There is an increasing need for new approach methodologies (NAMs) to generate relevant ecotoxicological data. This study demonstrates the strengths of calcein AM, a highly sensitive fluorescent stain for esterase activity, in an automated image-based multiwell plate assay for detecting sublethal effects in Daphnia magna. Sample processing and feeding conditions were optimized to ensure a uniform dye distribution. The protocol was validated using two esterase inhibitors, triphenyl phosphate and netilmicin sulfate, and subsequently applied to test the environmental contaminants methoxychlor, lindane, tributyltin chloride, pentachlorophenol, diuron, and ethofumesate. The test organisms were imaged in vivo using automated confocal microscopy, and fluorescence intensity was quantified to generate concentration–response curves. The effects of triphenyl phosphate and netilmicin sulfate were observed at concentrations 3-fold and 6-fold lower, respectively, than in the OECD 202 immobilization test. All tested contaminants also inhibited esterase activity, with concentrations resulting in no esterase activity at 48 h, correlating with mortality observed at 48 h. This method provides a new sensitive fluorescent tool for detecting sublethal chemical effects in D. magna, with the added advantage of visualizing intracellular processes in vivo.

This study demonstrated the strengths of invivo molecular staining coupled with automated imaging analysis in Daphnia magna. A multiwell plate protocol was developed to assess mitochondrial membrane potential using the JC-1 dye. The suitability of five common anesthetics was initially tested, and 5% ethanol performed best in terms of anesthetic effects and healthy recovery. The staining conditions were optimized to 30min staining with 2 μM JC-1 for best J-aggregate formation. The protocol was validated with the model compound carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and used to measure the effect of four environmental contaminants, 2,4-dinitrophenol, triclosan, n-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), and ibuprofen, on mitochondrial health. Test organisms were imaged using anautomated confocal microscope, and fluorescence intensities were automatically quantified. The effect concentrations for CCCP were lower by a factor of 30 compared with the traditional OECD 202 acute toxicity test. Mitochondrial effects were also detected at lower concentrations for all tested environmental contaminants compared to the OCED 202 test. For 2,4-dinitrophenol, mitochondria effects were detectable after 2h exposure to environmentally relevant concentrations and predicted organism death was observed after 24h. The high sensitivity and time efficiency of this novel automated imaging method make it a valuable tool for advancing ecotoxicological testing.