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Growth and Cu accumulation by plants grown on Cu containing mine tailings in Cyprus
Applied Geochemistry Applied Geochemistry 20 (2005) 101–107 www.elsevier.com/locate/apgeochem
Growth and Cu accumulation by plants grown on Cu containing mine tailings in Cyprus Lisa Johansson a, Constantinos Xydas b, Nikos Messios b, Eva Stoltz a, Maria Greger a,* a
Department of Botany, Stockholm University, SE-106 91 Stockholm, Sweden b Hellenic Copper Mines LTD, Cyprus Received 27 May 2003; accepted 3 July 2004 Editorial handling by R. Fuge
Abstract The Skouriotissa Cu mine in the northern part of Cyprus has produced large amounts of mine waste. Phytoremediation could stabilise the erosion or extract the metals of this waste. The aim of this study was to find out if Pistacia terebinthus, Cistus creticus, Pinus brutia and/or Bosea cypria could grow and tolerate or maybe accumulate Cu from the mine waste containing up to 787 mg Cu (kg DW) 1. Another aim was to see if the liquid wine waste product Vinassa, containing organic acids and having a low pH, or chicken fertilizer could improve plant growth and/or Cu accumulation. The four species were planted at the mine waste site untreated or with the addition of Vinassa or chicken fertilizer as mine waste modifiers. After 3 months, shoot length growth was measured and the plants were analysed for Cu concentration. The pH and Cu concentration of the mine waste mixture in the different treatments was also measured. To find out if plants accumulated Cu to the highest extent in roots or shoot, a greenhouse study was undertaken where the plant species were cultivated for 3 weeks in Cu spiked soil. The results showed that all of the tested species survived and grew on the mine waste site, which indicates that they tolerate the high level of Cu at the mine waste site. The leaves of C. creticus had the highest Cu accumulation of all tested species. Copper accumulation varied with plant species. They seemed to have different distribution strategies for Cu: in Pistacia terebinthus and C. creticus most of the Cu was found in the roots, while B. cypria accumulated most of the Cu in the leaves. Addition of Vinassa and chicken fertilizer did not increase plant growth or Cu accumulation, but did affect the Cu distribution in B. cypria. Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction The Skouriotissa Cu mine in the northern part of Cyprus has been exploited since ancient times (Pyatt, 2001).
*
Corresponding author. Fax: +46 8 162268. E-mail address: [email protected] (M. Greger).
It is situated in the Troodos Mountains, which are dominated by volcanic rock with limited outcrops of umbers, marls and limestone (Searle and Panayioto, 1980). The mining activities in this area have produced large amounts of mine waste, which is rich in pyrite and other sulfide minerals (Yukselen, 2002). The waste can create several environmental problems, of which the leaching of heavy metals, in this case Cu, is considered to be
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the most serious one. Another environmental problem caused by mining activities is erosion due to the absence of vegetation cover on the mine waste site (ClemenssonLindell et al., 1992). The climate in the area of the mine can be classified as semi-arid and the mean annual precipitation is 300 mm while during some years of drought, the annual precipitation can be as low as 200 mm. In Cyprus, there is a long dry hot summer, which lasts for 4–5 months. Several methods for rehabilitation of mining waste areas and the prevention of leaching of heavy metals have been discussed. One of them is phytoextraction, where metal tolerant plants exhibiting high metal accumulation extract and remove heavy metals from soil by harvesting the plants (Cunningham and Berti, 1993; Salt et al., 1998). Phytoextraction postulates that the plants used accumulate a vast amount of the metal of interest in their leaves. The criterion to define a plant as a Cu hyperaccumulator is that it should have a Cu, concentration in leaves of 5000 mg Cu (kg DW) 1 or higher (Brooks, 1998). To this date, Cu hyperaccumulators have been found in 15 plant families (Baker et al., 2000). Examples of hyperaccumulating plant species are the trees Acacia retinoides and Eucalyptus torquata, which when growing in a Cu contaminated site in Cyprus accumulated 0.19% and 0.21% Cu in their leaves, respectively (Pyatt, 2001). Another remediation method is phytostabilisation, where metal tolerant plants stabilise the metals in the soil and thus reduce their bioavailability by root uptake, precipitation or reduction (Salt et al., 1995). These plants should, along with Cu immobilization in soil, have a restricted translocation of the metals to the shoot. In addition, these methods can, if the vegetation cover is high, prevent erosion of the waste material. Copper is an essential micronutrient for plants. The critical deficiency level of Cu in plants is generally in the range of 1–5 mg (kg DW) 1 (Marschner, 1995) and the sufficient or normal level is approximately 5–30 mg (kg DW) 1 (Pais and Jones, 1997). However, Cu can be toxic to plants at concentrations which are too high. As a consequence, some plants have developed tolerance to high levels of Cu. Copper tolerance can be mediated by accumulation and storage (detoxification) or exclusion of Cu by the plant (Salt et al., 1998). Resistance to heavy metal uptake is usually highly specific and depends on the metals enriched in the soil (Verkleij and Schat, 1990). To get plants to grow on mine waste sites, improvements of the soil commonly have to be done since it often is acidic, low in nutrients, high in metal concentrations and retains water poorly (McCabe and Otte, 1997). Different kinds of additives in the surface layer of the metalliferous soils can be applied in order to raise its nutritional status. For example, sewage
sludge and horse dung have been tried out as fertilizers to promote vegetation growth on mine waste with positive results (Borgega˚rd and Rydin, 1989; Stoltz and Greger, 2002). In the present study, the liquid wine waste product Vinassa and chicken fertilizer were used as soil additives. The reason for using Vinassa was the possibility that it could lower the alkalinity (the pH of the studied mine waste was high due to the limestone content) in the soil and thereby increase the Cu availability and the capacity for phytoextraction. Soil from the mine vicinity was also added to provide some nutrients and organic material to the mine waste. The main purpose of the present study was twofold, firstly to find a method for phytoremediation of the Skouriotissa mine waste area, i.e., to find plant species that could be used for phytostabilization and/ or phytoextraction of Cu at the mine waste, and secondly to see if the soil additives could be used to improve the phytoremediation results. To test these, the authors investigated whether the chosen plants (Pi. terebinthus, Cistus creticus, Pinus brutia and Bosea cypria) could grow and tolerate the high Cu concentrations and possibly accumulate Cu from the mine waste. In addition, this study was also done to see if Vinassa or chicken fertilizer could improve plant growth and/or Cu accumulation. The first hypothesis was that the chosen plant species would grow, i.e., tolerate the high levels of Cu in the Skouriotissa mine waste and have different Cu distribution strategies. The second hypothesis was that the addition of Vinassa and chicken fertilizer could improve plant growth by adding nutrients to the soil and, in the case of Vinassa, lower the alkalinity in the soil and thereby increase Cu accumulation.
2. Materials and methods 2.1. Plant species Four local plant species were selected for the study. The selection was based on which species could possibly cope with the environment at the Skouriotissa mine waste dump in Cyprus, i.e., could grow on dry, rocky soil at an altitude of 300 m. The species were: Pi. terebinthus Bieberstein, C. creticus L., P. brutia Tenore and B. cypria Boissier. B. cypria and Pi. terebinthus were bought from the Athalassa nursery, P. brutia from the Platania nursery and C. creticus from the Phassouri nursery in Cyprus. In addition, Polygonum equisetiforme L. was collected at the mine waste dump of the Skouiriotissa mine. This plant was used in the field experiments, as a ground covering vegetation in order to prevent soil erosion and as a possible producer of organic material.
0.10 ND 0.78 0.23 (0.02) 0.25 (0.04) 0.17 (0.01) 0.15 3.9a 41.5 0.7 (0.1) 0.97 (0.26) 2.73 (0.38) 0.21 1.2a 31.7 0.27 (0.04) 0.30 (0.01) 1.54 (0.36) 1.95 ND 360 8.97 (1.45) 9.53 (1.99) 20.00 (2.15) – – – 6.8 (0.3) 7.1 (0.2) 6.3 (0.1) – – – 7.5 (0.1) 7.5 (0.1) 7.2 (0.1) 6.5 3.8 8.8 – – – Mine waste Vinassa Chicken fertilizer Mine waste + soil Mine waste + soil + Vinassa Mine waste + soil + chicken fertilizer
103 The cultivation material was mine waste mixed with soil (1:1) untreated or treated with chicken fertilizer or Vinassa. Samples were taken at 0–15 cm depth. Cutot = HNO3 extractable Cu fraction. ND = no data (n = 2–32), (SE). a In g l 1.
787 3.04a 83 519 (45) 520 (61) 490 (47)
1
Cutot mg kg DW 1
Ca g kg DW 1
N g kg DW 1
P g kg DW 1
C g kg DW
Pistacia terebinthus, C. creticus, P. brutia and B. cypria were planted at the waste dump of the Skouriotissa mine in Cyprus, February 14–15th, 2002. The plants were planted in squares (with an area of approximately 0.25 m2 and with 1 m in between each square) made by a digging machine. The 30-cm top layer of the squares consisted of soil from the mine vicinities mixed with the mine waste in the proportion 1:1. One hundred ml of the physical soil conditioner Terra Cottem (Terra Cottem, Belgium) was also added to all the squares up to a depth of 30 cm. Terra Cottem is a mixture of Kbased organic hydroabsorbent polymers, which is used for its water retaining properties. Vinassa or chicken fertilizer was added to some of the squares as soil additives (10 l of each additive in each square). The chicken fertilizer was added during the plantation and mixed with the topsoil, and the Vinassa was added after the plantation. Vinassa was added one more time (5 l per square) during the experiment, at March 15th, 2002. Samples of pure mine waste, chicken fertilizer and Vinassa were collected for analysis. Experimental set-up for each species: two plants in soil with no soil additives, two plants in soil with chicken fertilizer and two plants in soil with Vinassa. Each treatment was made in four replicates. In addition, two plants of Po. equisetiforme were planted in all of the squares. All of the plantation squares were placed randomly in the test area. The plants were watered once every 2 weeks. Samples of the leaves and stems were collected after approximately 3 months (May 15–16th, 2002) as well as samples of the mine waste soil from all squares (at the depths of 0–10 cm and 10–15 cm). The height of the above ground part of the plants was measured at the beginning and at the end of the experiment with a ruler. Shoot plant samples from Sinapis alba L., Vaccaria pyramidata Medicus, Po. equisetiforme L., Capparis spinosa L. and Ficus caria L. were also taken from different
pH (10–15 cm)
2.3. Field experiments
pH (0–10 cm)
Two different soil additives were used in the study: the liquid wine waste product Vinassa and chicken fertilizer. The Vinassa came from the Keo brewery in Limassol, Cyprus. It had a pH of 3.8 (Table 1) and contained amino acids, tannic acid, tartaric acid, traces of sugars, suspended solids and other natural constituents of wine, according to the brewery. The chicken fertilizer was bought from local farmers. Soil from the mine vicinities (crop field soil) was added to all of the squares to increase its nutritional value. Table 1 shows the concentration of macronutrients and Cu of the mine waste, mine waste + soil and soil additives.
pH
2.2. Soil and soil additives
Table 1 Nutritional status, pH, total and bioavailable Cu concentration of mine waste, Vinassa and chicken fertilizer as well as of samples taken of cultivation material after 3 months of plant growth
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locations at the mine waste site, in order to get background values for plants growing in the studied area. These plants had a maximum Cu concentration of 47.4, a median concentration of 30.5 and a minimum concentration of 12.4 mg Cu (kg DW) 1. 2.4. Greenhouse experiments Plants of Pi. terebinthus, C. creticus, P. brutia and B. cypria were planted in compost (Weibull, Hammenho¨g, Sweden) in a greenhouse equipped with supplementary lamps ((OSRAM daylight, HQI-BT 400 W) (18 h light) and with a temperature of 17–20 °C) and treated with Provado insecticide sticks (Bayer AG, Leverkusen, Denmark) for 2 weeks to prevent insect attack. At the experimental start, the plants were planted in soil spiked with copper chloride (approximately 7 weeks in advance) with a Cu concentration of 150 mg Cu (kg DW soil) 1, and in unspiked soil with a Cu content of 3 mg Cu (kg DW soil) 1. This was done to investigate the influence of Cu concentration in the soil on Cu uptake and distribution within plants. Samples of the plant leaves, stems and roots were taken after 3 weeks. The roots were washed twice in redistilled water, once in EDTA solution (20 mM) and twice in redistilled water. A study on the effect of Vinassa on Cu uptake by Po. equisetiforme was also conducted. Plants of Po. equisetiforme were planted in compost (Weibull), one plant per pot (with the volume 0.62 l). Four of them were planted in the spiked soil with a Cu concentration of 150 mg Cu (kg DW soil) 1, and Vinassa (0.08 l (l soil) 1) was added to two of these plants. The remaining three were control plants. Plant samples of the shoots and roots were taken after 3 weeks, and the roots were washed as above. 2.5. Sample analysis The plant samples were dried at 105 °C and thereafter wet digested in HNO3:HClO4 (7/3, V/V) and analysed for Cu concentration. All metal analysis was performed by atomic absorption spectroscopy (SpectrAA 100, GTA-100, Varian, Springvale, Australia) using both flame and furnace. The amount of Cu released from the mine waste soil by the addition of Vinassa was also measured by mixing the soil (0.5 g) with Vinassa or water (5 ml). The samples were then shaken for 16 h and analysed for Cu content. Additionally, total Cu concentration of all the soil samples from 10–15 cm depth was measured. The samples were dried in an oven at 105 °C, acid digested in HNO3 (7 M) and analysed for Cu. The pH of the soil samples was measured by mixing 5 g of soil with 12 ml of redistilled water. After 16 h of incubation, the pH was measured with a pH-meter (Metrohm 744).
The organic acid content in the Vinassa was analysed by using a Dionex ion chromatographic system consisting of a 4500-gradient pump, an anion micro membrane AMMS-ICE II suppressor, an ED50A electrochemical detector, a conductivity cell D23 detection stabilizer Model DS3-1, and an ionpac ICE-AS6 analytical column (Dionex corporation; Sunnyvale, CA, USA). Twenty ll of the sample were injected manually into the ion chromatograph and run for 20 min measuring conductivity. The sample was eluated isocratically with 0.4 mM heptafluorobutyric acid, and the flow rate of the eluent was 1.0 ml min 1. The regenerant was 5 mM tetrabutylammonium hydroxide with a flow rate of 5 ml min 1. Organic acids (tartaric, lactic and acetic acid) were used as standards (40 mg l 1). 2.6. Calculations and statistical methods The accumulation factor of the samples from the field experiment was calculated by dividing the Cu concentration in a given plant part with the Cu concentration in the soil. Statistical analysis of the data was performed using ttest, ANOVA followed by Post Hoc comparisons with TukeyÕs Honest significant difference test (HSD) and Simple Regression analysis. StatisticaÕ99 (Microsoft) was used for the statistical analysis.
3. Results All four species survived and grew during the test period at the Skouriotissa mine waste site. Biomass production, measured as shoot length growth varied with species. Pi. terebinthus grew most during the study and B. cypria the least (Fig. 1, p < 0.01). There was no significant difference in growth caused by the various additives. The background concentration of the four species, prior to the experiment, was 2–6 mg of Cu (kg DW) 1 in leaves and stems, except the leaves of P. brutia which had a significant higher Cu concentration in the needles compared to the other species (Table 2). Copper concentrations in the planted species shoots (5.5–44.7 mg (kg DW) 1) can be compared with those in shoots of Po. equisetiforme, S. alba, V. pyramidata, C. spinosa and F. caria, which were already growing at the mine waste site (12.4–47.4 mg Cu (kg DW) 1). Independent of soil additives, the Cu accumulation in both stems and leaves varied with plant species (Table 2). The leaves of C. creticus had the highest accumulation of Cu of all the species (p < 0.05) and a significantly higher Cu accumulation in the stems than Pi. terebinthus (p < 0.01). In the greenhouse control study on Cu accumulation in leaves, stems and roots of Pi. terebinthus, C. creticus and B. cypria, the first two spe-
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Fig. 1. Shoot length growth (%) of four plant species growing in mine waste mixed with soil (1:1) untreated or treated with chicken fertilizer or Vinassa, n = 4 ± SE.
cies accumulated most Cu in the roots while B. cypria accumulated most in its leaves (Fig. 2). Soil treatment did not have any significant effect on accumulation factors in leaves and stems, which were 0.02–0.06 in most cases. However, C. creticus had a significantly higher leaf accumulation factor than the other species (untreated 0.11 and treated with Vinassa 0.19) and a higher stem accumulation factor than Pi. terebinthus (p < 0.05) (not shown). The mean Cu accumulation in leaves and stems of the plant species Pi. terebinthus, C. creticus, P. brutia and B. cypria (Table 2) was not influenced by the addition of the soil additives Vinassa or chicken fertilizer, with the exception of B. cypria. Leaves of this plant growing in untreated squares with no soil additive showed a significantly higher Cu accumulation (p < 0.005) than leaves of plants of the same species growing in squares with
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Fig. 2. Cu accumulation (Cu (kg DW) 1) of leaves, stems and roots of three species growing for 3 weeks in soil spiked with 150 mg Cu (kg DW) 1 (n = 2). The accumulation value of the Pi. terebinthus stem is too small to be visible.
the addition of Vinassa or chicken fertilizer. Additionally, stems of B. cypria showed a lower Cu accumulation in the untreated squares compared with the stems of those growing in additive-treated squares (p < 0.05). In the greenhouse experiment, Cu accumulation by Po. equisetiforme was not affected by the addition of Vinassa (not shown). Vinassa did release Cu from the mine waste soil: 33.8 mg (kg DW) 1 compared with 0.4 mg (kg DW) 1 for redistilled water (not shown). Vinassa also contained certain organic acids such as tartaric and lactic acid (not shown). Some soil treatments affected pH in the top layer of the soil (0–10 cm down) (Table 1). The pH of the soil in both untreated and Vinassa-treated squares had a significantly higher pH than the soil in chicken fertilizertreated squares (p < 0.01). The pH in the top layer (0–10 cm down) of the soil in the field experiment
Table 2 Copper concentration mg (kg DW) 1 and Cu accumulation mg (kg DW) 1 by four plant species grown during 3 months in mine wasted mixed with soil (1:1) untreated or treated with chicken fertilizer or Vinassa Parameter Treatment
Pistacia terebinthus Leaf
Stem
Leaf
Stem
Leaf
Stem
Leaf
Stem
Background concentration Cu concentration Untreated mine waste +Vinassa +Chicken fertilizer
2.13 (0.62)
5.75 (2.76)
5.4 (0.28)
4.9 (0.96)
11.6 (0.61)
5.7 (0.60)
4.6 (1.00)
3.8 (0.65)
8.37 (1.85) 7.28 (0.61) 6.98 (0.95)
13.4 (1.60) 11.6 (2.20) 8.93 (1.10)
44.7 (10.3) 38.9 (8.24) 39.8 (7.33)
17.1 (1.79) 15.0 (1.37) 18.4 (4.71)
17.1 (2.14) 21.6 (3.51) 20.7 (2.10)
13.9 (1.41) 14.5 (1.41) 10.8 (0.82)
36.8 (4.70) 13.7 (1.94) 13.1 (0.90)
11.8 (1.10) 25.2 (3.03) 30.6 (4.66)
6.24 (1.85) 5.15 (0.61) 4.85 (0.95)
7.64 (1.60) 5.89 (2.20) 3.18 (1.10)
39.3 (10.3) 33.5 (8.24) 34.4 (7.33)
12.2 (1.79) 10.2 (1.37) 13.5 (4.71)
5.5 (2.14) 10.0 (3.51) 9.1 (2.10)
8.2 (1.41) 8.8 (1.41) 5.1 (0.82)
32.3 (4.70) 9.2 (1.94) 8.5 (0.90)
8.0 (1.10) 21.4 (3.03) 26.8 (4.66)
Cu accumulation Untreated mine waste +Vinassa +Chicken fertilizer
Cistus creticus
Pinus brutia
Bosea cypria
Background concentration of Cu is the concentration in plants before plantation. Accumulated Cu is Cu concentration after 3 months – background concentration. n = 4, (SE).
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affected Cu accumulation in stems (p < 0.01) in all four plant species and treatments together, i.e., Cu accumulation increased with a decrease in pH (not shown).
4. Discussion All the tested plant species survived and grew for 3 months at the mine waste site. This indicates, in accordance with the first hypothesis, that the plant species Pi. terebinthus, C. creticus, P. brutia and B. cypria tolerated the enhanced Cu concentration at the mine waste site to some extent. Copper concentrations in C. creticus leaves, untreated B. cypria leaves and treated B. cypria stems reached levels (Table 2) that are toxic to many other species according to Pais and Jones (1997). Concerning the amount of accumulated Cu in leaves and stems, none of the species were hyperaccumulators of Cu ( P5000 mg Cu (kg DW) 1) according to Brooks (1998). It is important to remember that a high Cu accumulation is not necessarily the only way for a plant to cope with a Cu rich environment. The plant may as well tolerate high Cu levels in the soil by preventing the Cu from entering the plant tissue. Metal exclusion is in fact a more common strategy for metal tolerance than metal accumulation (McGrath et al., 2001). In accordance with the first hypothesis, that the different plant species would have different Cu distribution strategies, Cu distribution within the plants varied with species (Table 2 and Fig. 2). This is probably because the studied species have different ways to cope with a metal-rich environment. The results of the greenhouse experiment on how the different plants accumulated and distributed Cu (Fig. 2) show that Pi. terebinthus, C. creticus and B. cypria have different Cu distribution strategies. While Pi. terebinthus and C. creticus accumulate or bind most Cu in their roots, B. cypria accumulates most Cu into its leaves. There are numerous factors that can affect Cu uptake and accumulation by plants. One of them is mycorrhiza. Mycorrhiza symbiosis, which most plants growing under natural conditions have, has been found in plants growing on metal contaminated soil (Pawlowska et al., 1996). Under normal conditions, mycorrhiza often increases the uptake of water and minerals by the plant. However, when the plant is growing in metal contaminated soil, the mycorrhizal interaction can also decrease the uptake of, e.g., Cu by the plant (Heggo and Angle, 1990). One should also keep in mind that Cu uptake and accumulation by plants can be specific not only for species, but also for populations within species. In contrast for the second hypothesis, that the addition of soil additives could improve plant growth and/ or Cu accumulation, the different soil additives Vinassa and chicken fertilizer neither increased Cu accumulation
within any species leaves or stems (Table 2) nor increased shoot length growth (Fig. 1). The absence of any growth effect by the additives is likely due to a too low nutrient content (Table 1). The explanation may also be that, especially in soils with toxic levels of Cu, some species growth is reduced as a consequence of the energy cost of metal tolerance (Wilson, 1988). The reason for the fact that Cu accumulation was not increased in all species and plant parts despite the low pH of Vinassa (Table 1) could be that Vinassa contains a lot of organic acids that may increase the release of Cu from minerals and soil colloids, but also form complexes with it (Jones, 1998). In that way, the addition of Vinassa does not increase Cu accumulation in the plants, which was also shown in the greenhouse experiment (not shown). Chicken fertilizer contains a lot of nutrient ions that can compete with Cu for uptake by the plant (Ernst et al., 1992; Lidon and Henriques, 1993), or bind Cu. It is possible that this is the reason for the unchanged Cu accumulation in the plants growing in squares with the addition of chicken fertilizer (Table 2). It is also possible that the plant-available Cu in the upper soil layer was initially diluted by the addition of soil additives, and that this explains why they did not influence the Cu accumulation. The fact that the untreated stems of B. cypria showed a lower Cu accumulation when growing in control soil compared to those growing in soil treated with Vinassa or chicken fertilizer and the opposite trend in Cu accumulation in leaves (Table 2), is likely to be a consequence of different Cu distribution strategies caused by the soil additives, and not by differences in Cu uptake.
5. Conclusions All of the tested species survived for 3 months and grew at the Skouriotissa mine waste dump, which indicates that they can tolerate Cu to some extent. Thus, it is hard to decide which one of the four species is most suitable for phytoremediation. It seems that B. cypria is a plant that could be suitable for phytoextraction, but this species growth is slow, which is not a good quality for a plant used in phytoextraction. Pi. terebinthus had a much higher growth, but not such a high Cu accumulation. Possibly this species can be used for phytostabilization, as according to the results of this study, it accumulates most Cu in its roots. Another species that might be suitable for further phytostabilization studies is C. creticus, because it had the second highest growth and also accumulated most Cu in its roots. The species that accumulates more Cu in leaves when untreated can be useful as phytoremediating plants, because of the economical advantages of using plants that do not need soil additives to be effective in extracting Cu
L. Johansson et al. / Applied Geochemistry 20 (2005) 101–107
from contaminated soil. Concerning the soil additives Vinassa and chicken fertilizer, the results indicate that these do not improve the plant growth or Cu accumulation considerably.
Acknowledgements This study was financed by Hellenic Copper Mines LTD and Stockholm University. We thank Mr. Demetris Vattis and Mrs. Kyriaki Georgaki at the Hellenic Copper Mines for their contribution to this study. We acknowledge Prof. Lena Kautsky for valuable comments on the manuscript, Per Ola Karis for his help in identification of the plant species and Mr. Takis Tsintides at the Cyprus Forest Department for advice concerning the choice of studied plant species.
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Growth and Cu accumulation by plants grown on Cu containing mine tailings in Cyprus Lisa Johansson a, Constantinos Xydas b, Nikos Messios b, Eva Stoltz a, Maria Greger a,* a
Department of Botany, Stockholm University, SE-106 91 Stockholm, Sweden b Hellenic Copper Mines LTD, Cyprus Received 27 May 2003; accepted 3 July 2004 Editorial handling by R. Fuge
Abstract The Skouriotissa Cu mine in the northern part of Cyprus has produced large amounts of mine waste. Phytoremediation could stabilise the erosion or extract the metals of this waste. The aim of this study was to find out if Pistacia terebinthus, Cistus creticus, Pinus brutia and/or Bosea cypria could grow and tolerate or maybe accumulate Cu from the mine waste containing up to 787 mg Cu (kg DW) 1. Another aim was to see if the liquid wine waste product Vinassa, containing organic acids and having a low pH, or chicken fertilizer could improve plant growth and/or Cu accumulation. The four species were planted at the mine waste site untreated or with the addition of Vinassa or chicken fertilizer as mine waste modifiers. After 3 months, shoot length growth was measured and the plants were analysed for Cu concentration. The pH and Cu concentration of the mine waste mixture in the different treatments was also measured. To find out if plants accumulated Cu to the highest extent in roots or shoot, a greenhouse study was undertaken where the plant species were cultivated for 3 weeks in Cu spiked soil. The results showed that all of the tested species survived and grew on the mine waste site, which indicates that they tolerate the high level of Cu at the mine waste site. The leaves of C. creticus had the highest Cu accumulation of all tested species. Copper accumulation varied with plant species. They seemed to have different distribution strategies for Cu: in Pistacia terebinthus and C. creticus most of the Cu was found in the roots, while B. cypria accumulated most of the Cu in the leaves. Addition of Vinassa and chicken fertilizer did not increase plant growth or Cu accumulation, but did affect the Cu distribution in B. cypria. Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction The Skouriotissa Cu mine in the northern part of Cyprus has been exploited since ancient times (Pyatt, 2001).
*
Corresponding author. Fax: +46 8 162268. E-mail address: [email protected] (M. Greger).
It is situated in the Troodos Mountains, which are dominated by volcanic rock with limited outcrops of umbers, marls and limestone (Searle and Panayioto, 1980). The mining activities in this area have produced large amounts of mine waste, which is rich in pyrite and other sulfide minerals (Yukselen, 2002). The waste can create several environmental problems, of which the leaching of heavy metals, in this case Cu, is considered to be
0883-2927/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.apgeochem.2004.07.003
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the most serious one. Another environmental problem caused by mining activities is erosion due to the absence of vegetation cover on the mine waste site (ClemenssonLindell et al., 1992). The climate in the area of the mine can be classified as semi-arid and the mean annual precipitation is 300 mm while during some years of drought, the annual precipitation can be as low as 200 mm. In Cyprus, there is a long dry hot summer, which lasts for 4–5 months. Several methods for rehabilitation of mining waste areas and the prevention of leaching of heavy metals have been discussed. One of them is phytoextraction, where metal tolerant plants exhibiting high metal accumulation extract and remove heavy metals from soil by harvesting the plants (Cunningham and Berti, 1993; Salt et al., 1998). Phytoextraction postulates that the plants used accumulate a vast amount of the metal of interest in their leaves. The criterion to define a plant as a Cu hyperaccumulator is that it should have a Cu, concentration in leaves of 5000 mg Cu (kg DW) 1 or higher (Brooks, 1998). To this date, Cu hyperaccumulators have been found in 15 plant families (Baker et al., 2000). Examples of hyperaccumulating plant species are the trees Acacia retinoides and Eucalyptus torquata, which when growing in a Cu contaminated site in Cyprus accumulated 0.19% and 0.21% Cu in their leaves, respectively (Pyatt, 2001). Another remediation method is phytostabilisation, where metal tolerant plants stabilise the metals in the soil and thus reduce their bioavailability by root uptake, precipitation or reduction (Salt et al., 1995). These plants should, along with Cu immobilization in soil, have a restricted translocation of the metals to the shoot. In addition, these methods can, if the vegetation cover is high, prevent erosion of the waste material. Copper is an essential micronutrient for plants. The critical deficiency level of Cu in plants is generally in the range of 1–5 mg (kg DW) 1 (Marschner, 1995) and the sufficient or normal level is approximately 5–30 mg (kg DW) 1 (Pais and Jones, 1997). However, Cu can be toxic to plants at concentrations which are too high. As a consequence, some plants have developed tolerance to high levels of Cu. Copper tolerance can be mediated by accumulation and storage (detoxification) or exclusion of Cu by the plant (Salt et al., 1998). Resistance to heavy metal uptake is usually highly specific and depends on the metals enriched in the soil (Verkleij and Schat, 1990). To get plants to grow on mine waste sites, improvements of the soil commonly have to be done since it often is acidic, low in nutrients, high in metal concentrations and retains water poorly (McCabe and Otte, 1997). Different kinds of additives in the surface layer of the metalliferous soils can be applied in order to raise its nutritional status. For example, sewage
sludge and horse dung have been tried out as fertilizers to promote vegetation growth on mine waste with positive results (Borgega˚rd and Rydin, 1989; Stoltz and Greger, 2002). In the present study, the liquid wine waste product Vinassa and chicken fertilizer were used as soil additives. The reason for using Vinassa was the possibility that it could lower the alkalinity (the pH of the studied mine waste was high due to the limestone content) in the soil and thereby increase the Cu availability and the capacity for phytoextraction. Soil from the mine vicinity was also added to provide some nutrients and organic material to the mine waste. The main purpose of the present study was twofold, firstly to find a method for phytoremediation of the Skouriotissa mine waste area, i.e., to find plant species that could be used for phytostabilization and/ or phytoextraction of Cu at the mine waste, and secondly to see if the soil additives could be used to improve the phytoremediation results. To test these, the authors investigated whether the chosen plants (Pi. terebinthus, Cistus creticus, Pinus brutia and Bosea cypria) could grow and tolerate the high Cu concentrations and possibly accumulate Cu from the mine waste. In addition, this study was also done to see if Vinassa or chicken fertilizer could improve plant growth and/or Cu accumulation. The first hypothesis was that the chosen plant species would grow, i.e., tolerate the high levels of Cu in the Skouriotissa mine waste and have different Cu distribution strategies. The second hypothesis was that the addition of Vinassa and chicken fertilizer could improve plant growth by adding nutrients to the soil and, in the case of Vinassa, lower the alkalinity in the soil and thereby increase Cu accumulation.
2. Materials and methods 2.1. Plant species Four local plant species were selected for the study. The selection was based on which species could possibly cope with the environment at the Skouriotissa mine waste dump in Cyprus, i.e., could grow on dry, rocky soil at an altitude of 300 m. The species were: Pi. terebinthus Bieberstein, C. creticus L., P. brutia Tenore and B. cypria Boissier. B. cypria and Pi. terebinthus were bought from the Athalassa nursery, P. brutia from the Platania nursery and C. creticus from the Phassouri nursery in Cyprus. In addition, Polygonum equisetiforme L. was collected at the mine waste dump of the Skouiriotissa mine. This plant was used in the field experiments, as a ground covering vegetation in order to prevent soil erosion and as a possible producer of organic material.
0.10 ND 0.78 0.23 (0.02) 0.25 (0.04) 0.17 (0.01) 0.15 3.9a 41.5 0.7 (0.1) 0.97 (0.26) 2.73 (0.38) 0.21 1.2a 31.7 0.27 (0.04) 0.30 (0.01) 1.54 (0.36) 1.95 ND 360 8.97 (1.45) 9.53 (1.99) 20.00 (2.15) – – – 6.8 (0.3) 7.1 (0.2) 6.3 (0.1) – – – 7.5 (0.1) 7.5 (0.1) 7.2 (0.1) 6.5 3.8 8.8 – – – Mine waste Vinassa Chicken fertilizer Mine waste + soil Mine waste + soil + Vinassa Mine waste + soil + chicken fertilizer
103 The cultivation material was mine waste mixed with soil (1:1) untreated or treated with chicken fertilizer or Vinassa. Samples were taken at 0–15 cm depth. Cutot = HNO3 extractable Cu fraction. ND = no data (n = 2–32), (SE). a In g l 1.
787 3.04a 83 519 (45) 520 (61) 490 (47)
1
Cutot mg kg DW 1
Ca g kg DW 1
N g kg DW 1
P g kg DW 1
C g kg DW
Pistacia terebinthus, C. creticus, P. brutia and B. cypria were planted at the waste dump of the Skouriotissa mine in Cyprus, February 14–15th, 2002. The plants were planted in squares (with an area of approximately 0.25 m2 and with 1 m in between each square) made by a digging machine. The 30-cm top layer of the squares consisted of soil from the mine vicinities mixed with the mine waste in the proportion 1:1. One hundred ml of the physical soil conditioner Terra Cottem (Terra Cottem, Belgium) was also added to all the squares up to a depth of 30 cm. Terra Cottem is a mixture of Kbased organic hydroabsorbent polymers, which is used for its water retaining properties. Vinassa or chicken fertilizer was added to some of the squares as soil additives (10 l of each additive in each square). The chicken fertilizer was added during the plantation and mixed with the topsoil, and the Vinassa was added after the plantation. Vinassa was added one more time (5 l per square) during the experiment, at March 15th, 2002. Samples of pure mine waste, chicken fertilizer and Vinassa were collected for analysis. Experimental set-up for each species: two plants in soil with no soil additives, two plants in soil with chicken fertilizer and two plants in soil with Vinassa. Each treatment was made in four replicates. In addition, two plants of Po. equisetiforme were planted in all of the squares. All of the plantation squares were placed randomly in the test area. The plants were watered once every 2 weeks. Samples of the leaves and stems were collected after approximately 3 months (May 15–16th, 2002) as well as samples of the mine waste soil from all squares (at the depths of 0–10 cm and 10–15 cm). The height of the above ground part of the plants was measured at the beginning and at the end of the experiment with a ruler. Shoot plant samples from Sinapis alba L., Vaccaria pyramidata Medicus, Po. equisetiforme L., Capparis spinosa L. and Ficus caria L. were also taken from different
pH (10–15 cm)
2.3. Field experiments
pH (0–10 cm)
Two different soil additives were used in the study: the liquid wine waste product Vinassa and chicken fertilizer. The Vinassa came from the Keo brewery in Limassol, Cyprus. It had a pH of 3.8 (Table 1) and contained amino acids, tannic acid, tartaric acid, traces of sugars, suspended solids and other natural constituents of wine, according to the brewery. The chicken fertilizer was bought from local farmers. Soil from the mine vicinities (crop field soil) was added to all of the squares to increase its nutritional value. Table 1 shows the concentration of macronutrients and Cu of the mine waste, mine waste + soil and soil additives.
pH
2.2. Soil and soil additives
Table 1 Nutritional status, pH, total and bioavailable Cu concentration of mine waste, Vinassa and chicken fertilizer as well as of samples taken of cultivation material after 3 months of plant growth
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locations at the mine waste site, in order to get background values for plants growing in the studied area. These plants had a maximum Cu concentration of 47.4, a median concentration of 30.5 and a minimum concentration of 12.4 mg Cu (kg DW) 1. 2.4. Greenhouse experiments Plants of Pi. terebinthus, C. creticus, P. brutia and B. cypria were planted in compost (Weibull, Hammenho¨g, Sweden) in a greenhouse equipped with supplementary lamps ((OSRAM daylight, HQI-BT 400 W) (18 h light) and with a temperature of 17–20 °C) and treated with Provado insecticide sticks (Bayer AG, Leverkusen, Denmark) for 2 weeks to prevent insect attack. At the experimental start, the plants were planted in soil spiked with copper chloride (approximately 7 weeks in advance) with a Cu concentration of 150 mg Cu (kg DW soil) 1, and in unspiked soil with a Cu content of 3 mg Cu (kg DW soil) 1. This was done to investigate the influence of Cu concentration in the soil on Cu uptake and distribution within plants. Samples of the plant leaves, stems and roots were taken after 3 weeks. The roots were washed twice in redistilled water, once in EDTA solution (20 mM) and twice in redistilled water. A study on the effect of Vinassa on Cu uptake by Po. equisetiforme was also conducted. Plants of Po. equisetiforme were planted in compost (Weibull), one plant per pot (with the volume 0.62 l). Four of them were planted in the spiked soil with a Cu concentration of 150 mg Cu (kg DW soil) 1, and Vinassa (0.08 l (l soil) 1) was added to two of these plants. The remaining three were control plants. Plant samples of the shoots and roots were taken after 3 weeks, and the roots were washed as above. 2.5. Sample analysis The plant samples were dried at 105 °C and thereafter wet digested in HNO3:HClO4 (7/3, V/V) and analysed for Cu concentration. All metal analysis was performed by atomic absorption spectroscopy (SpectrAA 100, GTA-100, Varian, Springvale, Australia) using both flame and furnace. The amount of Cu released from the mine waste soil by the addition of Vinassa was also measured by mixing the soil (0.5 g) with Vinassa or water (5 ml). The samples were then shaken for 16 h and analysed for Cu content. Additionally, total Cu concentration of all the soil samples from 10–15 cm depth was measured. The samples were dried in an oven at 105 °C, acid digested in HNO3 (7 M) and analysed for Cu. The pH of the soil samples was measured by mixing 5 g of soil with 12 ml of redistilled water. After 16 h of incubation, the pH was measured with a pH-meter (Metrohm 744).
The organic acid content in the Vinassa was analysed by using a Dionex ion chromatographic system consisting of a 4500-gradient pump, an anion micro membrane AMMS-ICE II suppressor, an ED50A electrochemical detector, a conductivity cell D23 detection stabilizer Model DS3-1, and an ionpac ICE-AS6 analytical column (Dionex corporation; Sunnyvale, CA, USA). Twenty ll of the sample were injected manually into the ion chromatograph and run for 20 min measuring conductivity. The sample was eluated isocratically with 0.4 mM heptafluorobutyric acid, and the flow rate of the eluent was 1.0 ml min 1. The regenerant was 5 mM tetrabutylammonium hydroxide with a flow rate of 5 ml min 1. Organic acids (tartaric, lactic and acetic acid) were used as standards (40 mg l 1). 2.6. Calculations and statistical methods The accumulation factor of the samples from the field experiment was calculated by dividing the Cu concentration in a given plant part with the Cu concentration in the soil. Statistical analysis of the data was performed using ttest, ANOVA followed by Post Hoc comparisons with TukeyÕs Honest significant difference test (HSD) and Simple Regression analysis. StatisticaÕ99 (Microsoft) was used for the statistical analysis.
3. Results All four species survived and grew during the test period at the Skouriotissa mine waste site. Biomass production, measured as shoot length growth varied with species. Pi. terebinthus grew most during the study and B. cypria the least (Fig. 1, p < 0.01). There was no significant difference in growth caused by the various additives. The background concentration of the four species, prior to the experiment, was 2–6 mg of Cu (kg DW) 1 in leaves and stems, except the leaves of P. brutia which had a significant higher Cu concentration in the needles compared to the other species (Table 2). Copper concentrations in the planted species shoots (5.5–44.7 mg (kg DW) 1) can be compared with those in shoots of Po. equisetiforme, S. alba, V. pyramidata, C. spinosa and F. caria, which were already growing at the mine waste site (12.4–47.4 mg Cu (kg DW) 1). Independent of soil additives, the Cu accumulation in both stems and leaves varied with plant species (Table 2). The leaves of C. creticus had the highest accumulation of Cu of all the species (p < 0.05) and a significantly higher Cu accumulation in the stems than Pi. terebinthus (p < 0.01). In the greenhouse control study on Cu accumulation in leaves, stems and roots of Pi. terebinthus, C. creticus and B. cypria, the first two spe-
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Fig. 1. Shoot length growth (%) of four plant species growing in mine waste mixed with soil (1:1) untreated or treated with chicken fertilizer or Vinassa, n = 4 ± SE.
cies accumulated most Cu in the roots while B. cypria accumulated most in its leaves (Fig. 2). Soil treatment did not have any significant effect on accumulation factors in leaves and stems, which were 0.02–0.06 in most cases. However, C. creticus had a significantly higher leaf accumulation factor than the other species (untreated 0.11 and treated with Vinassa 0.19) and a higher stem accumulation factor than Pi. terebinthus (p < 0.05) (not shown). The mean Cu accumulation in leaves and stems of the plant species Pi. terebinthus, C. creticus, P. brutia and B. cypria (Table 2) was not influenced by the addition of the soil additives Vinassa or chicken fertilizer, with the exception of B. cypria. Leaves of this plant growing in untreated squares with no soil additive showed a significantly higher Cu accumulation (p < 0.005) than leaves of plants of the same species growing in squares with
105
Fig. 2. Cu accumulation (Cu (kg DW) 1) of leaves, stems and roots of three species growing for 3 weeks in soil spiked with 150 mg Cu (kg DW) 1 (n = 2). The accumulation value of the Pi. terebinthus stem is too small to be visible.
the addition of Vinassa or chicken fertilizer. Additionally, stems of B. cypria showed a lower Cu accumulation in the untreated squares compared with the stems of those growing in additive-treated squares (p < 0.05). In the greenhouse experiment, Cu accumulation by Po. equisetiforme was not affected by the addition of Vinassa (not shown). Vinassa did release Cu from the mine waste soil: 33.8 mg (kg DW) 1 compared with 0.4 mg (kg DW) 1 for redistilled water (not shown). Vinassa also contained certain organic acids such as tartaric and lactic acid (not shown). Some soil treatments affected pH in the top layer of the soil (0–10 cm down) (Table 1). The pH of the soil in both untreated and Vinassa-treated squares had a significantly higher pH than the soil in chicken fertilizertreated squares (p < 0.01). The pH in the top layer (0–10 cm down) of the soil in the field experiment
Table 2 Copper concentration mg (kg DW) 1 and Cu accumulation mg (kg DW) 1 by four plant species grown during 3 months in mine wasted mixed with soil (1:1) untreated or treated with chicken fertilizer or Vinassa Parameter Treatment
Pistacia terebinthus Leaf
Stem
Leaf
Stem
Leaf
Stem
Leaf
Stem
Background concentration Cu concentration Untreated mine waste +Vinassa +Chicken fertilizer
2.13 (0.62)
5.75 (2.76)
5.4 (0.28)
4.9 (0.96)
11.6 (0.61)
5.7 (0.60)
4.6 (1.00)
3.8 (0.65)
8.37 (1.85) 7.28 (0.61) 6.98 (0.95)
13.4 (1.60) 11.6 (2.20) 8.93 (1.10)
44.7 (10.3) 38.9 (8.24) 39.8 (7.33)
17.1 (1.79) 15.0 (1.37) 18.4 (4.71)
17.1 (2.14) 21.6 (3.51) 20.7 (2.10)
13.9 (1.41) 14.5 (1.41) 10.8 (0.82)
36.8 (4.70) 13.7 (1.94) 13.1 (0.90)
11.8 (1.10) 25.2 (3.03) 30.6 (4.66)
6.24 (1.85) 5.15 (0.61) 4.85 (0.95)
7.64 (1.60) 5.89 (2.20) 3.18 (1.10)
39.3 (10.3) 33.5 (8.24) 34.4 (7.33)
12.2 (1.79) 10.2 (1.37) 13.5 (4.71)
5.5 (2.14) 10.0 (3.51) 9.1 (2.10)
8.2 (1.41) 8.8 (1.41) 5.1 (0.82)
32.3 (4.70) 9.2 (1.94) 8.5 (0.90)
8.0 (1.10) 21.4 (3.03) 26.8 (4.66)
Cu accumulation Untreated mine waste +Vinassa +Chicken fertilizer
Cistus creticus
Pinus brutia
Bosea cypria
Background concentration of Cu is the concentration in plants before plantation. Accumulated Cu is Cu concentration after 3 months – background concentration. n = 4, (SE).
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affected Cu accumulation in stems (p < 0.01) in all four plant species and treatments together, i.e., Cu accumulation increased with a decrease in pH (not shown).
4. Discussion All the tested plant species survived and grew for 3 months at the mine waste site. This indicates, in accordance with the first hypothesis, that the plant species Pi. terebinthus, C. creticus, P. brutia and B. cypria tolerated the enhanced Cu concentration at the mine waste site to some extent. Copper concentrations in C. creticus leaves, untreated B. cypria leaves and treated B. cypria stems reached levels (Table 2) that are toxic to many other species according to Pais and Jones (1997). Concerning the amount of accumulated Cu in leaves and stems, none of the species were hyperaccumulators of Cu ( P5000 mg Cu (kg DW) 1) according to Brooks (1998). It is important to remember that a high Cu accumulation is not necessarily the only way for a plant to cope with a Cu rich environment. The plant may as well tolerate high Cu levels in the soil by preventing the Cu from entering the plant tissue. Metal exclusion is in fact a more common strategy for metal tolerance than metal accumulation (McGrath et al., 2001). In accordance with the first hypothesis, that the different plant species would have different Cu distribution strategies, Cu distribution within the plants varied with species (Table 2 and Fig. 2). This is probably because the studied species have different ways to cope with a metal-rich environment. The results of the greenhouse experiment on how the different plants accumulated and distributed Cu (Fig. 2) show that Pi. terebinthus, C. creticus and B. cypria have different Cu distribution strategies. While Pi. terebinthus and C. creticus accumulate or bind most Cu in their roots, B. cypria accumulates most Cu into its leaves. There are numerous factors that can affect Cu uptake and accumulation by plants. One of them is mycorrhiza. Mycorrhiza symbiosis, which most plants growing under natural conditions have, has been found in plants growing on metal contaminated soil (Pawlowska et al., 1996). Under normal conditions, mycorrhiza often increases the uptake of water and minerals by the plant. However, when the plant is growing in metal contaminated soil, the mycorrhizal interaction can also decrease the uptake of, e.g., Cu by the plant (Heggo and Angle, 1990). One should also keep in mind that Cu uptake and accumulation by plants can be specific not only for species, but also for populations within species. In contrast for the second hypothesis, that the addition of soil additives could improve plant growth and/ or Cu accumulation, the different soil additives Vinassa and chicken fertilizer neither increased Cu accumulation
within any species leaves or stems (Table 2) nor increased shoot length growth (Fig. 1). The absence of any growth effect by the additives is likely due to a too low nutrient content (Table 1). The explanation may also be that, especially in soils with toxic levels of Cu, some species growth is reduced as a consequence of the energy cost of metal tolerance (Wilson, 1988). The reason for the fact that Cu accumulation was not increased in all species and plant parts despite the low pH of Vinassa (Table 1) could be that Vinassa contains a lot of organic acids that may increase the release of Cu from minerals and soil colloids, but also form complexes with it (Jones, 1998). In that way, the addition of Vinassa does not increase Cu accumulation in the plants, which was also shown in the greenhouse experiment (not shown). Chicken fertilizer contains a lot of nutrient ions that can compete with Cu for uptake by the plant (Ernst et al., 1992; Lidon and Henriques, 1993), or bind Cu. It is possible that this is the reason for the unchanged Cu accumulation in the plants growing in squares with the addition of chicken fertilizer (Table 2). It is also possible that the plant-available Cu in the upper soil layer was initially diluted by the addition of soil additives, and that this explains why they did not influence the Cu accumulation. The fact that the untreated stems of B. cypria showed a lower Cu accumulation when growing in control soil compared to those growing in soil treated with Vinassa or chicken fertilizer and the opposite trend in Cu accumulation in leaves (Table 2), is likely to be a consequence of different Cu distribution strategies caused by the soil additives, and not by differences in Cu uptake.
5. Conclusions All of the tested species survived for 3 months and grew at the Skouriotissa mine waste dump, which indicates that they can tolerate Cu to some extent. Thus, it is hard to decide which one of the four species is most suitable for phytoremediation. It seems that B. cypria is a plant that could be suitable for phytoextraction, but this species growth is slow, which is not a good quality for a plant used in phytoextraction. Pi. terebinthus had a much higher growth, but not such a high Cu accumulation. Possibly this species can be used for phytostabilization, as according to the results of this study, it accumulates most Cu in its roots. Another species that might be suitable for further phytostabilization studies is C. creticus, because it had the second highest growth and also accumulated most Cu in its roots. The species that accumulates more Cu in leaves when untreated can be useful as phytoremediating plants, because of the economical advantages of using plants that do not need soil additives to be effective in extracting Cu
L. Johansson et al. / Applied Geochemistry 20 (2005) 101–107
from contaminated soil. Concerning the soil additives Vinassa and chicken fertilizer, the results indicate that these do not improve the plant growth or Cu accumulation considerably.
Acknowledgements This study was financed by Hellenic Copper Mines LTD and Stockholm University. We thank Mr. Demetris Vattis and Mrs. Kyriaki Georgaki at the Hellenic Copper Mines for their contribution to this study. We acknowledge Prof. Lena Kautsky for valuable comments on the manuscript, Per Ola Karis for his help in identification of the plant species and Mr. Takis Tsintides at the Cyprus Forest Department for advice concerning the choice of studied plant species.
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