First detection of azaspiracid-2 in shellfish from the Croatian coast of the Adriatic Sea

Due to the ubiquitous anthropogenic and climatic changes altering the marine habitat and ecology of biotoxin producers, the aim of this study was to assess the risk of human consumption associated with the accumulation of lipophilic toxins in the commercially important bivalve mollusc (mussel Mytilus galloprovincialis Lamarck, 1819) in the Krka River estuary located in the central part of the Croatian Adriatic Sea coast. Shellfish samples were collected weekly at three sampling stations during 2024, with a focus on monitoring lipophilic biotoxins. This study confirmed the first occurrence of the azaspiracid biotoxin, namely azaspiracid-2 (AZA-2), in shellfish from the Croatian part of the eastern Adriatic Sea coast. However, the toxicity only occurred in the first five months of the investigated year, as no AZA-2 toxin could be detected in shellfish sampled after May. Shellfish soft tissue samples were subjected to liquid chromatography–mass spectrometry (LC–MS) analysis to determine the presence of okadaic acid, dinophysistoxins, pectenotoxins, azaspiracids, yessotoxins and spirolides. The presence of lipophilic toxins in the samples was confirmed by comparing the retention times in the chromatograms and the fragmentation spectra with those of certified reference materials from the National Research Council, Canada. In particular, levels of azaspiracid-2 in the range of 0.03 -146.90 µg/kg were determined. The highest AZA-2 concentrations were found in the January samples. Thereafter, the concentrations showed a decreasing trend until the end of May, when they were no longer detected for the rest of the year. The concentration of this toxin was below the maximum permitted level in all samples in accordance with the EU regulation. Azaspiracids (AZAs) are a group of polyether compounds with a spirocyclic structure that can cause symptoms such as nausea, vomiting, diarrhoea and stomach cramps in humans. This is the first report on the occurrence of AZA-2 in the Croatian part of the Adriatic Sea and proves that the occurrence of lipophilic biotoxins needs to be further investigated and monitored in order to protect public health, but also with regard to aquaculture activities and their socio-economic benefits.