N-HETEROCYCLIC COMPOUNDS USED AS NITRIFICATION INHIBITOR

Abstract

The use of an N-heterocyclic compound of the general formula (a) or (b) with the following definitions: X.sup.1 being S or X.sup.2 being S or O and at least one of X.sup.1 and X.sup.2 being S R.sup.2 H or C.sub.1-4-alkyl, R.sup.3 H or C.sub.1-4-alkyl R.sup.6 and R.sup.7 are hydrogen or together form a covalent carbon-carbon bond in general formula (a) R.sup.1 being H, C.sub.1-12-alkyl or CH.sub.2NR.sup.4R.sub.5 with R.sup.4 hydrogen or C.sub.1-4-alkyl, R.sup.5 C.sub.1-12-hydrocarbon residue which can contain one to three halogen atoms and/or one to four heteroatoms, selected from the group consisting of nitro-gen, oxygen and sulfur, it also being possible for R.sup.4 and R.sup.5, together with the nitrogen atom joining them, to form a 5- or 6-membered saturated or unsaturated heterocyclic radical, which optionally may also contain one or two further heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, in general formula (b) R.sup.1 being H or C.sub.1-17-hydrocarbon, preferably H, or CH.sub.2R.sup.5 with R.sup.5 being H or C.sub.1-16-hydrocarbon residue, which hydrocarbon can contain one to three halogen atoms and/or one to six heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, and preferably in general formula (a) and (b) X.sup.1 and X.sup.2 being S, as nitrification inhibitor.

##STR00001##

Claims

16. A method for inhibiting nitrification, comprising utilizing an N-heterocyclic compound of the general formula (a) or (b) ##STR00076## with the following definitions: X1 being S or O, X2 being S or O and at least one of X1 and X2 being S R2 H or C1-4-alkyl, R3 H or C1-4-alkyl R6 and R7 are hydrogen or together form a covalent carbon-carbon bond in general formula (a) R1 being H, C1-12-alkyl or CH2NR4R5 with R4 hydrogen or C1-4-alkyl, R5 C1-12-hydrocarbon residue which can contain one to three halogen atoms and/or one to four heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, it also being possible for R4 and R5, together with the nitrogen atom joining them, to form a 5- or 6-membered saturated or unsaturated heterocyclic radical, which optionally may also contain one or two further heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, in general formula (b) R1 being H or C1-17-hydrocarbon, preferably H, or CH2R5 with R5 being H or C1-16-hydrocarbon residue, which hydrocarbon can contain one to three halogen atoms and/or one to six heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, and preferably in general formula (a) and (b) X1 and X2 being S, as nitrification inhibitor, wherein the N-heterocyclic compound of the general formula (a) or (b) is an N-heterocyclic compound of the general formula (I) or (II) or (III) or (IV) ##STR00077## with the following definitions: X1 being S or O, X2 being S or O and at least one of X1 and X2 being S R2 H or C1-4-alkyl, R3 H or C1-4-alkyl in general formula (I) and (III) R1 being H, C1-12-alkyl or CH2NR4R5 with R4 hydrogen or C1-4-alkyl, R5 C1-12-hydrocarbon residue which can contain one to three halogen atoms and/or one to four heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, it also being possible for R4 and R5, together with the nitrogen atom joining them, to form a 5- or 6-membered saturated or unsaturated heterocyclic radical, which optionally may also contain one or two further heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, in general formula (II) and (IV) R1 being H or C1-17-hydrocarbon, preferably H, or CH2R5 with R5 being H or C1-16-hydrocarbon residue, which hydrocarbon can contain one to three halogen atoms and/or one to six heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, and preferably in general formula (I), (II), (III) and (IV) X1 and X2 being S.

17. The method of claim 16, wherein R2, R3 and R4 independently of one another are hydrogen, methyl or ethyl, wherein in general formula (I) and (III) X1 is S or O, and X2 is S, preferably wherein in general formula (I) and (III), X1 and X2 are S.

18. The method of claim 16, wherein in general formula (I) and (III) R5 is a C3-10-hydrocarbon residue which can contain one to two halogen atoms and/or one to three heteroatoms and which contains at least one cyclic structure, wherein preferably in general formula (I) and (III) R5 is a C3-8-hydrocarbon residue which can contain one to two halogen atoms and/or one to three heteroatoms and which contains a 5- or 6-membered cyclic structure which can be annellated to a second 5- or 6-membered cyclic structure, wherein more preferably in formula (III) R1, R2 and R3 are independently hydrogen, methyl or ethyl.

19. The method of claim 16, wherein in general formula (II) and (IV) R1 is C1-4-alkyl or CH2R5 with R5 C.sub.3-14-hydrocarbon residue, which can contain one or two halogen atoms and/or two to six heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, wherein preferably in general formula (II) and (IV) R1 is methyl, ethyl or CH2R5 with R5 C5-12-hydrocarbon residue, which can contain one halogen atom and/or two to four heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur wherein more preferably in formula (IV) R1 is hydrogen, methyl or ethyl.

20. The method of claim 16, wherein the N-heterocyclic compound is selected from the group consisting of: ##STR00078##

21. The method of claim 16, wherein the N-heterocyclic compound of the general formula (I) or (II) or (III) or (IV) is applied in the form of an additive or coating material for inorganic and/or organic and/or organomineral fertilizers, preferably inorganic fertilizers, more preferably ammonium- and/or urea-containing nitrogen fertilizers.

22. The method of claim 16, wherein the N-heterocyclic compound of the general formula (I) or (II) or (III) or (IV) is delivered in a form of a formulation, solution or dispersion, separately or simultaneously with a fertilizer, or is incorporated into the fertilizer or is applied to the fertilizer.

23. The method of claim 16, wherein the nitrogen or carbon losses in inorganic and/or organic and/or organomineral fertilizers or nitrogen- or carbon-containing compounds or materials and also on harvest refuse and on grazed land or during the storage of liquid manure are reduced and the ammonia load in animal stalls are lowered.

24. The method of claim 16, wherein the N-heterocyclic compound of the general formula (I) or (II) or (III) or (IV) is used together with at least one additional agrochemical agent, preferably selected from the group consisting of at least one further nitrification inhibitor, preferably selected from the group consisting of 2-(3,4-dimethyl-pyrazol-1-yl)-succinic acid (DMPSA), 3,4-dimethylpyrazole (DMP), 3,4-dimethylpyrazolephosphate (DMPP), dicyandiamide (DCD), 1H-1,2,4-triazole, 3-methylpyrazole (3-MP), 2-chloro-6-(trichloromethyp-pyridine, 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazol, 2-amino-4-chloro-6-methyl-pyrimidine, 2-mercapto-benzothiazole, 2-sulfanilamidothiazole, thiourea, sodium azide, potassium azide, 1-hydroxypyrazole, 2-methylpyrazole-1-carboxamide, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, 2,4-diamino-6-trichloromethyl-5-triazine, carbon bisulfide, ammonium thiosulfate, sodium trithiocarbonate, 2,3-dihydro-2,2-dimethyl-7-benzo furanol methyl carbamate and N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester, at least one urease inhibitor, preferably selected from N-n-butylthiophosphoric triamide (NBTPT) and/or N-n-propylthiophosphoric triamide (NPTPT), at least one customary agrochemical auxiliary agent, preferably selected from the group consisting of aqueous and/or organic solvents, pH-adjusting agents, surfactants, wetting agents, spreading agents, adhesion promoters, carriers, fillers, viscosity-adjusting agents, emulsifiers, dispersants, sequestering agents, anti-settling agents, coalescing agents, rheology modifiers, defoaming agents, photo-protectors, anti-freeze agents, biostimulants, pesticides, biocides, plant growth regulators, safeners, penetrants, anticaking agents, mineral and/or vegetable oils and/or waxes, colorants and drift control agents, and mixtures thereof.

25. A mixture, containing at least one N-heterocyclic compound as defined in claim 16, and at least one additional agrochemical agent, preferably selected from the group consisting of at least one further nitrification inhibitor, preferably selected from the group consisting of 2-(3,4-dimethyl-pyrazol-1-yl)-succinic acid (DMPSA), 3,4-dimethylpyrazole (DMP), 3,4-dimethylpyrazolephosphate (DMPP), dicyandiamide (DCD), 1H-1,2,4-triazole, 3-methylpyrazole (3-MP), 2-chloro-6-(trichloromethyp-pyridine, 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazol, 2-amino-4-chloro-6-methyl-pyrimidine, 2-mercapto-benzothiazole, 2-sulfanilamidothiazole, thiourea, sodium azide, potassium azide, 1-hydroxypyrazole, 2-methylpyrazole-1-carboxamide, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, 2,4-diamino-6-trichloromethyl-5-triazine, carbon bisulfide, ammonium thiosulfate, sodium trithiocarbonate, 2,3-dihydro-2,2-dimethyl-7-benzofuranol methyl carbamate and N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester, at least one urease inhibitor, preferably selected from N-n-butylthiophosphoric triamide (NBTPT) and/or N-n-propylthiophosphoric triamide (NPTPT), at least one customary agrochemical auxiliary agent, preferably selected from the group consisting of aqueous and/or organic solvents, pH-adjusting agents, surfactants, wetting agents, spreading agents, adhesion promoters, carriers, fillers, viscosity-adjusting agents, emulsifiers, dispersants, sequestering agents, anti-settling agents, coalescing agents, rheology modifiers, defoaming agents, photo-protectors, anti-freeze agents, biostimulants, pesticides, biocides, plant growth regulators, safeners, penetrants, anticaking agents, mineral and/or vegetable oils and/or waxes, colorants and drift control agents, and mixtures thereof.

26. A fertilizer mixture, containing A. an inorganic and/or organic and/or organomineral fertilizer and B. 10 to 10000 weight-ppm, based on the inorganic fertilizer, of at least one N-heterocyclic compound as defined in claim 16.

27. The fertilizer mixture as claimed in claim 26, wherein the fertilizer mixture is in solid form and the N-heterocyclic compound of the general formula (I) and/or (II) and/or (III) and/or (IV) is applied to the surface of the, preferably inorganic, fertilizer.

28. The fertilizer mixture as claimed in claim 26, wherein the fertilizer mixture contains at least one additional agrochemical agent, preferably selected from the group consisting of at least one further nitrification inhibitor, preferably selected from the group consisting of 2-(3,4-dimethyl-pyrazol-1-yl)-succinic acid (DMPSA), 3,4-dimethylpyrazole (DMP), 3,4-dimethylpyrazolephosphate (DMPP), dicyandiamide (DCD), 1H-1,2,4-triazole, 3-methylpyrazole (3-MP), 2-chloro-6-(trichloromethyp-pyridine, 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazol, 2-amino-4-chloro-6-methyl-pyrimidine, 2-mercapto-benzothiazole, 2-sulfanilamidothiazole, thiourea, sodium azide, potassium azide, 1-hydroxypyrazole, 2-methylpyrazole-1-carboxamide, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, 2,4-diamino-6-trichloromethyl-5-triazine, carbon bisulfide, ammonium thiosulfate, sodium trithiocarbonate, 2,3-dihydro-2,2-dimethyl-7-benzofuranol methyl carbamate and N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester, at least one urease inhibitor, preferably selected from N-n-butylthiophosphoric triamide (NBTPT) and/or N-n-propylthiophosphoric triamide (NPTPT), at least one customary agrochemical auxiliary agent, preferably selected from the group consisting of aqueous and/or organic solvents, pH-adjusting agents, surfactants, wetting agents, spreading agents, adhesion promoters, carriers, fillers, viscosity-adjusting agents, emulsifiers, dispersants, sequestering agents, anti-settling agents, coalescing agents, rheology modifiers, defoaming agents, photo-protectors, anti-freeze agents, biostimulants, pesticides, biocides, plant growth regulators, safeners, penetrants, anticaking agents, mineral and/or vegetable oils and/or waxes, colorants and drift control agents, and mixtures thereof.

29. A process for producing the fertilizer mixture as claimed in claim 26 by introducing the N-heterocyclic compound into the fertilizer, and/or applying the N-heterocyclic compound to the surface of the fertilizer.

Description

EXAMPLES

1. Heterocyclic Compounds of General Formula (I) to (IV)

[0267] Different heterocyclic compounds of general formula (I) to (IV) were obtained from ENAMINE Ltd., UkrOrgSynthesis Ltd., or Vitas-M Laboratory, Ltd. The respective compounds are shown in Table 1 below. 2-Thiazoline-2-thiol is also available from Sigma-Aldrich.

[0268] The compounds of formula (I) and (II) and (III)/(IV) were tested for their nitrification inhibition effect in two screens, using assays with the nitrifying bacteria N. europaea and N. multiformis. In practice, ammonium and candidate nitrification inhibitors (at 100 M) were added to a dense bacterial culture in multiwell culture plates and after 24 h of incubation, the nitrite level was measured. To normalize for differences between batches of cultures and multiwell culture plates, for each well a relative nitrification was calculated that was normalized towards both the negative and positive controls (see Material and Methods below for full details).

2. Material and Methods

2.1 Nitrite Measurements

[0269] Nitrite (NO2) was measured using the Griess reagent (Product number: G4410, Griess reagent (modified), Sigma-Aldrich). Equal volumes of the sample or a diluted sample and the Griess reagent were mixed a transparent flat bottom multi-well plate and incubated in the dark at room temperature for 10 minutes. Absorbance values at 540 nm were measured spectrophotometrically (EnVision, Perkin Elmer) and used to calculate the [NO2] by use of a standard curve.

2.2 Ammonium Measurements

[0270] Ammonium (NH4+) was measured via a modified Berthelot's reagent protocol. 8 l culture sample, 35 l reagent A (0.5 g NaOH with 8 ml NaClO (2.5%) in 92 ml MilliQ) and 33 l reagent B (1 g salicylic acid, 0.5 g NaOH and 1.0237 g sodium nitroprusside dihydrate in 100 ml MilliQ) were consecutively added to 160 l MilliQ in flat bottom 96-well plates (Cat. No. 353072, Falcon 96 Well Clear Microplate, Corning). Absorbance values at 635 nm were measured spectrophotometrically (EnVision, Perkin Elmer) after a min incubation and used to calculate [NH4+] by use of a standard curve.

2.3 Culture Maintenance

2.3.1 Nitrosomonas europaea

[0271] Nitrosomonas europaea growth medium was prepared by aseptically combining 900 mL of stock solution 1 (27.75 mM (NH4)2SO4, 3.35 mM KH2PO4, 0.83 M MgSO4, 0.22 CaCl2, 11 M FeSO4, 18.33 M EDTA and 0.56 M CuSO4) with 100 mL of stock solution 2 (400 mM KH2PO4 and 40 mM NaH2PO4, pH 8.0 (NaOH)) and 8 mL stock solution 3 (5% anhydrous Na2CO3) solution. All three stock solutions were autoclaved in advance. Nitrosomonas europaea (ATCC 25978) cells were grown in sterile Erlenmeyer flasks sealed with tape (Micropore Surgical Tape 1530-1, 3M) at 28 C. and shaken (150 rpm) in the dark. Cultures in the late-log phase ([NO2]=10-20 mM) were subcultivated by centrifugation (4000 rpm, 15 min, 5 C.) and complete medium refreshment by discarding the supernatant and resuspension of the bacterial cell pellets in freshly prepared Nitrosomonas europaea growth medium.

2.3.2 Nitrosospira multiformis

[0272] Nitrosospira multiformis (NCIMB 11849) cells were grown in Erlenmeyer flasks filled with autoclaved 181-medium for AOB (NCIMB Ltd) that contained 1.78 mM (NH4)2SO4, 1.47 mM KH2PO4, 272 M CaCl22 H2O, 162 M MgSO47 H2O, 1 mL stock solution 1 (1.8 mM FeSO47 H2O and 1.49 mM NaEDTA) and 1 ml stock solution 2 (0.5% phenol red, pH indicator) (pH 7.5-8). pH was maintained by regular additions of sterile 5% Na2CO3. Cultures flasks were sealed with tape (Micropore Surgical Tape 1530-1, 3M) and incubated in the dark at 30 C. while shaking (150 rpm). Late-log phase cultures ([NO2]32 3 mM) were subcultivated by centrifugation (4000 rpm, 15 min, 5 C.) and complete medium refreshment by discarding the supernatant and resuspension of the bacterial cell pellets in pH-adjusted 181-medium.

Nitrification Inhibition Assays

2.4.1 Nitrosomonas europaea

[0273] To prepare a high-throughput nitrification inhibition assay using N. europaea, late-log phase cultures were subcultivated two days before the assay. Just before the assay, the cultures were 5 times overconcentrated with fresh growth medium after centrifugation (4000 rpm, 15 min, 5 C.) in 50 mL centrifuge tubes (Cat. No. 430829, CentriStar Conical Centrifuge Tubes, Corning). Per batch, all the cultures were pooled in one sterile Schott bottle and subsequently dispensed into 384-well plates (Cat. No. 781086, CELL-STAR plate, Greiner Bio-One; 50 l/well) by use of a dispenser (Multidrop Combi Reagent Dispenser, Thermo Scientific) that was first flushed with growth medium. Specifically for the high-throughput screen for new nitrification inhibitors, 0.5 l of 99.99% DMSO (negative controlfinal concentration 1%) was added to the outer two columns on the left side of the plate and 0.5 l of 10 mM DMP (3,4-dimethylpyrazole, positive controlfinal concentration 100 M) was added to the outer two columns on the right side manually. Finally, 0.5 l of candidate nitrification inhibitors (5 mM stock solutions in 99.99% DMSOfinal concentration 50 M) were added to the central wells using a pin tool on a Tecan robot (Freedom EVO, Tecan). In between additions, the pins were washed in sequence with 99.5% DMSO, MilliQ water and 100% ethanol and air-dried. All plates were separately wrapped in Parafilm. Stacks of 4 plates were put on top of a 96-well plate filled with 100 l MilliQ per well, covered with aluminum foil and shaken at 150 rpm at 28 C. 24 h later, NO2 production was assessed. For this, the samples were first 200 times diluted by pipetting 1.5 l of samples into 300 l of fresh growth medium in intermediate plates (Cat. No. 353077, Falcon 96-well Clear Round Bottom Microplate, Corning), to then mix 15 l of the diluted samples with 15 l Griess reagent in the wells of a transparent, flat bottom 384-well plate (Cat. No. X.sup.7001, Low Profile Microplate, Molecular Devices) that was measured spectrophotometrically at 540 nm (EnVision, Perkin Elmer).

2.4.2 Nitrosospira multiformis

[0274] 500 mL cultures grown in 1-L Erlenmeyer flasks that reached the late-log phase ([NO2]3 mM) within 3 to 4 days and showed 500 M increase in [NO2] over the last 24 h were used for a high-throughput nitrification inhibition assay. Cultures were first 5 times overconcentrated in fresh 181-medium by centrifugation (4000 rpm, 15 min, 5 C.) in 50 mL centrifuge tubes (Cat. No. 430829, CentriStar Conical Centrifuge Tubes, Corning). Per batch, all the cultures were pooled in one sterile Schott bottle and subsequently dispensed in 384-well plates (Cat. No. 781086, CELLSTAR plate, Greiner BioOne; 50 l/well) by use of a dispenser (Multidrop Combi Reagent Dispenser, Thermo Scientific) that was first flushed with 181-medium. 0.5 l of 99.99% DMSO (negative controlfinal concentration 1 was added to the outer two columns on the left side of the plate and 0.5 l of 10 mM DMP (3,4-dimethylpyrazole, positive controlfinal concentration 100 M) was added to the outer two columns on the right side manually. Finally, 0.5 l of candidate nitrification inhibitors (5 mM stock solutions in 99.99% DMSOfinal concentration 50 M) were added to the central wells using a pin tool on a Tecan robot (Freedom EVO, Tecan). In between additions, the pins were washed in sequence with 99.5% DMSO, MilliQ water and 100% ethanol and air-dried. All plates were separately wrapped in Parafilm. Stacks of 4 plates were put on top of a 96-well plate filled with 100 l MilliQ per well, covered with aluminum foil and shaken (150 rpm) at 30 C. 24 h later, NO2 production was assessed. For this, the samples were first 100 times diluted by pipetting 3 l of samples into 300 l fresh growth medium in intermediate plates (Cat. No. 353077, Falcon 96-well Clear Round Bottom Microplate, Corning) to then mix 15 l of the diluted samples with 15 l Griess reagent in the wells of a transparent, flat bottom 384-well plate (Cat. No. X.sup.7001, Low Profile Microplate, Molecular Devices) that was measured spectrophotometrically at 540 nm (EnVision, Perkin Elmer).

2.4.3 Quantification of the Nitrification Inhibition

[0275] To assess the efficacy of the compounds (in terms of nitrification inhibition) and to enable comparison between plates and batches of cultures, we calculated the relative nitrification. More in detail, all nitrite results were normalized towards both a negative control, containing no compound, and a positive control, containing the benchmark (100 M DMP), using Equation 1. This normalization was done per multi-well plate and each plate contained 32 positive and 32 negative controls.

[00001]$\begin{array}{cc}\mathrm{Relative}\mathrm{nitrification}=\frac{\mathrm{Nitrite}\left(\mathrm{compound}\right)-\mathrm{Nitrite}\left(\mathrm{positive}\mathrm{control}\right)}{\mathrm{Nitrite}\left(\mathrm{negative}\mathrm{control}\right)-\mathrm{Nitrite}\left(\mathrm{positive}\mathrm{control}\right)}& \left(\mathrm{Equation}1\right)\end{array}$

[0276] As a result, compounds that allowed full nitrification (no nitrification inhibition) show a relative nitrification of 1 (or 100%). A compound that shows the same nitrification inhibition as the positive control shows a nitrification inhibition of 0.

[0277] IC50 values, that is the concentration that inhibits 50% of the nitrification, were calculated after fitting a logistic curve to 8-point dose response data, using doses between 2 and 100 M with 1.75 fold steps. If a concentration of 2 M already inhibited nitrification, the IC50 value could not be calculated. In that case, IC50 values represent the dose that gives inhibition closest to 50%.

Cell Growth Inhibition Assays

[0278] The studied structures have (at least in the tested assays) no toxic/aspecific effects. There is no effect on AOA systems (ABIL, from Avecom, and Nitrososphaera viennensis) and no effect on microtox.

2.6 Nitrosomonas europaea ammonia Vs. Hydroxylamine Oxidation Assays

[0279] To determine if compounds specifically inhibited NH3 or NH2OH oxidation, N. europaea cells were provided with either NH3 or hydroxylamine (NH2OH) as N-source. Two days old, late-log phase cultures with a NO.sub.2 concentration between 10 and 20 mM were washed 3 times in fresh growth medium (without N) and finally 4 times overconcentrated via centrifugation (4000 rpm, 15 min, 4 C.). Transparent, flat bottom 96-well plate (Cat. No. 353072, Falcon 96 Well Clear Flat Bottom Microplate, Corning) were filled with 150 l culture per well using a dispenser (Multidrop Combi Reagent Dispenser, Thermo Scientific). Compounds (final concentration 50 M) were added in triplicate to the multi-well plate by use of a Tecan robot (Freedom EVO, Tecan). Thiourea (positive control for inhibition of NH3 oxidationfinal concentration 100 M) and phenylhydrazine hydrochloride (positive control for inhibition of NH2OH oxidationfinal concentration 1 mM) were added to the outer two columns. Each plate was made in duplicate to add either 500 M (NH4)2SO4 (final [NH4+]=1 mM) or 1 mM NH2OH. All plates were separately wrapped in Parafilm. Stacks of 4 plates were put on top of a 96-well plate filled with 100 l MilliQ per well, covered with aluminum foil and shaken (150 rpm) at 28 C. To prevent read-out of secondary effects on NO.sub.2 production, NO.sub.2 was measured only 30 min later. For this, the samples were first 2 times diluted by pipetting 15 l in 15 l fresh growth medium in 96-well intermediate plates (Cat. Ref. PCR-96-FS-C, 96-well PCR Microplate, Axygen) to then mix 15 l of the diluted samples with 15 l Griess reagent in the wells of a transparent, flat bottom 384-well plate (Cat. No. X.sup.7001, Low Profile Microplate, Molecular Devices) that was measured spectrophotometrically at 540 nm (EnVision, Perkin Elmer).

2.7 In Soil Assays

[0280] Top layer (0 to 10 cm) soil samples were collected from different fields in Belgium (Merelbeke and Moorslede). Vegetation on the field trial was removed and samples were taken from different plots. All soil samples were mixed and sieved (mesh size 2.8 mm) to filter large debris and homogenize soil. The soil was stored at 5 C. in plastic containers covered with Saran foil to prevent changes in microbial community composition and to maintain the original soil water content.

[0281] Soil water content was determined by drying 20 g soil for 48 h in a 60 C. oven and measuring weight before and after drying. Based on the soil water content (20%), compound solutions were prepared so that addition of 200 l compound and 200 l NH4Cl solution would result in a final compound concentration of 50 M and a final NH4.sup.+ concentration of 10 mM. Per treatment, 5 small pots were filled with 20 g soil. Next, the soil was treated first with the respective compound solution and then with the NH4Cl solution. Per tray, positive (50 M DMP) and negative controls (DMSO) were included. Pots were incubated at 21 C. (6 a.m. to 10 p.m. light) for 7 days. Demineralized water was added to the soil every 2 to 3 days to a soil weight of 20 g. In the end, each sample of 20 g soil was dissolved in 100 ml 1M KCl and shaken for 1 hour, followed by filtration through Whatmann paper. The filtrate was used to measure pH, NH4.sup.+ and NO.sub.3 concentrations.

2.8. Cu-Binding Assay

[0282] DMSO solutions with compound at a final concentration of 2.5 mM or 10 mM and CuSO4 at a final concentration of 5 mM were compared with pure compound or CuSO4 solutions at the same concentrations. 100 l of the solution was added to a 96-well plate and the full absorbance spectrum was measured with a SpectraMex2550 plate reader (Molecular devices).

[0283] Two agricultural soils coming from fields in Moorslede and Sint-Laureins were used. Screw top vials were filled with 10 g soil. Vials were sealed with Parafilm and preincubated at 21 C. in the dark for 5 days. After pre-incubation, vials were ventilated using a fan. The 100 L of a 1 mM compound solution (10% DMSO) was added, immediately followed by the addition of 100 L 200 mM NH4Cl. Vials were closed gastight and 5 mL synthetic air was added to the headspace using a plastic syringe, followed by the transfer of 5 mL headspace to an evacuated 3 mL exetainer. Vials were incubated at 21 C. in the dark and background NO concentration was noted. At each sampling timepoint, 5 mL synthetic air was added again to the headspace using a plastic syringe, again followed by the transfer of 5 mL headspace to an evacuated 3 mL exetainer. Vials were ventilated and closed again, and new gas samples were transferred to texetainers like before. N2O, CO2, CH.sub.4 in the exetainers were measured via gas chromatography. NO concentration was measured directly.

3. Results

[0284] The compounds of formula (I) and (II) were tested for their nitrification inhibition effect in two screens, using assays with the nitrifying bacteria N. europaea and N. multiformis. In practice, ammonium and candidate nitrification inhibitors (at 100 M) were added to a dense bacterial culture in multiwell culture plates and after 24 h of incubation, the nitrite level was measured. To normalize for differences between batches of cultures and multiwell culture plates, for each well a relative nitrification was calculated that was normalized towards both the negative and positive controls (see Material and Methods above for full details). As such, a relative nitrification of 100% or 0% indicates no nitrification inhibition or nitrification inhibition at the level of the positive control (100 M 3,4-dimethylpyrazole (DMP)), respectively.

3.1 Results in Table 1

[0285] The results shown in Table 1 below are based on the use of the nitrifying bacteria Nitrosomonas europaea and Nitrosospira multiformis. The N-heterocyclic compounds of general formula (I) and (II) and (III) and (IV) inhibit the nitrification in at least one the screened systems. The inhibitory activity of this class of molecules was confirmed for a number of structures with varying sub-groups. Eight different doses and four biological repeats were employed. Table 1 shows that all (tested) substances clearly inhibit nitrification in the tested systems, and that some inhibitors show strong inhibition at very low doses (some show still complete inhibition at 2 M).

[0286] As a reference, 3,4-dimethylpyrazole was employed in Comparative Example C1. The IC50 values denote the estimated concentration causing 50% nitrification inhibition. The values were predicted by fitting a logistic curve on 8-point dose response data or based on the lowest tested dose that gives inhibition higher than 50%.

[0287] IC50 presents the concentration at which 50% inhibition occurs. Hence, lower values indicate stronger nitrification inhibition. All tested nitrification inhibitors show a stronger effect on Nitrosospira multiform is.

[0288] From Table 1 it becomes evident that all (tested) molecules were able to inhibit nitrification. Therefore, the presence of the general formula (I), (II), (III) or (IV) substructure is necessary and seems to be sufficient to achieve the nitrification inhibition. Different side groups on the aminomethyl group in general formula (I) and (III), bound via the nitrogen atom or on the thioether group in general formula (II) and (IV), hardly affect the nitrification inhibition. Addition of molecular structures on the groups R2 and R3 of the structures of general formula (I) and (II) are possible as well, but particularly complex substructures might tend to weaken the nitrification inhibitory capacity. Nevertheless, all molecules of the substructures of general formula (I) and (II) and (III) and (IV) inhibit nitrification to some extent in at least one of the two tested nitrifying bacteria.

[0289] Not all molecules shown in Table 1 were tested.

[0290] All tested molecules are strong nitrification inhibitors that act specifically on nitrifying bacteria. NA means not analyzed.

TABLE-US-00001 TABLE 1 Nitrosomonas Nitrosospira europaea multiformis Nitrification (%) Nitrification at tested at tested concentration concentration (0 = (0 = Tested maximum IC50 maximum IC50 concentration Example Structure activity) [M] activity) [M] (M) C1 [00007] 0 6 0 14 100 1 [00008] 0 <2 0 <0.8 100 2 [00009] 0 <2 0 <2 100 3 [00010] 0 <3.5 0 <2 100 4 [00011] 0 <2 0 <2 100 5 [00012] 0 11 0 <3.5 100 6 [00013] 0 14 0 <3.5 100 7 [00014] 0 <6 0 <10 100 8 [00015] 47 <100 31 <100 100 9 [00016] 68 >100 0 <6 100 10 [00017] 81 >100 6 <10 100 11 [00018] NA NA 0 <20 100 12 [00019] 0 <100 11 <20 100 13 [00020] 73 NA 0 NA 50 14 [00021] 0 <57 0 <20 100 15 [00022] 78 NA 13 NA 50 16 [00023] 17 [00024] 100 NA 36 NA 50 18 [00025] 19 [00026] 20 [00027] 79 NA 42 NA 50 21 [00028] 22 [00029] 23 [00030] 24 [00031] 1 13 0 1.3 100 25 [00032] 2 12 53 140 100 26 [00033] 0 38 0 2.1 100 27 [00034] 62 >100 77 >100 100 28 [00035] 65 >100 0 12 100 29 [00036] 0 28 61 >100 100 30 [00037] 21 42 0 36 100 31 [00038] 12 14 11 16 100 32 [00039] 46 <100 75 >100 100 33 [00040] 68 >100 64 >100 100 34 [00041] 0 52 0 11 100 35 [00042] 12 43 0 3 100 36 [00043] 0 16 15 49 100 37 [00044] 58 >100 7 44 100 38 [00045] 92 >100 53 >100 100 39 [00046] 22 26 52 >100 100 40 [00047] 0 4 0 3 100 41 [00048] 88 >100 68 >100 100 42 [00049] 40 68 12 <57 100 43 [00050] 0 7 0 20 100 44 [00051] 0 2 0 24 100

[0291] Particularly, the molecule of Example 1, 3-[[(6-chloroimidazo[2,1-b][1,3]thiazol-5-yl)methyl-methylamino]methyl]-1,3-thiazolidine-2-thione strongly inhibited nitrification on both tested bacteria. Two similar molecules, 3-[[1,3-benzothiazol-2-ylmethyl(methyl)amino]methyl]-1,3-oxazolidine-2-thione and 3-[[(2-chloro-6-fluorophenyl)methyl-methylamino]methyl]-1,3-oxazolidine-2-thione of Examples 5 and 6, respectively, showed a strong nitrification inhibition towards the two ammonia oxidizing bacteria.

[0292] These three structures of Examples 1, 5 and 6 that are particularly preferred are shown below:

##STR00052##

3.2 Ammonia vs Hydroxylamine Oxidation Assay

[0293] To get insights on the targeted metabolic pathway and to discriminate between compounds that specifically affect NH3 oxidation or that affect another pathway, three representative novel nitrification inhibitors (Examples 1, 5 and 6) were tested in the ammonia vs hydroxylamine oxidation assay in which NO.sub.2 production from NH3 was compared with NO.sub.2 production from NH2OH. As NH3 is converted into NH2OH before NO.sub.2 is produced, comparing nitrification inhibition of the new compounds towards NH3 (see 1. in the scheme below) versus NH2OH (see 2. in the scheme below) indicates which part of the pathway is inhibited.

[0294] No nitrification inhibitor:

##STR00053##

[0295] Non-specific or general metabolic inhibitor:

NH.sub.3.fwdarw.X

NH.sub.2OH.fwdarw.X

[0296] Ammonia oxidation-specific inhibitor:

NH.sub.3.fwdarw.X

NH.sub.2OH.fwdarw.NO.sub.2.sup.

[0297] Ammonia oxidation is an essential step for the metabolism of nitrifying bacteria. Therefore, inhibition of ammonia oxidation by a nitrification inhibitor will indirectly affect all other enzymatic steps and will affect the second step as well, albeit less strong than the first step. Indeed, even in a 30 minutes assay, DMP, known to target ammonia oxidation, also reduced NH2OH oxidation, but in a lesser extent than the effect on ammonia oxidation.

[0298] The below Table 2 shows that all three new nitrification inhibitors clearly inhibit ammonia oxidation, but hardly hydroxylamine oxidation. The limited effect on hydroxylamine oxidation is most likely an indirect effect as inhibiting ammonia oxidation will affect the whole metabolism. Indeed, DMP, known to target the first step, has also a limited effect on the second step in the assay (Table 2). Hence, the new nitrification inhibitors specifically target ammonia oxidation (the first step of nitrification), which further corroborates that they specifically act on AOBs and are not generally toxic.

TABLE-US-00002 TABLE 2 Nitrification from NH2OH Nitrification from NH3 (% compared to Example Structure (% compared to DMSO) DMSO) C1 [00054] 27 56 1 [00055] 12 81 5 [00056] 17 77 6 [00057] 18 80

3.3 Test Performed in Agricultural Soils

[0299] Finally, the effect of a representative set of the new nitrification inhibitors was tested in soil.

[0300] The nitrification inhibitors were applied in a nitrification inhibition assay using agricultural soil. In practice, ammonium with or without the new nitrification inhibitors was added to soil from a field in Merelbeke (Belgium). The ammonium level was measured at the start of the experiment and at the end of the experiment, after one week of incubation. A nitrification inhibition percentage was calculated by comparing the reduction in ammonium level with the nitrification inhibitor to the reduction in ammonium without the nitrification inhibitor.

[0301] Ammonium was added to soil with a high nitrification activity and treated with the nitrification inhibitors (final concentration of 50 M). Table 3 shows that, after one week of treatment, especially the thiazolidine-containing compounds of Example 1 strongly inhibited the ammonium consumption. Also the thiazolidine-containing compounds that form thio-ethers (Examples 9 and 10) and the oxazolidine-containing compound of Example 5 showed significant inhibition. This validates that the new nitrification inhibitors are effectively inhibiting ammonia oxidation by soil communities.

TABLE-US-00003 TABLE 3 % Nitrification inhibition in soil (7 days of incubation, Merelbeke Example Structure soil) 1 [00058] 60 5 [00059] 14 9 [00060] 10 10 [00061] 7

[0302] It was found that presence of a thiazolidine-thiol substructure can be associated to nitrification inhibition capacity. Evaluation of different structural variants show that thiazolidine-thiol- and oxazolidine-thiol-containing structures are preferred nitrification inhibitors. Preferred compounds have a substructure that is bound via an amino-methyl bound to a more complex structure. Such molecules can also form the thiol tautomers if R1 is hydrogen in general formula (I). Preferred are the compounds containing a thiazolidine-thiol substructure, an oxazolidine-thiol substructure, or a thiazolidine-thioether substructure.

[0303] A set of examples was tested in the same conditions (Moorslede soil, 3 days of incubation) at a concentration of 50 M. The results are shown in Table 4.

TABLE-US-00004 TABLE 4 % Nitrification inhibition in soil (3 days of incubation, Moorslede Example Structure soil) at 50 M 7 [00062] 100 24 [00063] 40 25 [00064] 73

[0304] Another set of examples was tested in different concentrations in Moorslede soil as well, and were sampled after 5 days. The results are shown in Table 5. Table 3-7 show that diverse variations within the claimed general formula effectively inhibit nitrification in soil.

TABLE-US-00005 TABLE 5 % Nitrification inhibition in soil (5 days of incubation, Moorslede soil) at indicated concentration Example Structure 50 M 20 M 5 M 7 [00065] 38 32 8 25 [00066] 30 14 3 39 [00067] 62 NA NA

3.4 Nitrification Inhibition Assay Employing 3H-1,3-thiazole-2-thione

[0305] The compound of Example 24, 3H-1,3-thiazole-2-thione was employed in a full dose-response assay in the two ammonia-oxidizing bacteria Nitrosomonas europaea and Nitrosospira multiformis in accordance with above sections 1., 2.4.1 to 2.4.3.

TABLE-US-00006 TABLE 6 Tested concentration [M] 2 3.5 6 11 19 33 57 100 Average nitrification [%] in Nitrosomonas euro- paea [00068] 79 78 76 74 60 43 20 1 [00069] 85 76 73 58 50 32 20 2 [00070] 91 84 80 83 78 71 50 0 Average nitrification [%] in Nitrosospira multifor- mis [00071] 25 4 0 0 0 0 0 0 [00072] 81 80 80 76 74 67 62 53 [00073] 51 27 6 2 0 0 0 0

[0306] Concentrations ranging from 2 to 100 M of 3H-1,3-thiazole-2thione (tautomeric form being 3H-1,3-thiazole-2-thiol), 2-(methylsulfanyI)-1,3-thiazole and 4-methyl-1,3-thiazole-2-thiol were tested. The results are shown in the following Table 5. The results for 100 M are also contained in Table 1.

[0307] 3H-1,3-thiazole-2-thione has a very strong effect towards Nitrosospira multiformis (only 25% nitrification after application of 2 M), but also still nearly completely inhibits nitrification in Nitrosomonas europaea using higher concentrations.

3.5 Test Performed in Agricultural Soil

[0308] The effect of 3H-1,3-thiazole-2-thione was tested in soil in accordance with the test described above in section 3.3. The results are summarized in the following Table 7.

TABLE-US-00007 TABLE 7 Average % nitrification inhibition in soil (5 Name Structure days of incubation, Moorslede soil) at 20 M 3H-1,3-thiazole-2-thione [00074] 014 1,3-thiazolidine-2-thione [00075] 7

[0309] 3H-1,3-thiazole-2-thione effectively inhibits nitrification in soil.

[0310] In the presented data, the extent of inhibition seems to be limited, but this experiment was intentionally conducted at a low concentration (20 M) to allow comparison of efficacy between different inhibitors.

3.6 Inhibition of Greenhouse Gas Emissions

[0311] To further confirm the inhibitory effect of the new class of nitrification inhibitors on nitrogen losses, gas emissions from soil fertilized with ammonium and treated with 1,3-thiazolidine-2-thione were captured. NO, N2O, CO2 and CH.sub.4 was measured. Table 7 shows the cumulative values during an incubation of 99 h, and shows that especially NO and N2O, but also CO2, are strongly reduced upon use of the new nitrification inhibitor, which was the case in two different agricultural soils. No significant effects could be detected on CH.sub.4.

TABLE-US-00008 TABLE 8 Green- Cumulative emission at indicated timepoint house [ppm] Soil gas Treatment 4 h 24 h 48 h 72 h 99 h Moorslede N2O DMSO (no inhibitor) 226 1895 3733 4466 4466 1,3-thiazolidine-2-thione 0 0 0 0 0 NO DMSO (no inhibitor) 263 614 1014 1162 1160 1,3-thiazolidine-2-thione 25 28 43 65 65 CO2 DMSO (no inhibitor) 724 2825 5175 7359 9431 1,3-thiazolidine-2-thione 619 2385 4261 6195 8131 CH4 DMSO (no inhibitor) 0.06 0.29 0.29 0.32 0.34 1,3-thiazolidine-2-thione 0.20 0.38 0.38 0.51 0.51 Sint- N2O DMSO (no inhibitor) 42 481 1065 1587 1730 Laureins 1,3-thiazolidine-2-thione 0 0 1 1 2 NO DMSO (no inhibitor) 92 228 423 631 713 1,3-thiazolidine-2-thione 17 52 86 140 161 CO2 DMSO (no inhibitor) 382 1367 3041 5330 7596 1,3-thiazolidine-2-thione 372 1179 2094 3101 4168 CH4 DMSO (no inhibitor) 0.18 0.22 0.25 0.35 0.35 1,3-thiazolidine-2-thione 0.00 0.10 0.16 0.25 0.25

4. New Nitrification Inhibitors are Cu-Chelators

[0312] The new nitrification inhibitors form complexes with Cu.

[0313] As multiple known nitrification inhibitors are Cu-chelators, the Cu-chelating ability was tested by analyzing the absorbance spectra with or without Cu for the new inhibitors. If Cu forms a complex with the nitrification inhibitor, the absorbance spectra are expected to be shifted. Indeed, the absorbance spectra of the new inhibitors clearly shifted if combined with Cu, indicating that they form complexes with Cu, and further corroborating their action as a nitrification inhibitor.