Mechanism of action of nitrification inhibitors based on dimethylpyrazole: A matter of chelation.

In agriculture, the applied nitrogen (N) can be lost in the environment in different forms because of microbial transformations. It is of special concern the nitrate (NO) leaching and the nitrous oxide (NO) emissions, due to their negative environmental impacts. Nitrification inhibitors (NIs) based on dimethylpyrazole (DMP) are applied worldwide in order to reduce N losses. These compounds delay ammonium (NH) oxidation by inhibiting ammonia-oxidizing bacteria (AOB) growth. However, their mechanism of action has not been demonstrated, which represent an important lack of knowledge to use them correctly. In this work, through chemical and biological analysis, we unveil the mechanism of action of the commonly applied 3,4-dimethyl-1H-pyrazole dihydrogen phosphate (DMPP) and the new DMP-based NI, 2-(3,4-dimethyl-1H-pyrazol-1-yl)-succinic acid (DMPSA). Our results show that DMP and DMPSA form complexes with copper (Cu) cations, an indispensable cofactor in the nitrification pathway. Three coordination compounds namely [Cu(DMP)Cl] (CuDMP1), [Cu(DMP)SO] (CuDMP2) and [Cu(DMPSA)]·HO (CuDMPSA) have been synthesized and chemical and structurally characterized. The CuDMPSA complex is more stable than those containing DMP ligands; however, both NIs show the same nitrification inhibition efficiency in soils with different Cu contents, suggesting that the active specie in both cases is DMP. Our soil experiment reveals that the usual application dose is enough to inhibit nitrification within the range of Cu and Zn contents present in agricultural soils, although their effects vary depending on the content of these elements. As a result of AOB inhibition by these NIs, NO-reducing bacteria seem to be beneficed in Cu-limited soils due to a reduction in the competence. This opens up the possibility to induce NO reduction to N through Cu fertilization. On the other hand, when fertilizing with micronutrients such as Cu and Zn, the use of NIs could be beneficial to counteract the increase of nitrification derived from their application.