Junguo Ma,1 Xiangyi Dong,1 Qian Fang,1 Xiaoyu Li,1 and Jianji Wang2 1 College of Life Science, Henan Normal University, Xinxiang, Henan Province, People’s Republic of China; E-mail: [email protected]; [email protected] 2 Key
Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan Province, People’s Republic of China Received 30 August 2013; revised 12 October 2013; accepted 16 October 2013
ABSTRACT: In the present study, the acute and developmental toxicities of imidazolium ionic liquids (ILs) with different alkyl chain lengths, as well as the antioxidant response and lipid peroxidation levels were evaluated in the snail, Physa acuta. Longer alkyl chains corresponded to increased IL toxicity in snails. Long-term IL exposure at lower concentrations inhibited snail growth and reproduction. We also found that IL inhibited the activities of superoxide dismutase (SOD) and glutathione S-transferase (GST), promoted the activity of catalase (CAT), and increased the glutathione content. However, SOD, GST, and CAT activities returned to control levels after 96 h of recovery. In addition, malondialdehyde levels were increased in treatment groups compared with the control and did not return to control levels even after a recovery period, indicating that ILs induced lipid peroxidation in snail viscera. These results suggest that oxidative stress and lipid peroxidation may be involved in the mechanism of toxC 2013 Wiley Periodicals, Inc. J. Biochem. icity for ILs. Mol. Toxicol. 28:69–75, 2014; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21537
KEYWORDS: Ionic Liquid; Physa Acuta; Toxicity; Antioxidant Enzymes; Lipid Peroxidation
INTRODUCTION Ionic liquids (ILs) are salts with relatively low melting points (below 100◦ C), and they are expected to Correspondence to: Xiaoyu Li. Contract Grant Sponsor: National Science Foundation of China. Contract Grant Number: 31172415, 20573019, and 20573034. Contract Grant Sponsor: Key Subjects of Biology and Ecology in Henan Province, People’s Republic of China. C 2013 Wiley Periodicals, Inc.
replace conventional volatile organic solvents to reduce air pollution [1, 2]. In recent years, ILs have attracted considerable attention due to their unique characteristics and properties, such as negligible vapor pressure, high thermal stability, and excellent solvation ability [3–5]. Therefore, they have been regarded as promising “green” solvents in manufacturing, processing, and cleaning technologies [6–10]. However, IL toxicity to aquatic organisms and possible environmental risks to aquatic ecosystems are a potential concern because most ILs are water soluble, poorly decomposed by microorganisms, and prone to entering the aquatic environment by accidental discharges [11–15]. Hence, the available literature regarding IL toxicity to aquatic organisms has been expanding; for example, IL-toxicity evaluations are available for algae [16, 17], cladocerans [18–20], and fish [21]. In contrast, limited reports regarding the toxicity of ILs to snails are available, including the works of Bernot et al. [22] and Li et al. [23]. The snail Physa acuta is a globally invasive species [24,25]. However, these snails are very sensitive to toxicant exposure and thus, appropriate for toxicity testing [26]. Imidazolium-based ILs were evaluated in the present study based on a two-point rationale. First, these chemicals usually have moderate or relatively decreased toxicity in organisms. Second, and more importantly, the toxic response of organisms to ILs is typically dose dependent, and the results from toxicity testing for common imidazolium ILs can be contextualized with the results of our previous studies [18,21,23,27,28]. The current study aimed to determine the acute toxicities of the imidazolium ILs with different alkyl chain lengths in adult snails, the reproductive and developmental toxicities of ILs in juvenile snails, and ILs’ effects on the snail antioxidant system to elucidate the biochemical mechanism of IL-induced toxicity.
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MATERIALS AND METHODS ILs and Chemicals The imidazolium-based ILs employed in the present study were 1-hexyl-3-methylimidazolium bromide ([C6 mim]Br), 1-octyl-3-methylimidazolium bromide ([C8 mim]Br), 1-decyl-3-methylimidazolium and 1-dodecyl-3bromide ([C10 mim]Br), methylimidazolium bromide ([C12 mim]Br). They were purchased from Hubei Hengshuo Chemical Company, Ltd., Wuhan, Hubei, People’s Republic of China, and their purities were 99%. Analytical grade reagents were purchased from Sigma (St Louis, MO). The IL solutions were prepared by dissolving the ILs in distilled water, followed by dilution to the desired concentrations with aerated tap water when tests were performed.
Snails P. acuta specimens were collected from a fishery pond in China. Specimens were maintained in our laboratory in glass jars (3 L in volume) with aerated tap water (total hardness 340 mg/L, pH 7.6, turbidity 1.5 NTU, total dissolved solid content 660 mg/L) at 20 ± 1◦ C and a photoperiod of 16 h:8 h light/darkness for several generations prior to the experiment. The snails were fed ad libitum with commercial goldfish food (Wannong Fishery Company, Xinxiang, Henan, People’s Republic of China) at a rate of 0.5%–1% of body weight per day, and the water was changed weekly. The adult snails had an average wet weight of 30.74 ± 2.57 mg and shell length of 7.26 ± 0.98 mm, and the juvenile snails (30 days after hatching) had an average wet weight of 8.85 ± 0.93 mg and shell length of 4.25 ± 0.36 mm. The adult and juvenile snails were used for the toxicity tests and antioxidant system assays described below. Generally, the first oviposition of the snail (sexual maturity) occurred at 45 to 60 days after hatching; following this developmental time point, the snails laid eggs (20–40) encapsulated in egg sacks once every 1 to 3 days. Snail longevity was approximately 12–18 months under our laboratory conditions.
Determination of the Acute Toxicity of Imidazolium ILs with Different Alkyl Chain Lengths in Adult Snails The acute toxicities of the imidazolium-based ILs with different alkyl chain lengths, that is, [C6 mim]Br, [C8 mim]Br, [C10 mim]Br, and [C12 mim]Br, were determined in adult snails (average wet body weight 30.74 ± 2.57 mg and average shell length 7.26 ±
FIGURE 1. Death rates in adult snails exposed to a concentration range of imidazolium ILs with different alkyl chain lengths. The acute toxicity tests were carried out according to the Spearman– K¨arber method [29], as described in section 2.3. Data are presented as the mean ± standard deviation (SD). Asterisks denote a response that is significantly different from controls (**P < 0.01). A, [C6 mim]Br; B, [C8 mim]Br; C, [C10 mim]Br; and D, [C12 mim]Br.
0.98 mm) according to the Spearman–K¨arber method [29]. Ten snails were placed in a 500-mL beaker containing 300 mL of a range of concentrations of IL solutions, as shown in Figure 1. The control snails were also bred temporarily in a beaker, but with aerated tap water only. The beakers were covered with cotton J Biochem Molecular Toxicology
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gauze to prevent the snails from escaping. Each test was conducted in triplicate. Any dead snails were removed immediately from the beaker, and the total numbers of dead and surviving snails were recorded in each group during the exposure period. The 96 h 50% of lethal concentration (LC50 ) values and their 95% confidence limits were calculated using SPSS 11.5.
Determination of the Subchronic Toxicity of [C8 mim]Br in Juvenile Snails Subchronic toxicity testing was designed to evaluate the effects of [C8 mim]Br exposure on the growth, development, and reproduction of juvenile snails. The tests included four groups, of which three served as IL-treatment groups (1, 4, 8 mg/L of [C8 mim]Br) and one served as a control. In the treatment groups, a total of 12 juvenile snails (30 days after hatching), with an average wet weight of 8.85 ± 0.93 mg and an average shell length of 4.25 ± 0.36 mm, were exposed to [C8 mim]Br solutions at concentrations of 1, 4, and 8 mg/L in a 500 mL beaker for 45 days. Control-group snails were placed in a beaker with aerated tap water for the same period. Each test was conducted in duplicate. During the testing, the snails were fed with commercial goldfish food, 7 mg/L of dissolved oxygen was maintained by aeration, and the water containing either the [C8 mim]Br solution for the treatment groups or water only for the control group was changed every other day. The numbers of egg sacks in every group were recorded once the snails began laying eggs. No snail deaths occurred during the IL exposure period. After 45 days of exposure, the body weight and body length of every snail in both the treatment and control groups were measured, and the total numbers of egg sacks in every group were tabulated.
Visceral Antioxidant System and Lipid Peroxidation Assays with Adult Snails A total of 20 adult snails (30.74 ± 2.57 mg) were exposed to 12.14 mg/L (approximately 1/10th of the LC50 for [C8 mim]Br in adult snails) or 24.28 mg/L (approximately 1/5th of the LC50 ) of [C8 mim]Br solution in a 500-mL beaker for 96 h. The beaker was covered with cotton gauze to prevent the snails from escaping. The control snails were bred temporarily in a beaker with aerated tap water. Each test was conducted in triplicate. Five snails were collected from each group after 12, 48, and 96 h of exposure for the antioxidant system and lipid peroxidation assays. The remaining five snails were moved to untreated, aerated tap water immediately after 96 h of IL exposure. These snails were allowed to recover in the IL-free water for another 96 h, J Biochem Molecular Toxicology
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and then they were also harvested for the biochemical assays. After sampling at each time point, the snail viscera were quickly isolated, washed with ice-cold 0.9% saline solution, blotted, and weighed. Then, they were homogenized in a 1/10 (w/v) ratio with cold phosphatebuffered saline (pH 7.2). Each homogenate sample was centrifuged at 15,000× g for 10 min at 4◦ C, and the resultant supernatant was stored at –20◦ C for biochemical assays. Visceral antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST), and the contents of glutathione (GSH) and malondialdehyde (MDA) were determined using a Diagnostic Reagent Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, People’s Republic of China), according to the manufacturer’s instructions. The results of antioxidant enzymatic assays are reported in units of enzymatic activity per milligram of protein (U/mg protein). Lipid peroxidation was expressed as nmoles MDA per milligram protein. The total protein content of each sample was determined using the Diagnostic Reagent Kit, with serum albumin as a standard.
Statistical Analyses Data were analyzed using a one-way analysis of variance followed by least significant difference determination using SPSS 11.5. P values less than 0.05 were considered statistically significant.
RESULTS Acute Toxicity in Adult Snails The results of the acute toxicity tests are reported in Figure 1. These results demonstrated that among the tested imidazolium ILs, [C6 mim]Br was the least toxic salt and [C12 mim]Br was the most toxic salt in adult snails (Figure 1). The 96 h LC50 values for [C6 mim]Br, [C8 mim]Br, [C10 mim]Br, and [C12 mim]Br in adult snails were 359.6 ± 11.6, 109.3 ± 4, 8.96 ± 5.66, and 1.35 ± 0.24 mg/L, respectively.
Subchronic Toxicity in Juvenile Snails The subchronic toxicity test results showed that [C8 mim]Br exposure significantly decreased the total number of egg sacks and the body length and weight gain at higher IL exposure concentrations (4 and 8 mg/L) when compared with the control groups (Figure 2A–2C). Further, a low IL exposure concentration (1 mg/L) also decreased body-weight gains
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observed between the treatment and control groups (Figure 3A–3C), suggesting that the IL-treated snails can recover from acute IL toxicity. In addition, GSH content in the IL-treated snails tended to increase following the exposure, even for snails allowed a recovery period (Figure 4A).
Lipid Peroxidation Induced by IL Exposure in Adult Snails MDA levels in the snail viscera in all treatment groups were significantly higher than that of the control groups, including the snails from the recovery test (Figure 4B), indicating that IL exposure induced lipid peroxidation in the snail viscera.
DISCUSSION
FIGURE 2. Total egg sacks, body length gain, and body weight gain of juvenile snails after 45 days of [C8 mim]Br exposure. Subchronic toxicity testing was conducted as described in section 2.4. Data are presented as the mean ± SD. Asterisks denote a response that is significantly different from controls (*P < 0.05, **P < 0.01). A, total egg sacks; B, gained body length; and C, gained body weight.
(Figure 2C). These results suggest that IL exposure may inhibit the growth and reproduction of snails.
Responses of the Visceral Antioxidant System to IL-Induced Toxicity in Snails CAT activities in snail viscera increased at 12 h of IL exposure, decreased at 48 h, and increased again at 96 h in the treatment groups when compared with the control groups (Figure 3A). SOD and GST activities increased after 12 and 48 h of IL exposure, followed by decreased activity at 96 h of exposure (Figure 3B and 3C). However, for snails allowed a 96 h recovery period in the IL-free water, no activity differences were
The results of the acute toxicity tests revealed that all imidazolium ILs with different alkyl chain lengths were toxic to snails. On the basis of the LC50 values obtained in this study, [C8 mim]Br and [C6 mim]Br were moderately toxic, whereas [C10 mim]Br and [C12 mim]Br were highly toxic. These results also indicated that ILs with longer alkyl chains were associated with greater toxicity, suggesting that IL toxicity may be determined mainly by the cationic component of the molecule, consistent with the results obtained previously in bacteria [30], alga [16], and daphnia [27]. Generally, ILs with longer alkyl chains have increased lipophilic properties and enhanced membrane permeability; therefore, they may cause greater toxicity in organisms. Subchronic toxicity testing is a very important determination of the harmful effects of chemicals at low doses on organisms over a long-term exposure. In this study, we found that 45 days of [C8 mim]Br exposure not only inhibited the growth of juvenile snails but also abated their reproductive ability. In addition, we also observed that [C8 mim]Br exposure decreased the hatching rate of P. acuta embryos and promoted the embryonic death rate [23]. These results suggest that the imidazolium ILs may cause reproductive and embryonic toxicity in snails. The antioxidant response system plays a key role in capturing and eliminating the excess toxicant-induced reactive oxygen species (ROS) that may damage intracellular macromolecules and initiate lipid peroxidation of the cellular membrane. The results of the current study showed that acute exposure to [C8 mim]Br induced changes in the activities of SOD, CAT, and GST and also altered the GSH content in snail viscera. This finding is consistent with our previous work regarding IL toxicity in Daphnia magna [27]. In addition, in J Biochem Molecular Toxicology
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FIGURE 3. Activities of antioxidant enzymes in the viscera of adult snails exposed to 12.14 or 24.28 mg/L of [C8 mim]Br, with and without a 96-h recovery period. [C8 mim]Br exposure procedures and the method of determination of CAT, SOD, and GST activities are described in section 2.5. Data are presented as the mean ± SD. Asterisks denote a response that is significantly different from controls (*P < 0.05, **P < 0.01). A, CAT activity; B, SOD; and C, GST.
this study, we also found that the antioxidant enzyme activities recovered to control levels after the animals were rescued from the stress of IL exposure. However, GSH levels failed to return to normal after 96 h of recovery. MDA is the product of the reaction between ROS and unsaturated fatty acids in cellular membranes, and increased concentrations of MDA in tissue indirectly reflect cellular membrane injury. Excess ROS induced by chemicals or a decrease in cellular antioxidant levels may lead to oxidative stress, culminating in cellular damage in organisms. In particular, lipid peroxidation is considered to be a deleterious result of ROS or oxidative stress. In this study, we observed that MDA levels J Biochem Molecular Toxicology
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in snail viscera from all treatment groups, including the recovery group, were significantly higher than that of the snail viscera from control group (Figure 4B), indicating that lipid peroxidation occurred in snail viscera during the IL exposure. The persistent change in the MDA levels in the snails from the recovery group suggests that the excess ROS were not successfully eliminated by the snail antioxidant system. The significantly increased MDA levels provide direct evidence for lipid peroxidation in snail viscera. This result also indicates that ROS and the antioxidant system are likely involved in the toxicity mechanism of ILs in snails. In conclusion, our results demonstrated that imidazolium ILs are toxic to P. acuta, and longer alkyl
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FIGURE 4. The GSH and MDA concentrations in the viscera of adult snails exposed to 12.14 or 24.28 mg/L of [C8 mim]Br, with and without a 96-h recovery period. [C8 mim]Br exposure procedures and the method of determination of GSH and MDA contents are described in section 2.5. Data are presented as the mean ± SD. Asterisks denote a response that is significantly different from controls (*P < 0.05, **P < 0.01). A, GSH contents; and B, MDA.
chains were associated with increased IL toxicity. Moreover, long-term IL exposure at low concentrations inhibited the growth and reproduction of the snails. Finally, IL exposure changed the activities of antioxidant enzymes and induced lipid peroxidation in snail viscera.
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