# Selecting Bioassay Test Species at the Screening Level of Soil Ecological Risk Assessments
## 2 Materials and Methods
### Bioassays  
For bioassays using soil samples, six test species were selected: _Vigna radiata_, _Oryza sativa_, _Eisenia andrei_, _Folsonia candida_, _Caenorhabditis elegans_, and _Chlorococcoun infusionum_. For the plant assays, dicotyledoneae mung bean (_V. radiata_) and monocotyledoneae rice (O. _sativa_) were selected as test species. The plant assay methods followed those of [PERSON] et al. [16] and the modified Organization for Economic Cooperation and Development (OECD) test guideline no. 208 [17]. For each pot, five seeds were exposed, and four replicates were run. For _V. radiata_, the experimental conditions were 25 \(\pm\) 1 \({}^{\circ}\)C, 16 h:8 h of light-dark photoperiod, and 80% humidity, while those for _O. sativa_ were 32 \(\pm\) 1 \({}^{\circ}\)C, 16 h:8 h of light-dark photoperiod, and 100% humidity. The experimental conditions were adjusted to reflect the growing environment of each species. After 7 days and 14 days of exposure, the length of the shoots were measured.  
In the earthworm assay, 310-600 mg of adult earthworm (_E. andrei_) was tested after removing gut contents on a moistened filter paper (3 h, 20 \(\pm\) 1 \({}^{\circ}\)C, darkness). The earth  
Figure 1: Image of the study site. The yellow circles show each sampling area. The aerial photograph was provided by the National Geographic Information Institute of Korea ([[https://www.ngi.go.kr](https://www.ngi.go.kr)]([https://www.ngi.go.kr](https://www.ngi.go.kr)), accessed 10 May 2021).  
worm soil acute assay followed a previous study [18]. Earthworms were maintained in the darkness at 20 \(\pm\) 1 \({}^{\circ}\)C for 14 days. Ten replicates for each soil sample were run and the assay was repeated twice. After the 14-day assessment, worms were moved to trays and the survival rate was assessed.  
The cellombola assays were conducted according to the OECD test guideline no. 232 using _F. candida_[19]. _F. candida_ exposed to soil samples were kept in 20 \({}^{\circ}\)C incubator with 16 h:8 h light-dark photoperiod for 14 days (acute), and 28 days (chronic). For chronic assays, ventilation was performed every week and food was supplied every 14 days. The survival rate (acute) and reproduction rate (chronic) of _F. candida_ were measured. Every test was run with four replicates.  
For the soil nematode assay, _C. elegans_ (wild type, Bristol strain N2) was used as the test species. The test was conducted according to a modified method of [PERSON] et al. [20]. Five young, adult _C. elegans_ (54-60 h after synchronization) were exposed to each soil sample and four replicates were run. Exposure conditions were 20 \(\pm\) 1 \({}^{\circ}\)C in the dark for 24 h; after exposure, the offspring were counted.  
In the soil algae assay, _C. infusionum_, obtained from SAG (Gottingen, Germany), was used as test species and precultured in [PERSON]'s basal medium (BBM). The tests were conducted in a 12-well plate following the method of [PERSON] et al. [21]. They were incubated at 24 \(\pm\) 2 \({}^{\circ}\)C under 16 h:8 h of light-dark photoperiod for 6 days. After 6 days, for the extraction of algae in the soil, the BBM medium was added to each well and plates were cultured for 24 h under exposed conditions. After 24 h, chlorophyll in the supernatant was extracted for 3 h and measured using a microplate reader (Variouskan LUX Multimode Microplate Reader, Thermo Fisher Scientific, MA, United States) at an excitation of 420 nm and emission of 671 nm.