Lantana camara–A Predictive Ecological Bioindicator Plant for Decontamination of Pb Impaired Soil under Supplemented Scenario

Accepted Manuscript Title: Lantana camara- A Predictive Ecological Bioindicator Plant for Decontamination of Pb Impaired Soil under Supplemented Scenario

Author: ALARIBE, Frank Ogenna and AGAMUTHU, Pariatamby PII: DOI: Reference:

S1002-0160(17)60365-5 10.1016/S1002-0160(17)60365-5 NA

To appear in: Received date: Revised date: Accepted date:

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Please cite this article as: ALARIBE, Frank Ogenna and AGAMUTHU, Pariatamby, Lantana camara- A Predictive Ecological Bioindicator Plant for Decontamination of Pb Impaired Soil under Supplemented Scenario, Pedosphere (2017), 10.1016/S1002-0160(17)60365-5. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT PEDOSPHERE Pedosphere ISSN 1002-0160/CN 32-1315/P

doi:10.1016/S1002-0160(17)60365-5

Lantana camara- A Predictive Ecological Bioindicator Plant for Decontamination of Pb Impaired Soil under Supplemented Scenario

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ALARIBE, Frank Ogenna.1, *and AGAMUTHU, Pariatamby.1 1 Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur(Malaysia). *Corresponding author.E-mail:[email protected]

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ABSTRACT Heavy-metal extraction and processing from ores releaseelements into the environment. Soil,being “unfortunate” sink, its bionomics are impaired and affected by metal pollution. Metals sneak into the food chain and pose risk to humans and other edaphic-dependent organisms. For decontamination, the use of ecosystemfriendly approach involving plants is known as phytoremediation. In this study, different Pb concentrations (80, 40, 20, and 10 mg/kg) wereused to contaminate a characterizedsoil, supplemented with empty fruit bunch (EFB) or spent mushroom compost (SMC),with non-edible plant-Lantana camara. Pb removal ranged from 45.51% to 88.03% for supplemented soil and 23.7% to 57.8% for unsupplemented soil (p < 0.05). EFB-supplemented and L. camara remediated soil showed the highest counts of heavy-metal-resistant bacteria “HMRB” (79.67 × 106 CFU/g soil to 56.0 × 106 CFU/g soil), followed by SMC (63.33 × 106 CFU/g soil to 39.0 × 106 CFU/g soil). Aerial metal uptake was 32.08 ± 0.8 to 5.03 ± 0.08 mg/kg dry wt., and bioaccumulation factor was 0.401 to 0.643 (p < 0.05). Half-life’s (t1/2) of 7.24–2.26 days (supplemented), 18.39–11.83 days (unsupplemented), and 123.75–38.72 days (soil controls) were respectively recorded. Freundlich isotherms showed that the intensity (n) of metal absorption ranged from 2.44 to 2.51 for supplemented soil with regression coefficients (R2) between 0.9012and0.9840. The computed free-energy change (ΔG)for Pb absorption ranged from−5.01 kJ mol−1 K−1 to 0.49 kJ mol−1 K−1 for EFB and−3.93 kJ mol−1 K−1to 0.49 kJ mol−1 K−1 for SMC. ______________________________ Key Words: heavy-metal, Lantana camara, phytoremediation, soil, supplements

INTRODUCTION

Lead (Pb)-contaminated soils, sludges, and sediments are the major causes of Pb contamination of surface water and groundwater (Gonzaga et al., 2006; Wang & Zhao, 2009),as well asdrinking water and into food chains(Xiaoe et al., 2005; Harmin et al., 2013). Pb contamination also induces oxidative stress (i.e., free-radical formation) by facilitating reactive oxygen species, which can undermine antioxidant defenses and even lead to cell destruction or death (Krystofova et al., 2009; Hazrat et al., 2013). Increasing industrialization and other anthropogenic activities (e.g., mining, smelting, and atmospheric deposition) that interfere with natural biogeochemical cycles are responsible for soil metal contamination (Khan et al., 2010; Luji et al., 2015; Zhou et al., 2007). The consequences of soil metal contamination include soil nutrient/quality degradation, diverse edaphic stress, ecosystem niche disturbances of native microflora, and trophic level metal biomagnification (Xia, 2004). Any metal species may be considered as contaminant, if it existswhere it is unwanted or in a form or concentration that triggers environmental emergencies irrespective of the source. Metal amounts in soil may 2

ACCEPTED MANUSCRIPT range from less than 1 mg/kg to 100 000 mg/kg (Long et al., 2002). However, heavy metals are the generic nomenclature forgroup of elements with atomic density greater than 6 g/m3 (Meera and Agamuthu, 2011). Jorge et al., (2005) reported that the normal concentration of heavy metals in soils, with the exception of metalliferous soils, is harmless to living organisms.

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The persistence of metals in the environment, with its potential ecological risks,demands remediation attention (Baylock and Huang, 2000). Pb was estimated to have a soil retention time of about 150 to5000 years, and also predicted to maintain a high concentration for approximately 150 years after soil had been supplemented with sludge, placing Pb among the most recalcitrant elements in soil metal-pollutions (Nandakumar et al., 1995; Xiaoe et al., 2005). Furthermore, the average biological half-life of Cd has been projected to be approximately 18 years, and 10 years once inside human body (Salt et al., 1995). In soil, heavy metals are stable and their chemical structures enables them bind to soil humic substances, which contains functional groups capable of interacting with metal ions to form complexes. This could facilitate environmental pollution, migration, and transformation of contaminants (Stevenson, 1985). Phytoremediation is the use of plants to remove, transfer, stabilize, or degrade contaminants in soil, sediments, and water (Greipsson, 2011). Technically, it is the capacity of the roots to absorb, concentrate, and translocate the toxic metals from the edaphic zone to above ground plant parts, before being harvested as a way of permanently removing the metal from the site. Phytoremediation is novel, applicablein situ, cost-effective, efficient, and ecosustainable (Lone et al., 2008; Sarma, 2011), hence, a solar-driven remediation strategy (Sing & Prasad 2011; Vithanage et al., 2012; Hazrat et al., 2013). Plant metal tolerance is the key prerequisite for metal accumulation during phytoremediation. In practice, the metal-accumulating plants are seeded or transplanted to metal-contaminated soil and cultured using conventional agricultural methods.

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The addition of organic supplements, such as green or agricultural waste compost, and sewage sludge to soil, improves their biological and physicochemical conditions, thereby increasing N for plant growth and microorganism metabolism. These conditions facilitate the transformation of trace elements by influencing their bioavailability and phytoabsorption (Walker et al., 2004). The pH, cation exchange capacity (CEC), soil type, and characteristics of the supplements are imperative in soil metal mobilization (Clemente et al., 2010).

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Detailed reviews and studies on plant-based remediation technology have been widely reported in Garbisu and Alkorta (2000), Alkorta et al., (2004), Satpathy and Reddy (2013), andNanthi et al., (2014). At the moment, Brassica juncea and Thlaspi caerulescens are among the most viable plant species considered for phytoextraction of some heavy metals, such as Cd, Cr, Cu, Pb, and Zn (Blaylock, 2000; Venesa & Elisa 2010). Previous investigations by Jiwan and Ajay (2012), Satpathy and Reddy (2013), and Gabriela et al., (2013) on the phytoremediation of various heavy metals with different plants, had limited information on extensive metal phytoabsorption,andextraction dynamics in the metal-contaminated soil. The present study explored the biodiversity of Lantana camara in the remediation of Pb-contaminated soil with validating models and kinetics. To the best of our knowledge, the remediation ability of this plant has not been widely studied.

MATERIALS AND METHODS Sample collection The soil samples and six-month-old L. camara plants were purchased from a nursery division at Sungai Buloh, Selangor, Malaysia. Spent mushroom compost (SMC) and empty fruit bunches (EFB) were collected from Gano Mushroom Farm, Tanjung Sepat, and Sime DarbySdn. Bhd,Bukit KerayongKapar Selangor, respectively.

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ACCEPTED MANUSCRIPT Reagents The salt PbCl2 (purity ≥ 98%) was supplied by Merck Shuchardt OHG (Hohenbrum, Germany). HNO3, (65% wt.), HCl (37% wt.), and H2O2 (30% wt.) were purchased from Merck (Darmstadt, Germany) and were all Suprapur® grade. Distilled water was used throughout the experiments. Plastic and glassware were soaked in a 10:1 mixture of distilled H2O and HNO3 overnight, washed, and kept at room temperature to dry.Stock solutions of analyte elements were used after suitable dilutions. Physicochemical parameters of the soil and supplemented organic waste

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The N content of the used soil and organic wastes were determined using the Kjeldahl method, whereas the Pb and P concentrations were analyzed by inductively coupled plasma (ICP) (USEPA 3050B) after acid digestion. The pH was determined in triplicate with a pH meter (Hanna Instruments:HI-8424, USA) on 1:2.5 (w/v) solid/distilled water after a 40 min equilibration. Metal soil-plant preliminary toxicity test

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Prior to final soil preparation, 3 kg of soil was induced with excess Pb greater than 80 ppm to achieve a considerable higher metal concentrations, order than the standard limits (national background concentration and intervention value) for use of soil under the Department of Environment (DOE) Malaysia 2009. Soil preparation and phytoremediation

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Following the data obtained on metal toxicity test on L. camara,different concentrations (10, 20, 40, and 80 mg/kg) of tolerance range were adopted and 3 kg soil aliquots were contaminated for this experiment. The soil samples were placed in high-density garden polyethylene bags and left to equilibrate for 21 days to undergo cycles of homogenization with distilled water (60% ± 2% strict moisture content), spontaneous air drying, and routine mixing. This procedure was adopted to reproduce soil metal sorption occurring in the field ecosystem. Subsequently, 6% organic wastes from (EFB: chopped into finer particles and SMC: properly segregated) were introduced and stirred again for another 3 days to attain stabilization prior to plant application (transplant approach) as shown in Table I, to form (soil-metal-organic waste and plant) phytoremediation system, and microcosm treatments enumerated every 14 days. All treatments were performed in triplicate (n = 3) at an average daytime temperature of 29 °C /302 K, within the greenhouse. Controls comprising of each specific concentrations with plants and zero organic waste, as well as another without plants and organic waste (i.e., soil–metal mixture only)was also set up to monitor biotic loss. Additional control treatment comprising autoclaved soil containing 0.5% NaN3 was also set up to determine abiotic loss of Pb from the soil. The plants were moderately hydrated once every two days with distilled water at 60% moisture capacity to prevent metal leaching from the nursery polyethylene bags, and monitored for 98 days, beginning from the plant inception date. L. camara was chosen as a model for this experiment, because its metal accumulation potential has not been widely investigated to the best of our knowledge. In addition, L. camara was selected for its rapid growth, good biomass, strong transpiration rates, elevated tolerance to stress, inedibility, and its repulsive smell that repels herbivores to avoid food chain contamination. Table I here Heavy-metal analysis Total metal concentration in soil and plant tissues samples were determined by ICP (USEPA 3050B) analysis following the methods of Doumett et al., (2008), after acid digestion using microwave system (ETHOS-1 MILESTONE). Plants were harvested and rinsed thoroughly with tap water followed by distilled water before the shoots and roots were separated. The plant parts were then dried for 2 days at 70 °C, and the dry matter was 4

ACCEPTED MANUSCRIPT analyzed. Dried plant tissues (0.4 g) were treated with 2 ml of 30% hydrogen peroxide and 7 ml of 65% HNO3. Soil samples (0.4 g) were treated with 3 ml of 30% H2O2, 3 ml of 65% HNO3, and 9 ml of HCl. Plant and soil samples were separately diluted to 50 ml with distilled water, filtered with 0.2 µm pore size of polytetrafluoroethylene filters, and analyzed for metal concentration using ICP 2060T (Skyray). Percentage phytoreduction efficiency (PE) of Pb in contaminated soil was evaluated as follows: % PE = [

Co−Ct Co

] x100

(1)

where Co (mg/kg) and Ct (mg/kg) are the initial and measured concentration at each withdrawal time in Pbcontaminated soil. Enumeration and Identification of Bacteria

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Samples (1.0 g) from each Pb-contaminated soil were removed every 14 days from the rhizosphere region, including the controls. Sampling was done in triplicate (n = 3). Serially diluted samples (0.1 ml) were inoculated on 0.5 mM of Pb as PbCl2 in nutrient agar to evaluate Pb-resistant bacteria (i.e., heavy-metalresistant bacteria or HMRB). To inhibit fungal growth, the media were supplemented with 5.0 mg/l fungicidin (USP, Amresco, USA) after autoclaving. Plates were incubated at 30 °C for 7 days respectively. Colonies that appeared on the plates were counted and expressed as colony forming units per gram of soil (cfu/g). The organisms were isolated and maintained on agar slants for further identification with IF-A (inoculation fluid) Biolog® Microstation system method, after being re-subcultured twice to obtain pure strains. Soil sample extraction

Instrumentation

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Soil was sampled from the rhizosphere region,for qualitative assessment of possible plant exudates, and metabolites that enhanced organic ligand formation which aided phytoremediation processes. Soil samples (20 g) were dried in a fume hood for 24 h and subsequently extracted by sonication with an acetone-water solution (4:1 v/v) in a 200 ml glass beaker for 20 min and stirred at 105 rad s−1 for extraction (Belmonte et al., 2005). The samples were passed through a 0.45 µm filter, and 1.5 ml of the extract was transferred into 2 autosampler vials for gas chromatography–mass spectrometry (GC–MS) analysis.

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GC–MS analysis was carried out using a Shimadzu (Tokyo, Japan) QP2010 GC–MS system equipped with a Shimadzu AOC-20i auto sampler and a DB-5 fused silica capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm) (J&W Scientific, Folsom, CA, USA). Helium was used as carrier gas at a flow rate of 1.8 ml/min. The GC conditions were as follows: initial oven temperature 80 °C for 2 min, increased to 300 °C at a rate of 10 °C/min, then held at 300 °C for 2 min. The injector temperature was 280 °C and all injections were in splitless mode.

Modeling uptake of Pb Pb uptake by L. camara was modeled based on the transfer of metal from the contaminated soil to plants. The simplest expressions of this process are bioaccumulation factor (BAF) and translocation factors (TF) and expressed as follows: 𝑷𝒃 𝑳𝒂𝒏𝒕𝒂𝒏𝒂 BAFPb = 𝑷𝒃 𝒔𝒐𝒊𝒍 (2) TFPb =

𝑃𝑏 𝑠ℎ𝑜𝑜𝑡 𝑃𝑏 𝑟𝑜𝑜𝑡

(3)

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ACCEPTED MANUSCRIPT where PbLantana is the concentration of Pb in harvested parts of L. camara (mg/kg), Pbsoilisthe concentration of Pb in soil(mg/kg), and Pbshoot and Pbroot were the concentrations (mg/kg) of Pb in the harvested shoot and root parts, respectively. More elaboration, on the underlying principles of this model aredetailed in (Hough et al., 2003; Maxted et al., 2007).

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Kinetic studies The data for metal removal by L. camara were fitted to first-order kinetics model: Ct = Co e-kt (4) where Ct is the concentration (mg/kg) of residual metal at aspecific time and Co is the initial metal concentration (mg/kg). A plot of ln [Ct/Co] vs. time, yields a straight line with slope k (day−1), which is the phytoextraction rate constant, and half-life (t1/2) = ln2/k, was also derived.

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Dynamic adsorption of Pb and equilibrium isotherms Adsorption of Pb by L. camarafor both supplemented and non-supplemented treatments were examined to fit into the experimental data. Hence, the following models were used for validation and interpretation: Freundlich isotherm model.The Freundlich isotherm is an empirical equation assuming that the metal adsorption process takes place on heterogeneous surfaces, and the adsorption capacity is related to the concentration of the metal at equilibrium. This isotherm model is defined by the equation below (Adediran et al., 2009): 1 Log qe = ( 𝑛 ) Log Ce + Log K (5) where qe is the Pb adsorbed on the biosorbents (mg/kg), Ce is the final concentration of Pb (mg/kg) in the contaminated soil, K and n are empirical constants that indicates the adsorption capacity and intensity of biosorbents, respectively. Intraparticle diffusion mechanism.During phytoremoval of Pb, solute transfer was characterized by external mass transfer (boundary layer diffusion), or intraparticle diffusion, or both. The adsorption process was identified by fitting an intraparticle diffusion plot. According to Reddy et al., (2010): qt = Kint𝑡1/2 + Ci (6) where Kintis the intraparticle diffusion rate constant (mg/kg day−0.5) and Ci is the intraparticle diffusion coefficient constant (mg/kg). The plot of qtvs.t0.5 at different initial and adsorbed metal concentrations gives the value of Kint and may exhibit multi-linearity, which indicates two or more steps occurring in the metal removal process. Statistical analysis Data was statistically analyzed with analysis of variance using SPSS software 21.0. RESULTS AND DISCUSSION Physicochemical properties of the soil and organic wastes used for phytoremediation The soil (Mollisol) used for phytoremediation was shown to contain low edaphic N (0.61%), 17.7% P, and pH 6.13 ± 0.51. For the organic wastes, EFB had 6.21% N and pH 6.21 ± 0.02, and SMC had 5.57% N and pH 6.21 ± 0.02. The soil had no pre-existing heavy-metal contamination based on the national background concentration stipulated by the DOE Malaysia, 2009. CEC was 10.2 meq/100 g for soil and 11.60 meq/100 g for EFB and SMC unknown. Response of plants to Pb concentration Visual assessment of L. camara in response to environmental stimuli at different concentrations was monitored throughout the experiment (98 days). No plant death (i.e., necrosis) was recorded in all the treatments contaminated with 10, 20, 40, and 80 mg/kg Pb. However, plants in 80 mg/kg Pb-contaminated soil showed 6

ACCEPTED MANUSCRIPT some induced phytotoxicity, such as yellowing of leaves toward the end of the experiment when compared to the control. Control plants also recorded some signs of chlorosis in at least one L. camara for each treatment (data not shown). These findings were in line with those of Sridhar et al., (2005) and Doumett et al., (2008), indicating a strong resistance of L. camara toward soilPb, even at high concentrations.

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Reduction of Pb in the different concentration of contaminated soil The percentages of phytoreduction of Pb in contaminated soil treatments are shown in Table II. Reduction of Pb at the end of 98 days for 80 mg/kg to 10 mg/kg Pb-contaminated soils ranged between 52.46 ± 0.3% to 88.03 ± 0.06% (EFB), 45.10 ± 1.25% to 82.73 ± 1.42% (SMC), and 23.69 ± 1.21% to 57.80 ± 0.35% (unsupplemented) (p < 0.05). Whereas in similar order, the biotic and abiotic controls recorded Pb reduction that ranged from 3.72 ± 0.01% to 12.20 ± 0.10% and 1.49 ± 0.13% to 5.56 ± 0.12% (autoclaved soil treated with sodium azide). High reduction of Pb in soils treated with EFB and L. camara may be due to the presence of appreciable N (1.3 ± 0.04%) and cation exchange capacity (11.6 meq/100 g) contents in EFB. Further reduction of Pb in SMC (N = 0.9 ± 0.8%) and L. camara treatments when compared to unsupplemented soil,implicated N to be the limiting factor. This result is in agreement with that of Agamuthu et al., (2010) that recorded 89.6% to 96.6% reduction in used oil-contaminated soil when supplemented with brewery spent grains. Soil reduction of Cr and Se were reportedly enhanced uponadding manure and crop residues (Bolan et al., 2003a; Hsu et al., 2009; Chiu et al., 2009). From another perspective, L. camaraeven without supplementation can reduce Pb load in soil, thus showing its ecological importance; Perumal et al., (2010) reported 60% reduction from Cr-contaminated soil using Cyperus rotundus and Ludwigea sp. without alterations. This implicated indicator plants to have that ability of growing and absorbing nourishment frommetal-polluted soil. Thus substantiating the entry of pollutants into the plant system during absorption were possible. Table II here

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Bacterial counts HMRB counts in Pb-contaminated soils showed that supplementation with EFB and L. camara remediation resulted in the highest counts of HMRB (79.67 × 106 to 56.0 × 106 CFU/g soil), followed by SMC (63.33 × 106 to 39.0 × 106 CFU/g soil). The lowest Pb concentration (10 mg/kg) recorded the highest HMRB counts, and may be attributed to lesser toxicity, whereas treatments with only L. camara without organic supplements resulted in HMRB counts ranging from 32.0 × 106 to 29.33 × 106 CFU/g soil. However, HMRB counts in unsupplemented control soils were low (23.33 × 106 to 21.31 × 106 CFU/g soil) at p < 0.05. The rationale for increase in HMRB in contaminated soil supplemented with organic wastes might be due to the presence of nutrients in the organic wastes especially N and P that enhanced the proliferation of bacteria in the soil. Moreover, the increased metal reduction in the supplemented soil compared with unsupplemented, might be due to the presence of organic wastes in the soil, which helped loosen the compactness and made optimal aeration available for indigenous bacteria present in the soil, thereby enhancing their metabolic activities in the contaminated soil environment. The HMRB isolated from the Pb-contaminated soil were identified as species of Enterobacter cloacae, Bacillus pumilus, and Lysinibacillus sphaericus. These bacterial species have been implicated in heavy-metal phytoremediation by different researchers (Dell’Amico et al., 2008; Kumar et al., 2008). Effects of pH and soil organic extraction on phytoextraction of heavy metal A pH value within 6.25 to 6.21 was recorded for metal-free soil with L. camara. The metal polluted soils with L. camara and supplements had three pH phases; 6.64 to 6.01 (0–42 days), 5.01 to 4.50 (56–70 days) optimal pH, and 4.79 to 5.95 (84–98 days), indicating an acidic medium. Decreasing soil pH in metal-contaminated soil 7

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has been implicated to cause metal desorption from soil matrices from the displacement of metal ions by H+ (protons), at soil binding sites and hence may increase metal solubility/bioavailability and subsequent uptake by plants roots (Brown et al., 1995; Maxted et al., 2007; Bolan et al., 2003b). This result is in agreement with Wang et al. (2006) who reported maximum uptake of Cd and Zn in the shoots of Thlaspi caerulescens at soil pH 5–6. The (GC-MS-QP2010) identified ligands from rhizosphere soil, as shown in Table III, which revealed exudates of organic acids and compounds suspected to be metal-binding peptides (phytochelatins), namely, 5amino-N-hydroxy [1,3,4] oxadiazol-2-carcoxamidine, glucofuranose-6-amino-6-deoxy-1,2:3,5-di-Oisopropylidene, and other metabolites were implicated in heavy-metal bioavailability and complexation. Critical review from Sheoran et al., (2011) suggested that organic acids and amino acids are ligands for chelation of heavy-metal ions because of the presence of donor atoms (S, N, and O) in their molecules (Marshner et al., 1986; Ma and Miyasaka, 1998;Wu et al., 2003), which is in agreement with this result. Table III here Accumulation of heavy metals in plants, Translocation Factor (TF), and Bioaccumulation Factor (BAF) The accumulation of Pb in roots and shoots across the different treatments were compared with the control. Estimates of TF and BAF are shown in Table IV. The metal accumulation at the roots and shoots, for EFB supplemented soils of 80, 40, 20, 10 mg/kg contaminants were 18.57 ± 0.06, 10.46 ± 0.05, 5.70 ± 0.10, 4.43 ± 0.06 mg/kg and 13.51 ± 0.01, 6.05 ± 0.05, 2.80 ± 0.01, 2.0 ± 0.12 mg/kg respectively. Results for the roots and shoots of SMC supplemented soil in same order were 15.36 ± 0.47, 7.17 ± 0.29, 3.87 ± 0.43, 3.95 ± 0.01 mg/kg and 11.07 ± 0.01, 4.96 ± 0.16, 2.60 ± 0.43, 1.98 ± 0.01 mg/kg. The control’s root and shoot ranged 8.01 ± 0.01 to 2.07 ± 0.06 mg/kg and 4.77 ± 0.12 to 1.0 ± 0.01 mg/kg, respectively. This result shows that L. camara exhibits a phytoextraction phenomenon where an increase in the concentration of metal in the soil also reflects the increase in its above ground plant metal bioaccumulation, especially at the root region. Similar findings were reported by Meera and Agamuthu (2011), on phytoextraction of As and Fe from soil contaminated with landfill leachate using Hibiscus cannabinus. This may be due to restricted metal translocation to other parts of the plant or concurrent roots interphase position between shoots and polluted soil, thereby forming a defense mechanism to protect shoots against phytotoxicity. Schmoger et al., (2002) opined that this trend might need further studies to unravel. However, the translocation of Pb from root to aerial L. camara parts was limited to TF values below 1 (Table IV). Metal accumulator plants have TF values above 1 (Jamil et al., 2009), and metal excluders have shown TF values lower than 1 (Baker, 1981). The TF of Pb to shoots was optimum at 40 mg/kg soil contamination at a range of 0.72 to 0.82, and Pb translocation was efficient at this control treatments. L. camara demonstrated high BAC and low TFroot at different pollutant concentrations in this study. These results suggest that the mechanism of Pb removal by L. camara plants may be via phytoextraction and phytostabilization, contrary to hyperaccumulators. Therefore, L. camara is a potential phytoremediator for Pb-contaminated soil in our ecosystem. Table IV here Phytoreduction/uptake rate First-order kinetics was used to determine the rate of metal reduction in the various treatments, as shown in Table V. In soils contaminated with 80, 40, 20, 10 mg/kg Pb, EFB-supplemented soil exhibited the highest uptake rates of 0.1256, 0.1358, 0.1395, and 0.3069 day−1, respectively. The SMC-supplemented soil exhibited 0.1023, 0.1075, 0.1118, and 0.2580 day−1 in similar order and unsupplemented soil with L. camara recorded 0.0377, 0.0398, 0.0586, and 0.1161 day−1, respectively. Control Pb and autoclaved soils recorded 0.0056, 0.0090, 0.0084, and 0.0179 day−1 and 0.0019, 0.0043, 0.0033, and 0.0080 day−1, respectively. These findings indicated optimum metal uptake rate at 10 mg/kg soil contamination, probably because of pollution level and tolerant biota associations. The EFB-supplemented treatments recorded half-life ranges of 5.52 to 2.26 days, 6.78 to 2.68 days for SMC, 18.39 to 5.97 days (unsupplemented), 123.75 to 38.72 days (control soil), and 364.74 to 86.63 days (autoclaved soil). High uptake rates in supplemented soils were significant (p < 0.05), 8

ACCEPTED MANUSCRIPT which might be due to the presence of N supporting nutrient availability for both plant growth and microbial activity, as was also reported by Adesodun et al. (2008). Table V here Freundlich isotherm model analysis and absorption kinetics

ΔG0 = -RTlnKc

(7)

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The Freundlich constants from plotted Log qe vs. Log Ce of the experimental data are shown in Table VI. The values n and K are the indicators of the absorption intensity and absorption capacity, respectively. Favorable absorption tends to give the Freundlich constant n a value between 1 and 10. Larger values of n imply strong interaction between sorbents and metal ions. Table VI shows that n values were positivefor all treatments. The order of interactionswereas follows: Soil (metal control) > Soil + L. camara (control) > Soil + L. camara + EFB > Soil + L. camara + SMC. The magnitude of n was higher in metal soil (7.59), because heavy metals have high binding affinities to soils (Shuman, 1991), but with the introduction of L. camara (control) and supplementation with EFB and SMC, n decreased to 2.59, 2.51, and 2.44, respectively. This indicated opposite metal movements towards the plants from the metal-contaminated soil caused by the effects of phytoextraction and organic waste complexation, which lessened metal affinities to the soil with regression coefficient R2 ranging from 0.9840– 0.9012. The plot of qtvs. t0.5 showed Kintvalues of 7.064 to 6.364 mg/kg day−0.5 (R2 = 0.9758 to 0.9233) and 3.014 to 1.407 mg/kg day−0.5 (R2 = 0.9740 to 0.9413) for supplemented treatments and controls, respectively. This high R2relationship implicates intraparticle diffusion as a factor in phytoremediation of soil metal pollutants. Table VI here The standard free energy for metal adsorption (kJ mol−1 K−1) during phytoremediation was evaluated and expressed as follows:

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where R is the universal gas constant (8.314 kJ mol−1 K−1), T is the temperature in Kelvin, and Kc is the equilibrium constant, expressed as follows:

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𝑞𝑒

Kc=∁𝑒

(8)

Ac

where qe is the amount of Pb adsorbed (mg/kg) and Ce is the final concentration in the soil (mg/kg) (Adediran et al, 2009). At the average greenhouse daytime temperature of 302 K, the equilibrium constant Kc of adsorption of Pb (10–80) mg/kg in L. camara supplemented with EFB soil ranged from 7.35 to 1.10 with corresponding free-energy change (ΔG) = −5.01 to 0.49 kJ mol−1 K−1, while SMC-supplemented soil had Kc ranging from 4.79 to 0.82 with corresponding ΔG = −3.93 to 0.49 kJ mol−1 K−1, control soil had Kc of 1.37 to 0.25 with ΔG = −0.79 to 3.49 kJ mol−1 K−1, and metal control soil had Kc of 0.14 to 0.04 with ΔG = 4.96 to 8.16 kJ mol−1 K−1, respectively. These results indicated that Pb removal in supplemented (i.e., catalyzed) phytoremediation was highly exothermic, and nearly to equilibrium process, whereas the unsupplemented (i.e., uncatalyzed) phytoremediation reflected less exothermic but far from equilibrium state. The control soil appears completely endothermic. The models and kinetics used in the validation of this study supported L. camara as a bioindicator metallophyte for phytoremediation, and EFB and SMC as good organic enhancers. CONCLUSIONS The present study explored the potential of L. camara in thephytoremediationofPb-contaminated soil. Given the current drive for industrialization, the soil ecosystem are faced with the challenges of being contaminated directly or indirectly, and the quest to remain green must be sustained. Therefore, the identification of emerging ecoreceptor plants through technical approach, and sourcing for biologically harmless waste to sustain their 9

ACCEPTED MANUSCRIPT metabolism while decontaminating the metal-polluted soil has become imperative. Our results demonstrated the capabilities of L. camara to grow in Pb-polluted soil, and its subsequent ability to decontaminate the soilmetalload. However, further research is needed as to explain this phenomenon through the application of advanced spectroscopic methods, which may investigate the in situ short and/or long-term effectiveness of these supplements.

ACKNOWLEDGEMENTS

cr ip

t

This research was supported by the “IPPP-University of Malaya Malaysia” under the grant no. [IPPP/PV054/2011A]. We thank the Solid Waste Laboratory, Institute of Graduate Studies University of Malaya and the graduate student colleagues who supported this research work.

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Xiaoe, Y, Ying, F, Zhenli, H., Peter, J.S. 2005. Molecular mechanisms of heavy metal hyperaccumulation and phytoremediation. J. of Trace element in Med. and Bio.18: 339--353. Zhou, J-M., Dang, Z., Cai, M-F., Liu, C-Q. 2007. Soil heavy metal pollution around the Dabaoshanmine, Guangdong Province, China. Pedosphere 17(5): 588--594. TABLEI: Experimental design Treatments

Details of treatments

A

3kg soil + [10/20/ 40/ 80 (mg/kg)] Pb +6%EFB+L.camara

B

3kg soil + [10 /20/ 40/ 80 (mg/kg)] Pb + 6% SMC + L.camara

C

3kg soil + [10 /20/ 40/ 80 (mg/kg)] Pb + L.camara 13

ACCEPTED MANUSCRIPT D

3kg soil + [10 /20/ 40/ 80 (mg/kg)] Pb only.

E

3kg autoclaved soil + [10 /20/ 40/ 80 (mg/kg)] Pb + 0.5%NaN3

F

3kg soil + L.camara

TABLE II: Percentage (%) phytoreduction efficiency of Pb in contaminated soils for (un)supplemented treatments for 98 days with L. camara and controls.

Soil(mg/kg)(a) EFB (%)

(b) SMC (%)

(c) Plant + Pb (%)(d) Pb soil(%)(e) Pb autoclaved soil (%)

80 →

45.10±1.25a

23.69±1.21ab

19.96±0.03ab6.70±0.03 abc 2.49±0.01 abcd

52.46±0.13

controls

t

un-supplemented

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Pb conc.Supplemented treatments

3.72±0.01 abc 1.49±0.13 abcd

40 →

56.66±0.02

46.73±0.14a

20 →

57.20±0.61

48.47±0.70a

32.99±0.01ab

10 →

88.03±0.06

82.73±1.42a

57.80±0.35ab12.20±0.10abc

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6.52±0.03 abc2.75±0.05 abcd 5.56±0.12 abcd

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[Pb (%) reduction in soil at (df: 4, 10); p<0.05; F-values = 2597.28 for (80mg/kg); 39711 for (40mg/kg); 10340 for (20mg/kg) and 10391 for (10mg/kg)],a, b, c, d and edenotes statistical significances as function for treatment codes.

TABLEIII: GC-MS-QP2010 possible metal complexing compounds in Pb contaminated soil during phytoremediation.

rhizosphere soil

ligands found

supplemented

Mol.

d

Organic

formula

ce pt e

Pb contaminated

L. camara +EFB/SMC

Oxalic acid

C6H12O6

Acetic acid

C10H20

5-Amino-N-hydroxy [1, 3, 4] oxadiazol-2-carcoxamidine

C6H12N5O2

Ac

Glucofuranose, 6-amino-6-deoxy-1,2:3,5-di-O-isopropylidene C6H12N5O2 Furan, tetrahydro-2, 2, 4, 4- tetramethyl*

C6H16O6

1, 6- Anhydro-3, 4-O- isopropylidene-2-tosyl-D-galactose*

C6H20N5O7S

O- Trimethylsilylpropargyalcohol*

C6H12N5OSi

3-hydroxy-2-methylpentanal**

C6H12O2

Un-supplemented L.camara + Pb

Glucofuranose, 6-amino-6-deoxy-1,2:3,5-di-O-isopropylidene C6H12N5O2 Oxalic acid

C6H12O6

Note: *EFB, ** SMC and none (* or **) indicates common ligands.

14

ACCEPTED MANUSCRIPT TABLE IV: Translocation Factor (TF) and Bioaccumulation Factor (BAC) of metal uptakes during Phytoremediation. Treatments 80(mg/kg) Pb [A

40(mg/kg) Pb 20(mg/kg) Pb 10(mg/kg) Pb

C] 80 [A

B

B

C] 40 [A

B

C] 20

[A

B

C] 10

TFroot 0.23

0.19

0.10

0.26

0.18

0.05

0.29

0.19

0.14

0.44

0.40 0.21

TFshoot 0.17

0.14

0.06

0.72

0.70

0.82

0.49

0.68

0.57

0.45

0.50 0.48

BAC

0.33

0.16

0.41

0.30

0.10

0.43

0.32

0.21

0.64

0.59 0.31

0.40

B:SMC+ L.amara

C:Pb+ L. camara (unsupplemented)

t

A: EFB + L. camara,

F-value for TFroot and TF shoot [ABC] 20 =136.2 and 28.8

;

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F-value for TFroot and TF shoot [ABC] 80 =549.9 and 9548 ; TFroot and TF shoot [ABC] 40 =822.9 and 826.8 (p<0.05)

TFroot and TF shoot [ABC] 10 =1121.7 and 101.2 (p<0.05)

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________________________________________________________________________________

TABLEV: Phytoreduction/uptake rates and half-life of L.camara in Pb contaminated soil Treatments

Uptake constant (k) day-1

Half-life (ln2/k)

(days)

a

0.1023

a

6.78

0.0377

b

18.39

0.0056

a

123.75

0.0019

a

364.74

0.1358

a

5.10

0.1075

a

6.45

0.0398

b

17.42

0.0090

a

77.02

Autoclaved soil +40 mg/kg Pb + 0.5%NaN3

0.0043

a

161.20

Soil +20 mg/kg Pb + EFB+ L.camara

0.1395a

4.97

0.1118

a

6.20

0.0586

b

11.83

0.0084

a

82.52

0.0033

a

210.01

0.3069

a

2.26

0.2580

b

2.68

Soil +80 mg/kg Pb + L.camara

ce pt e

Soil +80 mg/kg Pb only.

d

Soil +80 mg/kg Pb + SMC L. camara

M

0.1256

Soil +80 mg/kg Pb + EFB+ L. camara

Autoclaved soil +80mg/kg Pb + 0.5%NaN3 Soil +40 mg/kg Pb + EFB+ L.camara Soil +40 mg/kg Pb + SMC L.camara Soil +40 mg/kg Pb + L. camara

Ac

Soil +40 mg/kg Pb only.

Soil +20mg/kg Pb + SMC L.camara Soil +20mg/kg Pb+ L.camara Soil +20mg/kg Pb only. Autoclaved soil +20mg/kg Cr + 0.5%NaN3 Soil +10 mg/kg Pb + EFB+ L. camara Soil +10 mg/kg Pb + SMC L.camara

15

5.52

ACCEPTED MANUSCRIPT Soil +10 mg/kg Pb+ L. camara

0.1161b

5.97

Soil +10 mg/kg Pb only.

0.0179a

38.72

Autoclaved soil +10 mg/kg Pb + 0.5%NaN3

0.0080a

86.63

Values followed by letter b indicate sig. difference at p<0.05 level while “a” indicates not sig. at p<0.05 level.

TABLE VI: Freundlich isotherm parameters and correlation to phytoremediation Plant &

(mg/kg)

Treatment mode

Phytoremediation Freundlich isotherm parameters n

R2

K

t

Pb conc.

2.51

0.01

0.9012

Pb: 80 to 10 → Soil + L.camara + SMC

2.44

0.05

0.9327

Pb: 80 to 10 → Soil + L.camara (control)

2.59

0.30

0.9840

Pb: 80 to 10 → Soil (metal control)

7.59

-0.02

0.9505

Ac

ce pt e

d

M

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cr ip

Pb: 80 to 10 → Soil + L.camara + EFB

16