# Applicability Evaluation of Soil Algae Pipe Assay in Silver Nanoparticle-Contaminated Soils
## 3 Results and Discussion  
### Observed Effects of Soil Algae over the Exposure Duration  
The growth of _C. reinhardtii_ under different distributions of AgNP-contaminated soil after 1 week is shown in Figure 3. As displayed in Figure 3A,F,K, remarkable growth of _C. reinhardtii_ in all the pipes after 1 week was observed with a green color over the surface soil, compared to the soil background color. Figure 3C,H,M present examples of the _C. reinhardtii_ cells in the control and the different distributions of the AgNP-contaminated soil groups (hetero and homo) after 1 week. The formation of muclaginous sheaths enlarged from _C. reinhardtii_ cells were exhibited in the control and AgNP-contaminated soil groups after 1 week (dotted inset in Figure 3C,H,M), as observed in [PERSON] et al. [20] The formation of muclaginous sheath in soil algae has been known as a protective mechanism to provide moisture or nutrients under stressors (e.g., desiccation, predators, chemicals, and nutrient deficiencies) [26; 27; 28; 29; 30; 31; 32; 33; 34; 35]. In this case, the muclaginous sheath in _C. reinhardtii_ was observed in all control and AgNP-contaminated soil groups after 1 week. Therefore, the muclaginous sheath of _C. reinhardtii_ may be formed to provide moisture against desiccation and not only for protection against the toxicity of the AgNPs. Moreover, both the heterogeneous and homogeneous AgNP-contaminated soil groups exhibited more partially hollow _C. reinhardtii_ cells than the control group (Figure 3H,M). After exposure, the AgNPs may cause morphological changes in _C. reinhardtii_, thereby inducing the loss of photosynthetic pigments in undeveloped chloroplasts in non-viable or chlorotic cells [36], the expansion of mitochondria, and the weakening of plasma membrane integrity in necrotic cells [37]. As shown in Figure 3D,LN, noticeable growth of soil algae under different distributions of AgNP-contaminated soil after 4 weeks was observed with a dark green color over the dry surface soil which was greater extent compared to after 1 week. Figure 3E,J,O present examples of soil algae cells in the control and different distributions of AgNP-contaminated soil groups (hetero and homo) after 4 weeks. Unlike after 1 week, non-target organisms were observed in the control, and there were different distributions of AgNP-contaminated soil groups (orange arrows in Figure 3E,J,O). Soil algal suspension extracted from the pipes included non-target organisms, which is probably due to biological soil crusts. Biological soil crusts, which commonly appear in open and arid areas, are formed by combinations of soil particles and various groups of organisms (e.g., cyanobacteria, algae, fungi, bacteria, archaea, lichens, and bryophytes) [38; 39]. The appearance of biological soil crusts may be the result of desiccation resistance and the unintentional introduction of non-targetorganisms provided from the atmosphere, non-sterile tap water, and non-sterile soils, under an open soil algae pipe assay. Previous studies have shown that after 4 weeks of exposure, unintentionally formed biological soil crusts have potential interference effects on target soil algae analysis, because physicochemical characteristics of soil (e.g., aggregate stability, water retention, organic matters) can be altered by biological soil crusts [39; 40; 41; 42], and the target soil algae and non-target organisms can become mixed in the samples. Therefore, the soil algae pipe assay is not applicable for 4 weeks of exposure due to biological soil crusts unintentionally forming in an open system. These results indicated that evaluation period of soil contamination by applying soil algae pipe assay under the open system should be under 4 weeks in the environmental management view. Figure 3: Macroscopy and microscopy images of soil algae extracted from the pipe planted in the control, heterogeneous AgNP-contaminated soil (hetero), and homogeneous AgNP-contaminated soil (homo) at 1 and 4 weeks.