A major question in the field of ecotoxicology is how DOM affects copper accumulation and toxicity in planktonic organisms; copper acute toxicity and bioaccumulation in Ceriodaphnia silvestrii were investigated in the presence and absence of humic substances (HS) under controlled laboratory conditions. Copper was determined as free Cu2+ ions in the media and total copper in the animals; metal ion buffers were used for ion selective electrode calibration, extending the lower detection limit to 10(-11) mol l(-1). Groups of 20 adult females of similar sizes were exposed (24 h) to a range of nominal copper concentrations. Based on total added copper, LC50 was 4.4 x 10(-8) mol l(-1) without HS, whereas with 20 mg l(-1) HS, it was 25 times higher (1.1 x 10(-6) mol l(-1)). Based on free Cu2+ ions LC50 was statistically similar either with (2.8 x 10(-8) mol l(-1)) or without HS (3.3 x 10(-8 )mol l(-1)). The present results showed that natural DOM reduced copper toxicity and that free Cu2+ ions correlates to the bioavailable fraction to zooplankton. Nevertheless, copper bioaccumulation by C. silvestrii was similar either in the presence or absence of humic substances, suggesting that C. silvestrii regulates its body copper content up to 3.0 x 10(-8) mol l(-1) free Cu2+ ions in the media. The organisms were not able to deal with higher free Cu2+ ions concentrations in the media.