Zinc oxide, titanium dioxide and C60 fullerene nanoparticles, alone and in mixture, differently affect biomarker responses and proteome in the clam Ruditapes philippinarum.

Continuous release of nanoparticles (NPs) into marine coastal environments results in an increased risk of exposure to complex NP mixtures for marine organisms. However, to date, the information on the effects at molecular and biochemical levels induced by the exposure to NPs, singly and as a mixture, is still scant. The present work aimed at exploring the independent and combined effects and the mechanism(s) of action induced by 7-days exposure to 1 µg/L nZnO, 1 µg/L nTiO2 and 1 µg/L FC60 fullerene in the Manila clam Ruditapes philippinarum, using a battery of immunological and oxidative stress biomarkers in haemolymph, gills and digestive gland. In addition, proteomics analyses were performed in gills and the digestive gland, where NP bioaccumulation was also assessed. Increased bioaccumulation of single NPs and the mixture was linked with increased oxidative stress and higher damage to proteins, lipids and DNA in all tissues analysed. The proteomics approach highlighted protein modulation in terms of abundance and damage (higher redox-thiol and carbonylated groups content). In particular, the modulated proteins (16 in gills and 18 in digestive gland) were mostly related to cytoskeleton and energetic metabolism. The digestive gland was the tissue more affected. For all biomarkers measured, increased detrimental effects were observed in the mixture compared to single NP exposures.

Toxicological effects and bioaccumulation of fullerene C60 (FC60) in the marine bivalve Ruditapes philippinarum.

Fullerene C60 (FC60), with its unique physical properties, has been used in many applications in recent decades. The increased likelihood of direct release into the environment has raised interest in understanding the biological effects of FC60 to aquatic organisms. Nowadays, only few studies have analysed FC60 effects and bioaccumulation in marine organisms following in vivo exposure. To provide new data about FC60 toxicity, Ruditapes philippinarum was selected as target species to assess potential adverse effects of the contaminant. Clams were exposed for 1, 3 and 7 days to predicted environmental concentrations of FC60 (1 and 10 µg/L) and cellular and biochemical responses were evaluated in clams' gills, digestive gland and haemolymph. The FC60 content in gills and digestive gland was determined in all experimental conditions after 7 days of exposure. Results showed an increase in oxidative stress. In particular, a significant modulation in antioxidant enzyme activities, and changes in glutathione S-transferase activity were observed in gills. Moreover, damage to lipids and proteins was detected in FC60-treated (10 µg/L) clams. In digestive gland, slighter variations in antioxidant enzyme activities and damage to molecules were detected. CAT activity was significantly affected throughout the exposure, whereas damage to lipids was evident only at the end of exposure. FC60 accumulation was revealed in both gills and digestive gland, with values up to twelve-fold higher in the latter. Interestingly, haemolymph parameters were slightly affected by FC60 compared to the other tissues investigated. Indeed, only Single Cell Gel Electrophoresis and Neutral Red uptake assays showed increased values in FC60-exposed clams. Moreover, volume and diameter of haemocytes, haemocyte proliferation, and micronucleus assay highlighted significant variations in treated clams, but only in the first phases of exposure, and no changes were detected after 7 days. Our results suggested clam gills as the target tissue for FC60 toxicity under the exposure conditions tested: the high damage detected to lipids and proteins could contribute to long-term problems for the organism.

Bioaccumulation and effects of titanium dioxide nanoparticles and bulk in the clam Ruditapes philippinarum.

Biochemical and cellular responses to low concentrations of TiO2 nanoparticles (nTiO2, 1 and 10 µg/L) and bulk (bTiO2, 10 µg/L) were evaluated in gills, digestive gland and haemolymph of the clam Ruditapes philippinarum after1, 3 and 7 days' exposure. At 7 days, titanium content was determined in gills and digestive gland. nTiO2 significantly increased antioxidant enzyme activities in both tissues, and lipid peroxidation in digestive gland at 10 µg/L only, and affected glutathione S-transferase activity. Slighter variations were observed in bTiO2-treated clams. A significant Ti bioaccumulation was detected in both gills and digestive gland of 10 µg nTiO2/L-exposed clams. In haemolymph, nTiO2 affected total haemocyte count, haemocyte proliferation, haemocyte diameter and volume, and induced DNA damage. Overall, this study demonstrated that TiO2 alters most of the biomarkers measured in clams, although responses were differently modulated depending on tissues and exposure conditions, and indicated that nTiO2 can be accumulated by bivalves, suggesting a potential risk for filter-feeding animals.

Assessing the Effects of Amoxicillin on Antioxidant Enzyme Activities, Lipid Peroxidation and Protein Carbonyl Content in the Clam Ruditapes philippinarum and the Mussel Mytilus galloprovincialis.

In this study, we evaluated the capability of amoxicillin (AMX)-one of the most widely used antibiotics worldwide-to induce oxidative stress in both gills and digestive gland from two bivalve species, the clam Ruditapes philippinarum and the mussel Mytilus galloprovincialis. Superoxide dismutase (SOD) and catalase (CAT) activities, as well as the lipid peroxidation levels (LPO) and protein carbonyl content (PCC), were measured in bivalves exposed to 100, 200 and 400 µg AMX/L for 1, 3 and 7 days. The results obtained demonstrated that AMX affected slightly biomarker responses of molluscs.

Does the antibiotic amoxicillin affect haemocyte parameters in non-target aquatic invertebrates? The clam Ruditapes philippinarum and the mussel Mytilus galloprovincialis as model organisms.

Amoxicillin (AMX) is one of the most widely used antibiotics worldwide, and its levels in aquatic ecosystems are expected to be detectable. At present, information concerning the toxic effects of AMX on non-target aquatic organisms, such as bivalves, is scarce. Consequently, in this study, we investigated for the first time the effects of AMX on the haemocyte parameters of two bivalve species, the clam Ruditapes philippinarum and the mussel Mytilus galloprovincialis, which share the same habitat in the Lagoon of Venice, in order to compare the relative sensitivity of the two species. The bivalves were exposed to 100, 200 and 400 µg AMX/L for 1, 3 and 7 days, and the effects on the total haemocyte count (THC), the diameter and volume of the haemocytes, haemocyte proliferation, lactate dehydrogenase (LDH) activity in cell-free haemolymph, the haemolymph pH, and the formation of micronuclei were evaluated. The actual concentrations of AMX in the seawater samples from the experimental tanks were also measured. Overall, the obtained results demonstrated that AMX affected slightly the haemocyte parameters of bivalves. In addition, no clear differences in terms of sensitivity to AMX exposure were recorded between the two bivalve species.

In vivo exposure of the marine clam Ruditapes philippinarum to zinc oxide nanoparticles: responses in gills, digestive gland and haemolymph.

Potential nanoparticle (NP) toxicity poses a growing concern in marine coastal environments. Among NPs, zinc oxide nanoparticles (nZnO) are widely used in many common products that ultimately become deposited in coastal habitats from multiple non-point sources. In this study, we evaluated the in vivo effects of nZnO in the clam Ruditapes philippinarum. Animals were exposed to nZnO (1 and 10 µg/L) and ZnCl2 (10 µg/L) for 7 days. ZnCl2 was used to compare the effects of the NPs to those of Zn(2+) and to ascertain whether nZnO toxicity is attributable to the release of ions into the aquatic medium. At differing time intervals during the exposure, several biochemical and cellular responses were evaluated in the clam gills, digestive gland, and haemolymph. The results showed that nZnO, at concentrations close to the predicted environmental levels, significantly affected various parameters in clam tissues. Significant increases in catalase and superoxide dismutase activities and a decreasing trend of glutathione S-transferase activity indicated the involvement of oxidative stress in nZnO toxicity. In clams exposed to ZnCl2, slight variations in antioxidant enzyme activities were detected with respect to nZnO-treated clams. However, no damage to lipids, proteins or DNA was revealed in all exposure conditions, suggesting a protection of antioxidant enzymes in the tissues. Of the various haemolymph parameters measured, haemocyte proliferation increased significantly, in ZnCl2-treated clams in particular. Under nZnO (10 µg/L) and ZnCl2 exposure, DNA damage in haemocytes was also revealed, but it was lower in clams exposed to ZnCl2. A decreasing trend in gill AChE activity of treated clams proposed a possible role of zinc ions in nZnO toxicity. However, the dissimilar modulation of the responses in the nZnO- and ZnCl2-exposed clams suggested different mechanisms of action, with nZnO toxicity possibly depending not only on the release of zinc ions but also on NP-specific features. Changes in the biological parameters measured in the clams were consistent with Zn accumulation in their gills and digestive glands.

In vitro exposure of haemocytes of the clam Ruditapes philippinarum to titanium dioxide (TiO2) nanoparticles: nanoparticle characterisation, effects on phagocytic activity and internalisation of nanoparticles into haemocytes.

The continuous growth of nanotechnology and nano-industries, the considerable increase of products containing nanoparticles (NPs) and the potential release of NPs in aquatic environments suggest a need to study NP effects on aquatic organisms. In this context, in vitro assays are commonly used for evaluating or predicting the negative effects of chemicals and for understanding their mechanisms of action. In this study, a physico-chemical characterisation of titanium dioxide NPs (n-TiO2) was performed, and an in vitro approach was used to investigate the effects of n-TiO2 on haemocytes of the clam Ruditapes philippinarum. In particular, the effects on haemocyte phagocytic activity were evaluated in two different experiments (with and without pre-treatment of haemocytes) by exposing cells to P25 n-TiO2 (0, 1 and 10 µg/mL). In addition, the capability of n-TiO2 to interact with clam haemocytes was evaluated with a transmission electron microscope (TEM). In this study, n-TiO2 particles showed a mean diameter of approximately 21 nm, and both anatase (70%) and rutile (30%) phases were revealed. In both experiments, n-TiO2 significantly decreased the phagocytic index compared with the control, suggesting that NPs are able to interfere with cell functions. The results of the TEM analysis support this hypothesis. Indeed, we observed that TiO2 NPs interact with cell membranes and enter haemocyte cytoplasm and vacuoles after 60 min of exposure. To the best of our knowledge, this is the first study demonstrating the internalisation of TiO2 NPs into R. philippinarum haemocytes. The present study can contribute to the understanding of the mechanisms of action of TiO2 NPs in bivalve molluscs, at least at the haemocyte level.