Biodegradation Mechanism Of Triphenyl Phosphate By Microbial Consortium And It's Combined Toxicity With Microplastics In HepG2 Cells

Compared with the traditional flame retardant,organophosphorus flame retardants(OPFRs)with higher flame-retardant efficiency and relative environment protection are industrializedly produced and used.Several studies have demonstrated that OPFRs could induce various toxic effects such as neurotoxicity,reproductive toxicity and endocrine disruption,which are called as emerging contaminant.Microbial method is considered to be an important means to remove toxic and harmful environment pollutants.However,knowledges on OPFRs degradation characteristics and mechanism by microbial are rare.Moreover,organism will not just be exposed to a single pollutant in the natural environment,but a complex system composed of multiple pollutants.Due to the characterstics of larger specific surface area and strong hydrophobicity,microplastics become an effective carrier of organic pollutants,and the combined pollution formed by microplastics and organic pollutants may cause potential risks to ecological environment and health of organism.Yet,most studies focused on exploring the toxicity effects of organic pollutants combined with microplastics on aquatic species and mammals,little information is available as for the effects of these combined pollutants on human cell lines and the mechanisms of these action.In the present study,triphenyl phosphate(TPHP)was used as typical contaminant of OPFRs.The objective of this work was to obtain a robust TPHP degrading consortium GYY by screening and domesticating the microorganism in Guiyu electronic waste disassembly site,and to investigate the biodegradation characteristics and transformation mechanism of TPHP by consortium GYY.Additionally,polystyrene(PS)particles were used as representative pollutants of microplastics,and the combined toxic effects and mechanism of co-exposure of TPHP and PS particles on Hep G2 cells were analyzed.The detailed conclusions are as follows:(1)Microbial consortium GYY with efficient capacity to degrade TPHP has been isolated,which could degrade 92.25% of TPHP within 4 h under the optimal conditions(p H 6-7,bacteria dosage 1 g/L wet weight,30?,TPHP initial concentration 3 ?mo L/L).The low concentration of TPHP had no obvious effect on the apoptosis of consortium GYY,while the apoptosis rate increased significantly when the concentration of TPHP was 60 ?mo L/L.The intracellular enzymes functioned critically during TPHP degradation by consortium GYY.The major metabolites of TPHP were identified using high-resolution liquid chromatography-qualitative quadrupole time-of-flight mass spectrometry(LC-Q-TOF-MS),including diphenyl phosphate(DPHP),phenyl phosphate(PHP),monohydroxylated-TPHP(OH-TPHP),remethylated OHTPHP,remethylated dihydroxylated TPHP(remethylated 2OH-TPHP)and methylated DPHP.Consortium GYY degraded and transformed TPHP by hydrolysis,methoxylation after hydrolysis,and methoxylation after hydroxylation pathways.(2)The community structure of consortium GYY was mainly composed of 10 different types bacteria such as Pseudarthrobacter?Sphingopyxis?Methylobacterium?Arthrobacter and Ochrobacterum in view of genus level.There were no significant differences in the relative abundance and diversity index values of consortium GYY before and after enrichment,indicating that consortium GYY had a certain stability.According to the analysis of the composition of consortium GYY during TPHP degradation,the relative abundance of Sphingopyxis?Methylobacterium?Arthrobacter and Pseudomonas increased firstly then decreased,while that of Pseudarthrobacter was opposite with the extension of biodegradation time,suggesting that consortium GYY had the ability of se-regulation and adaptation to counter TPHP stress.In the other hand,Sphingopyxis strain GY-1 and Pseudarthrobacter strain GY-2 were isolated from consortium GYY.Strain GY-1 could degrade 98% of TPHP(3 ?mo L/L)within 7 days and utilize TPHP as the sole carbon to grow,while strain GY-2 degraded TPHP poorly(?10%).(3)The cytotoxicity of TPHP treatment,TPHP combined with PS particles treatment were compared.It was found that the exposure of these two treatments would inhibit Hep G2 cells viability to different degrees.Moreover,the addition of PS particles would cause higher levels of oxidative stress response in cells,the mitochondrial dysfunction,the release of lactate dehydrogenase,and ultimately induce the significant changes of Hep G2 cells apoptosis.PS particles of 0.07 ?m in diameter exhibited higher cytotoxicity than that of 1 ?m.The above results indicated that the apoptosis induced by TPHP and PS particles might be related to ROS-mediated mitochondrial signaling pathway,and the combined pollution of TPHP and PS particles would poison Hep G2 cells more severely.These results in this study revealed the mechanism of microbial degradation and transformation of TPHP,preliminarily explored the cytotoxicity of TPHP treatment,TPHP combined with PS particles treatment on Hep G2 cells,which provided theoretical basis for the development of OPFRs contaminated microbial remediation technology,clarification of the possible toxicity effects of OPFRs combined with microplastics on environment and human health.