Elevated Toxicity and High-Risk Impacts of Small Polycyclic Aromatic Hydrocarbon Clusters on Microbes Compared to Large Clusters

Polycyclic aromatic hydrocarbons (PAHs) are widespread contaminants that can accumulate in microorganisms, posing significant ecological risks. While previous studies primarily focused on PAH concentrations, the impacts of PAH self-clustering have been largely overlooked, which will lead to inaccurate assessments of their ecological risks. This study evaluates the toxic effects of four prevalent PAH clusters on microbes with an emphasis on comparing the cluster sizes. Results revealed that over 95% of PAHs can form clusters in the aquatic environment, with smaller clusters more likely to form at lower concentrations and with fewer benzene rings. To quantify the toxic effects and understand underlying mechanisms, single-cell Raman-D₂O was employed to link bacterial phenotypes with transcriptomic profiles. Bacteria exposed to smaller PAH clusters showed a 1%-10% reduction in metabolic activity, which was associated with a 1.8-2.9-fold increase in intracellular reactive oxygen species (ROS). Furthermore, when exposed to smaller PAH clusters, the expression of genes related to the ROS response and efflux pumps was upregulated by up to 6.33-fold and 4.97-fold, respectively, suggesting that smaller PAH clusters pose greater toxicity to microbes. These findings underscore the potentially overlooked risks of PAH clusters in environmental systems and deepen our understanding of the environmental fate and ecological risks of these contaminants.