Effects of imidazolium chloride ionic liquids and their toxicity to Scenedesmus obliquus

Effects of imidazolium chloride ionic liquids and their toxicity to Scenedesmus obliquus

Ecotoxicology and Environmental Safety 122 (2015) 83–90 Contents lists available at ScienceDirect Ecotoxicology and Environmental Safety journal hom...

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Ecotoxicology and Environmental Safety 122 (2015) 83–90

Contents lists available at ScienceDirect

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

Effects of imidazolium chloride ionic liquids and their toxicity to Scenedesmus obliquus Huijun Liu n, Xiaoqiang Zhang, Caidong Chen, Shaoting Du, Ying Dong School of Environmental Science and Engineering, Zhejiang Gongshang University, Hangzhou 310018, Zhejiang Province, China

art ic l e i nf o

a b s t r a c t

Article history: Received 4 May 2015 Received in revised form 7 July 2015 Accepted 7 July 2015

The low volatility of ionic liquids effectively eliminates a major pathway for environmental release and contamination; however, the good solubility, low degree of environmental degradation and biodegradation of ILs may pose a potential threat to the aquatic environment. The growth inhibition of the green alga Scenedesmus obliquus by five 1-alkyl-3-methylimidazolium chloride ionic liquids (ILs) ([Cn mim]Cl, n ¼ 6, 8, 10, 12, 16) was investigated, and the effect on cellular membrane permeability and the ultrastructural morphology by ILs ([Cnmim]Cl, n ¼ 8, 12, 16) were studied. The results showed that the growth inhibition rate increased with increasing IL concentration and increasing alkyl chain lengths. The relative toxicity was determined to be [C6mim]Cl o[C8mim]Cl o[C10mim]Cl o [C12mim]Cl o [C16mim]Cl. The algae were most sensitive to imidazolium chloride ILs at 48 h according to the results from the growth inhibition rate and cellular membrane permeability tests. The ultrastructural morphology showed that the ILs had negative effects on the cellular morphology and structure of the algae. The cell wall of treated algae became wavy and separated from the cell membrane. Chloroplast grana lamellae became obscure and loose, osmiophilic material was deposited in the chloroplast, and mitochondria and their cristae swelled. Additionally, electron-dense deposits were observed in the vacuoles. & 2015 Elsevier Inc. All rights reserved.

Keywords: 1-alkyl-3-methylimidazolium chloride ionic liquid Scenedesmus obliquus Growth inhibition rate Cellular membrane permeability Ultrastructure

1. Introduction With the increasing development of green chemistry, it seems extremely urgent to reduce or eliminate the use of hazardous reagents and solvents. Ionic liquids (ILs), a type of “considered” green and reusable solvent, have gained great attention as a promising alternative to traditional organic solvents (Shamsipur et al., 2010; Suarez et al., 1996; Zhuo et al., 2015). ILs are composed of large organic cations, such as ammonium, imidazolium, pyridinium, piperidinium or pyrrolidinium, with alkyl side chains that can vary in length, number, position and anion type (Ventura et al., 2010). The unique properties of ILs, such as their good solubility, catalytic properties, nonflammability, low vapor pressure, wide electrochemical window and high stability (Santos et al., 2014), make them prevalent in organic synthesis (Jiang et al., 2004), chemical separation (Chai et al., 2014; Kárászová et al., 2014), electrochemistry (Unemoto et al., 2014) and other fields. Although ILs are unlikely to act as air contaminants or inhalation toxins, they are potential water and soil contaminants as a result of accidental spills, a lack of waste water treatment effectiveness, leaching of landfill sites or of effluents (Cvjetko Bubalo n

Corresponding author. Fax: þ 86 571 28008215. E-mail address: [email protected] (H. Liu).

http://dx.doi.org/10.1016/j.ecoenv.2015.07.010 0147-6513/& 2015 Elsevier Inc. All rights reserved.

et al., 2014). The environmental impact and ecological activity of several ILs have been investigated in terrestrial invertebrates, vertebrates, algae, microorganisms, plants and human cells (Biczak et al., 2014; Egorova and Ananikov, 2014; Liu et al., 2015; Pham et al., 2010). Many ILs were found to be toxic or even highly toxic towards cells and living organisms (Egorova and Ananikov, 2014), and the aquatic (eco)toxicity of ILs plays a very important role in their environmental impact because of their good solubility and high stability (Cvjetko Bubalo et al., 2014; Pham et al., 2010). Over the past decade, the toxicity of ILs on aquatic animals (Bernot et al., 2005; Li et al., 2012; Samori et al., 2007; Yu et al., 2009), aquatic plants (Kumar et al., 2010, 2011; Latala et al., 2005) and aquatic microorganisms (Megaw et al., 2013; Ranke et al., 2004; Ventura et al., 2012; Viboud et al., 2012) has been extensively investigated. Studies have shown that the alkyl chain length of ILs have significant effects on ILs toxic ability (Bernot et al., 2005; Chen et al., 2014; Du et al., 2012; Luo et al., 2009; Radosevic et al., 2013; Ranke et al., 2004; Samori et al., 2007). Among the various aquatic organisms, algae are widely used to monitor water quality and aquatic toxicity. This is because algae are not only the primary producers in the food chain, but they also tend to be more sensitive to contaminants than fish and invertebrates (Boyle, 1984). Recently, some studies on the effects of ILs on algae were conducted, specifically relating to acute toxicity (Chen et al., 2014; Ma et al., 2010), photosynthesis (Pham et al., 2008), antioxidant

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systems (Deng et al., 2015; Kumar et al., 2011) and the enantioselective toxicities of chiral ILs to aquatic algae (Chen et al., 2014). The role of carrier solvent (acetone, salinity) on the induced toxicity, and the binary mixture of ILs were studied (Tsarpali et al., 2015a, 2015b; Tsarpali and Dailianis, 2015). The quantitative structure–activity relationship (QSAR) was also adopted to study the toxicity of ILs (Das and Roy, 2014, Peric et al., 2015). However, relatively few studies have examined the effect of ILs on cellular morphology and structure of the algae. To date, the most commonly used ILs are 1-alkyl-3-methylimidazolium salts, which remain in the fluid state over a broad range of temperatures (Latala et al., 2005), more attention should be paid to their ecological impact. In the present study, five 1-alkyl-3-methylimidazolium chloride ([Cnmim]Cl) ILs with different alkyl chain length were adopted. The toxicological sensitivity of Scenedesmus obliquus (S. obliquus) to the five ILs with varying chain length was evaluated. After exposure times of 24, 48, 72 and 96 h, the IC50 values of the five ILs (alkyl chain length n ¼6, 8, 10, 12 and 16) were measured, the cellular membrane permeabilities of algae exposed to three ILs (alkyl chain length n¼ 8, 12 and 16) were determined, and changes in the ultrastructural morphology were studied by transmission electron microscopy (TEM). The effects of ILs with varying chain length were compared, the toxicity of ILs at different exposure times was studied, and the possible mechanism of IL toxicity was discussed.

2. Materials and methods 2.1. Chemicals 1-Hexyl-3-methylimidazolium chloride ionic liquid ([C6mim]Cl) (99% purity), 1-octyl-3-methylimidazolium chloride ionic liquid ([C8mim]Cl) (99% purity), 1-decyl-3-methylimidazolium chloride ionic liquid ([C10mim]Cl) (99% purity), 1-dodecyl-3-methylimidazolium chloride ionic liquid ([C12mim]Cl) (99% purity) and 1-hexadecyl-3-methylimidazolium chloride ionic liquid ([C16mim]Cl) (99% purity) were purchased from Lanzhou Greenchem ILS, LICP, CAS, China. Fluorescein diacetate (FDA) was purchased from SigmaAldrich. All other reagents were of analytical grade purity. The structures of the ILs are shown in Fig. 1. 2.2. The algal cultures The initial stock of the microalga S. obliquus was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). The HB-4 medium for S. obliquus growth was prepared according to the Chinese National Environmental Protection Agency Guidelines 201 (CNEPA, 1990), which is composed of distilled water and the following chemical ingredients: (NH4)2SO4 200 mg/L; Ca(H2PO4)2  H2O þ(CaSO4  H2O) 30 mg/L; MgSO4  7H2O 80 mg/L; NaHCO3 100 mg/L; KCl 23 mg/L; FeCl3 1.5 mg/L; and

0.5 mL of soil leaching solution. The HB-4 medium was sterilized in a Hirayama HVE-50 autoclave. Standard lighting and temperature conditions (16:8 light:dark cycle; illumination 3000–4000 lx; 25 °C) were adopted (Liu and Xiong, 2009). Because they were growing in static cultures, the algae needed to be shaken manually 3–5 times at fixed times each day. 2.3. Growth inhibition Growth inhibition tests were conducted according to the Organization for Economic Cooperation and Development (OECD) (2006) guidelines with slight modifications. S. obliquus cells were pre-cultured in HB-4 to induce the exponential phase of growth and were then inoculated in 250-mL Erlenmeyer flasks at an initial density of 8.0  105 cells/mL and a final solution volume of 100 mL. The test concentrations were identified through preliminary range-finding tests. The S. obliquus cells were exposed to a series of IL concentrations (0–25 mg/L for [C6mim]Cl, 0–8 mg/L for [C8mim]Cl, 0–1 mg/L for [C10mim]Cl, 0–0.5 mg/L for [C12mim]Cl, and 0–0.1 mg/L for [C16mim]Cl). Three replicates were tested for each treatment. After exposure for 24, 48, 72 and 96 h, the biomasses in the cultures were determined according to linear equations based on direct cell counts and optical densities measured at 680 nm (OD680) using a Shimadzu UV-2401PC UV–visible spectrophotometer (Tokyo, Japan). The relative inhibitions of algal growth in each concentration were calculated as follows:

RI (%) = (X 0 − X n ) × 100%/X 0

(1)

where X0 represents the average algal biomass of control treatment and Xn represents the average algal biomass of each ILs treatment. The concentration of ILs leading to a 50% inhibition (IC50) of algal growth was calculated using probit analysis, and logistic model: y¼A2 þ(A1  A2)/ [1 þ (x/x0)P], where y represents the inhibition of alga growth (%), x represents the ILs concentration (mg/ L), x0 represents the IC50,5d (mg/L), and A1, A2 and P are constants. 2.4. Cell membrane permeability IL concentrations in the cell membrane permeability test were as follows: 0–1 mg/L for [C8mim]Cl, 0–0.03 mg/L for [C12mim]Cl, and 0–0.015 mg/L for [C16mim]Cl. Three replicates were tested for each treatment. FDA is a hydrophobic and nonfluorescent dye that exhibits esterase activity and cell membrane permeability (Wen et al., 2011). The cell membrane permeability was measured according to the method of Cai et al. (2008). FDA in acetone was prepared to a final FDA concentration of 1 mg/mL. The algal cell density of each treatment was diluted to 4  105 cells/mL with HB4 medium after 24, 48, 72 and 96 h. Then, the FDA solution was added to each treatment to achieve a final concentration of 2  10  6 mol/L. The hydrolysis rate of the samples was determined during the first 10 min using fluorescence spectrophotometry. The initial hydrolysis rates for FDA were obtained from the slope of the linear regression of fluorescence intensity versus time (r2 Z0.95). 2.5. Transmission electron microscopy Cells from the control, 2 mg/L [C8mim]Cl treatment, 0.04 mg/L [C12mim]Cl treatment, and 0.02 mg/L [C16mim]Cl treatment were collected by centrifugation (2080g, 10 min) for transmission electron microscopy (TEM) studies after 48 h using a method adapted from the work of Liu and Xiong (2009). 2.6. Statistical analysis

Fig. 1. Structure of ILs.

Each treatment was conducted in triplicate. The data were

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analyzed using Origin 8.5 software, and probit analysis was using SPSS19.0. Comparisons were made using one-way analyses of variance (ANOVAs), and the differences between individual treatments were determined by Duncan's new multiple range test at a significance level of p o0.05.

3. Results and discussion 3.1. Effect of ILs on growth inhibition of S. obliquus The growth inhibition rate of IL treatments significantly increased (po 0.05) with increasing concentration at the same exposure time, showing an obvious dose–response relationship (shown in Supplementary material Table 1). The growth inhibition rate of [C6mim]Cl treatments increased from 6.22% (0.5 mg/L) to 59.10% (25 mg/L) at 24 h, that of [C8mim]Cl treatments increased from 16.43% (0.2 mg/L) to 53.05% (8 mg/L), that of [C10mim]Cl treatments increased from 8.20% (0.005 mg/L) to 66.40% (1 mg/L), that of [C12mim]Cl treatments increased from 10.39% (0.002 mg/L) to 61.38% (0.5 mg/L), and that of [C16mim]Cl treatments increased from 8.26% (0.005 mg/L) to 59.99% (0.1 mg/L). The same trends were observed at the other exposure times. The growth inhibition rate decreased with increasing exposure time in low-concentration treatments. The growth inhibition rate of [C6mim]Cl treatments decreased from 6.22% (24 h) to 1.54% (96 h) with 0.5 mg/L treatment and from 49.0% (24 h) to 37% (96 h) with 6 mg/L treatment. The decrease in inhibition rate with exposure time was also observed with the [C8mim]Cl (0.2–0.6 mg/ L), [C10mim]Cl (0.005–0.03 mg/L), [C12mim]Cl (0.002–0.01 mg/L) and [C16mim]Cl (0.002–0.008 mg/L) treatments. The results suggest that the effect of ILs on algal growth can be reversed with increasing exposure time at low concentrations. The growth inhibition rate increased first and then decreased with increasing exposure time at intermediate concentration treatments. The maximum inhibition rate at 48 h (8 mg/L [C6mim]Cl, 0.8 and 1 mg/ L [C8mim]Cl, 0.02–0.05 mg/L [C10mim]Cl, 0.02 and 0.03 mg/L [C12mim]Cl, 0.01–0.02 mg/L [C16mim]Cl) or 72 h (10 mg/L [C6mim]Cl, 0.1 mg/L [C10mim]Cl, 0.05 and 0.08 mg/L [C12mim]Cl, 0.03 mg/L [C16mim]Cl) shows that the effect of ILs on algal growth can also be reversed with time at intermediate concentrations. The growth inhibition rate increased with exposure time in response to high concentration treatments (10–25 mg/L [C6mim]Cl, 2–8 mg/ L [C8mim]Cl, 0.1–1 mg/L [C10mim]Cl, 0.1–0.5 mg/L [C12mim]Cl, 0.05–0.1 mg/L [C16mim]Cl), suggesting that ILs had irreversible effects on algal growth at high concentrations. Similar results were obtained from the research conducted by Latala et al. (2005), which showed that during an 11-d exposure time, the growth ability of the Baltic alga Oocystis submarina was restored to the same level as that of the control at low concentrations of four imidazolium IL treatments but not at the highest concentrations. The compensation response in low concentration treatments over time may represent a modest allocation of resources for repairing damage, also may be integrated into other processes leading to a reduction in background damage, or to enhance resistance (Calabrese, 2015). The concentration–response fitting curves of ILs using a logistic model are shown in Fig. 2. Three methods were used to calculate the IC50 values. The IC50 values calculated by probit analysis, fitted by the logistic model and those obtained from the curves by drawing are shown in Table 1. These results showed that the 48-h, 72-h and 96-h IC50 values obtained by three methods were very similar, especially for [C10mim]Cl, [C12mim]Cl and [C16mim]Cl. For [C16mim]Cl, the 48-h, 72-h and 96-h IC50 values obtained using the drawing method were 0.010, 0.016, 0.018 mg/L, respectively, while those obtained using the logistic model were 0.010, 0.015,

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0.017 mg/L, respectively, and those obtained by probit analysis were 0.012, 0.018, 0.020 mg/L, respectively. However, there is a large difference between the 24-h values obtained by three methods. This may be because the maximum growth inhibition rate of algae exposed to ILs for 24 h was only approximately 60%, which is not suitable for the logistic model or probit analysis. The results indicated that the acute toxicity of the five imidazolium chloride ILs increased first and then decreased with increasing exposure time, reaching maximum toxicity at 48 h. The 24-h IC50 value of [C6mim]Cl treatment was 6.67 mg/L, while the 48-h value decreased to 5.61 mg/L. Then, the IC50 value increased gradually, with 72-h and 96-h IC50 values of 6.22 and 6.82 mg/L, respectively. The 24-h, 72-h and 96-h IC50 values of the [C8mim]Cl-treatment were 3.30, 1.06 and 1.09 mg/L, respectively, while the 48-h IC50 value was 0.86 mg/L. The same trend was also observed in the [C10mim]Cl, [C12mim]Cl and [C16mim]Cl treatments. Fu et al. evaluated the quality of the toxicity data for green algae using 2323 algal toxicity data (log 1/EC50) in different toxicity response endpoints for 1081 compounds and 26 algal species with different exposure periods, showing that an exposure period of 48 h is the most sensitive for algal growth inhibition tests, and its sensitivity is 0.25 log units greater than those of the 72 and 96 h exposure periods (Fu et al., 2015). While the IC50 decreased with the exposure time for the 1-alkyl-3-methylimidazolium bromide ILs on S. obliquus (Ma et al., 2010). Compared with the IC50 of [Cnmim]Br (Ma et al., 2010), the IC50–48 h of [C6mim]Br is 2.6 times of [C6mim]Cl, [C8mim]Br is 1.6 times of [C8mim]Cl, [C10mim]Br is 6 times of [C10mim]Cl, and [C12mim]Br is 3.8 times of [C10mim]Cl. The IC50 values were affected by ILs' anion, and [Cn mim]Cl is more toxic to S. obliquus than [Cnmim]Br. The results showed that the IC50 values of the five ILs are negatively correlated to the alkyl chain lengths of the cation ring at each incubation time (Table 2). The 24-h IC50 value of [C6mim]Cl is 2.0 times that of [C8mim]Cl, 60.7 times that of [C10mim]Cl, 162.9 times that of [C12mim]Cl and 417.5 times that of [C16mim]Cl. The same trend was also observed for the 48-h, 72-h and 96-h IC50 values. The results suggest that the toxicity of ILs to S. obliquus increases with increasing alkyl chain lengths of the ILs. The relative toxicity was as follows: [C6mim]Clo[C8mim]Clo[C10mim]Clo[C12mim]Clo[C16mim]Cl. The toxicity of [C16mim]Cl is approximately 417.5, 562, 389.4 and 379.4 times higher than that of [C6mim]Cl at 24, 48, 72 and 96 h, respectively. The effects of the imidazolium ILs with different alkyl chain lengths on various cell lines and organisms were extensively investigated. The studied effects include the cytotoxicity of imidazolium ILs with different alkyl chain lengths on fish CCO cell lines by WST-1 proliferation assay (Radosevic et al., 2013) and the effects of imidazolium and pyridinium ILs with different alkyl chain lengths on the photosynthesis response of the alga Pseudokirchneriella subcapitata (Pham et al., 2008) and of freshwater snails (Bernot et al., 2005). The studies showed that the toxicity of imidazolium and pyridinium ILs is positively correlated to the alkyl chain length of the corresponding ILs. This is because the increased lipophilic properties of ILs with longer alkyl chains increase the membrane permeability, resulting in higher toxicity (Ranke et al., 2004). 3.2. Effects of ILs on cellular membrane permeability of S. obliquus The effects of three ILs with varying chain lengths on the membrane permeability of algal cells are shown in Fig. 3. The membrane permeability of algal cells was positively related to the concentration of each IL treatments at the same exposure time. The cellular membrane permeability at 48 h was 1.50, 1.65, 2.18, 3.32 and 4.82 times that of the control group upon treatment with [C8mim]Cl. The cellular membrane permeability at 48 h was 1.11, 1.60, 1.74, 2.79 and 3.98 times that of the control group upon treatment with [C12mim]Cl. The cellular membrane permeability

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Fig. 2. The concentration–response curves for the effect of ILs treatment on the inhibition rate using a logistic model (Note: the logistic model was used for calculating the IC50: y¼ A2 þ(A1  A2)/[1 þ(x/x0)P], where y represents the inhibition of alga growth (%), x represents the ILs concentration (mg/L), x0 represents the IC50,5d (mg/L), and A1, A2 and P are constants. All of the parameters are estimated using Origin 8.1.).

at 48 h was 1.11, 1.53, 1.93, 2.69 and 3.66 times that of the control group upon treatment with [C16mim]Cl. The ratios of each treatment to the control group of cellular membrane permeability (expressed as MPx/MP0) were calculated,

as shown in Table 3. It can be seen that the MPx/MP0 value first increased and then decreased as the exposure time increased, reaching a maximum value at 48 h. The MPx/MP0 value of 1 mg/L [C8mim]Cl treatment was 1.67, 4.82, 3.94 and 3.09 at 24, 48, 72 and

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Table 1 IC50 values of five ILs determined by different method (mg/L). ILs

Method

24 h-IC50

48 h-IC50

72 h-IC50

96 h-IC50

[C6mim]Cl

L D P L D P L D P L D P L D P

1.99 6.67 9.40 0.49 3.30 4.05 0.066 0.110 0.157 0.005 0.041 0.075 0.002 0.016 0.024

4.84 (4.26, 5.41) 5.61 6.17 (5.33, 7.11) 0.69 (0.63, 0.74) 0.86 1.36 (0.95, 1.98) 0.031 (0.021, 0.041) 0.040 0.047 (0.033, 0.073) 0.020 (0.015, 0.022) 0.021 0.027 (0.018, 0.040) 0.010 (0.009, 0.011) 0.010 0.012 (0.009, 0.016)

5.61 6.22 6.58 0.97 1.06 1.44 0.046 0.048 0.060 0.031 0.032 0.035 0.015 0.016 0.018

6.27 6.82 7.25 1.06 1.09 1.25 0.062 0.062 0.072 0.038 0.039 0.041 0.017 0.018 0.020

[C8mim]Cl

[C10mim]Cl

[C12mim]Cl

[C16mim]Cl

(1.58, 2.40) (7.10, 13.15) (0.02, 0.96) (2.88, 6.48) (0.038, 0.094) (0.113, 0.236) (0.002, 0.009) (0.048, 0.130) (  0.001, 0.005) (0.017, 0.038)

(5.24, 5.97) (5.94, 7.28) (0.94, 1.00) (1.14, 1.84) (0.039, 0.054) (0.045, 0.082) (0.028, 0.034) (0.025, 0.048) (0.014, 0.016) (0.016, 0.022)

(5.87, 6.67) (6.46, 8.12) (1.02, 1.10) (1.05, 1.50) (0.054, 0.070) (0.055, 0.097) (0.032, 0.043) (0.031, 0.054) (0.016, 0.018) (0.018, 0.022)

Note: Method “L” means the IC50 value was obtained using the logistic model, method “D” means the IC50 value was determined from the curve, method “P” means the IC50 value was obtained using the probit analysis. The value within brackets represent 95% confidence intervals.

Table 2 The regression equations for the IC50 values and the length of alkyl chain of the tested ILs. Expose time (h)

Regression equationa

R2

Significance level

24 48 72 96

Y ¼242.743e  0.652x Y ¼103.225e  0.640x Y ¼91.292e  0.602x Y ¼97.693e  0.595x

0.871 0.836 0.816 0.833

0.020 0.030 0.036 0.031

a Y stands for IC50 value (D method from Table 1) and X stands for the number of carbon atom in the side chain.

96 h, respectively. The MPx/MP0 value of 0.03 mg/L [C12mim]Cl treatment was 1.53, 3.98, 3.05 and 2.20 at 24, 48, 72 and 96 h, respectively. The MPx/MP0 value of 0.015 mg/L [C16mim]Cl treatment was 1.47, 3.66, 2.35 and 2.34 at 24, 48, 72 and 96 h, respectively. It can be concluded that the cellular membrane permeability was affected most seriously at 48 h, suggesting that 48 h was the most sensitive exposure period for the effect of ILs on alga, which was consistent with the results of the growth inhibition test. The effect of ILs on cellular membrane permeability increased with chain length as follows: [C8mim]Clo[C12mim]Clo[C16mim]Cl. ILs with longer alkyl chain lengths tended to possess stronger lipophilicity, which increased the odds of contact with the lipid bilayer and hydrophobic proteins of the membrane, thus damaging the normal physiological function of the membrane or even resulting in cell death (Latala et al., 2005; Ranke et al., 2007). 3.3. Effects of ILs on cellular ultrastructure of S. obliquus 3.3.1. Effects of ILs on cell wall, membrane and morphology The ultrastructural changes of S. obliquus are presented in Fig. 4. The cell walls of green algae consist of polysaccharides and multilayered structures, such as cellulose, making it quite hard. It was reported that S. obliquus is more sensitive to ILs than Euglena gracilis, which lacks cell walls, because the cellulose fibers that constitute the cell walls of S. obliquus can dissolve in ILs (Chen et al., 2014). The cell wall and cell membrane damage has been reported in herbicide-treated alga (Liu and Xiong, 2009). In the present study, the intact structure with a sealed and smooth cell wall was observed for the control (Fig. 4a). The cell wall became wavy when exposed to 2 mg/L [C8mim]Cl treatment, but plasmolysis did not occur (Fig. 4c). The cell wall became wavy and vague,

and obvious plasmolysis was observed upon 0.04 mg/L [C12mim]Cl treatment (Fig. 4e). The cell wall became slightly wavy and no longer compact upon 0.02 mg/L [C16mim]Cl treatment, and obvious plasmolysis was observed (Fig. 4g). The results showed that the cell wall and cell membrane were damaged by ILs compared with the control group, probably due to the ILs binding with the enzyme responsible for synthesis of very long chain fatty acid (VLCFAs). This would lead to an imbalance in the fatty acid composition of the cell plasma membranes, resulting in the loss of cell rigidity and permeability (Liu and Xiong, 2009). The concentrations of these three ILs at an algal growth inhibition rate of approximately 70% were used in the ultrastructure test. There are no large differences among these three treatments with regards to the extent of damage to the cell wall and membrane. The algal cells became more round and plasmolysis was observed following the 0.04 mg/L [C12mim]Cl and 0.02 mg/L [C16mim]Cl treatments. 3.3.2. Effects of ILs on chloroplast ultrastructure The chloroplasts of the control group occupy almost one-third of the cell volume and are located peripherally (Fig. 4a). The lamella structure of thylakoid stacks are clearly regularly, and neatly arranged in parallel formations (Fig. 4b). The lamella structure of the chloroplast became loose, osmiophilic granules were observed in the chloroplasts, and the thylakoid became twisted upon 2 mg/L [C8mim]Cl treatment (Fig. 4d). The lamella structure of chloroplasts became vague and larger osmiophilic granules in the chloroplasts were observed upon 0.04 mg/L [C12mim]Cl treatment (Fig. 4f). The lamella structure of chloroplasts became loose and osmiophilic granules were also observed upon 0.02 mg/L [C16mim]Cl treatment (Fig. 4h). The three ILs significantly damaged the structures of the chloroplasts and thylakoids, suggesting that the functions of the chloroplasts could be affected by the three ILs to some degree. This is probably because the external stress leads to a decrease in the photosynthetic pigment content (Geoffroy et al., 2007). There is no significant difference among the three treatments in terms of the damage to chloroplasts. 3.3.3. Effects of ILs on mitochondria ultrastructure Mitochondria are double-membrane-enclosed eukaryotic organelles with a central role in numerous cellular functions (Zick et al., 2009). It was found that the high dose of UV-B radiation led to swollen, irregular mitochondria and disintegrated cristae of mitochondria in algae (Dunaliella salina) (Tian and Yu, 2009). We can observe from the control group that the mitochondria presented as oval-shaped with a well-developed and visible double

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Fig. 3. Effects of ILs on the membrane permeability of algal cells (a. [C8mim]Cl; b. [C12mim]Cl; c. [C16mim]Cl) Note: The bars represent M 7SD. The different letters indicate significant differences at p o 0.05 compared to the other treatment according to Duncan's multiple range test.

Table 3 The ratios of each treatment to the control group of cellular membrane permeability (expressed as MPx/MP0). ILs

[C8mim]Cl

[C12mim]Cl

[C16mim]Cl

Concentration (mg/L)

0.2 0.4 0.6 0.8 1.0 0.002 0.005 0.010 0.020 0.030 0.003 0.006 0.008 0.010 0.015

MPx/MP0 24 h

48 h

72 h

96 h

1.23 1.33 1.46 1.59 1.67 1.29 1.45 1.46 1.51 1.53 1.14 1.19 1.24 1.30 1.47

1.50 1.65 2.18 3.32 4.82 1.11 1.60 1.74 2.79 3.98 1.11 1.53 1.93 2.69 3.66

1.10 1.75 2.15 3.15 3.94 1.12 1.37 1.71 2.06 3.05 1.06 1.29 1.61 2.11 2.34

1.15 1.48 2.07 2.72 3.09 0.99 1.21 1.52 1.58 2.20 1.02 1.15 1.49 2.08 2.34

membrane, and the cristae of mitochondria are not very rich (Fig. 4b). There was no significant difference in the volume of mitochondria between the treatments and the control group, but

the cristae of mitochondria seemed to be slightly swollen following the 2 mg/L [C8mim]Cl treatment (Fig. 4d) and the 0.04 mg/L [C12mim]Cl-treatment (Fig. 4f). The volume of mitochondria increased and the cristae seemed to be slightly swollen upon 0.02 mg/L [C16mim]Cl treatment (Fig. 4h). The mitochondria were damaged by the three ILs, suggesting that the three ILs influence the cellular respiration and energy conversion of algae. 3.3.4. Effects of ILs on vacuole ultrastructure As a type of detoxification organelle, vacuoles allow the exchange of gases and nutrients between the protoplast and its surroundings. It can also store and digest some intracellular metabolites. It was observed that non-membranous, electron-opaque deposits of an undefined structure occupied almost the entire vacuole in Cd-treated algal cells (Tukaj et al., 2007). Vacuoles were not observed in the control groups (Fig. 4a); however, large electron-dense deposits appeared in vacuoles upon 2 mg/L [C8mim]Cl treatment (Fig. 4c and d). Some vacuoles were filled with more electron-dense deposits upon 0.04 mg/L [C12mim]Cl treatment (Fig. 4e and f). Those vacuoles appeared more light-colored than those of the 2 mg/L [C8mim]Cl-treated group and had no obvious boundaries with the cytoplasm, suggesting that the structure of vacuoles may be damaged. Similar to the 2 mg/L [C8mim]Cl treatment, large electron-dense deposits appeared in vacuoles following 0.02 mg/L [C16mim]Cl treatment (Fig. 4g and h). In

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Fig. 4. Transmission electron micrographs of S. obliquus cells after 48 h (a and b: control treatment, c and d: 2 mg/L [C8mim]Cl treatment, e and f: 0.04 mg/L [C12mim]Cl treatment, g and h: 0.02 mg/L [C16mim]Cl treatment. All of the concentrations were chosen at a growth inhibition rate of approximately 70%).

conclusion, electron-dense deposits in vacuoles were observed in all three IL-treated groups, and more deposits were observed in the [C12mim]Cl and [C16mim]Cl treatment groups. These electronopaque deposits may be a mechanism for detoxifying xenobiotics (Liu and Xiong, 2009), suggesting that the algal cells responded to stress with detoxification, and the increased deposits in vacuoles indicated stronger adaptation to ILs. However, the detailed components of the electron-dense deposits require further study.

4. Conclusion The results showed that the IL toxicity was influenced by two main factors: exposure time and the alkyl chain lengths of

imidazolium chloride ILs. The growth inhibition rate and cellular membrane permeability results showed that the algae were most sensitive to the ILs at an exposure time of 48 h. The effects of the ILs on the IC50 values showed that the order of toxicity was [C6mim]Cl o[C8mim]Cl o[C10mim]Cl o[C12mim]Cl o[C16mim]Cl. The three ILs with varying alkyl chain length had negative effects on the cellular morphology and ultrastructure of the algae. The cell walls of the treated algae became wavy and separated from the cell membrane, the chloroplast grana lamellae became obscure and loose, osmiophilic material was observed in the chloroplasts, the mitochondria and their cristae swelled, and electron-dense deposits were observed in vacuoles. There was no significant difference among the three treatments of different ILs when growth inhibitions were at the same level (70%).

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Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21377115), the Zhejiang Provincial Natural Science Foundation of China (No. Y5100376), University Student's Science and Technology Innovation Project of the Zhejiang Provincial (No. 3070KZN0215088), and the Graduate Student's Science and Technology Project of Zhejiang Gongshang University (No. 1260XJ1513145).

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.07.010.

References Bernot, R.J., Kennedy, E.E., Lamberti, G.A., 2005. Effects of ionic liquids on the survival, movement, and feeding behavior of the freshwater snail, Physa acuta. Environ. Toxicol. Chem. 24, 1759–1765. Biczak, R., Pawłowska, B., Bałczewski, P., Rychter, P., 2014. The role of the anion in the toxicity of imidazolium ionic liquids. J. Hazard. Mater. 274, 181–190. Boyle, T.P., 1984. The effect of environmental contaminants on aquatic algae. In: Shubert, L.E. (Ed.), Algae as Ecological Indicators. Academic Press, New York, pp. 237–256. Cai, X.Y., Liu, W.P., Sheng, G.Y., 2008. Enantioselective degradation and ecotoxicity of the chiral herbicide diclofop in three freshwater alga cultures. J. Agric. Food Chem. 56, 2139–2146. Calabrese, E.J., 2015. Hormesis: principles and applications. Homeopathy 104, 69–82. Chai, S.H., Fulvio, P.F., Hillesheim, P.C., Qiao, Z.A., Mahurin, S.M., Dai, S., 2014. “Brickand-mortar” synthesis of free-standing mesoporous carbon nanocomposite membranes as supports of room temperature ionic liquids for CO2  N2 separation. J. Membr. Sci. 468, 73–80. Chen, H., Zou, Y.Q., Zhang, L.J., Wen, Y.Z., Liu, W.P., 2014. Enantioselective toxicities of chiral ionic liquids 1-alkyl-3-methylimidazolium lactate to aquatic algae. Aquat. Toxicol. 154, 114–120. CNEPA (Chinese National Environmental Protection Agency), 1990. Algal GrowthInhibiting Test. Guidelines for Testing of Chemicals. The Chinese Chemical Industry Press, Beijing, China. Cvjetko Bubalo, M., Radoševié, K., Radojčić, I.R., Halambek, J., Srček, V.G., 2014. A brief overview of the potential environmental hazards of ionic liquids. Ecotoxicol. Environ. Saf. 99, 1–12. Das, R.N., Roy, K., 2014. Predictive modeling studies for the ecotoxicity of ionic liquids towards the green algae Scenedesmus vacuolatus. Chemosphere 104, 170–176. Deng, Y., Beadham, I., Wu, J., Chen, X.D., Hu, L., Gu, J., 2015. Chronic effects of the ionic liquid [C4mim][Cl] towards the microalga Scenedesmus quadricauda. Environ. Pollut. 204, 248–255. Du, Z.K., Zhu, L.S., Dong, M., Wang, J.H., Wang, J., Xie, H., Zhu, S.Y., 2012. Effects of the ionic liquid [Omim]PF6 on antioxidant enzyme systems, ROS and DNA damage in zebrafish (Danio rerio). Aquat. Toxicol. 124–125, 91–93. Egorova, K.S., Ananikov, V.P., 2014. Toxicity of ionic liquids: eco(cyto)activity as complicated, but unavoidable parameter for task-specific optimization. ChemSusChem 7, 336–360. Fu, L., Li, J.J., Wang, Y., Wang, X.H., Wen, Y., Qin, W.C., Su, L.M., Zhao, Y.H., 2015. Evaluation of toxicity data to green algae and relationship with hydrophobicity. Chemosphere 120, 16–22. Geoffroy, L., Gilbin, R., Simon, O., Floriani, M., Adam, C., Pradines, C., Cournac, L., Garnier-Laplace, J., 2007. Effect of selenate on growth and photosynthesis of Chlamydomonas reinhardtii. Aquat. Toxicol. 83, 149–158. Jiang, T., Gao, H.X., Han, B.X., Zhao, G.Y., Chang, Y.H., Wu, W.Z., Gao, L., Yang, G.Y., 2004. Ionic liquid catalyzed Henry reactions. Tetrahedron Lett. 45, 2699–2701. Kárászová, M., Kacirková, M., Friess, K., Izák, P., 2014. Progress in separation of gases by permeation and liquids by pervaporation using ionic liquids: a review. Sep. Purif. Technol. 132, 93–101. Kumar, M., Kumari, P., Gupta, V., Anisha, P.A., Reddy, C.R., Jha, B., 2010. Differential responses to cadmium induced oxidative stress in marine macroalga Ulva lactuca (Ulvales, Chlorophyta). Biometals 23, 315–325. Kumar, M., Trivedi, N., Reddy, C.R., Jha, B., 2011. Toxic effects of imidazolium ionic liquids on the green seaweed Ulva lactuca: oxidative stress and DNA damage. Chem. Res. Toxicol. 24, 1882–1890. Latala, A., Stepnowski, P., Nedzi, 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., Zeng, S.H., Dong, X.Y., Ma, J.G., Wang, J.J., 2012. Acute toxicity and responses of antioxidant systems to 1-methyl-3-octylimidazolium bromide at different developmental stages of goldfish. Ecotoxicology 21, 253–259. Liu, H.J., Xiong, M.Y., 2009. Comparative toxicity of racemic metolachlor and S-metolachlor to Chlorella pyrenoidosa. Aquat. Toxicol. 93, 100–106.

Liu, H.J., Zhang, S.X., Zhang, X.Q., Chen, C.D., 2015. Growth inhibition and effect on photosystem by three imidazolium chloride ionic liquids in rice seedlings. J. Hazard. Mater. 286, 440–448. Luo, Y.R., Wang, S.H., Yun, M.X., Li, X.Y., Wang, J.J., Sun, Z.J., 2009. The toxic effects of ionic liquids on the activities of acetylcholinesterase and cellulase in earthworms. Chemosphere 77, 313–318. Ma, J.M., Cai, L.L., Zhang, B.J., Hu, L.W., Li, X.Y., Wang, J.J., 2010. Acute toxicity and effects of 1-alkyl-3-methylimidazolium bromide ionic liquids on green algae. Ecotoxicol. Environ. Saf. 73, 1465–1469. Megaw, J., Busetti, A., Gilmore, B.F., 2013. Isolation and characterisation of 1-alkyl3-methylimidazolium chloride ionic liquid-tolerant and biodegrading marine bacteria. PLoS One 8, e60806. Organization for Economic Cooperation and Development (OECD), 2006. Guide line for testing of chemicals: algal growth inhibition test. In: OECD Guideline 201. Peric, B., Sierra, J., Martí, E., Cruañas, R., Garau, M.A., 2015. Quantitative structure– activity relationship (QSAR) prediction of (eco)toxicity of short aliphatic protic ionic liquids. Ecotoxicol. Environ. Saf. 115, 257–262. Pham, T.P.T., Cho, C.-W., Min, J., Yun, Y.-S., 2008. Alkyl-chain length effects of imidazolium and pyridinium ionic liquids on photosynthetic response of Pseudokirchneriella subcapitata. J. Biosci. Bioeng. 105, 425–428. Pham, T.P.T., Cho, C.W., Yun, Y.S., 2010. Environmental fate and toxicity of ionic liquids: a review. Water Res. 44, 352–372. Radosevic, K., Cvjetko, M., Kopjar, N., Novak, R., Dumic, J., Srcek, V.G., 2013. In vitro cytotoxicity assessment of imidazolium ionic liquids: biological effects in fish Channel Catfish Ovary (CCO) cell line. Ecotoxicol. Environ. Saf. 92, 112–118. Ranke, J., Mölter, K., Stock, F., Bottin-Weber, U., Poczobutt, J., Hoffmann, J., Ondruschka, B., Filser, J., Jastorff, B., 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., Muller, A., Bottin-Weber, U., Stock, F., Stolte, S., Arning, J., Stormann, R., Jastorff, B., 2007. Lipophilicity parameters for ionic liquid cations and their correlation to in vitro cytotoxicity. Ecotoxicol. Environ. Saf. 67, 430–438. Samori, C., Pasteris, A., Galletti, P., Tagliavini, E., 2007. Acute toxicity of oxygenated and nonoxygenated imidazolium-based ionic liquids to Daphnia magna and Vibrio fischeri. Environ. Toxicol. Chem. 26, 2379–2382. Santos, D., Costa, F., Franceschi, E., Santos, A., Dariva, C., Mattedi, S., 2014. Synthesis and physico-chemical properties of two protic ionic liquids based on stearate anion. Fluid Phase Equilib. 376, 132–140. Shamsipur, M., Beigi, A.A.M., Teymouri, M., Pourmortazavi, S.M., Irandoust, M., 2010. Physical and electrochemical properties of ionic liquids 1-ethyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium trifluoromethanesulfonate and 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide. J. Mol. Liq. 157, 43–50. Suarez, P.A.Z., Dullius, J.E.L., Einloft, S., De Souza, R.F., Dupont, J., 1996. The use of new ionic liquids in two-phase catalytic hydrogenation reaction by rhodium complexes. Polyhedron 15, 1217–1219. Tian, J., Yu, J., 2009. Changes in ultrastructure and responses of antioxidant systems of algae (Dunaliella salina) during acclimation to enhanced ultraviolet-B radiation. J. Photochem. Photobiol. B: Biol. 97, 152–160. Tsarpali, V., Belavgeni, A., Dailianis, S., 2015a. Investigation of toxic effects of imidazolium ionic liquids, [bmim][BF4] and [omim][BF4], on marine mussel Mytilus galloprovincialis with or without the presence of conventional solvents, such as acetone. Aquat. Toxicol. 164, 72–80. Tsarpali, V., Harbi, K., Dailianis, S., 2015b. Physiological response of the greenmicroalgae Dunaliella tertiolecta against imidazolium ionic liquids [bmim][BF4] and/or [omim][BF4]: the role of salinity on the observed effects. J. Appl. Phycol. 10.1007/s10811-015-0613-6 Tsarpali, V., Dailianis, S., 2015. Toxicity of two imidazoliumionic liquids, [bmim] [BF4] and [omim][BF4], to standard aquatic test organisms: role of acetone in the induced toxicity. Ecotoxicol. Environ. Saf. 117, 62–71. Tukaj, Z., Baścik-Remisiewicz, A., Skowroński, T., Tukaj, C., 2007. Cadmium effect on the growth, photosynthesis, ultrastructure and phytochelatin content of green microalga Scenedesmus armatus: a study at low and elevated CO2 concentration. Environ. Exp. Bot. 60, 291–299. Unemoto, A., Ogawa, H., Gambe, Y., Honma, I., 2014. Development of lithium-sulfur batteries using room temperature ionic liquid-based quasi-solid-state electrolytes. Electrochim. Acta 125, 386–394. Ventura, S.P., Goncalves, A.M., Goncalves, F., Coutinho, J.A., 2010. Assessing the toxicity on [C3mim][Tf2N] to aquatic organisms of different trophic levels. Aquat. Toxicol. 96, 290–297. Ventura, S.P., Marques, C.S., Rosatella, A.A., Afonso, C.A., Goncalves, F., Coutinho, J.A., 2012. Toxicity assessment of various ionic liquid families towards Vibrio fischeri marine bacteria. Ecotoxicol. Environ. Saf. 76, 162–168. Viboud, S., Papaiconomou, N., Cortesi, A., Chatel, G., Draye, M., Fontvieille, D., 2012. Correlating the structure and composition of ionic liquids with their toxicity on Vibrio fischeri: A systematic study. J. Hazard. Mater. 215–216, 40–48. Wen, Y.Z., Chen, H., Shen, C.S., Zhao, M.R., Liu, W.P., 2011. Enantioselectivity tuning of chiral herbicide dichlorprop by copper: roles of reactive oxygen species. Environ. Sci. Technol. 45, 4778–4784. Yu, M., Wang, S.H., Luo, Y.R., Han, Y.W., Li, X.Y., Zhang, B.J., Wang, J.J., 2009. Effects of the 1-alkyl-3-methylimidazolium bromide ionic liquids on the antioxidant defense system of Daphnia magna. Ecotoxicol. Environ. Saf. 72, 1798–1804. Zhuo, K., Du, Q., Bai, G., Wang, C., Chen, Y., Wang, J., 2015. Hydrolysis of cellulose catalyzed by novel acidic ionic liquids. Carbohydr. Polym. 115, 49–53. Zick, M., Rabl, R., Reichert, A.S., 2009. Cristae formation-linking ultrastructure and function of mitochondria. Biochim. Biophys. Acta 1793, 5–19.