Tolerance and bioaccumulation of cadmium by Phragmites australis grown in the presence of elevated concentrations of cadmium, copper, and zinc

Nadia Ait Alia,*, M. Pilar Bernalb, Mohammed Atera

a

des Sciences, UFR Biologie Ve ge tale, Laboratoire dEcologie et Biologie des Populations, Faculte Abdelmalek Essaadi, B.P. 2121, 93002 Te touan, Morocco Universite b Department of Soil and Water Conservation and Organic Waste Management, a y Biolog a Aplicada del Segura, CSIC, Campus Universitario de Espinardo, Centro de Edafolog Apartado 4195, 30080 Murcia, Spain Received 22 November 2002; received in revised form 3 August 2004; accepted 30 August 2004

Abstract The effects of cadmium and of combined treatments of cadmium, copper and zinc on growth, tolerance indices, mineral composition (N, P, K, Fe, Zn and Mn) and metal uptake of reed (Phragmites australis [(Cav.) Trin. ex Steudel]) were investigated in hydroponic experiments, with Cd concentrations ranging from 4.4 to 17.6 mM, either alone or combined with different treatments of Cu and Zn. Neither root nor shoot growth were affected by the treatments with 4.4 and 8.8 mM Cd alone. However, root and shoot length were signicantly reduced by 17.6 mM Cd. Combined metal treatments signicantly decreased shoot length, root number and plant fresh weight further than Cd alone, but not plant dry weight. Cd alone had no effect on tissue concentrations of N, P or K, while Mn concentration decreased in both shoot and root, and Zn concentration increased. As a result of the Cd, Cu and Zn (combined treatment) toxicity, the concentrations of N and K decreased in shoot and root tissues, respectively, and Mn concentration decreased in both shoot and root. Fe concentration increased in roots, leading to a signicant relationship between root tissue Fe and Cd concentrations with increasing individual concentration of Cd (log10 basis, P < 0.01) and in all the combined
du Que bec a ` Montre al, De partement de Chimie-Biochimie, * Corresponding author. Present address: Universite al, Que., Canada H3C 3P8. Laboratoire Popovic, Case postale 8888, Succursale Centre-Ville, Montre Tel.: +1 514 987 8467; fax: +1 514 987 4054. E-mail address: ait_ali.nadia@courrier.uqam.ca (N. Ait Ali).

treatments of Cd, Cu and Zn (P < 0.001). Metal bioconcentration (tissue concentration/solution concentration) was higher in roots than in shoots, ranging from 287.5 to 1193 for Cd, 849 to 1554 for Cu and 166 to 915 for Zn. The tolerance level of reed to Cd suggests that reed could be useful in wastewater treatments for the removal of Cd. However, the simultaneous presence of elevated concentrations of Cd, Cu and Zn in the efuents may limit the efciency. # 2004 Elsevier B.V. All rights reserved.
Keywords: Bioaccumulation; Cadmium; Metal tolerance; Mineral composition; Phragmites australis

1. Introduction The accumulation of heavy metal contaminants in the environment has become a concern due to the health risks to humans and animals. Heavy metals are elements that cannot be degraded by microbial or chemical process, and tend to accumulate in soils and aquatic sediments. The problem is not restricted to soils with high metal levels, such as mining areas, but also includes those with moderate to low contamination of metals. These toxic elements, such as cadmium (Cd), copper (Cu) and zinc (Zn), are present at elevated levels mainly through human activities, as smelting, rening of non-ferrous metals, electroplating, agricultural practices, and industrial and municipal waste disposal on land (Ross, 1994). Cu and Zn are micronutrients essential at low concentrations for normal plant growth. However, the presence of Cu and Zn at high concentrations induces a strong phytotoxicity and retarded plant growth (critical toxicity levels in leaves Cu > 2030 mg g1, Zn > 100 300 mg g1; Marschner, 1995). A recent study has shown that Cu in excess amounts caused chlorosis of leaves, inhibition of shoot and root growth and macronutrient (N, P and K) deciency in maize (Zea mays L.) seedlings (Ait Ali et al., 2002). Zn toxicity is associated with the reduction of root elongation, chlorosis in young leaves by an induced deciency of Mg or Fe, and, in bean plants, it has been found to inhibit photosynthesis at various steps and through different mechanisms (Marschner, 1995). Cd has become an increasing problem and its toxic effects on biological systems have been reported by various authors (Das et al., 1997). Although not essential for plant growth, Cd2+ ions are readily taken up by roots and translocated into the leaves in many plant species, with Cd depressing growth by affecting photosynthesis, chlorophyll uorescence and nutrient uptake by plants (Mendelssohn et al., 2001). In non-contaminated soils, total concentrations of Cd, Cu and Zn generally range from 0.02 to 2 mg kg1, 2 to 250 mg kg1, and 1 to 900 mg kg1, respectively (Alloway, 1995; Bowen, 1979). These ranges of concentrations depend predominantly on the parent material of soil and on the degree of weathering. Toxic levels in soils with respect to plant growth are reported as 38 mg kg1 Cd, 60125 mg kg1 Cu, and 70400 mg kg1 Zn (Kabata-Pendias and Pendias, 1984). To protect natural ecosystems when sewage sludge is applied to agricultural soil, the European Union has set the following range for heavy metal concentrations in soil, according to the soil pH (low value for pH 6, high value for pH 7): 13 mg kg1 Cd, 50140 mg kg1 Cu, and 150300 mg kg1 Zn (CEC, 1986). Recently, interest in using plants for soil restoration has increased due to the natural capacity of particular species to accumulate various heavy metals. Phytoremediation has

emerged as an alternative technology for removing heavy metals from soil and wastewater, using metal-accumulating plants (Meagher, 2000). Rhizoltration is a form of phytoremediation in which plant roots absorb and accumulate toxic metals from polluted water (Salt et al., 1998). Thus, for rhizoltration, metal accumulation in the shoot is not required. Phragmites australis [(Cav.) Trin. ex Steudel] is a wetland species, which, in the last few decades, has been widely used in the treatment of wastewaters containing various metals (Vrhovsek et al., 1996). Recent work has shown that P. australis is able to growth in the presence of 15.7 mM Cu in solution culture (Ait Ali et al., 2002), and 242 mM of Zn (Bernal et al., 2000). Another study demonstrated that P. australis is a good indicator of the zdemir and Sag irog lu, 2000). Cu concentrations in the soil (O The majority of published data concerning toxicity testing of heavy metals has focused on single metal effects, for example the effect of Cu on P. australis and Z. mays (Ait Ali et al., 2002), comparison of the toxicity of two heavy elements, such as Cu and arsenate on Holcus lanatus L. (Hartley-Whitaker et al., 2001), Zn and Cu in Brassica species (Ebbs and Kochian, 1997). Metal pollution of soil and waters, however, usually is due to the presence of several heavy metals (Walker et al., 2003). Thus, plants in the eld are generally exposed to mixture of heavy metals, which may exhibit toxicity simultaneously and interactively. The toxicity of metal mixtures is rarely studied, for example Sharma et al. (1999) studied the effects of Cu, Cd, and Zn, both alone and in binary mixtures, on root elongation, as toxicity endpoint, and metal uptake of roots of Silene vulgaris. Also, Ince et al. (1999) used a duckweed assay and a microtox assay to assess the interactive toxicity of Zn, Cu, Co and Cr in binary mixtures. With respect to P. australis, Ye et al. (1997a) studied the uptake of Zn, Pb, and Cd by plants raised from seeds collected from a contaminated and a clean site, using single metal treatments. Metal tolerance and accumulation of Zn was tested by Bernal et al. (2000), and of Cu by Ait Ali et al. (2002), who also studied the effect of Cu on the nutrient composition of the plants. Uptake and bioaccumulation of heavy metals by wetland plant species were studied by Stoltz and Greger (2002), comparing the accumulation properties of As, Cd, Cu, Pb and Zn in the combined presence of these elements in the growing medium. In short, studies on the interactive effects of heavy metals in mixtures are rare, and their effect on nutrient status of plants has remained unstudied so far. Therefore, the present investigation focused on the effect of elevated concentrations of Cd, both alone and in combination with Zn and Cu, on shoot and root growth, nutrient composition and metal uptake of P. australis, in order to evaluate its metal tolerance and the interactive effects of Cd, Cu and Zn, and thus to indicate its suitability for rhizoltration. Individual Cu and Zn treatments have been studied already for this population (Ait Ali et al., 2002; Bernal et al., 2000), which will allow the comparison with the multi-element (Cd, Cu and Zn) treatments.

2. Materials and methods 2.1. Selection and propagation of plants Rhizomes of reed (P. australis (Cav.) Trin. ex Steudel) were taken from adult plants growing along the banks of the river Guadalent n (Murcia, Spain). Rhizomes of approximately 20 cm were placed in plastic trays (50 cm 40 cm), with a 7 cm layer of

vermiculite, in a glasshouse with temperature ranging from 28 2 8C (day) to 15 2 8C (night), and watered with tap water every 48 h for 15 days, until roots and shoots had developed, then each plant was separated by cutting the old rhizomes. Plants obtained were transferred to hydroponic culture in plastic containers (15 cm 15 cm) (4 plants per container) with 1.5 l of modied Hoagland nutrient solution (Hoagland and Arnon, 1941) (10% of macronutrients and 100% of micronutrients, to prevent precipitation of Cu whilst avoiding a micronutrients deciency) for 7 days. Iron was added as 20 mM Fe-EDTA. The solution was aerated continuously and buffered to pH 6.0 with 0.5 mM MES (2-[NMorpholino] ethanesulphonic acid). 2.2. Hydroponic experiments Plants of a uniform size (30 cm tall), propagated as previously described, were transferred to the different treatments. Two experiments were run. In the rst, individual Cd treatments were used: a control, with modied Hoagland nutrient solution (0 mM Cd, 0.5 mM Cu and 0.8 mM Zn), and three individual Cd treatments: 4.4, 8.8 and 17.6 mM Cd (equivalent to 0.5, 1 and 2 mg Cd L1, respectively). The second experiment was designed using three mixture treatments of Cd, Cu and Zn, each with one heavy metal present in excessively high concentration. The treatments were: a control (as in the rst experiment), and three Cd:Cu:Zn combinations (in mM): 4.4:31.5:76.5, 17.6:31.5:30.6 and 4.4:78.7:30.6 (equivalent to 0.5:2:5, 2:2:2 and 0.5:5:2 mg L1 Cd:Cu:Zn, respectively). The pH of the nutrient solution was buffered to pH 6.0 with 0.5 mM MES (2-[NMorpholino] ethanesulphonic acid). CdCl2 was used as cadmium source, CuSO45H2O as copper source and ZnSO47H2O as zinc source. Three replicates were used per treatment, giving a total of 12 pots per experiment, which were arranged in a randomized design. All solutions were changed every 4 days during the 15 days of both experiments. The experiments were conducted in a glasshouse with temperature ranging from 28 2 8C (day) to 15 2 8C (night), without supplementary light in October (approximately 12 h of photoperiod per day). At the beginning of the experiments and after 15 days of growth, total fresh weight, shoot length, total root length (the sum of all root lengths) and the number of roots per pot were determined, and the changes in these parameters were used to evaluate Cd and Cd:Cu:Zn toxicity. At the end of the experiment, shoots were separated from roots (rhizomes had hardly developed). The plant roots were washed twice with distilled water (acidied to pH 4.0 with HCl) and then washed with deionised water. The shoots were washed twice with deionised water. The samples were oven-dried, at 70 8C for 24 h, to obtain the dry weight, and then ground to a ne powder. For analysis, dry plant material was digested in a mixture of HNO3/HClO4 (3/1, v/v), at 150 8C for 2 h and 210 8C for 1 h, and then dissolved in HCl (0.5N) (Abrisqueta and Romero, 1969). The concentrations of Cd, Cu, Zn, Fe, and Mn in the extracts were analysed by ame atomic absorption spectrometry, and K by ame photometry. P was analysed by a colorimetric method (Kitson and Mellon, 1944). The total nitrogen concentration of the plant tissues was determined by an elemental microanalyser (C/N/S Carlo Erba, NA 1500). The tolerance index (TI), at different individual concentrations of Cd and combined treatments of Cd, Cu and Zn, was calculated by dividing the root length at the different

metal concentrations by that obtained in the control solution, as suggested by Wilkins (1978). The following equation was used: TI (%) = 100 (root length in metal treatment)/ (root length in the control). Total metal accumulation rate, expressed as mg g1 DW day1, was calculated as accumulation rate metalshoot DWshoot metalroot DWroot =15 DWshoot DWroot The bioconcentration factor (BCF) was calculated as BCF metal mg g1 shoot or root =metal mg L1 nutrient solution The results were analysed using one-way ANOVA, and the Tukey HSD test to compare the means of treatments at P < 0.05.

3. Results 3.1. Metal tolerance and growth In treatments with Cd alone, root elongation and increase of both shoot length and fresh weight, decreased signicantly (P < 0.05) with increasing Cd concentration in the nutrient solution (!8.8 mM Cd; Fig. 1). However, even at the highest Cd concentration (17.6 mM) no signicant inhibition was obtained for the mean increase of root number or production of dry weight, compared to control plants (Fig. 1). In the combined treatments of Cd, Cu and Zn (Cd:Cu:Zn), all the growth parameters were signicantly less than controls in all metal treatments, except for dry weight production (Fig. 1). Among the measured growth parameters, root elongation was the most sensitive to both Cd alone and to Cd:Cu:Zn treatments. The tolerance index (TI), based on root length for different treatments of Cd and Cd:Cu:Zn, indicated that even at the same concentration of Cd (e.g. 4.4 and 17.6 mM), the combined effect of Cd:Cu:Zn was more toxic to reed than the individual Cd concentrations (Fig. 2). At 4.4 mM Cd, the TI was 100%, whereas at the same Cd concentration in the Cd:Cu:Zn treatments, the TI was less than 40%. In addition, the TI of reed was about 60% at 17.6 mM Cd, while it was only 20% at 17.6:31.5:30.6 mM of Cd:Cu:Zn. This suggests that reed can tolerate a relative excess of Cd ( 8.8 mM), however, it was more sensitive to the combined treatments of Cd:Cu:Zn. In particular, 4.4:78.7:30.6 mM appeared the most phytotoxic treatment compared to other combined concentrations of the three elements. 3.2. Concentrations and accumulation of cadmium, copper and zinc In individual cadmium treatments, Cd concentrations in reed tissues increased signicantly in both shoot (P < 0.001) and root (P < 0.01) with increasing external Cd concentration. Most of the Cd absorbed by reed plants was found in the root tissue (Table 1).

Fig. 1. Mean increase in root length (A), number of roots (B), shoot length (C), fresh weight (D) (based on difference between nal and initial mean values) and production of dry weight (E) for P. australis after 15 days growth in metal treatments (results of four plants per pot). Values are mean S.D. (n = 3). Mean values followed by the same letter are not statistically different according to the Tukey HSD test (P < 0.05), within individual Cd or combined Cd:Cu:Zn treatments.

Fig. 2. Tolerance indices (TI) of P. australis at different individual Cd treatments and combined Cd, Cu and Zn treatments, calculated using the total root length (n = 3). Mean values followed by the same letters are not statistically different according to the Tukey HSD test (P < 0.05), within individual Cd or combined Cd:Cu:Zn treatments.

In the combined supply of Cd, Cu and Zn, the most important observation is that no Cd accumulation (Cd < 0.2 mg g1) occurred in the shoots tissues of reed grown at 4.4:78.7:30.6 mM of Cd:Cu:Zn, probably due to the interference of the high Cu concentration in the nutrient solution (78.7 mM) with Cd absorption (Table 2). Cd absorption was conned to the roots. Likewise, Zn concentrations in the shoots and roots diminished with increasing Cu concentration in the growth medium. For example, in the 17.6:31.5:30.6 treatment, Zn concentration in the shoot was 211 mg g1, while it was only 84 mg g1 at 4.4:78.7:30.6 mM of Cd:Cu:Zn. Also, Cu concentrations of roots and shoot were lower in the 4.4:31.5:76.5 compared to the 17.6:31.5:30.6 treatment. The bioconcentration factor (BCF) differed according to metal and treatment (Tables 1 and 2). The highest value of BCF was observed at root level. In the Cd treatments BCF
Table 1 Concentration of Cd, bioconcentration factor (BCF), and total accumulation rate of P. australis grown in individual Cd treatments for 15 days Treatments of Cd (mM) Cd concentration (mg g1 DW) Shoots Control 4.4 8.8 17.6 P (ANOVA) <0.2 a 43 12 b 71 7 c 90 8 c <0.001 Roots <0.2 a 392 61 a 1193 371 b 1350 435 b <0.01 BCF Shoots 85 71 45 Roots 783 1193 675 Total accumulation rate (mg g1 DW day1)

5.9 14.7 16.5

Values are mean S.D. (n = 3). Mean values followed by the same letter are not statistically different according to the Tukey HSD test (P < 0.05).

Table 2 Concentrations of Cd, Cu and Zn (mg g1 DW), bioconcentration factor (BCF) and total accumulation rate (mg g1 DW day1) of P. australis grown in combined metal treatments for 15 days Treatments of Cd:Cu:Zn (mM) Cd Control 4.4:31.5:76.5 17.6:31.5:30.6 4.4:78.7:30.6 P (ANOVA) Cu Control 4.4:31.5:76.5 17.6:31.5:30.6 4.4:78.7:30.6 P (ANOVA) Zn Control 4.4:31.5:76.5 17.6:31.5:30.6 4.4:78.7:30.6 P (ANOVA) Metal concentration (mg g1) Shoots <0.2 a 12 1 b 51 7 c <0.2 a <0.001 Roots <0.2 a 321 61 b 899 239 c 144 13 b <0.01 BCF Shoots Roots Total accumulation rate (mg g1 DW day1)

25 26 <0.2

641 450 287

2.8 10.9 0.9

26 3 39 3 61 18 73 145 >0.05

41 12 a 2207 214 b 3108 659 bc 4248 431 c <0.01

19 31 14

1103 1554 849

16.8 31.2 31.9

152 23 ac 271 27 b 211 31 ab 84 7 c <0.01

86 10.9 a 2355 269 b 1829 171 b 331 24.4 a <0.001

54 106 42

471 915 166

31.6 28.5 7.1

Values are mean S.D. (n = 3). Mean values followed by the same letter are not statistically different according to the Tukey HSD test (P < 0.05).

ranged from 675 to 1193 and from 45 to 85, in roots and shoots, respectively. However, it was lower in the combined treatments of Cd:Cu:Zn, ranging from 287 to 641 and from 25 to 26 at root and shoot level, respectively. In the Cd:Cu:Zn treatments, the maximum BCF was 1554 for Cu and 915 for Zn. In addition, the total accumulation rate of Cd was very low in Cd:Cu:Zn treatments compared with that of the individual Cd treatments. 3.3. Effect of metals on plant mineral composition The effect of individual Cd treatments was not signicant (P > 0.05) with respect to the macronutrient concentrations (N, P and K) in reed tissues, as indicated by the ANOVA and the multiple range test (Table 3). However, the root and shoot concentrations of the micronutrients Zn and Fe increased in response to increasing external Cd treatment. On the other hand, Mn concentrations tended to decrease in both roots and shoots, while Cu increased at root level. The concentration of Cd in the root tissue was signicantly correlated with the Fe concentration, giving a linear regression equation when both were expressed on a log10 basis (r = 0.81, P < 0.01, Table 4). Linear regression equations were also obtained between concentrations of Cd and those of Zn and Cu (r = 0.98, P < 0.001; r = 0.97, P < 0.001, respectively). Such correlations were not signicant for shoot tissue.

Table 3 Nutrient concentrations in shoots and roots of P. australis grown in cadmium treatments for 15 days Treatments of Cd (mM) Shoots Control 4.4 8.8 17.6 P N (mg g1) (mg g1) 31 7 31 4 32 3 31 0.5 3.7 1 3.2 0.6 3.1 0.4 2.4 0.3 >0.05 K Cu (mg g1) (mg g1) 51 6 46 5 49 2 52 19 >0.05 17 0.2 a 16 3 ab 15 0.6 ab 12 2 b <0.05 Mn (mg g1) 51 23 56 2 40 7 29 3 >0.05 Zn (mg g1) 63 15 a 136 30 b 157 68 b 59 2 a <0.05 Fe (mg g1) 58 1 a 102 32 b 54 4 a 73 5 ab <0.05

P (ANOVA) >0.05 Roots Control 4.4 8.8 17.6

34 7 31 2 29 2 27 3

2.9 0.5 2.9 0.7 2.8 0.2 2.7 0.2 >0.05

38 8 36 3 42 11 42 2 >0.05

25 6 a 173 49 b 169 32 b 180 27 b <0.01

341 68 a 330 116 a 75 15 b 72 15 b <0.01

78 11 a 353 112 b 626 68 c 557 39 c <0.001

560 330 a 1802 788 ab 1757 578 ab 3517 1173 b <0.05

P (ANOVA) >0.05

Values are mean S.D. (n = 3). Mean values followed by the same letter are not statistically different according to the Tukey HSD test (P < 0.05).

The combined treatments of Cd, Cu and Zn did not have any signicant effect on the concentration of P in shoots and roots. However, a slight decrease was obtained for N in shoots and for K in roots (Table 5). In addition, Cd:Cu:Zn treatments induced a highly signicant decrease in both shoot and root tissue concentrations of Mn (P < 0.001), while the Fe concentrations in roots increased signicantly. Additionally, the relationship between the root tissue concentrations of Cd and Fe was linear, with a signicant coefcient of correlation (r = 0.94, P < 0.001, Table 4), as were those between Cd and Zn, and Cd and Cu, on a log10 basis. In roots, the concentration of Fe was also correlated with that of Zn and of Cu.

Table 4 Signicant relations between different metal concentrations in roots of P. australis grown in Cd and Cd:Cu:Zn treatments for 15 days y x Cd treatments a S.E. Fe Zn Cu Fe Fe Zn Cd Cd Cd Cu Zn Cu 0.22 0.05 0.28 0.01 0.28 0.02 0.74 0.17 0.77 0.17 0.95 0.09 b S.E. 2.68 0.12 1.87 0.04 1.40 0.05 1.66 0.37 1.24 0.43 0.57 0.18 r 0.81** 0.98*** 0.97*** 0.79** 0.81** 0.95*** Cd:Cu:Zn treatments a S.E. 0.39 0.04 0.47 0.07 0.69 0.07 0.54 0.04 0.62 0.15 0.53 0.14 b S.E. 2.67 0.09 1.88 0.16 1.68 0.17 1.78 0.13 1.68 0.44 1.18 0.45 r 0.94*** 0.89*** 0.94*** 0.97*** 0.78** 0.75**

a: slope; b: intercept; r: correlation coefcient; S.E.: standard error. Correlations were calculated on a log10 basis of concentration. ** P < 0.01. *** P < 0.001.

Table 5 Nutrient concentrations in shoots and roots of P. australis grown in combined metal treatments for 15 days Treatments of Cd:Cu:Zn (mM) Shoots Control 4.4:31.5:76.5 17.6:31.5:30.6 4.4:78.7:30.6 P (ANOVA) Roots Control 4.4:31.5:76.5 17.6:31.5:30.6 4.4:78.7:30.6 P (ANOVA) N (mg g1) P (mg g1) K (mg g1) Mn (mg g1) Fe (mg g1)

37 2 32 2 31 3 24 2 <0.001

a ab b c

4.6 0.2 3.5 0.2 3.6 1.0 3.1 0.9 >0.05

42 5 37 2 36 8 37 5 >0.05

119 27 a 27 2 b 23 6 b 21 4 b <0.001

55 3 118 66 91 21 87 9 >0.05

37 4 37 2 38 2 31 4 >0.05

5.2 0.7 5.1 0.2 5.8 1.2 3.9 0.6 >0.05

37 4 26 3 28 5 25 2 <0.05

a b ab b

267 25 a 44 14 b 31 3 b 29 20 b <0.001

424 38 a 4532 136 bc 4961 2305 bc 5279 2228 c <0.05

Values are mean S.D. (n = 3). Mean values followed by the same letter are not statistically different according to the Tukey HSD test (P < 0.05).

4. Discussion With respect to Cu toxicity, Ait Ali et al. (2002) found that root length of reed and maize seedlings was more sensitive than other measured growth parameters. Similar results were found in the present study, where Cd alone and Cd:Cu:Zn supply greatly affected root length. Single toxic effects of Cu and Zn on the growth of the same population of reed were studied previously (Ait Ali et al., 2002; Bernal et al., 2000). Based on root length, reed was tolerant to 15.7 mM Cu, with a tolerance index (TI) of 100%, but not to 78.7 mM Cu (TI = 10%) (Ait Ali et al., 2002). Likewise, reed was clearly tolerant to 30.6 mM Zn, with a TI of 94% (Bernal et al., 2000). Also, results from the present study show that reed was tolerant to 4.4 mM Cd, with a TI of 100%, whilst Cd concentrations above 8.8 mM reduced the TI to below 80%. In similar studies, P. australis (propagated from seeds) showed a 50% depression in root elongation when grown at 33.6965.75 mM Zn, and a supply of Cd ranging from 2.85 to 3.20 mM (Ye, 1995). The differing results may be due to the different propagation methods. Bernal et al. (2000) found that P. australis propagated from seeds was less tolerant to Zn than when propagated from rhizomes, although the Zn accumulation in shoots was not affected. Typha latifolia L., a wetland species, showed 30% inhibition of root length and 9% inhibition of leaf elongation when exposed to 0.79 mM Cu (Ye et al., 1997b), and decreases of 61% and 74% in root and leaf elongation, respectively, when grown at 15.29 mM Zn (Ye et al., 1998). Thus, reed was less sensitive to individual metal treatments than T. latifolia, for Cu and Zn. Otherwise, in the present study, 17.6 mM Cd decreased root length of reed by 37% compared to control plants, while, all the combined supply of Cd:Cu:Zn depressed root length by 7391% with a TI < 30%. Moreover, 15.75 mM Cu had no signicant effect on root length of reed (Ait Ali et al., 2002). For the same species, 30.6 mM Zn caused very

little inhibition (about 6.4%) of root growth (Bernal et al., 2000). These results suggest that the combination of the three elements (Cd, Cu and Zn) are more toxic for reed than their individual application. This suggested an additive toxicity of each element. According to Sharma et al. (1999), the effects of Cd + Zn, Cd + Cu, and Zn + Cu on root growth of S. vulgaris were response-additive at high metal treatments. Reed can be considered as tolerant to relatively excessive concentrations of Cd alone, as shown by its TI values, and also because the N, P and K composition was not affected. Cu concentration tended to decrease in the shoot and to increase at root level, suggesting an inhibition of Cu translocation to the shoot due to Cd. Sharma et al. (1999) also found an increasing concentrations of Cu in root of S. vulgaris, when the Cd concentration in the nutrient solution was increased up to 0.5 mM. However, reed was more sensitive to the Cd:Cu:Zn treatments, N, K, and Mn concentrations decreasing in plant tissues. In individual Cu treatments (15.75157.50 mM), N, P and K composition of reed was not affected (Ait Ali et al., 2002), but a signicant decrease in Zn and Mn concentrations was obtained in root and shoot tissues, respectively. The presence of high concentrations of Zn in the nutrient solution (244.7 mM) hardly changed the N, P, K nutrient composition of reed, while Mn concentration was reduced in shoots and roots, but Cu decreased in shoots and increased in roots (Bernal, unpublished), as occurred in the present Cd experiment. In other plant species, very high Cd concentrations (1 mM Cd) induced an important decline in tissue nutrient levels of Triticum aestivum L., especially those of Mg, Ca and K in leaves (Ouzounidou et al., 1997). Furthermore, both the individual Cd treatments and the combined Cd:Cu:Zn treatments induced a sharp increase in Fe concentration for shoot and root tissue of reed. Treatments with Cu previously have been shown to increase the Fe concentrations of shoot and root tissue of reed (Ait Ali et al., 2002), suggesting the presence of an iron plaque on the roots, which may protect the roots from metal toxicity (Greipsson, 1994). The reason for increasing Fe level in plant tissue is not clear. Greipsson (1995) suggested that an iron plaque might increase metal tolerance, leading to higher Fe levels within leaves, as well as absorbing and immobilizing other metals. This may explain the increased concentrations of Zn and Cu in root tissue of reed for individual Cd treatments. Interactions between plant Cd uptake and other micronutrients (Cu, Mn, Fe) have been demonstrated in solution culture (Cataldo et al., 1983). In the present study, no detectable accumulation of Cd was obtained in the shoot of reed, for the treatment of 4.4:78.7:30.6 (Cd:Cu:Zn), whilst the concentration in roots was reduced with respect to the other combined treatments. This may be explained by the interaction of excess Cu with Cd uptake (McLaughlin and Henderson, 1999). However, absorption of Cd and Zn by plants probably occur via independent uptake mechanisms, as was demonstrated in maize (Hinsley et al., 1978). This may explain why the uptake of Cd by reed seedlings was not inhibited when a high Zn concentration was added to the nutrient solution (e.g. the 4.4:31.5:76.5 mM Cd:Cu:Zn treatment). Nevertheless, pronounced interactions between Zn and Cd, with respect to Cd uptake and translocation have been demonstrated in many plant species (McLaughlin and Henderson, 1999). With respect to metal bioaccumulation, reed accumulated higher amounts of metal in root than in shoot. This was in agreement with other studies using various wetland plant species (Zhu et al., 1999; Stoltz and Greger, 2002), and in previous experiments of Cu, Pb

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and Zn in reed (Ait Ali et al., 2002; Bernal et al., 2000). In individual and combined metal treatments, reed roots reached hyperaccumulator concentrations of Cu and Cd, based on the dened thresholds for shoot hyperaccumulation in terrestrial plants: 10 000 mg g1 Zn, 1000 mg g1 Cu and 100 mg g1 Cd (Reeves et al., 1996). To evaluate the phytoextraction ability of any plant species, whole plant biomass, the metal concentration in the growth media, and the metal concentration in plant tissue must be taken into consideration. Zayed et al. (1998) dened a good accumulator as having: (i) a total tissue concentration of the metal higher than 0.5% of total DW and (ii) a BCF above 1000. According to our results, and the above criteria, for a low Cd treatment (8.8 mM), reed can be considered as a good accumulator with respect to roots (BCF = 1193), although Cd concentration was only 0.2% of root DW. For the combined treatments of Cd:Cu:Zn (4.4:31.5:76.5 and 17.6:31.5:30.6 mM), roots exhibited a Cu-BCF higher than 1000, but reed was very sensitive to these treatments, thus the tolerance level of reed was exceeded (TI < 30%), and the high tissue Cu concentration may result from passive transport. For rhizoltration, only the metal concentration in root tissues is of interest, while high metal accumulation in shoot tissues is not relevant. If we consider the normal biomass production of reed, about 23 Mg/ ha for the above ground biomass, and 29.7 Mg/ha for the underground biomass (Ennabili et al., 1998), the rhizoltration of Cd can be estimated to be 0.98 kg ha1 for the aerial part and 11.64 kg ha1 for the roots. This was estimated for the 4.4 mM Cd treatment, which give no signicant growth reduction of reed. Taking into account the less phytotoxic combined treatment of Cd:Cu:Zn (4.4:31.5:76.5 mM), the rhizoltration may be: Cd 0.29 and 9.52 kg ha1; Cu 0.91 and 65.52 kg ha1; and Zn 6.22 and 69.90 kg ha1, in shoot and root tissues, respectively. According to our results, it appears that the combined Cd:Cu:Zn treatments have a strong effect than Cd alone on morphometry (number of roots, shoot and root length) of reed, but not on dry weight production. Based on root elongation, we conclude that exposure to 8.8 mM Cd and to all the combined treatments with Cd, Cu and Zn signicantly decreased growth of reed plants. Therefore, reed can be considered as a Cd-tolerant plant at up to 8.8 mM Cd in the nutrient solution. This differential tolerance was also observed in the ability of reed to maintain tissue nutrient concentrations. Alone, Cd had no effect on macronutrient (N, P, K) concentrations, in shoot or root, however, the combined treatments of Cd, Cu and Zn (17.6:31.5:30.6 and 4.4:78.7:30.6) signicantly decreased N in the shoot and K in the root tissue. Cd, Cu and Zn translocation to the shoots was low, these elements mainly accumulated in roots. For the low Cd concentration (8.9 mM), the root bioconcentration factor (BCF = 1193) of reed was just higher than the threshold value of 1000 for a good accumulator, as dened by Zayed et al. (1998), which makes this species potentially interesting for rhizoltration treatment of wastewater. However, the presence of combined high concentrations of Cd, Cu and Zn, such as those used in this study, would limit its potential.

Acknowledgements The authors thank Dr. D.J. Walker for his comments on the manuscript and correction of the English. This study has been funded by the CSIC (Spain) and the CNCPRST

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