Technosol composition affects Lumbricus terrestris surface cast composition and production

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Ecological Engineering 67 (2014) 238–247

Contents lists available at ScienceDirect

Ecological Engineering

journa l h om epa ge: www.elsev ier .com/ locate /eco leng

echnosol composition affects Lumbricus terrestris surface castomposition and production

enjamin Peya,b,∗, Jérôme Corteta,b,c, Yvan Capowiezd, Johanne Nahmanie,ranc oise Watteaua,b,f, Christophe Schwartza,b

Université de Lorraine, Laboratoire Sols et Environnement, UMR 1120 BP 172, F-54505 Vandœuvre-lès-Nancy Cedex, FranceINRA, Laboratoire Sols et Environnement, UMR 1120 BP 172, F-54505 Vandœuvre-lès-Nancy Cedex, FranceUniversité Paul Valéry Montpellier III, Centre d’Ecologie Fonctionnelle et évolutive, UMR 5175 CEFE, route de Mende, F-34199 Montpellier Cedex 5, FranceINRA, UR 1115 “Plantes et Systèmes Horticoles”, Site Agroparc, F-84914 Avignon Cedex 09, FranceCNRS, Center of Functional Ecology and Evolution, 1919, route de Mende, F-34293 Montpellier Cedex 5, FranceCNRS, UMS 3562, F-54501 Vandœuvre-lès-Nancy Cedex, France

r t i c l e i n f o

rticle history:eceived 24 June 2013eceived in revised form 27 January 2014ccepted 29 March 2014

eywords:nthropogenic soilcological reclamationpi-anecic earthwormoil functioningoil constructionoil engineering

a b s t r a c t

Constructed Technosols deliberately combine technogenic materials to obtain specific services (e.g. plantbiomass production) in particular contexts (e.g. industrial wasteland reclamation). Their ecological recla-mation by key members of the soil fauna, such as earthworms, is a promising way to ensure such services.However, literature which treats these animals as a biological agent of constructed Technosol function-ing is very scarce. This work assesses the effects of the composition of a constructed Technosol (i.e. theproportions of the constituent materials) on earthworm (Lumbricus terrestris) surface cast productionand composition, and to a lesser extent on earthworm survival and biomass. Earthworms were placed inlaboratory microcosms made of different proportions of materials: green-waste compost (GWC) and ther-mally treated industrial soil mixed with paper mill sludge (TIS/PS), for 30 days. We found that 25% of GWCon the surface and 75% of TIS/PS below was the most beneficial composition in that it gave the highestbody mass gain. Furthermore, this composition generated a moderate surface cast production comparedto other compositions and an increase in microorganism activity and number compared to non-ingestedby earthworm soil. This composition also led to casts with bacteria of diverse morphologies and largest

microaggregates compared to compositions without GWC, resulting from the ingestion and mixing of thetwo materials in the earthworm gut. However, earthworms did not modify the carbon contents of surfacecasts. This laboratory approach was a necessary step before field assessment of ecological reclamation byearthworms. From a soil engineering point of view, this contributes to better-managed soil constructionfor ecological reclamation.

© 2014 Elsevier B.V. All rights reserved.

a

. Introduction

Urban and industrial activities create anthropogenic soils orechnosols. Their properties and pedogenesis are dominated by

Abbreviations: GISFI, French scientific group concerned with industrial waste-and; GWC, green waste compost; PS, paper mill sludge; TIS/PS, treated industrialoil with paper mill sludge; TIS, treated industrial soil; WHC, water holding capacity.∗ Corresponding author. Present address: INRA, UR 251 PESSAC, RD 10, F-78026ersailles Cedex, France. Tel.: +33 01 30 83 32 72; fax: +33 01 30 83 32 59.

E-mail addresses: [email protected] (B. Pey),[email protected] (J. Cortet), [email protected] (Y. Capowiez),[email protected] (J. Nahmani), [email protected]. Watteau), [email protected] (C. Schwartz).

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ttp://dx.doi.org/10.1016/j.ecoleng.2014.03.039925-8574/© 2014 Elsevier B.V. All rights reserved.

rtificial or transported materials which are called technogenicaterials (IUSS Working Group WRB, 2006). Soil construction com-

ines deliberately selected technogenic materials to form new soilorizons (Séré et al., 2008). Resulting constructed Technosols areesigned to fulfil chosen soil functions to obtain specific servicese.g. revegetation, plant biomass production) in a particular con-ext such as soil reclamation (e.g. industrial wasteland) (Séré et al.,010).

Soil reclamation traditionally focuses on vegetation establish-ent, as being both an agent and a final product of ecological

eclamation (Boyer and Wratten, 2010; Bradshaw, 2000). In spitef its acknowledged beneficial effects on “natural” soil function-ng (Kibblewhite et al., 2008), soil fauna was often overlooked inegraded soil reclamation (Boyer and Wratten, 2010; Frouz et al.,

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Table 1Main properties of the technogenic materials. GWC, green waste compost; TIS,treated industrial soil; PS, paper mill sludge.

Details Unit Measure GWC TIS/PS

– – Bulk density 0.45 0.86

– % Water holding capacity (WHC) 135.4 89.6

– g/kg Total nitrogen 10.6 2.23– – C/N ratio 16.6 38.2– g/kg Total carbon 234 134– g/kg Organic carbon 176 85.2– g/kg Organic matter 304 147

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007b; Snyder and Hendrix, 2008). Of this fauna, large animalsuch as earthworms have been identified as being good candidatesor such purposes (Boyer and Wratten, 2010; Bradshaw, 2000;utt, 2010; Haimi, 2000; Snyder and Hendrix, 2008). Indeed, thereation of burrows and the deposition of casts by earthwormsave been demonstrated to improve degraded soil functioning, for

nstance in post-mining soils. Earthworms contribute to litter dis-ppearance (Frouz et al., 2007a, 2001) and soil mixing (Frouz, 2008;rouz et al., 2008; Rutherford and Arocena, 2012). They also redis-ribute organic matter in the soil profile (Baker et al., 2006; Frouzt al., 2007a; Scullion and Malik, 2000). Aggregation stability wasromoted when earthworms were present (Marashi and Scullion,003; Scullion and Malik, 2000). Finally, earthworms influenceicrobial biomass and activity (Frouz et al., 2011; Scullion andalik, 2000). Localization (near the surface or at depth) of these

ffects depends very much on the context (for instance for car-on, see Frouz et al., 2007a). All of these changes can benefit plantsCheng and Wong, 2008; Roubickova et al., 2009). However earth-orms can sometimes hinder post-mining soil reclamation. For

nstance, soon after earthworm inoculation, the soil surface canecome waterlogged as a result of earthworm burrowing (Marashind Scullion, 2004).

To our knowledge only one study has so far explicitly taken intoccount effects of soil fauna on constructed Technosol function-ng. This study was based on the introduction of earthworms ofiverse species and observed an increase in cast production in soilsade of materials from a landfill and paper mill sludge (Piearce

t al., 2003). Furthermore, the success of earthworms in reclaim-ng degraded areas is strongly linked to successful colonizationnd/or introduction (Boyer and Wratten, 2010; Eijsackers, 2010,011; Haimi, 2000). Again, the literature on this topic for con-tructed Technosols is scarce. In several types of soil constructioncrushed bark, wastewater sludges, sand and/or mull), soil faunaommunity compositions were similar to those frequently found inoils or situations characterized by high contents of decomposablerganic matter and cattle manure, and very different from thosef arable soils. Indeed they were made up of macroarthropods (e.g.hilopods, beetles), some earthworms of all eco-morphological cat-gories and mesofauna (e.g. springtails of all eco-morphologicalategories) (Huhta et al., 1979). In some other constructed Tech-osols made of soil from a landfill and paper mill sludge, some

noculated earthworms survived (Piearce et al., 2003).We hypothesized that the ecological reclamation success by

arthworms of a given constructed Technosol must depend on itsomposition. In this preliminary work, only the proportions of theonstituent materials varied. The scientific aim of the present workas thus to assess the effects of various Technosol compositions

n (i) earthworm casting activity as a proxy of earthworm influ-nces on soil functioning and to a lesser extent on (ii) earthwormurvival and biomass. Experiments were carried out with the earth-orm Lumbricus terrestris Linnaeus 1758 in various compositions

f constructed Technosols under laboratory conditions.

. Materials and methods

.1. Preparation of materials

The materials used in this experiment were collected from ann situ constructed Technosol. It is a Spolic Garbic Hydric Tech-osol (Calcaric) (IUSS Working Group WRB, 2006; Séré et al., 2010)

he main idea of this in situ soil was to design a new soil pro-le to ensure some chosen major soil functions (e.g. vegetationevelopment) (Séré et al., 2008) by recycling materials consid-red as waste. Green-waste compost (GWC) and a mixture of

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Water pH – pH 8.41 8.25– g/kg Total carbonates (CaCO3) 124 402– g/kg Active carbonates (CaCO3) 44.4 89.3

aper mill sludge and thermally treated industrial soil (TIS/PS,:1 volumetric ratio) were collected before the in situ soil con-truction in September 2007 from the experimental site of therench Scientific Interest Group for Industrial Wasteland (GISFI)t Homécourt (north-eastern France, http://www.gisfi.fr). Thermalreatment consisted of boiling the soil at very high temperaturesfter excavation to extract the organic pollutants. They were driednd sieved to two millimetres to reduce their heterogeneity. Whenieving at 2 mm, hard pieces of TIS/PS >2 mm were crushed by handf possible. Some of these hard pieces were pieces of PS coated withIS powder. These pieces could be entirely crushed by hand. Somethers were TIS stones coated with PS. In that case, only the PS partsould be removed from stones and crushed. GWC was gently sievedwithout pushing the material through the sieve) to limit mechan-cal fragmentation. The main agronomic properties of these sievedechnogenic materials are presented in Table 1. They were thenefaunated by alternate drying and freezing (Krogh, 1995). Materi-ls were not moistened during the defaunation process. This wouldave meant manipulating water-soaked materials several timesue to their high water retention which would have led to techni-al problems (Pey et al., 2013). The contamination of the materialsill not be considered as the main driver explaining the earthworm

ffects and responses. Indeed, the values of contaminants of initialaterials are in the range of, or close to some reference values of

natural” soils geographically close to the area, where the industrialoil was sampled before being desorbed and used in the Technosolonstruction (Darmendrail, 2000) (Appendix).

.2. Microcosm management

The materials were introduced into 0.0018 m3 microcosms (PVCylinders of 10 cm height and 15 cm diameter). The size of theicrocosms was based on constraints coming from the in situ

onstructed Technosol. Even though the theoretical profile of then situ constructed Technosol is deep (1 m 50), the whole profiles usable with difficulty by earthworms. Apart from the surfaceompost layer, the rest of the profile made of TIS and PS mayither (i) be compacted or (ii) contain stones. The small heightf the microcosms was chosen to mimic these constraints in theaboratory. Treatments were designed to vary the proportions ofhe constituent materials of the Technosol. Five treatments (% ofhe total volume) were prepared: (i) 100%GWC (100GWC), (ii)5%GWC–25%TIS/PS (75GWC), (iii) 50%GWC–50%TIS/PS (50GWC),iv) 25%GWC–75%TIS/PS (25GWC) and (v) 100%TIS/PS (100TIS/PS).he materials were moistened to 35% of their water holding capac-ty (WHC). The hydrophobicity of materials led us to moisten them

lightly more than the literature recommendations (Lowe and Butt,005) and to do so before layering them in the microcosms. Indeed,revious attempts to moisten them when they were placed in theicrocosm failed. The GWC was always placed on the top of the

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IS/PS to form two layers. The microcosms were finally covered by 1-mm mesh for ventilation purposes and to prevent earthwormsrom escaping. A total of 25 microcosms were set up, correspond-ng to five treatments, five replicates and one sampling date (30ays after the microcosm preparation and earthworm inoculation).he PVC cylinders were initially weighed and stored at 18 ± 2 ◦CLowe and Butt, 2005) in the dark. After animal addition, water wasprayed onto the topsoil every week to compensate for water massvaporation (water was sprayed until the microcosms reachedheir initial weight, i.e. materials + earthworms + empty PVC micro-osm).

.3. Earthworms and casts

L. terrestris was chosen as a representative species of epi-aneciccosystem engineers, which are able to modify soil structure byurrowing sub-vertical galleries, feed on particulate organic matternd create stable organo-mineral aggregates (Brown et al., 2000).uch earthworms should be capable of initiating the mixing of theayers of the Technosol. They could also play a crucial role in theransformation of the surface GWC layer as they possess somepigeic behaviours. Furthermore, L. terrestris is known as a slowolonizer of virgin lands such as reclaimed mine soils or poldersBaker et al., 2006; Marinissen and Van den Bosch, 1992). Hencesing L. terrestris also helps to justify the inoculation of epi-anecicarthworms in such constructed Technosols. Finally, it is a veryommon large species used in laboratory experiments (Lowe andutt, 2005).

L. terrestris were bought from a biological supply company. Fourndividual earthworms (L. terrestris) were introduced into each

icrocosm, giving a density of 225 ind/m2, which was within theange of temperate pasture densities (Fründ et al., 2010). This den-ity was chosen since the in situ Technosol was sown with Lolium., Dactylis L. and Festuca L. to mimic a pasture. Earthworms wereashed in tap water, gently dried on paper and weighed (with-

ut gut voiding) at the beginning and end of the experiment. Allhe treatments of a given replicate had a comparable introducedarthworm fresh biomass (an average of 14.00 g with a standardeviation of 8.86%).

After 30 days, the number of living earthworms per microcosmas counted for four of the five replicates. Total live earthworm

iomass was measured. Indeed, weight variation can be consid-red as an indicator of general health of earthworms (Capowiezt al., 2005). Surface casts were sampled, dried and weighed, andetrieved for carbon analysis. For two of the treatments (100TIS/PSnd 75GWC) of the fifth replicate, a few surface casts and a fewilligrams of GWC and TIT/PS non-ingested by earthworms were

etrieved for ultrastructural analysis. We limited ultrastructuralnalysis to two contrasting treatments: 100TIS/PS and 75GWC ashere were not enough casts to carry out such an analysis for the00GWC treatment. Only surface casts were sampled since (i) L.errestris is known to cast mainly at the soil surface (Capowiezt al., 2010; Zicsi et al., 2011) and (ii) it is difficult to recognizeelow-ground casts.

.4. Analyses

Surface casts were weighed to calculate their dry matter after8 h at 50 ◦C. The total carbon content of surface casts was deter-ined using a CN analyser (Vario Micro Cube Elementar) from the

omogenously crushed and mixed surface casts of a given replicate

f a treatment (approximately between 8 and 10 mg).

For each of the treatments 100TIS/PS and 75GWC, five replicatesf 2–5 mm3 of surface casts, non-ingested GWC and non-ingestedIS/PS materials were preserved. All of these samples (five

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eplicates × two treatments × three material types: surface cast,WC, TIS/PS) were chemically fixed in osmium tetroxide, thenhysically preserved by a method that retains the structurePey et al., 2013; Villemin and Toutain, 1987). Each of them wasehydrated in graded acetone series and embedded in epoxy resinEpon 812) until complete polymerization. At least 3 ultra-thinections per replicate (80–100 nm) from at least 3 of the 5 repli-ates were obtained using a diamond knife on a Leica Ultracut Sltramicrotome.

In addition, for each treatment (100TIS/PS and 75GWC), oneram of the three material types (surface cast, GWC, TIS/PS) werelso used to provide some granulometric fractions. Each gramas shaken in 10 ml of deionised water for 5 min. After decant-

ng, the floating fraction and the heavier fraction were separated.ive replicates of both fractions were conditioned according tohe same protocol as described above (five replicates × two treat-

ents × three material types × two fractions: floating and heavier).All the ultra-thin sections were then stained with uranyl

cetate and lead citrate (Pey et al., 2013). Ultra-thin sections werexamined with a JEOL EMXII transmission electron microscopeperating at 80 kV. A description and observation frequency ofach type of organic, mineral or organo-mineral microstructuresere recorded (Watteau et al., 2006). A morphological descrip-

ion and observation frequency of micro-organisms were alsoecorded as well as their physiological state (alive, dead, spores),nd their enzymatic activity through the observation of organicatter lysis area. Similarity level between treatments was speci-

ed by comparing observation frequency of such elements. Manybservations of ultra-thin sections of a treatment were made so aso be able to detect a significant difference between treatments.nly few representative features of observed microstructures andicroorganisms were selected and presented as examples in the

aper.

.5. Statistical analyses

The survival rate was expressed as the ratio (%) of the final num-ers of living earthworms to that initially introduced into eachicrocosm. Biomass variation was expressed as the ratio (%) of theean final biomass of living individual earthworms to the mean ini-

ial biomass of individuals. Surface cast production was expresseds a volume, by dividing dry weights of surface casts of a treatmenty the theoretical bulk density of its initial constructed Technosol.heoretical bulk densities of the initial Technosol of a treatmentere calculated for each treatment as the sum of the products of

he proportions of each material (% of GWC and/or TIS/PS) and theirorresponding bulk density (dried and 2 mm sieved bulk densitiesf initial GWC and TIS/PS, Table 1). Carbon content variation of castsn each treatment was calculated by dividing carbon contents ofurface casts of a treatment by the theoretical carbon content of itsnitial constructed Technosol. This was done in a similar way, butsing the theoretical carbon contents of the initial Technosols (cal-ulated from dried and 2 mm sieved carbon contents of initial GWCnd TIS/PS, Table 1). For carbon content variations only, the valuef the 100TIS/PS treatment of the fourth replicate was aberrant andas therefore discarded. Aberrant means that the value is twice theighest value of the 100GWC treatment. Unfortunately we did notnow if it was due to a technical error or a local accumulation ofarbon (for instance, anthropic carbon). Differences between treat-ents for L. terrestris survival rates, biomass variations, surface cast

roductions and carbon content variations were tested with theruskal–Wallis tests (p < 0.05), followed by a non-parametric postoc test (“nparcomp” package in R) (R Development Core Team,010).

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Table 2Survival of Lumbricus terrestris after 30 days under the different treatments of theconstructed Technosol (Kruskal–Wallis test followed by a post hoc). GWC, greenwaste compost; TIS, treated industrial soil; PS, paper mill sludge.

Measure (unit) p-Value Treatments Means(standarderrors)

Survival rate (%) p = 0.08

100GWC 38 (±16)75GWC 75 (±14)50GWC 75 (±25)

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Table 3Surface cast production and carbon content variation of Lumbricus terrestris after 30days under the different treatments of the constructed Technosol (Kruskal–Wallistest followed by a post hoc). GWC, green waste compost; TIS, treated industrial soil;PS, paper mill sludge. Different letters indicate statistical differences.

Measure (unit) p-Value Treatments(statisticalgroups)

Means(standarderrors)

Surface cast production(g)

p = 0.007

100GWC (c) 0.9 (±0.5)75GWC (c) 2.3 (±0.7)50GWC (bc) 7.9 (±5.6)25GWC (b) 11.5 (±2.9)100TIS/PS (a) 45.7 (±8.9)

Carbon contentp = 0.09

100GWC 0 (±4)75GWC −12 (±16)50GWC −11 (±4)

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25GWC 100 (±0)100TIS/PS 88 (±7)

. Results and discussion

.1. Earthworm survival and biomass

Survival rates did not differ between treatments after 30 daysTable 2). They presented wide variations within a given treat-

ent, which rendered any interpretation dubious. This was partlyecause our experiment was not designed to test the effectsf Technosol composition on earthworm survival but rather theffects of earthworms on the Technosol, depending on the Tech-osol composition. To be able to draw conclusions on earthwormurvival, more than 4 earthworms by replicate should have beensed (Spurgeon et al., 2000).

Biomass variation was affected by the proportions of materials.iomass variations of the 25GWC treatment were statistically dif-

erent from treatments with high GWC contents (100% and 75%,ig. 1). The 25GWC treatment was the only one to show positiveiomass variations. These results were consistent with the liter-ture dealing with applying organic compost to mineral soils inhich earthworm biomass and density increased (Emmerling and

aulsch, 2001; Lapied et al., 2009). The pure GWC treatment pre-ented the greatest biomass loss, greater than on the 25GWC (nooss), 75GWC and 100TIS/PS treatments. Finally the intermediate

eight loss of the 50GWC was not different from the others (Fig. 1).In controlled abiotic laboratory experimental conditions, the

ood source strongly influences both consumption rates and castroduction of earthworms (Curry and Schmidt, 2007; Flegel et al.,

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ig. 1. Body mass variation of Lumbricus terrestris after 30 days under the differentreatments of the constructed Technosol (Kruskal–Wallis test followed by a postoc, p = 0.02). GWC, green waste compost; TIS, treated industrial soil; PS, paper millludge. Different letters indicate statistical differences.

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variation (%)25GWC +5 (±2)100TIS/PS +13 (±4)

998). It was demonstrated that L. terrestris preferred litter (putn the soil surface) with the lowest C/N ratio (Hendriksen, 1990;chönholzer et al., 1998). Furthermore, L. terrestris were often foundn soils with a low C/N ratio (mean = 11.4) (Bouché, 1972). The high/N ratio of the TIS/PS (Table 1) could explain the slight body mass

oss of the 100TIS/PS treatment. Yet the literature shows that theffects of pure paper mill sludge (Butt, 1993), alone or in mix-ures with mineral soil (Piearce et al., 2003), were beneficial to theiomass of the earthworm L. terrestris. In our experiment, mixingqual volumes of paper mill sludge with the treated industrial soilTIS) did not provide such biomass benefits for earthworms.

If earthworm biomass was only driven by the C/N ratio, the00GWC treatment should have led to less body mass losses than

n other treatments, as GWC was the material with the lowest C/Natio. Our assumptions were that (i) the GWC contained distastefulompounds such as phenolic materials (Curry and Schmidt, 2007)nd/or (ii) the GWC may not have had enough available water for L.errestris. Indeed, the literature indicates that earthworm biomassKretzschmar and Bruchou, 1991) and activities (burrowing andasting) are influenced by water availability and hence soil waterontent (Capowiez et al., 2009; Hindell et al., 1994; Kretzschmar,991). Our assumptions were confirmed by direct observationsf treatments with two materials, revealing that burrows in theWC part were coated with TIS/PS, whereas the burrows in theIS/PS were not coated with GWC. So, by displacing TIS/PS mate-ial, earthworms could reduce their direct exposure to potentiallyistasteful GWC compounds and/or could reduce adverse effectsf the low water availability in GWC. Indeed TIS/PS potentially hadore available water according to its WHC (Table 1). Since it was

ot significantly different from the other treatments because ofide variation, we assumed that the 50GWC treatment behaved

ike 75GWC.

.2. Earthworm casting activity

.2.1. Surface cast production and compositionSurface cast production and composition were affected by the

roportions of materials. Increasing proportions of GWC led to aignificant decrease in cast production (Table 3). This decreasedurface cast production could be explained by the distastefulnessnd/or dryness of the GWC.

The main results of the ultrastructural analysis were that aigher number of live bacteria and microaggregates were observed

n casts of the 75GWC and the 100TIS/PS treatments than in theulk materials (non-ingested GWC and TIS/PS). The literature indi-ates that 0–2 �m microaggregates are considered to be the mosteactive soil fractions due to their high organic carbon content,

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242 B. Pey et al. / Ecological Engineering 67 (2014) 238–247

Fig. 2. Micrographs of transmission electronic microscope of the GWC bulk material of the constructed Technosol. (a) Plant residue: degraded cell walls and polyphenolc n planb ; m, m

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ompounds; (b) ligneous tissue: degraded and non-degraded cell walls; (c) spores iacteria; br, bacterial residue; cw, cell wall; dc, degraded cell wall; ex, exopolymer

he resistance of organic matter to biodegradation and their largedsorption capacity, and that they thereby play a significant rolen soil structure (Six et al., 2004; Watteau et al., 2006). Further-

ore, these microaggregates are widely recognized as being aavourable habitat for bacteria (Ranjart and Richaume, 2001). Oth-rwise, microbial activity and diversity, as well as the size of theicroaggregates of the surface casts, were larger in the presence ofWC than for casts from pure TIS/PS treatment. Finally, when bothaterials were present, they were ingested.In full details, ultra-thin sections of the GWC bulk material

evealed a wide diversity of fragmented plant tissues with differ-ng degrees of decomposition (Fig. 2). They could be identified asissue fragments (Fig. 2a), cellular fragments (Fig. 2b and c), or asacked fine organic matter (Fig. 2d). The observed organic mat-er was of different biochemical constitution, i.e. pecto-cellulosiccell walls in Fig. 2b and c), ligneous (Fig. 2a) or polypheno-

ic (Fig. 2b–d). Tissues could have been partially degraded andresented a former bacterial activity (Fig. 2a, b and d), or werendergoing bacterial degradation (Fig. 2b). Spores (Fig. 2c) andacterial residues (Fig. 2b and d) were indicators of progressively

ssob

t cell residue; and (d) fine organic matter coming from plant tissue degradation. b,ineral; oc, organic compound; pc, polyphenol compound; s, spore.

ecreasing bacterial activity in the compost. A few minerals werelso detected as originating in the initial constituents of the GWCaterial (Fig. 2c).Ultra-thin sections revealed that TIS/PS bulk material was made

f minerals essentially originating in TIS (Fig. 3a–c) and cellulosicbres (Fig. 3c) originating in PS. Minerals were free (Fig. 3c) orssociated in micro-aggregates (Fig. 3a and b). However, individ-al cellulosic fibres did not participate in this aggregation (Fig. 3c).ew bacteria were present (Fig. 3a) and there was no indication oformer bacterial activity.

In the 100TIS/PS treatment, thin sections of surface casts pre-ented some similar aspects to the TIS/PS bulk material. Indeed, freeellulosic fibres (Fig. 4a), free minerals (Fig. 4b and c) and organo-ineral associations (Fig. 4b and d) were present. Nevertheless in

ome aggregates, some cellulosic fibres participated in aggregationFig. 4c), unlike in TIS/PS bulk material. More free bacteria were

een in the casts than in the bulk material (Fig. 4a), some still pre-enting cellulolytic activity (Fig. 4b). Bacterial colonies were alsobserved, some of which formed bacterial aggregates, as mineralsecame associated on bacterial exopolymers (Fig. 4d).

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B. Pey et al. / Ecological Engineering 67 (2014) 238–247 243

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ig. 3. Micrographs of transmission electronic microscope of the TIS/PS bulk materiineral aggregate; and (c) cellulosic fibre. b, bacteria; cf, cellulosic fibre; h, hole; m

Thin sections of casts of the 75GWC treatment contained ele-ents of both bulk materials. Minerals (Fig. 5a) and cellulosic fibres

Fig. 5b) indicated the ingestion of TIS/PS by earthworms, whilelant residues indicated (Fig. 5c and d) the ingestion of GWC. Liveacteria were degrading fibres (Fig. 5b) or plant cells (Fig. 5c and d).acteria were sometimes present as isolated colonies (Fig. 5e) or

oncentrated near plant cell intersections, where various bacteriaegraded the middle lamella (Fig. 5f). Furthermore, some bacte-ia displaying some of the morphological features characteristicf those involved in the nitrogen cycle (Nitrobacter) (Josserand

c

pa

he constructed Technosol. (a) Organo-mineral aggregate with bacteria; (b) organo-ral. TIS, treated industrial soil; PS, paper mill sludge.

nd Cleyet-Marel, 1979) (Fig. 5g), were gathered in large bacte-ial aggregates, where TIS minerals were adsorbed on bacterialxopolymers (Fig. 5g).

.2.2. Relating cast composition and production to surface cast

arbon content, soil stability and soil mixing

As literature on constructed Technosols is very scarce, we com-ared our results as far as possible with those of “natural” soilsnd post-mining soils. Post-mining soils are clearly constructed

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244 B. Pey et al. / Ecological Engineering 67 (2014) 238–247

Fig. 4. Micrographs of transmission electronic microscope of casts of Lumbricus terrestris (100TIS/PS treatment) of the constructed Technosol. (a) Mineral/cellulosic fibreaggregate; (b) organo-mineral associations; (c) detail of mineral association with cellulosic fibre; (d) organo-mineral associations with exopolymers. b, bacteria; cf, cellulosicfi iner

fomosemt2nednotn

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bre; cr, cellular residue; df, degraded cellulosic fibre; ex, exopolymer; h, hole; m, m

rom different materials but are newly created Technosols madef technogenic materials as constructed Technosols. Concerningicro-organism observations, our results appear similar to those

bserved in “natural” and post-mining soils. Indeed, in “naturaloils”, a microbial activation is observed in the presence of anecicarthworms at the ultrastructural scale (Rafidison, 1982). In post-ining soils, microbial biomass and respiration are influenced by

he presence of earthworms (Frouz et al., 2007a; Scullion and Malik,000). Furthermore, microbial biomass, respiration and bacteriaumber were higher in casts than in bulk soil aggregates (Frouzt al., 2011). Our results obtained from ultrastructural analyses,emonstrating that earthworms affect microorganism activity andumber, are thus consistent with literature findings for other kindsf soils. Furthermore, our work demonstrated that earthworms inhe presence of GWC led to more diverse microorganism commu-ities.

Otherwise, the observed microbial changes in our experiment

ould have led to carbon changes in casts, as has been demonstratedn other kinds of soils. Indeed, (i) in “natural” soils, biomass of bac-erial communities changes can affect the overall mineralization of

pe

al. TIS: treated industrial soil; PS: paper mill sludge.

oil organic matter (Postma-Blaauwa et al., 2006) and soil carbonontent and (ii) in post-mining soils, earthworm presence mod-fied carbon content at the cast scale (Frouz et al., 2011) and athe soil profile scale (Frouz et al., 2009; Scullion and Malik, 2000).his was not the case in our experiment as the surface cast rela-ive carbon variations were non-existent and consequently did noteveal any differences between treatments (Table 3). One expla-ation could be that the effects of earthworms on carbon coulde masked by the very rich carbon content of the Technosol ini-ial materials (Pey et al., 2013). Although the statistical test wasot significant, the surface casts of treatments with high contentsf TIS/PS seemed to be slightly enriched, whereas treatments withigh contents of GWC (except for pure GWC, whose carbon contentid not vary) seemed to be impoverished. Clearer effects might beetected much later, especially if the soil properties evolve withther simultaneous (physical/chemical or biological) processes orf earthworm biomass increases.

In addition, earthworms are known to promote soil mixing inost-mining soils, which eventually promotes soil formation (Frouzt al., 2001, 2008). That was also the case in our study, as in the

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B. Pey et al. / Ecological Engineering 67 (2014) 238–247 245

Fig. 5. Micrographs of transmission electronic microscope of casts of Lumbricus terrestris (75GWC treatment) of the constructed Technosol. (a) Minerals; (b) degradedc bacterb dc, degm

cptiaTc

e2

ellulosic fibre; (c) fine organic matter after degradation; (d) degraded tissues; (e)

acteria; br, bacterial residue; cf, cellulosic fibre; cr, cellular residue; cw, cell wall;

l, middle lamella; oc, organic compound; s, spore. GWC, green waste compost.

ase of treatments with both materials, these two materials wereresent in a same cast as organo-mineral associations. Earthwormshus promoted the Technosol micro-mixing. In addition, our exper-

ment demonstrated that surface cast production was significantnd increased with TIS/PS content. Thus, earthworms affected theechnosol macro-mixing, with intensity depending on Technosolomposition.

mged

ial colony; (f) bacterial diversity in middle lamella; and (g) bacterial aggregate. b,raded cell wall; df, degraded cellulosic fibre; ex, exopolymer; h, hole; m, mineral;

Finally, soil stability has been shown to be higher in the pres-nce of earthworms in post-mining soils (Marashi and Scullion,003; Scullion and Malik, 2000). In our study, casts contained

ore microaggregates than bulk soils. Furthermore, microaggre-

ates were larger in the presence of GWC. This suggested thatarthworms may promote soil stability in constructed Technosols,epending on the materials present.

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46 B. Pey et al. / Ecological E

.3. What would be the best composition on a field scale?

The results of our laboratory experiment lead us to believe that5% of green-waste compost on the surface and 75% of a mix-ure of treated industrial soil and papermill sludge below is the

ost favourable composition for constructed Technosol ecologicaleclamation by earthworm. Indeed, (i) it allows the earthwormsigher body mass gain compared to other compositions. Further-ore, this composition generated (ii) a moderate surface cast

roduction compared to other compositions and (iii) an increasen microorganism activity and number compared to non-ingestedy earthworm soil. By analogy with the 75GWC composition, thisomposition also led to casts with (iv) bacteria of diverse mor-hologies and largest microaggregates compared to compositionsithout GWC.

In spite of the limitations of our experiment (e.g. one samplingime with five replicates, dryness of the compost), we considerhat such a composition could allow L. terrestris to beneficiallyffect Technosol functioning on the field scale, such as by later car-on content changes, soil stability improvement and initiation ofoil formation. Such assertions are valid in the case of Technosolsonstructed from materials rich in organic matter and when anpi-anecic earthworm is used. In addition, the constructed Tech-osol in situ contains approximately 50% of stones which were not

ncluded in our study. They can impede earthworm survival andiomass. Another crucial non-considered parameter in our studyas the presence of surface litter and plants. But work on post-ining soils led us to think that their presence did not impede

oil reclamation in field conditions but enhances soil formationFrouz et al., 2008; Roubickova et al., 2009). Otherwise, no bioticnteractions with other soil fauna were considered in our study.

e employed an ecosystem engineer able to create new habitatshich will certainly favour establishment by other animals. Devel-

ping knowledge about effects of soil fauna diversity in constructedechnosols is required. Lastly, collateral damage could occur whenerforming ecological reclamation with earthworms (Marashi andcullion, 2004). For all these reasons, from an applied point of view,e recommend inoculating earthworms in small areas as a first

tep before attempting extensive inoculation.Our laboratory work was a necessary step before field assess-

ent of ecological reclamation by earthworms. From a soilngineering point of view, it contributes to better management ofoil construction for ecological reclamation, by orienting soil com-osition prior to construction, as well as managing constructedechnosols after construction (e.g. fauna introduction, suitablegricultural practices).

cknowledgments

This work was funded by the ADEME (Agence de’Environnement et de la Maîtrise de l’Energie) and the Régionorraine. We thank the staff of the laboratory (especially Stéphaneolin) for their technical contribution. We would also like to thankr. Daniel Cluzeau for fruitful discussions concerning the use ofumbricus terrestris.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.ecoleng.014.03.039.

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