Assessment of ionic quenching on soil ATP bioluminescence reaction

Co-extracted ions with ATP from soils may interfere with ATP luciferin–luciferase luminescence reaction when ATP is assayed. The effects were investigated in a typical concentration range of cations and anions potentially extractable in soils. A commercial ATP assay product (Sigma Chemical Co.) was used. Significant quenching is evidenced from a concentration of 0.10 mM with Cu2+ and Zn2+, and 1.00 mM with Ca2+. The order of quenching at 1.00 mM was: Cu2+>Zn2+>Ca2+=Na+=Mg2+, while Mg2+=Mn2+, both Ca2+ and Na+>Mn2+. The quenching was found to be much more severe with selected special heavy metal cations with quenching in the order: Ti3+>Hg2+>Cr3+. Because cation quenching can be alleviated by addition of EDTA, three forms of EDTA (Mg, Na and acid EDTA) were tested for their suitability for the assay. The Mg-EDTA was found superior to the other two. Presence of PO43− at concentrations of 0.01 and 0.05 mM, and NO3− at 0.01 and 0.10 mM, significantly enhanced ATP light emission (8–13%). However, SO42− at similar concentrations significantly decreased light emission. The quenching by CO32− and Cl− was only observed at high concentrations (3.20 mM and up). The order of quenching for the anions at a concentration of 6.4 mM was: PO43−>CO32−>SO42−>NO3−>Cl−. Enhanced or depressed light emission induced by ions would produce significant over or underestimation of soil ATP. While addition of Mg-EDTA may alleviate cation quenching, the interference from anions may require the ATP assay standards be prepared in a solution of similar chemical composition to that in soil ATP extracts.