The embryonic and postembryonic developmental toxicity of imidazolium-based ionic liquids on Physa acuta
Abstract
The embryonic and postembryonic developmental toxicity of imidazolium-based ionic liquids (ILs) to the snail Physa acuta was evaluated in this study. The results of embryonic toxicity tests showed that lower concentrations of 1-octyl-3-methylimidazolium bromide ([C8mim]Br) (1.5 and 2.1 mg/L) inhibited the hatching rate of snail embryos, and partial snails hatched normally and died, while all of the treated embryos died when the exposure concentration was higher than 4.16 mg/L, at which IL caused the deformation, death, and decay of snail embryos. Statistical analyses revealed obvious differences in the hatching rates between three developmental stages in the 2.1 and 2.94 mg/L groups, indicating that the veliger stage is more sensitive to [C8mim]Br exposure than the blastula and gastrula stages. Furthermore, the 96 h LC50 values of [C8mim]Br on the tested snails at three developmental stages (juvenile, subadult, and adult) were 70.83 ± 2.99, 97.59 ± 4.05, and 109.3 ± 2.22 mg/L, respectively, indicating that young snails were more sensitive to [C8mim]Br toxicity than adults. In addition, the 96 h LC50 values of ILs with different alkyl chain lengths, that is, [C12mim], [C10mim], [C8mim], and [C6mim], in adult snails were 1.35 ± 0.24, 8.96 ± 5.66, 109.3 ± 4, and 359.6 ± 11.6 mg/L, respectively, suggesting that longer alkyl chains can increase the toxicity of imidazolium ILs on snails. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 697–704, 2014.
INTRODUCTION
Conventional organic solvents used in the chemical industry tend to cause air pollution, and this drawback has motivated the development of new nonvolatile solvents to substitute for these volatile organic solvents. Ionic liquids (ILs) have come along at the right moment (Welton, 1999). ILs are salts with low melting points (below 100°C) and are composed solely of cations, mainly ammonium, imidazolium, pyridinium, piperidinium, and pyrrolidinium, and anions, such as chloride (Cl−), tetrafluoroborate (BF4−), hexafluorophosphate (PF6−), and bromide (Br−) (Ranke et al., 2007). In recent years, ILs have attracted considerable attention due to their unique characteristics and properties, such as negligible vapor pressure, thermal stability, and solvation ability (Wilkes, 2002; Yang et al., 2004; Jungnickel et al., 2008). ILs are claimed to be environmentally benign because they usually have negligible vapor (Sheldon, 2005). Currently, they have been regarded as promising “green” substitutes for conventional organic solvents in manufacturing, processing, and cleaning technologies by both commercial businesses and individual consumers (Brennecke and Maginn, 2001; Gordon, 2001; Rantwijk et al., 2003; Weyershausen and Lehmann, 2004; Hough et al., 2007).
However, concerns about IL toxicity to aquatic organisms and environmental risks to aquatic ecosystems have been raised recently (Pham et al., 2010) because most ILs are water-soluble and might enter the aquatic environment through accidental leakage or effluent. Meanwhile, many reports have indicated that most ILs are poorly decomposed by microorganisms (Gathergood et al., 2006; Romero et al., 2008; Czerwicka et al., 2009; Petkovic et al., 2010). There have been many studies regarding IL toxicity to aquatic organisms, such as algae (Cho et al., 2008; Latala et al., 2009, 2010; Pretti et al., 2009), cladocerans (Luo et al., 2008; Pretti et al., 2009; Ventura et al., 2010), mussels (Costello et al., 2009), and fish (Pretti et al., 2009; Li et al., b). In 2005, Bernot et al. studied the effects of ILs on the survival and behavior (movement and feeding rates) of the freshwater pulmonate snail Physa acuta (Bernot et al., 2005). Thereafter, however, to the best of our knowledge, there has been no literature available until now with regard to the ecotoxicity and embryonic developmental toxicity of ILs on snails, although our laboratory has studied the embryonic developmental toxicity of ILs on goldfish and frogs (Li et al., 2009; Wang et al., 2010).
Imidazolium ILs have been widely investigated as possible substitutes for organic solvents (Welton, 1999; Jungnickel et al., 2008). Furthermore, they are stable and conductive at room temperature and possess all of the properties of ILs. Additionally, the toxicity response of imidazolium ILs in animals was dose-dependent, and the results of toxicity testing would be representative of common ILs, according to previous reports (Luo et al., 2008; Latala et al., 2009, 2010; Li et al., b). Therefore, imidazolium-based ILs were chosen as the experimental material in this study.
Aquatic snails are important aquatic populations to maintain a balanced ecosystem. The snail P. acuta is an invasive species and is now widely distributed in Chinese freshwater environments (Albrecht et al., 2009; Guo, et al., 2009). They have a high reproductive capacity; for example, they can spawn every day when the environment (temperature, food, and light) is suitable, and they lay large amounts of eggs (Guo and Zhao, 2009). Therefore, it is easy to obtain and culture their eggs under laboratory conditions. Furthermore, their embryonic development is rapid (usually 1 week), and the developmental process can be observed with microscopy because their egg mass and chorion are transparent (Guo and Zhao, 2009). Above all, P. acuta is sensitive to toxicant exposure and thus appropriate for toxicity testing (Evans-White and Lamberti, 2009; Sánchez-Argüello et al., 2009; Lance et al., 2010; Musee et al., 2010). The aim of this study was to determine the embryonic and postembryonic developmental toxicity of imidazolium ILs on P. acuta and evaluate the consequences and environmental risks of ILs in aquatic ecosystems.
MATERIALS AND METHODS
ILs and Chemicals
The imidazolium-based ILs employed in this study were 1- hexyl-3-methylimidazolium bromide ([C6mim]Br), 1-octyl-3-methylimidazolium bromide ([C8mim]Br), 1-decyl-3-methylimidazolium bromide ([C10mim]Br), and 1-dodecyl-3-methylimidazolium bromide ([C12mim]Br). All of the ILs, which had a purity of more than 99%, were purchased from Hubei Hengshuo Chemical, China. The IL solutions were prepared by dissolving the IL in distilled water and then diluting it to the desired concentrations with distilled water or embryonic hatching solution when required. Analytical grade reagents were purchased from Sigma (St Louis, MO).
Culture of the Snails and Their Embryos
The snail P. acuta was collected from a fishery pond in China and maintained in glass jars (3 L in volume) with aerated tap water [total hardness of water 340 mg/L, pH 7.6, turbidity 1.5 nephelometric turbidity units, and total dissolved solid content 660 mg/L] at 25 ± 1°C and a photoperiod of 16 h:8 h light/darkness under laboratory conditions for several generations prior to the experiment, and outcrossing was dominant. The snails were fed ad libitum with commercial goldfish food (Wannong Fishery Company, China) at a rate of 0.5–1% of body weight/day, and the water was changed weekly. The adult snails used in the experiments had an average wet weight with shell of 30.74 ± 2.57 g and an average shell length of 7.26 ± 0.98 mm. After 1 month of acclimation, egg masses containing embryos were collected from the culture jar and transferred to a petri dish (60 mm in diameter) with embryo culture solution recommended by the ISO (International Organization for Standardization, 1999), which contained 65 mM NaHCO3, 6 mM KCl, 294 mM CaCl2·2H2O, and 123 mM MgSO4·7H2O, to complete their embryonic development. The embryonic developmental stages of the snail were determined according to the report by Guo et al. ( 2009). Once they hatched, larvae were immediately transferred to 120 mm-diameter petri dishes containing aerated tap water to complete their postembryonic development. After 10–15 days of hatching, the juveniles were fed with fine particulate fish food (Wannong Fishery Company, China) ad libitum. When the juveniles reached an appropriate size to be handled, they were transferred to glass jars (3 L) with aerated tap water. The food for this new generation was progressively increased up to 0.5% of their body weight/day, and the water was changed weekly. The first oviposition of the snail (sexual maturity) occurred 2 months after hatching under our culture conditions. Three months later, they gradually became completely adult. Their lifespan was ∼12–18 months according to our observation under laboratory conditions.
Embryonic Toxicity of [C8mim]Br
Approximately 50–100 adults were placed in three clean jars with aerated tap water to allow mating and spawning. The egg masses were collected from the jars the following morning and transferred to a 60 mm-diameter petri dish for embryonic toxicity tests. The toxicity test design and the exposure concentrations were based on the Spearman-Kärber method (Kärber, 1931) and the results of primary acute toxicity testing. Briefly, the test included six groups, five of which received IL treatment and one served as a control. For the treatment groups, two embryo masses (each contained 20–30 eggs) were placed in a culture dish containing 10 mL of different concentrations of test solution obtained by diluting the stock solution of [C8mim]Br with the embryonic hatching solution. Embryos at different development stages, that is, blastula, gastrula, and veliger, were exposed to 1.5, 2.1, 2.94, 4.16, or 5.76 mg/L of [C8mim]Br, and the control embryos were cultured under the same conditions as the treatment groups except without [C8mim]Br in their hatching solution. All of the embryonic hatching solutions were changed daily. Each test was conducted in triplicate. The numbers of hatched larvae and dead embryos were recorded in each group during the treatment period. Data acquisition was stopped and the LC50 was calculated when the embryonic development of control snails was completed (∼7 days at 25 ± 1°C). The acute toxicity of [C8mim]Br on snail embryos was assessed by calculating the percentage of dead embryos in each of the [C8mim]Br-treated groups, and the LC50 was expressed as the median concentration that would kill 50% of embryos after embryo development was complete. The mortality and hatching rate of snail embryos were expressed as percentage of the dead embryos and hatched larva, respectively, in the total tested embryos.
Postembryonic Developmental Toxicity of [C8mim]Br
Three postembryonic developmental stages, juvenile (1 month after hatching, with average wet body weights of 8.85 ± 0.93 g and shell length of 4.25 ± 0.36 mm), subadult (2 months, body weights of 18.07 ± 1.1 g and shell length of 5.35 ± 0.27 mm), and adult (3 months, body weights of 30.74 ± 2.57 g and shell length of 7.26 ± 0.98 mm), were chosen for toxicity tests. The numbers, body weights, and lengths of the tested snails and exposure concentrations of [C8mim]Br are described in Table 1. Acute toxicity tests were carried out using the Spearman-Kärber method (Kärber, 1931), with modifications, to obtain the 50% lethal concentration (LC50) of [C8mim]Br in snails after 96 h of exposure. Ten snails were placed in a 500 mL beaker containing 300 mL of [C8mim]Br solution, and the control snails were bred temporarily in a beaker with aerated tap water. The beaker was covered with cotton gauze to prevent snails from escaping. The cotton gauze must be on the surface of water in the beaker so that the snails cannot choke due to dryness if they attach to the cotton gauze. Each test was conducted in triplicate. The dead snails were immediately removed from the beaker, and the total number of dead and surviving snails was recorded in each group during the period of exposure.
| Developmental stage | Embryo numbers/group | IL concentrations (mg/L) | LC50 (mg/L) | 95% Confidence limits |
|---|---|---|---|---|
| Blastula | 40 | 0, 1.5, 2.1, 2.94, 4.16, 5.76 | 2.70 ± 0.69a | 2.10–3.47 |
| Gastrula | 40 | 0, 1.5, 2.1, 2.94, 4.16, 5.76 | 2.61 ± 0.69a | 2.02–3.39 |
| Veliger | 40 | 0, 1.5, 2.1, 2.94, 4.16, 5.76 | 2.21 ± 0.54a | 1.74–2.82 |
| Juvenile (1 month) | 10 | 0, 50, 60, 72, 86.4, 103.7 | 70.83 ± 2.99b | 58.83–70.81 |
| Subadult (2 months) | 10 | 0, 80, 88, 96.8, 106.5, 117.1 | 97.59 ± 4.05c | 89.49–105.69 |
| Adult (3 months) | 10 | 0, 90.9, 100, 110, 121, 133 | 109. 3 ± 2.22d | 104.8–114.3 |
- Toxicity test design and exposure concentrations were based on the Spearman-Kärber method (Kärber, 1931) and the results of primary acute toxicity testing, which are described in Section Materials and Methods. Each test was conducted in triplicate, and the results are expressed as the mean ± S.D. The values with the same superscript are not significantly different (P > 0.05).
Toxicity of ILs with Different Alkyl Chain Lengths on Adult Snails
This test was designed to determine the relationship between IL alkyl chain lengths and their toxicity, and four types of the imidazolium-based ILs, that is, [C6mim]Br, [C8mim]Br, [C10mim]Br, and [C12mim]Br, and adult snails (average wet body weight of 30.74 ± 2.57 g and shell length of 7.26 ± 0.98 mm) were employed in the test. Acute toxicity tests were carried out using the Spearman-Kärber method (Kärber, 1931), with modifications, to obtain the 96 h LC50 of the ILs on snails. The numbers, body weights, and lengths of the tested snails and exposure concentrations are described in Table 2. The methods for IL exposure, observation, and data acquisition were similar to section “Toxicity of ILs with different alkyl chain lengths on adult snails.”
| ILs | Snail numbers/group | IL concentrations (mg/L) | 96 h LC50 (mg/L) | 95% Confidence limits |
|---|---|---|---|---|
| [C6mim]Br | 10 | 0, 300, 330, 360, 399.3, 439.2 | 359.6 ± 11.6a | 346.2–369.3 |
| [C8mim]Br | 10 | 0, 90.9, 100, 110, 121, 133 | 109.3 ± 4b | 105.5–113.5 |
| [C10mim]Br | 10 | 0, 3.5, 5.95, 10.1, 17.2, 29.2 | 8.96 ± 5.66c | 6.81–18.13 |
| [C12mim]Br | 10 | 0, 0.5, 0.85, 1.45, 2.46, 4.18 | 1.35 ± 0.24d | 1.37–1.85 |
- The acute toxicity tests of imidazolium-based ILs with different alkyl chain lengths on adult snails (average wet body weight, 30.74 ± 2.57 g; shell length, 7.26 ± 0.98 mm) were carried out according to the Spearman-Kärber method (Kärber, 1931), which is described in Section Materials and Methods. Each test was conducted in triplicate, and the results are expressed as the mean ± S.D. The values with the same superscript are not significantly different (P > 0.05).
Statistical Analyses
Data were analyzed using a one-way analysis of variance followed by least significant difference determination using SPSS 11.5. P values <0.05 were considered statistically significant. LC50 values and their 95% confidence limits were calculated using SPSS 11.5.
RESULTS
Toxic Effects of [C8mim]Br on the Embryonic Development of P. acuta
Effects of [C8mim]Br on the embryonic hatching rate and mortality of the snails are displayed in Figure 1. Regardless of the stage exposed, a marked dose-dependent response was observed between the exposure concentration and the hatching rate and mortality, as shown in Figure 1(A–C). In the lower concentration groups (1.5 and 2.1 mg/L), a small number of embryos hatched normally, and partial snails died. Meanwhile, at concentrations higher than 4.16 mg/L, all [C8mim]Br-treated embryos died and then decayed, and no embryo hatched. Microscopic observation demonstrated that [C8mim]Br exposure caused the deformation, death, and decay of embryos (Fig. 2). In addition, no statistically significant difference in LC50 values of [C8mim]Br was observed between the three stages (Table 1). However, further statistical analysis revealed obvious differences in the hatching rates between the three stages in the 2.1 and 2.94 mg/L groups [Fig. 1(D)], indicating that the veliger stage was more sensitive to [C8mim]Br exposure than the blastula and gastrula stages.
Hatching rates and mortalities of P. acuta embryos exposed to different concentrations of [C8mim]Br at three developmental stages. The asterisk (*) denotes a response that is significantly different from the control (*P < 0.05; **P < 0.01). The values with the same superscript are not significantly different (P > 0.05). A: [C8mim]Br exposure at the blastula stage (48 h after fertilization). B: Gastrula stage (72 h). C: Veliger stage (144 h). D: Hatching rate comparison of P. acuta embryos at three developmental stages.
Developing P. acuta embryos at different stages and [C8mim]Br-treated embryos (all at 100 × of magnification). The embryo culture and [C8mim]Br exposure methods are described in Section Material and Methods. The embryonic development stages of the snail were determined according to the report by Guo et al. ( 2009). A: Normal embryo at theblastula developmental stage (48 h after fertilization). B: Gastrula embryo (72 h). C: Trochophore embryo (120 h). D: Veliger embryo (144 h). E: Hatching embryo (196 h). F: A deformed embryo treated with 1.5 mg/L of [C8mim]Br at the blastula stage (Image after 140 h of exposure). G: A decayed embryo treated with 5.76 mg/L of [C8mim]Br at the gastrula stage (Image after 76 h of exposure). H: A deformed embryo treated with 2.94 mg/L of [C8mim]Br at the veliger stage (Image after 48 h of exposure). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Acute Toxicity of [C8mim]Br on Snails at Three Developmental Stages
The results of acute toxicity tests showed that [C8mim]Br had toxic effects on the survivals of snails at three developmental stages, and a dose-dependent response is shown in Figure 3. Furthermore, the 96 h LC50 values of [C8mim]Br on the tested snails at three developmental stages, that is, juvenile, subadult, and adult, were 70.83 ± 2.99, 97.59 ± 4.05, and 109.3 ± 2.22 mg/L, respectively (Table 1), indicating that young snails were more sensitive to IL toxicity than adults. Additionally, behavioral observation showed that the poisoned snails in treatment groups tried to escape from the treatment solution, and thus, a number of snails adhered to the underside of the cotton gauze on the surface of the solution in the beaker. The dead snails usually sank to the bottom of the beaker and did not respond or move when touched.
Mortalities of three developmental stages of P. acuta snails exposed to different concentrations of [C8mim]Br. The asterisk (*) denotes a response that is significantly different from the control (**P < 0.01). A: Juvenile snail (1 month after hatching). B: Subadult snail (2 months). C: Adult snail (3 months).
Acute Toxicity of ILs with Different Alkyl Chain Lengths on Adult Snails
The 96 h LC50 values of ILs with different alkyl chain lengths, that is, [C12mim]Br, [C10mim]Br, [C8mim]Br, and [C6mim]Br, in the adult snails were 1.35 ± 0.24, 8.96 ± 5.66, 109.3 ± 4, and 359.6 ± 11.6 mg/L, respectively (Table 2). According to the results, [C6mim]Br was the least toxic salt and [C12mim]Br was the most toxic among the tested ILs, showing the following order of toxicity strength: [C12mim]Br > [C10mim]Br > [C8mim]Br > [C6mim]Br.
DISCUSSION
The results of embryonic-developmental toxicity tests in this study indicate that [C8mim]Br has toxic effects on the embryonic development of snails, suggesting that ILs may disturb aquatic ecosystems once they are released into water. Although there was no significant difference in the LC50 of [C8mim]Br between the three stages (Table 1), differences in the hatching rates between the three stages in the 2.1 and 2.94 mg/L groups [Fig. 1(D)] were found. This result indicates that the snail embryo sensitivity to [C8mim]Br exposure is developmental stage specific, which is consistent with our previous studies in freshwater fish and amphibians treated with [C8mim]Br (Li et al., 2009; Wang et al., 2010). Comparing the results of these embryonic toxicity tests (Table 3), we also found that the LC50 values of [C8mim]Br in blastula embryos from the three types of aquatic animals were different. The blastula embryos of snails are 10-fold or 100-fold more sensitive to [C8mim]Br than those of frogs and goldfish. In addition, snail embryonic malformation induced by [C8mim]Br was also observed in this study, which is similar to goldfish (Li et al., 2009), frogs (Wang et al., 2010), and zebrafish (Pretti et al., 2006).
| Animals | LC50 (mg/L) | References |
|---|---|---|
| Snail (Physa acuta) embryos | 2.70 ± 0.69 | This study |
| Goldfish (Carassius auratus) embryos | 187.10 ± 9.92 | Wang et al., 2010 |
| Frog (Rana nigromaculata) embryos | 43.4 ± 3.2 | Li et al., 2009 |
| Daphnia magna | 0.95 ± 0.25 | Yu et al., 2009 |
| Snail (P. acuta) | 109.3 ± 4.0 | This study |
| Goldfish (C. auratus) | 244 ± 51 | Li et al., 2012a |
| Brocarded carp (Cyprinus carpio) | 687.5 ± 12.5 | Li et al., 2012b |
In postembryonic developmental toxicity tests, we found that [C8mim]Br had lethal effects on the survival of snails at all three developmental stages (Fig. 3). The 96 h LC50 values of [C8mim]Br on the snails revealed that young snails were more sensitive to [C8mim]Br toxicity than adults (Table 1). Moreover, the [C8mim]Br LC50 in the postembryonic developmental snails was several 10-fold higher than in embryos (Table 1), suggesting that snail embryonic development may be much more sensitive to [C8mim]Br than postembryonic development.
Compared with our previous results of [C8mim]Br toxicity in Daphnia magna (Yu et al., 2009), goldfish (Li et al., 2012a), and brocarded carp (Li et al., 2012b), we found that the snail is more sensitive to [C8mim]Br than goldfish and brocarded carp but less sensitive than D. magna (Table 3), showing an evolution-related tendency.
Ranke et al. ( 2004) discovered that ILs with longer alkyl chains usually had higher toxicity when they determined the toxicity of methyl and some ethylimidazolium ILs on luminescent bacteria (Vibrio fischeri) and IPC-81 (leukemia cells) and C6 (glioma cells) rat cell lines. Thus, they hypothesized that IL toxicity seemed to be determined mainly by the cationic component (Ranke et al., 2004). Subsequently, Bernot et al. ( 2005), Cho et al. ( 2007), Pham et al. ( 2008), and Yu et al. ( 2009) verified this hypothesis by conducting toxicity tests in algae and Daphnia with imidazolium ILs. The results of this study are also in agreement with these reports, that is, longer alkyl chains promote IL toxicity, indicating a positive relationship between alkyl chain length and toxicity. Many ILs are structurally similar to cationic surfactants due to their structure and inherent charges (Roberts and Costello, 2003; Yang et al., 2004; Sheldon, 2005; Gathergood et al., 2006; Jungnickel et al., 2008). Therefore, it is possible that ILs have properties that are comparable to surfactants, such as adsorbing onto the surface of organisms, acting on biomembranes and increasing cellular membrane permeability (Roberts and Costello, 2003). This theory suggests that uptake into cells may be governed by lipophilicity, or more exactly, by membrane-water partitioning (Ranke et al., 2004). ILs with longer alkyl chains tends to possess more lipophilic capacity, and this property enhances membrane permeability and cause greater toxicity in organisms.
CONCLUSION
The results of this study show that [C8mim]Br have toxic effects not only on the embryonic development of the snail P. acuta but also on postembryonic development, and [C8mim]Br toxicity to embryos is higher than to juvenile or adult snails. Furthermore, the embryonic and developmental toxicity of [C8mim]Br to the snail is dose and developmental stage dependent. Additionally, the toxicity of imidazolium-based ILs seems to be determined mainly by the cationic component. The results also reveal that snail embryos and adults are more sensitive to [C8mim]Br exposure than fish and frogs.
