For effect assessment several extrapolation methods can be used to derive the concentration of toxic chemicals above which adverse effects on aquatic ecosystems may occur. These methods are based on single-species toxicity data. At present, however, it is uncertain whether the values calculated with these extrapolation methods really represent accurate estimations of concentrations harmless to ecosystems. Therefore a validation of extrapolation methods was carried out by comparing NOECs derived from multiple-species (semi-) field experiments with extrapolated values. In this study validation was restricted to the methods of Aldenberg and Slob and Wagner and Løkke and a modification of the method of the U.S. Environmental Protection Agency. Multiple-species experiments for organic compounds and metals in aquatic ecosystems were studied. For only 29 compounds, 19 organic compounds, and 10 metals, one or more multiple-species NOECs could be derived. For 11 of these compounds an insufficient amount of single-species toxicity data was available to apply the methods of Aldenberg and Slob and Wagner and Løkke. With reservations, due to this paucity of data, it is concluded that single-species toxicity data can be used to derive “safe” values for the aquatic ecosystem. Furthermore, extrapolation methods seem to be a good basis for determining these values. Based on the results of this study, the best correlation between multiple-species NOECs and extrapolated values can be obtained with the methods of Aldenberg and Slob and Wagner and Løkke, both with a 95% protection level and a 50% confidence level.