The acetylcholinesterase (AChE) inhibition assay has been frequently applied for environmental monitoring to capture insecticides such as organothiophosphates (OTPs) and carbamates. However, natural organic matter such as dissolved organic carbon (DOC) co-extracted with solid-phase extraction from environmental samples can produce false-negative AChE inhibition in free enzyme-based AChE assays. We evaluated whether disturbance by DOC can be alleviated in a cell-based AChE assay using differentiated human neuroblastoma SH-SY5Y cells. The exposure duration was set at an optimum of 3 h considering the effects of OTPs and carbamates. Because loss to the airspace was expected for the more volatile OTPs (chlorpyrifos, diazinon, and parathion), the chemical loss in this bioassay setup was investigated using solid-phase microextraction followed by chemical analysis. The three OTPs were relatively well retained (loss <34%) during 3 h of exposure in the 384-well plate, but higher losses occurred on prolonged exposure, accompanied by slight cross-contamination of adjacent wells. Inhibition of AChE by paraoxon-ethyl was not altered in the presence of up to 68 mgc /L Aldrich humic acid used as surrogate for DOC. Binary mixtures of paraoxon-ethyl and water extracts showed concentration-additive effects. These experiments confirmed that the matrix in water extracts does not disturb the assay, unlike purified enzyme-based AChE assays. The cell-based AChE assay proved to be suitable for testing water samples with effect concentrations causing 50% inhibition of AChE at relative enrichments of 0.5-10 in river water samples, which were distinctly lower than corresponding cytotoxicity, confirming the high sensitivity of the cell-based AChE inhibition assay and its relevance for water quality monitoring. Environ Toxicol Chem 2022;41:3046-3057. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.
Keywords: Acetylcholinesterase inhibitors; SH-SY5Y cells; dissolved organic matter; effects-based monitoring; neurotoxicity; volatility.
© 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.