Toxic Effect Of CuO Nanoparticles On Marine Medaka Oryzias Melastigma Under The Condition Of Ocean Acidification

Ocean acidification?OA?caused by human activities will lead to changes in the marine environment,which directly affect the living environment of marine organisms,and seriously risk the safety and stability of marine ecosystems.With the rapid development of nanotechnology,nanomaterials have been widely used in industry,energy,medical care and consumer products.As a result,nanoparticles?NPs?are inevitably released into marine environment,and finally pose a potential risk to the marine environments.More and more studies have shown that NPs are toxic to phytoplankton,zooplankton,nekton and benthic organisms.However,the effects of OA and NPs in natural marine environments always combine and act on marine ecosystems instead of existing alone.Furthermore,OA is likely to affect the environmental behavior and toxicity of NPs.To date,the combined toxic effect of CuO NPs with OA on marine organisms has not been reported.Therefore,in this study we selected Oryzias melastigma as experimental organism and copper oxide nanoparticles?CuO NPs?as experimental material.We set up a control group?pH:8.20,without CuO NPs?,two acidification groups?pH:7.70 and 7.40,without CuO NPs?,a copper ion group?pH:8.20,CuSO₄:0.016mg/L?,two copper ion-acidification groups?pH:7.70,CuSO₄:0.026 mg/L;pH:7.40,CuSO₄:0.045 mg/L?,a nano group?pH:8.20,CuO NPs:10 mg/L?,and two nano-acidification groups?pH:7.70,CuO NPs:10 mg/L;pH:7.40,CuO NPs:10 mg/L?.In addition,the concentration of CuSO₄ were set according to the equilibrium concentration of Cu²⁺released by CuO NPs.The following studies were conducted:?1?the environmental behaviors of CuO NPs under normal and acidified seawater such as suspension,hydrodynamic diameter and ion release;?2?the effects of OA and CuO NPs on Oryzias melastigma embryos mortality and developmental toxicity?including heart rate,hatching rate,hatching time and malformation?;?3?the effects of OA and CuO NPs on Oryzias melastigma embryos biochemical indexes,including the changes of superoxide dismutase?SOD?,catalase?CAT?and malondialdehyde?MDA?;?4?the effects of CuO NPs on to the surface morphology of Oryzias melastigma embryos and the internalization of CuO NPs by marine medaka embryos.Results are as follows:?1?CuO NPs would agglomerate rapidly after entering seawater?pH=8.20?.After 96h,the average hydraulic diameter was 1546.46 nm.Under acidification conditions?pH=7.70 and 7.40?,the average hydraulic diameter of CuO NPs were 1156.77 and 787.54nm.Due to the decrease of pH,CuO NPs were far away from the zero point of charge in seawater(pH_zpc=10.03),resulting in enhanced repulsive force between particles,smaller average hydraulic diameter,and more dispersed particles.At 120 h,the Cu²⁺concentration released by CuO NPs were 0.015 mg/L,0.026 mg/L and 0.042 mg/L at seawater pH 8.20,7.70 and 7.40.This showed that the Cu²⁺concentration released by CuO NPs increases under low pH conditions.?2?During the 25-day experiment,the mortality,heart rate,hatching rate,hatching time and juvenile fish malformation rate of the acidification groups and nano group were higher than the control group,and the hatching rates were lower than the control group,indicated that the early development of Oryzias melastigma was affected by OA and CuO NPs.Compared with the control group and acidification groups,there were no significant difference in mortality,heart rate,hatching rate and juvenile fish malformation rate between copper ion group and copper ion-acidification groups,indicated that Cu²⁺released by CuO NPs had no significant effect on the early development of Oryzias melastigma.It was observed that the malformations of Oryzias melastigma mainly include spinal deformities,fin rot,pericardial edema and craniofacial deformities.The analysis showed that OA significantly increased the mortality,reduced hatching rate and delayed hatching time of CuO NPs to medaka embryos.?3?The SOD activities of the medaka embryos were significantly induced in the acidification groups,and the CAT activities were significantly inhibited,but MDA contents did not change significantly.When CuO NPs were added,embryonic SOD and CAT activities were significantly inhibited,and the inhibitory effect in the nano-acidification groups were higher than the nano group,but MDA contents were significantly increased,and MDA contents were increased in the nano-acidification groups.Compared with the control group and the acidification groups,the copper ion group and the copper ion-acidification groups had no significant effect on SOD,CAT activity and MDA contents of Oryzias melastigma embryos.The analysis showed that pH and CuO NPs interacted with SOD activities,CAT activities at 12 h and MDA contents of seawater Oryzias melastigma embryos.?4?A large amount of CuO NPs were adsorbed on to the surface of Oryzias melastigma embryos exposure to nano group and nano-acidification groups.But the particle sizes in the nano-acidification groups were smaller than the nano group.The enrichment of Cu in the nano group and nano-acidification groups were significantly higher than other groups,and the nano-acidification group?pH 7.40?was significantly higher than the nano group.This was mainly due to the decrease in the hydraulic diameter of CuO NPs and the increase in the amount of Cu ions released under OA conditions.This also led to an enhanced toxic effect of CuO NPs on Oryzias melastigma under OA conditions.In addition,the enrichment of Cu in embryos was mainly derived from the particles of CuO NPs in the nano group and nano-acidification groups,indicating that the size effect was the main factor for the toxic effects of CuO NPs.However,the contribution of Cu ions released by CuO NPs to the enrichment of Cu condent in embryos were still lower than the particles of CuO NPs in the nano-acidification groups.Therefore,the size effect was still the main influencing factor of the toxic effects of CuO NPs under OA conditions.In summary,OA enhanced the early developmental toxicity of CuO NPs to Oryzias melastigma.This was mainly because the samaller hydraulic diameter and enhanced suspension of CuO NPs under OA conditions,thus increasing the biological effectiveness of CuO NPs to Oryzias melastigma.Although the ion release of CuO NPs was increased under OA conditions,it was not the main factor for enhancing the toxicity of CuO NPs under OA conditions.Therefore,the dominant factor in the early developmental toxicity of CuO NPs to Oryzias melastigma enhanced by OA was the size effect rather than its ion effect.