Using protonated alginate (HA) beads, copper (Cu²⁺) levels and toxicity were concurrently reduced in semiconductor wastewater produced from the chemical mechanical planarization process. The beneficial effect of protonation could be explained by a reduction in the release of Ca²⁺, which is a competitive cation during sorption of Cu²⁺, leading to an increased in the sorption capacity from 107 to 189 mg/g. Monitoring of the acute toxicity of two different types of semiconductor wastewater toward Daphnia magna suggested that Cu²⁺ was the cause of toxicity. The toxicity identification evaluation using D. magna indicated that Cu²⁺ was a major toxicant in the raw wastewater with concentrations of 1.97 and 3.37 mg/L for two different raw wastewater samples with initial toxicities of 14.2 and 23.6 toxic unit (TU), respectively. This relationship was verified by the correlation coefficients between Cu²⁺ concentration and acute toxicity (r² = 0.829 at P < 0.05 and 0.894 at P < 0.05 for two types of semiconductor wastewater) in mass balance tests. The laboratory continuous column test using HA beads showed that the Cu²⁺ concentration in effluent was proportional to the acute toxicity. In particular, the TU values increased sharply when the residual concentration of Cu²⁺exceeded 0.1 mg/L. The test battery results indicated that D. magna was more sensitive than other aquatic species, i.e., algae and bacteria, to Cu²⁺ in semiconductor wastewater.