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Soil Biology & Biochemistry 32 (2000) 497±509
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Nitrogen loss through denitri®cation in a soil under pasture in
New Zealand
J. Luo*, R.W. Tillman, P.R. Ball
The Institute of Natural Resources, Massey University, Palmerston North, New Zealand
Accepted 24 September 1999

Abstract
Denitri®cation on several contrasting topographical sites in a New Zealand dairy-farm pasture was measured periodically over
a year, using the acetylene inhibition technique, by incubating undisturbed soil cores in a closed system. The measured
denitri®cation rates varied considerably both spatially and temporally. High coecients of variation (CV) and log-normal
distributions of denitri®cation rate were often observed. The spatial variance in denitri®cation rate changed temporally and was
apparently related to soil moisture content and the grazing pattern. Denitri®cation rates followed a marked seasonal pattern,
with highest rates being measured during the wet winter and lowest rates during the dry summer and early autumn. Di€erences
in denitri®cation rates among sites were not consistent. However, slightly higher denitri®cation rates were usually detected in the
¯oor of a gully and in a gateway area than on other sites. Mean denitri®cation rates from individual dates were positively
correlated to soil moisture content. However, there was a negative correlation between denitri®cation rate and soil nitrate
concentration, respiration rate and temperature. An annual nitrogen loss of 4.5 kg N haÿ1 through denitri®cation was estimated
in this legume-based dairy-farm pasture. Low soil moisture content was the primary factor limiting denitri®cation during the dry

summer and early autumn. Low denitri®cation rates were also caused by lack of available soil NOÿ
3 -N. 7 2000 Elsevier Science
Ltd. All rights reserved.
Keywords: Denitri®cation; Pasture; Nitrous oxide; New Zealand; Soil

1. Introduction
Most of the previous research on nitrogen cycling
in grazed pastures has demonstrated the importance
of the grazing animal in returning N ingested in
the herbage to the soil in the form of urine and
dung (Ball and Tillman, 1994). Although early
research indicated that grazing animals had a bene®cial role in nutrient cycling through the transfer
of fertility in the form of excreta around the farm
(Sears, 1950), this viewpoint has since been modi®ed. Research has shown that the return of N to

* Corresponding author. Present address: Land and Environmental
Management, AgResearch, P.O. Box 3123, Hamilton, New Zealand.
Tel.: +64-7-838-5125; fax: +64-7-838-5155.
E-mail address: Luoj@agresearch.cri.nz (J. Luo).

the soil in the form of extremely concentrated urine
spots can lead to greater losses than originally indicated, particularly on intensively managed, high fertility farms (Ball and Keeney, 1981). The fate of
excretal N in pastures under New Zealand conditions has been investigated by a number of
workers (e.g. Ball et al., 1979; Carran et al., 1982;
Field et al., 1985; Williams et al., 1989). In several
of these studies, mass balance considerations indicated that signi®cant amounts of N were unaccounted for. Loss of this N through denitri®cation
is one possibility. Certainly, at ®rst glance the potential for denitri®cation from pastures would
appear to be high due to high amounts of organic
C in the surface soil and high concentrations of
NOÿ
3 -N present in the soil under urine and dung
patches (Haynes and Williams, 1993). Although studies

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 7 9 - 0

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J. Luo et al. / Soil Biology & Biochemistry 32 (2000) 497±509

on the potential denitri®cation in New Zealand pastures have been made by Limmer and Steele (1982),
Luo et al. (1996) and Crush (1998), the extent of denitri®cation and the factors a€ecting it in New Zealand
pastures requires study, and more information about
denitri®cation is needed to assess the contribution of
denitri®cation in New Zealand grasslands to regional
and global N cycling.
There are a number of reports of denitri®cation
in grassland soils, and most studies have been conducted in the northern hemisphere (e.g. Ryden,
1983; Jarvis et al., 1994; Schwarz et al., 1994). Investigations have included the e€ect of N fertilisers,
slurry application and irrigation on denitri®cation
rates. The measured annual denitri®cation rates in
the ®eld vary from 0 to >100 kg N haÿ1 (Jordan,
1989; Aulakh et al., 1992; Gro€man and Turner, 1995;
Ledgard et al., 1997).
An issue complicating the study of denitri®cation
in the ®eld is the marked spatial and temporal
variability (Ryden, 1983; Folorunso and Rolston,
1984). Spatial variability results from the pathy distribution of denitrifying `hot spots' in the soil. `Hot
spots' are caused by non-homogeneous distribution
of available C (Parkin, 1987) and other factors that

regulate denitri®cation, such as NOÿ
3 and soil aeration (Smith, 1980; Gro€man and Tiedje, 1989a). The
temporal variations can be explained mainly by corresponding variations in soil temperature and other denitri®cation regulating factors (Gro€man and Tiedje,
1989b). The general tendency is for highest denitri®cation rates to occur when soils are warm, wet and soil
NOÿ
3 and C are available. Denitri®cation rates are generally highest in spring, summer and autumn in northern temperate agricultural soils (e.g. Ryden, 1983;
Parsons et al., 1991; Estavillo et al., 1994; De Klein
and Van Logtestijn, 1994; Schnabel and Stout, 1994),
and in winter in New Zealand pasture soils (Ruz-Jerez
et al., 1994). Variations in N fertiliser application, irrigation and rainfall are also reasons for temporal variation in denitri®cation rate (Jarvis et al., 1991; Aulakh
et al., 1992; Schnabel and Stout, 1994). Animal grazing
in pastures also in¯uences the temporal variation of
denitri®cation rates (Carran et al., 1995; Luo et al.,
1999a).
We investigated the extent of denitri®cation in a
New Zealand pasture. The study site was located on
an intensive dairy-farm on a poorly-drained soil. The
combination of poor drainage and compaction from
high stocking rates was expected to restrict aeration.
The return of dung and urine should have ensured a

ready supply of NOÿ
3 -N and soluble C. It was felt that
such a site might provide a useful insight into the
`upper boundary' to denitri®cation N losses from pasture.

2. Materials and methods
2.1. Site description
The research was carried out using soil samples collected from a paddock of the Massey University No. 4
dairy-farm, Palmerston North, New Zealand. The size
of the paddock was about 2.5 ha. The paddock was
under ryegrass±white clover pasture (i.e. legume-based
pasture) and was periodically grazed by cows at stocking density of about 100 cows haÿ1. Throughout the
study, the paddock received no N fertilisers. The soil
at this site, Tokomaru silt loam (Cowie, 1974), is
classi®ed as a yellow grey earth (Taylor and Pohlen,
1968) or a pallic soil (Hewitt, 1992). It is a poorlydrained soil with wet conditions in winter, and relatively dry conditions in summer. The paddock was predominantly ¯at with a small gully (about 3 m deep)
running through it. Five contrasting sites were located
within the paddock. These were a ¯at land area,
north- and south-facing gully slopes, the gully bottom
and a fertile and compacted gateway area. Soil properties of the upper 7.5 cm of the pro®le are given in

Table 1.
2.2. Field denitri®cation measurement
2.2.1. Collection of samples
One sampling area (9  9 m) was usually selected
within each of the gateway, south-facing and northfacing slope sites and two sampling areas selected
within each of the ¯at land and gully bottom sites.
Denitri®cation measurements were made regularly at
all the sites from July 1992 to July 1993, with more
frequent sampling in the late summer and early
autumn of 1993. On measurement occasions, 16 soil
cores were taken from randomly-selected areas at each
site. The sampling points in each sampling were
arranged at 3-m intervals over the 9  9 m area.
2.2.2. Measurement of denitri®cation rate
The rate of denitri®cation was measured using the
acetylene-inhibition technique (Yoshinari et al., 1977),
using the individual soil core incubation system under
®eld conditions as described by Ryden et al. (1987).
The technique involved incubation of soil samples in
the presence of C2H2 to prevent conversion of N2O to

N2 (Yoshinari et al., 1977). N2O is the sole gaseous
product of denitri®cation in soils incubated in atmosphere containing 0.1±10% v/v C2H2 and the moles of
N2O produced (with C2H2) are equal to moles of
N2O+N2 (without C2H2) (Yoshinari et al., 1977). This
procedure simpli®es analytical procedures for denitri®cation assays, since denitri®cation can be estimated by
a single measurement of N2O using a gas chromatograph. Although there are areas of concern with the

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J. Luo et al. / Soil Biology & Biochemistry 32 (2000) 497±509

acetylene and core incubation method (Tiedje et al.,
1989), we believed that it is an appropriate technique
for measuring denitri®cation rates in grazed pasture
soils, because the uneven distribution of urine and
dung causes considerable spatial variability and this
technique allows numerous core incubations to be
quickly carried out to integrate the denitri®cation rate.
On each sampling date, soil cores were collected using
a soil corer. A previous study found that denitri®cation activities were maximal in the surface soil (0±5

cm) and generally decreased exponentially with depth
in the same pasture (Luo et al., 1998). Consequently,
surface soil samples (0±7.5 cm depth) were collected.
Each core was approximately 2 cm in diameter. Individual cores were transferred from corers into PVC
tubes (2.5 cm diameter by 15 cm in length). The tubes
were closed at both ends with rubber septa. 6 ml of air
was withdrawn from the tube and the same amount of
puri®ed C2H2 (by passing industrial C2H2 gas through
a high concentration of H2SO4) was injected into each
tube using a syringe. The syringe was pumped several
times to mix the C2H2 within the tube. Approximately
10% of the volume of the headspace was replaced with
C2H2.
All tubes were incubated for 24 h on the ground in
a shaded place close to the paddock. At 1 and 24 h
after the addition of C2H2, samples of the headspace
gases were collected in 5 ml `venoject' evacuated test
tubes (Becton Dickinson Vacutainer Systems). A gas
chromatograph, equipped with a 63Ni electron capture
detector, was used to measure the concentrations of

nitrous oxide (N2O) in the samples. The details of the
measurements and the calculations were described by
Luo et al. (1999a).
2.3. Soil moisture, NOÿ
3 , CO2 measurements and soil
temperature
On most occasions, soil cores were brought back to
the laboratory after 24 h of incubation and removed

from the tubes. The individual soil samples from each
of the tubes were then bulked. Soil moisture was determined from the weight loss of sub-samples dried overÿ
night at 1058C. Soil NOÿ
3 (including NO2 ) were
extracted by shaking a 5 g soil sample with 20 ml of 2
M KCl for 60 min and ®ltering through Whatman No.
42 ®lter paper. NOÿ
3 -N was determined colorimetrically on a Technicon Autoanalyser (Downes, 1978).
CO2 concentration was determined from the same
gas samples as those used for N2O analysis and was
measured in a gas chromatograph equipped with a

thermal conductivity detector.
Monthly data for soil temperature (10 cm depth),
rainfall and evaporation were obtained from the
nearby meteorological station of AgResearch Grasslands. The data for soil temperature, rainfall and evaporation over the study period are shown in Fig. 1.
2.4. Statistics
Coecients of skewness were calculated to quantify
departures from normality on both untransformed and
log-transformed data. The signi®cance of the di€erence
from zero of the coecients of skewness was evaluated
as described by Zar (1974). Frequency distributions of
denitri®cation rates and log-transformed rates at each
site on each sampling date were calculated by Wilk±
Shapiro statistics to assess whether these rates were
normally distributed (SAS Institute, 1982).
The mean soil denitri®cation rate and NOÿ
3 -N concentration were calculated using the Uniform Minimum Variance Unbiased Estimators (White et al.,
1987; Parkin and Robinson, 1992), when data were
highly skewed and log-normally distributed. Consequently, the comparisons of log-normally distributed
denitri®cation rates or NOÿ
3 -N concentrations of the

di€erent sampling sites and dates were carried out by
testing the overlaps of upper and lower 95% con®dence limits using the untransformed data (Parkin,
1993). Pearson correlation coecients were calculated

Table 1
Major characteristics of the soil at 0±7.5 cm depth
Site
gully bottom
pH (H2O)
Texture
% Sand
% Silt
% Clay
Bulk density (Mg mÿ3)
Total N (%)
Total P (%)
Total C (%)

N-facing slope

S-facing slope

¯at land area

gateway

6.06

6.00

5.91

6.04

5.94

23.90
63.79
12.31
0.83
0.47
0.13
5.16

22.98
62.39
14.63
0.87
0.36
0.09
4.80

21.26
63.08
15.66
0.88
0.37
0.08
4.91

22.71
67.32
9.97
0.84
0.42
0.09
5.22

22.74
62.83
14.55
0.93
0.48
0.16
5.50

500

J. Luo et al. / Soil Biology & Biochemistry 32 (2000) 497±509

among the measured variables. The log-transformed
data for denitri®cation rate and NOÿ
3 -N concentration
were used as input variables in the correlations.

3. Results and discussion
3.1. Spatial variation and frequency distribution
3.1.1. Denitri®cation
Measurements of ®eld denitri®cation rates by the

acetylene inhibition and soil core incubation technique
were made on 14, 13, 13, 20 and 13 occasions on
gully, south-facing slope, north-facing slope, ¯at land
and gateway sites, respectively. The measured denitri®cation rates exhibited a high degree of skewness and a
large spatial variation at all the sampling sites throughout the sampling periods. The frequency distributions
were positively skewed (P < 0.01) for 63 of the total
73 data sets for individual sites during the sampling
periods (Table 2). Statistical analysis con®rmed that
most of ®eld denitri®cation rates measured by this

Fig. 1. Monthly rainfall and evaporation (a) and mean soil temperature (10 cm depth) (b) during the ®eld denitri®cation study.

337
39.2
256
154
59.4
26.0
26.8
4.98
7.0
3.1
4.7
2.6
2.3
0.97
3.2
2.3
ÿ0.25
ÿ1.2
0.09
ÿ1.25
ÿ0.92
ÿ2.7
ÿ0.91
ÿ1.1
73
73
67
67
73
73
67
67
Denitri®cation (mg N2O-N kgÿ1dÿ1) no
yes
ÿ
ÿ1
NOÿ
no
3 (mg NO3 -N kg )
yes
no
CO2 (mg CO2-C kgÿ1dÿ1)
yes
Moisture (% w wÿ1)
no
yes

minimum maximum

63
24
51
17
18
0
11
7

5
73
4
67
51
81
57
67

68
0
63
0
22
0
10
0

20.9
5.09
19.3
17.3
9.80
3.55
3.10
0.79

minimum maximum

45
0
14
4
0
0
0
0

sampling number
(CV > 100)
range
sampling number
normal log-normal
(positive, P < 0.01)
range

Log-transformed Number of sampling events Coecient of skewness
Variables

Table 2
Statistical properties of soil parameters measured on individual soil cores at all sampling sites and dates

Distribution
(number of sampling)

Coecient of variation
(CV)
(%)

J. Luo et al. / Soil Biology & Biochemistry 32 (2000) 497±509

501

technique had a log-normal rather than a normal distribution, irrespective of sites and sampling dates
(Table 2). It was also observed that the values of the
coecients of skewness for log-transformed rates on
most occasions were not signi®cantly positive (Table 2).
The coecient of variation (CV) of denitri®cation
rates exceeded 100% in 45 of the total 73 data sets
(Table 2). The CVs for the log-transformed rates were
smaller than for the untransformed rates (Table 2).
Large CVs and skewed distributions occur when
most samples have low denitri®cation rates and a few
samples have very high rates. In the current study
about 25% of the soil cores contributed more than
50% of the total N loss through denitri®cation from
all the soil cores on each sampling occasion. Large
CVs and log-normal distribution patterns of denitri®cation rates in other ®eld studies have been often
reported (Christensen et al., 1990; Parsons et al., 1991;
Estavillo et al., 1994; Schnabel and Stout, 1994).
3.1.2. NOÿ
3N
The concentrations of soil NOÿ
3 -N were variable and
they also appeared to be highly skewed and in most
cases exhibited a log-normal distribution (Table 2).
This agrees with the ®ndings of White et al. (1987) and
Bramley and White (1991).
3.1.3. CO2
CO2 emission rates have also been found to be lognormally distributed (Focht et al., 1979). However, in
our study soil CO2 emission rates from core incubations did not often exhibit a high degree of spatial
variation and were ®tted better by normal than lognormal distributions on most sampling dates at most
sites (Table 2). This may indicate that a high proportion of the measured CO2 originated from the respiration of evenly-distributed soil C or possibly grass
roots in this pasture.
3.1.4. Moisture
Soil moisture content was relatively uniform and
could be described by normal distributions on almost
all sampling dates at all sites (Table 2). However,
skewed distributions for soil moisture content were
generally observed when the soil was relatively dry
(Table 3). It is possible that there may be a patchy distribution of moist soil under dry ®eld conditions due
to urine and dung deposits from dairy cattle.
3.1.5. Temporal patterns of spatial variability
The pattern of variation in denitri®cation rates in
this pasture showed a temporal dependence, that was
mostly in¯uenced by grazing events and rainfall. After
an intensive grazing event in the winter, soil NOÿ
3 -N
concentration and denitri®cation rates increased
(Table 4). The grazing also increased the skewness of

502

J. Luo et al. / Soil Biology & Biochemistry 32 (2000) 497±509

Table 3
E€ect of rainfall on the variation of denitri®cation rate, soil nitrate concentration and soil moisture content
Date (1993)a

Site

19 February

gully bottom
north slope
south slope
¯at land
gateway
gully bottom
north slope
south slope
¯at land
gateway
gully bottom
north slope
south slope
¯at land
gateway
gully bottom
north slope
south slope
¯at land
gateway
gully bottom
north slope
south slope
¯at land
gateway
gully bottom
north slope
south slope
¯at land
gateway

20 February

21 February

22 February

10 March

14 March

a

Soil moisture

Soil nitrate

Denitri®cation

content (% w wÿ1)

skewness

content (mg N kgÿ1)

skewness

rate (mg N kgÿ1dÿ1)

skewness

21.9
20.9
25.6
20.0
22.2
37.2
33.6
39.5
35.0
40.4
39.5
33.9
36.4
36.1
40.2
36.7
30.9
34.3
33.7
36.1
30.6
25.9
30.9
28.8
31.0
48.9
43.8
44.4
44.7
45.6

1.85
1.25
ÿ1.753
2.4
ÿ1.24
1.185
1.16
ÿ1.75
2.4
ÿ0.24
ÿ0.059
ÿ0.410
0.170
0.161
0.186
0.493
ÿ0.338
ÿ0.484
0.31
0.292
ÿ0.507
0.173
ÿ1.289
3.22
ÿ1.027
1.39
0.17
0.22
1.07
0.014

8.5
7.9
6.5
7.5
14.6
5.2
1.3
0.18
5.5
5.2
7.1
3.3
1.5
0.92
1.6
7.1
1.2
1.2
2.2
3.3
10.7
4.1
5.6
6.8
8.8
6.7
1.9
2.1
2.3
2.3

2.4
1.9
1.3
2.1
1.4
2.8
2.2
1.6
3.3
2.4
2.0
1.6
3.2
3.5
1.3
1.9
1.6
2.0
3.4
1.2
2.0
1.9
2.1
3.2
1.8
2.0
1.8
2.1
3.2
1.8

4.6
4.8
4.7
3.6
8.4
21.4
17.5
7.16
9.00
86.3
16.1
7.1
10.4
12.1
31.0
23.4
12.1
14.8
12.2
17.7
7.01
4.7
6.5
4.6
5.6
17.0
8.1
8.9
6.7
8.5

1.8
1.7
2.4
1.9
1.1
5.3
3.2
3.2
2.8
3.4
3.2
2.3
0.73
2.1
0.96
2.6
0.88
1.9
4.0
2.6
3.9
2.9
0.76
1.8
0.37
3.6
2.6
0.7
2.8
2.5

A rainfall (26 mm) started in the evening of 19 February 1993, stopped on 21 February. Another rainfall started on 12 March 1993.

the soil NOÿ
3 -N concentration and denitri®cation rates
(Table 4). These results suggest that the high skewness
of the denitri®cation rates was probably due to uneven
distribution of the soil NOÿ
3 -N from animal excreta in
the ®eld.
Our study also showed that denitri®cation rates
were low and the skewness of denitri®cation rates was
also low when the soil was relatively dry in the summer and early autumn (Table 3). After a rainfall event,
the soil moisture content and denitri®cation rates

increased and the skewness of denitri®cation rates also
increased initially, but then tended to decrease again as
the soil became saturated (Table 3). The coecients of
skewness for soil moisture content were large in relatively dry soil, and decreased rapidly when the soil
became wet (Table 3). Although the NOÿ
3 -N concentration decreased rapidly, the skewed distribution
remained after rainfall (Table 3).
It seems that following rainfall anaerobic conditions developed and caused the patchily-distributed

Table 4
E€ect of intensive grazing on denitri®cation and other soil variables (sampled on 5 August 1993)
Variable

Control site
Grazed sitea
a

Denitri®cation

Nitrate

Moisture

rate
(mg N2O-N kgÿ1dÿ1)

coecient of skewness

concentration
ÿ1
(mg NOÿ
3 -N kg )

coecient of skewness

content
(% w wÿ1)

coecient of skewness

21
42

1.58
2.67

0.79
3.12

2.56
3.88

47.03
48.54

0.44
0.56

10 d after grazing event (stocking rate at 300 cows haÿ1).

J. Luo et al. / Soil Biology & Biochemistry 32 (2000) 497±509

soil NOÿ
3 -N to become available to denitrifying microorganisms. Denitri®cation rates therefore increased
as did the skewness of the distribution. A few days
later, after the soil was saturated, the soil moisture
became less skewed, and hence a more uniform distribution of anaerobic sites was achieved in the soil,
resulting in less spatial variability in denitri®cation
rates (Table 3).
As discussed above, high soil moisture contents are
favourable to denitri®cation, and the variability of
denitri®cation rate appeared to decrease because of a
more even distribution of anaerobic sites in the soil
after the rainfall in the dry season. Christensen et al.
(1990) suggested that the distribution of denitri®cation
rates appeared to be less strongly skewed on soil
above ®eld capacity compared with soil at ®eld capacity. In our study, soil was above the ®eld capacity
for a period in the winter, but the variance and skewness of denitri®cation rate at this time did not tend to
be less than the rest of the sampling dates in the study
pasture (data not shown). It was found that the variability of NOÿ
3 -N or available-C are more important
factors controlling the spatial variability of denitri®cation and soil anaerobiosis alone may not determine the
skewed distribution of denitri®cation rate in this particular pasture when the soil was wet in the winter
(Luo et al., 1999b).

503

3.2. Temporal variation of denitri®cation rate
3.2.1. Seasonal pattern
The rates of denitri®cation on the gully bottom,
north-facing, south-facing, ¯at and gateway sites in the
study paddock are shown in Fig. 2. Denitri®cation
rates were highest in the winter (May to August), followed by a decrease during the spring (September to
November). Denitri®cation rates were generally lowest
in the summer (December to February) and then
increased during the autumn (March to April).
The seasonal pattern in denitri®cation rate under
®eld conditions we observed in this study (Fig. 2)
was similar to that found by Ruz-Jerez et al. (1994)
on a freely-drained, ®ne sandy loam in the same
locality. Both studies reveal that the highest N losses
by denitri®cation occurred in the winter and lowest
occurred during the summer. Changes in soil aeration, supply of NOÿ
3 -N and availability of C under
®eld conditions may all be implicated in seasonal
variations of denitri®cation activity, as the dominant
controlling factors a€ecting denitri®cation appeared to
vary temporally in the study pasture (Luo et al.,
1999b).
Peaks of denitri®cation occurred in late February
and mid-March, after rainfall events (Fig. 2). Formation of anaerobic sites by receipt of water from

Fig. 2. Temporal variation in the rate of denitri®cation.

504

J. Luo et al. / Soil Biology & Biochemistry 32 (2000) 497±509

rainfall was probably a fundamental requisite for denitri®cation in those periods. Increased denitri®cation
rates following rainfall events can also be attributed to
increased availability of C and NOÿ
3 -N in the soil (Luo
et al., 1999b).
3.2.2. Site di€erences in denitri®cation rate
There were di€erences in denitri®cation rate between
sampling sites, but these were not always consistent
(Fig. 2). Soil near the gateway exhibited slightly (but
not signi®cantly at P < 0.05) greater rates of denitri®cation than the other sites on most sampling dates
through the year. When the denitri®cation rate was

generally low during the summer (December to February), no di€erences in the rates among the ¯at, slopes
and gully bottom were found. However, from July to
October the denitri®cation rates in both sloping sites
were low compared to the rates at other sites. This observation emphasises the importance of animal e€ects
on denitri®cation activity at various points in a pasture. More excreta from animals is deposited in the
paths of animal movement (Barrow, 1967) and on hill
pasture the animals can transport signi®cant quantities
of nutrients to ¯at areas, because they tend to camp
there (Saggar et al., 1988). Loss of N by denitri®cation
may, thus, be enhanced around gateways or in camp-

Fig. 3. Seasonal variation in the soil nitrate concentration (a), the soil moisture content (b) and the soil respiration rate (c) (the soil moisture content and respiration rate are the average over all sites).

505

J. Luo et al. / Soil Biology & Biochemistry 32 (2000) 497±509

site areas, due to higher amounts of deposition of
urine and dung.
A slightly higher (not signi®cantly at P < 0.05) denitri®cation rate was observed in the gully bottom than
in ¯at and sloping sites after a rainfall event in February (Fig. 2). A slightly higher peak of denitri®cation
rate was also observed in the gully bottom compared
with the other sites after a rainfall event in March, but
it was not as high as that in February (Fig. 2). These
di€erent responses of denitri®cation to soil wetting in
various areas of landscape could be a result of substrate (NOÿ
3 -N or C) redistribution. Substrates were
most likely to accumulate in the gully bottom after
rainfall events (Fig. 3); presumably they were transported from slopes to the gully bottom in water.
3.3. Characteristics of denitri®cation and its regulators
3.3.1. Factors related to denitri®cation rates measured
in individual soil cores
Examination of the correlation-coecient matrix
revealed that at some individual sampling times and in
some topographical sites, denitri®cation rates were closely related to one or more of the other measured variables. However, these relationships were not consistent
over time or between topographical sites (data not presented). When the data for each sampling time were
combined for each individual sites or the whole paddock, denitri®cation rates were always weakly, but signi®cantly (P < 0.01), correlated to soil moisture
content. The correlations between denitri®cation rates
and soil NOÿ
3 -N concentrations and soil respiration
rate (CO2 emission rate) were not consistent. When the
entire data sets for the whole year were evaluated, correlations between denitri®cation rate and NOÿ
3 -N concentration, or respiration rate, were improved, when
the pooled data were partitioned according to soil
moisture (Table 5). The highest correlation between
denitri®cation rate and NOÿ
3 -N concentration was 0.38
and this was obtained when the soil moisture content
was over 45% (w wÿ1) (about ®eld capacity). In contrast, the highest correlation coecient between denitri®cation rate and soil respiration rate was 0.44, and
this was obtained when the soil moisture content was
less than 30% (w wÿ1). That the strength of corre-

lation between denitri®cation rate and soil NOÿ
3 -N
concentration or soil respiration rate were dependent
on the soil moisture content suggests that the availability of NOÿ
3 -N or readily biodegradable C can be
a€ected by soil moisture in this particular pasture.
3.3.2. Associations among means of measured variables
Among the edaphic conditions, soil moisture content
had a pattern similar to the denitri®cation rate (Figs. 2
and 3). Changes in soil NOÿ
3 concentration and respiration rate had opposite temporal patterns to changes
in denitri®cation rate (Figs. 2 and 3). Correlation coef®cients were computed between the mean values of
denitri®cation rate and the other edaphic properties
for each sampling date for both the individual sites
and the whole paddock. In all cases, the closest relationships were obtained between denitri®cation rate
and soil moisture content (Table 6). Soil NOÿ
3 -N concentration, respiration rate and temperature appeared
to be negatively correlated with denitri®cation rate,
however, the signi®cance of the correlations varied
among sites (Table 6).
3.3.3. Periods of relatively high denitri®cation rates
A relatively high denitri®cation rate was observed in
the winter (Fig. 2), although the soil temperature was
only about 88C (Fig. 1). The active denitri®cation in
the winter appears to have been associated mainly
with high soil moisture contents. Due to frequent rainfall and low evaporation (Fig. 1), the moisture content
in the soil can readily reach amounts greater than
`®eld capacity' in this poorly-drained soil in the winter
(Fig. 3). Most soil pores would then be ®lled with
water and O2 di€usion through water is considerably
slower than through air. It has long been recognised
that O2 concentrations can a€ect both synthesis and
activity of the denitri®cation enzyme system (Firestone,
1982). Therefore, soil denitri®cation can be increased
by an increase in the number of anaerobic sites in the
soil in the winter. Previous ®eld studies have demonstrated that the rate of denitri®cation often remains
negligible during dry periods, but then increases considerably when soil water content exceeds a certain
critical amount (e.g. Aulakh and Rennie, 1985; De

Table 5
Correlations between soil denitri®cation (mg N2O-N kgÿ1 dÿ1) and measured variables using data from individual soil coresa,b
Moisture content (w wÿ1)

Sample number

ÿ1
Nitrate (mg NOÿ
3 -N kg )

Respiration (mg CO2-C kgÿ1 dÿ1)

> 45%
30±45%
< 30%

438
931
267

0.38
0.12
0.17

0.13
0.10
0.44

a

Log-transformed data for denitri®cation rate and NOÿ
3 -N concentration were used in correlation.
, Signi®cant at P < 0.05 and 0.01, respectively.

b  

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J. Luo et al. / Soil Biology & Biochemistry 32 (2000) 497±509

Klein and Van Logtestijn, 1996; Nelson and Terry,
1996).
In the study pasture, the low concentration of soil
NOÿ
3 -N, especially during wet periods of the year, was
an important factor which limited the rate of denitri®cation (Luo et al., 1999b). The good correlation
obtained between denitri®cation rates and NOÿ
3 -N
concentrations at the high soil moisture contents
(Table 5) also suggests that in this unfertilised pasture
NOÿ
3 -N become a more important limiting factor for
denitri®cation when the potential rate of denitri®cation
is increased at high soil moisture contents. Other studies also indicate that soil NOÿ
3 -N availability generally limit denitri®cation in unfertilised grassland soils
(e.g. Tenuta and Beauchamp, 1996). The availability of
ÿ
NOÿ
3 -N could be in¯uenced by soil NO3 -N concenÿ
trations or NO3 -N movement to active denitri®cation
sites. In the study pasture, high soil water contents in
the winter provide an optimum medium for di€usion
and, therefore, NOÿ
3 originating from nitri®cation can
more easily move to anaerobic denitri®cation sites. It
has been suggested that nitri®cation and denitri®cation
can occur simultaneously on opposite sides of an
aerobic±anaerobic interface (Knowles, 1978). So NOÿ
3N could be denitri®ed rapidly and would not accumulate in a soil with high moisture content. The weak relationships between denitri®cation rate and CO2 at
high soil moisture contents may suggest that soil C
was not an important regulatory factor for denitri®cation in the winter, as there would be plenty of anaerobic sites and low concentrations of NOÿ
3 -N in the wet
soil in this pasture.
The rate of denitri®cation can undoubtedly be limited by low temperature in the winter (Luo et al.,
1999b). However, the mean soil temperature (Fig. 1) in
the winter in which the study was carried out was
always above the critical temperature for denitri®cation, as the lowest temperature at which ®eld denitri®cation can occur has been reported to be 58C (Ryden,
1986). The e€ect of temperature on denitri®cation in
the natural environment is complicated by other factors. In the present study, high denitri®cation rates at
relatively low temperature in the winter were caused
by an opposing, seasonal relationship between tem-

perature and water content in the soil. The temperature e€ect on denitri®cation may also be a€ected by
simultaneous changes in plant growth in the ®eld. The
relatively high denitri®cation rate observed during the
winter may have been a€ected by the limited uptake of
available NOÿ
3 from the soil by pasture, since the
growth of grass slowed as daylight and temperature
decreased in the winter.

3.3.4. Periods of relatively low denitri®cation rates
Rates of denitri®cation were generally very low in
the summer (Fig. 2), although soil NOÿ
3 -N concentration and respiration rate were relatively high during
this period (Fig. 3). The most obvious factor limiting
denitri®cation was the low moisture content of the soil
(Fig. 3). Other studies have also found that soil moisture content can limit denitri®cation rates in grassland
soils, in which NOÿ
3 -N and C availability would be
considered adequate for denitri®cation (e.g. Jarvis et
al., 1991). The better correlation between denitri®cation and CO2 production at low soil moisture content
than at high soil moisture content in the present study
(Table 5) may suggest that the role of C in relatively
dry soil may involve O2 consumption by respiration,
which would produce increased number of anaerobic
sites suitable for denitri®cation.
A study by Luo et al. (1999b) at this site determined
that denitri®cation was limited by the slow rate of diffusion of NOÿ
3 -N to active denitri®cation sites when
soil was relatively dry in the summer. It appears that
the weak correlations between denitri®cation rate and
NOÿ
3 -N concentration observed at low soil water contents (Table 5) is a further evidence that the NOÿ
3 -N
concentration was not a major limiting factor for denitri®cation when soil moisture was low. Other workers
have also reported that slow di€usion rates can limit
NOÿ
3 -N availability to denitri®cation even at a high
concentration of soil NOÿ
3 -N (Ryden, 1983). The low
denitri®cation rates in the summer may have also been
a€ected by the activity of plant roots, since water and
NOÿ
3 -N uptake by rapidly-growing pasture would be
expected to be substantial. High rates of plant uptake
decrease the availability of NOÿ
3 -N to denitrifying

Table 6
Correlations between denitri®cation (mg N2O-N kgÿ1 dÿ1) and measured variables using means from individual datesa,b
Site

Gully bottom

N-facing slope

S-facing slope

Flat site

Gateway

Whole paddock

ÿ1
Nitrate (mg NOÿ
3 -N kg )
Respiration (mg CO2-C kgÿ1 dÿ1)
Moisture (% w wÿ1)
Temperature (8C)

ÿ0.41
ÿ0.33
0.82
ÿ0.57

ÿ0.64
ÿ0.49
0.74
ÿ0.60

ÿ0.44
ÿ0.33
0.59
0.37

ÿ0.48
ÿ0.58
0.80
ÿ0.75

ÿ0.61
ÿ0.44
0.60
ÿ0.63

ÿ0.33
ÿ0.41
0.67
ÿ0.57

a

Log-transformed data for denitri®cation rate and NOÿ
3 -N concentration were used in correlation.
, Signi®cant at P < 0.05 and 0.01, respectively.

b  

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J. Luo et al. / Soil Biology & Biochemistry 32 (2000) 497±509
Table 7
Estimated annual nitrogen loss through denitri®cation
Site

Gully bottom

N-facing slope

S-facing slope

Flat site

Gateway

Whole paddock

Relative area (%)
Nitrogen loss (kg N haÿ1 yÿ1)

10
4.70

5
3.56

5
3.70

77
4.54

3
5.80

100
4.50

microorganisms, particularly in the rhizospheric zones,
where the availability of C may be high.

3.4. Nitrogen loss through denitri®cation
Denitri®cation rates measured on soil cores were
expressed on an areal basis, using bulk density values,
and were interpolated over the period between
sampling dates to estimate annual N loss by denitri®cation. Cumulative annual N losses monitored in this
study were 4.70, 3.56, 3.70, 4.54 and 5.80 kg N haÿ1 at
the gully bottom, north-facing slope, south-facing
slope, ¯at and gateway sites, respectively (Table 7).
The overall estimate was 4.5 kg N haÿ1 yÿ1 for the
study pasture. This value was determined by using
weighted averages for the losses at the di€erent sites in
this paddock. This annual N loss of 4.5 kg N haÿ1 by
denitri®cation from the pasture ®eld in the current
study is of the same order as the N losses found by
both Ruz-Jerez et al. (1994) and Ledgard et al. (1997)
in pastures without fertiliser in New Zealand. The N
loss by denitri®cation does not appear to be substantial in terms of N balances for pasture and is lower
than the expected values for denitri®cation that have
been derived from previous N mass balance studies
(Ball et al., 1979; Field et al., 1985).
As the study area is in a temperate region, high soil
water conditions necessary for the denitri®cation process are typically found in the winter and therefore associated with low temperatures. During this period, a
soil NOÿ
3 -N supply for denitri®cation is also restricted
in this unfertilised pasture. Therefore, denitri®cation is
limited. Although soil temperature increases in the
summer, the low soil moisture content limits denitri®cation and therefore N loss. Most of the soil mineral
N in this paddock was associated with excreta from
the grazing animals. So the e€ects of animal grazing
on denitri®cation would be signi®cant. The direct
e€ects of grazing on denitri®cation in the study area
were reported by Luo et al. (1999a). Overall, the limited availability of soil NOÿ
3 -N or low soil water content during most times of the year are probably the
main cause of the small loss of N by denitri®cation
observed from this legume-based pasture.

4. Conclusions
Denitri®cation rates exhibited marked spatial variability in this pasture, with coecient of variation frequently being larger than 100%. The distribution of
rates was generally skewed. A log-normal distribution
was the most appropriate for describing the spatial
variation among denitri®cation rates measured in the
various topographical areas. Rainfall and animal grazing events a€ected the spatial variation of denitri®cation rates. Rainfall in the warm-dry season increased
the skewness of the denitri®cation rate initially, but
then decreased as the soil became more uniformly wet.
An intensive grazing event in the winter increased the
skewness of the frequency distribution of denitri®cation rates.
Denitri®cation rates in this legume-based dairy-farm
pasture were highest in the wet winter and lowest
during the dry summer and early autumn. However,
high denitri®cation rates did occur for brief periods
after rainfall events in the dry season. This pasture
showed a characteristic relationship between denitri®cation rate and soil moisture content. Low soil moisture content was the primary factor limiting
denitri®cation during the summer and early autumn.
Changes in soil NOÿ
3 concentration, respiration rate
and soil temperature had opposite temporal patterns
to changes in denitri®cation rate. About 4.5 kg N haÿ1
annual N loss by denitri®cation was estimated. Denitri®cation does not appear to be a major pathway for
loss of N from this pasture.

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