AGRICULTURAL AND FOOD SCIENCE Emissions of nitrous acid (HONO), nitric oxide (NO), and nitrous oxide (N 2 O) from horse dung

Abstract

Horse dung contains considerable amounts of nitrogen which is partly lost during the storage period. Leaching of nitrogen from the dung can be prevented with constructions but also gaseous N-emissions occur. However, the emission rates are not reported in the literature. We measured in laboratory conditions nitrous oxide (N 2 O), nitric oxide (NO) and nitrous acid (HONO) emissions from fresh, one month old and one year old horse dung samples. NO and HONO emissions increased with the storage time of the dung. The mean emission rates of HONO and NO were from 36 to 280 ng N kg dw-1 h-1 and from 15 to 3500 ng N kg dw-1 h-1 , respectively. N 2 O emissions were more variable showing also highest emissions (20.3 µg N kg dw-1 h-1) from the oldest samples. Thus, the longer storage of horse dung increases gaseous N losses which should be taken into account when planning an environmental friendly way to handle horse dung.

Full text

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Manuscript received September 2016
Emissions of nitrous acid (HONO), nitric oxide (NO), and nitrous
oxide (N2O) from horse dung
Marja Maljanen, Zafar Gondal, Hem Raj Bhattarai
University of Eastern Finland, Department of Environmental and Biological Sciences, P.O. Box 1627, 70211 Kuopio
email: Marja.Maljanen@uef.fi
Horse dung contains considerable amounts of nitrogen which is partly lost during the storage period. Leaching of
nitrogen from the dung can be prevented with constructions but also gaseous N-emissions occur. However, the
emission rates are not reported in the literature. We measured in laboratory conditions nitrous oxide (N2O), nitric
oxide (NO) and nitrous acid (HONO) emissions from fresh, one month old and one year old horse dung samples.
NO and HONO emissions increased with the storage time of the dung. The mean emission rates of HONO and NO
were from 36 to 280 ng N kg dw-1 h-1 and from 15 to 3500 ng N kg dw-1 h-1, respectively. N2O emissions were more
variable showing also highest emissions (20.3 µg N kg dw-1 h-1) from the oldest samples. Thus, the longer storage
of horse dung increases gaseous N losses which should be taken into account when planning an environmental
friendly way to handle horse dung.
Key words: nitrogen, carbon, nitrification, greenhouse gas, animal excreta
Introduction
The number of horses is increasing in the EU countries including Finland. There are currently 75 000 horses in
Finland and these horses are producing more than one million m3 manure annually, of which a small portion is
left on the pastures during the summer (Manninen et al. 2016). Annually about 770 000 m3 of manure is collected
from the stables, and it is mainly used as fertilizer in the fields. However, at the moment most of the new
stables are established in the urban areas/without connection to agricultural fields, which causes problems with the
manure management and the cost for disposing of the manure is significant (Manninen et al. 2016).
At horse stables manure is stored in stockpiles on e.g. concrete bottom structure or in a skip before trans-
portation or further processing. There are requirements for the stockpiling the manure to prevent leach-
ing of nutrients, e.g. nitrate, by Government Decree on the Restriction of Discharge of Nitrates From Ag-
riculture into Waters based on the EU Nitrates Directive (http://www.finlex.fi/en/laki/kaannokset/2000/
en20000931.pdf). However, during this storage period also the gaseous N-losses e.g. ammonia (NH3) or ni-
trous oxide (N2O) from the manure are taking place (Thomsen 2000, Garlipp et al. 2011). Emissions from
manure may vary with storage time and moisture (Pratt et al. 2015) and depend also of the bedding material
(e.g. straw, wood chips, peat) used (Garlipp et al. 2011). However, these direct emission rates from horse manure
or dung itself are not reported in the literature.
Nitrous oxide (N2O) is a strong greenhouse gas (about 300 times stronger than CO2) mainly originating from
agriculture and livestock manure management accounts for almost 10% of greenhouse gas emissions from agricul-
ture globally (Owen and Silver 2015). Another N-gas which is produced in the same microbial processes as N2O is
nitric oxide (NO). NO is not a greenhouse gas but it reacts to form smog and acid rain and it is also important to the
formation of tropospheric ozone (Heil et al. 2016). Nitrous acid (HONO), also a reactive gas in the atmosphere, is
linked to nitrogen cycle processes and thus also the production of N2O and NO (Su et al. 2011, Maljanen et al. 2013).
It is not a greenhouse gas, instead, it contributes to formation of hydroxyl radicals (OH–), which are strong oxidizing
molecules and can oxidize e.g. atmospheric methane (CH4) (Riedel and Lassey 2008). Formation of aerosols (e.g. sul-
furic acid and volatile organic compounds), can speed up the HONO emissions, which are linked to air pollution and
climate change (Kulmala and Petäjä 2011). The soil related sources and formation pathways of HONO are not well
known. HONO is produced in the atmosphere by photolytic reaction of nitrite (NO2
–) or humic acid with nitrogen
dioxide (Stemmler et al. 2006) but also in soil processes in N-rich soils (Su et al. 2011, Maljanen et al. 2013, Oswald
et al. 2013, Scharko et al. 2015). Large HONO emissions have been reported from N-rich organic soils with low C/N
ratio (Maljanen et al. 2013) and our hypothesis is that animal dung with high organic matter and N-content can
also emit significant amount of HONO as well as the other N-gases; NO and N2O.

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To our knowledge there are no published studies on the gaseous N emissions (e.g. N2O, NO or HONO) from horse
dung but several research papers report N2O emissions from cattle, sheep or poultry manure (e.g. Larios et al. 2016).
Since horse dung has different composition and structure than e.g. dairy cow dung, the gaseous emissions may
differ significantly. These emissions should be known when planning the environmental friendly way to handle the
horse dung. To study the effect of storage time we measured these gaseous N-emissions from fresh, short term
stored and long term stored horse dung.
Materials and methods
The horse dung was sampled in February 2016 from a stable located in Eastern Finland (mean annual T 3.2. °C,
annual precipitation 630 mm, Pirinen et al. 2012). The stable is housing about 40 horses and the diet consists
mainly oats and hay. Fresh dung (F) samples (< 12 h) were collected directly from several boxes inside the stable,
short term stored (about 1 month, M) and older samples (stored at least one year, Y) were collected from several
sampling points from outdoor manure storage piles on concrete floor which were covered with a roof. Each type
of sample was first collected in a 20 l plastic bucket, covered with a lid and transported to laboratory within 2
hours. The bedding material used in the stable is saw dust pellets. Dung was sampled as free from bedding material
as possible. Before manual mixing and splitting into sub samples the remaining bedding material was re-
moved manually. Four replicate sub samples (~150 g dw) were placed in PVC rings (diameter 18 cm) which
were sealed from the bottom with an aluminium foil. Rings were incubated over night at room temperature
(20 °C) before gas flux measurements. Gas fluxes (N2O, NO and HONO) were measured first at original moisture
content (Table 1) and after adjusting all samples to same moisture level (2 g H2O g dw-1) and incubation of 13 days.
For adjusting the moisture the F and M samples were let to dry at room temperature whereas milliQ H2O was
added in the Y samples to increase moisture content. However, water was evaporating faster from the Y samples
during the gas flux measurements and the final moisture of Y samples was therefore less than the targeted value.
pH and electrical conductivity (EC) were measured from sample:water slurry (30:50 v/v). Total C and N were ana-
lysed with vario MAX elementar analyser (Elementar Analysensysteme GmbH, Germany). For analysis of nitrate
(NO3
–), nitrite (NO2
–) and dissolved organic carbon (DOC) a 30 ml sample and 100 ml milliQ-H2O were shaken at
175 rpm for one hour and then filtered and analyzed with an ion chromatograph (DX 120, Dionex Corporation,
USA) for NO3
–and NO2
– and with Shimadzu TOC-VCPH/CPN analyzer (Shimadzu, Japan) for DOC. To extract ammo-
nium (NH4
+) a sample of 30 ml dung and 100 ml 1M KCl were used and NH4
+ was analyzed with spectrophotometer.
Gravimetric moisture was determined by drying the samples for 24 h at 65 °C. Organic matter (OM) concentration
was determined by loss on ignition at 550 °C (e.g. Maljanen et al. 2013).
HONO emissions were measured by dynamic flow chamber connected with a commercial HONO analyzer (LOPAP,
QUMA Elektronik & Analytik GmbH, Germany), see supplementary information for details. NO fluxes were meas-
ured by dynamic chamber connected with Thermo 42i NOx analyzer (Thermo Fisher Scientific). N2O fluxes were
measured with a static chamber system and samples were taken with syringes from the headspace of the cham-
ber at intervals of 5, 10, 15 and 20 min after closing the chamber. Gas samples were injected into 12 ml Exetainers
(Labco, UK) and were analyzed with a gas chromatograph (Agilent 7890B, Agilent Technologies, USA). In addi-
tion to N2O also CH4 and CO2 fluxes at initial moisture content were measured with the same method. Statistical
differences in the N-gas fluxes were tested with non-parametric Mann Whitney U-test since the data was not nor-
mally distributed. Gas fluxes before and after adjusting moisture were treated separately. Other parameters were
analyzed by One-Way ANOVA and Tukey’s test.
Results
OM and C concentration and C/N ratio were significantly lower in Y samples than in the other samples (Table 1).
DOC concentration decreased with increasing age of dung. The initial moisture content was higher in F and M
samples than in Y sample, as expected. All dung samples had pH above 7 and EC above 800 µS cm-1 .
All studied horse dung samples emitted both HONO and NO, but only F and Y samples emitted N2O. Storage time of
dung increased clearly the emissions of NO and HONO and there was a clear statistical difference between F, M and Y
samples (Fig. 1). The emission rates of NO were 56 ± 22, 900 ± 77 and 3000 ± 670 and emission rates of HONO were
35 ± 19, 53 ± 20 and 113 ± 34 ng N kg dw-1 h-1 in F, M and Y samples at original moisture content, respectively (Fig. 1).

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The statistical difference between sample types were analyzed by One-Way ANOVA; ** = p < 0.01. Values with a common letter
as superscript do not differ as a statistical significance of p < 0.05 in Tukey’s test.
Table 1. Sample properties (average ± standard deviation) measured from four replicate dung samples. EC = electrical
conductivity (µS cm-1), OM = organic matter concentration (% of dw), C = total carbon concentration (% of dw), N = total
nitrogen concentration (% of dw), C/N = carbon to nitrogen ratio, DOC = water extractable dissolved organic carbon
(mg g dw-1), GM = initial and adjusted gravimetric moisture (g H2O g dw-1), CH4 = methane production rate (µg g dw-1
h-1) and CO2 = carbon dioxide production rate (mg g dw-1 h-1) measured at initial moisture.
Fresh (F) One month old (M) One year old (Y) F-value
pH 7.5 7.9 7.7 1.64
EC 810 ± 180a1670 ± 330b1630 ± 350b10.43**
OM 90.1 ± 0.4a90.6 ± 1.0a65.7 ± 8.1b36.42**
C44 ± 0.1a43 ± 0.4a31.8 ± 4.6b27.31**
N 1.8 ± 0.1 1.6 ± 0.1 1.8 ± 0.3 1.07
C/N 25.0 ± 0.7a27.6 ± 0.8b18.1 ± 1.5c83.61**
DOC 18.8 ± 0.6a13.5 ± 0.9b11.3 ± 1.8b97.43**
GMinitial 3.63 ± 0.12a3.05 ± 0.15a0.56 ± 0.13b12.29**
GMadjusted 1.98 ± 0.10 1.94 ± 0.05 1.58 ± 0.40 1.85
CH4 20.8 ± 4.1a10.0 ± 3.3b0.61 ± 2.4c36.76**
CO21140 ± 280a1940 ± 400a120 ± 54b41.61**
Fig. 1. HONO, NO and N2O emissions and mineral nitrogen concentrations from samples in a) original
moisture content and b) adjusted moisture content. *) The average N2O emission from M sample was
negative (mean N2O consumption rate 510 ± 330 and 350 ± 280 ng N kg dw -1 at original and adjusted
moisture) and therefore it is not shown in the figure with a log-scale on y-axis. Different letters indicate
statistical difference between sample types (Mann Whitney U-test).

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HONO emissions were five times and NO emissions 60 times higher in one year old sample than in fresh dung
at original moisture level but 30 and 500 times higher after adjusting the moisture, respectively. N2O emissions
were more variable but also the highest N2O emissions (20.3 ± 2 µg N kg dw-1 h-1) were measured from the oldest
samples after adjusting the moisture (Fig. 1). F and Y samples emitted N2O but M samples had negative mean N2O
emission (uptake). The mean N2O uptake rate in M samples was 510 ± 333 and 353 ± 277 ng N kg dw -1 at original
and adjusted moisture levels, respectively (not shown in Fig. 1 with log-scale on y-axis). The mean N2O emission
from Y samples were from 40 to 50 times higher than those from F samples.
NO3
– concentration in dung increased with age of the manure at initial moisture content (Fig. 1). After adjusting
the moisture content and incubation of 13 days concentrations were lower than the detection limit (0.01 µg g-1)
in F and M samples. NO2
– was detectable (> 0.01 µg g-1) only from Y samples (Fig. 1). Ammonium concentration
was lower in F samples than in M or Y at original moisture content but did not differ significantly after adjusting
the moisture.
Methane emissions at initial moisture content decreased with dung age (Table 1). Some of the Y samples even
consumed CH4. CO2 emissions from the dung were significantly higher from younger F and M samples than from
Y samples (Table 1).
Discussion
All dung sample types emitted NO and HONO and emission rates increased with storage time. Also N2O was emit-
ted from F and Y samples, but surprisingly not from M samples where net N2O uptake was measured. The reason
for low N2O emission from M samples is not clear, M samples were collected in winter and perhaps the storage
of dung at low temperature (from 2 to –15 °C) had decreased nitrogen mineralization. We assume that higher
emissions from Y samples is a result of increased N mineralization/ nitrification rate during long term storage.
When manure is stored, a portion of the organic N is converted by soil microbes to NH4. NH4 is then oxidized by
different microbes to nitrate and then converted to gaseous N (including N2, N2O, NO and HONO) (Oswald et al.
2013, Owen and Silver 2015, Scharko et al. 2015). However, in storage piles dung is mixed with urine and bed-
dings which can also affect the amount of available N. Ammonium was not accumulating in the dung samples
indicating that it was used rapidly in nitrification. Detectable amounts on NO2
– was found only from Y dung, this
could be linked to higher HONO emissions which can be formed chemically from NO2
– in acidic conditions (Su et
al. 2011). Since the pH of horse dung is neutral or alkaline the microbial pathway (mainly via nitrifiers) of HONO is
also possible. The C/N ratio in our study was between 18 and 27, which is slightly lower than reported by Swinker
et al. (1997) for horse manure but optimal for microbial processes. Thus, gaseous N emissions from the horse
dung can be linked with both physio-chemical (Su et al. 2011, Heil et al. 2016) and microbial processes (Oswald
et al. 2013, Scharko et al. 2015).
There are no published results about direct GHG emissions from horse dung. Some studies (Garlipp et al. 2011,
Borhan et al. 2014) are showing that horse dung is emitting N2O and CH4 but actual flux rates are not reported.
In some reports reference N2O emission factor of 0.25% of total N concentration have been used (e.g. Manninen
et al. 2016). If we use the N2O emissions rate from Y samples, it gives 0.038 g of N2O yr-1 from 1 kg of dung, corre-
sponding 0.22% of total N calculated for one year, which is close to the reported emission factor.
If we compare the highest mean Y horse dung emissions calculated per area (182 µg N2O-N m-2 h-1 and 52 µg NO-N
m-2 h-1) with those from dairy cow dung patches measured with similar chamber and analyzer systems in the field
in Eastern Finland (110 ± 20 µg N2O–N m-2 h-1 and 15 ± 2 µg NO–N m-2 h-1) we can see that NO and N2O emissions
from horse dung are higher than the mean emissions from dairy cow dung (Maljanen et al. 2007). However, the
methane emissions from horse dung are lower than those from dairy cow dung (Maljanen et al. 2012) as a result
of lower moisture content and differences in digestion (ruminant – non ruminant). Methane emissions decreased
with the age of the dung because CH4 from fresh dung originates from the anaerobic conditions in the digestion
and no CH4 is produced in aerobic conditions. CO2 production rate was lowest in the oldest sample and highest
in M samples which could be associated to fast decomposition of M samples at room temperature after storage
below 0 °C temperatures.

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Our study was made in laboratory conditions in constant temperature and humidity. Field measurements from
stockpiles or from croplands where dung is spread as fertilizer would be necessary to estimate the real N-gas
field emissions. It seems that horse dung is very potential source of N-gases, including still rather unknown HONO
gas. The mean HONO-N emissions were only about 4% of the mean N2O-N emissions and therefore the positive
climate effect of HONO cannot compensate the warming effect of N2O from horse dung. However, the HONO emis-
sions measured here from the one Y dung samples were among the highest “soil” related HONO sources published
so far (Su et al. 2011, Maljanen et al. 2013).
This study shows that nitrogen is lost from horse dung in addition to ammonia also in forms of HONO, NO and
N2O. The storage of horse dung enhances gaseous N losses which should be taken into account in life cycle analysis
and when planning an environmental friendly way to handle horse dung.
Acknowledgements
The study was funded by Marjatta and Eino Kolli foundation. We thank all the horses at Tapsan Talli Ranta-Toivala,
Finland for providing material for the study.
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