A Hotspot of TTX Contamination in the Adriatic Sea: Study on the Origin and Causative Factors.

Tetrodotoxins (TTXs), the pufferfish venom traditionally associated with Indo-Pacific area, has been reported during last decades in ever wider range of marine organisms and ever more geographical areas, including shellfish in Europe. Wild mussels (Mytilus galloprovincialis) grown in the Marche Region (N Adriatic Sea, Italy) were shown to be prone to TTX contamination during the warm season, with a suspected role of Vibrio alginolyticus characterized by non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS)-encoding genes. This work aimed to deepen the knowledge about the toxin's origin and the way through which it accumulates in mussels. A two-year study (spring-summer 2020-2021) confirmed the recurrent presence of TTX (11-68 µg kg-1) in the official monitored natural mussel beds of the Conero Riviera. During 2021, a supplementary nonroutine monitoring of a natural mussel bed in the same area was carried out weekly from June until August for TTXs and/or the presence of V. alginolyticus. Biotic (mussels, mesozooplankton, worms and phytoplankton); abiotic (water and sediment) matrices and phytoplankton assemblage characterizations were studied. Mussels showed relevant TTX contamination levels (9-296 µg kg-1) with extremely rapid TTX accumulation/depletion rates. The toxin presence in phytoplankton and its distribution in the different mussel tissues supports its possible exogenous origin. The V. alginolyticus count trend overlaps that of TTX contamination in mussels, and similar trends were reported also for some phytoplankton species. The role of V. alginolyticus carrying NRPS or PKS genes as a possible TTX source and of phytoplankton as a "potential vector" should therefore be further investigated.

A High Throughput Screening HPLC-FLD Method for Paralytic Shellfish Toxins (PSTs) Enabling Effective Official Control.

Paralytic Shellfish Toxins (PSTs) are marine biotoxins, primarily produced by dinoflagellates of the genera Gymnodinium spp., Alexandrium spp. They can accumulate in shellfish and, through the food chain, be assimilated by humans, giving rise to Paralytic Shellfish Poisoning. The maximum permitted level for PSTs in bivalves is 800 µg STX·2HCl eqv/kg (Reg. EC N° 853/2004). Until recently, the reference analytical method was the Mouse Bioassay, but Reg. EU N° 1709/2021 entered into force on 13 October 2021 and identified in the Standard EN14526:2017 or in any other internationally recognized validated method not entailing the use of live animals as official methods. Then the official control laboratories had urgently to fulfill the new requests, face out the Mouse Bioassay and implement instrumental analytical methods. The "EURLMB SOP for the analysis of PSTs by pre-column HPLC-FLD according to OMA AOAC 2005.06" also introduced a simplified semiquantitative approach to discriminate samples above and below the regulatory limit. The aim of the present paper is to present a new presence/absence test with a cut-off at 600 µg STX·2HCl eqv/kg enabling the fast discrimination of samples with very low PSTs levels from those to be submitted to the full quantitative confirmatory EN14526:2017 method. The method was implemented, avoiding the use of a large number of certified reference standards and long quantification procedures, resulting in an efficient, economical screening instrument available for official control laboratories. The protocol was fully validated, obtaining good performances in terms of repeatability (<11%) and recovery (53−106%) and accredited according to ISO/IEC 17025. The method was applied to mollusks collected from March 2021 to February 2022 along the Marche region in the frame of marine toxins official control.

Tetrodotoxins (TTXs) and Vibrio alginolyticus in Mussels from Central Adriatic Sea (Italy): Are They Closely Related?

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.

Inorganic Elements in Mytilus galloprovincialis Shells: Geographic Traceability by Multivariate Analysis of ICP-MS Data.

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 >90% of individuals sampled to their respective harvesting locations and for being applied to counteract fraud.

Single sample preparation for brominated flame retardants in fish and shellfish with dual detection: GC-MS/MS (PBDEs) and LC-MS/MS (HBCDs).

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, ß 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.

Biological Effects of the Azaspiracid-Producing Dinoflagellate Azadinium dexteroporum in Mytilus galloprovincialis from the Mediterranean Sea.

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' 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.

Exploring Perfluoroalkyl Substances (PFASs) in Aquatic Fauna of Lake Trasimeno (Italy): Insights from a Low-Anthropized Area.

This study investigated the concentrations and profiles of 19 perfluoroalkyl substances (PFASs) in the muscle and liver of four freshwater species from Lake Trasimeno (Italy): Anguilla anguilla (European eel), Carassius auratus (goldfish), Perca fluviatilis (European perch), and Procambarus clarkii (red swamp crayfish). In livers, the amount of PFASs ranged from 3.1 to 10 µg kg-1, significantly higher than that in muscle (0.032-1.7 µg kg-1). The predominant PFASs were perfluorooctane sulfonic acid (PFOS) and long-chain carboxylic acids (C8-C14). Short-chain compounds (C4-C5), as well as the long-chain sulfonic acids (C9-C12), were not quantified. The contamination patterns were similar among species with few differences, suggesting the influence of species-specific accumulation. The PFAS concentrations in livers were comparable among species, while in muscle, the higher values were measured in European eel, followed by goldfish, European perch, and red swamp crayfish. The levels were generally lower than those reported for fish from Northern Italian lakes and rivers. The concentrations of regulated PFASs were lower than the maximum limits set by Regulation EU 2023/915 and did not exceed the Environmental Quality Standards (PFOS in biota). This study provides the first valuable insights on PFASs in freshwater species from Lake Trasimeno.

Subcellular effects and lipid metabolism alterations in the gilthead seabream Sparus aurata fed on ovatoxins-contaminated mussels.

The marine microalgae Ostreopsis cf. ovata are a well-known producer of palytoxin (PlTXs) analogues, i.e. ovatoxins (OVTXs) among others, which arouse concern for animal and human health. Both in field and laboratory studies, presence of OVTXs, detected in species directly feeding on O. cf. ovata, was frequently correlated with impairment on organisms' physiology, development and behaviour, while similar knowledge is still lacking for animals feeding on contaminated preys. In this study, transfer and toxicity of OVTXs were evaluated in an exposure experiment, in which gilthead seabream Sparus aurata was fed with bivalve mussel Mytilus galloprovincialis, contaminated by a toxic strain of O. cf. ovata. Mussels exposed to O. cf. ovata for 21 days accumulated meanly 188 ± 13 µg/kg OVTXs in the whole tissues. Seabreams fed with OVTX-contaminated mussels started to reject the food after 6 days of contaminated diet. Although no detectable levels of OVTXs were measured in muscle, liver, gills and gastro-intestinal tracts, the OVTX-enriched diet induced alterations of lipid metabolism in seabreams livers, displaying a decreased content of total lipid and fatty acid, together with overexpression of fatty acid biosynthetic genes, downregulation of ß-oxidation genes and modulation of several genes related to lipid transport and regulation. Results from this study would suggest the hypothesis that OVTXs produced by O. cf. ovata may not be subject to bioaccumulation in fish fed on contaminated preys, being however responsible of significant biological effects, with important implications for human consumption of seafood products.

Marine phycotoxin levels in shellfish-14 years of data gathered along the Italian coast.

Along the Italian coasts, toxins of algal origin in wild and cultivated shellfish have been reported since the 1970s. In this study, we used data gathered by the Veterinary Public Health Institutes (IZS) and the Italian Environmental Health Protection Agencies (ARPA) from 2006 to 2019 to investigate toxicity events along the Italian coasts and relate them to the distribution of potentially toxic species. Among the detected toxins (OA and analogs, YTXs, PTXs, STXs, DAs, AZAs), OA and YTX were those most frequently reported. Levels exceeding regulatory limits in the case of OA (≤2,448 µg equivalent kg-1) were associated with high abundances of Dinophysis spp., and in the case of YTXs (≤22 mg equivalent kg-1) with blooms of Gonyaulax spinifera, Lingulodinium polyedra, and Protoceratium reticulatum. Seasonal blooms of Pseudo-nitzschia spp. occur all along the Italian coast, but DA has only occasionally been detected in shellfish at concentrations always below the regulatory limit (≤18 mg kg-1). Alexandrium spp. were recorded in several areas, although STXs (≤13,782 µg equivalent kg-1) rarely and only in few sites exceeded the regulatory limit in shellfish. Azadinium spp. have been sporadically recorded, and AZAs have been sometimes detected but always in low concentrations (≤7 µg equivalent kg-1). Among the emerging toxins, PLTX-like toxins (≤971 µg kg-1 OVTX-a) have often been detected mainly in wild mussels and sea urchins from rocky shores due to the presence of Ostreopsis cf. ovata. Overall, Italian coastal waters harbour a high number of potentially toxic species, with a few HAB hotspots mainly related to DSP toxins. Nevertheless, rare cases of intoxications have occurred so far, reflecting the whole Mediterranean Sea conditions.

Perfluoroalkyl and polyfluoroalkyl substances (PFASs): An optimized LC-MS/MS procedure for feed analysis.

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 < 0.10 ng g-1) spiked at five different concentrations (0.10, 0.50, 1.0, 5.0 and 10 ng g-1). Apparent recoveries (R%) were generally between 88 and 111%; R% < 80% were obtained only at the lower validation levels for those molecules not having the corresponding labelled analogues. Relative standard deviations in repeatability conditions (RSDr) and within-lab reproducibility conditions (RSDwR) were lower or equal to 11% and 22% respectively. Limits of quantification were set for most of the analytes at 0.10 ng g-1 (LOQs) and verified with repeated analysis on fortified samples (0.10 ng g-1). Limits of detection (LODs) were calculated as 1/3 LOQ. Finally the method was applied to 23 feed samples of different type (compound feed, vegetable feed material, complementary feed, pre-mixture and mineral feed) and all of them did not show PFAS above LOQ.

Heavy metal spatial and temporal trends (2008-2018) in clams and mussel from Adriatic Sea (Italy): Possible definition of forecasting models.

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.

Monitoring of cyanobacterial blooms and assessing polymer-enhanced microfiltration and ultrafiltration for microcystin removal in an Italian drinking water treatment plant.

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 (always <0.15 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 removal >99%. 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.

BFRs (PBDEs and HBCDs) in freshwater species from Lake Trasimeno (Italy): The singular case of HBCDs in red swamp crayfish.

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-, ß-, 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 ß- (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%.

Morphological Variability of Pseudo-nitzschia pungens Clade I (Bacillariophyceae) in the Northwestern Adriatic Sea.

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 µm), 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' 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) in Mussels from North-Central Adriatic Sea: First Evidence of Gymnodimine A in Italy.

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 2014-2015. 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 µg 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 µg 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 µg 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.

Two-year study of lipophilic marine toxin profile in mussels of the North-central Adriatic Sea: First report of azaspiracids in Mediterranean seafood.

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.