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A seven-chamber reactor was developed for a 2-h batch toxicity assessment in water using sulfur-oxidizing bacteria (SOB) based bioassay. The optimum sulfur to water ratio (g:mL) was evaluated using a constant 20 g of sulfur and varying volumes of water at ratios of 1:1, 1:1.5, 1:2, and 1:2.5. Sulfur particles with 20 g sulfur and 20 mL water (1:1 ratio) proved the most efficacious and led to higher electrical conductivity (EC) values. Air flow rates may hinder the effectiveness of EC as the indicator since rates greater than 300 mL/min were shown to lead to higher ECs, most likely through evaporation resulting in higher concentrations. Once optimal conditions were established, the toxicity of Cr⁶⁺, Cu²⁺, Zn²⁺, Hg²⁺, and Ag²⁺ were assessed. The toxicity values (EC₅₀) were decreased as exposure time was increased. A 2-h EC₅₀ of 1.78 mg/L for Cr⁶⁺, 5.0 mg/L for Cu²⁺, 1.55 mg/L for Zn²⁺, 0.21 mg/L for Hg² …