The challenge of replacing animals in shellfish toxin testing.
Tore Aune
Department of Pharmacology, Microbiology and Food Hygiene, Norwegian School of Veterinary Science, Oslo, Norway.
Shellfish feed on microalgae in seawater. Some algal species have the ability to produce toxins. These toxins do not harm the molluscs, and therefore, one cannot distinguish between toxic and wholesome mussels. Consequently, shellfish may pose a threat to consumers. To avoid this, seafood must undergo analysis to ensure tolerable levels of algal toxins before marketing. Mouse bioassays have traditionally been used to verify safe levels of the well known paralytic and diarrhetic shellfish toxins. Much effort has gone into studies of marine algal toxins in recent years. By combining biological, analytical chemical and in vitro methods, an increasing number of toxins have been discovered. The biological methods have proven their usefulness in detecting unknown toxins. On the other hand, one of the drawbacks associated with biological methods, aside from the very important ethical issue, is their lack of sensitivity and specificity. It has become increasingly clear that assays for shellfish toxins based on live animals should be replaced by alternative methods.
A large step in the right direction was taken in 2001, when an expert group suggested separate tolerance levels for each group of shellfish toxins, based on available toxicological data. For the first time, shellfish toxins were divided into groups, according to their mechanism of toxicity. The expert group strongly urged for production of reference materials and standards for the actual toxins, enabling international validation of alternative methods to the mouse bioassays.
In Europe, the directives will be changed according to the recent developments. All relevant toxin groups have to be analysed, and alternatives to the mouse bioassays will be accepted as soon as the validation process is successfully completed.
In Norway, alternatives to the mouse bioassays have been used in parallel to the biological methods for the last two years for all shellfish harvested for commercial marketing. In addition, two national surveillance programs have been in operation, with sampling stations scattered along the entire coastline. In the latter programs, the main methods have been based on analytical techniques (high pressure liquid chromatography and liquid chromatography-mass spectrometry). The correlation between the biological and the analytical methods is very good. Newly detected toxins have been included in the analytical methods, and it is obvious that the precision is far better with these methods, compared with the mouse bioassays. In other words, routine analysis of shellfish concerning marine algal toxins should be performed with analytical methods as soon as the methods are validated. In addition, a whole range of rapid test methods are being developed, and they may be used for pre-screening purposes. The biological methods should only be used for small scale screening towards unknown toxins.
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