Bioassay techniques validation

There are literally hundreds of publications that, directly or indirectly, have contributed to the development, validation and refinement of bioassay techniques both for liquid and solid media assessment. These papers incorporate initiatives that... 

Electrochemical methods have been used for determinations of species of elements in natural waters. Of the many electrochemical techniques, only a few have proved to be useful for studies of speciation in complex samples, and to possess the sensitivity required for environmental applications. The greatest concern is the measurement of the toxic fraction of a metal in an aqueous sample. The definition of a toxic fraction of a metal is that fraction of the total dissolved metal concentration that is recognised as toxic by an aquatic organism. Toxicity is measured by means of bioassays. Elowever, a universally applicable bioassay procedure cannot be adopted because the responses of different aquatic species to metal species vary. Nevertheless, bioassays should be used as means of evaluation and validation of speciation methods. A condition is that the test species (of the bioassay) should be very sensitive to the metals being studied so as to simulate a worst case situation (Florence, 1992). 

An in vitro bioassay can be designed in several ways, but requires statistical validity. A one point assay is not valid. The bioassay should be designed to consider factors that introduce variability, and the analysis should test such variability. A measurement series of a test sample should be compared to an equivalent series of the reference material, carefully considering the comparisons between the linear portions of the dose-response curves (Mire-Sluis et al., 1996). To test validity of a bioassay inter- and intra-assay variability should be considered in both preparation, and in the case of multiwell plates, the variability between each plate. To reduce the positional effect in plate tests, it is advisable to distribute the points on the curves randomly and also to include a reference standard in each plate (Gaines-Das and Meager, 1995). One of the most widely used techniques to validate a bioassay s performance is to include internal duplicates. The data arising from the comparison can be important in assessing the test s variability. 

The results have shown that we have developed a sensitive and specific ELISA assay for the analysis of clomazone residues from soil samples. The procedures have demonstrated good recovery of clomazone from soil, and excellent correlation of the ELISA test results with standard GLC methodology. In addition, the results of the ELISA tests demonstrate good correlation between the observed soil levels of clomazone, and crop injury when the bioassay is performed under controlled, greenhouse conditions. This assay could, therefore, be used as a more rapid and convenient analytical method over the standard GLC technique after further validation. 

After the library has been synthesized, the product analysis must be completed to validate the library members. During the development of HTOS technologies, two philosophies have developed regarding the characterization of products. One view is that the components of a library need not be analyzed. The compounds are synthesized, then screened for activity in a bioassay. If a sample shows activity, it can be resynthesized and fully characterized using traditional techniques. This methodology provides the most effective way to screen large numbers of compounds. 

Over the past few years, the LC-MS and LC-MS/MS techniques have been successfully applied to the quantification and identification of YTXs. Several drafts for methods have already been published, which would facilitate immediate replacement of the mouse bioassay. However, Decision 2002/225/EC (Annex) requires validation of alternative methods by use of reference materials for all the individual compounds specified. So, the lack of pure reference samples of most YTXs prevents the substitution of the mouse bioassay as the routine or reference method for years to come. 

We can therefore conclude that the three different techniques should be available to laboratories involved in arachidonic acid metabolites studies RIA for routine assays. Bioassay to measure unstable compounds and to reveale the presence of new active metabolites and Mass Fragmentography to validate the other assays and to develop in reasonable time quantitative assays for these new compounds. 

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