The presence of marine toxins in shellfish products could lead to fatal food poisoning. Monitoring programs to control levels of toxins establish monthly or even weekly sampling in production areas. Opening or closure of harvest areas would depend upon the results obtained. Post-harvest and end product testing is also carried out in some countries. Currently, paralytic (saxitoxins), ammesic (domoic acid) and lipophilic toxins (okadaic acid and derivatives, yessotoxins, pectenotoxins and azaspirazids) are legislated.
Mouse bioassay (MBA) has been used for long time for monitoring lipophilic marine toxins. However, concerns about the ethical problems and analytical results obtained were raised some years ago among different stakeholders.
Three mice per sample are injected with a solvent solution and the death of 2 out of 3 mice in 24 h determines the presence of lipophilic toxins above the regulatory limits (160 micrograms per kilogram of okadaic acid, derivatives and pectenotoxins together).
Social awareness and legal requirements to avoid animal experiments together with the proven unreliable results produced by mouse assay have lead to ban this method for monitoring of lipophilic marine toxins. In 2011 Commission Regulation (EU) No. 15/2011 established the liquid chromatography mass spectrometry as reference method. Member Estates must replace the mouse assay by the current reference method or any other method complying with the legislation before the 31st of December 2014.
The European legislation allows several analytical methods for monitoring of lipophilic toxins providing they fulfil the method performance criteria stipulated by the EU-RL (European Reference laboratory) and offer the same level of public health.
OkaTest, a phosphatase inhibition assay, fulfils the requirements of Chapter III A (4) a, b, and c of Annex III to Regulation (EC) 2074/2005 and can be used for the detection of Okadaic Acid group toxins OA, DTX1, DTX2, DTX3 including their esters. METHOD FOR DETECTION OF OKADAIC ACID TOXINS GROUP IN EDIBLE MOLLUSCS – click here.
OkaTest is an enzymatic kit for quantitative determination of Okadaic Acid (OA) and other carboxylic toxins of the OA group including DTX1, DTX2 and DTX3 by a colorimetric phosphatase inhibition assay. This method is applicable to shellfish species such as mussels, clams, oysters and scallops. More info here.
The toxicity of Okadaic Acid and DTXs is directly related to their inhibitory activity against a family of structurally related serine/threonine protein phosphatases (PP), in particular PP1 and PP2A. This strong inhibitory property is used to determine Okadaic Acid content in shellfish by means of a microplate assay, using the enzyme PP2A and a chromogenic substrate for this enzyme. The enzyme hydrolyses the substrate and the product can be measured by an absorbance measurement at 405 nm using a microplate reader. As the ability of the PP2A to hydrolyse the substrate depends on the presence of Okadaic Acid and analogues in the samples, the toxin concentration can be calculated by using a standard curve.The kit includes five standards and has a working range from 63 µg/kg to 352 µg/kg. The LOD and LOQ are 44 µg/kg and 56 µg/kg, respectively.
OkaTest has been single and inter-laboratory validated (Smienk et al. Toxins 2012 and Smienk. et al. 2013, JAOAC, 96,1,77-85).
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