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Agriculture, Ecosystems and Environment 149 (2012) 64– 73

Contents lists available at SciVerse ScienceDirect

Agriculture, Ecosystems and Environment

jo u r n al hom ep age: www.elsev ier .com/ locate /agee

aseous emissions of N2O and NO and NO3− leaching from urea applied with

rease and nitrification inhibitors to a maize (Zea mays) crop

lberto Sanz-Cobena ∗, Laura Sánchez-Martín, Lourdes García-Torres, Antonio VallejoTSI Agrónomos, Technical University of Madrid, Ciudad Universitaria, 28040 Madrid, Spain

r t i c l e i n f o

rticle history:eceived 10 September 2011eceived in revised form9 December 2011ccepted 20 December 2011vailable online 20 January 2012

eywords:rease inhibitoritrogen losses

rrigationitrification

a b s t r a c t

Urea has become the predominant source of synthetic nitrogen (N) fertilizer used throughout the world.Among the various available mitigation tools, urease inhibitors like NBPT have the most potential toimprove efficiency of urea by reducing N losses, mainly via ammonia volatilization. However, there is alack of information on the effect of N-(n-butyl) thiophosphoric triamide (NBPT) on other N losses such asgaseous emissions of N2O and NO and NO3

− leaching. A two-year field experiment using irrigated maize(Zea mays) crop was carried out under Mediterranean conditions to evaluate the effectiveness of ureacoated with NBPT (0.4%, w/w) alone and with both NBPT and nitrification inhibitor dicyandiamide (DCD)(0.4 and 3%, w/w, respectively) to mitigate N2O–N, NO–N and NO3

−–N losses. The different treatmentsof U, U+NBPT and U+NBPT+DCD were applied to the maize crop in 2009 and then in 2010. The 2010maize crop followed a fallow period, during which the 2009 crop residues were incorporated into thesoil. Two different irrigation regimes were followed each year. In 2009, irrigation was controlled for thefirst 2 weeks following urea fertilization; whereas, the 2010 crop period was characterized by increasedirrigation in the same period. After each treatment application, measurements of the changes in soilmineral N, gaseous emissions of N2O and NO, nitrate leaching and biomass production were made. N2Oemissions were effectively abated by NBPT and NBPT+DCD and were reduced by 54 and 24%, respectively,in 2009. A reduction in nitrification rate by the inhibitors was also observed during 2009. In 2010 croppingperiod, NBPT reduced N2O emissions by 4%; while the combination of NBPT and DCD treatment reducedN2O emission by 43%. Yield-scaled N2O emissions were reduced by 50 and 18% by NBPT and the mixtureof NBPT+DCD, respectively, in 2009. Applying inhibitors did not have any significant effect on yield-scaled N2O emissions in the 2010 crop period. Total NO losses from urea were 2.25 kg NO–N ha−1 inthe 2009 crop period and 5 times lower in the following year; this may provide an indicator of the

prevalence of nitrification as the main process in the production of N2O in the 2009 maize crop. Most ofthe NO3

− was lost within the fallow period (i.e. 92, 81 and 75% of the total NO3− leached for U, U+NBPT

and U+NBPT+DCD, respectively), so the incorporation of crop residues was not as effective as expected atreducing these N losses. Our study suggests that the effectiveness of NBPT and combination of NBPT+DCDin reducing N losses from applied urea is influenced by management practices, such as irrigation, andclimatic conditions.

. Introduction

The use of synthetic N fertilizers has increased in response tontensification of agricultural systems (Erisman et al., 2007), buthis brings with it high associated N losses from agricultural soilso the environment (Bouwman et al., 2002). Of the different types of

fertilizer that exist, urea has become the predominant source ofnorganic N used throughout the world (Harrison and Webb, 2001).owever, its use has been associated with large ammonia (NH3)

∗ Corresponding author. Tel.: +34 913363257; fax: +34 913365639.E-mail address: a.sanz@upm.es (A. Sanz-Cobena).

167-8809/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.agee.2011.12.016

© 2011 Elsevier B.V. All rights reserved.

losses, especially when applied at less than optimum conditionssuch as low moisture and high soil temperature. This reduces theeffectiveness of the fertilizer applied and also poses environmentaland health problems associated with processes of acidification, Nenrichment of natural ecosystems, and the formation of air-bornefine particulate matter, respectively (Turner et al., 2010). Addition-ally, the application of urea to agricultural systems also enhancesthe release of other N forms to both the atmosphere, such as nitricoxide (NO) and nitrous oxide (N2O) (Meijide et al., 2009) and to the

water bodies, such as nitrate (e.g. Díez et al., 2000; Menéndez et al.,2009). Nitric oxide is a precursor of tropospheric ozone and alsoa source of N in atmospheric deposition (Davidson and Kingerlee,1997). Nitrous oxide is an important greenhouse gas (GHG), which

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A. Sanz-Cobena et al. / Agriculture, Eco

ontributes to both global warming and the destruction of thezone layer (Crutzen, 1979; IPCC, 2007) whereas nitrate leachings a major worldwide cause of groundwater N pollution.

The development of N abatement techniques from urea-ertilized soils has become a priority, but their efficiency for

itigating each emitted N form in agroecosystems is highly relatedo the mechanism producing and consuming these compounds inoils. Therefore, a unique strategy does not exist for mitigating allmitted N compounds in all situations. In this context, the use ofrease inhibitors has been the most encouraged strategy to miti-ate NH3 volatilization in urea-fertilized soils (UNECE, 2001). Theseompounds effectively abate NH3 emissions by slowing the hydrol-sis of urea, which limits the pool of NH4

+ potentially lost througholatilization (Sanz-Cobena et al., 2008; Turner et al., 2010). Of thearious types of urease inhibitors that have been identified andested, N-(n-butyl) thiophosphoric triamide (NBPT) has been the

ost widely used (Xiang et al., 2008) and has already been foundo be highly effective at mitigating NH3 emissions at relatively lowoncentrations under both laboratory (Gill et al., 1997) and fieldonditions (Sanz-Cobena et al., 2008). However, there have beenery few studies showing the effect of urease inhibitors on theelease of other N forms (i.e. NO, N2O and NO3

−) from urea-basedertilizers. Zaman et al. (2008, 2009) found a small effect of NBPTn the reduction of N2O emissions in urine-fertilized pastures. Byontrast, Menéndez et al. (2009) did not find any effect on N2Omissions from grassland fertilized with urea under different WPFS,lthough they did find a positive effect of NBPT reducing NO emis-ions when WFPS was below 60%. In cropland, the effectivenessf NBPT in mitigating N2O losses was recently evaluated by Dingt al. (2011), who observed a mitigating effect of 37.7% for NBPT in

urea-fertilized irrigated maize crop. Furthermore, to our knowl-dge, no research has been found into the effects of NBPT on thebatement of N losses from urea-fertilized croplands to either thetmosphere or to groundwater in the form of NO, N2O or NO3

−.ur main hypothesis was that a lower pool of NH4

+ in soils as con-equence of a urease inhibitor could diminish the nitrification rateffecting both emission of N2O and NO, which are very dependentn this mechanism in urea-fertilized soils (Sánchez-Martín et al.,010).

Nitrification inhibitors, such as dicyandiamide (DCD), have beenffectively used to reduce N2O, NO and NO3

− losses from urea fertil-zed soils (e.g. McCarty et al., 1990). These compounds can depresshe activity of ammonia monooxygenase enzyme in soil, therebyelaying NH4

+ oxidation to hydroxylamine, which is further oxi-ized into first nitrite (NO2

−) and then nitrate (NO3−) (Prasad and

ower, 1995). In consequence, a reduction in the N2O producedhrough this pathway would be expected to occur (Firestone andavidson, 1989). However, since the application of DCD to ureaoes not affect the urea hydrolysis rate, ammonia volatilization isot reduced and it could even be increased due to the extendedresence of NH4

+ in the upper part of soil. In this sense, a possibletrategy to enhance the effectiveness of urea is the combination ofBPT with DCD, therefore maintaining the potential benefit of each

nhibitor.The efficiency of both nitrification and urease inhibitors in soils

s very dependent on the water-filled pore space (WFPS) of the soiluring the period after application (Zaman et al., 2009). In irrigatedgroecosystems, the addition of water may promote soil conditionshat could affect the capacity of NBPT and DCD to mitigate N lossesrom urea fertilization. In this sense, controlled irrigation during theays following urea application would prevent an increase in soiloisture, favoring nitrifying conditions and probably increasing

he efficiency of these inhibitors to mitigate emissions of N oxides.In irrigated maize crops under Mediterranean climate, nitrate

eaching occurs during the irrigation period, and following the crop,n autumn and winter (fallow period), when rainfall is often high

s and Environment 149 (2012) 64– 73 65

(Díez et al., 2000; Arregui and Quemada, 2006). In order to miti-gate N leaching in the fallow period, a recommended strategy is toincorporate crop residues with a high C:N ratio, which promotesN immobilization (e.g. Gentile et al., 2008). However, the incorpo-ration of crop residues could also affect mechanisms of N oxidesproduction and/or consumption, especially denitrification. Addi-tionally, a higher amount of available organic C, slowly mineralizedby microorganisms, could enhance N2O emission.

In this context, a two-year field study was carried out with amaize (Zea mays) crop fertilized with urea in a semiarid soil in Cen-tral Spain. The main objective of the study was to evaluate the effectof irrigation on the effectiveness of the urease inhibitor NBPT toincrease the N-use efficiency of urea-based fertilizers (i.e. by reduc-ing N losses via N2O and NO emissions and NO3

− leaching withcrop residues) and to compare this abatement effect with that of acombination of NBPT and the nitrification inhibitor DCD.

2. Materials and methods

2.1. Experimental location

The field experiment was located at the ‘El Encín’ field station,38 km east of Madrid, Central Spain (latitude 40◦32′N, longitude3◦17′W). The mean annual temperature and rainfall in this area are13.2 ◦C and 430 mm, respectively. The soil type is a Calcic Haplox-erepts (Soil Survey Staff, 1992) with a sandy clay loam texture (clay,28%; silt, 17%; sand, 55%) in the upper (0–28 cm) horizon. Someof the physico-chemical properties of the top 0–28 cm of the soillayer, measured by standard methods of soil analysis (Burt, 2004),were: total organic C, 8.2 ± 0.4 g kg−1; pHH2O, 7.9; bulk density,1.4 ± 0.1 g cm−3 and; CaCO3, 13.1 ± 0.3 g kg−1.

2.2. Experimental design

This study focused on two consecutive crop periods(April–October 2009 and April–October 2010) and the fallowperiod between them (October 2009 to April 2010). Twelve(8 m × 8 m) plots were selected and arranged, according totreatments, in a randomized complete block design with threereplicates per treatment. The same plots were maintained for eachtreatment within the two consecutive crop periods. The fertilizertreatments used were: urea (U); urea coated with the ureaseinhibitor NBPT (commercial name Agrotain®) (U+NBPT) to give aweight-based proportion of inhibitor in the mixture of 0.4% (i.e.1 kg NBPT ha−1); urea coated with a mixture of the nitrificationinhibitor dicyandiamide (DCD; 3%, w/w urea) and NBPT (1:7)(U+DCD+NBPT) (i.e. 7.5 kg DCD ha−1 and 1 kg NBPT ha−1); and asoil without any N fertilizer application (i.e. control, C). A blanketbasal application of 50 kg P ha−1, applied as triple superphosphate,and 50 kg K ha−1, applied as potassium sulfate, was applied tothe surface as seedbed. Nitrogen fertilizers were homogeneouslyapplied by hand on 19th June 2009 and 23rd June 2010 at a rateof 250 kg N ha−1. Maize was sown on 27th April and 28th April for2009 and 2010, respectively, at a density of 115,942 seeds ha−1

each year. A sprinkler was set up for irrigation in the middle ofeach plot. Two different irrigation regimes were followed in anattempt to promote contrasting soil moisture conditions underwhich the capacity of the inhibitor to effectively abate N losseswas subsequently evaluated. In 2009, irrigation was controlled forthe first 2 weeks following urea fertilization (1 irrigation eventand total amount of 9 mm in the 2 weeks following fertilization);

whereas, the 2010 crop period was characterized by standardirrigation (5 irrigation events and total amount of 46 mm in the 2weeks following fertilization). Irrigation was split into 43 and 36irrigation events, in the 2009 and 2010 crop periods, respectively.

66 A. Sanz-Cobena et al. / Agriculture, Ecosystem

Table 1Characteristics of the crop residues incorporated after the harvest of 2009.

Treatments C/N Hemicellulose(%)

Cellulose(%)

Lignin(%)

Control 73.38 38.83 32.86 3.83NBPT 71.43 36.15 30.93 4.14

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DCD 58.85 37.21 32.07 3.96U 63.11 37.41 32.46 3.79

Maize was harvested after each crop period (on 15th Octobern 2009 and on 21st October in 2010), with the 2009 crop residueseing equally incorporated into the soil in all of the plots duringhe fallow period. The main characteristics of the maize straw arehown in Table 1.

.3. Soil mineral N

Soil samples from the upper soil layer (0–10 cm) were takenor soil analysis using a soil corer (vol. 0.07 dm3). Three samplesf soil were randomly collected from each plot and mixed in theaboratory. Soil nitrate (NO3

−–N), dissolved organic carbon (DOC)nd ammonium nitrogen (NH4

+–N) contents were determined byxtracting 8 g of freshly mixed soil with 50 ml of water and with0 ml of 1 M KCl solution, respectively. After the soil extraction, theO3

−–N concentration was determined based on its reduction toitrite and a subsequent colorimetric determination of nitrite with

diazo-coupling reaction (Griess reaction) (AOAC, 1997). Ammo-ium was determined by automated colorimetry (Searle, 1984).he DOC content was determined as described by Mulvaney et al.1997).

Water-filled pore space (WFPS) was estimated by dividing theolumetric water content by total soil porosity. Total soil poros-ty was calculated by measuring the bulk density of the soilccording to the relationship: soil porosity = 1 − (soil bulk den-ity/2.65), assuming a particle density of 2.65 Mg m−3 (Danielsonnd Sutherland, 1986). Volumetric water content was determinedy oven-drying soil samples at 105 ◦C.

Rainfall and air temperature data were collected using a meteo-ological station located at the experimental site. Soil temperatureas monitored using a temperature probe inserted 10 cm into the

oil. Mean hourly temperature data were stored on a data logger.

.4. Sampling and analysis of N2O and NO fluxes

Fluxes of N2O were measured using manually operated circulartatic chambers (Sánchez-Martín et al., 2010). Two chambers perlot were used, each with a volume of 19.24 l (inner diameter andeight: 35 cm and 20 cm, respectively). The chambers were closedy placing them on stainless steel frames, which were inserted intohe soil at the beginning of the experiment (10 cm depth) withhe aim of preventing soil disturbances. Thermometers were putnside three randomly selected chambers during the closure periodf each measurement in order to correct the N2O fluxes for tem-erature. Gas samples (vol. 20 ml) were taken by syringe 0, 30 and0 min after the closure of the chambers in order to measure thevolution of the N2O concentration inside them. The gas samplesere stored in chromatography vials and measured by a Hewlett

ackard (HP6890) gas chromatograph equipped with a headspaceutoanalyzer (HT3), both from Agilent Technologies (Barcelona,pain). HP-Plot Q capillary columns transported gas samples fromhe injector to a 63Ni electron-capture detector. During the crop

easons, gas samples were taken four times in the first two weeksfter fertilizer application (1, 2, 3, 5, 8, 10, 12, 14 days after applica-ion of treatments) and then twice per week during the following

onths. In the fallow period, samples were taken two to three

s and Environment 149 (2012) 64– 73

times per week during rainfall periods and once per month duringnon-rainfall periods.

A gas flow-through system was used to measure NO fluxes.The chamber used for this analysis had the same characteristicsas that used for N2O sampling, but it was covered with Teflon®

and had both inlet and outlet holes and a transparent lid (Sánchez-Martín et al., 2010). Gas samples were pumped from the outletto chemiluminescence detection instrument (AC31 M-LCD, Envi-ronment S.A., Poissy, France) through 5 m of Teflon tubing, at aconstant flow rate of 40 l min−1. An ambient air sample was takenand measured between each gas sampling. For the gas sampling,concentrations were measured continuously until they gave a con-stant value; this normally took approximately 5 min per sample.Under these steady-state conditions, the change of concentrationwith respect to the time is zero and so the NO flux can be calculatedfollowing the equation proposed by Kaplan et al. (1988):

J

h=

(L

h+ q

V

)Ceq − q

(Cair

V

)(1)

where h is the internal height of the chamber, J is the emissionflux per unit area, q is the flow rate through the chamber, V is thevolume of the chamber, Ceq is the concentration measured at thechamber outlet and Cair is the NO concentration in the air imme-diately adjacent to the chamber (i.e. inlet concentration). L is thesum of the loss of NO through reactions with the chamber walls andthe chemical reactions of NO with existing oxidants in the carriergas, such as O3 and peroxyl radicals (Kim et al., 1994). The value ofL/h = 0.013 l min−1 was experimentally calculated based on Kaplanet al. (1988) and agreed with those reported by Roelle et al. (1999).

Intensive NO measurements were carried out during the firstmonth following fertilizer application in order to study the highestemissions associated with the different treatments applied. Afterthe first month, NO emissions were measured monthly.

The total N2O–N and NO–N fluxes per plot were estimated bysuccessive linear interpolations of the flux measurements. It wasassumed that emissions followed a linear trend during the periodsin which no samples were taken.

The N2O emission factor (i.e. EFN2O) refers to the N2O–N emit-ted per N applied (%), which was calculated as the sum of the N2Oemitted (discounting N2O losses from the control soil). Yield scaledN2O and NO emissions were calculated based on van Groenigenet al. (2010), considering total N uptake (i.e. by grain and straw)and cumulative N2O and NO emissions, respectively.

2.5. Drainage and leaching

Changes in soil moisture every hour were monitored using fourfrequency domain reflectometry (FDR) probes (EnviroScan system,Sentek Sensor Technologies, Stepney, SA, Australia) (i.e. one probeper treatment). Each probe consisted of sensors located at depthsof 10, 30, 50 and 80 cm inside a thin-walled PVC tube (with anexternal diameter of 5.6 cm) and measurements were made asdescribed in Sánchez-Martín et al. (2010). Daily drainage at a depth(D) of 0.8 m was calculated by applying the following simplifiedone-dimensional (vertical) water balance equation for each mea-surement position (Arregui and Quemada, 2006):

D = R + I − ETc ± �S (2)

where R is the rainfall (mm), I is the irrigation (mm), ET0 is theevapotranspiration (mm) and �S is the change in soil water (mm)between depths of 0 and 80 cm. ET0 was estimated using the cropcoefficient Kc and the Penman-Monteith model (Allen et al., 2006)

based on data provided by a meteorological station located at theexperimental site. Ceramic cups (0.8 m depth and 40◦ inclination)were used to sample the soil solution at depth. Samples of leachatewere taken as described in Sánchez-Martín et al. (2010) at 13

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A. Sanz-Cobena et al. / Agriculture, Eco

ifferent times during the experiment and they were analyzed forO3

− and dissolved organic carbon (DOC) in the laboratory follow-ng the method described above. The quantities of NO3

− and DOChat leached to depths greater than 0.8 m were calculated as theroduct of the mean concentrations in the soil solution and theuantity of drained water, which was calculated from the wateralance between two consecutive samples (Díez et al., 2000).

.6. Analysis of crop residues and yield calculation

One line (10 m length) of maize in each plot was harvested byand and weighed in the field after first separating the grain andtraw. Crop yield values represent the production of above-groundiomass (i.e. grain and straw) as a dry weight. Total nitrogen waseasured following a Kjeldahl digestion. The composition of the

rop residues (i.e. hemicellulose, cellulose and lignin) was also ana-yzed following the methodology proposed by van Soest (1963)Table 1).

.7. Statistical analysis

All the experiments were carried out with three replicates.tatistical analysis was performed using Statgraphics Plus 5.1Manugistics, 2000). The data distribution normality of the N2Ouxes, soil NO3

−, NH4+ and DOC and the NO3

− and DOC concentra-ions in the leachates were verified using the Kolmogorov–Smirnovest. In some cases, the data were log-transformed before anal-sis. Differences between treatments for each sampling eventnd for the cumulative emissions (for normally distributed data)ere analyzed using an analysis of variance (ANOVA, P < 0.05).

or non-normally distributed data, the Kruskal–Wallis test wassed on non-transformed data to evaluate differences at P < 0.05.he Schaich–Hamerle’s analysis was also carried out as a post hocest. Linear regression analyses (P < 0.05) were performed to deter-

ine relationships between N2O and NO fluxes and the soil DOC,H4

+–N, NO3−–N and DOC and NO3

−–N lost through the leachedater.

. Results

.1. Environmental conditions

The monthly average soil temperature ranged between 25 and9 ◦C in both the 2009 and 2010 crop periods (Fig. 1a). The aver-ge soil moisture for the 2009 crop period increased from 54 to9% WFPS in June and August. In 2010, June was the month withighest soil moisture content (65% of WFPS), remaining constantntil harvest. The soil moisture values for the 2 weeks after apply-

ng the treatments differed between the two years. In 2009, WFPSecreased to a minimum of 52% by the 5th day after fertiliz-

ng, whereas 70% WFPS was measured at the same time in 2010Fig. 1b).

The fallow period (October 2009 to April 2010) was character-zed by an average daily soil temperature ranging from 5 and 15 ◦CFig. 1a). October and November were the months with the low-st soil moisture levels (i.e. 33 and 30% WFPS, respectively) whileebruary and March registered the highest values of WFPS (77 and6%, respectively). Total rainfall was 403 mm from October 2009 toay 2010, with 75% of the total rainfall occurring from December

o March (Fig. 1).

.2. Soil mineral N and soil organic carbon

Urea fertilizer increased the ammonium content of the soil solu-ion (0–10 cm depth) within 24 h after application (Fig. 2a). Theighest concentrations were measured in the U and U+NBPT+DCD

s and Environment 149 (2012) 64– 73 67

plots 4 days after fertilization and no significant differences(P > 0.05) between them were measured. The U+NBPT-fertilized soilalso registered its highest value at the same time. Although higherthan the control, NBPT lowered NH4

+ concentration in the firstweek after the fertilization. This effect extended for the following2 weeks (Fig. 2a).

The nitrate concentration in the upper soil layers peaked twicewithin the measurement period for the 2009 maize crop (Fig. 2c). Inall cases, the first increase was noticed 48 h after urea application.The U+NBPT+DCD fertilized soil showed the highest concentra-tion at that time, without there being any significant differencesbetween the other treatments. A second peak was observed 15days after applying the fertilizers. Urea and U+NBPT were the treat-ments with the highest NO3

− concentrations measured at that time(Fig. 2c).

In 2010, the NH4+ concentration peaked once after treatment

application (Fig. 2b). The U and U+NBPT+DCD-treated soils showedthe highest NH4

+ concentrations 1 week after fertilization (60.2and 51.8 mg NH4

+ kg−1, respectively). The ammonium concentra-tion was 30% lower in the U+NBPT fertilized plots than in the Uplots, at the same sampling time. Then, after a progressive decreaseover a 5-week period, the NH4

+ concentration reached the valuesof the control soil in all cases (Fig. 2b).

Presence of DCD significantly reduced NO3− content of the

upper soil. Within two weeks of fertilization, the NO3− contents had

increased to 158.4 mg NO3− kg−1 and 126.1 mg NO3

− kg−1, respec-tively, in the U+NBPT and U-treated soils. By contrast, NO3

− peakedat 83.03 mg NO3

− kg−1 one week later on the U+NBPT+DCD plots(Fig. 2d).

In 2009, the dissolved organic carbon in the soil behaved in asimilar way in all of the treatments, except in the U-treated soil amonth after fertilizer application (28th July). In all cases, the con-centration of DOC ranged from 4 to 20 mg DOC–C kg−1. There wasan initial decrease followed by an increase after fertilizer applica-tion, and then a final decrease at the end of the measurement period(Fig. 3a). In 2010, DOC concentration in the soil ranged from 8 to29 mg C kg−1 (Fig. 3b). The DOC concentration fluctuated through-out the 2010 crop period, with a sharp measured decrease, in allcases, immediately after sowing (Fig. 3b). Thereafter, DOC startedto increase and peaked 5 days after fertilization on the U, U+NBPTand U+NBPT+DCD plots.

The dissolved organic carbon content ranged from 15 to29 mg DOC–C kg−1 during the fallow period. In this case, DOCincreased during the 2 months following the incorporation of the2009 crop residues (Fig. 3b).

3.3. Leached nitrate and DOC

During the two crop periods, measured drainage was 108.9 mm(August and September) and 74.9 mm for 2009 and 2010, respec-tively. The total drained water in the fallow period (i.e. from 29thSeptember 2009 to 28th April 2010) was 334.6 mm (Fig. 4a).

Nitrogen losses through NO3− leaching during the 2009 crop

period ranged from 8.7 ± 0.36 to 17.3 ± 1.97 kg NO3−–N ha−1 for the

U and U+NBPT+DCD treatments, respectively (Fig. 4b). By contrast,the fallow period was characterized by a significantly greater quan-tity of N lost through NO3

− leaching. In this case, the urea-onlyfertilized soil (U) experienced the greatest associated NO3

− lossesbetween 9th December and 21st January 2010 (Fig. 4b). At the endof the fallow period, the total amount of leached NO3

−–N was sig-nificantly smaller for the soil fertilized with U+NBPT than for the

one treated with U. By contrast, the application of U+NBPT+DCDseemed to enhance NO3

− losses by 33%. Cumulative NO3−–N losses

from the unfertilized soil were significantly lower than those reg-istered in the other treatments (Table 2).

68 A. Sanz-Cobena et al. / Agriculture, Ecosystems and Environment 149 (2012) 64– 73

Fig. 1. Soil temperature (◦C) and evapotranspiration “ET0” (mm) (a) and evolution of soil moisture (WFPS), irrigation (mm) and rainfall (mm) (b) during 2009 and 2010.

Fig. 2. NH4+–N (mg NH4

+–N kg−1) and NO3−–N (mg NO3

−–N kg−1) concentrations in the upper soil layer (0–10 cm) during the two crop periods 2009 (a–c) and 2010 (b–d).Verticals bars indicate standard errors. Black arrows show the fertilization date. Note differences in the scale of the “y” axis between figures.

A. Sanz-Cobena et al. / Agriculture, Ecosystems and Environment 149 (2012) 64– 73 69

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ig. 3. DOC (mg DOC–C kg−1) in the upper soil layer (0–10 cm) during the two crop period has been incorporated in (b).

As occurred in the previous crop period, the quantity of NO3−–N

eached from May to October 2010 was very small in comparisonith that of the fallow period and there were no significant differ-

nces between treatments (Table 2). The total NO3−–N losses by

he end of the experimental period are shown in Table 2. Lossesrom U+NBPT and U+NBPT+DCD plots were 18% lower and 32%igher than those from the U soil, respectively. Most of these lossesccurred in the fallow period (i.e. 79, 71 and 76% for U, U+NBPT and+NBPT+DCD fertilized plots, respectively). By contrast, the distri-ution of leached NO3

− was more homogeneous in the unfertilizedoil, with 42% being lost in the fallow period and 28 and 30% in the009 and 2010 crop periods, respectively (Fig. 4b).

The concentration of DOC in the leachate was generally low<1.2 mg C l−1) (Fig. 4c), with the highest daily concentrations being

easured in the fallow period. The total quantity of leached C forhe whole drainage period was never greater than the 4.5 kg C ha−1

bserved in the U-treated plots and there were no significant dif-erences between treatments (Fig. 4c).

.4. Gaseous N losses

.4.1. Fluxes of NODaily NO emissions peaked twice in the U plots, 2 and 3

eeks after the fertilizer application (3rd and 9th of July) (Fig. 5a).BPT and NBPT+DCD reduced emissions by 60% with respect to U

32.3 and 15.4 mg NO–N d−1 m−2 during the first and second peaks,espectively). In the 2010 crop, NO emissions were significantlyower than those from the previous crop, with the highest emissionf 1.1 mg NO–N d−1 m−2 being registered for the U treatment. Noignificant differences were found between the other two fertilizer

reatments (Fig. 5b).

The highest cumulative emissions of NO in 2009 were measuredor the U treatment (0.9% EF). This value was within the range of.5–10% proposed by Stehfest and Bouwman (2006) in a review

able 2otal N losses emitted in the form of N2O, NO and NO3

− leached in the experimental peri

Treatments N2O(kg N2O–N ha−1)

NO(kg NO–N h

Crop2009

Fallow2009–10

Crop2010

Crop2009

C 0.19 ± 0.03a 0.01 ± 0.01a 0.05 ± 0.01a 0.02 ± 0.12U+NBPT+DCD 1.21 ± 0.10bc 0.12 ± 0.02a 0.62 ± 0.08ab 0.37 ± 0.96U+NBPT 0.72 ± 0.03ab 0.03 ± 0.02a 1.03 ± 0.11b 0.74 ± 0.26U 1.59 ± 0.16c 0.15 ± 0.03a 1.08 ± 0.08b 2.25 ± 0.06

ata are averages of means from three replicates ± standard deviation. Different letters wignificant difference (LSD) test at P < 0.05.

s 2009 (a) and 2010 (b). Verticals bars indicate standard errors. Note that the fallow

of available NO emission data relating to European experimentalsites. The two inhibitor treatments, NBPT and NBPT+DCD, reducedNO emissions by 67 and 84%, respectively. The U+NBPT+DCD-treated and control soils had the lowest NO emissions by the endof the measurement period (Table 2). In 2010, total NO emis-sions were 0.45 ± 0.18 kg NO–N ha−1 for the U treatment (0.2% EF),with the next largest emissions being from the control soil andthose fertilized with the inhibitors (a mitigation of 36 and 53% forNBPT and NBPT+DCD, respectively). No significant differences werefound between NO emissions from the different treatments in 2010(Table 2).

3.4.2. N2O emissionsApplication of NBPT significantly reduced N2O losses in 2009

(P < 0.05), but this was not the case in 2010. The combined useof NBPT and DCD mitigated these emissions during the two yearperiod. Daily emissions of N2O peaked twice within the 2009 cropperiod for all fertilized soils (Fig. 6a). Application of U+NBPT didnot delay the emission peak but reduced N2O losses by 35% atthat time (the 1st of July, 11 days after fertilizing). Emissions fromU+NBPT+DCD peaked 2 days later, being 72% lower than those fromU (Fig. 6a). After 6–10 days, N2O emissions peaked again in all cases,with the emissions from the U+NBPT+DCD treatment being twice ashigh as those from the U+NBPT treatment and not significantly dif-ferent than U (P > 0.05). A decrease in N2O fluxes was then observedin all cases until the end of the measurement period (Fig. 6a). Con-sidering total emissions, application of the inhibitors reduced N2Olosses by 54 and 24%, for NBPT and NBPT+DCD, respectively. Thecalculated EF was 0.3 and 0.5% for both treatments, respectively.

Only one N2O peak was registered in the fertilized plots in 2010

(Fig. 6c). The highest emissions from the U and U+NBPT fertilizedsoils were observed on the 2nd of July, 9 days after fertilization. Atthis time, NBPT and NBPT+DCD reduced emissions by 33 and 87%,respectively. In 2010, the use of NBPT only abated N2O emissions

od.

a−1)NO3

− leached(kg NO3

−–N ha−1)

Crop2010

Crop2009

Fallow2009–10

Crop2010

a 0.34 ± 0.07a 10.81 ± 0.36b 16.8 ± 8.7a 11.72 ± 10.9cab 0.21 ± 0.03a 17.33 ± 1.97b 74.74 ± 22.3c 7.04 ± 3.3abb 0.29 ± 0.08 a 11.81 ± 4.20a 43.32 ± 3.20b 6.21 ± 4.3abc 0.45 ± 0.18a 8.7 ± 3.13a 59.72 ± 9.21c 6.61 ± 1.70a

ithin columns indicate significant differences applying Fischer’s unprotected least

70 A. Sanz-Cobena et al. / Agriculture, Ecosystems and Environment 149 (2012) 64– 73

Fig. 4. Drainage (mm) (a), NO3− (kg NO3

−–N ha−1) (b) and DOC (kg DOC–C ha−1)l

bb0wo

tgst

Fig. 6. Emissions of N2O (mg N2O–N d−1 m−2) during the two crop periods, 2009 (a)

Fa

eached (c) within the experimental period. Verticals bars indicate standard errors.

y 4%. The combination of NBPT and DCD reduced these N lossesy 33%. The calculated N2O emission factors for U were 0.64 and.43%, respectively, for the 2009 and 2010 crop periods; both ofhich were within the range proposed by the IPCC (i.e. 0.2–2.25%

f the N applied with mineral fertilizer) (IPCC, 2007).No significant differences (P > 0.05) were observed between

reatments in the fallow period and N2O fluxes were never

reater than 0.5 mg N2O–N d−1 m−2 (Fig. 6b). Total N2O emis-ions at the end of the fallow period were no higher thanhe 0.15 ± 0.03 kg N2O–N ha−1 observed for the U treatment and

and 2010 (c), and the fallow period (b). Vertical bars indicate standard errors. Notedifferences in the scale of the “y” axis between figures.

ig. 5. Emissions of NO (mg NO–N d−1 m−2) in the 2009 (a) and 2010 (b) measurement periods. Vertical bars indicate standard errors. Note differences in the scale of the “y”xis between figures.

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A. Sanz-Cobena et al. / Agriculture, Eco

uccessively lower for the U+NBPT and U+NBPT+DCD treatmentsnd the control soil (Table 2).

.5. Grain yield, N uptake and yield-scaled N emissions

The highest grain yield in the 2009 maize crop was harvestedrom the U+NBPT fertilized soil (14,483 ± 942 kg ha−1), with theext largest yields corresponding to the U+NBPT+DCD and U treat-ents (Table 3). There were no significant differences between

he different fertilizer treatments. By contrast, the control plotsroduced the lowest measured grain yield. The nitrogen uptake

ncreased in the soil fertilized with inhibitors (174 kg N ha−1 foroth the U+NBPT+DCD and U+NBPT treatments), with increasesf 18 and 38.5% with respect to the U-treated and control soils,espectively (data not shown).

Yield-scaled N2O emissions were highest for the U-ertilized plots (4.37 ± 0.9 g N2O–N kg−1 N uptake), followedy U+NBPT+DCD and U+NBPT (Table 3). In the case of NO, theighest yield-scaled emissions for the 2009 crop were registered

or the U plots; the next highest corresponded to the U+NBPTnd U+NBPT+DCD treatments (Table 3). These differences weretatistically significant (P < 0.05), in contrast to the situation in010, when significantly lower yield-scaled NO emissions wereeasured for all of the treatments. The highest grain yields after

he 2010 crop period were registered for the U+NBPT+DCD-treatedlots (10,867 ± 1675 kg ha−1), but there were no significant differ-nces between the yield-scaled N2O emissions from plots fertilizedith U plus inhibitors and with U only (Table 3).

. Discussion

.1. Nitrogen losses from urea fertilization

Urea fertilization increased emissions of N2O and NO to thetmosphere. Nitrous oxide emissions increased immediately after

application, first peaking one week later in the 2009 croppingeriod and coinciding with the intensive oxidation of NH4

+ to NO3−

y nitrifiers (Figs. 3 and 6a). Several authors (e.g. Meijide et al.,007; Sánchez-Martín et al., 2010) have previously reported peaksf N2O and NO associated with high levels of nitrification in irri-ated soils fertilized with urea.

The NO/N2O ratio has traditionally been used as an indirectndex for evaluating the main source of N oxides (i.e. nitrificationr denitrification) (Anderson and Levine, 1986; FAO, 2001). Theseuthors suggested that the molar NO/N2O ratio was >1 for culturesf nitrifiers, while it was less than 0.01 for denitrifiers. The NO/N2Oatio for the gases emitted during the 4 weeks after fertilizer appli-ation (i.e. NO/N2O = 2.9 ± 0.5), confirming that nitrification was theain pathway producing N oxides in that period.A second N2O peak was then reported a week later on the U-

reated plots (i.e. on 15th July 2009), when the NH4+ concentration

n the soil was below 5 mg N kg−1 and WFPS had increased (>76%)ue to irrigation. Under these conditions, NO fluxes were reduced,ith the NO/N2O ratio remaining below 0.1. These gases could

herefore have mainly been produced through the denitrificationrocess. A decreasing trend in N2O fluxes was then observed, indi-ating that the emission of N2O as a result of denitrification slowedown. Under denitrification conditions, Meijide et al. (2010) foundhat N2O fluxes only occurred during a short period, although N2as continually emitted over a longer period.

In 2010, the application of a greater quantity of water, at stan-

ard irrigation rates for maize grown in the area, during the 2eeks after fertilizer application, helped to maintain WFPS at lev-

ls above those for the same period of 2009 (e.g. >65%). This mayave changed the trend of daily N2O and NO emissions for this short

s and Environment 149 (2012) 64– 73 71

period of the 2010 maize crop, and could have made the emissionpattern differ from that observed in the previous year. In this sense,only one N2O peak was registered, which occurred the 2nd of July,7 days after U fertilization. This accounted for a similar quantity ofN2O as that emitted during the two emission events monitored in2009 (Fig. 6).

The lowest daily NO emissions in 2010, which were 30 timeslower than in 2009, may support the hypothesis that denitrifica-tion was the most important pathway for N2O production in thisurea-fertilized soil (NO/N2O = 0.1 ± 0.05 in the 4 weeks following Ufertilization).

Nitrate leaching was unexpectedly low in the 2009 and 2010crop periods (Table 2); this was probably because the drainageperiod was short and the quantity of leached water was small inboth 2009 and 2010 (Fig. 4a). This pattern of leaching losses couldalso have been closely associated with the soil texture and the lowhydraulic conductivity of the soil at depth.

Nitrate leaching in the fallow period was responsible for the highNO3

− losses registered by the end of the experiment (Table 2 andFig. 4). Driven by higher drainage (334.6 mm), associated with highrainfall rates and low temperatures (Fig. 1a), NO3

− losses from ureaaccounted for 59.7 ± 9.2 kg NO3

−–N ha−1 by the end of the fallowperiod. These results suggest that, despite the fact that it had beenexpected to have a mitigating effect on NO3

−–N losses, the incor-poration of crop residues after harvesting the 2009 maize crop didnot reduce them at all. The lack of any significant effect of the cropresidues on the NO3

− leaching losses could be due to the differ-ence in soil depths at which crop residues and available NO3

− werepresent in soil profile at that given time. If the NO3

− was presentfar below the incorporation depth of the crop residues, then oneshould not expect any impact of the residues on immobilizing theavailable NO3

−. However, this could not be confirmed by this studysince there was not a control plot without maize straw.

4.2. Effect of inhibitors on abating N losses from urea fertilization

In 2009, emissions of nitrogen oxides (i.e. N2O and NO) fromurea fertilization were significantly reduced by applying NBPT andNBPT+DCD (Table 1). Zaman et al. (2009) similarly observed areduction in N2O emissions when urine was mixed with NBPT andapplied to grassland instead of using urine alone. However, in ourcase, the abating effect of NBPT was greater than that previouslyreported elsewhere. NBPT delayed urea hydrolysis during 16 daysafter application and this would explain the lower concentrationof easily exchangeable NH4

+–N observed in this soil (Fig. 2a). Thisreduction in the size of the NH4

+–N pool may have reduced thenitrification rate in such a way that the production of N2O andNO was also affected. The reduction of urea hydrolysis caused byapplying NBPT was in line with previous research in which thesame urease inhibitor was shown to be effective at decreasing thehydrolysis process (i.e. Zhengping et al., 1991; Zaman et al., 2009).

An important additional finding presented here, which has notbeen previously studied, is that NBPT could be used to effectivelymitigate NO emissions from urea fertilized croplands. Emissionsof this gas were mainly produced through nitrification in the daysfollowing fertilization, and were particularly fostered by applyingurea. According to these results, these emissions were reduced byusing NBPT. In the 2009 experiment, the abatement effect of usingNBPT was responsible for reducing total NO emissions from ureaby around 67%.

In 2009, despite the presence of the nitrification inhibitor,emissions of N2O from the U+NBPT+DCD treatment were not sig-

nificantly different to those from U+NBPT treatment, with theobserved fluxes being even higher in the former case. There isno easy explanation for this. In contrast to the behavior of NBPTin its single form, the combination of NBPT with the nitrification

72 A. Sanz-Cobena et al. / Agriculture, Ecosystems and Environment 149 (2012) 64– 73

Table 3Grain yield, yield scaled N2O and NO emissions considering the N uptake by the aboveground biomass of maize at harvest.

Treatment Grain yield (t ha−1) Yield scaled N2O emissions(g N2O–N kg−1 N uptake)

Yield scaled NO emissions(g NO–N kg−1 N uptake)

2009 2010 2009 2010 2009 2010

C 10.46 ± 2.37a 2.68 ± 3.31a 0.8 ± 0.3a 1.5 ± 0.1a 0.1 ± 0.05a 0.17 ± 0.1aU 13.42 ± 3.36b 10.69 ± 3.52b 4.4 ± 0.9c 3.9 ± 0.2b 6.4 ± 0.8c 0.14 ± 0.1aU+NBPT 14.48 ± 1.13b 10.32 ± 1.26b 2.2 ± 0.4b 4.6 ± 0.3b 1.0 ± 0.7b 0.09 ± 0.05aU+NBPT+DCD 13.59 ± 2.86b 10.86 ± 2.33b 3.6 ± 0.6b 4.3 ± 0.6b 1.7 ± 0.4b 0.08 ± 0.02a

D ters ws

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ata are averages of means from three replicates ± standard deviation. Different letignificant difference (LSD) test at P < 0.05.

nhibitor, DCD, did not reduce NH4+ but, in fact, increased its

oncentration to values not significantly different from those reg-stered in the U-treated soil. Furthermore, the delaying effect ofCD on NH4

+ oxidation, which depressed the activity of ammo-ia monooxygenase enzyme (Skiba et al., 1993; Prasad and Power,995), may have increased the accumulation of NH4

+–N in thepper soil (Fig. 2). Based on this and the lowered NO3

− (Fig. 2d), weonclude that DCD may have decreased the nitrification rate. How-ver, no effect was noticed over the N gaseous losses. We speculatehat some change could have occurred in the effectiveness of NBPTs urease inhibitor, when combined with DCD.

Managing irrigation in the days immediately after fertilizerpplication could be used as a strategy to reduce emissions of Nxides. In general, lower emissions of both N2O and NO were pro-uced when large quantities of water were applied following ureapplication. In this case, the use of inhibitors contributed to a signif-cant reduction in NO emissions, but not for those of N2O; this wasrobably because a dilution of the concentration of the inhibitor in

greater volume of soil reduced its efficiency. These results coulde extrapolated to other irrigated crops, especially when urea isapidly transformed into NO3

−, since the effect of N uptake overhe N pool is not crucial for such a short period. A general emissionattern could also be established for this type of cropping systemnd its relation to emissions of N oxides.

In 2010, the application of DCD mitigated both N2O and NOmission for 14 and 10 days, respectively. Some authors (Menéndezt al., 2006) noticed a residual effect of nitrification inhibitors. Theseuthors observed a mitigating effect over NO emissions after aeriod of ca. 2 months, although N2O emissions were not affectedy the inhibitor from the 3rd week after fertilizing. No resid-al effect of DCD on N2O or NO emissions was measured in ourtudy.

Another important finding of this experiment was related tohe significant effect of NBPT on reducing NO3

− leaching. In theiterature reviewed, there is very little information about the effectf urease inhibitors on N leaching losses. Zaman et al. (2009) alsoecently reported a beneficial effect of NBPT in combination withCD for reducing these losses. However, the main question here ishy a urease inhibitor should influence leaching losses, particularly

onsidering its short period of effectiveness for reducing ureasectivity (normally less than 2 weeks).

In our experiment, this effect was mainly observed within therst drainage event within the fallow period (December 2009),hich was 6 months after the application of fertilizers. Underediterranean conditions, it is very frequent for the greatest

rainage to take place in the fallow period (i.e. autumn and winter)Sánchez-Martín et al., 2010). Along these lines, the unusually highevel of drainage (334.6 mm) occurred within this period and pro-uced intensive leaching of residual NO3

− to deeper soil layers. This

was not used by the crops but was transferred from the soil to

he groundwater. Most of the leaching losses occurred in January;his coincided with intensive drainage following the first importantainfall period in winter and a low ET0 (Fig. 1a).

ithin columns indicate significant differences applying Fischer’s unprotected least

From a crop yield perspective, it is very important to synchronizeN release and N uptake in order to ensure optimal crop productiv-ity. In this experiment, the urease inhibitor NBPT, whether appliedalone or mixed with DCD, had been well synchronize with N plantdemand and it had no effect on grain yield in either 2009 or 2010,compared with applying urea alone.

According to our findings, and based only on grain yield results,it is not possible to establish general fertilizing recommendationsfor farmers based on the use of NBPT. Nevertheless, the positiveeffect of this urease inhibitor in mitigating NO and N2O emissionsand NO3

− leaching losses does offer a new technological oppor-tunity to improve the N efficiency of urea fertilizers. Even so, ifwe consider its abatement effect with respect to other gaseousemissions of reactive N from sources other than NH3 volatiliza-tion, which have been very well demonstrated by other authors(e.g. Sanz-Cobena et al., 2008; Zaman et al., 2009; Zaman andBlennerhassett, 2010), it is evident that increased N efficiency couldmostly be achieved in crop systems to which relatively small quan-tities of water are normally added within 2 weeks of applying urea.This management strategy may promote a good distribution of theurease inhibitor in the upper part of the soil profile. Sanz-Cobenaet al. (2011) showed how administering an irrigation rate of 7 mmimmediately after applying U+NBPT to the soil produced a higherabatement effect on NH3 volatilization than that obtained by addingU+NBPT without irrigation.

Under similar circumstances, our results suggested than NBPTdid not significantly abate N2O emissions when the managementpractices and soil conditions promoted denitrification as the mainpathway for the production of this GHG. As we initially hypoth-esized, the mitigating effect of this urease inhibitor was onlydetected when nitrification was the dominant process in the pro-duction of N2O. Since NO is mostly produced through nitrificationunder these conditions, using NBPT was also effective in the abate-ment of this reactive N gas (i.e. emission reductions of 67.1 and35.7% were observed in the 2009 and 2010 crops, respectively)(Table 1).

5. Conclusions

The use of the urease inhibitor NBPT, used in single form, waseffective in the abatement of N losses (i.e. N2O and NO emissionsand NO3

− leaching) from a urea-fertilized maize crop. However,the effectiveness of this compound was significantly influencedby management practices and their impact on soil conditions, andespecially soil moisture. Under nitrifying conditions (ca. 55% WFPS),NBPT significantly abated N2O and NO losses by 54 and 67.1%,respectively. In contrast, an increase in soil moisture (WFPS ≥ 65%)led to a loss of effectiveness of NBPT, abating N2O emissions by 4%with respect to plots that were only fertilized with U. The com-

bined application of NBPT and DCD did not improve the efficiencyachieved by NBPT in abating N2O and NO emissions under nitrifyingconditions; this could have been associated with a negative inter-action between the two inhibitors, thereby reducing the efficiency

system

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A. Sanz-Cobena et al. / Agriculture, Eco

f NBPT. Most of the NO3− losses that occurred through leaching

ere observed during the fallow period. This was mainly due to thenusual and intensive drainage during that period. Nevertheless, athe end of the experiment, the application of NBPT and NBPT+DCDeduced NO3

− losses by 47.3 and 22.7%, respectively.

cknowledgements

The authors are grateful to the Spanish Ministry of Sci-nce and Innovation and the Autonomous Community of Madridor their economic support through Projects AGL2006-05208,GL2009-08412-AGR and the Agrisost Project (S2009/AGR-1630),espectively. They would also like to give thanks to Agrotainnternational for partially funding this research and providing theertilizer treatments used. Finally, this study would not have beenossible without technical assistance from the technicians andesearchers at the Department of Chemistry and Agricultural Analy-is of the Agronomy Faculty (Technical University of Madrid, UPM).pecial thanks to Ana Ros, Pilar Ortiz and the technicians workingith us at “El Encín” field station (IMIDRA), and to Mark Theobald

or checking the English spelling.

eferences

nderson, I.C., Levine, J.S., 1986. Relative rates of nitric oxide and nitrous oxide pro-duction by nitrifiers, denitrifiers, and nitrate respirers. Appl. Environ. Microbiol.51, 938–945.

llen, R.G., Pereira, L.S., Raes, D., Smith, M., 2006. Crop evapotranspiration: guide-lines for computing crop requirements. Irrigation and Drainage, vol. 56. FAO,Rome, Italy.

OAC, 1997. AOAC Official Methods of Analysis, 16th ed. Association of OfficialAnalytical Chemists, Gaithersburg.

rregui, L.M., Quemada, M., 2006. Drainage and nitrate leaching in a crop rotationunder different N-fertilizer strategies: application of capacitance probes. PlantSoil 288, 57–69.

ouwman, A.F., Boumans, L.J.M., Batjes, N.H., 2002. Emissions of N2O and NO fromfertilized fields: summary of available measurement data. Global Biogeochem.Cycles 16, 1080.

urt, R., 2004. Soil survey laboratory methods manual. NRCS Soil Survey Investiga-tions Report No. 42.

rutzen, P.J., 1979. The role of NO and NO2 in the chemistry of the troposphere andthe stratosphere. Ann. Rev. Earth Planet. Sci. 7, 333–372.

anielson, R.E., Sutherland, P.L., 1986. Porosity. In: Klute, A. (Ed.), Methods of SoilAnalysis, Part 1, Physical and Mineralogical Methods. Soil Sci. Soc. Am. Inc. Madi-son, Wisconsin, pp. 443–460.

avidson, E.A., Kingerlee, W., 1997. A global inventory of nitric oxide emissions fromsoils. Nutr. Cycl. Agroecosyst. 48, 37–50.

íez, J.A., Caballero, R., Román, R., Tarquis, A., Cartagena, M.C., Vallejo, A., 2000.Integrated fertilizer and irrigation management to reduce nitrate leaching inCentral Spain. J. Environ. Qual. 29, 1539–1547.

ing, W.X., Yu, H.Y., Zucong, C.C., 2011. Impact of urease and nitrification inhibitorson nitrous oxide emissions from fluvo-aquic soil in the North China Plain. Biol.Fertil. Soils 47, 91–99.

risman, J.W., Bleeker, A., Galloway, J., Sutton, M.S., 2007. Reduced nitrogen in ecol-ogy and the environment. Environ. Pollut. 150, 140–149.

AO, 2001. Global estimates of gaseous emissions of NH3, NO and N2O from agricul-tural Land. FAO, Rome, Italy.

irestone, M.K., Davidson, E.A., 1989. Microbiological basis of NO and N2O productionand consumption in soil. In: Andreae, M.O., Schimel, D.S. (Eds.), Exchange of theTrace Gases Between Terrestrial Ecosystems and the Atmosphere. John Wileyand Sons, Chichester, UK, pp. 7–21.

entile, R., Vanlauwe, B., Chivenge, P., Six, J., 2008. Interactive effects from combin-ing fertilizer and organic residue inputs on nitrogen transformations. Soil Biol.Biochem. 40, 2375–2384.

ill, J.S., Bijay-Singh, Khind, C.S., Yadvinder-Singh, 1997. Efficiency of N-(n-butyl)thiophosphoric triamide in retarding hydrolysis of urea and ammonia volatiliza-tion losses in a flooded sandy loam soil amended with organic materials. Nutr.Cycl. Agroecosyst. 53, 203–207.

arrison, R., Webb, J., 2001. A review of the effect of N fertilizer type on gaseous

emissions. Adv. Agric. 73, 65–108.

ntergovernmental Panel on Climate Change (IPCC), 2007. Climate change. In: Syn-thesis report of the fourth assessment report of IPCC, p. 49 (Chapter 3).

aplan, W.A., Wofsy, S.C., Keller, M., Da Costa, J.M., 1988. Emission of NO and depo-sition of O3 in a tropical forest system. J. Geophys. Res. 93, 1389–1395.

s and Environment 149 (2012) 64– 73 73

Kim, D.S., Aneja, V.P., Robarge, W.P., 1994. Characterization of nitrogen oxide fluxesfrom soil of a fallow field in the central Piedmont of North Carolina. Atmos.Environ. 28, 1129–1137.

Manugistics, 2000. Statgraphic Plus Version 5.1. Manugistics, Rockville, USA.McCarty, G.W., Bremner, J.M., Lee, J.S., 1990. Inhibition of plant and microbial ureases

by phosphoroamides. Plant Soil 127, 269–283.Meijide, A., Díez, J.A., Sánchez-Martín, L., López-Fernández, S., Vallejo, A., 2007. Nitro-

gen oxide emissions from an irrigated maize crop amended with treated pigslurries and composts in a Mediterranean climate. Agric. Ecosyst. Environ. 121,383–394.

Meijide, A., García-Torres, L., Arce, A., Vallejo, A., 2009. Nitrogen oxide emissionsaffected by organic fertilization in a non-irrigated Mediterranean barley field.Agric. Ecosyst. Environ. 132, 106–115.

Meijide, A., Cardenas, L.M., Bol, R., Bergstermann, A., Goulding, K.W.T., Well, R.,Vallejo, A., Scholefield, D., 2010. Dual isotope and isotopomer fractionation forthe understanding of N2O production and consumption during denitrificationin an arable soil. Eur. J. Soil Sci. 61, 364–374.

Menéndez, S., Merino, P., Pinto, M., González-Murua, C., Estavillo, J.M., 2006. 3,4-Dimethylpyrazol phosphate effect on nitrous oxide, nitric oxide, ammonia, andcarbon dioxide emissions from grasslands. J. Environ. Qual. 35, 973–981.

Menéndez, S., Merino, P., Pinto, M., González-Murua, C., Estavillo, J.M., 2009. Effectof N-(n-butyl) thiophosphoric triamide and 3,4-dimethylpyrazole phosphateon gaseous emissions from grasslands under different soil water contents. J.Environ. Qual. 38, 27–35.

Mulvaney, R.L., Khan, S.A., Mulvaney, C.S., 1997. Nitrogen fertilizers promote deni-trification. Biol. Fertil. Soils 24, 211–220.

Prasad, R., Power, J.F., 1995. Nitrification inhibitors for agriculture, health, and theenvironment. Adv. Agron. 54, 233–281.

Roelle, P., Aneja, V.P., O‘Connor, J., Robarge, W., Kim, D., Levine, J.S., 1999. Mea-surement of nitrogen oxide emissions from an agricultural soil with a dynamicchamber system. J. Geophys. Res. 104, 1609–1619.

Sánchez-Martín, L., Dick, J.L., Bocary, K., Vallejo, A., Skiba, U.M., 2010. Residual effectof organic carbon as a tool for mitigating nitrogen oxides emissions in semi-aridclimate. Plant Soil 326, 137–145.

Sanz-Cobena., A., Misselbrook, T.H., Arce, A., Mingot, J.I., Diez, J.A., Vallejo, A., 2008.An inhibitor of urease activity effectively reduces ammonia emissions from soiltreated with urea under Mediterranean conditions. Agric. Ecosyst. Environ. 126,243–249.

Sanz-Cobena, A., Misselbrook, T., Camp, V., Vallejo, A., 2011. Effect of water additionand the urease inhibitor NBPT on the abatement of ammonia emission fromsurface applied urea. Atmos. Environ. 45, 1517–1524.

Searle, P.L., 1984. The Berthelot or indophenol reaction and its use in the analyticalchemistry of nitrogen: a review. Analyst 109, 549–568.

Skiba, U., Smith, K.A., Fowler, D., 1993. Nitrification and denitrification as sourcesof nitric oxide and nitrous oxide in a sandy loam soil. Soil Biol. Biochem. 11,1527–1536.

Soil Survey Staff, 1992. Keys to soil taxonomy. In: SMSS Technical Monograph 19,5th ed. Pocahontas Press, Blacksburg, VI, USA.

Stehfest, E., Bouwman, L., 2006. N2O and NO emission from agricultural fields andsoils under natural vegetation: summarizing available measurement data andmodelling of global annual emissions. Nutr. Cycl. Agroecosyst. 74, 1385–1314.

Turner, D.A., Edis, R.B., Chen, D., Freney, J.R., Denmead, O.T., Christie, R., 2010. Deter-mination and mitigation of ammonia loss from urea applied to winter wheatwith N-(n-butyl) thiophosphorictriamide. Agric. Ecosyst. Environ. 137, 261–266.

UNECE, 2001. UNECE framework code for Good Agricultural Practices forreducing ammonia. Expert Group on Ammonia Abatement. Geneva, UnitedNations Economic Commission for Europe. http://www.clrtap-tfrn.org/sites/clrtaptfrn.org/files/documents/EPMAN%20Documents/eb.air.wg.5.2001.7.e.pdf.

van Groenigen, J.W., Velthof, G.L., Oenema, O., van Groenigen, K.J., van Kessel, C.,2010. Towards an agronomic assessment of N2O emissions: a case study onarable crops. Eur. J. Soil Sci. 61, 903–913.

van Soest, P.J., 1963. Use of detergents in the analysis of fibrous feeds: II. A rapidmethod for the determination of fibre and lignin. J. Assoc. Off. Anal. Chem. 46,829–835.

Xiang, S., Doyle, A., Holden, P.A., Schimel, J.P., 2008. Drying and rewetting effects on Cand N mineralization and microbial activity in surface and subsurface Californiagrassland soils. Soil Biol. Biochem. 40, 2281–2289.

Zaman, M., Nguyen, M.L., Blennerhassett, J.D., Quin, B.F., 2008. Reducing NH3, N2Oand NO3

−–N losses from a pasture soil with urease or nitrification inhibitors andelemental S-amended nitrogenous fertilizers. Biol. Fertil. Soils 44, 693–705.

Zaman, M., Saggar, S., Blennerhassett, J.D., Singh, J., 2009. Effect of urease and nitri-fication inhibitors on N transformation, gaseous emissions of ammonia andnitrous oxide, pasture yield and N uptake in grazed pasture system. Soil Biol.Biochem. 41, 1270–1280.

Zaman, M., Blennerhassett, J.D., 2010. Effects of the different rates of urease and nitri-fication inhibitors on gaseous emissions of ammonia and nitrous oxide, nitrate

leaching and pasture production from urine patches in an intensive grazed pas-ture system. Agric. Ecosyst. Environ. 136, 236–246.

Zhengping, W., Van Cleemput, O., Demeyer, P., Baert, L., 1991. Effect of ureaseinhibitors on urea hydrolysis and ammonia volatilization. Biol. Fertil. Soils 11,43–47.