Study On The Pollution Level Of Typical Phthalates In Environmental Water And Reproductive Toxicity Of Combined Exposure In Zebrafish

Phthalate esters?PAEs?are a class of environmental hormones.Their ecological risks,toxic effects and mechanisms of action have attracted widespread attention.It has been reported that PAEs can impair the reproductive capacity of the organism and affect the development of offspring.At present,there are few studies on the reproductive toxicity of PAEs to aquatic organisms,and most of them focus on the effects of single PAEs exposure.However,there are multiple combinations of PAEs in actual water environment.The reproductive toxicity and their mechanisms of combined PAEs on aquatic organisms need to be studied urgently.Therefore,the pollution level,source distribution and ecological risks of typical PAEs in the main rivers of Zhenjiang were studied.Based on the above study,zebrafish was used as a model organism to investigate the reproductive toxicity of combined exposure to dibutyl phthalate?DBP?and diisobutyl phthalate?DiBP?.Transcriptome sequencing combined with morphological,pathological and other molecular biological methods were used to explore the potential reproductive toxicity mechanism of combined exposure to DBP and DiBP.This study will provide theoretical basis for the ecology and health risks of PAEs pollutants.The main contents of this study are given below:?1?Pollution level,source and ecological risk assessment of PAEs in the main rivers of Zhenjiang were studied.Six typical PAEs?DMP,DEP,DiBP,DBP,DEHP and DOP?in water,suspended particulates?SPM?and sediment from the Yunliang River,the ancient canal,and the Beijing-Hangzhou Grand Canal during the wet and dry seasons were detected by the solid-phase extraction?SPE?coupled with gas chromatography-mass spectrometry?GC-MS?.The distribution,sources and ecological risk of six PAEs were investigated.The results indicated that the total concentrations of six PAEs??₆PAEs?spanned a range of 2.65-39.31?g/L in water,1.97-34.10?g/g in SPM and 0.93-34.70?g/g in sediment.DiBP,DBP and DEHP were the dominant PAEs in water,while DiBP,DEHP and DOP were the dominant PAEs in SPM and sediment.K_d1 of PAEs in water and SPM phase ranged from 0.004 to 3.36 L/g in the wet season and from 0.12 to 2.84 L/g in the dry season.K_d2 of PAEs in water and sediment phase was 0.001-9.75 L/g in the wet season and 0.006-8.05 L/g in the dry season.Multivariate statistical analysis showed that the discharge of domestic sewage and industrial wastewater might be the main potential sources of PAEs in the main rivers of Zhenjiang.The risk quotient?RQ?method used for the risk assessment revealed that DBP?0.01<RQ<1?posed a medium risk,while DiBP and DEHP?RQ>1?posed a high environmental risk in water,DiBP?RQ>1?also showed a high risk in sediment.It can be seen that there was no significant seasonal difference in the distribution of?₆PAEs,DBP and DiBP coexist in water,SPM and sediment samples,and may present exposure risks.?2?The reproductive toxicity and its mechanism of combined exposure to DBP and DiBP on adult male zebrafish were studied.Plasma sex hormone assay results showed that testosterone?T?levels of combined exposure were higher than single DBP and DiBP exposure.The pathological analysis of testis showed that combined exposure not only reduced the sperm count but also expanded intercellular spaces when comparing with DBP,DiBP single exposure.Transcriptome analysis revealed that there were 4570DEGs in the combined exposure,while DEGs in single DBP and single DiBP exposure were 2795 and 1613,respectively.The GO function annotation of DEGs found that there were 25 reproduction-related DEGs in the combined exposure,while reproduction-related DEGs in single DBP and single DiBP exposure were 10 and 5,respectively.KEGG pathway analysis showed that both single and combined exposure could act on the cytokine-cytokine receptor interaction signaling pathway.The difference was that combined exposure also affected steroid hormone synthesis,retinol metabolism,PPAR signaling pathway and Extracellular matrix receptor interaction signaling pathways.It can be seen that combined exposure of DBP and DiBP presented reproductive toxicity on male zebrafish,which may be produced by acting on cytokines and cytokine receptors interaction,steroid hormone synthesis,retinol metabolism,extracellular matrix receptor interactions and PPAR signaling pathways.?3?The reproductive toxicity and its mechanism of combined exposure to DBP and DiBP on adult female zebrafish were studied.Ovarian pathological analysis showed that single DBP exposure induced loss of contact between the oocyte cell membrane and the follicular cell layer,and the proportion of late/mature oocytes?LO?decreased to 17.3%.Single DiBP exposure induced loss of contact between oocyte membrane and follicular cell layer and oocyte atresia,and the proportion of LO decreased to 16.2%.No lesions were observed in the combined exposure.Plasma sex hormone assay results showed that the estradiol?E2?levels of single DBP exposure and single DiBP exposure were decreased by 38.9%and 41.0%,respectively,while E2 level of combined exposure were not significantly affected.However,ovarian transcriptome sequencing revealed that DEGs in single DBP and DiBP exposure were 3135 and 3645,respectively,while DEGs in combined exposure were 2564.The GO function annotation of DEGs found that the reproduction-related DEGs in single DBP and single DiBP exposure were 16 and 27,respectively,while reproduction-related DEGs in the combined exposure were 13.KEGG pathway analysis showed that both single and combined exposure could act on the neuroactive ligand-receptor interactions,gonadotropin-releasing hormone,progestin-mediated oocyte maturation,oocyte meiosis and steroid hormone biosynthetic signaling pathways.The difference was that single DBP and single DiBP exposure could also affect choline metabolism,extracellular matrix-receptor interaction and cell adhesion signaling pathways.It can be seen that combined exposure of DBP and DiBP showed potential reproductive toxicity on female zebrafish at the molecular level.?4?The early development of zebrafish offspring and its mechanism of parental combined exposure to DBP and DiBP were studied.The results showed that both parental single exposure and parental combined exposure induced significant changes in early developmental indicators?heart rate,hatchability,mortality and malformation?of the offspring.The spawning rate of zebrafish were significantly decreased in parental single DBP and single DiBP exposure,while those in parental combined exposure were not significantly changed.Transcriptome sequencing revealed that DEGs of the offspring in parental single DBP and single DiBP exposure were 701 and 1492,respectively,while those in combined exposure were 139.The GO function annotation of DEGs found that DEGs related to visual development appeared in both parental single and combined exposure.The difference was that DEGs in parental single DBP exposure also involved metabolism,redox and sterol transport,and those in parental single DiBP exposure also involved in polymer cytoskeleton fiber,shrink fiber and various enzyme activities.KEGG pathway analysis showed that parental single DBP and single DiBP exposure could act on the light transduction signal pathway.The difference was that parental single DBP exposure also acted on the fat digestion and absorption,protein digestion,vitamin digestion and absorption signaling pathways,and parental single DBP exposure also acted on proteasomes,circadian rhythms and bile secretion signaling pathways.It can be seen that parental combined exposure of DBP and DiBP could affect the eye development of the offspring by interfering with the expression of genes related to visual perception.In summary,there were gender differences in the reproductive toxicity of adult zebrafish after single and combined exposure to DBP and DiBP,male zebrafish had higher reproductive toxicity than female zebrafish.The toxicity of parental combined exposure to DBP and DiBP on the early development of zebrafish offspring was consistent with the reproductive toxicity of female zebrafish exposed to DBP and DiBP,both of which were lower than the single exposure.Transcriptomics analysis speculated that combined exposure to DBP and DiBP produced reproductive toxicity by changing the expression levels of key genes in signal pathways related to reproduction.Parental combined exposure of DBP and DiBP changed the expression levels of genes related to visual perception during early development of progeny,then affected the eye development of the offspring.