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The Mediterranean basin is the second largest global biodiversity hotspot in the world, which coexists with a plethora of anthropogenic stress. This study examines the risks that marine litter poses to Mediterranean biodiversity, using the Special Protection Area of Mediterranean Importance (SPAMI) "Pelagos Sanctuary" as a case study. In this paper, a new survey method and data analysis strategies to assess the impact of marine litter, including microplastics, on Mediterranean organisms, is proposed. A total of 23 species, from invertebrates to cetaceans, were analysed using two main monitoring approaches. In the first approach, the core density distribution areas of 11 megafauna species (elasmobranchs, sea turtles, seabirds, and cetaceans) and 4 invertebrate species were evaluated through an extended field survey. Simultaneously, monitoring of floating macro- and micro-litter was performed to be overlapped with the species distribution and abundance. The second monitoring approach assessed the ingestion of marine litter, microplastics and the levels of plastic additives in 10 stranded megafauna species and in 7 invertebrate and fish species. The final data processing, merging the data on biodiversity and marine litter abundance and distribution in the environment, with the data of ingested marine litter, microplastics, and plastic additives in sentinel species, allowed to: a) create risk maps for the species inhabiting the Pelagos Sanctuary, identifying critical areas for biodiversity conservation; b) identify the most exposed species to the risk of marine litter by an "exposure score"; c) to select sentinel species for pelagic environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

Marine litter, particularly microplastics, is a growing threat to the Mediterranean Sea, impacting biodiversity and ecosystem health. However, most studies conducted in the Mediterranean Sea have focused on monitoring of only specific environmental compartments, and rarely have highlighted the overall impacts affecting an area. Therefore, using a new multi-compartment monitoring approach and a standardized methodology, this study investigates the abundance, distribution, composition and impact of marine litter on beaches, surface waters, fish and mussels in a coastal area of Tuscany (Italy). Concerning beach macro litter values, significant differences were found among the three beaches analysed, with the highest amount in the Feniglia beach (mean value = 1245 items/100 m). The top items found are cotton bud sticks (32.3 %), which in the winter survey at Feniglia beach, reached a remarkable density of 1983 items/100 m. Microlitter (1-5 mm), was detected in the beach sediments with a mean abundance of 130 items/m 2 . Regarding floating macrolitter, the transect with the highest values was the Feniglia site at 3 nautical miles in autumn (1083 items/km 2 ) while for floating microlitter the highest concentration (832,683 MPs/km 2 ) was found in front of Scarlino. Of the 234 fish analysed from 5 different species, 67 contained microplastics in the gastrointestinal tract (28 %) with a mean value of 0.4 items per individual. The species with the highest occurrence was the European anchovy (Engraulis encrasicolus) (53 %) followed by the bogue (Boops boops) (40 %). Through the application of the Marine Litter Impact Index (MLII), considering all the compartments analysed, the Feniglia area emerges as the ecosystem most impacted by marine litter (mean MLII = 3.5, high). This study highlights how a multi-compartment monitoring approach is crucial for understanding the complex interactions between land, sea, and biota., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2024. Published by Elsevier Ltd.)

Plastic materials contain additives such as plasticizers and flame retardants, which are not covalently bound to plastic polymers and can therefore be unintentionally released into the marine environment. This study investigated three families of compounds, phthalates (PAEs), organophosphate esters (OPEs), and non-phthalate plasticizers (NPPs) currently used as plastic additives, in 48 muscle samples of bogue (Boops boops), European hake (Merluccius merluccius), red mullet (Mullus barbatus), and European pilchard (Sardina pilchardus) sampled in the Central Adriatic and the Ligurian Seas. The additional goal of this study is to assess the potential risk to human health from fish consumption with the objective of determining whether the detected levels might potentially pose a concern. PAEs represent the majority of the plastic additives detected in the selected species, with ubiquitous distribution across the study areas, whereas for OPEs and NPPs, there is a more pronounced difference between the two study areas, suggesting that these compounds may represent different exposure levels in the two seas. Among PAEs, bis(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), and diisobutyl phthalate (DIBP) were the most abundant compounds, reaching levels up to 455 ng/g ww. OPEs were detected at higher concentrations in samples from the Ligurian Sea, and triethyl phosphate (TEP) was the most abundant compound. Among the NPPs, acetyl tributyl citrate (ATBC) was most frequently detected. From the results obtained, fish consumption may not pose a risk to human health (Hazard Quotient<1) but needs to be considered in future studies. Given the limited number of studies on PAEs, OPEs and NPPs in the Mediterranean Sea, further research is necessary to understand their potential bioaccumulation in marine organisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Sea turtles can host a wide range of parasitic taxa, some of which may lead to severe diseases, weakening and endangering their health. Assessing free-ranging sea turtles' responses to these potential pathogens using biomarkers can provide valuable insights into the cellular and molecular impacts of parasites. This information can serve as a crucial tool for conservation efforts. During rehabilitation, hematological, genotoxic and innate immune parameters were evaluated in 70 free-ranging sea turtles rescued along the Southern Adriatic coast (Eastern Mediterranean Sea) from 2021 to 2023, 24 of which were found to have parasitic infections. Hapalotrema mistroides, Sulcascaris sulcata and Neospirorchis sp. were identified by coprological and molecular techniques. Some significant differences for biomarkers were associated with these parasites. These findings highlight the importance of considering the infection status of free-ranging sea turtles when evaluating biomarker results, as major pathogens like parasites can influence certain parameters., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest and that they have no actual or potential competing financial interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Biodegradable polymers have been proposed as an alternative to conventional plastics to mitigate the impact of marine litter, but the research investigating their toxicity is still in its infancy. This study evaluates the potential ecotoxicological effects of both virgin and marine-incubated microparticles (MPs), at environmentally relevant concentration (0.1 mg/l), made of different biodegradable polymers (Polycaprolactone, Mater-Bi, cellulose) and conventional polymers (Polyethylene) on Mytilus galloprovincialis by using transcriptomics. This approach is increasingly being used to assess the effects of pollutants on organisms, obtaining data on numerous biological pathways simultaneously. Whole hepatopancreas de novo transcriptome sequencing was performed, individuating 972 genes differentially expressed across experimental groups compared to the control. Through the comparative transcriptomic profiling emerges that the preponderant effect is attributable to the marine incubation of MPs, especially for incubated polycaprolactone (731 DEGs). Mater-Bi and cellulose alter the smallest number of genes and biological processes in the mussel hepatopancreas. All microparticles, regardless of their polymeric composition, dysregulated innate immunity, and fatty acid metabolism biological processes. These findings highlight the necessity of considering the interactions of MPs with the environmental factors in the marine ecosystem when performing ecotoxicological evaluations. The results obtained contribute to fill current knowledge gaps regarding the potential environmental impacts of biodegradable polymers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Monitoring microplastics (MPs) in the marine environment is an ongoing process, and our understanding of their impact on marine organisms is limited. The present study evaluates the effects of ingested MPs on the marine MP pollution bioindicator fish species Mullus surmuletus. The study follows a three-fold approach to assess the impact of MPs on marine organisms by investigating: 1) the ingestion of MPs, 2) the bioaccumulation of phthalate compounds as plastic additives, and 3) the evaluation of toxicological biochemical and cellular biomarkers. Striped red mullets were sampled in the marine protected area (MPA) of the National Marine Park of Zakynthos and coastal sites with high touristic pressure in Zakynthos Island in the Ionian Sea, Greece. Fewer ingested MPs and lower phthalate concentrations were found in fish inside the MPA compared to those sampled outside the marine park. However, no relationship was found between either phthalate concentrations or biomarker levels with the ingested MPs in the red striped mullets. Biomarker levels were influenced by season and site, but no effect could be attributed to the ingested MPs. The lack of association of biomarker responses and plasticizer bioaccumulation to MP ingestion can be explained by the low number of ingested MPs in the fish from Zakynthos coastal area as MP abundance ranged from 0.15 to 0.55 items per individual fish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study delves into the magnitude and attributes of plastic pollution in the salt marshes of the Bahía Blanca Estuary, Argentina, with a specific focus on its spatial distribution. The investigation included the evaluation of microplastics (1-5 mm), mesoplastics (5-25 mm) and macroplastics (25-100 mm), discovering elevated levels along the high salt marsh strandline compared to low salt marsh and mudflat areas. Notably, the abundance of plastic reached staggering levels, reaching up to 20,060 items/m 2 in the vicinity of an illegal dumpsite. Microplastics, particularly in the 2-4 mm range, were dominant, and the main plastic components were high-density polyethylene (HDPE), low-density polyethylene (LDPE), polypropylene (PP), and polystyrene (PS). Plastic films emerged as the predominant plastic type, while the presence of pellets hinted at potential sources such as illegal dumping and port-related activities. This contamination could be largely attributed to inappropriate waste management practices and urban runoff, which pose a substantial ecological threat to these ecosystems. Urgent remedial action is essential to protect these marshes, underscoring the critical need for comprehensive wetland management and educational initiatives to ensure their long-term sustainability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Marine litter concentration in the Mediterranean Sea is strongly influenced both by anthropogenic pressures and hydrodynamic factors that locally characterise the basin. Within the Plastic Busters MPAs (Marine Protected Areas) Interreg Mediterranean Project, a comprehensive assessment of floating macro- and microlitter in the Pelagos Sanctuary and the Tuscan Archipelago National Park was performed. An innovative multilevel experimental design has been planned ad-hoc according to a litter provisional distribution model, harmonising and implementing the current sampling methodologies. The simultaneous presence of floating macro- and microlitter items and the potential influences of environmental and anthropogenic factors affecting litter distribution have been evaluated to identify hotspot accumulation areas representing a major hazard for marine species. A total of 273 monitoring transects of floating macrolitter and 141 manta trawl samples were collected in the study areas to evaluate the abundance and composition of marine litter. High mean concentrations of floating macrolitter (399 items/km 2 ) and microplastics (259,490 items/km 2 ) have been found in the facing waters of the Gulf of La Spezia and Tuscan Archipelago National Park as well in the Genova canyon and Janua seamount area. Accordingly, strong litter inputs were identified to originate from the mainland and accumulate in coastal waters within 10-15 nautical miles. Harbours and riverine outfalls contribute significantly to plastic pollution representing the main sources of contamination as well as areas with warmer waters and weak oceanographic features that could facilitate its accumulation. The results achieved may indicate a potentially threatening trend of litter accumulation that may pose a serious risk to the Pelagos Sanctuary biodiversity and provide further indications for dealing with plastic pollution in protected areas, facilitating future management recommendations and mitigation actions in these fragile marines and coastal environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Anthropogenic debris, including plastics, has recently been identified as a major threat for marine mammals and the Marine Strategy Framework Directive aims to achieve the good environmental status of European waters by addressing among other criteria, the effects of marine litter on biota. This study implemented for the first time a non-invasive technique for collecting monk seal samples to assess microdebris ingestion in combination with identifying plastic additives and porphyrins biomarkers. A total of 12 samples of monk seal faeces were collected from marine caves in Zakynthos Island, Greece. A total of 166 microplastic particles were identified; 75 % of the particles were smaller than 3 mm. Nine phthalates and three porphyrins were detected. A strong correlation was found between the number of microplastics and the concentration of phthalates. The values of both phthalates and porphyrins were found lower than in other marine mammal tissues, suggesting that seals might not be impacted by them yet., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The impacts of microplastics on filter feeders megafauna have recently received increased attention. These organisms are potentially exposed to plastic ingestion and the release of added/sorbed contaminants during feeding activities. An assessment of microplastic abundance and the chemical impact of Phthalates esters (PAEs) were performed in neustonic samples and skin biopsies of Balaenoptera physalus and Rhincodon typus inhabiting the Gulf of California (Mexico). Sixty-eight percent of the net tows contained plastics with a maximum of 0.24 items/m 3 mainly composed of polyethylene fragments. PAE levels were detected both in environmental and skin biopsy samples, with the highest values in the fin whale specimens (5291 ng/g d.w). Plasticizer fingerprint showed a similar distribution pattern between neustonic samples and filter-feeding species, with DEHP and MBP having the highest concentrations. The detection of PAE levels confirmed their potential role as plastic tracers and give preliminary information about the toxicological status of these species feeding in La Paz Bay., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Most studies examining the occurrence of plastics and microplastics in marine organisms have identified anthropogenic fibres, of natural and synthetic origin, as the most commonly occurring category. Anthropogenic fibres may have been chemically treated with additives making them more persistent and a potential threat to marine organisms. However, fibres have often been excluded from analytical data for the difficulties related to the sampling and analytical procedures, including potential overestimation of the results due to airborne contamination. This review aimed to collect and analyse all studies focusing on the interaction between anthropogenic fibres and marine organisms worldwide, highlighting critical issues that need to be overcome for the analysis fibres on marine organisms. Furthermore, emphasis was placed on the species studied in the Mediterranean Sea, which is particularly affected by this type of pollution. Overall, this review shows that fibre pollution is an underestimated threat to marine organisms and that a specific, harmonised protocol for the analysis of different anthropogenic fibres needs to be developed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

This study investigates for the first time the presence of microplastics in sediment, water, and benthic organisms (foraminifera) of a marine cave in the Gulf of Orosei (Sardinia, Italy). Microplastics were found in all water, and sediment samples with similar shapes, sizes, and compositions; identified items were mainly fragments and fibers constituted by PVC and polyethylene. Their provenance was supposed to be predominantly from the sea than from the seasonal freshwater supplies from the karst system. Foraminiferal assemblages were mainly constituted by calcareous hyaline taxa in the outer station, while in the inner ones, the agglutinated Eggerelloides advenus was dominant. FTIR analyses on agglutinated shells identified polyethylene. Microplastic items are collected by the foraminifers and sediment grains building the shell chambers. This is the first study providing evidence that marine caves may be collectors of microplastics and that, in these habitats, microplastics enter the biotic matrix at the protist's level., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Few studies evaluated long-term effects of polyvinyl chloride (PVC) microplastics (MPs) ingestion in fish. The present study aimed to investigate the integrated biomarker responses in the liver and blood of 162 European seabass, Dicentrarchus labrax, exposed for 90 days to control, virgin and marine incubated PVC enriched diets (0.1 % w/w) under controlled laboratory condition. Enzymatic and tissue alterations, oxidative stress, gene expression alterations and genotoxicity were examined. Additives and environmental contaminants levels in PVC-MPs, control feed matrices and in seabass muscles were also detected. The results showed that the chronic exposure at environmentally realistic PVC-MPs concentrations in seabass, cause early warning signs of toxicological harm in liver by induction of oxidative stress, the histopathological alterations and also by the modulation of the Peroxisome proliferator-activated receptors (PPARs) and Estrogen receptor alpha (ER-α) genes expression. A trend of increase of DNA alterations and the observation of some neoformations attributable to lipomas suggest also genotoxic and cancerogenic effects of PVC. This investigation provides important data to understand the regulatory biological processes affected by PVC-MPs ingestion in marine organisms and may also support the interpretation of results provided by studies on wild species., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Plastic pollution in the Mediterranean Sea has been widely reported, but its impact on biodiversity has not been fully explored. Simultaneous sampling of microplastics (MP) with a manta net and surveys of large marine vertebrates were conducted along the coastal waters of Sicily (Western Ionian Sea). A total of 17 neustonic samples have been collected and 17 marine species (cetaceans, sea turtles, seabirds, and fish) have been sighted in the target area. Kernel density estimation was evaluated to highlight a possible overlap between the presence of large marine fauna and MP densities to provide a preliminary risk assessment. The highest biodiversity and MP concentration (0.197 ± 0.130 items/m 2 ) were observed in the southernmost part of the studied area. The overlap between biodiversity hotspots and the occurrence of MP, potential contribute to the identification of sensitive areas of exposure in a poorly studied region., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The phenotypic plasticity of many organisms is mediated in part by epigenetics, the heritable changes in gene activity that occur without any alterations to DNA sequence. A major mechanism in epigenetics is the DNA methylation (DNAm). Hypo- and hyper-methylation are generalized responses to control gene expression however recent studies have demonstrated that classes of contaminants could mark specific DNAm signatures, that could usefully signal prior environmental exposure. We collected skin and blubber from 6 free-ranging fin whale (Balaenoptera physalus) individuals sampled as a part of a previous published study in the northern Mediterranean Sea. Genomic DNA extracted from the skin of the fin whales and levels of contaminants measured in the blubber of the same individuals were used for DNAm profiling through reduced representation bisulfite sequencing (RRBS). We tested the hypothesis that differences in the methylation patterns could be related to environmental exposure to contaminants and load in the whale tissues. The aims of this study were to determine the DNAm profiles of the methylation contexts (CpGs and non-CpGs) of differently contaminated groups using the RRBS, and to identify potential contaminant exposure related genes. Amount and proportion of methylcytosines in CpG and non-CpG regions (CHH and CHG) was very similar across the 6 samples. The proportion of methylcytosines sites in CpG was n = 32,682, the highest among all the sequence contexts (n = 3216 in CHH; n = 1743 in CHG). The majority of the methylcytosine occurred in the intron regions, followed by exon and promoter regions in CpG, CHH and CHG. Gene Ontology results indicated that DNAm affected genes that take place in cell differentiation and function in cutaneous, vascular and nervous systems. The identification of cellular response pathways allows a better understanding of the organism biological reaction to a specific environmental challenge and the development of sensitive tools based on the predictive responses. Eco-epigenetics analyses have an extraordinary potential to address growing issues on pollution biomonitoring, ecotoxicity assessment, conservation and management planning., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In the framework of the Plastic Busters MPAs project, a harmonization exercise on two methods of microplastic extraction from biological samples i.e. 15% H 2 O 2 digestion and 10% KOH digestion was carried out. The two methods were tested in four laboratories on fish gastrointestinal tracts and mussel tissues spiked with polyethylene, polypropylene and polyethylene terephthalate. The recovery percentage of microplastics for each method, species and polymer tested were overall similar among laboratories, and interlaboratory coefficient of variation was less than 11% for the majority of samples. Microplastic recovery rates for the two methods were similar for each sample tested, but overall mean interlaboratory recovery rate using KOH (96.67%) was higher than H 2 O 2 (88.75%) . Results validate the use of both methods for extracting microplastics from biota tissues. However, when comparing the two methods in terms of microplastic recovery rate, time consumed, technical difficulties and cost, digestion with 10% KOH is considered optimal., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In this study, we characterized plastic debris (PD) found on beaches from Concepción Bay in central Chile during spring 2017 and summer 2018. The identification of polymers was carried out using FT-IR. Persistent organic pollutants (POPs) were extracted with hexane using an ultrasonic bath and further quantified through GC-MS. The highest abundance of PD was obtained during the summer (4.1 ± 3.7 items/m 2 ), with the most common size range between 2.5 and 10 cm (42%) and the most frequent shape were plastic fragments (44%). FT-IR analysis showed that polypropylene was the most recurrent plastic polymer found. The ∑10PBDEs ranged from 2.1 to 1300 ng/g in spring 2017 and 392 to 3177 ng/g in summer 2018. ∑7PCBs ranged from 0.9 to 93 ng/g during the spring 2017 and 0.3 to 4.5 ng/g for summer 2018. This study is the first with information on POPs occurrence in the plastic debris of central Chile., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

We determined levels of mercury, cadmium, lead and selenium in muscle and liver of 29 specimens of a large pelagic fish rarely investigated, the Mediterranean spearfish Tetrapturus belone (Raf., 1810). The following element concentration ranking (mean ± S.D.; espressed in mg/kg dry weight) was recorded in muscle: Hg (3.401 ± 1.908) > Se (1.727 ± 0.232) > Pb (0.532 ± 0.322) > Cd (0.019 ± 0.015), and Se (6.577 ± 1.789) > Cd (5.815 ± 3.038) > Hg (2.698 ± 2.214) > Pb (0.661 ± 1.334) in liver. Levels of Hg, Se and Cd were compared to those reported for other Istiophoridae from oceanic areas and for other large predators of Mediterranean Sea, like swordfish and tuna. Organotropism of trace elements and their relation to size was discussed. Ecophysiological considerations regarding the Se-Hg relationship as well as Se-Cd indicate a possible detoxification mechanism. The implications for human consumption are briefly discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Citizen science programs carried out by volunteers are fundamental for the collection of scientific data on a wide spatial scale. From 2011 to 2018, 468 survey dives were conducted in 172 coastal locations of the Mediterranean Sea through Project AWARE's citizen science program, Dive Against Debris®. During the dives, information was collected on quantity, typology and distribution of seafloor litter in shallow coastal waters. Overall, the observed average density was 43.55 items/100 m 2 and plastic was largely the dominant material (55% of the total collected items) on basin scale. The most abundant seafloor litter items were plastic fragments with 9.46%, followed by beverage cans (7.45%). Single-use plastic items constituted 33% of the total marine litter. The results of the study can be used to inform policymakers of the European Community towards specific management action to contrast the marine litter in relation to the distribution of the recorded litter category., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The gastrointestinal tracts of 229 demersal fish belonging to two species (Mullus barbatus, Merluccius merluccius) were examined for microplastic ingestion. Samples were collected in 3 different FAO Geographical Sub-Areas (GSA-9, GSA-17, GSA-19) of the Mediterranean Sea. Ingested microplastics were characterized using a stereo-microscope: observed, photographed, measured and categorized according to size class, shape and colour. Plastic fragments (ranging from 0.10 to 6.6 mm) were detected in 23.3% of the total investigated fish; a total of 65 plastic particles (66% constituted by fibers) were recorded. The percentage of plastic ingestion shows high variability between the two species and among the different sampling area. The highest frequency (48%) was found in European hake from GSA-19. These preliminary results represent a baseline for the implementation of the Marine Strategy Framework Directive descriptor 10 in Italy as well as an important step for detecting microplastics in bioindicator species from different GSAs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In this study we have identified and characterized microplastic particles (MPs) found in six fish species of commercial importance in central Chile. The fish species belong to different trophic levels and were obtained from the oceanic and coastal habitats. To analyze MPs, the fish gastrointestinal content was extracted, analyzed and characterized using a microscopy equipped with Fourier-transform infrared spectroscopy (FT-IR). The MPs found in fish samples were mainly constituted by red microfibers (70-100%) with sizes ranging between 176 and 2842 μm. Polyester, polyethylene (PE) and polyethylene terephthalate (PET) were identified as the prevalent polymers detected. The coastal species showed the presence of microfibers with a higher size and abundance (71%) compared to oceanic species (29%), suggesting there is a greater exposure risk. These findings are consistent with results found in other investigations worldwide. However, further research is still needed to accurately establish the potential exposure risk for the public consuming these fish and the impact of MPs in the Chilean fishery activities., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Few studies have focused so far on plastic ingestion by sharks in the Mediterranean Sea. The aim of this paper was to determine, for the first time, the plastic litter ingested by blue sharks (Prionace glauca), categorized as "Critically Endangered" in the Mediterranean Sea by IUCN, caught in the Pelagos Sanctuary SPAMI (North-Western Mediterranean Sea). The analysis of the stomach contents was performed following the MSFD Descriptor 10 standard protocol implemented with FT-IR spectroscopy technique. The results showed that 25.26% of sharks ingested plastic debris of wide scale of sizes from microplastics (<5 mm) to macroplastics (>25 mm). The polyethylene sheetlike user plastics, widely used as packaging material, are the most ingested debris. This research raises a warning alarm on the impact of plastic debris on a threatened species, with a key role in the food web, and adds important information for futures mitigation actions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The loggerhead sea turtle (Caretta caretta) is a long-lived carnivorous reptile included in the IUCN Red List. The IUCN assessment for this species underlines the lack of information regarding pollution and pathogens and indicates as a priority effort to investigate and reduce the impacts of these threats. In this contest, the aim of our study was to conduct the first ecotoxicological assessment of this species in the Mediterranean Sea using a nondestructive integrated methodology. We set up and applied a monitoring protocol which includes endpoints, such as CYP1A, LPO, ENA assay, B esterases, never investigated before in this species. Seventy-five loggerhead turtles were sampled in a nondestructive way in Italian Sea Turtle Rescue Centers or free-ranging along the Spanish coasts. Blood, skin and carapace samples were used to test biomarker responses (CYP1A, VTG, LPO, Comet and ENA assay, BChE, GGT) and contaminant (OCs, PAHs, Pb, Cd, Hg) levels. Elaboration of experimental results was carried out taking also into consideration different age classes of the specimens. Among the main results obtained we should underline the statistically significant correlation between carcinogenic PAHs in blood and DNA fragmentation as well as between Cd in carapace and GGT in plasma. We also evidenced as youngest animals showed significantly higher DNA fragmentations, BChE inhibition and increase of GGT. Older specimens showed the highest levels of erythrocyte nuclear abnormalities which may indicate a long term toxicological stress. This study contributed to expand the knowledge about the ecotoxicology of C. caretta in the Mediterranean. The nondestructive protocol could also be applied to other marine ecosystems and other sea turtle species., (Copyright © 2018. Published by Elsevier B.V.)

Monitoring efforts are required to understand the sources, distribution and abundance of microplastic pollution. To verify the abundance of microplastics along the Tuscan coastal waters (Italy), water-column and surface samples were collected in two seasons across four transects at different distances to the coast (0.5, 5, 10 and 20 km), within the implementation of the European Marine Strategy Framework Directive. The results show an average concentration of 0.26 items/m 3 in the water-column samples and 41.1 g/km 2 and 69,161.3 items/km 2 of floating microplastics, with an increase with the distance to the coast The seasonality and the sampling area do not affect the abundance of microplastics. The most abundant size class is 1-2.5 mm as fragments and sheets suggesting that fragmentation of larger polyethylene and polypropylene items could be the main source of microplastics. These data represent the application of a harmonized protocol to make the data on microplastics comparable and reliable., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Chemicals discovered in water at levels that may be significantly different than expected are referred to as contaminants of emerging concern (CECs) because the risk to environmental health posed by their occurrence/frequency is still unknown. The worldwide distributed compounds perfluorooctanoic acid (PFOA) and bisphenol A (BPA) may fall into this category due to effects on endocrine receptors. We applied an ex vivo assay using small slices of bioptic skin from the bottlenose dolphin, Tursiops truncatus, cultured and treated for 24 h with different PFOA or BPA concentrations to analyze global gene expression. RNA was labeled and hybridized to a species-specific oligomicroarray. The skin transcriptome held information on the contaminant exposure, potentially predictive about long-term effects on health, being the genes affected involved in immunity modulation, response to stress, lipid homeostasis, and development. The transcriptomic signature of dolphin skin could be therefore relevant as classifier for a specific contaminant., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This study focuses, for the first time, on the presence of plastic debris in the stomach contents of large pelagic fish (Xiphias gladius, Thunnus thynnus and Thunnus alalunga) caught in the Mediterranean Sea between 2012 and 2013. Results highlighted the ingestion of plastics in the 18.2% of samples. The plastics ingested were microplastics (<5mm), mesoplastics (5-25mm) and macroplastics (>25mm). These preliminary results represent an important initial phase in exploring two main ecotoxicological aspects: (a) the assessment of the presence and impact of plastic debris on these large pelagic fish, and (b) the potential effects related to the transfer of contaminants on human health., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The impact of microplastics (plastic fragments smaller than 5 mm) on large filter feeding marine organisms such as baleen whales and sharks are largely unknown. These species potentially are ingesting micro-litter by filter feeding activity. Here we present the case studies of the Mediterranean fin whale (Balaenoptera physalus) and basking shark (Cetorhinus maximus) exploring the toxicological effects of microplastics in these species measuring the levels of phthalates in both species. The results show higher concentration of MEHP in the muscle of basking shark in comparison to fin whale blubber. These species can be proposed as indicators of microplastics in the pelagic environment in the implementation of Descriptor 8 and 10 of the EU Marine Strategy Framework Directive (MSFD)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The Marine Strategy Framework Directive (MSFD) requires the assessment of the environmental status in relation to human pressures. In this study the biodiversity of the cetacean community is proposed as MSFD descriptor of the environmental status and its link with anthropogenic pressures is investigated. Functional groups are generally favoured over indicator species since they are thought to better reflect to anthropogenic stressors. Cetaceans are in many situations the most well known component of pelagic ecosystems. Their habitat requirements are known and can be used to evaluate the theoretical biodiversity that should be expected in a certain area. The deviations between the theoretical biodiversity and the actual biodiversity may be used to detect the impacts of human activities. Based on this analysis fishery resulted to be by far the most significant of the existing pressures. Among all the species, bottlenose dolphin was found the most correlated with the fishery sector dynamics., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A plethora of different sampling methodologies has been used to document the presence of micro-plastic fragments in sea water. European Marine Strategy suggests to improve standard techniques to make future data comparable. We use Manta Trawl sampling technique to quantify abundance and distribution of micro-plastic fragments in Sardinian Sea (Western Mediterranean), and their relation with phthalates and organoclorine in the neustonic habitat. Our results highlight a quite high average plastic abundance value (0.15 items/m(3)), comparable to the levels detected in other areas of the Mediterranean. "Site" is the only factor that significantly explains the differences observed in micro-plastic densities. Contaminant levels show high spatial and temporal variation. In every station, HCB is the contaminant with the lowest concentration while PCBs shows the highest levels. This work, in line with Marine Strategy directives, represents a preliminary study for the analysis of plastic impact on marine environment of Sardinia., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Perfluorinated compounds (PFCs), synthetic musks compounds (SMCs), bisphenol A (BPA), para-nonylphenol (p-NP) and polybrominated diphenyl ethers (PBDEs) are known for their toxicity and ability to interfere with the endocrine system. The aim of this study was to determine levels and distribution of the above mentioned compounds in liver samples of Caretta caretta and levels of porphyrins that have been proposed as sensitive biomarkers of exposure to contaminants. This paper reports the results for 9 specimens yet analysed. Musk ketone was never detected, PFOA was found in one sample, while PFOS was the prevalent contaminant. For PFCs the levels are lower than the results of studies of comparison. The porphyrins profile showed a predominance of protoporphyrins on coproporphyrins and uroporphyrins, with a positive statistical correlation between levels of PFOS and uroporphyrins. These data represent, for several parameters, the first evidence of contaminant levels and biomarker responses in loggerhead turtles., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In the present study, the genotoxic potential of nanosized TiO2 anatase and micro-sized rutile on bottlenose dolphin (Tursiops truncatus) fibroblasts and leukocytes was investigated. Human and mouse cells were also studied in order to compare susceptibility to TiO2 in different mammalian species. Cell lines were exposed for 4, 24, and 48 h to different concentrations of TiO2 (20, 50, 100, 150 μg/ml) and DNA damage was investigated by single cell gel electrophoresis (Comet assay). Both anatase and rutile induced increased DNA damage, even though statistically significant effects were scattered according to species and cell lines. Bottlenose dolphin leukocytes and murine fibroblasts exhibited increased DNA damage after rutile exposure at some doses/times, while human fibroblasts showed a significant dose-response effect after a 4 h exposure to anatase. Human leukocytes were tolerant to both anatase and rutile. Ultrastructural investigation showed that TiO2 particles entered the cell and were compartmentalized within membrane-bound vesicles., (Copyright © 2014. Published by Elsevier Ltd.)

The aim of this study was to validate the comet assay in erythrocytes of Caretta caretta, a species never investigated for genotoxicity. We studied 31 loggerhead sea turtles from three Italian marine rescue centres. Peripheral blood samples were collected from all the animals and the comet assay applied. All comet cells were analysed using two methods: visual scoring and computer image analysis. The % DNA in tail mean value ± SD and Damage Index were 21.56 ± 15.41 and 134.83 ± 94.12, respectively. A strong and statistically significant statistically correlation between the two analytical methods was observed (r = 0.95; p < 0.05). These results demonstrate that the comet assay is a useful method to detect the possible effects of genotoxic agents in loggerhead sea turtle and to increase the knowledge about the ecotoxicological health status of this threatened species., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This work evaluated the presence and the frequency of occurrence of marine litter in the gastrointestinal tract of 31 Caretta caretta found stranded or accidentally bycaught in the North Tyrrhenian Sea. Marine debris were present in 71% of specimens and were subdivided in different categories according to Fulmar Protocol (OSPAR 2008). The main type of marine debris found was user plastic, with the main occurrence of sheetlike user plastic. The small juveniles showed a mean±SD of marine debris items of 19.00±23.84, while the adult specimens showed higher values of marine litter if compared with the juveniles (26.87±35.85). The occurrence of marine debris observed in this work confirms the high impact of marine debris in the Mediterranean Sea in respect to other seas and oceans, and highlights the importance of Caretta caretta as good indicator for marine litter in the Marine Strategy Framework Directive (MSFD) of European Union., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Perfluorinated compounds (PFCs), widely used for their hydro-oil repellent properties, are almost non-degradable in the environment; there is scientific evidence that indicate bioaccumulation. They represent a threat to many organisms, because they are toxic and are endocrine disruptors. Scientific studies have demonstrated the presence of PFCs in blood and liver samples of fish, turtles, birds and mammals of marine ecosystems in different geographical areas. The aim of this study was to determine the distribution of PFOS and PFOA in blood samples of the marine turtle Caretta caretta, using a minimally invasive sampling procedure. 49 blood samples of marine turtle, taken from several Italian marine turtle rescue centers, were analyzed. While PFOA was never detected, measurable concentrations of PFOS were found in 15 blood samples; the values show a range from 1.14 ng/g to 28.51 ng/g (wet wt.). No differences between groups of samples taken from different areas were found., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The concurrence of man-made pressures on cetaceans in the Mediterranean Sea is potentially affecting population stability and marine biodiversity. This needs to be proven for the only pelagic marine protected area in the Mediterranean Sea: the Pelagos Sanctuary for Mediterranean Marine Mammals. Here we applied a multidisciplinary tool, using diagnostic markers elaborated in a statistical model to rank toxicological stress in Mediterranean cetaceans. As a case study we analyzed persistent, bioaccumulative and toxic chemicals combined with a wide range of diagnostic markers of exposure to anthropogenic contaminants and genetic variation as marker of genetic erosion in striped dolphin (Stenella coeruleoalba) skin biopsies. Finally, a statistical model was applied to obtain a complete toxicological profile of the striped dolphin in the Pelagos Sanctuary and other Mediterranean areas (Ionian Sea and Strait of Gibraltar). Here we provide the first complete evidence of the toxicological stress in cetaceans living in Pelagos Sanctuary., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Baleen whales are potentially exposed to micro-litter ingestion as a result of their filter-feeding activity. However, the impacts of microplastics on baleen whales are largely unknown. In this case study of the Mediterranean fin whale (Balaenoptera physalus), we explore the toxicological effects of microplastics on mysticetes. The study included the following three steps: (1) the collection/count of microplastics in the Pelagos Sanctuary (Mediterranean Sea), (2) the detection of phthalates in surface neustonic/planktonic samples, and (3) the detection of phthalates in stranded fin whales. A total of 56% of the surface neustonic/planktonic samples contained microplastic particles. The highest abundance of microplastics (9.63 items/m(3)) was found in the Portofino MPA (Ligurian Sea). High concentrations of phthalates (DEHP and MEHP) were detected in the neustonic/planktonic samples. The concentrations of MEHP found in the blubber of stranded fin whales suggested that phthalates could serve as a tracer of the intake of microplastics. The results of this study represent the first warning of this emerging threat to baleen whales., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The Marine Strategy Framework Directive (MSFD) establishing a framework for community action in the field of marine environmental policy has been developed and is being implemented, with the objective to deliver "Good Environmental Status" by 2020. A pragmatic way forward has been achieved through the development of 11 "qualitative descriptors". In an attempt to identify gaps in MSFD, regarding the data on large marine vertebrates, the SETAC--Italian Branch organised a workshop in Siena (IT). Particular attention was paid to the qualitative descriptors 8 (contaminants and pollution effects) and 10 (marine litter). The specific remit was to discuss the potential use of large marine vertebrates (from large pelagic fish, sea turtles, sea birds and cetaceans) in determining the environmental status of pelagic marine ecosystems. During the workshop it emerged that large pelagic fish may be especially useful for monitoring short- to medium-term changes in pelagic ecosystems, while cetaceans provided a more integrated view over the long-term. A theme that strongly emerged was the broad recognition that biomarkers offer real potential for the determination of good ecological status detecting the "undesirable biological effects" (indicator for descriptor 8)., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The main objective of this study was to apply a set of sensitive non-lethal biomarkers in skin biopsies of fin whales (Balaenoptera physalus) to evaluate the toxicological status of this mysticete in the Pelagos Sanctuary (Mediterranean Sea) and in the Gulf of California (Sea of Cortez-Mexico). We developed a "multi-trial diagnostic tool" (based on field and in vitro studies), combining molecular biomarkers (western blot of CYP1A1, CYP2B) and gene expression (qRT-PCR of HSP70, ERα, AHR, E2F-1) with the analysis of OCs, PAHs and PBDEs. The study revealed a higher level of toxicological stress in the Mediterranean fin whales., (Copyright © 2009 Elsevier Ltd. All rights reserved.)

The aim of the present study was to propose the immunofluorescence technique in cultured fibroblasts from Mediterranean cetaceans as a new "in vitro" tool to explore the susceptibility of these marine mammals to different xenobiotic compounds. The cell lines were cultured from integument biopsies of free-ranging and stranded cetaceans (dead within 12h). Using the indirect immunofluorescence assay, we detected endogenous proteins induced by different contaminants. Here we present the method used for qualitative and quantitative evaluation of cytochromes P450 (CYP1A1 and CYP2B) induced by some POPs (DDTs and PCBs) and emerging contaminants (PBDEs) in fibroblast cell cultures of striped dolphin (Stenella coeruleoalba) and bottlenose dolphin (Tursiops truncatus). Immunofluorescence was quantified with a specially designed Olympus macro, DetectIntZ. A major result was the possibility of using this "in vitro" assay to quantify induction of endogenous proteins.

The need to develop powerful tools to detect exposure and effects of POPs and emerging contaminants in Mediterranean cetaceans led us to develop a suite of sensitive non-lethal biomarkers in integument biopsies of free-ranging animals. In order to propose induction of CYP1A1 and CYP2B, detected by Western blot analysis, as biomarkers of exposure to OCs, PAHs and PBDEs, a three-phase experimental protocol (in vitro experiments, calibration experiments and field applications) was followed using fibroblast cell cultures and biopsies of Mediterranean Stenella coeruleoalba and Tursiops truncatus. This methodology was confirmed to be sensitive and stable in comparison to previous methods used to detect CYP1A1 in biopsies, enabling analysis of several inducible proteins in non-lethal samples and analysis of material from stranded animals.

The main aim of this study was to develop and validate a suite of sensitive responses (biomarkers) for monitoring conservation status and ecotoxicological impact in Posidonia oceanica meadows. Analytical methods were developed for NADPH cytochrome c reductase, ethoxycoumarin-o-deethylase (ECOD), guaiacol peroxidase (GPOX) and superoxide dismutase (SOD) assays. A preliminary proteomic approach using 2-D electrophoresis was also proposed as a biomarker. These techniques were initially tested on samples of posidonia exposed experimentally to various contaminants. Once validated, this approach was applied to posidonia in a field study. Samples of the seagrass were collected at four sites with potentially different environmental impact along the northern Tyrrhenian coast. The results showed that reductase activity was significantly induced in the various sampling areas with respect to the reference site. GPOX and SOD showed a similar trend; the highest activities were found in samples collected off a chlor-alkali plant and near a river estuary. Analysis of trace elements, polycyclic aromatic hydrocarbons (PAHs) and organochlorine compounds (OCs) in posidonia leaves showed differences between sites. A significant correlation was found between Hg concentrations and GPOX activity and between Cr, Al and As concentrations and reductase activity. The results validated these biomarkers in posidonia for the assessment of ecotoxicological impact on the coastal ecosystem.