Acute immobilization assay with Daphnia similis: A miniaturized protocol

To reduce volume of samples, residues, costs and time, ecotoxicologists are seeking for new approach methodologies. Miniaturized versions of already well-established protocols are arising as a promising tool to diminish costs and residues in the laboratory. The aim of this study was to establish and validate a miniaturized protocol of the acute immobilization assay with Daphnia similis, using 96-well microplates. For that, conventional and miniaturized assays were performed, exposing the test organism to organic and inorganic substances and environmental samples, and results compared. The correlation of results obtained with both protocols revealed that they were not influenced by the different volumes of liquid, used in each version. Tests with negative controls confirmed that the reduced volume did not negatively interfere in the fitness of D. similis and were sufficient to perform tests with quality, validating the miniaturized protocol. In addition to the reduction of production of residues from the test and faster scoring, the reduced volume of samples used to perform the miniaturized version is particularly advantageous when low amount of sample is available (i.e., extracts, synthesized environmental samples, new formulated products), allowing testing of different concentrations and repetitions. These peculiarities make the miniaturized test with D. similis an effective and feasible version of the conventional one and can be used as an alternative protocol in ecotoxicological investigations.