Miniaturisation of the Daphnia magna immobilisation assay for the reliabletesting of low volume samples

International standard test guidelines for the ecotoxicological characterisation of various substances use organisms like algae, daphnids and fish embryos. These guidelines use relatively high volumes of water for the process of testing. However, for various samples such as extracts from environmental monitoring or leachates from microplastic aging experiments, the amount of available sample volume is limited. Using the exposure volumes as recommended in test guidelines would not allow to test a range of different concentrations or to repeat tests. Lower media volumes would allow the testing of more samples (more concentrations per sample, more test repetitions for statistical robustness) but it may also decrease the possible number of organisms tested in the same volume. Here, we aimed at reducing the test volumes in the acute daphnia assay without impacting animals’ sensitivity towards toxicants. A literature review on existing miniaturisation approaches was used as a starting point. Subsequently, assays employing conventional as well as reduced test volumes were compared for 15 selected test substances with a diverse spectrum of lipophilicity. Results showed that there are differences in EC 50 between the two approaches, but that these differences were overall only within a range of a factor of two to three. Further, by retrieving EC50 values for the genus Daphnia and 15 test substances from the US EPA database, we demonstrated that our results are well inline with the general differences in sensitivities.