The Mediterranean Sea is particularly endangered by microplastics (MPs), polymers, and additives. These contaminants can be ingested by fishes and, hence, translocate into tissues. We aimed to quantify MPs, polyethylene terephthalate (PET), polycarbonate (PC), bisphenol A (BPA), and p-phthalic acid (PTA) in the edible muscles of swordfish (Xiphias gladius) and bluefin tuna (Thunnus thynnus) caught in the Mediterranean Sea. The MPs were extracted from muscles and characterized by stereomicroscopy and Raman microspectroscopy, while the polymers (PET and PC) and additives (BPA and PTA) were identified by LC-MS/MS. The number of MPs ranged from 140 to 270 no. kg−1 in swordfish and from 160 to 270 no. kg−1 in tuna. The most frequent MP polymer was polypropylene in swordfish (33%) and in tuna (34.7%), while the most abundant pigments were PB115, PB116, PBr101/102. A similar level of plastic contamination was revealed in these two fish species with differences in shapes, colors, pigments and polymers of MPs.

Highly populated coastal environments receive large quantities of treated and untreated wastewater from human and industrial sources. Bivalve molluscs accumulate and retain contaminants, and their analysis provides evidence of past contamination. Rivers and precipitation are major routes of bacteriological pollution from surface or sub-surface runoff flowing into coastal areas. However, relationships between runoff, precipitation, and bacterial contamination are site-specific and dependent on the physiographical characteristics of each catchment. In this work, we evaluated the influence of precipitation and river discharge on molluscs' Escherichia coli concentrations at three sites in Central Italy, aiming at quantifying how hydrometeorological conditions affect bacteriological contamination of selected bivalve production areas. Rank-order correlation analysis indicated a stronger association between E. coli concentrations and the modelled Pescara River discharge maxima (r = 0.69) than between E. coli concentration and rainfall maxima (r = 0.35). Discharge peaks from the Pescara River caused an increase in E. coli concentration in bivalves in 87% of cases, provided that the runoff peak occurred 1-6 days prior to the sampling date. Precipitation in coastal area was linked to almost 60% of cases of E. coli high concentrations and may enhance bacterial transportation offshore, when associated with a larger-scale weather system, which causes overflow occurrence.

In the present study, a 4-year surveillance plan for the detection of lipophilic marine biotoxins in Mytilus galloprovincialis and Callista chione from different aquaculture farms and natural beds along the Central Adriatic coasts of Croatia was reported. The samples were analyzed by a validated LC–MS/MS method in accordance with the Regulation (EU) No 2019/627. Lipophilic marine biotoxins belonging to the okadaic acid group, except for azaspiracids, as well as yessotoxins, were found at different concentrations, always below the maximum limits established by the Regulation (EC) No 853/2004. The seasonal distribution showed the highest values in summer months, and an interannual variability was also observed, probably due to environmental conditions aging on the density of harmful algal blooms. The comprehensive data reported in this study should help the future research in making advancing prediction models along the Eastern Adriatic coast.

Emerging contaminants could exert combined toxic effects, including synergetic and antagonistic ones, that cannot be identified by chemical analysis tools. The biological early warning systems (BEWS) perform a real-time and continuous (24 h) monitoring of physiological and/or behavioural parameters of organism alterations, potentially correlated to water pollution. They are based on the response of living sentinels (i.e. molluscs, algae, crustaceans, fish) to a contaminant or mixture of them. Early warnings can be sent by SMS, e-mail, etc. to operators, in order to activate response actions. Belonging to different trophic levels, the crustacean D. magna, the alga C. vulgaris and the mollusc P. casertanum have been used to control Gran Sasso-Sirente aquifer in three different locations. Drinkable water of Teramo province and irrigation water of L’Aquila have been continuously monitored by the commercial tools “bbe® Daphnia Toximeter (DTOX)” and “bbe® Algae Toximeter (ATOX)”, respectively. In Tirino river spring, a novel sensor “SmartShell” has registered for the first time the valve movements of the autochthonous bivalve. After the first testing period, DTOX and ATOX did not register any typology of alarms in the potable and irrigation water. The valve movements of P. casertanum have been examined through spectral analysis in order to evaluate the behavioural rhythms useful for further investigation on their alterations as early warnings. The objective has been to reinforce the aquifer protection by installing instruments internationally recognised as efficient tools and exploring new proposals for guaranteeing human and ecosystem health.

Biological early warning systems (BEWS) are installed worldwide for the continuous control of water intended for multiple uses. Sentinel aquatic organisms can alert us to contaminant presence through their physiological or behavioural alterations. The present study is aimed at sharing the experience acquired with water biomonitoring of the Gran Sasso-Sirente (GS-S) aquifer. It represents the major source of the Abruzzi region surface water, also intended as drinkable and for irrigation use. Besides the biomonitoring of drinkable water of the Teramo Province made by Daphnia Toximeter and irrigation water of the L'Aquila Province by Algae Toximeter, a novel sensor named "SmartShell" has been developed to register the behaviour of the "pea clam" P. casertanum, an autochthonous small bivalve living in the Nature 2000 site "Tirino River spring". The valve movements have been recorded directly on the field. Its behavioural rhythms have been analysed through spectral analyses, providing the basis for further investigations on their alterations as early warnings and allowing us to propose this autochthonous bivalve species as a novel sentinel organism for spring water.

Loggerhead sea turtle ( C. caretta ) is the official European bioindicator of marine litter in the Mediterranean Sea. In 2019, 8 sea turtles, out of 28 specimens loggerhead on the Adriatic coast of Molise, were subjected to necropsy. The intestinal contents were collected and the microplastics until 0.45 μm were extracted. Qualitative and quantitative assessments were performed by stereomicroscope observation and spectroscopic analyses (attenuated total reflection-Fourier transform infrared spectroscopy, ATR-FTIR and Raman microspectroscopy, RMS). Moreover, the analytical quantification of polyethylene terephthalate (PET), polycarbonate (PC), para phthalic acid (PTA) and bisphenol A (BPA) in fat and liver tissues was performed by LC-MS/MS. Microparticles ranging from 0.45 μm to 1 mm were found in all turtles, for a total of 623, while plastic litter greater than 1 mm were found only in 4 specimens (ranging from 0.03 to 0.11 g). Nineteen different polymers and 10 pigments, including polyester (100% of animals), high-density polyethylene (50%) and polypropylene (50%) were identified. BPA, PTA and PET were detected in fat and liver tissues of all animals, while PC was found only in 50%. A major prevalence was registered in the abdominal fat tissue, although only PC compounds were significantly higher in abdominal tissue ( p < 0.05), except for free PTA with liver tissue being the most contaminated ( p < 0.05). Microplastics and additives surely impact the health status of turtles that showed gastrointestinal impairment and an important level of contamination in tissues. Graphical abstract

Human Norovirus has been reported as the major non-bacterial cause of human gastroenteritis due to the consumption of contaminated bivalve mollusks. The European legislation established microbiological criteria only for bacteria (Salmonella spp. and Escherichia coli), while no viruses have still been considered. In this study, samples of Chamelea gallina were harvested along the Central Adriatic coasts (Italy) and artificially contaminated with Murine norovirus-1 (MNV-1) up to a final concentration of 10(3) TCID50/ml in water. They were subject to a depuration process in a closed-circuit system using both ozone and ultraviolet light. Four experimental trials (100 specimens/trial) were performed and, at the end of depuration, the digestive glands of mollusks were examined by means of two methods - namely, RT-PCR and tissue culture. The results of RT-PCR ranged from 10(3.17) to 10(4.60) TCID50/ml, and the constant presence of MNV-1 was confirmed by the tissue culture as well. In conclusion, no significant viral reduction was obtained, but the contaminated bivalve mollusks remained infectious until the end of the depuration treatment. The proper cooking of live bivalve mollusks could be considered the most important preventive measure against this sanitary risk.

Marine litter is one of the most important human-caused pressures on the marine environment, it is defined as “any persistent, manufactured or processed solid material discarded, disposed of or abandoned in the marine and coastal environment” [1, 2]. The most abundance part of marine litter is composed by plastic [3]. The main and most remarkable risks for the species inhabiting the marine environment are entanglement and accidental ingestion. Moreover, from the direct release of particles of plastic and as a fragmentation of large items consequence, originate microplastic. The presence of microplastics in the marine environment has raised scientific interest during the last decade. As demonstrated, ingestion of marine litter can have lethal and sub-lethal effects on wildlife that accidentally ingests it, and sea turtles are particularly susceptible. For this reason loggerhead turtle (C. caretta) has been identified by the European Commission (EC), in the Marine Strategy Framework Directive (MSFD) [4, 5], as indicator for monitoring the amount of Marine Litter ingested by marine animals in Mediterranean basin. The Istituto Zooprofilattico Sperimentale dell’Abruzzo e Molise “G. Caporale” (IZSAM) is contributing to monitor the impact of marine litter on sea turtles and biota for MSFD, thanks to the participation of EC funded-project “Implementation Of Indicators Of Marine Litter On Sea Turtles And Biota In Regional Sea Conventions And Marine Strategy Framework Directive Areas – INDICIT” ( https://indicit-europa.eu/ ). This is an European program to implement novel protocols and indicators of litter impact on marine turtles [6] Data from Adriatic coast are provided by IZSAM that is a member of Abruzzo and Molise stranding network instituted by Regional Government [7, 8], whose main objectives is diagnostic investigation of dead cause of stranded marine animals with the aim conservation of protected species, the indirect control of environmental status and the protection of public health.

Lipophilic marine biotoxins include okadaic acid, pectenotoxin, yessotoxin and azaspiracid groups. The consumption of contaminated molluscs can lead to acute food poisoning syndromes depending on the exposure level. Regulatory limits have been set by Regulation (European Community, 2004a) No 853/2004 and LC-MS/MS is used as the official analytical method according to Regulation (European Community, 2011) No 15/2011. In this study specimens of mussels (Mytilus galloprovincialis) were collected along the coasts of the central Adriatic Sea during the years 2015-2017 and analyzed by the European harmonized Standard Operating Procedure. The method was validated for linearity, specificity, repeatability and reproducibility and it revealed able to be used for the detection of the lipophilic marine biotoxins. Levels of okadaic acid, pectenotoxin, yessotoxin and its analogs were detected at different concentrations in 148 (37%) out of a total of 400 samples, always below the maximum limits, except for 11 (4.3%) of them that were non-compliant because they exceeded the regulatory limit. Moreover, some samples were exposed to a multi-toxin mixture with regards to okadaic acid, yessotoxin and 1-Homo yessotoxin. Following these results, the aquaculture farms from which the non-compliant samples derived were closed until the analytical data of two consecutive samplings returned favorable. Besides the potential risk of consumption of mussels contaminated by lipophilic marine biotoxins, these marine organisms can be considered as bio-indicators of the contamination status of the marine ecosystem.

Harmful algal blooms are natural phenomena caused by the massive growth of phytoplankton that may contain highly toxic chemicals, the so-called marine biotoxins causing illness and even death to both aquatic organisms and humans. Their occurrence has been increased in frequency and severity, suggesting a worldwide public health risk. Marine biotoxins can accumulate in bivalve molluscs and regulatory limits have been set for some classes according to European Union legislation. These compounds can be distinguished in water-and fat-soluble molecules. The first group involves those of Paralytic Shellfish Poisoning and Amnesic Shellfish Poisoning, whereas the toxins soluble in fat can cause Diarrheic Shellfish Poisoning and Neurotoxic Shellfish Poisoning. Due to the lack of long-term toxicity studies, establishing tolerable daily intakes for any of these marine biotoxins was not possible, but an acute reference dose can be considered more appropriate, because these molecules show an acute toxicity. Dietary exposure assessment is linked both to the levels of marine biotoxins present in bivalve molluscs and the portion that could be eaten by consumers. Symptoms may vary from a severe gastrointestinal intoxication with diarrhea, nausea, vomiting, and abdominal cramps to neurological disorders such as ataxia, dizziness, partial paralysis, and respiratory distress. The official method for the detection of marine biotoxins is the mouse bioassay (MBA) showing some limits due to ethical restrictions and insufficient specificity. For this reason, the liquid chromatography-mass spectrometry method has replaced MBA as the reference technique. However, the monitoring of algal blooms producing marine biotoxins should be regularly assessed in order to obtain more reliable, accurate estimates of bloom toxicity and their potential impacts.

Samples of Mytilus galloprovincialis collected from shellfish aquaculture plans located in the Adriatic Sea were analysed for yessotoxin (YTX) by three methods, in vivo (Mouse Bioassay, MBA), in vitro (functional assay) and chemical test (Liquid Chromatography-Mass Spectrometry, LC-MS/MS). As YTX coexists with other phycotoxins in shellfish, namely the diarrhoetic shellfish poisoning, okadaic acid, dinophysistoxins, pectenotoxins, and azaspirazids, the MBA is not completely satisfactory because it is difficult to identify which toxin causes the death of the mice. So, the two other techniques were proposed to detect and quantify YTX and its analogues in order to avoid this problem. The global results showed no difference among the three methods and the correlation between the functional assay and LC-MS/MS was positive (Spearman r=0.72). Both analytical methods demonstrated advantages; the functional assay is specific, very sensitive and correlates well with real toxicity, whereas LC-MS/MS is convenient because it allows the detection of YTX and some analogues which are currently included in the EU regulation. For this reason LC-MS/MS will become the official method starting 1st January 2015 (Regulation 15/2011/EU). Only four samples exceeded the current EU regulation limit of 1mg of YTX equivalent kg(-1). However, all samples belonged to a monitoring program and they were not suitable for consumers.

Yessotoxins (YTXs) are polycyclic ether compounds produced by phytoplanktonic dinoflagellates and accumulated in filter-feeding shellfish. Mouse bioassay is still the official method to detect these toxins, even if it is lacking of specificity and sensitivity. Moreover, there is growing resistance against the use of animal experiments. Many efforts have been made to determine YTXs with other methods. The detection of YTX using a functional assay allows its quantification with an automated and repetitive technique at concentrations in the range of the 1 mg of YTX equivalent/kg European regulatory limit. In this study, an in vitro functional assay based on YTX treatment of MCF-7 cells and resulting in the accumulation of a 100-kDa fragment of E-cadherin was developed on samples of Mytilus galloprovincialis collected from the Adriatic Sea, Italy, along the coasts of Abruzzo, Molise, and Emilia Romagna regions. The YTX concentrations ranged from 0.2 to 1.8 mg of YTX equivalent/kg. The occurrence of levels exceeding the above mentioned limit was observed only in samples of Emilia Romagna region. This last result could represent a risk for human health, but these shellfish were not intended to consumers, because they belonged to a preventive monitoring program.

The swimming behaviour of adult Daphnia largely governs their depth, which has a direct effect on both individual foraging success and predation avoidance. We treated individual swimming behaviour as a threshold character and used directional changes in average clonal depth within experimental tubes as a test for character plasticity. We compared the swimming behaviours of two cohabiting, phenotypically similar Daphnia (Daphnia galeata and Daphnia galeata–Daphnia rosea hybrid) to determine (1) whether there is inherited variation (H2) for different traits (responses to hunger and predator cues); (2) whether changes in genetic parameters (norm of reaction and character state) across four environments could be simulated by combinations of the presence or absence of a predator cue and high or low hunger levels; and (3) whether these Daphnia would respond to directional selection, particularly in a trade-off environment (high hunger and predator cue treatments). Responses of both D. galeata and theD. galeata –rosea hybrid to hunger and a predator cue and the trade-off environment were plastic.Daphnia galeata expressed significant genetic variation within (H2) and between environments (heritability of plasticity). Both the character state and norm of reaction estimates of heritable variation in the hybrid were close to zero. Genetic correlations were positive and stable across six environmental pairs in Daphnia galeata. Most hybrid genetic correlations were not significant. The phenotypic distributions of both D. galeata and the hybrid were bimodal in the trade-off environment. The D. galeata distribution was partly due to between-clone variation and the hybrid distribution was almost entirely due to within-clone variation. Genetic variation expressed byD. galeata in the trade-off environment appears to depend on both clone by environment interactions and stable inherited differences. These results indicate that while plastic phenotypic responses cause a similar opportunity for selection inD. galeata and the hybrid, their responses to selection would be different in the trade-off and in related environments.

Common sole (Solea solea) is one of the most interesting species for diversification of aquaculture production in the Mediterranean area, but production of sole on an industrial scale has not yet begun. In the present study, larval rearing was performed in 1 cubic meter tanks with a re-circulating system, using the "green water" technique. After an initial mortality occurred during the first 10 days of larval rearing, no particular mortality was registered during weaning period until 69 dph (day post hatching). On 66 dph, because of technical problems, fish were transferred in a new tank with open system where temperature control was not possible and water temperature increased from 19 degrees C to 24 degrees C in three days. Four days later and for the following ten days we registered a mortality rate of 84.5%. Microbiological analysis revealed the absence of Betanodavirus and the presence of Vibrio anguillarum O2 and of Tenacibaculum maritimum; the former represents the first isolation on common sole in Italy. Histology revealed necrotic-haemorrhagic lesions in brain, kidney and gut, and severe skin erosions, the latter especially on fish collected at the end of the outbreak. Moreover the pathogens have been detected by immunohistochemistry (IHC). This severe mortality could be interpreted as a multi-factorial event where post-weaning weakness, manipulation, higher temperature and pathogens could have had a synergic effect. The extreme stress susceptibility of S. solea, pointed out in this and previous papers is discussed.

Based on previous studies, in the East Adriatic Sea (Croatia) the Neretva River estuary has been identified as area of high scientific and commercial interest. This area is particularly suitable for natural shellfish bed especially for clams (Chamelea gallina). A study has been carried out for the evaluation of C. gallina resource with the participation of Croatian and Italian experts and fishermen who have worked on the possibility of initiating of the natural shellfish bed activity through the professional fishery gears.