In vitro methodologies in ecotoxicological hazard assessment: the case of bioaccumulation testing for fish.

Worldwide programmes for the regulation of chemicals require an assessment of the risks of chemicals to human and environmental health based on three categories of concern: Persistence, Bioaccumulation and Toxicity (PBT). Among these three categories, bioaccumulation refers to the enrichment of environmental chemicals in organisms. It encompasses the absorption, distribution, metabolism and excretion (ADME) of a chemical inside an organism, and ultimately determines the internal toxic dose. For the aquatic environment, the most widely used parameter to estimate the bioaccumulation potential of a chemical is the so-called Bioconcentration Factor (BCF). The BCF represents the ratio of the steadystate chemical concentration in the organism and the chemical concentration in the respiratory medium, i.e. water. For the experimental determination of the BCF, the test procedure as described in OECD Test Guideline 3051 represents the current ‘gold standard’. In this test, fish are exposed to a chemical for 28 days, to reach an equilibrium of chemical concentration between fish and water, followed by a 28-day depuration period to measure the elimination rate. This test, in addition to being lengthy and costly, requires a high number of animals (> 100 fish per test). Regulatory programmes require bioaccumulation information for chemicals which are lipophilic (for example, those with a log Kow > 3), and which are produced at a certain tonnage (for example, the European Community REACH Regulation requires BCF information for lipophilic chemicals that are produced at > 100 tonnes per year). Experi mentally determined BCF data are not available for the vast majority of existing compounds. For instance, in a Canadian investigation of 23,000 existing chemicals, it was found that bioaccumulation data existed for less than 4% of them (cf. Nichols et al.2). If the missing BCF data had to be generated by means of the OECD 305 test, this would entail a drastic increase in animal use.3,4 Therefore, there is an urgent need to develop alternative methods to reduce the number of fish used for in vivo bioaccumulation testing. The bioconcentration of chemicals in fish results from the competing rates of chemical uptake via the gills and skin (k1) and chemical elimination via respiratory exchange (k2), faecal egestion (ke) and metabolic biotransformation (km). 5 In addition, dilution as a result of growth (kd) can influence bioconcentration. With the involvement of these different processes, it is clear that non-animal approaches to bioconcentration assessment cannot be based on one single method, but have to rely on an array of methodologies.2,4,6 An initial non-animal based approximation of the bioconcentration potential of an organic chemical in aquatic organisms can be obtained from an in silico hydrophobicity model, which considers bioconcentration as a passive partitioning process resulting from the competing uptake and elimination processes. In this model, bioconcentration can be predicted from the lipophilicity of a chemical, as estimated from its octanol–water partition coefficient, Kow. 5 Also, it can actually be measured by using artificial membranes which simulate the passive diffusion processes across the respiratory epithelia.7