Cadmium tolerance and accumulation in eight

Biotechnology Advances 27 (2009) 555–561

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Biotechnology Advances j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v

Cadmium tolerance and accumulation in eight potential energy crops Gangrong Shi a,b, Qingsheng Cai a,⁎ a b

College of Life Sciences, Nanjing Agricultural University, Nanjing, 210095, China The Anhui Provincial Key Laboratory of the Resource Plant Biology in Department of Biology, Huaibei Coal Industry Teachers College, Huaibei, 235000, China

a r t i c l e

i n f o

Available online 22 April 2009 Keywords: Energy crops Cadmium Tolerance Accumulation Bioconcentration factor Translocation index

a b s t r a c t The production of energy crops that can be used for biodiesel production is a sustainable approach for the removal of metal pollutants by phytoremediation. This study investigated the cadmium (Cd) accumulation and tolerance of eight potential energy crops. After growth for 28 days in substrates containing 0, 50, 100 or 200 mg Cd·kg− 1, seedlings were evaluated for growth parameters, chlorophyll content, chlorophyll fluorescence parameters and Cd accumulation. All eight crops were moderately tolerant to Cd toxicity, with four [i.e., hemp (Cannabis sativa), flax (Linum usitatissimum), castor (Ricinus communis) and peanut (Arachis hypogaea)] being more tolerant than the others. Three of these crops (hemp, flax and peanut) had higher Cd accumulation capacities. The roots of peanut and hemp had high bioconcentration factors (BCF N 1000), while flax shoots accumulated a higher concentration of Cd (N 100 mg/kg). These results demonstrate that it is possible to grow energy crops on Cd-contaminated soil. Hemp, flax and peanut are excellent candidates for phytoremediation. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Recent increases in crude oil prices, the limited availability of fossil oil, and environmental concerns have prompted a renewed focus on vegetable oils for the production of biodiesel. Biodiesel is regarded as the best alternative to fossil fuel for reducing greenhouse gas release, as well as being beneficial for agriculture (Agarwal, 2007). Several countries are actively investigating the potential of different vegetable oils for diesel fuel production, depending on their climate and soil conditions. For example, soybean (Glycine max) oil in the USA, rapeseed (Brassica rapa) and sunflower (Helianthus annuus) oils in Europe, palm (Trachycarpus fortunei) oil in Southeast Asia (mainly Malaysia and Indonesia), and coconut (Cocos nucifera) oil in the Philippines have all been considered as possible substitutes for fossil diesel (Agarwal, 2007). The promotion of biodiesel, however, requires huge amounts of arable land that are also needed for traditional purposes, such as food production, so the ability to use land that is currently set aside or polluted for the production of energy crops for biodiesel is an important consideration. Another environmental problem is the widespread cadmium (Cd) contamination of land caused by the application of sludge or urban composts, pesticides, fertilizers, emissions from waste incinerators, waste water irrigation, and residues from metalliferous mining and the metal smelting industry (McGrath et al., 2001; Yang et al., 2004). Cd is a heavy metal that is highly toxic to plants, animals and humans. ⁎ Corresponding author. College of Life Sciences, Nanjing Agricultural University, Nanjing, 210095, China. Tel.: +86 25 84395187; fax: +86 25 84396542. E-mail address: [email protected] (Q. Cai). 0734-9750/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2009.04.006

This pollution is especially important when the contaminated lands are used for crop cultivation, since Cd is easily transferred from the soil into the food chain, so threatening human and animal health (Gupta and Gupta, 1998; Jarup, 2003). Clean-up of Cd-contaminated soils is difficult. Existing methods such as mechanical removal and chemical engineering are expensive, and are often incompatible with maintaining soil structure and fertility (Pulford and Watson, 2003). Phytoremediation, i.e. the use of plant systems to remove toxic elements from the soils, has recently attracted a great deal of attention as an alternative means of soil decontamination, since it is a cost-effective, environmentally-friendly approach, applicable to large areas. However, phytoremediation also has some disadvantages. The plants that can be used for phytoremediation are rare herbs with small biomass, which have little economic value (Linger et al., 2002). The process of phytoremediation is quite slow and usually takes several years, or even decades, to halve the levels of heavy metal contaminants in the soil (McGrath and Zhao, 2003). During phytoremediation, the contaminated land cannot be sold or rented, which can cause problems for local economic development (Peuke and Rennenberg, 2005). Methods for the disposal of the metal-enriched biomass have not yet been well developed (Sas-Nowosielska et al., 2004), though the reuse of the harvest biomass in non-food industries is being investigated (Citterio et al., 2003). In order to fully utilize Cd-contaminated soils and to overcome the disadvantage of phytoremediation, we postulate a new strategy of combining phytoremediation with oil crop cultivation, with a view to achieving low cost decontamination of soil through the production of biodiesel. This process could be of particular importance in countries with less arable land, such as China and Japan. Implementation of this strategy requires the selection of energy crops that can tolerate heavy

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metals. This study aimed to (1) evaluate the Cd tolerance and Cd accumulation capacity of eight energy crops, including rapeseed, sunflower, soybean, hemp (Cannabis sativa), flax (Linum usitatissimum), castor (Ricinus communis), safflower (Carthamus tinctorius) and peanut (Arachis hypogaea); (2) screen for energy crops that can be planted in Cd-contaminated areas for use in biodiesel production; (3) identify crops that can be used for phytoremediation of Cdcontaminated land. An understanding of the Cd tolerance of the potential energy crops is necessary for studying the plant–metal interactions before application of these crops for phytoremediation and biodiesel production. 2. Materials and methods 2.1. Plants After an initial screening, eight energy crops were selected for this study: rapeseed (cv. Jujia1), sunflower (cv. G101), soybean (cv. Xudou9), hemp (cv. YM12), castor (cv. Zibi3), safflower (cv. Honghua1), flax (cv. Longya7) and peanut (cv. Luhua11). 2.2. Experimental set-up Pot tests were carried out in a greenhouse at the Huaibei Coal Industry Teachers College, Huaibei, China. The average temperature throughout the test period was between 24.6 ± 0.4 °C (daytime) and 22.0 ± 0.4 °C (night), and the relative humidity was 61.5 ± 1.3% (daytime) and 68.0 ± 1.9% (night). Seeds were directly sown into pots (16 cm × 18 cm) filled with a mixture of acid-washed sand and perlite (5:4, v/v), supplemented with Cd as CdCl2·2.5H2O at 0, 50, 100 or 200 mg/kg (dry weight). Each Cd treatment was replicated in three pots, and five uniform plants were allowed to grow in each pot, at a uniform spacing. The pot was irrigated daily so that water holding capacity remained at 60%. Every 3 days, the plants were fertilized with 100 ml of Hoagland's nutrient solution: 5 mM Ca(NO3)2, 5 mM KNO3, 1 mM KH2PO4, 1 mM MgSO4, 50 μM H3BO3, 4.5 μM MnCl2, 3.8 μM ZnSO4, 0.3 μM CuSO4, 0.1 mM (NH4)6 Mo7O24 and 10 μM FeEDTA. 2.3. Evaluation of plant growth and metal accumulation Seedlings were harvested after 28 days and the root and shoot lengths were measured. The plants were then washed with running tap water and rinsed with deionized water to remove any sand and perlite particles attached to the plant surfaces. The roots and shoots were separated and oven-dried for 30 min at 105 °C, then at 70 °C, until they reached constant weights. The dried tissues were weighed and ground into a powder. Cd concentrations were then measured using flame atomic absorbance spectrometry after digestion with mixed acid [HNO3 + HClO4 (3:1, v/v)]. The tolerance index (TI) was expressed on the basis of plant growth parameters including root length, shoot length, root weight and shoot weight, calculated as the following (Wilkins, 1978): TI = 100 × ½Growth parameters Cd = ½Growth parameterscontrol :

ð1Þ

The translocation factor (TF) of Cd from root to shoot, bioconcentration factor (BCF), and shoot total Cd uptake were calculated as follows (Ali et al., 2002; Monni et al., 2000): TF = ½Cdshoot = ½Cdroot

ð2Þ

BCF = ½Cdshoot or root = ½Cdsoil

ð3Þ

Total Cd uptake = total shoot biomass

ð4Þ

× total Cd concentration in the shoot:

2.4. Chlorophyll concentration analysis Mature leaves (0.2 g) from five plants in each pot were extracted in the dark at 4 °C in a 5-ml mixture of acetone and ethanol (v/v = 1:1) until the color had completely disappeared. Light absorbance at 663 and 645 nm was determined by spectrophotometry. Chlorophyll a and b contents were calculated according to Arnon (1949). 2.5. Chlorophyll fluorescence measurement Before harvesting, all seedlings were analyzed for photosynthetic activity by measuring chlorophyll a fluorescence parameters (Ruley et al., 2006). This was performed using a Mini PAM (Walz, Effeltrich, Germany). Plants were dark-adapted for 30 min and then subjected to a 1-s pulse of red light. The following fluorescence parameters were measured: F0, the minimum chlorophyll a fluorescence after darkadaptation, and Fm, the maximum fluorescence after the pulse of red light. Fv/Fm (the ratio of variable to maximal fluorescence, which is a measure of the quantum yield of photosystem II photochemistry) values were determined based on these measurements. Steady state chlorophyll a fluorescence yield (F) was monitored under ambient irradiation (150 μmol quanta m2 s− 1). A saturating light pulse (5000 μmol quanta m2 s− 1, 1 s) was applied for closing all reaction centers, and the maximum fluorescence yield (Fm′) was subsequently measured. The effective quantum yield of PS II (ΦPS II) was calculated as follows: ΦPS II = ΔF / Fm′ = (Fm − F) / Fm. 2.6. Statistical analysis Statistical analyses were performed using SPSS Version 11.5 software (SPSS Inc., USA). The data were subjected to ANOVA, and differences between means were determined using the least squares deviation (LSD) test. 3. Results 3.1. Plant growth and TI At day 28, peanut had the largest biomass, followed sequentially by soybean, sunflower, castor, rapeseed, safflower, and flax (Table 1). Treatment with low concentrations of Cd (50 mg/kg) did not suppress root length in rapeseed, hemp, sunflower, soybean or castor, and even promoted root growth in rapeseed. This hormesis effect of Cd has also been observed in Indian mustard (Brassica juncea) (Singh and Tewari, 2003), barley (Hordeum vulgare) (Aery and Rana, 2003) and tumbleweed (Salsola kali) (de la Rosa et al., 2004). However, root growth in the other plant species, namely safflower, flax, and peanut, was repressed, even at this low Cd concentration. Treatment with 100 mg/kg Cd repressed root growth in six out of the eight crops, but not in rapeseed and hemp. Treatment with high Cd concentrations (200 mg/kg) markedly reduced root growth in all plant species. The reduction in root growth was smallest in castor (32%), followed by flax (38%), hemp (43%) and sunflower (47%) (Table 1). In all species except hemp, shoot length decreased as Cd concentration increased (P b 0.05). At a Cd concentration of 200 mg/ kg, the smallest reduction in shoot length was seen in flax (35%), followed by hemp (43%), soybean (49%), castor (56%) and peanut (58%) (Table 1). In general, total biomass and shoot biomass of the eight crops decreased when soil Cd concentration increased (Table 1). Similarly, root biomass decreased as Cd concentration increased, except in hemp and peanut, which showed no change in root biomass (P N 0.05) when treated with 50 mg/kg. Root biomass was reduced least in peanut, followed by hemp, flax and castor. The least reduction in shoot biomass was seen in flax, followed by peanut, castor and hemp.


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Table 1 Root length (cm), shoot length (cm), root biomass (g/plant), and shoot biomass (g/plant) of eight energy crops grown in Cd-treated substrates (mean ± S.E., n = 15). Treatments (mg/kg)

Arachis hypogaea

Root length 0 50 100 200

Brassica rapa

Cannabis sativa

28.7 ± 1.6a 23.1 ± 1.3b 15.2 ± 1.1c 9.8 ± 0.6d

15.8 ± 0.6b 17.8 ± 0.6a 15.7 ± 0.7b 7.43 ± 0.3c

15.9 ± 1.3ab 13.9 ± 0.5b 16.9 ± 0.6a 9.2 ± 0.7c

Shoot length 0 50 100 200

15.2 ± 0.2a 9.5 ± 0.2b 7.2 ± 0.2c 6.4 ± 0.2c

nd nd nd nd

28.4 ± 2.0a 26.5 ± 1.6a 25.5 ± 1.4a 16.3 ± 0.7b

Root biomass 0 50 100 200

0.32 ± 0.02a 0.28 ± 0.02a 0.19 ± 0.01b 0.14 ± 0.01c

0.041 ± 0.002a 0.031 ± 0.001b 0.020 ± 0.002c 0.006 ± 0.001d

0.081 ± 0.012a 0.074 ± 0.004a 0.062 ± 0.004b 0.024 ± 0.003c

Shoot biomass 0 50 100 200

0.98 ± 0.05a 0.56 ± 0.04b 0.44 ± 0.03c 0.37 ± 0.02c

0.15 ± 0.01a 0.11 ± 0.00b 0.07 ± 0.01c 0.02 ± 0.00d

Total biomass 0 50 100 200

1.30 ± 0.06a 0.85 ± 0.05b 0.63 ± 0.04c 0.51 ± 0.03c

Root/shoot 0 50 100 200

0.33 ± 0.01c 0.51 ± 0.02a 0.43 ± 0.01b 0.38 ± 0.02b

Carthamus tinctorius

Glycine max

Helianthus annuus

Linum usitatissimum

Ricinus communis

18.1 ± 0.9a 12.5 ± 0.9b 10.8 ± 0.8bc 8.0 ± 0.5c

21.2 ± 0.5a 23.5 ± 1.7a 10.2 ± 0.7b 5.2 ± 0.3c

17.4 ± 1.0a 17.8 ± 1.2a 13.8 ± 0.7b 9.2 ± 0.7c

18.6 ± 0.5a 15.9 ± 0.5b 16.5 ± 0.7b 11.6 ± 0.4c

23.7 ± 1.4a 24.3 ± 1.1a 20.2 ± 0.9b 16.1 ± 0.9c

13.5 ± 0.6a 7.0 ± 0.4b 6.1 ± 0.4c 5.1 ± 0.3d

31.0 ± 0.6a 21.1 ± 0.5b 20.7 ± 0.5b 15.9 ± 0.5c

38.9 ± 1.1a 20.0 ± 0.7b 15.1 ± 1.0c 7.7 ± 0.5d

14.4 ± 0.4a 12.6 ± 0.4b 12.1 ± 0.4b 9.3 ± 0.3c

13.9 ± 0.4a 9.7 ± 0.3b 7.6 ± 0.3c 6.1 ± 0.2d

0.058 ± 0.016a 0.014 ± 0.002b 0.015 ± 0.002b 0.013 ± 0.001c

0.37 ± 0.01a 0.15 ± 0.01b 0.11 ± 0.01c 0.05 ± 0.00d

0.19 ± 0.01a 0.13 ± 0.01b 0.08 ± 0.01c 0.07 ± 0.01c

0.027 ± 0.000a 0.020 ± 0.001b 0.014 ± 0.001c 0.011 ± 0.001d

0.23 ± 0.03a 0.15 ± 0.01b 0.15 ± 0.01b 0.09 ± 0.01c

0.31 ± 0.04a 0.15 ± 0.01b 0.17 ± 0.01b 0.09 ± 0.02c

0.13 ± 0.01a 0.06 ± 0.00b 0.06 ± 0.00b 0.05 ± 0.01b

0.82 ± 0.03a 0.43 ± 0.01b 0.41 ± 0.02b 0.23 ± 0.01c

0.69 ± 0.03a 0.27 ± 0.02b 0.20 ± 0.01c 0.16 ± 0.01c

0.030 ± 0.002a 0.022 ± 0.001b 0.020 ± 0.001bc 0.017 ± 0.000c

0.58 ± 0.03a 0.33 ± 0.01b 0.26 ± 0.01c 0.18 ± 0.01d

0.19 ± 0.01a 0.14 ± 0.00b 0.09 ± 0.01c 0.03 ± 0.00d

0.39 ± 0.05a 0.19 ± 0.01bc 0.23 ± 0.01b 0.12 ± 0.02c

0.19 ± 0.03a 0.07 ± 0.00b 0.08 ± 0.00b 0.07 ± 0.01b

1.19 ± 0.04a 0.58 ± 0.02b 0.53 ± 0.02b 0.28 ± 0.02c

0.88 ± 0.04a 0.40 ± 0.03b 0.28 ± 0.02c 0.23 ± 0.02c

0.06 ± 0.00a 0.04 ± 0.00b 0.03 ± 0.00c 0.03 ± 0.00d

0.82 ± 0.06a 0.49 ± 0.01b 0.41 ± 0.02b 0.28 ± 0.01c

0.28 ± 0.03a 0.29 ± 0.02a 0.29 ± 0.01a 0.34 ± 0.01a

0.26 ± 0.02b 0.27 ± 0.01b 0.38 ± 0.02a 0.31 ± 0.04b

0.42 ± 0.08a 0.24 ± 0.03b 0.24 ± 0.01b 0.25 ± 0.02b

0.46 ± 0.01a 0.35 ± 0.01b 0.28 ± 0.01c 0.20 ± 0.01d

0.28 ± 0.01c 0.48 ± 0.03a 0.42 ± 0.03b 0.40 ± 0.02b

0.90 ± 0.06a 0.90 ± 0.01a 0.72 ± 0.03b 0.66 ± 0.07b

0.38 ± 0.03b 0.46 ± 0.02ab 0.55 ± 0.04a 0.50 ± 0.04a

nd, not determinable. Means in the same column for each crop followed by the same letter are not significantly different at P b 0.05 based on LSD test.

The effect of Cd stress on the root/shoot ratio was speciesspecific. Cd treatment increased the root/shoot ratio in peanut, sunflower, and castor, while it decreased it in safflower, soybean and flax. Cd treatment had no effect on the root/shoot ratio in

rapeseed and hemp (with the exception of 100 mg/kg Cd treatment) (Table 1). TIs of the eight energy crops are shown in Table 2. Three crops (hemp, flax and castor) showed higher TIs, regardless of Cd

Table 2 Tolerance index (TI) for eight energy crops subjected to different Cd concentrations for 28 days (mean ± S.E., n = 3). nd, not determinable. Treatments (mg/kg)

Arachis hypogaea

Brassica rapa

Cannabis sativa


Glycine max

Helianthus annuus

Linum usitatissimum

Ricinus communis

Root length 50 100 200

81 ± 0a 53 ± 7b 34 ± 3c

112 ± 5a 99 ± 3b 47 ± 2c

87 ± 7b 106 ± 5a 57 ± 7c

68 ± 8a 60 ± 5a 44 ± 1b

111 ± 9a 48 ± 4b 25 ± 2c

102 ± 1a 79 ± 5b 53 ± 8c

85 ± 2a 89 ± 7a 62 ± 3b

103 ± 2a 85 ± 7ab 68 ± 6b

Shoot length 50 100 200

62 ± 2a 47 ± 2b 42 ± 1b

nd nd nd

94 ± 12a 90 ± 8a 57 ± 2b

52 ± 3a 46 ± 6ab 38 ± 1b

68 ± 2a 67 ± 2a 51 ± 1b

51 ± 4a 38 ± 4a 20 ± 2b

87 ± 2a 84 ± 3a 65 ± 3b

70 ± 1a 54 ± 2b 44 ± 2c

Root biomass 50 100 200

90 ± 6a 58 ± 7b 44 ± 1b

75 ± 3a 48 ± 6b 15 ± 2c

91 ± 9a 76 ± 5b 29 ± 4c

25 ± 4a 26 ± 3a 23 ± 2a

40 ± 1a 31 ± 0a 13 ± 1b

68 ± 6a 44 ± 6b 35 ± 6b

72 ± 4a 53 ± 2b 40 ± 4c

66 ± 5a 62 ± 5a 40 ± 4b

Shoot biomass 50 100 200

58 ± 4a 45 ± 4ab 38 ± 1b

72 ± 3a 45 ± 6b 13 ± 1c

47 ± 5a 53 ± 5a 30 ± 7b

46 ± 2a 49 ± 2a 41 ± 4a

52 ± 1a 51 ± 2a 28 ± 0b

40 ± 4a 29 ± 2b 24 ± 2b

72 ± 4a 65 ± 2b 55 ± 1b

57 ± 1a 45 ± 4b 31 ± 1c

Total biomass 50 100 200

66 ± 4a 48 ± 4b 39 ± 1b

72 ± 2a 46 ± 6b 13 ± 1c

47 ± 6ab 58 ± 5a 30 ± 6b

39 ± 3a 42 ± 2a 35 ± 3a

48 ± 1a 44 ± 2b 23 ± 0c

46 ± 4a 32 ± 3b 26 ± 3b

72 ± 4a 59 ± 2b 48 ± 2c

60 ± 2a 50 ± 5a 34 ± 2b

Means in the same column for each crop followed by the same letter are not significantly different at P b 0.05 based on LSD test.

558


Fig. 1. Chlorophyll a, chlorophyll b, and total chlorophyll content of eight energy crops subjected to different Cd concentrations. Each value represents the mean ± S.E. (n = 3). Means for each crop followed by the same letter are not significantly different at the level of P b 0.05 based on LSD test.

concentration. Rapeseed had a higher TI only at Cd concentrations of 50 mg/kg and 100 mg/kgCd. Root and shoot biomasses were more effectively suppressed by Cd treatment than was length (except in peanut). Shoots were more sensitive to Cd.

3.2. Photosynthetic activity Chlorophyll and chlorophyll a contents decreased as Cd concentration increased (Fig.1). This change was also reflected by the change in color of

Table 3 The Fv/Fm and ΦPS II of energy crops grown in soils with different Cd concentrations (mean ± S.E., n = 15). Treatments (mg/kg)

Arachis hypogaea

Brassica rapa

Cannabis sativa


Glycine max

Helianthus annuus

Fv/Fm 0 50 100 200

0.839 ± 0.004a 0.820 ± 0.008a 0.816 ± 0.014a 0.823 ± 0.007a

0.807 ± 0.001a 0.797 ± 0.002a 0.796 ± 0.002a 0.762 ± 0.010b

0.797 ± 0.003a 0.794 ± 0.003a 0.781 ± 0.003a 0.719 ± 0.030b

0.806 ± 0.002a 0.792 ± 0.003b 0.776 ± 0.006c 0.769 ± 0.006c

0.813 ± 0.003a 0.807 ± 0.002a 0.734 ± 0.017c 0.613 ± 0.027d

0.809 ± 0.003a 0.810 ± 0.002a 0.775 ± 0.011a 0.692 ± 0.037b

0.78 ± 0.004a 0.79 ± 0.002a 0.76 ± 0.007a 0.61 ± 0.035b

ΦPS II 0 50 100 200

0.660 ± 0.011a 0.601 ± 0.019a 0.568 ± 0.032a 0.621 ± 0.024a

0.512 ± 0.023a 0.341 ± 0.012b 0.305 ± 0.012b 0.341 ± 0.030b

0.635 ± 0.010a 0.662 ± 0.005a 0.666 ± 0.010a 0.594 ± 0.017b

0.526 ± 0.018a 0.455 ± 0.017b 0.388 ± 0.024c 0.446 ± 0.021b

0.559 ± 0.017a 0.490 ± 0.018b 0.355 ± 0.023c 0.301 ± 0.032c

0.483 ± 0.015a 0.429 ± 0.012ab 0.378 ± 0.029bc 0.298 ± 0.014c

0.392 ± 0.022ab 0.420 ± 0.017a 0.329 ± 0.033b 0.229 ± 0.030c

The leaves of flax were too small to determine the chlorophyll fluorescence. Means in the same column for each crop followed by the same letter are not significantly different at P b 0.05 based on LSD test.

Ricinus communis


559

BCFs were high in all eight crops, with the highest in hemp, followed by peanut, rapeseed, and safflower. The shoot BCF was highest in rapeseed, followed by safflower N flaxN peanutN sunflowerN hemp. Translocation factors (TFs) of the 8 crops were low (Table 4), suggesting that these crops have a low capacity to move Cd from root to shoot. The highest TF was found in flax (54%–66%), followed by rapeseed, safflower and sunflower. Shoot Cd uptake is an indicator of ability of phytoextraction. Peanut had the largest total Cd uptake (56.0–68.9 μg/ plant), with sunflower being the next (20.7–24.1 μg/plant). Although their BCF was not as great as that of accumulator plants, peanut and sunflower still have high extraction potentials because of their large biomasses. Seven crops showed no significant difference in total Cd uptake among different treatments. But rapeseed had a relatively higher total Cd uptake at low substrate Cd concentrations (50, 100 mg/kg), and lower uptake at higher substrate concentrations (200 mg/kg). Hemp and peanut had a higher Cd accumulation because of their high root Cd contents and large biomasses.

the leaves. Changes in chlorophyll b were species-specific. Treatment with Cd at 50 mg/kg and 100 mg/kg had no effect on the chlorophyll content of castor and hemp, and 50 mg/kg Cd did not alter the total chlorophyll and chlorophyll b contents of rapeseed did not change either. The chlorophyll a fluorescence parameters are shown in Table 3. Although Fv/Fm did not change in all plants after treatment with 50 or 100 mg/kg Cd, there was a tendency for it to decrease as Cd concentration increased. At high Cd concentrations (200 mg/kg), all crops except peanut showed dramatic reductions in Fv/Fm. ΦPS II also showed a tendency to decrease as Cd concentration increased, but peanut showed no change in ΦPS II at any of the three Cd concentrations. Hemp and castor showed no changes in ΦPS II at Cd concentrations of 50 and 100 mg/kg. 3.3. Cd concentrations and accumulation in plants Root and shoot Cd contents are shown in Table 4. In general, plant Cd content increased as Cd concentration increased in the culture substrate. Most of the Cd absorbed by the plants was found in the roots, the root Cd concentration was the highest in hemp, followed by peanut N safflower N rapeseed. The shoot Cd content was highest in rapeseed, followed by safflowerN flaxN peanut. It was very low in castor and soybean following treatment with the three Cd concentrations. Except for castor, the bioconcentration factor (BCF) for all other crops decreased as substrate Cd concentration increased (Table 4). The root

4. Discussion 4.1. Cd tolerance of the eight energy crops Previous studies have suggested that plants can suffer toxic effects when the soil total Cd concentration is N8 mg/kg, the soluble (bioavailable) Cd concentration is N0.001 mg/kg, or when the tissue

Table 4 Content of Cd in plant tissues, bioconcentration factor (BCF), translocation factor (TF), and total Cd uptake of eight energy crops grown in Cd-treated substrates for 28 days (mean± S.E., n = 3). Treatments (mg/kg)

Roots

Shoot

Roots

Shoot

TF (%)

Total Cd uptake (μg/plant)

A. hypogaea 50 100 200

616.9 ± 24.8b 2013.7 ± 67.6a 2124.5 ± 52.1a

97.4 ± 5.6c 157.7 ± 3.0b 177.4 ± 5.2a

1234 ± 50b 2014 ± 68a 1062 ± 26b

195 ± 11a 158 ± 3b 89 ± 3c

15.8 ± 0.3a 7.8 ± 0.3b 8.4 ± 0.1b

56.0 ± 4.0a 68.9 ± 5.5a 65.6 ± 1.5a

B. rapa 50 100 200

682.3 ± 16.3c 899.6 ± 30.0b 993.4 ± 38.7a

235.6 ± 0.9c 266.3 ± 8.1b 287.7 ± 5.4a

1365 ± 33a 900 ± 30b 497 ± 19c

471 ± 2a 266 ± 8b 144 ± 3c

34.6 ± 0.9a 29.7 ± 1.2b 29.0 ± 0.7b

25.4 ± 1.1a 18.1 ± 1.9b 5.4 ± 0.5c

C. sativa 50 100 200

1549.7 ± 101.4c 2349.0 ± 114.3b 4052.8 ± 225.6a

56.7 ± 6.4c 82.9 ± 2.7b 101.5 ± 8.0a

3099 ± 203a 2349 ± 114b 2026 ± 112b

113 ± 13a 83 ± 3b 51 ± 4c

3.7 ± 0.6a 3.6 ± 0.3a 2.5 ± 0.1a

8.5 ± 1.9a 13.8 ± 1.7a 9.9 ± 3.0a

C. tinctorius 50 100 200

616.3 ± 18.4c 838.2 ± 9.6b 1076.6 ± 29.9a

191.4 ± 4.1b 231.8 ± 9.6a 256.9 ± 11.5a

1232 ± 36a 838 ± 9b 538 ± 14c

382 ± 11a 231 ± 10b 128 ± 6c

35.8 ± 5.6a 27.6 ± 0.8a 29.1 ± 2.4a

11.5 ± 0.3a 14.9 ± 0.2a 13.8 ± 1.6a

G. max 50 100 200

458.7 ± 9.9b 618.6 ± 8.0a 614.3 ± 27.2a

26.8 ± 1.0c 34.7 ± 0.7b 68.9 ± 2.3a

917 ± 20a 619 ± 8b 307 ± 14c

54 ± 2a 35 ± 1b 34 ± 1b

5.9 ± 0.3b 5.6 ± 0.2b 11.2 ± 0.4a

11.5 ± 0.6b 14.4 ± 0.4a 15.8 ± 0.8a

H. annuus 50 100 200

341.5 ± 1.5b 374.7 ± 2.4c 408.3 ± 5.4d

88.1 ± 4.1b 105.5 ± 4.9c 146.8 ± 0.7d

683 ± 3a 375 ± 3b 204 ± 3c

176 ± 8a 106 ± 5b 74 ± 4c

25.8 ± 1.26b 28.1 ± 1.18b 36.0 ± 0.39a

24.1 ± 1.9a 20.7 ± 1.0a 23.8 ± 2.2a

203 ± 10b 293 ± 5ab 372 ± 78a

109 ± 5b 144 ± 7b 231 ± 36a

405 ± 20a 293 ± 5b 186 ± 39c

218 ± 11a 144 ± 7b 115 ± 18b

54 ± 3a 49 ± 2a 66 ± 13a

2.4 ± 0.1a 2.9 ± 0.1a 3.9 ± 0.7a

341.4 ± 2.1b 386.0 ± 4.6b 2517.9 ± 178.7a

14.7 ± 1.9b 28.0 ± 4.0b 59.8 ± 7.5a

683 ± 4b 386 ± 5c 1259 ± 89a

25 ± 7a 24 ± 7a 30 ± 4a

4.3 ± 0.5b 7.3 ± 1.1a 2.4 ± 0.2b

4.9 ± 0.6a 7.6 ± 2.0a 11.0 ± 1.4a

L. usitatissimum 50 100 200 R. communis 50 100 200

Cd content (μg/g)

BCF (%)

Means in the same column for each crop followed by the same letter are not significantly different at P b 0.05 based on LSD test.

560


Cd concentration reaches 3–10 mg/kg dry weight (Balsberg-Pahlsson, 1989; Polle and Schützendübel, 2003; Ghosh and Singh, 2005). The results of the current study suggest that the eight tested energy crops were moderately tolerant to Cd stress. All plants survived, even at soil Cd concentrations of 200 mg/kg. In contrast, Indian mustard and several grass species were unable to grow on soils with the same Cd concentrations (Ghosh and Singh, 2005). Our results suggest that the energy crops used in the current study have innate resistance to Cd stress. Many indicators, such as root biomass, shoot biomass and growth rate, have been used to evaluate metal toxicity in plants. Ali et al. (2002) reported that root growth was more sensitive to Cu than were shoot growth and biomass. The current study suggests that biomass is a better indicator of Cd toxicity than root length or shoot length, but that shoots are more sensitive than roots. Thus, the Cd sensitivities of different parameters are species-specific. We found that hemp, flax, castor, and peanut were relatively tolerant to Cd, based on measures of plant growth (Table 1) and TI (Table 2). Plant tolerance to environmental stress is reflected in chlorophyll content and chlorophyll a fluorescence parameters. These parameters are also indicators of damage to the photosynthetic system induced by environmental stressors (Lichtenthaler and Miehe, 1997; Maxwell and Johnson, 2000). In the current study, the chlorophyll content and chlorophyll a fluorescence parameters were relatively high in hemp, castor, peanut and flax, suggesting that the photosynthetic organs in these plants were tolerant to Cd stress, to varying degrees. These results are consistent with those from growth indicators. The mechanisms of metal tolerance in plants include metal exclusion and accumulation (Baker, 1987). Metal exclusion is the avoidance of absorption and the restriction of translocation to the shoots. Metal accumulation is an extreme type of physiological response whereby plants absorb and accumulate high concentrations of metals (Dahmani-Muller et al., 2000). As shown in Table 4, Cd absorbed in the eight crops in this study was retained in the root, suggesting that these plants limited translocation of Cd to the shoots. Such metal immobilization in root cells, as emphasized by TF values of b1, implies an exclusion mechanism (Baker, 1987). 4.2. Cd accumulation in the eight energy crops It has long been recognized that metal accumulation capacity varies greatly between different species and varieties, and is affected by various edaphic conditions. Shu et al. (2002) showed that roots accumulated much higher concentrations of heavy metals than shoots. The present study demonstrated that, even though the majority of Cd was found in the roots, substantial amounts were still found in the shoots, especially in shoots of rapeseed, safflower and flax, where the Cd concentration was N100 mg/kg dry mass. This level reached the threshold concentration defined by Baker et al. (2000) for a Cd hyperaccumulator. This observation warrants further study of these crops. The BCF is an excellent indicator of metal accumulation capacity of plants, because it takes into account the trace element concentration in the substrate (Zayed et al., 1998; Odjegba and Fasidi, 2004). Zayed et al. (1998) proposed that a good metal accumulating plant should have a BCF N 1000. The eight crops studied here had shoot BCFs far lower than 1000, and were therefore not considered to be metal accumulators. Hemp and peanut, however, had root BCFs N 1000 at all Cd concentrations. At a Cd concentration of 50 mg/kg, root BCFs of safflower and rapeseed also exceeded 1000. These data suggest that these plants have high capacities for Cd phytostabilization. An ideal plant for phytoextraction applications should have high metal tolerance and high accumulation capacity in its harvestable parts (Salt et al., 1998). Phytoremediation efficiency depends on shoot biomass and the ability of metal to be translocated to the shoots. The results of the present study showed a high root BCF and a high shoot Cd accumulation in peanut and hemp. Flax showed a high shoot Cd

accumulation. These crops have high Cd stress tolerance. Among the eight crops, peanut accumulated the most Cd in the shoots and could therefore be more efficient than the other crops for phytoremediation of Cd pollution. 5. Conclusions The eight energy crops were moderately tolerant to high levels of Cd contamination, and appeared to reduce Cd toxicity via an exclusion strategy. Variations in Cd tolerance existed among the species. Four energy crops with higher Cd tolerances (hemp, flax, castor and peanut) could be planted on Cd polluted land for use in biodiesel production. Three of these crops (peanut, hemp and flax) were better Cd accumulators, and may therefore be the best candidates for phytoremediation and fuel production on Cd-contaminated soil.

Acknowledgements Financial support from the Natural Science Foundation of Jiangsu Province (BK2006148) and the Natural Science Foundation for College of Anhui Province (KJ2009B073) is gratefully acknowledged.

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