Ecotoxicological Effects And Mechanisms Of DEHP On Earthworms

Phthalates（phthalic acid esters,PAEs）,are typical plasticizers that can increase the stability,plasticity,and durability of plastic products,while offering good cost performance and a wide range of uses.Since PAEs are not polymerized into the plastic matrix,but are linked to it by hydrogen bonds or van der Waals forces,they can easily escape from the plastic and pose a health risk to the soil ecosystem.Currently,PAEs have become the second largest environmental pollutant worldwide and are the most abundant semi-volatile organic pollutants in Chinese agricultural soils.Therefore,pollution issues and ecotoxic effects caused by PAEs are receiving increasing attention and gradually become a hot spot for research in the environmental field.Earthworms can be used as model organisms to assess the soil ecological risk of target pollutants,but current studies have mainly focused on the description of effects of PAEs,while studies on the toxic mechanisms are still unclear,especially at the molecular level,which are relatively lacking and need to be enriched.On the other hand,the toxicological studies carried out so far mainly use Eisenia foetida and Eisenia andrei as model organisms recommended by OECD and ISO,but as representatives of Epigeic earthworms,these two earthworms do not accurately reflect the ecotoxic effects of other ecotypes of contaminants,such as Anecic earthworms（Metaphire guillelmi）.Therefore,in the face of the increasingly serious problem of PAEs pollution,it is of great importance to comprehensively reveal their toxic effects and toxic mechanisms,and to investigate the sensitivity of different earthworms to typical PAEs in order to accurately assess the ecological risk of pollutants and protect soil health.Based on the above findings,di 2-ethylhexyl phthalate（DEHP）,which is used in large quantities and present high-risk,was selected as the target contaminant in this study,E.foetida and M.guillelmi were used as typical soil model organisms.The ecotoxic effects and mechanisms of DEHP on earthworms were evaluated and reveal at the individual,tissue,cellular,genetic and molecular levels using a combination of enzymatic,molecular biological and multi-omics approaches,respectively.The major findings were obtained:（1）Acute toxicity risk of DEHP to both earthworms was relatively low at the individual level,but direct exposure to contaminants induced different levels of toxicity symptoms in earthworms.Results showed that the LC₅₀ for M.guillelmi were 3534.26μg/cm² and 1221.15mg/kg for the filter paper and soil methods,respectively,while the acute lethal effects and body weight inhibition for E.foetida were significantly lower than those for M.guillelmi.At the tissue level,DEHP exposure could also cause tissue damage such as epidermal depression and detachment and thinning of midgut tissue in earthworms.（2）DEHP induced oxidative stress and DNA damage in two earthworms.Contaminant exposure activated ROS levels in earthworms,altered antioxidant enzyme activities,and further induced lipid peroxidation damage in earthworm cell membranes.At the DNA damage level,DEHP exposure significantly increased cellular DNA damage and 8-OHd G content.Additionally,DEHP disrupted the relative expression of functional genes such as ATP-6,NADH1,and COX1/2 in the worm’s mitochondrial respiratory pathway,which posed a potential threat to the normal mitochondrial biological functions and the expression of downstream antioxidant functional enzymes.Ecological risk assessment indicated that DEHP exhibited higher toxic effects on M.guillelmi,and the biological response of sensitive biomarkers to DEHP contaminants was also dependent on earthworm and treatment time.（3）DEHP may influence key enzyme activities by altering protein structure.The results suggest that DEHP contaminants can disrupt the protein backbone structure of SOD/CAT at the molecular level and alter the endogenous fluorescent microenvironment,which in turn affects the structural properties of proteins.Molecular docking further validated the possibility of DEHP binding to two key antioxidant enzymes and revealed that the two binding processes were primarily mediated by hydrogen bonding and hydrophobic interactions.（4）DEHP exposure affected the gut microbial community structure and gut microbial homeostasis in both M.guillelmi and E.foetida.DEHP could alter the diversity and stability of the gut microbial communities of two earthworms and disrupt the balance of the earthworm gut microbial ecological network.In addition,it was shown that two worms were in a position to enhance degradation of contaminants in response to environmental stresses by increasing the abundance of specific microorganisms and making adaptive changes.（5）In this study,the molecular mechanisms underlying the toxic effects of DEHP on earthworms were further investigated by transcriptomic and metabolomic analyses.Transcriptomic analysis showed that significantly more DEGs were altered in M.guillemi than in E.foetida after DEHP exposure,and these significantly DEGs were mainly involved in biological functions such as cellular and metabolic processes and stability of membrane components.Metabolomic analysis showed that DEHP significantly affected three metabolic pathways,namely arachidonic acid metabolism,cysteine and methionine metabolism and unsaturated fatty acid biosynthesis in E.foetida,while 31 metabolic pathways were significantly altered in M.guillemi.These results suggest that the toxic effects of DEHP on M.guillemi were stronger than those of E.foetida,with the former screening for more differentially expressed genes and more complex metabolic pathways.（6）Enrichment capacity of M.guillelmi for DEHP was greater than that of E.foetida.DEHP concentration in the soil of earthworms gradually decreased with increasing treatment time during the exposure phase and showed some time dependence;in the recovery phase,DEHP were also found in the soil to varying degrees,indicating that the undegraded contaminants may have re-entered the soil through earthworm feces.Earthworms were all significantly enriched for DEHP contaminants,with BCFs of 1.48±1.27（M.guillelmi）and1.17±0.93（E.foetida）,indicating that M.guillelmi has a slightly greater enrichment capacity for DEHP contaminants than E.foetida and its bioavailability is also higher,suggesting that DEHP exhibited more intense toxic effects on possibly M.guillelmi.In summary,this study comprehensively assessed the ecotoxic effects of typical phthalate DEHP on earthworms at multiple levels and perspectives,comparing the biological responses of different earthworms to pollutants and screening several sensitive biomarkers,using two earthworms as soil model organisms,respectively.At the same time,the molecular mechanism of the toxic effects of DEHP on earthworms was comprehensively revealed in terms of protein structure,gut microbial community,differential gene expression,and metabolic function and metabolic processes by using a combination of innovative tools such as molecular docking simulation,microbiomes,transcriptomics and metabolomics,and the related research results provide an important basis for accurate assessment of the ecological risk of phthalate esters and soil environmental health.