Antibiotics represent a unique and diverse group of drugs, which are known to exert deleterious effects on non-target species and contribute to the phenomenon of antimicrobial resistance. With central inclusion on the EU Surface Water Watch List, and reported known affects in multiple model organisms, the importance of the sufficient monitoring of antibiotics in the aquatic environment has been highlighted. Most studies report the impact of individual antibiotics following exposure for a single generation in animals. In this study, we assessed the impact of four antibiotics with different modes of action (amoxicillin, trimethoprim, erythromycin, and sulfamethoxazole) and their mixture on the sentinel species Daphnia magna over three generations, via biochemical markers and a targeted metabolomic analysis of central metabolic pathways. No mortality was observed at 50 mg/L of each selected antibiotic and their composite mixture. Thus, a working concentration of 1 mg/L was chosen to progress this study. Results indicated that enzyme activity was particularly sensitive to exposure to amoxicillin and the mixture, whereas trimethoprim and the mixture induced the most metabolic changes in glycolysis and the TCA cycle. Additionally, the quaternary mixture had a stronger impact on the first generation of daphnids, altering the activity of β-galactosidase, glutathione S-transferase, and acid and alkaline phosphatase, suggesting that Daphnia can adapt to stress caused by antibiotics.

Daphnids, commonly known as water fleas, are freshwater planktonic microcrustacean species used as model organisms in ecotoxicology, particularly in regulatory frameworks that adhere to OECD and ISO standards. Mortality is the most common endpoint in toxicity testing; however, more sensitive indicators are required to assess sublethal acute effects of pollutants. The use of feeding impairment as a toxicity phenotypic endpoint in daphnids is considered a cost-effective approach that aligns with the 3Rs principle (Replace, Reduce, Refine) and is more physiologically and environmentally relevant. Current feeding methods are inefficient due to the large test volumes and extended incubation periods required. In this paper, we present a miniaturised protocol to assess feeding behaviour following exposure to chemicals in daphnids. The method is based on the consumption of algae, which is measured with chlorophyll fluorescence. The optimised protocol is more robust and rapid, and results can be obtained in 30 min and in a 96-well plate. Responses in feeding rate were investigated using this miniaturised protocol following exposure to a range of prevalent pollutants, which include two metals and, as a more realistic sample, a leachate from smoked cigarette filters. All three pollutants were tested at sublethal concentrations. This method provides an efficient approach to assess the toxicity of chemicals and water quality.