Repeated Exposure Enhanced Toxicity of Clarithromycin on Microcystis aeruginosa Versus Single Exposure through Photosynthesis, Oxidative Stress, and Energy Metabolism Shift

Antibiotics are being increasingly detected in aquatic environments, and their potential ecological risk is of great concern. However, most antibiotic toxicity studies involve single-exposure experiments. Herein, we studied the effects and mechanisms of repeated versus single clarithromycin (CLA) exposure on Microcystis aeruginosa. The 96 h effective concentration of CLA was 13.37 μg/L upon single exposure but it reduced to 6.90 μg/L upon repeated exposure. Single-exposure CLA inhibited algal photosynthesis by disrupting energy absorption, dissipation and trapping, reaction center activation, and electron transport, thereby inducing oxidative stress and ultrastructural damage. In addition, CLA upregulated glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Repeated exposure caused stronger inhibition of algal growth via altering photosynthetic pigments, reaction center subunits biosynthesis, and electron transport, thereby inducing more substantial oxidative damage. Furthermore, repeated exposure reduced carbohydrate utilization by blocking the pentose phosphate pathway, consequently altering the characteristics of extracellular polymeric substances and eventually impairing the defense mechanisms of M. aeruginosa. Risk quotients calculated from repeated exposure were higher than 1, indicating significant ecological risks. This study elucidated the strong influence of repeated antibiotic exposure on algae, providing new insight into antibiotic risk assessment.