Comparative chelate-assisted phytoextraction of heavy metals by Jatropha curcas, Jatropha gossypifolia and Jatropha multifida cultivated on soil collected from selected dumpsites in Ekiti State, Nigeria.

Background: It is needful to design a process that will lead to the use of fast growing tropical plants for phytoextraction studies. Study Design: It is an analytical study. Place and Duration of Study: The study was conducted between October 2010 and June 2012 on selected dumpsites located in Ekiti State, Nigeria. Methodology: Bulk soil samples were collected from selected dumpsites on seven points at interval of 10 m, starting from top of the slope. Physicochemical and heavy metals content of soil were determined on bulk and fractional soil samples, using sequential extraction technique. The heavy metals were analyzed using atomic adsorption spectrophotometer, prior to plants’ cultivation. Heavy metal concentration of plant with and without 1g/kg EDTA was determined in different sections of plant by flame atomic absorption spectrophotometer, in other to determine Original Research Article Awokunmi; BJAST, 5(4): 353-370, 2015; Article no.BJAST.2015.033 354 their phytoextraction potentials. Results: The result revealed the mean value of pH (6.02±0.11-6.31±0.12), organic matter content (3.00±0.08-7.00±0.13%) and the CEC (59.20-62.40 mmol/kg) to be highest at Aba Egbira dumpsite. The result of sequential extraction revealed that heavy metal were distributed in fraction soil samples in the order 75>150>350>495>1000 μm, showing the effect of particulate site on the availability of heavy metals. Application of EDTA increased mobility of heavy metals from soil to shoot of plants with concentration of Cd in J. curcas, J. gossypifolia and J. multifida in the following range; (372.0-440.1; experiment, 150.2-186.6; control), (377.5-418.2; experiment, 142.2202.4; control) and (455.0-530.6; experiment, 245.0-259.2; control) while Cu and Pb ranged from (921.0-118.0; experiment, 405.0-821.0; control), (818.0-962.0; experiment, 442.0-650.0; control) and (1079.0-1138.0; experiment, 644.0-686.0; control) and Pb (384.4-426.2; experiment, 242.2283.0; control), (328.0-376.0; experiment, 159.0-186.2; control) and (417.0-436.0; experiment, 330.0-370.7; control) mg/kg respectively, in all dumpsites investigated. Bioaccumulation factor (BF), translocation factor (TF) and remediation ratio (RR) greater than one showed that they are effective in chelant-assisted phytoextraction. Conclusion: Therefore, the use the species of Jatopha are advocated for phytoextraction Cd, Pb


INTRODUCTION
The indiscriminate dumping of waste, most especially metallic and hazardous waste have become common practice in Nigeria, where most waste dumpsites are located close to residential area, markets and road side [1]. Accumulation of toxic heavy metals such as Cd, Zn Cu, Pb, Cr, As and their potential effects on human health, agriculture and ecosystem has since generated concern by environmentalist [2,3]. It is generally believed that these heavy metals are sometimes hazardous to health. Such health risks and hazards have been widely reported [4,5]. However, the clean-up of heavy metals from polluted soil is emergent and imperative. Leon et al. [6] reported that some plant species are endemic to metalliferous soils and can tolerate greater than usual amounts of heavy metals or other toxic compounds. Such plants have been employed in phytoextraction studies; and those that can accumulate high levels of heavy metals in their harvestable portion without symptoms of phtotoxicity [1,4,7]. Hyper accumulators are plants capable of accumulating extraordinarily high metal levels; such plant has genetic potential to clean-up heavy metals contaminated soils [8]. Ultimately, the plant's potential for phytoextraction depends on several factors, including the extent of soil contamination, metal availability for uptake into roots (bioavailabilty) and plant ability to intercept absorbs and accumulates metals in shoot [7]. Alvarez et al. [9] reported the potential use of fast growing pioneer plant species for phytoremediation strategies. Though, some of these are limited in scope due to low biomass production; as a candidate plant for phytoextraction must produce large biomass and resists phytotoxicity [1,10]. However, the phytoextraction efficiencies of these fast growing plants have been enhanced with chelating agents such as ethylene diamine tetracetic acid (EDTA), ethylene glycol tetra acetic acid (EGTA), citric acid; just to mention a few [1,11,12]. Jatropha curcas, Jatropha gossypifolia and Jatropha multifida are fast growing tropical plants, which are found to grow naturally on dumpsites. Therefore, the objective of the research was to investigate the role of EDTA on comparative phytoextraction of heavy metals by J. curcas, J. gossypifolia and J. multifida, with a view to applying them in cleaning up heavy metals contamination from the environment.

Collection, Preparation and Analysis of Soil Samples
Bulk soil samples were collected from four different dumpsites located at Aba Egbira (7.41° 288 1 N; 5.15° 661 1 E), Atikankan (7.37° 032 1 N, 5.13° 263 1 E) at Ado Ekiti ( Fig. 1) and Igbehin Street (7.29° 729 1 N; 5.13° 263 1 E), and Moshood road (7.39° 363 1 N; 5.13° 931 1 E) at Ikere Ekiti (Fig. 2). The samples were collected at the depth of 0-15 cm from seven points at interval of 10 m down the slope, using calibrated soil auger. Control samples were taken 200 m away from the last sampling point on each dumpsite. Soil samples were separately oven dried at 40°C grounded with a wooden roller and was allowed to pass through 4 mm mesh. Each sample was immediately placed in a previously leached polythene bags and tightly sealed.
In the pH determination, 2 g of each sample was weighed into a 50 ml beaker and 20 ml distilled/deionized water was added. The beaker was allowed to stand for 30 minutes with occasional stirring. The pH meter that had been standardized with potassium hydrogen phthalate and ammonium buffer was inserted into each solution and pH determined.
Organic matter content of soil samples were determined by loss on ignition according to AOAC [13].

Cation
Exchange Capacity (CEC) was determined by a method described by Jackson [14] by measuring 1 mol/L acetic acid solution, which pH was determined. The 2.5 g of soil was placed in a 20 ml beaker with 25 ml acetic acid; the solution was stirred for a period of one hour using a mechanical shaker. After agitation the mixture was left undisturbed for complete sedimentation of the soil particles. The pH of the supernatant was read, which the same as that of the mixture was practically. CEC was calculated by, CEC= mmol c /kg of exchangeable metal cations M equals 0.1kg of dry soil.

Sequential Extraction of Heavy Metals
The stepwise extraction of heavy metals in different phase of soil was carried out by a method prescribed by Tessier et al. [15] modified by Campanella et al. [16]. The soil had already been sieved by atomic testing sieve shaker to following sizes; >1000, >495, >350. >150 and >75 µm. The metal in aqueous phase of soil was extracted with 45 ml of 1M ammonium acetate at pH 5 with acetic acid under stirring for 24 hours. The exchangeable fraction was determined through extraction with 22.5 ml of hydroxyl ammonium chloride (1M) and 22.5 ml acetic acid (25%), with stirring at room temperature. Metal adsorbed on inorganic soil constituent was extracted with 12.5 ml of 0.1M HCl and stirring for 24 hours. Those associated with organic matter was treated with 12.5 ml of 0.5 M NaOH and stirring for 24 hours, later dried under IR lamp at 60°C and then digested with 4 ml of 65% HNO 3 and 2 ml of 40% HF in a microwave oven. The metals precipitated as pure or mixed solids were extracted using 12.5 ml of 8M HNO 3 and digested for 3 hours at 80°C. Lastly, the residual solid was digested with 4 ml of oxidizing mixture (HNO 3 : HCl) and 6 ml HF in Teflon recipient put in microwave oven. The sample was also shaken with 5.6g HBO 3 to avoid silica evaporation and diluted to 100 ml by deionized water. Concentration of heavy metals was determined using flame atomic absorption spectrophotometer (Perkin Elmer, model 306). The flame atomic absorption spectrophotometer was calibrated by preparing different concentrations of each metal by dilution from the stock (standard) solution for each cation. These were used in preparing linear curves, passing through the origin, for each metal. The wavelength (nm), lamp current (mA) and band width (nm) were adjusted for each metals as follows; Pb (217.0, 5.0, 0.4), Zn (213.9, 5.0, 0.4), Cd (228.8, 5.0, 0.4), Cr (357.9, 5.0, 0.4) and Cu (324.7, 5.0, 0.4) respectively. The flame is oxidizing blue air/ acetylene. These experimental procedures were repeated in all cases, where heavy metals concentrations were determined with flame atomic absorption spectrophotometer.

Plant Cultivation
Plastic pots each 15 cm high and 20 cm wide were filled with 5 kg soil samples from dumpsites and control sites. The Uniform seedlings of Jatropha curcas, Jatropha gossypifolia and Jatropha multifida obtained from Plant Science Department Herbarium of Ekiti State University, Ado Ekiti were planted on each pot marked 'experiment' (with application of 1.0 g/kg EDTA) and control (without EDTA application). EDTA application was carried out at preflowering, flowering and mature stages according to Sun et al. [7]. These plastic pots were place in green house, where the plants were watered throughout lifespan and no fertilizer was added. A Petri dish was placed under each pot to collect potential leachates, which were immediately added to each pot to prevent loss of target heavy metals.

Analysis of Plants
Matured plants were harvested, treated with 0.01 M HCl to remove external heavy metals; they were later separated into root, stem, leaf and fruit. The plants were air-dried and subsequently dried at 700°C in an oven to complete dryness [17]. They were digested with a solution of 3:1 HNO 3 :HClO 4 (v/v). Heavy metals concentration was determined using atomic absorption spectrophotometer.

Data Analysis
All data collected were replicates of three determinations and were analyzed statistically by calculating the mean and significant difference between multiple treatments by LSD test. Translocation factor (TF), the quotient of heavy metals concentration in plant to soil; bioaccumulation factor (BF), the ratio of contaminant in plant to soil; and remediation ratio (RR) were calculated. The remediation ratio was calculated according to this equation: where Mshoot is concentration of metals in the shoots of plants (mg/kg), Wshoot is the plant dry plant shoot (g); Msoil is concentration metals in soil measured in each pot (mg/kg) Wsoil is the mass of soil in the pot (g). The RR reflects the amount of metals extracted by a plant from soil, which indicate phytoextraction efficiency under chelant-induced experiments.

Physicohemical Characteristics of Soil Samples Collected from Dumpsites and Control Sites
The results of physicochemical characteristics of soil collected from selected dumpsites and control sites are depicted in (Table 1). Soil pH was observed to be comparatively lower in control sites than all dumpsites under investigation. The pH values were obtained to range between 6.02±0.11 and 6.31±0.12, where the highest was pH obtained at Aba Egbira dumpsite. It has been reported that lower pH value will favour availability, mobility and redistribution of heavy metals in various soil fractions [1].
The organic matter content of these dumpsites ranged from 3.00±0.08 to 7.00±0.13, with Aba Egbira having the highest. These values were higher compare to what was obtained on control sites (1.50±0.03 to 5.80±0.08), which could be as a result of dumping of organic wastes on these dumpsites. Moderate levels of organic matter content have been reported to indicate high mineral content [4].
Apart from high mineral content indicated by high organic matter content of dumpsites compared to control sites, higher cation exchange capacity (CEC) values of dumpsites compared to control sites ( Table 1) are characteristic of soil ionic elements contents, concentration of clay and mud, texture, degree of compression, levels of porosity and permeability [18]. Moderate values of CEC obtained indicated the redistribution potentials of ionic element in the soil horizon, though the ionic element may be depleted during rainfall; this was prevented by cultivating plants in green house [18].

Sequential Extraction of Heavy Metals from Dumpsites
(Tables 2 to 5) present the sequential extraction of heavy metals from bulk and fractional samples of soil from Aba Egbira, Atikankan, Igbehin Street and Moshood road dumpsites respectively. Information obtained from all dumpsites showed that particulate size of soil samples has significant effect on the concentration of total metals. The heavy metals concentration in the size fractional samples were found to vary in the order 75>150>350>495>1000 µm, showing that the size 75 µm has the highest concentration of heavy metals in all extractive steps. This same observation was reported by Pagnanelli et al. [19], while trying to explore the same principle on river sediments of abandoned pyrite mining area of Bocchegiano. The classification adopted here (75 to 1000 µm) was based on Dia-zorita and Grosso [20] conventional classification; >2000 µm; gravel, 50-200 µm; sand, 20-50; silt and, 2 µm; clay. This kind of classification was based on texture only not accounting for the mineralogical composition of different particle sizes of samples.
Sequential extraction revealed that antropogenic and mineralogical composition of heavy metals in all extractive phases of soil from all dumpsite investigated (Tables 2, 3, 4, 5 and 6). The model prescribed by Tessier et al. [15] and modified by Campanella et al. [16] was employed. The results revealed that all heavy metals investigated were distributed in all extractive phases, with over 50% of Cd and Pb found in anthropogenic phases of soil, in all dumpsites investigated. (Table 6) provided information on the distribution patterns of heavy metals in each extractive phase, thereby helping in the source identification of heavy metals from the selected dumpsites (lithogenic or anthropogenic). These metals were classified depending on their degree of association with each extractive phase; information regarding bioavailability of these metals is needful in planning phytoextraction The reproducibility of the sequential extraction procedures were carried out by comparing the total amount of metals extracted by different reagents during the sequential extraction procedure with the results of the total digestion according to Yuan et al. [21]. The recovery of sequential extraction was calculated as follows:   It was also observed that there was a good agreement between the concentration of metals from the total digestion and sums of six fractions (Table 6), showing reliability and reproducibility of results. The recovery a is the percentage of heavy metals present as result of human activities while Recovery b is the percentage of heavy metal in the mineralogical matrix of soil.

The Effect of EDTA on Plant Biomass
The heights of three species of Jatropha cultivated on soil of dumpsites with and without application of EDTA are shown in (Fig. 3  Hyperaccumulators are species capable of accumulating metals at levels 100-fold greater than those typically measured in the shoot of non-accumulators plants. Thus, a hyperaccumulator of Cd will concentrate more than 100mg/kg Cd [4]. From the results presented, the three species of Jatropha could be potentially good candidate for phytoextraction of Cd from contaminated sites.     The results presented were lesser than the threshold value reported by Baker et al. [4], for Cu and Pb except at Abaegbira and Atikankan dumpsites, where J. curcas accumulated Cu in its shoot at levels greater than 1000 mg/kg. Also J. multifida accumulated Cu in its shoot at levels greater than 1000 mg/kg in all dumpsites investigated.

Comparative EDTA Accumulation of Metals by Plants
However, there is possibility of proposing the three species of Jatropha considered in this report for phytoextraction; a candidate for plant for phytoextraction must accumulate high concentrations of target metals in their above ground masses [24].
Conversely, comparatively lower concentrations of Cr and Zn were observed in all Jatropha species in all dumpsites investigated (Tables 7,  8 , 9), these values fell below the threshold values reported by Baker et al. [4]. However, heavy metals accumulation in the harvestable tissues of plant does not only confer hyperaccumulation tendencies on plant, phytoextraction efficiency should also be considered [1,7,25].

Phytoextraction Efficiency
The phytoextraction efficiency of a supposed hyperaccumulators is evaluated by bioaccumulation factor (BF), translocation factor (TF) and remediation ratio (RR) [7]. According to the results presented in (Tables 10, 11 and 12), BF, TF and RR values were greater than one on the application of EDTA for Cd, Cu and Pb indicating the hyperaccumulative tendencies of the three species of Jatropha by the target heavy metals. These values were lesser than the ones observed in control.          However EDTA and other synthetic chelators had been reported to have positive impact on the phytoextraction of heavy metals from contaminated sites [1,10]. The concentration of heavy metals in different tissues of plant on the four dumpsites varied significantly at p≤0.05 (LSD test), showing the effect of different soil conditions on these dumpsites. Also there were gradual reductions in the BF, TF and RR values in the order of moshood road < Igbehin street < Aba Egbira.