Environmental history determines biofilm tolerance to short-term pulse exposure towards chemical stress

Periphytic biofilms are key mediators of freshwater ecosystem processes but are increasingly exposed to anthropogenic stressors such as antibiotics. We investigated how prior exposure to wastewater effluent, a press disturbance, affects biofilm tolerance to chemical stress. Biofilms were sampled upstream and downstream of a wastewater treatment plant and exposed in the laboratory to increasing concentrations of the antibiotic ciprofloxacin (CIP). We assessed biofilm biomass, pigment composition, fatty acid profiles, and microbial community structure using qPCR and metabarcoding. Downstream biofilms, shaped by chronic wastewater exposure, exhibited higher biomass stability, reduced pigment and fatty acid losses, and smaller shifts in bacterial and eukaryotic community composition under CIP stress compared to upstream biofilms. Upstream communities, despite higher baseline pigment and fatty acid concentrations, showed pronounced sensitivity to CIP, including biomass declines and reduced prokaryotic diversity. Taxon-specific responses revealed that downstream biofilms harbored stress-tolerant Chlorophyceae, cyanobacteria, and heterotrophic taxa, which likely contributed to their functional resilience. Our findings demonstrate that environmental history and community composition strongly modulate biofilm responses to antibiotic exposure, supporting the pollution-induced community tolerance framework. These results highlight the importance of considering legacy effects and spatial heterogeneity when assessing ecosystem vulnerability and resilience to chemical stressors.