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Planktonic diatoms of the family Bacillariaceae, including Nitzschia spp, Pseudo-nitzschia spp and Cylindrotheca closterium, are common and often highly abundant in the Adriatic Sea. During the 30 ...

Species of the genus Pseudo-nitzschia are common inhabitants of phytoplankton communities of the northern Adriatic Sea, reaching high abundances. During 2018–2019, a total of 138 cultured Pseudo-ni...

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are extremely stable highly fluorinated aliphatic compounds, widely used in chemical and industrial applications since 1950s. They are ubiquitously detected in the environment and diet is the main route for human exposure. To ensure human safety, it is necessary to follow the whole food production chain, including animal feed. Still PFASs are not regulated as undesirable substances in feed, although several studies have shown they can be transferred from feed to livestock and thereafter to food. This paper describes the development, optimization and the full validation of a sensitive and reliable analytical protocol enabling the quantification of 19 PFASs in animal feeds by liquid chromatography - mass spectrometry (LC-MS/MS). The method was optimized, assessing the possible matrix interferences, and submitted to comprehensive validation (55 independent spiking experiments). Validation experiments were conducted on blank fish feed samples (natural levels of PFASs

The international seafood trade is based on food safety, quality, sustainability, and traceability. Mussels are bio-accumulative sessile organisms that need regular control to guarantee their safe consumption. However, no well-established and validated methods exist to trace mussel origin, even if several attempts have been made over the years. Recently, an inorganic multi-elemental fingerprint coupled to multivariate statistics has increasingly been applied in food quality control. The mussel shell can be an excellent reservoir of foreign inorganic chemical species, allowing recording long-term environmental changes. The present work investigates the multi-elemental composition of mussel shells, including Al, Cu, Cr, Zn, Mn, Cd, Co, U, Ba, Ni, Pb, Mg, Sr, and Ca, determined by inductively-coupled plasma mass-spectrometry in Mytilus galloprovincialis collected along the Central Adriatic Coast (Marche Region, Italy) at 25 different sampling sites (18 farms and 7 natural banks) located in seven areas. The experimental data, coupled with chemometric approaches (principal components analysis and linear discriminant analysis), were used to create a statistical model able to discriminate samples as a function of their production site. The LDA model is suitable for achieving a correct assignment of &gt, 90% of individuals sampled to their respective harvesting locations and for being applied to counteract fraud.

Tetrodotoxins (TTXs), potent neurotoxins, have become an increasing concern in Europe in recent decades, especially because of their presence in mollusks. The European Food Safety Authority published a Scientific Opinion setting a recommended threshold for TTX in mollusks of 44 µg equivalent kg−1 and calling all member states to contribute to an effort to gather data in order to produce a more exhaustive risk assessment. The objective of this work was to assess TTX levels in wild and farmed mussels (Mytilus galloprovincialis) harvested in 2018–2019 along the coastal area of the Marche region in the Central Adriatic Sea (Italy). The presence of Vibrio spp. carrying the non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes, which are suspected to be involved in TTX biosynthesis, was also investigated. Out of 158 mussel samples analyzed by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS), 11 (7%) contained the toxins at detectable levels (8–26 µg kg−1) and 3 (2%) contained levels above the EFSA safety threshold (61–76 µg kg−1). Contaminated mussels were all harvested from natural beds in spring or summer. Of the 2019 samples, 70% of them contained V. alginolyticus strains with the NRPS and/or PKS genes. None of the strains containing NRPS and/or PKS genes showed detectable levels of TTXs. TTXs in mussels are not yet a threat in the Marche region nor in Europe, but further investigations are surely needed.

Pseudo-nitzschia pungens is a common component of the phytoplankton community of the northern Adriatic Sea. In this study, an in-depth morphological analysis of P. pungens was carried out in both cultured strains isolated in different periods and field samples, revealing a surprisingly wide variability in a number of details, with both the gross morphology and ultrastructural levels deviating from the nominal P. pungens. Colonies showed an overlap (from one-third to one-sixth) and a transapical axis (rarely reaching 3 &micro, m), strongly differing from the original description of the species. Moreover, valves may be either symmetrical or slightly asymmetrical, with striae almost always biseriate but sometimes uniseriate or triseriate. Poroids&rsquo, morphology in cingular bands was characterized by a wide variability (square, circular, or rectangular poroids without or with up to two hymen sectors), with several combination of them, even within the same cingular band. Phylogenetic analyses based on ITS rDNA showed that the P. pungens of the northern Adriatic Sea belonged to clade I. Domoic acid was not detected.

Cyclic imines (CIs) are emerging marine lipophilic toxins (MLTs) occurring in microalgae and shellfish worldwide. The present research aimed to study CIs in mussels farmed in the Adriatic Sea (Italy) during the period 2014&ndash, 2015. Twenty-eight different compounds belonging to spirolides (SPXs), gymnodimines (GYMs), pinnatoxins (PnTXs) and pteriatoxins (PtTXs) were analyzed by the official method for MLTs in 139 mussel samples collected along the Marche coast. Compounds including 13-desmethyl spirolide C (13-desMe SPX C) and 13,19-didesmethyl spirolide C (13,19-didesMe SPX C) were detected in 86% of the samples. The highest levels were generally reported in the first half of the year reaching 29.2 &micro, g kg-1 in January/March with a decreasing trend until June. GYM A, for the first time reported in Italian mussels, was found in 84% of the samples, reaching the highest concentration in summer (12.1 &micro, g kg-1). GYM A and SPXs, submitted to tissue distribution studies, showed the tendency to accumulate mostly in mussel digestive glands. Even if SPX levels in mussels were largely below the European Food Safety Authority (EFSA) reference of 400 &mu, g SPXs kg-1, most of the samples contained CIs for the large part of the year. Since chronic toxicity data are still missing, monitoring is surely recommended.

The water intake of a drinking water treatment plant (DWTP) in Central Italy was monitored over six bloom seasons for cyanotoxin severity, which supplies drinking water from an oligo-mesotrophic lake with microcystin levels up to 10.3 μg/L. The historical data showed that the water temperature did not show extreme/large seasonal variation and it was not correlated either with cyanobacterial growth or microcystin concentration. Among all parameters, the cyanobacteria growth was negatively correlated with humidity and manganese and positively correlated with atmospheric temperature. No significant correlation was found between microcystin concentration and the climatic parameters. Polymer(chitosan)-enhanced microfiltration (PEMF) and ultrafiltration (PEUF) were further tested as an alternative microcystin removal approach from dense cyanobacteria-rich flows. The dominant cyanobacteria in the water intake, Planktothrix rubescens, was isolated and enriched to simulate cyanobacterial blooms in the lake. The PEMF and PEUF were separately applied to enriched P. rubescens culture (PC) (microcystin = 1.236 μg/L) as well as to the sand filter backwash water (SFBW) of the DWTP where microcystin concentration was higher than 12 μg/L. The overall microcystin removal rates from the final effluent of PC (always0.15 μg/L) were between 90.1-94.7% and 89.5-95.4% using 4 and 20 mg chitosan/L, respectively. Meanwhile, after the PEMF and PEUF of SFBW, the final effluent contained only 0.099 and 0.057 μg microcystin/L with an overall removal99%. The presented results are the first from the application of chitosan to remove P. rubescens as well as the implementation of PEMF and PEUF on SFBW to remove cyanobacterial cells and associated toxins.

Tetrodotoxins (TTXs), potent neurotoxins, have become an increasing concern in Europe in recent decades, especially because of their presence in mollusks. The European Food Safety Authority published a Scientific Opinion setting a recommended threshold for TTX in mollusks of 44 µg equivalent kg−1 and calling all member states to contribute to an effort to gather data in order to produce a more exhaustive risk assessment. The objective of this work was to assess TTX levels in wild and farmed mussels (Mytilus galloprovincialis) harvested in 2018–2019 along the coastal area of the Marche region in the Central Adriatic Sea (Italy). The presence of Vibrio spp. carrying the non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes, which are suspected to be involved in TTX biosynthesis, was also investigated. Out of 158 mussel samples analyzed by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS), 11 (7%) contained the toxins at detectable levels (8–26 µg kg−1) and 3 (2%) contained levels above the EFSA safety threshold (61–76 µg kg−1). Contaminated mussels were all harvested from natural beds in spring or summer. Of the 2019 samples, 70% of them contained V. alginolyticus strains with the NRPS and/or PKS genes. None of the strains containing NRPS and/or PKS genes showed detectable levels of TTXs. TTXs in mussels are not yet a threat in the Marche region nor in Europe, but further investigations are surely needed.

An analytical method for the simultaneous determination of polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecanes (HBCDs) in fish, shellfish and muscle of terrestrial animals was developed as an extension of a previously validated method for PBDE analysis. A single sample preparation based on QuEChERS-like extraction and a two-step clean-up, followed by a dual instrumental detection, was implemented. GC-MS/MS was used for PBDEs and LC-MS/MS for HBCDs. The method allows the quantification of fifteen PBDEs (28, 47, 49, 66, 77, 85, 99, 100, 138, 153, 154, 183, 197, 206, 209) and three HBCD isomers (α, β and γ), reaching 10 pg/g LOQs for all the analytes except BDE 206 and 209 (100 pg/g LOQ). The validated method was applied to the analysis of 12 fish and shellfish species (sole, spiny dogfish, smooth-hound, mackerel, swordfish, grey mullet, cod, anchovy, red mullet, Atlantic horse mackerel, tuna fish and mussel) collected in central Italian markets accounting for a total of 44 samples. Generally, ΣPBDEs showed higher concentration than ΣHBCDs except in the case of mussels in which ΣHBCDs > ΣPBDEs. Cod and smooth-hound are the least contaminated species among those analysed in the present study, whereas the highest brominated flame retardant (BFR) levels were found in spiny dogfish samples. The measured contamination levels were generally comparable to or lower than those already published in European studies. Literature data for HBCDs in European fish and shellfish were hard to find; therefore, PBDE and HBCD levels were also compared with European Environmental Quality Standard (EQS) in biota (Directive 2013/39/EU). EQSs for HBCDs are six orders of magnitude higher than those for PBDEs; therefore, while no exceedance was observed for the first, almost all the samples analysed for PBDEs were above EQSs. The presented preliminary data on PBDEs and HBCDs are among the first published in marine fish and shellfish commercialised in Central Italy. Graphical abstrac.

Eighty-six samples belonging to five different species (crucian carp, Carassius carassius; European perch, Perca fluviatilis; tench, Tinca tinca; eel, Anguilla anguilla; red swamp crayfish, Procambarus clarkii) collected from Lake Trasimeno (Italy) were analyzed to assess polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecanes (HBCDs) contamination. The Trasimeno is the largest Italian peninsular lake located in Umbria (Central Italy), in a rural area with low anthropogenic impact. All the samples were analyzed by an in-house developed analytical procedure involving a single sample preparation with dual detection: Gas- and Liquid-Chromatography coupled to tandem Mass Spectrometry (GC–MS/MS for PBDEs and LC-MS/MS for HBCDs). BFRs levels in crucian carp, tench and European perch were negligible and mostly below quantification limits (LOQs). In eel, the species with the higher fat content, PBDE sum (15 congeners) ranged from 0.269 to 0.916 ng/g w.w. BDE-47, -100 and -154 accounted for roughly 57%, 16% and 11% of the PBDE sum, respectively, while BDE-99 (usually one of the most abundant congeners in biota), only for 3%. HBCDs (sum of α-, β-, γ-isomers) were found between 0.157 and 1.14 ng/g w.w. with α- as predominant isomer (92% of the sum), followed by γ- (5%) and β- (2%). Peculiar was the contamination in red swamp crayfish characterized by negligible PBDEs and very high HBCDs levels with a singular contamination pattern. In female pools (n = 9) the mean HBCDs sum was 0.150 ng/g w.w., while in males higher concentrations were measured (mean = 2.77 ng/g w.w.). A significant correlation seems to exist between the contamination level and the seasonal cycle only in male crayfish. Interestingly, among the HBCDs, the γ-isomer was the highest (67% of the total) while α- contributes only for 20%.

In 2008–2018, 1458 georeferenced samples of clams and 343 of mussels were harvested in Italy from classified areas along Marche coast and analyzed within food-safety monitoring plans. Pb, Cd, V, Ni, Cr, and As median levels (mg kg−1) were 0.09, 0.08, 0.29, 0.77, 0.35, and 2.35 in clams and 0.16, 0.15, 0.46, 0.48, 0.25, and 3.34 in mussels. The reported levels were comparable with the published ones, and Hg always fell below LOQ (0.025 mg kg−1). Pb and Cd datasets, based on quarterly results, show a decreasing trend in clams and constant baseline value for mussels. Time-trends and forecasting models represent an asset to predict emerging food safety/security risks. The metal levels in mussels and clams from the same area allow environmental safety assessment and thus enable the evaluation of contaminants in both water column and sediment. Monitoring of elements that are not regulated may help in preserving food and environmental safety.

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

Massive blooms of the harmful benthic dinoflagellate Ostreopsis cf. ovata are of growing environmental concern in the Mediterranean, having recently caused adverse effects on benthic invertebrates and also some intoxication episodes to humans. The toxicological potential of produced palytoxin-like compounds was investigated in the present study on a typical marine sentinel species, the mussel Mytilus galloprovincialis . Organisms were sampled during various phases of a O . cf. ovata bloom, in two differently impacted sites. The presence of the algal toxins was indirectly assessed in mussels tissues (mouse test and hemolysis neutralization assay), while biological and toxicological effects were evaluated through the measurement of osmoregulatory and neurotoxic alterations (Na + /K + -ATPase and acetylcholinesterase activities), oxidative stress responses (antioxidant defences and total oxyradical scavenging capacity), lipid peroxidation processes (level of malondialdehyde), peroxisomal proliferation, organelle dysfunctions (lysosomal membrane stability, accumulation of lipofuscin and neutral lipids), immunological impairment (granulocytes percentage). Obtained results demonstrated a significant accumulation of algal toxins in mussels exposed to O . cf. ovata. These organisms exhibited a marked inhibition of the Na + /K + -ATPase activity and alterations of immunological, lysosomal and neurotoxic responses. Markers of oxidative stress showed more limited variations suggesting that toxicity of the O. cf. ovata toxins is not primarily mediated by an over production of reactive oxygen species. This study provided preliminary results on the usefulness of a multi-biomarker approach to assess biological alterations and toxicological events associated to blooms of O. cf. ovata in marine organisms.

Since the late 1980s, the North-central Adriatic Sea has frequently experienced blooms of harmful algal species, producing marine lipophilic toxins (MLTs) which accumulate in mussels and pose a serious threat to consumer health. Here, we present a 2-year LC-MS/MS study (2012-2014) of the MLT profile in mussels from the North-central Adriatic Sea in the context of the presence of toxic phytoplankton concentrations in seawater. Okadaic acid increased in mussels from all areas during the summer and autumn-winter periods with a rising trend between 2012 and 2014. In the same periods, Dinophysis sp. increased in abundance in seawater, but the highest densities of algae did not always coincide with the highest levels of toxins in mussels. Yessotoxins (YTXs) content in mussel increased sharply in the autumn-winter periods even exceeding the legal limit; although this accumulation did not always correlated with the YTX-producers in water (such as Lingulodinium polyedrum and Protoceratium reticulatum) a massive bloom of Gonyaulax spinifera was reported in November 2013, suggesting the role of this species in YTXs shellfish contamination. Traces of Azaspiracid 2 (AZA-2) were observed often in mussels during the study period, confirming for the first time the presence of this biotoxin in Mediterranean seafood.

The activities of several enzymes and the levels of metabolites have been measured in the digestive gland of Mytilus galloprovincialis exposed to increasing hypoxia from 7 to 168 hr. A sharp decrease of pyruvate kinase was observed after 7 hr. The anoxic enzyme showed increased Km for phosphoenolpyruvate and decreased apparent Ki for alanine. Glyoxalase I was constant after up to 72 hr of exposure and then decreased. Phosphoenolpyruvate carboxykinase and alanopine dehydrogenase decreased. The metabolites alanine and succinate increased with hypoxia time, whereas D- and L-lactic and aspartic acids were undetectable and constant respectively. Mitochondrial formation of pyruvate from D-lactate was demonstrated in intact mitochondria isolated from the digestive gland of Mytilus galloprovincialis. The significance of the observed enzyme and metabolite changes in hypoxia is discussed in comparison with other invertebrate organisms. The role of mitochondria in the overall adaptive strategy of Mytilus galloprovincialis is discussed. J. Exp. Zool. 286:107–113, 2000. © 2000 Wiley-Liss, Inc.

In the last decade massive blooms of the Ostreopsis cf. ovata have occurred in the Mediterranean basin, posing great concern to both environmental and human health. Biotoxicological and chemical studies demonstrated that O. cf. ovata produces palytoxin and ovatoxins; besides direct respiratory effects on humans due to inhalation of marine toxic aerosols, O. cf. ovata blooms can cause adverse effects on benthic invertebrates. The main aim of this study was to highlight the role of immunological, cellular and oxidative mechanisms in modulating the toxicity induced by O. cf. ovata in mussels Mytilus galloprovincialis . Organisms were exposed in laboratory condition to O. cf. ovata and analysed after 7 and 14 days of exposure. Obtained results demonstrated a clear involvement of the immune system with a significant decrement of granulocytes respect to the hyalinocytes type cells, a diminished phagocytosis activity and a reduced lysosomal membrane stability in haemocytes, after both 7 and 14 days of exposure. Histological analyses showed a decrease of the digestive gland wall thickness, dilatation of the tubules, haemocytes infiltration into the digestive gland and a decrement of neutral lipid levels in exposed mussels; similar results suggest a possible inhibition of the feeding activity, with a consequent induction of authophagic phenomena and utilization of stored reserve products such as neutral lipids. Antioxidant parameters revealed a limited role of O. cf. ovata to induce oxidative stress in mussels exposed under laboratory conditions excepting for a certain increase of catalase, glutathione reductase and glutathione peroxidases activities, and a significantly higher capability to neutralize peroxyl radicals in mussels exposed for 14 days. Although the obtained results suggest a non-specific response of mussels to the O. cf. ovata exposure, observed effects on the general health status of exposed mussels should be adequately considered when assessing the ecological relevance of these algal blooms.

The official biotoxicological (MBA) method for the determination of lipophilic marine toxins shows important gaps with regard to reproducibility, specificity and quality of the data provided and serious ethical contraindications. The Commission Regulation (EU) No 15/2011 establishes the date of December 31, 2014 as the deadline for replacement of the mouse test with a LC-MS/MS method by the laboratories of the member states responsible for the monitoring of marine biotoxins. In order to facilitate the interpretation of the MBA during this transition period, we further evaluated the specificity and selectivity of this assay in comparison with a LC-MS/MS method in detecting lipophilic marine toxins on mussels of the middle Adriatic Sea. Data show a high percentage of false-positive results on MBA due to interference by yessotoxins. It was also possible to evaluate the toxic profile of the samples analyzed.