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Phosphate-Induced Lead Immobilization in Contaminated Soil

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PAGE 1

PHOSPHATE-INDUCED LEAD IMMOBILI ZATION IN CONTAMINATED SOIL By JOONKI YOON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

PAGE 2

Copyright 2005 by Joonki Yoon

PAGE 3

This thesis is dedicated to my gra nd parents, Jinhyung Lee and Youngsook Kim.

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ACKNOWLEDGMENTS I wish to extend my thanks to Dr. Lena Q. Ma for her assistance and guidance of my research. I would also like to thank members of my committee, Drs. Willie G. Harris and Jean Claude Bonzongo, for their advice, support and critical review of this thesis. I gratefully acknowledge Dr. Xinde Cao who worked with me and helped me a lot. I wish to thank Thomas Luongo for laboratory analysis and being a good instructor. Special thanks go to Drs. Mrittunjai Srivastava and Gregory E. MacDonald for their assistance with plant identification procedure. I would like to thank my parents Byungkyu Yoon and Sookjae Lee for their love, encouragement and support throughout my studies. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES ............................................................................................................vii LIST OF FIGURES .........................................................................................................viii ABSTRACT .......................................................................................................................ix CHAPTER 1 INTRODUCTION...........................................................................................................1 Lead in the Environment..............................................................................................1 Health Effects of Lead..................................................................................................3 Lead Remediation Techniques.....................................................................................6 Phosphate Induced Lead Immobilization..............................................................6 Phosphoric acid..............................................................................................8 Synthetic apatite.............................................................................................8 Bonemeal apatite............................................................................................9 Phosphate rock.............................................................................................10 Rhizosphere.........................................................................................................11 Phytoremediation.................................................................................................12 Phytoextraction.............................................................................................13 Phytostabilization.........................................................................................13 Summary.....................................................................................................................14 2 EFFECTS OF APPLICATION METHODS ON P-INDUCED Pb IMMOBILIZATION IN A SOIL COLUMN.............................................................16 Introduction.................................................................................................................16 Materials and Methods...............................................................................................18 Soil Characterization...........................................................................................18 Soil Sample Preparation......................................................................................18 Column Leaching................................................................................................19 Analytical Procedure...........................................................................................20 Results.........................................................................................................................21 Soil Characterization...........................................................................................21 Effects of P Application on Soil pH....................................................................22 v

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TCLP Pb..............................................................................................................23 Lead Bioavailability............................................................................................24 Distribution of Pb in Soil Column.......................................................................25 Leachate Analysis................................................................................................29 3 THE EFFECTS OF PLANTS ON PHOSPHATE-INDUCED Pb IMMOBILIZATION IN THE RHIZOSPHERE SOIL..............................................33 Introduction.................................................................................................................33 Materials and Methods...............................................................................................34 Results.........................................................................................................................37 Soil Characterization...........................................................................................37 Bioavailable Pb....................................................................................................38 Formation of Lead Phosphate..............................................................................39 Conclusion..................................................................................................................43 4 SCREENING OF PLANTS FOR ACCUMULATION OF Pb, Cu, Zn FROM A CONTAMINATED SITE...........................................................................................45 Introduction.................................................................................................................45 Materials and Methods...............................................................................................47 Site Characterization...........................................................................................47 Sample Preparation and Chemical Analysis........................................................48 Results.........................................................................................................................48 Metal Concentrations in Soils.............................................................................48 Metal Concentrations in Plants............................................................................50 Accumulation and Translocation of Metals in Plants..........................................52 Conclusion..................................................................................................................60 5 CONCLUSION..............................................................................................................62 LIST OF REFERENCES...................................................................................................64 BIOGRAPHICAL SKETCH.............................................................................................72 vi

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LIST OF TABLES Table page 1-1 Theoretical solubility of Pb in different.....................................................................7 2-1 Soil characterization at the contaminated site..........................................................21 2-2 pH of soil after treatment.........................................................................................22 2-3 Pb concentration in column leachate (g L -1 )..........................................................29 3-1 Selected characteristics of the lead-contaminated soil.............................................37 3-2 Water soluble phosphorous in plant rhizosphere soil (ppm)....................................42 3-3 pH of rhizosphere soil treated with phosphate rock after 4-weeks growth..............43 4-1 Selected properties of soil samples collected from the contaminated site at Jacksonville, Florida.................................................................................................49 4-2 Lead concentrations in soil and plant samples (mg kg -1 ) collected from the contaminated site at Jacksonville, Florida................................................................52 4-3 Copper concentrations in soil and plant samples (mg kg -1 ) collected from the contaminated site at Jacksonville, Florida................................................................54 4-4 Zinc concentrations in soil and plant samples (mg kg -1 ) collected from the contaminated site at Jacksonville, Florida................................................................55 4-5 Accumulation and translocation of Pb, Cu and Zn in selected plants......................56 4-6 Plants with high BCF and low TF for phytostabilization.........................................59 vii

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LIST OF FIGURES Figure page 2-1 TCLP metal contents in sectioned soil columns after leaching...............................24 2-2 PBET extractable Pb contents in sectioned soil columns after leaching..................26 2-3 Total Pb concentration in sectioned soil columns (bottom section).........................28 2-4 P concentration in column leachate (mg L -1 )...........................................................30 3-1 Lolium rigidum after 4 weeks of growth..................................................................36 3-2 Agrostis capillaries after 4 weeks of growth...........................................................36 3-3 Brassica napus after 4 weeks of growth..................................................................37 3-4 Lead concentrations using the physiologically based extraction test in the rhizosphere soil with phosphoric acid and with phosphate rock treatment..............38 3-5 Scanning electron microscopy elemental dot map of Agrostis capillaries..............41 3-6 Scanning electron microscopy elemental dot map of Brassica napus.....................42 3-7 Scanning electron microscopy elemental dot map of Lolium rigidum.....................42 4-1 Map of Jacksonville site with lead concentration contour map and sampling point..........................................................................................................................47 viii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science PHOSPHATE-INDUCED LEAD IMMOBILIZA TION IN CONTAMINATED SOIL By Joonki Yoon May 2005 Chair: Lena Ma Major Department: Soil and Water Science Activities such as mining and manufacturing, and the use of synthetic products result in heavy metal contamination of soil and water resources. The objectives of this research were to 1) examine the effectiveness of five P application methods on Pb immobilization in soil using a column study; 2) explore the effects of plants on the effectiveness of P-induced Pb immobilization in soil using a pot study; and 3) evaluate metal accumulation in plants growing on a contaminated soil, based on field screening. The soil column test was conducted to evaluate the effectiveness of different application methods using phosphate rock (PR) and phosphoric acid (PA). Mixing both PA and PR with the soil was the most effective method of Pb immobilizati on, reducing TCLP-Pb by up to 95% (Toxicity Characteristic Leaching Procedure) and bioavailable Pb by 25 to 42% in the soil. The application of PR as a layer in the soil column was effective in reducing Pb migration in the soil column by 73 to 79%. In a gree nhouse study, all th ree plants (Agrostis capillaries, Lolium rigidum, and Brassica napus) were effective in enhancing ix

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Pb immobilization in the rhizosphere soil when P was applied as insoluble PR (13 to 20% reduction in bioavailable Pb) but not when applied as a mixture of PR and PA. A fieldstudy was conducted to screen 17 native plants for their potential for accumulation of Pb, Cu and Zn from a heavy metal contaminated site. Elevated metal concentrations were observed in the 17 plants collected from the experimental site, with total concentrations ranging from 20 to 1,183 mg kg -1 for Pb, from 5 to 460 mg kg -1 for Cu, and from17 to 598 mg kg -1 for Zn. Though no metal hyperaccumulator was identified, G. pennelliana was the most effective in taking up all three metals, while Cyperus was the most effective in translocating all three metals from the roots to the shoots. Our study demonstrated that the most effective P-application method coupled with plant growth can maximize the effectiveness of phosphate-induced lead immobilization in soils. x

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CHAPTER 1 INTRODUCTION Lead in the Environment Lead (Pb), from the Latin plumbum, has an atomic number of 82 and an atomic weight of 207.19. While lead is the 36th most abundant element, it is the most abundant heavy metal in the earths crust with an average concentration of 13 mg kg -1 (Brown and Elliott, 1992; Nriagu, 1978). The relatively low Pb concentration in soil solutions confirms reports of leads low mobility among the heavy metals (Kabata-pendias and Pendias, 1984). Natural mobilization of lead into the environment occurs principally from the erosion of lead-containing rocks and through gaseous emissions during volcanic activity (Waldron, 1980). Important physical attributes of lead (i.e. high density, softness, flexibility, and electric potential) ensure its widespread industrial application. An Anthropogenic source of lead includes flanking paint, mining operations, smelter and industrial emissions, and application of insecticides, which have contributed to elevated lead levels in environment. Lead is also used in the production of ammunition, and has been used in other metallic products (such as solder, some brass, bronze products, and piping), and ceramic glazes, but its use in these capacities has been outlawed in the U.S. (Scheuhammer and Norris, 1995). Chemicals such as tetraethyl lead and tetramethyl lead were once used as gasoline additives to increase octane rating. However, their use was phased out in the 1980s, and lead was banned from use in gasoline for transportation beginning January 1, 1996 (USEPA, 2001a). 1

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2 Lead occurs naturally in all soils in concentration ranging from 1 to 200 mg kg -1 but rarely at levels that are considered to be toxic. However, increases of anthropogenic sources of Pb in the environment have caused the Pb loading rate in soil to exceed its natural removal rate by approximately 20-fold (Nriagu, 1978). There are several thousands of Pb contaminated sites in United States. Currently there are 440 sites listed on the EPA National Priority List (NPL), in which the main contamination of concern is lead. Many studies show that there are high levels of Pb in various environments. A national survey in the U.K indicates that total Pb concentrations in garden soils ranges from 13 to 14,100 mg kg -1 (Cotter-Howells, 1996). Soil samples obtained from a former battery-cracking site in Florida had Pb concentrations up to 13,500 mg kg -1 (Cao et al., 2001). Pichtel et al. (2000) reported Pb concentrations of a Superfund site and a Pb battery dump that ranged from 195-140,500 mg kg -1 A recent survey found Pb concentration in soils adjacent to homes and near expressways in Tampa, Florida as high as 2,000 mg kg -1 (Cao et al., 2001). Also uptake of heavy metals by humans, largely through the respiratory and digestive pathways, has contributed to elevated blood Pb concentration, which can cause impaired mental development, nervous system disorders, immune-system dysfunction and DNA damage. Presence of hazardous levels of Pb in the environment and the toxicity of Pb requires remedial action to protect the health of the public. Considerable attention is now being paid to develop cost effective and less disruptive remediation technologies to reduce human exposure via direct ingestion and dust inhalation. Ionic lead (Pb 2+ ) is the dominant form of lead in the soil and groundwater. However, Pb 4+ occasionally can be found in some highly oxidized soils. With the

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3 exception of a few sporadic measurements in urban air, marine fish, and in human brains there is relatively little information available on organometallic lead compounds (Nriagu, 1978). Because lead enters the soil in various complex compounds, its reactions may differ widely between geographic areas. Lead is found in soils most commonly as galena (PbS) and in smaller quantities as cerrusite (PbCO 3 ), anglesite (PbSO 4 ) and crocoite (PbCrO 4 ). Lead added to soil may react with available soil anions such as SO 4 2, PO 4 3, or CO 3 2to form sparingly soluble salts (Waldron, 1980). Compounds such as lead carbonate [Pb 3 (OH) 2 (CO 3 ) 2 ] and chloropyromorphite [Pb 5 (PO 4 ) 3 Cl] provide the least soluble inorganic salts at near neutral pHs. There are several other mechanisms by which lead may be immobilized in soils, such as complexation by soil organic matter (humic and fulvic acids), which are then adsorbed onto soil solids, or by ion exchange at sites on the solid material in the soil. These mechanisms may facilitate attachment of substantial amounts of lead, but cannot account entirely for the low mobility of lead in soils (Harrison and Laxen, 1981). Health Effects of Lead Since lead is a natural constituent in the environment, man has been under inescapable exposure to Pb (Waldron, 1980), which originates from an array of sources that include Pb contaminated soil, atmospheric Pb particles, Pb paint, Pb water pipes, and Pb solder used to seal food cans. With such an array of potential sources, it is no wonder that lead poisoning is ranked as the number one environmental health threat, by both the U.S Agency for Toxic Substance and Disease Registry (ATSDR) and the Center for Disease Control (CDC). Dust and soil have emerged as important lead ingestion pathways in recent years. Although their amounts are small, once Pb enters the blood

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4 only approximately 10% is excreted and the remainder stays in the body, affecting the nervous system, blood cells, and metabolic processes and eventually residing in bone material for decades (Scheckel and Ryan, 2003). The major health effects of lead are manifest in three organ systems; the hematological system, the central nerve system and the renal system (Needleman, 1992). The hematological effects of lead have been recognized for many years. Such effects are related to the derangement of hemoglobin synthesis, the shortened life span of circulating erythrocytes and the secondary stimulation of erythropoiesis, which, may result in anemia. Higher lead concentration may affect the degree of maturation of red blood cells, globin synthesis, and the morphology and stability of cells (Nriagu, 1978). Acute effects of lead on nervous system are both structural and functional, involving the cerebellum as well as the spinal cord and the motor and sensory nerves leading to specific areas of the body (Nriagu, 1978). Effects may also lead to the general deterioration of intellectual functions, sensory functions, neuromuscular functions and psychological functions. If calcium is deficient, synaptic transmission is impaired. In addition, lead has also been reported to interfere with the normal metabolism of some central neurotransmitters, including acetylcholine and catecholamines. Other effects, which have been noted, include inhibition of cytoplasmic NAD(P)H oxidation by cerebral mitochondria and the inhibition of adenylcyclase. Lead also affects the intrinsic muscles and oculomotor nerves of the visual system. Changes in the character of the blood cells and supporting fluids may lead to changes in the intraocular tension. Such changes may lead to mydriasis or visual paralysis. Neuritis may occur within the visual system itself and a scotoma, which may be limited to certain colors, is usually present in

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5 optic neuritis. The actual effects of lead on auditory dysfunctions are not clear (Nriagu, 1978). Nriagu (1978) summarized the functional and morphological effects of lead on the kidney as follows: Stage 1: swollen proximal tubular lining cells; mitochondrial changes; intranuclear inclusion bodies (mostly lead-protein complexes); proximal tubular dysfunction, Stage 2: fewer inclusion bodies; intense interstitial fibrosis; tubular atrophy and dilation Stage 3: renal tumor (observed in rats only). The first stage is usually seen in children with short-term heavy exposure to lead. The functional effects include the fanconi syndrome manifested by an increase in urinary excretion of glucose, amino acids and phosphate. The second stage nephropathy can be produced in experimental animals fed a high dose of lead and in workmen with excessive lead exposure for several years. The duration and degree of lead exposure necessary to produce irreversible kidney damage is not known. Dingwall-Fordyce and Lane (1963) followed 425 retirees and others with long exposure to lead in a battery factory. They found no increase in cancer incidence but saw excess in cerebrovascular accidents. Lead can effect chromosomes, reproduction, immune system and endocrine system, but usually is not considered as a human carcinogen. Toxicity of lead to plants has not been frequently observed. Plants do not translocate absorbed lead into the shoots while they are growing; instead, they accumulate it in their roots. The health concern surrounding plants is that lead containing

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6 soil particles can contaminate plant surfaces, which might be ingested by animals (McBride, 1994). Lead Remediation Techniques In contrast to many organic contaminants, the immobile and non-degradable nature of lead necessitates active remediation because natural attenuation will do little to reduce its concentration (Nedwed and Clifford, 1997). There are several technologies available for the remediation of land contaminated by heavy metals (USDA, 2000; Mulligan et al., 2001). The selection of the most appropriate soil remediation method depends on the site characteristics, concentration and types of pollutants of concern, and end use of the contaminated medium (Mulligan et al., 2001). However, many of these technologies are costly (e.g. excavation of contaminated material and chemical/physical treatment) or do not achieve a permanent or aesthetic solution (e.g. encapsulation and vitrification). Formation of insoluble heavy metal compounds immobilizes the metal and reduces their bioavailability (Cotter-Howells and Caporn, 1996). Phosphate amendment has been suggested as a cost-effective remediation option for Pb contaminated soils. Along with that, phytoremediation, in which plants are used to do the remediation work, can be another cost-effective and less disruptive approach. Phosphate Induced Lead Immobilization When considering potential Pb-immobilizing anions(Table 1) phosphates are less soluble under earth surface equilibrium conditions than oxides, hydroxides, carbonates, and sulfates (Ruby et al., 1994). In its reactions with lead, phosphorus forms sparingly soluble orthophosphates (pyromorphites) (Nriagu, 1972). Experimental evidence supports that Pb phosphates can

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7 form rapidly when adequate Pb and phosphate are present in aqueous system (Zhang and Ryan, 1999a; Ma et al., 1994b). Phosphate minerals have been used to immobilize Pb in situ from aqueous solutions and Pb contaminated soils (Cao et al., 2001, 2002; Ma et al., 1993, 1995; Mench et al., 1994). The primary mechanism of Pb immobilization appears to be through phosphate-mineral dissolution and subsequent precipitation of pyromorphite-like minerals, though mechanisms such as cation substitution, adsorption and precipitation as other Pb minerals are also possible. Hence, solubility of phosphate minerals largely determines the effectiveness of in situ Pb immobilization in soil (Ma, 1996). Table 1-1. Theoretical solubility of Pb in different phases Pb Phase Stoichiometry log Ksp Litharge PbO 12.9 Anglesite PbSO 4 -7.7 Cerrusite PbCO 3 -12.8 Galena PbS -27.5 Chloropyromorphite Pb 5 (PO 4 ) 3 Cl -84.4 Hydroxypyromorphite Pb 5 (PO 4 ) 3 OH -76.8 Fluoropyromorphite Pb 5 (PO 4 ) 3 F -71.6 BromoPyromorphite Pb 5 (PO 4 ) 3 Br -78.1 Corkite PbFe 3 (PO 4 )(SO 4 )(OH) 6 -112.6 Drugmanite Pb 2 (Fe,Al)(PO 4 ) 2 (OH) 2 NA Hindsdalite PbAl 3 (PO 4 )(SO 4 )(OH) 6 -99.1 Plumbogummite PbAl 3 (PO 4 ) 2 (OH) 5 H 2 O -99.3 Lead-metal sulfate (Fe,Zn,Pb)SO 4 NA Iron-nanganese-lead oxide (Fe,Mn,Pb)O NA Lead-metal oxides (Fe,Al,Zn,Pb)O NA

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8 Phosphoric acid The rate of chloropyromorphite, one of pyromorphite minerals, precipitation is kinetically rapid and controlled by the availability of soluble Pb and P as shown in Eq (1) 5Pb 2+ + 3H 2 PO 4 +Cl Pb 5 (PO 4 ) 3 Cl (s) + 6H + (1) Conversion of soil Pb to pyromorphite could immobilize soil Pb and reduce its bioavailability. If sufficient soluble P is provided, the dissolution of Pb solids would then be the limiting factor for pyromorphite formation (Yang et al., 2001). Dissolution of soil Pb increases with decreasing pH (Zhang and Ryan, 1999b). Neutralization of protons generated during chloropyromorphite precipitation shown in Eq (1) could favor the reaction toward chloropyromorphite formation. Thus, initially acidifying the soil followed by a gradual increase of soil pH should enhance the transformation of soil Pb to pyromorphite. It is believed that adding H 3 PO 4 to calcareous soil would lower soil pH to facilitate dissolution of soil Pb and increase the activity of soluble P, thereby enhancing pyromorphite formation (Yang et al., 2001). Synthetic apatite Hydroxyapatite [HA, Ca 10 (PO 4 ) 6 OH 2 ], a member of the apatite family, is the prototype of the inorganic constituent of bones and teeth, thus its crystal and solution chemistry have been researched extensively. Most research has centered on synthetic HA, because relatively pure natural HA is uncommon. Hydroxyapatite effectively and rapidly attenuates Pb from aqueous solutions, exchange sites, and Pb contaminated soils (Ma et al., 1993)

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9 Ma et al. (1993) have examined the utility of natural and synthetic apatite for in situ stabilization of Pb in contaminated soils. The immobilization of Pb by HA was attributed to HA dissolution followed by precipitation of hydroxypyromorphite [HP, Pb 10 (PO 4 ) 6 (OH) 2 ]: as shown in Eqs. (2) and (3): Ca 10 (PO 4 ) 6 (OH) 2 (s) + 14 H + (aq) 10Ca 2+ (aq) + 6H 2 PO 4 (aq) + 2H 2 O (2) Dissolution 10Pb 2+ (aq) + 6H 2 PO 4 (aq) + 2H 2 O Pb 10 (PO 4 ) 6 (OH) 2 (s) + 14 H + (aq) (3) Precipitation Laperche et al. (1996) investigated the use of HA as a soil additive with the goal of converting soil Pb to HP. Hydroxyapatite was mixed with contaminated soil to test its feasibility in reducing aqueous Pb and in converting the solid phase forms of Pb to HP. The concentration of dissolved Pb in the suspension was reduced from 0.82 to 0.71 mg L -1 at solution pH of 7.7 and to 0.22 mg L -1 at pH 5. Dissolved Pb concentrations in the control samples increased with reaction time and decreasing pH. Also, the addition of HA to a Pb polluted soil led to a substantial decrease of Pb concentrations in the shoots of Gradd sudax (Boisson et al., 1999). Bonemeal apatite The mineral portions of animal bones and teeth are composed of HA. Bonemeal is a less soluble source of phosphorous than synthetic apatite, but it is much more economically feasible for field scale use. The ability of bonemeal (finely ground, poor-crystalline apatite) to immobilize pollutant metals in soils and reduce metal bioavailability through the formation of metal phosphates has been evaluated by Hodson et al. (2000). The researchers used a 1:50 bonemeal:soil mix. The mixture was packed into columns and leached with synthetic rainwater. The pH increase appears to be due to

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10 the dissolution of the bonemeal. Monitoring of leachates over a three-month period indicated that bonemeal additions resulted in the immobilization of metals and an increase in the pH of the column leachate, the soil pore water and the soils themselves. Analytical scanning electron microscopy (SEM) and X-ray diffraction (XRD) identified Pb and Ca-Zn phosphates as the reaction products. Assessment of metal bioavailability by chemical extraction indicates that bonemeal additions to acidic soils reduced metal bioavailability in soils via the formation of highly insoluble Pb and Zn phosphates. Phosphate rock Ma et al. (1995) have shown that phosphate rock [PR, primarily fluorapatite (Ca 10 (PO 4 ) 6 F 2 )] effectively immobilized Pb from aqueous solutions, with Pb immobilization ranging from 39 to 100%. The primary mechanism of Pb immobilization is via dissolution of PR and subsequent precipitation of a fluorpyromorphite-like mineral [Pb 10 (PO 4 ) 6 F 2 ], although precipitation of Pb as hydrocerussite also occurred in some instances. Phosphate rock immobilizes lead according to the following simplified equations (Ma and Rao, 1999). Ca 9.5 (PO 4 ) 5 CO 3 FOH +10H + 9.5Ca 2+ + 5H 2 PO 4+ CO 3 2+ F + OH (4) 9.5Pb 2+ + 5H 2 PO 4 + CO 3 2+ F + OH Pb 9.5 (PO 4 ) 5 CO 3 FOH + 10H + (5) Ma et al. (1995) demonstrated the potential of Florida PR to immobilize aqueous Pb from Pb contaminated soils. The two most effective Florida PRtypes in immobilizing Pb [Chemical (CF) and Occidental Chemical (OC)] were chosen for the column experiments to evaluate the feasibility of using PR to immobilize Pb from a contaminated soil and to evaluate the effects of different methods of mixing PR and soil and incubation

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11 time on Pb immobilization efficiency. A lead contaminated soil was mixed with PR at a ratio of 0:10, 1:10, 2:10 and 4:10. After uniform mixing, the soil-PR mixture was placed in columns. All PR types were effective in reducing Pb concentration from an initial concentration of 4.82 mol L -1 to below 72.4 nmol -1 (the current EPA action level for Pb) after 16 days of reaction and at a 4:10 PR:soil ratio. Incubation time and mixing methodology had little effect on the efficiency of Pb immobilization by PR. Hettiarachchi et al. (2000) reported that PR was equally or more effective than triple super phosphate fertilizer. Rhizosphere Phosphorous is an essential plant nutrient and is of the most limiting factors in plant growth.. Because roots can only absorb free phosphate, several mechanisms exist to increase the soil P available to them (Cotter-Howells, 1996). Root exudates contain phosphatase enzymes that can convert organic P to phosphate in the rhizosphere (Haeussling and Marschner, 1989), as do rhizosphere microorganisms (bacteria and fungi), not those infecting the roots (Cosgrove, 1967). This free phosphate would also be available to heavy metal compounds to form metal phosphates. Cotter-Howells et al. (1996) showed that the formation of metal phosphates could also be induced by the biochemical action of the roots of Agrostis capillaries. However, the exact mechanism is not clear. Agrostis capillaries roots in these experiments were free from mycorrhizal infection. Thus, root exudates containing phosphatase enzymes or non-root infecting microorganisms could be responsible for the conversion of organic P to phosphate in the rhizosphere.

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12 Another study by Hinsinger and Gilkes (1997) indicated that additional dissolution of PR occurred in the presence of plant roots. The largest root-induced dissolution was achieved by ryegrass (Lolium rigidum) and rape (Brassica napus), amounting to 19 to 32% of the PR present in the first two mm of the rhizosphere. Phytoremediation Phytoremediation is a general term for using plants to remove, degrade, or contain soil pollutants such as heavy metals, pesticides, polyaromatic hydrocarbons, and landfill leachates. These processes include (1) modifying the physical and chemical properties of contaminated soil; (2) releasing root exudates, thereby increasing organic carbon; (3) improving aeration by releasing oxygen directly to the root zone as well as increasing the porosity of the upper soil zones; (4) intercepting and retarding the movement of chemicals; (5) effecting co-metabolic microbial and plant enzymatic transformation of recalcitrant chemicals; and (6) decreasing vertical and lateral migration of pollutants to the ground water by extracting available water and reversing the hydraulic gradient (Susarla et al., 2002). Recent researches have demonstrated that plants are effective in cleaning up heavy metal contaminated soils (Wenzel and Jockwer, 1999; Pichtel et al., 2000; Baker and Brooks, 1989). In many remediation projects, phytoremediation is seen as a final polishing step following the initial treatment of the high-level contamination. However, when contaminants are low in concentration, phytoremediation alone may be the most economical and effective remediation strategy (Susarla et al., 2002). Plants have been used to stabilize or remove metals from soil and water. The main methods used include phytoextraction and phytostabilization (USDA, 2000).

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13 Phytoextraction Many higher plant species have adaptations that enable them to survive and to reproduce in soils heavily contaminated with heavy metals. Such species are divided into two main groups: the so called pseudometallophytes that grow on both contaminated and non-contaminated soils, and the absolute metallophytes that grow only on metal contaminated and naturally metal-rich soils (Baker and Brooks, 1989). Phytoextraction involves growing plants in metal contaminated soil and harvesting the metal-rich plant biomass, which can then be incinerated or composted to recycle the metals. Several crop growth cycles are needed to decrease contaminant levels to allowable limits. If the plants are incinerated, the ash must be disposed of in a hazardous waste landfill, but the volume of such ash is far smaller than the volume of waste generated from direct soil manipulation techniques (i.e. soil removal and incineration or chemical treatment) (USDA, 2000). Phytoextraction is often done with plants called hyperaccumulators, which absorb unusually large amounts of particular metals in comparisons to other plants. Hyperaccumulators can tolerate, uptake, and translocate high levels of certain heavy metals that would be toxic to most other organisms. They are defined as plants whose leaves may contain >100 mg kg -1 of Cd, >1000 mg kg -1 of Ni and Cu, or >10,000 mg kg -1 of Zn and Mn (dry weight) when grown in a metal-rich medium. Hyperaccumulators of Co (26 species), Cu (24), Mn (8), Ni (145), Pb (4), and Zn (14) have been reported (Baker and Brooks, 1989). Phytostabilization Root induced changes in rhizosphere soil properties may have significant influence on the mobility and bioavailability of nutrients and trace metals (Arienzo et al., 2003).

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14 Phytostabilization is the use of perennial, non-harvested plants to stabilize or immobilize contaminants in the soil and groundwater. This process takes advantage of the ability of plant roots to alter soil conditions, such as pH and soil moisture content by exudation (Susarla et al., 2002). Metals are absorbed in and accumulated by the roots, adsorbed onto the roots, or precipitated within rhizosphere. Phytostabilization reduces the mobility of the contaminant and prevents further movement of the contaminant into the groundwater or the air and reduces its bioavailability for entry into the food chain (USDA, 2000). One advantage of this strategy over phytoextraction is the disposal of metal-laden plant material is not required. By choosing and maintaining an appropriate cover of plant species, coupled with appropriate soil amendments, it may be possible to stabilize certain contaminants in the soil and reduce the interaction of contaminants with associated biota (Susarla et al., 2002). In order to achieve a successful phytoremediation of soil polluted with metals, a strategy of combining a rapid screening of plant species possessing the ability to tolerate and accumulate heavy metals with agronomic practices that enhance shoot biomass production and increase metal bioavailability in the rhizosphere must be adapted (Kamal et al., 2004). Summary Lead occurs naturally in the earths crust, but due to increased release of anthropogenic Pb into the environment, contamination of soil by Pb constitutes a great environmental concern. In particular, the ubiquity of Pb, its toxicity even in trace quantities and tendency to bioaccumulate in the food chain make lead poisoning a leading environmental health threat.

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15 Presence of hazardous levels of Pb in the environment and the toxicity of Pb requires remedial action to protect public health. Remediation technologies considered to be cost effective and less disruptive, namely phosphorous induced Pb immobilization and phytoremedation have been discussed in this review. This study was conducted for the better understanding of phosphorous induced Pb immobilization mechanisms and the feasibility of phytoremediation in Pb contaminated site.

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CHAPTER 2 EFFECTS OF APPLICATION METHODS ON P-INDUCED Pb IMMOBILIZATION IN A SOIL COLUMN Introduction Activities such as mining and manufacturing, and the use of synthetic products (e.g. pesticides, leaded paints, batteries, and industrial wastes have resulted in many Pb-contaminated sites. Over time, the Pb loading rate in soil has exceeded its natural removal rate by more than 20-fold (Nriagu, 1978). Contamination of soil by lead is of major concern due to not only its high toxicity to humans and animals, but also to its ease of exposure through ingestion or inhalation. A soil is generally considered contaminated with Pb when its total Pb concentration exceeds 400 mg kg -1 (USEPA, 1996), and remediation is required at this level (Ma and Gao, 1999). Various remediation technologies have been developed to clean up metal contaminated soils. Among those, in situ stabilization of heavy metals using binding agents is a promising approach due to its sustainability and cost-effectiveness. Other remediation technologies, including excavation, solidification and chelation/extraction, are either very costly or only partially effective. The estimated cost of solidification is about $750/m 3 and that of stabilization $250/m 3 Stabilization seems to be the more feasible option due to its ease of operation and relatively low cost (Wang et al., 2001). In situ immobilization of soil lead using phosphate has been considered a cost-effective and environmentally benign remediation technology. When phosphorous reacts with lead in soil, it transforms the reactive and bioavailable Pb species into a more stable 16

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17 form. Pyromorphite is one of the least soluble forms of lead found in soils under a wide range of environment conditions. The bioavailability and mobility of soil Pb can be drastically reduced when unstable Pb forms, such as cerrusite (PbCO 3 ), are converted into pyromorphite (Zhang and Ryan, 1999a). During the last decade, many researchers have successfully demonstrated the effectiveness of phosphate-induced Pb immobilization by mixing phosphate minerals with Pb contaminated soils (Ma et al., 1993, 1995; Cotter-Howells, 1996). The immobilization mechanism is considered to be the dissolution of the lead compounds followed by the precipitation of lead phosphate. Thus, successful immobilization of lead in soil requires enhanced solubility of soil Pb and P by decreasing soil pH and applying sufficient phosphorous. Among the various phosphorous sources (apatite, phosphate rock, phosphatic clay, and soluble P), the feasibility of using phosphate rock (PR) to immobilize Pb in soils has been rarely researched. Phosphate rock, a complex assemblage of phosphate minerals, is mainly composed of microcrystalline carbonate fluorapatite (Ma et al., 1995). In addition to reducing metal solubility, phosphate amendments are also effective at reducing metal bioavailability associated with incidental ingestion of soil by humans (Basta and McGowen, 2004). The effectiveness of PR in immobilizing Pb primarily depends on its ability to provide soluble P (Ma et al., 1993, 1995). To compensate for the low solubility of PR in soil soluble phosphoric acid (PA) has been used in combination with PR, which effectively immobilized Pb from a Pb-contaminated soil and facilitated precipitation of Pb-phosphate compounds (Cao et al., 2001). The role of PA in the mixture was to

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18 solubilize lead minerals in soils and PR, thereby increasing the readily available Pb and P in the soil. This, in turn, facilitates more precipitation of Pb-phosphate compounds. The objective of this study was to evaluate the effectiveness of different application methods on lead immobilization in soil using PR and PA as P sources. This was accomplished by (1) determining Pb leaching characteristics and bioavailability; and (2) to evaluate Pb distribution in soil after P application. Materials and Methods Soil Characterization The soil used for this study was collected from a lead-contaminated site in an urban area of northwest Jacksonville, Florida. The site is located in a vacant, fenced rectangular area (4,100 m 2 ), and covered by vegetation, mainly grasses. Past industrial activities, which included a gasoline station, salvage yard, auto body shop, and the recycling of lead batteries, have all contributed to the contamination of this site. Total lead concentrations in the soil ranged from 36 to 21,074 mg kg -1 Lead concentration decreased with soil depth, with the majority of the Pb present near the soil surface (0-20 cm). Mineralogical characterization of the site by x-ray diffraction (XRD) reveals that PbCO 3 (cerussite) is the predominant Pb mineral on the site (Cao et al., 2001). Soil Sample Preparation Soil samples were collected from the top 20 cm at the Jacksonville site. They were air-dried, sieved through a 2-mm stainless steel screen and stored at room temperature. The soil sample was thoroughly mixed to ensure uniformity. They were then digested using the hot-block digestion procedure (USEPA Method 3050A) for total Pb concentration.

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19 Contaminated soil samples were collected from a location where high concentrations of lead are present (Cao et al., 2001). The clean soil sample was collected from outside the fence directly adjacent to the site. The Pb content in the contaminated soil was greater than 5,000 mg kg -1 and the soil collected from outside contained 77 mg kg -1 of Pb. Column Leaching The column test was conducted to simulate field conditions. 400 g of soil was packed into a PVC column (40 cm height by 3.5cm diameter), with the top half (0-20 cm) being filled with the contaminated soil and the lower half (20-40 cm) the clean soil to simulate Pb distribution on the site. The bulk density of packed soil was 1.2 g cm -3 A total of 18 columns were mounted with each treatment replicated three times. The phosphate application rate was based on a P/Pb molar ratio =4, which was the optimum rate for Pb-immobilization in the contaminated soil (Cao et al., 2001). Phosphorous was applied half as PR and half as PA. Both PR and PA were applied only to the first 20-cm of the soil column. The PR was either mixed (M) with the contaminated soil or added as a layer (L) at the bottom of the contaminated soil portion of the column, while the PA solution was added to the top of the column at 75% field capacity. Five different P application methods were used in this experiment and they were as follows: R M A S1 : P was added as P R and P A where PR was m ixed with the contaminated soil and PA was added from the top of the column at the s ame time;

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20 R M A W1 : P was added as P R and P A where PR was m ixed with the contaminated soil and DI was added to the top of the column at 75% field capacity. PA was added from the top of the column one w eek later; R L A S1 : P was added as P R and P A where PR was added as a l ayer at the bottom of the contaminated soil and PA was added from the top of the column at the s ame time; R M A W2 : P was added as P R P A and PA where PR was m ixed with the contaminated soil and PA was added from the top of the column two times with one w eek apart; R L A W2 : P was added as P R P A and PA where PR was added as a l ayer at the bottom of the contaminated soil and PA was added from the top of the column two times with one w eek apart; A fine textured glass wool was applied at the end of the column, acting as a filter for the leachate. The soil columns were leached with deionized distilled water (DDW) twice up to 2 pore-volumes 1 and 4 weeks after the application of PR. Analytical Procedure Soil pH was measured with deionized water at a 1:1 soil:solution ratio after 24 h of equilibrium. Total P was measured colorimetrically with a Shimadzu 160U spectrometer using the molybdate ascorbic acid method. At the end of the experiment, the soil columns were divided into 4 sections (0-10, 10-20, 20-30, and 30-40 cm). The modified physiologically based extraction test (PBET) and Toxicity Characteristic Leaching Procedure (TCLP) were conducted on each section to determine the effectiveness of P-induced Pb-immobilization in the soil. Also total Pb in each soil layer was determined to compare the downward movement of Pb among

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21 different treatments. After leaching columns with up to 2 pore volumes of DDW twice, column leachates were analyzed for Pb and P concentrations. Pb analysis was performed using a flame atomic absorption spectrophotometer or graphite furnace atomic absorption spectrophotometer, depending on the analytic needs of the samples. Soil clay fractions were separated by centrifugation and analyzed using XRD to detect the formation of pyromorphite in the soil. Results Soil Characterization Selected chemical properties of the collected surface soil (0-20 cm) are listed in Table 2-1. Table 2-1 Soil characterization at the contaminated site Soil pH Total Pb (mg kg -1 ) Total Cu (mg kg -1 ) Total Zn (mg kg -1 ) Background soil 6.7 77 20 195 Contaminated soil 6.2 5,017 990 2,200 *Detection limit in soil concentration = 20 mg kg -1 (Pb); 5 mg kg -1 (Cu); 1 mg kg -1 (Zn) The soil is very sandy, with a pH of 6.2-6.7, which is within the range typical of Florida soils (Chen et al., 1999). Lead was the main contaminant with a concentration of 5,017 mg kg -1 which exceeds the critical level for industrial soils (1,400 mg kg -1 ). Also, elevated concentrations of Cu and Zn were observed (990 and 2,200 mg kg -1 respectively). The complete details of the metal contamination at this site have been described by Cao et al. (2001). Generally, Pb, Cu and Zn were concentrated on the surface soil (0-20 cm) and their concentrations decreased with soil depth. However, a substantial amount of Pb (>2,000 mg kg -1 ) was found at depths below 30 cm. In the long

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22 run, it is possible that the metals may leach downward to the subsurface soil (Cao et al., 2002) Effects of P Application on Soil pH The pHs of the P treated soils were measured to evaluate the degree of soil acidification. As seen from Table 2-2, reduced soil pH in the surface soil (0-20 cm) was observed in all the P treated soils due primarily to the addition of phosphoric acid (H 3 PO 4 ). Significant reduction in pH, approximately 1 pH unit, was limited to only the top 10 cm. Table 2-2 pH of soil after treatment Soil layer(cm) Control R M A S1 R M A W1 R L A S1 R M A W2 R L A W2 0-10 6.210.12 5.20.16 5.250.07 5.120.17 5.210.08 5.260.06 10-20 6.160.08 5.750.12 5.990.07 5.890.05 5.870.02 5.890.04 20-30 6.780.02 5.930.02 6.080.04 6.120.06 6.350.01 6.460.02 30-40 6.740.16 6.070.06 6.110.01 6.180.05 6.410.04 6.460.06 Of all of the treatments, R L A S1 induced the greatest decrease in soil pH. The pH of the surface soil was reduced from 6.21 in the control to 5.12 in treatment R L A S1 which is typical of Florida soils (Chen et al., 1999). Yang et al. (2001) added 5,000 mg kg -1 of P as phosphoric acid to immobilize Pb and reported a reduction of soil pH by 3 units from 7.22 to 4.34. Reduction in soil pH was mostly limited to the top 20 cm of the soil column (Table 2-2), indicating limited movement of P in the soil profile (data not shown). Without the application of phosphate rock, soil pH may have further decreased to levels where increased metal solubility may become an issue. Reduction of soil pH to near 5.5 is necessary for efficient Pb immobilization in soil. Since a large fraction of the Pb in the soil is associated with carbonate (Cao et al., 2001),

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23 addition of PA to the soil is essential for the dissolution of PR and Pb carbonate. Soluble P and Pb may enhance precipitation of insoluble pyromorphite-like minerals in the soil, which at given lead and phosphate concentrations is formed a greater rate at pH 5 than at pH 6 or 7 (Laperche et al., 1996). Other researchers have used liming materials along with phosphate treatments to buffer against a decrease in pH (Basta and McGowen, 2004; Brown and Elliot, 1992). Also the application of phosphoric acid to acidify soil may increase the risk of groundwater contamination with P and other heavy metals and, therefore, caution should be exercised. TCLP Pb The toxicity characteristic leaching procedure (TCLP) is designed to evaluate whether hazardous constituents may migrate through the vadose zone soils to the water table by simulating landfill conditions. Higher Pb concentration in TCLP extracts means higher Pb mobility in the soil. TCLP-Pb concentrations in the control soil without P treatment were as high as 127 mg L -1 significantly exceeding the regulatory level of 5 mg L -1 (Fig. 2-1). This is possibly because most of the Pb is in the carbonate fraction, which would readily dissolve in the acidic TCLP solution (Melamed et al., 2003). Phosphate treated soils, on the other hand, reduced TCLP-Pb by >95% compared to the control (Fig. 2-1). As a result of P application, TCLP-Pb in the surface soil (0-20 cm) was reduced to below 5 mg L -1 for all five treatments (Fig. 2-1). Among the five P treatments, the highest reduction in TCLP-Pb was observed for treatment R M A S1 where PR and PA were applied at the same time, which had less than 2 mg L -1 in the 0-10 cm fraction of the soil column (Fig.2-1). On the other hand, treatment R M A W2 where PA was added in two aliquots one week apart was the least effective in

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24 reducing TCLP-Pb in the soil. This result may indicate that spontaneous reaction of PA with PR to solubilize both Pb and P is essential in reducing TCLP-Pb in the soil. Soil Depth TCLP Pb(mg L -1 ) Fig.2-1 TCLP metal contents in sectioned soil columns after leaching *Detection limit = 0.2 mg L -1 Data for lower half section (20-40 cm) are not shown due to no significant difference Hettiarachchi et al. (2000) and Chen et al. (2003) reported the addition of P significantly reduced TCLPPb in contaminated soil to below the critical level of 5 mg L -1 Lead Bioavailability Incidental ingestion of Pb-contaminated soil has been proposed as a primary exposure pathway to humans for elevated blood Pb levels (Chen et al., 2003). Physiologically based extraction test (PBET) has been used to estimate Pb bioavailability (in vivo), which simulates Pb dissolution under gastrointestinal conditions using a

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25 chemical extraction (Yang et al., 2001). Lead bioavailability in contaminated soils has been shown to vary with its mineralogical forms (Davis et al., 1993). In vivo and in vitro assays have indicated that the mammalian gastrointestinal availability of Pb is controlled by the form and relative solubility of Pb solids (Ruby et al., 1996). Reduction in Pb bioavailability was measured by PBET after various amounts and sources of P were added to a Pb contaminated soil (Hettiarachchi et al., 2000). The bioavailability of soil Pb is associated with its solubility and dissolution rate in the gastrointestinal tract. The data in Fig. 2-2 indicate that bioavailable Pb in the contaminated soil based on PBET measurements was reduced after P application in all five treatments. The control soil showed 24-25 mg kg -1 of bioavailable Pb while P-treated soils showed reduction of PBET-Pb by 25-42%, which was similar to the 25-35% reduction reported by Hettiarachchi et al. (2000) and 39% by Yang et al. (2001). The greatest reduction was obtained from treatment R M A S1 where PR and PA were added to the soil at the same time, this result is similar to the TCLP results (Fig.2-1). Distribution of Pb in Soil Column In most contaminated soils, metals do not appear to leach downward in significant quantities in the short run primarily due to their strong interactions with the soil. However, in the long run, metals can leach downward in a soil due to their complexation with solubilized organic mater especially in an alkaline environment where organic matter is more soluble (Marschner and Wilczynski, 1991). This may be true at the Jacksonville site where soil organic matter (3.91%) and pH (6.95) are much higher than those of typical Florida soil (Chen et al., 1999).

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26 0102030ControlRMAS1RMAW1RLAS1RMAW2RLAW2Treatments 0-10 cm 10-20 cm PBET Pb(mg L -1 ) Fig. 2-2 PBET extractable Pb contents in sectioned soil columns after leaching *Detection limit = 0.2 mg L -1 Data for lower half section (20-40 cm) are not shown due to no significant difference To determine the effectiveness of P treatment to prevent Pb downward migration, total Pb concentrations in bottom half of the column (20-40 cm) were determined (Fig. 2-3). As expected, significant downward movement of Pb was observed in the control soil, especially in the 20-30 cm fraction, which was located directly under the Pb-contaminated (0-20 cm). The initial Pb concentration in the lower half of the column (20-40 cm), the control soil, was 77 mg kg -1 (Table 2-1). After being in contact with the Pb-contaminated soil for six weeks under 75% field capacity, the Pb concentration in the 20-30cm-soil column fraction increased to 365 mg kg -1 However, Pb migration was mostly limited to that section of the column as Pb concentration in the 30-40 cm fraction

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27 remained at 77 mg kg -1 and little difference was observed among the five treatments (Fig. 2-3). All treatments were effective in reducing Pb migration except R M A W2 where PR was mixed with the contaminated soil in the top 20-cm, and PA was applied two times with one week between applications. Since PA was added in two aliquots, the amount of acidity was probably insufficient to dissolve enough Pb from the soil to induce the precipitation of Pb-phosphate minerals. This was confirmed by the P data where P concentration in the leachate of R M A W2 was the lowest (Fig. 2-4). This was also consistent with the highest TCLP-Pb (Fig. 2-1) and PBET-Pb (Fig. 2-2) observed in treatment R M A W2 Treatments with PR as a layer (e.g. R L A S1 R L A W2 ) were more effective in reducing Pb migration than those where PR was mixed with the soil (R M A S1 R M A W1 and R M A W2 ; Fig.2-3). This may be due to the formation and/or containment of stable Pb minerals within the PR layer. Among the treatments where PR was mixed with the soil, treatment R M A S1, where PA was applied at the same time as when PR was mixed with the contaminated soil, was most effective. To determine the mechanism of reduction in Pb downward movement, soil around the PR layer was separated and analyzed with XRD. Previous analysis of mineralogical changes over time resulting from P addition using XRD showed formation of stable pyromorphite-like minerals in P-treated soil (Cao et al., 2002). However, no Pb-phosphate was identified in the clay fractions in this research (data not shown). With the Pb concentration the studied soil being near the MDL for XRD the lack of lead-phosphate

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28 confirmation cannot be ruled out. Further investigation is needed to verify the mechanism within the PR layer. 0100200300400500ControlRMAS1RMAW1RLAS1RMAW2RLAW2Treatments 20-30 cm 30-40 cm Total Pb(mgkg -1 ) Fig. 2-3 Total Pb concentration in sectioned soil columns (bottom section) *Detection limit=20mg kg -1 Data for upper half section (0-20 cm) are not shown due to no significant difference All P-treated soil columns showed that leachable Pb had been reduced to below the EPA drinking water regulatory level of 15 gL -1 or non-detectable (Table 2-3). The second highest Pb concentration was observed in treatment R M A W2 and was comparable to the control soil. Lead concentrations in the second leachate were lower than those in the first leachate. Significant differences were seen in the release of Pb between treatments with different application methods of PA, where treatment R M A W2 was observed to be the least effective and yet still below the EPA regulatory level (15 gL -1 ). All five treatments proved to be effective in reducing Pb contamination from soil.

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29 Table 2-3 Pb concentration (ppb) in column leachate (g L -1 ) Treatment 1 st leaching 2 nd leaching Control 33.6.5 14.5.74 R M A C1 nd nd R M A S1 nd nd R L A C1 nd nd R M A s2 11.0.9 5.7.91 R L A S2 2.3.45 nd *Detection Limit = 1 gL -1 Leachate Analysis Column studies more closely simulate field conditions than the TCLP extraction does. To simulate field conditions, soil columns were leached twice with DDW and the leachate was collected after one and five weeks of incubation. The leachate was analyzed for total Pb and P concentrations (Table 2-3, Figure 2-4). The addition of large amounts of P to a contaminated soil may increase the risk of eutriphication to water bodies (Basta and McGowen, 2004). Taking into account P leaching in the soil after P application is important with respect to secondary contamination. Although it is well recognized that phosphate amendment is an effective method for immobilizing metals in contaminated soils, some elevated soluble phosphorous may enhance eutrophication risk (Cao et al., 2001).

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30 00.20.40.60.811.21.4ControlRMAS1RMAW1RLAS1RMAW2RLAW2Treatments 1st leaching 2nd leaching Leachate P (mg L -1 ) Fig. 2-4. P concentration in column leachate (mg L -1 ) *Detection limit = 1 gL -1 This was not the case in this study as phosphorous leached from the P-treated soil columns was less than 1 mg L -1 for all treatments (Fig. 2-4). Treatments where PA was applied in two aliquots (R M A W2, R L A W2 ) had lower P concentrations in the leachate than in the other treatments. This may indicate that the application of P in small quantities can limit the downward movement of PA, but with correspondingly lower efficiency in immobilizing Pb in the soil (Figures 2-1 and 2-2). Though phosphorous release from P-treated soil was minimal, some of the P was released as it was not converted to lead phosphate. Therefore, caution is needed to assess the P movement within P-treated fields to prevent eutrophication risk, and consideration given to soil type and the amount of P added.

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31 Conclusion The efficiency of in situ P-induced Pb immobilization depends on the type and rate of P amendment, along with appropriate application methods. Preliminary laboratory studies to assess its mechanism and efficiency are important. This study has shown that applying a mixture of PA and PR effectively reduced TCLP-extractable Pb, bioavailable Pb and vertical Pb migration in the Pb-contaminated soil studied. All P-treated soil resulted in significant reductions in bioavailable and leachable Pb as compared to the control soil. The effective immobilization of Pb was attributed to the formation of stable Pb minerals after P application. Of all the treatments, the mixture of PR and soil coupled with the simultaneous application of PA (R M A S1 ) was the most effective in decreasing leachable and bioavailable Pb with the least impact on soil pH and lowest eutrophication risk. Treatments with PA being applied in two aliquots and PR being applied as a layer was the least effective overall. Possible explanations of these results are: 1) the reaction between soil and P amendments before PA application (after one week) inhibited the formation of lead phosphate; 2) less PR was mixed with soil (R L A S1 and R L A W2 ), which caused lower efficiency in reducing leachable and bioavailable Pb in soil. These results also indicate that a minimal amount of simultaneously applied PA with the PR is necessary for effective (maximal) immobilization of Pb in soil to take place. The fact that layered PR showed lower efficiency in Pb immobilization as compared to the other treatments may be due to an insufficient amount of P supplied to the soil. However, the PR layer showed improved reduction of Pb migration than other mixture treatments. Furthermore, these results suggest layered PR below contaminated soil may serve as a reactive barrier to prevent Pb

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32 from migrating into the groundwater. Although the effects of PA on pH and leachibility P were acceptable in this experiment, competitive heavy metal leaching and eutrophication may be potential drawbacks of its indiscriminate utilization. Further studies are needed to determine the mechanism of Pb-migration reduction by the PR layer. Also, the combination of the soil-PR mixture and PR layer can be considered as well for remediation of Pb contaminated soils, and with reduced leaching, bioavailability and mobility of Pb.

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CHAPTER 3 THE EFFECTS OF PLANTS ON PHOSPHATE-INDUCED Pb IMMOBILIZATION IN THE RHIZOSPHERE SOIL Introduction Of the various technologies for the remediation of lead contaminated sites, phosphate-induced Pb immobilization has received much attention in recent years. Lead phosphates have low solubility, and are generally several orders of magnitude less soluble than analogous carbonates and sulfates (Cotter-Howells, 1996). Various forms of phosphate have been used to immobilize Pb in situ from aqueous solutions and Pb contaminated soils (Ma et al., 1995; Hodson et al., 2000; Yang et al., 2001). The primary mechanism of Pb immobilization appears to be through phosphate mineral dissolution and subsequent precipitation of pyromorphite-like minerals. Hence, the solubility of phosphate-mineral applied largely determines the effectiveness of in situ Pb immobilization in soils (Ma, 1996). For soil P to be available to react with Pb to form lead phosphate, it must be present as free phosphate (H n PO 4 n-3 where n=1 to 3) in the soil solution. However, phosphate minerals in soils also have relatively low solubilities. In a study on Pb immobilization using phosphorous, a mixture of phosphoric acid (PA) and phosphate rock (PR) as the sources of P were applied to enhance solubilization of P and Pb-carbonate in soil (Cao et al., 2001). Phosphorous is one of the essential macronutrients, and among the most limiting nutrients for plant growth in soils. The average P-content in soil is about 0.05 % (w/w) 33

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34 but only 0.1% of that P is available to plants (Illmer and Scinner, 1995). It has been shown that organic acids can greatly increase the concentration of plant-available P in the soil solution through acidification, chelation and anion exchange (Reyes et al., 2001). Root exudates containing organic acid or phosphatase enzymes play an important role in the liberation of phosphate ions from organic and inorganic compounds in soils (Cotte-Howells, 1996). Through roots-induced chemical modifications in the rhizosphere, higher plants may be directly responsible for the dissolution of PR. In particular, rhizosphere pH has been shown to differ by 2 units as compared to that of the bulk soil. Several researchers have concluded that excretion of protons or organic acids by the roots is a major process by which plants can acquire P from PR present in soil (Hinsinger and Gilkes, 1997). Free phosphate in the soil solution and available to plant roots may be available to react with lead and form lead phosphate. The overall objective of this experiment was to determine the effectiveness of phosphate-induced Pb immobilization in the rhizosphere soil of different plant species. Specific objectives were, 1) to evaluate the effect of plant roots on P-induced reduction of Pb bioavailability; and 2) to determine the effect of plant roots on formation of stable Pb phosphates. Materials and Methods Surface (0-20 cm) soil samples were collected from a heavy-metal contaminated site in Jacksonville, Florida. Soils were air-dried for 2 weeks, sieved through a 2-mm stainless steel screen and stored at room temperature prior to the experiment. The soil sample was mixed thoroughly to ensure uniformity. Soil pH was measured using a 1:1 soil:water ratio. Soil samples were digested using the hot-block digestion procedure

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35 (USEPA Method 3050a) for total lead concentration. Ground rock phosphate [PR, primarily Ca 10 (PO 4 ) 6 F 2 ] and PA were used as the sources of phosphorous. Three plant species were selected to determine the effects of plant root exudates on phosphate-induced Pb immobilization in the rhizosphere soil. The first, Agrostis capillaries, has proven to be effective in inducing the formation of metal-phosphates (Cotter-Howells, 1996). The other two, Lolium rigidum and Brassica napus, may also enhance root-induced PR dissolution in the rhizosphere (Hinsinger and Gilkes, 1997). The following treatments were considered: Control 1: Soil + Phosphate rock (PR) Control 2: Soil + PR + Phosphoric acid (PA) Treatment 1: Soil + PR + Plant Treatment 2: Soil + PR + PA + Plant Pots of 4 inches in diameter were used and filled with 520 g of the Pb-contaminated soil collected from Jacksonville. 10-20 plants seeds were sown into each pot. The plant seeds were sown directly onto the surface soil, with the rhizosphere soil separated from the bulk soil. This was accomplished by using a circle of plastic netting 9.5cm in height and 5.5cm in diameter, which was covered with a nylon mesh cloth (mesh size 45 m) placed in the center of the pot, and filled with what would become the rhizosphere soil. This limited root growth to this area of the pot. The plants (10 plants per pot) were grown in a greenhouse for 4 weeks (plant heights were approximately 10 cm) and watered as necessary. At the end of four weeks, the rhizosphere soil was separated from the bulk soil. The roots were washed gently to remove the adhered soil particles and placed in a glass vial containing approximately 10 ml of deionized distilled

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36 water (DDW). This container was then ultrasonicated for 30 seconds to remove any remaining soil particles. The resulting solution in the glass vial was filtered using a 45 m filter paper and collected. The residues on the filter containing both soil and plant root samples were mounted on a carbon stub for further analysis. Fig. 3-1 Lolium rigidum after 4 weeks of growth Fig. 3-2 Agrostis capillaries after 4 weeks of growth Examination of the rhizosphere soil was conducted with a scanning electron microscope (SEM) to investigate formation of Pb-P minerals (i.e. pyromorphite). The

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37 presence of pyromorphite like minerals was further analyzed by X-ray diffraction (XRD). For XRD analysis, the clay fractions of the soil samples were separated and characterized. Fig. 3-3 Brassica napus after 4 weeks of growth Also PBET (physiologically-based extraction test) was conducted on the rhizosphere soil to see the effects of plant roots on bioavailable Pb in the soil. Results Soil Characterization The lead contaminated soil had a pH of 6.2 (Table 3-1), which was high compared to the average value for Florida soils (pH 5.04) (Chen et al., 1999). Lead was the main contaminant at the site, with a concentration of 5,017 mg kg -1 In addition, elevated concentrations of Cu and Zn were also observed (990 and 2,200 mg kg -1 each). Table 3-1. Selected characteristics of the lead-contaminated soil Soil pH Total Pb (mg kg -1 ) Total Cu (mg kg -1 ) Total Zn (mg kg -1 ) Contaminated soil 6.2 5,017 990 2,200 *Detection limit in soil concentration = 20 mg kg -1 (Pb); 5mg kg -1 (Cu); 1mg kg -1 (Zn)

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38 Bioavailable Pb In vitro metal bioavailability is an estimate of metal bioavailability to humans and animals by simulating metal dissolution under gastrointestinal condition using a chemical extraction (Yang et al., 2001). 05101520253035PR onlyPR+PATreatment Control Agrostis capillaries Brassica napus Lolium rigidum PBET Pb ( m g L -1 ) *Detection limit = 0.2 mg L -1 Fig. 3-4. Lead concentrations using the physiologically based extraction test in the rhizosphere soil with phosphoric acid and with phosphate rock treatment. Bioavailable Pb in the rhizosphere soil as measured by PBET was reduced in the presence of all three plants when growing in soils treated with PR (Fig. 3-4). The rhizosphere soil in the control had 26 mg kg -1 of bioavailable Pb while those with plants had 21-23 mg kg -1 an 11-20% reduction. Such a reduction in the bioavailable Pb in the rhizosphere soil may result from the formation of Pb phosphate, which was supported by

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39 the SEM observation of the plant roots (Figs. 3-5, 6, 7). In the treatment with PR+PA, no significant reduction of bioavailable Pb was found. Formation of Lead Phosphate The main mechanism of Pb immobilization in soil is via dissolution of P and/or meta-stable Pb compounds and the subsequent precipitation of pyromorphite-like minerals (Cao et al., 2001). The analysis of mineralogical changes in the rhizosphere soil that were affected by root exudates using SEM helps to determine whether root-induced formation of Pb phosphate occurred. The SEM element dot maps of root samples after growing in a soil for 4-weeks show (Figs. 3-5, 6, 7) close association of Pb with P and Ca, which may suggest the formation of lead phosphate in the rhizosphere soil For A. capillaries, no significant associations between Pb with P and Ca were observed in the SEM elemental dot map (Fig. 3-5), but broad association of the three elements may indicate Pb phosphate in the phosphate rock treated rhizosphere soil (Fig. 3-5b,c,d). For B. napus, the SEM data suggests a close association between Pb, P and Ca. (Fig 3-6c, d, and e). For L. rigidum, association of P and Pb was observed but not a strong association with Ca. The association of Ca, P and Pb may suggest that Pb precipitation occurred on the surface of the phosphate rock particle, mainly as Ca 10 (PO 4 ) 6 F 2 (Cao et al., 2001). Formation of stable lead-phosphate minerals may be enhanced due to the biochemical actions of plant roots, i.e. root exudates containing phosphatase or organic acid. In XRD analysis, no pyromorphite-like mineral was identified in the clay fractions of the rhizosphere soil (data not shown). However, this is because the Pb concentration in the soil was barely detectable by XRD, and the lack of confirmation of this technique does not prove that no pyromorphite was formed. However, root induced phosphorous

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40 dissolution is proved by analyzing water-soluble phosphorous in the rhizosphere soil. As seen in Table 3-2, higher water soluble phosphorous was observed in the soil with plants growing as compared to the control soil. This increase ranged from 37-46% in the PR-only treated soils to 8-9% in the PR+PA treatment. These results indicate that more phosphorous solubilization occurred in the presence of plant roots and that higher available phosphorous may enhance the formation of stable lead-phosphates in the rhizosphere. Root exudates containing phosphatase enzymes and/or root-infecting microorganisms may be responsible for this dissolution of P and precipitation of lead phosphates. Another possible explanation for lead phosphate formation in the rhizosphere soil may be that the plant roots created localized acidity, which may have enhanced the dissolution of the phosphorous minerals and caused precipitation on the root surface (Cao et al., 2002). Soil pH was analyzed to evaluate the effects its reduction by root exudates on phosphate-induced Pb immobilization. Numerous studies have shown that soil pH may also be critical in the dissolution of PR in soil (Ma et al., 1993, 1995; Singh et al., 2000). The dissolution of PR may be enhanced by the supply of protons and removal of dissolution products in particular Ca and P. Acidic soils are likely to yield an extensive PR dissolution because they often combine a low pH, with a high P sorption capacity due to the abundance of Al and Fe oxyhydroxides (Hinsinger and Gilkes, 1997). As seen in Table 3-3, slightly reduced soil pH was observed in the rhizosphere soils treated with PR but no significant differences were found in those treated with the PR+PA mixture. Solubilization of phosphorous is thought to be caused by the release of organic acids and/or phosphatase, which has been reported to be

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41 exclusively acid (George et al., 2002). The reduction of pH induced by root exudatation (i.e. organic acid, phophatase) may enhance the dissolution of PR and Pb-carbonate, promoting the formation of lead phosphate. Several authors have concluded that proton excretion by the roots was the major process by which plants such as buckwheat, rape or various legumes can acquire P from PR (Bekele et al., 1983; Hinsinger and Gilkes, 1997). Youssef and Chino (1989) have shown that a single species of rape can strongly acidify its rhizosphere when grown in an alkaline soil. a b c d Fig. 3-5. Scanning electron microscopy elemental dot map of Agrostis capillaries Hoffland et al. (1989) considered the dissolution of PR by rape was due mainly to the excretion of organic acid by the roots. Significant reduction of bioavailable Pb and pH, and higher PR solubilization in soil was observed only in PR-treatments whereas no significant differences in bioavailable Pb was found for soils treated with PR+PA. It is possible that the addition of PA provided both the necessary acidity and soluble P to the plants so no effect from plant roots was observed.

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42 Fig.3-6. Scanning electron microscopy elemental dot map of Brassica napus Fig. 3-7. Scanning electron microscopy elemental dot map of Lolium rigidum Table 3-2 Water soluble phosphorous in plant rhizosphere soil (ppm) PR Only PR+PA Control 0.170.01 5.791.16 Agrostis capillaries 0.320.05 6.310.51 Lolium rigidum 0.280.04 6.360.57 Brassica napus 0.270.04 6.320.55 d c a d c a b e b e

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43 Table 3-3 pH of rhizosphere soil treated with phosphate rock after 4-weeks growth PR Only PR+PA Control 5.720.21 5.010.23 Agrostis capillaries 5.310.41 4.970.34 Lolium rigidum 5.340.07 4.890.43 Brassica napus 5.230.13 4.900.23 Conclusion This study has shown that all three plant species had the ability to induce the formation of lead phosphate in the rhizosphere soil as supported by the SEM analysis of plant root samples (Figs. 3-5, 6, 7). Association between Pb, P and Ca in the SEM dot maps suggests formation of lead phosphates induced by the biochemical action of the plant roots. This experiment established that the biochemical effects of plant roots can promote additional dissolution of phosphorous minerals and formation of lead phosphates. Reduction of the rhizosphere soil pH may indicate that plant roots enhance the dissolution of PR and Pb-carbonate by excreting H + thereby facilitating the formation of lead phosphate. Reduction in bioavailable Pb in the rhizosphere soil may due to enhanced dissolution of PR and formation of lead phosphate by biochemical reaction in the rhizosphere soil. Using various leachates to examine the bioavailability of Pb in a contaminated soil, Rabinowitz (1993) found a 43% decrease in Pb concentration extracted with 10% citric acid in phosphate-amended soils. Our results indicate that all three plants may have the potential to be applied for the reclamation of Pb-contaminated sites in combination with the application of PR. Also soil microorganisms are involved in this process and their role in the solubilization of phosphate-bearing materials has been the subject of many studies. There is a considerable number of phosphate-solubilizing bacteria present in soil and in the rhizospheres of plants, and their concentration much

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44 higher in the rhizosphere as compared to non-rhizosphere soil (Rodriguez and Fraga, 1999). The combination of plant root exudates and microorganisms may increase the amount of free phosphate, thus enhancing the formation of lead phosphate in the rhizosphere soil. However, the extent to which either of these factors could re-solublize heavy metal phosphate for subsequent phosphate extraction is unknown. Further research should investigate the stability of the phosphate dissolution mechanisms of both plants and soil microorganisms. Vegetative techniques have been widely used in the reclamation of mine waste, mainly to stabilize the soil and promote nutrient cycling. This work suggests that the growth of plants, which has the ability to induce formation of insoluble lead phosphates, can enhance the reclamation of a contaminated site. The successive growth and decay of species over several seasons could eventually produce enough lead phosphates (Cotter-Howells, 1996) to achieve this end. Substantial amounts of pyromorphite present in soils can reduce bioavailable Pb. The increased effectiveness in phosphate-induced Pb immobilization as a result of plant root activity would be more ecologically acceptable than the addition of a large amount of soluble phosphate alone because of less concern with soil acidification and eutrophication. However, additional research is needed to further assess the effects of plant-root enhanced phosphate-induced Pb immobilization in soil on a field scale.

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CHAPTER 4 SCREENING OF PLANTS FOR ACCUMULATION OF Pb, Cu, Zn FROM A CONTAMINATED SITE Introduction Heavy metals are one of the most serious environmental concerns today. They are harmful to humans and animals and tend to bioaccumulate in the food chain. Past usage of fossil fuels, mining and smelting of metal ores, industrial emissions and the application of insecticides and fertilizers all have contributed to elevated heavy metal levels in the environment. The threat that heavy metals pose to human and animal health is aggravated by their elemental nature and long-term persistence. There are many technologies available for the remediation of land contaminated by heavy metals (USDA, 2000). However, many of these technologies are costly (e.g. excavation of contaminated material and chemical/physical treatment) or do not achieve either a long term or aesthetic solution (Cotter-Howells, 1996). A new approach, termed phytoremediation, offers an alternative technology. Phytoremediation can provide a cost-effective, long-lasting and aesthetic solution for the remediation of contaminated sites. One of the strategies of phytoremediation with respect to metal contamination is phytoextraction, i.e. the uptake and accumulation of metals into plant shoots, and their subsequent harvest. Metal accumulating plants are not only of scientific interest, but they also serve to protect several aspects of the environment, such as phytoremediation of lands polluted with heavy metals, long-term stabilization of wastelands and the reduction of potentially 45

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46 toxic elements (Raskin and Enseley, 2000). There are over four hundreds plants known to be hyperaccumulators of metals, which accumulate high concentrations of metals into their aboveground biomass. These plants include trees, vegetable crops, grasses and weeds. Based on Baker and Brooks (1989), hyperaccumulators are defined as plants that accumulate >1,000 mg kg -1 of Cu, Co, Cr, Ni or Pb, or >10,000 mg kg -1 of Mn or Zn. Hyperaccumulators of Co (26 species), Cu (24), Mn (8), Ni (145), Pb (5), and Zn (4) have been reported. The five hyperaccumulators of lead include Armeria martima, Thlaspi rotundifolium, Thlaspi alpestre, Alyssum wulfenianum, and Polycarpaea synandra. It is important to use indigenous native plants species for the phytoremediation process as these plants are better adapted for survival, growth and reproduction under the particular environmental stresses encountered than plants introduced from another environment. However, few studies have evaluated the phytoremediation potential of natural hyperaccumulators under field conditions (McGrath and Zhao, 2003). This study was conducted to screen plants that were growing in a contaminated site for their potential in accumulating Pb, Cu and Zn. The overall objectives were: 1) to determine the concentrations of Pb, Cu and Zn in the plant biomass; 2) to compare metal concentrations in the aboveground biomass to those in the roots and in the soils; and 3) to assess the feasibility to use these plants for the purpose of phytoremediation The information obtained from this study should provide insight for the use of native plants to remediate metal contaminated sites.

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47 Materials and Methods Site Characterization The plant and soil samples for this study were collected from a known lead contaminated site located at an urban area of northwest Jacksonville, Florida. The fenced vacant site is rectangular in shape, occupying approximately one acre. The site is covered by vegetation, mainly grasses. Past industrial activities include being home to a gasoline station, salvage yard, auto body shop, and recycler of lead batteries, which all presumably, contributed to its contamination. Total lead concentration in the soils ranged from 90 to 4,100 mg kg (Fig. 4-1 and Table 4-1) -1 Fig. 4-1. Map of Jacksonville site with lead concentration contour map and sampling point In addition to Pb contamination, the site also contained elevated levels of Cu and Zn, i.e. 20-990 and 195-2,200 mg kg -1 (Table 4-1). The contamination by heavy metals is concentrated in the top 20 cm of the site (data not shown).

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48 Sample Preparation and Chemical Analysis Plant samples, together with associated soil samples were collected from the site. The selection of plant samples was based on their coverage at the site. In total, 36 plant samples of 17 different species were collected from 10 locations at the site in December of 2002 (Fig. 4-1). Soil samples from the rooting zone (0-20 cm) were taken from each location. Soil samples were air-dried at room temperature for two weeks, and then sieved through a 2-mm stainless steel sieve. They were then digested using the hot-block digestion procedure (USEPA Method 3050a) for total metal concentration. For water-soluble Pb concentration, 25 ml of deionized distilled water (DDW) was mixed thoroughly with 2 g of soil for 12 h. The mixture was then centrifuged for 15 min. at 3,500 rpm. The supernatant was analyzed for Pb by flame atomic absorption spectrophotometry (FAAS). Plants samples were divided into roots and shoot, washed gently with DDW for approximately 1 minute to remove the soil. After washing, plant samples were air-dried at room temperature for two weeks. They were then ground to a powder before digestion via USEPA Method 3050a. Results Metal Concentrations in Soils Characteristics of the soil samples collected from this study are shown in Table 4-1. Soil pH ranged from 6.62 to 7.20, relatively high compared to typical Florida soils (Chen et al., 1999). The high soil pH has been attributed to the presence of cerrusite (PbCO 3 ), the predominant form of the Pb minerals at the site (Cao et al., 2001).

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49 Table 4-1. Selected properties of soil samples collected from the contaminated site at Jacksonville, Florida Site # Soil pH Water soluble Pb (mg L -1 ) Total Pb (mg kg -1 ) Total Cu (mg kg -1 ) Total Zn (mg kg -1 ) 1 7.03 1 90 20 200 2 7.00 3 143 21 195 3 6.62 32 4,100 990 2,200 4 7.06 82 1,375 980 900 5 7.2 65 1,886 860 683 6 7.08 20 767 314 551 7 6.95 234 2,405 746 1,000 8 6.97 96 1,451 300 572 9 7.01 2 333 29 532 10 6.63 1 145 26 720 *Detection limit = 0.1mg kg -1 (Pb); 5mg kg -1 (Cu); and 1mg kg -1 (Zn) Total lead concentrations in the soil samples collected from 10 different locations were variable, ranging from 90 mg kg -1 at site 1 to 4,100 mg kg -1 at site 3 (Fig. 4-1). The mean Pb concentration in Florida soils is 77 mg kg -1 The global baseline level of Pb in natural surface soils is reported to be 20 mg kg -1 (Chen et al., 1999). In addition to total Pb, water-soluble Pb was also determined (Table 4-1). As expected, they were much lower than total Pb concentrations. However, total Pb correlated with water-soluble Pb (r 2 = 0.48). There were also elevated concentrations of Cu and Zn, ranging from 20 to 990 mg kg -1 for Cu, and 195 to 2,200 mg kg -1 for Zn. Metal concentrations in the soil samples collected from different locations were highly correlated with r 2 = 0.72-90, i.e. a site that had a high Pb concentration also tended to have high Zn and Cu concentrations. Among the 10 locations sampled, sites 3, 4, 5, and 7 were the most contaminated with all three metals (Table 4-1).

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50 Metal Concentrations in Plants A total of 17 plant species were collected from 10 locations at the site. They were then identified and analyzed for heavy metal (Pb, Cu, and Zn) concentrations in their biomass. Concentrations of Pb, Cu and Zn in the soil and in the plant biomasses are listed in Tables 4-2, 3, and 4. It is generally agreed that metal concentrations in plants vary with plant species (Alloway, 1994; Alloway et al., 1990). Plant uptake of heavy metals from soil occurs either passively with the mass flow of water into the roots or through active transport, crossing the plasma membrane of roots epidermal cells. Under normal growing conditions, plants can potentially accumulate certain metal ions an order of magnitude greater than the surrounding medium (Kim et al., 2003). Lead concentrations in the plants ranged from non-detectable to as high as 1,183 mg kg -1 with the maximum value found in the roots of Phyla nodiflora collected from site 4 (Table 4-2). In addition, other species such as Bidens alba, Rubus frutocosus and Gentiana pennelliana also contained significantly higher Pb concentrations than the rest plant species. None of the plants accumulated Pb at 1,000 mg kg -1 in the aboveground biomass, which is the criterion for a Pb hyperaccumulator (Baker and Brooks, 1989). In 95% of the plant samples, the roots Pb concentration was much greater than that of the shoots, indicating a low mobility of Pb from the roots to the shoots and immobilization of heavy metals in roots. No relationship between water soluble Pb and plant-boimass Pb was found. Pichtel et al. (2000) also analyzed Pb in native plants, which was collected from a dumpsite and got similar results (non-detectible to 1,800 mg kg -1 ). Stoltz and Greger (2002) reported a range of 3.4-920 mg kg -1 in different plant species collected from mine trailings.

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51 Copper concentrations in the plants varied from non-detectable to 460 mg kg -1 (Table 4-3), with the maximum value found in the roots of P. nodiflora. Similar to Pb, no plant species accumulated Cu above 1,000 mg kg -1 the criterion for a Cu hyperaccumulator (Baker and Brooks, 1989). As expected, the Cu concentrations in the roots were greater than those in the shoots. In addition to P. nodiflora, G. pennelliana,, Bidens alba also contained significantly higher Cu concentrations than other plant species. Copper concentrations of 6.4-160 mg kg -1 in the plant biomass were reported by Stoltz and Greger (2002), which were lower than those in our research. Shu et al. (2002) reported Cu concentrations of 8-45 mg kg -1 in the biomass of Paspalum distichum and Cynodon dactylon. The Zn content in the plants ranged from 17 to 598 mg kg -1 (Table 4-4), with the maximum value found in the roots of P. nodiflora collected from site 4, which also contained the highest Pb and Cu concentrations in the roots. Similar to Pb and Cu, no plant species accumulated Zn at 10,000 mg kg -1 the criterion for a Zn hyperaccumulator (Baker and Brooks, 1989). Generally, Zn concentrations were greater in the roots than the shoots. Also Paspalum notatum, G. pennelliana, Bidens alba and Stenotaphrum secundatum showed significantly higher Zn concentrations than the rest of plant species collected. Research conducted by Stoltz and Gerger (2002) found Zn concentrations of 68-1,630 mg kg -1 in the plants they collected while those by Shu et al. (2002) contained 66-1,015 mg kg -1 Phyla. nodiflora collected from site 4 accumulated the highest Pb, Cu and Zn in the roots (1183, 460, and 598 mg kg -1 respectively). Other than P. nodiflora, G. pennelliana and Bidens alba had higher concentrations of heavy metals (Pb, Cu and Zn) than other

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52 species collected from the contaminated site. No species satisfied the criterion for a heavy metal hyperaccumulator, but ability of the above species to accumulate heavy metals in their biomass needs further investigations. Although total metal concentrations in soils play an important role in the uptake of metals by plants, this can be influenced by several factors. In general, a negative correlation was found between soil pH and metal concentrations in the plants. Other soil factors such as cation exchange capacity also influence metal uptake by plants in some cases (Jung and Thornton, 1996). Accumulation and Translocation of Metals in Plants In this study, none of the plant species showed metal concentrations >1,000 mg kg -1 in the shoots (Tables 4-2, 3, 4), i.e. none of them are hyperaccumulators based on Baker and Brooks (1989). Bioconcentration factor (BCF) and translocation factor (TF) can be used to estimate a plants potential for phytoremediation effectiveness. A plants ability to accumulate metals from soils can be estimated using BCF, which is defined as the ratio of metal concentration in the roots to that in the soil. A plants ability to translocate metals from the roots to the shoots is measured using TF, which is defined as the ratio of metal concentration in the shoots to that of the roots. Phytoextraction is the removal of a contaminant from soil, groundwater or surface water by live plants. Enrichment occurs when a contaminant taken up by a plant, is not degraded rapidly or completely accumulated in the plant. The process of phytoextraction generally requires the translocation of heavy metals to the easily harvestable plant parts, i.e. the shoots.

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53 Table 4-2. Lead concentrations in soil and plant samples (mg kg-1) collected from the contaminated site at Jacksonville, Florida Common name Scientific name Site # Roots Shoots Soil Bahia grass Paspalum notatum Flugge 4 575 428 1,375 5 397 92 1,886 9 nd* nd 333 Wire grass Gentiana pennelliana Fern 1 968 453 90 8 881 491 1,451 Romerillo Bidens alba (L.) DC 2 947 91 143 3 149 23 4,100 5 660 77 1,886 Bermudagrass Cynodon dactylon (L.) DC 5 293 88 1,886 6 75 52 767 Flatsedge Cyperus esculentus L. 1 28 18 90 2 16 26 143 8 417 26 1,451 Ticktrefoil Desmodium paniculatum (L.) 2 130 20 143 Horsetail Equisetum arvense L. 3 284 38 4,100 Hydrocotyle Hydrocotyle americana L. 8 99 8 1,451 10 nd nd 145 Turkey tangle Phyla nodiflora (L.) Greene 1 44 24 90 4 1,183 73 1,375 7 117 83 2,405 7 451 55 2,405 Plantain Plantago major L. 1 9 52 90 5 294 67 1,886 Blackberry Rubus frutocosus L. 3 825 22 4,100 6 127 12 767 9 51 nd 333 Goldenrod Solidago altissima L. 6 59 49 767 10 nd nd 145 Sowthistle Sonchus asper (L.) Hill 7 146 39 2,405 St. Augustine Stenotaphrum secundatum 1 31 14 90 3 68 32 4100 Bluejacket Tradescantia ohiensis Raf. 5 206 140 1,886 Tuberous Verbena rigida Spreng. 1 23 11 90 8 35 11 1,451 Bigpod Sesbania exaltata (Raf.) Cory 2 150 nd 143 *Detection limit for Pb = 0.1mg kg -1

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54 Table 4-3. Copper concentrations in soil and plant samples (mg kg -1 ) collected from the contaminated site at Jacksonville, Florida Common name Scientific name Site # Roots Shoots Soil Bahia grass Paspalum notatum Flugge 4 250 352 980 5 360 60 860 9 10 11 30 Wire grass Gentiana pennelliana Fern 1 432 200 20 8 375 210 300 Romerillo Bidens alba (L.) DC 2 10 8 21 3 44 17 990 5 400 32 860 Bermudagrass Cynodon dactylon (L.) DC 5 310 52 860 6 36 21 314 Flatsedge Cyperus esculentus L. 1 16 10 20 2 10 28 21 8 150 20 300 Ticktrefoil Desmodium paniculatum (L.) 2 6 6 21 Horsetail Equisetum arvense L. 3 110 23 990 Hydrocotyle L. Hydrocotyle americana L. 8 32 13 300 10 21 16 26 Turkey tangle Phyla nodiflora (L.) Greene 1 31 14 20 4 460 20 516 7 nd* 47 746 7 180 23 746 Plantain Plantago major L. 1 23 10 20 5 150 27 860 9 24 12 29 Blackberry Rubus frutocosus L. 3 30 46 990 6 65 13 314 9 47 265 29 Goldenrod Solidago altissima L. 6 277 241 314 Sowthistle Sonchus asper (L.) Hill 7 46 34 746 St. Augustine grass Stenotaphrum secundatum 1 22 17 20 3 42 15 990 Bluejacket Tradescantia ohiensis Raf. 5 194 117 860 Tuberous vervain Verbena rigida Spreng. 1 14 10 20 8 25 18 300 Bigpod sesbania Sesbania exaltata (Raf.) Cory 2 12 48 21 *Detection limit for Cu=5mg kg -1

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55 Table 4-4. Zinc concentrations in soil and plant samples (mg kg -1 ) collected from the contaminated site at Jacksonville, Florida Common name Scientific name Site # Roots Shoots Soil Bahia grass Paspalum notatum Flugge 4 450 316 900 5 260 166 683 9 250 200 532 Wire grass Gentiana pennelliana Fern 1 524 250 200 8 310 359 572 Romerillo Bidens alba (L.) DC 2 25 20 195 3 143 230 2,200 5 462 17 683 Bermudagrass Cynodon dactylon (L.) DC 5 244 171 683 6 231 162 551 Flatsedge Cyperus esculentus L. 1 172 80 200 2 162 165 195 8 260 290 572 Ticktrefoil Desmodium paniculatum (L.) 2 44 63 195 Horsetail Equisetum arvense L. 3 246 160 2,200 Hydrocotyle L. Hydrocotyle americana L. 8 267 50 572 10 62 36 720 Turkey tangle Phyla nodiflora (L.) Greene 1 191 86 200 4 598 110 906 7 32 200 1,000 7 400 453 1,000 Plantain Plantago major L. 1 137 70 200 5 256 161 683 9 213 169 532 Blackberry Rubus frutocosus L. 3 340 400 2,200 6 173 93 551 Goldenrod Solidago altissima L. 6 111 86 551 10 90 200 720 Sowthistle Sonchus asper (L.) Hill 7 134 250 1,000 St. Augustine grass Stenotaphrum secundatum 1 164 100 200 3 516 320 2,200 Bluejacket Tradescantia ohiensis Raf. 5 220 211 683 Tuberous vervain Verbena rigida Spreng. 1 176 130 200 8 155 198 572 Bigpod sesbania Sesbania exaltata (Raf.) Cory 2 283 239 195 9 213 169 532 *Detection limit for Zn=1mg kg -1

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56 By comparing BCF and TF, we can assess the ability of different plants in taking up metals from soils and translocating them to the shoots. Tolerant plants tend to restrict soil-root and root-shoot transfers, and therefore has much lower accumulation in their biomasses, while hyperaccumulators actively take up and translocate metals into their aboveground biomass. Plants exhibiting TF and particularly BCF values less than 1 are ineffective for use in phytoextraction (Fitz and Wenzel, 2002). A few plants growing at the site were capable of accumulating heavy metals in their roots, but most of them had Table 4-5 Accumulation and translocation of Pb, Cu and Zn in selected plants Bioconcentration factor Translocation factor Site # Pb Cu Zn Pb Cu Zn Gentiana pennelliana Fern 1 10.76 21.6 2.62 0.47 0.46 0.48 8 0.61 1.25 0.54 0.56 0.56 1.16 Cyperus esculentus L. 2 0.11 0.48 0.83 1.6 2.8 1.02 8 0.29 0.5 0.45 0.06 0.13 1.12 Phyla nodiflora L. Greene 1 0.5 1.55 0.96 0.55 0.45 0.45 7 0.05 0.01 0.03 0.71 11.75 6.25 7 0.19 0.24 0.4 0.12 0.13 1.13 Rubus fruticosus L. 3 0.2 0.03 0.15 0.03 1.53 1.18 9 0.15 1.62 0.4 NA 5.64 0.79 Sesbania exaltata (Raf.) Cory 2 1.05 0.57 1.45 NA 4 0.84 Plantago major L. 1 0.1 1.15 0.69 5.96 0.43 0.51 Bidens alba (L.) DC 2 6.62 0.48 0.13 0.1 0.8 0.8 3 0.04 0.04 0.07 0.15 0.39 1.61 Paspalum notatum Flugge 4 0.42 0.26 0.5 0.47 1.41 0.7 9 NA 0.33 0.47 NA 1.1 0.8 Stenotaphrum secundatum 1 0.34 1.1 0.82 0.45 0.77 0.61 Verbena rigida Spreng 8 0.02 0.08 0.27 0.31 0.72 1.28 Sonchus asper (L.) Hill 7 0.06 0.06 0.13 0.27 0.74 1.87 Solidago altissima L. 10 NA 0.77 0.13 NA 0.5 2.22 Desmodium paniculatum (L.) DC 2 0.91 0.29 0.23 0.15 1 1.43

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57 low TFs and BCFs, which means they have limited ability to accumulate and translocate the metals studied (Tables 4-5). Among the 17 plants screened, G. pennelliana, growing at site 1, had the highest BCF of Pb (BCF=10.8), though its Pb concentrations in the plant body were <1,000 mg kg -1 On the other hand, P. nodiflora had the highest Pb concentration in the roots (1,183 mg kg -1 ), but its BCF was less than 1 (Table 4-5). Cyperus esculentus L.and Plantagomajor L. had TFs greater than 1 (TF=1.6, and 6.0, respectively). The BCF value of Pb found in this research was lower than that of Kim et al. (2003) in P. thunbergii (5 58), and higher than from those (0.3-0.45) reported by Stoltz and Greger (2002). Shallari et al. (1998) reported a high BCF of Pb in Koeleria eriostachya (31), Setaria viridis (18) and Verbascum blattaria (3). Similar to Pb, no plant species accumulated Cu above 1,000 mg kg -1 (Table 4-3). The maximum value for Cu was 460 mg kg -1 in the roots of P. nodiflora. Though several plant species showed BCFs or TFs greater than 1 for Cu, only Rubus fruticosus L. growing at site 9 had both a BCF (1.6) and a TF (5.6) greater than 1 (Table 4-5). However, soil Cu concentration at this site was relatively low, at 29 mg kg -1 The BCF values for these species were lower than those found in P. thunbergii (41-168) by Stoltz and Greger(2002). The highest BCF for Cu was 6.3 in Silene gallica by Shallari et al. (1998). The highest Zn concentration was 598 mg kg -1 which was in the roots of P. nodiflora (Table 4-4). Both G. pennelliana. and S. exaltata had a BCF greater than 1, and several species had a TF greater than 1, with P. nodiflora having the highest TF of 6.3. The highest BCF for Zn was 2.6 in G. pennelliana, lower than those reported by Kim et

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58 al. (2002) in P. thunbergii (22 136) but higher than those (0.004-0.11) reported by Stoltz and Greger (2002). Research conducted by Shallari et al. (1998) reported the highest Zn BCF for Alyssim markgrafii at 5.9. Though none of the plants sampled were metal hyperaccumulators, some interesting observations were noted. Based on the average BCFs of all plant samples, plant roots were most efficient in taking up Cu (BCF=1.1), followed by Pb (0.79) and Zn (0.50) (data not shown). Based on the average TFs of all the plant samples, the plants were most efficient in translocating Cu (TF=1.2), followed by Zn (0.98) and Pb (0.54). From a general point of view, among the three metals tested, the plants growing on the Jacksonville site were most efficient in taking up and translocating Cu. Low translocation of Pb indicates that plants are unwilling to transfer Pb from their roots to shoots due to toxicity of Pb. Lead can be toxic to photosynthetic activity, chlorophyll synthesis and antioxidant enzyme production (Kim et al., 2003). Baker and Brooks (1989) also discussed the restriction of metal uptake by plants from contaminated soils and the presence of exclusion mechanisms in such species. Since Zn and Cu are essential nutrients for plant systems, their higher translocation factors can be supported. Thomas and Eong (1984) treated established Rhizophora mucronata L.am. and Avicennia alba B.l seedlings in sediment with Pb and Zn. For these species, root accumulation and reduced translocation to the leaf tissues were observed for Zn and Pb. In general, all three heavy metals occurred at elevated levels in the plant biomass as a result of previous industrial activity at the site. Normal and phytotoxic concentrations of Pb, Zn and Cu were reported by Levy et al. (1999), which were 0.5-10, and 30-300 mg kg -1 for Pb, 3-30 and 20-100 mg kg -1 for Cu, and 10-150 and >100 mg kg -1 for Zn.

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59 Almost all collected species had heavy metal concentrations higher than the normal or at phytotoxic levels. These results may indicate that plant species growing in heavy metal contaminated site were tolerant of these metals. Restriction of upward movement into the shoots can be considered a tolerance mechanism, as can the increase in metal-binding capacity of their cell walls (Verkleij and schat, 1990). In addition, the relationships of BCFs and TFs among the three metals were determined through simple correlation. A few studies have been published to show that high Pb levels reduce the uptake of other elements such as Fe, Mn and Zn. The correlation of BCFs of all plant samples between two of the metals ranged from 0.63 to 0.84 (data not shown), i.e. a plant, which was effective in taking up Pb, was very likely to be effective in taking up Cu and Zn. However, the relationship between TFs was different. Only the TFs of Zn and Cu were correlated (r 2 =0.79), whereas no correlation of TFs was found between Pb and Cu, or Pb and Zn. In other words, a plant, which was effective in translocating Zn, was also effective in translocating Cu and vice versa. However, Cu and Zn translocation in these plants were not related to Pb translocation. These relationships may indicate that elevated Pb concentration can inhibit the transfer of essential micronutrients to the plant biomass. Table 4-6. Plants with high BCF and low TF for phytostabilization Bioconcentration factor Translocation factor Applicable Phystabilazation Site # Pb Cu Zn Pb Cu Zn Gentiana pennelliana Flugge 1 10.8 21.6 2.62 0.47 0.46 0.48 Pb, Cu Zn 8 0.61 1.25 0.54 0.56 0.56 1.16 Cu Bidens alba (L.) DC 2 6.62 0.48 0.13 0.10 0.80 0.80 Pb Sesbania exaltata (Raf.) Cor y 2 1.05 0.57 1.45 0.01 4.00 0.84 Pb ,Zn Plantago major L. 1 0.10 1.15 0.69 5.96 0.43 0.51 Cu Stenotaphrum secundatum 1 0.34 1.10 0.82 0.45 0.77 0.61 Cu

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60 Phytostabilization is another technology that can be used to minimize migration of contaminants in soils (Susarla et al., 2002). This process uses the ability of plant roots to change soil conditions via root exudation. Plants can immobilize heavy metals through absorption and accumulation by the roots, adsorption onto roots, or precipitation within the rhizosphere. This process reduces metal mobility and leaching into the ground water, and also reduces metal bioavailability for entry into the food chain. One advantage of this strategy over phytoextraction is the disposal of the metal-laden plant material is not required (Susarla et al., 2002). By using metal-tolerant plant species for stabilizing contaminants, particularly metals, in soil, could also provide improved conditions for natural attenuation or stabilization of contaminant in the soil. Metals accumulated in the roots are not considered to be a threat for release into environment. Studies are needed regarding the turnover of nutritive roots and the potential release of metals from decomposing roots (Weis and Weis, 2004). Also effects of plant-bacteria and/or plant-mycorrhizae interactions, which might affect the metal uptake and translocation need further investigation. Although no heavy metal hyperaccumulators were found in our study, heavy metal tolerant plant with high BCF and low TF can be used for phytostabilization of contaminated site (Table 4-6). Conclusion This study was conducted to screen plants growing on a contaminated site to determine their potential for metal accumulation. A total of 17 plant species were collected and analyzed for heavy metal concentrations. Only plants with both a BCF and a TF greater than 1 have potential to be used for the purpose of phytoextraction. In this study, no plant species were identified as metal hyperaccumulators based on Baker and Brooks (1989). However, several plants had BCFs or TF >1. G. pennelliana. was the

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61 most effective in taking up all three metals, with BCFs at 1.1-22 with one exception (Table 4-5). Cyperus esculentus was the most effective in translocating all three metals (Table 4-5). Among those plant species collected from the contaminated site, G. pennelliana was considered as the most promising species for phytoextraction. Further clarification of the phytoremediation potential of these plant species needs to be investigated. Also the plant sampling was conducted randomly, and a more systematic sample collection could yield more definitive results.

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CHAPTER 5 CONCLUSION This study has shown that applying a mixture of phosphoric acid and phosphate rock effectively reduced TCLP-extractable Pb (Toxicity Characteristic Leaching Procedure), bioavailable Pb and Pb leachability in the Pb-contaminated soil studied. Collected soil form the field had TCLP-extractable Pb < 127mg L -1 bioavailable Pb <26 mg L -1 Application of phosphoric acid and phosphate rock reduced TCLP-Pb by >95% and bioavailable Pb by 25-42% and reduced Pb leachability from the contaminated soil. A layer of phosphate rock resulted in improved reduction of Pb migration. Result suggests layered phosphate rock below a contaminated soil may serve as a reactive barrier to prevent Pb from migrating into the groundwater. No significant soil acidification or phosphorous leaching was observed by the application of phosphoric acid. Of all the treatments, mixture of phosphate rock and soil with co-application of phosphoric acid (R M A S1 ) was the most effective in decreasing leachable and bioavailable Pb with acceptable impact on soil pH and eutrophication risk. A greenhouse study showed that the three plants (Agrostis capillaries, Lolium rigidum, and Brassica napus) were effective in enhancing Pb immobilization in the rhizosphere soil. Plant rhizosphere soil showed 13-20% reduction in bioavailable-Pb as compared to the control soil. Reduction of the bioavailable Pb in the rhizosphere soil may result from the formation of Pb phosphates, which was supported by the SEM observation of the plant roots. Formation of stable lead-phosphate minerals may be enhanced via biochemical actions of roots (i.e. organic acid, phosphatase exudation). This 62

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63 study demonstrated that using the most effective P-application method coupled with plant growth can maximize the effectiveness of phosphate-induced lead immobilization in soils. Elevated metal concentrations were observed in the 17 plants collected from the experimental site located in Jacksonville, Florida, with total Pb concentrations ranging from non-detectable to 1,183, Cu 5 to 460, and Zn 17 to 598 mg kg -1 .Among the17 collected species, G. pennelliana. was the most effective in taking up all three metals, with BCFsbeing 1.1-22 and Cyperus was most effective in translocating all three metals with TFs being 1.0-2.8. Though no metal hyperaccumulator was identified, heavy metal tolerant plants with a high BCF and a low TF can be used for phytostabilization to minimize the migration of a particular contaminant in a soil.

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LIST OF REFERENCES Alloway, B.J., 1994. Toxic metals in soil-plant systems. John Wiley and Sons, Chichester, UK. Alloway, B.J., Jackson, A.P., Morgan, H., 1990. The accumulation of cadmium by vegetables grown on soils contaminated from a variety of sources. The Science of Total Environment. 91, 223-236. Arienzo, M., Adamo, P., Cozzolino, V., 2003. The potential of Lolium perenne for revegetation of contaminated soil from a metallurgical site. The Science of Total Environment. 319, 13-25. Baker, A.J.M., Brooks, R.R., 1989. Terrestrial higher plants which hyperaccumulate metallic elements-a review of their distribution, ecology and phytochemistry. Biorecovery.1, 81-126. Baker, A.J.M., Reeves, R.D., Hajar, A.S.M., 1994. Heavy metal accumulation and tolerance in British populations of the metallophyte Thlaspi caerulescens J. & C. presl (Brassicaceae). New Phytologist. 127, 61-68. Basta, N.T., McGowen, S.L., 2004. Evaluation of chemical immobilization treatments for reducing heavy metal transport in a smelter-contaminated soil. Environmental Pollution. 127, 73-82. Bekele, T., Cino, B.J., Ehlert, P.A.I., Van der Maas A.A., Van Diest A., 1983. An evaluation of plant-borne factors promoting the solubilization of alkaline rock phosphate. Plant and Soil. 75, 361-378. Boisson, J., Ruttens, A., Mench, M., Vangronsveld, J., 1999. Evaluation of hydroxyapatite as a metal immobilizing soil additive for the remediation of polluted soils. Part 1. Influence of hydroxyapatite on metal exchangeability in soil, plant growth and plant metal accumulation. Environmental Pollution. 104, 225-233. Brown, G.A., Elliott, H.A., 1992. Influence of electrolytes on EDTA extraction of Pb from polluted Soil. Water, Air, and Soil Pollution. 62, 157-165. Callender, E., Rice, K.C., 2000. The urban environmental gradient, Anthropogenic influences on the spatial and temporal distributions of lead and zinc in sediments. Environmental Science and Technology. 34, 232-238. 64

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65 Cao, X., Ma, L.Q., Chen, M., Singh, S.P., Harris, W.G., 2002a. Phosphate induced metal immobilization in a contaminated site. Environmental Pollution. 122, 19-28. Cao, X., Ma, L.Q., Chen, M., Singh, S.P., Harris, W.G., 2002b. Impacts of Phosphate amendments on lead biogeochemistry at a contaminated site. Environmental Science and Technology. 36, 5296-5304. Cao, X., Ma, L.Q., Harris, W.G., Nkedi-kizza P., 2001. Field demonstration of metal immobilization in contaminated soils using P amendments, FIPR Project #97-01-148R. Final Report. University of Florida, Gainesville, FL. Chen, M., Ma, L.Q., Harris, W.G., Hornsby, A., 1999. Background concentrations of 15 trace metals in Florida soil. Journal of Environmental Quality. 28, 1173-1181. Chen, M., Ma, L.Q., Singh, S.P., Cao, X., Melamed, R., 2003. Field demonstration of in situ immobilization of soil Pb using P amendments. Advances in Environmental Research. 8, 93-102. Chen, X., Wright, J., Conca, J., and Peurrung, L., 1997. Effects of pH on heavy metal sorption on mineral apatite. Environmental Science and Technology. 31, 624-631. Cosgrove, D.J., 1967. Metabolism of organic phosphate in soil. Soil Biochemistry. 1, 216-228. Cotter-Howells, J., Caporn, S., 1996. Remediation of contaminated land by formation of heavy metal phosphates. Applied Geochemistry. 11, 335-342. Cotter-Howells, J., 1996. Lead phosphate formation in soils. Environmental Pollution. 93, 9-16. Dahmani-Muller, H., Van Oort, F., Gelie, B., Balabane, M., 2000. Strategies of heavy metal uptake by three plant species growing near a metal smelter. Environmental Pollution. 109, 231-238. Davis, A., Drexler, J.W., Ruby, M.V., Nicholson, A., 1993. Micromineralogy of mine wastes in relation to lead bioavailability, Butte, Montana. Environmental Science and Technology. 23, 1415-1425. Dingwall-Fordyce, I., Lane, R.E., 1963. A follow-up study of lead workers. Br. J. Ind. Med. 20, 213. Fitz, W.J., Wenzel, W.W., 2002. Arsenic transformation in the soil-rhizosphere-plant system, fundamentals and potential application of phytoremediation. Journal of Biotechnology. 99, 259-278.

PAGE 76

66 Gardea-Torresdy, J.L., Peralta-Videa, J.R., Montes, M., de La Rosa, G., Corral-Diaz, B., 2003. Bioaccumulation of cadmium, chromium and copper by Convolvulus arvensis L., impact in plant growth and uptake of nutritional elements. Bioresource Technology. 92, 229-235. George, T.S., Gregory, P.J., Wood, M., Read, D., Buresh, R.J., 2002. Phosphatase activity and organic acid in the rhizosphere of potential agroforestry species and maize. Soil Biology and Biochemistry. 34, 1487-1494. Haeussling, M., Marschner, H., 1989. Organic and inorganic soil phosphates and acid phosphatase ativity in the rhizosphere of 80-year-old Norway spruce [Picea abies (L.) Karst trees.] trees. Biology and Fertility of Soils. 8, 128-133. Harrison, R.M., Laxen, D.P.H., 1981. Lead pollution, Causes and Control. Chapman and Hall, London. Henry, J., 2000. An Overview of the phytoremediation of lead and mercury. EPA NNEMS Report. www.clu-in.org (accessed March 2005) Hettiarachchi, G., Pierzynski, G., Ransom, M., 2000. In situ stabilization of soil lead using phosphorus and manganese oxide. Environmental Science and Technology. 34, 4614-4619. Hinsinger, P., Gilkes, R.J., 1997. Dissolution of phosphate rock in the rhizosphere of five plant speies grown in an acid, P-fixing mineral substrate. Geoderma. 75, 231-249. Hodson, M., Valsami-Jones, E., Cotter-Howells, J., 2000. Bonemeal additions as a remediation treatment for metal contaminated soil. Environmental Science and Technology. 34, 3501-3507. Hoffland, E., Findenegg, G.R., Nelemans, J.A., 1989. Solubilization of rock phosphate by rape. Local root exudation of organic acids as a response to P-starvation. Plant and Soil. 113, 161-165. Hutchison, T.C., Meema, K.M., 1987. Lead, mercury, cadimum and arsenic in the Environment. John Wiley and Sons, New York. Illmer, P., Schinner, F., 1995. Solubilization of inorganic calcium-phosphates-solubilization mechanisms. Soil Biology and Biochemistry. 27, 257-263. Jung, M.C., Thornton, I., 1996. Heavy metal contamination of soils and plants in the vicinity of a lead-zinc mine, Korea. Applied Geochemistry. 11, 53-59. Kabata-Pendias, A., Pendias, H., 1984. Trace elements in soils and plants. CRC Press Inc., Boca Raton, FL. Kamal, M., Ghaly, A.E., Mahmoud, N., Cote, R., 2004. Phytoaccumulation of heavy metals by aquatic plant. Environmental International. 29, 1029-1039.

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67 Kim, I.S, Kang, H.K, Johnson-Green, P, Lee E.J., 2003. Investigation of heavy metal accumulation in Polygonum thunbergii for phytoextraction. Environmental Pollution. 126, 235-243. Laperche, V., Logan, T., Gaddam, P., Traina, S., 1997. Effect of apatite amendments on plant uptake of lead from contaminated soil. Environmental Science and Technology. 31, 2745-2753. Laperche, V., Traina, S., Gaddam, P., Logan, T., 1996. Chemical and mineralogical characterizations of Pb in a contaminated soil, reactions with synthetic apatite. Environmental Science and Technology. 30, 3321-3326. Levy, D.B., Redente, E.F., Uphoff, G.d., 1999 Evaluating the phytotoxicity of Pb-Zn tailings to big bluesteam (Andropogon gerardii vitman) and switchgrass (Panicum virgatum L.). Soil Science 164: 363-375 Ma, L., 1996. Factors influencing the effectiveness and stability of aqueous lead immobilization by hydroxyapatite. Journal of Environmental Quality. 25, 1420-1429. Ma, L., Choate, A., Rao, G., 1997. Effects of incubation and phosphate rock on lead extractability and speciation in contaminated soils. Journal of Environmental Quality. 26, 801-807. Ma, Q., Logan, T., Traina, S., and Ryan, J., 1994a. Effects of NO 3 Cl F SO 4 2, and CO 3 2on Pb 2+ immobilization by hydroxyapatite. Environmental Science and Technology. 28, 408-418. Ma, Q., Logan, T., Traina, S., 1995. Lead immobilization from aqueous solutions and contaminated soils using phosphate rocks. Environmental Science and Technology. 29, 1118-1126. Ma, L. Rao, G., 1997. Effects of phosphate rock on sequential chemical extraction of lead in contaminated soils. Journal of Environmental Quality. 26, 788-794. Ma, L. Rao, G., 1999. Aqueous Pb reduction in Pb-contaminated soils by florida phosphate rocks. Water, Air, and Soil Pollution. 110, 1-16. Ma, Q., Traina, S., Logan, T., 1993. In situ lead immobilization by apatite. Environmental Science and Technology. 27, 1803-1810. Ma, Q., Traina, S., Logan, T., Ryan, J., 1994b. Effects of aqueous Al, Cd, Cu, Fe(II), Ni, and Zn on Pb immobilization by hydroxyapatite. Environmental Science and Technology. 28, 1219-1228. Marschner, B., Wilczynski, A.M., 1991. The effects of liming on quantity and chemical composition of soil organic matter in a pine forest in Berlin, Germany. Plant and Soil. 137, 229-236.

PAGE 78

68 MacFarlane, G.R., Burchett, M.D., 2002. Toxicity, growth and accumulation relationships of copper lead and zinc in the grey mangrove Avicennia marina (Forsk) Vierh. Marins Environmental Research. 54, 65-84. Madejon, P., Murillo, J.M., Maranon,T., Cabrera, F., Lopez, R., 2002. Bioaccumulation of As, Cd, Cu, Fe and Pb in wild grasses affected by the Aznalcollar mine spill (SW Spain). The science of the Total Environment. 290, 105-120. Mavropoulos, E., Rossi, A., Costa A., Perez, C., Moreira, J., Saldanha, M., 2002. Studies on the mechanisms of lead immobilization by hydroxyapatite. Environmental Science and Technology. 36, 1625-1629. McBride, M., 1994. Environmental chemistry of soils. Oxford University Press, New York, NY. Mcgrath, S.P., Zhao, F.J., 2003. Phytoextraction of metals and metalloids from contaminated soils. Current Opinion in Biotechnology. 14, 1-6. Melamed, R., Cao, X., Chen, M., Ma, L.Q., 2003. Field assessment of lead immobilization in a contaminated soil after phosphate application. The Science of the Total Environment. 305, 117-127. Mench, M., Vangronsveld, J., Didier, V., Clijsters, H., 1994. Evaluation of metal mobility, plant availability and immobilization by chemical agents in a limed-silty soil. Environmental Pollution. 86, 279-286. Mulligan, C.N., Yong, R.N., Gibbs, B.F., 2001 Remediation technologies for metal-contaminated soils and groundwater, an evaluation. Engineering Geology. 60, 193-207. Mercedes D. R., Rafael F., Concepcion A., Dinoraz V., Rosa M., Antonio D.H. B., 2002. Heavy metals and arsenic uptake by wild vegetation in the Guadiamar river area after the toxic spill of the Aznalcollar mine. Journal of Biotechnology. 98, 125-137. Nedwed, T., Clifford, D.A., 1997. A survey of lead battery recycling sites and soil remediation processes. Waste Management. 17, 257-269. Needleman, H.L., 1992. Human lead exposure. CRC Press Inc., Boca Raton, FL. Nelson, D.W., Sommers, L.E., 1982. Total Carbon, organic carbon, and organic matter. In, Page, A.L., Moller R.H., Keeney, D.R.(Eds.), Methods of soil analysis, Part 2 Chemical and micro-biological properties. ASA, Madison, WI. 539-577. Nriagu, J.O., 1972. Lead Orthophosphates-I. Solubility and hydrolysis of secondary lead orthophosphate. Inorganic Chemistry. 11, 2499-2503. Nriagu, J.O., 1974. Lead orthophosphates-IV. Formation and Stability in the Environment. Geochimica et Chomochimica Acta. 38, 887-808.

PAGE 79

69 Nriagu J.O., 1978. The biogeochemistry of lead in the environment Part A, B. Elsevier/North-Holland Biomedical Press, Amsterdam Pearson, M., Maenpaa, K., Pierzinsky, G., Lydy, M., 2000. Effects of soil amendments on the bioavailability of lead, zinc, a nd cadmium to earthworms. Journal of Environmental Quality. 29, 1611-1617. Piechalak, A., Tomaszewska, B., Baralkiewicz, D., Malecka A., 2002. Accumulation and detoxification of lead ions in legumes. Phytochemistry. 60, 153-162. Pichtel, J., Kuroiwa, K., Sawyerr, H.T., 2000. Distribution of Pb, Cd, and Ba in soils and plants of two contaminated sites. Environmental Pollution. 110, 171-178. Pugh, R.E., Dick, D.G., Freedeen, A.L., 2002. Heavy metal (Pb, Zn, Cd, Fe, and Cu) contents of plant foliage near the Anvil Range lead/zinc mine Faro, Yukon territory. Ecotoxicology and Environmental Safety. 52, 273-279. Pulford, I.D., Watson, C., 2003. Phytoremediation of heavy metal contaminated land by trees-a review. Environment International. 29, 529-540. Rabinowitz, M.B., 1993. Modifying soil lead bioavailability by phosphate addition. Bulletin of Environmental Contamination and Toxicology. 515, 438-444 Raskin, I., Ensley, B., 2000. Phytoremediation of toxic metals, using plants to clean up the environment. John Wiley and Sons, Inc., New York. Reddy, M.S., Kumar, S., Babita, K., 2002. Biosolubilization of poorly soluble rock phosphates by Aspergillus tubingensis and Aspergillus niger. Bioresource Technology. 84, 187-189. Reyes, I., Baziramakenga, R., Bernier, L., Antoun, H., 2001. Solubilization of phosphate rocks and minerals by a wild-type strain and two UV-induced mutants of Penicillum rugulosum. Soil Biology and Biochemistry. 33, 1741-1747. Rio, M.D., Rafael, F., Almela, C., Velez, D., Montoro, R., Bailon, A.D.H., 2002. Heavy metals and arsenic uptake by wild vegetation in the Guadiamar river area after the toxic spill of the Aznalcollar mine. Journal of Biotechnology. 98,125-137. Rodriguez, H., Fraga, R., 1999. Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnology Advances. 17, 319-339. Ruby, M., Davis, A., Nicholson, A., 1994. In situ formation of lead phosphates in soils as a method to immobilize lead. Environmental Science and Technology. 28, 646-654. Ruby, M., Davis, A., Schoof, R., Eberle, S., Sellstone, C., 1996. Estimation of lead and arsenic bioavailability using a physiologically based extraction procedure. Environmental Science and Technology. 30, 422-430.

PAGE 80

70 Sauve, S., Mcbride, M., Hendershot, W., 1997. Speciation of lead in contaminated soil. Environmental Pollution.98, 149-155. Sayer, J.A., Cotter-Howells, J.D., Watson, C., Hiller, S., Gadd, G.M., 1999. Lead mineral transformation by fungi. Current Biology. 9, 691-694. Scheckel, K.G., Ryan, J.A., 2003. In vitro formation of pyromorphite via reaction of Pb sources with soft-drink phosphoric acid. The Science of Total Environment. 302, 253-265. Scheuhammer, A.M., Norris S.L., 1995. A review of the environmental impacts of lead shotshell ammunition and lead lishing weights in canada Minister of Environment Canadian Wildlife Service, Ottawa, Ontario. Shallari, S., Schwartz, C., Hasko, A., Morel, J.L., 1998. Heavy metals in soils and plants of serpentine and industrial sites of Albania. The Science of Total Environment. 209, 133-142. Shu, W.S., Ye, Z.H., Lan, C.Y., Zhang, Z.Q., Wong, M.H., 2002. Lead, zinc and copper accumulation and tolerance in populations of Paspalum distichum and Cynodon dactylon. Environmental Pollution. 120, 445-453. Singh, S.P., Ma, L.Q., Harris, W.G., Nkedi-kizza, P., 2000. Field demonstration of metal immobilization in contaminated soils using P amendments, FIPR Project #97-01-148R. Quarterly Project Report No.6. University of Florida, Gainesville, FL. Stoltz, E., Greger, M., 2002. Accumulation properties of As, Cd, Cu, Pb and Zn by four wetland plant species growing on submerged mine tailings. Environmental and Experimental Botany. 47, 271-280. Susarla, S, Medina, V.F., McCutcheon, S.C., 2002. Phytoremediation, An ecological solution to organic contamination. Ecological Engineering. 18, 647-658. Thomas, C., Eong O.J. 1984. Effects of the heavy metals Zn and Pb on R. mucronata and A. alba seedlings. In E. Soepadmo, A. M. Rao, & M. D. MacIntosh (Eds.), Proceedings of the Asian symposium on mangroves and environment; research and management (pp. 568). ISME, Malaysia. United States Department of Agriculture (USDA), 2000. Heavy metal soil contamination. Soil quality-Urban Technical note No.3 United States Environmental Protection Agency (USEPA), 1996. Soil screening guidance, users guidance, EPA 540/R96/018. Office of Solid and Emergency Response, Washington, DC. United States Environmental Protection Agency (USEPA), 2001a. Lead in human health. www.epa.gov/superfund/programs/lead/lead.htm Technical Review Workgroup for Lead (accessed March 2005)

PAGE 81

71 United States Environmental Protection Agency (USEPA), 2001b. EPA-902-B01-001, Best management practices for lead at outdoor shooting ranges. United States Environmental Protection Agency Region 2, Washington, DC. Vassiliva, M., Azcon, R., Barea, J.M., Vassilev, N., 2000. Rock phosphate solubilization by free and encapsulated cells of Yarowia lipolytica. Process Biochemistry. 35, 693-697. Verkleij, J.A.C., Schat H., 1990 Mechanisms of metal tolerance in plants. Heavy metal tolerance in plants-evolutionary aspects. CRC Press, Boca Raton, FL. Waldron, H.A., 1980. Metals in environment. Academic Press Inc, London. Wang, Y.M., Chen, T.C., Yeh, K.J., Shue, M.F., 2001. Stabilization of an elevated heavy metal contaminated site. Journal of Hazardous Materials. 88, 63-74. Weis, J.S., Weis, P., 2004. Metal uptake, transport and release by wetland plants, implications for phytoremediation and restoration. Environmental International. 30, 685-700. Wenzel, W.W., Jockwer, F., 1999. Accumulation of heavy metals in plants growing on mineralized soils of the Austrian Alps. Environmental Pollution. 104, 145-155. Yang, J., Mosby, D.E., Castell, S.W., Blanchar R.W., 2001. Lead immobilization using phosphoric acid in a smelter-contaminated urban soil. Environmental Science and Technology. 35, 3553-3559. Youssef, R.A., Chino M., 1989. Root induced changes in the rhizosphere of plants. I. pH changes in relation to the bulk soil. Soil Science and Plant Nutrition. 35, 461-468. Zhang, P., Ryan, J.A., 1999a. Formation of chloropyromorphite from galena (PbS) in the presence of hydroxyapatite. Environmental Science and Technology. 33, 618-624. Zhang, P., Ryan, J.A., 1999b. Transformation of Pb(II) from cerrusite to chloromorphite in the presence of hydroxyapatite under varying conditions of pH. Environmental Science and Technology. 33, 625-630.

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BIOGRAPHICAL SKETCH Joonki Yoon was born on May 27, 1976, in Seoul, Korea, to ByungKyou Yoon and SookJae Lee. He attended Yonsei University where he received Bachelor of Science degree in geology with minors in biology in February of 2002. Upon completion of his B.S., he joined the University of Florida in the fall of 2002 to pursue a Master of Science degree in soil and water Science specializing in remediation of Pb-contaminated soils with Dr. Lena Ma. 72


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Physical Description: Mixed Material
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PHOSPHATE-INDUCED LEAD IMMOBILIZATION IN CONTAMINATED SOIL


By

JOONKI YOON














A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Joonki Yoon

































This thesis is dedicated to my grand parents, Jinhyung Lee and Youngsook Kim.
















ACKNOWLEDGMENTS

I wish to extend my thanks to Dr. Lena Q. Ma for her assistance and guidance of

my research. I would also like to thank members of my committee, Drs. Willie G. Harris

and Jean Claude Bonzongo, for their advice, support and critical review of this thesis. I

gratefully acknowledge Dr. Xinde Cao who worked with me and helped me a lot. I wish

to thank Thomas Luongo for laboratory analysis and being a good instructor. Special

thanks go to Drs. Mrittunj ai Srivastava and Gregory E. MacDonald for their assistance

with plant identification procedure.

I would like to thank my parents Byungkyu Yoon and Sookj ae Lee for their love,

encouragement and support throughout my studies.





















TABLE OF CONTENTS


page


ACKNOWLEDGMENT S ................. ................. iv.............


LIST OF TABLES ................ ..............vii .......... ....


LI ST OF FIGURE S ................. ................. viii............


AB STRAC T ................ .............. ix


CHAPTER


1 INTRODUCTION ................. ...............1.......... ......


Lead in the Environment .............. ...............1.....
Health Effects of Lead ................. ...............3.......... .....
Lead Remediation Techniques .............. ... ...............6...
Phosphate Induced Lead Immobilization ....._____ ..... ...___ .. ........_......6
Phosphoric acid .............. ...............8.....
Synthetic apatite .............. ...............8.....
Bone meal apatite ................. ...............9...._._._ .....
Phosphate rock ........._.._.. ...._... ...............10....
Rhizosphere ........._.. ..... ._ ._ ...............11....
Phytoremediation............... ............1
Phytoextracti on ................. ................. 13..............
Phytostabilization ................. ...............13......._ ......
Sum m ary ................. ...............14......... ......


2 EFFECTS OF APPLICATION METHODS ON P-INDUCED Pb
IMMOBILIZATION IN A SOIL COLUMN ................. ..............................16


Introducti on ................. ...............16.................
Materials and Methods .............. ...............18....
Soil Characterization ................ ...............18.................
Soil Sample Preparation .............. ...............18....
Column Leaching .............. ...............19....
Analytical Procedure .............. ...............20....
Re sults ................ ............ ...............21.......
Soil Characterization ................. ..... ...............21
Effects of P Application on Soil pH ................. ...............22........... ..













T CLP Pb ................. ...............23...

Lead Bioavailability .............. ...............24....
Distribution of Pb in Soil Column............... ...............25.

Leachate Analysis............... ...............29


3 THE EFFECTS OF PLANTS ON PHOSPHATE-INDUCED Pb
IMMOBILIZATION IN THE RHIZOSPHERE SOIL .............. .....................3


Introducti on ............. ...... ._ ...............33...
Materials and Methods .............. ...............34....
R e sults.............. ......._ ...............37...
Soil Characterization .............. ...............37....
Bioavailable Pb.................... ...............38

Formation of Lead Phosphate ................. ...............39 ........._....
Conclusion ................. ...............43......... ......


4 SCREENING OF PLANTS FOR ACCUMULATION OF Pb, Cu, Zn FROM A
CONTAMINATED SITE ................. ...............45.................


Introducti on ................. ...............45.................
Materials and Methods .............. ...............47....
Site Characterization ............... .... .... ................4

Sample Preparation and Chemical Analysis............... ...............48
R e sults................ ...... .... .. .. ......... .............4
Metal Concentrations in Soils .............. ...............48....
Metal Concentrations in Plants................. ..... ...............5
Accumulation and Translocation of Metals in Plants ................. ................ ...52
Conclusion ................ ...............60.................


5 CONCLUSION ................. ...............62.................


LIST OF REFERENCES ................. ...............64................


BIOGRAPHICAL SKETCH .............. ...............72....

















LIST OF TABLES


Table pg

1-1 Theoretical solubility of Pb in different ................. ...............7............ ..

2-1 Soil characterization at the contaminated site ........._.._.. ...._... ......._.._......21

2-2 pH of soil after treatment. ........... ..... .._ ...............22..

2-3 Pb concentration in column leachate (Clg L1) ..........._.._ ....... .........._.......29

3-1 Selected characteristics of the lead-contaminated soil ................ ......................37

3-2 Water soluble phosphorous in plant rhizosphere soil (ppm) ................. ................42

3-3 pH of rhizosphere soil treated with phosphate rock after 4-weeks growth..........._...43

4-1 Selected properties of soil samples collected from the contaminated site at
Jacksonville, Florida. ................. ...............49......... .....

4-2 Lead concentrations in soil and plant samples (mg kg- ) collected from the
contaminated site at Jacksonville, Florida............... ...............52

4-3 Copper concentrations in soil and plant samples (mg kg- ) collected from the
contaminated site at Jacksonville, Florida............... ...............54

4-4 Zinc concentrations in soil and plant samples (mg kg- ) collected from the
contaminated site at Jacksonville, Florida............... ...............55

4-5 Accumulation and translocation of Pb, Cu and Zn in selected plants ................... ...56

4-6 Plants with high BCF and low TF for phytostabilization............... ...........5

















LIST OF FIGURES


Figure pg

2-1 TCLP metal contents in sectioned soil columns after leaching .............. ................24

2-2 PBET extractable Pb contents in sectioned soil columns after leaching..................26

2-3 Total Pb concentration in sectioned soil columns (bottom section) ................... ......28

2-4 P concentration in column leachate (mg L 1) .............. ...............30....

3-1 Lolium rigidum after 4 weeks of growth ....._.._................ .............. ....3

3-2 Agrostis capillaries~~~1111~~~~111 after 4 weeks of growth ................. ............... .............36

3-3 Bra~ssica napus after 4 weeks of growth .............. ...............37....

3-4 Lead concentrations using the physiologically based extraction test in the
rhizosphere soil with phosphoric acid and with phosphate rock treatment. .............38

3-5 Scanning electron microscopy elemental dot map of Agrostis capillaries~~~1111~~~~111 ..............41

3-6 Scanning electron microscopy elemental dot map of Bra~ssica napus...................42

3-7 Scanning electron microscopy elemental dot map of Lolium rigidum .....................42

4-1 Map of Jacksonville site with lead concentration contour map and sampling
point............... ...............47.
















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

PHOSPHATE-INDUCED LEAD IMMOBILIZATION INT CONTAMINATED SOIL


By

Joonki Yoon

May 2005

Chair: Lena Ma
Major Department: Soil and Water Science

Activities such as mining and manufacturing, and the use of synthetic products

result in heavy metal contamination of soil and water resources. The obj ectives of this

research were to 1) examine the effectiveness of Hyve P application methods on Pb

immobilization in soil using a column study; 2) explore the effects of plants on the

effectiveness of P-induced Pb immobilization in soil using a pot study; and 3) evaluate

metal accumulation in plants growing on a contaminated soil, based on Hield screening.

The soil column test was conducted to evaluate the effectiveness of different application

methods using phosphate rock (PR) and phosphoric acid (PA). Mixing both PA and PR

with the soil was the most effective method of Pb immobilization, reducing TCLP-Pb by

up to 95% (Toxicity Characteristic Leaching Procedure) and bioavailable Pb by 25 to 42%

in the soil. The application of PR as a layer in the soil column was effective in reducing

Pb migration in the soil column by 73 to 79%. In a greenhouse study, all three plants

(Agrostis capillariesll1~~~111~~~11 Lolium rigidum, and Bra~ssica napus) were effective in enhancing









Pb immobilization in the rhizosphere soil when P was applied as insoluble PR (13 to 20%

reduction in bioavailable Pb) but not when applied as a mixture of PR and PA.

A fieldstudy was conducted to screen 17 native plants for their potential for accumulation

of Pb, Cu and Zn from a heavy metal contaminated site. Elevated metal concentrations were

observed in the 17 plants collected from the experimental site, with total concentrations

ranging from 20 to 1,183 mg kg-l for Pb, from 5 to 460 mg kg-l for Cu, and froml7 to

598 mg kg-l for Zn. Though no metal hyperaccumulator was identified, G. pennelliana

was the most effective in taking up all three metals, while Cyperus was the most effective

in translocating all three metals from the roots to the shoots. Our study demonstrated that

the most effective P-application method coupled with plant growth can maximize the

effectiveness of phosphate-induced lead immobilization in soils.















CHAPTER 1
INTTRODUCTION

Lead in the Environment

Lead (Pb), from the Latin plumbum, has an atomic number of 82 and an atomic

weight of 207. 19. While lead is the 36th most abundant element, it is the most abundant

heavy metal in the earth' s crust with an average concentration of 13 mg kgl (Brown and

Elliott, 1992; Nriagu, 1978). The relatively low Pb concentration in soil solutions

confirms reports oflead's low mobility among the heavy metals (Kabata-pendias and

Pendias, 1984). Natural mobilization of lead into the environment occurs principally from

the erosion of lead-containing rocks and through gaseous emissions during volcanic

activity (Waldron, 1980).

Important physical attributes of lead (i.e. high density, softness, flexibility, and

electric potential) ensure its widespread industrial application. An Anthropogenic source

of lead includes flanking paint, mining operations, smelter and industrial emissions, and

application of insecticides, which have contributed to elevated lead levels in

environment. Lead is also used in the production of ammunition, and has been used in

other metallic products (such as solder, some brass, bronze products, and piping), and

ceramic glazes, but its use in these capacities has been outlawed in the U.S.

(Scheuhammer and Norris, 1995). Chemicals such as tetraethyl lead and tetramethyl lead

were once used as gasoline additives to increase octane rating. However, their use was

phased out in the 1980s, and lead was banned from use in gasoline for transportation

beginning January 1, 1996 (USEPA, 2001a).









Lead occurs naturally in all soils in concentration ranging from 1 to 200 mg kg- ,

but rarely at levels that are considered to be toxic. However, increases of anthropogenic

sources of Pb in the environment have caused the Pb loading rate in soil to exceed its

natural removal rate by approximately 20-fold (Nriagu, 1978). There are several

thousands of Pb contaminated sites in United States. Currently there are 440 sites listed

on the EPA National Priority List (NPL), in which the main contamination of concern is

lead. Many studies show that there are high levels of Pb in various environments. A

national survey in the U.K indicates that total Pb concentrations in garden soils ranges

from 13 to 14,100 mg kg-l (Cotter-Howells, 1996). Soil samples obtained from a former

battery-cracking site in Florida had Pb concentrations up to 13,500 mg kg-l (Cao et al.,

2001). Pichtel et al. (2000) reported Pb concentrations of a Superfund site and a Pb

battery dump that ranged from 195-140,500 mg kg- A recent survey found Pb

concentration in soils adj acent to homes and near expressways in Tampa, Florida as high

as 2,000 mg kg-l (Cao et al., 2001). Also uptake of heavy metals by humans, largely

through the respiratory and digestive pathways, has contributed to elevated blood Pb

concentration, which can cause impaired mental development, nervous system disorders,

immune-system dysfunction and DNA damage. Presence of hazardous levels of Pb in the

environment and the toxicity of Pb requires remedial action to protect the health of the

public. Considerable attention is now being paid to develop cost effective and less

disruptive remediation technologies to reduce human exposure via direct ingestion and

dust inhalation.

Ionic lead (Pb2+) is the dominant form of lead in the soil and groundwater.

However, Pb4+ Occasionally can be found in some highly oxidized soils. With the









exception of a few sporadic measurements in urban air, marine fish, and in human brains

there is relatively little information available on organometallic lead compounds (Nriagu,

1978).

Because lead enters the soil in various complex compounds, its reactions may differ

widely between geographic areas. Lead is found in soils most commonly as galena (PbS)

and in smaller quantities as cerrusite (PbCO3), angleSite (PbSO4) and crocoite (PbCrO4).

Lead added to soil may react with available soil anions such as SO42-, PO43-, Or CO32- to

form sparingly soluble salts (Waldron, 1980). Compounds such as lead carbonate

[Pb3(OH)2(CO3)2] and chloropyromorphite [Pb5(PO4)3C1] prOVide the least soluble

inorganic salts at near neutral pH's. There are several other mechanisms by which lead

may be immobilized in soils, such as complexation by soil organic matter humicc and

fulvic acids), which are then adsorbed onto soil solids, or by ion exchange at sites on the

solid material in the soil. These mechanisms may facilitate attachment of substantial

amounts of lead, but cannot account entirely for the low mobility of lead in soils

(Harrison and Laxen, 1981).

Health Effects of Lead

Since lead is a natural constituent in the environment, man has been under

inescapable exposure to Pb (Waldron, 1980), which originates from an array of sources

that include Pb contaminated soil, atmospheric Pb particles, Pb paint, Pb water pipes, and

Pb solder used to seal food cans. With such an array of potential sources, it is no wonder

that lead poisoning is ranked as the number one environmental health threat, by both the

U.S Agency for Toxic Substance and Disease Registry (ATSDR) and the Center for

Disease Control (CDC). Dust and soil have emerged as important lead ingestion

pathways in recent years. Although their amounts are small, once Pb enters the blood










only approximately 10% is excreted and the remainder stays in the body, affecting the

nervous system, blood cells, and metabolic processes and eventually residing in bone

material for decades (Scheckel and Ryan, 2003).

The maj or health effects of lead are manifest in three organ systems; the

hematological system, the central nerve system and the renal system (Needleman, 1992).

The hematological effects of lead have been recognized for many years. Such effects are

related to the derangement of hemoglobin synthesis, the shortened life span of circulating

erythrocytes and the secondary stimulation of erythropoiesis, which, may result in

anemia. Higher lead concentration may affect the degree of maturation of red blood cells,

globin synthesis, and the morphology and stability of cells (Nriagu, 1978).

Acute effects of lead on nervous system are both structural and functional,

involving the cerebellum as well as the spinal cord and the motor and sensory nerves

leading to specific areas of the body (Nriagu, 1978). Effects may also lead to the general

deterioration of intellectual functions, sensory functions, neuromuscular functions and

psychological functions. If calcium is deficient, synaptic transmission is impaired. In

addition, lead has also been reported to interfere with the normal metabolism of some

central neurotransmitters, including acetylcholine and catecholamines. Other effects,

which have been noted, include inhibition of cytoplasmic NAD(P)H oxidation by

cerebral mitochondria and the inhibition of adenylcyclase. Lead also affects the intrinsic

muscles and oculomotor nerves of the visual system. Changes in the character of the

blood cells and supporting fluids may lead to changes in the intraocular tension. Such

changes may lead to mydriasis or visual paralysis. Neuritis may occur within the visual

system itself and a scotoma, which may be limited to certain colors, is usually present in










optic neuritis. The actual effects of lead on auditory dysfunctions are not clear (Nriagu,

1978).

Nriagu (1978) summarized the functional and morphological effects of lead on the

kidney as follows:

Stage 1: swollen proximal tubular lining cells; mitochondrial changes; intranuclear

inclusion bodies (mostly lead-protein complexes); proximal tubular dysfunction,

Stage 2: fewer inclusion bodies; intense interstitial fibrosis; tubular atrophy and

dilation

Stage 3: renal tumor (observed in rats only).

The first stage is usually seen in children with short-term heavy exposure to lead.

The functional effects include the fanconi syndrome manifested by an increase in urinary

excretion of glucose, amino acids and phosphate. The second stage nephropathy can be

produced in experimental animals fed a high dose of lead and in workmen with excessive

lead exposure for several years. The duration and degree of lead exposure necessary to

produce irreversible kidney damage is not known. Dingwall-Fordyce and Lane (1963)

followed 425 retirees and others with long exposure to lead in a battery factory. They

found no increase in cancer incidence but saw excess in cerebrovascular accidents. Lead

can effect chromosomes, reproduction, immune system and endocrine system, but usually

is not considered as a human carcinogen.

Toxicity of lead to plants has not been frequently observed. Plants do not

translocate absorbed lead into the shoots while they are growing; instead, they

accumulate it in their roots. The health concern surrounding plants is that lead containing









soil particles can contaminate plant surfaces, which might be ingested by animals

(McBride, 1994).

Lead Remediation Techniques

In contrast to many organic contaminants, the immobile and non-degradable nature

of lead necessitates active remediation because natural attenuation will do little to reduce

its concentration (Nedwed and Clifford, 1997). There are several technologies available

for the remediation of land contaminated by heavy metals (USDA, 2000; Mulligan et al.,

2001). The selection of the most appropriate soil remediation method depends on the site

characteristics, concentration and types of pollutants of concern, and end use of the

contaminated medium (Mulligan et al., 2001). However, many of these technologies are

costly (e.g. excavation of contaminated material and chemical/physical treatment) or do

not achieve a permanent or aesthetic solution (e.g. encapsulation and vitrification).

Formation of insoluble heavy metal compounds immobilizes the metal and reduces

their bioavailability (Cotter-Howells and Caporn, 1996). Phosphate amendment has been

suggested as a cost-effective remediation option for Pb contaminated soils. Along with

that, phytoremediation, in which plants are used to do the remediation work, can be

another cost-effective and less disruptive approach.

Phosphate Induced Lead Immobilization

When considering potential Pb-immobilizing anions(Table 1) phosphates are less

soluble under earth surface equilibrium conditions than oxides, hydroxides, carbonates,

and sulfates (Ruby et al., 1994).

In its reactions with lead, phosphorus forms sparingly soluble orthophosphates

(pyromorphites) (Nriagu, 1972). Experimental evidence supports that Pb phosphates can









form rapidly when adequate Pb and phosphate are present in aqueous system (Zhang and

Ryan, 1999a; Ma et al., 1994b).

Phosphate minerals have been used to immobilize Pb in situ from aqueous

solutions and Pb contaminated soils (Cao et al., 2001, 2002; Ma et al., 1993, 1995;

Mench et al., 1994). The primary mechanism of Pb immobilization appears to be through

phosphate-mineral dissolution and subsequent precipitation of pyromorphite-like

minerals, though mechanisms such as cation substitution, adsorption and precipitation as

other Pb minerals are also possible. Hence, solubility of phosphate minerals largely

determines the effectiveness of in situ Pb immobilization in soil (Ma, 1996).

Table 1-1. Theoretical solubility of Pb in different phases
Pb Phase Stoichiometry log Ksp
Litharge PbO 12.9
Anglesite PbSO4 -7.7
Cerrusite PbCO3 -12.8
Galena PbS -27.5
Chloropyromorphite Pb5(PO4)3Cl -84.4
Hydroxypyromorphite Pb5(PO4)30H -76.8
Fluoropyromorphite Pb5(PO4)3F -71.6
BromoPyromorphite Pbs(PO4)3Br -78.1
Corkite PbFe3(PO4)(SO4)(OH)6 -112.6
Drugmanite Pb2(Fe,Al)(PO4)2(OH)2 NA
Hindsdalite PbAl3(PO4)(SO4)(OH)6 -99.1
Plumbogummite PbAl3(PO4)2(OH)sH20 -99.3
Lead-metal sulfate (Fe,Zn,Pb)SO4 NA
Iron-nanganese-lead oxide (Fe,Mn,Pb)O NA
Lead-metal oxides (Fe,Al,Zn,Pb)O NA









Phosphoric acid

The rate of chloropyromorphite, one of pyromorphite minerals, precipitation is

kinetically rapid and controlled by the availability of soluble Pb and P as shown in Eq (1)



5Pb2+ + 3H2PO4- +Cl- O Pbs (PO4) 3l(s) + 6H+ (1)


Conversion of soil Pb to pyromorphite could immobilize soil Pb and reduce its

bioavailability. If sufficient soluble P is provided, the dissolution of Pb solids would then

be the limiting factor for pyromorphite formation (Yang et al., 2001). Dissolution of soil

Pb increases with decreasing pH (Zhang and Ryan, 1999b). Neutralization of protons

generated during chloropyromorphite precipitation shown in Eq (1) could favor the

reaction toward chloropyromorphite formation. Thus, initially acidifying the soil

followed by a gradual increase of soil pH should enhance the transformation of soil Pb to

pyromorphite. It is believed that adding H3PO4 to calcareous soil would lower soil pH to

facilitate dissolution of soil Pb and increase the activity of soluble P, thereby enhancing

pyromorphite formation (Yang et al., 2001).

Synthetic apatite

Hydroxyapatite [HA, Calo(PO4)60H2], a member of the apatite family, is the

prototype of the inorganic constituent of bones and teeth, thus its crystal and solution

chemistry have been researched extensively. Most research has centered on synthetic HA,

because relatively pure natural HA is uncommon. Hydroxyapatite effectively and rapidly

attenuates Pb from aqueous solutions, exchange sites, and Pb contaminated soils (Ma et

al., 1993)









Ma et al. (1993) have examined the utility of natural and synthetic apatite for in

situ stabilization of Pb in contaminated soils. The immobilization of Pb by HA was

attributed to HA dissolution followed by precipitation of hydroxypyromorphite [HP,

Pblo(PO4)6(OH)2]: aS shown in Eqs. (2) and (3):



Calo(PO4)6(OH)2(S) + 14 H (aq) O 10Ca2+(aq) + 6H2PO4-(aq) + 2H20 (2)
Dissolution

10Pb2+(aq) + 6H2PO4-(aq) + 2H20 OPblo(PO4)6(OH)2(S) + 14 H (aq) (3)
Precipitation

Laperche et al. (1996) investigated the use of HA as a soil additive with the goal

of converting soil Pb to HP. Hydroxyapatite was mixed with contaminated soil to test its

feasibility in reducing aqueous Pb and in converting the solid phase forms of Pb to HP.

The concentration of dissolved Pb in the suspension was reduced from 0.82 to 0.71 mg L~

Sat solution pH of 7.7 and to 0.22 mg L^1 at pH 5. Dissolved Pb concentrations in the

control samples increased with reaction time and decreasing pH.

Also, the addition of HA to a Pb polluted soil led to a substantial decrease of Pb

concentrations in the shoots of Gradd sudax (Boisson et al., 1999).

Bonemeal apatite

The mineral portions of animal bones and teeth are composed of HA. Bonemeal is

a less soluble source of phosphorous than synthetic apatite, but it is much more

economically feasible for field scale use. The ability ofbonemeal (finely ground, poor-

crystalline apatite) to immobilize pollutant metals in soils and reduce metal

bioavailability through the formation of metal phosphates has been evaluated by Hodson

et al. (2000). The researchers used a 1:50 bonemeal:soil mix. The mixture was packed

into columns and leached with synthetic rainwater. The pH increase appears to be due to









the dissolution of the bonemeal. Monitoring of leachates over a three-month period

indicated that bonemeal additions resulted in the immobilization of metals and an

increase in the pH of the column leachate, the soil pore water and the soils themselves.

Analytical scanning electron microscopy (SEM) and X-ray diffraction (XRD)

identified Pb and Ca-Zn phosphates as the reaction products. Assessment of metal

bioavailability by chemical extraction indicates that bonemeal additions to acidic soils

reduced metal bioavailability in soils via the formation of highly insoluble Pb and Zn

phosphates.

Phosphate rock

Ma et al. (1995) have shown that phosphate rock [PR, primarily fluorapatite (Calo

(PO4) 6F2)] effectively immobilized Pb from aqueous solutions, with Pb immobilization

ranging from 39 to 100%. The primary mechanism of Pb immobilization is via

dissolution of PR and subsequent precipitation of a fluorpyromorphite-like mineral [Pb l0

(PO4)6F2], although precipitation of Pb as hydrocerussite also occurred in some instances.

Phosphate rock immobilizes lead according to the following simplified equations (Ma

and Rao, 1999).


Ca9.5(PO4)5CO3FOH +10H+ 4 9.5Ca2+ + 5H2PO4-+ CO32- + F- + OH- (4)

9.5Pb2+ + 5H2PO4- + CO32- + F- + OH- 4 Pb9.5 (PO4)5CO3FOH + 10H+ (5)

Ma et al. (1995) demonstrated the potential of Florida PR to immobilize aqueous

Pb from Pb contaminated soils. The two most effective Florida PRtypes in immobilizing

Pb [Chemical (CF) and Occidental Chemical (OC)] were chosen for the column

experiments to evaluate the feasibility of using PR to immobilize Pb from a contaminated

soil and to evaluate the effects of different methods of mixing PR and soil and incubation









time on Pb immobilization efficiency. A lead contaminated soil was mixed with PR at a

ratio of 0: 10, 1:10, 2: 10 and 4: 10. After uniform mixing, the soil-PR mixture was placed

in columns. All PR types were effective in reducing Pb concentration from an initial

concentration of 4.82 Clmol L1 to below 72.4 nmol-l (the current EPA action level for Pb)

after 16 days of reaction and at a 4: 10 PR: soil ratio. Incubation time and mixing

methodology had little effect on the efficiency of Pb immobilization by PR. Hettiarachchi

et al. (2000) reported that PR was equally or more effective than triple super phosphate

fertilizer.

Rhizosphere

Phosphorous is an essential plant nutrient and is of the most limiting factors in

plant growth.. Because roots can only absorb free phosphate, several mechanisms exist to

increase the soil P available to them (Cotter-Howells, 1996). Root exudates contain

phosphatase enzymes that can convert organic P to phosphate in the rhizosphere

(Haeussling and Marschner, 1989), as do rhizosphere microorganisms (bacteria and

fungi), not those infecting the roots (Cosgrove, 1967). This free phosphate would also be

available to heavy metal compounds to form metal phosphates. Cotter-Howells et al.

(1996) showed that the formation of metal phosphates could also be induced by the

biochemical action of the roots of Agrostis capillaries.ll1~~~111~~~11 However, the exact mechanism is

not clear. Agrostis capillaries~~~1111~~~~111 roots in these experiments were free from mycorrhizal

infection. Thus, root exudates containing phosphatase enzymes or non-root infecting

microorganisms could be responsible for the conversion of organic P to phosphate in the

rhizosphere.









Another study by Hinsinger and Gilkes (1997) indicated that additional dissolution

of PR occurred in the presence of plant roots. The largest root-induced dissolution was

achieved by ryegrass (Lolium rigidum) and rape (Bra~ssica napus), amounting to 19 to

32% of the PR present in the first two mm of the rhizosphere.

Phytoremediation

Phytoremediation is a general term for using plants to remove, degrade, or contain

soil pollutants such as heavy metals, pesticides, polyaromatic hydrocarbons, and landfill

leachates. These processes include (1) modifying the physical and chemical properties of

contaminated soil; (2) releasing root exudates, thereby increasing organic carbon; (3)

improving aeration by releasing oxygen directly to the root zone as well as increasing the

porosity of the upper soil zones; (4) intercepting and retarding the movement of

chemicals; (5) effecting co-metabolic microbial and plant enzymatic transformation of

recalcitrant chemicals; and (6) decreasing vertical and lateral migration of pollutants to

the ground water by extracting available water and reversing the hydraulic gradient

(Susarla et al., 2002). Recent researches have demonstrated that plants are effective in

cleaning up heavy metal contaminated soils (Wenzel and Jockwer, 1999; Pichtel et al.,

2000; Baker and Brooks, 1989). In many remediation proj ects, phytoremediation is seen

as a final polishing step following the initial treatment of the high-level contamination.

However, when contaminants are low in concentration, phytoremediation alone may be

the most economical and effective remediation strategy (Susarla et al., 2002). Plants have

been used to stabilize or remove metals from soil and water. The main methods used

include phytoextraction and phytostabilization (USDA, 2000).









Phytoextraction

Many higher plant species have adaptations that enable them to survive and to

reproduce in soils heavily contaminated with heavy metals. Such species are divided into

two main groups: the so called pseudometallophytes that grow on both contaminated and

non-contaminated soils, and the absolute metallophytes that grow only on metal

contaminated and naturally metal-rich soils (Baker and Brooks, 1989). Phytoextraction

involves growing plants in metal contaminated soil and harvesting the metal-rich plant

biomass, which can then be incinerated or composted to recycle the metals. Several crop

growth cycles are needed to decrease contaminant levels to allowable limits. If the plants

are incinerated, the ash must be disposed of in a hazardous waste landfill, but the volume

of such ash is far smaller than the volume of waste generated from direct soil

manipulation techniques (i.e. soil removal and incineration or chemical treatment)

(USDA, 2000).

Phytoextraction is often done with plants called hyperaccumulators, which absorb

unusually large amounts of particular metals in comparisons to other plants.

Hyperaccumulators can tolerate, uptake, and translocate high levels of certain heavy

metals that would be toxic to most other organisms. They are defined as plants whose

leaves may contain >100 mg kg-l of Cd, >1000 mg kg-l of Ni and Cu, or >10,000 mg kgl

of Zn and Mn (dry weight) when grown in a metal-rich medium. Hyperaccumulators of

Co (26 species), Cu (24), Mn (8), Ni (145), Pb (4), and Zn (14) have been reported

(Baker and Brooks, 1989).

Phytostabilization

Root induced changes in rhizosphere soil properties may have significant influence

on the mobility and bioavailability of nutrients and trace metals (Arienzo et al., 2003).










Phytostabilization is the use of perennial, non-harvested plants to stabilize or immobilize

contaminants in the soil and groundwater. This process takes advantage of the ability of

plant roots to alter soil conditions, such as pH and soil moisture content by exudation

(Susarla et al., 2002). Metals are absorbed in and accumulated by the roots, adsorbed onto

the roots, or precipitated within rhizosphere. Phytostabilization reduces the mobility of

the contaminant and prevents further movement of the contaminant into the groundwater

or the air and reduces its bioavailability for entry into the food chain (USDA, 2000). One

advantage of this strategy over phytoextraction is the disposal of metal-laden plant

material is not required. By choosing and maintaining an appropriate cover of plant

species, coupled with appropriate soil amendments, it may be possible to stabilize certain

contaminants in the soil and reduce the interaction of contaminants with associated biota

(Susarla et al., 2002).

In order to achieve a successful phytoremediation of soil polluted with metals, a

strategy of combining a rapid screening of plant species possessing the ability to tolerate

and accumulate heavy metals with agronomic practices that enhance shoot biomass

production and increase metal bioavailability in the rhizosphere must be adapted (Kamal

et al., 2004).

Summary

Lead occurs naturally in the earth's crust, but due to increased release of

anthropogenic Pb into the environment, contamination of soil by Pb constitutes a great

environmental concern. In particular, the ubiquity of Pb, its toxicity even in trace

quantities and tendency to bioaccumulate in the food chain make lead poisoning a leading

environmental health threat.









Presence of hazardous levels of Pb in the environment and the toxicity of Pb

requires remedial action to protect public health. Remediation technologies considered to

be cost effective and less disruptive, namely phosphorous induced Pb immobilization and

phytoremedation have been discussed in this review. This study was conducted for the

better understanding of phosphorous induced Pb immobilization mechanisms and the

feasibility of phytoremediation in Pb contaminated site.















CHAPTER 2
EFFECTS OF APPLICATION METHODS ON P-INTDUCED Pb IMMOBILIZATION
IN A SOIL COLUMN

Introduction

Activities such as mining and manufacturing, and the use of synthetic products (e.g.

pesticides, leaded paints, batteries, and industrial wastes have resulted in many Pb-

contaminated sites. Over time, the Pb loading rate in soil has exceeded its natural removal

rate by more than 20-fold (Nriagu, 1978). Contamination of soil by lead is of maj or

concern due to not only its high toxicity to humans and animals, but also to its ease of

exposure through ingestion or inhalation. A soil is generally considered contaminated

with Pb when its total Pb concentration exceeds 400 mg kgl (USEPA, 1996), and

remediation is required at this level (Ma and Gao, 1999).

Various remediation technologies have been developed to clean up metal

contaminated soils. Among those, in situ stabilization of heavy metals using binding

agents is a promising approach due to its sustainability and cost-effectiveness. Other

remediation technologies, including excavation, solidification and chelation/extraction,

are either very costly or only partially effective. The estimated cost of solidification is

about $750/m3 and that of stabilization $250/m3. Stabilization seems to be the more

feasible option due to its ease of operation and relatively low cost (Wang et al., 2001).

In situ immobilization of soil lead using phosphate has been considered a cost-

effective and environmentally benign remediation technology. When phosphorous reacts

with lead in soil, it transforms the reactive and bioavailable Pb species into a more stable









form. Pyromorphite is one of the least soluble forms of lead found in soils under a wide

range of environment conditions. The bioavailability and mobility of soil Pb can be

drastically reduced when unstable Pb forms, such as cerrusite (PbCO3), are COnverted into

pyromorphite (Zhang and Ryan, 1999a). During the last decade, many researchers have

successfully demonstrated the effectiveness of phosphate-induced Pb immobilization by

mixing phosphate minerals with Pb contaminated soils (Ma et al., 1993, 1995; Cotter-

Howells, 1996). The immobilization mechanism is considered to be the dissolution of the

lead compounds followed by the precipitation of lead phosphate. Thus, successful

immobilization of lead in soil requires enhanced solubility of soil Pb and P by decreasing

soil pH and applying sufficient phosphorous.

Among the various phosphorous sources (apatite, phosphate rock, phosphatic clay,

and soluble P), the feasibility of using phosphate rock (PR) to immobilize Pb in soils has

been rarely researched. Phosphate rock, a complex assemblage of phosphate minerals, is

mainly composed of microcrystalline carbonate fluorapatite (Ma et al., 1995). In addition

to reducing metal solubility, phosphate amendments are also effective at reducing metal

bioavailability associated with incidental ingestion of soil by humans (Basta and

McGowen, 2004).

The effectiveness of PR in immobilizing Pb primarily depends on its ability to

provide soluble P (Ma et al., 1993, 1995). To compensate for the low solubility of PR in

soil soluble phosphoric acid (PA) has been used in combination with PR, which

effectively immobilized Pb from a Pb-contaminated soil and facilitated precipitation of

Pb-phosphate compounds (Cao et al., 2001). The role of PA in the mixture was to









solubilize lead minerals in soils and PR, thereby increasing the readily available Pb and P

in the soil. This, in turn, facilitates more precipitation of Pb-phosphate compounds.

The obj ective of this study was to evaluate the effectiveness of different application

methods on lead immobilization in soil using PR and PA as P sources. This was

accomplished by (1) determining Pb leaching characteristics and bioavailability; and (2)

to evaluate Pb distribution in soil after P application.

Materials and Methods

Soil Characterization

The soil used for this study was collected from a lead-contaminated site in an urban

area of northwest Jacksonville, Florida. The site is located in a vacant, fenced rectangular

area (4, 100 m2), and covered by vegetation, mainly grasses. Past industrial activities,

which included a gasoline station, salvage yard, auto body shop, and the recycling of lead

batteries, have all contributed to the contamination of this site. Total lead concentrations

in the soil ranged from 36 to 21,074 mg kg l. Lead concentration decreased with soil

depth, with the maj ority of the Pb present near the soil surface (0-20 cm). Mineralogical

characterization of the site by x-ray diffraction (XRD) reveals that PbCO3 (COTUSsite) is

the predominant Pb mineral on the site (Cao et al., 2001).

Soil Sample Preparation

Soil samples were collected from the top 20 cm at the Jacksonville site. They were

air-dried, sieved through a 2-mm stainless steel screen and stored at room temperature.

The soil sample was thoroughly mixed to ensure uniformity. They were then digested

using the hot-block digestion procedure (USEPA Method 3050A) for total Pb

concentration.









Contaminated soil samples were collected from a location where high

concentrations of lead are present (Cao et al., 2001). The clean soil sample was collected

from outside the fence directly adj acent to the site. The Pb content in the contaminated

soil was greater than 5,000 mg kg- and the soil collected from outside contained 77 mg

kg-l of Pb.

Column Leaching

The column test was conducted to simulate field conditions. 400 g of soil was

packed into a PVC column (40 cm height by 3.5cm diameter), with the top half (0-20 cm)

being filled with the contaminated soil and the lower half (20-40 cm) the clean soil to

simulate Pb distribution on the site. The bulk density of packed soil was 1.2 g cm-3

A total of 18 columns were mounted with each treatment replicated three times.

The phosphate application rate was based on a P/Pb molar ratio =4, which was the

optimum rate for Pb-immobilization in the contaminated soil (Cao et al., 2001).

Phosphorous was applied half as PR and half as PA. Both PR and PA were applied only

to the first 20-cm of the soil column. The PR was either mixed (M) with the

contaminated soil or added as a layer (L) at the bottom of the contaminated soil portion of

the column, while the PA solution was added to the top of the column at 75% field

capacity .

Five different P application methods were used in this experiment and they were as

follows:

RMAs1: P was added as '/ PR and '/ PA where PR was mixed with the

contaminated soil and PA was added from the top of the column at the same time;









R\I.An I: P was added as '/ PR and '/ PA where PR was mixed with the

contaminated soil and DI was added to the top of the column at 75% field capacity. PA

was added from the top of the column one week later;

RLAs1: P was added as '/ PR and '/ PA where PR was added as a l er at the

bottom of the contaminated soil and PA was added from the top of the column at the

same time;

R\I.111 2: P was added as '/ PR, '/ PA and '/ PA where PR was mixed with the

contaminated soil and PA was added from the top of the column two times with one week

apart;

RLAW2: P was added as '/ PR, '/ PA and '/ PA where PR was added as a la er at

the bottom of the contaminated soil and PA was added from the top of the column two

times with one week aart;

A fine textured glass wool was applied at the end of the column, acting as a filter

for the leachate. The soil columns were leached with deionized distilled water (DDW)

twice up to 2 pore-volumes 1 and 4 weeks after the application of PR.

Analytical Procedure

Soil pH was measured with deionized water at a 1:1 soil:solution ratio after 24 h of

equilibrium. Total P was measured colorimetrically with a Shimadzu 160U spectrometer

using the molybdate ascorbic acid method.

At the end of the experiment, the soil columns were divided into 4 sections (0-10,

10-20, 20-30, and 30-40 cm). The modified physiologically based extraction test (PBET)

and Toxicity Characteristic Leaching Procedure (TCLP) were conducted on each section

to determine the effectiveness of P-induced Pb-immobilization in the soil. Also total Pb

in each soil layer was determined to compare the downward movement of Pb among








































Contaminated soil 6.2 5,017 990 2,200

*Detection limit in soil concentration = 20 mg kg- (Pb); 5 mg kg- (Cu); 1 mg kg- (Zn)

The soil is very sandy, with a pH of 6.2-6.7, which is within the range typical of

Florida soils (Chen et al., 1999). Lead was the main contaminant with a concentration of

5,017 mg kg- which exceeds the critical level for industrial soils (1,400 mg kg- ). Also,

elevated concentrations of Cu and Zn were observed (990 and 2,200 mg kg- ,

respectively). The complete details of the metal contamination at this site have been

described by Cao et al. (2001). Generally, Pb, Cu and Zn were concentrated on the

surface soil (0-20 cm) and their concentrations decreased with soil depth. However, a

substantial amount of Pb (>2,000 mg kg- ) was found at depths below 30 cm. In the long


Total Pb Total Cu Total Zn
Soil pH
(mg kg-') (mg kg-') (mg kg- )

Background soil 6.7 77 20 195


different treatments. After leaching columns with up to 2 pore volumes of DDW twice,

column leachates were analyzed for Pb and P concentrations. Pb analysis was performed

using a flame atomic absorption spectrophotometer or graphite furnace atomic absorption

spectrophotometer, depending on the analytic needs of the samples. Soil clay fractions

were separated by centrifugation and analyzed using XRD to detect the formation of

pyromorphite in the soil.

Results

Soil Characterization

Selected chemical properties of the collected surface soil (0-20 cm) are listed in


Table 2-1.

Table 2-1 Soil characterization at the contaminated site










run, it is possible that the metals may leach downward to the subsurface soil (Cao et al.,

2002)

Effects of P Application on Soil pH

The pHs of the P treated soils were measured to evaluate the degree of soil

acidification. As seen from Table 2-2, reduced soil pH in the surface soil (0-20 cm) was

observed in all the P treated soils due primarily to the addition of phosphoric acid

(H3PO4). Significant reduction in pH, approximately 1 pH unit, was limited to only the

top 10 cm.

Table 2-2 pH of soil after treatment
Soil layer(cm) Control R~MAsi RLAsi ,.1 RLAw2
0-10 6.2110.12 5.210.16 5.25+0.07 5.1210.17 5.2110.08 5.2610.06

10-20 6. 1610.08 5.7510.12 5.9910.07 5.8910.05 5.8710.02 5.8910.04

20-30 6.7810.02 5.9310.02 6.0810.04 6. 1210.06 6.3 510.01 6.4610.02

30-40 6.7410. 16 6.0710.06 6.1110. 01 6. 1810.05 6.4110.04 6.4610.06


Of all of the treatments, RLAs1 induced the greatest decrease in soil pH. The pH of

the surface soil was reduced from 6.21 in the control to 5.12 in treatment RLAs1, which is

typical of Florida soils (Chen et al., 1999). Yang et al. (2001) added 5,000 mg kg-l of P as

phosphoric acid to immobilize Pb and reported a reduction of soil pH by 3 units from

7.22 to 4.34. Reduction in soil pH was mostly limited to the top 20 cm of the soil column

(Table 2-2), indicating limited movement ofP in the soil profile (data not shown).

Without the application of phosphate rock, soil pH may have further decreased to levels

where increased metal solubility may become an issue.

Reduction of soil pH to near 5.5 is necessary for efficient Pb immobilization in soil.

Since a large fraction of the Pb in the soil is associated with carbonate (Cao et al., 2001),









addition of PA to the soil is essential for the dissolution of PR and Pb carbonate. Soluble

P and Pb may enhance precipitation of insoluble pyromorphite-like minerals in the soil,

which at given lead and phosphate concentrations is formed a greater rate at pH 5 than at

pH 6 or 7 (Laperche et al., 1996). Other researchers have used liming materials along

with phosphate treatments to buffer against a decrease in pH (Basta and McGowen, 2004;

Brown and Elliot, 1992). Also the application of phosphoric acid to acidify soil may

increase the risk of groundwater contamination with P and other heavy metals and,

therefore, caution should be exercised.

TCLP Pb

The toxicity characteristic leaching procedure (TCLP) is designed to evaluate

whether hazardous constituents may migrate through the vadose zone soils to the water

table by simulating landfill conditions. Higher Pb concentration in TCLP extracts means

higher Pb mobility in the soil. TCLP-Pb concentrations in the control soil without P

treatment were as high as 127 mg L^1, significantly exceeding the regulatory level of 5

mg L^1 (Fig. 2-1). This is possibly because most of the Pb is in the carbonate fraction,

which would readily dissolve in the acidic TCLP solution (Melamed et al., 2003).

Phosphate treated soils, on the other hand, reduced TCLP-Pb by >95% compared to the

control (Fig. 2-1). As a result of P application, TCLP-Pb in the surface soil (0-20 cm) was

reduced to below 5 mg L^1 for all five treatments (Fig. 2-1). .

Among the five P treatments, the highest reduction in TCLP-Pb was observed for

treatment RhlAs1 where PR and PA were applied at the same time, which had less than 2

mg L^1 in the 0-10 cm fraction of the soil column (Fig.2-1). On the other hand, treatment

RMAW2 where PA was added in two aliquots one week apart was the least effective in









reducing TCLP-Pb in the soil. This result may indicate that spontaneous reaction of PA

with PR to solubilize both Pb and P is essential in reducing TCLP-Pb in the soil.


Fig.2-1 TCLP metal contents in sectioned soil columns after leaching

*Detection limit = 0.2 mg L^1
* Data for lower half section (20-40 cm) are not shown due to no significant difference

Hettiarachchi et al. (2000) and Chen et al. (2003) reported the addition of P

significantly reduced TCLP- Pb in contaminated soil to below the critical level of 5 mg

L^1

Lead Bioavailability

Incidental ingestion of Pb-contaminated soil has been proposed as a primary

exposure pathway to humans for elevated blood Pb levels (Chen et al., 2003).

Physiologically based extraction test (PBET) has been used to estimate Pb bioavailability

(in vivo), which simulates Pb dissolution under gastrointestinal conditions using a









chemical extraction (Yang et al., 2001). Lead bioavailability in contaminated soils has

been shown to vary with its mineralogical forms (Davis et al., 1993). In vivo and in vitro

assays have indicated that the mammalian gastrointestinal availability of Pb is controlled

by the form and relative solubility of Pb solids (Ruby et al., 1996). Reduction in Pb

bioavailability was measured by PBET after various amounts and sources of P were

added to a Pb contaminated soil (Hettiarachchi et al., 2000).

The bioavailability of soil Pb is associated with its solubility and dissolution rate in

the gastrointestinal tract. The data in Fig. 2-2 indicate that bioavailable Pb in the

contaminated soil based on PBET measurements was reduced after P application in all

five treatments. The control soil showed 24-25 mg kg-l of bioavailable Pb while P-treated

soils showed reduction of PBET-Pb by 25-42%, which was similar to the 25-3 5%

reduction reported by Hettiarachchi et al. (2000) and 39% by Yang et al. (2001). The

greatest reduction was obtained from treatment RMAs 1 where PR and PA were added to

the soil at the same time, this result is similar to the TCLP results (Fig.2-1).

Distribution of Pb in Soil Column

In most contaminated soils, metals do not appear to leach downward in significant

quantities in the short run primarily due to their strong interactions with the soil.

However, in the long run, metals can leach downward in a soil due to their complexation

with solubilized organic mater especially in an alkaline environment where organic

matter is more soluble (Marschner and Wilczynski, 1991). This may be true at the

Jacksonville site where soil organic matter (3.91%) and pH (6.95) are much higher than

those of typical Florida soil (Chen et al., 1999).













30
S0-10 cani 10-20 cani


-~20










Co ntrol1 RMAS1 RMAW1 RLAS1 RMAW2 RLAW2

Treatments

Fig. 2-2 PBET extractable Pb contents in sectioned soil columns after leaching

*Detection limit = 0.2 mg L^1
* Data for lower half section (20-40 cm) are not shown due to no significant difference


To determine the effectiveness of P treatment to prevent Pb downward migration,

total Pb concentrations in bottom half of the column (20-40 cm) were determined (Fig. 2-

3). As expected, significant downward movement of Pb was observed in the control soil,

especially in the 20-30 cm fraction, which was located directly under the Pb-

contaminated (0-20 cm). The initial Pb concentration in the lower half of the column (20-

40 cm), the control soil, was 77 mg kg-l (Table 2-1). After being in contact with the Pb-

contaminated soil for six weeks under 75% field capacity, the Pb concentration in the 20-

30cm-soil column fraction increased to 365 mg kg- However, Pb migration was mostly

limited to that section of the column as Pb concentration in the 30-40 cm fraction









remained at 77 mg kg-l and little difference was observed among the five treatments (Fig.

2-3 ).

All treatments were effective in reducing Pb migration except RMAW2 where PR

was mixed with the contaminated soil in the top 20-cm, and PA was applied two times

with one week between applications. Since PA was added in two aliquots, the amount of

acidity was probably insufficient to dissolve enough Pb from the soil to induce the

precipitation of Pb-phosphate minerals. This was confirmed by the P data where P

concentration in the leachate of RMAW2 WAS the lowest (Fig. 2-4). This was also

consistent with the highest TCLP-Pb (Fig. 2-1) and PBET-Pb (Fig. 2-2) observed in

treatment RMAW2.

Treatments with PR as a layer (e.g. RLAs1, RLAW2) Were mOre effective in reducing

Pb migration than those where PR was mixed with the soil (RhlAs1, RhlAw1, and RMAW2,

Fig.2-3). This may be due to the formation and/or containment of stable Pb minerals

within the PR layer. Among the treatments where PR was mixed with the soil, treatment

RhlAs1, where PA was applied at the same time as when PR was mixed with the

contaminated soil, was most effective.

To determine the mechanism of reduction in Pb downward movement, soil around

the PR layer was separated and analyzed with XRD. Previous analysis of mineralogical

changes over time resulting from P addition using XRD showed formation of stable

pyromorphite-like minerals in P-treated soil (Cao et al., 2002). However, no Pb-

phosphate was identified in the clay fractions in this research (data not shown). With the

Pb concentration the studied soil being near the MDL for XRD the lack of lead-phosphate






















5 20-30 can 5 30--10 can


confirmation cannot be ruled out. Further investigation is needed to verify the mechanism


within the PR layer.


h
oa
re
oa 300
E
V
a
PI
200
c,
o


Control RMAS1 RMAW1 RLAS1

Tre atme nts


RMAW2 RLAW2


Fig. 2-3 Total Pb concentration in sectioned soil columns (bottom section)

*Detection limit=20mg kg-l
* Data for upper half section (0-20 cm) are not shown due to no significant difference



All P-treated soil columns showed that leachable Pb had been reduced to below the


EPA drinking water regulatory level of 15 ClgL^1 or non-detectable (Table 2-3). The


second highest Pb concentration was observed in treatment RhlAW2, and was comparable

to the control soil. Lead concentrations in the second leachate were lower than those in


the first leachate. Significant differences were seen in the release of Pb between


treatments with different application methods of PA, where treatment RMAW2 WAS


observed to be the least effective and yet still below the EPA regulatory level (15 ClgL^)~.


All Hyve treatments proved to be effective in reducing Pb contamination from soil.









Table 2-3 Pb concentration (ppb) in column leachate (Clg L 1)

Treatment 1st leaching 2nd leaching

Control 33.611.5 14.511.74

RMAc1 nd nd

RMAs1 nd nd

RLAc1 nd nd

RMAs2 11.011.9 5.710.91

RLAS2 2.310.45 nd

*Detection Limit = 1 CgL^1

Leachate Analysis

Column studies more closely simulate field conditions than the TCLP extraction

does. To simulate field conditions, soil columns were leached twice with DDW and the

leachate was collected after one and five weeks of incubation. The leachate was analyzed

for total Pb and P concentrations (Table 2-3, Figure 2-4).

The addition of large amounts of P to a contaminated soil may increase the risk of

eutriphication to water bodies (Basta and McGowen, 2004). Taking into account P

leaching in the soil after P application is important with respect to secondary

contamination. Although it is well recognized that phosphate amendment is an effective

method for immobilizing metals in contaminated soils, some elevated soluble

phosphorous may enhance eutrophication risk (Cao et al., 2001).













1.4
1. 5Ist letiching 2ndl letiching
1.







0.


Control RMAS1 RMAW1~ RLAS1 RMAW2 RLAW2

Tre atme nts

Fig. 2-4. P concentration in column leachate (mg L^1)

*Detection limit = 1 CgL^

This was not the case in this study as phosphorous leached from the P-treated soil

columns was less than 1 mg L^1 for all treatments (Fig. 2-4). Treatments where PA was

applied in two aliquots (RMAW2, RLAW2) had lower P concentrations in the leachate than

in the other treatments. This may indicate that the application of P in small quantities can

limit the downward movement of PA, but with correspondingly lower efficiency in

immobilizing Pb in the soil (Figures 2-1 and 2-2).

Though phosphorous release from P-treated soil was minimal, some of the P was

released as it was not converted to lead phosphate. Therefore, caution is needed to assess

the P movement within P-treated fields to prevent eutrophication risk, and consideration

given to soil type and the amount of P added.









Conclusion

The efficiency of in situ P-induced Pb immobilization depends on the type and rate

of P amendment, along with appropriate application methods. Preliminary laboratory

studies to assess its mechanism and efficiency are important.

This study has shown that applying a mixture of PA and PR effectively reduced

TCLP-extractable Pb, bioavailable Pb and vertical Pb migration in the Pb-contaminated

soil studied. All P-treated soil resulted in significant reductions in bioavailable and

leachable Pb as compared to the control soil. The effective immobilization of Pb was

attributed to the formation of stable Pb minerals after P application. Of all the treatments,

the mixture of PR and soil coupled with the simultaneous application of PA (RhlAs1) was

the most effective in decreasing leachable and bioavailable Pb with the least impact on

soil pH and lowest eutrophication risk. Treatments with PA being applied in two aliquots

and PR being applied as a layer was the least effective overall. Possible explanations of

these results are: 1) the reaction between soil and P amendments before PA application

(after one week) inhibited the formation of lead phosphate; 2) less PR was mixed with

soil (RLAs1 and RLAW2), which caused lower efficiency in reducing leachable and

bioavailable Pb in soil. These results also indicate that a minimal amount of

simultaneously applied PA with the PR is necessary for effective (maximal)

immobilization of Pb in soil to take place. The fact that layered PR showed lower

efficiency in Pb immobilization as compared to the other treatments may be due to an

insufficient amount ofP supplied to the soil. However, the PR layer showed improved

reduction of Pb migration than other mixture treatments. Furthermore, these results

suggest layered PR below contaminated soil may serve as a reactive barrier to prevent Pb









from migrating into the groundwater. Although the effects of PA on pH and leachibility P

were acceptable in this experiment, competitive heavy metal leaching and eutrophication

may be potential drawbacks of its indiscriminate utilization.

Further studies are needed to determine the mechanism of Pb-migration reduction

by the PR layer. Also, the combination of the soil-PR mixture and PR layer can be

considered as well for remediation of Pb contaminated soils, and with reduced leaching,

bioavailability and mobility of Pb.















CHAPTER 3
THE EFFECTS OF PLANTS ON PHOSPHATE-INDUCED Pb IMMOBILIZATION INT
THE RHIZOSPHERE SOIL



Introduction

Of the various technologies for the remediation of lead contaminated sites,

phosphate-induced Pb immobilization has received much attention in recent years. Lead

phosphates have low solubility, and are generally several orders of magnitude less

soluble than analogous carbonates and sulfates (Cotter-Howells, 1996).

Various forms of phosphate have been used to immobilize Pb in situ from

aqueous solutions and Pb contaminated soils (Ma et al., 1995; Hodson et al., 2000; Yang

et al., 2001). The primary mechanism of Pb immobilization appears to be through

phosphate mineral dissolution and subsequent precipitation of pyromorphite-like

minerals. Hence, the solubility of phosphate-mineral applied largely determines the

effectiveness of in situ Pb immobilization in soils (Ma, 1996). For soil P to be available

to react with Pb to form lead phosphate, it must be present as free phosphate (HnPO4n-3,

where n=1 to 3) in the soil solution. However, phosphate minerals in soils also have

relatively low solubilities. In a study on Pb immobilization using phosphorous, a mixture

of phosphoric acid (PA) and phosphate rock (PR) as the sources of P were applied to

enhance solubilization of P and Pb-carbonate in soil (Cao et al., 2001).

Phosphorous is one of the essential macronutrients, and among the most limiting

nutrients for plant growth in soils. The average P-content in soil is about 0.05 % (w/w)









but only 0. 1% of that P is available to plants (Illmer and Scinner, 1995). It has been

shown that organic acids can greatly increase the concentration of plant-available P in the

soil solution through acidification, chelation and anion exchange (Reyes et al., 2001).

Root exudates containing organic acid or phosphatase enzymes play an important role in

the liberation of phosphate ions from organic and inorganic compounds in soils (Cotte-

Howells, 1996). Through roots-induced chemical modifications in the rhizosphere, higher

plants may be directly responsible for the dissolution of PR. In particular, rhizosphere pH

has been shown to differ by 2 units as compared to that of the bulk soil. Several

researchers have concluded that excretion of protons or organic acids by the roots is a

maj or process by which plants can acquire P from PR present in soil (Hinsinger and

Gilkes, 1997). Free phosphate in the soil solution and available to plant roots may be

available to react with lead and form lead phosphate.

The overall obj ective of this experiment was to determine the effectiveness of

phosphate-induced Pb immobilization in the rhizosphere soil of different plant species.

Specific obj ectives were, 1) to evaluate the effect of plant roots on P-induced reduction of

Pb bioavailability; and 2) to determine the effect of plant roots on formation of stable Pb

phosphates.

Materials and Methods

Surface (0-20 cm) soil samples were collected from a heavy-metal contaminated

site in Jacksonville, Florida. Soils were air-dried for 2 weeks, sieved through a 2-mm

stainless steel screen and stored at room temperature prior to the experiment. The soil

sample was mixed thoroughly to ensure uniformity. Soil pH was measured using a 1:1

soil:water ratio. Soil samples were digested using the hot-block digestion procedure










(USEPA Method 3050a) for total lead concentration. Ground rock phosphate [PR,

primarily Calo (PO4)6F2] and PA were used as the sources of phosphorous.

Three plant species were selected to determine the effects of plant root exudates

on phosphate-induced Pb immobilization in the rhizosphere soil. The first, Agrostis

capillariesll1~~~111~~~11 has proven to be effective in inducing the formation of metal-phosphates

(Cotter-Howells, 1996). The other two, Lolium rigidum and Bra~ssica napus, may also

enhance root-induced PR dissolution in the rhizosphere (Hinsinger and Gilkes, 1997).

The following treatments were considered:

Control 1: Soil + Phosphate rock (PR)

Control 2: Soil + PR + Phosphoric acid (PA)

Treatment 1: Soil + PR + Plant

Treatment 2: Soil + PR + PA + Plant

Pots of 4 inches in diameter were used and filled with 520 g of the Pb-

contaminated soil collected from Jacksonville. 10-20 plants seeds were sown into each

pot. The plant seeds were sown directly onto the surface soil, with the rhizosphere soil

separated from the bulk soil. This was accomplished by using a circle of plastic netting

9.5cm in height and 5.5cm in diameter, which was covered with a nylon mesh cloth

(mesh size 45 Clm) placed in the center of the pot, and filled with what would become the

rhizosphere soil. This limited root growth to this area of the pot. The plants (10 plants

per pot) were grown in a greenhouse for 4 weeks (plant heights were approximately 10

cm) and watered as necessary. At the end of four weeks, the rhizosphere soil was

separated from the bulk soil. The roots were washed gently to remove the adhered soil

particles and placed in a glass vial containing approximately 10 ml of deionized distilled









water (DDW). This container was then ultrasonicated for 30 seconds to remove any

remaining soil particles. The resulting solution in the glass vial was filtered using a 45

Clm filter paper and collected. The residues on the filter containing both soil and plant

root samples were mounted on a carbon stub for further analysis.


Fig. 3-1 Lolium rigidum after 4 weeks of growth


Fig. 3-2 Agrostis capillaries~~~1111~~~~111 after 4 weeks of growth

Examination of the rhizosphere soil was conducted with a scanning electron

microscope (SEM) to investigate formation of Pb-P minerals (i.e. pyromorphite). The










presence of pyromorphite like minerals was further analyzed by X-ray diffraction (XRD).

For XRD analysis, the clay fractions of the soil samples were separated and

characterized.


















Fig. 3-3 Bra~ssica napus after 4 weeks of growth

Also PBET (physiologically-based extraction test) was conducted on the

rhizosphere soil to see the effects of plant roots on bioavailable Pb in the soil.


Results

Soil Characterization

The lead contaminated soil had a pH of 6.2 (Table 3-1), which was high compared

to the average value for Florida soils (pH 5.04) (Chen et al., 1999). Lead was the main

contaminant at the site, with a concentration of 5,017 mg kg- In addition, elevated

concentrations of Cu and Zn were also observed (990 and 2,200 mg kg-l each).


Table 3-1. Selected characteristics of the lead-contaminated soil
Soi pH Total Pb (mg Total Cu (mg Total Zn (mg
Soi pHkg-') kg-') kg- )
Contaminated
6.2 5,017 990 2,200
soil
*Detection limit in soil concentration = 20 mg kg-l (Pb); 5mg kg- (Cu); Img kg-l (Zn)










Bioavailable Pb

In vitro metal bioavailability is an estimate of metal bioavailability to humans and

animals by simulating metal dissolution under gastrointestinal condition using a chemical

extraction (Yang et al., 2001).


35
5 Control

30 5 AgolYS tiS Cap illar Iie s
O Bnissica l naipus
25 O Loliumll rigidumll

20

15

S10





PR only PR+PA
Tre atme nt

*Detection limit = 0.2 mg L^1


Fig. 3-4. Lead concentrations using the physiologically based extraction test in the
rhizosphere soil with phosphoric acid and with phosphate rock treatment.

Bioavailable Pb in the rhizosphere soil as measured by PBET was reduced in the

presence of all three plants when growing in soils treated with PR (Fig. 3-4). The

rhizosphere soil in the control had 26 mg kg-l of bioavailable Pb while those with plants

had 21-23 mg kg- an 11-20% reduction. Such a reduction in the bioavailable Pb in the

rhizosphere soil may result from the formation of Pb phosphate, which was supported by









the SEM observation of the plant roots (Figs. 3-5, 6, 7). In the treatment with PR+PA, no

significant reduction of bioavailable Pb was found.

Formation of Lead Phosphate

The main mechanism of Pb immobilization in soil is via dissolution of P and/or

meta-stable Pb compounds and the subsequent precipitation of pyromorphite-like

minerals (Cao et al., 2001). The analysis of mineralogical changes in the rhizosphere soil

that were affected by root exudates using SEM helps to determine whether root-induced

formation of Pb phosphate occurred. The SEM element dot maps of root samples after

growing in a soil for 4-weeks show (Figs. 3-5, 6, 7) close association of Pb with P and

Ca, which may suggest the formation of lead phosphate in the rhizosphere soil

For A. capillariesll1~~~111~~~11 no significant associations between Pb with P and Ca were

observed in the SEM elemental dot map (Fig. 3 -5), but broad association of the three

elements may indicate Pb phosphate in the phosphate rock treated rhizosphere soil (Fig.

3-5b,c,d). For B. napus, the SEM data suggests a close association between Pb, P and Ca.

(Fig 3-6c, d, and e). For L. rigidum, association of P and Pb was observed but not a

strong association with Ca.

The association of Ca, P and Pb may suggest that Pb precipitation occurred on the

surface of the phosphate rock particle, mainly as Calo (PO4)6F2 (CRO et al., 2001).

Formation of stable lead-phosphate minerals may be enhanced due to the biochemical

actions of plant roots, i.e. root exudates containing phosphatase or organic acid. In XRD

analysis, no pyromorphite-like mineral was identified in the clay fractions of the

rhizosphere soil (data not shown). However, this is because the Pb concentration in the

soil was barely detectable by XRD, and the lack of confirmation of this technique does

not prove that no pyromorphite was formed. However, root induced phosphorous









dissolution is proved by analyzing water-soluble phosphorous in the rhizosphere soil. As

seen in Table 3-2, higher water soluble phosphorous was observed in the soil with plants

growing as compared to the control soil. This increase ranged from 37-46% in the PR-

only treated soils to 8-9% in the PR+PA treatment. These results indicate that more

phosphorous solubilization occurred in the presence of plant roots and that higher

available phosphorous may enhance the formation of stable lead-phosphates in the

rhizosphere.

Root exudates containing phosphatase enzymes and/or root-infecting

microorganisms may be responsible for this dissolution of P and precipitation of lead

phosphates. Another possible explanation for lead phosphate formation in the

rhizosphere soil may be that the plant roots created localized acidity, which may have

enhanced the dissolution of the phosphorous minerals and caused precipitation on the

root surface (Cao et al., 2002). Soil pH was analyzed to evaluate the effects its reduction

by root exudates on phosphate-induced Pb immobilization. Numerous studies have

shown that soil pH may also be critical in the dissolution of PR in soil (Ma et al., 1993,

1995; Singh et al., 2000). The dissolution of PR may be enhanced by the supply of

protons and removal of dissolution products in particular Ca and P. Acidic soils are likely

to yield an extensive PR dissolution because they often combine a low pH, with a high P

sorption capacity due to the abundance of Al and Fe oxyhydroxides (Hinsinger and

Gilkes, 1997). As seen in Table 3-3, slightly reduced soil pH was observed in the

rhizosphere soils treated with PR but no significant differences were found in those

treated with the PR+PA mixture. Solubilization of phosphorous is thought to be caused

by the release of organic acids and/or phosphatase, which has been reported to be










exclusively acid (George et al., 2002). The reduction of pH induced by root exudatation

(i.e. organic acid, phophatase) may enhance the dissolution of PR and Pb-carbonate,

promoting the formation of lead phosphate. Several authors have concluded that proton

excretion by the roots was the maj or process by which plants such as buckwheat, rape or

various legumes can acquire P from PR (Bekele et al., 1983; Hinsinger and Gilkes, 1997).

Youssef and Chino (1989) have shown that a single species of rape can strongly acidify

its rhizosphere when grown in an alkaline soil.

IMArGE, 255 Si~jKa, 28 IPKa, II








PbMal, 12 CllaKa, 11











Fig. 3-5. Scanning electron microscopy elemental dot map of Agrostis capillaries~~~1111~~~~111

Hoffland et al. (1989) considered the dissolution of PR by rape was due mainly to

the excretion of organic acid by the roots. Significant reduction of bioavailable Pb and

pH, and higher PR solubilization in soil was observed only in PR-treatments whereas no

significant differences in bioavailable Pb was found for soils treated with PR+PA. It is

possible that the addition of PA provided both the necessary acidity and soluble P to the

plants so no effect from plant roots was observed.




























































Control 0. 1710.01 5.7911.16
Agrostis capillaries 0.3210.05 6.3110.51
Lolium rigidum 0.2810.04 6.3610.57
Brassica napus 0.2710.04 6.3210.55


Fig.3-6. Scanning electron microscopy elemental dot map of Bra~ssica napus
II~wlAR 755 IlblKa 11 ||SiKa 24


Fig. 3-7. Scanning electron microscopy elemental dot map of Lolium rigidum


Table 3-2 Water soluble phosphorous in plant rhizosphere soil (ppm)


PR+PA


PR Only









Table 3-3 pH of rhizosphere soil treated with phosphate rock after 4-weeks growth
PR Only PR+PA
Control 5.7210.21 5.0110.23
Agrostis capillaries 5.3110.41 4.9710.34
Lolium rigidum 5.3410.07 4.8910.43
Brassica napus 5.2310.13 4.9010.23

Conclusion

This study has shown that all three plant species had the ability to induce the

formation of lead phosphate in the rhizosphere soil as supported by the SEM analysis of

plant root samples (Figs. 3-5, 6, 7). Association between Pb, P and Ca in the SEM dot

maps suggests formation of lead phosphates induced by the biochemical action of the

plant roots. This experiment established that the biochemical effects of plant roots can

promote additional dissolution of phosphorous minerals and formation of lead

phosphates.

Reduction of the rhizosphere soil pH may indicate that plant roots enhance the

dissolution of PR and Pb-carbonate by excreting H thereby facilitating the formation of

lead phosphate. Reduction in bioavailable Pb in the rhizosphere soil may due to enhanced

dissolution of PR and formation of lead phosphate by biochemical reaction in the

rhizosphere soil. Using various leachates to examine the bioavailability of Pb in a

contaminated soil, Rabinowitz (1993) found a 43% decrease in Pb concentration

extracted with 10% citric acid in phosphate-amended soils. Our results indicate that all

three plants may have the potential to be applied for the reclamation of Pb-contaminated

sites in combination with the application of PR. Also soil microorganisms are involved in

this process and their role in the solubilization of phosphate-bearing materials has been

the subj ect of m any studi es. There i s a con si derable numb er of pho sphate- solub ili zi ng

bacteria present in soil and in the rhizospheres of plants, and their concentration much










higher in the rhizosphere as compared to non-rhizosphere soil (Rodriguez and Fraga,

1999). The combination of plant root exudates and microorganisms may increase the

amount of free phosphate, thus enhancing the formation of lead phosphate in the

rhizosphere soil. However, the extent to which either of these factors could re-solublize

heavy metal phosphate for subsequent phosphate extraction is unknown. Further research

should investigate the stability of the phosphate dissolution mechanisms of both plants

and soil microorganisms.

Vegetative techniques have been widely used in the reclamation of mine waste,

mainly to stabilize the soil and promote nutrient cycling. This work suggests that the

growth of plants, which has the ability to induce formation of insoluble lead phosphates,

can enhance the reclamation of a contaminated site. The successive growth and decay of

species over several seasons could eventually produce enough lead phosphates (Cotter-

Howells, 1996) to achieve this end. Substantial amounts of pyromorphite present in soils

can reduce bioavailable Pb. The increased effectiveness in phosphate-induced Pb

immobilization as a result of plant root activity would be more ecologically acceptable

than the addition of a large amount of soluble phosphate alone because of less concern

with soil acidification and eutrophication. However, additional research is needed to

further assess the effects of plant-root enhanced phosphate-induced Pb immobilization in

soil on a field scale.















CHAPTER 4
SCREENING OF PLANTS FOR ACCUMULATION OF Pb, Cu, Zn FROM A
CONTAMINATED SITE

Introduction

Heavy metals are one of the most serious environmental concerns today. They are

harmful to humans and animals and tend to bioaccumulate in the food chain. Past usage

of fossil fuels, mining and smelting of metal ores, industrial emissions and the application

of insecticides and fertilizers all have contributed to elevated heavy metal levels in the

environment. The threat that heavy metals pose to human and animal health is aggravated

by their elemental nature and long-term persistence.

There are many technologies available for the remediation of land contaminated by

heavy metals (USDA, 2000). However, many of these technologies are costly (e.g.

excavation of contaminated material and chemical/physical treatment) or do not achieve

either a long term or aesthetic solution (Cotter-Howells, 1996). A new approach, termed

phytoremediation, offers an alternative technology. Phytoremediation can provide a cost-

effective, long-lasting and aesthetic solution for the remediation of contaminated sites.

One of the strategies of phytoremediation with respect to metal contamination is

phytoextraction, i.e. the uptake and accumulation of metals into plant shoots, and their

subsequent harvest.

Metal accumulating plants are not only of scientific interest, but they also serve to

protect several aspects of the environment, such as phytoremediation of lands polluted

with heavy metals, long-term stabilization of wastelands and the reduction of potentially









toxic elements (Raskin and Enseley, 2000). There are over four hundreds plants known to

be hyperaccumulators of metals, which accumulate high concentrations of metals into

their aboveground biomass. These plants include trees, vegetable crops, grasses and

weeds. Based on Baker and Brooks (1989), hyperaccumulators are defined as plants that

accumulate >1,000 mg kg-l of Cu, Co, Cr, Ni or Pb, or >10,000 mg kg-l of Mn or Zn.

Hyperaccumulators of Co (26 species), Cu (24), Mn (8), Ni (145), Pb (5), and Zn (4)

have been reported. The five hyperaccumulators of lead include Armeria martimza

Thlaspi rotundifolium, Thlaspi alpestre, Alyssum wulfenianum, and Polycalpaea

synandra.

It is important to use indigenous native plants species for the phytoremediation

process as these plants are better adapted for survival, growth and reproduction under the

particular environmental stresses encountered than plants introduced from another

environment. However, few studies have evaluated the phytoremediation potential of

natural hyperaccumulators under field conditions (McGrath and Zhao, 2003).

This study was conducted to screen plants that were growing in a contaminated site

for their potential in accumulating Pb, Cu and Zn. The overall objectives were: 1) to

determine the concentrations of Pb, Cu and Zn in the plant biomass; 2) to compare metal

concentrations in the aboveground biomass to those in the roots and in the soils; and 3) to

assess the feasibility to use these plants for the purpose of phytoremediation The

information obtained from this study should provide insight for the use of native plants to

remediate metal contaminated sites.









Materials and Methods

Site Characterization

The plant and soil samples for this study were collected from a known lead contaminated

site located at an urban area of northwest Jacksonville, Florida. The fenced vacant site is

rectangular in shape, occupying approximately one acre. The site is covered by

vegetation, mainly grasses. Past industrial activities include being home to a gasoline

station, salvage yard, auto body shop, and recycler of lead batteries, which all

presumably, contributed to its contamination. Total lead concentration in the soils ranged

from 90 to 4,100 mg kgl (Fig. 4-1 and Table 4-1).















0 10 20 30 40 5


pomto
In~r addtio toP otmntotest locotie lvtdlvl fC n

Zn ie.2090 nd19-,20 g g-(abe -1.Th cntmiaio b hay etlsi

conentate intetp2 mo h st dt o hw)










Sample Preparation and Chemical Analysis

Plant samples, together with associated soil samples were collected from the site.

The selection of plant samples was based on their coverage at the site. In total, 36 plant

samples of 17 different species were collected from 10 locations at the site in December

of 2002 (Fig. 4-1). Soil samples from the rooting zone (0-20 cm) were taken from each

location.

Soil samples were air-dried at room temperature for two weeks, and then sieved

through a 2-mm stainless steel sieve. They were then digested using the hot-block

digestion procedure (USEPA Method 3050a) for total metal concentration. For water-

soluble Pb concentration, 25 ml of deionized distilled water (DDW) was mixed

thoroughly with 2 g of soil for 12 h. The mixture was then centrifuged for 15 min. at

3,500 rpm. The supernatant was analyzed for Pb by flame atomic absorption

spectrophotometry (FAAS).

Plants samples were divided into roots and shoot, washed gently with DDW for

approximately 1 minute to remove the soil. After washing, plant samples were air-dried

at room temperature for two weeks. They were then ground to a powder before digestion

via USEPA Method 3050a.

Results

Metal Concentrations in Soils

Characteristics of the soil samples collected from this study are shown in Table 4-1.

Soil pH ranged from 6.62 to 7.20, relatively high compared to typical Florida soils (Chen

et al., 1999). The high soil pH has been attributed to the presence of cerrusite (PbCO3),

the predominant form of the Pb minerals at the site (Cao et al., 2001).









Table 4-1. Selected properties of soil samples collected from the contaminated
site at Jacksonville, Florida
Water soluble Total Pb Total Cu Total Zn

Site # Soil pH Pb (mg L ) (mg kg-') (mg kg-') (mg kg- )
1 7.03 1 90 20 200
2 7.00 3 143 21 195
3 6.62 32 4,100 990 2,200
4 7.06 82 1,375 980 900
5 7.2 65 1,886 860 683
6 7.08 20 767 314 551
7 6.95 234 2,405 746 1,000
8 6.97 96 1,451 300 572
9 7.01 2 333 29 532
10 6.63 1 145 26 720

*Detection limit = 0.1Img kg- (Pb); 5mg kg- (Cu); and Img kg- (Zn)


Total lead concentrations in the soil samples collected from 10 different locations

were variable, ranging from 90 mg kg-l at site 1 to 4,100 mg kg-l at site 3 (Fig. 4-1). The

mean Pb concentration in Florida soils is 77 mg kg- The global baseline level of Pb in

natural surface soils is reported to be 20 mg kg-l (Chen et al., 1999). In addition to total

Pb, water-soluble Pb was also determined (Table 4-1). As expected, they were much

lower than total Pb concentrations. However, total Pb correlated with water-soluble Pb (r

= 0.48). There were also elevated concentrations of Cu and Zn, ranging from 20 to 990

mg kg-l for Cu, and 195 to 2,200 mg kg-l for Zn. Metal concentrations in the soil

samples collected from different locations were highly correlated with r2= 0.72-90, i.e. a

site that had a high Pb concentration also tended to have high Zn and Cu concentrations.

Among the 10 locations sampled, sites 3, 4, 5, and 7 were the most contaminated with all

three metals (Table 4-1).









Metal Concentrations in Plants

A total of 17 plant species were collected from 10 locations at the site. They were

then identified and analyzed for heavy metal (Pb, Cu, and Zn) concentrations in their

biomass. Concentrations of Pb, Cu and Zn in the soil and in the plant biomasses are listed

in Tables 4-2, 3, and 4. It is generally agreed that metal concentrations in plants vary with

plant species (Alloway, 1994; Alloway et al., 1990). Plant uptake of heavy metals from

soil occurs either passively with the mass flow of water into the roots or through active

transport, crossing the plasma membrane of roots epidermal cells. Under normal growing

conditions, plants can potentially accumulate certain metal ions an order of magnitude

greater than the surrounding medium (Kim et al., 2003).

Lead concentrations in the plants ranged from non-detectable to as high as 1,183

mg kg- with the maximum value found in the roots of Phyla nodiflora collected from

site 4 (Table 4-2). In addition, other species such as Bidens alba, Rubus frutocosus and

Gentianapennelliana also contained significantly higher Pb concentrations than the rest

plant species. None of the plants accumulated Pb at 1,000 mg kg-l in the aboveground

biomass, which is the criterion for a Pb hyperaccumulator (Baker and Brooks, 1989). In

95% of the plant samples, the roots Pb concentration was much greater than that of the

shoots, indicating a low mobility of Pb from the roots to the shoots and immobilization of

heavy metals in roots. No relationship between water soluble Pb and plant-boimass Pb

was found. Pichtel et al. (2000) also analyzed Pb in native plants, which was collected

from a dumpsite and got similar results (non-detectible to 1,800 mg kg- ). Stoltz and

Greger (2002) reported a range of 3.4-920 mg kg-l in different plant species collected

from mine trailing.









Copper concentrations in the plants varied from non-detectable to 460 mg kg-l

(Table 4-3), with the maximum value found in the roots ofP. nodi~ora. Similar to Pb, no

plant species accumulated Cu above 1,000 mg kg- the criterion for a Cu

hyperaccumulator (Baker and Brooks, 1989). As expected, the Cu concentrations in the

roots were greater than those in the shoots. In addition to P. nodi~ora, G. pennelliana,,

Bidens alba also contained significantly higher Cu concentrations than other plant

species. Copper concentrations of 6.4-160 mg kg-l in the plant biomass were reported by

Stoltz and Greger (2002), which were lower than those in our research. Shu et al. (2002)

reported Cu concentrations of 8-45 mg kg-l in the biomass ofPaspahtna distichunt and

Cynodon dactylon.

The Zn content in the plants ranged from 17 to 598 mg kg-l (Table 4-4), with the

maximum value found in the roots of P. nodi~ora collected from site 4, which also

contained the highest Pb and Cu concentrations in the roots. Similar to Pb and Cu, no

plant species accumulated Zn at 10,000 mg kg- the criterion for a Zn hyperaccumulator

(Baker and Brooks, 1989). Generally, Zn concentrations were greater in the roots than the

shoots. Al so Paspahtna notatuns, G. pennelliana, Bidens alba and Stenotaphrunt

secundatunt showed significantly higher Zn concentrations than the rest of plant species

collected. Research conducted by Stoltz and Gerger (2002) found Zn concentrations of

68-1,630 mg kg-l in the plants they collected while those by Shu et al. (2002) contained

66-1,015 mg kg-l

Phyla. nodi~ora collected from site 4 accumulated the highest Pb, Cu and Zn in the

roots (1183, 460, and 598 mg kg- respectively). Other than P. nodi~ora, G. pennellian2a

and Bidens alba had higher concentrations of heavy metals (Pb, Cu and Zn) than other










species collected from the contaminated site. No species satisfied the criterion for a heavy

metal hyperaccumulator, but ability of the above species to accumulate heavy metals in

their biomass needs further investigations. Although total metal concentrations in soils

play an important role in the uptake of metals by plants, this can be influenced by several

factors. In general, a negative correlation was found between soil pH and metal

concentrations in the plants. Other soil factors such as cation exchange capacity also

influence metal uptake by plants in some cases (Jung and Thornton, 1996).

Accumulation and Translocation of Metals in Plants

In this study, none of the plant species showed metal concentrations >1,000 mg kgl

in the shoots (Tables 4-2, 3, 4), i.e. none of them are hyperaccumulators based on Baker

and Brooks (1989).

Bioconcentration factor (BCF) and translocation factor (TF) can be used to estimate

a plant's potential for phytoremediation effectiveness. A plant' s ability to accumulate

metals from soils can be estimated using BCF, which is defined as the ratio of metal

concentration in the roots to that in the soil. A plant' s ability to translocate metals from

the roots to the shoots is measured using TF, which is defined as the ratio of metal

concentration in the shoots to that of the roots. Phytoextraction is the removal of a

contaminant from soil, groundwater or surface water by live plants. Enrichment occurs

when a contaminant taken up by a plant, is not degraded rapidly or completely

accumulated in the plant. The process of phytoextraction generally requires the

translocation of heavy metals to the easily harvestable plant parts, i.e. the shoots.









Table 4-2. Lead concentrations in soil and plant samples (mg kg-1) collected from the
contaminated site at Jacksonville, Florida
Common name Scientific name Site # Roots Shoots Soil
Bahia grass Paspalum notatum Flugge 4 575 428 1,375
5 397 92 1,886
9 nd* nd 333
Wire grass Gentianapennellia~na Fern 1 968 453 90
8 881 491 1,451
Romerillo Bidens alba (L.) DC 2 947 91 143
3 149 23 4,100
5 660 77 1,886
Bermudagrass Cynodon dactylon (L.) DC 5 293 88 1,886
6 75 52 767
Flatsed ge Cyperus esculentus L. 1 28 18 90
2 16 26 143
8 417 26 1,451
Ticktrefoil Desmodium pan2iculatum (L.) 2 130 20 143
Horsetail Equisetum arvense L. 3 284 38 4,100
Hydrocotyle Hydrocotyle amnericana L. 8 99 8 1,451
10 nd nd 145
Turkey tangle Phyla nodiflora (L.) Greene 1 44 24 90
4 1,183 73 1,375
7 117 83 2,405
7 451 55 2,405
Plantain Plantago major L. 1 9 52 90
5 294 67 1,886
Blackberr Rubus frutocosus L. 3 825 22 4,100
6 127 12 767
9 51 nd 333
Goldenrod Solidago altissima L. 6 59 49 767
10 nd nd 145
S owthi stl e Sonchus asper (L.) Hill 7 146 39 2,405
St. Augustine Stenotaphrum secundatum 1 3 1 14 90
3 68 32 4100
Bluej acket Tradescantia ohiensis Raf: 5 206 140 1,886
Tuberous Verbena rigid Spreng. 1 23 11 90
8 35 11 1,451
Bigpod Sesbania exaltata (Raf.) Cory 2 150 nd 143
*Detection limit for Pb 0 .1mg kgl









Table 4-3. Copper concentrations in soil and plant samples (mg kg- ) collected from the
contaminated site at Jacksonville, Florida
Common name Scientific name Site # Roots Shoots Soil
Bahia grass Paspalum notatum Flugge 4 250 352 980
5 360 60 860
9 10 11 30
Wire grass Gentianapennellia~na Fern 1 432 200 20
8 375 210 300
Romerillo Bidens alba (L.) DC 2 10 8 21
3 44 17 990
5 400 32 860
Bermudagrass Cynodon dactylon (L.) DC 5 310 52 860
6 36 21 314
Flatsed ge Cyperus esculentus L. 1 16 10 20
2 10 28 21
8 150 20 300
Ticktrefoil Desmodium paniculatum (L.) 2 6 6 21
Horsetail Equisetum arvense L. 3 110 23 990
Hydrocotyle L. Hydrocotyle amnericana L. 8 32 13 300
10 21 16 26
Turkey tangle Phyla nodiflora (L.) Greene 1 3 1 14 20
4 460 20 516
7 nd* 47 746
7 180 23 746
Plantain Plantago major L. 1 23 10 20
5 150 27 860
9 24 12 29
Blackberr Rubus frutocosus L. 3 30 46 990
6 65 13 314
9 47 265 29
Goldenrod Solidago altissima L. 6 277 241 314
S owthi stl e Sonchus asper (L.) Hill 7 46 34 746
St. Augsie ras Stenotaphrum secundatum 1 22 17 20
3 42 15 990
Bluej acket Tradescantia ohiensis Raf: 5 194 117 860
Tuberous vervain Verbena rigid Spreng. 1 14 10 20
8 25 18 300
Bipo sesbania Sesbania exaltata (Raf.) Cory 2 12 48 21
*Detection limit for Cu=5mg kg- .









Table 4-4. Zinc concentrations in soil and plant samples (mg kg l) collected from the
contaminated site at Jacksonville, Florida
Common name Scientific name Site # Roots Shoots Soil
Bahia grass Paspalum notatum Flugge 4 450 316 900
5 260 166 683
9 250 200 532
Wire grass Gentianapennellia~na Fern 1 524 250 200
8 310 359 572
Romerillo Bidens alba (L.) DC 2 25 20 195
3 143 230 2,200
5 462 17 683
Bermudagrass Cynodon dactylon (L.) DC 5 244 171 683
6 231 162 551
Flatsedge Cyperus esculentus L. 1 172 80 200
2 162 165 195
8 260 290 572
Ticktrefoil Desmodium paniculatum (L.) 2 44 63 195
Horsetail Equisetum arvense L. 3 246 160 2,200
Hydrocotyle L. Hydrocotyle amnericana L. 8 267 50 572
10 62 36 720
Turkey tangle Phyla nodiflora (L.) Greene 1 191 86 200
4 598 110 906
7 32 200 1,000
7 400 453 1,000
Plantain Plantago major L. 1 137 70 200
5 256 161 683
9 213 169 532
Blaker Rubus frutocosus L. 3 340 400 2,200
6 173 93 551
Goldenrod Solidago altissima L. 6 111 86 551
10 90 200 720
S owthi stl e Sonchus asper (L.) Hill 7 134 250 1,000
St. Augsie ras Stenotaphrum secundatum 1 164 100 200
3 516 320 2,200
Blue acket Tradescantia ohiensis Raf: 5 220 211 683
Tuberous vervain Verbena rigid Spreng. 1 176 130 200
8 155 198 572
Bipo sesbania Sesbania exaltata (Raf.) Cory 2 283 239 195
9 213 169 532
*Detection limit for Zn=1mg kg-










By comparing BCF and TF, we can assess the ability of different plants in taking

up metals from soils and translocating them to the shoots. Tolerant plants tend to restrict

soil-root and root-shoot transfers, and therefore has much lower accumulation in their

biomasses, while hyperaccumulators actively take up and translocate metals into their

aboveground biomass. Plants exhibiting TF and particularly BCF values less than 1 are

ineffective for use in phytoextraction (Fitz and Wenzel, 2002). A few plants growing at

the site were capable of accumulating heavy metals in their roots, but most of them had

Table 4-5 Accumulation and translocation of Pb, Cu and Zn in selected plants
Bioconcentration
factor Transl location factor
Site # Pb Cu Zn Pb Cu Zn
G~entiana ennelliana Fern 1 10.76 21.6 2.62 0.47 0.46 0.48
8 0.61 1.25 0.54 0.56 0.56 1.16
Cyperus esculentus L. 2 0.11 0.48 0.83 1.6 2.8 1.02
8 0.29 0.5 0.45 0.06 0.13 1.12
Phyla nolra L. Greene 1 0.5 1.55 0.96 0.55 0.45 0.45
7 0.05 0.01 0.03 0.71 11.75 6.25
7 0.19 0.24 0.4 0.12 0.13 1.13
Rubus frticosus L. 3 0.2 0.03 0.15 0.03 1.53 1.18
9 0.15 1.62 0.4 NA 5.64 0.79
Sesbania exaltata (Raf.)Cr 2 1.05 0.57 1.45 NA 4 0.84
Plan2tago major L. 1 0.1 1.15 0.69 5.96 0.43 0.51
Bidens alba (L.) DC 2 6.62 0.48 0.13 0.1 0.8 0.8
3 0.04 0.04 0.07 0.15 0.39 1.61
Pas lum notatm Flugge 4 0.42 0.26 0.5 0.47 1.41 0.7
9 NA 0.33 0.47 NA 1.1 0.8
Stenotahrum secundatum 1 0.34 1.1 0.82 0.45 0.77 0.61
Verbena rigid Spreng 8 0.02 0.08 0.27 0.31 0.72 1.28
Sonchus asper (L.) Hill 7 0.06 0.06 0.13 0.27 0.74 1.87
Solidago altissima L. 10 NA 0.77 0.13 NA 0.5 2.22
Desmodium panicula~tum (L.) DC 2 0.91 0.29 0.23 0.15 1 1.43









low TF's and BCF's, which means they have limited ability to accumulate and

translocate the metals studied (Tables 4-5).

Among the 17 plants screened, G. pennelliana, growing at site 1, had the highest

BCF of Pb (BCF=10.8), though its Pb concentrations in the plant body were <1,000 mg

kg- On the other hand, P. nodi~ora had the highest Pb concentration in the roots (1,183

mg kg- ), but its BCF was less than 1 (Table 4-5). Cyperus esculentus L.and

Plan2tagomajor L. had TF's greater than 1 (TF=1.6, and 6.0, respectively). The BCF

value of Pb found in this research was lower than that of Kim et al. (2003) in P.

thunbergii (5 58), and higher than from those (0.3-0.45) reported by Stoltz and Greger

(2002). Shallari et al. (1998) reported a high BCF of Pb in Koeleria eriostachya (31i),

Setaria viridis (18) and Verba~scum blattaria (3).

Similar to Pb, no plant species accumulated Cu above 1,000 mg kg-l (Table 4-3).

The maximum value for Cu was 460 mg kg-l in the roots ofP. nodi~ora. Though several

plant species showed BCFs or TFs greater than 1 for Cu, only Rubus fruticosus L.

growing at site 9 had both a BCF (1.6) and a TF (5.6) greater than 1 (Table 4-5).

However, soil Cu concentration at this site was relatively low, at 29 mg kg- The BCF

values for these species were lower than those found in P. thunbergii (41-168) by Stoltz

and Greger(2002). The highest BCF for Cu was 6.3 in Silene gallica by Shallari et al.

(1998).

The highest Zn concentration was 598 mg kg- which was in the roots of P.

nodi~ora (Table 4-4). Both G. pennelliana. and S. exaltata had a BCF greater than 1, and

several species had a TF greater than 1, with P. nodi~ora having the highest TF of 6.3.

The highest BCF for Zn was 2.6 in G. pennelliana, lower than those reported by Kim et










al. (2002) in P. thunbergii (22 136) but higher than those (0.004-0. 11) reported by Stoltz

and Greger (2002). Research conducted by Shallari et al. (1998) reported the highest Zn

BCF for Alyssim markgurafii at 5.9.

Though none of the plants sampled were metal hyperaccumulators, some

interesting observations were noted. Based on the average BCFs of all plant samples,

plant roots were most efficient in taking up Cu (BCF=1.1), followed by Pb (0.79) and Zn

(0.50) (data not shown). Based on the average TFs of all the plant samples, the plants

were most efficient in translocating Cu (TF=1.2), followed by Zn (0.98) and Pb (0.54).

From a general point of view, among the three metals tested, the plants growing on the

Jacksonville site were most efficient in taking up and translocating Cu. Low translocation

of Pb indicates that plants are unwilling to transfer Pb from their roots to shoots due to

toxicity of Pb. Lead can be toxic to photosynthetic activity, chlorophyll synthesis and

antioxidant enzyme production (Kim et al., 2003). Baker and Brooks (1989) also

discussed the restriction of metal uptake by plants from contaminated soils and the

presence of exclusion mechanisms in such species. Since Zn and Cu are essential

nutrients for plant systems, their higher translocation factors can be supported. Thomas

and Eong (1984) treated established Rhizophora mucronata L.am. and Avicennia alba B.1

seedlings in sediment with Pb and Zn. For these species, root accumulation and reduced

translocation to the leaf tissues were observed for Zn and Pb.

In general, all three heavy metals occurred at elevated levels in the plant biomass as

a result of previous industrial activity at the site. Normal and phytotoxic concentrations of

Pb, Zn and Cu were reported by Levy et al. (1999), which were 0.5-10, and 30-300 mg

kg-l for Pb, 3-30 and 20-100 mg kg-l for Cu, and 10-150 and >100 mg kg-l for Zn.










Almost all collected species had heavy metal concentrations higher than the normal or at

phytotoxic levels. These results may indicate that plant species growing in heavy metal

contaminated site were tolerant of these metals. Restriction of upward movement into the

shoots can be considered a tolerance mechanism, as can the increase in metal-binding

capacity of their cell walls (Verkleij and schat, 1990).

In addition, the relationships of BCFs and TFs among the three metals were

determined through simple correlation. A few studies have been published to show that

high Pb levels reduce the uptake of other elements such as Fe, Mn and Zn. The

correlation of BCFs of all plant samples between two of the metals ranged from 0.63 to

0.84 (data not shown), i.e. a plant, which was effective in taking up Pb, was very likely to

be effective in taking up Cu and Zn. However, the relationship between TFs was

different. Only the TFs of Zn and Cu were correlated (r2=0.79), whereas no correlation of

TFs was found between Pb and Cu, or Pb and Zn. In other words, a plant, which was

effective in translocating Zn, was also effective in translocating Cu and vice versa.

However, Cu and Zn translocation in these plants were not related to Pb translocation.

These relationships may indicate that elevated Pb concentration can inhibit the transfer of

essential micronutrients to the plant biomass.

Table 4-6. Plants with high BCF and low TF for phytostabilization
Bioconcentration Translocation Applicable
factor factor Phystabilazation
Site # Pb Cu Zn Pb Cu Zn
Gentiana pennelliana Flugge 1 10.8 21.6 2.62 0.47 0.46 0.48 Pb, Cu Zn
8 0.61 1.25 0.54 0.56 0.56 1.16 Cu
Bidens alba (L.) DC 2 6.62 0.48 0.13 0.10 0.80 0.80 Pb
Sesbania exaltata (Raf.) Coryl 2 1.05 0.57 1.45 0.01 4.00 0.84 Pb,Zn
Plantago major L. 1 0.10 1.15 0.69 5.96 0.43 0.51 Cu
Stenotaphrum secundatum 1 0.34 1.10 0.82 0.45 0.77 0.61 Cu










Phytostabilization is another technology that can be used to minimize migration of

contaminants in soils (Susarla et al., 2002). This process uses the ability of plant roots to

change soil conditions via root exudation. Plants can immobilize heavy metals through

absorption and accumulation by the roots, adsorption onto roots, or precipitation within

the rhizosphere. This process reduces metal mobility and leaching into the ground water,

and also reduces metal bioavailability for entry into the food chain. One advantage of this

strategy over phytoextraction is the disposal of the metal-laden plant material is not

required (Susarla et al., 2002). By using metal-tolerant plant species for stabilizing

contaminants, particularly metals, in soil, could also provide improved conditions for

natural attenuation or stabilization of contaminant in the soil. Metals accumulated in the

roots are not considered to be a threat for release into environment. Studies are needed

regarding the turnover of nutritive roots and the potential release of metals from

decomposing roots (Weis and Weis, 2004). Also effects of plant-bacteria and/or plant-

mycorrhizae interactions, which might affect the metal uptake and translocation need

further investigation. Although no heavy metal hyperaccumulators were found in our

study, heavy metal tolerant plant with high BCF and low TF can be used for

phytostabilization of contaminated site (Table 4-6).

Conclusion

This study was conducted to screen plants growing on a contaminated site to

determine their potential for metal accumulation. A total of 17 plant species were

collected and analyzed for heavy metal concentrations. Only plants with both a BCF and

a TF greater than 1 have potential to be used for the purpose of phytoextraction. In this

study, no plant species were identified as metal hyperaccumulators based on Baker and

Brooks (1989). However, several plants had BCFs or TF >1. G. pennellian2a. was the









most effective in taking up all three metals, with BCFs at 1.1-22 with one exception

(Table 4-5). Cyperus esculentus was the most effective in translocating all three metals

(Table 4-5). Among those plant species collected from the contaminated site, G.

pennelha~na was considered as the most promising species for phytoextraction. Further

clarification of the phytoremediation potential of these plant species needs to be

investigated. Also the plant sampling was conducted randomly, and a more systematic

sample collection could yield more definitive results.














CHAPTER 5
CONCLUSION

This study has shown that applying a mixture of phosphoric acid and phosphate

rock effectively reduced TCLP-extractable Pb (Toxicity Characteristic Leaching

Procedure), bioavailable Pb and Pb leachability in the Pb-contaminated soil studied.

Collected soil form the Hield had TCLP-extractable Pb < 127mg L- bioavailable Pb <26

mg L^1. Application of phosphoric acid and phosphate rock reduced TCLP-Pb by >95%

and bioavailable Pb by 25-42% and reduced Pb leachability from the contaminated soil.

A layer of phosphate rock resulted in improved reduction of Pb migration. Result

suggests layered phosphate rock below a contaminated soil may serve as a reactive

barrier to prevent Pb from migrating into the groundwater. No significant soil

acidification or phosphorous leaching was observed by the application of phosphoric acid.

Of all the treatments, mixture of phosphate rock and soil with co-application of

phosphoric acid (RhlAs1) was the most effective in decreasing leachable and bioavailable

Pb with acceptable impact on soil pH and eutrophication risk.

A greenhouse study showed that the three plants (Agrostis capillariesll1~~~111~~~11 Lolium

rigidum, and Bra~ssica napus) were effective in enhancing Pb immobilization in the

rhizosphere soil. Plant rhizosphere soil showed 13-20% reduction in bioavailable-Pb as

compared to the control soil. Reduction of the bioavailable Pb in the rhizosphere soil may

result from the formation of Pb phosphates, which was supported by the SEM

observation of the plant roots. Formation of stable lead-phosphate minerals may be

enhanced via biochemical actions of roots (i.e. organic acid, phosphatase exudation). This










study demonstrated that using the most effective P-application method coupled with plant

growth can maximize the effectiveness of phosphate-induced lead immobilization in

soils.

Elevated metal concentrations were observed in the 17 plants collected from the

experimental site located in Jacksonville, Florida, with total Pb concentrations ranging

from non-detectable to 1,183, Cu 5 to 460, and Zn 17 to 598 mg kg l.Among thel17

collected species, G. pennelliana. was the most effective in taking up all three metals,

with BCFsbeing 1.1-22 and Cyperus was most effective in translocating all three metals

with TFs being 1.0-2.8. Though no metal hyperaccumulator was identified, heavy metal

tolerant plants with a high BCF and a low TF can be used for phytostabilization to

minimize the migration of a particular contaminant in a soil.
















LIST OF REFERENCES


Alloway, B.J., 1994. Toxic metals in soil-plant systems. John Wiley and Sons,
Chichester, UK.

Alloway, B.J., Jackson, A.P., Morgan, H., 1990. The accumulation of cadmium by
vegetables grown on soils contaminated from a variety of sources. The Science of
Total Environment. 91, 223-236.

Arienzo, M., Adamo, P., Cozzolino, V., 2003. The potential of Lolium perenne for
revegetation of contaminated soil from a metallurgical site. The Science of Total
Environment. 319, 13-25.

Baker, A.J.M., Brooks, R.R., 1989. Terrestrial higher plants which hyperaccumulate
metallic elements-a review of their distribution, ecology and phytochemistry.
Biorecovery.1i, 81-126.

Baker, A.J.M., Reeves, R.D., Hajar, A.S.M., 1994. Heavy metal accumulation and
tolerance in British populations of the metallophyte Thlaspi caerulescens J. & C.
pres1 (Brassicaceae). New Phytologist. 127, 61-68.

Basta, N.T., McGowen, S.L., 2004. Evaluation of chemical immobilization treatments for
reducing heavy metal transport in a smelter-contaminated soil. Environmental
Pollution. 127, 73-82.

Bekele, T., Cino, B.J., Ehlert, P.A.I., Van der Maas A.A., Van Diest A., 1983. An
evaluation of plant-borne factors promoting the solubilization of alkaline rock
phosphate. Plant and Soil. 75, 361-378.

Boisson, J., Ruttens, A., Mench, M., Vangronsveld, J., 1999. Evaluation of
hydroxyapatite as a metal immobilizing soil additive for the remediation of polluted
soils. Part 1. Influence of hydroxyapatite on metal exchangeability in soil, plant
growth and plant metal accumulation. Environmental Pollution. 104, 225-233.

Brown, G.A., Elliott, H.A., 1992. Influence of electrolytes on EDTA extraction of Pb
from polluted Soil. Water, Air, and Soil Pollution. 62, 157-165.

Callender, E., Rice, K.C., 2000. The urban environmental gradient, Anthropogenic
influences on the spatial and temporal distributions of lead and zinc in sediments.
Environmental Science and Technology. 34, 232-238.










Cao, X., Ma, L.Q., Chen, M., Singh, S.P., Harris, W.G., 2002a. Phosphate induced metal
immobilization in a contaminated site. Environmental Pollution. 122, 19-28.

Cao, X., Ma, L.Q., Chen, M., Singh, S.P., Harris, W.G., 2002b. Impacts of Phosphate
amendments on lead biogeochemistry at a contaminated site. Environmental
Science and Technology. 36, 5296-5304.

Cao, X., Ma, L.Q., Harris, W.G., Nkedi-kizza P., 2001. Field demonstration of metal
immobilization in contaminated soils using P amendments, FIPR Proj ect #97-01-
148R. Final Report. University of Florida, Gainesville, FL.

Chen, M., Ma, L.Q., Harris, W.G., Hornsby, A., 1999. Background concentrations of 15
trace metals in Florida soil. Journal of Environmental Quality. 28, 1173-1181.

Chen, M., Ma, L.Q., Singh, S.P., Cao, X., Melamed, R., 2003. Field demonstration of in
situ immobilization of soil Pb using P amendments. Advances in Environmental
Research. 8, 93-102.

Chen, X., Wright, J., Conca, J., and Peurrung, L., 1997. Effects of pH on heavy metal
sorption on mineral apatite. Environmental Science and Technology. 31, 624-631.

Cosgrove, D.J., 1967. Metabolism of organic phosphate in soil. Soil Biochemistry. 1,
216-228.

Cotter-Howells, J., Caporn, S., 1996. Remediation of contaminated land by formation of
heavy metal phosphates. Applied Geochemistry. 11, 335-342.

Cotter-Howells, J., 1996. Lead phosphate formation in soils. Environmental Pollution.
93, 9-16.

Dahmani-Muller, H., Van Oort, F., Gelie, B., Balabane, M., 2000. Strategies of heavy
metal uptake by three plant species growing near a metal smelter. Environmental
Pollution. 109, 231-238.

Davis, A., Drexler, J.W., Ruby, M.V., Nicholson, A., 1993. Micromineralogy of mine
wastes in relation to lead bioavailability, Butte, Montana. Environmental Science
and Technology. 23, 1415-1425.

Dingwall-Fordyce, I., Lane, R.E., 1963. A follow-up study of lead workers. Br. J. Ind.
Med. 20, 213.

Fitz, W.J., Wenzel, W.W., 2002. Arsenic transformation in the soil-rhizosphere-plant
system, fundamentals and potential application of phytoremediation. Journal of
Biotechnology. 99, 259-278.










Gardea-Torresdy, J.L., Peralta-Videa, J.R., Montes, M., de La Rosa, G., Corral-Diaz, B.,
2003. Bioaccumulation of cadmium, chromium and copper by Convolvulus
arvensis L., impact in plant growth and uptake of nutritional elements. Bioresource
Technology. 92, 229-235.

George, T.S., Gregory, P.J., Wood, M., Read, D., Buresh, R.J., 2002. Phosphatase
activity and organic acid in the rhizosphere of potential agroforestry species and
maize. Soil Biology and Biochemistry. 34, 1487-1494.

Haeussling, M., Marschner, H., 1989. Organic and inorganic soil phosphates and acid
phosphatase activity in the rhizosphere of 80-year-old Norway spruce [Picea abies
(L.) Karst trees.] trees. Biology and Fertility of Soils. 8, 128-133.

Harrison, R.M., Laxen, D.P.H., 1981. Lead pollution, Causes and Control. Chapman and
Hall, London.

Henry, J., 2000. An Overview of the phytoremediation of lead and mercury. EPA
NNEMS Report. www.clu-in.org (accessed March 2005)

Hettiarachchi, G., Pierzynski, G., Ransom, M., 2000. In situ stabilization of soil lead
using phosphorus and manganese oxide. Environmental Science and Technology.
34, 4614-4619.

Hinsinger, P., Gilkes, R.J., 1997. Dissolution of phosphate rock in the rhizosphere of five
plant species grown in an acid, P-fixing mineral substrate. Geoderma. 75, 231-249.

Hodson, M., Valsami-Jones, E., Cotter-Howells, J., 2000. Bonemeal additions as a
remediation treatment for metal contaminated soil. Environmental Science and
Technology. 34, 3501-3507.

Hoffland, E., Findenegg, G.R., Nelemans, J.A., 1989. Solubilization of rock phosphate by
rape. Local root exudation of organic acids as a response to P-starvation. Plant and
Soil. 113, 161-165.

Hutchison, T.C., Meema, K.M., 1987. Lead, mercury, cadimum and arsenic in the
Environment. John Wiley and Sons, New York.

Illmer, P., Schinner, F., 1995. Solubilization of inorganic calcium-phosphates-
solubilization mechanisms. Soil Biology and Biochemistry. 27, 257-263.

Jung, M.C., Thornton, I., 1996. Heavy metal contamination of soils and plants in the
vicinity of a lead-zinc mine, Korea. Applied Geochemistry. 11, 53-59.

Kabata-Pendias, A., Pendias, H., 1984. Trace elements in soils and plants. CRC Press Inc.,
Boca Raton, FL.

Kamal, M., Ghaly, A.E., Mahmoud, N., Cote, R., 2004. Phytoaccumulation of heavy
metals by aquatic plant. Environmental International. 29, 1029-1039.










Kim, L S, Kang, H.K, Johnson-Green, P, Lee E.J., 2003. Investigation of heavy metal
accumulation in Polygonum thunbergii for phytoextraction. Environmental
Pollution. 126, 235-243.

Laperche, V., Logan, T., Gaddam, P., Traina, S., 1997. Effect of apatite amendments on
plant uptake of lead from contaminated soil. Environmental Science and
Technology. 31, 2745-2753.

Laperche, V., Traina, S., Gaddam, P., Logan, T., 1996. Chemical and mineralogical
characterizations of Pb in a contaminated soil, reactions with synthetic apatite.
Environmental Science and Technology. 30, 3321-3326.

Levy, D.B., Redente, E.F., Uphoff, G.d., 1999 Evaluating the phytotoxicity of Pb-Zn
tailings to big bluesteam (Andropogon gerardii vitman) and switchgrass (Panicum
virgatum L.). Soil Science 164: 363-375

Ma, L., 1996. Factors influencing the effectiveness and stability of aqueous lead
immobilization by hydroxyapatite. Journal of Environmental Quality. 25, 1420-
1429.

Ma, L., Choate, A., Rao, G., 1997. Effects of incubation and phosphate rock on lead
extractability and speciation in contaminated soils. Journal of Environmental
Quality. 26, 801-807.

Ma, Q., Logan, T., Traina, S., and Ryan, J., 1994a. Effects of NO3-, Cl-, F SO42-, and
CO32- On Pb2+ immobilization by hydroxyapatite. Environmental Science and
Technology. 28, 408-418.

Ma, Q., Logan, T., Traina, S., 1995. Lead immobilization from aqueous solutions and
contaminated soils using phosphate rocks. Environmental Science and Technology.
29, 1118-1126.

Ma, L. Rao, G., 1997. Effects of phosphate rock on sequential chemical extraction of lead
in contaminated soils. Journal of Environmental Quality. 26, 788-794.

Ma, L. Rao, G., 1999. Aqueous Pb reduction in Pb-contaminated soils by florida
phosphate rocks. Water, Air, and Soil Pollution. 110, 1-16.

Ma, Q., Traina, S., Logan, T., 1993. In situ lead immobilization by apatite.
Environmental Science and Technology. 27, 1803-1810.

Ma, Q., Traina, S., Logan, T., Ryan, J., 1994b. Effects of aqueous Al, Cd, Cu, Fe(II), Ni,
and Zn on Pb immobilization by hydroxyapatite. Environmental Science and
Technology. 28, 1219-1228.

Marschner, B., Wilczynski, A.M., 1991. The effects of liming on quantity and chemical
composition of soil organic matter in a pine forest in Berlin, Germany. Plant and
Soil. 137, 229-236.










MacFarlane, G.R., Burchett, M.D., 2002. Toxicity, growth and accumulation
relationships of copper lead and zinc in the grey mangrove Avicennia marina
(Forsk) Vierh. Marins Environmental Research. 54, 65-84.

Madejon, P., Murillo, J.M., Maranon,T., Cabrera, F., Lopez, R., 2002. Bioaccumulation
of As, Cd, Cu, Fe and Pb in wild grasses affected by the Aznalcollar mine spill
(SW Spain). The science of the Total Environment. 290, 105-120.

Mavropoulos, E., Rossi, A., Costa A., Perez, C., Moreira, J., Saldanha, M., 2002. Studies
on the mechanisms of lead immobilization by hydroxyapatite. Environmental
Science and Technology. 36, 1625-1629.

McBride, M., 1994. Environmental chemistry of soils. Oxford University Press, New
York, NY.

Mcgrath, S.P., Zhao, F.J., 2003. Phytoextraction of metals and metalloids from
contaminated soils. Current Opinion in Biotechnology. 14, 1-6.

Melamed, R., Cao, X., Chen, M., Ma, L.Q., 2003. Field assessment of lead
immobilization in a contaminated soil after phosphate application. The Science of
the Total Environment. 305, 117-127.

Mench, M., Vangronsveld, J., Didier, V., Clij sters, H., 1994. Evaluation of metal
mobility, plant availability and immobilization by chemical agents in a limed-silty
soil. Environmental Pollution. 86, 279-286.

Mulligan, C.N., Yong, R.N., Gibbs, B.F., 2001 Remediation technologies for metal-
contaminated soils and groundwater, an evaluation. Engineering Geology. 60, 193-
207.

Mercedes D. R., Rafael F., Concepcion A., Dinoraz V., Rosa M., Antonio D.H. B., 2002.
Heavy metals and arsenic uptake by wild vegetation in the Guadiamar river area
after the toxic spill of the Aznalcollar mine. Journal of Biotechnology. 98, 125-137.

Nedwed, T., Clifford, D.A., 1997. A survey of lead battery recycling sites and soil
remediation processes. Waste Management. 17, 257-269.

Needleman, H.L., 1992. Human lead exposure. CRC Press Inc., Boca Raton, FL.

Nelson, D.W., Sommers, L.E., 1982. Total Carbon, organic carbon, and organic matter.
In, Page, A.L., Moller R.H., Keeney, D.R.(Eds.), Methods of soil analysis, Part 2
Chemical and micro-biological properties. ASA, Madison, WI. 539-577.

Nriagu, J.O., 1972. Lead Orthophosphates-I. Solubility and hydrolysis of secondary lead
orthophosphate. Inorganic Chemistry. 11, 2499-2503.

Nriagu, J.O., 1974. Lead orthophosphates-IVr. Formation and Stability in the
Environment. Geochimica et Chomochimica Acta. 38, 887-808.










Nriagu J.O., 1978. The biogeochemistry of lead in the environment Part A, B.
El sevier/North-Holland Biomedical Press, Amsterdam

Pearson, M., Maenpaa, K., Pierzinsky, G., Lydy, M., 2000. Effects of soil amendments
on the bioavailability of lead, zinc, and cadmium to earthworms. Journal of
Environmental Quality. 29, 1611-1617.

Piechalak, A., Tomaszewska, B., Baralkiewicz, D., Malecka A., 2002. Accumulation and
detoxification of lead ions in legumes. Phytochemistry. 60, 153-162.

Pichtel, J., Kuroiwa, K., Sawyerr, H.T., 2000. Distribution of Pb, Cd, and Ba in soils and
plants of two contaminated sites. Environmental Pollution. 110, 171-178.

Pugh, R.E., Dick, D.G., Freedeen, A.L., 2002. Heavy metal (Pb, Zn, Cd, Fe, and Cu)
contents of plant foliage near the Anvil Range lead/zinc mine Faro, Yukon
territory. Ecotoxicology and Environmental Safety. 52, 273-279.

Pulford, I.D., Watson, C., 2003. Phytoremediation of heavy metal contaminated land by
trees-a review. Environment International. 29, 529-540.

Rabinowitz, M.B., 1993. Modifying soil lead bioavailability by phosphate addition.
Bulletin of Environmental Contamination and Toxicology. 515, 438-444

Raskin, I., Ensley, B., 2000. Phytoremediation of toxic metals, using plants to clean up
the environment. John Wiley and Sons, Inc., New York.

Reddy, M. S., Kumar, S., Babita, K., 2002. Biosolubilization of poorly soluble rock
phosphates by Aspergillus tubingensis and Aspergillus niger. Bioresource
Technology. 84, 187-189.

Reyes, I., Baziramakenga, R., Bernier, L., Antoun, H., 2001. Solubilization of phosphate
rocks and minerals by a wild-type strain and two UV-induced mutants of
Penicillum rugulosum. Soil Biology and Biochemistry. 33, 1741-1747.

Rio, M.D., Rafael, F., Almela, C., Velez, D., Montoro, R., Bailon, A.D.H., 2002. Heavy
metals and arsenic uptake by wild vegetation in the Guadiamar river area after the
toxic spill of the Aznalcollar mine. Journal of Biotechnology. 98, 125-137.

Rodriguez, H., Fraga, R., 1999. Phosphate solubilizing bacteria and their role in plant
growth promotion. Biotechnology Advances. 17, 319-339.

Ruby, M., Davis, A., Nicholson, A., 1994. In situ formation of lead phosphates in soils as
a method to immobilize lead. Environmental Science and Technology. 28, 646-654.

Ruby, M., Davis, A., Schoof, R., Eberle, S., Sellstone, C., 1996. Estimation of lead and
arsenic bioavailability using a physiologically based extraction procedure.
Environmental Science and Technology. 30, 422-430.










Sauve, S., Mcbride, M., Hendershot, W., 1997. Speciation of lead in contaminated soil.
Environmental Pollution.98, 149-155.

Sayer, J.A., Cotter-Howells, J.D., Watson, C., Hiller, S., Gadd, G.M., 1999. Lead mineral
transformation by fungi. Current Biology. 9, 691-694.

Scheckel, K.G., Ryan, J.A., 2003. In vitro formation of pyromorphite via reaction of Pb
sources with soft-drink phosphoric acid. The Science of Total Environment. 302,
253-265.

Scheuhammer, A.M., Norris S.L., 1995. A review of the environmental impacts of lead
shotshell ammunition and lead lishing weights in canada Minister of Environment
Canadian Wildlife Service, Ottawa, Ontario.

Shallari, S., Schwartz, C., Hasko, A., Morel, J.L., 1998. Heavy metals in soils and plants
of serpentine and industrial sites of Albania. The Science of Total Environment.
209, 133-142.

Shu, W.S., Ye, Z.H., Lan, C.Y., Zhang, Z.Q., Wong, M.H., 2002. Lead, zinc and copper
accumulation and tolerance in populations ofPaspalum distichum and Cynodon
dactylon. Environmental Pollution. 120, 445-453.

Singh, S.P., Ma, L.Q., Harris, W.G., Nkedi-kizza, P., 2000. Field demonstration of metal
immobilization in contaminated soils using P amendments, FIPR Proj ect #97-01-
148R. Quarterly Proj ect Report No.6. University of Florida, Gainesville, FL.

Stoltz, E., Greger, M., 2002. Accumulation properties of As, Cd, Cu, Pb and Zn by four
wetland plant species growing on submerged mine tailings. Environmental and
Experimental Botany. 47, 271-280.

Susarla, S, Medina, V.F., McCutcheon, S.C., 2002. Phytoremediation, An ecological
solution to organic contamination. Ecological Engineering. 18, 647-658.

Thomas, C., Eong O.J. 1984. Effects of the heavy metals Zn and Pb on R. mucronata and
A. alba seedlings. In E. Soepadmo, A. M. Rao, & M. D. MacIntosh (Eds.),
Proceedings of the Asian symposium on mangroves and environment; research and
management (pp. 568-574). ISME, Malaysia.

United States Department of Agriculture (USDA), 2000. Heavy metal soil contamination.
Soil quality-Urban Technical note No.3

United States Environmental Protection Agency (USEPA), 1996. Soil screening
guidance, user' s guidance, EPA 540/R96/018. Office of Solid and Emergency
Response, Washington, DC.

United States Environmental Protection Agency (USEPA), 2001a. Lead in human health,
www.epa.gov/superfund/programs/lead/leadht Technical Review Workgroup for
Lead (accessed March 2005)










United States Environmental Protection Agency (USEPA), 2001b. EPA-902-B01-001,
Best management practices for lead at outdoor shooting ranges. United States
Environmental Protection Agency Region 2, Washington, DC.

Vassiliva, M., Azcon, R., Barea, J.M., Vassilev, N., 2000. Rock phosphate solubilization
by free and encapsulated cells of Yarowia~YYY~~~~YYY~~~YYY lipolytica. Process Biochemistry. 35,
693-697.

Verkleij, J.A.C., Schat H., 1990 Mechanisms of metal tolerance in plants. Heavy metal
tolerance in plants-evolutionary aspects. CRC Press, Boca Raton, FL.

Waldron, H.A., 1980. Metals in environment. Academic Press Inc, London.

Wang, Y.M., Chen, T.C., Yeh, K.J., Shue, M.F., 2001. Stabilization of an elevated heavy
metal contaminated site. Journal of Hazardous Materials. 88, 63-74.

Weis, J.S., Weis, P., 2004. Metal uptake, transport and release by wetland plants,
implications for phytoremediation and restoration. Environmental International. 30,
685-700.

Wenzel, W.W., Jockwer, F., 1999. Accumulation of heavy metals in plants growing on
mineralized soils of the Austrian Alps. Environmental Pollution. 104, 145-155.

Yang, J., Mosby, D.E., Castell, S.W., Blanchar R.W., 2001. Lead immobilization using
phosphoric acid in a smelter-contaminated urban soil. Environmental Science and
Technology. 35, 3553-3559.

Youssef, R.A., Chino M., 1989. Root induced changes in the rhizosphere of plants. I. pH
changes in relation to the bulk soil. Soil Science and Plant Nutrition. 35, 461-468.

Zhang, P., Ryan, J.A., 1999a. Formation of chloropyromorphite from galena (PbS) in the
presence of hydroxyapatite. Environmental Science and Technology. 33, 618-624.


Zhang, P., Ryan, J.A., 1999b. Transformation of Pb(II) from cerrusite to chloromorphite
in the presence of hydroxyapatite under varying conditions of pH. Environmental
Science and Technology. 33, 625-630.
















BIOGRAPHICAL SKETCH

Joonki Yoon was born on May 27, 1976, in Seoul, Korea, to ByungKyou Yoon and

SookJae Lee. He attended Yonsei University where he received Bachelor of Science

degree in geology with minors in biology in February of 2002. Upon completion of his

B.S., he j oined the University of Florida in the fall of 2002 to pursue a Master of Science

degree in soil and water Science specializing in remediation of Pb-contaminated soils

with Dr. Lena Ma.