Environmental Pollution 258 (2020) 113691

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Environmental Pollution

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Microplastics impair digestive performance but show little effects on antioxidant activity in under low pH conditions*

Xinghuo Wang a, b, c, 1, Wei Huang b, d, 1, Shuaishuai Wei a, c, Yueyong Shang a, c, Huaxin Gu a, b, c, Fangzhu Wu b, d, Zhaohui Lan f, Menghong Hu a, c, Huahong Shi e, * Youji Wang a, b, c, d, a International Research Center for Marine Biosciences at Shanghai University, Ministry of Science and Technology, b Key Laboratory of Satellite Ocean Environment Dynamics, Second Institute of Oceanography, Ministry of Natural Resources, Hangzhou, China c Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, 201306, China d Laboratory of Marine Ecosystem and Biogeochemistry, Second Institute of Oceanography, Ministry of Natural Resources, Hangzhou, China e State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai, 200062, China f Department of Neurology, University of California Davis, USA article info abstract

Article history: In the marine environment, microplastic contamination and acidification may occur simultaneously, this Received 25 September 2019 study evaluated the effects of ocean acidification and microplastics on oxidative stress responses and Received in revised form digestive enzymes in mussels. The thick shell mussels coruscus were exposed to four concen- 27 November 2019 trations of polystyrene microspheres (diameter 2 mm, 0, 10, 104 and 106 particles/L) under two pH levels Accepted 28 November 2019 (7.7 and 8.1) for 14 days followed by a 7-day recovery acclimation. Throughout the experiment, we found Available online 29 November 2019 that microplastics and ocean acidification exerted little oxidative stress to the digestive gland. Only catalase (CAT) and glutathione (GSH) showed a significant increase along with increased microplastics Keywords: Microplastics during the experiment, but recovered to the control levels once these stressors were removed. No sig- fi Acidification ni cant effects of pH and microplastics on glutathione peroxidase (GPx) and superoxide dismutase (SOD) Oxidative stress were observed. The responses of digestive enzymes to both stressors were more pronounced than Digestive enzyme antioxidant enzymes. During the experiment, pepsin (PES), trypsin (TRS), alpha-amylase (AMS) and lipase (LPS) were significantly inhibited under microplastics exposure and this inhibition was aggravated by acidification conditions. Only PES and AMS tended to recover during the recovery period. Lysozyme (LZM) increased significantly under microplastic exposure conditions, but acidification did not exacer- bate this effect. Therefore, combined stress of microplastics and ocean acidification slightly impacts oxidative responses but significantly inhibits digestive enzymes in mussels. © 2019 Elsevier Ltd. All rights reserved.

1. Introduction (Wright et al., 2013a; Wright et al., 2013b), especially in filter- feeding bivalves which represent an important element in coastal There is an increasing concern about the ecological effects of systems (Cole et al., 2013; Wright et al., 2013b; Gardon et al., 2018). microplastics (MPs), which are defined as plastic particles with size Moreover, Browne et al. (2008) found that microplastics (9.6 mm) below 5 mm in marine environments (Thompson et al., 2009). Due could be translocated into the circulatory system of mussels. The to their small size, MPs are reported to be ingested and accumu- biological effects of MPs on bivalves have been reported in various lated in numerous marine from invertebrates to fish aspects, including disturbed energy metabolism (Gardon et al., 2018), reduced anti-oxidative capacity, inhibited immune defense (Canesi et al., 2015), induced genotoxicity and neurotoxicity, and * This paper has been recommended for acceptance by Eddy Y. Zeng. histological damage (von Moos et al., 2012; Ribeiro et al., 2017). * Corresponding author. International Research Center for Marine Biosciences at Except for the emergent MPs contaminants, marine ecosystems Shanghai Ocean University, Ministry of Science and Technology, China. have also been impacted by other global threats such as ocean E-mail address: [email protected] (Y. Wang). acidification (OA). OA primarily is caused by elevated atmospheric 1 These authors contributed equally to this work. https://doi.org/10.1016/j.envpol.2019.113691 0269-7491/© 2019 Elsevier Ltd. All rights reserved. 2 X. Wang et al. / Environmental Pollution 258 (2020) 113691 carbon dioxide concentrations (pCO2), and has attracted extensive microplastics have been reported, and these contaminants may attentions in the past decade (Fabry et al., 2008; Kroeker et al., affect the physiology of marine (Ashton et al., 2010; 2010). Compared to pre-industrial times, alterations in seawater Holmes et al., 2012, 2014; Gandara et al., 2016; Nobre et al., 2015). carbonate chemistry have caused a decrease in average oceanic Thus, to reduce noises and ensure the exact effects of the target surface water (pH 8.1) by 0.1 pH units (Intergovernmental Panel on type of microplastics, the natural particles from the environment Climate Change IPCC, 2014). Moreover, experts have predicted that were not studied in this study. by the end of the century, the average ocean pH will decline further to 7.7e7.8 (Caldeira and Wickett, 2003; Feely et al., 2009; 2. Materials and methods Intergovernmental Panel on Climate Change IPCC, 2014). It is well established that OA can impair performances of marine bivalves 2.1. Experimental mussels through affecting growth, metabolism, reproduction and calcifica- tion (Kroeker et al., 2010), implying that OA will have a profound The thick shell mussels M. coruscus (dry weight 1.5 ± 0.90 g; impact on future marine ecosystems (Nagelkerken and Connell, shell length 7.95 ± 0.32 cm) used in this experiment were sampled 2015). from the Shengsi island of China. Before experimentation, they The thick shell mussels Mytilus coruscus is an economically were kept in aquarium for 2 weeks under controlled conditions important marine bivalve, mainly distributed in waters around the (temperature: 25 ± 0.4 C; pH: 8.1 ± 0.05; salinity: 25 ± 0.8 psm; East China Sea, where a large pH fluctuation in summer can be photoperiod (12:12 h)) and fed with Chlorella vulgaris (concentra- found (Wei et al., 2017) and is predicted to be susceptible to OA tion: 5 104 cells/ml) every day. because of the combined impacts of augmentation of atmospheric CO2 and eutrophication (Chou et al., 2013; Lui et al., 2015). At the 2.2. Experimental design and system same time, the occurrence of MPs has been simultaneously re- ported in the surface water, seafloor sediment as well as the To determine the interactive effects of MPs and OA, four con- farming bivalves of the Yangtze estuary system (Zhao et al., 2014; centrations (0, 10, 104 and 106 particles/L) of 2 mm polystyrene Peng et al., 2017). However, no information is available on the microspheres (micro-PS) and two pH levels (8.1 and 7.7) were 9 interactive effects of MPs and CO2-induced pH reduction posed for selected. The micro-PS (5.69 10 particles/ml, contains 0.05% mussels. sodium azide, 2.5% w/v, 10 mL) used in this experiment were pur- Marine bivalves are a vulnerable group which are more likely to chased from Tianjin BaseLine ChromTech Research Centre, Tianjin, uptake small particles including MPs and then accumulate them in China. The MPs were validated with a micro-Fourier Transform their tissues (Newell, 2004; Li et al., 2015b, 2016; Cho et al., 2019). A Infrared (m-FT-IR) spectroscope (Nicolet iN10 MX, Thermo-Fisher higher accumulation of MPs in the digestive gland than gill was Scientific). The surface morphology and particle size of the micro- observed in different bivalves (e.g., Mytilus trossulus, Macoma PS at different pH conditions were analyzed by scanning electron balthica)(Setala et al., 2016). Seawater acidification can promote microscope (SEM, Hitachi JSM-7500F). The particle distribution of the accumulation of some metals in mussel tissues, among which the micro-PS under different pH conditions was analyzed by Mul- the digestive gland is the main target organ (Cao et al., 2018). In tisizer 3 (Beckman, Irvine, CA, United States) daily, MPs concen- addition, relevant studies have shown that the digestive gland is trations in each treatment were stable every day, and no difference also the main organ for the accumulation of microplastics (Faggio of MPs behaviour was observed between the two pH treatments. et al., 2018). It is very necessary to study the effect of micro- Polystyrene as one of the most commonly used plastic polymers plastics on the digestive gland of mussels, especially under the worldwide is often found in marine ecosystems. Low concentration acidic conditions which may occur in their habitats, e.g., the (10 particles/L) of micro-PS was chosen in accordance with the MPs Yangtze River estuary. The thick shell mussels are important eco- concentrations observed from the Yangtze River estuary offshore nomic mussels along the and East China Sea in China (Zhao et al., 2014). In addition, medium (104 particles/L) and high (Liu et al., 2015). One of the largest mussel farm areas in China is the (106 particles/L) concentrations were chosen according to previous Shengsi Islands (Liao et al., 2013). The mussels there may experi- reported toxicological effects of MPs on marine organism (Magni ence fluctuations in pH conditions during rainy season (Sui et al., et al., 2018; Capolupo et al., 2018). The current seawater pH 8.1 2015) and also encounter heavy MPs pollution, and the concen- was set as the control pH and pH 7.7 was set as low pH because it tration of microplastics can reach up to 900e4000 particles/m3 in can occur in the Shengsi island in summer (Li et al., 2015a) and also some areas of the Yangtze Estuary and East China Sea (Luo et al., is predicted to reach for surface seawater by 2100 (Feely et al., 2019; Zhao et al., 2014). Moreover, the microplastics pollution in 2009; Intergovernmental Panel on Climate Change IPCC, 2014). the rainy season is more serious in estuary waters (Cheung et al., After the acclimatization, the mussels were randomly assigned 2016). We hypothesize that the degree of stress effect may be to eight treatment groups (2 pH values 4 concentrations of micro- enhanced when mussels were exposed to MPs pollution under PS). Each treatment contained three 30-L tanks (twenty mussels acidification conditions. Therefore, it is of great necessity to explore per tank) as three replicates. Diluting the stock aqueous suspension the potential interactive effects of MPs under ocean acidification (5.69 109 particles/ml) to gain micro-PS concentrations required conditions in marine bivalves. for this experiment. Half of the seawater was changed daily for each To explore the combined effects of ocean acidification and MPs tank (15 L), while adding micro-PS to maintain concentration to on oxidative stress responses (catalase (CAT), superoxide dismutase meet the exposure test requirements. Chlorella spp. (5 104 cells/ (SOD), glutathione peroxidase (GPx) and glutathione (GSH)), ml) was fed to mussels daily. The MPs concentration was counted digestive enzymes (pepsin (PES), trypsin (TRS), alpha-amylase by Multisizer 3. Pure CO2 was applied to set pH which was (AMS) and lipase (LPS)) and lysozyme (LZM) in mussels, Mussels controlled by the pCO2/pH system (DAQ-M, 4 Channel, Tjele, were exposed to two levels of pH (7.7 and 8.1) and four concen- Denmark) (Kong et al., 2019a). Salinity, pH, total alkalinity (AT), 4 6 trations of 2 mm polystyrene microspheres (0, 10, 10 and 10 par- dissolved inorganic carbon (DIC), pCO2, calcite saturation state ticles/L) for 21 days followed by 7 days recovery acclimation. (Ucal) and aragonite saturation state (Uara) was measured based on Cheung and Shin (2005) found that natural particles with the method of Huang et al. (2018). different size can have negative impacts on mussels. In addition, A 14-day exposure experiment was performed under above recently some contaminants carried or released by environmental conditions. Then the seawater in all tanks was completely changed X. Wang et al. / Environmental Pollution 258 (2020) 113691 3 and subjected to a 7-day recovery experiment under normal con- principal component analysis (PCA) was carried out for all ditions (pH 8.1 0 particles/L). Sampling of the digestive gland was biochemical parameters using XLSTAT® 2014, and a double plot performed on day 1, 7, 14, and 21 during the experiment. was drawn using the parameters and observations. The results were presented as means ± SD, and P < 0.05 was considered as a 2.3. Digestive gland sampling significant difference between the treatments.

At each sampling point, three mussels were randomly sampled 3. Results from one tank for subsequent assays of biochemical parameters. To reduce individual differences, three digestive glands from one tank 3.1. Characteristics of MPs were pooled as a replicate per treatment and placed in cryotubes. Then, samples were immediately placed in liquid nitrogen. After all According to m-FT-IR analysis, the spectrum match of the sampling work was completed, tissues were later homogenized in microplastics was 94% (Fig. 1A). The surface morphology of the MPs buffer containing 0.1 M Tris-HCl (pH 7.5). The sample was ground at different pH conditions were analyzed by scanning electron with a Potter-Elvehjem tissue grinder, then the homogenate sample microscope (SEM, Hitachi JSM-7500F) (Fig. 1B and C). The particle was treated with a Braun Labsonic Usonifier at 0 C. After centri- size of the MPs was 2.31 ± 0.02 and 2.30 ± 0.03 at pH 8.1 and pH 7.7 fugation at 10,000 g for 25 min (4 C), collecting the supernatant based on SEM (n ¼ 10), respectively. The particle distribution of the was collected for subsequent biochemical measurements. MPs under different pH conditions was similar, and the distribution was concentrated at 2.28 ± 0.04 and 2.30 ± 0.05 at pH 8.1 and pH 2.4. Biochemical measurements 7.7 based on Multisizer 3 (n ¼ 21), respectively (Fig. 1D). There was no significant difference of particle diameter between the two pH The biochemical parameters were measured with commercially treatments (P < 0.05). available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). A microplate reader (Flexstation® 3, Molecular Devices, 3.2. Seawater chemistry California, USA) was used to measure the optical density values, and the total protein (TP) content of the crude extract was determined Seawater carbonate chemistry (salinity: 25.1 ± 0.4 psm; tem- using the Coomassie Brilliant Blue (G-250) method (Bradford, perature: 25.0 ± 0.3 C; AT: 2212e2434 mmol/kg) for each treat- 1976). ment was listed in Supplementary Table 1. No experimental animals died during the experiment. 2.4.1. Antioxidant enzyme activities According to Beauchamp and Fridovich (1971), the nitro blue 3.3. Antioxidant enzyme activities tetrazolium method was applied to estimate the SOD activity. The CAT activity was determined according to the method of Goth There was no significant difference in SOD activity among all (1991). The levels of GSH activeity was measured based on the treated groups (Table S2 and Fig. 2A). Throughout the experiment, method of Ringwood et al. (1999). The GPx activity was measured no significant interaction was observed between pH and micro-PS by the method of Lawrence and Burk (1976). U/mg protein was used in CAT, which was significantly affected by micro-PS at day 1 and to express SOD, CAT and GPx activities, and mmol/g protein was day 7 (Table S2). During the exposure period, the mussels exposed used to express GSH level. to high concentration of micro-PS (106 particles/L) had significantly higher CAT activity than low (10 particles/L) and control (0 parti- 2.4.2. Digestive enzyme activities cles/L) groups under pH 7.7 conditions (Fig. 2B). During recovery The PES activity was measured based on the method of period, there were no significant differences among all groups Rungruangsak and Utne (1981). The TRS activity was determined (Fig. 2B). For GSH, pH showed significant effect at day 7, and micro- according to the method of Chao et al. (2018). The AMS activity was PS showed significant effect during the whole exposure period. measured according to the method of Xiao et al. (2006). The levels Interaction between pH and micro-PS was observed at day 1, day 14 of LPS activity was measured by the method of Graca et al. (2005). and day 21 (Table S2). During the exposure period, at day 1, the GSH U/mg protein was used to express all the digestive enzyme activity in the medium and high concentration micro-PS (104 par- activities. ticles/L, 106 particles/L) were significantly increased under pH 7.7 conditions (Fig. 2C). High concentration micro-PS (106 particles/L) 2.4.3. Lysozyme (LZM) activity significantly promoted GSH activity when individuals were According to Kong et al. (2019b), the enzyme activity was exposed to pH 7.7 at day 7 and pH 8.1 at day 14 (Fig. 2C). During determined by a reaction between the bacterial fluid (Micrococcus recovery period, there were no significant differences among all lysodeik) and samples. The LZM activity was expressed as U/mg groups (Fig. 2C). As for GPx activity, pH showed significant effect at protein. day 1, and micro-PS showed no significant effect throughout the experiments, but there was no significant interaction between pH 2.5. Statistical analysis and micro-PS (Table S2). There was no significant difference in GPx activity among all treated groups (Fig. 2D). All data were processed through SPSS software. Before analysis, the Levene’s test was used to test the homogeneity of the variance 3.4. Digestive enzyme activities and the Shapiro-Wilk’s test was used to test the normality of the data. The influences of pH, MPs and their interactive effects were PES activity was significantly affected by pH and micro-PS at day analyzed by two-way analysis of variance (ANOVA). Then the ef- 7 and 14, but there was no significant interaction between pH and fects of MPs at each fixed pH value were determined through one- micro-PS (Table S3). During the exposure period, at day 7 and 14, way ANOVA followed by Tukey’s HSD tests. Student’s t-test was the PES activity in the medium and high concentration micro-PS carried out for analyzing the significant effects of different pH on (104 particles/L, 106 particles/L) were significantly reduced at both the biochemical parameters at a fixed MPs concentration and the pH conditions (Fig. 3A). At day 7 and 14, pH 7.7 significantly significant effects of different pH on the particle size. Finally, inhibited PES activity under four concentrations of micro-PS 4 X. Wang et al. / Environmental Pollution 258 (2020) 113691

Fig. 1. Micro-Fourier Transform Infrared (m-FT-IR) spectroscopy of micro-PS (A); Scanning electron microscope (SEM) image of micro-PS at pH 8.1 (B) and pH 7.7 (C); Particle size analysis of micro-PS at pH 8.1 and pH 7.7 (D).

(Fig. 3A). During recovery period, there were no significant differ- conditions (Fig. 3B). At day 7 and 14, pH 7.7 significantly inhibited ences among all groups (Fig. 3A). Regarding TRS activity, it was TRS activity under four concentrations of micro-PS (Fig. 3B). During significantly affected by pH and micro-PS from day 7e21, and there the recovery period, the TRS activity in the medium and high was a significant interaction between pH and micro-PS at day 21 concentration micro-PS (104 particles/L, 106 particles/L) were (Table S3). During the exposure period, at day 7 and 14, the TRS significantly reduced at both pH conditions, and pH 7.7 significantly activities in the medium and high concentration micro-PS (104 inhibited the TRS activity only in the treatment without micro-PS particles/L, 106 particles/L) were significantly reduced at both pH (Fig. 3B). About AMS activity, it was significantly affected by pH X. Wang et al. / Environmental Pollution 258 (2020) 113691 5

Fig. 2. SOD activity (A), CAT activity (B), GSH activity (C) and GPx activity (D) in the mussel M. couruscus digestive gland during 21 days’ exposure to different pH (8.1, 7.7) and micro- PS concentrations (0, 10, 104 and 106 particles/L). Different lowercase letters represent significant difference between different micro-PS concentrations under the same pH con- ditions (P < 0.05). * indicates significant differences between different pH under the same micro-PS concentration (P < 0.05).

Fig. 3. PES activity (A), TRS activity (B), AMS activity (C) and LPS activity (D) in the mussel M. couruscus digestive gland during 21 days’ exposure to different pH (8.1, 7.7) and micro- PS concentrations (0, 10, 104 and 106 particles/L). Different lowercase letters represent significant difference between different micro-PS concentrations under the same pH con- ditions (P < 0.05). * indicates significant differences between different pH under the same micro-PS concentration (P < 0.05). and micro-PS at day 7 and day 14, but there was no significant and high concentration micro-PS (104 particles/L, 106 particles/L) interaction between pH and micro-PS (Table S3). During the were significantly reduced under each pH (Fig. 3C). Under the exposure period, at day 7 and 14, the AMS activity in the medium medium and high concentration micro-PS (104 particles/L, 106 6 X. Wang et al. / Environmental Pollution 258 (2020) 113691

Fig. 4. LZM activity in the mussel M. couruscus digestive gland during 21 days’ Fig. 5. Biplot originating from principal component analysis (PCA) integrating all exposure to different pH (8.1, 7.7) and micro-PS concentrations (0, 10, 104 and 106 measured variables (SOD, CAT, GSH, GPx, AMS, PES, TRS, LPS, LZM) and four time points particles/L). Different lowercase letters represent significant difference between at eight different treatments (▪epH 8.1 0 particles/L, ,epH 7.7 0 particles/L, different micro-PS concentrations under the same pH conditions (P < 0.05). AepH 8.1 10 particles/L, ⋄epH 7.7 10 particles/L, :epH 8.1 104 particles/L, △epH 7.7 104 particles/L, CepH 8.1 106 particles/L, BepH 7.7 106 particles/L). Both the loadings of the variables (C) and the scores of the experimental conditions particles/L), pH 7.7 significantly inhibited AMS activity at day 7 and were displayed. 14 (Fig. 3C). During recovery period, there were no significant dif- ferences among all groups (Fig. 3C). As for LPS activity, it was significantly affected by pH and micro-PS from day 7e21, and there exposure (Revel et al., 2019). Magara et al. (2018) found that CAT in was a significant interaction between pH and micro-PS during the gills and digestive gland of Mytilus edulis increased significantly exposure (Table S3). During the exposure period, at day 7 and 14, under microplastics exposure, but SOD had no significant effect, the LPS activity in the medium and high concentration micro-PS and it is speculated that superoxide anion production may not be 4 6 (10 particles/L, 10 particles/L) was significantly reduced the main factor of oxidative damage. During the experiment, only (Fig. 3D). And pH 7.7 significantly inhibited LPS activity when in- GSH and CAT showed a significant response to microplastics 4 dividuals were exposed to 10 particles/L and 10 particles/L micro- exposure. Probably GSH and CAT are more sensitive to micro- 4 6 PS at day 7 and exposed to 10 particles/L and 10 particles/L micro- plastics than SOD and GPX. Magni et al. (2018) studied the effect of PS at day 14 (Fig. 3D). During recovery period, the LPS activity in the microplastics on freshwater zebra mussels and found that mixed 4 6 medium and high concentration micro-PS (10 particles/L, 10 exposure of 1 and 10 mm polystyrene microplastics significantly particles/L) were significantly reduced at both pH conditions, and affected CAT activity in soft tissue of Dreissena polymorpha, but pH 7.7 significantly inhibited the LPS activity under four concen- there was no significant difference in SOD activity. Similar to our trations of micro-PS (Fig. 3D). study, Browne et al. (2008) showed that microplastic exposure did not affect antioxidant system as well as the viability and phagocytic 3.5. Lysozyme (LZM) activities activity of hemocytes of M. edulis. Moreover, Santana et al. (2018) found that microplastic exposure had no significant effect in LZM activity was significantly affected by pH at day 7 and micro- physiological functions, biomarkers and health condition of the PS at day 14 (Table S4). During the exposure period, at pH 8.1, a green mussel Perna perna. At the molecular level, Detree and significant increased LZM occurred in the high concentration Gallardo-Escarate (2017) found that the expression of CAT gene in 6 micro-PS (10 particles/L) group at day 14 (Fig. 4). During recovery the digestive gland of Mytilus galloprovincialis was significantly period, there were no significant differences among all groups upregulated under exposure to polystyrene microplastics. Based on (Fig. 4). the above information, generally microplastics are not effective drivers for the oxidative stress in mussels. From the trend of GSH on 3.6. Principal component analysis day 14, acidification significantly increased the negative impact of high concentration microplastics on mussels. It may be due to the PCA of the effects of micro-PS and pH on oxidative stress limited ability of GSH to regulate pressure, and low pH exposure the response and digestive enzymes during the experiment showed 14 days has exceeded the regulatory limit of GSH. In the study of that the two principal components accounted for 71.07% of the total Huang et al. (2018), it was also found that acidification can signif- composition. 54.76% of the total variance was accounted by PC1, icantly affect the antioxidant system of M. coruscus gland. At day 14, this axis represents the specific reaction of micro-PS treatment, SOD showed a downward trend under micro-plastic exposure which separates control group and medium/high concentration conditions, and CAT showed an upward trend. We speculate that micro-PS groups. PC2 representing 16.31% of total variance, this SOD was no longer able to maintain a balance between pro- axis represents the specific reaction of time, which separate day 1 oxidation and anti-oxidation in the cell, and CAT was needed to and day 7 (Fig. 5). continue this balance. Although no significant effects of pH 7.7 and microplastics on SOD and GPx were observed, microplastics ex- 4. Discussion posures has caused damage to the antioxidant defense of M. couruscus from the results of CAT and GSH. Moreover, acidifi- In this study, microplastics exerted a slight impact on the anti- cation increased the oxidative damage of the body exposed to oxidant system of the thick shell mussels. It was found that the SOD micro-PS environments. and CAT activities of the digestive gland of blue mussels Mytilus spp. As for digestive enzyme activity, compared with the other Increased significantly under the conditions of microplastics digestive enzymes (PES, AMS, LPS), TRS has the highest activity, so X. Wang et al. / Environmental Pollution 258 (2020) 113691 7 it may take a major place in the digestive function of thick-shell enzymes than antioxidant enzymes. Although the negative effects mussels. For microplastics exposure, environmentally- on certain digestive enzymes showed a reversible trend, more concentrated microplastics did not affect TRS activity, but high- biochemical indicators have not recovered. concentration microplastics continued to inhibit TRS activity throughout the experiment. Green et al. (2017) reported that Declaration of competing interest microplastics exposure significantly impaired the filtration of M. edulis. The results of this study support this phenomenon, The authors declare that they have no known competing probably because microplastics exposure disrupts the homeostasis financial interests or personal relationships that could have of digestive enzymes in the digestive gland, resulting in a reduction appeared to influence the work reported in this paper. in filtration. Similarly, Capolupo et al. (2018) investigated the effect of 3 mm polystyrene microplastics on the early developmental stage CRediT authorship contribution statement of M. galloprovincialis. It was found that the ingested microplastics retained in larva gut for up to 192 h, which had a physical impact on Xinghuo Wang: Methodology, Investigation, Writing - original digestive function. Moreover, Paul-Pont et al. (2016) observed his- draft. Wei Huang: Conceptualization, Methodology, Resources. topathological damage in the digestive glands of Mytilus spp. after 7 Shuaishuai Wei: Software, Formal analysis. Yueyong Shang: days of polystyrene microplastics exposure. At pH 7.7 conditions, Investigation, Writing - review & editing. Huaxin Gu: Investigation. TRS activity was significantly inhibited. Moreover, acidification Fangzhu Wu: Formal analysis. Zhaohui Lan: Software. Menghong significantly enhanced the negative impact of microplastics on TRS. Hu: Project administration. Huahong Shi: Writing - review & Similar studies have shown that low pH significantly reduced the editing. Youji Wang: Conceptualization, Resources, Supervision. feeding rate of juvenile clams (Jose Fernandez-Reiriz et al., 2011). However, at day 14, there was an upward trend, presumably due to Acknowledgements the adaptive mechanism of M. couruscus under acidification con- ditions. PES, AMS and LPS were significantly impacted by micro- This work was supported by the Open Research Fund of State plastics throughout the experiment, and acidification significantly Key Laboratory of Estuarine and Coastal Research. (Grant number aggravated this negative effect. However, PES and AMS tended to SKLEC-KF201706). The authors acknowledge funding from the recover during the recovery period, indicating that the negative Natural Science Foundation of China (31872587), the Shanghai combined effects of acidification and microplastics composite Pujiang Talent Program (18PJ1404000), Shanghai Municipal Natural stress on PES and AMS may be reversible. In mollusks, LZM not only Science Foundation (17ZR1412900), State Key Laboratory of Satel- has digestive capacity, but also has a sterilizing role (Nilsen et al., lite Ocean Environment Dynamics (No. SOEDZZ1902) and China- 2003; Nilsen et al., 1999). At day 7, LZM activity increased signifi- APEC Cooperation Fund (No. 2029901). This work was also sup- cantly at high concentrations of microplastics and acidification ported by Guangdong South China Sea Key Laboratory of Aqua- conditions. Canesi et al. (2015) studied the immunomodulation of culture for Aquatic Economic Animals, Guangdong Ocean hemocytes of M. galloprovincialis exposed to cationic polystyrene University (KFKT2019ZD04) and the Open Fund of Shandong Key nanoparticles and also found a significant upregulated of LZM ac- Laboratory of Disease Control in Mariculture (KF201802). tivity. However, at day 14, the LZM activity decreased under the combined stress conditions. It is speculated that the external stress Appendix A. Supplementary data has exceeded the LZM regulation ability. In this study, the use of polystyrene microplastics produced a Supplementary data to this article can be found online at significant negative effect on the digestive function of mussels. https://doi.org/10.1016/j.envpol.2019.113691. 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