Validation of the paper-disc soil method using soil alga Chlorococcum infusionum to quantitatively determine the toxicity of heavy metals.

The paper-disc soil method is a soil algal bioassay used to assess soil water transfer in polluted soils. Although this ecotoxicological method was first established in 2016 to assess soil pollution, quantitative toxicity assessments of Cu- or Ni-treated soil and in situ or ex situ soils polluted with other heavy metals (HMs) have not been widely conducted. In this study, the paper-disc soil method was validated under different test conditions. The validated paper-disc soil method was used to quantitatively investigate the toxicity of four HMs (As, Cd, Cr, and Zn). Based on the results, the test species, initial inoculation concentration in the algal-dispersed solution, water content, light intensity, and exposure duration were proposed as Chlorococcum infusionum, 3 × 10 cells/mL in Bold's basal medium, 80% water-holding capacity, 4000 ± 500 lx, and 6 days, respectively. The toxicity of HMs was quantified using the validated paper-disc soil method, and the half-maximal effective concentration values for biomass were calculated as 22.49 (21.26-23.78) mg Cr/kg dry weight (dw) soil, 42.72 (38.64-47.24) mg Cd/kg dw soil, 57.79 (55.46-60.21) mg As/kg dw soil, and 183.06 (175.38-191.06) mg Zn/kg dw soil. The paper-disc soil method using the soil alga C. infusionum was validated by quantitatively evaluating the test conditions and toxicity of HMs. Our results provide important quantitative toxicity data for soil algae exposed to HMs and a basis for standardizing the paper-disc soil method using soil algae.