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Chemical Remediation of Copper-Contaminated Soils Using Calcium Water Treatment Residue

Permanent Link: http://ufdc.ufl.edu/UFE0042469/00001

Material Information

Title: Chemical Remediation of Copper-Contaminated Soils Using Calcium Water Treatment Residue
Physical Description: 1 online resource (122 p.)
Language: english
Creator: Fan, Jinghua
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: bioavailability, chemical, copper, sandy, soil, water
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Copper (Cu) contamination to agricultural soils is on the rise worldwide due to extensive use of Cu-containing fertilizers, fungicides, farm manures, and waste disposal in agricultural lands. In south Florida, Cu accumulation in soils under citrus production has been accelerated as a result of increased application of Cu-based fungicides for battling canker disease. In this study, laboratory analysis, greenhouse experiments, and field trials were conducted to investigate the status of soil Cu contamination in south Florida, the effectiveness of chemical remediation using lime-based water treatment residue (Ca-WTR) and the biogeochemical processes controlling Cu availability in Ca-WTR amended soils. Soil samples were collected from 18 representative commercial citrus groves in the Indian River area of South Florida and characterized for Cu chemistry and availability. Total Cu in the surface soils ranged from 4.74 (4 yrs grove) to 228 mg kg-1 (30 yrs grove), with approximately 50% of the soil samples having a total recoverable Cu concentration above or close to the critical Cu level of total Cu: 85 mg kg-1. On average, 48% of the total recoverable Cu was Mehlich-3 extractable, indicating high availability of the soil Cu. The largest proportion (25 to 58% of total Cu) of soil Cu was present as organically-bound. Both total recoverable Cu and Mehlich-3 extractable Cu were significantly correlated with this Cu fraction (P < 0.001). Most of the Cu was accumulated in the surface soil layer (0-15 cm), but vertical leaching occurred in some soils with pH < 6.5. Two typical soils (a Spodosol and an Alfisol with a total recoverable Cu ~ 100 mg kg-1), which are dominant under citrus production in the Indian River area, were selected for remediation studies. The results from incubation studies indicated that amendment of Ca-WTR (pH 9.1, containing mainly CaCO3 and minor CaO) significantly raised soil pH. Water soluble and exchangeable Cu fraction decreased by 62% in the original and by 90% in the Cu-enriched soils (added with 400 mg Cu kg-1), whereas oxides-bound and residual Cu in the soils increased accordingly. Similar results were obtained from column leaching experiments. The cumulative amount of Cu in the leachates from 10 leaching events was reduced by 80% and 73%, respectively for Alfisol and Spodosol at the highest Ca-WTR amendment rates (20 g kg-1 for Alfisol and 100 g kg-1 for Spodosol). Greenhouse studies with ryegrass (Lolium perenne L.) and lettuce (Lactuca sativa L.) as indicator crop plants indicated that the growth of plants was inhibited in the slightly Cu-contaminated soils (~100 mg kg-1), but no plant survived in the severely Cu-contaminated soils (added with 1000 mg kg-1). Amendment of Ca-WTR at 5-20 g kg-1 for the Alfisol (pH 5.45) and 5-100 g kg-1 for the Spodosol (pH 4.66) significantly reduced plant Cu concentrations and thus improved plant growth as evidenced by a significant increase in plant biomass yield (P < 0.01). The effectiveness of Ca-WTR amendment (at three metric ton per hectare) in reducing Cu loading in surface runoff water was evaluated at two commercial citrus grove field sites for three years. The results showed that soil amendment with Ca-WTR generally raised soil pH and Mehlich-3 extractable Ca, but decreased Mehlich-3 extractable Cu. The mean concentration of Cu in surface runoff water was reduced by 35% in the naval orange site and 14% in the grapefruit site during the 2006-2009 period. Ion speciation analysis using the MINTEQ model for the runoff water samples from the amended and control sites indicated that Cu complexes with dissolved organic matter dominated Cu speciation and Ca-WTR application decreased the concentrations of free Cu2+ by 49.6% and 21.2%, respectively for the two sites. The field observation agreed with the results from lab simulation experiments that Ca-WTR was effective in reducing Cu bioavailability and leaching/runoff losses from Cu-contaminated soils.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jinghua Fan.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: He, Zhenli.
Local: Co-adviser: Ma, Lena Q.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042469:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042469/00001

Material Information

Title: Chemical Remediation of Copper-Contaminated Soils Using Calcium Water Treatment Residue
Physical Description: 1 online resource (122 p.)
Language: english
Creator: Fan, Jinghua
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: bioavailability, chemical, copper, sandy, soil, water
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Copper (Cu) contamination to agricultural soils is on the rise worldwide due to extensive use of Cu-containing fertilizers, fungicides, farm manures, and waste disposal in agricultural lands. In south Florida, Cu accumulation in soils under citrus production has been accelerated as a result of increased application of Cu-based fungicides for battling canker disease. In this study, laboratory analysis, greenhouse experiments, and field trials were conducted to investigate the status of soil Cu contamination in south Florida, the effectiveness of chemical remediation using lime-based water treatment residue (Ca-WTR) and the biogeochemical processes controlling Cu availability in Ca-WTR amended soils. Soil samples were collected from 18 representative commercial citrus groves in the Indian River area of South Florida and characterized for Cu chemistry and availability. Total Cu in the surface soils ranged from 4.74 (4 yrs grove) to 228 mg kg-1 (30 yrs grove), with approximately 50% of the soil samples having a total recoverable Cu concentration above or close to the critical Cu level of total Cu: 85 mg kg-1. On average, 48% of the total recoverable Cu was Mehlich-3 extractable, indicating high availability of the soil Cu. The largest proportion (25 to 58% of total Cu) of soil Cu was present as organically-bound. Both total recoverable Cu and Mehlich-3 extractable Cu were significantly correlated with this Cu fraction (P < 0.001). Most of the Cu was accumulated in the surface soil layer (0-15 cm), but vertical leaching occurred in some soils with pH < 6.5. Two typical soils (a Spodosol and an Alfisol with a total recoverable Cu ~ 100 mg kg-1), which are dominant under citrus production in the Indian River area, were selected for remediation studies. The results from incubation studies indicated that amendment of Ca-WTR (pH 9.1, containing mainly CaCO3 and minor CaO) significantly raised soil pH. Water soluble and exchangeable Cu fraction decreased by 62% in the original and by 90% in the Cu-enriched soils (added with 400 mg Cu kg-1), whereas oxides-bound and residual Cu in the soils increased accordingly. Similar results were obtained from column leaching experiments. The cumulative amount of Cu in the leachates from 10 leaching events was reduced by 80% and 73%, respectively for Alfisol and Spodosol at the highest Ca-WTR amendment rates (20 g kg-1 for Alfisol and 100 g kg-1 for Spodosol). Greenhouse studies with ryegrass (Lolium perenne L.) and lettuce (Lactuca sativa L.) as indicator crop plants indicated that the growth of plants was inhibited in the slightly Cu-contaminated soils (~100 mg kg-1), but no plant survived in the severely Cu-contaminated soils (added with 1000 mg kg-1). Amendment of Ca-WTR at 5-20 g kg-1 for the Alfisol (pH 5.45) and 5-100 g kg-1 for the Spodosol (pH 4.66) significantly reduced plant Cu concentrations and thus improved plant growth as evidenced by a significant increase in plant biomass yield (P < 0.01). The effectiveness of Ca-WTR amendment (at three metric ton per hectare) in reducing Cu loading in surface runoff water was evaluated at two commercial citrus grove field sites for three years. The results showed that soil amendment with Ca-WTR generally raised soil pH and Mehlich-3 extractable Ca, but decreased Mehlich-3 extractable Cu. The mean concentration of Cu in surface runoff water was reduced by 35% in the naval orange site and 14% in the grapefruit site during the 2006-2009 period. Ion speciation analysis using the MINTEQ model for the runoff water samples from the amended and control sites indicated that Cu complexes with dissolved organic matter dominated Cu speciation and Ca-WTR application decreased the concentrations of free Cu2+ by 49.6% and 21.2%, respectively for the two sites. The field observation agreed with the results from lab simulation experiments that Ca-WTR was effective in reducing Cu bioavailability and leaching/runoff losses from Cu-contaminated soils.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jinghua Fan.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: He, Zhenli.
Local: Co-adviser: Ma, Lena Q.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042469:00001


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CHEMICAL REMEDIATION OF COPPER-CONTAMINATED SOILS USING CALCIUM WATER TREATMENT RESIDUE By JINGHUA FAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010 1

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2010 Jinghua Fan 2

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To my parents, Guangyi Fan and Shuyu Lin 3

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ACKNOWLEDGMENTS First of all, I would like to thank my adv isor, Dr. Zhenli He, fo r his expert guidance and support. Not only was he readily available for me providing comprehensive instructions in the lab, but he always read and responded to the drafts of my work in time and with patience. He reviewed my multip le versions of this dissertation, and helped me to improve my presentation and cl arify my writing. His comments are always perceptive and helpful, without which this disse rtation would not have been finished. My thanks go out to my co-advisor Dr. Lena Q. Ma and colleges from the Biogeochemistry of Trace Metals group fo r the hospitality and support I had received during my first two semesters of course studies in Gainesvill e. I had benefited a lot from Dr. Mas insightful and pertinent suggestions for my research, especially in writing manuscripts. I would also like to give my sincere than ks to Drs. Peter J. Stoffella, Patrick C. Wilson and Maria L. Silveira for serving on my advisory committee. I am grateful for their advice, assistance and encouragement in every step towards my PhD degree. I deeply express my appreciation to my colleges in Soil and Water Science Laboratory: Drs. Xiaoe Yang, Yuangen Yang, Wenrong Chen, Zhanbei Liang, Yangbo Wang, Yunlong Liu, Xuxia Zhou, Shengke Tian, PhD students Qin Lu, Bruno Pereira, Thiago Nogueira, Santanu Bakshi Alex Merlin, Eloise Mello Mr. Brian Cain, Mr. Douglas J. Banks, Ms. Shaoqin Lu, and Mr. Diangao Z hang for providing useful academic and social assistance. We studi ed and relaxed together. Without their help and friendship, the successful completion of my PhD study would be impossible. I have always felt fortunate to be part of Dr. He s group where I have learned, enjoyed and benefited from team work. 4

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I also appreciate the people on the facu lty and staff at Indian River Research Center of University of Fl orida for providing me wonderfu l environment for both studying and living for three years. Everyo ne from the center is so kind to me that I feel at home in the center. I am particul arly thankful to Dr. Peter J. Stoffella, Ms. Laura McKeon, and Ms. Velma Spencer for their timely and e fficient assistance and arrangement in the student dorm. I am also grateful to Ms. Jackie White for her help with my English language practice. It is lu cky for me to have Dr. Juanju an Qu as a roommate for one year, who taught me not only cooking but also the way to balance the research and family. Last but not least, I would like to thank my dearest parents for their parts in making me who I am today. They will always love me and be my source of strength. My enormous debt of gratitude can hardly be repaid to them for their unconditional support. 5

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ..................................................................................................4 LIST OF TABLES ............................................................................................................8 LIST OF FIGURES ..........................................................................................................9 CHAPTER ABSTRACT ...................................................................................................................10 1 LITERATU RE REVI EW...........................................................................................13 Copper Application in Agriculture ............................................................................13 Copper Concentrations and Distribut ions in Lithosphere and Soils ........................14 Copper Speciation and Behavior in Soils ................................................................15 Potential Impact on Soil Quality and Crop Production ............................................19 Cu Release from Soil and Transport from Land to Aqueous Environment ..............21 Remediation of Cu -contaminated Soils ...................................................................23 Conclusions and Perspectives ................................................................................27 2 ACCUMULATION AND AVAILABILITY OF COPPER IN CITRUS-GROVE SOILS AS AFFECTED BY FU NGICIDE APPL ICATION.........................................30 Introduction .............................................................................................................30 Materials and Methods ............................................................................................31 Soil Collection and Characterization .................................................................31 Soil Cu Analysis and Fractionation ...................................................................31 Data Analysis ...................................................................................................33 Results and Discussion ...........................................................................................33 Copper Accumulation in Relati on to Citrus Planting History .............................33 Effects of pH on Downward Movement of Copper in Soils ...............................34 Copper Availability as Affected by Cu Accumulation and Soil pH .....................35 Soil Cu Fractionation and Availability ...............................................................37 Conclusions ............................................................................................................39 3 IMMOBILIZATION OF CU IN CONTAMINATED SANDY SOILS USING CALCIUM-WATER TREAT MENT RESI DUE..........................................................45 Introduction .............................................................................................................45 Materials and Methods ............................................................................................46 Sampling and Characterization ........................................................................46 Incubation Study ...............................................................................................47 Column Leaching Study ...................................................................................48 Statistical Analysis ............................................................................................49 6

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Results and Discussion ...........................................................................................49 Addition of Ca-WTR Increased So il pH and Decreased Extractable Cu ...........49 Addition of Ca-WTR Converted Labi le Cu to More Stable Cu Forms ...............51 Addition of Ca-WTR Reduced Leachable Cu in Soil Columns .........................52 Conclusions ............................................................................................................55 4 AMENDMENT OF CALCIUM WA TER TREATMENT RESIDUE (CA-WTR) REDUCES CU BIOAVAILABILITY IN CU-CONTAMINAT ED SOILS......................65 Introduction .............................................................................................................65 Materials and Methods ............................................................................................67 Soil Samples and Ca-WTR Collection ..............................................................67 Greenhouse Experiments .................................................................................67 Chemical Analyses of Soil and Plant Samples .................................................68 Statistical Analysis ............................................................................................69 Results and Discussion ...........................................................................................69 Effects of Ca-WTR Amendment on Soil pH and Extractable Cu ......................69 Effects of Ca-WTR Amendment on Plant Cu Concentration ............................71 Effects of Ca-WTR Amendment on Plant Growth/Dry Matter Yields and Cu Uptake ...........................................................................................................73 Effects of Ca-WTR Amendment on Uptake of Other Nutrients .........................75 Conclusion ..............................................................................................................76 5 AMENDMENT OF CALCIUM-WATER TREATMENT RESIDUE (CA-WTR) ON CITRUS GROWTH AND COPPER LOADING IN SURFACE RUNOFF IN SOUTH FLOR IDA...................................................................................................82 Introduction .............................................................................................................82 Materials and Methods ............................................................................................83 Site Description and Amendment Characterization ..........................................83 Sample Collection and Analysis .......................................................................84 Results and Discussion ...........................................................................................87 Soil Quality Characterization and Monitoring ....................................................87 Water Quality and Cu Speciation .....................................................................88 Fruit Nutrient Contents and Yields ....................................................................90 Conclusions ............................................................................................................91 6 SUMMARY, CONCLUS IONS AND PERSPE CTIVE............................................101 LIST OF REFERENCES .............................................................................................107 BIOGRAPHICAL SKETCH ..........................................................................................122 7

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LIST OF TABLES Table page 1-1 Use of copper as a fungicide ...............................................................................29 1-2 Copper concentrations in selected rocks ............................................................29 1-3 Summary of previously reported concentrations of Cu in top soils of agriculture fields .................................................................................................29 2-1 General properti es of the evaluated soils ............................................................40 2-2 Selected chemical properti es and Cu concentration of the soils .........................41 3-1 Physicochemical properties of the soils used in this study ..................................56 3-2 Mehlich-3 extr actable Cu in the soils after 10 leaching events ............................57 4-1 Selected physical and chemical properties of tested soils and Ca-WTR .............77 4-2 Total recoverable soil elements in soils ...............................................................77 4-3 Effect of Ca-WTR am endment on soil pH and extractable Cu ............................78 4-4 Correlation coefficients (r) between plants (ryegrass and lettuce) shoots tissue concentration and soil pH and extrac table Cu as estimated by Mehlich3, and 0.01 M CaCl2 extraction procedures ........................................................79 4-5 Total Cu uptake in shoots of ryegrass and lettuce determined at the end of 8 weeks of growth. Letters indicate significant differences between least squares means within soil at a 95% confidence level (LSD) ..............................79 5-1 Soil selected properties of different field sites befor e initiation of the project ......93 5-2 Mean concentrations of water qua lity related constituents in surface runoff samples from the four field sites from 2006 to 2009 ...........................................94 5-3 Percentage distribution of various Cu species in surface runoff samples from the four field sites ...............................................................................................95 5-4 Selected elemental composition of citrus leaf samples from and the Ca-WTR amended and the control field sites ....................................................................95 5-5 Selected elemental composition of citrus frui t samples from the Ca-WTR amended and the control field sites ....................................................................96 5-6 External and inter nal quality parameters of fruit samples from the Ca-WTR amended and the control field sites ....................................................................97 8

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LIST OF FIGURES Figure page 2-1 Distribution of Cu along soil profiles. ...................................................................42 2-2 Relationship between Mehlich-3 extractable and NH4OAc extractable Cu and total recoverable Cu concentration in soils. ........................................................43 2-3. Rela tionship between CaCl2 extractable Cu and total recoverable concentration Cu in soils. ...................................................................................43 2-4 Copper fractions as a percentage of total recoverable Cu in surface soils. .........44 3-1 Relationship between soil pH and WTR application rates. .................................58 3-2 Changes of Mehlich-3 extractable Cu in soils with incubation time as affected by Ca-WTR application rates.. ............................................................................59 3-3 Relationship between M 3 extractable Cu and soil pH. .......................................60 3-4 Cu Fractionation in soils amended with different Ca-WTR rates.. .......................61 3-5 Changes of water soluble /exchang eable Cu with Ca-WTR application rates.. ...62 3-6 The concentrations of Cu, in leac hate from two soils in ten leaching events ......63 3-7 Cumulative loss of Cu after 10 leaching events as a function of Ca-WTR treatment rates. ..................................................................................................64 4-1 Relationship between plant shoot dr y matter yields after 8 weeks of growth and Ca-WTR treatment rates ..............................................................................80 4-2 Relationship between plants (ryegr ass and lettuce) shoots Cu concentration and Ca-WTR treatment rates.. ............................................................................81 5-1 Dynamic changes of soil pH in the Ca-WTR amended and the control field sites. ...................................................................................................................98 5-2 Dynamic change of Mehlich-3 extractable Cu in soils of the Ca-WTR amended and the control field sites. ...................................................................99 5-3 Citrus fruit yiel d of the WTR amended and the cont rol field sites during the 2006-2009 period. ............................................................................................100 9

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Abstract of Dissertation Pr esented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Doctor of Philosophy CHEMICAL REMEDIATION OF COPPER-CONTAMINATED SOILS USING CALCIUM WATER TREATMENT RESIDUE By Jinghua Fan December 2010 Chair: Zhenli He Co-chair: Lena Q. Ma Major: Soil and Water Science Copper (Cu) contami nation to agricultural soils is on the rise worldwide due to extensive use of Cu-containing fertilizers, f ungicides, farm manures, and waste disposal in agricultural lands. In south Florida, Cu accumulation in soils u nder citrus production has been accelerated as a result of increas ed application of Cu-based fungicides for battling canker disease. In this study, laboratory analysis, greenhouse experiments, and field trials were conducted to investigate t he status of soil Cu co ntamination in south Florida, the effectiveness of chemical re mediation using lime-bas ed water treatment residue (Ca-WTR) and the biogeoc hemical processes controllin g Cu availability in CaWTR amended soils. Soil samples were collected from 18 r epresentative commercial citrus groves in the Indian River area of South Fl orida and characterized for Cu chemistry and availability. Total Cu in the surface soils ranged from 4.74 (4 yrs grove) to 228 mg kg -1 (30 yrs grove), with approximately 50% of the soil samples having a total recoverable Cu concentration above or close to the crit ical Cu level of total Cu: 85 mg kg -1 On average, 48% of the total recoverable Cu was Mehlich-3 extractable, indicating high availability of 10

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the soil Cu. The largest proportion (25 to 58% of total Cu) of soil Cu was present as organically-bound. Both total recoverable Cu and Mehlich-3 extractable Cu were significantly correlated with this Cu fraction ( P <0.001). Most of the Cu was accumulated in the surface soil layer (0-15 cm), but vertical leaching occurred in some soils with pH < 6.5. Two typical soils (a Spodosol and an Alfisol with a total recoverable Cu ~ 100 mg kg -1 ), which are dominant under citrus produc tion in the Indian River area, were selected for remediation studies. The result s from incubation studies indicated that amendment of Ca-WTR (pH 9.1, containing mainly CaCO 3 and minor CaO) significantly raised soil pH. Water soluble and exchangeable Cu fraction decreased by 62% in the original and by 90% in the Cuenriched soils (added with 400 mg Cu kg -1 ), whereas oxides-bound and residual Cu in the soils in creased accordingly. Similar results were obtained from column leaching experiments. The cumulative amount of Cu in the leachates from 10 leaching events was reduced by 80% and 73%, respectively for Alfisol and Spodosol at the highes t Ca-WTR amendment rates (20 g kg -1 for Alfisol and 100 g kg -1 for Spodosol). Greenhouse st udies with ryegrass ( Lolium perenne L .) and lettuce ( Lactuca sativa L .) as indicator crop plants indicated that the growth of plants was inhibited in the slightly Cu-contaminated soils (~100 mg kg -1 ), but no plant survived in the severely Cucontaminated soils (added with 1000 mg kg -1 ). Amendment of Ca-WTR at 5-20 g kg -1 for the Alfisol (pH 5.45) and 5-100 g kg -1 for the Spodosol (pH 4.66) significantly reduced plant Cu concentrations and thus improved pl ant growth as evidenced by a significant increase in plant biomass yield (P<0.01). 11

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The effectiveness of Ca-WTR am endment (at three metric ton per hectare) in reducing Cu loading in surface runoff wate r was evaluated at two commercial citrus grove field sites for three years. The re sults showed that soil amendment with Ca-WTR generally raised soil pH and Mehlich-3 extractable Ca, but decreased Mehlich-3 extractable Cu. The mean concentration of Cu in surface runoff water was reduced by 35% in the naval orange site and 14% in the grapefruit site during the 2006-2009 period. Ion speciation analysis using the MINTEQ model for the runoff water samples from the amended and control sites indi cated that Cu complexes with dissolved organic matter dominated Cu speciation and Ca-WTR application decreased the concentrations of free Cu 2+ by 49.6% and 21.2%, respectively for the two sites. The field observation agreed with the results from lab simulation experiments that Ca-WTR was effective in reducing Cu bioavailability and leaching/runoff losses from Cu-contaminated soils. 12

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CHAPTER 1 LITERATURE REVIEW Copper Application in Agriculture Copper compounds were the first fungicide used for agricultural crops, starting in the vineyards of Bordeau, France in 1885. The mixture of Cu and lime (Bordeaux mixture), applied to vines to control downy mildew, was the basis of a wide range of commercial Cu formulations and represented the beginning of chemical application in crops. Since 1920, a number of inorganic and organic Cu fungicides have been developed and used in horticulture and agriculture They include basic copper sulfate, copper hydroxide, and copper oxychloride, copper carbonate, copper ammonium carbonate, copper naphthenate, copper oleate, copper oxide, copper 8-quinolinolate, copper salts of fatty and rosin acids, and copper-zinc sulfate complex (Gianessi and Puffer, 1992). Copper compounds have been the key chemicals in numerous fungal and bacterial diseases as foliar sprays in many crops. Their use, over the past 100 years, has contributed to substantial improvement in yields and qua lity of many horticultural crops such as citrus, almonds, wal nuts, tomatoes, peaches, peanuts, peppers, and beans (Delas, 1963; Epstein and Bassein, 2001; Reuther and Smith, 1953; Reuther et al., 1952). Copper compounds are recommended fo r the control of brown rot, greasy spot, melanose, blast, brown leaf spot, sheath blight, shothole, anthracnose, bacterial canker, speck and spot, early and late blight, leaf curl, cercospora leaf spot, rust, bacterial leaf spot, frogeye spot, common blight, halo blight, walnut blight, and downy and powdery mildew (Gianessi and Puffer, 1992). However, repeated use of Cucontaining fungicides, pesticides and herbicides resulted in Cu accumulation in soils 13

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and negatively impacted on the environment (He et al., 2004; Van-Zwieten et al., 2004). To control heavy metal contamination of soil and food, the USEPA and the European Union have established maximum heavy metal limits for soil. The current soil cleanup criteria for Cu in the USA is 600 mg kg -1 (USEPA, 1999), and the upper limit of total soil Cu set by the European Union for receiv ing Cu-containing sewage sludge is 140 mg kg 1 (Department of Environment, 1993). Copper accumulation in soil is mostly resulted from anthropogenic inputs. The use of Cu as fungicides has been a continuing source of soil Cu pollution, especially in the tropical and subtropical regions. In the United States, the annual use of Cu fungicides is estimated at 4.5 million kilograms. Copper us e is heavily concentrated in the fruit, nut, and vegetable producing regions, including Cali fornia and Florida, which account for 85% of copper use in agricultural fungicides (Gianessi and Puffer, 1992) (Table 1-1). Citrus production ranked number one in Cu us e, which accounts for 25% of total Cu use in the US crop production. These f ungicides are used to manage various citrus diseases including brown rot, melanose, ci trus scab, and greasy spot (Mozaffari, 1996). No other alternative pesticide is as effectiv e as Cu to treat citrus melanose (Gianessi and Puffer, 1992). Compared with disinfectants and antibiotics, which induce resistance in plants, Cu products were more effective in canker control, especially in preventing canker infection of fruit (Timmer et al., 2006; Willi ams et al., 2006). Copper Concentrations and Distributions in Lithosphere and Soils For natural soils, Cu is inherited from parent materials. Baker and Senft (1995) reported 70 mg kg -1 as the average Cu concentration in the lithosphere. Different types of rocks contain different Cu concentrations (Table 1-2). 14

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Soils differ widely in Cu concentrations depending on parent materials, land use, and distance from pollution sources (Reimann and Caritat, 1998). For world soils, Cu concentrations in uncontaminated so ils range from 2 to 30 mg kg -1 which falls in the lower end of concentration range in rocks (Adri ano, 1986; Ma et al., 1997; Wang et al., 2003). In soils, Cu is associated with organic matter, oxides, clays and other silicate minerals. Copper shows high mobility in ac id and oxidizing environments, while it has limited mobility in reducing and alkaline environment s. Therefore, Cu is relatively mobile among heavy metals during t he hypergenic processes. Copper Speciation and Behavior in Soils Since most of the applied Cu is reta ined in soil, total soil Cu increases proportionally with the age of agricultural pr oduction resulting from Cu fungicides application (Mirlean et al. 2007). Although the annual contribut ions are small, long-term production results in high Cu concentration in soils, which is significantly greater than soils with native vegetation (He et al., 2006b; Wightwick et al., 2008). Total Cu concentrations in top soils under agricultura l use in various countries are presented in Table 1-3. When determining potential risks associated wit h soil contamination, total Cu in soil is a poor measure. This is because Cu is reactive and interacts with other soil components in soil. Copper compounds from agricultural sources have higher water solubility than those from smelting and mi ning sources (Sauve et al., 1997; Sauve, 2002). Bioavailability and potentia l toxicity of Cu in soil and its ecotoxicological significance are determined by its chemical forms and association with various soil components, and by solid-solution equilibrium at the solid-water interface. These chemical processes, such as precipitat iondissolution, adsorptiondesorption, and 15

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chelation, depend on soil properties the nature of metal associ ations with other soluble ionic species and organic matter, plant and microbiological processes, and environmental factors (Carillo-Gonzal ez et al., 2006; Yu et al., 2002). Plant-availability of Cu in soils is la rgely influenced by the free activity of Cu 2+ in the soil solution. Soil pH is critical in many chemical processes controlling Cu availability, including precipitation and chemisorptions (Alva et al., 2000). Increasing soil pH increases the proportion of insoluble Cu such as Cu hydroxide, carbonate and phosphate (Alva et al., 2000). Three types of soluble complexes can be formed between metal ions and ligands: ion pairs, soluble metal-organic ligand complexes, and chelation. Soil pH also affects ion pairs, complex formation, surface charge, and organic matter solubility. Cu-based antifungal treatment can increase the local concentration of soluble Cu in acid soils but to a less extent in moderately ac idic or neutral soils. Sauve et al. (1998) concluded that the relation between soil solution Cu activity (pCu= -log [Cu]), pH, and total Cu concentration (Cu T ) can be expressed as: pCu = 3.42 + 1.4 pH 1.7 log Cu T (n = 64, r 2 = 0.85) (1) The well-known high affinity of copper for organic matter has two consequences. In the presence of dissolved organic matter (OM), Cu mobility increases as Cu form organic complexes (Hsu and Lo, 2000). Higher Cu mobility and availability to earthworms was reported in soil amended with sewage sludge (Kizilkaya, 2004). On the other hand, particulate OM, by virtue of its high CEC, can effectively adsorb Cu ion. High molecular weight organic compounds can al so bind and strip Cu from the solution, because they can be insoluble and therefor e, semi-immobile (M oolenaar et al., 1998; Spark et al., 1997). For instanc e, Temmingghoff et al. (1998) reported that humic acids 16

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enhanced Cu mobility, but the process was st rongly affected by Ca concentration and pH of the soil solution. Soil pH and organi c matter accounted for approximately 70% of the variability in Cu partitioning and 80% of th e variability in bioavailable Cu in 40 soils collected from the USA, Canada, and UK (Impe lliteri et al., 2003). McBr ide et al. (1997) reported the following equation for Cu, wh ich takes dissolved OM into account: pCu= 1.28+ 1.37 pH 1.95 log Cu T + 1.95 log OM (n = 68, r 2 = 80) (2) Clay content and soil structure also affect soil Cu retention. Clay-rich soils generally have higher retention c apacity than soils with minimal or no clay. Selectivity of Cu 2+ adsorption varies with clay minerals. Vermiculite is more effective in adsorbing Cu 2+ than montmorillonite, apparently due to mo re specific adsorption sites (Malla, 2002). Abd-Elfattah and Wada (1981) observed t he following selective adsorption for Cu ion: Fe-oxides, Halloysite, Imogolite > Humus, Kaolinite, Allophane > Montmorillonite. In extremely reducing envir onment, it may occur as metallic metal (Cu 0 ). But in most cases, Cu solubility is influenced indirectly by ot her processes. As stat ed above, Cu associates with Mn oxides through co-precipitation and subs titution. When Mn is reduced, this part of Cu will be released (Reimann and Caritat, 1998). Parat et al. (2002) showed that Fe contributed to the bind ing of Cu through its inclusion in Fe oxyhydroxides by sequential extraction procedure. As Fe is the main agent in the formation of clayhumus complexes, it could also be indirectly responsib le for most of the Cu present in the clay fraction. However, clay content and redox pot ential are less important in affecting Cu release than soil pH, organic matter, and Cu speciation in agricultural soils. Many chemical and biochemical processes are involved in the partition of heavy metals between the solid and liquid phase in a soil (Islam et al. 2001). A number of 17

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chemical equilibrium models are available to predict solution phase speciation of Cu (Cancs et al. 2003; Tipping et al. 2003; W eng et al., 2002). All these models have the database of parameters for the sorptiondesorption and dissolution-precipitation reactions of metals (Hayes and Traina, 1998 ). Chemical fractionation, which is determined by sequential extraction using se lective chemical reagents, has proven useful in investigating the metal fractionation in soils an d the result can support the hypotheses derived from models. Water soluble and exchangeable fractions are considered to be bioavailable, oxide-, carbonate-, and organic matter-bound fractions may be potentially bioavailable, and the residual fraction is mostly unavailable to either plants or microorganisms (Shuman, 1991). Delineating these metal fractions in soils is considered essential for understanding Cu availability and mobility in soils. Recent advancements in microscopic and molecular-scale tools and data analysis have made it possible to investigate trace element specia tion in natural samples (Strawn and Baker, 2008). These powerful modern techniques su ch as X-ray absorption fine structure (XAFS) provide precise determinati on of Cu fractionation in soil. In addition to chemical fractionation, anot her approach to study relations between Cu and soil constituents and how Cu is disse minated in the environment by runoff is physical fractionation. Over the last decade, these methods were used successfully for studying soil organic matter dynamics (Christens on, 1992). More recently, particle size fractionation has been used to analyze the distribution of metal elements among different soil particles. Flores-Velez et al (1996) studied the ac idic vineyard soils contaminated by Cu and emphasized the import ance of the coarse plant residue larger than 50 m forming the particulate organic matter (POM) fraction for the retention of 18

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metals. Research of Besnard et al. (2001) in champagne vineyard so ils showed that two fractions were mainly responsible for Cu rete ntion in soils, i.e., the clay-size fraction less than 2 m and the organic debris larger than 50 m or coarse POM. The POM contained up to 2000 mg Cu kg -1 and the clay fraction contained up to 500 mg Cu kg -1 The most Cu-contaminated pl ots showed the highest accumulation of organic carbon and Cu in the coarse sand and fine sand fractions. Zhang et al. (2003b) found Cu retention in various fractions in Florida sandy soils under commercial citrus production tends to increase with decreasing aggregate size, suggesting that surface retention mechanisms control the distribution of Cu among different size fractions, and they are readily transported to surface waters through suspended fine particles. Potential Impact on Soil Quality and Crop Production Excessive Cu has potential negative effect s on soil flora and fauna. Copper toxicity to citrus has been studied since the early stage of last centur y. Westgate (1952) reported Cu problem in old vegetable fields that had received years of Cu sprays with the symptoms of plants having brown, stubby roots, iron chlorosis of the leaves, and a general stunting. Ford (1953) r eported that where t here were high Cu concentrations and a pH below 5.0 citrus feeder roots of seve rely iron deficient trees were ashy gray, rather shriveled, and highly corked with no visi ble evidence of an actively growing root tip. Copper precipitation is the basis for t he decrease in Cu toxicity with an increase in soil pH. Leonard and Stewart (1952) stated that Cu is less available to plants on limed than unlimed soils. They reported that grapefruit leaves from the plots with soil pH at 4.0 had 76 mg kg -1 Cu but where the pH was 5.6 the leaves contained only 14 mg kg -1 Reuther et al. (1953) c oncluded that toxicity effect of high Cu concentration in soils of high exchange capacity was not as great as in soils with a low exchange capacity. They 19

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also reported that t he negative effect of high Cu was less in old grove soils than in a virgin soil as high phosphate concentrations in old grove soils reduced Cu toxicity. Particularly in sandy acidic soil, 100 to 150 mg total Cu kg -1 (Alva et al., 1993) or 20 mg kg -1 of either diethylenetriami ne pentaacetic (DTPA) or 0.1 M HCl extractable Cu (Walsh et al., 1972) decreased productivity of Cu-sensitive crops. Bacteria, fungi, and mollusks are generally t he most sensitive to Cu, as compared with flowering plants and vertebrate animals (Giller et al., 1998). Soil respiration was reduced in soil amended with 50 mg Cu kg -1 (Chang and Broadbent, 1981). Lukas et al. (2004) concluded that appr oximately 300 Cu mg kg -1 were responsible for significant reductions in microbial biomass carbon ev en though the orchard soils had similar or elevated levels of total organic carbon. The ratio of microbial biomass carbon to soil total organic carbon, defined as microbial quotient, a measure of soil organic matter quality, was significantly lower in Cu-contam inated soils. Solutions containing 1 mg Cu L -1 are toxic to fungal spores. The mycorrhizal fungus Glomus mosseae colonized fewer onion ( Allium cepa L.) roots in soil amended with 15 mg Cu kg -1 (Gildon and Tinker, 1983) and G.intraradices colonized fewer citrus roots in soil amended with 34 mg Cu kg 1 (Graham et al., 1986). Concentrations of 80 to 110 mg Cu kg -1 in orchard and other soils reduced earthworm populations (Edw ards and Bohlen, 1996). Earthworms ( Eisenia feuda ) in soils containing 53 mg Cu/kg DW showed a 50% reduction in cocoon production in 56 d (Spurgeon et al. 1994). Concent rations for toxic Cu levels may vary, which depend on several factors including organisms, acute or chronic toxicity, extraction method, and soil characteristics such as pH, exchange capacity, and organic matter (Driscoll, 2004). 20

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Cu Release from Soil and Transpor t from Land to Aqueous Environment Increased Cu concentrations were detected in waters and sediments, which may have negative impact on water quality and ecosystem functions (He et al., 2006a; Hoang et al. 2008; Ribolzi et al., 2002). Transpor t of Cu from land to surface waters through surface runoff and/or leaching has b een suspected to be one of the nonpoint sources. The concentrations and loads of Cu in runoff water from agric ultural fields were related to soil Cu accumulation due to r epeated use of Cu-containi ng fungicides (Zhang, et al., 2003a). The applied Cu fungicide is only partially trapped in the soil surface because of its transport to aquatic environment through surface runoff and leaching. The inputs of Cu into surface water from agricultural produc tion systems is a potential problem since the aquatic organisms are more sensitive to Cu. High Cu concentrations can cause a significant decrease in populations of aquat ic invertebrates and fish. In aquatic invertebrates, Cu disrupts gill epithelium at hi gh concentrations and in fish, it interferes with osmoregulations; death is caused by tissue hypoxia associated with disrupted ATP synthesis (Eisler, 1998). Adverse sublethal e ffects of Cu on behavior, growth, migration, and metabolism occur in different species of fishes at nominal water concentrations as low as 4 mg L -1 (Eisler, 1998). The sensory ph ysiology and predator avoidance behaviors of juvenile coho were both signific antly impaired by Cu at concentrations as low as 2 g L -1 (Sandahl et al., 2007). Copper can c ause demise of trout and other fish, which are acid-sensitive, especially in soft or acidic waters. Cupric ion (Cu 2+ ) is the most readily available and toxic inorganic species, although it accounts for <1% of total Cu in the surface water body (Eisler, 1998). There is an environmental caution in Cu fungicide 21

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labels produced in U.S, suggesting toxici ty to fish and aquatic organisms and phytotoxicity to Cu-sensitive cultivars. In recent years, Cu concentrations as high as 300 mg kg -1 have been measured in the sediments of the St. Lucie Estuary, an important surfac e water body in South Florida (He et al, 2004; 2006), which is 15 times greater than its background level (20 mg kg -1 ). The high Cu concentrations are suspected to pot entially affect fish health in the Estuary and the Indian River Lagoon. Florida Departm ent of Environment Protection (FDEP) (1999) reported that Cu concentrations exceeded Class III standards in two citrus runoff water samples and has attracted public att ention. Zhang et al. (2003a) studied the dissolved Cu losses in runoff in Florida sandy soils under commercial citrus and vegetable production and the re lationship between soil-extractable Cu forms and dissolved Cu concentrations in runoff water. They found that mean dissolved Cu in the field runoff water was signific antly correlated with the extr actable Cu by 0.01 M CaCl 2 The results indicate that 0.01 M CaCl 2 extractable Cu can be us ed to predict readily released Cu in sandy soils. This may be due to the fact that the 0.01 M CaCl 2 test is the closest approximation of the pool of Cu in the soil that is susceptible to leaching loss. Apparently, the Cu in the runoff came mainly from the water-soluble fraction in soil, which was extractable with the 0.01 M CaCl 2 During intense surface runoff and erosi on processes, Cu can be predominantly transported by suspended particles in runoff wa ter (Gilbin et al., 2000; Xue et al., 2000), which can be related to the affinity of Cu to some components of suspended sediments like organic matter, clay minerals and hydr ous metal oxides (Flemming and Trevors, 1989). Ribolzi et al. (2002) studied the specia tion and origin of particulate Cu in runoff 22

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water from a Mediterranean viney ard catchment. Their research indicated that fungicide treatments in vineyards can l ead to contamination of a large area of surface water by particulate copper during storm events. The mean Cu concentration of the suspended particles was 245 mg kg -1 of which 1% was exchangeable, 4% acid-soluble, 10% oxidizable, 23% reducible, and 63% residual, which indicated the importance of soil erosion during a storm flow. The residual Cu fraction, i.e., the one firmly bound to the minerals since it is incorporated in the cryst alline structure, constitutes the essential element of particulate Cu in t he catchment. Since the residual Cu is not very soluble it is therefore not likely toxic to organisms. Howeve r, the reducible fraction, which is mainly associated with iron (Fe) and manganese (Mn) oxides, may cause environment impacts, since the reducible fraction is particularly sensitive to reducing conditions and can therefore be solubilized in the aquatic ecosystems. Remediation of Cu-contaminated Soils To address the potential Cu contamination at agricultural sites, In situ stabilization is advantageous over other remediation methods as it is low-cost and less disruptive to agricultural production. The stabilization tec hnique, which reduces element mobility in soils by adding chemical agents is termed as chemical stabilization, is effective for a wide range of polluted sites (Alvarenga et al., 2009; Bolan and Duraisamy, 2003). Chemical stabilization in soil can be achieved by adding ameliorants able to adsorb, complex, or precipitate contaminants. The goal is to reduc e elemental phytotoxicity to the crops and element release to the aquatic environment by decreasing their mobility and bioavailability (Kumpiene et al. 2007). Stability of Cu in soil is strongly pH depen dent the mobility of Cu is usually the lowest at slightly alkaline pH but increases under highly alkaline conditions (pH>10) due 23

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to the formation of OHcomplexes (van der Sloot et al., 1997). Application of coal fly ash to soil, which usually increases soil pH a nd the amount of carbonates, is efficient for controlling Cu mobility (Jackson and Miller, 2000). However, in the same study, increasing soil pH to above 8 by the application of coal fly ash (15% by weight) and red mudgypsum (15% by weight) showed less t han 50% and 10% efficiency, respectively. Soil organic matter plays a dual role in controlling Cu solubility. Peat and high organic soils can retain significant amounts of Cu at lower pH while Cu mobility increases as Cu humic acids (HA) and Cu fulvic acids (FA) complexes are formed in the presence of dissolved OM, especially at high pH (Kabata-Pendias and Pendias, 2000). Recent studies indicate that the mobility and availabili ty of Cu can be reduced by OM applications. Kiikkila et al. (2002) incorporated a range of organic amendments including woodchips and bark chips mixed with compost, sewage sludge, leaf material, and peat into a metal polluted forest soil. They reported that compost, compost mixtures, and sewage sludge prov ided new exchange sites for cations as well as other binding sites in the soil without affecting microbial community. It is assumed that the added organic matter provides a dditional sites for binding metals and this process will continue as long as the organic matter is not decomposed. Diaz-Barientos et al. (2003) reported an increased retention of Cu in a sandy soil amended with either olive mill wastewater or urban compost. Acid extracta ble Cu fraction decreased in soils amended with four different mixtures of sewage sludge and cotton waste, and composting of sewage sludge prior to the application was recommended (Sanchez-Monedero et al., 2004). Zhang et al. (2004) investigated the solubility and fractionation of Cu, Cd, Ni, Pb, Zn from a potting soil that was amended with 0 to 100% yard-waste compost. Both 24

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water solubility and sequential extraction studies revealed that concentrations of watersoluble heavy metals were very low. Application of fly-ash stabilized sewage sludge also reduced Cu leaching and availability to corn and significantly in creased dry mass of corn (Su and Wong, 2004). Mixing organic matter and fly ash can offset a decrease in soil pH due to the decomposition of OM (J ackson and Miller, 2000). As assessed by a batch test, the amount of leached Cu decr eased by 98.2% and Pb by 99.9% in soil induced by the addition of coal fly ash and natural organic matter ( peat). Metal leaching from the treated soil was lowe r by two orders of magnitude compared to the untreated soil in the field lysimeters (Kumpiene et al., 2007). Clay minerals have also been tested as an amendment to stabilize soil polluted with Cu. Soil treated with palygor skite (>4%) could decrease Cu mobility by 59% after the percolation of a water volume corres ponding to the annual rainfall and greatly reduce the risk of groundwater pollution (Alvarez-Ayuso and Garcia-Sanchez, 2003). The suggested mechanism of Cu retention is the reaction with silanol groups, which is consistent with the facility of cations to hy drolyze. Industrial by-products like sugar foam, dolomitic residue and to a lesser extent, gypsum and phosphogypsum, can also reduce the mobility and availability of Cu in soil (Garrido et al., 2005). The addition of Ca caused the release of the mo st exchangeable fraction to the soil solution and the precipitation as metal carbonat es, oxy-hydroxides or both, especially at the increased favorable soil pH. In addition, the formation and retention of Fe and Al-hydroxy polymers also increased the metal sorption capac ity of the soils. Lot henbach et al. (1997) reported Cu immobilization by montmorillonite, but a subsequent field experiment with 25

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clay-rich gravel sludge (Lothenbach et al ., 1999) showed that soil pH limited the increases in available Cu in the treated soils. Minimal research was conducted on Fe, Al and Mn oxides amendment. McBride and Martinez (2000) reported that both Mn ox ides and Fe hydroxides with large surface areas significantly reduced free Cu 2+ activity in soil solution, probably due to changes in dissolved organic carbon (DOC) concentration caused by amendments-induced changes in soil pH. However, Hartley et al. (2004) tested several iron amendments, such as goethite, iron grit (Fe( 0)), iron(II)/(III) sulfates pl us lime, but they were not effective for Cu stabilization. Phytoremediation is the use of special and engineered metal-accumulating plants for extraction or immobili zation of metals in soils (Baker et al., 1994). Yoon et al. (2006) evaluated the potential of 36 plant s (17 species) growing on a site where total Cu varied from 20 to 990 mg kg -1 in North Florida. No hyperaccu mulator species were identified, which are suitable for phytoextraction. However, some plants with a high bioconcentration factor (BCF, metal concentra tion ratio of plant roots to soil) and low translocation factor (TF, me tal concentration ratio of pl ant shoots to roots) have a potential for phytostabilization. Some plants that can extract heavy metals from soils and concentrate them in their harvestable pa rts are recognized as hyperaccumulators. Chinese native herbs E. splendens, growing dominantly on old Cu-mine deposit, has been identified as a Cu tolerant and accumulating plant species in the mined areas (Yang et al., 2005). It has a local nicknam e copper flower because it grows only on highly Cu-contaminated soils. In a field of a mined area, this pl ant accumulated 2,288 mg kg 1 Cu in the roots and 304 mg kg 1 Cu in the shoots. Phytoextraction using E. 26

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splendens can effectively reduce the plant-availabl e Cu level in the polluted soils and the shoot biomass yield of this plant as high as 11,000 kg ha 1 can be achieved when grown in the field with favorable water and nut rient supplies. These characteristics make the plant a great candidate for the phytoremediation of Cu-contaminated soils (Jiang et al., 2004). Phytoextraction by hyperaccumula tors has also been proposed for soils with low to moderate Cu concentrations. Mura kami and Ae (2005) found there is a great potential for Cu phytoextraction by the Gold Dent maize and the Milyang 23 rice from paddy soils with low to moderate contam ination under aerobic soil conditions. Conclusions and Perspectives Copper functions as an essential micronutrient to plants, but is toxic when present in excess amounts. Soil pollution with Cu has become an increasingly concerned challenge due to the continued dependence of Cu fungicides to control plant diseases. Bioavailability and potential toxicity of Cu in soil and its ecotoxicological significance are determined by the specific fo rms and solid-solution equilibrium of Cu at the solid-water interface, which depends on the soil properties, the nature of Cu associations with other soluble ionic species and organic matter, plant and microbiological processes, and environmental factors. Soil acidification, ofte n caused by fertilization or acid deposit, can greatly enhance Cu activity in soils, thus exacerbating it s phytotoxicity. Generally, copper input from agricultural prac tice can be a significant source of Cu pollution to surface waters. Problems arise from runoff following fungicide applications. The majority of particulate Cu is associated with the residual fraction, which is not very soluble and is therefore not likely to be to xic to organisms. The dissolved Cu released from soils can be predicted by the extrac tion method. Modern analytical techniques allow for precise determination of the binding of Cu to soil components. This facilitates 27

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better risk analysis and improved remediation strategies, and helps explain results from recent studies that have investigated Cu mobility and bioavailability in the soil Chemical stabilization mostly achieved by raising soil pH or adding organic ameliorants can reduce Cu phytotoxicity to crops and release to the aquatic environment by decreasing mobility and bioavaila bility of Cu. Phyt oremediation of soil Cu has achieved some progress in rec ent years and appears promising as a costeffective approach. However, hyperaccumulators still have several shortcomings to be introduced into fields contaminated with low to moderate toxic-metals. Further studies are needed to obtain more information on phyto remediation for the development of best management practices to control the availability and mobility of Cu in soils. 28

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Table 1-1. Use of copper as a fungicide (Gianessi and Puffer, 1992) Crop Treated area (ha) Rate (kg/ha) Total quantity (kg) Citrus 221,433 2.1 1,158,629 Rice 59,774 4.9 718,205 Almond 96,926 2.0 488,872 Walnut 69,972 2.6 445,376 Tomato 81,758 1.8 370,392 Peach 28,112 7.6 212,717 Peanut 49,263 3.4 166,712 Grape 38,484 2.6 99,613 Others 256,277 844,690 Total 901,998 4,505,207 Table 1-2. Copper concentrations in selected rocks (Reimann and Caritat, 1998) Rock type Cu concentration (mg kg -1 ) Gabbro, Basalt 90 Shale, Schist 45 Ultramafic rock 40 Greywacke 24 Coal 20 Granite, Granodiorite 12 Limestone 6 Sandstone 2 Table 1-3. Summary of previously report ed concentrations of Cu in top soils of agriculture fields Cu concentration (mg kg -1 ) Country mean range Crops Years of Cu use References France 458 100 grapes NR a FloresVelez et al., 1996 Italy 297 NR grapes NR Delusia et al., 1996 Germany 1280 NR grapes NR Tiller and Merry, 1981 Spain 144 40 grapes >100 Arias et al., 2004 Brazil 2198 1214 grapes 100 Mirlean et al., 2007 Australia 73 24 grapes 40 Wightwick et al., 2008 US NR 67 citrus >100 He et al., 2006b Australia NR 103 orchard 255 Zwietena et al., 2004 Costa Rica. NR 20 Banana >100 Thrupp, 1991 a NR, not reported. 29

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CHAPTER 2 ACCUMULATION AND AVAILABILITY OF COPPER IN CITRUS-GROVE SOILS AS AFFECTED BY FUNGICIDE APPLICATION Introduction Florida is well known for its citrus produc tion. In the last two decades, increased amounts of Cu-containing fungicides have been used to treat canker diseases, especially for grapefruit trees (Timmer et al ., 1998; Stover et al., 2002). As a result, significant amounts of Cu have been accumulated in the soils, causing phytotoxicity and accelerating transport to the environment, parti cularly in low pH soils. Hence, it is important to monitor the extent of Cu a ccumulation in soils under citrus and other horticultural crop production. In soil, Cu is subject to various chemic al reactions including sorption on colloidal surfaces, complex with humic substances and precipitation as sparingly soluble compounds (Alloway, 1995; Sauve et al., 1997) Several one-step extraction methods can be used to determine available Cu in so il, including Mehlich-3, Mehlich-1, CaCl 2 NH 4 OAc, and HCl (Brun et al., 2001; Yu et al., 2002; Zhang et al., 2003a). These procedures allow defining pools of extractable metals in particular chemical conditions. Sequential extractions are used to predict the chemical fractionation of Cu in soils, which can help understand Cu behavior in soils and subsequently the mobility and availability of Cu to the environment (He et al., 2006; Pi etrzak and McPhail, 2004; Yang et al., 2005). Although significant research has been conducted on the transformation and availability of Cu in soils, lim ited information is available in citrus soils. This study used representative soils under ci trus production in Florida to improve understanding of the impacts of agricultural practices on Cu dynamics in soil. The specific objectives of this 30

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study were to a) estimate and compare Cu av ailability in soils using various extraction procedures, and b) understand the processes and soil properties that control Cu availability and mobility in sandy soils. Materials and Methods Soil Collection and Characterization Eighteen soils (9 Alfisols and 9 Spodosol s) were collected from representative commercial citrus groves in the Indian River area, south Florida. They were selected based on their use of Cu-based fungicide, citrus cultivar and planting history, and soil properties. General characterist ics of the study sites are presented in Table 2-1. For each location triplicate samples were randomly collected at 0-15 cm depth, with 3 soil cores being combined to make a composite sa mple. To study the vertical movement of Cu, profile soil samples were collected at 0-5, 5-15, 15-30 cm and then every 30 cm increment up to 120 cm depth. A total of 6 representative prof ile samples were collected. The soil samples were air-dried, ground, and passed through a 2-mm sieve prior to physical and chemical analyses. Soil pH (1 :1 soil: water ratio) and electrical conductivity (1:2 soil: water ratio) we re measured in deionized water using a pH/ion/conductivity meter (Denver Inst rument, CO). Total soil carbon (C) was determined by combustion using a C/N anal yzer (Vario MAX CN Macro Elemental Analyzer; Elemental Analysensys tem GmbH, Hanau, Germany). Soil Cu Analysis and Fractionation Total recoverable Cu in the soils wa s determined following EPA method 3050B. In brief, 1.0 g of soil was digested with repeated additions of nitric acid (HNO 3 ) and hydrogen peroxide (H 2 O 2 ). Labile Cu in the soils was estimated by three extraction methods: (i) 0.01 M CaCl 2 [1:10 ratio of soil/0.01 M CaCl 2 ]; 60-min reaction time (Zhang 31

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et al., 2003a), extracting water-soluble and readily exchangeable Cu; (ii) 1 M ammonium acetate (NH 4 OAc) [1:4 ratio of soil/1 M NH 4 OAc solution]; 60-min reaction time (Reed and Martens, 1996), including water soluble and exchangeable Cu; and (iii) Mehlich-3 [1:10 ratio of soil/Mehlich-3 solution (0.2 M CH 3 COOH + 0.25 M NH 4 NO 3 + 0.015 M NH 4 F + 0.013 M HNO 3 + 0.001 M EDTA, pH 2.0)]; 5-min reac tion time (Mehlich, 1984), including water soluble, exchangeable, and part of CaCO 3 and organically-bound Cu. At the end of extractions, the soil sus pensions were centrifuged at 7500 g for 30 min and the supernatant was passed through a What man #42 filter paper to remove any suspended materials. To understand the relationship between chemic al fractions and availability of Cu, soil Cu was fractionated into five different fractions following the modified procedure of Amacher (1996). Soil samples (2.0 g) were sequentially extract ed with 20 mL of 0.1 M Mg(NO 3 ) 2 20 mL of 1 M NaOAc, 40 mL of 0.1 M Na 4 P 2 O 7 and 40 mL of 0.2 M ammonium oxalate + 0.2 M oxalic acid, and 0.1 M ascorbic acid (pH 3) for water soluble and exchangeable, carbonate-bound, organically bound, and ox ides-bound Cu fractions, respectively. After eac h extraction, the suspension was centrifuged at 7500 g for 30 min, and the supernatant was filtered through a Whatman # 42 filt er paper. The soil residues were rinsed three times with 5 ml of ethanol and evaporated to dryness before next extraction. Residual Cu wa s calculated by subtracting th e sum of the four fractions from their total recoverable soil concentration. Copper concentrations in the extracts and digested solution were determined using an inductively coupled plasma optical emission spectrometer (ICPOES) (Ultima, JY Horiba Group, Edison, NJ) following EPA method 200.7. The National Environmental 32

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Laboratory Accreditation Conference (NELAC ) 2003 standards were followed for quality assurance and quality control of chemical an alyses. For Cu analysis with ICP-OES, the detection limit was 0.8 g L -1 with a recovery of 95-105%. Data Analysis All data were analyzed using SAS program (version 8.2, SAS Institute, 2004). Correlation analyses were conducted between total recoverable Cu, extractable Cu concentrations and different Cu fractions. Diffe rences in the citrus soil Cu concentration among the sites were tested by means of a one-way analysis of variance (ANOVA). Tukeys test was performed to determine significant differences among the soils. Stepwise regression analysis was also used to calculate the relationships between total recoverable Cu and extractable Cu concentrati on. Statistical significance is accepted at = 0.05. Results and Discussion Copper Accumulation in Relation to Citrus Planting History The dominant soils in the Indian River area under commercial citrus production are Spodosols and Alfisols. The soils are mostly sandy with sand content above 85%, acidic to neutral, with low organic matter content and low buffering capacity (Zhang et al., 2003b). Surface soil pH varied from 3.8 to 7.3, with an average of 5.5. Total organic carbon content ranged fr om 3.3 to 11.1 g kg -1 (Table 2-2). Total recoverable Cu included all Cu in t he soil except for those residual bound in the mineral crystal structure and theref ore, is generally lower than total Cu concentration. Total recoverable Cu concentra tions in the soils ranged from 4.74 to 228 mg kg -1 which were all higher than the background level of 3.7 mg kg -1 in Florida soils (Ma et al., 1997). Copper accumulation in the soils was related to citrus planting history. 33

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The low total recoverable Cu (4.74 mg kg -1 ) was measured in the soil from the youngest citrus grove (since 2004) whereas the highest total recoverable Cu (228 mg kg -1 ) was observed in the soil from an old grove (sin ce 1975) (Table 2-2). Apparently Cu has accumulated in the soils with an increase of 27% in the 4-yr grove and by 60 times in the 33-yr grove. In addition to plant history, citrus cultivar also affects Cu accumulation in the soil. In general, grapefruit production systems receiv ed more Cu in fungicides than orange production systems as the former is more susceptible to fungal diseases such as canker and melanose. Among different products Cu-containing fungicides have been considered the most effective (Timmer et al ., 2006). One single application of Cu-based fungicides may introduce 3 to 5 kg Cu ha -1 Furthermore, three to six applications per year would correspond to an annual l oad of approximately 10-30 kg Cu ha -1 Soil number 8 9, 10 and 11 were all planted with r ed grapefruit and belonged to the same soil type with similar management practices and t herefore, their significant difference in total recoverable Cu concentration can be attributed to citrus production. It was estimated by simple linear regression that to tal recoverable Cu in the soil increased by 1.9 mg kg -1 or 4.3 kg Cu ha -1 yr -1 Considering that fruit harvest removes only a small fraction of Cu from the soil, the majority of the applied Cu may have been lost into surface runoff water or deeper soil layers along soil profiles. Effects of pH on Downward Movement of Copper in Soils Based on the soil properties and Cu cont ent, 6 soils were selected for vertical distribution study. Soil pH affected the downw ard movement of Cu (Fig. 2-1). Total recoverable Cu decreased more rapidly with depth in relatively high-pH soils (Soils 4 and 12) than low-pH soils (Soil 2). Up to 441 mg kg -1 Cu in Soil 12 was accumulated in 34

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the top 5 cm with minimal dow nward movement and decreased ma rkedly with soil depth, whereas Soil 2 had lower Cu concentration in the top 5 cm but Cu had been significantly leached through the soil profile, accu mulating at the 90 cm depth. These results suggest that pH play a critical role in Cu downward movement in these soils, since these soils had similar properties except for pH (USDA, 1980) (Tables 2-1 and 2-2). Soils that had pH>6.5 (Soils 4 and 12) had most of the Cu concentrated in the top 5 cm soil layer with minimal amount being leached downward even after over 30 years of citrus cultivation (Fig.2-1). In these soils, Cu downward migration along soil profiles was restrained and ther efore its pollution to groundwater was minimal. Low pH enhances Cu leaching losses. When soils had pH <6.5, particularly in very acidic soils (pH<5.5) such as Soils 1 and 10, downwar d movement of Cu was significant. For instance, >50 mg kg -1 of total recoverable Cu was measured at the 15-30 cm soil layer in Soil 10 (Fig. 2-1). Copper Availability as Affected by Cu Accumulation and Soil pH Soil available Cu as estimated by M ehlich-3 ranged from 2.31 to 119 mg kg -1 with a mean of 31.3 mg kg -1 while NH 4 OAc-extractable Cu ranged from 0.20-10.9 mg kg -1 and CaCl 2 -extractable Cu were 0.01-1.50 mg kg -1 (Table 2-2). The soil available-Cu extracted by the three methods was correlated among themselves in addition to total re coverable Cu. Except for CaCl 2 -extractable Cu, significant correlation was observed for all pairs (r = 0.892-0. 941, p<0.001; data not shown). Based on the regre ssion slope of Mehlich-3 and NH 4 OAc extractable to total recoverable Cu (Fig. 2-2), 48.9% of the total recover able Cu was extractable by Mehlich-3 and 4.5% of the total reco verable Cu was extractable by NH 4 OAc. When CaCl 2 -extractable Cu was divided into two groups based on soil pH, it was highly 35

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correlated to total recoverable Cu. The regr ession slope to total recoverable Cu was greater at pH < 6.5 than pH>6 .5 (Fig. 2-3). This suggest s that water-soluble soil Cu was affected by soil pH, which decreased rapidly at high pH. Total recoverable Cu alone may not pr ovide adequate information regarding Cu bioavailability in soils as only a small portion of the total recoverable Cu is available to plants or subject to leaching. Pl ants influence Cu availability, especially in the rhizosphere. For example, root activity lowers rhizosphere pH, and root exudates mobilize Cu by forming Cu-OM complex (Dessureault-Rompr et al., 2008), and transform Cu into metallic nanoparticles nea r the roots to reduce Cu toxicity (Manceau et al. 2008). Various extraction methods have been used to estimate available Cu in soils. Mehlich-3 procedure is commonly used to measure labile Cu in soil, which includes water soluble, exc hangeable, and a variable portion of soil Cu bound to organic matter, oxides and carbonates (Mehlich, 19 84). These fractions of Cu are readily dissolved in mild acidic/chelation (containing EDTA) conditions like the rhizosphere. CaCl 2 is an analog of soil solution and extracts mainly water-soluble plus a part of exchangeable Cu that can be readily taken up by the roots. The NH 4 OAC extraction is intermediate between t he two procedures. In this study, Mehlich-3 and NH 4 OAc extractable Cu were largely explained by the total recoverable Cu, as attested by their high correlation coeffi cient. However, the correlation of CaCl 2 extractable and total recoverable Cu was affected by soil pH. The CaCl 2 extraction method is appropriate for estima ting Cu availability in polluted soils. Brun et al. (2001) recommended 0.1 M CaCl 2 extraction as the best suitable for the determination of Cu availability to plants in acid-neutral soils. Zhang et al. (2003a) 36

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studied the losses of dissolved Cu in runo ff in Florida sandy soils under commercial citrus. They reported that mean dissolved-Cu concentration in surface runoff water from agricultural fields was significantly correlated with 0.01 M CaCl 2 extractable Cu. The results from this study toget her with those from previous reports indicate that CaCl 2 extractable Cu may be a better indicator of Cu av ailability in soil as it reflects the effects of both total Cu and pH. Soil Cu Fractionation and Availability Sequential fractionation has proven useful for distingui shing Cu association with different soil fractions (Ma and Rao, 1997). The Cu fractions in the surface soil decreased in the order of organic bound ( 48.2%) > oxides-bound (29.8%) > residual (16.5%) > carbonate-bound (4.2%) > exchangeable (1.3%) (Fig. 2-4).The fact that organic-bound Cu dominated soil Cu (from 43% to 62%) indicates its role in controlling the fate and transport of Cu in soils. In addi tion, oxides-bound Cu was also significantly correlated to total recoverable Cu and accounted for one-third of Cu in the soils. To understand which fraction is bioavail able, Mehlich-3 extractable Cu was correlated with various Cu fractions. M ehlich-3 extractable Cu was significantly correlated with organic-bound Cu fraction (r= 0.96, P<0.001). In addition, Mehlich-3 extractable Cu was also highly correlat ed with carbonateand oxides-bound Cu but less with exchangeable Cu. This occurred sinc e a larger proportion of the former two fractions is associated with t he labile Cu pool in the sandy soils. In comparison, NH 4 OAc and CaCl 2 extracted a smaller amount of Cu than the Mehlich-3 procedure (Table 2-2), but the CaCl 2 -extractable Cu was more closel y related to water soluble and exchangeable Cu than the other fractions (r=0.83, P<0.01) which agrees with the mild 37

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nature of the reagent, implying that this part of Cu was more readily leachable into groundwater or surface waters. Copper fractions were similar among different soils, indicating that Cu further conversion from available to less available fractions (aging) takes longer than the current citrus cultivation history in this st udy. In the sandy soils under citrus production, Cu was mainly bound to organic materials, oxides, and clay minerals (residual). Approximately half of the soil Cu was tied wi th organic matter; which is unexpected in sandy soil with low organic matter but consistent with other reports on acidic soils (Flores-Veles et al., 1996; Fernandez-Calvino et al., 2009). Similar results were also obtained by Strawn and Baker (2008) who exam ined Cu speciation in a contaminated agricultural soil via advanced x-ray spectroscopy. This may be attributed to the formation of inner-sphere complexes of Cu with humic acids (Komarek et al., 2010), which are very stable. The ot her possible reasons include low concentration of clay and oxides in the sandy soils. Similarly, Cu can be specifically adsorbed on the surfaces of Fe, Al and Mn oxides, thereby Cu being al so concentrated in the oxide fraction. Generally, the water soluble and exchangeable Cu is readily bioavailable whereas the oxide-, carbonate-, and organic matter-bound fr actions are potentially bioavailable because they are sensitive to environmental changes such as decomposition of organic matter, redox potential and pH, and can be mobilized under changing chemical environment (Alva et al., 2000; Shuman, 1991) Therefore, development of best management practices (BMPs), like raising soil pH etc., for preventing Cu loss into the environment and phytotoxicity needs to take c onsideration of reducing the release and transport of the readily and potentially available Cu from land to water. 38

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Conclusions The repeated uses of Cu-based fungicides have caused significant accumulation of Cu in the soils under citrus production. The available Cu as estimated by chemical extraction was enhanced by the increase in total recoverable Cu and the decrease in soil pH as a result of soil contamination by the anthropogenic input of Cu. However, the downward movement of Cu al ong soil profiles was mainly controlled by soil pH. Among the commonly used soil tests, CaCl 2 extractable Cu was a improved indicator of Cu availability in the sandy soils as it reflect ed the effects of both soil Cu accumulation and soil pH change. Most of the Cu in t he soils was associated with organic matter and oxides. The organicand oxides-bound Cu were highly correlated with extractable Cu and are potentially available or subjected to leaching loss. These findings merit attention in the development of BMPs for remediating Cu-contaminated soils. 39

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40 *GF=grapefruit, NAV=navel orange, VAL=Valencia orange No. Citrus culltivar Cu use hi story Soil orders Soil texture 1 Red GF 1988 Alfisol Sand 2 NAV 1988 Alfisol Sand 3 Red GF 1973 Alfisol Sand 4 Red GF 1966 Alfisol Sand 5 NAV 1982 Alfisol sand 6 VAL 1970 Alfisol Loamy sand 7 VAL 1970 Alfisol Loamy sand 8 Red GF 2005 Spodosol Sand 9 Red GF 1973 Spodosol Sand 10 Red GF 1994 Spodosol Sand 11 Red GF 1975 Spodosol Sand 12 White GF 1975 Alfisol Sand 13 VAL 1975 Alfisol Loamy sand 14 White GF 1989 Spodosol Sand 15 Murcott 1989 Spodosol Sand 16 Red GF 1989 Spodosol Sand 17 White GF 1955 Spodosol Sand 18 Murcott 1959 Spodosol Sand Table 2-1. General properti es of the evaluated soils

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41 Cu Soils pH (H 2 O) EC Total Organic C Total recoverable Mehlich-3 Extractable NH 4 OAc extractable CaCl 2 Extractable ( S cm -1 ) __ g kg -1 __ ______________________________________________ mg kg -1 _____________________________________________ 1 5.03.03 129.2 3.29 1.96 43.17.41 20.75. 86 1.43.52 0.89.04 2 5.34.49 155.6 7.82 1.36 96.47.13 30.52. 12 2.73.42 1.50.46 3 5.82.61 142.7 5.29.14 102.5520.22 42.94.40 3.61.73 1.41.28 4 6.65.91 194.7 4.26.64 122.60 18.57 60.18.50 2.79.31 0.19.09 5 5.09.06 212.8 5.26 0.49 48.75.03 26.27. 01 2.16.53 0.42.20 6 7.34.65 118.2 7.20.54 54.43 1.53 24.52.64 0.85.12 0.02.01 7 7.31.33 130.4 6.46.61 32.45 2.34 14.36.95 0.59.08 0.01.00 8 6.38.05 107.9 5.29.47 4.74. 02 2.31.14 0.20.10 0.01.00 9 4.81.17 268.0 8.59.19 70.12 6.66 44.80.75 1.20.56 0.99.11 10 3.85.06 194.8 7.241.77 44.67.52 18.00. 04 1.05.14 0.96.15 11 4.92.21 129.8 11.05 0.45 67.53.49 40.34.06 0.55.06 0.65.05 12 7.29.58 113.4 5.64.44 228.49 97.73 119.08.75 10.90.11 0.26.64 13 6.00.74 224.9 6.45. 77 60.44.49 41.53.98 0.93.31 0.78.00 14 4.39.54 164.7 3.37. 43 27.84.96 19.06.71 0.62.22 0.14.02 15 4.55.09 278.3 4.19.13 19.28 1.09 13.91.44 0.46.10 0.12.09 16 3.91.25 195.4 6.22.03 39.00 6.02 24.62.58 0.98.21 1.32.58 17 4.42.35 147.0 5.31 0.74 63.40.88 36.73.10 1.22.99 0.47.66 18 4.19.91 105.3 7.570.08 41.47.82 20.02. 48 0.59.26 0.36.08 Table 2-2. Selected chemical properties and Cu concentration of the soils

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Figure 2-1. Distribution of Cu along soil profiles. 42

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Figure 2-2. Relationship betw een Mehlich-3 extractable and NH 4 OAc extractable Cu and total recoverable Cu concentration in soils. Figure 2-3. Relationship between CaCl 2 extractable Cu and total recoverable concentration Cu in soils. 43

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Figure 2-4. Copper fractions as a percentage of total recoverable Cu in surface soils. 44

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CHAPTER 3 IMMOBILIZATION OF CU IN CONT AMINATED SANDY SOILS USING CALCIUM-WATER TREATMENT RESIDUE Introduction Soil contamination with Cu causes soil degradation, phytotoxic ity, and increased Cu transport from land to su rface and ground waters (Allowa y, 1995). Because of its persistent nature, remediation of Cu-contaminated soils is of great challenge. Physical methods such as scavenging or burial are e ffective but often too expensive for a large scale remediation. Phytoremediation is cost-effective, but requires a long time to accomplish desired results. Amendment-induced chemical stabilization is often used to remediate agricultural soils. The amendm ents decrease Cu leachability and bioavailability via chemical processes such as pr ecipitation, adsorption, and/or chelation. Amendment selection is often based on its affi nity for the contaminants. In soils, Cu is often associated wit h carbonates, phosphates, organic matter, oxides, clays and other silicate minerals. Copper stability in soil is strongly pH dependent. Copper is mobile in acidic and highly alka line conditions, but its mobility is usually the lowest at neutral to slightly alkaline pH (Celardin et al., 2004; Boudesocque et al., 2007 ). A range of amendments have been tested for Cu immobilizat ion, including fly ash, clay minerals, compost, sewage sludge, peat, phosphates and lime (Jackson and Miller, 2000; Stuckey et al 2008; Alvarez-Ayuso and Ga rcia-Sanchez, 2003; Alva et al. 2005; Besnard et al. 2001; Kiikkila et al. 2002; Pa ramasivam et al 2009; Cao et al 2004; Liu and Zhao, 2007; Nzegbule, 2007). These material s reduce Cus mobility and toxicity to biota by raising soil pH and chemisorbing or precipitating Cu in soils (McBride and Martinez, 2000). 45

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Application of industrial byproducts to agricultural soils to enhance plant growth has been reported extensively (Jackson and Miller, 2000). Water treatment residue (WTR) is a byproduct of wate r purification process to re move suspended and dissolved solids, organic matter, and other contaminants. It has a rela tively simple composition, which radically differs from wastewater treatment biosolids. A number of studies have examined the effect of various WTR on pl ant growth and nutrient uptake, with few focusing on remediation, especially the mechanisms of heavy metal immobilization (Basta et al., 2000; Elliott and Dempsey, 1991; Rengasamy et al., 1980; Silveira et al., 2006). Calcium WTR, containing mainly CaCO 3 and minor CaO, has potential for reducing Cu loading in surface runoff water in Florida soils due to its strong acidneutralizing capacity based on our field obser vation (He et al., 2009); however, its mechanisms are not well understood. In this study, laboratory incubation and column leaching experiments were conducted to understand the mechanisms of Cu immobilization by Ca-WTR and to estimate t he optimal rates of Ca-WTR to remediate Cu-contaminated soils. Materials and Methods Sampling and Characterization Alfisol and Spodosol are the dominant soils under citrus production in the Indian River area, South Florida, which received increased amounts of Cu-containing fungicide (He et al. 2004). Soil samples were collected at 0-15 cm from existi ng citrus groves. The soils used were: Riviera fine sand (Loam y, siliceous, active, hyperthermic Arenic Glossaqualfs) and Wabasso sand (sandy, silic eous, hyperthermic alfic alaquods). After collection, the soil samples were air-dried and passed through a 2-mm sieve prior to use. 46

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The Ca-WTR used in this study is a bypr oduct of drinking water purification collected from Utility Authority facilit y, Fort Pierce, FL. The pH and EC of soil and Ca-WTR were measured in deionized water at the soil: water ratio of 1:1 and 1:2 using a pH/ion/ conductivity meter (DIM 200, Denver Instrument, Denver, CO). Particle compos ition of the soil sample was determined with the micropipette method (Miller and Mille r, 1987). Total organic carbon (C) was determined using a C/N analyzer (Vario Max, Elemental Analysensystem GmbH, Hanau, Germany). Total recoverable Cu in so il was determined follo wing EPA method 3050B. Soil extractable Cu and nutrients were det ermined by Mehlich-3 extraction (Mehlich, 1984). Copper concentrations in the digest ed solutions or extracts were determined using an inductively coupled plasma optical emission spectrometer (ICPOES, Ultima; J. Y. Horiba Group., Edison, NJ, USA) following EPA method 200.7. The NELAP 2003 standards were followed for quality assuranc e and quality control of chemical analyses. For Cu analysis with ICP-OES, t he detection limit was 0.8 g L -1 with the recovery of 90110% for internal quality check and 95-105% for 2 nd source QC samples. Their physical and chemical properties are presented in Table 3-1. Incubation Study The Spodosol had lower pH and higher buffer capacity than Alfisol collected in this study (Table 3-1). In the batch experiment, 2 kg of soil was mixed with different rates of Ca-WTR, with half of the soil being used in the column experiment. The Alfisol was amended with 0, 5, 10, and 20 g kg -1 and the Spodosol with 0, 5, 50, and 100 g kg -1 CaWTR. In addition to the original soil, a Cu-enriched soil at 400 mg kg -1 Cu as Cu(NO 3 ) 2 was included. Ca-WTR was applied to the Cu-enriched soil immediately to simulate field condition of high Cu concentration in the top soil after fungi cide application. There 47

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were three replications fo r each treatment. The amended so ils were then incubated at room temperature (23C) fo r 70 d with soil moisture being maintained at 70% of water holding capacity (WHC) by adding water to compensate for water lo ss. At the end of incubation, subsamples were taken to deter mine pH, and Mehlich-3 extractable Cu. In addition, soil Cu was fractionated into wa ter soluble and exchangeable, carbonatebound, organic-bound, oxide-bound, and resi dual Cu fraction following the modified procedure of Amacher (1996). Column Leaching Study The column leaching experiment was conducted using a randomized complete block experimental design with three replic ates of each treatment. Soil (1 kg) was packed into a PVC column (40 cm height by 7.5 cm diameter) to reach approximately 16 cm high. The bulk density of t he packed soil column was 1.4 g cm The bottom of the column consisted of a Plexiglas plate containing several 5-mm-wide holes. The plate was covered with a nylon cloth to prevent soil loss during l eaching. Two disks of filter paper (Whatman # 42) were placed on the top of soil to prevent disturbance by applied water. The soil columns were saturated with dei onized water from the bottom for three days to reach field-holding capacity prior to leaching. Deionized water (350 mL) (approximately 1 pore volume) of was applied to the top of each soil column at a rate of 2 mL min -1 every 3 days using a peristaltic pum p. Leaching was continued for 30 days with a total of 10 leaching ev ents. This application rate of leaching solution did not cause water ponding on the top of the column For each leaching event, leachates were collected into a 1000-mL beaker below the so il columns and filtered through a 0.45-m membrane filter for the analysis of pH, EC, DOC and Cu concentrations. At the end of 48

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the leaching experiment, soils were remove d from the columns and mixed thoroughly. Subsamples (each 300 g) of the soils were air-d ried and analyzed for 0.01 M CaCl 2 and Mehlich-3 extractable Cu and other metals. Statistical Analysis All results were expressed as a mean of three replicates with a standard error, and treatment effects were determined by the analysis of variance according to the general linear model procedure of the Statistical Analysis System (version 8.2, SAS Institute, 2004). Differences among the treatment means were separated by the Least Significant Difference, at the 0.05 probability level. Results and Discussion Addition of Ca-WTR Increased Soil pH and Decreased Extractable Cu Both the Alfisol and Spodosol soil were acidic, sandy and with low organic carbon. Addition of Ca-WTR significantly raised soil pH (Fig. 3-1). At 5 g kg-1, soil pH was elevated to 6.4-6.9 in the Alfisol and to 6.2 in the Spodosol soil. The Ca-WTR was a byproduct of drinking water purif ication containing mainly CaCO 3 and minor CaO with minimum contaminant: pH 9.06, electrical conductivity (EC) 659 S cm -1 Mehlich-3 extractable Ca 292 g kg -1 and Cu 0.32 mg kg -1 and total recoverable Cu 0.40 mg kg -1 Copper loading at 400 mg kg -1 decreased soil pH by 0.26-0.45 units (Fig. 3-1). As a consequence, the mobility and phytotoxici ty of Cu in soil may be enhanced. The mechanisms of pH decrease from external Cu loading or contamination involves the replacement of exchangeable H + /Al 3+ on clay minerals and oxides. Similar results were reported by Yu et al. (2002) in red soils, in which adsorption of one mole of Cu 2+ resulted in the release of 1.1 to 2.6 mole of proton (H + ), depending on so il properties such as contents of clay minerals and oxides, and on Cu 2+ adsorption mechanisms. 49

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Theoretically, one mole of Cu 2+ adsorbed can release one mole of protons if only monodentate adsorption mechanism is involved, two moles of protons if a bidentate adsorption occurs on hydroxylated surfaces or through cation exchange reaction (one Cu 2+ replacing two H + ) on exchange complex (Padmanabham, 1983), and three moles of protons if a concurrent reaction of Cu 2+ hydrolysis and a bidentate adsorption of Cu 2+ and/or hydroxyl Cu happens on the hydroxyl ated surfaces of oxides (Summer, 1998). Higher application rates of Ca-WTR resulted in higher soil pH (F ig. 3-1). At 20 g kg -1 and 100 g kg -1 (equivalent to field applicat ion rates of ~44.8 and 224 Mg ha -1 incorporated into a soil depth of 15 cm), the soil pHs were increas ed to 6.7-7.2 in the Alfisol soil and 7.1-7.2 in the Spodosol soil. The initial pH of Spodosol (4.18) was lower than the Alfisol (5.83) while the buffer capacit y was higher (Table 31). Therefore, more amendments were required in the Spodosol than in the Alfisol. In general, high application rates should be avoided, which ma y lead to negative effect on soil properties or crop yields (Titshall et al., 2007). Furthermore Cu mobility in soils can increase at pH values above ~7.5 due to increased solub ility of soil organic matter (SOM) and the formation of Cu-SOM complexes (Fernndez-Calvio et al., 2008 and FernndezCalvio et al., 2009). Application of coal fly ash (pH = 12.2) was effective in reducing Cu mobility, however, increasing soil pH to above 8 (15% by weight) decreased its efficiency (Ciccu et al., 2003). To determine the long-term effectiveness of Cu-immobilization by Ca-WTR, the soils were incubated for 70 d under 70% WFC. Melich 3 extractable Cu in the Ca-WTR amended soils decreased during the 70-d incubati on (Fig. 3-2), indicating that a portion of Mehlich-3 extractable Cu (water soluble, exchangea ble, part of CaCO 3 and 50

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organically-bound Cu) (Mehlich, 1984) was c onverted into more strongly bound fractions. The extractable Cu generally dec reased with increasing appl ication rate (Fig. 3-2), which resulted in increasing pH (Fig. 3-3). Amendment at 5 g kg -1 Ca-WTR reduced the extractable Cu by 24-36% in t he Alfisol and 22-47% in the Spodosol. Based on a linear model of the extractable Cu vs soil pH, increasing soil pH by one unit decreased the extractabl e Cu by 12-90 mg kg -1 At pH 6.5, the extractable Cu was reduced by 28-30% in the Alfisol and by 42-44% in the Spodosol. Ca-WTR amendment has a strong acid-neutra lizing capacity. It is likely that less water soluble Cu compounds such as Cu(OH) 2 were formed at the raised soil pH. In addition, exchangeable Al 3+ and Fe 3+ were replaced by Ca 2+ and precipitated as Al(OH) 3 and Fe(OH) 3 when soil pH is above 5.5 (Lindsay, 1979). These newly formed oxides and hydroxides provide additional sites for sorbing Cu As pH increased, there is an increase in negative surface charge of metal oxides, thus reducing Cu concentration in soil solution. In addition, some adsor bed Cu may be also occluded by the newly formed oxides and become inaccessible to chemical extraction (Ma et al., 2006; Sayen et al., 2009). Addition of Ca-WTR Converted Labile Cu to More Stable Cu Forms Sequential fractionation was c onducted to further distinguish different Cu forms in soil matrix. Among the five Cu fractions bas ed on sequential extraction, water soluble and exchangeable Cu (WE-Cu) is more liabl e (Ma and Rao, 1997). Addition of Ca-WTR drastically reduced WE-Cu while increased oxi de-bound and residual Cu fractions (Fig. 3-4). Although the best results were obtained at the highest dose, lower dose at 5 g kg -1 was effective in reducing WE-Cu fractions. At 5 g kg -1 Ca-WTs reduced WE-Cu by 62% in the original soils, and by 90% in the Cu-enriched soils (Fig. 3-5). Ca-WTR introduced 51

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a significant amount of CaCO 3 /CaO, which can control Cu mobi lity to a great extent by surface precipitation of CuCO 3 (Lindsay, 1979). The retention of Cu through coprecipitation with carbonates is associated with the release of Ca 2+ Mg 2+ Na + and H + into the soil solution at equal molar ratios The precipitation of newly-formed Cu phases in a soil represents an important mechanism of Cu immobilization. These phases may include Cu(OH) 2 CuCO 3 /Cu 2 (OH) 2 CO 3 CuO and various Cu-hydroxysulfates if sulfates are present (Komrek et al., 2009; Ma et al., 2006, Ponizovsky et al., 2007, and Strawn and Baker, 2009). The carbonate-bound Cu was reduced by Ca-WTR at the high Ca-WTR rates (10-100 g kg -1 ), probably because the binding of Cu is stronger to oxides than carbonates (Jalali, 2008), as evidenced by t he increase in the oxi de fraction (Fig. 3-4). The conversation of labile Cu to more stable Cu forms was also supported by an increase in the residua l fraction (Fig. 3-4). Addition of Ca-WTR Reduced L eachable Cu in Soil Columns The incubation studies provided insight into Cu transformations as affected by CaWTR amendment, whereas column studies were to determine Cu mobility, particularly downward movement in term of its environment al risks (Tyler and McBride, 1982). As expected, the highest leachable Cu concentrati ons occured in the first leaching event for all samples (Fig. 3-5). In the absence of Ca-WTR, higher Cu concentrations were leached during the leachi ng period. At the b eginning, 0.71-0.97 mg L -1 Cu in the original soils, and 12.12-18.1 mg L -1 Cu in the Cu-enriched soils were detected (Fig. 35). For the original soils, the decrease in leachable Cu concentration consisted of two phases. It involved an initial rapid decrease, followed by a slow but steady decrease (Fig. 3-5). In comparison, leachable Cu co ncentrations were constantly low in the 52

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amended soils, even at the lowest rate of 5 g kg -1 For the unamended Cu-enriched soils, Cu concentrations in the leachate were above the U.S. EPA drinking water limit of 1.30 mg L -1 (USEPA, 2004). In comparison, Cu concentrations in the amended soils were below the limit at the end of the 10 leaching events even at the lowest treatment rates (Fig. 3-5). After ten leaching events, the cumulative loss of Cu decreased with increasing CaWTR application rates for all soils (Table 3-2). Compared with the original soils, application of Ca-W TR at 5.0 g kg -1 decreased Cu leaching loss by 56-66% in the original soils and by 55-59% in the Cu-enr iched soils. In comparison, at the highest rates (20 g kg -1 for Alfisol and 100 g kg -1 for Spodosol), the cumu lative amount of Cu lost in the 10 leaching events decreased by 35% in the original soils and by 73-80% in the Cu-enriched soils. These results suggest the amendment of Ca-WTR can significantly reduce Cu leaching losses from sandy soils. This agrees with the incubation studies results, which showed a signi ficant portion of labile Cu was converted to more stable forms in the amended soils (Fig. 3-4). The decrease in cumulative losses of Cu with Ca-WTR application rate followed a quadratic model (P<0.05) (Fig. 3-6). With increasing application rates the cumulative losses of Cu dramatically decreased, parti cularly for the Cu-enriched soils, but the decrease diminished with further increases in WTR. This indicates there was an optimal level of WTR application rate, above which the benefit may be limited. The cumulative losses of Cu in the Cu-enriched soils was gr eater in Alfisol than Spodosol, indicating higher Cu leaching potential in Alfisol soils likely because of its lower organic matter and clay contents for holding Cu (Table 3-1). 53

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A great challenge to chemical remediatio n of metal-contaminated soils is to determine the optimal amendment rates (K umpiene et al., 2007). The calculated CaWTR rate for achieving the lowest Cu loss was ~15 g kg -1 for the Alfisol and 75 g kg -1 for the Spodosol. However, high amendmen t rates are not recommended for economic consideration. In addition, high rates may lead to negative effects on soil quality and/or crop yield. The results from our field survey indicate that the relationship between readily available Cu (by CaCl 2 extraction) and total recoverabl e Cu is affected by soil pH, and a pH value of 6.5 is optimum for reduci ng Cu availability in soils (Fan et al., unpublished). Based on this criterion and the response curve of pH to Ca-WTR application rates (Fig. 3-1) the optimal rates of Ca -WTR were 3-8, and 30 g kg -1 for the Alfisol and Spodosol soils. In addition to total Cu in the leachate, Mehlich-3 extractable Cu in the leached soils decreased with increasing Ca-WTR application rates. Application of Ca-WTR at 20 g kg -1 to the Alfisol soil and 100 g kg -1 to the Spodosol soil decreased Mehlich-3 extractable Cu by 3466% and 53 to 64%, respectively. For CaCl 2 extractable Cu, the reduction was 55-89% and 50-52%, respectively (Table 3-2). Both Mehlich-3 extractable Cu and CaCl 2 extractable Cu were signi ficantly correlated with the leaching loss of Cu (r = 0.86 and 0.73, respectively). There was no si gnificant difference in the proportion of extractable to the total Cu in the soils before and after the leachings (Fig. 3-2). These results suggest that leaching had a minimal e ffect on the overall Cu distribution in the soils and the amendment of Ca-WTR in creased soil Cu holding capacity and established a relatively stable equilibrium of Cu in the contaminated soils. 54

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55 Conclusions Copper input to agricultural soils resulted in soil pH decrease and Cu loss acceleration. Application of Ca-WTR effect ively increased soil pH and converted labile Cu into more stable forms. Ca-WTR significantly reduced leachate Cu concentrations and the cumulative amounts of Cu loss. The cumulative loss of Cu as a function of CaWTR treatment rates can be described by a quadratic model. From the environmental and economic considerations, the optical Ca -WTR application rates can be estimated based on soil pH of 6.5 for significant reducti on of available Cu in the soil. As a byproduct from drinking water treatment fac ility, Ca-WTR can be obtained at minimum cost and contain minimum detectable contam inants, and therefore has a potential for the remediation of Cu contaminated soils, pa rticularly in those acidic sandy soils.

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Table 3-1. Physicochemical properties of the soils used in this study Property Cropping history pH (H 2 O) EC Sand (> 0.05 mm) Silt (> 0.002 and < 0.05mm) Clay (< 0.002 mm) Organic matter Recoverable Cu Mehlich-3 Extractable Cu S cm -1 -------------------------------g kg -1 ---------------------------------------mg kg -1 --------Alfisol Red GF a /1988 5.83.14 b 132 986 5 3 1 11 1 3.29.97 63.2.34 39.9.86 Spodosol Red GF /1994 4.18.16 194 928 52 1 20 2 7.24.77 114.41 72.3.04 Ca-WTR 9.06.01 659 12 4 136 5 852 5 -0.40.05 0.32.05 b Mean SE (n = 3). a GF=grapefruit

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Table 3-2. Mehlich-3 extractable Cu (mg kg -1 ) in the soils after 10 leaching events Treatments rate Mehlich-3 extractable Cu (mg kg -1 ) CaCl 2 extractable Cu (mg kg -1 ) g kg -1 Original soil Cu enriched soil Original soil Cu enriched soil Alfisol 0 27.8a 299.0a 0.063a 0.503a 5 21.4ab 225.6b 0.023b 0.432b 10 13.8b 213.8b 0.020b 0.263c 20 9.6b 198.1b 0.007c 0.244c Spodosol 0 60.2a 215.1a 0.035a 0.799a 5 33.8b 211.2a 0.026b 0.791a 50 30.1b 105.5b 0.020c 0.362b 100 21.9c 101.6b 0.016c 0.349b

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AlfisolCa-WTR treatment rate (g kg-1) 0 5 10 15 20Soil pH (H2O) 5.0 5.5 6.0 6.5 7.0 7.5 8.0 Original Soil Soil + 400 mg Cu Kg-1 SpodosolCa-WTR treatment rate (g kg-1) 020406080100Soil pH (H2O) 3 4 5 6 7 8 Figure 3-1. Relationship between so il pH and WTR application rates.

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Figure 3-2. Changes of Mehlich-3 extract able Cu in soils with incubation time as affected by Ca-WTR application rates. A) Alfisol. B) Spodosol. C) Alfisol + 400mg Cu kg -1 soil. D) Spodosol + 400mg Cu kg -1 soil.

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Soil pH 4567Mehlich 3 extractable Cu (mg kg-1) 0 10 20 30 40 50 60 70 80 Alfisol y = -20.73x + 162.8 R2 = 0.903 Spodosol y = -12.34x + 117.8 R2 = 0.957 Soil pH 4567Mehlich 3 extractable Cu (mg kg-1) 50 100 150 200 250 300 350 400 Alfisol + 400 mg Cu kg-1 y = -96.98x + 888.5 R2 = 0.984 Spodosol + 400 mg Cu kg-1 y = -50.988x + 501.42 R2 = 0.879 Figure 3-3. Relationship between Meh lich-3 extractable Cu and soil pH.

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Figure 3-4. Cu Fractionation in soils amended with different Ca-W TR rates. A) Alfisol. B) Spodosol. C) Alfisol + 400mg Cu kg -1 soil. D) Spodosol + 400mg Cu kg 1 soil.

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ACa-WTR rates (g kg-1) 0 5 10 20Cu concentration (mg kg-1) 0 1 2 3 4 5 CCa-WTR rates (g kg-1) 0 5 10 20Cu concentration (mg kg-1) 0 20 40 60 80 100 a a b b b b c c BCa-WTR rates (g kg-1) 0 5 50 100Cu concentration (mg kg-1) 0 1 2 3 4 5 DCa-WTR rates (g kg-1) 0 5 50 100Cu concentration (mg kg-1) 0 20 40 60 80 100 a a b b b b cc Figure 3-5. Changes of water soluble /exc hangeable Cu with Ca-WTR application rates. A) Alfisol. B) Spodosol. C) Alfisol + 400 mg Cu kg -1 D) Spodosol + 400 mg Cu kg -1

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Figure 3-6. The concentrations of Cu, in leachate from two soils in ten leaching events. A) Alfisol. B) Spodosol. C) Alfisol + 400 mg kg -1 Cu. D) Spodosol + 400 mg kg -1 Cu.

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AlfisolCa-WTR rates (g kg-1) 0 5 10152025Cumulative loss of Cu (mg kg-1) 0 5 10 15 20 Original Soil y = 0.0046x2 0.1314x + 1.0417 R2 = 0.942 Soil + 400 mg Cu kg-1y = 0.072x2 2.1649x + 18.379 R2 = 0.9847 SpodosolCa-WTR rates (g kg-1) 020406080100120Cumulative loss of Cu (mg kg-1) 0 2 4 6 8 10 12 14 Original Soil y = 0.0002x2 0.0293x + 1.3432 R2 = 0.9376 Soil + 400 mg Cu kg-1y = 0.0017x2 0.2526x + 11.491 R2 = 0.9595 Figure 3-7. Cumulative loss of Cu after 10 leaching events as a function of Ca-WTR treatment rates.

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CHAPTER 4 AMENDMENT OF CALCIUM WATER TREA TMENT RESIDUE (CA-WTR) REDUCES CU BIOAVAILABILITY IN CU-CONTAMINATED SOILS Introduction Contamination of agricultural soils with Cu as a result of fungicide application reduces soil quality for crop growth. Only a small fraction of the total Cu present in the soil is available for plant uptake. Although most Cu-based fungicides are highly water soluble, the Cu can be strongly bonded, adsorbed, or precipitated to soil particles when it is applied to soil (He, et al. 2005). These processes are, in turn, influenced by soil factors such as pH, organic matter content, redox potential, and composition of clay minerals. The leaching potentia l of Cu, though low in almost all soils, could be high in sandy soils. The phytotoxicity of Cu depends on the level of acidity or alkalinity of the soil given the same level of total concentration (Ginocchio et al. 2002; Gray et al., 2006). Generally, Cu has higher phytotoxicity in acidic soils with a low cation exchange capacity than in slightly alkaline conditions. Fi ne texture soils with high concentrations of organic matter, carbonates, clay, and oxides can have a larger holding capacity for Cu. Li et al. (2010) studied a wide range of soils with different properties and climate characteristics and concluded that soil pH organic carbon (OC) content, and cation exchange capacity (CEC) can explain over 80% of the variance in Cu toxicity across soils from Asia and Europe. When determining potential risks associated wit h soil contamination, total Cu in soil is often a poor measure. In general, the availability of Cu in soil can be estimated using chemical extraction procedures. Most extracting solutions contain multiple reagents including chemically aggressive stro ng acids, reducing an d oxidizing agents, metal chelators and dilute salts, e.g. Mehlich-1, and Mehlich-3, EDTA, DTPA, and 65

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0.01M CaCl 2 solution (Lindsay and Norwell, 1978; Aten and Gupta, 1996; McBride, et al. 2003). The extracting solution should thus be chosen carefully according to the purpose and specific soil characteristics Soil excavation and landfilling, the tradi tional approaches for remediating metalcontaminated soils, are not economically feasible or environmentally sound for a large scale remediation. Phytoremediation is cost-effective, but requires longer time to accomplish desired results as compared to other approaches. Ther efore, chemical approaches are frequently adopted for the remediation of agricultural soils. Soil amendment is one of the bes t management practices and sustainable approaches to remediate metal-contaminated surface soils from agricultural production systems. The reduction of the mobile metal content in t he soil solution will minimize metal leaching into the groundwater, and transporting to neighboring areas. Liming materials are perhaps the most common and safe materials for in situ soil remediation (Gray et al., 2006). Calcium water treatment residue (Ca-WTR) is a byproduct of water purification process to remove suspended and dissolv ed solids, organic matter, and other contaminants. It has a rela tively simple composition (pH 9.06, containing mainly CaCO 3 and minor CaO). In previous work, treatment with Ca-WTR was shown to significantly reduce mobile Cu levels of acidic Cu-contam inated soils in a batch study (Chapter 3). In the present study, Ca-WTR was mix ed at different proportions: from 5 g kg -1 to 100 g kg -1 soil to two representative sandy acidic soils in south Florida. Cu-enriched soils (incubated with 1000 mg Cu kg -1 soil) were also investigated as a highly contaminated site for comparison. Ryegrass ( Lolium perenne L.) and lettuce ( Lactuca sativa L.) were grown on the treated and control soils for eight weeks. The main 66

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objectives of the current study were to determine if Ca-WTR would (a) reduce Cu accumulation in plant and (b) improve plant growth. Materials and Methods Soil Samples and Ca-WTR Collection Alfisol and Spodosol are representative citr us grove soils in South Florida, which received increased amounts of Cu -containing fungicide. Soil samples were collected to a depth of 15 cm from the su rface of a Riviera fine sand (Loamy, siliceous, active, hyperthermic Arenic Glossaqualfs) and a W abasso sand (sandy, siliceous, hyperthermic alfic alaquods) in the Indian River area, S outh Florida. The soil samples were air-dried and passed through a 2-mm sieve prior to physical and chemical analyses and green house study. The Ca-WTR, a byproduct of drinking water purification containing mainly CaCO 3 and minor CaO, was collected from the Fort Pierce Utility Authority facility. Selected properties of the so ils and Ca-WTR are presented in Tables 4-1 and 4-2. Greenhouse Experiments Pot experiments were conducted in a gr eenhouse with a mean of 10-h sunlight photoperiod. According to the properties of the two soils (Spodosol had lower pH and higher buffer capacity), 2 kg oven dried basis) soil amended with Ca-WTR at the rates of 5, 10, 20, 50 g kg -1 for the Alfisol and 5, 10, 50, 100 g kg -1 for the Spodosol was weighed and homogenized into a 2-L plastic c ontainer. Soil moisture was adjusted to 70% field-holding capacity. There were two levels of Cu for each soil: with or without being enriched with 1000 mg kg -1 Cu in the form of Cu(NO 3 ) 2 Non-amended soils were carried through the experiment as contro ls. After three days, water-soaked and sterilized ryegrass seeds were planted at a rate of 100 seeds per pot after one week germination. Similarly, 4 seedlings (30 da ys old) were grown per pot for lettuce. 67

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Fertilizers were applied at the beginning to each pot at the rate of 250 ppm N (as NH 4 NO 3 ), 200 ppm K (KNO 3 ), and 50 ppm P (as KH 2 PO 4 ). All treatments were replicated three times. The lost moistu re was supplemented every other day with deionized water by weighing and evaluatin g the fresh weight of the plants. After 8 weeks growth, the plant shoot s and roots were harvested (cutting the shoots, taking the soil column with the roots from the pot, loosening soil by carefully crushing the column, and washing away any soil particles clinging to the roots), rinsed with tap water followed by deionized water, and oven dried at 70 C for 72 h. Fresh and dried biomass was recorded. Dried plants we re ground using a micro stainless ball mill 0.4 mm prior to digestion. The soils in the pots were sampl ed at the end of the experiment, air-dried, and pa ssed through 2-mm sieve prior to analysis of available nutrients, Cu, and related chemical properties. Chemical Analyses of Soil and Plant Samples The pH and EC of soil and Ca-WTR were measured using a pH/ion/conductivity meter (DIM 200, Denver Inst rument, Denver, CO). Total organic carbon (C) and total N was determined using a C/N analyzer (Vario Max, Elemental Analysensystem GmbH, Hanau, Germany). Percent organic matter was determined by multiplying percent organic C by 1.724. Total recoverable Cu in soil was determined following EPA method 3050B. And soil extractable Cu and nutrient analyses were performed by 0.01 M 0.1 M CaCl 2 and Mehlich-3 extraction method (1:10 so il:solution ratio) (Mehlich-1984). Ground plant samples (0.4 g) were digested with 5 ml of concentrated HNO 3 using an A.I. digestion system (A.I. scientific, Inc., USA) The concentrations of Cu and other elements in soil extracts and digested samples were determined using inductively 68

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coupled plasma optical emission spectrometry (ICP-OES, Ultima, J. Y. Horiba, Edison, NJ, USA) following USEPA me thod 200.7. Quality Assurance/Quality Control (QA/QC) plan included a blank, a duplicate, a spike and a Standard Reference Material (SRM) analysis every 20 samples with an acceptable recovery of 95-105%. Statistical Analysis All treatments were replicated three times. Treatment effects on pH, extractable Cu, plant Cu, and plant biomass were det ermined by analysis of variance using the Statistical Analysis System software (releas e 9.1 for Windows; SAS In stitute, Cary, NC, USA). Least significant difference (LSD ) analysis was conducted to determine the differences among the treatments at the 0.05 probability level. A type I error ( ) of 5% was used for all statistical analyses. Results and Discussion Effects of Ca-WTR Amendment on Soil pH and Extractable Cu Both soils were acidic, but the Spodosol had lower pH (pH=4.66) than Alfisol (pH=5.45). The Ca-WTR was al kaline and had a higher electric al conductivity (EC) than the soils. The organic matter content of the Spodosol was approximately two times higher than that of the Alfi sol. Both soils had high amount s of sand (>90%). The CaWTR consisted of mainly clay and silt ( 13.6 and 85.2%, respectively), a minimal amount of organic carbon and non-detectable heavy metals. Higher Cu concentration was measured in the spodosol due to the long period of fungicide application (Table 4-1). On the other hand, the Alfisol c ontained higher concentrations of Zn, Fe and Al than the Spodosol. Other properties of the tw o soils were similar (Table 4-2). Copper loading at 1000 mg kg -1 reduced soil pH by 0.6 uni ts for the Alfisol and 0.48 pH units for the Spodosol (Table 4-3) The mechanisms of pH decrease from 69

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external Cu loading or contamination may re sult from the replacem ent of exchangeable H + /Al 3+ on clay minerals and oxides. Similar results were reported by Yu et al. (2002) in red soils, in which adsorption of one mole of Cu 2+ resulted in the release of 1.1 to 2.6 mole of proton (H + ), depending on soil properties such as contents of clay minerals and oxides and on Cu 2+ adsorption mechanisms (Padm anabham, 1983; Summer, 1998). The application of Ca-WTR significantly in creased soil pH from acidic to neutral (Table 4-3). At 5 g kg -1 soil pH was elevated by 0.93 to 1.01 units in the Alfisol and 1.24 to 1.71 units in t he Spodosol soil. At 50 g kg -1 and 100 g kg -1 (equivalent to field application rates of ~89.6 and 224 Mg ha -1 incorporated into a soil depth of 15 cm), the soil pHs were increased to 7.25-7.3 in the Alfi sol soil and 6.98 -7.25 in the Spodosol soil. The initial pH of Spodosol (4.66) is lower t han the Alfisol (5.45) while the buffer capacity was higher (Table 4-1). Ther efore, higher rates of Ca-W TR amendments were applied in the Spodosol than in the Alfisol. During the plant growing peri od, soil pH values had minimal variation, remaining at relatively constant levels for both soils of each treatment. Furthermore, the standard errors of mean pH values for each treatment were generally small and in the range of 0.1 to 0.3 pH units. Soil extraction with Mehlich-3 and CaCl 2 was used to gain insight into the mobility and bioavailability of Cu in soils. Analysis of variance indicated that the Application of Ca-WTR to soil significantly reduced availabl e Cu as estimated by both methods. The extractable Cu generally decreased with increasing Ca-WTR application rate. Mehlich-3 extractable Cu was reduced by 20-40% whereas CaCl 2 extractable Cu was reduced by over 70% as compared to the control. CaCl 2 (0.01M) is a mild extractant for Cu and it measures the soluble pool of Cu in soil (Sau ve et al., 1996). Mehlich-3 solution is a 70

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more aggressive extractant for Cu due to it s strongly acidic nature. Chaignon et al. (2003) observed significantly higher Cu concentrations in tomato (Lycopersicon esculentum Mill.) roots grown on strongly acidic soils as compared to calcareous or mildly acidic soils with the same Cu conc entration. Organic matte r (OM) plays a very important role in Cu retention in soils by means of complexation with humic and fulvic acids (Strawn and Baker, 2009). These result s indicate that Ca-WTR amendment not only has a strong acid-neutralizing capacity but also can convert labile Cu to more stable Cu forms in the amended soils. As the pH of the soil increases more Cu in the soil solution is adsorbed onto the soil surfac e due to an increase in surface negative charge (Naidu et al., 1998); Cu may form innerand outer-sphere complexes with newly deprotonated functional groups present in soil colloids (Bolan and Duraisamy, 2003); or may form precipitates at high metal loadings and high pH (Lindsay, 1979). Perhaps less water soluble Cu compounds such as Cu(OH) 2 and CuCO 3 were formed at the raised soil pH. In addition, exchangeable Al 3+ and Fe 3+ were replaced by Ca 2+ and precipitated as Al(OH) 3 and Fe(OH) 3 when soil pH is above 5.5. These newly formed oxides and hydroxides provide additiona l sites for sorbing Cu. As pH increased, there is an increase in negative surface charge of metal oxides, therefore reducing Cu concentration in soil solution. Moreover, some adsorbed Cu may be also occluded by the newly formed oxides and become inaccessi ble to chemical ex traction (Ma et al., 2006; Sayen et al., 2009). Effects of Ca-WTR Amendment on Plant Cu Concentration Copper concentrations in ryegrass shoots decreased significant ly with increasing Ca-WTR application rates (Figure 4-2). The highest concentrations of Cu occurred in the control for the original Alfi sol and Cu enriched Alfisol with 5g kg -1 Ca-WTR as no 71

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plant could grow in the Cuenriched Alfisol. The results for Spodosol were similar, except that the highest Cu concentration in the plant was greater in Alfisol than Spodosol. These observations confirm the results of soil analysis where similar conclusions about the Cu binding effect of soil organic matter were drawn. Nevertheless, less Ca-WTR was needed for reduc ing plant Cu concentration in Alfisol than Spodosol below the upper critical toxicity level (30-35 mg kg -1 Macnicol and Beckett, 1985). For lettuce, the plant Cu c oncentration decreased only at the high CaWTR rates. The concentration of plant Cu in Cu-enriched soils was slightly higher than that in the original soils. However, there were no signific ant differences in plant Cu concentration between the treatment of low Ca-WTR rates (5-10 g kg -1 ) and the control. Besides, plant Cu concentrations were much lower in lettuce than in ryegrass under the same treatment level. Similar results were reported by other researchers (Sauve etal. 1996; Kulli et al. 1999; Krebs et al. 1999). This may be explained by the selective transport mechanism of ions within the plants (Kabata-Pendias & Pendias, 1992). However, more research is needed to investigate this mechanism. Shoot Cu concentration of both ryegrass and lettuce was negatively correlated with soil pH (p<0.05), indicating increased Cu availability with lowering soil pH. Shoot Cu concentration of ryegrass was closely related to Mehlich-3 extractable Cu (p<0.01) and yielded a higher leve l of statistical sign ificance to 0.01M CaCl 2 extractable Cu in soil (p<0.001). However, no correlation was found between shoot Cu concentration of lettuce and extractable Cu in the soils (T able 4-4). Obviously there were varietal differences in Cu acquisition between these two plant species, but the mechanism was not well understood. Mulchi et al. (1991) reported a fair ly strong correlation between 72

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Mehlich-3 extractable soil Zn and Cu and the concentrations of these metals in tobacco. However McBride et al. (2003) reported no relationship bet ween Cu concentration in a clover and CaCl 2 or M3-extractable Cu in soil probably because of a soil-plant barrier to the accumulation of Cu in excess of normal levels. Nevertheless, they still recommended dilute CaCl 2 as a universal soil extractant for estimating short-term trace metal availability to crop plants. The study of Aten and Gupta (1996) also showed that the concentrations of Cu, Zn, Cd, and Pb in plants grown on 35 soils in Switzerland were more correlated with soil extractable Cu obtained with weak extractants (0.1 M NaNO 3 and 0.05 M CaCl 2 ). Brun et al. (1998) recommended the 0.1 M CaCl 2 extraction as the best suitable for the determination of Cu (bio)availability in acid-neutral soils; on the other hand, 0.05 M EDTA or 0.005 M DTPA are preferable for alkaline soils (Brun et al., 2001, Chaignon et al., 2003 and Komarek et al., 2008). Effects of Ca-WTR Amendmen t on Plant Growth/Dry Matter Yields and Cu Uptake Ryegrass was chosen as an indicator plant because of its use as animal fodder and tolerance to low fertility. Lettuce is an important leafy vegetabl e consumed directly by humans. The overall shoot growth for the two plant species affected by the amendments was very similar. Regression analyses of shoot dry matter yield (DMY) with Ca-WTR application rate had similar quadr atic relationships associated with the two soils for both ryegrass and lettuce (Fig. 4-1). Overall, plant biomass yields increas ed with WTR application rates at the low levels (5-20 g kg -1 for Alfisol and 5-50 g kg -1 for Spodosol), reached maximum at the moderate level (10-50 g kg -1 ) and started to decrease at the high levels (>20 g kg -1 for Alfisol and >50 g kg -1 for Spodosol) (Fig. 4-1). However, the differences in plant yields between the treatments were smaller in the or iginal soils than the Cu enriched soils. 73

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For the original Alfisol and Spodosol, only lettuce grown in the control original Spodosol had some chlorosis (old leaves turn yellow), but the roots were normal, indicating a slight Cu-contaminati on condition. The amendment at10 g kg -1 Ca-WTR resulted in the maximum growth for ryegras s (133% of the control for Alfisol and 149% of the control for spodosol), while the maximu m yield of lettuce occurred at the Ca-WTR rates of 20 g kg -1 and 50 g kg -1 respectively for the Alfisol and Spodosol (145% of the control for Alfisol and 206% of the control fo r Spodosol). These results indicate that the application of Ca-WTR at proper rates is beneficial to plant growth even in slightly Cu contaminated soils. Both ryegrass and lettuce grew very poorly with minimal biomass yield in the Cuenriched soils (Fig. 4-1). The loading of 1000 mg kg -1 Cu caused death of most plants and severe Cu toxic effects on plants were indicated by strong chlorosis and necrosis of plant leaves and impairment of root growth as described by Lepp (1981). Chlorosis initially appeared in the older leaves, and mo ved progressively up to the youngest. The amendment of Ca-WTR significantly improved plant growth with a dramatic increase in biomass yield at the application rate of 5g kg -1 but the increase became less at higher application rates, and the yield of ryegrass star ted to decrease at the WTR rate of 20 g kg -1 for both soils. The yield of lettuce reache d the maximum at the WTR rate of 50 g kg -1 for both soils (Fig. 4-1). A negative relationship occurred between the shoot dry matter yields and shoot Cu concentrations for both plant species. The c oefficients of correlation (r) were -0.47 and 0.55, respectively for ryegrass and lettuce (p<0.05). This is in agreement with the 74

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results of Gharbi et al. (2005) regarding plant response to Cu toxicity. The addition of Ca-WTR changed soil pH and the availabi lity of Cu and other nutrients. The uptake of Cu by ryegrass decreased with increasing amendme nts rate for all soils (Table 4-5). In comparison, Cu uptake by lettuce was less affected by Ca-WTR amendment, probably due to its soil-plant barrier to accumula tion of Cu in excess of normal levels. The positive effect of Ca-WTR on reduced uptake of heavy metals by plants has generally been explained by the increase in soil pH. Moreover, according to the Terrestrial Biotic Ligand Model (TBLM), Ca and Mg ions can compete with divalent metals for biotic ligan ds (e.g., roots) ( Thakali et al., 2006 ). Alva et al. (1993) reported that increased Ca availability in the rooting environment, applied either as CaCO 3 or CaSO 4 significantly decreased concentrations of Cu in the fibrous roots and therefore ameliorated the effects of Cu phytotoxicity The Ca-WTR introduced a large amount of Ca ions, which may compete with Cu for plant uptake. Effects of Ca-WTR Amendment on Uptake of Other Nutrients The Ca-WTR increased soil pH and subsequent ly influenced the availability of other nutrients. Phosphate deficie ncy is often a liming factor, as soluble P can react with Ca to form a series of products of phosphates with decreasing solubility (i.e. slightly soluble dicalcium phosphate, very low solubilit y tricalcium phosphate). In this study, no symptoms of P deficiency (purpling of some leaves) were observed. Ahmed et al. (1997) reported that long-term P deficiency problems were not evident at the field scale, although P concentrations in lawn grass tissue decreased in neutral to alkaline pH in the pot experiment. We also observed that increasing amending ra tes led to a decrease of zinc (Zn) in the tissue. The concentration in Cu-enriched soils at t he highest WTR treatment rates 75

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was 6-8 mg kg -1 dry matter, which was lower than t he sufficient Zn concentration (10 mg/kg) (Reuter and Robinson, 1997). In most st udies, Zn availability is considered more sensitive to soil pH increase (Smith, 1994). And this might partly explain the yield reduction at high treatment rates. No other deficiency symptoms were observed. Conclusion Overall, Ca-WTR proved to be a promising am endment for the in situ immobilization of Cu in slightly to se verely Cupolluted soils. Application of Ca-WTR at adequate amounts could effectively improve plant growth and reduce plant uptake of Cu in Cu-contaminated soils. In general appropriate amounts of WTR addition to soils should not lead to serious effects on either soil chemical properties or the crop. Ca-WTR is available in very large quantities and at a low price, making it an economically viable option. Questions relating to possible adverse effects on soil structure or other soil properties, plant nutrient imbalance from superfluous amendments application, and the long-term stability of heavy metal binding, in particular under acidifying conditions still remain to be answered. 76

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Table 4-1. Selected physical and chemical properties of tested soils and calcium type water treatment residue (Ca-WTR) Property Alfisol Spodosol Ca-WTR (H 2 O) 5.45 4.66 9.05 pH (KCl) 4.48 3.5 8.69 EC (S cm -1 ) 140 151 659 Total C (g kg -1 ) 3.29 9.24 112 Total N (g kg -1 ) 0.29 0.69 0.19 Organic matter (g kg -1 ) 5.7 15.9 -Particle distribution ( g kg -1 ) Sandy 945 909 12 Silt 43 51 136 Clay 12 40 852 Table 4-2. Total recoverable soil elements in soils Soils Al Ca Fe K Mg Na P Cu Ni Mn Pb Zn ----------------------------g kg -1 ------------------------------------------mg kg -1 ---------------Alfisol 1.87 1.04 1.62 0.06 0.45 0.06 0.22 84.7 0.59 62.5 7.49 59.2 Spodosol 1.02 1.19 1.03 0.05 0.4 0.07 0.18 134 0.87 59.4 5.29 26.9 77

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Table 4-3. Effect of Ca-WTR am endment on soil pH and extractable Cu Mix Ca-WTR application Rate pH (H 2 O) Mehlich-3 extractable Cu CaCl 2 extractable Cu g kg -1 mg kg -1 Original Alfisol 0 5.45 39.9 0.63 5 6.38 35.6 0.33 10 6.56 26.3 0.20 20 7.00 24.8 0.17 50 7.30 25.1 0.20 Cu enriched Alfisol 0 4.85 929 1.01 5 5.86 853 0.86 10 6.50 779 0.53 20 7.07 758 0.28 50 7.25 743 0.29 Original Spodosol 0 4.66 68.3 0.035 5 5.90 46.4 0.026 10 6.39 52.9 0.030 50 6.66 53.2 0.020 100 7.25 46.1 0.011 Cu enriched Spodosol 0 4.18 882 0.82 5 5.89 853 0..51 10 6.18 599 0.29 50 6.69 636 0.17 100 6.98 656 0.15 78

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Table 4-4. Correlation coefficients (r ) between plants (ryegrass and lettuce) shoots tissue concentration and soil pH and extrac table Cu as estimated by Mehlich3, and 0.01 M CaCl 2 extraction procedures pH (H2O) Mehlich-3 extractable Cu CaCl 2 extractable Cu Ryegrass tissue Cu concentration -0.53* 0.59** 0.80*** Lettuce tissue Cu concentration -0.55* 0.40 NS 0.20 NS *p<0.05; **p<0.01; ***p<0.001; NS not significant. Table 4-5. Total Cu uptake in shoots of ryegrass and lettuce determined at the end of 8 weeks of growth. Letters indicate significant differences between least squares means within soil at a 95% confidence level (LSD) Ca-WTR application rate Ryegrass ( g pot -1 ) Lettuce ( g pot -1 ) g kg -1 Original soil Cu enriched soil Original soil Cu enriched soil Alfisol 0 591a 0d 150a 0c 5 366b 359a 179a 23.0b 10 290bc 315a 173a 70.3a 20 230cd 216ab 189a 65.7a 50 185d 156bc 136a 43.8ab Spodosol 0 161a 0d 57.5a 0c 5 146a 339a 59.4a 40.1b 10 166a 174b 89.1a 72.7a 50 110b 131b 80.5a 87.2a 100 107b 64.5c 77.8a 68.3ab 79

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051050100Shoot DMY for lettuce (g pot-1) 0 1 2 3 4 5 6 Shoot DMY for ryegrass (g pot-1) 0 1 2 3 4 5 6 Original soil Cu enriched soil A B C DAlfisol Spodosol Shoot DMY for ryegrass (g pot-1) 0 2 4 6 8 10 12 14 05102050Shoot DMY for lettuce (g pot-1) 0 2 4 6 8 10 12 Ca-WTR application rate (mg kg-1 soil) Ca-WTR application rate (mg kg-1 soil) Figure 4-1. Relationship between plant shoot dry matter yields afte r 8 weeks of growth and Ca-WTR treatment rates. A) ryegrass in Alfisol. B) lettuce in Alfisol. C) ryegrass in Spodosol. D) lettuce in Spodosol. 80

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A Ca-WTR amendment rate (mg kg-1 soil) 0 5 10 50 100Cu in shoot (mg kg -1 dry matter) 0 50 100 150 200 b 0 5 10 20 50Cu in shoot (mg kg -1 dry matter) 0 50 100 150 200 Ryegrass in original soil Ryegrass in Cu enriched soil Lettuce in original soil Lettuce in Cu enriched soil B Figure 4-2. Relationship between plants (ryegr ass and lettuce) shoots Cu concentration and Ca-WTR treatment rates. A) Alfisol. B) Spodosol. 81

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CHAPTER 5 AMENDMENT OF CALCIUM-WATER TREATM ENT RESIDUE (CA-WTR) ON CITRUS GROWTH AND COPPER LOADING IN SU RFACE RUNOFF IN SOUTH FLORIDA Introduction There is a growing environmental concer n about the extent of Cu pollution in agricultural soils resulting from utilization of fertilizer, fungicides, and pesticides. Copper based products have been used to fight against diseases and sustain adequate yields in citrus growing fields for over 100 years in t he Indian River area, Sout h Florida. Since the early 1900's, Cu has been applied for controlling citrus dieback in the rates ranging from 10 to 25 kg Cu ha -1 yr -1 (Alva and Graham, 1991). The application dose has increased in recent years as a common practice to pr event and cure citrus canker (Albrigo et al., 2005). Background concentration of Cu in the unpol luted surface layer of Floridian soils is averaged 3.7 mg kg -1 (Ma et al., 1997), whereas the concentrations ranging from 100 to 1200 mg Cu kg -1 soil have been documented in the Indian River area (SFWMD, 2001). Alva et al. (1993) reported that the half maxima l effective concentration (EC50) values for citrus ( Citrus limon and Citrus reticulata ) seedlings are approximately 200 mg kg -1 for aboveground biomass, grown in a sandy soil with a low CEC. Soil Cu has also been reported to be trans ported into the surface water through surface runoff and there is increasing concern that raised Cu levels in sediment may cause the degradation of aquatic ecosystems such as the St. Lucie River (Zhang et al. 2003a; He et al., 2004; 2005). The stability of Cu in soil is strongly pH dependent Cu is most mobile in an acidic soil and the mobility is usually the lowest at neutral to slightly alkaline pH, and increases under highly alkaline conditions. Most of th e soils under commercial citrus production in the Indian River area of Florida are sandy, acidic, and low in organic matter in the 82

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surface layer, but have a impermeable spodic or argillic horizon in the B horizon. As a result, Cu released from soil to surfac e runoff can be a nonpoint source of Cu for receiving water bodies. Considerable effort has been directed at developing reliable and cost-effective technologies to control metal bioavailability and mobility in contaminated soils. Physical remediation approaches, such as soil excava tion and landfilling, are costly and affect soil quality and crop production. Phytoremediation is cost-effective but requires more time to achieve remediation goals. In com parison, chemical remediation can accomplish remediation goals within a short term at a reasonable cost. Compared to those ex situ approaches involving excavation, in situ stabilization can limit their mobility and bioavailability by inducing changes in meta l biogeochemistry. In sites where the metal contamination is concentrated near the soil su rface, the application of materials capable of immobilizing metals would often be appropr iate. The most common practice is liming in the remediation of Cu-contaminat ed acidic soils (Gray et al., 2006). In laboratory simulation studies, treatment with Ca-water plant residue (Ca-WTR, pH 9.06, containing mainly CaCO 3 and minor CaO) was shown to significantly reduce the leaching of Cu in acidic sandy soils (C hapter 3). In this study, field trials were conducted in parallel to evaluate the effectiveness of Ca-WTR amendment in reducing Cu loading in surface runoff and the effects on soil quality, Cu availability, and citrus growth. Materials and Methods Site Description and Amendment Characterization Two field sites located in the Indian River area, South Florida were selected for this study, one with navel or ange and the other with grapefru it in a representative 83

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commercial citrus grove with citrus trees planted in 1988. The soil was a Riviera fine sand (Loamy, siliceous, active, hyperthermic Arenic Glossaqualfs). Each field site consisted of a control plot (number 31 for navel orange and 81 for grapefruit, respectively and a plot amended with Ca-WTR (C36 for navel orange and C86 for grapefruit, respectively). Selected properties of the soils from the two field sites are given in Table 5-1. Ca-WTR was collected from the Fort Pier ce Utility Authority facility (FPUA), Fort Pierce, Florida. It is a byproduct of drinking water purification, containing mainly CaCO 3 and minor CaO, with a pH of 9.05 and negligib le amounts of nutrients and heavy metals. This material is safe for agricultural use. Florida Department of Environmental Protection does not require permit for use of this type of material as a soil amendment. The FPUA annually generates approximately 5000 me tric tons of this material, which is available at a minimal cost. This soil amendment was applied to the citrus groves annually using a mechanic spreader at the rate of 5 ton ha -1 (oven dry basis). The material was evenly applied to the soil surface under the citrus trees. Sample Collection and Analysis Soil samples were collected every six-mont hs from 0-15 and 15-30 cm soil depths of each field plot. The soils were air-d ried, ground, and passed through a 2-mm sieve prior to physical and chemical analyses. Soil pH (1:1 soil: water ratio) and electrical conductivity (1:2 soil: water ratio) we re measured in deionized water using a pH/ion/conductivity meter (Denver Inst rument, CO). Total soil carbon (C) was determined by combustion using a C/N anal yzer (Vario MAX CN Macro Elemental Analyzer; Elemental Analysensystem GmbH, Hanau, Germany). Soil available metals were determined by extracting the soil wit h Mehlich-3 solution (Mehlich, 1984) and 84

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measuring the concentrations of metal usin g an inductively coupled plasma optical emission spectrometer (ICPO ES) (Ultima, JY Horiba Group, Edison, NJ) following EPA method 200.7. Briefly, 4.0 g air-dried soil sa mples were weighed, into 50-ml polystyrene centrifuge tubes, and 40 ml of Mehlich-3 extractant were added. At the end of extractions, the soil suspensions were centrifuged at 7500 g for 30 min and the supernatant was filtered th rough a Whatman #42 f ilter paper to remove any suspended materials. Surface runoff water samples were co llected by autosamplers (SIGMA 900MAX portable sampler), which were installed at the end of PVC drainage pipe that leads water from a furrow to t he canal for each plot. The autosamplers were checked periodically to ensure normal performance and to collect water samples from the sampler to the laboratory for immediate analysis. Prior to filtration, pH and EC of the water samples were determined using a pH/ion/conductivity meter following EPA methods 120.1 and 150.1, respectively. Turbidit y of water samples was measured using a turbidity meter (DRT-100B, HF Scientific Inc. Fort Myers, FL). Solid concentrations of the water samples were measured using a gravimetric method wit h oven drying. Subsamples were filtered through Whatman #42 f ilter paper. Portions of the sub-samples were further filtered through a 0.45 m membrane filter for the measurement of anions, total dissolved P, and metals. The concentrations of anions (including F, Cl, Br, NO 3 -N, PO 4 3-P, and SO 4 2 S) were measured within 24 hrs a fter sample collection using an ionic chromatograph (ICS 2000; Dionex Corporation Sunnyvale, Calif.) following EPA Method 300.0. The concentrations of disso lved macroand microelements in water were determined using the ICP-OES. Ammoni um was determined using an N/P discrete 85

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autoanalyzer (EasyChem, Systea Scientific, Italy), and dissolved organic carbon (DOC) was determined using a TOC/ TN analyzer (liquiTOC/TN b Elemental Analyzer, Elemental Analysensystem GmbH, Hanau, Germany). The information obtained from the measurements of solution was used to estimate chemical speciation using the chemical equi librium model Visual MINTEQ, version 2.40 (Gustafsson, 2010). The requir ed input parameters included t he major ionic species, pH, redox potential, temperature and ionic st rength. The MINTEQ model contains a dissolved organic matter (DOM) submodel to compute the complexation of metals with DOM; it is a composite ligan d model with a Gaussian affinity distribution (Christensen and Christensen, 1999). This study assumed that all DOM was DOC (Dean et al. 2005). The ionic strength was related to the conduc tivity measurements (Snoeyink and Jenkins 1980) as according to the following equation: I 1.6 10 -5 EC Six-month old spring flush leaves were randomly sampled at mid-July each year from healthy trees in each field site for determining mineral concentrations. The leaf samples were washed in dilu te detergent, rinsed several ti mes in tap water, soaked in 5% HCl for 20 seconds, and rinsed in distilled water. The plant samples were dried at 70C for 72 hours, ground using a micro st ainless ball mill and passed through a 0.4mm sieve. Ground plant sample (0.40 g) wa s digested with 5 ml of concentrated HNO 3 using an A.I. digestion system (A.I. scientific, In c., USA). The concentrations of P, Ca, Mg, K, Na, Cu, Zn, Pb, Cd, Cr, Fe, Mn, and Ni in the digester were determined using the ICPAES. Total N content was determined us ing a CN-analyzer (Vario MAX CN Macro Elemental Analyzer, Elemental Anal ysensystem GmbH, Hanau, Germany). 86

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Representative citrus fruit samples were collected in the period of DecemberJanuary, each year for quality and nutrient analysi s. External (fruit size) and internal quality properties of the fruit including juice, Brix, acid, and soluble solids were analyzed following the standard procedures at the postharvest laborator y, Indian River Research and Education Center, Fort Pierce. Subsam ples of the fruit were cleaned using deionized water, cut into small pieces, and oven dried at 70C, and the dried fruit material was ground to <1 mm prior to analysi s. Fruit N, P, and metals were analyzed using the same procedure as leaf samples. Results and Discussion Soil Quality Characterization and Monitoring The soils were representative of domi nant soils under agricultural production systems in the Indian River area. Most of the soils were very sandy, consisting of >9095% of sand, with low organic matter in surf ace layer, and with pH ranging from 4.4 to 6.6 (Table 5-1). Soil available Cu, as estimated by Mehlich-3, was 112 and 102 mg kg -1 in the surface layer for the control fiel d and amended sites, respectively; whereas the corresponding values for the total recover able Cu concentrations were 162 and 160 mg kg -1 The subsurface layer had significantly lower Cu concentrations than the surface soil. Application of Ca-WTR increased soil pH. However, soil amendment generally decreased Mehlich-3 extractabl e Cu, which was likely due to raised pH by the applied WTR. The effect of soil amendment on the availability of Cu in soil will be further discussed based on the data obtai ned from leaf analysis. Theoretically, lime materials application can increase soil pH and thereby cause metal immobilization through (a) an increase in negative surface c harge of organic matter and me tal oxides (resulting in 87

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increased cation adsorption), (b) promotion of hydrolysis reactions leading to the formation of precipitates, and/or (c) promot ion of metal coprecip itation with carbonate (Naidu et al., 1994; Mench et al., 1998) Water Quality and Cu Speciation The physical and chemical properties in cluding pH, EC, and concentrations of inorganic ligands and metals of the surfac e runoff water samples collected during the project life period are summarized in Table 5-2. These quality characteristics varied seasonally and among the field sites, as affected by soil proper ties, rainfall, irrigation, crop species, and management practices. The annual mean pH values of the runoff water samples from Ca-WTR treated plots (s ites 36 and 86) were gener ally higher than those from the control plots (sites 31 and 81), due to the limi ng effect of the amendment. For instance, the mean pH value of runoff water samples is 7.64 for the C36 (Ca-WTR treated plot), and 7.05 for site 31 (the corresponding control pl ot). Similar results were obtained from the grapefruit fiel d site (site 86 vs. 81) (Table 5-2). The application of CaWTR tended to increase EC and decreased the concentrations of N and P and metals (Cu, Fe, Zn) in surface runoff waters w hereas the concentrations of other elements were comparable between the Ca-WTR treated and the control plot (Table 5-2). These results indicate that Ca-WTR is effective in reducing Cu loading in surface runoff for the Cu-contaminated acidic soils, thus decreasin g Cu transport from land to water Copper speciation in runoff waters wa s calculated using the thermodynamic equilibrium model MINTEQ. In the Gaussian model it is assumed that the concentration of individual ligands of the metalDOM co mplex are normally distributed with respect to their log K (equilibrium const ant) value (Allison et al. 199 1). The equilibrium distribution of metal-DOM complexes is dictated by the relative log K values and the relative free 88

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activities of all competing reactants. All reactions for protonation and metal complexation assume 1:1 stoichiometry bet ween the complexing anions and the ligand (Allison et al. 1991). The concentrations of Cu -DOM complexes in runoff waters were on average reduced by 49.6% and 21.2%, respective ly for the navel orange and grapefruit field sites, whereas the corresponding va lues for the concentrations of free Cu 2+ were 56.4% and 28.8% (Table 5-3). Among the aqueous Cu species, Cu 2+ is considered to be most bioavailable to aquatic organisms in acute exposures (Santore et al. 2001). Increasing evidence indicates that bioavailability is not only c ontrolled by the free metal concentrations but also by the kinetics of dissociation of me tal complexes (Vasconcelos et al., 1997). Among complexed species of copper hydroxyl species Cu(OH) + and Cu(OH) 2 (aq) display some toxicity (Meador, 1991) while copper -inorganic carbon species (e.g. CuHCO 3 + CuCO 3 and Cu(CO 3 ) 2 2 ) and chloro species (CuCl n ( n 2) ) are much less toxic or not toxic at all (Devez et al., 2005; Huang and Wang, 2003; Wang et al., 2002). Most of the dissolved Cu (> 50%) in t he runoff water was complexed with DOM, which is compatible with the results from soil Cu fractionation that organic-bound Cu was the dominant fraction in these soils (see Chapter 2). Similar results were reported by Sajwan et al. (2006) who reported that the fraction of organi c Cu and exchangeable Cu (free Cu 2+) in soils from Florida citrus gr oves and Savannah (Georgia) soils ranged from 32% to 59% and 1% to 4% of total Cu, respectively. He et al. (2006) also reported up to 70% of total dissolved Cu released from Florida soils was in organic complexes. The fraction of organic Cu complexes varies, depending on soil organic matter content. 89

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Saeki et al. (2002) reported t hat the fraction of organic Cu complexes ranged from 1% to 56% of total Cu in soil solution that had soluble organic carbon from 1 to 102 mg/L. Fruit Nutrient Contents and Yields The concentrations of macro and micro-el ements in citrus leaf and fruit samples collected from the 2006-2008 period are presented in Tables 56 and 5-7, respectively. Leaf N concentrations were 26.0-26.8 g kg -1 for the navel orange and 20.9-21.2 g kg -1 for the grapefruit, which are withi n the optimal N range (25-27 g kg -1 for orange and 2022 g kg -1 for grapefruit) for the two crops. Soil amendment of Ca-WTR had a minimal effect on leaf N concentration of the citrus trees. Leaf P concentrations were slightly decreased by Ca-WTR application, but still within the opti mal range (18-20 g kg -1 for orange and 11-13 g kg -1 for grapefruit). Soil amendment with Ca-WTR increased Ca and Mg concentrations in plant, which may be important for improving fruit quality and yield, particularly in acidic soils. The concentra tions of Cu, Fe, Zn and Mn in citrus leaf samples were in the normal range (Reuter and Robinson 1997). No significant difference was found in leaf Cu concentra tion between the cont rol and amended plots while leaf Fe concentration was decreased by Ca-WTR application (Table 5-4). Soil amendment with Ca-WTR tended to increase Ca, K, and Mg concentrations in fruit, but had a minimal influence on N and P concentrations in fruit (Table 5-5). The increase in Ca, Mg and K concentration in fruit may improve fruit storage life. The concentrations of heavy metals including Cd, Co, Cr, Ni, Pb and Mn in fruit were mostly not detectable, and soil amendment with Ca-WTR appeared to increase Cu, Fe and Zn concentrations in fruit, which may be beneficial to human heal th. These results indicate that soil amendment with Ca-WTR generally improve mineral nutrition of citrus trees, particularly 90

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Ca and Mg and improve fruit mineral nutriti on value by increased Ca, Mg, Cu, Fe, and Zn density in fruit. The data of fruit quality and yields for the 2006-2008 production period are presented in Table 5-6 and Fig. 5-3, re spectively. Soil amendment with Ca-WTR increased fruit size by 9% for orange and 11% for grapefruit (Table 56). Internal quality of fruit was also improve as reflected in the increases in juice and solids content and Brix/acid ratio and a decrease in acidity. These results indicate that Ca-WTR amendment can improve fruit quality and enhance fruit ma turity because of the improved nutrition condition with Ca, Mg, an d K. Alva et al. (1993) reported that increasing rates of Ca, applied either as CaCO 3 or CaSO 4 significantly decreased the concentrations of Cu in the fibrous roots, whic h indicate that increased Ca availability in the rooting environment ameliorates the effect s of Cu phytotoxicity. Based on the yield data of grapefruit yield for th ree years, and navel orange yield for one year (Fig. 5-3), it was quite convincing that Ca-WTR amendment increased fruit yield by 10 to 50%. Conclusions By integrating all the information from the analyses of soil and water quality, plant nutrition and fruit quality, it can be concl uded that the amendment of Ca-WTR to Cucontaminated sandy acidic soils can reduc e Cu loading in surface runoff water and nutrient (N, P) losses and improve soil cond itions such as raised soil pH and Ca concentration and reduced Cu toxic stress. These in turn improve citrus nutrition and subsequently fruit quality and yield. Further research should continue to focus on the sustainability of the treatment by investigating the long-term efficiency of Ca-WTR and the effect of the soil properties on the treatment efficiency. In addition, monitoring 91

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92 should be conducted over longer periods, an d toxicity assays could be developed to determine genotoxicity both in vitro and in vivo.

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Table 5-1. Soil selected properties of different field sites before initiation of the project Site Crop Site area Depth Texture (g kg -1 ) pH (H 2 O) Organic C CEC Cu (g kg -1 ) m 2 cm Sand Silt Clay g kg -1 cmol kg -1 M3 extractable Total recoverable 0-15 951 33.2.1 15.6.2 4.4.1 21.1.5 7.6.4 112.95 162 31 / 36 Navel orange 1822 15-30 986 10.5.9 3.0.1 4.3. 1 7.9.5 2.8.5 80.0.2 156 0-15 966 22.6.0 11.5.2 5.3.2 5.7.1 4.6.7 102.81 160 81 / 86 Red flame Grapefruit 2242 15-30 976 17.2.6 7.1.8 5.6.1 1.1.1 1.4.4 42.71.65 79.9 93

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94 Table 5-2. Mean concentrations of water quality related cons tituents in surface runoff sample s from the four field sites from 2006 to 2009 Site pH EC TSS DOC Inorganic ligands F Cl Br NO 3 -N PO 4 3-P SO 4 2-S NH 4 + -N S cm -1 g L -1 mg l -1 --------------------------------------------mmol L -1 -------------------------------------------31 7.05 921 0.51 30.0 0.0300 4.1467 0.0083 0.0849 0.0297 0.9355 0.0560 36 7.64 1548 0.85 39.5 0.0270 7.2854 0.0126 0.0441 0.0158 2.3558 0.0340 81 7.16 1296 0.71 33.2 0.0260 9.3217 0.0114 0.0534 0.0236 1.4511 0.0305 86 7.49 1277 0.74 27.6 0.0172 8.1182 0.0124 0.0900 0.0069 0.9738 0.0201 Metals Ca K Mg Na B Cd Co Cr Cu Fe Mn Mo Ni Pb Zn -----------------------------------------------------------------------------mmol L -1 ---------------------------------------------------------------------------31 1.76 0.78 0.69 2.80 0.0088 ND 0.0002 0.0002 0.0024 0.0048 0.0014 ND 0.0003 0.0001 0.0013 36 2.99 0.78 0.98 4.03 0.0071 0.0001 0.0003 0.0002 0.0015 0.0033 0.0050 ND 0.0005 0.0002 0.0009 81 2.53 0.42 0.83 4.38 0.0099 ND 0.0001 0.0001 0.0023 0.0097 0.0046 ND 0.0003 0.0002 0.0015 86 2.23 0.39 0.71 3.77 0.0065 0.0002 0.0003 0.0002 0.0020 0.0032 0.0005 0.0001 0.0006 0.0003 0.0007

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Table 5-3. Percentage distribution of various Cu species in surface runoff samples from the four field sites Sites Concent. Cu Cu +2 CuDOM CuOH + Cu(OH) 2 Cu 2 (OH) 2 +2 CuSO 4 CuHPO 4 CuCO 3 CuHCO 3 + mM ----------------------------------------------------------%-----------------------------------------------------------31 0.0024 15.0 77.3 3.7 0.1 <0.1 1.0 0.9 1.8 <0.1 36 0.0015 12.1 53.9 10.7 0.7 0.1 1.6 0.4 20.0 0.1 81 0.0023 17.1 71.8 5.2 0.1 <0.1 1.5 0.8 3.2 <0.1 86 0.0020 15.5 58.8 10.0 0.5 0.2 0.9 0.3 13.4 0.1 Table 5-4. Selected elemental composition of citrus leaf samples from and the Ca-WTR amended and the control field sites Site N P K Ca Mg Fe Mn Zn Cu --------------------------g kg-1-------------------------------------------mg kg-1--------------------2006 31 28.6 1.79 27.39 44.77 2.69 50.6 41.9 52.4 16.5 36 29.2 1.71 25.76 47.14 3.07 49.2 84.0 109.9 27.2 81 21.6 1.37 11.58 40.39 4.10 45.6 82.0 116.0 57.8 86 21.4 0.53 9.46 43.00 5.13 34.1 62.4 100.4 53.7 2007 31 25.3 1.89 19.40 19.50 2.64 69.3 12.6 21.0 24.5 36 27.0 1.98 20.50 17.00 2.65 45.2 9.0 21.4 18.4 81 22.1 1.24 10.80 38.00 3.51 28.6 10.0 22.6 61.2 86 22.3 1.39 13.20 47.10 3.77 18.1 8.1 17.0 73.5 2008 31 24.0 2.01 18.80 17.70 2.54 46.3 20.4 22.1 15.2 36 24.3 1.92 19.30 22.90 2.80 41.9 33.0 29.3 25.0 81 19.8 1.54 11.80 40.70 3.74 51.2 102.2 110.7 27.1 86 19.1 1.53 12.50 53.90 4.29 46.0 54.2 57.9 30.5 95

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Table 5-5. Selected elemental composition of citrus fruit samples from the Ca-WTR amended and the control field sites Site N P K Ca Mg Fe Mn Zn Cu --------------------------g kg-1--------------------------------------------mg kg-1-----------------2006 31 11.57 1.22 11.46 4.65 0.86 25.69 U<0.1 3.82 5.63 36 9.38 1.08 9.88 4.83 0.78 20.00 0.02 10.17 8.32 81 8.39 0.96 10.51 6.67 0.84 12.98 U<0.1 8.30 8.66 86 8.74 1.22 10.42 6.92 1.29 10.53 U<0.1 2.91 7.06 2007 31 7.66 1.22 11.84 2.86 0.74 29.20 U<0.1 8.62 6.26 36 6.95 1.11 13.74 3.82 0.75 15.60 U<0.1 5.38 3.82 81 7.75 1.14 11.49 4.05 0.89 16.90 U<0.1 5.22 2.36 86 5.82 1.01 12.44 6.04 0.74 35.20 U<0.1 2.92 6.23 2008 31 11.60 1.18 10.28 2.83 0.86 26.68 U<0.1 5.26 5.15 36 9.38 1.25 10.72 2.30 0.64 31.61 U<0.1 3.77 4.67 81 8.39 0.99 9.80 4.55 0.75 25.43 0.03 6.64 4.36 86 8.74 1.13 10.48 5.25 0.72 46.84 U<0.1 5.53 4.07 96

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Table 5-6. External and internal quality parameters of fruit samples from the Ca-WTR amended and the control field sites Field ID Fruit size (kg/fruit) Juice content (%) Brix (%) Acid (%) Ratio Solids 2006 31 0.40 42.5 11.5 0.65 17.7 4.89 36 0.46 40.2 11.6 0.57 20.4 4.66 81 0.32 51.8 11.1 1.30 8.6 5.75 86 0.33 51.0 11.3 1.30 8.7 5.76 2007 31 0.30 35.1 11.2 0.60 18.5 3.93 36 0.31 40.6 11.7 0.59 19.9 4.73 81 0.40 45.9 10.8 1.08 10.0 4.96 86 0.47 49.7 10.8 1.24 8.7 5.34 2008 31 0.33 51.2 11.9 0.64 18.7 5.49 36 0.35 52.9 11.9 0.61 19.5 5.66 81 0.36 53.3 10.3 1.25 8.2 4.50 86 0.41 54.1 10.2 1.10 9.2 4.50 97

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2006 Jul2007 Jan2007 Jul2008 Jan2008 Jul2009 JanpH (H2O) 3 4 5 6 7 31 0-15cm 36 0-15cm 31 15-30cm 36 15-30cm 2006 Jul2007 Jan2007 Jul2008 Jan2008 Jul2009 JanpH (H2O) 3 4 5 6 7 8 81 0-15cm 86 0-15cm 81 15-30cm 86 15-30cm Figure 5-1. Dynamic changes of soil pH in the Ca-WTR amended and the control field sites. 98

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2006 Jul2007 Jan2007 Jul2008 Jan2008 Jul2009 JanCu mg kg-1 20 40 60 80 100 120 31 0-15cm 31 15-30cm 36 0-15cm 36 15-30cm 2006 Jul2007 Jan2007 Jul2008 Jan2008 Jul2009 JanCu mg kg-1 20 40 60 80 100 81 0-15cm 81 15-30cm 86 0-15cm 86 15-30cm Figure 5-2. Dynamic change of Mehlich-3 extractable Cu in soils of the Ca-WTR amended and the control field sites. 99

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Figure 5-3. Citrus fruit yi eld of the WTR amended and the control field sites during the 2006-2009 period. 100

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CHAPTER 6 SUMMARY, CONCLUSIONS AND PERSPECTIVE Soil pollution by Cu is on the rise worldwide due to anthropogenic inputs of both point and non-point sources. Florida has a long history of citr us production. Coppercontaining fungicides have been increasingly used for preventing diseases and sustaining desired yields of citrus for over 100 years. As a result, substantial amounts of Cu have been accumulated in agricultural soil s, with total Cu concentrations being over 1000 ppm. Copper contamination causes so il degradation with reduced microbial activities and phytotoxicity with reduced fruit yield and quality. It also poses a severe threat to the aquatic environment as Cu c an be transported from land to water by surface runoff water or leaching. The mobility and availability of Cu in soils are controlled by many chemical processes, su ch as precipitationdissolution, adsorption desorption, and chelation. Transport of Cu fr om land to surface waters through surface runoff and/or leaching has been identified as a problem, since aquatic organisms are more susceptible to Cu contamination t han terrestrial plants. The concentrations and loads of Cu in runoff water from agricult ural field are often correlated with soil Cu accumulation. There is increasing public conc ern over Cu pollution to land and water. Research is needed to develop cost-effect ive remediation strategies for Cucontaminated soils in south Florida and elsewhere in the world. The remediation of heavy metal (such as Cu) contaminated soils includes physical, chemical and biological approaches. Physi cal approach involves scavenging of contaminated soils or washing out of contam inants using large quant ity of water, which is often costly and causes degradation of t he soil. Phytoremediation is cost-effective and environmentally friendly, but it takes longer time to accomp lish the remediation 101

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goals. In comparison, chemical remediation is less costly than physical but it can reach the goals within a short period. In this study, field survey, l aboratory incubation and column leaching, greenhouse experiments and fiel d trials were conducted to understand chemical transformation of Cu during the contamination and remediation processes and to evaluate the effectiveness of Ca-WTR for remediating Cu-contaminated soils, including reduction in the availability to plants, leaching potential, and loading of Cu in surface runoff as well as effects on citrus yield and quality. The results are summarized as follows. Copper transformation, availability and l eaching potential in contaminated soils Significant accumulation and increased avail ability and mobility of Cu occurred in soils with increasing citrus production period. Copper contamination causes, to variable degree soil acidification, which in turn enhanced Cu leaching along soil profiles. Available Cu estimated by Mehlich-3, or NH 4 OAc extraction was highly correlated with total recoverable Cu, whereas the relationship between CaCl 2 extractable Cu and total recoverable Cu was affected by soil pH, with a greater slope of the re lationship curve at soil pH <6.5. Vertical movement of Cu in a soil profile was more pronounced at pH <6.5. The largest proportion of soil Cu was presen t as organically-bound, ranging from 43% to 62%. Both recoverable Cu and Mehlich-3 extr actable Cu were significantly correlated with this Cu fraction (P<0.001). These result s indicate that significant accumulation of Cu occurred in citrus grove soils due to f ungicide application, and the availability and mobility of Cu in the soils were increased by Cu accumulation and soil acidification as a result of soil Cu contamination. However, the elevated soil pH could be a significant factor in decreasing Cu migrati on of from soil to water body. 102

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Ca-WTR amendment affects Cu fr actionation and leaching potential In laboratory incubation and column leac hing studies, amendment of Ca-WTR (pH 9.06, containing mainly CaCO3 and minor CaO), an industrial by-product was found to be effective to reduce Cu mobility and trans port to water body by laboratory incubation and column leaching experiments. The incubation study showed that Ca-WTR amendment significantly raised soil pH and de creased water soluble and exchangeable Cu by >62% in control soil and >90% in a Cu-enriched soils due to increases in oxidesbound and residual Cu in the soils. The results from column leaching agreed with those from incubation study. The cumulative amount of Cu in the leachate after 10 leaching events was reduced by 80% and 73%, respecti vely for Alfisol and Spodosol at the highest Ca-WTR amendment rate (20 g kg -1 for Alfisol and 100 g kg -1 for Spodosol). These results indicate that Ca-WTR can effe ctively raise soil pH and convert labile Cu to more stable Cu forms in the soils. A pH value of 6.5 was found to be critical for lowering Cu availability in soils. Based on th is criterion and pH response curve to CaWTR application, the optimal rates of Ca -WTR can be estimated for different Cucontaminated soils. Ca-WTR amendment reduces Cu toxici ty to plants and enhances crop growth A greenhouse study was conducted to test the ability of Ca-WTR to reduce the toxicity and uptake of Cu by crop plants in Cu-contaminated sandy soils. Ryegrass ( Lolium perenne L.) and lettuce ( Lactuca sativa L.) were used as indicator crop plants and grown for eight weeks in the Cu-contaminated soils (Alfisol and Spodosol with and without incubation of 1000 mg Cu kg -1 soil) amended with different levels of Ca-WTR (from 5 g kg-1 to 100 g kg1 soil). The growth of plants wa s inhibited in the lightly Cu103

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contaminated soils (about100 mg kg -1 ), but both the plants were killed in the severely Cu-contaminated soils (added with 1000 mg kg -1 ). Overall, plant biomass yields increased with WTR application rates at the low levels (5-20 g kg -1 for Alfisol and 5-50 g kg -1 for Spodosol), reached maximum at the moderate level (10-50 g kg -1 ) and started to decrease at the high levels (>20 g kg -1 for Alfisol and >50 g kg -1 for Spodosol). Copper concentrations in ryegrass shoots decreased significantly with increasing Ca-WTR application rates. For lettuce, plant Cu c oncentration decreased only at the high CaWTR rates. In addition, Ryegrass has a relatively higher potential for Cu uptake and translocation than lettuce in both soils. Effects of Ca-WTR amendment on copper loading in surface runoff and citrus growth Field trials were conducted in represent ative commercial citrus groves in the Indian River area, South Florida to evaluat e the effectiveness of Ca-WTR for reducing Cu loading in surface runoff and subsequent influence on soil Cu availability and citrus growth. The concentrations of Cu in soils a nd surface runoff were m onitored over a 3-yr period at two field sites on an Alfisol under navel orange and grapefruit production. Soil amendment with Ca-WTR generally raised soil pH and Mehlich-3 extractable Ca, but decreased Mehlich-3 extractabl e Cu. The mean concentrations of Cu in surface runoff water were reduced by 35% and 14% durin g the 2006-2009 period for the navel orange and grapefruit site, respectively. The results of ion speciation of Cu in the runoff water using MINTEQ model indicated that Cu complexes with dissolved organic matter dominated Cu speciation and the application of Ca-WTR decreased the concentrations of free Cu 2+ by 49.6% and 21.2%, respectively fo r the naval orange and grapefruit site. The field observation agreed with the results fr om our lab leaching studies that raised 104

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pH increased Cu retention, likely th rough surface precipitation of CuCO 3 and Fe and Al hydrolysis reactions leading to the co-precipitation of Cu with oxides or occlusion by oxides. Fruit quality was, to certain degree, improved and fruit yields in the plots receiving Ca-WTR were 10 to 50% higher than the control. This is probably because Ca-WTR amendment raised the pH of acidic soils and thus improved nutrient availability and other soil conditions. These results indicate that in situ application of Ca-WTR may provide a cost-effective remediation for the Cu-contaminated soils wit hout affecting crop production. In conclusion, Ca-WTR is a promising so il amendment for chemical remediation of Cu contaminated soils. It is effective in r educing Cu toxicity to plants and decreasing leaching potential of Cu to surface and gr ound waters. In addition, Ca-WTR amendment creates a favorable environment for plant gr owth such as raised soil pH and increased Ca availability in the acidic soils, etc. However, Ca-WTR is highly water soluble and cannot last long in the acidic sandy soils su ch as those under citrus production in south Florida. Therefore, it is necessary to apply this material annually to maintain soil pH at desired levels (>6.5). Fortunatel y, Ca-WTR is produced in la rge quantities in the local water treatment plants as byproducts, which ar e available to the growers at a minimal cost. Since soil Cu contamination is ever increasing concern in the Indian River area, South Florida, the data and information from this study should be useful for the development of best management practices for citrus management. Future research is needed to address emergent concerns with due attention to the following aspects. 105

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Firstly, recent advancements in micro scopic and molecular-scale tools and data analysis have made possible to investigate trac e element speciation in natural samples. While chemical fractionation can provide the rmodynamic information on Cu speciation in contaminated or remediated soils, microscopically focused XAFS allows for analysis of Cu in molecular scale due to the intens ive X-rays generated by sy nchrotron sources. These results will provide direct evidence to s how how Cu is distributed in contaminated soil and how this distribution and subsequently bioavailability and mobility of Cu in soil can be modified by WTR amendment. Secondly, part of Cu loss may be transpor ted by suspended matter in runoff water, which is related to the affinity of copper to the surfaces of suspended constituents such as organic matter, clay minerals and hydrou s metal oxides. Further study needs to characterize the amounts and forms of c opper transported by suspended matter in contaminated or remediated soils, and identif y the major pathways of losses from land to waters. Finally, a further analysis over a longer monitoring period is needed to determine the chemical, physical, mineralogical, and bi ological influence of Ca-WTR under field condition, which will give an approximation of what could be expected in the future of the remediated soils. 106

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BIOGRAPHICAL SKETCH Jinghua Fan was born in Tianjin, China. She received a bachelors degree in environmental planning and managem ent from Nankai University, China, in 2002 and a Master of Science degree in environmental e ngineering from Resear ch Center for EcoEnvironmental Sciences, Chinese Academy of Sciences in 2005. She began her Ph.D. study in the Soil and Water Science Department, University of Florida, in 2006 and received her degree in 2010. Since 2007, she has worked as research assistant at the University of Floridas Soil and Water Science Laboratory (SWSL) at the Indian River Research and Education Center in Ft. Pierce. She conducted research on the biogeochemistry of Copper c ontamination and remedi ation, specifically the chemical immobilization with calcium water treatment s residue (Ca-WTR). At the SWSL, She was also appointed as the deputy Quality Assurance/Quality Control manager, and provided analytical and technical support and traini ng regarding the National Environmental Laboratory Accreditation Conference (NELAC ). She received the Grinter Fellowship from University of Florida. She is a member of the American Society of Agronomy (ASA), Crop Science Society of America (CSSA) Soil Science Society of America (SSSA), Soil and Crop Science Society of Florida (SCSSF), and Florida State Horticultural Society (FSHS). She has presented posters and talks about her research at the ASA-CSSASSSA internationa l annual meetings and FSHS-SCSSF annual meetings and received a FSHS Graduate Student Sc holarship in 2010. 122