Single-cell Analysis Reveals The Accumulation Characteristics Of Silver Nanoparticles In Freshwater Algae

Silver nanoparticles are the most productive and widely used nano materials in the world.Their large amount of environmental emissions and bioaccumulation pose a potential threat to the ecological environment and even human health.At present,the bioaccumulation research of nanoparticles is based on the traditional quantitative determination of elements,ignoring the unique physical and chemical properties of nanoparticles(such as small particle size effect)and the characteristics of different cell individuals(morphology and size),so it is difficult to clearly explain the unique accumulation characteristics and laws of nanoparticles in organisms.Therefore,in this study,single cell inductively coupled plasma mass spectrometry(Single-cell ICP-MS)technology was used,and sodium citrate coated silver nanoparticles(Ag NPs-50 nm,Ag NPs-70 nm)with particle sizes of 50 nm and 70 nm were selected as representatives to reveal the effects of particle size,concentration,exposure time and morphological characteristics of algae on the bioaccumulation level of nano silver in algae cells at the single cell level.The main results are as follows:(1)The particle size of nanoparticles dominates the accumulation level of silver nanoparticles in Chlorella vulgaris.In the 24 h,48 h and 72 h stages of nano silver exposure,the mean mass of Ag per algal cell and the number of algal cells containing nano silver / total number of algal cells(hereinafter referred to as the percentage of algal cells containing nanoparticles)in the large particle size Ag NPs-70 nm group were significantly higher than those in the small particle size Ag NPs-50 nm group.With the increase of exposure time,the mean mass of Ag per algal cell of Ag NPs-70 nm group also increased gradually,and the mean mass of Ag per algal cell increased from 65068.61 ag / cell(24 h)to 110099.45 ag / cell(72 h);In Ag NPs-50 nm group,the mean mass of Ag per algal cell increased first and then decreased.The mean mass of Ag per algal cell changed from 11793.84 ag / cell(24 h)to 17147.32 ag / cell(72 h).At the same time,the percentage of algal cells containing nanoparticles of Ag NPs-70 nm group and Ag NPs-50 nm group decreased from 26.3 % and 16.1 %(24 h)to 6.9 %and 1.2 %(72 h),respectively.In the removal stage(48 h)in medium without silver,the mean mass of Ag per algal cell and the percentage of algal cells containing nanoparticles in large particle size Ag NPs-70 nm group were still significantly higher than those in small particle size Ag NPs-50 nm group.The number of algal cells increased exponentially during exposure and recovery,which significantly reduced the percentage of algal cells containing nanoparticles.(2)At the level of environmental related concentration of nano silver,the concentration of nano particles had no significant effect on the internalization and cell accumulation of nano silver in Chlorella vulgaris.First,the concentration of silver nanoparticles did not significantly affect the accumulation of silver nanoparticles(Ag NPs-70 nm)by algal cells at 24 h,48 h and 72 h.In the low concentration nano silver treatment group(number of algal cells / number of nanoparticles = 1:5),the accumulated silver in algal cells at 24 h and 48 h was 47549.10 ag/cell and 20767.39ag/cell,respectively.In the high concentration treatment group(number of algal cells /number of nanoparticles = 1:10),the accumulated silver in algal cells at 24 h and 48 h were 88536.69 ag/cell and 31635.06 ag/cell,respectively.Secondly,the percentage of algae cells containing nanoparticles in the total number of cells decreased with the extension of exposure time.The percentage of algal cells containing nanoparticles in low concentration(1:5)treatment group and high concentration(1:10)treatment group at 24 h,48 h and 72 h were 30.5%,13.7%,0.8%,31.7%,14.3% and 1.3%,respectively.In conclusion,there was no significant difference in the single cell accumulation of nano silver and the percentage of algal cells containing nanoparticles between the two concentration treatment groups.(3)Algal morphology significantly affected the accumulation level of nano silver in algal cells.The accumulation of nano silver in three kinds of freshwater algal cells(Chlorella vulgaris,Raphidocelis subcapitata and Chlorella ellipsoidea)is mainly affected by the morphology of algal cells.At 24 h of exposure,the accumulation rule of Ag NPs-70 nm in algal cells is that the larger the surface area of algal cells,the greater the amount of silver accumulation,and the order is: R.subcapitata > C.vulgaris > C.ellipsoidea.Relatively speaking,the accumulation rule of Ag NPs-50 nm in algal cells is that the larger the algal volume,the greater the enrichment amount,and the order is: C.vulgaris > R.subcapitata > C.ellipsoidea.Pearson correlation analysis showed that the surface area of algal cells had the greatest influence on the accumulation of Ag NPs-70 nm in algal cells,while the specific surface area(surface area / volume)had the greatest influence on the percentage of algal cells containing nanoparticles.The volume of algal cells had the greatest influence on the accumulation of Ag NPs-70 nm in algal cells,and specific surface area(surface area / volume)has the greatest influence on the percentage of algal cells containing nanoparticles.In conclusion,this study revealed the accumulation characteristics of nanosilver in freshwater algal cells at the single-cell level for the first time.Nanoparticle size and cell morphology dominated the bioaccumulation level of silver nanoparticles in algal cells.The results of this study provide a new perspective for clarifying the interaction and bioaccumulation characteristics of nanoparticles and cells at the single-cell level.