# Applicability Evaluation of Soil Algae Pipe Assay in Silver Nanoparticle-Contaminated Soils
## 3 Results and Discussion
### Evaluation of Different Distributions of AgNP-Contaminated Soil in Assay  
Figure 4 shows the in vivo chlorophyll \(a\), cell granularity, cell size, and lipid contents of _C. reinhardtii_ exposed to different distributions of AgNP-contaminated soil at 1 week. _C. reinhardtii_ cells in both heterogeneous and homogeneous AgNP-contaminated soil groups showed a significant decrease in in vivo chlorophyll \(a\) (_p_ < 0.05), as indicated by left shifts in the histograms in Figure 4B. This indicated a decrease in chlorophyll contents in each _C. reinhardtii_ cells due to the AgNPs, and we observed partially hollow _C. reinhardtii_ cells showing the loss of photosynthetic pigments in undeveloped chloroplasts in Section 3.1. A decrease in chlorophyll content in algal cells has been commonly observed with the growth inhibition of algae exposed to nanomaterials [20; 43; 44; 45; 46; 47; 48]. Meanwhile, cell granularity, cell size, and lipid contents were significantly changed in the homogeneous AgNP-contaminated soil group (_p_ < 0.05), as indicated by left or right shifts in the histograms in Figure 4C-E. In a previous study of nanomaterials toxicity, the characterization of the nano-bio interaction between nanomaterials and algae has been analyzed by flow cytometry. Changes in cell granularity and cell size were explained as shifts in the SSC and FSC in flow cytometry [49]. An increase in SSC indicates the internalization of nanomaterials into algal cells [50; 51; 52] and an increase in FSC indicates the adsorption of nanomaterials to the algal cell wall [53] or the production of a mucilaginous sheath enlarged from the algal cell wall [20]. In this study, the decrease in cell granularity and cell size was significant in the homogeneous AgNP-contaminated soil group (Figures 3D and 4A,C). This phenomenon may be induced by necrosis and immaturity. Necrosis is cell death resulting from the leakage of cell components or the damage of plasma membrane, as reflected by a decrease in SSC and FSC [37]. In particular, the loss of photosynthetic pigments in undeveloped chloroplasts by AgNPs has a decisive effect on the decrease in cell granularity, organizing microscopic and SSC analysis. In addition, immaturity by AgNPs may influence the decrease in cell size, thereby distributing immature cells (e.g., motile zoosporangia or zoospores, and non-motile aplanosporangia or aplanospores) instead of mature vegetative cells [7], along with the disappearance of big _C. reinhardtii_ cells exposed to silica-coated quantum dots (with a decrease in average cell size and increase in cell count) [54]. A significant increase in the lipid contents was observed in the homogeneous AgNP-contaminated soil group (Figure 4A,E). Lipid accumulation commonly appears in algal cells as a stress-dependent mechanism [55], as storing lipids excludes toxic molecules during the main metabolism [56] and acts as an electron sink to maintain cellular redox homeostasis and prevent oxidative damage [57]. Lipid was accumulated under AgNPs stress, which agreed with the results of other stressors, such as nutrients [58; 59; 34], and nanomaterials [56; 60; 61; 62]. When the _C. reinhardtii_ cells were exposed to homogeneous 20 mg/kg AgNP-contaminated soil, a decrease in in vivo chlorophyll \(a\), cell granularity, and cell size, and an increase in lipids showed significant differences compared to the control. On the other hand, when the _C. reinhardtii_ cells were exposed to heterogeneous 20 mg/kg AgNP-contaminated soil, only the decrease in in vivo chlorophyll \(a\) was significantly different compared to the control group (_p_ < 0.05) and a significant difference in in vivo chlorophyll \(a\) was observed compared to the homogeneous 20 mg/kg AgNP-contaminated soil, as illustrated in Figure 4A. This difference in toxicity sensitivity may have resulted from the heterogeneous and homogeneous distribution range of AgNP-contaminated soils (Figure 5).