Nanoplastics are bioaccumulated in fish liver and muscle and cause DNA damage after a chronic exposure.

The extent of the widespread, planetary contamination by plastic waste is difficult to fully capture. Nanoplastics (NPs) are currently in the center of research concerning plastic litter, both for the analytical challenges they pose and for their potential to provoke hazardous effects in organisms. However, there are still many unanswered questions in this multidisciplinary field, with a crucial missing piece being the quantification of NPs in fish tissues after in vivo exposures. Another relevant question that is still greatly unexplored is how a chronic exposure to NPs will affect fish health. This study aims to provide answers to both of these relevant knowledge gaps. To this end, goldfish (Carassius auratus) were exposed to 44 nm polystyrene (PS)-NPs via water for 30 days. Following the exposure, gastrointestinal tract, liver and muscle were sampled for PS-NPs analysis by means of size exclusion chromatography coupled to high resolution mass spectrometry. PS-NPs were detected in all liver and muscle samples of exposed fish, with higher concentrations in liver than in muscle, whereas no PS-NPs were detected in the gastrointestinal tract. Nevertheless, exposure to PS-NPs did not induce changes in hematology parameters nor in cortisol and glucose levels in plasma. On the other hand, even a relatively low concentration of PS-NPs was able to cause DNA damage, measured by an increase in erythrocyte nuclear abnormalities, suggesting that PS-NPs can reach the cell nucleus and cause genotoxicity. These results show for the first time that PS-NPs find their way to fish muscle after chronic exposure, where they bioaccumulate, but do not alter fish survival nor hematological or physiological stress indicators. The accumulation of PS-NPs in fish muscle can represent a threat to human health as a possible route of exposure to small-sized plastics. The present results in a model fish species open windows for future studies in edible fish species.

A baseline study on the impact of nanoplastics on the portals of entry of xenobiotics in fish.

Mediterranean waters are particularly vulnerable to plastic pollution, with plastic particles concentrations comparable to those found in oceanic gyres. This work aimed to assess the impact of polymethylmethacrylate nanoplastics (PMMA-NPs) on the most important mucosal barriers of the gilthead seabream (Sparus aurata), a highly consumed fish species in the Mediterranean area. Fish were waterborne exposed to NPs (0.001-10 mg/L) for 24 and 96 h, and biochemical parameters associated with oxidative status (total oxidative status and total antioxidant capacity) and immune function (adenosine deaminase, ADA, acetylcholinesterase activity, AChE, and esterase activity, EA) were assessed in gills, intestine, and skin. In intestine, PMMA-NPs led to oxidative status alterations and decreased ADA and EA. In gills, PMMA-NPs induced EA decrease and AChE activity increase. Total protein values were significantly increased in skin. Overall, more alterations were observed in intestine, suggesting it may be one of the most affected tissues by exposure to NPs.

Short-term exposure to polymethylmethacrylate nanoplastics alters muscle antioxidant response, development and growth in Sparus aurata.

Polymethylmethacrylate (PMMA) plastic fragments have been found abundant in the environment, but the knowledge regarding its effects on the physiology of aquatic animals is still poorly studied. Here the short-term (96 h) effects of waterborne exposure to PMMA nanoplastics (PMMA-NPs) on the muscle of gilthead sea bream (Sparus aurata) fingerlings was evaluated at a concentration range that includes 0.001 up to 10 mg/L. The expression of key transcripts related to cell stress, tissue repair, immune response, antioxidant status and muscle development, together with several biochemical endpoints and metabolic parameters. Results indicate that exposure to PMMA-NPs elicit mildly antioxidant responses, enhanced the acetylcholinesterase (AChE) activity, and inhibited key regulators of muscle development (growth hormone receptors ghr-1/ghr-2 and myostatin, mstn-1 transcripts). However, no effects on pro-inflammatory cytokines (interleukin 1ß, il1ß and tumor necrosis factor α, tnfα) expression nor on the levels of energetic substrates (glucose, triglycerides and cholesterol) were found.

Immuno-modulatory effects of nanoplastics and humic acids in the European seabass (Dicentrarchus labrax).

Pernicious effects of plastic particles, emergent contaminants worldwide, have been described in different species. In teleost species, alterations of immune function after exposure to nanoplastics (NPs) have been reported, but the interaction with cortisol - hypothalamic-pituitary-adrenal (HPI) axis has not yet been explored. Furthermore, the role of dissolved organic matter on the effects of NPs is poorly known. Thus, the aims of this research were to assess if polystyrene NPs (PSNPs) acted as a stressor on juvenile European seabass (Dicentrarchus labrax), interfering with the immune response, as well as to elucidate if humic acids (HA) modulated the potential effects of PSNPs. A short-term exposure to PSNPs and HA elicited an immuno-modulatory response, with an activation of steroidogenic stress-related pathways. An upregulation of anti-inflammatory cytokine (il10, tgfb) and stress-related (mc2r, gr1) transcripts were observed after exposure to HA and PSNPs both individually and in co-exposure. No notable alteration of inflammatory markers was consistently found, which may reflect a protective anti-inflammatory effect of HA in the presence of PSNPs. Nevertheless, there seems to be a more complex interaction between both components. Overall, data show that understanding the interaction of NPs with dissolved organic substances is key to deciphering their environmental risks.

Waterborne exposure of gilthead seabream (Sparus aurata) to polymethylmethacrylate nanoplastics causes effects at cellular and molecular levels.

This study evaluated the effect of a short-term exposure to 45 nm polymethylmethacrylate nanoplastics (PMMA-NPs) on the gilthead seabream (Sparus aurata), by assessing biomarkers at different levels of biological organization in liver and plasma. Fish were exposed via water to PMMA-NPs (0, 0.001, 0.01, 0.1, 1 and 10 mg L-1) and sampled after 24 and 96 h. Results showed a general up-regulation of mRNA levels of key genes associated with lipid metabolism (e.g. apolipoprotein A1 and retinoid X receptor). Together with the modulation of the lipid pathway genes we also found a global increase in cholesterol and triglycerides in plasma. Antioxidant-related genes (e.g. glutathione peroxidase 1) were also up-regulated after 24 h of exposure, but their expression levels returned to control afterwards. Total antioxidant capacity (TAC) was increased throughout the experiment, however at 96 h the antioxidant capacity became less efficient, reflected by an increase in the total oxidative status (TOS). Concomitantly, we found an increase in the erythrocytic nuclear abnormalities (ENAs) throughout the trial. Altogether, PMMA-NPs activated the organism's antioxidant defenses and induced alterations in lipid metabolism pathways and genotoxicity in the blood cells of gilthead seabream.

Divergent personalities influence the myogenic regulatory genes myostatin, myogenin and ghr2 transcript responses to Vibrio anguillarum vaccination in fish fingerlings (Sparus aurata).

Myogenic regulators of muscle development, metabolism and growth differ between fish species in a context-specific manner. Commonly, the analysis of environmental influences on the expression of muscle-related gene regulators in teleosts is based on differences in swimming performance, feeding behaviour and stress-resistance, but the evaluation of behavioural phenotyping of immune and stress-related responsiveness in skeletal muscle is still scarce. Here we challenge proactive and reactive fingerlings of gilthead sea bream (Sparus aurata), one of the most commonly cultured species in the Mediterranean area, with highly pathogenic O1, O2α and O2ß serotypes of Vibrio anguillarum, a widespread opportunistic pathogen of marine animals, to analyse skeletal muscle responses to bath vaccination. Transcripts related to inflammation (interleukin 1ß, il1ß; tumour necrosis factor-α, tnfα; and immunoglobulin M, igm), and muscle metabolism and growth (lipoprotein, lpl; myostatin, mstn-1; myogenin; and growth hormone receptors type I and II, ghr1 and ghr2, respectively) were analysed. Biochemical indicators of muscle metabolism and function (creatine kinase, CK, aspartate aminotransferase, AST; esterase activity, EA; total antioxidant status, TAC and glucose) were also determined. Our results indicate that proactive, but not reactive, fish respond to Vibrio vaccination by increasing the expression levels of mstn-1, myogenin and ghr2 transcripts at short-/medium- term (1 to 3 days' post vaccination). No effect of vaccination was observed in immune indicators or biochemical parameters in either phenotypes, except for elevated levels of EA in reactive fish one-week post vaccination. This suggests that behavioural divergence should be taken into account to evaluate the crosstalk between immune, metabolic and growth processes in muscle of immune-challenged fish.

Variations in the immune and metabolic response of proactive and reactive Sparus aurata under stimulation with Vibrio anguillarum vaccine.

Environmental insults, such as exposure to pathogens, modulate the behavioural coping style of animals to stressors, and repeated exposure to stressful environments may lead to species-specific infection phenotypes. To analyse the influence of stress behavioural phenotypes on immune and metabolic performance, gilthead sea bream (Sparus aurata L.) were first screened for proactive and reactive coping styles. Once characterized, both behavioural phenotypes fish groups were bath vaccinated with bacterin from Vibrio anguillarum, an opportunistic widespread pathogen of fish. Gills and liver were sampled at 0 (control group), 1, 3 and 7 days post-vaccination. Immune-, oxidative stress- and metabolic-related transcripts (il1ß, tnfα, igm, gpx1, sod, cat, lpl, ghr1 and ghr2), metabolic endpoints (glucose, cholesterol and triglycerides), hepatic health indicators (aspartate aminotransferase, alanine transaminase and alkaline phosphatase), oxidative stress status (esterase activity, total antioxidant capacity and total oxidative status) and stress biomarkers (cortisol) were determined. Present results indicate that screening for coping styles in the gilthead sea bream segregated the two distinct phenotypes as expected: proactive and reactive. Results also indicate that under bath vaccination proactive fish show high immune response and lower metabolism, whereas reactive fish show low immune and higher metabolic responses.

Effects of polymethylmethacrylate nanoplastics on Dicentrarchus labrax.

The present study aimed to evaluate the effects of ~45 nm nanoplastics (NPs) on the marine fish Dicentrarchus labrax after a short-term exposure. Animals were exposed to a concentration range of NPs for 96 h and liver, plasma and skin mucus were sampled. Assessed endpoints included biochemical biomarkers and expression of genes related to lipid metabolism, immune system and general cell stress. Abundance of mRNA transcripts related to lipid metabolism, pparα and pparγ, were significantly increased after exposure to NPs. Biochemical endpoints revealed decreased esterase activity levels in plasma, suggesting that the immune system of fish might be compromised by exposure to NPs. Moreover, significantly lower levels of alkaline phosphatase were found in the skin mucus of animals exposed to NPs. The present results suggest that NPs may represent a hazard to this marine fish, potentially interfering with the metabolism of lipids and the correct function of the immune response.

Effects of nanoplastics on Mytilus galloprovincialis after individual and combined exposure with carbamazepine.

Plastic pollution is a worldwide problem, highlighted by the fact that plastic materials degrade into nano-size particles (<100 nm), potentially becoming more bioavailable as well as a source of entry of other contaminants into organisms. The present study aimed to assess the effects of polystyrene nanoplastics (PS), individually or combined with carbamazepine (Cbz), on the Mediterranean mussel, Mytilus galloprovincialis. For this purpose, mussels were exposed for 96 h to a concentration range of PS (from 0.05 up to 50 mg L-1), to Cbz (6.3 µg L-1) alone and to the mixture of PS + Cbz (0.05 mg L-1+ 6.3 µg L-1). Molecular and biochemical biomarkers were assessed in the digestive glands, gills and haemolymph. The abundance of mRNA in the digestive glands and gills revealed significant alterations in the expression of genes associated with biotransformation, DNA repair, cell stress-response and innate immunity. Combined exposure of PS + Cbz induced significant downregulation in gene expression (e.g., hsp70) when compared to individual exposure. Total oxidant status increased in digestive glands after exposure to 0.5 mg L-1 PS. Moreover, increased total antioxidant capacity and esterase activity were observed for PS 50 mg L-1, in digestive glands and gills, respectively. The PS induced effects on neurotransmission, measured as inhibition of cholinesterase activity in haemolymph. Genotoxicity was found in haemocytes after exposure to PS, Cbz and their mixture. Moreover, lipid peroxidation was observed for 0.05 mg L-1 PS exposure, showing that nanoplastics can induce oxidative damage. The present study demonstrated that PS, even at low concentrations, led to alterations on the assessed mussels' endpoints.