Toxic Effects Of Polystyrene Nanoplastics On Nannochloropsis Oceanica Under Elevated PCO₂

With the rapid development of industrial science and technology and the wide application of plastic products,a large number of plastic waste and chemical products will inevitably enter the marine environment,after oxidation,mechanical force,the formation of nanoplastics（NPs）.Studies have shown that NPs can produce toxic effects on marine microalgae,and then pose a potential threat to the whole marine ecosystem.Therefore,the marine environmental effects of NPs have become a hot issue in the research at home and abroad.At the same time,the ocean acidification caused by excessive CO₂ released by human activities has attracted much attention.Marine microalgae,as the main primary producers in the ecosystem,play a vital role in the balance and stability of the whole marine ecosystem.Current studies mainly focus on the toxic effects of NPs on marine microalgae,but the toxic effects of NPs on marine microalgae under the conditions of ocean acidification have not been reported,and the mechanism of toxicity is still unclear.Therefore,in this paper,Nannochloropsis oceanica,a single-celled eukaryotic microalgae,was used as the experimental organism,and PS-NH₂ NPs was used as the experimental material.The control group（C,pCO₂～400μatm,no NPs）and the short-term acidification group（SA,pCO₂～1,000μatm,no NPs）,long-term acidizing group（LA,pCO₂～1,000μatm,no NPs）,low-concentration nanoplastics group（m PS,pCO₂～400μatm,0.5 mg/L NPs）,high-concentration nanoplastics group（h PS,pCO₂～400μatm,1.5 mg/L NPs）,short-term acidification low-concentration nanoplastics group（SAm PS,pCO₂～1,000μatm,0.5 mg/L NPs）,short-term acidification high-concentration nanoplastics group（SAh PS,pCO₂～1,000μatm,1.5 mg/L NPs）,long-term acidification low concentration nanoplastics group（LAm PS,pCO₂～1,000μatm,0.5 mg/L NPs）,and long-term acidification high-concentration nanoplastics group（LAh PS,pCO₂～1,000μatm,1.5 mg/L NPs）.These groups were used to analyze changes in the environmental behavior of PS-NH₂ NPs under normal and acidified conditions;The effects of PS-NH₂ NPs on the physiology and biochemistry of N.oceanica were studied under the condition of increasing pCO₂;Morphological changes of algal cells were observed by scanning electron microscopy（SEM）and transmission electron microscopy（TEM）;Transcriptome methods were used to analyze the molecular regulatory mechanism of N.oceanica in response to PS-NH₂ NPs stress under acidified conditions.The toxic effect of PS-NH₂ NPs on N.oceanica under the condition of pCO₂ increase was clarified,and the mechanism of the effect of pCO₂ increase on the biological toxic effect of PS-NH₂ NPs was revealed,which provided a theoretical basis for scientific assessment of the risk of NPs in marine environment.The main findings are summarized as follows:（1）Through SEM observation and DLS particle size analysis,it was found that PS-NH₂ NPs significantly aggregated in acidified seawater,the particle size increased significantly,and the dispersion decreased,indicating that the increase of pCO₂ would promote the agglomeration of PS-NH₂ NPs,reduce its suspension performance,and thus reduce the number of NPs in the form of nano in the experimental seawater.（2）It was found that PS-NH₂ NPs significantly inhibited the growth and photosynthesis of N.oceanica,and the inhibition rate increased with the increase of PS-NH₂ NPs concentration,showing an obvious dose-effect relationship.However,after acidification treatment,the inhibitory effect of PS-NH₂ NPs on the growth and photosynthesis of N.oceanica was reduced,and even the cell density in LAm PS was significantly higher than that in control group.In conclusion,increasing pCO₂ can alleviate the inhibition of PS-NH₂ NPs on N.oceanica.In addition,the antioxidant oxidase test results showed that,compared with the control group,SOD,CAT and POD activities in N.oceanica cells treated with NPs increased,and N.oceanica cells reacted to PS-NH₂ NPs interference with oxidative stress.After PS-NH₂ NPs acidified,SOD,CAT and POD activities were generally lower than those of single PS-NH₂ NPs stress.In conclusion,the increase of pCO₂ can reduce the oxidative stress of N.oceanica in response to PS and NPs.In terms of EPS,EPS production in low-concentration（0.5 mg/L）experimental groups（m PS,SAm PS,LAm PS）was significantly higher than that of the control group,but EPS production after acidified PS-NH₂ NPs was significantly lower than that of single PS-NH₂NPs.（3）Scanning electron microscopy（SEM）showed that PS-NH₂ NPs induced cell surface morphology changes,cell wall shrinkage,serious surface pits,and reduced smoothness.After acidification,the cell wall of PS-NH₂ NPs experimental group was reduced,and the smoothness was much improved compared with that of nano plastic group.At the same time,transmission electron microscopy（TEM）showed that PS-NH₂ NPs could separate the cytoplasmic walls of N.oceanica,but after acidification,the cytoplasmic wall separation was greatly changed.These results indicated that ocean acidification weakened the damage of PS-NH₂ NPs on the surface morphology and cell structure of N.oceanica cells.（4）Transcriptome analysis revealed that the pathway of the TCA cycle was significantly stimulated by PS-NH₂ NPs exposure,and several key enzymes involved in the TCA cycle were significantly down-regulated.These results indicated that PS-NH₂ NPs,as an exogenous stressor,induced oxidative stress and interfered with energy and substance metabolism by inhibiting the TCA cycle,thus inhibiting detoxification reaction and leading to the decrease of the growth of N.oceanica.In addition,the most significant pathway by which elevated pCO₂ mitigates the toxicity of PS-NH₂ NPs to N.oceanica is during ribosomes and their synthesis,which contains 99 upregations of DEGs.These results indicated that the increase of pCO₂ promoted the synthesis of related enzymes and proteins by up-regulating the expression of ribosome and RNA transport-related genes,and alleviated the damage of PS-NH₂ NPs to N.oceanica cells.