|Other Abstract||Microplastics (MPs) are ubiquitous in the marine environment, and because of their small size, can be ingested by a variety of marine organisms, thus causing adverse effects on marine organisms. Therefore, microplastic contamination is attracting increasing attention worldwide. As reported by previous studies, the coastal area of China, especially the inner Bohai Sea, is one of the most seriously contaminated regions by microplastic pollution. However, the microplastic pollution characteristics in the bay area of the Bohai Sea have not been revealed. Laizhou Bay is one of the typical bays in the Bohai Sea, there are many rivers flowing into Laizhou Bay, including China's second largest river, the Yellow River, and more than 10 other rivers. Around Laizhou Bay, rapid urbanization and industrialization, as well as large-scale raft aquaculture and greenhouse vegetable cultivation bases, have led to a large input of various pollutants. Given the small size and high availability of microplastics, they are likely to interact with marine organisms. Therefore, an increasing number of laboratory exposure experiments involved the bioavailability of microplastics in the marine environment, showing the potential impacts of microplastics on marine animals. However, in most laboratory studies, commercialized microplastics with a spherical shape, single polymer and precise size are used. Moreover, the selected exposure concentrations are often very high and not representative of the expected microplastics concentrations in coastal waters. In addition, many of these studies used microplastics smaller than those reported from the field and without taking into account that microplastics are usually present in different sizes in the marine environment. In recent years, the accumulation of microplastics by marine bivalves has attracted widespread attention due to their widespread existence, high level of filter-feeding activity and edibility. Previous studies have confirmed the accumulation of microplastics in bivalves around the world and their potential toxicity toward these organisms. However, there is a lack of comparative studies on microplastics exposure to bivalves from different ecological niches. Therefore, in this study, the patterns of microplastic contamination in surface water and sediment from 58 sites, and living fish from 31 sites, and oysters (Crassostrea gigas), clams (Ruditapes philippinarum) and scallops (Chlamys farreri) were investigated in a semi-closed bay (Laizhou Bay, China). Then, the toxicities of typical microplastics (polyethylene (PE) and polyethylene terephthalate (PET)) at concentrations of 10 and 1000 μg/L and the modes of action were investigated in oysters, clams and scallops. Furthermore, the integrated biomarker response (IBR) and weight of evidence (WOE) model were employed to evaluate the potential toxic risk of microplastics on bivalves. In addition, the response of oyster digestive gland tissues to microplastics was analyzed using metabolomic and proteomic techniques to provide information on the toxic effects of PE and PET microplastics on oysters at the molecular level. The results were summarized as followed:
(1) Microplastics in Laizhou Bay were pervasively distributed, particularly in the form of fibers. Microplastic abundance exhibited no significant differences among regions in either surface waters or sediment, indicating multiple sources of microplastics pollution in the bay. Spatial hotspot (Getis-Ord Gi*) analysis demonstrated that microplastic pollution was mainly concentrated in the Laizhou-Weifang area, which in turn was mainly affected by ocean current dynamics. Although the spatial distribution of microplastics in sediment was different from surface water, it was also affected by geology, hydrogeology, and anthropogenic activities. The most common polymer in the surface waters was PET, while cellophane (CP) was the most frequently observed polymer in sediment, suggesting different sinking behaviors of these microplastics. The proportion of low-density microplastics (PE and PP) in surface water was approximately 19.9%, but these microplastics accounted for only approximately 1.7% in the sediment, suggesting that low-density microplastic particles preferentially migrate to open sea. There were significant differences in shape, size and polymer type of the microplastics among surface water, sediment and biota (p < 0.05). Cluster analysis suggested that the Gudong, Yellow River Estuary and Laizhou-Weifang regions are three sources of microplastics, which might originate from river input, plastic recycling and marine raft aquaculture. Furthermore, microplastic particle diversity was greater in sediment at offshore sites, suggesting that these sites received microplastics from multiple sources. Our results characterized the microplastic pollution pattern, and will provide important information for risk evaluation and pollution control in this area.
(2) In the present study, PE and PET microplastics were detected in the gills and digestive gland of oyster C. gigasfollowing exposure to both tested concentrations, confirming ingestion of microplastics by the organisms. Both PE and PET microplastic exposure decreased the clearance and respiration rates and induced oxidative stresses in the oysters. Moreover, both PE and PET microplastics inhibited lipid metabolism, while energy metabolism enzyme activities were activated in the oysters. Histopathological damage of exposed oysters was also observed in this study. Integrated biomarker response (IBR) results and the weight of evidence (WOE) model analysis showed that the toxicity induced by microplastics increased with increasing microplastics concentration, and the toxic effects of PET microplastics on oysters were greater than PE microplastics. In this study, there were significant differences in the abundance of microplastics in bivalves. The abundance of microplastics in R. philippinarum clams was highest per unit weight of soft tissues, while C. gigas oysters had the highest microplastic abundance per individual. The reasons for this phenomenon may be related to the size of individual organisms, feeding mechanisms and the degree of microplastic contamination in the environment. This study reports new insights into the consequences of microplastics exposure in marine bivalves at environmentally relevant concentrations, providing valuable information for ecological risk assessment of microplastics in realistic conditions.
(3) Metabolomics analysis suggested that microplastics exposure induced alterations in metabolic profiles in oysters, with changes in energy metabolism and inflammatory responses. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of DEPs revealed that microplastic exposure mainly disturbed the "arachidonic acid metabolism", "linoleic acid metabolism" and "glycerophospholipid metabolism" processes in oysters. Gene Ontology enrichment analysis revealed that microplastics affected the oxidation-reduction process, lipid metabolism process and pentose phosphate pathway. Moreover, a significant increase in the mRNA expression of genes related to lipid and aerobic metabolism and apoptosis pathways was observed after microplastic exposure. In summary, our results indicated that microplastics could alter lipid and glucose metabolism processes in oysters. This study provided the effects of PE and PET microplastic toxicity on oysters at the molecular level, which will contribute to better understanding the defense mechanisms of oysters against microplastic exposure.
(4) In this study, microplastics were observed in the digestive glands and gills of clam R. philippinarum and scallop C. farreri. The clearance rates and respiration rate of both species of bivalves were not significantly affected by different MPs treatments. However, the MPs exposure had caused oxidative stress, energy and lipids metabolism disorder in clams and scallops. Histopathological damage was also observed in both gills and digestive gland of bivalves. Analysis of IBR values showed that a trend of increased stress with increasing MPs concentrations and the toxic effects of PET MPs on bivalves were greater than PE MPs. Additionally, the weight of evidence (WOE) model analysis suggested that the MP hazard increased with increasing MP concentration and the toxic effects of PET MPs on clam digestive glands were greater than those of PE MPs. However, in the gills of clams and scallops, the PE MPs hazard increased with increasing MP concentration, while the trend of PET MPs was the opposite. These results reported the response of different bivalves exposed in MPs at environmentally relevant concentrations. In terms of their histopathological damage and WOE analysis, the clams R. philippinarum are more sensitive to MPs than scallops C. farreri. This study provides new insights for the ecological risk assessment of MPs in realistic conditions.
In conclusion, microplastics were ubiquitously detected in surface water, sediment and organism samples from Laizhou Bay. Its potential pollution sources mainly include river input, plastic recycling and marine raft culture. The hydrologic process is the main reason for the heterogeneity of microplastics spatial distribution in Laizhou Bay. By choosing PE and PET as representative microplastics, multi-level exploration towards toxicological effects of PE and PET to typical bivalves was conducted. It was found that exposure to PE and PET could cause oxidative stress, tissue damage, and disturbance of energy and lipid metabolism in bivalves. The potential toxicity risk of microplastics to bivalves was evaluated by integrated biomarker response (IBR) and the weight of evidence (WOE) model. It was found that the stress response of bivalves showed an increasing trend with increasing microplastic exposure concentration, and the toxic effect of PET microplastics was higher than that of PE microplastics. This research might provide an important basis for the potential ecological risk assessment of microplastics pollution in the environment of Laizhou Bay.|