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AbstractDuring its aquatic life cycle, nanosized titanium dioxide (nTiO2) may interact with natural organic matter (NOM) ultimately altering the ecotoxicity of co-occurring chemical stressors such as heavy metals (e.g. copper (Cu)). In this context, the following hypotheses were tested: (1) aging of nTiO2along with Cu reduces Cu toxicity, (2) nTiO2agglomerates have a lower potential to reduce Cu toxicity and (3) aging of nTiO2in presence of NOM reduces Cu toxicity further. A multifactorial test design crossing three nTiO2levels (0.0, 0.6 and 3.0 mg/L) with two levels of NOM (0 versus 8 mg total organic carbon (TOC)/L) and seven nominal Cu concentrations (ranging from 0 to 1536 μg/L) aged in ASTM medium for 0, 1, 3 and 6 days was realised, while two aging scenarios were applied (type 1: nTiO2jointly aged with Cu; type 2: Cu added after nTiO2aging). Subsequently, Cu toxicity was assessed using the immobility ofDaphnia magnaafter 48 h of exposure as response variable. The experiments revealed that neither aging duration nor the extent of agglomeration (type 1 vs. type 2 aging) has a substantial impact on Cu induced toxicity. Moreover, it was confirmed that the presence of NOM substantially reduced Cu induced toxicity, independent of the aging scenario and duration. More importantly, the data suggest the ingestion of Cu loaded nTiO2as additional exposure pathway contributing to Cu toxicity. In conclusion, it seems unlikely that nTiO2concentrations currently detected in or predicted for aquatic ecosystems, which are at least one order of magnitude below the concentration tested here, influence Cu toxicity meaningfully.