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Assessment of nitrogen losses from urea and an organic manure with and without nitrification inhibitor, dicyandiamide, applied to lettuce under glasshouse conditions.

Introduction

Nitrogen (N) is the major nutrient element that regulates plant growth but is also contributor to environmental degradation. In New Zealand, urea is a widely used N fertiliser and its application has increased by 27%, i.e. from 314000 to 399 000 t, between 2002 and 2004. This increase is attributed to intensification in horticultural and dairy farming (Statistics 2006). Additionally, a significant proportion (up to 50%) of New Zealand's waste stream made up of biowaste is used as organic manure and soil amendment. Organic manure not only provides organic matter but also a blend of nutrients that is beneficial to soil properties and a function of a diverse group of soil microorganisms (Zaman et al. 1999). One such organic manure made from sheep manure and a natural organic pelletised fertiliser named 'Garden galore' (GG) is widely used in vegetable fanning in New Zealand.

One of the major concerns with application of urea fertilisers and recycled animal and poultry manures is the loss of N through either emissions of ammonia (N[H.sub.3]) and nitrous oxide ([N.sub.2]O) or leaching of nitrate (N[O.sub.3.sup.-]) and the subsequent impact on the environment. Nitrous oxide emissions contribute not only to global warming but also to the destruction of ozone layer (Crutzen 1981), while N[H.sub.3] emissions result in acid rain and act as a secondary source of [N.sub.2]O emissions (Bouwman 1990; Mosier et al. 1998). Nitrate leaching results in surface and groundwater pollution (Bolan et al. 2004). Because of these environmental concerns and New Zealand's commitment to the Kyoto Protocol, there is an urgent need to reduce the N losses from N inputs. Recently, in New Zealand, there has been an increasing interest in the use of N inhibitors to mitigate gaseous and leaching losses of N. Dicyandiamide (DCD) helps to delay the oxidation of N[H.sub.4.sup.+] to N[O.sub.3.sup.-] by depressing the activities of Nitrosomonas bacteria in soil (Prasad and Power 1995); thus, it can reduce [N.sub.2]O emissions directly by decreasing nitrification or indirectly by reducing the availability of N[O.sub.3.sup.-] for denitrification and leaching. As a consequence, DCD can increase N use efficiency (NUE) by increasing plant growth and N uptake. Most research in New Zealand is confined to quantifying the effects of DCD on N losses from urea fertiliser (Zaman et al. 2007), urine deposition in legume-based pasture (Di and Cameron 2003, 2004), and dairy shed effluent (Williamson and Jarvis 1997). Most of the New Zealand research on DCD has focused on pasture and there is little information on the effect of DCD in reducing N losses from vegetable cultivation where N is the key nutrient input. Therefore, the objectives of this study were to: (i) assess the effect of DCD on N transformation; (ii) quantify changes in N losses from urea and organic manure (GG) with DCD; and (iii) determine the effect of DCD on plant growth. Lettuce was used as a test crop because of its sensitivity to N[H.sub.4.sup.+].

Materials and methods

Soil preparation

Manawatu fine sandy loam weathered fluvial recent soil (Hewitt 1998) was used in this study. Field soil samples (0-100 mm depth) were collected randomly from a sheep-grazed, permanent legume-based pasture at Massey University Frewen's Research Block, Turitea Campus, Palmerston North, New Zealand. Bulk soil samples were air-dried and sieved to < 2 mm. Subsamples of the sieved soil were analysed for pH and total C and N in another study (Singh 2007). Soil pH was measured at a 1 : 2.5 soil : water ratio using a combined electrode pH meter (Blakemore et al. 1987), while total C and N in the soil were measured by combustion in a Leco FP-2000 CNS (LECO Corp.; St Joseph, MI).

Glasshouse trial

The equivalent of 1 kg oven-dried and sieved soil was used in a 2-L container. The requisite amounts of urea (46% N) or GG (4% N, 1.4% P, 4% K) to provide 9g N/[m.sup.2], with and without DCD (1.3 g/[m.sup.2]) treatments, were mixed with the soil. The 4 treatments were GG, GG + DCD, urea, and urea+ DCD. One seedling of fast-growing and short-cycle lettuce (Lactuca saliva L.) was transplanted per container and placed in each chamber. The temperature in the glasshouse was maintained regularly between 15 and 20[degrees]C. The moisture content in the soil was maintained at 80% of the potted soil crop moisture capacity by monitoring the water loss and adding water to it. Each treatment was replicated 3 times.

Lettuce dry matter yield

Lettuce plants were harvested after 5 weeks of growth. The shoots and roots materials were separated and rinsed with deionised water to remove all the soil and dried at 65[degrees]C to a constant weight. Plant samples were ground in a cutting mill and analysed for total N by the Kjeldahl digestion method using an auto analyser (McKenzie and Wallace 1954).

Gas sampling

Each container was placed in a closed base chamber. These chambers are described in Saggar et al. (2004). Ammonia emission was measured for 15 days after application of the treatments using the active flux method, a constant air supply at 1 [dm.sup.3]/min and 0.05 M [H.sub.2]S[O.sub.4] acid traps to capture N[H.sub.3] (Singh et al. 2003). Each chamber has 2 removable lids with 1 or 2 ports for sampling [N.sub.2]O or N[H.sub.3] fluxes, respectively. The lid with 1 port had a 100-mm-long tubing (3.2 mm diameter) with a 3-way stopcock attached to it to take [N.sub.2]O gas samples using 60-mL polypropylene syringes. The lid with 2 ports had 1 input port connected to a compressed air supply and other exhaust port connected to a chemical trap to absorb N[H.sub.3]. Fifty mL of 0.05 M [H.sub.2]S[O.sub.4] (Wulf et al. 2001) was used to trap N[H.sub.3]. Ammonia measurements were taken daily, and the N[H.sub.3] flux (mg N/[m.sup.2].h) was then calculated using the following equation:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (1)

where C is N[H.sub.3] concentration in the acid trap (mg/[dm.sup.3]), V is the volume of the acid ([dm.sup.3]), a is total cross-sectional area ([m.sup.2]) of soil cores in the chamber, and D is duration (h) of each sampling. After taking N[H.sub.3] measurements, the lids were removed and the chambers were kept open to achieve equilibrium with ambient conditions.

For [N.sub.2]O sampling, the chambers were closed using the lid with 1 port for 1 h, and air in the chamber was sampled through a 3-way tap on the chamber lid, using a 60-mL syringe. The [N.sub.2]O flux was measured daily during the first week, twice during the second and third weeks, and once during the fourth and fifth weeks. At each sampling time, 3 gas samples were taken from each chamber at 0, 30, and 60min, after closing the chamber. The gas samples collected were transferred to evacuated vials, analysed using a Shimadzu GC-17A gas chromatograph with a [sup.63]Ni-electron capture detector, and gas fluxes calculated as described in Saggar et al. (2004, 2007).

Laboratory incubations

Plastic containers containing 1 kg air-dried and sieved soil receiving the same treatments as mentioned in the glasshouse study were incubated at 18[degrees]C for 5 weeks and the moisture content was maintained at 80% field capacity. Each treatment was carried out in 3 replicates. A 30-g soil subsample was taken daily during the first week, twice during second and third weeks, and once during the last fourth and fifth weeks from each container and analysed for pH and mineral N.

The pH of incubated soil samples was measured at a 1 : 2.5 soil: water ratio using a combined electrode pH meter. The incubated soil subsamples were extracted with 2M KC1 solution (1:6 soil: extractant ratio) and the extract was analysed calorimetrically for mineral N by an auto analyser (Blakemore et al. 1987).

Leaching experiment

At the end of the glasshouse lettuce growth experiment, a 100-g soil subsample from each replicate treatment was packed into polyvinyl chloride pipes of 200 mm height and 50 mm internal diameter. The bottom of the pipes was tightly covered with muslin cloth. The columns were arranged vertically with plastic funnels at the bottom, to facilitate the collection of leachate. The soils were then leached by adding 100 mL of deionised water, and the volume of leachates was measured. Subsamples of leachates were analysed for mineral N by an auto analyser.

Statistics

Data were analysed using analysis of variance (ANOVA) and the differences between treatment means were compared by l.s.d. and significant differences are expressed at P [less than or equal to] 0.05 (SAS 2003).

Results

Effect of DCD on N losses

N[H.sub.3] emissions

Ammonia emissions were observed immediately after the application of urea and GG with and without DCD. These emissions peaked on day 3 and gradually declined to zero levels by day 15 (Fig. 1). No emissions were observed after day 15. The N[H.sub.3] emission from urea and GG treatments with DCD showed higher peaks of 199.4 and 65.7 mg N[H.sub.3]-N/[m.sup.2]. day compared with 126.3 and 43.7 mg N[H.sub.3]-N/[m.sup.2].day in urea and GG alone. The cumulative fluxes over the 15-day emission period were significantly higher in both DCD-treated soils with urea (988.7 mg N[H.sub.3]-N/[m.sup.2]) and GG (346.9 mg N[H.sub.3]-N/[m.sup.2]) (Table 1). Overall, there was 58% and 38% increase in N[H.sub.3] emission with DCD compared with treatments without DCD. More N[H.sub.3] was emitted from soils with urea (626.1 mg N/[m.sup.2]) than with GG (251.4mg N/[m.sup.2]).

[N.sub.2]O emissions

The [N.sub.2]O emissions also peaked at day 3 and declined subsequently to reach the background levels within 4 weeks (Fig. 2). Nitrous oxide emissions in soils with urea and GG alone were substantially higher than those with urea and GG. A peak [N.sub.2]O emission flux of 2.79 and 0.70mg [N.sub.2]O-N/[m.sup.2].day was detected at day 3 with urea and GG, respectively, while 0.70 and 0.16 mg [N.sub.2]O-N/[m.sup.2].day of [N.sub.2]O flux was obtained with the addition of DCD. Overall, 3.9 and 1.0mg [N.sub.2]O-N/[m.sup.2] was emitted, amounting to 0.04 and 0.01% of the applied N in the treatments with urea and GG with DCD (Table 1). These emissions were about 65% and 66% of those measured without DCD.

[FIGURE 1 OMITTED]

N[H.sub.4.sup.+] and N[O.sub.3.sup.-] leaching

The amount of N[H.sub.4.sup.+] and N[O.sub.3.sup.-] leached from soils fertilised with GG with and without DCD was almost negligible (2.0 and 0.4mg/kg) (Fig. 3). Application of urea induced higher soil N[O.sub.3.sup.-]-N leaching. The addition of DCD to urea decreased N[O.sub.3.sup.-] leaching but enhanced N[H.sub.4.sup.+] leaching. The reduction in induced N[O.sub.3.sup.-] leaching from urea and GG with the DCD addition was 77 and 80%.

Changes in mineral-N content with and without DCD

Soil N[H.sub.4.sup.+]-N concentration increased with urea and GG application, both with and without DCD, with peak values measured on day 3 (Fig. 4a). Higher N[H.sub.4.sup.+]-N concentration was obtained in soils fertilised with urea than with GG. These concentrations of N[H.sub.4.sup.+] further increased significantly with the addition of DCD to both urea and GG. The N[O.sub.3.sup.-] concentrations increased gradually over lime in soils treated with urea and GG; however, lower amounts of N[O.sub.3.sup.-] were produced with GG than urea. The addition of DCD to urea and GG decreased the soil N[O.sub.3.sup.-] concentrations (Fig. 4b).

Highest levels of soil pH were measured on day 3 in all treatments, with subsequent decline. The addition of urea increased the pH to 6.7, and the pH increased to 6.4 in GG treatment. The addition of DCD with both urea and GG significantly (P<0.05) enhanced the soil pH, with a maximum pH of 7.2 and 6.8 obtained at day 3 (Fig. 4c).

Effect of DCD on lettuce growth and N uptake

Lettuce total dry matter (DM) yield with GG application (2.35 g) was significantly higher (P<0.05) than with urea application (0.90 g) (Fig. 5). The addition of DCD to both GG and urea resulted in slightly lower DM yield. Higher N concentrations were measured in both shoot and root of lettuce with urea than with GG treatment (Table 2). The N content of lettuce in all treatments was 2.05-3.60% in the shoots and 1.07-2.80% in the roots. Addition of DCD increased N concentrations in both shoots and roots from 3.42 to 3.60% and 2.48 to 2.80% with urea and from 2.05 to 2.65 and 1.07 to 1.77% with GG. The N uptake by lettuce with GG and urea with DCD was 0.08 and 0.02 g/[m.sup.2], and without DCD was 0.07 and 0.05g/[m.sup.2]; however, the treatments differences were not significant (Table 3).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Discussion

Effect of DCD on N[H.sub.3], [N.sub.2]O emissions and N[O.sub.3.sup.-] leaching

This glasshouse experiment showed that there was a greater loss of N as N[H.sub.3] than as [N.sub.2]O, and the effect was more pronounced with urea than with GG. Low N[H.sub.3] emissions and small fluxes of [N.sub.2]O obtained from GG were probably attributed to the slow-release nutrient characteristics of organic GG and could be due to the lower soil pH produced in GG compared with urea during the hydrolysis of urea. Addition of DCD resulted in a significant (P < 0.05) increase in N[H.sub.3] loss from urea, but there was no significant difference in N[H.sub.3] emission from GG with and without DCD, despite a small increase. Soil temperature, soil pH, type of the soil, and rate and placement of N fertiliser are among the contributing factors that influence the loss of N[H.sub.3]. The enhanced N[H.sub.3] emissions from urea in the presence of DCD obtained in our study could be attributed to its effect on delaying the conversion of N[H.sub.4.sup.+] to N[O.sub.3.sup.-],thus resulting in accumulation of large amounts of N[H.sub.4.sup.+]. Similar increase in N[H.sub.3] emission with DCD has previously been reported by several authors (Prakasa Rao and Puttanna 1987; Fox and Bandel 1989; Gioacchini et al. 2002).

Nitrification inhibitors have been shown to reduce [N.sub.2]O emissions (Di and Cameron 2002; Hatch et al. 2005). Our results show that the inhibition effect of DCD has decreased the [N.sub.2]O emissions with GG and urea. However, no significant difference was obtained when DCD was added to GG, while [N.sub.2]O emission decreased significantly with urea amended with DCD than urea alone. In other field studies, it has been demonstrated that no significant differences were detected in [N.sub.2]O emissions when DCD was added to cow dung (Williamson and Jarvis 1997). However, Merino et al. (2002) showed that DCD effectively reduced [N.sub.2]O losses to control levels, and significantly smaller cumulative emission losses were found from slurry treated with DCD than slurry alone. Dobbie and Smith (2003) reported that DCD was effective in reducing [N.sub.2]O emissions when DCD was applied with urea and ammonium sulfate from an intensively managed grassland site in the UK.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

The addition of DCD influences both N[O.sub.3.sup.-] and N[H.sub.4.sup.+] leaching. Although DCD reduced N[O.sub.3.sup.-] leaching losses in both urea and GG treatments, it also enhanced N[H.sub.4.sup.+] leaching, which could be attributed to accumulation of N[H.sub.4.sup.+] in the soil from the effect of DCD. Addition of DCD to various N sources such as fertiliser, slurry, and urine showed higher accumulation of N[H.sub.4.sup.+]-N in soil (Puttanna et al. 2001; Cookson and Cornforth 2002; Merino et al. 2002). Vogeler et al. (2007) demonstrated that DCD decreased both N[O.sub.3.sup.-] and N[O.sub.3.sup.-]-induced N[H.sub.4.sup.+] leaching in the urea-treated soil. Most of the soil lysimeters studies reported by Di and Cameron (2002, 2003, 2004, 2005) show that the N[O.sub.3.sup.-] leaching losses were significantly reduced when DCD was applied in solution and fine particle suspension form in combination with high urine (1000 kg N/ha) and high urea (200kg N/ha) inputs.

N transformation

The laboratory incubation experiment indicated higher mineral N production in urea-treated soils than GG-treated soils. Lower concentrations of N[H.sub.4.sup.+]-N and N[O.sub.3.sup.-]-N obtained from GG-treated soil throughout the experiment indicate that GG releases N slowly. The lower N[H.sub.3] and [N.sub.2]O emissions obtained from this treatment were due to the limited substrate present in the GG; thus, it provides a greater chance of N uptake by lettuce plants as indicated by the higher N uptake with GG than urea (Table 3). Soil N[H.sub.4.sup.+] content in DCD-amended soils was significantly greater than that in soils without DCD and remained higher throughout the experimental period of 29 days. A similar trend was observed in the laboratory incubation experiments carried out by Vogeler et al. (2007), in which urea amended with DCD showed higher and significant increases in soil N[H.sub.4.sup.+] compared with urea alone, remaining higher after the 8 days; however, there was no significant difference, although there was an increase in the amount of N[O.sub.3.sup.-] produced. The soil N[O.sub.3.sup.-] concentration in our soil incubation study was significantly lower in the presence of DCD with both GG and urea. These results suggest that DCD delayed the oxidation of N[H.sub.4.sup.+] to N[O.sub.3.sup.-].

The pH of the treated soils was generally higher than the initial soil pH. However, after an initial increase in pH of the treated soils, there was a slow decrease with time of incubation. The initial increases in pH following urea and GG applications were due to hydrolysis of urea to ammonium carbonate via the action of urease enzyme. The pH in urea- and GG-treated soil subsequently decreased due to production of nitrate through the nitrification process. A similar trend of pH changes has been observed following the applications of urea and manure in soil (Gameh et al. 1990; Wang and Alva 1996). The presence of DCD with both urea and GG retarded the nitrification process, resulting in higher accumulation of N[H.sub.4.sup.+] near the soil surface. This caused an increase in soil pH, which is likely to have induced N[H.sub.3] volatilisation (Fig. 1).

Effect of DCD on lettuce growth and N uptake

Higher DM yield was obtained from GG than from urea, suggesting that 90 kg N/ha of GG was sufficient to produce a good yield of lettuce in Manawatu soil. Various beneficial effects of GG such as improvement in soil physical structure, nutrient availability, and water-bolding capacity might have contributed to better yield production of lettuce. The root formation in GG-treated soil was higher than that in urea-treated soil, probably because of phosphorus (P) contribution from GG. P is essential for energy storage and transfer, and sufficient P supply has been associated with increased root growth (Havlin et al. 2005).

Addition of DCD in both urea and GG did not significantly affect lettuce yields, although there was a trend to reduction in yields. However, the N concentration in lettuce shoots and roots was enhanced with DCD (Table 2). Reduced growth of lettuce with DCD in either urea or GG treatments may be attributed to N[H.sub.4.sup.+] toxicity, which showed as shoot tip burning, and the symptoms were more severe with urea. It is also noted that lettuce plants died, resulting in low yields for this treatment. The common symptoms of N[H.sub.4.sup.+] toxicity are growth suppression and yield depressions among sensitive plant species ranging from 15 to 60% (Chaillou et al. 1986), and even death (Magalhaes and Wilcox 1984a, 1984b; de Graaf et al. 1998). Similar results were obtained when DCD was applied with cattle slurry and calcium ammonium nitrate (Belastegni Macadam et aL 2003), resulting in chlorosis and further necrosis at the border of the leaves of white clover after 21 days of application, thereby causing a 16% yield reduction. In other study by Zerulla et al. (2001) comparing the effect of DCD and 3,4-dimethylpyrazole phosphate (DMPP) applied at 8-fold the recommended rate on lettuce, the DCD caused pronounced phytotoxic effect, while no effect was observed with DMPP.

In this study, fewer root hairs with shorter roots were formed with urea than GG, and with the addition of DCD, which may be attributed to the increase in N[H.sub.4.sup.+] concentration resulting in salt injury. Similar symptoms of N[H.sub.4.sup.+] toxicity affecting plant roots have been reported by several authors, e.g. lowering ratios of root to shoot (Atkinson 1985; Blacquiere et al. 1987; Wang and Below 1996) and a decrease in the fine to coarse root ratio (Boxman et al. 1991). The total N content in both shoots and roots of the lettuce plants were also enhanced by DCD inhibitor. This was attributed to greater N[H.sub.4.sup.+] accumulation in the soil in the presence of DCD, which caused the lettuce plants to take more N as N[H.sub.4.sup.+] and could be attributed to a small amount of additional N from the breakdown of DCD (67% N) in the soil that may have entered the lettuce plants (Belastegui Macadam et al. 2003).

Conclusions

The results of these study showed that higher N losses were obtained from urea than GG. DCD decreases N[O.sub.3.sup.-] production, [N.sub.2]O emission, and N[O.sub.3.sup.-] leaching. On the other hand N[H.sub.4.sup.+] production and N[H.sub.3] emission were enhanced with DCD. The presence of DCD also caused a decrease in shoot and root growth, resulting in lower lettuce total DM yield.

Acknowledgement

The authors thank Landcare Research, Palmerston North, New Zealand, for equipment and analytical facilities.

Manuscript received 27 November 2007, accepted 26 May 2008

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Janice Asing (A,D), S. Saggar (B), Jagrati Singh (B), and Nanthi S. Bolan (C)

(A) Institute of Natural Resources, Massey University, New Zealand.

(B) Landcare Research, Palmerston North, New Zealand.

(C) Centre for Environmental Risk Assessment and Remediation (CERAR), University of South Australia, Mawson Lakes, SA 5095, Australia.

(D) Corresponding author. Email: J.Asing@massey.ac.nz
Table l. Cumulative N[H.sub.3] and [N.sub.2]0 losses and the
proportion of the applied N lost as N[H.sub.3] and [N.sub.2]0 from
treatments with area and GG with and without DCD during 5 weeks of
plant growth

Treatments N added N[H.sub.3]-N % Added N
 emitted as
 (mg N/[m.sup.2]) N[H.sub.3]

Urea 9000 626.1 6.96
Urea + DCD 9000 988.7 10.99
GG 9000 251.4 2.79
GG + DCD 9000 346.9 3.85
l.s.d. (P = 0.05) 265.7

Treatments [N.sub.2]O-N % Added N Total % of
 (mg M/[m.sup.2]) emitted as N emitted
 [N.sub.2]O

Urea 11.3 0.13 7.1
Urea + DCD 3.9 0.04 11.0
GG 2.9 0.03 2.8
GG + DCD 1.0 0.01 3.9
l.s.d. (P = 0.05) 3.98

Table 2. Total N (%) in shoots and roots of lettuce after 5 weeks of
plant growth

Treatments Shoots Roots

Urea 3.42 2.48
Urea + DCD 3.60 2.80
GG 2.05 1.07
GG + DCD 2.65 1.77
l.s.d. (P = 0.05) 0.41 0.97

Table 3. Total dry matter (DM) yield, percent of added N in DM and N
uptake by lettuce from potted soil receiving area and GG with and
without DCD

Treatments Total DM N in DM N uptake
 (g/[m.sup.2]) (%) (g/[m.sup.2])

Urea 0.90 5.90 0.05
Urea + DCD 0.34 6.40 0.02
GG 2.35 3.12 0.07
GG + DCD 1.78 4.42 0.08
l.s.d. (P=0.05) 1.13 0.06
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Author:Asing, Janice; Saggar, S.; Singh, Jagrati; Bolan, Nanthi S.
Publication:Australian Journal of Soil Research
Article Type:Report
Geographic Code:1USA
Date:Sep 1, 2008
Words:5723
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