Engineered nanoparticles (NPs) have realistic potential of reaching natural waterbodies and of exerting toxicity to freshwater organisms. The toxicity may be influenced by the composition of natural waters as crucial NP properties are influenced by water constituents. To tackle this issue, a case study was set up in the framework of EU FP7 NanoValid project, performing an interlaboratory hazard evaluation of NPs in natural freshwater. Ag and CuO NPs were selected as model NPs because of their potentially high toxicity in the freshwater. Daphnia magna (OECD202) and Danio rerio embryo (OECD236) assays were used to evaluate NP toxicity in natural water, sampled from Lake Greifen and Lake Lucerne (Switzerland). Dissolution of the NPs was evaluated by ultrafiltration, ultracentrifugation and metal specific sensor bacteria. Ag NP size was stable in natural water while CuO NPs agglomerated and settled rapidly. Ag NP suspensions contained a large fraction of Ag⁺ ions and CuO NP suspensions had low concentration of Cu²⁺ ions. Ag NPs were very toxic (48 h EC₅₀ 1-5.5 µg Ag/L) to D. magna as well as to D. rerio embryos (96 h EC50 8.8-61 µg Ag/L) in both standard media and natural waters with results in good agreement between laboratories. CuO NP toxicity to D. magna differed significantly between the laboratories with 48 h EC50 0.9-11 mg Cu/L in standard media, 5.7-75 mg Cu/L,in Lake Greifen and 5.5-26 mg Cu/L in Lake Lucerne. No toxicity of CuO NP to zebrafish embryos was detected up to 100 mg/L independent of the medium used. The results show that Ag and CuO NP toxicity may be higher in natural water than in the standard media due to differences in composition. NP environmental hazard evaluation can and should be carried out in natural water to obtain more realistic estimates on the toxicity.