Short-term exposure to polystyrene microplastics hampers the cellular function of gills in the Mediterranean mussel Mytilus galloprovincialis.

Plastic is undoubtedly the most useful and versatile polymeric material that man has developed in the last two centuries Despite the societal benefits, plastic is now a serious global issue because it is persistent and may bioaccumulate into aquatic biota as microplastics (MPs). This study was designed to evaluate the daily uptake and cellular effects due to a short-term (up to 72 h) exposure to 3 µm red polystyrene MPs (50 beads/mL) in the gills of the Mediterranean mussel Mytilus galloprovincialis, chosen as model species for its ecological and commercial relevance. After measuring the daily uptake of MPs and detecting their presence within the branchial epithelium at all the exposure time-points (T24, T48, T72), some cleaning mechanisms were observed by neutral and acid mucous secretions at mussel gills. The protonic Nuclear Magnetic Resonance (1H NMR)-based metabolomics, combined with chemometrics, allowed to comprehensively explore the time-dependent metabolic disorders triggered by MPs in mussel gills over the short-term trial. Specifically, the clear clustering between MP-treated mussel gills and those from control, together with the grouping for experimental time-points as depicted by the Principal Component Analysis (PCA), were due to changes in the amino acids and energy metabolism, disturbances in the osmoregulatory processes, as well as in the cholinergic neurotransmission. Moreover, as evidenced by enzymatic assays, even the oxidative defense systems and lipid metabolism were hampered by MP exposure. Overall, these findings provides the first insights into the early time-dependent mechanisms of toxicity of polystyrene MPs in marine mussels, and underline the potential environment and human health risk posed by MPs contamination.

Single and combined effects of caffeine and salicylic acid on mussel Mytilus galloprovincialis: Changes at histomorphological, molecular and biochemical levels.

Caffeine (CAF) and salicylic acid (SA) are frequently detected in waterbody, though information on their biological impact is poor. This work assesses the effects of CAF (5 ng/L to 10 µg/L) and SA (0.05 µg/L to 100 µg/L) alone and combined as CAF+SA (5 ng/L+0.05 µg/L to 10 µg/L+100 µg/L) on mussel Mytilus galloprovincialis under 12-days exposure by histomorphology of digestive gland and oxidative stress defense at molecular and biochemical levels. Besides evaluating tissue accumulation, absence of histomorphological damage and haemocyte infiltration highlighted activation of defensive mechanisms. Up-regulation of Cu/Zn-sod, Mn-sod, cat and gst combined with increased catalase and glutathione S-transferase activity were found in CAF-exposed mussels, while SA reduced ROS production and mitochondrial activity. CAF+SA exposure induced differential responses, and the integrated biomarker response (IBR) revealed more pronounced effects of SA than CAF. These results enlarge knowledge on pharmaceuticals impact on non-target organisms, emphasizing the need for proper environmental risk assessment.

Differential Cell Metabolic Pathways in Gills and Liver of Fish (White Seabream Diplodus sargus) Coping with Dietary Methylmercury Exposure.

Mercury (Hg) is a dangerous and persistent trace element. Its organic and highly toxic form, methylmercury (MeHg), easily crosses biological membranes and accumulates in biota. Nevertheless, understanding the mechanisms of dietary MeHg toxicity in fish remains a challenge. A time-course experiment was conducted with juvenile white seabreams, Diplodus sargus (Linnaeus, 1758), exposed to realistic levels of MeHg in feed (8.7 µg g-1, dry weight), comprising exposure (E; 7 and 14 days) and post-exposure (PE; 28 days) periods. Total Hg levels increased with time in gills and liver during E and decreased significantly in PE (though levels of control fish were reached only for gills), with liver exhibiting higher levels (2.7 times) than gills. Nuclear magnetic resonance (NMR)-based metabolomics revealed multiple and often differential metabolic changes between fish organs. Gills exhibited protein catabolism, disturbances in cholinergic neurotransmission, and changes in osmoregulation and lipid and energy metabolism. However, dietary MeHg exposure provoked altered protein metabolism in the liver with decreased amino acids, likely for activation of defensive strategies. PE allowed for the partial recovery of both organs, even if with occurrence of oxidative stress and changes of energy metabolism. Overall, these findings support organ-specific responses according to their sensitivity to Hg exposure, pointing out that indications obtained in biomonitoring studies may depend also on the selected organ.

Time- and dose-dependent biological effects of a sub-chronic exposure to realistic doses of salicylic acid in the gills of mussel Mytilus galloprovincialis.

Among nonsteroidal anti-inflammatory drugs (NSAIDs) commonly found in seawater and wastewater, salicylic acid (SA) represents one of the most persistent and hazardous compounds for aquatic organisms. This study was therefore designed to elucidate the biological effects of SA in mussel Mytilus galloprovincialis. During a sub-chronic exposure (12 days), mussels were exposed to five realistic concentrations of SA (C1: 0.05 µg/L; C2: 0.5 µg/L; C3: 5 µg/L; C4: 50 µg/L; C5: 100 µg/L) and gills, selected as the target organ, were collected at different time points (T3: 3 days; T5: 5 days; T12: 12 days). Exposure to SA induced no histological alterations in mussel gills, despite a relevant hemocyte infiltration was observed throughout the exposure as a defensive response to SA. Temporal modulation of glutathione S-transferase (GST), catalase (CAT), and superoxide dismutase (SOD) activities suggested the occurrence of antioxidant and detoxifying responses against SA exposure, while lipid peroxidation (LPO), except for a partial increase at T3, was prevented. Inhibition of the cholinergic system was also reported by reduced acetylcholinesterase (AChE) activity, mainly at T12. Overall, findings from this study contribute to enlarge the current knowledge on the cytotoxicity of SA, on non-target aquatic organisms, and might for the enhancement of new ecopharmacovigilance programs and optimization of the efficacy of wastewater treatment plants for mitigation of pharmaceutical pollution in coastal areas.

Comparison of cellular mechanisms induced by pharmaceutical exposure to caffeine and its combination with salicylic acid in mussel Mytilus galloprovincialis.

Urban and hospital-sourced pharmaceuticals are continuously discharged into aquatic environments, threatening biota. To date, their impact as single compounds has been widely investigated, whereas few information exists on their effects as mixtures. We assessed the time-dependent biological impact induced by environmental concentrations of caffeine alone (CAF; 5 ng/L to 10 µg/L) and its combination with salicylic acid (CAF+SA; 5 ng/L+0.05 µg/L to 10 µg/L+100 µg/L) on gills of mussel Mytilus galloprovincialis during a 12-day exposure. Although no histological alteration was observed in mussel gills, haemocyte infiltration was noticed at T12 following CAF+SA exposure, as confirmed by flow cytometry with increased hyalinocytes. Both the treatments induced lipid peroxidation and cholinergic neurotoxicity, which the antioxidant system was unable to counteract. We have highlighted the biological risks posed by pharmaceuticals on biota under environmental scenarios, contributing to the enhancement of ecopharmacovigilance programmes and amelioration of the efficacy of wastewater treatment plants.

Embryotoxicity of polystyrene microplastics in zebrafish Daniorerio.

In the recent years, increasing scientific and societal concern has been raised over the presence and accumulation of plastic debris in the environment and the effects of microplastics (MPs) that can easily interact with biota. In order to elucidate the impact of MPs at the critical development stages of freshwater fish species, a fish embryo toxicity test was herein performed on the zebrafish Danio rerio, exposed to 10 µm polystyrene MPs at 200 particles/mL for 120 hpf. After exposure, accumulation of MPs in larvae was measured, survival, hatching and larvae development were monitored and the oxidant/anti-oxidant responses and cellular detoxification evaluated. No impact on survival of developing zebrafish was revealed, but a moderate delay in hatching was observed. Alterations in larvae development were recorded with zebrafish exhibiting serious deformities, mainly at the level of column and tail, as well as a compromised integrity of the visual structure of the eyes. Moreover, increased levels of gene transcription involved in the oxidative stress (sod1, sod2 and cat) and in cellular detoxification (gst and cyp) were also detected in MPs-exposed zebrafish larvae. Overall, this research work provides new insights on the ecotoxicological impact of polystyrene MPs on the critical developmental stages of a freshwater fish species, therefore enhancing the current knowledge of the environmental risk posed by MPs to the aquatic ecosystem.