| Microplastics can be enriched in a large amount in the water environment due to its stable nature and difficult degradation.In particular,the application of plastic cultivation equipment and the cultivation method of placing plastic film at the bottom of pond have aggravated the threat of microplastics to aquatic organisms.In this paper,benthic omnivorous juvenile Paramisgurnus dabryanus was used to investigate the growth effects of microplastics(0.5 μm and 5 μm)with different particle sizes at concentrations of 100 μg/L and 1,000 μg/L and the degree of enrichment in the liver,intestine and gills of the loach;The injury of live tissue and that apoptosis of hepatocytes by microplastics were investigated;The effects on the enzymes related to the antioxidant system in the liver,the ATPase in the gills and the genes related to the Keapl-Nrf2 signaling pathway were also analyzed.These results will provide valuable reference for studying the toxic effects of microplastics with different particle sizes on freshwater fish.The main experimental results the paper are as follows:(1)In this experiment,the fluorescence polystyrene microplastics with the particle size of 0.5 μm and 5 μm was used for one week enrichment experiment in the water environment with the concentration of 1,000 μg/L.The degree of enrichment in liver,intestine and gill was determined by the tissue digestion method.The results showed that the enrichment of 5 μm microplastics in liver was 1.53 times and 1.25 times that in intestine and gill,respectively;The enrichment of 0.5 μm microplastics in liver was 1.77 times and 2.3 times that in intestine and gill,respectively.In liver and intestine,the enrichment of 0.5 μm microplastics was 1.56 times and 1.39 times that of 5 μm microplastics,respectively.In this experiment,the microplastics with the particle size of 0.5 μm and 5 μm was used for 21 days toxicity experiment in the water environment with the concentration of 100 μg/L and 1000 μg/L.The results showed that under the exposure of 100 μg/L microplastics,the survival rates of loach in the 5 μm and 0.5 μm groups were 5.7%and 11.6%lower than that of the control group,respectively;The weight gain rates were 43.02%and 28.93%lower than that of the control group,respectively;And the specific growth rates were 0.85%and 0.78%lower than that of the control group,respectively.Under the exposure of microplastics at 1,000 μg/L,the survival rates of loach in the 5μm and 0.5 μm groups were 13.0%and 17.7%lower than that of the control group,respectively;The weight gain rates were 47.44%and 61.08%lower than that of the control group,respectively;And the specific overgrowth rate was 0.93%and 1.02%lower than that of the control group,respectively.The survival rates of loach in the 0.5μm microplastics group at two concentrations(100 μg/L and 1,000 μg/L)were 5.9%and 4.7%lower than those in the 5 μm microplastics group,respectively.(2)In order to study the tissue damage of microplastics on the liver of loach,histopathological observation and apoptosis experiment were conducted in this experiment.The results showed that microplastics could induce liver injury,such as blood sinus congestion and dilation,cell vacuoles and cell necrosis,and the degree of injury increased with the increase of concentration;Even liver tissue bleeding and cytoplasmic PH changes were observed after exposure to 1,000 μg/L high concentration of microplastics.TUNEL staining showed that under the exposure of 1,000 μg/L high concentration of microplastics,the apoptotic index of hepatocytes in the 5 μm group and 0.5 μm group was 3.25 times and 5.12 times that of the control group,respectively.(3)In order to explore the effects of microplastics on the liver and gills of juvenile loach,the content of lipid peroxidation(MDA)in the liver,the activities of antioxidant system-related enzymes(SOD,CAT,AChE,GSH-PX)and the activities of gill ATP enzymes(Ca2+-ATP,Mg2+-ATP,Na+/K+-ATP)were measured.Results showed that compared with the control group,the activities of antioxidant system-related enzymes in different experimental groups increased in the early stage of the experiment and decreased significantly at the end of the experiment(p<0.05);The content of MDA at the end of the experiment was also significantly higher than that in the control group(p<0.05);On day 21,ATPase in most of the experimental groups was inhibited under the exposure of 1,000 μg/L high concentration of microplastics.In addition,under the exposure to high concentration of 1,000 μg/L microplastics,the degree of inhibition of CAT,GSH-PX,Ca2+-ATPase and Mg2+-ATPase in the 0.5 μm microplastics group was greater than that in the 5 μm microplastics group on day 21,and the content of MDA was also higher than that in the 5 μm microplastics group.(4)In this experiment,we further measured the relative expression levels of genes related to Keapl-Nrf2 signaling pathway(Nrf2,Keap1,SOD,CAT,GSH-PX)that defend the body against oxidative stress from the molecular perspective.The results showed that,compared with the control group,the expression levels of Nrf2 and Keap1 in most experimental group tended to increase at the early stage of the experiment and decrease at the end of the experiment.The change trend of the expression levels of downstream antioxidant enzyme genes in the signaling pathway was generally consistent with the change trend of the expression levels of Nrf2 and Keapl.And the change trend of antioxidant enzyme genes was basically consistent with the change trend of antioxidant enzyme activities.In summary,microplastics could be enriched in the liver,intestine and gills of Paramisgurnus dabryanus,reducing its survival rate and adversely affecting the growth of juvenile loach.Microplastics could induce oxidative stress in loach,thereby activating the Keapl-Nrf2 signaling pathway related to the antioxidant system of the body and promoting the expression of antioxidant enzyme genes.However,with the prolongation of exposure time,the structural integrity of the liver and gills of loach was damaged,and the apoptosis of hepatocytes was aggravated.The activities of antioxidant enzymes,gill ATPase and the expression of genes related to Keapl-Nrf2 signaling pathway were all inhibited.In addition,the differences in survival rate,cell apoptosis,and gene expression and enzyme activity between the experimental groups can all prove from the side that the toxicity of 0.5 μm microplastics is greater than that of 5 μm microplastics.On the basis of the existing research,it provides some references for studying the harm of microplastics to freshwater fish and exploring the relationship between the particle size and toxicity of microplastics. |
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