Heavy metal accumulation and growth responses in ... - CiteSeerX

Environmental and Experimental Botany 52 (2004) 79–88

Heavy metal accumulation and growth responses in poplar clones Eridano (Populus deltoides × maximowiczii) and I-214 (P. × euramericana) exposed to industrial waste Luca Sebastiani a,∗ , Francesca Scebba a,1 , Roberto Tognetti b a b

Scuola Superiore “Sant’Anna” di Studi Universitari e di Perfezionamento, Piazza Martiri della Libertà 33, 56127 Pisa, Italy Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università del Molise, Via De Sanctis, 86100 Campobasso, Italy Accepted 13 January 2004

Abstract In this study, the effects of non-hazardous levels of heavy metal (Zn, Cu, Cr and Cd)-enriched organic waste on biomass partitioning and heavy metal accumulation in plant organs in July and October were determined for two poplar clones (Populus deltoides × maximowiczii—clone Eridano and P. × euramericana—clone I-214) commonly used in Italian poplar plantations. Soil amended with the industrial organic waste did not exert any toxic effects on plants. Leaf, stem, root and woody cutting biomasses of treated plants were significantly greater than in the controls in both clones, except for stem biomass at the beginning of October. Leaf area of Eridano and I-124 treated plants was significantly higher than that of control plants, both in July and October, while specific leaf area (SLA) did not show any significant changes due to treatment. Shoot (SMR) and root mass ratios (RMR) were not significantly affected by the treatment in July, while in October plants grown in treated soil showed significant differences in stem and root biomass allocation with respect to controls. Among the four heavy metals (Zn, Cu, Cr and Cd) contained in the industrial organic waste, only Zn, Cu and Cr concentrations in plants differed consistently between clones or soil treatments, while Cd levels were always below the detection limits. Both phytoextraction and phytostabilisation strategies were observed in the two clones studied. The results suggested that only non-hazardous industrial biosolid levels might be environmentally sustainable for poplar plantations. © 2004 Elsevier B.V. All rights reserved. Keywords: Cadmium; Chromium; Copper; Growth analysis; Phytoremediation; Zinc

1. Introduction Heavy metals are natural constituents of the Earth’s crust, but human activities have drastically altered ∗ Corresponding author. Tel.: +39-050-883070; fax: +39-050-883495. E-mail address: [email protected] (L. Sebastiani). 1 Present address: CNR, Via Moruzzi 1, 56124, 56127 Pisa, Italy.

their geochemical cycles and biochemical balance. Heavy metal pollution is responsible for several environmental problems and risks to human health, including decreased soil microbial activity and fertility, and yield losses (McGrath et al., 1995). Actually, large areas of land are contaminated with heavy metals deriving from urban activities (municipal sewage and waste incinerators), agricultural operations (fertilisers and pesticides) and industrial processing (met-

0098-8472/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2004.01.003

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L. Sebastiani et al. / Environmental and Experimental Botany 52 (2004) 79–88

alliferous mining, smelting industry, paint factory and tannery). Heavy metal-contaminated waste can be disposed in landfills, but this solution negatively affects human health, wild flora and fauna, as well as productivity of crop plants and livestock (Lasat, 2002). The management of heavy metal-contaminated water or biosolids is becoming more challenging as stricter regulations to improve water quality and soil fertility are imposed. In Italy, many paint factories and tanneries need a sustainable alternative to expensive technologies, such as treatment plants or large landfills, for treating and/or immobilising heavy metal-enriched organic waste. Phytoremediation is an emerging cleanup technology, which uses grasses or higher plants for treating environmental contaminants such as heavy metals, trace elements, organic or radioactive compounds in soils, groundwater, and industrial waste (Baker et al., 1991; Raskin et al., 1997; Wenzel et al., 1999). Phytoremediation includes the overall biological, chemical, and physical processes that enable the uptake, sequestration, degradation, and metabolisation of contaminants, either by plants or by organisms that constitute the plant’s rhizosphere. Compared to current remediation technologies (landfill disposal or in situ chemical and physical treatments) phytoremediation provides an in situ solution at a relative low level of financial and technical input (Baker and Brooks, 1989; Cunningham and Berti, 1993; Raskin et al., 1994). The early phytoremediation studies used hyperaccumulator species (Baker and Brooks, 1989; Baker et al., 1994), which are plants able to accumulate unusually high levels of metals in their tissues (McGrath et al., 1997). In addition to hyperaccumulators, plants such as trees and grasses are now being actively evaluated (Entry et al., 1996; Burken and Schnoor, 1998; Robinson et al., 2000; Stoltz and Greger, 2002), though their metal bioconcentrating capability is well below that of hyperaccumulator plants (Chaney et al., 1997). Fast-growing tree species, such as poplar, could be a suitable candidate to treat heavy metal-polluted soils (Cunningham and Ow, 1996; Schnoor, 2000) and to produce economically valuable non-food biomass exploitable for energy production. Compared to herbaceous species, poplar trees have several advantageous characteristics, such as a deeper root system, a higher productivity and transpiration activity. Poplar trees are already being studied for clean

up of soil or water polluted by atrazine- (Burken and Schnoor, 1998), trichloroethylene- (Newman et al., 1997), chloroacetanilide herbicides- (Gullner et al., 2001), Cd- (Robinson et al., 2000), Se- (Pilon-Smits et al., 1998), and Zn-polluted (Di Baccio et al., 2003) soil or water, as well as to store excess atmospheric CO2 (Tognetti et al., 1999). The removal of heavy metals by plants is based on their ability to take up these potentially harmful chemicals into their tissues, i.e. phytoextraction. Alternatively, contaminated sites and sediments can be stabilised using vegetation, which mitigates the migration of toxic contaminants in the soil profile, i.e. phytostabilisation. In particular, poplars provide promising examples in both approaches (Di Baccio et al., 2003; Schnoor, 2000; Tognetti et al., 2004). The potential beneficial reuse of heavy metal-enriched organic waste in poplar plantations will, however, depend on the genetic background of plants. Besides the uptake of metals into tree organs, the phytotoxicity of specific wastes and the performance of poplar clones are of concerns and they need to be studied before large-scale field practices of phytoremediation can be applied. In this study, the effects of heavy metal-enriched organic waste on biomass partitioning and heavy metals accumulation into plant organs were determined for two poplar clones (I-214 and Eridano). These two clones were selected for their wide use in existing poplar plantations and for their adaptability to different environmental conditions. We hypothesised that differential clonal response, in terms of growth traits, to soil application of biosolid containing non-hazardous levels of heavy metals would be reflected in different accumulation potential.

2. Materials and methods 2.1. Plant material and treatments Woody cuttings of two hybrid poplar clones (Populus deltoides × maximowiczii—clone Eridano and P. × euramericana—clone I-214) were rooted in perlite in a nursery. In early spring (1 March), 24 homogeneously rooted and uniform in size (height ranged between 18 and 20 cm) cuttings (of each clone) were selected for the experiments and randomly assigned to pots.


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Table 1 Particle size distribution and physico-chemical properties of the soil used for the experiment

Table 2 Physical and chemical properties of the industrial organic waste used to amend the soil

Clay (%, w/w) Silt (%, w/w) Sand (%, w/w) pH (in water) Organic matter (%) Total CaCO3 (%) Active CaCO3 (%) Cation exchange capacity (meq./100 g) C/N Total N (‰) Assimilable P (ppm) Exchangeable K (ppm) Exchangeable Mg (ppm) Exchangeable Ca (ppm) Assimilable Cu (ppm) Assimilable Zn (ppm) Soluble B (ppm) Total Fe (%) Total Cu (ppm) Total Zn (ppm) Total Cr (ppm) Total Cd (ppm)

Organic matter (%) Relative humidity (%) Total N (%) Total P (ppm) Total K (ppm) Total Ca (ppm) Total Fe (ppm) Total Zn (ppm) Total Cr (ppm) Soluble Cl (ppm) Total Cu (ppm) Total Cd (ppm)

20.4 30.7 48.9 7.9 1.91 34.4 6.3 13.1 8.6 1.3 12 136 228 2188 2.63 7.98 0.08 2.02 25.3 112 106 5.5

Analyses followed the standard procedures described by Italian Society of Soil Science (AAVV, 2000) and Soil Science Society of America (AAVV, 1982).

30.3 62.9 4.49 4900 700 109000 54000 10300 14800 6200 102 4.4

Analyses followed the standard procedures described by Italian Society of Soil Science (AAVV, 2000) and Soil Science Society of America (AAVV, 1982).

was determined using an area meter (Delta-T Devices Ltd., Cambridge, UK). Leaf (LMR), shoot (SMR), root (RMR) and woody cuttings mass ratios (WcMR) were calculated as the ratio of the corresponding component biomass to the total plant biomass. Specific leaf area (SLA) was calculated as the ratio of leaf area to corresponding leaf biomass. 2.3. Heavy metals determination

Each pot was filled with a sandy-loam soil (Table 1) amended with 0 g (control soil) or 150 g (treated soil) of an industrial organic waste (63% dry mass) containing, on a dry weight basis: 10,300 ppm Zn, 102 ppm Cu, 14,800 ppm Cr, and 4.4 ppm Cd; while total N, P and K were 44.9, 4.9 and 0.7 mg kg−1 , respectively (Table 2). As a result of mixing “manure” in soil, total Cr content increased from 4770 to 6169 mg (on a dry mass basis), and total Zn content from 5040 to 6013 mg, while total Cu and Cd content did not vary consistently. All pots were fertilised with a slow-release commercial fertiliser, manually weeded and daily drip irrigated throughout the experiment to avoid leaching of nutrients and heavy metals.

Leaves, stem and roots samples were obtained from three plants of each clone treatment combination at the beginning of October for heavy metals determination. Oven-dried plant organs were homogenised to a fine powder in a blender (Waring) for subsequent analysis. One gram of leaves, stem and roots powder was weighed and mineralised in an HCl–HNO3 (3:1) solution. The clarified extract was diluted with ultra-pure water and used for atomic absorption measurements of heavy metals (PU 9200X, Philips, The Netherlands). Uptake ratio was calculated as the ratio between the total amount of each heavy metal in whole plant divided by the mean total amount value of heavy metal in the corresponding soil (control and treated).

2.2. Biomass partitioning 2.4. Statistical analysis In mid-July and at the beginning of October, six plants of each clone and treatment combination were harvested for biomass analysis of roots, stem, leaves and woody cutting. Plant material was oven-dried at 70 ◦ C until constant weight was reached. Leaf area

Data were subjected to a two-way analysis of variance (ANOVA) to statistically examine the effects of clone and waste treatment. Statistical analysis was conducted using SYSTAT statistical package (SYS-

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TAT Inc., Evanston, IL, USA). Separation of means was performed by post-hoc (LSD test) comparison at the 0.05 significance level.

3. Results Leaf, stem, root and woody cutting biomasses (Table 3) of treated plants were significantly greater than in the controls in both clones, except (P-value = 0.1990) for stem biomass at the beginning of October. In particular, the biomass of leaves, stems and roots collected in July were 78, 113 and 126% (Eridano) and 49, 64 and 44% (I-214) higher in treated than in control plants, respectively. However, at the beginning of October, the biomass of leaves, stems and roots were only 32, 11 and 77% (Eridano) and 39, 32 and 69% (I-214) higher in treated than in control plants, respectively. Changes in total biomass (data not shown) for both clones also reflected this trend, with 91 and 40% (July) and 45 and 52% (October) increase, respectively, for Eridano and I-214 grown in contaminated soil compared to control plants. Leaf area (Table 4) of Eridano and I-124 treated plants was significantly higher than that of control plants, both in July and October, while SLA did not show any significant changes due to treatment. In July, LMR did not differ between treatments: 0.345 and 0.342 g g−1 in Eridano and 0.276 and 0.291 g g−1

in I-214, for control and treated plants, respectively. In October, LMR showed a slight decrease (P-value = 0.0578) induced by the treatment in both clones: 0.210 g g−1 versus 0.187 g g−1 in Eridano and 0.186 g g−1 versus 0.169 g g−1 in I-214, in control and treated plants, respectively. SMR and RMR were not significantly affected by the treatment in July, while in October plants grown in treated soil showed significant differences in stem (P-value = 0.0375) and root (P-value = 0.0008) biomass allocation with respect to controls. In particular, SMR decreased (0.262 g g−1 versus 0.207 g g−1 in Eridano and 0.279 g g−1 versus 0.245 g g−1 in I-214, respectively, in control and treated plants), while RMR values increased (from 0.379 to 0.461 g g−1 in Eridano and from 0.352 to 0.389 g g−1 in I-214, for control and treated plants, respectively). Among the four heavy metals (Zn, Cu, Cr and Cd) contained in the industrial organic waste, only Zn, Cu and Cr concentrations in plants differed consistently between clones or soil treatments, while Cd levels were always below the detection limits. Plants grown in treated soil showed consistently higher Zn concentrations in leaves (Fig. 1A) of both clones, while no differences were observed in stem (Fig. 1B) and roots (Fig. 1C). Differences in Zn concentrations between clones were also observed. In particular, I-214 plants had significantly higher Zn concentrations in leaves, while Eridano showed higher Zn concentrations in stem and roots.

Table 3 Leaves, stem, roots and woody cutting biomass of Eridano and I-214 poplar clones grown in soil treated with industrial waste and control soil, measured in July and at the beginning of October Period

Organ

Clone

P-value (ANOVA)

Eridano

I-214

Control July

October

Treated

Clone

Control

Soil

Clone × soil

Treated

Leaves Stem Roots Woody cuttings

6.99 2.12 4.12 7.17

± ± ± ±

0.96 0.22 1.09 1.90

12.41 4.51 9.32 12.73

± ± ± ±

4.57 2.29 4.76 10.32

5.86 2.46 5.12 7.94

± ± ± ±

1.00 0.47 0.84 1.99

8.73 4.03 7.39 9.79

± ± ± ±

3.41 2.15 3.35 3.53

Leaves Stem Roots Woody cuttings

11.42 15.10 21.06 8.07

± ± ± ±

1.54 6.42 4.90 0.95

15.03 16.71 37.17 11.63

± ± ± ±

1.26 2.78 4.35 0.88

8.27 12.64 15.57 8.14

± ± ± ±

0.75 3.64 0.76 0.96

11.47 16.64 26.27 13.24

± ± ± ±

1.68 2.41 1.74 1.60

n.s. n.s. n.s. n.s.

∗ ∗ ∗

n.s.

∗∗∗

∗∗∗

n.s.

n.s.

n.s.

∗∗∗

∗∗∗

∗∗∗

n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

Values are mean ± standard deviation for each soil treatment (n = 4 individuals). Units are grams. ANOVA values for clone and soil, and the interactions between factors (clone × soil) are shown: n.s. = not significant; ∗ = significant at P ≤ 0.05; ∗∗∗ = significant at P ≤ 0.001.


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Table 4 Leaf area, specific leaf area (SLA), leaf (LMR), stem (SMR), root (RMR) and woody cuttings (WcMR) mass ratio of Eridano and I-214 poplar clones grown in soil treated with industrial waste and control soil, measured in July and at the beginning of October Period

Organ

Clone

P-value (ANOVA)

Eridano

July

October

I-214

Clone

Control

Treated

Control

Treated

Leaf area SLA LMR SMR RMR WcMR

0.0863 ± 0.0111 0.0123 ± 0.0002 0.345 ± 0.026 0.105 ± 0.011 0.202 ± 0.037 0.348 ± 0.033

0.1484 ± 0.0376 0.0121 ± 0.0007 0.342 ± 0.058 0.117 ± 0.015 0.242 ± 0.032 0.299 ± 0.074

0.0586 ± 0.0117 0.0101 ± 0.0015 0.276 ± 0.021 0.115 ± 0.004 0.240 ± 0.015 0.369 ± 0.036

0.1049 ± 0.0470 0.0122 ± 0.0009 0.291 ± 0.026 0.128 ± 0.025 0.245 ± 0.060 0.336 ± 0.096

Leaf area SLA LMR SMR RMR WcMR

0.1104 ± 0.0160 0.0097 ± 0.0010 0.210 ± 0.026 0.262 ± 0.052 0.379 ± 0.027 0.149 ± 0.025

0.1549 ± 0.0103 0.0103 ± 0.0003 0.187 ± 0.010 0.207 ± 0.026 0.461 ± 0.024 0.145 ± 0.010

0.0917 ± 0.0082 0.0111 ± 0.0004 0.186 ± 0.015 0.279 ± 0.045 0.352 ± 0.035 0.183 ± 0.018

0.1161 ± 0.0228 0.0102 ± 0.0014 0.169 ± 0.021 0.245 ± 0.022 0.389 ± 0.020 0.196 ± 0.025

∗ ∗ ∗∗

n.s. n.s. n.s.

Soil

Clone × soil

∗∗

n.s.

n.s. n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s.

∗∗

∗∗∗

n.s.

n.s. n.s. (0.0578)

∗

n.s.

∗

∗∗

∗∗∗

∗∗

n.s.

∗

n.s. n.s. n.s. n.s. n.s. n.s.

Values are mean ± standard deviation for each soil treatment (n = 4 individuals). Units are m2 for leaf area, m2 g−1 for SLA and g g−1 for LMR, SMR, RMR and WcMR. ANOVA values for clone and soil, and the interactions between factors (clone × soil) are shown: n.s. = not significant; ∗ = significant at P ≤ 0.05; ∗∗ = significant at P ≤ 0.01; ∗∗∗ = significant at P ≤ 0.001.

Copper concentrations in stem (Fig. 1B) of control plants were significantly higher than in treated ones (10.3 mg kg−1 versus 8.1 mg kg−1 in Eridano and 11.2 mg kg−1 versus 7.6 mg kg−1 in I-214). Chromium concentrations in roots (Fig. 1C) of treated plants were significantly higher than in controls only in both clones: 5.7 mg kg−1 versus 15.3 mg kg−1 in Eridano and 8.2 mg kg−1 versus 14.0 mg kg−1 in I-214, in control and treated plants, respectively. The total amount of heavy metals accumulated in leaves, stem and roots of the two poplar clones are shown in Fig. 2. Zinc uptake in leaves, stem and roots of both clones was significantly higher in treated plants in comparison to controls. Moreover, while uptake of Zn in leaves did not differ between clones (3019 ␮g in Eridano versus 2883 ␮g in I-214), Zn accumulation in stem and roots of Eridano was much higher compared to I-214 (1203 and 2579 ␮g versus 786 and 1230 ␮g, respectively). Copper uptake in leaves (Fig. 2A) of treated plants was higher than in controls, and it was higher in Eridano leaves (239 ␮g) compared to I-214 leaves (172 ␮g). Chromium uptake in stem and roots (Fig. 2B and C) was higher in treated plants of both clones: 214 ␮g versus 402 ␮g (stem) and 113 ␮g versus 587 ␮g (roots) in Eridano and 226 ␮g

versus 292 ␮g (stem) and 126 ␮g versus 382 ␮g (roots) in I-214, respectively. The total amount of heavy metals in poplar clones are shown in Fig. 3A. Whole plant Zn uptake in both clones was significantly higher in treated plants in comparison to controls. Whole plant Zn uptake in clone Eridano was much higher compared to I-214 (9.13 mg versus 4.48 mg in Eridano and 5.97 mg versus 3.83 mg in I-214 in treated and control soils, respectively). Whole plant Cu and Cr uptake of treated plants were higher than in controls, and while clone Eridano took up more Cu than I-214, both clones behaved similarly for Cr. Uptake ratios in poplar clones are shown in Fig. 3B. Higher values were obtained for Zn and Cu, while for Cr values remained very low. The biosolid treatment increased heavy metals uptake coefficients in poplars, and both clones showed a similar trend except for Zn.

4. Discussion The beneficial reuse of organic waste enriched in heavy metals is not an easy task to achieve due to the stricter regulations, which have been recently im-

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Fig. 1. Zinc (Zn), copper (Cu) and chromium (Cr) concentration in leaves (A), stem (B) and roots (C) of I-214 and Eridano poplar clones grown in soil treated with industrial waste and control soil at the beginning of October. Values are mean + standard deviation for each soil treatment combination (n = 4 individuals). ANOVA values for clone and soil, and the interactions between factors (clone × soil) are shown: n.s. = not significant; ∗ = significant at P ≤ 0.05; ∗∗ = significant at P ≤ 0.01; ∗∗∗ = significant at P ≤ 0.001.

Fig. 2. Total amount of zinc (Zn), copper (Cu) and chromium (Cr) uptake in leaves (A), stem (B) and roots (C) of I-214 and Eridano poplar clones grown in soil treated with industrial waste and control soil at the beginning of October. Values are mean + standard deviation for each soil treatment combination (n = 4 individuals). ANOVA values for clone and soil, and the interactions between factors (clone×soil) are shown: n.s. = not significant; ∗ = significant at P ≤ 0.05; ∗∗ = significant at P ≤ 0.01; ∗∗∗ = significant at P ≤ 0.001.


Fig. 3. Zinc (Zn), copper (Cu) and chromium (Cr) whole plant uptake (A) and uptake ratio (B) (ratio between the total amount of heavy metal in plant and in soil) of I-214 and Eridano poplar clones grown in soil treated with industrial waste and control soil at the beginning of October. Values are mean + standard deviation for each soil treatment combination (n = 4 individuals). ANOVA values for clone and soil, and the interactions between factors (clone × soil) are shown: n.s. = not significant; ∗ = significant at P ≤ 0.05; ∗∗ = significant at P ≤ 0.01; ∗∗∗ = significant at P ≤ 0.001.

posed. Indeed, the effect of heavy metals on soil is becoming a widespread problem on dump sites and agricultural lands. Metals do not degrade and large-scale recovery of contaminated soil is difficult to achieve. The organic waste of industrial origin containing several heavy metals (Zn, Cu, Cr and Cd) are considered particularly dangerous to plants with respect of other causes of pollution, such as those determined by the application of fertilisers and pesticides containing heavy metals. In this experiment, the addition to soil of industrial biosolids from tanneries was deliberately designed to

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reach non-lethal and below risk threshold of heavy metal concentrations (total Cr and Zn amount in soil increased of about 30 and 20%, respectively) to verify the hypothesis that the application of biosolids would enhance plant growth and heavy metal uptake in poplars at the same time. Plants were subjected to the amended soil for one growing season, which may be considered a short time for woody plants. This relatively short-term exposure was nevertheless sufficient to achieve early evidence of positive effects on leaf functioning and structure (Tognetti et al., 2004) and biomass partitioning. Soil treatment with the industrial organic waste did not exert any toxic effect on the plants and, despite this short-term exposure, positive effects on growth were observed for both poplar clones (Table 3). Additional N and other nutrients contained in the industrial waste (Table 2) may have caused such growth responses, though plants were fertilised during the experiment. Enhanced growth in treated plants compared to controls was observed in both clones, although differences were greater in Eridano. The biomass produced by poplar plants grown in amended soil was mainly partitioned toward roots: 46% versus 38% of total biomass in Eridano and 39% versus 35% of total biomass in I-214, for amended and control soils, respectively. This result was unexpected because increased nutrients from the added organic waste should have resulted in lower RMR. It is possible that heavy metal enrichment has increased partitioning to roots, though this trait needs further investigation. In plants heavy metals can play different roles and can be roughly divided into: (a) essential (i.e. Zn and Cu), which are required for a variety of metabolic processes; and (b) non-essential (i.e. Cr and Cd) (Taiz and Zeiger, 1998). Independent from their biological function, both essential and non-essential heavy metals can be toxic above a certain threshold and plants have evolved a complex metal homeostasis network system, which regulates their uptake and distribution enabling an effective protection of the metabolic processes (Clemens, 2001; Clemens et al., 2002). Different mechanisms or strategies have been proposed to explain plant tolerance to toxic levels of metals: “exclusion”, restricting metal uptake and/or root to shoot transport; “inclusion” sequestrating and compartmenting metals in organs and/or organelles (Baker,

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1981; Baker and Walker, 1990; Salt et al., 1998). All these aspects of plant–heavy metal interactions are well documented for herbaceous and hyperaccumulator species, but a lack of knowledge exists for woody plants (Lindberg and Greger, 2002). In experiments with I-214 clone exposed to increasing Zn concentration (from 0.1 to 1000 ␮M), Di Baccio et al. (2003) suggested that poplar adopts both strategies. Although the concentration and the type of heavy metals used in this study were different, it is possible to hypothesise a similar behaviour in our experimental plants. Zinc was actively transported and accumulated in leaves of both clones, while Cu was almost entirely confined to roots, particularly in Eridano, and Cr behaved intermediately. Phytoremediation of heavy metal-polluted sites have been studied in terms of: (a) removal (phytoextraction) of heavy metals from soil by concentrating the metals in above ground plant organs (stems and leaves); (b) absorption, concentration and precipitation (rhizofiltration); and (c) stabilisation (phytostabilisation) by roots (Ensley, 2000). Regardless of phytoremediation strategies, a better understanding of uptake, transport and tolerance to heavy metal in plants remains of great importance for planning of large-scale field applications of this technique. Recently, fast-growing hybrid poplar trees have been suggested as a valuable solution to stabilise heavy metals in contaminated soils (Schnoor, 2000), showing some potential for Zn phytoextraction (Di Baccio et al., 2003). In this study, the combined effects of enhancement in plant growth and heavy metals concentrations in plant organs, following the application of biosolids, resulted in significant increases of heavy metal uptake in both tested clones. Phytoextraction of Zn (leaves and stem) and Cr (stem) as well as phytostabilisation (roots) of Zn, Cu and Cr were observed in both Eridano and I-214 (Fig. 2). Heavy metals were removed at a smaller rate compared to application, a few mg versus thousand mg, respectively. Our experiment confirms the observation of Moffat et al. (2001) on 3-year-old poplar clones (Populus trichocarpa × P. deltoides, clone Beaupré and P. trichocarpa, clone Trichobel) grown in the field. Indeed, while plants treated with industrial waste increased their uptake ratios for heavy metals (Fig. 3B), the content of heavy metals in the soil re-

mained almost stable, at least in the initial phase. At final harvest, biosolids treatment increased the proportion of non-harvestable plant parts (roots) compared to above ground potential yield. Considering heavy metal contents of the harvestable portion (stem) of poplar plants treated with biosolids and hypothesising a plantation density of 10,000 plants ha−1 (short rotation forestry), producing 119 t of dry biomass ha−1 in an 11-year cycle (Bonari, 2001), our experimental values correspond to 6623 and 9657 g ha−1 of Zn, 902 and 962 g ha−1 of Cu, 2058 and 2700 g ha−1 of Cr, respectively, for I-214 and Eridano. If biosolid amendments would increase by 10 ppm the level of Zn, Cu and Cr (in the case of Cr, which is present at the level of 14,800 mg kg−1 , this means 27 t ha−1 of industrial organic waste) in 1.5 × 107 kg of soil (1 ha surface × 1 m depth and 1.5 kg dm−3 apparent bulk density), the recovery of original heavy metal contents could be attained in 22.7 and 15.5 years for Zn, in 166.2 and 155.9 years for Cu and in 72.9 and 55.5 years for Cr, respectively, in I-214 and Eridano. These hypothetical estimates suggest that only non-hazardous industrial biosolid levels, which maintain total heavy metals concentrations close to background values, may be environmentally sustainable, non-lethal and below risk thresholds for poplar plantations. Heavier contamination levels could pose severe risks to plant, soil and environment. In conclusion, these poplar clones responded to industrial waste treatment through increasing plant growth and metal accumulation, although the two clones differed in their response, implying some degree of acclimation potential. Differential responses between clones suggest the need for trials with increasing level of industrial waste and for breeding programs that incorporate traits enhancing heavy metal uptake into poplar.

Acknowledgements The authors wish to thank Dr. Giuseppe Palumbo (Università del Molise) for his assistance in heavy metals analysis. This research was in part supported by Scuola Superiore “Sant’Anna” (grant ARIS-00-CV) and Cassa di Risparmio di San Miniato Foundation.


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