Growth inhibition and oxidative stress induced by 1-octyl-3-methylimidazolium bromide on the marine diatom Skeletonema costatum

Growth inhibition and oxidative stress induced by 1-octyl-3-methylimidazolium bromide on the marine diatom Skeletonema costatum

Ecotoxicology and Environmental Safety 132 (2016) 170–177 Contents lists available at ScienceDirect Ecotoxicology and Environmental Safety journal h...

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Ecotoxicology and Environmental Safety 132 (2016) 170–177

Contents lists available at ScienceDirect

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

Growth inhibition and oxidative stress induced by 1-octyl-3methylimidazolium bromide on the marine diatom Skeletonema costatum Xiang-Yuan Deng n, Xiao-Li Hu, Jie Cheng, Zhi-Xin Ma, Kun Gao College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212003, China

art ic l e i nf o

a b s t r a c t

Article history: Received 22 March 2016 Received in revised form 4 June 2016 Accepted 6 June 2016

Marine diatom Skeletonema costatum is an important prey in the marine food web and is often used as a standard test organism in ecotoxicological studies. In this study, in vivo experiments were performed to analyze the effects of 1-octyl-3-methylimidazolium bromide ([C8mim]Br) on the growth, photosynthetic activity, and oxidative stress in S. costatum using 96 h growth tests with a batch-culture system. The growth of S. costatum was significantly inhibited by [C8mim]Br with 48 and 96 h-EC50 of 17.9 and 39.9 mg L  1, respectively. The maximum quantum yield (Fv/Fm) and the light use efficiency (α) were inhibited by [C8mim]Br, which affected the growth of S. costatum. Subsequent biochemical assays in S. costatum revealed that [C8mim]Br induced changes of Chl a content, soluble protein content, and SOD activity, which had significant increases in low [C8mim]Br treatments (r 20 mg L  1), but decreased in high [C8mim]Br exposures ( Z 40 mg L  1). The increase of SOD activity at low concentrations (r 20 mg L  1) may be considered as an active defense of S. costatum against [C8mim]Br stress by reactive oxygen species (ROS) quenching. In addition, [C8mim]Br increased ROS level and malondialdehyde (MDA) content in S. costatum, suggesting that the physiological effects of [C8mim]Br are resulted from ROS generation. & 2016 Elsevier Inc. All rights reserved.

Keywords: Ionic liquid Skeletonema costatum 1-Octyl-3-methylimidazolium bromide Photosynthetic activity Antioxidant enzymes

1. Introduction With increasing development of green chemistry, ionic liquids (ILs) have gained more attention due to their excellent properties, such as very low vapor pressure, high solvency ability, high thermal stability and density, and large electrical conductivity (Freire et al., 2007; Grishina et al., 2013). Recently, ILs have been applied in many existing biological and chemical reactions to replace volatile organic solvents (Grishina et al., 2013; Deng et al., 2015). Numerous studies have implied that ILs could become persistent contaminants in water and soil due to low degree of their biodegradability (Docherty et al., 2007; Bruzzone et al., 2011; Cvjetko Bubalo et al., 2014b), and have significantly toxic effects on aquatic organisms, such as algae (Cho et al., 2008; Latała et al., 2009; Pretti et al., 2009; Latała et al., 2010), cladocerans (Bernot et al., 2005; Couling et al., 2006; Luo et al., 2008; Pretti et al., 2009; Ventura et al., 2010), mussels (Costello et al., 2009), and fish (Pretti et al., 2006, 2009). The toxic effects of ILs on aquatic organisms present “alkyl side chain” effect (increase in antimicrobial activity with n

Corresponding author. E-mail address: [email protected] (X.-Y. Deng).

http://dx.doi.org/10.1016/j.ecoenv.2016.06.009 0147-6513/& 2016 Elsevier Inc. All rights reserved.

elongation of the alkyl chain) and “cut-off” effect (beyond a given chain length, the effects cannot increase any further) (Ventura et al., 2012). To date, toxic mechanisms of ILs are not yet fully understood, although membrane disruption induced by lipid peroxidation, as a consequence of ROS generation, is proposed as a major toxic mechanism (Liu et al., 2010; Thuy Pham et al., 2010; Nancharaiah et al., 2012; Wu et al., 2013; Zhang et al., 2013; Cvjetko Bubalo et al., 2014a). As important primary producers in aquatic ecosystem, diatoms contribute to 20–25% of global primary productivity (Mann, 1999; Montsant et al., 2005). A cosmopolitan marine diatom Skeletonema costatum plays an important ecological role in supporting the primary productivity to higher trophic levels, and has been found in great abundance in coastal and oceanic waters (Thompson and Ho, 1981). S. costatum is also often used as a standard test organism in ecotoxicological assays (Macedo et al., 2008; Onduka et al., 2013; Brandt et al., 2015). Although some studies concerning the toxicity of ILs against diatoms have been reported (Ma et al., 2010; Samorì et al., 2011), to our knowledge, there is still little information about toxic mechanisms of ILs to diatoms. In this study, 1-octyl-3-methylimidazolium bromide ([C8mim]Br) was selected as the IL to be tested for the following

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reasons: (1) it is one of the representative imidazolium ILs (Luo et al., 2008; Yu et al., 2008; Ma et al., 2010; Li et al., 2012); (2) it is easily synthesized and widely used in chemical industry (Bonhôte et al., 1996); (3) it has been commonly used in previous studies, with toxicity levels between that of [C6mim]Br and [C10mim]Br (Luo et al., 2008; Yu et al., 2008; Ma et al., 2010; Li et al., 2012). Our objectives were to evaluate the effects of [C8mim]Br on the growth, photosynthetic activity, soluble protein content, antioxidant enzyme activity, and lipid peroxidation levels in S. costatum, and to analyze the toxic mechanisms of ILs to diatoms.

2. Materials and methods 2.1. Test chemicals and solutions The ionic liquid [C8mim]Br (CAS number: 61,545-99-1, purity 499.9%) was purchased from Chengjie Chemical Co., Ltd. (Shanghai, China); and other chemicals used in this study were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Stock solutions of [C8mim]Br were prepared in distilled water at a concentration of 5 g L  1. Test solutions were obtained by diluting the stock solution in f/2 medium (Guillard and Ryther, 1962). Concentrations of [C8mim]Br used in this study were 0, 5, 10, 20, 40, and 80 mg L  1 according to our previous investigations. 2.2. Test organism The unicellular marine diatom S. costatum was obtained from the State Key Laboratory of Marine Environmental Science (Xiamen University), and grown photoautotrophically in 500 mL Erlenmeyer flasks containing 200 mL of f/2 medium. The inoculum was pre-cultured aseptically in 250 mL Erlenmeyer flasks with 100 mL of f/2 medium. The flasks were placed in a 20 71 °C incubator (Jiangnan Instrument Factory, Ningbo, China), and illuminated from two sides by vertical cool white fluorescent lamps placed parallel to the flasks with a 12 h light/12 h dark photoperiod and a light density of 40 μmol photons m  2 s  1. 2.3. Experimental setup After pre-cultivation for 7 days, inoculum of S. costatum reached the exponential growth phase with a cell density of 1.6  107 cells mL  1. A total of 5 mL of the inoculum was collected by centrifugation (4000  g, 4 °C, 15 min). The collected cells of S. costatum were washed twice with sterile seawater, and then inoculated into 250 mL Erlenmeyer flasks to reach a final cell density of 8  105 cells mL  1 in 100 mL of f/2 medium with different [C8mim]Br concentrations, which were identified through preliminary range-finding tests. Three replicates were set up for each treatment and six for the controls, which were incubated as described above. 2.4. Microalgal growth analysis Cell density of S. costatum was determined spectrophotometrically at 680 nm using a multi-mode microplate reader (SpectraMax M5, Molecular Devices Company, CA, USA). The relationship between cell density of S. costatum (X, cells mL  1) and optical density of S. costatum culture at 680 nm (OD680) was determined experimentally, and is shown in the following linear equation:

(

)

X cells mL−1 = ( 3.52 × OD680 + 0.0089) × 107 , ( r = 0.997) The growth inhibition rate (IR, %) of S. costatum was calculated

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as follows:

IR (%) =

Xc − Xi × 100% Xc

where Xc and Xi are the average cell densities (cells mL  1) of S. costatum in the controls and ILs treatments, respectively. Furthermore, the specific growth rate (ri, h  1) was calculated according to the following formula:

ri (h−1) =

ln Xt − ln X 0 t

where X0 and Xt are the average cell densities (cells mL  1) of S. costatum at the beginning and at t h of exposure, respectively. The effect concentration of ILs leading to a 50% growth inhibition (EC50) of S. costatum was calculated using Marquardt's algorithm with the following theoretical mathematical function (Deng et al., 2012):

y=

c 1 + exp [b (x − a)]

where y is the specific growth rate of S. costatum in the test medium i, ri; x is the logarithm of test chemical concentration; a is the logarithm of EC50; b is the slope of the function; c is the specific growth rate of S. costatum in the controls. 2.5. Measurements of photosynthetic parameters The photosynthetic parameters of S. costatum exposed to different [C8mim]Br concentrations (0, 5, 10, 20, 40, and 80 mg L  1) at different times (0, 12, 24, 48, 72 and 96 h) were measured by pulse-amplitude modulation (PAM) fluorometry (Phyto-PAM, Heinz Walz GmbH, Effeltrich, Germany). Prior to measurements, samples were kept in the dark for 15 min. Then, the maximum quantum yield (Fv/Fm) and the light use efficiency (α) were determined and calculated according to Schreiber (1998). 2.6. Chlorophyll a content measurements To determine chlorophyll a (Chl a) content, triplicates of 5 mL of well-blended cultures were centrifuged at 4000  g for 15 min to remove the supernatants. The pellets were homogenized with 5 mL of high-performance liquid chromatography (HPLC)-grade methanol for pigment extraction. The mixtures were vigorously shaken with a vortex and placed in a refrigerator in the dark at 4 °C for 24 h. The methanol-extracted samples were centrifuged at 10,000  g for 5 min to remove the pellets, and the supernatants were transferred into 96-well plates (Corning Incorporated Life Sciences, Tewksbury, USA) and measured for Chl a at 750, 665, and 652 nm using a multi-mode microplate reader. All absorbance values were corrected using HPLC-grade methanol as control. Concentrations of Chl a (μg mL  1) (Ca) were calculated by the following equation (Porra, 2002):

Ca (μg mL−1) = 16.3 × ( A665 − A750 ) − 8.54 × ( A652 − A750 ) where A652, A665 and A750 are the absorbance values at 652, 665 and 750 nm, respectively. The final value of Chl a content was expressed as micrograms of Chl a per 107 cells (μg  107 cell  1) ( Ca′ ), and calculated using the following equation:

Ca′ (μg × 107cell−1) =

Ca × VMeOH X × Vsample

where VMeOH is the methanol volume (5 mL), Vsample is the volume of sample (5 mL) and X is the cell density of S. costatum (cells mL  1). 2.7. Protein content and superoxide dismutase activity

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determination After 96 h of [C8mim]Br exposure, 40 mL of well-blended cultures were harvested by centrifugation (4000  g, 15 min, 4 °C). The harvested cells of S. costatum were placed in 1.5 mL extraction buffer containing 0.05 mol L  1 sodium phosphate buffer (pH 7.8), and then lysed by sonication (Scientz Biotechnology Co. Ltd., Ningbo, China) for 10 min with a repeating duty cycle of 5 s in ice bath. The cellular homogenate was centrifuged at 12,000  g for 10 min at 4 °C, and the liquid supernatant was stored at  70 °C for protein determination and enzyme assay. Total soluble protein content was measured using the Bradford method with bovine serum albumin as standard (Bradford, 1976). Results were expressed as micrograms of protein per 107 cells (μ g  107 cell  1). Superoxide dismutase (SOD) activity was determined using a reagent kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions and Wang and Zheng (2008). Results of SOD activity were given as units of enzyme activity per gram of total soluble protein (U g  1 protein). 2.7.1. Reactive oxygen species level and malondialdehyde content determination Reactive oxygen species (ROS) and malondialdehyde (MDA) assay kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China). ROS level was determined using a 2,7-dichlorofuorescin diacetate probe. ROS level was detected by a multi-mode microplate reader (SpectraMax M5, Molecular Devices Company, CA, USA) at the emission and excitation wavelengths of 525 and 485 nm, respectively. Results of ROS level were given as units of fluorescence per 107 cells (FU 107 cell  1). MDA was extracted and detected from the supernatant liquids (above) according to the manufacturer's instructions and Wang and Zheng (2008). Results of MDA content were given as nanomole per 107 cells (nmol 107 cell  1), respectively. 2.8. Statistical analysis All results were presented as mean value 7standard deviation (SD). Statistical analysis was performed using SPSS PASW Statistics 18 software. One-way analysis of variance was used to establish differences among treatments, with a significance level setting at 5% (p ¼0.05).

3. Results and discussion 3.1. Growth inhibition of S. costatum by [C8mim]Br One of the representative imidazolium ILs ([C8mim]Br) was used in this study to evaluate effects of ILs on the growth of S. costatum. Results showed that in the controls, S. costatum had a normal exponential growth pattern with the maximum specific growth rate of 0.012 h  1 at 72 h. Growth patterns of S. costatum had no significant differences with the controls and the maximum specific growth rates were 0.011 and 0.011 h  1 in 5 and 10 mg L  1 [C8mim]Br treatments, respectively. However, the growth of S. costatum in 20, 40 and 80 mg L  1 [C8mim]Br treatments showed a significant decrease (p o0.05) and the maximum specific growth rates were 0.010, 0.006 and  0.009 h  1, respectively (Fig. 1). Similar findings were obtained by Nancharaiah and Francis (2015), who reported that the growth of bacteria (Clostridium sp. and Pseudomonas putida) was stimulated at up to 2.5 g L  1 and inhibited at 4 2.5 g L  1 of 1-ethyl-3-methylimidazolium acetate. Thus, it is indicated that [C8mim]Br can be a threat to the growth of phytoplankton once it is accidentally leaked into aquatic

Fig. 1. Growth curves of Skeletonema costatum under different [C8mim]Br concentrations during 96 h of exposure. The points represent the means of 3 replicates; the error bars represent standard deviation (SD).

ecosystems. The growth inhibition rate (IR, %) of S. costatum increased with increasing [C8mim]Br concentration at the same exposure time. For example, IR increased from 6.56% (5 mg L  1) to 84.7% (80 mg L  1) at 96 h of exposure. However, IR increased first and then decreased with increasing exposure time in low-concentration of [C8mim]Br treatments. In 5, 10 and 20 mg L  1 [C8mim]Br treatments, IR increased from 14%, 23.8% and 30.9% (24 h) to 17.5%, 25.3% and 34.6% (48 h), and then decreased to 6.55%, 13.6% and 24.8% at 96 h, respectively. These results suggested that inhibition effects of [C8mim]Br on the growth of S. costatum could be decreased with increasing exposure time at low concentrations. The IR increased with increase of exposure time in response to highconcentration of [C8mim]Br treatments. The IR increased from 37.8% (24 h) to 58.5% (96 h) and from 57.2% (24 h) to 84.7% (96 h) in 40 and 80 mg L  1 [C8mim]Br treatments, respectively. These results indicated that [C8mim]Br had toxic effects on the growth S. costatum at high concentrations. Similar results have also been described for the Baltic algae Oocystis submarina treated with [C6mim]BF4 (Latała et al., 2005), and Scenedesmus obliquus treated with [C8mim]Cl (Liu et al., 2015). The growth of O. submarina and S. obliquus could be recovered by increasing exposure time at low concentrations, but not at high concentrations of ILs. In low-concentration treatments, recovery mechanism of microalgae may be due to a modest allocation of resources for repairing damage, which may be integrated into other processes leading to a reduction in background damage, or to enhance resistance (Calabrese, 2015). In addition, the EC50 values of [C8mim]Br against S. costatum were calculated by Marquardt's algorithm method (Deng et al., 2012), and compared with data on [C8mim]Br toxicity to microalgae in previously published literatures (Table 1). In this study, the 24, 48, 72 and 96 h EC50 values were 24.4, 17.9, 39.7, 39.9 mg L  1, respectively, which suggested that the toxicity of [C8mim]Br increased first and then decreased with increasing exposure time, reaching its maximum toxicity at 48 h. The maximum toxic effects of [C8mim]Cl was also presented at 48 h against S. obliquus, while it was more toxic to S. obliquus than [C8mim]Br (Liu et al., 2015). Previous reports indicated that an exposure time 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 of exposure (Fu et al., 2015). In this study, there were about 0.35 log units differences between the toxicity of 48 h and 72 h or 96 h of exposure to S. costatum. The decrease in toxicity with

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Table 1 Comparison of the toxicity of [C8mim]Br to microalgae. EC50: concentration for 50% of the maximal effects; nr: not reported. Microalgae

Freshwater species Chlamydomonas reinhardtii Chlorella ellipsoidea Selenastrum capricornutum Scenedesmus obliquus Scenedesmus quadricauda Marine species Skeletonema costatum Phaeodactylum tricornutum Oocystis submarina Cyclotella meneghiniana

EC50 (mg L  1)

Reference

96

50.7

96 96

6.37 7.24–15.1

Kulacki and Lamberti, 2008 Ma et al., 2010 Cho et al., 2007

96 96

0.34 0.005

Ma et al., 2010 Kulacki and Lamberti, 2008

96 96

39.9 8.89

This study Deng et al., 2015

nr nr

Latała et al., 2005 Latała et al., 2005

Exposure time (h)

288 288

increase of exposure time probably resulted from the acclimation of algae to chemicals (Ertürk and Saçan, 2012). Furthermore, there were several orders of magnitude differences between EC50 values of [C8mim]Br to microalgae, which may help to explain the mechanism of ILs toxicity. One clue to this mechanism may be related to the cell wall structures of different microalgal species, since the cell wall plays an important role in transport of materials in and out of the cell (Kulacki and Lamberti, 2008; Samorì et al., 2011; Nancharaiah and Francis, 2011). 3.2. Photosynthetic activity and pigments of S. costatum after exposure to [C8mim]Br The efficiency of light energy conversion into chemical energy and its subsequent use by plants and algae may give useful information on the physiological state of these organisms (Schreiber et al., 1995). The maximum quantum yield (Fv/Fm) and the light use efficiency (α) of S. costatum were determined after dark adaptation, and presented in Fig. 2. Fv/Fm is often used as a sensitive indicator of plant photosynthetic performance, at which light absorbed by PSII is used for reduction of the primary acceptor (quinone QA). As seen in Fig. 2A, Fv/Fm increased sharply from 0 to 12 h of exposure, and then remained constant (about 0.52) after 12 h of exposure in the controls, which suggested that there was an initial

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lag period (12 h) at the beginning of S. costatum cultivation. In 5, 10 and 20 mg L  1 [C8mim]Br treatments, Fv/Fm increased from 0.39, 0.40 and 0.41-0.53, 0.50 and 0.41 with increase of exposure time from 0 to 48 h of exposure, and then decreased to 0.40, 0.31 and 0.27 at 96 h of exposure, respectively. In addition, Fv/Fm decreased from 0.37 and 0.40-0.24 and 0.18 from 0 to 96 h of exposure in 40 and 80 mg L  1 [C8mim]Br, respectively, while there was a slight increase from 0 to 24 h of exposure. Fv/Fm measurements are recommended as a simple and rapid way to abiotic and biotic stressors on plants and algae (Baker, 2008). The decrease of Fv/Fm observed by increasing [C8mim]Br concentrations could be the result of an increased proportion of QB-non-reducing PSII reaction centers. Moreover, [C8mim]Br may also induce a state analogous to photoinhibition in S. costatum, wherein trapping of an exciton in the PSII reaction center is followed by its non-radiative dissipation. In addition, effects of [C8mim]Br on the light use efficiency of S. costatum are shown in Fig. 2B. The α increased from 0.17 to 0.24 from 0 to 24 h of exposure, and then remained constant (0.24) after 24 h of exposure in the controls. Although increases of α were observed from 0 to 24 h of exposure, α gradually decreased to 0.22, 0.19, 0.17 and 0.15 at 96 h of exposure in 5, 10, 20 and 40 mg L  1 [C8mim]Br treatments, respectively. In 80 mg L  1 [C8mim]Br treatments, α decreased from 0.18 to 0.09 during 96 h of exposure. At 96 h of exposure, the α decreased by 5.67%, 18.9%, 28.4%, 38.4% and 62.4% in 5, 10, 20, 40, and 80 mg L  1 [C8mim]Br treatments relative to the controls, respectively. As we know, α represents the light use efficiency of plants. Reduction of α indicates that [C8mim]Br restricted light use efficiency of S. costatum perhaps as a result of the blockage of electron transport, and finally caused growth inhibition of S. costatum. During the microalgal growth and reproduction, Chl a can transfer photons to the reaction center of photosystem II (Kalaji and Guo, 2008), which is also a sensitive parameter to evaluate the microalgae response to toxic organic contaminants (Bi et al., 2012). The change of Chl a content in S. costatum under [C8mim]Br exposure is illustrated in Fig. 3. Relative to the controls, Chl a content increased significantly in 5 mg L  1 [C8mim]Br treatments (p o0.05), but did not change significantly in [C8mim]Br treatments of 10 and 20 mg L  1 (p 40.05). It is very interesting to note that the Chl a content in S. costatum under 5 mg L  1 [C8mim]Br exposure was even higher than that of the controls. This phenomenon was attributed to the conversion of other pigments and Chl a under stressors (Fang et al., 1998). We suggested that high accumulation of Chl a content in S. costatum is a response to the

Fig. 2. Photosynthetic activity changes in Skeletonema costatum under different levels of [C8mim]Br stress during 96 h of exposure, showing changes of (A) the maximum photochemical efficiency of PSII (Fv/Fm) and (B) the light use efficiency (α). The points represent the means of 3 replicates; the error bars represent standard deviation (SD).

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Fig. 3. Effects of [C8mim]Br on the mean values (mean 7SD; number of replicates (n)¼ 3) of chlorophyll a content in Skeletonema costatum after 96 h of exposure. Each bar is the mean of 3 replicates. The error bar represents standard deviation (SD). Datasets that are significantly different from each other are represented by different letters (p o 0.05).

stress of [C8mim]Br according to previous studies, which showed that pollutants can significantly promote the activity of nitrate reductase to accelerate the conversion of inorganic nitrogen (NO3-N and NH4 þ -N) to organic nitrogen (chlorophyll) (Yang et al., 2006). Furthermore, Chl a content had a significant decrease (43.8% and 27.2% of that in the controls) in 40 and 80 mg L  1 [C8mim]Br treatments, respectively (p o0.05). It is also reported that the primary productivity and total chlorophyll content of S. obliquus decreased significantly under [C10mim]Br exposure (Ma et al., 2010). This could be attributed to that the long alkyl chain possessed by [C8mim]Br could be incorporated into the polar head groups of the phospholipid bilayer. It would cause the disruption of the membrane-bound proteins and the structural integrity of chloroplasts (Couling et al., 2006). It is well known that any interference or damage to photosynthesis will negatively affect the microalgal growth. 3.3. Soluble protein content and SOD activity of S. costatum after exposure to [C8mim]Br As an important indicator in metabolism, soluble protein content is known to respond to a wide variety of environmental stressors (Bajguz and Piotrowska-Niczyporuk, 2014). Fig. 4 shows

that total soluble protein content in S. costatum had a slight increase (1.15 and 1.48 times of that in the controls) in 5 and 10 mg L  1 [C8mim]Br treatments, respectively, and reached the maximum value (20.3 μg 107 cell  1) in 20 mg L  1 [C8mim]Br treatments. Then total soluble protein content had a significant decrease in 40 and 80 mg L  1 [C8mim]Br treatments (p o0.05). Enzymes, including antioxidant and biotransformation enzymes, are important components of soluble protein, and may serve as an active defense mechanism to protect cells from abiotic and biotic stressors (Kumar et al., 2008). Thus, we suggest that the increase of total soluble protein content, especially enzymes, may help S. costatum to tolerate the toxicity of [C8mim]Br as well as to compensate the need for energy to help in defense against [C8mim]Br. As a key antioxidant defense enzyme, alteration in the SOD activity is also an important biomarker to estimate oxidative stress. SOD plays an important role in normal cellular metabolism and is capable of eliminating redundant reactive oxygen species (ROS) and decreasing the potential toxic effects to organisms (Blokhina et al., 2003). In the present study, [C8mim]Br concentrations of 5 and 10 mg L  1 caused a slight increase (1.44 and 1.65 times greater than that in the controls) of SOD activity in S. costatum; and the maximum SOD activity of 26.1 U g  1 protein (2.67 times of that in the controls) was obtained in 20 mg L  1 [C8mim]Br treatments (Fig. 4). A significant increase of SOD activity compared with the controls was also observed in the freshwater crustacean Daphnia magna treated with [C8mim]Br (Yu et al., 2009). When an aquatic plant duckweed (Lemna minor) was exposed to [C8mim]Br for 28 d, SOD activity was also increased at 7 d of exposure, while it decreased at the termination of exposure (Zhang et al., 2013). According to Rana and Shivanandappa (2010), ROS are induced when foreign substances enter into an organism. Thus, the increase of SOD activity indicated its elevated capacity to scavenge ROS. Higher SOD activity could also efficiently protect against potential increased production of ROS and would reflect an adaptation in response to the oxidative conditions to which S. costatum was exposed. However, SOD activity in S. costatum decreased from 26.1 to 8.31 U g  1 protein when [C8mim]Br concentrations increased from 20 to 80 mg L  1 (Fig. 4). Similar results were also found in Lemna minor (Zhang et al., 2013). This phenomenon may be because of the accumulation of ROS in S. costatum induced by [C8mim]Br. High production of ROS would overwhelm cellular antioxidants due to the inhibition of antioxidant enzymes such as SOD. Once the excess of ROS overwhelm

Fig. 4. Effects of [C8mim]Br on the mean values (mean7 SD; number of replicates (n)¼ 3) of total soluble protein content and SOD activity in Skeletonema costatum after 96 h of exposure. Each bar is the mean of 3 replicates. The error bar represents standard deviation (SD). Datasets that are significantly different from each other are represented by different letters (p o 0.05).

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Fig. 5. Effects of [C8mim]Br on the mean values (mean7 SD; number of replicates (n) ¼3) of ROS level and MDA content in Skeletonema costatum after 96 h of exposure. Each bar is the mean of 3 replicates. The error bar represents standard deviation (SD). Datasets that are significantly different from each other are represented by different letters (po 0.05).

the antioxidant system, the protective role of SOD would be minimized and oxidative stress would occur (Jihen et al., 2009; Dong et al., 2013; Deng et al., 2015). 3.4. ROS level and MDA content of S. costatum after exposure to [C8mim]Br Under normal circumstances, ROS are generated as natural byproducts of normal cellular metabolism, which is important to maintain homeostasis (D’Autréaux and Toledano, 2007). But excessive ROS may be produced when cells are exposed to external stimulus such as pollutants. As shown in Fig. 5, ROS level of S. costatum was enhanced significantly over the controls when [C8mim]Br concentrations are Z10 mg L  1 at 96 h exposure; while there was not significant difference between the controls and the treatments with 5 mg L  1 [C8mim]Br. Now, it is well known that damaging effects on chloroplast and photosynthesis can cause an increase of ROS level in plants (Okpodu et al., 1996). In photosynthetic organisms, ROS is mainly produced through the transfer of electrons on molecular oxygen (O2) from photosystem I (PSI) during the photosynthesis process (Asada, 1999). An increase of ROS level in this study may be the consequence of the inhibition on photosynthesis as mentioned above. Furthermore, the excess ROS in S. costatum may lead to oxidative stress, which can aggressively attack other molecules, such as DNA, proteins and fatty acids (Finkel and Holbrook, 2000; Kim et al., 2008). These results are consistent with other reports which also found increased ROS level in ILs treatments relative to the controls (Zhang et al., 2013; Wu et al., 2013; Chen et al., 2014). One of the major consequences of ILs treatment is the enhanced level of ROS, which can induce the oxidation of polyunsaturated fatty acids and lead to lipid peroxidation (Dong et al., 2013). MDA is a cytotoxic product of lipid peroxidation and often used as an indicator of ROS production and severity of cellular damage. In this study, MDA content in S. costatum did not increase significantly in 5 mg L  1 [C8mim]Br treatments (p 40.05), but it significantly increased by 132% and 207% in 10 and 20 mg L  1 [C8mim]Br treatments (p o0.05), respectively, relative to the controls. And then, MDA content decreased to 1.51 and 1.10 nmol 107 cell  1 in S. costatum exposed to 40 and 80 mg L  1 [C8mim]Br, respectively (Fig. 5). It is deduced that the increased MDA content under [C8mim]Br exposure may be attributed to the induction of ROS, which enhances the oxidation of polyunsaturated fatty acids

and leads to lipid peroxidation (Valavanidis et al., 2006; Dong et al., 2013). And more MDA would further damage the microalgal cells because MDA can readily interact with several functional groups of molecules, such as proteins, lipoproteins and DNA (Maes et al., 2006). Thus, it can be deduced that the physiological effects of [C8mim]Br on S. costatum were resulted from ROS generation.

4. Conclusion Results of this study showed that the growth of S. costatum was significantly inhibited by [C8mim]Br. The inhibition of [C8mim]Br could be decreased with increasing exposure time at low concentrations (r20 mg L  1), but not at high concentrations (Z 40 mg L  1). It is also indicated that an exposure time of 48 h was the most sensitive for algal growth inhibition tests. Simultaneously, remarkable physiological and biochemical responses were occurred in S. costatum. Chl a content, soluble protein content and SOD activity had significant decreases in high [C8mim]Br treatments ( Z40 mg L  1), but they were increased in low [C8mim]Br treatments ( r20 mg L  1). The increase of SOD activity at low concentrations ( r20 mg L  1) may be considered as an active defense of S. costatum against moderate [C8mim]Br stress by ROS quenching. Furthermore, the general increase of ROS level and MDA content suggests that the physiological effects of [C8mim]Br on S. costatum were caused by ROS generation.

Acknowledgments This manuscript was supported by the National Natural Science Foundation of China (No. 31200381), China Postdoctoral Science Foundation Funded Project (Nos. 2013M531370, 2014T70532), Key Laboratory for Ecological Environment in Coastal Areas, State Oceanic Administration (201209).

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