Oxidative Stress and Damage Biomarkers in Clam Ruditapes decussatus Exposed to a Polluted Site: The Reliable Biomonitoring Tools in Hot and Cold Seasons.

In the present study, a multi-biomarker approach was used to assess the biological effects of metal pollution in the southern lagoon of Tunis, on clam Ruditapes decussatus both in "hot" (in summer) and "cold" (in winter) seasons. Clams were collected in August 2015 and February 2016 from three sites of the lagoon and from Louza considered a reference site. The concentrations of five trace metals (cadmium, copper, iron, lead, and zinc) in the soft tissues of R. decussatus were evaluated at the sampling sites. A core of biomarkers indicative of (a) neurotoxicity (acetylcholinesterase, AChE); (b) biotransformation (glutathione S-transferase, GST); (c) oxidative stress (catalase, CAT; total glutathione peroxidase, T-GPx; total glutathione peroxidase, T-GPx; selenium-dependent glutathione peroxidase, Se-GPx; glutathione reductase, GR; superoxide dismutase, SOD) (d) lipid peroxidation (malondialdhyde, MDA level), and (e) apoptotic process (caspase 3-like, CSP3) was selected for measurements of environmental effects on the populations of clams collected from the different sampling sites. The results of metal bioaccumulation in soft tissues of Ruditapes decussatus revealed a high pollution in the South Lagoon of Tunis with spatial variation and relatively high levels at the navigation channel. Anthropogenic pollutants in the lagoon led to the activation of antioxidant defense and biotransformation enzymes to oxidative damage of the membrane and activation of apoptosis, and revealed neurotoxicity. Among this core of biomarkers, the antioxidants enzymes (CAT, SOD, GR, and GPx) were very sensitive, allowing the discrimination among sites and pointing to the navigation channel as the most impacted site in the southern lagoon of Tunis. Moreover, a significant effect of season was recorded on biomarkers responses (e.g., CAT, GR, SOD, AChE, and CSP3 activities and MDA levels) with higher levels in winter than in summer, probably influenced by the reproductive stage and food availability. Finally, the measurement of the selected core of biomarkers in the whole soft tissues of clams was considered as an integrated indicator of environmental stress. Moreover, R. decussatus proved to be a remarkable sentinel species capable to establish a reliable diagnosis of the health status of the marine environment in different areas of the southern lagoon of Tunis, both in "hot" and "cold" seasons.

Cadmium sulfide quantum dots induce oxidative stress and behavioral impairments in the marine clam Scrobicularia plana.

Cadmium sulfide (CdS) quantum dots have a number of current applications in electronics and solar cells and significant future potential in medicine. The aim of the present study was to examine the toxic effects of CdS quantum dots on the marine clam Scrobicularia plana exposed for 14 d to these nanomaterials (10 µg Cd L(-1) ) in natural seawater and to compare them with soluble Cd. Measurement of labile Cd released from CdS quantum dots showed that 52% of CdS quantum dots remained in the nanoparticulate form. Clams accumulated the same levels of Cd regardless of the form in which it was delivered (soluble Cd vs CdS quantum dots). However, significant changes in biochemical responses were observed in clams exposed to CdS quantum dots compared with soluble Cd. Increased activities of catalase and glutathione-S-transferase were significantly higher in clams exposed in seawater to Cd as the nanoparticulate versus the soluble form, suggesting a specific nano effect. The behavior of S. plana in sediment showed impairments of foot movements only in the case of exposure to CdS quantum dots. The results show that oxidative stress and behavior biomarkers are sensitive predictors of CdS quantum dots toxicity in S. plana. Such responses, appearing well before changes might occur at the population level, demonstrate the usefulness of this model species and type of biomarker in the assessment of nanoparticle contamination in estuarine ecosystems.

Biochemical and behavioural responses of the marine polychaete Hediste diversicolor to cadmium sulfide quantum dots (CdS QDs): waterborne and dietary exposure.

Cadmium sulfide (CdS) quantum dots are widely used in medical imaging. The aim of this study was to examine toxicity effects of CdS engineered nanoparticles (CdS NPs) compared to soluble Cd, on marine ragworms (Hediste diversicolor) exposed for 14 d to these contaminants (10 µg Cd L(-1)) in seawater or via their food (contaminated worm tissue). In our experimental media, Dynamic Light Scattering studies showed that the majority of CdS remained in the nanoscale (1-10 nm) with the exception of few aggregates (100-300 nm). Labile Cd fractions released from CdS NPs were estimated by diffusive gradient in thin films, showing that about 50% of CdS NPs remained in nanoparticulate form. Ragworms accumulated Cd in both soluble Cd and CdS NPs in waterborne exposures only. Greater significant changes of biochemical responses were observed in worms exposed to CdS NPs in seawater compared to contaminated food. Catalase and glutathione-S-transferase activities were the most sensitive biochemical biomarkers responding to both Cd treatments for waterborne exposure. Inductions of CAT were higher in diet-exposed worms to Cd as NPs vs soluble form suggesting a specific "nano" effect. Caspase activities increased in worms exposed to soluble Cd and Cd NPs for the two routes of exposure compared to controls. Defences, may be insufficient to prevent reactive oxygen species generation and the associated apoptosis. Behaviour of invertebrates inside sediment showed impairments of body movements in worms exposed to CdS NPs. This study points out oxidative processes as the main consequences of exposure to Cd based NPs in worms.

A marine mesocosm study on the environmental fate of silver nanoparticles and toxicity effects on two endobenthic species: the ragworm Hediste diversicolor and the bivalve mollusc Scrobicularia plana.

Silver nanoparticles are widely used in a range of products and processes for their antibacterial properties, electrical and thermal conductivity. The fate and effects of Ag nanoparticles were examined in two endobenthic species (Scrobicularia plana, Hediste diversicolor), under environmentally realistic conditions in outdoor mesocosms exposed to Ag at 10 µg L(-1) in nanoparticulate (Ag NPs) or soluble salt (AgNO3) forms for 21 days. Labile Ag was determined in water and sediment by using diffusive gradient in thin films. Ag levels were equivalent in contaminated Ag NPs mesocosms to those contaminated with the soluble form. Bioaccumulation of Ag was observed for both species exposed to either Ag in the nanoparticulate or ionic forms. Concerning biomarker responses, both soluble and nanoparticulate Ag forms, induced defenses against oxidative stress, detoxification, apoptosis, genotoxicity and immunomodulation. Nevertheless, DNA damages measured by the comet assay in the digestive gland of S. plana, and Phenoloxidase and lysozyme activities in S. plana and H. diversicolor, respectively, were higher in the presence of Ag NPs compared to soluble Ag suggesting a specific nano effect.

A mesocosm study of fate and effects of CuO nanoparticles on endobenthic species (Scrobicularia plana, Hediste diversicolor).

The fate and effects of CuO nanoparticles (CuO NPs) were examined in endobenthic species (Scrobicularia plana , Hediste diversicolor), under environmentally realistic conditions in outdoor mesocosms (exposure to Cu at 10 µg L(-1) in particulate (CuO NPs) or soluble salt (CuNO(3)) forms) for 21 days. Labile Cu was determined in water and sediment by using diffusive gradient in thin films. No labile Cu being detected from CuO NPs; the observed effects in invertebrates exposed to CuO NPs were mainly attributed to the toxicity of nanoparticulate rather than dissolved Cu toxicity. Bioaccumulation of CuO NPs was observed in both species. Biomarkers were examined at different levels of biological organization: biochemical markers of defense and damage, biomarkers of genotoxicity (comet assay), and behavioral biomarkers (feeding and burrowing). Behavioral biomarkers, antioxidant defenses (catalase, glutathion S-transferase, metallothionein), and genotoxicity are the most sensitive tools to highlight the effect of soluble or nanoparticulate metal forms. Concerning other biomarkers of defense (superoxide dismutase, lactate dehydrogenase, laccase) and damage (thiobarbituric acid reactive substances, acetylcholinesterase, acid phosphatase), no significant effects were detected. This experiment shows the suitability of mesocosms for studying the environmental effects of nanoparticles.

Biochemical and behavioural responses of the endobenthic bivalve Scrobicularia plana to silver nanoparticles in seawater and microalgal food.

Because of their bactericidal effects, Ag nanoparticles (Ag NPs) have promising industrial development but could lead to potential ecological risks. The aim of this study was to examine the uptake and effect of silver (soluble or as lactate Ag NPs of 40 nm) at low concentrations (10 µg L(-1)) in the endobenthic bivalve Scrobicularia plana exposed, for 14 days, directly (water) or via the diet (microalgae). The stability of Ag NPs in seawater was examined using dynamic light scattering. Release of soluble Ag from Ag NPs in the experimental media was quantified by using diffusive gradient in thin film. Bioaccumulation of Ag in bivalves was measured by electrothermal atomic absorption spectrometry. Behavioural and biochemical biomarkers were determined in bivalves. Aggregation of Ag NPs and the release of soluble Ag from Ag NPs were observed in the experimental media. For both forms of Ag, bioaccumulation was much more important for waterborne than for dietary exposure. The response of oxidative stress biomarkers (catalase, glutathion S-transferase, superoxide dismutase) was more important after dietary than waterborne exposure to Ag (soluble and NPs). These defences were relatively efficient since they led to a lack of response of damage biomarkers. Burrowing was not affected for bivalves exposed directly or through the diet to both Ag forms but feeding behaviour was impaired after 10 days of dietary exposure. Since no differences of responses to Ag either soluble or nanoparticulate were observed, it seems that labile Ag released from Ag NPs was mainly responsible for toxicity.

Fate of isotopically labeled zinc oxide nanoparticles in sediment and effects on two endobenthic species, the clam Scrobicularia plana and the ragworm Hediste diversicolor.

Although it is reported that metal and metal oxide nanoparticles, which are among the most rapidly commercialized materials, can cause toxicity to organisms, their fate in the environment and toxicity to marine organisms are not well understood. In this study, we used a stable isotope labelling approach to trace the fate of nanoparticles (NPs) in sediments and also investigated bio-uptake in two estuarine intra-sedimentary invertebrates Scrobicularia plana and Nereis diversicolor. We selected exposure to 3 mg kg(-1) sediment ZnO NPs since this level is a realistic prediction of the environmental concentration in sediments. 67ZnO NPs (DLS: 21-34 nm, positively charged: 31.3 mV) suspensions were synthesised in diethylene glycol (DEG). We explored the fate of 67ZnO NPs in sediment, 67Zn bioaccumulation and the biochemical (biomarkers of defence and damage) and behavioural (burrowing kinetics and feeding rates) biomarkers in both species to 67ZnO NPs and DEG on its own during a 16 d laboratory exposure. After exposure, 67Zn concentrations in sediment showed higher levels in the upper section (1cm: 2.59 mg kg(-1)) decreasing progressively (2 cm: 1.63 mg kg(-1), 3 cm: 0.90 mg kg(-1), 4 cm: 0.67 mg kg(-1)) to a minimum value at the bottom (5 cm: 0.31 mg kg(-1)). 67Zn bioaccumulation was observed in both organisms exposed to 67ZnO NPs in DEG but no major inter-species differences were found. At the biochemical level, 67ZnO NPs exposure significantly induced increased glutathione-S-transferase activity in worms and catalase activity in clams whereas superoxide dismutase activity and thiobarbituric acid reactive substance levels were not affected in any species. Exposure to DEG on its own leads to a significant increase of metallothionein-like protein levels in clams compared with those exposed to 67ZnO NPs or controls. Burrowing behaviour as well as feeding rate were significantly impaired in both species exposed to 67ZnO NPs. Concerning exposure to DEG on its own, burrowing behaviour impairments were also shown in both species and feeding rate was impaired in bivalves. At environmentally realistic concentration of 67ZnO NPs in sediment, there is no strong evidence for a severe nanoparticle effect since most effects were also observed in the presence of DEG alone.

Size dependent bioaccumulation and ecotoxicity of gold nanoparticles in an endobenthic invertebrate: the Tellinid clam Scrobicularia plana.

Gold nanoparticles (AuNPs) have important technological applications resulting in an increased potential for release to the environment, and a greater possibility of toxicological effects. The marine bivalve Scrobicularia plana was exposed to AuNPs of size 5, 15 and 40 nm during a 16 d laboratory exposure at 100 µg Au L(-1). After exposure to AuNPs forming aggregates (>700 nm), the clams accumulated Au in their soft tissues. Biochemical (biomarkers) and behavioral (burrowing and feeding) responses were investigated. Au NPs were responsible of metallothionein induction (5, 40 nm), increased activities of catalase (15, 40 nm) and superoxide dismutase (40 nm) and of glutathione S-transferase by the three sizes of AuNPs indicating defense against oxidative stress. Exposure to AuNPs impaired burrowing behavior. However, it must be underlined that these effects were observed at a dose much higher than expected in the environment.

An ecotoxicological approach with transplanted mussels (Mytilus galloprovincialis) for assessing the impact of tyre reefs immersed along the NW Mediterranean Sea.

Tyre artificial reefs were deposited in a marine protected area (Vallauris-Golfe Juan Bay, France) located along the NW Mediterranean coast, during the early 80's. The potential toxic effects of the tyre artificial reefs were investigated using transplantation of marine mussels, Mytilus galloprovincialis, to stations located above tyre blocks (St1, St2) or reference site (StR). Mussels transplanted to different stations presented the following sequence of mortality: St1 > St2 > StR. Principal Component Analysis, taking into account metal accumulation (cadmium, copper and zinc) and biomarker (SOD, CAT, GST and AChE activities, TBARS and MT levels, Condition Index) responses in mussel tissues indicated a clear separation between the three stations. St1 organisms were significantly more affected by tyre reefs than those from other stations. Such an integrated monitoring study represents a key approach to assess in situ the biological impact of >25 year-old tyre artificial reefs.