A fluorescence-based hydrolytic enzyme activity assay for quantifying toxic effects of Roundup® to Daphnia magna

Daphnia magna is a widely used model organism for aquatic toxicity testing. In the present study, we investigated the hydrolytic enzyme activity of D. magna after exposure to toxicant stress. In vivo enzyme activity was quantified using 15 fluorogenic enzyme probes based on 4-methylumbelliferyl or 7-amino-4-methylcoumarin. Probing D. magna enzyme activity was evaluated using short-term exposure (24-48 h) to the reference chemical K2Cr2O7, or the herbicide formulation Roundup®. Toxicant induced changes in hydrolytic enzyme activity were compared to changes in mobility (ISO 6341). The results showed that hydrolytic enzyme activity was quantifiable as a combination of whole body fluorescence of D. magna, and fluorescence of the surrounding water. Exposure of D. magna to lethal and sublethal concentrations of Roundup® resulted in loss of whole body enzyme activity, and release of cell constituents including enzymes and DNA. Roundup® caused comparable inhibition of mobility and alkaline phosphatase activity with EC50 values at 20 °C of 8.7-11.7 mg a.i./L. Inhibition of alkaline phosphatase activity by Roundup® was lowest at 14 °C and greater at 20 and 26 °C. The results suggested that the fluorescence based hydrolytic enzyme activity assay (FLEA assay) can be used as an index of D. magna stress. Combining enzyme activity with fluorescence measurements may be applied as a simple and quantitative supplement for toxicity testing with D. magna.