Study Of Toxic Effects And Mechanism Of ZnO NPs On Two Ciliates

ZnO NPs is widely used in all walks of life,such as food,medicine,cosmetics,electronics,and hygiene because of its special physical and chemical properties.Which makes ZnO NPs has a greater probability to enter the hydrographic,soil and atmospheric environment during the processing,using and treatment after using,therefore the issue about if ZnO NPs has toxic effects on creatures should be taken seriously by researchers.Euplotes aediculatus and Paramecium bursaria were selected as tested organisms for the first time in this study,and the toxicity of ZnO NPs in hydrographic environment was measured by the medial lethal concentration(LC₅₀)and median effective concentration(EC₅₀)of the two tested organisms within 24 h.The toxicity mechanism of ZnO NPs was explored by studing the ultrastructural destruction,enzyme activity changes and reactive oxygen species(ROS)accumulation in Euplotes aediculatus and Paramecium bursaria after ZnO NPs treatment.The toxic contribution of zinc ions in ZnO NPs solution was also studied by Inductively coupled plasma atomic emission spectrometry(ICP-AES),the determination of toxicological data and electron microscope observation.1.Toxic effects of ZnO NPs on two ciliates.The 24 h-LC₅₀ of ZnO NPs against Euplotes aediculatus and Paramecium bursaria were 1.67?g/m L and 0.39?g/m L respectively.The 24 h-EC₅₀ of ZnO NPs against Euplotes aediculatus and Paramecium bursaria were 0.24?g/m L and 0.04?g/m L respectively.The regression equations and fitted curves showed that the mortality and reproductive inhibition rates of two ciliates within 24 h had good linear relationship with the logarithm of ZnO NPs concentration.Compared with other tested aquatic organisms,ciliates showed lower tolerance to ZnO NPs.24 h-LC₅₀ ZnO NPs could cause the swelling of Euplotes aediculatus,but there would be no effect on pellicle and cilia.TEM results showed that 24 h-LC₅₀ ZnO NPs treatment resulted cytoplasmic vacuolation of Euplotes aediculatus,and cytoplasm changed from dense to loose.The structures of mitochondria,pharyngeal disk and nucleus were destroyed,and lots of lysosomes and lysosomal residues were observed at the same time.The Paramecium bursaria in 24 h-LC₅₀ ZnO NPs treatment groups swelled into drop-shaped,accompanied by the fall off of cilia,grid structure of surface was distorted and shrank,or the grid structure maintain its normal shape but the pellicle of surface was damaged seriously.After 24 h-LC₅₀ ZnO NPs treatment,TEM showed that the endosymbiosis system of Paramecium bursaria and chlorella was destroyed,scattered membrane structure appeared in pellicular alveolus,the number of lysosomes increased,and the structure of mitochondria and nuclear were abnormal.2.Study on the toxicity mechanism of ZnO NPsThe results of DHE showed that the mean fluorescence intensity in ZnO NPs treatment groups of Euplotes aediculatus and Paramecium bursaria were significantly increased compared with the control group.Which indicated ZnO NPs could induce the accumulation of ROS in Euplotes aediculatus and Paramecium bursaria,and then led to oxidative stress.After 24 h-EC₅₀ ZnO NPs treatment,the changes of SOD and CAT enzyme activities,the accumulation of MDA content and the damage of mitochondrial structure showed that the antioxidant system in the cells was not able to resist the oxidative stress induced by ZnO NPs.In summary,this study suggests that oxidative stress is one of the ways of ZnO NPs exerting toxic effects in an aqueous environmentAccording to the dissociation degree of ZnO NPs in a solution,different concentrations of zinc ions were prepared with zinc vitriol to study the toxic contribution of zinc ions to ciliates in ZnO NPs solution.The results showed that zinc ions had apparent lethal effect on both Euplotes aediculatus and Paramecium bursaria.Especially for Paramecium bursaria,zinc ion would be the main factor leading to mortality.After the treatment with zinc ions,the accumulation of ROS in Euplotes aediculatus and Paramecium bursaria was detected by dihydroethidium(DHE).By comparing the average fluorescence intensity of zinc ion treatment groups and ZnO NPs treatment groups,It would be found that zinc ions in ZnO NPs solution played a great role in ROS production for Euplotes aediculatus and played a partial role in ROS production for Paramecium bursaria.5.6?g/m L and 1.67?g/m L(24 h-LC₅₀)ZnO NPs solution has the same concentration of zinc ions,however,the former has higher concentration of nanoparticles than the later.Euplotes aediculatus treated with 5.6?g/m L ZnO NPs solution,after the observation of SEM,there was no obvious damage to pellicle and cilia in treatment groups.But the cytoplasm looser than the 24 h-LC₅₀ treatment groups.what's more,few organelles and cellular structures would be seen in Euplotes aediculatus.According to the above phenomena,it is speculated that the toxicity of ZnO NPs particles is related to autophagy.In this study,results shown that the ciliates,especially Paramecium bursaria has high sensitivity to ZnO NPs.And we guess the toxic mechanism of ZnO NPs in aqueous environment involves two aspects.On the one hand,ZnO NPs particles can generate ROS spontaneously under light conditions,which then damages surface ultrastructures of ciliates.Besides,ZnO NPs particles can destroy mitochondria,pharyngeal fins,nucleus and other cellular structures directly after entering into cells,and induce severe autophagy and self-depletion.On the other hand,zinc ions which ZnO NPs particles dissociate in aqueous environment can induce the production of intracellular ROS.Which makes cells suffer oxidative stress.According to the results of this study,in aqueous environment,zinc ion is an important inducement for ZnO NPs to cause oxidative stress in the tested organisms.Combined with the results of acute toxicity of zinc ions to two ciliates,our study suggests that surface modification which can inhibit solubility will improve significantly the safety of ZnO NPs in water.Environmental factors such as p H value,ion type and concentration which may affects the degree of of nanomaterials dissociation should be taken into account While studying the toxicity of other nanomaterials.Our study will provide directions and ideas for in-depth research on the toxic mechanism of ZnO NPs and provide theoretical basis for improvement of the safety of ZnO NPs in water.