Replacement of the mouse bioassay: Development of SPE and LC-MS for detection of paralytic shellfish poisoning toxins

Bivalve molluscs such as mussels, oysters and scallops feed by filtering algae from the water. Some algae are toxic, therefore at times the molluscs consume toxins as well as nutrients. The toxins are seemingly harmless to the shellfish but toxic to organisms further up the food chain including humans. The most prevalent of the shellfish poisoning syndromes caused by bioaccumulated toxins is paralytic shellfish poisoning (PSP) with instances occurring world-wide. Each year in the UK levels of PSP toxins which exceed the legal limit (80 µg STX equivalents per 100g of flesh) are detected and concentrations greater than those quoted as causing illness/death (approx. 300 µg STX equivalents per 100g of flesh) are found. Current EC legislation (Directive 91/492/EEC) requires Competent Authorities to monitor shellfish for the presence of these toxins. Currently, the regulatory method for monitoring PSP toxins in shellfish is the AOAC mouse bioassay, which involves the extraction of shellfish meat in heated, acidified water followed by injection of an aliquot of the extract intraperitoneally into mice. If PSP toxins are present, the death time of the mice is the means of quantification. A particularly contentious issue is the use of death as an end-point as it causes considerable animal suffering. Therefore, for ethical reasons, there now exists considerable pressure to replace this test in the UK and across Europe. As such it is the objective of this project, if funded, to develop analytical tests in the form of solid phase extraction (SPE) with liquid chromatography and mass spectrometry (LC-MS) for the detection and quantification of PSP toxins in shellfish. LC-MS detection methods, which currently exist for PSP toxins in shellfish are not sufficiently sensitive, therefore this proposal will aim to develop a procedure, which increases sensitivity through improved sample preparation and optimisation of detection techniques. This will initially involve investigations into SPE and chromatographic techniques with subsequent validation of an optimised quantitative method. It is envisaged that these methods can replace the mouse bioassay in the UK (and potentially EU) shellfish monitoring programmes.