In the analysis of ecotoxicological bioassays the concentration of the test compounds is assumed to be constant. In many situations this assumption is questionable, as various processes may cause a substantial decline in the concentration during exposure. This leads to difficulties in the estimation of parameters that characterise the toxicity of the test compound. As a solution to this problem, time-varying concentrations are often replaced by their mean values for the estimation of toxicity parameters. However, Monte-Carlo simulations show that this approach results in biased estimates of the toxicity parameters. As an alternative approach, we propose models to estimate one important toxicity parameter, the no effect concentration, in situations where the concentration of the compound varies in time. These models are extensions of the DEBtox model (Kooijman and Bedaux, 1996) which is based on biological assumptions about toxicokinetics and toxic effects. We also propose a new approach for the estimation of toxicity parameters for strongly accumulating non-metabolisable compounds. This approach does not require any kinetics assumption. Computer simulation and experimental data confirm the relevance of the different proposals. © Elsevier Science Ltd. All rights reserved.