Contaminants of Emerging Concern: Antibiotics Research in Mussels from the Coasts of the Tyrrhenian Sea (Sardinia, Italy).

Contaminants of emerging concern (CECs) are compounds found in several environmental compartments whose ubiquitous presence can cause toxicity for the entire ecosystem. Several personal care products, including antibiotics, have entered this group of compounds, constituting a major global threat. It is essential to develop simple and reliable methods by which to quantify these contaminants in several matrices. In this work, mussels were chosen as sentinel organisms to assess environmental pollution and the safety of bivalve mollusk consumption according to the "One Health perspective". A liquid chromatographic tandem mass spectrometry method (LC-MS/MS) was developed for the quantification of two macrolides, erythromycin (ERY) and azithromycin (AZI), in mussels. This new method was validated according to international guidelines, showing high selectivity, good recoveries (>60% for both of them), sensitivity, and precision. The method was successfully applied for ERY and AZI research in mussels farmed along the Sardinian coasts (Italy), demonstrating itself to be useful for routine analysis by competent authorities. The tested macrolides were not determined in the analyzed sites at concentrations above the limits of detection (LODs). These results demonstrate the food safety of mussels (as concerns the studied antibiotics) and a negligible amount of pollution derived from these drugs in the studied area.

Trend of Pathogenic Vibrio parahaemolyticus Occurrences in Bivalve Molluscs Harvested in Sardinian Coastal Environments Between 2011 and 2018.

The aim of the present study was to evaluate Vibrio parahaemolyticus occurrences in bivalve molluscs harvested from Sardinian coastal environments between 2013 and 2015. The prevalence of pathogenic V. parahaemolyticus isolates is based on the detection of the two major virulence genes thermostable direct hemolysin (tdh) and thermolabile hemolysin (trh) To assess changes between 2011 and 2018 in the prevalence of V. parahaemolyticus in bivalve molluscs, we compared our results with those of previous investigations. In total, 2,933 samples were collected: 1,079 in 2013, 1,288 in 2014, and 566 in 2015. The mean prevalence of V. parahaemolyticus in shellfish was 3.5% in 2013, 1.7% in 2014, and 3.5% in 2015. The highest percentage of positive samples in 2013 and 2014 was observed in clams (3.5% and 2.7%, respectively), whereas in 2015, it was reported in oysters (15.1%). By comparing the sampling period of 2011-2014 with that of 2015-2018, an increase in the prevalence of V. parahaemolyticus was observed in shellfish (p < 0.05). In parallel, 208 potentially enteropathogenic V. parahaemolyticus strains were identified through the years 2011-2018 and, in particular, 10 trh+ and six tdh+ isolates. Our present study provides information regarding trends of V. parahaemolyticus occurrences in bivalve molluscs harvested from Sardinian coastal environments between 2011 and 2018 suggesting that the prevalence varies depending on the sampling period and shellfish species.

Determination of phytoplankton in water samples, algal biotoxins, microbiological parameters and microplastics in Mediterranean mussels (Mytilus galloprovincialis Lamarck, 1819) from an experimental pilot farm in the Calich Lagoon (Sardinia, Italy).

The aims of this paper were to collect and analyse preliminary data of phytoplankton in the water, biotoxins, Escherichia coli, Salmonella spp., Vibrio spp. and microplastic eventually present in farmed mussels, and to acquire information about the production capability from an experimental pilot farm of the Calich Lagoon. Two sampling sessions were carried out, in February and in May 2019, also monitoring the water condition (pH, temperature, salinity, dissolved oxygen, chlorophyll a). No potentially toxic algae were detected, and moreover no biotoxins (Paralytic Shellfish Poison, Diarrheic Shellfish Poison, Amnesic Shellfish Poison) were found in mussels. E.coli was present with the highest concentration in February (16000 MPN/100g e.p.). Salmonella and Vibrio spp. have not been detected. Almost a microplastic per grams was found, mainly fiber of different colours. Further studies, carried out for several months, will allow to better understand the possible problems related to the production of mussels in a lagoon not yet classified as a shellfish production area.

Identification and quantification of potential microplastics in shellfish harvested in Sardinia (Italy) by using transillumination stereomicroscopy.

Plastics are non-biodegradable polymers made up of different groups of petrochemical materials. Several biotic and abiotic factors can change the density of plastic fragmenting it and originating microplastics (MPs). MPs have been defined as small pieces of plastic less than 5 mm in size. Due to their small size, they are an emerging concern in the marine environment since they can be ingested by aquatic organisms, especially filter-feeding organisms, such as bivalve mollusks. Impacts of MPs exposure have been shown at various levels of biological organization, from cellular to tissue to individual and population levels. For example, oxidative stress and inflammation have been observed in copepods and mussels, obstruction and physical damage of the digestive tract were found in fish and swimming behavior alterations, disruption of foraging and feeding behavior and overall reduced fitness and survival were observed in fish and oysters. In addition, MPs can act as a vector for the transfer of chemicals to marine biota. The aim of the present study was the identification and quantification of potential MPs in shellfish harvested in Sardinia (Italy) by using transillumination stereomicroscopy. Bivalves were collected from 4 of the main production areas located along the Sardinian coast and selected according to the principles of the risk assessment. The results of the present study demonstrated the presence of potential MPs in 70% of the analyzed samples: the presence of MPs in bivalve mollusks may pose a threat to food safety, and there is an urgent need to evaluate the potential risks of MPs to human health.

New evidence of pectenotoxins in farmed bivalve molluscs from Sardinia (Italy).

Several planktonic dinoflagellates can produce lipophilic phycotoxins that represent a significant threat to public health as well as to shellfish and fish farming. Poisoning related to some of these toxins is categorised as diarrhetic shellfish poisoning. We analysed 975 shellfish samples from Tortoli in the central-eastern region of Sardinia (Italy) from January 2016 to March 2020, to investigate the prevalence of different lipophilic marine biotoxins in mollusc bivalves. The results highlighted the predominant presence of toxins belonging to the okadaic acid group in all samples with toxin concentrations exceeding legal limits, and revealed the new occurrence of pectenotoxins in oysters and clams with a winter seasonality in recent years. The origin of shellfish toxicity was associated with the same Dinophysis species, mainly D. acuminata. Based on both these results and other precedents, monitoring and recording systems are strongly recommended.

Presence, Seasonal Distribution, and Biomolecular Characterization of Vibrio parahaemolyticus and Vibrio vulnificus in Shellfish Harvested and Marketed in Sardinia (Italy) between 2017 and 2018.

ABSTRACT: In the present study, we investigated the presence, seasonal distribution, and biomolecular characteristics of Vibrio parahaemolyticus and Vibrio vulnificus in samples of bivalve mollusks (Mytilus galloprovincialis, Crassostrea gigas, and Ruditapes decussatus) harvested and marketed in Sardinia (Italy) between 2017 and 2018. A total of 435 samples were submitted for qualitative determination of Vibrio spp., V. parahaemolyticus, and V. vulnificus. Potentially enteropathogenic isolates were detected with biomolecular methods. The overall prevalence of Vibrio spp. was 7.6%. The highest Vibrio prevalence was found in R. decussatus (8.3%). The prevalences of V. parahaemolyticus and V. vulnificus were 2.7 and 4.8%, respectively. Higher prevalences of V. parahaemolyticus and V. vulnificus were found in R. decussatus (4.2%) and C. gigas (6.2%), respectively. Only two pathogenic V. parahaemolyticus strains were recovered (genotypes: tdh- and trh+; tdh+ and trh-), both from M. galloprovincialis. None of the isolates were tdh+ and trh+. Pathogenic Vibrio infections are often underestimated, and human infections are increasing in Europe. European data on the true distribution of Vibrionaceae are scarce, and the results of the present study highlight the need of constant monitoring to update the distribution of pathogenic vibrios.

Influence of seasonality on the presence of okadaic acid associated with Dinophysis species: A four-year study in Sardinia (Italy).

In Sardinia (Italy), bivalve molluscs production plays an important role in the trade balance. Diarrhoetic shellfish poisoning (DSP), an intoxication caused by the ingestion of bivalve molluscs that have accumulated high levels of Okadaic acid (OA), may represent a serious risk for the public health and a remarkable economic loss for the producers. Aim of this work was to improve knowledge about the repeatability of OA accumulation phenomena in various seasons trying to understand whether or not there was a trend. Also, the interaction between toxic algae and OA accumulation was examined. In this study, data of lipophilic toxins, water temperature and abundance of DSP-producing microalgal species were collected in a four-year period (2015-2018) in coastal production areas of Sardinia. Several episodes of OA positive values (>160 eq µgAO/Kg pe, Reg 853/04) were recorded during the study period in different production areas of Sardinia and in different seasons. A seasonal repeatability of OA accumulation in molluscs was observed in some production areas; moreover, different temporal gaps between the presence of toxic algae and OA accumulation were reported. Toxicity was observed almost exclusively in Mytilus galloprovincialis Lamark (99%), being this matrix the most abundant species bred in Sardinia.

Recent findings of paralytic shellfish toxins linked to the genus Alexandrium Halim in Mediterranean mollusc production areas.

Paralytic shellfish poisoning is a human intoxication syndrome associated with the consumption of seafood that has been contaminated with paralytic shellfish toxins (PSTs), a group of natural neurotoxic alkaloids produced by marine dinoflagellates, including some Alexandrium species. This study presents findings of PSTs in mussels (Mytilus galloprovincialis) during 2018-2019 in several mollusc production areas of Sardinia (Italy, western Mediterranean). Investigations of the presence and abundance of PST-producing microalgal species in marine water and of the toxins associated with shellfish were carried out concomitantly. Overall, the results suggested a spatio-temporal expansion of Alexandriumpacificum and Alexandriumminutum in recent years, with an increasing number of PSTs present in molluscs and increased occurrences of toxicity cases. Liquid chromatography with fluorescence detection determined the toxin profile to be composed primarily of the carbamate gonyautoxin-5 and N-sulphocarbamoyltoxins 1 and 2. The study highlights the potential high risk to consumers of poisoning by bivalve molluscs bred in Sardinia, where shellfish production is a very important industrial sector. For this reason, routine monitoring is strongly recommended in order to mitigate any harm to human health as well as negative socio-economic consequences.

Bacterial and Viral Investigations Combined with Determination of Phytoplankton and Algal Biotoxins in Mussels and Water from a Mediterranean Coastal Lagoon (Sardinia, Italy).

Calich Lagoon is a Mediterranean coastal lagoon located along the northwestern coast of Sardinia (Italy). The connection to marine and fresh water determines the high productivity of this coastal lagoon. Despite its great potential and the presence of natural beds of bivalve mollusks (Mytilus galloprovincialis), the lagoon has not yet been classified for shellfish production. In this study, through a multidisciplinary approach, the presence of several bacterial pathogens (Escherichia coli, Salmonella spp., and Vibrio spp.) and viral pathogens (hepatitis A virus and norovirus genogroups I and II) was evaluated from March 2017 to February 2018. In addition, phytoplankton composition in lagoon waters and associated algal biotoxins (paralytic and diarrhetic shellfish poisoning) in mussels were also monitored. The aim of this study was to provide useful data to improve knowledge about their seasonal presence and to assess the potential risk for public health, as well as to provide input for future conservation and management strategies. In mussels, Salmonella spp. were found in spring, along with E. coli, but Salmonella spp. were not found in autumn or winter, even though E. coli was detected in these seasons. Vibrio parahaemolyticus was found in autumn and winter, but not in spring. Norovirus genogroups I and II were found in winter samples. None of the bacteria were found in summer. Algal biotoxins have never been detected in mussel samples. Among potentially harmful phytoplankton, only Pseudo-nitzschia spp. were present, mainly in summer. The results showed that a possible bacterial and viral contamination, together with the presence of potentially toxic microalgae, is a real problem. Therefore, the development of natural resource management strategies is necessary to ensure the good quality of waters and guarantee the protection of consumers.

Detection of Dinophysis Species and Associated Okadaic Acid in Farmed Shellfish: a Two-year Study from The Western Mediterranean Area.

INTRODUCTION: Diarrhoetic shellfish poisoning (DSP), an alimentary intoxication known to lead to intestinal symptoms, and caused by toxins produced by some dinoflagellates (including several Dinophysis), represents a serious threat to public health. The aim of this paper was to provide information about the occurrence and abundance of potentially toxic harmful algal species causing DSP, and the associated concentration of okadaic acid (OA) toxins. The departing assumption was that in the study area there was an increase in the presence both of Dinophysis species and OA and its derivates that could result in a risk to the health of seafood consumers. MATERIAL AND METHODS: During 2015-2016, water and shellfish samples were collected in the Mediterranean area (Sardinia, Italy). Dinophysis cells were counted according to Utermöhl's method from water samples, while mass spectrometry was used to identify lipophilic toxins in molluscs. RESULTS: A total of 46 non-compliant samples of Mytilus galloprovincialis were observed. Their non-compliance concerned their OA levels above the legal limit. Among toxic dinoflagellates, D. acuminata and D. sacculus were the species found mostly during DSP events. CONCLUSION: No cases of human intoxication have been reported, but continuous surveillance of toxic phytoplankton is necessary to predict and prevent its harmful effects on human health.

Correction: Occurrence of Harmful Algal Species and Shellfish Toxicity in Sardinia (Italy).

[This corrects the article on p. 6095 in vol. 5, PMID: 28058244.].

Yessotoxin detection in bivalve molluscs: A case study from coastal mussel farms (Sardinia, Italy).

This work reports the first communication relating to the presence of yessotoxins in Mytilus galloprovincialis from coastal mussel farms (Sardinia, western Mediterranean) detected during 2008 and 2013 through a monitoring programme. The paper emphasizes how the changes both in yessotoxin permitted limits and used methods, established by legislation, have influenced the interpretation of the obtained results. Consequently, the samples that resulted negative during 2008 would have been positive until August 2013 and negative from September 2013 up to now, and the samples that were positive in 2013 would have been positive in 2008 and negative nowadays, according to Regulation currently in force. Regular monitoring of biotoxins demonstrated that, although yessotoxins have been rarely present in the past in Sardinia, they may cause toxicity in shellfish. So, it's important to keep up on legislation's changing and laboratory methods.

Determination of microcystin-LR in clams (Tapes decussatus) of two Sardinian coastal ponds (Italy).

The presence of microcystin-LR (MC-LR) was monitored in Tapes decussatus harvested in two Sardinian ponds (Cabras and Tortolì, Italy) in spring and summer. After solid phase extraction, samples were analyzed using a screening enzyme-linked immunosorbent assay (ELISA) followed by a liquid chromatographic coupled to tandem mass spectrometer (LC-MS/MS) analysis. Results obtained through the ELISA test showed the presence of microcystins with a maximum concentration in August for Cabras pond (0.55ng/g) and in September for Tortolì pond (0.85ng/g). The LC-MS/MS analysis did not confirm the presence of MC-LR suggesting that results obtained with the ELISA technique could be due to the presence of other microcystins. According to the tolerable daily intake suggested by the World Health Organization, these results hint that clams harvested in these ponds are safe for human health. These data can contribute to enrich the knowledge about the healthiness of Sardinian ponds and of their products.

Occurrence of Harmful Algal Species and Shellfish Toxicity in Sardinia (Italy).

Sardinia (Italy, north-western Mediterranean) is a commercially important producer of edible bivalve molluscs. Since the early 2000s, it was subjected to recurring cases of mussel farm closures due to toxic algal poison. Here, we present the studies on toxin concentrations and the associated potentially toxic phytoplankton distribution and abundances carried out by a regular monitoring programme in Sardinian shellfish areas, from January to May 2015. Diarrheic shellfish poisoning (DSP) toxins were detected in several bivalve molluscs samples, while paralytic shellfish poisoning (PSP) and paralytic shellfish poisoning toxins were present just once, without exceeding the legal limits. Potentially toxic algal species have been constantly present. Pseudo-nitzschia species were present during the entire study often with high abundances, while Dinophysis species reached high densities sporadically. Among PSP phytoplankton, only Alexandrium minutum Halim was found. The data obtained in this study showed an increase in the DSP toxicity in mussels in Sardinia. No clear relation between the occurrence of toxins in shellfish and the presence of potentially toxic algal species was found, although a slight correlation between DSP toxins and Dinophysis species could be supported.