Antidepressants and their metabolites primarily affect lysosomal functions in the marine mussel, Mytilus galloprovincialis.

Antidepressants widely occur as emerging contaminants in marine coastal waters, with concentrations reported in the low ng/L range. Although at relatively lower levels with respect to other pharmaceuticals, antidepressants - fluoxetine (FLX) in particular - have attracted attention because of their striking effects exerted at low doses on marine invertebrates. In this study, the effects of four antidepressants including FLX, sertraline (SER), and citalopram, as members of the selective serotonin reuptake inhibitor (SSRI) class, and venlafaxine (VEN) as a member of the serotonin and norepinephrine reuptake inhibitor (SNRI) class, were evaluated in the mussel Mytilus galloprovincialis. In addition, the effects of two main metabolites of FLX and VEN, i.e., norfluoxetine (NFL) and O-desmethylvenlafaxine (ODV) respectively, were compared to those of the parent compounds. Eight concentrations of each drug (0.5-500 ng/L range) were tested on the early life stage endpoints of gamete fertilization and larval development at 48 h post fertilization (hpf). Egg fertilization was reduced by all compounds, except for VEN. Larval development at 48 hpf was affected by all SSRIs, but not by SNRIs. The above effects were significant but never exceeded 20 % of control values. Adult mussels were exposed in vivo for 7 days to environmental concentrations of the drugs (0.5, 5, and 10 ng/L) and a battery of eight biomarkers was assessed. Antidepressants primarily targeted lysosomal functions, decreasing haemocyte lysosome membrane stability (up to 70 % reduction) and increasing of the lysosome/cytosol ratio (up to 220 %), neutral lipid (up to 230 %), and lipofuscin (up to 440 %) accumulation in digestive gland. Only SER and NFL significantly affected catalase and glutathione-S-transferase activities in gills and digestive gland. NFL and ODV, were effective and sometimes more active than the parent compounds. All compounds impaired mussel health status, as indicated by the low to high stress levels assigned using the Mussel Expert System.

Bioplastic leachates characterization and impacts on early larval stages and adult mussel cellular, biochemical and physiological responses.

Bioplastics are promoted as safer alternatives to tackle the long-term persistence of conventional plastics. However, information on the potential release of additives and non-intentionally added substances (NIAS) in the surrounding environment is limited, and biological effects of the leachates have been little studied. Leachates produced from three bioplastics, i.e. compostable bags (CB), bio-polyethylene terephthalate bottles (bioPET) and polylactic acid cups (PLA), and a control polymeric material, i.e. rubber tire (TR), were examined. The chemical nature of bioplastic polyesters PET, PLA and poly (butylene adipate-co-terephthalate) (PBAT) in CB, was confirmed by analytical pyrolysis. Fragments were incubated in artificial sea water for 14 days at 20 °C in darkness and leachate contents examined by GC-MS and HPLC-MS/MS. Catalysts and stabilizers represented the majority of chemicals in TR, while NIAS (e.g. 1,6-dioxacyclododecane-7,12-dione) were the main components of CB. Bisphenol A occurred in all leachates at a concentration range 0.3-4.8 µg/L. Trace metals at concentrations higher than control water were found in all leachates, albeit more represented in leachates from CB and TR. A dose response to 11 dilutions of leachates (in the range 0.6-100%) was tested for biological effects on early embryo stages of Mytilus galloprovincialis. Embryotoxicity was observed in the whole range of tested concentrations, the magnitude of effect depending on the polymers. The highest concentrations caused reduction of egg fertilization (CB, bioPET, TR) and of larvae motility (CB, PLA, TR). TR leachates also provoked larvae mortality in the range 10-100%. Effects on adult mussel physiology were evaluated after a 7-day in vivo exposure to the different leachates at 0.6% concentration. Nine biomarkers concerning lysosomal functionality, neurotransmission, antioxidant and immune responses were assessed. All lysosomal parameters were affected, and serum lysozyme activity inhibited. Harmonized chemical and biological approaches are recommended to assess bioplastic safety and support production of sustainable bioplastics.

A Comparative Assessment of the Chronic Effects of Micro- and Nano-Plastics on the Physiology of the Mediterranean Mussel Mytilus galloprovincialis.

The ocean contamination caused by micro- and nano-sized plastics is a matter of increasing concern regarding their potential effects on marine organisms. This study compared the effects of a 21-day exposure to 1.5, 15, and 150 ng/L of polystyrene microplastics (PS-MP, 3-µm) and nanoplastics (PS-NP, 50-nm) on a suite of biomarkers measured in the Mediterranean mussel Mytilus galloprovincialis. Endpoints encompassed immunological/lysosomal responses, oxidative stress/detoxification parameters, and neurotoxicological markers. Compared to PS-MP, PS-NP induced higher effects on lysosomal parameters of general stress. Exposures to both particle sizes increased lipid peroxidation and catalase activity in gills; PS-NP elicited greater effects on the phase-II metabolism enzyme glutathione S-transferase and on lysozyme activity, while only PS-MP inhibited the hemocyte phagocytosis, suggesting a major role of PS particle size in modulating immunological/detoxification pathways. A decreased acetylcholinesterase activity was induced by PS-NP, indicating their potential to impair neurological functions in mussels. Biomarker data integration in the Mussel Expert System identified an overall greater health status alteration in mussels exposed to PS-NP compared to PS-MP. This study shows that increasing concentrations of nanoplastics may induce higher effects than microplastics on the mussel's lysosomal, metabolic, and neurological functions, eventually resulting in a greater impact on their overall fitness.

The sub-lethal impact of plastic and tire rubber leachates on the Mediterranean mussel Mytilus galloprovincialis.

Ocean contamination by synthetic polymers can represent a risk for the fitness of marine species due to the leaching of chemical additives. This study evaluated the sub-lethal effects of plastic and rubber leachates on the mussel Mytilus galloprovincialis through a battery of biomarkers encompassing lysosomal endpoints, oxidative stress/detoxification parameters, and specific responses to metals/neurotoxicants. Mussels were exposed for 7 days to leachates from car tire rubber (CTR), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS) and polyvinyl chloride (PVC), containing organic additives and metals in the ng-µg/L range. The leachate exposure affected general stress parameters, including the neutral lipid content (all leachates), the lysosomal membrane stability (PS, PP, PVC and CTR leachates) and lysosomal volume (PP, PVC and TR leachates). An increased content of the lipid peroxidation products malondialdehyde and lipofuscin was observed in mussels exposed to PET, PS and PP leachates, and PP, PVC and CTR leachates, respectively. PET and PP leachates increased the activity of the phase-II metabolism enzyme glutathione S-transferase, while a decreased acetylcholinesterase activity was induced by PVC leachates. Data were integrated in the mussel expert system (MES), which categorizes the organisms' health status based on biomarker responses. The MES assigned healthy status to mussels exposed to PET leachates, low stress to PS leachates, and moderate stress to PP, CTR and PVC leachates. This study shows that additives leached from selected plastic/rubber polymers cause sub-lethal effects in mussels and that the magnitude of these effects may be higher for CTR, PVC and PP due to a higher content and release of metals and organic compounds.

Integration of physical, geochemical and biological analyses as a strategy for coastal lagoon biomonitoring.

Coastal lagoons are complex environments threatened by natural and anthropogenic stressors. Here, we tested the effectiveness of combining physical, geochemical and chemical measurements with biomarker data obtained in field-exposed marine mussels (Mytilus galloprovincialis) as a biomonitoring strategy for a highly pressured lagoon (Pialassa Baiona, Ravenna, Italy). Data showed a spatial trend of sediment contamination by Hg, Pt, Au, Ag, Mo, Re, Cd, Pd and Zn. Local conditions of high water temperature/low conductivity were detected among selected sites. After a 30-day in situ exposure, Ag and Hg were the most bioaccumulated elements (10 and 5 folds, respectively) in mussels followed by Sb, Al, Ti and Fe. Decreased survival, lysosomal dysfunctions, increased metallothionein content and peroxisome proliferation were observed in mussels in relation to metal spatial distribution and physico-chemical fluctuations. Overall, this study provides a further confirmation of the role of biomonitoring to reliably assess the environmental quality of highly pressured lagoons.

The hidden threat of plastic leachates: A critical review on their impacts on aquatic organisms.

Plastic products are made from the essential polymer mixed with a complex blend of substances including catalyst remnants, polymerization solvents, and a wide range of other additives deliberately added to enhance the desirable characteristics of the final product. Additives include bisphenols, phthalates, flame retardants, and further emerging and legacy contaminants. With a few exceptions, additives are not chemically bound to the polymer, and potentially migrate within the material reaching its surface, then possibly leach out to the environment. Leachates are mixtures of additives, some of which belong to the list of emerging contaminants, i.e. substances that show the potential to pose risks to the environment and human health, while are not yet regulated. The review discusses the state of the art and gaps concerning the hidden threat of plastic leachates. The focus is on reports addressing the biological impacts of plastic leachates as a whole mixture. Degradation of plastics, including the weathering-driven fragmentation, and the release of additives, are analysed together with the techniques currently employed for chemically screening leachates. Because marine plastic litter is a major concern, the review mainly focuses on the effects of plastic leachates on marine flora and fauna. Moreover, it also addresses impacts on freshwater organisms. Finally, research needs and perspectives are examined, to promote better focused investigations, that may support developing different plastic materials and new regulations.

Chemical composition and ecotoxicity of plastic and car tire rubber leachates to aquatic organisms.

Synthetic polymer-based materials are ubiquitous in aquatic environments, where weathering processes lead to their progressive fragmentation and the leaching of additive chemicals. The current study assessed the chemical content of freshwater and marine leachates produced from car tire rubber (CTR), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS) and polyvinyl chloride (PVC) microplastics, and their adverse effects on the microalgae Raphidocelis subcapitata (freshwater) and Skeletonema costatum (marine) and the Mediterranean mussel Mytilus galloprovincialis. A combination of non-target and target chemical analysis revealed a number of organic and metal compounds in the leachates, including representing plasticizers, antioxidants, antimicrobials, lubricants, and vulcanizers. CTR and PVC materials and their corresponding leachates had the highest content of tentatively identified organic additives, while PET had the lowest. The metal content varied both between polymer leachates and between freshwater and seawater. Notable additives identified in high concentrations were benzothiazole (CTR), phthalide (PVC), acetophenone (PP), cobalt (CTR, PET), zinc (CTR, PVC), lead (PP) and antimony (PET). All leachates, except PET, inhibited algal growth with EC50 values ranging from 0.5% (CTR) and 64% (PP) of the total leachate concentration. Leachates also affected mussel endpoints, including the lysosomal membrane stability and early stages endpoints as gamete fertilization, embryonic development and larvae motility and survival. Embryonic development was the most sensitive parameter in mussels, with EC50 values ranging from 0.8% (CTR) to 65% (PET) of the total leachate. The lowest impacts were induced on D-shell larvae survival, reflecting their ability to down-regulate motility and filtration in the presence of chemical stressors. This study provides evidence of the relationship between chemical composition and toxicity of plastic/rubber leachates. Consistent with increasing contamination by organic and inorganic additives, the leachates ranged from slightly to highly toxic to mussels and algae, highlighting the need for a better understanding of the overall impact of plastic-associated chemicals on aquatic ecosystems.

The Multixenobiotic resistance system as a possible protective response triggered by microplastic ingestion in Mediterranean mussels (Mytilus galloprovincialis): Larvae and adult stages.

The emerging paradigm on plastic pollution in marine environments is that microsize particles (MPs) have far more subtle effects than bigger fragments, given their size range overlapping with that of particles ingested by filter-feeders. The impacts include gut blockage, altered feeding and energy allocation, with knock-on effects on widespread physiological processes. This study investigated whether ingestion of polystyrene MPs (PS-MPs) triggers protective processes in marine mussels. The Multixenobiotic resistance (MXR) system is a cytoprotective mechanism acting as an active barrier against harmful xenobiotics and a route of metabolite detoxification. Both larvae and adults were employed in laboratory experiments with different concentrations of 3-µm PS-MPs (larvae), and 3-µm and 45-µm PS-MPs (adults) matching size range of planktonic food through the mussel lifecycle. Embryos grown in the presence of 3-µm PS-MPs showed significant reduction of MXR activity and down-regulation of ABCB and ABCC transcripts encoding the two main MXR-related transporters P-glycoprotein and the Multidrug resistance-related protein, respectively. In adults, effects of PS-MPs were assessed in haemocytes and gills, which showed different modulation of MXR activity and ABCB/ABCC expression according to MP size (haemocyte and gills) or particle concentration (haemocyte). These data showed that modulation of MXR activity is part of a generalized response triggered by particle ingestion.

The impact of propranolol, 17α-ethinylestradiol, and gemfibrozil on early life stages of marine organisms: effects and risk assessment.

Pharmaceuticals are ubiquitously detected in the marine environment at the ng-µg/L range. Given their biological activity, these compounds are known to induce detrimental effects on biota at relatively low exposure levels; however, whether they affect early life stages of marine species is still unclear. In this study, a set of bioassays was performed to assess the effects of propranolol (PROP), 17-α ethinylestradiol (EE2), and gemfibrozil (GEM) on gamete fertilization and embryonic development of mussels (Mytilus galloprovincialis) and sea urchins (Paracentrotus lividus), and on the survival of seabream (Sparus aurata) larvae. Treatments of PROP (500, 5000, 50,000 ng/L), EE2 (5, 50, 500 ng/L), and GEM (50, 500, 5000 ng/L) were selected to encompass levels comparable or superior to environmental concentrations. Obtained data were tested for dose-response curve fitting and the lowest EC10/LC10 used to calculate risk quotients (RQs) based on the MEC/PNEC. No alteration was induced by PROP on the mussel gamete fertilization, while inhibitory effects were observed at environmental levels of EE2 (500 ng/L) and GEM (5000 ng/L). Fertilization was significantly reduced in sea urchin at all PROP and EE2 dosages. The 48-h exposure to all pharmaceuticals induced the onset of morphological abnormalities in either mussel or sea urchin embryos. Alterations were generally observed at environmentally relevant dosages, except for PROP in mussels, in which alterations occurred only at 50,000 ng/L. A decreased survival of seabream larvae was recorded after 96-h exposure to PROP (all treatments), EE2 (50-500 ng/L), and GEM (500 ng/L). A median RQ > 1 was obtained for all pharmaceuticals, assigning a high risk to their occurrence in marine environments. Overall, results showed that current levels of contamination by pharmaceuticals can impact early stages of marine species, which represent critical junctures in the resilience of coastal ecosystems.

Uptake and transcriptional effects of polystyrene microplastics in larval stages of the Mediterranean mussel Mytilus galloprovincialis.

The widespread occurrence of microplastics (MP) in the marine environment is cause of increasing concerns about the safety of the exposed ecosystems. Although the effects associated to the MP uptake have been studied in most marine taxa, the knowledge about their sub-lethal impacts on early life stages of marine species is still limited. Here, we investigated the uptake/retention of 3-µm polystyrene MP by early stages of the Mediterranean mussel Mytilus galloprovincialis, and the related effects on gut clearance, feeding efficiency, morphological and transcriptional parameters involved in embryo-larval development. Uptake measurements were performed on larvae at 48 h, 3, 6 and 9 days post fertilization (pf) after exposure to a range of 50-10,000 particles mL-1. At all tested pf periods, treatments resulted in a significant and linear increase of MP uptake with increasing concentrations, though levels measured at 48 h pf were significantly lower compared to 3-9 d pf. Ingested MP were retained up to 192 h in larvae's gut, suggesting a physical impact on digestive functions. No change was noted between the consumption of microalgae Nannochloropsis oculata by larvae when administered alone or in the presence of an identical concentration (2000 items mL-1) of MP. The exposure to 50-10,000 MP mL-1 did not alter the morphological development of mussel embryos; however, transcriptional alterations were observed at 50 and 500 MP mL-1, including the up-regulation of genes involved in shell biogenesis (extrapallial protein; carbonic anhydrase; chitin synthase) and immunomodulation (myticin C; mytilin B), and the inhibition of those coding for lysosomal enzymes (hexosaminidase; ß-glucorinidase; catepsin-L). In conclusion, though not highlighting morphological or feeding abnormalities, data from this study revealed the onset of physical and transcriptional impairments induced by MP in mussel larvae, indicating sub-lethal impacts which could increase their vulnerability toward further environmental stressors.

Characterization of a ß2 adrenergic receptor protein precursor in the European eel (Anguilla anguilla) and its tissue distribution across silvering.

This study provides the characterization and tissue distribution of a ß2-AR in the female European eel during silvering, aiming to better understand the adrenergic system involvement in this critical maturation event. A putative ß2-AR (ADRB2) mRNA was cloned and sequenced. Amino acid residues and motifs important for ligand binding are generally conserved across fish and between fish and mammals, although the occurrence of some sequence variabilities may explain the noted peculiarities of eel AR interaction with pharmacological ligands. The tissue distribution of the ADRB2 gene product was analyzed in five tissues of the eel at different silvering stages and compared with that of the ADRA1 mRNA encoding an α1-AR subtype. On the whole, data suggested that relative ADRA1/ADRB2 tissue expression across silvering is part of the preparatory (molecular) adjustments required to face changes in habitats and migration efforts.

A comprehensive evaluation of the environmental quality of a coastal lagoon (Ravenna, Italy): Integrating chemical and physiological analyses in mussels as a biomonitoring strategy.

This study aimed at evaluating the environmental quality of a coastal lagoon (Pialassa Piomboni, NW-Adriatic, Italy) by combining analyses of biomarkers of environmental stress and bioaccumulation of contaminants in marine mussels (Mytilus galloprovincialis) transplanted for 28days to six selected sites. Assessed biomarkers encompassed lysosomal endpoints, oxidative stress and detoxification parameters, specific responses to metals, neuro- and genotoxic substances; chemical analyses focused on PAHs, metals, pesticide and pharmaceuticals. Results showed up to a 67-fold bioaccumulation of 4- to 6-ring PAHs, including pyrene, fluoranthene, chrysene and benzo(ghi)perylene in transplanted mussels compared to reference conditions (T0). A 10-fold increase of Fe, Cr and Mn was observed, while pesticides and pharmaceuticals were not or slightly detected. The onset of a significant (p<0.05) general stress syndrome occurred in exposed mussels, as outlined by a 50-57.7% decrease in haemocytes lysosomal membrane stability and an increased lysosomal volume (22.6-26.9%) and neutral lipid storage (18.9-48.8%) observed in digestive gland. Data also revealed a diffuse lipofuscin accumulation (86.5-139.3%; p<0.05) in digestive gland, occasionally associated to a catalase activity inhibition in gill, indicating an increased vulnerability toward pro-oxidant factors. Higher levels of primary DNA damage (258%; p<0.05) and PAH accumulation were found in mussels exposed along the eastern shoreline, hosting a petrochemical settlement. Bioaccumulated metals showed a positive correlation with increased metallothionein content (85-208%; p<0.05) observed in mussels from most sites. Overall, the use of physiological and chemical analyses detected chronic alterations of the mussel health status induced by specific toxicological pathways, proving a suitable approach in the framework of biomonitoring programs of coastal lagoons.

Use of an integrated biomarker-based strategy to evaluate physiological stress responses induced by environmental concentrations of caffeine in the Mediterranean mussel Mytilus galloprovincialis.

The occurrence of caffeine (CF), a biologically active drug, has widely been documented in coastal waters, and whether its environmental concentrations do represent a threat for marine organisms is unclear. The present study aimed at assessing sub-lethal effects induced by a 7-day exposure to environmentally relevant concentrations of CF (5, 50 and 500ng/L) in the Mediterranean mussel, Mytilus galloprovincialis. CF in water and mussel tissues, and a battery of biomarkers, including lysosomal parameters of general stress, oxidative stress responses and endpoints of neurological and genetic damages, were evaluated and tested for significance vs controls (p<0.05). CF exposure triggered a significant decrease of lysosomal membrane stability in both haemocytes and digestive gland (at 50 and 500ng/L CF) and a significant increase of lysosomal content of neutral lipids (at 500ng/L CF), indicating the onset of a stress syndrome. No effects were noted on lipid peroxidation parameters, such as malondialdehyde and lipofuscin content. The activity of the antioxidant enzymes glutathione S-transferase (GST) and catalase was unmodified in gills, while a significant increase of GST activity was observed in digestive gland (at 5 and 500ng/L CF), suggesting the occurrence of GST-mediated phase II detoxifying processes. CF did not induce geno/neurotoxicity, as shown by the lack of effects on primary DNA damages and acetylcholinesterase activity. In line with its high hydrophilicity, CF did not bioaccumulate in mussel tissues. Data were integrated using the Mussel Expert System, which assigned a low stress level to mussels exposed to 500ng/L CF, whereas no alterations of animal health status were highlighted at lower dosages. This study revealed a low profile of toxicity for environmental concentrations of CF, and confirmed the suitability of an integrated biomarker-based approach to provide a comprehensive picture of the degree of stress induced by emerging contaminants in marine invertebrates.

An exploratory investigation of various modes of action and potential adverse outcomes of fluoxetine in marine mussels.

The present study investigated possible adverse outcome pathways (AOPs) of the antidepressant fluoxetine (FX) in the marine mussel Mytilus galloprovincialis. An evaluation of molecular endpoints involved in modes of action (MOAs) of FX and biomarkers for sub-lethal toxicity were explored in mussels after a 7-day administration of nominal FX concentrations encompassing a range of environmentally relevant values (0.03-300ng/L). FX bioaccumulated in mussel tissues after treatment with 30 and 300ng/L FX, resulting in bioconcentration factor (BCF) values ranging from 200 to 800, which were higher than expected based solely on hydrophobic partitioning models. Because FX acts as a selective serotonin (5-HT) re-uptake inhibitor increasing serotonergic neurotransmission at mammalian synapses, cell signaling alterations triggered by 5-HT receptor occupations were assessed. cAMP levels and PKA activities were decreased in digestive gland and mantle/gonads of FX-treated mussels, consistent with an increased occupation of 5-HT1 receptors negatively coupled to the cAMP/PKA pathway. mRNA levels of a ABCB gene encoding the P-glycoprotein were also significantly down-regulated. This membrane transporter acts in detoxification towards xenobiotics and in altering pharmacokinetics of antidepressants; moreover, it is under a cAMP/PKA transcriptional regulation in mussels. Potential stress effects of FX were investigated using a battery of biomarkers for mussel health status that included lysosomal parameters, antioxidant enzyme activities, lipid peroxidation, and acetylcholinesterase activity. FX reduced the health status of mussels and induced lysosomal alterations, as suggested by reduction of lysosomal membrane stability in haemocytes and by lysosomal accumulation of neutral lipids in digestive gland. No clear antioxidant responses to FX were detected in digestive gland, while gills displayed significant increases of catalase and glutathione-s-transferase activities and a significant decrease of acetylcholinesterase activity. Though AOPs associated with mammalian therapeutic MOAs remain important during assessments of pharmaceutical hazards in the environment, this study highlights the importance of considering additional MOAs and AOPs for FX, particularly in marine mussels.