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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.

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.

Environmental pharmaceuticals represent a threat of emerging concern for marine ecosystems. Widely distributed and bioaccumulated, these contaminants could provoke adverse effects on aquatic organisms through modes of action like those reported for target species. In contrast to pharmacological uses, organisms in field conditions are exposed to complex mixtures of compounds with similar, different, or even opposing therapeutic effects. This review summarizes current knowledge of the main cellular pathways modulated by the most common classes of environmental pharmaceuticals occurring in marine ecosystems and accumulated by nontarget species-including nonsteroidal anti-inflammatory drugs, psychiatric drugs, cardiovascular and lipid regulator agents, steroidal hormones, and antibiotics-and describes an intricate network of possible interactions with both synergistic and antagonistic effects on the same cellular targets and metabolic pathways. This complexity reveals the intrinsic limits of the single-chemical approach to predict the long-term consequences and future impact of pharmaceuticals at organismal, population, and community levels. Expected final online publication date for the Annual Review of Marine Science, Volume 14 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Aquatic animals are vulnerable to arsenic (As) toxicity. However, rarely does a contaminant occur alone in the aquatic environment. For this reason, this study was conducted to evaluate whether titanium dioxide nanoparticles (nTiO2) can interfere with the effects induced by As in Litopenaeus vannamei. Arsenic accumulation and metabolic capacity; expression and enzymatic activity of GSTΩ (glutathione-S-transferase omega isoform); antioxidant responses such as GSH, GR, and GST (reduced glutathione levels, glutathione reductase, and glutathione-S-transferase activity, respectively); and lipid peroxidation in the gills and hepatopancreas of shrimp were evaluated. The results are summarized as follows: (1) higher accumulation of As occurred in both tissues after exposure to As alone; (2) co-exposure to nTiO2 affected the capacity to metabolize As; (3) GSTΩ gene expression was not modified, but its activity was decreased by co-exposure to both contaminants; (4) As alone increased the GSH levels in the hepatopancreas, and co-exposure to nTiO2 reduced these levels in both tissues; (5) a decrease in the GST activity in the gills occurred with all treatments; (6) in the gills, GR activity was increased by As, and nTiO2 reversed this increase, whereas in the hepatopancreas co-exposure inhibited enzyme activity; (7) only in the hepatopancreas lipid damage was observed when animals were exposed to As or nTiO2 but not in co-exposure. The results showed that the As induces toxic effects in both tissues of shrimp and that co-exposure to nTiO2 can potentiate these effects and decrease the capacity to metabolize As, favoring the accumulation of more toxic compounds.

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 polychaetesPlatynereis dumeriliiandP. 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 CO2vent 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 (acidifiedvs.control). Here, individuals of bothPlatynereisspecies, sampled from a natural CO2vent 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 highpCO2–low pH with slight seasonal variations of their antioxidant efficiency and the absence of disturbances of the oxidative status ofPlatynereisspp. tissues.

The correlation between endocrine active contaminants in the environment and alterations in reproductive development of Sarotherodon melanotheron from Lagos lagoon has been investigated. Sediment and a total of 155 fish (74 males and 81 females) were collected between November 2014-March 2015 from selected contaminated sites (Ikorodu, Oworonshoki, Makoko and Idumota) and a putative control site (Igbore) along the lagoon. Sediment contaminant analysis revealed, significantly higher concentration of lindane, dieldrin, 4-iso-nonylphenol, 4-t-octylphenol and monobutyltin cation at the contaminated sites. Examination of gross morphological and histological changes of fish gonads showed a 27.4% prevalence of intersex in the sampled fish, of which 78% were males (testes-ova) and 22% were females (ovo-testis). Quantitative PCR (qPCR) of liver transcripts revealed the presence of vitellogenin (vtg) levels in male fish from contaminated sites. Zona radiata proteins (zrp) mRNA levels were significantly higher in females, compared to male fish. In general, significantly lower vtg and zrp transcripts levels were recorded at Igbore (control site), compared with contaminated sites. Principal component analysis (PCA) showed site and sex relationship in biological responses and contaminants, including trace metals, demonstrating that measured endocrine responses in fish were associated with contaminant burden in sediment. In addition, positive relationships were observed in male fish from Idumota, Oworonshoki and Ikorodu with vtg and dieldrin/4-iso-nonyphenol, with higher levels in male fish, compared to females. Further, contaminants from the Makoko, Oworonshoki and Ikorodu sites were positively associated with higher GSI and zrp in females. More importantly, the severity of intersex and changes in vtg transcripts imply a progressive feminization of male fish with concomitant alteration in the reproductive health of fish inhabiting the Lagos lagoon.

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.

Microplastics are present in all parts of the ocean and can have deleterious effects on marine resources. The aim of this work was to map the presence of microplastics in commercial marine species such as bivalves (mussels Mytilus galloprovincialis and clams Scrobicularia plana), crabs (Carcinus maenas) as well as fish (Mullus surmuletus) to relate microplastics levels to pollution sources, assess possible impact on marine food chains and on human health. These species were collected from several sites of the Ria Formosa lagoon and along the south coast of Portugal. A quantitative assessment (number, size and color) and typology of microplastics were made in these species. Only one green fragment of polypropylene was detected in the gills of the crabs, while a blue polyethylene fragment was detected in the hepatopancreas of the mullets. Moreover, no microplastics were present in S. plana nor in the crabs whole soft tissues. Among mussels, 86% of microplastics were present from all sites and the number, size and color were site specific. Mussels from the west side of the coast (Sites 1–3) had the highest levels of MPs per mussel and per weight compared to the other sites, probably related to the impact of touristic activity, fishing gears, fresh water and sewage effluents along with the hydrodynamics of the area. FCT JPI OCEANS MICROPLAST/0005/2018; UID/00350/2020 info:eu-repo/semantics/publishedVersion

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.

Studies that aim to characterise the susceptibility of the ecologically relevant and non-model fish polar cod (Boreogadus saida) to model carcinogens are required. Polar cod were exposed under laboratory conditions for six months to control, 0.03 μg BaP/g fish/week and 0.3 μg BaP/g fish/week dietary benzo(a)pyrene (BaP), a reference carcinogen. The concentrations of the 3-OH-BaP bile metabolite and transcriptional responses of genes involved in DNA adduct recognition (xpc), helicase activity (xpd), DNA repair (xpf, rad51) and tumour suppression (tp53) were assessed after 0, 1, 3 and 6 months of exposure, alongside body condition indexes (gonadosomatic index, hepatosomatic index and condition factor). Micronuclei and nuclear abnormalities in blood and spleen, and liver histopathological endpoints were assessed at the end of the experiment. Fish grew steadily over the whole experiment and no mortality was recorded. The concentrations of 3-OH-BaP increased significantly after 1 month of exposure to the highest BaP concentration and after 6 months of exposure to all BaP concentrations showing the biotransformation of the mother compound. Nevertheless, no significant induction of gene transcripts involved in DNA damage repair or tumour suppression were observed at the selected sampling times. These results together with the absence of chromosomal damage in blood and spleen cells, the subtle increase in nuclear abnormalities observed in spleen cells and the low occurrence of foci of cellular alteration suggested that the exposure was below the threshold of observable effects. Taken together, the results showed that polar cod was not susceptible to carcinogenesis using the BaP exposure regime employed herein.

Polybrominated diphenyl ethers (PBDEs) are flame retardants, characterized by elevated stability in the marine environment, where are accumulated by organisms, inducing a wide panel of negative effects. In this study, some biochemical patterns related to toxicity, biotransformation and oxidative stress, were studied in the marine model system, Mytilus galloprovincialis, exposed to BDE-47. Mussels were fed with microalgae, previously treated with increasing concentrations of PBDEs (maximum dose 100 ng L-1 of BDE-47 per day). After 15 days of treatment, mussels were fed with the same diet without BDE-47, for additional 15 days. Gills and digestive glands were analyzed at T 0, at 15 and 30 days. Histopathological lesions were assessed in digestive glands of contaminated mussels, while expression of genes, related to cell cycle, multidrug resistance, oxidative stress and detoxification was evaluated on both gills and digestive glands. After 15 days, BDE-47 exposure significantly affected the cell activity in digestive gland and, at 30 days, only mussels exposed to the lower doses showed a certain recovery. Regarding the gene expression, both gills and digestive glands showed a significant down-regulation of the target genes at 15 days, although most of them were up-regulated at 30 days in digestive gland. The results on BDE-47 accumulation in mussels revealed a dose-dependent concentration in tissues, which remained elevated after further 15 days of depuration. This trend supports the responses of the biomarkers, indicating that exposure, at environmentally realistic concentrations of BDE-47, strongly modulates oxidative stress and related patterns of gene expression, suggesting concerns for long-term effect in the biota.

In this study, we have investigated biotransformation and oxidative stress responses in relation to tissue contaminant burden in the African sharptooth catfish (Clarias gariepinus) exposed to simulated leachate from a solid waste dumpsite in Calabar, Nigeria. Fish were exposed to simulated leachate, diluted to 0:0 (negative control), 1:10, 1:50, 1:100 and phenanthrene (a PAH: 50 μg/L used as a positive control) for 3, 7 and 14 days. Hepatic transcripts for cat, sod1, gpx1, gr, gst, cyp1a, cyp2d3, and cyp27 were analyzed by real-time PCR, while enzymatic assays for ethoxyresorufin O-deethylase (EROD), buthoxyresorufin O-deethylase (BROD), methoxyresorufin O-deethylase (MROD), pentoxyresorufin O-deethylase (PROD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), uridine diphospho-glucuronosyltransferase (UDPGT) and lipid peroxidase (LPO) were measured using standard methods. In addition, protein expression for CYP1A, CYP3A and metallotheionin (MT) were measured by immunoblotting. Fish muscle samples were analyzed for selected group of contaminants after 14 days exposure showing significantly high uptake of heavy metals (Cd, Hg and Pb), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), chlorophenols, organochlorine (OC) and organophosphate pesticides in exposed fish. We observed significant concentration- and time-specific increases in biotransformation and oxidative stress responses at transcript and functional (enzyme and protein) levels, that paralleled tissue contaminants bioaccumulation patterns, after exposure to the simulated leachates. Our results highlighted the potential environmental, wildlife and public health consequences from improper solid waste disposal. In addition, it also provides a scientific basis for local sensitization and inform legislative decisions and policy formulation towards sustainable environmental management of solid wastes in Nigeria and other developing countries.

The sea cucumbers are common members of marine benthic communities, widespread distributed, easily available and handled. Nevertheless, no data are available on embryo toxicity assays using sea cucumbers, despite some of these species could fully meet the requirements for model test organisms. Holothuria polii is a key species in soft sediments and seagrass meadows; the aim of the present study was the standardization of a new embryo bioassay with this species, as an ecologically relevant test to evaluate the effects of environmental stressors. Sequential experiments were carried out, allowing to define the test acceptability, and a minimum sample size of 240 embryos. Temperature of 26 °C, salinity at 36‰ and a density of 60 eggs/ml were identified as optimum experimental conditions for performing the bioassay. The EC50 calculated for Cd2+ and Cu2+ in dose-response experiments indicated a good sensitivity of H. polii, with comparable values with those obtained in embryo toxicity bioassays of other marine invertebrates. An Integrative Toxicity Index (ITI) was calculated by integrating the frequency of abnormal embryos with the severity of observed abnormalities. The index allows to better discriminate different levels of toxicity, appearing particularly relevant for validating the usefulness of H. polii in embryo assays and ecotoxicological studies on environmental quality.

Although some studies have showed the effects of different crystalline structures of nTiO2 (anatase and rutile) and their applicability in several fields, few studies has analyzed the effect of coexposure with other environmental contaminants such as copper. Thus, the objective of this study was to evaluate if the coexposure to nTiO2 (nominal concentration of 1 mg/L; anatase or rutile) can increase the incorporation and toxic effect induced by Cu (nominal concentration of 56 μg/L) in different tissues of Linmoperna fortunei after 120 h of exposure. Our results showed that the coexposure increased the accumulation of Cu in the gills and adductor muscle independently of the crystalline form and can positively or negatively modulate the antioxidant system, depending on the tissue analyzed. However, exposure only to rutile nTiO2 induced damage in the adductor muscle evidenced by the infiltration of hemocytes in this tissue. Additionally, histomorphometric changes based on fractal dimension analysis showed that coexposure to both forms of nTiO2 induced damage in the same tissue. These results suggest that both crystalline forms exhibited toxicity depending on the analyzed tissue and that coexposure of nTiO2 with Cu may be harmful in L. fortunei, indicating that increased attention to the use and release of nTiO2 in the environment is needed to avoid deleterious effects in aquatic biota.

Non-steroidal anti-inflammatory drugs (NSAIDs) represent a growing concern for marine ecosystems due to their ubiquitous occurrence and documented adverse effects on non-target organisms. Despite the remarkable efforts to elucidate bioaccumulation and ecotoxicological potential under short-term conditions, limited and fragmentary information is available for chronic exposures. In this study bioavailability, molecular and cellular effects of diclofenac (DIC), ibuprofen (IBU) and ketoprofen (KET) were investigated in mussels Mytilus galloprovincialis exposed to the realistic environmental concentration of 2.5 μg/L for up to 60 days. Results indicated a significant accumulation of DIC and IBU but without a clear time-dependent trend; on the other hand, KET concentrations were always below the detection limit. Analyses of a large panel of molecular, biochemical and cellular biomarkers highlighted that all investigated NSAIDs caused alterations of immunological parameters, genotoxic effects, modulation of lipid metabolism and changes in cellular turn-over. This study provided the evidence of long-term ecotoxicological potential of NSAIDs, further unraveling the possible hazard for wild marine organisms.

The sea urchin embryo-toxicity test is widely used to assess the toxicity of contaminants and environmental matrices. In standard guideline and literature studies, the classical toxicity criteria are based on distinguishing between normal and abnormal embryos at pluteus stage. The aim of this research was to further expand the potentiality of the recently developed Integrative Toxicity Index (ITI), investigating the reversibility of the effects induced by various trace metals (cadmium, copper, lead and zinc) on sea urchin development. For this purpose, embryos were observed after different periods of exposure and recovery to metals. Results were analysed comparing ITI with standard criteria, thus moving from the simple observation of general effects to the classification of their severity. The onset and reversibility of effects by trace metals were more efficiently discriminated by the use of the ITI, which recognized and weighted the delay and degree of various abnormalities. Above, this study was expected to provide new insights into the capability of each metal to induce anomalies leading to a block or delay in embryogenesis of the embryos to recover normal development after metal exposure, thus adding further ecological value to the sea urchin bioassay.

Sea cucumbers are widely distributed deposit-feeders that represent an important component of benthic communities worldwide. These echinoderms were recently proposed as candidates in embryo bioassays to provide a new tool in the toxicity assessment of pollutants in marine water and sediments. The aim of this study is to evaluate the usefulness of a new species, Holothuria tubulosa (Gmelin, 1788), as a model organism for sensitive embryo bioassays, defining the acceptability of controls, minimum sample size, embryo density and salinity range. Cadmium (Cd), copper (Cu), lead (Pb), mercury (Hg), sodium dodecyl sulphate (SDS) and 4-n-nonylphenol (NP) were used as reference toxicants to assess specific embryotoxicity endpoints. Sea cucumber sensitivity to marine sediment elutriates were finally assessed by comparing their responsiveness in tandem with that of routinely employed sea urchin embryos. The results showed an acceptability threshold of 10% (abnormal embryos), a minimum sample size of 200 embryos, an embryo density of 200 embryos/mL and an optimal salinity range of 36–37‰. The sensitivity to the environmental pollutants and matrices tested revealed values (expressed as EC50) comparable with those of embryos belonging to other marine invertebrates commonly used in bioassays, indicating that this species has a good level of responsiveness. A specific integrative toxicity index (ITI) was applied, combining the frequency of developmental anomalies and weighting their severity. ITI data demonstrated good discrimination of sample toxicity, with a dose-dependent increase of teratogenic effects for all the tested substances, indicating H. tubulosa as a promising species for future assessments of marine pollution.

To elucidate the dynamics of a suite of organochlorine contaminants (PCBs, HCB), PAHs and Hg and verify the potential of these pollutants as reliable fingerprints of sources, an ensemble of marine sediments and organisms (finfish, shellfish species and Mytilus galloprovincialis) were analysed from the contaminated Augusta Bay (Southern Italy). The Hg and HCB concentration in the sediments exceeded the EQS of the Directive 2000/60/EU. Similarly, ∑PCB and selected PAHs were above the threshold limit set by regulation. The marine organisms showed Hg concentrations above CE 1881/2006. Contaminants in transplanted mussel evidenced an increased accumulation overtime and different distribution patterns between sampling sites. Analysis of the homolog composition of PCB congeners revealed comparable patterns between sediments and marine organisms and offered the opportunity to define a robust fingerprint for tracing contaminants transfer from the abiotic to the biotic compartments. These results were confirmed by the Fluoranthene/Pyrene, Hg and HCB distribution modes.

Despite the great interest in the consequences of climate change on the physiological functioning of marine organisms, indirect and interactive effects of rising temperature and pCO2 on bioaccumulation and responsiveness to environmental pollutants are still poorly explored, particularly in terms of cellular mechanisms. According to future projections of temperature and pH/pCO2, this study investigated the main cellular pathways involved in metal detoxification and oxidative homeostasis in Mediterranean mussels, Mytilus galloprovincialis, exposed for 4 weeks to various combinations of two levels of pH/pCO2 (8.2/∼400 μatm and 7.4/∼3000 μatm), temperature (20 and 25 °C), and cadmium addition (0 and 20 μg/L). Bioaccumulation was increased in metal exposed organisms but it was not further modulated by different temperature and pH/pCO2 combinations. However, interactions between temperature, pH and cadmium had significant effects on induction of metallothioneins, responses of the antioxidant system and the onset of oxidative damages, which was tissue dependent. Multiple stressors increased metallothioneins concentrations in the digestive gland revealing different oxidative effects: while temperature and cadmium enhanced glutathione-dependent antioxidant protection and capability to neutralize peroxyl radicals, the metal increased the accumulation of lipid peroxidation products under acidified conditions. Gills did not reveal specific effects for different combinations of factors, but a general stress condition was observed in this tissue after various treatments. Significant variations of immune system were mainly caused by increased temperature and low pH, while co-exposure to acidification and cadmium enhanced metal genotoxicity and the onset of permanent DNA damage in haemocytes. Elaboration of the whole biomarker data in a cellular hazard index, corroborated the synergistic effects of temperature and acidification which increased the toxicological effects of cadmium. The overall results confirmed that climate change could influence ecotoxicological effects of environmental contaminants, highlighting the importance of a better knowledge of cellular mechanisms to understand and predict responsiveness of marine organisms to such multiple stressors.

Contamination status of coastal areas of Finland (northern Baltic Sea) markedly affected by anthropogenic activities (harbours, 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 harbour 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.

International audience; The Ephemare project was supported in the period 2015–2018 by JPI Oceans, as one of 4 sister projects in the joint action on ecological aspects of microplastics. Ephemare investigated several issues concerning the ecotoxicological effects of microplastics (MPs) in marine organisms. Ephemare included 16 European Institutions from 10 Countries and was organized into seven, highly complementary Work Packages (WPs) with the aim to elucidate adsorption and release of chemicals to/from MPs, coupled with MP ingestion rates, translocation in different tissues, trophic transfer and egestion, potential toxicological effects and mechanisms of action, as well as real distributions of MPs in marine organisms from several European areas.

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.

Ingestion of microplastics (MPs) has been documented in several marine organisms, but their occurrence in deep-sea species remains almost unknown. In this study, MPs were investigated in two economically and ecologically key crustaceans of the Mediterranean Sea, the Norwegian lobster Nephrops norvegicus and the shrimp Aristeus antennatus. Both the species were collected from 14 sites around Sardinia Island, at depths comprised between 270 and 660 m. A total of 89 and 63 stomachs were analysed for N. norvegicus and A. antennatus respectively, and more than 2000 MPs-like particles were extracted and sorted for identification and characterization by μFT-IR. In N. norvegicus, 83% of the specimens contained MPs, with an average abundance of 5.5 ± 0.8 MPs individual−1, while A. antennatus showed a lower frequency of ingestion (67%) and a lower mean number of MPs (1.66 ± 0.1 MPs individual−1). Composition and size of particles differed significantly between the two species. The non-selective feeding strategy of N. norvegicus could explain the 3–5 folds higher numbers of MPs in its stomach, which were mostly composed of films and fragments derived by polyethylene and polypropylene single-use plastic items. Contrarily, most MPs in the stomachs of A. antennatus were polyester filaments. The MPs abundance observed in N. norvegicus is among the highest detected in Mediterranean species considering both fish and invertebrates species, and provides novel insights on MPs bioavailability in deep-sea habitats. The overall results suggest that both N. norvegicus and A. antennatus, easily available in common fishery markets, could be valuable bioindicators and flagship species for plastic contamination in the deep-sea.

The aim of the present investigation was to provide new insights on accumulation and possible adverse effects of various non-steroidal anti-inflammatory drugs (NSAIDs) in mussels, Mytilus galloprovincialis, exposed to an environmentally realistic concentration (0.5 μg/L) of individual compounds, Acetaminophen (AMP), Diclofenac (DIC), Ibuprofen (IBU), Ketoprofen (KET) or Nimesulide (NIM). The measurement of drugs in mussel tissues was integrated with both functional alterations at cellular level and transcriptomic responses. Results indicated the capability of mussels to accumulate DIC and NIM, while AMP, IBU and KET were always below detection limit. A large panel of ecotoxicological biomarkers revealed the early onset of alterations induced by tested NSAIDs on immunological responses, lipid metabolism and DNA integrity. The gene transcription analysis through DNA microarrays, supported cellular biomarker results, with clear modulation of a large number of genes involved in the arachidonic acid and lipid metabolism, immune responses, cell cycle and DNA repair. The overall results indicated an ecotoxicological concern for pharmaceuticals in M. galloprovincialis, with transcriptional responses appearing as sensitive exposure biomarkers at low levels of exposure: such changes, however, are not always paralleled by corresponding functional effects, suggesting caution when interpreting observed effects in terms of perturbed cellular pathways. Fascinating similarities can also be proposed in the mode of action of NSAIDs between bivalves and vertebrate species.

In the present study, the developmental (including fertility) and endocrine-disruptive effects in relation to chemical burden in male and female Nile crocodiles (Crocodylus niloticus), from a commercial crocodile farm in the Brits district, South Africa, exposed to various anthropogenic aquatic contaminants from the natural environment was investigated. Hepatic transcript levels for vitellogenin (Vtg), zona pellucida (ZP) and ERα (also in gonads) were analyzed using real-time PCR. Plasma estradiol-17β (E2), testosterone (T) and 11-ketotestosterone (11-KT) were analyzed using enzyme immunoassay. Gonadal aromatase and hepatic testosterone metabolism (6β-hydroxylase (6β-OHase)) were analyzed using biochemical methods. Overall, there is high and abnormal number (%) of infertile and banded eggs during the studied reproductive seasons, showing up to 57 and 34% of infertile eggs in the 2009/2010 and 2013/2014 seasons, respectively. In addition, the percentage of banded eggs ranged between 10 and 19% during the period of 2009-2014 seasons. While hepatic ERα, Vtg, ZP mRNA and testosterone 6β-OHase, were equally expressed in female and male crocodiles, gonadal ERα mRNA and aromatase activity were significantly higher in females compared to male crocodiles. On the other hand, plasma T and 11-KT levels were significantly higher in males, compared to female crocodiles. Principal component analysis (PCA) produced significant grouping that revealed correlative relationships between reproductive/endocrine-disruptive variables and liver contaminant burden, that further relates to measured contaminants in the natural environment. The overall results suggest that these captive pre-slaughter farm crocodiles exhibited responses to anthropogenic aquatic contaminants with potentially relevant consequences on key reproductive and endocrine pathways and these responses may be established as relevant species endocrine disruptor biomarkers of exposure and effects in this threatened species.

The sea urchin embryo toxicity test is classically used to assess the noxious effects of contaminated marine waters and sediments. In Italian guidelines on quality of dredged sediments, the standard toxicity criteria used for this assay are based on a single endpoint at 48 hours of development, corresponding to the pluteus stage. Different typologies of abnormalities, including those which occur at earlier stages, are not categorized, thus preventing the evaluation of the actual teratogenic hazards. A new integrative toxicity index has been developed in this study based on the analysis of two developmental stages, at 24 and 48h post-fertilization, and the differentiation between development delays and germ layers impairments: the new toxicity index is calculated by integrating the frequency of abnormal embryos with the severity of such abnormalities. When tested on dredged sediments, the evaluation of increasing levels of toxicity affecting embryonic outcomes enhanced the capability to discriminate different samples, appearing particularly relevant to validate the sea urchin embryo toxicity assay, and supporting its utility in practical applications such as the sediments classification in harbor areas.

Despite the increasing interest for pharmaceuticals in the marine environment, their accumulation in wild organisms and consequent environmental hazards are still poorly known. The Mediterranean Sea is highly challenged by the density of coastal populations, large consumption of pharmaceuticals and their often limited removal by Wastewater Treatment Plants (WWTPs). In this respect, the present study aims to provide the first large-scale survey on the distribution of such contaminants of emerging concern in native mussels, Mytilus galloprovincialis from Italian coasts. Organisms were collected from 14 sites representative of relatively unpolluted marine waters along the Adriatic and Tyrrhenian Sea and analysed for 9 common pharmaceuticals including Non-Steroidal Anti-Inflammatory Drugs (NSAIDs: Diclofenac DIC, Ibuprofen IBU, Ketoprofen KET and Nimesulide NIM), the analgesic Acetaminophen AMP, the antiepileptic Carbamazepine CBZ, the antihypertensive Valsartan VAL, the anxiolytic Lormetazepam LOR and the antidepressant Paroxetine PAR. Results indicated the widespread occurrence of the majority of pharmaceuticals in mussel tissues: CBZ was measured in >90% of analysed samples, followed by VAL (>50%), PAR (>40%), and DIC (>30%), while only AMP and KET were never detected. Heterogeneous tissue concentrations ranged from a few units up to hundreds of ng/g (d.w.), while seasonal and interannual variability, investigated over 4 years, did not highlight any clear temporal trend. Limited differences obtained between the Adriatic and Tyrrhenian Sea, as well as coastal versus off-shore sampling sites, suggest that analysed levels of pharmaceuticals in mussels tissues should be considered as baseline concentrations for organisms collected in unpolluted areas of the Mediterranean. This study provided the first unambiguous evidence of the widespread occurrence of pharmaceuticals in marine mussels from Italian coasts, giving novel insights on the potential ecotoxicological hazard from such compounds in marine species.

Arsenic (As) is one of the most widespread contaminants; it is found in almost every environment. Its toxic effects on living organisms have been studied for decades, but the interaction of this metalloid with other contaminants is still relatively unknown, mainly whether this interaction occurs with emerging contaminants such as nanomaterials. To examine this relationship, the marine shrimp Litopenaeus vannamei was exposed for 48 h to As, graphene oxide (GO; two different concentrations) or a combination of both, and gills, hepatopancreas and muscle tissues were sampled. Glutathione S-transferase (GST)-omega gene expression and activity were assessed. As accumulation and speciation (metabolisation capacity) were also examined. Finally, a molecular docking simulation was performed to verify the possible interaction between the nanomaterial and GST-omega. The main finding was that GO modulated the As toxic effect: it decreased GST-omega activity, a consequence related to altered As accumulation and metabolism. Besides, the molecular docking simulation confirmed the capacity of GO to interact with the enzyme structure, which also can be related to the decreased GST-omega activity and subsequently to the altered As accumulation and metabolisation pattern.

Despite the important ecosystem and hydrological roles of coastal and inland waters, there are no established biomonitoring protocols for evaluating environmental, wildlife and human health for these coastlines in Nigeria. In the present study, contaminants tissue burden and effects at molecular and physiological levels, were investigated in Tilapia guineensis from a man-made lake (Eleyele Lake) that is used for municipal domestic water supply and compared to a reference site (Igboho Lake). Gene expression of phase I and II biotransformation systems, oxidative stress and obesogen responses were analyzed using real-time PCR, and these results were related to general health index (condition factor: CF) and muscle burden of trace metals, aliphatic and polycyclic aromatic hydrocarbons (PAHs). We observed a significant increase in phase I and II biotransformation systems, oxidative stress and obesogen responses in male and female fish from Eleyele lake compared with the reference site. Overall, our data showed significant relationships between biological responses and tissue concentrations of metals and PAHs for the Eleyele lake compared with the reference site. Given that a positive influence on genes and pathways associated with metabolic status has been previously associated with peroxisome proliferator activated receptors (PPARs), xenobiotic compounds that activate PPARs may produce changes in energy and metabolic processes, leading to obesity. The high CF (>1 = good health condition) observed, coupled with the high muscle burden of inorganic and organic contaminants in fish from Eleyele lake, suggest a potential obesogenic effect in these fishes. These findings represent co-relational evidence that the Eleyele lake is contaminated and consequently affecting biological and physiological integrity of organisms inhabiting the lake. These findings also suggest potential health risks for humans, since the lake is extensively used for domestic water supply and fisheries.

Ocean warming and acidification could represent an additional threat to marine organisms already coping with other anthropogenic impacts, such as chemical contamination in coastal areas. In this study, interactions between such multiple stressors and their synergistic effects in terms of accumulation, detoxification and biological effects of metals were investigated in the Mediterranean mussel Mytilus galloprovincialis. Organisms sampled during the winter period were exposed for 28 days to different combinations of two temperatures (10 °C and 15 °C), two pH/pCO2 (8.20/∼400μatm and 7.4/∼3000μatm) and two cadmium concentrations (0 and 20 μg/L). Cadmium concentrations increased in digestive glands and gills of metal-exposed mussels and were further enhanced by co-exposure at higher temperature. Interactive effects of temperature and/or pH were observed on Cd-mediated metallothionein induction, responsiveness of antioxidant system and onset of oxidative damages in lipids, with tissue-specific effects. Immunological effects showed a generalized sensitivity of lysosomal membrane stability toward the investigated stressors with major effects in co-exposed organisms. Cadmium and temperature affected phagocytosis efficiency and composition of haemocyte populations probably influencing the micronucleus frequency through varied mitotic rate. Several differences were highlighted between these results and those previously obtained from mussels exposed in summer, supporting the importance of season when addressing the tolerance of temperate organisms to variations of environmental factors. The elaboration of the whole biomarker results through weighted criteria allowed to summarize specific hazard indices, highlighting tissue-specific sensitivity toward multiple stressors and the need of improving the knowledge on interactions between multiple stressors.

The exponential production and use of plastics has generated increasing environmental release over the past decades, and microplastics (MPs) have been reported across all the oceans. Field studies have documented the occurrence of MPs in several species, but important knowledge gaps still remain. In the present study, we characterized the distribution of MPs in ten sediment-dwelling and epibenthic species representative of different habitat, feeding modes and trophic levels within the inner Oslofjord (Oslo, Norway), an area subjected to moderate anthropogenic pressures. Analysed species included fish, bivalves, echinoderms, crustaceans and polychaetes. MPs were present in all the species with a frequency up to 65% of positive individuals for some species. In most cases, 1 or 2 MPs were found per individual, but some organisms contained up to 7 particles. A total of 8 polymer typologies were identified, with PE and PP being the most common according to our extraction protocol. MP sizes ranged from 41 μm to lines as long as 9 mm. Our results indicate that occurrence of MPs in analysed biota is not influenced by organism habitat or trophic level, while characteristics and typology of polymers might be significantly affected by feeding mode of organisms.

Pharmaceuticals are nowadays recognized as a threat for aquatic ecosystems. The growing consumption of these compounds and the enhancement of human health in the past two decades have been paralleled by the continuous input of such biologically active molecules in natural environments. Waste water treatment plants (WWTPs) have been identified as a major route for release of pharmaceuticals in aquatic bodies where concentrations ranging from ng/L to μg/L are ubiquitously detected. Since medicines principles are designed to be effective at very low concentrations, they have the potential to interfere with biochemical and physiological processes of aquatic species over their entire life cycle. Investigations on occurrence, bioaccumulation and effects in non target organisms are fragmentary, particularly for marine ecosystems, and related to only a limited number over the 4000 substances classified as pharmaceuticals: hence, there is a urgent need to prioritize the environmental sustainability of the most relevant compounds. The aim of this review is to summarize the main adverse effects documented for marine species exposed in both field and laboratory conditions to different classes of pharmaceuticals including non-steroidal anti-inflammatory drugs, psychiatric, cardiovascular, hypocholesterolaemic drugs, steroid hormones and antibiotics. Despite a great scientific advancement has been achieved, our knowledge is still limited on pharmaceuticals behavior in chemical mixtures, as well as their interactions with other environmental stressors. Complex ecotoxicological effects are increasingly documented and multidisciplinary, integrated approaches will be helpful to clarify the environmental hazard of these "emerged" pollutants in marine environment.

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)&mdash, a bioactive component of the green alga Caulerpa cylindracea that has invaded the entire Mediterranean basin&mdash, 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.

The presence of microplastics in the marine environment has raised scientific interest during the last decade. Several organisms can ingest microplastics with potentially adverse effects on the digestive tract, respiratory system and locomotory appendages. However, a clear evidence of tissue accumulation and transfer of such microparticles in wild organisms is still lacking, partially hampered by technical difficulties in isolation and characterization protocols from biological samples. In this work, we compared the efficacy of some existing approaches and we optimized a new protocol allowing an extraction yield of microplastics from fish tissues ranging between 78% and 98%, depending on the polymer size. FT-IR analyses confirmed that the extraction procedure did not affect the particles characteristics. The method was further validated on the fish mullet, Mugil cephalus, exposed under laboratory conditions to polystyrene and polyethylene; the particles were isolated and quantified in stomach and liver, and their presence in the hepatic tissue was confirmed also by histological analyses. A preliminary characterization revealed the presence and distribution of microplastics in various fish species collected along the Adriatic Sea. FT-IR analyses indicated polyethylene as the predominant polymer (65%) in the stomach of fish. The overall results confirmed the newly developed method as a reliable approach to detect and quantify microplastics in the marine biota.

Oxidative stress biomarkers are widely used in marine ecotoxicology. Environmental pollutants enhance intracellular formation of oxyradicals through several mechanisms, but complex oxidative interactions occur in response to chemical mixtures. Metabolism of individual classes of pollutants can be influenced by a sophisticated network of prooxidant relationships, reciprocal and cascade effects, changes of redox-sensitive signaling proteins, and transcription factors. Chemically mediated pathways can affect antioxidant responses at different levels, including pretranscriptional, transcriptional, protein, and catalytic functions; such mechanisms remain largely unexplored in marine organisms. Molecular responses of antioxidants are frequently not paralleled by expected biochemical changes or cellular effects, and caution is needed when interpreting the effects of environmental pollutants. Results on antioxidant variations can be influenced by mRNA stability and protein turnover, different timing for transcriptional and translational mechanisms, metabolic capability of tissues, posttranscriptional modifications of proteins, biphasic responses of antioxidant enzymes, and adaptation mechanisms to chronic pollution.

Ocean acidification (OA) may affect sensitivity of marine organisms to metal pollution modulating chemical bioavailability, bioaccumulation and biological responsiveness of several cellular pathways. In this study, the smooth scallop Flexopecten glaber was exposed to various combinations of reduced pH (pH/pCO2 7.4/∼3000 μatm) and Cd (20 μg/L). The analyses on cadmium uptake were integrated with those of a wide battery of biomarkers including metallothioneins, single antioxidant defenses and total oxyradical scavenging capacity in digestive gland and gills, lysosomal membrane stability and onset of genotoxic damage in haemocytes. Reduced pH slightly increased concentration of Cd in scallop tissues, but no effects were measured in terms of metallothioneins. Induction of some antioxidants by Cd and/or low pH in the digestive gland was not reflected in variations of the total oxyradical scavenging capacity, while the investigated stressors caused a certain inhibition of antioxidants and reduction of the scavenging capacity toward peroxyl radical in the gills. Lysosomal membrane stability and onset of genotoxic damages showed high sensitivity with possible synergistic effects of the investigated factors. The overall results suggest that indirect effects of ocean acidification on metal accumulation and toxicity are tissue-specific and modulate oxidative balance through different mechanisms.

Accepted manuscript version. Published version available at https://doi.org/10.1016/j.marenvres.2017.06.005. Accepted manuscript version, licensed CC BY-NC-ND 4.0. Increasing anthropogenic activities in the Arctic pose the risk for accidental oil spills but our knowledge of ecotoxicological effects is still limited for Arctic organisms. This study aimed to provide insights in baseline levels of antioxidants in polar cod (Boreogadus saida) from different Arctic locations and investigate the susceptibility of this species to oxidative stress during a 32 day dietary crude oil exposure. Baseline levels of individual antioxidants and total oxyradical scavenging capacity (TOSC) varied among different Arctic fjords. Upon crude oil exposure, dose- and time-dependent polycyclic aromatic hydrocarbons (PAHs) biotransformation was evident through hepatic ethoxyresorufine-O-deethylase activity, glutathione S-transferase mRNA expression and biliary PAH metabolites. Changes in the activity of individual antioxidants and TOSC was, however, slight and transient upon exposure. In conclusion, biotransformation was clearly induced by the sub-lethal levels of crude oil in polar cod, while the antioxidant defense responded with high variability.

Despite the key importance of Nrf2-Keap1 in regulating antioxidant system in vertebrates, this system is still poorly investigated in marine species. The present study focused on the Antarctic silverfish Pleuragramma antarctica which, during the final phases of embryo development in platelet ice, is challenged by a sudden enhancement of environmental oxidative conditions associated to ice melting. Partial coding sequences were identified for Nrf2, its repressor Keap1 and for typical Nrf2-target antioxidant genes, like catalase, glutathione peroxidase isoform 1 and Cu/Zn-dependent superoxide dismutase. Compared to temperate homologues, the protein sequences showed an elevated conservation of amino acids essential for catalytic functions, while a few specific substitutions in non-essential regions may represent a molecular adaptation to improve flexibility and accessibility to active site at cold temperatures. The role of the Nrf2-Keap1 pathway in modulating the activation of antioxidant defences was demonstrated at both transcriptional and functional levels with a clear temporal increase of antioxidant protection in embryos before the hatching. Such findings confirm the importance of Nrf2 and highlight regulation of antioxidants as an adaptive strategy in P. antarctica to protect the early life stages toward the environmental changes of pro-oxidant pressure.

Hydrothermal vent systems are inhabited by dense benthic communities adapted to extreme conditions such as high temperature, hydrogen sulphide (H 2 S) and elevated fluxes of metals. In the present work, a wide range of trace elements (Ag, Al, As, Ba, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Sb, Se, V and Zn) were measured in tissues of three tube dwelling annelids, Alvinella pompejana , Alvinella caudata and Riftia pachyptila , which colonize distinct habitats of the East Pacific Rise (EPR) at 2500 m depth. Metals concentrations in alvinellids were often 2–4 orders of magnitude higher than those commonly found in marine organisms, while much lower values were observed in the vestimentiferan polychaete. Mobility of trace elements was further characterized in tissues of A. pompejana where metals appeared mostly in insoluble forms, i.e. associated with hydrated oxides and sulphides. Arsenic was mainly present in a weakly insoluble form and with concentrations in the branchial tentacles of alvinellids, approximately 5–15 fold higher than those measured in the thorax. Chemical speciation of this element in tissues of the three polychaete species revealed a major contribution of methylated arsenic compounds, like dimethylarsinate (DMA) and, to a lower extent, monomethylarsonate (MMA) and trimethylarsine oxide (TMAO). Although the biotransformation of inorganic arsenic might represent a detoxification mechanism in polychaetes from hydrothermal vents, the elevated levels of methylated forms of arsenic in branchial tissues also suggest an ecological role of this element as an antipredatory strategy for more vulnerable tissues toward generalist consumers.

Antarctic organisms developed specific adaptation mechanisms making these species able to survive to extreme environment conditions. Among fishes, Pleuragramma antarctica presents a specific peculiarity due to the occurrence of eggs with fully developed yolk-sac embryos below the platelet ice layer. This ice is an environment with strong pro-oxidant characteristics at the beginning of austral spring, when the rapid growth of algal ice communities, the massive release of nutrients and the photoactivation of dissolved organic carbon and nitrates represent an important sources for oxyradical formation. Such processes are concentrated in a short period of a few weeks, which overlaps with the final stage of development of P. antarctica embryos in platelet ice. For this reason, embryonated eggs of P. antarctica, before hatching, should possess adequate protection toward the marked and sudden increase of reactive oxygen species exposure. In this respect, molecular and functional characteristics of antioxidants in P. antarctica provide new insights on the modulation of the antioxidant defence pathway in response to varied environmental pro-oxidant challenge. To this aim, the main antioxidant components have been characterized in P. antarctica sampled from platelet ice in its nursery area in the Ross Sea, and data on nucleotide and protein sequences have been integrated with the analysis of regulation at transcriptional and functional levels. The results revealed a marked temporal increase of antioxidants in embryos of P. antarctica as adaptive counteracting response to oxidative conditions of platelet ice.

Gaseous and liquid hydrocarbons are seeping from sandy sea bottom similar to 10 m deep, about 2.4 km NNE of Civitanova Marche harbour, in central Adriatic Sea (Italy). We investigated the origin of the gas, the presence of a wide range of aromatic and aliphatic hydrocarbons and trace metals in shallow sediments, as well as the stable carbon and oxygen isotope composition of benthic foraminifera. In absence of detailed seismic images and subsurface geochemical data, we tried to estimate the source rock type and maturity based only on seep gas geochemistry. Molecular and isotopic composition of gas bubbles showed that the CH4-rich gas is thermogenic (delta C-13(CH4 similar to) -55 parts per thousand; delta H-2(CH4 similar to) -280 parts per thousand; C-1/(C-2 + C-3) < 100) with isotopic features that are compatible with low maturity source rocks belonging to the Emma-Scaglia (carbonate source rocks) Petroleum System (Upper Trias to Paleocene). Gas could then be stored in a biodegraded hydrocarbon pool, as suggested by C-13 enrichment in propane (delta C-13(3): -24 parts per thousand) and CO2 (delta C-13(CO2): +12 parts per thousand). Fluid seepage might be due to a local fracture zone corresponding to the intersection of NNW-SSE thrust faults with a NE-SW regional transversal deformation belt. Compared to other shallow marine seeps in Europe, the amount of methane released into the atmosphere is negligible (10(2)-10(3) kg of CH4 per year); but the seep also releases ethane and propane (10(3)-10(4) L per year), which are photochemical pollutants and are not emitted by microbial gas seeps. Compared to a reference site one nautical mile far from the seep, the seabed sediments show higher concentrations of various classes of chemicals, such as benzene, toluene and ethylbenzene, semivolatile and non volatile aliphatic hydrocarbons (C-10-C-40), and phenols (2-methylphenol and 2,4-dichlorophenol). These compounds likely derive from the oil seepage. The sediments at the seepage site and those at the reference site have similar concentrations of trace metals (arsenic, barium, cadmium, chromium, copper, iron, manganese, nickel, lead, vanadium, zinc, mercury), typical of uncontaminated and shallow coastal areas. Finally, we provided the first data on foraminifera associated to thermogenic hydrocarbons. No endemic foraminifera species or authigenic carbonates occur in the sediments. Carbon isotopic composition of Quinqueloculina padana where oil slick prevails is less variable than in the gas bubbling site. However, thermogenic methane and oil do not apparently decrease the delta C-13 value of foraminifera carbonate shell. (C) 2014 Elsevier Ltd. All rights reserved.

A complex framework of chemical, biological and oceanographic activities was immediately activated after the Costa Concordia shipwreck, to assess possible contamination events and the environmental impact during both emergency and wreck removal operations. In the present paper, we describe the results obtained with caged mussels, Mytilus galloprovincialis, chosen as bioindicator organisms to detect variations of bioavailability and the early onset of molecular and cellular effects (biomarkers). Seven translocation experiments were carried out during the first year from the incident, with organisms deployed at 2 depths in 3 different sites. After 4-6 weeks, tissue concentrations were measured for the main classes of potentially released chemicals (trace metals, polycyclic aromatic hydrocarbons, volatile and aliphatic hydrocarbons, polychlorinated biphenyls, halogenated pesticides, organotin compounds, brominated flame retardants, anionic surfactants); a wide battery of biomarkers covered responses indicative of exposure, detoxification, oxidative stress, cell damage and genotoxic effects. Results excluded serious contamination events or a consistent increase of environmental pollution although some episodic spills with reversible effects were detected. Data were elaborated within a quantitative weight of evidence (WOE) model which provided synthetic hazard indices for each typology of data, before their overall integration in an environmental risk index, which generally ranged from slight to moderate. The proposed WOE model was confirmed 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.

The precision-cut tissue slices (PCTS) represent a largely used biological model in mammalian research. This ex vivo approach offers the main advantages of in vitro systems, while maintaining the natural architecture of the tissue. The use of PCTS in toxicological research has been proposed for investigating the cellular effects of xenobiotics or bioactive compounds mostly in mammalian models. Their application is increasing also in marine organisms, but still limited to fish. This work validates the use of PCTS in an invertebrate species, the Mediterranean mussel Mytilus galloprovincialis. Intact tissue slices of different thicknesses (300, 350 and 400 μm) were successfully obtained from the digestive gland. The slices maintained the histological integrity and the viability after 6 h and 24 h incubation in culture medium, with some differences depending on the thickness. The enzymatic activities and mRNA levels of catalase and glutathione S-transferase, chosen as model biological endpoints, were measured until 24 h incubation, revealing the functionality of such systems. This work demonstrates the suitability of mussel PCTS for investigating molecular and cellular responses in ecotoxicological research.

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&rsquo, 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.

The monitoring of organisms’ health conditions by the assessment of their immunocompetence may serve as an important criterion for the achievement of the Good Environmental Status (GES) as defined in the Marine Strategy Framework Directive (EU). In this context, the complex role of natural environmental stressors, e.g. salinity, and interfering or superimposing effects of anthropogenic chemicals, should be carefully considered, especially in scenarios of low to moderate contamination. Organisms from the Baltic Sea have adapted to the ambient salinity regime, however energetically costly osmoregulating processes may have an impact on the capability to respond to additional stress such as contamination. The assessment of multiple stressors, encompassing natural and anthropogenic factors, influencing an organisms’ health was the main aim of the present study. Immune responses of Mytilus edulis, collected and kept at natural salinities of 12‰ (LS) and 20‰ (MS), respectively, were compared after short-term exposure (1, 7 and 13 days) to low copper concentrations (5, 9 and 16 μg/L Cu). A significant interaction of salinity and copper exposure was observed in copper accumulation. LS mussels accumulated markedly more copper than MS mussels. No combined effects were detected in cellular responses. Bacterial clearance was mostly achieved by phagocytosis, as revealed by a strong positive correlation between bacterial counts and phagocytic activity, which was particularly pronounced in LS mussels. MS mussels, on the other hand, seemingly accomplished bacterial clearance by employing additional humoral factors (16 μg/L Cu). The greatest separating factor in the PCA biplot between LS and MS mussels was the proportion of granulocytes and hyalinocytes while functional parameters (phagocytic activity and bacterial clearance) were hardly affected by salinity, but rather by copper exposure. In conclusion, immune responses of the blue mussel may be suitable and sensitive biomarkers for the assessment of ecosystem health in brackish waters (10–20‰ S).

Antioxidant and biotransformation pathways are widely studied in marine organisms exposed to environmental stressors. However, mechanisms of responses and links between different intracellular levels are not always easy to elucidate and conflicting results are frequently observed between molecular and enzymatic data. In this study, transcriptional and catalytic responses of antioxidant and biotransformation parameters were analyzed after a 4-week exposure of a marine invertebrate, Mytilus galloprovincialis, to chemical mixtures from low polluted and highly polluted sediments. A significant, dose-dependent bioaccumulation was observed for polycyclic aromatic hydrocarbons, especially low molecular weight compounds. Among antioxidant defences, catalase and glutathione peroxidases did not exhibit variations in enzymatic activity, while the corresponding gene transcriptions were up- and down-regulated, respectively; unchanged mRNA levels of superoxide dismutase confirmed the non-synchronous pathways of variations for such antioxidants. Biotransformation responses also revealed inconsistent trends between transcriptional and catalytic variations of glutathione S-transferases, and a significant increase in mRNA levels for cytochrome P450 3A1. The overall results indicated that transcriptional responses might be sensitive but do not necessarily correspond to functional changes, being more useful as "exposure" rather than "effect" biomarkers. Data on gene transcription and catalytic activities should be carefully interpreted when assessing the impact of chemical pollutants and additional studies are needed on modulation of post-transcriptional mechanisms by environmental stressors.

Pharmaceuticals represent a major environmental concern since the knowledge on their occurrence, distribution and ecotoxicological potential is still limited particularly in coastal areas. In this study, bioaccumulation and cellular effects of various non steroidal anti-inflammatory drugs (NSAIDs) were investigated in mussels Mytilus galloprovincialis to reveal whether common molecules belonging to the same therapeutic class might cause different effects on non target organisms. Organisms exposed to environmental concentrations of acetaminophen (AMP), diclofenac (DIC), ibuprofen (IBU), ketoprofen (KET) and nimesulide (NIM) revealed a significant accumulation of DIC, IBU and NIM, while AMP and KET were always below detection limit. Nonetheless, for all tested NSAIDs, measurement of a large panel of ecotoxicological biomarkers highlighted impairment of immunological parameters, onset of genotoxicity and modulation of lipid metabolism, oxidative and neurotoxic effects. Laboratory results were integrated with a field study which provided the first evidence on the occurrence of DIC, IBU and NIM in tissues of wild mussels sampled during summer months from an unpolluted, touristic area of Central Adriatic Sea. Overall results demonstrated M. galloprovincialis as a good sentinel species for monitoring presence and ecotoxicological hazard of pharmaceuticals in the Mediterranean.