Biol Fertil Soils (1996) 22:196-21 9 Springer-Verlag 1996 L. Vittori Antisari 9 C. Marzadori 9 P. Gioacchini S. Ricci 9 C. Gessa Effects of the urease inhibitor N-(n-butyl)phosphorothioic triamide in low concentrations on ammonia volatilization and evolution of mineral nitrogen Received: 1 March 1995 Abstract Laboratory incubation experiments were conducted to study the influence of increasing concentrations of N-(n-butyl)phosphorothioic triamide (NBPT) on NH3 volatilization and rate of urea hydrolysis and evolution of mineral N in Ozzano, and soils with different physicochemical characteristics. Low concentrations of NBPT reduced NH3 losses due to volatilization after urea fertilization and the effectiveness of the inhibitor was related to the soil characteristics (e.g. high concentrations of organic C and sand). After 15 days of incubation, no significant reductions of losses were found for any of the NBPT concentrations employed in soil. The application of NBPT led to a considerable reduction of the formation of nitrite. This process was completely annulled with the highest dose of NBPT (.5% w/wurea) in the soil after 15 days. n soil, however, the use of NBPT was less effective in influencing nitrite formation. The use of NBPT favoured accumulation of nitrate proportional to the NBPT concentration employed while it had no influence on the NH~ fixation by 2:1 layer silicates. The data obtained support previous evidence that NBPT is effective in reducing the problems encountered in using urea as fertilizer. However, environmental conditions and soil physicochemical characteristics may have an important influence on the effectiveness of NBPT. Key words Urease inhibitors - N-(n-butyl)thiophosphorothioic triamide 9 Urea hydrolysis 9 Ammonia volatilization 9 Nitrite accumulation L. Vittori Antisari (~) 9 C. Marzadori 9 R Gioacchini - S. Ricci C. Gessa nstituto di Chimica Agraria, Universit~t di Bologna, Via S. Giacomo, 7, 1-4126 Bologna, taly ntroduction The rapid conversion of urea to ammonia and carbon dioxide by urease in soil leads to an increase in ph and accumulation of N H 4 + and NO2, if the oxidation of nitrite to nitrate by Nitrobacter sp. is inhibited (Bremner and Chai 1989). Ammonia accumulation can cause NH3 losses by volatilization and these losses can exceed 5% of the applied fertilizer N (Terman 1979). Nitrite accumulation can be toxic for plants and soil microflora (Bremner and Chai 1989), and may result in losses of gaseous N by chemical denitrification; these problems may render urea less efficient than ammonia fertilizers (Hauck 1984). n order to reduce the problems associated with the use of urea as a fertilizer, attention has been focused on compounds that are able to retard urea hydrolysis. These compounds are applied to soil, together with the fertilizer, in low concentrations with respect to the rate of the fertilizer applications. Many organic and inorganic compounds have been tested as inhibitors of soil urease (Mulvaney and Bremner 1981). Test results have shown that N-(n-butyl)phosphorothioic trianaide (NBPT) is one of the more efficient inhibitors of urea hydrolysis, and its use reduces NH 3 losses due to volatilization (Carmona et al. 199). Little is known concerning the factors controlling NBPT effectiveness. Christianson et al. (199) have reported that NBPT is not an active urease inhibitor but must be converted in the soil to its oxygen analog N-(n-butyl)phosphoric triamide (BNPO), which is the actual urease inhibitor. The distribution of the urea N in the various N pools of soil is a problem related to the rationalization of nitrogen fertilizer applications (Nelson et al. 198; Mengel et al. 1982); for this reason it is important to have a better understanding of the influence of various concentrations of NBPT on the transformations of urea-n that may take place. The objectives of this work were to study, under laboratory conditions, the effect of increasing concentrations of NBPT on NH3 volatilization, rate of urea hydrolysis, accumulation of NO~, NO~, exchangeable NH~, and interlayer NH~, in different soils treated with urea N.
197 Materials and methods The soils (Ozzano, and, according to the sites in taly from which they were collected) were superficially sampled (- 2 cm), air dried and sieved (<2-mm screen). The physicochemical properties of these soils are reported in Table 1. The urease inhibitor N-(n-butyl)phosphorotioic triamide (NBPT) was added with urea fertilizer to give.1%,.5% and.1% (w/w,rea), for the Ozzano and soils, and.5%,.1% and.5% (w/wurst) for the soil NBPT-urea mixtures, respectively. The urea-nbpt mixtures were prepared by dissolving appropriate amounts of urea and NBPT in methanol, drying the methanol solution in an oven and then grinding the resulting material to pass a.2-mm screen (Carmona et al. 199). Soil (1 g dry wt.) was placed into cylindrical glass containers (6 cm in diameter and 9 cm high), brought to 5% of the water-holding capacity and incubated for 15 days at 23~ to stabilize the microbial activity of the soil. After this preincubation, either 1 mg urea N g a soil (control) or NBPT urea in the established concentrations [.t-.5% (W/Wurea)] were applied to the soil. Statistical analyses of inorganic nitrogen were carried out using a personal computer and STATGRAPHCS 4. software. Analysis of variance to evaluate the effect of the increasing NBPT concentrations was carried out using ANOVA and applying the LSD test at the 95% probability level. Results and discussion NH3 volatilization and urea hydrolysis The N losses due to volatilization of NH3 (expressed as a percentage of the urea-n applied) after the application of the urea-nbpt to soils are reported in Fig. 1. n agreement with the results of Carmona et al. (199), these losses are generally lower in the presence than in the ab- NH3 volatilization 1 Ozza~o The NH3 loss by volatilization was determined periodically (1, 3, 6, 9, 12, and 15 days after the fertilizer applications), following the method reported by Zhengping et al. (1991). The glass cylinders containing the treated soils were hermetically connected to a line of compressed air. The compressed air, provided by a commercial compressor, was humidified and purified from any NH3 before entering the gas-exchange chamber. The resulting air flow was sufficient to change the air in the chamber above the soil at the rate of 5 times min n. The volatilized NH3 was collected in 2 ml 2% boric acid. Two repetitions were carried out for each treatment. Analyses of mineral nitrogen Treated soil (5 g) was placed in plastic containers (3 cm in diameter and 5 cm high) and stored in a thermostatted chamber at 23~ and 1% relative humidity. The air in the thermostat was changed once a day in order to avoid saturation of the atmosphere with volatilized NH3. Periodically (1, 3, 6, 9, 12 and 15 days after the urea-nbpt applications) for each treatment, three containers were removed and the soil samples were extracted with 1 N KCl-phenyl mercuric acetate to block the urease activity, making it possible to determine the residual urea of soil as reported by Mulvaney and Bremner (1979). Exchangeable NH~ and NO2+NO3 were determined in the extract of 2 M KC1 by steam distillation in the presence of MgO and MgO-Devarda's alloy, respectively (Keeney and Nelson 1982). Nitrite in the 2 M KC1 extract was determined by the colorimetric method as described by Brernner (1965). Determination of interlayer NH~ was carried out as reported by Silva and Bremner (1966). Analysis of soil properties (Table 1) were canied out according to the Metodi Ufficiali di Analisi Chimica del Suolo (Ministero delle Risorse Agricole, Alimentari e Forestali 1994), and the mineralogical composition of the clay fraction was determined as reported by Whitting (1965). Analysis of urease activity was carried out according to Tabatabai (1982). 8 6 4 2 1 N ~ so > 6 4 ~; 2 25 1 8 6 4 2 2 4 6 8 1 12 14 2 4 6 8 1 12 14 /---"~J ~ ~.... 2 4 6 8 1 12 14 Fig. 1 Cumulative N losses due to NH3 volatilization with respect to the amount of urea-n applied to soil with and without NBPT. Ozzano and soils: urea alone O, NBPT.1% @. NBPT.5% V, NBPT.1% V, soil: urea alone O, NBPT.5% V, NBPT.1% V, NBPT.5% [] jv Table 1 Some characteristics of soils selected Location ph Sand Clay of soil (H2) (%) (%) Type of clay mineral (% of total clay) Kaolinite llite Smectite Cloriti Organic Urease Total N nterlayer carbon activity 1 (mg kg ~) N-NH+4 (%) (fig N g- ) Ozzano 8.2 59 14 1 55 15 2 8.5 81 8 33 67 Capri 8. 21 35 1 2 6 1 1.53 62.4 748 54.2 2.3 178.6 2287 2.3 1.76 159.9 1599 151.5
198 sence of NBPT; after 9 days of incubation the N losses by NH3 volatilization in the Ozzano, and soils treated with urea alone were 38.5%, 57.%, and 49.7%, respectively; the corresponding values for the soils treated with.5% NBPT were 2.6%, 48.3%, and 24.5%. n Ozzano soil the.1% NBPT treatment had little effect (N loss of 31.8%, compared with 38.5% for urea alone), while it markedly differed with respect to the.5% NBPT treatment (31.8% vs 2.6%); a small difference was observed between the.1% and the.5% NBPT treatments (18.% vs 2.6%). n contrast the N losses of the soil due to N-NH3 volatilization in the presence of.1%,.5% and.1% NBPT were nearly the same (5.2%, 48.3%, and 44.%, respectively). After 15 days of incubation, the N losses of the.1% NBPT Ozzano soil were 45.8% as compared to the 52.% for the control. Only the.5%, and.1% NBPT treatments led to a iarge reduction in ammonia losses (33.1%, and 3.2%, respectively, vs 52.% for the control). n soil after 15 days of incubation, the NH3 losses were greater than those in Ozzano soil; however, small reductions in the N losses were observed among the NBPT treatments (6.4% for the control vs 58.1%, 54.5%, and 5.8% for.1%,.5%, and.1% NBPT, respectively). Not all the soils, therefore, responded in the same way to the addition of NBPT at low concentrations with urea fertilizer. Carmona et al. (199) showed that the presence of high percentages of organic carbon in soil reduced the effectiveness of NBPT. t has also been shown that NH3 volatilization losses are greater in sandy soils (Fenn and Hossner 1985). Since the soil is rich in both organic matter and sand (Table 1), the poor effectiveness of NBPT in this soil can probably be attributed to its particular physicochemical characteristics. n the soil, therefore, it can be expected that high NBPT concentrations are required to reduce NH3 losses by volatilization. n soil the.1% NBPT concentration was not employed because it was judged to be too low to be effective and it was therefore replaced by a higher NBPT concentration (.5%). n soil, after 15 days of incubation, the N losses by volatilization for the.5% NBPT treatment were extremely low (23.2%) and significant reductions were also obtained with.5% and.1% NBPT treatments (45.7% and 39.3%, respectively) as compared to the control (59.4%). nhibition of urea hydrolysis by NBPT increases with increasing amounts of the applied inhibitor and decreases with the incubation period (Fig. 2). The degradation of urea, in the absence of inhibitor, was more or less complete after 3-5 days and was faster in than in Ozzano and soils. ndeed, urease activity of soil was greater than that of Ozzano and soils (Table 1). n the latter soil treated with the.5% NBPT, the percentage of urea hydrolysed after 1 and 15 days was.% and 79.5%, respectively (Fig. 2), while in the soil treated with.5% NBPT the values were 3% and 87%, respectively (not shown). 1~ 8 6 4 2 ~" 1i ~; 8 ~d 6 qj 4(3 2 ~ Ozzano 2 4 6 8 1 12 14 2 4 6 8 1 12 14,,. _ ~, - - n - - - - ~ i ~ 2 4 6 8 1 12 14 Fig. 2 Residual urea-n as percentage of the total amount applied to soil. For explanation of symbols, see Fig. 1 Evolution of mineral N The effects of the NBPT concentrations (.1%,.5%, and.1% in Ozzano and soils;.5%,.1%, and.5% in soil) on exchangeable NH~, NO~ and NO~ are shown in Figs. 3, 4 and 5, respectively. n the absence of the inhibitor, exchangeable NH2 accumulates in the soil after 3-6 days to reach concentrations as high as 5 mg NH~, and the presence of NBPT slows down the formation of exchangeable NH~, due to the inhibition of urea hydrolysis (Fig. 3). Up to 9 days, there was little difference in the values of exchangeable NH~ in Ozzano soil treated with.1% and.5% NBPT. For longer incubation times, the values of exchangeable NH~ tend to differ (Fig. 3). n soil similar values of exchangeable NH~ were obtained at.1% and.5% NBPT up to 3 days; differences were then observed and they increased up to 9 days and then decreased. n the.1% NBPT treated soil, the amount of exchangeable NH~ increased throughout. However, at the end of the incubation period the same concentrations of exchangeable NH~ were found in all three NBPT treatments. After 6 days of incubation, the.5% and.1% NBPT treated soil and the respective control showed similar values of exchangeable NH~. The amount of exchangeable N-NH~ in the.5% NBPT soil was very low compared with the other inhibitor treatments due to a low rate of urea hydrolysis.
199 6 Ozzano 5 Ozzeno / ~ 2 4 1 O 2 v 6 2 4 6 8 1 12 14 t~ t~ 3 2 2 4 6 8 1 12 14 Z Z 4 1 N F-...q 2 6 2 4 6 8 1 12 14 Z 3 2 2 4 6 8 1 12 14 loo /~ ~ 2OO / _ 2 4 6 8 1 12 14 Fig. 3 Effect of NBPT concentration on the amount of exchangeable NH~ in Ozzano, and soils. For explantation of symbols, see Fig. 1 2 4 6 8 12 14 Fig. 4 Effect of NBPT concentration on the amount of nitrite in Ozzano, and soils. For explanation of symbols, see Fig. 1 1 8 Ozzano X n and Ozzano soils, the NO[ concentration was the highest when urea alone was applied, while lower values were found in the presence of NBPT also when it was applied at the lowest rate,.1% (Fig. 4). ndeed the oxidation of NO[ to NO3 is inhibited by the accumulation of NH3 and the consequent increase in ph (Schmidt 1982). n both soils, NBPT retarded the urea hydrolysis and, controlling the ammonia concentration in soil, decreased the NO[ concentration (Bremner and Chai 1989). n soil, after 15 days of incubation, high values of NO[ were observed both in the presence and in the absence of NBPT (Fig. 4). The accumulation of NO[ during the period of experiment confirms the lower efficiency of NBPT in this kind of soil. After 15 days of incubation the amount of NO[ in the.5% and.1% NBPT treatments was significantly lower than in the control, but this reduction was smaller than in Ozzano and soils. The NO[ concentration in the soil was similar in all treatments up to 12 days. After this period, however, the values of NO~ increased with increasing NBPT concentration, and in Ozzano and soils higher values were found in NBPT-treated soil than in the control. Christianson et al. (1993) indicated that the nitrification process was proceeding more rapidly in the zone of the lowest NH~ concentration. n Fig. 6 is reported the variation of interlayer NH~ during the incubation period. soil 6 4 2 ~ 3,-.M 24 18 12 2; 6 5 i r r t r ; i 2 4 6 8 1 12 14 i F 2 4 6 8 1 12 14 4 /~ 3 2 1 r ~2-2 4 6 8 1 12 14 Fig. 5 Effect of NBFF concentration on the amount of nitrate in Ozzano, and soils. For explanation of symbols, see Fig. 1 T i
2 + 2 12 8 4O 5 4 Z 3 i z 2O >~ 1 3 Ozzarlo 9 2 4 6 8 1 12 14 ~imini 2 4 6 8 1 12 14 The fixation in the interlayer silicates of the soils probably did not influence the exchangeable NH~ concentration. ndeed at the end of the incubation period the same amount of interlayer NH~ was found for the different treatments in each soil studied. This behaviour in all three soils may depend on the presence of fillosilicates characterized by low fixing capacity, as shown by their mineralogical composition (Table 1). Also the microbial immobilization could influence the exchangeable NH~ at the end of the incubation period. Hendrickson et al. (1987) found that urease inhibitor totally prevented the apparent immobilization of urea for 7 days, then N immobilization proceeded at a rate comparable to that without inhibitor. NBPT has a considerable inhibitory effect on urea hydrolysis and influences the formation of NO2, NO3 and exchangeable N H 4, + but its efficiency also depends on the physicochemical characteristics of the soil. ndeed the soils with high organic C content need higher rates of NBPT to reduce the urea hydrolysis and NH3 volatilization. 25O 2 15 1 Acknowledgements Work was carried out under a contract between the Ministry of Universities and Scientific and Technological Research and Enichem Agricoltura SPA as part of National Research Program for Advanced Biotechnologies. 1 i f f i i i J 2 4 6 8 1 12 14 Fig. 6 Effect of NBPT concentration on the amount of interlayer ammonium in Ozzano, and soils. For explanation of symbols, see Fig. 1 was not able to fix large amounts of N H + 4. n this soil the presence of clays characterized by low fixing capacity did not lead to significant variations of interlayer NH~ during the incubation period in any treatment. n Ozzano and soils the maximum value of interlayer NH~ was reached after 3-9 days of incubation in the presence of urea alone. No particular differences were found when different rates of the inhibitor were applied to these soils. n conclusion these data emphasize that NBPT influences the formation and final concentration of NO2 and NO~ in soil. ndeed the highest concentration of NBPT produced a significant decrease in NO2 and a significant increase in NO~ in all soils employed after 15 days of incubation. Therefore the combined application of urea and NBPT to the soil can reduce the toxic effect due to the NO~ accumulation, but, on the other hand, it could cause groundwater pollution because of the NO3 leaching. Thus it is suggested that NBPT be used with one nitrification inhibitor. The interpretation of exchangeable NH~ data is more complicated because, at the end of the incubation period in the and soils, the greatest amount of exchangeable NH~ was found in the control, whereas in Ozzano soil it was found with the highest NBPT rate. However, the concentration of exchangeable NH~ can be influenced by several factors such as volatilization, which was greater in the presence of urea alone, microbial immobilization and fixation into organic form and interlayer silicates. References Bremner JM (1965) norganic forms of nitrogen. n: Black CA, et al (eds) Methods of soil analysis, part 2. American Society of Agronomy- SSSA, Madison, pp 1179-1237 Bremner JM, Chai HS (1989) Effects of phosphoroamides on ammonia volatilization and nitrite accumulation in soils treated with urea. Biol Fertil Soils 8:227-23 Carmona G, Christianson CB, Bymes BD (199) Temperature and low concentration effects on the urease inhibitor N(n-butyl)thiophosphoric triamide (nbtpt) on ammonia volatilization from urea. Soil Biol Biochem 22:933-937 Christianson CB, Byrnes BH, Carmona G (199) A comparison of the sulfur and oxygen analogs of phosphoric triamide urease inhibitors in reducing urea hydrolysis and ammonia volatilization. Fert Res 26:21-27 Christianson CB, Baethgen WE, Carmona G, Howard RG (1993) Microsite reactions of urea-nbtpt fertilizer on the soil surface. Soil Biol Biochem 25:117-1117 Fenn LL, Hossner HS (1985) Ammonia volatilization from ammonium or ammonium forming nitrogen fertilizers. Adv Soil Sci 1:123-9 Hauk RD (1984) Technical approaches to improving the efficiency of nitrogen fertilizer use by crop plants. n: Hauck RD (ed) Nitrogen in crop production. American Society of Agronomy - SSSA, Madison, pp 551-56 Hendrickson LL, Omholt TE, O'Connor MJ (1987) Effect of phenylphosphorodiamidate on immobilization and ammonia volatilization. Soil Sci Soc Am 51:7-171 Keeney DR, Nelson DW (1982) Nitrogen inorganic forms. n: Page AL, et al (eds) Methods of soil analysis, part 2. American Society of Agronomy - SSSA, Madison, pp 643-698 Mengel DB, Nelson DW, Huber DM (1982) Placement of nitrogen fertilizers for no-till and conventional com. Agron J 74:515-518 Ministero delle Risorse Agricole, Alimentari e Forestali (1994) Metodi ufficiali di analisi chimica del suolo con cormnenti e interpretazioni, pp 1-152 Mulvany RL, Bremner JM (1979) A modified diacetyl monoxime method for colorimetric determination of urea in soil extracts. Commun Soil Sci Plant Anal 1:13 117
21 Mulvaney RL, Bremner JM (1981) Control of urea transformations in soils. n: Paul EA, Ladd JN (eds) Soil biochemistry, voi 5. Marcel Decker, New York, pp 153-196 Nelson KE, Turgeon A, Street JR (198) Thatch influence on mobility and transformation of nitrogen carriers applied to turf. Agron J 72:487-492 Schmidt EL (1982) Nitrification in soil. n: Stevenson FJ (ed) Nitrogen in agricultural soils. American Society of Agronomy - SSSA, Madison, pp 253-288 Silva JA, Bremner JM (1966) Determination and isotope-ratio analysis of different forms of nitrogen in soil, 5. Fixed ammonium. Soil Sci Soc Am J 3:587-594 Tabatabai MA (1982) Soil enzymes. n: Page AL (ads) Methods of soil analysis, part 2, 2nd edn. Agronomy 9. American Society of Agronomy-SSSA, Madison, pp 93-947 Terman GL (1979) Volatilization losses of nitrogen as ammonia from surface-applied fertilizers, organic amendments, and crop residues. Adv Agron 31:189-223 Zhengping W, Van Cleemput O, Boert L (1991) Effect of urease inhibitors on urea hydrolysis and ammonia volatilization. Biol Fertil Soils 11:43-47 Whitting LD (1965) X-ray diffraction technique for mineral identification and mineralogical composition. n: Black CA, et al. (eds) Methods of soil analysis, part 1. Agronomy 9, American Society of Agronomy-SSSA, Madison, pp 671-698