Effects of copper and cadmium on heavy metal polluted waterbody restoration by duckweed (Lemna minor)

Plant Physiology and Biochemistry 45 (2007) 62e69 www.elsevier.com/locate/plaphy

Research article

Effects of copper and cadmium on heavy metal polluted waterbody restoration by duckweed (Lemna minor) Wenhua Hou a,*, Xiao Chen b, Guanling Song c, Qunhui Wang d, Chein Chi Chang e a

State Environmental Protection Key Laboratory for Lake Pollution Control, Research Center of Lake Eco-environment, Chinese Research Academy of Environment Sciences, Da Yang Fang No. 8, An Wai, Beijing 100012, China b Department of Environmental Science and Engineering, Harbin Institute of Technology, Harbin 150090, China c Department of Life Science, Shandong University of Technology, Zibo 255049, China d Department of Environmental Engineering, Beijing University of Science and Technology, Beijing 100083, China e Department of Civil and Environmental Engineering, University of Maryland, Baltimore County, Baltimore, MD 21250, USA Received 4 May 2006; accepted 20 December 2006 Available online 28 December 2006

Abstract Aquatic plants have been identified as a potentially useful group for accumulating and bioconcentrating heavy metals. In the study, we investigated changes in the contents of soluble protein and photosynthetic pigments as well as the activity of antioxidant enzymes caused by copper sulfate and cadmium dichloride, respectively in duckweed (Lemna minor) during concentration-dependent exposure (0.05e20 mg l1) to metal salt. The results demonstrated that exposure to high concentration heavy metals (Cu > 10 mg l1, Cd > 0.5 mg l1) could result the disintegration of antioxidant system in duckweed. Also, the significant decrease of contents of soluble protein and photosynthetic pigments was observed to high-level metal stress. Additionally, cadmium was found to be more toxic than copper on plants. The outcome of this study corroborate that Lemna minor is a suitable candidate for the phytoremediation of low-level copper and cadmium contaminated waterbody. Ó 2006 Elsevier Masson SAS. All rights reserved. Keywords: Duckweed; Copper; Cadmium; Soluble protein; Photosynthetic pigment; Antioxidant enzyme

1. Introduction Over the last two decades there has been an ever-increasing awareness of how heavy metals are as environmental pollutants. Their presence in the atmosphere, soil, and water, even in trace concentrations, can cause serious problems to organisms [23]. Copper and cadmium are reported to be widespread heavy metal pollutants in natural and wastewaters in China resulting from agriculture and industrial activities such as pigments, mining, smelting and electroplating, etc. [23,37]. Cu is an essential micronutrient and a component of several enzymes

* Corresponding author. Tel.: þ86 10 8493 3058. E-mail address: [email protected] (W. Hou). 0981-9428/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2006.12.005

mainly participating in electron flow and catalyzing the redox reactions [6]. But it becomes toxic at high concentrations, whereas Cd has no known biological function and is a highly toxic metal to aquatic organism [50]. A variety of technologies, including chemical, physical and biological methods, have been applied to prevent and treat water pollutions. However, these methods present different efficiencies for different metals and they can be very expensive especially if large volumes, low metal concentration and high standards of cleaning are required [32]. In recent years, interest has been focused on using aquatic plants, such as Lemna minor, Microspora, and Pistia stratiotes et al., as a promising approach to take up heavy metals from water body [8,13,19,26,27,36]. A study by Maine et al. [27] showed that cadmium separation by Pistia stratiotes could reach 85% for the concentrations of 1e6 mg l1. Duckweed (Lemna

W. Hou et al. / Plant Physiology and Biochemistry 45 (2007) 62e69

minor) also has been found to have good ability to accumulate heavy metals at certain concentration. In the experiment Lemna minor could remove 75e90% of lead from water after three weeks’ exposure to concentration of 5 mg l1 [13]. Moreover, duckweed is widely distributed and commonly found year-round in fresh and brackish waters in China and therefore the bioremediation of metal pollution can be inexpensive compared to the traditional means of heavy metal removal. However, the removal of highly polluted water body by Lemna minor may not be a success owing to physiological characteristic of duckweed. In this paper we studied the responses of duckweed to the increasing concentrations of copper and cadmium, respectively with reference to: (1) changes in soluble protein content; (2) changes in contents of chlorophyll a, chlorophyll b and carotenoid; and (3) changes in the contents of antioxidant enzymes such as, peroxidase (POD), catalase (CAT) and dismutase (SOD), and malondialdehyde (MDA). So this can determine the concentration extent to which Lemna minor is suitable for remediation of water body polluted by heavy metals. 2. Materials and methods 2.1. Plant materials and growth condition Lemna minor used in this study was collected from the region of Taihu lake in China, located at 31 17.4080 N, 119 55.1010 E. The duckweed fronds were maintained in glass aquariums (0.5  0.8  0.6 m3), which were placed in a controlled room at 26  2  C, under illumination provided by metal halide lamps with a light intensity of 72 mmol m2 s1 and a LD cycle of 16:10 h for three months as a pre-treatment before experiments. The medium of the pre-treatment was 1/10 Hutner medium [18] with a pump to keep circulation. The medium was replaced every 1.5 months. 2.2. Experiments Before the experiment, pre-treated Lemna fronds from aquariums were disinfected by immersing them in 1% (v/v) NaClO for 3e5 min and then rinsed with distilled water for three times. About 2 g fronds, determined after 5 min blotting on dry tissue paper (hereafter referred to as fresh weight), were incubated in 500 ml beakers containing 300 ml medium, which were placed in a growth chamber (SANYO, MLR350H, Japan). The incubation condition is as follows: temperature, 28  C in the light and 26  C in the dark; light intensity, 36 mmol m2 s1; LD cycle, 16 h: 8 h; humidity, 60%. Modified Steinberg medium is the medium for incubation [31] (Table 1) for the study of influences of copper and cadmium on Lemna. Lemna fronds are with Cu2þ (copper sulfate) and Cd2þ (cadmium dichloride) of concentrations 0, 0.05, 0.5, 5, 10 and 20 mg l1 in modified Steinberg medium, respectively. After four days exposure to heavy metals, the fresh weight (FW), soluble protein, photosynthetic pigments, POD, CAT,

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Table 1 The composition of modified Steinberg medium Substance

Concentration

Substance

Concentration

KNO3 KH2PO4 K2HPO4 MgSO4$7H2O Ca (NO3)2$4H2O

350 90 12 100 295

MnCl2$4H2O H3BO3 Na2MoO4 ZnSO4$7H2O FeCl3$6H2O Na2EDTA$2H2O

0.18 0.12 0.044 0.18 0.76 1.5

Units: Concentration: mg l1.

SOD, and MDA of fronds were determined. A minimum of three replicates were performed in each experiment. 2.3. Photosynthetic pigments and soluble protein determination Lemna fronds without roots (approximately 150 mg fresh weight) were homogenized on ice with mortar and pestle in 3 ml of 66 mM phosphate buffer, pH 7.2 with 10 mM KCl. The homogenate was then extracted with cold acetone (80%). The absorbance of pigment extract was measured at wavelength of 470, 626, 645, 663 and 730 nm with spectrophotometer (HITACHI, U-3210, Japan). The contents of Chl a, Chl b and carotenoid were calculated in accordance with experimental equations as described by Lichtenthaler [20]. Protein content was determined using bovine albumin for calibration [22]. 2.4. Enzyme extraction and assay To obtain the enzyme extract, Lemnor fronds (approximately 500 mg fresh weight) was homogenized in 5 ml cold potassium phosphate buffer (0.1 M, pH 7.8). The homogenate was centrifuged at 15,000  g (4  C) for 15 min. The supernatant was used as the enzyme extract which was saved for analysis. All the work for preparation of enzyme extract was carried out at 4  C. POD activity was determined spectrophotometrically by measuring the increase in absorbance at 470 nm after 20 min incubation at room temperature. The reaction mixture contained potassium phosphate buffer (50 mM, pH 7.0, 1 ml), H2O2 (0.2%, v/v, 2 ml), guaiacol (0.2%, v/v, 0.95 ml) and enzyme extract (50 ml). The reaction started by adding H2O2. CAT activity was evaluated spectrophotometrically by measuring the consumption of H2O2 at 240 nm [1] where the testing medium contained in final volume of potassium phosphate buffer (50 mM, pH 7.5, 750 ml), H2O2 (200 mM, 100 ml), and enzyme extract (150 ml) in a final volume of 1 ml. SOD was determined using Superoxide Dismutase detection kit which was produced in Nanjing Jiancheng Bioengineering Institute. Assay was carried out according to the specification of the detection kit. MDA activity was determined to indicate the level of lipid peroxidation of fronds as described by Zhao [52]. Enzyme extract (1.5 ml) and thiobarbituric acid (0.5%, v/v, 2.5 ml) were boiled for 20 min and then centrifuged at 10,000  g for

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5 min. The supernatant was measured spectrophotometrically at wavelength of 532 nm, 600 nm and 450 nm. 2.5. Statistic All data presented in the paper are the means of at least three replicates. Significance of differences of samples was calculated by Student’s t-test. Results of testing were considered significant if calculated p-values were 0.05. 3. Results 3.1. Effects of copper and cadmium on soluble protein in Lemna minor Under the condition of heavy metal menace, changes of soluble protein content in Lemna minor were shown Fig. 1, which indicated that exposure to 0.05 mg l1 of Cu2þ slightly decreased soluble protein content after 4 day of treatment, while 0.5 mg l1 of Cu2þ decreased protein content significantly. Beyond the concentration, Cu2þ inhibited soluble protein content steadily. While the presence of 0.05 and 0.5 mg l1 of Cd2þ in growth medium could result in a fast and strong inhibition of protein content, and beyond that concentration a slight and steady decline of protein content occurred as shown in Fig. 1. 3.2. Effects of copper and cadmium on photosynthetic pigments in Lemna minor The amounts of chlorophyll pigments in the tissue of Lemna fronds grown under different concentrations of copper and cadmium are shown in Table 2. When Lemna fronds were exposed to 0.05 mg l1 of Cu2þ, a slight increase of Chl a and

Soluble protein content (mg g-1)

9.8

9.6

3.3. Effects of copper and cadmium on antioxidant enzymes and MDA in Lemna minor

9.4

9.2

9.0

8.8

8.6

Chl b content occurred (non-significant, p  0.05) (Table 2). Presence of 5 mg l1 of Cu2þ resulted in a significant decrease of Chl a. Beyond that concentration, a steady decrease of Chl a content was observed with elevated concentration level of Cu2þ. When Lemna fronds were exposed to 20 mg l1 of Cu2þ, the amount of Chl a reached a minimum value of 0.284  0.010 mg g1 fresh weight (62.0% of control) (Table 2), while the content of Chl b decreased steadily and was less inhibited than that of Chl a beyond 0.05 mg l1 of Cu2þ (Table 2). A concentration-dependent decline of Chl a occurred as a consequence of exposure to Cd2þ concentrations of 0 w 20 mg l1, at which concentrations a minimum decrease of 46.3% from the control was reached (Table 2), while the content of Chl b decreased at a relatively slower rate resulting from the presence of Cadmium. The value of Chl b content was 68.3% of the control level at the highest Cd2þ concentration (Table 2). The concentration-dependent trends of content of total chlorophyll (Chl a þ Chl b) were presented in Fig. 2. The total chlorophyll content steadily declined with the increase of Cu2þ and Cd2þ in growth medium, respectively and the values of total chlorophyll under two heavy metals stress were close. However, the ratios of Chl a/Chl b for Lemna fronds under two metals differed much from each other. The curve for ratio of Chl a/Chl b under Cd2þ exposure declined regularly with the increasing metal concentration while the ratio under Cu2þ stress was not that regular (Fig. 2). The changing trends of carotenoid content of Lemna minor exposed to a range concentration of copper and cadmium were similar to those of Chl a (Tables 2 and 3). Carotenoid content of Lemna fronds treated with Cu2þ displayed a biphasic responds as the content of Chl a did. The carotenoid content was reduced steadily with the increased Cd2þ concentration (Table 3). By comparison, it is found that cadmium exerted more influences on the content of photosynthetic pigments than copper by the reason that the amounts of Chl a, b and carotenoid of Lemna treated with cadmium decreased more significant than those of Lemna treated with copper (Tables 2 and 3).

0

5

10

15

20

Concentration (mg l-1) Fig. 1. Effects of Cu2þ and Cd2þ on soluble protein content of Lemna minor. Standard deviations are represented by error bars. (,) Cu2þ; (C) Cd2þ.

When Lemna minor was exposed to copper, a concentration-dependent increase of POD activity was observed with metal ion as low as 0.05 mg l1 up to 10 mg l1 (Fig. 3). Beyond that concentration, a significant decrease of POD activity occurred as shown in Fig. 3. From the lowest tested concentration (0.05 mg l1) a significant stimulation (47.5%) of POD activity appeared as a consequence of cadmium addition (Fig. 3). The activity increased up to a maximum activity of 1530.45 U mg protein1 min1 with the concentration of Cd2þ reaching 0.5 mg l1. Beyond that concentration, POD activity decreased to reach lower than that of the control (Fig. 3).

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Table 2 The toxic effects of Cu2þ and Cd2þ on the chlorophyll a and chlorophyll b content in Lemna minor Cu2þ treatment

Concentration

Control 0.05 0.5 5 10 20

Cd2þ treatment

Chlorophyll a (% of control)

Chlorophyll b (% of control)

Chlorophyll a (% of control)

Chlorophyll b (% of control)

0.457  0.049 (100) 0.459  0.051 (100.3) 0.381  0.020 (83.3) 0.360  0.030* (78.7) 0.291  0.021* (63.7) 0.284  0.010* (62.0)

0.256  0.014 (100) 0.257  0.017 (100.4) 0.246  0.035 (96.1) 0.224  0.021 (87.4) 0.204  0.011* (79.5) 0.177  0.013* (69.9)

0.460  0.015 (100) 0.430  0.022 (93.4) 0.374  0.028* (81.3) 0.341  0.030* (74.0) 0.270  0.023* (58.6) 0.247  0.017* (53.7)

0.285  0.013 (100) 0.281  0.027 (98.7) 0.276  0.017 (96.7) 0.258  0.012 (90.6) 0.220  0.022* (77.3) 0.195  0.006* (68.3)

Units: Concentration: mg l1. Chlorophyll a, b: mg g1 of fresh weight. *p  0.05, significantly different from control.

1.8

increased gradually with the increased concentration of heavy metals, while responses of MDA activity exposed to cadmium was more significant to that exposed to copper (Fig. 6). 4. Discussion 4.1. Effects of copper and cadmium on soluble protein and photosynthetic pigments The present results clearly indicate that the presence of heavy metals, copper and cadmium ions brought about the toxicity to Lemna fronds. Heavy metals had a strong inhibition effects on soluble protein and photosynthetic pigments of Lemna minor. Soluble protein content in organisms, an important indicator of reversible and irreversible changes in metabolism, is known to respond to a wide variety of stressors such as natural and xenobiotic [44]. It was reported that Cd or pesticides resulted in a significant inhibition of protein level in Brassica juncea L and root tips of barley seedlings [25,44]. In the

1.7

1.6

Table 3 The toxic effects of Cu2þ and Cd2þ on the carotenoid content in Lemna minor

1.6

1.4

1.5

1.2 1.4 1.0 1.3

0.8

Concentration

Chl a / Chl b

Total Chlorophyll content (mg g FW-1)

The CAT activity of Lemna minor treated with copper was similar to that of POD activity (Figs. 3 and 4). When the concentration of Cu2þ increased to 10 mg l1, the CAT activity reached a maximum value of 96.93 U mg protein1 min1 (Fig. 4). Beyond the concentration, great decrease of CAT activity occurred as shown in Fig. 4. The curve of CAT activity of Lemna minor treated with cadmium reached summit when the concentration of Cd2þ reached 0.5 mg l1 and beyond that concentration, CAT activity decreased with the increased amount of Cd2þ (Fig. 4). The SOD activity of Lemna treated with copper was similar to that of treated with cadmium. The presence of the two metals in growth medium both stimulated the SOD activity, the activity reaching maximum value at the concentration of 20 mg l1 (Fig. 5). The curve of MDA content of Lemna fronds after Cu2þ treatment was similar to that of Cd2þ treatment. MDA content

1.2

0.6

1.1

0.4 0

5

10

Concentration (mg 2þ

15

20

l-1)

Fig. 2. Effects of Cu and Cd2þ on the total chlorophyll content and ratio of Chl a/Chl b. (,) Cu2þ and (C) Cd2þ for total chlorophyll content; (-) Cu2þ and (B) Cd2þ for Chl a/Chl b.

Control 0.05 0.5 5 10 20

Carotenoid content (% of control) Cu2þ treatment

Cd2þ treatment

0.178  0.021 (100) 0.180  0.015 (100.7) 0.159  0.013 (89.4) 0.134  0.007* (75.2) 0.114  0.011* (64.2) 0.110  0.010* (62.1)

0.212  0.013 (100) 0.197  0.017 (96.1) 0.194  0.007 (91.6) 0.155  0.003* (73.0) 0.122  0.007* (57.4) 0.111  0.014* (52.2)

Units: Concentration: mg l1. Carotenoid: mg g1 of fresh weight. *p  0.05, significantly different from control.

W. Hou et al. / Plant Physiology and Biochemistry 45 (2007) 62e69

66

120

1500

SOD activity (U mg protein-1 min-1)

POD activity (U mg protein-1 min-1)

1600

1400 1300 1200 1100 1000 900

0

5

10

15

20

90

80

105

4.0

60

10

15

20

of protein. Also, the accumulation of cadmium in plants could inhibit the uptake of Mg and K on which protein synthesis system relied [12]. The mechanism of cadmium inhibition of protein content is complex and need further study. For fronds treated with copper, as shown in Table 2, 0.05 mg l1 of Cu2þ stimulated the activity of photosynthetic pigments, but 0.5 mg l1 of Cu2þ was sufficient to induce a decrease of pigments, indicating that, although copper was an essential micronutrient for the growth and development of plants at low levels, it could be a strong inhibitor of photosynthesis when Cu2þ in excess [10,49]. The loss in chlorophyll content could be due to peroxidation of chloroplast membranes mediated or replacing magnesium in chlorophyll

4.5

75

5

Fig. 5. Effects of Cu2þ and Cd2þ on SOD activity in Lemna minor. Standard deviations are represented by error bars. (,) Cu2þ; (C) Cd2þ.

120

90

0

Concentration (mg l-1)

MDA content (mmol g FW-1)

CAT activity (U mg protein-1 min-1)

experiment, changes in soluble protein content of Lemna fronds exhibited inverse relationships with increased Cu2þ and Cd2þ concentrations. Also results showed that cadmium inhibited soluble protein content faster and stronger than copper did. The inability of Lemna fronds to synthesize protein after copper treatment might be caused by acute oxidative stress induced by Cu excess in plant cells (Mazhoudi et al., 1997). While cadmium could induce DNA damage such as singleand double-strand breaks, modified bases, abasic sites, DNAprotein cross-links, oxidized bases, 8-hydroxyguanine, and even bulky adducts etc. in organisms [4]. The presence of the above types of DNA lesions might induce important structural changes that could significantly affect the synthesization

100

70

Concentration (mg l-1) Fig. 3. Effects of Cu2þ and Cd2þ on POD activity in Lemna minor. Standard deviations are represented by error bars. (,) Cu2þ; (C) Cd2þ.

110

3.5

3.0

2.5

2.0 45 0

5

10

15

20

Concentration (mg l-1) Fig. 4. Effects of Cu2þ and Cd2þ on CAT activity in Lemna minor. Standard deviations are represented by error bars. (,) Cu2þ; (C) Cd2þ.

0

5

10

15

20

Concentration (mg l-1) Fig. 6. Effects of Cu2þ and Cd2þ on MDA content in Lemna minor. Standard deviations are represented by error bars. (,) Cu2þ; (C) Cd2þ.

W. Hou et al. / Plant Physiology and Biochemistry 45 (2007) 62e69

molecule by copper [29,41]. Numerous sites were identified as targets of copper action in chloroplast and therefore excess of cupric ions may result in decrease in the electron transfer rates consequent to its binding to the sites [28]. Cadmium, as non-essential metal ions, was found to be more toxic to pigments than copper to Lemna fronds. Even under low concentration, 0.05 mg l1 of Cd2þ had adverse effects on photosynthetic pigments. Also, with the increase of Cd2þ concentration in growth medium, the decline rate of pigments value was higher than that of copper-treated medium. Cadmium ions inhibited the formation of chlorophyll by interfering with protochlorophyllide reduction and the synthesis of aminoevulinic acid; it might interfere with different steps of Calvin cycle, resulting in the inhibition of photosynthetic CO2 fixation [34,51]. Also, cadmium could do great harm to chloroplast envelope and thylakoid via increased production of free radicals [15]. The mechanism of heavy metals on photosynthetic pigments may be owed to three reasons: 1. Heavy metals enter frond chloroplast and may be over-accumulated in local causing oxidative stress which will cause damages like peroxidation of chloroplast membranes [39,40]. Also they can directly destroy the structure and function of chloroplast by binding with SH group of enzyme and over all chlorophyll biosynthesis through Mg2þ, Fe2þ or Zn2þ [43]. 2. Heavy metal ions inhibit uptake and transportation of other metal elements such as Mn, Zn and Fe by antagonistic effects and therefore the fronds lose the capacity of synthesis of pigments [5,12,24]. 3. Heavy metals may activate pigment enzyme and accelerate the decomposition of pigment. From Tables 2, 3 and Fig. 2, we can draw a conclusion that the degradation rate of Chl b under heavy metal stress was slower than that of Chl a and carotenoid, respectively. The results suggest that the damage of heavy metals on Chl a is greater than that on Chl b. As is known, Chl a is one of the most important center pigments in photosynthesis and therefore the decrease of Chl a can inhibit the photosynthesis greatly. Carotenoid, which plays the part of guard of chlorophyll, also serves as an antioxidant to quench or scavenge the free radicals and reduce the damage of cell, cell membrane, and its main genetic composition [16]. The experimental results could suggest the function of carotenoid under stress.

4.2. Effects of copper and cadmium on MDA and antioxidant enzymes Normally, reactive oxygen intermediates (ROIs) are unavoidable byproducts of aerobic metabolism in plants, such as photosythesis and respiration [2]. Whereas under environmental stresses, such as desiccation, salt stress, heavy metals, ultraviolet radiation, air pollution such as ozone and SO2, enhanced production of ROIs occurs [33]. Meanwhile, antioxidant enzymes of plants are activated to scavenge excess ROIs and play the part of detoxification.

67

MDA is the decomposition product of polyunsaturated fatty acids (PUFA) of biomembranes and its increase shows plants are under high-level antioxidant stress. Cell membranes stability has been widely used to differentiate stress tolerant and susceptible cultivars of many crops [38] and in some cases higher membrane stability could be correlated with better performance [45]. In the experiments, stimulating effects of increased heavy metals on MDA activity of fronds indicated plants encountered enhanced lipid peroxidation (Fig. 6). Similar results were obtained in the effects of Cu2þ on Ceratophyllum demersum L. [6]. Results indicated that under the same concentrations, fronds treated with cadmium were confronted with higher oxidative stress than those with copper (Fig. 6). POD belongs to enzymes involved in growth, development and senescence processes of plants. They affect lignin and ethylene synthesis, decomposition of IAA, and involves in resistance against pathogens and wound healing [3]. CAT belongs to most important enzymes scavenging the active oxygen species in plant cells. CAT participates in the main defense system against accumulation and toxicity of hydrogen peroxide and can play the role in controlling H2O2 level in cells. It acts on H2O2 and converts it to water and oxygen [35]. The activity of POD and CAT of Lemna minor treated with heavy metals mainly displayed biphasic responses with increased metal concentration. When Lemna was faced with low-level metal stress, fronds could activate POD and CAT activities, which led to a strengthening of fronds, to scavenge ROIs responsible for lipid peroxidation [11], while the activities decreased distinctly under too acute stress which overloaded cellular defense system of fronds [14,21]. Also the declines in the activities of POD and CAT might be due to the formation of protein complex with metals, to change the structure or integrity of proteins [9,34]. Similar results were obtained in effects of copper and folpet on CAT activity, respectively [46,48] and effect of diuron on P-POD and G-POD activity [47]. For fronds treated with increased metals, CAT and POD activities appeared to be inhibited exposed to 10 mg l1 of Cu2þ, whereas CATand POD activities began to decline under 0.5 mg l1 Cd2þ stress (Figs. 4 and 5). The results suggested that cadmium was more toxic to Lemna minor than copper for the reason that at relatively lower concentrations, antioxidant system appeared to be in disorder. SOD is an essential component of antioxidative defense system in plants and it dismutates two superoxide radicals (O2) to water and O2. SOD appears to play a pivotal role in combating oxidative stress in plant [42]. In the experiment, SOD activity under elevated heavy metal stress was steadily stimulated with the increasing metal ion value in medium. The results indicated that SOD activity could suffer from high-level heavy metal concentration (Fig. 5). Similarly, responses of SOD activity under cadmium stress came out to be more acute than under copper stress. With increased heavy metal concentrations, antioxidant enzymes POD, CAT and SOD activities appeared to be complex; under high metal stress, POD and CAT activities were inhibited while SOD activity could put up with the high stress. By the reason that those enzymes were located at different

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cellular sites, which had different resistances to heavy metals, and the deterioration of cellular system functions by high metal stress might result in the inactivation of enzyme activity [7,33]. However, the mechanism of antioxidant enzyme inactivation is not clear and need further investigation. 5. Conclusion The toxic effects of copper and cadmium on Lemna minor were discussed in this paper. Based on the physiological responses of duckweed under increasing concentrations of heavy metals, following conclusions can be achieved. a. Of two heavy metals, cadmium was more toxic than copper. The antioxidant system had become disordered when Lemna fronds were exposed to 0.5 mg l1 of cadmium in grown medium. b. At the lowest concentration (0.05 mg l1), copper did little harm to duckweed. When the concentration reached up to 10 mg l1, the antioxidant system of plants began to break down. Our experiments showed that Lemna minor could tolerate low level heavy metal stress (Cu2þ<10 mg l1, Cd2þ < 0.5 mg l1). Also the removal of low-level toxic metals from aqueous solution by duckweed was quite efficient [17]. Therefore, combined with advisable harvesting, the bioremediation of low-level polluted waterbody by copper and cadmium is available. Acknowledgements Financial assistance provided by Chinese basal keystone research project (973 project) (2002CB412300) and International Science and Technology Cooperation Key research project (2003DFB00018) is fully acknowledged. References [1] H. Aebi, Catalase in vitro, Methods Enzymol 105 (1984) 121e176. [2] K. Asada, M. Takahashi, Production and scavenging of active oxygen in photosynthesis, in: D.J. Kyle, et al. (Eds.), Photoinhibition, Elsevier, 1987, pp. 227e287. [3] K. Asada, Ascorbate peroxidase-a hydrogen peroxide-scavenging enzyme in plants, Physiol. Plant 85 (1992) 235e241. [4] I. Atesi, H.S. Suzen, A. Aydin, et al., The oxidative DNA base damage in tests of rats after intraperitoneal cadmium injection, Biometals 17 (4) (2004) 371e377. [5] P. Das, S. Samantaray, G.R. Rout, Studies on cadmium toxicity in plants: a review, Environ. Pollut. 98 (1) (1997) 29e36. [6] S.R. Devi, M.N.V. Prasad, Copper toxicity in Ceratophyllum demersum L. (Coontail), a free floating macrophyte: response of antioxidant enzymes and antioxidants, Plant Sci. 138 (1998) 157e165. [7] D. Dewez, L. Geoffroy, G. Vernet, et al., Determination of photosynthetic and enzymatic biomakers sensitivity used to evaluate toxic effects of copper and fludioxonil in alga Scenedesmus obliquus, Aquatic Toxicol. 74 (2005) 150e159. [8] V.P. Dushenkov, B.A. Nanda Kumar, H. Motto, et al., Rhizofiltration: the use of plants to remove heavy metals from aqueous streams, Environ. Sci. Technol. 29 (1995) 1239e1245.

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