# Rapid In Situ Biomonitoring of Subsoil Contamination by Applying an Algae-Soaked Disc Seeding Assay
## 1 Introduction  
Monitoring soil pollution is very important for protection of other environmental media because there is potential behavior of soil pollutants via hydrological transport or volatilization to other environmental media (e.g., groundwater, surface water, sediments, or air) [1]. For monitoring soil pollution effectively, it is necessary to consider an integrated approach with practical bioavailability by biomonitoring and measured chemicals by analytic chemistry [2]. The soil quality of a target site is investigated by biological indicators as well as physicochemical indicators, such as plants [3, 4], earthworms [5, 6], enzyme activity [7], and soil algae [6, 8]. However, these chemistry and toxicity data of the target soil resulted from temporarily short-term experiments. Only in situ soil dehydrogenase activity in soils treated with tebuconazole or azoxystrobin and amended with spent mushroom substrate was investigated in a periodic long-term experiment, over 355 or 378 days [9, 10]. This traced the change of in situ soil dehydrogenase activity induced from tebuconazole or azoxystrobin over time (0, 36, 92, 124, 355 days or 0, 28, 84, 181, 378 days). Therefore, it is necessary to conduct periodic subsoil contamination monitoring for prevention of environmental effects resulting from unpredictable subsoil contamination (e.g., oil leaks, radioactive effluents, or illegal disposal).  
Soil algae are a key food source for mesothelioma and microfauna [11]. In addition, they synthesize organic matter and form mucaginous sheaths that are essential to maintain soil fertility and aggregation [11]. In a previous study [12], we developed an algae-soaked disc seeding assay as a rapid in situ assessment for predicting soil quality and presented decrease of chlorophyll fluorescence of the alga _Chlamydomonas reinhardtii_ after exposure to contaminated, remediated, and amended farm and paddy soils in the field using the algae-soaked disc seeding assay. The results of that study identified that the algae-soaked disc seeding assay is convenient for rapidly assessing in situ soil contamination. However,Nam et al. [12] only assessed chlorophyll fluorescence of _C. reinhardtii_ in solely heterogenous soil (30 cm of clean topsoil plus 70 cm of target subsoil) collected from the field at 2 days. Soil algae, as terrestrial organisms lived on and beneath the soil surface [11], can be a terrestrial test species as a periodic toxicity indicator in the long-term experiment. In this study, we extended our experiment to assess its applicability to periodic evaluation for subsoil contamination under different contamination configurations of heterogenous soils, together with examination of cell morphology, esterase activity, oxidative stress, and membrane permeability using an algae-soaked disc seeding assay. Silver nanoparticles (AgNPs) were used as a target chemical because of their broad use in our life [13,14] and the ecotoxicity of AgNPs on soil organisms: plants, isopods, earthworms, soil algae, and exoenzymes [15, 16, 17, 18, 19]. _C. reinhardtii_, which is conventionally living in freshwater and soil, has been used as a biological indicator [6,12]. To the best of our knowledge, this is the first study to assess applicability of an algae-soaked disc seeding assay considering periodic evaluation of subsoil contamination under different contamination configurations of heterogenous soils, as part of an efficient algae-soaked disc seeding assay for rapid in situ biomonitoring of subsoil contamination over time.