Toxicity of Three Crystalline TiO₂ Nanoparticles in Activated Sludge: Bacterial Cell Death Modes Differentially...

The eco-toxicities of different crystalline phases of TiO₂-NPs are controversial, and the effects and mechanisms on activated sludge are unclear. Therefore, we assessed the acute-toxicities (8-h exposure) of P25, anatase, and rutile TiO₂-NPs in activated sludge using flow cytometry under simulated sunlight (hereafter-sun) and evaluated the relationship between sludge dewatering and bacterial cell death modes using Pearson's correlation coefficients ( r). Additionally, the response of the microbial community structure was examined by high throughput sequencing. Bacterial survival and death were observed by confocal laser scanning microscopy. Toxicity indicators (e.g., lactate dehydrogenase (LDH) and reactive oxygen species (ROS)) were determined. Overall, TiO₂-NPs toxicity was concentration-dependent and crystalline-phase-dependent. The responses of bacterial communities to crystalline phases were more obvious than that of dosage. P25-sun and anatase-sun caused necrosis-like cell death via strong photo-oxidation confirmed by 131%/123% (1 mg/L) and 301%/254% (50 mg/L) LDH released by the control, while rutile-sun induced apoptosis-like death via intracellular ROS production increased to 165% (1 mg/L) and 420% (50 mg/L) of the control. P25 and anatase NPs had higher protein and polysaccharide affinities, while rutile NPs exhibited stronger attachment onto phospholipids. TiO₂-NPs-sun reduced activated sludge dewaterability. Specific resistance to filtration (SRF) showed the strongest positive correlation with tightly bound extracellular polymeric substances (EPS) and total soluble microbial byproducts ( r = 0.974, p < 0.01) and was closely related to EPS content and composition, especially the increased bound water (BW) content and sludge protein concentrations. High Pearson correlation coefficients were observed between early apoptotic cells and BW content ( r = 0.952, p < 0.01) resulting from massive polysaccharides and between necrotic (including late apoptotic) cells and SRF ( r = 0.959, p < 0.01) resulting from high protein and EPS concentrations. Thus, in response to TiO₂-NPs, bacterial cell death modes differentially weakened sludge dewatering.