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DIVISION S-3-SOIL BIOLOGY & BIOCHEMISTRY

Effect of Long-Term, Biennial, Fall-Applied Anhydrous Ammonia and Nitrapyrin on Soil Nitrification

^a Dep. of Biological Sciences, 1392 Lily Hall of Life Science, Purdue Univ., West Lafayette, IN 47907-1392 USA
^b Dep. of Plants, Soils, and Biometeorology, Utah State Univ., Logan, UT 84322-4820 USA

jennyn{at}cc.usu.edu

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

 
Long-term dryland wheat plots were established in northwestern Utah and maintained for 8 yr in a 2-yr wheat-fallow rotation. Nitrapyrin [2-chloro-6-(trichloromethyl)pyrindine] was applied with anhydrous ammonia (NH₃) in the fall preceding wheat growth to retard nitrification. Our objective was to determine the effects of long-term, biennial application of anhydrous NH₃ with and without nitrapyrin on soil nitrification. We were particularly interested in the potential residual effects of the long-term repeated applications of anhydrous NH₃ and nitrapyrin. Nitrification potentials were measured in control (no added N) soil, or soil fertilized with anhydrous NH₃ with or without nitrapyrin for both rotation phases. Nitrification potentials were higher in soils receiving anhydrous NH₃ than in the control soil during the cropped rotation. Nitrification potentials in soils receiving anhydrous NH₃ with nitrapyrin were similar to those of the control soils during the entire wheat fallow rotation period. Further, nitrification potentials in soils with a history of nitrapyrin use were significantly lower than in soils without nitrapyrin use when measured after 2 yr. We observed a transient increase in nitrification potentials with the application of anhydrous NH₃ that did not last in the fallow year, suggesting that the long-term, biennial application of anhydrous NH₃ at a rate of 50 kg ha^-1 had no residual effect on soil nitrifier population size. In contrast, our results suggest that the long-term, biennial application of nitrapyrin did have a residual effect on soil nitrifier populations that lasted at least 2 yr.

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

 
AMMONIUM-BASED NITROGEN FERTILIZERS combined with nitrification inhibitors are commonly applied to winter wheat (Triticum aestivum L.) in the fall. Anhydrous NH₃, a major N fertilizer source, has been widely applied with nitrapyrin, a nitrification inhibitor, in winter wheat in the northwestern USA (Papendick and Engibous, 1980). Nitrapyrin retards nitrification (Keeney, 1986), therefore, the applied NH⁺₄-plNH⁺₄-based N will be retained in NH⁺₄ form, which is less susceptible to loss by leaching or denitrification than NO^-₃. It is expected that N fertilizer use efficiency and crop yields will be increased for systems treated with nitrapyrin. The potential for long-term, repeated use of anhydrous NH₃ with nitrapyrin to have a residual effect on soil nitrification has not previously been investigated.

Since nitrapyrin was first introduced in 1962 by C.A.I. Goring of The Dow Chemical Company, its inhibition of nitrification has been extensively tested in laboratory and field experiments (Briggs, 1975; Gomes and Loynachan, 1984; Powell and Prosser, 1986; Sahrawat et al., 1987; McCarty and Bremner, 1990; Walters and Malzer, 1990). Factors affecting the efficacy of nitrapyrin and other nitrification inhibitors have been reviewed by Keeney (1980, 1986). The general belief about nitrapyrin and other nitrification inhibitors is that their inhibition of nitrification is short term, usually lasting for a few days to a few months (Briggs, 1975; Gomes and Loynachan, 1984; Malhi and Nyborg, 1988; McCarty and Bremner, 1990; Glasscock et al., 1995; Rochester et al., 1996). The functional period of nitrapyrin depends on its bioactivity and persistence in soil; these are related to soil type, organic matter content, temperature, moisture, and soil management practice (Keeney, 1980, 1986). Once nitrification inhibitors are degraded, the nitrification rate may recover. Because the persistence and the efficacy of nitrification inhibitors are interrelated, the degradation of nitrification inhibitors has also been studied. The reported half life of nitrapyrin ranges from less than 2 to 13 wk (Keeney, 1986). In contrast to the above observation with nitrapyrin, Klemedtsson and Mosier (1994) reported that long-term exposure of soil to acetylene, a nitrification inhibitor, had a long lasting effect on soil nitrification. They observed that the nitrification potential of the soil that was exposed to acetylene was lower than that of the control soil even 1 yr later.

Autotrophic ammonium oxidizers get their metabolic energy solely from the oxidation of NH⁺₄ to NO^-₂. Nitrification rate and nitrifier populations respond to NH⁺₄ availability (Belser, 1979). The short-term effect of NH⁺₄ substrate concentration on increased nitrification rate and nitrifier populations has been studied in the laboratory (Darrah et al., 1985; Nishio and Fujimoto, 1990). However, relatively few studies (Eaton and Patriquin, 1988; Biederbeck et al., 1996) have documented the residual effect of long-term application of NH⁺₄-based fertilizers on soil nitrification. We have used a long-term dryland wheat experiment to investigate the residual effect of the repeated use of anhydrous NH₃ and nitrapyrin on soil nitrification.

Generally, NO^-₃ or NH⁺₄ pool sizes are used to evaluate the effects and efficacy of nitrification inhibitors. The assumption is that if nitrification inhibitors block nitrification, the NH⁺₄ pool size will be larger or the NO^-₃ pool size will be smaller in soils treated with nitrification inhibitors than in those without nitrification inhibitors. Therefore, two general indices to evaluate nitrification inhibitors are: (i) the percentage of difference of NH⁺₄– or NO^-₃–N pool size between soils with or without a nitrification inhibitor in relation to the NH⁺₄– or NO^-₃–N pool size of the respective control soil (McCarty and Bremner, 1990; Goos and Johnson, 1992), and (ii) the recovery of applied NH⁺₄–N in soil (Gomes and Loynachan, 1984; Zourarakis and Killorn, 1990). However, the effect of NH⁺₄ substrate concentrations cannot be differentiated from those due to changes in the nitrifier population by using the indices of NH⁺₄– or NO^-₃–N pool sizes, because the changes of these pool sizes are the confounded effects of both NH⁺₄ availability and nitrifer population activity. Long-term residual effects of anhydrous NH₃ and nitrapyrin on soil nitrifiers need to be investigated by isolating the effect of NH⁺₄ substrate concentrations. In this study, we used nitrification potential as an index to evaluate a long-term residual effect of anhydrous NH₃ and nitrapyrin on soil nitrification.

The aim of this study was to test if the long-term (8 yr), biennial, fall-applied anhydrous NH₃ and nitrapyrin have residual effects on soil nitrification. We compared soils that were untreated (control) and treated with anhydrous NH₃ or anhydrous NH₃ plus nitrapyrin. The NH⁺₄– and NO^-₃–N pool sizes were used to evaluate short-term effects of anhydrous NH₃ and nitrapyrin. Nitrification potentials and nitrifier sensitivity to nitrapyrin were used to evaluate long-term, residual effects of anhydrous NH₃ and nitrapyrin.

	Materials and methods

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The experimental site was located at the Blue Creek Farm of Utah State University in northwestern Utah. The soil is Timpanogos silt loam (fine-loamy, mixed, superactive, mesic Calcic Argixeroll). Average annual precipitation was 381 mm. Average annual temperature was 7.4°C (Utah Climate Center, Logan, UT).

Experimental Design
The experiment was set up in the late 1980s for testing the effects of fall-applied anhydrous NH₃ with nitrapyrin on winter wheat yields. The experiment involved a 2-yr, wheat-fallow rotation and was carried out in two fields. Field I was cropped, and Field II was fallow. Next year, Field I was fallow, and Field II was cropped. Each field involved 14 treatments, which were arranged as a randomized complete block design with two replications. We only sampled the soil from the treatments as follows: (i) Control, without application of anhydrous NH₃ or nitrapyrin; (ii) 50A, 56 kg N ha^-1 of anhydrous NH₃; (iii) 50AN, 56 kg N ha^-1 of anhydrous NH₃ plus 0.56 kg nitrapyrin ha^-1; and (iv) 70AN, 79 kg N ha^-1 of anhydrous NH₃ plus 0.56 kg nitrapyrin ha^-1. The treatment 70AN was changed to 70A (79 kg N ha^-1 of anhydrous NH₃) in fall 1994. The plots for each treatment were 4 m wide and 180 m long. Anhydrous NH₃ with or without nitrapyrin was contained in a pressurized tank and injected into soil in bands 30 cm apart and 8 to 10 cm deep by an applicator equipped with banding knife shanks. During each cropping year, soil was tilled three or four times to less than a 15-cm depth.

The study was conducted in the fields from 1995 to 1997. The dates of fertilization, planting, harvesting, and soil samplings are given in Table 1 . The plots were divided into four sub-plots along their lengths with each about 90 m long to stratify sampling. Soil was collected by coring (5-cm diam.) from both 0- to 15-cm and 15- to 30-cm depths in each sub-plot. Four cores were located randomly within each subplot. Tillage decreased the localization of the bands of NH₃ and nitrapyrin except for soil samplings in the fall immediately after the fertilizer application. Therefore, no attempt was made to locate the application bands when collecting soil cores.

Nitrification potentials were measured by the soil shaken slurry method (Hart et al., 1994). Fresh soils were sieved (<2 mm) and 15 g of moist soils were weighed into 250-mL flasks. The flasks received 100 mL of phosphate buffer (1 mM potassium phosphate, pH 7.2) and were continuously shaken for 24 h at the high speed (200 RPM) (Stark, 1996). Ten-milliliter aliquots were sampled at 2, 4, 22, and 24 h and centrifuged at 8000 x g for 10 min. The (NO^-₃+NO^-₂)–N in the liquid was analyzed colorimetrically as described above. Soil nitrification potential was expressed on soil dry weight basis.

Nitrifier sensitivity to nitrapyrin was determined by a modified nitrification potential method. The soils were sampled from 0- to 15-cm depth on 2 Oct. 1996, from the 50A and 50AN treatments in the fallow field. After the shaken soil slurries were sampled at 3, 6, and 18 h, different concentrations of nitrapyrin at 0. 0.1, 0.2, 0.5, and 1.0 mg kg^-1 soil were added to the individual flasks. Soil slurries were then sampled at 22, 27, 36, and 48 h. The (NO^-₃+NO^-₂)–N in soil slurries was analyzed by the method described above.

Ammonium oxidation kinetics was determined by a modified nitrification potential method. Ammonium N at 0, 0.05, 0.1, 0.2, 0.5, 0.8, 1.0, or 2.0 mM in 100 mL of phosphate buffer (Hart et al., 1994) was added to 250-mL flasks that contained 15 g of fresh soils. Because nitrification potentials were not equal to zero under the conditions of no added NH⁺₄, initial soil NH⁺₄ expressed as milligrams N per kilogram of soil was converted to millimolarity and summed to the NH⁺₄ concentration in 100 mL of buffer. The measured nitrification rates at different NH⁺₄ concentrations were fit to the nonlinear regression of the Michaelis-Menten equation (SigmaPlot 3.0, Jandel Scientific, 1995) for determining the apparent V_max (maximum nitrification rate, i.e., nitrification potential) and apparent K_m (Michaelis-Menten rate constant).

Degradation of nitrapyrin was measured in a laboratory incubation experiment. Composited soil was collected from the control plots of the fallow field on 2 Oct. 1996. Moist soils (10 g) were placed into 20-mL vials, and 20 mg nitrapyrin kg^-1 soil in emulsion was injected into the soil. Soils were incubated at 18°C and soil water content was adjusted to 10% every week. Three vials were withdrawn randomly at 0, 2, 7, 14, 30, 47, 64, and 93 d. The nitrapyrin was extracted with a solution containing 10 mL water, 1 g sodium sulfate, and 5 mL hexane (Touchton et al., 1978). The nitrapyrin dissolved in the hexane layer was determined by absorbance at 270 nm (Bremner et al., 1978). The measured nitrapyrin concentrations were fit to the exponential model, , where NI₀ is initial nitrapyrin concentration, NI is nitrapyrin concentration at time t, k is the decomposition rate constant (Keeney, 1980). We used the nonlinear regression program (see above) to fit the data. The half-life of nitrapyrin was calculated from the equation .

The pH of soil shaken slurry in nitrification potential assay was measured for convenience. Soil pH (1:2 H₂O) was measured only for soils sampled from the cropped field on 2 Oct. 1996. The pH of soil shaken slurry was simply regressed with soil pH (1:2 H₂O) ( , , P < 0.001).

Statistical Analysis
Inorganic N pool sizes, nitrification potentials, and pH of soil shaken slurry in different fields, blocks, treatments, sampling locations, soil depths, and sampling times were statistically analyzed by ANOVA using a nested multiple split-plot design, in which blocks were nested in the fields, treatments were the main plot; sampling locations, soil depths, and sampling times were multiple sub-plots.

The patterns of NO^-₃–N accumulation with time in nitrapyrin sensitivity analyses were statistically analyzed by ANOVA with a multiple split-plot design with treatments as main plot, concentrations as sub-plot and sampling times as sub-subplot.

The patterns of nitrification rates in NH⁺₄ oxidation kinetics analysis were analyzed by a split-plot design with treatment as main plot and NH⁺₄–N concentrations as sub-plot. The parameters of V_max and K_m were compared among three treatments from their t-values calculated from the best fit values and standard errors (GraphPad Software 1996, GraphPad Software Inc., San Diego, CA).

	Results

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Soil NH⁺₄–N Pool Sizes
Generally, NH⁺₄–N pool sizes were larger in soils fertilized with 50A or 50AN than those of control soils ( ); this difference was only significant at the 0- to 15-cm depth. Figure 1 shows the dynamic patterns of NH⁺₄–N pool sizes among the differently fertilized soils in the fallow and the cropped fields. The NH⁺₄–N pool sizes in the control soils were consistently small (<2 mg N kg^-1 soil) throughout all soil sampling dates, whereas the NH⁺₄–N pool sizes in the soils fertilized with anhydrous NH₃ significantly fluctuated with the soil sampling dates. The lower NH⁺₄ pool size in the 50AN treated soils compared to the 50A treated soil for the cropped field, Fall 1996 (Fig. 1), may be due to soil sampling inadvertently between the application bands. In general, the highest NH⁺₄–N concentrations were observed in the fall close to the application of anhydrous NH₃, then soil NH⁺₄–N concentrations decreased to the level of the control soils in the next spring and were maintained at that low level thereafter. However, NH⁺₄–N applied combined with nitrapyrin was significantly retained until the next spring.

View larger version (28K): [in this window] [in a new window]   Fig. 1 Time course of NH+4–N pool sizes at 0- to 15-cm soil depth in the control soil (Control), the soil fertilized with anhydrous NH3 plus nitrapyrin (50AN), and the soil fertilized with anhydrous NH3 (50A). Values are means and standard errors for . Arrows indicate the application time of anhydrous NH3 and nitrapyrin

View larger version (32K): [in this window] [in a new window]   Fig. 2 Time course of NO-3–N pool sizes at 0- to 15-cm soil depth in the control soil (Control), the soil fertilized with anhydrous NH3 plus nitrapyrin (50AN), and the soil fertilized with anhydrous NH3 (50A). Values are means and standard errors for . Arrows indicate the application time of anhydrous NH3 and nitrapyrin

View larger version (34K): [in this window] [in a new window]   Fig. 3 Soil nitrification potentials at soil 0- to 15 and 15- to 30-cm depths. Nitrification potentials of the fallow field were compared with those of the cropped field by averaging the four sampling dates and the three soil treatments; values are means and standard errors for . Nitrification potentials were compared among the three soil treatments by averaging four sampling dates and two fields, values are means and standard errors for

View larger version (39K): [in this window] [in a new window]   Fig. 4 Time course of nitrification potentials at 0- to 15-cm soil depth in the control soil (Control), the soil fertilized with anhydrous NH3 and nitrapyrin (50AN), and the soil fertilized with anhydrous NH3 (50A). Values are means and standard errors for . Arrows indicate the application time of anhydrous NH3 and nitrapyrin

View larger version (18K): [in this window] [in a new window]   Fig. 5 The response of nitrifier in the soil fertilized with anhydrous NH3 plus nitrapyrin (50AN) or the soil fertilized with anhydrous NH3 (50A) to the fresh addition of nitrapyrin at different concentrations. Arrows indicate the application time of nitrapyrin

	Discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

Nitrapyrin successfully blocked nitrification from late fall to early spring, and the applied NH⁺₄ was significantly retained in soil until next spring (Fig. 1). This short-term effect of nitrapyrin on soil nitrification was consistent with the field work of other researchers (Gomes and Loynachan, 1984; Malhi and Nyborg, 1988; Rao, 1996). However, NO^-₃–N pool size in soil fertilized with anhydrous NH₃ but with or without nitrapyrin was not different (Fig. 2). Leaching of NO^-₃–N from the 50A treatment may explain this observation, because NO^-₃–N rapidly decreased from late fall to early spring when N uptake by winter wheat would likely be low. Papendick and Engibous (1980) indicated that drier upper soil layers in fall would favor water penetration, and extensive leaching of NO^-₃–N might occur during winter. At the 15- to 30-cm depth, the NO^-₃–N pool from soils which were fertilized with anhydrous NH₃ was higher than that from the control soils (data not shown), which may further indicate the occurrence of downward movement of NO^-₃–N. These observations substantiate the benefits of nitrapyrin use for retention of inorganic N in the soil root of winter wheat.

Nitrogen fertilization and organic N mineralization are the major sources of soil NH⁺₄– and NO^-₃–N pools. The inorganic N in the control soils (20 mg N kg^-1 soil in the fall, see Fig. 1 and 2) was attributed to the soil organic N mineralization. Nitrogen mineralization could contribute more inorganic N in long-term N fertilized soils than in control soils, because more plant materials may remain in soils because of higher primary production. Yields of winter wheat from the NH₃, fertilized soils, indeed, were much higher than those from the unfertilized control soils (unpublished data, 1976–1997, Ray Cartee). Consequently, the measured inorganic N pool size was higher than the expected from the applied NH₃ (Fig. 1 and 2).

Application of NH⁺₄ will increase nitrification rate and nitrifier activity under the condition of NH⁺₄ limitation (Belser, 1979). The NH⁺₄–enhanced nitrification has been reported in agricultural soil (Berg and Rosswall, 1985). The short-term effect of NH⁺₄ on soil nitrification was obvious in soil at the 0- to 15-cm depth where anhydrous NH₃ was placed (Fig. 1, Fig. 4). We did not observe a residual effect of repeated, biennial application of anhydrous NH₃ on soil nitrification, however. Nitrification potential is an index of active nitrifier population size (Belser, 1979). The established higher active nitrifier population by application of anhydrous NH₃ was not maintained in soil (Fig. 4). During the fallow period, the enhanced nitrifier activity decreased to that of the control soil. Davidson et al. (1996) reported that intensive repeated use of NH⁺₄–based N early in a single cropping season increased soil nitrifier activity, and this activity remained high even without further N fertilization. The residual effect of a 10-yr, annual application of anhydrous NH₃ on soil nitrification was documented by Biederbeck et al. (1996). In their study, they found that the nitrifier populations were higher in the soil receiving anhydrous NH₃ than in the control soil until the next year's fertilization. Our observation that there was no residual effect of anhydrous NH₃ on soil nitrification may be due to the less intensive use of anhydrous NH₃ with every second year.

In contrast, repeated, biennial application of nitrapyrin had a residual effect on soil nitrification. Nitrification potential in soil fertilized with both anhydrous NH₃ and nitrapyrin was similar to that of the control soil through both cropped and fallow rotation phases (Fig. 4). Even without further use of nitrapyrin, nitrification potential was still lower in soil with a history of nitrapyrin use than in soil without this history (Table 2). This residual effect is not irreversible, however. Soil nitrifiers can finally recover after 3 or 4 yr without nitrapyrin application (Table 2). Belser and Schmidt (1981) indicated that nitrifier communities had different sensitivities to nitrapyrin. They suggested that long-term repeated use of nitrapyrin might select for less sensitive strains. Our data (Fig. 5) showed that the dominant strains of nitrifiers in soils that received nitrapyrin and those that did not, had similar sensitivity to nitrapyrin. Therefore, the residual effect of nitrapyrin is not explained by changes in nitrifier sensitivity alone. The parameters of Michaelis-Menten kinetics also indicated that the residual effect depended on the differences in the active nitrifier populations (V_max). In laboratory experiments, we observed evidence of substrate inhibition of ammonia oxidation at NH⁺₄ levels greater than 1 mM for all the soils. Levels of NH⁺₄ likely to cause substrate inhibition in field soils may be attained for a limited time in localized soil close to fertilizer application bands.

The degradation of nitrapyrin in this soil followed the exponential model of NI . The half-life of nitrapyrin was calculated as 41 d, which is in the range previously reported (Keeney, 1980, 1986). With this high degradation rate, we do not expect that nitrapyrin itself stays in the soil several months after the application to block nitrification. Biederbeck et al. (1996) showed that a long-term, repeated application of anhydrous NH₃ decreased soil pH, which influenced on nitrifier activity. In our study, the repeated use of NH₃ did decrease soil pH to 6.9, compared with the control soil of pH 7.0. However, the pH of the differently fertilized soils was still neutral, which should not significantly influence the nitrifier population activity.

	Conclusions

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

 
Application of nitrapyrin with NH₃ in fall successfully retained applied NH⁺₄ in soil until next spring. A long-term, biennial application of nitrapyrin had a residual effect on soil nitrification. After NH₃ was applied to soil, nitrification potential in soil with a history of nitrapyrin use was lower than in soil without this history. However, this effect is not irreversible; nitrification potentials recovered after 3 or 4 yr without the use of nitrapyrin. In contrast, in our system the long-term, biennial application of NH₃ had no residual effect on soil nitrification.

	ACKNOWLEDGMENTS

 
This research was financially support by the Project no. 323 of the Utah Agricultural Experiment Station and approved as journal paper no.7151. We thank Mr. Ray Cartee for allowing us to use the N fertilizer experimental plots at the Blue Creek Farm and for his informative discussion. We appreciate the advice of Dr. Donald Sisson for statistical analysis. We thank Tracy Shiozawa and Scott Perrin for their hard work in the field.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

 
Contribution from the Utah Agric. Exp. Stn. as Journal no. 7151.

Received for publication March 11, 1999.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

 

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