Study On The Toxic Effect Of Nanoparticles Of Cu And Cuo On Two Species Of Euplotes

A great number of nanoparticles have been widely used in various fields for the past years.However,nanoparticles inevitably flow into water due to improper handling,which has a certain impact on water environment ecosystem.The ecotoxicological effect of nanoparticles on aquatic organisms has attracted extensive attention from scientists.Protozoa are the most primitive,lowest and simplest single-celled eukaryotic animals,whose cell membranes are in direct contact with the environment.And their responses can reflect environmental changes in a timely manner.As a result,protozoa are often used as environmental indicator animals.Of this study was to choose Cu nanoparticles?CuNPs?and CuO nanoparticles?CuONPs?as the experimental material,both were 30 nm.And we chose Euplotes aediculatus lived in fresh water and E.vannus lived in sea water for the toxicology tests.Cell morphology was observed through the optical microscope and transmission electron microscopy.We used the mitochondria,lysosomes fluorescent staining method and Attenuated Total Reflection Flourier Transformed Infrared Spectroscopy?ATR-FTIR?,determining antioxidant enzyme activity at the same time involving the superoxide dismutase?T-SOD?,peroxidase?CAT?and glutathione peroxidase?GPx?,to investigate the toxicity and mechanism of two nanoparticles on two species of Euplotes in different water environments.1.Toxicological test results of CuNPs and CuONPs on Euplotes.Acute toxicology results showed:the 24 h-LC₅₀ of CuNPs and CuONPs on E.aediculatus were 0.46?g/L and 1.24×10³?g/L,respectively.Chronic toxicology results showed:the 6 h-LC₅₀ of CuNPs and CuONPs on E.vannus were 345.32?g/L and1.34×10⁶?g/L,respectively.The 24 h-EC₅₀,48 h-EC₅₀ and 72 h-EC₅₀ of CuNPs on E.aediculatus were 2.10×10^-3?g/L,7.92×10^-4?g/L and 2.77×10^-4?g/L,respectively.The EC₅₀0 at same period of CuONPs were 7.20?g/L,0.86?g/L and 0.19?g/L,respectively.The 6 h-EC₅₀,12 h-EC₅₀ and 18 h-EC₅₀ of CuNPs on E.vannus were 43.32?g/L,6.72?g/L and 0.30?g/L,respectively.The EC₅₀0 at same period of CuONPs were 6.64×10³?g/L,262.26?g/L and 72.13?g/L,respectively.Compared with the control group,the growth and reproduction of the two Euplotes could be inhibited by the two NPs with different concentrations.It was dose-dependent and the inhibition of CuNPs was stronger.Accordingly,the toxicity of CuNPs was stronger than that of CuONPs.2.The toxic effect of CuNPs and CuONPs on Euplotes.After the treatment of two NPs,the movement ability of Euplotes was decreased and the cilia were shaken violently.Vacuoles appeared in the cell and gradually merged and expanded.The cell body of E.aediculatus swelled and finally ruptured.E.vannus was slightly swollen and cytoplasm outflow to death.The extent of nuclear and mitochondrial damage varies.The chromatin in E.aediculatus was condensed and showed a circular structure,the mitochondria were wrinkled and their shape were irregular,and some cristae were broken and disintegrated in CuNPs group.However,the changes of materials in the nucleus were not obvious in CuONPs group.And most of the mitochondrial shape were irregular,a few were swollen,and the cristae were broken in CuONPs group.Nucleoli in the nucleus disintegrated and disappeared,and chromatin condensed in E.vannus with CuNPs.The nuclei in E.vannus were deformed with round nucleoli and the chromatin condensed into irregular clumps of different volumes in CuONPs group.The mitochondria in CuNPs group were swollen and round or oval with broken cristae,while in the CuONPs group,the morphology of mitochondria was irregular with normal cristae.Fluorescent staining results showed that the two NPs had a destructive effect on the mitochondrial function of Euplotes.CuNPs caused more damage and produced more acidic materials such as lysosomes.The cell membrane was relatively intact by TEM,but the oxidation of some functional groups,e.g.PO₂^-,C-O-C,??COH?of carbohydrates,for the Euplotes membrane in CuNPs group was detected by ATR-FTIR.CuONPs has little effect on them.The enzyme activities of T-SOD,CAT and GPx in Euplotes were increased after the two NPs treated with different concentrations,which were dose-dependent.3.A preliminary study on the mechanism of CuNPs and CuONPs on Euplotes.CuNPs and CuONPs mainly caused cell death by causing oxidative stress in cells,damaging the antioxidant system,and damaging cell structures such as mitochondria and nucleus.Oxidative stress and the production of reactive oxygen species may be the most important toxic mechanism.The oxidized functional groups in the cell membrane components and lysosomes predicted the antioxidant and defense behavior of cells against external stress.For E.aediculatus or E.vannus,the toxicity of CuNPs was greater than that of CuONPs,and the damage to cell structure was more serious with CuNPs.This may be due to the lively chemical nature of CuNPs and its surface oxides that cause cells to generate free radicals,resulting in cellular antioxidant activity.And it caused cell structure damage.The results of this study provide a new theoretical basis for the research of cytotoxicology of nanomaterials.