A new multisubstance potentially affected fraction (msPAF)-based method for calculating ecotoxicological effect factors for life-cycle assessment is introduced and compared to two other available methods of calculation. The new method is based on marginal increase of the msPAF of species. The method follows concentration-additive rules for pollutants with the same toxic mode of action (TMoA) and response-additive calculation rules for pollutants with independent action, and it combines a TMoA-specific factor, which is calculated differently in different methods, and a substance-specific factor, which is common to all methods. For 261 substances in 22 toxic modes of action, ecotoxicological effect factors for freshwater ecosystems have been calculated by different methods. Method-related differences appear to be rather small. Intersubstance differences in effect factors stem from differences in substance-specific toxic potencies, which span eight orders of magnitude, rather than from differences in TMoA-specific factors, which span only three orders of magnitude. Based on these insights, the choice of a calculation method seems to be a matter of personal (scientific) preference. The new hybrid msPAF method was greatly sensitive to data that usually are not known with sufficient certainty. Its use is recommended, entirely for reasons of scientific consistency, under strict conditions. When such conditions are not met or if necessary parameter values are unavailable, use of a fixed value for the TMoA-specific component is recommended.