This study investigates erythromycin toxicity toward activated sludge as a function of exposure time and antibiotic concentration. Batch experiments were conducted and microscopic techniques ranging from bright-field microscopy to epifluorescence and confocal laser scanning microscopy (CLSM), combined with a fluorescent viability indicator (BacLight (R) Bacterial Viability Kit, Molecular Probes), allowed us to study erythromycin time-kill activity. The erythromycin toxicity was observed at lower concentration when exposure time increased. A 4 mu g/L erythromycin concentration was toxic to heterotrophic bacteria on a 5-day time exposure, and a 5 mg/L concentration inhibited nitrification. These findings are in agreement with the microscopic studies, which showed a latency time before the lower antibiotic concentrations began to kill bacteria. Microscope slide wells were used as micro-reactors in which erythromycin concentration ranged from 0.1 to 1 mg/L. After 45 min there were 94% (SD 3.8) of living bacteria in control micro-reactors, 67% (SD 3.1) in micro-reactors that contained 0.1 mg/L erythromycin and 37% (SD 18.6) in micro-reactors that contained 1 mg/L erythromycin. CLSM allowed visualization of isolated stained cells in the three-dimensional structure of damaged flocs. (C) 2010 Elsevier Ltd. All rights reserved.