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Nanoparticle decoration with noble metal represents a promising alternative to improve their photocatalytic and photovoltaic properties. However, toxicity can be influenced by such modification, as the bioavailability of these substances may be influenced. To understand how decoration influences the NP impacts in marine ecosystems, we exposed suspension-feeding clams, Ruditapes decussatus, to two photocatalyst nanocomposites, TiO 2 NPs and AuTiO 2 NPs, over 2 concentrations, 50 μg L -1 and 100 μg L -1 , in a laboratory experiment. Accumulation of Au and Ti in gills and digestive gland was noted in clams after exposure to TiO 2 NPs and AuTiO 2 NPs using inductively coupled plasma optic emission spectroscopy (ICP-OES). TiO 2 and AuTiO 2 NPs alter the behavior of the clams Ruditapes decussatus by reducing filtration and respiration rates. Furthermore, the highest concentration of TiO 2 NPs induces an overproduction of H 2 O 2 in gills and digestive gland and NO production only in gills. Superoxide dismutase (SOD), Catalase (CAT), Glutathione-S-transferase (GST) and acetylcholinesterase (AChE) activities were induced in gills and digestives gland in concentration and nanocomposite type dependent manner. Decorated form presented higher Malondialdehyde (MDA) levels in gills and digestive gland than the undecorated form, suggesting different mechanisms of action that may be mediated through oxidative stress. In conclusion, the considered parameters could represent reliable biomarkers for the assessment of NP toxicity on R. decussatus as biological biomonitoring model. In addition, based on the obtained results, nanoparticle decoration influences the toxicity of metal nanoparticles in marine organism., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The increased use of gold nanoparticles (AuNPs) in several applications has led to a rise in concerns about their potential toxicity to aquatic organisms. In addition, toxicity of nanoparticles to aquatic organisms is related to their physical and chemical properties. In the present study, we synthesize two forms of gold octahedra nanoparticles (Au_ 0.03 and Au_ 0.045 ) in 1.3-propandiol with polyvinyl-pyrrolidone K 30 (PVPK 30 ) as capping agent using polyol process. Shape, size and optical properties of the particles could be tuned by changing the molar ratio of PVP K 30 to metal salts. The anisotropy in nanoparticles shape shows strong localized surface plasmon resonance (SPR) in the near infrared region of the electromagnetic spectrum. Environmental impact of Oct-AuNPs was determined in the marine bivalve, Ruditapes decussatus exposed to different concentrations of Au_ 0.03 and Au_ 0.045 . The dynamic light scattering showed the stability and resistance of Au_ 0.03 and Au_ 0.045 in the natural seawater. No significant modification in vg-like proteins, MDA level and enzymatic activities were observed in treated clams with Au_ 0.03 even at high concentration. In contrast, Au_ 0.045 induced superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GST) activities, in a concentration dependent manner indicating defense against oxidative stress. Enhanced lipid peroxidation represented by malondialdehyde content confirmed oxidative stress of Au_ 0.045 at high concentration. These results highlight the importance of the physical form of nanomaterials on their interactions with marine organisms and provide a useful guideline for future use of Oct-AuNPs. In addition, Vitellogenin is shown not to be an appropriate biomarker for Oct-AuNPs contamination even at high concentration. We further show that Oct-AuNPs exhibit an important antioxidant response without inducing estrogenic disruption., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Oxidative stress is often implicated in nanoparticle toxicity. Several studies have highlighted the role of internalization routes in determining nanotoxicity. Here, we investigate how two endocytotic mechanisms (clathrin- and caveolae-mediated) impact on redox balance in gill and digestive gland of the mussel, Mytilus galloprovincialis. Animals were exposed (for 3, 6 and 12 h) to two sizes of silver nanoparticles (AgNP: <50 nm and <100 nm) prior to and after blockade of two endocytic pathways (amantadine blocks clathrin-mediated endocytosis while nystatin blocks caveolae-mediated endocytosis). Redox-proteomic tools were used to determine effects. Our results demonstrate the ability of both sizes of AgNP (<50 and <100 nm) to cause protein thiol oxidation and/or protein carbonylation. However, blockade of endocytotic routes mitigated AgNP toxicity. Differential ROS-related toxicity of AgNP to mussel tissues seemed to be linked to tissue-specific mode of action requirements. Cell uptake mechanism strongly influences toxicity of AgNPs in this filter-feeder., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The synthesis of hybrid nanomaterials has greatly increased in recent years due to their special physical and chemical properties. However, information regarding the environmental toxicity associated with these chemicals is limited, in particular in the aquatic environment. In the present study, an experiment was performed in which the marine bivalve (Ruditapes decussatus) was exposed for 14days to 2 concentrations of zinc oxide-decorated Au nanoparticles (Au-ZnONPs: Au-ZnONP50=50μg/L; Au-ZnONP100=100μg/L). The stability and resistance of Au-ZnONPs in the natural seawater were assessed by combining transmission electron microscopy and dynamic light scattering. Inductively coupled plasma-atomic emission spectroscopy revealed uptake of these nanoparticles within clams and their ability to induce metallic deregulation. The results obtained indicate that Au-ZnONPs induce biochemical and histological alterations within either the digestive gland or gill tissues at high concentration. This was deduced from the significant increase in H 2 O 2 level, superoxide dismutase and catalase activities and malondialdehyde content. Furthermore, the toxicity of Au-ZnO nanoparticles was linked with the increase of intracellular iron and calcium levels in both tissues. Histological alterations in gill and digestive gland were more pronounced with Au-ZnONP100 and this is likely related to oxidative mechanisms. Gill and digestive gland are differentially sensitive to Au-ZnONPs if the exposure concentration is higher than 50μg/L. In conclusion, the parameters considered here could constitute reliable biomarkers for evaluation of hybrid nanoparticles toxicity in environmental model organisms. In addition, based on the results obtained, gill and digestive gland of R. decussatus could be proposed as models to detect harmful effects of hybrid nanoparticles., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Anthracene (ANT) and permethrin (PER) are two of the more toxic compounds reaching the marine environment. This study aimed to determine the impact of these molecules on Venerupis decussata, an economically important species cultured on the Tunisian coast. Shell structure and its possible transformation upon exposure to the two contaminants were studied by X-ray diffraction and gravimetric analyses. Results revealed a phase transition in shell composition from aragonite to calcite after PER exposure, to a mixture of PER and ANT (Mix) but not for ANT alone. Catalase (CAT), superoxide dismutase (SOD) and glutathione transferase (GST) activities were determined in digestive gland and gills after exposure to ANT, PER and Mix to assess the impact of the contamination on the oxidative status of V. decussata. Enzyme activities increased in the digestive gland after PER treatment and in the gills after ANT treatment. PER exposure significantly reduced the levels of free thiols and increased levels of carbonylated proteins in the digestive gland, as compared to controls. In contrast, ANT exposure significantly reduced free thiols and increased the number of carbonylated proteins in the gills. Mix induced additive effects as measured by both enzymatic and proteomic approaches. The present study suggests that PER has a strong effect on shell structure; that PER and ANT exposure generate compound-dependent oxidative stress in the tissues of V. decussata and that a mixture of the two compounds has synergistic effects on biochemical response., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The increasing industrial use of nanomaterials during the last decades poses a potential threat to the environment and in particular to organisms living in the aquatic environment. In the present study, the toxicity of zinc oxide nanoparticles (ZnONP) was investigated in Pacific oysters Crassostrea gigas. The nanoscale of ZnONP, in vehicle or ultrapure water, was confirmed, presenting an average size ranging from 28 to 88 nm. In seawater, aggregation was detected by TEM and DLS analysis, with an increased average size ranging from 1 to 2 μm. Soluble or nanoparticulated zinc presented similar toxicity, displaying a LC50 (96 h) around 30 mg/L. High zinc dissociation from ZnONP, releasing ionic zinc in seawater, is a potential route for zinc assimilation and ZnONP toxicity. To investigate mechanisms of toxicity, oysters were treated with 4 mg/L ZnONP for 6, 24 or 48 h. ZnONP accumulated in gills (24 and 48 h) and digestive glands (48 h). Ultrastructural analysis of gills revealed electron-dense vesicles near the cell membrane and loss of mitochondrial cristae (6 h). Swollen mitochondria and a more conspicuous loss of mitochondrial cristae were observed after 24 h. Mitochondria with disrupted membranes and an increased number of cytosolic vesicles displaying electron-dense material were observed 48 h post exposure. Digestive gland showed similar changes, but these were delayed relative to gills. ZnONP exposure did not greatly affect thiol homeostasis (reduced and oxidized glutathione) or immunological parameters (phagocytosis, hemocyte viability and activation and total hemocyte count). At 24 h post exposure, decreased (-29%) glutathione reductase (GR) activity was observed in gills, but other biochemical responses were observed only after 48 h of exposure: lower GR activity (-28%) and levels of protein thiols (-21%), increased index of lipid peroxidation (+49%) and GPx activity (+26%). In accordance with ultrastructural changes and zinc load, digestive gland showed delayed biochemical responses. Except for a decreased GR activity (-47%) at 48 h post exposure, the biochemical alterations seen in gills were not present in digestive gland. The results indicate that gills are able to incorporate zinc prior (24 h) to digestive gland (48 h), leading to earlier mitochondrial disruption and oxidative stress. Our data suggest that gills are the initial target of ZnONP and that mitochondria are organelles particularly susceptible to ZnONP in C. gigas., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Gold nanoparticles (AuNP) have potential applications in drug delivery, cancer diagnosis and therapy, food industry and environment remediation. However, little is known about their potential toxicity or fate in the environment. Mytilus edulis was exposed in tanks to 750 ppb AuNP (average diameter 5.3 ± 1 nm) for 24h to study in vivo biological effects of nanoparticles. Traditional biomarkers and an affinity procedure selective for thiol-containing proteins followed by two-dimensional electrophoresis (2DE) separations were used to study toxicity and oxidative stress responses. Results were compared to those obtained for treatment with cadmium chloride, a well known pro-oxidant. M. edulis mainly accumulated AuNP in digestive gland which also showed higher lipid peroxidation. One-dimensional SDS/PAGE (1DE) and 2DE analysis of digestive gland samples revealed decreased thiol-containing proteins for AuNP. Lysosomal membrane stability measured in haemolymph gave lower values for neutral red retention time (NRRT) in both treatments but was greater in AuNP. Oxidative stress occurred within 24h of AuNP exposure in M. edulis. Previously we showed that larger diameter AuNP caused modest effects, indicating that nanoparticle size is a key factor in biological responses to nanoparticles. This study suggests that M. edulis is a suitable model animal for environmental toxicology studies of nanoparticles., (Copyright 2010 Elsevier B.V. All rights reserved.)

Blue mussels (Mytilus edulis L.) collected at three sampling sites in each of three geographical regions (South, Middle, North) along the permanent longitudinal South-North salinity gradient of the Baltic Sea, were exposed for 10 days to copper (35ppb) or 95 octane petrol (0.3 per thousand). During the experiment, they were maintained at the respective sampling site salinity. Scope for growth (SFG) was determined, and biochemical stress markers (protein carbonyl groups, disulfide bond formation, and glutathione transferase (GST), and catalase (CAT) activities) were investigated in gill tissue upon termination of the experiment. Treatment and regional effects for SFG and protein carbonyl groups were all significant for petrol. The largest increase in protein carbonyl groups was observed in the North. Mussels from the southern, more saline ( approximately 7 per thousand) region had the highest SFG, and displayed the largest SFG decrease in response to treatment, indicating that they had the most energy available for allocation to stress response. They also displayed the least increase in the level of protein carbonyl groups. Mussels from the Northern, less saline ( approximately 5%) region had the highest degree of protein carbonyl groups in response to both treatments, and lowest average SFG. Silver stained diagonal gels for samples from one sampling site in South and North, respectively, demonstrated differences in disulfide bond profiles for both stress treatments. There was also a regional difference in the number of protein disulfides observed on diagonal gels. The most diverse protein disulfide response was found in South. No treatment related effects on GST and CAT activities were observed. We suggest that both SFG and protein carbonyl groups show that geographical difference in stress susceptibility, previously established between the North and the Baltic Seas, also apply on a regional scale within the Baltic Sea, along the salinity gradient.

Mytilus edulis was exposed under controlled conditions to a panel of model pro-oxidants (H(2)O(2), CdCl(2) and menadione) for 24h. Protein extracts of gill, mantle and digestive gland were analysed by immunoblotting in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) separations. Immunoblotting revealed extensive and comparable levels of protein carbonylation across the pro-oxidant panel with approximately 1.5-fold higher levels in gill than digestive gland. Ubiquitination in gill was modest in response to H(2)O(2), but increased in response to menadione and CdCl(2). High ubiquitination levels were found for all pro-oxidants in digestive gland with levels comparable to the highest found in gill. Two-dimensional (2D) SDS-PAGE confirmed specific targeting of individual proteins by ubiquitin against a generally stable protein expression signature. Spot matching suggested that carbonylation is not a pre-requisite for ubiquitination. While gill showed consistently higher constitutive levels of glutathione transferase, glucose 6-phosphate dehydrogenase and glutathione reductase activity, pro-oxidant treatment had only modest effect on these enzymes and on the ratio of reduced/oxidised glutathione. In digestive gland, this latter ratio was higher than in gill and increased in response to menadione and CdCl(2). Ubiquitination may provide a marker for acute onset of oxidative stress in bivalves.

Protein carbonylation and levels of heat shock proteins (hsp; 60, 70 and 90 kDa) were measured in gill, mantle and digestive gland of Ruditapes decussatus following exposure to p,p'-dichlorodiphenyldichloroethylene (DDE). Heat shock response was measured by immunoblotting using antibodies specific to heat shock proteins (hsps). Densitometry analysis of individual bands revealed no difference between control and treated samples except appearance of hsp90 in DDE-treated mantle. Carbonylated protein content was determined following 2,4-dinitrophenylhydrazine derivatization and two-dimensional electrophoresis coupled with western blotting. Immunoblotting with dinitrophenol-specific antibody revealed extensive differences in both extent and number of carbonylated proteins in mantle and digestive gland in response to DDE while gill was unaffected. These results demonstrate for the first time that DDE causes tissue-specific formation of reactive oxygen species in clams.

Protein expression profiles (PEPs) were generated by two-dimensional electrophoresis (2-D SDS-PAGE) for gill and digestive glands of Mytilus edulis sampled from a polluted and reference site in Cork Harbour, Ireland. Similar patterns and expression levels were found for both sites in silver stained gels. However, Western blotting for carbonylated proteins demonstrated higher levels of specific carbonylation of proteins in tissues from animals in the polluted site. Animals from the reference site were acclimated in holding tanks, exposed to 1 mM H2O2 for 24 h, dissected and analysed by 2-D SDS-PAGE. Again, generally similar PEPs were found in control and exposed animals for gill and digestive gland but carbonylation was more pronounced in polluted and exposed animals. Western blotting of extracts after one-dimensional electrophoresis with antibodies to glutathione and actin revealed that gill proteins are glutathionylated more strongly than digestive gland and that this process is more pronounced in polluted animals than in controls. We conclude that carbonylation and glutathionylation can occur in gill and digestive gland in response to oxidative stress in M. edulis. Actin is a major target for both glutathionylation and carbonylation under oxidative stress conditions.

Multixenobiotic resistance (MXR) is a mechanism analogous to the mammalian multidrug resistance (MDR) phenotype, whereby, simultaneous resistance is conferred against the intracellular accumulation of structurally and functionally diverse, natural, endogenous and environmental toxicants. Expression of P-glycoproteins (P-gp), ATP-dependent transporters encoded for by the mdr1 gene that have been implicated in this xenobiotic efflux mechanism, have previously been detected in normal teleost tissues involved in a secretory, absorption or a barrier function. The presence of these proteins in the epidermis of fish species has not to our knowledge previously been investigated. In the present study, primary cultures of epidermis from the rainbow trout Oncorhynchus mykiss were employed to investigate whether an MXR mechanism is functional in the epidermis of fish. The efflux of the fluorescent mdr1 substrate rhodamine 123 from the cells was significantly inhibited by verapamil, a compound known to interfere with P-gp mediated transport. The cultured epidermal cells were also observed to accumulate this fluorescent dye in a verapamil sensitive manner, thus indicating the presence of an mdr1-like mechanism. Immunocytochemical analysis, using a monoclonal antibody (JSB1) directed against a conserved cytoplasmic P-gp epitope, also demonstrated the presence of P-gp-like proteins. Sediment elutriate extracts were employed as models of environmental complex mixtures to evaluate the potential of the epidermal cultures to discriminate between samples of varying contaminant burden using MXR activity as an endpoint. The induction of P-gp expression was found to be in accordance with the level of contamination detected in the sediments from which the elutriates were extracted. The findings of the functional study also demonstrated that environmental pollutants, which interfere with P-gp function, could be identified using this model.