Science of the Total Environment 410-411 (2011) 146–153

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Phytotoxicity testing of lysimeter leachates from aided phytostabilizedCu-contaminated soils using duckweed (Lemna minor L.)

Lilian Marchand a,b,⁎, Michel Mench a,b, Charlotte Marchand a,b, Philippe Le Coustumer c,d,Aliaksandr Kolbas a,b, Jean-Paul Maalouf a,b

a UMR BIOGECO INRA 1202, Ecologie des Communautés, Université Bordeaux 1, Bât. B2 RDC Est, Avenue des facultés, 33405 Talence, Franceb UMR BIOGECO INRA 1202, INRA, 69 route d'Arcachon, FR-33612 Cestas cedex, Francec Université de Bordeaux, UFR des Sciences de la Terre et de la Mer, B18, avenue des Facultés, 33405 Talence, Franced EA 4592 Géoressources & Environnement, Institut EGID, 1 allée F. Daguin, 33607 Pessac, France

Abbreviations: LDS, Linz–Donawitz slag; DL, dolomOMDL, compost+0.2% w/w dolomitic limestone; Z, zepost+2% w/w zerovalent iron grit; PTTE, potentiallyuntreated soil planted with Agrostis gigantea and PopBeaupré; UNT, untreated soil without plants; CCA, Chrdry weight; NOEC, No observed effect concentration; ECEC50, 50% effective concentration.⁎ Corresponding author at: UMR BIOGECO INRA 120

Université Bordeaux 1, Bat B2 RDC Est, Avenue des facuFax: +33 5 40 00 36 57.

E-mail addresses: lilian.marchand@hotmail.fr (L. Mamench@bordeaux.inra.fr (M. Mench), plc@lnet.fr (P. Le

0048-9697/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.scitotenv.2011.09.049

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 June 2011Received in revised form 19 September 2011Accepted 19 September 2011Available online 13 October 2011

Keywords:CompostDolomitic limestoneIn situ stabilizationLinz–Donawitz slagPhytoremediationZerovalent iron grit

Aided phytostabilization of a Cu-contaminated soil was conducted at a wood preservation site located in south-west France using outdoor lysimeters to study leaching from the root zone and leachate ecotoxicity. The effects ofCu-tolerant plants (Agrostis gigantea L. and Populus trichocarpa x deltoides cv. Beaupré) and four amendmentswere investigated with seven treatments: untreated soil without plants (UNT) and with plants (PHYTO),and planted soils amended with compost (OM, 5% per air-dried soil weight), dolomitic limestone (DL, 0.2%),Linz–Donawitz slag (LDS, 1%), OMwith DL (OMDL), and OMwith 2% of zerovalent iron grit (OMZ). Total Cu con-centrations (mg kg−1) in lysimeter topsoil and subsoil were 1110 and 111–153, respectively. Lysimeter leach-ates collected in year 3 were characterized for Al, B, Ca, Cu, Fe, Mg, Mn, P, K and Zn concentrations, free Cuions, and pH. Total Cu concentration in leachates (mg L−1) ranged from 0.15±0.08 (LDS) to 1.95±0.47(PHYTO). Plants grown without soil amendment did not reduce total Cu and free Cu ions in leachates. Lemnaminor L.was used to assess the leachate phytotoxicity, and based on its growth, theDL, LDS, OMandOMDL leach-ates were less phytotoxic than the OMZ, PHYTO and UNT ones. The LDS leachates had the lowest Cu, Cu2+, Fe,and Zn concentrations, but L. minor developed less in these leachates than in a mineral water and a river fresh-water. Leachate Mg concentrations were in decreasing order OMDLNDLNPHYTO=OM=LDSNUNT=OMZ andinfluenced the duckweed growth.

itic limestone; OM, compost;rovalent iron grit; OMZ, com-toxic trace elements; PHYTO,ulus trichocarpa x deltoides cvomated Copper Arsenate; DW,10, 10% effective concentration;

2, Ecologie des Communautés,ltés, F-33405 Talence, France.

rchand),Coustumer).

rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

The soils at many current and former wood preservation sites areCu-contaminated soils as many Cu-based wood preservatives wereused to control insects and fungi (Mills et al., 2006; Mench and Bes,2009; Karjalainen et al., 2009). Excessive Cu concentrations in top-soils usually affect plant communities and performance (Bes et al.,2010; Verdugo et al., 2011). In addition, due to runoff and leaching,Cu in dissolved and solid forms may migrate to aquatic ecosystemswhere it accumulates in sediments and living organisms (Ma et al.,

2003; Kanoun-Boule et al., 2009; Karjalainen et al., 2009). Such accu-mulation may cause physiological and biochemical changes in macro-phytes, animals and microbes (Megateli et al., 2009; Cvjetko et al.,2010). Copper toxicity is mainly due to the existence of two readilyinterconvertible oxidation states that make Cu highly reactive and acatalyst of the formation of free radicals through the Haber–Weiss reac-tion. Free Cu ions can initiate oxidative breakdown of polyunsaturatedlipids (Kanoun-Boule et al., 2009). Dispersion of inorganic soil contam-inants can be quenched by the in situ stabilization technique and/or therestoration of a vegetation cover. Stabilization henceforth refers to thephysico-chemical stabilization of potentially toxic trace elements(PTTE) caused by addition of soil conditioners (Mench et al., 2003;Kumpiene et al., 2008). Incorporation of amendments, e.g. lime, com-post (OM), basic slag, activated carbon, and zerovalent iron grit (Z),into metal and metalloid-contaminated topsoils induces changes inthe physico-chemical state and/or chemical speciation of metals suchas Cu (Mench et al., 2003; Mench et al., 2006; Kumpiene et al., 2006;2011; Bes and Mench, 2008). Formation of insoluble, sorbed, or boundchemical species of metals such as Cu may reduce their leaching fromthe root zone and their labile pool available for biological action(Ruttens et al., 2006a, 2006b; Bes and Mench, 2008; Lagomarsinoet al., 2011). Increasing root uptake and storage in the root system is

147L. Marchand et al. / Science of the Total Environment 410-411 (2011) 146–153

another key step in removingmetals from the soil solution and keepingthem in the rhizosphere. Phytostabilization uses tolerant plants withexcluder phenotypes and associated microbes for long-term contain-ment of contaminants such as PTTE in solid matrices. This worksthrough mechanical and (bio)chemical stabilization that either pre-vents or minimizes pollutant linkages such as PTTE transfer in thefood chain, leaching from the root zone and downward percolation togroundwater, migration to aquatic systems, and re-entrainment of con-taminated particulates for direct inhalation or ingestion (Mench et al.,2010). In the case of aided phytostabilization, single or combinedamendments are incorporated into the soil to decrease the labile con-taminant pool and phytotoxicity by inducing various sorption and/orprecipitation processes prior to planting tolerant excluder plants(Adriano et al., 2004; Mench et al., 2010; Lizama Allende et al., 2011).The vegetation cover minimizes wind dispersion of inorganic contami-nants and limits their leaching from the root zone through evapotrans-piration and root uptake. Plant roots prevent water erosion and storea fraction of the labile metal (Cu) pool during their lifespan. Con-versely, root channels may create pathways for enhanced leachingwhereas decayed products and dissolved organic matter from organicamendments such as compost may change metal (Cu) speciation andmobility (Mench et al., 2003; Ruttens et al., 2006a, 2006b; Marchandet al., 2010). Consequently, the composition and ecotoxicity of leach-ates from the root zone of aided phytostabilized Cu-contaminatedsoils remain unclear.

The context of this study is the appraisal of aided phytostabiliza-tion at a former wood preservation site through successive steps.Soil ecotoxicity was first assessed and stabilizing amendments wereselected in pot experiments (Bes and Mench, 2008; Mench and Bes,2009; Negim et al., 2009). In parallel, plants were assessed for theirCu-tolerance and potential usefulness for Cu phytostabilization(Aulen et al., 2007; Bes et al., 2007; Mench et al., 2008; Bes et al.,2010). Field plots and outdoor lysimeters were then established toassess several aided phytostabilization options, notably using grassyand woody species (Bes et al., 2007; Mench et al., 2009; Mench andBes, 2009).

The aim of this study was to assess the phytotoxicity of lysimeterleachates from the root zone of a Cu-contaminated soil with three op-tions: (1) bare soil, (2) phytostabilization: untreated soil planted withtwo non-native Cu-tolerant plant species, i.e. Agrostis gigantea L. and

Table 1Main characteristics of the soil layers used to fill the lysimeters and main soil characteristic

0–15 cm 15–30 cm 30–60 cm 60–1

Sand% – – – –

Silt% – – – –

Clay% – – – –

Organic carbon (g C/kg) 13.6 4.4 3.13 1.57Organic matter (g/kg) 23.5 7.62 5.42 2.71Total nitrogen (g N/kg) 0.78 0.44 0.36 0.21C/N 17.3 9.95 8.63 7.5EC (dS/m) 0.13 0.44 0.39 0.29CEC cmol/kg 3.36 1.39 1.17 1.09pH 7.05 4.69 4.04 4.09As (mg/kg) 15.5 3.53 4.71 6.11Cd (mg/kg) – – – –

Co (mg/kg) 1.66 1.87 2.28 2.67Cu (mg/kg) 1110 772 153 111Cr (mg/kg) 34.1 16.3 18.4 22.5Fe (mg/kg) 6300 6800 7900 8700Mn (mg/kg) 172 238 185 107Ni (mg/kg) 5.22 5.35 7.97 10.6Pb (mg/kg) – – – –

Tl (mg/kg) – – – –

Zn (mg/kg) 54.9 30.5 28.7 29.3pCu2+ 7.96 4.78 5.12 5.45

aBackground values in French sandy soils are median and upper whisker values, excepconcentrations above which a negative impact can be observed on plants and animals (Bai

Populus trichocarpa x deltoides cv. Beaupré, and (3) aided phytostabiliza-tion: incorporation of one amendment into the soil, i.e. Linz–Donawitzslag (LDS), dolomitic limestone without (DL) and with compost(OMDL), compost (OM), or compost with zero-valent iron grit (OMZ),followed by the plantation with the two plants mentioned above.

Phytotoxicity of lysimeter leachates was assessed using Lemnaminor L. (duckweed) as a bioindicator of water quality (US EPA,1996). The test was also carried out on samples of a mineral waterand freshwater from an uncontaminated river (Jalle d'Eysine river,Gironde, France) for the purpose of comparison. The questions askedwere (1) what remediation options minimize PTTE concentrations inlysimeter leachates? (2) are the lysimeter leachates phytotoxic com-pared to a mineral water or a river freshwater?

2. Materials and methods

2.1. Soils and amendments

The wood preservation site (6 ha) is located in southwest France(44°43′N; 0°30′W) and has been used for over a century to preserveand store timbers, posts and utility poles (Mench and Bes, 2009).The industrial facility dates back to 1846. Creosote, Cu sulfate (from1913 to 1980), CCA (from 1980 to 2006), and Cu hydroxycarbonateswith benzylalkonium chlorides (since 2006) were successively used(Mench and Bes, 2009). Established vegetation and site characteristicswere previously assessed (Mench and Bes, 2009; Bes et al., 2010). An-thropogenic soils developed on an alluvial soil (Fluviosol). Soil investi-gation pits (0–1.5 m) revealed major contamination of topsoils by Cuwith spatial variation (65 to 2400 mg Cu kg−1 soil DW), whereas totalsoil As and Cr, i.e. 10–53 mg As and 20–87 mg Cr kg−1 in topsoils,remained relatively low in all soil layers. In February 2007, a sandysoil was collected with a steel spade in a trench at the P3 sub-site (forsite details, see Mench and Bes, 2009). Soil samples made of six inde-pendent sub-samples were taken from the soil layers, air-dried, andsieved at 2 mm prior to analysis (Table 1). All soil analyses were per-formed at the INRA Laboratoire d'Analyses des Sols (LAS, Arras, France)using standard methods (LAS INRA, 2007).

InMarch 2007, large vats (75 dm3, 0.5 m diameter) were filled withthree successive layers: 5 cm of coarse gravels (1–3 cm, diameter),22 cm of sub-soil (from the 30–60 cm soil layer), and 25 cm of topsoil

s at the P3 sub-site (0–25 cm soil layer).

00 cm P3 (0–25 cm) Background valuesin French sandy soilsa

Threshold valuesc

85.8 – –

8.3 – –

5.9 – –

– – –

16 – –

– – –

17.2 – –

– – –

3.5 – –

7 – –

9.8 1.0–25b –

0.12 0.03–0.24 –

b 2 1.4–6.8 –

1460 3.2–8.4 3523 14.1–40.2 1006090 6000–14300 –

181 72–376 –

5 4.2–14.5 5027 16.4–58.7 –

0.24 0.29 –

46 17–48 1507.66 – –

t bfrequent total As concentrations for all French soil types; cthreshold values areze, 1997). pCu2+=3.20+1.47 pH −1.84 log10 (total soil Cu) (Sauvé, 2003).

Mn

PK

ZnCu

2+

EC

5±

0.33

ab0.42

±0.07

a0.94

±0.16

bc3.77

±0.67

ab0.10

±0.01

c0.44

±0.06

b70

.7±

34.9bc

1±

0.17

a0.32

±0.02

a0.60

±0.11

c3.07

±0.34

ab0.07

±0.02

b0.33

±0.1b

45.2±

4.6c

5±

0.2a

b0.24

±0.06

a0.76

±0.29

bc8.51

±1.75

c0.06

±0.01

b0.32

±0.17

b89

.5±

0.7b

6±

0.66

bc0.11

±0.04

a0.28

±0.08

ac1.69

±0.22

ab0.03

±0.01

a0.19

±0.1b

92.4±

20.1b

0±

0.29

a0.12

±0.04

a0.20

±0.0a

c10

.73±

2.98

c0.02

±0.01

a0.14

±0.09

b79

.9±

7.2b

4±

0.21

cd0.49

±0.69

a0.23

±0.05

ac7.23

±4.77

bc0.02

±0.01

a0.02

3±

0.01

7ab

112.5±

19.2ab

1±

0.2a

b0.55

±0.57

a0.20

±0.0a

1.01

±0.77

a0.01

±0.0a

0.01

2±

0.00

2a15

8.6±

11.6a

5±

0.23

db0.02

⁎b0.2⁎

4.24

±1.32

abb0.00

7⁎0.00

6±

0.00

2a38

0±

80d

b0.02

⁎b0.2⁎

1b0.00

7⁎–

571

mg/L

12.8

μg/L

–2.3mg/L

2.27

μg/L

––

ocTu

keyHSD

test).Stars(*)indicate

values

below

thede

tectionlim

it.aSa

lpeteu

ret

al.(20

06).

148 L. Marchand et al. / Science of the Total Environment 410-411 (2011) 146–153

(from the 0–30 cm soil layer) (Table 1). Gravels and the sub-soil wereseparated by a geotextile. Total Cu concentrations (mg kg−1) were1110 in the topsoil and 111–153 in the subsoil (Table 1). Amendmentswere carefully mixed with the topsoil using a vat, singly and in combi-nation (% air-dried soil DW, w/w), before filling the lysimeters, toform the seven soil treatments that were conducted in triplicate: bareuntreated soil (UNT), untreated soil with plants (PHYTO), 5% compostmade of wood chips and poultry manure (OM, Orisol, Cestas, France),0.2% dolomitic limestone (DL, Bes and Mench, 2008), OM with DL(OMDL), OM with 2% zerovalent iron grit (Z, GH120, particle sizeb0.1 mm, Wheelabrator, Allevard, France) (OMZ) and 1% P-spikedLinz–Donawitz slag (LDS, Centre Technique et de Promotion des LaitiersSidérurgiques, La Plaine Saint-Denis, France). LDS is mainly composedof Ca (30.7 wt.% CaO), Fe (21.4 wt.% Fe2O3), Si (14.6 wt.% SiO2), P(14.0 wt.% P2O5), Mg (9.5 wt.% MgO), Al (5.5 wt.% Al2O3) and Mn(2.5 wt.%MnO2). Other compounds such as TiO2 and K2Owere detectedat low concentrations, 1.09 and 0.53 wt.%, respectively (Negim et al.,2009). A. gigantea L. (2 patches, 5 cm in diameter) and one poplar(P. trichocarpa x deltoides cv. Beaupré, initial shoot length: 30±5 cm) were transplanted in all lysimeters except for the UNT treat-ment. Lysimeters (n=21) were placed in situ (March, 2007). Ly-simeter leachates were periodically collected in plastic bottles(1.5 dm3) from March 5, 2007 after each major precipitation event(N30 mm, leachate volumeN1.5 L). They were collected in year 3(March, 2010) for this experiment, and kept at 4 °C for no morethan 48 h prior to the test. Both leachate and soil pH (1:1 soil:water suspension, Jackson, 1967) were determined (Hanna instru-ments, pH 210, combined electrode Ag/AgCl — 34), and electricalconductivity (EC, WTW Cond 340i and TetraCon 325) of leachatesas well.

Table2

Elem

entalc

ompo

sition

(mgL−

1),pH

,and

EC(μScm

−1)of

leacha

tesin

year

3an

dof

fresh-

andmineral

waters.

Trea

tmen

tspH

Al

BCa

CuFe

Mg

PHYT

O6.58

±0.27

b31

.70±

5.13

c0.02

±0.01

ab4.82

±1.63

b2.53

±0.47

b19

.00±

2.86

c1.8

UNT

6.55

±0.09

b20

.47±

3.33

c0.02

±0.0a

4.70

±1.33

b1.40

±0.16

ab12

.10±

1.97

b1.2

OM

6.77

±0.28

b24

.97±

3.07

c0.03

±0.0b

5.15

±0.57

b1.56

±0.12

b13

.37±

2.39

bc1.8

DL

6.80

±0.5b

10.85±

3.26

b0.02

±0.0a

8.78

±4.39

b0.82

±0.28

ab5.96

±1.53

ab2.6

OMZ

6.76

±0.15

b2.70

±0.72

ab0.02

±0.0a

4.13

±1.76

b0.25

±0.13

ab1.64

±0.46

a0.8

OMDL

6.73

±0.25

b4.06

±4.58

ab0.02

±0.01

ab10

.25±

2.21

b0.40

±0.15

ab3.19

±4.2a

3.1

LDS

7.62

±0.1a

0.05

±0.0a

0.02

±0.0a

31.80±

4.1a

0.15

±0.08

a0.02

±0.01

a1.9

Jalle

freshw

ater

6.90

±0.48

bb0.05

⁎0.03

2±

0.01

b29

.47±

7.09

ab0.00

8⁎0.12

±0.09

a4.5

Mineral

water

7.2

b0.05

⁎–

78b0.00

8⁎–

24Med

ianva

lues

insu

rface

water

forFren

chrive

rsa

86.6μg

/L24

.1μg

/L10

0mg/L

0.88

μg/L

24.1

μg/L

4.6

Value

saremea

ns±

SD(n

=3)

,letters

indicate

sign

ificant

differen

cesbe

twee

ntrea

tmen

tsforea

chpa

rameter

atpb0.05

(posth

2.2. Plant material and toxicity test

The L. minor L. population used was collected in the Jalle d'Eysinesriver (Bordeaux, Gironde, southwest France) in April 2010. Plantswere washed with sterile distilled water, placed in a plastic container(10 dm3) filled with modified Hoagland medium (Hewitt, 1966)(pH=4.9±0.04), and grown for six weeks in a greenhouse at 22 ±2 °C with a 12 h photoperiod before the tests. Container solutionwas replaced once a week to provide nutrients and oxygen for theLemna fronds and to prevent the development of root fungal diseases(Kamal et al., 2004). Fronds are defined here as single Lemna “leaf-like” structures (US EPA, 1996).

In the second step, duckweed fronds (2 colonies with 3 fronds and 2colonies with 4 fronds=14 fronds in all), randomly selected from thestorage container, were cultivated in triplicate for 12 days in250 mL Erlenmeyer flasks containing 150 mL of one of the followingsolutions (adapted from US EPA, 1996): lysimeter leachates (triplicatescorresponding to the three lysimeter replicates, for all treatmentsdescribed above), the freshwater from the Jalle d'Eysine river, andEvian mineral water (France). An additional experiment was carriedout in the same way, but 30 mL of Hoagland solution were addedto 120 mL of each treatment solution. Leachates and waters wereanalyzed at the INRA Usrave laboratory, Villenave d'Ornon, France(ICP-AES, Varian Liberty 200). The free Cu ion concentration inleachates and waters was measured with an mV-meter (Hanna instru-ments, pH 210) and a selective ion electrode (Fischer Bioblock CupricIon Electrode, N83921) (Table 2).

Fronds were counted every three days, as the number of frondshas been used as a relevant surrogate for biomass (Radic et al.,2010), to assess the phytotoxicity of the growth media. Relativegrowth rate (RGR) was then determined after 3, 6, 9, and 12 days ofexperimentation using the following equation: RGR=[ln(final frondnumber)− ln(initial frond number)] /days, the initial number of frondsbeing n=14 at the beginning of the experiment.

149L. Marchand et al. / Science of the Total Environment 410-411 (2011) 146–153

2.3. Statistical analyses

The effects of treatments on the leachate element concentrations,leachate pH and the RGR of L. minorwere tested using one-way analysesof variance (ANOVAs). Normality and homoscedasticity of residualsweremet for all tests. Post hoc TukeyHSD testswere performed to assessmulti-comparison of means. Differences were considered statisticallysignificant at pb0.05. A Principal Component Analysis (PCA) was con-ducted on leachate composition and pH. Pearson correlation coefficientswere calculated between growth (RGR) and each of the trace- andmacro-element concentrations in leachates and leachate pH. All statisti-cal analyseswere performed using R software (version 2.12.0, R Founda-tion for Statistical Computing, Vienna, Austria).

3. Results and discussion

3.1. Composition of lysimeter leachates

Concentrations of trace elements, pH and EC in leachates dependedon soil treatments, notably on the amendments used to stabilize the

a

b c

tota

Free.

Dimension 1 (53.47%)

Factor Correlation P value

Al 0.98 <0.0001

Fe 0.97 <0.0001

Zn 0.93 <0.0001

Total Cu 0.92 <0.0001

P 0.92 <0.0001Free Cu ion 0.86 <0.0001

B 0.49 <0.0001

pH -0.68 <0.0001

Ca -0.67 <0.0001

Dimension 2 (14.52%)

Factor Correlation P value

Ca 0.63 0.002

pH 0.51 0.01

Mg 0.45 0.04

K -0.69 <0.001

Dim

Dim 2 (14.5%) OM

PHYTO

UNT

Fig. 1. Principal Component Analysis (PCA) of the treatments accounting for Al, B, Ca, total(a) PCA, (b) variable contributions to axes, and (c) correlation circle.

contaminated soil (Table 2). The first axis (53.5%) of the PCA corre-sponded to the PTTE concentrations and the second axis (14.5%) corre-sponded to Ca and Mg concentrations and pH (Fig. 1). Based on thePCA, leachates can be divided into four groups. Group 1 (PHYTO, UNTand OM) included leachates with the highest P and PTTE concentrationsbutwith lowCa concentrations. Group 2 (OMDL and DL) included leach-ates with lower PTTE concentrations, low Ca concentration, and highMg concentration. Group 3 (OMZ) had low PTTE concentrations, thehighest K concentration, and the lowest Ca and Mg concentrations.Group 4 (LDS) had low PTTE concentrations, the highest pH and Caconcentrations, but the lowest P and K concentrations. The leachateMg concentration differed significantly across treatments (Table 2). Itsvalue (in mg L−1) peaked in the OMDL treatment (3.1), was the lowestin the OMZ treatment (0.8), and varied between 1.2 (UNT) and 2.6 (DL)in the other treatments. This agrees with the results of Kumpiene et al.(2008) who reported that soil treatments with zerovalent iron, e.g.OMZ, immobilized macro-elements such as Mg, whereas the use of do-lomitic limestone ensured adequate Mg concentrations (Riggs et al.,1995). However, leachate Mg concentrations in the amended soilswere all below the Jalle freshwater value (4.55 mg L−1, pb0.05).

pH

Al B

Ca

l.Cu Fe

Mg Mn

P

K

Zn

Cu.ion

1 (53.5%)

d = 2

DL

LDS

OMDL

OMZ

Cu, free Cu ion, Fe, Mg, Mn, P, K, and Zn concentrations and pH in lysimeter leachates:

150 L. Marchand et al. / Science of the Total Environment 410-411 (2011) 146–153

3.1.1. Untreated (UNT)Leachates from the UNT soil (Table 2) presented high Cu and Zn

concentrations compared to the upper threshold values used by theFrenchwater agency (Adour-Garonne region) for superficial freshwaters(respectively 10–15 and 43–98 μg L−1). Total Cu, Cu2+, Fe, Al, and Znconcentrationswould render UNT leachates toxic for the aquatic ecosys-tems (Karjalainen et al., 2009). Total Cu concentration in UNTleachates largely exceeded frequent values reported for Europeanwaters(in μg Cu L−1: minimum 0.08, median 0.88, maximum 14.6) (Salpeteurand Angel, 2010; INERIS, 2010), the NOEC/CE10 value for algae in fresh-water (0.01 mg Cu L−1, INERIS, 2011), the chronic ecotoxicity parame-ters for L. minor, i.e. 14-day NOEC value (0.06 mg Cu L−1, Jenner andJanssen-Mommen, 1993), the EC10 value (0.057 mg Cu L−1 based onfrond number, Naumann et al., 2007), and all the EC50 values cited inthe literature for Lemna sp. (in mg Cu L−1: 0.16, Teisseire et al., 1998;0.33, Naumann et al., 2007; 0.47, Khellaf and Zerdaoui, 2009; 0.60,Drost et al., 2007; 1.1, Wang, 1986) – except Lakatos et al. (1993) andInce et al. (1999) (1.5 mg Cu L−1). Leachate Zn concentration in UNTwas four times higher than the EC10 value for L. minor (0.017 mg ZnL−1 based on frond number, Naumann et al., 2007) (Table 2).

3.1.2. Linz–Donawitz slag (LDS)The lowest total Al, Cu, Fe and Zn concentrations occurred in the LDS

leachates (Table 2), which led to their individualization from the otherleachates (Fig. 1). As an alkaline by-product of steel-making, LDSmainlyconsists of Al, Ca, Fe, Mg, Mn, and Si oxides, but may also contain rela-tively high Cr, Mn and V concentrations (Negim et al., 2009; Sjöberget al., 2010). It provides a high neutralizing capacity at low cost, whichlikely induces metal hydrolysis reactions and/or co-precipitation withcarbonates and acts as a precipitating agent for metals in the solution(Bes and Mench, 2008). Al, Cu, Mn, Ni and Zn are co-precipitated in Fe(hydr)oxides and Co, Fe, Ni and Zn are co-precipitated inMn (hydr)oxides,dissolved from the slag, whose overall mean surface charge switches frompositive to negative value as pH increases (Sheoran and Sheoran, 2006;Marchand et al., 2010). Here, metals co-precipitated with Fe and Mn(hydr)oxides, and LDS leachates presented the highest pH across the treat-ments (pH ranged from 6.5 for UNT to 7.6 for LDS, Tukey HSD test,pb0.05). Bes and Mench (2008) reported an increase in pH from 6.25(untreated soil) to 8.1 in a contaminated soil from the same site amendedwith 0.1% calcium oxide. High pH and Ca concentrations decreased Cumobility in the soil (Fig. 1), its lowest value being at slightly alkalinepH (Kumpiene et al., 2008). Across treatments, the lowest total Cuand Cu2+ concentrations (0.15 and 0.012 mg L−1, respectively) occurredin LDS leachates (Table 2). However, these concentrations remainedhigher than concentrations measured in the Jalle freshwater (totalCu=b0.008 mg L−1 and Cu2+=b0.006 mg L−1). Total Cu in LDS leach-ates exceeded theEC10 value for L.minor (Table 2). The LDS treatmentwas

ab

a

b

ββ

α α

β

DL LDS OM

Tr

Cu2+

Total Cu

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Cop

per

conc

entr

atio

n (m

g/L)

Fig. 2. Total copper and free copper ion (Cu2+) concentrations in lysimeter leachates. Valulevel with the Tukey HSD test.

thus the most efficient in reducing PTTE concentrations in lysimeterleachates, but did not decrease them to the levels of an uncontaminatedfreshwater. The LDS leachates had the lowest K concentration(1.0 mg L−1, Tukey post hoc test, pb0.05) and reduced P leaching(Table 2) whichmay indicate an increase in phosphate sorption. Applica-tion of slag can decrease exchangeable soil K content due to a reduction inthe percentage of K saturation in the exchange complex (Negim et al.,2009). Release of LDS-born vanadium and its leaching from the rootzone may constitute a potential environmental threat that needs to befurther investigated (Sjöberg et al., 2010).

3.1.3. Dolomitic limestone (DL)Dolomitic limestone has been widely used to reduce PTTE mobility,

mainly Cu and Pb, by raising soil pH. In this study, it increased pH to 6.8andMg concentration in leachates (Table 2). Leachate Ca concentrationincreased less in DL than in LDS (Table 2). The DL treatment reducedPTTE mobility (e.g. Cu total=0.82 mg L−1 in DL leachates, Table 2)but it was less efficient than the LDS treatment. As initial soil pH (6.5)was nearly neutral, limingmay be not sufficient to reduce PTTEmobilityas it does in an acid soil, and an alkaline amendment containing Fe/Mnoxides such as LDSwas shown to bemore appropriate. As for LDS, DL in-corporation led to a low leachate K concentration (Table 2).

3.1.4. Organic matter (OM), with dolomitic limestone (OMDL), and withzerovalent iron grit (OMZ)

Incorporation of OM into the soil, singly or combined with anotheramendment, can be used to reduce PTTE leaching (Ruttens et al.,2006a, 2006b). The Al, total Cu, Cu2+ , Fe and Zn concentrationswere higher in the OM leachates than in the OMDL and OMZ ones(Table 2). Except for Cu2+, which was lower in OMDL than in OMZ(respectively 0.023 and 0.14 mg L−1), PTTE concentrations in OMDLand OMZ leachates were similar (Table 2). Insoluble high molecularweight organic acids can retain Cu in soil upon soil acidification (ChirenjeandMa, 1999) and the OM-bound Cu fraction can account for 96% of thetotal Cu in a CCA-contaminated soil (Balasoiu et al., 2001). However, theOM treatment did not reduce Cu and Zn leaching from the root zone inthe soil tested here. Dissolved OM may partly promote Cu mobility(Kumpiene et al., 2008). The combination of OM with zerovalent irongrit (OMZ) wasmore efficient than OM alone, especially for Cu stabiliza-tion (Fig. 2). Newly formed Fe andMn oxides after Z corroded in the soillikely enhancemetal sorption by the poorly crystalline Fe oxyhydroxidesand crystalline Fe–Mn oxides of the OMZ soil (Bes and Mench, 2008;Kumpiene et al., 2011). The OMDL treatment numerically reduced Culeaching more than the DL and OM (Fig. 2; Table 2) confirming previousfindings with acid and contaminated soils (Mench et al., 2000a, 2000b).The OMDL and OMZ treatments were less efficient than LDS (Figs. 1, 2)in reducing metal concentrations in lysimeter leachates.

abab

ab

b

ββ

ββ

OMDL OMZ PHYTO UNT

eatment

es are means±SD (n=3); different letters stand for statistical significance at the 0.05

3 6 9 12

b b b b b b b b b b b b b b b b b b b b b b b b b c c c

a aa a

a a

a a

Day

-0.1

0.0

0.1

0.2

0.3

Rel

ativ

e G

row

th R

ate

Jalle river Mineral waterDLLDS

OMOMDLOMZPHYTOUNT

Fig. 3. Relative growth rate (RGR) of Lemna minor across treatments over the 12-day period (without addition of Hoagland solution). Values are means±SD (n=3), different lowercase letters stand for statistical significance at the 0.05 level with the Tukey HSD test.

151L. Marchand et al. / Science of the Total Environment 410-411 (2011) 146–153

3.1.5. Untreated soil planted (PHYTO)The presence of A. gigantea and poplar may reduce metal concentra-

tions in leachates by sorbing metals onto the root plaque, root uptake,and increasing soil CEC (Vangronsveld et al., 1996; Doyle and Otte, 1997;Marchand et al., 2010). Conversely, through root exudation of organicacids (Ryan et al., 2001) and grass phytosiderophores (Kidd et al., 2009),as well as root-induced porosity, plants may maintain metal solubilityand promotemetal leaching from the root zone. Indeed, Fe andZn concen-trations were significantly higher in PHYTO leachates (Table 2, Tukey HSDtest, pb0.05), and a similar numerical trend was observed for total Cu,Cu 2+, and Al (Table 2, Fig. 1). This increase in metal concentrations ofleachates may result from rhizosphere activities and the binding of soilmetals with dissolved organic matter, including phytosiderophores ofA. gigantea and organic acids. Thus for other soil treatments, e.g. LDS,amendments rather than plants would be responsible for reduction ofleachate metal concentrations.

3.2. Phytotoxicity and duckweed growth

Duckweed is a ubiquitousfloating freshwatermonocotyledon andoneof the world's smallest flowering plants. It is widely used in water qualitytests to monitor contaminants such as PTTE because of its physiologicaltraits, i.e. small size, rapid growth between pH 5–9 with a doubling timeof 1–4 days or less, and vegetative reproduction (Horvat et al., 2007;Kanoun-Boule et al., 2009). An excess of PTTE, such as Cu, inhibits duck-weed growth (Kanoun-Boule et al., 2009; Megateli et al., 2009).

On day 3 of this experiment, the RGR of plants growing in DL, LDS,OM, OMDL, OMZ, PHYTO, and UNT leachates were in the same range(from −0.03 in UNT and DL to 0.02 in PHYTO and OMZ). These RGRvalues were significantly lower (Tukey HSD test, pb0.05) than thoseof plants growing in the Jalle and mineral waters (respectively 0.19and 0.20, Fig. 3). The same effect was found at days 6 and 9. On day6, RGR of plants growing in lysimeter leachates ranged from −0.02(OMDL) to 0.03 (OMZ), whereas RGR of plants growing in the Jalleand mineral waters reached 0.1. On day 9, RGR of plants growing onlysimeter leachates ranged from 0–0.01 (UNT, OMDL, OM) to 0.03(LDS), while RGR of plants growing on the Jalle and mineral watersincreased to 0.08 and 0.09. On day 12, RGR of plants growing in the

Table 3Correlations based on Pearson coefficients between the Relative Growth Rate (RGR) of Lleachates (total Al, B, Ca, Cu, Fe, Mg, Mn, P, K, and Zn concentrations, free Cu ion concent

pH Al B Ca Cu tot Cu 2+

RGR 0.31⁎⁎ −0.24⁎ 0 0.37⁎⁎ −0.17⁎⁎ −0.30

⁎ Statistical significance at the 0.05 level.⁎⁎ At the 0.01 level; Cu tot: total Cu concentration; Cu2+: free Cu ion concentration.

Jalle and mineral waters reached 0.09 and 0.12, respectively, whereasthe other treatments split in two groups (Tukey HSD, pb0.05). In thefirst group, i.e. OM, DL, OMDL and LDS, duckweed growth remainedstable or increased slightly in the leachates (RGR values 0, 0.02, 0,and 0.02, respectively). In the second group, i.e. PHYTO, UNT andOMZ, duckweed growth was inhibited (leaf discoloration, and RGRrespectively=−0.05, −0.1, and −0.04). Changes in plant growthwere significantly negatively correlated with total Cu, Cu2+, Al, Fe,and Zn concentrations in the solutions tested (Table 3). ExcessiveCu exposure, notably free Cu ions, induces oxidative stress in plantsleading to peroxidation of polyunsaturated lipids (Mocquot et al.,1996; Kanoun-Boule et al., 2009). The free radicals produced damagethe photosynthetic apparatus (Kanoun-Boule et al., 2009) and maycatalyze protein degradation through oxidative modification and in-creased proteolytic activity (Romero-Puertas et al., 2002). Copperaccumulation in plant tissues can also cause changes in nitrogenmetabolism and increase free amino acid concentrations (Llorenset al., 2000; Megateli et al., 2009). Previous studies on Lemna sp.exposed to Cu showed a wide range of EC50, i.e. from 2.52 μM(0.16 mg L−1) to 23.6 μM (1.5 mg L−1) (Table 2). Babu et al.(2003) reported that duckweed growth decreased above 4 μM Cu(0.25 mg L−1). Our results (Table 2, Figs. 1 and 3) agree with thelow EC50 values. However, the OMZ leachate, which had the lowest Cuconcentration after LDS (Cu=3.94 μM, 0.25 mg L−1), inhibited duck-weed growth at day 12 (Fig. 3). Al, Fe, and Zn concentrations in theleachate were relatively low in this treatment (respectively 2.70, 1.64,and 0.02 mg L−1). Therefore additional factors may be involved. Onone hand, Z may increase Ni exposure (Mench et al., 2000a, 2000b),while on the other hand, several interactions between Mg and Cahomeostasis and metal exposure may drive duckweed growth. Mag-nesium limited duckweed growth in the leachates tested (Table 3,Pearson coefficients: 0.41). In plant cells, Mg2+ is vital for membranestabilization, ATP utilization, and nucleic acid biochemistry. It is a cofac-tor for many enzymes (including ribosome enzymes) and the coordi-nating ion in the chlorophyll molecule (Schaul, 2002). With excessivemetal accumulation, Mg2+ ions associated with the tetrapyrrole ringof chlorophyll molecules can be replaced (Radic et al., 2010), especiallyby Cu2+ ions (Kupper et al., 2002). In addition, in plants exposed to

emna minor (leachates without Hoagland solution) and the chemical parameters ofration, and pH) across the treatments (n=21).

Fe Mg Mn P K Zn

⁎⁎ −0.28⁎ 0.41⁎⁎ 0 0.21⁎ 0 −0.38⁎⁎

Table 4Relative growth rate (RGR) of Lemna minor on lysimeter leachates, with addition ofHoagland solution, and on freshwater and mineral waters over the 12-day period.

Hoagland solution Modality RGR

(day) 3 6 9 12

Jalle River 0.186 a 0.101 a 0.083 a 0.089 aMineral Water 0.197 a 0.099 a 0.092 a 0.123 a

PHYTO 0.009 b 0.045 b −0.052 c −0.072 cUNT −0.056 c 0.037 b −0.088 c −0.109 cOM −0.027 b 0.040 b 0.025 b 0.030 b

Leachates with DL 0.002 b 0.065 ab 0.024 b 0.021 bHoagland solution OMZ 0.008 b 0.068 ab 0.025 b 0.050 b

OMDL −0.036 b 0.043 b 0.001 b 0.017 b

Values are means (n=3), different lower case letters stand for statistical significance atthe 0.05 level with Tukey HSD test.

152 L. Marchand et al. / Science of the Total Environment 410-411 (2011) 146–153

PTTE, protein levels are often lower due toMg and K deficiencies, whichcause loss of protein synthesis (Hou et al., 2007; Cvjetko et al., 2010). InMg2+ deficient plants, other secondary effects include carbohydrateimmobility and loss of RNA transcription. In particular, in our study,Mg could have been immobilized in the OMZ soil, decreasing Mg con-centration in the OMZ leachates (Table 2) and duckweed growth. Con-versely, duckweed growth was higher in the Jalle and mineral waters –richer in Mg – than in leachates. In the additional experiment, Mg wasadded with the Hoagland solution to the OMZ leachates and their neg-ative effect was completely neutralized, OMZ being the most efficienttreatment at day 6 and day 12 (Table 4). High Al concentration in leach-ates, especially in PHYTO andOM,may impact duckweed growth. Al3+ isa strong inhibitor of Mg2+ uptake (Rengel, 1990). In our data, Cu2+/Mgratio in lysimeter leachates from the root zone was a relevant indicator(y=−251.51x+21.14, R²=0.59, pb0.05) of duckweed growth (RGR).

Duckweed growth was correlated with Ca concentrations (Table 3).Calcium increases soil and leachate pH and hence playing a role inmetalimmobilization, but it is also implicated in cell wall development andcell membrane integrity of duckweeds (Huebert and Shay, 1991), Casub-cellular homeostasis and the detoxification of oxidative stress(Cuin, 2006). Calcium can influence Cu uptake and rhizotoxicity(Wang et al., 2010). This makes this macro element, which was morepresent in the DL, OMDL, and LDS leachates, essential to enhance duck-weed growth in leachates from contaminated soils. Duckweed growthdepends on interactions between trace elements and between PTTEand macro-elements. This cannot be explained by simple antagonismand/or synergism but has to be considered as a whole (Cvjetko et al.,2010; Upadhyay and Panda, 2010).

4. Conclusions

Composition of lysimeter leachates from the root zone of a Cu-contaminated soil, collected in year 3 after the incorporation of soilconditioners, depended on the amendments used for in situ metalstabilization and, to a lesser extent, on the presence of Cu tolerantplants. The growth of A. gigantea L. and P. trichocarpa x deltoides cv.Beaupré – Cu tolerant plants – did not reduce total Cu and free Cuions in leachates compared to the bare soil. Conversely, aided phytost-abilization based on the incorporation of Linz–Donawitz slag (LDS, 1%w/w) led to the lowest leachate Cu, Cu2+, Fe, and Zn concentrations.In such amended soils, amendments rather than plants would beresponsible for reduction of leachate metal concentrations. At similarsoil pH (~6.7), compost with zerovalent iron grit (OMZ), and dolomiticlimestone with (OMDL) and without compost (DL) numerically re-duced total Cu and free Cu ions in leachates, but differences were notsignificant compared to the unamended soil with (PHYTO) andwithout(UNT) plants. The Mg concentration was lower in the OMZ leachatethan in the other leachates, and this negatively affected duckweedgrowth. Further investigation of the enrichment of the OMZ treatmentwith Mg2+ would be necessary to enhance its efficacy. The duckweed

growth in leachates in increasing order was UNT, PHYTO, OMZbOM,OMDL, DL, LDS. However, duckweed developed less in the LDS leachatesthan in a mineral water and a river freshwater.

Acknowledgments

This work was financially supported by AXA foundation (PhDgrant of L. Marchand), ADEME, Department Brownfields and PollutedSites, Angers, France, and the European Commission under the SeventhFramework Programme for Research (FP7-KBBE-266124, GREEN-LAND). Authors thank FlorienNsangwimana for his technical assistance.We are grateful to Daphne Goodfellow for improving the English.

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