Responses of Cynara cardunculus L. to single and combined cadmium and nickel treatment conditions

Ecotoxicology and Environmental Safety 74 (2011) 195–202

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Responses of Cynara cardunculus L. to single and combined cadmium and nickel treatment conditions E.G. Papazoglou n Department of Natural Resources Management and Agricultural Engineering, Agricultural University of Athens, 75 Iera Odos Street, 11855 Athens, Greece

a r t i c l e in fo

abstract

Article history: Received 24 November 2009 Received in revised form 28 June 2010 Accepted 29 June 2010 Available online 24 August 2010

A greenhouse pot experiment was carried out to study the responses of Cynara cardunculus L. (cardoon) to cadmium and nickel. Three groups of 12 pots each were planted with cardoon plants and spiked with single and combined cadmium and nickel aqueous solutions. The bioavailable metal concentrations, measured in soil, ranged widely and were up to 246.7 mg kg  1 for Cd and 61.1 mg kg  1 for Ni. Under Cd treatment, cardoon growth remained unaffected, while increased Ni soil concentrations inhibited plant growth and were lethal to the highly treated plants. In the combined Cd and Ni treatments, an antagonistic effect was observed between the two metals. Cadmium and nickel concentrations in cardoon tissues rose with increasing metal concentrations in the soil. Cadmium and nickel contents in shoots reached 169.3 and 342.3 mg kg  1 in the single treatments while, under the combined Cd and Ni treatments, they were up to 235.0 and 440.7 mg kg  1, respectively. Generally, mean contents of both metals in the shoots were higher than in the roots and the translocation factor was greater than 1. A possible enhancing effect of nickel on cadmium uptake was observed. Cardoon showed characteristics of a Cd accumulator. & 2010 Elsevier Inc. All rights reserved.

Keywords: Cadmium Nickel Cynara cardunculus Uptake Plant toxicity Cd accumulator Phytoremediation Tolerance Food crops Energy crops

1. Introduction Soils used for growing food crops in commercial horticulture and agriculture can be contaminated with potentially toxic heavy metals due to agricultural practices (e.g., application of mineralbased fertilizers, use of pesticides and herbicides, sewage sludge, waste water irrigation) (Alloway, 1995; Kabata-Pendias and Pendias, 2001). In areas where the contamination is not so great as to cause phytotoxicity and/or possible crop failure, there is possible risk for livestock or people when consuming large quantities of the produced crops. They might suffer illness and even death due to long-lasting intake of metals (Alloway, 2005; Huang et al., 2008). Food consumption is identified as the major pathway for human exposure to environmental contaminants, accounting for 4 90% of intake compared to inhalation or dermal routes of exposure (Fries, 1995). About 30% of human cancers are caused by low exposure to initiating carcinogenic contaminants in the diet (Mansour et al., 2009). In areas where soil is contaminated at toxic levels with heavy metals, certain plant species, which are adapted to these extremely harsh environments, can grow. These metal-tolerant

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species possess the ability to tolerate very high levels of heavy metals in the soil by two basic strategies, as proposed by Baker (1981): (i) In exclusion, transport of metals is low or restricted. The heavy metal contents accumulated in the aboveground biomass and roots are very low, or else only low percentages of the metals are transported to the aboveground parts, even though the concentrations in roots may be very high. (ii) In accumulation, metals are accumulated in nontoxic form in the upper plant parts at a wide range of soil-metal concentrations. According to Verkleij et al. (2009), there is a duality in plant tolerance to pollutants and its response to pollutant stress: (i) Metal-tolerant plants may have the ability to accumulate or hyperaccumulate metals in their shoots, and this could be beneficial for phytoremediation purposes to clean up soil and water and (ii) metal-tolerant food crops, grown in contaminated soils and accumulating metals in their tissues, represent an important route of toxic metals into the human food chain. For this experiment, it has been decided to work with Cynara cardunculus L. (cardoon), which is a food crop and, at the same time, a very promising energy crop. The investigation of the effects of heavy metals on this species could give useful results as to: (i) its potential to transfer metals in the food chain and (ii) its combined use in phytoremediation and bioenergy production, which constitutes a quite interesting additional duality.

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If cardoon has the potential to accumulate metals in its tissues, this should be seriously taken into consideration whenever it will be used as a food crop. However, this very same potential of cardoon could be exploited for economically feasible phytoextraction using this energy crop, thus contributing to an increase of income for farmers, who could cultivate them in contaminated and/or marginal areas. C. cardunculus is a promising high-yielding perennial crop of the family Asteraceae, native to the Mediterranean region. Traditionally, cardoon is used as a food crop (Fernandez et al., 2006; Gominho et al., 2009). The extracted proteases have been used for centuries in the Iberian peninsula as the enzymatic source for milk coagulation in traditional cheesemaking and its blanched leaves, fleshy leaf petioles, and receptacle are used in soups, stews, and salads. Kukic et al. (2008) reported several pharmaceutical uses for C. cardunculus, such as a diuretic, cardiotonic, choleretic, and antihemorrhodial agent, for treatment of dyspepsia, and as an antidiabetic. Moreover, its biomass can be used for multiple purposes, e.g., energy from biomass combustion, biodiesel from the seed oil, or fiber supply for pulp and paper industries (Gominho et al., 2009; Angelini et al., 2009). Cadmium and nickel are widespread pollutants and are considered public health threats (Reeves and Baker, 2000; Kabata-Pendias and Pendias, 2001). This experiment was conducted to study the effects of these metals on C. cardunculus. Three treatments were investigated, namely addition of Cd alone, Ni alone, and combined Cd and Ni to the soil. Four different metal concentrations were used per treatment and selected morphological parameters of the plants were measured. Moreover, the uptake ability of cardoons was determined.

2. Materials and methods 2.1. Soil sampling and characterization The soil used in this experiment was collected (at depth 0–20 cm) from a field of the Agricultural University of Athens (371590 N, 231420 E, and altitude 33 m), which had remained uncultivated for several years. The soil sample was homogenized and air-dried and then passed through a 1-cm sieve. Selected soil characteristics were determined following standard procedures (Table 1). Particle size distribution was measured by the sieving and pipette method (Day, 1965). Soil pH was determined in 1:1 soil/distilled water suspensions after 1 h with a combination pH electrode. Organic matter content was measured by the Walkley– Black method (Nelson and Sommers, 1982), the equivalent carbonate calcium by the Rowell method (Das, 1990), and the permeability by the constant-head method (Rowell, 1997). The total and diethylenetriaminepentaacetic acid (DTPA)-extractable metal concentrations of the soil were measured (Table 1). Total concentrations of Cd and Ni were determined by nitric acid digestion. Soil samples (1.0 g) were digested with 10 mL of HNO3 and 10 mL of H2O2 and the samples were heated on a hot plate at 120 1C for approximately 1 h, until the color became clear. After cooling,

Table 1 Physical and chemical properties of the initial soil. Physical Size fractions (%) Clay Silt Sand Texture Permeability (cm s  1)

15.5 13.4 71.1 Sandy loam 2.1  10  2

Chemical pH Organic matter (%) Equal carbonate (%) Cd total concentration (mg kg  1) Ni total concentration (mg kg  1) Cd bioavailable concentration (mg kg  1) Ni bioavailable concentration (mg kg  1)

8.00 0.5 23.7 0.5 1.3 0.2 0.7

the digests were brought up to 50 mL with distilled water, filtered through a Macherey–Nagel MN 6141/4, and analyzed by an atomic absorption spectrometer (AAS) (908 AA, GBC Scientific Equipment Pty. Ltd., Australia). Bioavailable fractions of Cd and Ni were determined using DTPA (Lindsay and Norvell, 1969; Walter et al., 2002; Walker et al., 2003). This extraction method provides more information about metal availability and tends to correlate with metal uptake by plants (Hooda and Alloway, 1994). The extraction was performed as described in Section 2.3.

2.2. Experimental setup and measurements The experiment was conducted inside a greenhouse, in plastic pots of capacity 11.8 L. Each pot was filled with 13 kg of the soil sample and was sown with eight cardoon seeds. After 45 days, the plantlets were manually thinned to one seedling per pot. During the experiment, the plants were irrigated every 2 days with deionized water. The soil moisture content was maintained at 75% of the waterholding capacity by weighing the pots and recalculating the dosage, in order to avoid drainage from the pots. By 4 months after the cardoon seeds were sown, the artificial contamination of the soil was performed. Metal application was timed to increase its effects on cardoon plants, when biomass was well developed. Three treatments were applied:

(i) Contamination with Cd alone (CdTREATMENT): Four different quantities of the salt Cd(NO3)2  4H2O were used, namely 0.0, 0.5, 5.0, and 10.0 g, which will be referred to as Cd0 (control), Cd0.5, Cd5, and Cd10, respectively. Each quantity was diluted in 500 mL of distilled water and was spiked into each pot. The actual metal concentrations of the aqueous solutions were determined by AAS: Not detected, 90.1, 884.5, and 1657.8 mg L  1, respectively. (ii) Contamination with Ni alone (NiTREATMENT): The total amounts of the salt Ni(NO3)2  4H2O used for Ni contamination were 0.0, 1.0, 10.0, and 20.0 g, and will be referred to as Ni0 (control), Ni1, Ni10, and Ni20, respectively. Each quantity was diluted in 500 mL of distilled water and was spiked into each pot. The actual Ni concentrations of the aqueous solutions were determined by AAS: Not detected, 100.2, 982.3, and 1843.1 mg L  1, respectively. (iii) Combined Cd and Ni contamination (Cd–NiTREATMENT): The mixture concentrations were prepared by adding half the amount of each metal used for their individual experiments. The quantities of Cd and Ni salts were 0+ 0, 0.25+ 0.5, 2.5 + 5, and 5+ 10, which will be referred to as Cd0–Ni0 (control), Cd0.25–Ni0.5, Cd2.5–Ni5, and Cd5–Ni10, respectively. Each binary mixture was diluted in 500 mL of distilled water and was spiked into each pot. The actual metal concentrations of the aqueous solutions for both metals were determined by AAS: (a) For Cd, not detected, 45.5, 440.6, and 814.0 mg L  1, respectively, and (b) for Ni, not detected, 50.0, 488.9, and 902.7 mg L  1, respectively.

The pots within each treatment were arranged according to a completely randomized design, with three replicates. After metal application (February 20, 2007), the plant height and the number of leaves were recorded every 7 days and the plants were observed, at regular intervals, in order to detect visible injuries. Plants were harvested 5 weeks after the artificial contamination of the soil, on March 27, 2007.

2.3. Plant and soil analysis At harvest, the plants were carefully removed from each pot and were divided into shoots and roots. Shoots were washed with deionized water, weighed, ovendried (72 h at 70 1C), and weighed again. Roots were washed thoroughly with tap water and twice with distilled water and were immersed in 0.01 M HCl for approximately 5 s in order to remove external metal from the root surface (Gardea-Torresday et al., 2004). Fresh and dry matter weights were recorded. All plant samples were ground using a cross-hammer beater mill and sieved with a 1-mm sieve. Plant samples (1-g dry weight) were digested with 10 mL concentrated HNO3 and placed on a hot plate. The digests were allowed to evaporate to near dryness and H2O2 was added till the solution became limpid. The sample volumes were adjusted to 10 mL using deionized water and the resulting solutions were filtered through Macherey–Nagel MN 6141/4. The filtrates were analyzed for heavy metals using AAS. Soil samples were collected from each pot, air-dried at room temperature, and ground to pass a 2.0-mm mesh. The bioavailable Cd and Ni forms were determined by extraction with DTPA. The extraction was performed by mixing 10 g of ground soil with 20 mL of DTPA solution (0.005 M DTPA, adjusted to pH 7.3). The metal concentrations were quantified by AAS. To ensure the accuracy and precision in the analyses, standard reference materials of metals (E-Merck, Germany) were run with samples.

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2.4. Statistical analysis Analysis of variance (one-way and multifactor ANOVA) was performed. Statistical analysis was conducted using the statistical packages STATISTICA 6.0 (Stat Soft, Inc.) and STATGRAPHICS Plus (Stat. Graph. Corp.).

3. Results 3.1. Bioavailable Cd and Ni concentrations in soil The DTPA-extractable Cd and Ni soil concentrations ranged widely (Table 2). Cadmium concentrations were higher than nickel concentrations and varied from 8.7 (Cd0.5) to 246.7 mg kg  1 (Cd10) for cadmium and from 3.4 (Cd0.25–Ni0.5) to 61.1 mg kg  1 (Ni20) for nickel, respectively. Stepwise multiple regression analysis was carried out, showing that both the levels of metal concentrations added to the soil and the metal species have a statistically significant effect on the DTPA-extractable concentrations (po0.0001), at the 95% confidence level. 3.2. Cd and Ni effects on cardoon growth In CdTREATMENT cardoon growth remained unaffected; no visible toxicity symptoms were observed and measured parameters were not influenced by the treatment. Plant height and leaf number increased during the cultivation period, but no significant differences were observed (p o0.05) between treated and control plants (Fig. 1(A1, A2)). At the end of the experiment, the mean height of the control plants was 57.6 cm, while the height of the treated plants was 58.3 cm (Cd0.5), 59.2 cm (Cd5), and 53.3 cm (Cd10); the mean number of leaves for the control plants was 10 and for the treated plants was 9–10 leaves per plant. Plant biomass was relatively stable (not significantly different) and fresh and dry weights of shoots were not significantly decreased (Fig. 2). Mean fresh weight of the control plants was 84.2 g and of the treated plants was 95.2 g (Cd0.5), 116.2 g (Cd5), and 76.0 g (Cd10). As can be observed, the medium (Cd5) treated plants showed an increasing trend in their growth (but not statistically significant), most probably due to the elevated concentrations of NO3 from the salt used, Cd(NO3)2. In Cd10 treatment, the elevated Cd concentrations reduced the positive reaction of the plants to the presence of NO3 . In NiTREATMENT the growth and development of the plants were affected by the bioavailable soil concentrations of 40.5 and 61.1 mg kg  1 (Figs. 1(B1, B2) and 2). In low (Ni1) treatment, phytotoxicity symptoms were not observed and plants were healthy with growth similar to that of the controls; measured parameters did not differ significantly between Ni0 and Ni1 (po0.05). At the end of the experiment, the mean height of the control plants was 60.3 cm and of the low-treated plants 63.3 cm,

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while the mean number of leaves was 9 per plant for both treatments and biomass remained unaffected in Ni1. In medium (Ni10) treatment, plant growth was inhibited; 2 weeks after Ni addition to the pots, plant height differed significantly from Ni0 and Ni1 (p o0.05) and did not increase further by the end of the experiment. Moreover, new leaves did not emerge and the existing ones became grayish-green, with twisted margins; several leaves were dried and the plants became generally weaker. On the last measurement date, the mean plant height remained at 36.5 cm and 6 leaves per plant were counted. Fresh and dry weights of the aboveground biomass decreased significantly (po0.05) compared to untreated plants and were 37.0 g and 4.4 g for Ni10-treated plants, while corresponding values for Ni0 were 85.1 g and 6.6 g, respectively. Thus, a reduction of over 50% was observed. Treatment Ni20 led to severe reduction of plant growth; all measured parameters differed significantly (po0.05) from control plants, and the cardoons were dried and dead. In Cd–NiTREATMENT plant height, leaf number, and fresh and dry biomass weights did not differ in low Cd0.25–Ni0.5 treatment from the control Cd0–Ni0 treatment (Figs. 1(C1, C2) and 2). An antagonistic effect was observed between the two metals for medium (Cd2.5–Ni5) and high (Cd5–Ni10) treatment. Plant height was affected only in the highly treated plants (Cd5–Ni10); a reduction in height was observed from the second week after the soil contamination (as in NiTREATMENT), but the differences became statistically significant (p o0.05) 4 weeks after metal application (March 20, 2007) and, at the end of the experiment, the height of Cd5–Ni10 plants was 35.3 cm while the corresponding height of control plants was 54.9 cm. The number of leaves differed significantly (p o0.05) from the untreated plants (10 leaves per plant) only in treatment Cd5–Ni10 (7 leaves per plant). Determined values of the fresh and dry weights of shoots for Cd2.5–Ni5 and Cd5–Ni10 treatments were between the corresponding values for CdTREATMENT and NiTREATMENT (Fig. 2); mean fresh and dry weights of the control plants (Cd0–Ni0) were 87.6 and 10.4 g, respectively; in Cd2.5–Ni5 mean shoot fresh weight was 97.9 g and mean dry weight was 12.7 g (not significantly different, p o0.05), while corresponding values for Cd5–Ni10 treatment differed significantly (po0.05) when compared to the control and were up to 17.2 and 2.2 g, respectively. Generally, plant growth was significantly reduced only in highly treated plants (Cd5–Ni10).

3.3. Cd and Ni uptake The amounts of Cd and Ni accumulated in C. cardunculus plants increased with increasing bioavailable metal concentrations in the soil for all treatments (Table 3). For both metals, the analysis of variance gave p¼ 0.0001 for the factor ‘‘bioavailable metal concentration in soil", indicating that soil concentrations have a statistically significant effect on metal accumulation in cardoon

Table 2 Cadmium and nickel DTPA-extractable concentrations in soil (mg kg  1) (mean values 7 SD, n ¼3, p o 0.05). Group A/CdTREATMENT Cd0 0.2 7 0.0a

Cd0.5 8.7 71.8a

Cd5 93.67 10.4b

Cd10 246.7 7 44.1c

Group B/NiTREATMENT Ni0 0.6 7 0.0a

Ni1 3.8 70.7a

Ni10 40.57 11.7b

Ni20 61.1 714.7b

Group C/Cd–NiTREATMENT Cd0–Ni0 Cd Ni 0.2 7 0.0a 0.57 0.0A

Cd0.25–Ni0.5 Cd 10.0 71.3a

Ni 3.4 70.9A

Cd2.5–Ni5 Cd 70.77 15.0a

Note: Different lower case and capital letters show significant differences within treatments of each group.

Ni 28.37 9.5AB

Cd5–Ni10 Cd 157.0 734.0b

Ni 43.37 9.6B

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CdTREATMENT

CdTREATMENT

80

Plant height (cm)

60

Cd 0 Cd 0.5 Cd 5 Cd 10

10

Number of leaves

70

12

50 40 30

Cd 0 Cd 0.5 Cd 5 Cd 10

8

6

4

20 2

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20Feb07

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6Mar07 13Mar07

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12 Ni0 Ni1 Ni10 Ni20

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NiTREATMENT

NiTREATMENT 80 70

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Ni 0 Ni 1 Ni 10 Ni 20

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27Feb07

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20Feb07

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Cd-NiTREATMENT

27Mar07

Cd-NiTREATMENT

Cd0 -Ni0 Cd0.25 -Ni0.5 Cd2.5 -Ni5 Cd5 -Ni10

10 a

50

a a

a

b

a a b

40 30

Number of leaves

Plant height (cm)

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Cd0 -Ni0 Cd0.25 -Ni0.5 Cd2.5 -Ni5 Cd5 -Ni10

a a b a

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20 2

10 0

20Feb07

6Mar07 27Feb07

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0

20Mar07 27Mar07

20Feb07

6Mar07 27Feb07

13Mar07

20Mar07 27Mar07

Fig. 1. Effects of single Cd (A1, A2), single Ni (B1, B2), and binary Cd and Ni (C1, C2) treatments on cardoon height and number of leaves (mean values7 SD, n¼ 3, po 0.05).

tissues. For the factor ‘‘metal species’’ the p-value was 0.0095, indicating that Cd and Ni concentrations in cardoon shoots and roots for all treatments were metal-dependent.

In CdTREATMENT, metal concentrations in shoots and roots were elevated and, in Cd10, were up to 169.3 and 33.2 mg kg  1, respectively, suggesting considerable bioaccumulation of Cd from

E.G. Papazoglou / Ecotoxicology and Environmental Safety 74 (2011) 195–202

the soil. In Cd–NiTREATMENT, cadmium concentrations in plant tissues were generally lower in low- and medium-treated plants but, in Cd5–Ni10 treatment, they were higher and reached 235.0 and 70.4 mg kg  1, respectively. Moreover, in treatment with Cd alone, metal concentrations in soil were generally higher than concentrations in shoots (almost 2 and 1.5 times those in Cd5 and Cd10, respectively); in combined Cd and Ni treatment, a reverse result was observed, since shoot Cd concentration was 1.5 higher than cadmium concentration in soil, indicating a possible enhancing effect of nickel. This can be confirmed also by the results presented in Fig. 3, where the accumulative characteristics of C. cardunculus are shown. In Cd5 treatment, cadmium concentration in shoots was 50.3 mg kg  1 (Fig. 3(A1)), while in Cd5–Ni10 treatment (where the same amount (5 g) of Cd salt was added to the soil, combined with 10 g Ni salt), shoot Cd

140

FW Cd

DW Cd

FW Ni

DW Ni

FW Cd-Ni

DW Cd-Ni

Weights (g)

120 100 80 60 40 20 0 CONTROL

LOW MEDIUM Treatment

HIGH

Fig. 2. Cardoon fresh (FW) and dry (DW) weights of the aboveground biomass, as affected by single and binary Cd and Ni treatments (mean values 7 SD, n¼3, p o0.05). LOW refers to the treatments Cd0.5, Ni1, and Cd0.25–Ni0.5. MEDIUM refers to the treatments Cd5, Ni10, and Cd2.5–Ni5. HIGH refers to the treatments Cd10, Ni20, and Cd5–Ni10.

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concentration was almost five times higher, namely 235.0 mg kg  1 (Fig. 3(B1)). Cadmium concentrations in roots were generally lower than in shoots for both Cd alone and combined Cd and Ni treatments and the corresponding translocation factors, calculated as the ratio of concentration in shoots to that of roots, were higher than 1 (Table 3). In NiTREATMENT, metal concentration in shoots and roots of the treated C. cardunculus plants were higher than those in control plants and, in Ni10, they reached 342.3 and 52.3 mg kg  1, respectively. For highly treated plants (Ni20) dried and Ni concentration were not possible to measure. In the binary Cd–NiTREATMENT, nickel concentrations were increased by the treatments and they were up to 440.7 and 133.4 mg kg  1 for shoots and roots, respectively. Both in Ni alone and in combined Cd and Ni treatments, root concentrations were lower than in shoots and the translocation factors were higher than 1 (Table 3). Metal uptake by cardoons was calculated by multiplying plant biomass by the metal concentration in the plant. Large Cd uptake was generally associated with higher bioavailable Cd in soil. The amounts of Cd taken up by plants were higher in CdTREATMENT (Fig. 3(A2)) than in Cd–NiTREATMENT (Fig. 3(B2)). Lower Cd uptake in the combined metal treatments was strongly correlated with biomass reduction (p o0.01), since the presence of Ni affected the growth of the plants. Therefore, even though Cd concentration in shoots in Cd5–Ni10 was approx. five times higher than in Cd5, the corresponding final uptakes were similar, 0.5 mg plant  1 for Cd5–Ni10 and 0.6 mg plant  1 for Cd5. In NiTREATMENT, the inhibition of plant growth significantly affected Ni uptake (p o0.05): from Ni1 to Ni10 treatments, plant biomass was reduced by approx. 70% (Fig. 2), while shoot Ni concentration was increased by 37 times (Table 3, Fig. 4(A1)), resulting in uptake increased by 12 times (Fig. 4(A2)), namely, from 0.13 (Ni1) to 1.51 (Ni10) mg plant  1. Similar results were observed in Cd–NiTREATMENT, where, despite the elevated shoot concentrations in highly treated plants (440.7 mg kg  1 in Table 3 and Fig. 4(B1)), the uptake was not accordingly increased due to the reduction of biomass (Fig. 4(B2)).

4. Discussion Table 3 Cadmium and nickel concentrations (mg kg  1) in cardoon shoots and roots and translocation factor (mean values 7SD, n¼ 3, p o0.05). Shoot

Root

Translocation factor

Cadmium treatment Cd0 0.47 0.1a Cd0.5 13.6 7 1.9a Cd5 50.3 7 3.1b Cd10 169.3 7 21.9c

0.5 70.1a 2.5 71.1a 4.6 70.7a 33.2 75.4b

0.8 5.4 10.9 5.1

Nickel treatment Ni0 Ni1 Ni10 Ni20

1.3 7 0.1a 9.3 7 1.9a 342.3 7 82.7b –

1.6 70.2a 7.8 72.0b 52.3 73.7c –

0.8 1.2 6.5 –

Cadmium in Cd–Ni treatment 0.57 0.1a Cd0–Ni0 Cd0.25–Ni0.5 8.1 7 1.2a Cd2.5–Ni5 13.4 7 2.9a Cd5–Ni10 235.0 7 47.9b

0.8 70.3a 1.9 70.5a 2.6 70.4a 70.4 78.1b

0.6 4.3 5.2 3.3

Nickel in Cd–Ni treatment 2.8 7 0.9a Cd0–Ni0 Cd0.25–Ni0.5 5.7 7 1.0a Cd2.5–Ni5 37.1 7 7.9a Cd5–Ni10 440.7 7 56.3b

1.1 70.3a 3.1 70.5a 20.5 71.9a 133.4 746.7b

2.5 1.8 1.8 3.4

Note: Different letters show significant differences among treatments of each group.

In this work, the effects of Cd and Ni alone and combined on cardoons were investigated, since it is known from several studies that the presence of one metal influences the uptake of another metal (Peralta-Videa et al., 2002; An et al., 2004; Ouzounidou et al., 2006; Rooney et al., 2007). Cadmium and nickel were added as soluble salts to the pots and their solubility was higher than for long-term contaminated soil. Plant availability was probably very high, but this experiment aimed to investigate, among other matters, the tolerance of cardoons for Cd and Ni and to highlight the response of plants in control conditions. Cadmium and nickel are among the most toxic heavy metals and can exert primary and secondary effects on plants at different integration levels (molecular, cellular, and tissue) (Ernst et al., 1992). Cadmium toxicity is largely determined by its affinity for sulfhydryl groups in proteins, peptides, and amino acids, while Ni toxicity is largely based on a high affinity for oxygen- and nitrogen-containing ligands (Verkleij et al., 2009). Growth inhibition and yield depression, stunted roots, red-brownish coloration, chlorosis, and necrosis are the general toxicity symptoms of Cd and Ni at the whole plant level (Kabata-Pendias and Pendias, 2001; Das et al., 1997; Clemens, 2006). The results of this experiment showed that growth and development of C. cardunculus under treatment with Cd alone was normal and no phytotoxicity symptoms appeared. In fact, it was not possible to distinguish the treated from the control

E.G. Papazoglou / Ecotoxicology and Environmental Safety 74 (2011) 195–202

Cd concentration (mg kg-1)

Cd concentration (mg kg-1)

200

250 200 150 100 50 0 Cd0

Cd0.5

Cd5

250 200 150 100 50 0 Cd0-Ni0

Cd10

Cd2.5-Ni5

Cd5-Ni10

2

Cd uptake (mg pot-1)

2

Cd uptake (mg pot-1)

Cd0.25-Ni0.5

Cd-NiTREATMENT

CdTREATMENT

1.5 1 0.5 0

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CdTREATMENT

Cd0.25-Ni0.5

Cd2.5-Ni5

Cd5-Ni10

Cd-NiTREATMENT

Ni concentration (mg kg-1)

Ni concentration (mg kg-1)

Fig. 3. Cadmium accumulative characteristics of Cynara cardunculus grown under treatment with Cd alone and binary Cd and Ni: (i) Cd concentration in shoots (A1, B1) and (ii) Cd uptake into shoots (A2, B2).

400 300 200 100 0 Ni0

Ni1

Ni10

400 300 200 100 0

Ni20

Cd0-Ni0

Cd2.5-Ni5

Cd5-Ni10

Cd-NiTREATMENT

NiTREATMENT 2

Ni uptake (mg pot-1)

2

Ni uptake (mg pot-1)

Cd0.25-Ni0.5

1.5 1 0.5

1. 5 1 0. 5 0

0 Ni0

Ni1

Ni10

Ni20

NiTREATMENT

Cd0-Ni0

Cd0.25-Ni0.5

Cd2.5-Ni5

Cd5-Ni10

Cd-NiTREATMENT

Fig. 4. Nickel accumulative characteristics of Cynara cardunculus grown under treatment with Ni alone and binary Cd and Ni: (i) Ni concentration in shoots (A1, B1) and (ii) Ni uptake into shoots (A2, B2).

plants. All measured growth parameters remained unaffected even at a bioavailable soil concentration of 246.7 mg kg  1 (Cd10), indicating tolerance characteristics.

In treatment with Ni alone, measured growth parameters of cardoons grown at a bioavailable soil concentration of 3.8 mg kg  1 (Ni1) showed a tendency to increase (not significantly); this

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trend may have resulted from a stimulating effect of Ni on plant growth. Similar effects of low Ni concentrations have been observed in wheat and upland cotton (Dalton et al., 1988), maize and white clover (Yang et al., 1996), fenugreek (Parida et al., 2003), and tomatoes (Rooney et al., 2007). The significant decrease of measured parameters for Ni10 treatment, with a soil Ni concentration of 40.5 mg kg  1, seemed to be the result of a depressing effect of the elevated levels of Ni. Similar depressing effects were observed in fenugreek (Parida et al., 2003) and wheat (Ouzounidou et al., 2006). For Ni10 treatment, the growth of the plants declined and Ni toxicity was manifested in a significant reduction of plant height, number of leaves, and fresh and dry biomass production. Growth inhibition in the presence of Ni was also reported for wheat (Ouzounidou et al., 2006), fenugreek (Parida et al., 2003), radishes (Simon et al., 2000), barley and tomatoes (Rooney et al., 2007), and sunflowers (Zornoza et al., 1999). Highly treated plants (Ni20), grown at a bioavailable Ni concentration of 61.1 mg kg  1, were totally dried. Root growth was also seriously restricted in plants suffering from Ni toxicity (data not shown). Under combined Cd and Ni treatment, there was evidence of an antagonistic effect upon cardoon plants between these metals, since the effects on all measured growth parameters were lower than the sum of the effects of the metals when applied individually. Thus, the presence of Cd decreased the effects of Ni on plant growth. Such antagonistic effects have been found in barley for Cu and Zn (Beckett and Davis, 1978), in lettuce, spinach, wheat, endive, and maize for Zn and Cd (Smilde et al., 1992), in railway beggartick for Cd and As (Sun et al., 2009), and in cucumbers for Cu, Cd, and Pb (An et al., 2004). A plant species can be defined as a metal accumulator if it has the following characteristics (Chaney et al., 1997; Reeves and Baker, 2000; Wei et al., 2008): (i) It should have high tolerance for toxic contaminants, namely being able to tolerate high plant-metal and soil-metal concentrations and to maintain high metal levels in tissues across a wide range of metal concentrations in the soil. (ii) It should have the ability to translocate the metal from roots to shoots at high levels and the translocation factor should be greater than 1. (iii) It should have bioaccumulation factor, the metal concentration ratio of plant to soil, greater than 1. Moreover, if concentrations in shoots (stems or leaves) exceed the critical levels defined by Baker and Brooks (1989) of 100 mg kg  1 for Cd and of 1000 mg kg  1 for Ni, then the plant species could be characterized as a Cd or Ni hyperaccumulator. In the present study, Cd concentrations in plants in treatments with Cd alone and combined Cd and Ni were higher than for control plants at a wide range of soil-metal concentrations; in highly treated plants shoot concentrations were up to 169.3 (Cd10) and 235.0 mg kg  1 (Cd5–Ni10), respectively, indicating that C. cardunculus had a strong capability to endure the toxicity of cadmium. Similarly high values of shoot Cd accumulation in cardoon were reported by Hernandez-Allica et al. (2008). Cadmium accumulation in aboveground biomass of lowand medium-treated cardoon, in both CdTREATMENT and Cd– NiTREATMENT, ranged from 8.1 to 50.3 mg kg  1. They did not exceed the critical level for a Cd hyperaccumulator, but they exceeded the concentrations of 0.01–0.2 mg kg  1 found in most plants (Kabata-Pendias and Mukherjee, 2007). Only for highly treated plants (Cd10 and Cd5–Ni10) did measured concentrations exceed the critical level of 100 mg kg  1 for a Cd hyperaccumulator. Moreover, the translocation factors determined were higher than 1 for treatment with both Cd alone and combined Cd and Ni,

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even though, in most plant species, Cd accumulates mainly in the roots. Normally, Cd root concentration is 10 or more times higher than shoot concentration (Chaney et al., 1997). Significant immobilization of Cd in the roots was reported for Thlaspi caerulescens (Lombi et al., 2000), Arabidopsis halleri (Kupper et al., 2000), Zea mays, Triticum aestivum and Sorghum bicolor (An, 2004), Agrostis tenuis (Dahmani-Muller et al., 2000), and Allium sativum (Jiang et al., 2001). Therefore, cardoon has the ability to translocate Cd from root to shoot at high rates, suggesting that it might have more efficient transport of Cd from roots to shoots than other plants. These results are in accordance with the findings of Hernandez-Allica et al. (2008), who indicate that cardoon has a very efficient Cd translocation system from root to shoot. According to current knowledge, plants do not possess specific transporters for nonessential metals such as cadmium, and its transport across a membrane is mediated by systems for essential cations. According to available data, Cd is transported by Fe, Zn, and Ca transporters or channels of broad substrate specificity (Verkleij et al., 2009). Hernandez-Allica et al. (2007) reported nonselective, apoplastic uptake of metal (Pb, Zn, Cd)–EDTA complexes by cardoon roots. Even at high metal chelate concentrations, the water status and transpiration capacity of cardoons remained effective for allowing translocation of metal chelate complexes to the shoots. Total soil concentrations of a metal are needed for the determination of the bioaccumulation factor. It is a common conception nowadays that total concentrations are not a representative indicator of bioavailability, or a good tool for potential risk assessment, due to the different and complex distribution patterns of metals among various chemical species or solid phases. In this experimental work, only the bioavailable fractions (DTPA extractable) were determined and, thus, the bioaccumulation factor could not be evaluated. Nevertheless, since bioavailable soil concentrations are generally higher than shoot concentrations (apart from Cd5–Ni10), it could be speculated that the bioaccumulation factor should be higher than 1. Therefore, summarizing the above-mentioned results, it could be said that C. cardunculus showed Cd-accumulator properties. In treatments with Ni alone and combined Cd and Ni, plants did not show tolerance for high plant-metal and soil-metal concentrations. In NiTREATMENT, concentrations in shoots were 9.3 (Ni1) and 342.3 (Ni10) mg kg  1, the latter resulting in substantial inhibition of plant growth. In Cd–NiTREATMENT, nickel concentrations ranged from 5.7 to 440.7 mg kg  1, causing significant reduction of plant growth only in highly treated plants. The measured Ni concentrations in shoots, in both NiTREATMENT and Cd–NiTREATMENT, exceeded the concentrations of 0.1–5 mg kg  1 found in most plants (Kabata-Pendias and Mukherjee, 2007) and were well below the critical level of 1000 mg kg  1 for a Ni hyperaccumulator (Baker and Brooks, 1989). Kabata-Pendias and Mukherjee (2007) reported that Ni is a metal very easily extracted from soils and very mobile within plants. The determined Ni translocation factors in both single Ni and binary Cd–Ni treatment were higher than 1. Summarizing the results for C. cardunculus responses to Ni, it could be said that cardoon cannot be considered a Ni accumulator. Investigation under field conditions is needed to further assess the cadmium and nickel phytoextraction potential of C. cardunculus.

5. Conclusions Based on the findings of this experimental work, it is concluded that C. cardunculus grown under single and binary cadmium and nickel treatment conditions can be considered

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initially as a Cd accumulator, since (i) it demonstrated a high tolerance for elevated Cd soil concentrations, (ii) it accumulated the metal in plant tissues, and (iii) the translocation factor was higher than one. Plant growth remained unaffected after Cd uptake and transport into cardoon tissues; thus this energy crop could be a promising candidate for Cd phytoextraction and, at the same time, for biofuel production. In contrast, cardoon plants could not tolerate elevated Ni soil concentrations and, even though metal concentrations in plant tissues were elevated and the translocation factor was higher than one, they could not be considered Ni accumulators and could not be used for Ni phytoremediation. An antagonistic effect between Cd and Ni upon cardoon was observed. Finally, the results of this work indicate that cardoon exposed to Cd and Ni in its growth medium could be dangerous as a carrier of these metals in the food chain.

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