Molecular clustering enhances perfluorocarboxylic acid bioaccumulation in lactuca sativa by disrupting the tricarboxylic acid cycle

Molecular clustering of perfluorocarboxylic acids (PFCAs) is chain-length dependent, yet its biological consequences in crops remain unclear. The plants were hydroponically exposed to PFCA clusters at concentrations of 1, 10, and 100 μmol/L for 14 days, with untreated controls. To elucidate the underlying mechanisms, they were analyzed using atomic force microscopy, molecular dynamics simulations, and integrated omics approaches to examine how PFCA clustering affects bioaccumulation and metabolic disruption. The results indicate that longer-chain PFCA (C10) formed larger, more stable clusters (122–194 nm) than shorter chain (C4, 35–95 nm). C10 clusters exhibited 5.5-fold higher root accumulation but 50% reduced shoot translocation compared to C4 clusters. Metabolomics revealed 664 significantly altered metabolites for C10 versus 545 for C4, predominantly downregulated. Further proteomic evidence showed that C10 exposure induced a greater number of differentially expressed proteins (DEPs) compared to C4, indicating broader protein-level disruption and more profound impairment of mitochondrial function. Most critically, C10 clusters severely impaired the tricarboxylic acid (TCA) cycle, reducing intermediates by up to 48% and downregulating associated enzymes. Molecular docking confirmed strong binding between PFCA clusters and metabolic enzymes (−6.3 to −6.4 kcal·mol⁻¹). These findings indicate that larger PFCA clusters are associated with greater phytotoxicity compared to smaller clusters, suggesting important implications for crop safety. Results highlight urgent needs for cluster-aware monitoring strategies and revised risk assessment frameworks accounting for aggregation phenomena.