Introduction
Agricultural soils can significantly contribute to the global greenhouse gas
(GHG) exchange, but the contribution varies among the gases. For nitrous
oxide (N2O), the emissions from agricultural soils account for 60 %
of the anthropogenic emissions (Smith et al., 2007), whereas for methane
(CH4), mineral agricultural soils are usually sinks as the aerobic
topsoil favours methanotrophic bacteria (Hütsch, 2001). For carbon
dioxide (CO2), soils can be either sinks or sources, depending on the
balance of carbon input and output (Stockmann et al., 2013). N2O
emissions are mainly regulated by soil oxygen status, but also by the
availability of nitrogen and organic carbon (Granli and Bøckman, 1994).
The oxygen availability varies with soil structure and moisture and the
potential for N2O emissions is greatest when the water-filled pore space
(WFPS) is 60–70 % (Davidson, 1991) as this enables both nitrification
and denitrification. When the WFPS is above 70 %, only denitrification
takes place due to the shortage of oxygen and the dominating end product is
the N2 gas.
The application of no-till practice has recently increased in the agriculture
(Derpsch et al., 2010). No-till often increases carbon sequestration to soils
and is therefore considered as a useful cultivation technique in climate
change mitigation (Lal, 1997). Elevated N2O emissions may, however,
decrease the atmospheric benefits of no-till (Li et al., 2005; Sheehy et al.,
2013; Palm et al., 2014) as the denser physical structure (Tebrügge and
Düring, 1999; Schjønning and Rasmussen, 2000) and higher moisture
content (e.g. Sharratt, 1996; Gregorich et al., 2008) of no-tilled soils lead
to higher N2O emissions. The abundance and diversity of earthworms can
also be markedly higher under no-till than conventional tillage (Edwards and
Lofty, 1982; Chan, 2001; Rothwell et al., 2011) and the role of earthworms in
the regulation and enhancement of GHG emissions has recently gained
increasing attention. Field results are still scarce, but a recent
meta-analysis of laboratory studies suggests that the presence of earthworms
can increase N2O and CO2 emissions by 42 and 33 %, respectively
(Lubbers et al., 2013a). A number of factors potentially contribute to this
phenomenon. For instance, by burrowing, casting and mixing crop residues into
the soil, the earthworms change soil organic carbon cycling, porosity,
aggregation and gas diffusivity, enhance decomposition and increase the
amount of mineral nitrogen in the soil (e.g. Subler and Kirsch, 1998; Lubbers
et al., 2011). Earthworm casts and burrow linings also have higher microbial
activity and more denitrifying bacteria than the bulk soil (Svensson et al.,
1986; Brown et al., 2000; Elliott et al., 1990) and the moist anaerobic
environment in the earthworm gut can stimulate microbial N2O production
(Karsten and Drake, 1997; Drake and Horn, 2006). On the other hand,
earthworms can increase microaggregate formation and the stability of soil
carbon (Fonte et al., 2007; Six and Paustian, 2014), and it is still unclear
whether earthworms increase or decrease soil organic carbon stocks in the
long term (Lubbers et al., 2013a; Blouin et al., 2013; Zhang et al., 2013).
Reduced tillage and no-till increase the densities of anecic, deep-burrowing
earthworms in arable fields (Whalen and Fox, 2007). In the temperate and
boreal fields, this group is mainly represented by the dew-worm,
Lumbricus terrestris L. (Chan, 2001; Kladivko, 2001). In Finland,
L. terrestris is the second most common earthworm species in arable
fields, lagging only behind Aporrectodea caliginosa Sav. (Nieminen
et al., 2011), and has the typical positive association with non-inversion
cultivation (Nuutinen, 1992; Nuutinen et al., 2011). It is a large earthworm,
which efficiently forages on crop residues (Subler and Kirsch, 1998; Shuster
et al., 2000) and builds middens (i.e. small mounds of collected litter and
surface castings) at the openings of its permanent burrows, often penetrating
deeper than 1 m (e.g. Nuutinen and Butt, 2003). The middens are biological
hot spots with high microbial activity (Schrader and Seibel, 2001; Aira et
al., 2009), diverse invertebrate populations (Hamilton and Sillman, 1989;
Maraun et al., 1999; Butt and Lowe, 2007) and higher nutrient and organic
carbon contents than the surrounding soil (Subler and Kirsch, 1998; Wilcox et
al., 2002; Aira et al., 2009). By transferring plant litter into the subsoil,
L. terrestris may also increase the subsoil carbon stocks; for
example, Don et al. (2008) estimated that L. terrestris sequestrates
carbon in the burrow linings at the rate of 22 g C m-2 yr-1. On
the other hand, the turnover time of burrow wall carbon can be only 3–5
years (Don et al., 2008). This is because the well-aerated burrow walls allow
the expansion of high microbial activity down the soil profile (Loquet et
al., 1977 in Devliegher and Verstraete, 1997) and the interactions among
microbes and their feeders in the burrow walls are intense and accelerate
carbon and nutrient mineralization (Tiunov and Scheu, 1999; Görres et
al., 1999, 2001). The burrows of L. terrestris are also bypass flow
routes for percolating water, and depending on arable soil management, they
may increase leaching of topsoil nitrogen to the subsoil (Shuster et al.,
2003).
Most of the investigations of earthworm effects on GHG emissions have been
carried out in the laboratory (Bertora et al., 2007; Rizhiya et al., 2007;
Giannopoulos et al., 2010; Lubbers et al., 2011; Augustenborg et al., 2012)
and to our knowledge, only three field experiments have been conducted
(Borken et al., 2000; Amador and Avizinis, 2013; Lubbers et al., 2013b).
Recent reviews have underlined the need for field studies with all major
gases (N2O, CO2 and CH4) to provide a more comprehensive
picture of earthworm contribution to soil GHG emissions (Lubbers et al.,
2013a; Blouin et al., 2013). In this study, we aimed at filling this research
gap by measuring the small-scale spatial variation of soil biological and
chemical properties and N2O, CO2 and CH4 fluxes caused by
L. terrestris in a northern, arable no-till field. We hypothesized
that (1) the N2O and CO2 emissions are greater in L. terrestris midden areas (higher earthworm activity) compared to adjacent
non-midden areas (lower earthworm activity), while CH4 emissions remain
unaffected; (2) the middens contribute to gas production and their removal
from the soil surface decreases instant gas emissions; and (3) the biological
and chemical soil properties essential for gas balance differ between the
midden and non-midden areas. Moreover, to test how well the earthworm effects
on GHG emissions in the field can be predicted by laboratory experiments, we
established a controlled laboratory study with a L. terrestris
treatment and measurements of response variables identical to those in the
field. Our aim was not to establish a laboratory experiment that would
perfectly mimic our field situation, but to establish a typical laboratory
experiment to test whether laboratory studies in general can produce results
that resemble the field results. This is an important aspect as most earlier
experiments have been carried out in the laboratory and e.g. the review by
Lubbers et al. (2013a) is entirely based on laboratory studies.
Methods
Field measurements
Field measurements of N2O, CO2 and CH4 emissions were
conducted in a long-term, no-till field (11 years of no-till cultivation) in
Säkylä (60∘58′ N, 22∘31′ E), south-western
Finland, in October 2008. The soil at the site (depth 0–20 cm) is fine sand
with 15 % clay, 29 % silt and 56 % sand. Soil pH (H2O) is
6.1 and the N and C concentrations 0.1 and 2.1 %, respectively. The
topsoil (0–5 cm) bulk density is 1.37 g cm-3. The annual crops
cultivated in the field in 2007 and 2008 were turnip rape and barley,
respectively. Ten large middens and their adjacent non-midden areas were
randomly chosen within two 20 m2 areas (called sites A and B; five
pairs in both) 1 month after crop harvest, which according to our experience
is a time of high L. terrestris activity. The two sites, 30 m
apart, were needed to obtain a sufficient number of treatment pairs, but they
also provide data for testing whether the treatment effect varies in space at
the field scale. For this purpose, the site was included in the statistical
models as an explaining factor. In order to minimize the environmental
variation within treatment pairs, the distance between the midden and
non-midden areas within a pair was kept short; the average distance between
the outer rims of measurement chambers within a pair was 13 cm (min 3 cm,
max 34 cm), while the average distance between a pair and its closest
counterpart was 1.35 m (min 0.37 m, max 3.00 m).
The gas measurements were accomplished using round PVC chambers (diameter
15 cm, height 10 cm). Five gas measurements were carried out at varying
intervals over a period of 2 weeks. Chambers were pressed into the soil to
the depth of approximately 2 cm and the soil was compressed by hand around
the chambers. Permanent installations were not established in order to avoid
the disturbance of earthworms, and since the experiment was conducted after
harvest, it was not necessary to take into account the decrease of CO2
flux that may follow when live roots are cut by the chamber (see Heinemeyer
et al., 2011). In each measurement, 20 mL of chamber air was sampled through
a rubber septum using a polypropylene syringe (BD Plastipak, Becton,
Dickinson and Company, Franklin Lakes, NJ, USA) immediately and 60 min after
the placement of the chamber. The air was then transferred into pre-evacuated
12 mL glass vials (Exetainer, Labco Ltd., High Wycombe, UK). Before each gas
sample, the air in the chamber was mixed by one syringe flush.
The air temperature, which was measured using a Fluke 52 II thermometer
(Fluke Corp., USA), fluctuated between 7.2 and 11.8 ∘C during the
gas measurements. Air temperature, instead of the chamber temperature, was
used to define the gas volume for flux calculation as chamber warming due to
radiation is minimal in October. Soil moisture was measured next to each
“midden–non-midden” pair at the depth of 0–15 cm during each gas
measurement using a TRASE system I moisture meter and time domain
reflectometry (TDR; Soil Moisture Equipment Corp., Goleta, CA, USA). The
changes in soil temperature were followed using thermologgers (ElcoLog,
Elcoplast Oy, Finland), which were installed at the depth of 5 cm outside
the gas sampling areas (this data is missing for the two first gas
measurements).
At the last measurement, gas samples were first taken as described above. The
middens (surface cast mounds and the associated residues) and the straw
litter of the non-midden areas were then removed and the gas measurements
were repeated to evaluate the effect of midden and straw material on gas
emissions. After these measurements, soil cores (diameter 5 cm, depth 5 cm)
were collected from the entrance of L. terrestris burrows and the
adjacent non-midden areas. The removed midden and straw material and the soil
samples were stored at -18 ∘C for 7.5 months before being analysed
for gravimetric moisture content, potential denitrification and mineral N
concentrations. To estimate earthworm abundances in the area of the gas
measurement, the measurement chamber was pushed deeper into the soil and the
earthworms were hand-sorted out of the obtained soil sample (diameter 15 cm,
depth 15 cm). Deep-residing earthworms were extracted from the bottom of the
pit by pouring 0.5–0.75 L formalin solution (0.5 %) into the pit and
collecting individuals that emerged within 30 min. Slugs, which were
abundant in the middens, were hand-sorted from the midden and non-midden area
samples and together with the earthworms were stored in 85 % ethanol,
weighted and identified into the species or genus level (Sims and Gerard,
1999; Kerney and Cameron, 1979).
Laboratory experiment
The soil, barley stubble straw and L. terrestris individuals were
collected for the laboratory mesocosms in the beginning of November 2008 from
the same no-till field that was used for field measurements. The 15-week
experiment was designed to simulate the post-harvest autumn conditions of a
no-till field and during the set-up; all unnecessary manipulation of soil,
straw and earthworms was avoided to preserve the natural communities of
microbes and soil micro- and meso-fauna. The moist soil (moisture content
27 % of fresh mass) was first sieved (6 mm) and mixed to ensure soil
homogeneity. Any earthworms found were removed. Thirty PVC tubes (diameter
15 cm, height 45 cm, bottoms enclosed with plastic lids) were then filled
with the soil to the height of 43 cm. During filling, the soil was compacted
to achieve even bulk density among the tubes (mean 1.43 g cm-3, min
1.40 and max 1.46 g cm-3, n= 30). The tubes were weighted (before
and after filling) and placed in an incubation room at 15–17 ∘C,
chosen as favourable temperature for L. terrestris activity (Butt,
1991), with a rhythm of 10 h day (fluorescent lamps providing on average
1102 lx) and 14 h night (no illumination). Air humidity was maintained
using a moistener, but varied from 26 to 81 % during the experiment. Soil
moisture content was adjusted to 28 % and kept approximately constant by
adding deionized water once a week (always 2 days before gas samplings) and
spraying the soil surface with water after gas measurements.
The L. terrestris individuals used in the experiment were extracted
from the field using a mustard mixture (Gunn, 1992) and immediately washed in
tap water. Individuals were kept in moist soil for 9 days (dark,
4 ∘C) before one large individual was added to each of the 15
randomly chosen mesocosms. Each individual was weighted (mean fresh mass
4.5 g, min 3.7 g, max 5.5 g) and the settling into the soil was
facilitated by creating an artificial burrow (depth 8.5 cm, diameter
0.5 cm) in the centre of the soil column. The remaining 15 mesocosms were
left without worms and served as controls. The L. terrestris and
control mesocosms were randomly placed in the incubation room as treatment
pairs. An even layer of chopped straw was added on the top of the soil in
each mesocosm (straw length 2 cm, total fresh mass 5 g), and to prevent
animal escape, the mesocosms were covered by a mesh. Emerging plant seedlings
were removed from the mesocosms during the experiment, whereas juvenile
earthworms, noticed to hatch from the cocoons, were not, as the removal would
have disturbed the experiment.
The gas measurements were started 1 month after mesocosm establishment and
were repeated twelve times, at 1-week intervals, from December 2008 to
February 2009. The sampling was always carried out within 1 day. For the
measurements, air-proof plastic lids (diameter 15 cm, height 10 cm) were
first placed on the tubes air-tightly. The incubation lasted for 60 min and
the samples were collected according to the field protocol described above.
At the final date, gas fluxes were measured before and after removing
L. terrestris midden and straw residues. The soil samples for soil
moisture, potential denitrification and mineral N measurements were taken as
in the field. The tubes were emptied and the L. terrestris
individuals and earthworm juveniles, hatched from the cocoons during the
experiment, were hand-sorted out of the soil. A 100 g subsample was taken
from the mixed soil to estimate the mineral N content of the entire soil
column. At the end of the experiment, three of the L. terrestris
mesocosms had 1–3 and seven of the control mesocosms 1–2 small earthworm
juveniles (both dark and light pigmented unidentified species) having a
maximum individual fresh mass of 0.16 g. All earthworms were washed in
deionized water and weighted and, in order to determine their GHG production,
incubated in 210 mL flasks for 60 min (separately for experimental
L. terrestris and the group of juveniles). The GHG production was
estimated using 10 mL gas samples taken in the beginning and at the end of
the incubation. Three incubations of L. terrestris produced deviant
fluxes of N2O, CO2 and CH4, and the results were excluded from
the data set.
Analyses of gases, potential denitrification and mineral
nitrogen
The gas samples were always analysed within 48 h after sampling using a gas
chromatograph (GC) equipped with a flame ionizer (FID), an electron capture
detector (ECD) and a nickel catalyst for converting CO2 to CH4.
The precolumn and analytical columns consisted of 1.8 and 3 m long steel
columns, respectively, packed with 80/100 mesh Hayesep Q (Supelco Inc.,
Bellefonte, PA, USA). The GC (HP 6890 Series, GC System, Hewlett Packard,
USA) had a 10-way valve with a 2 mL sample loop and a backflush system for
separating water from the sample and for flushing the precolumn between the
runs. A six-way valve was used to lead the flow to either the FID or ECD.
The temperature of the GC oven, FID and ECD was 70, 300 and 350 ∘C, respectively. Nitrogen was used as the
carrier gas and a mixture of argon and methane (5 %) as a make-up gas (1.4 mL min-1) to increase the ECD sensitivity. A standard gas mixture (AGA
Gas AB, Lidingö, Sweden) of known N2O, CH4 and CO2
concentrations was used for the calibration curve. The flux rate of each gas
was calculated using the gas accumulation rate during the 60 min enclosure
period. Cumulative fluxes were calculated by assuming linear changes between
subsequent measurement dates. The net gas balance as a global warming
potential (GWP) was determined using the factor 298 for N2O and 25 for
CH4 (Myhre et al., 2013).
The denitrification potentials of the midden soil and the straw of the
L. terrestris middens and the adjacent non-midden areas were
determined as in Klemedtsson et al. (1988) and Henault et al. (1998) with
some modifications. In brief, the defrosted and sieved 10 g (d.m.) soil
samples (moisture was on average 26 % in the field and 21 % in the
laboratory samples) were placed in 120 mL bottles and 4 mL of distilled
water was added. The straw samples were combined within treatments (midden
vs. non-midden, separately for areas A and B), because the amount of material
in one sample was not enough for the analysis, and then divided into
2.5–5.5 g (d.m.) subsamples. After one night at 6 ∘C, the samples
were transferred to 25 ∘C and treated with 5 mL of potassium
nitrate (KNO3) solution and 5 mL of glucose solution (corresponding to
amendments of 200 mg N and 500 mg C kg-1 soil). The bottles were
then sealed using butyl rubber septa and crimp seals, evacuated, and flushed
three times with dinitrogen gas. The overpressure in the bottles was released
through a 0.5 mm needle, pierced through the septum, and to prevent the
entry of oxygen into the bottle, the needle was mounted on a 1 mL plastic
syringe (without piston) filled with 0.1 mL distilled water. The bottles
were then amended with 12 mL of acetylene (C2H2) to block the
N2O reduction step of denitrification, which was regarded as the start
of the incubation (t= 0). Three mL gas samples were then taken after 15
and 45 min, followed by 1 mL samples after 75, 105, 135, 165, 195, 225 and
255 min, and these were injected into 12 mL evacuated vials. All samples
were diluted with N2 to a volume of 18 mL to ensure that the
concentrations were in the range of the calibration curve. Samples were
analysed using the Hewlett Packard GC as described above.
For the analyses of soil ammonium and nitrate concentrations, samples were
first homogenized manually using a steel spatula, and from each sample 50 g
of fresh soil was mixed with 125 mL of 2 M KCl and shaken for 2 h on an
orbital shaker. The amount of straw material in one sample was too small for
the analysis, so straw samples were combined within treatments. The combined
samples were then divided into 6–21 g (f.w.) subsamples and treated
similarly as the soil samples. The extracts of soil and straw samples were
filtered through filter paper (130 g m-2, Tervakoski Oy, Tervakoski,
Finland) and analysed for nitrate and ammonium the next day after storage at
6 ∘C. A colorimetric autoanalyser (QuikChem AE, Lachat Instruments,
Loveland, CO, USA) was used for the simultaneous analysis of nitrate and
ammonium.
Statistical analyses
The field data of N2O, CO2 and CH4 emissions were obtained
from a randomized complete block design with repeated measurements.
Altogether, there were ten pairs (blocking factor) of midden–non-midden
areas (treatment factor) from the two sites (A and B). The measurements at
the same experimental site were correlated, which was taken into account in
the statistical models through appropriate covariance structures. The
statistical model thus became
yijkl=μ+si+βj(i)+tk+(st)ik+εijk+dl+(sd)il+(βd)jl(i)+(td)kl+(std)ikl+γijkl,
where μ is the constant intercept, and si, tk, (st)ik,dl, (sd)il, (td)kl and (std)ikl are fixed main and
interaction effects for site (s), treatment (t) and date (d). The
βj(i) is the random effect for block j within site i and
εijk is random plot-to-plot variation, all mutually
independent with variances var(βj(i))=σ2β and
var(εijk)=σ2ε. The (βd)jl(i)
represents the random date-specific contribution for block i within site
j, and γijkl represents the random error effect for observations
on the same plot (Gumpertz and Brownie, 1993). This model was used for
CH4. For N2O and CO2, a simplified model was used as the site
had no effect on the fluxes of either gas. The effect of removing middens and
straw litter from the soil surface on N2O, CO2 and CH4
emissions was analysed using a similar model as for the repeated gas
measurements, except that the repeated measurement effect of date was
replaced with the repeated measurement effect of before and after removal.
Analogously to the earlier models, the site effect was included in the model
for CH4, but not for N2O and CO2. In the case of N2O, log
transformation was used to meet the normality assumption.
The background variables were measured at the last measurement date
(Table 4). Since these measurements were not repeated, the statistical models
used were simplified analogues of the model presented above, except for the
number of slugs, which was analysed using the non-parametric Wilcoxon sign
rank test as the assumptions of the parametric methods were not met. The
cumulative emissions of N2O, CO2 and CH4 were analysed using a
simplified non-repeated analogue of the model presented above. The analysis
of laboratory data followed the analysis of field data, except that the site
effect and interactions were not included in the models. Log transformations
were used for N2O and mineral nitrogen (top 5 cm soil samples) and in
addition, two outliers were excluded from the mineral nitrogen data due to
exceptionally high values in comparison to the other 13 observations in the
control mesocosms.
For all the parametric models, REML (restricted maximum likelihood) was used
as the estimation method, degrees of freedom were calculated by the
Kenward–Roger method (Kenward and Roger, 1997), and model assumptions were
checked using appropriate graphs. The models were fitted using the MIXED
procedure of SAS 9.2 (SAS Institute Inc., Cary, NC, USA) and pairwise
comparisons were performed using two-sided t-type tests.
Results
Field measurements
In the field, the N2O and CO2 emissions were significantly higher
in the midden than non-midden areas (Table 1; Fig. 1a, b). The overall (all
repeated measurements included) model-based mean estimates of N2O fluxes
were 0.23 (95 % CI 0.18–0.27) and 0.13 (0.09–0.17) µg N
chamber area-1 h-1 for the midden and non-midden areas,
respectively. The corresponding figures for CO2 were 1754 (1568–1941)
and 1201 (1015–1388) µg CO2 chamber area-1 h-1,
respectively. Based on these estimates, the chamber area with one midden
produced on average 43 % more N2O and 32 % more CO2 than an
equivalent non-midden area. N2O and CO2 emissions varied among the
dates (Fig. 1a, b; Table 1), but this variation was apparently not explained
by soil moisture or temperature, which fluctuated little among the dates
(min–max 37.2–38.3 % and 6.5–8.5 ∘C, respectively). The
CH4 fluxes differed between the midden and non-midden areas at two
measurement dates, but the effects were specific to the measurement site
(Table 1), i.e. the flux was higher in the midden than non-midden areas in
site B at the first measurement (t14.1=-4.02, p= 0.001), but lower in
site A at the fourth measurement (t12.4= 2.44, p= 0.031; Fig. 1c,
d). The model-based mean estimates of cumulative emissions were significantly
higher in the midden than non-midden areas for N2O and CO2
(F1,7.34= 16.91, p= 0.004; F1,7.66= 43.80,
p < 0.001, respectively), but not for CH4
(F1,7.74= 3.24, p= 0.111) (Table 2). The removal of middens and
other residues from the soil surface had no effect on N2O and CO2
emissions in either the midden or non-midden areas (Table 3; Fig. 1a, b). For
CH4, the removal decreased the flux in site A (t9.1= 2.86,
p= 0.019), but not in site B (t7.87=-0.65, p= 0.532), and no
difference was found between the responses of midden and non-midden areas
(Table 3, Fig. 1c, d).
Fixed effect (treatment and site) P values of general linear mixed
models with repeated measurements (date) for N2O, CO2 and
CH4 emissions in the field and laboratory measurements. Treatment is
“midden area vs. non-midden area” in the field and “L. terrestris vs. control” in the
laboratory mesocosms.
Model term
N2O
CO2
CH4
Field
Site
0.008
Treatment
< 0.001
< 0.001
0.043
Treatment × site
0.072
Date
0.004
< 0.001
0.029
Site × date
< 0.001
Treatment × date
0.289
0.588
< 0.001
Treatment × site × date
0.007
Laboratory
Treatment
< 0.0001
< 0.0001
0.482
Date
< 0.0001
< 0.0001
0.144
Treatment × date
0.159
0.401
0.039
The number of earthworms was 125 % and their biomass 150 % higher in
the midden than in the non-midden areas (Table 4). However, only in four
midden and two non-midden areas, a large (> 0.8 g) L. terrestris was found and the majority of earthworms were juveniles. In the
midden areas, 18 % of individuals belonged to Lumbricus,
51 % to Aporrectodea and 31 % remained unidentified. In the
non-midden areas, the corresponding figures were 16, 58 and 26 %,
respectively. The soil surrounding the burrow entrance (within 5 cm
diameter) was on average 1 % unit moister, contained 23 % more
mineral N and had 20 % higher potential denitrification than the topsoil
of the non-midden areas (Table 4), but the denitrification potential of the
midden and non-midden straw did not differ (2.7 vs. 2.8
µg N2O–N g-1 straw d.m. h-1, respectively). The
mineral N content of the straw was 28 and 69 mg kg-1 straw d.m. in the
midden and non-midden areas, respectively, while the midden areas had more
straw litter on the soil surface (visual observation). In total, 31 slugs
(Arion fasciatus N.) were found from the midden areas after the
final gas measurement, while only three were found from the non-midden areas
(Table 4). In the midden areas, 77 % of the slugs were found in the
midden, 23 % in the soil beneath the midden.
The mean (±SE) estimates of statistical models for (a) N2O, (b) CO2 and (c, d) CH4 (separately for field sites A and
B) emissions in L. terrestris midden (•) and non-midden (∘) areas and the effect of the
removal of middens and surface residues on the emissions. For CH4, the
differences between the midden and non-midden areas at p < 0.05 are
marked with * (for effects on N2O and CO2, see Table 1).
![]()
The mean estimates (SE) of statistical models for cumulative
N2O, CO2 and CH4 fluxes in the field (duration 2 weeks) and
laboratory (15 weeks) measurements.
N2O
CO2
CH4
µg N chamber area-1
mg chamber area-1
µg chamber area-1
Field:
Midden area
74.2 (5.1)
591.4 (28.4)
-2.6 (1.1)
Non-midden area
47.6 (5.1)
394.4 (28.4)
-4.8 (1.1)
Laboratory:
L. terrestris
111.3 (7.1)
3224 (157)
-230.7 (9.2)
Control
90.3 (6.2)
2729 (152)
-224.7 (8.1)
Laboratory experiment
In the laboratory, N2O and CO2 emissions were significantly higher
with than without L. terrestris (Table 1; Fig. 2a, b). The
model-based mean estimates (with all repeated measurements included) of
N2O emissions with and without L. terrestris were 0.060
(95 % CI 0.053–0.067) and 0.044 (0.039–0.049) µg N chamber
base area-1 h-1. The corresponding figures for CO2 flux were
1769 (1600–1937) and 1536 (1367–1704) µg CO2 chamber base
area-1 h-1, respectively. Based on these values, one L. terrestris individual increased the mesocosm emission of N2O and
CO2 by 27 and 13 %, respectively. On average, the fluxes of N2O
and CO2 decreased in the course of the experiment (Fig. 2a, b). The
CH4 flux fluctuated during the experiment without a clear trend
(Table 1b, Fig. 2c) and, only at day 98, the emission rate differed between
the treatments, being then higher with than without L. terrestris
(t171=-2.12, p= 0.035). The model-based mean estimates of the
cumulative emissions were significantly higher with than without L. terrestris for N2O and CO2 (F1,12.9= 5.09, p= 0.042;
F1,9.65= 29.21, p < 0.001, respectively), but not for
CH4 (F1,11.5= 0.33, p= 0.579) (Table 2).
The removal of middens and straw residues from the soil surface affected the
N2O and CO2 emissions, but not the CH4 emissions (Table 3; Fig. 2a–c). The N2O emissions increased after the removal in all
mesocosms, whereas the response of CO2 flux depended on the treatment:
the removal increased CO2 emissions in the presence (t26=-3.36, p= 0.002), but had no effect in the absence of L. terrestris (t26=-0.64,
p= 0.525).
Fixed effect (site and treatment) P values of general linear mixed
models with repeated measurements (midden and residue removal) for N2O,
CO2 and CH4 emissions in the field and laboratory measurements.
Treatment is “midden area vs. non-midden area” in the field and “L. terrestris vs.
control” in the laboratory mesocosms.
Model term
N2O
CO2
CH4
Field
Site
0.007
Treatment
0.012
0.009
0.015
Treatment × site
0.080
Removal
0.401
0.980
0.139
Site × removal
0.034
Treatment × removal
0.845
0.338
0.176
Treatment × site × removal
0.894
Laboratory
Treatment
0.083
0.002
0.886
Removal
0.004
0.008
0.440
Treatment × removal
0.449
0.054
0.317
At the end of the experiment, mesocosms with L. terrestris had less
straw litter on the soil surface (visual observation) and 4 % more
mineral N in the 0–43 cm soil column (excluding the soil core collected
around the burrow) than the mesocosms without L. terrestris
(Table 5). In all except two mesocosms the resident worm had created a burrow
that reached the bottom of the soil column. The soil that surrounded the
L. terrestris burrow entrance (diameter 5 cm) was 0.3 % unit
moister, contained 16 % more mineral N and had a 17 % greater
potential denitrification rate than the topsoil of the control treatment
(Table 5). The potential denitrification of the straw collected from
L. terrestris and control mesocosms was 0.24 and 0.19 µg
N2O–N g-1 straw d.m. h-1 and its mineral N content 664 and
122 mg kg-1 d.m., respectively.
The mean estimates (±SE) of statistical models for (a) N2O, (b) CO2 and (c) CH4 emissions in
L. terrestris (•) and control (∘)
mesocosms and the effect of the removal of middens and surface residues on
the emissions. For CH4, the differences between treatments at
p < 0.1 are marked with * (for effects on N2O and CO2, see
Table 1).
![]()
Characteristics of L. terrestris midden (n= 10) and
adjacent non-midden (n= 10) areas at the end of the field measurements
(model-based mean estimates with 95 % confidence intervals presented for
all other variables except for the slug Arion fasciatus, which has
medians with a minimum and maximum). F- and P-statistics show the
statistical significance of the difference between the midden and non-midden
areas (for slugs the values are from the non-parametric Wilcoxon signed rank
test).
Midden area
Non-midden area
df
F
P
Earthworm numbera
3.6 (2.6–4.6)
1.6 (0.6–2.6)
1, 8
8.51
0.019
Earthworm mass (g f.w.)a
2.0 (1.4–2.7)
0.8 (0.1–1.5)
1, 16
7.81
0.013
Slug numbera
3.0 (0, 6)
0 (0, 1)
22.5
0.004
Soil moisture (% of f.w.)b
26.5 (25.8–27.2)
25.4 (24.8–26.1)
1, 8
7.66
0.024
Mineral N (mg kg-1 soil d.w.)b
9.2 (7.9–10.5)
7.1 (5.7–8.4)
1,8
8.24
0.021
Potential denitrification
1.2 (1.1–1.4)
1.0 (0.9–1.2)
1,8
4.16
0.076
(µg N2O–N g-1 soil d.w. h-1)b
a Sample covers the chamber base area (diameter
15 cm). b Soil core (depth 5 cm, diameter 5 cm) in the midden
area taken around the L. terrestris burrow entrance.
Two of the 15 L. terrestris individuals had died and the remaining
13 had lost on average 1.0 g or 22 % weight during the 15-week
experiment. When incubated in glass flasks at the end of the experiment, the
mean emission rate of one L. terrestris individual (mean fresh mass
3.6 g, min 3.1 g and max 4.2 g) was 0.006 (SE
0.001) µg N2O–N, 425 (41) µg CO2 and
-0.001 (0.002) µg CH4 h-1. Mean emissions per unit
fresh mass (min, max) for the three gases were 0.06 (0.03, 0.12), 2678 (1501,
4197) and -0.03 (-0.19, 0.12) nmol gas g-1 f.w. h-1,
respectively. Based on these values, the proportion emitted by L. terrestris of the total N2O, CO2 and CH4 fluxes at the last
gas measurement was 16, 36 and 0.7 %, respectively.
Discussion
In agreement with our first hypothesis, field N2O and CO2 emissions
were greater in L. terrestris midden than non-midden areas. CH4
fluxes were variable without a clear effect, but there was a slight
indication that the presence of L. terrestris decreased the CH4
oxidation rate of the soil. Against our second hypothesis, the removal of
middens and residues from the soil surface did not decrease N2O and
CO2 emissions. This indicates that the effect of L. terrestris
on GHG emissions results from changes in soil conditions at its living site,
not from the surface midden. Following our third hypothesis, most of the
investigated biological, chemical and physical soil variables differed
between the midden and non-midden areas, telling of the significance of
L. terrestris as an ecosystem engineer in arable fields. The fact
that we found an equally positive effect of L. terrestris on
N2O and CO2 emissions in the laboratory further indicates that the
observed effects in the field cannot be purely explained by confounding
factors such as the burrows acting as a chimney for gas emissions from a
larger area than the chamber, the worms selecting sites of high microbial
activity, or L. terrestris affecting the emissions of the adjacent
control area by collecting straw from it. However, the magnitude of the
effect was significantly smaller in the laboratory than in the field, i.e. a
27 % vs. 43 % increase for N2O and 13 % vs. 32 %
increase for CO2. It also appeared that the laboratory test could not
fully simulate the role of L. terrestris middens in gas emissions as
the removal of middens increased the emissions. These results underline the
value of comparing the measurements in the laboratory to those in natural
field sites with established earthworm populations.
Characteristics of L. terrestris (n= 13) and control
mesocosms (n= 15) at the end of the laboratory experiment (model-based
mean estimates and 95 % confidence intervals presented for all
variables). F- and P-statistics show the statistical significance of the
difference between the L. terrestris and control mesocosms.
L. terrestris
Control
df
F
P
Mineral N (mg kg-1 soil d.w.)a
21.9 (20.9–23.0)
21.0 (20.0–21.9)
1, 12.3
8.71
0.012
Soil moisture (% of f.w.)b
20.7 (20.6–20.8)
20.4 (20.3–20.5)
1, 14.1
13.46
0.003
Mineral N (mg kg-1 soil d.w.)b
23.1 (21.0–25.4)
19.3 (17.6–21.2)
1, 24
7.74
0.010
Potential denitrification
0.30 (0.27–0.32)
0.25 (0.23–0.27)
1, 26
10.55
0.003
(µg N2O–N g-1 soil d.w. h-1)b
a Sample represents the entire soil column (excluding
the soil core). b Soil core (depth 5 cm, diameter 5 cm) in the
L. terrestris mesocosm taken around the burrow entrance.
Our results show that L. terrestris can create sites of elevated
N2O emissions in arable no-till soils: in the field, the cumulative
N2O emissions were 36 % higher in the midden than non-midden areas
and, in the laboratory, 19 % higher in mesocosms with than without
L. terrestris. These results are in good agreement with earlier
laboratory studies (e.g. Matthies et al., 1999; Giannopoulos et al., 2010),
but also with field studies, such as the study by Borken et al. (2000), which
reported a 57 % increase in N2O emissions in beech forest mesocosms
due to L. terrestris. The recent meta-analysis of laboratory studies
by Lubbers et al. (2013a) also suggested a 42 % increase in soil N2O
emissions in the presence of earthworms. Few opposite findings exist (e.g.
Speratti and Whalen, 2008), although some studies suggest that the
contribution of earthworms to N2O emissions could be transient (Amador
and Avizinis, 2013; Lubbers et al., 2013b). In general, the contribution of
earthworms to GHG emissions is composed of direct and indirect emissions.
Direct emissions originate from earthworm metabolism and indirect from those
changes the earthworms induce in their environment. Living earthworms have
been found to emit N2O (Drake et al., 2006; Karsten and Drake, 1997) and
our incubation measurements support these findings (Table 6). The reported
values of direct N2O emissions emitted by L. terrestris vary
from 0.05 to 0.95 nmol N2O–N g-1 f.w. h-1 (Matthies et
al., 1999; Horn et al., 2006; Wüst et al., 2009), so our value,
0.06 nmol of N2O–N g-1 f.w. h-1, is at the lower end of
this range.
Although the direct N2O emissions have been quantified in many studies,
there are few estimations of their proportion of total emissions. In our
laboratory experiment, the proportion emitted by L. terrestris of
the total N2O flux was on average 16 %, which is in good agreement
with that reported by Karsten and Drake (1997) for beech forest soil
(16 %), but significantly higher than their value for oak–beech forest
soil (0.25 %). Our estimate is high and it may overestimate the
proportion in the field because the time interval L. terrestris was
able to shape the soil was short in our laboratory trial. In the field, the
soil is subjected to a long-term earthworm impact and it is likely that this
leads to a greater contribution of indirect emissions from the environment.
It should also be noted that part of the N2O produced by the earthworms
may be reduced to N2 while diffusing from the soil to the atmosphere and
the significance of direct emissions may also for this reason in the field be
lower than estimated based on laboratory measurements. Consequently, it is
likely that the enhanced N2O emissions in the presence of L. terrestris are also due to the changes in topsoil conditions and creation of
hot spots of high biological activity, including the elevated macrofaunal
densities, in the vicinity of the middens. For instance, the higher content
of mineral nitrogen and soil moisture favour denitrification, which was
manifested as elevated values of potential denitrification in our
measurements. In our field site, soil moisture was nearly 40 %,
corresponding to 80 % WFPS, which is suitable for earthworm N2O
contribution (Evers et al., 2010). Another potential mechanism for increased
N2O emissions in the field are the burrows that may act as large pores
that ease the diffusion of N2O from the bottom soil and allow more of
the N2O ending up in the atmosphere without being reduced to N2.
The laboratory soil was dryer than the field soil, which could be one reason
for the less noteworthy earthworm effect as soil moisture can significantly
modify the earthworm-induced N2O emissions (Chen et al., 2014).
The increase in soil cumulative CO2 emissions due to the presence of
L. terrestris was 33 and 15 % in our field and laboratory
measurements, respectively. These results echo the meta-analysis by Lubbers
et al. (2013a), which suggests a 33 % increase in soil CO2 emissions
in the presence of earthworms. When we estimated the respiration of
individual earthworms in the laboratory, the mean CO2 emission
(425 µg h-1) was almost double the mean difference between
the mesocosms with and without L. terrestris (230 µg
chamber area h-1) and the proportion of the total CO2 flux
explained by earthworm respiration was 36 %. These values suggest that
the increased emissions of CO2 from the soils occupied by L. terrestris were fully explainable by the respiration of the animal itself.
If this is true in general, the discrepancy between the observations of
increased CO2 emissions vs. increased carbon stability (Lubbers et al.,
2013a) would be explained by earthworm respiration counteracting the enhanced
carbon sequestration. However, this conclusion has to be treated cautiously
as we do not know how well the measurements of earthworm respiration in the
laboratory represent the respiration in the field. In the field, the elevated
slug densities of the middens also likely contributed to increased CO2
emissions as snail castings and mucus have been observed to increase the
efflux from leaf litter (Theenhaus and Scheu, 1996). Snail activity
accelerates N cycling, too (Theenhaus and Scheu, 1996), but we are not aware
of any studies of snail impacts on N2O emissions.
Unlike the effects on N2O and CO2 fluxes, the effects of L. terrestris on
CH4 flux were variable and mostly inconsequential and there was only a
slight indication in the cumulative field fluxes that the presence of L. terrestris might
decrease soil CH4 oxidation rate. Such a decrease could be a
consequence of increased moisture and N content in the vicinity of middens
(Hütsch, 2001). Small and varying earthworm effects on net CH4
fluxes have also been reported earlier (Borken et al., 2000; Aira et al.,
2009; Bradley et al., 2012), and our estimate of 0.7 % L. terrestris contribution to
the total CH4 flux is in good agreement with the earlier statement that
L. terrestris is not a source of CH4 (Šustr and Šimek 2009). As CH4
fluxes are also in general non-significant in the context of carbon cycling
in boreal arable soils (Regina et al., 2007), it appears that the effects of
earthworms on the GWP of these soils are driven by their effects on N2O
and CO2 emissions.
Recent studies suggest that Finnish no-till fields are characterized by both
high population densities of L. terrestris (Nuutinen et al., 2011)
and elevated N2O emissions (Sheehy et al., 2013). Higher N2O
emissions are usually explained by denser soil structure and higher soil
moisture compared to tilled soils. Our results suggest that increased
population densities of L. terrestris can also contribute to the
elevated N2O emissions. We found on average 20 L. terrestris
middens per m2 in our no-till field and when compared to a square metre
of equal field with no middens, such a density would increase the N2O
emissions by 27 % (estimated using mean values of midden and non-midden
areas). Although this estimate has to be treated with caution as the
non-midden areas were not completely out of the reach of L. terrestris activity, it appears that enhanced earthworm activity may explain
a substantial part of the 60–150 % increase in N2O emissions
observed in Finnish no-till fields (Sheehy et al., 2013). Moreover, when all
three gases were considered together, L. terrestris increased the
GWP of the soil by 50 and 18 % in our field and laboratory
investigations, respectively. These values, and particularly the field
estimate, exceed the 16 % mean increase in the net GWP of laboratory
soils reported by Lubbers et al. (2013a) in their meta-analysis based on 33
observations from individual earthworm studies that reported the cumulative
emissions of both N2O and CO2. However, the temporal variation in
emissions is probably high, mainly due to soil moisture variation. For
example, in a field study by Lubbers et al. (2013b), earthworms increased
N2O emissions of managed grassland in the autumn when the WFPS of soil
was 61–65 %, but had no effect in the dry spring when the WFPS was
16–25 %. Our field experiment represents the conditions that prevail for
approximately 3 months in the autumn when L. terrestris is highly
active and it is possible that during other seasons, the gas emissions are
less affected by the species. Moreover, the field estimate may exaggerate the
earthworm effect as part of the straw in the non-midden areas and was likely
transferred and consumed in the midden area. In contrast to what we expected,
the contributions of earthworm-induced N2O and CO2 emissions to the
net increase in GWP were 6 and 94 % in the field and 2 and 98 % in
the laboratory, respectively. This indicates that the elevated N2O
actually has a minor significance in the total balance despite its high GWP
value.
One of our aims was to test whether the earthworm effects on GHG emissions
that are found in laboratory trials can be generalized to field conditions.
For this purpose, we established a mesocosm experiment using soil and
L. terrestris individuals collected from the field site. The
mesocosms had generally higher CO2 and lower N2O emission rates
than the field soil, which probably was due to soil sieving increasing the
availability of microbial resources and microbial respiration (Hartley et
al., 2007) and drier mesocosm soil supporting lower N2O production.
Unlike in the field, the flux rates also steadily decreased in the
laboratory, which probably indicates diminishing resource availability after
the initial resource pulse (Hartley et al., 2007). Despite these differences
in the level and dynamics of the flux rates, a clear, positive effect of
L. terrestris on N2O and CO2 emissions was found in both
systems. The magnitude of the L. terrestris effect was smaller in
the laboratory, which could be related to soil moisture and the loss of
earthworm weight over the experiment, but also to the significantly elevated
faunal abundance and activity in the long-lived L. terrestris living
sites in the field. The size of the effect on CO2 emissions also
decreased in the laboratory as the experiment proceeded. Such a decrease is
common in laboratory studies (Borken et al., 2000; Lubbers et al., 2013a) and
is most probably related to the lack of fresh plant input to the soil, which
has a negative impact on L. terrestris metabolism. The distinct
difference between the field and laboratory emissions in their response to
the removal of middens and residues from the soil surface can possibly be
explained by the lack of air current in laboratory conditions, which may have
led to GHG accumulation in the soil pores and release of gases when the
midden and straw were removed. All these findings suggest that while the
general influence of L. terrestris on GHG emissions can be
approximated in laboratory conditions, field measurements are needed for more
accurate estimates and proper mechanistic understanding.
To conclude, our study contributes to filling the gap of field studies of the
effects of earthworms on GHG emissions, particularly in soils long occupied
by earthworms (Lubbers et al., 2013a). Our results emphasize the significance
of L. terrestris in the gas balance of agricultural soils, and
especially in no-till fields. We showed that L. terrestris
respiration can explain the observed increase in CO2 emissions in the
presence of earthworms and that a substantial part of the increase of
N2O emissions in no-till arable lands can be explained by earthworm
contribution. The gap of knowledge that still remains after our study is that
the effects of earthworms have almost solely been studied in the absence of
plants and without considering plant growth. As the effects of earthworms on
plant growth are generally positive (van Groenigen et al., 2014), the
disservice of increased N2O emissions may be counteracted by enhanced
plant growth to the degree that no increase in yield-scaled emissions results
(Wu et al., 2015). Extrapolation from our results to field scale may not be
simple either as the effect of midden density on GHG production is not
necessarily linear due to resource competition among earthworm individuals.
However, considering that field soils with active L. terrestris
middens had 50 % higher global warming potential than non-midden areas,
it is clear that L. terrestris, and potentially other earthworm
species as well, are among the key players that need to be taken into
consideration when the role of agricultural soils and cultivation practises
are evaluated for climate change mitigation. All in all, our study points out
how studies on the effects of conservation practices are necessary to fully
understand their effects on the environment.