Unraveling IPPD-Induced Phytotoxicity in White Clover (Trifolium repens L.): Integrating Physiological Impairment with Metabolic Disruption

N-isopropyl-N′-phenyl-1,4-phenylenediamine (IPPD), a widely used high-performance substituted p-phenylenediamine antioxidant, is commonly used in tires and other industrial rubber products. Currently, there are few studies investigating the toxicological effects of such antioxidants on plant species. In this study, white clover (Trifolium repens L.) was exposed to IPPD to evaluate its phytotoxic effects on plants. White clover was exposed to varying concentrations of IPPD via hydroponic culture, and plant growth physiology and metabolic indicators were measured after 15 days. Results showed that IPPD significantly inhibited plant growth and disrupted root cell membranes leading to cellular necrosis, reducing root biomass by 82.2% compared to control group (CK). At an exposure concentration of 5 mg L⁻¹, white clover demonstrated significant absorption and translocation of IPPD, with root concentrations reaching 354 ± 52.33 ng g⁻¹, which were much higher than those in shoot. As the IPPD exposure increased, photosynthesis was inhibited and photosynthetic pigment content decreased. IPPD triggered oxidative stress responses in plants, as evidenced by a significant increase in reactive oxygen species (ROS) content compared to CK, with malondialdehyde (MDA) levels in roots rising by 719.9%. Furthermore, antioxidant enzyme activities were enhanced and the levels of osmoregulatory substances (soluble sugars and soluble proteins) increased significantly. Simultaneously, a greater number of differentially expressed metabolites were detected in the roots, potentially attributable to the higher accumulation of IPPD in root tissue, which may have triggered more changes in metabolic processes. By KEGG analysis, IPPD disrupted the metabolic pathways of white clover, especially glycolysis and tricarboxylic acid cycle. These results suggest that IPPD-induced oxidative stress and metabolic dysregulation collectively impair plant energy metabolism and growth.