1 Global budget of major nitrification inhibitors (1980~2015) 2 3 Yong Zhang (yongzhang.eco@outlook.com)1, Diego Abalos (d.abalos@agro.au.dk)2, 4 Xiaoli Cheng (xlcheng@ynu.edu.cn)1,3* 5 6 1 7 School of Ecology and Environmental Science, Yunnan University, Kunming, China 8 2 Department of Agroecology, Land-CRAFT, Aarhus University, Tjele, Denmark 9 3 Yunnan Key Laboratory of Soil Erosion Prevention and Green Development, Yunnan Ministry of Education Key Laboratory for Transboundary Ecosecurity of Southwest China, 10 University, Kunming, China 11 * Corresponding author. Email: xlcheng@ynu.edu.cn 12 13 ORCID: 14 Yong Zhang, https://orcid.org/0000-0002-1181-032X 15 Diego Abalos, https://orcid.org/0000-0002-4189-5563 16 Xiaoli Cheng, https://orcid.org/0000-0002-0346-675X 17 The three most important nitrification inhibitors (NIs) applied worldwide are 18 Dicyandiamide (DCD), 3,4-Dimethylpyrazole-phosphate (DMPP), and 2-Chloro-6- 19 (trichloromethyl)-pyridine (Nitrapyrin) [1,2]. To assess the environmental fate of these 20 NIs, a top-down model was developed to make a rough estimate of their global budget: 21 3 𝑌𝑗,𝑡 = ∑ 𝑁𝑡 ⋅ 𝐴𝑡 ⋅ 𝐵𝑖,𝑡 ⋅ (𝑊𝑖 ± 𝜀𝑖 ) ⋅ 𝐹𝑖,𝑗,𝑡 ⋅ 1000 𝑖=1 22 where i, j, and t denote the species of NIs use (i = 1 to 3; i.e., DCD, DMPP, and 23 Nitrapyrin), the composition of NIs budget (j = 1 to 3; i.e., transport/degradation in 24 biosphere, residue in soil, and leaching/runoff from soil), and the year (t = 1980 to 2015), 25 respectively. Yj,t indicates the annual budget (Gg) of j. Other symbols: Nt, the amount 26 (Tg) of N-fertilizers application; At, the percent (mass %) of N-fertilizers containing NIs; 27 Bi,t, the share (mass %) of each NI use; Wi, the mixing ratio (mass %) of each NI to N- 28 fertilizers; εi, the standard deviation (mass %) of Wi; Fi,j,t, the fraction (mass %) of 29 different NI-flows for i. Please see details on the parameters in Tables 1~4. The results for 30 global budget of major NIs (1980~2015) have been showed in Figure 1. 31 Table 1. The percent of nitrogen (N) fertilizers containing nitrification inhibitors (NIs). Percent (%) of N-fertilizers Code Query Number of records N-fertilizers #1 192,958 NA NIs #2 5,497 2.85 containing NIs 32 Notes: The percent of N-fertilizers containing NIs was estimated by the observation- 33 based occurrences approach (according to probability theory), which is commonly used 34 for global biodiversity estimations [3-6]. The literature search was performed at the Web 35 of Science (2023-12-18) by using the following search terms: 36 Query #1: TS = ("nitrogen" AND "fertili*") and Preprint Citation Index (Exclude – Database) 37 Query #2: TS = (“nitrification” AND “inhibitor*”) and Preprint Citation Index (Exclude – Database) 38 Table 2. The share of each nitrification inhibitor (NI) use. Number of Share (%) of each records NI use #1 9,743 61.62 DMPP #2 1,883 11.91 Nitrapyrin #3 508 3.21 Others (#5) NOT (#6) 3,678 23.26 All DCD + DMPP + Nitrapyrin + Others 15,812 100 Code Query DCD 39 Notes: We focused on three major NIs worldwide: Dicyandiamide (DCD), 3,4- 40 Dimethylpyrazole-phosphate (DMPP), and 2-Chloro-6-(trichloromethyl)-pyridine 41 (Nitrapyrin) [1,2]. The share of each NI use was estimated by the observation-based 42 occurrences approach (according to probability theory), which is commonly used for 43 global biodiversity estimations [3-6]. The literature search was performed at the Web of 44 Science (2023-12-18) by using the following search terms: 45 Query #1: TS = (“dicyandiamide”) and Preprint Citation Index (Exclude – Database) 46 Query #2: TS = (“DMPP”) and Preprint Citation Index (Exclude – Database) 47 Query #3: TS = (“nitrapyrin”) and Preprint Citation Index (Exclude – Database) 48 Query #4: TS = (“nitrification” AND “inhibitor*”) and Preprint Citation Index (Exclude – Database) 49 Query #5: #1 OR #2 OR #3 OR #4 and Preprint Citation Index (Exclude – Database) 50 Query #6: #1 OR #2 OR #3 and Preprint Citation Index (Exclude – Database) 51 Table 3. The parameters used for nitrification inhibitors (NIs) in the top-down model. Fraction of different NI-flows Percent of NSpecies of fertilizers NIs use containing NIs (A) Share of each NI use (B) Mixing ratio (F) after 30 days of NIs use of each NI to Transport and N-fertilizers degradation in (W) biosphere (j = 1) Unit Residue in soil (j = 2) Leaching and runoff from soil (j = 3) mass % DCD 61.62 3.4 ± 1.1 48.7 44.3 7.0 (i = 1) (Table 2) [7] [ 8] [ 8] [ 9] DMPP 2.85 11.91 1.2 ± 0.4 69.8 30.2 0 (i = 2) (Table 1) (Table 2) [7] [10] [10] [11] Nitrapyrin 3.21 0.65 ± 0.15 85.7 14.3 0 (i = 3) (Table 2) [12] [13] [13] [11] 52 Notes: Where i and j denote the species of NIs use (i = 1 to 3; i.e., DCD, DMPP, and 53 Nitrapyrin), and the composition of NIs budget (j = 1 to 3; i.e., transport/degradation in 54 biosphere, residue in soil, and leaching/runoff from soil), respectively. DCD, 55 Dicyandiamide; DMPP, 3,4-Dimethylpyrazole-phosphate; Nitrapyrin, 2-Chloro-6- 56 (trichloromethyl)-pyridine. To be comparable for the test data of different NIs, we 57 specified the test duration (i.e., 30 days, because this value is available for test 58 experiments of DCD, DMPP, and Nitrapyrin [8,10,13]). The fraction of different NI-flows 59 (after 30 days of NIs use) was estimated by experimental test data based on 60 recommended doses for DCD (15.4 mg kg soil-1) [8], DMPP (1.54 mg kg soil-1) [10], and 61 Nitrapyrin (2 mg kg soil-1) [2,13]. The DCD was subject to leaching/runoff (7%) [9,14] 62 while no leaching/runoff of DMPP and Nitrapyrin was observed in lysimeter experiments 63 [11], thus the leaching/runoff for DMPP and Nitrapyrin was set to 0. 64 Table 4. The parameters used for nitrogen (N) fertilizers [15] in the top-down model. Year Amount of N-fertilizers application Year Amount of N-fertilizers application (t; unit: yr) (N; unit: Tg) (t; unit: yr) (N; unit: Tg) 1980 95.33 1998 127.81 1981 96.24 1999 129.74 1982 97.23 2000 126.66 1983 101.54 2001 128.84 1984 106.95 2002 130.02 1985 107.90 2003 135.96 1986 109.72 2004 138.50 1987 113.54 2005 141.45 1988 117.75 2006 143.09 1989 118.57 2007 148.05 1990 117.15 2008 149.46 1991 116.13 2009 151.84 1992 114.60 2010 158.70 1993 113.07 2011 161.71 1994 115.10 2012 167.88 1995 121.14 2013 173.48 1996 125.56 2014 177.30 1997 125.10 2015 178.50 65 Notes: The three nitrification inhibitors (DCD, DMPP, and Nitrapyrin) were registered 66 for use before 1980s [2], thus data on global use of N-fertilizers after 1980 were selected 67 to match that on nitrification inhibitors. DCD, Dicyandiamide; DMPP, 3,4- 68 Dimethylpyrazole-phosphate; Nitrapyrin, 2-Chloro-6-(trichloromethyl)-pyridine. 69 Table 5. Experimental evidences for side-effects of major synthetic nitrification inhibitors (SNIs). SNIs Side-effects on Description Refer ence “However, the use of nitrification inhibitors is not free of side-effects. Some may be absorbed by the plant and cause phytotoxicity or even affect the food chain”. Plants “The use of high DCD concentrations affected the leaf chlorophyll content and plant growth”. “The action of dicyandiamide is found to be toxic to germination of seed in beans and of growth generally in other plants such as flax”. “DCD decreased plant dry weight and leaf chlorophyll concentration”. DCD [16] [17] [18] “Dicyandiamide (DCD), considered to be a nitrification inhibitor, poses threat to human’s health with exposure from milk, infant formula and other food products”. Animals [19] “The pepsin interacted with DCD at a hydrophobic cavity, leading to a conformational changes in the pepsin”. “Multisystemic granulomatous and haemorrhagic syndrome resembling cell-mediated hypersensitivity, associated with DCD ingestion”. [20] “DMPP translocates to leaves and high doses may cause visual phytotoxicity symptoms”. Plants “When DMPP accumulates in leaves it induces stress responses, notably provoking changes in cell redox [21] balance, hormone signaling, protein synthesis and turnover and carbon and nitrogen metabolism”. “DMPP only provoked detrimental effects in plants at very high dose”. [22] “DMPP, DMOP, and DMMP commonly inhibited the total reproduction while stimulated lifespan”. “DMPP, DMOP, and DMMP caused different regulations on the expressions of vab-1, ceh-18, and gsa-1 which DMPP are involved in oocyte growth, ovulation, and maturation. Meanwhile, they commonly downregulated those of Animals [23] rme-2 and rcy-4 that regulate germ cells and gonads and lipid metabolism in oocytes”. “Reproductive toxicities of DMPs depended on the exposure life stages and also the chemical structures”. “Exposure to DMPP caused the lethality at the L2/L3 molt, and this toxicity was concentration-dependent”. “These observations imply the potential sensitivity of L2/L3 molting time to certain environmental toxicants or stresses in nematodes”. [24] Microbes “DMPP application increased the number of colony forming units of various fungi (off-target microbes)”. [25] “DMPP imposed significant changes in the composition of fungal communities (off-target microbes)”. [10] “Nitrapyrin toxicity appeared as a curling of leaf margin and a tendril type of stem growth”. “Toxicity of Nitrapyrin to sunflower under different N nutrition regimes”. Plants Nitrapyrin “Nitrapyrin toxicity appeared as leaf chlorosis in cow peas, and interveinal chlorosis in chick peas”. “A suppression in plant growth was associated with Nitrapyrin application”. Microbes [27] “Visual symptoms of Nitrapyrin toxicity appeared as leaf curling and tendril type of stem growth”. [28] “According to Nitrapyrin labels, it has an acute toxicity to aquatic invertebrates and algae/aquatic plants”. [29] “Therefore, the data support the conclusion that Nitrapyrin-induced liver tumors in female mice are mediated Animals [26] via a CAR MoA”. [30] “According to Nitrapyrin labels, it has an acute and chronic toxicity to fish”. [29] “Nitrapyrin inhibited growth, CH4 oxidation (involved in off-target microbes)”. [31] “Piadin and Vizura (two commercial NIs) showed ecotoxic effects throughout all experiments”. Plants Microbes 71 [32] species L. gibba and on the root development of several terrestrial plant species”. Others 70 “All findings indicate ecotoxic effects of Piadin and Vizura (two commercial NIs), especially on the aquatic “QI (quinone imine, a derivate from ethoxyquin) induced significant changes (off-target microbes) in the composition of the bacterial and fungal communities”. [33] 72 73 Figure 1. Global budget of major NIs (after 30 days of NIs use). Shading area indicates 74 standard deviations of top-down estimated NI-flows. 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. FAO (2023) Food Safety Implications from the Use of Environmental Inhibitors in Agrifood Systems (1st edn), FAO. https://doi.org/10.4060/cc8647en Wiley-VCH (2011) Fertilizers: 4.5. Nitrification and urease inhibitors. In Ullmann's Encyclopedia of Industrial Chemistry (7th edn), Wiley-VCH Heberling, J.M. et al. (2021) Data integration enables global biodiversity synthesis. Proceedings of the National Academy of Sciences 118, e2018093118. https://doi.org/10.1073/pnas.2018093118 Tamme, R. et al. (2021) Global macroecology of nitrogen-fixing plants. Global Ecology and Biogeography 30, 514-526. https://doi.org/10.1111/geb.13236 Guralnick, R.P. et al. (2007) Towards a collaborative, global infrastructure for biodiversity assessment. Ecology Letters 10, 663-672. https://doi.org/10.1111/j.14610248.2007.01063.x Sahr, K. et al. (2003) Geodesic discrete global grid systems. Cartography and Geographic Information Science 30, 121-134. https://doi.org/10.1559/152304003100011090 Byrne, M.P. et al. (2020) Urease and nitrification inhibitors—as mitigation tools for greenhouse gas emissions in sustainable dairy systems: A review. Sustainability 12, 6018. https://doi.org/10.3390/su12156018 Papadopoulou, E.S. et al. (2022) The effects of quinone imine, a new potent nitrification inhibitor, Dicyandiamide, and Nitrapyrin on target and off-target soil microbiota. Microbiology Spectrum 10, 1-17. https://doi.org/10.1128/spectrum.02403-21 Cahalan, E. et al. (2015) The effect of precipitation and application rate on dicyandiamide persistence and efficiency in two Irish grassland soils. Soil Use and Management 31, 367-374. https://doi.org/10.1111/sum.12194 Bachtsevani, E. et al. (2021) Effects of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on the activity and diversity of the soil microbial community under contrasting soil pH. Biology and Fertility of Soils 57, 1117-1135. https://doi.org/10.1007/s00374-021-01602-z Barth, G. et al. (2001) Influence of soil parameters on the effect of 3,4-dimethylpyrazolephosphate as a nitrification inhibitor. Biology and Fertility of Soils 34, 98-102. https://doi.org/10.1007/s003740100382 Degenhardt, R.F. et al. (2016) Application of nitrapyrin with banded urea, urea ammonium nitrate, and ammonia delays nitrification and reduces nitrogen loss in Canadian soils. Crop, Forage & Turfgrass Management 2, 1-11. https://doi.org/10.2134/cftm2016.03.0027 Rakhi, R. et al. (2019) Dissipation of nitrapyrin (nitrification inhibitor) in subtropical soils. Polish Journal of Soil Science 52, 173-182. https://doi.org/10.17951/pjss/2019.52.2.173 Matthaei, C. et al. (2014) Enduser Guide: The effects of dicyandiamide (DCD) leaching and runoff on aquatic environments (1 edn), University of Otago. https://doi.org/10.13140/RG.2.1.4227.7843/1 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. Zhang, X. et al. (2021) Quantification of global and national nitrogen budgets for crop production. Nature Food 2, 529-540. https://doi.org/10.1038/s43016-021-00318-5 Padash, A. et al. (2022) Use of symbiotic fungi to reduce the phytotoxic effect of DCD nitrification inhibitors in lettuce. Agriculture 12, 251. https://doi.org/10.3390/agriculture12020251 Petrosini, G. (1947) Tests on the toxicity of Dicyandiamide in plant nutrition. Bollettino della Societa italiana di biologia sperimentale 23, 570-572 Reeves, D.W. and Touchton, J.T. (1986) Relative phytotoxicity of Dicyandiamide and availability of its nitrogen to cotton, corn, and grain sorghum. Soil Science Society of America Journal 50, 1353-1357. https://doi.org/10.2136/sssaj1986.03615995005000050054x Yue, Y. et al. (2017) Probing the binding properties of dicyandiamide with pepsin by spectroscopy and docking methods. Chemosphere 185, 1056-1062. https://doi.org/10.1016/j.chemosphere.2017.07.115 Cayzer, J. et al. (2018) Systemic granulomatous disease in dairy cattle during a dicyandiamide feeding trial. New Zealand Veterinary Journal 66, 108-113. https://doi.org/10.1080/00480169.2017.1412840 Rodrigues, J.M. et al. (2019) Multi-omic and physiologic approach to understand Lotus japonicus response upon exposure to 3,4 dimethylpyrazole phosphate nitrification inhibitor. Science of The Total Environment 660, 1201-1209. https://doi.org/10.1016/j.scitotenv.2019.01.047 Rodrigues, J.M. et al. (2018) 3,4-Dimethylpyrazole phosphate and 2-(N-3,4-dimethyl-1Hpyrazol-1-yl) succinic acid isomeric mixture nitrification inhibitors: Quantification in plant tissues and toxicity assays. Science of The Total Environment 624, 1180-1186. https://doi.org/10.1016/j.scitotenv.2017.12.241 Zhang, J. et al. (2024) Reproductive toxicities of dimethyl phthalates on Caenorhabditis elegans with alteration in responses over life stages. Land Degradation & Development 35, 2635-2642. https://doi.org/10.1002/ldr.5087 Wang, D. (2020) Exposure stages of environmental toxicants or stresses. In Exposure Toxicology in Caenorhabditis elegans (Wang, D., ed), pp. 23-39, Springer Ruiz, C.A. et al. (2018) Evaluation of in vitro effect of DMPP (3, 4-Dimethylpyrazole phosphate) on fungal microbiota isolated from a soil. ITEA 114, 2-16. https://doi.org/10.12706/itea.2018.001 Maftoun, M. et al. (1982) Toxicity of nitrapyrin to sunflower under different N nutrition regimes. Plant and Soil 65, 411-414. https://doi.org/10.1007/BF02375061 Maftoun, M. et al. (1981) Comparative phytotoxicity of nitrapyrin and ATC to several leguminous species. Plant and Soil 63, 303-306. https://doi.org/10.1007/BF02374610 Maftoun, M. and Sheibany, B. (1979) Comparative phytotoxicity of several nitrification inhibitors to soybean plants. Journal of Agricultural and Food Chemistry 27, 1365-1368. https://doi.org/10.1021/jf60226a013 Brickler, C.A. (2020). Innovative Soil Additives to Mitigate Agricultural Runoff 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 30. 31. 32. 33. Contamination and Nitrous Oxide Emissions. Florida Agricultural and Mechanical University Murphy, L. et al. (2021) Bridging sex-specific differences in the CAR-mediated hepatocarcinogenesis of Nitrapyrin using molecular and apical endpoints. Frontiers in Toxicology 3. https://doi.org/10.3389/ftox.2021.766196 Topp, E. and Knowles, R. (1984) Effects of Nitrapyrin [2-Chloro-6-(Trichloromethyl) Pyridine] on the Obligate Methanotroph Methylosinus trichosporium OB3b. Applied and Environmental Microbiology 47, 258-262. https://doi.org/10.1128/aem.47.2.258262.1984 Kösler, J.E. et al. (2019) Evaluating the ecotoxicity of nitrification inhibitors using terrestrial and aquatic test organisms. Environmental Sciences Europe 31, 91. https://doi.org/10.1186/s12302-019-0272-3 Papadopoulou Evangelia, S. et al. (2022) The effects of quinone imine, a new potent nitrification inhibitor, Dicyandiamide, and Nitrapyrin on target and off-target soil microbiota. Microbiology Spectrum 10, e02403-02421. https://doi.org/10.1128/spectrum.02403-21