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The present research was carried out to investigate the accumulation potential of heavy metals Fe, Zn, Cu, Pb, Ni and Cd and biomass, chlorophyll content, photosynthetic activity were determined for Brassica juncea L. in response to grown on different amendments of 0, 10, 25, 50, and 100% of fly ash (FA), National Thermal Power Plant Corporation (NTPC), Badarpur, New Delhi, India. The result showed a decrease in the levels of chlorophyll content, photosynthetic area to exposure at 25-100% and PSI & PSII activity increase for the 25% and then decrease exposure to 100% FA. The plant exhibited a well growth of biomass at the exposure to 25% and decline in biomass over the 100% exposure in all treatments. B. juncea Pb, Zn, Cu and Fe metal of the metals were suitable for phytoextraction compared with Ni and Cd because no hyperaccumulation was observed in plant. The HMs concentration in B. juncea after 90 d of experiment, was in the order of FeÂ >Â ZnÂ >Â CuÂ >Â Pb > Ni > Cd. The B. juncea, was the suitable in accumulating Fe, Zn and Cu its translocation factors in shoot (TFs=1.5, 1.3 and 1.05), while Pb, Ni and Cd was most efficient its translocation factors in its shoot (TFs=8.5, 4.3, 3.3). Plant uptake of the HMs Pb, Zn and Cu was highly correlationship, whereas Fe, Ni and Cd was lowest correlationship though translocation in shoot of Pb, Zn and Cu were strong. Our study showed that B. juncea growing on FA may have the potential for phytoremediation.
Keywords: HMs, heavy metal toxicity, Fly-ash, Hyper accumulator, Phytoremediation, Biocentration factors (BCFs), Translocation Factors (TFs)
HMs is ubiquitous environmental contaminants in industrial wastes. Soil and water pollution by metals differs from air pollution, because HMs persists in soil and water in longer life than in other compartments of the biosphere . Excess HMs due to discharge of industrial effluents and fly-ash wastes are the general contamination of soil, water and air has caused serious environmental haphazard in the biosphere. Properties of fly-ash is high basic in nature and contains many essential elements like Cl-, Ca, Mg, Fe, Cu, Zn, and P along with highly toxic HMs, such as Pb, Ni, As and Cd for improving soil biosphere by phytoextraction [2, 3, 4]. FA application to soil at low amendments has been reported to promote the growth of the plants  that mean improvement of soil porosity, conductivity, organic carbon and microbial activity , soil conditioning and water holding capacity . This raises application the possibility and implications of metal accumulation mechanisms to phytoremediation of using phytoremediation as an sustainable technology to remediate the FA contaminated areas as well as to restore soil biosphere these for beneficial purposes [8, 9, 10]. The technology uses plants to remediate toxic HMs and elements from the contaminated soil through their accumulation in harvestable shoot parts (i.e. phytoextraction) or to immobilize heavy metals in soils or sediments by root uptake and shoot adsorption onto roots or precipitation in the rhizosphere (i.e. phytostabilization) [11, 12, 13].
In this background, this present study included the analysis of physico-chemical properties of FA and the evaluation of heavy metals, such as, Fe, Zn, Cu, Pb, Ni and Cd in FA, FA effluent as well as in various FA collected from the selected contaminated area. Compartmentalization of metals in the vacuole and physiological systems is also part of the tolerance mechanism of some metal hyperaccumulators and phytoremediation (14, 15]. The Pb, Zn, Ni hyperaccumulator Thlaspi goesingense and herbaceous plants, enhances its tolerance by compartmentalizing most of the intracellular into the vacuole . HMs causes major environmental and human health problem hence, need our immediate attention for effective and affordable technological techniques to phytoremediation of metal pollution . It has been reported that plants could accumulate considerable amount of toxic metals and thus, play significant role in cleaning of metal pollutants .
Brassica juncea belongs to family brassicaceae and is a very important oil crop. Mustard oil is one of the major edible oils in India. Mustard oil has also got medicinal importance. Residual part of seeds is used as cattle feed and in fertilizer. Indian mustard (Brassica juncea L.) is a fast growing plant which produces a high biomass even in heavy metal polluted soils [11, 19]. Thus this plant might be a potential candidate for phytofiltration and /or phytostabilization of heavy metal contaminated waste waters and soil from fly-ash. So far this plant species has been used in studies of the translocation and phytoremediation of HMs like Fe, Zn, Cu, Pb, Ni, Cd [20, 21, 22, 23, 24] and arsenic stresses on plants. Brassica juncea, Brassica nigra, Trifolium repens, Lolium perenne, Thlaspi, Beta Vulgaris plants could tolerate toxic and translocation a large amount of HMs but effects on development grwoth of plants [25, 26, 27, 28, 29]. The increase in the level of HMs on the earth, because of industrial wastes are released invariably into nearby soil and water body(ies). The accumulation of phytotoxic amounts of metals by plants causes retardation of growth, reduction of biomass, loss of membrane integrity and inhibition of enzymes, stress chlorophyll contents, photosynthetic area [8, 30, 31, 32, 33]. Subsequently, the HMs is leached in its surrounding biosphere like in soils including that effect to the agricultural fields . Owing to serious cost of toxicity that can result due to excess of HMs contamination from fly-ash, there have been increasing efforts that were being made to look for appropriate techniques means to clean the toxic heavy metals from the biosphere environment (both from soil and water) [35, 36, 37]. Similarly, the metal accumulating potential of a number of aquatic plants, i.e. Pistia stratiotes, Cladophora frata, Ceratophyllum demersum, Bacopa monnieri, Spirulina platensis etc. has been demonstrated (38, 39].
In accumulation, some recent reports demonstrate high metal accumulation ability of algal species like Hydrilla spp., Oscillatoria spp., Phormedium spp., Spirogyra spp., etc. . Thus, terrestrial and aquatic plants, and algal species possess the capability to remove the toxicants and may be used for restoration of the degraded ecosystems. In recent times, restoration of FA affected tracts of land through transplantation of Dalbergia sisso, Eucalyptus, Acacia, Cassia and Tamarindus has been attempted . An initial success in this direction has pushed the researchers to find out other plant species, both aquatic as well as terrestrial, to cover the FA with dense mat of plantation and make the site aesthetically pleasing and to exploit their metal accumulation ability at the same time . According to Yoon et al. , plants growing naturally on a contaminated site respond better under stress conditions than plants introduced from other areas in terms of survival, growth and reproduction. Thus, there is a need to evaluate the metal accumulation ability of various native plants growing in FA affected sites to better use them for phytoremediation purposes in future and restoration of degraded lands.
Plants ideal for phytoremediation should posses multiple traits. They must be fast growing, have high biomass, deep roots, be easy to harvest and should tolerate and accumulate a range of heavy metals in their aerial and harvestable parts. To date, no plant has been described that fulfils all these criteria. However, a rapidly growing non-accumulator could be engineered so that it achieves some of the properties of hyperaccumulators. In contrast, phytoremediation is considered a cost-effective and environment-friendly technology for the treatment of waters contaminated by heavy metals [44, 45, 46]. One of the strategies of phytoremediation of metal-contaminated soil is phytoextraction, i.e. through uptake and accumulation of metals into plant shoots, which can then be harvested and removed from the site. Another application of phytoremediation is phytostabilization where plants are used to minimize metal mobility in contaminated soils [47, 48].
The present investigations were undertaken with an aim to evaluate mustard (Brassica juncea, family Brassicaceae) plants have the potential to accumulate heavy metals and thus can help in the process of phytoremediation of heavy metals in soils contaminated with through chemical effluents. So new technology "Phytoremediation" was introduced, which is not only cost effective but also protective to humans as well as to the environment [11, 20].
Materials and methods
The samples collected from the identified locations of FA were from FA dumping sites near NTPC, Badarpur, and New Delhi, India. After collection, all the samples were brought in polythene bag to the laboratory for analysis. The pH was analyzed by Orion ion meter (USA). Total nitrogen (%) was estimated by Kjeldhal method, organic carbon and total phosphorus (%) by Olsen method given by Jackson was used . Level of sulfate, potassium, carbonate, chloride, magnesium and porosity were estimated following standard procedures according to APHA . Water holding capacity was measured by hydrometry. The fly-ash used in this experiment was collected from the field of FA dumping sites near NTPC, Badarpur, India. The FA was oven dried at 80 oC for 5 d and sieved through 6 mm mesh. Physico-chemical parameters of the soil were analyzed according to the method . The soil has pH-7.9, Electrical conductivity-1.13 dsm-1, total nitrogen (%) 0.09, total phosphorus (%) 0.78, organic carbon (%) 0.487, respectively.
Seeds of Brassica juncea cv. Pusa Jaikisan were obtained from Indian Agricultural Research Institute (IARI, New Delhi). The seeds were sown and the plants were raised in 12"/12" earthen pots. 30 d after sowing the seed, Seeds were sown in pots (2000 in diameter) containing 15 kg of 100% FA and different amendments of GS (control), with 0%, 10%, 25%, 50% and 100% FA, in three replicates for each exposure and were kept under natural conditions. For convenience, the amendments are denoted as 10% FA (10% FA + 90% soil), 25% FA (25% FA + 75% soil), 50% FA (50% FA + 50% soil) and 100% FA (100% FA). Garden soil was used as a control. The plants were irrigated with tap water at regular intervals avoiding leakage of water from the pots. Plants were harvested at 30 and 90 d of growth and used for the determination of various parameters of physico-chemical and heavy metals. The plants from each treatment were placed under natural conditions. The plants were treated daily and care was taken to avoid leaching of water from the pots. A plastic tub was placed below each pot to collect the leachates. The collected leachates were again returned to the experimental pot. No rainfall was recorded during the period of experiment. The roots from each plant were detached and washed repeatedly into tap water to remove unwanted debris and blotted. Fresh biomass was record on dry weight (DW) basis.
2.2 HMs analysis and quality assurance
For metal analysis in soils, dried (1g) soil from each of the treatments was grounded and digested overnight with 5 ml of 1:3 :: HNO3:HCl in conical flasks. The samples were digested further with HF:H2O2. The samples were suitably diluted with tripled distilled water to measure concentration of toxic heavy metals. For metal analysis, dried (1g) plant samples were ground in a grinder and digested in HNO3:HClO4 (3:1, v/v) at 80 0C and metals (Fe, Zn, Cu, Pb, Ni and Cd) were estimated by Atomic Absorption Spectrophotometer (Perkin Elmer 3100). The standard reference materials (E-Merck, Germany) of Fe, Zn, Cu, Pb, Ni and Cd were used to provide calibration and quality assurance for each analytical batch. The efficiency of digestion of plant samples was determined by adding standard reference material of metals, samples were digested and metals were estimated. The detection limits of Fe, Zn, Cu, Pb, Ni and Cd were 0.5, 0.05, 0.02, 0.01, 0.01 and 0.002 Âµg/ml, respectively. Replicate (n=3) analysis was conducted to assess the precision of the analytical techniques. Triplicate analysis for each metal varied by no more than 5 %. Photosynthetic pigments and chlorophyll contents were extracted in 80%-chilled acetone as per procedure of Arnon et al. . PI & PSII was estimated by the method of Lowry et al. .
2.3 Translocation Factors (TFs) and Biocencentrations Factors (BCFs)
However, with a high bioconcentration factor (BCF, metal concentration ratio of plant roots to soil) and low translocation factor (TF, metal concentration ratio of plant shoots to roots) have the potential for phytostabilization. Root-to-shoot translocation factor was described as the ratio of heavy metals in plant shoot to that in plant root, while enrichment coefficient (R) was calculated as follows:
R = C above ground/C soil (1)
Where, C aboveground and C soil, represent the metal concentrations in the above ground parts of the plant and soil on dry weight basis, respectively. Enrichment coefficient basically depends on the soluble fraction of metals and organic matters in soils, where, C aerialÂ =Â Conc. in plant's aerial part (mg/kg) and C rootÂ =Â Conc. in plant's root (Î¼gÂ gâˆ’1). BCF is the ratio of metal concentration in plant tissues at harvest and initial concentration of metals in external environment.
2.4 Statistical Analysis
Two-way analysis of variance (ANOVA) was done on all the data to confirm the variability of data and validity of results. Differences among means were determined by analyses of variance. The SPSS (Statistic Program for Social Sciences) statistical program package (Release 12.0) was used for statistical analyses of data Pearson product moment correlation coefficients (r) were used to express the associations of quantitative variables and was performed to determine the significant difference between treatments that p < 0.05 significant and p < 0.01 strongly significant.
3 Results and Discussion
The analysis of physico-chemical of FA and levels of HMs in FA and effluent coming through NTPC Badarpur, FA composition has been presented in Table 1. The pH of the FA was in alkaline nature, i.e. 7.9. The various analysis data and levels of total nitrogen, phosphorus and organic carbon were low but the porosity and water holding capacity were high. Due to very minute size of particles, FA showed high porosity and water holding capacity . The alkaline pH of FA and low level of nitrogen and phosphorus found in this research are in according with the previous findings . In addition, the level of sulfate, an important nutrient for B. juncea plants under FA stress conditions, was also high. Previously, it has been demonstrated that FA may enhance the level of sulfur in plants growing on FA amended soils .
The uptake of HMs (Fe, Zn, Cu, Pb and Ni) was found to be significantly high in FA than control (garden soil) except for Fe. The analysis of the results showed that the level of all the HMs was found to be increased, except for Fe with an increase in FA amendments from 25% FA to 100% FA. In previous findings, the levels of total HMs in soil/fly ash had been reported (47, 48). However, the total HMs present in the soil is not available to the plant grown therein a metals can be used as an indicator of bioavailability and toxicity of the HMs (47, 49). The accumulation of HMs (Fe, Zn, Cu, Pb, Ni and Cd) in the plants grown on fly ash amended soil is given in (Fig. 1-6). However, the concentrations and relative distribution of the metals in the plant differed . Among all the metals studied, the accumulation of Fe and Pb was found to be the highest showing the strong correlationship (r=0.99), whereas Ni accumulation was found minimum corelationship (r=0.48), among all the exposure of FA in different parts of the plant. At 100% FA, metal accumulation was found to be in the order Fe > Zn > Pb > Cu > Ni > Cd after 90 d of exposure and the metal translocations was found higher in root and shoot and lesser in leaves. With the increasing percentage of FA amendments in soil the HMs concentrations increased in different parts of plants with all exposures.
The accumulation of HMs in the B. juncea varies from root, stem and leaf. In all parts of the plant, the accumulation of Fe (Fig. 1) was found to increase significantly (P < 0.01) over all the amendments of fly ash for all the exposure periods as compared to their respective control. In 100% FA, Fe content decreased non-significantly (P < 0.05) in root, stem and leaves of the plant as compared to their respective. At 25% at all exposure periods, whereas Fe increased significantly in roots stem and leaves. At 90 d, the accumulation of Zn (Fig. 2) increased significantly (P<0.01) in all amendments of FA in the root, stem and leaves compared to control. At 100% FA, Zn content decreased significantly in leaves (P < 0.05), whereas it increased significantly in the stem and the root after 90 d as compared to the control. In the case of Cu (Fig. 3), an increase in the accumulation was observed with an increase in the fly ash amendment ratio up to 100% FA in all parts of the plant for all the exposure periods as compared to control. However, at 100% FA, a non-significant stem (P < 0.05) was observed in Cu accumulation in all the parts for all the exposure periods except a significant increase in roots and leaves (P<0.01) (90 d), compared to control. The analysis of the data showed that Pb content (Fig. 4) in roots, stem and leaves was found to increase significantly (p < 0.01) in the plants grown on fly ash amended soil and 100% FA as compared to control after 90 d except for a non-significant increase in leaves (p < 0.05). The accumulation in stem of Pb was found more than two folds higher compared with root an exposure 50% to 100% amendment. As compared to control, the accumulation of Pb (Fig. 4) was found to be significantly high (p < 0.01) in all parts of the plant grow on fly ash amended soil and 100% FA after 90 d of exposure. Out of all the tested metals, the lowest accumulation of Ni non significant (p < 0.05) (Fig. 5) was found in all parts of the plant. The level of Cd was found to increase significantly for all the exposure periods as compared to control at 100% FA and the highest accumulation of Cd with lowest significant (p < 0.05) (Fig. 6) was found in all parts of the plant respective with control. The correlation coefficient (r) was calculated between FA amendments metals and metals in tissues (leaves, stem and roots) after 90 d of exposure which have shown the values Fe (r=0.995); Zn, (r=0.886); Cu, (r=0.581); Pb, (r=0.856); Ni, (r=0.462); Cd, (r=0.389), that mean Cu, Ni and Cd were weekly correlated with FA metal concentrations .
In this study, a significantly high uptake of essential metals (Fe, Zn, Cu) in different parts of the plants was found which is in according several other findings (52, 54, 54]. In all the tested metals, generally the level of metals (Fe, Zn, Cu) were found more in the plants grown on 10% FA amendment as compared to other amendments. However, metal uptake depends on the bioavailability of the metal from the soil and the metabolic requirement of the metals in plant, but when present in excess, these and non-essential metals such as Pb and Ni can became extremely toxic . Thus, mechanisms exist to not only to satisfy the requirement of cellular metabolism but also to protect cells from toxic effects. On the other hand, the accumulation and translocation of Cu, Ni and Pb in the roots was found to be more than that in leaves which may be due to complexation and compartmentilization of HMs in root and stem and same trends was observed of accumulation and translocation of metals in naturally growing vegetation on fly-ash lagoons [54, 55]. In the present study, the level of toxic metals (Pb, Ni and Cd) was found to be more in the plants grown in fly ash and its different amendments. There is a remarkable difference in the metal-concentration in the leaf and root tissue with respect to different elements. The data the level of biomass was found to be an increased up to 58% with an increase up to 25% fly ash amendments, after that decrease the biomass up to 100% FA. At 25% at all exposure periods, whereas it increased biomass significantly (P < 0.01) as compared with respect to control [Fig 7]. However, a non-significant (P < 0.05) was observed in photosynthetic area in all the parts for all the exposure periods except in control. Same trend were observed in chlorophyll contents, a non significant (P < 0.05) in all the treatment and in all exposure with FA respective with control. In order to evaluate the level of PSI and PSII was found to be an increased significant (P < 0.01), with an increase up to 10% fly ash amendments in all the exposure and in all treatments and were non-significantly (P < 0.05), decreased lowered than those of control. Moreover the extent of suppression is PSI and PSII activities increase significantly with increase in the 25 to 100% FA exposure.
The accumulation and translocation of HMs in the B. juncea tissues are important technique to evaluate the role in remediation of HMs from contaminated sites. Among the metals, Fe, Zn and Cu had the highest BCF (BCF=934; 725 and 710), where the other Pb, Ni and Cd had the lowest BCF (BCF=45, 95 and 151). The BCF values found for the plants assessed in the previous studies by Kamal et al. , Mishra and Tripathi , and Peng et al.  were 55-152, 138-1000, 147, 93-876, and 191-228, respectively, showing equal trends for the values calculated for B. juncea in the present study. This finding suggests that B. juncea shows great potential for accumulating Fe, Zn, Cu, Pb, Ni and Cd from FA. The translocation of Pb, Ni and Cd showed the highest TFs concentration in the shoot (TFs=8.1, 4.3, 3.3), its TFs was higher than two and showing more translocation HMs compared with TFs of Fe, Zn and Cu (TFs=1.5, 1.3, 1.05), while in B. juncea was found higher than those (TFs=0.004-0.45) found by other researcher [58, 59] reported a finding was TFs 0.1 for Fe. Though its total Pb concentration in the plant was <700 mg kg-1 (Fig. 4). Though B. juncea showing the highest Pb concentration in the stem (167.3 mg kg-1; (Fig. 4). The BCF of Pb and Cu in this study was lower than that found by [57, 60] in P. thunbergii (BCF=5-58), and higher than those (BCF=0.004-0.45) reported by [56, 60]. According to Yoon , was mentioned BCF of 0.1 for Pb in P. distichum. Similar to Pb and Cu no plant species accumulated above 1000 mg kg-1. Though several plant species showed BCFs or TFs values were greater than one. The present studies for HMs Fe, Zn, Cu, Pb, Ni and Cd, growing on several exposure of FA were showing both the BCF and TF greater than one. However, the FA Pb and Cu concentration at this experiment over the 90 d was relatively, 295 and 267 Âµg g-1 and BCF values for these HMs Pb and Cu were (BCF=45 and 151) lower than those found in P. thunbergii (BCF=41-160) and higher than those obtained for (BCF=0.1-0.2) reported by Yoon . The present result research showed that high accumulation of metals in roots to transfer in shoot in its TFs was found more in Pb, in the upper part of the plant. However, the relationship between the two groups TFs (Fe, Zn, Cu) and TFs (Pb, Ni, Cd) was different [56-60]. Only TFs of all plants between Pb, Ni, and Cd were correlated with p < 0.01, where as no correlations p < 0.05, of TFs were found between Fe, Zn and Cu. This means a B. juncea, which was effectively translocating Pb, Ni and Cd, was also effective translocating Fe, Zn, and Cu compared less. However, (Fe, Zn, Cu) translocation in the plant were not related with (Pb, Ni, Cd) translocation. Poor correlation between the TFs of group of HMs and good correlation between the TFs of Fe, Zn, and Cu may indicate the elevated Pb, Ni, and Cd concentration can inhibit transfer of essential elements In B. juncea biomass . Among the six metals were tested, the plants growing on the FA were most efficient in taking up and translocating Fe, Zn, Cu and Pb. Low translocation of Ni and Cd indicates that plants were unwilling to transfer Ni and Cd from their roots to shoots possibly due to toxicity . The results obtained in the present study indicate that B. juncea is superior to other plant species assessed by Liu et al. , Kamal et al. , Mishra and Tripathi , and Peng et al. , Yoon et a. , Kim et al. .
We conclude that (i) B. juncea is quite good in resisting they heavy metals accumulated considerable amounts of Fe, Zn, Cu, Pb, Ni, and Cd in different plants parts and exhibited high tolerance to these metals. HMs Fe, Zn, Cu, Pb, Ni, and Cd analyses in plant tissues indicate that B. juncea is a good accumulator of these metals. Therefore, it could be used as heavy metal filter and remediation for metal polluted sites. Heavy metals are known to be accumulated by plants into vacuoles and thus keep the toxic metals away from the regions of the cell where essential metabolic events are taking place (iii) Brassica family come under the category of hyper accumulators of heavy metals. Earlier, B. juncea has been reported to have good potential to withstand higher levels of heavy metals. This ability of B. juncea to withstand well in spite of watering the plants with effluents containing very high levels of toxic heavy metals could be due to the potential 90% of their cells to compartmentalize the heavy/toxic metals in the vacuoles.