Biogeosciences, 12, 5481–5493, 2015
www.biogeosciences.net/12/5481/2015/
doi:10.5194/bg-12-5481-2015
© Author(s) 2015. CC Attribution 3.0 License.
Impact of earthworm Lumbricus terrestris living sites on the
greenhouse gas balance of no-till arable soil
M. Nieminen1, T. Hurme1, J. Mikola2, K. Regina1, and V. Nuutinen1
1Natural Resources Institute Finland (Luke), Natural Resources and Bioproduction, 31600 Jokioinen, Finland
2Department of Environmental Sciences, University of Helsinki, 15140 Lahti, Finland
Correspondence to: V. Nuutinen (visa.nuutinen@luke.fi)
Received: 27 March 2015 – Published in Biogeosciences Discuss.: 29 April 2015
Accepted: 8 September 2015 – Published: 23 September 2015
Abstract. We studied the effect of the deep-burrowing earth-
worm Lumbricus terrestris on the greenhouse gas (GHG)
fluxes and global warming potential (GWP) of arable no-till
soil using both field measurements and a controlled 15-week
laboratory experiment. In the field, the emissions of nitrous
oxide (N2O) and carbon dioxide (CO2) were on average 43
and 32 % higher in areas occupied by L. terrestris (the pres-
ence judged by the surface midden) than in adjacent, unoc-
cupied areas (with no midden). The fluxes of methane (CH4)
were variable and had no consistent difference between the
midden and non-midden areas. Removing the midden did not
affect soil N2O and CO2 emissions. The laboratory results
were consistent with the field observations in that the emis-
sions of N2O and CO2 were on average 27 and 13 % higher in
mesocosms with than without L. terrestris. Higher emissions
of N2O were most likely due to the higher content of min-
eral nitrogen and soil moisture under the middens, whereas
L. terrestris respiration fully explained the observed increase
in CO2 emissions in the laboratory. In the field, the signifi-
cantly elevated macrofaunal densities in the vicinity of mid-
dens likely contributed to the higher emissions from areas oc-
cupied by L. terrestris. The activity of L. terrestris increased
the GWP of field and laboratory soil by 50 and 18 %, but
only 6 and 2 % of this increase was due to the enhanced N2O
emission. Our results suggest that high N2O emissions com-
monly observed in no-till soils can partly be explained by
the abundance of L. terrestris under no-till management and
that L. terrestris can markedly regulate the climatic effects of
different cultivation practises.
1 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 top-
soil favours methanotrophic bacteria (Hütsch, 2001). For car-
bon dioxide (CO2), soils can be either sinks or sources, de-
pending on the balance of carbon input and output (Stock-
mann 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 oxy-
gen 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 in-
creases carbon sequestration to soils and is therefore consid-
ered as a useful cultivation technique in climate change mit-
igation (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 phys-
ical 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
Published by Copernicus Publications on behalf of the European Geosciences Union.
5482 M. Nieminen et al.: Impact of earthworm Lumbricus terrestris living sites
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 increas-
ing 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 fac-
tors potentially contribute to this phenomenon. For instance,
by burrowing, casting and mixing crop residues into the soil,
the earthworms change soil organic carbon cycling, poros-
ity, 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 stim-
ulate 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 or-
ganic 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. (Niemi-
nen 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 micro-
bial activity (Schrader and Seibel, 2001; Aira et al., 2009), di-
verse invertebrate populations (Hamilton and Sillman, 1989;
Maraun et al., 1999; Butt and Lowe, 2007) and higher nu-
trient 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. ter-
restris may also increase the subsoil carbon stocks; for exam-
ple, 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 mi-
crobial 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 con-
ducted (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 un-
affected; (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 proper-
ties 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 labora-
tory 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 labora-
tory 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 car-
ried out in the laboratory and e.g. the review by Lubbers et
al. (2013a) is entirely based on laboratory studies.
2 Methods
2.1 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
Biogeosciences, 12, 5481–5493, 2015 www.biogeosciences.net/12/5481/2015/
M. Nieminen et al.: Impact of earthworm Lumbricus terrestris living sites 5483
large middens and their adjacent non-midden areas were ran-
domly chosen within two 20 m2 areas (called sites A and B;
five pairs in both) 1 month after crop harvest, which accord-
ing 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 test-
ing whether the treatment effect varies in space at the field
scale. For this purpose, the site was included in the statisti-
cal 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 pe-
riod 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 (El-
coLog, 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 re-
peated to evaluate the effect of midden and straw material
on gas emissions. After these measurements, soil cores (di-
ameter 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 sam-
ples were stored at−18 ◦C for 7.5 months before being anal-
ysed for gravimetric moisture content, potential denitrifica-
tion and mineral N concentrations. To estimate earthworm
abundances in the area of the gas measurement, the measure-
ment chamber was pushed deeper into the soil and the earth-
worms were hand-sorted out of the obtained soil sample (di-
ameter 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 individ-
uals that emerged within 30 min. Slugs, which were abun-
dant 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).
2.2 Laboratory experiment
The soil, barley stubble straw and L. terrestris individuals
were collected for the laboratory mesocosms in the begin-
ning 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 manip-
ulation of soil, straw and earthworms was avoided to pre-
serve 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 illu-
mination). Air humidity was maintained using a moistener,
but varied from 26 to 81 % during the experiment. Soil mois-
ture 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 in-
dividual was added to each of the 15 randomly chosen meso-
cosms. 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 facil-
itated by creating an artificial burrow (depth 8.5 cm, diam-
eter 0.5 cm) in the centre of the soil column. The remain-
ing 15 mesocosms were left without worms and served as
www.biogeosciences.net/12/5481/2015/ Biogeosciences, 12, 5481–5493, 2015
5484 M. Nieminen et al.: Impact of earthworm Lumbricus terrestris living sites
controls. The L. terrestris and control mesocosms were ran-
domly 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 earth-
worms, noticed to hatch from the cocoons, were not, as the
removal would have disturbed the experiment.
The gas measurements were started 1 month after meso-
cosm 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 incu-
bation lasted for 60 min and the samples were collected ac-
cording 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 mea-
surements were taken as in the field. The tubes were emp-
tied 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 en-
tire 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 in-
dividual 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 ju-
veniles). The GHG production was estimated using 10 mL
gas samples taken in the beginning and at the end of the in-
cubation. Three incubations of L. terrestris produced deviant
fluxes of N2O, CO2 and CH4, and the results were excluded
from the data set.
2.3 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 Se-
ries, GC System, Hewlett Packard, USA) had a 10-way valve
with a 2 mL sample loop and a backflush system for separat-
ing 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. Nitro-
gen 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 concentra-
tions 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 calcu-
lated by assuming linear changes between subsequent mea-
surement dates. The net gas balance as a global warming po-
tential (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 modifica-
tions. In brief, the defrosted and sieved 10 g (d.m.) soil sam-
ples (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, sepa-
rately for areas A and B), because the amount of material
in one sample was not enough for the analysis, and then di-
vided 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 glu-
cose 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 bot-
tles 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 sam-
ples 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 de-
scribed above.
For the analyses of soil ammonium and nitrate concen-
trations, 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 or-
bital 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
Biogeosciences, 12, 5481–5493, 2015 www.biogeosciences.net/12/5481/2015/
M. Nieminen et al.: Impact of earthworm Lumbricus terrestris living sites 5485
AE, Lachat Instruments, Loveland, CO, USA) was used for
the simultaneous analysis of nitrate and ammonium.
2.4 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 co-
variance structures. The statistical model thus became
yijkl = µ+ si +βj (i)+ tk + (st)ik + εijk + dl + (sd)il
+ (βd)j l(i)+ (td)kl + (std)ikl + γijkl, (1)
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 ran-
dom 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)j l(i) repre-
sents the random date-specific contribution for block i within
site j , and γijkl represents the random error effect for ob-
servations on the same plot (Gumpertz and Brownie, 1993).
This model was used for CH4. For N2O and CO2, a simpli-
fied model was used as the site had no effect on the fluxes
of either gas. The effect of removing middens and straw lit-
ter 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 mea-
surement date (Table 4). Since these measurements were not
repeated, the statistical models used were simplified ana-
logues of the model presented above, except for the num-
ber 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 labo-
ratory 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 ex-
cluded 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 maxi-
mum 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.
3 Results
3.1 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 cor-
responding 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 varia-
tion was apparently not explained by soil moisture or tem-
perature, 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 mea-
surement site (Table 1), i.e. the flux was higher in the mid-
den than non-midden areas in site B at the first measure-
ment (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 re-
sponses of midden and non-midden areas (Table 3, Fig. 1c,
d).
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 major-
ity 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 di-
ameter) was on average 1 % unit moister, contained 23 %
more mineral N and had 20 % higher potential denitrification
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5486 M. Nieminen et al.: Impact of earthworm Lumbricus terrestris living sites
Table 1. Fixed effect (treatment and site) P values of general lin-
ear mixed models with repeated measurements (date) for N2O, CO2
and CH4 emissions in the field and laboratory measurements. Treat-
ment is “midden area vs. non-midden area” in the field and “L. ter-
restris 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
Table 2. The mean estimates (SE) of statistical models for cumula-
tive 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)
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, re-
spectively). 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 fi-
nal 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.
3.2 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 mea-
surements 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 corre-
sponding figures for CO2 flux were 1769 (1600–1937) and
1536 (1367–1704) µg CO2 chamber base area−1 h−1, respec-
tively. Based on these values, one L. terrestris individual in-
creased 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
9 14 16 17 22
-0.10
-0.05
0.00
0.05
*
CH4 (site B)
µg
 
CH
4 
ch
am
be
r 
ba
se
 
a
re
a-
1 h
-
1
Midden/ 
residues 
removed
22
Day of measurement in October
µg
 
CH
4 
ch
am
be
r 
ba
se
 
ar
ea
-
1 h
-
1
-0.10
-0.05
0.00
0.05 CH4 (site A)
*
µg
 
CO
2 
ch
a
m
be
r 
ba
se
 
a
re
a
-
1 h
-
1  
0
500
1000
1500
2000
2500
µg
 
N
2O
-
N
 
ch
am
be
r 
ba
se
 
a
re
a-
1 h
-
1
0.00
0.10
0.20
0.30
0.40
N2O-N
CO2
(a)
(b)
(c)
(d)
Figure 1. 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).
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 sig-
nificantly 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).
Biogeosciences, 12, 5481–5493, 2015 www.biogeosciences.net/12/5481/2015/
M. Nieminen et al.: Impact of earthworm Lumbricus terrestris living sites 5487
Table 3. Fixed effect (site and treatment) P values of general linear
mixed models with repeated measurements (midden and residue re-
moval) 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
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).
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 (exclud-
ing the soil core collected around the burrow) than the meso-
cosms without L. terrestris (Table 5). In all except two meso-
cosms 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.
Two of the 15 L. terrestris individuals had died and the re-
maining 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.
Day from the beginning of the experiment
30 35 40 47 56 63 70 77 84 91 98 105
µg
 
CH
4 
ch
a
m
be
r 
ba
se
 
ar
ea
-
1 
h-
1  
-0.20
-0.15
-0.10
-0.05
0.00
Midden/
residues 
removed
* *
105
CH4
µg
 
N
2O
-
N
 
ch
a
m
be
r 
ba
se
 
a
re
a
-
1  
h-
1
0.00
0.02
0.04
0.06
0.08
0.10
N2O-N
(a)
(b)
(c)
µg
 
CO
2 
ch
a
m
be
r 
ba
se
 
a
re
a
-
1 
h-
1  
0
500
1000
1500
2000
2500
CO2
Figure 2. 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 be-
tween treatments at p < 0.1 are marked with ∗ (for effects on N2O
and CO2, see Table 1).
4 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 ef-
fect, 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 emis-
sions in the laboratory further indicates that the observed ef-
fects in the field cannot be purely explained by confounding
factors such as the burrows acting as a chimney for gas emis-
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5488 M. Nieminen et al.: Impact of earthworm Lumbricus terrestris living sites
Table 4. 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.
sions from a larger area than the chamber, the worms select-
ing 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 signifi-
cantly 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 re-
sults underline the value of comparing the measurements in
the laboratory to those in natural field sites with established
earthworm populations.
Our results show that L. terrestris can create sites of ele-
vated 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 re-
sults are in good agreement with earlier laboratory stud-
ies (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. Di-
rect emissions originate from earthworm metabolism and in-
direct 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 aver-
age 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 at-
mosphere 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 in-
stance, the higher content of mineral nitrogen and soil mois-
ture 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 contri-
bution (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 bot-
tom soil and allow more of the N2O ending up in the atmo-
sphere 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 signif-
icantly modify the earthworm-induced N2O emissions (Chen
et al., 2014).
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M. Nieminen et al.: Impact of earthworm Lumbricus terrestris living sites 5489
Table 5. 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.
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 individ-
ual 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 cham-
ber area h−1) and the proportion of the total CO2 flux ex-
plained 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 respi-
ration of the animal itself. If this is true in general, the dis-
crepancy between the observations of increased CO2 emis-
sions 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 ele-
vated 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 (Theen-
haus 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 inconsequen-
tial 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 conse-
quence 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 to-
tal 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 char-
acterized 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 den-
sities 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 esti-
mate 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). More-
over, when all three gases were considered together, L. ter-
restris increased the GWP of the soil by 50 and 18 % in our
field and laboratory investigations, respectively. These val-
ues, and particularly the field estimate, exceed the 16 % mean
increase in the net GWP of laboratory soils reported by Lub-
bers et al. (2013a) in their meta-analysis based on 33 ob-
servations 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 emis-
sions 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 au-
tumn 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 ar-
eas and was likely transferred and consumed in the midden
area. In contrast to what we expected, the contributions of
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5490 M. Nieminen et al.: Impact of earthworm Lumbricus terrestris living sites
earthworm-induced N2O and CO2 emissions to the net in-
crease 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 de-
spite 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 estab-
lished 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 respira-
tion (Hartley et al., 2007) and drier mesocosm soil support-
ing lower N2O production. Unlike in the field, the flux rates
also steadily decreased in the laboratory, which probably in-
dicates diminishing resource availability after the initial re-
source 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 mois-
ture 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; Lub-
bers 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 gen-
eral influence of L. terrestris on GHG emissions can be ap-
proximated 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, par-
ticularly in soils long occupied by earthworms (Lubbers et
al., 2013a). Our results emphasize the significance of L. ter-
restris 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 pres-
ence 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 re-
mains after our study is that the effects of earthworms have
almost solely been studied in the absence of plants and with-
out 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 sim-
ple 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 con-
sideration 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 conser-
vation practices are necessary to fully understand their effects
on the environment.
Acknowledgements. We thank Mirva Céder, Leena Seppänen,
Kirsikka Sillanpää, Ari Seppänen and Taisto Sirén for their help in
field and laboratory work, Timo Rouhiainen for the kind permission
to carry out the field work on his land and two anonymous referees
for helpful comments. M. Nieminen gratefully acknowledges a per-
sonal grant from the Kone Foundation. The study was conducted as
a part of the VILMA and ZERO-TILMA projects of MTT Agrifood
Research Finland.
Edited by: X. Wang
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