Bioaccumulation, cellular and molecular effects in adult zebrafish after exposure to cadmium sulphide nanoparticles and to ionic cadmium.

Few works have addressed the effects provoked by the exposure to cadmium containing nanoparticles (NPs) on adult zebrafish (Danio rerio). We studied the effects of CdS NPs (5 nm) or ionic cadmium (10 µg Cd/L) after 3 and 21 d of exposure and at 6 months post-exposure (mpe). Acute toxicity was recorded after exposure to both forms of cadmium. Significant cadmium accumulation was measured in the whole fish after both treatments and autometallography showed a higher accumulation of metal in the intestine than that in the liver. Histopathological alterations, such as inflammation in gills and vacuolization in the liver, were detected after the exposure to both cadmium forms and, in a lower extent, at 6 mpe. X-ray analysis proved the presence of CdS NPs in these organs. The hepatic transcriptome analysis revealed that gene ontology terms such as "immune response" or "actin binding" were over-represented after 21 d of exposure to ionic cadmium respect to CdS NPs treatment. Exposure to CdS NPs caused a significant effect on pathways involved in the immune response and oxidative stress, while the exposure to ionic cadmium affected significantly pathways involved in DNA damage and repair and in the energetic metabolism. Oxidative damage to liver proteins was detected after the exposure to ionic cadmium, while a stronger destabilization of the hepatocyte lysosomal membrane was recorded under exposure to CdS NPs. In summary, although ionic cadmium provoked stronger effects than CdS NPs, both cadmium forms exerted an array of lethal and sublethal effects to zebrafish.

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.

Waterborne exposure of adult zebrafish to silver nanoparticles and to ionic silver results in differential silver accumulation and effects at cellular and molecular levels.

Effects of silver nanoparticles (Ag NPs) on freshwater species have been reported in several studies, but there is not information on the potential long-term consequences of a previous exposure. In this work, we investigated the long-term effects of maltose-coated Ag NPs (20 nm) and of ionic silver (10 µg/L) after 21 days of exposure and at 6 months post-exposure (mpe) in adult zebrafish. Exposure resulted in significant silver accumulation in the whole body of fish exposed to ionic silver, but not in those exposed to Ag NPs. However, autometallography revealed metal accumulation in the liver and intestine of fish treated with the two silver forms and especially in the intestine of fish exposed to Ag NPs. X-ray microanalysis showed the presence of silver in gills, liver and intestine and of Ag NPs in gill and liver cells. Inflammation and hyperplasia were evident in the gills after both treatments and these histopathological conditions remained at 6 mpe. According to the hepatic transcriptome analysis, at 3 days ionic silver regulated a larger number of transcripts (410) than Ag NPs (129), while at 21 days Ag NPs provoked a stronger effect (799 vs 165 regulated sequences). Gene ontology terms such as "metabolic processes" and "response to stimulus" appeared enriched after all treatments, while "immune system" or "reproductive processes" were specifically enriched after the exposure to Ag NPs. This suggests that the toxicity of Ag NPs may not be solely related to the release of Ag ions, but also to the NP form. No evident effects were found on protein oxidation or on hepatocyte lysosomal membrane stability during exposure, but effects recorded on liver lysosomes and persistent damage on gill tissue at 6 mpe could indicate potential for long-term effects in exposed fish.

Developmental and reproductive toxicity of PVP/PEI-coated silver nanoparticles to zebrafish.

Cellular and molecular mechanisms of toxicity of silver nanoparticles (NPs) and their toxicity to fish embryos after waterborne exposure have been widely investigated, but much less information is available regarding the effect of Ag NPs on physiological functions such as growth or reproduction. In this work, the effects of waterborne exposure of adult zebrafish (Danio rerio) to PVP/PEI coated Ag NPs (~5nm) on reproduction (fecundity) were investigated. Moreover, the development of the embryos after parental exposure was compared with the development of embryos after direct waterborne exposure to the NPs. For this, two experiments were run: 1) embryos from unexposed parents were treated for 5days with Ag NPs (10µgAgL-1-10mgAgL-1) and development was monitored, and 2) selected breeding zebrafish were exposed for 3weeks to 100ngAgL-1 (environmentally relevant concentration) or to 10µgAgL-1 of Ag NPs, fecundity was scored and development of resulting embryos was monitored up to 5days. Waterborne exposure of embryos to Ag NPs resulted in being highly toxic (LC50 at 120h=50µgAgL-1), causing 100% mortality during the first 24h of exposure at 0.1mgAgL-1. Exposure of adults, even at the environmentally relevant silver concentration, caused a significant reduction of fecundity by the second week of treatment and resulting embryos showed a higher prevalence of malformations than control embryos. Exposed adult females presented higher prevalence of vacuolization in the liver. These results show that Ag NPs at an environmentally relevant concentration are able to affect population level parameters in zebrafish.

Acute toxicity, bioaccumulation and effects of dietary transfer of silver from brine shrimp exposed to PVP/PEI-coated silver nanoparticles to zebrafish.

The extensive use and release to the aquatic environment of silver nanoparticles (NPs) could lead to their incorporation into the food web. Brine shrimp larvae of 24h showed low sensitivity to the exposure to PVP/PEI-coated Ag NPs (5nm), with EC50 values at 24h of 19.63mgAgL-1, but they significantly accumulated silver after 24h of exposure to 100µgL-1 of Ag NPs. Thus, to assess bioaccumulation and effects of silver transferred by the diet in zebrafish, brine shrimp larvae were exposed to 100ngL-1 of Ag NPs as an environmentally relevant concentration or to 100µgL-1 as a potentially effective concentration and used to feed zebrafish for 21days. Autometallography revealed a dose- and time-dependent metal accumulation in the intestine and in the liver of zebrafish. Three-day feeding with brine shrimps exposed to 100ngL-1 of Ag NPs was enough to impair fish health as reflected by the significant reduction of lysosomal membrane stability and the presence of vacuolization and necrosis in the liver. However, dietary exposure to 100µgL-1 of Ag NPs for 3days did not significantly alter gene transcription levels, neither in the liver nor in the intestine. After 21days, biological processes such as lipid transport and localization, cellular response to chemical stimulus and response to xenobiotic stimulus were significantly altered in the liver. Overall, these results indicate an effective dietary transfer of silver and point out to liver as the main target organ for Ag NP toxicity in zebrafish after dietary exposure.

Effects of metal-bearing nanoparticles (Ag, Au, CdS, ZnO, SiO2) on developing zebrafish embryos.

Due to the increasing commercialization of consumer and industrial products containing nanoparticles (NPs), an increase in the introduction of these materials into the environment is expected. NP toxicity to aquatic organisms depends on multiple biotic and abiotic factors, resulting in an unlimited number of combinations impossible to test in practice. The zebrafish embryo model offers a useful screening tool to test and rank the toxicity of nanomaterials according to those diverse factors. This work aims to study the acute and sublethal toxicity of a set of metal-bearing NPs displaying different properties, in comparison to that of the ionic and bulk forms of the metals, in order to establish a toxicity ranking. Soluble NPs (Ag, CdS and ZnO) showed the highest acute and sublethal toxicity, with LC50 values as low as 0.529 mg Ag l(-1) for Ag NPs of 20 nm, and a significant increase in the malformation prevalence in embryos exposed to 0.1 mg Cd l(-1) of CdS NPs of ∼4 nm. For insoluble NPs, like SiO2 NPs, acute effects were not observed during early embryo development due to the protective effect of the chorion. But effects on larvae could be expected, since deposition of fluorescent SiO2 NPs over the gill lamella and excretion through the intestine were observed after hatching. In other cases, such as for gold NPs, the toxicity could be attributed to the presence of additives (sodium citrate) in the NP suspension, as they displayed a similar toxicity when tested separately. Overall, the results indicated that toxicity to zebrafish embryos depends primarily on the chemical composition and, thus, the solubility of the NPs. Other characteristics, such as size, played a secondary role. This was supported by the observation that ionic forms of the metals were always more toxic than the nano forms, and bulk forms were the least toxic to the developing zebrafish embryos.