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- LC-HRMS profiling of paralytic shellfish toxins in Mytilus galloprovincialis after a Gymnodinium catenatum bloomPublication . Lage, Sandra; Reis Costa, Pedro; Canario, Adelino; Da Silva, José PauloSaxitoxin and its more than 50 analogues are a group of naturally occurring neurotoxins collectively designated as paralytic shellfish toxins (PSTs). PSTs are toxic to humans and maximum legal limits in seafood have been implemented by regulatory authorities worldwide. In the European Union, monitoring of PSTs is performed using the AOAC Official Method 2005.06, based on liquid chromatography coupled with fluorescence detection (LC- FLD). However, this method has been suggested to not effectively detect the emerging C-11 hydroxyl (M-toxins) and benzoate (GC-toxins) analogues, with these analogues currently not being surveyed in monitoring programs. In this study, a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was used to search for these emerging PSTs in mussels, Mytilus galloprovincialis, contaminated following an intense Gymnodinium catenatum bloom in the Tagus estuary (Lisbon, Portugal). Five M-toxins (M1, M2, M6, dcM6, and dcM10), but no GC-toxins, were detected in the mussels’ whole-soft body tissue. Moreover, the classical PSTs (C1 to C4, GTX 4 to GTX6, dcGTX1 to dcGTX4, dcSTX, dcNEO, and STX) were also found and comprised the largest fraction of the PSTs’ profile. The presence of unregulated PSTs in edible mussel samples suggests potential seafood safety risks and urges further research to determine the frequency of these analogues in seafood and their contribution to toxicity
- 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 daThe 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.
- New vectors of TTX analogues in the North Atlantic Coast: the edible crabs Afruca tangeri and Carcinus maenasPublication . Lage, Sandra; ten Brink, Felicitas; Canário, Adelino V. M.; Silva, José Paulo daTetrodotoxin (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.
- Chemical composition and species identification of microalgal biomass grown at pilot-scale with municipal wastewater and CO2 from flue gasesPublication . Lage, Sandra; Gentili, Francesco G.The production potential of a locally isolated Chlorella vulgaris strain and a local green-algae consortium, used in municipal wastewater treatment combined with CO2 sequestration from flue gases, was evaluated for the first time by comparing the elemental and biochemical composition and heating value of the biomass produced. The microalgae were grown in outdoor pilot-scale ponds under subarctic summer conditions. The impact of culti-vation in a greenhouse climate was also tested for the green-algae consortium; additionally, the variation in species composition over time in the three ponds was investigated. Our results showed that the biomass produced in the consortium/outdoor pond had the greatest potential for bioenergy production because both its carbohy-drates and lipids contents were significantly higher than the biomasses from the consortium/greenhouse and C. vulgaris/outdoor ponds. Although greenhouse conditions significantly increased the consortium biomass's monounsaturated fatty acid content, which is ideal for biodiesel production, an undesirable increase in ash and chemical elements, as well as a reduction in heating value, were also observed. Thus, the placement of the pond inside a greenhouse did not improve the production potential of the green-algae consortium biomass in the current study infrastructure and climate conditions.
- 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, SandraThe 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.
- Gymnodinium catenatum paralytic Shellfish toxin production and photobiological responses under marine heat wavesPublication . Lopes, Vanessa M.; Court, Mélanie; Seco, Martim Costa; Borges, Francisco O.; Vicente, Bernardo; Lage, Sandra; Braga, Ana Catarina; Duarte, Bernardo; Santos, Catarina Frazão; Amorim, Ana; Reis Costa, Pedro; Rosa, RuiMarine heatwaves (MHWs) have doubled in frequency since the 1980s and are projected to be exacerbated during this century. MHWs have been shown to trigger harmful algal blooms (HABs), with severe consequences to marine life and human populations. Within this context, this study aims to understand, for the first time, how MHWs impact key biological and toxicological parameters of the paralytic shellfish toxin (PST) producer Gymnodinium catenatum, a dinoflagellate inhabiting temperate and tropical coastal waters. Two MHW were simulated—category I (i.e., peak: 19.9 ◦C) and category IV (i.e., peak: 24.1 ◦C)—relative to the estimated baseline in the western coast of Portugal (18.5 ◦C). No significant changes in abundance, size, and photosynthetic efficiency were observed among treatments. On the other hand, chain-formation was significantly reduced under category IV MHW, as was PSP toxicity and production of some PST compounds. Overall, this suggests that G. catenatum may have a high tolerance to MHWs. Nevertheless, some sublethal effects may have occurred since chain-formation was affected, suggesting that these growth conditions may be sub-optimal for this population. Our study suggests that the increase in frequency, intensity, and duration of MHWs may lead to reduced severity of G. catenatum blooms.
- 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, VítorThe 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.
- Tissue accumulation of tetrodotoxin (TTX) and analogues in trumpet shell Charonia lampasPublication . Lage, Sandra; Afonso, Inês I.; Reis Costa, Pedro; Canario, A.V.M.; Da Silva, José PauloTetrodotoxin (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.
- Tissue accumulation of tetrodotoxin (TTX) and analogues in trumpet shell Charonia lampasPublication . Afonso, Inês I.; Lage, Sandra; Reis Costa, Pedro; Canario, Adelino; Silva, José Paulo daTetrodotoxin (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.
- Effect of temperature on growth and yessotoxin production of protoceratium reticulatum and lingulodinium polyedra (Dinophyceae) isolates from the Portuguese coast (NE Atlantic)Publication . Barbosa, Miguel; Reis Costa, Pedro; David, Helena; Lage, Sandra; Amorim, AnaThe dinoflagellates Protoceratium reticulatum and Lingulodinium polyedra are potential yessotoxin (YTX) producers, which have been associated with blooms responsible for economic, social, and ecological impacts around the world. They occur in Iberian waters, but in this region, little is known of their ecophysiology and toxin profiles. This study investigated the growth and toxin production of two strains of each species, from the Portuguese coast, at 15 degrees C, 19 degrees C, and 23 degrees C. Growth curves showed higher growth rates at 19 degrees C, for both species. YTX and three analogs (homo YTX; 45-OH YTX; 45-OH homo YTX) were investigated by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), and the presence of other analogs was investigated by Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS). No evidence of toxin production was found in L. polyedra. By contrast, YTX and 45,55-diOH-YTX were detected in both strains of P. reticulatum. These results confirm P. reticulatum as a source of yessotoxins along the Portuguese coast and add to the observed high intraspecific variability on YTX production of both species, at a global scale.