Toxicological Effects Of Zinc Oxide Nanoparticles In Mytilus Galloprovincialis

Metal nanomaterials have their unique optical,electrical and biological properties,and have been widely used in many industrial and scientific fields such as catalysis,biosensors,nano-self-assembly and drug delivery.Among these metal nanomaterials,zinc oxide nanoparticles（ZnO NPs）has been widely used as an antibacterial agents in aquaculture due to their high antibacterial activity.However,in the process of it’s production and utilizationuse,ZnO NPs inevitably enter into the environment and eventually converge into the water environment,causing potential adverse effects on aquatic organisms and human health.Therefore,it is necessary to study the ecotoxicological effects of nanomaterials.In this study,PVP-coated ZnO NPs were prepared by microwave radiation method,and characterized by transmission electron microscopy,cold field emission scanning electron microscopy,energy dispersive spectroscopy,and nanoparticle size potential analyzer.Then,the typical marine environmental monitoring organism Mytilus galloprovincials was used as the stress organism,the toxicity of ZnO NPs were predicted by a two-step toxicity kinetic model.The dissolution of ZnO NPs in seawater and the enrichment in mussel tissues were determined by inductively coupled plasma mass spectrometry.The toxicological effects of ZnO NPs in Mytilus galloprovincials were investigated by tissue section,enzyme activities detection and real-time fluorescence quantitative PCR.The results provide a theoretical basis for the study of the toxicity of nanomaterials to bivalves,and also provide a scientific basis for the safety evaluation of ZnO NPs as an aquatic antibacterial agent.The main results were as follows:1.The prepared ZnO NPs were nanomaterials.Transmission electron microscopy images and scanning electron microscopy images showed that the shape distribution of ZnO NPs was uniform,and the particle size did not change much,with an average of 20 nm.The results of energy spectrum analysis showed that the main components of nanomaterial particles were Zn and O,respectively.The double distilled water suspension of nano-dry powder was analyzed by nano-particle size analysis,and the DLS diameter obtained was 363.3 nm.The Zeta potential test results showed that the absolute value of the Zeta potential of the double distilled water suspension was less than 30 mv.Therefore,the ZnO NPs we prepared belonged to nanomaterials,which laid a good foundation for subsequent experiments.2.TK-TD two-compartment model was used to analyze the toxicity of ZnO NPs The toxicity of ZnO NPs was analyzed by TK-TD two-compartment model.The biological toxicity prediction of Zn was in accordance with the one-compartment model(first compartment,k₁₂=k₂₁=0).That is,the organism had no obvious detoxification effect on Zn,and only responded to the potential toxicity of metals through the metabolism and excretion activities in the first compartment.Many studies have also shown that the toxicity of ZnO NPs mainly comes from the release of Zn²⁺.Therefore,ZnO NPs had potential biological toxicity to organisms,and its toxicity depended on the metabolic rate and excretion rate of Zn²⁺（ke₁）.3.The dissolution of ZnO NPs in seawater and the enrichment of ZnO NPs inMytilus galloprovincials tissues（1）Distribution of ZnO NPs in seawaterAfter 24 h of ZnO NPs stress,the content of Zn in seawater was 1.564μg/L,and the ingestion rate was 12.8%.Although the solubility in seawater was high,it was easy to form a larger coagulation in seawater,which affected its ingestion rate by marine organisms.（2）Enrichment of ZnO NPs in Mytilus galloprovincials tissuesThe order of Zn content in Mytilus galloprovincials tissues from high to low was:gill>hepatopancreas>adductor muscle>mantle.Among them,the Zn content in the mantle and adductor muscle fluctuated less.4.The biological toxicity of ZnO NPs（1）Pathological changes of gill tissue of Mytilus galloprovincialsThe arrangement of epithelial cells in the hepatopancreas of Mytilus galloprovincials in the ZnO NPs stress group were damaged to varying degrees,and a small amount entered the lumen of the digestive tract,and the surrounding connective tissue structure was not significantly affected.In the gill tissue,the swelling of the gill filaments was more serious at 96 h,and the shrinkage and infiltration occurred at 30 d.The damage degree of gill tissue was higher than that of hepatopancreas.（2）Effects of antioxidant enzymes and antioxidants in Mytilus galloprovincialsThere was no significant change in antioxidant enzyme activities in hepatopancreas and gill at 96 h.At 30 d,the content of MDA in hepatopancreas increased,but had no significant change（p=0.079）,and the activities of CAT and GPx in gill increased,but had no significant change（p=0.098 and 0.061）.The results showed that ZnO NPs did not cause excessive oxidative stress to Mytilus galloprovincials.（3）Changes of acetylcholinesterase（ACh E）activity in Mytilus galloprovincialsAfter 30 days of exposure,ACh E activity in gills increased,but had no significant change（p=0.060）.It is inferred that within the time range we monitored,the ACh E activity in the hepatopancreas and gills of the mussels was basically unaffected,and it may be a longer process for the damage of the nervous system.（4）Expression changes of immune-related genes in Mytilus galloprovincialsThe expression levels of four heat shock protein genes（HSPA12A,TCTP,s HSP22and s HSP24.1）and three apoptotic genes（P63,Bcl-2 and Ras）showed that s HSP22,s HSP24.1,TCTP,Ras and Bcl-2 played an anti-apoptotic role under ZnO NPs exposure.（5）Immune response of mussel hemocytesChanges of LMS in blood cells:LMS did not change significantly at 96 h.At 30 d,LMS was not significantly affected.It was concluded that ZnO NPs did not cause serious cytotoxicity to mussels.Respiratory burst of blood cells:At 96 h,the ROS content in the blood cells of the three experimental groups increased significantly.At 30 d,there was no significant change in ROS content in ZnO NPs group.It can be inferred that the Mytilus galloprovincials blood cells could have a significant stress response to ZnO NPs.Apoptosis of blood cells:At 30 d,the early apoptosis rate of blood cells in ZnO NPs group was significantly increased（p<0.05）.The early apoptotic stress response of blood cells basically echoes the respiratory burst.