Toxicity of sulfamethazine and sulfamethoxazole and their removal by a green microalga, Scenedesmus obliquus

Chemosphere 218 (2019) 551e558

Contents lists available at ScienceDirect

Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Toxicity of sulfamethazine and sulfamethoxazole and their removal by a green microalga, Scenedesmus obliquus Jiu-Qiang Xiong a, Sanjay Govindwar a, *, Mayur B. Kurade a, Ki-Jung Paeng b, Hyun-Seog Roh c, Moonis Ali Khan d, Byong-Hun Jeon a, ** a

Department of Earth Resources and Environmental Engineering, Hanyang University, Seoul, 04763, South Korea Department of Chemistry, Yonsei University, 1 Yonseidae-gil, Wonju, 26493, South Korea Department of Environmental Engineering, Yonsei University, 1 Yonseidae-gil, Wonju, Gangwon, 26493, South Korea d Chemistry Department, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia b c

h i g h l i g h t s
S. obliquus can withstand high doses of SMZ and SMX.
EC50 of SMZ, SMX and their mixture for S. obliquus was 1.23, 0.12, and 0.15 mg L1.
S. obliquus removed 62.3 and 46.8% of SMZ and SMX, respectively.
A greater biodegradation was observed in higher SMZ and SMX concentration.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 August 2018 Received in revised form 21 November 2018 Accepted 23 November 2018 Available online 24 November 2018

A comprehensive ecotoxicological evaluation of a sulfamethazine (SMZ) and sulfamethoxazole (SMX) mixture was conducted using an indicator microalga, Scenedesmus obliquus. The toxicological effects of this mixture were studied using microalgal growth patterns, biochemical characteristics (total chlorophyll, carotenoid, carbohydrate, fatty acid methyl ester), and elemental and Fourier-transform infrared spectroscopy analyses. The 96-h half maximal effective concentration (EC50) of the SMZ and SMX mixture was calculated to be 0.15 mg L1 according to the dose-response curves obtained. The chlorophyll content decreased with elevated SMZ and SMX concentrations, while the carotenoid content initially increased and then decreased as concentration raised. The unsaturated fatty acid methyl esters (FAMEs) content was enhanced with higher SMZ and SMX concentrations, while that of saturated FAMEs simultaneously decreased due to SMZ and SMX stress. Elemental analyses showed an improved percentage of nitrogen and sulfur in the microalgal biomass as SMZ and SMX concentrations increased. The microalga S. obliquus was shown to biodegrade the chemicals tested and removed 31.4e62.3% of the 0.025e0.25 mg SMZ L1 and 27.7e46.8% of the 0.025e0.25 mg SMX L1 in the mixture after 12 days of cultivation. The greater biodegradation observed at higher SMZ and SMX concentrations indicates that microalgal degradation of SMZ and SMX could act as an efficient adaptive mechanism to antibiotics. © 2018 Published by Elsevier Ltd.

Handling Editor: Jian-Ying Hu Keywords: Emerging contaminants Pharmaceutical contaminants Microalgae Biodegradation Toxicity Bioremediation

1. Introduction Environmental pollution with pharmaceutical contaminants (PCs) is an emerging concern, since PCs can have adverse ecological effects on the growth, nitrification and denitrification, and

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] hanyang.ac.kr (B.-H. Jeon). https://doi.org/10.1016/j.chemosphere.2018.11.146 0045-6535/© 2018 Published by Elsevier Ltd.

(S.

Govindwar),

bhjeon@

community composition of microbes (Khetan and Collins, 2007; Schwarzenbach et al., 2006; Xiong et al., 2018a). Steroidal PCs are known to induce widespread feminization in fish due to their endocrine disruptor properties (Lange et al., 2009). The presence of PCs in the environment facilitates the development of bacterial resistance to various drugs, which is one of most critical problems currently facing human health (Martínez, 2008). Conventional wastewater treatment plants cannot effectively remove these PCs, leading to their frequent detection in various environments, such as streams, lakes, rivers, groundwater, wastewater, and soil (Hughes

552

J.-Q. Xiong et al. / Chemosphere 218 (2019) 551e558

et al., 2012). Considering their implications to the health of humans and aquatic ecosystems, a comprehensive ecotoxicological analysis of PCs and effective, cost-efficient technologies for their remediation are needed for use in risk evaluations and treatment of contaminated sites. The use of growth inhibition tests of contaminants involving microalgae, specifically freshwater algae and cyanobacteria, has been suggested as a standard protocol to analyze the toxicity of contaminants by the Organization for Economic Co-operation and Development (OECD, 2011). Additionally, interest in microalgaebased remediation biotechnologies has been growing in recent years, as these green technologies have many benefits, including being driven by solar energy, eco-friendly, and potentially themselves producing valuable products and biomass. Although many studies have successfully utilized microalgae for the removal of nutrients and organic matter from wastewater in different locations (Salama et al., 2017; Hom-Diaz et al., 2017), only a limited number of studies have been conducted on microalgae-based methods for the removal of PCs. Matamoros et al. (2016) demonstrated that mixed microalgal species (mainly Chlorella sp. and Scenedesmus sp.) removed numerous pharmaceuticals from wastewater, including carbamazepine, galaxolide, ibuprofen, tris(2-chloroethyl) phosphate, 4-octylphenol, tributyl phosphate, and caffeine. Hom-Diaz et al. (2017) showed that microalgae can remove 30e98% of fifteen different PCs from lake water and pharmaceutical wastewater. Nevertheless, information on the effects of different PCs and their capacity for removal by microalgae require further investigation. Sulfonamides have been developed as the first antibiotics to systemically treat infectious diseases of humans and animals. Sulfonamides contamination has been frequently found in groundwater, surface water, wastewater, and soil. Adverse ecological effects and related human health issues have been demonstrated because of the accumulation properties and toxicity of sulfonamides. Sulfamethazine (SMZ) and sulfamethoxazole (SMX) are two of the most frequently found sulfonamides in environment with a concentration range of 10 mg L1 to 231 mg L1 (SMZ), and 4e12 mg L1 (SMX), respectively in wastewaters (Bielen et al., 2017; Loos et al., 2009). Limited literatures are available to illustrate the toxicity of SMZ and SMX, and their removal in microalgal culture. In this study, the effects of SMZ and SMX in a mixture, and their removal in a freshwater microalga, Scenedesmus obliquus were investigated. The toxicity of the SMZ and SMX mixture (w/w 1:1) was carefully evaluated in terms of its effects on the growth and biochemical composition of S. obliquus, and the removal kinetics of

the SMZ and SMX mixture by S. obliquus were monitored over the course of 12 days of microalgal cultivation. 2. Materials and methods 2.1. Chemicals Sulfonamides (sulfamethazine and sulfamethoxazole) were acquired from Sigma-Aldrich (St. Louis, MO, USA), and their physicochemical properties are presented in Table 1. Other chemicals used were of analytical grade, and obtained from Duksan (Seoul, South Korea). 2.2. Toxicity and SMZ and SMX removal experiment The microalgal species S. obliquus HM103383 used in this study was previously isolated from a local river in Wonju, South Korea (Abou-Shanab et al., 2011). A 250-mL capacity Erlenmeyer flask with sterilized Bold's Basal Medium (BBM, 150 mL) was used for the inoculum preparation of S. obliquus in a shaking incubator (under a light intensity of 45e50 mmol photons m2 s1) for 14 days. The light/dark ratio, cultivation temperature, and shaking speed were set at 16:8 h, 27  C, and 150 rpm, respectively. The toxicity of different concentrations of the SMZ and SMX mixture (0, 0.05, 0.15, 0.25, 0.35, and 0.5 mg L1) with a SMZ: SMX ratio of 1:1 (w/w) to S. obliquus, as well as their removal by the microalga, were investigated. The abiotic removal (without inoculum of microalgae) of SMZ and SMX was conducted, which was monitored through amending the culture flasks with the same concentrations of SMZ and SMX mixture without inoculum of microalgae. Flasks containing 1.0% concentrations (Vinoculum/Vmedia) of the microalgal culture at an optical density of ~1.0 at 680 nm (OD680 ¼ 1.0) were held in the abovementioned incubation conditions and different SMZ and SMX mixture concentrations for 12 days. The desired optical density of the microalgal suspension was achieved through diluting the initial 14-day-old microalgal culture with sterilized BBM. 2.3. Growth, biochemical characteristics, elemental analyses, and FT-IR of S. obliquus The dry cell weight (DCW) of S. obliquus was measured by following methods used in our previous study (Xiong et al., 2016, 2017a). Pigments were extracted using 90% methanol, and their content was calculated according to equations reported elsewhere

Table 1 Physicochemical properties of sulfamethazine and sulfamethoxazole. Properties

Sulfamethazine (SMZ)

Sulfamethoxazole (SMX)

Chemical structure

Molecular formula CAS reg. no. Molecular mass Water solubility logKow pKa Therapeutic class Mode of action

C12H14N4O2S C10H11N3O3S 57-68-1 723-46-6 278.33 253.279 1.5 mg mL1 0.61 mg mL1 0.14 0.89 2.07; 7.49 1.6; 5.7 Sulfonamides Inhibiting bacterial synthesis of dihydrofolic acid by competing with para-aminobenzoic acid for binding to dihydropteroate synthetase (dihydrofolate synthetase). Inhibition of dihydrofolic acid synthesis decreases the synthesis of bacterial nucleotides and DNA.

J.-Q. Xiong et al. / Chemosphere 218 (2019) 551e558

(Xiong et al., 2017b). The percentage compositions of different elements (carbon, hydrogen, nitrogen, and sulfur) and major functional groups in S. obliquus were quantified using an elemental analyzer (FLASH EA1112, Thermo Electron Corporation, USA) and a Fourier-transform infrared (FT-IR) spectroscope (Nicolet iS50, Thermo Scientific, USA), respectively at the Hanyang LINC þ Analytical Equipment Center (Hanyang University, Seoul, South Korea). Fatty acids profile of S. obliquus was determined using a previously published method (Lepage and Roy, 1984). Briefly, freezedried algal biomass (10 mg) was dissolved in 2.0 mL of solvent containing chloroform and methanol (2:1, v/v). Additionally, 1.0 mL of chloroform containing an internal standard and transmethylation reagents was added. The samples were incubated at 100  C for 10 min and are separated into two layers through addition of 1.0 mL de-ionized water. The bottom chloroform layer containing fatty acid methyl esters (FAMEs) was sampled after centrifugation for 10 min and was used for determination by gas chromatography with a flame ionization detector and a HP-INNO Wax capillary column (Agilent Technologies, USA). The total carbohydrate content of S. obliquus was assessed using a phenol-sulfuric acid method (Rao and Pattabiraman, 1989). In brief, suspensions (10 mL) containing 10 mg of freeze-dried algal biomass were sonicated for 30 min in a water bath. Sonicated samples (1.0 mL) were mixed with sulfuric acid (96%, 5.0 mL) and phenol (5%, 1.0 mL) in a water bath and allowed to react for 5 min at 90  C. The absorbance of the samples at 490 nm was then measured with a spectrophotometer, and the total carbohydrate concentration was calculated according to a standard curve based on glucose. 2.4. Analysis of antibiotic removal by HPLC The removal of SMZ and SMX by either S. obliquus or abiotic factors (in mixtures without an inoculum of microalgae) were investigated over the course of 12 days of cultivation. Suspensions (2.0 mL) of samples from each flask were sampled at 2, 4, 6, 8, 10, and 12 days after cultivation began, and centrifugated to remove microalgal cells at 15,000 rpm for 10 min. The resulting supernatant was analyzed to determine the residual SMZ and SMX concentrations after filtration with a 0.20 mm membrane filter (Pall Life Sciences, USA) using high-performance liquid chromatography (HPLC) (Alliance 2695 system, Waters, USA) with a C18 column (250  4.6 mm, 5 mm). A 20-mL injection of sample and a flow rate of 1 mL min1 were used. The mobile phase contained acetonitrile and water (30:70 v/v) with 0.1% (v/v) formic acid. The absorbance at 271 nm of SMZ and SMX were monitored during these procedures. The temperature of the column was kept constant at 30  C. The removal kinetics of SMZ and SMX by S. obliquus were analyzed using a first-order reaction equation as follows:

lnCt ¼ kt þ lnC0 where C0 is the SMZ and SMX concentration at time zero, Ct is the SMZ and SMX concentration at time t, k is the removal rate constant (day1), and t is the remediation time in days. 2.5. Statistical analyses All experiments were conducted in triplicate. The Graph Pad Prism version 5.0 software for Windows (USA) was used to analyze the experimental data. One-way analyses of variance (ANOVAs) followed by Tukey-Kramer multiple comparison tests were used to perform statistical analyses on the data. Differences between groups were considered significant when p < 0.05. The half maximal (50%) effective concentration (EC50) of the SMZ and SMX mixture after

553

96 h was determined by performing a linear regression between the log of the concentration of the SMZ and SMX mixture (x) and growth inhibition (y) of the microalga in Graph Pad Prism version 5.0 software, and then using the regression equation to calculate the concentration at which growth was inhibited by 50%. 3. Results and discussion 3.1. Growth rates and surface characteristics of S. obliquus under antibiotic stress The toxicity of SMZ and SMX to S. obliquus was assessed according to the algal growth inhibition guidelines of the OECD (2011). The growth inhibition of S. obliquus (in terms of DCW) by concentrations  0.05 mg L1 of the SMZ and SMX mixture was insignificant over the 12 days of cultivation after the lag phase (first 2 days) (Fig. 1A). An increase in the SMZ and SMX mixture concentration (0.15e0.5 mg L1) significantly (p < 0.05) decreased the growth rate of S. obliquus. The highest growth inhibition of S. obliquus by the SMZ and SMX mixture was observed at day 4, whereas after this S. obliquus started to adapt to the toxicity shock and for the remainder of the 12 days of cultivation showed 9.52, 30.84, 40.01, and 73.44% inhibition compared to the control (without SMZ and SMX) at 0.15, 0.25, 0.35, and 0.5 mg L1 of the SMZ and SMX mixture (w/w 1:1), respectively (Fig. 1B). As shown in Fig. 1C, we also compared the effects of SMZ and SMX in isolation to each other and their mixture and found that S. obliquus was the most sensitive to SMX, followed by the SMZ and SMX mixture (w/w 1:1), and then SMZ (Fig. 1C). These results are consistent with previous studies, which showed that SMZ and SMX are very toxic to microalgae because they can damage cell structures and organelles by disturbing the homeostasis of reactive oxygen species (ROS) control and energy transduction (Eguchi et al., 2004; Isidori et al., 2005; Yang et al., 2008). The dose-response curve of a contaminant is used to predict its 50% effective concentration (EC50). As shown in Table 2, the EC50 of the SMZ and SMX mixture increased with cultivation time, which suggests that S. obliquus adapted to contaminant stress through the biodegradation of contaminants over the course of cultivation. The predicted no-effect concentration was also determined in this study, in which there was no significant effect on S. obliquus when the SMZ and SMX mixture concentration was < 1 mg L1. The toxicity of contaminants has previously been categorized into three different levels by the EU Directive 93/67/EEC (1996), which are: very toxic (EC50 < 1 mg L1), toxic (EC50 ¼ 1e10 mg L1), and harmful (EC50 ¼ 10e100 mg L1). Based on the data in Table 2, we can conclude that the SMZ and SMX mixture is very toxic to aquatic organisms. The FT-IR spectra of S. obliquus cells obtained showed absorption bands over the wavenumber range of 3600e1000 cm1 (Fig. S1). The bands were identified as specific functional groups through referencing biochemical standards and the literature (Table S1). The hydroxyl (O-H) group (with a broad absorption peak) always appears in the high-energy region (3500-3200 cm1) with an expansive peak. Peaks at 3000-2850 cm1 can signal the stretching of the methyl group (C-H) of alkanes, and bends in alkanes result in characteristic bands for -CH3 (at 1450 and 1375 cm1) and -CH2 (1465 cm1). The carbonyl groups (C¼O) of amides form bands at 1680-1630 cm1. The N-H group of primary amines appears in the range from 1640 to 1560 cm1, whereas the band moves to near 1500 cm1 in aromatic secondary amines. The -C-O functional group of alcohol, ethers, esters, carboxylic acids, and anhydrides shows an absorbance band near 1300-1000 cm1. Comparing the microalgal biomass of control samples (without SMZ and SMX) and the treated microalgal biomass, there was no significant change in

554

J.-Q. Xiong et al. / Chemosphere 218 (2019) 551e558 Table 2 Half maximal effective concentration (EC50) of the SMZ and SMX mixture (w/w 1:1) on S. obliquus at different cultivation times. Values were calculated based on linear regression equations of the relationship between the log concentration of contaminants and growth inhibition derived from analyses in Graph Pad Prism version 5.0 software. Time (days)

EC50 (mg L1)

R2

95% confidence interval

PNECa (ng L1)

2 4 6 8 10

0.124 0.151 0.187 0.280 0.691

0.99 0.99 0.99 0.99 0.98

0.094e0.164 0.143e0.160 0.172e0.204 0.212e0.368 0.623e0.766

124 151 187 280 691

a PNEC: predicted no-effect concentration. The PNEC value of the contaminant was the ratio between the EC50 values and an assessment factor of 1000 (Gonz
alezPleiter et al., 2013).

S. obliquus, including total chlorophyll (TChl) and carotenoid (Cx þ c) content, was investigated to assess the effect of these contaminants on the photosynthetic activity of microalgae. We found that the TChl was not influenced by a relatively low concentration of the SMZ and SMX mixture (<0.05 mg L1), but the TChl content of S. obliquus then significantly decreased with increasing contaminant concentration (0.15e0.5 mg L1) (Fig. 2). This observation was consistent with other studies, which showed that the TChl of microalgae exposed to contaminants significantly decreased (Ding et al., 2017; Xiong et al., 2016). Contrary to this considerable decrease in TChl content, the ratio of chlorophyll a (Chl a) to chlorophyll b (Chl b) was significantly increased by higher contaminant concentrations (Fig. 2). It has been reported that Chl a is primarily responsible for photosynthesis, while Chl b can absorb energy and transfer it to Chl a, and thus acts as an accessory photosynthetic pigment. The increased ratio of Chl a to Chl b may indicate an imbalance in the photosynthetic activity of S. obliquus due to SMZ and SMX stress. The carotenoid (Cx þ c) content was significantly increased by concentrations of 0.15e0.35 mg L1 of the SMZ and SMX mixture, which suggested that carotenoids can act as protective agents to scavenge the excessive ROS produced in the chloroplasts under stress (Fig. 2). Carotenoids include carotenes and xanthophylls, which are essential to photosynthesis because of their important roles in light harvesting, free radical scavenging, extra energy dissipation, structural maintenance, and antioxidant pathways (Jahns and Holzwarth, 2012). We also calculated the ratio between carotenoid and total chlorophyll content and found that this ratio significantly increased as the SMZ and SMX mixture concentration increased (Fig. 3). It has previously been concluded

Fig. 1. Effects of the SMZ and SMX mixture (w/w 1:1) on S. obliquus in terms of algal dry cell weight (A) and relative growth inhibition during 12 days of cultivation (B), and comparison of the individual and mixture toxicity of SMZ and/or SMX (C) at 96 h. Error bars represent the standard error of the mean (n ¼ 3). Columns with the symbol ‘*’ indicate significant differences (p < 0.05) between these samples exposed to the contaminants and controls (without the addition of SMZ and SMX).

FT-IR spectra, which indicates that the contaminant does not cause structural changes in this microalga.

3.2. Effects of SMZ and SMX on pigments content The effects of SMZ and SMX mixture on the pigment content of

Fig. 2. Effects of the SMZ and SMX mixture (w/w 1:1) on the total chlorophyll and carotenoid content of S. obliquus after 12 days of cultivation. Error bars represent the standard error of the mean (n ¼ 3). Columns with the symbol ‘*’ indicate significant differences (p < 0.05) between these samples exposed to the contaminants and controls (without the addition of SMZ and SMX). Chl a: chlorophyll a; Chl b: chlorophyll b; Cx þ c: carotenoid xanthophyll plus carotene; TChl: total chlorophyll.

J.-Q. Xiong et al. / Chemosphere 218 (2019) 551e558

555

antioxidant enzymes in Chlorella vulgaris were increased during cypermethrin stress (Gao et al., 2016). More interestingly, the sulfur content of S. obliquus was elevated when the SMZ and SMX mixture concentration increased. Greater sulfur uptake may indicate the potential bioaccumulation of the sulfur contained in SMZ and SMX through different sulfur transporters in cell membranes, including membrane ports, ATP binding proteins, and periplasmic sulfate binding components. Increased sulfate absorption by eukaryotic algae has previously been reported under different environmental conditions (Rüdiger et al., 2008). It is important to note that ROS can react with the thiol groups in proteins and metabolites, causing potential damage, although microalgae can produce redox signal cascades that activate protection reactions and adaptive mechanisms to deal with such effects of environmental stress. Fig. 3. Effects of the SMZ and SMX mixture (w/w 1:1) on the elemental composition of S. obliquus after 12 days of cultivation. Error bars represent the standard error of the mean (n ¼ 3).

that the ratios of Chl a to Chl b, and/or of Cx þ c to TChl, can be used as indicators of the capacity of microalgal cells to protect themselves from the toxicity induced by different environmental stressors (Ding et al., 2017). 3.3. Elemental composition of S. obliquus exposed to antibiotics The elemental (carbon, hydrogen, nitrogen, sulfur) composition of freeze-dried S. obliquus biomass samples were analyzed after 12 days of cultivation with and without exposure to SMZ and SMX (Fig. 3). There were only minor changes in algal carbon, nitrogen, and hydrogen content under the influence of SMZ and SMX. However, the carbon and nitrogen ratio (C/N) significantly increased at low SMZ and SMX concentrations and decreased at high concentrations of SMZ and SMX. Both unicellular algae and benthic plants utilize light energy to fix carbon and absorb other elements such as nitrogen and phosphorus at relatively constant stoichiometric ratios. The C/N ratio can be affected by different environmental stressors, leading to different light absorption and nutrient uptake rates (Baird and Middleton, 2004). Although the difference in nitrogen content of S. obliquus observed due to the contaminants was not significant, it gradually varied from 8.00 to 8.24% with increasing concentrations of the SMZ and SMX mixture. This indicated that the protein content of S. obliquus must have increased, as the total protein content (wt. %) equals 4.92e6.25 times the total nitrogen content (wt. %) (Lourenco et al., 2002). The increased protein content of S. obliquus may indicate that the synthesis of defensive proteins, such as stress responsive proteins occurred under conditions of environmental stress. Gao et al. (2016) reported that the activities of chaperone proteins and

3.4. Changes of fatty acids composition and total carbohydrate content Fatty acids (including FAMEs) are the products of the conserved acetyl CoA pathway and have been suggested as stress responsive biomarkers in microalgae (Guschina and Harwood, 2006). The FAMEs we identified in S. obliquus included myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1n9c), linolelaidic acid (C18:2n6t), g-Linolenic acid (C18:3n6), and arachidic acid (C20:0), as presented in Table 3. The percent compositions of the saturated (C14:0, C16:0, C18:0 and C20:0), monounsaturated (C16:1 and C18:1), and polyunsaturated (C18:2 and C18:3) fatty acids in S. obliquus ranged from 48.8 to 42.0%, 11.5e17.9%, and 39.8e40.1%, respectively, with increasing concentrations of SMZ and SMX (0e0.5 mg L1) (Table 3). The changes in the total amount of saturated, mono-unsaturated, and poly-unsaturated FAMEs are shown in Fig. 4A. These results indicated that the percentage of fatty acids made up of saturated FAMEs decreased from 48 to 42% with increasing SMZ and SMX mixture concentrations, while that made up by unsaturated FAMEs conversely increased from 50 to 58%. The amount and composition of FAMEs has been shown to be influenced by many different factors, such as trace metals, nitrogen, phosphorous, temperature, pH, and light (Guschina and Harwood, 2006). Similarly decreased saturated and increased unsaturated compositions of FAMEs were previously reported in the microalgae Chlamydomonas mexicana and Chlorella vulgaris when exposed to bisphenol A, as well as in a cyanobacterium, Microcystis aeruginosa, when it was treated with the allelochemical eathyl-2-methyl acetoacetate (Ji et al., 2014; Li et al., 2007). Unsaturated fatty acids play essential roles in the maintenance of cell membrane fluidity. Cell membrane fluidity is important in the interactions between membrane proteins and small membrane-bound molecules, particularly in mitochondria. It

Table 3 Effects of different concentrations of the SMZ and SMX mixture on the fatty acid methyl ester (FAME) composition of S. obliquus after 12 days of cultivation. Type of FAME

SMZ and SMX mixture concentration (mg L1) Control

0.05

0.15

0.25

0.35

0.5

0 16.67* 5.15* 16.64* 11.05 32.07 8.00 10.42*

0 16.45* 3.87* 17.84* 11.79* 32.00 7.82 10.21*

0 16.63* 5.52* 16.46* 11.65** 32.40 7.77 9.57

0 16.66* 5.78* 16.10* 12.16 31.69 8.37 9.24

% of each FAME out of the total FAMEs Myristic acid methyl ester (C14:0) Palmitic acid methyl ester (C16:0) Palmitoleic acid methyl ester (C16:1) Stearic acid methyl ester (C18:0) Elaisic acid methyl ester (C18:1n9t) Linolelaidic acid methyl ester (C18:2n6t) g-Linolenic acid methyl ester (C18:3n6) Arachidic acid methyl ester (C20:0)

3.6 13.76 2.13 21.90 9.36 31.21 8.54 9.55

0 13.73 4.90* 19.85 10.14 33.47* 8.56 9.35

Note: each data point represents the mean value of duplicate. * Represents statistical significance of the difference between treatments and control.

556

J.-Q. Xiong et al. / Chemosphere 218 (2019) 551e558

2014). 3.5. Removal of SMZ and SMX by S. obliquus

Fig. 4. Effects of the SMZ and SMX mixture (w/w 1:1) on the fatty acid methyl ester (FAME) composition (A) and carbohydrate content (B) of S. obliquus after 12 days of cultivation. Error bars represent the standard error of the mean (n ¼ 3).

is also widely accepted that the activity of integral membrane proteins (e.g., proliferative signal proteins) is closely related to the lipids of the membrane bilayer (Burdon, 1994). The observed increase in unsaturated fatty acid content, indicating a change in membrane fluidity, may also suggest a protective and/or stress resistance mechanism of S. obliquus toward SMZ and SMX. Carbohydrates are one of the three main components of microalgal biomass (the others are proteins and lipids), and mainly comprise the cellulose and starch present in the cell wall and the plastids. In our study, the carbohydrate content of S. obliquus decreased gradually from 25.04% to 20.08% as the SMZ and SMX mixture concentration increased from 0 to 0.5 mg L1 (Fig. 4B). The decreased carbohydrate content of S. obliquus observed was consistent with the decreased total chlorophyll content, as carbon fixation by photosynthesis is the main source of carbohydrates in microalgal cells. The decreased carbohydrate content also indicates that SMZ and SMX can disrupt the metabolic activities of S. obliquus that store energy (e.g., as starch) or synthesize the main components of cell walls (e.g., cellulose). Numerous studies have investigated the effects of irradiance, nitrogen depletion, temperature, carbon sources, and phosphorus on the accumulation of carbohydrate in microalgal biomass (Abreu et al., 2012; Aikawa et al., 2012). However, few studies have been done on the effects of PCs such as SMZ and SMX on the synthesis of FAMEs and carbohydrates in microalgae. Our study demonstrated that SMZ and SMX have adverse effects on microalgae that include decreased photosynthetic activity and resultantly lower carbohydrate production, which is consistent with previous report on the effect of organic pollutants on microalgae (Guschina and Harwood, 2006; Ji et al.,

The removal of SMZ and SMX from mixtures of these PCs at concentrations of 0e0.5 mg L1 (w/w 1:1) by S. obliquus was investigated in this study (Fig. 5). The microalga S. obliquus was able to remove 31, 35, 49, 55, and 62% of SMZ (0.025, 0.075, 0.125, 0.175, and 0.25 mg L1), and 28, 29, 35, 41, and 47% of SMX (0.025, 0.075, 0.125, 0.175, and 0.25 mg L1) from the SMZ and SMX mixture after 12 days of cultivation (Fig. 5A and B). The removal of individual contaminants by S. obliquus ranged from 15 to 17% (SMZ) and 16e29% (SMX) (data not shown). Abiotic removal of 0.1e1 mg L1 SMZ was observed to be around 10e13%, whereas only 1e2% abiotic removal was achieved of 0.05e0.2 mg L1 SMX. The abiotic removal of SMZ and SMX was around 10% and 3%, respectively, which agrees with a previous study (Reis et al., 2014). Negligible removal induced by the abiotic factors indicated that the removal of SMZ and SMX should be accomplished by biosorption, bioaccumulation, and biodegradation by S. obliquus (Ding et al., 2017; Peng et al., 2014; Xiong et al., 2018). Removal of the SMZ and SMX mixture by S. obliquus can be explained by an apparent first order kinetic reaction (Fig. 5C and D). The k and T1/2 of SMZ (0.025e0.25 mg L1) were 0.037e0.093 day1 and 18.73e7.45 day, respectively, while those of SMX (0.025e0.25 mg L1) were from 0.035 to 0.061 day1 and 19.80e11.42 day. The R2 values for regression equations fit to the data for SMZ and SMX removal were 0.92e0.97 and 0.86e0.96, respectively. Although the removal (30%) of the single contaminant SMX by a microalga, Nannochloris sp., was previously investigated (Bai and Acharya, 2016), our study represents the first report of the removal of a SMZ and SMX mixture by a microalga, as well as the first comprehensive assessment of these removal kinetics. More interestingly, we observed relatively greater removal of SMZ and SMX in higher concentration mixtures than in those of lower concentrations. A similar observation was previously reported for bacterial degradation of SMZ and SMX (Vila-Costa et al., 2017). The authors of that study stated that the increased degradation of SMZ and SMX at relative higher concentrations occurred because degradation of antibiotics is an efficient resistance mechanism in bacteria. Enzymatic transformation of antibiotics, development of antibiotic resistance genes, and physical removal of intracellular antibiotics by activating membrane efflux pumps are the three main antibiotic resistance pathways in natural microbial communities (Vila-Costa et al., 2017). Although the occurrence and function of these mechanisms are widely accepted, the specific role of each mechanism and the factors affecting their occurrence in natural environments is not well understood, especially in microalgae. In our study, the greater biodegradation of the SMZ and SMX mixture observed at higher concentrations may indicate the ability of microalgal enzymatic degradation of SMZ and SMX to potentially act as an efficient antibiotic resistance mechanism. We observed that the biodegradation of SMZ and SMX was performed by different enzymatic reactions, including hydroxylation, methylation, nitrosation, and deamination, based on the identification of metabolites of SMZ and SMX produced after microalgal degradation (Xiong et al., 2018b). Although we did observe direct evidence of the activities of the potential degradation enzymes used by the microalgae in our study, it is generally accepted that microalgae have a complex enzyme system that consists of phase I and phase II enzyme families. The initial degradation begins with the phase I enzyme, usually of a cytochrome 450 type such as aminopyrine Ndemethylase or aniline hydroxylase, which makes the target compound more hydrophilic by adding or unmasking a hydroxyl group, and usually is involved in performing hydrolysis, oxidation, or reduction reactions (Foflonker et al., 2016; Khona et al., 2017). The

J.-Q. Xiong et al. / Chemosphere 218 (2019) 551e558

557

Fig. 5. Removal, and kinetic analyses thereof, of SMZ (A, C) and SMX (B, D) by S. obliquus in mixture (w/w 1:1) over 12 days of cultivation. Error bars represent the standard error of the mean (n ¼ 3).

presence of genes coding for different known catalytic enzymes, such as mono(di)oxygenase, soluble inorganic pyrophosphatase, glutamyl-tRNA reductase, malate/pyruvate dehydrogenase, uroporphyrinogen III carboxylase/decarboxylase, dehydratase, alkaline and acid phosphatase, transferase, catalase, laccases, violaxanthin de-epoxidase, and hydrolases, have also been previously reported in microalgae (Foflonker et al., 2016; Khona et al., 2017). Moreover, all microorganisms have large numbers of multidrug-resistant efflux pumps (Martínez, 2008). Therefore, it can be concluded that enzymatic transformation of antibiotics and physical removal of intracellular antibiotics by activating membrane efflux pumps are two likely resistance mechanisms used by the microalga S. obliquus to resist SMZ and SMX antibiotics. However, more indepth studies using metatranscriptomics, metaproteomics, and metabolomics to reveal the specific resistance mechanism of this and other microalgae should be conducted to achieve a more comprehensive understanding of microalgal resistance mechanism to antibiotics. 4. Conclusion The effects of a SMZ and SMX mixture on a microalga, S. obliquus, were investigated in this study, and the results showed that S. obliquus can withstand high concentrations of SMZ and SMX. The biochemical characteristics (total chlorophyll, carotenoid, carbohydrate, and FAMEs) of S. obliquus were significantly influenced by SMZ and SMX. Elemental analyses showed a significant increase in the sulfur content of the biomass of microalgae exposed to SMZ and SMX compared to controls. The degradation of SMZ and SMX was relatively greater at higher concentrations of SMZ and SMX, indicating that biodegradation may be an efficient mechanism of microalgal adaptation to antibiotics. Further molecular approaches are needed to more clearly show the specific steps and components

involved in the defense, degradation, and adaptation mechanism of microalgae toward various antibiotics. Acknowledgement This study was supported by the Korea Environmental Industry and Technology Institute (KEITE) grants funded by the Ministry of Environment (ME) of the South Korean government (No. RE201805203). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2018.11.146. References Abreu, A.P., Fernandes, B., Vicente, A.A., Teixeira, J., Dragone, G., 2012. Mixotrophic cultivation of Chlorella vulgaris using industrial dairy waste as organic carbon source. Bioresour. Technol. 118, 61e66. Abou-Shanab, R.A.I., Hwang, J.H., Cho, Y.C., Min, B., Jeon, B.H., 2011. Characterization of microalgal species isolated from fresh water bodies as a potential source for biodiesel production. Appl. Energy 88, 3300e3306. Aikawa, S., Izumi, Y., Matsuda, F., Hasunuma, T., Chang, J.S., Kondo, A., 2012. Synergistic enhancement of glycogen production in Arthrospira platensisby optimization of light intensity and nitrate supply. Bioresour. Technol. 108, 211e215. Bai, X., Acharya, K., 2016. Removal of trimethoprim, sulfamethoxazole, and triclosan by the green alga Nannochloris sp. J. Hazard. Mater. 315, 70e75. Baird, M.E., Middleton, J.H., 2004. On relating physical limits to the carbon: nitrogen ratio of unicellular algae and benthic plants. J. Mar. Syst. 49, 169e175. 
Bielen, A., Simatovi c, A., Kosi
c-Vuksi
c, J., Senta, I., Ahel, M., Babi
c, S., Jurina, T., Plaza, J.J., Milakovi
c, M., Udikovi
c-Koli
c, N., 2017. Negative environmental impacts of antibiotic-contaminated effluents from pharmaceutical industries. Water Res. 126, 79e87. Burdon, R.H., 1994. Chapter 6 - free radicals and cell proliferation. N. Compr. Biochem. 28, 155e185. Ding, T., Yang, M., Zhang, J., Yang, B., Lin, K., Li, J., Gan, J., 2017. Toxicity, degradation and metabolic fate of ibuprofen on freshwater diatom Navicula sp. J. Hazard.

558

J.-Q. Xiong et al. / Chemosphere 218 (2019) 551e558

Mater. 330, 127e134. EU Directive 93/67/EEC, 1996. Commission of the European Communities. Technical Guidance Document in Support of Commission Directive 93/67/EEC on Risk Assessment for New Notified Substances and Commission Regulation (EC) No 1488/94 on Risk Assessment for Existing Substances. Part II; Environmental Risk Assessment. Office for Official Publications of the European Communities, Luxembourg. Eguchi, K., Nagase, H., Ozawa, M., Endoh, Y.S., Goto, K., Hirata, K., Miyamoto, K., Yoshimura, H., 2004. Evaluation of antimicrobial agents for veterinary use in the ecotoxicity test using microalgae. Chemosphere 57, 1733e1738. Foflonker, F., Ananyev, G., Qiu, H., Morrison, A., Palenik, B., Dismukes, G.C., Bhattacharya, D., 2016. The unexpected extremophile: tolerance to fluctuating salinity in the green alga Picochlorum. Algal Res 16, 465e472. Guschina, I.A., Harwood, J.L., 2006. Lipids and lipid metabolism in eukaryotic algae. Prog. Lipid Res. 45, 160e186.
lez-Pleiter, M., Gonzalo, S., Rodea-Palomares, I., Legane
s, F., Rosal, R., Gonza
ndez-Pin ~ as, F., 2013. Toxicity of five antibiotics and Boltes, K., Marco, E., Ferna their mixtures towards photosynthetic aquatic organisms: implications for environmental risk assessment. Water Res. 47, 2050e2064. Gao, Y., Lim, T.K., Lin, Q., Li, S.F.Y., 2016. Identification of cypermethrin induced protein changes in green algae by iTRAQ quantitative proteomics. J. Proteom. 139, 67e76.
n-Gil, A., Bello-Laserna, I., Rodríguez-Mozaz, S., Vicent, T., Hom-Diaz, A., Jae
, D., Bl
Barcelo anquez, P., 2017. Performance of a microalgal photobioreactor treating toilet wastewater: pharmaceutically active compound removal and biomass harvesting. Sci. Total Environ. 592, 1e11. Hughes, S.R., Kay, P., Brown, L.E., 2012. Global synthesis and critical evaluation of pharmaceutical data sets collected from river systems. Environ. Sci. Technol. 47, 661e677. Isidori, M., Lavorgna, M., Nardelli, A., Pascarella, L., Parrella, A., 2005. Toxic and genotoxic evaluation of six antibiotics on non-target organisms. Sci. Total Environ. 346, 87e98. Ji, M.K., Kabra, A.N., Choi, J., Hwang, J.H., Kim, J.R., Abou-Shanab, R.A.I., Oh, Y.K., Jeon, B.H., 2014. Biodegradation of bisphenol A by the freshwater microalgae Chlamydomonas mexicana and Chlorella vulgaris. Ecol. Eng. 73, 260e269. Jahns, P., Holzwarth, A.R., 2012. The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II. Biochim. Biophys. Acta 1817, 182e193. Khona, D.K., Shirolikar, S.M., Gawde, K.K., Hom, E., Deodhar, M.A., D'Souza, J.S., 2017. Characterization of salt stress-induced palmelloids in the green alga, Chlamydomonas reinhardtii. Algal Res 6, 434e448. Khetan, S.K., Collins, T.J., 2007. Human pharmaceuticals in the aquatic environment: a challenge to green chemistry. Chem. Rev. 107, 2319e2364. Lange, A., Paull, G.C., Coe, T.S., Katsu, Y., Urushitani, H., Iguchi, T., Tyler, C.R., 2009. Sexual reprogramming and estrogenic sensitization in wild fish exposed to ethinylestradiol. Environ. Sci. Technol. 43, 1219e1225. Li, F.M., Hu, H.Y., Chong, Y.X., Men, Y.J., Guo, M.T., 2007. Effects of allelochemical EMA isolated from Phragmites communis on algal cell membrane lipid and ultrastructure. Environ. Sci. 28, 1534e1538. Lepage, G., Roy, C.C., 1984. Improved recovery of fatty acid through direct transesterification without prior extraction or purification. J. Lipid Res. 25, 1391e1396. Loos, R., Gawlik, B.M., Locoro, G., Rimaviciute, E., Contini, S., Bidoglio, G., 2009. EUwide survey of polar organic persistent pollutants in European river waters. Environ. Pollut. 157, 561e568. Lourenço, S.O., Barbarino, E., De-Paula, J.C., Pereira, L.O., Marquez, U.M.L., 2002.

Amino acid composition, protein content and calculation of nitrogen-to-protein conversion factors for 19 tropical seaweeds. Phycol. Res. 50, 233e241. Martínez, J.L., 2008. Antibiotics and antibiotic resistance genes in natural environments. Science 321, 365e367. Matamoros, V., Uggetti, E., García, J., Bayona, J.M., 2016. Assessment of the mechanisms involved in the removal of emerging contaminants by microalgae from wastewater: a laboratory scale study. J. Hazard. Mater. 301, 197e205. Organization for Economic Co-operation and Development (OECD), 2011. Organization for Economic Co-operation and Development (OECD) Guidelines for the Testing of Chemicals, Section 2: Effects on Biotic Systems Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test OECD. OECD ILibrary. https://www.oecd-ilibrary.org/environment/test-no-201-alga-growth-inhibition-test_9789264069923-en. Peng, F.Q., Ying, G.G., Yang, B., Liu, S., Lai, H.J., Liu, Y.S., Chen, Z.F., Zhou, G.J., 2014. Biotransformation of progesterone and norgestrel by two freshwater microalgae (Scenedesmus obliquus and Chlorella pyrenoidosa) transformation kinetics and products identification. Chemosphere 95, 581e588. Rüdiger, H., Dahl, C., Knaff, D., Leustek, T., 2008. Sulfur metabolism in phototrophic organisms. Adv. Photosynth. Respir. 27, 15e26. Rao, P., Pattabiraman, T.N., 1989. Reevaluation of the phenol-sulfuric acid reaction for the estimation of hexoses and pentoses. Anal. Biochem. 181, 18e22. Reis, P.J.M., Reis, A.C., Ricken, B., Kolvenbach, B.A., Manaia, C.M., Corvini, P.F.X., Nunes, O.C., 2014. Biodegradation of sulfamethoxazole and other sulfonamides by Achromobacter denitrificans PR1. J. Hazard. Mater. 280, 741e749. Salama, E.S., Kurade, M.B., Abou-Shanab, R.A.I., El-Dalatony, M.M., Yang, I.S., Min, B., Jeon, B.H., 2017. Recent progress in microalgal biomass production coupled with wastewater treatment for biofuel generation. Renew. Sustain. Energy Rev. 79, 1189e1211. Schwarzenbach, R.P., Escher, B.I., Fenner, K., Hofstetter, T.B., Johnson, C.A., Gunten, U., Wehrli, B., 2006. The challenge of micropollutants in aquatic systems. Science 313, 1072e1077. ~ a, J., Pe
rez, S., Casamayor, E.O., Dachs, J., 2017. Vila-Costa, M., Gioia, R., Acen Degradation of sulfonamides as a microbial resistance mechanism. Water Res. 115, 309e317. Xiong, J.Q., Kurade, M.B., Abou-Shanab, R.A.I., Ji, M.K., Choi, J., Kim, J.O., Jeon, B.H., 2016. Biodegradation of carbamazepine using freshwater microalgae Chlamydomonas mexicana and Scenedesmus obliquus and the determination of its metabolic fate. Bioresour. Technol. 205, 183e190. Xiong, J.Q., Kurade, M.B., Jeon, B.H., 2017a. Ecotoxicological effects of enrofloxacin and its removal by monoculture of microalgal species and their consortium. Environ. Pollut. 226, 486e493. Xiong, J.Q., Kurade, M.B., Kim, J.R., Roh, H.S., Jeon, B.H., 2017b. Ciprofloxacin toxicity and its co-metabolic removal by a freshwater microalga Chlamydomonas mexicana. J. Hazard. Mater. 323, 212e219. Xiong, J.Q., Kurade, M.B., Jeon, B.H., 2018a. Can microalgae remove pharmaceutical contaminants from water? Trends Biotechnol. 36, 30e45. Xiong, J.Q., Kim, S.J., Kurade, M.B., Govindwar, S., Abou-Shanab, R.A.I., Kim, J.R., Roh, H.S., Khan, M.A., Jeon, B.H., 2018b. Combined effects of sulfamethazine and sulfamethoxazole on a freshwater microalga, Scenedesmus obliquus: toxicity, biodegradation, and metabolic fate. J. Hazard. Mater. in press. https://doi.org/10. 1016/j.jhazmat.2018.07.049. Yang, L.H., Ying, G.G., Su, H.C., Stauber, J.L., Adams, M.S., Binet, M.T., 2008. Growthinhibiting effects of 12 antibacterial agents and their mixtures on the freshwater microalga Pseudokirchneriella subcapitata. Environ. Toxicol. Chem. 27, 1201e1208.