Metabolomic Alterations Reveal Toxic Mechanisms of Single and Combined 6PPD and Arsenic Exposure on Mung Bean (Vigna radiata)

Abstract

N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), a typical antioxidant present in rubber products, is detected at significant concentrations across multiple environmental compartments. However, there is no existing knowledge on the single and combined effect of 6PPD and arsenic (As) on terrestrial plants. This study examined the effects of 6PPD (1, 5, 10 and 20 µg L⁻¹) and its combination with arsenic (500 µg L⁻¹ As + 6PPD) on physio-biochemical characteristics and metabolomics of mung beans (Vigna radiata) after 21 days of exposure. Our results demonstrated that although 6PPD and arsenic individually affect plant growth, their combined exposure significantly reduced shoot biomass to 0.5 g in (As + 20 µg L⁻¹ 6PPD) compared to 1.5 g in controls, and root biomass to 0.15 g exposed to (As + 10 µg L⁻¹ 6PPD), indicating a synergistic toxic effect. A significant decline in chlorophyll content (20.6) was observed under the combined treatment (As + 20 µg L⁻¹ 6PPD). This was accompanied by substantial reductions in photosynthetic parameters: transpiration rate (E) decreased to 3.9 µmol m⁻² s⁻¹ under (As 500 µg L⁻¹) and further to 2.1 µmol m⁻² s⁻¹ in (As + 20 µg L⁻¹ 6PPD); stomatal conductance (gₛw) and assimilation rate (A) were reduced to 0.085 mol m⁻² s⁻¹ and 0.0064 µmol m⁻² s⁻¹, respectively, under the combined treatment. Oxidative stress indicated a significant antioxidant enzyme modulation and a 60% and 70% increase in ROS and MDA levels. Metabolomic profiling demonstrated that the treatments disrupted amino acid metabolism, impairing protein synthesis and growth, while concurrently elevating carbohydrate and fatty acid metabolism to compensate for energy deficits and enhance stress tolerance. These findings highlight the major phytotoxicity risk to the agricultural system and emphasize the need for risk assessment and monitoring for these contaminants.