|Microplastics (MPs) have become an emerging new pollutant in the marine environment. MPs have attracted remarkable attention due to their toxic effects on marine organisms and potential ecological risks. At present, most of the studies collected mollusk samples only once. Seasonal investigations of MPs pollution levels in mollusks are relatively few, and MPs uptake characteristics in mollusks have not been studied systematically. Previous studies showed that the toxicity of MPs on marine organisms is also related to MPs characteristics (e.g. particle size, shape, and polymer type). Nevertheless, few studies have investigated the stress effects of MPs size on marine organisms in combined exposure experiments. As coastal environments are complex and changeable, MPs and environmental factors may cause combined stress effects on marine organisms. However, relatively few studies have examined combined stress effects of MPs and environmental factors on marine organisms, and the mechanism of the combined stress effects is still unclear. Therefore, in this study, the seasonal MPs abundance characteristics of C. gigas from five aquaculture farms in Shandong Peninsula were investigated. In addition, we detected MPs abundance and environmental factors (temperature, pH, salinity, and nutrient salts) in surface seawater around oysters in different seasons to reveal the comprehensive influence of actual environmental conditions on the seasonal MPs abundance of C. gigas. Moreover, the combined exposure experiments of environmental factors (temperature, pH, and salinity) and MPs to C. gigas were conducted. Specifically, we investigated the combined effects of polystyrene MPs of two sizes (small polystyrene microplastics, SPS-MPs: 6 μm; large polystyrene microplastics, LPS-MPs: 50~60 μm) and environmental factors (temperature, pH, and salinity) to C. gigas at the physiological, biochemical and molecular levels. The combined stress effects mechanism was revealed based on the integrated biomarker response index (IBR) and Principal Component Analysis (PCA). The influential mechanisms of different MPs sizes on the immunity, oxidative stress, and energy metabolism of oysters under combined exposure conditions were also investigated. In addition, untargeted metabolomics techniques were used to elucidate the combined stress effects of temperature, pH, salinity, and SPS-MPs on C. gigas. The results were summarized as follows:
（1）Field investigation revealed the seasonal characteristics and influencing factors of MPs abundance in C. gigas. In the present study, the ingestion rate of MPs in C. gigas was the highest in autumn and the lowest in spring, and the MPs ingestion rate in C. gigas at different sites was the highest at the Yantai site and the lowest at the Laizhou site. According to the statistics on MPs abundance of C. gigas at all sites in different seasons, the MPs abundance of C. gigas ranged from 2.40 (winter) to 3.28 (autumn) items/individual, or from 0.22 (spring) to 0.45 (summer) items/g (ww). According to the statistics on MPs abundance of C. gigas at different sites, the MPs abundances of C. gigas were relatively lower at the Changdao and Rongcheng sites when expressed as items/individual and MPs abundance of C. gigas was highest at the Changdao site and lowest at the Laizhou site when expressed as items/g (ww). In oysters, the dominant type was cellophane (CP), followed by polyethylene terephthalate (PET), polyacrylonitrile (PAN), nylon (PA), and polyethylene/polypropylene (PE/PP); the main shape of MPs was fiber, followed by fragment and film; the most abundant size class of MPs was 0.5~1 mm. In surface seawater, the abundance of MPs was significantly lower in spring than in summer and autumn (p < 0.05) and was relatively lower at the Changdao and Rongcheng sites. In surface seawater, the dominant type was PET, followed by CP, PE, PA, PP, and PE/PP; the main shape of MPs was fiber, followed by fragment and film; the most abundant size class of MPs was 0.5~1 mm. Redundancy (RDA) analysis showed that NH4-N and SiO3-Si were the main environmental factors affecting MPs abundance of oysters and surface seawater in summer and winter, respectively. Spearman correlation analysis showed that the MPs abundance of C. gigas in different seasons was significantly positively correlated with environmental factors (temperature and NO2-N) when expressed as items/individual (p < 0.05). From the above, MPs characteristics in oysters are related to MPs characteristics in surface seawater.
（2）The combined stress effects of low salinity and polystyrene MPs of different sizes on C. gigas were investigated. The results showed that low salinity exposure decreased the MPs accumulation in C. gigas, inhibited the expression of Hexokinase (HK) and Phosphoenolpyruvate carboxylase (PEPCK) genes in digestive glands, induced the expression of Pyruvate kinase (PK) genes in gills, and increased Superoxide dismutase (SOD) and Glutathione S-transferase (GST) activity in digestive glands and gills; low salinity (21 psu) exposure reduced Malondialdehyde (MDA) levels in digestive glands and gills, and increased the apoptosis rate of hemolymph. MPs can induce the apoptosis rate of hemolymph, and can induce SOD and GST activity in digestive glands; SPS-MPs can increase MDA levels in digestive glands compared with LPS-MPs. The combined effects of MPs and low salinity were mostly antagonistic effects, followed by synergistic effects, and the synergistic effects were mainly induced by SPS-MPs and low salinity; in the combined exposure groups, the MDA levels of C. gigas tended to decrease by improving antioxidant enzyme activity and regulating energy metabolism. The combined effects of MPs and low salinity on PEPCK gene expression in digestive glands were influenced by salinity levels: low salinity (26 psu) combined with MPs had an antagonistic effect, and low salinity (21 psu) combined with MPs had a synergistic effect. IBR analysis showed that the combined exposure group of low salinity and SPS-MPs had the strongest stress effects, suggesting that a smaller size may cause severe damage. The metabolomics results showed that salinity was the main factor affecting metabolism in the gills of C. gigas. In addition, the results suggested that the complex stress effects might be related to osmotic regulation, energy metabolism, and antioxidant defense. The results of PCA analysis showed that low salinity was related to antioxidant enzymes, while MPs exposure was related to lipid peroxidation levels and hsp genes.
（3）The combined stress effects of seawater acidification and polystyrene MPs of different sizes on C. gigas were investigated. The results showed that seawater acidification had no effect on MPs accumulation in C. gigas, but increased the apoptosis rate of hemolymph and increase the clearance rate (CR) of C. gigas to enhance energy reserves for stress adaptation; seawater acidification inhibited antioxidant enzymes and inhibited and induced lysosomal enzyme activity in digestive glands and gills, respectively; seawater acidification increased p53 gene expression in gills and induced the immune response. The exposure to LPS-MPs increased the production of ROS in hemolymph, while the exposure to SPS-MPs increased the activity of Catalase (CAT) in gills, and increased MDA levels. The combined effect of MPs and seawater acidification was mostly antagonistic, followed by a synergistic effect, and the synergistic effect was mainly the combined effect of SPS-MPs and seawater acidification. Combined exposure influenced the energy metabolism of oysters by regulating glycolysis gene expression, inhibited antioxidant enzyme activity, and induced oxidative damage in gills. The IBR analysis showed that the IBR value of the combined exposure group of seawater acidification and SPS-MPs was higher than that of the single exposure group of SPS-MPs or seawater acidification, suggesting that the stress effects of the combined exposure of seawater acidification and SPS-MPs were the strongest. The metabolomic results indicated that SPS-MPs were the main factors affecting the digestive gland tissue metabolism of C. gigas, and complex stress effects might be related to osmoregulation, energy metabolism, and antioxidant defense. PCA analysis also showed that MPs was related to antioxidant enzymes and seawater acidification was related to hsp genes and lysosomal enzymes.
（4）The combined stress effects of elevated temperature and polystyrene MPs of different sizes on C. gigas were investigated. The results showed that elevated temperature significantly reduced LPS-MPs accumulation in the digestive glands of C. gigas, but it had no effect on SPS-MPs accumulation; elevated temperature increased Glutathione S-transferase (GST) activity, and induced immune response. MPs exposure increased the gene expression of Inhibitor of NF-κB (IκB), and induced GST activity in the gills; SPS-MPs exposure increased CR, inhibited ACP activity in the gills, increased the MDA content in the digestive glands, and induced ROS production of hemolymph; LPS-MPs exposure decreased AKP activity in digestive glands. There was a significant synergistic effect between elevated temperature and MPs on glycogen content in digestive glands, indicating that the combined exposure group had a higher energy demand. The results of combined exposure of elevated temperature and MPs on GST activity, MDA content, and gene expression of p53 and IκB in gills of oysters, as well as p53 gene expression in digestive glands, suggested that antagonistic interactions between MPs and elevated temperature may occurred. The combined effects of elevated temperature and MPs on hsp90 and IκB gene expression in digestive glands were influenced by MPs size: SPS-MPs combined with elevated temperature had a synergistic effect, and LPS-MPs combined with elevated temperature had an antagonistic effect. The IBR analysis showed that the IBR value of combined exposure to SPS-MPs and the elevated temperature was higher than that of single SPS-MPs and elevated temperature, suggesting that the stress effects of the combined exposure of SPS-MPs and the elevated temperature to oysters were the strongest. The metabolomics results showed that the metabolic profile of the combined exposure group was significantly different from that of the single SPS-MPs or single elevated temperature group, which mainly involved changes in metabolites such as energy metabolism, antioxidant defense, and osmotic regulation. PCA analysis showed that elevated temperature was related to oxidative stress, and MPs was related to oxidative stress and immune defense.