In vitro cytotoxicity assessment of imidazolium ionic liquids: Biological effects in fish Channel Catfish Ovary (CCO) cell line

Ecotoxicology and Environmental Safety 92 (2013) 112–118

Contents lists available at SciVerse ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

In vitro cytotoxicity assessment of imidazolium ionic liquids: Biological effects in fish Channel Catfish Ovary (CCO) cell line Kristina Radošević a, Marina Cvjetko a, Nevenka Kopjar b, Rudjer Novak c, Jerka Dumić c, Višnja Gaurina Srček a,n a

Laboratory of Cell Culture Technology and Biotransformation, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, HR-10000 Zagreb, Croatia Institute for Medical Research and Occupational Health, Ksaverska cesta 2, HR-10000 Zagreb, Croatia c Department for Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, HR-10000 Zagreb, Croatia b

art ic l e i nf o

a b s t r a c t

Article history: Received 17 January 2013 Received in revised form 22 February 2013 Accepted 1 March 2013 Available online 3 April 2013

Increasing interest in the application of ionic liquids as green replacement for volatile organic solvents emphasized the need for the evaluation of their toxic effects at different biological systems in order to reduce the risk for human health and environment. To our knowledge, effects of imidazolium ionic liquids on cellular level of fish cell lines have not been studied yet. The cytotoxicity of imidazolium ionic liquids containing different anions and alkyl chain lengths as the substituent at the cation ring towards the fish CCO cell line was determined by WST-1 proliferation assay. Morphological alterations were examined by fluorescent microscopy using acridine orange/ethidium bromide staining and flow cytometry analysis was also performed. The results showed concentration-dependent cytotoxicity of ionic liquids in CCO cells, related to the type of anion and alkyl chain length, while EC50 values showed moderate to high cytotoxicity of tested imidazolium ionic liquids. Distinct morphological changes observed under fluorescence microscope and data obtained by flow cytometry suggest that the toxicity of imidazolium ionic liquids with longer alkyl chains could be related to necrosis. Results presented in here may be helpful for filling existing gaps of knowledge about ionic liquids toxicity and their impact on aquatic environment. & 2013 Elsevier Inc. All rights reserved.

Keywords: CCO cells Cytotoxicity Imidazolium ionic liquids Cell death

1. Introduction In recent years, ionic liquids (ILs) gained considerable attention due to their unique properties of great dissolving power, nonflammability, and thermal, chemical and electrochemical stability. These properties make them favorable for different types of processes, including organic synthesis, biocatalysis and biotransformation, electrochemistry, as well as for extraction and isolation processes of biologically important compounds (Pham et al., 2010; Cvjetko and Žnidaršič-Plazl, 2011) but also suggest potential problems with their degradation and persistence in the environment. The risk of air pollution by ILs application is negligible, due to their insignificant vapour pressure, which ensured those attributes such as “green” and “eco-friendly”. However, ILs possesses significant solubility in water (McFarlane et al., 2005) and might enter into aquatic environments by effluents or accidental spills therefore affecting different aquatic organisms (Samorı et al., 2010). Establishment of a sensitive biological monitoring system for an early detection and ecotoxicological evaluation of different

n

Corresponding author. Fax: þ 385 1 46 05 065. E-mail address: [email protected] (V.G. Srček).

0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.03.002

pollutants including ILs is strongly recommended. Application of cell cultures in ecotoxicological assessment offers numerous advantages in comparison to in vivo tests (Castaño and GómezLechón, 2005) and makes them feasible to examine a large variety of pollutants in the environment. Recently, toxicological effects of different ILs were investigated in different human and mammalian cell lines (Stepnowski et al., 2004; Ranke et al., 2004; Frade et al., 2009; Samorı et al., 2010) indicating that ILs caused cellular and subcellular alterations. The evaluation of ILs toxicity in aquatic environment is mostly based on the in vivo inhibition assays using different aquatic species such as marine bacteria Vibrio fischeri (Ventura et al., 2012), Daphnia magna (Samorı et al., 2010) and algae (Latała et al., 2005). According to the literature, toxic effects of different ILs to fish were performed as acute toxicity tests to zebrafish (Pretti et al., 2006, 2009) and goldfish (Li et al., 2012a) showing different effects of ILs depending on their chemical structures. The use of fish cell lines satisfies the societal desire to reduce the number of animals used in aquatic toxicology testing (Schirmer, 2006). Also, it takes into consideration that toxic effects are often species-specific and therefore toxicity toward fish can be better studied in fish specific models. Recent studies have indicated that CCO fish cell line was good in vitro model in cytotoxicity

K. Radošević et al. / Ecotoxicology and Environmental Safety 92 (2013) 112–118

studies of different metals (Tan et al., 2008) and environmental estrogens (Radošević et al., 2011). Our previous results on usefulness of CCO cells in ILs cytotoxicity testing (Cvjetko et al., 2012) encouraged us to further investigate the effects of imidazolium ILs on cellular level since such experiments on fish cells have not been performed yet. Considering that, the aim of the present study was to investigate the in vitro cytotoxicity induced by seven imidazolium ILs with different anions and lengths of alkyl chains on the viability and cellular changes in fish CCO cells. Spectrophotometric WST-1 assay was used to evaluate cell viability, while morphological changes were analyzed by fluorescence microscopy. Finally, flow cytometry was used to quantify the type of cell death induced by selected ILs. 2. Materials and methods 2.1. Ionic liquids The ionic liquids: 1-n-butyl-3-methylimidazolium tetrafluoroborate [C4MIM] [BF4] and 1-n-butyl-3-methylimidazolium hexafluorophosphate [C4MIM][PF6] used in the experiments were purchased from Acros Organics (USA). 1-n-Butyl-3methylimidazolium bromide [C4MIM][Br], 1-n-butyl-3-methylimidazolium bis(trifluoromethylsulphonyl)imide [C4MIM][Tf2N], 1-n-pentyl-3-methylimidazolium bis (trifluoromethylsulphonyl)imide [C5MIM][Tf2N], 1-n-heptyl-3-methylimidazolium bis(trifluoromethylsulphonyl)imide [C7MIM][Tf2N] and 1-n-decyl-3-methylimidazolium bis(trifluoromethylsulphonyl)imide [C10MIM][Tf2N] were synthesized according to standard procedures (Bonhôte et al., 1996; Huddleston et al., 1998). Purity was confirmed by NMR and silver nitrate test. The structures of ionic liquids used in this study are showed in Table 1. 2.2. Cell culture Fish ovary CCO cell line, purchased from American Type Culture Collection (ATCC: CRL-2772) was cultured in 25 cm2 T-flasks in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, UK) supplemented with ten percent (v/v) fetal bovine serum (FBS, Gibco, UK) and maintained in a humidified atmosphere of five percent CO2 at 30 1C. 2.3. Cytotoxicity assay The effect of ILs on cell proliferation was examined by the WST-1 assay (Roche, Germany). CCO cells from the exponential growth phase were trypsinized and plated out in 96-well plates at a density of 5  104 cells/well in 100 μL of media. After overnight cell growth, the media was replaced with fresh one containing different concentrations of individual ionic liquid (0.1–10 mM) and cells were incubated for 72 h. Following exposure, 10 μL of tetrazolium salt WST-1 {4-[3-(4iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) was added to each well and cells were incubated for further 4 h. The absorbance was measured at 450 nm on the microplate reader (Tecan, Switzerland). The experiments were performed three times with eight parallels for each concentration and data were expressed as the means 7 S.D. Cell viability was presented as percentage of control cells. The EC50 value, defined as the concentration of ionic liquid that resulted in 50 percent growth inhibition, was calculated from the dose–response curves using equations of best-fitted trend-lines. 2.4. Morphological assessment by fluorescent microscopy CCO cells were seeded in six-well plates (Corning, USA) at initial concentration of 1  105 cells/mL, allowed to attach for 24 h and then exposed to different concentrations of selected ionic liquid (1–5 mM) for 72 h. Following exposure, the cells were washed with sterile PBS and stained by a mixture of acridine orange (AO) (100 μg/mL in PBS) and ethidium bromide (EB) (100 μg/mL in PBS) for 10 min. The cells were examined using a fluorescent microscope Olympus BX51 (Olympus, Japan) with integrated camera. 2.5. Flow cytometry analysis Cytotoxic effects induced by ionic liquids were analyzed by flow cytometry. Briefly, CCO cells were seeded in T-flasks (Corning, USA) at initial concentration of 1  105 cells/mL, allowed to attach for 24 h and then exposed to different concentrations of selected ionic liquid (1–5 mM) for 72 h. Both attached and detached CCO cells were collected after 72 h of treatment, centrifuged, washed twice with DMEM

113

medium and resuspended in cold DMEM with ten percent of FBS to a final concentration of 1  106 cells/mL. Samples were incubated for 10 min at room temperature with Annexin-V-FITC and propidium iodide (PI) and analyzed by Cytomics FC500 MPL flow cytometer (Beckman Coulter, USA) to detect viable (Annexin-V-FITC-negative and PI-negative), apoptotic (Annexin-V-FITC-positive and PI-negative) and necrotic cells (Annexin-V-FITC-negative and PI-positive). A minimum of 10,000 cells were analyzed per sample.

3. Results 3.1. Viability of CCO cells exposed to ILs with different anions and alkyl chain lengths To evaluate the cytotoxicity of imidazolium ILs in CCO cells following 72 h-exposure we used WST-1 cell proliferation assay. Fig. 1 showed dose–response cytotoxicity of tested ILs with regard of anionic part (Fig. 1A) and length of alkyl chain on imidazolium cation (Fig. 1B). Corresponding EC50 values of tested ILs were calculated and are showed in Table 2. When comparing the anionic part of ILs containing [C4MIM] cation, the highest cytotoxicity was observed for [C4MIM][Tf2N] (EC50 ¼2.88 mM), while [C4MIM][PF6] exhibited the lowest cytotoxic effect (EC50 410 mM). Furthermore, the elongation of alkyl chain length negatively influenced cell proliferation, whereby [C10MIM][Tf2N] proved to be the most toxic (EC50 o0.1 mM) among tested ILs. 3.2. Morphological changes of CCO cells induced by ILs Morphological changes in cell structure were observed by AO/ EB double staining after the cells were exposed to different concentrations of ILs. The AO enters in both viable and nonviable cells intercalating into DNA, while EB is excluded from viable cells with intact cell membrane. Untreated CCO cells showed intact DNA and nucleus and had a round and green nuclei (Fig. 2A). Cells treated with 5 mM [C4MIM][PF6], [C4MIM][BF4], [C4MIM][Br] and [C4MIM][Tf2N] are shown in Fig. 2B–E. Early apoptotic cells had fragmented DNA which gives several green colored nuclei (Fig. 2B). Late apoptotic and necrotic cells had fragmented DNA and were stained orange and red as shown in Fig. 2C and D. The most toxic anion [Tf2N] destroyed cellular monolayer as can be seen in Fig. 2E. CCO cells treated with 1 mM [C5MIM][Tf2N], [C7MIM][Tf2N] and [C10MIM][Tf2N] are shown in Fig. 2F–H. Late apoptotic and necrotic cells are showed in Fig. 2F and G, while [C10MIM][Tf2N] completely destroyed cell structure (Fig. 2H). It was clear that with increasing the length of alkyl chain of ionic liquid the number of viable cells decreased tremendously, which is in agreement with obtained cytotoxicty data. 3.3. Flow cytometry analysis of CCO cells exposed to ILs Apoptotic and necrotic cells could be differentiated from the healthy ones by Annexin-V-FITC and PI staining and evaluated by flow cytometry (Fig. 3A–D). [C4MIM][BF4] at 5 mM concentration induced a slight increase in percentage of necrotic cells (3.3 percent) compared to control cells (1.1 percent). Furthermore, 1 mM of [C7MIM][Tf2N] induced more necrotic cells (14.3 percent) than the same concentration of [C4MIM][Tf2N] (2.0 percent), which confirms alkyl chain length dependent toxicity. The results of flow cytometry support cytotoxicity results and are in good agreement with those obtained by fluorescence microscopy.

4. Discussion In order to predict the effects of environmental pollutants on human health and environment, it is necessary to develop simple

114

K. Radošević et al. / Ecotoxicology and Environmental Safety 92 (2013) 112–118

and reliable methods and models for their assessment. Recent EU regulation REACH (EC 1907/2006; Registration, Evaluation, Authorization and Restriction of CHemical substances) emphasizes the use of alternative in vitro models, since this approach reduces the number of laboratory animals used in the toxicological studies. ILs, as relatively new chemicals with tremendous potential of industrial applications, should be critically investigated with regarded of their toxicity at different biological levels and environmental impact. In the aquatic risk assessment, a large number of acute in vivo fish toxicity tests of newly synthesized and/or existing chemicals are performed. Permanent fish cell lines represent a promising test system for the assessment of chemical risk and in particular the implementation of the REACH regulations in reducing animal testing (Tanneberger, 2010). On the basis of the above considerations, we studied the cytotoxicity and morphological changes of several commercial and synthesized imidazolium ILs in fish CCO cells to evaluate the contribution of anionic part as well as alkyl chain length to the observed toxicity.

In this study, results of cell viability demonstrated that ILs exhibit cytotoxicity effects to CCO cells dependent on anion and alkyl chain length. [Tf2N] anion showed the highest toxic effect to CCO cells which is in correlation with results observed in HeLa cells (Stepnowski et al., 2004; Wang et al., 2007; Cvjetko et al., 2012). Some studies suggested that the increase in toxicity of ILs paired with [Tf2N] anion could be due to the hydrolytic cleavage that resulted in the formation of free fluoride ions (Stolte et al., 2006). Fluoride ion is a potent inhibitor of Na þ K þ ATP-ase, located at the surface of the cell and may interfere with essential cellular processes. The influence of various lengths of alkyl chains on imidazolium ring showed that ILs with longer alkyl chains caused stronger toxic effects in CCO cell line. Obtained results on the influence of both anionic and cationic parts of ILs on CCO cell viability are consistent with results obtained in leukemia IPC-81 cells (Ranke et al., 2004). According to UFT Merck Ionic Liquids Biological Effects Database (http://www.il-eco.uft.uni-bremen.de), where classification of cytotoxicity is given toward IPC-81 cell line,

Table 1 Systematic and trade names, molecular weights and chemical formula of ionic liquids used in the study. Systematic name

Trade name

Molecular weight

1-n-Butyl-3-methylimidazolium hexafluorophosphate

[C4MIM][PF6]

284.18

1-n-Butyl-3-methylimidazolium tetrafluoroborate

[C4MIM][BF4]

226.02

1-n-Butyl-3-methylimidazolium bromide

[C4MIM][Br]

219.12

1-n-Butyl-3-methylimidazolium bis(trifluoromethylsulphonyl) imide

[C4MIM][Tf2N]

419.36

1-n-Pentyl-3-methylimidazolium bis(trifluoromethylsulphonyl) imide

[C5MIM][Tf2N]

433.38

1-Heptyl-3-methylimidazolium bis(trifluoromethylsulphonyl) imide

[C7MIM][Tf2N]

461.45

1-Decyl-3-metilimidazolijev bis(trifluormetilsulfonil) imide

[C10MIM][Tf2N]

503.53

Chemical formula

K. Radošević et al. / Ecotoxicology and Environmental Safety 92 (2013) 112–118

115

Fig. 1. Effects of different anions [PF6], [BF4], [Br] and [Tf2N] (A) and different lengths of alkyl chains (butyl, pentyl, heptyl and decyl) on imidazolium ring (B) on CCO cell viability assessed by the WST-1 assay. Data are expressed as a percentage of unexposed control cells 7 S.D. of three replicates for each exposure concentration. Table 2 EC50 values (mM) for CCO cells following exposure to selected imidazolium ionic liquids as derived by the WST-1 assay. Ionic liquid

EC50 (mM)

[C4MIM][PF6] [C4MIM][BF4] [C4MIM][Br] [C4MIM][Tf2N] [C5MIM][Tf2N] [C7MIM][Tf2N] [C10MIM][Tf2N]

410 4.46 7 0.39 3.81 7 0.36 2.88 7 0.43 0.26 7 0.05 o 0.1 o 0.1

tested ILs [C4MIM][PF6], [C4MIM][BF4], [C4MIM][Br], [C4MIM] [Tf2N] and [C5MIM][Tf2N] possess moderate (0.1 mMo EC50 o5 mM) or low (EC50 45 mM) cytotoxicity toward CCO cells. High level of cytotoxicity (10−3 mM oEC50 o0.1 mM) was observed in CCO cells only for [C7MIM][Tf2N] and [C10MIM] [Tf2N]. Our results on sensitivity of CCO cells to imidazolium ILs, with EC50 values in the millimolar range, are in good agreement with those obtained in vivo with bacteria V. fischeri (0.45–9.8 mM) and cladoceran D. magna (0.05–1.14 mM) (Samorı et al., 2010). Acute toxicity of ILs was assessed in zebra fish Danio rerio measuring their lethal effect after 96 h exposure in a static test (Pretti et al., 2006) wherein most of fifteen tested ILs proved to be

practically harmless with LC50 4 100 mg/L. If we recalculate LC50 values for [C4MIM][PF6], [C4MIM][BF4], and [C4MIM][Tf2N] into millimolar concentration we come to conclusion that our EC50 values are about ten times higher than LC50 values obtained by the same author. The difference between our in vitro results and in vivo results by Pretti et al. (2006) can be elucidated by the fact that fish cell lines are about ten times less sensitive than in vivo assays (Schirmer, 2006). Therefore, we have proved that CCO cell line, used in this work, could be a good model for preliminary screening of ILs in order to assess possible harmful impacts on aquatic organisms and environment. The toxicity of ILs is likely to occur through several different modes of action and observed effects can be attributed to reduced metabolic activity, inhibited cell proliferation or to cell death. The cell viability measured as WST-1 reduction potential directly indicates the metabolic state of cells. The biochemical evidence of ILs toxicity is accompanied by morphological changes seen in CCO cells after treatment with selected ILs. An absolute feature to discriminate between healthy (or early apoptotic) and necrotic cells is the physical integrity of plasma membrane. Due to lipophilic (i.e. hydrophobic) character of tested ILs we have presumed that observed cytotoxic effect would be related to the impact on plasma membrane, as assessed by AO/EB staining. The uptake of EB, which readily crosses the damaged plasma membrane, was observed in the case of all tested ILs. Apoptotic, late apoptotic and necrotic cells,

116

K. Radošević et al. / Ecotoxicology and Environmental Safety 92 (2013) 112–118

Fig. 2. Photomicrographs of CCO cells stained with acridine orange and ethidium bromide. Control cells (A) and cells treated for 72 h with 5 mM [C4MIM][PF6], [C4MIM][BF4], [C4MIM][Br] and [C4MIM][Tf2N] (B–E) and 1 mM [C5MIM][Tf2N], [C7MIM][Tf2N] and [C10MIM][Tf2N] (F–H). Viable cells shown in green (A) while apoptotic/necrotic cells were observed in treated samples (B–H). Magnification 400  . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

with yellow, reddish and intense orange fluorescence were visible in CCO cells treated with 5 mM [C4MIM][BF4] and [C4MIM][Br] compared to control cells, while the most toxic anion [Tf2N] produced advanced degeneration and disruption of cell monolayer. The enhanced membrane permeability and evident membrane disruption were more pronounced than the longer alkyl chain on imidazolium ring, where [C10MIM][Tf2N] was the most “invasive” for CCO cells. Results of fluorescence microscopy are consistent with the observation that one of the possible routes of ionic liquidinduced cytotoxicity can be ;\due to the interaction of lipophilic long chain alkyl substituents with the cell membrane (McClelland et al., 2003) or enhanced membrane permeability which alter the lipid bilayer as reported by some other authors (Stepnowski et al., 2004; Latała et al., 2005 Ranke et al., 2007). In order to confirm interactions of ILs with cell membrane, Cornmell et al. (2008) analyzed subcellular fractions of Escherichia coli after exposure to trihexyltetradecylphosphonium bis(trifluoromethylsulfonyl)imide and detected that tested ILs accumulated specifically in the membrane fraction of the cells. Even though cytototoxicity mechanism of ILs is not fully understood yet, it has been proposed that the mode of toxic action for ionic liquids takes place through membrane disruption because of the structural similarities of imidazoliumbased ILs to detergent, pesticides and antibiotics (Docherty and Kulpa, 2005). Apoptotic cell death caused by 1-ethyl-3methylimidazolium tetrafluoroborate [C2MIM][BF4] exposure and detected by AO/EB fluorescence staining has been reported in HeLa cell line, where increase in concentration of ionic liquid caused

increase in number of apoptotic/necrotic cells probably due to direct cell membrane destruction (Wang et al., 2007). Also, Kumar et al. (2009) reported MCF7 cell death after treatment with 1-methyl-1propylpyrrolidinium bromide [MPPyrro][Br] at EC50 value concentration which is explained by loss of membrane integrity. Imidazolium-based ILs showed dose-dependent cytotoxicity against PC12 cells and it was found that [C8MIM][Br] induces apoptosis associated with excessive ROS production and mitochondrial depolarization (Li et al., 2012b). Cells generally undergo morphological changes in an asynchronous manner so microscopic analysis of a relatively small number of cells thus did not reveal information on the whole cell population. To confirm visual results obtained by fluorescent microscopy and accumulate information on the cell population, we have applied flow cytometry to detect membrane alternation in dying cells. One of the earliest changes in apoptosis is the movement of negatively charged phosphatidylserine from the inside of the cell membrane to the outside, so we have used Annexin-V-FITC and PI staining to discriminate live, apoptotic and necrotic cells. The flow cytometry analysis showed increased proportion of necrotic CCO cells indicating that toxicity of [C7MIM][Tf2N] was more characterized by necrosis than apoptosis. This results may be due to the fact that strong lipophilic character of [C7MIM][Tf2N] tends to affect plasma membrane of CCO cells which was consistent with results observed by fluorescent microscopy. Therefore, data on cytotoxicity assay, as well as fluorescent microscopy and flow cytometry can provide better insight into the

K. Radošević et al. / Ecotoxicology and Environmental Safety 92 (2013) 112–118

117

Fig. 3. Flow cytometric analysis of control CCO cells (A) and CCO cells treated with 5 mM [C4MIM][BF4] (B), 1 mM [C4MIM][Tf2N] (C) and 1 mM [C7MIM][Tf2N] (D) is shown as percentage of apoptotic, necrotic and live cells in each sample.

examination of biological impacts of ILs in fish cell lines. By such approach we can study in more details the alterations on cellular level which provides us preliminary insights into the mechanisms of ILs-induced toxicity.

5. Conclusion We can conclude that treatment with selected imidazolium ILs decreased CCO cell viability on dose-dependent manner and showed dependency on anion and alkyl chain length. Induced morphological alterations observed by fluorescence microscopy are related to the structure and concentration of tested IL while flow cytometry results showed that ILs with longer alkyl chain increased the proportion of necrotic cells. Complementary use of WST-1 cell proliferation assay, fluorescent microscopy and flow cytometry can provide better insight into interrogation of biological impact of ILs in (fish) cell lines. By such approach we can study in more details the alterations in cells and their membranes, which are probably related to the mechanism of actions of ILs. Results presented in here could be relevant for filling existing gaps of knowledge about ILs toxicity in aquatic environment, as guidance for design of safer and sustainable ILs. Acknowledgments This work was supported by the Ministry of Science, Education and Sports of the Republic of Croatia (Grant nos. 058-05821842414, 058-0582261-2256 and 006-0061194-1218).

References Bonhôte, P., Dias, A.P., Papageorgiou, N., Kalyanasundaram, K., Grätztel, M., 1996. Hydrophobic, highly conductive ambient-temperature molten salts. Inorg. Chem. 35, 1168–1178. Castaño, A., Gómez-Lechón, M.J., 2005. Comparison of basal cytotoxicity data between mammalian and fish cell lines: a literature survey. Toxicol. In Vitro 19, 695–705. Cornmell, R.J., Winder, C.L., Tiddy, G.J.T., Goodacre, R., Stephens, G., 2008. Accumulation of ionic liquids in Escherichia coli cells. Green Chem. 10, 836–841. Cvjetko, M., Žnidaršič-Plazl, P., 2011. Ionic liquids within microfluidic devices. In: Kokorin, A. (Ed.), Ionic Liquids: Theory, Properties, New Approaches. Intech, Rijeka, pp. 681–700. Cvjetko, M., Radošević, K., Tomica, A., Slivac, I., Vorkapić-Furač, J., Gaurina Srček, V., 2012. Evaluation of cytotoxicity induced by ionic liquids in fish and human cell lines. Arh. Hig. Rada Toksikol. 63, 15–20. Docherty, K.M., Kulpa, C.F.J., 2005. Toxicity and antimicrobial activity of imidazolium and pyridinium ionic liquids. Green Chem. 7, 185–189. Frade, R., Rosatella, A., Marques, C., Branco, L., Kulkarni, P., Mateus, N., Afonso, C., Duarte, C., 2009. Toxicological evaluation on human colon carcinoma cell line (CaCo-2) of ionic liquids based on imidazolium, guanidinium, ammonium, phosphonium, pyridinium and pyrrolidinium cations. Green Chem. 11, 1160–1165. Huddleston, J.G., Willauer, H.D., Swatloski, R.P., Visser, A.E., Rogers, R.D., 1998. Room temperature ionic liquids as novel media for “clean” liquid–liquid extraction. Chem. Commun., 1765–1766. Kumar, A., Papaiconomou, N., Lee, J., Salminen, J., Clark, D., Prausnitz, J., 2009. In vitro cytotoxicity of ionic liquids: effect of cation rings, functional groups, and anions. Environ. Toxicol. 24, 388–395. Latała, A., Stepnowski, P., Nędzi, M., Mrozik, W., 2005. Marine toxicity assessment of imidazolium ionic liquids: acute effects on the Baltic algae Oocystis submarina and Cyclotella meneghiniana. Aquat. Toxicol. 73, 91–98. Li, X.Y., Zheng, S.H., Dong, X.Y., Ma, J.G., Wang., J.J., 2012a. Acute toxicity and responses of antioxidant systems to 1-methyl-3-octylimidazolium bromide at different developmental stages of goldfish. Ecotoxicology 21, 253–259. Li, X.Y., Jing, C.Q., Lei, W.L., Li, J., Wang, J.J., 2012b. Apoptosis caused by imidazoliumbased ionic liquids in PC12 cells. Ecotoxicol. Eviron. Saf. 83, 102–107.

118

K. Radošević et al. / Ecotoxicology and Environmental Safety 92 (2013) 112–118

McClelland, D., Evans, R.M., Abidin, I., Sharma, S., Choudhry, F.Z., Jaspars, M., Sepcic, M., Scot, R.H., 2003. Irreversibile and reversibile pore formation by polymeric alkylpyridinium salts (poly-APS) from the sponge Reniera sarai. Br. J. Pharmacol. 139, 1399–1408. McFarlane, J., Ridenour, W.B., Luo, H., Hunt, R.D., Depaoli, D.W., Ren, R.X., 2005. Room temperature ionic liquids for separating organics from produced water. Sep. Sci. Technol. 40, 1245–1265. Pham, T.P., Cho, C.W., Yun, Y.S., 2010. Environmental fate and toxicity of ionic liquids: a review. Water Res. 44, 352–372. Pretti, C., Chiappe, C., Pieraccini, D., Gregori, M., Abramo, F., Monni, G., Intorre, L., 2006. Acute toxicity of ionic liquids to 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. Eviron. Saf. 72, 1170–1176. Radošević, K., Tonković, T., Slivac, I., Kniewald, Z., Gaurina Srček, V., 2011. Comparison of cytotoxicity induced by 17α-ethynylestradiol and diethylstilbestrol in fish CCO and mammalian CHO-K1 cell lines. Bull. Environ. Contam. Toxicol. 86, 252–257. Ranke, J., Molter, K., Stock, F., Bottin-Weber, U., Poczobutt, J., Hoffmann, J., 2004. Biological effects of imidazolium ionic liquids with varying chain lengths in acute Vibrio fischeri and WST-1 cell viability assays. Ecotoxicol. Environ. Saf. 58, 396–404. Ranke, J., Müller, A., Bottin-Weber, U., Stock, F., Stolte, S., Arning, J., Störman, R., Jastroff, B., 2007. Lipophilicity parameters for ionic liquid cations and their correlation to in vitro cytotoxicity. Ecotoxicol. Environ. Saf. 67, 430–438.

Samorı, C., Malferrari, D., Valbonesi, P., Montecavalli, A., Moretti, F., Galletti, P., Sartor, G., Tagliavini, E., Fabbri, E., Pasteris, A., 2010. Introduction of oxygenated side chain into imidazolium ionic liquids: evaluation of the effects at different biological organization levels. Ecotoxicol. Environ. Saf. 73, 1456–1464. Schirmer, K., 2006. Proposal to improve vertebrate cell cultures to establish them as substitutes for the regulatory testing of chemicals and effluents using fish. Toxicology 224, 163–183. Stepnowski, P., Skladanowski, A.S., Ludwiczak, A., Laczynska, E., 2004. Evaluating the cytotoxicity of ionic liquids using human cell line HeLa. Hum. Exp. Toxicol. 23, 513–517. Stolte, S., Arning, A., Bottin-Weber, U., Muller, A., Pitner, W.R., Welz-Biermann, U., Jastroff, B., Ranke, J., 2006. Anion effects on the cytotoxicity of ionic liquids. Green Chem. 8, 621–629. Tan, F., Wang, M., Wang, W., Lu, Y., 2008. Comparative evaluation of the cytotoxicity sensitivity of six fish cell lines to four heavy metals in vitro. Toxicol. In Vitro 22, 164–170. Tanneberger, K., 2010. Assessment of chemicals: fish cells as an alternative to whole fish. Eawag News 68e, 25–27. Ventura, S.P.M., Marques, C.S., Rosatella, A.A., Afonso, C.A.M., Gonçalves, F., Coutinho, J.A.P., 2012. Toxicity assessment of various ionic liquid families towards Vibrio fischeri marine bacteria. Ecotoxicol. Environ. Saf. 76, 162–168. Wang, X.F., Ohlin, A.C., Lu, Q., Fei, Z., Hu, J., Dyson, P.J., 2007. Cytotoxicity of ionic liquids and precursor compounds towards human cell line HeLa. Green Chem. 9, 1191–1197.