Abstract The effect of long-term copper acclimation of the freshwater green algae Pseudokirchneriella subcapitata to copper was investigated using different physiological and toxicological endpoints. The algae were exposed to seven—five of which are ecologically relevant for European surface waters—copper concentration ranging from 0.5 to 100 μg Cu l −1 during a 3-month period. A standard medium was used as culture and test medium with an addition of 2 mg DOC l −1 (replacing EDTA). At certain intervals, experiments were performed to assess algal biomass, growth rate, chlorophyll and carotenoid content, pigment diversity, autotrophic index, intracellular and adsorbed copper, and the sensitivity of the algae to copper. Chronic copper tolerance (mean ± standard deviation) increased significantly from 88±15 to 124±25 μg Cu l −1 for P. subcapitata acclimated to 0.5 and 100 μg Cu l −1, respectively. Based on the algal biomass, the growth rate, the pigment diversity and the autotrophic index, an optimal concentration range was observed between 1 and 35 μg Cu l −1. Significant decreases in algal biomass, pigment diversity and autotrophic index were observed in algal cultures acclimated to 0.5 μg Cu l −1 and 100 μg Cu l −1. Chlorophyll a content (mean ± standard deviation) increased from 8.4±3.1 to 28.6±7.5×10 −14 g per cell and carotenoid content (mean ± standard deviation) increased from 3.7±0.8 to 7.1±1.2×10 −14 g per cell for algae exposed to 1 and 100 μg Cu l −1, respectively. Intracellular copper increased from 0.099 to 20.6×10 −15 g Cu per cell and adsorbed copper increased from 0.026 to 1.8×10 −15 g Cu per cell for algae acclimated for 12 weeks to 0.5 and 100 μg Cu l −1, respectively. This research demonstrates that the use of standard culture media, some of which may be deficient in copper, can result in sub-optimal performance of the organisms, which in turn may affect toxicity test results. Additionally, this work also established an optimal concentration range for copper for this algal species. This phenomenon should be taken in consideration when performing environmental risk assessments of essential elements.