Metal accumulation, growth, antioxidants and oil yield of Brassica juncea L. exposed to different metals

Ecotoxicology and Environmental Safety 73 (2010) 1352–1361

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Metal accumulation, growth, antioxidants and oil yield of Brassica juncea L. exposed to different metals Sarita Sinha n, Geetgovind Sinam 1, Rohit Kumar Mishra 1, Shekhar Mallick 1 Ecotoxicology and Bioremediation, National Botanical Research Institute (Council of Scientific and Industrial Research), Lucknow-226 001, India

a r t i c l e in f o

a b s t r a c t

Article history: Received 1 February 2010 Received in revised form 22 June 2010 Accepted 24 June 2010

In agricultural fields, heavy metal contamination is responsible for limiting the crop productivity and quality. This study reports that the plants of Brassica juncea L. cv. Pusa bold grown on contaminated substrates [Cu, Cr(VI), As(III), As(V)] under simulated field conditions have shown translocation of metals to the upper part and its sequestration in the leaves without significantly affecting on oil yield, except for Cr and higher concentration of As(V), compared to control. Decrease in the oil content in As(V) treated plants was observed in a dose dependent manner; however, maximum decrease was recorded in Cr treated plants. Among all the metal treatments, Cr was the most toxic as evident from the decrease in oil content, growth parameters and antioxidants. The accumulation of metals was below the detection limit in the seeds grown on 10 and 30 mg kg  1As(III) and Cr(VI); 10 mg kg  1 As(V)) and thus can be recommended only for oil cultivation. & 2010 Elsevier Inc. All rights reserved.

Keywords: Brassica juncea Cr(VI) As(V) As(III) Cu Oil content Antioxidants Growth parameters Accumulation

1. Introduction Unplanned disposal of sewage and industrial effluents to land has been implicated as the major source of heavy metal contamination of agricultural fields and eventually in the plants growing therein. However, little is known about the level of translocation of metals to the edible parts of the plants grown on contaminated soil, which is aimed at producing socio-economic benefits; however, it is not safe and may not be sustainable in the long term (Sinha et al., 2006; Mapanda et al., 2007). Most species of Brassicaceae including Indian mustard (Brassica juncea) has been reported to be tolerant towards heavy metal and, therefore, advocated as a species suitable for the phytoextraction of heavy metals from contaminated soil (Qadir et al., 2004, Salido et al., 2003). B. juncea is an important oil bearing crop that contributes to the third largest edible oil production in the world after soy and palm oil. At a production level of 13–14 million tons, it accounts for about 12% of the world’s total edible oil production. India annually produces 4–7, 1–2 and 2–4 million tons of seed, oil and mustard oil cake, respectively. Owing to the importance of the crop and its tolerance towards heavy metals, many studies have n

Corresponding author. E-mail addresses: [email protected], [email protected] (S. Sinha). 1 Contributed equally to this work. 0147-6513/$ - see front matter & 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2010.06.025

been devoted to study the translocation of toxic metals to the various parts of Brassica sp. in different varieties and its tolerance mechanism (Pickering et al., 2000; Salido et al., 2003; Han et al., 2004; Qadir et al., 2004; Pandey et al., 2005; Singh and Sinha, 2005; Gupta et al., 2009; Khan et al., 2009; Sinha et al., 2009). However, many of such studies were conducted for a short span of time and in hydroponics under laboratory conditions. There is a need to investigate the response of the plants towards stress under natural conditions, where the plants are exposed to various factors along with metals. Thus, the study conducted in solution culture is not appropriate to extrapolate for field conditions, as the bioavailability of metals are guided by several physicochemical properties of the soil. To the best of our knowledge this is one of the first reports where a comparative account of the bioavailability and subsequent fate of metals (Cu, As(III), As(V) and Cr(VI)) in B. juncea has been made under natural condition in order to assess the translocation of the metals to the seed, effect on the metabolism and its oil production. Among these metals, Cr (Shanker et al., 2005) and As (Chakraborti et al., 2003) are nonessential metals which are toxic and have been reported for their carcinogenic properties. Arsenic enters the environment through geological and anthropogenic activities such as smelting operations, fossil fuel combustion and widespread groundwater pollution. Arsenic contaminated soils, sediments and water supplies are major sources of food chain contamination, eventually endangering human health. Due to its

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carcinogenic property, it has raised serious concern especially in India and Bangladesh (Chakraborti et al., 2003). Arsenate [As(V)] is the predominant As species in aerobic soils, whereas arsenite [As(III)] dominates under anaerobic conditions. Arsenate acts as a phosphate analog and can disrupt phosphate metabolism, whereas As(III) reacts with sulfhydryl groups of enzymes and tissue proteins, leading to inhibition of cellular functions (Meharg and Hartley-Whitaker, 2002). In industries, Cr (VI) compounds are used for tanning of hides and metal plating. These anthropogenic activities have led to the widespread contamination of Cr in the environment and have increased its bioavailability and biomobility (Singh et al., 2004; Sinha et al., 2006). Evidence also indicates that chromosomal abnormalities and genomic instability are possibly involved in the induction of cancer by Cr(VI). Copper is an essential micronutrient; however, it is toxic at higher concentrations. Recently, there have been reports about heavy metal pollution in various parts of mustard growing on contaminated soil (Sinha et al., 2006; Mapanda et al., 2007). Eventually, these metals (Cu, As, Cr) are redox metals, which accumulate in tissues and cause toxicity, both directly by damaging cell structure and indirectly via replacement of other essential nutrients. One of the major consequences of redox metals toxicity is oxidative stress mediated by increased levels of reactive oxygen species (ROS). Intracellular Cr(VI) mediates Fenton-like reaction and produces ROS, which are responsible for the toxicity and genotoxicity of Cr(VI) (Shanker et al., 2005). In case of As, ROSs may be generated through the conversion of As(V) to As(III), which may result in damage to DNA, proteins and lipids (Mascher et al., 2002). Arsenic can also block enzymatic centers in its trivalent form by binding to free sulfhydryl groups; however, As(V) is structurally very similar to phosphate, which can replace in different reactions, for example in the synthesis of ATP from ADP. The deleterious effects resulting from the cellular oxidative state may be alleviated by the enzymatic and nonenzymatic antioxidants of the plant. Exposure to toxic metals leads to loss of agricultural produce; besides many biosynthetic pathways are affected, along with alteration in nutrition and contamination of the edible produce. In view of this, this study was undertaken to assess accumulation of metals in the different parts of B. juncea cv. Pusa bold, growth and antioxidative responses on exposure to Cu at two levels (30, 50 mg g  1) and Cr (VI), As (III) and As (V) at three levels (10, 30, 50 mg g  1) under simulated natural field conditions. In addition, the effect on the oil content of the seeds has been made. The seeds of Brassica sp. are used throughout the Indian subcontinent as a food source; therefore, the transport of metal to seeds was assessed.

2. Materials and methods

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two treatments (30 and 50 mg kg  1 dw) of Cu as CuSO4. For convenience, the different concentrations (10, 30, 50 mg kg  1 dw) of different metals (Cu, As (III), As (V), Cr (V)) were abbreviated as Cu(30) and Cu(50), As(III)10, As(III)30, As(III) 50 and As(V)10, As(V)30, As(V)50 and Cr(10), Cr(30) and Cr(50). The soil in the pots were tilled for aeration and weeded for proper growth of the plants. Care was taken to avoid leaching from the pots and watered with about 500 ml of water. The plants were harvested after 15 d (first harvest) and 30 d (second harvest) of treatment. The plants were grown till maturity of the seeds for the analysis of metal level, seed weight and oil content. After each harvest, the soil adhered to the plant roots were removed, by tapping against a hard surface and followed by washing with tap water and finally rinsing with RO water. The leaves of the plants were washed with RO water and blotted dry for biochemical analysis.

2.2. Growth and biochemical parameters and oil content Fresh weight (FW) of root and shoot (stem and leaves), root and shoot lengths were taken immediately after harvesting. Chlorophyll content in the leaves of the plant (100 mg) was estimated by the method of Arnon (1949). Protein content in the leaves of the plants was estimated using BSA as a standard protein (Lowry et al., 1951). Lipid peroxidation in the plant tissue was measured in terms of malondialdehyde (MDA) content, determined by thiobarbituric acid (TBA) reaction (Heath and Packer, 1968). Non-protein thiol (acid soluble thiol) content was measured (Ellman, 1959) using Ellman’s reagent (5,50 -dithiobis 2-nitrobenzoic acid). The activity of superoxide dismutase (SOD) was measured in the leaves of the plant by the method of Nishikimi and Rao (1972), using the enzyme extract. Inhibition of 50% shows the expression of one unit (1 U) enzyme. A reaction mixture devoid of enzymes served as blank. The ascorbate peroxidase (APX) activity was measured by the method of Nakano and Asada (1981), estimating the rate of ascorbate oxidation at 290 nm. The activity was calculated using its extinction coefficient (2.8 mM  1 cm  1). Guiacol peroxidase (GPX) was measured in leaves, following the method of Kato and Shimizu (1987). Activity was calculated using the extinction coefficient (26.6 mM  1 cm  1 at 470 nm) for oxidized tetraguiacol polymer. One unit of peroxidase activity was defined as the calculated consumption of 1 mol of H2O2 min  1 g  1 fresh weight. Crushed seeds (5 g) were extracted with hexane (35 ml) and solvent was evaporated after filtration. The weight of the oil was determined by weighing till constant weight.

2.3. Metal accumulation Oven-dried tissue samples (leaves, stem, roots and seeds) were ground and digested in HNO3 (70%) on a hot plate to dryness and the volume was made up using MilliQ water. The digested solution was filtered across 42 No. Whatman filter paper before analyzing on AAS (GBC S Avanta). Analyses of As (III, V) in seeds of the plants were performed on AAS fitted with a hydride generator using NaBH4 and HCl.

2.4. Bioavailable metals EDTA extractable fraction was obtained by mechanical shaking of sample (5 g) with 0.05 M Na-EDTA for 1 h (Quevauviller et al., 1997). For bioavailable As, the As fractionation procedure of Onken and Adriano (1997) was used where 2 g of soil was shaken with 40 ml of 1 M NH4Cl for 0.5 h.

2.1. Plant material and experimental design In India, B. juncea (Indian mustard) belonging to Brassicaceae family is cultivated widely as a major oilseed crop. Seeds of B. juncea L. were purchased from commercial seed supplier. Seeds were subjected to 30% H2O2 treatment for 10 min, thereafter washed thrice with MilliQ water and left in a beaker for imbibing water for 24 h in dark. The imbibed seeds (10 nos.) were sown in an earthen pot (23 cm in diameter) to a depth of 0.5 cm, containing garden soil (9 kg) for germination. The plants were allowed to grow in the institute’s experimental garden under natural sunlight and temperature (15–25 1C) in a randomized block design with four replicates (separate pot). The pots were watered daily till seed germination and thinned out to retain five uniform seedlings after 10 d of germination. The plants (5 nos.) were further allowed to grow for 20 d. After 30 d of germination, the plants were treated with different concentrations. Both metals and metalloid have been used in the study; however, for convenience, the word metal has been used wherever there is a mention of all the elements. Three treatments (10, 30 and 50 mg kg  1 dw) of the different metals, i.e. As(III), As(V) and Cr, were made using NaAsO2, Na2HAsO4  7 H2O and K2Cr2O7, respectively, and

2.5. Quality control and statistical analysis Analytical data quality of metals was ensured through repeated analysis of sewage sludge samples of Resource Technology Corporation (EPA Certified Reference material) (Catalog No. CRM029-050; Lot No. JC029a) and the measured results were recorded within the range of reference values (Cu and Cr¼ 72.9%; As and Zn ¼ 7 2.2%; Fe ¼ 7 5%; Mn ¼ 7 1%). For plants, spiked samples of the plants were used in order to check the precision and accuracy of metal estimation. The blanks were run in triplicate to check the precision of the method with each set of samples. The experiment was set up as randomized block design. To confirm the variability of data and validity of results, analysis of variance (ANOVA) was conducted. To determine whether differences between treatments were significant as compared to control, Duncan multiple range test (DMRT) was determined. In tables and figures, the values are marked by an asterisk for the significance level as compared to the control.

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3. Results 3.1. Bioavailable metals The total metal present in the soil is not available to the plants growing therein. Thus, EDTA and DTPA extractable metals have been widely used as an indicator of bioavailable metals. It was observed that Cr was not bioavailable in DTPA extractant (data not shown). Among these two extractants, EDTA (Fig. 1) has shown better extractability for all the tested metals after 30 d. In EDTA extraction, the level of Mn and Fe was quite consistent between different metal treatments and C, which ranged between 60.78 and 61.99 mg kg  1 dw Mn and 132.73 and 140.84 mg kg  1 dw Fe. The level of bioavailable Cu, Cr, As(III) and As(V) increased with increase in soil metal treatments. The level of bioavailable As(III) was found to increase from 7.81 to 12.81 mg kg  1 dw and As(V) from 11.14 to 15.39 mg kg  1 dw, as the level of spiked As(III) and As(V)) increased in the soil from 10 to 50 mg kg  1 dw. Thus, it was observed that As(V) was more bioavailable than As (III); however, a similar level of As was spiked to the soil. 3.2. Accumulation of metals in the plant tissue The accumulation of the metals in different parts of the plant after 30 d of growth is shown in Table 1. The accumulation of Cu increased in different parts of the plant in a dose dependent manner. In As treated plants, the accumulation of As in the roots was found to be more than in upper parts. With increase in As concentration, the translocation of As(III) from root to upper part did not follow any trend; in contrast, an increase was observed in the translocation of As(V). Maximum accumulation (mg g  1 dw) of As(III) and As(V) in the roots was recorded as 438 and 617, in stems 234 and 430, in leaves 184 and 334, respectively, in the plants grown on As50. Thus, the comparison of As accumulation in the plants showed that more accumulation was found in As(V) treated plants than As(III). In Cr treated plants, the accumulation in all parts of the plants increased in a dose dependent manner. In general, the uptake of Cr by the plants is relatively low in comparison with other metals. Chromium accumulation (mg g  1 dw) in the leaves was observed to be 26 at Cr 10, which

increased to 63 at Cr 50. The accumulation of Cr was found to be more in roots than in upper parts, as the trend was observed in many other plants species. In roots of all the treated plants, Mn accumulation was observed to increase except for As (III)30, As(V)50 and Cr10 with maximum increase of 104% in As(III)50 as compared to C. In leaves, the accumulation of Mn increased in As(V) treated plants and in Cr30 and Cr50; however, decrease was observed in As(III) as compared to C. As compared to C, Zn accumulation in the roots increased in all the treatments with maximum increase of 214% in As(III)10 and 209% in As(V)10. The decrease was observed in Zn accumulation along with increase of As concentration in the soil. The increase in Zn accumulation was observed in As treated leaves as compared to C; however, no such trend was observed in Cu and Cr treated leaves. The accumulation of Zn decreased along with increase of As concentration in the soil. Fe accumulation in the roots of As treated plants increased with increase in the dose of metal in the soil except for As(III)50. The accumulation of Fe in the roots of Cr treated plants increased more than three times (from 247 to 809 mg g  1 dw) and also in leaves (636.24–766.9 mg g  1 dw) along with the increase of Cr concentration in the soil. Similarly, Fe accumulation increased five-fold (179.43–534.23) in the leaves along with increase in As(V) concentration in soil. Cu accumulation in the root was found to increase in all the treatments, except for Cr10 and Cr50 as compared to C. The accumulation of Cu in the roots at treatment variant Cu50 increased almost four times as compared to C. Two-fold increase in Cu accumulation was observed in Cr treated leaves (5.14 in Cr10 to 10.37 in Cr50). On the contrary, decrease in Cu accumulation in the leaves was observed with increase in As(V) treatments, whereas no change was observed among the As(III) treated plants. 3.3. Metal accumulation in seeds, seed weight and oil content There was not much difference in the accumulation of essential metal (Cu, Fe, Mn, Zn) content among all the treatments (Table 2). Interestingly, no difference in the level of Cu in the seeds was observed in metal spiked soil as compared to C. At As concentrations [As(III)10, As(III)30 and As(V)10], the accumulation of the metal in seeds was found to be below the detection limit. Similarly, least

Fig. 1. Level (mg kg  1) of extracted metals (As with NH4Cl) and other metals (with EDTA) from soil spiked with metal after harvesting of the plants.

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Table 1 Accumulation of metals in different parts of the plants after 60 d of growth. All the values are mean of four replicates 7SD. Treatments/plant’s part

CR CS CL Cu30R Cu30S Cu30L Cu50R Cu50S Cu50L As(III)10R As(III)10S As(III)10L As(III)30R As(III)30S As(III)30L As(III)50R As(III)50S As(III)50L As(V)10R As(V)10S As(V)10L As(V)30R As(V)30S As(V)30L As(V)50R As(V)50S As(V)50L Cr10R Cr10S Cr10L Cr30R Cr30S Cr30L Cr50R Cr50S Cr50L

Accumulation of metals (mg g  1 dw) Mn

Zn

Fe

Cu

As

16.037 0.33 12.15 7 1.49 31.14 7 1.68 21.507 2.99 19.11 7 1.57 47.37 7 8.34 17.78 7 0.92 10.677 1.67 34.34 7 7.31 20.327 0.61 7.147 0.43 18.88 7 1.38 14.13 7 1.23 11.55 7 1.26 22.16 7 3.40 32.68 7 3.79 14.72 7 3.22 21.76 7 3.95 32.33 7 8.95 11.53 7 1.52 37.61 7 10.45 18.75 7 3.17 6.667 1.11 47.607 10.38 14.027 1.35 9.317 1.47 45.22 7 11.84 14.87 7 4.83 17.53 7 1.03 28.11 7 8.02 22.32 7 1.30 12.307 1.62 48.907 11.10 21.89 7 1.25 16.77 7 4.84 47.807 12.03

33.35 79.39 23.74 75.09 31.01 71.77 37.80 712.38 23.92 71.08 32.12 74.46 56.41 718.29 18.24 71.27 17.74 73.69 104.79 730.00 39.86 73.83 66.03 723.57 71.64 74.15 44.26 78.48 46.35 74.68 74.88 74.00 47.77 72.42 47.17 73.82 103.17 716.70 44.19 71.56 49.02 725.91 99.69 713.01 31.48 72.77 37.09 714.05 59.18 74.07 24.02 71.33 34.78 74.77 47.70 70.77 31.40 71.16 23.32 74.70 50.97 77.09 21.57 71.65 37.01 71.53 38.23 74.57 16.60 71.03 28.47 74.44

374.987 68.76 161.827 40.40 744.657 153.3 265.837 23.65 146.287 13.56 320.32 7 31.40 368.90 7 88.62 116.237 28.90 236.667 25.11 305.20 7 56.98 107.27 7 14.21 515.647 91.05 399.50 7 45.96 143.537 30.69 273.367 48.88 327.50 7 24.75 161.247 1.36 223.367 28.88 226.757 43.70 146.00 7 28.39 179.437 28.90 367.80 7 25.17 78.25 7 15.84 428.00 7 66.78 390.83 7 94.14 94.13 7 3.79 534.237 84.24 247.137 45.22 213.467 14.47 636.247 51.25 405.21 7 42.51 94.96 7 6.50 758.09 7 44.69 809.34 7 31.59 173.01 7 14.67 766.90 7 72.07

5.207 0.27 7.087 0.95 9.107 2.56 12.88 7 0.88 10.92 7 4.68 21.98 7 3.11 19.31 7 0.61 16.53 7 0.15 16.03 7 1.79 19.007 1.11 13.27 7 2.45 7.67 7 4.16 8.017 2.75 13.08 7 0.45 8.56 7 1.00 8.28 7 2.52 13.08 7 0.45 8.56 7 1.00 16.94 7 1.26 6.86 7 1.54 8.62 7 1.02 13.98 7 3.57 8.58 7 0.69 5.25 7 0.92 22.78 7 6.75 7.31 7 1.87 4.53 7 1.97 1.47 7 0.04 3.73 7 1.11 5.14 7 2.89 7.36 7 3.57 4.78 7 1.32 7.207 2.15 3.31 7 2.07 6.97 7 0.51 10.37 7 .0.03

264.117 12.72 159.217 7.36 215.877 23.88 388.187 18.64 275.547 23.95 177.377 17.50 438.187 52.07 233.50 7 47.38 183.877 8.31 295.05 7 1.00 166.857 16.76 194.04 7 25.52 491.50 7 61.50 406.85 7 12.52 290.007 49.25 617.04 7 37.73 430.36 7 48.10 334.137 55.90

Cr

30.79 74.47 28.96 76.52 26.04 72.21 99.77 718.48 47.66 71.08 40.67 75.51 221.67 731.34 38.97 73.09 63.97 714.80

Table 2 Accumulation of metals in seeds, its weight and oil content at maturity. All the values are means of four replicates 7SD. Different letters indicate significantly different values (DMRT, p o 0.05). BDL ¼below detection limits,  ¼ not present. Treatments

C Cu30 Cu50 As(III)10 As(III)30 As(III)50 As(V)10 As(V)30 As(V)50 Cr10 Cr30 Cr50

Accumulation of metals (mg g  1 dw) in seeds As

Cr

Cu

Fe

Zn

Mn

– – – BDL BDL 3.71 7 0.52 BDL 5.91 7 2.16 6.84 7 0.57 – – –

– – – – – – – – – BDL BDL 0.33 70.19

3.09 7 1.44 3.45 7 0.29 4.67 7 0.42 3.400 7 0.31 5.33 7 1.96 4.26 7 1.51 3.14 7 1.68 5.10 7 1.32 2.48 7 1.25 3.45 7 0.01 2.57 7 1.37 3.21 7 0.86

62.97 7 22.20 52.73 7 2.37 96.80 7 7.56 47.70 7 22.91 72.79 7 12.65 47.36 7 15.23 92.13 7 37.42 61.15 7 10.92 65.34 7 0.54 95.67 7 12.27 60.70 7 19.65 62.59 7 7.02

42.67 7 10.30 58.24 7 10.52 51.99 7 0.59 45.16 7 8.33 64.36 7 15.34 47.77 7 5.02 49.037 3.68 45.73 7 6.51 43.907 6.18 79.61 7 8.04 44.45 7 3.23 43.58 7 2.36

18.58 74.14 62.90 75.77 30.38 72.95 27.12 72.20 28.40 72.25 26.15 74.34 32.03 73.60 25.04 72.95 23.62 72.87 35.80 73.72 21.96 72.19 20.20 70.93

accumulation of Cr (0.33 mg g  1 dw) was recorded in the seeds at Cr50. The weight of seeds (100 nos.) varied in all sets of metal treatments (Table 2). In As(V) and Cr treated plants, the seed weight decreased in a dose dependent manner. However, there was no difference in seed weight with increase in As(III) concentrations. In case of Cu, maximum increase of 17% in seed weight was recorded in Cu30 as compared to C followed by a decrease. Similar to seed

Wt. (g) of 100 seeds

Oil (g)/100 g seeds

408.85 74.39b 459.5 730.44d 405.1 730.15bc 408.1 75.95bc 409.1 711.79bc 406.7 73.55bc 404.7 75.66bc 324.5 724.62a 310.2 716.37a 438.4 730.16cd 400.0 758.8bc 386.4 729.48bc

27.52 7 1.87c 26.43 7 3.45c 26.32 7 1.41c 26.94 7 2.26c 26.57 7 1.35c 26.89 7 1.06c 25.56 7 0.05bc 23.65 7 1.28abc 22.24 7 1.99ab 20.207 0.66a 22.30 7 0.55ab 21.39 7 0.56a

weight, seed oil content (Table 2) did not show much change among all the As(III) treatments. However, the decrease was observed in seed oil collected from the plants grown on As(V) and Cr spiked soils with the increase in metal concentration. Among different Cu treatments, no significant difference in oil content was observed. As compared to C, maximum decrease of 19% and 22% in oil content was observed in As(V)50 and Cr50 plants.

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Fig. 2. The effect on shoot and root lengths (cm) and fresh weight (g) in the plants grown on metals spiked soil after 15d of metal treatments. All the values are means of four replicates 7 SD. Different letters indicate significantly different values at a particular duration (DMRT, p o 0.05).

3.4. Plant growth parameters, photosynthetic pigments and lipid peroxidation Among all the treatments, growth parameters (Fig. 2) were most affected in the plants grown on Cr spiked soil and maximum decrease of 63%, 34% and 42% was recorded in fresh weight, shoot (po0.05) and root lengths (p o0.05) at Cr50 as compared to C after 15 d. As compared to C, shoot length decreased significantly in all the treatments, whereas, there was non-significant increase in root length of As treated plants except As(V)50 after 15 d. In Cr and Cu treated plants, root length decreased non-significantly. Among both the species of As (III, V) and Cr, no significant difference was observed in growth parameters among the different metal treatments. In contrast, shoot and root lengths decreased significantly at Cu50 as compared to Cu30. The total chl content (Fig. 3A) increased non-significantly in all the metal treatments at 15 d as compared to C except for Cr 50. At 30 d, the chl content has shown decrease as compared to C as well as within each treatment with increase in concentration except for As(V) treated plants where no difference was noticed. Maximum decrease of 266% was observed in As(III)50, followed by Cr50 after 30 d as compared to C. No significant difference was recorded in

carotenoid content (Fig. 3B) at 15 d, whereas at 30 d, it decreased with increase in concentration except for As(V) treated plants where non-significant increase was observed as compared to C. The level of protein content (Fig. 3C) decreased significantly in all the treatments at 15 d and non-significantly increased at 30 d as compared to C except for Cr50 and As(V)30. Within treatments, no significant difference was found with increase in metal concentration in all the treatments. The thiobarbituric assay, evaluating the degree of lipid peroxidation, resulted in increase in MDA content (Fig. 3D) in the leaves of B. juncea at both the harvesting periods, compared to C. Among all the treatments, the increase in As(III) treated leaves was more pronounced at both the treatment durations and all the concentrations. The maximum increase of 57%, 48% and 40% was recorded in As(III)50, As(V)50 and Cr50, respectively, after 15 d as compared to C. Within each treatment, no significant difference was noticed in all the treatments at both the treatment durations with increase in metal concentration. Overall, among all the metal treatments, MDA level was observed to be lowest in Cu spiked plants and highest in As(III) treated plants at both the harvesting periods; however, increase was observed as compared to C.

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Fig. 3. The effect on total chlorophyll, carotenoid and protein contents (mg g  1 fw) and MDA content (mmol g  1 fw) in the plants grown on metals spiked soil after 15 and 30 d of metal treatments. All the values are means of four replicates 7SD. Different letters indicate significantly different values at a particular duration (DMRT, p o0.05).

3.5. Effect on antioxidants SOD activity (Fig. 4A) of all the treated plants exhibited no significant change after 15 d whereas it decreased at higher metal concentrations in all the treatments as compared to C at 30 d. Within each metal treatment after 30 d, SOD activity decreased significantly with increase in As(V), Cr and Cu concentrations as compared to the respective lowest metal concentration at 30 d. Among all the treatments after 30 d, SOD (Fig. 4A) and APX activities (Fig. 4B) were found to be lowest in Cr treated plants except Cr10. The APX activity decreased in a dose dependent manner after 15 d; however, no difference was observed with increase in metal concentration after 30 d. APX activity also decreased significantly in Cr treated plants as compared to C after 30 d. Within treatments of As(III) and As(V), decline in APX activity in the leaves was observed; in general, there was more decline in As(V) than in As(III) treated leaves of the plants after 30 d at higher concentration. In As(III), As(V) and Cu treated plants, there was no significant difference in APX activity except for a significant increase in As(III)10 and a significant decrease in As(V)50 after 30 d, compared to C. As compared to C, significant increase in GPX activity (Fig. 4C) was observed at a lower

concentration of As (III, V) as compared to C after 15 d and the rest of the treatments have shown non-significant increase. At 30 d, no significant difference was recorded in GPX activity except for a significant increase in Cr50, compared to C. At 30 d, within each metal treatment, no significant difference was observed, except for a significant increase in Cr 50. GR activity (Fig. 4D) decreased in all the treatments at both the treatment durations as compared to C. The decrease in activity was more pronounced (significant) in Cr treated leaves at both the treatment durations, compared to C. Within each metal treatment at 30 d, a declining trend in GR activity was recorded with increase in concentration of metals except for As(V). In Cu treated plants, GR activity was found to decrease with increase in metal concentration after both the harvesting periods. Overall the activities of antioxidant enzymes were found to exhibit a maximum decrease in the order of 24%, 75% and 60% for SOD, GR and APX, respectively, in Cr50 treated leaves as compared to C after 30 d. In contrast to these three activities, significant increase (85%) was found in GPX activity in Cr 50 treated leaves of the plants at 30 d. Decrease in GSH content (Fig. 5) was observed in the leaves of B. juncea in all the treatments as compared to C except in Cu30 and As(V)10 and at 15 d. Maximum decrease of 37% was recorded

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Fig. 4. The effect on activities of SOD (U min  1 g  1 fw), APX (mmol min  1 g  1 fw), GPX (mmol min  1 g  1 fw) and GR (mmol min  1 g  1 fw) in the plants grown on metals spiked soil after 15 and 30 d of metal treatments. All the values are means of four replicates 7 SD. Different letters indicate significantly different values at a particular duration (DMRT, po 0.05).

Fig. 5. The effect on GSH content (mmol g  1 fw) in the plants grown on metals spiked soil after 15 and 30 d of metal treatments. All the values are means of four replicates 7 SD. Different letters indicate significantly different values at a particular duration (DMRT, po 0.05).

in Cr50 treated leaves after 15 d. Within each metal treatment at 15 d, significant decrease was observed at the highest metal concentration as compared to the lowest metal concentration

except As(III). At 30 d, no significant difference in GSH content was observed within all the metal treatments with increase in metal concentrations as well as compared to C.

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4. Discussion The total metal content of the soil is not appropriate to assess the bioavailability and mobility of the toxic metals to the plants. Fayiga et al. (2007) reported that As concentrations in the watersoluble plus exchangeable As (WE-As, using NH4Cl) fraction in all the treatments significantly correlated with As removed by the plant after 8 weeks of growth. Similar to this finding, the accumulation of As in different parts of the plants was correlated with exchangeable As. For the extraction of other metals, Gupta and Sinha (2007) reported that extraction capacity of EDTA was found to be a suitable extractant for most of the metals from tannery wastewater contaminated soil to B. juncea and followed the following order: EDTA4DTPA4NH4NO3 4CaCl2 4NaNO3. In this study, EDTA has also shown better extraction capacity for the studied metals than DTPA. Phyto-toxicity of As depends on the relative distribution of its different chemical forms, solubility and bioavailability of As, which is a function of soil properties, such as pH, CEC, organic matter, phosphorus concentration and soil redox conditions (Alva et al., 2000). Similarly, phyto-toxicity of all the metals also depends on physico-chemical characteristics of the soil (Gupta and Sinha, 2009). Besides, the plants have differential uptake mechanisms for different oxidation states of the same metal. Pickering et al. (2000) reported that As(V) is taken up by the roots of Indian mustard, possibly via the phosphate transport channel, a small fraction of which is transported to the shoot via the xylem as the oxyanions of As(V) and As(III). The accumulation of As in the plants of B. juncea as observed in this study was more in As(V) than As(III) in spiked plants, which may be due to the difference in the transport mechanism. However, the majority of the metalloid remain in the roots possibly as an As III-tris-thiolate complex, whereas in the shoot it is mostly found as As III-tris-glutathione. Recently, Zhao et al. (2009) also reported that As(V) is taken up by phosphate transporters whereas As(III) is transported through a number of the aquaporin nodulin 26-like intrinsic proteins (NIPs). The pathway of Cr(VI) transport in the plants is an active mechanism involving carriers of essential anions such as sulfate. Studies on the uptake of heavy metals by the plants have shown that heavy metals can also be transported passively from roots to shoots through the xylem vessel. The high accumulation of Cr(VI) in roots of the plants could be due to immobilization of Cr in the vacuoles of the root cells (Shanker et al., 2005). Lower transport of Cr from root to aerial parts in B. juncea is in accord with other observations on Indian mustard (Han et al., 2004). In contrast to increase in Mn accumulation in Cu and higher concentrations of Cr treated plants in this study, GardeaTorressdey et al. (2004) reported the decrease in the accumulation of Mn and Zn in Convolvulus arvensis L. when exposed to Cu(II) and Cr. However, Zn accumulation increased in the roots of B. juncea. Similar to the findings of Mokgalaka-Matlala et al. (2008), the accumulation of Mn decreased in As(III) treated leaves of the plants. As(III) and As(V) treated plants significantly reduced Fe accumulation in the leaves of B. juncea, which has also been reported earlier (Mokgalaka-Matlala et al., 2008; Tu and Ma, 2005). In case of Fe accumulation in Cr treated plants, the accumulation in roots and leaves was not significant in the lower concentration of Cr; however, increase was recorded at Cr30 and Cr50. A similar finding was also reported by GardeaTorressdey et al. (2004) in Convolvulus arvensis exposed to Cr(VI). Overall, Cr treatment played a positive role in the uptake of nutrients like Mn, Zn and Fe in B. juncea. However, in Cr treated plants, Fe accumulation has shown antagonistic relationship with Cu treatments. Mallick et al. (2010) also reported decrease in the level of Cu in leaves and roots of Cr treated Zea mays. Turner and

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Rust (1971) had assumed that excess of Cr can affect the uptake of nutrients in the plant. However, it can be said from our observation that the uptake of nutrients under the interaction effect of other metals can vary from plant to plant and genotypes within the same species. In addition, plant parts (fruit and seed), which are storage organs and have low transpiration rates, did not accumulate heavy metals, as these parts are largely phloem-loaded and heavy metals are generally less mobile in it. In the present study, this may be the reason for the least translocation of metals in the seeds of the plants. There are many other reports where accumulation of Cr was found below the detection limit in the seeds of oil bearing plants grown on Cr contaminated soil (Singh et al., 2004; Gupta and Sinha, 2009; Singh and Sinha, 2005). Similarly, Sinha et al. (2006) also reported that leafy vegetables have greater potential of accumulating heavy metals in their edible parts than grain or fruits, which follow a general order of translocation: roots 4leaves4seeds. There are many reports where Brassica sp. (leafy vegetables) collected from the field irrigated with wastewater indicated widespread contamination of heavy metals despite showing luxuriant growth. In general, cultivation of food crops on contaminated soil is a major concern for mass consumption of the agricultural produce without studying its implications (Sinha et al., 2006; Mapanda et al., 2007). This demonstrates that the transport of metal to seeds of B. juncea warrants more attention, as most of the studies are based on hydroponic findings (Pandey et al., 2005; Gupta et al., 2009). However, there are very few reports where the effect on oil content in the plants grown on contaminated soil was reported under simulated field condition (Singh et al., 2004; Singh and Sinha, 2005; Gupta and Sinha, 2009). In all these studies, it was observed that the level of oil content increased in the plants grown on lower amendment of tannery sludge, which may be due to the presence of essential metals and high organic matter and decreased at higher levels. Jones et al. (1987) reported that metals could affect lipid synthesis/composition which are based on the hypothesis that metal toxicity is mediated by metal inactivation of physiologically essential thiol-containing enzymes and cofactors which may affect the biosynthesis of oil. This could be a possible reason for decrease in oil content observed in this study in metal treated plants. In this study, it was observed that there was no difference in oil content with increase in As(III) treatment, whereas it decreased in case of As(V) in a dose dependent manner. It is thus speculated that As(V) has the same oxidation state as inorganic phosphate (P5 + ) and shares many chemical properties with P5 + which can substitute for P5 + in ATP production. In the process of generation of ATP though glycolysis, 1-arseno-3phosphoglycerate is produced rather than 1,3-bisphosphoglycerate, an enzyme vital for ATP synthesis in the ATP generation phase of the process. 1-Arseno-3-phospho-glycerate hydrolyzes spontaneously, so that ATP continues to be produced. However, the arsenic–oxygen bond is significantly weaker than the phosphorus–oxygen bond, and 1-arseno-3-phosphoglycerate hydrolysis yields dramatically less energy (Lai et al., 2005). Acetyl CoA is the precursor of fatty acid synthesis, which is a product of CoA catalyzed by citrate lyase using ATP. Probably, the fatty acid synthesis is hindered in an ATP deficient system as the case in As(V) treated plants where decrease in the oil content was observed. In contrast, the plants treated with As(III) did not show any decrease in the oil content possibly due to the fact that As(III), unlike As(V), quickly binds with thiol containing compounds such as glutathione present in the plant. Among all the treatments, Cr spiked plants exhibited prominent morphological response towards dose. Similar to the present study, Shankar et al. (2005) reported that Cr (VI) treatments in the plants have shown significant reduction in growth parameters. In

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contrast, the plants grown on tannery waste contaminated soil containing high level of Cr have shown increase in growth parameters, which may be due to the presence of other nutrients and organic matter (Sinha et al., 2009; Singh and Sinha, 2004). Similar to the present study, Khan et al. (2009) reported reduction in plant growth parameters in As treated Indian mustard (cv. Pusa Jai Kisan) grown hydroponically. Although Cu is an essential metal, it is toxic at higher concentrations, which is evident from the decrease in growth parameters of the Cu treated plants in this study. Gupta et al. (1996) also reported toxicity of Cu in the plants at higher concentrations. The higher total chlorophyll content after 15 d in all treatments, both as compared to C and that of their corresponding 30 d values, explains that photosynthetic efficiency of younger plants exposed to metal stress is more than the older plants. A similar observation was also reported where the level of chlorophyll content in the plants of Helianthus annuus grown on contaminated soil has shown higher values after 60 d than longer treatment duration (Singh et al., 2004). Carotenoid, a non-enzymatic antioxidant, is a part of photosynthetic pigment, which plays an important role in protection of chlorophyll pigment under stress condition by quenching the photodynamic reactions and replacing peroxidation. The decrease in chlorophyll content in As(III) treated plants was more pronounced at higher concentration after 30 d, which may be due to binding with essential thiol groups, in contrast to As(V) where no change was observed. Similar to the present findings in case of Cr treated plants, Shanker et al. (2005) reported the inactivation of enzymes involved in the chlorophyll biosynthetic pathway, which also contribute to the general reduction in chlorophyll content in most of the plants under Cr stress. Similar to our findings where decrease in chlorophyll content at 30 d was almost the same in Cu50 and Cr50, GardeaTorressdey et al. (2004) also observed that the reduction in chlorophyll content was more when the plants was exposed to higher concentrations of Cu than Cr. It is known that chloroplast is the main site for Cu accumulation in higher plants, which in turn could affect chlorophyll synthesis (Fernandes and Henriques, 1991). In Cu treated plants, the decrease in chlorophyll content was reported earlier (Gupta et al., 1996). There was no change in protein content in all the treatments, which is in congruence with many similar studies (Singh et al., 2004; Sinha et al., 2009). The level of malondialdehyde (MDA) content has been considered as an indicator of oxidative stress. Pickering et al. (2000) reported that As was complexed to sulfur and stored within the root tissue of hydroponically grown Indian mustard plants. A small proportion of As was translocated in the xylem sap to the shoots and present as the oxyanions As(V) and As(III), which was not coordinated by sulfur. Thus, reactive oxygen species may be generated through the conversion of As(V) to As(III), which may result in damage to DNA, proteins and lipids (Mascher et al., 2002). This may be the reason of the higher level of MDA content ion As treated plants. In plant cells, the other transition metals have demonstrated the potential to induce toxic oxygen species, which play an important role in the onset of lipid peroxidation, which varies from metal to metal (Gupta et al., 1996; Halliwell and Gutteridge, 2004; Shanker et al., 2005). The plant cells develop an antioxidant defense mechanism to cope with reactive oxygen species that vary at various cellular and subcellular levels in different plants; however, the level of response also varies with plant species/cultivars and intensity of stress. The level of oxygen free radicals in the plants depends on the activities of the enzymatic antioxidants involved in their detoxification such as SOD, CAT, GR or peroxidases. SOD is a key enzyme in protecting cell against oxidative stress, which catalyzes the dismutation of O2  to H2O2 and O2. Plants exposed to different metals respond differently with reference to enzymatic activities.

In the present study, no major role of SOD was observed in combating metal induced toxicity in the leaves at lower metal concentrations; however, it decreased at higher concentration of Cr and As(V) treated plants. Similar to the present study, SOD activity decreased in B. juncea cv. Pusa Jaikisan exposed to Cr in hydroponic conditions (Pandey et al., 2005). Similar to the present study, these authors also reported a decrease in APX activity in Cr treated B. juncea. Recently, Gupta et al. (2009) reported insignificant increase in GPX activity in B. juncea cv. Pusa bold in As(III) treated plants under hydroponic conditions. In this study, SOD and GPX in general showed a similar pattern; this reflects that they are functioning together for metal tolerance. GR catalyzes the reduction of oxidized glutathione (GSSG) in an NADPHdependent reaction and plays an essential role in the protection of chloroplasts against oxidative damage. In the present investigation, GR activity decreased in most of the treatments. The interconversion of reduced and oxidized forms of glutathione to maintain redox status of the cell as well as to scavenge free radicals could have caused a non-significant decrease of GSH in Cr treated plants at higher metal treatment. The decrease in GR activity as the concentration of the external Cr increased might be due to its inhibitory effect on the enzyme system itself (Shanker et al., 2005). Among all the treatments, more pronounced decrease in the activities of APX, GR was observed in Cr treated plants. Overall, the presented results suggest that, among all the studied metals, Cr-induced oxidative damage in B. juncea leaves was severe as a result of enhanced production of toxic oxygen species and the activities of the antioxidant enzymes were not sufficient to scavenge them and subsequently resulted in toxicity. Thus, comparing the activities of the antioxidant enzymes, i.e. SOD, APX, GPX and GR, it is evident that As induced a strong antioxidative response at lower metal concentrations, while Cr inflicted maximum decrease in the activities of APX and GR. The sequestration of the metals and binding to the sulphydryl group may be the reason for the high level of tolerance exhibited by the As(III) treated plants and its least mobility to the seeds of the plants.

5. Conclusion Metal accumulation in the leaves of B. juncea suggests transportation of the metals from roots to shoots and its sequestration in the leaves without significant effect on oil yield except for Cr and higher concentration of As(V), compared to the control. Among the various treated plants, accumulation of Cu, Fe and Zn in the leaves increased along with increase of Cr concentration in the soil; similar was the accumulation of Mn and Fe in As (V) treated plants. Fe and Zn were found to decrease along with increase in dose of As(III) treated plants, similar to Cu in the leaves of As(V) treated plants. Among all the metal treated plants, the decrease in APX and GR activities was more pronounced in Cr treated plants and resulted in higher toxicity as evident from growth parameters and oil yield. The oil content in the As(V) treated plants decreased in a dose dependent manner, whereas no decrease was found in the case of As(III) treated plants Maximum decrease was found in Cr treated plants. The metal accumulation was below the detection limit in the seeds grown on lower metal concentration, and hence can be recommended for oil cultivation only.

Acknowledgments The authors are thankful to Dr. Rakesh Tuli, Director, NBRI, for necessary help and financial support by Council of Scientific and

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