Introduction: Copper (Cu) is a toxic and one of the most common heavy metals. Even at low concentrations, this pollutant negatively impacts the aquatic environment. This study aimed to characterize the mechanisms underlying Cu toxicity in the freshwater microalga Raphidocelis subcapitata at environmentally relevant concentrations.

Methods: Algal cultures were exposed to Cu (33 or 53 µg L⁻¹) in OECD medium up to 72 hours. Growth, photosynthetic pigments content, photosynthetic efficiency (by PAM fluorometry), intracellular ROS levels, antioxidant enzyme activity (SOD and CAT), lipid peroxidation (MDA levels), and membrane integrity were assessed over time.

Results: Both Cu concentrations significantly inhibited growth, with cell density reductions of 53% and 93%, respectively. At 33 µg L⁻¹, chlorophyll a level decreased transiently, with partial recovery by 72 hours. In contrast, exposure to 53 µg L⁻¹ led to sustained chlorophyll loss and photoinhibition, as indicated by a decrease in F_v/F_m. ROS and MDA levels increased significantly for both concentrations, with membrane damage for 53 µg L⁻¹ (5% of cells showed compromised membrane integrity). Enhanced catalase (CAT) activity indicates activation of antioxidant defences.

Conclusions: Exposure to environmentally relevant Cu concentrations caused a severe physiological disruption in R. subcapitata, as translated by the modification of different biomarkers of toxicity. This work provides insights into the algal response to Cu contamination and presents a global proposal of the toxicity pathway of Cu on R. subcapitata at environmental concentrations.