In the first year of the ETOXPT project, as part of Work Package 1 objective, we developed methods to analyze various toxins, both regulated and emerging, using advanced techniques. These toxins included okadaic acid (OA), dinophysistoxins (DTX-1 and DTX-2), pectenotoxins (PTXs), yessotoxins (YTXs), azaspiracids (AZAs), amnesic shellfish toxin (AST), paralytic shellfish toxins (PSTs), tetrodotoxin (TTX), and β-N-methylamino-L-alanine (BMAA). With these methods, we could also identify related compounds (analogues) that don’t have commercial standards and whose toxicity is unknown. Developing these methods was essential for the project's success.
In Work Package 2, we began with a preliminary study that confirmed the presence of the emerging toxin TTX and its analogues (collectively named as TTXs) in the gastropod trumpet shell, Charonia lampas, found along the Algarve Coast. We discovered that contaminated trumpet shells had reached local markets. High concentrations of TTX in the digestive and excretory tissues suggested that these toxins accumulate through food ingestion.
These findings led us to conduct a year-long sampling campaign, working with local fishermen to collect trumpet shells and their primary prey, the starfish Astropecten aranciacus, both often caught as by-catch. We found consistently high TTX concentrations in trumpet shells throughout the year, with no seasonal variation. The toxins were mainly in gastrointestinal tissues but were also found in muscle tissue. Throughout the campaign, 60% of the gastrointestinal tissues of the trumpet shells had TTX levels above the European Food Safety Authority (EFSA) recommended limit of 44 µg TTX/kg. Proper cleaning (evisceration) of these organisms is essential to avoid poisoning, as TTXs are heat-stable and water-soluble, meaning they do not degrade with cooking or preservation. This is particularly important in the Algarve region, where trumpet shells are sold whole in markets and served as delicacies in restaurants. TTXs were also found in the starfish, but less frequently and in lower concentrations, supporting the idea that TTX accumulates in trumpet shells through their diet. Moreover, we discovered that edible crabs Afruca tangeri and Carcinus maenas, also found along the Algarve coast, contain TTX analogues. This indicates that TTXs are present in multiple marine species in the region. Therefore, it is advisable to implement TTX monitoring programs in Portugal, especially along the southern coast.
We also looked into the presence of BMAA, another toxin, in top marine predators in the Iberian Peninsula. According to bioaccumulation theory, high levels of BMAA would be expected in these predators. However, we did not find BMAA in 22 stranded cetaceans of three different species in North-West Spain, nor in 12 common gulls from Málaga, Spain, which had shown symptoms of a paralytic syndrome of unknown origin. This suggests that the presence of BMAA in these top predators is likely low.
In Work Package 3, we aimed to study how exudates from the macrophyte Zostera marina affect the profile of both classical (regulated and monitored) and emerging (M-toxins and GC-toxins) PST analogues from the dinoflagellate Alexandrium minutum. However, the samples will be analyzed at a later stage.
The results of the ETOXPT project were published in several open-access, peer-reviewed journals, which can be easily accessed on the project's website. We also presented our findings at 13 international and national conferences, including the ASLO Aquatic Sciences Meeting 2023, the SETAC Europe 34th Annual Meeting, the XIV and XV Reunião Ibérica sobre Microalgas Tóxicas e Biotoxinas Marinhas, and the Encontro Ciência 2024 + Science for Health and Global Well-being.In addition, we shared our results with 8th and 9th graders at schools in the Faro district through the Cientificamente Provável program.
