# Rapid In Situ Biomonitoring of Subsoil Contamination by Applying an Algae-Soaked Disc Seeding Assay
## 3 Results and Discussion
[29] reported that silver ions dissolved from NPs in 50 nm untreated AgNPs increased the toxicity of the latter for earthworms over time. [PERSON] et al. [27] reported that silver ions released from AgNPs at the end of their experiment were ten times higher than those at the beginning of the experiment. Figure 4: _Cont_. In terms of toxicity endpoints, a significant increase in cell size, cell granularity, oxidative stress, and membrane permeability and a decrease in esterase activity of _C. reinhardtii_ were observed in all exposure scenarios at the end of the experiment. Some results related to cell size, membrane permeability, and esterase activity of this study were similar to those in our other recent study [17]. After six days of exposure to the AgNP-treated soil, _C. reinhardtii_ showed an increase in cell size and membrane permeability and a decrease in esterase activity. According to prior literature, increase of cell size has been interpreted as production of exopolysaccharideacharide enlarged from the algal cell wall as a protective barrier [17; 30] or adhesion of nanomaterials to the algal cell wall [31]. Uptake of nanomaterials into algal cells has been interpreted as an increase of cell granularity [32; 33]. The increase of oxidative stress has been regarded as a cellular antioxidant defense system to nanomaterials after disturbance of balance between oxidant/antioxidant processes [34]. Damage of membrane integrity and disturbance of electron and ion transport chains of the cell membrane after the attachment of nanomaterials has been regarded as increase of membrane permeability [35]. Decrease of esterase activity has been recognized as a reduction of metabolic activity in algal cells [36]. In AgNP-treated soils, NPs themselves or silver ions dissolved from the NPs may respond to exposed algal cell walls, thereby increasing cell size. Subsequently, membrane permeability may be increased due to attachment of NP or ions to algal cell walls and physical damage to the cell walls. After that, esterase activity may be reduced by toxicity of NP or ions internalized via permeable membrane, thereby increasing cell granularity. Finally, oxidative stress may operate against the toxicity of NP or ions in algal cells. In other words, the algae-soaked disc seeding assay can sense a change of subsoil contamination by periodic evaluation and multiple toxicity endpoints over time, although the algae-soaked disc is set on the surface soil. However, further analysis on the toxic effects caused by AgNPs or dissolved Ag ions on _C. reinhardtii_ is needed to clarity mechanism, as the mechanism of the experiment could not be determined in this study. In addition, an extended experiment using various soil algae and soil textures is needed to study, as the prior literature using an algae-soaked disc seeding assay shows morphological differences on _C. reinhardtii_ and _PseudokircInteriella subcapitata_ exposed to nickel [20] and inapplicability on loamy sand soil due to high soil infiltration rate in a contaminated field [11]. Figure 4: Physiological effects of _Chlamydomonas reinhardtii_ dependent on the time and contamination configuration in soil treated with silver nanoparticles (AgNPs). (**a**) esterase activity, (**b**) oxidative stress, and (**c**) membrane permeability. Asterisks (*) represent results that are significantly different from the control (\(p<0.05\)). N stands for “not applicable” due to low cell density.