Toxicity Of Graphene And 17β-estradiol At Environmentally Relevant Concentrations To Zebrafish

Graphene is a two-dimensional carbon nanomaterial composed of carbon atoms with sp2 hybrid orbitals.Because of its excellent physical and chemical properties,graphene has received extensive attention since its discovery and has been widely used in electrochemistry and biomedical fields.During production,processing,and use,graphene inevitably enters the environment and produces toxic effects on organisms.The toxic effects of graphene are not only related to its own concentration,size,functional groups,etc.,but also to the combined biological effects of graphene and other pollutants.Because graphene has large specific surface,it easily adsorb pollutants in the environment,especially organic pollution with benzene rings throughπ-π interaction.17β-estradiol(E2)is a widely naturally occurring estrogen.Studies have reported that E2 has multiple toxic effects on organisms,especially endocrine disrupting effects.In the environment,once graphene coexists with E2,a combined biological effect may occur.In literature,the concentration of graphene used in studies on toxic effects was often relatively high,which was far from the predicted environmentally relevant concentrations of graphene-based nanomaterials,and the study on combined pollution of graphene-based nanomaterials and organic pollutants was still scarce.The combined biological toxicity is unclear,and the assessment on the potential ecological risks is not difficult.Therefore,this study first explored thethe developmental toxicity to zebrafish embryos of three graphene and one carbon quantum dot at low concentrations,including,and subsequently investigated the single and combined effects to zebrafish juveniles of a few-layer graphene(FLG),as a representative of graphene-based nanomaterials,and E2,as a representative of organic pollutants.Finally,the transcriptome method was used to analyze the single and combined effects of FLG and E2 to zebrafish juveniles at genetic level.The main results are as follows:This study first investigated the developmental toxic effects of three graphene and one carbon quantum dot on zebrafish embryos at different low concentrations(<10 mg/L),and sizes,with a focus on survival rate,malformation rate,and hatching rate.The four nanomaterials did not have significant developmental toxicity to zebrafish embryos and did not affect the survival rate of zebrafish embryos,but had a certain degree of effects on the malformation and hatching of zebrafish embryos.Secondly,the toxic effects of FLG and E2 on zebrafish juveniles under single and combined conditions were studied,focusing on the lethality,body weight,body length,and fullness,and the VTG synthesis.The study found that FLG and E2 at environmental concentrations did not have a lethal effect on the juveniles under single and combined conditions,nor did they affect the juvenile’s body length,weight,and fullness.However,E2 and the combined pollution of FLG and E2 at environmental concentration affected the synthesis of VTG in juveniles.And under combined pollution,E2 at environmental concentration induced a synthesis of lower amount of VTG in zebrafish juveniles than under single E2.This could be because under combined conditions,the adsorption of E2 on FLG reduced the bioavailability of E2 and therefore reduced the induced VTG synthesis by E2.Finally,the toxicity of FLG and E2 on zebrafish juveniles under single and combined conditions were studied at the molecular level using transcriptome analysis,with focuses on differential gene expression and enrichment.Analysis of differentially expressed genes through a variety of statistic methods showed that low concentrations of FLG or/and E2 at environmentally relevant concentrations affected protein,lipid,and carbohydrate metabolism,the immune,endocrine,and digestive systems,as wellas the growth and development of zebrafish juveniles.Among them,FLG also affected a series of genes related to respiration.E2 affected ribosomal structure,peptide synthesis,and response pathway of extracellular matrix,and also affected the pathways related to gender differentiation.The presence of FLG reduced the toxicity of E2 on ribosomes and increased the effects of E2 on the pathways related to gender differentiation.