Nitrification inhibitors have been coformulated with nitrogen fertilizers since the 1970s to modulate the microbiological conversion of nitrogen in agricultural soils. 3,4-Dimethyl-1H-pyrazole (DMP) and dicyandiamide (DCD) are currently the most used commercial nitrification inhibitors, but their mode of action is not well understood. This work seeks to fill this void by assessing for the first time in detail their mechanism of inhibition, efficacy, and acute toxicity with pure cell cultures of Nitrosomonas europaea. Bacterial assays based on the quantification of the nitrite (NO2 –) production showed that both inhibitors reversibly target ammonia monooxygenase (AMO), which catalyzes the first step of the nitrification process. Michaelis–Menten kinetics suggest that both DMP and DCD act as uncompetitive inhibitors. Real-time measurements of the oxygen (O2) consumption confirmed the nonmechanistic mode of inhibition and showed that DMP reduced the O2 uptake rate by AMO much more at considerably lower concentrations than DCD, in line with the lower inhibitory efficiency of the latter. Acute toxicity tests revealed that DCD has a 10% higher toxicity than DMP when comparing treatments at the same inhibition efficacy (i.e., DMP at 10 ppm, DCD at 100 ppm), indicating that the inhibition of the nitrification process cannot simply be achieved by increasing the inhibitor concentration. The methods presented in this study could assist the development of more reliable nitrification inhibitors in the future.

The microbial conversion of ammonia to nitrite in soils involves three enzymatic steps. Nitrification inhibitors (NIs) are designed to inhibit ammonia monooxygenase (AMO), the enzyme performing the initial oxidation of ammonia to hydroxylamine, to mitigate excessive nitrogen fertilizer losses in agricultural systems. Because the efficiency of the current commercial NIs is highly unreliable, novel, better performing compounds need to be developed. Previously, time-consuming soil incubation studies were required as the first step to test new potential NIs. We present here a simple and cost-efficient colorimetric assay that has been developed for the rapid assessment of the efficiency of new synthetic NIs to identify the most promising compounds for subsequent soil studies. This protocol enables screening of the inhibitor activity of multiple compounds at the same time with high reproducibility and can be manipulated to determine pH and temperature-dependent effects on NIs.