Animal treading stimulates denitrification in soil under pasture

John C. Menneera,b,*, Stewart Ledgardb, Chris McLayc, Warwick Silvestera

aUniversity of Waikato, Private Bag, Hamilton, New ZealandbAgResearch Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand

cEnvironment Waikato, P.O. Box 4010, Hamilton, New Zealand

Received 5 December 2003; received in revised form 24 November 2004; accepted 27 January 2005

Abstract

The effects of animal treading on denitrification in a mixed ryegrass-clover pasture were studied. A single treading event of moderate

or severe intensity was applied in plots during spring by using dairy cows at varying stocking rates (4.5 cows 100 mK2 for 1.5 or 2.5 h,

respectively). Treading caused a significant short-term 21 days) increase in denitrification. Denitrification rates reached a maximum of

52 g N2O-N haK1 dayK1 at 8 days after severe treading compared to 2.3 g N2O-N haK1 dayK1 under nil treading. Thereafter,

denitrification rates declined, and were similar to non-trodden control plots after 28 days. Soil aeration, was significantly reduced by

treading as expressed by water-filled porosity. In addition, soil NH4C-N and NO3

K-N concentrations were also increased by treading. We

propose that the underlying processes involved in increasing denitrification under treading were two-fold. Firstly, treading caused a

temporary (e.g. 3 days after treading) reduction in soil aeration through soil physical damage, and secondly, reduced soil N utilisation

prompted by reduced plant growth led to increased soil NH4C-N and NO3

K-N availability. This study shows that treading, without the

influence of other grazing animal factors (e.g. excretion), can cause a large short-term stimulation of denitrification in grass-clover

pastures.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Denitrification; Animal treading; Soil nitrate; Water-filled porosity; Soil aeration; Plant N uptake

1. Introduction

Losses of nitrogen (N) from soil by denitrification are

known to be affected by many soil properties, such as soil

water content, forms and amounts of N, soil temperature, pH

and available carbon (Groffman et al., 1987). In intensively

grazed pastures, the principal regulators of denitrification

are soil aeration and soil NO3K-N availability (Barton et al.,

1999; Groffman et al., 1987, 1993; Jarvis et al., 1991). In

legume-based systems not receiving fertiliser N, inputs of

excreta N by grazing animals potentially provide a large

source of soil NO3K-N for denitrifying microbes. Indeed,

studies using synthetic urine applied to soil cores,

lysimeters, and directly onto pasture soils (e.g. Clough

et al., 1994; Fraser et al., 1994; de Klein and van Logtestijn,

0038-0717/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.soilbio.2005.01.023

* Corresponding author. C64 7 8385130.

E-mail address: john.menneer@agresearch.co.nz (J.C. Menneer).

1994; Ryden, 1986) have shown greatly enhanced rates of

denitrification.

Under field conditions, various workers (Carran et al.,

1995; Luo et al., 1999; Ruz-Jerez et al., 1994) have

measured high denitrification rates soon after grazing. In

these studies the stimulation of denitrification has been

mainly attributed to the direct influence of the N returned in

excreta. However, other factors associated with grazing are

often thought to contribute to higher denitrification after

grazing, and include increased soil NO3K-N due to limited

plant N uptake after defoliation, increased soil carbon

availability by deposition of animal excreta, and reduced

soil aeration resulting from animal treading and soil

compaction (e.g. Carran et al., 1995; Luo et al., 1999;

Ruz-Jerez et al., 1994). Very little is known about the effect

of these grazing related factors on denitrification or the

underlying regulating processes involved.

The aims of the current study were, firstly, to determine

the effect of treading on denitrification without the influence

of animal excreta inputs, and secondly, to describe the

underlying processes that regulate denitrification in soils

affected by treading.

Soil Biology & Biochemistry 37 (2005) 1625–1629

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J.C. Menneer et al. / Soil Biology & Biochemistry 37 (2005) 1625–16291626

2. Materials and Methods

2.1. Experimental site and soil characteristics

The experiment was established on a Te Kowhai silt

loam (Typic Ochraqualf, Soil Survey Staff, 1994; Typic

Orthic Gley Soil, Hewitt, 1993) with impeded subsoil

drainage. Measured soil properties are presented in Table 1.

The pasture was a long-term (O 30 years) permanent mixed

stand of perennial ryegrass (Lolium perenne L.) and white

clover (Trifolium repens L.). Fertiliser N had not been

applied to the site for at least 5 months. Daily mean soil

temperature (at 100 mm depth) increased from 11 to 16 8C

over the 28 days of the study.

Plate 1. Soil surface characteristics 6 days after treading at 4.5 cows 100

mK2 for 1.5 and 2.5 hours, for moderate and severe treading, respectively.

2.2. Experimental design

Treatments consisted of a single treading event of three

severities (nil, moderate and severe). A randomised block

experimental design was used with eight replicates. Plots

were 2.5!7 m long with a 0.5 m buffer strip around each

plot. The site was taken out of grazing 5 months prior to

commencing the study, and all dung pats removed. Two pre-

conditioning harvests were taken from the site during the

month prior to commencing the study to reduce variability.

In early spring (21st September) 1999, moderate and severe

treading treatments were imposed (after the site had been

harvested) by walking dairy cows (c. 500 kg liveweight per

cow) through the plots at a typical grazing intensity of 4.5

cows 100 mK2 for 1.5 or 2.5 h, respectively, to achieve

levels of treading damage (Plate 1) that can occur in dairy

pasture during wet spring conditions. Rainfall and light

irrigation in the days before commencing the experiment

meant that the soil was near saturation when the treading

treatments were applied. Prior to applying the treading

treatments the cows were kept in stockyards overnight to

avoid inputs of dung and urine onto the plots during the

treading event. In the rare event that animal excretion did

occur during the treading event, these were intercepted

using a large bucket and removed from the site. This level of

cow contact was achieved by allocating three cows per

person to observe and intercept excreta. As a result, no urine

or dung was deposited on the experimental site. Following

the treading event, animals were excluded from the site for

the remainder of the study.

Table 1

Soil properties (0–75 mm) at the experimental site measured immediately

prior to treatment application

Soil

texture pHa

Organic-C

(g kgK1)

Total N

(g kgK1) C:N ratio

Silt loam 5.4 68 5.8 11.7

a pH in 1:2.1 soil:water extract.

2.3. Denitrification measurements

Denitrification rates were determined over 28 days (on

days 3, 8, 14, 21 and 28) after treading using a field soil

incubation system, involving acetylene inhibition (Aulakh

et al., 1992; Ryden et al., 1987). Minimally disturbed soil

cores (65 mm diameter!70 mm depth) were taken from

four replicate plots in each treatment and wrapped in tinfoil

(to minimise diffusion of atmospheric O2 into the core), but

with the soil surface still exposed, and placed in 10 l

preserving jars. Three jars containing two cores were taken

from each plot. The jars were closed with a steel lid, fitted

with a silicon septum, and sealed with a rubber gasket and a

screw band. Acetylene (50 ml, 10% v/v) was injected into

each jar through the septum after ejecting an equal volume

of headspace to maintain atmospheric pressure. The jars

were then incubated in situ in a shallow covered trench on

site for 24 h.

After incubation, the headspace of the jars was sampled

in duplicate using double-ended sampling needles and pre-

evacuated Vacutainer (Becton, Dickinson and Co.,

0 5 10 15 20 25 30

0

5

10

15Severe treadingNil Mod

NH4+-N

NO3--N

NO3--N

NH4+-N

a

b

a

a

b

a a aaa

a

a

D

b

C *

Wat

er-f

illed

por

osity

(%

)

40

50

60

70

B NS* NSNS *

Den

itrif

icat

ion

rate

(m

g N

kg

soil-

1 da

y-1 )

0.02

0.04

0.06

0.08

0.10

Nil treadingModerate treadingSevere treading

NS *** ** NSA NS

Soil

moi

stur

e (%

)

20

25

30

35

40

45

50

NS* NSNS

Field capacity

Wilting point

NS

Days after treading

Soil

inor

gani

c N

(m

g N

kg

dry

soil-

1 )

Nil treadingModerate treading

Fig. 1. The effect of treading on denitrification rate (A), and water-filled

porosity (B), soil moisture (C), and soil nitrate concentration (D). For (A)

and (D), geometric means are presented, and LSRs are 1.93–3.06 and 2.78,

respectively; P!0.05–0.1. For (D), bar plots with different letters are

significantly different from each other; P!0.05. For (B) and (C) error bars

represent SED; P!0.05 unless designated NS, not significant (four

replicates).

J.C. Menneer et al. / Soil Biology & Biochemistry 37 (2005) 1625–1629 1627

Rutherford, NJ, USA) blood-sampling tubes (10 ml

volume). Direct analysis of the gas sample for N2O

concentration was carried out immediately after collection

by gas chromatography.

2.4. Soil analysis

After sampling the headspace of each jar, the incubated

soil was thoroughly mixed, and 20 g of fresh soil was

extracted with 50 ml 2 M KCl. Soil NH4C-N and NO3

K-N

concentrations were determined using a flow injection

analyser (Blakemore et al., 1987). Soil water content was

determined gravimetrically after drying fresh subsamples at

105 8C for at least 24 h.

For soil physical measurements, five intact soil cores

(0–50 mm; 380 cm3) were randomly taken from the topsoil

of four plots in each treatment at day 3. Bulk density,

particle density and total porosity were estimated using the

methods of the New Zealand Soil Bureau (1972). Water

contents were gravimetrically determined and multiplied by

bulk density to calculate volumetric water contents. Water-

filled porosity (WFP; Fig. 1b) in soil was calculated for days

3, 8, 14, 21 and 28 by dividing the volumetric water content

(Fig. 1c) on that day by the initial total pore space measured

on day 3 (Table 2).

2.5. Plant analysis

At the end of the denitrification measurement period (day

28), plots were harvested by mower, and pasture dry

matter yield determined. Immediately before harvesting,

herbage was sampled from within each plot for plant species

separation. Clover and weed species comprised only a small

proportion of the pasture composition, which was domi-

nated by ryegrass. Herbage, sampled from within a 3!1 m

microplot in each plot, was dried and analysed for total N.

2.6. Statistical analysis

Analysis of variance was carried out using Genstat 4.2,

Fifth Edition. Data was log-transformed where necessary to

satisfy the assumptions of analysis of variance. SED or LSR

values were used to compare means, when main effects were

found to be significant.

Table 2

Effect of treading severity on soil physical properties 3 days after a single

treading event by dairy cows in spring

Treading severity

Nil Moderate Severe SED

Soil particle

density (g cmK3)

2.30 2.32 2.30 0.015NS

Soil bulk

density (g cmK3)

0.77 0.85 0.83 0.037NS

Total porosity (%) 66.4 63.2 64.3 1.46NS

NSZnot significant at P!0.05 (four replicates).

Table 3

Main effects of treading on grass dry matter and N yield, grass N

concentration, and total soil inorganic N (0–70 mm depth) at 28 days after

treading

Treading severity

Nil Moderate Severe SED/LSR

Grass DM yield

(kg haK1)

852 342 67 232*

Grass N yield (kg haK1) 23.9 10.1 1.4 7.0*

Grass N concentration

(%N)

2.9 3.0 3.6 0.17***

Total soil inorganic Na

(kg haK1)

4.0 7.7 11.4 2.4†

***, *, † significant at P!0.001, P!0.05, P!0.1, respectively, unless

designated NS not significant (eight replicates for grass measurements; four

replicates for soil N data).a Data is geometric mean, and relevant error term is LSR; total inorganic

NZNH4C-NCNO3

K-N.

J.C. Menneer et al. / Soil Biology & Biochemistry 37 (2005) 1625–16291628

3. Results and discussion

3.1. Denitrification as affected by treading

Denitrification rates showed significant (P!0.001–0.1)

increases up to 21 days after both moderate and severe

treading (Fig. 1a). The highest rates of denitrification were

measured 8 days after treading, reaching a maximum rate of

52 g N2O-N haK1 dayK1 under severe treading. Sub-

sequently, denitrification rates in treading treatments

declined sharply, and were similar to control soils after

21 days. Similarly, Luo et al. (1999) reported a short-term

increase (14 days) of denitrification in a grass-clover pasture

after grazing.

Integration of the daily rates of denitrification over time

resulted in total losses of about 105, 376, and 593 g N haK1

under nil, moderate and severe treading, respectively, for

the 4 week study. Thus, total losses of N through

denitrification were 3–6 fold greater under treading than

under nil-treading.

In pasture soils denitrification is regulated mainly by soil

aeration and soil NO3-N (Groffman et al., 1987; Jarvis et al.,

1991; Mosier et al., 1986; Smith and Tiedje, 1979).

However, both these soil properties are strongly influenced

by pasture management and site-dependent environmental

factors. In intensively grazed systems, deposited excreta can

cover a large proportion of pasture, and make a significant

contribution to the soil N pool (Haynes, and Williams, 1993;

Menneer et al., 2003). Increased soil NO3K-N from excreta

has been suggested as the main cause of increased

denitrification rates following animal grazing (e.g. Carran

et al., 1995; Luo et al., 1999; Ruz-Jerez et al., 1994).

However, these authors also speculated that treading and

compaction may have reduced soil aeration creating an

environment more conducive to denitrification. In our study,

pasture had not been grazed for 5 months prior to

commencing the experiment, and had been under a cutting

regime. Hence, any influences of excreta on soil inorganic N

and carbon availability was avoided, thereby enabling the

potential effects of treading on soil aeration and denitrifica-

tion to be determined. This experiment, therefore, separated

the two factors relating to grazing, that of treading and

compaction, from the addition of N in excreta.

3.2. Regulators of denitrification under treading

Soil aeration was reduced by treading, with an immediate

(within 3 days) increase in water-filled porosity (WFP) and

soil moisture compared to the untrodden control treatment

(Fig. lb and c). For example, water-filled porosity was 58%

in the control but averaged 70% under moderate and severe

treading (Fig. 1b). Notably, elevated (P!0.001) values of

WFP on day 3 were not associated with any increase in

denitrification between treatments. However, a relatively

high denitrification rate in control plots on day 3 compared

to subsequent days, suggests that a WFP value of between

55–60% in this soil is adequate to stimulate denitrification.

After day 3, values of WFP gradually declined in all

treatments, and although values were greatest under

treading, this difference was only significant on day 21.

By day 28, WFP had reached an average lower limit in

treading treatments of 43%. Reported threshold values of

WFP causing denitrification differ according to soil type,

but for soils similar to those used in our study (silt loams),

threshold WFP ranging from 50 to 74% have been reported

(e.g. Nelson and Terry, 1996; Sexstone et al., 1988). Soil

bulk density was not significantly affected by animal

treading (Table 2).

Grass dry matter production was dramatically reduced by

60 and 94% under moderate and severe treading, respect-

ively (Table 3). In contrast, soil NH4C-N and NO3

K-N

concentrations increased (P!0.05–0.10) during the first

28 days of the study (Fig. 1d, and Table 3). Within 3 days of

treading, soil NH4C-N had increased under severe treading

to a value 2.5 fold greater than that of the control (P!0.l;

Fig. 1d). However, soil NO3K-N concentrations on day 3

were similar in all treatments. Notwithstanding, by day 28

soil NO3K-N concentration had increased (P!0.05) from

4.0 mg NO3K-N kgK1 dry soil, in the control treatment, to 10

and 16 mg NO3K-N kgK1 dry soil, for the moderate and

severe treading treatments, respectively, (Fig. 1d). Greater

ryegrass N concentrations in severely trodden plots on day

28 also indicate that plant-available soil N concentrations

had increased in these lower yielding treatments (Table 3).

Apparently, nitrification was not markedly inhibited by the

reduction in soil aeration, and soil NH4C-N was readily

converted to NO3K-N. Given the much reduced grass growth

and therefore low soil N uptake under treading, it is not

surprising that soil NH4C-N and NO3

K-N increased in these

treatments. Although soil NO3K-N concentrations on day

28 were above reported values that generally limit

denitrification in clay loams (1–2 mg NO3K-N kg dry

soilK1; Estavillo et al., 1994; Jordan, 1989), denitrification

rates were similar between treatments, and probably

J.C. Menneer et al. / Soil Biology & Biochemistry 37 (2005) 1625–1629 1629

reflected the low WFP (43%) at the time (spring). Further

research in wetter seasons (e.g. autumn/winter) is necessary

to determine if treading-induced reductions of soil aeration

will have more prolonged effects on denitrification under

less aerated conditions.

The data presented in this study shows that treading

alone, without any influence of excreta, can lead to an

increase in denitrification in grass-clover pastures. We

propose that the underlying processes involved are two-fold.

Firstly, treading causes a temporary reduction in soil

aeration and, secondly, reduced soil N utilisation prompted

by reduced plant growth increases soil NH4C-N and NO3

K-N

availability. Future research is required to determine the

relative effects of grazing factors (e.g. excreta and treading)

on denitrification and the amounts of total N loss involved.

Acknowledgements

M. Sprosen and G. Rajendram for valuable assistance in

the field and laboratory. J. Lou for helpful and constructive

comments on the original manuscript. The first author is

grateful to the Agricultural Marketing and Research

Development Trust for providing scholarship funding for

this study.

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