Geoderma 160 (2011) 517–523

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Greenhouse and field studies on Cr, Cu, Pb and Zn phytoextraction by Brassica napusfrom contaminated soils in the Apulia region, Southern Italy

Gennaro Brunetti a,1, Karam Farrag b,c,d,⁎, Pedro Soler Rovira e,2, Franco Nigro f, Nicola Senesi a,3

a Dipartimento di Biologia e Chimica Agroforestale e Ambientale, Università di Bari, Via Amendola, 165/A, 70126 Bari, Italyb Central Lab for Environmental Quality Monitoring (CLEQM), Egyptc National Water Research Center (NWRC), Egyptd Ministry of Water Resources and Irrigation (MWRI), Egypte Centro de Ciencias Medioambientales, Consejo Superior de Investigaciones Científicas, Serrano 115 dpdo., 28006 Madrid, Spainf Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università di Bari, Via Amendola, 165/A, 70126 Bari, Italy

⁎ Corresponding author. Central Lab for EnvironmentNational Water Research Center (NWRC), Egypt. Tel.: +201

E-mail address: Karam_farrag@hotmail.com (K. Farr1 Tel.: +39 080 5442953; fax: +39 080 5442850.2 Tel.: +34 917452500.3 Tel.: +39 080 5442853; fax: +39 080 5442850.

0016-7061/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.geoderma.2010.10.023

a b s t r a c t

a r t i c l e i n f o

Article history:Received 23 June 2010Received in revised form 23 September 2010Accepted 16 October 2010Available online 26 November 2010

Keywords:PhytoextractionHeavy metalsBrassica napus, compostBacillus licheniformis

In the framework of a project aiming to phytoremediate heavy metal contaminated soils in the Apulia region,Southern Italy, a series of greenhouse experiments followed by field trials were performed in order tooptimize heavy metal phytoextraction by Brassica napus. The effects of root colonization by Bacilluslicheniformis BLMB1 and of addition of municipal solid waste (MSW) composts on the capacity of B. napus totolerate and accumulate Cr, Cu, Pb and Zn were evaluated. B. napus was able to accumulate high amount ofmetals in greenhouse conditions, whereas it grew with difficulty or not at all in the open field, and metalaccumulation in plant fractions was relatively low. The accumulation of metals in the plant fractions was inthe order: CrNZnNCuNPb. The presence of either compost or B. licheniformis BLMB1 strain enhanced metalaccumulation, Cr in particular, in the experimental conditions used. This effect can be useful in thephytoextraction of Cr from contaminated soils.

al Quality Monitoring (CLEQM),01022229; fax: +20222035083.ag).

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

The accumulation of heavy metals in agricultural soils is ofincreasing concern due to food safety issues and potential healthrisks as well as detrimental effects on soil ecosystems (McLaughlinet al., 1999). Phytoremediation can be defined as the combined use ofplants, soil amendments and agronomic practices to remove pol-lutants from the environment or decrease their toxicity (Salt et al.,1998). This technique may be employed using various approaches,including, phytoextraction, phytovolatilization and phytostabilization(Chaney et al., 1997). In particular, phytoextraction refers to theability of hyperaccumulator plants to uptake metals from soil andtransport them to the above ground parts, which are able to accu-mulate concentrations up to 100-fold greater than those normallyfound in non-accumulators species (Baker and Brooks, 1989; Bakeret al., 2000; McGrath and Zhao, 2003).

Finding optimal plant species for remediating a specific soil andselection of appropriate soil amendments able to improve soilconditions allowing plant survival and growth, are the key aspect toachieve this objective (Clemente et al., 2005). Among many fast-growing and high biomass-accumulating plant species suitable forphytoextraction brassicaceae have received considerable attention(Prasad and Freitas, 2003) based on their capacity to uptake andaccumulate Cr and other heavy metals in amounts higher than thoseof other plant species (Kumar et al., 1995). B. juncea is one of the mostpromising, non hyperaccumulating plant species for extracting heavymetals from contaminated soils, however other species of the Brassicagenus, such as B. campestris, B. carinata, B. napus, B. nigra, B. oleraceaand B. rapa, have also been studied (Gisbert et al., 2006; Kumar et al.,1995; Marchiol et al., 2004; Meers et al., 2005).

The effect of organic amendment on heavy metal bioavailabilitydepends on its nature, microbial degradability, salt content andinfluence on soil pH and redox potential, as well as on the particularsoil type and metal concerned (Shuman, 1999a,b; Walker et al., 2003,2004). In particular, the effect of different kinds ofmunicipal solidwaste(MSW) composts has been studied for their capacity of increasingmetalavailability in soil and their uptake by plants (Maftoun et al., 2004;Murphy and Warman, 2001; Pinamonti et al., 1999; Sebastiao et al.,2000; Warman and Rodd, 1998; Zheljazkov and Warman, 2004a,b).

Another possibility to enhance metal bioavailability is the use ofsoil microorganisms and plant root-associated bacteria, which are

518 G. Brunetti et al. / Geoderma 160 (2011) 517–523

stimulated by root exudates including a wide range of organicmolecules (Kamnev and van der Leile, 2000; van der Lelie, 1998). Theuse of rhizobacteria in combination with plants is expected to providehigh efficiency in phytoremediation (Abou-Shanab et al., 2003;Whiting et al., 2001). In particular, plant growth promotingrhizobacteria (PGPR) are now being considered to play an importantrole in phytoremediation technologies (Mayak et al., 2004). The PGPRcan promote plant growth in contaminated sites and enhance arid soildetoxification by conferring resistance to water stress (Burd et al.,2000; Mayak et al., 2004). According to Khan (2005), PGPR canimprove the growth of plants used for phytoremediation, e.g., byincreasing plant nutrition and health and biomass production, andreducing the level of contaminant uptake.

Bacillus licheniformis, a Gram-positive, spore-forming soil bacterium,is regarded as a plant growth promoting rhizobacterium and has beenclassified as GRAS (generally recognized as safe). Previous researchesdemonstrated that different microbial strains of B. licheniformis canimprove the growth and development of the host plant in heavy metalcontaminated soils by mitigating toxic effects of heavy metals on theplants (McLean et al., 1990; 1992; Ramos et al., 2003; Yakimov et al.,1995). However, research in this area is very limited and requires fieldstudies to support greenhouse or growth chamber results (Lucy et al.,2004). Thus, combining the use of PGPR with MSW composts may be agood means for increasing the efficiency of phytoremediation.

This work is part of a series of studies initiated in 2007 to evaluatethe feasibility of the phytoremediation technique to remediate soilscontaminated by heavy metals (Cr in particular) in the Park of AltaMurgia, in Apulia region, Southern Italy (Brunetti et al., 2009a,b;Farrag et al., 2008, 2009). The main objective of this study was toevaluate the potential role of a MSW compost and B. licheniformisBLMB1 on the metal phytoextraction capacity of Brassica napus. Inparticular, the availability, accumulation, uptake and removal of Cr,Cu, Pb and Zn from polluted soils will be evaluated.

2. Materials and methods

2.1. Soil and pot experiment

The contaminated soil (A) used in this experiment was a TypicHaploxeralfs, fine-loamy, mixed, thermic (Soil Taxonomy, 2003)collected in the Park of AltaMurgia, which features total concentrationsof Cr, Cu, Pb and Zn largely exceeding the Italian maximum levelspermitted for agricultural soils (Italy, 2006). An uncontaminatedagricultural soil (B) collected from the same area was used as a ref-erence. Soils were collected after grass cover removal from the top20 cm, air-dried, gently ground to pass through a 2-mm sieve, ho-mogenised and used to fill the pots (3 kg soil per pot).

A MSW compost (10% w/w) and an aqueous cell suspension(108 cells ml−1) of B. licheniformis BLMB1 isolated from a semi-commercial formulate (10% v/w) were used as amendments bythorough mixing with the soil. The heavy metal content of thecompost used was within the Italian limits (Iwegbue et al., 2007).

The pot experiment was carried out from July to October 2007 onB. napus plants grown in a greenhouse covered with a screen withoutsupplementary light or heat. The average temperature of the green-house ranged from 29.6±5.60 °C (day) to 14.5±4.32 °C (night), andthe relative humidity was 65.5±10.9%, with an average of 12 hphotoperiod per day. To prevent emergence failures, twenty seedswere sown in each pot initially. Then, when the first pair of true leavesappeared only 10 uniform seedlings per pot were allowed to grow for90 days after sowing, and were then harvested.

The set-up consisted of 15 pots made of polyvinyl chloride (PVC)with a diameter of 20 cm and a depth of 20 cm. The design included T1(uncontaminated soil B), T2 (contaminated soil A), T3 (T2+compostat 10%), T4 (T2+B. licheniformis BLMB1 10%), and T5 (T2+compostat 10%+B. licheniformis BLMB1 10%). All treatments were carried out

in triplicate. All pots were watered and kept at the field capacitymoisture throughout the growing season.

2.2. Field experiment

The field experiment was carried out in the Altamura site of thePark of Alta Murgia from February to August 2008 in plots of35 m×35 m which were established in two contaminated (A1, A2)and one non-contaminated (B1) locations. Each plot was divided intotwo portions, an uphill one (35 m×60 m) with no amendment, and adownhill one (35 m×20 m) treated with B. licheniformis BLMB1 (1 Ldiluted in 10 L of water then sprayed on the soil). A distance of 5 mseparates the two portions. The experiments were performed intriplicate. The B. napus plants grown on these plots were harvestedafter 150 days from sowing. For each selected plot n. 7 soil subsampleswere collected and composite.

2.3. Analytical procedures

2.3.1. SoilSoil analyses were carried out following internationally recom-

mended procedures and the Italian official methods (Italy, 1999). SoilpH was determined by a glass electrode in distilled water (pH H2O)suspensions at 1:2.5 soil to liquid ratio. Electrical conductivity (EC) wasmeasured using a conductimeter in filtrates from suspensions of 1:2 soilto water ratio. Texture and particle size distribution were determinedby the pipette method after dispersing the soil sample in a sodiumhexametaphosphate and sodium carbonate solution. Total organiccarbon (TOC) was measured by the Walkley–Black method (Walkleyand Black, 1934). Available phosphorus (Pava) was determined using aspectrophotometer UV/VIS on sodium bicarbonate and sodium hydrox-ide soil extracts according to the Olsen method (Olsen et al., 1954).

The total content of heavy metals were determined in microwaveassisted digests (Multiwave Perkin Elmer 3000) of soil samples addedwith a suprapure HNO3:H2O2:HCl mixture (5:1:1 v/v). The metalextractable fraction in soil was estimated on soil extracts bydiethylenetriamine pentaacetic acid (DTPA)–CaCl2–triethanolamine(TEA) buffered at pH 7.3 (Lindsay and Norvell, 1978). This protocol isgenerally recommended for alkaline calcareous soils and excludes theeffects of carbonate dissolution. The contents of heavy metals (Cd, Cr,Cu, Ni, Pb and Zn) in both acid-digested and DTPA extracts weredetermined using an inductively coupled plasma optical emissionspectrometer (ICP-OES ICAP 6300 Thermo Electron).

All chemicals were of analytical reagent grade and distilled waterMilli-Qwas used for solution preparation and dilution. Reagent blanksand laboratory NIST certified standards were used and routinelychecked during ICP-OES determinations.

2.3.2. PlantTo evaluate the heavy metal concentrations, B. napus plants were

harvested and separated into roots and shoots (all the above groundparts). Roots collected extended down to 0–20 cm soil depth.Successively, the separated fractions were thoroughly washed withtap water to remove all visible fine soil particles, rinsed withdeionised water, oven dried at 60 °C for 3 days, and finally groundto a powder using a Retsch MM200 mixer mill and kitchen miller. Todetermine the total heavy metal contents the plant fractions weresubjected to microwave assisted digestion with a suprapure HNO3:H2O2:HCl mixture (5:1:1 v/v) and spectroscopic measurement asdescribed for soil samples.

The Bioconcentration Factor (BF) of each metal in plants wascalculated by dividing the total content in shoots by the total contentin soil (Brooks, 1998). Further, the Translocation Factor (TF) wascalculated by dividing the total metal content in shoots by the totalmetal content in roots (Brooks, 1998). Both factors were calculated ona dry mass basis.

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2.3.3. CompostThe compost has been analyzed using standard methods (Collana

Ambiente, 1998; Italy, 1999) after air-drying, grinding with a mixermill, and passing through a 1 mm sieve. The pH was measured indistilled water at a 1:10 sample to water ratio using a pH meter.Electrical conductivity (EC) was determined on the filtrate at 1:10sample to water ratio by a conductimeter. The total organic carbonwas determined using the Walkley–Black method. Total nitrogen(Ntot) was determined by the Kjeldahl method. Heavy metalconcentrations were determined as described for soil samples.

2.3.4. BacteriumB. licheniformisMBBL1 was grown in nutrient agar plate for 48 h at

30 °C. The agar medium consisted of beef extract (5 g), peptone (10 g)and agar (20 g) in 1 L distilled water. The pH value of the mediumwasadjusted to 7.2 with 10% (w/v) NaOH and 10% (w/v) HCl. A loop of thebacterial culture was then inoculated in 300 ml of the nutrientmedium and the flasks were incubated at 30 °C for 48 h. The cellsgrown were centrifuged at 6000 rpm for 10 min, washed three timeswith phosphate buffer, and suspended in 300 ml of the same buffer.

To assess the population of sporogenous bacillaceae (expressed aslog CFU g−1, CFU=Colony Forming Unit), soils samples werecollected from the treated pots both before B. napus seeding and atthe end of its vegetative cycle. According to Chilcott and Wigley(1993), soil samples were taken from the sub-surface (2–15 cm —

plant's root zone) using a sterile spatula. All samples (250 g each)were transported in sterile plastic bags to the laboratory, allowed todry for 3 days in the air, and then 2 mm — sieved to remove all largeparticles and plant debris. Successively, 1 g of each soil sample wassuspended in 10 ml of sterile distilled water and shaken vigorously for2 min. Then the samples were treated at 70 °C for 60 min in a waterbath. Aliquots (100 μl) from several 10-fold serial dilutions of theheat-treated sample were spread onto nutrient agar plates andincubated in the dark at 37 °C for 24 h. Pure colonies were obtainedby repetitive dilution and triple streaking on nutrient agar. Among

Table 1General properties and contents of total and DTPA-extractable heavy metals in soils and co

Parameters Units Soilsa

A B A1

Texture – SL SL SLpH H2O – 8.5 8.7 8.2±EC ds m−1 0.32 0.22 0.41CO3tot g kg−1 89 85 135CO3act g kg−1 83 30 108TOC g kg−1 65.9 41.7 89.9Ntot mg kg−1 8.4 6.1 11.3Pava mg kg−1 83 71 150

Total concentrationsCd mg kg−1 1.6 (2)b 0.9 1.2±Cr mg kg−1 1977.8 (150) 66.3 181Cu mg kg−1 188.8 (120) 28.7 351Ni mg kg−1 74.8 (120) 39.2 59.0Pb mg kg−1 202.3 (100) 58.5 257Zn mg kg−1 679.5 (150) 114.7 851

DTPA-extractable contentsCd mg kg−1 0.18 0.09 0.14Cr mg kg−1 0.06 0.002 0.09Cu mg kg−1 6.93 0.67 8.56Ni mg kg−1 1.37 0.18 1.25Pb mg kg−1 4.57 0.60 4.92Zn mg kg−1 60.45 0.44 82.5

EC, electrical conductivity; CO3, carbonates; TOC, total organic carbon; Ntot, total nitrogen;a Soil values of A1, A2 and B1 are mean±STD (n=7), a distance of 5 km separates the fie

experiment (A, B).b Values in parentheses are the maximum total contents in soils for public and private gc Values in parentheses are the maximum metal total contents in compost according to t

the colonies developed, those belonging to the Bacillus genuswere identified based on morphological characters and biologicalprocedures.

2.4. Statistical analyses

Statistical analysis of data was made by using the Statgraphics Plus5.1. software. One-way analysis of variance (ANOVA) and Tukey's testwere applied. Means and standard deviations were calculated usingMicrosoft Excel.

3. Results and discussion

3.1. Total and available metal contents in soils

The main properties, total and DTPA-extractable heavy metallevels of the soils used, A, B, A1, A2 and B1 and compost, are shown inTable 1. Data show that the soil texture was silty loam (SL), except thesilty clay loam (SCL) from the B2 site. The slightly alkaline pH values,the high percentages of OM and the presence of carbonates suggestthat an important retention of heavy metals is to be expected in thesesoils (Greger, 2003; Kabata-Pendias and Pendias, 1992; Larlson et al.,2000; Shuman, 1999a,b). More details on these soils can be found inBrunetti et al., 2009a.

The total contents of Cr, Cu, Pb and Zn in the polluted soils werehigher and that of Ni and Cd lower than the maximum admissiblelevels for agricultural soils (Italy, 2006). In particular, the valuerecorded for Cr (1977.8 mg kg−1) was 13 times higher than themaximum admissible limits in Italy.

Based on their content in soils, the metals studied can be classified,from the higher to the lower pollution level, as follows: CrNZnNPbNCu. However, metal availability in these soils appeared to be low(Brunetti et al., 2009a), as shown by the DTPA-extractable contents(Table 1), and in the order: ZnNCuNPbNCr.

mpost studied.

Compost

A2 B1

SL SCL –

0.2 8.0±0.1 8.0±0.3 8.7±0.04 0.42±0.04 0.22±0.02 1.3±22 76±38 36±13 *±38 69±29 31±8 *±8.3 71.2±17 21.5±2 232±2.8 12.1±3.1 2.2±0.2 14.8±24 147±18 23±5 83

0.1 0.9±0.2 0.7±0.1 b0.001(105)c

2.4±183 1391.1±38.2 54.5±4.4 48(100).4±20.8 162.5±27.8 32.2±2.4 105(300)±4.7 52.4±6.3 37.8±2.3 39(50).4±42 810.9±29.6 46.4±2.7 67(140).4±64.3 514.9±167.5 119.1±7.4 230(500)

±0.03 0.12±0.02 0.08±0.01±0.01 0.11±0.02 0.004±0.001±1.80 5.22±1.20 0.48±0.05±0.36 1.12±0.28 0.22±0.03±0.71 6.68±1.20 0.52±0.06±18.2 76.64±14.20 0.64±0.09

Pava, available phosphorus; SL, silty loam; SCL, silty clay loam; *, not determined.ld experiment locations (A1, A2, B1) and the locations of soils collected for greenhouse

reen areas and residential sites (Italy, 2006).he Italian limits (Vander derf et al., 2002).

Fig. 1. Population of sporogenous bacillaceae (expressed as log CFU g−1) in control soil(B) and polluted soil (A) before seeding B. napus, and control soil (T1), polluted soil (T2),polluted soil+compost (T3), polluted soil+B. licheniformis MBBL1 (T4), and soil+compost 10%+B. licheniformis MBBL1 (T5) at the end of the vegetative cycle of theplants. One-way variance analysis (ANOVA), Tukey test, 95% confidence level (p≤0.05).

520 G. Brunetti et al. / Geoderma 160 (2011) 517–523

3.2. Metal concentrations in plants grown in greenhouse pots

In general, the total concentration of heavy metals in plants grownon polluted soil substrate in greenhouse pots (T2) was relatively high(Table 2), thus showing accumulation and high tolerance of B. napusas it was expected. The highest values measured (mg kg−1) in shootsand roots for Cr were 254.8 and 51.3 followed by Zn with values of156.1 and 39.1, respectively. Further, data in Table 2 indicated that thetype of soil amendment affected significantly and differently theconcentration of heavy metals in B. napus. Although treatment T5showed the highest concentrations of metals in shoots, no statisticallysignificant effect was found among T3, T4 and T5, except for Ni. Inagreement with Baker (1981), the accumulation of metals wasgenerally higher in shoots than in roots, which is a typical behaviorof accumulator species. The content of metals in the roots of B. napus,for treatments T3, T4 and T5 were significantly higher than in T2. Thehighest metal contents and the highest differences were found fortreatment T4 and T5 where bacillus was added.

Further, at the end of .B. napus vegetative cycle, a significantlyhigher population of sporogenous bacillaceae was found in the soilamended with bacillus (T4), as compared to the untreated pollutedsoil (T2) (Fig. 1). The highest population was found in the soilamendedwith both the bacillus and compost (T5), as compared to theother treatments (Fig. 1), whereas a similar population of sporoge-nous bacillaceae was found for treatments T3 and T4.

These results suggested that B. licheniformis BLMB1 is a promisingcandidate strain for enhancing plant metal accumulation in multimetal contaminated soils. This effect may be achieved by variousmechanisms including: increased mobilization of metals (Chen et al.,2005; Gadd, 1990; Idris et al., 2004), reduced toxicity of metalstransformed into less toxic forms, immobilization of metals on the cellsurface or in intracellular polymers, and metal precipitation orbiomethylation (Silver, 1996). Similar to bacillus, compost additioncaused significant metal accumulation in shoots and roots of B. napus(Table 2), as previously reported by various authors (Maftoun et al.,2004; Murphy and Warman, 2001; Pinamonti et al., 1999; Warmanand Rodd, 1998; Zheljazkov and Warman, 2004a,b).

For all treatments the BF values of B. napuswere lower than 0.2 forCr, Cu, Ni and Pb, between 0.35 and 0.43 for Cd, and between 0.23 and0.29 for Zn (Table 3). In general, BF was reported to decrease withincreasing soil metal concentration (Zhao et al., 2003), and valueslower than 0.2 are considered normal when plants are grown onpolluted soils (McGrath and Zhao, 2003). In contrast, the TF valueswere higher than 1 for treatments T2, T3, T4 and T5 (Table 3), with amaximum value of 5.04 for Cr and Pb. As tolerant plants have TFvalues less than 1 and hyperaccumulators higher than 1 (Baker,1981), the TF valuesmeasured here showed that B. napus behaved as ahighly metal accumulator plant in greenhouse conditions.

Table 2Concentration of heavy metals (mg kg−1 d.w.) in shoots and roots of Brassica napus grown

Treat. Cd Cr Cu

ShootsT1 0.39±0.02 a 1.9±0.3 a 5.6±0.9 aT2 0.56±0.05 b 254.8±24.5 b 23.2±1.7 bT3 0.67±0.01 c 349.3±3.5 c 27.2±0.4 cT4 0.68±0.02 c 366.7±13.4 c 27.4±0.8 cT5 0.67±0.02 c 372.2±7.7 c 28.4±0.6 c

RootsT1 0.30±0.03 a 4.9±1.2 a 9.1±1.4 aT2 0.32±0.03 a 51.3±6.5 a 10.9±0.7 aT3 0.43±0.03 b 129.5±15.2 b 16.3±1.2 bT4 0.57±0.03 c 229.8±23.2 c 20.7±1.5 cT5 0.61±0.06 c 254.2±31.2 c 22.0±2.4 c

Analysis of variance (ANOVA): Tukey's test with 95% of significance (values in the same vedifferent at p≤0.05).

3.3. Metal concentrations in plants grown in the field

In the field experiment an acceptable growth of B. napus wasobserved in experiment B1, whereas it failed to grow properly inexperiment A2 and no growth was observed in experiment A1. Onepossible explanation for these results is that the contamination levelsof soil A1 (e.g., Cr mean concentration was 1812.4 mg kg−1) washigher than that of soil A2.

Although B. napus successfully accumulated relatively highamounts of metals in greenhouse conditions, it failed to uptake thesame amount of metals in field conditions, possibly because plantsexplore potted soil more intensely (Delorme et al., 2000). Accordingto Conesa et al. (2007), the differences in metal uptake between fieldand controlled pot conditions may be attributed to the differentphysiological state of the plant and/or to some modification of soilproperties and climate parameters in pot conditions. B. napusappeared to accumulate most metals in the root system, whereasthe shoots content was relatively low. Thus B. napus cannot beconsidered an accumulator plant in field conditions but a metaltolerant plant that possesses mechanisms allowing to cope with highmetal concentration in soil.

The order of accumulation of the six metals analyzed by B. napuswas: ZnNCrNCuNPbNNiNCd. Generally, the metal contents in shootsand roots of B. napus collected from soil A2 were higher than thosecollected from soil B1(Table 4). Cr uptake was relatively low in thefield experiment where B. napus, like many other plants, may expressan exclusion strategy (Khan, 2001) accumulating Cr in roots more

in greenhouse pots.

Ni Pb Zn

0.7±0.1 a 0.6±0.1 a 30.7±2.1 a10.7±0.6 b 19.6±1.9 b 156.1±12.3 b13.2±0.4 bc 23.4±0.4 c 186.9±2.0 c14.0±0.7 c 23.6±0.8 c 190.4±5.5 c15.0±2.1 c 24.6±0.7 c 195.6±2.1 c

1.9±0.4 a 1.2±0.2 a 19.7±1.8 a2.9±0.2 a 3.9±0.4 a 39.1±3.1 a5.2±0.5 b 9.1±1.1 b 78.9±11.7 b7.9±0.5 c 15.2±1.3 c 118.8±11.4 c8.2±0.8 c 16.6±2.2 c 127.5±14.0 c

rtical column and same plant tissues followed by the same letter are not significantly

Table 3BF and TF of studied metals in Brassica napus for different treatments in greenhouse pots.

Metal Factors T1 T2 T3 T4 T5

Cd BF 0.43±0.02 b 0.35±0.03 a 0.41±0.01 b 0.42±0.01 b 0.43±0.01 bTF 1.32±0.19 a 1.78±0.28 b 1.57±0.10 ab 1.21±0.04 a 1.16±0.12 a

Cr BF 0.03±0.00 a 0.13±0.01 b 0.18±0.00 c 0.19±0.01 c 0.19±0.00 cTF 0.41±0.10 a 5.04±0.94 c 2.73±0.35 b 1.60±0.11 ab 1.48±0.18 a

Cu BF 0.19±0.03 b 0.12±0.01 a 0.14±0.00 a 0.15±0.00 a 0.15±0.00 aTF 0.62±0.15 a 2.13±0.24 c 1.67±0.10 b 1.33±0.06 b 1.30±0.12 b

Ni BF 0.02±0.00 a 0.14±0.01 ab 0.18±0.01 b 0.19±0.01 c 0.20±0.03 cTF 0.41±0.08 a 3.73±0.53 c 2.55±0.26 b 1.77±0.07 b 1.84±0.35 b

Pb BF 0.01±0.00 a 0.10±0.01 ab 0.12±0.00 b 0.12±0.00 c 0.12±0.00 cTF 0.51±0.11 a 5.04±0.95 c 2.60±0.38 b 1.55±0.08 ab 1.50±0.20 ab

Zn BF 0.27±0.02 b 0.23±0.02 a 0.28±0.00 b 0.28±0.01 b 0.29±0.00 bTF 1.57±0.23 a 4.02±0.57 b 2.41±0.37 a 1.61±0.11 a 1.55±0.16 a

Analysis of variance (ANOVA): Tukey's test with 95% of significance (values in the same horizontal row and same factor followed by the same letter are not significantly different atpb0.05).

521G. Brunetti et al. / Geoderma 160 (2011) 517–523

than shoots, thus behaving as a typical tolerant plant. The Cu level inboth roots and shoots of B. napus grown on soil A2was lower than thaton soil B1 showing no effect due to bacillus amendment, whereas Pbconcentration was slightly greater in A2. Zn concentration in shootsand roots of B. napus grown on B1wasmuch lower than that of A2 and2–3 times greater than that in B.

Statically, different effects of the bacillus were observed on theaccumulation efficiency of metals in B. napus grown on soils A2 and B1(Table 4). In soil B1, the bacillus enhanced shoots uptake of Cu,whereas in soil A2 the bacillus enhanced the shoots accumulation ofCr and Pb. No significant effect was observed in metal uptake by rootsin B1 and A2 soils after the addition of bacillus. Thus, more specificstudies are needed to distinguish the role of this strain on metalmobility in field conditions.

The BF values of B. napus were lower than 1 for all tested metals(Table 5), whereas with few exceptions, the TF values were generallylower than 1, except for Cd. This result explains the high concentra-tion of metals found in roots. In general, B. napus in field appeared tobehave as a tolerant plant with relatively high efficiency inaccumulating and translocating different metals.

3.4. Suitability of B. napus for metals phytoextraction

Based on the results of greenhouse experiments, the metalphytoextraction by B. napus for one hectare of contaminated soil hasbeen estimated to need several centuries (Table 6). The amount ofmetals that B. napus is able to extract from soil was calculated from themetal concentration in the plant and the total plant biomass,considering 5 t ha−1 per year as the average harvest recorded for B.napus in the studied area, and one crop per year. The amount of metalsto be extracted from soil was calculated as the difference between themetal concentration (Cr, Cu, Pb and Zn) in soil, considering 20 cm topsoil (bulk density 1.3 g cm−1), and the limits established by the Italianlaw. According to McGrath and Zhao (2003), the efficiency ofphytoextraction is determined by two key factors: biomass produc-

Table 4Concentration of heavy metals (mg kg−1) in shoots and roots of Brassica napus grown in fi

Plots Cd Cr Cu

Shoots B1-uphill 0.52±0.08 b 0.50±0.13 a 5.4B1-dowhill 0.65±0.10 b 0.58±0.09 a 9.8A2-uphill 0.18±0.05 a 22.59±10.71 b 6.8A2-dowhill 0.21±0.0 a 32.76±1.6 c 7.2

Roots B1-uphill 0.28±0.02 b 0.84±0.13 a 15.3B1-dowhill 0.36±0.08 b 0.41±0.04 a 14.4A2-uphill 0.19±0.01 a 57.04±18.43 c 7.6A2-dowhill 0.19±0.02 a 39.02±2.71 b 7.4

The values in the table are mean±STD. B1-dowhill: control soil+bacillus; A2-dowhill: csignificance (values in the same column followed by the same letter are not significantly d

tion and metal BF. These authors concluded that phytoextraction isnot feasible using plants having a low BF for metals, regardless of howlarge is the achievable biomass. In particular, the BF should be 20 orgreater to achieve a halving of soil metal contents in less than 10 cropharvests, assuming that the metal is taken up by plants from the top20-cm soil. Further, with a biomass of 20 t ha−1 per crop, a BF greaterthan 10 would be required (McGrath and Zhao, 2003). Data in Table 6show that hundreds of years would be needed to respect Italianregulation limits. Due to this, phytoremediation by B. napus cannotbe considered the appropriate choice for metal polluted soils in thestudied area.

4. Conclusions

B. napus showed a different metal tolerance and accumulationin field conditions compared to the pot experiment. In the potexperiment, a significantly high accumulation of Cr, Cu, Pb and Znwasfound in shoots and roots of B. napus. The accumulation of metals wasfound to be higher in shoots than in roots, which is the typicalbehavior of accumulator species. In the field experiment, B. napusfailed to achieve the same amount of accumulated metals achievedunder the greenhouse conditions. The accumulation of studied metalsin plant parts was relatively low, roots accumulated more than shootsshowing tolerance mechanisms allowing B. napus to cope with highmetal concentration in soil.

Data obtained also indicated that the type of soil amendmentaffected significantly the concentrations of metals in B. napus. Bothcompost and B. licheniformis BLMB1 strain enhanced B. napusaccumulation of metals, especially Cr, in pot experiment. Generally,B. licheniformis BLMB1 showed high resistance to high concentrationsof Cr. Further researches are needed to elucidate the mechanisms ofthis strain against toxic elements, Cr in particular, in soil and plant, toimprove the knowledge about the potential for field application, andto reduce time needed for soil recovery.

eld conditions.

Ni Pb Zn

8±0.89 a 0.58±0.05 a 0.96±0.10 a 25.96±6.48 a2±0.52 c 0.64±0.05 ab 0.93±0.10 a 31.92±1.46 a9±1.09 ab 0.77±0.15 b 1.73±0.37 b 73.79±7.05 b0±0.9 b 0.80±0.1 b 2.25±0.4 c 69.60±1.7 b8±1.48 b 1.25±0.16 b 1.49±0.26 a 23.44±0.95 a9±1.01 b 0.65±0.05 a 1.42±0.08 a 26.44±2.31 a6±0.99 a 1.45±0.28 b 2.57±0.61 b 100.72±15.29 c2±0.50 a 1.42±0.07 b 2.35±0.25 b 71.09±2.08 b

ontaminated soil+bacillus. Analysis of variance (ANOVA): Tukey's test with 95% ofifferent at pb0.05).

Table 6Time remediation (years) needed by Brassica napus for different treatments ingreenhouse pots.

Treatment Cr Cu Pb Zn

T2 (polluted soil) 3730 1540 2715 1764T3 (polluted soil+compost) 2721 1317 2277 1473T4 (polluted soil+bacillus) 2592 1305 2256 1446T5 (polluted soil+compost+bacillus) 2554 1260 2161 1408

Table 5BF and TF of studied metals in Brassica napus in field condition.

Plots Cd Cr Cu Ni Pb Zn

BF B1-uphill 0.72±0.16 b 0.01±0.00 a 0.17±0.04 b 0.02±0.00 a 0.02±0.00 b 0.22±0.05 bB1-dowhill 1.01±0.16 c 0.01±0.00 a 0.32±0.02 c 0.02±0.00 a 0.02±0.00 b 0.28±0.01 cA2-uphill 0.19±0.05 a 0.02±0.01 b 0.04±0.01 a 0.02±0.00 a 0.002±0.004 a 0.14±0.03 aA2-dowhill 0.23±0.02 a 0.03±0.00 c 0.05±0.01 a 0.01±0.00 a 0.003±0.004 a 0.14±0.03 a

TF B1-uphill 1.75±0.30 b 0.64±0.10 ab 0.42±0.05 a 0.49±0.08a 0.66±0.04 a 1.13±0.23 aB1-dowhill 1.80±0.09 b 1.35±0.08 c 2.16±0.26 c 0.96±0.20 b 0.68±0.03 a 1.14±0.27 aA2-uphill 0.93±0.23 a 0.40±0.02 a 0.90±0.11 b 0.57±0.15 a 0.65±0.04 a 0.74±0.07 aA2-dowhill 1.17±0.18 a 0.84±0.06 b 0.97±0.20 b 0.56±0.05 a 0.96±0.15 b 0.98±0.12 a

The values in the table are mean±STD. B1-dowhill: control soil+bacillus; A2-dowhill: contaminated soil+bacillus. Analysis of variance (ANOVA): Tukey's test with 95% ofsignificance (values in the same column followed by the same letter are not significantly different at pb0.05).

522 G. Brunetti et al. / Geoderma 160 (2011) 517–523

Acknowledgements

This work has been funded by the Regione Puglia (Italy) throughthe research project POR Puglia 2000–2006, Misura 1.8-Azione 4:“Monitoraggio siti inquinati.” Supporto scientifico alle attività direcupero funzionale ed il ripristino ambientale del sito inquinatodell'Alta Murgia. P. Soler-Rovira is a recipient of a contract from JAE-Doc program of CSIC.

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