# Rapid In Situ Biomonitoring of Subsoil Contamination by Applying an Algae-Soaked Disc Seeding Assay
## 3 Results and Discussion  
Figures 3 and 4 show the biomass, cell size, cell granularity, esterase activity, oxidative stress, and membrane permeability of _C. reinhardtii_ grown on four exposure scenarios after 1, 4, 7, 16, 13, 16, 19, and 22 weeks. All but one endpoint showed no significant effects at the beginning of the experiment. The exception was the _C. reinhardtii_ grown on the fourth soil layer-treated group; it showed a decrease in cell size and cell granularity and an increase in membrane permeability. The third, second, and first soil layer-treated groups showed initial significant effects after seven, 10, and 10 weeks, respectively. Over time, all exposure scenarios consistently showed significant effects compared to the control. The fourth soil layer-treated group showed a decrease in biomass after four and seven weeks. Cell size and cell granularity decreased after 1 week and increased after 13 weeks (cell size) and 16 and 19 weeks (cell granularity), respectively. The esterase activity decreased continuously after 10, 13, 16, 19, and 22 weeks. Oxidative stress increased after 10 and 13 weeks. Membrane permeability also increased after 1, 13, 16, and 19 weeks. In the third soil layer-treated group, cell size and cell granularity increased after 10 weeks (cell size) and 16 and 19 weeks (cell granularity), respectively. Esterase activity decreased continuously after 10, 13, 16, 19, and 22 weeks. Oxidative stress increased after 7, 13, 16, and 19 weeks. Membrane permeability also increased after 7, 10, 13, 16, and 19 weeks. In the second soil layer-treated group, cell granularity increased after 10 and 16 weeks. Esterase activity decreased after 13, 16, and 22 weeks. Oxidative stress increased after 13 and 16 weeks and membrane permeability increased after 10, 13, and 16 weeks. In the first soil layer-treated group, cell size and cell granularity increased after 10, 19, and 22 weeks (cell size) and 19 weeks (cell granularity), respectively. The esterase activity decreased after 10, 13, 19, and 22 weeks. Oxidative stress increased after 16 weeks, and membrane permeability increased after 13 and 16 weeks. Figure 3: _Cont._The difference in the toxicity of the contamination configuration-dependent AgNP-treated soils can be attributed to the distance between the stressor (the soil layer) and the receptor (the _C. reinhardtii_ grown on the disc that adhered to the topsoil). The initial effect on the _C. reinhardtii_ grown on the fourth soil layer-treated group was faster than the other exposure scenarios, and third, second, and first soil layer-treated groups showed initial significant effects in that order. The toxic effects of AgNPs are known to be induced by the reactive surface area of nanoparticles (NPs) or silver ions dissolved from them [17; 25; 26]. In this study, the initial significant effect on the _C. reinhardtii_ grown on the fourth soil layer-treated group may be related to how _C. reinhardtii_ adhered to nanoparticles in the topsoil. [PERSON] et al. [27] reported strong sorption of Ag on soil ligands in the first few hours. The significant effect of AgNPs over time on the _C. reinhardtii_ grown on the fourth, third, second, and first soil layer-treated groups may be related to the leaching of silver ions dissolved from the NPs. Previous studies also reported on the fate or toxicity of silver ions  
Figure 3: Biomass and cell morphology of _Chlamydomonas reinhardtii_ dependent on the time and contamination configuration in soil treated with silver nanoparticles ( AgNPs). (**a**) Biomass, (**b**) cell size, and (**c**) cell granularity. Asterisks (***) represent results that are significantly different from the control (_p_ < 0.05). N stands for “not applicable” due to low cell density. dissolved in soil from NPs over time [27; 28; 29]. [PERSON] et al. [28] reported that 25 and 75 nm PVP-AgNPs have low retention and high mobility in tropical soils. [PERSON] et al.