Accumulation and distribution of lead (Pb) in plant tissues of guar (Cyamopsis tetragonoloba L.) and sesame (Sesamum indicum L.): profitable phytoremediation with biofuel crops

Abstract Contamination of lead indicates one of the major threats to soil system. Phytoremediation technique utilized plants which are able to tolerate and accumulate metals within in their tissues. It has recently been suggested that biofuel plants are more suitable for both utilization and remediation of metal contaminated soil. This study reported Pb phytoremediation potential of Cyamopsis tetragonoloba L. in comparison with Sesamum indicum L. in the framework of a pot-experiment. Plants were subjected to seven Pb concentrations (0, 100, 200, 400, 600, 800 and 1000 mg kg-1 soil) for 12 weeks. Our results demonstrated that both C. tetragonoloba and S. indicum were able to tolerate Pb concentrations up to 1000 mg kg-1 which confirms the plant ability to grow well in higher Pb levels. Significant metal accumulation was observed in root along with reduced biomass for both plants species. Furthermore, both plant species could possibly be used for phytostabilization, with success in marginally polluted soils where their growth would not be impaired and decontamination of Pb could be maintained at satisfying levels. However, bioconcentration factor (BCF), bioaccumulation coefficient (BAC) and translocation factor (TF) values proposed that C. tetragonoloba was more efficient for phytoremediation than S. indicum at higher Pb levels.


Introduction
Soil pollution with toxic trace metals and their accumulation in soil is of great concern in agricultural production owing to the adverse effects on crop growth i.e., metal phytotoxicity, and soil micro-organisms (Nagajyoti, Lee, & Sreekanth, 2010). Toxic trace metals have entered into agricultural soils primarily because of rapid industrialization, inappropriate utilization and disposal of toxic trace metal containing wastes, excessive use of fertilizers and pesticides (Amel et al., 2016;Bashmakov, Lukatkin, Anjum, Ahmad, & Pereira, 2015), which become hazardous to human and environmental health. Trace metals, such as lead (Pb), cadmium (Cd), nickel (Ni), cobalt (Co), iron (Fe), zinc (Zn), chromium (Cr), iron (Fe) (Nagajyoti et al., 2010), are major environmental pollutants, particularly in areas with high anthropogenic activities.
Among soil pollutant, lead (Pb) is one of the toxic metal pollutants (Shahid, Pinelli, Pourrut, Silvestre, & Dumat, 2011); widely used in many industrial processes and occurs as a contaminant in all environmental compartments including soil, water, and living organisms (Punamiya et al., 2010); known to induce a broad range of toxic effects to morphological, physiological, and biochemical activities of living organisms. Lead impairs plant growth such as root elongation, seed germination, seedling development, transpiration, chlorophyll production, lamellar organization in the chloroplast, and cell division (Gupta, Huang, Yang, Razafindrabe, & Inouhe, 2010;Maestri, Marmiroli, Visioli, & Marmiroli, 2010). However, the extent of these effects varies and depends on the lead concentrations, the duration of exposure, the intensity of plant stress, and the particular organs studied.
Lead occurs naturally in the earth's crust (Arias et al., 2010) and its natural levels remain below 50 mg kg −1 (Pais & Jones, 2000), but anthropogenic activities often modify the amount and nature of lead species present in soil. Anthropogenic sourced lead (Pb) include leadbased paints, lead arsenate pesticide application, gasoline, coal burning, explosives, lead batteries and from the disposal of municipal sewage sludge (Tian, Lu, Yang, et al., 2010;Zheng, Liu, Lütz-Meindl, & Peer, 2011).
In soils, lead may occur as a free metal ion, complexed with inorganic constituents (e.g., and Cl − ), may exist as organic ligands (e.g., amino acids, fulvic acids, and humic acids); or may be adsorbed on to particle surfaces (e.g., Fe-oxides, biological material, organic matter, and clay particles) (Uzu, Sobanska, Aliouane, Pradere, & Dumat, 2009;Vega, Andrade, & Covelo, 2010). Generally, lead may accumulates on the surface layer of soil (Cecchi et al., 2008) and caused disturbance of soil system. Thus, in order to reduce the level of lead from the natural environment many developed countries has tried to reduce lead emissions using leadfree fuels (Adriano, 2001).
Remediation of lead-polluted soils by traditional physical and chemical methods are not suitable for agricultural lands (Oh, Li, Cheng, Xie, & Yonemochi, 2013), required large investments and technological resources. Phytoremediation is a promising technique to remediate metal-contaminated soils because it offers in situ advantages, cost effective, and less energy-intensive than high-cost traditional clean-up methods. Phytoremediation involves the use of plants to reclaim high amount of metals from soil into the harvestable parts (i.e., underground roots and aboveground shoots) (Mahmood, 2010). The harvested biomass then can be safely processed by drying, ashing, composting, and storage at a landfill, anaerobic digestion, pure plant oil production, microbial, physical, or other chemical means (Ginneken et al., 2007).
The capacities of heavy metal uptake and accumulation, mechanisms of metal concentration, exclusion and compartmentation vary among different plant species and also between various parts of plants (Lone, He, Stoffella, & Xe, 2008;Sharma, Singh, & Manchanda, 2014). Various plant species belongs to botanical families, in particular the Brassicaceae, Asteraceae, Fabaceae, Poaceae, and Chenopodiaceae showing phytoremediation potential, are well documented in the literature (Stanislaw & Gawronski, 2007;Anjum, Umar, & Iqbal, 2014) but few or less work has been reported on Cyamopsis tetragonoloba L. and Sesamum indicum L.
Guar (Cyamopsis tetragonoloba L.) is one of the important annual legume crops belongs to family Fabaceae. The endosperm of guar seeds contain gum, which is gaining importance as non-food item (Ashraf, Akhtar, Sarwar, & Ashraf, 2002). Recently, the oil industry has started using guar gum in hydraulic fracturing in which high pressure is used to crack rock. The increased use of guar gum in oil fracking has boosted the demand of guar woldwide (Abidi et al., 2015;Deepak, Sheweta, & Bhupendar, 2014). A key feature of legumes as a resource for phytoremediation is their role in providing additional N-compounds to the soil, thus improving soil fertility and ability to support biological growth (Xiuli et al., 2013;Hao et al., 2014). On the other hand, sesame (Sesamum indicum L.) is an economically important oil seed crop of family Pedaliaceae, planted in arid and semi-arid regions of the world (Elleuch, Besbes, Roiseux, Blecker, & Attia, 2007) including Pakistan, used as an alternative feedstock for the production of a biodiesel fuel (Saydut, Duz, Kaya, Kafadar, & Hamamci, 2008). The methyl ester extracted from sesame seeds can effectively be utilized as petrodiesel (Ahmad et al., 2011).
This study was set up to investigate a plant species that could tolerate high toxicity of Pb treatments in soil. Both the plant species i.e., C. tetragonoloba and S. indicum have fast growth and development rate also high aboveground biomass. It was estimated that plant species with high tolerance could then be utilized for phytoremediation. Considering above facts, the objectives of this research were: (1) to study the phytotoxicity of lead on plant growth parameters; (2) to determine the accumulation and distribution of lead in plant parts growing on a lead-polluted soil, and (3) to identify a potential candidate for effective phytoremediation practice.

Seeds procurement and sterilization
Seeds of the guar variety BR-99, from the Institute of Fodder Research Program and sesame variety TH-6, were collected from the Institute of Oilseeds Research Program, National Agricultural Research Centre (NARC), Islamabad (Table 1). In order to avoid any microbial contamination, seeds were surface sterilized with 0.1% HgCl 2 for 10 min and washed 7 times with sterilized water (Pourakbar, Khayami, Khara, & Farbidina, 2007).

Experimental soil collection
Soil samples were collected from Pb-free agricultural fields located in Jamshoro, Sindh, Pakistan, at depth of 0-15 cm using hand shovel. Equidistant (2 m) collected samples were homogenized to prepare one bulk sample. For the greenhouse experiment, soil was air dried at room temperature for 15 days and ground to a final particle size of 2 mm. To enhance soil porosity, sand was mixed in 3:1 proportion with soil samples soil.

Pot experiment
Plastic pots were filled with 5 kg sieved soil, after which soil was artificially spiked with Pb (aqueous solution) using Pb(NO 3 ) 2 salt to each pot in increasing concentrations (100, 200, 400, 600, 800, 1000 mg kg −1 ), each with three replicates and kept for 2 weeks to attain equilibrium. The clean soil without Pb spiking was used as control. Pots were arranged in a completely randomized design. After 15 days of equilibration, 20 surface-sterilized seeds were sown per pot. One week after seed germination, plants were thinned to 5 per pot. A plastic tray was kept below the treatment pot to collect any leachate, which was returned to the pots at next watering. The experiment was conducted in a greenhouse for 3 months. Any symptoms of metal toxicity exhibited by plants were visually noted during the experimental period. Plants were harvested 12 weeks after germination. Soil samples (in triplicate) were also collected for analysis of Pb content by an Atomic Absorption Spectrophotometer (Perkin-Elmer, AAnalyst 800). Plant growth and biochemical parameters were also measured.

Morphological parameters
Plants were taken from each replicate pot to measure morphological parameters. Root length, shoot length were measured with the help of scale. Root and shoot fresh weights were also measured with the help of analytical balance. Plant samples were air dried for one week. Then, oven-dried at 80 °C to a constant weight and dry weights were recorded.

Estimation of photosynthetic pigments
Photosynthetic pigments, in fully expanded leaves from each treatment, were extracted using 0.5 g of fresh material, ground with 10 mL of 80% aqueous acetone. After filtering, 1 mL of the suspension was diluted with a further 2 mL of acetone, and optical density were determined with a UV-visible spectrophotometer (Biochrom Libra S22), using two wavelengths (663 and 645 nm) against blank. Chlorophyll a (Chl a), chlorophyll b (Chl b) and total chlorophyll contents (mg g −1 f.w) were obtained by calculation, following the method of Arnon (1949).

Determination of tolerance index
Tolerance Index (TI) is expressed as the ratio between the growth parameters (root/shoot length, root/shoot fresh, and dry weight) of the plants in contaminated soil in relation to the growth parameters of plants from non-polluted soil calculated according to Chen et al., 2011.

Quality control and quality assurance
All the glassware used during the present experimentation was of high quality, acid resistant Pyrex glass. The analytical grade reagents with a certified purity of 99% and stock metal standard solution (1000 ppm) for (2) Chlorophyll a = (12.7 × OD663) − (2.69 × OD645) Tolerance index (%) = Growth parameter Pb contaminated soil Growth parameter Control soil × 100  method. To quantify the Pb content in soil, sample of 1 g soil was digested by means of wet acid digestion method through HNO 3 along with HCl in proportion of 3:1 (v/v) and heated on a hot plate for 2 h until the solution becomes clear. After cooling, the volume was completed to 50 mL by adding distilled water. The solution was filtered through Whatman's filter paper and consequently, examine for Pb contents with Atomic Absorption Spectrophotometer.

Evaluation of phytoremediation efficiency
To evaluate the phytoextraction/phytostabilization potential of C. tetragonoloba and S. indicum, the following factors were calculated:

Bioconcentration factor (BCF)
The bioconcentration factor (BCF) was represented as the Pb concentration ratio in plant roots to soil, calculated as follow:

Bioaccumulation coefficient (BAC)
The bioaccumulation coefficient (BAC) was expressed as a ratio of Pb in shoot to that in soil, calculated as follow:

Translocation factor (TF)
The translocation factor (TF) was determined as a ratio of heavy metals in plant shoot to that in plant root, calculated as follow:

Statistical analysis
All experiments were conducted with three replicates and the data collected were analyzed statistically using PASW ® Statistics 18 (SPSS Inc., Chicago, IL, U.S.A.). To compare the means of the treatments, analysis of variance (ANOVA) was performed followed by Duncan's multiple range Post Hoc tests at significance level of p < 0.05, to observe significance difference among means.

Soil characterization
The soil was sandy loam with slightly acidic to neutral pH (6.89), organic carbon (2.20%), organic matter contents (3.79%), and electrical conductivity (1662 μS cm -1 ). Among soil properties, soil pH has strong effects on solubility and speciation of metals both in the soil and (8)

Plant samples preparation and Pb determination
To determine Pb accumulation in different plant tissues (i.e., root, stem, leaf, and pod), harvested plant parts were rinsed thoroughly by means of tap water, and after that with deionized (DI) water to clean adhered components of soil, then oven-dried at 80 °C till steady weight. The oven-dried plant tissues were ground carefully using an electric grinder and passed through a 1.0 mm mesh strainer. The ground plant tissue samples (0.5 g) were digested with 12 mL of 3:1 (v/v) HNO 3 /HClO 4 mixtures on a hot plate for 2 h. After cooling, the digested solution was filtered through Whatman's filter paper and finally makes up the volume up to mark 50 mL by adding deionized (DI) water. The quantification of lead (Pb) in respective tissues was carried out using atomic absorption spectrometer (Perkin-Elmer, AAnalyst 800) equipped with a lead cathode lamp, under optimum analytical conditions for the estimation of lead. The optimum conditions for AAS used throughout these studies given in Table 4. The standard calibration method was adopted for the quantification of results and triplicate samples were run to ensure the precision of quantitative results. The Pb concentration and accumulation in plant root and shoot were calculated by the following formula: (Monni, Salemaa, & Millar, 2000)

Soil sample preparation and Pb determination
Soil samples were air dried at room temperature, ground, mixed well, and kept in plastic (polyethylene) sealed lock bags used for subsequent metal analysis. Digestions of soil samples were done using aqua regia (6) Pb Conc. mg∕kg = AAS interpretation (reading) × dilution factor dry wt.of plant tissues Pb Acc. μg∕plant = Pb conc. × dry wt.of plant tissues of Pb was significantly (p < 0.05) inhibited growth in terms of root and shoots length in C. tetragonoloba and S. indicum (Table 5). In C. tetragonoloba and S. indicum, the longest roots (17.85 and 13.76 cm) and shoots (134.30 cm and 119.04 cm) were observed in control treatments with 0 mg kg −1 Pb, respectively. Pb is not an essential nutrient and at high concentrations inhibits plant growth. At 1000 mg Pb kg −1 concentration, root length decreased by 13.60 cm and 10.67 cm while shoot length reduced by 100.47 cm and 85.33 cm in both C. tetragonoloba and S. indicum, respectively. Higher concentration of Pb-induced morphological changes in plants i.e., root and shoot elongation in different plants showed a great sensitivity to excessive Pb. Lead exposure in plants strongly limits the growth and development of seedlings (Gopal & Rizvi, 2008). At high concentrations, lead inhibits the growth of roots which directly influences root growth and decreasing the capacity of water and nutrients absorption, ultimately leading to reduction in growth of the plant species. Plant growth is an important parameter used to assess the survival and adaptation of a given species to environmental factors that decisively control biomass production. Addition of Pb-inhibited biomass growth of C. tetragonoloba and S. indicum. Pb contamination showed significant (p < 0.05) negative impacts on both fresh and dry biomass of C. tetragonoloba and S. indicum (Table 5). Compared to control treatments, Pb stress at 1000 mg kg −1 reduced root fresh weight (8.15 g plant −1 and 5.92 g plant −1 ) and shoot fresh weight (21.13 g plant −1 and 9.15 g plant −1 ) in C. tetragonoloba and S. indicum, respectively. The dry biomass follows the same trend as fresh weight. Compared to control treatments, Pb stress at 1000 mg kg −1 reduced root dry weight from (5.09 g plant −1 and 3.70 g plant −1 ) and shoot dry weight (10.90 g plant −1 and 5.87 g plant −1 ) in C. tetragonoloba and S. indicum, respectively. Plant biomass is a significant indicator for characterizing the growth performance of plants in the presence of heavy metal. Under severe lead toxicity stress, plants displayed obvious symptoms of growth inhibition. Plant biomass can be restricted by high doses particularly in the soil solution (Fanrong et al., 2011), whereas, organic matter content has a strong influence on cation exchange capacity, buffer capacity as well as on the retention of heavy metals. Thus, metals present in organic rich soils contaminated with heavy metals are less mobile and less bioavailable than metals present in mineral soils (Olaniran, Balgobind, & Pillay, 2013). The mobility and availability of heavy metals in the soil are generally low, especially when the soil is high in pH, clay, and organic matter (Rosselli, Keller, & Boschi, 2003). Dede et al. (2012) reported that Pb is an immobile metal in soil, since it readily forms a precipitate with a low aqueous solubility within the soil matrix, and in many cases it is not readily bioavailable. So, in accordance with soil properties such factors may act individually or in combination with each other and may alter the soil behaviour of the lead present, as well as the rate of uptake by plants.

Pb-induced phytotoxic effects
Gradual increase in Pb concentration significantly (p < 0.05) reduced all tested growth and biochemical parameter in two plant species. In the current investigation, the germination percentage of C. tetragonoloba and S. indicum seeds were significantly (p < 0.05) affected at 1000 mg Pb kg −1 as compared to control ( Table 5). The reduced germination percentages (64.76 and 80.00%) were recorded at 1000 mg Pb kg −1 in C. tetragonoloba and S. indicum, respectively. It has been well documented in the literature that germination is an essential process to determine the effects of Pb toxicity on different plant species. Germination is strongly inhibited at very low concentrations of Pb, even at micromolar levels (Kopittke, Asher, Kopittke, & Menzies, 2007). In this study, C. tetragonoloba and S. indicum seeds were able to germinate in the presence of low to moderate level of Pb concentrations in soil. The inhibition of seed germination may also result from the interference of lead with protease and amylase enzymes (Sengar et al., 2009).
Seedling's height (root and shoot length) is also among primary determinants of plant growth. Addition Table 5. phytotoxicity of pb on germination and growth parameters of Cyamopsis tetragonoloba l. and Sesamum indicum l.
notes: similar letters in same column are statistically non-significant according to duncan's Multiple Range Test (p < 0.05), data are means (n = 3 ± sd), a in superscript represent significantly highest followed by later alphabets for lower means. of lead exposure. Decrease in plant biomass may be associated with disturbed metabolic activities due to reduced uptake of essential nutrients when grown under Pb stress (Gopal & Rizvi, 2008;Kopittke et al., 2007). Lead, if present at high levels, can interfere with normal enzyme functions in plants and especially photosynthesis which is one of the plant processes most drastically affected by Pb toxicity. It is evident that chlorophyll content in leaves of C. tetragonoloba and S. indicum were influenced by Pb treatments. Chlorophyll contents decreased significantly (p < 0.05) with gradual increase in Pb concentration from 0 to 1000 mg kg −1 (Figure 1). In C. tetragonoloba and S. indicum, the maximum amount of chlorophyll contents were measured at control, while the lowest concentration of chlorophyll a (0.63 and 0.51 mg g −1 ), chlorophyll b (0.35 and 0.21 mg g −1 ), and total chlorophyll (0.98 and 0.72 mg g −1 ) was at 1000 mg Pb kg −1 , respectively. A decreased rate of photosynthetic pigment accumulation in association with Pb treatment may be the consequence of peroxidation of chloroplast membranes due to increased level of ROS generation (Srinivasan, Sahi, Paulo, & Venkatachalam, 2014). Lead may inhibit chlorophyll biosynthesis by impairing the uptake of essential photosynthetic pigment elements, such as magnesium, potassium, calcium, and iron (Gopal & Rizvi, 2008) which disturbed photosynthesis with the substitution of divalent cations by lead.
Tolerance indices (TIs) were also affected by Pb toxicty. Plant tolerance to heavy metal stress is estimated based on their root and/or shoot growth inhibition by the metal present in a medium (Srinivasan et al., 2014). According to Audet and Charest (2007), if TI values less than 1, this indicates that the plant suffered a stress due to metal pollution with a net decrease in biomass. By contrast, if TI values greater than 1, suggest that plants have developed tolerance with a net increase in biomass (hyper accumulator). If TI values equal to 1, the plant is unaffected by metal pollution, indicate no difference relative to control treatments. In this study, both the plant species (C. tetragonoloba and S. indicum) had notes: similar letters are statistically non-significant according to duncan's Multiple Range Test (p < 0.05), data are means (n = 3 ± sd), a in superscript represent significantly highest followed by later alphabets for lower means. Table 6. effect of pb stress on the growth tolerance indices (TIs) of Cyamopsis tetragonoloba l. and Sesamum indicum l.
notes: similar letters in same column are statistically non-significant according to duncan's Multiple Range Test (p < 0.05), data are means (n = 3 ± sd), a in superscript represent significantly highest followed by later alphabets for lower means.

Plant species
Pb applied  Srinivasan et al. (2014) reported that growth inhibition is a common response to heavy metal stress and is also one of the most important agricultural indices of heavy metal tolerance.

Pb concentration in plant tissues
Concentration trends of Pb uptake among the different plant tissues (root, stem leaf, and pod) in both C. tetragonoloba and S. indicum are presented in

Pb accumulation in root and shoot
Beside concentrations, a total amount of metals accumulated in the shoots is considered as the most important parameter to evaluate the potential of phytoextraction in plants (Hanen et al., 2010). A significant rise in Pb accumulation per plant in root and shoot of both the plant species varied with respect to Pb treatments are expressed in (Figure 2

Conclusions
From this study, it has been concluded that none of the plant species were identified as metal hyperaccumulators and not suitable for phytoextraction. Considering the rapid growth, biomass, accumulation efficiency, adaptive properties, tolerance, restoration potential towards Pb, and being leguminous and fast growing crop, C. tetragonoloba can be used as an effective tool to decontaminate Pb-polluted soils in quick and successive flushes than S. indicum. Furthermore, the value of BCFs, BACs, and TFs suggests that both the plant species were suitable for Pb phytostabilization but C. tetragonoloba was more potential candidates for phytoremediation than S. indicum. Moreover, both plants species have economic and ecological values. These plants can both remediate metal-contaminated sites and produce valuable biomass, which can bring income for the owners of the sites. The harvested biomass could then be incinerated and disposed off or the accumulated metal could also be recovered for commercial uses and thus reused as biofuel. Further work is needed to understand the mechanisms of metal absorption and tolerance in plants.

Disclosure statement
No potential conflict of interest was reported by the authors.
to them are dependent on the metal, its bioavailability, the plant species and its metabolism (D'Souza, Varun, Pratas, & Paul, 2013).

Phytoremediation potential
The bioconcentration factor (BCF), bioaccumulation coefficient (BAC), and translocation factor (TF) values (Table 7) help to identify the suitability of plants for phytoremediation (i.e., phytoextraction or phytostabilzation) by explaining the accumulation characteristics and translocation behaviours of metals in plants. Plants with BCF, BAC, and TF values > 1 are considered promising phytoextractor, suitable for phytoextraction, while those with BCF and TF < 1 are not suitable for phytoextraction/phytostabilzation (Fitz & Wenzel, 2002). However, plants with BCF > 1 and TF < 1 are considered potential phytostabilizers (Mendez & Maier, 2008), suitable for phytostabilziation (immobilization). Between tested plant species, C. tetragonoloba had BCF values > 1 from 100 to 600 mg Pb kg −1 while, BAC and TF values < 1 at all Pb treatments, indicating that C. tetragonoloba could not be a high efficiency plant for Pb translocation from root to the shoot; but suitable for Pb phytostabilzation. In contrast, S. indicum had the BCF > 1 from 100 to 400 mg Pb kg −1 , while also have the BAC and TF values < 1 at all Pb treatments, notes: similar letters are statistically non-significant according to duncan's Multiple Range Test (p < 0.05), data are means (n = 3 ± sd), a in superscript represent significantly highest followed by later alphabets for lower means.