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AGEE 5500 No. of Pages 9

Agriculture, Ecosystems and Environment xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Agriculture, Ecosystems and Environment

journal homepage: www.elsevier.com/locate/agee

Effect of nitrification and urease inhibitors on nitrous oxide and

methane emissions from an oat crop in a volcanic ash soil
Sara Hubea , Marta A. Alfaroa,* , Clemens Scheerb , Christian Brunkb , Luis Ramíreza ,
David Rowlingsb , Peter Graceb
a
Instituto de Investigaciones Agropecuarias, INIA Remehue. P.O. Box 24-O, Osorno, Chile
b
Institute for Future Environments, Queensland University of Technology, Brisbane, QLD 4000, Australia

A R T I C L E I N F O A B S T R A C T

Article history:
Received 4 February 2016 Nitrous oxide (N2O) emissions from the Chilean agricultural sector are primarily associated with the use
Received in revised form 7 May 2016 of nitrogen (N) fertilizers, so that the use of nitrification and urease inhibitors may represent a potential
Accepted 27 June 2016 mitigation option. However, their effectiveness in volcanic ash soils with high organic matter is still
Available online xxx uncertain. In this study, the effect of Dicyandiamide (DCD) and N-(n-butyl) thiophosphoric triamide
(NBPT) on soil N2O and CH4 emissions from an oat crop was determined using a fully automated
Keywords: greenhouse gas measuring system. Three urea treatments were evaluated: 120 kg N ha
1, 120 kg N
Emission factor ha
1 + DCD and 120 kg N ha
1 + NBPT (n = 3). A zero treatment (-N) was also considered. Annual N2O
NBPT
emissions ranged from 0.36 to 1.00 kg N2O-N ha
1 (P > 0.05) whereas the net uptake of CH4 ranged from
DCD
6.72 to 7.00 kg CH4-C ha-1 (P > 0.05). Increases in N2O emissions were associated with rainfall events, as
Emission intensity
Greenhouse gases characteristic of Mediterranean rain fed ecosystems. Soil CH4 uptake did not vary among treatments but
had a significant role offsetting N2O emissions, resulting in total emissions ranging from
17.1 to 239.0 kg
CO2eq ha
1 (P < 0.001). There was no significant effect of DCD or NBPT on average soil mineral N
concentration, and total N2O and CH4 emissions. NBPT significantly increased crop yield and crop N
uptake by 27% and 33% respectively (P < 0.05), resulting in greater N use efficiency. The use of DCD did not
increase productive parameters, but resulted in a 35% reduction of the N2O-N emission intensity
(P < 0.05). The study shows that volcanic ash soils have a relatively low N2O emission potential due to
physic-chemical characteristics and that NBPT could be used to increase N use efficiency in crops, while
DCD has a potential to reduce N2O losses per unit yield.
ã 2016 Elsevier B.V. All rights reserved.

1. Introduction is reactive and participates in the destruction of the ozone layer

Agriculture plays a major role in the global nitrous oxide (N2O)
fluxes, as this gas is produced mainly by soil microbial processes
occurring in fertilized soils (Paustian et al., 1995). Main sources of
N2O are nitrogen (N) fertilizers, urine and dung deposition during
grazing, and manure application onto grasslands (Snyder et al.,
2009). Globally, 3.3 Tg N2O-N yr
1 and 1.8 Tg N2O-N yr
1 are
emitted from fertilized croplands and grasslands, respectively.
Moreover, according to US-EPA (2006), an increase of 50% on N2O
emissions is expected worldwide by 2020 due to the increasing use
of N fertilizer and animal manure.
In the troposphere, N2O is stable for about 120 years and
contributes to the greenhouse effect, while in the stratosphere N2O
* Corresponding author.
E-mail addresses: malfaro@inia.cl, martita.alfaro@gmail.com (M.A. Alfaro).
(Crutzen, 1981). In soils, direct N2O emissions result from two
microbial processes: nitrification and denitrification. Nitrification
is the conversion of ammonium (NH4+) to nitrite (NO2
) and then
nitrate (NO3
); and denitrification is the conversion of NO3
to N2O
and then dinitrogen gas (N2). The conversion of NH4+ to N2 could be
complete, but a small and variable portion of the N escapes to the
environment, primarily as intermediaries, like N2O (Robertson and
Groffman, 2007; Snyder et al., 2009). Indirect N2O emissions can
also be produced by secondary deposition of ammonia volatiliza-
tion (NH3; Erisman et al., 2007).
During the last years several countries have evaluated options
to mitigate N losses and increase N-use efficiency (NUE) in crops
and pastoral lands using inhibitors of the N cycle, such as urease
and nitrification inhibitors (Di and Cameron, 2006; Subbarao et al.,
2006; Saggar et al., 2009; Zaman and Blennerhassett, 2010; Sanz-
Cobena et al., 2012).

http://dx.doi.org/10.1016/j.agee.2016.06.040
0167-8809/ã 2016 Elsevier B.V. All rights reserved.

Please cite this article in press as: S. Hube, et al., Effect of nitrification and urease inhibitors on nitrous oxide and methane emissions from an oat
crop in a volcanic ash soil, Agric. Ecosyst. Environ. (2016), http://dx.doi.org/10.1016/j.agee.2016.06.040
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Urease inhibitors reduce NH3 loses from surface applications Table 1

Initial soil characteristics (2nd of March 2012, 0–20 cm, n = 3,  standard error of the
of urea fertilizer, inhibiting urease activity and thereby retarding
mean).
the urea hydrolysis when applied to soils with the fertilizer,
increasing NUE. Ammonia itself is not a greenhouse gas; however, Parameter Results
it acts as a secondary source of N2O in the atmosphere and thus Clasificationa Typic Hapludands
contributes to global warming (Martikainen, 1985). One of the Series Osorno
Soil texture Loamy
most efficient urease inhibitors is the N-(n-butyl) thiophosphoric
pH H2O (soil:water, 1:2.5) 5.61  0.04
triamide (NBPT; Bremner and Chai 1986; Bronson et al., 1990; pH CaCl2 (soil:CaCl2, 1:2.5) 4.73  0.03
Gioacchini et al., 2002; Zaman et al., 2009). This inhibitor is Organic Matter, g kg
1 168.8  14.2
transformed in the soil to its oxon analog N-(n-butyl) phosphoric Available N, mg kg
1 19.99  0.65
triamide (NBPTO), which then forms a tridentate ligand with the Olsen P, mg kg
1 16.39  2.06
Available S, mg kg
1 8.06  1.43
urease enzyme, thus inactivating the enzyme (Manunza et al.,
Exchangeable Ca, cmol (+) kg
1 3.07  0.37
1999), slowing urea hydrolysis. Among the nitrification inhibitors, Exchangeable Mg, cmol (+) kg
1 0.76  0.10
dicyandiamide (DCD) has also been successfully tested (Zaman Exchangeable K, cmol (+) kg
1 0.73  0.03
et al., 2009; Di and Cameron, 2016). This odourless powder when Exchangeable Na, cmol (+) kg
1 0.10  0.02
Al Saturation, %b 7.27  0.29
applied to the soil inhibits ammonia oxidation, the first step of the
Initial bulk density, g cm
3 0.66  0.02
nitrification process, slowing the activity of nitrifying bacteria Finalc bulk density, g cm
3 0.72  0.01
responsible for the oxidation of NH4+ to NO2
, reducing NO3

a
concentration, and thus reducing N2O emissions. The NH4+ can be b
Acording to CIREN (2003).
Al Saturation: Proportion of available Al in relation to total cation content
adsorbed onto the soil cation exchange complex and then
(Ca + Mg + K + Na + Al).
gradually utilized for plant growth, favoring an increase of NUE c
Bulk density determination carried out at the site, in the natural grassland
by plants (Abbasi and Adams, 2000; Cameron et al., 2005; Di and established after harvest.
Cameron, 2016).
Agricultural soils can also produce or capture methane (CH4),
another greenhouse gas (GHG). Methane is emitted under and 13.1  C in winter, and 7.3 and 20.4  C in summer, respectively,
anaerobic soil conditions, like in paddy rice soils, by methanogenic as average of the last 39 years.
bacteria (Chan and Parkin, 2001), and it is consumed through the Three representative soil samples (0–20 cm) were collected
CH4 oxidation of methanotrophic bacteria in well-drained aerobic prior to tillage, for chemical and physical characterization
soils (McLain and Martens, 2006). following the methods compiled by Sadzawka et al. (2006), and
Chilean agricultural sector is the second largest emitter, outlined by Rowell (1997), respectively. Briefly, soil pH was
accounting for 15.1% of total GHG emissions in 2010 (13,825.6 measured in water and CaCl2 solution by potentiometry, organic
GgCO2 eq). Emissions from this sector increased by 29% since 1990, matter concentration was estimated using a modified Walkley-
mainly caused by the steady increase in the use of synthetic N- Black method by wet digestion. Exchangeable cations (Ca, Mg, K,
based fertilizers (MMA, 2014). The methodology used to estimate and Na) and exchangeable Al were extracted with a solution of
GHG from agricultural soils for the national GHG Inventory in Chile ammonium acetate (NH4Ac) 1 M at pH 7.0 and potassium chloride
includes the use of default Emission Factors (EF) given by the (KCl) 2 M, respectively, an analysed by atomic absorption
Intergovernmental Panel on Climate Change (IPCC). This approach spectrophotometry (AAS). Sulfate (SO4
) and phosphate (PO4
)
has the risk of not properly reflecting national circumstances, and anions were extracted in a solution of sodium bicarbonate
the development and improvement of country-specific emission (NaHCO3) 0.5 M at pH 8.5 and calcium dihydrogen phosphate
factors for major categories, such as agricultural soils are urgently (Ca(H2PO4)2) 1 M and analysed by the Murphy and Riley method
needed and prioritized by Chile (MMA, 2014). and turbidimetry, respectively (Tabatabai, 1982).
Little information is available on the effect of inhibitors on N2O Conventional tillage was used (0–20 cm), and lime (1 t ha
1,
emissions on cropping soils in Chile (Muñoz et al., 2010, 2011), and Soprocal) was applied 15 days before the experiment started, to
only one preliminary study reports CH4 capture in a grassland soil increase the initial soil pH (Table 1).
(Vistoso et al., 2014). We hypothesize that the use of N cycle
inhibitors, combined with the capacity of the volcanic soil to 2.2. Experimental design
capture CH4, will significantly reduce the overall GHG emissions of
an oat cereal crop. Thus, the aim of this study was to quantify the An oat cereal crop (Avena sativa cv Nehuén) was seeded on
effect of a urease and nitrification inhibitors on N2O and CH4 3  4 m plots on March 20th of 2012. Four treatments were
emissions, and its secondary effect on crop yield parameters. considered: Zero N (0 N kg N ha
1), Urea (120 kg N ha
1), Urea + N-
(n-butyl) thiophosphoric triamide (NBPT) as urease inhibitor
2. Materials and methods (120 kg N ha
1 + 0.25 g NBPT kg N
1), and Urea + Dicyandiamide
(DCD) as nitrification inhibitor (120 kg N ha
1 + 10 kg DCD ha
1),
2.1. Study site following a completely randomized block design (n = 3). The N rate
used is representative of that used by farmers for an oat crop in
The field experiment was conducted during the 2012/13 southern Chile, while the inhibitors rates used were based on the
cropping season at the Instituto de Investigaciones Agropecuarias respective commercial recommendations.
(INIA), Centro Regional de Investigación Remehue (40 310 S, 73 Each experimental plot received a basal fertilizer application at
030 W), in southern Chile. The soil at the experimental site was an seeding, including 150 kg P2O5 ha
1 (Triple Superphosphate, 46%
Andisol from the Osorno soil series (Typic hapludans; CIREN, P2O5), 150 kg K2O ha
1 (Potassium chloride, 62% K2O), 40 kg S ha
1
2003), of adequate soil fertility conditions for oat production (Gypsum, 18% CaSO4) and 40 kg MgO ha
1 (Magnesium oxide, 85%
(Table 1). Prior to the establishment of the experiment, the site had MgO). Also at this time all +N treatments received an initial N dose
been under permanent pasture (Lolium perenne L., Holcus lanatus L. of 30 kg N ha
1 (Urea, 46% N). The difference to complete the total N
and Dactylis glomerata L.) for a period of 25 years. rate was top dressed onto the plots at tillers stage (11th May 2012).
The mean annual rainfall at the experimental site is 1253.6 mm The inhibitors treatments were sprayed immediately after this
and the mean daily minimum and maximum temperatures are 4.3 second fertilizer application with the use of gardening water

Please cite this article in press as: S. Hube, et al., Effect of nitrification and urease inhibitors on nitrous oxide and methane emissions from an oat
crop in a volcanic ash soil, Agric. Ecosyst. Environ. (2016), http://dx.doi.org/10.1016/j.agee.2016.06.040
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sprayers. Only enough water to allow inhibitors spraying was used
( < 0.5 mm m
2), to avoid effects on urea dissolution.
Experimental plots were imaginary divided in half, one-half
was used for N2O and CH4 measuring, the remainder half was used
for destructive soil and crop sampling.
2.3. Soil environmental and climatic measurements
Soil temperature (10 cm depth) and chamber temperature was
measured every minute in conjunction with the automatic
sampling system using a PT100 probe (Temperature Controls
Pty, Australia). Daily values of air temperature (maximum,
minimum, and average), relative humidity, wind speed and rainfall
were recorded with an automatic weather station (CR10X-1 M
Model, Campbell Scientific Inc., Logan, Utah, USA) placed within
200 m of the experimental site.
2.4. Soil and plant analysis
Available soil N (NO3
-N and NH4+-N) and the soil gravimetric
water content were measured (0–20 cm) every 15 days after crop
establishment to explain seasonal variations of N2O and CH4 fluxes.
At each sampling date, two soil samples were collected from each
plot, and individually analysed for NO3
and NH4+ by extraction
with 2 M KCl (Sadzawka et al., 2006). Soil water content was
determined gravimetrically after drying the sample at 105  C for
24 h (Rowell, 1997). The water-filled pore space (WFPS) was
calculated from the gravimetric water content results, using results
of the initial soil bulk andparticle densities (Table 1). Bulk density
was again measured after harvest on the grassland naturally
developed at the site. This information was used to analyse the
implications for WFPS.
Plots were harvest on 22th January 2013 and samples from
straw, cape (including seed shell, glumes and the spike’s rachis)
and grain were collected from each plot to determine dry mater
(DM) concentration and total yield (t DM ha
1), at 60  C for 48 h
(Sadzawka et al., 2007). Total N in each component was
determined by the Kjeldahl digestion method (Sadzawka et al.,
2007) and used to calculate total N uptake per treatment. Using N
uptake values the apparent NUE was estimated according to:
NþN
NZ
NUE ¼  100
NA
where NþN is the nitrogen accumulation in the total aboveground
biomass (grain + straw + cape) in the +N treatment (kg), NZ is the
nitrogen accumulation in the total aboveground biomass (grain +
straw + cape) in the Zero N treatment (kg) and NA is the amount of
nitrogen applied (kg).
Yield information was also used to estimate the N2O emission
intensity of each treatment, calculated as the ratio of total N2O
emissions per unit of DM yield.
2.5. Continuous N2O and CH4 flux measurement
th
Fluxes of N2O and CH4 were measured from 20 of March 2012
to the 20th of March 2013 with a fully automated measuring system
(Scheer et al., 2012, 2013). Briefly, the system consists of twelve
acrylic static chambers (50 cm x 50 cm x 15 cm) fixed on stainless
steel bases inserted permanently into the soil (0.1 m). Two
chamber bases were located in each plot and the measuring
chambers moved between the two bases every two weeks in order
to minimise the effect of the chamber on crop growth and soil
properties, in agreement with Barton et al. (2008) and Scheer et al.
(2013). To reduce the impact of this management on the
quantification of cumulative N2O emissions, both bases areas
received the same area-equivalent rate of N at each fertilizer
Please cite this article in press as: S. Hube, et al., Effect of nitrification and urease inhibitors on nitrous oxide and methane emissions from an oat
crop in a volcanic ash soil, Agric. Ecosyst. Environ. (2016), http://dx.doi.org/10.1016/j.agee.2016.06.040
3
application. Acrylic extensions of 50 cm height were used to
increase the height of the chamber to a total height of 65 cm.
Extensions were added when the oat crop reached 15 cm height
(07th June 2012).
The automated chambers were sealed airtight during sampling
by two lids that opened and closed via pneumatic actuators. During
a measurement cycle, a set of four chambers closed (one chamber
per treatment) for 60 min and each chamber was sequentially
sampled for 3 min followed by a known calibration standard. This
process was repeated for each chamber four times over the closure
period before the chambers reopened and the next set of four
chambers closed. A complete full cycle of twelve chambers lasted
for 3 h, during which each chamber was sampled for 1 h and
remained opened for 2 h to restore ambient conditions, allowing
eight single fluxes to be determined per chamber each day.
To minimise a potential climatic impact of the chamber,
transparent tinted plastic coating was placed on the lids to reduce
heat build-up within the chambers when closed. A tipping bucket
rain gauge (Davis Instruments Corp. CA, USA) and a temperature
sensor inside of one chamber per experimental block were
connected to the system, to allow for automated opening of the
lids during rainfall events (more than 0.4 mm over a 5 min
1
period) and increases of temperature (over 40  C).
After chamber closure, air samples were automatically pumped
from the chamber’s headspace into the sampling unit with a flow
rate of 200 ml min
1. At the end of a 3 min sampling period a 3 ml
gas sample was injected into the carrier stream of a gas
chromatograph (SRI 8610C, Torrance/USA) equipped with a 63Ni
electron capture detector (ECD) for N2O analysis and a flame
ionization detector (FID) for CH4 analysis. To minimize the
interference of moisture vapour and CO2 on N2O measurement,
a pre-column filled with sodium-hydroxide-coated silica was
installed upstream of the ECD and changed regularly. Sample gas
measurements were calibrated automatically by a single point
calibration using certified gas standards (Air Liquide, Dallas, TX,
USA) of 0.5 ppm N2O and 2.5 ppm CH4. The detection limit of the
system was c. 2.0 mg N2O-N and 2.0 mg CH4-C m
2 h
1; and
10.0 mg N2O-N and CH4-C mg m
2 h
1 with extensions in placed,
for N2O. Sample dilution via leakage was considered negligible.
2.6. Flux calculations
Fluxes of N2O and CH4 from the automated chambers were
calculated from the slope of the linear increase or decrease of the
four concentrations measured over the enclosure time, similarly to
the procedure outlined by Barton et al. (2008). Flux rates were
expressed on an elemental weight basis as both mg N2O-N m
2 h
1
and g N2O-N ha
1 day
1 for N2O, and mg CH4-C m
2 h
1 and g CH4-
C ha
1 day
1 for CH4. The flux rates were calculated and corrected
for air temperature, atmospheric pressure and the ratio of chamber
volume to surface area as follows:
b  V CH  MW  60  106
F¼
ACH  MV corr  109
where F is the hourly flux rate (mg m
2 h
1), b is the increase in
headspace concentration (ppb min
1), VCH is the volume of the
measuring chamber (m3), MW is the molecular weight of the gas
(28 for N2O-N and 12 for CH4-C), 60 converts minutes to hours, 106
converts g to mg, ACH is the basal area of the measuring chamber
(m2), MVcorr is the pressure and temperature corrected molecular
volume (m3 mol
1), 109 converts ppb to mL m
3.
 
273:15 þ T p0
MV corr ¼ 0:02241  
273:15 p1
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where 0.02241 m3 is 22.41 L mol volume, T is the chamber
temperature during the measurement (Kelvin), p0 is air pressure
at sea level and p1 is the air pressure at the experimental site. Air
pressure at the site was estimated from the height above sea level
using a barometric equation.
Daily fluxes were calculated by averaging sub-daily measure-
ments for each chamber over the 24 h period (midnight to
midnight) before averaging across the three replicate chambers. To
calculate seasonal cumulative fluxes, calculated daily fluxes were
then summed according to the measurement period before
averaging across the three replicate chambers. Gaps in the dataset
were filled by linear interpolation across missing days.
2.7. Equivalent CO2 emissions (CO2 eq) and emission factor
The concept of CO2 eq allows assessing the radiative forcing of
different GHG’s relative to the reference gas, in this case CO2, over a
specific time horizon. The study only refers to the equivalent
emissions of CH4 and N2O emissions from the investigated
agroecosystems. Soil-borne CO2 emissions were not taken into
account for the calculation. The equivalence was calculated using
the CO2-equivalents of 25 and 298 for CH4 and N2O, respectively
(IPCC, 2007), and expressed in kilograms of carbon dioxide
equivalents per hectare per day. The CO2 eq concept allows
comparing N2O and CH4 fluxes and assessing the relative
contribution of these two gases on a reference scale, according
to following formulas:
gN2 O
N 44
GWPðN2 OÞ½gCO2 eq=ha=day ¼ x1   298
haday 28
gCH4
C 16
GWPðCH4 Þ½gCO2 eq=ha=day ¼ x2   25
haday 12
where x1 = average daily N2O-N emission rate (g N2O-N ha
1 day
1)
x2 = average daily CH4-C emission rate (g CH4-C ha
1 day
1)
The EF for N-N2O, this is the fraction of N applied lost as N2O,
discounted the emission in the Zero N treatment, was estimated as
follow:
 
A
B
Emission Factor ðEFÞ ¼  100
C
where A is N2O-N emitted (kg) in any of the +N treatment, B is N2O-
N emitted (kg) by the Zero N treatment, and C is the amount of N
applied (kg).
and moderately acid water pH (Table 1), typical characteristics in
most Andisols of this area (Vistoso et al., 2012; Alfaro et al., 2006).
A total of 814.6 mm rain fell during the study period, lower than
the 39-yr average (1253.2 mm) for this site for the same period. The
minimum, maximum and mean air daily temperature over the
experimental period was 5.9, 16.4 and 10.7  C respectively, similar
to the 39-yr average (5.8, 16.8, 11.2  C, respectively). The minimum
air temperature was registered during June (-4.1  C) and the
maximum air temperature was registered during January (33.4  C).
Average soil temperature (0–10 cm) was 12  C with the lowest soil
temperatures in the June to August period and minimum soil daily
temperature of 4.9  C during July. Highest soil temperatures were
observed in the January–February period with a maximum of
20.7  C during January (Fig. 1b).
The WFPS (0–20 cm) varied over the season in response to
rainfall events, ranging from 33.0 to 85.3%, with the lowest value
registered in the summer (end of January) and the highest value
registered in winter (June) (Fig. 1a), but did not differ among
treatments (P > 0.05). The final bulk density check was carried out
after the experiment was finished providing a final value of
0.72  0.01 g cm
3 (0–20 cm depth). When bulk density was used
to re-estimate the WFPS, the new values obtained ranged between
37.6 to 97.4%, because of a reduction in total pore space from 62.7 to
59.6% over the period.
3.2. Soil NO3
and NH4+
Soil available N (NO3
-N + NH4+-N) varied during year, with the
highest concentrations prior to the tiller stage N fertilizer
application (Figs. 2a and b).
At the beginning of the cropping season soil NH4+-N varied
between 11.32  1.78 and 63.71 11.94 mg kg
1, with higher

2.8. Statistical analysis

Effects of treatments on soil available N, total N2O and CH4

emissions, C balance, crop yield, NUE and EF were assessed by
ANOVA. Normality was evaluated using the Shapiro-Wilk analysis.
For N2O calculations, linearity was checked in all cycles and only
linear fluxes were used to estimate cumulative N2O emissions
(>90% of flux cycles). Fisher test was used to determine the effect of
specific treatments at 95% confidence level using Genstat 12.2
(Genstat, 2003).

3. Results

3.1. Soil conditions and seasonal variability for environmental

parameters
Soil at the experimental site had a high organic matter (OM)
concentration (169 g kg
1) with a medium range of P, low S content
Fig. 1. Minimum and maximum daily air temperature ( C) and average soil
temperature ( C, 10 cm depth) (a), and daily precipitation (mm), plus water-filled
pore space (WFPS, %) by treatments (b) during the experimental period (March
2012–March 2013).

Please cite this article in press as: S. Hube, et al., Effect of nitrification and urease inhibitors on nitrous oxide and methane emissions from an oat
crop in a volcanic ash soil, Agric. Ecosyst. Environ. (2016), http://dx.doi.org/10.1016/j.agee.2016.06.040
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Fig. 2. Soil ammonium (NH4+-N; a) and nitrate (NO3
-N; b) concentrations

(mg N kg dry soil
1, 0–20 cm) over time during the experimental period (March
2012–March 2013). The arrows indicate the timing of fertilizer application. Initial
soil available N was 20  0.65 mg kg
1 dry soil.

concentrations in the fertilized plots (P < 0.05). Fifty days after
seeding, concentrations increased up to 64.40  11.64 and
204.95  37.73 mg kg
1 (Fig. 2a), for the Zero N and +N treatments,
respectively (P > 0.001). Twelve days after the second urea
application a significant increase of concentration was observed
in the Urea + DCD treatment (204.44  24.40 mg kg
1), in compar-
ison to the Urea + NBPT and urea treatments (100.65  7.14 and
79.86  7.04 mg kg
1, respectively). All +N treatments were higher
than the Zero N (4.39  4.60 mg kg
1) (P < 0.001) (Fig. 2a). During
winter (June-September), soil N-NH4 was low (Fig. 2a).
Soil NO3
-N concentrations after seeding were low, varying
between 27.85  6.88 and 37.73  4.7 mg kg
1 (P > 0.05), with a
significant increase after 50 days of seeding for all treatments
(Fig. 2b) up to 58.87  11.68 to 210.81 11.95 mg kg
1, in the Zero N
and +N treatments, respectively (P < 0.001). After tiller stage
application (12 and 40 days), the Urea + NBPT treatment showed an
increase in concentration in relation to the other treatments
Fig. 3. Daily N2O-N fluxes (a) and daily CH4-C fluxes (b) over time for the different
treatments during the experimental period (March 2012–March 2013). Original
data with no interpolation. The arrows indicate the timing of fertilizer application.
(P < 0.05), similarly to what was observed in February 2013
(P < 0.05; Fig. 2b).
3.3. Plant yield and N uptake
Total aboveground biomass was in the range of 7.03–16.13 t
ha
1, with the highest biomass in the Urea + NBPT treatment
(Table 2; P < 0.001). This resulted in a greater NUE in this treatment
(Table 2; P < 0.05).
3.4. Daily and cumulative N2O and CH4 fluxes
3.4.1. N2O fluxes
Daily N2O-N emissions showed high temporal and spatial
variability (Fig. 3). Values ranged from
4.56  0.03 to 26.19  5.31

Table 2
Crop productive parameters, greenhouse gases emission and carbon balance for the different treatments (n = 3,  standard error of the mean).

Parameter Zero N Urea Urea + DCD Urea + NBPT

Crop Total yield t ha
1 7.03  0.69 c 12.70  1.29 b 11.26  0.67 b 16.13  0.54 a
N grain g kg
1 17.00  1.00 a 16.33  0.33 a 13.00  1.73 a 13.33  1.76 a
N straw g kg
1 6.67  0.88 a 6.00  0.00 a 6.33  0.33 a 6.33  0.88 a
N uptake kg N ha
1 47.45  6.72 b 78.35  8.02 ab 72.18  1.23 ab 104.21  17.30 a
N use efficiency % No 25.75  7.19 b 20.61  5.28 b 47.30  9.29 a

GHG emissions Annual N2O-N emission kg N2O-N ha
1 0.36  0.12 a 1.00  0.30 a 0.55  0.25 a 0.98  0.36 a
N2O emissions intensity g N2O-N t DM
1 53.92  22.95 b 76.39  16.31 a 49.28  21.27 b 61.59  23.55 a
N emission factor % No 0.54  0.30 a 0.16  0.19 a 0.52  0.21 a
CH4-C uptake kg CH4-C ha
1 -6.72  0.45 a
7.00  0.11 a
6.96  0.03 a
6.99  0.31 a
Carbon balance kg CO2 eq ha
1
17.08  77.90 c 238.96  143.25 a 28.72  115.69 b 263.67  188.48 a

Different letters in columns indicate significant differences among treatments (P < 0.05).

Please cite this article in press as: S. Hube, et al., Effect of nitrification and urease inhibitors on nitrous oxide and methane emissions from an oat
crop in a volcanic ash soil, Agric. Ecosyst. Environ. (2016), http://dx.doi.org/10.1016/j.agee.2016.06.040
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g N2O-N ha
1 day
1, with one emission peak in all +N treatments
22 days after the tiller stage application and the highest values for
Urea + NBPT (26.19  5.31 g N2O-N ha
1 day
1) followed by Urea
and Urea + DCD (21.215  4.05 and 11.36  0.77 g N2O ha
1 day
1,
respectively). Emission decreased in all treatments during winter
until background values. Annual N2O-N emissions did not differ
among treatments (P > 0.05; Table 2).
3.4.2. CH4 fluxes
Daily CH4 fluxes varied between
1.86  1.54 and
66.38
 21.65 g CH4-C ha
1 day
1, without differences between treat-
ments (P > 0.05). During spring, CH4-C uptake increased, with an
average uptake rate of 63.1 1.43 g CH4-C ha
1 day
1.
Annual CH4-C uptake ranged between 6.72  0.45 to
7.00  0.11 kg CH4-C ha
1 (Table 2), without differences between
treatments (P > 0.05).
3.5. Intensity of emissions, CO2 eq and Emissions Factors
3.5.1. Intensity of N2O-N emission
Intensity of N2O-N emission varied between 49.3  21.3 to
76.4  16.3 g N2O-N t
1 (Table 2). The largest emission intensity
was observed in the Urea and Urea + NBPT treatments (76.4  16.3
and 61.6  23.5 g N2O-N t
1, respectively; P < 0.05). The use of DCD
reduced emission intensity, in comparison to other +N treatments.
3.5.2. CO2 eq emissions and Emission Factors
Equivalent CO2 emissions were positive in all +N treatments
(Fig. 4), however a significant reduction of emissions was observed
when including the capture provided by CH4 (P < 0.001).
Emission factors were lower than 1% in all treatments (Table 2),
with no differences among +N treatments (P > 0.05).
4. Discussion
Daily N2O-N emissions ranged from
4.56 to 26.19 g N2O-
N ha
1 day
1, with a total loss ranging from 0.4 to 1 kg N2O-
N ha
1 yr
1, and no effect of N addition on overall emissions. These
values are in the lower range of emissions reported globally across
a variety of climatic regions, with values usually varying from 0.3 to
16.8 kg N-N2O ha
1 yr
1 and lower values in low N inputs systems
(Stehfest and Bouwman, 2006), suggesting a low potential of
cropping areas for N2O emissions under Mediterranean-rain fed
conditions. This agrees with the low EF reported by Cayuela et al.
(2016) in a meta-analysis carried out for Mediterranean areas.
Fig. 4. Equivalent greenhouse gas emissions (CO2 eq) per treatment for the
experimental period (March 2012–March 2013).
Please cite this article in press as: S. Hube, et al., Effect of nitrification and urease inhibitors on nitrous oxide and methane emissions from an oat
crop in a volcanic ash soil, Agric. Ecosyst. Environ. (2016), http://dx.doi.org/10.1016/j.agee.2016.06.040
Annual emissions reported in this paper are based on one year of
measurements, although a greater length would have been
desirable to account for climate variability (Barton et al., 2008).
The impact of irrigation on N2O emissions remains as an area of
interest for crops production in southern Chile, as it is expected
this management would increase emissions (Saggar et al., 2013;
Cayuela et al., 2016).
Highest N2O emissions were associated with the start of the
rainfall season rather than to N application (Figs. 1 and 3), in
agreement with Mummey et al. (1997) and Barton et al. (2008) for
annual N2O losses from rain fed systems such as those under
Mediterranean conditions. We recorded eighteen 10 mm rainfall
after seeding, and 14 of them occurred during the 60 days after
fertilizer application (Fig. 1), corresponding to 39% of the total
annual rainfall. Our results could be related to the increase in N
mineralization during the summer period, which would lead to
higher soil available NO3
with the first rainfall of the autumn, as
described for andisoils in the area of the study (Lagos et al., 2014),
and thus, to greater potential N losses as N2O at this time of the
year.
After the tiller stage fertilizer application the most significant
increases in soil available N were found in all +N treatments with a
significant effect of the NBPT for soil NH4+-N, consistent with
results of Vistoso et al. (2012) for N autumn application (April) on a
grassland Andisol. Moreover, soil NO3
-N increased 12 days after
the tiller stage application in all +N treatments, in agreement with
nitrification periods reported previously by Cartes et al. (2009) and
Cardenas et al. (2013) for this soil type. Nevertheless, this was not
associated to further N2O peaks.
The low N2O emissions and its lack of relationship with soil
available N has been previously described for volcanic ash soils of
southern Chile (Vistoso et al., 2012; Alfaro et al., 2016). This would
agree with previous studies by Huygens et al. (2007) and Cardenas
et al. (2013), who show reduce soil N availability, due to high
dissimilatory nitrate reduction to ammonium (DNRA) activity and
NH4+ adsorption on clay minerals, respectively, as mechanisms
limiting the oxidation of NH4+ to NO3
and thereafter. This results
in reduce leaching (Salazar et al., 2012) and gas (Alfaro et al., 2016)
losses. It is also possible than in this soil, a grater abundance of
nosZI denitrifying genes in soil bacteria (Cardenas et al., 2013)
would result in a complete (N2) rather than incomplete (N2O)
denitrification of the small amounts of soil available NO3
. In
addition, preliminary results of our group show that nosZII is
present in bacterial DNA from these soils (data not shown),
contributing again to total denitrification, although future studies
are necessary to clarify the importance and function of this gene in
Chilean volcanic ash soils.
The frequency of soil N sampling could have prevented the
detection of differences among treatments for soil available N
concentrations closer to N fertilizer addition, or it might have
resulted in incomplete soil N peaks detection. We suggest that in
further studies, soil N sampling frequency after N addition should
be increased to avoid this bias.
The EF calculated for the use of urea in this experiment was half
the 1% default IPCC value (IPCC, 2006) currently being used in the
Chilean GHG Inventory. Thus, this information would contribute to
the accuracy of the Chilean inventory. These low values agree with
those reported by Cayuela et al. (2016) for rain fed Mediterranean
regions (>450 mm rainfall per year). Furthermore, although no
significant, the use of DCD reduced the EF by 70%, from 0.54%
(Urea) to 0.16%, representing a potential alternative for further
reductions, in concordance with Akiyama et al. (2010) and Cayuela
et al. (2016), having positive and significant implications for the
overall CO2 balance of the system. The lack of statistical
significance was associated to the high variability of N2O emissions
registered, in agreement with Saggar et al. (2009). The addition of
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NBPT did not reduced the EF, probably because the hydrolysis of
urea is not directly related to N2O emissions, in contrast to
nitrification. Thus, the use of NBPT would only delay the hydrolysis
of urea, but all the fertilizer would eventually be hydrolysed
(Akiyama et al., 2010). The EF values reported here, although low
are higher than those generated by Vistoso et al. (2012) for the use
of urea in a grassland Andisol. This was probably associated to the
added effect of i) timing of application, as a result of increasing
losses at the beginning of the winter period in this experiment and,
ii) increasing rates (84 versus 45 kg N ha
1 applied in the present
versus the grassland study, respectively).
Our results for CH4-C uptake are substantially higher than
values reported for other arable cropping systems. In a meta-
analysis, Dalal et al. (2008) reported mean rates of CH4
consumption of 3.1 1.6 g CH4-C ha
1 day
1 for arable cropping
soils. This is 10–20 times lower than the average uptake rates
observed in our study. This high CH4 uptake provides evidence of a
natural high uptake capacity of the Andisol, related to its high
porosity and low bulk density, allowing for high rates of gas
diffusion into the soil. It has been demonstrated that soil gas
diffusivity is the main regulator of CH4 uptake, being influenced by
a combination of dynamic (soil moisture content and soil
temperature) and structural (soil bulk density and soil porosity)
factors (Fest et al., 2009). The annual uptake rates reported here are
also 1.9 times higher than those reported for and andisoil in Japan
by Yonemura et al. (2013), who registered an uptake of 3.6 kg CH4-
C ha
1 yr
1. In both cases, a low uptake over the winter period was
measured associated to an increase in soil moisture and the
corresponding lack of adequate aerobic conditions, which together
with low temperatures, would reduce bacterial activity. In the case
of the current study, an increase in CH4-C uptake during the crop-
growing season was observed, probably related to C sequestration
as plant biomass and its transformation into soil organic matter, in
agreement with Johnson et al. (2007).
The high CH4 uptake registered in all treatments provides
evidence of the soil natural capacity to capture C, with a significant
contribution to N2O emissions offsetting, especially when DCD was
applied, as in this treatment N2O emissions were reduced to
background levels (Fig. 4), resulting in a significant reduction in the
overall CO2 emissions for this cropping system.
High CH4 uptake is a potential opportunity for N2O mitigation
within the agricultural sector of Chile, and for Mediterranean
cropping systems with increasing soil activity. It would also
contribute to reach the target of 30% reduction in emissions per
unit of economic growth, in relation to the 2007 reference value,
submitted by Chile to the United Nations Framework on Climate
Change (UNFCC) as it Intended Nationally Determined Contribu-
tion (iNDC) for the 2020–2030 period (Gobierno de Chile, 2015).
The lack of significant effect of the use of DCD on crop yield and
NUE could limit farmers’ adoption of this technology because of
the additional cost associated to the inclusion of this practice and
the lack of direct benefit. As discussed previously, this would be
associated to the NH4+ adsorption and limited availability in soil
solution, so that nitrification (and then denitrification) would be
naturally limited in the soil with no effect of external inhibitors
additions. Additionally, this could also be associated to the use of a
cereal for the experiment and the low N input applied, as impacts
of inhibitors on productivity and NUE have been reported to be
significant (25% increase in yield and 32% increase in NUE) in
permanent grasslands (Di and Cameron, 2016), and with applica-
tion rates >300 kg N ha
1 (Abalos et al., 2014). However, its effect
on N2O emissions intensity reduction and overall CO2 balance, if
compared with the other +N treatments, opens a window of
opportunity as a potential mitigation option in Mediterranean
cropping ecosystems, especially in vegetables or industrial crops (
Abalos et al., 2014).
Please cite this article in press as: S. Hube, et al., Effect of nitrification and urease inhibitors on nitrous oxide and methane emissions from an oat
crop in a volcanic ash soil, Agric. Ecosyst. Environ. (2016), http://dx.doi.org/10.1016/j.agee.2016.06.040
7
In contrast, the yield increase observed in the N + NBPT
treatment creates an opportunity to increase NUE in such cropping
systems, in agreement with Abalos et al. (2014), who also reported
an increase in crop yield with the use of this inhibitor, in
comparison with the use of nitrification inhibitors. Areas of further
work include the application of reduced N rates associated to the
use of this inhibitor, in response to an increase of 1.8 times in the
NUE. It is possible that the proposed reduction for rain fed areas
could account for the additional cost of using this product for
farmers, favouring the adoption of this technology. Furthermore,
the use of NBPT did not directly reduce N2O emissions but it can
reduce NH3 volatilization, as studies on grassland soils in the area
have shown (Salazar et al., 2014; Alfaro et al., 2016), which would
reduce indirect N2O losses by reducing secondary N2O deposition
after NH3 volatilization. In these field experiments, the reduction
on NH3 volatilization has ranged between 8% (cold and wet
conditions) and 30% (dry and warm conditions) of the N
application (100 kg N ha
1 equivalent, urea application; Salazar
et al., 2014). Because in the present experiment the N + NBPT
application was carried out under wet and cooler conditions (May),
and a lower N input was used (84 kg N ha
1), it is expected that NH3
volatilization losses would be found at the lower end of this range.
This would also agree with the NH4+ soil data, which were lower in
the Urea + NBPT treatment at the beginning of the experiment,
immediately after the second N and inhibitors addition. Our results
suggest that the use of NBPT represents a clear opportunity to
increase NUE because of the potential significant impact of this loss
on effective N availability for the crop. Thus, an integrated strategy
considering both inhibitors throughout the year may improve both
the crop NUE, and thus the economic benefit for famers, and the
environmental performance of agricultural systems.
Further studies are required to develop mechanisms to enhance
CH4 capture in these volcanic soils, as together with the low
potential for N2O losses and the mitigation effect of DCD, it
represents an opportunity for the development of low C emitting
cropping systems in volcanic ash soils.
5. Conclusions
Total N2O losses were low and ranged from 0.36 to 1.00 kg N2O-
N ha
1 yr
1. The highest peak of emissions was associated with the
start of the rainfall season rather than the time of the N application,
probably associated to soil processes limiting the oxidation of NH4+
to NO3
and thereafter. The EF calculated for the use of urea as
fertilizer in crops under rainfed conditions was half the 1% default
IPCC value. The high CH4 uptake registered in all treatments
provides evidence of the soil natural capacity to capture C, which
almost completely offset N2O emissions, resulting in a significant
reduction in the overall CO2 eq emissions. The use of N + DCD
resulted in a significant reduction of the N2O emission intensity.
The increase in yield observed in the N + NBPT treatment creates an
opportunity to increase NUE in cropping systems, while having a
significant reduction on indirect N2O losses by reducing secondary
N2O deposition after NH3 volatilization. The study shows that
volcanic ash soils have almost neutral emissions due to their
unique nature of high methane CH4 uptake and limited NH4+ soil
availability. The combination of these processes provides a
potential opportunity for GHG mitigation within the agricultural
sector of Chile.
Acknowledgements
We thank to the Australian Government for the donation of the
equipment, and to INIA for funding this research. We also thank to
Mrs. Ana Rosas for her help in laboratory matters.
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Please cite this article in press as: S. Hube, et al., Effect of nitrification and urease inhibitors on nitrous oxide and methane emissions from an oat
crop in a volcanic ash soil, Agric. Ecosyst. Environ. (2016), http://dx.doi.org/10.1016/j.agee.2016.06.040