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Saxitoxin and its derivatives, the paralytic shellfish toxins (PSTs), are well known to be toxic to humans, and maximum permitted levels in seafood have been established by regulatory authorities in many countries. Monitoring of PSTs is typically performed using chemical methods which quantify the concentration of the individual PST analogues, of which there are many. However, since the toxicities of analogues are different, they do not equally contribute to the overall toxicity of the sample. To account for these differences, toxicity equivalency factors (TEFs) need to be determined for each analogue and applied. Currently there are no established TEFs for decarbamoyl gonyautoxin 1&4 (dcGTX1&4), which occurs in some clam species such as Mactra chinensis contaminated with PSTs due to metabolism within the shellfish. In this study the median lethal dose of purified, equilibrated epimeric mixture of dcGTX1&4 has been determined by intraperitoneal injection (i.p.) (4.75 μmol/kg) and by feeding (34.9 μmol/kg). The most relevant route of exposure is orally with feeding being more representative of human consumption and more reliable than gavage. Based on the median lethal dose by feeding, a TEF of 0.1 is recommended for dcGTX1&4. Receptor binding activity and i.p. toxicity results showed dcGTX1&4 to be much less toxic than STX (140–170-fold). However, by feeding a much smaller difference in toxicity was observed with dcGTX1&4 being only 11-fold less toxic than STX. Analysis of the gut contents of mice dosed with dcGTX1&4 showed the presence of decarbamoyl gonyautoxin 2&3, decarbamoyl saxitoxin and decarbamoyl neosaxitoxin, all of which are of greater toxicity. This conversion of dcGTX1&4 within the digestive track to more toxic congeners may explain the high relative toxicity of dcGTX1&4 by feeding compared to that determined by i.p. and by sodium channel activity.

For the first time wild-caught Tasmanian abalone, Haliotis rubra, have been reported to contain paralytic shellfish toxins (PSTs). This observation followed blooms of the toxic dinoflagellate Gymnodinium catenatum. No illnesses were reported, but harvesting restrictions were enforced in commercial areas. Abalone were assayed using HPLC-FLD methodology based on AOAC official method 2005.06. An uncommon congener, deoxydecarbamoyl-STX (doSTX), was observed in addition to regulated PSTs as unassigned chromatographic peaks. A quantitative reference material was prepared from contaminated Tasmanian abalone viscera and ampouled at 54.2 μmol/L. The LD50 of doSTX via intraperitoneal injection was 1069 nmol/kg (95% confidence limits 983-1100 nmol/kg), indicating it is nearly 40 times less toxic than STX. A toxicity equivalence factor of 0.042 was generated using the mouse bioassay. Levels of PSTs varied among individuals from the same site, although the toxin profile remained relatively consistent. In the foot tissue, STX, decarbamoyl-STX and doSTX were identified. On a molar basis doSTX was the dominant congener in both foot and viscera samples. The viscera toxin profile was more complex, with other less toxic PST congeners observed and was similar to mussels from the same site. This finding implicates localised dinoflagellate blooms as the PST source in Tasmanian abalone.

A toxic dinoflagellate, Karenia brevisulcata, devastated almost all marine life in Wellington Harbour, New Zealand during the late summer of 1998. Brevisulcatic acids (BSXs) and brevisulcenals (KBTs), both polycyclic ether toxins, have been identified as the causative agents. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the sensitive and specific determination of BSXs and KBTs in culture medium, seawater and shellfish. Acidified algal culture, or seawater, was extracted using reverse phase solid phase extraction cartridges. Shellfish tissue homogenate was blended with methanol-water (9:1) and partitioned with hexane to remove non-polar lipids. This extraction protocol is similar to that used for analysis of lipophilic shellfish toxins. LC-MS/MS (triple quadrupole) was used for quantitative analysis with gradient elution (acidic buffer), positive electrospray ionization and multiple-reaction monitoring. Purified toxins were available for 4 KBTs (KBT-F, -G, -H and -I) and 4 BSXs (-1, -2, -4, and -5), and were used to calibrate the instrument responses. Relative response factors were used for semi-quantitative analysis of BSX-3 and BSX-6, using BSX-1 and BSX-4 respectively. Calibration curves for all toxins monitored were linear over the concentration range tested (5-200 ng mL(-1)) with r(2) values >0.99. The method limit of quantitation was determined to be 2 ng mL(-1) for BSXs and KBTs, except KBT-I, which was 5 ng mL(-1). Validation data was generated for culture medium and shellfish. Toxin recoveries were typically >70% with relative standard deviations

Palytoxin is a highly toxic non-proteinaceous marine natural product that can pass through the food chain and result in human illnesses. A recent review by the European Food Safety Authority concluded that palytoxin requires regulation in seafood and a limit of 30 μg kg⁻¹ for shellfish flesh was suggested. Current methods based on LC-MS detection of intact palytoxins do not have sufficient sensitivity to enforce this limit for palytoxin. To improve sensitivity for trace analysis, a novel screen approach has been developed that uses LC-MS/MS analysis of substructures generated by oxidative cleavage of vicinal diol groups present in the intact toxin. Oxidation of palytoxins, ovatoxins or ostreocins using periodic acid generates two nitrogen-containing aldehyde fragments; an amino aldehyde common to these toxins, and an amide aldehyde that may vary depending on toxin type. Conditions for micro-scale oxidation of palytoxin were optimised, which include a novel SPE cleanup and on-column oxidation step. Rapid analysis of cleavage fragments was established using LC-MS/MS. Linear calibrations were established for the amino aldehyde from a palytoxin reference standard, which is suitable for all known palytoxin-like compounds, and for the confirmatory amide aldehydes of palytoxin and ostreocin-D. Palytoxin recoveries (at 10 μg kg⁻¹) from shellfish and fish tissues were 114-119% (as amine aldehyde) and 90-115% (as amide aldehyde) with RSDs for both of ≤ 18% (all tissues, n = 12). The method LOD was determined to be approximately 1 ng mL⁻¹ and the LOQ 4 ng mL⁻¹, which corresponds to 10 μg kg⁻¹ in tissue (flesh of shellfish or fish). The method has potential for use in research and is sufficiently sensitive for regulatory testing, should it be required.