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Abstract Human pharmaceuticals represent a major challenge in natural environment. A better knowledge on their mechanisms of action and adverse effects on cellular pathways is fundamental to predict long-term consequences for marine wildlife. The FTIRI Imaging (FTIRI) spectroscopy represents a vibrational technique allowing to map specific areas of non-homogeneous biological samples, providing a unique biochemical and ultrastructural fingerprint of the tissue. In this study, FTIRI technique has been applied, for the first time, to characterize (i) the chemical building blocks of digestive glands of Mytilus galloprovincialis, (ii) alterations and (iii) resilience of macromolecular composition, after a 14-days exposure to 0.5 µg/L of carbamazepine (CBZ), valsartan (VAL) and their mixture, followed by a 14-days recovery period. Spectral features of mussels digestive glands provided insights on composition and topographical distribution of main groups of biological macromolecules, such as proteins, lipids, and glycosylated compounds. Pharmaceuticals caused an increase in the total amount of protein and a significant decrease of lipids levels. Changes in macromolecular features reflected the modulation of specific molecular and biochemical pathways thus supporting our knowledge on mechanisms of action of such emerging pollutants. Overall, the applied approach could represent an added value within integrated strategies for the effects-based evaluation of environmental contaminants.

Chemical pollution in marine ecosystems is a factor of stress interacting in multiple and complex ways with other major causes of deterioration, such as warming seas due to climate change. Here we surveyed epibenthic communities from a shipwreck in the Levantine Basin for temporal and spatial changes in the community in relation to chronic oil pollution, comparing results collected from an area of the wreck characterized by chronic oil leakage with another area not affected by oil. Polycyclic aromatic hydrocarbons (PAHs) bioaccumulation analyses were integrated with characterization of the efficiency of xenobiotics biotransformation processes and antioxidant network of the scleractinian coral Madracis pharensis, chosen as bioindicator species. Results highlighted the two areas hosting different epibenthic communities over a period of 11 years. Significant changes in the percentage cover of M. pharensis could be the result of recent mass mortality associated to Marine Heat Waves. Biological investigation conducted in M. pharensis tissues revealed an increased content of PAHs in specimens collected from the oil-impacted area, coupled with an increased capability of oxyradicals scavenging capacity and a lower functionality of phase II biotransformation mechanisms associated to glutathione S-transferase. Overall, the results suggest that M. pharensis has the capability to develop cellular and physiological adaptations to chemical-mediated stress, with yet unknown possible energy trade-offs to sustain stress response.

The effective management of marine ecosystems in the face of growing anthropogenic pressures requires the integration of data from different ecological components. Holistic approaches to evaluate the ecological status of marine ecosystems are still scarce, likely due to the challenge of integrating the complex information from a variety of indicators. In this study, we provided an application of a quantitative Weight-Of-Evidence (WOE) model based on the Sediqualsoft® software, combining environmental and biological data to assess ecological risk in soft-bottom habitats within Natura sites 2000 in the Northern Adriatic Sea (Mediterranean Sea). Here, the WOE approach combined three lines of evidence (LOE): chemical characterization (LOE1), ecotoxicological properties (LOE4), and benthic community status (LOE5). A separate hazard quotient was derived for each LOE prior to a weighted integration into a synthetic WOE assessment. The chemical analysis of the sediments revealed concentrations of pollutants far lower the reference limits, except for As and Hg and for polycyclic aromatic hydrocarbons which determined a ‘Slight’ to ‘Severe’ chemical hazard in coastal sites. Ecotoxicological hazard was rated as ‘Absent’ at all sampling stations, and the analysis of benthic communities indicated ‘undisturbed’ conditions for most sites. The WOE approach classified the overall ecological risk to be ‘Absent’ for offshore sites and ‘Slight’ in nearshore sites. Although results suggested a general low ecological risk, the potential for future risks is recognized, especially in coastal areas, due to well-known sediment pollution in the region. The application of the WOE approach may represent a valuable tool for managing marine protected sites, and to characterize the overall ecological status of these areas and improve conservation strategies in highly anthropized environmental contexts.

Cigarette butts (CBs), one of the most common litter items found on beaches, represent a still unexplored environmental hazard. This study aimed at a multidisciplinary characterization of their toxicological risks on marine organisms integrating chemical analyses of released compounds with a wide panel of biological responses, such as ecotoxicological bioassays on species of different trophic positions, molecular responses in an ex vivo model (Precision-Cut Tissue Slices, PCTS of mussels digestive glands), bioavailability and cellular biomarkers in mussels exposed to CBs in laboratory experiments. Trace metals, aliphatic and polycyclic aromatic hydrocarbons, nicotine and cotinine were released in artificial seawater after 24 h which determined a significant inhibition of bacterial bioluminescence, oyster embryo development and growth in different algal species. Modulation of peroxisomal proliferation and antioxidant gene expression was observed in mussels PCTS, while the in vivo exposure determined accumulation of chemicals and significant alterations of immune system, antioxidant and neurotoxic responses, peroxisomal proliferation and genotoxic damage. Using a quantitative Weight of Evidence model, the risks of CBs to the marine environment were summarized, highlighting the importance of integrating chemical analyses, batteries of ecotoxicological bioassays, molecular and cellular biomarkers to assess the impact of these hazardous materials on marine environment.

Abstract Chronic exposure to pollutants affects natural populations, creating specific molecular and biochemical signatures. In the present study, we tested the hypothesis that chronic exposure to pollutants might have substantial effects on the Manila clam hologenome long after removal from contaminated sites. To reach this goal, a highly integrative approach was implemented, combining transcriptome, genetic and microbiota analyses with the evaluation of biochemical and histological profiles of the edible Manila clam Ruditapes philippinarum, as it was transplanted for 6 months from the polluted area of Porto Marghera (PM) to the clean area of Chioggia (Venice lagoon, Italy). One month post‐transplantation, PM clams showed several modifications to its resident microbiota, including an overrepresentation of the opportunistic pathogen Arcobacter spp. This may be related to the upregulation of several immune genes in the PM clams, potentially representing a host response to the increased abundance of deleterious bacteria. Six months after transplantation, PM clams demonstrated a lower ability to respond to environmental/physiological stressors related to the summer season, and the hepatopancreas‐associated microbiota still showed different compositions among PM and CH clams. This study confirms that different stressors have predictable effects in clams at different biological levels and demonstrates that chronic exposure to pollutants leads to long‐lasting effects on the animal hologenome. In addition, no genetic differentiation between samples from the two areas was detected, confirming that PM and CH clams belong to a single population. Overall, the obtained responses were largely reversible and potentially related to phenotypic plasticity rather than genetic adaptation. The results here presented will be functional for the assessment of the environmental risk imposed by chemicals on an economically important bivalve species.

In the last century, many Mediterranean coastal areas have been subjected to anthropogenic disturbances from industrial activities, uncontrolled landfills, shipyards, and high maritime traffic. The Augusta Bay (eastern Sicily, Italy) represents an example of a strongly impacted coastal environment with an elevated level of sediments contamination due to the presence of one of the largest European petrochemical plants, combined with an extensive commercial and military harbor. The most significant contaminants were represented by mercury (Hg) and hexachlorobenzene (HCB), derived from a former chlor-alkali plant, and other organic compounds like polycyclic aromatic hydrocarbons (PAHs) and polychlorobiphenyls (PCBs). Since the 1970s, Augusta Bay has become internationally recognized as a contaminated marine environment, although very little information is available regarding the temporal trend of contaminants bioavailability and biological impacts on aquatic organisms. In this study, the Hg and HCB concentrations were investigated over 10 years (from 2003 to 2013) in sediments and invertebrate and vertebrate organisms; these two contaminants' ecotoxicity was further evaluated at a biochemical and cellular level by analyzing the induction of organic biotransformation processes and DNA damages. The results showed high concentrations of Hg and HCB in sediments and their strong bioaccumulation in different species with significantly higher values than those measured in reference sites. This trend was paralleled by increased micronuclei frequency (DNA damage biomarker) and activity of the biotransformation system. While levels of chemicals in sediments remained elevated during the time course, their bioavailability and biological effects showed a gradual decrease after 2003, when the chlor-alkali plant was closed. Environmental persistence of Hg and HCB availability facilitates their bioaccumulation and affects the health status of marine organisms, with possible implications for environmental risk, pollutants transfer, and human health.

Textile microfibers (MFs) have natural (e.g. cotton, wool and silk) or synthetic origin (e.g. polyester and polyamide), and are increasingly documented in the marine environment. Knowledge on their biological effects in marine organisms is still limited, and virtually unexplored is their capability to modulate the responsiveness toward other stressors, including those of emerging relevance under global changes scenario. With such background, the aims of this study were to i) determine the ingestion and biological effects of MFs, discriminating between synthetic and natural ones, and ii) elucidate the possibility that MFs alter the responsiveness toward additional stressors occurring at a later stage, after exposure. Adult mussels Mytilus galloprovincialis were exposed for 14 days to a high but still environmentally realistic concentration of 50 MFs L-1 of either polyester (618 ± 367 µm length, 13 ± 1 µm diameter), polyamide (566 ± 500 µm length, 11 ± 1 µm in diameter) or cotton (412 ± 342 µm length, 16 ± 4 µm diameter). After the exposure, mussels were left for 7 days to recover at control temperature (23°C) or exposed to a heatwave condition (27°C). At the end of each phase (exposure – recovery – heat stress), MFs ingestion-elimination was evaluated, along with a wide panel of biological responses, including neuro-immune and antioxidant systems alterations, lipid metabolism and onset of cellular damages. Results were elaborated through a Weight of Evidence approach to provide synthetic hazard indices based on both the magnitude and toxicological relevance of observed variations. Beside limited differences in retention and elimination of MFs, biological analyses highlighted disturbance of the immune system and demand of protection toward oxidative insult, particularly evident in mussels exposed to synthetic-MFs. Carry-over effects were observed after 7 days of recovery: organisms that had been previously exposed to MFs showed a higher susceptibility of the neuroendocrine-immune system and lipid metabolism to thermal stress compared to un-exposed mussels. Overall, this study provided evidence of direct cellular effects of MFs, emphasizing differences between synthetic and natural ones, and highlighted their capability to modulate organisms’ susceptibility toward additional stressors, as those predicted for future changes in marine ecosystems.

Microplastics (MPs) and brominated flame retardants (BFRs) represent a synergic threat for aquatic environments and organisms' health status, with an additional concern over food quality and food security for species of commercial interest. In this study, the ingestion of MPs, levels of polybrominated diphenyl ethers (PBDEs), and hexabromocyclododecane (HBCDs) were assessed in organisms from two lakes of Central Italy, with the aims of exploring the bioavailability of these pollutants in freshwater environments, the possible translocation of MPs from digestive to edible tissues, and the relationship between MPs ingestion and bioaccumulation of BFRs. The fish Perca fluviatilis, Anguilla anguilla, Carassus auratus, and the crayfish Procambarus clarkii, all species commercialized for human consumption, were caught in Trasimeno Lake; moreover, P. fluviatilis and Rutilus rutilus were also sampled in Piediluco, a lake strongly influenced by industrial and anthropogenic activities, where fishing for commercial purpose is forbidden. With the exception of C. auratus which showed the highest frequency of MPs ingestion (75%), species from Piediluco Lake exhibited a more elevated percentage of organisms positive to MPs ingestion (45%) and higher levels of PBDEs and HBCDs (mean values of 343 and 792 pg/g, respectively, in P. fluviatilis; 445 and 677 pg/g, respectively, in R. rutilus) than Trasimeno species (25% frequency of MPs ingestion, mean values between 6 and 163 pg/g for PBDEs and 5-107 pg/g for HBCDs). Polyester fibers dominated among MP typologies, and a high occurrence of man-made natural fibers was recorded. The number of MPs extracted in the gastrointestinal tracts of fish and soft bodies of crayfishes positive to MPs ingestion ranged between 1 and 2, whereas no MPs were found in fish filets. Given these results, the risk related to human consumption of Trasimeno organisms appears very low, whereas further investigations are required to better elucidate the possible role of MPs pollution in modulating chemical bioaccumulation in edible tissues. This study contributed to assess both environmental quality and food safety, reinforcing the use of bioindicator species for monitoring plans, in accordance with European recommendations.

The use of multidisciplinary investigations for the evaluation of aquatic ecosystems status is recommended by the European Directives, but it is still a challenging practice. In this study, we apply a quantitative weight of evidence (WOE) approach (Sediqualsoft) for the integration of extensive data obtained from different typologies of investigations, obtained over a 4-year monitoring study of dredging activities in the harbor of Leghorn (Italy). During different phases of such operations, selected sites have been characterized in terms of levels of trace metals and polycyclic aromatic hydrocarbons in sediments, bioaccumulation of contaminants, and a wide battery of biomarkers in transplanted mussels, ecotoxicological effects of sediments through a battery of bioassays (algal growth inhibition, bioluminescence inhibition, and embryotoxicity tests), and the status of benthic communities. Each typology of data, line of evidence (LOE), has been initially elaborated through dedicated logical flowcharts and algorithms providing specific hazard indices, followed by their overall integration based on different weights assigned to each LOE. This approach allowed to summarize more than 10,000 results, reaching robust conclusions on environmental impact during various phases of dredging operations. This approach was confirmed as a useful tool for monitoring the risk, supporting a “site-oriented” decision making process by providing stakeholders simple interpretation of complex data.

Marine bivalves are frequently exposed to multiple co-occurring challenges such as temperature extremes and anthropogenic pollution. These stressors can elicit negative effects on several biological pathways, including antioxidant and neuroendocrine-immune (NEI) systems, leading to immune disorders and altered immunocytes functionality. Since interactive mechanisms of action and resulting outcomes are still scarcely explored, we examined the single and combined effects of increased temperature (+5°C) and cadmium (20 μg/L) in the Mediterranean mussel Mytilus galloprovincialis. Analyzed parameters included cholinergic system in gills and hemolymph (acetylcholinesterase activity, AChE), total oxyradical scavenging capacity in gills and key functional processes in hemocytes, including lysosomal membrane stability, hemocytes subpopulations ratio, phagocytosis capacity, and onset of genotoxic damage. Results highlighted interactive inhibition of AChE activity along to a concomitant increased total oxyradical scavenging capacity, confirming neuroendocrine-immune system (NEI) disturbance and oxidative pressure. In hemocytes, lysosomal membrane stability and granulocytes:hyalinocytes ratio revealed additive effects of stressors, while a consistent reduction of phagocytosis was caused by temperature stress, with a slightly antagonistic effect of cadmium. Pearson’s correlation statistics provided either positive or negative relationships between investigated parameters and stressors, allowing to hypothesize putative mechanism of immune system functional alterations. The overall results suggest that the occurrence of short-term events of increased temperature and concomitant metal exposure could elicit interactive and negative effects on immune system efficiency of marine organisms.

DNA methylation is an essential epigenetic mechanism influencing gene expression in all organisms. In metazoans, the pattern of DNA methylation changes during embryogenesis and adult life. Consequently, differentiated cells develop a stable and unique DNA methylation pattern that finely regulates mRNA transcription during development and determines tissue-specific gene expression. Currently, DNA methylation remains poorly investigated in mollusks and completely unexplored in Mytilus galloprovincialis. To shed light on this process in this ecologically and economically important bivalve, we screened its genome, detecting sequences homologous to DNA methyltransferases (DNMTs), methyl-CpG-binding domain (MBD) proteins and Ten-eleven translocation methylcytosine dioxygenase (TET) previously described in other organisms. We characterized the gene architecture and protein domains of the mussel sequences and studied their phylogenetic relationships with the ortholog sequences from other bivalve species. We then comparatively investigated their expression levels across different adult tissues in mussel and other bivalves, using previously published transcriptome datasets. This study provides the first insights on DNA methylation regulators in M. galloprovincialis, which may provide fundamental information to better understand the complex role played by this mechanism in regulating genome activity in bivalves.

During 2006–2008, a pipeline was buried in Vallona lagoon in the Northern Adriatic Sea (Italy). A Before-During-After environmental monitoring programme was scheduled to monitor possible alterations. Bioaccumulation of metal(loid)s, BTs (butyltins) and HMW-PAHs (High Molecular Weight Polycyclic Aromatic Hydrocarbons), and biological responses (Condition index, air Survival—LT50, Acetylcholinesterase, Micronuclei—MN, acyl-CoA oxidase, catalase, malondialdehyde—MDA, and the total oxyradical scavenging capacity—TOSCA) were investigated in Manila clams (Ruditapes philippinarum) from November 2005 to June 2015. In opera (IO) results showed higher levels of HMW-PAHs (73 ± 13 ng/g), BTs (90 ± 38 ng Sn/g) and increasing levels of Pb (6.7 ± 0.7 mg/kg) and Zn (73.6 ± 6.08 mg/kg) probably linked to works. Other contaminant alterations, especially metal(loid)s, before (AO) and after (PO) the burial, were attributed to a general condition of the area and mostly unrelated to works. In addition, LT50, MN and TOSCA showed alterations, probably due to hotspots occurring in IO. TOSCA and MDA increases, right after the burial, were considered delayed responses of IO, whilst other biological responses detected later were connected to the general condition of the area. Comparisons between results of Principal Component Analyses (PCAs) highlighted partial overlapping of AO and IO, whilst PO differed only for contaminants. Visual correlations between PCAs highlighted the biomarkers’ latter response.

Contaminants of emerging concern and ocean changes are key environmental stressors for marine species with possibly synergistic, but still unexplored, deleterious effects. In the present study the influence of a simulated ocean acidification scenario (pH = 7.6) was investigated on metabolism and sub-lethal effects of carbamazepine, CBZ (1 µg/L), chosen as one of the most widely diffused pharmaceuticals in marine organisms. A multidisciplinary approach was applied on mussels, M. galloprovincialis, integrating measurement of drug bioaccumulation with changes in the whole transcriptome, responsiveness of various biochemical and cellular biomarkers including immunological parameters, lipid and oxidative metabolism, onset of genotoxic effects. Chemical analyses revealed a limited influence of hypercapnia on accumulation and excretion of CBZ, while a complex network of biological responses was observed in gene expression profile and functional changes at cellular level. The modulation of gamma-aminobutyric acid (GABA) pathway suggested similarities with the Mechanism of Action known for vertebrates: immune responses, cellular homeostasis and oxidative system represented the processes targeted by combined stressors. The overall elaboration of results through a quantitative Weight of Evidence model, revealed clearly increased cellular hazard due to interactions of CBZ with acidification compared to single stressors.

European legislative framework supports a multidisciplinary strategy of environmental monitoring because the environment is a complex system of abiotic and biotic interactions, and it should not be studied and protected by looking at one single aspect. The resulting heterogeneous data request to be carefully processed, and the application of Weight of Evidence (WOE) approaches is, thereby, an integrated validated tool. In this perspective, the present study aims to: (i). apply a specific model (Sediqualsoft) based on the WOE approach for processing multidisciplinary data related to four Lines Of Scientific Evidence (LOEs: chemical analyses, ecotoxicological bioassays, bioaccumulation tests and biomarkers) regarding sediments from an area of the Adriatic Sea; (ii). evaluate the usefulness of this specific integrated approach to estimate the potential environmental hazard due to the presence of gas production platforms respect to the traditional approach of sediment chemical characterization. This latter recognized a more contaminated area within 100 m of the platforms in which the Sediqualsoft model showed the presence of a chemical hazard, ranging from moderate to severe, and identified the contaminants (e.g., some metals, benzo(a)pyrene and acenaphthene) most responsible for it. A significant hazard also appeared in some of the sampled stations by analyzing the LOEs dedicated to the biological responses. The choice of different reference values (regulatory limits, threshold values or concentrations measured in the control area) influenced only the chemical hazard but not the overall integration with other LOEs, showing a moderate hazard for the majority of stations. Here, the concentrations measured in a control area are firstly proposed as possible reference values in Sediqualsoft model applications; this could be of particular relevance when Sediment Quality Guidelines are not available for all the measured substances. Moreover, the limitations of a conventional pass-to-fail approach or worst-case scenario were overcoming interpreting whole chemical and ecotoxicological results. All data analyzed and discussed confirm Sediqualsoft as a suitable tool for processing environmental data, including those first processed here on a monitoring scenario of gas platforms that discharge Produced Water into the sea.

Contamination status of coastal areas of Finland (northern Baltic Sea) markedly affected by anthropogenic activities (harbors, shipyards and maritime activity, industry, municipal and agricultural inputs, legacy contamination) was assessed for the first time using the weight of evidence (WOE) approach. The key element of the study was the caging (transplantation) of Baltic mussels (Mytilus trossulus) for the measurement of tissue accumulation of polycyclic aromatic hydrocarbons (PAHs) and applying a suite of biomarkers of biological effects of contaminants. Additional variables included in the assessment were trace metals in seawater, macrozoobenthos, near-bottom oxygen levels and eutrophication indicators. The chemical parameters were supported by passive sampling of PAHs and organotins at the study sites. The integrated approach combining all the line of evidence (LOE) variables into the WOE showed separation of some sites as more affected by hazardous substances than others, with the most contaminated areas found around harbor and ship yard areas. The contaminant levels measured in the different matrices were not alarmingly high at none of the areas compared to many other areas within or outside the Baltic Sea under more heavy anthropogenic impact, rarely exceeding any given threshold values for Good Environmental Status of the EU Marine Strategy Framework Directive. However, significant biological effects were recorded in mussels in the most contaminated sites, signifying that the combined effects caused by the contaminants and other environmental factors are disturbing the health of marine organisms in the area. The results of this successful combined application based on the mussel transplantation method and the WOE approach are highly encouraging for further trials in developing the monitoring of chemical contamination in the Baltic Sea.

Multidisciplinary investigations based on integration of chemical and biological measurements, represent an added value to monitoring and management protocols, and their use is recommended by European Directives to evaluate the environmental status of aquatic ecosystems. However, assessing the overall significance of results obtained in different typologies of studies is often a difficult challenge. The aim of this work was to present a quantitative Weight Of Evidence (WOE) model (Sediqualsoft) to integrate huge amounts of heterogeneous data and to validate this approach in complex monitoring scenarios. Using the case-study of an off-shore platform field in the Adriatic Sea, procedures are presented to elaborate different typologies of data (lines of evidence, LOEs), including chemical characterization of sediments, bioavailability, biomarkers, ecotoxicological bioassays and benthic communities around three platforms. These data are initially evaluated by logical flowcharts and mathematical algorithms, which provide specific hazard indices for each considered LOE, before their different weighting and overall integration in an environmental risk index. The monitoring study selected for the WOE elaboration consisted on chemical analyses of trace metals, aliphatic hydrocarbons, polycyclic aromatic hydrocarbons carried out on 60 sediment samples; the same samples were also characterized for the status of benthic communities; bioavailability of metals from sediments was assessed in laboratory conditions on the polychaete Hediste diversicolor, while bioaccumulation of inorganic and organic chemicals and biomarker responses were measured in native and transplanted mussels; ecotoxicological properties of sediments were evaluated through a battery of bioassays determining algal growth of the diatom Phaeodactylum tricornutum, bioluminescence of the marine bacterium Vibrio fischeri, survival of the copepod Acartia tonsa and embryotoxicity of sea urchin Paracentrotus lividus. Overall, almost 7000 analytical results were elaborated and summarized in specific hazard indices. The WOE integration of multiple typologies of data allowed more robust and weighted conclusions compared to the use of individual LOEs, highlighting the feasibility of this procedure for multidisciplinary monitoring and risk assessment approaches. On a practical side, the WOE evidences also suggested a revision of actual monitoring procedures. Overall, the proposed WOE model appeared as a useful tool to summarize large datasets of complex data in integrative indices, and to simplify the interpretation for stakeholders and decision makers, thus supporting a more comprehensive process of “site-oriented” management decisions.

Marine organisms are exposed to a pH decrease and to alteration of carbonate chemistry due to ocean acidification (OA) that can represent a source of oxidative stress which can significantly affect their antioxidant defence systems efficiency. The polychaetes Platynereis dumerilii and P. massiliensis (Nereididae) are key species of the benthic community to investigate the effect of OA due to their physiological and ecological characteristics that enable them to persist even in naturally acidified CO2 vent systems. Previous studies have documented the ability of these species to adapt to OA after short- and long-term translocation experiments, but no one has ever evaluated the basal antioxidant system efficiency comparing populations permanently living in habitat characterized by different pH conditions (acidified vs. control). Here, individuals of both Platynereis species, sampled from a natural CO2 vent system and from a nonventing “control” site in three different periods (April 2016, October 2016, and February 2017), were compared highlighting signals which suggested the ability of both species to acclimatize to high pCO2–low pH with slight seasonal variations of their antioxidant efficiency and the absence of disturbances of the oxidative status of Platynereis spp. tissues.

Ocean-warming and acidification jeopardize Antarctic marine species, adapted to cold and constant conditions and naturally exposed to high pro-oxidant pressures and cadmium (Cd) bioavailability. The aim of this study was to investigate if projected temperature increase and pH reduction may affect the accumulation and the effects of Cd in the rockcod Trematomus bernacchii. Organisms were exposed for 14 days to six scenarios, combining environmental or increased temperature (−1 °C, +1 °C) and control or reduced pH (8.05, 7.60), either with or without Cd (40 µg/L). Responses in liver and gills were analyzed at different levels, including mRNA and functional measurements of metallothioneins and of a wide battery of antioxidants, integrated with the evaluation of the total antioxidant capacity and onset of oxidative damages. In the gills, metallothioneins and mRNA of antioxidant genes (nrf2, keap1, cat, gpx1) increased after Cd exposure, but such effects were softened by warming and acidification. Antioxidants showed slighter variations at the enzymatic level, while Cd caused glutathione increase under warming and acidified scenarios. In the liver, due to higher basal antioxidant protection, limited effects were observed. Genotoxic damage increased under the combined stressors scenario. Overall results highlighted the modulation of the oxidative stress response to Cd by multiple stressors, suggesting the vulnerability of T. bernacchii under predicted ocean change scenarios.

The ubiquitous occurrence of microplastics (MPs) in the marine environment is raising concern for interactions with marine organisms. These particles efficiently adsorb persistent organic pollutants from surrounding environment and, due to the small size, they are easily available for ingestion at all trophic levels. Once ingested, MPs can induce mechanical damage, sub-lethal effects, and various cellular responses, further modulated by possible release of adsorbed chemicals or additives. In this study, ecotoxicological effects of MPs and their interactions with benzo(a)pyrene (BaP), chosen as a model compound for polycyclic aromatic hydrocarbons (PAHs) were investigated in Mediterranean mussels, Mytilus galloprovincialis. Organisms were exposed for 4 weeks to 10 mg/L of low-density polyethylene (LDPE) microparticles (2.34 * 107 particles/L, size range 20–25 μm), both virgin and pre-contaminated with BaP (15 μg/g). Organisms were also exposed for comparison to BaP dosed alone at 150 ng/L, corresponding to the amount adsorbed on microplastics. Tissue localization of microplastics was histologically evaluated; chemical analyses and a wide battery of biomarkers covering molecular, biochemical and cellular levels allowed to evaluate BaP bioaccumulation, alterations of immune system, antioxidant defenses, onset of oxidative stress, peroxisomal proliferation, genotoxicity, and neurotoxicity. Obtained data were elaborated within a quantitative weight of evidence (WOE) model which, using weighted criteria, provided synthetic hazard indices, for both chemical and cellular results, before their integration in a combined index. Microplastics were localized in hemolymph, gills, and especially digestive tissues where a potential transfer of BaP from MPs was also observed. Significant alterations were measured on the immune system, while more limited effects occurred on the oxidative status, neurotoxicity, and genotoxicity, with a different susceptibility of analyzed pathways, depending on tissue, time, and typology of exposure. Molecular analyses confirmed the general lack of significant transcriptional variations of antioxidant and stress genes. The overall results suggest that microplastics induce a slight cellular toxicity under short-term (28 days) exposure conditions. However, modulation of immune responses, along with bioaccumulation of BaP, pose the still unexplored risk that these particles, under conditions of more chronic exposure (months to years) or interacting with other stressors, may provoke long-term, subtle effects on organisms' health status.

Azaspiracids (AZAs) are marine biotoxins including a variety of analogues. Recently, novel AZAs produced by the Mediterranean dinoflagellate Azadinium dexteroporum were discovered (AZA-54, AZA-55, 3-epi-AZA-7, AZA-56, AZA-57 and AZA-58) and their biological effects have not been investigated yet. This study aimed to identify the biological responses (biomarkers) induced in mussels Mytilus galloprovincialis after the bioaccumulation of AZAs from A. dexteroporum. Organisms were fed with A. dexteroporum for 21 days and subsequently subjected to a recovery period (normal diet) of 21 days. Exposed organisms accumulated AZA-54, 3-epi-AZA-7 and AZA-55, predominantly in the digestive gland. Mussels’ haemocytes showed inhibition of phagocytosis activity, modulation of the composition of haemocytic subpopulation and damage to lysosomal membranes; the digestive tissue displayed thinned tubule walls, consumption of storage lipids and accumulation of lipofuscin. Slight genotoxic damage was also observed. No clear occurrence of oxidative stress and alteration of nervous activity was detected in AZA-accumulating mussels. Most of the altered parameters returned to control levels after the recovery phase. The toxic effects detected in M. galloprovincialis demonstrate a clear biological impact of the AZAs produced by A. dexteroporum, and could be used as early indicators of contamination associated with the ingestion of seafood.

Although the chemical warfare between invasive and native species has become a central problem in invasion biology, the molecular mechanisms by which bioactive metabolites from invasive pests influence local communities remain poorly characterized. This study demonstrates that the alkaloid caulerpin (CAU)—a bioactive component of the green alga Caulerpa cylindracea that has invaded the entire Mediterranean basin—is an agonist of peroxisome proliferator-activated receptors (PPARs). Our interdisciplinary study started with the in silico prediction of the ligand-protein interaction, which was then validated by in vivo, ex vivo and in vitro assays. On the basis of these results, we candidate CAU as a causal factor of the metabolic and behavioural disorders observed in Diplodus sargus, a native edible fish of high ecological and commercial relevance, feeding on C. cylindracea. Moreover, given the considerable interest in PPAR activators for the treatment of relevant human diseases, our findings are also discussed in terms of a possible nutraceutical/pharmacological valorisation of the invasive algal biomasses, supporting an innovative strategy for conserving biodiversity as an alternative to unrealistic campaigns for the eradication of invasive pests.

Objective: Methodological issues are major reasons preventing the use of endothelial function testing in clinical practice This study aimed to address the relationship between two non-invasive techniques, brachial artery flow-mediated dilation (FMD) and as reactive hyperemia index (RHI), comparing also lower (forearm, L) and upper (arm, U) cuff occlusion. Methods: In 17 young healthy subjects (9 males, age 29±4 years) FMD (Cardiovascular Suite, Quipu s.r.l., Pisa, Italy). and RHI (EndoPAT 2000, Itamar Medical, Israel) were measured simultaneously in two separate occasions using 5 minutes of L- or U-ischemia. Baseline and Hyperemic Shear rate (SR) were also computed. Results: L-FMD (7.32±4.87%) and L-RHI (0,61±0.29%) were significantly lower (p

In the present study, the biotransformation and oxidative stress responses in relation to chemical burden in the liver of male and female Nile crocodiles--Crocodylus niloticus--from a commercial crocodile farm passively exposed to various anthropogenic aquatic pollutants was investigated. In general, the data showed that male crocodiles consistently produced higher biotransformation and oxidative stress responses compared to females. Relationships between these responses and concentrations of aliphatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs) were also observed. Specifically, the catalytic assays for EROD and BROD (not PROD and MROD) showed sex-differences between male and female crocodiles and paralleled immunochemically determined CYP1A and CYP3A protein levels; the relatively similar levels of PAHs in both sexes suggest an estrogen-mediated reduction of this pathway in females. The antioxidant system exhibited higher levels in male crocodiles with slight or significant higher values for catalase (CAT), glutathione reductase (GR), glutathione peroxidases-H2O2 (GPx-H2O2), glutathione peroxidases-Cu (GPx-Cu), total antioxidant capacity towards peroxyl radicals (TOSC-ROO) and hydroxyl radicals (TOSC-HO), total glutathione (GSH) and malondialdehyde (MDA). On the other hand, the activities of acyl-CoA oxidase (AOX) and glutathione S-transferases (GST) were significantly higher in females. Principal component analysis (PCA) produced significant groupings that revealed correlative relationships (both positive and negative) between biotransformation/oxidative stress variables and liver PAHs and aliphatic hydrocarbon burden. The overall results suggest that these captive pre-slaughter crocodiles exhibited adverse exposure responses to anthropogenic aquatic contaminants with potentially relevant effects on key cellular pathways, and these responses may be established as relevant species biomarkers of exposure and effects in this endangered species.

The green alga Caulerpa racemosa var. cylindracea has invaded Mediterranean seabed including marine reserves, modifying the structure of habitats and altering the distributional patterns of associated organisms. However, the understanding of how such invasion can potentially affect functional properties of Mediterranean subtidal systems is yet to be determined. In this study, we show that C. racemosa changes foraging habit of the native white seabream, Diplodus sargus. In invaded areas, we found a high frequency of occurrence of C. racemosa in the stomach contents of this omnivorous fish (72.7 and 85.7%), while the alga was not detected in fish from a control area. We also found a significant accumulation of caulerpin, one of the main secondary metabolites of C. racemosa, in fish tissues. The level of caulerpin in fish tissues was used here as an indicator of the trophic exposure to the invasive pest and related with observed cellular and physiological alterations. Such effects included activation of some enzymatic pathways (catalase, glutathione peroxidases, glutathione S-transferases, total glutathione and the total oxyradical scavenging capacity, 7-ethoxy resorufin O-deethylase), the inhibition of others (acetylcholinesterase and acylCoA oxidase), an increase of hepatosomatic index and decrease of gonadosomatic index. The observed alterations might lead to a detrimental health status and altered behaviours, potentially preventing the reproductive success of fish populations. Results of this study revealed that the entering of alien species in subtidal systems can alter trophic webs and can represent an important, indirect mechanism which might contribute to influence fluctuations of fish stocks and, also, the effectiveness of protection regimes.