ABSTRACT. Aquaculture is the fastest growing and highly traded global food sector - set to dominate the supply of aquatic protein by 2050. To deliver significantly enhanced volumes of food sustainably, appropriate account needs to be taken of its impacts on the environment, the health and welfare of organisms cultured, and human health outcomes of consuming seafood from the sector. The One Health Aquaculture concept argues for an evidence-policy refresh, bringing a wider array of industry, government and societal stakeholders together to ensure key ‘success metrics’ spanning human, environmental and organismal health are ‘designed-in’ to supply chains - effectively operationalising a One Health approach to food production from the sector. Success metrics, applied to given sub-sectors of the industry (e.g., seaweed, molluscs, shrimp, finfish) and assessed according to availability of research, evidence, policy, and legislation provides a framework around which potential negative impacts of production can be defined, reduced or averted. Enhanced focus on identifying and controlling chemical and pathogen hazards which interact with, and impact, aquatic food supply chains will form a particularly important element of this as we increasingly rely on intact and healthy aquatic systems as locations in which we wish to grow our food. As aquaculture positions to dominate supply of seafood, application of One Health principles provides a means to optimise the benefits associated with food production from water. In addition, recognition of aquaculture in broader food systems necessitates new debate on its interaction with land-based food production sectors – and appropriate national and international science and policy strategies which support improved food system design from land and water. Intricate relations between aquatic animals and their environment should drive closer alignment of environmental and food policy especially relating to the impact of aquatic chemical and pathogen hazards on safe and sustainable aquatic food supply.

ABSTRACT. Endocrine Disrupting Chemicals (EDCs) affect organisms by disrupting their endocrine systems through the interference with any aspect of hormone action. The most common mechanism of action of EDCs in vertebrates is to mimic endogenous hormones and compete with their Nuclear Receptors (NRs) as agonists or antagonists. Although there is key and accumulating evidence that EDCs impact on almost all groups of invertebrates, the lack of detailed description of their endocrine signaling pathways and Nuclear Receptors function prevents the understanding of endocrine disruption in these organisms. In this work, the mechanism of action of selected model EDCs during the early larval development of the Mediterranean mussel Mytilus galloprovincialis (Mg) were characterized and summarized into plausible Adverse Outcome Pathways (AOP) that could be linked to mechanisms of endocrine disruption. We first characterized the neuroendocrine elements regulating larval development and established the morphogenetic process of shell biogenesis as susceptible to neuroendocrine disruption. Consequently, plausible neuro-/endocrine AOPs of the model EDCs BPA and TBBPA were established in relation to the observed adverse biological outcomes on Mg larval shell biogenesis and neurogenesis. In addition, by characterizing the effects of TBT in Mg larvae, a plausible relationship between NR signaling and the neuroendocrine system was also established. What is more, the analysis of the transcriptome assembly of Mg early larval stages showed that the expression dynamics of gene networks defining biological processes sensitive to EDCs are in line with the phenotypic and morphological observations, thus corroborating the theorized pathways of neuro-/endocrine disruption. The results of this work represent a further step towards a better understanding of endocrine disruption in invertebrate organisms.

ABSTRACT. Endocrine disruption has raised concern for public health, but also seriously affects wildlife. Crustaceans constitute an important component of the aquatic fauna, but it remains unclear how xenobiotics may interfere with their endocrine systems. Because of the public health concern and because oestrogenic and anti-oestrogenic compounds are amongst the bulk endocrine disrupting chemicals, the endocrine disruption debate has primarily focussed on steroid hormones and molecules that may interfere with the vertebrate hormone system. It has been suggested that oestrogens and oestrogen mimics could also affect reproductive functions or moulting in crustaceans. The mechanistic basis for such action remains, however, obscure, as crustaceans have no oestrogen receptors as well as androgen and progestogen receptors. On the contrary, much of the crustacean endocrinology is neuroendocrine in nature and relies heavily on the release of peptide hormones via neurosecretory cells. This offers a different perspective on endocrine disruption in crustaceans connecting it with pollutants that modulate neurological and neuroendocrine functions. It is concluded that the issue of endocrine disruption in crustaceans should primarily consider the impacts of neuroendocrine disruptors on reproduction, development, growth and behaviours to identify environmental pollutants that could modify endocrine processes specific to this phylum.

ABSTRACT. An increasing number of studies demonstrates that exposure to certain environmental chemicals during early life stages may disrupt development and other endpoints across multiple subsequent non-exposed generations. Many of these chemicals are widely used in consumer products and pharmaceuticals, such as tributyltin, fluoxetine, phthalates, carbamazepine, simvastatin, among others. These findings have major implications for understanding disease etiology and adverse outcomes. Most evidences indicate that the mode of action (MOA) associated with transgenerational inheritance of negative phenotypes involves epigenetic modifications. Here, we will discuss how biomarkers of epigenetic modifications can be integrated into the current hazard and risk assessment frameworks associated the endocrine disrupting chemicals.

ABSTRACT. The application of biogenic wastes as fertilizers and amendments can improve soil properties and resilience. Nevertheless, growing concerns are related to their potential hazard to natural ecosystems and human health, due to the presence of toxic chemicals either hydrophobic or hydrophilic. Therefore, there is an urgent need to develop effect-based tools, suitable in assessing quality of environmental complex matrices, to guarantee their safe application into the environment. In this scenario, we assessed the ecotoxicity of different matrices such as sludge, manure, compost and digestate using zebrafish embryo (Danio rerio) as model species. We tested acute toxicity of elutriates as well as sub-lethal effects, applying a multi-biomarker suite related to oxidative stress, neurotoxicity, energetic metabolism and behavioral alterations. Exposure to elutriates boosted basal respiration and affected swimming performances of embryos. Besides, elutriates from digestate and sewage sludge were also able to inhibit Acetylcholinesterase activity. The zebrafish embryotoxicity test, coupled with sub-lethal endpoints, proved to be a suitable screening tool for integrating the effects of a complex matrices -such as elutriates- linking first interactions at biochemical level with the modification of key physiological functions of zebrafish embryos. Acknowledgements: This study was financed by Cariplo Foundation project number 2020-1019.

ABSTRACT. Multiple reaction monitoring (MRM) mass spectrometry is emerging as a relevant tool for measuring customized molecular markers in freshwater sentinel species. However, it is typically used for the validation of a limited number of molecular markers preselected from shotgun experiments and at a whole-individual level in small invertebrates, limiting the physiological interpretation of these markers due to a lack of organ specificity. . In this study, we show that recent gains of MRM multiplexing capacity offers new possibilities to conduct large-scale screening of animal proteomes at the organ level, through a proof of concept in freshwater amphipod Gammarus fossarum, a sentinel species frequently used in ecotoxicology. By combining laboratory and field (in situ caging) strategies and MRM technologies, this study proposes a new strategy for the discovery of candidate molecular markers in the caeca, one of the main detoxification and metabolically active organs in amphipods. Seven hundred and sixty-five peptides representing 450 distinct proteins were monitored in MRM, resulting in a comprehensive coverage of biological functions. This MRM method was then successfully applied to analyze the caeca of gammarids either exposed in the laboratory to several environmental concentrations of cadmium (Cd), silver (Ag) and zinc (Zn) or caged in river sites across French with various types and levels of metal contamination. Differential proteomics and functional analyses indicate that the proteome of Gammarus fossarum caeca show metal specific responses to different metal exposures, paving the way to define metal-specific molecular signatures of exposure. This confirmed the interest of developing an organ-centred ecotoxicological assessment of contamination using highly multiplexed MRM mass spectrometry.

ABSTRACT. The widespread use of Plant protection products (PPP) in agriculture leads to the exposure of non-target organisms, including species of ecological relevance, such as insect pollinators, to a large number/mix of contaminants. While lethal effects of PPP active principles are tested, there is still a strong need to investigate the sublethal effects of commercial formulations and to monitor farmed and natural populations. Also, fungicides and herbicides need a particular focus being much less investigated up to now. Our work aims to investigate the sublethal effect of PPP and other environmental contaminants on the honeybee, Apis mellifera, an insect pollinator subjected to a rapid decline in the last decades. We developed and applied a multi-biomarker approach that includes several biochemical and cellular endpoints encoding for neurotoxicity (AChE and CaE), genotoxicity (NA assay), metabolic alterations (ALP and GST) and immunotoxicity (LYS, differential haemocyte count). Laboratory treatments with fungicides, herbicides and trace elements were carried out as well as beehives monitoring in rural and urban areas. The multidimensional perspective provided by the use of a large set of biomarkers was used to develop, for the first time in honeybees, an Integrated Biological Response index (IBRv2). Lab experiments showed a modulation in the biomarker response to different tested compounds, even within the same PPP class. The fungicide Amistar®Xtra was found to cause neurotoxic, genotoxic and immunotoxic effects. Field monitoring, also, showed a modulation in the responses depending on the different anthropic impacts and the different agricultural activities. Neurotoxicity, metabolic alterations and genotoxicity were found in agricultural areas while immunosuppression was found in urban areas. The multi-biomarker approach proved to be a sensitive tool to investigate, in laboratory and field, the potential risk deriving from anthropic contamination to A. mellifera.

ABSTRACT. Pesticides, in addition to other classes of contaminants, can cause alterations or damage to avian species. Great tit (Parus major), the most representative of the Passeriformes, has a good ecological relevance and its territoriality provide precise information on a given territory. Common kestrel (Falco tinnunculus) is a raptor considered resident in the southern Europe. The aim of the study is to investigate the effects of environmental contaminants on the two avian species by an approach that integrates chemical analysis, enzymatic responses, and behavioural traits. From an idea of Prof. Matteo Griggio, several nest boxes for great tit were installed in different areas of Veneto region (northern Italy). Nest boxes for common kestrel, collocated in different area of Lazio region (central Italy), were also monitored. The areas are subjected to different impact: agricultural, urban and wooded environments. Neurotoxicity (esterases inhibition), genotoxicity (ENA and comet assay), immunotoxicity (complement system) and oxidative stress (TAS assay) effects were evaluated on blood and excreta. Different contaminant levels (heavy metal, etc.) were evaluated. Parental care (nest defence, pair coordination over offspring provisioning) of the species breeding, and cognitive abilities, phenotypic endpoints and reproductive success were assessed. Responses to contaminants at different biological levels were modulated depending on the characteristics of the areas. For great tit, a strong increase of the TAS was found in specimens from agricultural areas, as well as genotoxic effects, also in urban one. Responses of tonic immobility showed differences between urban and agricultural sites. Genotoxic effects were also observed in common kestrels from the agricultural area, while immunotoxicity effects were highest in specimens from the urban area. The developed protocol was proved to be a useful tool to obtain a more complete picture of the ecotoxicological status of the species.

ABSTRACT. Synthesized to cure and save human lives, nowadays pharmaceuticals are present everywhere in the environment, threating seriously the nontarget aquatic wildlife. Amphibians represent a particularly vulnerable group of organisms, which are currently at risk of extinction due to several anthropogenic interventions, with pharmaceuticals among the most important ones. Considering the limited studies on the negative effects of pharmaceuticals in amphibians, a better understanding of the mechanisms underlying the sublethal effects of drug mixtures in wildlife is an urgent call. To address this existing research gap, the effects of the two mostly found drugs in Albanian freshwater ecosystems, the antidepressant fluoxetine and the anti-inflammatory ibuprofen, as well as their combination has been studied. Tadpoles of Bufo bufo were exposed for seven days to two environmentally realistic concentrations of fluoxetine, ibuprofen and their mixture. The development behavior and erythron profile were evaluated as endpoints of exposure response. Furthermore, to elucidate the molecular mechanisms of drug-induced erythrocyte apoptosis based on drug interactions with DNA, in-silico molecular docking and multi-biomarkers approach were used. Both drugs negatively affected tadpoles’ growth and development by significantly delaying their time to metamorphosis and reduced body weight, and impared their behaviour by increasing unresponsiveness to different stimuli. A significant increase in the frequency of cellular and nuclear abnormalities strongly confirms the toxicological potential of fluoxetine and ibuprofen in B. bufo tadpoles. This is the first study unraveling the molecular mechanisms of nonsteroidal anti-inflammatory drug mixture using amphibians as model organism. Both drugs interacted in impairing animal health which in turn might affect species population dynamic.

ABSTRACT. The use of innovative materials to be used for applications in the field of aquaculture is an important alternative to favor the reduction of the impact due to the use of recalcitrant plastic materials. Indeed, in addition to causing direct damage to marine organisms, plastics and microplastics can favor quick colonization by microorganisms on their surface by hosting a real microbial community, called plasticsphere [1]. Plastics and microplastics could also act as transport vectors for toxin-producing and pathogenic microbial species [2,3], thus promoting the spread of potential infectious agents. During this study, the bio-based material Mater-Bi (Novamont S.p.A., Italy) was tested as an alternative material to the classic polypropylene (PP) stockings for mussel farming (Mytilus galloprovincialis) in the Gulf of Taranto, for an experimental period of 6 months. At pre-established interval times, three fragments of each sock (with and without mussels) have been collected and stored. The total bacterial abundance was determined with DAPI-staining. For the enumeration of cultivable bacteria, aliquots of diluted suspensions were spread plated on common medium for marine heterotrophic microrganisms. The presence of microbial fecal pollution indicator (i.e. total coliforms, Enterococci, E. coli, Shigella, Salmonella, Proteus, Klebsiella, V. cholerae, V. parahaemolyticus, P. aeruginosa, S. aureus and C. perfringens) have been also assessed by using specific media. All bacterial strains were also tested for biofilm production, to elucidate the different adhesion and affinity level to the two matrices. The results indicated a clear diversification of the microbial colonization on the PP and Mater-Bi in terms of abundance and presence of bacterial pathogens. Further analysis are planned to complete the study with determination of total microbial communities by metabarcoding approach.

ABSTRACT. Aquacultured bivalves are increasingly subjected to recurrent disease outbreaks resulting in mass mortalities. Although oyster juveniles (Crassostrea gigas) are most severely affected, with dramatic economic/ecological consequences, mortalities occur in other species1. These episodes are polymicrobial and multifactorial, involving both host and environmental factors1, where the interactions between potential pathogens and the immune system of the host may play a key role2. Arcobacter-like bacteria represent emerging foodborne zoonotic pathogens, primarily transmitted through contaminated food and water; their increasing presence in seafood is raising environmental concern3. Although Arcobacter are detected in diseased oysters and stressed bivalves, no data are available far on its potential pathogenicity to the bivalve host. In this work, a strain of Malaciobacter marinus, isolated from an oyster mortality event in 2019 in Spain (IRTA-19-131) was investigated for its in vitro interactions with the hemolymph of Mytilus galloprovincialis and C. gigas in different conditions (106-108 CFU/mL; 30-90 min) and immune responses were evaluated. Although M. marinus-induced lysosomal stress in hemocyteswas associated with efficient killing of bacteria in mussel and oysters, distinct mechanisms were involved. In mussels, the defense response was due to activation of extracellular defences and serum activity. In oysters, bactericidal activity was fully due to intracellular mechanisms. In mussels, absence of pathogenicity was confirmed in vivo in adults and larvae. These are the first data on interactions between Arcobacter environmental isolates and bivalve immune system. The results are compared with those obtained with pathogenic bacteria (Vibrios). In different bivalve host, distinct interactions of Arcobacter with the immune system may be sensitive to environmental factors or pathogen infections, resulting in a strongly opportunistic behavior or pathogenicity.

ABSTRACT. Paracentrotus lividus roes represent the most famous food delicacies in the whole world. The increase in the demand of this seafood caused increased pressure on the wild stocks of this species. Fishery management failures could gradually lead to an exponential decrease and eventually to the local extinction of populations in impacted coastal areas. In this scenario, aquaculture facilities could meet the effects of anthropogenic pressures on the wild stocks of P. lividus. For this reason, experimental studies should be conducted to enhance techniques aimed at improving efficient aquaculture practices for these animals. In this work, we performed for the first time a molecular analysis, measuring by Real-Time qPCR the expression levels of several genes involved in stress response and skeletogenesis. We aimed at understanding if material influences may significantly impact the life of future offsprings and if the culture conditions may be as well important. Our findings demonstrated that the outcomes of the in vitro fertilization of P. lividus were influenced by maternal influences, but these effects are largely tuned by the culture conditions. In fact, twenty-three genes, involved in the response to stress and skeletogenesis, were differently expressed in sea urchins cultured in two experimental conditions, and the results were largely modified in offspring deriving from two group of females.

ABSTRACT. Aquaculture faces an ongoing threat from bacterial diseases and antibiotic therapy plays a key role in protecting fish. However, the widespread use of antibiotics causes their eventual release in the aquatic environment, with negative implications for the ecosystem. Hence, alternative solutions to reduce the antibiotic load in the water are needed and nanotechnology could help in this regard. Among the available magnetic nanomaterials, Surface Active Maghemite Nanoparticles (SAMNs) stand out for their peculiar surface chemistry, offering advantages, such as the high colloidal stability in the absence of any coating and the direct functionalization by simple wet reactions. Flumequine (FLU), a widely used antibiotic in fish, was bound to SAMNs and the resulting nanocarrier (SAMN@FLU) showed potential for application in aquaculture[1]. The present work aims at a preliminary evaluation of the therapeutic efficacy of SAMN@FLU using D.magna as a model organism. Firstly, the antimicrobial efficacy of SAMN@FLU was tested on the opportunistic pathogen of fish Aeromonas hydrophila, isolated from naturally infected daphnids, using a quantitative assay of the minimum inhibitory concentration (MIC). Noteworthy, the MIC of SAMN@FLU was lower than that of FLU. Daphnids were then experimentally infected with bacteria and the therapeutic effect of SAMN@FLU was compared with that of the soluble FLU. SAMN@FLU slightly improved the survival of infected daphnids, though to a lesser extent than FLU. Despite this, SAMN@FLU potentially allows to recover the antibiotic in excess from the aquatic environment. Although the feasibility of using SAMN@FLU as nanoantibiotic in aquaculture requires a deeper investigation, this study highlights the need of new drug delivery systems aimed to ensure the sustainable development of aquaculture. [1]Bortoletti et al.Nano-immobilized flumequine with preserved antibacterial efficacy.Colloids Surf B Biointerfaces,2020;191:111019

ABSTRACT. Plastic is a widely used material in aquaculture facilities that may become marine litter (ML). In Italy a thousand tonnes of polypropylene (PP) socks per year are used in mussel farm. Thus, it is important to consider alternative solutions such as the employment of bio-based materials, but the studies assessing their behaviour are still poor. In this context, the present study aims to replace PP with Mater-Bi socks (Novamont S.p.A., Italy) and to assess: i) the polymer degradation along the production cycle; ii) the mussel’s biomass (weight, size), iii) the macrofaunal community associated to Mytilus galloprovincialis in a farm located in the Ionian Sea (Mar Piccolo, Italy). In October 2021, six socks in PP and six Mater-Bi were deployed in the study area (T0) and two different treatments were considered for each type of socks: socks with mussels vs socks without mussels (n=3). Socks samples from each replica were collected monthly (T1-T2-T3) while mussels and the associated biodiversity were sampled seasonally (winter and spring). Fourier transform infrared (FT-IR) spectroscopy analysis was performed using a self-generated polymer library characterised by only the reference spectra of the Mater-Bi (T0) before the start of the analysis, to assess the possible polymers alterations. The polymer identification was accepted when the match with the reference spectra had a level of certainty >70%. Taxonomic identification was carried out through microscopy observation and biometric parameters of each specimen of blue mussel were recorded. Preliminary FT-IR analysis did not show evident alterations in the different experimental levels. From preliminary results we observed that the dominant taxon in the macrofaunal community changed across seasons. Further studies, considering longer observation time, are needed to better understand the suitability of bio-based materials in aquaculture and to improve the sustainability of this crucial production sector.

ABSTRACT. Pollution particularly affects coastal ecosystems due to their proximity to anthropic sources. Among those environments, harbour areas are subjected to marine traffic but also to accidental and chronic pollution. These areas are thus exposed to complex mixtures of contaminants such as trace elements and organic pollutants which can impact marine species, habitats, and ecosystem services. The monitoring of these contaminants and their impact on ecosystems are priority actions within the Marine Strategy Framework Directive. In this context, the aim of the present work was to evaluate the environmental contamination of several harbours in Corsica. For two years, the bioaccumulation of trace elements, polycyclic aromatic hydrocarbons, and polychlorinated biphenyls was measured in marine molluscs, mussels Mytilus galloprovincialis and limpets Patella caerulea. Moreover, potential pollution-induced effects were assessed in these organisms by conducting multi-biomarker analyses related to oxidative stress, energetic metabolism, neurotoxicity, and immunity. The results reveal a relatively low contamination in the Corsican harbours studied compared to larger Mediterranean ports and suggest that limpets are suitable bioindicators for monitoring chronic pollution in these coastal areas.

ABSTRACT. Evaluation of organism responses to environmental stressors at the molecular level can help understanding mechanisms of toxicity and adaptation. In this light, lipids are a class of molecules with unique promise for environmental sciences, since they are highly abundant and almost ubiquitous across all organisms, and lipid profiles often change drastically in response to external stimuli. One of the major lipid classes, sphingolipids, play essential structural and functional roles in cell membranes, and modulate multiple cellular functions. In particular, ceramides show a variety of biological roles, suggesting their importance across a variety of life stages and environmental conditions. In bivalve molluscs, lipidomics has been so far applied only in studies on their nutritional value as aquaculture seafood. Like many invertebrates, bivalves possess characteristic ceramide classes such as ceramide phosphoethanolamines (CPE), and ceramide aminoethylphosphonates (CAEP). In oysters, changes in expression of ceramide-related genes induced by bacterial challenge suggests their role in stress and/or immune responses. In this work, data are presented on membrane lipidomics in Mytilus galloprovincialis immune cells (hemocytes), eggs and early larval stages (24 and 48 hpf). Lipid extracts were analyzed by 1H-NMR and 31P-NMR to identify main lipid classes, and by LC-electrospray ionization-MS for qualitative/quantitative analyses of membrane phospholipids. In parallel, a search for Mytilus genes involved in ceramide homeostasis and expression evaluated across early development was performed. The results show that CAEP represent 29.0 and 13 mol%, respectively, in mussel hemocytes and larvae. These data represent the basis for understanding the role of lipids, ceramides in particular, in mussel immunity and early development, and identifying specific molecular components of lipid homeostasis as possible targets for environmental stressors.

ABSTRACT. Many animals display rapid colour changes that may be controlled either nervously or hormonally. In cephalopods, serotonin induces muscle relaxation leading to pigment concentration in the chromatophores. In crustaceans, dopamine stimulates pigment concentration via red pigment concentrating hormone, whereas serotonin ensures pigment dispersion via pigment dispersing hormone. Environmental pollutants can potentially interfere with these mechanisms of physiological colour change and cryptic behaviour. We exposed Carcinus maenas, Crangon crangon and Sepia officinalis to waterborne selective serotonin and/or norepinephrine reuptake inhibitors, which are known to alter the levels of serotonin and other biogenic amines in wildlife. Venlafaxine provoked pigment concentration in chromatophores of S. officinalis at the same concentration (10-4M) as did serotonin. Fluoxetine alone (5 ng L–1) or in combination with venlafaxine (2.5 or 5 ng L–1) improved uniform, but not disruptive body patterns in newly hatched cuttlefish. C. crangon exposed to 10-1000 ng L–1 fluoxetine for up to one week showed a significant increase in darkening following 30 min on black substrates. Similarly, pigment dispersion in juvenile C. maenas transferred from a white to a dark background tended to increase when exposed to fluoxetine at 5 ng L–1, but when exposed to a combination of fluoxetine and venlafaxine at 5 ng L–1 each, darkening was significantly impaired. These studies demonstrate that physiological colour changes are susceptible to environmental chemicals that interfere with the neuronal or neurohormonal control of chromatophore movements. Hence, they may serve as indicators of environmental pollution and could point to detrimental effects on behavioural ecology that are important for the animals’ survival. Colour change bioassays, therefore, provide a promising tool for environmental testing with the advantage of being non-invasive, inexpensive and relatively straightforward.

ABSTRACT. Large marine mammals fill important ecological niches as mid or top predators in marine food webs. Their high energy intake is often accompanied with high intake of contaminants having bioaccumulating and biomagnifying properties. They can thus act as barometers for environmental pollution. The global burden of chemical pollution and new entities is now considered to have exceeded the planetary boundary, and there is thus a need for continuous and increasing efforts to investigate impacts of legacy persistent organic pollutants (POPs) and chemicals of emerging concern (CECs) in wildlife. Killer whales are by far the most contaminated marine mammal species, while the lowest concentrations of POPs are measured in blue whales. Both legacy POPs and CECs are endocrine and/or metabolic disruptors, and can disturb the physiology, growth, and reproduction, as well as immune function of animals and hence impair overall health of individuals and populations. To date, only a handful of studies have given mechanistic insights in contaminant response in marine mammals. Over the last couple of years, we have established alternative approaches to overcome these hurdles using in vitro and in silico methods. The goal of this work is to characterize functional properties of key molecular targets for environmental contaminants in killer whale (Orcinus orca), fin whale (Balaenoptera physalus), and polar bear (Ursus maritimus), and to establish fibroblast cell cultures from skin biopsies that can be further used in reprogramming into mesenchymal stem cell lines and in characterizing toxicological responses in these animals. Results from these ongoing studies will be presented and discussed. This work is supported by the Fram Centre Flagship-project “Cellular responses to contaminant exposure in marine mammals from the Arctic”, project nr. 462/602019 to the Norwegian Polar Institute.

ABSTRACT. Artificial barriers in rivers cause a substantial decline in endemic and migratory fish as habitat fragmentation, changed reproduction environments, and blocked migratory routes. Fishways can allow safe passage across these obstructions while also preserving river biodiversity. Hydrodynamic variables influence fish swimming ability and behaviour, which must be understood to construct appropriate fishways. When fish cannot consist or balance, high velocities at passages result in physical stress. School formation results in a hydrodynamic benefit and improves individual performance. The hydrodynamic use of swimming alongside other people is observed in schools, improving performance. A multidisciplinary research strategy was used to investigate the effects of group size at various hydraulic conditions. As a result, collective behaviour in physiological stress reactions was investigated. In a portable flume, wild vairone (Telestes muticellus) was examined in groups of 1, 2, and 6 fish. The stress response was studied by analysing the hypothalamic-pituitary-interrenal axis and the cellular antioxidant defence system. Oxidative stress was also evaluated, analysing lipid peroxidation and protein oxidation in the skeletal muscle. Preliminary results suggest that oxidative damage is lower in grouped fish than in a single fish. Furthermore, increased oxidative stress risk in this tissue will likely enhance cytoplasmatic and mitochondrial gene expression of various antioxidant enzymes, assessed by qRT PCR. Specific antioxidant responses result in lower muscle cell damage and a higher homeostatic capacity. (Supported by the European Union Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie Actions, Grant Agreement No. 860800).

ABSTRACT. Metal pollution is a major concern for aquatic environments. Diverse patterns and levels of metal contamination are observed in rivers, which can lead to impacts on the maintenance of animal populations. Metallothioneins are a family of proteins conserved across metazoan evolution, with low molecular weight and rich in cysteine, whose main physiological functions are the maintenance of the homeostasis of essential metals, detoxification of non-essential metals and the protection against oxidative stress. From the acquisition of RNAseq and the assembly of individual transcriptomes in the amphipod Gammarus fossarum, we identified two metallothioneins (namely MT-1 and MT-2) in this species commonly used in aquatic ecotoxicology. Based and experimental exposures, our results show that the induction of MT-1 expression is stronger following exposure to Cd compared to 2 other metals tested (Zn and Ag), suggesting it is probably a Cd-thioneine. Expression patterns of these two metallothioneins at the organ level (caecum, gills, gonads, embryos) were respectively studied in tolerant and Cd-sensitive populations. A specific expression of MT1 was observed in the caeca and gills of individuals in tolerant populations, suggesting that cadmium sensitivity is probably mediated by organ-specific MT-1 expression, particularly in the caeca and gills of amphipods. These results corroborates previous observations that cadmium tolerance in gammarids is a plastic phenotype and is not determined by a genetic adaptation.

ABSTRACT. The number and size of offshore wind facilities (OWFs) is increasing rapidly to meet the demand for renewable energy. When OWFs operate, they produce low-frequency noise at a higher intensity than that naturally present in the sea soundscape. This noise can travel far from the source location, and potentially impact marine organisms. The energy produced by OWFs is transported through high voltage Direct Current (DC) subsea cables. These produce magnetic fields (MFs) that could impact marine organisms transiting through the network of DC cables. There is very little knowledge of the impact of operational noise and MFs on the early life history stages of marine fish. We present a novel approach to investigating these questions that couples in situ and laboratory-based experiments to assess the impacts of OWFs’ low-frequency noise and MFs on the swimming, orientation and dispersal of the larvae of commercially important species that could be impacted by large-scale wind farms. Using cutting-edge technology, we observed the behavior of larval cod (Gadus morhua) swimming in drifting chambers at sea. Using a low-frequency sound projector, larvae were exposed to 100 Hz signal in the intensity range of that produced by OWFs. Using hydrophones and a 3D-particle velocimeter, we assessed the behavioral response of cod larvae in situ to the sound pressure and particle motion generated by the signal. We also conducted lab-based experiments using electric coils to simulate the scenario of larvae swimming by a MF gradient generated by DC cables. We tested potential attraction/avoidance to MF and its impacts on swimming of both Atlantic haddock (M. aeglefinus) and lesser sandeel (A. marinus). Both low-frequency noise and MF significantly impacted the behavior of these larvae. We show the impacts of each stressor and how, on a large scale, they could significantly modify the larval dispersal of these species

ABSTRACT. Humane endpoints (HE) have become a major element in animal experiments to fulfill ethical issues. This study aimed to define a clear HE scoring system in a streptozotocin (STZ)-induced diabetes model using male Sprague-Dawley rats. The rats were divided into control (n=8) and induced (n=16) groups. The induced group started drinking water with fructose (10%,w/v) for 14 days. Then, the induced and control animals were intraperitoneally administered with STZ (40mg/kg) and 0.1M citrate buffer, respectively. The following parameters were evaluated: body weight, body posture, hair/tail appearance, grooming, grimace scale, mental status, response to external stimuli, hydration status, stool appearance, convulsions, and response to abdominal palpation. A score was given to each parameter and animals were sacrificed if their sum reaches the value of four or more. Several nutritional parameters were registered (ie, Lee index, body weight index (BWI), and abdominal and thoracic perimeters). The animals were euthanized after 8 weeks of experiments. All ethical issues followed the Portuguese Direção Geral de Alimentação e Veterinária guidelines. No deaths were recorded, no animal reached the HE score of four. No changes were observed in control group. Induced animals showed after fructose administration, dehydration(14/16). After STZ injection, were observed lack of grooming(8/16), narrowing of the orbital area(1/16), curved posture(10/16), liquid(3/16), pasty diarrhea(1/16), and abdomen distention(1/16). The total mean score registered in induced animals was 0.53±0.42. The Lee index, BWI, and abdominal and thoracic perimeters were significantly lower than controls (p<0.01). Induced animals showed polydipsia, polyphagia and polyuria, and negative weight growth one week after STZ administration. Given the lack of published research concerning HE evaluation in diabetes animal models, this work will help researchers to apply the 3Rs in the implementation of this protocol.