Miniaturising acute toxicity and feeding rate measurements in Daphnia magna

- Acute toxicity is studied as function of exposure vessel size and animal density.
- No significant change in toxicity occurs with miniaturisation.
- Daphnia feeding rate test is also miniaturised in volume and animals used.
- Both endpoints provide automation compatible single animal phenotypic markers.

Phenotypic markers of animal health form an essential component of regulatory toxicology. Immobilisation of neonate water fleas - Daphnia magna - as a surrogate measure of their mortality following exposure to a chemical for 24-48 h forms the basis of the internationally utilised OECD acute toxicity test 202. A second important marker of animal physiology and health is feeding rate, which in Daphnia is determined by measuring the algae feeding rate.Given the widespread use of OECD test 202 for acute toxicity as well as the quantification of feeding rate in toxicological studies of daphniids, significant benefits could result from miniaturising this assay. In particular, miniaturisation would use fewer animals, less media and chemicals, less laboratory space and make the tests more compatible with automation, and therefore could result in considerable time savings. Furthermore, miniaturising phenotypic markers to the ultimate level of a single animal per well would facilitate multiple measurements of other phenotypic markers, such as behavioural responses, which could be integrated at the individual level.In this study we used a wide range of exposure vessels to evaluate the impacts of systematically varying total media volume, surface to volume ratio and animal density for the acute toxicity testing of cadmium. We demonstrate that Daphnia acute toxicity tests using single animals within 24- or 48-well plates produce equivalent results as for traditional test configurations, for different chemicals. Considering algae feeding rates by Daphnia, we studied the impacts of varying algae concentration, total volume and animal density. After having demonstrated that multiwell plates can again yield equivalent test results as traditional experimental setups, we used miniaturised test vessels to show the impact of metals on the feeding activity on daphniids for both neonates and adult animals. Overall we confirm the feasibility of a multiwell approach for Daphnia toxicity testing that requires less time and materials than a traditional assay and can provide phenotypic characterisation at a single animal level.