Interactive effects of roxithromycin and freshwater microalgae, Chlorella pyrenoidosa: Toxicity and removal mechanism

Ecotoxicology and Environmental Safety 191 (2020) 110156

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Interactive effects of roxithromycin and freshwater microalgae, Chlorella pyrenoidosa: Toxicity and removal mechanism

T

Jiping Lia,b, Zhongfang Mina, Wei Lia,b,∗, Lijie Xua, Jiangang Hana,b, Pingping Lia,∗∗ a

Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Longpan Road 159, Nanjing, 210037, Jiangsu, China b State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Science, Nanjing, 210008, Jiangsu, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Antibiotics Roxithromycin Ecotoxicity Algae Removal mechanism

Roxithromycin (ROX) has received increasing concern due to its large usage, ubiquitous detection in environment and high ecotoxicology risk. This study investigated the acute and chronic effects of ROX on the growth, chlorophyll, antioxidant enzymes, and malonaldehyde (MDA) content of Chlorella pyrenoidosa, as well as the removal mechanism of ROX during microalgae cultivation. The calculated 96 h median effective concentration of ROX on yield (EyC50) and specific growth rate (ErC50) of C. pyrenoidosa was 0.81 and 2.87 mg/L, respectively. After 96 h exposure, 1.0 ~ 2.0 mg/L of ROX significantly inhibited the synthesis of chlorophyll and promoted the activities of SOD and CAT (p < 0.05). The MDA content increased with the ROX concentration increasing from 0.5 ~ 1.0 mg/L, and then decreased to 105.76% of the control exposure to 2.0 mg/L ROX, demonstrating the oxidative damage could be moderated by the upregulation of SOD and CAT activities. During the 21 d chronic exposure, low concentration of ROX (0.1 and 0.25 mg/L) showed no significant effect on the growth and chlorophyll content of algae during the first 14 d, but significantly inhibited the growth of algae and the synthesis of chlorophyll at 21 d (p < 0.05 or p < 0.01). 1.0 mg/L ROX significantly inhibited the growth of microalgae during 3 ~ 21 d and the synthesis of chlorophyll at 7 ~ 21 d. High concentration and long-term exposure of low concentration of ROX caused the SOD and CAT activities and MDA content to increase, demonstrating a higher level of oxidative damage of microalgae. During the first 14 d, abiotic removal of ROX played a more important role, contributing about 12.21% ~ 21.37% of ROX removal. After 14 d, the biodegradation of ROX by C. pyrenoidosa gradually became a more important removal mechanism, contributing about 45.99% ~ 53.30% of ROX removal at 21 d. Bio-adsorption and bioaccumulation both played minor roles in the removal of ROX during algae cultivation.

1. Introduction

2015). Roxithromycin (ROX) is semi-synthetic MCLs widely used to treat respiratory tract, urinary and soft tissue infections (Ding et al., 2015; Zhang et al., 2019b). Due to the widespread application and in-effective removal of wastewater treatment plant, ROX was frequently detected in the influent and effluent of wastewater treatment plant (Lin et al., 2016), surface water (Yan et al., 2013), and even drinking source water (Sun et al., 2015). Although the concentration level was usually low at ng/L to μg/L in aquatic environment, ROX still poses adverse effects on target and non-target organisms, and more worrying, harms human health by the bio-magnification through food chains (Mo et al., 2017). Considerable efforts have been made on the ecotoxicity of ROX. ROX has been observed to affect the growth and biochemical

During the past three decades, the occurrence, migration, transformation and ecotoxicology of antibiotics have been the research hotspots in the field of environment. Antibiotics are widely used in human and veterinary medicine to prevent or treat infectious diseases, as well as in aquaculture operations (Du et al., 2018). Among various antibiotics, macrolides (MCLs) are the second most used antibiotics after the βlactam family. In 2013, the usage of MCLs in China was 42,200 tons and accounted for 26% of the total antibiotics consumption (Zhang et al., 2015). Generally, MCLs and other antibiotics used by human and livestock can't be fully absorbed by the body, most of them will be excreted and eventually enter into the environment (Mandaric et al.,

∗

Corresponding author. Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Longpan Road 159, Nanjing, 210037 Jiangsu, China. ∗∗ Corresponding author. E-mail addresses: [email protected] (W. Li), [email protected] (P. Li). https://doi.org/10.1016/j.ecoenv.2019.110156 Received 13 August 2019; Received in revised form 30 December 2019; Accepted 31 December 2019 0147-6513/ © 2020 Elsevier Inc. All rights reserved.

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conical flasks containing 100 mL sterile BG11 culture medium at 10% concentration (Vinoculum/Vmedia) in an illumination incubator (MLR352H-PC, Panasonic, Japan) under the following conditions: 3000 lx; 25 ± 1 °C; alternate light/dark periods of 12 h/12h and humidity 60%. The flasks were shaken twice a day to avoid precipitation of biomass. The microalgae suspension was cultured to reach the algae cell density of 107 cells/mL (measured by microscopy) for further experiments.

characteristics of aquatic organisms such as algae, daphnia and fish (Xiong et al., 2019b; Yang et al., 2008; Zhang et al., 2019a, 2019b), and it was found that algae is more sensitive than daphnia and fish to ROX. For instance, Choi et al. (2008) reported that the 48 h and 96 h median effective concentration (EC50) of ROX on the mobilization of Daphnia magna were 74.3 and 7.1 mg/L, respectively. Zhang et al. (2019b) found the 48 h EC50 and the median lethal concentration (LC50) values of ROX to Daphnia magna were 20.28 and 22.03 mg/L, respectively. Yang et al. (2008) reported the 72 h EC50 of ROX to Pseudokirchneriella subcapitata was 0.047 mg/L. Overall, previous researches mainly focused on the acute toxicity of ROX. The chronic effects of low concentration of ROX to the aquatic organism are still not clear. Recent researches demonstrated that green algae had the ability to remove antibiotics and other emerging contaminants. Xiong et al. (2017b) found Chlamydomonas mexicana was able to remove 13% of 2 mg/L ciprofloxacin after 11 d of cultivation by biodegradation, bioaccumulation and/or bio-adsorption. Xiong et al. (2019a) reported that Scenedesmus obliquus exhibited 17.3% removal of 0.1 mg/L sulfamethazine and 29.3% removal of 0.2 mg/L sulfamethoxazole after 11 d of cultivation. Yu et al. (2017) found green algae C. pyrenoidosa removed 92.70% of ceftazidime and 96.07% of 7-aminocephalosporanic acid by adsorption, cell wall-transmission and biodegradation after a certain treatment time. Therefore, we have a hypothesis that ROX exposure may affect the growth and biochemical characteristics of green algae, but meanwhile, green algae may cause the adsorption or degradation of ROX. However, to the best of our knowledge, the removal mechanism of ROX by the green algae is still unknown. To test the hypothesis, green alga C. pyrenoidosa was employed as test organism because of its high sensitivity and the characteristic of easy cultivation. The objectives of this present paper were: (1) to examine the acute toxicity effect of ROX on the growth, chlorophyll content, antioxidant enzyme activities and malondialdehyde (MDA) content of C. pyrenoidosa; (2) to study the chronic effects of low concentration of ROX on the growth and biochemical characteristics of C. pyrenoidosa; (3) to explore the removal mechanism of ROX during the green algae cultivation.

2.3. Acute and chronic toxicity experiment The acute toxic effects of ROX on green algae were conducted according to the guideline 201 of Organization for Economic Co-operation and Development (OECD, 2011) with a slightly change on the exposure time. The preliminary test was first performed to determine the ROX concentration causing about 75% inhibition of algal growth rate, which is 2.0 mg/L in this study. Accordingly, in the final definitive acute experiment, the initial exposure concentrations of ROX were set at 0, 0.0625, 0.125, 0.25, 0.5, 1.0, 2.0 mg/L, arranged in a geometric series with a factor of 2. In the chronic experiment, 1.0 mg/L (close to the 96 h EyC50), 0.25 mg/L (close to 96 h EyC10) based on the acute experiment and 0.1 mg/L (higher than but close to environmental concentration) based on the environmental concentration were chosen to study the chronic effects of ROX on the green algae. The ROX exposure solution was prepared by mixing certain amount of ROX stock solution (10 mg/L, stirred in sterile ultrapure water for 12 h to dissolve), 50 mL BG11 at twice concentration (2 × BG11), sterile ultrapure water, and 10 mL microalgae suspension. The total volume of the medium was 100 mL and the initial cell density of C. pyrenoidosa was about 106 cells/mL. All experiments were conducted in triplicate. 2.4. Measurement of microalgae growth The dry cell weight was measured according to a previously established method (Gao et al., 2011; Huo et al., 2015). In brief, an algae suspension of 30 mL was filtered through a pre-dried and pre-weighed Whatman filter paper (GF-52) and then dried at 105 °C for 24 h. After cooling to room temperature, the filter papers with algae cells were weighed again and the dry weight was calculated and expressed as g/L. The optical density at 680 nm (OD680) of C. pyrenoidosa was measured using a spectrophotometer (Lambda 365, PerkinElmer, USA). The relationship between OD680 and the dry cell weight (g/L) was established and shown as the following equation: Dry cell weight of C. pyrenoidosa (g/L) = 0.1585 × OD680 – 0.0016 (R2 = 0.9943) The specific growth rate (μ) was obtained by fitting the dry cell weight to a function by using the following equation (Converti et al., 2009).

2. Materials and methods 2.1. Chemical reagents ROX (80,214-83-1) with the purity of > 98% was purchased from J &K Chemical Ltd. (China). The 95% ethanol (64-17-5), dichloromethane (75-9-2), tertiary butyl alcohol (75-65-0), KH2PO4 (7778-77-0), K2HPO4·3H2O (16,788-57-1), NaNO3 (7631-99-4), MgSO4·7H2O (10,034-99-8), CaCl2·2H2O (10,035-04-8), citric acid (5949-29-1), ferric ammonium citrate (1185-57-5), EDTANa2 (139-333), Na2CO3 (497-19-8), H3BO3 (10,043-35-3), MnCl2·4H2O (13,446-349), ZnSO4·7H2O (7446-20-0), Na2MoO4·2H2O (10,102-40-6), CuSO4·5H2O (7758-99-8), Co(NO3)2·6H2O (10,026-22-9) were purchased from Sinopharm Chemical Reagent Co. Ltd. (China). Glutaraldehyde (111-30-8, electron microscope reagent) was purchased from Shanghai Macklin Biochemical Co. Ltd. (China). HPLC grade acetonitrile (75-05-8) and methanol (67-56-1) were purchased from Fisher Scientific (USA). HPLC grade ammonium acetate (631-61-8) and acetic acid (64-19-7) were purchased from Aladdin (China). All chemical reagents used in this study are at least analytical grade and used as received. All solutions were prepared using ultrapure water (18.2 MΩ) produced by a Milli-Q Advantage A10 system (Millipore, USA).

μ= 

lnN2 − lnN0 t2 − t0

where, N2 is the cell density at time t2 and N0 is the cell density at time t0 (hour or day 0). 2.5. Measurement of physiological biochemical indexes 2.5.1. Chlorophyll content The chlorophyll content was measured based on Wintermans and De Mots (1965) with some improvements. Briefly, a 20 mL microalgae suspension was harvested by centrifugation at 10,000 rpm for 10 min. The pellet was re-suspended in 20 mL of 95% ethanol after discarding the supernatant, incubated at 4 °C for 24 h in dark and centrifuged again for 10min. The absorbance of the supernatant at 665 nm and 649 nm was measured with a spectrophotometer. The concentrations of chlorophyll were calculated using the following equations:

2.2. Cultivation of green algae The green algae C. pyrenoidosa (FACHB-11) was purchased from the Institute of Hydrobiology at the Chinese Academy of Sciences (Hubei, China). The cultivation of C. pyrenoidosa was performed in 250 mL 2

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Fig. 1. Effects of ROX on the growth of C. pyrenoidosa in terms of dry cell weight (a) and specific growth rate (b) during 96 h of cultivation. Error bars represent standard deviation (n = 3). * and ** indicate significant differences (p < 0.05 and p < 0.01, respectively) compared to the control.

Chlorophyll-a (mg/L) = 13.7 × OD665 – 5.76 × OD649 Chlorophyll-b (mg/L) = 25.80 × OD649 – 7.60 × OD665 Total chlorophyll (mg/L) = 6.10 × OD665 + 20.04 × OD649

adsorbed onto the microalgae cell surface, Ac is the quantity of ROX accumulated into the microalgae cells. 2.7. Analysis of ROX

2.5.2. Antioxidant indicators After certain exposure time, 30 mL of the algae suspension were collected and centrifuged at 8000 rpm for 10 min at 4 °C and the supernatant was discarded. Then, the microalgae pellet was re-suspended in 20 mL of 0.01 M phosphate buffer (pH 7.4, precooled at 4 °C) and sonicated for 15 min (900 W, ultrasonic time 2s, rest time 2s) in ice bath. The mixture was centrifuged at 10,000 rpm for 10 min at 4 °C, and the algae cell lysate supernatant was used to determine the superoxide dismutase (SOD) and catalase (CAT) activities and MDA content using the commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The measurements were conducted according to the operation instructions of the assay kits.

ROX concentration was determined using HPLC/MS system that included an HPLC (Ultimate 3000, Dionex, USA) and a LTQ-orbitrap XL mass spectrometer (Thermo Scientific, Germany) equipped with an Agilent Poroshell 120, EC-C18 column (100 mm × 2.1 mm, 2.7 μm). The mobile phase consisted of 1:1 methanol-acetonitrile and 10 mM ammonium acetate added with 0.05% acetate (60:40, v/v). The flow rate was 0.2 mL/min, and the sample injection volume was 5 μL. The mass spectrometer was operated in the positive-ion mode using a heated electrospray ionization (HESI). The HESI conditions were as follows: sheath gas flow rate 40 arbitrary; auxiliary gas flow rate 10 arbitrary; heater temperature 350 °C; capillary temperature of 325 °C; spray voltage of 3.5 kV; capillary voltage of 9 V, tube lens voltage of 100 V. The scan event cycle used a full scan mass spectrum at a resolution of 30,000 and a corresponding data-dependent MS/MS event also at a resolution of 30,000 operated in collision induced dissociation (CID) mode. MS/MS activation parameters used an isolation width of 1.0 Da, normalized collision energy of 35% and an activation time of 30 ms.

2.6. Metabolic fate of ROX The abiotic control containing the same concentration of ROX in BG11 medium without microalgae was incubated in the same conditions as chronic exposure experiment. 2 mL sample was collected from the abiotic control at certain time intervals (0, 1, 3, 7, 14, and 21 d), filtered with a 0.2 μm membrane filter and used to determine the abiotic removal of ROX. The bio-adsorption, bioaccumulation and biodegradation of ROX by C. pyrenoidosa were studied during the chronic exposure (section 2.3) according to previous publication (Xiong et al., 2017a). Aliquots (30 mL) of the microalgae suspension were withdrawn and centrifuged at 8000 rpm for 10 min. The supernatant filtered with a 0.2 μm membrane filter was used to analyze the residual concentration of ROX in the culture medium. The cell pellets on the wall of centrifuge tube were washed and suspended in ultrapure water (10 mL) and then centrifuged again, and the supernatant was recovered for analyzing of the concentration of ROX adsorbed onto the cell surface. The cell pellets were suspended with 10 mL of dichloromethane: methanol (1:2 v/v) and sonicated for 30 min (80 kHz, 0.58 kW). The supernatant recovered after centrifugation at 10,000 rpm for 10 min was used to determine the concentration of ROX that was accumulated into the microalgae cells. The biodegradation percentage (Ab) of ROX by C. pyrenoidosa was calculated according to the following equation:

Ab (%)  =  (Ai  −  A a  −  Ar  −  A d  −  A c)

2.8. Statistical analysis All the experiments were operated in triplicates. The data were analyzed with a one-way analysis of variance using the Least Significant Difference (LSD) multiple comparison test by SPSS version 18.0 for Windows. The significance level for all tests was set at p < 0.05. 3. Results and discussion 3.1. Acute toxicity of ROX on C. pyrenoidosa 3.1.1. Effects of ROX on the growth of microalgae The growth of C. pyrenoidosa in terms of biomass (dry cell weight) and specific growth rate exposed to different concentration of ROX was shown in Fig. 1. Obviously, ROX impacted the growth of C. pyrenoidosa at all detection time, and this effect gradually expanded with the extension of culture time. Compared to the control group, low concentration of ROX (0.0625 mg/L) slightly promoted the growth of C. pyrenoidosa at 24 h ~ 96 h, but the promotion effect did not achieve a significant level. On the contrary, high concentration of ROX inhibited the yield and specific growth rate of C. pyrenoidosa, and the inhibition

100 Ai

where, Ai is the initial concentration of ROX added to the medium, Aa is the quantify of ROX removed by abiotic process, Ar is the residual quantity of ROX in the culture medium, Ad is the quantity of ROX 3

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Table 1 Inhibition of yield and specific growth rate of C. pyrenoidosa, and the calculated EC50 of ROX. Inhibition

Inhibition (%) of yield

Inhibition (%) of specific growth rate

Exposure duration

24 48 72 96 24 48 72 96

h h h h h h h h

ROX concentration (mg/L) 0.0625

0.125

0.25

0.5

1.0

2.0

−5.55 −0.08 −5.25 −2.94 −4.13 −0.77 −2.32 −1.36

2.53 10.12 0.28 3.22 2.41 5.08 0.64 1.43

−2.35 14.46 8.53 11.47 −5.81 3.17 0.29 1.21

21.68 39.08 42.81 44.11 7.38 15.46 14.89 13.48

23.37 48.22 53.29 58.20 11.83 24.20 23.47 23.42

45.09 60.17 68.80 73.76 31.14 35.75 37.76 37.62

EyC50 or ErC50

95% confidence interval

2.59 1.12 0.93 0.81 4.39 3.44 2.56 2.87

1.86~4.45 0.90~1.50 0.53~2.55 0.55~1.36 2.84~10.84 2.33~6.67 1.46~27.99 2.12~4.59

Fig. 2. Effects of ROX on the chlorophyll content (a), SOD activity (b), CAT activity (c) and MDA content (d) of C. pyrenoidosa at 96 h of cultivation. Error bars represent standard deviation (n = 3). * and ** indicate significant differences (p < 0.05 and p < 0.01, respectively) compared to the control.

can use ROX as nutrients (Wan et al., 2015). It is also worthy to mention that all the specific growth rate of control and treatment group gradually decreased with the increase of cultivation time, which may be caused by the nutrient decrease in medium by the metabolism of C. pyrenoidosa (Gan et al., 2014). Based on the probit regression analysis, the median effective concentration on yield (EyC50) and specific growth rate (ErC50) of ROX at 24 h, 48 h, 72 h and 96 h were calculated (Table 1). The former went from 2.59 to 0.81 mg/L, and the later went from 4.39 to 2.87 mg/L, indicating ROX was highly toxic to C. pyrenoidosa according to the toxicity classification standard of the Chinese EPA (2002). It should be mentioned that EyC50 was always lower than ErC50, demonstrating

increased with the increasing of ROX exposure concentration. For example, 2.0 mg/L ROX significantly inhibited the growth of C. pyrenoidosa, with the inhibition rate of the yield and specific growth rate achieving up to 73.76% and 37.62%, respectively, at culture time of 96 h (p < 0.01). The results indicated that ROX had a dual effect (promotion and inhibition) on the growth of C. pyrenoidosa, probably causing hormesis effect on the green algae (Liu et al., 2015). This was consistent with that of another macrolide erythromycin (ERY) (Wan et al., 2015), which promoted the growth of M. flos-aquae at low concentration (0.001 ~ 1 μg/L), but inhibited the growth of M. flos-aquae at high concentration (> 10 μg/L). In this study, low concentration of ROX also promoted the growth of algae probably because green algae 4

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2016). MDA is a byproduct of lipid peroxidation caused by excessive ROS. Therefore, the activities of SOD and CAT as well as the content of MDA were used to assess the oxidative stress of ROX to C. pyrenoidosa. The activities of SOD and CAT after exposure to ROX for 96 h both showed the increasing tendency with the increase of ROX concentration (Fig. 2b and c). No significant differences were observed at ROX concentration of 0.0625 ~ 0.5 mg/L compared with the control group. The activities of SOD and CAT significantly increased at ROX concentration of 1.0–2.0 mg/L (p < 0.05 or p < 0.01), achieving 1.82 times and 2.44 times of that of control at ROX concentration of 2.0 mg/L. This demonstrated that high concentration of ROX caused oxidative stress to algae and stimulate the activities of SOD and CAT. The results were consistent with previous research reported that CLA and ERY induced the SOD and CAT activities of algae to increase (Aderemi et al., 2018; Wang et al., 2019a). The content of MDA in algae cells after exposure to ROX for 96 h was shown in Fig. 2d. It can be seen that the MDA level at ROX concentration of 0.0625 mg/L was significantly lower than that of control. This was consistent with the results that low concentration of ROX promoted the growth of algae (Fig. 1) and slightly simulated the activity of SOD and CAT (Fig. 2b and c). The MDA content at ROX exposure concentration of 0.125 ~ 0.25 mg/L showed no significant difference from control. When ROX concentration increased to 0.5 ~ 1.0 mg/L, the MDA content increased to 1.30 times and 1.39 times of that of control, which showed significant differences with the control group (p < 0.05 or p < 0.01). This implied that the SOD and CAT insufficiently eliminated the ROS caused by 0.5 ~ 1.0 mg/L ROX, and caused the oxidative damage to algae. On the contrary, the MDA content remained close to the control group at ROX concentration of 2.0 mg/L. This demonstrated that the membrane lipid peroxidation damage caused by ROX could be moderated by the increased activities of SOD and CAT (Wang et al., 2017; Wu et al., 2016).

EyC50 was a more sensitive indicator in the ecotoxicity assessment of antibiotics than ErC50. The 96 h EC50 values of ROX to the growth of four algae Pseudokirchneriella subcapitata, Scenedesmus quadricauda, Scenedesmus obliquus, and Scenedesmus acuminatus were previously reported to be 0.733, 0.129, 0.077 and 2.876 mg/L, respectively (Xiong et al., 2019b). The difference of EC50 values in this and previous study may be caused by the different sensitivity of various algae species (Ma, 2005), which is related to the species-specific morphology, cytology, physiology and phylogenetic of different algae species (Xiong et al., 2017b). In addition, compared with other macrolide antibiotics, ROX seems to be less toxic to green algae. For example, the 72 h ErC50 of clarithromycin (CLA) to Desmodesmus subspicatus and Anabaena flos-aquae were 37.1 and 12.1 μg/L, while the EyC50 of CLA to two algae were 32.1 and 5.6 μg/L, respectively (Baumann et al., 2015). Isidori et al. (2005) and Nie et al. (2013) reported that the 72 h EyC50 of ERY to Pseudokirchneriella subcapitata was 0.020 mg/L and the 96 h EyC50 values was 0.20 mg/L. High concentration of ERY can affect the growth of cells, because it induced stronger lipid peroxidation and more serious damage to the cell membrane (Wan et al., 2015). ERY and other MCLs inhibited protein synthesis by binding to the 50S subunit of the ribosome (Sendra et al., 2018; Waiser et al., 2016). Moreover, oxidative damage caused by ERY exposure may result in DNA damage (Rodrigues et al., 2016). ROX has the similar structure with ERY and CLA, and the toxic mechanism of ROX to green algae can also attribute to the alteration in the composition and structure of lipid, protein and DNA (Xiong et al., 2019b). 3.1.2. Effects of ROX on the chlorophyll content of microalgae The chlorophyll content after exposure to different concentration of ROX for 96 h was shown in Fig. 2a. Similar with effect of ROX on the growth of C. pyrenoidosa, low concentration of ROX (0.0625 mg/L) slightly promoted the synthesis of chlorophyll-a, chlorophyll-b and the total chlorophyll, causing hormesis effects on the photosynthesis system of microalgae (Liu et al., 2015), however, no significance difference between low concentration ROX treatments and the control group was observed. When the concentration of ROX increased from 0.125 to 2.0 mg/L, the chlorophyll-a, chlorophyll-b and the total chlorophyll content all gradually decreased, and high concentration of ROX (0.5 ~ 2.0 mg/L) significantly inhibited the synthesis of chlorophyll-a, chlorophyll-b and total chlorophyll (p < 0.05 or p < 0.01). The concentration of chlorophyll-a, chlorophyll-b and total chlorophyll was reduced to 42.52%, 41.82% and 42.30% of that of control group, respectively, exposured to 2.0 mg/L ROX. Generally, antibiotics affect algae photosynthesis capacity, cell proliferation and growth via inhibiting chloroplast formation and protein biosynthesis and damaging chlorophyll (Liu et al., 2018). To the best of our knowledge, the toxic mechanism of ROX inhibiting the chlorophyll formation has not been reported. However, previous research demonstrated that another MCLs ERY may inhibit chloroplast gene translation process and the synthesis of membrane protein in thylakoid of algae (Liu et al., 2011). ROX have a similar molecular structure with ERY, so we suspect that ROX has a similar toxic mechanism on the chlorophyll of algae.

3.2. Chronic ecotoxicity of ROX on C. pyrenoidosa 3.2.1. Effects of ROX on the growth of microalgae Chronic effects of ROX (0 ~ 1.0 mg/L) on the growth represented as dry cell weight of C. pyrenoidosa was depicted in Fig. 3a. The dry cell weight of C. pyrenoidosa of the control group and ROX exposure groups all gradually expanded with the extension of the culture time. The dry cell weight of C. pyrenoidosa exposure to 0.1 and 0.25 mg/L ROX was slightly higher than that of control during the first 7 d. This is similar with the acute results that low concentration of ROX can slightly promote the growth of algae. However, when the exposure time prolonged to 14 d and longer, the dry cell weight of C. pyrenoidosa exposure to 0.1 and 0.25 mg/L ROX was inhibited, with the inhibition rate of 23.10% and 21.57% at 21 d. On the other hand, high concentration of ROX (1.0 mg/L) inhibited the growth of algae during the entire cultivation period, and the inhibition rate decreased from 42.84% at 3 d to 23.78% at 21 d. Effect of ROX on the specific growth rate of C. pyrenoidosa calculated based on the dry cell weight showed a similar trend with that of dry cell weight (Fig. 3b). 0.1 and 0.25 mg/L of ROX showed no significant effect on the specific growth rate of C. pyrenoidosa during the first 14 d, while significantly inhibited the specific growth rate at 21 d (p < 0.01). 1.0 mg/L of ROX significantly inhibited the specific growth rate of C. pyrenoidosa during the entire cultivation time. It should be emphasized that long-term exposure to low concentration of ROX can significantly inhibit the growth of green algae. Similar phenomenon was previously reported by Niu et al. (2019), who found that low concentration (0–100 ng/L) of sulfamethoxazole and norfloxacin showed no significant effect on the growth of Chlorella sp. during the first 7 d, but significantly inhibited the growth of Chlorella sp. during 7 ~ 14 d. Galdiero et al. (2015) reported that no mortality of Daphnia magna was observed during the first 5 d exposure to melittin, while the survival of daphnia magna was 60% after 6 ~ 7 d and decreased to 40% between 7 ~ 21 d. On the other hand, the toxic effect of

3.1.3. Effects of ROX on the antioxidant system of microalgae Toxic compounds stress may break the dynamic equilibrium of the generation and scavenging of reactive oxygen species (ROS) in algae cells, and induce the excessive production of ROS including singlet oxygen (1O2), superoxide (⋅O2−), hydrogen peroxide (H2O2), and hydroxyl radical (•OH), which will result in oxidative damage and apoptosis of algae cells (Xu et al., 2019). On the other hand, algae and other plants possess their own defense system to prevent the oxidative stress. For example, the antioxidant enzyme SOD can convert ∙O2− to H2O2, while CAT can further convert H2O2 to H2O, and eventually partly or completely eliminate the oxidative damage (Qin et al., 2012; Wu et al., 5

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Fig. 3. Effects of ROX on the growth of C. pyrenoidosa in terms of dry cell weight (a) and specific growth rate (b) during 21 d of cultivation. Error bars represent standard deviation (n = 3). * and ** indicate significant differences (p < 0.05 and p < 0.01, respectively) compared to the control.

research also upregulated, demonstrating that a higher antioxidative capacity was necessary for C. pyrenoidosa to scavenge ROS when the algae exposed to ROX for a relative long time (Nie et al., 2013). The MDA concentration of C. pyrenoidosa exposure to 0.1 and 0.25 mg/L ROX for 14 and 21 d and exposure to 1.0 mg/L of ROX for 7 and 21 d significantly increased than that of control (Fig. 4d). This increasing tendency of MDA concentration indicated that the alteration of antioxidant enzymes was unable to prevent and counteract the formation of ROS caused by ROX. It was similar to the literature that a high content of MDA was induced by ROX in daphnia magna and fish (Zhang et al., 2019b). Overall, the SOD and CAT activities and MDA content in the ROX treatments were significantly higher than that of the control after 21 d cultivation. Especially, low concentration of ROX (0.1 mg/L) after longterm exposure can also induce significant change in the antioxidant enzyme and MDA content. It is common that antibiotics resulted in an increase of ROS, which in turn stimulated the response of antioxidant defenses, impaired the physiological function of green algae, and finally resulted in decreased cell growth rate (Xu et al., 2019).

ROX on the green algae may be also affected by the internal exposure caused by bioaccumulation (Jeong et al., 2016; Straub, 2016). In this study, the accumulation of ROX in algae cells in 0.1 and 0.25 mg/L treatments was observed from the 14th day, and the inhibition effect of ROX on the growth of algae started to increase at the same time. The accumulation of ROX in algae cells in 1.0 mg/L treatment was observed in 3 d and caused significant inhibition of the growth of algae. Therefore, the accumulation of ROX in algae cells seemed to be correlated with the effect of ROX on the growth of green algae. And the accumulation of ROX in green algae will be illustrated in section 3.3 in more detail. 3.2.2. Effects of ROX on the biochemical characteristics of microalgae The chronic effects of ROX on the physiological and biochemical characteristics of C. pyrenoidosa including chlorophyll content, SOD and CAT activities and MDA content were shown in Fig. 4. As shown in Fig. 4a, the chlorophyll-a contents of 0.1 and 0.25 mg/L ROX treatments showed no significant difference with that of control during the first 14 d of exposure, however, when the exposure time increased to 21 d, the chlorophyll-a contents of 0.1 and 0.25 mg/L treatments significantly decreased, with the inhibition of 13.06% and 24.56%, respectively. This was consistent with growth effect and indicated that low concentration of ROX (0.1 ~ 0.25 mg/L) can also cause significant damage to the photosynthetic system of green algae after long-term exposure (Teixeira and Granek, 2017; Wang et al., 2019b). High concentration of ROX significantly inhibited the synthesis of chlorophyll-a, the inhibition rates was 27.92% ~ 35.06% after exposed for 7 ~ 21 d. Effect of ROX on the total chlorophyll content show a similar trend with chlorophyll-a, that is, 1.0 mg/L ROX significantly decreased the total chlorophyll, and 0.1 and 0.25 mg/L ROX significantly decreased the total chlorophyll only at 21 d. There was a distinct change in SOD activity between 7 d and 14 d or 21 d (Fig. 4b). The SOD activities of 0.25 and 1.0 mg/L ROX treatments significantly lower than that of control at 7 d (p < 0.01), however, when the cultivation time prolonged to 14 and 21 d, the SOD activities of all ROX treatments increased. On the other hand, the CAT activities of 0.1 and 0.25 mg/L ROX treatments were significantly higher than that of control at 14 and 21 d (p < 0.05 or p < 0.01, Fig. 4c), and the CAT activity of 1 mg/L treatment was significantly higher than that of control at all exposure time (p < 0.01). Yan et al. (2017) founded the SOD and CAT activities in the liver of crucian carp exposure to 4 and 20 μg/L ROX significantly increased. In fact, the upregulation of SOD and CAT activity of green algae is a normal phenomenon to resist or relieve the oxidative stress caused by antibiotics and other organic pollutants (Wu et al., 2016; Xiong et al., 2016; Xu et al., 2019). Similar to the previous researches, the SOD and CAT activities in current

3.3. Removal mechanism of ROX by C. pyrenoidosa The removal of ROX in the BG11 medium with and without C. pyrenoidosa were shown in Fig. 5. As shown in Fig. 5a, the abiotic removal of 0.1, 0.25 and 1.0 mg/L ROX reached 12.21%, 18.88% and 21.37% after 14 d cultivation, with the pseudo-first order reaction rate constants of 0.0211, 0.0226 and 0.0176 d−1, respectively (R2 > 0.85). However, when the cultivation time increased to 21 d, the removal of ROX did not further proceed and the removal reaction rate constants decreased to 0.0180 (R2 = 0.89), 0.0150 (R2 = 0.78) and 0.0117 d−1 (R2 = 0.80), respectively. This may be attributed to the degradation products of ROX (Batchu et al., 2014; Li et al., 2019), which may hinder the further photo-degradation of ROX. As there were no algae in the solution, the removal of ROX was mainly caused by photo-degradation. Batchu et al. (2014) reported the photo-degradation rate constant of ROX in pure water was 0.1368 d−1 under the irradiation of simulated solar light, which is about 10 times of that reported in present study. It is reasonable because the energy of fluorescence light used in the illumination incubator is much lower than that of simulated solar. Also, it should be noted that the degradation rate constant of ROX deceased with the increase of the ROX concentration, which was in accordance with the previous reports (Chen et al., 2013; Jin et al., 2017). The removal of 0.1, 0.25 and 1.0 mg/L ROX in the presence of microalgae achieved 80.45%, 76.35% and 64.81% after 21 d cultivation (Fig. 5b). This is similar with the previous research that reported more than 80% removal of ROX by four algae species (Zhou et al., 6

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Fig. 4. Effects of ROX on the chlorophyll content (a), SOD activity (b), CAT activity (c) and MDA content (d) of C. pyrenoidosa at 7, 14 and 21 d of cultivation. Error bars represent standard deviation (n = 3). * and ** indicate significant differences (p < 0.05 and p < 0.01, respectively) compared to the control.

0.0580 d−1 (R2 = 0.69) and 0.0574 d−1 (R2 = 0.72), respectively. This may be explained by the accumulation of ROX and the upregulation of enzyme activities. Yu et al. (2017) reported that the algal removal of antibiotic can be separated into three steps: a rapid adsorption, a slow cell wall-transmission and the final biodegradation. This is consistent with our results that adsorption was observed at 3 d and the accumulation was observed since 14 d in the 0.1 and 0.25 mg/L ROX treatments. On the other hand, C. pyrenoidosa might gain the resistance mechanism to antibiotics by improving enzyme systems, extracellular polymeric substances and cell membrane structure, which is powerful for degrading antibiotics (Xiong et al., 2017b). The SOD and CAT

2014). However, there was an interesting phenomenon in the present study, the degradation rate of 0.1 and 0.25 mg/L ROX with microalgae were slower than that of without microalgae during the first 14 d cultivation, with the rate constants of 0.0192 d−1 (R2 = 0.94) and 0.0218 d−1 (R2 = 0.81), respectively. This demonstrated the removal of lower concentration of ROX during the first 14 d cultivation was predominantly removed by photo-degradation, and the degradation rate was inhibited by the light screening effect of algae cells (Norvill et al., 2017). However, when the cultivation time prolonged to 21 d, the degradation of 0.1 and 0.25 mg/L ROX was significantly accelerated by C. pyrenoidosa and the degradation rate constants of ROX increased to

Fig. 5. The removal of ROX without (a) and with (b) microalgae. 7

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treatment technology for eliminating antibiotics, further research is needed to explore the degradation mechanism of antibiotics, to optimize the growth condition of green algae and to enhance the removal of antibiotics (Sutherland and Ralph, 2019). 4. Conclusions This paper mainly studied the interactive effects of ROX and fresh algae C. pyrenoidosa. The 96 h EyC50 and ErC50 of ROX on the growth of C. pyrenoidosa was 0.81 and 2.87 mg/L, respectively, demonstrating that ROX is a high toxic compound to C. pyrenoidosa. Low concentration of ROX (< 0.25 mg/L) showed no significant effect on the growth of microalgae and chlorophyll content during the acute exposure and the first 14 d of chronic exposure, however, when the exposure time prolonged to 21 d, low concentration of ROX also caused significant inhibition on the growth and chlorophyll content of microalgae. Similarly, low concentration of ROX showed no significant effect on the SOD and CAT activities and MDA content, but the increasing of SOD and CAT activities and MDA content were observed since 14 d. On the other hand, high concentration of ROX (≥1.0 mg/L) significantly inhibited the growth of microalgae and synthesis of chlorophyll during the entire exposure period, and showed a trend of upregulation of SOD and CAT activities and accumulation of MDA. The results indicated that high concentration of ROX and long-term exposure to low concentration of ROX could cause inhibition of algae growth and oxidative damage. In real aquatic environment, the organisms are always exposed to low concentration of antibiotics for a really long term, even for several generations. Therefore, the effects of long-term exposure to low concentration of antibiotics should be paid more attention and the toxicity mechanism of low concentration of ROX on green algae should be further studied. ROX can be removed predominantly by photo-degradation and biodegradation. After exposure for 21 d, about 18.81% ~ 27.16% of ROX was removed by photo-degradation, while about 45.99% ~ 53.30% of ROX was removed by biodegradation at ROX concentration of 0.1 ~ 1.0 mg/L. This research proved that algae have the capacity to remove ROX after long-term exposure, and this may provide a new insight in the removal of ROX and other antibiotic in environment by green algae. Therefore, to develop the technology of antibiotics treatment by algae, the removal mechanism of ROX and other antibiotics by green algae, for instance, the impact factors, the degradation products of antibiotics and their ecotoxicity should be thoroughly investigated in the future.

Fig. 6. The contribution of different removal mechanism on the initial concentration of ROX.

activities were significantly higher than that of control since 14 d, therefore, it can be inferred that the accumulation of ROX and upregulation of SOD and CAT activities promoted the biodegradation of ROX since 14 d. The removal of 1.0 mg/L ROX followed pseudo-first order kinetics with a consistent rate constant of 0.053 d−1 during the 21 d cultivation. The accumulation and upregulation of enzyme activities observed since 3 d's exposure to 1.0 mg/L ROX further proved the removal mechanism of ROX mentioned above. Fig. 6 summarized the contribution of abiotic-degradation, biodegradation, bio-adsorption and bioaccumulation on the initial exposure concentration of ROX. It can be seen that photo-degradation played a major role in the first 14 d in the lower concentration, while biodegradation became the predominant removal mechanism of ROX in the last 7 d. In contrast, bio-adsorption and bioaccumulation both played minor roles in the removal of ROX. The bio-adsorption could reach the highest value at cultivation time of 3 or 7 d. For instance, when exposure to 0.1 mg/L ROX, 1.39% of ROX absorbed onto the surface of algae cells at 3 d, then the contribution decreased to 0.69% at 21 d. This may demonstrate that the antibiotics absorbed on the surface of cell penetrated into the cell. Moreover, the adsorption is related to the concentration of antibiotics present in the solution (Guarín et al., 2018). As the concentration of residual ROX decreased, the ROX adsorbed on the algae and contribution of adsorption also decreased. There was no ROX accumulated in algae cells during the first 10 d when exposure to 0.1 and 0.25 mg/L ROX, however, when the cultivation time increased to 14 d, ROX began to accumulated in algae cells and the accumulation increased with the extension of cultivation time. When exposure to 1.0 mg/L ROX, the bioaccumulation of ROX in algae cell was detected at 3 d, with the contribution increasing from 0.09% at 7 d to 0.28% at 21 d. Previous research also reported that bio-adsorption and bioaccumulation showed a smaller or negligible contribution to the removal of emerging contaminants (Xiong et al., 2016, 2017c), they reported bio-adsorption and bioaccumulation contributed 0.36% and 0.80% to the removal of levofloxacin by S. obliquus after 11 d of cultivation. Overall, C. pyrenoidosa have the ability to remove ROX and other antibiotics. Sun et al. (2017) found C. pyrenoidosa showed a great ability to deplete sulfamethazine (SMZ) from the culture media, with the biotic degradation of 48.45%, 60.20% and 69.93% of 2, 4 and 8 mg/L of SMZ, respectively. Song et al. (2020) reported Chlorella sp. UTEX 1602 and L38 removed about 95% and 97% of 46.2 mg/L thiamphenicol. However, high concentration of antibiotic may also cause toxic effects on green algae. Therefore, to develop the algae

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