Cadmium (Cd) is being frequently detected in marine organisms. However, dose-dependent effects of Cd challenged unraveling the toxicological mechanisms of Cd to marine organisms and developing biomarkers. Here, the dose-dependent effects of Cd on clams Ruditapes philippinarum following exposure to 5 doses of Cd (3, 9, 27, 81, 243 μg/L) were investigated using benchmark dose (BMD) method. By model fitting, calculation of BMD values was performed on transcriptomic profiles, metals concentrations, and antioxidant indices. Cd exposure induced not only significant Cd accumulation in clams, but also marked alterations of essential metals such as Ca, Cu, Zn, Mn, and Fe. Gene regulation posed little influence on essential metal homeostasis, indicated by poor enrichment of differentially expressed genes (DEGs) associated with metal binding and metal transport in lower concentrations of Cd-treated groups. BMD analysis on biological processes and pathways showed that peptide cross-linking was the most sensitive biological process to Cd exposure, followed by focal adhesion, ubiquitin mediated proteolysis, and apoptosis. Occurrence of apoptosis was also confirmed by TUENL-positive staining in gills and hepatopancreas of clams treated with Cd. Furthermore, many DEGs, such as transglutaminases (TGs), metallothionein (MT), STEAP2-like and laccase, which presented linear or monotonic curves and relatively low BMD values, were potentially preferable biomarkers in clams to Cd. Overall, BMD analysis on transcriptomic profiles, metals concentrations and biochemical endpoints unraveled the sensitiveness of key events in response to Cd treatments, which provided new insights in exploring the toxicological mechanisms of Cd in clams as well as biomarker selection.

Cadmium is one of the most serious metal pollutants in the Bohai Sea. Previous studies revealed that mitochondrion might be the target organelle of Cd toxicity. However, there is a lack of a global view on the mitochondrial responses in marine animals to Cd. In this work, the mitochondrial responses were characterized in clams Ruditapes philippinarum treated with two concentrations (5 and 50 μg/L) of Cd for 5 weeks using tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining, ultrastructural observation and quantitative proteomic analysis. Basically, a significant decrease of mitochondrial membrane potential (△Ψm) was observed in clams treated with the high concentration (50 μg/L) of Cd. Cd treatments also induced specific morphological changes indicated by elongated mitochondria. Furthermore, iTRAQ-based mitochondrial proteomics showed that a total of 97 proteins were significantly altered in response to Cd treatment. These proteins were closely associated with multiple biological processes in mitochondria, including tricarboxylic acid (TCA) cycle, oxidative phosphorylation, fatty acid β-oxidation, stress resistance and apoptosis, and mitochondrial fission. These findings confirmed that mitochondrion was one of the key targets of Cd toxicity. Moreover, dynamical regulations, such as reconstruction of energy homeostasis, induction of stress resistance and apoptosis, and morphological alterations, in mitochondria might play essential roles in Cd tolerance. Overall, this work provided a deep insight into the mitochondrial toxicity of Cd in clams based on a global mitochondrial proteomic analysis.

CO2-driven acidification and emerging contaminants, such as pharmaceuticals, pose new threats for the maintenance of natural populations of marine organisms by interfering with their normal biochemical pathways and defences. The combined effects of seawater acidification, as predicted in climate change scenarios, and an emerging contaminant (the non-steroidal anti-inflammatory drug, NSAID, diclofenac) on oxidative stress-related parameters were investigated in the Mediterranean mussel Mytilus galloprovincialis and the Manila clam Ruditapes philippinarum. A flow-through system was used to carry out a three-week exposure experiment with the bivalves. First, the animals were exposed to only three pH values for 7 days. The pH was manipulated by dissolving CO2 in the seawater to obtain two reduced pH treatments (pH −0.4 units and pH −0.7 units), which were compared with seawater at the natural pH level (8.1). Thereafter, the bivalves were concomitantly exposed to the three experimental pH values and environmentally relevant concentrations of diclofenac (0.00, 0.05 and 0.50 μg/L) for an additional 14 days. The activities of superoxide dismutase, catalase and cyclooxygenase, and lipid peroxidation and DNA strand-break formation were measured in both the gills and digestive gland after 7, 14 and 21 days of exposure to each experimental condition. The results show that the biochemical parameters measured in both the mussels and clams were more influenced by the reduced pH than by the contaminant or the pH*contaminant interaction, although the biomarker variation patterns differed depending on the species and tissues analysed. Generally, due to increases in its antioxidant defence, M. galloprovincialis was more resistant than R. philippinarum to both diclofenac exposure and reduced pH. Conversely, reduced pH induced a significant decrease in COX activity in both the gills and digestive gland of clams, possibly resulting in the increased DNA damage observed in the digestive gland tissue.

Several biomarkers were measured to evaluate the effects of Trimethoprim (TMP; 300, 600 and 900 ng/L) in the clam Ruditapes philippinarum after exposure for 1, 3 and 7 days. The actual TMP concentrations were also measured in the experimental tanks. The total haemocyte count significantly increased in 7 day-exposed clams, whereas alterations in haemocyte volume were observed after 1 and 3 days of exposure. Haemocyte proliferation was increased significantly in animals exposed for 1 and 7 days, whereas haemocyte lysate lysozyme activity decreased significantly after 1 and 3 days. In addition, TMP significantly increased haemolymph lactate dehydrogenase activity after 3 and 7 days. Regarding antioxidant enzymes, only a significant time-dependent effect on CAT activity was recorded. This study demonstrated that environmentally realistic concentrations of TMP affect haemocyte parameters in clams, suggesting that haemocytes are a useful cellular model for the assessment of the impact of TMP on bivalves.

POP bioaccumulation pathways in the clam Tapes philippinarum were examined for two years from juveniles to adult size. Two polluted sites, one with sandy sediment, the other muddy were compared with a reference site characterized by low contamination levels. Juvenile clams coming from a hatchery were reared both on the sediment and in nets suspended at 30 cm from the bottom. POP changes in clam tissue were related to the concentrations recorded in sediments and in the particulate matter during the entire fattening period. Results provided interesting data on the relationships between environmental contamination and bioaccumulation. Contrary to studies on the decontamination times of the clams collected in polluted areas, this work investigates the preferential clam bioaccumulation pathways during growth under different environmental conditions. In general POP bioaccumulation resulted to be correlated to concentrations in SPM rather than in sediments and was higher in S-clams rather than in B-clams.

Benzo[a]pyrene (B[a]P) has a high carcinogenic potential. B[a]P concentrations and molecular biomarkers (mRNA expressions of Pgp, AhR, CYP4, CYP414A1, GST-pi, GST-S2, Cu/Zn-SOD and Mn-SOD) were assayed in gills and digestive glands of the clam Ruditapes philippinarum exposed to 0.03, 0.3 and 3 μg/L B[a]P for 21 days and then exposed to natural seawater for 15 days. Results showed that B[a]P was rapidly accumulated in and then eliminated from tissues of the clams. All gene mRNA expressions in the treated groups were induced significantly with the exception of CYP414A1 and Cu/Zn-SOD in the 0.03 μg/L B[a]P group. According to correlation analysis, mRNA expressions of AhR, GST-pi and Mn-SOD in gills and GST-pi in digestive glands had good correlations with B[a]P concentrations and could be used as molecular biomarkers of B[a]P exposure. This study investigated the molecular response of the genes mentioned above and selected useful molecular biomarkers for B[a]P pollution monitoring.

Biomarkers comprising activities of biotransformation enzymes (ethoxyresorufin-O-deethylase –EROD–, dibenzylfluorescein dealkylase –DBF–, glutathione S-transferase –GST), antioxidant enzymes (glutathione reductase –GR– and glutathione peroxidase –GPX), lipid peroxidation –LPO– and DNA strand breaks were analyzed in the clam Ruditapes philippinarum caged at Cádiz Bay, Santander Bay and Las Palmas de Gran Canaria (LPGC) Port (Spain). Sediments were characterized. Digestive gland was the most sensitive tissue to sediment contamination. In Cádiz Bay, changes in LPO regarding day 0 were related with metals. In LPGC Port, DBF, EROD, and GST activity responses suggested the presence of undetermined contaminants which might have led to DNA damage. In Santander Bay, PAHs were related with EROD activity, organic and metal contamination was found to be associated with GR and GST activities and DNA damage presented significant (p < 0.05) induction. R. philippinarum was sensitive to sediment contamination at biochemical level. Biomarkers allowed chemical exposure and sediment quality assessment.

In estuarine ecosystems, bivalves experience large pH fluctuations caused by the anthropogenic elevation of atmospheric CO₂ and Cu pollution. This study investigates whether Cu toxicity increases indiscriminately in two bivalve species from different estuarine habitats as a result of elevated Cu bioaccumulation in acidified seawater. This was carried out by evaluating the effects of Cu exposure on two bivalve species (clams and scallops) for 28 d, at a series of gradient pH levels (pH 8.1, 7.8, and 7.6). The results demonstrated an increase in the Cu content in the soft tissues of clams and scallops in acidified seawater. Cu toxicity increased under acidified seawater by affecting the molecular pathways, physiological function, biochemical responses, and health status of clams and scallops. An iTRAQ-based quantitative proteomic analysis showed increased protein turnover, disturbed cytoskeleton and signal transduction pathways, apoptosis, and suppressed energy metabolism pathways in the clams and scallops under joint exposure to ocean acidification and Cu. The integrated biomarker response results suggested that scallops were more sensitive to Cu toxicity and/or ocean acidification than clams. The proteomic results suggested that the increased energy metabolism and suppressed protein turnover rates may contribute to a higher resistivity to ocean acidification in clams than scallops. Overall, this study provides molecular insights into the distinct sensitivities between two bivalve species from different habitats under exposure to ocean acidification and/or Cu. The findings emphasize the aggravating impact of ocean acidification on Cu toxicity in clams and scallops. The results show that ocean acidification and copper pollution may reduce the long-term viability of clams and scallops, and lead to the degradation of estuarine ecosystems.

Bivalves are useful bioindicators of microplastic contamination in the marine environment for several reasons, such as extensive filter feeding activity, broad geographical distribution, and limited movement capability. This study conducted a nationwide monitoring of microplastic pollution along the Korean coasts using filter-feeding bivalves (including oyster, mussel, and Manila clam) as bioindicators to identify the national contamination level and characteristics of microplastics. Seawater sample was collected from the same sampling stations of oyster and mussel for comparison. Microplastics were widely distributed in both coastal bivalves and waters with mean concentrations of 0.33 ± 0.23 n/g (1.21 ± 0.68 n/individual) in oyster/mussel, 0.43 ± 0.32 n/g (2.19 ± 1.20 n/individual) in Manila clam, and 1400 ± 560 n/m³ in seawater. Despite the lack of significant relationship in the abundance of microplastics, their dominant features such as size, shape, color and polymer type were similar between bivalves and seawater. Fragments (69% for oyster/mussel, 72% for Manila clam, and 77% for seawater), particles smaller than 300 μm (96% for oyster/mussel, 83% for Manila clam, and 84% for seawater) and colorless (79% for oyster/mussel, 85% for Manila clam, 75% for seawater) were the dominant shape, size and color, respectively. The major polymer types were polypropylene, polyethylene, and polyester. The microplastic level in bivalves was relatively high in urbanized areas with a wide diversity of polymer types compared with those in non-urbanized areas, and the proportion of polystyrene in the Korean samples was abundant compared with other regions due to wide use of polystyrene products in Korea. Our result suggests that microplastic contamination is widespread in the Korean coastal environment, and bivalves can reflect the microplastic pollution characteristics of the surrounding waters where they live.

Plastic pollution, which is one of the most important environmental problems at the present time, has been understood recently, and the effects of this pollution on ecosystem and biota are becoming a growing problem, especially in the aquatic ecosystems. Direct or indirect exposure to those particles leads to adverse effects on marine organisms. In the marine environment, plastic materials interact with other pollutants such as metals, thereby affecting the uptake levels of those pollutants in marine organisms. In the present study, the Manila clam Ruditapes philippinarum was exposed to polyethylene microbeads and mercury chloride in single, combined and incubated form at environmentally relative concentrations for one week in controlled laboratory conditions. The uptake and tissue distribution of both stressors as well as the vector role of microplastics on mercury uptake in the organisms were investigated. Filtration rates, biomarkers for immunomodulation and oxidative stress, and histological alterations were also evaluated. Microplastics were ingested by the clams, and translocated to the various tissues. However, contaminated microplastics displayed a negligible vector role in terms of mercury bioaccumulation in the clams. The single and interactive exposure of the stressors reduced the filtration rate in the clams. Both pollutants affected the immune system of the organisms. Histological alterations were determined in the gill and digestive gland tissues of the clams among the treatment groups, although oxidative stress biomarkers remained unchanged. This study suggests that the vector role of polyethylene microplastics in mercury uptake is negligible and reveals that the single and interactive one-week exposure of two pollutants induce toxicity in the manila clams.