Adaptation of the Daphnia sp. acute toxicity test: miniaturization and prolongation for the testing of nanomaterials

Manufacturing of nanomaterials (NMs) is often complex and expensive, and their environmental risks are poorly understood or even unknown. An economization of testing NMs is therefore desirable, which can be achieved by miniaturizing test systems. However, the downsizing of test vessels and volumes can enlarge the surface/volume ratio (SVR) which in turn can affect the bioavailable concentration of adsorbing substances like NMs. The present study focused on the miniaturization of the acute toxicity test with Daphnia magna. The adaptations were verified with three reference substances, the non-adsorbing potassium dichromate (K₂Cr₂O₇) and as potentially highly-adsorbing substances silver nanoparticles (AgNPs) and silver nitrate (AgNO₃). The miniaturized test was conducted in 24-well microtiter plates (MT) and simultaneously compared to the OECD standard test (ST). Furthermore, the test duration was prolonged from 48 to 96 h since NMs tend to show effects only after extended exposure. The toxicity of K₂Cr₂O₇ and AgNPs continued to increase within the prolonged test span. The test comparisons with K₂Cr₂O₇ did not reveal any significant differences between ST and MT. AgNO₃ toxicity was significantly decreased in MT compared to ST due to the enlarged SVR. The toxicity of AgNPs in MT after 24 h was equal to ST. Contrary to our expectations an exposure longer than 24 h resulted in an increase of AgNP toxicity in MT, possibly due to enhanced dissolution of silver. Microtiter plates are appropriate alternative test vessels for the Daphnia sp. acute toxicity test; thus, its miniaturization is feasible. The enlarged SVR has to be taken into account since it can affect the toxicity of potentially adsorbing substances. Furthermore, the standard test duration of 48 h might underestimate the toxicity of many substances, especially of NMs.