Improvements in and relating to nitrification inhibitors

Abstract

The present invention relates generally to nitrification inhibitors and their use to prevent nitrate leaching or nitrous oxide emissions and to increase forage or crop yield. The present invention also relates to nitrification inhibitors and formulations comprising the same for direct or indirect application to soil or pasture.

Description

TECHNICAL FIELD

[0001] The present invention relates to improvements in and relating to nitrification inhibitors. BACKGROUND

[0002] The present invention relates to the global problem of the negative impact on the environment of modern agricultural by-products.

[0003] In particular, nitrate (NO3 - ) leaching from intensive agricultural land, such as livestock production systems, cropping systems and intensive horticultural systems, has been shown to be a major cause of groundwater, river and lake water pollution and indeed is a major global environmental problem.

[0004] Furthermore, nitrous oxide (N2O) is a potent greenhouse gas with a long-term global warming potential 298 times greater than CO2.

[0005] In grazing pastures, most of the N2O and NO3 - comes from nitrogen (N) returned to the land through excreta, particularly urine from grazing animals, which can have a N load of about 300 to 1000 kg N ha -1 Most of the N in animal urine is urea (NH2)2CO and once in the soil is rapidly hydrolyzed to plant available ammonium (NH4 + ) by urease as shown in the following equation (Tisdale et. al. 1985):

[0006] (NH2)2CO + H2O + urease → NH3 + H2NCOOH → 2NH 3(气体) + CO 2(气体)

[0007] NH 3(气体) + H2O → NH4 + + OH -

[0008] and then nitrification occurs by microorganisms including Nitrosomonas , Nitrosospira, Nitrosococcus , Nitrobacter and Nitrococcus species, where NH3 is oxidized to hydroxylamine (NH2OH) and then to nitrite (NO2 - ) and nitrate (NO3 - ) to gain energy (Di and Cameron 2016).

[0009] Keeping N as NH4 +It is also beneficial to keep the nitrogen in the form of ammonium, as the top layer of soil colloid is negatively charged and thus helps to retain the positively charged NH4 + in place. This will reduce nitrogen leaching. Nitrate, which is negatively charged, is not strongly retained by soil colloid and thus is easily leached as long as there is drainage through the soil (Di and Cameron 2016).

[0010] Furthermore, keeping nitrogen in the form of ammonium by slowing down the nitrification process is also beneficial in reducing the emission of the greenhouse gas nitrous oxide (N2O), as N2O is produced mainly from nitrification (oxidation of ammonia to nitrate, known as nitrification) and denitrification (reduction of nitrate to gaseous forms, including N2O).

[0011] Thus, an effective way to reduce nitrate leaching and nitrous oxide emission in agricultural soils is to slow down or stop the nitrification process, as the nitrification process leads to the production of nitrate that can be leached and to the production of N2O.

[0012] Previous studies have shown that nitrate leaching and nitrous oxide emission can be reduced by inhibiting the nitrification process (Di and Cameron, 2016).

[0013]

[0014]

[0015]

[0016] Furthermore, in agricultural fields, NO3 - It can also come from nitrogen fertilisers, such as urea, which is hydrolysed in the soil to produce ammonia / ammonium; ammonium from nitrogen fertilisers can be nitrified to produce nitrate in the soil in a similar way to ammonium in animal urine patches in the soil.

[0017] Research over the past two decades has clearly shown that N2O emission and nitrate leaching can be effectively reduced by treating grazing pasture soils, in which animal urine patches or nitrogen fertilisers are deposited, with nitrification inhibitors (see for example Di and Cameron, 2002; de Klein et al., 2011; Dai et al., 2013).

[0018] NZ patent 520549 teaches a soil treatment method that applies nitrification inhibitors in the form of a solution and / or fine particulate suspension to substantially cover the entire pasture to reduce, among other things, nitrate leaching and nitrous oxide emission.

[0019] In particular, NZ 520549 teaches the use of dicyandiamide (DCD) as a nitrification inhibitor to treat soil. DCD was chosen as the preferred nitrification inhibitor for commercial agricultural use because DCD is:

[0020] - partially soluble, thus delivery to pasture systems is both easy and cost effective; and

[0021] - importantly, non-toxic.

[0022] DCD is 10 times less toxic than table salt.

[0023] For example, the oral LD -1 in rats for DCD is greater than 30,000 mg kg 50 body weight, compared to another well-known commercial nitrification inhibitor, DMPP (3,4-dimethylpyrazole phosphate), which has an oral lethal dose (LD -1 ) in rats ranging from 300 to 2,000 mg kg 50 body weight. This is why DCD is the preferred nitrification inhibitor in NZ 520549.

[0024] However, due to the risk of DCD detection in dairy products and the lack of international standards for DCD residues in food, New Zealand stopped using DCD as a nitrification inhibitor in 2013. This risk is partly due to the relatively high rate of application of DCD (i.e. 10 kg / ha) to achieve the desired environmental outcome.

[0025] Therefore, there is still a clear need for other nitrification inhibitors as alternatives to DCD that:

[0026] - are low toxicity, high efficacy (so low rate of application), and do not present food safety concerns, and

[0027] - can be easily and cost-effectively applied to farmland to reduce the adverse environmental effects of nitrate and nitrous oxide caused by modern agriculture described above.

[0028] There is also a need for nitrification inhibitors that are non-volatile (or low volatile); not flammable; otherwise, non-hazardous; or safe to manufacture or handle.

[0029] There is also a need for new nitrification inhibitors to help prevent the effectiveness of known nitrification inhibitors from decreasing due to continued long-term use.

[0030] Furthermore, there is also a need for different nitrification inhibitors as some can be more suitable for different soil and environmental conditions.

[0031] Furthermore, in relation to nitrification inhibitor formulations, there is a need for new compounds that can be used as active ingredients.

[0032] Importantly, to help feed the growing world population, agricultural intensity can increase, including:

[0033] - the use of nitrogen fertilisers or other organic fertilisers as a source of nutrients; and

[0034] - more livestock globally.

[0035] Accordingly, to mitigate environmental impact, there will be an increased demand for effective, safe nitrification inhibitors.

[0036] It is an object of the present invention to address the foregoing problems or at least provide the public with a useful choice.

[0037] All references, including any patents or patent applications cited in this specification, are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in New Zealand or in any other country.

[0038] Throughout this specification, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0039] Further aspects and advantages of the present invention will become apparent from the ensuing description, which is given by way of example only.

[0040] Definitions

[0041] As used herein, the term 'NNI' refers to a novel nitrification inhibitor of the present invention that exhibits nitrification inhibition at the same or preferably a lower dosage rate than DCD.

[0042] As used herein, the term'sufficient amount' refers to the dosage of NNI that gives the required percentage of nitrification inhibition. Figure 2-5 Possible dosages for various NNI's, for example, outlined herein are listed.

[0043] As used herein, the term 'dosage quantity' refers to a determined amount of NNI that corresponds to the required amount applied to:

[0044] - to a determined land / plant area to reduce nitrification and / or increase the amount of plant available nitrogen;

[0045] - to a determined amount of nitrogen fertiliser to reduce the nitrification rate of ammonium from the nitrogen fertiliser when applied to the soil;

[0046] - to a determined liquid volume to ensure that the liquid is able to inhibit the nitrification process if the liquid is re-applied to the land and / or plants;

[0047] - to a determined farm effluent volume to inhibit the nitrification process if the effluent is re-applied to the land and / or plants.

[0048] By way of representative example only, 1 kg of NNI is the dose for 1 hectare of land / plants to be treated, where the nitrification inhibitor is in the form of 2-ethynyl 1,3 diazine.

[0049] As used herein, the term 'nitrogen efficiency' refers to the ability of nitrogen to be retained in the soil in a plant assimilable form.

[0050] The term 'N' when used alone as a symbol (i.e. not as part of a chemical formula of a compound) denotes the element nitrogen. It should be borne in mind that NO3 - is only temporarily available to the plant as it is subject to nitrate leaching, and unlike NH4 + it is not retained by the negatively charged soil exchange surfaces in the soil.

[0051] As used herein, the term 'agricultural land' refers to a land area used for agricultural / horticultural purposes, including but not limited to: a pasture field; or a farmland; and in relation thereto: nitrogen application is beneficial or occurs as a result of the agricultural use of the land, for example, through animal urine deposition or application of nitrogen fertiliser.

[0052] As used herein, the term 'urban land' refers to a land area that requires nitrogen application to fertilise grass or plants growing or to be grown thereon, such as parks, gardens, sports fields and golf courses, and the like.

[0053] As used herein, the term 'urine patch' refers to a land area on which animal urine has been deposited. The term 'urine patch' can include a specific patch of land on which urine is actually deposited by an animal when it urinates, or can cover a determined land area, such as a paddock, or other bounded / delineated area, where an animal is placed for a period of time during which one or more animals can have urinated.

[0054] As used herein, the term 'topsoil' refers to the top 0cm to 30cm of soil which has the highest concentration of organic matter and is where most of the soil biological activity occurs. SUMMARY

[0055] Having seen the fate of DCD (as outlined above), there is an unmet need to find new environmentally and food safe commercially suitable nitrification inhibitors to produce more food for the ever-growing world population without polluting the earth.

[0056] Thus, while in vitro laboratory tests can provide some preliminary indications for possible candidate compounds that can have potential as nitrification inhibitors, this does not give an in-depth understanding of whether a compound will work as a commercially useful nitrification inhibitor in the field.

[0057] Soil testing is the only definitive way to determine whether something has potential to be a nitrification inhibitor. Thus, finding new potential commercial nitrification inhibitors is a time-consuming and very expensive task.

[0058] Depending on the intended use of the nitrification inhibitor, certain attributes of the nitrification inhibitor are preferred over others. For example, water-soluble nitrification inhibitors are easier to make into aqueous solutions for land application. Furthermore, nitrification inhibitors with lower volatility are preferred over nitrification inhibitors with high volatility, as low volatility improves storage and extends shelf life.

[0059] Known commercially accepted nitrification inhibitors

[0060] Despite all the research that has been done in this field to date, only a few nitrification inhibitors have been successfully commercialized, which include:

[0061] • DCD (aka dicyandiamide)

[0062]

[0063] • DMPP (aka 3,4-dimethylpyrazole phosphate)

[0064]

[0065] • Chloride (aka 2-chloro-6-(trichloromethyl)pyridine)

[0066]

[0067] • DMPSA (aka 34-dimethylpyrazole succinic acid)

[0068]

[0069] The diversity and contrast of the chemical structures of these known commercially used nitrification inhibitors makes it impossible to predict a chemical structure that will have the desired nitrification inhibitor properties.

[0070] Broadest aspect of the invention

[0071] According to a first broad aspect of the application, there is provided the use of a compound of the formula selected from the group consisting of:

[0072] Formula 1

[0073]

[0074] Pyrimidines

[0075] • wherein X 1 and X 5 = N, X 2 and X 4 = CH, and X 3 = C-R; wherein R can be selected from H and OMe; or

[0076] • wherein X 1 and X 5 = CH, X 2 and X 4 = N, and X 3 = C-R; wherein R can be selected from H and OMe; or

[0077] • wherein X 1 and X 3 = N, and X 2 , X 4 and X 5 = CH;

[0078] or

[0079] Pyridines

[0080] • wherein X 1 , X 4 and X 5 = CH, X 2 may be selected from N and N=O, and X 3 = C-R; wherein R can be selected from H and OMe; or

[0081] • wherein X 1 = N, X 2 , X 4 and X 5 = CH, and X 3 = C-R; wherein R can be selected from OMe and C≡CH;

[0082] or

[0083] Pyridazines

[0084] • wherein X 1 and X 2 = N, X 3 = C-R, and X 4and X 5 = CH; wherein R can be selected from H and OMe;

[0085] or

[0086] pyrazines

[0087] • wherein X 1 and X 4 = N; X 2 and X 5 = CH; and X 3 = C-R; wherein R can be selected from H and OMe;

[0088] or

[0089] benzenes

[0090] • wherein X 1 , X 2 , X 4 and X 5 = CH; X 3 = C-R; wherein R can be selected from OMe, OEt and C≡CH.

[0091] According to a second broad aspect of the present application, there is provided a method for reducing the rate of nitrification in an agricultural soil, characterized by the steps of:

[0092] a) using at least one compound of the following formula, selected from the group consisting of:

[0093] Formula 1

[0094]

[0095] pyrimidines

[0096] • wherein X 1 and X 5 = N; X 2 and X 4 = CH; and X 3 = C-R; wherein R can be selected from H and OMe; or

[0097] • wherein X 1 and X 5 = CH; X 2 and X 4 = N; and X 3 = C-R; wherein R can be selected from H and OMe; or

[0098] • wherein X 1 and X 3 = N; and X 2 , X 4 and X 5 = CH;

[0099] or

[0100] pyridines

[0101] • wherein X 1 , X 4 and X 5 = CH; X 2 may be selected from N and N=0; and X 3 = C-R; wherein R can be selected from H and OMe; or

[0102] • wherein X 1 = N; X 2 , X 4 and X 5 = CH; and X 3 = C-R; wherein R can be selected from OMe and C≡CH;

[0103] or

[0104] pyridazines

[0105] • wherein X 1 and X 2 = N; X 3 = C-R; and X 4 and X 5 = CH; wherein R can be selected from H and OMe;

[0106] or

[0107] pyrazines

[0108] • wherein X 1 and X 4 = N; X 2 and X 5 = CH; and X 3 = C-R; wherein R can be selected from H and OMe;

[0109] or

[0110] benzenes

[0111] • wherein X 1 , X 2 , X 4 and X 5 = CH; X 3 = C-R; wherein R can be selected from OMe, OEt and C≡CH;

[0112] as nitrification inhibitors, or for use in the preparation of nitrification inhibitors, for direct or indirect application to soil.

[0113] Preferably, indirect application to soil comprises application of the compound to soil via a carrier.

[0114] Preferably, the carrier can be water or effluent.

[0115] Preferably, the carrier can be a fertilizer granule.

[0116] Preferably, the fertilizer granule can be urea.

[0117] A method substantially as described above which can reduce nitrate leaching or nitrous oxide emissions.

[0118] A method substantially as described above which can also increase pasture or crop yield.

[0119] A use or method substantially as described above wherein the compound is selected from the group consisting of:

[0120] - 2-ethynyl 1,3 diazine;

[0121] - 3-ethynyl 1,5 diazine;

[0122] - 4-ethynyl pyrimidine;

[0123] - 2-ethynyl-5-methoxy pyrimidine;

[0124] - 5-ethynyl-2-methoxy pyrimidine;

[0125] - 2-ethynyl-5-methoxy pyridine;

[0126] - 5-ethynyl-2-methoxy pyridine;

[0127] - 3-ethynyl pyridine 1-oxide;

[0128] - 2,5-diethynyl pyridine

[0129] - 3-ethynyl pyridazine;

[0130] - 3-ethynyl-6-methoxy pyridazine;

[0131] - 2-ethynyl pyrazine;

[0132] - 2-ethynyl-5-methoxy pyrazine;

[0133] - 4-ethynyl anisole;

[0134] - 1-ethoxy-4-ethynyl benzene;

[0135] - 1,4-diethynyl benzene;

[0136] and having a dosage rate substantially between 1 kg / ha to 9 kg / ha.

[0137] A use or method wherein the compound has a dosage rate of substantially 2 kg / ha.

[0138] A use or method substantially as hereinbefore described wherein the compound is selected from the group consisting of:

[0139] - 2-ethynyl 1,3 diazine;

[0140] - 3-ethynyl 1,5 diazine;

[0141] - 4-ethynyl pyrimidine;

[0142] - 2-ethynyl-5-methoxy pyrimidine;

[0143] - 5-ethynyl-2-methoxy pyrimidine;

[0144] - 2-ethynyl-5-methoxy pyridine;

[0145] - 3-ethynyl pyridine 1-oxide;

[0146] - 3-ethynyl pyridazine;

[0147] - 3-ethynyl-6-methoxy pyridazine;

[0148] - 2-ethynyl-5-methoxy pyrazine;

[0149] - 4-ethynyl anisole;

[0150] and has a dosage rate of substantially 1 kg / ha.

[0151] A use or method substantially as hereinbefore described wherein the compound is selected from the group consisting of:

[0152] - 2-ethynyl 1,3 diazine;

[0153] - 3-ethynyl 1,5 diazine;

[0154] - 2-ethynyl-5-methoxy pyridine;

[0155] - 3-ethynyl pyridine 1-oxide;

[0156] - 3-ethynyl pyridazine;

[0157] and has a dosage rate of substantially 0.5 kg / ha.

[0158] According to a third broad aspect there is provided a use of a compound to treat urine stains substantially as hereinbefore described.

[0159] Preferably, the compound can be applied to the urine stains by an agricultural spray cart or autonomous robot.

[0160] Preferably, the agricultural spraying vehicle or autonomous robot has a device for detecting urine stains thereon.

[0161] The first set of aspects of the present invention: 2-ethynyl 1,3-diazine (also known as 2-ethynylpyrimidine) (CAS 37972-24-0)

[0162] In one set of aspects, the present invention relates to the surprising new use of pyrimidine 2-ethynyl 1,3-diazine having the same or substantially similar structure to that shown in the following formula:

[0163]

[0164] The use is in relation to its reduction of NO3 - leaching and N2O emissions. Figure 1 The relationship between inhibition of nitrification in soil and reduction of nitrate leaching and nitrous oxide emissions is illustrated (Di and Cameron 2016). It is ultimately shown that by inhibiting the nitrification process using nitrification inhibitors, nitrate leaching and N2O emissions can be reduced (Di and Cameron 2016).

[0165] According to a first part of a first aspect of the present invention, there is provided the use of 2-ethynyl 1,3-diazine as a nitrification inhibitor.

[0166] Preferably, the use of 2-ethynyl 1,3-diazine substantially as described above, wherein the effective application rate (in kg / ha) is substantially less than the effective application rate of DCD.

[0167] According to a second part of the first aspect of the present invention, there is provided the use of 2-ethynyl 1,3-diazine as an active ingredient in a nitrification inhibitor formulation.

[0168] According to a third part of the first aspect of the present invention, there is provided the use of 2-ethynyl 1,3-diazine in the manufacture of a nitrification inhibitor.

[0169] According to a fourth part of the first aspect of the present invention, there is provided the vending of 2-ethynyl 1,3-diazine as a nitrification inhibitor, or for use in the manufacture of a nitrification inhibitor.

[0170] Preferably, the vending can be in a dose or a multiple thereof.

[0171] According to a fifth part of the first aspect of the present invention, there is provided the use of 2-ethynyl 1,3-diazine in the manufacture and / or vending of a soil treatment to achieve nitrification inhibition and related effects.

[0172] Preferably, the soil treatment reduces nitrification and / or increases plant nitrogen use efficiency.

[0173] According to a sixth part of the first aspect of the application, there is provided use substantially as hereinbefore described, wherein the nitrification inhibition reduces one or more of the following in the soil:

[0174] - nitrous oxide emissions;

[0175] - nitrate leaching; or

[0176] - a combination thereof.

[0177] According to a seventh part of the first aspect of the application, there is provided use of 2-ethynyl 1,3-diazine to treat soil to achieve nitrification inhibition and associated effects.

[0178] According to an eighth part of the first aspect of the application, there is provided soil treatment substantially as hereinbefore described, wherein 2-ethynyl 1,3-diazine is co-applied with urea granules or other nitrogen-containing fertilizer granules.

[0179] According to a ninth part of the first aspect of the application, there is provided use of 2-ethynyl 1,3-diazine to coat urea fertilizer granules or other nitrogen fertilizer granules.

[0180] According to a tenth part of the first aspect of the application, there is provided a nitrogen fertilizer granule coated with 2-ethynyl 1,3-diazine.

[0181] Preferably, the nitrogen fertilizer granule can be urea (or other ammonium form of nitrogen or nitrogen fertilizer that releases ammonium in the soil).

[0182] According to an eleventh part of the first aspect of the application, there is provided use of 2-ethynyl 1,3-diazine as a nitrogen-containing liquid fertilizer additive.

[0183] According to a twelfth part of the first aspect of the application, there is provided use of 2-ethynyl 1,3-diazine as a soil treatment agent in granular and / or liquid form.

[0184] According to a thirteenth part of the first aspect of the application, there is provided manufacture and / or sale of 2-ethynyl 1,3-diazine coated nitrogen fertilizer.

[0185] According to a fourteenth part of the first aspect of the application, there is provided use of 2-ethynyl 1,3-diazine to increase nitrogen use efficiency in a crop / pasture system or to increase nitrogen use efficiency of a fertilizer.

[0186] According to a fifteenth part of the first aspect of the application, there is provided use of 2-ethynyl 1,3-diazine to interrupt the nitrification process by soil microbes.

[0187] According to Part 16 of the first aspect of the application, there is provided the use of 2-ethynyl 1,3-diazine to improve the growth of pasture / plant.

[0188] According to Part 17 of the first aspect of the application, there is provided the use of 2-ethynyl 1,3-diazine as a treatment for urine patch areas in a pasture.

[0189] According to Part 18 of the first aspect of the application, there is provided the use of 2-ethynyl 1,3-diazine and a nitrogen source applied together to a crop or pasture.

[0190] According to Part 19 of the first aspect of the application, there is provided the sale of 2-ethynyl 1,3-diazine for use substantially as described above in relation to the uses of Parts 6, 7, 9, 11, 12, 14, 15, 16, 17 and 18 of the first aspect of the application.

[0191] According to Part 20 of the first aspect of the application, there is provided a method of treating agricultural land or other land having problems with nitrate leaching or nitrous oxide emissions, the method comprising applying 2-ethynyl 1,3-diazine directly or indirectly thereto.

[0192] According to Part 21 of the first aspect of the application, there is provided the co-application of 2-ethynyl 1,3-diazine with a nitrogen fertiliser to inhibit nitrification in soil.

[0193] According to Part 22 of the first aspect of the application, there is provided a method of treating an animal urine patch by applying 2-ethynyl 1,3-diazine thereto before, at the same time as, or after, urine deposition.

[0194] According to Part 23 of the first aspect of the application, there is provided a method of treating soil by applying 2-ethynyl 1,3-diazine to an area of soil that is to be or has been subjected to urea, fertiliser, effluent, or other nitrogen source.

[0195] According to Part 24 of the first aspect of the application, there is provided the application of 2-ethynyl 1,3-diazine to soil, farmland or pasture.

[0196] According to Part 25 of the first aspect of the application, there is provided the use of 2-ethynyl 1,3-diazine substantially as described above, wherein the dosage rate is selected from 0.5 kg / ha to 9 kg / ha.

[0197] According to Part 26 of the first aspect of the application, there is provided the use of 2-ethynyl 1,3-diazine substantially as described above, wherein the dosage rate is 0.5 kg / ha.

[0198] According to Part 27 of the first aspect of the application, there is provided the use of 2-ethynyl 1,3-diazine to retard the corrosion of architectural stone or statuary.

[0199] According to Part 28 of the first aspect of the invention, there is provided a method of treating agricultural land or urban land, the method comprising applying 2-ethynyl 1,3 diazine sold in one or more of the following ways:

[0200] - dosage;

[0201] - formulation type;

[0202] - co-delivery with a nitrogen source; or

[0203] - combinations thereof,

[0204] This makes 2-ethynyl 1,3 diazine suitable for treating land to reduce the environmental impact of: animal urine; nitrogen fertiliser; animal manure; or effluent.

[0205] Further information on 2-ethynyl 1,3 diazine (including chemical suppliers) can be accessed at:

[0206] https: / / www.sigmaaldrich.com / catalog / product / aldrich / 802956

[0207] Second set of aspects of the invention: 3-ethynyl 1,5 diazine (aka 5-ethynyl pyrimidine) (CAS 153286-94-3)

[0208] In the second set of aspects, the invention also relates to the surprising new use of pyrimidine 3-ethynyl 1,5 diazine, which has the same or substantially similar structure to that shown in the following formula:

[0209]

[0210] The use is in relation to its reduction of NO3 - leaching and N2O emissions. Figure 1 The relationship between inhibiting nitrification in soil and reducing nitrate leaching and nitrous oxide emissions is illustrated (Di and Cameron 2016). It was ultimately shown that by inhibiting the nitrification process using a nitrification inhibitor, nitrate leaching and N2O emissions can be reduced (Di and Cameron 2016).

[0211] According to Part 1 of the second aspect of the invention, there is provided the use of 3-ethynyl 1,5 diazine as a nitrification inhibitor.

[0212] Preferably, the use of 3-ethynyl 1,5 diazine substantially as described above, wherein the effective application rate in kg / ha is significantly less than the effective application rate of DCD.

[0213] According to a second part of the second aspect of the application, there is provided the use of 3-ethynyl 1,5 diazine as an active ingredient in a nitrification inhibitor formulation.

[0214] According to a third part of the second aspect of the application, there is provided the use of 3-ethynyl 1,5 diazine in the manufacture of a nitrification inhibitor.

[0215] According to a fourth part of the second aspect of the application, there is provided the sale of 3-ethynyl 1,5 diazine as a nitrification inhibitor, or for use in the manufacture of a nitrification inhibitor.

[0216] Preferably, the sale can be in doses or multiples thereof.

[0217] According to a fifth part of the second aspect of the application, there is provided the use of 3-ethynyl 1,5 diazine in the manufacture and / or sale of a soil treatment to achieve nitrification inhibition and associated effects.

[0218] Preferably, the soil treatment reduces nitrification and / or increases plant nitrogen use efficiency.

[0219] According to a sixth part of the second aspect of the application, there is provided the use substantially as hereinbefore described, wherein the nitrification inhibition reduces one or more of the following in the soil:

[0220] - nitrous oxide emissions;

[0221] - nitrate leaching; or

[0222] - a combination thereof.

[0223] According to a seventh part of the second aspect of the application, there is provided the use of 3-ethynyl 1,5 diazine treatment to achieve nitrification and associated effects.

[0224] According to an eighth part of the second aspect of the application, there is provided a soil treatment substantially as hereinbefore described, wherein the 3-ethynyl 1,5 diazine is co-applied with urea granules or other nitrogen-containing fertilizer granules.

[0225] According to a ninth part of the second aspect of the application, there is provided the use of 3-ethynyl 1,5 diazine to coat urea fertilizer granules or other nitrogen fertilizer granules.

[0226] According to a tenth part of the second aspect of the application, there is provided a nitrogen fertilizer granule coated with 3-ethynyl 1,5 diazine.

[0227] Preferably, the nitrogen fertilizer granule can be urea (or other ammonium form of nitrogen or nitrogen fertilizer that releases ammonium in the soil).

[0228] According to an eleventh part of the second aspect of the application, there is provided the use of 3-ethynyl 1,5 diazine as an additive for nitrogen-containing liquid fertilizers.

[0229] According to Part 12 of the second aspect of the application, there is provided the use of 3-ethynyl 1,5 diazine as a soil treatment agent in granular and / or liquid form.

[0230] According to Part 13 of the second aspect of the application, there is provided the manufacture and / or sale of 3-ethynyl 1,5 diazine coated nitrogen fertiliser.

[0231] According to Part 14 of the second aspect of the application, there is provided the use of 3-ethynyl 1,5 diazine to increase nitrogen use efficiency in a crop / pasture system or to increase the nitrogen use efficiency of a fertiliser.

[0232] According to Part 15 of the second aspect of the application, there is provided the use of 3-ethynyl 1,5 diazine to interrupt the nitrification process of soil microbes.

[0233] According to Part 16 of the second aspect of the application, there is provided the use of 3-ethynyl 1,5 diazine to improve pasture / plant growth.

[0234] According to Part 17 of the second aspect of the application, there is provided the use of 3-ethynyl 1,5 diazine as a treatment agent for urine patch areas in a pasture.

[0235] According to Part 18 of the second aspect of the application, there is provided the use of 3-ethynyl 1,5 diazine and a nitrogen source co-applied to a crop or pasture.

[0236] According to Part 19 of the second aspect of the application, there is provided the sale of 3-ethynyl 1,5 diazine for use substantially as described above in relation to the use of the 6th, 7th, 9th, 11th, 12th, 14th, 15th, 16th, 17th and 18th parts of the second aspect of the application.

[0237] According to Part 20 of the second aspect of the application, there is provided a method of treating agricultural land or other land having a problem with nitrate leaching or nitrous oxide emissions, the method comprising applying 3-ethynyl 1,5 diazine directly or indirectly thereto.

[0238] According to Part 21 of the second aspect of the application, there is provided the co-application of 3-ethynyl 1,5 diazine with a nitrogen fertiliser to inhibit nitrification in soil.

[0239] According to Part 22 of the second aspect of the application, there is provided a method of treating an animal urine patch by applying 3-ethynyl 1,5 diazine thereto before, at the same time as, or after urine deposition.

[0240] According to Part 23 of the second aspect of the application, there is provided a method of treating soil by applying 3-ethynyl 1,5 diazine to an area of soil that is to be or has been subjected to urea, fertiliser, effluent, or other nitrogen source.

[0241] According to Part 24 of the second aspect of the application, there is provided the application of a 3-ethynyl 1,5 diazine to soil, tillage or pasture.

[0242] According to Part 25 of the second aspect of the application, there is provided the use of a 3-ethynyl 1,5 diazine substantially as hereinbefore described, wherein the dosage rate is selected from the group consisting of 0.5 kg / ha to 9 kg / ha.

[0243] According to Part 26 of the second aspect of the application, there is provided the use of a 3-ethynyl 1,5 diazine substantially as hereinbefore described, wherein the dosage rate is 0.5 kg / ha.

[0244] According to Part 27 of the second aspect of the application, there is provided the use of a 3-ethynyl 1,5 diazine to retard the corrosion of architectural stone or statuary.

[0245] According to Part 28 of the second aspect of the application, there is provided a method of treating agricultural or urban land, the method comprising the sale of a 3-ethynyl 1,5 diazine in one or more of the following:

[0246] - dosage;

[0247] - formulation type;

[0248] - co-delivery with a nitrogen source; or

[0249] - combinations thereof,

[0250] This makes the 3-ethynyl 1,5 diazine suitable for use in treating land to reduce the environmental impact of: animal urine; nitrogen fertilisers; animal manure; or effluent.

[0251] Further information on 3-ethynyl 1,5 diazines (including chemical suppliers) can be accessed at:

[0252] https: / / www.sigmaaldrich.com / catalog / product / aldrich / 802980

[0253] Third group of aspects of the application: 4-ethynylpyrimidine (CAS 1196146-58-3)

[0254] In the third group of aspects, the present application also relates to the surprising new use of a 4-ethynylpyrimidine having the same or substantially similar structure to that shown in the following formula:

[0255]

[0256] The use is in relation to its application in reducing NO3 - leaching and N2O emissions by preventing / inhibiting the conversion of ammonia to nitrate by soil microbes. Figure 1The relationship between inhibition of nitrification in soil and reduction of nitrate leaching and nitrous oxide emissions is illustrated (Di and Cameron 2016). Ultimately, it is shown that by using nitrification inhibitors to inhibit the nitrification process, nitrate leaching and N2O emissions can be reduced (Di and Cameron 2016).

[0257] According to a first part of the third aspect of the application, there is provided the use of 4-ethynylpyrimidine as a nitrification inhibitor.

[0258] Preferably, the use of 4-ethynylpyrimidine substantially as described above, wherein the effective application rate in kg / ha is substantially less than the effective application rate of DCD.

[0259] According to a second part of the third aspect of the application, there is provided the use of 4-ethynylpyrimidine as an active ingredient in a nitrification inhibitor formulation.

[0260] According to a third part of the third aspect of the application, there is provided the use of 4-ethynylpyrimidine in the manufacture of a nitrification inhibitor.

[0261] According to a fourth part of the third aspect of the application, there is provided the sale of 4-ethynylpyrimidine as a nitrification inhibitor, or for use in the manufacture of a nitrification inhibitor.

[0262] Preferably, the sale can be in a dose or a multiple thereof.

[0263] According to a fifth part of the third aspect of the application, there is provided the use of 4-ethynylpyrimidine in the manufacture and / or sale of a soil treatment to achieve nitrification inhibition and associated effects.

[0264] Preferably, the soil treatment reduces nitrification and / or improves plant nitrogen use efficiency.

[0265] According to a sixth part of the third aspect of the application, there is provided the use substantially as described above, wherein the nitrification inhibition reduces one or more of the following in the soil:

[0266] - nitrous oxide emissions;

[0267] - nitrate leaching; or

[0268] - a combination thereof.

[0269] According to a seventh aspect of the application, there is provided a treatment to achieve nitrification and associated effects using 4-ethynylpyrimidine.

[0270] According to an eighth part of the third aspect of the application, there is provided a soil treatment substantially as described above, wherein the 4-ethynylpyrimidine is co-applied with urea granules or other nitrogen-containing fertilizer granules.

[0271] According to a ninth aspect of the third aspect of the application, there is provided the use of a 4-ethynylpyrimidine to coat urea fertilizer particles or other nitrogen fertilizer particles.

[0272] According to a tenth aspect of the third aspect of the application, there is provided a nitrogen fertilizer particle coated with a 4-ethynylpyrimidine.

[0273] Preferably, the nitrogen fertilizer particle can be urea (or other ammonium form of nitrogen or nitrogen fertilizer that releases ammonium in the soil).

[0274] According to an eleventh aspect of the third aspect of the application, there is provided the use of a 4-ethynylpyrimidine as a nitrogen-containing liquid fertilizer additive.

[0275] According to a twelfth aspect of the third aspect of the application, there is provided the use of a 4-ethynylpyrimidine as a soil treatment agent in granular and / or liquid form.

[0276] According to a thirteenth aspect of the third aspect of the application, there is provided the manufacture and / or sale of a 4-ethynylpyrimidine-coated nitrogen fertilizer.

[0277] According to a fourteenth aspect of the third aspect of the application, there is provided the use of a 4-ethynylpyrimidine to increase nitrogen use efficiency in a crop / pasture system or to increase the nitrogen use efficiency of a fertilizer.

[0278] According to a fifteenth aspect of the third aspect of the application, there is provided the use of a 4-ethynylpyrimidine to interrupt the nitrification process of soil microbes.

[0279] According to a sixteenth aspect of the third aspect of the application, there is provided the use of a 4-ethynylpyrimidine to improve pasture / plant growth.

[0280] According to a seventeenth aspect of the third aspect of the application, there is provided the use of a 4-ethynylpyrimidine as a treatment agent for urine patch areas in a pasture.

[0281] According to an eighteenth aspect of the third aspect of the application, there is provided the use of a 4-ethynylpyrimidine and a nitrogen source co-applied to a crop or pasture.

[0282] According to a nineteenth aspect of the third aspect of the application, there is provided the sale of a 4-ethynylpyrimidine for use substantially as described above in relation to the use of the sixth, seventh, ninth, eleventh, twelfth, fourteenth, fifteenth, sixteenth, seventeenth and eighteenth aspects of the third aspect of the application.

[0283] According to a twentieth aspect of the third aspect of the application, there is provided a method of treating agricultural land or other land having a problem with nitrate leaching or nitrous oxide emissions, the method comprising applying a 4-ethynylpyrimidine directly or indirectly thereto.

[0284] According to Part 21 of the third aspect of the application, there is provided the co-application of 4-ethynylpyrimidine with a nitrogen fertilizer to inhibit nitrification in soil.

[0285] According to Part 22 of the third aspect of the application, there is provided a method of treating animal urine patches by applying 4-ethynylpyrimidine to urine before, at the same time as, or after it is deposited.

[0286] According to Part 23 of the third aspect of the application, there is provided a method of treating soil by applying 4-ethynylpyrimidine to an area of soil that is about to be or has been subjected to urea, fertilizer, effluent, or other source of nitrogen.

[0287] According to Part 24 of the third aspect of the application, there is provided the application of 4-ethynylpyrimidine to soil, farmland, or pasture.

[0288] According to Part 25 of the third aspect of the application, there is provided the use of 4-ethynylpyrimidine substantially as described above, wherein the dosage rate is selected from 1 kg / ha to 9 kg / ha.

[0289] According to Part 26 of the third aspect of the application, there is provided the use of 4-ethynylpyrimidine substantially as described above, wherein the dosage rate is 1 kg / ha.

[0290] According to Part 27 of the third aspect of the application, there is provided a method of treating agricultural or urban land, the method comprising selling 4-ethynylpyrimidine in one or more of the following ways:

[0291] - dosage;

[0292] - formulation type;

[0293] - co-delivery with a source of nitrogen; or

[0294] - combinations thereof,

[0295] This makes 4-ethynylpyrimidine suitable for use in treating land to reduce the environmental impact of: animal urine; nitrogen fertilizer; animal manure; or effluent.

[0296] Further information about 4-ethynylpyrimidine (including chemical suppliers) can be accessed at:

[0297] https: / / pubchem.ncbi.nlm.nih.gov / compound / 4-Ethynylpyrimidine

[0298] Fourth group of aspects of the application: 2-ethynyl-5-methoxypyrimidine (CAS 2400905-32-8)

[0299] In the fourth group of aspects, the present application also relates to the surprising new use of 2-ethynyl-5-methoxypyrimidine, which has the same or substantially similar structure to that shown in the following formula:

[0300]

[0301] The stated use relates to its ability to reduce NO3 by preventing / inhibiting soil microorganisms from converting ammonia into nitrate. - Applications of leaching and N2O emissions. Figure 1 This study demonstrated the relationship between inhibiting nitrification in soil and reducing nitrate leaching and nitrous oxide emissions (Di and Cameron 2016). Ultimately, it showed that inhibiting nitrification by using nitrification inhibitors can reduce nitrate leaching and N2O emissions (Di and Cameron 2016).

[0302] According to a first part of the fourth aspect of the present invention, the use of 2-ethynyl-5-methoxypyrimidine as a nitration inhibitor is provided.

[0303] Preferably, the use of 2-ethynyl-5-methoxypyrimidine is substantially as described above, wherein the effective application rate in kg / ha is significantly less than the effective application rate of DCD.

[0304] According to a second part of the fourth aspect of the present invention, the use of 2-ethynyl-5-methoxypyrimidine as an active ingredient in nitration inhibitor formulations is provided.

[0305] According to the third part of the fourth aspect of the present invention, the use of 2-ethynyl-5-methoxypyrimidine in the preparation of nitration inhibitors is provided.

[0306] According to a fourth part of the fourth aspect of the invention, 2-ethynyl-5-methoxypyrimidine is provided for sale as a nitration inhibitor or for use in the preparation of a nitration inhibitor.

[0307] Preferably, the product can be sold in doses or multiples thereof.

[0308] According to the fifth part of the fourth aspect of the present invention, 2-ethynyl-5-methoxypyrimidine is provided for use in the preparation and / or sale of soil treatment agents to achieve nitrification inhibition and related effects.

[0309] Preferably, the soil treatment agent reduces nitrification and / or increases the efficiency of nitrogen use by plants.

[0310] According to the sixth part of the fourth aspect of the invention, the use of 2-ethynyl-5-methoxypyrimidine, substantially as described above, is provided, wherein nitrification inhibition reduces one or more of the following in the soil:

[0311] - Nitrous oxide emissions;

[0312] Nitrate leaching; or

[0313] - Their combination.

[0314] According to a seventh aspect of the fourth aspect of the application, there is provided a treatment to effect nitrification and associated effects using 2-ethynyl-5-methoxy pyrimidine.

[0315] According to an eighth aspect of the fourth aspect of the application, there is provided a soil treatment substantially as hereinbefore described, wherein 2-ethynyl-5-methoxy pyrimidine is co-applied with urea granules or other nitrogen-containing fertiliser granules.

[0316] According to a ninth aspect of the fourth aspect of the application, there is provided the use of 2-ethynyl-5-methoxy pyrimidine to coat urea fertiliser granules or other nitrogen fertiliser granules.

[0317] According to a tenth aspect of the fourth aspect of the application, there is provided a nitrogen fertiliser granule coated with 2-ethynyl-5-methoxy pyrimidine.

[0318] Preferably, the nitrogen fertiliser granule can be urea (or other ammonium form of nitrogen or nitrogen fertiliser that releases ammonium in the soil).

[0319] According to an eleventh aspect of the fourth aspect of the application, there is provided the use of 2-ethynyl-5-methoxy pyrimidine as a nitrogen-containing liquid fertiliser additive.

[0320] According to a twelfth aspect of the fourth aspect of the application, there is provided the use of 2-ethynyl-5-methoxy pyrimidine as a soil treatment agent in granular and / or liquid form.

[0321] According to a thirteenth aspect of the fourth aspect of the application, there is provided the manufacture and / or sale of 2-ethynyl-5-methoxy pyrimidine coated nitrogen fertiliser.

[0322] According to a fourteenth aspect of the fourth aspect of the application, there is provided the use of 2-ethynyl-5-methoxy pyrimidine to increase nitrogen use efficiency in a crop / pasture system or to increase the nitrogen use efficiency of a fertiliser.

[0323] According to a fifteenth aspect of the fourth aspect of the application, there is provided the use of 2-ethynyl-5-methoxy pyrimidine to interrupt the nitrification process of soil microbes.

[0324] According to a sixteenth aspect of the fourth aspect of the application, there is provided the use of 2-ethynyl-5-methoxy pyrimidine to improve pasture / plant growth.

[0325] According to a seventeenth aspect of the fourth aspect of the application, there is provided the use of 2-ethynyl-5-methoxy pyrimidine as a treatment for urine patch areas in a pasture.

[0326] According to an eighteenth aspect of the fourth aspect of the application, there is provided the use of 2-ethynyl-5-methoxy pyrimidine and a nitrogen source co-applied to a crop or pasture.

[0327] According to Part 19 of the fourth aspect of the application, there is provided the use of 2-ethynyl-5-methoxy pyrimidine for the sale of use substantially as hereinbefore described with respect to Parts 6, 7, 9, 11, 12, 14, 15, 16, 17 and 18 of the fourth aspect of the application.

[0328] According to Part 20 of the fourth aspect of the application, there is provided a method of treating agricultural land or other land having a problem with nitrate leaching or nitrous oxide emissions, the method comprising applying 2-ethynyl-5-methoxy pyrimidine directly or indirectly thereto.

[0329] According to Part 21 of the fourth aspect of the application, there is provided the co-application of 2-ethynyl-5-methoxy pyrimidine with a nitrogen fertiliser to inhibit nitrification in soil.

[0330] According to Part 22 of the fourth aspect of the application, there is provided a method of treating an animal urine patch by applying 2-ethynyl-5-methoxy pyrimidine thereto before, at the same time as, or after, urine deposition.

[0331] According to Part 23 of the fourth aspect of the application, there is provided a method of treating soil by applying 2-ethynyl-5-methoxy pyrimidine to an area of soil that is about to be or has been subjected to urea, fertiliser, effluent, or other source of nitrogen.

[0332] According to Part 24 of the fourth aspect of the application, there is provided the application of 2-ethynyl-5-methoxy pyrimidine to soil, farmland or pasture.

[0333] According to Part 25 of the fourth aspect of the application, there is provided the use of 2-ethynyl-5-methoxy pyrimidine substantially as hereinbefore described, wherein the dosage rate is selected from 1 kg / ha to 9 kg / ha.

[0334] According to Part 26 of the fourth aspect of the application, there is provided the use of 2-ethynyl-5-methoxy pyrimidine substantially as hereinbefore described, wherein the dosage rate is 1 kg / ha.

[0335] According to Part 27 of the fourth aspect of the application, there is provided a method of treating agricultural land or urban land, the method comprising the sale of 2-ethynyl-5-methoxy pyrimidine in one or more of the following ways:

[0336] - dosage;

[0337] - formulation type;

[0338] - co-delivery with a source of nitrogen; or

[0339] - combinations thereof,

[0340] This makes 2-ethynyl-5-methoxymethylpyrimidine suitable for treating land to reduce the environmental impact of: animal urine; nitrogen fertilisers; animal manure; or effluent.

[0341] Further information on 2-ethynyl-5-methoxymethylpyrimidine (including chemical suppliers) can be accessed at:

[0342] https: / / chem-space.com / search / text / 2-Ethynyl-5-methoxypyrimidine / 0b8 4d50603c89ce6fdb0f732e78b0105.

[0343] A fifth group of aspects of the present invention: 5-ethynyl-2-methoxymethylpyrimidine (CAS 1059705-07-5)

[0344] In the fifth group of aspects, the present invention also relates to the surprising new use of 5-ethynyl-2-methoxymethylpyrimidine, which has the same or substantially similar structure to that shown in the following formula:

[0345]

[0346] The use is in relation to its reduction of NO3 - leaching and N2O emissions. Figure 1 The relationship between inhibition of nitrification in soil and reduction of nitrate leaching and nitrous oxide emissions is illustrated (Di and Cameron 2016). Ultimately, it is shown that by inhibiting the nitrification process using a nitrification inhibitor, nitrate leaching and N2O emissions can be reduced (Di and Cameron 2016).

[0347] According to a first part of the fifth aspect of the present invention, there is provided the use of 5-ethynyl-2-methoxymethylpyrimidine as a nitrification inhibitor.

[0348] Preferably, the use of 5-ethynyl-2-methoxymethylpyrimidine substantially as described above, wherein the effective application rate in kg / ha is significantly less than the effective application rate of DCD.

[0349] According to a second part of the fifth aspect of the present invention, there is provided the use of 5-ethynyl-2-methoxymethylpyrimidine as an active ingredient in a nitrification inhibitor formulation.

[0350] According to a third part of the fifth aspect of the present invention, there is provided the use of 5-ethynyl-2-methoxymethylpyrimidine in the manufacture of a nitrification inhibitor.

[0351] According to a fourth part of the fifth aspect of the present invention, there is provided the sale of 5-ethynyl-2-methoxymethylpyrimidine as a nitrification inhibitor, or for use in the manufacture of a nitrification inhibitor.

[0352] Preferably, the sale can be in a dose or a multiple thereof.

[0353] According to a fifth part of the fifth aspect of the application, there is provided the use of 5-ethynyl-2-methoxypyrimidine in the manufacture and / or sale of a soil treatment to achieve nitrification inhibition and related effects.

[0354] Preferably, the soil treatment reduces nitrification and / or increases plant nitrogen use efficiency.

[0355] According to a sixth part of the fifth aspect of the application, there is provided the use of 5-ethynyl-2-methoxypyrimidine substantially as hereinbefore described, wherein the nitrification inhibition reduces one or more of the following in the soil:

[0356] - nitrous oxide emissions;

[0357] - nitrate leaching; or

[0358] - a combination thereof.

[0359] According to a seventh part of the fifth aspect of the application, there is provided a treatment using 5-ethynyl-2-methoxypyrimidine to achieve nitrification and related effects.

[0360] According to an eighth part of the fifth aspect of the application, there is provided a soil treatment substantially as hereinbefore described, wherein 5-ethynyl-2-methoxypyrimidine is co-applied with urea granules or other nitrogen-containing fertilizer granules.

[0361] According to a ninth part of the fifth aspect of the application, there is provided the use of 5-ethynyl-2-methoxypyrimidine to coat urea fertilizer granules or other nitrogen fertilizer granules.

[0362] According to a tenth part of the fifth aspect of the application, there is provided a nitrogen fertilizer granule coated with 5-ethynyl-2-methoxypyrimidine.

[0363] Preferably, the nitrogen fertilizer granule can be urea (or other ammonium form of nitrogen or nitrogen fertilizer that releases ammonium in the soil).

[0364] According to an eleventh part of the fifth aspect of the application, there is provided the use of 5-ethynyl-2-methoxypyrimidine as a nitrogen-containing liquid fertilizer additive.

[0365] According to a twelfth part of the fifth aspect of the application, there is provided the use of 5-ethynyl-2-methoxypyrimidine as a soil treatment agent in granular and / or liquid form.

[0366] According to a thirteenth part of the fifth aspect of the application, there is provided the manufacture and / or sale of 5-ethynyl-2-methoxypyrimidine coated nitrogen fertilizer.

[0367] According to a fourteenth part of the fifth aspect of the application, there is provided the use of 5-ethynyl-2-methoxypyrimidine to increase nitrogen use efficiency in a crop / pasture system or to increase the nitrogen use efficiency of a fertilizer.

[0368] According to Part 15 of the fifth aspect of the application, there is provided the use of 5-ethynyl-2-methoxymethylpyrimidine to interrupt the nitrification process of soil microorganisms.

[0369] According to Part 16 of the fifth aspect of the application, there is provided the use of 5-ethynyl-2-methoxymethylpyrimidine to improve the growth of pasture / plants.

[0370] According to Part 17 of the fifth aspect of the application, there is provided the use of 5-ethynyl-2-methoxymethylpyrimidine as a treatment for urine patch areas in a paddock.

[0371] According to Part 18 of the fifth aspect of the application, there is provided the use of 5-ethynyl-2-methoxymethylpyrimidine and a nitrogen source applied together to a crop or pasture.

[0372] According to Part 19 of the fifth aspect of the application, there is provided the sale of 5-ethynyl-2-methoxymethylpyrimidine for use substantially as described above in relation to the uses of Parts 6, 7, 9, 11, 12, 14, 15, 16, 17 and 18 of the fifth aspect of the application.

[0373] According to Part 20 of the fifth aspect of the application, there is provided a method of treating agricultural land or other land having a problem with nitrate leaching or nitrous oxide emissions, the method comprising applying 5-ethynyl-2-methoxymethylpyrimidine directly or indirectly thereto.

[0374] According to Part 21 of the fifth aspect of the application, there is provided the co-application of 5-ethynyl-2-methoxymethylpyrimidine with a nitrogen fertiliser to inhibit nitrification in soil.

[0375] According to Part 22 of the fifth aspect of the application, there is provided a method of treating an animal urine patch by applying 5-ethynyl-2-methoxymethylpyrimidine thereto before, at the same time as, or after, urine deposition.

[0376] According to Part 23 of the fifth aspect of the application, there is provided a method of treating soil by applying 5-ethynyl-2-methoxymethylpyrimidine to an area of soil that is to be or has been subjected to urea, fertiliser, effluent, or other nitrogen source.

[0377] According to Part 24 of the fifth aspect of the application, there is provided the application of 5-ethynyl-2-methoxymethylpyrimidine to soil, farmland or a paddock.

[0378] According to Part 25 of the fifth aspect of the application, there is provided the use of 5-ethynyl-2-methoxymethylpyrimidine substantially as described above, wherein the dosage rate is selected from 1 kg / ha to 9 kg / ha.

[0379] According to Part 26 of the fifth aspect of the invention, there is provided the use of 5-ethynyl-2-methoxypyrimidine substantially as hereinbefore described, wherein the dosage rate is 1 kg / ha.

[0380] According to Part 27 of the fifth aspect of the invention, there is provided a method of treating agricultural land or urban land, the method comprising selling 5-ethynyl-2-methoxypyrimidine in one or more modes selected from:

[0381] - dosage;

[0382] - formulation type;

[0383] - co-delivery with a nitrogen source; or

[0384] - combinations thereof,

[0385] This makes 5-ethynyl-2-methoxypyrimidine suitable for use in treating land to reduce the environmental impact of: animal urine; nitrogen fertilisers; animal manure; or effluent.

[0386] Further information on 5-ethynyl-2-methoxypyrimidine (including chemical suppliers) can be accessed at:

[0387] https: / / www.achemblock.com / 5-ethynyl-2-methoxypyrimidine.html

[0388] Sixth set of aspects of the invention: 2-ethynyl-5-methoxypyridine (CAS 1196155-18-6)

[0389] In the sixth set of aspects, the invention also relates to the surprising new use of 2-ethynyl-5-methoxypyridine, which has the same or substantially similar structure to that shown in the following formula:

[0390]

[0391] The use is in relation to its reduction of NO3 - leaching and N2O emissions. Figure 1 The relationship between inhibition of nitrification in soil and reduction of nitrate leaching and nitrous oxide emissions is illustrated (Di and Cameron 2016). It was ultimately shown that by inhibiting the nitrification process using a nitrification inhibitor, nitrate leaching and N2O emissions can be reduced (Di and Cameron 2016).

[0392] According to Part 1 of the sixth aspect of the invention, there is provided the use of 2-ethynyl-5-methoxypyridine as a nitrification inhibitor.

[0393] Preferably, the use of 2-ethynyl-5-methoxypyridine substantially as hereinbefore described, wherein the effective application rate in kg / ha is substantially less than the effective application rate of DCD.

[0394] According to a second part of the sixth aspect of the application, there is provided the use of 2-ethynyl-5-methoxypyridine as an active ingredient in a nitrification inhibitor formulation.

[0395] According to a third part of the sixth aspect of the application, there is provided the use of 2-ethynyl-5-methoxypyridine in the manufacture of a nitrification inhibitor.

[0396] According to a fourth part of the sixth aspect of the application, there is provided the sale of 2-ethynyl-5-methoxypyridine as a nitrification inhibitor or for use in the manufacture of a nitrification inhibitor.

[0397] Preferably, the sale can be in doses or multiples thereof.

[0398] According to a fifth part of the sixth aspect of the application, there is provided the use of 2-ethynyl-5-methoxypyridine in the manufacture and / or sale of a soil treatment to achieve nitrification inhibition and related effects.

[0399] Preferably, the soil treatment reduces nitrification and / or increases plant nitrogen use efficiency.

[0400] According to a sixth part of the sixth aspect of the application, there is provided the use substantially as hereinbefore described, wherein the nitrification inhibition reduces one or more of the following in the soil:

[0401] - nitrous oxide emissions;

[0402] - nitrate leaching; or

[0403] - a combination thereof.

[0404] According to a seventh part of the sixth aspect of the application, there is provided a treatment using 2-ethynyl-5-methoxypyridine to achieve nitrification and related effects.

[0405] According to an eighth part of the sixth aspect of the application, there is provided a soil treatment substantially as hereinbefore described, wherein 2-ethynyl-5-methoxypyridine is co-applied with urea granules or other nitrogen-containing fertilizer granules.

[0406] According to a ninth part of the sixth aspect of the application, there is provided the use of 2-ethynyl-5-methoxypyridine to coat urea fertilizer granules or other nitrogen fertilizer granules.

[0407] According to a tenth part of the sixth aspect of the application, there is provided a nitrogen fertilizer granule coated with 2-ethynyl-5-methoxypyridine.

[0408] Preferably, the nitrogen fertilizer granule can be urea (or other ammonium form of nitrogen or nitrogen fertilizer that releases ammonium in the soil).

[0409] According to Part 11 of the sixth aspect of the application, there is provided the use of 2-ethynyl-5-methoxypyridine as a nitrogen-containing liquid fertilizer additive.

[0410] According to Part 12 of the sixth aspect of the application, there is provided the use of 2-ethynyl-5-methoxypyridine as a soil treatment agent in granular and / or liquid form.

[0411] According to Part 13 of the sixth aspect of the application, there is provided the manufacture and / or sale of 2-ethynyl-5-methoxypyridine coated nitrogen fertilizer.

[0412] According to Part 14 of the sixth aspect of the application, there is provided the use of 2-ethynyl-5-methoxypyridine to increase nitrogen use efficiency in a crop / pasture system or to increase the nitrogen use efficiency of a fertilizer.

[0413] According to Part 15 of the sixth aspect of the application, there is provided the use of 2-ethynyl-5-methoxypyridine to interrupt the nitrification process by soil microbes.

[0414] According to Part 16 of the sixth aspect of the application, there is provided the use of 2-ethynyl-5-methoxypyridine to improve pasture / plant growth.

[0415] According to Part 17 of the sixth aspect of the application, there is provided the use of 2-ethynyl-5-methoxypyridine as a treatment agent for urine patch areas in a pasture.

[0416] According to Part 18 of the sixth aspect of the application, there is provided the use of 2-ethynyl-5-methoxypyridine and a nitrogen source co-applied to a crop or pasture.

[0417] According to Part 19 of the sixth aspect of the application, there is provided the sale of 2-ethynyl-5-methoxypyridine for use substantially as described above in relation to the uses of Parts 6, 7, 9, 11, 12, 14, 15, 16, 17 and 18 of the sixth aspect of the application.

[0418] According to Part 20 of the sixth aspect of the application, there is provided a method of treating agricultural land or other land having nitrate leaching or nitrous oxide emissions problems, the method comprising applying 2-ethynyl-5-methoxypyridine directly or indirectly thereto.

[0419] According to Part 21 of the sixth aspect of the application, there is provided the co-application of 2-ethynyl-5-methoxypyridine with a nitrogen fertilizer to inhibit nitrification in soil.

[0420] According to Part 22 of the sixth aspect of the invention, there is provided a method of treating animal urine stains by applying 2-ethynyl-5-methoxypyridine to urine deposits before, at the same time as, or after they are deposited.

[0421] According to Part 23 of the sixth aspect of the invention, there is provided a method of treating soil by applying 2-ethynyl-5-methoxypyridine to an area of soil that is about to be, or has been, subjected to urine, manure, effluent, or other source of nitrogen.

[0422] According to Part 24 of the sixth aspect of the invention, there is provided the application of 2-ethynyl-5-methoxypyridine to soil, farmland, or pasture.

[0423] According to Part 25 of the sixth aspect of the invention, there is provided the use of 2-ethynyl-5-methoxypyridine substantially as described above, wherein the dosage rate is selected from the group consisting of 0.5 kg / ha to 9 kg / ha.

[0424] According to Part 26 of the sixth aspect of the invention, there is provided the use of 2-ethynyl-5-methoxypyridine substantially as described above, wherein the dosage rate is 0.5 kg / ha.

[0425] According to Part 27 of the sixth aspect of the invention, there is provided a method of treating agricultural or urban land, the method comprising selling 2-ethynyl-5-methoxypyridine in one or more of the following ways:

[0426] - dosage;

[0427] - formulation type;

[0428] - co-delivery with a source of nitrogen; or

[0429] - combinations thereof,

[0430] This makes 2-ethynyl-5-methoxypyridine suitable for use in treating land to reduce the environmental impact of: animal urine; nitrogen fertiliser; animal manure; or effluent.

[0431] Further information on 2-ethynyl-5-methoxypyridine (including chemical suppliers) can be accessed at:

[0432] https: / / www.sigmaaldrich.com / catalog / product / aldrich / cds023940

[0433] Seventh group of aspects of the invention: 5-ethynyl-2-methoxypyridine (CAS 663955-59-7)

[0434] In the seventh group of aspects, the invention also relates to the surprising new use of 5-ethynyl-2-methoxypyridine, which has the same or substantially similar structure to that shown in the following formula:

[0435]

[0436] The use in relation to its reduction of NO3 - leaching and N2O emissions. Figure 1 The relationship between inhibition of nitrification in soil and reduction of nitrate leaching and nitrous oxide emissions is illustrated (Di and Cameron 2016). Ultimately, it is shown that by inhibiting the nitrification process using a nitrification inhibitor, nitrate leaching and N2O emissions can be reduced (Di and Cameron 2016).

[0437] According to a first part of the seventh aspect of the present application, there is provided the use of 5-ethynyl-2-methoxypyridine as a nitrification inhibitor.

[0438] Preferably, the use of 5-ethynyl-2-methoxypyridine substantially as described above, wherein the effective application rate in kg / ha is substantially less than the effective application rate of DCD.

[0439] According to a second part of the seventh aspect of the present application, there is provided the use of 5-ethynyl-2-methoxypyridine as an active ingredient in a nitrification inhibitor formulation.

[0440] According to a third part of the seventh aspect of the present application, there is provided the use of 5-ethynyl-2-methoxypyridine in the manufacture of a nitrification inhibitor.

[0441] According to a fourth part of the seventh aspect of the present application, there is provided the sale of 5-ethynyl-2-methoxypyridine as a nitrification inhibitor or for use in the manufacture of a nitrification inhibitor.

[0442] Preferably, the sale can be in doses or multiples thereof.

[0443] According to a fifth part of the seventh aspect of the present application, there is provided the use of 5-ethynyl-2-methoxypyridine in the manufacture and / or sale of a soil treatment agent to achieve nitrification inhibition and associated effects.

[0444] Preferably, the soil treatment agent reduces nitrification and / or improves plant nitrogen use efficiency.

[0445] According to a sixth part of the seventh aspect of the present application, there is provided the use substantially as described above, wherein the nitrification inhibition reduces one or more of the following in the soil:

[0446] nitrous oxide emissions;

[0447] nitrate leaching; or

[0448] a combination thereof.

[0449] According to a seventh aspect of the seventh part of the seventh aspect of the application, there is provided a treatment to effect nitrification and related effects using 5-ethynyl-2-methoxypyridine.

[0450] According to an eighth part of the seventh aspect of the application, there is provided a soil treatment substantially as hereinbefore described, wherein 5-ethynyl-2-methoxypyridine is co-applied with urea granules or other nitrogen-containing fertilizer granules.

[0451] According to a ninth part of the seventh aspect of the application, there is provided the use of 5-ethynyl-2-methoxypyridine to coat urea fertilizer granules or other nitrogen fertilizer granules.

[0452] According to a tenth part of the seventh aspect of the application, there is provided a nitrogen fertilizer granule coated with 5-ethynyl-2-methoxypyridine.

[0453] Preferably, the nitrogen fertilizer granule can be urea (or other ammonium form of nitrogen or nitrogen fertilizer that releases ammonium in the soil).

[0454] According to an eleventh part of the seventh aspect of the application, there is provided the use of 5-ethynyl-2-methoxypyridine as a nitrogen-containing liquid fertilizer additive.

[0455] According to a twelfth part of the seventh aspect of the application, there is provided the use of 5-ethynyl-2-methoxypyridine as a soil treatment agent in granular and / or liquid form.

[0456] According to a thirteenth part of the seventh aspect of the application, there is provided the manufacture and / or sale of 5-ethynyl-2-methoxypyridine coated nitrogen fertilizer.

[0457] According to a fourteenth part of the seventh aspect of the application, there is provided the use of 5-ethynyl-2-methoxypyridine to increase nitrogen use efficiency in a crop / pasture system or to increase the nitrogen use efficiency of a fertilizer.

[0458] According to a fifteenth part of the seventh aspect of the application, there is provided the use of 5-ethynyl-2-methoxypyridine to interrupt the nitrification process by soil microbes.

[0459] According to a sixteenth part of the seventh aspect of the application, there is provided the use of 5-ethynyl-2-methoxypyridine to improve pasture / plant growth.

[0460] According to a seventeenth part of the seventh aspect of the application, there is provided the use of 5-ethynyl-2-methoxypyridine as a treatment for urine patch areas in a pasture.

[0461] According to an eighteenth part of the seventh aspect of the application, there is provided the use of 5-ethynyl-2-methoxypyridine co-applied with a nitrogen source to a crop or pasture.

[0462] According to Part 19 of the seventh aspect of the application, there is provided the use of 5-ethynyl-2-methoxypyridine for the sale of use substantially as hereinbefore described with respect to Parts 6, 7, 9, 11, 12, 14, 15, 16, 17 and 18 of the seventh aspect of the application.

[0463] According to Part 20 of the seventh aspect of the application, there is provided a method of treating agricultural land or other land having a problem with nitrate leaching or nitrous oxide emissions, the method comprising applying 5-ethynyl-2-methoxypyridine directly or indirectly thereto.

[0464] According to Part 21 of the seventh aspect of the application, there is provided the co-application of 5-ethynyl-2-methoxypyridine with a nitrogen fertiliser to inhibit nitrification in soil.

[0465] According to Part 22 of the seventh aspect of the application, there is provided a method of treating an animal urine patch by applying 2-ethynyl-5-methoxypyridine thereto before, at the same time as, or after, urine deposition.

[0466] According to Part 23 of the seventh aspect of the application, there is provided a method of treating soil by applying 5-ethynyl-2-methoxypyridine to an area of soil that is about to be or has been subjected to urea, fertiliser, effluent, or other source of nitrogen.

[0467] According to Part 24 of the seventh aspect of the application, there is provided the application of 5-ethynyl-2-methoxypyridine to soil, farmland or pasture.

[0468] According to Part 25 of the seventh aspect of the application, there is provided the use of 5-ethynyl-2-methoxypyridine substantially as hereinbefore described, wherein the dosage rate is selected from 2 kg / ha to 9 kg / ha.

[0469] According to Part 26 of the seventh aspect of the application, there is provided the use of 5-ethynyl-2-methoxypyridine substantially as hereinbefore described, wherein the dosage rate is 2 kg / ha.

[0470] According to Part 27 of the seventh aspect of the application, there is provided a method of treating agricultural land or urban land, the method comprising the sale of 5-ethynyl-2-methoxypyridine in one or more of the following ways:

[0471] - dosage;

[0472] - formulation type;

[0473] - co-delivery with a source of nitrogen; or

[0474] - combinations thereof,

[0475] This makes 5-ethynyl-2-methoxypyridine suitable for treating land to reduce the environmental impact of: animal urine; nitrogen fertilisers; animal manure; or effluent.

[0476] Further information on 5-ethynyl-2-methoxypyridine (including chemical suppliers) can be accessed at:

[0477] https: / / www.sigmaaldrich.com / catalog / product / aldrich / rni00198

[0478] NIC 142

[0479] Eighth set of aspects of the invention: 3-ethynylpyridine 1-oxide (CAS 49836-11-5)

[0480] In the eighth set of aspects, the invention also relates to the surprising new use of 3-ethynylpyridine 1-oxide, which has the same or substantially similar structure to that shown in the following formula:

[0481]

[0482] The use is in relation to its reduction of NO3 - leaching and N2O emissions. Figure 1 The relationship between inhibiting nitrification in soil and reducing nitrate leaching and nitrous oxide emissions is illustrated (Di and Cameron 2016). Ultimately, it is shown that by inhibiting the nitrification process using a nitrification inhibitor, nitrate leaching and N2O emissions can be reduced (Di and Cameron 2016).

[0483] Aspects of the invention

[0484] According to a first part of the eighth aspect of the invention, there is provided the use of 3-ethynylpyridine 1-oxide as a nitrification inhibitor.

[0485] Preferably, the use of 3-ethynylpyridine 1-oxide substantially as described above, wherein the effective application rate in kg / ha is significantly less than the effective application rate of DCD.

[0486] According to a second part of the eighth aspect of the invention, there is provided the use of 3-ethynylpyridine 1-oxide as an active ingredient in a nitrification inhibitor formulation.

[0487] According to a third part of the eighth aspect of the invention, there is provided the use of 3-ethynylpyridine 1-oxide in the manufacture of a nitrification inhibitor.

[0488] According to a fourth part of the eighth aspect of the invention, there is provided the sale of 3-ethynylpyridine 1-oxide as a nitrification inhibitor, or for use in the manufacture of a nitrification inhibitor.

[0489] Preferably, the sale can be in doses or multiples thereof.

[0490] According to a fifth part of the eighth aspect of the application, there is provided the use of 3-ethynylpyridine 1-oxide in the manufacture and / or sale of a soil treatment to achieve nitrification inhibition and related effects.

[0491] Preferably, the soil treatment reduces nitrification and / or increases plant nitrogen use efficiency.

[0492] According to a sixth part of the eighth aspect of the application, there is provided the use substantially as hereinbefore described, wherein the nitrification inhibition reduces one or more of the following in the soil:

[0493] - nitrous oxide emissions;

[0494] - nitrate leaching; or

[0495] - a combination thereof.

[0496] According to a seventh part of the eighth aspect of the application, there is provided a treatment using 3-ethynylpyridine 1-oxide to achieve nitrification and related effects.

[0497] According to an eighth part of the eighth aspect of the application, there is provided a soil treatment substantially as hereinbefore described, wherein 3-ethynylpyridine 1-oxide is co-applied with urea granules or other nitrogen-containing fertilizer granules.

[0498] According to a ninth part of the eighth aspect of the application, there is provided the use of 3-ethynylpyridine 1-oxide to coat urea fertilizer granules or other nitrogen fertilizer granules.

[0499] According to a tenth part of the eighth aspect of the application, there is provided a nitrogen fertilizer granule coated with 3-ethynylpyridine 1-oxide.

[0500] Preferably, the nitrogen fertilizer granule can be urea (or other ammonium form of nitrogen or nitrogen fertilizer that releases ammonium in the soil).

[0501] According to an eleventh part of the eighth aspect of the application, there is provided the use of 3-ethynylpyridine 1-oxide as a nitrogen-containing liquid fertilizer additive.

[0502] According to a twelfth part of the eighth aspect of the application, there is provided the use of 3-ethynylpyridine 1-oxide as a soil treatment agent in granular and / or liquid form.

[0503] According to a thirteenth part of the eighth aspect of the application, there is provided the manufacture and / or sale of 3-ethynylpyridine 1-oxide coated nitrogen fertilizer.

[0504] According to Part 14 of Aspect 8 of the present application, there is provided the use of 3-ethynylpyridine 1-oxide to increase nitrogen use efficiency or increase nitrogen use efficiency of fertilizers in a crop / pasture system.

[0505] According to Part 15 of Aspect 8 of the present application, there is provided the use of 3-ethynylpyridine 1-oxide to interrupt the nitrification process by soil microbes.

[0506] According to Part 16 of Aspect 8 of the present application, there is provided the use of 3-ethynylpyridine 1-oxide to improve pasture / plant growth.

[0507] According to Part 17 of Aspect 8 of the present application, there is provided the use of 3-ethynylpyridine 1-oxide as a treatment for urine patch areas in a pasture.

[0508] According to Part 18 of Aspect 8 of the present application, there is provided the use of 3-ethynylpyridine 1-oxide and a nitrogen source co-applied to a crop or pasture.

[0509] According to Part 19 of Aspect 8 of the present application, there is provided the sale of 3-ethynylpyridine 1-oxide for use substantially as described above in relation to the uses of Parts 6, 7, 9, 11, 12, 14, 15, 16, 17 and 18 in relation to Aspect 10 of the present application.

[0510] According to Part 20 of Aspect 8 of the present application, there is provided a method of treating agricultural land or other land having a problem with nitrate leaching or nitrous oxide emissions, the method comprising applying 3-ethynylpyridine 1-oxide directly or indirectly thereto.

[0511] According to Part 21 of Aspect 8 of the present application, there is provided the co-application of 3-ethynylpyridine 1-oxide with a nitrogen fertilizer to inhibit nitrification in soil.

[0512] According to Part 22 of Aspect 8 of the present application, there is provided a method of treating an animal urine patch by applying 3-ethynylpyridine 1-oxide thereto before, at the same time as, or after urine deposition.

[0513] According to Part 23 of Aspect 8 of the present application, there is provided a method of treating soil by applying 3-ethynylpyridine 1-oxide to an area of soil that is to be or has been subjected to urea, fertilizer, effluent or other nitrogen source.

[0514] According to Part 24 of Aspect 8 of the present application, there is provided the application of 3-ethynylpyridine 1-oxide to soil, to a field, or to a pasture.

[0515] According to Part 25 of 8thaspect of the invention, there is provided the use of 3-ethynylpyridine 1-oxide substantially as hereinbefore described wherein the dosage rate is selected from the group consisting of 0.5 kg / ha to 9 kg / ha.

[0516] According to Part 26 of 8thaspect of the invention, there is provided the use of 3-ethynylpyridine 1-oxide substantially as hereinbefore described wherein the dosage rate is 0.5 kg / ha.

[0517] According to Part 27 of 8thaspect of the invention, there is provided a method of treating agricultural land or urban land, the method comprising selling 3-ethynylpyridine 1-oxide in one or more of the following:

[0518] - dosage;

[0519] - formulation type;

[0520] - co-delivery with a nitrogen source; or

[0521] - combinations thereof,

[0522] This makes 3-ethynylpyridine 1-oxide suitable for use in treating land to reduce the environmental impact of: animal urine; nitrogen fertilisers; animal manure; or effluent.

[0523] Further information on 3-ethynylpyridine 1-oxide (including chemical suppliers) can be accessed at:

[0524] https: / / pubchem.ncbi.nlm.nih.gov / compound / 3-Ethynyl-pyridine-1-oxide

[0525] 9thgroup of aspects of the invention: 2,5-diethynylpyridine (CAS 137000-75-0)

[0526] In the 9thgroup of aspects, the invention also relates to the surprising new use of 2,5-diethynylpyridine, which has the same or substantially similar structure to that shown in the following formula:

[0527]

[0528] The use is in relation to its reduction of NO3 - leaching and N2O emissions. Figure 1 The relationship between inhibition of nitrification in soil and reduction of nitrate leaching and nitrous oxide emissions is illustrated (Di and Cameron 2016). It was ultimately shown that by inhibiting the nitrification process using nitrification inhibitors, nitrate leaching and N2O emissions can be reduced (Di and Cameron 2016).

[0529] According to a first part of the 9th aspect of the application, there is provided the use of 2,5-diethynylpyridine as a nitrification inhibitor.

[0530] Preferably, the use of 2,5-diethynylpyridine substantially as hereinbefore described, wherein the effective application rate in kg / ha is substantially less than the effective application rate of DCD.

[0531] According to a second part of the 9th aspect of the application, there is provided the use of 2,5-diethynylpyridine as an active ingredient in a nitrification inhibitor formulation.

[0532] According to a third part of the 9th aspect of the application, there is provided the use of 2,5-diethynylpyridine in the manufacture of a nitrification inhibitor.

[0533] According to a fourth part of the 9th aspect of the application, there is provided the sale of 2,5-diethynylpyridine as a nitrification inhibitor, or for use in the manufacture of a nitrification inhibitor.

[0534] Preferably, the sale can be in doses or multiples thereof.

[0535] According to a fifth part of the 9th aspect of the application, there is provided the use of 2,5-diethynylpyridine in the manufacture and / or sale of a soil treatment to achieve nitrification inhibition and related effects.

[0536] Preferably, the soil treatment reduces nitrification and / or increases plant nitrogen use efficiency.

[0537] According to a sixth part of the 9th aspect of the application, there is provided the use substantially as hereinbefore described, wherein the nitrification inhibition reduces one or more of the following in the soil:

[0538] - nitrous oxide emissions;

[0539] - nitrate leaching; or

[0540] - combinations thereof.

[0541] According to a seventh part of the 9th aspect of the application, there is provided a treatment using 2,5-diethynylpyridine to achieve nitrification and related effects.

[0542] According to an eighth part of the 9th aspect of the application, there is provided a soil treatment substantially as hereinbefore described, wherein 2,5-diethynylpyridine is co-applied with urea granules or other nitrogen-containing fertilizer granules.

[0543] According to a ninth part of the 9th aspect of the application, there is provided the use of 2,5-diethynylpyridine to coat urea fertilizer granules or other nitrogen fertilizer granules.

[0544] According to a tenth part of the 9th aspect of the application, there is provided a nitrogen fertilizer granule coated with 2,5-diethynylpyridine.

[0545] Preferably, the nitrogen fertilizer granule can be urea (or other ammonium form of nitrogen or nitrogen fertilizer that releases ammonium in the soil).

[0546] According to Part 11 of Aspect 9 of the present application, there is provided the use of 2,5-diethynylpyridine as a nitrogen-containing liquid fertilizer additive.

[0547] According to Part 12 of Aspect 9 of the present application, there is provided the use of 2,5-diethynylpyridine in granular and / or liquid form as a soil treatment agent.

[0548] According to Part 13 of Aspect 9 of the present application, there is provided the manufacture and / or sale of 2,5-diethynylpyridine coated nitrogen fertilizer.

[0549] According to Part 14 of Aspect 9 of the present application, there is provided the use of 2,5-diethynylpyridine to increase nitrogen use efficiency in a crop / pasture system or to increase the nitrogen use efficiency of a fertilizer.

[0550] According to Part 15 of Aspect 9 of the present application, there is provided the use of 2,5-diethynylpyridine to interrupt the nitrification process by soil microbes.

[0551] According to Part 16 of Aspect 9 of the present application, there is provided the use of 2,5-diethynylpyridine to improve pasture / plant growth.

[0552] According to Part 17 of Aspect 9 of the present application, there is provided the use of 2,5-diethynylpyridine as a treatment agent for urine patch areas in a pasture.

[0553] According to Part 18 of Aspect 9 of the present application, there is provided the use of 2,5-diethynylpyridine and a nitrogen source co-applied to a crop or pasture.

[0554] According to Part 19 of Aspect 9 of the present application, there is provided the sale of 2,5-diethynylpyridine for use substantially as described above in relation to the use of the 6th, 7th, 9th, 11th, 12th, 14th, 15th, 16th, 17th and 18th Parts of Aspect 11 of the present application.

[0555] According to Part 20 of Aspect 9 of the present application, there is provided a method of treating agricultural land or other land having a problem with nitrate leaching or nitrous oxide emissions, the method comprising applying 2,5-diethynylpyridine directly or indirectly thereto.

[0556] According to Part 21 of Aspect 9 of the present application, there is provided the co-application of 2,5-diethynylpyridine with a nitrogen fertilizer to inhibit nitrification in soil.

[0557] According to Part 22 of the 9th aspect of the application, there is provided a method of treating animal urine stains by applying 2,5-diethynylpyridine to urine deposits before, at the same time as, or after they are deposited.

[0558] According to Part 23 of the 9th aspect of the application, there is provided a method of treating soil by applying 2,5-diethynylpyridine to an area of soil that is about to be or has been subjected to urea, manure, effluent or other source of nitrogen.

[0559] According to Part 24 of the 9th aspect of the application, there is provided the application of 2,5-diethynylpyridine to soil, farmland or pasture.

[0560] According to Part 25 of the 9th aspect of the application, there is provided the use of 2,5-diethynylpyridine substantially as described above, wherein the dosage rate is selected from 2 kg / ha to 9 kg / ha.

[0561] According to Part 26 of the 9th aspect of the application, there is provided the use of 2,5-diethynylpyridine substantially as described above, wherein the dosage rate is 2 kg / ha.

[0562] According to Part 27 of the 9th aspect of the application, there is provided a method of treating agricultural or urban land, the method comprising selling 2,5-diethynylpyridine in one or more of the following ways:

[0563] - dosage;

[0564] - formulation type;

[0565] - co-delivery with a source of nitrogen; or

[0566] - combinations thereof,

[0567] This makes 2,5-diethynylpyridine suitable for use in treating land to reduce the impact on the environment of: animal urine; nitrogen fertiliser; animal manure; or effluent.

[0568] Further information on 2,5-diethynylpyridine (including chemical suppliers) can be accessed at:

[0569] http: / / www.chemspider.com / Chemical-Structure.23500007.html

[0570] 10th group of aspects of the application: 3-ethynylpyridazine (CAS 1017793-08-6)

[0571] In the 11th group of aspects, the application also relates to the surprising new use of 3- ethynylpyridazine, which has the same or substantially similar structure to that shown in the following formula:

[0572]

[0573] The use in relation to its reduction of NO3 - Application of leaching and N2O emissions. Figure 1 The relationship between inhibition of nitrification in soil and reduction of nitrate leaching and nitrous oxide emissions is illustrated (Di and Cameron 2016). Ultimately, it is shown that by inhibiting the nitrification process using a nitrification inhibitor, nitrate leaching and N2O emissions can be reduced (Di and Cameron 2016).

[0574] Aspects of the invention

[0575] According to a first part of the 10th aspect of the invention, there is provided the use of 3-ethynylpyridazine as a nitrification inhibitor.

[0576] Preferably, the use of 3-ethynylpyridazine substantially as described above, wherein the effective application rate in kg / ha is substantially less than the effective application rate of DCD.

[0577] According to a second part of the 10th aspect of the invention, there is provided the use of 3-ethynylpyridazine as an active ingredient in a nitrification inhibitor formulation.

[0578] According to a third part of the 10th aspect of the invention, there is provided the use of 3-ethynylpyridazine in the manufacture of a nitrification inhibitor.

[0579] According to a fourth part of the 10th aspect of the invention, there is provided the sale of 3-ethynylpyridazine as a nitrification inhibitor, or for use in the manufacture of a nitrification inhibitor.

[0580] Preferably, the sale can be in a dose or a multiple thereof.

[0581] According to a fifth part of the 10th aspect of the invention, there is provided the use of 3-ethynylpyridazine in the manufacture and / or sale of a soil treatment agent to achieve nitrification inhibition and associated effects.

[0582] Preferably, the soil treatment agent reduces nitrification and / or improves plant nitrogen use efficiency.

[0583] According to a sixth part of the 10th aspect of the invention, there is provided the use substantially as described above, wherein the nitrification inhibition reduces one or more of the following in the soil:

[0584] - nitrous oxide emissions;

[0585] - nitrate leaching; or

[0586] - a combination thereof.

[0587] According to a seventh part of the 10th aspect of the invention, there is provided a treatment using 3-ethynylpyridazine to achieve nitrification and associated effects.

[0588] According to an eighth aspect of the tenth aspect of the application, there is provided a soil treatment substantially as hereinbefore described, wherein the 3-ethynylpyridazine is applied in conjunction with urea granules or other nitrogen-containing fertilizer granules.

[0589] According to a ninth aspect of the tenth aspect of the application, there is provided the use of 3-ethynylpyridazine to coat urea fertilizer granules or other nitrogen fertilizer granules.

[0590] According to a tenth aspect of the tenth aspect of the application, there is provided a nitrogen fertilizer granule coated with 3-ethynylpyridazine.

[0591] Preferably, the nitrogen fertilizer granule can be urea (or other ammonium form of nitrogen or nitrogen fertilizer that releases ammonium in the soil).

[0592] According to an eleventh aspect of the tenth aspect of the application, there is provided the use of 3-ethynylpyridazine as a nitrogen-containing liquid fertilizer additive.

[0593] According to a twelfth aspect of the tenth aspect of the application, there is provided the use of 3-ethynylpyridazine in granular and / or liquid form as a soil treatment agent.

[0594] According to a thirteenth aspect of the tenth aspect of the application, there is provided the manufacture and / or sale of 3-ethynylpyridazine-coated nitrogen fertilizers.

[0595] According to a fourteenth aspect of the tenth aspect of the application, there is provided the use of 3-ethynylpyridazine to increase nitrogen use efficiency in a crop / pasture system or to increase the nitrogen use efficiency of a fertilizer.

[0596] According to a fifteenth aspect of the tenth aspect of the application, there is provided the use of 3-ethynylpyridazine to interrupt the nitrification process of soil microbes.

[0597] According to a sixteenth aspect of the tenth aspect of the application, there is provided the use of 3-ethynylpyridazine to improve pasture / plant growth.

[0598] According to a seventeenth aspect of the tenth aspect of the application, there is provided the use of 3-ethynylpyridazine as a treatment agent for urine patch areas in a pasture.

[0599] According to an eighteenth aspect of the tenth aspect of the application, there is provided the use of 3-ethynylpyridazine and a nitrogen source applied in conjunction to a crop or pasture.

[0600] According to a nineteenth aspect of the tenth aspect of the application, there is provided the sale of 3-ethynylpyridazine for use substantially as hereinbefore described in relation to the uses of the sixth, seventh, ninth, eleventh, twelfth, fourteenth, fifteenth, sixteenth, seventeenth and eighteenth aspects of the twelfth aspect of the application.

[0601] According to Part 20 of Aspect 10 of the present application, there is provided a method of treating agricultural land or other land having a problem with nitrate leaching or nitrous oxide emissions, the method comprising applying 3-ethynylpyridazine directly or indirectly thereto.

[0602] According to Part 21 of Aspect 10 of the present application, there is provided the co-application of 3-ethynylpyridazine with a nitrogen fertiliser to inhibit nitrification in soil.

[0603] According to Part 22 of Aspect 10 of the present application, there is provided a method of treating animal urine patches by applying 3-ethynylpyridazine to them before, at the same time as, or after urine deposition.

[0604] According to Part 23 of Aspect 10 of the present application, there is provided a method of treating soil by applying 3-ethynylpyridazine to an area of soil that is about to be or has been subjected to urea, a fertiliser, effluent or other source of nitrogen.

[0605] According to Part 24 of Aspect 10 of the present application, there is provided the application of 3-ethynylpyridazine to soil, farmland or pasture.

[0606] According to Part 25 of Aspect 10 of the present application, there is provided the use of 3-ethynylpyridazine substantially as hereinbefore described, wherein the dosage rate is selected from the group consisting of 0.5 kg / ha to 9 kg / ha.

[0607] According to Part 26 of Aspect 10 of the present application, there is provided the use of 3-ethynylpyridazine substantially as hereinbefore described, wherein the dosage rate is 0.5 kg / ha.

[0608] According to Part 27 of Aspect 10 of the present application, there is provided the use of 3-ethynylpyridazine to retard the corrosion of architectural stone or statuary.

[0609] According to Part 28 of Aspect 10 of the present application, there is provided a method of treating agricultural land or urban land, the method comprising selling 3-ethynylpyridazine in one or more of the following ways:

[0610] - dosage;

[0611] - formulation type;

[0612] - co-delivery with a source of nitrogen; or

[0613] - combinations thereof,

[0614] This makes 3-ethynylpyridazine suitable for use in treating land to reduce the environmental impact of: animal urine; nitrogen fertilisers; animal manure; or effluent.

[0615] Further information about 3-ethynylpyridazine (including chemical suppliers) can be accessed at:

[0616] https: / / www.achemblock.com / 3-ethynyl-pyridazine.html

[0617] The 11th aspect of the present application: 3-ethynyl-6-methoxypyridazine (CAS 1019331-16-8)

[0618] In the 11th aspect, the present application also relates to the surprising new use of 3-ethynyl-6-methoxypyridazine, which has the same or substantially similar structure to the structure shown in the following formula:

[0619]

[0620] The use is with respect to its reduction of NO3 - leaching and N2O emissions. Figure 1 The relationship between inhibition of nitrification in soil and reduction of nitrate leaching and nitrous oxide emissions is illustrated (Di and Cameron 2016). It was ultimately shown that by inhibiting the nitrification process using nitrification inhibitors, nitrate leaching and N2O emissions can be reduced (Di and Cameron 2016).

[0621] Aspects of the present application

[0622] According to a first part of the 11th aspect of the present application, there is provided the use of 3-ethynyl-6-methoxypyridazine as a nitrification inhibitor.

[0623] Preferably, the use of 3-ethynyl-6-methoxypyridazine substantially as described above, wherein the effective application rate in kg / ha is substantially less than the effective application rate of DCD.

[0624] According to a second part of the 11th aspect of the present application, there is provided the use of 3-ethynyl-6-methoxypyridazine as an active ingredient in a nitrification inhibitor formulation.

[0625] According to a third part of the 11th aspect of the present application, there is provided the use of 3-ethynyl-6-methoxypyridazine in the manufacture of a nitrification inhibitor.

[0626] According to a fourth part of the 11th aspect of the present application, there is provided the sale of 3-ethynyl-6-methoxypyridazine as a nitrification inhibitor, or for use in the manufacture of a nitrification inhibitor.

[0627] Preferably, the sale can be in a dose or a multiple thereof.

[0628] According to a fifth part of the 11th aspect of the present application, there is provided the use of 3-ethynyl-6-methoxypyridazine in the manufacture and / or sale of a soil treatment agent to achieve nitrification inhibition and associated effects.

[0629] Preferably, the soil treatment agent reduces nitrification and / or increases plant nitrogen use efficiency.

[0630] According to a sixth part of the 11th aspect of the application, there is provided use substantially as hereinbefore described, wherein the nitrification inhibition reduces one or more of the following in the soil:

[0631] - nitrous oxide emissions;

[0632] - nitrate leaching; or

[0633] - a combination thereof.

[0634] According to a seventh part of the 11th aspect of the application, there is provided treatment to achieve nitrification and associated effects using 3-ethynyl-6-methoxypyridazine.

[0635] According to an eighth part of the 11th aspect of the application, there is provided soil treatment substantially as hereinbefore described, wherein 3-ethynyl-6-methoxypyridazine is co-applied with urea granules or other nitrogen-containing fertilizer granules.

[0636] According to a ninth part of the 11th aspect of the application, there is provided use of 3-ethynyl-6-methoxypyridazine to coat urea fertilizer granules or other nitrogen fertilizer granules.

[0637] According to a tenth part of the 11th aspect of the application, there is provided nitrogen fertilizer granules coated with 3-ethynyl-6-methoxypyridazine.

[0638] Preferably, the nitrogen fertilizer granules can be urea (or other ammonium form of nitrogen or nitrogen fertilizer that releases ammonium in the soil).

[0639] According to an eleventh part of the 11th aspect of the application, there is provided use of 3-ethynyl-6-methoxypyridazine as a nitrogen-containing liquid fertilizer additive.

[0640] According to a twelfth part of the 11th aspect of the application, there is provided use of 3-ethynyl-6-methoxypyridazine as a soil treatment agent in granular and / or liquid form.

[0641] According to a thirteenth part of the 11th aspect of the application, there is provided manufacture and / or sale of 3-ethynyl-6-methoxypyridazine-coated nitrogen fertilizer.

[0642] According to a fourteenth part of the 11th aspect of the application, there is provided use of 3-ethynyl-6-methoxypyridazine to increase nitrogen use efficiency in a crop / pasture system or to increase nitrogen use efficiency of a fertilizer.

[0643] According to a fifteenth part of the 11th aspect of the application, there is provided use of 3-ethynyl-6-methoxypyridazine to interrupt the nitrification process by soil microbes.

[0644] According to Part 16 of Aspect 11 of the present application, there is provided the use of 3-ethynyl-6-methoxypyridazine to improve the growth of pasture / plant.

[0645] According to Part 17 of Aspect 11 of the present application, there is provided the use of 3-ethynyl-6-methoxypyridazine as a treatment for urine patch areas in a paddock.

[0646] According to Part 18 of Aspect 11 of the present application, there is provided the use of 3-ethynyl-6-methoxypyridazine and a nitrogen source applied together to a crop or pasture.

[0647] According to Part 19 of Aspect 12 of the present application, there is provided the sale of 3-ethynyl-6-methoxypyridazine for use substantially as described above in relation to the use of Part 6, 7, 9, 11, 12, 14, 15, 16, 17 and 18 of Aspect 12 of the present application.

[0648] According to Part 20 of Aspect 11 of the present application, there is provided a method of treating agricultural land or other land having problems with nitrate leaching or nitrous oxide emissions, the method comprising applying 3-ethynyl-6-methoxypyridazine directly or indirectly thereto.

[0649] According to Part 21 of Aspect 11 of the present application, there is provided the co-application of 3-ethynyl-6-methoxypyridazine with a nitrogen fertiliser to inhibit nitrification in soil.

[0650] According to Part 22 of Aspect 11 of the present application, there is provided a method of treating an animal urine patch by applying 3-ethynyl-6-methoxypyridazine thereto before, at the same time as, or after, urine deposition.

[0651] According to Part 23 of Aspect 11 of the present application, there is provided a method of treating soil by applying 3-ethynyl-6-methoxypyridazine to an area of soil that is to be or has been subjected to urea, fertiliser, effluent, or other nitrogen source.

[0652] According to Part 24 of Aspect 11 of the present application, there is provided the application of 3-ethynyl-6-methoxypyridazine to soil, to a field, or to a paddock.

[0653] According to Part 25 of Aspect 11 of the present application, there is provided the use of 3-ethynyl-6-methoxypyridazine substantially as described above, wherein the dosage rate is selected from 2 kg / ha to 10 kg / ha.

[0654] According to Part 26 of Aspect 11 of the present application, there is provided the use of 3-ethynyl-6-methoxypyridazine substantially as described above, wherein the dosage rate is 2 kg / ha.

[0655] According to Part 27 of 11thaspect of the invention, there is provided the use of 3-ethynyl-6-methoxypyridazine to retard the corrosion of architectural stone or statuary.

[0656] According to Part 28 of 11thaspect of the invention, there is provided a method of treating agricultural land or urban land, the method comprising selling 3-ethynyl-6-methoxypyridazine in one or more of the following ways:

[0657] - dosage;

[0658] - formulation type;

[0659] - co-delivery with a nitrogen source; or

[0660] - combinations thereof,

[0661] This makes 3-ethynyl-6-methoxypyridazine suitable for use in treating land to reduce the environmental impact of: animal urine; nitrogen fertiliser; animal manure; or effluent.

[0662] Further information on 3-ethynyl-6-methoxypyridazine (including chemical suppliers) can be accessed at:

[0663] https: / / chem-space.com / CSSB00011011728-FA5855

[0664] Twelfth group of aspects of the invention: 2-ethynylpyrazine (CAS 153800-11-4)

[0665] In the twelfth group of aspects, the invention also relates to the surprising new use of 2-ethynylpyrazine, which has the same or substantially similar structure to that shown in the following formula:

[0666]

[0667] The use is in relation to its reduction of NO3 - leaching and N2O emissions. Figure 1 The relationship between inhibiting nitrification in soil and reducing nitrate leaching and nitrous oxide emissions is illustrated (Di and Cameron 2016). It is ultimately shown that by inhibiting the nitrification process using a nitrification inhibitor, nitrate leaching and N2O emissions can be reduced (Di and Cameron 2016).

[0668] According to Part 1 of 12thaspect of the invention, there is provided the use of 2-ethynylpyrazine as a nitrification inhibitor.

[0669] Preferably, the use of 2-ethynylpyrazine substantially as described above, wherein the effective application rate in kg / ha is substantially less than the effective application rate of DCD.

[0670] According to a second part of the 12th aspect of the application, there is provided the use of 2-ethynylpyrazines as an active ingredient in a nitrification inhibitor formulation.

[0671] According to a third part of the 12th aspect of the application, there is provided the use of 2-ethynylpyrazines in the manufacture of a nitrification inhibitor.

[0672] According to a fourth part of the 12th aspect of the application, there is provided the sale of 2-ethynylpyrazines as a nitrification inhibitor, or for use in the manufacture of a nitrification inhibitor.

[0673] Preferably, the sale can be in doses or multiples thereof.

[0674] According to a fifth part of the 12th aspect of the application, there is provided the use of 2-ethynylpyrazines in the manufacture and / or sale of a soil treatment to achieve nitrification inhibition and associated effects.

[0675] Preferably, the soil treatment reduces nitrification and / or increases plant nitrogen use efficiency.

[0676] According to a sixth part of the 12th aspect of the application, there is provided the use of 2-ethynylpyrazines substantially as hereinbefore described, wherein the nitrification inhibition reduces one or more of the following in the soil:

[0677] - nitrous oxide emissions;

[0678] - nitrate leaching; or

[0679] - combinations thereof.

[0680] According to a seventh part of the 12th aspect of the application, there is provided a treatment using 2-ethynylpyrazines to achieve nitrification and associated effects.

[0681] According to an eighth part of the 12th aspect of the application, there is provided a soil treatment substantially as hereinbefore described, wherein 2-ethynylpyrazines are co-applied with urea granules or other nitrogen-containing fertilizer granules.

[0682] According to a ninth part of the 12th aspect of the application, there is provided the use of 2-ethynylpyrazines to coat urea fertilizer granules or other nitrogen fertilizer granules.

[0683] According to a tenth part of the 12th aspect of the application, there is provided a nitrogen fertilizer granule coated with 2-ethynylpyrazines.

[0684] Preferably, the nitrogen fertilizer granule can be urea (or other ammonium form of nitrogen or nitrogen fertilizer that releases ammonium in the soil).

[0685] According to an eleventh part of the 12th aspect of the application, there is provided the use of 2-ethynylpyrazines as an additive for nitrogen-containing liquid fertilizers.

[0686] According to Part 12 of Aspect 12 of the present application, there is provided the use of 2-ethynylpyrazines as soil treatment agents in granular and / or liquid form.

[0687] According to Part 13 of Aspect 12 of the present application, there is provided the manufacture and / or sale of 2-ethynylpyrazine coated nitrogen fertilisers.

[0688] According to Part 14 of Aspect 12 of the present application, there is provided the use of 2-ethynylpyrazines to increase nitrogen use efficiency in crop / pasture systems or to increase the nitrogen use efficiency of fertilisers.

[0689] According to Part 15 of Aspect 12 of the present application, there is provided the use of 2-ethynylpyrazines to interrupt the nitrification process in soil microorganisms.

[0690] According to Part 16 of Aspect 12 of the present application, there is provided the use of 2-ethynylpyrazines to improve pasture / plant growth.

[0691] According to Part 17 of Aspect 12 of the present application, there is provided the use of 2-ethynylpyrazines as a treatment for urine patch areas in pastures.

[0692] According to Part 18 of Aspect 12 of the present application, there is provided the use of 2-ethynylpyrazines and a nitrogen source co-applied to crops or pastures.

[0693] According to Part 19 of Aspect 12 of the present application, there is provided the sale of 2-ethynylpyrazines for use substantially as described above in relation to the use of the 6th, 7th, 9th, 11th, 12th, 14th, 15th, 16th, 17th and 18th Parts of Aspect 14 of the present application.

[0694] According to Part 20 of Aspect 12 of the present application, there is provided a method of treating agricultural land or other land having nitrate leaching or nitrous oxide emissions problems, the method comprising applying 2-ethynylpyrazines directly or indirectly thereto.

[0695] According to Part 21 of Aspect 12 of the present application, there is provided the co-application of 2-ethynylpyrazines with nitrogen fertilisers to inhibit nitrification in soil.

[0696] According to Part 22 of Aspect 12 of the present application, there is provided a method of treating animal urine patches by applying 2-ethynylpyrazines thereto before, at the same time as, or after urine deposition.

[0697] According to Part 23 of Aspect 12 of the present application, there is provided a method of treating soil by applying 2-ethynylpyrazines to soil areas that are to be or have been subjected to urea, fertilisers, effluent or other nitrogen sources.

[0698] According to Part 24 of Aspect 12 of the present application, there is provided the application of a 2-ethynylpyrazine to soil, tillage or pasture.

[0699] According to Part 25 of Aspect 12 of the present application, there is provided the use of a 2-ethynylpyrazine substantially as hereinbefore described, wherein the dosage rate is selected from 2 kg / ha to 9 kg / ha.

[0700] According to Part 26 of Aspect 12 of the present application, there is provided the use of a 2-ethynylpyrazine substantially as hereinbefore described, wherein the dosage rate is 2 kg / ha.

[0701] According to Part 27 of Aspect 12 of the present application, there is provided a method of treating agricultural or urban land, the method comprising selling a 2-ethynylpyrazine in one or more of the following:

[0702] - dosage;

[0703] - formulation type;

[0704] - co-delivery with a nitrogen source; or

[0705] - combinations thereof,

[0706] This makes the 2-ethynylpyrazine suitable for use in treating land to reduce the environmental impact of: animal urine; nitrogen fertilisers; animal manure; or effluent.

[0707] Further information on 2-ethynylpyrazine (including chemical suppliers) can be accessed at:

[0708] https: / / pubchem.ncbi.nlm.nih.gov / compound / 15639205

[0709] Thirteenth Group of Aspects of the Invention: 2-Ethynyl-5-methoxypyrazine (CAS 1374115-62-4)

[0710] In the Thirteenth Group of Aspects, the present application also relates to the surprising new use of 2-ethynyl-5-methoxypyrazine, which has the same or substantially similar structure to that shown in the following formula:

[0711]

[0712] The use is in relation to its reduction of NO3 - leaching and N2O emissions. Figure 1 The relationship between inhibition of nitrification in soil and reduction of nitrate leaching and nitrous oxide emissions is illustrated (Di and Cameron 2016). It was ultimately shown that by inhibiting the nitrification process using a nitrification inhibitor, nitrate leaching and N2O emissions can be reduced (Di and Cameron 2016).

[0713] According to a first part of the 13th aspect of the present application, there is provided the use of 2-ethynyl-5-methoxypyrazine as a nitrification inhibitor.

[0714] Preferably, the use of 2-ethynyl-5-methoxypyrazine substantially as hereinbefore described, wherein the effective application rate in kg / ha is substantially less than the effective application rate of DCD.

[0715] According to a second part of the 13th aspect of the present application, there is provided the use of 2-ethynyl-5-methoxypyrazine as an active ingredient in a nitrification inhibitor formulation.

[0716] According to a third part of the 13th aspect of the present application, there is provided the use of 2-ethynyl-5-methoxypyrazine in the manufacture of a nitrification inhibitor.

[0717] According to a fourth part of the 13th aspect of the present application, there is provided the sale of 2-ethynyl-5-methoxypyrazine as a nitrification inhibitor, or for use in the manufacture of a nitrification inhibitor.

[0718] Preferably, the sale can be in doses or multiples thereof.

[0719] According to a fifth part of the 13th aspect of the present application, there is provided the use of 2-ethynyl-5-methoxypyrazine in the manufacture and / or sale of a soil treatment to achieve nitrification inhibition and associated effects.

[0720] Preferably, the soil treatment reduces nitrification and / or increases plant nitrogen use efficiency.

[0721] According to a sixth part of the 13th aspect of the present application, there is provided the use of 2-ethynyl-5-methoxypyrazine substantially as hereinbefore described, wherein the nitrification inhibition reduces one or more of the following in the soil:

[0722] - nitrous oxide emissions;

[0723] - nitrate leaching; or

[0724] - combinations thereof.

[0725] According to a seventh part of the 13th aspect of the present application, there is provided a treatment using 2-ethynyl-5-methoxypyrazine to achieve nitrification inhibition and associated effects.

[0726] According to an eighth part of the 13th aspect of the present application, there is provided a soil treatment substantially as hereinbefore described, wherein 2-ethynyl-5-methoxypyrazine is co-applied with urea granules or other nitrogen-containing fertilizer granules.

[0727] According to a ninth part of the 13th aspect of the present application, there is provided the use of 2-ethynyl-5-methoxypyrazine to coat urea fertilizer granules or other nitrogen fertilizer granules.

[0728] According to Part 10 of Aspect 13 of the application, there is provided a nitrogen fertiliser particle coated with 2-ethynyl-5-methoxypyrazine.

[0729] Preferably, the nitrogen fertiliser particle can be urea (or other ammonium form of nitrogen or nitrogen fertiliser that releases ammonium in the soil).

[0730] According to Part 11 of Aspect 13 of the application, there is provided the use of 2-ethynyl-5-methoxypyrazine as a nitrogen-containing liquid fertiliser additive.

[0731] According to Part 12 of Aspect 13 of the application, there is provided the use of 2-ethynyl-5-methoxypyrazine as a soil treatment agent in granular and / or liquid form.

[0732] According to Part 13 of Aspect 13 of the application, there is provided the manufacture and / or sale of a nitrogen fertiliser coated with 2-ethynyl-5-methoxypyrazine.

[0733] According to Part 14 of Aspect 13 of the application, there is provided the use of 2-ethynyl-5-methoxypyrazine to improve nitrogen use efficiency in a crop / pasture system or to improve the nitrogen use efficiency of a fertiliser.

[0734] According to Part 15 of Aspect 13 of the application, there is provided the use of 2-ethynyl-5-methoxypyrazine to interrupt the nitrification process by soil microbes.

[0735] According to Part 16 of Aspect 13 of the application, there is provided the use of 2-ethynyl-5-methoxypyrazine to improve pasture / plant growth.

[0736] According to Part 17 of Aspect 13 of the application, there is provided the use of 2-ethynyl-5-methoxypyrazine as a treatment agent for urine patch areas in a pasture.

[0737] According to Part 18 of Aspect 13 of the application, there is provided the use of 2-ethynyl-5-methoxypyrazine and a nitrogen source co-applied to a crop or pasture.

[0738] According to Part 19 of Aspect 13 of the application, there is provided the sale of 2-ethynyl-5-methoxypyrazine for use substantially as described above in relation to the use of the 6th, 7th, 9th, 11th, 12th, 14th, 15th, 16th, 17th and 18th parts of Aspect 15 of the application.

[0739] According to Part 20 of Aspect 13 of the application, there is provided a method of treating agricultural land or other land having a problem with nitrate leaching or nitrous oxide emissions, the method comprising applying 2-ethynyl-5-methoxypyrazine directly or indirectly thereto.

[0740] According to Part 21 of Aspect 14 of the present application, there is provided the co-application of 2-ethynyl-5-methoxypyrazine with a nitrogen fertilizer to inhibit nitrification in soil.

[0741] According to Part 22 of Aspect 13 of the present application, there is provided a method of treating an animal urine stain by applying 2-ethynyl-5-methoxypyrazine to it before, at the same time as, or after urine deposition.

[0742] According to Part 23 of Aspect 13 of the present application, there is provided a method of treating soil by applying 2-ethynyl-5-methoxypyrazine to an area of soil that is about to be or has been subjected to urea, fertilizer, effluent, or other source of nitrogen.

[0743] According to Part 24 of Aspect 13 of the present application, there is provided the application of 2-ethynyl-5-methoxypyrazine to soil, farmland, or pasture.

[0744] According to Part 25 of Aspect 13 of the present application, there is provided the use of 2-ethynyl-5-methoxypyrazine substantially as described above, wherein the dosage rate is selected from 2 kg / ha to 10 kg / ha.

[0745] According to Part 26 of Aspect 14 of the present application, there is provided the use of 2-ethynyl-5-methoxypyrazine substantially as described above, wherein the dosage rate is 2 kg / ha.

[0746] According to Part 27 of Aspect 13 of the present application, there is provided a method of treating agricultural or urban land, the method comprising selling 2-ethynyl-5-methoxypyrazine in one or more of the following ways:

[0747] - dosage;

[0748] - formulation type;

[0749] - co-delivery with a source of nitrogen; or

[0750] - combinations thereof,

[0751] This makes 2-ethynyl-5-methoxypyrazine suitable for use in treating land to reduce the environmental impact of: animal urine; nitrogen fertilizer; animal manure; or effluent.

[0752] Further information on 2-ethynyl-5-methoxypyrazine (including chemical suppliers) can be accessed at:

[0753] https: / / chem-space.com / CSSB00010369260-B1A2BE

[0754] Aspect 14 of the present application: 4-ethynylanisole (CAS 768-60-5)

[0755] In a 14th aspect, the present invention relates to the surprising new use of 4-ethynylanisole (also known as 1-ethynyl-4-methoxybenzene) which has the same or substantially similar structure to that shown in the following formula:

[0756]

[0757] The use is in relation to its reduction of NO3 - leaching and N2O emissions. Figure 1 The relationship between inhibition of nitrification in soil and reduction of nitrate leaching and nitrous oxide emissions is illustrated (Di and Cameron 2016). Ultimately, it is shown that by inhibiting the nitrification process using a nitrification inhibitor, nitrate leaching and N2O emissions can be reduced (Di and Cameron 2016).

[0758] According to a first part of the 14th aspect of the present invention, there is provided the use of 4-ethynylanisole as a nitrification inhibitor.

[0759] Preferably, the use of 4-ethynylanisole substantially as described above, wherein the effective application rate in kg / ha is substantially less than the effective application rate of DCD.

[0760] According to a second part of the 14th aspect of the present invention, there is provided the use of 4-ethynylanisole as an active ingredient in a nitrification inhibitor formulation.

[0761] According to a third part of the 14th aspect of the present invention, there is provided the use of 4-ethynylanisole in the manufacture of a nitrification inhibitor.

[0762] According to a fourth part of the 14th aspect of the present invention, there is provided the sale of 4-ethynylanisole as a nitrification inhibitor, or for use in the manufacture of a nitrification inhibitor.

[0763] Preferably, the sale can be in a dose or a multiple thereof.

[0764] According to a fifth part of the 14th aspect of the present invention, there is provided the use of 2-4-ethynylanisole in the manufacture and / or sale of a soil treatment agent to achieve nitrification inhibition and associated effects.

[0765] Preferably, the soil treatment agent reduces nitrification and / or improves plant nitrogen use efficiency.

[0766] According to a sixth part of the 14th aspect of the present invention, there is provided the use substantially as described above, wherein the nitrification inhibition reduces one or more of the following in the soil:

[0767] - nitrous oxide emissions;

[0768] - nitrate leaching; or

[0769] - combinations thereof.

[0770] According to a seventh part of the 14th aspect of the application, there is provided a soil treatment using 4-ethynylanisole to achieve nitrification inhibition and associated effects.

[0771] According to an eighth part of the 14th aspect of the application, there is provided a soil treatment substantially as hereinbefore described, wherein 4-ethynylanisole is co-applied with urea granules or other nitrogen-containing fertilizer granules.

[0772] According to a ninth part of the 14th aspect of the application, there is provided the use of 4-ethynylanisole to coat urea fertilizer granules or other nitrogen fertilizer granules.

[0773] According to a tenth part of the 14th aspect of the application, there is provided a nitrogen fertilizer granule coated with 4-ethynylanisole.

[0774] Preferably, the nitrogen fertilizer granule can be urea (or other ammonium form of nitrogen or nitrogen fertilizer that releases ammonium in the soil).

[0775] According to an eleventh part of the 14th aspect of the application, there is provided the use of 4-ethynylanisole as a nitrogen-containing liquid fertilizer additive.

[0776] According to a twelfth part of the 14th aspect of the application, there is provided the use of 4-ethynylanisole as a soil treatment agent in granular and / or liquid form.

[0777] According to a thirteenth part of the 14th aspect of the application, there is provided the manufacture and / or sale of 4-ethynylanisole-coated nitrogen fertilizer.

[0778] According to a fourteenth part of the 14th aspect of the application, there is provided the use of 4-ethynylanisole to increase nitrogen use efficiency in a crop / pasture system, or to increase the nitrogen use efficiency of a fertilizer.

[0779] According to a fifteenth part of the 14th aspect of the application, there is provided the use of 4-ethynylanisole to interrupt the nitrification process by soil microbes.

[0780] According to a sixteenth part of the 14th aspect of the application, there is provided the use of 4-ethynylanisole to improve pasture / plant growth.

[0781] According to a seventeenth part of the 14th aspect of the application, there is provided the use of 4-ethynylanisole as a treatment for urine patch areas in a pasture.

[0782] According to an eighteenth part of the 14th aspect of the application, there is provided the use of co-applying 4-ethynylanisole and a nitrogen source to a crop or pasture.

[0783] According to Part 19 of Aspect 14 of the application, there is provided 4-ethynylanisole for use in the sale of substantially as hereinbefore described with respect to the uses of Part 6, Part 7, Part 9, Part 11, Part 12, Part 14, Part 15, Part 16, Part 17 and Part 18 of Aspect 16 of the application.

[0784] According to Part 20 of Aspect 14 of the application, there is provided a method of treating agricultural land or other land having a problem with nitrate leaching or nitrous oxide emissions, the method comprising applying 4-ethynylanisole directly or indirectly thereto.

[0785] According to Part 21 of Aspect 14 of the application, there is provided the co-application of 4-ethynylanisole with a nitrogen fertiliser to inhibit nitrification in soil.

[0786] According to Part 22 of Aspect 14 of the application, there is provided a method of treating an animal urine patch by applying 4-ethynylanisole thereto before, at the same time as, or after, urine deposition.

[0787] According to Part 23 of Aspect 14 of the application, there is provided a method of treating soil by applying 4-ethynylanisole to an area of soil that is about to be or has been subjected to urea, a fertiliser, effluent or other source of nitrogen.

[0788] According to Part 24 of Aspect 14 of the application, there is provided the application of 4-ethynylanisole to soil, farmland or pasture.

[0789] According to Part 25 of Aspect 14 of the application, there is provided the use of 4-ethynylanisole substantially as hereinbefore described, wherein the dosage rate is selected from 1 kg / ha to 9 kg / ha.

[0790] According to Part 26 of Aspect 14 of the application, there is provided the use of 4-ethynylanisole substantially as hereinbefore described, wherein the dosage rate is 1 kg / ha.

[0791] According to Part 27 of Aspect 14 of the application, there is provided the use of 4-ethynylanisole to retard the corrosion of architectural stone or statuary.

[0792] According to Part 28 of Aspect 14 of the application, there is provided a method of treating agricultural land or urban land, the method comprising the sale of 4-ethynylanisole in one or more of the following ways selected from:

[0793] - dosage;

[0794] - formulation type;

[0795] - co-delivery with a source of nitrogen; or

[0796] - combinations thereof,

[0797] This makes 4-ethynylanisole suitable for treating land to reduce the environmental impact of: animal urine; nitrogen fertilizers; animal manure; or effluents.

[0798] Further information on 4-ethynylanisole (including chemical suppliers) can be accessed at:

[0799] https: / / www.sigmaaldrich.com / catalog / product / aldrich / 206490

[0800] Fifteenth group of aspects of the present invention: 1-ethoxy-4-ethynylbenzene (CAS 79887-14-2)

[0801] In the fifteenth group of aspects, the present invention also relates to the surprising new use of 1-ethoxy-4-ethynylbenzene, which has the same or substantially similar structure to the structure shown in the following formula:

[0802]

[0803] The use is in relation to its reduction of NO3 - leaching and N2O emissions. Figure 1 The relationship between inhibition of nitrification in soil and reduction of nitrate leaching and nitrous oxide emissions is illustrated (Di and Cameron 2016). It was ultimately shown that by inhibiting the nitrification process using a nitrification inhibitor, nitrate leaching and N2O emissions can be reduced (Di and Cameron 2016).

[0804] According to a first part of the fifteenth aspect of the present invention, there is provided the use of 1-ethoxy-4-ethynylbenzene as a nitrification inhibitor.

[0805] Preferably, the use of 1-ethoxy-4-ethynylbenzene substantially as described above, wherein the effective application rate in kg / ha is substantially less than the effective application rate of DCD.

[0806] According to a second part of the fifteenth aspect of the present invention, there is provided the use of 1-ethoxy-4-ethynylbenzene as an active ingredient in a nitrification inhibitor formulation.

[0807] According to a third part of the fifteenth aspect of the present invention, there is provided the use of 1-ethoxy-4-ethynylbenzene in the manufacture of a nitrification inhibitor.

[0808] According to a fourth part of the fifteenth aspect of the present invention, there is provided the sale of 1-ethoxy-4-ethynylbenzene as a nitrification inhibitor, or for use in the manufacture of a nitrification inhibitor.

[0809] Preferably, the sale can be in a dose or a multiple thereof.

[0810] According to a fifth part of the 15th aspect of the application, there is provided the use of 1-ethoxy-4-ethynylbenzene in the manufacture and / or sale of a soil treatment to achieve nitrification inhibition and related effects.

[0811] Preferably, the soil treatment reduces nitrification and / or increases plant nitrogen use efficiency.

[0812] According to a sixth part of the 15th aspect of the application, there is provided the use of 1-ethoxy-4-ethynylbenzene substantially as hereinbefore described, wherein the nitrification inhibition reduces one or more of the following in the soil:

[0813] - nitrous oxide emissions;

[0814] - nitrate leaching; or

[0815] - a combination thereof.

[0816] According to a seventh part of the 15th aspect of the application, there is provided a treatment using 1-ethoxy-4-ethynylbenzene to achieve nitrification inhibition and related effects.

[0817] According to an eighth part of the 15th aspect of the application, there is provided a soil treatment substantially as hereinbefore described, wherein 1-ethoxy-4-ethynylbenzene is co-applied with urea granules or other nitrogen-containing fertilizer granules.

[0818] According to a ninth part of the 15th aspect of the application, there is provided the use of 1-ethoxy-4-ethynylbenzene to coat urea fertilizer granules or other nitrogen fertilizer granules.

[0819] According to a tenth part of the 15th aspect of the application, there is provided a nitrogen fertilizer granule coated with 1-ethoxy-4-ethynylbenzene.

[0820] Preferably, the nitrogen fertilizer granule can be urea (or other ammonium form of nitrogen or nitrogen fertilizer that releases ammonium in the soil).

[0821] According to an eleventh part of the 15th aspect of the application, there is provided the use of 1-ethoxy-4-ethynylbenzene as a nitrogen-containing liquid fertilizer additive.

[0822] According to a twelfth part of the 15th aspect of the application, there is provided the use of 1-ethoxy-4-ethynylbenzene as a soil treatment agent in granular and / or liquid form.

[0823] According to a thirteenth part of the 15th aspect of the application, there is provided the manufacture and / or sale of 1-ethoxy-4-ethynylbenzene to coat nitrogen fertilizers.

[0824] According to a fourteenth part of the 15th aspect of the application, there is provided the use of 1-ethoxy-4-ethynylbenzene to increase nitrogen use efficiency in a crop / pasture system or to increase nitrogen use efficiency of a fertilizer.

[0825] According to Part 15 of Aspect 15 of the present application, there is provided the use of 1-ethoxy-4-ethynylbenzene to interrupt the nitrification process of soil microorganisms.

[0826] According to Part 16 of Aspect 15 of the present application, there is provided the use of 1-ethoxy-4-ethynylbenzene to improve pasture / plant growth.

[0827] According to Part 17 of Aspect 15 of the present application, there is provided the use of 1-ethoxy-4-ethynylbenzene as a treatment for urine patch areas in a pasture.

[0828] According to Part 18 of Aspect 15 of the present application, there is provided the use of 1-ethoxy-4-ethynylbenzene and a nitrogen source applied together to a crop or pasture.

[0829] According to Part 19 of Aspect 15 of the present application, there is provided the sale of 1-ethoxy-4-ethynylbenzene for use substantially as described above in relation to the uses of Parts 6, 7, 9, 11, 12, 14, 15, 16, 17 and 18 in relation to Aspect 17 of the present application.

[0830] According to Part 20 of Aspect 15 of the present application, there is provided a method of treating agricultural land or other land having a problem with nitrate leaching or nitrous oxide emissions, the method comprising applying 1-ethoxy-4-ethynylbenzene directly or indirectly thereto.

[0831] According to Part 21 of Aspect 15 of the present application, there is provided the co-application of 1-ethoxy-4-ethynylbenzene with a nitrogen fertiliser to inhibit nitrification in soil.

[0832] According to Part 22 of Aspect 15 of the present application, there is provided a method of treating an animal urine patch by applying 1-ethoxy-4-ethynylbenzene thereto before, at the same time as, or after, urine deposition.

[0833] According to Part 23 of Aspect 15 of the present application, there is provided a method of treating soil by applying 1-ethoxy-4-ethynylbenzene to an area of soil that is to be or has been subjected to urea, fertiliser, effluent or other nitrogen source.

[0834] According to Part 24 of Aspect 15 of the present application, there is provided the application of 1-ethoxy-4-ethynylbenzene to soil, farmland or pasture.

[0835] According to Part 25 of Aspect 15 of the present application, there is provided the use of 1-ethoxy-4-ethynylbenzene substantially as described above, wherein the dosage rate is selected from 2 kg / ha to 9 kg / ha.

[0836] According to Part 26 of 15thaspect of the invention, there is provided the use of 1-ethoxy-4-ethynylbenzene substantially as hereinbefore described wherein the dosage rate is 2 kg / ha.

[0837] According to Part 27 of 15thaspect of the invention, there is provided a method of treating agricultural land or urban land, the method comprising applying 1-ethoxy-4-ethynylbenzene sold in one or more of the following:

[0838] - dosage;

[0839] - formulation type;

[0840] - co-delivery with a nitrogen source; or

[0841] - combinations thereof,

[0842] This makes 1-ethoxy-4-ethynylbenzene suitable for use in treating land to reduce the environmental impact of: animal urine; nitrogen fertilisers; animal manure; or effluent.

[0843] Further information on 1-ethoxy-4-ethynylbenzene (including chemical suppliers) can be accessed at:

[0844] https: / / pubchem.ncbi.nlm.nih.gov / compound / 4-Ethoxyphenylacetylene

[0845] Sixteenth set of aspects of the invention: 1,4-diethynylbenzene (CAS 935-14-8)

[0846] In the sixteenth set of aspects, the invention also relates to the surprising new use of 1,4-diethynylbenzene, which has the same or substantially similar structure to that shown in the following formula:

[0847]

[0848] The use is in relation to its reduction of NO3 - leaching and N2O emissions. Figure 1 The relationship between inhibition of nitrification in soil and reduction of nitrate leaching and nitrous oxide emissions is illustrated (Di and Cameron 2016). It is ultimately shown that by inhibiting the nitrification process using a nitrification inhibitor, nitrate leaching and N2O emissions can be reduced (Di and Cameron 2016).

[0849] According to Part 1 of 16thaspect of the invention, there is provided the use of 1,4-diethynylbenzene as a nitrification inhibitor.

[0850] Preferably, the use of 1,4-diethynylbenzene substantially as hereinbefore described wherein the effective application rate in kg / ha is significantly less than the effective application rate of DCD.

[0851] According to a second part of the 16th aspect of the application, there is provided the use of 1,4-diethynylbenzene as an active ingredient in a nitrification inhibitor formulation.

[0852] According to a third part of the 16th aspect of the application, there is provided the use of 1,4-diethynylbenzene in the manufacture of a nitrification inhibitor.

[0853] According to a fourth part of the 16th aspect of the application, there is provided the sale of 1,4-diethynylbenzene as a nitrification inhibitor, or for use in the manufacture of a nitrification inhibitor.

[0854] Preferably, the sale can be in doses or multiples thereof.

[0855] According to a fifth part of the 16th aspect of the application, there is provided the use of 1,4-diethynylbenzene in the manufacture and / or sale of a soil treatment to achieve nitrification inhibition and associated effects.

[0856] Preferably, the soil treatment reduces nitrification and / or increases plant nitrogen use efficiency.

[0857] According to a sixth part of the 16th aspect of the application, there is provided the use of 1,4-diethynylbenzene substantially as hereinbefore described, wherein the nitrification inhibition reduces one or more of the following in the soil:

[0858] - nitrous oxide emissions;

[0859] - nitrate leaching; or

[0860] - combinations thereof.

[0861] According to a seventh part of the 16th aspect of the application, there is provided a treatment to achieve nitrification inhibition and associated effects using 1,4-diethynylbenzene.

[0862] According to an eighth part of the 16th aspect of the application, there is provided a soil treatment substantially as hereinbefore described, wherein 1,4-diethynylbenzene is co-applied with urea granules or other nitrogen-containing fertilizer granules.

[0863] According to a ninth part of the 16th aspect of the application, there is provided the use of 1,4-diethynylbenzene to coat urea fertilizer granules or other nitrogen fertilizer granules.

[0864] According to a tenth part of the 17th aspect of the application, there is provided a nitrogen fertilizer granule coated with 1,4-diethynylbenzene.

[0865] Preferably, the nitrogen fertilizer granule can be urea (or other ammonium form of nitrogen or nitrogen fertilizer that releases ammonium in the soil).

[0866] According to Part 11 of Aspect 16 of the present application, there is provided the use of 1,4-diethynylbenzene as a nitrogen-containing liquid fertilizer additive.

[0867] According to Part 12 of Aspect 16 of the present application, there is provided the use of 1,4-diethynylbenzene in granular and / or liquid form as a soil treatment agent.

[0868] According to Part 13 of Aspect 16 of the present application, there is provided the manufacture and / or sale of 1,4-diethynylbenzene coated nitrogen fertilizers.

[0869] According to Part 14 of Aspect 16 of the present application, there is provided the use of 1,4-diethynylbenzene to increase nitrogen use efficiency in a crop / pasture system or to increase the nitrogen use efficiency of a fertilizer.

[0870] According to Part 15 of Aspect 16 of the present application, there is provided the use of 1,4-diethynylbenzene to interrupt the nitrification process of soil microbes.

[0871] According to Part 16 of Aspect 16 of the present application, there is provided the use of 1,4-diethynylbenzene to improve pasture / plant growth.

[0872] According to Part 17 of Aspect 16 of the present application, there is provided the use of 1,4-diethynylbenzene as a treatment agent for urine patch areas in pastures.

[0873] According to Part 18 of Aspect 16 of the present application, there is provided the use of 1,4-diethynylbenzene and a nitrogen source co-applied to a crop or pasture.

[0874] According to Part 19 of Aspect 16 of the present application, there is provided the use of 1,4-diethynylbenzene for the sale of use substantially as hereinbefore described with respect to Parts 6, 7, 9, 11, 12, 14, 15, 16, 17 and 18 of Aspect 17 of the present application.

[0875] According to Part 20 of Aspect 16 of the present application, there is provided a method of treating agricultural land or other land having nitrate leaching or nitrous oxide emissions problems, the method comprising applying 1,4-diethynylbenzene directly or indirectly thereto.

[0876] According to Part 21 of Aspect 16 of the present application, there is provided the co-application of 1,4-diethynylbenzene with a nitrogen fertilizer to inhibit nitrification in soil.

[0877] According to Part 22 of Aspect 16 of the present application, there is provided a method of treating animal urine patches by applying 1,4-diethynylbenzene thereto prior to, simultaneously with or after urine deposition.

[0878] According to Part 23 of Aspect 16 of the present application there is provided a method of treating soil by applying 1,4-diethynylbenzene to an area of soil that is to be or has been subjected to urea, manure, effluent or other source of nitrogen.

[0879] According to Part 24 of Aspect 16 of the present application there is provided the application of 1,4-diethynylbenzene to soil, farmland or pasture.

[0880] According to Part 25 of Aspect 16 of the present application there is provided the use of 1,4-diethynylbenzene substantially as described above, wherein the dosage rate is selected from 2 kg / ha to 9 kg / ha.

[0881] According to Part 26 of Aspect 16 of the present application there is provided the use of 1,4-diethynylbenzene substantially as described above, wherein the dosage rate is 2 kg / ha.

[0882] According to Part 27 of Aspect 16 of the present application there is provided a method of treating agricultural or urban land, the method being with 1,4-diethynylbenzene sold in one or more of the following ways:

[0883] - dosage;

[0884] - formulation type;

[0885] - co-delivery with a source of nitrogen; or

[0886] - combinations thereof,

[0887] This makes 1,4-diethynylbenzene suitable for use in treating land to reduce the impact on the environment of: animal urine; nitrogen fertiliser; animal manure; or effluent.

[0888] Further information on 1,4-diethynylbenzene (including chemical suppliers) can be accessed at:

[0889] https: / / www.sigmaaldrich.com / catalog / product / aldrich / 632090 BRIEF DESCRIPTION OF DRAWINGS

[0890] Further aspects of the present application will become apparent from the following description, given by way of example only, and with reference to the accompanying drawings:

[0891] Context for aspects of the present application

[0892] Figure 1 is a diagram illustrating the nitrification process and how nitrate leaching and the greenhouse gas N20 are formed in soil, and how nitrification inhibitors can reduce nitrate leaching and nitrous oxide emissions (Di and Cameron 2016).

[0893] Figure 2-1 Nitrate-N concentration in soil at the end of 30 days incubation showing the high efficiency of inhibition of nitrification by 2-ethynyl 1,3 diazine applied at 2 kg / ha, as shown, nitrate-N concentration in soil treated with urine + 2-ethynyl 1,3 diazine was lower compared to urine alone or urine + dimethyl sulfoxide (DMSO) control treatment. Error bars represent one standard error (SEM) of the mean.

[0894] Figure 3-1 Nitrate-N concentration in soil at the end of 30 days incubation showing the high efficiency of inhibition of nitrification by 2-ethynyl 1,3 diazine applied at 1 kg / ha, as shown, nitrate-N concentration in soil treated with urine + 2-ethynyl 1,3 diazine was lower compared to urine alone or urine + DMSO control treatment. Error bars represent one standard error (SEM) of the mean.

[0895] Figure 4-1 Nitrate-N concentration in soil at the end of 30 days incubation showing the high efficiency of inhibition of nitrification by 2-ethynyl 1,3 diazine applied at 0.5 kg / ha, as shown, nitrate-N concentration in soil treated with urine + 2-ethynyl 1,3 diazine was lower compared to urine alone or urine + DMSO control treatment. Error bars represent one standard error (SEM) of the mean.

[0896] Figure 5-1 Effect of 2-ethynyl 1,3 diazine in reducing N2O-N emission when sprayed on the surface of soil in a field with pasture that has received animal urine. When 2-ethynyl 1,3 diazine was applied at 10 kg / ha and 2 kg / ha, respectively, N2O-N emission in soil treated with urine + 2-ethynyl 1,3 diazine was reduced by about 96% and 91% compared to urine alone control treatment. This shows the efficiency of 2-ethynyl 1,3 diazine in reducing N2O emission in soil. The nitrous oxide reduction by 2-ethynyl 1,3 diazine was greater than the nitrous oxide reduction by DCD. Error bars in the figure represent one standard error (SEM) of the mean.

[0897] Figure 2-2 Nitrate-N concentration in soil at the end of 30 days incubation showing the high efficiency of inhibition of nitrification by 3-ethynyl 1,5 diazine applied at 2 kg / ha, as shown, nitrate-N concentration in soil treated with urine + 3-ethynyl 1,5 diazine was lower compared to urine alone or urine + DMSO control treatment. Error bars represent one standard error (SEM) of the mean.

[0898] Figure 3-2 Nitrate-N concentration in soil at the end of 30 days incubation showing the high efficiency of 3-ethynyl 1,5 diazine in inhibiting nitrification, as shown, the nitrate-N concentration in soil treated with urine + 3-ethynyl 1,5 diazine was lower compared to urine alone or urine + DMSO control treatment. Error bars represent one standard error (SEM) of the mean.

[0899] Figure 4-2 Nitrate-N concentration in soil at the end of 30 days incubation showing the high efficiency of 3-ethynyl 1,5 diazine in inhibiting nitrification, as shown, the nitrate-N concentration in soil treated with urine + 3-ethynyl 1,5 diazine was lower compared to urine alone or urine + DMSO control treatment. Error bars represent one standard error (SEM) of the mean.

[0900] Figure 5-2 Effect of 3-ethynyl 1,5 diazine in reducing N2O-N emissions when sprayed on the surface of a field having pasture soil that has received animal urine. When 3-ethynyl 1,5 diazine was applied at 10 kg / ha or 2 kg / ha, N2O-N emissions in soil treated with urine + 3-ethynyl 1,5 diazine was reduced by about 93% compared to urine alone control treatment. This shows the efficiency of 3-ethynyl 1,5 diazine in reducing N2O emissions in soil. The nitrous oxide reduction by 3-ethynyl 1,5 diazine was greater than the nitrous oxide reduction by DCD. Error bars in the graph represent one standard error (SEM) of the mean.

[0901] Figure 2-3 Nitrate-N concentration in soil at the end of 30 days incubation showing the high efficiency of 4-ethynyl pyrimidine in inhibiting nitrification, as shown, the nitrate-N concentration in soil treated with urine + 4-ethynyl pyrimidine was lower compared to urine alone or urine + dimethyl sulfoxide (DMSO) control treatment. Error bars represent one standard error (SEM) of the mean.

[0902] Figure 3-3 Nitrate-N concentration in soil at the end of 30 days incubation showing the high efficiency of 4-ethynyl pyrimidine in inhibiting nitrification, as shown, the nitrate-N concentration in soil treated with urine + 4-ethynyl pyrimidine was lower compared to urine alone or urine + DMSO control treatment. Error bars represent one standard error (SEM) of the mean.

[0903] Figure 2-4 Nitrate-N concentrations in soil at the end of 30 days incubation showing the high efficiency of inhibition of nitrification by 2-ethynyl-5-methoxypyrimidine applied at 2 kg / ha, as shown, nitrate-N concentrations in soil treated with urine + 2-ethynyl-5-methoxypyrimidine were lower compared to urine alone or urine + dimethyl sulfoxide (DMSO) control treatments. Error bars represent one standard error (SEM) of the mean.

[0904] Figure 3-4 Nitrate-N concentrations in soil at the end of 30 days incubation showing the high efficiency of inhibition of nitrification by 2-ethynyl-5-methoxypyrimidine applied at 1 kg / ha, as shown, nitrate-N concentrations in soil treated with urine + 2-ethynyl-5-methoxypyrimidine were lower compared to urine alone or urine + DMSO control treatments. Error bars represent one standard error (SEM) of the mean.

[0905] Figure 2-5 Nitrate-N concentrations in soil at the end of 30 days incubation showing the high efficiency of inhibition of nitrification by 5-ethynyl-2-methoxypyrimidine applied at 2 kg / ha, as shown, nitrate-N concentrations in soil treated with urine + 5-ethynyl-2-methoxypyrimidine were lower compared to urine alone or urine + dimethyl sulfoxide (DMSO) control treatments. Error bars represent one standard error (SEM) of the mean.

[0906] Figure 3-5 Nitrate-N concentrations in soil at the end of 30 days incubation showing the high efficiency of inhibition of nitrification by 5-ethynyl-2-methoxypyrimidine applied at 1 kg / ha, as shown, nitrate-N concentrations in soil treated with urine + 5-ethynyl-2-methoxypyrimidine were lower compared to urine alone or urine + dimethyl sulfoxide (DMSO) control treatments. Error bars represent one standard error (SEM) of the mean.

[0907] Figure 2-6 Nitrate-N concentrations in soil at the end of 30 days incubation showing the high efficiency of inhibition of nitrification by 2-ethynyl-5-methoxypyridine applied at 2 kg / ha, as shown, nitrate-N concentrations in soil treated with urine + 2-ethynyl-5-methoxypyridine were lower compared to urine alone or urine + dimethyl sulfoxide (DMSO) control treatments. Error bars represent one standard error (SEM) of the mean.

[0908] Figure 3-6 Nitrate-N concentration in soil at the end of 30 days incubation showing the high efficiency of inhibition of nitrification by 2-ethynyl-5-methoxypyridine applied at 1 kg / ha, as shown, nitrate-N concentration in soil treated with urine + 2-ethynyl-5-methoxypyridine was lower compared to urine alone or urine + DMSO control treatment. Error bars represent one standard error (SEM) of the mean.

[0909] Figure 4-6 Nitrate-N concentration in soil at the end of 30 days incubation showing the high efficiency of inhibition of nitrification by 2-ethynyl-5-methoxypyridine applied at 0.5 kg / ha, as shown, nitrate-N concentration in soil treated with urine + 2-ethynyl-5-methoxypyridine was lower compared to urine alone or urine + DMSO control treatment. Error bars represent one standard error (SEM) of the mean.

[0910] Figure 5-6 Effect of 2-ethynyl-5-methoxypyridine in reducing N2O-N emissions when sprayed on the surface of soil in a field with pasture that has received animal urine. When 2-ethynyl-5-methoxypyridine was applied at 10 kg / ha and 2 kg / ha, respectively, N2O-N emissions in soil treated with urine + 2-ethynyl-5-methoxypyridine were reduced by about 96% and 91% compared to urine alone control treatment. This shows the efficiency of 2-ethynyl-5-methoxypyridine in reducing N2O emissions in soil. The nitrous oxide reduction by 2-ethynyl-5-methoxypyridine was greater than the nitrous oxide reduction by DCD. Error bars in the graph represent one standard error (SEM) of the mean.

[0911] Figure 2-7 Nitrate-N concentration in soil at the end of 30 days incubation showing the high efficiency of inhibition of nitrification by 5-ethynyl-2-methoxypyridine applied at 2 kg / ha, as shown, nitrate-N concentration in soil treated with urine + 5-ethynyl-2-methoxypyridine was lower compared to urine alone or urine + dimethyl sulfoxide (DMSO) control treatment. Error bars represent one standard error (SEM) of the mean.

[0912] Figure 2-8 The nitrate-N concentration in soil at the end of 30 days incubation under the influence of 3-ethynylpyridine 1-oxide applied at 2 kg / ha is shown, demonstrating the high efficiency of 3-ethynylpyridine 1-oxide in inhibiting nitrification, as shown, the nitrate-N concentration in soil treated with urine + 3-ethynylpyridine 1-oxide is lower compared to urine alone or urine + dimethyl sulfoxide (DMSO) control treatments. Error bars represent one standard error (SEM) of the mean.

[0913] Figure 3-8 The nitrate-N concentration in soil at the end of 30 days incubation under the influence of 3-ethynylpyridine 1-oxide applied at 1 kg / ha is shown, demonstrating the high efficiency of 3-ethynylpyridine 1-oxide in inhibiting nitrification, as shown, the nitrate-N concentration in soil treated with urine + 3-ethynylpyridine 1-oxide is lower compared to urine alone or urine + DMSO control treatments. Error bars represent one standard error (SEM) of the mean.

[0914] Figure 4-8 The nitrate-N concentration in soil at the end of 30 days incubation under the influence of 3-ethynylpyridine 1-oxide applied at 0.5 kg / ha is shown, demonstrating the high efficiency of 3-ethynylpyridine 1-oxide in inhibiting nitrification, as shown, the nitrate-N concentration in soil treated with urine + 3-ethynylpyridine 1-oxide is lower compared to urine alone or urine + DMSO control treatments. Error bars represent one standard error (SEM) of the mean.

[0915] Figure 5-8 The effect of 3-ethynylpyridine 1-oxide in reducing N2O-N emissions when sprayed on the surface of a field of pasture soil that has received animal urine is shown. When 3-ethynylpyridine 1-oxide was applied at 10 kg / ha and 2 kg / ha, respectively, N2O-N emissions in soil treated with urine + 3-ethynylpyridine 1-oxide was reduced by about 96% and 91% compared to urine alone control treatment. This demonstrates the efficiency of 3-ethynylpyridine 1-oxide in reducing N2O emissions in soil. The nitrous oxide reduction by 3-ethynylpyridine 1-oxide is greater than the nitrous oxide reduction by DCD. Error bars in the figure represent one standard error (SEM) of the mean.

[0916] Figure 2-9 Nitrate-N concentrations in soil at the end of 30 days incubation affected by application of 2,5-diethynylpyridine at 2 kg / ha, showing the high efficiency of 2,5-diethynylpyridine in inhibiting nitrification, as shown, nitrate-N concentrations in soil treated with urine + 2,5-diethynylpyridine were lower compared to urine alone or urine + dimethyl sulfoxide (DMSO) control treatments. Error bars represent one standard error (SEM) of the mean.

[0917] Figure 2-10 Nitrate-N concentrations in soil at the end of 30 days incubation affected by application of 3-ethynylpyridazine at 2 kg / ha, showing the high efficiency of 3-ethynylpyridazine in inhibiting nitrification, as shown, nitrate-N concentrations in soil treated with urine + 3-ethynylpyridazine were lower compared to urine alone or urine + dimethyl sulfoxide (DMSO) control treatments. Error bars represent one standard error (SEM) of the mean.

[0918] Figure 3-10 Nitrate-N concentrations in soil at the end of 30 days incubation affected by application of 3-ethynylpyridazine at 1 kg / ha, showing the high efficiency of 3-ethynylpyridazine in inhibiting nitrification, as shown, nitrate-N concentrations in soil treated with urine + 3-ethynylpyridazine were lower compared to urine alone or urine + DMSO control treatments. Error bars represent one standard error (SEM) of the mean.

[0919] Figure 4-10 Nitrate-N concentrations in soil at the end of 30 days incubation affected by application of 3-ethynylpyridazine at 0.5 kg / ha, showing the high efficiency of 3-ethynylpyridazine in inhibiting nitrification, as shown, nitrate-N concentrations in soil treated with urine + 3-ethynylpyridazine were lower compared to urine alone or urine + DMSO control treatments. Error bars represent one standard error (SEM) of the mean.

[0920] Figure 2-11 Nitrate-N concentrations in soil at the end of 30 days incubation affected by application of 3-ethynyl-6-methoxypyridazine at 2 kg / ha or 1 kg / ha, showing the high efficiency of 3-ethynyl-6-methoxypyridazine in inhibiting nitrification, as shown, nitrate-N concentrations in soil treated with urine + 3-ethynyl-6-methoxypyridazine were lower compared to urine alone or urine + dimethyl sulfoxide (DMSO) control treatments. Error bars represent one standard error (SEM) of the mean.

[0921] Figure 2-12 The nitrate-N concentration in soil at the end of 30 days incubation showing the high efficient inhibition of nitrification by 2-ethynylpyrazine applied at 2 kg / ha, as shown, the nitrate-N concentration in soil treated with urine + 2-ethynylpyrazine is lower compared to urine alone or urine + dimethyl sulfoxide (DMSO) control treatment. Error bars represent one standard error (SEM) of the mean.

[0922] Figure 2-13 The nitrate-N concentration in soil at the end of 30 days incubation showing the high efficient inhibition of nitrification by 2-ethynyl-5-methoxypyrazine applied at 2 kg / ha or 1 kg / ha, as shown, the nitrate-N concentration in soil treated with urine + 2-ethynyl-5-methoxypyrazine is lower compared to urine alone or urine + dimethyl sulfoxide (DMSO) control treatment. Error bars represent one standard error (SEM) of the mean.

[0923] Figure 2-14 The nitrite production rate of Nitrosomonas europaea is significantly reduced when 4-ethynylanisole is applied at a wide range of concentrations from 1.56 mM to 400 mM, indicating the efficient inhibition of the nitrification process by 4-ethynylanisole. The OD519nm reading is an indicator of the nitrite concentration in the test liquid, the higher the reading, the higher the nitrite concentration.

[0924] Figure 3-14 The change in nitrate-N concentration in soil as affected by different ratios of 4-ethynylanisole application. Error bars represent one standard error (SEM) of the mean. The graph shows that the increase in nitrate-N concentration in soil treated with urine + 4-ethynylanisole is significantly slower compared to the nitrate-N concentration in soil treated with urine (control), indicating a significant inhibition of the nitrification process by 4-ethynylanisole. The lower the nitrate concentration, the lower the risk of nitrate leaching and nitrous oxide emission.

[0925] Figure 4-14 The nitrate-N concentration in soil at the end of 30 days incubation showing the high efficient inhibition of nitrification by 4-ethynylanisole as affected by different ratios of 4-ethynylanisole treatment, as shown, the nitrate-N concentration in soil treated with urine + 4-ethynylanisole is lower compared to urine control treatment alone. Error bars represent one standard error (SEM) of the mean.

[0926] Figure 5-14 Nitrate concentrations in soil at the end of 60 days incubation showing the effect of treatment with 4-ethynylanisole applied at a rate equivalent to 10 kg / ha. The graph shows that 4-ethynylanisole was much more effective than DCD in inhibiting nitrate production in urine by inhibiting the nitrification process. Solvent (DMSO) did not result in a statistically significant decrease in nitrates concentrations compared to urine treatment alone (P>0.05). Error bars represent one standard error of the mean (SEM).

[0927] Figure 6-14 The effect of 4-ethynylanisole in reducing N2O-N emissions when sprayed on the surface of soil that had received animal urine is shown. N2O-N emissions were reduced by 93.3% in the soil treated with urine + 4-ethynylanisole compared to N2O-N emissions in the urine control treatment alone. This shows the efficiency of 4-ethynylanisole in reducing N2O emissions in soil. The reduction in nitrous oxide was greater for 4-ethynylanisole than for DCD (giving a 43.4% reduction in N2O emissions). Error bars in the graph represent one standard error of the mean (SEM).

[0928] Figure 7-14 Nitrates concentrations in soil at the end of 30 days incubation showing the effect of treatment with 4-ethynylanisole applied at a rate equivalent to 1 and 2 kg / ha. The graph shows that 4-ethynylanisole was much more effective than DCD applied at 10 kg / ha in inhibiting nitrate production in urine by inhibiting the nitrification process. Error bars represent one standard error of the mean (SEM).

[0929] Figure 8A-14 、 8B-14 The reduction of oxygen consumption driven by ammonium to zero ( Figure 8A-14 ) in the presence of 4-ethynylanisole, but not the oxygen consumption driven by hydroxylamine (H3NO) ( Figure 8B-14 ) in the presence of 4-ethynylanisole, indicates that the ammonia monooxygenase (AMO) of ammonia oxidizing bacteria is the potential target of inhibition by 4-ethynylanisole, and not the hydroxylamine oxidoreductase.

[0930] Figure 2-15 Nitrates-N concentrations in soil at the end of 30 days incubation showing the effect of treatment with 1-ethoxy-4-ethynylbenzene applied at 2 kg / ha, demonstrating the high efficiency of 1-ethoxy-4-ethynylbenzene in inhibiting nitrification. Nitrates-N concentrations were lower in soil treated with urine + 1-ethoxy-4-ethynylbenzene compared to urine or urine + dimethyl sulfoxide (DMSO) control treatments as shown. Error bars represent one standard error of the mean (SEM).

[0931] Figure 2-16 The nitrate-N concentration in soil at the end of 30 days incubation showing the high efficiency of inhibition of nitrification by 1,4-diethynylbenzene applied at 2 kg / ha, as indicated, the nitrate-N concentration in soil treated with urine + 1,4-diethynylbenzene was lower compared to urine alone or urine + dimethyl sulfoxide (DMSO) control treatments. Error bars represent one standard error (SEM) of the mean. DETAILED DESCRIPTION

[0932] Figure 1 is a simplified diagram showing the sources of animal urine-N as N2O emissions and NO3 - leaching, and the point of intervention of nitrification inhibitors. If nitrogen fertilizers are applied to soil instead of animal urine, such as urea or other fertilizers that release ammonium in soil, similar reactions as shown in the figure can occur. The nitrification process, sources of nitrate leaching and nitrous oxide emissions, and the point of intervention of using nitrification inhibitors to treat soil that has been or will be or substantially simultaneously subjected to animal urine, nitrogen fertilizers or animal effluents are illustrated using animal urine N as an example.

[0933] The problem to be solved by the present invention is to find new nitrification inhibitors - with the required properties that enable the inhibitors to be used instead of DCD for agricultural applications to address the above-mentioned nitrogen-related problems.

[0934] In their search to solve this problem, the present inventors screened over 25,000 compounds by computer screening against a model of the structure of the ammonia monooxygenase enzyme.

[0935] From there, about 5000 compounds were then screened in vitro using a high-throughput phenotypic screening method optimized for the identification of compounds that specifically inhibit the oxidation of ammonium to nitrite. The phenotypic screening involved assessing the nitrification inhibitory potency of the selected 5000 compounds against a key ammonia-oxidizing bacterium responsible for ammonia oxidation in soil (Nitrosomonas europaea) in a 96-well microtiter plate. The N. europaea cells were grown in the presence of an ammonium source and the potential of the putative nitrification inhibitors to inhibit the production of nitrite (NO2 - ) was measured relative to untreated cells.

[0936] Some key required features of the new potential nitrification inhibitor compounds include:

[0937] • high nitrification inhibitory potency at low (i.e. much lower than the 10 kg / ha used for DCD) application rates;

[0938] • low toxicity;

[0939] • in addition, meeting various food and environmental regulatory requirements or standards.

[0940] Of the 5000 compounds screened, approximately 150 showed inhibitory properties in vitro against N. europaea. Subsequent screening of the compounds that inhibited nitrification in the phenotypic screen and met these criteria, and testing of their nitrification inhibition potency in the critical soil test, and approximately 30 compounds showed sufficient inhibition of nitrification in the soil test.

[0941] The cost of testing potential compounds is not inexpensive, and to date this discovery program has cost in excess of $3 million.

[0942] Table 1 below details a summary of selected compounds initially found to act as or not act as nitrification inhibitors.

[0943] Table 1

[0944]

[0945]

[0946] Test 2-Ethynyl 1,3 Diazine

[0947] Study 1-1

[0948] A laboratory incubation study was conducted to determine the potency of 2-ethynyl 1,3-diazine to inhibit nitrification in soil. This experiment used Templeton sandy loam soil. 500 g of soil (dry weight basis) was packed into jars. 50 mL of synthetic bovine urine with an N concentration of 7 g N / L (comprising approximately 87% urea-N and 13% glycine-N) was applied to the soil (equivalent to 700 kg N / ha by weight assuming a bulk density of 1 g / cm 3 ) in the top 10 cm of the soil profile). An application of 700 kg N / ha was used to simulate the urine-N application rate under a typical patch of cow urine in a grazed pasture. 5 mg of DCD was dissolved in 0.9 mL of DMSO to give a concentration of 10 kg / ha; and 1 mg of 2-ethynyl 1,3-diazine was dissolved in 0.9 mL of DMSO to give a concentration of 2 kg / ha; both were added to the urine-applied soil.

[0949] Control treatments also added to the soil were:

[0950] - urine (50 mL) alone;

[0951] - DMSO (equivalent to 0.9 mL / 500 g soil) + urine (50 mL) to determine the effect of DMSO on nitrification rate;

[0952] - water (50 mL) alone (to simulate soil areas without urine deposition).

[0953] Urine, water, DMSO, DCD and 2-ethynyl 1,3-diazine treatment were applied to the soil surface and mixed thoroughly. The jars were covered with a lid with a breathing hole to allow gas exchange during incubation. The jars were incubated at 12°C. The soil moisture content was maintained at field capacity by adjusting according to weight twice a week.

[0954] Soil samples were collected and then thoroughly mixed, sub-samples were extracted with potassium chloride solution and analysed for mineral-N. The soil moisture content was also determined. Sampling was carried out 30 days after the start of incubation.

[0955] Figure 2-1 The nitrate-N concentration in the soil at the end of 30 days incubation is shown, showing a significant reduction in nitrate-N concentration in the urine + (2-ethynyl 1,3-diazine dissolved in DMSO) treatment, demonstrating the ability of 2-ethynyl 1,3-diazine at 2 kg / ha to inhibit nitrification in the soil. These results show that a significant reduction in nitrification rate can be achieved when applying 2-ethynyl 1,3-diazine at 2 kg / ha to treat urine patches in soil. In particular, 2-ethynyl 1,3-diazine at a concentration of just 2 kg / ha was more effective in reducing nitrification rate than DCD at a higher concentration of 10 kg / ha.

[0956] Study 2-1

[0957] A second laboratory incubation study was carried out to determine the efficacy of 2-ethynyl 1,3-diazine in nitrification inhibition when applied at a lower rate of 1 kg / ha. The experimental procedure was the same as described in Study 1 except that 2-ethynyl 1,3-diazine was applied at 1 kg / ha.

[0958] Figure 3-1 The nitrate-N concentration in the soil at the end of 30 days incubation is shown, showing a significant reduction in nitrate-N concentration in the urine + (2-ethynyl 1,3-diazine dissolved in DMSO) treatment, demonstrating the ability of 2-ethynyl 1,3-diazine at 2 kg / ha to inhibit nitrification in the soil. These results show that a significant reduction in nitrification rate can be achieved when applying 2-ethynyl 1,3-diazine at 2 kg / ha to treat urine patches in soil. In particular, 2-ethynyl 1,3-diazine at a concentration of just 2 kg / ha was more effective in reducing nitrification rate than DCD at a higher concentration of 10 kg / ha.

[0959] Study 3-1

[0960] A third laboratory incubation study was carried out to determine the efficacy of 2-ethynyl 1,3-diazine in nitrification inhibition when applied to soil at a lower rate of 0.5 kg / ha. The experimental procedure was the same as described in Study 1 except that 2-ethynyl 1,3-diazine was applied at 0.5 kg / ha.

[0961] Figure 4-1 The nitrate-N concentration in soil at the end of 30 days incubation showing the effect of application of 2-ethynyl 1,3 diazine at 0.5 kg / ha, illustrates the high efficiency of inhibition of nitrification by 2-ethynyl 1,3 diazine at this very low application rate, as shown, the nitrate-N concentration in soil of urine + 2-ethynyl 1,3 diazine treatment was lower compared to urine alone or urine + DMSO control treatments.

[0962] Study 4-1

[0963] To determine the effect of soil treatment with 2-ethynyl 1,3 diazine on nitrous oxide emissions under field soil conditions, a field study was carried out using the static chamber method. The study was carried out at the University of Lincoln Research Farm, the soil was a Templeton sandy loam with an established ryegrass (Lolium perenne) and white clover (Trifolium repens.) sward. Metal rings (diameter 500 mm, height 200 mm) were inserted into the ground. A water trough located on top of the metal ring allowed the placement of a static chamber located on top of the water trough to provide an air tight seal to allow N20 gas sampling.

[0964] Synthetic urine with a nitrogen concentration of 7 g N / L was applied to soil plots confined within the metal rings at an equivalent rate of 700 kg N / ha. 196.25 mg or 39.2 mg of 2-ethynyl 1,3 diazine was dissolved in 2 mL of DMSO, mixed with 1000 mL of water and then sprayed onto the small plots at a rate of 196.25 mg / plot and 39.2 mg / plot, equivalent to 10 kg / ha and 2 kg / ha respectively. The plots were irrigated with irrigation water in accordance with local dairy farming practice.

[0965] The chamber (diameter 500 mm, height 120 mm) was constructed from a metal cylinder insulated with 2.5 cm thick polystyrene foam to avoid a heating atmosphere within the chamber during sampling. The rim of the chamber was placed within a small water trough installed around the top of each metal ring for gas sampling during N20 measurements. At each sampling time, the chamber was placed on top of the soil ring for a total of 40 minutes and 3 samples were withdrawn through a rubber septum on the top of the chamber using a syringe, spaced 20 minutes apart. Sampling was carried out twice per week. Each sampling was carried out between 12:00 h and 14:00 h of the day (Di et al., 2007). The N20 concentration in the samples was analysed using a gas chromatograph (SRI 8610C with an Electron Capture Detector (ECD) (SRI Instruments, USA) connected to a Gilson 222XL auto-sampler (Gilson, France).

[0966] Figure 5-1 The effect of 2-ethynyl 1,3-diazine to reduce N2O-N emissions was shown when 2-ethynyl 1,3-diazine was sprayed on the surface of a field having pasture grass that had received animal urine. When 2-ethynyl 1,3-diazine was applied at 10 kg / ha and 2 kg / ha, respectively, N2O-N emissions from the soil in the urine + 2-ethynyl 1,3-diazine treatments were reduced by about 96% and 91% compared to the urine only control treatment. This shows the efficiency of 2-ethynyl 1,3-diazine to reduce N2O emissions from the soil. The reduction of N2O by 2-ethynyl 1,3-diazine was greater than the reduction of N2O by DCD.

[0967] The effect of 2-ethynyl 1,3-diazine on nitrate concentration and N2O emissions in Studies 1-4 is shown in Table 1-1 below:

[0968] Table 1-1

[0969]

[0970]

[0971] Test 3-Ethynyl 1,5 Diazine

[0972] Study 1-2

[0973] A laboratory incubation study was conducted to determine the efficacy of 3-ethynyl 1,5-diazine in nitrification inhibition when applied at a rate of 2 kg / ha. The experimental procedure was the same as described in Study 1-1.

[0974] Figure 2-2 The nitrate-N concentration in the soil at day 30 of incubation is shown, showing that the nitrate-N concentration in the urine + (3-ethynyl 1,5-diazine dissolved in DMSO) treatment was significantly reduced, demonstrating the ability of 3-ethynyl 1,5-diazine at 2 kg / ha to inhibit nitrification in the soil. These results show that a significant reduction in nitrification rate can be achieved when urine patches in the soil are treated with 3-ethynyl 1,5-diazine at 2 kg / ha. In particular, 3-ethynyl 1,5-diazine at just 2 kg / ha concentration was more effective in reducing nitrification rate than DCD at a higher concentration of 10 kg / ha.

[0975] Study 2-2

[0976] A second laboratory incubation study was conducted to determine the efficacy of 3-ethynyl 1,5-diazine in nitrification inhibition when applied at a lower rate of 1 kg / ha. The experimental procedure was the same as described in Study 1-1.

[0977] Figure 3-2 The nitrate-N concentration in soil at the end of 30 days incubation showing the effect of applying 3-ethynyl 1,5 diazine to soil at 1 kg / ha, illustrates the high efficiency of 3-ethynyl 1,5 diazine in inhibiting nitrification, as shown, the nitrate-N concentration in soil treated with urine + 3-ethynyl 1,5 diazine was lower compared to urine alone or urine + DMSO control treatments.

[0978] Study 3-2

[0979] A third laboratory incubation study was conducted to determine the efficacy of 3-ethynyl 1,5 diazine in inhibiting nitrification when applied to soil at a lower rate of 0.5 kg / ha. The experimental procedure was the same as described in Study 1-1.

[0980] Figure 4-2 The nitrate-N concentration in soil at the end of 30 days incubation showing the effect of applying 3-ethynyl 1,5 diazine to soil at 0.5 kg / ha, illustrates the high efficiency of 3-ethynyl 1,5 diazine in inhibiting nitrification at this very low application rate, as shown, the nitrate-N concentration in soil treated with urine + 3-ethynyl 1,5 diazine was lower compared to urine alone or urine + DMSO control treatments.

[0981] Study 4-2

[0982] To determine the effect of treating soil with 3-ethynyl 1,5 diazine on nitrous oxide emissions under field soil conditions, a field study was conducted using the static chamber method. The study was conducted at the University of Lincoln research dairy farm, the soil was a Templeton sandy loam with established ryegrass (Lolium perenne) and white clover (Trifolium repens.) pastures. A metal ring (500 mm diameter, 200 mm high) was inserted into the ground. A water trough located on top of the metal ring allowed the placement of a static chamber on top of the water trough to provide an air tight seal to allow N2O gas sampling.

[0983] Synthetic urine with a nitrogen concentration of 7 g N / L was applied to soil plots confined within the metal rings at an equivalent rate of 700 kg N / ha. 196.25 mg or 39.2 mg of 3-ethynyl 1,5 diazine was dissolved in 2 mL of DMSO, mixed with 1000 mL of water and then sprayed onto the small plots at a rate of 196.25 mg / plot and 39.2 mg / plot, equivalent to 10 kg / ha and 2 kg / ha respectively. The plots were irrigated with irrigation water in accordance with local dairy farming practices.

[0984] The gas chamber (diameter 500 mm, height 120 mm) was constructed from a metal cylinder insulated with 2.5 cm thick polystyrene foam to avoid a heating atmosphere in the chamber during sampling. During N20 measurements, the rim of the chamber was placed in a small water bath installed around the top of each metal ring for gas sampling. At each sampling time, the chamber was placed on top of the soil rings for a total of 40 minutes and 3 samples were withdrawn through a rubber septum on top of the gas chamber using a syringe, spaced 20 minutes apart. Sampling was performed twice a week. Each sampling was performed between 12:00 h and 14:00 h of the day (Di et al., 2007). The N20 concentration in the samples was analyzed using a gas chromatograph (SRI 8610C with an Electron Capture Detector (ECD) (SRI Instruments, USA) connected to a Gilson 222XL autosampler (Gilson, France).

[0985] Figure 5-2 The effect of 3-ethynyl 1,5 diazine to reduce N20-N emissions when sprayed on the surface of a field of pasture having soil that has received animal urine is shown. When 3-ethynyl 1,5 diazine was applied at 10 kg / ha and 2 kg / ha, respectively, N20-N emissions from the soil of the urine + 3-ethynyl 1,5-diazine treatment was reduced by about 96% and 91% compared to the urine only control treatment. This shows the efficiency of 3-ethynyl 1,5 diazine to reduce N20 emissions from soil. The reduction of nitrous oxide by 3-ethynyl 1,5 diazine was greater than the reduction of nitrous oxide by DCD.

[0986] The effect of 3-ethynyl 1,5 diazine on nitrate concentration and nitrous oxide emissions in studies 1-4 is shown in Table 1-2 below:

[0987] Table 1-2

[0988]

[0989]

[0990] Test 4-Ethynyl Pyrimidine

[0991] Study 1-3

[0992] A laboratory incubation study was performed to determine the efficacy of 4-ethynyl pyrimidine in nitrification inhibition when applied at a rate of 2 kg / ha. The experimental procedure was the same as described in Study 1-1.

[0993] Figure 2-3 The results show the nitrate-N concentration in the soil on day 30 of incubation, demonstrating a significant reduction in nitrate-N concentration during the urine + (4-ethynylpyrimidine dissolved in DMSO) treatment, illustrating the ability of 4-ethynylpyrimidine at 2 kg / ha to inhibit nitrification in the soil. These results show that a significant reduction in nitrification can be achieved when urine spots in the soil are treated with 4-ethynylpyrimidine at 2 kg / ha. In particular, 4-ethynylpyrimidine at only 2 kg / ha is more effective in reducing nitrification than the higher concentration of DCD at 10 kg / ha.

[0994] Study 2-3

[0995] A second laboratory incubation study was conducted to determine the efficacy of 4-ethynylpyrimidine in nitration inhibition when applied at a lower rate of 1 kg / ha. The experimental procedure was the same as described in Study 1-1.

[0996] Figure 3-3 The effect of applying 1 kg / ha of 4-ethynylpyrimidine to the soil was shown, and the nitrate-N concentration in the soil at the end of 30 days of incubation was recorded, demonstrating the highly effective inhibition of nitrification by 4-ethynylpyrimidine. As shown, the nitrate-N concentration in the soil treated with urine + 4-ethynylpyrimidine was lower compared with the urine alone or urine + DMSO control treatment.

[0997] Test 2-Ethynyl-5-methoxy Pyrimidine

[0998] Studies 1-4

[0999] Laboratory incubation studies were conducted to determine the efficacy of 2-ethynyl-5-methoxypyrimidine in nitration inhibition when applied at a rate of 2 kg / ha. The experimental procedure was the same as described in Study 1-1.

[1000] Figure 2-4 The results show the nitrate-N concentration in the soil after 30 days of incubation, demonstrating a significant reduction in nitrate-N concentration during urine + (2-ethynyl-5-methoxypyrimidine dissolved in DMSO) treatment, illustrating the ability of 2-ethynyl-5-methoxypyrimidine at 2 kg / ha to inhibit nitrification in the soil. These results indicate that a significant reduction in nitrification can be achieved when urine spots in the soil are treated with 2 kg / ha of 2-ethynyl-5-methoxypyrimidine. In particular, 2-ethynyl-5-methoxypyrimidine at only 2 kg / ha is more effective in reducing nitrification than the higher concentration of DCD at 10 kg / ha.

[1001] Studies 2-4

[1002] A second laboratory incubation study was performed to determine the efficacy of 2-ethynyl-5-methoxymirimidine in nitration inhibition when applied at the lower rate of 1 kg / ha. The experimental procedure was the same as described in Study 1-1.

[1003] Figure 3-4 The nitrate-N concentration in soil at the end of the 30 day incubation, showing the effect of applying 2-ethynyl-5-methoxymirimidine to soil at 1 kg / ha, demonstrates the high efficiency of inhibition of nitrification by 2-ethynyl-5-methoxymirimidine, as shown, the nitrate-N concentration in soil treated with urine + 2-ethynyl-5-methoxymirimidine was lower compared to the urine or urine + DMSO control treatments.

[1004] Route to 2-ethynyl-5-methoxymirimidine

[1005]

[1006] A suspension of 2-bromo-5-methoxymirimidine (1.57 g, 8.3 mmol, 1.0 eqv.), ethynyltrimethylsilane (1.8 mL, 12.5 mmol, 1.5 eqv.), triethylamine (5.8 mL, 41.5 mmol, 5.0 eqv.), bis(triphenylphosphine)palladium(II) dichloride (292 mg, 0.42 mmol, 0.05 eqv.) and copper(I) iodide (158 mg, 0.84 mmol, 0.1 eqv.) in degassed dry tetrahydrofuran (30 mL) was heated under reflux under argon for 15 hours.

[1007] The mixture was cooled to room temperature and filtered through a Celite® pad. The filtrate was concentrated in vacuo. Purification by column chromatography (petroleum ether / ethyl acetate 19:1) gave 5-methoxy-2-((trimethylsilyl)ethynyl)rimidine (FL1460) as an off-white solid (1.68 g, 98%).1H NMR (400 MHz, CDCI3) δ 0.27 (9H, s), 3.92 (3H, s), 8.34 (2H, s);13C NMR (100 MHz, CDCI3) δ -0.3 (CH3), 56.2 (CH3), 92.4 (C), 102.2 (C), 143.6 (CH), 145.3 (C), 152.5 (C).

[1008] A mixture of 5-methoxy-2-((trimethylsilyl)ethynyl)rimidine (FL1460, 1.27 g, 6.2 mmol, 1.0 eqv.) and potassium hydroxide (345 mg, 6.2 mmol, 1.0 eqv.) in methanol-water (24 mL, 5:1 v / v) was stirred at room temperature for 10 minutes.

[1009] The mixture was then diluted with ethyl acetate (100 mL) and water (100 mL). The separated aqueous layer was further extracted with ethyl acetate (2 x 50 mL), the combined organic extracts were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo.

[1010] Purification by column chromatography (petroleum ether / ethyl acetate 4:1) gave 2-ethynyl-5-methoxy- pyrimidine (HDDR326 / FL1462) as an off-white solid (720 mg, 87%). Melting point 56.2-57.2 °C;1H NMR (400 MHz, CDCI3) δ 3.01 (1 H, s), 3.90 (3 H, s), 8.32 (2 H, s);13C NMR (100 MHz, CDCI3) δ 56.2 (CH3), 74.3 (CH), 81.6 (C), 143.6 (CH), 144.6 (C), 152.8 (C); HRMS (ESI+): N / A.

[1011] Test 5-Ethynyl-2-methoxy Pyrimidine

[1012] Study 1-5

[1013] A laboratory incubation study was performed to determine the efficacy of 5-ethynyl-2-methoxy-pyrimidine in nitration inhibition when applied at a rate of 2 kg / ha. The experimental procedure was the same as described in Study 1-1.

[1014] Figure 2-5 The nitrate-N concentration in soil at the end of 30 days incubation showing the effect of applying 5-ethynyl-2-methoxy-pyrimidine to soil at 2 kg / ha, demonstrates the high efficiency of inhibition of nitrification by 5-ethynyl-2-methoxy-pyrimidine, as shown, the nitrate-N concentration in the urine + 5-ethynyl-2-methoxy-pyrimidine treated soil is lower compared to the urine or urine + DMSO control treatments alone.

[1015] Study 2-5

[1016] A second laboratory incubation study was performed to determine the efficacy of 5-ethynyl-2-methoxy-pyrimidine in nitration inhibition when applied at a lower rate of 1 kg / ha. The experimental procedure was the same as described in Study 1-5.

[1017] Figure 3-5 The nitrate-N concentration in soil at the end of 30 days incubation showing the effect of applying 5-ethynyl-2-methoxy-pyrimidine to soil at 1 kg / ha, demonstrates the high efficiency of inhibition of nitrification by 5-ethynyl-2-methoxy-pyrimidine, as shown, the nitrate-N concentration in the urine + 5-ethynyl-2-methoxy-pyrimidine treated soil is lower compared to the urine or urine + DMSO control treatments alone.

[1018] Route to 5-ethynyl-2-methoxymidazole

[1019]

[1020] A suspension of 5-bromo-2-methoxymidazole (4.72 g, 23.7 mmol, 1.0 eqv.), ethynyltrimethylsilane (4.9 mL, 35.6 mmol, 1.5 eqv.), triethylamine (16.6 mL, 118.5 mmol, 5.0 eqv.), bis(triphenylphosphine)palladium(II) dichloride (832 mg, 1.2 mmol, 0.05 eqv.) and copper(I) iodide (451 mg, 2.4 mmol, 0.1 eqv.) in degassed dry tetrahydrofuran (75 mL) was heated under reflux under argon for 15 hours.

[1021] The mixture was cooled to room temperature, filtered through a Celite® pad and the filtrate concentrated in vacuo. Purification by column chromatography (petroleum ether / ethyl acetate 49:1) gave 2-methoxy-5-((trimethylsilyl)ethynyl)imidazole (FL1457) as an off-white solid (3.97 g, 81%). The mixture was cooled to room temperature, filtered through a Celite® pad and the filtrate concentrated in vacuo. Purification by column chromatography (petroleum ether / ethyl acetate 49:1) gave 2-methoxy-5-((trimethylsilyl)ethynyl)imidazole (FL1457) as an off-white solid (3.97 g, 81%). 1 H NMR (300 MHz, CDC13) δ 0.24 (9H, s), 4.01 (3H, s), 8.56 (2H, s); 13 CNMR (75 MHz, CDC13) δ -0.1 (CH3), 55.3 (CH3), 98.1 (C), 100.1 (C), 113.0 (C), 161.9 (CH), 164.1 (C).

[1022] A mixture of 2-methoxy-5-((trimethylsilyl)ethynyl)imidazole (FL1457, 3.97 g, 23.5 mmol, 1.0 eqv.) and potassium hydroxide (1.08 g, 23.5 mmol, 1.0 eqv.) in methanol-water (96 mL, 5:1 v / v) was stirred at room temperature for 30 minutes.

[1023] The methanol was removed under reduced pressure and the crude mixture diluted with ethyl acetate (200 mL) and water (200 mL).

[1024] The separated aqueous layer was further extracted with ethyl acetate (2 x 100 mL) and the combined organic extracts washed with brine (200 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo.

[1025] Purification by column chromatography (petroleum ether / ethyl acetate 9:1) afforded 5-ethynyl-2-methoxypyrimidine ( / HDDR327 / FL1459) as a white solid (1.07 g, 34%). Melting point 82.3-83.1 °C (lit. 3 Melting point 82 °C); 1 H NMR (400 MHz, CDC13) δ 3.26 (1H, s), 4.01 (3H, s), 8.59 (2H, s); 13 C NMR (100 MHz, CDC13) δ 55.4 (CH3), 77.2 (C), 82.4 (CH), 111.9 (C), 162.1 (CH), 164.4 (C); HRMS (ESI+): N / A.

[1026] Test 2-Ethynyl-5-methoxy Pyridine

[1027] 2-Ethynyl-5-methoxypyridine is a commercially available compound, which is available from many suppliers, which can be found online using (1196155-18-6), see for example:

[1028] https: / / www.sigmaaldrich.com / catalog / product / aldrich / cds023940?lang=en®ion=NZ Figure 2-6

[1029] Study 1-6

[1030] A laboratory incubation study was performed to determine the efficacy of 2-ethynyl-5-methoxypyridine in nitration inhibition when applied at a rate of 2 kg / ha. The experimental procedure was the same as described in Study 1-1.

[1031] Figure 3-6 The nitrate-N concentration in soil at the 30th day of incubation is shown, showing that the nitrate-N concentration in the urine + (2-ethynyl-5-methoxypyridine dissolved in DMSO) treatment was significantly reduced, demonstrating the ability of 2-ethynyl-5-methoxypyridine at 2 kg / ha to inhibit nitrification in soil. These results show that a significant reduction in the rate of nitrification can be achieved when applying 2-ethynyl-5-methoxypyridine at 2 kg / ha to urine patches in soil. In particular, 2-ethynyl-5-methoxypyridine at a concentration of just 2 kg / ha was more effective in reducing the rate of nitrification than DCD at a higher concentration of 10 kg / ha.

[1032] Study 2-6

[1033] A second laboratory incubation study was performed to determine the efficacy of 2-ethynyl-5-methoxypyridine in nitration inhibition when applied at a lower rate of 1 kg / ha. The experimental procedure was the same as described in Study 1-1.

[1034] Figure 4-6 The nitrate-N concentration in soil at the end of 30 days incubation showing the effect of applying 2-ethynyl-5-methoxypyridine to soil at 1 kg / ha, illustrates the high efficiency of inhibition of nitrification by 2-ethynyl-5-methoxypyridine, as shown, the nitrate-N concentration in the urine + 2-ethynyl-5-methoxypyridine treated soil is lower compared to the urine alone or urine + DMSO control treatments.

[1035] Study 3-6

[1036] A third laboratory incubation study was conducted to determine the efficacy of 2-ethynyl-5-methoxypyridine in nitrification inhibition when applied to soil at a lower rate of 0.5 kg / ha. The experimental procedure was the same as described in Study 1-1.

[1037] Figure 5-6 The nitrate-N concentration in soil at the end of 30 days incubation showing the effect of applying 2-ethynyl-5-methoxypyridine to soil at 0.5 kg / ha, illustrates the high efficiency of inhibition of nitrification by 2-ethynyl-5-methoxypyridine at this very low application rate, as shown, the nitrate-N concentration in the urine + 2-ethynyl-5-methoxypyridine treated soil is lower compared to the urine alone or urine + DMSO control treatments.

[1038] Study 4-6

[1039] To determine the effect of treating soil with 2-ethynyl-5-methoxypyridine on nitrous oxide emissions under field soil conditions, a field study was conducted using the static chamber method. The study was conducted at the University of Lincolnshire Research Dairy Farm, the soil was a Templeton sandy loam with established ryegrass (Lolium perenne) and white clover (Trifolium repens.) pastures. A metal ring (500 mm diameter, 200 mm high) was inserted into the ground. A water trough located on top of the metal ring allowed the placement of a static chamber on top of the water trough to provide an air tight seal to allow N20 gas sampling.

[1040] Synthetic urine with a nitrogen concentration of 7 g N / L was applied to soil plots confined within the metal rings at an equivalent rate of 700 kg N / ha. 196.25 mg or 39.2 mg of 2-ethynyl-5-methoxypyridine was dissolved in 2 mL of DMSO, mixed with 1000 mL of water and then sprayed onto the small plots at a rate of 196.25 mg / plot and 39.2 mg / plot, equivalent to 10 kg / ha and 2 kg / ha respectively. The plots were irrigated with irrigation water in accordance with local dairy farming practices.

[1041] The gas chamber (diameter 500 mm, height 120 mm) was constructed from a metal cylinder insulated with 2.5 cm thick polystyrene foam to avoid a heating atmosphere in the chamber during sampling. During N20 measurements, the rim of the chamber was placed in a small water bath installed around the top of each metal ring for gas sampling. At each sampling time, the chamber was placed on top of the soil rings for a total of 40 minutes and 3 samples were withdrawn through a rubber septum on top of the gas chamber using a syringe, spaced 20 minutes apart. Sampling was performed twice a week. Each sampling was performed between 12:00 h and 14:00 h of the day (Di et al., 2007). The N20 concentration in the samples was analyzed using a gas chromatograph (SRI 8610C with an Electron Capture Detector (ECD) (SRI Instruments, USA) connected to a Gilson 222XL autosampler (Gilson, France).

[1042] Table 1-6 The effect of 2-ethynyl-5-methoxypyridine to reduce N20-N emissions was shown when 2-ethynyl-5-methoxypyridine was sprayed on the soil surface of a field with pasture that had received animal urine. When 2-ethynyl-5-methoxypyridine was applied at 10 kg / ha and 2 kg / ha, respectively, N20-N emissions in the soil of the urine + 2-ethynyl-5-methoxypyridine treatment were reduced by about 85% compared to the urine only control treatment. This shows the efficiency of 2-ethynyl-5-methoxypyridine to reduce N20 emissions in soil.

[1043] The effect of 2-ethynyl-5-methoxypyridine on nitrate concentration and nitrous oxide emissions in studies 1-4 is shown in Tables 1-6 below:

[1044] Test 5-Ethynyl-2-methoxy Pyridine

[1045]

[1046] Figure 2-7

[1047] Study 1-7

[1048] A laboratory incubation study was performed to determine the efficacy of 5-ethynyl-2-methoxypyridine in nitrification inhibition when applied at a rate of 2 kg / ha. The experimental procedure was the same as described in study 1-1.

[1049] Test 3-Ethynyl Pyridine 1-oxide The nitrate-N concentration in soil at the end of 30 days incubation showing the effect of applying 5-ethynyl-2-methoxypyridine to soil at 2 kg / ha, demonstrates the high efficiency of 5-ethynyl-2-methoxypyridine in inhibiting nitrification, as shown, the nitrate-N concentration in soil treated with urine + 5-ethynyl-2-methoxypyridine is lower compared to urine alone or urine + DMSO control treatments.

[1050] Figure 2-8

[1051] Study 1-8

[1052] A laboratory incubation study was conducted to determine the efficacy of 3-ethynylpyridine 1 -oxide in nitrification inhibition when applied at a rate of 2 kg / ha. The experimental procedure was essentially the same as described in Study 1-1.

[1053] Figure 3-8 The nitrate-N concentration in soil at the end of 30 days incubation showing the effect of applying 5-ethynyl-2-methoxypyridine to soil at 2 kg / ha, demonstrates the high efficiency of 5-ethynyl-2-methoxypyridine in inhibiting nitrification, as shown, the nitrate-N concentration in soil treated with urine + 5-ethynyl-2-methoxypyridine is lower compared to urine alone or urine + DMSO control treatments.

[1054] Study 2-8

[1055] A second laboratory incubation study was conducted to determine the efficacy of 3-ethynylpyridine 1 -oxide in nitrification inhibition when applied at a lower rate of 1 kg / ha. The experimental procedure was the same as described in Study 1-1.

[1056] Figure 4-8 The nitrate-N concentration in soil at the end of 30 days incubation showing the effect of applying 5-ethynyl-2-methoxypyridine to soil at 2 kg / ha, demonstrates the high efficiency of 5-ethynyl-2-methoxypyridine in inhibiting nitrification, as shown, the nitrate-N concentration in soil treated with urine + 5-ethynyl-2-methoxypyridine is lower compared to urine alone or urine + DMSO control treatments.

[1057] Study 3-8

[1058] A third laboratory incubation study was conducted to determine the efficacy of 3-ethynylpyridine 1 -oxide in nitrification inhibition when applied to soil at a lower rate of 0.5 kg / ha. The experimental procedure was the same as described in Study 1-1.

[1059] Figure 5-8 The nitrate-N concentration in soil at the end of 30 days incubation showing the effect of 3-ethynylpyridine 1-oxide applied at 0.5 kg / ha, illustrates the inhibition of nitrification by 3-ethynylpyridine 1-oxide at this very low application rate, as shown, the nitrate-N concentration in soil of the urine + 3-ethynylpyridine 1-oxide treatment was lower compared to the urine or urine + DMSO control treatments alone.

[1060] Study 4-8

[1061] To determine the effect of treating soil with 3-ethynylpyridine 1-oxide on nitrous oxide emissions under field soil conditions, a field study was conducted using the static chamber method. The study was conducted at the University of Lincoln’s research dairy farm, the soil was a Templeton sandy loam with established ryegrass (Lolium perenne) and white clover (Trifolium repens.) pastures. Metal rings (500 mm diameter, 200 mm high) were inserted into the ground. A water trough located on top of the metal ring allowed the placement of a static chamber located on top of the water trough to provide an air tight seal to allow N2O gas sampling.

[1062] Synthetic urine with a nitrogen concentration of 7 g N / L was applied to soil plots confined within the metal rings at an equivalent rate of 700 kg N / ha. 196.25 mg or 39.2 mg of 3-ethynylpyridine 1-oxide was dissolved in 2 mL of DMSO, mixed with 1000 mL of water and then sprayed onto the small plots at a rate of 196.25 mg / plot and 39.2 mg / plot, equivalent to 10 kg / ha and 2 kg / ha respectively. The plots were irrigated with irrigation water in accordance with local dairy farming practice.

[1063] The chamber (500 mm diameter, 120 mm high) was constructed from a metal cylinder insulated with 2.5 cm thick polystyrene foam to avoid a heating atmosphere within the chamber during sampling. The rim of the chamber was placed within a small water trough installed around the top of each metal ring for gas sampling during N2O measurements. At each sampling time, the chamber was placed on top of the soil ring for a total of 40 minutes and 3 samples were withdrawn through a rubber septum on the top of the chamber using a syringe, spaced 20 minutes apart. Sampling was carried out twice per week. Each sampling was carried out between 12:00 h and 14:00 h of the day (Di et al., 2007). The N2O concentration in the samples was analysed using a gas chromatograph (SRI 8610C with an Electron Capture Detector (ECD) (SRI Instruments, USA) connected to a Gilson 222XL auto-sampler (Gilson, France).

[1064] Table 1-8 The effect of 3-ethynylpyridine 1 -oxide on reducing N2O-N emissions is shown when 3-ethynylpyridine 1 -oxide is sprayed on the surface of a field having pasture grass that has received animal urine. N2O-N emissions in the soil of the urine + 3-ethynylpyridine 1 -oxide treatment were reduced by about 94% when 3-ethynylpyridine 1 -oxide was applied at 10 kg / ha, and by 72% when 3-ethynylpyridine 1 -oxide was applied at 2 kg / ha, compared to the urine alone control treatment. This shows the efficiency of 3-ethynylpyridine 1 -oxide in reducing N2O emissions in soil. This shows the efficiency of 3-ethynylpyridine 1 -oxide in reducing N2O emissions in soil.

[1065] The effect of 3-ethynylpyridine 1 -oxide on nitrate concentration and nitrous oxide emissions in Studies 1-4 is shown in Tables 1-8 below:

[1066] Test 2,5-Diethynyl Pyridine

[1067]

[1068]

[1069] Figure 2-9

[1070] Study 1-9

[1071] A laboratory incubation study was conducted to determine the efficacy of 2,5-diethynylpyridine in nitrification inhibition when applied at a rate of 2 kg / ha. The experimental procedure was the same as described in Study 1-1.

[1072] Test 3-Ethynyl Pyridazine The effect of applying 2,5-diethynylpyridine to soil at 2 kg / ha on nitrate-N concentration in the soil at the end of 30 days of incubation is shown, demonstrating the high efficiency of inhibition of nitrification by 2,5-diethynylpyridine, as shown, the nitrate-N concentration in the soil of the urine + 2,5-diethynylpyridine treatment was lower compared to the urine alone or urine + DMSO control treatments.

[1073] https: / / www.chemicalbook.com / ChemicalProductProperty_EN_CB72570313.htm

[1074] 3-ethynylpyridazine is a commercially available compound that can be purchased from many suppliers, which can be found online using (CAS 1017793-08-6), for example, see:

[1075] Figure 2-10 Figure 3-10

[1076] Study 1-10

[1077] A laboratory incubation study was conducted to determine the efficacy of 3-ethynylpyridazine in nitrification inhibition when applied at a rate of 2 kg / ha. The experimental procedure was the same as described in Study 1-1.

[1078] Figure 4-10 The nitrate-N concentration in the soil at the end of the 30 day incubation is shown, demonstrating that the nitrate-N concentration in the urine + (3-ethynylpyridazine dissolved in DMSO) treatment was significantly reduced, illustrating the ability of 3-ethynylpyridazine at 2 kg / ha to inhibit nitrification in the soil. These results show that a significant reduction in nitrification rate can be achieved when the urine patch in the soil is treated with 3-ethynylpyridazine at 2 kg / ha. In particular, 3-ethynylpyridazine at a concentration of just 2 kg / ha was more effective in reducing nitrification rate than DCD at a higher concentration of 10 kg / ha.

[1079] Study 2-10

[1080] A second laboratory incubation study was conducted to determine the efficacy of 3-ethynylpyridazine in nitrification inhibition when applied at a lower rate of 1 kg / ha. The experimental procedure was the same as described in Study 1-1.

[1081] Table 1-10 The nitrate-N concentration in the soil at the end of the 30 day incubation is shown, demonstrating that the nitrate-N concentration in the urine + (3-ethynylpyridazine dissolved in DMSO) treatment was significantly reduced, illustrating the ability of 3-ethynylpyridazine at 2 kg / ha to inhibit nitrification in the soil. These results show that a significant reduction in nitrification rate can be achieved when the urine patch in the soil is treated with 3-ethynylpyridazine at 2 kg / ha. In particular, 3-ethynylpyridazine at a concentration of just 2 kg / ha was more effective in reducing nitrification rate than DCD at a higher concentration of 10 kg / ha.

[1082] Study 3-10

[1083] A third laboratory incubation study was conducted to determine the efficacy of 3-ethynylpyridazine in nitrification inhibition when applied to the soil at a lower rate of 0.5 kg / ha. The experimental procedure was the same as described in Study 1-1.

[1084] Test 3-Ethynyl-6-methoxy Pyridazine The nitrate-N concentration in the soil at the end of the 30 day incubation is shown, demonstrating that the nitrate-N concentration in the urine + (3-ethynylpyridazine dissolved in DMSO) treatment was significantly reduced, illustrating the ability of 3-ethynylpyridazine at 2 kg / ha to inhibit nitrification in the soil. These results show that a significant reduction in nitrification rate can be achieved when the urine patch in the soil is treated with 3-ethynylpyridazine at 2 kg / ha. In particular, 3-ethynylpyridazine at a concentration of just 2 kg / ha was more effective in reducing nitrification rate than DCD at a higher concentration of 10 kg / ha.

[1085] The effect of 3-ethynylpyridazine on nitrate concentration in the soil in Study 1-3 is shown in Table 1-10 below:

[1086] Figure 2-11

[1087] The effect of 3-ethynylpyridazine on nitrate concentration in the soil in Study 1-3 is shown in Table 1-10 below:

[1088] Test 2-Ethynyl Pyrazine

[1089] Studies 1-11

[1090] A laboratory incubation study was conducted to determine the efficacy of 3-ethynyl-6-methoxypyridazine in nitrification inhibition when applied at rates of 2 kg / ha and 1 kg / ha. The experimental procedure was the same as described in Study 1-1.

[1091] Figure 2-12 The effect of applying 3-ethynyl-6-methoxypyridazine to soil at 2 kg / ha or 1 kg / ha on the concentration of nitrate-N in the soil at the end of 30 days incubation is shown, demonstrating the high efficiency of inhibition of nitrification by 3-ethynyl-6-methoxypyridazine, as shown, the concentration of nitrate-N in the soil of the urine + 3-ethynyl-6-methoxypyridazine treatment was lower compared to the urine or urine + DMSO control treatments.

[1092] Route to 3-ethynyl-6-methoxypyridazine

[1093]

[1094] A suspension of 3-bromo-6-methoxypyridazine (3.00 g, 15.9 mmol, 1.0 eqv.), ethynyltrimethylsilane (3.3 mL, 23.8 mmol, 1.5 eqv.), triethylamine (11.1 mL, 79.4 mmol, 5.0 eqv.), bis(triphenylphosphine)palladium(II) dichloride (557 mg, 0.79 mmol, 0.05 eqv.) and copper(I) iodide (303 mg, 1.6 mmol, 0.1 eqv.) in degassed dry tetrahydrofuran (50 mL) was heated under reflux under argon for 18 hours.

[1095] The mixture was cooled to room temperature and concentrated in vacuo. The residue was filtered and the filtrate was concentrated in vacuo.

[1096] Purification by column chromatography (petroleum ether / ethyl acetate 19:1) gave 3-methoxy-6-((trimethylsilyl)ethynyl)pyridazine (FL1521) as a brown solid (560 mg, 17%). 1 H NMR (400 MHz, CDCI3) δ 0.26 (9H, s), 4.13 (3H, s), 6.89 (1 H, d, J = 9.3 Hz), 7.42 (1 H, d, J = 8.8 Hz); 13C NMR (100 MHz, CDC13) δ -0.2 (CH3), 55.1 (CH3), 98.4 (C), 100.8 (C), 116.6 (CH), 132.6 (CH), 143.5 (C), 163.5 (C).

[1097] A mixture of 3-methoxy-6-((trimethylsilyl)ethynyl)pyridazine (FL1521, 560 mg, 2.7 mmol, 1.0 eqv.) and potassium carbonate (750 mg, 5.4 mmol, 2.0 eqv.) in diethyl ether-methanol (13.5 mL, 4:1 v / v) was stirred at room temperature for 2 hours.

[1098] The mixture was then filtered and the filtrate was diluted with diethyl ether (50 mL) and saturated aqueous ammonium chloride solution (50 mL).

[1099] The separated aqueous layer was further extracted with diethyl ether (2 x 30 mL) and the combined organic extracts were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo.

[1100] Purification by column chromatography (hexane / ethyl acetate 19:1) gave 3-ethynyl-6-methoxypyridazine (HDDR325 / FL1525) as a brown solid (260 mg, 71%). Melting point 51.7-53.1 °C; 1 H NMR (400 MHz, CDC13) δ 3.29 (1H, s), 4.12 (3H, s), 6.91 (1H, d, J = 9.4 Hz), 7.44 (1H, d, J = 9.4 Hz); 13 C NMR (100 MHz, CDC13) δ 55.2 (CH3), 80.0 (C), 80.3 (CH), 116.6 (CH), 132.6 (CH), 142.7 (C), 163.8 (C); HRMS (ESI+): [M+Na] for C7H6N2NaO + Calculated 157.0372; Found 157.0374.

[1101] Test 2-Ethynyl-5-methoxy Pyrazine

[1102] Study 1-12

[1103] A laboratory incubation study was performed to determine the efficacy of 2-ethynylpyrazine in nitrate inhibition when applied at a rate of 2 kg / ha. The experimental procedure was the same as described in Study 1-1.

[1104] Figure 2-13The results show the nitrate-N concentration in the soil on day 30 of incubation, demonstrating a significant reduction in nitrate-N concentration during the urine + (2-ethynylpyrazine dissolved in DMSO) treatment, illustrating the ability of 2-ethynylpyrazine at 2 kg / ha to inhibit nitrification in the soil. These results show that a significant reduction in nitrification rate can be achieved when 2-ethynylpyrazine is applied to urine spots in the soil at 2 kg / ha. In particular, 2-ethynylpyrazine at only 2 kg / ha is more effective in reducing nitrification rate than the higher concentration of DCD at 10 kg / ha.

[1105] Test 4-Ethynyl Anisole

[1106] Studies 1-13

[1107] Laboratory incubation studies were conducted to determine the efficacy of 2-ethynyl-5-methoxypyrazine in nitration inhibition when applied at ratios of 2 kg / ha and 1 kg / ha. The experimental procedures were the same as those described in Study 1-1.

[1108] Figure 2-14 The effects of applying 2-ethynyl-5-methoxypyrazine to the soil at 2 kg / ha or 1 kg / ha on nitrate-N concentration at the end of 30 days of incubation were shown, demonstrating the highly effective inhibition of nitrification by 2-ethynyl-5-methoxypyrazine. As shown, the nitrate-N concentration in the soil treated with urine + 2-ethynyl-5-methoxypyrazine was lower compared to the urine alone or urine + DMSO control treatment.

[1109] Towards the route for the synthesis of 2-ethynyl-5-methoxypyrazine

[1110]

[1111] A suspension of 2-bromo-5-methoxypyrazine (410 mg, 2.2 mmol, 1.0 eqv.), ethynyltrimethylsilane (0.5 mL, 3.3 mmol, 1.5 eqv.), triethylamine (1.5 mL, 11.0 mmol, 5.0 eqv.), bis(triphenylphosphine)palladium(II) dichloride (76 mg, 0.11 mmol, 0.05 eqv.), and copper(I) iodide (41 mg, 0.22 mmol, 0.1 eqv.) was stirred under argon at room temperature for 1 hour in degassed anhydrous tetrahydrofuran (8 mL).

[1112] pass The mixture was filtered and the filtrate was concentrated in vacuo. Purification by column chromatography (hexane / ethyl acetate 49:1) gave 2-methoxy-5-((trimethylsilyl)ethynyl)pyrazine (FL1528) as a pale yellow liquid (295 mg, 66%).1H NMR (400 MHz, CDCI3) δ 0.27 (9H, s), 3.97 (3H, s), 8.16 (1H, d, J = 1.5 Hz), 8.22 (1H, d, J = 1.5 Hz);13C NMR (100 MHz, CDCI3) δ -0.1 (CH3), 54.1 (CH3), 96.8 (C), 100.9 (C), 131.5 (C), 135.6 (CH), 144.4 (CH), 159.3 (C).

[1113] A mixture of 2-methoxy-5-((trimethylsilyl)ethynyl)pyrazine (FL1528, 295 mg, 1.4 mmol, 1.0 eqv.) and potassium carbonate (395 mg, 2.8 mmol, 2.0 eqv.) in diethyl ether-methanol (10 mL, 4:1 v / v) was stirred at room temperature for 3 hours.

[1114] The mixture was then filtered and the filtrate was diluted with diethyl ether (30 mL) and saturated aqueous ammonium chloride solution (30 mL).

[1115] The separated aqueous layer was further extracted with diethyl ether (2 x 15 mL) and the combined organic extracts were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo.

[1116] Purification by column chromatography (n-pentane / ethyl ether 49:1) gave 2-ethynyl-5-methoxypyrazine (HDDR328 / FL1530) as a white solid (140 mg, 73%). Melting point 41.3-42.7 °C;1H NMR (400 MHz, CDCI3) δ 3.20 (1H, s), 3.96 (3H, s), 8.16 (1H, d, J = 1.4 Hz), 8.24 (1H, d, J = 1.4 Hz);13C NMR (100 MHz, CDCI3) δ 54.1 (CH3), 79.0 (CH), 80.1 (C), 130.6 (C), 135.8 (CH), 144.5 (CH), 159.5 (C); HRMS (ESI+): N / A.

[1117] Figure 3-14

[1118] Figure 4-14The significant reduction in nitrite production by N. europaea when 4-ethynylanisole was applied at a wide concentration range of 1.56 μΜ to 400 μΜ indicates effective inhibition of the nitrification process by 4-ethynylanisole. The OD5i9 nm reading is an indicator of the nitrite concentration in the test liquid, with higher readings indicating higher nitrite concentrations.

[1119] The minimum inhibitory concentration (MIC) of 4-ethynylanisole for nitrite production was determined to be 25-50 μΜ. The IC 50 The inhibitor concentration at which nitrite production was reduced by half (IC50) was determined to be a very low value of 2.4 μΜ, indicating the high potency of 4-ethynylanisole for nitrification.

[1120] Study 1-14

[1121] A laboratory incubation study was conducted to determine the efficacy of 4-ethynylanisole for inhibiting nitrification in soil. The experiment used Templeton sandy loam soil. 500 g of soil (dry weight basis) was packed into jars. Synthetic bovine urine with an N concentration of 7 g N / L (comprising approximately 87% urea-N and 13% glycine-N) was applied to the soil (equivalent to 700 kg N / ha assuming a bulk density of 1 g / cm 3 , top 10 cm). The application of 700 kg N / ha was used to simulate the urine-N application rate under a typical patch of cow urine in a grazed pasture. 4-ethynylanisole dissolved in DMSO was applied to the soil at a range of rates equivalent to 0, 2, 5, 10 and 20 kg / ha. Control treatments also added to the soil were:

[1122] - urine alone;

[1123] - DMSO (equivalent to 0.9 mL / 500 g soil) + urine to determine the effect of DMSO on nitrification rate;

[1124] - water alone (to simulate soil areas without urine deposition).

[1125] The urine, water, DMSO and 4-ethynylanisole treatments were applied to the soil surface and the soil mixed thoroughly. The jars were covered with lids with breath holes to allow for gas exchange during incubation. The jars were incubated at 12 °C. The soil moisture content was maintained at field capacity by adjusting by weight twice per week.

[1126] Soil samples were collected and then mixed thoroughly, sub-samples were extracted with potassium chloride solute and analysed for mineral-N. The soil moisture content was also determined. Sampling was done on day 1, day 14 and day 30.

[1127] Figure 5-14 The change in soil nitrate-N concentration with time is shown for the different treatments, indicating the significant inhibition of nitrate in the soil by 4-ethynylanisole at a range of rates.

[1128] Figure 6-14 The change in soil nitrate-N concentration with time is shown for the different treatments, indicating the significant inhibition of nitrate in the soil by 4-ethynylanisole at a range of rates.

[1129] Study 2-14

[1130] To determine the effect of treating soil with 4-ethynylanisole on the rate of nitrification and nitrous oxide emissions, a soil column study in static chambers was conducted. The experiment was conducted in cylindrical PVC containers (diameter = 186 mm, height = 240 mm). The containers were filled with soil to half the height of the container (120 mm), leaving a headspace of 120 mm. The soil was Templeton sandy loam collected from the University of Lincolnshire Research Dairy Farm. It was sieved using a 5 mm sieve, then air-dried to the required moisture level and packaged to a bulk density of 1 g / cm3.

[1131] The experiment was conducted under controlled conditions. The temperature of the soil columns was maintained at approximately 12°C by using a water bath.

[1132] The treatments included control (water), control (animal urine), control (urine + DMSO), control (urine + DCD, 10 kg / ha) and 4-ethynylanisole (10 kg / ha) in urine + DMSO. Synthetic bovine urine (comprising approximately 87% urea-N and 13% glycine-N) was applied at 700 kg N / ha and 4-ethynylanisole was applied at 10 kg / ha. There were two sets of cores for each treatment, one set fitted with a removable lid static chamber for nitrous oxide sampling and the other set for soil sampling. The urine was sprayed onto the soil surface to simulate urine deposition by grazing animals; the DMSO or DCD or 4-ethynylanisole mixed with DMSO was also sprayed onto the urine-treated soil to simulate treatment of the urine patch. Nitrous oxide gas measurements were taken after these treatments - as described in further detail below.

[1133] For soil columns set up for mineral nitrogen analysis of collected soil samples, different treatments were mixed with soil for soil sampling. Soil moisture was adjusted to 100% field capacity (FC) and maintained between 80% and 100% FC during the experiment.

[1134] Nitrous oxide (N2O) gas was sampled twice weekly using a standard procedure (Di et al., 2007). The vials were evacuated before sampling. Three gas samples were collected at 20-minute intervals between each sampling. As the N2O flux decreased, the sampling frequency was reduced to once a week.

[1135] Soil samples were taken on days 1, 7, 14, 30, and 60. The mineral nitrogen in the soil samples was analyzed.

[1136] Figure 7-14 The figure shows the soil nitrate-N concentration after 60 days of incubation. The significant decrease in nitrate-N concentration observed in the urine + 4-ethynylanisole treatment demonstrates the ability of 4-ethynylanisole to inhibit nitrification in the soil. This figure indicates that 4-ethynylanisole is far more effective than DCD in inhibiting nitrate production by suppressing the nitrification process. Compared to urine treatment alone, DMSO did not result in a statistically significant reduction in nitrate concentration (P > 0.05).

[1137] Figure 1 This study demonstrates the effectiveness of 4-ethynylanisole in reducing nitrous oxide (N₂O₃) emissions when sprayed onto soil surfaces that have been treated with animal urine. The N₂O₃ emissions in the urine + 4-ethynylanisole treatment soil were reduced by 93.3% compared to the urine (control) treatment soil. This demonstrates the efficiency of 4-ethynylanisole in reducing N₂O emissions in soil. The reduction in nitrous oxide by 4-ethynylanisole (93.3%) is significantly greater than the DCD showing only a 43.4% reduction in N₂O emissions.

[1138] Study 3-14

[1139] To determine the effects of soil treatment with low ratios of 4-ethynylanisole, such as 1 kg / ha and 2 kg / ha, soil column studies were conducted. Experiments were carried out in cylindrical PVC containers (diameter = 186 mm, height = 240 mm). The containers were filled with soil to half their height (120 mm), leaving a 120 mm headspace. The soil was Templeton sandy loam collected from a research dairy farm at the University of Lincoln. It was sieved using a 5 mm sieve, then air-dried to the desired moisture level and packed to a bulk density of 1 g / cm³.

[1140] The experiment was conducted under controlled conditions. The temperature of the soil column was maintained at approximately 12°C by using a water bath.

[1141] Treatments included control (water), control (animal urine), control (urine + DMSO), control (urine + DCD, 10 kg / ha), 4-ethynylanisole in urine + DMSO (1 kg / ha) and 4-ethynylanisole in urine + DMSO (2 kg / ha). Synthetic cattle urine (comprising approximately 87% urea-N and 13% glycine-N) was applied at 700 kg N / ha and 4-ethynylanisole was applied at 1 or 2 kg / ha equivalent. Treatments were mixed with soil. Soil moisture was adjusted to 100% field capacity (FC) and maintained between 80% and 100% FC during the experiment. Soil sampling was performed at days 1, 7, 14, 30 and 60. Mineral N was analysed in soil samples.

[1142] Figure 8A-14 Soil nitrate-N concentrations at day 60 of incubation are shown, showing a significant reduction in nitrate-N concentration in urine + 4-ethynylanisole at 1 or 2 kg / ha, demonstrating the ability of 4-ethynylanisole at these low rates to inhibit nitrification in soil. This graph shows that 4-ethynylanisole is much more effective at inhibiting nitrate production by inhibiting the nitrification process than DCD applied at the higher rate of 10 kg / ha.

[1143] Study 4-14

[1144] In addition, the present inventors have also determined the oxygen consumption of 4-ethynylanisole to inhibit ammonium-driven metabolism, but not hydroxylamine-driven metabolism (see Figure 8B-14 ).

[1145] To begin to understand the mechanism of action of 4-ethynylanisole, oxygen consumption rates were measured using an Oroborus oxygraphy-2k (Oroboros Instruments). Prior to the experiment, P. europium cells were grown in ATCC 2265 media for four days. Thereafter, whole cells of P. europium were washed thoroughly as previously described and resuspended in ATCC 2265 wash buffer (without ammonium) to an OD 600 of 0.25. Prior to the start of each experiment with the addition of a metabolisable energy source (ammonium or hydroxylamine), the test suspension (2 ml of cells at an OD 600Oxygen concentration was allowed to stabilize for 2-3 minutes, until a stable signal was obtained. Under normal experimental conditions, this usually resulted in an oxygen concentration of approximately 220 mM. After the oxygen concentration in the Oroboros oxygraphy-2k reaction cell had stabilized, a rubber septum was subsequently inserted to close the system. To initiate respiration, ammonium sulfate (12.5 mM) or hydroxylamine (6.25 mM) was injected directly into the washed cell suspension of N. europaea. Once a stable oxygen consumption rate was achieved in the presence of ammonium or hydroxylamine, 4-ethynylanisole was injected through the rubber septum at the indicated concentrations. All measurements were performed at 25 °C with stirring at 750 rpm and a data recording interval of 1 s -1 .

[1146] Test 1-Ethoxy-4-ethynyl Benzene It is shown that in the presence of 4-ethynylanisole, the ammonium-driven oxygen consumption is reduced to zero, while the hydroxylamine (H3NO)-driven oxygen consumption is not affected by 4-ethynylanisole Figure 2-15 . This indicates that the ammonia monooxygenase (AMO) of ammonia-oxidizing bacteria is the potential inhibitory target of 4-ethynylanisole, rather than the hydroxylamine oxidoreductase (see above equations 2 and 3).

[1147] Investigations 5-14

[1148] In addition, the present inventors have also identified that 4-ethynylanisole does not inhibit other bacteria found in soil.

[1149] To determine whether 4-ethynylanisole specifically targets the ammonium-oxidizing bacterium N. europaea, we examined the antibacterial properties of 4-ethynylanisole against selected bacterial species. Bacteria were grown in lysogeny broth (LB) (E. coli and B. subtilis), LB-Tween (M. smegmatis), or Rhizobium-determined medium (G RDM) (M. loti R7A). E. coli, B. subtilis, and M. smegmatis were grown overnight (18 hours) at 37 °C with shaking (200 rpm), while M. loti R7A was grown statically at 28 °C for 48 hours. The 96-well microtiter plates for antibacterial susceptibility testing were set up as follows: 200 mΐ of medium (or as appropriate) was added to column 1 (A-H), and 100 mΐ of medium was added to the remaining wells of the polystyrene 96-well plate. 4-ethynylanisole was added at a concentration of 512 mM (or as required) to column 1 and serially diluted 2-fold (100 mΐ transfer) into adjacent wells, ensuring that 100 mΐ was discarded from the last well. This resulted in a serial dilution of each compound. Prior to adding 100 mΐ of culture to each well of the MIC plate, the overnight bacterial culture was diluted in fresh medium to achieve ~5 x 105 CFU / ml. Plates were incubated at 37°C or 28°C (shaking as required) for 18-48 hours prior to determining MIC. The MIC was determined as the lowest concentration where no growth occurred.

[1150] The results show that at the concentrations of 4-ethynylanisole used to inhibit nitrification in soil to reduce nitrate leaching and nitrous oxide emissions, 4-ethynylanisole does not inhibit other soil bacteria such as Mycobacterium smegmatis, Escherichia coli, Bacillus subtilis and Mesorhizobium loti R7A.

[1151] Test 1,4-Diethynyl Benzene

[1152] Study 1-15

[1153] A laboratory incubation study was conducted to determine the efficacy of 1-ethoxy-4-ethynylbenzene in nitrification inhibition when applied at a rate of 2 kg / ha. The experimental procedure was the same as described in Study 1-1.

[1154] Figure 2-16 The nitrate-N concentration in soil at day 30 of incubation is shown, demonstrating that the nitrate-N concentration in the urine + (1-ethoxy-4-ethynylbenzene dissolved in DMSO) treatment was significantly reduced, demonstrating the ability of 1-ethoxy-4-ethynylbenzene at 2 kg / ha to inhibit nitrification in soil. These results indicate that a significant reduction in nitrification rate can be achieved when a urine patch in soil is treated with 1-ethoxy-4-ethynylbenzene at 2 kg / ha. In particular, 1-ethoxy-4-ethynylbenzene at just 2 kg / ha concentration was more effective in reducing nitrification rate than DCD at a higher concentration of 10 kg / ha.

[1155] ​

[1156] Study 1-16

[1157] A laboratory incubation study was conducted to determine the efficacy of 1,4-diethynylbenzene in nitrification inhibition when applied at a rate of 2 kg / ha. The experimental procedure was the same as described in Study 1-1.

[1158] ​ The nitrate-N concentration in the soil at day 30 of incubation is shown, showing that the nitrate-N concentration in the urine + (1,4-diethynylbenzene dissolved in DMSO) treatment was significantly reduced, demonstrating the ability of 1,4-diethynylbenzene at 2 kg / ha to inhibit nitrification in the soil. These results indicate that a significant reduction in nitrification rate can be achieved when applying 1,4-diethynylbenzene at 2 kg / ha to treat urine patches in soil. In particular, 1,4-diethynylbenzene at just 2 kg / ha concentration was more effective in reducing nitrification rate than DCD at a higher concentration of 10 kg / ha.

[1159] Further description of the invention and ways in which it can be implemented

[1160] Preparation of the NNI according to the invention can include direct packaging into bulk storage / transport containers at the site of preparation.

[1161] Furthermore, the packaged amount of NNI can correspond to the dose required for a particular size of application area. For example, g / m 2 or kg / ha.

[1162] In some embodiments, the storage / transport container can include instructions on how to apply to the land / plant area in kg / ha amounts (or other amounts greater than 200g). The instructions can include information on the amount / type of solvent / solute to provide the required application dose and preferred method of application.

[1163] For areas of about half an acre (i.e. about 2023m 2 ) or less, urban use amounts can typically be sold in 200g amounts. For example, selling such amounts can be useful for lawns (such as parks) that require nitrogen fertiliser to be applied at a particular time of year.

[1164] The NNI can be sold in dissolvable bags or containers to facilitate ready delivery. The invention can also be said broadly to include the parts, elements and features referred to or indicated in the specification of this application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features.

[1165] Importantly, the present inventors have found that the NNI can be delivered to the soil using an aqueous medium (such as water, DMSO or effluent) as a carrier in which the nitrification inhibitor is dissolved and / or suspended. For example, the NNI can simply be sprayed on the surface of soil that has or will receive animal urine, or added to a flowing liquid stream for application to the soil.

[1166] Alternatively, the NNI can be mixed with or coated onto a nitrogen fertiliser, which is then co-applied to the soil, as is known in the art.

[1167] Alternatively, in another example, the NNI can be added in dissolvable bags containing kg / ha dosages for addition to a water source (such as an irrigation source or effluent pond), mixed with a pond pump, and then delivered when the pond is used to irrigate a farm.

[1168] Alternatively, in another example, the NNI can be added to a solution using a dosing pump to provide kg / ha dosages for addition to a water source (such as an irrigation source or effluent pond), mixed with a pond agitator or pump, and then delivered when the pond is used to irrigate a farm.

[1169] Thus, the NNI described herein truly represents an alternative to DCD, which is one of the most widely commercially used known nitrification inhibitors in the world.

[1170] The soil can be treated (i.e. impregnated) with the NNI in any convenient manner, taking into account the nature of the particular NNI and / or the area of soil to be treated.

[1171] For example, depending on the nature of the NNI (or formulation comprising the NNI), it can be:

[1172] - mixed with the topsoil machinery;

[1173] - applied to the surface of the soil;

[1174] - applied to the surface of the soil and then physically introduced / mixed with the topsoil (e.g. ploughed / disced or otherwise moved / mixed into the soil or washed into the soil by irrigation water);

[1175] - coated on a fertilizer granule and co-applied to the soil / pasture;

[1176] - sprayed directly on the land to be treated;

[1177] - introduced into irrigation water;

[1178] - granulated and delivered to the soil area by land or aerial hopper devices or the like;

[1179] - delivered specifically to urine patches or specific areas of soil (e.g. paddocks) shortly after animals have grazed on them as part of a rotational grazing program.

[1180] However, the above list of how the NNI can be applied to treat the soil should not be seen as limiting.

[1181] By way of example, if the NNI is applied directly to the pasture, the grass is cut or grazed to have an average height of about 40mm to 50mm. After application, the treated pasture requires rain or irrigation, ideally within 24 hours to 48 hours, to wash the NNI off the grass and into the topsoil.

[1182] Advantages of the present invention can also include one or more of the following:

[1183] - preparation and handling safety;

[1184] - provision of an alternative nitrification inhibitor to DCD;

[1185] - ability to be formulated into a solution or suspension formulation for direct spraying onto soil / land / plants;

[1186] - improved nitrification efficacy at significantly lower kg / ha dose compared to DCD;

[1187] - ability to be co-applied with fertilisers;

[1188] - ability to be co-applied with irrigation water;

[1189] - ability to be co-applied with effluent;

[1190] - ability to vary the dose to meet specific regulatory targets relating to nitrate leaching or nitrous oxide emissions;

[1191] - ability to reduce the dose and still be at least as effective as DCD;

[1192] - ability to reduce N20 emissions more than DCD;

[1193] - ability to reduce NO3 - leaching more than DCD;

[1194] - ability to be delivered through an aqueous carrier;

[1195] - ability to be applied through an agricultural spray vehicle or autonomous robot;

[1196] - ability to be granulated and delivered through a hopper;

[1197] - ability to be mechanically mixed with soil;

[1198] - ability to coat fertilizer granules.

[1199] It will be appreciated that the above list is not intended to be limiting and that other advantages can also be inherent in the present invention and aspects thereof detailed herein.

[1200] The present invention can also be said broadly to include the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features.

[1201] It should be noted that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the application and without diminishing its attendant advantages.

[1202] Aspects of the application have been described by way of example only and it should be appreciated that modifications and additions can be made thereto without departing from the scope thereof as defined in the appended claims.

[1203] References

[1204] Dai, Y., Di, H. J., Cameron, K. C. and He, J. Z. (2013). Effects of nitrogen application rate and a nitrification inhibitor dicyandiamide on ammonia oxidizers and N2O emissions in a grazed pasture soil. Science of the Total Environment. 465: 125-135.

[1205] Di, Hong Jie and Cameron, Keith C. (2016). Inhibition of nitrification to mitigate nitrate leaching and nitrous oxide emissions in grazed grassland: a review. J Soils Sediments (2016) 16: 1401-1420.

[1206] Di HJ, Cameron KC (2002) The use of a nitrification inhibitor, dicyandiamide (DCD), to reduce nitrate leaching and nitrous oxide emissions in a simulated grazed and irrigated grassland. Soil Use Manag 18: 395-403.

[1207] Di, H. J., Cameron, K. C. and R. R. Sherlock (2007). Comparison of the effectiveness of a nitrification inhibitor, dicyandiamide (DCD), in reducing nitrous oxide emissions in four different soils under different climatic and management conditions. Soil Use and Management 23: 1-9.

[1208] De Klein, C, Cameron, K. C, Di, H. J., Rys, G., Monaghan, R. and Sherlock, R. R. (2011). The effect of long-term use of the nitrification inhibitor DCD on reducing N2O emissions from cow urine. Animal Feed Science and Technology 166-167, 480-491.

[1209] Griess, P. (1879). Bemerkungen zu der abhandlung der H. H. Weselsky und Benedikt "Ueber einige azoverbindungen." Chem. Ber. 12, 426-8.

[1210] Tisdale, Samuel L.; Nelson, Werner L.; Beaton, James D. (1985), Soil fertility and fertilizers, New York: Macmillan, pp. 161-168, ISBN 0-02-420830-2.

Claims

1. The use of the compound of formula 1 as a nitration inhibitor, in, X 1 and X 5 = N, X 2 and X 4 = CH, and X 3 = CR, where R is selected from H and OMe; or Among them, X 1 and X 5 = CH, X 2 and X 4 = N, and X 3 = CR, where R is selected from H and OMe; or Among them, X 1 and X 3 = N, and X 2 X 4 and X 5 = CH; or Among them, X 1 X 4 and X 5 = CH, X 2 Selected from N and N=O, and X 3 = CR, and where, if X 2 If N=O, then R is H, and where X 2 If N is true, then R is OMe; or Among them, X 1 = N, X 2 X 4 and X 5 = CH, and X 3 = CR, where R is OMe; or Among them, X 1 and X 2 = N, X 3 = CR, and X 4 and X 5 = CH, where R is selected from H and OMe; or Among them, X 1 and X 4 = N, X 2 and X 5 = CH, and X 3 = CR, where R is selected from H and OMe; or Among them, X 1 X 2 X 4 and X 5 = CH, X 3 = CR, where R is selected from OMe, OEt, and .

2. The use according to claim 1, wherein, The compound is selected from the group consisting of: - 2-ethynyl-1,3-diazine; - 3-ethynyl-1,5-diazine; - 4-ethynylpyrimidine; - 2-ethynyl-5-methoxypyrimidine; - 5-ethynyl-2-methoxypyrimidine; - 2-ethynyl-5-methoxypyridine; - 5-ethynyl-2-methoxypyridine; - 3-ethynylpyridine 1-oxide; - 3-ethynylpyridazine; - 3-ethynyl-6-methoxypyridazine; - 2-ethynylpyrazine; - 2-ethynyl-5-methoxypyrazine; - 4-ethynyl anisole; - 1-Ethoxy-4-ethynylbenzene; - 1,4-Diethynylbenzene; It also has a dosage rate between 1 kg / ha and 9 kg / ha.

3. The use according to claim 1 or 2, wherein, The compound has a dosage rate of 2 kg / ha.

4. The use according to claim 1 or 2, wherein, The compound is selected from the group consisting of: - 2-ethynyl-1,3-diazine; - 3-ethynyl-1,5-diazine; - 4-ethynylpyrimidine; - 2-ethynyl-5-methoxypyrimidine; - 5-ethynyl-2-methoxypyrimidine; - 2-ethynyl-5-methoxypyridine; - 3-ethynylpyridine 1-oxide; - 3-ethynylpyridazine; - 3-ethynyl-6-methoxypyridazine; - 2-ethynyl-5-methoxypyrazine; - 4-ethynyl anisole; It also has a dosage rate of 1 kg / ha.

5. The use according to claim 1, wherein, The compound is selected from the group consisting of: - 2-ethynyl-1,3-diazine; - 3-ethynyl-1,5-diazine; - 2-ethynyl-5-methoxypyridine; - 3-ethynylpyridine 1-oxide; - 3-ethynylpyridazine; It also has a dosage rate of 0.5 kg / ha.

6. A method for reducing nitrification rate in agricultural soils, the method characterized by the following steps: a) Using at least one compound selected from claim 1 as a nitration inhibitor; b) Prepare a nitration inhibitor from the compound of formula 1 as defined in claim 1; For use in applying directly or indirectly to the soil.

7. The method according to claim 6, wherein, Indirect application to the soil includes applying the compound to the soil via a carrier.

8. The method according to claim 7, wherein, The carrier is water or an effluent.

9. The method according to claim 7, wherein, The carrier is fertilizer granules.

10. The method according to claim 9, wherein, The fertilizer granules are urea.

11. The method according to claim 6, used to reduce nitrate leaching or nitrous oxide emissions.

12. The method according to claim 6 is also used to increase the yield of pasture or crops.

13. The method according to claim 6, wherein, The compound is selected from the group consisting of: - 2-ethynyl-1,3-diazine; - 3-ethynyl-1,5-diazine; - 4-ethynylpyrimidine; - 2-ethynyl-5-methoxypyrimidine; - 5-ethynyl-2-methoxypyrimidine; - 2-ethynyl-5-methoxypyridine; - 5-ethynyl-2-methoxypyridine; - 3-ethynylpyridine 1-oxide; - 3-ethynylpyridazine; - 3-ethynyl-6-methoxypyridazine; - 2-ethynylpyrazine; - 2-ethynyl-5-methoxypyrazine; - 4-ethynyl anisole; - 1-Ethoxy-4-ethynylbenzene; - 1,4-Diethynylbenzene; It also has a dosage rate between 1 kg / ha and 9 kg / ha.

14. The method according to claim 6 or 13, wherein, The compound has a dosage rate of 2 kg / ha.

15. The method according to claim 6 or 13, wherein, The compound is selected from the group consisting of: - 2-ethynyl-1,3-diazine; - 3-ethynyl-1,5-diazine; - 4-ethynylpyrimidine; - 2-ethynyl-5-methoxypyrimidine; - 5-ethynyl-2-methoxypyrimidine; - 2-ethynyl-5-methoxypyridine; - 3-ethynylpyridine 1-oxide; - 3-ethynylpyridazine; - 3-ethynyl-6-methoxypyridazine; - 2-ethynyl-5-methoxypyrazine; - 4-ethynyl anisole; It also has a dosage rate of 1 kg / ha.

16. The method according to claim 6, wherein, The compound is selected from the group consisting of: - 2-ethynyl-1,3-diazine; - 3-ethynyl-1,5-diazine; - 2-ethynyl-5-methoxypyridine; - 3-ethynylpyridine 1-oxide; - 3-ethynylpyridazine; It also has a dosage rate of 0.5 kg / ha.

17. A method of treating urine stains, comprising the step of applying a compound of formula 1 as defined in claim 1 to the urine stains.

18. The method according to claim 17, wherein, The compound is applied to the urine stains by an agricultural sprayer or an autonomous robot.

19. The method according to claim 18, wherein, The agricultural spraying vehicle or autonomous robot is equipped with a device for detecting urine stains.

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