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.

Impacts of dietary exposure to different sized polystyrene microplastics alone and with sorbed benzo[a]pyrene on biomarkers and whole organism responses in mussels Mytilus galloprovincialis.

Due to their hydrophobicity and relatively large surface area, microplastics (MPs) can act as carriers of hydrophobic pollutants in the ocean and may facilitate their transfer to organisms. This study examined effects of dietary exposure to polystyrene MPs of 0.5 and 4.5 µm alone and with sorbed benzo[a]pyrene (BaP) on mussels Mytilus galloprovincialis in order to elucidate the effects of MP size and the presence of sorbed BaP on the organism. MPs were provided daily, mixed with algae, during 26 days at equivalent mass (0.058 mg/L), corresponding to 1000 particles/mL for 4.5 µm MPs and to 7.44 × 105 particles/mL for 0.5 µm MPs. Effects were determined on early cellular biomarkers in hemocytes, structure and cell type composition of digestive tubules (DTs), histopathology and whole organism responses (condition index (CI), clearance rate (CR), food absorption efficiency (AE), respiration rate (RR) and scope for growth (SFG)). BaP concentrations in mussels increased with time, in particular when sorbed to smaller MPs. Large MPs were abundant in the lumen of stomach and DTs, but were also occasionally found within epithelial cells. Effects in all treatments increased with exposure time. MPs with sorbed BaP were more toxic than MPs alone according to hemocyte viability and catalase activity and to the quantitative structure of DT epithelium. Higher toxicity of small MPs compared to larger ones was recorded for DNA damage and cell composition of DTs. At tissue level a slight increase in prevalence of inflammatory responses occurred in all exposed groups. At whole organism level a compensatory effect was observed on absorption efficiency across MP treatments at day 26, resulting in increased SFG in mussels exposed to small MPs with sorbed BaP. This could be related to an increased energy need to deal with stress observed in biomarkers. Further work is required to understand the Trojan horse effect of a variety of plastic type, size, shape combinations together with a wide variety of pollutants.

Season influences the transcriptomic effects of dietary exposure to PVP/PEI coated Ag nanoparticles on mussels Mytilus galloprovincialis.

Toxicity of AgNPs has been widely studied in waterborne exposed aquatic organisms. However, toxic effects caused by AgNPs ingested through the diet and depending on the season are still unexplored. The first cell response after exposure to xenobiotics occurs at gene transcription level. Thus, the aim of this study was to assess transcription level effects in the digestive gland of female mussels after dietary exposure to AgNPs both in autumn and in spring. Mussels were fed daily for 21 days with Isochrysis galbana microalgae previously exposed for 24 h to a dose close to environmentally relevant concentrations of 1 µg Ag/L PVP/PEI coated 5 nm AgNPs (in spring) and to a higher dose of 10 µg Ag/L of the same AgNPs both in autumn and in spring. After 1 and 21 days, mussels RNA was hybridized in a custom microarray containing 7806 annotated genes. Mussels were more responsive to the high dose compared to the low dose of AgNPs and a higher number of probes were altered in autumn than in spring. In both seasons, significantly regulated genes were involved in the cytoskeleton and lipid transport and metabolism COG categories, among others, while genes involved in carbohydrate transport and metabolism were specifically altered in autumn. Overall, transcription patterns were differently altered depending on the exposure time and season, indicating that season should be considered in ecotoxicological studies of metal nanoparticles in mussels.

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.