Chronic Toxicity and Intergenerational Effects of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) Exposure Alone and in Combination with Zn on Daphnia Magna (Cladocera)

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and Zn, 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 Zn 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 Zn (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 Zn exacerbated the reproductive toxicity of 6PPD. Across three generations, low-dose (0.02 µg/L) 6PPD alone and combined with Zn induced a cumulative degenerative maternal effect. Conversely, high-dose (20 µg/L) 6PPD, both independently and in combination with Zn, 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 Zn, with no such effect in the group exposed to 20 μg/L 6PPD alone, suggesting that Zn 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.