Effects Of Diisononyl Phthalate Exposure On The Oxidative Stress And Gut Microorganisms In Earthworm

Phthalate acid esters are the most widely used type of plasticizers,which are commonly applied to agriculture plastic films,food packaging,daily necessities,and medical equipment.In recent years,because of the implementation of regulation measures,new polymer phthalate plasticizers,represented by di-isononyl phthalate(DINP),are gradually replacing the highly toxic di(2-ethylhexyl)phthalate(DEHP).DINP is characterized by its high molecular weight and long half-life.Moreover,like other PAEs,DINP and carbon chains are bound through hydrogen bonding and van der Waals forces,making it easy for the DINP to be released into the surrounding environment.Thus,the extensive use of DINP may have potential toxic effects on organisms.Currently,most studies focused on the toxicity effects of DINP on model organisms,such as rats,Danio rerio,and Sparus aurata.However,the influences of DINP on terrestrial invertebrates have rarely been reported.In this study,we selected Eisenia fetida as a bioindicator and investigated the subchronic toxicity of DINP exposure(7,14,21,and 28 d)on Eisenia fetida by an artificial soil test.Meanwhile,Illumina high-throughput sequencing technology was applied to study the influence of DINP on variations in diversity and differences between groups of the bacterial community in Eisenia fetida gut and surrounding soil.This study shed light on the toxic mechanism of DINP at biochemical,molecular and omics levels,which contribute to providing important information for the ecological risk assessment of DINP in soil.The main results are as follows:(1)At the biochemical levels,DINP exposure can induce Eisenia fetida to produce oxidative stress and DNA damage.After exposure to different concentrations of DINP,reactive oxygen species(ROS)levels in Eisenia fetida significantly increased throughout exposure period,causing varying degrees of activation of antioxidant enzymes(SOD and CAT)and detoxifying enzyme(GST).DINP exposure can also lead to lipid peroxidation and DNA damage in Eisenia fetida,which is specifically manifested by the increase of malondialdehyde(MDA)and 8-hydroxydeoxyguanosine(8-OHd G)contents.(2)At the molecular level,DINP exposure can regulate the expression progress of the corresponding antioxidative and detoxifying enzyme genes(SOD gene,CAT gene and GST gene).After DINP exposure,the SOD gene expressions of most treatments downregulated in the earlier stage(7,14 and 21 d),while the SOD gene expressions upregulated on 28 d.Compared with the control,the CAT gene expressions of most treatments upregulated on 7,21 and 28 d,while the CAT gene expressions downregulated on 14 d.However,compared with the control,the GST gene expressions of most treatments downregulated throughout exposure period.(3)According to the consequence of Illumina high-throughput sequence,DINP exposure can influence the bacteria community diversity and structure in Eisenia fetida gut and surrounding soil.The bacteria communities of Eisenia fetida gut and surrounding soil showed different responses to DINP exposure.Compared with the control,Shannon index had no significant difference in all treatments of Eisenia fetida gut and surrounding soil,Chao index remarkably decreased only in the highest concentration treatment of Eisenia fetida gut.DINP significantly increased the abundance of Chloroflexi and reduced the abundance of Bacteroidota in Eisenia fetida gut.In surrounding soil,the abundance of Proteobacteria significantly increased after DINP exposure,while the abundance of Bacteroidota and Firmicutes remarkably decreased.At genus level,no significant variations were observed in Eisenia fetida gut,but two genera(Ottowia and unclassified?f?Comamonadaceae)showed significant changes in surrounding soil.