Zinc accumulation in plant species indigenous to a portuguese polluted site: relation with soil contamination

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Zinc Accumulation in Plant Species Indigenous to a Portuguese Polluted Site: Relation

with Soil Contamination

Ana P. G. C. Marques, Anto´nio O. S. S. Rangel, and Paula M. L. Castro* ABSTRACT

The levels of zinc accumulated by roots, stems, and leaves of two plant species, Rubus ulmifolius and Phragmites australis, indigenous to the banks of a stream in a Portuguese contaminated site were inves-tigated in field conditions. R. ulmifolius, a plant for which studies on phytoremediation potential are scarce, dominated on the right side of the stream, while P. australis proliferated on the other bank. Hetero-geneous Zn concentrations were found along the banks of the stream. Zn accumulation in both species occurred mainly in the roots, with poor translocation to the aboveground sections. R. ulmifolius presented Zn levels in the roots ranging from 142 to 563 mg kg21, in the stems from 35 to 110 mg kg21, and in the leaves from 45 to 91 mg kg21, vs. average soil total Zn concentrations varying from 526 to 957 mg kg21. P. australis showed Zn concentrations in the roots from 39 to 130 mg kg21, in the stems from 31 to 63 mg kg21, and in the leaves from 37 to 83 mg kg21, for the lower average soil total Zn levels of 138 to 452 mg kg21found on the banks where they proliferated. Positive correlations were found between the soil total, available and extractable Zn fractions, and metal accumulation in the roots and leaves of R. ulmifolius and in the roots and stems of P. australis. The use of R. ulmifolius and P. australis for phytoextraction purposes does not appear as an effective method of metal removing, but these native metal tolerant plant species may be used to reduce the effects of soil contamination, avoiding further Zn transfer to other environmental compartments.

H

EAVYmetal contamination in ecosystems poses

ma-jor environmental problems worldwide with sub-stantial economic consequences. Phytoremediation—the use of plants to remove (phytoextraction) or immobilize (phytostabilization) contaminants (Salt et al., 1998)— may offer a safe and low cost method for the remediation of metal-contaminated soil. The relatively low potential cost of phytoremediation allows the treatment of sites that cannot be addressed with currently available en-gineering methods, while preserving the topsoil. Plants can be grown and harvested economically, leaving only residual levels of pollutants (Ensley, 2000).

Heavy metals play an important role in plant met-abolic functions and some, such as zinc, are essential for plant growth and are involved in numerous physi-ological processes (Rengel, 1999). However, at high concentrations they are strongly toxic and may impair the growth of some plant species. The metal phytoavail-ability—fraction of the total contaminant mass in soil actually available for the receptor plant (National Re-search Council, 2003)—has emerged as an important

paradigm, replacing the old belief that biological response by receptor organisms could be predicted by the total concentration in the soil (Adriano et al., 2004). During the investigation of metal bioavailability different soil extrac-tion techniques should be used (Lasat, 2002). In general, aqueous extraction seems to provide an estimate of the amount of bioavailable metal in the soil solution. Estimates of the total bioavailable metal, which includes not only the metal available in soil solution but also the metal ions bound to soil exchange sites, are obtained by extracting the soil with organic compounds.

Many studies have been conducted to identify plant species capable of accumulating Zn. Thlaspi caerules-cens is probably the best known Zn hyperaccumulator species (Whiting et al., 2001). Schwartz et al. (2001) showed evidence that the plants Armeria maritima and Arabidopsis halleri are Zn and Pb hyperaccumulators. Peralta-Videa et al. (2004) presented Medicago sativa plants as an option for cleaning up soils through Zn tis-sue accumulation. Brassica juncea (Ebbs and Kochian, 1998) and several willow species (Vandecasteele et al., 2004) have also been reported amongst the plants with potential for the phytoremediation of Zn.

It is often difficult to predict the behavior and fate of a metal on a contaminated matrix only through an ex-trapolation of results obtained from laboratory ecotoxic-ity experiments (Straalen and Denneman, 1989). In the field, differences in the metal availability for plant up-take, operation of ecological compensation mechanisms, exposure to metal mixtures, and adaptation to metal stress may occur (Lock et al., 2003). Field studies are necessary to obtain information on how indigenous plants behave under the conditions installed. The use of fast-growing pioneer species capable of colonizing poor, contaminated soils is potentially very useful for phy-toremediation strategies (Alvarez et al., 2003). Stud-ies identifying specStud-ies with potential capacity for heavy metal accumulation are necessary and valuable when selecting the most appropriate plants for phytoremedia-tion strategies. In addiphytoremedia-tion, it is important to study the accumulation of the contaminant in the different plant sections (root and shoot). A plant which accumulates higher levels of the contaminant in its harvestable sec-tions (usually stems and leaves) is considered a good can-didate for phytoextraction (Blaylock and Huang, 2000), while a species which restricts the accumulation to its roots will be useful for the stabilization of the contami-nated soil, reducing the human health and environmental hazards by a different but yet equally protective strategy– phytostabilization (Berti and Cunningham, 2000). Escola Superior de Biotecnologia, Universidada Cato´lica Portuguesa,

Rua Dr. Anto´nio Bernardino de Almeida, 4200-072 Porto, Portugal. Received 18 July 2006. *Corresponding author (plcastro@esb.ucp.pt).

Abbreviations: BCR, Community Bureau of Reference; EDTA, ethilenediaminetetraacetic acid; FA-AAS, flame atomic absorption spectroscopy; HCl, hydrochloric acid; NH4–Ac, ammonium acetate.

Reproduced from Journal of Environmental Quality. Published by ASA, CSSA, and SSSA. All copyrights reserved.

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A large industrial complex, composed essentially by chemical facilities, surrounds the area considered in the present study. For many years, several of these in-dustries have discharged their solid residues in an im-provised sediment basin in the surrounding area, and released its wastewaters into a nearby stream—“Esteiro de Estarreja”—which is a small and almost stagnated watercourse (Oliveira et al., 2001). Profiting from the high permeability of the site, the percolates proceeding from this improvised sediment basin infiltrated in the soil of the area. Therefore, the levels of heavy metals in the sediments of this stream are above the limits established by EC Directive 86/278/EC (Atkins, 1999). Among the metals present at higher levels in those sediments— average levels of 835 mg Pb kg21

, 66 mg Hg kg21, 26 mg Cr kg21, 37 mg Ni kg21, 16 800 mg Fe kg21were measured for the site (Oliveira et al., 2001)—Zn appears as one of the main contaminants, with the soils presenting levels of up to 3620 mg Zn kg21 _{(total Zn)}

(Oliveira et al., 2001). The area near the former exit of contaminated wastewaters is the most polluted one (to a distance of ca 50 m further from the exit), although the contamination along the banks and between banks is very heterogeneous, and is mainly occurring in the top 20 cm layer of the soil (Atkins, 1999; Oliveira et al., 2001). The banks of the stream (with a slope of approximately 45j), with approximately 2-m width, are periodically flooded with rainwater, from late October to late February, and the ditch of the stream (with approxi-mately 1.5-m depth) remains almost dry during the remaining months. Despite the high levels of metals in the sediments, the vegetation in the banks of the stream remains prolific, yet heterogeneously distributed. Two plants appeared as the main colonizers: Rubus ulmifolius Schott dominating the side where the wastewaters were discharged and Phragmites australis (Cav.) Trin. ex Steudel being the main colonizer on the opposite side. Both R. ulmifolius and P. australis are plant species widely distributed throughout this region of Portugal. Remediation studies using P. australis, namely for Cd (Ali et al., 2004) and also for Pb- and Zn-contaminated mine tailings (Ye et al., 1997) have shown that the plant is able to accumulate these metals. R. ulmifolius was mentioned as a vascular plant growing on mine refuse (Freitas and Prasad, 2003), but studies evaluating metal accumulation are not common.

The aim of this study was to determine the accu-mulation of Zn in different sections—roots, stems, and leaves—of plants colonizing a contaminated site (P. australis and R. ulmifolius), and relate that feature to the Zn content of the soil. Accordingly to the accu-mulation patterns and abilities of R. ulmifolius and P. australis, the analysis of their possible application in soil phytoremediation was evaluated.

MATERIALS AND METHODS Research Site and Sampling Techniques Plant and soil sampling was made at seven different spots in each bank, starting near the conduct exit and following the stream course, with each of the species being collected from

the bank in which they were predominant. The seven collec-tion spots in each bank were separated by 10 m from each other, as performed by Atkins (1999), to cover a high range of soil metal concentrations potentially toxic to plants, profiting from the heterogeneity of the site contamination. At each sampling location, 0.5 m sided squares delimited the location from which plant samples were collected. In each square three plants were collected randomly—selected plants were approx-imately 1.5 and 1 m in height, for P. australis and R. ulmifolius, respectively. A soil sample from each plant rooting zone (0- to 20-cm depth) was also collected. Sampling was made in the dry season, particularly in the flowering season for each one of the plant species in the site—April for R. ulmifolius and August for P. australis. In addition, soil and R. ulmifolius and P. australis plant samples, in a similar development stage, from uncon-taminated areas were collected, using the same procedure.

Soil Analysis

Soil samples were oven-dried at 40jC for 48 h and passed through a 2-mm sieve. The soil pH was measured using a 1:2.5 soil/water ratio. Water content was determined by drying pre-oven-dried (40jC) soil at 105jC until constant mass was achieved. Organic matter content was determined by loss on ignition. Samples for total phosphorous and nitrogen were digested at high temperatures (up to 330jC) with a Se and salicylic and sulfuric acids mixture. For total N colorimetric determination, two reagents were added to the digests: reagent 1, consisting of a mixture of a 5 3 1022_{M dissodium hydrogen}

phosphate buffer (pH 5 12.3) and a 4% bleach solution, and reagent 2 consisting of a mixture of a 1 M salicylate solution, a 1 3 1023_{M sodium nitroprusside solution and a 3 3 10}23_M

EDTA solution. For total phosphorous colorimetric determi-nation, two different reagents were added to the digests: reagent 1, consisting of a 3 3 1022_{M ascorbic acid solution and}

reagent 2 consisting of a mixture of a 6 3 1023_{M antimonyl}

tartarate solution, a 5 3 1023_{M ammonium molybdate}

solu-tion, 0.7 M sulfuric acid, and an anticoagulation agent (Wetting aerosol 22, Cytek, New Jersey, USA). The elements concen-tration on the resulting preparations was determined on an UNICAM HELIOS spectrophotometer (Waltham, USA), at 660 nm for nitrogen and 880 nm for phosphorous. For total soil Zn determination, soil samples were digested at high tempera-tures (up to 140jC) with concentrated nitric and hydrochloric acids (1:1). All previous methods were based on Houba et al. (1995). The water (De Koe, 1994), exchangeable (Thomas, 1982)—ethilenediaminetetraacetic acid (EDTA)-extractable—, and available (Thomas, 1982)—ammonium acetate (NH4

–Ac)-extractable—Zn fractions were determined using, respectively, 1:5 soil/water (De Koe, 1994), 1:5 soil/1M NH4–Ac (De Koe,

1994), and 1:10 soil/0.05 M EDTA (Houba et al., 1995) ratios. The resulting solutions were incubated for 2 h at 20jC, after which they were filtrated through a 0.45-mm cellulose acetate filter. The Zn content of the resulting digests and extracts were determined using flame atomic absorption spectroscopy (FA-AAS) in an UNICAM 960 spectrophotometer (Waltham, USA) (Houba et al., 1995). BCR (Community Bureau of Reference) reference sample CRM 141 R (calcareous loam soil) was analyzed through the referred total Zn determination analytical method. The value obtained by FA-AAS (281 6 1 mg Zn kg21_sample)

confirmed the accuracy and precision of the method by comparison with the certified value (283 6 5 mg Zn kg21_sample).

Plant Analysis

Entire plants were washed with tap water, followed by washing with HCl 0.1 M, and with demineralized water, after

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which they were separated in roots, stems, and leaves, oven-dried at 70jC for 48 h, grinded, and sieved to ,1 mm. The resulting samples were digested at high temperature (up to 205jC) with a mixture of concentrated nitric, perchloric, and sulfuric acids (40:4:1). Zinc content was determined using FA-AAS of the digests (Wallinga et al., 1989). BCR (Community Bureau of Reference) reference sample CRM 279 (sea lettuce) was analyzed using the above-described total Zn determina-tion analytical method. The value obtained by FA-AAS (52.3 6 1.5 Zn kg21 _{sample) confirmed the accuracy and}

precision of the method by comparison with the certified value (51.3 6 0.2 mg Zn kg21_sample).

Statistical Analysis

Statistical analysis was performed using the SPSS program (SPSS Inc., 2003). The data were analyzed through analy-sis of variance (ANOVA). To detect the statistical signifi-cance of differences (P , 0.05) between means, the Tukey test was performed. Regression analyses were performed with different variables and Spearman’s correlation coefficients were determined.

Chemicals

The chemicals used were analytical grade and were obtained from Pronalab (liquid reagents), and Merck (solid reagents).

RESULTS AND DISCUSSION

Soils

The soil collected from the banks of the stream pre-sented a range of pH from 5.3 to 7.5, a water content ranging from of 1.1 to 2%, and organic matter contents ranging from 7.5 to 12% (Table 1). The nutrient content of the soils was variable. The levels of nitrogen in the

contaminated soils were higher than the levels found in the control sites (Table 1).

The total Zn concentration in the soil was variable along the stream, with some significant (P , 0.05) dif-ferences registered within the sampling points (Tables 2 and 3), corroborating the data obtained by Atkins (1999) and allowing us to fulfil the objective of achieving a range of soil metal concentrations. The bank from where P. australis proliferated presented a lower level of Zn— ranging from 138 to 452 mg kg21_{dry soil—than the bank}

where the population of R. ulmifolius appeared, which ranged from 526 to 957 mg kg21 dry soil. According to Kabata-Pendias and Pendias (1984) a total fraction of 70 to 400 mg kg21 Zn in the soil would already be considered as toxic to plants. The metal content in the banks, especially on the R-side, exceeded this range.

The water, EDTA, and ammonium acetate soil-extractable fractions were also different from point to point and between banks (Tables 3 and 4). The metals considered readily available for plant uptake are those that exist as soluble components in the soil solution or that are easily desorbed or solubilized by root exu-dates, representing often only a small portion of the to-tal meto-tal content of the soil (Blaylock and Huang, 2000), as was found in the present study. Regression analysis of the levels of total Zn in the soils vs. the water-extractable, available (EDTA-extractable), and ex-changeable (NH4–Ac-extractable) Zn fractions in the

soil was performed (Table 4). For the R. ulmifolius collection spots, strong positive correlations were found between total Zn concentrations in the soil and the Zn levels in all the other fractions, with the exception of the water-extractable Zn. For the P. australis collection spots, stronger positive correlations were always found between total Zn concentrations in the soil and the Zn

Table 1. Soil properties of the study sites.†

Collection site pH Water content Organic content N P

% mg kg21 R1 to R7 (n 5 21) 6.9 6 0.4 1.5 6 0.2 9.0 6 0.9 2684 6 1306 219 6 79 (6.3–7.5) (1.1–2.0) (7.5–10.3) (1105–4984) (41–284) R. ulmifolius control (n 5 3) 6.60 6 0.1 1.00 6 0.09 8.3 6 0.4 885 6 9 202 6 11 P1 to P7 (n 5 21) 6.3 6 0.8 1.5 6 0.3 9 6 2 2640 6 978 147 6 90 (5.3–7.5) (1.0–2.0) (8–12) (1615–3893) (6–244) P. australis control (n 5 3) 6.80 6 0.1 1.8 6 0.2 9 6 2 936 6 3 157 6 16

† Results are expressed as means 6 SD. For the samples collected at the stream banks, the range (minimum to maximum) is presented. R and P refer to samples taken, respectively, from the sides where R. ulmifolius and P. australis proliferated.

Table 2. Total and extractable Zn concentrations in soils from different sampling locations of P. australis.†

Zn

Collection site Total H2O extract EDTA extract NH4–Ac extract

mg kg21 P1 452 6 24a 8.0 6 0.4a 113 6 3a 20 6 2a P2 328 6 46bd 7.8 6 0.5a 91 6 1b 14.9 6 0.4b P3 257 6 6d 3.2 6 0.7b 57 6 0.8c 12 6 1c P4 360 6 34df 4.7 6 0.4c 102 6 9d 12.0 6 0.4c P5 138 6 4e 1.0 6 0.2d 36 6 0.5e 11 6 1c P6 387 6 44f 9.2 6 0.03e 101 6 7d 27 6 2d P7 296 6 9b 1.7 6 0.06f 67 6 3f 21 6 1a Pcontrol 97 6 2g 1.6 6 0.1f 15 6 2g 7.5 6 0.4e

† Results are expressed as means 6 SD (n 5 3). Means in the same column with different letters are significantly different from each other (P , 0.05) according to the Tukey test.

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levels in all the other fractions (Table 4), which seems to indicate that in this bank the availability of Zn (pro-portionally to the total amount) is higher than in the bank where R. ulmifolius proliferates.

The inexistence of a correlation between total and water-extractable Zn and the lower correlation coeffi-cients obtained for the R. ulmifolius-derived soil, may be explained by the slightly higher average pH in the first case. In the soil, metal availability to plant roots in-creases as the soil pH dein-creases (McBride, 1994). As correlation coefficients were always positive, increasing total Zn levels in the soil will result in higher bioavail-ability of the metal.

Plants

The Zn content in the sections of plants collected in the banks of the stream varied among sites of collection, for both species (Fig. 1). In R. ulmifolius, Zn concentra-tions (mg kg21) ranged from 142 to 563 in the roots, 35.5 to 110 in the stems, and 45.1 to 90.8 in the leaves. In P. australis Zn concentrations (mg kg21) ranged from 39.4 to 130 in the roots, 31.2 to 63.3 in the stems, and 37.3 to 82.6 in the leaves. P. australis presented accumu-lation levels similar to those registered by Aksoy et al. (2005) for P. australis growing in a polluted wetland in Turkey—average Zn accumulation of approximately 70, 41, and 22 mg kg21_{dry wt., for respectively roots, stems,}

and leaves were reported.

The Zn levels determined for all the plant sections— roots, stems, and leaves—of the P. australis and R. ulmifolius individuals collected at the noncontami-nated (control) sites (Pcontroland Rcontrol, respectively)

were always lower than the ones obtained for the plants grown in the banks of the stream—Zn levels ranged from 21.6 to 26.4 mg kg21for P. australis (Table 5) and 30 to 59 mg kg21_{for R. ulmifolius (Table 6). None of the}

plants presented visual toxicity symptoms (chlorosis of the leaves or necrosis of any of the plant sections), both for individuals derived from contaminated and con-trol sites.

The Zn accumulation levels reported in this study are similar, and sometimes lower, to others registered in the literature for other plants, especially in what relates to aboveground accumulation. Mbila and Thompson (2004) evaluated the uptake of Zn by Osmorhiza longi-stylis and Sanicula marilandica plants present in mine spoils, reporting accumulation levels ranging from 114 to 159 mg Zn kg21_{dry wt. in the roots and 42 to 55 mg}

Zn kg21 dry wt. in the shoots. Del Rio et al. (2002) conducted a field survey covering contaminated sites affected by the Aznalco´llar mine spill and found several wild species, such as Scolymus hispanicus, Lupinus an-gustifolius, Fumaria agraria, Solanum nigrum, Anagallis arvensis, and Amaranthus blitoides, obtaining a range of Zn accumulation levels from 76 to 930 mg kg21_{dry wt.}

Armeria maritima ssp. halleri, Arabidopsis halleri and Arrhenatherum elatius were found to proliferate in a former smelter site in France, accumulating in their aboveground tissues, respectively, up to 966, 6269, and 752 mg Zn kg21dry wt. (Schwartz et al., 2001). Leersia hexandra, Juncus effuses, and Equisetum ramosisti plants growing in a swale receiving wastewaters from a mine in China, accumulated up to approximately 300 mg Zn kg21_{dry wt. in their shoots and 1500 mg Zn kg}21_{dry wt.}

in their roots (Deng et al., 2004).

In this study, despite the clear influence of the soil contamination in the accumulation of Zn by P. australis and R. ulmifolius, the amounts were almost always below the Zn phytotoxic levels reported for plants—500 to 1500 mg kg21, according to Chaney (1989). Excep-tions were registered in some cases for R. ulmifolius roots (according to the range presented in Table 6). The roots of both plant species, especially of R. ulmifolius, presented Zn levels generally above the considered normal levels of Zn in plant tissues—10 to 100 mg kg21, according to Frisberg et al. (1986), which may indicate that these plant species growing on the contaminated site were tolerant to Zn. Significant (P , 0.05) differ-ences between the Zn levels in the roots and the above-ground sections (stems and leaves) were observed for both plant species (Tables 5 and 6). Zinc was mainly accumulated in the roots of both plant species, indicating poor metal translocation into stems and leaves. Similar

Table 3. Total and extractable Zn concentrations in soils from different sampling locations of R. ulmifolius.†

Zn

Collection site Total H2O extract EDTA extract NH4–Ac extract

mg kg21

R1 957 6 74a 0.8 6 0.2ab 87 6 2a 4 6 0.2a

R2 992 6 30a 1.1 6 0.3c 102 6 22b 12 6 1b R3 715 6 47b 1.1 6 0.2bc 71 6 2c 12 6 1b R4 588 6 23c 0.9 6 0.2abc 42.3 6 0.3c 3.5 6 0.3a R5 853 6 13d 0.7 6 0.3a 42 6 2cd 10.7 6 0.3c R6 526 6 12e 1.17 6 0.06c 29.0 6 0.2d 3.9 6 0.2a R7 713 6 55b 1.0 6 0.3bc 35 6 2d 2.8 6 0.2d

Rcontrol 196 6 4f 0.80 6 0.07ab 7.77 6 0.05e 0.90 6 0.07e

† Results are expressed as means 6 SD (n 5 3). Means in the same column with different letters are significantly different from each other (P , 0.05) according to the Tukey test.

Table 4. Spearman’s correlation coefficients between total Zn concentration in the soil and available levels.

Zn soil factors Total H2 O-extractable EDTA-extractable NH4 –Ac-extractable P. australis (n 5 24) 0.73** 0.95** 0.71** R. ulmifolius (n 5 24) ns 0.85** 0.67** ** Correlation is significant at the 0.01 level; * correlation is significant at

the 0.05 level; ns, no significant correlation.

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differences between root and aboveground concentra-tions of Zn have been observed for other plant species (Armeria maritima ssp. halleri and Agrostis tenuis) (Dahmani-Muller et al., 2000), and Osmorhiza long-istylis (Mbila and Thompson, 2004) and may suggest a Zn exclusion strategy from stems and reproductive tissue by retaining the metal in the roots (Baker, 1981). The mechanism behind the successful growth of these species in the site may also be a reason to disregard the use of these plants in a phytoextraction strategy. The harvest of the aboveground sections will not be an ef-fective source of metal removal from a contaminated soil matrix, as the plant is excluding the metal from these sections, and concentrating it at the root zone. Tolerant plants tend to restrict soil-root and root-shoot transfers,

having much less accumulation in their biomass (Yoon et al., 2006). However, from a toxicological point of view, this may be a desirable property, as Zn would not pass into the food chain via herbivores thus avoiding further environmental risks (Deng et al., 2004).

Soil–Plant Relations

The metal uptake by plants is largely influenced by the bioavailability of the metal in the soil. In previous studies it has been shown that the accumulation of met-als, both by roots and aboveground tissues, was not linear in correlation to the soil metal concentration in-crease (Keller et al., 1998; Greger, 1999). Nevertheless, Cardwell et al. (2002) have reported that aquatic macro-Fig. 1. Zinc levels in (a) P. australis and (b) R. ulmifolius sections.

Table 5. Zinc concentration in the different plant parts for P. australis.†

Zn

Plant part Estarreja (n 5 21) Control site (n 5 3) mg kg21

Roots 72 6 27a 21.6 6 0.5a

Stems 50 6 9b 25.0 6 0.5b

Leaves 48 6 14b 26.4 6 0.7b

† Results are expressed as means 6 SD. Means in the same column with different letters are significantly different from each other (P , 0.05) according to the Tukey test.

Table 6. Zinc concentration in the different plant parts for R. ulmifolius.†

Zn

Plant part Estarreja (n 5 21) Control site (n 5 3) mg kg21

Roots 312 6 150a 59 6 2a

Stems 71 6 22b 32 6 1b

Leaves 63 6 16b 30 6 2b

† Results are expressed as means 6 SD. Means in the same column with different letters are significantly different from each other (P , 0.05) according to the Tukey test.

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phytes exhibited an increasing Zn accumulation with an increasing sediment metal concentration.

Regression analyses were performed with the lev-els of Zn in plants roots, stems, and leaves vs. the total, water-extractable, available (EDTA-extractable), and exchangeable (NH4–Ac-extractable) Zn concentrations

in the soil (Table 7). For P. australis, positive correlations were found between Zn concentrations in the soil with all the different extractants and the Zn levels in the roots and stems sections. As to the levels of Zn in the leaves, no correlation was seen with the soil concentration (Table 7). An increase in Zn soil concentration (total and EDTA, H2O, and ammonium acetate-extractable

fractions) resulted in an increasing accumulation in the stems and especially in the roots. For R. ulmifolius, positive correlations were found between Zn concentra-tions in the soil with different extractants (with the exception of water-extractable Zn) and the Zn levels in the roots and leaves, with stronger correlations being found for the roots. Regarding the levels of Zn in the stems, no correlation with the soil concentrations was found (Table 7). This indicates that an increasing Zn soil

concentration (total and EDTA and ammonium acetate-extractable fractions) results in an increasing accumula-tion in the stem and especially in the root.

Positive correlations exist between Zn levels in dif-ferent plant sections and Zn concentrations in the soil. For example, correlation coefficients of 0.95 for Zn in R. ulmifolius roots vs. EDTA-extractable fraction and 0.82 for Zn in P. australis roots vs. ammonium acetate-extractable fraction (Table 7) have been reported by Deng et al. (2004) for the total and available fraction of Zn in the soil, and indicate that Zn concentrations in plant different sections increase with an increasing con-centration of the metal in the soil. For P. australis and R. ulmifolius a mechanism of aboveground exclusion seems to occur and thus such relations are more notice-able at the root level.

Regression analyses were performed only between metal concentrations in plant root, stem, and leaves, and in the soil (with different extractants). The lack of corre-lation obtained in some cases may imply that in isocorre-lation these factors are not dominant in determining metal ac-cumulation by that plant part. It is possible that variation

Table 7. Spearman’s correlation coefficients between Zn concentration in the plant parts and in the soil.

Zn soil factors

Plant part Species Total H2O-extractable EDTA-extractable NH4–Ac-extractable

Roots P. australis (n 5 24) 0.54** 0.43* 0.47** 0.82** R. ulmifolius (n 5 24) 0.85** ns 0.95* 0.78** Stems P. australis (n 5 24) 0.72** 0.79** 0.74* 0.49* R. ulmifolius (n 5 24) ns ns ns ns Leaves P. australis (n 5 24) ns ns ns ns R. ulmifolius (n 5 24) 0.56** ns 0.51* 0.69**

** Correlation is significant at the 0.01 level; * correlation is significant at the 0.05 level; ns, no significant correlation.

Fig. 2. Bioconcentration factors (BCF) for (a) P. australis and (b) R. ulmifolius sections.

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and interaction of these factors with parameters such as organic content (Wright and Otte, 1999), pH (van der Merwe et al., 1990), soil nutrient status (Deng et al., 2004), and salinity (Fitzgerald et al., 2003) obscures any possible relationship between Zn in the soil and in the plant.

Bioconcentration factors (BCF values) based on dry weight have been determined for the different plant sections (root, stems, and leaves), expressed as the ratio between the metal concentration in the plant section and in soil (Fig. 2). The BCF has been used to evaluate plant availability of heavy metals in contaminated soils (Li et al., 2006; Yoon et al., 2006). In this study, the highest BCF value registered was 0.54, obtained for R. ulmi-folius growing on collection site 2. Plants exhibiting BCF values less than one are referred to be unsuitable for phytoextraction (Fitz and Wenzel, 2002). Nevertheless, these species established successfully on the Zn-polluted soil, whether or not metal was taken up into their above-ground tissues, and their application may be interesting in the rehabilitation of Zn-contaminated soils via phy-tostabilization. Metals accumulated in the roots are considered relatively stable concerning their release to other environmental compartments, such as water and biota (Yoon et al., 2006).

CONCLUSIONS

R. ulmifolius and P. australis colonize a Zn-contam-inated site and accumulate Zn, mainly in the roots. A positive correlation was found between the total Zn concentration in the soil and the extractable Zn frac-tions. The plant individuals found in the area did not show visual toxicity symptoms, indicating Zn tolerance and possibly metal resistance. In both species, highest levels of Zn were found in the roots, which indicate a low metal translocation to aboveground tissues and the probable use of an exclusion strategy by the plant species. The use of R. ulmifolius and P. australis for phytoextraction purposes does not appear as an effective method of metal extraction, but these native metal-tolerant plant species may be used to reduce the effects soil contamination, avoiding further Zn move-ment to other environmove-mental compartmove-ments.

ACKNOWLEDGMENTS

The authors wish to thank Caˆmara Municipal de Estarreja for the provision of access to the site. Ana Marques had the support of a Fundaça˜o para a Cieˆncia e a Tecnologia grant SFRH/BD/7030/2001. The work was funded by Project MICOMETA- POCI/AMB/60131/2004 (Fundaça˜o para a Cieˆncia e Tecnologia).

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