Elevated Toxicity\nand High-Risk Impacts of Small Polycyclic\nAromatic Hydrocarbon Clusters on Microbes Compared to Large Clusters

Polycyclic aromatic hydrocarbons (PAHs) are widespread\ncontaminants\nthat can accumulate in microorganisms, posing significant ecological\nrisks. While previous studies primarily focused on PAH concentrations,\nthe impacts of PAH self-clustering have been largely overlooked, which\nwill lead to inaccurate assessments of their ecological risks. This\nstudy evaluates the toxic effects of four prevalent PAH clusters on\nmicrobes with an emphasis on comparing the cluster sizes. Results\nrevealed that over 95% of PAHs can form clusters in the aquatic environment,\nwith smaller clusters more likely to form at lower concentrations\nand with fewer benzene rings. To quantify the toxic effects and understand\nunderlying mechanisms, single-cell Raman-D<sub>2</sub>O was employed\nto link bacterial phenotypes with transcriptomic profiles. Bacteria\nexposed to smaller PAH clusters showed a 1%–10% reduction in\nmetabolic activity, which was associated with a 1.8–2.9-fold\nincrease in intracellular reactive oxygen species (ROS). Furthermore,\nwhen exposed to smaller PAH clusters, the expression of genes related\nto the ROS response and efflux pumps was upregulated by up to 6.33-fold\nand 4.97-fold, respectively, suggesting that smaller PAH clusters\npose greater toxicity to microbes. These findings underscore the potentially\noverlooked risks of PAH clusters in environmental systems and deepen\nour understanding of the environmental fate and ecological risks of\nthese contaminants.