Novel Solid-Phase Bioassay Kit with Immobilized Chlorella vulgaris Spheres for Assessing Heavy Metal and Cyanide Toxicity in Soil

Heavy metal and cyanide contamination in soil presents serious environmental and ecological concerns due to their persistence, bioavailability, and toxicity to soil biota. In this study, a novel solid-phase direct contact bioassay kit was developed using immobilized Chlorella vulgaris spheres to evaluate the toxicity of soils contaminated with mercury (Hg2+), silver (Ag+), copper (Cu2+), and cyanide (CN−). The assay was designed using 25 mL glass vials in which algal spheres were directly exposed to spiked soils for 72 h without the need for pollutant extraction. Oxygen evolution in the headspace was measured as the primary endpoint, alongside optical density and chlorophyll a fluorescence (OJIP) to assess photosynthetic inhibition. The assay demonstrated high sensitivity and reproducibility, with strong correlations (R2 > 0.93) between oxygen evolution and optical density. EC50 values based on oxygen evolution were 4.43, 4.18, 3.10, and 61.3 mg/kg for Hg2+, Ag+, CN−, and Cu2+, respectively, and 7.8, 7.4, 2.9, and 29.7 mg/kg based on optical density. The relatively higher EC50 for copper was attributed to its biological role as an essential micronutrient. OJIP transient profiles supported the observed photosynthetic inhibition, particularly under Hg2+, Ag+, and CN− exposure. The present study overcomes the limitations of conventional chemical analyses by providing a rapid, low-cost, and ecologically relevant tool for direct soil toxicity assessment, with potential applications in environmental monitoring and contaminated site evaluation.