# Applicability Evaluation of Soil Algae Pipe Assay in Silver Nanoparticle-Contaminated Soils
## 2 Materials and Methods
### Experimental Design and Set Up  
To evaluate the applicability of the soil algae pipe assay for its dependence on exposure duration and the heterogeneous distribution of AgNP contaminants, three distributed soils were used: Control, heterogeneous AgNP-contaminated soil (hetero), and homogeneous AgNP-contaminated soil (homo). A schematic of the differently distributed soil is provided in Figure 2. To prepare the heterogeneous AgNP-contaminated soil, 1350 g portions of untreated soil were placed in open plastic pots (15 \(\times\) 15 \(\times\) 15 cm), and 450 g of 20 mg AgNPs/kg soil was covered over the untreated soil. To prepare the homogeneous AgNP-contaminated soil, 1800 g portions of 20 mg AgNPs/kg soil were placed in the pots. Subsequently, all soils in the pots were hydrated with a 30% (\(p/w\)) N-free Hoagland solution [23] and were aged for 7 days in a greenhouse (15-25 \({}^{\circ}\)C, humidity 50-60%).  
This study referred to the soil algae pipe assay from [PERSON] et al. (2019). Green alga, _Chlamydomonas reinhardtii_ (UTEX No. 2244), was purchased from the University of Texas, Austin (USA). Incubation was conducted in a Tris-acetate-phosphate medium at 24 \(\pm\) 2 \({}^{\circ}\)C and shaken at 100 rpm under a 16:8 h light:dark photoperiod provided by cool-white fluorescent lamps (54 \(\mu\)mol photons/m\({}^{2}\cdot\)s\({}^{1}\)). Two UV-sterilized plastic pipes (diameter \(\times\) length, 2 \(\times\) 3 cm) were planted on both sides of each pot at a 2 cm depth in the surface soil in four replicates. Pre-cultured soil alga (1 mL) in the exponential growth phase (OD\({}_{660}\) = 2.0) was inoculated on the surface soil in each pipe. To moisten the soil, tap water was provided at intervals of 2-3 days. After 1 and 4 weeks, the pipes were collected from each pot, and a portion of soil alga grown on the surface soil was sampled. The sampled soil algae were detached from the soil during the 24 h incubation  
Figure 2: Schematic of different distributions of AgNP-contaminated soil. (**A**) The upper image of test pot, (**B**) The side images of control, heterogeneous AgNP-contaminated soil, and homogeneous AgNP-contaminated soil.  
period in Bold's Basal medium and then analyzed using flow cytometry (FACSCalibur, BD Biosciences, NJ, USA). Microscopic images were observed by light microscopy (BX-51; Olympus, Tokyo, Japan). Flow cytometric analysis was only applied to the soil algae sampled after 1 week exposure. Cell size, cell granularity, and in vivo chlorophyll \(a\) were detected via forward scattered light (FSC), side scattered light (SSC), and FL3 (670 nm band pass filter, excitation at 488 nm blue laser, 15 mW, argon ion laser), respectively. The lipid contents were detected via FL2 (564-606 nm band pass filter) after staining with Nile red for the determination of intracellular neutral lipid in algae [24]. Nile red powders were dissolved in acetone to obtain a 250 \(\upmu\)g/mL solution, and the stock solution was stored at 4 \({}^{\circ}\)C until use. Subsequently, a working solution was prepared by diluting the stock solution 200 times with a mixture of dimethyl sulfoxide and Bold's Basal medium (1:3). Finally, soil algae were stained with Nile red at the rate of 1:1 for 10 min (dark, room temp.). The geometric means of the FSC, SSC, FL3, and FL2 intensities were normalized based on 10,000 events in the gated soil alga population.