Biodegradation of naproxen by freshwater algae Cymbella sp. and Scenedesmus quadricauda and the comparative toxicity

Accepted Manuscript Biodegradation of Naproxen by freshwater algae Cymbella sp. and Scenedesmus quadricauda and the comparative toxicity Tengda Ding, Kunde Lin, Bo Yang, Menting Yang, Juying Li, Wenying Li, Jay Gan PII: DOI: Reference:

S0960-8524(17)30499-6 http://dx.doi.org/10.1016/j.biortech.2017.04.018 BITE 17913

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

22 February 2017 30 March 2017 5 April 2017

Please cite this article as: Ding, T., Lin, K., Yang, B., Yang, M., Li, J., Li, W., Gan, J., Biodegradation of Naproxen by freshwater algae Cymbella sp. and Scenedesmus quadricauda and the comparative toxicity, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.04.018

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Biodegradation of Naproxen by freshwater algae Cymbella sp. and Scenedesmus quadricauda and the comparative toxicity

ξ

Tengda Ding†, ‡, Kunde Lin‡, Bo Yang†, , Menting Yang†, Juying Li†,

ξ,*

, Wenying Li ¶,

Jay Gan§

†

College of Chemistry and Environmental Engineering, Shenzhen University,

Shenzhen 518060, P.R. China ‡

State Key Laboratory of Marine Environmental Science, Key Laboratory of the

Ministry of Education for Coastal and Wetland Ecosystem, College of the Environment and Ecology, Xiamen University, Xiamen 361005, China ¶

Institute of Agricultural Resources & Environment, Guangdong Academy of

Agricultural Sciences, Guangzhou 510640, PR China §

Department of Environmental Sciences, University of California, Riverside, CA

92521 ξ

Shenzhen Key Laboratory of Environmental Chemistry and Ecological Remediation, ,

Shenzhen University, Shenzhen 518060, P.R. China

*Corresponding Author: Juying Li, College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen 518060, China Tel.: +86-0755-26538657 E-mail: [email protected]

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Abstract: Naproxen is one of the most prevalent pharmaceuticals and of great environment concern. Information about bioremediation of naproxen by algae remains limited and no study has been reported on the degradation mechanism and the toxicity of NPX on algae. In this study, both Cymbella sp. and Scenedesmus quadricauda showed complete growth inhibition (100%) at 100 mg L-1 within 24 h. Biochemical characteristics including chlorophyll a, carotenoid contents and enzyme activities for these two microalgae were affected by NPX at relatively high concentrations after 4 d of exposure. Degradation of naproxen was accelerated by both algae species. Cymbella sp. showed a more satisfactive effect in the bioremediation of NPX with higher removal efficiency. A total of 12 metabolites were identified by LC-MS/MS and the degradation pathways of naproxen in two algae were proposed. Hydroxylation, decarboxylation, demethylation, tyrosine conjunction and glucuronidation contributed to naproxen transformation in algal cells. Keywords: Naproxen; Biodegradation; Degradation pathway; Algae; Toxicity 1. Introduction Naproxen (NPX) is one of the most commonly used nonsteroidal antiinflammatory drugs. Continuous discharge of NPX into the aquatic environment could pose high risks to non-target organisms (Isidori et al., 2005; Singh et al., 2014). For example, NPX can affect mRNA expression and cause gastrointestinal or renal effects in zebrafish at environmentally relevant concentrations (Stancová et al., 2015; Chattopadhyay et al., 2016). NPX was found to be toxic to Chlorella vulgaris and Ankestrodesmus falcatus with the 24 h EC 50 values of approximately 40 mg L-1 (EI-

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Bassat et al., 2012). In addition, NPX is frequently detected in surface water with concentrations ranging from few ng L-1 to several µg L-1 (Kasprzyk-Hordern et al., 2008; Marco-Urrea et al., 2010). For instance, Loos et al. (2009) found that NPX was detected in 69% of more than 100 water samples from over 100 European rivers in 27 European Countries with concentrations up to 2, 027 ng/L. The high detection frequency and levels of NPX in natural waters is likely due to the low removal efficiency (< 15%) by wastewater treatments such as conventional activated sludge processes and granular activated carbon processes (Vélez et al., 2016; Paredes et al., 2016). Though the membrane bioreactor can achieve high NPX removal rate (Arriaga et al., 2016; Prasetkulsak et al., 2016), the high operational costs and membrane fouling have limited its widespread application (Remy et al., 2010). Photodegradation is also an effective way to remove NPX, but the byproducts are usually found to be more toxic (Isidori et al., 2005; Avetta et al., 2016). Algae can usually uptake organic contaminants and are used in bioremediation of organic pollutants (Chan et al., 2006; Caceres et al., 2008; Guo et al., 2016). For instance, 17αethinylestradiol is highly resistant to degradation in STPs (< 10%) (Paredes et al., 2016). Maes et al. (2014) found that up to 75% of 17α-ethinylestradiol could be removed by freshwater microalgae Desmodesmus subspicatus. Thus, algae are considered as a potential candidate for environmentally sustainable reclamation strategy for contaminated waters. Interest in the capabilities of algae for NPX degradation is growing. However, information on biodegradation and the interactions of NPX with algae (i.e., ecotoxicological effects) are limited.

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The aim of this study was to investigate the biodegradation and toxicity potential of NPX by using two freshwater algae. The degradation pathway of NPX was elucidated with an emphasis on the identification of transformation products by UPLCMS/MS. 2. Materials and methods 2.1 Chemicals Naproxen was purchased from Sigma-Aldrich (Shanghai, China). A stock solution of NPX (10 g L-1) was prepared in methanol. All organic solvents and other chemicals used were of analytical or HPLC grade. 2.2 Algal growth assay The freshwater algae Cymbella sp. and Scenedesmus quadricauda were obtained from the Center of Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences (FACHB-Collection, Wuhan, China). The algae were inoculated into 500 mL sterile D1 and BG11 medium, respectively. The specific recipe for D1 and BG11 medium can be found in our previous studies (Zhang et al., 2013, 2014). A range of NPX (0, 0.1, 0.5, 1, 10, 50 and 100 mg L-1) was prepared in the D1 and BG11 medium, which were then sterilized at 121 0C for 20 min. The algal cells were then inoculated in 50 mL algal cultures and incubated for 96 h at 23 ± 1 0C in an incubator with illumination by fluorescent lamps (4000 lux, light: dark of 12: 12 h). Each treatment was performed in triplicate. The algal cells were calculated every 24 h by the optical densities at 680 nm in a UV-2550 spectrophotometer (Shimadzu, Japan). The cellular growth rates (d -1) were

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calculated by fitting the cell numbers to an exponential function as described in our previous work (Zhang et al., 2013). More detailed descriptions can be found in the Supporting Information (Text S1). The inhibition rate (I %) was calculated based on the specific growth rate of algae between the treatments and control, i.e. I % = (µ1-µ2) / µ1×100 (µ1, µ2 are the growth rate of algae in the control and in the treatments at the same time, respectively). At the end of incubation, all samples were collected to analyze the pigment content, including chlorophyll a, chlorophyll b/c, and carotenoid contents of two algae, which were extracted by 10 mL methanol/H2O (9/1, v/v) at 60 0

C in a water bath for 15 min, and measured as described by Xiong et al. (2016).

2.3 Determination of antioxidant enzymes and lipid peroxidation A 10 mL algal suspension was harvested after 96 h of cultivation and centrifuged at 4000 rpm for 20 min at 4 oC. The pellet was washed with distilled water and centrifuged again. The recovered algal pellet was resuspended in 0.1 M Tris-HCl (pH 7.4), sonicated for 5 min at 4 0C, and centrifuged at 10000 rpm for 10 min at 4 0C. The supernatant of cell lysate was used to determine the activity of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (MDA). The SOD, CAT, and MDA were determined by the assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocol. One unit of SOD activity is defined as the amount that caused a 50% decrease of SOD, whereas one unit of CAT is the amount that decompose 1 µmol of H2O2 per second. 2.4 Uptake of NPX by Cymbella sp. and S. quadricauda The uptake of NPX (1, 10 and 100 mg L-1) in Cymbella sp. and S. quadricauda

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were performed in 100 mL Erlenmeyer flasks containing 50 mL D1 and BG11 medium for 30 days, respectively. Two types of controls were included: untreated algae controls without IBU in the medium and NPX-spiked medium controls without algae, which were used to detect any NPX contamination emanating from elsewhere and assess the abiotic removal of NPX in the medium, respectively. At 0, 2, 4, 7, 10 and 30 days after treatment, three replicates of 10 mL solution were transferred to 10 ml centrifuge tubes and centrifuged at 4000 rpm for 20 min. The concentration of NPX in the supernatant was determined. The pellet left was extracted by sonication with 4 mL dichloromethane: methanol (1:2, v/v) for 1 h to determine the concentration of NPX within the microalgal cells. The algal cell biomass was calculated by counting the cell numbers of each algal species. An algal cell density of 1 × 105 cells mL-1 was equivalent to an algal cell biomass of 0.0184 mg dry biomass mL-1 (Zhang et al., 2013). Based on the algal dry biomass, the uptake of NPX was determined. The bioconcentration factor (BCF) was calculated by ratio of NPX concentration in algae to that in water. 2.5 Chromatographic analysis The samples were filtrated through a 0.22 µm membrane filter and subjected to high performance liquid chromatography (HPLC) (Ultimate 3000 system, Thermo Scientific, Waltham, MA) equipped with a UV-Vis detector for the determination of NPX. The NPX metabolites were detected by an Agilent 1290 HPLC system (Agilent Technologies, Santa, CA) coupled with an electrospray ionization tandem mass spectrometry (LC-MS/MS). The detailed chromatographic information can be found in

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Supporting Information (Text S2). 2.6 Data analysis All of the experiments in this study were carried out in triplicates. One-way ANOVA was used to evaluate the significance of differences in growth rate, chlorophyll and carotenoid content in two algae between control and NPX treatment. A difference was considered statistically significant at a level of 0.05. All statistical analyses were performed using SPSS. 3. Results and discussion 3.1 Comparative toxicity of NPX on the growth of two algae The inhibition rate of two microalgal species Cymbella sp. and S. quadricauda by NPX was evaluated in culture medium spiked with different NPX concentrations (0.1, 0.5, 1, 10, 50, 100 mg L-1) during 96 h (Figure 1). The toxicity of NPX on algae was concentration- and time-dependent. The inhibition rates of both algae increased as the NPX concentration increased and both microalgae showed complete growth inhibition (100.0%) at 100 mg L-1 NPX within 24 h. The toxicity of NPX to algae decreased when the exposure time prolonged. For example, the growth inhibition rate in two algae decreased from 100.0% at 24 h to about 45.0% at 72h under the exposure of 100 mg L-1 NPX. It may be ascribed to the peroxidases, which have the catalytic ability to remove pollutants and were continuously produced by algae (Li et al., 2016). Furthermore, the NPX toxicity varied in the two algae tested. For instance, Cymbella sp. was found to be more tolerant to NPX than S. quadricauda at 24 h exposure, implying that the diatom frustules of Cymbella sp. may play a protective role in

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preventing NPX from entering into algal cells. Interestingly, at the 48 h exposure, the growth inhibition rate of Cymbella sp. (28.7%) was significantly higher than that of S. quadricauda (13.5%) under exposure of 1 mg L-1 NPX, indicating that the detoxification mechanism of S. quadricauda (e.g., the production of peroxidases or catalase) was more effective, leading to a higher tolerance to intracellular NPX. The effective concentration (EC50) was also used to evaluate the toxicity of xenobiotics. NPX showed high toxic effect to S. quadricauda and harmful effect to Cymbella sp. during the incubation time of 24 h. The 24 h, 48 h and 72 h EC50 values of Cymbella sp. were 29.20, 37.93 and 102.76 mg L-1, respectively, whereas the 24 h, 48 h and 72 h EC50 values in S. quadricauda were 0.70, 69.86 and 101.45 mg L-1, respectively (Table S1). Similarly, NPX was found to show significant growth inhibition of green algae Pseudokirchneriella subcapitata, Chlorella vulgaris, and Ankestrodesmus falcatus, with the EC50 of 31.82, 42, and 40 mg L-1, respectively (Isidori et al., 2005; EI-Bassat et al., 2012). The photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids) of the two tested algae were measured to assess the influence of NPX on photosynthetic process of algae (Figure 2). Chlorophyll-a (Chl-a) can prevent the cell damage caused by reactive oxygen species (ROS) under environmental stress (Tsiaka et al., 2013; Xiong et al., 2016). The Chl-a and carotenoid contents in two algae were significantly reduced (p < 0.05) at 100 mg NPX L-1, and the Chl a contents in all treatments were correlated with carotenoids contents. This could be due to the fact that carotenoids can deactivate excited chlorophyll to scavenge the accumulated ROS in chloroplast

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(Paliwal et al., 2015). Ratios of Chl a/b or carotenoids/total Chl are indicators of the integrity protection ability for algal cells against photo oxidative damage (Pancha et al., 2015). Significantly lower Chl a/b or carotenoids/total Chl ratios were observed in Cymbella sp. cultivated under 100 mg NPX L-1 compare to the NPX-free control (Figure 2), suggesting that the Cymbella sp. cells may be destroyed under high NPX exposure. In contrast, S. quadricauda was more tolerant to NPX in the chronic exposure as the Chl a/b or carotenoids/total Chl ratios kept unchanged. 3.2 Effects of NPX on antioxidant enzymes (SOD, CAT) and lipid peroxidation Reactive oxygen species, such as hydroxyl radical (HO·), superoxide radical (O2· -) or hydrogen peroxide (H2O2), are harmful to organisms at high concentrations and can be accumulated in algal cells. Enhanced generation of ROS can overwhelm intrinsic antioxidant defenses of cells, and result in lethal damages to cell organelles (Lin et al., 2010; Xiong et al., 2016). The ROS induced toxicity can be reduced via scavenging or detoxification of excess ROS by the enzymic antioxidants (e.g., SOD and CAT) (Zhang et al., 2012). For instance, the reactive superoxide anion (O2-) can be converted to H2O2 by SOD, and further degraded to water and oxygen by CAT (Regoli, 1998). After 96 h exposure of NPX, the enzyme activities including SOD and CAT activities measured in two algae are shown in Figure 3a. The SOD activity in Cymbella sp. was significantly increased at 1 mg NPX L-1 (195.15 U mg -1) and decreased at higher concentrations (33.43 and 60.64 U mg-1 at 50 and 100 mg L-1, respectively) compared to the control without NPX (135.74 U mg-1). The CAT activity was significantly increased at the NPX concentration of > 10 mg L-1 ( > 35.65 U mg-1) (Figure 3a, p <

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0.05), as compared to 20.62 U mg-1 in the control. In S. quadricauda, the SOD activity showed a similar trend, while significantly higher CAT activity was only observed when NPX was added at 1 and 50 mg L-1 (Figure 3a, p < 0.05). These observations were consistent with the report by Gómez-Oliván et al. (2014) that the SOD activity was increased at low NPX concentrations (0.018 mg L-1) in Daphnia magna. The decreased SOD and CAT activity in algae exposed to higer NPX levels led to more OH- and H2O2 accumulation in algal cells, which are consistent with the decreased algal growth rate at higher NPX concentrations. In diatom Cymbella sp., the MDA content was positively correlated with SOD and CAT activities. A significant increase in MDA content was observed at the NPX concentration of > 50 mg L-1 (Figure 3b). The induced end-products, e.g., MDA, could interact with biomolecules such as proteins, lipoproteins and DNA to impair the algal cells (Maes et al., 2006). This observation was in agreement with the higher lipid peroxidation found in algae Chlorella vulgaris and Ankestrodesmus falcatus at NPX concentration of > 40 mg L-1 (EI-Bassat et al., 2012). However, the MDA content of S. quadricauda was significantly increased at the exposure of 0.1 mg L-1 NPX and then decreased when the NPX concentration increased (Figure 3b). Similar trends have been previously observed for the effect of 1-octyl-3-methylimidazolium bromide stress on diatom Skeletonema costatum (Deng et al., 2016). Chen et al (2016) also found that the MDA contents in the green algae Chlorella vulgaris were significantly decreased at high copper concentration. It has been reported that the higher NPX stress could reduce the production of ROS (Kimura, 1997), mitigating the oxidative stress on the algae.

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This may be the reason for the decreasing MDA contents at higher NPX concentration. 3.3 Removal of naproxen by Cymbella sp. and S. quadricauda The two algae showed different trends in terms of removal efficiency of NPX during the 30 d of incubation (Figure 4). In general, higher removal of NPX was obtained by Cymbella sp. For example, after 30 days of incubation, significant enhanced removal was found in Cymbella sp. culture (97.1%) spiked with 1 mg L-1 NPX as compared to that in pure medium (76.7%) and S. quadricauda cultures (58.8%). The removal rates of NPX in algae free cultures added with 10 and 100 mg L1

NPX after 30 days incubation were 72.6% and 1.7%, respectively. It is noteworthy

that the diatom Cymbella sp. increased the NPX removal rate to 83.0% and 58.1%, respectively, whereas only 2.4% of NPX was removed by green algae S. quadricauda when NPX was added with a concentration of 100 mg L-1 and S. quadricauda even showed an inhibition effect on degradation of NPX with a lower removal efficiency of 60%, implying that Cymbella sp. showed a more satisfactive effect in the bioremediation of NPX in natural waters. Bioaccumulation in aquatic organisms is a fundamental process for the dissipation of contaminants in aquatic environment, and contributes to the toxicity of pollutants (Mackay and Fraser, 2000). The cellular accumulation of NPX showed an upward trend as the concentrations and cultivation time of NPX increased in Cymbella sp. and S. quadricauda (Figure 5). The cellular NPX concentration in S. quadricauda was higher than that in Cymbella sp. at different concentrations within 96 h of exposure, but after 10 d of exposure, NPX accumulated in S. quadricauda was lower than that in

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Cymbella sp. For example, 125.3 and 1548.0 mg g-1 of NPX were found in Cymbella sp. and S. quadricauda added with 100 mg L-1 NPX on day 2, respectively. On day 10, the accumulated NPX increased to 888.6 mg g-1 in Cymbella sp. and decreased to 260.4 mg g-1 in S. quadricauda. The growth rate of S. quadricauda significantly decreased during first 24 h and recovered in the subsequent exposure time, which is consistent with the accumulation trend of NPX in S. quadricauda, suggesting that the growth of S. quadricauda was influenced by the accumulated NPX. The log BCF of NPX in both algae spiked with 1, 10, and 100 mg L-1 of NPX during the incubation was obtained (Figure 5c). The log BCF increased from 0.5 ± 0.08 on day 2 to 0.81 ± 0.21 on day 10 in Cymbella sp. exposed to 1 mg NPX L-1, whereas it decreased from 0.95 ± 0.05 on day 2 to 0.34±0.13 on day 10 in S. quadricauda. Similar trends were also observed at higher NPX concentrations, implying that NPX may be easily accumulated in Cymbella sp. during the chronic exposure and potentially threaten the aquatic ecosystem through food web. 3.4 The metabolic fate of NPX in Cymbella sp. and S. quadricauda 3.4.1

Formation of degradation intermediates in two algae The degradation products of NPX were extracted and identified by UPLC-MS/MS

in the medium amended with 1 mg L-1 NPX. A total of 12 metabolites were found in algal cells during 10 d of incubation. These metabolites are labeled herein as TP 341, TP 220, TP 441, TP 423, TP 431, TP 216, TP 265, TP 339, TP 323, TP 353, TP 333 and TP 369 with the increasing retention times (Figure S1). Because of the lack of standard chemicals for reference, structural identification of the intermediates was based on the

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analysis of the total ion chromatogram (TIC) and the corresponding mass spectrum, as shown in Figures S2-13. The structural and fragmentation information for NPX and its degradation metabolites in algal cells are shown in Table 2. In Cymbella sp., a total of 10 metabolites were detected. TP 341 was tentatively determined to be a tyrosine conjugated metabolite (6-(2-amino-1-hydroxy-3-(4hydroxyphenyl)propoxy) naphthalene-2, 3-diol). The fragments m/z 179 and 160.8 may be attributed to the dissociation of the ester side chain. The further cleavage of tyrosine with the loss of CO2 and hydroxyl group and electron rearrangement could form the ion clusters at m/z 119.2 (Figure S2). TP 216 (6-O-desmethylnaproxen) was tentatively identified as a demethylated derivative of naproxen, which was previously detected in wastewater treatment plants (Quintana et al., 2005). The ion cluster at m/z 170 was likely due to a loss of carboxyl group and electron rearrangement (Figure S3). Fragment ions of TP 265 in the spectrum represent the loss of a carboxyl group and a subsequent ring fracture, resulting in m/z 217 and 97, respectively. The proposed structure for TP 265 is 2-(7-hydroxy-6-methoxynaphthalen-2-yl)propane-1,1,3-triol. The metabolites TP 353 and TP 369 were also considered to be tyrosine conjugated metabolites. For TP 353, m/z 177 and 163 corresponded to fragment peaks after the cleavage of the ester group (Figure S5). It was identified as 6-hydroxy-7methoxynaphthalen-2-yl-2-amino-3-(4-hydroxyphenyl) propanoate. The metabolite TP 369 was tentatively identified as 6-hydroxy-7-methoxynaphthalen-2-yl 2-amino-3-(4hydroxyphenyl)-3-(4-hydroxyphenyl)propanoate. The fragment ions m/z 337 corresponded to the loss of –OH and methyl group. The fragment ions m/z 193

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corresponded to the cleavage of the ester group and an electron transfer in TP369, and m/z 163 was indicative of consecutive losses of a hydroxyl group (Figure S6). For TP 333, the fragment ions m/z 275.1, 233.3 and 175.1 were indicative of the consecutive losses of hydroxyl groups and fracture of naphthalene ring. The loss of 42 mass units likely corresponded to the loss of a hydroxyl and a methyl group (Figure S7). The proposed structure for this product is 1-(hydroxy(6-hydroxynaphthalen-2yl)methyl)naphthalene-2, 6-diol. The metabolite TP 220 was tentatively determined to be 2-(1-hydroxy)-6-O-desmethylnaproxen. The loss of a mass unit of 18 from m/z 219.9 to 201.9 corresponded to a loss of H2O. The fragment ion m/z 167 was indicative of further loss of two hydroxyl groups and the formation of oxygen heterocycle (Figure S8). The fragment ion m/z 227 for TP 441 is mostly attributed to losses of glucuronide and H2O (Figure S9). TP 441 may correspond to 6-(2-(6,7-dimethoxynaphthalen-2-yl)1-hydroxypropoxy)-3,4,5,6-tetrahydroxyhexanoic acid. TP 423 and TP 431 presented a similar fragment pattern on LC-ESI-MS with TP 441, indicating they were structurally similar and may correspond to the derivatives of TP 441.The molecular ion of TP 423 could lose a carboxyl group to give the ion at m/z 381, and lose hydroxyl groups and a methoxyl group to give the ion at m/z 283 (Figure S10). For TP 431, the peak at m/z 385 and 355 corresponded to the loss of a carboxyl group and a further loss of a methoxyl group (Figure S11). Thus, TP 423 and TP 431 were tentatively identified as 3,4,5-trihydroxy-6-((2-(7-hydroxy-6-methoxynaphthalen-2yl)propanoyl)oxy)tetrahydro-2H-pyran-2-carboxylic acid and 6-((2-(6,7dimethoxynaphthalen-2-yl)propanoyl)oxy)-3,4,5-trioxotetrahydro-2H-pyran-2-

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carboxylic acid, respectively. Six metabolites including TP 341, TP 216, TP 265, TP 353, TP 333, and TP 369 were also detected in S. quadricauda, suggesting that NPX showed certain similar metabolisms in the two algae tested. Three additional metabolites were found in S. quadricauda. TP 339 was tentatively determined to be a tyrosine conjugated metabolite, which was 6, 7-dihydroxynaphthalen-2-yl 2-amino-3(4-hydroxyphenyl)propanoate. The fragment ions at m/z 163 corresponded to the residue of tyrosine (Figure S12). TP 323 was tentatively identified as an oxalic acid conjugated compound of hydroxyl naproxen. The ion peaks at m/z 305 and 261 corresponded to loss of H2O and COOH, respectively. The fragment ion at m/z 217 indicated a further loss of a methoxyl and hydroxyl group (Figure S13). We presumed that TP 323 was 2-(6-((carboxycarbonyl)oxy)-7-hydroxynaphthalen-2-yl)-3hydroxypropanoic acid. 3.4.2

Proposed pathways for degradation of NPX in two algae

On the basis of the identified metabolites and their kinetics during incubation, possible degradation pathways of NPX in two algae are schematically shown in Figure S14. In our studies, demethylation is presumably the first step of degradation of NPX in Cymbella sp. The demethylated metabolite (TP 216) increased from day 4 to day 7 (Figure S15a). Hydroxylation is considered as a main degradation pathway of contaminants in algae because of the presence of cytochrome P-450 (CYP 450) (Quintana et al., 2005). For instance, hydroxylation and formation of desmethylnaproxen comprised the initial step in the degradation of NPX by enzymes such as CYP 450 (Rodarte-Morales et al., 2012). The metabolite 6-O-

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desmethylnaproxen (TP 216) was also formed in the microbiological transformation of NPX by CYP 450 (Domaradzka et al., 2015). Demethylation and hydroxylation are followed by loss of -CH3, yielding intermediate TP220. NPX proceeds to a hydroxylation process at the ortho-position of the methoxy group on the naphthalene ring and the methyl group on the propanoic acid to give TP 265. TP 216 was also likely further metabolized via conjugation with amino acid, yielding a series of tyrosine conjugated metabolites (e.g., TP 341, TP 353, and TP369). A transformation product 6methoxy-1-(1-(6-methoxynaphthalen-2-yl)ethyl)naphthalen-2-ol was found previously (Isidori et al., 2005). It may undergo subsequent hydroxylation to yield TP 333. However, 6-methoxy-1-(1-(6-methoxynaphthalen-2-yl)ethyl)naphthalen-2-ol was likely a transient intermediates that quickly underwent further transformations as it was not detected during the incubation. In addition, the hydroxylation of NPX could be also combined with glucuronide compounds, leading to the formation of TP 441, TP 423, and TP 431. Brozinski et al. (2011) reported that glucuronides-like compounds were found to be the main metabolites of NPX in fish. The metabolism pathway of NPX in S. quadricauda was similar to that in Cymbella sp. as most of metabolites were detected in both algae (Figure S14b). In addition, TP 339 was a tentatively transient intermediate for TP 341. TP 323 emerged only on day 4, suggesting it may be rapidly transformed. Given that tyrosine- or glucuronide-like compounds could activate the detoxification processes in algae (Xu et al., 2013; Pietrini et al., 2015), the conjugation products of glucuronide or tyrosine compounds with NPX may be included in the detoxification mechanism of NPX in two algae.

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4. Conclusions The biochemical characteristics including chlorophyll a, carotenoid contents and enzyme activities show that Cymbella sp. and S. quadricauda can be affected by NPX. This inhibitory effect was concentration- and time-dependent. Cymbella sp. showed a more satisfactive effect in bioremediation of NPX than S. quadricauda with higher removal efficiency and bioaccumulation of NPX. Twelve metabolites were identified in algal cells and the degradation pathways were proposed. The conjugation products (e.g., glucuronide or tyrosine compounds with NPX) may be included in the detoxification mechanism of NPX. The potential effects of other NPX metabolites in aquatic environment should be further investigated. Acknowledgements This research was financially supported by the National Natural Science Foundation of China (Grants Nos. 21407108 and 21607106), the Shenzhen Science and Technology Project (Grant Nos. KQJSCX20160226200315 and ZDSYS201606061530079), the Science and Technology Planning Project of Guangdong Province, China (Grant Nos. 2014A020216020 and 2012B031000028), and the Natural Science Foundation of SZU (Grant Nos. 827-000077 and 201444). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at…. Reference [1] Arriaga, S., De Jonge, N., Nielsen, M.L., Andersen, H.R., Borregaard, V., Jewel,

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Table 1 LC-MS/MS data for the identification of naproxen and its metabolites in two algae Retention time (min)

ESI(-)MS2 (m/z)

230

4.6

229>185>170

TP 341

341

1.4

341>179>161>119>89>59

2-(1-hydroxy)-6-O-desmethylnaproxen

TP 220

220

1.6

218>202>167

6-O-desmethylnaproxen

TP 216

216

1.9

216>170>130>112

6-(2-(6,7-dimethoxynaphthalen-2-yl)-1hydroxypropoxy)-3,4,5,6tetrahydroxyhexanoic acid

TP 441

441

2.6

440>237>139>97

3,4,5-trihydroxy-6-((2-(7-hydroxy-6methoxynaphthalen-2yl)propanoyl)oxy)tetrahydro-2H-pyran-2carboxylic acid

TP 423

423

4.3

422>381>283>139

Compound

Product

Mw

Naproxen

----

6-(2-amino-1-hydroxy-3-(4hydroxyphenyl)propoxy)naphthalene-2, 3diol

Chemical structure

24

6-((2-(6,7-dimethoxynaphthalen-2yl)propanoyl)oxy)-3,4,5-trioxotetrahydro2H-pyran-2-carboxylic acid

TP 431

431

5.1

430>385>355>205>149

2-(7-hydroxy-6-methoxynaphthalen-2yl)propane-1,1,3-triol

TP 265

265

5.5

262>217>97> 80

6,7-dihydroxynaphthalen-2-yl 2-amino-3(4-hydroxyphenyl)propanoate

TP 339

339

2-(6-((carboxycarbonyl)oxy)-7hydroxynaphthalen-2-yl)-3hydroxypropanoic acid

TP 323

323

5.8

320>305>261>217>165>1 09

6-hydroxy-7-methoxynaphthalen-2-yl 2amino-3-(4-hydroxyphenyl)propanoate

TP 353

353

6.8

353>177>163

1-(hydroxy(6-hydroxynaphthalen-2yl)methyl)naphthalene-2,6-diol

TP 333

333

6.9

332>275>233>175

6-hydroxy-7-methoxynaphthalen-2-yl 2amino-3-(4-hydroxyphenyl)-3-(4hydroxyphenyl)propanoate

TP 369

369

7.4

367>337>193>163

5.6

25

339>163

Figure captions: Figure 1 The inhibition rate of Cymbella sp. (a) and S. quadricauda (b) at different NPX concentrations. Different letters above adjacent bars indicate a significant difference (p<0.05) between the treatments, whereas the same letter indicates no significant difference. Figure 2 Effect of NPX on the chlorophyll, carotenoid content and pigment ratios of Cymbella sp. (a) and S. quadricauda (b) after 96 h exposure. Error bars represent the standard error of the mean (n=3). Columns with asterisk * and ** indicate significant (p<0.05) and very significant (p<0.01) differences between the control and treatment, respectively. Figure 3 Effect of NPX on the SOD and CAT activity in two algal cells (a) at 96 h exposure, as well as MDA levels in two algae (b). Error bars represent the standard error of the mean (n=3). Columns with asterisk *, **, and *** indicate significant (p<0.05), very significant (p<0.01) and extremely significant (p<0.001) differences between the control and treatment, respectively. Figure 4 Total removal of NPX in pure medium, Cymbella sp. and S. quadricauda at different concentrations. Error bars represent the standard error of the mean (n=3). Columns with asterisk * indicate significantdifferences between the control and treatment (p<0.05). Figure 5 The cellular accumulation of NPX by Cymbella sp. and S. quadricauda at different incubation time (a), and different NPX concentrations (b), as well as the bioconcentration factors at different incubation time (c). Error bars represent the

26

standard error of the mean (n=3).

27

Figure 1

28

Figure 2

29

Figure 3

Figure 4

30

Figure 5 Highlights:


100% degradation of 100 ppm naproxen obtained by Cymbella sp. and S. quadricauda




Cymbella sp. showed a more satisfactive effect in the bioremediation of naproxen




Naproxen affected biochemical characteristics of both algae




Naproxen degradation mechanisms are proposed based on 12 identified metabolites

31

33