With the rapid development of maritime transport industry and the huge emission of domestic and industrial wastewater, marine pollution has posed a serious threat to the marine environment and ecological safety. Heavy metals, well-known for their toxicity, persistence and bioaccumulation characteristics, are considered a major anthropogenic contaminant in coastal environment. Persistent organic pollutants are highly toxic and displayed a wide range of chronic effects, such as mutagenicity and carcinogenicity. In the marine environment, the heavy metals and organic pollutants not only have a single toxicity to marine organisms, but also interact with each other.

    The Pacific oysters Crassostrea gigas are widely distributed in offshore areas, and often used as bio-indicator in marine environmental assessment due to their sessile and filter-feeding lifestyle, wide geographical distribution and ability to accumulate contaminants from the surrounding water. In the present study, the individual and combined toxicities of typical heavy metals cadmium (Cd) and lead (Pb), and organic pollutants benzo[a]pyrene (BaP) and 2,2’,4,4’-Tetrabromodiphenyl ether (BDE-47) on the early development (acute) and adult period (chronic) of C. gigas were investigated, hopefully sheding light on the environmental monitoring and ecological risk assessment. Furthermore, an integrative biomarker approach was employed to evaluate the environmental quality of the coastal areas of Bohai Sea and Northern Yellow Sea using native oysters. Additonally, we applied iTRAQ-based proteomics to characterize the differential proteomic changes and elucidate the differential toxicological effects of oysters collected from different stations. The results were summarized as followed:

    (1) The individual and combined toxicities of dissolved Cd²⁺ and Pb²⁺ on early life stages of the oyster was investigated. For embryotoxicity, the median effective concentration (EC₅₀) values for individual Cd²⁺, Pb²⁺ and their mixture were of 272.2µg/L, 660.3µg/L and 373.1 µg/L, respectively. The median lethal concentrations (LC₅₀) for 96 h larval mortality were determined to be 353.3 µg/L, 699.5 µg/L and 205.5 µg/L for individual Cd²⁺, Pb²⁺ and their mixture, respectively; Moreover, the Marking-Dawson additive toxicity indices indicated an additive effect with a trend to synergism for the Cd²⁺ and Pb²⁺ combination. Furthermore, DNA strand breaks were observed in oyster embryos after individual Cd²⁺, Pb²⁺ and their mixture exposure, and a significant positive correlation was demonstrated between embryotoxicity and genotoxicty. These data will be helpful to predict the toxicity of metal mixtures, and provide biological criteria for the implementation of marine water quality standards.

    (2) The individual and combined toxicities of BaP and BDE-47 on early life stages of the C. gigas were investigated. The EC₅₀ values of BaP, BDE-47 and their mixture on embryogenesis were 18.4 µg/L, 203.3 µg/L and 72.0 µg/L respectively, while the LC₅₀ values for 96 h larval mortality were 26.8 µg/L, 244.5 µg/L and 108.9 µg/L respectively. The Marking-Dawson additive toxicity indices indicated an additive effect with a trend to antagonism. The results indicated that BaP and BDE-47 expoure may cause potential toxic effect to oyster embryos and larvae, especially at high-concentration areas.

    (3) The individual and combined chronic toxic effects (7, 14, 28 days) of dissolved Cd²⁺ and Pb²⁺ on the C. gigas were investigated. There was a strong accumulation of heavy metals in the gills and digestive glands of C.gigas, indicating a significant dose-dependent relationship. Bioconcentration factor (BCF) decreased generally with the increase of heavy metal concentrations in ambient seawater. Significant histopathological changes such as hemocyte infiltration, cilium eletion and filaments tumidness were observed in gills; while hemocyte infiltration, thinning and exfoliation of digestive gland tubule epithelium into lumen and tubule atrophy were found in digestive gland of C. gigas after Cd²⁺ and Pb²⁺ exposure. An integrated biomarker response (IBR) approach was employed to integrate the biomarkers of oxidative stress (superoxide dismutase, SOD, catalase catalase, CAT, lipid peroxidation, LPO and glutathione S-transferase, GST). The results showed that the combined toxicity effects of Cd²⁺ and Pb²⁺ were synergistic, especially in the high concentration treatment.

    (4) The individual and combined chronic toxic effects (7, 14, 28 days) of dissolved BaP and BDE-47 on the C. gigas were investigated. The gills and digestive glands of oysters showed a strong accumulation of organic pollutants, and presented a significant dose-dependent pattern. BCF decreased generally with the increase of POPs exposure concentration. After BaP and BDE-47 exposure, the gills and digestive glands of C. gigas displayed significant histopathological changes, such as hemocyte infiltration, cilium eletion and filaments tumidness were found in gills; hemocyte infiltration, thinning and exfoliation of digestive gland tubule epithelium into lumen and tubule atrophy were found in digestive glands indicating microstructure damage and dysfunetion in oysters. IBR method was employed to integrate the biomarkers of oxidative stress (SOD, CAT, LPO and GST). The results showed that the combined toxicity effects of BaP and BDE-47 were mainly antagonistic.

    (5) The integrated biomarker (IBR) approach was used to evaluate the environmental quality of the Bohai Sea and Northern Yellow Sea coastal area. There was a spatial difference of heavy metal concentration and biomarker responses in C. gigas collected from different sites. Among them, XZ and HLD sites were the most severely contaminated area with the highest IBR/n value (more than 2), and the XSD, PL and DL sites were the less-polluted area with the lowest IBR/n value (less than 0.2). Moreover, iTRAQ-based proteomics was applied to elucidate the perturbance of heavy metals in digestive glands of C. gigas from different coastal areas of Bohai Sea and Northern Yellow Sea. The alteration of proteins involved in signaling pathway, oxidative stress, energy metabolism and immune regulatory was observed in oyster digestive glands between the polluted areas (MP, XZ, HLD site) and less-polluted station (XSD site).