Shellfish aquaculture has become an increasingly important factor in Bulgarian economy in the recent years. Marine biotoxins, produced by some phytoplankton species, may accumulate in mussels and present an important challenge in commercialization of shellfish. The aim of this study was to determine the occurrence and variability of hydrophilic toxinsparalytic shellfish poisoning (PSP) and amnesic shellfish poisoning (ASP) as well as lipophilic toxinsincluding diarrheic shellfish poisoning (DSP), pectenotoxins (PTXs) and yessotoxins (YTXs) in plankton, wild and farmed mussel samples from the Southern Black Sea coast, an important shellfish farming area in spring 2017. PSP toxins were determined by HPLC with postchromatographic oxidation and fluorescence detection and domoic acid and lipophilic toxins by liquid chromatograph coupled to tandem mass spectrometry. DA and pectenotoxin-2 were detected in plankton, wild and farmed mussel samples. Yessotoxins were detected only in cultivated mussels and no PSP toxins were detected. The occurrence of phycotoxins differed over both space and time. Toxin profile includes prevalent domoic acid, scarce presence of pectenotoxin-2 and yessotoxins in the samples.

AbstractIntroductionLipophilic phycotoxins, produced by toxic phytoplankton species, accumulate in digestive glands of mussels. If contaminated mussels containing marine biotoxins over regulatory levels are consumed, it is assumed that this could result in e.g. gastrointestinal disorders.ObjectivesThe aim of the study was to report contamination level of lipophilic marine biotoxins and to assess the potential health risk based on dietary intake for regular consumers and recreational harvesters from Bulgaria.Method/DesignWild and cultivated mussels were sampled in spring seasons of 2017 and 2018 from the North Black Sea coast. Positive phycotoxins concentrations determined via LC-MS/MS in extracts from digestive glands of the mussels were converted in phycotoxin levels for whole mussel meat (mm) by applying a factor of 5 proposed by EFSA. Dietary intakes were calculated by multiplying the mean positive phycotoxin levels (mg /kg mm) by the food consumption data for an average body weight (bw) of the investigated population group.Results and discussionIn total 20 farmed and 15 wild mussel samples were investigated. Yessotoxins and pectenotoxin-2 were detected. The mean YTX levels in cultivated mussels were measured about two times higher in spring 2018 (1.64 mg/kg) than in 2017 (0.70 mg/kg). The mean YTX level in wild mussels was found much lower - 0.46 mg/kg (spring 2017). In this regard, estimation of mean dietary YTX intake show that exposure of both population groups by consumption of cultivated mussels from spring 2018 is highest. Nevertheless, the regulatory threshold – acute reference dose of 25 μg/kg bw, is much above the highest calculated dietary intake (0.006 μg/kg female consumers). The mean PTX2 level in wild mussel samples from spring 2017 (0.71 mg/kg) was found about six times higher than in farmed mussels.The calculation of mean dietary PTX2 intake indicate that most exposed are females if wild mussels from spring 2017 were consumed. Still, the calculated value – 0.003 μg/kg bw were much lower than the ARfD - 0.8 μg/kg bw. All wild mussel samples from spring 2018 were negative. Lowest exposure to detected toxins were established for recreational harvesters.ConclusionThis study showed in general low contamination level with YTX and PTX2 of investigated mussel samples. Thereon, the estimated dietary intake of selected population groups is also much lower than the regulatory thresholds.

Mussels accumulate marine biotoxins (phycotoxins) produced by certain phytoplankton species. In EU are set limits for toxin concentration (e.g. domoic acid, okadaic acid, yessotoxins etc.) beyond that mussels are safe for consumption. Marine biotoxins tend to accumulate in the digestive gland (hepatopancreas) of the mussel. Consequently, this tissue is preferred for toxin concentration determination. Normally the whole shellfish is consumed and therefore the occurrence data for phycotoxins need to be expressed in terms of whole shellfish meat. A theoretical factor of 5 is used to convert the value to whole shellfish meat. The aim of this study was to determine an empirical factor in order to convert phycotoxin concentrations from hepatopancreas to whole shellfish meat of main marine aquaculture in Bulgaria- mussels Mytilus galloprovincialis. Wild and cultivated mussels were collected from the north Black Sea coast of Bulgaria in 2017. In total 13 mussel samples were studied whereas in each sample subsamples of hepatopancreas only and whole mussel meat are prepared. Phycotoxins were extracted for all types of phycotoxins by means of liquid-liquid extraction and their concentration was determined via LC/MS. Yessotoxins appeared in most of the samples and therefore seemed most suitable for empirical conversion factor determination. It is calculated as ratio between determined concentration in hepatopancreas and whole shellfish meat. Mean defined value was 5.36. Determination and application of empirical conversion factor is important when establishing very low concentration for chronic exposure assessment. It will be advantageous when taking into account interspecies differences and to avoid phycotoxin omission if preparing whole shellfish sample.

INTRODUCTION: Marine biotoxins can be accumulated in shellfish and in turn can lead to severe illness or chronical consequences in human shellfish consumers. AIM: The aim of this study was to assess the levels of hydrophilic marine biotoxins in both farmed and wild mussels from the Bulgarian coast sampled in 2017 and to estimate the exposure (acute and chronic) of Bulgarian consumers to detected toxins if investigated mussels were consumed. To the group of hydrophilic marine toxins belong amnesic toxins (domoic acid, isodomoic acid) and paralytic toxins (neosaxitoxin, gonyautoxins and their decarbamoyl and N-sulfocambamoyl analogs). MATERIALS AND METHODS: The hydrophilic toxin-domoic acid (DA) was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). Paralytic toxins (saxitoxin (STX), neosaxitoxin (NEO), gonyautoxin-1 (GTX1), gonyautoxin-2 (GTX2), gonyautoxin-3 (GTX3), gonyautoxin-4 (GTX4), gonyautoxin-5 (B1), decarbamoyl gonyautoxin-2 (dcGTX2), decarbamoyl gonyautoxin-3 (dcGTX3), decarbamoyl saxitoxin (dcSTX), N-sulfocarbamoyl gonyautoxin-1 (C1), N-sulfocarbamoyl gonyautoxin-2 (C2)) were investigated via high performance liquid chromatography with fluorescence detection (HPLC-FD). RESULTS: Among all hydrophilic toxins investigated DA and GTX2 were detected in the studied samples. Mean domoic acid in whole mussel meat was estimated to be 0.139 mg/kg mm which is below the regulatory limit of 20 mg/kg mm. Mean GTX2 level in whole mussel meat was calculated to be 0.151 μg saxitoxin dihydrochloride equivalent (STX.2HCl eq)/kg which is far beneath the legislative limit of 800 μg STX.2HCl eq/kg mm. Estimation of acute exposure for both detected toxins-DA and GTX2, and of chronic exposure to domoic acid showed similar results among male and female, as well as among wild and cultivated mussel consumers. CONCLUSION: This study showed an overall low contamination level of wild and farmed mussels with hydrophilic marine biotoxins compared to the regulatory limits. This leads to the conclusion that there is low acute and chronic exposure via consumption of contaminated mussels.

Anthropogenic pollution of marine ecosystems is one of the main sources of polycyclic aromatic hydrocarbons (PAHs). Marine bivalves are often used as bioindicators of environmental pollution due to their wide distribution and capability of xenobiotic bioaccumulation. The aim of the present study was to assess the presence of PAHs in soft tissues of wedge clams Donax trunculus (Linnaeus, 1758), collected from sublittoral sandy habitats at different locations off the Bulgarian Black Sea coast. Wedge clams from the different locations showed variations in the content of accumulated PAHs’ compounds. The concentrations of PAHs were measured by gas chromatography system with mass spectrometry detection. The total PAHs content (sum of 13 PAHs’ compounds) measured was in the range from 5.59 to 50.50 ng/g wet weight and was comparable with other European studies. The compounds phenanthrene and fluorene were most abundant in all analyzed samples. The results showed that low molecular weight (LMW) PAHs (2 and 3 aromatic rings) were predominant, accounting for 91% of the total PAHs levels, while high molecular weight (HMW) PAHs (4–5- and 6- rings) presence was 8.9% on average. The ratio LMW/HMWPAHs was higher than one, suggesting predominant pollution of petrogenic origin. The concentrations of benzo (a)pyrene did not exceed the limit set in EC Regulation although it was detected in 20% of the analyzed samples. In conclusion, maximum overall PAHs content was found in clams from Arkutino, while minimum PAHs content was present in samples from Elenite. The Sum PAH4 (sum of four polycyclic aromatic hydrocarbons: benzo[a]pyrene, chrisene, benzo[a]anthracene, and benzo[b]fluoranthene) in the wedge clams for all localities studied was below legislation limits. Data from the present research can be used for assessing pollution levels in the marine environment and also risk of human exposure to PAHs using D. trunculus as bioindicator species.

Polychlorinated biphenyls (PCBs) can still be a problem for the aquatic environment. Fish species are a suitable indicator for the environmental pollution monitoring because they concentrate pollutants in their tissues directly from water. Concentrations of PCBs were measured in marine fish, collected from Bulgarian Black Sea coast in order to monitor the dynamics of these pollutants in 2007, 2010 and 2015. The fish species: goby (Neogobius melanostomus), sprat (Sprattus sprattus sulinus), horse mackerel (Trachurus Mediterraneus ponticus) and grey mullet (Mugil cephalus) were chosen because of their characteristic feeding behavior. The PCBs were determined by gas chromatography system with mass spectrometry detection. The Total PCBs ranged from 93.8 to 513.3 ng/g lipid weight (in grey mullet and goby, respectively). Levels of PCBs in goby and grey mullet decreased in 2010 and 2015. In order to assess the safety of fish as food were calculated TEQ. They are determined by the results of dioxin - like (dl) PCBs. TEQs were calculated from 0.01 to 0.04 pg TEQ/g ww and did not exceed the EC limit of 3 pg TEQ/g ww. The levels of PCBs in fish from Bulgarian Black Sea were comparable to those found in neighboring seas.

Plastic pollution in seawaters is ubiquitous, but quantitative estimates on the floating microplastics in the Black Sea are still limited. Plastics may adsorb persistent environmental contaminants, thus representing a potential risk for marine organisms. Aim: Thе aim of the study was evaluation of the presence and characteristics of microplastic particles (MPs) in waters from the Black Sea coast of Bulgaria. Materials and methods: Samples of coastal waters were collected from March 2021 to April 2022 from different stations on the Black Sea coast, including protected, aquaculture and industrial areas. In order to determine the number of plastic particles, 23 samples were collected from the surface waters at depth of 1–3 m close to the Bulgarian shore. Samples were treated with H2O2, plastic particles were isolated by density separation and filtered over a membrane filter. Identification analysis of micro particles (< 5 mm) was performed visually by microscopy. Main results: Results indicated widespread presence of microplastics in coastal waters. Mean MPs concentration was calculated 7.3 ± 4.9 pt/l. The comparison of the North, Varna and South sampling area showed that there is no significant difference in the abundance of plastic particles. The most dominant type forms were fibres followed by fragments. The most abundant size class of fragments was 101–500 μm Ferret diameter. Conclusion: Further studies are needed in order to fill knowledge gap and to evaluate distribution of plastic particles in the Black Sea and their potential ecological risk.

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