Red Tides and Phycotoxins: the Problems are in the Details

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Study on risk of exposure of seafood consumers in Bulgaria to hydrophilic marine toxins

Marine biotoxins can be accumulated in shellfish and in turn can lead to severe illness or chronical consequences in human shellfish consumers.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 detected toxins to Bulgarian consumers if investigated mussels were consumed.The hydrophilic toxin - domoic acid was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). Mean domoic acid in whole mussel meat was estimated 0.139 mg/kg which is below the regulatory limit of 20 mg/kg.Among all paralytic shellfish (PSP) toxins evaluated only gonyautoxin-2 (GTX2) was detected via high performance liquid chromatography with fluorescence detection (HPLC-FD). Mean GTX2 level in whole mussel meat was calculated to be 0.151 mg STX.2HCl eq/kg which is far beneath the legislative limit of 800 mg STX.2HCl eq/kg.Estimation of acute exposure for both detected toxins - domoic acid 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.This study showed an overall low contamination level of wild and farmed mussels with hydrophilic marine biotoxins compared to the regulatory limits. This is concluding in low acute and chronic exposure via consumption of contaminated mussels

Study on the risk exposure of seafood consumers in Bulgaria to hydrophilic marine toxins

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

Method optimization of the simultaneous detection of B12 congeners leading to the detection of a novel isomer of hydroxycobalamin in seawater

Rationale More than half of surveyed microalgae and over 90% of harmful algae have an obligate requirement for vitamin B12, but methods for directly measuring dissolved B12 in seawater are scarce due to low concentrations and rapid light-induced hydrolysis. Methods We present a method to detect and measure the four main congeners of vitamin B12 dissolved in seawater. The method includes solid-phase extraction, separation by ultrahigh-performance liquid chromatography and detection by triple-quadrupole tandem mass spectrometry utilizing an electrospray ion source. This method was applied to coastal field samples collected in the German Bay, Baltic Sea and the Danish Limfjord system. Results The total dissolved B12 pool ranged between 0.5 and 2.1 pM. Under ambient conditions methyl-B12 and adenosyl-B12 were nearly fully hydrolyzed to hydroxy-B12 in less than 1 h. Hydroxy-B12 and a novel, corresponding isomer were the main forms of B12 found at all field sites. This isomer eluted well after the OH-B12 peak and was also detected in commercially available OH-B12. Both compounds showed very high similarity in their collision-induced dissociation spectra. Conclusions The high instability of the biologically active forms of Me-B12 and Ado-B12 towards hydrolysis was shown, highlighting the importance of reducing the duration of the extraction protocol. In addition, the vitamin B12 pool in the study area was mostly comprised of a previously undescribed isomer of OH-B12. Further studies into the structure of this isomer and its bioavailability are needed

A Mediterranean Alexandrium taylorii (Dinophyceae) Strain Produces Goniodomin A and Lytic Compounds but Not Paralytic Shellfish Toxins

Species of the dinophyte genus Alexandrium are widely distributed and are notorious bloom formers and producers of various potent phycotoxins. The species Alexandrium taylorii is known to form recurrent and dense blooms in the Mediterranean, but its toxin production potential is poorly studied. Here we investigated toxin production potential of a Mediterranean A. taylorii clonal strain by combining state-of-the-art screening for various toxins known to be produced within Alexandrium with a sound morphological and molecular designation of the studied strain. As shown by a detailed thecal plate analysis, morphology of the A. taylorii strain AY7T from the Adriatic Sea conformed with the original species description. Moreover, newly obtained Large Subunit (LSU) and Internal Transcribed Spacers (ITS) rDNA sequences perfectly matched with the majority of other Mediterranean A. taylorii strains from the databases. Based on both ion pair chromatography coupled to post-column derivatization and fluorescence detection (LC-FLD) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis it is shown that A. taylorii AY7T does not produce paralytic shellfish toxins (PST) above a detection limit of ca. 1 fg cell−1, and also lacks any traces of spirolides and gymnodimines. The strain caused cell lysis of protistan species due to poorly characterized lytic compounds, with a density of 185 cells mL−1 causing 50% cell lysis of cryptophyte bioassay target cells (EC50). As shown here for the first time A. taylorii AY7T produced goniodomin A (GDA) at a cellular level of 11.7 pg cell−1. This first report of goniodomin (GD) production of A. taylorii supports the close evolutionary relationship of A. taylorii to other identified GD-producing Alexandrium species. As GD have been causatively linked to fish kills, future studies of Mediterranean A. taylorii blooms should include analysis of GD and should draw attention to potential links to fish kills or other environmental damage

SELECTED CONTAMINANTS IN FISH AND MUSSELS FROM THE BULGARIAN BLACK SEA

The presence of polychlorinated biphenyl congeners, organochlorine pesticides and marine biotoxins in the marine environment is important for the evaluation of a potential risk to human health. The purpose of the present study was to determine concentrations of polychlorinated biphenyl congeners (PCBs) and organochlorine pesticides (DDT and its metabolites) in three fish species and mussels (Mytilus galloprovincialis) and marine biotoxins in mussels from the Black Sea, Bulgaria. Concentration of six Indicator PCB congeners, DDT and its metabolites were determined by gas chromatography coupled to mass spectrometry. The mean levels of I-PCBs ranged between 6.78 ng/g ww and 16.33 ng/g ww (garfish and bluefish respectively). The sum of I-PCBs in all seafood studied did not exceed the EU maximum level. Hydrophilic marine biotoxins determination was performed by HPLC with postchromatographic oxidation. Lipophilic marine toxins were determined on liquid chromatograph coupled to mass spectrometry. The analyzed marine biotoxins were under the limit of detection.</jats:p

Comparison of seasonal and spatial phycotoxin profiles of mussels from South Bulgarian coast

Phycotoxins (marine algal toxins) are toxic metabolites released by certain phytoplankton species. They can be responsible for seafood poisoning outbreaks because filter-feeding mollusks, such as mussels, can accumulate these toxins throughout the food chain and present a threat for consumers’ health. A wide range of symptoms, from digestive to nervous, are associated to human intoxication by biotoxins, characterizing different and specific syndromes, called shellfish poisoning. The aim of this study is to compare the seasonal and spatial phycotoxin profiles of mussels (wild and farmed) harvested from South Bulgarian coast in the period 2017-2018. Analyzed were 57 samples by different analytical techniques - liquid chromatography tandem mass spectrometry (LC-MS/MS) and high-performance liquid chromatography with fluorescent detection followed by postcolumn derivatization. Domoic acid (DA), yessotoxin (YTX), pectenotoxin-2, PTX-2sa/ epi-PTX-2sa and gonyautoxin-2 (GTX2) were detected in the studied samples. Results revealed huge seasonal variations in the phycotoxin profiles of the mussels investigated. Spring 2017 profile is dominated by domoic acid present in 67% of the samples and reaching highest level of 618.9 ng. g-1. In summer 2017 samples YTX is prevalent (60%) reaching a level of 8.3 ng.g-1. No phycotoxins were detected in samples from fall 2017. The epimer pair PTX-2sa/ epi-PTX-2sa was with highest seasonal abundance in winter-spring 2018 – 47%. Its maximum detected level was 7.1 ng.g-1. No statistically significant differences in mean phycotoxin levels of different sampling locations were determined. Generally, the herein reported marine toxins levels are comparable or even lower than in other European studies and much lower than legislative limits set in EU. Nevertheless, the huge seasonal variations in the phycotoxin profile show that for protection of consumers’ health a further surveillance on marine toxins content in edible mussels is required

Accumulation, transformation and breakdown of DSP toxins from the toxic dinoflagellate Dinophysis acuta in blue mussels, Mytilus edulis

Okadaic acid (OA), dinophysistoxins (DTX) and pectenotoxins (PTX) produced by the dinoflagellates Dinophysis spp. can accumulate in shellfish and cause diarrhetic shellfish poisoning upon human consumption. Shellfish toxicity is a result of algal abundance and toxicity as well as accumulation and depuration kinetics in mussels. We mass-cultured Dinophysis acuta containing OA, DTX-1b and PTX-2 and fed it to the blue mussel, Mytilus edulis under controlled laboratory conditions for a week to study toxin accumulation and transformation. Contents of OA and DTX-1b in mussels increased linearly with incubation time, and the net toxin accumulation was 66% and 71% for OA and DTX-1b, respectively. Large proportions (≈50%) of both these toxins were transformed to fatty acid esters. Most PTX-2 was transformed to PTX-2 seco-acid and net accumulation was initially high, but decreased progressively throughout the experiment, likely due to esterification and loss of detectability. We also quantified depuration during the subsequent four days and found half-life times of 5–6 days for OA and DTX-1b. Measurements of dissolved toxins revealed that depuration was achieved through excreting rather than metabolizing toxins. This is the first study to construct a full mass balance of DSP toxins during both accumulation and depuration, and we demonstrate rapid toxin accumulation in mussels at realistic in situ levels of Dinophysis. Applying the observed accumulation and depuration kinetics, we model mussel toxicity, and demonstrate that a concentration of only 75 Dinophysis cells l−1 is enough to make 60 mm long mussels exceed the regulatory threshold for OA equivalents