Effects Of Tris (1,3-dichloro-2-propyl) Phosphate On Growth And Reproduction Of Daphnia Magna At Environmentally Relevant Concentrations

In recent years,brominated flame retardants have been phased out in many countries and regions,and as their substitutes,organic phosphorus flame retardants have been broadly utilized in various industries such as construction,electronics,textile,funitures,transportation and others.Tris(1,3-dichloro-2-propyl)phosphate(TDCIPP)is a typical organic phosphorus flame retardant with excellent flame retardancy,good oil resistance and hydrolysis resistance,whose total production and consumption amounts have increased significantly during the last decades.TDCIPP is usually physically-bonded to the related materials,therefore it is easy to released into the environment through wear and volatilization.As a result,the presence of TDCIPP can be detected in natural water and domestic water in various countries around the world currently,with its concentrations range from tens to thousands of ng/L.Previous studies have indicated that TDCIPP is rather toxic to aquatic organisms.However,these studies mainly focused on toxicity tests with TDCIPP above the environmentally relevant concentrations,and the toxic effects of environmentally relevant concentrations of TDCIPP on aquatic organisms remain unknown.In order to provide more scientific data to study the potential risks of TDCIPP to the environment and human health,freshwater planktonic organism Daphnia magna(D.magna)was used as test organism in this study,and environmentally relevant concentrations of TDCIPP were used to conduct sub-chronic exposure(28 d)and whole-life-stage exposure experiments and to evaluate developmental and reproductive toxicities and possible mechanisms of TDCIPP to D.magna through examinations of phenotypic indicators and gene transcription.The main works and findings are as follows:1.Sub-chronic exposure experiment was conducted to D.magna with environmentally relevant concentrations(50,500,5000 ng/L)of TDCIPP to evaluate the dose-and time-dependent effects on development and reproduction.Nucleic acid sequences and EST sequences of D.magna were obtained from the National Center for Biotechnology Information(NCBI),then key pathways of D.magna were obtained and PCR array was established by sequence stitching,de-redundancy and KEGG pathway analysis to study transcriptional response of D.magna to TDCIPP.The results indicated that exposure to environmentally relevant concentrations of TDCIPP significantly 2020 inhibited growth of D.magna and reduced its accumulated offspring number and body length of the neonates.Transcription response results showed that expressions of 57 of the155 genes involved in the PCR array were altered after TDCIPP exposure,and these genes were mainly enriched in protein synthesis and metabolic-related pathways and cell phagocytosis-related pathways.Time-effect experiment indicated that changes in gene expressions preceded changes in phenotypic indicators as exposure time prolonged,and the developmental and reproductive toxicities of TDCIPP to D.magna were mainly caused through altering gene expressions and further interfering with normal protein anabolic metabolism and cell phagocytosis.2.A method to evaluate the whole-life-stage developmental and reproductive toxicity of chemicals to D.magna was established by characterizing its basic biology with the growth,reproduction and survival curves of the whole-life-stage and transcriptome sequencing.According to the curves,the whole-life-stage of D.magna was divided into birth,growth,reproduction and death stage.Day 0,6,32 and 62 were selected as the representative sampling time-points for the four life stages,respectively.Stage-specific PCR array was established by comparing the transcriptome profiles of two continuous life stages and filtering key pathways that regulate each life stage and locating the most enriched genes in each pathway.In the whole-life-stage exposure experiment,by sampling at the time-points at the growth,reproduction and death stage and examining developmental and reproductive phenotypic indicators and gene transcription at each life stage,the relationship between the lowest observed effect concentration(LOEC)and the exposure time was obtained.By examining the expressions of vital genes that regulating each life stage,the potential toxic mechanisms at each life stage were understood,and thus it is useful to comprehensively evaluate the developmental and reproductive toxicity effects of chemicals on the whole-life-stage of D.magna.3.D.magna was exposed to environmentally relevant concentrations(300,3000ng/L)of TDCIPP for the whole-life-stage and effects on development,reproduction and gene transcription were evaluated at different life stages.The results illustrated that environmentally relevant concentrations of TDCIPP significantly inhibited growth and reproduction of D.magna and as exposure period prolonged,the LOEC decreased by an order of magnitude.Besides,TDCIPP significantly reduced survival rate on day 62,however the adverse effects were not observed on day 6 and day 32.The results suggested that partial-life-stage exposure experiments might underestimate the environmental risksof TDCIPP to some extent.In addition,gene transcription results revealed expressions of genes involved in the DNA replication and repair pathways were significantly down-regulated on day 6 after TDCIPP exposure;The expressions of genes involved in the pathways associated with cell cycle,splice and cell transcription were significantly down-regulated on day 32;On day 62 of TDCIPP exposure,the expressions of all genes contained in the pathways associated with dilated cardiomyopathy(DCM)and hypertrophic cardiomyopathy(HCM)were significantly up-regulated.Gene trascription results indicated that the developmental toxicity of TDCIPP to D.magna at the growth stage might be related to the inhibition of DNA replication and repair;the reproductive toxicity of TDCIPP to D.magna at the reproduction stage might be the results of inhibition of cell cycle,splicing and cell transcription pathways;and TDCIPP might promote the process of loss of heart function at the death stage of D.magna and further shortened its average life expectancy.