# 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
In this exposure system, the exposure concentration of homogeneous 20 mg/kg AgNP-contaminated soil is regarded as 20 mg/kg just as it was in the pot. However, the average exposure concentration of heterogeneous 20 mg/kg AgNP-contaminated soil is simply converted into 5 mg/kg as it was composed of 25% AgNP-contaminated soil and 75% uncontaminated soil. Although the soil algae pipe was planted in 20 mg/kg AgNP-contaminated soil, the effect of less than 20 mg/kg AgNP in the contaminated soil in the heterogeneous pot may be induced by the _C. reinhardtii_ cells. Figure 4: Flow cytometric analysis of soil algae extracted from the pipe planted in the control, heterogeneous AgNP-contaminated soil (hetero), and homogeneous AgNP-contaminated soil (homo) at 1 week. (**A**) Flow cytometric statistics of soil alga (\(n=3\)–\(4\)), (**B**–**E**) flow cytometric histogram of soil alga ((**B**) chlorophyll, (**C**) cell granularity, (**D**) cell size, and (**E**) lipid contents). Asterisks (*) indicate results that are significantly different from the control (\(p<0.05\)). Crosshatches (\(\#\)) indicate results that are significantly different from the topsoil (\(p<0.05\)). Blue or red arrows indicate significant difference of each parameter from the control. The alleviated toxicity in heterogeneous AgNP-contaminated soil compared to homogeneous AgNP-contaminated soil may be due to pore water absorption of the upper soil layer from the lower layer as shown in tomato and alfalfa plants under salt stress [63; 64]. [PERSON] et al. [63] reached similar results with tomato plants exposed to heterogeneous salt distributed soils, where the toxicity of heterogeneous T\({}_{1.5}\) (soil salt contents in the upper and lower soil layer were 1% and 5%, respectively) was alleviated as shown by the biomass and photosynthesis of the tomato plants in comparison to homogeneous T\({}_{3.3}\) (soil salt contents in the upper and lower soil layer were 3% and 3%). In homogeneous AgNP-contaminated soil, AgNPs adsorbed in the lower soil surface may be re-released into the pore water due to the low retention of polyvinylpyrrolidone capping AgNPs in soil [65]. The pore water from the lower soil layer may be absorbed by _C. reinhardtii_ over time, significantly affecting it in the end. Meanwhile, in the heterogeneous AgNP-contaminated soil, non-AgNPs (e.g., nutrients) adsorbed in the lower soil surface may be re-released into the pore water and may be advantageously taken in by _C. reinhardtii_. The reactive surface area of AgNPs themselves or silver ions dissolved from AgNPs are known to be the main causes of the adverse effects of AgNPs on soil organisms [17; 18; 19; 20; 66; 67]. In other words, a  
Figure 5: A schematic diagram representing transport of nutrients or stressors via water absorption and formation of mucilaginous sheath enlarged from algal cells under desiccation in the control, heterogeneous AgNP-contaminated soil (hetero), and homogeneous AgNP-contaminated soil (homo) after 1 week, and appearance of non-target organisms (biological soil crusts) provided from the atmosphere, non-sterile tap water, and non-sterile soils after 4 weeks. soil algae pipe assay may be eventually influenced by the average exposure concentration due to pore water absorbed vertically from the other soil layers, even in heterogeneous soil. Even though the present study focused on applicability to evaluation of soil algae pipe assay, the limitations of analyses of toxic mechanisms of AgNPs to soil algae can be identified as follows. First, unclear bioaccumulated silver concentration in the algae remained; nevertheless, some previous studies reported the effects AgNPs themselves or silver ions dissolved from AgNPs on soil organisms including plants, isopod, springtail, worm, and soil alga [17; 18; 19; 20; 66; 67]. Next, toxicity contribution of PVP (polyvinylpyrrolidone) to soil algae were not investigated. It is estimated that 20 mg AgNPs/kg soil contained 0.6-0.8 mg PVP/kg soil.