Toxic Effects And Transcriptomic Mechanisms Of Oil Spill Pollution On The Early Development Of Zebrafish

Over recent years,oil spill accidents occasionally occur worldwide,which could be involved in various types of crude oil and its refined products.The physicochemical properties and chemical compositions of crude oils are varying depending on their geological sources and types.It might result in various toxic effects on aquatic organisms.However,knowledge is limited about the impact of crude oils from different geological sources on the developed fish embryos and their potential mechanisms,based on the combination of phenotypic,physiological,and transcriptomic techniques.Therefore,in the present study,two typical crude oils:Oman crude oil（OCO,a light crude oil）and Merey crude oil（MCO,a heavy crude oil）derived from different geological sources,were selected as the test crude oils.As one of the classical teleost fish model organisms,zebrafish（Danio rerio）embryos were selected as the test organism.Through the 120 h semi-statically acute embryonic exposure test,the present study systematically compared and analyzed the developmental effects and their underlying mechanisms induced by the water-accommodated fractions（WAFs）of two crude oils on zebrafish embryo-larvae,based on the observation,detection,and analysis of morphological development,physiological function,gene expression,transcriptomics sequencing（RNA-Seq）,and chemical analysis.The main obtained results are as follows:（1）The determination for physical and chemical properties of crude oils indicated that there were obvious differences between OCO and MCO.The results showed that the asphaltene content,viscosity,and density of OCO and MCO were 1.75%and 10.73%,13.26 and 164.90mm²?s^-1,and 0.8669 g?cm^-3 and 0.9486 g?cm^-3,respectively.Through the ultraviolet spectrophotometry and the gas chromatography-mass spectrometry（GS-MSD）analysis,total petroleum hydrocarbons（TPH）and polycyclic aromatic hydrocarbons（ΣPAHs）concentrations in MCO WAF solution were 1.54-fold and 1.28-fold higher than those in OCO WAF solutions,respectively.However,through the analysis of the proportions of each PAH,low molecular weight PAHs accounted for the highest proportion both in OCO and MCO WAFs solutions.And the proportion of low molecular weight PAHs in MCO WAF solution was significantly higher than that in OCO WAF solution;while the proportion of high molecular weight PAHs in OCO WAF solution was significantly higher than that in MCO WAF solution correspondingly,especially 3 ring phenanthrene and 4-ring benzo[a]anthracene.Additionally,ΣPAHs levels that were biologically enriched in zebrafish embryo-larvae following exposure to OCO and MCO WAFs were also detected by the GC-MSD.The results showed that theΣPAHs levels in zebrafish embryo-larvae were 522.14±173.26 ng?g^-1 and 867.25±237.36ng?g^-1 for OCO and MCO exposure(oil loading rate:25 g?L^-1),respectively,which indicated that the bioaccumulation contents ofΣPAHs in zebrafish embryo-larvae exposed to MCO WAF was 1.66-fold higher than that exposed to OCO WAF.（2）Both OCO and MCO WAFs exposure caused a significant dose-dependent decrease in the survival and hatching rate of zebrafish embryo-larvae based on the increases of petroleum hydrocarbon compounds concentration.For survival,based on the TPH concentration,the median lethal concentration（LC50）values of OCO and MCO WAFs were 3.58 mg?L^-1 and 4.30mg?L^-1,respectively;or based on theΣPAHs contents,LC50 values were 5.17μg?L^-1 and 6.81μg?L^-1.Results indicated that OCO exposure could be more lethal toxic to zebrafish embryo-larvae than MCO exposure.For the hatching rate,the impact of OCO exposure on the hatching rate of embryos was more significant than that of MCO exposure.（3）OCO and MCO WAFs exposure caused severe developmental delay and teratogenic effects of zebrafish embryo-larvae in a dose-dependent manner.OCO exposure caused severer development delay and teratogenic effects than MCO.The effects of OCO and MCO WAFs exposure on morphological development and teratogenic endpoints were similar to a certain degree.The significant endpoints of developmental delay were mainly involved with hatching delay or hatching failure,abnormal inflation of swim bladder,absence of spontaneous movement,unresorbed yolk sac,and poor blood circulation.The significant endpoints of teratogenic effects were mainly related to pericardial edema,chorda malformation,tail malformation,and scoliosis.（4）OCO and MCO WAFs exposure induced a significant reduction in the indicators of cardiac function（including heartbeat,stroke volume,and cardiac output）in a dose-dependent manner with the increase of petroleum hydrocarbon concentration.Results showed that the cardiac dysfunctions in zebrafish embryo-larvae induced by OCO exposure were significantly higher than those induced by MCO.Cardiac output was much more sensitive than heart rate and stroke volume in zebrafish embryo-larvae to crude oil exposure,which indicated that cardiac output could be a better biochemical indicator for evaluating the cardiac dysfunctions in fish early-life stages following exposure to crude oil pollution.Through the Spearman’s rank-order correlation analysis between cardiac function indicators and low/high molecular weight PAHs levels,the results showed that compared to low molecular weight PAHs,the cardiac dysfunctions induced by exposure to two crude oils had a stronger negative correlation with the content of high molecular weight PAHs,which indirectly indicated that the differences in cardiotoxicity caused by crude oils from different geological sources could be mainly attributed to the differences in the proportion of high and low molecular weight PAHs.Besides,OCO and MCO WAFs exposure down-regulated the gene expressions related to the cardiomyocyte excitation-contraction coupling pathway,including ryr2,atp2a2a,atp2a2b,ncx1h,and kcnh2,in a dose-dependent manner with the increase of petroleum hydrocarbon concentration.It could be one of the main molecular mechanisms involved with cardiac dysfunctions in zebrafish embryo-larvae following crude oil exposure.（5）Both OCO and MCO WAFs exposure caused a significant increase in the frequencies of swim bladder non-inflation and a significant decrease in the eye areas of zebrafish embryo-larvae,and the effects of OCO WAFs exposure were much higher than that of MCO WAFs exposure.Through the quantitative real-time PCR analysis,we found that crude oil could inhibit the gene expressions related to swimming bladder tissue layer structure,including anxa5,acta2,and hb9,resulting in the disruption of swim bladder development and inflation.The results also showed that crude oil could significantly inhibit the gene expressions related to eye development,including notch1a and notch1b,leading to a decrease in eye areas.（6）RNA-Seq analysis manifested that OCO and MCO WAFs sublethal exposure(oil loading rate:1 g?L^-1)induced 961 and 957 genes that were differentially expressed in zebrafish embryo-larvae,respectively.Based on the function analysis and pathway enrichment analysis of the differentially expressed genes,the gene ontology GO terms and the KEGG enrichment pathway of the differentially expressed genes induced by crude oil exposure had a high degree of similarity between the two crude oils.Among these KEGG enrichment pathways,visual phototransduction and retinol metabolism were one of the significantly enriched pathways in the two crude oil exposure groups,which indicated that crude oil exposure could disrupt the expression of key genes related to visual phototransduction and retinol metabolism,subsequently leading to abnormal eye development and disrupting the rhythmic photodynamic response during the early development of zebrafish.Additionally,sublethal exposure to crude oil WAFs could also disrupt the Ah R-mediated activation of CYP1A signaling pathways,steroid biosynthesis processes,and immune-inflammatory related pathways（including cytokine-cytokine receptor interactions,NOD-like receptor signaling pathway,and Toll-like receptor signaling pathway）,and other biological metabolic pathways,which could in turn affect the development and metabolism processes in the early life stage of zebrafish.The results of the present study could provide primary experimental data for the more accurate assessment of the toxic effects of oil spill pollution on aquatic organisms and also provide a solid theoretical scientific basis for the environmental impact assessments of oil spill accidents that may occur in the future.