Matrix effects on a cell-based assay used for the detection of paralytic shellfish toxins in bivalve shellfish samples.

Detecting marine biotoxins such as paralytic shellfish toxins (PSTs) is essential to ensuring the safety of seafood. The mouse bioassay is the internationally accepted method for monitoring PSTs, but technical and ethical issues have led to a search for new detection methods. The mouse neuroblastoma cell-based assay (Neuro-2a CBA) using ouabain and veratridine (O/V) has proven useful for the detection of PSTs. However, CBAs are sensitive to shellfish-associated matrix interferences. As the extraction method highly influences matrix interferences, this study compared three extraction protocols: Association of Official Analytical Chemists (AOAC) 2005.06, AOAC 2011.02 and an alternative liquid-liquid method. These methods were used to assess the matrix effect of extracts from four commercially important bivalve species (Chilean mussel, Magellan mussel, clam and Pacific oyster) in Neuro-2a CBA. Extracts from all three protocols caused a toxic effect in Neuro-2a cells (without O/V) when tested at a concentration of 25 mg of tissue-equivalent (TE) ml(-1). The greatest toxicity was obtained through the AOAC 2011.02 protocol, especially for the Chilean mussel and Pacific oyster extracts. Similar toxicity levels (less than 15%) were observed in all extracts at 3.1 mg TE ml(-1). When assessed in Neuro-2a CBA, AOAC 2005.06 extracts presented the lowest matrix interferences, while the highest interferences were observed for AOAC 2011.02 in Magellan mussel and clam extracts. Finally, the AOAC 2005.06 and alternative protocols were compared using Chilean mussel samples fortified with 40 and 80 µg STX per 100 g meat. The AOAC 2005.06 method demonstrated better results. In conclusion, the AOAC 2005.06 extracts exhibited the fewest interferences in the Neuro-2a CBA. Therefore, this extraction method should be considered for the implementation of Neuro-2a CBA as a high-throughput screening methodology for PST detection.

Differences in the toxin profiles of Alexandrium ostenfeldii (Dinophyceae) strains isolated from different geographic origins: Evidence of paralytic toxin, spirolide, and gymnodimine.

Among toxin-producing dinoflagellates of the genus Alexandrium, Alexandrium ostenfeldii is the only species able to produce paralytic shellfish poisoning (PSP) toxins, spirolides (SPXs) and gymnodimines (GYMs). In this study we characterized and compared three A. ostenfeldii strains isolated from the Baltic, Mediterranean, and southern Chile Seas with respect to their toxin profiles, morphology, and phylogeny. Toxin analyses by HPLC-FD and LC-HRMS revealed differences in the toxin profiles of the three strains. The PSP toxin profiles of the southern Chile and Baltic strains were largely the same and included gonyautoxin (GTX)-3, GTX-2, and saxitoxin (STX), although the total PSP toxin content of the Chilean strain (105.83 ± 72.15 pg cell(-1)) was much higher than that of the Baltic strain (4.04 ± 1.93 pg cell(-1)). However, the Baltic strain was the only strain that expressed detectable amounts of analogues of GYM-A and GYM-B/-C (48.27 ± 26.12 pg GYM-A equivalents cell(-1)). The only toxin expressed by the Mediterranean strain was 13-desmethyl SPX-C (13dMeC; 2.85 ± 4.76 pg cell(-1)). Phylogenetic analysis based on the LSU rRNA showed that the studied strains belonged to distinct molecular clades. The toxin profiles determined in this study provide further evidence of the taxonomic complexity of this species.