The Effects And Toxic Mechanisms Of Combined Exposure Of Polystyrene Microplastics And Lead On Neurobehavior In Zebrafish

Microplastics（MPs）are plastic particles less than 5 mm in diameter.MPs are widespread in the environment and co-occur with multiple pollutants.Polystyrene has large yields and are widely used,and the polystyrene microplastics（PS-MPs）produced by their crushing have been detected in many surface waters.PS-MPs accumulate in the brain of fish,threatening the nervous system of fish.Similarly,it has been reported that Lead（Pb）can damage nervous system in living organisms.In aquatic environments,Pb often co-occur with PS-MPs,However,the current studies on PS-MPs and Pb have mainly focused on single toxicity.Studies on the combined toxicity of PS-MPs and Pb are rarely reported,especially for neurobehavioral toxicity.In this study,zebrafish（Danio rerio）were used as experimental animal.The embryos and adult fish were exposed by Pb（10 μg/L）in combination with PS-MPs at different concentrations（25 μg/L,250 μg/L）and sizes（50 nm,25 μm）.The toxic effects of combined exposure to PS-MPs and Pb on neurobehaviors（including light-dark stimulation and percussion response,motor behavior,anxiety behavior,etc.）were investigated by means of behavioral detection,apoptosis detection,expression of genes related to neurodevelopment,and quantification of PS-MPs and Pb in tissues.Whether PS-MPs affected the combined toxicity by interfering with lead uptake in zebrafish was verified by adding desferrioxamine（DFO）to increase the expression of the divalent cation transporter（dmt1）gene.The study clarified the effect and mechanism of PS-MPs and Pb on the neurobehavior of zebrafish and provided new data for the effect of microplastics and heavy metal composite pollution.Through the research,the following results are obtained:1.PS-MPs could accumulate in zebrafish.50 nm PS-MPs could penetrate cells and accumulate in the fish brain,however 25 μm PS-MPs could not enter the brain.2.PS-MPs and Pb could affect the neurobehavior of zebrafish such as light-dark stimulus response,movement,anxiety and so on.Exposure to 50 nm PS-MPs at a concentration of 250 μg/L for 5 days affected the ability of zebrafish to respond to light-dark stimuli and tapping,however,exposure to 25 μm PS-MPs or Pb alone did not affect the locomotion of zebrafish.Combined treatment of PS-MPs and Pb compared with single treatment of lead,PS-MPs at 50 μm affected the ability of larvae to respond to stimuli and increased anxiety behavior in adults,while 25 μm PS-MPs did not produce similar effects.3.PS-MPs and Pb could affect apoptosis in zebrafish brain.For embryos and larvae,PS-MPs and Pb increased the expression ratio of bax/bcl2 genes at each treatment time.Combined treatment of PS-MPs and Pb compared with single treatment of Pb,50 nm PS-MPs increased the gene expression of apoptotic factors and increased the number of apoptotic bodies to upregulate apoptosis level,while 25 μm PS-MPs reduced the level of apoptosis by Pb.4.PS-MPs and Pb could affect the expression of neurodevelopment-related genes in zebrafish.The expression of gap43,syn2 a and α1-tubulin genes was affected by the treatment of 50 nm PS-MPs or Pb.Combined treatment of PS-MPs and Pb compared with single treatment of Pb,50 nm PS-MPs reduced gene expression in larve and increased expression in adult fish,while 25 μm PS-MPs did not produce similar effects.5.In the combined treatment group,PS-MPs reduced the content of Pb in zebrafish.PS-MPs could reduce the concentration of Pb in the water to reduce the availability of Pb in zebrafish,and at the same time downregulated the expression of dmt1 gene in zebrafish and reduced the transport of Pb,thereby reducing the contents of Pb in zebrafish.6.PS-MPs could alleviate Pb-induced neurotoxicity by downregulated the expression of dmt1 gene.Up-regulation of dmt1 gene expression with DFO increased the uptake of Pb and the gene expression of gap43 and α1-tubulin was affected.In conclusion,the neurobehavioral effects of PS-MPs and Pb combined exposure on zebrafish were as follows: The 50 nm PS-MPs aggravated neurobehavioral damage of Pb,while 25 μm PS-MPs alleviated neurobehavioral damage of Pb.The mechanism of 25 μm PS-MPs alleviating neurobehavioral damage was to reduce the concentration of lead in water and the expression of dmt1 gene.Although the 50 nm PS-MPs had similar effects,they could enter the zebrafish brain and had neurobehavioral toxicity due to their small size,and the combined treatment showed an increase in neurobehavioral toxicity in zebrafish.In brief,this study investigated the neurobehavioral effects and toxicity mechanisms of PS-MPs and Pb on zebrafish and provided evidence and data on the neurobehavioral toxicity of combined treatment of microplastics and heavy metal pollutants on fish.