Accumulation,Distribution And Transformation Of Organophosphate Flame Retardants In Fish And Their Underlying Mechanism

Organophosphate flame retardants(OPFRs)are a series of chemicals which have been used as flame retardants or plasticizer in a wide range of industries,including textiles,construction,electronics and chemical industries.Many OPFRs are mixed into material and not chemically bonded to the substrates,and thus are easily released into surrounding environment.Through urban sewage pipe networks,these OPs could be finally spread into various water ecosystems.A growing number of toxicological studies have demonstrated that OPFRs such as triphenyl phosphate(TPHP),tris(1,3-dichloro-2-propyl)phosphate(TDCIPP)and tris(2-butoxyethyl)phosphate(TBOEP)elicit effects on embryonic development,heart beating,hormonal secretion,and neurological system in fish or other aqueous organism.Investigations on the in vivo bioaccumulation and bio transformation of compounds are essential for evaluating their toxicity.Up to date,only limited studies have focused on the accumulation of OPFRs in fish,and the transformation or fate of OPFRs in fish is substantially unknown.In the present study,the tissue-specific accumulation,depuration and transformation of seven typical OPFRs(tripropyl phosphate(TPRP),tri-n-butyl phosphate(TNBP),TBOEP,tris(2-chloroethyl)phosphate(TCEP),TDCIPP,TPHP,and tri-p-cresyl phosphate(p-TCP))in adult zebrafish were investigated under laboratory conditions.The underlying mechanisms of accumulation and transformation of OPFRs in fish were explored by the physiologically based toxicokinetic(PBTK)model and the molecular calculation and docking.Results and conclusions drawn from the experiments have been described as follows.(1)A protocol for the extraction and quantification of OPFRs and thier major metabolites from fish tissues was developed.Solution of cyclohexane/ethyl acetate(1:1 v/v)was used for the extraction of OPFRs or metabolites from fish tissues,followed by cleanup procedure including gel permeation chromatography(GPC)and solid phase extraction(SPE)cartridges.Gas chromatography-mass spectrometry(GC-MS)and liquid chromatography-mass spectrometry(LC-MS/MS and LC-Q-TOF MS)were used for the identification and quantification of OPFRs and their major metabolites.(2)The difference in the accumulaiton process of OPFRs in the tissues(brain,intestine,gills,liver,eggs and muscle)of zebrafish was elucidated by exposure zebrafish to three levels of mixed OPFRs solution(0,1/150 LC50,and 1/30 LC50).Aryl OPFRs(TPHP and p-TCP)tended to accumulate in gills and intestines;alkyl or alkoxy OPFRs(TPRP and TNBP or TBOEP)or short chain chlorinated alkyl OPFRs(TCEP)tended to accumulate in roes,brains,livers,and muscles;and polychloroalkyl OPs(TDCIPP)have more proportion in the muscles than those of other OPFRs.For TDCIPP,TPHP,and TCP,larger amount of accumulation and the longest half-lives or slowest depuration rates in roes than those in other tissues indicated that the three OPFRs were potentially stored in roes and transferred to the next generation.The bioconcentration factors(BCFs)of OPFRs increased with the logKow and/or tissue lipid content.The high values of BCFlw were observed in the TPHP-and TCP-exposed zebrafish,ranging from 1925 to 5474 and 3136 to 8909,respectively.These results indicated that,from the weight perspective of lipid,TPHP and TCP might be highly bioaccumulative.The transformation effect of the OPFRs in zebrafish was evaluated useing PBTK model.In the liver of zebrafish,the transformation proportions of TDCIPP,TPHP,and TNBP were approximately 42.3%and 13.7%and 3.4%,indicating that biotransformation had significantly affected the accumulation of TDCIPP,TPHP,and TNBP in fish.However,the remaining approximately 57.7%,86.3%,and 96.6%of the total accumulated TDCIPP,TPHP,and TNBP in liver demonstrated that the partition process between the tissues and water might finally dominate the accumulation process.(3)Potential metabolites of six OPFRs were screened in liver using an Agilent 1200 liquid chromatography coupled with an AB Sciex high resolution qualitative quadrupole time-of-flight mass spectrometry TOF5600(LC-Q-TOF MS).20 metabolites,including organic phosphate diesters,hydroxylated organic phosphate diesters,hydroxylated OPFR triesters,and glucuronic acid conjugated,were detected and successfully identified on the basis of their mass-to-charge ratios and the fragment and isotope patterns.The reaction pathways of OPFRs in zebrafish were proposed based on the detected metabolites,and were further confirmed by the frontier electron density and point charge calculations.Tissue distribution of the 20 metabolites revealed that liver and intestine with the highest levels of metabolites were the most active organs for OPFRs biotransformation,and brain,roe,and muscle might have no contribution to the transformation of OPFRs in zebrafish.Except the high levels of metabolites in tissues,the widely detected organic phosphate diesters and hydroxylated organic phosphate diesters in water indicated that the formed metabolites may be easily released into water,and special attention should be paid to their environmental monitoring and toxic effect assessment.(4)Activities of representative CYP450(ECOD),carboxylesterase(CESE),glutathione transferase(GST),acetylcholinesterase(ACHE)and possible phosphotriesterase(PTE)were studied in the liver of fish after exposure to seven targent OPFRs in water.It was found that the seven OPFRs had no significant effect on PTE activities at the designed exposure concentration of 1/30 LC50.However,TPHP and p-TCP showed sustained significant inhibition of CESE from day 3.Homologous modeling and molecular docking results suggested that the inhibitory effect might be related to the hydrophobic interaction,electrostatic interaction and hydrogen bonding of TPHP or p-TCP with tyrosine 407,glutamic acid 404 and arginine 371 of CESE,respectively.This study provides critical information on the bioaccumulation,distribution,and metabolization of OPFRs in the tissue of fish,which might give some hints for the exploration of their toxic mechanism in aquatic life.