Pesticide exposure sequence and duration modify the effects of heatwaves and water level reduction on river biofilms

Fluvial ecosystems are increasingly affected by stressors from anthropogenic activities and climate change-related pressures. Here we examined the potential relevance of stressor interaction, sequence and timing of exposure through a controlled laboratory experiment. We selected both chemical (mixture of pesticides) and physical (temperature heatwave and decreased water level) stressors and we performed two 96 h microcosm experiments designed to assess the effects on the structure and function of epilithic biofilms. Biofilms respond rapidly to these stressors, making them valuable indicators of global change. Our results showed that pesticides negatively impacted photosynthetic activity, while heatwaves increased both structural (chlorophyll concentration) and functional (glucosidase activity) biofilm descriptors. In contrast, water level reduction had comparatively mild effects. The impacts of pesticides were more pronounced when exposure was continuous or occurred during the final 48 h of the experiment, whereas early exposure combined (first 48 h) with a water level reduction resulted in weaker effects, accompanied by an increase and partial recovery of microbial abundance. Furthermore, adverse pesticide effects were more pronounced at 8 °C than at 15 °C. Overall, our results highlight that the sequence, timing and duration of stressor exposure play a critical role in the biofilm structure and function, with likely effects on the whole river ecosystem. • Stressor sequence strongly shapes biofilm structure and function. • Late/continuous pesticide exposure impairs biofilm; early exposure allows recovery. • Heatwaves mitigate pesticide effects; low water levels have milder impacts. • Temporal stressor dynamics are key for biofilm resilience under global change.