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.

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.

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.

Study of challenging calcium alkoxides in solution by electrospray ionisation mass spectrometry (Part 2).

Calcium alkoxides in solution give rise to oligomers with different reactivity and solubility and, as observed in a previous investigation, small differences in the ligand structure lead to strong differences in the cluster composition. Electrospray ionisation mass spectrometry (ESI-MS) gives evidence of this behaviour, allowing the identification of these oligomers. In this paper, ESI was applied in the study of calcium alkoxides with different steric hindrances and with additional donor atoms on the ligands. A systematic analysis was conducted by varying the ESI instrumental parameters (voltages, temperatures) to identify the best conditions for the analysis of this class of compounds. Furthermore, particular attention was paid to the study of the best solvent to be employed, considering the possible occurrence of alcoholysis and decomplexation phenomena.