Effects of combined zinc and 2 mercaptobenzothiazole exposure on growth and physiological characteristics of Lolium perenne cultivars.

Heavy metal and organic pollutant co-contamination is increasingly being detected in soils and water systems, particularly due to tire wear-derived compounds such as zinc (Zn) and 2-mercaptobenzothiazole (MBT), yet their combined effects on forage grasses remain poorly understood. This hydroponic study investigated the varied responses of 20 Lolium perenne cultivars to elevated zinc, 2-mercaptobenzothiazole, and their combined stress (Zn 20 mg L-1 (Zn treatment group), MBT20 mg L-1 (MBT treatment group), and MBT20 mg L-1 + Zn 20 mg L-1 (MBT-Zn treatment group)). Fifteen physiological and biochemical markers, including biomass, photosynthetic pigments, osmoregulatory chemicals, and antioxidant enzyme activity, were examined. The results demonstrated specific varietal adaptation mechanisms in response to both individual and combined stresses. Most varieties exhibited significant alterations in 13 of 15 traits compared to controls, except for underground biomass and soluble protein content. Across varieties, plant height, aerial/underground biomass, chlorophyll a/b, carotenoids, and root activity decreased under stress. In contrast, the levels of soluble protein, malondialdehyde (MDA), superoxide dismutase (SOD), and free proline exhibited significant increases. These changes were consistent with altered oxidative regulation and protective physiological responses under contaminant exposure. Combined Zn and MBT exposure produced trait-dependent and cultivar-dependent responses. Although combined exposure generally intensified oxidative responses, its effects on growth and photosynthetic traits were not consistently more severe than those of the individual stressors. Several cultivars showed partial attenuation of specific growth effects under combined exposure, indicating a nonadditive interaction between Zn and MBT rather than uniformly greater or lower toxicity. The findings provide a physiological basis for identifying L. perenne cultivars that maintain growth and protective metabolism under simultaneous Zn and MBT exposure. The identified tolerant cultivars represent candidates for further studies of contaminant uptake, translocation, and removal.