Phthalate esters (PAEs) are widely used as plasticizers and can be ubiquitously detected in environment. However, the toxic effects and mechanisms of diisononyl phthalate (DINP) on earthworms are still poorly understood. In this study, earthworms (Eisenia fetida) were exposed to DINP at various doses (0, 300, 600, 1200, and 2400 mg/kg) to investigate their subchronic toxicity. The results demonstrated that the reactive oxygen species (ROS) levels displayed an “increase-decrease” trend with the increasing DINP doses after DINP exposure on days 7, 14, 21, and 28. The malondialdehyde (MDA) content increased with increasing DINP doses on days 7, 14, and then decreased on days 21, 28. The values of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) showed similar variation patterns and reached a maximum level on 21 d. Moreover, on day 28, the SOD and CAT gene expression levels were upregulated, while the GST gene expression levels were downregulated. Meanwhile, 16S rRNA genes of E. fetida gut bacteria and surrounding soil bacteria were measured after 28 days of exposure to DINP. The Chao index of E. fetida gut bacteria decreased when the treatment with the highest concentration (2400 mg/kg) was applied. At the phylum level, the abundance of Chloroflexi was significantly lower in the gut of E. fetida. In addition, the abundance of Proteobacteria at the phylum level and Ottowia at the genus level significantly increased in the surrounding soil. Overall, our results shed light on the toxic mechanism of DINP at biochemical, molecular, and omics levels, and contributed to a better understanding of the ecotoxicity of DINP.

邻苯二甲酸酯 (PAE) 广泛用作增塑剂，并且可以在环境中无处不在地检测到。然而，邻苯二甲酸二异壬酯 (DINP) 对蚯蚓的毒性作用和机制仍知之甚少。在这项研究中，蚯蚓（Eisenia fetida) 以不同剂量（0、300、600、1200 和 2400 mg/kg）暴露于 DINP 以研究其亚慢性毒性。结果表明，在 DINP 暴露后第 7、14、21 和 28 天，随着 DINP 剂量的增加，活性氧 (ROS) 水平呈现“升高-降低”趋势。丙二醛 (MDA) 含量随着 DINP 剂量的增加而增加第7、14天下降，第21、28天下降。超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽S-转移酶(GST)的变化规律相似，21 d达到最大值。此外，在第 28 天，SOD和CAT基因表达水平上调，而GST基因表达水平下调。同时，16S rRNA 基因在暴露于 DINP 28 天后测量E. fetida肠道细菌和周围土壤细菌。施以最高浓度（2400 mg/kg）的处理时， E. fetida肠道细菌的 Chao 指数下降。在门水平上， E. fetida肠道中绿屈菌的丰度显着降低。此外，在周围土壤中，门水平的变形菌和属水平的Ottowia的丰度显着增加。总体而言，我们的研究结果揭示了 DINP 在生化、分子和组学水平上的毒性机制，并有助于更好地了解 DINP 的生态毒性。