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Research Project
Emergent toxins on the Portuguese coast: occurrence, transfer kinetics and toxicity
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New vectors of TTX analogues in the North Atlantic Coast: the edible crabs Afruca tangeri and Carcinus maenas
Publication . Lage, Sandra; ten Brink, Felicitas; Canário, Adelino V. M.; Silva, José Paulo da
Tetrodotoxin (TTX) and its analogues are naturally occurring toxins historically responsible for human poisoning fatalities in Eastern Asia. It is typically linked to the consumption of pufferfish and, to a lesser extent, marine gastropods and crabs. In the scope of a comprehensive project to understand the prevalence of emergent toxins in edible marine organisms, we report, for the first time, the detection of TTX analogues in the soft tissues of edible crabs, the European fiddler crab (Afruca tangeri) and green crab (Carcinus maenas), harvested in southern Portugal. No TTX was detected in the analyzed samples. However, three TTX analogues were detected—an unknown TTX epimer, deoxyTTX, and trideoxyTTX. These three analogues were found in the European fiddler crab while only trideoxyTTX was found in the green crab, suggesting that the accumulation of TTX analogues might be influenced by the crabs’ different feeding ecology. These results highlight the need to widely monitor TTX and its analogues in edible marine species in order to provide adequate information to the European Food Safety Authority and to protect consumers.
Metabolomic and taxonomic characterization of Haloleptolyngbya lusitanica sp. nov . (Cyanobacteria, Synechococcales)
Publication . Cordeiro, Rita; Luz, Rúben; Lage, Sandra; Menezes, Carina; Dias, Elsa; Flores, Cintia; Fonseca, Amélia; Gonçalves, Vítor
The morphological plasticity of cyanobacteria and their widespread ecological dominance in a wide range of habitats highlights the need for in-depth taxonomic studies. This work focused on the taxonomical revision of Leptolyngbya (Cyanophyceae) strains deposited in the ESSACC culture collection and their metabolomic characterization. Although the studied ESSACC strains were morphologically identified as Leptolyngbya sp., the 16S rRNA gene and 16S-23S rRNA ITS analysis revealed that two strains (LMECYA 079 and LMECYA 173) belong to Haloleptolyngbya and represent a new taxonomical unit, genetically unique, ecologically plastic and adapted to both freshwater and thermal habitats, here described as Haloleptolyngbya lusitanica sp. nov. To perform a suspect screening of cyanometabolites in these strains, we used a non-targeted liquid chromatography-high resolution mass spectrometry (LC-HRMS) metabolomic approach. Several metabolites were identified in Haloleptolyngbya lusitanica: micropeptin MM978, spumigin 640, oscillatoxin A and anabaenopeptin D. Strains were maintained and grown under the same conditions, revealing the common production of oscillatoxin A by both H. lusitanica strains. Other identified metabolites, however, were strain-specific, such as anabaenoptin D, which was only detected in LMECYA 173. The different cyanometabolite profiles reinforce the notion that cyanobacteria have the ability to adapt to different habitats, which is maintained under long-term culturing conditions.
Quantitation overcoming Matrix effects of Lipophilic toxins in Mytilus galloprovincialis by liquid chromatography-full scan high resolution mass spectrometry analysis (LC-HR-MS)
Publication . Costa, Camila Q. V. da; Afonso, Inês I.; Lage, Sandra; Costa, Pedro Reis; Canario, Adelino; Silva, José Paulo da
The analysis of marine lipophilic toxins in shellfish products still represents a challenging task due to the complexity and diversity of the sample matrix. Liquid chromatography coupled with mass spectrometry (LC-MS) is the technique of choice for accurate quantitative measurements in complex samples. By combining unambiguous identification with the high selectivity of tandem MS, it provides the required high sensitivity and specificity. However, LC-MS is prone to matrix effects (ME) that need to be evaluated during the development and validation of methods. Furthermore, the large sample-to-sample variability, even between samples of the same species and geographic origin, needs a procedure to evaluate and control ME continuously. Here, we analyzed the toxins okadaic acid (OA), dinophysistoxins (DTX-1 and DTX-2), pectenotoxin (PTX-2), yessotoxin (YTX) and azaspiracid-1 (AZA-1). Samples were mussels (Mytilus galloprovincialis), both fresh and processed, and a toxin-free mussel reference material. We developed an accurate mass-extracted ion chromatogram (AM-XIC) based quantitation method using an Orbitrap instrument, evaluated the ME for different types and extracts of mussel samples, characterized the main compounds co-eluting with the targeted molecules and quantified toxins in samples by following a standard addition method (SAM). An AM-XIC based quantitation of lipophilic toxins in mussel samples using high resolution and accuracy full scan profiles (LC-HR-MS) is a good alternative to multi reaction monitoring (MRM) for instruments with HR capabilities. ME depend on the starting sample matrix and the sample preparation. ME are particularly strong for OA and related toxins, showing values below 50% for fresh mussel samples. Results for other toxins (AZA-1, YTX and PTX-2) are between 75% and 110%. ME in unknown matrices can be evaluated by comparing their full scan LC-HR-MS profiles with those of known samples with known ME. ME can be corrected by following SAM with AM-XIC quantitation if necessary.
No β-N-Methylamino-L-alanine (BMAA) was detected in stranded cetaceans from Galicia (North-West Spain)
Publication . Soliño, Lucia; Kim, Sea-Yong; López, Alfredo; Covelo, Pablo; Rydberg, Sara; Reis Costa, Pedro; Lage, Sandra
The neurotoxin β-N-methylamino-L-alanine (BMAA), a non-proteinogenic amino acid produced by several species of both prokaryotic (cyanobacteria) and eukaryotic (diatoms) microorganisms, has been proposed to be associated with the development of neurodegenerative diseases. At first, BMAA appeared to be ubiquitously present worldwide in various organisms, from aquatic and terrestrial food webs. However, recent studies, using detection methods based on mass spectrometry, instead of fluorescence detection, suggest that the trophic transfer of BMAA is debatable. This study evaluated BMAA in 22 cetaceans of three different species (Phocoena phocoena, n = 8, Delphinus delphis, n = 8, and Tursiops truncatus, n = 6), found stranded in North-West Spain. BMAA analysis of the liver, kidney, or muscle tissues via sensitive liquid chromatography with tandem mass spectrometry did not reveal the presence of this compound or its isomers. The absence recorded in this study highlights the need to better understand the trophic transfer of BMAA and its anatomical distribution in marine mammals.
Tissue accumulation of tetrodotoxin (TTX) and analogues in trumpet shell Charonia lampas
Publication . Lage, Sandra; Afonso, Inês I.; Reis Costa, Pedro; Canario, A.V.M.; Da Silva, José Paulo
Tetrodotoxin (TTX) is a potent neurotoxin responsible for a human intoxication event in Spain associated with the consumption of trumpet shell Charonia lampas. In Europe, TTX is not regulated or monitored, and there is little knowledge about its presence in seafood. Here, we investigated the tissue distribution of TTX and analogues in three specimens of trumpet shell C. lampas bought in a market in southern Portugal. Toxin concentration was above the EFSA recommended limit in the non-edible tissues of all specimens and within the limit in the edible tissues of two specimens. 4,9-AnhydroTTX and 13 additional TTX analogues were detected in tissues, the most abundant being anhydrotrideoxyTTX and trideoxyTTX. These results suggest that although thorough evisceration may lower the amount of TTX consumed, it may not be sufficient to ensure consumer safety. Regular monitoring of TTX and analogues in trumpet shell and other edible gastropods is therefore recommended to avoid poisoning incidents.
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Funding agency
European Commission
Funding programme
H2020
Funding Award Number
101003376