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Soil Biology & Biochemistry 53 (2012) 82e89

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Soil Biology & Biochemistry

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Efficiency of nitrification inhibitor DMPP to reduce nitrous oxide emissionsunder different temperature and moisture conditions

Sergio Menéndez a,*, Iskander Barrena b, Igor Setien b, Carmen González-Murua b, José María Estavillo b

a Institute of Agro-biotechnology (IdAB), UPNa-CSIC-GN, 31192 Mutilva Baja, Navarra, SpainbDepartment of Plant Biology and Ecology, University of the Basque Country (UPV/EHU), Apdo. 644, E-48080 Bilbao, Spain

a r t i c l e i n f o

Article history:Received 18 January 2012Received in revised form19 April 2012Accepted 23 April 2012Available online 21 May 2012

Keywords:Carbon dioxide (CO2)3,4-Dimethylpyrazol phosphate (DMPP)Methane (CH4)Nitrification inhibitorNitrous oxide (N2O)Water filled pore space (WFPS)

* Corresponding author. Fax: þ34 94 816 89 30.E-mail addresses: sergio.menendez@unavarra.e

(S. Menéndez).

0038-0717/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.soilbio.2012.04.026

a b s t r a c t

Agricultural intensification has led to the use of very high inputs of nitrogen fertilizers into cultivatedland. As a consequence of this, nitrous oxide (N2O) emissions have increased significantly. Nowadays, thechallenge is to mitigate these emissions in order to reduce global warming. Addition of nitrificationinhibitors (NI) to fertilizers can reduce the losses of N2O to the atmosphere, but field studies have shownthat their efficiency varies depending greatly on the environmental conditions. Soil water content andtemperature are key factors controlling N2O emissions from soils and they seem to be also key param-eters responsible for the variation in nitrification inhibitors efficiency. We present a laboratory studyaimed at evaluating the effectiveness of the nitrification inhibitor 3,4-dimethylpyrazol phosphate(DMPP) at three different temperatures (10, 15 and 20 �C) and three soil water contents (40%, 60% and80% of WFPS) on N2O emissions following the application of 1.2 mg N kg�1 dry soil (equivalent to140 kg N ha�1). Also the CO2 and CH4 emissions were followed to see the possible side effects of DMPP onthe overall microbial activities. Nitrogen was applied either as ammonium sulfate nitrate (ASN) or asENTEC 26 (ASN þ DMPP). The application of ENTEC 26 was effective reducing N2O losses up to the levelsof an unfertilized control treatment in all conditions. Nevertheless, the percentage of reduction inducedby DMPP in the ENTEC treatment with respect to the ASN varied from 3% to 45% depending ontemperature and soil water content conditions. At 40% of WFPS, when nitrification is expected to be themain process producing N2O, the increase of N2O emissions in ASN together with temperature provokedan increase in DMPP efficiency reducing these emissions from 17% up to 42%. Contrarily, at 80% of WFPS,when denitrification is expected to be the main source of N2O, emissions after ASN application decreasedwith temperature, which induced a decrease from 45% to 23% in the efficiency of DMPP reducing N2Olosses. Overall, the results obtained in this study suggest that DMPP performance regarding N2O emis-sions reduction would be the best in cold and wet conditions. Neither CO2 emissions nor CH4 emissionswere affected by the use of DMPP at the different soil water contents and temperatures.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Agricultural intensification has led to the use of high inputs ofnitrogen fertilizers into cultivated land. As a consequence of this,losses by nitrate leaching and N2O emissions have increasedsignificantly (Bouwman et al., 2002). Regarding gaseous emissions,soil microbial processes produce gases like CO2, N2O and CH4 whichare emitted to the atmosphere and play an important role in envi-ronmental terms due to their global warming potential (IPCC,1997).

Nitrous oxide (N2O) has great importance as a greenhouse gasbecause it has amean atmospheric residence time ofmore than 100

s, sergio.menendez@ehu.es

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years. N2Owarming potential depends on its life time. When a timehorizon of 100 years for N2O is considered, its warming potentialhas been estimated to be 310 times higher than the CO2 warmingpotential (Prather et al., 2001). Moreover, N2O is not only involvedin the global warming effect, but it also contributes to thedestruction of the ozone layer. Approximately 35% of the globalannual N2O emission is attributed to agriculture (Isermann, 1994),being agricultural soils the major source of these emissions, whicharise mainly from both anaerobic denitrification and aerobic nitri-fication microbial processes.

Methane (CH4) is also a greenhouse gas which contributes ina 15% to global warming (Chistiansen and Cox, 1995). Its concen-tration is expected to rise from 1.72 ppb in 1994 to about 1.82 ppb in2034 (IPCC, 1995) with the added risk that its warming potential ina time horizon of 100 years is 21 times higher than the CO2

S. Menéndez et al. / Soil Biology & Biochemistry 53 (2012) 82e89 83

warming potential. The net soil � atmosphere CH4 flux is the resultof the balance between the offsetting processes of methanogenesis(CH4 produced during decomposition of organic matter in anoxicconditions) (Woese et al., 1990) and methanotrophy (microbial CH4consumption in aerobic conditions) (Whalen and Reeburgh, 1996),although there is some evidence of an anaerobic pathway for CH4oxidation (Segers, 1998). So, depending on the environmentalconditions (oxygen availability) soils can be a source or a sink ofmethane (Nykänen et al., 1995; Maljanen et al., 2003).

Over thepastyears,nitrification inhibitorshavebeenpresentedasa tool to reduceN losses and increase fertilizeruse efficiency (Slangenand Kerkhoff, 1984; Dittert et al., 2001; Di and Cameron, 2003;Boeckxet al., 2005; Pereira et al., 2010). Oneof these inhibitors is 3,4-dimethylpyrazole phosphate (DMPP) which has become a verypopular and used inhibitor in the world in the last decade (Zerullaet al., 2001; Hatch et al., 2005). Several studies have demonstratedthat DMPP not only increases crop yield in barley, maize and wheat(Linzmeier et al., 2001; Pasda et al., 2001), but it also reduces nitrousoxide emissions (Weiske et al., 2001; Hatch et al., 2005;Merino et al.,2005) without increasing the risk of enhanced ammonia volatiliza-tion (Menéndezet al., 2006, 2009; Li et al., 2009). Regarding the othergreenhouse gases (CO2 and CH4), opposite results have beendescribed in the literature. While Menéndez et al. (2006, 2009)reported that CO2 emissions were unaffected by DMPP, Weiskeet al. (2001) described an unexpected reduction in CO2 and CH4

emissions induced by DMPP. In fact, these authors could not find anexplanation for these reductionsandreported that theywerenotableto confirm the reduction in CO2 emission they have observed in thefield when they performed a laboratory study with the same soil.There is no expected reason why DMPP should decrease CO2 emis-sions. Even in the case that DMPP affected to CO2 production/consumption by nitrifiers, it would be unlikely to observe any effectof DMPP on overall CO2 soilfluxes, as nitrifiers represent only a smallproportion of soil microorganisms. So, unless its application induceda great change in the soil activity and/or growth of microbial pop-ulations other than the nitrifiers, which is neither expected, CO2emissions are not presumed to change. In the case of CH4 emissions,we could contemplate that DMPP may have an effect on methanemonooxygenase activity. In this sense, it has been reported thatmethane monooxygenase structural similarity with ammoniummonooxygenase could lead to its inhibition induced by some nitri-fication inhibitors (Bronson and Mosier, 1994). Furthermore, it hasbeen described that high soil ammonium contents can induce aninhibition of methanotrophic activity (Jassal et al., 2011). So, theapplication of a nitrification inhibitor that induces an increase in soilammonium content, could reduce CH4 consumption rates, and anincrease in CH4 emission could be foreseen. Moreover, this effectwould be alsomodulated in a differentmanner at different soilwatercontents, as methanotrophy is dominant in drier soils and meth-anogenesis in wetter soils.

The efficiency of DMPP in reducing N2O emissions in fieldexperiments has been shown to vary from 0% up to 60% (Barth et al.,2001; Dittert et al., 2001; Zerulla et al., 2001; Macadam et al., 2003;Menéndez et al., 2006, 2009; Chen et al., 2010; Pereira et al., 2010)although the reason for this variation in efficiency is not fullyunderstood. Both the fluctuations with time in the environmentalconditions along these field experiments as well as the differentexperimental designs in each case make difficult to find the reasonsfor these variations in results. Two environmental factors have beendescribed as key parameters responsible for this variation: soilwatercontent and temperature. In this sense, the literature suggests thatthe efficiency of DMPP in reducing N2O emissions decreases at highsoilwater contents and temperature.With respect to the influence ofsoil water content Menéndez et al. (2009) described that DMPP wasnot effective reducing losseswhen soilwater contentwasover60%of

WFPS, and they attributed this lack of efficiency to the low emissionsmeasured in comparison to those reported when WFPS was lower.Regarding the effect of temperature, Merino et al. (2005) describedthat in our edaphoclimatic conditions the duration of the effect ofDMPP inautumnwas longer than in spring, suggesting thatapossiblefaster degradation of themolecule at higher soil temperaturesmightbe responsible for a lower efficiency. So, themechanismwhereby soilwater content and temperature influence the efficiency of DMPPreducing greenhouse gas emissions needs to be thoroughly studied.In this sense, a matter that needs to be analyzed is to what extentDMPP has been able to reduce losses up to unfertilized levels in thedifferentexperiments and this fact hasnot been taken into account atthe time of interpreting the differences in the percentages of emis-sion reductions observed with respect to conventional fertilizer. Inthe literature, authors might have not always used properly theterminology that distinguishes betweenDMPP’s efficiency inhibitingnitrification and DMPP’s efficiency reducing emissions.

In field conditions, it is difficult to ascertain the mechanismwhereby soil water content and temperature influence the effi-ciency of DMPP reducing greenhouse gas emissions, due to thenatural fluctuations in temperature and rainfall. Consequently, theaim of the present work was to evaluate the effect of temperatureand soil water content on the performance of DMPP under labo-ratory conditions where both temperature and soil water contentwere thoroughly controlled.

2. Materials and methods

2.1. Experiment set-up

Soil was collected from a 0e20 cm layer of a typical cutgrassland in the Basque Country (Northern Spain, 43�1802000 N,3�530000 W; 30 m a.s.l.). The soil was a poorly drained clay loam(34% fine sand, 3% coarse sand, 34% silt, and 29% clay) with a pH(1:2 H2O) of 6.6 and an organic matter content of 1.72%. Rootsand stones were removed and the soil sieved at 4 mm beforebeing dried at air temperature. The apparent density of the soilafter sieving was also determined. One hundred g of dried soilwere weighed and introduced in a 300 mL open pot. In order toreactivate soil microorganisms (Anderson and Domsch, 1973), soilwas rehydrated and 0.5 g of glucose and 85 mg N kg�1 dry soil(equivalent to 10 kg N ha�1) as ammonium sulfate nitrate (ASN)were added to each pot 21 days before treatments application.

The trial was designed as a split plot arrangement. Main factorwas incubation temperature. Soil water content and fertilizertreatments were subfactors. Three fertilizer treatments wereapplied: a control treatment without fertilizer, a second one withammonium sulfate nitrate (ASN 26%) and a third one consisting inthe combination of ASN with DMPP, available in the market asENTEC 26 (developed by BASF (Ludwigshafen, Germany) andcommercialized by K þ S Nitrogen (Mannheim, Germany). Fertil-izers were applied at a rate of 1.2 mg N kg�1 dry soil (equivalent to140 kg N ha�1). Nitrogen in ASN consisted of 7.5% nitric and 18.5%ammoniacal. In order to get a homogenous distribution of fertil-izers in soil, they were dissolved inwater and 5 mL volume of thosesolutions was applied with a pipette to the corresponding pots inorder to get a rate of 140 kg N ha�1 and a homogenous distributionof fertilizers in soil. Concurrently, each incubation temperature(10 �C,15 �C and 20 �C) was subdivided into three sub-treatments atdifferent soil water contents (40%, 60% and 80% of WFPS). Eachsubtreatment was sub-subdivided into other three groups,according to the fertilizer applied.Water was added to all pots up toreach the humidity defined for each soil water content. Pots werecovered with Parafilm in order to maintain soil humidity. Every 3days, pots were weighted and water was added if it was necessary.

Table 1Statistical significance of the effect of the different main factors (soil water content,temperature and fertilizer) and their interactions on the cumulative emissions ofN2O, CO2 and CH4 (P < 0.05; n ¼ 3).

N2O CO2 CH4

Temperature 0.021 0.000 n.s.WFPS 0.000 0.007 n.s.Fertilizera 0.048 n.s. n.s.Temperature*WFPS 0.002 n.s. n.s.Temperature*Fertilizer n.s. n.s. n.s.WFPS*Fertilizer n.s. n.s. n.s.Temp*WFPS*Fertilizer n.s. n.s. n.s.

a Fertilizer factor includes only ASN and ENTEC treatments.

S. Menéndez et al. / Soil Biology & Biochemistry 53 (2012) 82e8984

Altogether, there were 27 different possible combinations and 3replicates for each combination (a total of 81 pots per day ofmeasurement). In order to avoid the possibility of measuringrepeatedly in the time unusual gas emissions corresponding to hotspots that might be present in some pots, each pot was measuredonly one time. So, the initial number of pots was 972, with 81 potsper measurement day.

2.2. Gaseous emissions measurements

N2O, CO2 and CH4 emissions weremeasured every 2 days duringthe first two weeks after treatments application. Afterwards,measurements continued at a frequency of once or twice per weekfor 51 days. Pots were incubated in hermetic jars with a volume of1.1 L at 20 �C during 1 h. Emission rates were calculated taking intoaccount the increase in the concentration of each gas duringincubation time (Menéndez et al., 2008). Gas samples (20 mL) fromthe atmosphere of the hermetic jars were stored in vials of 12 mLand later analyzed by gas chromatography (GC) (Agilent, 7890A)equippedwith an electron capture detector (ECD) for N2O detectionand a flame ionization detector (FID) for CH4. For the determinationof CO2, the GC was equipped with a methanizer to reduce CO2 up toCH4. A capillary column (IA KRCIAES 6017: 240 �C, 30 m � 320 mm)was used and the samples were injected by means of a headspaceautosampler (Teledyne Tekmar HT3) connected to the gas chro-matograph. Standards of N2O, CO2 and CH4 were stored andanalyzed at the same time as the samples.

Cumulative gas production during the experiment was esti-mated by averaging the fluxes of two successive determinations,multiplying that average flux by the length of the period betweenthe measurements, and adding that amount to the previouscumulative total.

2.3. DMPP determination

On days 11, 23, 37 and 51 after treatments application, thethree replicates of each treatment with ENTEC were mixed, and150 g of fresh soil were used to determine DMPP content in thesoil. Soil was extracted with methyl-butyl-ether, and evaporatedin the presence of HCL. Extracts were later analyzed byHPLCeDAD.

2.4. Data analysis

The effect of the main factors on cumulative gas emissions wascompared by ANOVA and the Duncan test was used for separationof means between treatments with the statistical software SPSS17.0. T-student tests between ASN and ENTEC treatments were alsocarried out for each combination of temperature and soil watercontent. Significant differences were conducted at p < 0.05.

3. Results

3.1. Nitrous oxide emissions

Cumulative N2O losses showed to be significantly affected bytemperature, soil water content and fertilizer type (Table 1).Mean cumulative N2O losses for all the combinations of watercontent and temperature of the experiment were of 1.19 mgN2OeN kg�1 dry soil and 1.25 mg N2OeN kg�1 dry soil in thecontrol and ENTEC treatments respectively, while in ASN treat-ments they were of 1.80 mg N2OdN kg�1 dry soil. So, theapplication of fertilizer as ASN induced a significant increase inN2O cumulative losses with respect to both the control andENTEC treatments, which were not statistically different among

them. These cumulative losses for the three main fertilizationtreatments resulted as consequence of daily emission rates whichnever were higher than 65 and 312 mg N2OeN kg�1 dry soil d�1

throughout the assay in the control and ENTEC treatmentsrespectively, while maximum daily emission rates of up to 527 mgN2OeN kg�1 dry soil d�1 were determined in the ASN treatment.While DMPP application reduced the daily fluxes of N2O withrespect to ASN treatment, it did not induce any delay in time orchange in the temporal pattern of daily fluxes of ENTEC incomparison to ASN treatment.

The maximum losses of N2O after ASN application occurred at80% of WFPS, when a decrease with temperature was observedfrom 4.33 mg N2OeN kg�1 dry soil at 10 �C down to 1.47 mgN2OeN kg�1 dry soil at 20 �C (Table 2). These losses in ASNtreatment ranged from 2.4% at 20 �C to 11.3% at 10 �C of the Napplied. When WFPS was 60% or lower, losses did not exceed the2.5% of the N applied. ENTEC treatment showed lower losses thanASN, being the difference between both treatments alwayssignificant except for 10 �C and 60% of WFPS (Table 2). Thepercentage of reduction in cumulative losses induced by DMPP inthe ENTEC treatment with respect to the ASN treatment variedfrom 3% to 45% depending on the incubation conditions. At thelowest soil water content (40% of WFPS) the percentage ofreduction of N2O emissions after DMPP application increased withtemperature from 17% at 10 �C up to 42% at 20 �C, while at thehighest soil water content (80% of WFPS) the percentage ofreduction of N2O emissions by the application of DMPP decreasedwith temperature being of 45%, 39% and 23% at 10 �C, 15 �C and20 �C respectively.

3.2. Carbon dioxide emissions

Neither ASN application nor ENTEC showed any effect on cumu-lative CO2 emissions with respect to the control (Tables 1 and 3).A significant effect ofWFPS and soil temperaturewasobserved. Thus,CO2 emissions in the ASN treatment increased significantly withWFPS, showing maximum losses at 80% of WFPS, while theydecreased with temperature, being CO2 fluxes statistically higherat 10 �C and 15 �C than at 20 �C (Table 3). The daily fluxes of CO2ranged from 3 mg CO2eC kg�1 dry soil d�1 up to 69 mg CO2eC kg�1

dry soil d�1. These fluxes were in the same range as thosedescribed by Menéndez et al. (2006) in a field study with the samegrassland soil.

3.3. Methane emissions

The soil acted as a sink or a source of CH4 depending on thetreatment, with daily fluxes ranging from �38 mg CH4eC kg�1 drysoil d�1 up to þ47 mg CH4eC kg�1 dry soil d�1. Cumulative CH4losses showed no significant effect of fertilization, DMPP, soil watercontent or temperature (Tables 1 and 4).

Table 2Cumulative N2O emissions after treatments application up to day 51 and the percentage of reduction induced by DMPP in each condition. Mean values have been calculated forASN treatment.

10 �C

40% WFPS mg N2OeN kg�1

dry soil60% WFPS mg N2OeN kg�1

dry soil80% WFPS mg N2OeN kg�1

dry soilASN mean value at 10 �C

Control 1077 � 139 1167 � 24 1324 � 105ASN 1109 � 40 17% * 1300 � 77 3% n.s. 4331 � 988 45% * 2247 aENTEC 920 � 46 1267 � 166 2395 � 733

15 �C

40% WFPS mg N2OeN kg�1

dry soil60% WFPS mg N2OeN kg�1

dry soil80% WFPS mg N2OeN kg�1

dry soilASN mean value at 15 �C

Control 923 � 48 1168 � 60 1534 � 261ASN 1234 � 114 21% * 1469 � 70 26% * 2487 � 588 39% * 1730 bENTEC 970 � 41 1088 � 66 1520 � 207

20 �C

40% WFPS mg N2OeN kg�1

dry soil60% WFPS mg N2OeN kg�1

dry soil80% WFPS mg N2OeN kg�1

dry soilASN mean value at 20 �C

Control 1070 � 53 1139 � 25 1299 � 159ASN 1611 � 436 42% * 1195 � 41 12% ** 1473 � 132 23% * 1420 cENTEC 930 � 26 1048 � 35 1129 � 27ASN mean value at

different WFPS1318 b 1413 b 2757 a

Different letters indicate significantly different rates using Duncan Test (P < 0.05; n ¼ 3).Significance of the reduction induced by DMPP (ENTEC vs ASN treatments) was analyzed using a T-student test.*P < 0.05.**P < 0.01.

S. Menéndez et al. / Soil Biology & Biochemistry 53 (2012) 82e89 85

3.4. DMPP in soil

DMPP’s concentration decreased significantly in all treatmentsby 32% in average between day 11 and 23 after fertilizer application,being steady from day 21 until the end of the experiment. Soilwater content showed to have a significant effect on the persistenceof DMPP molecule (P < 0.043). Thus, treatments at 80% of WFPSshowed significantly higher concentrations than treatments at 40%of WFPS (Fig. 1), while treatments at 60% of WFPS showed inter-mediate values. By the contrary, temperature did not show anyeffect on DMPP’s persistence (P < 0.991).

Table 3Cumulative CO2 emissions after treatments application up to day 51. Mean values have b

10 �C

40% WFPS mg CO2eC kg�1

dry soil60% WFPS mg CO2eC kgdry soil

Control 1325 � 47 1715 � 92ASN 1401 � 102 1683 � 29ENTEC 1444 � 178 1623 � 115

15 �C

40% WFPS mg CO2eC kg�1

dry soil60% WFPS mg CO2eC kgdry soil

Control 1484 � 20 1572 � 101ASN 1493 � 32 1523 � 49ENTEC 1444 � 85 1481 � 93

20 �C

40% WFPS mg CO2eC kg�1

dry soil60% WFPS mg CO2

dry soil

Control 1123 � 65 1276 � 14ASN 1118 � 168 1071 � 23ENTEC 1065 � 45 1172 � 22ASN mean value at

different WFPS1337 b 1426 b

Different letters indicate significantly different rates using Duncan Test (P < 0.05; n ¼ 3

4. Discussion

4.1. Effect of soil water content

It is widely accepted that N2O emissions are closely related tosoil water content (Davidson, 1991) for which a value of 60% ofWFPS appears to be the threshold between water-limited andaeration limitedmicrobial processes in a range of soils. In our study,ASN treatment showed double mean N2O emission rates at 80% ofWFPS than at 40% and 60% of WFPS (Table 2). Nitrification wasexpected to be the main source of N2O at 40% and 60% of WFPS,

een calculated for ASN treatment.

�1 80% WFPS mg CO2eC kg�1

dry soilASN mean value at 10 �C

1618 � 531552 � 126 1545 a1624 � 98

�1 80% WFPS mg CO2eC kg�1

dry soilASN mean value at 15 �C

1728 � 911714 � 53 1577 a1513 � 58

eC kg�1 80% WFPS mg CO2eC kg�1

dry soilASN mean value at 20 �C

1428 � 891358 � 54 1182 b1228 � 71541 a

).

Table 4Cumulative CH4 emissions after treatments application up to day 51 . Mean values have been calculated for ASN treatment.

10 �C

40% WFPS mg CH4eC kg�1

dry soil60% WFPS mg CH4eC kg�1

dry soil80% WFPS mg CH4eC kg�1

dry soilASN mean value at 10 �C

Control �184 � 117 �70 � 163 �2 � 47ASN �208 � 109 �180 � 128 64 � 21 �108 aENTEC �152 � 119 �195 � 69 �180 � 75

15 �C

40% WFPS mg CH4eC kg�1

dry soil60% WFPS mg CH4eC kg�1

dry soil80% WFPS mg CH4eC kg�1

dry soilASN mean value at 15 �C

Control �150 � 12 �202 � 138 �139 � 33ASN �65 � 27 57 � 90 �204 � 56 �71 aENTEC �109 � 130 �147 � 63 �13 � 35

20 �C

40% WFPS mg CH4eC kg�1

dry soil60% WFPS mg CH4eC kg�1

dry soil80% WFPS mg CH4eC kg�1

dry soilASN mean value at 20 �C

Control �26 � 100 �183 � 89 �182 � 183ASN �206 � 155 �86 � 71 �149 � 39 �147 aENTEC �39 � 112 �126 � 166 �134 � 31ASN mean value at

different WFPS�160 a �70 a �96 a

Different letters indicate significantly different rates using Duncan Test (P < 0.05; n ¼ 3).

S. Menéndez et al. / Soil Biology & Biochemistry 53 (2012) 82e8986

when the soil is in aerobic conditions. Even though at 80% of WFPSthe soil is supposed to be in hypoxic conditions when denitrifica-tion is expected to be the dominating process responsible for N2Oproduction, as it will be further discussed, the fact that DMPPreduces N2O losses in those conditions suggests that nitrificationwas also taking place albeit slightly. In this sense, several authors(McTaggart and Tsuruta, 2003; Menéndez et al., 2008) havereported that nitrification can occur at WFPS values around 85% indifferent soil types. Moreover, according to Seifert (1962), theintensity of nitrification is influenced by soil structure, whichregulates aeration. So, the fact that in our experimental set-up soilwas broken and sieved previously, should have enhanced a higherdegree of O2 availability at 80% of WFPS.

Regarding CO2 emissions, several authors have reported a posi-tive correlation between soil WFPS and CO2 emission rates whenWFPS is lower than 60%, and a negative correlation when WFPS ishigher than 60% (Davidson et al., 1998; Kiese and Butterbach-Bahl,2002). In our study, CO2 fluxes in the ASN treatments increasedwith soil water content, being the maximum losses at 80% of WFPS.This confirms that in our conditions at 80% of WFPS there was stillO2 available for microorganisms as it has been discussed previouslyfor N2O emissions.

The mean consumption of CH4 in ASN treatment was notstatistically affected by the soil water content (Table 4) although at60% and 80% of WFPS the CH4 consumption was slightly lower.

0

100

200

300

400

500

0 10 20 30 40 50 60

Days

µg D

MP

P k

g-1

dry

so

il

Fig. 1. DMPP presence in soil after treatments application during the following 51days. (C) 40% WFPS, (-) 60% WFPS, (:) 80% WFPS.

Several authors (Yavitt et al., 1995; Whalen and Reeburgh, 1996;van den Pol-van Dasselaar et al., 1998) have described a decrease ofCH4 consumption when the soil water content increased in otherstudies with soils from grasslands and forests. As CH4 oxidation bymethanotrophs requires molecular O2 (Dutaur and Verchot, 2007)an increase of soil water content would have reduced O2 availabilityand as consequence the oxidation of CH4. As we have discussedbefore, at 80% of WFPS in our study soil was still in aerobicconditions with O2 available, favoring CH4 oxidation.

4.2. Effect of temperature

When soil water content was at 80% of WFPS, N2O emissions inASN treatment decreased with temperature, the highest emissionstaking place at 10 �C (Table 2). Provided that at 80% of WFPS themain source of N2O is expected to be the denitrification process, thereason for this decrease with temperature would be related withthe N2/N2O emission ratio of this process. Several authors havedescribed that the N2/N2O ratio in the denitrification processincreases with temperature (Nommik, 1956; Keeney et al., 1979;Maag and Vinther, 1996), therefore diminishing N2O losses to theatmosphere. This could be due to the different kinetics of theenzymes involved in denitrification (nitrate reductase and nitrousoxide reductase) at different temperatures. Nevertheless, our studywas designed in such a way that all gas measurements were per-formed at 20 �C in order to prevent this, so that we could distin-guish specifically the effect of the incubation temperaturemaintained in the time from the instantaneous effect thattemperature would have at the moment of emission (i.e. theintrinsic different kinetics of the enzymes at the different temper-atures). Consequently the different emission rates measured wouldprobably reflect changes in soil microbial metabolism and/orpopulations. Thus, the different incubation temperatures mightinduce changes in soil microbial populations (Braker et al., 2010) orin the differential expression of the enzymes responsible for thedifferences in N2/N2O emissions (Saleh-Lakha et al., 2009), favoringthe reduction of N2O to N2 at high temperatures. Moreover, not onlysoil populations can be quantitatively affected, but genetic diversityis also correlated with environmental conditions. As the kinetics ofbacterial enzymes are affected by environmental conditions such as

S. Menéndez et al. / Soil Biology & Biochemistry 53 (2012) 82e89 87

temperature, pH, nutrients, etc., these environmental factors areselective agents on genes controlling enzyme systems; therefore,a relationship should exist between various components of geneticdiversity among populations of bacteria and variability associatedwith their environment (McArthur et al., 1988).

Opposite to the expected, CO2 fluxes at 10 �C and 15 �C werestatistically higher than at 20 �C. In relation to this, it has beenreported that CO2 emissions should not be always expected toincrease with temperature. Giardina and Ryan (2000) describedthat the decomposition of organic matter in soil did not increaseCO2 emissions with temperature ranging from 5 �C to 35 �C. In ourstudy CO2 daily fluxes in treatments at 20 �C were slightly lowerthan treatments at 10 �C and 15 �C, which induced significantlylower CO2 cumulative emissions at 20 �C than at lower tempera-tures. This could be due to a greater depletion and degradation ofcarbon pools at 20 �C than at 10 �C and 15 �C, as was reported byGrisi et al. (1998).

Several authors have described an increase of CH4 emissionswithtemperature, with an optimum at about 35 �C in paddy fields(Oremland, 1988; Sass et al., 1991; Parashar et al., 1993) and at25e30 �C in neutral landfill cover soils (Boeckx and Van Cleemput,1996). Nevertheless, we did not observe any effect of temperatureon CH4 emissions. In our experimental conditions, the oxygenavailabilitywas not so limited even at 80% ofWFPS, so CH4 oxidationwouldbe themainprocess responsible for theCH4fluxesdeterminedas discussed before. Dunfield et al. (1993) described that CH4 oxida-tion showed a lower temperature-dependence than CH4 production.This would explain why no effect of temperature was observed.

4.3. Effect of DMPP

Our results show that the persistence of the molecule of DMPPin soil is not influenced by temperature in the range between 10and 20 �C, as had been suggested byMerino et al. (2005). This rangeof temperature in soil is the approximated range between winterand summer in our edaphoclimatic conditions. Consequently, anydifference observed in the behavior of DMPP under the differenttemperatures of the different seasons of the year must be due toother reasons than a differential degradation of the molecule.Instead of temperature, it is soil water content which has shown toinfluence the persistence of the DMPP molecule in soil, witha higher persistence at higher water contents. The higher O2availability at lower soil water contents would have favored theoxidation of the molecule. Nevertheless, the different levels ofpersistence achieved have shown to be of minor importance, beingthe lowest level enough to be efficient under the tested conditions,as further discussed. In fact, it is in the conditions where the lowestlevel of inhibitor persistence was achieved (i.e. 40% WFPS) whenthe efficiency of DMPP reducing N2O emissions increased withtemperature, while in the conditions where the highest level ofinhibitor persistence was achieved (i.e. 80% WFPS) the efficiency inreducing N2O emissions decreased with temperature. Thus, ataWFPS of 80%, the percentage of reduction of N2O emissions by theapplication of DMPP was of 45%, 39% and 23% at 10 �C, 15 �C and20 �C respectively. These results seem to show a decrease in DMPPefficiency with increasing temperature. But if control and ENTECtreatments are compared, ENTEC treatment presented the same oreven lower losses than the control at 15 �C and 20 �C, demon-strating that in spite of the variability in the percentage of reductionof N2O losses, DMPP has the maximum possible performance atboth temperatures. The explanation for this is that, as describedpreviously, temperature was probably affecting the N2/N2O ratio oflosses coming from denitrification, masking the efficiency of DMPPin reducing N2O losses at 15 �C and 20 �C with respect to 10 �C. At10 �C, the differences between control and ENTEC treatments

suggest that denitrification of the applied nitrate is mainly takingplace up to N2O.

At 40% of WFPS, when nitrification is the main processproducing N2O, the opposite effect of temperature on N2O emis-sionswas observed. Under this soil water content, the percentage ofreduction of N2O emissions after DMPP application increased withtemperature, from 17% at 10 �C up to 42% at 20 �C. Nevertheless,ENTEC always reduced the losses up to the levels of the unfertilizedtreatment or even more, indicating that the efficiency of DMPP atthe three different temperatures was the same. So, the differencesin the percentages of reduction are proportional to the increase ofemissions with temperature in the ASN treatment.

At 60% of WFPS, in spite that nitrification is supposed to havea main role, denitrification would be also slightly occurring,increasing N2O cumulative losses with respect to those at 40% ofWFPS. As described previously, temperature affected the N2/N2Oratio of losses coming from denitrification, disguising the efficiencyof DMPP in reducing losses at 15 �C and 20 �C with respect to 10 �C.According to these results, the terms “efficiency” of DMPP inhibit-ing nitrification and “efficiency” of DMPP reducing emissionsshould be distinguished carefully. Additionally, our results indicatethat DMPP shows its best performance under extreme environ-mental conditions (cold and wet conditions or hot and dry condi-tions). So, in our edaphoclimatic conditions, to achieve the bestperformance of DMPP regarding N2O emissions, ENTEC should beapplied either at the early spring when temperatures are still lowand soil water content high or in summer when temperature hasincreased and rainfalls are limited. This is in agreement with theresults of Merino et al. (2005), who described that inwet conditionsa greater reduction of N2O losses took place at low temperatures(autumn) than at high temperatures (later spring). Moreover, if wetake into account not only the percentage in reduction of N2O lossesachieved by DMPP but the absolute quantity of emission reduced,our results show that the application of ENTEC would be quanti-tatively more effective when applied in cold wet conditions that inhot dry conditions, as higher N2O losses have to be diminished froma wetter and colder soil than from a drier and hotter soil. So,although more studies should be performed to confirm theseresults in a wider range of temperatures than those assayed in thisstudy, our results indicate that DMPP performance regarding N2Oemissions reduction would be the best in cold and wet climates.

With respect to CH4 emissions, several authors have suggestedthat some nitrification inhibitors (nitrapyrin and etridiazole) mayaffect methane monooxygenase, presumably because of the closestructural relationship between ammonium monooxygenase andmethane monooxygenase activities (Bérdard and Knowles, 1989;Topp, 1993; Bronson and Mosier, 1994). On the other hand, Bérdardand Knowles (1989) described that NH4

þ can inhibit methanemonooxygenase, increasing CH4 emissions. In this sense, thegreater availability of NH4

þ in the presence of nitrification inhibitorshas been described to inhibit CH4 oxidation in arable and grasslandsoils (Tlustos et al., 1999). Results described in the literature arecontradictory with respect to the effect of DMPP on CH4 emissions.Hatch et al. (2005) described that under aerobic conditions DMPPcould enhance CH4 emissions. Opposite to those results, Weiskeet al. (2001) described a positive effect of DMPP reducing CH4

emissions. In our experimental conditions we did not observe anyeffect on CH4 emissions, and, as expected, we did neither observeany effect of DMPP on CO2 emissions at the different temperaturesand soil water contents.

5. Conclusions

ENTEC 26 inhibited efficiently the nitrification process reducingN2O losses up to control levels in all tested conditions. However, the

S. Menéndez et al. / Soil Biology & Biochemistry 53 (2012) 82e8988

percentages of reduction with respect to ASN varied depending onincubation conditions. At 40% of WFPS, when nitrification isexpected to be themain process producing N2O, the increase of N2Oemissions in ASN together with temperature provoked an increasein DMPP efficiency reducing these emissions. Contrarily, at 80% ofWFPS,whendenitrification is expected tobe themain sourceofN2O,emissions after ASN application decreasedwith temperature,whichinduced a decrease in the efficiency of DMPP reducing N2O losses.Neither CO2 emissions nor CH4 emissionswere affected by the use ofDMPP at the different temperatures and soil water contents.

Acknowledgments

This project was funded by the Spanish Government (AGL2009-13339-CO2-01), by the Basque Government (K-EGOKITZEN, ETOR-TEK 2010-2012 and GV IT-526-10). Technical and human support byPhD. Azucena González, Phytotron Service, SGIker (UPV/EHU) isgratefully acknowledged.

References

Anderson, J.P.E., Domsch, K.H., 1973. Quantification of bacterial respiration, selectiveinhibition and biovolume measurements of and fungal contributions to soilrespiration. Arch. Mikrobiol. 93, 113e117.

Barth, G., von Tucher, S., Schmidhalter, U., 2001. Influence of soil parameters on theeffect of 3,4-dimethylpyrazole-phosphate as a nitrification inhibitor. Biol. Fertil.Soils 34, 98e102.

Bérdard, C., Knowles, R., 1989. Physiology, biogeochemistry, and specific inhibitorsof CH4, NH4

þ, and CO oxidation by methanotrophs and nitrifiers. Microbiol. Rev.53, 68e84.

Boeckx, P., Van Cleemput, O., 1996. Methane oxidation in a neutral landfill coversoil: influence of moisture content, temperature, and nitrogen-turnover.J. Environ. Qual. 25, 178e183.

Boeckx, P., Xu, X., Van Cleemput, O., 2005. Mitigation of N2O and CH4 emission fromrice and wheat cropping systems using dicyandiamide and hydroquinone. Nutr.Cycl. Agroecosyst. 72, 41e49.

Bouwman, A.F., Boumans, L.J.M., Batjes, N.H., 2002. Emissions of N2O and NO fromfertilized fields: summary of available measurement data. Global Biogeochem.Cy. 16, 1080. doi:10.1029/2001GB001812.

Braker, G., Schwarc, J., Conrad, R., 2010. Influence of temperature on the composi-tion and activity of denitrifying soil communities. FEMS Microbiol. Ecol. 73,134e148.

Bronson, K.F., Mosier, A.R., 1994. Suppression of methane oxidation in aerobic soilby nitrogen fertilizers, nitrification inhibitors, and urease inhibitors. Biol. Fertil.Soils 17, 263e268.

Chen, D., Suter, H.C., Islam, A., Edis, R., 2010. Influence of nitrification inhibitors onnitrification and nitrous oxide (N2O) emission from a clay loam soil fertilizedwith urea. Soil. Biol. Biochem. 42, 660e664.

Chistiansen, T.R., Cox, P., 1995. Response of methane emission from artic tundra toclimatic change: results from a model stimulation. Tellus 47 (B), 301e309.

Davidson, E.A., Belk, E., Boone, R., 1998. Soil water content and temperature asindependent or confounded factors controlling soil respiration in a temperatemixed hardwood forest. Glob. Change Biol. 4, 217e227.

Davidson, E.A., 1991. Fluxes of nitrous oxide and nitric oxide from terrestrialecosystem. In: Rogers, J.E., Whitman, W.B. (Eds.), Microbial Production andConsumption of Greenhouse Gases: Methane, Nitrogen Oxides, and Halo-methanes. Am. Soc. Microbiol., Washington, DC, pp. 219e235.

Di, H.J., Cameron, K.C., 2003. Mitigation of nitrous oxide emissions in spray-irrigated grazed grassland by treating the soil with dicyandiamide, a nitrifica-tion inhibitor. Soil Use Manage. 19, 284e290.

Dittert, K., Bol, R., King, R., Chadwick, D., Hatch, D., 2001. Use of a novel nitrificationinhibitor to reduce nitrous oxide emission from 15N-labelled dairy slurryinjected into soil. Rapid Com. Mass Spectr. 15, 1291e1296.

Dunfield, P., Knowles, R., Dumont, R., Moore, T.R., 1993. Methane production andconsumption in temperate and subarctic peat soils: response to temperatureand pH. FEMS Microbio. Lett. 102 (3e4), 185e195.

Dutaur, L., Verchot, L.V., 2007. A global inventory of the soil CH4 sink. Global Bio-geochem. Cy. 21, GB4013. doi:10.1029/2006GB002734.

Giardina, C.P., Ryan, M.G., 2000. Evidence that decomposition rates of organiccarbon in mineral soil do not vary with temperature. Nature 404,858e861.

Grisi, B., Grace, C., Brookes, P.C., Benedetti, A., Dell’abate, M.T., 1998. Temperatureeffects on organic matter and microbial biomass dynamics in temperate andtropical soils. Soil Biol. Biochem. 30, 1309e1315.

Hatch, D., Trindade, H., Cardenas, L., Carneiro, J., Hawins, J., Scholefield, D.,Chadwick, D., 2005. Laboratory study of the effects of two nitrification inhibi-tors on greenhouse gas emissions from a slurry-treated arable soil: impact ofdiurnal temperature cycle. Biol. Fertil. Soils 41, 225e232.

Intergovernmental Panel on Climate Change, 1995. Climate Change Scientific andTechnical Analysis of Impacts, Adaptions and Mitigation. In: Contribution ofWorking Group II to the Second Assessment Report of the IntergovernmentalPanel on Climate Change. Cambridge Univ. Press, Cambridge.

Intergovernmental Panel on Climate Change, 1997. Revised 1996 IPCC Guidelines forNational Greenhouse Gas Inventories. OECD, Paris.

Isermann, K., 1994. Agriculture’s share in the emission of trace gases affecting theclimate and some cause-oriented proposals for sufficiently reducing this share.Environ. Pollut. 83, 95e111.

Jassal, R.S., Black, T.A., Roy, R., Ethier, G., 2011. Effect of nitrogen fertilization on soilCH4 and N2O fluxes, and soil and bole respiration. Geoderma 162, 182e186.

Keeney, D.R., Fillery, I.R., Marx, G.P., 1979. Effect of temperature on the gaseousnitrogen products of denitrification a silt loam soil. Soil Sci. Soc. Am. J. 43,1124e1128.

Kiese, R., Butterbach-Bahl, K., 2002. N2O and CO2 emissions from three differenttropical forest sites in the wet tropics of Queensland, Australia. Soil Biol. Bio-chem. 34, 975e987.

Li, H., Chen, Y., Liang, X., Lian, Y., Li, W., 2009. Mineral-nitrogen leaching andammonia volatilization from a rice-rapeseed system as affected by 3,4-dimethylpyrazole phosphate. J. Environ. Qual. 38 (5), 2131e2137.

Linzmeier, W., Schmidhalter, U., Gutser, R., 2001. Effect of DMPP on Nitrification andN Losses (nitrate, NH3, N2O) from Fertilizer Nitrogen in Comparison to DCD. In:VDLUFA-Institution Series Congress Volume (in German), vol. 52, pp. 485e488.

Maag, M., Vinther, F.P., 1996. Nitrous oxide emissions by nitrification and denitri-fication in different soil types and at different moisture contents and temper-atures. Appl. Soil Ecol. 4, 5e14.

Macadam, X.M.B., del Prado, A., Merino, P., Estavillo, J.M., Pinto, M., González-Murua, C., 2003. Dicyandiamide and 3,4-dimethyl pyrazole phosphate decreaseN2O emissions from grassland but dicyandiamide produces deleterious effectsin clover. J. Plant Physiol. 160, 1517e1523.

Maljanen, M., Liikanen, A., Silvola, J., Martikainen, P.J., 2003. Methane fluxes onagricultural and forested boreal organic soils. Soil Use Manag. 19, 73e79.

McArthur, J.V., Kovacic, D.A., Smith, M.H., 1988. Genetic diversity in natural pop-ulations of a soil bacterium across a landscape gradient. Proc. Natl. Acad. Sci. 85,9621e9624.

McTaggart, I.P., Tsuruta, H., 2003. The influence of controlled release fertilisers andthe form of applied fertiliser nitrogen on nitrous oxide emissions from anandosol. Nutr. Cycl. Agroecosys. 67, 47e54.

Menéndez, S., Merino, P., Pinto, M., González-Mura, C., Estavillo, J.M., 2006. 3,4-Dimethylpyrazol phosphate effect on nitrous oxide, nitric oxide, ammonia,and carbon dioxide emissions from grasslands. J. Environ. Qual. 35, 973e981.

Menéndez, S., López-Bellido, R.J., Benítez-Vega, J., González-Murua, C., López-Bellido, L., Estavillo, J.M., 2008. Long-term effect of tillage, crop rotation and Nfertilization to wheat on gaseous emissions under rainfed Mediterraneanconditions. Eur. J. Agron. 28, 559e569.

Menéndez, S., Merino, P., Pinto, M., González-Murua, C., Estavillo, J.M., 2009. Effectof N-(n-butyl) thiophosphoric triamide and 3,4 dimethylpyrazole phosphate ongaseous emissions from grasslands under different soil water contents.J. Environ. Qual. 38, 27e35.

Merino, P., Menéndez, S., Pinto, M., González-Murua, C., Estavillo, J.M., 2005. DMPPreduces nitrous oxide emissions from grassland after slurry application. Soil UseManag. 21, 53e57.

Nommik, H., 1956. Investigations on denitrification in soil. Acta Agric. Scand. 6,195e228.

Nykänen, H., Alm, J., Lång, K., Silvola, J., Martikainen, P.J., 1995. Emissions of CH4,N2O and CO2 from a virgin fen and a fen drained for grassland in Finland.J. Biogeogr. 22, 351e357.

Oremland, R.S., 1988. Biogeochemistry of methanogenic bacteria. In: Zehnder, A.(Ed.), Biology of Anaerobic Microorganism. Wiley, New York, pp. 405e447.

Parashar, D.C., Gupta, P.K., Rai, J., Sharma, R.C., Singh, N., 1993. Effect of soil temper-ature on methane emissions from paddy fields. Chemosphere 26, 246e250.

Pasda, G., Hähndel, R., Zerulla, W., 2001. Effect of fertilizers with the new nitrifi-cation inhibitor DMPP (3,4 dimethylpyrazole phosphate) on yield and quality ofagricultural and horticultural crops. Biol. Fertil. Soils 34, 85e97.

Pereira, J., Fangueiro, D., Chadwick, D., Misselbrook, T.H., Coutinho, J., Trindade, H.,2010. Effect of cattle slurry pre-treatment by separation and addition of nitri-fication inhibitors on gaseous emissions and N dynamics: a laboratory study.Chemosphere 79 (6), 620e627.

Prather, M., Ehhalt, D., Dentener, F., Derwent, R., Dlugokencky, D., Holland, E.,Isaksen, I., Katima, J., Kirchhoff, P., Matson, P., Midgley, P., Wang, M., 2001.Atmospheric chemistry and green house gases. In: Houghton, J.T., Ding, Y.,Griggs, D.J., Noguer, M., van der Linden, P.J., Dai, X., Maskell, K., Johnson, C.A.(Eds.), Climate Change 2001: The Scientific Basis. Contribution of WorkingGroup I to the Third Assessment Report of the Intergovernmental Panel onClimate Change. Cambridge University Press, New York, pp. 240e287.

Saleh-Lakha, S., Shannon, K.E., Henderson, S.L., Goyer, C., Trevors, J.T., Zebarth, B.J.,Burton, D.L., 2009. Effect of pH and temperature on denitrification geneexpression and activity in Pseudomonas mandelii. Appl. Environ. Microbiol. 75(12), 3903e3911.

Sass, R.L., Fisher, F.M., Harcombe, P.A., Turner, F.T., 1991. Methane production andemission in a Texas rice field. Global Biogeochem. Cy. 4, 47e68.

Segers, R., 1998. Methane production and methane consumption: a review ofprocesses underlying wetland methane fluxes. Biogeochemistry 41, 23e51.

Seifert, J., 1962. The influence of the soil structure and moisture content on thenumber of bacteria and the degree of nitrification. Foliar Microbiol. 7, 234e238.

S. Menéndez et al. / Soil Biology & Biochemistry 53 (2012) 82e89 89

Slangen, J.H.G., Kerkhoff, P., 1984. Nitrification inhibitors in agriculture and horti-culture: a literature review. Fert. Res. 5, 1e76.

Tlustos, P., Willison, T.W., Baker, J.C., Murphy, D.V., Pavlikova, D., Goulding, K.W.T.,Powlson, D.S., 1999. Short-term effects of nitrogen on methane oxidation insoils. Biol. Fertil. Soils 28, 64e70.

Topp, E., 1993. Effects of selected agrochemicals on CH4 oxidation by an organicagricultural soil. Can. J. Soil Sci. 73, 287e291.

van den Pol-van Dasselaar, A., van Beusichem, M.L., Oenema, O., 1998. Effects of soilmoisture content and temperature on methane uptake by grasslands on sandysoils. Plant Soil 204, 213e222.

Weiske, A., Benckiser, G., Herbert, T., Ottow, J.C.G., 2001. Influence of the nitrificationinhibitor 3,4-dimethylpyrazole phosphate (DMPP) in comparison to dicyan-diamide (DCD) on nitrous oxide emissions, carbon dioxide fluxes and methane

oxidation during 3 years or repeated application in field experiments. Biol.Fertil. Soils 34, 109e117.

Whalen, S.C., Reeburgh, W.S., 1996. Moisture and temperature sensitivity of CH4oxidation in boreal soils. Soil Biol. Biochem. 28 (10e11), 1271e1281.

Woese, C.R., Kandler, O., Wheelis, M.L., 1990. Towards a natural system of organ-isms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc. Natl. Acad.Sci. USA 87, 4576e4579.

Yavitt, J.B., Fahey, T.J., Simons, J.A., 1995. Methane and carbon dioxide dynamics ina northern hardwood ecosystem. Soil Sci. Soc. Am. J. 59, 796e804.

Zerulla, W., Barth, T., Dressel, J., Erhardt, K., von Locquenghien, K.H., Pasda, G.,Rädle, M., Wissemeier, A.H., 2001. 3,4-dimethylpyrazole phosphate (DMPP)-a new nitrification inhibitor for agriculture and horticulture. Biol. Fert. Soils 34,79e84.