Authors
Amira Perez-Liñan, Cedric Abele, Paula Pierozan, Magnus Breitholtz, Oskar Karlsson
Published in
Environmental science & technology. Aug 21, 2025. Epub Aug 21, 2025.
Abstract
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.
PMID:
40841535
Bibliographic data and abstract were imported from PubMed on 22 Aug 2025.
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