agronomy

Article
Greenhouse Gas Emissions as Affected by
Fertilization Type (Pig Slurry vs. Mineral) and Soil
Management in Mediterranean Rice Systems
Beatriz Moreno-García †, * , Mónica Guillén and Dolores Quílez
Unidad de Suelos y Riegos (asociada a EEAD-CSIC), Centro de Investigación y Tecnología Agroalimentaria
de Aragón, Avda Montañana 930, 50059 Zaragoza, Spain; [email protected] (M.G.); [email protected] (D.Q.)
* Correspondence: [email protected]
† Present address 220 ENGR Hall, Biological & Agricultural Engineering, University of Arkansas, Fayetteville,
AR 72701, USA.




Received: 19 February 2020; Accepted: 30 March 2020; Published: 1 April 2020


Abstract: The great increase in livestock production in some European areas makes it necessary
to recycle organic slurries and manures and to integrate them in crop production. In Northeast
Spain, the application of pig slurry (PS) is being extended to alternative crops such as rice due to the
great increase in pig production. However, there is a lack of information of the effect of substitution
of synthetic fertilizers with pig slurry on greenhouse gas (GHG) emissions in rice crop, and this
information is key for the sustainability of these agricultural systems. The aim of this study was to
evaluate the effect of the substitution of mineral fertilizers by PS on GHG emissions in Mediterranean
flooded rice cultivation conditions under optimal nitrogen (N) fertilization. Two field experiments
were carried out in two different (contrasting) soil types with different land management. Site 1
had been cultivated for rice in the previous three years with no puddling practices. Site 2 had been
cultivated for rice for more than 15 years with puddling tillage practices and had higher organic
matter content than site 1. The cumulative nitrous oxide emissions during the crop season were
negative at both sites, corroborating that under flooded conditions, methane is the main contributor
to global warming potential rather than nitrous oxide. The substitution of mineral fertilizer with
PS before seeding at the same N rate did not increase emissions in both sites. However, at site 1
(soil with lower organic matter content), the higher PS rate applied before seeding (170 kg N ha−1 )
increased methane emissions compared to the treatments with lower PS rate and mineral fertilizer
before seeding (120 kg N ha−1 ) and complemented with topdressing mineral N. Thus, a sustainable
strategy for inclusion of PS in rice fertilization is the application of moderate PS rates before seeding
(≈120 kg N ha−1 ) complemented with mineral N topdressing.

Keywords: flooded rice; organic fertilization; pig slurry; methane; nitrous oxide;
Mediterranean conditions

1. Introduction
Agriculture contributes to approximately 10%–12% of the total anthropogenic greenhouse gas
(GHG) emissions [1] and accounts for 60% and 59% of the total anthropogenic emissions of methane
(CH4 ) and nitrous oxide (N2 O), respectively [2]. Rice paddies are considered to be responsible for 11%
of the methane anthropogenic emissions [2]. Although rice paddies also emit N2 O, methane emissions
contribute to almost 90% of the global warming potential (GWP) in flooded rice systems [3]. Despite
the low contribution of N2 O to GWP, both gases have to be considered together when mitigation
practices are developed, since the mitigation practices that focus on CH4 emission reduction tend to
increase N2 O emissions [4–6].

Agronomy 2020, 10, 493; doi:10.3390/agronomy10040493 www.mdpi.com/journal/agronomy

Agronomy 2020, 10, 493 2 of 19

The emission of nitrous oxide (N2 O) into the atmosphere from agricultural soils is mainly related
to two biological processes, nitrification and denitrification [7,8]. Methane emission is a result of two
opposite mechanisms, production (methanogenesis) and oxidation (methanotrophy) [9,10].
Agricultural soils are also a source of carbon dioxide (CO2 ), which is emitted as a result of the
decomposition of organic matter [11] and respiration processes. However, only agricultural non-CO2
sources are considered as anthropogenic GHG emissions because the CO2 emitted is considered neutral
due to annual cycles of carbon fixation and oxidation [1]. Despite of that, practices to increase the soil
organic carbon diminish the atmospheric CO2 concentration and thus, mitigate climate change, as well
as increase fertility and health of soils [12].
Rice flooded systems are different from other cropping systems because in flooded conditions
soil processes are dominated by the anaerobic conditions created under flooding [13,14] and thus,
denitrification and methanogenesis are two of the main processes taking place.
Fertilization is essential to obtain high rice yields, but fertilization may affect GHG emission [15].
In addition, when mineral fertilizers are replaced by organic amendments, the additional carbon (C)
source could enhance soil processes such as denitrification and methanogenesis [8,16] and hence, the
application of these products could imply higher GHG emissions in comparison with mineral fertilizers.
Farmers in Northeast Spain have traditionally applied mineral fertilizers (urea and ammonium
sulfate) to rice crop, but in the last few years, they have started to include pig slurry (PS) in the
fertilization plans, initially due to the cost of mineral fertilizers and later because of the pressure to
recycle the high amount of PS produced.
Studies focusing on the influence of organic fertilizers on GHG emissions under flooded rice
systems have been carried out in different regions for evaluating products such as straw [6,17,18], green
manure [19,20], pig manure [21], pig slurry and chicken manure [22], and anaerobically digested pig
slurry (ADPS) [23]. The most consistent result found in the literature is that the incorporation of crop
residues increases methane emissions due to the additional C input [6,17,24]. However, the effect of
incorporation of crop residues on N2 O emissions is not clear, although it might be the opposite [18,25].
Nevertheless, pig slurry (PS) composition is very different than straw; the straw C content is about
30% [26], while C content in PS is below 5% [27], thus the effect of PS fertilization on GHG emissions is
expected to be different compared to the effect of crop residue incorporation.
There are a few studies that focused on the effect of PS application to rice, as a substitute for
mineral fertilizers, on GHG emissions and results are not consistent. In Asia, Sasada et al. [28] found no
significant differences in CH4 and N2 O emissions between plots fertilized with a chemical fertilizer and
plots fertilized with anaerobically digested pig slurry (ADPS). Win et al. [23] reported that cumulative
methane emissions for the growing season were 1.6 times higher for ADPS-fertilized plots than for
plots with chemical fertilization, but with no significant differences, while no differences in N2 O fluxes
were found between the two types of fertilizers. Huang et al. [29] found significant increases in CH4
emissions by applying ADPS. Under Mediterranean rice conditions, only Maris et al. [22] studied the
application of PS to flooded rice, and their results showed no significant differences in GHG emissions
and GWP for PS fertilization compared to ammonium fertilization. Thus, more studies focusing on the
substitution of mineral fertilizers with organic fertilizers to evaluate the effect on GHG emissions are
needed in the framework of a more sustainable agriculture.
Our objective was to generate information on the modification of GHG emissions due to the
substitution of mineral fertilizers with PS in Mediterranean flooded rice cultivation conditions under
optimal N fertilization. To achieve this objective, CH4 , N2 O, and CO2 emissions from the soil were
quantified during the whole crop season in two different (contrasting) soil types with different land
management in Northeast Spain. We hypothesize that, due to the low organic C content of PS,
greenhouse gas cumulative emissions during the crop season under PS fertilization will not be higher
than that under mineral fertilization.
Agronomy 2020, 10, 493 3 of 19

2. Materials and Methods

2.1. Sites Description and Experimental Design

The study was carried out in two flooded rice fields in Northeast Spain (Figure 1) with different
soil characteristics and crop management practices.

Figure 1. Locations and experimental designs of the two experiments at Villanueva de Sigena and
Grañén.
Agronomy 2020, 10, 493 4 of 19

Site 1, located at Villanueva de Sigena, was sampled in 2013 and site 2, located at Grañén (40 km
from site 1), was sampled in 2014. The climate of the two experimental fields is semiarid continental
Mediterranean, with high temperatures during the summer and low precipitation. The main climatic
characteristics for both sites are detailed in Table 1.

Table 1. Main site and soil characteristics in the 0–0.3 m soil depth at the beginning of the experiments
at the two experimental sites. ET0 is the Penman- Monteith reference evapotranspiration.

Site 1 Site 2
Site and Soil Characteristics
Villanueva de Sigena Grañén
Previous years growing rice 3 >15
Puddling No Yes
Latitude 41◦ 45’ 31.87” N 41◦ 57’ 29.97” N
Longitude 0◦ 2’ 18.16” W 0◦ 22’ 37.56” W
Elevation (m) 250 332
Annual precipitation (mm) † 347 334
Mean annual air temperature (◦ C) † 14.6 13.5
Annual ET0 (mm) † 1201 1194
pH (1:2.5, water extract) 8.5 8.3
Electrical conductivity of saturated paste extract
0.8 4.9
(ECe , dS m−1 )
Organic matter (Walkley–Black; % dry matter) 1.01 2.06
Calcium carbonate eq. (% dry matter) 29 24
NO3 − (potassium chloride extract; mg kg−1 dry soil) 11.79 14.99
NH4 + (potassium chloride extract; mg kg−1 dry soil) 6.07 10.87
Olsen P (mg kg−1 dry soil) 6 38.2
K (ammonium acetate extract; mg kg−1 dry soil) 81 224
Particle size distribution (%)
Sand (2000–50 µm) 13.4 16.4
Silt (50–2 µm) 66.2 54.1
Clay (<2 µm) 20.4 29.5
USDA textural class Silty loam Silty clay loam
† Climatic data are average values over the last ten years

Site 1 had been cultivated for rice in the previous three years with no puddling practices. Site
2 had been cultivated for rice for more than 15 years with puddling tillage practices. In puddling,
plowing and harrowing are carried out at high soil water content with straw incorporation in order to
destroy soil aggregates and create an impermeable layer. There were also differences in soil properties
between the two sites; organic matter content, salinity (ECe) , and the main nutrients’ content (N, P, and
K) were higher in site 2 than in site 1 (Table 1), and soil clay content in site 2 was also higher than in
site 1.
The samplings in site 1 were carried out in the experimental field (Figure 1) described in
Moreno-García et al. [30]. Four fertilization treatments were selected to evaluate the effect of PS
versus mineral fertilization on GHG emissions (Table 2): control (C) with no N fertilization; M120M60
(mineral treatment) with a rate of 120 kg N ha−1 (ammonium sulfate) before seeding complemented
with 60 kg N ha−1 (ammonium sulfate) at topdressing; and two PS strategies, PS120M60 with a rate of
PS equivalent to 120 kg NH4 + -N ha−1 before seeding complemented with 60 kg N ha−1 (ammonium
sulfate) at topdressing, and PS170M0 with a rate of PS equivalent to 170 kg NH4 + -N ha−1 before
seeding and no topdressing N. In PS120M60, mineral N before seeding was replaced by PS, while in
PS170M0, the crop N total requirements were covered by PS.
Agronomy 2020, 10, 493 5 of 19

Table 2. N fertilization treatments in the two experimental sites. N rates for pig slurry (PS) treatments
are the actual N rates applied in the field.

NH4 + -N Organic N Organic C NH4 + -N NH4 + -N Total N

kg ha−1 kg ha−1 kg ha−1
Site Treatments Before Seeding Topdressing Growing Season
1 Control (C) – – – – – –
M120M60 120 † – – 60 † 180 180
PS120M60 109 ‡ 92 663 60 † 169 261
PS170M0 165 ‡ 140 1007 – 165 305
2 Control (C) – – – – – –
M170M0 170 ¥ – – – 170 170
PS170M0 171 ‡ 45 824 – 171 216
† Ammonium sulfate, ‡ pig slurry, ¥ urea

Small plots specific for GHG emission measurements were established out of the main experimental
design, in order not to disturb the experimental plots during GHG sampling (Figure 1). The experiment
was arranged as a randomized block design with four replications and the plot size was 6 m x 6 m for
PS plots and 6 m x 3 m for the control and mineral plots.
In site 2, GHG emissions were evaluated in three different N treatments of an agronomic experiment
comparing PS and mineral fertilization. Selected treatments were (Table 2): control (C) with no N
fertilization, PS170M0 with a rate of PS equivalent to 170 kg NH4 + -N ha−1 before seeding and no
topdressing N, and M170M0 (mineral treatment) with a rate of 170 kg N ha−1 (urea) before seeding
and no topdressing N. Similar to site 1, small plots specific for GHG emission measurements were
established out of the main experimental design in site 2, in order not to disturb the experimental plots
during GHG sampling (Figure 1). The experiment was arranged as a randomized block design with
three replications and the plot size was 2 m x 1 m.
In both experiments, the selected treatments (except the control) were considered to be optimum
N treatments [30] (Table S1). In site 1, significant differences between yield values were only observed
between the control treatment and the three fertilization treatments. However, in site 2, the mineral
treatment had a lower yield compared with the PS treatment due to a fungal infection during grain
filling. This fungal infection decreased grain yield, but rice growth and biomass values were similar
between PS and mineral plots.
Pig slurry was collected from the closest fattening farm to each experimental field. PS application
rates were established according to PS ammonium N concentration measured in situ by Quantofix®
N-volumeter (Terraflor GmbH, Iserlohn, Germany) [31] and conductimetry [32]. Pig slurry was band
spread on the soil surface. Although machinery was calibrated before application in order to apply
target rates, the slurry tank was weighed before and after application to know the actual PS rates
applied (Table 2). Slurry samples were collected at the two sites for laboratory characterization (Table 3).
On the same day of PS application, basal mineral N was applied to the mineral treatments together
with P (100 kg P2 O5 ha−1 ) and K (100 kg K2 O ha−1 ) to ensure that these two nutrients were not
limiting since in site 1, P and K levels were suboptimal (Table 1). Slurry and mineral fertilizers were
incorporated into the soil in the afternoon of the same day.
Agronomy 2020, 10, 493 6 of 19

Table 3. Physicochemical characteristics of the PS applied at each site.

Site 1 Site 2
Villanueva de Sigena Grañén
Specific weight (g L−1 ) 1045 1021
Dry matter (kg mg−1 ) 94 23
Organic C (kg mg−1 ) 18.58 † 9.13
Ammonium N (kg mg−1 ) 3.05 1.89
Total N (Kjeldahl, kg mg−1 ) 5.63 2.39
P2 O5 (acid extraction, kg mg−1 ) 4.09 0.3
K2 O (acid extraction, kg mg−1 ) 3.57 1.96
† Organic C in site 1 was not measured and was estimated based on the average C/N ratio, from Yagüe et al. [27] for
fattening farms, equal to 3.3.

Table S2 shows the amounts of N, P2 O5 (Olsen), and K2 O (ammonium acetate) in the first 0–0.3 m
of the soil at the beginning of the experiment in each site, together with the amount of the nutrients
applied as a fertilizer in each treatment.
For both experimental fields, typical land preparation was carried out by the farmer in April
2013 and 2014 before fertilization, seeding, and flooding. In both sites, rice straw and stubbles from
the previous crop were incorporated into the soil during puddling in site 2 and with ploughing on
dry soil in site 1. Rice (Oryza sativa L. spp. Japónica cv. Guadiamar) was broadcast seeded in both
fields once they were flooded at a rate of 180 kg ha−1 . Water was applied at the top of the field and
cascaded down the paddies through levee gates. A water layer of 5 cm was maintained during the
first few days to improve rice germination; after that, a water layer of 10–15 cm was maintained until
approximately one month before harvest, when fields were drained. Moreover, the fields were briefly
drained for several days for the application of herbicides, pesticides, and fungicides, according to
habitual practices in the area (usually twice during the flooded period). Topdressing N was applied on
the water at the end of the tillering stage in site 1.

2.2. Greenhouse Gas Measurements and Analyses

The emissions of N2 O, CH4 , and CO2 from the soil into the atmosphere were measured using the
static non-vented chamber method.
At the beginning of each experiment, one (site 1) or two (site 2) polyvinyl chloride collars (19.5 cm
inner diameter) were inserted in each plot into the soil to a depth of 13 cm. Chambers (37 cm height)
were fitted into the collars at the time of sampling.
Gas sampling occurred between 09:00 and 12:00. Samples were taken through a three-way valve
placed on the top of the chamber and adjusted with a metal fitting. Gas samplings were performed
every 7–10 days or more frequently when fields were eventually drained. Air samples were obtained,
through a Teflon® (Chemours, Wilmington, Delaware, USA) tube connected to the three-way valve
and into a 100 mL propylene syringe adapted with a valve, at 0, 15, 30, and 45 min after closing
the chamber. The air inside the chamber was mixed by filling and emptying the syringe three times
before withdrawing the sample. Once the sample was taken, the valve connected to the syringe was
closed [22].
In site 1, 100 mL of air samples was taken from the individual chamber installed in each plot. In
site 2 (two chambers per plot), syringes were filled with 50 mL of air samples from each of the two
chambers in each plot (composite sample) [33]; for doing that, the tygon tube connected to the syringe
allowed the system to be closed, while moving between chambers. In both sites, 100 mL syringe
duplicates were taken in each plot per sampling time. Samples were transported to the laboratory
and N2 O, CH4 , and CO2 concentrations were quantified using the photoacoustic technique (Innova
1412i Photoacoustic Multigas Monitor, LumaSense Technologies, Ballerup, Denmark). Average GHG
concentrations in duplicates were used for the mass flux calculations.
Agronomy 2020, 10, 493 7 of 19

Soil temperature in the uppermost 0.05 m and floodwater depth (to quantify chamber headspace
volume) were measured at each sampling date.
Emissions fluxes were calculated using the linear increase/decrease in the concentration inside the
chamber over time, considering the headspace volume of the chamber. Figure S1 shows an example of
the linear regression for N2 O and CH4 on 6 August 2014 for a replicate of the PS170M0 treatment in
site 2.

2.3. Soil Sampling and Analysis

Soil (0–0.1 m) was sampled in 80% of GHG sampling dates, and moisture content and nitrate
and ammonium concentration were determined. Soil extracts were prepared using 10 g of fresh soil
and 30 mL of KCl 2N solution. Nitrate [34] and ammonium [35] concentrations were determined by
colorimetry with a continuous flow analyzer (AutoAnalyzer 3, Bran+Luebbe, Norderstedt, Germany).

2.4. Calculations and Statistical Analysis

The cumulative emissions of N2 O, CH4 , and CO2 for the studied period were quantified by
integrating the emissions over time. For doing that, the average gas fluxes between dates were
multiplied by the time interval between sampling dates.
The effects of treatments and sampling dates on GHG fluxes were evaluated by repeated measures
of analysis of variance (ANOVA). The effect of treatments on GHG cumulative emissions was evaluated
by analysis of variance. When the analysis was significant, a comparison among the treatment means
was performed using Tukey’s multiple range test at p = 0.05 (SAS 9.4 software, SAS Institute Inc., Cary,
North Caroline, USA). When necessary, data were log transformed prior to analysis in order to fulfill
ANOVA assumptions (homogeneity of variance and normality).

3. Results

3.1. Meteorological Conditions and Drainage of the Plots

Rainfall and air temperature during the sampling period (Figures 2a and 3a) were obtained
from the closest meteorological station to the experimental site (Red SIAR (Sistema de Información
Agroclimática para el Regadío), site 1 Alcolea de Cinca Station and site 2 Grañén Station). Both
sites showed similar meteorological conditions, with low precipitation during the summer and high
temperatures. The average air temperature in August was 23.9 ◦ C in site 1 (2013) and 22.9 ◦ C in site 2
(2014). The soil temperatures measured at the sampling dates were similar to the daily average air
temperatures. In both sites, high rainfall events during fall were registered.
Puddling was conducted only in site 2, not in site 1. When puddling is conducted, an impermeable
layer (plough pan) to stop percolation is created. This plough pan controls vertical water losses toward
the subsoil and it has been found to be less permeable for the older and more developed paddy
soils compared to young paddy fields [36,37]. This fact combined with the different soil particle size
distribution and the higher clay percentage of site 2 compared with site 1 (Table 1) were responsible for
the differences in the drainage speed once irrigation stopped (final drainage before harvest). Site 1
(Figure 2a) drained rapidly, while site 2 (Figure 3a) remained flooded much longer after irrigation was
stopped. In site 1, water inflow was closed on 14 September 2013, the floodwater disappeared one
week later and soil started to dry up. However, in site 2, the water inflow was closed on 1 October
2014 and the field remained flooded for almost one month; when the floodwater disappeared, the soil
remained always saturated and a very thin water layer was always present over the soil surface. In
addition, a 40 mm rainfall event on 29 November 2014 flooded the field again.
Agronomy 2020, 10, 493 8 of 19

Figure 2. Site 1 (Villanueva de Sigena) year 2013. (a) Soil and air temperature, rainfall, and floodwater
depth during the studied period; and (b) N2 O, (c) CH4 , and (d) CO2 emissions as affected by the
fertilization treatment. Vertical arrows indicate the dates of fertilization applications (F) and harvest
(H). The grey shaded areas represent the periods in which the water inflow to the field was open. Note
that once water was stopped, the field remained flooded for several days.
Agronomy 2020, 10, 493 9 of 19

Figure 3. Site 2 (Grañén) year 2014. (a) Soil and air temperature, rainfall, and floodwater depth
during the studied period; and (b) N2 O, (c) CH4 , and (d) CO2 emissions as affected by the fertilization
treatments. Vertical arrows indicate the dates of fertilization applications (F) and harvest (H). The grey
shaded areas represent the periods in which the water inflow to the field was open. Note that once
water was stopped, the field remained flooded for several days.
Agronomy 2020, 10, 493 10 of 19

3.2. Nitrous Oxide Fluxes and Cumulative Emissions

At site 1, N2 O fluxes ranged from −27.5 to 44.4 g N2 O-N ha−1 d−1 and the different N treatments
showed similar patterns (Figure 2b). No differences between fertilization treatments were noted in the
mean N2 O fluxes (Table 4). The fluxes were negative or close to 0 until the final drainage of the plot,
when N2 O emissions increased and became positive in some cases (Figure 2b).
When integrating all the sampling periods, cumulative emissions were not significantly different
between fertilization treatments (Table 4).
At site 2, N2 O fluxes ranged between −14.7 and 6.3 g N2 O-N ha−1 d−1 , the N treatments showed
similar patterns (Figure 3b), and average fluxes were not affected by treatments (Table 5). Although an
increase in N2 O fluxes was observed after the final drainage before harvest (the fluxes were negative,
but lower in absolute values, i.e., consumption of N2 O was lower than that previously observed), the
fluxes remained negative for the whole studied period unlike site 1 (Figure 3b).
At site 2, nitrous oxide cumulative emissions in the study period showed no effect of the treatments
in the same manner as at site 1 (Table 5).

Table 4. Site 1 (Villanueva de Sigena, 2013). Average fluxes of N2 O, CH4 , and CO2 and cumulative
N2 O, CH4 , and CO2 emissions during the studied period, for the different N fertilization treatments,
indicating the effects of treatment (T), date of sampling (D), and their interaction (T × D).

Gas Fluxes Cumulative Emissions

N2 O-N CH4 -C CO2 -C N2 O-N CH4 -C CO2 -C
g ha−1 d−1 kg ha−1 d−1 kg N ha−1 kg C ha−1
Treatments (T) n.s *** n.s. n.s. n.s. n.s.
Control (C) −3.92 2.47 b 6.76 −0.71 549.1 1571.0
M120M60 −2.33 2.99 b 7.87 −0.45 665.1 1959.1
PS120M60 −0.45 3.52 b 6.52 −0.04 869.9 1619.6
PS170M0 −3.24 6.31 a 9.15 −0.51 1336.4 2036.1
Date (D) *** *** ***
T*D n.s. n.s. n.s.
Note: n.s., not significant; *** p < 0.001. Different letters in the same column indicate significant differences
between treatments.

Table 5. Site 2 (Grañén, 2014). Average fluxes of N2 O, CH4 , and CO2 and cumulative N2 O, CH4 , and
CO2 emissions during the studied period, for the different N fertilization treatments, indicating the
effects of treatment (T), date of sampling (D), and their interaction (T * D).

Gas Fluxes Cumulative Emissions

N2 O-N CH4 -C CO2 -C N2 O-N CH4 -C CO2 -C
g ha−1 d−1 kg ha−1 d−1 kg N ha−1 kg C ha−1
Treatments (T) n.s. n.s. n.s. n.s. n.s. n.s.
Control (C) −5.45 18.81 7.04 −1.20 3986.8 1329.1
M170M0 −5.20 16.43 6.68 −1.09 3701.9 1330.7
PS170M0 −5.05 18.92 6.45 −1.10 4326.3 1254.1
Date (D) *** *** ***
T*D n.s. n.s. n.s.
Note: n.s., not significant; *** p < 0.001.
Agronomy 2020, 10, 493 11 of 19

3.3. Methane Fluxes and Cumulative Emissions

At site 1, CH4 fluxes varied from −0.1 to 26.6 kg CH4 -C ha−1 d−1 (Figure 2c). Nitrogen fertilization
affected the average CH4 fluxes for the studied period, with a greater mean CH4 flux for the PS170M0
treatment (Table 4). However, no differences in CH4 fluxes between M120M60 and PS120M60
(treatments with equivalent N rates before seeding) were found.
Overall, the highest emissions were observed at the end of August (at heading stage), but for the
PS170M0 treatment, high emissions were also observed at the beginning of the season (with a peak
on 19 June 2013). An emission peak also occurred in all treatments during a short drainage of the
field for an herbicide treatment (1 July 2013). Once the field was drained, CH4 emissions decreased
dramatically, reaching values lower than 100 g CH4 -C ha−1 d−1 (Figure 2c).
Methane cumulative emissions in the study period in site 1 did not show significant differences
among the fertilization treatments (Table 4), but the PS170M0 treatment showed higher (although not
significant) CH4 emission than the other three treatments.
At site 2, CH4 fluxes were higher than those at site 1. The values ranged from 0.01 to 57.8 kg
CH4 -C ha−1 d−1 (Figure 3c). The CH4 fluxes increased over time during the growing season, except
during a brief drainage episode (25 July 2014). Maximum CH4 fluxes were reached in late September
2014, during the rice ripening phase and just before the plot was drained. CH4 emissions decreased
after the beginning of final drainage; however, in contrast to site 1, the decrease in CH4 fluxes was
slower and did not reach values lower than 1 kg CH4 -C ha−1 d−1 . In contrast to site 1, treatments had
no significant effect on the average CH4 fluxes (Table 5) and cumulative CH4 emission during the
sampling period (Table 5).

3.4. Carbon Dioxide Fluxes and Cumulative Emissions

At site 1, CO2 fluxes ranged between −0.10 and 40 kg CO2 -C ha−1 d−1 (Figure 2d). The average
CO2 fluxes did not show differences among N fertilization treatments; however, a higher (but
non-significant) average flux was observed in the PS170M0 treatment (Table 4). The evolution of
CO2 over time (Figure 2d) showed CO2 emissions lower than 5 kg CO2 -C ha−1 d−1 until the plot was
drained to harvest (with the exception of an emission peak on 1 July 2013 during a short drainage).
Once the field was drained in mid-September 2013, CO2 emissions increased rapidly.
At site 2, CO2 emissions varied from −0.10 to 16.7 kg CO2 -C ha−1 d−1 (Figure 3d) and N fertilization
treatments did not affect the average CO2 flux (Table 5). During the flooded period, a small emission
peak during a short drainage period was observed. Once the field was drained for harvest, CO2
fluxes decreased slightly in site 2 in contraposition to site 1 where CO2 fluxes increased rapidly after
drainage started.
Carbon dioxide cumulative emissions for the studied period did not show significant differences
among fertilization treatments either in site 1 (Table 4) or in site 2 (Table 5).

3.5. Soil Ammonium and Nitrate Concentration

At site 1, soil (0–0.1 m) nitrate concentration ranged from 0.1 to 18.4 mg NO3 − -N kg dry soil−1
(Figure 4a). At the beginning of the experiment, immediately after the field was flooded, the nitrate
content decreased, reaching values lower than 1 mg kg−1 . In early June 2013, there was an increment
in all treatments associated with shallow floodwater depth (Figure 2a), but immediately, the nitrate
content lowered again. After that, the values remained below 1 mg NO3 − -N kg dry soil−1 until the
field was drained (14 September 2013), then nitrate content started to increase steadily (Figure 4a).
At site 1, soil ammonium concentration (0–0.1 m) ranged between 4.1 and 25.4 kg NH4 + -N kg dry
soil−1 (Figure 4b). Soil ammonium concentration was high in the first sampling dates in treatments
with N application (M120M60, PS120M60, and PS170M0), then decreased in early June 2013 (Figure 4b)
at the same time that nitrate concentration increased (Figure 4a) and after that, increased again at the
same time that nitrate decreased. From July 2013 until the plot was drained, ammonium concentration
Agronomy 2020, 10, 493 12 of 19

decreased gradually. Once the field was drained, a slight increase was observed, but again NH4
lowered (Figure 4b), matching with an increase in nitrate concentration (Figure 4a).
At site 2, soil nitrate and ammonium concentrations (data not shown) were high at the beginning
of the experiment (Table 1) with small oscillations along the crop season and similar for the different
treatments, even for the control treatment, and they did not provide useful information to be related to
gas emissions.

Figure 4. Site 1 (Villanueva de Sigena) year 2013. Soil (0–0.1 m) (a) nitrate and (b) ammonium
concentration during the studied period as affected by the fertilization treatment. Vertical arrows
indicate the dates of fertilization applications (F) and harvest (H). The grey shaded areas represent the
periods in which the water inflow to the plot was open.

4. Discussion

4.1. Nitrous Oxide Fluxes and Cumulative Emissions

Nitrous oxide emissions ranged from -27.5 to 44.4 g N2 O-N ha−1 d−1 in site 1 (Figure 2b) and from
-14.7 to 6.3 g N2 O-N ha−1 d−1 in site 2 (Figure 3b).
In site 1, there was N2 O consumption during the crop season until the field was drained (Figure 2b).
Many researchers have reported the consumption of N2 O in rice under flooded conditions [23,38,39]
and the reason is that, although nitrate is usually the starting point of denitrification, other nitrogen
oxides (NO2 − , NO, and N2 O) can serve as terminal electron acceptors for denitrifying bacteria due to a
lack of nitrate [8,40] (Equation 1).

2 NO3 - → 2 NO2 - → 2 NO → N2 O → N2 (1)

Once the field was drained (14 September 2013), N2 O emissions increased rapidly and reached
positive values (Figure 2b). The formation of N2 O in the soil is due to both nitrification [7,41] and
Agronomy 2020, 10, 493 13 of 19

denitrification processes. When soil starts to dry and O2 penetrates into the soil, the denitrification
process can stop with N2 O (third step of reaction in Equation 1), since the enzymes that operate in the
early part of the reaction are less sensitive to the availability of O2 than the reductase enzyme that
operates in the last step (N2 O to N2 ) [42]. In addition, the N2 O emissions following soil drainage
could be due to the release of dissolved and entrapped N2 O formed before drainage [4,13]. An
increase in N2 O fluxes after draining plots is well-known and it has been reported by many other
authors [4,5,25,43].
The N2 O flux pattern in site 1 was related to changes in soil nitrate and ammonium concentrations
(Figure 4a,b). During the flooded period, soil nitrate was very low, with the exception of a short period
in June 2013. Denitrifying organisms used N2 O as an electron acceptor because of the lack of nitrate,
resulting in N2 O consumption. After the field was drained, the nitrate concentration increased steadily
due to nitrification and denitrification processes. The increase in nitrate concentration in June 2013
was associated with a reduction in floodwater depth to improve rice seedling growth (Figure 2a),
which promoted O2 diffusion to the soil and hence, nitrification. This matched the decrease in soil
ammonium concentration, at the same time that nitrate increased (Figure 4a,b). After that, nitrate
concentration dramatically dropped, probably because of denitrifying process, and the ammonium
concentration increased. The cause of the increment in soil ammonium concentration might be
the release of ammonium previously fixed in the clay minerals. Anaerobic conditions promote the
temporary fixation of NH4 + in the interlayers of clay minerals [44–46] because of a net increase in the
negative surface charge of the clay [47]. Then, that fixed ammonium can be released, influenced by the
concentration of ammonium in the soil solution [44,48]. At the end of the crop season, when the plot
was drained, an increase in soil ammonium content was observed, probably due to ammonification,
but the ammonium content rapidly decreased at the same time that the nitrate increased, verifying that
nitrification took place.
In site 2, the N2 O fluxes were negative for the whole period, but values were less negative (close to
0) after the plot was drained (Figure 3b). In site 2, the field was kept flooded for more days than in site 1
(Figures 2a and 3a) and the floodwater only disappeared for a few days, but the soil was kept saturated
and a very thin water layer (several millimeters) remained over the soil surface. These findings agree
with the study reported by Iida et al. [49], who found that N2 O emission can be mitigated considerably
by even a thin film of floodwater on paddy fields.
In both sites, the cumulative N2 O emissions were negative ranging between −0.04 and −1.10 kg N
ha season−1 (Tables 4 and 5). Simmonds et al. [50] also found negative values of cumulative N2 O
−1

emissions in a field study conducted in California with the lowest value of −0.19 kg N ha−1 season−1 .
Cumulative N2 O emissions in both sites were not significantly different between the PS and
mineral treatments, thus, in this study, PS fertilization did not increase N2 O emissions compared to
inorganic fertilization. Other researchers have reported lower N2 O emissions from plots fertilized
with straw or green manure than those observed in plots fertilized with mineral fertilizers due to the
addition of C substrates, which may enhance the final reduction of N2 O to N2 by the denitrification
process [18,19,25,51]. However, PS has a low C content and thus, its application would be not expected
to have a strong influence on N2 O emissions in comparison to synthetic N. Indeed, in our study,
significant differences in the mean values of N2 O fluxes or in the cumulative N2 O emissions were not
found between treatments with the chemical fertilizer and PS (with low C content). Sasada et al. [28] and
Win et al. [23] found similar results in rice experiments with anaerobically digested pig slurry (ADPS)
in Japan, where no differences in N2 O emissions were found between ADPS and chemical fertilizers.

4.2. Methane Fluxes and Cumulative Emissions

Methane emissions ranged between −0.1 and 26.6 kg CH4 -C ha−1 d−1 and from 0.01 to 57.8 kg
CH4 -C ha−1 d−1 in site 1 and site 2, respectively (Figures 2c and 3c).
Agronomy 2020, 10, 493 14 of 19

Organic matter applied to the fields such as rice straw, soil organic matter (SOM), and organic
matter from rice plants (exudates and sloughed tissues) are the carbon sources for CH4 emissions [52,53].
Likewise, organic C contained in PS (Table 3) is an additional source of carbon.
In the present study, at site 1, the average CH4 flux was significantly higher for the PS170M0
treatment (Table 4). This treatment showed an emission peak during the first few weeks after flooding;
moreover, another peak was observed in all treatments later in the season. The results suggested that
the additional C source applied in the PS170M0 plots promoted higher emissions early in the season.
Our results agree with those of Wassmann et al. [54] and Neue et al. [16] in rice field experiments. They
found that early in the season, organic amendments provide substrates for methanogenesis, while root
exudates become more important at the later growth stages. However, in our study, the PS120M60
treatment did not show higher CH4 emissions than M120M60 (Table 4), even though an additional C
source was applied to the soil (Table 2); thus, organic C applied at moderate rates of PS did not seem to
be enough to increase the CH4 emissions.
After the final drainage of the plot, methane emission decreased rapidly, as soil started to dry up
and oxygen promoted aerobic decomposition of organic matter to carbon dioxide and less methane.
Methane emissions were higher in site 2 than in site 1. Puddling was performed in site 2, but
not in site 1. Puddling disperses soil colloids and increases the water-to-soil ratio, resulting in very
low bulk densities, promoting reduction. In contrast, high soil bulk density from less intense field
preparation retards organic matter decomposition and reduces the speed of potential redox changes as
well as CH4 formation [55]. Moreover, SOM in site 2 was higher than that in site 1 (Table 1). Puddling
and a higher SOM content could explain the higher CH4 emissions in site 2.
At site 2, methane fluxes were not significantly affected by N fertilization (Table 5), despite the
additional C source (Table 2) in the PS treatment compared to the mineral treatment. Although organic
amendments may increase CH4 production by providing readily mineralizable carbon sources, these
changes are more pronounced when organic substrates are added to soils with low organic matter
content [23,55]. Soil at site 1 had lower organic matter content than soil at site 2 (Table 1); therefore,
organic C contained in the PS had a stronger influence at site 1 because of the lower SOM content
compared to site 2. The results suggested that, in site 2, emissions were more influenced by SOM and
land management (puddling) than by C content of fertilizers. These results are in agreement with
those reported in the studies by Sasada et al. [28] and Win et al. [23], where differences in the effects of
anaerobically digested pig slurry (ADPS) on methane emissions were attributed to the differences in
soil C content, suggesting that the application of ADPS might have a higher stimulating effect on CH4
emissions when soil C content is lower.
In contrast to site 1, CH4 fluxes did not decrease immediately after the drainage of the plot, but the
emissions decreased slowly (Figure 3c) and the reason was the difference in the drying speed. The field
at site 2 remained flooded for more days than that at site 1 (Figures 2a and 3a) and hence, favorable
conditions for methanogenesis were maintained.
The mean daily CH4 fluxes ranging between 2.5 and 6.3 kg CH4 -C ha−1 d−1 and the maximum
flux (27 kg CH4 -C ha−1 d−1 ) reported at site 1 were similar to those reported in the literature for paddy
rice fields [24,28,56]. The cumulative emissions for the season are also in accordance with the values in
the review by Sanchis et al. [57]. However, the mean daily CH4 fluxes at site 2, ranging between 16.4
and 18.9 kg CH4 -C ha−1 d−1 were higher than those in site 1 and higher than those reported in the
literature, and hence, cumulative CH4 losses were higher than expected. The reason for these high
values could be the absence of the methane oxidation process. Methane emission is a result of two
opposite mechanisms, production and oxidation [9,10]. Aerobic oxidation of methane takes place in
the soil–water interface of the submerged paddy soil and in the rhizosphere where oxygen is available
in a shallow layer around the rice roots [9]. Some studies have reported significant methane oxidation
rates during the crop maximum development stages [9,58,59]. In our experiments, plants were cut
inside the chambers since the purpose was to measure soil emissions. Thus, methane oxidation in the
rhizosphere inside the collars was reduced, increasing methane emissions. Another reason could be the
Agronomy 2020, 10, 493 15 of 19

technique used for the quantification of fluxes (photoacoustic spectroscopy—PAS). While some authors
reported good results when comparing PAS with gas chromatography (GC) [60,61], other authors
found a large effect of water vapor on PAS CH4 readings, concluding that manufacturer calibration
for moisture was not sufficient [62,63]. Although we are aware that the absolute CH4 values could be
overestimated, we consider that the comparative between PS and mineral treatments, which was the
main objective of this study, is reliable.
As expected, the comparative analysis showed that PS applied before seeding at the same rates as
the mineral fertilizer (PS120M60 vs. M120M60 in site 1, PS170M0 vs. M170M0 in site 2; Tables 4 and 5)
did not increase methane cumulative emissions.
Other studies have also found no differences in methane emissions when mineral fertilizers are
replaced by PS [22] or ADPS [28] at the same N rates. However, opposite results were reported by
Huang et al. [29] who found significant CH4 increases when mineral fertilizer was replaced by ADPS
and the application was fractioned (40% base, 25% tillering, and 35% heading), but no differences were
found when ADPS was applied in a unique application (base fertilization).

4.3. Carbon Dioxide Fluxes and Cumulative Emissions

Carbon dioxide emissions varied from 0.10 to 40 kg CO2 -C ha−1 d−1 and between −0.10 and
16.7 kg CO2 -C ha−1 d –1 (Figures 2d and 3d) in site 1 and site 2, respectively, and followed an opposite
pattern compared to methane emissions.
At site 1, low emissions were recorded during the flooded period and higher emissions were
recorded immediately after the drainage of the plot (Figure 2d), which were clearly associated with
the aerobic mineralization of organic matter. Unlike CH4 emissions, significant differences between
treatments in the mean CO2 flux were not observed; nevertheless, the mean CO2 flux for the PS170M0
treatment was higher (not significant) than that observed for the rest of the treatments (Table 4); and
similar to CH4 emissions, the reason was the addition of C with PS.
At site 2, similar to CH4 emissions, the pattern of the CO2 fluxes after the drainage for harvest was
totally different than that observed in site 1. While CO2 emissions increased at site 1 (Figure 2d), CO2
fluxes decreased slowly at site 2 (Figure 3d). We consider the soil moisture content to be responsible
for this fact, as the soil did not dry up after drainage at site 2, O2 diffusion was restricted, and hence,
aerobic decomposition.
It is important to mention that in the present study, emissions were measured only during the
crop season and one month after harvesting, thus, further studies including measurements during
the whole year would be necessary to evaluate whether there are differences between fertilization
treatments in the intercrop period.

5. Conclusions
The characteristics of the soil and land management have a strong influence on GHG emissions,
as methane fluxes are higher in paddy fields with higher organic matter content or with continuous
puddling tillage practices.
The cumulative nitrous oxide emissions during the crop season were negative at both sites,
corroborating that under flooded conditions, methane was the main contributor to GWP rather than
nitrous oxide. GHG emissions were not affected by the application of pig slurry at the same N rate as
the mineral fertilizer. However, application of high PS rates before seeding to a soil with low SOM
increased methane emissions in comparison to mineral fertilization. Therefore, application of PS
before seeding at rates to cover about 70% of crop N needs and N topdressing to complement crop
requirements are recommended in order not to increase methane emissions. Taking into account this
consideration, PS might be an excellent fertilizer for replacing synthetic fertilizers without jeopardizing
the sustainability of rice systems.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4395/10/4/493/s1,

Table S1: Grain yield (14% moisture) for the different N fertilization treatments in site 1 (Villanueva de Sigena) and
Agronomy 2020, 10, 493 16 of 19

site 2 (Grañén). Table S2: N, P2 O5 (Olsen), and K2 O (ammonium acetate) amounts in the 0–0.3 m soil depth at the
beginning of each experiment and the nutrient amounts applied with the fertilizers (PS or mineral). Figure S1: (a)
N2 O and (b) CH4 concentrations over time on 6 August 2014 for a replicate of the PS170M0 treatment in site 2
(Grañén).
Author Contributions: B.M.-G. was responsible for field data acquisition, laboratory analysis, data analysis, and
wrote the manuscript draft. M.G. was responsible for field data acquisition and laboratory analysis. D.Q. was
responsible for funding acquisition and directed the study. All authors have read and agreed to the published
version of the manuscript.
Funding: This study was funded by the National Institute for Agricultural and Food Scientific Research and
Technology of Spain (INIA), the Government of Aragón, FEDER, and FEADER funds (RTA2010-0126-C02-01,
RTA2013-0057-C05-04 and DRU-2014-02-50-541-00-IFO-00740020007). B. Moreno-García was granted with an
FPI-INIA fellowship.
Acknowledgments: We thank the field and laboratory personnel of the Soils and Irrigation Department of CITA.
Conflicts of Interest: The authors declare no conflicts of interest.

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