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Effect of mixed cadmium, copper, nickel and zinc at different pHs upon alfalfa growth and heavy metal uptake
Environmental Pollution 119 (2002) 291–301 www.elsevier.com/locate/envpol
Research papers
Effect of mixed cadmium, copper, nickel and zinc at different pHs upon alfalfa growth and heavy metal uptake J.R. Peralta-Videaa,b, J.L. Gardea-Torresdeya,c,*, E. Gomezc, K.J. Tiemanna,c, J.G. Parsonsa,c, G. Carrillod a
Environmental Science and Engineering PhD Program, University of Texas at El Paso, El Paso, TX 797968, USA b ITESM Campus Ciudad Jua´rez, PMB 10482, El Paso, TX 79995, USA c Chemistry Department, University of Texas at El Paso, El Paso TX 79968, USA d IREGEP, Colegio de Postgraduados, Montecillo, Mexico 56230 Received 5 October 2001; accepted 28 December 2001
‘‘Capsule’’: Alfalfa (Medicago sativa) plants were able to take up metals from a mixture of Cd(II), Cu(II), Ni(II) and Zn(II) in soils. Abstract Alfalfa plants were grown in soil-pots contaminated with a mixture of Cd(II), Cu(II), Ni(II), and Zn(II), (at 50 mg/kg each) at pHs of 4.5, 5.8, and 7.1. The plants were fertilized using a nutrient solution, which was adjusted appropriately to the same pH. Plants in the control treatment were grown in the absence of the heavy metals mixture. The growth of the control plants was the same at the three pHs studied and the heavy metal stressed plants also showed similar behavior at each pHs. There were statistically significant differences (P< 0.05) between the shoot length of the control treatment plants and the length of plants grown in the presence of the heavy metal mixture. Under the effects of the heavy metal mixture, nickel was the most accumulated element in the shoot tissue, with 437, 333, and 308 ppm at pH 7.1, 5.8, and 4.5, respectively. Cadmium was found to be second in accumulated concentrations with 202 ppm, 124 ppm, and 132 ppm at pH 7.1, 5.8, and 4.5, respectively; while zinc was third, followed by copper. The maximum relative uptakes (element in plant/element in soil–water–solution) were found to be 26 times for nickel, 23 times for cadmium, 12 times for zinc, and 6 times for copper. We considered these relations as indicative of the ability of alfalfa plants to take up elements from a soil matrix contaminated with a mixture of cadmium, copper, nickel, and zinc. # 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction It is almost impossible to visualize a soil without trace levels of heavy metals. However, anthropogenic activities have concentrated some of these elements in certain areas up to dangerous levels for living organisms (Chatterjee and Chatterjee, 2000). Activities such as mining and agriculture have polluted extensive areas throughout the world (Herawati et al., 2000; Brun, 2001; Zanthopoulos et al., 1999; Shallari et al., 1998; Gardea-Torresdey et al., 1996; Smith et al., 1996). The advancement of science in instrumental and analytical methods towards the end of the twentieth-century has allowed for the determination * Corresponding author. Tel.: +1-915-747-5359; fax: +1-915-7475748. E-mail address: [email protected] (J.L. Gardea-Torresdey).
of the role certain transition elements in the metabolism of some plant species. It is well known that elements such as Cu, Mo, Ni, Cl, and Zn, among others, are essential for plant growth in low concentrations (Reeves and Baker, 2000; Kahle, 1993; Brady and Weil, 1999; Taiz and Zeiger, 1998). Nevertheless, beyond certain threshold concentrations, these same elements become toxic for most plant species (Monni et al., 2000; Blaylock and Huang, 2000; Brady and Weil, 1999). The discovery that certain plant species are capable of tolerating high concentrations of heavy metals opened new possibilities to use these plants to remediate contaminated soils (phytoremediation). Many studies have been conducted to determine the toxic levels of heavy metals for certain plants, especially those metals considered as public health threats (Reeves and Baker, 2000; Fernandes and Henriques, 1991; Terry and
0269-7491/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0269-7491(02)00105-7
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Ban˜uelos, 2000). Several studies have been performed to investigate plants that accumulate different metals separately or in combinations. Some seaweed species for example, have been found to tolerate metal ions both individually (up to 1000 g l 1 of zinc) and in multi-metal solutions (Filho et al., 1997, Sanchez et al., 2001). Some varieties of Thlaspi and certain ecotypes of Silene vulgaris have also been found to be resistant to Zn and Cd (Ernst, et al., 2000, Brown et al., 1994). Reeves and Baker (2000) presented a detailed compilation of plant species that accumulate nickel, zinc, cadmium, lead, cobalt, copper, manganese, and selenium, individually, as well as in mixtures containing zinc and cadmium; zinc and lead; and cobalt and copper. Ouzounidou et al. (1995) studied the interaction of Cu with Ca and Fe assimilation and Bates (1971) studied the interaction and differential uptake of macro and microelements by several plant species. Also, Luo and Rimmer (1995) demonstrated that the absorption of Zn and Cu in barley is affected by the presence of Cd and Pb in the soil. In a previous study conducted with live alfalfa plants, it was determined that they were able to accumulate approximately 26,600 mg/kg of zinc, 8500 mg/kg of nickel, 12,000 mg/kg of copper, 6000 mg/kg of chromium, and 10,700 mg/kg of cadmium, individually (Peralta et al., 2001a). However, the objective of the present study is to evaluate the combined effects of cadmium, copper, nickel, and zinc applied to montmorillonite containing soils [abbreviated chemical formula: (Al,Mg)2(OH)2Si4O10] at three pHs, on seed germination and plant growth.
2. Materials and methods 2.1. Experimental setup This study was conducted under controlled light and temperature conditions. General-purpose plastic pots were filled with 300 g of a mixture of 70% of sand 0.25 mm of diameter (f), 25% gravel (0.81 mm f), and 5% bentonite (montmorillonite clay). Gravel and sand were soaked for two days in HCl (0.01 M) to remove any metals that may be present, then rinsed three times with deionized water and oven dried at 100 C. The soil in each pot was watered with 100 mL of a mixed metal solution made from Cd(NO3)2 4(H2O), Cu(NO3)2 2.5(H2O), Ni(NO3)2 6(H2O), and Zn(NO3)2 6(H2O) salts diluted in deionized water. The mix contained a concentration of 50 ppm of Cd(II), Cu(II), Ni(II), and Zn(II). The metal mixture was then separated in three aliquots. One aliquot was adjusted to pH 4.5, and the other aliquots to pH 5.8 and 7.1. The solution was slowly mixed thoroughly with the soil using a glass rod. Each treatment was replicated three times for statistical purposes. After 2 days, each pot received 50 ml of a
nutrient solution, which was adjusted to the respective pH of 4.5, 5.8 and 7.1. The nutrient solution consisted of Ca(NO3)2 4(H2O), 3.5710 4M; H3BO3, 2.3110 5 M; Ca(Cl2) 2(H2O), 2.1410 3 M; KH2PO4, 9.68 10 4 M; KNO3, 2.5510 4 M; Mg(ClO4)2, 1.0410 3 M; FeCl3, 6.8310 5 M; MnSO4 H2O, 7.6910 6 M; MoO3, 110 5 M; CuSO4 5(H2O); and Zn (NO3)2 6(H2O). The control treatment consisted of the nutrient solution adjusted to the same respective pHs as the heavy metal mixture. The deionized water used for watering all the treatments was also adjusted to pHs of 4.5, 5.8, and 7.1. Approximately 75 seeds of alfalfa (Mesa variety) were soaked in formaldehyde 3% v/v for 10 min to reduce fungal contamination, washed three times with deionized water and put into pots. These pots were provided with a photoperiod of 12 h at 1500 luxes. These were covered with transparent plastic bags to avoid excessive desiccation and prevent airborne contamination. 2.2. Evaluation of treatments Seedlings were harvested 15 days after planting. A sample of five plants per treatment was utilized to determine the shoot length, by measuring them from the crown to the main shoot apex. The alfalfa growth data were further analyzed through analysis of variance (ANOVA). All the plants were washed with deionized water, oven dried at 70 C for 72 h, weighed and acid digested. The acid digestion was performed with a microwave oven (CEM MarsX) at 100 C for 15 min with 10 ml of concentrated nitric acid (HNO3). The volume was subsequently adjusted to 10 ml and the samples were analyzed for elemental content using a Perkin–Elmer Optima 4300 Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES). 2.3. Soil analysis A sample of approximately 1 g of dry soil was taken from each treatment-pot to determine the amount of element present in the water-soluble fraction. The volume of each sample was brought up to 8 ml with deionized water, left to equilibrate for 1 day and centrifuged at 3000 rpm for 5 min. Then, each supernatant was analyzed using the ICP-OES to determine the water-soluble concentration of heavy metals tested (Cd, Cu, Ni, and Zn) as well as the water-soluble macro and micronutrients (Ca, Mg, S, P, Mn, Mo, Fe, and Cl) present in the soil.
3. Results Originally, the mixture of heavy metals tested included: Cr(VI), Cd(II), Cu(II), Ni(II), and Zn(II), but the
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alfalfa seeds hardly germinated. Further screenings of different combinations of the metals in Petri dishes were performed in order to identify which one of the metals was causing detrimental effects. Results from these procedures identified Cr(VI) as the metal responsible of germination inhibition and thus Cr(VI) was excluded from the metal mixture tested. 3.1. Evaluation of alfalfa shoot growth Table 1 shows the shoots’ length of the alfalfa plants grown with and without the heavy metal treatments. Our results show that there were no statistical significant differences among the shoot length of the control treatment plants, although the plants grown at pH 5.8 had the highest elongation (4.1 cm). Likewise, there were no statistical significant differences between the shoots’ lengths of plants that grew in the presence of the mixed heavy metals. Overall, the plants grown at pH 7.1 had the highest elongation (2.1 cm). However, the differences between the shoot length of the control treatment plants and the plants cultivated with the mixed heavy metals were found to be statistically significant (P < 0.01; See Table 1). 3.2. Concentration of heavy metals in plant tissues Fig. 1 shows the amount of heavy metals found in the alfalfa shoot tissues depending on the concentration of heavy metals in the soil and the initial pH. This figure shows that when the heavy metals were at trace levels in the soil (Fig. 1a), the amounts of Zn in the shoot plant tissues (160 ppm) were significantly higher than Cd, Cu, and Ni at all pHs studied (P < 0.01). These results agree with the previous results found in alfalfa plants cultivated in agar (Peralta at al., 2001b). Nevertheless, when the soil was contaminated with the 50 ppm mixture of Cd, Cu, Ni, and Zn, nickel was found to be the heavy metal present in the highest amount in shoot plant tissue (Fig. 1b). The concentrations of nickel were found to be 437, 333, and 308 ppm at pH 7.1, 5.8, and 4.5, respectively. The differences among the concentration of nickel in shoot tissues (average of the three pHs), as compared with the
Table 1 Shoot length (cm) of alfalfa plants Mesa variety after 2 weeks of exposure to heavy metals at three pHsa pH
Control
Heavy metal mixture
4.5 5.8 7.1
3.9 4.1 4.0
2.0 2.0 2.1
a
Data are average of five plants.
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maximum concentration at any pH of cadmium (202 ppm), copper (105 ppm), and zinc (160 ppm) were statistically significant (P < 0.05). In all cases the highest amount of heavy metals in shoot tissues were found in those plants that grew in soil watered with the initial solution adjusted at pH 7.1. These results were slightly different from those found in maize, in which there was no pH-dependence for copper uptake (Brun et al., 2001). 3.3. Concentration of microelements in plant tissues Fig. 2a,b shows the concentration of the micronutrients found in the shoot plant tissues depending on the presence of the heavy metals in the soil. Fig. 2a shows that Fe, Mn, and Mo were present in higher amounts in the tissues of those plants that grew at pH 7.1 in the control treatment. The concentration of Fe found in the tissues of the control treatment plants grown at pH 7.1 was 229 ppm (Fig. 2a), while the Fe level was 151 ppm in the plants that grew with the mixed heavy metals (Fig. 2b). The difference was found to be statistically significant (P < 0.05). Similar results were found with Fe in maize (Ouzounidou et al., 1995). The presence of chlorine in plant tissues was significantly higher (P < 0.0001) in those plants that grew in the presence of mixed heavy metals (1600 ppm at pH 5.8; Fig. 2b). Aluminum was present in the soil at higher levels in all the treatments, as compared with the other elements studied (Fig. 2a,b). It is known that montmorillonite clay contains Al as a structural element. However, no significant aluminum uptake by alfalfa plant was observed (Fig. 2a,b). 3.4. Concentration of macronutrient in plant tissues Fig. 3a,b shows that phosphorus and sulfur were found to be the macronutrients with the highest uptake in the plant tissues (P < 0.01). Under growth conditions with heavy metals at trace level (Fig. 3a), the amount of P in the plant tissue was higher at pH 4.5 and pH 7.1 (33,311 and 36,111 ppm, respectively), while lower (18,135 ppm) at pH 5.8. In the case of sulfur, the tissues had 41,595 ppm at pH 4.5, while 30,828 ppm at pH 7.1 and 11,753 ppm at pH 5.8. When the soil received 50 ppm of the heavy metal mixture (Fig. 3b), the concentrations of P and S in plant tissues increased as the pH was raised from 4.5 to 7.1 (refer to Fig. 3b). The presence of the heavy metal mixture also caused a reduction in the amount of Ca in the plant tissue at pH 4.5. Similar results were found in maize grown with 80 mM of Cu(II) (Ouzounidou et al., 1995). These investigators found that Cu(II) caused a reduction in Ca and Fe concentration in maize roots. Also, excess of Cu(II) concentration causes a reduction in Ca uptake by Lotus purshianus (Lin and Wu, 1994).
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Fig. 1. Heavy metal concentration in soil and shoot tissues of alfalfa plant after 15 days of growth at three different pHs. (a) Control treatments when the dose of heavy metals in soil was at trace levels; (b) the results after the soil received a mixture of 50 ppm of each heavy metal. (s indicates soil, p indicates plant).
3.5. Relative uptake of the elements The concentration of the elements present in the plant tissues was related to the elemental concentration in the soil. Fig. 4a shows the ratio of heavy metal in plant tissues to heavy metal in the soil (P/S) in the control treatment while Fig. 4b shows the ratio P/S found in those plants grown in the presence of the mixture of the
heavy metals. In the control treatment, the ratios for Zn at pH 7.1 and the ratios for Cd at the three pHs could not be calculated since Zn and Cd were not detected in the soil extract solution (Zn and Cu were present in the nutrient solution and traces of Ni were detected in the bentonite, while Cd was not a component of the nutrient solution and was not detected in the bentonite). There were no statistical differences between the P/S
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Fig. 2. Micronutrient concentrations in soil and shoots tissues of alfalfa plant after 15 days of growth at three different pHs. (a) Control treatments when the dose of heavy metals in soil was at trace level; (b) the results after the soil received a mixture of 50 ppm of Cd(II), Cu(II), Ni(II), and Zn(II); (s indicates soil, p indicates plant).
ratios for Cd and Ni at any pH (Fig. 4b). Nickel presented P/S ratios of 22, 23, and 26 times, and cadmium presented P/S ratios of 23, 18, and 23 times at the pHs of 4.5, 5.8, and 7.1, respectively. Cu and Zn presented P/ S ratios close to 10 times at the three pHs. The differences among the ratios found for Ni and Cd, and those found for Cu and Zn where statistically significant (P < 0.05). A study performed with rice by Herawati et
al. (2000), did not showed correlation among the content of Cd, Cu, and Zn in plant tissue and the amount of those metals found in the soil. Fig. 5a,b show the magnitude of the P/S ratios for some micronutrients considered in this study. Fe did not present changes in the P/S ratio for the control treatment plants (Fig. 5a) and for the plants grown under heavy metal stress (Fig. 5b). At pH 7.1, molybdenum
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Fig. 3. Macronutrient concentrations in soil and shoots tissues of alfalfa plant after 15 days of growth at three different pHs. (a) Control treatments when the dose of heavy metals in soil was at trace level; (b) the results after the soil received a mixture of 50 ppm of Cd(II), Cu(II), Ni(II), and Zn(II); (s indicates soil, p indicates plant).
showed P/S ratios of 121 times in the plants of the control treatment (Fig. 5a) and 43 times in those plants grown in the soil treated with the heavy metal mixture (Fig. 5b). At pH 4.5, the ratios were 19 times (Fig. 5a) and 64 times (Fig. 5b). Manganese presented P/S ratios of 56 times (Fig. 5a) and 52 times (Fig. 5b). At pH 5.8 the ratios were 12 times (Fig. 5a) and 23 times (Fig. 5b).
At pH 4.5 there were ratios of 36 times (Fig. 5a) and 32 times (Fig. 5b). It was not possible to calculate the ratio of Cl in the plant tissue related to Cl in the soil extract solution because Cl was not detected in the soil extract solution. Even though the macronutrients S and P were found in comparable quantities within tissues of both the
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Fig. 4. Ratio of Plant to soil concentration of the heavy metals in shoot tissues of alfalfa after 15 days of growth at three different pHs. (a) Control treatments when the dose of heavy metals in soil was at trace level; (b) the results after the soil received a mixture of 50 ppm of Cd(II), Cu(II), Ni(II), and Zn(II); (s indicates soil, p indicates plant).
control treatment plants and mixed heavy metal stressed plants (Fig. 3a,b), the amount of S in the plant related to the amount of S in the soil, was significantly lower (P < 0.00001) than the P/S ratio of P in both the control treatment plants and the plants grown in the presence of the heavy metal mixture (Fig. 6a,b, respectively). In contrast with the other elements studied, the P/S ratios presented by phosphorus were higher in those plants
grown under the effects of the heavy metal mixture (Fig. 6a,b, respectively). Significant linear correlations (P < 0.001) were found between 48 pairs of combinations among the elements detected in the soil and plant tissue (Table 2). Only aluminum (data not shown) and iron presented significant negative correlation coefficients (P < 0.001) with some of the elements in the plant tissues and the solution soil
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Fig. 5. Ratio of Plant to soil concentration of the microelements in shoot tissues of alfalfa after 15 days of growth at three different pHs. (a) Control treatments when the dose of heavy metals in soil was at trace level; (b) the results after the soil received a mixture of 50 ppm of Cd(II), Cu(II), Ni(II), and Zn(II); (s indicates soil, p indicates plant).
extracts. In addition, Fe in the plant tissue was negatively correlated with Fe in the soil (r2= 0.8237) and with Mg in the soil (r2= 0.8696). The amounts of heavy metals in the plant tissues were found to have a high correlation with the amount of these metals in the solution soil extracts (P < 0.001) with an r2 close to 1.00. Ozdemir and Sagiroglu (2000) reported positive significant correlations between Cu in soil and in plant tissues for several species of plants. However, Herawati et al. (2000) did not find the same correlation among the content of Cu, Cd, and Zn in soil and rice plant tissues.
4. Discussion These studies have shown that at least 100 ppm of Cd, Cu, Ni, and Zn, were present in the shoot tissues of alfalfa plant cultivated in the soil contaminated with a mixture of heavy metals (at 50 ppm each metal). Our results indicate a lack of specificity for metal uptake in the alfalfa plant, which is a common phenomenon in other plant species (Baker et al., 2000). Comparing the shoot length of the plants grown under the effects of the heavy metal mixture and that of the control treatment (Table 1), it is evident that a combined stress is
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Fig. 6. Ratio of Plant to soil concentration of the macroelements in shoot tissues of alfalfa after 15 days of growth at three different pHs. (a) Control treatments when the dose of heavy metals in soil was at trace level; (b) the results after the soil received a mixture of 50 ppm of Cd(II), Cu(II), Ni(II), and Zn(II); (s indicates soil, p indicates plant).
occurring due to the heavy metal mixture, because the alfalfa plant has demonstrated the ability to grow well in soils individually contaminated with more than 50 ppm of heavy metals (Peralta et al., 2001b). Similar detrimental effects have also been observed in other plant species (Monni et al., 2000). These researchers found that the mixture of copper and nickel affected the elongation of shoots, as well the root dry weight of Empetrum nigrum. Additionally, the bentonite clay
(montmorillonite) used in this study possesses overall negative charges on its surface, which made it possible to adsorb the Cd(II), Cu(II), Ni(II), and Zn(II) in the soil micelle (Brady and Weil, 1999), which results in low extraction of the metals from the soil. However, the concentration of Ni found in the alfalfa plant shoots (Fig. 1b) and the P/S ratio seen for Ni, as compared to the ratios of Zn, and Cu indicate that the alfalfa plant successfully competes with the bentonite and uptakes
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Table 2 Pearson product–moment correlation coefficient for some elements measured in this researcha Variable Cap Cap Mgp Mnp Mop Cup Cdp Nip Znp Fep Clp Fes Mgs Zns Nis Cds Cus Pp a
1.0000 0.8893 0.6418 0.6526 0.6033 0.5908 0.5633 0.6051 0.3839 0.4089 0.0757 0.0244 0.4569 0.4770 0.5484 0.5816 0.9093
Mgp
1.0000 0.8819 0.8978 0.5241 0.4313 0.4001 0.4903 0.6205 0.2489 0.1525 0.2408 0.2790 0.3065 0.3977 0.4242 0.9901
Mnp
1.0000 0.9555 0.4753 0.2971 0.2641 0.3840 0.7041 0.1626 0.3379 0.3952 0.1724 0.2015 0.2521 0.2763 0.8364
Mop
1.0000 0.2514 0.0869 0.0533 0.1702 0.8620 0.0674 0.5095 0.5743 0.0569 0.0267 0.0439 0.0701 0.8568
Cup
1.0000 0.9701 0.9467 0.9859 0.2465 0.8803 0.6220 0.5754 0.9220 0.9308 0.9431 0.9616 0.5435
Cdp
1.0000 0.9924 0.9944 0.3994 0.9415 0.7271 0.6815 0.9765 0.9804 0.9911 0.9987 0.4582
Nip
1.0000 0.9850 0.4307 0.9726 0.7071 0.6664 0.9907 0.9935 0.9977 0.9931 0.4147
Znp
1.0000 0.3317 0.9307 0.6773 0.6309 0.9631 0.9694 0.9846 0.9922 0.5099
Fep
1.0000 0.5290 0.8237 0.8696 0.5268 0.5015 0.4466 0.4183 0.5786
Clp
Fes
1.0000 0.6786 0.6579 0.9891 0.9884 0.9670 0.9428 0.2416
1.0000 0.9945 0.7405 0.7261 0.7310 0.7438 0.0677
Zns
Nis
Cds
Cus
Pp
1.0000 0.9995 0.9867 0.9767 0.2904
1.0000 0.9893 1.0000 0.9801 0.9947 1.0000 0.3167 0.4156 0.4525 1.0000
Figures in bold represent significant values (P <0.001).
this metal vigorously. Therefore the alfalfa plant actively removes Ni from the soil matrix and translocates it from the roots to the shoots in a similar fashion to the mechanism suggested for heavy metal hyperaccumulator species (Salt and Kramer, 2000). Furthermore, the elevated levels of Cl and S (Figs. 2b,3b) found within the shoots of the alfalfa plants grown in soils contaminated with heavy metals have been suggested to be due to the powerful complexation of Cl and S to the heavy metals, which results in their common translocation with the metals taken up by the plant (McLaughlin et al., 1998a,b). The concentrations of Ca, Mg, and K in the tissues of the control treatment plants did not show appreciable differences when compared with the concentrations of Ca, Mg, and K found in the tissues of the plants grown with the heavy metal mixture. According to Brady and Weil (1999) the order of strength of adsorption of these macronutrients is Ca > Mg> K. This was indeed observed with the alfalfa plants in this study. Furthermore, the magnitude of the ratio of the metal concentration in the plant tissues to the metal concentration in the soil followed the same order. A negative correlation coefficient was found between Fe in soil and Fe in the plant tissue (Table 2). The pH effects may explain this since Fe is available to complex with many ligands within the pH ranges of this study. The Fe may form microcrystalline structures that make it unavailable in order for the plant to uptake it (Filep, 1999). Nevertheless, the data from these studies show that the concentrations of some of the metals were considerably higher than the original soil concentrations, indicating that the plant is capable of competing with
the bentonite clay to remove and fix metal ions from the soil matrix.
5. Conclusions Based on these results, we concluded that alfalfa plant can successfully uptake Cd(II), Cu(II), Ni(II), and Zn(II) in a montmorillonite-clay medium. The concentrations of the elements in the plant tissues were affected by the concentrations of the heavy metal in the soil and original pH. In the present conditions this plant did not show capabilities to tolerate Cr(VI) in the cultivation matrix. The concentration of Ni found in the alfalfa shoot tissues and the P/S ratio seen for Ni, as compared to the ratios of Zn and Cu, indicate that the alfalfa plant successfully competes with the bentonite and take up this metal vigorously. Live alfalfa plants were able to accumulate up to 202 ppm of Cd(II), 103 ppm of Cu(II), 437 ppm of Ni(II), and 160 ppm of Zn(II) when these heavy metals were applied in a mixture at the dose of 50 ppm each. Therefore, the alfalfa plant actively removes Ni from the soil matrix and translocates it from the roots to the shoots in a similar fashion to the mechanism suggested for heavy metal hyperaccumulator species.
Acknowledgements The authors would like to thank the National Institute of Health (grant S06 GM8012-30), and the University of Texas at El Paso’s Center for Environmental
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Resource Management (CERM) through funding from the Office of Exploratory Research of the US Environmental Protection Agency (cooperative agreement CR819849-01). We also acknowledge the support from the HBCU/MI Environmental Technology consortium, which is funded by the Department of Energy. Also J. L. Gardea-Torresdey acknowledges the financial support from the BEST Program, Department of Defense, Army Corps of Engineers. References Baker, A.J., Mcgrath, S.P., Reeves, R.D., Smith, A.J.C., 2000. Metal hyperaccumulator plants: a review of the ecology and physiology of biological resource for phytoremediation of metal-polluted soils. In: Terry, N., Ban˜uelos, G. (Eds.), Phytoremediation of Contaminated Soil and Water. Lewis Publishers, CRC Press LLC, Boca Raton Florida, pp. 85–108. Bates, T.E., 1971. Factors affecting critical nutrient concentrations in plants and their evaluation: a review. Soil Science 112, 116–130. Blaylock, M.J., Huang, J.W., 2000. Phytoextraction of metals. In: Raskin, I., Ensley, B.D. (Eds.), Phytoremediation of Toxic Metals: Using Plants to Clean Up the Environment. John Wiley, pp. 53–70. Brady, N.C., Weil, R.R., 1999. The Nature and Properties of Soils, twelfth ed. Prentice Hall, Upper Saddle River, New Jersey. Brown, S.L., Chaney, R.L., Angle, J.S., Baker, J.M., 1994. Phytoremediation potential of Thlaspi caerulescens and Bladder Campion for zinc and cadmium-contaminated soil. Journal of Environmental Quality 23, 1151–1157. Brun, L.A., Maillet, J., Hinsinger, P., Pe´pin, M., 2001. Evaluation of copper availability in copper-contaminated vineyards soils. Environmental Pollution 111, 293–302. Chatterjee, J., Chatterjee, C., 2000. Phytotoxicity of cobalt, chromium and copper in cauliflower. Environmental Pollution 109, 69–74. Ernst, W.H.O., Nelisse, H.J.M., Ten Brookum, W.M., 2000. Combination toxicology of metal-enriched soils: physiological responses of Zn and Cd-resistant ecotype of Silene vulgaris on polymetallic soils. Environmental and Experimental Botany 43, 55–71. Fernandes, J.C., Henriques, F.S., 1991. Biochemical, physiological, and structural effects of excess copper in plants. The Botanical Review 57, 247–273. Filep, G., 1999. Soil Chemistry Processes and Constituents. Akademiai Kiado, Budapest. Filho, G.M.A., Karez, C.S., Andrade, L.R., Yoneshigue-Valentin, Y., Pfeiffer, W.C., 1997. Effects on growth and accumulation of zinc in six seaweed species. Ecotoxicology and Environmental Safety 37, 223–228. Gardea-Torresdey, J.L., Polette, L., Arteaga, S., Tiemann, K.J., Bibb, J., Gonzalez, J.H., 1996. Determination of the content of hazardous heavy metals on Larrea tridentata grown around a contaminated area. In: Erickson, L.R., Tillison, D.L., Grant, S.C., McDonald, J.P. (Eds.), Proceedings of the Eleventh Annual EPA Conference on Hazardous Waste Research Substances. Albuquerque, NM, p. 660. Herawati, N., Susuki, S., Hayashi, K., Rivai, I.F., Koyama, H., 2000. Cadmium, copper, and zinc levels in rice and soil of Japan, Indonesia, and China by soil type. Bulletin of Environmental Contamination and Toxicology 64, 33–39. Kahle, H., 1993. Response of roots of trees to heavy metals. Environmental and Experimental Botany 33, 99–119.
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Lin, S.L., Wu, L., 1994. Effects of copper concentration on mineral nutrient uptake and copper accumulation in protein of coppertolerant and non-tolerant Lotus purshianus L. Ecotoxicology and Environmental Safety 29, 214–228. Luo, Y., Rimmer, D.L., 1995. Zinc-copper interaction affecting plant growth on a metal-contaminated soil. Environmental Pollution 88, 79–83. McLaughlin, M.J., Smolders, E., Merckx, R., 1998a. Soil-root interface: physicochemical processes. In: Huang, P.M. (Ed.), Soil Chemistry and Ecosystem Health. SSSA Special Publication Number 52, Madison, WI, USA, pp. 233–277. McLaughlin, M.J., Andrews, S.J., Smart, M.K., Smolders, E., 1998b. Effects of sulfate on cadmium uptake by Swiss chard: I. Effects of complexation and calcium competition in nutrient solutions. Plant and Soil 202, 211–216. Monni, S., Salemaa, M., Millar, N. The tolerance of Empetrum nigrum to copper and nickel. Environmental Pollution 109, 221–229. Ouzounidou, G., Ciamporova, M., Moustakas, M., Karataglis, S., 1995. Responses of maize (Zea mays L.) plants to copper stress—I. Growth, mineral content and ultrastructure of roots. Environmental and Experimental Botany 35, 167–176. Ozdemir, Z., Sagliroglu, A., 2000. Biogeochemical zinc anomalies along the Maden Cayi Valley, Maden-Elazig, Turkey. Zeitschrift fuer Angewandte Geologie 46, 218–222. Peralta, J.R., Gardea-Torresdey, J.L., Tiemann, K.J., Gomez, E., Arteaga, S., Rascon, E., Parsons, J.G., 2001a. Uptake and effects of five heavy metals on seed germination and plant growth in alfalfa (Medicago sativa L.). Bulletin of Environmental Contamination and Toxicology 66, 727–734. Peralta, J. R., Gomez, E., Gardea-Torresdey, J. L., Tiemann, K. J., Armenda´riz, V., Herrera, I., Walton, J., Carrillo, G., Parsons, J. G., 2001b. Effect of metal concentration and soil pH upon heavy metal uptake and plant growth in alfalfa. Hazardous Substance Research Center 2001 Conference, Manhattan, Kansas (Abstract). Reeves, R., Baker, A.J.M., 2000. Metal-accumulating plants. In: Raskin, I., Ensley, B.D. (Eds.), Phytoremediation of Toxic Metals: Using Plants to Clean Up the Environment. John Wiley, pp. 193–229. Sanchez-Rodriguez, I., Huerta-Diaz, M.A., Choumiline, E., Holguin-Quin˜ones, O., Zertuche-Gonzalez, J.A., 2001. Elemental concentrations in different species of seaweeds from Loreto Bay, Baja California Sur, Mexico: implications for the geochemical control of metals in algal tissue. Environmental Pollution 114, 145–160. Salt, D.E., Kra¨mer, U., 2000. Mechanisms of metal hyperacculation in plants.. In: Raskin, I., Ensley, B.D. (Eds.), Phytoremediation of Toxic Metals: Using Plants to Clean Up the Environment. John Wiley, pp. 231–246. Shallari, S., Schwartz, C., Hasko, A., Morel, J.L., 1998. Heavy metals in soils and plants of a serpentine and industrial sites of Albania. The Science of the Total Environment 209, 133–142. Smith, C.J., Hopmans, P., Cook, F.J., 1996. Accumulation of Cr, Pb, Cu, Ni, Zn and Cd in soil following irrigation with treated urban effluent in Australia. Environmental Pollution 94, 317–323. Taiz, L., Zeiger, E., 1998. Plant physiology, second ed. Sinauer Associates, Sunderland, Massachusetts. Terry, N., Ban˜uelos, G. (Eds.), 2000. Phytoremediation of Contaminated Soil and Water. Lewis Publishers, CRC Press LLC, Boca Raton Florida. Zantopoulos, N., Antoniou, V., Nikolaidis, E., 1999. Copper, zinc, cadmium, and lead in sheep grazing in North Greece. Bulletin of Environmental Contamination and Toxicology 62, 691–699.
Research papers
Effect of mixed cadmium, copper, nickel and zinc at different pHs upon alfalfa growth and heavy metal uptake J.R. Peralta-Videaa,b, J.L. Gardea-Torresdeya,c,*, E. Gomezc, K.J. Tiemanna,c, J.G. Parsonsa,c, G. Carrillod a
Environmental Science and Engineering PhD Program, University of Texas at El Paso, El Paso, TX 797968, USA b ITESM Campus Ciudad Jua´rez, PMB 10482, El Paso, TX 79995, USA c Chemistry Department, University of Texas at El Paso, El Paso TX 79968, USA d IREGEP, Colegio de Postgraduados, Montecillo, Mexico 56230 Received 5 October 2001; accepted 28 December 2001
‘‘Capsule’’: Alfalfa (Medicago sativa) plants were able to take up metals from a mixture of Cd(II), Cu(II), Ni(II) and Zn(II) in soils. Abstract Alfalfa plants were grown in soil-pots contaminated with a mixture of Cd(II), Cu(II), Ni(II), and Zn(II), (at 50 mg/kg each) at pHs of 4.5, 5.8, and 7.1. The plants were fertilized using a nutrient solution, which was adjusted appropriately to the same pH. Plants in the control treatment were grown in the absence of the heavy metals mixture. The growth of the control plants was the same at the three pHs studied and the heavy metal stressed plants also showed similar behavior at each pHs. There were statistically significant differences (P< 0.05) between the shoot length of the control treatment plants and the length of plants grown in the presence of the heavy metal mixture. Under the effects of the heavy metal mixture, nickel was the most accumulated element in the shoot tissue, with 437, 333, and 308 ppm at pH 7.1, 5.8, and 4.5, respectively. Cadmium was found to be second in accumulated concentrations with 202 ppm, 124 ppm, and 132 ppm at pH 7.1, 5.8, and 4.5, respectively; while zinc was third, followed by copper. The maximum relative uptakes (element in plant/element in soil–water–solution) were found to be 26 times for nickel, 23 times for cadmium, 12 times for zinc, and 6 times for copper. We considered these relations as indicative of the ability of alfalfa plants to take up elements from a soil matrix contaminated with a mixture of cadmium, copper, nickel, and zinc. # 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction It is almost impossible to visualize a soil without trace levels of heavy metals. However, anthropogenic activities have concentrated some of these elements in certain areas up to dangerous levels for living organisms (Chatterjee and Chatterjee, 2000). Activities such as mining and agriculture have polluted extensive areas throughout the world (Herawati et al., 2000; Brun, 2001; Zanthopoulos et al., 1999; Shallari et al., 1998; Gardea-Torresdey et al., 1996; Smith et al., 1996). The advancement of science in instrumental and analytical methods towards the end of the twentieth-century has allowed for the determination * Corresponding author. Tel.: +1-915-747-5359; fax: +1-915-7475748. E-mail address: [email protected] (J.L. Gardea-Torresdey).
of the role certain transition elements in the metabolism of some plant species. It is well known that elements such as Cu, Mo, Ni, Cl, and Zn, among others, are essential for plant growth in low concentrations (Reeves and Baker, 2000; Kahle, 1993; Brady and Weil, 1999; Taiz and Zeiger, 1998). Nevertheless, beyond certain threshold concentrations, these same elements become toxic for most plant species (Monni et al., 2000; Blaylock and Huang, 2000; Brady and Weil, 1999). The discovery that certain plant species are capable of tolerating high concentrations of heavy metals opened new possibilities to use these plants to remediate contaminated soils (phytoremediation). Many studies have been conducted to determine the toxic levels of heavy metals for certain plants, especially those metals considered as public health threats (Reeves and Baker, 2000; Fernandes and Henriques, 1991; Terry and
0269-7491/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0269-7491(02)00105-7
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Ban˜uelos, 2000). Several studies have been performed to investigate plants that accumulate different metals separately or in combinations. Some seaweed species for example, have been found to tolerate metal ions both individually (up to 1000 g l 1 of zinc) and in multi-metal solutions (Filho et al., 1997, Sanchez et al., 2001). Some varieties of Thlaspi and certain ecotypes of Silene vulgaris have also been found to be resistant to Zn and Cd (Ernst, et al., 2000, Brown et al., 1994). Reeves and Baker (2000) presented a detailed compilation of plant species that accumulate nickel, zinc, cadmium, lead, cobalt, copper, manganese, and selenium, individually, as well as in mixtures containing zinc and cadmium; zinc and lead; and cobalt and copper. Ouzounidou et al. (1995) studied the interaction of Cu with Ca and Fe assimilation and Bates (1971) studied the interaction and differential uptake of macro and microelements by several plant species. Also, Luo and Rimmer (1995) demonstrated that the absorption of Zn and Cu in barley is affected by the presence of Cd and Pb in the soil. In a previous study conducted with live alfalfa plants, it was determined that they were able to accumulate approximately 26,600 mg/kg of zinc, 8500 mg/kg of nickel, 12,000 mg/kg of copper, 6000 mg/kg of chromium, and 10,700 mg/kg of cadmium, individually (Peralta et al., 2001a). However, the objective of the present study is to evaluate the combined effects of cadmium, copper, nickel, and zinc applied to montmorillonite containing soils [abbreviated chemical formula: (Al,Mg)2(OH)2Si4O10] at three pHs, on seed germination and plant growth.
2. Materials and methods 2.1. Experimental setup This study was conducted under controlled light and temperature conditions. General-purpose plastic pots were filled with 300 g of a mixture of 70% of sand 0.25 mm of diameter (f), 25% gravel (0.81 mm f), and 5% bentonite (montmorillonite clay). Gravel and sand were soaked for two days in HCl (0.01 M) to remove any metals that may be present, then rinsed three times with deionized water and oven dried at 100 C. The soil in each pot was watered with 100 mL of a mixed metal solution made from Cd(NO3)2 4(H2O), Cu(NO3)2 2.5(H2O), Ni(NO3)2 6(H2O), and Zn(NO3)2 6(H2O) salts diluted in deionized water. The mix contained a concentration of 50 ppm of Cd(II), Cu(II), Ni(II), and Zn(II). The metal mixture was then separated in three aliquots. One aliquot was adjusted to pH 4.5, and the other aliquots to pH 5.8 and 7.1. The solution was slowly mixed thoroughly with the soil using a glass rod. Each treatment was replicated three times for statistical purposes. After 2 days, each pot received 50 ml of a
nutrient solution, which was adjusted to the respective pH of 4.5, 5.8 and 7.1. The nutrient solution consisted of Ca(NO3)2 4(H2O), 3.5710 4M; H3BO3, 2.3110 5 M; Ca(Cl2) 2(H2O), 2.1410 3 M; KH2PO4, 9.68 10 4 M; KNO3, 2.5510 4 M; Mg(ClO4)2, 1.0410 3 M; FeCl3, 6.8310 5 M; MnSO4 H2O, 7.6910 6 M; MoO3, 110 5 M; CuSO4 5(H2O); and Zn (NO3)2 6(H2O). The control treatment consisted of the nutrient solution adjusted to the same respective pHs as the heavy metal mixture. The deionized water used for watering all the treatments was also adjusted to pHs of 4.5, 5.8, and 7.1. Approximately 75 seeds of alfalfa (Mesa variety) were soaked in formaldehyde 3% v/v for 10 min to reduce fungal contamination, washed three times with deionized water and put into pots. These pots were provided with a photoperiod of 12 h at 1500 luxes. These were covered with transparent plastic bags to avoid excessive desiccation and prevent airborne contamination. 2.2. Evaluation of treatments Seedlings were harvested 15 days after planting. A sample of five plants per treatment was utilized to determine the shoot length, by measuring them from the crown to the main shoot apex. The alfalfa growth data were further analyzed through analysis of variance (ANOVA). All the plants were washed with deionized water, oven dried at 70 C for 72 h, weighed and acid digested. The acid digestion was performed with a microwave oven (CEM MarsX) at 100 C for 15 min with 10 ml of concentrated nitric acid (HNO3). The volume was subsequently adjusted to 10 ml and the samples were analyzed for elemental content using a Perkin–Elmer Optima 4300 Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES). 2.3. Soil analysis A sample of approximately 1 g of dry soil was taken from each treatment-pot to determine the amount of element present in the water-soluble fraction. The volume of each sample was brought up to 8 ml with deionized water, left to equilibrate for 1 day and centrifuged at 3000 rpm for 5 min. Then, each supernatant was analyzed using the ICP-OES to determine the water-soluble concentration of heavy metals tested (Cd, Cu, Ni, and Zn) as well as the water-soluble macro and micronutrients (Ca, Mg, S, P, Mn, Mo, Fe, and Cl) present in the soil.
3. Results Originally, the mixture of heavy metals tested included: Cr(VI), Cd(II), Cu(II), Ni(II), and Zn(II), but the
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alfalfa seeds hardly germinated. Further screenings of different combinations of the metals in Petri dishes were performed in order to identify which one of the metals was causing detrimental effects. Results from these procedures identified Cr(VI) as the metal responsible of germination inhibition and thus Cr(VI) was excluded from the metal mixture tested. 3.1. Evaluation of alfalfa shoot growth Table 1 shows the shoots’ length of the alfalfa plants grown with and without the heavy metal treatments. Our results show that there were no statistical significant differences among the shoot length of the control treatment plants, although the plants grown at pH 5.8 had the highest elongation (4.1 cm). Likewise, there were no statistical significant differences between the shoots’ lengths of plants that grew in the presence of the mixed heavy metals. Overall, the plants grown at pH 7.1 had the highest elongation (2.1 cm). However, the differences between the shoot length of the control treatment plants and the plants cultivated with the mixed heavy metals were found to be statistically significant (P < 0.01; See Table 1). 3.2. Concentration of heavy metals in plant tissues Fig. 1 shows the amount of heavy metals found in the alfalfa shoot tissues depending on the concentration of heavy metals in the soil and the initial pH. This figure shows that when the heavy metals were at trace levels in the soil (Fig. 1a), the amounts of Zn in the shoot plant tissues (160 ppm) were significantly higher than Cd, Cu, and Ni at all pHs studied (P < 0.01). These results agree with the previous results found in alfalfa plants cultivated in agar (Peralta at al., 2001b). Nevertheless, when the soil was contaminated with the 50 ppm mixture of Cd, Cu, Ni, and Zn, nickel was found to be the heavy metal present in the highest amount in shoot plant tissue (Fig. 1b). The concentrations of nickel were found to be 437, 333, and 308 ppm at pH 7.1, 5.8, and 4.5, respectively. The differences among the concentration of nickel in shoot tissues (average of the three pHs), as compared with the
Table 1 Shoot length (cm) of alfalfa plants Mesa variety after 2 weeks of exposure to heavy metals at three pHsa pH
Control
Heavy metal mixture
4.5 5.8 7.1
3.9 4.1 4.0
2.0 2.0 2.1
a
Data are average of five plants.
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maximum concentration at any pH of cadmium (202 ppm), copper (105 ppm), and zinc (160 ppm) were statistically significant (P < 0.05). In all cases the highest amount of heavy metals in shoot tissues were found in those plants that grew in soil watered with the initial solution adjusted at pH 7.1. These results were slightly different from those found in maize, in which there was no pH-dependence for copper uptake (Brun et al., 2001). 3.3. Concentration of microelements in plant tissues Fig. 2a,b shows the concentration of the micronutrients found in the shoot plant tissues depending on the presence of the heavy metals in the soil. Fig. 2a shows that Fe, Mn, and Mo were present in higher amounts in the tissues of those plants that grew at pH 7.1 in the control treatment. The concentration of Fe found in the tissues of the control treatment plants grown at pH 7.1 was 229 ppm (Fig. 2a), while the Fe level was 151 ppm in the plants that grew with the mixed heavy metals (Fig. 2b). The difference was found to be statistically significant (P < 0.05). Similar results were found with Fe in maize (Ouzounidou et al., 1995). The presence of chlorine in plant tissues was significantly higher (P < 0.0001) in those plants that grew in the presence of mixed heavy metals (1600 ppm at pH 5.8; Fig. 2b). Aluminum was present in the soil at higher levels in all the treatments, as compared with the other elements studied (Fig. 2a,b). It is known that montmorillonite clay contains Al as a structural element. However, no significant aluminum uptake by alfalfa plant was observed (Fig. 2a,b). 3.4. Concentration of macronutrient in plant tissues Fig. 3a,b shows that phosphorus and sulfur were found to be the macronutrients with the highest uptake in the plant tissues (P < 0.01). Under growth conditions with heavy metals at trace level (Fig. 3a), the amount of P in the plant tissue was higher at pH 4.5 and pH 7.1 (33,311 and 36,111 ppm, respectively), while lower (18,135 ppm) at pH 5.8. In the case of sulfur, the tissues had 41,595 ppm at pH 4.5, while 30,828 ppm at pH 7.1 and 11,753 ppm at pH 5.8. When the soil received 50 ppm of the heavy metal mixture (Fig. 3b), the concentrations of P and S in plant tissues increased as the pH was raised from 4.5 to 7.1 (refer to Fig. 3b). The presence of the heavy metal mixture also caused a reduction in the amount of Ca in the plant tissue at pH 4.5. Similar results were found in maize grown with 80 mM of Cu(II) (Ouzounidou et al., 1995). These investigators found that Cu(II) caused a reduction in Ca and Fe concentration in maize roots. Also, excess of Cu(II) concentration causes a reduction in Ca uptake by Lotus purshianus (Lin and Wu, 1994).
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Fig. 1. Heavy metal concentration in soil and shoot tissues of alfalfa plant after 15 days of growth at three different pHs. (a) Control treatments when the dose of heavy metals in soil was at trace levels; (b) the results after the soil received a mixture of 50 ppm of each heavy metal. (s indicates soil, p indicates plant).
3.5. Relative uptake of the elements The concentration of the elements present in the plant tissues was related to the elemental concentration in the soil. Fig. 4a shows the ratio of heavy metal in plant tissues to heavy metal in the soil (P/S) in the control treatment while Fig. 4b shows the ratio P/S found in those plants grown in the presence of the mixture of the
heavy metals. In the control treatment, the ratios for Zn at pH 7.1 and the ratios for Cd at the three pHs could not be calculated since Zn and Cd were not detected in the soil extract solution (Zn and Cu were present in the nutrient solution and traces of Ni were detected in the bentonite, while Cd was not a component of the nutrient solution and was not detected in the bentonite). There were no statistical differences between the P/S
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295
Fig. 2. Micronutrient concentrations in soil and shoots tissues of alfalfa plant after 15 days of growth at three different pHs. (a) Control treatments when the dose of heavy metals in soil was at trace level; (b) the results after the soil received a mixture of 50 ppm of Cd(II), Cu(II), Ni(II), and Zn(II); (s indicates soil, p indicates plant).
ratios for Cd and Ni at any pH (Fig. 4b). Nickel presented P/S ratios of 22, 23, and 26 times, and cadmium presented P/S ratios of 23, 18, and 23 times at the pHs of 4.5, 5.8, and 7.1, respectively. Cu and Zn presented P/ S ratios close to 10 times at the three pHs. The differences among the ratios found for Ni and Cd, and those found for Cu and Zn where statistically significant (P < 0.05). A study performed with rice by Herawati et
al. (2000), did not showed correlation among the content of Cd, Cu, and Zn in plant tissue and the amount of those metals found in the soil. Fig. 5a,b show the magnitude of the P/S ratios for some micronutrients considered in this study. Fe did not present changes in the P/S ratio for the control treatment plants (Fig. 5a) and for the plants grown under heavy metal stress (Fig. 5b). At pH 7.1, molybdenum
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Fig. 3. Macronutrient concentrations in soil and shoots tissues of alfalfa plant after 15 days of growth at three different pHs. (a) Control treatments when the dose of heavy metals in soil was at trace level; (b) the results after the soil received a mixture of 50 ppm of Cd(II), Cu(II), Ni(II), and Zn(II); (s indicates soil, p indicates plant).
showed P/S ratios of 121 times in the plants of the control treatment (Fig. 5a) and 43 times in those plants grown in the soil treated with the heavy metal mixture (Fig. 5b). At pH 4.5, the ratios were 19 times (Fig. 5a) and 64 times (Fig. 5b). Manganese presented P/S ratios of 56 times (Fig. 5a) and 52 times (Fig. 5b). At pH 5.8 the ratios were 12 times (Fig. 5a) and 23 times (Fig. 5b).
At pH 4.5 there were ratios of 36 times (Fig. 5a) and 32 times (Fig. 5b). It was not possible to calculate the ratio of Cl in the plant tissue related to Cl in the soil extract solution because Cl was not detected in the soil extract solution. Even though the macronutrients S and P were found in comparable quantities within tissues of both the
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297
Fig. 4. Ratio of Plant to soil concentration of the heavy metals in shoot tissues of alfalfa after 15 days of growth at three different pHs. (a) Control treatments when the dose of heavy metals in soil was at trace level; (b) the results after the soil received a mixture of 50 ppm of Cd(II), Cu(II), Ni(II), and Zn(II); (s indicates soil, p indicates plant).
control treatment plants and mixed heavy metal stressed plants (Fig. 3a,b), the amount of S in the plant related to the amount of S in the soil, was significantly lower (P < 0.00001) than the P/S ratio of P in both the control treatment plants and the plants grown in the presence of the heavy metal mixture (Fig. 6a,b, respectively). In contrast with the other elements studied, the P/S ratios presented by phosphorus were higher in those plants
grown under the effects of the heavy metal mixture (Fig. 6a,b, respectively). Significant linear correlations (P < 0.001) were found between 48 pairs of combinations among the elements detected in the soil and plant tissue (Table 2). Only aluminum (data not shown) and iron presented significant negative correlation coefficients (P < 0.001) with some of the elements in the plant tissues and the solution soil
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Fig. 5. Ratio of Plant to soil concentration of the microelements in shoot tissues of alfalfa after 15 days of growth at three different pHs. (a) Control treatments when the dose of heavy metals in soil was at trace level; (b) the results after the soil received a mixture of 50 ppm of Cd(II), Cu(II), Ni(II), and Zn(II); (s indicates soil, p indicates plant).
extracts. In addition, Fe in the plant tissue was negatively correlated with Fe in the soil (r2= 0.8237) and with Mg in the soil (r2= 0.8696). The amounts of heavy metals in the plant tissues were found to have a high correlation with the amount of these metals in the solution soil extracts (P < 0.001) with an r2 close to 1.00. Ozdemir and Sagiroglu (2000) reported positive significant correlations between Cu in soil and in plant tissues for several species of plants. However, Herawati et al. (2000) did not find the same correlation among the content of Cu, Cd, and Zn in soil and rice plant tissues.
4. Discussion These studies have shown that at least 100 ppm of Cd, Cu, Ni, and Zn, were present in the shoot tissues of alfalfa plant cultivated in the soil contaminated with a mixture of heavy metals (at 50 ppm each metal). Our results indicate a lack of specificity for metal uptake in the alfalfa plant, which is a common phenomenon in other plant species (Baker et al., 2000). Comparing the shoot length of the plants grown under the effects of the heavy metal mixture and that of the control treatment (Table 1), it is evident that a combined stress is
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Fig. 6. Ratio of Plant to soil concentration of the macroelements in shoot tissues of alfalfa after 15 days of growth at three different pHs. (a) Control treatments when the dose of heavy metals in soil was at trace level; (b) the results after the soil received a mixture of 50 ppm of Cd(II), Cu(II), Ni(II), and Zn(II); (s indicates soil, p indicates plant).
occurring due to the heavy metal mixture, because the alfalfa plant has demonstrated the ability to grow well in soils individually contaminated with more than 50 ppm of heavy metals (Peralta et al., 2001b). Similar detrimental effects have also been observed in other plant species (Monni et al., 2000). These researchers found that the mixture of copper and nickel affected the elongation of shoots, as well the root dry weight of Empetrum nigrum. Additionally, the bentonite clay
(montmorillonite) used in this study possesses overall negative charges on its surface, which made it possible to adsorb the Cd(II), Cu(II), Ni(II), and Zn(II) in the soil micelle (Brady and Weil, 1999), which results in low extraction of the metals from the soil. However, the concentration of Ni found in the alfalfa plant shoots (Fig. 1b) and the P/S ratio seen for Ni, as compared to the ratios of Zn, and Cu indicate that the alfalfa plant successfully competes with the bentonite and uptakes
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Table 2 Pearson product–moment correlation coefficient for some elements measured in this researcha Variable Cap Cap Mgp Mnp Mop Cup Cdp Nip Znp Fep Clp Fes Mgs Zns Nis Cds Cus Pp a
1.0000 0.8893 0.6418 0.6526 0.6033 0.5908 0.5633 0.6051 0.3839 0.4089 0.0757 0.0244 0.4569 0.4770 0.5484 0.5816 0.9093
Mgp
1.0000 0.8819 0.8978 0.5241 0.4313 0.4001 0.4903 0.6205 0.2489 0.1525 0.2408 0.2790 0.3065 0.3977 0.4242 0.9901
Mnp
1.0000 0.9555 0.4753 0.2971 0.2641 0.3840 0.7041 0.1626 0.3379 0.3952 0.1724 0.2015 0.2521 0.2763 0.8364
Mop
1.0000 0.2514 0.0869 0.0533 0.1702 0.8620 0.0674 0.5095 0.5743 0.0569 0.0267 0.0439 0.0701 0.8568
Cup
1.0000 0.9701 0.9467 0.9859 0.2465 0.8803 0.6220 0.5754 0.9220 0.9308 0.9431 0.9616 0.5435
Cdp
1.0000 0.9924 0.9944 0.3994 0.9415 0.7271 0.6815 0.9765 0.9804 0.9911 0.9987 0.4582
Nip
1.0000 0.9850 0.4307 0.9726 0.7071 0.6664 0.9907 0.9935 0.9977 0.9931 0.4147
Znp
1.0000 0.3317 0.9307 0.6773 0.6309 0.9631 0.9694 0.9846 0.9922 0.5099
Fep
1.0000 0.5290 0.8237 0.8696 0.5268 0.5015 0.4466 0.4183 0.5786
Clp
Fes
1.0000 0.6786 0.6579 0.9891 0.9884 0.9670 0.9428 0.2416
1.0000 0.9945 0.7405 0.7261 0.7310 0.7438 0.0677
Zns
Nis
Cds
Cus
Pp
1.0000 0.9995 0.9867 0.9767 0.2904
1.0000 0.9893 1.0000 0.9801 0.9947 1.0000 0.3167 0.4156 0.4525 1.0000
Figures in bold represent significant values (P <0.001).
this metal vigorously. Therefore the alfalfa plant actively removes Ni from the soil matrix and translocates it from the roots to the shoots in a similar fashion to the mechanism suggested for heavy metal hyperaccumulator species (Salt and Kramer, 2000). Furthermore, the elevated levels of Cl and S (Figs. 2b,3b) found within the shoots of the alfalfa plants grown in soils contaminated with heavy metals have been suggested to be due to the powerful complexation of Cl and S to the heavy metals, which results in their common translocation with the metals taken up by the plant (McLaughlin et al., 1998a,b). The concentrations of Ca, Mg, and K in the tissues of the control treatment plants did not show appreciable differences when compared with the concentrations of Ca, Mg, and K found in the tissues of the plants grown with the heavy metal mixture. According to Brady and Weil (1999) the order of strength of adsorption of these macronutrients is Ca > Mg> K. This was indeed observed with the alfalfa plants in this study. Furthermore, the magnitude of the ratio of the metal concentration in the plant tissues to the metal concentration in the soil followed the same order. A negative correlation coefficient was found between Fe in soil and Fe in the plant tissue (Table 2). The pH effects may explain this since Fe is available to complex with many ligands within the pH ranges of this study. The Fe may form microcrystalline structures that make it unavailable in order for the plant to uptake it (Filep, 1999). Nevertheless, the data from these studies show that the concentrations of some of the metals were considerably higher than the original soil concentrations, indicating that the plant is capable of competing with
the bentonite clay to remove and fix metal ions from the soil matrix.
5. Conclusions Based on these results, we concluded that alfalfa plant can successfully uptake Cd(II), Cu(II), Ni(II), and Zn(II) in a montmorillonite-clay medium. The concentrations of the elements in the plant tissues were affected by the concentrations of the heavy metal in the soil and original pH. In the present conditions this plant did not show capabilities to tolerate Cr(VI) in the cultivation matrix. The concentration of Ni found in the alfalfa shoot tissues and the P/S ratio seen for Ni, as compared to the ratios of Zn and Cu, indicate that the alfalfa plant successfully competes with the bentonite and take up this metal vigorously. Live alfalfa plants were able to accumulate up to 202 ppm of Cd(II), 103 ppm of Cu(II), 437 ppm of Ni(II), and 160 ppm of Zn(II) when these heavy metals were applied in a mixture at the dose of 50 ppm each. Therefore, the alfalfa plant actively removes Ni from the soil matrix and translocates it from the roots to the shoots in a similar fashion to the mechanism suggested for heavy metal hyperaccumulator species.
Acknowledgements The authors would like to thank the National Institute of Health (grant S06 GM8012-30), and the University of Texas at El Paso’s Center for Environmental
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