Comparative Analysis of Chernozem Microbiome Resistance to Toxic Heavy Metals (Co²⁺, Cu²⁺, Zn²⁺, Ni²⁺) under Individual and Pairwise Exposure

<title>Abstract</title> <p>Anthropogenic polymetallic contamination threatens high-fertility chernozem ecosystems, disrupting microbiome-mediated nutrient cycling. This study evaluated the systemic functional stability of the native chernozem microbiome exposed to Cu²⁺, Co²⁺, Zn²⁺, and Ni²⁺. Resilience to individual stressors and 1:1 binary mixtures was quantified over 15 days using OD540 and CFU assays as thermodynamic proxies for active metabolic load. Logistic dose-response modeling established a strict acute toxicity hierarchy: Co²⁺ (EC50 = 130.8 mg/L) &gt; Ni²⁺ (EC50 = 167.6 mg/L) &gt; Zn²⁺ (EC50 = 290.0 mg/L) &gt; Cu²⁺ (EC50 = 331.2 mg/L). Crucially, the Bliss Independence model demonstrated severe synergistic inhibition across all binary mixtures. At 400 mg/L total concentration, combinations yielded Synergistic Ratios up to 7.1, with viability reductions drastically exceeding additive predictions. These findings quantitatively prove that single-metal ecological risk assessments critically underestimate co-contamination hazards, emphasizing the urgent need to integrate synergistic factors into environmental safety standards and bioremediation strategies.</p>