Miniaturisation of the Daphnia magna immobilisation assay for the reliable testing of low volume samples.

Abstract

International standard test guidelines for the ecotoxicological characterisation of various substances use organisms such as algae, daphnids and fish embryos. These guidelines recommend or use relatively high volumes of water for the process of testing, for example, 200 mL for a complete dose-response relationship in a daphnia assay. However, for various samples such as concentrated extracts from environmental monitoring or leachates from microplastic ageing experiments, the amount of available sample volume is limited, that is, rather in the range of 10-50 mL/biotest. Using the exposure volumes as recommended in test guidelines would not allow to test a range of different concentrations or to repeat tests or use multiple different organismic bioassays. Lower media volumes would allow the testing of more samples (more concentrations per sample, more test repetitions for statistical robustness, etc.) 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 (using a maximum of 30 mL for a complete dose-response relationship) 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 16 selected test substances with a diverse spectrum of lipophilicity. Results showed that there are differences in EC₅₀ 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 16 test substances from the United States Environmental Protection Agency database, we demonstrated that our results are well in line with the general differences in sensitivities.