Synthesis of PHYTENER programme · integrated into works of a critical, pedagogical or informational nature, subject to compliance with the stipulations of articles L 122-10 – L

Synthesis of PHYTENER programme

PHYTOSTABILISATION DEVELOPMENT ON METAL-CONTAMINATED SOILS

TO PRODUCE ENERGY: ECOLOGICAL VIABILITY, SOCIAL ADVANTAGES

AND ECONOMIC ASSESSMENT

March 2015

Study realised for the ADEME by: ISA Lille

Contract number: 0972C0052

Technical coordination: Frédérique Cadière, Bastien Collet Direction\Service : Service Friches Urbaines et Sites Pollués, ADEME Angers

SYNTHESIS

Synthesis of PHYTENER programme Page 2 sur 32

ACKNOWLEDGMENT FOR THEIR PARTICIPATION IN THE SCIENTIFIC COMMITTEE

Frédérique Cadière (ADEME, Angers) Olivier Faure (Université Jean Monnet, Saint-Etienne) Isabelle Lamy (UR251 PESSAC, INRA Centre de Versailles – Grignon, Versailles)

QUOTATION OF THIS REPORT

Douay F., Bidar G. 2015. Synthesis of PHYTENER program – Final report. ADEME. 31.

Any representation or reproduction of the contents herein, in whole or in part, without the consent of the author(s) or their assignees or successors, is illicit under the French Intellectual Property Code (article L 122-4) and constitutes an infringement of copyright subject to penal sanctions. Authorised copying (article 122-5) is restricted to copies or reproductions for private use by the copier alone, excluding collective or group use, and to short citations and analyses integrated into works of a critical, pedagogical or informational nature, subject to compliance with the stipulations of articles L 122-10 – L 122-12 incl. of the Intellectual Property Code as regards reproduction by reprographic means.


This document summarizes the results obtained as part of a multidisciplinary research programme called Phytener and realized between 2009 and 2013 with the financial support of ADEME. The participants are:

Groupe ISA

LGCgE : Géraldine Bidar, Sébastien Détriché, Francis Douay, Hervé Fourrier, Brice Louvel, Aurélie Pelfrêne, Bertrand Pourrut, Florien Nsanganwimana, Christelle Pruvot, Christophe Waterlot

Equipe BioGAP – Institut Charles Viollette : Jérôme Muchembled

GRECAT : Eric Comont, Corinne Statnik

Université Lille 1

LGCgE : Sylvain Demuynck, Fabien Grumiaux, Sébastien Lemière, Alain Leprêtre, Céline Pernin

Physicochimie des Processus de Combustion et de l’Atmosphère : Jean-François Pauwels, Eric Therssen

Université Lille 2

Laboratoire des Sciences Végétales et Fongiques : Annabelle Deram, Audrey Hayet

Université du Littoral Côte d’Opale

Unité de Chimie Environnementale et Interaction sur le Vivant : Sylvain Billet, Stéphane Firmin, Joël Fontaine, Sonia Labidi, Anissa Lounès-Hadj Sahraoui, Pirouz Shirali, Benoît Tisserand, Anthony Verdin

Centre Commun de Mesures : Fabrice Cazier, Dewaele Dorothée, Paul Genevray

Université de Franche Comté

Laboratoire Chrono-Environnement : Renaud Scheifler

INRA

Laboratoire d’Analyses des Sols d’Arras : Nicolas Proix, Julien Retailleau, Antoine Richard

Chambre Régionale d’Agriculture Nord – Pas de Calais

Jacques Blarel

Exploitation du Lycée Agricole de Tilloy-lès-Mofflaines

Benoît Lefevre, Geoffrey Billaut


SUMMARY 1. Context and objectives .............................................................................................................................. 7

2. Experimental approach ............................................................................................................................. 7

2.1. Woody plots ...........................................................................................................................................8

2.2. Miscanthus plots ................................................................................................................................. 10

2.3. Miscanthus harvesting and combustion tests ..................................................................................... 11

2.4. Economic and social balance of the management methods studied .................................................. 12

3. Main results............................................................................................................................................. 13

3.1. Woody plots ........................................................................................................................................ 13

3.1.1. Degree of contamination and soil functioning ............................................................................. 13

3.1.2. Tree vegetation ............................................................................................................................ 14

3.1.3. Soil animal communities .............................................................................................................. 17

3.1.4. Toxicity tests ................................................................................................................................. 18

3.1.5. Oral bioaccessibility of metals ...................................................................................................... 18

3.2. Miscanthus plots ................................................................................................................................. 19

3.2.1. Degree of contamination and soil functioning ............................................................................. 19

3.2.2. Miscanthus ................................................................................................................................... 20

3.2.3. Soil animal community ................................................................................................................. 23

3.2.4. Toxicity tests ................................................................................................................................. 26

3.2.5. Oral bioaccessibility of metals ...................................................................................................... 26

3.3. Miscanthus harvesting and combustion tests ..................................................................................... 26

3.4. Economic advantages of the sectors studied ...................................................................................... 27

3.5. Perception and social advances of the management methods studied .............................................. 28

4. Conclusion and prospects ........................................................................................................................ 28

References ....................................................................................................................................................... 30


1. Context and objectives

In Northern France, a former lead smelter, called Metaleurop Nord and located at Noyelles-Godault (North of France), released large quantities of metal-contaminated dust into the atmosphere for more than a century until its closure in 2003. This atmospheric discharge highly contaminated the soils around the smelter with cadmium (Cd), lead (Pb), and zinc (Zn). Other features of this area are landscape fragmentation by the anthropogenic pressures (past industrial and mining activities, high population density, dense communication network) and a precarious socio-economic situation.

In agricultural fields, the contamination is mainly limited to the ploughed horizon. The crops harvested on soils contaminated with more than 4 and/or 200 mg kg-1 of Cd and/or Pb, respectively, exceed legislation thresholds for food or feedstuff. Overall, this involves 750 ha, which cannot be economically managed by conventional physico-chemical methods.

Phytoremediation could be a relevant and an environmentally friendly technique to manage large and highly contaminated sites. To provide scientific and technical arguments on the advantages of phytomanagement, a multidisciplinary research programme, called Phytener and supported by ADEME, was set up in 2009. The management methods were based on Cd, Pb and Zn phytostabilisation, assisted or unassisted, and involved two non-food biomass productions: wood (Robinia pseudoacacia, Alnus glutinosa, Quercus ilex, and Acer pseudoplatanus) and herbaceous vegetation (Miscanthus x giganteus). The programme was expected to assess the sustainability of the proposed management plan and to contribute to a sustainable conversion of agriculture in a disadvantaged area by developing innovative technologies using plant biomass from metal-contaminated soils to produce energy. The aim was to give an economic value to a disqualified farmland to meet environmental, economic and social expectations.

The approach is a part of the national SAFIR network (http://safir-network.com/). It offers a demonstration site to promote the management methods studied and to assist prescribers and contractors in charge of management of trace element (TE) -contaminated sites and soils.

2. Experimental approach

The approach included ex situ experiments in a controlled environment (greenhouse, breeding room or laboratory room) but also in situ experimental devices (Figure 1). The size of these devices had integrated environmental and operational constraints. The age of plantations had also made it possible to establish a mid-term appraisal of the effects of management methods on the ecosystem soil and the behaviour of crops for each of the metallic pollutants. Indeed, most of the experimental plots were set up between 2000 and 2008 in scientific, institutional and industrial partnerships. This programme benefited from scientific and technical data acquired previously.

Figure 1. Localisation of the experimental device around the former lead smelter (references plots, setting up dates

and nature of mineral soil amendments before afforestation)


2.1. Woody plots

The management of the wood plots consisted in associating (i) amendment ashes on topsoil which came from thermal circulating fluidised bed boiler using coal schlamms as combusts (FA1) or a mix of lignite and schlamms (FA2) and (ii) a tree plantation. This treatment was applied on a former agricultural field, located about 1 ha from Metaleurop Nord, under the prevailing wind.

In 2000, three plots were used, named R, F1, and F2 (Figure 1). The first one did not receive amendment and was the reference in this approach. The soil of the second plot was amended with an aluminosilicate ash amendment (FA1) and the third one received a sulfo-calcic ash amendment (FA2). The composition of the ashes is given in Table 1. The amendment mass ratio was 6% considering a plow depth of 30 cm.

Table 1. Composition of two studied mineral amendments

Parameters Unit FA1 FA2

pH (H2O) 9.9 12.6

CaCO3 total g kg-1

21 67

N total g kg-1

0.22 1.04

CEC cmol+ kg

-1 5.1 6.4

CaO g kg-1

28.4 184.5

MgO g kg-1

2.93 1.99

K2O g kg-1

0.91 0.21

Na2O g kg-1

0.18 0.06

P2O5 g kg-1

0.09 0.24

S (H2O) g kg-1

5.5 13.9

Fe total g kg-1

38 31

Al total g kg-1

122 47

After amendment, the soils were ploughed, grassed and planted in a regular arrangement of various species: Robinia pseudoacacia L. (black locust), Alnus glutinosa L. (black alder), Quercus robur L. (English oak), Acer pseudoplatanus L. (sycamore) and Salix alba L. (white willow). The species planted were consistent with those used by the Etablissement Public Foncier Nord - Pas de Calais for the requalification of industrial brownfields. The choice of integrating S. alba into the experimental device attempted to mimic usual practices in the region, even if this species is not suitable for Cd, Pb and Zn phytostabilisation. The trees were spaced 2 m apart in the row, 2.5 m between rows. Each plot was spaced 4 m apart. In total, nearly 1,800 trees were planted (Picture 1).

Picture 1. Woody plots (December 2011)


During the 9 years before the Phytener programme, the experimental site had been subjected to regular monitoring: development and health of vegetation, TE concentrations in soils and aerial parts of trees, environmental availability of the metals, etc. Data acquired on the R plot was the subject of a doctoral thesis (Bidar, 2007). During the present programme, this database was supplemented with new samples of soils and vegetation. Investigations also incorporated the neighbouring plots of the site to take particular account of their potential influences on faunal communities. Analyses focused on mineral amendments, soils, aerial organs of trees, and meso and macro fauna.

The knowledge of the physical parameters of the fly ashes studied was complemented by observations by SEM-EDX, the characterisation of particle size and the specific surface area.

Conventional soil parameters were measured (particle size, pH, cationic exchange capacity, total carbonates, organic carbon, total nitrogen, available phosphorus, exchangeable cations, Fe and Mn oxide and hydroxide contents). The concentrations of organic (16 polycyclic hydrocarbons [PAHs] from the EPA list, dioxins/furans) and inorganic (Bi, Cd, Co, Cr, Cu, Hg, In, Mo, Ni, Pb, Sb, Sn, Th, Tl, U, V, and Zn) contaminants were also determined. Chemical extractions (single or sequential) were done to evaluate the extractability of major pollutants (Cd, Pb, and Zn) and their distribution in the different soil fractions.

The activities of soil microorganisms were evaluated by measuring enzyme activities, quantification of the total biomass and microbial respiration. Fungal and bacterial biomass were quantified with lipid biomarkers (dosage of fatty acids and ergosterol). The mycorrhisation rate was evaluated and arbuscular mycorrhisal fungi were identified.

The vegetation on the surroundings of the experimental site has been inventoried. The approach was complemented by a survey of dormant seeds in the soil with aiming to evaluate the potential for revegetation of each forested plot. The plantation development was followed by regularly measuring the trunk diameter of each tree in the R, F1, and F2 plots. The Cd, Pb, and Zn concentrations in the leaves, twigs, fine and large branches of the trees were measured. Concentration profiles were determined on ten cores from tree trunks. Tree health was evaluated through measurements of oxidative stress. In general, plants can undergo external aggression which can be biotic (microorganisms, fungi, insects, etc.) and abiotic (metals, drought, salinity, etc.) in origin. These agents may affect, directly or indirectly, the different constituents of plant cells (cell wall, proteins, DNA, etc.). The indirect effects on plants mainly stem from the excessive formation of highly reactive oxygen species which can damage macromolecules (lipids, proteins) and disrupt many physiological processes. Oxidative stress is defined by this imbalance between pro-oxidant (formation of chemical species) and antioxidant (defense mechanisms) systems. At the membrane level, oxidative stress results in a degradation of the lipid layer of the cell wall and the formation of a compound called malondialdehyde (MDA). At the nucleus level, one of the most common types of DNA damage is deoxyguanosine hydroxylation resulting in the formation of an oxidative adduct 8-OHdG. Plants set up defense mechanisms which can reduce the quantity of oxidative species. These mechanisms use antioxidant enzymes such as superoxide dismutase (SOD) and peroxidase (PO) and also use peptides such as glutathione which can be oxidised (GSSG) or reduced (GSH). In this study, the enzymatic activities of SOD and PO, the content of MDA, the GSSG and GSH content ratio and also the formation of 8-OHdG were determined to evaluate the toxicity induced by soil contamination and/or the management method on trees.

The soil fauna was studied to assess the quality of the ecosystem resulting from the management method studied. Animal communities were estimated with samples and catches at different times of the year. Captured individuals (springtails, mites, worms, ground beetles, molluscs and micromammals) were counted and identified to variable taxonomic levels depending on the species. When possible, the populations were characterised by parameters such as descriptor abundance, species richness (number of species in an ecosystem) and taxonomic diversity. The TE concentrations were measured in earthworms (Lumbricus terrestris), molluscs and micromammals.


Soil quality was also evaluated with tests based on the avoidance response manure worm Eisenia fetida when it was subjected to a choice between two soils: one as a reference, the other to evaluate and present little or less favourable conditions to this species. The toxicity of pollutants in soils was studied using the Microtox® test, which measures bioluminescence reduction of the marine bacterium Vibrio fischeri, when it is affected by soil metals.

Finally, human exposure related to the ingestion of contaminated soil particles was quantified using the unified UBM (Unified Bioaccessibility Method) test developed by the BARGE (Bioaccessibility Research Group of Europe; Cave et al., 2006) group. This test consists of two TE extractions done at the same time and simulating the physiological mechanisms of human digestion, in particular physico-chemical reactions taking place in the gastrointestinal tract.

In 2011, a preliminary synthesis of the data acquired on soil and tree vegetation at the experimental site was drawn up (Lopareva-Pohu, doctoral thesis).

2.2. Miscanthus plots

The experimental device "Miscanthus" included six plots planted between 2007/2008 and 2010. Two plots, called M2007 and M2010, were located respectively 75 and 50 km away from the former smelter, in Linzeux and Bucquoy townships. These two plots can be considered in the experiment as control plots because of their distance from any source of contamination. Four other plots (MV, M1000, M500 and M200) were located around the smelter (Figure 1, Picture 2). Because of textural soil variability within the M200 plot, two zones were distinguished: M200-1 and M200-3. The M200-1 ploughed horizon is characterised by a slightly clayey texture (25 % clay versus 19 % for M200-3).

The ploughed horizons of these six plots showed the following TE (Pb, Cd and Zn) contamination gradient: M2007 ≈ M2010 < M200 (M200-1 ≈ M200-3) < M500≤ M1000 < MV.

Picture 2. The MV plot (Spring 2012)

Depending on the plot, different crop conditions were studied: three origins of miscanthus plants (called A, B and I), plantation of rhizomes or plantlets, two plantation densities, addition or not of a commercial endomycorrhizal inoculum (SolRize®, Agrauxine, Saint Evarzec, France) to plantlets, nitrogen fertilisation (50 units of ammonium nitrate 27 %) or no fertilisation during the second growing season. Table 2 summarises the methods studied for each plot.

Like the woody plots, investigations also incorporated the neighbouring plots of the experimental device.

The analytical approach, which evaluates the ecological sustainability of the Miscanthus plots, was inspired from that described above for the wood plots. It also integrated the measurement of Cd, Pb and Zn concentrations in Miscanthus roots and rhizomes to clarify the behaviour of this plant to soil contamination. The results were expressed in terms of bioaccumulation assessed using the


bioconcentration factor (BCF). This is the ratio of metal concentrations in plant organs to metal concentrations in topsoils.

Table 2. Description of the methods studied for each miscanthus plots

Plots Miscanthus

origin Year of

plantation Nature of plantation

Plantation density1

Addition or not of a endomycorrhizal

inoculum2

Nitrogen fertilization or

not3

Co

nta

min

ated

MV

B 2010 Plant D1 et D2 M0 et M1 F0 et F1

A 2010 Plant D1 et D2 M0 et M1 F0 et F1

I 2010 Plant D2 M0 et M1 F0 et F1

M1000 A 2010 Rhizome D1 M0 F0

B 2010 Plant D2 M0 F0

M500 B 2008 Rhizome D1 M0 F0


Co

ntr

ols


M2010 B 2010 Plant D2 M0 et M1 F0

A 2010 Plant D2 M0 et M1 F0

1 D1 : high density (20,000 plant ha

-1)

D2 : low density (15,000 plant ha-1

) In spring 2010, in M200 and M2007 plots, miscanthus plants fragmented and rhizome fragments dispersed in order to increase the density of plant.

2 M0 : no addition of endomycorrhizal inoculum

M1 : addition of 10 g of endomycorrhizal inoculum 3

F0 : no fertilisation during the four years of this program

F1 : nitrogen fertilisation in June 2012.

The experiments, in situ and ex situ, were studied within a doctoral thesis (Nsanganwimana, 2014).

2.3. Miscanthus harvesting and combustion tests

Dust generated by the miscanthus harvest could contribute to dispersion of pollutants in the environment. To test this hypothesis, sensors were installed on the mechanical equipment (tractor, harvester) and around the plots harvested (Picture 3). These sensors were installed for the harvest of the M200 and M500 plots in March 2011 and April 2013, respectively. The Cd, Pb, and Zn concentrations were measured on each sample of dust collected.

Fixed sampler Mobile sampler installed on the tractor Picture 3. Dust sampler at the miscanthus harvest

The combustion of miscanthus biomass (20–40 mg) from the M200 plot was studied in the laboratory with a thermogravimetric analyser coupled to a mass spectrometer by a supersonic expansion (Figure 2).


Figure 2. Schema of a thermobalance equipped with the supersonic expansion system

coupled to a mass spectrometer

Sample mass variations were followed depending on the temperature and the combustion time (temperature rise from 15 to 105 °C at 5 °C min-1, held for 20 min at 105 °C, again a rise in temperature from 105 °C to 900 °C at 40 °C min-1, held for 30 min at 900 °C). Air was used to activate the combustion. The gases emitted during combustion were instantly cooled, diluted and then analysed.

These tests were completed with an automatic multi-fuel biomass boiler 40 kW (HKRST-FSK, REKA; Figure 3). The products from miscanthus combustion from the M200 and a control plot were quantified. The latter plot was not integrated into the Phytener experimental device. It was a field of the Tilloy-lès-Mofflaines High School farm, which is located near Arras, France.

In comparison with the thermogravimetric analyser, the use of the boiler was designed to provide results that were more realistic in terms of the combustion of contaminated biomass. The pollutant concentrations in emissions and ashes obtained during these combustion tests were compared.

Figure 3. Polycombustible boiler

2.4. Economic and social balance of the management methods studied

The economic balance of miscanthus was compared to a very short rotation willow coppice process, based on bibliographic data.


Surveys and interviews with the different stakeholders of the study area (residents, farmers, communities) were undertaken to clarify how soil pollution problems and miscanthus cultures are perceived by the local population and to determine their expectations.

Finally, depending on the results and research perspectives raised, but also the administrative resolutions around the Metaleurop smelter, the conditions necessary for phytomanagement were defined to meet expectations in terms of sustainable management and contribution to restructuring agriculture in the study area.

3. Main results

3.1. Woody plots

3.1.1. Degree of contamination and soil functioning

The results confirmed the high TE contamination of the organic-mineral horizon (0–25 cm) as well as the existence of spatial heterogeneity (Figure 4). The Cd concentrations varied between 14 and 20 mg kg-1, Pb concentrations between 696 and 1,150 mg kg-1 and Zn concentrations between 916 and 1,400 mg kg-1. There was an excellent correlation between the Cd, Pb, and Zn concentrations as observed for the soils of the Metaleurop site (Sterckeman et al., 2002). These major pollutants were also accompanied, in smaller quantities, by other pollutants such as Ag, As, Bi, Cu, Hg, In, Ni, Sb, Se, Sb, Sn, and Tl.

Figure 4. Pb isoconcentration curves (mg kg-1

) in soil (0-25 cm) of wooded experimental device

For all PAHs, no difference in soil concentrations was observed between the plots. Except for naphthalene, PAH concentrations exceeded the regional reference values proposed by Douay et al. (2005) 1.7–4 times. The proximity of the former smelter, but more generally high industrialisation of the former mining area, could explain these overruns. However, the dioxin/furan concentrations were within the concentration ranges for most countries and land uses.

The rare earth element contents of soils were homogeneous and on the same order of magnitude as those reported in the literature for the earth crust.

The amendments influenced some soil parameters from their incorporation in 2000 until 2011. The amendments increased soil pH, between +0.4 to +0.7 pH units for FA1 and between +0.4 to +0.5 pH units for FA2, throughout the experiment. Knowing the composition of the FA2 ashes (Table 1), the organic-mineral horizon of the F2 plot differed in its higher contents of CaCO3, Ca, S and total P and reduced Cd, Pb and Zn mobility. With the exception of total P, these observations were also made to a lesser extent on the F1 plot.

Since the experiment began, a significant temporal evolution of these different parameters has been observed. On the three plots, an organic carbon increase has been shown (+21, 23, and 25.5 %,


respectively, for the R, F1, and F2 plots) and a slight pH decrease in the organic-mineral soil layer (−0.7 for parcel R and −0.5 for the F1 and F2 plots). This could be explained by natural processes and highlighted the passage of an agricultural soil functioning to young carbonated forest soils. Furthermore, a vertical redistribution of some elements (CaCO3, P2O5, Ca, and total S) was also observed in the amended topsoils, including F2, suggesting leaching of the soluble components contained in the ashes (Figure 5).

Despite the solubility of some constituents of the amendments, the ashes maintained their ability to reduce Cd, Pb and Zn mobility and availability 10 years after soil amendment. The amendment by FA2 ashes generated slower mineralisation and humification of organic matter in the F2 soil. This effect went with an imbalance in the microbial communities. Regarding the observations made on the R and F1 plots, FA2 ashes favoured bacteria communities to the detriment of fungal communities.

Figure 5. Distribution of agronomic parameters (CaCO3, P2O5, Ca, and S total) measured in R, F1 and F2 soils taken every 5 cm between 0 and 55 cm deep in 2009

3.1.2. Tree vegetation

Despite acclimatisation difficulties, especially for R. pseudoacacia and Q. robur, whose dead trees had to be replaced in 2001, tree development has shown a very strong acceleration since 2009. However, Q. robur was the essence with the highest mortality rates over time for all plots. In general, the results obtained in 2008 and 2011 showed that the ashes could respectively decrease Cd, Pb, and Zn


concentrations in aerial organs of trees from 31 % to 65 %, 27 % to 61 %, and 23 % to 67 %, respectively. This effect was stronger in 2008 than in 2011. However, in 2011, when an effect was observed, it resulted in a reduction in TE accumulation (between 14 and 67 %), except for Pb in the thin branches of the trees planted on the F1 plot and Cd in the thin branches of acacia (Table 3).

Table 3. Cd, Pb and Zn concentrations in the leaves (n = 3), twigs (n = 3) and fine branches (n = 9) of alder, maple, locust and willow sampled in 2011 on the three plots (R, F1, F2). The results are expressed as their mean and their

standard errors, in brackets. The letters a, b and c indicate a significant difference between the plots R, F1, F2 (Tukey's test, p


Fe, Al, Ca, K, and Mg, which came into competition with Cd, Pb, and Zn present in soil (Benavides et al., 2005). Differences between the physico-chemical parameters could explain that FA2 ashes were more effective than FA1 ashes in reducing TE mobility in the soil and decreasing the accumulation of pollutants in the aerial parts of plants. The leaves and twigs of A. glutinosa, A. pseudoplatanus, R. pseudoacacia, and S. alba were very little contaminated compared to the literature on these species cultivated on uncontaminated soils (André et al., 2006; Çelik et al., 2005; Lorenc-Plucinska et al., 2013; Mertens et al., 2004; Migeon, 2009; Pourrut et al., 2011; Vandecasteele et al., 2008; Vandecasteele et al., 2002). These observations suggested that A. pseudoplatanus, A. glutinosa and R. pseudoacacia were suitable species for Cd, Pb and Zn phytostabilisation of agricultural soils at the Metaleurop site. This was not the same for S. alba and specifically for Cd. Furthermore, the Zn and Cd concentrations in S. alba were 3 and 1.6 times higher, respectively, closer to the bark than at the heart of the trunk. Inversely, Pb concentrations were 1.5 times higher in the trunk heart.

Although the amendments studied could reduce TE mobility and phytoavailability, some species were more or less affected by these amendments. Indeed, the measurement of the tree diameters of the three plots (Figure 6) showed that the maples and willows planted on the amended soils had significantly lower growth compared with those planted on the unamended soils, whatever the measurement period. Inversely, the ashes did not seem to affect the growth of alders for any plantation age. For locust, the negative effect of ashes was only observed on growth for the FA2 amended soil, until 2004. After that time, ashes had no impact on the growth of this species.

Figure 6. Evolution between 2003 and 2012 of the average diameter of trees per species per plot (R, F1 and F2). A double asterisk indicates significant differences between plots R / F1 and R / F2; a single asterisk indicates a

significant difference between the R plots and the two amended plots shown in parenthesis (Tukey test, p


Table 4. Alder, maple, locust and willow growth calculated in cm yr-1

(mean and standard deviation) for the 2003 -2009 and 2009 -2012 periods on the three plots

R F1 F2

2003-2009 2009-2012 2003-2009 2009-2012 2003-2009 2009-2012

Alder 1.43 (0.33) 11.18 (2.63) 1.36 (0.42) 10.56 (3.19) 1.28 (0.33) 9.72 (2.72)

Maple 0.87 (0.26) 9.31 (3.02) 0.60 (0.24) 6.68 (2.59) 0.37 (0.19) 5.06 (2.04)

Locust 2.09 (0.49) 15.48 (3.99) 2.25 (0.47) 16.08 (3.86) 2.17 (0.50) 16.06 (3.85)

Willow 1.33 (0.63) 16.05 (6.67) 0.92 (0.44) 11.58 (5.88) 0.84 (0.47) 11.31 (6.61)

The effects of ashes on tree growth were also observed at the cellular level. The dosage of lipid degradation products of DNA and the evaluation of defense mechanisms (SOD, PO, GSH / GSSG) revealed the existence of a stress for trees sampled on the F2 plot. These analyses suggested a toxic effect of FA2 ashes. This effect was more moderate for maple since it was the least sensitive species.

Finally, the analysis of fresh litter showed that the quantities of Cd, Pb and Zn brought to the soil surface represented a tiny portion of the stock of organic horizons. However, these elements could supply the TE labile pool during the organic matter breakdown.

3.1.3. Soil animal communities

For all soil amendments, the tree plantation positively influenced the richness of mesofauna (springtails and mites) despite the short period of time since afforestation, the lack of a shrub layer and the presence of a nettle community that was dominant but sparse. The plantation also gave favourable living conditions for beetles and earthworms (multi-specific nature of afforestation, low level of light, soil humidity, vegetation cover consisting of litter and herbaceous vegetation). The presence of forest species such as the Dendrobaena rubida worm and species of general ground beetles revealed a positive evolution of the forest ecosystem created, although it was insufficiently rich to be a real source of biodiversity. The woody plots were an unfavourable environment for molluscs and micromammals (low grass cover, lack of shrub layer).

Ashes had variable effects depending on species. There was a negative effect of FA1 ashes on mites, which were less abundant on the F1 plot compared to the R plot (50 vs 85 %). None of the ashes had an influence on ground beetles. However, there was a positive effect on earthworm density and biomass. The alkalinity of the ashes, particularly FA2, was not a favourable factor for worm reproduction.

Among the earthworms, L. terrestris was the only one collected in sufficient quantity to determine Cd, Pb and Zn. It is an anecic worm, meaning that it lives in deep galleries and feeds on the surface, in the contaminated organic-mineral horizons. The Cd, Pb, and Zn concentrations were significantly higher in the worms sampled in woody plots than those sampled in the soil of the Lille 1 University campus, which was the reference soil (Table 5).

Table 5. Cd, Pb and Zn concentrations Lumbricus terrestris (mg kg-1

dry mass). The results are expressed as the mean and standard deviations are in parentheses. The letters (a, b, c) indicate significant differences between Lille 1

campus, R, F1, and F2 plots (p


None of the ashes had an effect on the accumulation of Pb and Zn in L. terrestris. The differences were not significant for both metals even if the worm concentrations tended to be higher in the F1 plot than the F2 and R plots. Cd concentrations in worms sampled in the F1 plots were greater than those from the R and F2 plots.

The management effects on TE accumulation in micromammals could not be measured due to the small

number of individuals caught (one wood mouse (Apodemus sylvaticus) in 2010 and three in 2012).

3.1.4. Toxicity tests

Avoidance tests with the manure worm E. fetida showed that: - the forest soils were more favourable to oligochaetes than agricultural soils located in the same

environmental context. - F1 and F2 amended soils were avoided by worms in favour of the R soil. The addition of ashes

caused significant changes in the physico-chemical parameters of the soil, making them less attractive to earthworms. Furthermore, the addition of the ashes to the soil and more particularly the FA2 ashes caused a very strong but transient rise in pH, resulting in very high oligochaete mortality (Grumiaux et al., 2007).

Compared to the results in the avoidance test worm E. fetida, the Microtox® test indicated a beneficial effect of the amendments regarding the results on the R soil. Indeed, after 30 min contact between the bacteria and metals extracted from the amended soils, 20% of the luminescence was inhibited versus 60% in the R soil. These results supported those with chemical extractions which revealed that the FA2 ashes reduced mobility and availability of metals in the soils studied.

3.1.5. Oral bioaccessibility of metals

The human bioaccessibility of Cd, Pb, and Zn was determined in topsoil samples collected in 2001 and 2011 on the R, F1 and F2 plots. The results expressed as the percentage of pseudototal soil TE concentrations were compared by differentiating gastric (G) and gastrointestinal (GI) phases (Figure 7).

Figure 7. Bioaccessible concentrations of Cd, Pb and Zn in gastric (G) and gastrointestinal (GI) phases of R, F1, and F2 soils in 2001 and 2011 (Results expressed as the percentage of pseudototal metals concentrations). Mean ± SD; n = 3

in 2001 and n = 12 in 2011; a and b note significant differences between soil in 2001, A and B note significant differences between soil in 2011, * notes the significant differences between years for each soil (Mann-Whitney test,

p


The bioaccessibility was higher for Cd and Pb than for Zn in the gastric and gastrointestinal phases (Cd ≥ Pb > Zn), most likely due to the different chemical forms in which the TE were bound to the soil constituents (Pelfrêne et al., 2011). Conventionally, the results recorded by the gastric phase (where the pH during extraction is 1.5) were higher than the data obtained in the gastrointestinal phase (where the pH is 6.3). These results might result from complexation or re-adsorption of TE onto the soil components or chemical precipitation of elements due to the higher pH environment of the intestinal compartment (Ellickson et al., 2001).

Comparison of the results obtained in 2001 and 2011 showed that: - the Cd and Pb bioaccessible concentrations in the G phase significantly increased in the R, F1, and F2

soils compared to those measured in 2001, but there was no significant difference for Zn (except for R). - the Cd bioaccessible concentrations in the GI phase increased in the F1 soil, but there was no

significant difference for the other soils. - the Pb bioaccessible concentrations in the GI phase significantly decreased in the F1 soil, but there

was no significant difference for the other soils. - the Zn bioaccessible concentrations in the GI phase decreased in the R and F2 soils.

The results suggest a strong influence of the proposed management method (woody plots with or without soil amendment) on the oral bioavailability of Cd and Pb in the gastric phase.

3.2. Miscanthus plots

3.2.1. Degree of contamination and soil functioning

The study plots presented a TE contamination gradient of topsoils as follows: M2007 ≈ M2010 < M200 < M500 ≤ M1000 < MV. Compared to regional agricultural values of loamy soils, M2007 and M2010 were reference plots. For the other plots, Cd concentrations varied between 3.2 and 15.5 mg kg-1, Pb concentrations between 190 and 798 mg kg-1 and Zn concentrations between 293 and 1,088 mg kg-1.

The results excluded a high PAH contamination of miscanthus soils wherever they were located in relation to the former smelter. The dioxin/furan concentrations were included in the concentration ranges in most countries and land uses.

In addition to their metallic contamination gradient, the soils differed on some physico-chemical parameters (pH, clay, carbonate, and organic carbon contents). The pH of contaminated and M2010 soils were slightly alkaline, but the M2007 soil was slightly acid (Table 6). Moreover, in comparison with the other soils, the M500 soil differed with substantial waterlogging during the wet season, with CEC, organic carbon and carbonate contents 2–3, 2–2.6 and 2–20 times higher, respectively.

Table 6. Physico-chemical parameters of M2007, M200, M500, M2010, M1000 and MV soils (0 – 25 cm). Results are expressed according to their means and their standard deviations (n = 3, except M2010).

Granulometry

Plot Clay Silt Silt Sand Sand Organic

carbon C/N pH CEC CaCO3 P2O5

fine coarse fine coarse

g kg-1 g kg-1 cmol+ kg-1 g kg-1 g kg-1

M2007 189 ± 11 272 ± 6 426 ± 8 108 ± 7 5 ± 2 18.3 ± 3.7 10.2 ± 0.4 5.96 ± 0.51 11.2 ± 2.2 1.13 ± 0.15 0.07 ± 0.03

M200 213 ± 47 177 ± 19 321 ± 71 259 ± 35 30 ± 8 16.4 ± 1.8 13.4 ± 0.7 7.76 ± 0.51 16.3 ± 3.0 7.73 ± 7.92 0.16 ± 0.02

M500 311 ± 19 183 ± 21 339 ± 18 147 ± 46 21 ± 6 35.7 ± 1.6 12.8 ± 0.1 7.96 ± 0.003 32.8 ± 1.3 22.73 ± 2.80 0.11 ± 0.03

M2010 208 250 418 112 12 13.7 ± 2.0 - 7.40 ± 0.11 10.7 2.10 ± 1.13 0.18

M1000 175 ± 12 191 ± 12 378 ± 26 227 ± 25 30 ± 3 16.5 ± 0.7 13.6 ± 0.4 8.13 ± 0.06 14.8 ± 0.7 6.63 ± 1.10 0.12 ± 0.01

MV 195 ± 20 180 ± 3 350 ± 22 234 ± 10 40 ± 3 18.2 ± 0.4 15.2 ± 0.4 8.20 ± 0.09 14.9 ± 1.6 10.2 ± 3.3 0.16 ± 0.01

In contaminated soils, the metals were classified according to a gradient of decreasing mobility: Cd > Zn >> Pb. In 2011, the plantation of miscanthus on the MV plot did not affect the ranking. In general, metal


mobility decreased at the end of the 1st year of cultivation and then increased after the 2nd year, exceeding the mobility measured before plantation. In 2011 and 2013, no effect of the conditions studied was observed on TE mobility in soils.

The mobility of Cd in the M500 soil is 1.4–1.5 times lower than that found in the MV and M200 soils, although the Cd total concentrations in the M500 soil were 2.6 times higher than those measured in the M200 soil. This was explained by the higher carbonate and organic carbon contents in the M500 soil than in the M200 and MV soils (respectively, 22.7 vs 7.7 and 10.2 g kg-1 and 35.7 vs 16.4 and 18.2 g kg-1, Table 6). According to the results of sequential extraction, a classification of the TE mobility was determined: MV > M200 > M500 for Cd, MV > M200 ≈ M500 for Pb, MV > M500 > M200 for Zn. Although for the M200 and M500 soils the classification of the TE mobility was not only dependent on the contamination degree of soils, this degree appeared to be an essential factor to consider in the mobility of Cd, Pb, and Zn in the MV soil (the most contaminated).

For the soil biological activities, the M500 soil had a higher respiratory activity, suggesting the presence of a larger number of microorganisms. This confirmed the special nature of this plot. However, although in general higher organic carbon and labile organic carbon contents were measured for the "high density" modality (20,000 feet ha-1) of the MV plot, no genotype influence was found on soil biological activities. Compared to a bare floor, the presence of miscanthus influenced the biological functioning of the soil in favour of the fungal over the bacterial component. In the MV plot, none of the modalities studied affected the diversity and biomass of mycorrhizal fungi. In contrast, the mycorrhization rate, the lower contents of fatty acid and ergosterol in the MV than in the M2010 plot suggest a negative effect of soil contamination on mycorrhisal fungi. Although the addition of the inoculum had no effect on fungal biomass, and it increased the mycorrhisation rate of roots for the genotypes "B" and "I".

3.2.2. Miscanthus

The differences observed in the first 2 years on the height and the number of stems per plant according to the genotype ("B"> "I"> "A") faded during the 3rd year. The average stem height was 3.5 m and their number per plant was between 10 and 36 stems for inoculated modalities and between 20 and 38 stems for noninoculated modalities. Biomass produced after 2 years of cultivation by the genotype "B", with or without addition of endomycorrhisal inoculum, was greater than that of the "A" genotype. For example, the highest yields on noninoculated modalities were 13 t DM ha-1 for the noninoculated genotype "B" and 8 t DM ha-1 for the inoculated genotype (Figure 8).

Figure 8. Miscanthus yield evaluated two years after plantation in the different modalities (B, I, A: Genotype; HD:

High density, F: Nitrogen fertilization)


In general, whatever the degree of soil contamination and the season, the Cd, Pb and Zn concentrations were higher in the roots than in the rhizomes and aerial parts (leaves and stems). In addition, the aerial parts of miscanthus cultivated on contaminated soils have similar Cd, Pb and Zn concentrations to those measured in the plants grown on uncontaminated soils (Figure 9).

Figure 9. TE concentrations measured in November 2011 in miscanthus organs sampled on M2007, M200 and M500 plots. Histograms represent means and associated standard deviations. Different letters represent significant

differences at p > Zn > Pb (roots), Cd > Zn > Pb (rhizomes), Cd = Zn > Pb (stems) and Zn ≥ Cd > Pb (leaves). The no accumulator quality of miscanthus in aerial parts for Cd, Pb and Zn was also highlighted. The TE accumulation in the miscanthus organs was related to the degree of soil contamination as well as the soil’s physical and chemical parameters. This was particularly true for the M500 plot for which TE accumulation was lower than that observed in M200 in all organs for Pb, in the aerial parts for Cd and in the rhizomes for Zn.

At senescence, the concentrations of Cd, Pb and Zn increased in the leaves sampled from the contaminated M200 and M500 plots, which was not the same for the other miscanthus organs.

On the MV plot, there was increased Cd accumulation in the genotype "B" leaves and stems and Pb in genotype "I" rhizomes, after 2 years of cultivation and with the addition of an endomycorrhisal inoculum (Figure 10).


Cd BCF Pb BCF Zn BCF

Leaves

Stems

Rhizome

Roots

Figure 10. Between-treatment comparison of bioconcentration factors (BCFs) of Cd, Pb, and Zn in the organs of the studied miscanthus cultivars during the 2

nd growing season. Values represent means ± standard deviations. Different

letters refer for significant differences depending on organs and treatments. (LD: low density; HD: high density; NM: not adding an inoculum; M: addition of an inoculum)

Furthermore, multivariate analysis genotype x planting density x addition of an endomycorrhisal inoculum showed that for 34 % of cases, the inoculum significantly affected miscanthus behaviour, increasing the accumulation of at least one element in the least one of its organs. In 2 % of cases, the soil inoculation significantly affected the behaviour of miscanthus with a decrease of Cd accumulation in roots. During winter 2013 (3rd-year crop), no significant difference in Cd and Pb accumulation in harvested biomass was shown for the different modalities. For Zn, slight accumulation increases were observed in some modalities combining low density and nitrogen fertilisation. The TE accumulation in the miscanthus organs induced an increase in oxidation markers, confirming the emergence of a metallic stress in miscanthus. The antioxidant defenses (SOD, PO ratio GSH: GSSG) were particularly active in genotype "B" but not sufficient to prevent the development of oxidative damage (MDA, 8-OHdG) in leaves. Genotype "A" seemed to be resistant and did not develop oxidative damage in the nucleus of the plant cell in the aerial parts. The addition of endomycorrhizal inoculum promoted the establishment of antioxidant defenses in plants and prevented oxidative damage induced indirectly by the metals. This suggested a beneficial effect


of the endomycorrhisal fungal community on the health of miscanthus growing in highly contaminated TE soils.

3.2.3. Soil animal community

On contaminated plots, the mesofauna community was dominated by mites (mean, 78.1 %). Collembola and the other organisms represented 15.3 % and 6.6 % of populations, respectively (Figure 11). These were mainly represented by adult beetles and larvae, as well as Hemiptera (aphids). These communities were similar to those on uncontaminated cultivated plots close to the Metaleurop site: 59.7 % dust mites, 16.2 % springtails, and 24.1 % other organisms.

Figure 11. Relative abundance of springtails, mites (Oribatida, Gamasida and Actinedida) and various organisms on miscanthus plots.

However, the abundance of Collembola varied greatly depending on the plot: 391 individuals m-2 on M1000 and 2,095 individuals m-2 on M2010. The abundance of mites varied from 2,247 individuals m-² on M200 to 3,008 individuals m-² on M2010. M500 had a greater abundance of mites, with an average of 11,246 individuals m-². Finally, the abundance of various organisms was lowest on M2007 (90 individuals m-²) and highest on M500 (542 individuals m-²).

Proportions of the three groups of mites differed depending on the plot and the age of plantation. On the M2010 and M1000 plots, planted in 2010, Oribatid mites accounted for only 3.3–4.3 % of mites, while they accounted for 55.7 and 56.2 % of communities sampled on M200 and M500 and more than 90% on M2007, planted in 2007/2008. On M1000 and M2010, Actinedida mites accounted for the highest number of mites (88.5 and 91.9 %, respectively). The Gamasida, mostly predators, accounted for between 3.7 and 14.6 % of the mites collected.

In general, the M500 plot had a significantly more abundant mesofauna community than the other miscanthus plots. This result underscored, firstly, that there was no effect of soil TE contamination on this community and, secondly, the specific characteristics of the M500 soil. Neighbouring stations showed an average similarity with miscanthus plots in terms of community composition, with greater similarity with each other.

The relatively high C/N ratio in the contaminated soils of miscanthus (Table 6) could induce a low rate of colonisation by microorganisms and disadvantage mesofauna which fed mainly in these soils. Thus, the miscanthus plots were not food sources for mesofauna. However, the M500 plot presented the largest mesofauna community. This was explained by wetter soils and abundant weed plant species, most likely to be quickly decomposed by microorganisms and mesofauna. In the balance sheet, the abundance and the specific composition and trophic mesofauna communities depended more on factors such as vegetation


cover, the technical route1, the age of plantations and the physico-chemical parameters of soil rather than soil contamination.

Concerning earthworms, the communities were very poor on contaminated miscanthus plots. These were mainly composed of species belonging to the genus Lumbricus, which is known to be resistant to metal pollution (Table 7).

Table 7. Biomass, total and detailed density of earthworm and species richness of miscanthus plots

plots M2007 M2010 M200 M500 M1000 MV n 10 6 10 18 28 9

Biomass (g m-2

) 74.7 26.2 43.6 11.7 23.8 3.1 Density (ind m

-2) 82.8 32.4 30.8 16.2 26.9 4.9

Allolobophora chlorotica 0.0 23.6 0.0 0.0 0.0 0.0 Aporrectodea caliginosa 6.1 0.0 0.0 0.0 0.0 0.0

Aporrectodea icterica 25.0 0.0 0.0 0.0 0.0 0.0 Aporrectodea longa 2.8 0.0 0.0 0.0 0.0 0.0 Aporrectodea rosea 1.1 0.0 0.0 0.0 0.0 0.0

Lumbricus castaneus 2.2 0.0 2.8 4.0 0.6 0.0 Lumbricus rubellus 3.3 0.0 0.0 0.0 0.0 0.0 Lumbricus terrestris 41.1 7.4 28.1 12.2 26.3 4.9 Octolasion cyaneum 0.0 1.4 0.0 0.0 0.0 0.0 Octolasion lacteum 1.1 0.0 0.0 0.0 0.0 0.0

Species richness 8 3 2 2 2 1

Endogenic species2 (except for some rare individuals) were absent from areas polluted with metals, much like the surrounding area, which did not show much earthworm diversity. On contaminated soils, no difference in species richness in earthworms was observed between miscanthus and traditional agricultural crops. However, miscanthus litter allowed the installation of an epigean species3, Lumbricus castaneus.

Crop of miscanthus was not favourable to carabid beetles (low richness and abundance). This might be explained by the young age of the plots and the fact that miscanthus was not a habitat which could have a reservoir function for beetles.

Regarding molluscs, generic richness was low, with only slugs of the genus Arion and snails of the genus Cepaea, which were only found in the MV plot. The Cd and Pb concentrations in the soft tissues of molluscs generally increased along the soil contamination gradient, but in a nonlinear manner (Table 8).

Table 8. Average Cd and Pb concentrations ± standard deviations (ug g-1

DM) in the soft tissues of Arion sp. slugs and Cepaea sp snails. (probably C. nemoralis) caught in September 2012, in Miscanthus plots.

Plots number Arion sp. Cepaea sp.

slugs snails Cd Pb Cd Pb

M200 6 99.60 ± 15.30 8.50 ± 2.63

M500 7 48.08 ± 7.00 8.99 ± 1.74

M1000 15 111.42 ± 29.26 22.87 ± 7.44

MV 11 13 129.65 ± 37.94 17.79 ± 8.44 120.99 ± 40.50 10.77 ± 7.98

Micromammals were captured during two campaigns: 83 individuals in 2010 and 32 in 2012. In 2010, wood mice (A. sylvaticus) largely dominated the community (69/83 individuals). This dominance was not significantly different from that observed (64 %) in autumn 2006 in woody habitats (Scheifler et al., 2010).

1 All specific farming techniques for a plant production.

2 Nonpigmented worms of medium size living in the first centimeters of soil where they build a subhorizontal gallery

network. They feed on organic matter in the soil. 3 Pigmented small worms living in the litter and feeding on decomposing organic matter.


Eleven common voles (Microtus arvalis), two white-toothed shrews (Crocidura russula) and a house mouse (Mus musculus) were also captured. In 2012, only wood mouse (A. sylvaticus) was captured.

During the first campaign, a significant disparity in catches was noted between the plots in a landscape context of intensive agriculture (M2007 and M2010), with a 29 % rate, and plots close to the former smelter (M200, M500, M1000 and MV), with an 8% catch rate. Miscanthus crops could therefore provide a shelter for micromammals in the context of intensive agriculture, but not in a more diversified landscape context such as around the former smelter.

The densities of rodent populations showed a large amplitude of variation over time, and these fluctuations could be annual or multiannual (Le Quéré and Louarn, 2011). On the Metaleurop site, a higher abundance in autumn than in spring has already been highlighted (Fritsch, 2010) and the results of the present research pointed in the same direction. However, the physiognomy of miscanthus plantations, which radically changes between autumn and spring, might also explain these results. In the autumn, miscanthus had a very high vegetation cover and high and dense vegetation. At the end of winter, the plant is harvested, leaving mulch on the soil surface in spring. Therefore, miscanthus plots did not make up a safe zone, contrary to autumn.

The Cd and Pb concentrations in the kidneys and livers of micromammals were very heterogeneous in the same plot, whatever the capture session (Table 9). This finding was consistent with data obtained in the other programs on the Metaleurop site (Fritsch et al., 2010 and 2011).

Table 9. Average Cd and Pb concentrations ± standard deviations (ug g-1

DM) in the kidneys and livers of wood mouse captured in autumn 2010 and spring 2012 in miscanthus plots (CV, coefficient of variation).

Plot n Cd Pb

kidneys liver kidneys liver

Mean ± SD CV (%) Mean ± SD CV (%) Mean ± SD CV (%) Mean ± SD CV (%)

20

10

M2007 18 0.36 ± 0.33 92 0.09 ± 0.09 100 0.64 ± 0.44 69 0.04 ± 0.01 25

M2010 24 0.61 ± 0.55 90 0.17 ± 0.14 82 0.95 ± 0.65 68 0.09 ± 0.06 67

M200 3 3.08 ± 3.21 104 1.05 ± 1.05 100 1.89 ± 0.95 50 0.23 ± 0.12 52

M500 12 5.27 ± 7.81 148 1.29 ± 1.33 103 103.98 ± 144.64 139 3.23 ± 2.72 84

MV 11 30.78 ± 31.81 103 10.82 ± 10.48 97 131.57 ± 106.98 81 3.20 ± 2.16 68

20

12

M2010 1 0.90 - 0.34 - 0.42 - 0.08* -

M500 4 17.03 ± 8.04 47 5.97 ± 5.51 92 1.89 ± 1.74 92 0.08* -

M1000 14 77.67 ± 72.81 94 24.99 ± 23.22 93 7.36 ± 3.72 51 1.30 ± 1.49 115

MV 8 36.91 ± 25.88 70 10.19 ± 7.44 73 9.81 ± 8.49 87 1.87 ± 2.16 116

*: value of ½ detection limit

Significant differences in the Cd and Pb concentrations in micromammal tissues were observed between miscanthus plots and plots with other types of land use (other agricultural productions, afforestation). Therefore, the average Cd and Pb concentration values in the livers of mice captured on reference miscanthus plots were lower than those observed in individuals captured in uncontaminated woody habitats (STARTT program; Fritsch et al., 2010). On the MV plot, the Cd concentrations in animal livers were approximately twice as high in in mice as in animals captured on woody habitats among the most contaminated of the Metaleurop site. This was not the case for Pb. In the kidneys, the median Cd concentrations were less than half in individuals from the MV plot compared to animals captured in woody habitats, whereas the Pb concentrations were much higher (between 30 and 110 times; Fritsch et al., 2010). Although statistically significant, these differences need to be weighted considering the low number of individuals captured.


3.2.4. Toxicity tests

With the results obtained during avoidance tests, it appeared that the worm E. fetida was not sensitive to the degree of TE soil contamination. An attraction of E. fetida for the M200 soil was even observed. In contrast, the M2007 soil was significantly avoided by the worms. The most likely reason was the soil’s acid pH (Table 6). The quality of organic matter could also affect the attractiveness of worms.

The results of Microtox® tests showed that the M200, M500, M1000, and MV contaminated soils had no toxic effect on the biosphere.

3.2.5. Oral bioaccessibility of metals

After 3 years of cultivation on the M200, M500, and MV plots, the results showed that Pb, Cd, and Zn bioaccessible fractions in the gastric phase (G) ranged from 70 to 98 % for Cd, 75 to 90 % for Pb and 30 to 85 % for Zn. These greatly reduced the gastrointestinal phase (GI): 32–52 % for Cd, 2–30 % for Pb and 8–26 % for Zn.

On the different management methods applied to the MV plot, an influence of the management method studied was shown to influence oral TE bioavailability with (i) variable increases according to the methods used in the G and GI phases and (ii) a sharp reduction of Pb bioaccessibility in the GI phase for all management methods.

Furthermore, comparison of the TE bioaccessible fractions in miscanthus soils (M200, M500 and MV) with those obtained on these soils without crop highlighted the influence of miscanthus on oral TE bioaccessibility (Table 10; Pelfrene et al., 2015.). Considering the gastric phase of in vitro tests, which is most often used for risk assessment because it is more conservative, it appeared that miscanthus was ineffective in reducing exposure to Pb, not very effective for Cd, and showed a variable effectiveness for Zn. These results could be nuanced over the long term to take into account the effects of miscanthus on certain physico-chemical parameters (pH, organic matter content, etc.) of soil, which could affect TE extractability. Indeed, the complexes formed between the metal and the various components of soil were more or less bioaccessible compared to chemical extractions (G and/or GI phases).

Table 10. Human oral bioavailability of Cd, Pb and Zn (mean ± standard deviation in mg kg-1

in gastric G gastrointestinal GI phases) in M200, M500 and MV soils) without culture and after three years of miscanthus crop.

The different letters represent significant differences at p


The multi-fuel boiler used had a cyclic functioning with a full-speed phase until 73 °C circulating water temperature was reached, followed by a pause phase and the cessation of the biomass feed screw. During combustion with the boiler, combustion gases and TE contained in air emissions (mostly in soot) and bottom ashes were analysed.

The composition of the combustion gases was correlated with changes in the temperature profiles in the chimney (T °C max – min of gas: 245 and 81 °C). Thus, when the combustion was slowed, the CO and hydrocarbon contents in gases increased sharply due to poor combustion. The O2 content was also 1.5–3 times higher in the pause periods than in full-speed periods, and the contents of CO2, NOx and SO2 were lower: 3.5–7, 3 and 5 times, respectively. Among the COV, benzene, and some light PAHs (naphthalene, acenaphthylene) were mainly present in the flue gas. Low levels of chlorinated compounds were detected. Few metals were present in the gas phase. Zn and Cd, vaporised in the flame and/or volatilised in very hot areas during combustion, re-condense on any cooler surfaces. In conditions of incomplete miscanthus combustion, soot was undoubtedly very attractive for re-condensing for these chemical species with a high molecular weight (Zn, Cd and their oxides). This was not the case for Pb in its elemental form because of its very high boiling point (1,750 °C) and therefore did not vaporise in the flame. However, it might vaporise in other chemical forms such as oxides and be re-condensed on the soot. The Pb concentrations in the soot were comparable depending on the origin of the biomass studied. Testing with the boiler showed that the metals present in miscanthus were concentrated mostly in the soot and ashes. The Cd, Pb and Zn concentrations in the combustion ashes were 0.53, 34.7 and 176 mg kg-1, respectively (Table 11).

Table 11. Cd, Pb and Zn concentrations measured in miscanthus samples used for combustion, ash and flue discharges to the multifuel boiler

Cd Pb Zn

miscanthus

(mg kg-1)

Ash

(mg kg-1)

flue discharges (mg m-3)

miscanthus (mg kg-1)

Ash

(mg kg-1)


miscanthus (mg kg-1)

Ash

(mg kg-1)


miscanthus of Tilloy-lès-

Moflaines High School

1.03 0.33 0.023 6.7 6.6 0.18 249 908 4.8

miscanthus of M200

0.39 0.53 0.011 2.4 34.7 0.22 42 176 1.6

With a 2.8 % ash content, it appeared that 1–4 % of the Cd concentration from the biomass was in ashes, 3–6 % for Pb and 10–13 % for Zn. The Cd, Pb, and Zn concentrations in the ashes from the combustion with the boiler were, respectively, 10 times, between 6.7 and 23 times, and 3.8–5.3 times lower than those measured with the thermogravimetric analyser. In conclusion, the Cd and Pb concentrations in the ashes from the combustion of miscanthus with the boiler from the M200 plot ranked in the range of concentrations in the ashes from the combustion of various plant biomasses (wheat straw, bark and spruce chips, etc.). Moreover, biomass from the M200 and M500 plots had a very good calorific value (17 MJ kg-1).

3.4. Economic advantages of the sectors studied

The ability of R. pseudoacacia, A. glutinosa and M. x giganteus for Cd, Pb, and Zn phytostabilisation in agricultural soils was evaluated. The plot management studied replied to the expected criteria for phytostabilisation (assisted or unassisted): reduction of environmental danger, contribution to improvement of functional biodiversity, etc. Moreover, the environmental and health advantages as well as phytomanagement need to integrate valorisation of the biomass produced.

For woody plots, the question of the use of biomass was not a concern when the experimental site was established in 2000. Furthermore, plot management was not implemented for logging. Even if the area was sufficiently large for an in situ experiment, it remained modest for a biomass production. At the end of the programme, many questions remained on the quality of wood products, their possible uses, the economic viability of the management, etc. Afforestation of the site’s highly contaminated soils could have


substantial advantages for renaturation and landscape reconquest of land that has been highly degraded by past industrial activities.

Miscanthus culture showed many agronomical advantages (sustainability, absence of inputs and pesticides). However, it was costly due to the purchase of rhizomes / plantlets (about EUR 3,000 ha-1) and requires special care the first 2 years (weed and rodent control, drought). However, this culture was easily integrated into regional agricultural practices in terms of planting equipment, harvesting and crop periodicity. On the Metaleurop site, unobstructed margins with miscanthus were limited. Therefore, the return on investment was faster (8 years) with rhizome plantation and for a biomass use for energy production without transformation. This return was 10–11 years with plantlets, due to their high initial purchase cost. This economic record can also be explained by a possible first harvest after 2–3 years of culture, the absence of local consumers and the lack of support from local and regional authorities. The culture of miscanthus was an investment culture whose profitability was established over time and could contribute to diversification of income sources in an agricultural reconversion context.

3.5. Perception and social advances of the management methods studied

The surveys conducted in the study area highlighted the awareness of the area’s pollution by the local population. The establishment of afforestation, miscanthus crops and a boiler fueled by the biomass produced received a rather favourable opinion. In general, the Phytener programme has been well received by the agricultural community, which is aware of soil pollution and its impacts. However, farmers were quite reluctant to establish this culture because they thought that miscanthus was not profitable and there was a significant financial risk.

For many politicians, agriculture remained the "poor relation" in the Hénin Carvin Urban Community in terms of visibility and understanding. However, this finding did not differ from those made in other regional urban areas. Elected representatives found it difficult to consider how farmers could contribute to the development of miscanthus crop at the service of the urban community. The problems resulted from the lack of a local agricultural organisation which would be able to make proposals, give visibility to the agricultural community and demonstrate its ability to act as a true player in its own right in the area. These difficulties were also strongly increased by a forthcoming prefectural order regulating the use of highly contaminated soils around the former smelter.

4. Conclusion and prospects

The Phytener programme aimed to contribute to the assessment of the advantages of phytomanagement on highly TE-contaminated soils integrating ecological and socioeconomic issues. Two methods were proposed to remedy problems present at the end of the 1990s.The aim was to reduce the environmental and health risks in an environmental, social and economic context degraded by industrial activities associated with coal mining and metallurgy.

With the woody plots 12 years after the soil amendment and tree plantation, we noted: - an accumulation of organic matter in topsoil, a beginning of acidification and a redistribution of

certain elements; - the effects of the ashes studied on the reduction of the natural acidification process in soils, a

limitation of the degradation of organic matter, and the functioning of microbial communities; - the ability of the locust, alder and maple for phytostabilisation with a low TE accumulation in the

aerial parts, including the amended plots; - the positive effects of afforestation on the richness of the meso- and macrofauna, which could be an

ecological reservoir in a degraded environment over the medium term.

Although the results contributed pragmatic answers on this management strategy, more fundamental issues nonetheless remained, related, for example, to the interactions between a species - soil - TE - ashes - pedofauna. The proposed afforestation of 50 ha of the most contaminated land at the Metaleurop site


could help provide answers to these questions. Discussion should also integrate the selection of adapted species to the purposes at hand, the maintenance modes, the future of the wood (thicket, short rotation coppice) and the approval of the population of these phytomanaged areas. The health component cannot be excluded because of possible berry picking or mushrooms gathering by the local population. Nevertheless, this management could be, in such a difficult context, an essential landscape component of the restoration of functional biodiversity.

With miscanthus plots, this study showed: - difficulties during the installation related more to cultivation requirements than soil contamination; - a development of miscanthus unaffected by high Cd, Pb, and Zn concentrations in the plowed

horizon of agricultural plots; - a yield of about 8–14 t DM ha-1 at the end of winter depending on the genotypes; - no significant difference in the Cd, Pb, and Zn concentrations of harvest depending on the degree of

soil contamination; - an accumulation of Cd, Pb, and Zn preferentially in roots, a small accumulation of these elements in

miscanthus rhizomes and aerial parts; - an accumulation of the TEs studied not dependent on plantation density, or mycorrhizal inoculation

of plants or genotype, even if oxidative stress levels differed according to genotype and if inoculation endomycorrhisal reduced stress;

- few effects of miscanthus on mesofauna (springtails and mites) on the plots studied, earthworm communities that were poor and made up of species resistant to metal contamination of soils, low attractiveness of miscanthus plots for beetles because the site had few prey;

- a confirmation of TE impregnation of micromammals.

The observations made over the 4 years of the first part of the Phytener programme need to be validated and developed in greater detail. These include understanding the interactions between the modalities implemented, the ability of miscanthus to accumulate Cd and Zn in particular and the effects on the health of the plant.

Although the use of miscanthus for energy is relevant and meets the environmental requirements in terms of releases, development of phytomanagement through culture and its contribution to the restructuring of agriculture on the Metaleurop site are not sufficiently engaged. Tools have been set up to answer questions from farmers, the local population, communities, site managers and manufacturers. On the four hectares, miscanthus plots came to a mature stage. A multi-fuel boiler using miscanthus has been functional for a year on the Tilloy-lès-Mofflaines High School farm. Several limiting factors for the development of miscanthus culture were identified:

- the high cost of plantation; - the lack of income for the 1st or 2nd year; - the small economic margin generated with this crop; - the lack of support from local, regional and national authorities; - uncertainties about the measures to be taken to manage the most contaminated farmland in the

study area.

Undoubtedly, the development of phytomanagement in this territory depends on the enactment of the prefectural order and its contents. As of now, "serious discussion needs to take place between the agricultural profession and the local and regional communities to identify the guidelines and conditions that need to be set up to support this change. The future of agriculture in this area requires a firm commitment of local and regional authorities, the definition of a medium-term strategy and assistance in both economic and scientific transformation" (Douay et al., 2011). Moreover, this can only be done with an organised local farming community, which is not the case today.

This section of the Phytener programme provided answers on the feasibility and advantages of phytomanagement on highly TE-contaminated soils. Plots have been put in place to promote the


advantages of this management. The support of ADEME during the next 2 years will maintain these tools and continue to contribute to discussions through further research.

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ABOUT ADEME

The French Environment and Energy Management

Agency (ADEME) is a public agency under the joint

authority of the Ministry of Ecology, Sustainable

Development and Energy, and the Ministry for Higher

Education and Research. The agency is active in the

implementation of public policy in the areas of the

environment, energy and sustainable development.

ADEME provides expertise and advisory services to

businesses, local authorities and communities,

government bodies and the public at large, to enable

them to establish and consolidate their environmental

action. As part of this work the agency helps finance

projects, from research to implementation, in the

areas of waste management, soil conservation,

energy efficiency and renewable energy, air quality

and noise abatement.

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Documents

Synthesis of PHYTENER programme · integrated into works of a critical, pedagogical or informational nature, subject to compliance with the stipulations of articles L 122-10 – L