Seasonal variations of caffeine and antibiotic concentrations were investigated along the selected sites of the Danube using solid-phase extraction followed by highperformance liquid chromatography (HPLC). The potential risks on the aquatic organisms were evaluated. HPLC methods for caffeine and antibiotic determination were modified and validated showing good accuracy, repeatability, selectivity, and robustness. The obtained results confirmed the presence of caffeine at four sampling points, near the wastewater discharges. The mean caffeine concentrations for summer, autumn, winter, and spring were 24.78, 26.83, 24.61, and 86.29 ng/L, respectively. The analyzed antibiotics (sulfamethoxazole, chloramphenicol, and tiamuline) were under the limit of detection in all tested samples taken from the Danube. The maximum risk indexes (MaxRIs) were calculated for caffeine at each sampling site. Four out of seven selected sampling sites showed sublethal effects on the aquatic organisms with 10 < MaxRI < 100 (class II), while the other three had no potential risk for aquatic organisms. The highest potential risk was calculated for the sampling site in the vicinity of one of the biggest wastewater discharges.
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