Copper Toxicity to Bioluminescent Nitrosomonas europaea in Soil Is Explained by the Free Metal Ion Activity in Pore Water

The terrestrial biotic ligand model (BLM) for metal toxicity in soil postulates that metal toxicity depends on the free metal ion activity in solution and on ions competing for metal sorption to the biotic ligand. Unequivocal evidence for the BLM assumptions is most difficult to obtain for native soil microorganisms because the abiotic and biotic compartments cannot be experimentally separated. Here, we report copper (Cu) toxicity to a bioluminescent Nitrosomonas europaea reporter strain that was used in a solid phase-contact assay and in corresponding soil extracts and artificial soil solutions. The Cu²⁺ ion activities that halve bioluminescence (EC50) in artificial solutions ranged 10⁻⁵ to 10⁻⁷ M and increased with increasing activities of H⁺, Ca²⁺ and Mg²⁺ according to the BLM concept. The solution based Cu²⁺ EC50 values of N. europaea in six contaminated soils ranged 2 × 10⁻⁶ to 2 × 10⁻⁹ M and these thresholds for both solid phase or soil extract based assays were well predicted by the ion competition model fitted to artificial solution data. In addition, solution based Cu²⁺ EC50 of the solid phase-contact assay were never smaller than corresponding values in soil extracts suggesting no additional solid phase toxic route. By restricting the analysis to the same added species, we show that the Cu²⁺ in solution represents the toxic species to this bacterium.