The Immunopathological Effects Of Microplastics From Zhanjiang Offshore Sediments On Zebrafish

Microplastics（MPs）are a persistent organic pollutant.Because of its small size and difficulty to degrade,it is widely present in nature.The majority of MPs will finally join the water body and its sediments after passing through the natural atmosphere and water bodies.It is very likely to reach the animal body via the digestive or respiratory tract due to its multiple modes of diffusion and migration.It is also easy to adsorb other harmful substances in the atmosphere due to its small particles,heavy adsorption,and agglomeration,posing a danger to species.In this study,MPs were extracted from Zhanjiang offshore sediments to explore the contamination status of MPs in Zhanjiang offshore sediments and their impact on zebrafish immunopathology.The main research contents of this experiment are as follows:（1）The aim of this experiment was to obtain MPs in Zhanjiang offshore sediments using density method flotation,acid digestion,and other steps,and then use Fourier Transform Infrared Spectroscopy（FT-IR）and a laser particle size analyzer to determine the species composition and particle size distribution of MPs in the sediment.（2）The adult zebrafish fish were randomly divided into blank control group,positive control group,low-dose group,middle-dose group and high-dose group,and zebrafish were exposed to MPs extracted from Zhanjiang offshore sediments.The histopathological changes of immune organs（liver,gills,intestine）of adult zebrafish were examined by using H&E staining.（3）The levels of humoral immune factors（mucus IgM and lysozyme）were measured using ELISA to investigate the effects of MPs on adult zebrafish.Results showed:（1）Ultrasonic-assisted flotation method using ZnCl₂as the solute in the salt solution+acid digestion method combined with 65%HNO₃ and 30%H₂O₂solutions is suitable for the extraction of MPs in offshore sediments.The main monomers of the polymer are ethylene and propylene,and contain a small amount of diene and acrylic monomers.（2）Compared with the blank control group,the liver index,gill index,and intestinal index increased significantly in all treatment groups.Among them,the intestinal index of the high-dose group had a significant difference on day 14 and 21（P<0.01）.（3）The other test groups displayed varying degrees of gill injury,the majority of which were swelling of the epithelial cells of the gill fragments and rod-shaped lesions at the center,and the degree of damage increased as exposure time and MPs concentration increased,and gill fragments emerged.Distortion,epithelial cell edema,and apex enlargement were also observed.On day 21,both the high-dose and the low-dose groups showed basal hyperplasia and adhesion,fractures,and severe damage to the gill structure.On day 7 of exposure to MPs,all the treatment groups have different degree of fat vacuoles accumulation around hepatocytes as compared to the blank control group.On day 14 of MPs exposure,all the treatment groups had slight dilatation of hepatic sinusoids,enlarged hepatocyte size,fat accumulation around hepatocytes.These changes were more specifically observed in positive control,middle dose and high dose groups.On the day 21 of the experiment,the liver tissue structure of the low-dose,middle-dose,and high-dose groups was damaged in different degrees,and nuclei were aggregated and intercellular.The boundaries were blurred and cavitation was increased.Compared to the blank control group,the intestinal histology of each test group showed a decrease in the height of intestinal villi,an increase in the height of the mucosal epithelium,and lateral longitudinal muscle layer.（4）Compared with the blank control group,the level of IgM in the low-dose,middle-dose,and high-dose groups showed a significant increase（P<0.05）in the first 7 days of the test.On the day 14,the IgM level of the middle-dose and the high-dose groups showed a significant increase（P<0.05）as compared to the control group.Compared with the blank control group,the IgM concentration of the middle-dose and the high-dose groups increased significantly（P<0.05）on day 21.The LyZ level of the low-dose group increased significantly in the first 7 days（P<0.01）as compared with the blank control group,and the LyZ concentration of the middle-dose and the high-dose groups showed a significant increase（P<0.05）on day 14,and this increase was more significant on day 21.The LyZ concentration of the low-dose group increased significantly（P<0.01）on day 21 as compared to the control group.In conclusion,the key components of Zhanjiang Offshore MPs are high molecular polymers mainly made up of ethylene and propylene monomers,with some diene and acrylic monomers.MPs have a potential to increase the immune organs index,and increased LyZ and IgM concentration in body surface mucus of the zebrafish as compared to the control group.These findings suggest that MPs can affect the immune function of zebrafish.