Ecotoxicity Effect And Mechanism Of Organophosphorus Esters (OPEs) On Earthworm Eisenia Fetida

Organophosphorus esters(OPEs),a class of organophosphorus flame retardants,have been widely applied in a range of fields including furniture,textile,building materials and electronic industries.Given that OPEs are mainly used as additive chemicals that just mixed and not chemically bonded to polymer matrices,they tend to be migrated into the surrounding environment by volatilization,leaching and abrasion.Increasing concentrations of OPEs have been detected in variousenvironmental compartments such as water,indoor dust,outdoor air and soil.Thus,OPEs have drawn growing concern about characterizing the potential risk on environmental health.However,study on ecotoxicity effects of OPEs were mainly confined to fish and birds.This study focus on ecotoxicity of OPEs with different substituents(chlororalkyl-OPEs:tris(1,3-dichloro-2-propyl)phosphate(TDCP)and tris(2-chloroethyl)phosphate(TCEP),non-chlorinated alkyl-OPEs:trimethyl phosphate(TMP)and Tri-n-butyl phosphate(TnBP),aryl-OPEs:tricresyl phosphate(TCP))on earthworm Eisenia fetida.Filter contact test and artificial soil test were applied to study the acute toxicity and toxicity effects of OPEs.Further,omics,including transcriptomics and metabolomics were employed to investigate the toxic mechanism of TnBP on earthworm Eisenia fetida.Results and conclusions drawn in this study have been described below in details.(1)LC50 was determined to uncover the acute toxicity of 5 OPEs on Eisenia fetida including TDCP,TCEP,TMP,TnBP and TCP with filter contact test.Their toxicity are arranged as follows:TnBP>TCP?TMP>TDCP?TCEP.(2)We further studied toxicity effects of TCEP,TCP and TnBP on Eisenia fetida by conducting artificial soil tests.Results showed that OPEs(10 mg/kg)had no significant inhibition on weight growth rate but could cause tissue injury.Three OPEs could all induce antioxidant enzymes and cause oxidative stress.Comet assays showed that DNA strand break might occur under the exposure concentration of 1 mg/kg for all 3 OPEs.OPEs showed no significant influence of the activity of AChE.By calculating Integrated Biomarker Response v2(IBRv2),we obtained the integrated toxicity of 3 OPEs and found that the toxicity of TnBP ranked the first,followed by TCP and TCEP.(3)TnBP accumulation in intestinal tissues caused the structure and digestive function damage.Combined the methods of transcriptome and metabolomics,the potential mechanism of intestinal damage caused by TnBP was explored,then the corresponding proteins and genes were selected for verification.It was found that TnBP destroyed the stability of the cell membrane for the decrease HEFS concentration.Moreover,the activity of Ca2+-ATPase decreased,causing Ca2+ unable to pump out the cells through Ca2+-ATPase.The increase of osmotic pressure in the membrane lead to the destruction of cellular structure.The tight junction between small intestinal epithelial cells were destroyed for the down-regulation of occludin and Zonula occludens-1,ZO-1 genes.The membrane damage and tight junction break of small intestinal epithelial cells caused the intestinal epidermal cells exfoliation.In addition,TnBP exposure significantly reduced the diversity of intestinal microorganisms and altered the intestinal microbial community structure.Among them,the abundance of two genus producing short-chain fatty acid(SCFA)significantly reduced and the ability to produce intestinal short-chain fatty acids decreased,making the intestinal tract prone to inflammatory response.(4)TnBP accumulation in nerve tissues disrupted the delivery of excitatory neurotransmitter glutamate and caused excitatory neurotoxicity.The abdominal nerve cord and head ganglion were studied respectively through the transcriptome and metabolomics methods,we explored the potential neurotoxicity mechanism caused by TnBP and the corresponding protein and the expression of genes was conducted for further verification.We found that TnBP increased expression of glutaminase and produce a large number of glutamate in the abdominal nerve cord.The transport of glutamate through synaptic vesicles was accelerated and the release of large glutamate in presynaptic membrane caused excessive stimulation of ionic receptor NMDARs.During the procedure,calcium accumulation was also caused in postsynaptic membrane.As the reduction of Ca2+-ATPase activity,the calcium in postsynaptic membrane cannot be transported out of the cell in time,resulting in the calcium overload.On the other hand,TnBP exposure reduced the expression of glutaminase and the content of glutamate decreased in the head ganglion.The reduction of Cal2+-ATPase activity caused the calcium in postsynaptic membrane cannot be transported out of the cell in time,resulting in the calcium overload.In addition,the reduction of Na+/K+-ATPase and Ca2+-ATPase activity caused the accumulation of Na+ and Cal2+ in the cells,resulting in the osmotic imbalance and neuronal injury.This study systematically investigate the ecotoxicity effects of OPEs with 3 different substitutes on earthworm Eisenia fetida and uncover the potential toxic mechanism,which provided theoretical support for its pollution control benchmark and environmental assessment.