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This document specifies a method for the determination of fish acute toxicity using the permanent cell line from rainbow trout (Oncorhynchus mykiss) gill, RTgill-W1. Cells in confluent monolayers in 24-well tissue culture plates are exposed to water samples, such as surface waters or different kinds of effluents, or to chemicals for 24 h and, thereafter, cell viability is assessed based on fluorescent cell viability indicator dyes (see 4.1). Data are then expressed as a percentage of unexposed control and toxicity quantified based on the percentage of cell viability versus the percentage of effluent or the chemical concentration in response curves (see Clause 9).