Cell and tissue level responses in mussels Mytilus galloprovincialis dietarily exposed to PVP/PEI coated Ag nanoparticles at two seasons.

Silver nanoparticles (Ag NPs) are present in numerous consumer products due to their antimicrobial and other unique properties, thus concerns about their potential input into aquatic ecosystems are increasing. Toxicity of Ag NPs in waterborne exposed aquatic organisms has been widely investigated, but studies assessing the potential toxic effects caused after ingestion through the food web, especially at low realistic concentrations, remain scarce. Moreover, it is not well known whether season may influence toxic effects of Ag NPs. The main objective of this study was to determine cell and tissue level responses in mussels Mytilus galloprovincialis dietarily exposed to poly-N-vinyl-2-pirrolidone/polyethyleneimine (PVP/PEI) coated 5 nm Ag NPs for 1, 7 and 21 days both in autumn and spring. Mussels were fed every day with microalgae Isochrysis galbana exposed for 24 h to a low dose (1 µg Ag/L Ag NPs) in spring and to a higher dose (10 µg Ag/L Ag NPs) in spring and autumn. Mussels fed with microalgae exposed to the high dose accumulated Ag significantly after 21 days in both seasons, higher levels being measured in autumn compared to spring. Intralysosomal metal accumulation measured in mussel digestive gland and time- and dose-dependent reduction of mussels health status was similar in both seasons. DNA strand breaks increased significantly in hemocytes at both exposure doses along the 21 days in spring and micronuclei frequency showed an increasing trend after 1 and 7 days of exposure to 1 µg Ag/L Ag NPs in spring and to 10 µg Ag/L in both seasons. Values decreased after 21 days of exposure in all the cases. In conclusion, PVP/PEI coated 5 nm Ag NPs ingested through the food web were significantly accumulated in mussel tissues and caused adverse cell and tissue level effects both in autumn and in spring.

Changes in protein expression in mussels Mytilus galloprovincialis dietarily exposed to PVP/PEI coated silver nanoparticles at different seasons.

Potential toxic effects of Ag NPs ingested through the food web and depending on the season have not been addressed in marine bivalves. This work aimed to assess differences in protein expression in the digestive gland of female mussels after dietary exposure to Ag NPs in autumn and spring. Mussels were fed daily with microalgae previously exposed for 24 h to 10 µg/L of PVP/PEI coated 5 nm Ag NPs. After 21 days, mussels significantly accumulated Ag in both seasons and Ag NPs were found within digestive gland cells and gills. Two-dimensional electrophoresis distinguished 104 differentially expressed protein spots in autumn and 142 in spring. Among them, chitinase like protein-3, partial and glyceraldehyde-3-phosphate dehydrogenase, that are involved in amino sugar and nucleotide sugar metabolism, carbon metabolism, glycolysis/gluconeogenesis and the biosynthesis of amino acids KEGG pathways, were overexpressed in autumn but underexpressed in spring. In autumn, pyruvate metabolism, citrate cycle, cysteine and methionine metabolism and glyoxylate and dicarboxylate metabolism were altered, while in spring, proteins related to the formation of phagosomes and hydrogen peroxide metabolism were differentially expressed. Overall, protein expression signatures depended on season and Ag NPs exposure, suggesting that season significantly influences responses of mussels to NP exposure.

Dietary exposure of mussels to PVP/PEI coated Ag nanoparticles causes Ag accumulation in adults and abnormal embryo development in their offspring.

Toxicity of silver nanoparticles (Ag NPs) to aquatic organisms has been widely studied. However, the potential toxic effects of Ag NPs ingested through the food web, especially at environmentally relevant concentrations, as well as the potential effects on the offspring remain unknown. The aims of this work were to screen the cytotoxicity of Poly N­vinyl­2­pirrolidone/Polyethyleneimine (PVP/PEI) coated 5 nm Ag NPs in hemocytes exposed in vitro and to assess the effects of dietary exposure to Ag NPs on mussels growth, immune status, gonad condition, reproductive success and offspring embryo development. For this, mussels Mytilus galloprovincialis were fed daily with microalgae Isochrysis galbana previously exposed for 24 h to a dose close to environmentally relevant concentrations (1 µg Ag/L Ag NPs) and to a high dose of 10 µg Ag/L Ag NPs. After 24 h of in vitro exposure, Ag NPs were cytotoxic to mussel hemocytes starting at 1 mg Ag/L (LC50: 2.05 mg Ag/L). Microalgae significantly accumulated Ag after the exposure to both doses and mussels fed for 21 days with microalgae exposed to 10 µg Ag/L Ag NPs significantly accumulated Ag in the digestive gland and gills. Sperm motility and fertilization success were not affected but exposed females released less eggs than non-exposed ones. The percentage of abnormal embryos was significantly higher than in control individuals after parental exposure to both doses. Overall, results indicate that Ag NPs taken up through the diet can significantly affect ecologically relevant endpoints such as reproduction success and embryo development in marine mussels.

Assessment of the effects of Cu and Ag in oysters Crassostrea gigas (Thunberg, 1793) using a battery of cell and tissue level biomarkers.

Oysters are considered sentinel organisms in environmental water quality monitoring programs in which cell and tissue level biomarkers are reliable tools. Copper (Cu) and silver (Ag) are present in relatively high concentrations in several estuaries, potentially affecting environmental and human health. Crassostrea gigas oysters were exposed during 28 days to a range of environmentally relevant concentrations of Cu and Ag alone or in mixture. Effects were studied through cell and tissue level biomarkers approach. Results indicated: changes in the Condition Index (CI), altered digestive gland epithelium and presence of histopathological alterations in the gonad and digestive gland of exposed oysters. A time-dependent increase in lipofuscin contents in exposed oysters and an increase in intralysosomal metal accumulation in digestive cells through the experiment were also recorded. The Integrative Biological Response (IBR) Index showed that even at low exposure levels, Ag and Cu can produce alterations on oysters' health status.