Physicochemical substance properties as indicators for unreliable exposure in microplate-based bioassays

In the last years many efforts were made to transform standardized algal test protocols into low-cost microplate assays. While advantages were pointed out frequently, limitations are not systematically addressed, thus hindering a widespread utilisation. In this study a group of organic substances with a wide distribution of volatility (logKAW from -6.53 to -2.13) and lipophilicity (logKOW from 1.26 to 4.92) was investigated with respect to the influence of these physicochemical properties on their algal toxicity in different assays. Therefore the EC50 values were determined with a microplate assay based on ISO 8692 protocol and the results were compared with those of an established algal growth inhibition test conducted in air tight glass vessels. Using the ratio of the EC50 values, a clear connection between biological response and volatility as well as lipophilicity of test substances could be detected. Chemicals with a logKOW higher than 3 or a Henry coefficient logKAW higher than -4 were identified as less effective in the microplate assay than in the comparative assay. The loss in nominal concentration due to physicochemical properties could be shown to contribute to this using HPLC analysis. Consequently, when using microplate assay's one should be aware that lipophilic and volatile chemicals might be underestimated in their toxicity, which could be indicated from evaluating related physicochemical properties modelled from structural information prior to an experimental investigation.