Influence of 2-chloro-6 (trichloromethyl) pyridine and dicyandiamide on nitrous oxide emission under different soil conditions

Abstract

Two experiments were conducted to evaluate the inhibitory effects of 2-chloro-6 (trichloromethyl) pyridine (nitrapyrin) and dicyandiamide on nitrous oxide (N₂O), a greenhouse gas, emission from soils amended with ammonium sulfate. In the two experiments, samples of an Andosol and a Gray Lowland soil were kept in glass vessels sealed with a butyl rubber cap and incubated at 25°C. In the first experiment, nitrapyrin (1 µg g⁻¹ dry soil) and dicyandiamide (10 µg g⁻¹ dry soil) were applied to samples of a water-saturated Andosol and a Gray Lowland soil to which ammonium sulfate had been applied at a rate of 0.1 mg N g⁻¹ dry soil. Nitrapyrin decreased N₂O emissions from the Andosol and the Gray Lowland soil by 71% and 24%, respectively. Dicyandiamide decreased N₂O emissions from the Andosol and Gray Lowland soil by 31% and 18%, respectively. In the second experiment, nitrapyrin (1 µg g⁻¹ dry soil) was applied to samples of an Andosol at 51% water-filled pore space to which ammonium sulfate had been applied at rates of 0.01, 0.1 and 0.5 mg N g⁻¹ dry soil. Nitrapyrin decreased N₂O emissions by 62%, 83% and 74%, respectively. Changes in the NH⁺ ₄ and NO⁻ ₂ + NO⁻ ₃ concentrations in soil showed that nitrapyrin and dicyandiamide slowed down the nitrification process, but did not completely stop the process at any time. The results reveal the potential of nitrification inhibitors to decrease N₂O emission from fertilized soil in a wide range of moisture conditions and nitrogen levels.

Key words:

• greenhouse gas
• nitrification
• nitrification inhibitor
• nitrous oxide

INTRODUCTION

The atmospheric concentration of nitrous oxide (N₂O) has increased by 46 p.p.b. (17%) since 1750 and continues to increase at a rate of 0.25% year⁻¹ (1980–1998). Radiative forcing amounts to 0.15 W m⁻², which accounts for 6% of the total from all of the long-lived and globally mixed greenhouse gases (Intergovernmental Panel on Climate Change 2001). Agricultural soil, a major source, accounts for approximately 6.2 Tg N₂O-N year⁻¹, or 35% of the annual global emission (Kroeze et al. 1999). Thus, it is necessary to reduce N₂O emission from agricultural soil to mitigate global warming.

Nitrification inhibitors (ammonia oxidation inhibitors) contribute to the decrease in N₂O emission from fertilized agricultural soil (McTaggart et al. 1997; Minami 1994; Wolt 2004). Nitrification inhibitors such as nitrapyrin suppress the accumulation of nitrite-N in soil and thus are likely to indirectly decrease N₂O emission via chemodenitrification or microbial denitrification of nitrite-N (Sahrawat 1989). Nitrification inhibitors also decrease nitrate leaching (Cookson and Cornforth 2002). However, the use of nitrification inhibitors on agricultural land is still limited because the cost of nitrification inhibitors is higher than the economical benefit for farmers. As the adverse effects of global warming are becoming evident, the demand for a decrease in N₂O emission will increase. Practical techniques to decrease N₂O emission from agricultural soil should be developed, and the use of nitrification inhibitors may achieve this objective. For that purpsose, it is necessary to elucidate the mechanisms whereby nitrification inhibitors affect N₂O emission under various conditions.

In the present study, the effect of nitrapyrin and dicyandiamide on N₂O emission was examined. Although there are numerous nitrification inhibitors (Slangen and Kerkhoff 1984), most commonly inhibit NH₃ monooxygenase (McCarty 1999). Nitrapyrin and dicyandiamide, which are two of the most common nitrification inhibitors (Bolan et al. 2004; Prasad and Power 1995), were selected in the present study as typical nitrification inhibitors. Studies have shown that N₂O emission increases as soil moisture increases (Bateman and Baggs 2005; Inubushi et al. 1999; McTaggart and Tsuruta 2003). However, it has not been determined whether nitrification inhibitors decrease N₂O emission from soil under water-saturated conditions. Soil moisture conditions have been shown to affect the effectiveness of nitrification inhibitors (Slangen and Kerkhoff 1984). The present study was conducted to clarify the effect of nitrification inhibitors on the decrease in N₂O emission from water-saturated soils. Nitrification inhibitors were applied to samples of an Andosol and a Gray Lowland soil under water-saturated conditions. Blackmer et al. (1980) reported that part of nitrified N evolved as N₂O, and increased with increasing NH⁺ ₄ concentration up to approximately 1 g NH⁺ ₄-N L⁻¹. Thus, the effect of nitrification inhibitors on N₂O emission may change depending on the NH⁺ ₄ concentration in the soil. Attempts were also made to test the effectiveness of nitrapyrin in decreasing N₂O emission in samples of an Andosol amended with three different application rates of ammonium sulfate under aerobic conditions.

MATERIALS AND METHODS

Soil sample

Two soil types, an Andosol and a Gray Lowland soil, were used for the experiments. Samples of the Andosol were taken from the surface layer (0–10 cm) of the Hachiman-tai experimental upland field of the Japan International Research Center for Agricultural Sciences (JIRCAS; Tsukuba, Ibaraki) in March. Soybean was cultivated in the summer season, but no crops had been grown in the winter season for more than 5 years. There was no vegetation at the time of soil sampling. Total C and N concentrations were 33.5 g kg⁻¹ and 2.8 g kg⁻¹, respectively. The soil pH (KCl) was 5.44. Samples of the Gray Lowland soil were collected from the Hachiman-tai experimental paddy field of JIRCAS from the surface layer (0–10 cm) at the end of April, before puddling for rice cultivation was carried out. Rice had been cultivated for more than 20 years, and no crops were cultivated in the winter season. Total C and N concentrations were 17.5 g kg⁻¹ and 1.7 g kg⁻¹, respectively. The soil pH (KCl) was 4.55. Fresh samples of the two soil types were partially dried at room temperature and sieved using a 2 mm mesh sieve.

N₂O analysis

The concentration of N₂O in the vessel headspace was determined using a gas chromatograph fitted with an electron capture detector at 340°C (GC-14B, Shimadzu, Kyoto, Japan). The separating columns were packed with Porapak Q and the carrier gas consisted of 5% methane in argon. Three glass vessels for each treatment were sealed using butyl rubber caps. After measurement, the caps were removed immediately and air in the headspace was ventilated. The vessels were then resealed for further incubation.

NO⁻ ₂ + NO⁻ ₃ and NH⁻ ₄ analysis

NO⁻ ₂ + NO⁻ ₃ and NH⁺ ₄ in soils were extracted with a 2 mol L⁻¹ KCl solution. The extract was filtered, kept in a freezer and the concentrations of NO⁻ ₂ + NO⁻ ₃ and NH⁺ ₄ were determined using an auto-analyzer (Auto Analyzer II, Bran+Luebbe, Tokyo, Japan).

Experiment 1

Experiment 1 examined the use of nitrapyrin and dicyandiamide in samples of an Andosol and a Gray Lowland soil under water-saturated conditions. Fresh soil samples (20 g on a dry weight basis) were placed in glass vessels with an inner diameter of 36.5 mm and 130 mm in height. Three types of treatments for each soil type were prepared as follows: ammonium sulfate (no-Ni), ammonium sulfate and nitrapyrin (1 mg g⁻¹ dry soil) (Np), and ammonium sulfate and dicyandiamide (10 mg g⁻¹ dry soil) (Dd). Ammonium sulfate was applied to all treatments at a rate of 0.1 mg N g⁻¹ dry soil. After the application of the reagents, the moisture content of all samples was adjusted to the saturated condition using distilled water. These samples were incubated at 25°C and N₂O emissions were determined at 2–4-day intervals for 45 days.

For the analysis of NO⁻ ₂ + NO⁻ ₃ and NH⁺ ₄, treatment vessels were prepared in the same ways as the samples used to examine N₂O emission. Two samples were extracted every week after the onset of incubation.

The preparation and incubation of the samples amended with nitrapyrin started 11 days after the other treatments. During that period, the fresh soil samples were put into plastic bags and kept in a refrigerator.

Experiment 2

Experiment 2 examined the use of nitrapyrin in samples of an Andosol amended with three levels of ammonium sulfate under aerobic conditions. Fresh Andosol samples (20 g on a dry weight basis) were placed in glass vessels as in experiment 1. Seven treatments were set up, including three levels of ammonium sulfate at application rates of 0.01, 0.1 and 0.5 mg N g⁻¹ dry soil with or without 1 mg g⁻¹ of nitrapyrin and a control treatment (no N and no nitrapyrin). The moisture content of all the samples was adjusted to 51% water-filled pore space (WFPS). Three glass vessels for each treatment were incubated at 25°C and the N₂O concentration in the vessel headspace was determined using gas chromatography every 3 or 4 days until the NH⁺ ₄ concentration in the soil decreased to the same level as that in the control.

For the analysis of NO⁻ ₂ + NO⁻ ₃ and NH⁺ ₄, treatment vessels were prepared in the same way as samples examining N₂O emission. Two samples were extracted with a 2 mol L⁻¹ KCl solution every week after the onset of incubation.

RESULTS

Experiment 1

Nitrapyrin and dicyandiamide decreased the N₂O emissions from the two soil types under water-saturated conditions (Fig. 1). In the Andosol, N₂O emission from the Np treatment was always lower than that from the no-Ni treatment for 45 days, whereas the N₂O emission from the Dd treatment was lower during the first 3 weeks

Figure 1  Nitrous oxide emission from an Andosol and a Gray Lowland soil amended with nitrification inhibitors under water-saturated conditions. (Bars represent the standard deviation.)

and then became the same as that from the no-Ni treatment. The total amount of N₂O-N emitted during the 45-day period of incubation was 4.06 ± 0.08, 1.18 ± 0.05 and 2.80 ± 0.10 mg g⁻¹ dry soil (average ± standard deviation), for the no-Ni, Np and Dd treatments, respectively. The amounts from the Np and Dd treatments were significantly smaller than the amount from the no-Ni treatment (P < 0.01; tested using Student's t-test). In the Gray Lowland soil, N₂O emission was not appreciably different among the three treatments for approximately the first 2 weeks, after which the N₂O emission decreased in the order of Np, Dd and no-Ni treatments. The total amount of N₂O-N emitted during the 45-day period of incubation was 12.5 ± 1.05, 9.49 ± 0.21 and 10.22 ± 1.12 mg g⁻¹, for the no-Ni, Np and Dd treatments, respectively. The amount from the Np and Dd treatments was significantly smaller than that from the no-Ni treatment (P < 0.01 and P < 0.05, respectively).

Changes in the NH⁺ ₄ and NO⁻ ₂+NO⁻ ₃ concentrations showed that nitrapyrin and dicyandiamide retarded, but did not stop, nitrification in both soil types (Fig. 2). Although the concentration was lower, nitrapyrin inhibited nitrification more strongly than dicyandiamide. The decrease in the total inorganic nitrogen concentration (NH⁺ ₄, NO⁻ ₂ and NO⁻ ₃) indicated that denitrification occurred in the Gray Lowland soil. However, the total inorganic nitrogen concentration in the Andosol did not decrease.

Experiment 2

Nitrapyrin decreased the emission of N₂O at all three levels of ammonium sulfate treatment (Fig. 3). The ratio of emitted N₂O to applied ammonium sulfate increased as the application rate increased (Table 1).

Changes in the NH⁺ ₄ and NO⁻ ₂ + NO⁻ ₃ concentrations in soil showed that nitrapyrin did not stop, but slowed down, the nitrification process at any time (data not shown). Nitrification was completed within 7, 14 and 28 days in the Andosol amended with 0.01, 0.1 and

Table 1 Emitted N2O and N2O-N/applied nitrogen from soil amended with ammonium sulfate and nitrapyrin

AS (mg g^−1)	NP^†	Emitted N2O^‡	N2O-N/applied N × 100^§
0	–	1.6 ± 0.1
0.01	–	2.9 ± 0.1	0.013
0.1	–	64.6 ± 2.2	0.06
0.5	–	934 ± 28	0.19
0.01	+	2.1 ± 0.1	0.005
0.1	+	11.3 ± 0.2	0.01
0.5	+	268 ± 16	0.05
^†+, applied; –, not applied.
^‡N2O-N pg (g dry soil)^−1 (mean ± standard deviation).
^§Emitted N2O-N (Treatment − Control)/applied N × 100. AS, ammonium sulfate; NP, nitrapyrin.

Figure 2  Changes in ammonium, nitrate plus nitrite and total inorganic nitrogen concentrations in an Andosol and a Gray Lowland soil amended with nitrification inhibitors under water-saturated conditions. (Each symbol is the average of two samples.)

0.5 mg N g⁻¹ dry soil of ammonium sulfate, respectively. It took more than 7, 21 and 41 days for nitrification to be completed in soil amended with 0.01, 0.1 and 0.5 mg N g⁻¹ dry soil of ammonium sulfate with 1 mg g⁻¹ of nitrapyrin, respectively. When the N₂O emission was higher than that of the control treatment, nitrification was also more active than that of the control treatment.

DISCUSSION

Nitrification inhibitors decreased N₂O emissions from the Andosol and Gray Lowland soils under water-saturated conditions. Poth and Focht (1985) reported that N₂O was produced by autotrophic NH₃ oxidizers through a reductive process in which organisms use as an electron acceptor, particularly when O₂ is a limiting factor. Williams et al. (1992) suggested that although energy is required to reduce NO⁻ ₂ to N₂O, this mechanism enables organisms to conserve limited O₂ for the oxidation of NH₃, from which they gain energy for growth and regeneration while avoiding the possible accumulation of toxic levels of NO⁻ ₂. It appears that the nitrification inhibitors slowed down the conversion rate of NH⁺ ₄ to in the soil, which contributed to a lower N₂O emission ratio to nitrified nitrogen.

The N₂O emitted from soil is mainly produced through nitrification and denitrification by soil microorganisms (Granli and Bockma 1994; Williams et al. 1992). Inubushi et al. (1996) observed that most N₂O was derived from autotrophic nitrification in two Andosols at 60% and 80% water-holding capacity with NH⁺ ₄ amendment, whereas large amounts of N₂O were emitted at 100% water-holding capacity mostly by denitrification. The decrease in the amount of total

Figure 3  Influence of nitrapyrin on nitrous oxide emission in an Andosol amended with different levels of ammonium sulfate. (Bars represent the standard deviation.)

inorganic nitrogen in the Gray Lowland soil in experiment 1 indicated that denitrification occurred, although the amount of total inorganic nitrogen in the Andosol did not decrease. Although both soil types were saturated with water, it appeared that the conditions in the Gray Lowland soil were more reductive than those in the Andosol. The higher N₂O emission from the Gray Lowland soil compared with the Andosol and the lower decrease in N₂O emission by the nitrification inhibitors in the water-saturated Gray Lowland soil were ascribed to the fact that some of the N₂O emitted from the Gray Lowland soil was produced by denitrification. In the present study, nitrapyrin (0.5 mg g⁻¹ dry soil) and dicyandiamide (2 mg g⁻¹ dry soil) retarded nitrification and decreased N₂O emission from the Andosol that was amended with ammonium sulfate (0.1 mg g⁻¹ N dry soil) at 51% WFPS (data not shown). It appeared that nitrification inhibitors decreased N₂O emission in soils over a wide range of moisture regimes.

In experiment 2, nitrapyrin inhibited the oxidation of NH⁺ ₄ to NO⁻ ₂ and decreased the ratio of N₂O emission to accumulated NO⁻ ₃ in the Andosol amended with ammonium sulfate (0.01, 0.1 and 0.5 mg N g⁻¹ dry soil). A higher ratio of N₂O was emitted when more NH⁺ ₄-N was applied to the soil (Table 1). Blackmer et al. (1980) reported that part of the nitrified N evolved as N₂O increased with increasing NH⁺ ₄ concentration up to approximately 1 g NH⁺ ₄-N L⁻¹. The NH⁺ ₄-N concentration in the soil water in experiment 2 was less than 1 g NH⁺ ₄-N L⁻¹ and this result supported previous results (Blackmer et al. 1980). Nitrapyrin decreased N₂O emission regardless of the NH⁺ ₄-N concentration within the prepared range in the Andosol.

Nitrification inhibitors contribute to the alleviation of N₂O emission from fertilized agricultural soil. Wolt (2004) indicated that in cornfields in Midwestern USA, nitrapyrin decreased greenhouse gas emissions by an average of 51%. However, the use of nitrification inhibitors on agricultural land is still limited. In addition to the cost-benefit problem, the limited use of nitrification inhibitors may be ascribed to variations in their effectiveness. It was observed that the temperature, pH value, organic matter content, clay content and moisture conditions of soil affect the effectiveness of nitrification inhibitors (Slangen and Kerkhoff 1984). To enhance the effectiveness and reliability of nitrification inhibitors, the conditions under which the inhibitors decrease N₂O emission and leaching loss of nitrogen need to be clarified. The present study showed that nitrapyrin effectively decreased N₂O emission. However, nitrapyrin has not been used in Japan. Nitrapyrin has a relative high vapor pressure (Slangen and Kerkhoff 1984) that excludes its addition to solid fertilizers. Alternative nitrification inhibitors that would be more effective than dicyandiamide and nitrapyrin need to be identified (Zerulla et al. 2001). It is also necessary to develop other methods of decreasing N₂O emissions from fertilized soil, including improvement of land management, organic matter management and the use of controlled-release fertilizers (Akiyama and Tsuruta 2003; Koga et al. 2004; Smith et al. 1997). Ishikawa et al. (1999) reported that a tropical grass growing in tropical pastures inhibits nitrification and decreases N₂O emission from soil where the grass grows. We may utilize that biological resource to breed crops that inhibit nitrification and decrease N₂O emission from arable land soil.