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Soil Biology & Biochemistry 42 (2010) 618e625

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Soil Biology & Biochemistry

journal homepage: www.elsevier .com/locate/soi lbio

Interactions between residue placement and earthworm ecological strategyaffect aggregate turnover and N2O dynamics in agricultural soil

Georgios Giannopoulos, Mirjam M. Pulleman, Jan Willem Van Groenigen*

Department of Soil Quality, Wageningen University, PO BOX 47, 6700AA Wageningen, The Netherlands

a r t i c l e i n f o

Article history:Received 4 May 2009Received in revised form11 December 2009Accepted 29 December 2009Available online 9 January 2010

Keywords:EarthwormLumbricus rubellusAporrectodea caliginosaNitrous oxideDenitrificationResidue management15N-labeled residuesStable aggregate fractions

* Corresponding author. Tel.: þ31 317 484784; fax:E-mail address: JanWillem.vanGroenigen@wur.nl

0038-0717/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.soilbio.2009.12.015

a b s t r a c t

Previous laboratory studies using epigeic and anecic earthworms have shown that earthworm activitycan considerably increase nitrous oxide (N2O) emissions from crop residues in soils. However, theuniversality of this effect across earthworm functional groups and its underlying mechanisms remainunclear. The aims of this study were (i) to determine whether earthworms with an endogeic strategy alsoaffect N2O emissions; (ii) to quantify possible interactions with epigeic earthworms; and (iii) to link theseeffects to earthworm-induced differences in selected soil properties. We initiated a 90-day 15N-tracermesocosm study with the endogeic earthworm species Aporrectodea caliginosa (Savigny) and the epigeicspecies Lumbricus rubellus (Hoffmeister). 15N-labeled radish (Raphanus sativus cv. Adagio L.) residue wasplaced on top or incorporated into the loamy (Fluvaquent) soil. When residue was incorporated, onlyA. caliginosa significantly (p< 0.01) increased cumulative N2O emissions from 1350 to 2223 mg N2OeN kg�1

soil, with a corresponding increase in the turnover rate of macroaggregates. When residue was appliedon top, L. rubellus significantly (p < 0.001) increased emissions from 524 to 929 mg N2OeN kg�1, anda significant (p < 0.05) interaction between the two earthworm species increased emissions to1397 mg N2OeN kg�1. These effects coincided with an 84% increase in incorporation of residue 15N intothe microaggregate fraction by A. caliginosa (p ¼ 0.003) and an 85% increase in incorporation into themacroaggregate fraction by L. rubellus (p ¼ 0.018). Cumulative CO2 fluxes were only significantlyincreased by earthworm activity (from 473.9 to 593.6 mg CO2eC kg�1 soil; p ¼ 0.037) in the presence ofL. rubellus when residue was applied on top. We conclude that earthworm-induced N2O emissions reflectearthworm feeding strategies: epigeic earthworms can increase N2O emissions when residue is appliedon top; endogeic earthworms when residue is incorporated into the soil by humans (tillage) or by otherearthworm species. The effects of residue placement and earthworm addition are accompanied bychanges in aggregate and SOM turnover, possibly controlling carbon, nitrogen and oxygen availabilityand therefore denitrification. Our results contribute to understanding the important but intricate rela-tions between (functional) soil biodiversity and the soil greenhouse gas balance. Further research shouldfocus on elucidating the links between the observed changes in soil aggregation and controls on deni-trification, including the microbial community.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Agricultural soils are a major contributor to global emissions ofthe greenhouse gas nitrous oxide (N2O). Approximately 62% of totalglobal N2O emissions is thought to be emitted from natural andagricultural soils (6.0 and 4.2 Tg NeN2O yr�1, respectively) (IPCC,2001, 2007). Ultimately, these emissions originate from soilmicrobial activity (mostly nitrification, denitrification and nitrifierdenitrification) (Wrage et al., 2001). Microbial activity in its turn iscontrolled by substrate availability (mineral nitrogen (N) and soil

þ31 317 419000.(J.W. Van Groenigen).

All rights reserved.

organic matter) and physico-chemical properties of the soil, inparticular anaerobicity, pH and soil temperature (Granli andBøckman, 1994). Changes in these soil properties resulting from theactivities of soil macrofauna may affect N2O emissions, and therebyplay an important role in the greenhouse gas balance of soils.

Earthworms have been described as soil ecosystem engineers(Jones et al., 1994; Lavelle et al., 1997). They affect physico-chemicaland biological soil properties by their feeding, burrowing andcasting activities. Through the mixing of soil particles and plantresidues they create a hierarchy of soil pores and aggregates,thereby affecting soil moisture dynamics and soil gas diffusivity(Oades, 1992; Kretzschmar and Ladd, 1993; Ketterings et al., 1997;Lavelle and Spain, 2003; Six et al., 2004) Earthworm-made(biogenic) aggregates are often enriched in particulate organic

Table 1Treatments included in the mesocosm study.a

Parallel experiments Factor Treatment code

A. caliginosa L. rubellus

1. Residue incorporated � � Iþ � IC� þ IRþ þ IRC

2. Residue placed on top � � Tþ � TC� þ TRþ þ TRC

a A treatment without residue addition nor earthworms was included and servedas a blank for both parallel experiments.

G. Giannopoulos et al. / Soil Biology & Biochemistry 42 (2010) 618e625 619

matter compared to physicogenic aggregates. Aerobic and anaer-obic microsites within biogenic aggregates may affect rates ofdecomposition and denitrification (Shipitalo and Protz, 1988;Zhang and Schrader, 1993) and it has been suggested that biogenicaggregates induce C stabilization in the longterm (Bossuyt et al.,2006).

The effect of earthworms on soil properties depends on theirecological strategy. Earthworms can be classified in three ecologicalgroups; i) epigeic species generally live in the litter layer where theyfeed on litter and its associated microflora, ingesting relatively littlesoil, ii) endogeic species live in the mineral soil, creating non-permanent burrows, feeding on soil organic matter, and iii) anecicspecies live in the mineral soil creating permanent vertical burrowsthat are created as a result of transporting litter from the surfaceinto the soil (Bouché, 1977; Lee and Foster, 1991; Edwards andBohlen, 1996). The abundance of earthworms in agricultural soilsand the presence and relative abundance of different functionalgroups depend strongly on agricultural management (especiallytillage and organic inputs) (Lee and Foster, 1991; Cook and Linden,1996; Chan, 2001). In a reduced or zero-tillage system, where moreorganic matter is available in the top soil and less mechanicaldisturbance takes place, higher activity of earthworms that mixcrop residues and soil particles can alter the soil structure andassociated soil properties (Lal and Akinremi, 1983; Hendrix et al.,1986; Thevathasan and Gordon, 2004).

The effect of earthworms on emissions of N2O is complex.Previous research has shown that earthworm activity can increasemicrobial populations, especially in the drilosphere, in earthwormcasts, and it the earthworms gut. This has been shown to lead toincreased rates of mineralization, nitrification, and denitrification(Svensson et al., 1986; Elliott et al., 1991; Parkin and Berry, 1999;Borken et al., 2000; Horn et al., 2003; Drake and Horn, 2006;Postma-Blaauw et al., 2006).

Earthworms have also been reported to increase N2O emissionsfrom soil. Rizhiya et al. (2007) reported that the epigeic earthwormspecies Lumbricus rubellus increased the N2O flux from 55.7 to789 mg N2OeN kg�1 soil while the anecic species Aporrectodea longaincreased the N2O flux from 55.7 to 227.2 mg N2OeN kg�1 soil ina mesocosm study.

However, several important questions with respect to this N2Oeffect remain unanswered. First, it has not been establishedwhether earthworms with endogeic strategies also increase N2Oemissions. This is of particular interest as these earthworms arethought to be instrumental in stabilizing C in the soil (Gilot, 1997;Bossuyt et al., 2005, 2006; Pulleman et al., 2005). Second, thenature of interactions between different functional earthwormgroups with regard to N2O emissions remains to be determined.This pertains especially to the interactions between epigeic andendogeic earthworms, as they have been shown for N mineraliza-tion (Postma-Blaauw et al., 2006) and C stabilization (Bossuyt et al.,2005). Third, the mechanisms by which earthworms affect N2Oemissions remain unclear.

In line with the questions outlined above, the objectives of thisstudy were (i) to determine the effect of endogeic earthworms onN2O emissions from crop residues; (ii) to determine interactionsbetween endogeic and epigeic earthworms; and (iii) to link theobserved earthworm effects to changes in soil aggregation. Wehypothesized that (i) due to their feeding strategy, endogeicearthworms result in elevated N2O emissions when the appliedcrop residue is incorporated into the soil; (ii) interactions betweenepigeic and endogeic earthworm activity may result in elevatedN2O emissions when residues are applied on top due to incorpo-ration of residues into the soil by the epigeics; and (iii) thatincreased N2O emission effects can be linked to faster incorporationand turnover of fresh organic residue in soil macroaggregates.

2. Materials and methods

2.1. Experimental setup

We quantified the effects of two different earthworm species onCO2 and N2O emissions and soil aggregation in a 90-day 15N labo-ratory study. The study consisted of two parallel experiments,differing only in residue application method: residue incorporatedand residue applied on top. Both parallel experiments weresubjected to four different earthworm treatments, which werethe result of a combination of two factors: presence or absence ofAporrectodea caliginosa present as a single species, and presenceor absence of L. rubellus (Table 1). Both experiments were laid outas a randomized block design with five replicates. A treatmentwithout residue or earthworms was included as a blank and alsoreplicated five times.

The loamy soil was collected from the former experimentalfarm “De Lovinkhoeve” in Marknesse, Noord-Oost Polder, TheNetherlands (52�430N, 5�520E). Soil collection took place from 0 to25 cm depth in September 2007. This soil was classified asa calcareous Typic fluvaquent (USDA, 1999) with 29% sand, 54% silt,17% clay, 1.24 g N kg�1, 17.5 g organic C kg�1 and a pH-KCl of 7.1.Field moist soil was passed through an 8mm screen and air dried at20 �C until further use. The soil was repeatedly mixed to ensurehomogeneity.

Individuals of the epigeic earthworm species L. rubellus(Hoffmeister) were collected from the floodplains of the riverRhine (51� 590 N, and 5� 390 E), and the endogeic species A caliginosa(Savigny) was collected in a park area (51� 590 N and 5� 390 E).Both locations are in the vicinity of the town Wageningen. Bothspecies are the most common species of their ecological groupsin Dutch soils (Didden, 2001). For 5 days prior to the start of theexperiment, earthworms were stored under dark conditions at 5�Cin containers with the loamy soil and clover (Trifolium pratense L.)residue.

The mesocosm setup was based on experiments by Rizhiya et al.(2007). In short, the mesocosms consisted of 6.1 L polyethylenebuckets, filled with 4.00 kg of air-dried soil and packed to a bulkdensity of 1.27 g cm�3. Soil moisture was brought to a gravimetricsoil moisture content of 250 g kg�1 soil, or 61% water filled porespace (WFPS). This level was considered to be optimal for earth-worm activity in this soil (Bertora et al., 2007; Rizhiya et al., 2007).Forty-fivemesocosmswere prepared and pre-incubated for 10 daysat 16 �C in the dark until initial N2O and CO2 emissions hadsubsided. Subsequently, 10.0 g of 15N-labeled radish residues(Raphanus sativus cv. Adagio L.; grown in the field with 15N-labeledfertilizer), chopped in 2 cm pieces, was applied (21.7 g N kg�1,371 g C kg�1 and 8.84 15N atom% excess). This corresponded to anapplication rate of 2630 kg dry matter ha�1, based on the surfacearea of the mesocosms (0.038 m2). The treatments with residue

G. Giannopoulos et al. / Soil Biology & Biochemistry 42 (2010) 618e625620

incorporated were repacked to the original bulk density, and theearthworms were applied to their respective treatments.

Earthworm treatments received 4 individuals of L. rubellus(3.8 g fresh weight) and/or 7 individuals of A. caliginosa(3.6 g fresh weight), corresponding to 80 and 150 individuals m�2

respectively (Table 1). The earthworms were adults or largejuveniles with their intestines voided for 48 h following the filterpaper method of Dalby et al. (1996). Each mesocosm was coveredwith a black polyethylene cloth. It was secured over the rim of themesocosm with two rubber bands. That cover allowed gaseousexchange with air, decreased water evaporation and preventedearthworms from escaping. No additional food source was addedto the mesocosms during the experiment. Mesocosms werekept in a climate-controlled room at 16 �C at 60% humidity. Soilmoisture content was corrected gravimetrically for each individualmesocosm every 3e5 days.

2.2. Flux measurements

Flux measurements of N2O and CO2 were taken daily in the firstweek, every second day in week 2, every third day in weeks 3e4,every fourth day in weeks 5e9 and once a week for the remainderof the experiment until the end of the experiment (90 days). Forflux measurements, the mesocosms were sealed for approximately30 min using a polyethylene lid equipped with two rubber septa.Gas measurements were taken with a photo-acoustic infrared gasanalyzer (Innova 1312), using two teflon tubes. When sampling forN2O, a soda-lime filter was used to minimize interference with CO2(Velthof et al., 2002). Fluxes were calculated assuming a linearincrease of N2O concentration over time during the closing of thelid. This was checked occasionally for each treatment during theexperiment. Values were corrected for ambient N2O concentrationand for mixing of the gas samplewith the previousmeasurement inthe internal volume of the gas monitor. CO2 emissions weremeasured using the same gas analyzer and a similar setup, butwithout the soda-lime filter and after a separate closing period(approximately 30 min) of the mesocosms. Cumulative emissionsfor both N2O and CO2 were calculated assuming linear changesbetween subsequent measurements (Kool et al., 2006a,b). Addi-tional gas samples of 15 mL for isotopic analyses of N2O were takenprior to the gas analyzer measurements on day 1, 4, 18 and 45 fromthe headspace volume using a syringe. Each sample was stored inhelium-flushed and evacuated 12 mL exetainers (Labco) and sendfor 15NeN2O analysis to the Stable Isotope Facility of the Universityof California at Davis (PDZ Europa 20e20, Cryoprep). An N2O/N2mixture was used as an internal standard. As no internationalcertified isotope standards are available for N2O, we calibrated 15Nby reacting the N2Owith glassy carbon at 1400 �C to convert N2O toN2. The resulting N2 was calibrated against the Oztech N2 standard.

2.3. Soil analyses

After the pre-incubation, before the start of the experiment,three additional mesocosms were destructively sampled to quan-tify initial soil conditions. Ammonium and nitrate concentrationswere determined colorimetrically after extraction with 1 M KCl(Kool et al., 2006b). Soil samples were also prepared for analysis oftotal N and background levels of 15N, at the Stable Isotope Facility ofUC Davis (see below).

At the end of the 90 d experiment intact core samples (100 cm3)were destructively taken from the center of each mesocosm at0e5 cm and 5e10 cm depth (the total depth of the mesocosms wasapproximately 12e13 cm) in order to quantify the effects of theearthworm functional groups on soil compaction at differentdepths in the profile. In these samples, air permeability and bulk

density were measured. Air permeability was measured using thetransient method of Grover (1955). A representative mixed samplefrom the complete depth profile of each mesocosm was taken forNeNH4

þ and NeNO3� analysis as described above.

Another representative subsample was taken from each meso-cosm for water-stable aggregate analysis. Aggregates were isolatedby wet sieving according to Elliott (1986) as modified by Sixet al. (2002) to obtain four size classes: large macroaggregates(2000e8000 mm), small macroaggregates (250e2000 mm), micro-aggregates (53e250 mm) and the silt and clay fraction (<53 mm). Inshort, 80 g of dried soil (30 �C for 2 days) was placed on top of the2000 mm sieve and submerged in 1800 mL of demi-water. Soilsampleswere left to slake for 2min prior to sieving. The sievingwasdone mechanically, moving the sieve up and down 60 times in2 min. The macroaggregate fraction remaining on the 2000 mmsieve was carefully backwashed, collected in aluminum pans, driedovernight at 50 �C and weighed. Floating OM particles > 2000 mmwere removed. Similarly, the small macroaggregate and micro-aggregate fraction were obtained by sieving the suspension thathad passed through the 2000 mm sieve over a 250 mm sieve andthe suspension that had passed through the 250 mm sieve over the53 mm sieve, respectively, repeating the same procedure. The<53 mm fraction was determined upon drying and weighing ofa representative subsample of 250mL from the suspension that hadpassed through the 53 mm sieve.

Very little material was found for the large macroaggregatefraction (<2%). Therefore, the large and the small macroaggregatefractions were combined for further analyses into one macroag-gregate fraction (250e8000 mm). A subsample of each fraction wasball-milled and oven-dried overnight at 60 �C. Approximately30 mg was weighed out in tin cups, the precise weight was recor-ded and the samples were sent to the Stable Isotope Facility ofUC Davis for measurement of total N and 15N atomic excess (PDZEuropa). Two internal laboratory standards were used, that werepreviously calibrated against NIST standard reference materials(IAEA-N1, IAEA-N2, IAEA-N3, IAEA-CH7, and NBS-22). Soil organic Cwas not measured in this study as the soil is calcareous and wouldhave to be fumigated with HCl in order to separate organic C frominorganic C (Harris et al., 2001). As the focus of the paper is on Ndynamics, we considered this extra analysis to be outside the scopeof this study.

2.4. Earthworm analyses

At the end of the experiment, simultaneously with the soilsampling, the earthworms were carefully collected from the mes-ocoms. The numbers of live earthworms were recorded and freshweights were determined after voidance of the guts during 48 h onwet filter paper. Subsequently, 15N recovery in earthworms wasdetermined following Schmidt et al. (2004): earthworms werefreeze-dried, ball-milled and oven-dried at 60 �C. A subsample ofapproximately 2 mg was weighed out as described above and sentto UC Davis for measurements of C, N and 15N atomic excess.

2.5. Statistical analyses

Our data were statistically analyzed using the general ANOVAmodule of the GenStat statistical package (9th Edition, VSN Inter-national Ltd.). For both experiments, gas flux data and soilparameters were analyzed using a two-way ANOVA with block-ing, with the two independent factors being the presence ofA. caliginosa and the presence of L. rubellus (e.g. Wootton, 1994).Earthworm biomass, recovery and enrichment of 15N in earthwormtissue was, for both experiments, tested as a one-way ANOVA withblocking, with presence of the other earthworm species as factor.

Table 3Cumulative N2O and CO2 fluxes during the incubation, with standard errors (n ¼ 5)and average percentages of residue-derived N2OeN. Treatment codes refer toTable 1.

G. Giannopoulos et al. / Soil Biology & Biochemistry 42 (2010) 618e625 621

Cumulative N2O fluxes had to be log-transformed beforestatistical analysis. For all analyses a p value of 0.05 or smaller wasconsidered significant.

Treatment mg N2OeN kg�1 soil mg CO2eC kg�1 soil Residue-derivedN2OeN (%)

Average (�St. Error) Average (�St. Error)

Blank �16.7 (�15.1) 108.5 (�33.5)

Experiment 1: Residue incorporatedI 1350.1 (�191.8) 610.1 (�75.9) 2.5IC 2223.1 (�223.7) 633.8 (�31.1) 4.1IR 1426.4 (�134.6) 642.2 (�54.3) 2.7IRC 1694.8 (�124.2) 637.1 (�23.3) 3.2

ANOVAL. rubellus 0.446 0.624A. caliginosa 0.010** 0.936L. rubellus �

A. caliginosa0.170 0.673

Block 0.736 0.193

Experiment 2: Residue on topT 524.3 (�22.1) 487.5 (�32.0) 1.0TC 441.1 (�33.3) 460.2 (�48.6) 0.8TR 928.7 (�69.3) 538.0 (�25.4) 1.7TRC 1396.5 (�277.3) 649.2 (�92.8) 2.6

3. Results

3.1. Earthworm survival and growth

Earthworm mortality during the experiment was 15% forL. rubellus and 14% for A. caliginosa. Live earthworm biomassdecreased for all treatments and for both species during the incu-bation, and the decrease was greater when both earthworm specieswere present (Table 2). However, this effect was only significant forthe weight change of L. rubellus when residue was applied on top(p ¼ 0.026). At the end of the incubation all residue had visuallydisappeared from the surface of those mesocosms that containedearthworms.

When residue was incorporated, 15N recovery in earthwormtissue was for both species significantly negatively affected by thepresence of the other species (Table 2). When residue was appliedon top, 15N enrichment of L. rubellus body tissue was significantlydecreased by the presence of A. caliginosa.

ANOVAL. rubellus <0.001*** 0.037*A. caliginosa 0.506 0.426L. rubellus �

A. caliginosa0.045* 0.199

Block 0.527 0.179

Levels of significance: * < 0.05; ** < 0.01; *** < 0.001.

3.2. Effects of earthworms on N2O and CO2 dynamics

Cumulative N2O emissions were in both experiments signifi-cantly affected by the presence of earthworms (Table 3; Fig. 1).When residue was incorporated, A. caliginosa increased cumulativeN2O emissions from 1350 to 2223 mg N2OeN kg�1 soil (p¼ 0.01) butL. rubellus had no significant effect. When residue was applied ontop, A. caliginosa had no significant effect, but L. rubellus increasedemissions from 524 to 929 mg N2OeN kg�1 (p < 0.001). Presence ofboth species increased emissions further to 1397 mg N2OeN kg�1

(species interaction effect: p ¼ 0.045).Carbon dioxide emissions were only significantly affected by the

presence of L. rubellus when residue was applied on top (Table 3).No interaction between L. rubellus and A. caliginosa was observed.

Fig. 2 illustrates the atom% 15N excess of N2O emissions from themesocosms. Based on the residue enrichment of 8.84 atom% excess,the maximum contribution of residue-N to total N2O emissions per

Table 2Changes in earthworm weight during the incubation, as well as 15N enrichment and 15Nexpressed as percentage change in fresh weight during the course of the incubation. ANOVweigh: the influence of L. rubellus on weight loss of A. caliginosa, and vice versa. Treatme

Treatment A. cal. L. rub. A. cal.

Weight change, % 15N en

Experiment 1: Residue incorporatedIC �10.9 � 5.4 2.05 �IR �35.4 � 20.0IRC �27.1 � 12.4 �62.6 � 3.2 1.05 �ANOVAL. rubellus 0.344 0.093A. caliginosa 0.273Block 0.685 0.591 0.509

Experiment 2: Residue on topTC �27.9 � 5.9 1.33 �TR �4.4 � 4.9TRC �52.1 � 5.8 �49.4 � 9.9 1.19 �ANOVAL. rubellus 0.073 0.759A. caliginosa 0.026*Block 0.806 0.837 0.284

Levels of significance: * < 0.05; ** < 0.01; *** < 0.001.

treatment ranged between approximately 2 and 18%. Highestpercentages of residue-derived N2OeN were reached on day 4,irrespective of residue placement method. On day 45, the 15Nsignature of N2OeN was significantly increased by L. rubellus(p ¼ 0.01), when residue was applied on top (Fig. 2b). With theresidues being incorporated, L. rubellus also positively affected the15N signature of N2OeN at day 45, but only if A. caliginosawas alsopresent, indicating a positive interaction.

When we calculated total N2OeN emissions as a percentage ofthe amount of N applied, it ranged from 2.5 to 4.1% when residues

recovery in earthworm tissue, with standard errors (n ¼ 5). Weight changes areA results test interactions between the two earthworm species with respect to bodynt codes refer to Table 1.

L. rub. A. cal. L. rub.

richment, at% excess 15N recovery, mg mesocosm�1

0.45 0.12 � 0.032.27 � 0.17 0.11 � 0.01

0.08 2.30 � 0.30 0.06 � 0.01 0.07 � 0.01

0.043*0.933 0.029*0.486 0.368 0.466

0.09 0.07 � 0.013.91 � 0.08 0.21 � 0.01

0.49 3.02 � 0.16 0.05 � 0.02 0.13 � 0.03

0.5290.009** 0.0650.577 0.283 0.613

Residue incorporated

0

500

1000

1500

2000

2500

0 10 20 30 40 50 60 70 80 90

liosgk

N-O

Ngµ

21-

Residue on Top

0

500

1000

1500

2000

2500

0 10 20 30 40 50 60 70 80 90

Days after start of experiment

liosgk

N-O

Ng µ

21-

No earthworms (I)A. caliginosa (I )C

L. rubellus (I )R

A. caliginosa + L. rubellus (I ) RC

No earthworms (T)A. caliginosa (T )C

L. rubellus (T )R

A. caliginosa + L. rubellus ( T )RC

a

b

Fig. 1. Cumulative N2O emissions during the incubation period: a) experiment 1: theeffect of earthworm species when residue was incorporated and b) experiment 2:the effect of earthworm species when residues were left on the surface. Error barsindicate standard errors (n ¼ 5).

Residue incorporated

0.0

0.5

1.0

1.5

2.0

0 10 20 30 40 50

ON-

Nssecxe

%motA

512

Residue on top

0.0

0.5

1.0

1.5

2.0

0 10 20 30 40 50Days after start of experiment

ON-

Nssecxe

%motA

512

L. rubellus (I )R

A. caliginosa + L. rubellus (I )RC

No earthworms (I)

A. caliginosa (I )C

No earthworms (T)

A. caliginosa (T )C

L. rubellus (T )R

A. caliginosa + L. rubellus (T )RC

a

b

Fig. 2. Atom% 15N excess for N2O daily flux on the 1st, 4th, 18th and 45th day ofthe mesocosm incubation: a) experiment 1: the effect of earthworm species whenresidue was incorporated and b) experiment 2: the effect of earthworm species whenresidue was left on the surface. Only presence of L. rubellus on the last sampling datefor the residue on top treatment showed a significant effect (p ¼ 0.01**). Error barsindicate standard errors (n ¼ 5).

G. Giannopoulos et al. / Soil Biology & Biochemistry 42 (2010) 618e625622

were incorporated, and from 0.8 to 2.6% when residue was appliedon top (Table 3).

3.3. Effect of earthworms on soil parameters

No significant changes in bulk density or air permeability due toearthworm treatments were detected (results not shown). Averagebulk density changed from 1.27 g cm�3 at the start to 1.23 at the endof the experiment. Air permeability averaged 1.93�10 m2 s�1.Similarly, NH4

þeN and NO3�eN concentrations in the bulk soil did

not change significantly due to earthworm treatments (resultsnot shown). The NO3

� concentration increased from 10.9 on day0 to 39.5 and 44.1 mg N kg�1 soil at the end of the incubationexperiment for experiment 1 and 2 respectively, averaged across alltreatments.

When residues were incorporated in the soil in the absence ofearthworms, the percentages of the aggregate size fractions were27%, 65% and 6% for the 250e8000 mm, 53e250 mm and <53 mmfractions, respectively. Earthworms did not significantly change thisdistribution, with the exception of A. caliginosa which increasedthe macroaggregate percentage to 32% (p ¼ 0.045; results notshown). No significant effect of earthworms on the size distributionof water-stable aggregates was detected when residue was appliedon top (results not shown). The percentages of the aggregate sizefractions, averaged across all earthworm treatments were 28%, 64%,and 5% for the 250e8000 mm, 53e250 mm and <53 mm fractions in

experiment 1, and 30%, 62% and 7% for experiment 2, respectively(data not shown).

Earthworm treatments significantly affected 15N dynamics inaggregate fractions for both residue treatments (Table 4). Whenresidue was incorporated, A. caliginosa significantly decreased bothatom% 15N excess in the macroaggregate fraction, and total 15Nrecovery in this fraction (p¼ 0.007 and 0.038, respectively; Table 4).When residue was applied on top, L. rubellus increased 15N excessand 15N recovery in the macroaggregate fraction (p ¼ 0.03 and0.018, respectively), and A. caliginosa increased 15N excess and 15Nrecovery in the silt and clay fraction (p ¼ 0.006 and 0.003,respectively).

4. Discussion

4.1. Earthworm interactions and N2O emissions

Our results show that both endogeic and epigeic earthwormshave the potential to substantially increase N2O emissions fromcrop residues. Their effect on N2O emissions, and the interactiveeffects of the two species, largely reflect their foraging behavior(Briones et al., 2005). Therefore, their effect is also dependent onresidue placement.

Earthwormmortality was relatively low for a 90-day incubationexperiment. The decrease in biomass by, on average, 34% whenresiduewas incorporated and 33%when residuewas applied on topis not unusual in studies where the food supply is limited (Rizhiyaet al., 2007) or absent (Speratti and Whalen, 2008). In our experi-ment the food supplywas clearly limited (2630 kg drymatter ha�1).This resulted in increased competition for food in the two-speciestreatments, since more individuals were present than in the one-species treatments. As ANOVA analyses require different factors inthe experimental setup (in this case: presence of the two earth-worm species) to be varied independently of each other, this was anessential aspect of our experimental setup (Wootton,1994; Johnson

Table 4The effect of earthworms on 15N enrichment and 15N recovery in the water-stable soil aggregate fractions at the end of the experiment. Treatment codes refer to Table 1.

Treatment Enrichment, % 15N excess Recovery, mg 15N in aggregate kg�1 bulk soil

250e8000 mm 53e250 mm < 53 mm 250e8000 mm 53e250 mm <53 mm

Experiment 1: Residue incorporatedI 0.286 0.102 0.782 1.197 0.763 0.813IC 0.212 0.103 0.587 0.948 0.704 0.795IR 0.309 0.094 0.635 1.289 0.695 0.881IRC 0.208 0.100 0.611 0.996 0.656 0.785

ANOVAL. rubellus 0.725 0.415 0.462 0.557 0.252 0.774A. caliginosa 0.007** 0.585 0.199 0.038* 0.329 0.570L. rubellus � A. caliginosa 0.627 0.653 0.306 0.852 0.841 0.699Block 0.597 0.436 0.799 0.548 0.146 0.341

Experiment 2: Residue on topT 0.116 0.038 0.399 0.426 0.250 0.543TC 0.208 0.058 0.602 0.894 0.417 0.863TR 0.316 0.060 0.365 1.236 0.452 0.433TRC 0.255 0.064 0.757 1.151 0.465 0.936

ANOVAL. rubellus 0.030* 0.294 0.509 0.018* 0.278 0.868A. caliginosa 0.768 0.345 0.006** 0.349 0.428 0.003**L. rubellus � A. caliginosa 0.155 0.537 0.308 0.184 0.498 0.415Block 0.162 0.240 0.205 0.159 0.542 0.224

Levels of significance: * < 0.05; ** < 0.01; *** < 0.001.

G. Giannopoulos et al. / Soil Biology & Biochemistry 42 (2010) 618e625 623

et al., 2009). Similar competition is likely to occur under fieldconditions in arable land where there is no continuous supply oflarge quantities of crop residues throughout the year and wheredifferent earthworm groups compete for food.

Different effects of the two earthworm species in the twoparallel experiments (residues on top vs. residues incorporated)with respect to earthworm biomass and 15N incorporation in bodytissue reflect earthworm foraging strategy. Although both specieshad access to fresh residue as a food source in both residue treat-ments (as shown by 15N incorporation in their body tissue; Table 2),L. rubellus performed best when residue was applied on top (4.4%body weight loss), and A. caliginosawhen residue was incorporated(10.9% body weight loss). These results are consistent with 15Nanalyses of earthworm body tissue, which showed highest recoveryin treatments IC and TR for A. caliginosa and L. rubellus, respectively.It should be noted that, although the guts were voided during 48 hbefore isotopic analysis of the earthworm, the presence of smallquantities of remaining soil in other parts of the body (e.g. thegizzard) may have influenced isotopic analyses. However, weconsider this a relatively small potential source of error.

With residues being incorporated into the soil A. caliginosasignificantly increased N2O emissions, whereas L. rubellus did not(Table 3 and Fig. 1a). With the residues applied on top, L. rubellusincreased N2O emissions compared to the treatment withoutearthworms, but A. caliginosa resulted in elevated N2O emissionsonly when L. rubellus was also present (Table 3 and Fig. 1b). ForL. rubellus, this pattern corroborates with other reports wheresurface-feeding earthworm species increased N2O emissions(Borken et al., 2000; Bertora et al., 2007; Rizhiya et al., 2007). To thebest of our knowledge, this is the first time that increased N2Oemissions are reported for endogeic earthworms. Our results aredifferent from the results of Speratti et al. (Speratti et al., 2007,Speratti and Whalen, 2008), who did not find a significant effect.However, these differences can be explained by the absence of anyresidue inputs as a source of C and N for N2O production. It remainsdifficult to determine whether C or N was limiting N2O emissionsduring the experiment. Soil NO3�N concentrations increasedbetween the start and the end of the experiment. However,temporary immobilization during the experiment cannot be ruled

out, especially when residue was mixed into the soil. When theresidue (which had a relatively low C:N ratio) was applied on top,the relatively low incorporation rate by earthworms compared tomanual incorporation in the other experiment is unlikely to haveresulted in N immobilization as amounts of mineral N in the soilappeared to have been high enough to support decomposition.Therefore, it is more likely that N2O production was limited byavailability of fresh organic C.

4.2. Water-stable aggregate and organic matterdistribution and 15N recovery

It has been firmly established in the literature that earthwormactivity, particularly of endogeic and anecic species, affects soilstructure (Six et al., 2004). In our experiment, changes in aggregatesize distribution due to earthworm treatments were very limited,which is in linewith Fonte et al. (2007), who reported no significanteffect of earthworms on stable aggregate size distribution in fieldmesocosms in which he used intact (uncrushed) soil as we did. Ina mesocosm experiment, Bossuyt et al. (2006) found a highlysignificant effect of A. caliginosa and L. rubellus on the formation oflarge macroaggregates (>2000 mm) at the expense of smallmacroaggregates (250e2000 mm). However, they used crushed soilmaterial (<250 mm) for their mesocosms and a less disruptivemethod for measuring aggregate stability; they used pre-wettedsoil in stead of air-dried soil as we did (the so-called slaking-method) (Beare and Bruce, 1993; Haynes, 2000).

In contrast to the amount of stable aggregate size fractions,stable isotope analysis did reveal significant differences betweenearthworm and residue treatments with respect to the incorpora-tion of freshly added residue into stable aggregate size fractions.Earthworm-induced changes were probably mostly related tochanges in aggregate turnover rates (Six et al., 1998; Fonte et al.,2007). When residues were incorporated, A. caliginosa reduced 15Nrecovery in macroaggregates, indicating that A. caliginosa increasedthe turnover of residue-derived N. When residue was applied ontop, L. rubellus and A. caliginosa significantly increased 15N recoveryin macroaggregates and in the silt and clay fraction, respectively(Table 4). This indicates that when residue is applied on top,

G. Giannopoulos et al. / Soil Biology & Biochemistry 42 (2010) 618e625624

earthworms considerably increase residue-N incorporation,possibly through a step-wise process in which A. caliginosaconsumes the excrements of L. rubellus as suggested by Bossuytet al. (2006). Although we did not distinguish between aggregateoccluded OM and free OM, we argue that earthworms incorporateingested and partly decomposed residues into their casts whichthey excrete to form soil aggregates. However, elevated CO2 emis-sions in the presence of L. rubellus or both species (Table 3) suggeststhat this species increased both incorporation of new SOM inaggregates and also net SOM mineralization. This finding suggeststhat earthworms may enhance net C mineralization in the short-term (e.g. in a 90 d incubation), an observation that is supported bythe findings of Coq et al. (2007) in a 5-month incubation experi-ment, rather than increase C stabilization as has been suggested inprevious studies by Bossuyt et al. (2006), Pulleman et al. (2005) andFonte et al. (2007). It remains to be determined whether thisincreased SOM turnover was the controlling factor for the effects ofboth residue incorporation and earthworm species on N2O emis-sions, or whether changes in the microbial community (Parkin andBerry, 1999; Drake and Horn, 2006) induced by other changes in(earthworm-mediated) soil properties dominated. Moreover, thelonger-term effects of earthworms on C mineralization remainpoorly known (Blanchart et al., 2007).

4.3. Other soil parameters

The absence of a significant effect on bulk density and airpermeability might partly be due to the fact that we sampled in thecenter of each mesocosm. During soil sampling we observed thatthe soil structure of the mesocosm appeared to be more affected byearthworm activity close to the edges of the mesocosms than in thecenter. The earthworms probably used side cracks as entry pointswhen they were colonizing the mesocosm at the start of theexperiment. The fact that the initial bulk density was already in thelower range of typical bulk densities (1.27 g m�2) for arable soils(Riley et al., 2008) might also partially explain the lack of a signifi-cant effect.

4.4. Implications for field-scale processes

Our study was designed to mimic as closely as possible condi-tions in the field after harvesting of crops. For this reason, theamount of crop residue was limited, no plants were present in themesocosms, and the soil was coarsely sieved to mimic disturbanceby soil tillage but avoid total destruction of soil structure. This isessential when extrapolating our results to the field, or comparingthem to other studies.

Earthworm densities in our mesocosms were 80, 150 and230 individuals m�2 for L. rubellus, A. caliginosa and both L. rubellusand A. caliginosa present, respectively. These earthworm densitiesare within normal ranges for Dutch soils (Didden, 2001). Further-more, A. caliginosa and L. rubellus represent the dominant speciesfor their respective functional group in Dutch soils (Didden, 2001;van Vliet et al., 2007; Rutgers et al., 2008). Therefore, although weshould be careful when extrapolating the results of one species toall species of their ecological group, we consider our experimentalsetup to reflect relatively close the processes actually occurringin the field.

Although to some extent speculative, we think that our resultsmay shed some light on differences between conventional andzero-tillage systems. It has been reported in the literature that zero-tillage systems have a more diverse and larger population ofearthworms, mainly due to the presence of epigeic species (Houseand Parmelee, 1985; Hendrix et al., 1986). If we associate theresidue incorporated treatments without earthworms or with

endogeic species only (I and IC) to a conventional tillage system andthe residue applied on top treatment with a diverse population(TRC) to a no-tillage system, N2O emissions are almost equal forI and TRC (1350.1 and 1396 mg NeN2O kg�1, respectively) and muchhigher for IC (2223 mg NeN2O kg�1). This would suggest higher orequal absolute N2O emissions in tilled systems than in no-tillsystems. Under the stipulation that this theoretical comparisondoes not take into account previously reported differences betweenzero-tillage and conventional tillage systems in soil moisturestorage and organic matter distribution. These may affect N2Oemissions in ways not tested in our setup.

In order to fully understand the intricate interactions betweendifferent functional earthworm groups, a future study includingcombinations of epigeic, endogeic and anecic earthworm specieswould be especially interesting. These studies should focus in moredetail on N and C dynamics during incubation, as well as to changesin the microbial community.

5. Conclusions

We conclude (i) that endogeic earthworm species can signifi-cantly increase N2O emissions, but only when residue is incorpo-rated in the soil; (ii) that this incorporation can be done by bothhuman intervention (tillage) and by epigeic earthworm species;and (iii) that the earthworm effect on N2O emissions appears to belinked with changes in soil OM distribution and incorporation. Itremains to be determined whether the effect of earthworms isultimately controlled by C and N availability, by changes in the soilmicrobial community, or by both. Our results contribute to theunderstanding the important but intricate relations between(functional) biodiversity and the greenhouse gas balance of the soil.

Acknowledgments

This study was supported by a personal VIDI grant from theNetherlands Organization for Scientific Research/Earth and LifeSciences (NWO-ALW) to Jan Willem van Groenigen. Additionalfunding was supplied by the Dutch Ministry of Agriculture, NatureConservation and Food Quality (project number KB-02-001-068).We would like to thank Eduard Hummelink, Gerlinde Vink, IngridLubbers, Jaap Nelemans, Willem Menkveld, Tamás Salanki, EefVelthorst, Willem Hoogmoed, and Annemariet van der Hout fortheir assistance, Bram van Putten for his advice on statistics andRon de Goede for his expert advise on earthworms. Finally weacknowledge Lijbert Brussaard for his comments on a previousversion of this paper.

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