Multiple physiological response analyses of <i>Chlorella vulgaris</i> exposed to silver nanoparticles, ciprofloxacin, and their combination

The combination of silver nanoparticles (AgNPs) and ciprofloxacin (CIP) can be considered an alternative to combat multidrug-resistant microbial infections. However, knowledge about their combined toxicity after being released in an aquatic environment is scarce. This study evaluated the individual toxicity of AgNPs and CIP and their combined toxicity on the unicellular green microalga Chlorella vulgaris, evaluating cellular responses and conducting metabolomic analysis. The median effect concentrations at 96 h (EC50-96h) for AgNPs, CIP, and the mixture were 132 µg L-1, 7,000 µg L-1, and 452 µg L-1, respectively. Ciprofloxacin exhibited a synergistic effect with AgNPs. The toxic ranking for C. vulgaris was AgNPs > AgNPs + CIP > CIP. The growth rate was the most evident parameter of toxicity. Cell diameter significantly increased (p < 0.001) at 96 h for the highest concentrations tested of AgNPs, CIP, and the mixture, with increases of 24%, 41%, and 19%, respectively, compared with the control. Photosynthetic pigment analyses revealed that C. vulgaris upregulated chlorophyll, carotenoids, and pheophytin. Cell exposure to CIP caused an emergency response involving increased protein and carbohydrate concentrations to tolerate antibiotic stress. Exposure to AgNPs and CIP increased catalase and glutathione S-transferase activity, but the mixture decreased the activity. Silver nanoparticles increased malondialdehyde content in exposed cells due to fatty acid peroxidation. These pollutants revealed their potential risks in interfering with survival and metabolism. Our findings highlight the possible hazards of copollutants at environmentally relevant quantities, providing insights into the individual and combined ecotoxicity of AgNPs and CIP.