**2.1. Assay establishment**

Within the economic costs, that caused due to diminished N use efficiency stands out. Among the environmental costs, the contribution of some N-based gaseous compounds that play a role in the greenhouse effect [10], acid rain [23, 24] and the contamination and eutrophication of waters due to nitrate leaching further than the root zone [1, 17] are notable. In this sense, in Álava, Arrate et al. [6] describe a series of changes in the management of arable land in the years 1967–1997 (wetland drainage, application of large quantities of fertilizers, phytosanitary products etc.) that progressively increased the concentration of N compounds in subsurface waters. Due to such reasons and by application of the European Directive 88/778/ EEC related to water for human consumption [13], the zone related to the Eastern sector of the quaternary aquifer of Vitoria was designated as vulnerable to nitrate pollution in the year 1999 [14]. This zone comprises 38% of the area where wheat is grown in Alava, 9500 ha approximately. In this zone, the N fertilization is as much as 140 kg N ha−1 depending on the previous culture and soil richness; N fertilization is not allowed at a distance closer than 3 m

150 years [47]. This gas adsorbs electromagnetic radiation in various wavelengths in the infrared region between 7.7 and 17 μm [35] and its greenhouse effect per mass unit is some 300

has contributed approximately 5% to the warming up of the planet [42, 43]. The origin of 90%

be produced both due to nitrification and denitrification (**Figure 1**). Nitrification is a microbial aerobic process in which ammonium first oxidizes to nitrite and then to nitrate. In this

other hand, denitrification is a microbial anaerobic process in which organic carbon is used as the energy source and the nitrate as the last electron acceptor so that it reduces to the last

O emissions is anthropogenic, and agriculture is its main source [22]. N<sup>2</sup>

O and N<sup>2</sup>

O) is not a very reactive gas; it persists in the atmosphere for as much as

[36]. In this sense, it is estimated that in the last 100 years, N<sup>2</sup>

O can be released into the atmosphere [46]. On the

. The nitrification and denitrification processes

O

O in soils can

of any water course.

times larger than that of CO<sup>2</sup>

ammonium to nitrate oxidation process, N<sup>2</sup>

**Figure 1.** Transformations of mineral nitrogen in the soil [46].

nitrogenous gaseous compounds N<sup>2</sup>

Nitrous oxide (N<sup>2</sup>

80 Global Wheat Production

of the N<sup>2</sup>

A nitrogen fertilization experiment was carried out in Gauna, Álava (average annual rainfall of 779 mm and average annual temperature of 11.5°C) from November 2001 to February 2004 in three consecutive seasons. The assay was conducted in the Western Sector of the quaternary aquifer of Vitoria, adjacent to the area vulnerable to the contamination of nitrates of agricultural origin. The trial was organized in random blocks with four repetitions in which each elementary plot covered an area of 50 m<sup>2</sup> . The soil on which the trial was established was classified as Aquertic Eutrudept [40] and was planted with wheat. Some of the soil properties are shown in **Table 1**. Data regarding when sowing, the first, second, and third N broadcastings, and the harvest took place are shown in **Table 2**. Before sowing, 90 kg ha−1 of P<sup>2</sup> O5 ha−1 and 90 kg ha−1 of K<sup>2</sup> O ha−1 were applied as 0-14-14. Nitrogen doses of 0, 140, and 220 kg N ha−1


**Table 1.** Soil properties of the experiment in Gauna (Álava).


**Table 2.** Data for sowing, first, second, and third N broadcasts, harvest and tilling.

were applied as ammonium nitrate (33.51% N g/g) in two or three broadcasts as described in **Table 3**. A control treatment in which no N was applied was included. The treatment in which 140 kg N ha−1 were broadcast was applied in two or three amendments (**Table 3**) to observe the splitting effect and to evaluate a low fertilization strategy in three broadcasts suggested as a possible N fertilization management in vulnerable zones.

**2.3. Leached nitrate**

flow injection [3, 4].

**Figure 2.** Ceramic capsule in the soil for leached liquid sampling.

Two ceramic cups were inserted per block and treatment. They were buried at a depth of 60 cm (**Figure 2**). As a test, in the year 2003, a hole in the soil was performed after harvest. It was observed that wheat roots reached a depth of 60 cm as a maximum; so the depth at which the ceramic cups were inserted was considered appropriate and it can be considered that the gathered nitrogen was not profitable for plants. Liquid samples were collected from ceramic cups when it had rained 20–40 mm or every fortnight. The first liquid sample after every insertion was extracted but then discarded. The sampling data occurred between December 20, 2002 and September 18, 2003 and between January 9 and September 27, both data in 2004. At both periods, the ceramic cups were removed at harvest and then inserted again. After every sampling session, a vacuum of approximately 50 kPa was performed with a manual pump. Afterwards, the nitrate concentration in the sampled water was analyzed through segmented

**Depth (cm) Soil apparent density (g cm−3) Coarse elements (% g/g) Stone free apparent density (g cm−3)**

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**Table 4.** Soil apparent density (g cm−3) and coarse elements (% g/g) at 0–20, 20–40, and 40–60 cm depths.

0–20 1.57 20.8 1.4 20–40 1.59 21.5 1.4 40–60 1.93 42.6 1.6

The water balance was determined in layers 0–20, 20–40, and 40–60 cm depth following Campbell's [11] simplified waterfall method. In this method, it is considered that waters fills

#### **2.2. Mineral nitrogen (Nmin)**

The first Nmin measures, (ammonic and nitric N), took place at start of tillering and at the end of winter 2001. They were determined from a mixture of eight samples throughout the trial taken at the depths 0–30 and 30–60 cm. Subsequently, all Nmin values were determined from a mixture of two samples per treatment, block and depth. These samples were taken before sowing, at the end of winter, at Z20, and also after harvest in all years and all treatments except at the end of winter of year 2003, when only samples of the control treatment were taken.

The soil samples were shredded manually. Stones, roots, and any other type of plant material were discarded. Then, the humidity of the samples was gravimetrically ascertained.

200 mL of 1 M KCl were added to 100 g of soil and the mixture was stirred for 30 min. After filtering, the nitrogen of nitric origin in the filtrate was analyzed by segmented flow injection [3, 4]. The calculation of Nmin per hectare was corrected according to the content of coarse elements of the soil (**Table 4**).


**Table 3.** Dose and broadcasts of the N fertilization treatments.


**Table 4.** Soil apparent density (g cm−3) and coarse elements (% g/g) at 0–20, 20–40, and 40–60 cm depths.

#### **2.3. Leached nitrate**

were applied as ammonium nitrate (33.51% N g/g) in two or three broadcasts as described in **Table 3**. A control treatment in which no N was applied was included. The treatment in which 140 kg N ha−1 were broadcast was applied in two or three amendments (**Table 3**) to observe the splitting effect and to evaluate a low fertilization strategy in three broadcasts suggested as

**Sowing First broadcast Second broadcast Third broadcast Harvest Tilling** 2001-11-30 2002-03-04 2002-04-08 2002-05-13 2002-07-24 2002-11-17

2002-10-29 2003-01-20 2003-03-24 2003-05-12 2003-07-08 2003-11-20 2004-03-16 2004-04-15 2004-05-20 2004-07-28

**Table 2.** Data for sowing, first, second, and third N broadcasts, harvest and tilling.

The first Nmin measures, (ammonic and nitric N), took place at start of tillering and at the end of winter 2001. They were determined from a mixture of eight samples throughout the trial taken at the depths 0–30 and 30–60 cm. Subsequently, all Nmin values were determined from a mixture of two samples per treatment, block and depth. These samples were taken before sowing, at the end of winter, at Z20, and also after harvest in all years and all treatments except at the end of winter of year 2003, when only samples of the control treatment

The soil samples were shredded manually. Stones, roots, and any other type of plant material

200 mL of 1 M KCl were added to 100 g of soil and the mixture was stirred for 30 min. After filtering, the nitrogen of nitric origin in the filtrate was analyzed by segmented flow injection [3, 4]. The calculation of Nmin per hectare was corrected according to the content of coarse

**Start of tillering (Z20)\* Start of jointing (Z30) Flag leaf (Z37)**

were discarded. Then, the humidity of the samples was gravimetrically ascertained.

0 0 0 0 0 40 + 100 40 100 0 40 + 60 + 40 40 60 40 80 + 140 80 140 0

a possible N fertilization management in vulnerable zones.

**Total dose (kg N ha−1) Treatment Broadcasts (kg N ha−1)**

Z20, Z30 and Z37 correspond to Zadoks' scale [50].

**Table 3.** Dose and broadcasts of the N fertilization treatments.

**2.2. Mineral nitrogen (Nmin)**

elements of the soil (**Table 4**).

were taken.

82 Global Wheat Production

\*

Two ceramic cups were inserted per block and treatment. They were buried at a depth of 60 cm (**Figure 2**). As a test, in the year 2003, a hole in the soil was performed after harvest. It was observed that wheat roots reached a depth of 60 cm as a maximum; so the depth at which the ceramic cups were inserted was considered appropriate and it can be considered that the gathered nitrogen was not profitable for plants. Liquid samples were collected from ceramic cups when it had rained 20–40 mm or every fortnight. The first liquid sample after every insertion was extracted but then discarded. The sampling data occurred between December 20, 2002 and September 18, 2003 and between January 9 and September 27, both data in 2004. At both periods, the ceramic cups were removed at harvest and then inserted again. After every sampling session, a vacuum of approximately 50 kPa was performed with a manual pump. Afterwards, the nitrate concentration in the sampled water was analyzed through segmented flow injection [3, 4].

The water balance was determined in layers 0–20, 20–40, and 40–60 cm depth following Campbell's [11] simplified waterfall method. In this method, it is considered that waters fills

**Figure 2.** Ceramic capsule in the soil for leached liquid sampling.

up every layer of the soil before it flows to the following layer (Eqs. (1)–(3)). Every time a sample was taken, the humidity of each of the three layers was measured so that the variation of the water reservoir was measured. For every layer, the humidity was measured gravimetrically as well as with an IMKO TDR (time domain reflectrometry) so as to calibrate the later. However, no relation between both measures was observed; thus, only the gravimetrical measures were considered afterward.

The water balance in the 0–20 cm deep layer was calculated as specified in Eq. (1):

$$D\_{\rm 20} = \Pr{-\rm ETc} \pm \rm VR\_{\rm 20} \tag{1}$$

where *D*20 is drainage (mm) below 20 cm, Pr stands for rain, ETc is the culture's evapotranspiration (mm) determined according to FAO methodology [2] and VR20 (mm) is the variation of the water reservoir in the 0–20 cm layer.

Drainage below 40 and 60 cm was, respectively, ascertained as described in Eqs. (2) and (3):

$$D\_{40} = D\_{20} \pm \text{VR}\_{40} \tag{2}$$

this period of time, samples were taken every 2 days in treatments 0, 40 + 100, 40 + 60 + 40, and 80 + 140. In the year 2003, more exhaustive measures were taken so as to assess not only

the field after harvest and laboring and to determine the effect of the temperature and humidity on emissions. Therefore, that year, samples were taken every 2 days after N broadcasting and every fortnight from January 20, 2003 to February 4, 2004 in treatments 0, 40 + 100, and

To perform such measurements, 3 L polyvinyl chloride (PVC) hermetic chambers with a rubber septum were inserted in the soil in every block at a depth of 2 cm (**Figure 3**). Before their placement, four 10 mL samples from the atmosphere of the essay were taken at a height of 2 m. After 45 min, four 10 mL samples of the air of each chamber were taken and kept in

chromatograph (Unicam 8925) equipped with an electron capture detector (ECD). When the described measurements were performed in the field, the temperatures of the air at a height of 2 m and that of the soil at a depth of 10 cm were also measured. In 2003, the humidity of the

With the soil humidity measure, the percentage of soil water-filled pore space was assessed

WFPS = H ρSFS /(1– ρSFS /2.65) (5)

where WFPS stands for the water-filled pore space (% ml/ml), H, the water percentage in dry soil for the 0–30 cm soil layer (% g/g), and ρSFS: stone free soil apparent density (g cm−3).

Soil apparent density is the quotient between the weight of the soil solid particles and the in situ total volume of the soil. The in situ volume of the soil was assessed through the excavation method [9] with the adaptation of using polyurethane resin (Wolf, [45]) as the average value obtained at four holes dug in the essay at depths 0–20, 20–40, and 40–60 cm. Due to the

first 30 cm soil layer was also gravimetrically measured at 4 points of the essay.

O emissions of

O in the samples was analyzed with a gas

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the differences in the emissions after N applications but also to study the N<sup>2</sup>

80 + 140. Measurement frequency intensified around laboring time.

Vacutainer® blood sampling tubes. Then, the N<sup>2</sup>

using Eq. (5).

**Figure 3.** Chamber in the soil.

where VR40 is the water reservoir variation in the 20–40 cm (mm) layer and *D*40 is drainage beyond 40 cm (mm).

$$D\_{\epsilon0} = D\_{40} \pm \text{VR}\_{\epsilon0} \tag{3}$$

where VR60 is the water reservoir variation in the 40–60 cm (mm) layer and *D*60 is drainage beyond 60 cm (mm).

Finally, the N mass drained during the sampling period was calculated with Eq. (4).

$$N\_{\!\!\!\/} = D\_{\!\!\/ 0} \cdot \left[ \text{N} \!\!\!/ \_{\!\!\/} \cdot 10^{\text{-}2} \right] \tag{4}$$

where *Ni* is the N mass of nitric origin drained per period and treatment (kg N ha−1), *D*60 is the water loss due to deep percolation in the period between sampling days (L m−2) and [N] is the nitric nitrogen concentration in the leachate sampled at the end of the period *i* (mg L−1).

Finally, the values obtained per day in Eq. (4) for each treatment and sampling day in the period of time comprehended between the days the capsules were inserted and when they were removed were summed up so as to assess the N mass leached per hectare in that period.

A piezometer was installed next to the assay so as to detect the moments when the level of water was beyond 60 cm. This occurred on the days February 5 and May 7, 2003 and March 11, and April 1 and 30, 2004.

#### **2.4. Gaseous losses**

#### *2.4.1. N2 O emissions*

To assess the fertilizer application effect on N<sup>2</sup> O emissions (N2 Oem), in the year 2002, those emissions were measured after the N fertilizer applications, from March 4 to May 17. During

**Figure 3.** Chamber in the soil.

up every layer of the soil before it flows to the following layer (Eqs. (1)–(3)). Every time a sample was taken, the humidity of each of the three layers was measured so that the variation of the water reservoir was measured. For every layer, the humidity was measured gravimetrically as well as with an IMKO TDR (time domain reflectrometry) so as to calibrate the later. However, no relation between both measures was observed; thus, only the gravimetrical

*D*<sup>20</sup> = Pr–ETc ± VR20 (1)

where *D*20 is drainage (mm) below 20 cm, Pr stands for rain, ETc is the culture's evapotranspiration (mm) determined according to FAO methodology [2] and VR20 (mm) is the variation of

Drainage below 40 and 60 cm was, respectively, ascertained as described in Eqs. (2) and (3):

*D*<sup>40</sup> = *D*<sup>20</sup> ± VR40 (2)

where VR40 is the water reservoir variation in the 20–40 cm (mm) layer and *D*40 is drainage

*D*<sup>60</sup> = *D*<sup>40</sup> ± VR60 (3)

where VR60 is the water reservoir variation in the 40–60 cm (mm) layer and *D*60 is drainage

the water loss due to deep percolation in the period between sampling days (L m−2) and [N] is the nitric nitrogen concentration in the leachate sampled at the end of the period *i* (mg L−1). Finally, the values obtained per day in Eq. (4) for each treatment and sampling day in the period of time comprehended between the days the capsules were inserted and when they were removed were summed up so as to assess the N mass leached per hectare in that period. A piezometer was installed next to the assay so as to detect the moments when the level of water was beyond 60 cm. This occurred on the days February 5 and May 7, 2003 and March

emissions were measured after the N fertilizer applications, from March 4 to May 17. During

is the N mass of nitric origin drained per period and treatment (kg N ha−1), *D*60 is

O emissions (N2

<sup>i</sup> · 10−<sup>2</sup> (4)

Oem), in the year 2002, those

Finally, the N mass drained during the sampling period was calculated with Eq. (4).

The water balance in the 0–20 cm deep layer was calculated as specified in Eq. (1):

measures were considered afterward.

84 Global Wheat Production

the water reservoir in the 0–20 cm layer.

*Ni* = *D*<sup>60</sup> · [N]

beyond 40 cm (mm).

beyond 60 cm (mm).

11, and April 1 and 30, 2004.

*O emissions*

To assess the fertilizer application effect on N<sup>2</sup>

**2.4. Gaseous losses**

*2.4.1. N2*

where *Ni*

this period of time, samples were taken every 2 days in treatments 0, 40 + 100, 40 + 60 + 40, and 80 + 140. In the year 2003, more exhaustive measures were taken so as to assess not only the differences in the emissions after N applications but also to study the N<sup>2</sup> O emissions of the field after harvest and laboring and to determine the effect of the temperature and humidity on emissions. Therefore, that year, samples were taken every 2 days after N broadcasting and every fortnight from January 20, 2003 to February 4, 2004 in treatments 0, 40 + 100, and 80 + 140. Measurement frequency intensified around laboring time.

To perform such measurements, 3 L polyvinyl chloride (PVC) hermetic chambers with a rubber septum were inserted in the soil in every block at a depth of 2 cm (**Figure 3**). Before their placement, four 10 mL samples from the atmosphere of the essay were taken at a height of 2 m. After 45 min, four 10 mL samples of the air of each chamber were taken and kept in Vacutainer® blood sampling tubes. Then, the N<sup>2</sup> O in the samples was analyzed with a gas chromatograph (Unicam 8925) equipped with an electron capture detector (ECD). When the described measurements were performed in the field, the temperatures of the air at a height of 2 m and that of the soil at a depth of 10 cm were also measured. In 2003, the humidity of the first 30 cm soil layer was also gravimetrically measured at 4 points of the essay.

With the soil humidity measure, the percentage of soil water-filled pore space was assessed using Eq. (5).

$$\text{WFFS} = \text{H} \, \mathfrak{p}\_{\text{ges}} / \text{(1} - \mathfrak{p}\_{\text{SFS}} / 2.65) \tag{5}$$

where WFPS stands for the water-filled pore space (% ml/ml), H, the water percentage in dry soil for the 0–30 cm soil layer (% g/g), and ρSFS: stone free soil apparent density (g cm−3).

Soil apparent density is the quotient between the weight of the soil solid particles and the in situ total volume of the soil. The in situ volume of the soil was assessed through the excavation method [9] with the adaptation of using polyurethane resin (Wolf, [45]) as the average value obtained at four holes dug in the essay at depths 0–20, 20–40, and 40–60 cm. Due to the fact that the soil was quite stony, the apparent density required a correction which was executed considering the percentage of gross elements (those that did not pass through a 2 mm sieve) (**Table 4**) and their density, which is acknowledged as 2.65 g cm−3 [37].

were ground and sieved through 0.5 and 1 mm sieves respectively. The total N was determined both for straw and grain by Kjeldahl procedure [5] with a Kjeltec Auto sampler System 1035 (Tecator). The N absorption by the aerial part of the plant (Nab) was determined as the sum of the products of the N concentration at grain and straw times their respective biomasses.

The yielded grain quantity datum, needed for the previous calculation was determined by harvesting the central 1.5 m wide aisle of each plot. Yield was referred to a humidity of

The N balances are based on the application of the mass conservation principle to the cultures. Thus, the variation of N stocked in a system equals the difference among the inputs and outputs to the system [31]. Often, the N balance is inferred considering the inputs and outputs in relation to the main source, the soil Nmin. This is considered available to plants in spite of the fact that it can also be consumed by microorganisms, dissipate as gas, or leach through the soil

NminS + Min + F = Nab + Nabr + Nlix + N2 Oem + NminAH + Nc (6)

where, NminS: Nmin quantity in soil before seeding (kg N ha−1), Min: soil N fraction in the soil due to the conversion of organic nitrogen to ammonium (kg N ha−1) (It is calculated from the adjustment of the balance for the control treatment, i.e., 0 (kg N ha−1)), F: fertilizer N applied (kg N ha−1), Nab: N absorbed by the aerial part of the plant (kg N ha−1), Nabr: N absorbed by the roots of the plant (kg N ha−1) (It was estimated that this was 25% of Nab [38]),

Thus, a positive Nc means that: (i) the inputs have been overestimated due to an error in its calculation or/and experimental error, (ii) the outputs have been underestimated for the same reasons as in (i), (iii) there are other N outputs that have not been considered, and (iv) a com-

For the assessment of N use efficiencies, the following parameters were defined (Huggins and

Efficiency in the use of the fertilizer (NUE): difference between Nab of the fertilized treatment

Harvest index (HI): quotient between the N in the grain and the N extracted by the aerial part

O (kg N ha−1), NminAH: Nmin in soil after

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Oprod or Ndeni since these last two refer

Oem: N emitted as N<sup>2</sup>

[34]. The balance was assessed as described in Eq. (6) [29]:

bination of all or some of the situations described here occurs.

to gases produced in the arable layer and thus may not exit the system.

and the non-fertilized, divided by the quantity of fertilizer applied.

Oem was considered in the balance instead of N<sup>2</sup>

120 g kg−1.

**2.6. N balance**

Nl: leached N (kg N ha−1), N<sup>2</sup>

**2.7. N use efficiency**

of the culture (dimensionless).

Pan, 1993):

N2

harvest (kg N ha−1), Nc: Not accounted for.

#### *2.4.2. N2 O production and total denitrification at the arable layer*

From January 23 to September 18, 2003, the N<sup>2</sup> O production rate (N<sup>2</sup> Oprod) was measured in the arable layer at the same treatments and times described for N<sup>2</sup> Oem. To achieve this, two 30 cm long and 2.65 cm diameter wide cylindrical samples were taken at the plots of the studied treatments. These two samples were transferred to a hermetic 2 L pot with a rubber septum (**Figure 4**). Then, the pots of the different plots were inserted in a hole next to the studied plot and were covered with the soil of the same hole and kept to incubate for 24 hours. In such a manner, the actual soil temperature and its changes could be mimicked. After 24 hours, 10 mL samples of the atmosphere of each pot were stocked in Vacutainer® tubes for their ulterior N<sup>2</sup> O determination by gas chromatography (Unicam 8925). The same process was followed for other pots to which 100 mL of acetylene (C<sup>2</sup> H2 ) (Air Liquide, SA) were injected making the atmosphere of the pot rich in acetylene by 5% (**Figure 4**). C<sup>2</sup> H2 blocks N<sup>2</sup> O reduction to N2 in the denitrification process and the ammonium oxidation in the nitrification process [33]. Therefore, the N<sup>2</sup> O produced in the incubation with an atmosphere of 5% C<sup>2</sup> H2 comprises the joint production rate of N<sup>2</sup> O + N<sup>2</sup> due to denitrification (Ndeni) [8] (Knowles, [25]).

The cumulated N loss was calculated by the integration of the diary rates of N<sup>2</sup> Oem, N<sup>2</sup> Oprod, and Ndeni over time.
