biostudies-literatureCoors AEuropean Federation of Pharmaceutical Industries and AssociationsNational Centre for the Replacement, Refinement and Reduction of Animals in ResearchH2020 HealthMedical Research CouncilNatural Environment Research CouncilBiotechnology and Biological Sciences Research Council1721-1730https://www.ebi.ac.uk/biostudies/studies/S-EPMC9893720biostudies-literatureUnknown57(4)There was no regulatory requirement for ecotoxicological testing of human pharmaceuticals authorized before 2006, and many of these have little or no data available to assess their environmental risk. Motivated by animal welfare considerations, we developed a decision tree to minimize in vivo fish testing for such legacy active pharmaceutical ingredients (APIs). The minimum no observed effect concentration (NOEC_min, the lowest NOEC from chronic <i>Daphnia</i> and algal toxicity studies), the theoretical therapeutic water concentration (TWC, calculated using the fish plasma model), and the predicted environmental concentration (PEC) were used to derive API risk quotients (PEC/NOEC_min and PEC/TWC). Based on a verification data set of 96 APIs, we show that by setting a tEnvironmental science & technologyMinimizing Experimental Testing on Fish for Legacy Pharmaceuticals.PMC9893720NE/H013172/1BB/V000411/1MC_PC_17189875508NC/R001421/1NE/L007371/1Nimrod Perkins AMaynard SKBrown ARCoors ATyler CROwen SfalseMinimizing Experimental Testing on Fish for Legacy Pharmaceuticals.There was no regulatory requirement for ecotoxicological testing of human pharmaceuticals authorized before 2006, and many of these have little or no data available to assess their environmental risk. Motivated by animal welfare considerations, we developed a decision tree to minimize in vivo fish testing for such legacy active pharmaceutical ingredients (APIs). The minimum no observed effect concentration (NOEC_min, the lowest NOEC from chronic <i>Daphnia</i> and algal toxicity studies), the theoretical therapeutic water concentration (TWC, calculated using the fish plasma model), and the predicted environmental concentration (PEC) were used to derive API risk quotients (PEC/NOEC_min and PEC/TWC). Based on a verification data set of 96 APIs, we show that by setting a t2023-01-01T00:00:00Z2023 Jan2026-07-16T20:11:38.827Z2025-06-01T01:57:23.401ZS-EPMC98937203665301910.1021/acs.est.2c07222