Uptake-related parameters as indices of phytoremediation potential

Biologia 65/6: 1004—1011, 2010 Section Botany DOI: 10.2478/s11756-010-0106-7

Uptake-related parameters as indices of phytoremediation potential Hema Diwan1, Altaf Ahmad1* & Muhammad Iqbal1,2 Molecular Ecology Laboratory, Department of Botany, Faculty of Science Jamia Hamdard, New Delhi 110062, India; e-mail: [email protected] 2 Department of Plant Production, College of Food & Agricultural Sciences, King Saud University, PO Box 2460, Riyadh 11451, Saudi Arabia 1

Abstract: Phytoremediation is emerging as an alternative agriculture-based technology because remediation of metalpolluted sites can be brought about utilizing the ability of plants to uptake and store contaminants in them. A field study was conducted to assess the role of Indian mustard in phytoremediation of chromium-contaminated substrata. Uptake parameters, namely, bio-concentration factor, translocation index, Cr distribution within plant, and tolerance index were used in determining the remediation potential of the crop. A significant increase in Cr accumulation (0.64–4.19 mg g−1 DW, stem; and 0.77–1.1 mg Cr g−1 DW, root), coupled with high tolerance indices, was observed in response to Cr stress, thus showing that Indian mustard is a potential hyperaccumulator. Movement and subsequent distribution of metal ions in the plant were assessed by studying the translocation index which showed a consistent increase (27–87% at T5) with time, and bioconcentration factor, where also an increase over a time period was observed in stem (1.3–11.4, T1) and root (1.96–5.56, T1), thereby, depicting the strong ability of Indian mustard for phytoextraction. A significant decline, however, was observed in the bioconcentration factor with increase in the dose of Cr application. Key words: chromium; metal uptake; phytoremediation; tolerance index; bioconcentration factor

Introduction Metals, radionuclides and inorganic contaminants are among the most prevalent forms of environmental contaminants, and their removal from soils and sediments is a difficult task. Heavy-metal contamination is one of the most serious current environmental problems (Singh & Sinha 2005; Anjum et al. 2005; Ansari et al. 2009). Chromium (Cr) is one such heavy metal (HM) that has turned into a major environmental contaminant (Nriagu 1988). Chromium concentration in the environment is rising tremendously because of a widespread Cr discharge from tanning, leather, paint, and metallurgical industries (Zayed & Terry 2003; Mohan & Pittman 2006). Chromium exists in multiple oxidation states, with Cr-III and Cr-VI being the most stable forms. Of these, Cr-VI is considered to be more toxic and lethal than Cr-III. Whether Cr can be considered as an essential element for the growth of plants is a subject of dispute since it exerts both inhibitory and stimulatory effects on plant growth. Nevertheless, being a broadline element with high redox potential, Cr-VI proves highly deleterious for plant growth. The unregulated release of heavy metals poses a significant threat to environment and public health because of the consequent HM accumulation and toxicity in the food chain. A variety of biological resources have been em* Corresponding author

c 2010 Institute of Botany, Slovak Academy of Sciences


ployed in the developed and developing countries to clean the metal-polluted sites. The classic technologies of land filling, lagooning or pyrolysis have proved to be both destructive and disruptive in nature (Srivastava & Thakur 2006); so focus has now shifted to the emerging concept of plants being used as efficient hyperaccumulators. Plants have been shown to take up and sequester HM in roots and/or shoots and, therefore, contribute significantly to HM removal from the environment through the mechanism of phytoextraction. Plants like water hyacinth (Lytle et al. 1998), Arabidopsis thaliana (Salt et al. 1998), buckwheat, cabbage, cauliflower and Thalpsi have shown potential for accumulating Cr in their tissues and thus serving as a useful tool for remediation. Efforts are directed to optimize plants’ efficiency for high uptake and tolerance for chromium. A number of attempts have been made with varying degrees of success in the prediction of uptake and concentration of HM in plants. For successful utilization of phytoremediation technology, analysis of factors governing the uptake, translocation and accumulation of metals in various plant parts becomes very crucial (Begonia et al. 2005; Audet & Charest 2007). Since the literature available on parameters defining uptake, translocation and distribution of metal in plants is scarce and scattered, we have made an attempt to put together, in one frame, all parameters relevant in

Uptake-related parameters as indices of phytoremediation potential deciding the phytoremediation candidature of Indian mustard, owing to its high biomass-producing efficiency, hardy nature and its ability to survive under rain-fed conditions with no intense irrigation. Moreover, this plant possesses a well-developed tap-root system, with deep penetrating roots, therby helping in enhanced uptake and above all since it is a seasonal crop, the status of contaminant removal can be examined at the end of each crop season.

Material and methods Seeds of Indian mustard [Brassica juncea (L.) Czern. Coss. cv. Pusa Jai Kisan] were procured from Indian Agricultural Research Institute, New Delhi and sown in prepared pots containing sandy soil from the experimental field of Hamdard University, New Delhi, India (28.38N, 77.11E, 228 m altitude) during the winter season. The climate of Delhi is semi-arid and subtropical with the mean annual rainfall of about 650 mm. Three pots for each treatment were used for cultivating the plants. The soil was amended with recommended levels of nitrogen (1.15 g kg−1 ), phosphorus (0.75 g kg−1 ) and potassium (0.27g kg−1 ), supplied in the form of urea, triple super phosphate and muriate of potash, respectively. The soil of the pots was contaminated with five levels of Cr, viz., 100 (T1), 200 (T2), 300 (T3), 400 (T4), and 800 (T5) mg Cr kg−1 soil (Diwan et al., 2010a). Chromium supplied in the form of potassium dichromate (a source of CrVI) dissolved in double distilled water (DDW), was mixed thoroughly in the soil. The prepared pots were placed in field conditions (in Jamia Hamdard herbal garden) to expose the growing plants to natural environment. The pots were arranged in randomized plot design, with three replications of each treatment. Five plants per pot were maintained. Forty-day-old plants were analyzed at 7 days interval for investigating the uptake potential. Biomass accumulation The plants were harvested and washed by deionized water followed by proper blotting between filter papers. Plants were dried separately in a hot air oven at 65 ◦C ± 2 ◦C for 72 h. The samples were weighed on an electronic top pan balance (Sartorius BL-210S, Germany) so as to obtain the biomass accumulation, which was expressed in g per plant.

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Translocation index Translocation index is used to work out the ability of plants to translocate heavy metal from roots to harvestable aerial plant parts (Zu et al. 2005). High translocation factor is always favourable for phytoremediation. It is calculated by the following formula: Translocation Index = Cr content in the leaves (mg g−1 DW) × 100 = Cr content in the root (mg g−1 DW) Tolerance index Tolerance Index (TI) is calculated as the mean weight (biomass) of a plant grown in the presence of a metal divided by the mean weight of a control (Baker et al. 1994). Tolerance Index was expressed as Tolerance Index = Biomass of the treated plants (g plant−1 ) = Biomass of the control plants (g plant−1 ) TI values greater than 1 reflect a net increase in biomass and suggest that plants have developed tolerance, whereas TI values lower than 1 indicate a net decrease in biomass and a stressed condition of plants. TI values equal to 1 indicate no difference relative to non-HM control treatments (Wilkins 1957, 1978). Bioconcentration factor (BCF) Bioconcentration factor (BCF) is defined as the ratio of the metal concentration in plant to the metal concentration in the soil (Zayed et al. 1998). It is the defining parameter in phytoremediation providing information on the uptake of metal, its mobilization into the plant tissues and storage in the aerial plant parts (Newman & Unger 2003). BCF has been used as a measure of HM-accumulation efficiency in plants; BCF values greater than 1 are indicative of a potential HM-hyperaccumulator species (Zhang et al. 2002). BCF =

Cr in the plant tissue (mg kg−1 DW) Cr in the soil (mg kg−1 DW)

Statistical analysis The experiment was conducted in randomized block design, taking three replications (n = 3) of each treatment. To confirm the variability of data and validity of results, analysis of variance (ANOVA) was conducted. In order to determine whether differences among the treatments within respective DAS were significant as compared to control, Tukey’s test was applied.

Results Chromium accumulation After harvesting, plant material was rinsed thoroughly in Milli Q water, oven-dried at 65 ◦C ± 2 ◦C for 72 h, and then ground to a fine powder. A 0.25 g of dry material from each treatment was added to 3 mL of concentrated HNO3 in a 50 mL digestion tube and mixed gently by swirling. The digestion tubes were placed in a heating block set at 150 ◦C for 1 h. Two ml of 30% H2 O2 was added to each digestion tube after cooling. They were heated for further 3 hours at 150 ◦C and then cooled to room temperature. Upon complete digestion of plant tissue, the solution was diluted to 50 mL and the upper clear part was separated from the lower sand-grit portion. The upper portion was sampled for determination of Cr content by using the atomic absorption spectrometer (Model ZEEnit 600/650, Analytik Jena, Germany).

Biomass accumulation Biomass increased with increasing age of plants. A linear increase in the biomass was evident with increase in time period with the maximum biomass accumulation evident at 75 DAS (Days After Sowing). At 82 DAS, a decline was observed as compared with growth observed at 68 and 75 DAS. However, a differential response towards Cr treatments was observed with Cr doses, i.e. increasing at lower doses (T1–T3) and decreasing at higher treatments (T4–T5). The increase in biomass with T1, T2 and T3 was significant when compared with control at all days of sampling but 75 DAS. Effect of Cr on biomass accumulation was significant with T5 only (Fig. 1).

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Fig. 1. Effect of chromium treatments on the biomass accumulation in Indian mustard as observed in different samples collected at 7-day intervals. T0, T1, T2, T3, T4 and T5 are the treatments with 0, 100, 200, 300, 400 and 800 mg Cr kg−1 soil, respectively. Results are presented as means ± standard error (n = 3). Values followed by same letters are not significantly different at a particular DAS (p < 0.05).

Chromium accumulation Chromium accumulation in roots progressed with age of the crop as well as with increase in the Cr dose. In general, metal accumulation in roots, stem and leaves increased when metal concentration and exposure times were raised. Cr levels in the tissue increased by 0.29 to 0.89 mg g−1 dry weight (DW) with T1. Similarly, Cr accumulation ranged from 0.38–1.1 mg, 0.53–1.3 mg, 0.63–1.6 mg and 0.77–1.1 mg Cr g−1 DW with T2, T3, T4 and T5, respectively (Fig. 2). These results showed relatively higher accumulation of Cr in the above-ground parts than in roots. Chromium accumulation in the stem increased in a dose- and timedependent manner. Maximum Cr accumulation (0.64– 4.19 mg g−1 DW) was evident with T5 dose of treatment (Fig. 2). Leaf Cr accumulation also seemed to depend on time and dose. With increase in the treatment dose, it got elevated from 0.05–0.37 mg g−1 DW with T1 and from 0.10–0.40 mg g−1 DW with T2, and showed the highest (0.20–0.99 mg Cr g−1 DW) gain with T5 (Fig. 2). Translocation Index Translocation index for chromium increased with time of Cr application. It ranged from 17–42%, 22–41%, 19– 43%, 21–39% and 27–87% with T1, T2, T3, T4 and T5 treatments, respectively. The increase over the control was statistically significant with T5 as observed at all

days after sowing. When compared among the treatments, the maximum translocation index was obtained with T5 at all the days of sampling (Fig. 3). Tolerance Index Indian mustard turned out to be most tolerant at T1, T2 and T3 doses of Cr application, having tolerance index (TI) around 1.01–1.29, 1.00–1.17 and 0.85–0.98 respectively. With T4 and T5 treatments, a decline in TI was evident. With increase in days of sampling, a nonsignificant effect of Cr was evident on tolerance (Fig. 4). Bioconcentration Factor The maximum BCF was noted in the stem, followed by roots and then by leaves. In general, the BCF values for Cr in roots, stem and leaves increased (p ≤ 0.05) with the passage of time. The minimum and maximum bioconcentration in the Indian mustard was evident at 40 DAS and 82 DAS respectively. The BCF values significantly decreased (p ≤ 0.05) with Cr concentration in the soil at each exposure time (data not shown). In roots and leaves, a significant decline was observed with reference to the T1 in all the doses of treatment except at 40 DAS in leaf (Fig. 5). Discussion For successful phytoremediation, the first requisite is

Uptake-related parameters as indices of phytoremediation potential

Cr accumulation (mg g -1DW)

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Fig. 2. Effect of chromium treatments on the Cr contents of root, stem and leaves in Indian mustard as observed in different samples collected at 7–day intervals. T1, T2, T3, T4 and T5 are the treatments with 100, 200, 300, 400 and 800 mg Cr kg−1 soil, respectively.

high production of plant biomass. Since metal removal is a function of metal concentration in the harvestable biomass, the plant should be able to produce enormous biomass so that it can grow successfully at the contaminated site. Low Cr concentrations caused an initial increase in the biomass of Indian mustard, followed by a

significant decline at higher concentrations. This kind of growth pattern observed in Indian mustard at different Cr concentrations is explained by the phenomenon of hormesis, which is defined as the ability of living organisms to adapt to low-level exposures of chemical agents – a phenomenon widely observed in nature (Calabrese

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Fig. 3. Effect of chromium treatments on the translocation index of Indian mustard as observed in different samples collected at 7-day intervals. T1, T2, T3, T4 and T5 are the treatments with 100, 200, 300, 400 and 800 mg Cr kg−1 soil, respectively. Results are presented as means ± standard error (n = 3). Values followed by same letters are not significantly different at a particular DAS (p < 0.05).

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Fig. 4. Effect of chromium treatments on the tolerance index of Indian mustard as observed in different samples collected at 7-day intervals. T1, T2, T3, T4 and T5 are the treatments with 100, 200, 300, 400 and 800 mg Cr kg−1 soil, respectively. Results are presented as means ± standard error (n = 3). Values followed by same letters are not significantly different at a particular DAS (p < 0.05).

& Baldwin 2003). One of the attributes of hormesis is stimulation of growth at low concentrations and inhibition at higher concentrations. Decreased growth of Indian mustard at higher levels of Cr is because it possibly interacts with certain micronutrients essential for plant growth and limits their availability by way of a decreased uptake or immobilization in roots. That Cr interacts synergistically with other environmental components to impair plant growth can also be not ruled out. Inhibition of biomass accumulation may also result from inhibition of cell division in general (Goldbold & Kettner 1991). Decreased biomass yield due to Cr accumulation has been observed in Vallisneria and

cauliflower (Chatterjee & Chatterjee 2000). Cr uptake is an important parameter in understanding the cellular responses of high HM concentration in plants and is one of the requisites contributing to the success of phytoremediation. The ambient metal concentration in the soil was the major factor influencing the metal-uptake efficiency as the metal uptake was observed to increase with increase in treatment doses (Ghosh & Rhyne 1999; Begonia et al. 2005). Considering the definition of hyperaccumulator given by Foy (1984), Baker & Brooks (1989), Baker et al. (2000) and Reeves & Baker (2000), Indian mustard behaves as a potential Cr hyperaccumulator. Fully matured plants

Uptake-related parameters as indices of phytoremediation potential

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0b0 0 0 0a 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 0 0 0 0 00 00 00 00 00 00 00 00 00 00 00 00 00c00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00d0 0 0 0000000000000000000 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 0000000000000000000

61DAS

68DAS

75DAS

82DAS

12 10 BCF

8 6 4 2 0

00000000 a00 00 00 00bd 00 00 00a00 00ac ad 0 0 0 0 0 0 0 0 0 0 0 0000 aab b 00 00a00 00 00 a00 00 00 00a00 00 a00 00 00 00 00 0b0 0 000 000 000 000 000 000 000 000 000 000 000 000 000c000 000 0 d0 0 0 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 0000000000000000000 0000000000000000000 0000000000000000000 40DAS

47DAS

54DAS

00 00 00 00 00 T1

LEAF

00 00 00 00 T2

00 00 00 00 T3

a

00 00 00 00 00 T4

00 00 00 00 00 T5

4.5 a

00 00 00 00 00 00 00 00 00 00 00000 00000 0 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00

00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00000 00 00 00 00 00 000000000000 0 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00

00 0 0 0 0 0 000 000 000 000 00 0 0 0 0 00 00 00 00 00 000000000 00 00 00 00 00 00 00 00 00 0 0 0 0 0 0 0 00000000000000000000 0 00 00 00 00 00 00 00 0 00 00 00 00 00 00 00 00 00 00 00 00 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00

0000 00 00 00 00 0 00 00 00 00 0 0 0 00 00 00 00 0 0 0 0 000000000000 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 0 0 0 0 00000000000000000000 0 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00

61DAS

68DAS

75DAS

82DAS

4.0 3.5

a

3.0 2.5 2.0 1.5 1.0 0.5 0.0

a

0000 00 00 00 00 0 00 00 00 00 00 00 00 00 00 00 00 0 0 0 0 0 0 0 0 0 00 00 00 00 0 0 0 0 0 00 00 00 0 00 00 00 00 00 00 00 00 00 00 00 0 0 0 0 00 00 00 00 0 00 00 00 0 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 00 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 a

a

a

ab

ab

a

a

c

40DAS

b bc

b

b

bd

b

b

47DAS

54DAS

b

b

b

b

bc

b bd

b

a

b

bc

bc

c

b

bc

cd

d

Fig. 5. Effect of chromium treatments on bioconcentration factor (BCF) of root, stem and leaf of Indian mustard as observed in different samples collected at 7-day intervals. T1, T2, T3, T4 and T5 are the treatments with 100, 200, 300, 400 and 800 mg Cr kg−1 soil, respectively. Results are presented as means ± standard error (n = 3). Values followed by same letters are not significantly different at a particular DAS (p < 0.05).

accumulated a lesser amount of Cr in roots (1.62 mg Cr g−1 DW) than in the above-ground parts (4.2 mg Cr g−1 DW). Known hyperaccumulator plants, namely, Convolvulus arvensis, Sutera fodina and Dicoma niccolifera could accumulate in leaves up to 3.8, 2.4 and 1.5 mg Cr g−1 DW, respectively (Gardea-Torresdey et al. 2004; Ghosh & Singh 2005). The phytoremediation performance of a plant is determined not only by its capacity to extract high metal concentration, but also by its

ability to translocate the metal to aerial parts and simultaneously produce a high biomass (Ximenez-Embun et al. 2001). At initial stage of plant growth, the Cr content was higher in the root than other plant parts. However, Cr content was higher in the stem at later stages of plant growth. This also finds support from high translocation index and bioconcentration factor. In order to continue absorption of Cr from the substrate, plants must be capable to translocate Cr from roots to the

1010 stem, or to compartmentalize it. Our test plant qualifies this criterion as is evident from the elevated translocation index. Chromium as an anion moves fast inside the plant resulting in high Cr levels in the stem at later stages of growth (Skeffington et al. 1976; Wallace et al. 1977; Shahandeh & Hossner 2000; Davies et al. 2001; Gardea-Torresdey et al. 2005). High translocation is advantageous to phytoextraction, as it can reduce Cr saturation, and the consequent toxicity potential in any given part of the root. Similar results were obtained by Ghosh & Singh (2005) from Cr-treated Phragmites karka. Tolerance is another trait that influences the process of phtoremediation (Bluskov et al. 2005). Chromium tolerance of Indian mustard was assessed by determining tolerance indices at varying treatment levels. Tolerance Index (TI) value, which represents effect on relative plant growth, increased with treatments T1 and T2 so as to be more than 1, thus depicting that Indian mustard was able to tolerate Cr at these treatment levels, and that it was very effective in extracting Cr and also protecting itself from adverse effects of the metal. However, beyond these treatments, the TI declined gradually, indicating that Cr had become deleterious for plants. Similar results were obtained for Lycopersicon esculentum (Madhaiyan et al. 2007) and Echinochloa polystachya (Solis-Dominguez et al. 2007) with reference to cadmium toxicity. The high TI (greater than one) of Indian mustard was also substantiated by a high translocation index displaying an efficient Cr transport from roots to stem. This might owe to the existence of some tolerance mechanism, such as the presence of a strong antioxidant defence system (Diwan et al. 2008; Khan et al. 2009; Diwan et al. 2010b), which helps the plant in coping with high concentrations of metals accumulated in its tissues. Many hyperaccumulators are also reported to sequester toxic metals in the vacuole of their epidermal cells. The ability of the leaves of B. juncea to accumulate Cr preferentially in the epidermis and lower spongy mesophyll (perhaps in the vacuole) might have increased the plant’s tolerance to the metal by protecting photosynthesis, which predominantly takes place in palisade cells (Bluskov et al. 2005). Bioconcentration factor (BCF) is another useful parameter for evaluating the potential of plants for metal accumulation. It helps in evaluating the potential of plants in accumulating metals (Lu et al. 2004). It expresses the ability of a plant to accumulate metals against a concentration gradient. A high bioconcentration factor (BCF) for metal elements at low external concentrations is important for phytoremediation because the process becomes more cost efficient than other conventional technique for treating large volumes of wastewater with low concentrations of pollutants (Wang et al. 2002; Kamal et al. 2004). The BCF in our study varied in response to different Cr levels and depended on the strength of Cr in the soil. Increase in the BCF with increase in time, as shown in our study with reference to Cr, means that Indian mus-

H. Diwan et al. tard accumulated Cr up to maturity and that its metalconcentrating ability was not impaired by the rising Cr concentrations in plant tissues both with time and treatment dose (except in extreme cases). Translocation of metal from roots to the aerial plant parts can become a limiting factor for bioconcentration in the aerial parts. BCF finely estimates the metal-accumulation power of plants at different time periods against a given background metal concentration. A stable BCF across a concentration range signifies that the plant may be used efficiently for phytoremediation across a wide and fluctuating HM concentration range (Zurayk 2001). According to Lu et al. (2004), increase in the treatment level leads to enhancement in metal accumulation by plants, thereby leading to a decrease in the BCF values. Our results verify the same, as BCF increased with time but decreased with Cr treatments. On the other hand, Cr accumulation was enhanced in various plant parts with time. In general, when metal concentration was increased, the amount of metal accumulation in plants increased. Similar results were obtained for Lemna minor (Jain et al. 1990) and water hyacinth (Zhu et al. 1999). In conclusion, Indian mustard displayed enhanced tolerance towards a range of Cr concentration besides accumulating high Cr in its various parts, thus serving as a suitable contender for phytoremediation. This study demonstrates the role of tolerance index, translocation index and bioconcentration factor in assessing the potential of the plant for effective phytoremediation. It is suggested that the remediation potential of Indian mustard can be tapped to the utmost by periodically harvesting the plant from the site being remediated, avoiding chances of the plant attaining lethal concentration of metal that could lead to oversaturation and hence the damage. The harvested biomass could then be incinerated and disposed off or the accumulated metal could also be recovered for commercial uses and thus recycled and reused. References Audet P. & Charest C. 2007. Heavy metal phytoremediation from a meta-analytical perspective. Environ. Pollut. 147: 231–237. Anjum N.A., Umar S., Ahmad A., Iqbal M. & Khan N.A. 2009. Ontogenic variation in response of Brassica campestris L. to cadmium toxicity. J. Plant Interac. 3: 189–198. Ansari M.K.A., Ahmad A., Umar S. & Iqbal M. 2009. Mercuryinduced changes in growth variables and antioxidative enzyme activities in Indian mustard. J. Plant Interac. 4: 131– 136. Baker A.J.M. & Brooks R.R. 1989. Terrestrial higher plants which hyperaccumulate metallic elements a review of their distribution, ecology and phytochemistry. Biorecovery. 1: 81–126. Baker A.J.M., Mc Grath S.P., Sideli C.M.D. & Reeves R.D. 1994. The possibility of in-situ heavy metal decontamination of polluted soils using crops of metal-accumulating plants. Resour. Conserv. Recycl. 11: 41–49. Baker A.J.M., McGrath S.P., Reeves R.D. & Smith J.A.C. 2000. Metal hyperaccumulator plants: a review of the ecology and physiology of a biological resource for phytoremediation of metal-polluted soils, pp. 85–107. In: Terry N. & Baelos G. (eds), Phytoremediation of Contaminated Soil and Water. Lewis Publishers, Boca Raton, FL.

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