Assessing the Efficacy, Acute Toxicity, and Binding Modes of the Agricultural Nitrification Inhibitors 3,4-Dimethyl-1H-pyrazole (DMP) and Dicyandiamide (DCD) with Nitrosomonas europaea

Abstract

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.

Insights into the Efficacy and Binding Mode of 1,4-Disubstituted 1,2,3-Triazoles─A New Class of Agricultural Nitrification Inhibitors

“…Thus, inhibition of AMO disrupts this electron shuttle and, therefore, also prevents to some extent the oxidation of NH 2 OH, as has previously been reported for DMP. 35,46 In the case of N. multiformis, a small to moderate percentage inhibition of NO 2 − production was also found (Figure 2d), which is again likely a consequence of AMO inhibition. One notable exception is compound 1, where the inhibition was lower in the experiment targeting AMO than in that targeting HOA (21% and 46%, respectively; Figure 2c vs Figure 2d).…”

“…The recovery of enzyme activity indicates that the inhibition is likely to be reversible, similar to previous findings for DMP and DCD. 35 The observation that complete recovery of activity did not occur is not unexpected, considering the high [NI] used in this experiment, which could cause some degree of cell loss (Table S8 for compound 1 as an example). Determining the Acute Toxicity of Inhibitor 1.…”

“…The bacterial assay, including growth and harvest of the cells, measurements of the O 2 consumption, and the Michael−Menten kinetics, as well as the activity recovery assay, were conducted as previously reported. 35 All bacterial incubations were performed in sodium phosphate buffer (NaPB) with 1% (v/v) DMSO at pH = 7.5 and 30 °C with the different inhibitors and concentrations, as indicated. Experimental details are provided in the Supporting Information (SI).…”

“…32−34 Our recent studies with pure cells of Nitrosomonas europaea (N. europaea) have demonstrated that both DMP and DCD reversibly inhibit AMO through an uncompetitive mechanism. 35 Unfortunately, the development of more reliable NIs is hampered by the fact that the catalytically active site of AMO, a transmembrane enzyme, is not known, as its isolation leads to loss of structure and function. However, comparison with the recently crystallized particulate methane monooxygenase (pMMO), which is evolutionally similar to AMO, suggests that a cupredoxin-like unit could be involved in the oxidation process.…”

Rapid and Inexpensive Assay for Testing the Efficiency of Potential New Synthetic Nitrification Inhibitors

“…NUEs can be increased by using fertilizers amended with nitrification inhibitors (NIs). , These additives inhibit nitrifying microorganisms in the soil, such as ammonia oxidizing bacteria (AOB), that are responsible for the conversion of NH 3 to NO 3 – via hydroxylamine (NH 2 OH), nitric oxide (NO), and nitrite (NO 2 – ), thereby increasing the residence time of ammonium (NH 4 + ) and reducing undesired N losses. − AOB are ubiquitous, and NIs are designed to target ammonia monooxygenase (AMO), , the key enzyme in the nitrification process, which is responsible for the initial (and rate determining) transformation NH 3 → NH 2 OH …”