Chronic toxicity and intergenerational effects of N-(1,3-dimethylbutyl)-N’-phenyl-p-phenylenediamine (6PPD) exposure alone and in combination with Zn²⁺ on Daphnia magna (Cladocera)

N-(1,3-dimethylbutyl)-N’-phenyl-p-phenylenediamine (6PPD) and Zn²⁺, extensively used in the tire manufacturing process, are frequently detected in freshwater environments. However, the intergenerational effects of isolated 6PPD exposure and joint 6PPD and Zn²⁺ exposure at concentrations approximating environmental levels remain unknown. This study assessed the chronic toxicity and intergenerational effects of 6PPD (0.02–20 μg/L) and a mixture of 6PPD and Zn²⁺ (5 μg/L) over three generations in Daphnia magna bioassays. In the F0 generation, a dose-dependent decline in total offspring number was observed with 6PPD exposure alone, while co-exposure with Zn²⁺ exacerbated the reproductive toxicity of 6PPD. Across three generations, low-dose (0.02 µg/L) 6PPD alone and combined with Zn²⁺ induced a cumulative degenerative maternal effect. Conversely, high-dose (20 µg/L) 6PPD, both independently and in combination with Zn²⁺, exhibited an adaptive maternal effect. Notably, the grandmaternal effect emerged exclusively in the co-exposure group treated with 20 μg/L 6PPD and 5 μg/L Zn²⁺, with no such effect in the group exposed to 20 μg/L 6PPD alone, suggesting that Zn²⁺ may enhance the potential toxicity of 6PPD. Overall, this study provides novel insight into the intergenerational impacts of environmentally relevant levels of 6PPD alone and in combination with a heavy metal, elucidating the environmental risks posed by tire-derived chemicals through their synergistic effects on transgenerational toxicity.