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.

Soil toxicity resulting from either natural or anthropogenic heavy metal contamination was evaluated through a nitrifying bacteria bioassay focused on the inhibition of oxygen consumption. Every contaminated soil sample inhibited the nitrifying bacteria bioassay, with inhibition levels ranging from 71% to 100%. The optimal conditions for maximizing O₂ consumption during the test procedure were established as follows: a test culture volume of 1 mL, a soil sample weight of 1 g, a rotation rate of 100 revolutions per minute, and a reaction duration of 48 h. In low- or uncontaminated soils, oxygen consumption ranged from 3.2 mL to 3.0 mL from a headspace volume of 1 mL filled with O₂. In contrast, contaminated soils exhibited a lower range, with values between 0.1 mL and 1.0 mL. EC50 levels for NB O₂ consumption were: Cr⁶⁺ 1.21 mg/kg; Cu²⁺ 6.92 mg/kg; Ag⁺ 8.38 mg/kg; As³⁺ 8.99 mg/kg; Ni²⁺ 10.35 mg/kg; Hg²⁺ 11.01 mg/kg; Cd²⁺ 31.33 mg/kg; Pb²⁺ 129.62 mg/kg. Values for inherent test variability (CVi), variation resulting from the natural characteristics of soil (CVns), and minimal detectable difference (MDD) were found to range between 1.6% and 4.7%, 7.8% and 14.6%, and 2.9% and 5.9%, respectively. A 10% toxicity threshold was set as the maximal tolerable inhibition (MTI) for effective soil toxicity assessment. Nitrifying bacteria bioassays offer a fast, affordable, and user-friendly tool for real-time soil toxicity assessment, boosting soil health monitoring and ecosystem protection.