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.

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.

A mobile pilot plant was set up in a wastewater treatment plant (WWTP) in southwest Spain to address potential adverse effects of effluents as a whole contaminant, which are discharging into marine environments. Ruditapes philippinarum specimens were exposed to different effluent concentrations (50%, 25%, 12.5%, 6.25%, and 3.15%) during seven days. After effluent exposure, lysosomal membrane stability alterations (LMS), changes in the energy status storage (total lipids content (TLP) and in the mitochondrial electron transport (MET), inhibition of inflammatory mechanisms (cyclooxygenase activity (COX)), and neurotoxic effects (acetylcholinesterase (AChE) were determined in exposed organisms. Furthermore, potential toxic reduction in the effluent was analysed by the application of an additional microalgae tertiary treatment called photobiotreatment (PhtBio). Results after PhtBio confirmed the toxic effect reduction in exposed organisms. Neuroendocrine effects, alterations in energy budget and in lipid storage revealed alterations in clam's health status causing stress conditions after effluent exposure.

Several works evaluated the toxicity of pharmaceutical drugs and climate related changes in invertebrates but few explored the combined effects of both stressors, namely considering their mode of action (MoA). Carbamazepine (CBZ) and cetirizine (CTZ) are pharmaceutical drugs detected in the environment and the toxicity derived from the combined effects of these drugs with ocean acidification (OA) is poorly explored. Thus, the present study investigated the biochemical parameters related to an oxidative stress response and the transcription of genes related to the MoA of CBZ (1.0 μg/L) and CTZ (0.6 μg/L) in the clam Ruditapes philippinarum chronically exposed (28 days) to control (7.8) and low (7.5) pH conditions. The results obtained showed that despite the clams accumulated both drugs, at low pH the clams exposed to CTZ decreased drug concentration and BCF values (CTZ uptake: 2.0 ± 0.5 ng/g fresh weight; BCF: 3.8 ± 0.9) in comparison with clams exposed to control pH (CTZ uptake: 2.9 ± 0.3 ng/g fresh weight; BCF: 5.5 ± 0.6). No oxidative stress was induced by the exposure to CBZ or CTZ at each pH level, but the transcription of several genes related with the MoA (neurotransmission, immunity and biomineralization) was altered by low pH, drug exposure and the combination of both stressors. At both pH conditions, CBZ increased the transcription of GABA receptor gene (neurotransmission) and CTZ led to a decrease of Perlucin gene (biomineralization) transcription. The transcription of MyD88 gene (immunity) decreased at low pH (7.5) combined with drug exposure (CBZ or CTZ). Thus, it was highlighted that the interaction of drug exposure and low pH conditions can change bivalves’ sensitivity to drugs or alter drugs toxicity.

Coastal lagoons are constantly subjected to releases of chemical pollutants, and so organisms may be exposed to such toxicants. This study investigated through a multivariate approach the physiological status of bivalve Ruditapes philippinarum, farmed in Sacca di Goro lagoon. Biomarkers at different levels of biological organization (catalase, superoxide dismutase, genotoxicity, reburrowing behavior) were evaluated at three sites exposed to different environmental conditions. A seasonal trend was observed, and micronucleus frequency was significantly lowest at the relatively pristine reference site. Enzymatic activity toward oxyradicals be quite efficient since variations in responsiveness were not consistent. However, behavioral impairment was observed in reburrowing rates. Sediment concentrations showed low PAH levels and high natural levels of trace metals Cr and Ni. DistLM statistical analysis revealed a non-significant relationship between selected biomarkers and xenobiotics. Therefore other potentially toxic compounds in admixture at low doses may be involved in driving differing spatial distribution of physiological impairment.

The degree of aggregation and/or coalescence of Au-citrate nanoparticles (AuNPs, mean size 21.5 ± 2.9 nm), after delivery in simulated seawater, are shown to be concentration-dependent. At low concentrations no coalescence and only limited aggregation of primary particles were found. Experiments were performed in which the marine bivalve (Ruditapes philippinarum) was exposed to AuNPs or dissolved Au and subsequently, bivalve tissues were studied by Scanning and Transmission Electron Microscopy and chemical analyses. We show that the bivalve accumulates gold in both cases within either the digestive gland or gill tissues, in different concentrations (including values of predicted environmental relevance). After 28 days of exposure, electron-dense deposits (corresponding to AuNPs, as proven by X-ray microanalysis) were observed in the heterolysosomes of the digestive gland cells. Although non-measurable solubility of AuNPs in seawater was found, evidence is presented of the toxicity produced by Au3+ dissolved species (chloroauric acid solutions) and its relevance is discussed.

Differences in the accumulation of mercury (Hg) in five species of marine bivalves, including scallops Chlamys nobilis, clams Ruditapes philippinarum, oysters Saccostrea cucullata, green mussels Perna viridis, and black mussels Septifer virgatus, were investigated. The bivalves displayed different patterns of Hg accumulation in terms of the body concentrations of methylmercury (MeHg) and total Hg (THg), as well as the ratio of MeHg to THg. Parameters of the biodynamics of the accumulation of Hg(II) and MeHg could reflect the species-dependent Hg concentrations in the bivalves. With the exception of black mussels, we found a significant relationship between the efflux rates of Hg(II) and the THg concentrations in the bivalves. The interspecific variations in the MeHg to THg ratio were largely controlled by the relative difference between the elimination rates of Hg(II) and MeHg. Stable isotope (δ¹³C) analysis indicated that the five bivalve species had contrasting feeding niches, which may also affect the Hg accumulation.

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.