Toxic Effects Of Polystyrene Nanoplastics On The Early Development Of Oryzias Melastigma Under Ocean Acidification Conditions

With the wide application of plastic products,more and more plastic waste enters the marine environment.These plastics are decomposed into microplastics and nanoplastics under physical,chemical,and biological effects.Among them,nanoplastics（NPs）have small particle sizes and large specific surface areas,which pose a potential threat to the marine ecological environment and have become a major global concern.At the same time,the carbon dioxide（CO₂）emitted by human activities has led to a decrease in the p H of seawater,making ocean acidification（OA）another major environmental issue that has attracted much attention.Current studies mainly focus on the single effects of OA or NPs on marine organisms,but their joint toxicity and the toxicity difference of different surface-modified NPs have not been considered.In fact,in the marine environment,marine organisms face the co-stress of OA and NPs at the same time,and the impact on them is likely to be more complex and serious than that under a single condition.This is mainly because OA may alter the environmental behavior of NPs and further affect their biological toxicity.At present,research on the toxic effect of NPs on marine organisms under the condition of OA is extremely scarce,and its mechanism of action is still unclear,which cannot meet the needs of marine environmental risk assessment of NPs under the condition of OA.In this study,the marine model organism O.melastigma embryo was selected as the research object,and the effects of amino-modified PS-NPs（PS-NH₂,80nm）and carboxyl-modified PS-NPs（PS-COOH,80nm）on O.melastigma embryos were studied.The experiment set up a control group and 5 treatment groups:seawater control group（p H 8.2,no PS-NPs）,acidified seawater group（p H 7.4,no PS-NPs）,amino-nanoplastics group（p H 8.2,PS-NH₂ 10μg/L）,carboxy-nanoplastic group（p H8.2,PS-COOH 10μg/L）,acidified amino-nanoplastic group（p H 7.4,PS-NH₂ 10μg/L）,acidified carboxy-nanoplastic group（p H 7.4,PS-COOH 10μg/L）.Meanwhile,the environmental behavior of two kinds of PS-NPs under normal seawater and acidified seawater conditions were studied,and the interaction mode of PS-NPs with O.melastigma embryos and their transfer in O.melastigma larvae were explored.In addition,the early developmental toxicity of NPs with different surface modifications on O.melastigma embryos under OA conditions was studied,and the effects of PS-NPs on O.melastigma growth,tissue surface morphology,microscopic and ultrastructure were clarified under OA conditions.The research results are as follows:（1）In normal seawater,compared with PS-COOH,PS-NH₂ is easier to disperse and suspend,because the average hydrodynamic diameter and polydispersity index（PDI）of PS-NH₂ are smaller.However,in acidified seawater（p H 7.4）,the average hydrodynamic diameter and PDI of PS-COOH are smaller than those of PS-NH₂,and p H 7.4 is far from the isoelectric point of PS-COOH in seawater（p H 10.3）,which indicates that in acidified seawater,compared with PS-NH₂,PS-COOH is easier to disperse and suspend.（2）Under OA condition,NPs led to an increase in mortality,heart rate,malformation rate,hatchability,and exercise capacity（swimming speed and distance）of O.melastigma embryos,among which the acidified carboxyl group had the greatest toxic effect.Through the analysis of Integrated Biomarker Responses（IBR）on the above indicators,it was found that NPs had a toxic effect on the early development of O.melastigma in normal seawater,and the toxicity of PS-NH₂ was higher than that of PS-COOH.In acidified seawater,the toxic effect of NPs is enhanced,and the toxicity of PS-COOH is higher than that of PS-NH₂.Thus,the toxic effects of positively and negatively charged NPs on O.melastigma embryos under ocean acidification conditions were reversed.（3）Non-invasive micro-test technology（NMT）was used to observe the changes in ion flow rate on the surface of O.melastigma embryos.In normal seawater,NPs led to a significant increase in the flow rate of Ca²⁺and H⁺on the surface of the embryos,and a decrease in the ion content in the body,among which PS-NH₂ has a significant impact.The effect of NPs on the ion flow rate on the surface of embryos was enhanced under seawater acidification conditions,and PS-COOH had a significant effect.Therefore,NPs cause a certain degree of damage to the ionocytes of O.melastigma embryos,hinder ion channels,and affect the transport and absorption of Ca²⁺and H⁺of embryos.The decrease of Ca²⁺content in the embryos led to abnormal nerve and muscle development,increased deformities,and decreased exercise ability in O.melastigma larva.The decrease of H⁺content in the embryos leads to an imbalance of acid-base in the body fluid in the embryos,affecting material metabolism and cell survival.（4）Laser scanning confocal microscope（CLSM）observed that NPs adsorbed on the surface chorion of the embryos and entered the embryos,accumulating in the yolk sac.Observation of O.melastigma larva by CLSM and transmission electron microscope（TEM）revealed that NPs were transferred from the embryos into the intestine of the larva and caused damage to the microvilli of intestinal epithelial cells.Through the analysis of the fluorescence intensity of NPs inside and outside the embryos and in the larva by Image J software,it was found that PS-NH₂ was more adsorbed on the surface of the embryos,in addition,in normal seawater,more PS-NH₂entered the embryos and transferred to the larva,while in acidified seawater,more PS-COOH entered the embryos and transferred to the larva.Therefore,the environmental concentration of NPs can enter the embryos,and the content of PS-COOH and PS-NH₂entering the body in different p H seawater is different,which has different effects on the early development of O.melastigma.In conclusion,the toxic effects of PS-COOH and PS-NH₂ on O.melastigma were reversed under ocean acidification compared to normal seawater conditions.This is mainly due to the changes in the environmental behavior of PS-NH₂ and PS-COOH in acidified seawater compared with normal seawater,thus changing the bioavailability of O.melastigma.The research results provide new insights for future assessment of marine ecological risks of NPs under climate change conditions.