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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2009-12-31.

Permanent Link: http://ufdc.ufl.edu/UFE0021681/00001

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Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2009-12-31.
Physical Description: Book
Language: english
Creator: Cheng, Yun
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, Ph.D.
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theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
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Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Yun Cheng.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Ogram, Andrew V.
Electronic Access: INACCESSIBLE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021681:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021681/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2009-12-31.
Physical Description: Book
Language: english
Creator: Cheng, Yun
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: 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

Statement of Responsibility: by Yun Cheng.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Ogram, Andrew V.
Electronic Access: INACCESSIBLE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021681:00001


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1 EXPLORATION OF BIODEGRADATION OF TOXAPHENE AND OTHER MAJOR PESTICIDES IN THE NORTH SHORE RESTORATION AREA SOILS OF LAKE APOPKA, FL By YUN CHENG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Yun Cheng

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3 To my parents

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4 ACKNOWLEDGMENTS I would lik e to especially recognize Dr. Andrew Ogram, chair of my graduate committee, for his excellent advice, unconditional support, a nd introducing me to the fascinating world of bioremediation. Further, I am thankful for the fi nancial support provided to me from his grant, and foremost for his moral support and encouragement to do my best. I would like to extend my sincere gratitude to Drs. Li-Tse Ou, Clayton Clark, Ramon Littell and Roy Rhue, members of my graduate committee, for their helpful comments and advice through my Ph.D. study. Particularly, I wo uld thank Dr. Clayton Clark for offering GCECD throughout the project. I also acknowledge St. John River Water Mana gement District and National Institute of Health for their financial s upport through my Ph .D. pursuing. I am grateful to Drs. Yongping Duan a nd Huaguo Wang for their encouragement and helpful suggestions in decision moments in my career. I am also grateful to Dr. Abid Al-Agely fo r his assistance with se tting up the mesocosm study. I wish to thank my current and former labmates, Drs. Hector Ca stro, Ashvini Chauhan, Ilker Uz, and Yannis Ipsilantis, Puja Jasrotia, and Lisa Stanle y for their generous help and support during my graduate studies Special thanks go to Jason Sm ith who made long days in the lab more funny. I want to thank all the people who were in strumental in the success of this study; especially, Drs. Huaguo Wang and Xiaosong Ch en, Bill Reve and Kafui Awuma for their willingness to help me.

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5 I also want to thank the faculty, staff, and all graduate students in the Soil and Water Soil Department for their support. Especial thanks go to Drs. Nicholas Comerford, Willie Harris and Peter Nkedi-Kizza for their suggestions and funny talks. I wish to thank Dr. Wei Hou for his friendshi p and helpful discussi on about statistics. I also wish to thank Steven Richter for his advice about extracting contaminants. I am greatly indebted to my parents, Guiz hen Ma and Renbing Cheng, my brother Xinqi Cheng, for their constant love and encouragement in various ways. Finally, I would like to thank my long time friends and unconditional supporters both in China and the States, who have made my life in Gainesville special, even through the hardest of times.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES.........................................................................................................................9 ABSTRACT...................................................................................................................................11 CHAP TER 1 INTRODUCTION..................................................................................................................14 Toxaphene and DDT in the Environment............................................................................... 14 Toxicity and Biochemistry of Toxaphene and DDT....................................................... 15 Environmental Fate of Toxaphene and DDT..................................................................17 Extraction of Toxaphene and DDT from Soils.......................................................................19 Microbiology of Toxaphene and DDT Degradation.............................................................. 21 Reductive Dechlorination of Organochlorine Contaminants.......................................... 21 Microbial Dechlorination and Degrad ation of toxaphene, DDT, DDD, an d DDE......... 22 Redox Potential and Biodegradatio n of Organochlorine Compounds ............................25 Bioremediation of Toxaphene, DDT, DDD and DDE........................................................... 26 Site Characteristics.................................................................................................................29 Research Hypotheses............................................................................................................ ..30 Study Overview................................................................................................................. .....31 2 EVALUATION OF EXTRACTION OF TOXAP HENE, DDT, DDD, AND DDE FROM NSRA SOILS WITH HI GH ORGANIC CARBON CONTENT.............................. 41 Materials and Methods...........................................................................................................43 Samples: Collection and Preparation............................................................................... 43 Extraction Procedures...................................................................................................... 44 Cleanup and Analysis......................................................................................................44 Statistical Analysis.......................................................................................................... 45 Results and Discussion......................................................................................................... ..46 Correlation of ASE and Soxhlet Rec overies to S oil Characteristics............................... 46 Effect of Extraction Solvents........................................................................................... 47 Comparison of ASE and Soxhlet..................................................................................... 49 Effect of Volumetric Water Content............................................................................... 50 3 BIODEGRADATION OF TOXAPHENE, DD T, AND ITS METABOLITES IN MICROCOSMS......................................................................................................................56 Materials and Methods...........................................................................................................58 Sampling..........................................................................................................................58

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7 Microcosm.......................................................................................................................58 OCP Determination......................................................................................................... 59 Isolation of Biodegraders................................................................................................ 61 DNA Sequencing Analysis.............................................................................................. 62 Statistical Analysis.......................................................................................................... 63 Results and Discussion......................................................................................................... ..63 Degradation of Toxaphene, DDT and its Metabolites.....................................................63 Establishment of Degradation of Toxaphene, DDT and its Metabolites......................... 67 Isolation of Toxaphene Degraders................................................................................... 70 4 BIOLOGICAL BREAKDOWN OF PESTICIDES IN LAKE APOPKA NORTH SHORE RESTORATION AREA SOIL IN A MESOCOSM EXPERIMENT ...................... 87 Materials and Methods...........................................................................................................89 Site...................................................................................................................................89 Mesocosm........................................................................................................................89 DNA Extraction Procedure..............................................................................................91 Redox Potential Measurement......................................................................................... 92 Temperature Measurement.............................................................................................. 92 Water Content Measurement...........................................................................................92 Sample Collection...........................................................................................................92 Organic Acid Analysis.................................................................................................... 93 OCP Analysis..................................................................................................................93 Statistical Analysis.......................................................................................................... 94 Results and Discussion......................................................................................................... ..95 Physical Conditions Related to OCPs Degradation within Mesocosms........................ 95 Degradation of Toxaphene in Mesocosm........................................................................96 Degradation of DDT and its Metabolites in Mesocosm.................................................. 99 5 SUMMARY AND CONLUSIONS...................................................................................... 116 LIST OF REFERENCES.............................................................................................................122 BIOGRAPHICAL SKETCH.......................................................................................................135

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8 LIST OF TABLES Table page 1-1 Physical and chemical properties of toxaphene, p,p -DDT, DDD and DDE .................... 33 1-2 Half-life of toxaphene, DDT, DD D a nd DDE in different matrices.................................. 34 1-3 Effect of Eh on CT biodegradation by Providencia stuartii ..............................................34 1-4 Gibbs free energy values and redox potentials of selected chlorinated compounds and inorganic redox couples ..................................................................................................... 35 2-1 Soil characteristics for low, modera tely, and highly contam inated soils........................... 52 2-2 Correlation of soil properties to r ecovery rate in Soxhlet and ASE .................................. 52 2-3 Comparison of extraction solvents for extr acting toxaphene, DDT and its metabolites using Soxhlet and ASE from highly contaminated soil..................................................... 53 2-4 Comparison of ASE and Soxhlet for ex tracting to xaphene, DDT and its metabolites from highly contaminated soil. Methylene chlo ride/acetone (4:1, v/v) was used as the solvent........................................................................................................................ ........53 2-5 Comparison of ASE and Soxhlet for ex tracting to xaphene, DDT and its metabolites from moderately contaminated soil. Methyl ene chloride/acetone (4:1, v/v) was used as the solvent......................................................................................................................54 2-6 Correlation of water content in the soil to extraction efficiency by Soxhlet and ASE. Methylene chloride/acetone (4:1, v/v) was used as the solvent. ........................................54 3-1 Soil characteristics for moderate ly and highly contam inated soils.................................... 73 3-2 Statistical contrast analyses of roles of various electron donors in biodegradation of OCPs in m icrocosms after two-month incubation............................................................. 74 4-1 Experimental design of mesocosm studies...................................................................... 103 4-2 Pair-wise Comparison of the treatmen ts on the degradation of pesticides ...................... 103

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9 LIST OF FIGURES Figure page 1-1 A: Toxaphene: C10H10Cln (n=5-12); B: DDT: C14H9Cl5; C: DDD: C14H10Cl4; D: DDE: C14H8Cl4...................................................................................................................36 1-2 Anaerobic degradation pathway of DDT (A, D, E and F are multiple steps whose inte rmediates are not identified yet.). (http://umbbd.ahc.umn.edu/ddt2/ddt2_map.html)............................................................. 37 1-3 Aerobic degradation pathway of DDT (A, B, C, D, and E are multiple steps whose intermediates are not identif ied yet.). (http://umbbd.ahc .umn.edu/ddt/ddt_map.html).....38 1-4 Six environmentally relevant toxa phene com ponents; I: 2,2,5-endo,6-exo,8c,9b,10aheptachlorobornane (Tox B); IIa: 2, 2,5-endo,6-exo,8b,8c,9c,10a-octachlorobornane (Tox A1); IIb: 2,2,5-endo,6-exo,8c,9b,9c,10a -octachlorobornane (Tox A2); III: 2,2,5endo,6-exo,8c,9b,10a,10b-octachlo robornane; IV: 2,2,5-endo,6exo,8b,8c,9c,10a,10c-nonachlorobornane; V: 2,2,5-endo,6-exo,8c,9b,9c,10a,10bnonachlorobornane............................................................................................................. 39 1-5 The North Shore Restoration area of Lake Apopka in FL................................................. 40 2-1 Effect of water content on the averag e m eans of extracted pesticides in the intermediate level of contaminated soils. Methylene chloride/acetone (4:1, v/v) was used as the solvent............................................................................................................ .55 3-1 Degradation of toxaphene in microcos m s containing different electron donors and sulfate concentrations.........................................................................................................75 3-2 Degradation of DDT in microcosms c ontaining different elec tron donors and sulfate concentrations ....................................................................................................................76 3-3 Degradation of DDE in microcosms c ontaining different elec tron donors and sulfate concentrations ....................................................................................................................77 3-4 Degradation in DDD in microcosms cont aining different electron donors and sulfate concentrations ....................................................................................................................78 3-5 Degradation of toxaphene in microcos m s containing different electron donors and sulfate concentrations.........................................................................................................79 3-6 Degradation of DDE in microcosms c ontaining different elec tron donors and sulfate concentrations ....................................................................................................................80 3-7 Degradation of DDD in microcosms c ontaining different elec tron donors and sulfate concentrations ....................................................................................................................81

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10 3-8 Degradation in DDT in microcosms c ontaining different elec tron donors and sulfate concentrations ....................................................................................................................82 3-9 Dechlorination of toxaphene with lact ate as the electron donors enriched from moderately contaminated soils........................................................................................... 83 3-10 Dechlorination of toxaphene with lactat e as the electron donors enriched from highly contaminated soils............................................................................................................. .84 3-11 Phylogenetic tree of dominant sequences (K13s, L12, L1s, K10, K19, L13s, L19, L19s, K1s, K19s, L13) enriched from NSRA soils ...........................................................85 4-1 Soil collection site ZSS0750 in the Nort h Shore R estoration Area at Lake Apopka, Orange County, Florida...................................................................................................104 4-2 Soil mixed in a 13 ft x 13 ft plot at si te ZSS0750 by a trackhoe pr ior to collection and transport to the greenhouses at the Departm e nt of Soil and Water Science, University of Florida, Gainesville. ................................................................................................... 105 4-3 Mesocosm tanks in the greenhouse at th e Departm ent of Soil and Water Science, University of Florida, Gainesville....................................................................................106 4-4 Permanently installed redox el ectrodes in m esocosm tank.............................................. 107 4-5 Temperatures within mesocosms at a depth of 5 cm....................................................... 108 4-6 Redox potentials with mesocosm tanks during flooded phase........................................ 109 4-7 Redox potentials following draining................................................................................110 4-8 Volumetric water cont ents following draining. ...............................................................111 4-9 Concentrations of toxaphene during study. ..................................................................... 112 4-10 Concentrations of DDT during study............................................................................... 113 4-11 Concentrations of DDD during study.............................................................................. 114 4-12 Concentrations of DDE during study............................................................................... 115

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EXPLORATION OF BIODEGRADATION OF TOXAPHENE AND OTHER MAJOR PESTICIDES IN THE NORTH SHORE RESTORATION AREA SOILS OF LAKE APOPKA, FL By Yun Cheng December 2007 Chair: Andrew V. Ogram Major: Soil and Water Science In order to accurately eval uate the bioremediation strategy of the Lake Apopka North Shore Restoration Area (NSRA) FL, the optim al conditions for extracting toxaphene, 1,1,1trichloro-2,2-bis( p -chlorophenyl)ethane (DDT ), 1,1-dichloro-2,2-bis( p-chlorophenyl)ethylene (DDE), and 1,1-dichloro-2,2-bis( p-chlorophenyl)ethane (DDD) from this area with high organic carbon were firstly established. Ac celerated solvent extraction (ASE) was compared to Soxhlet extraction (SOX) and the effects of solvent systems, and soil characteristics such as volumetric water content, and carbon content on extraction efficiencies were investigated. The results indicated that only the toxaphene recovery in ASE was highly corr elated to soil carbon content. Using ASE, methylene chloride/acetone (4:1, v/v) extracted toxaphene, DDE, and DDT more efficiently than hexane/acetone (1:1, v/v); while the extraction for DDD and DDE was extracted by SOX with higher efficiency using hexane /acetone (1:1, v/v) than using methylene chloride/acetone (4:1, v/v). Volumetric water content was significantly correlated to extraction efficiency for DDT by SOX, and for DDE, DDT, and toxaphene by ASE. In both ASE and SOX, intermediate water content yielded the greatest overall efficiencies for extracting toxaphene, DDT and its toxic metabolites simultaneously from the soils. Finally, with the exception of DDT,

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12 toxaphene, DDE, and DDD can be ex tracted by ASE with statistica lly significant efficiency in compared with by SOX from highly contaminated soils, while ASE produced at least comparable efficiencies with SOX for extr acting DDT family from moderate ly contaminated soils with a statistically significant highe r efficiency for toxaphene. A series of laboratory microcosms were fi rstly set up with organochlorine pesticidecontaminated soils taken from NSRA to identify anaerobic processes leading to the decomposition of the recalcitrant pesticides toxa phene and DDT family. In separate replicated (two) microcosms, lactate, butyrate, H2, formate, acetate, and propionate were used as electron donors in the presence of varying co ncentrations of sulfate and a c onstant level of the pesticides. After about one month, all microc osms were sacrificed and orga nochlorine pesticides (OCPs) were extracted and determined using the established methods. Among the tested electron donors, lactate, butyrate, and hydrogen showed potential capability of stimula ting the biodegradation. Their roles in OCPs degradation were further established with another series of laboratory microcosms with three replicates. After a twomonth incubation, significant loss of the parent compounds were observed in the lactate, butyrate, and H2 microcosms relative to autoclaved controls and controls with no exogenous electron donor. In order to understand the characteristics of microorganisms participa ting the contaminants metabolism, indigenous bacteria with the capability to dechlorinate toxaphene were enriched using lactate as the electron donor in the presence of 20 ppm toxaphene After five transfers, release of Clwas measured by ion chromatography with time. A rapid and si gnificant rise in concentrations of Clwas observed in all replicates relative to controls, indi cating biological dechlorination mechanism of toxaphene. The possibility that dechlorination wa s affected by sulfate reducing bacteria was investigated by isolation of DNA followed by polymerase chain reaction (PCR) with primers

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13 specific to the dissimilatory sulfite reductase gene, dsrA No dsrA amplicons were observed, which meant other group(s) of bacteria besides sulfate reducing bacteria may be responsible for degrading toxaphene. Phylogenetic analysis demonstrated that some sequences most abundant in the clone libraries were related to the genus Citrobacter (99.9% similarity), some closely related to Gram positive Sporomusa (98% similarity) Finally, the concepts for remediation of th e contaminated area were confirmed and extended up to the mesocosm scale using easily availa ble plant materials, cattail, or lactate as the electron donors plus a set of control with a cycle of anoxic followed by oxic conditions promoting degradation. Concentrations of OCPs were monitored every two weeks. Both lactate and cattail treated mesocosms showed significant disappear ance of toxaphene and DDT by the end of the anoxic phase. More importantly, biodegradation of OCPs in cattail treated mesocosms was as obvious as that in lactate treated ones, which will greatly decrease the expense without any adverse effect on the efficiency. However, ove rall concentrations of toxaphene, DDT, DDD and DDE did not show significant difference by the end of the oxic treatment. The present study contributed to establish a efficient guideline for simultaneously extracting toxaphene, DDD, DDE, and DDT from soils with high organic matter and a greater understanding of bioremediation of toxaphene a nd DDT family contaminated farmland using a cost efficient strategy.

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14 CHAPTER 1 INTRODUCTION Toxaphene and DDT in the Environment Occurrence and Chemical Structu re of Toxaphene and DDT Even though it was first synthesized in 1847, 1, 1, 1-trichloro -2,2-bis(4chlorophenyl)ethane, (DDT) was not widely used as an insecticide unti l World War II, when it was used to protect the troops and civilians from some diseases (Parr and Smith, 1974). Afterwards DDT was used for agricultural app lication to controlling disease vectors. The investigations on toxicological effects of DDT and the persistence of its hazardous metabolites, 1,1-dichloro-2,2-bis( p-chlorophenyl)ethylene (DDE), and 1,1-dichloro-2,2-bis( p chlorophenyl)ethane (DDD), resulted in some c ountries banning its use in the 1970s, forcing some countries to turn to alternatives. Toxaphene was one of the most im portant pesticides in USA following the national ban on DDT, with more than 409,000 metric tons of toxaphene applied over ten years (Hooper et al., 1979). It was registered for use on more than 277 agricultural commodities and crops to control 16 7 major insect pests. Toxaphene was also applied by fisheries managers in Canada and USA to get rid of undesirable fish (Saleh, 1991). In 1982, EPA banned its use after toxaphe ne was shown to cause cancer in rats and mice (Reuber, 1979; Saleh, 1991). In addition to being carcinogenic, toxaphene is highly persistent (Nash and Woolson, 1967; Menzie, 1972) in the environment. Neither toxaphene nor DDT occurs naturally in the environment. Fig. 1-1 and Table 1-1 show the structure, physical and chemical properties of p,p' -DDT, p,p' -DDD, p,p' -DDE, and toxaphene (with the general formula C10H10Cln, n=5-10). The widest us e of DDT was to kill disease vectors such as mosquitoes carrying malaria. It is cheap and efficient, and was used as an insecticide in agricultural applications, part icularly for cotton. Toxaphene is a complex

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15 compound with a broad spectrum of pesticidal ac tivity. This compound consists at least of 670 congeners, whose composition varies with the manufact urer. It is generally considered to have an average formula of C10H10Cl8 (Hooper et al., 1979). Both of these two contaminants exhibit low aqueous solubilities (Table 1-1) and dissolve in many organic solv ents. Because 80 to 90% of all toxaphene and DDT use was estimated to be used on cotton, their residues are predominantly in the southern and southeastern U.S. (Sale h, 1991), where cotton was primarily planted. Toxaphene was also used on other crops such as soybeans and peanuts. Toxicity and Biochemistry of Toxaphene and DDT Num erous investigations have b een carried out on the toxicity of toxaphene to a range of organisms, including mammals, birds, aquatic orga nisms, insects, and plants. Among the reports, those testifying its carcinogenici ty, and mutagenicity gained increasing attention. Until 1979, a few decades after its first production and appli cation, toxaphene was shown to cause not only neoplasms at all sites but also the malignant ne oplasms in both female and male rats and mice (Reuber, 1979). Although some researchers (Eps tein et al., 1972) repor ted that there is no significant evidence that shows the mutagenicity of toxaphene Hooper et al. (Hooper et al., 1979), Zeiger (Zeiger, 1987), and Saleh (Sale h, 1991) confirmed that toxaphene has both mutagenic and carcinogenic effects. Among the tested organisms, fish are the most sensitive to DDT. Generally, behavioral development of hatched eggs is very vulnerable. For example, very high dose levels of DDT, 50 and 100 g/L, impaired the balance and delayed the occurrence of normal be havior after 30-day growth after hatching in Atlantic salmon eggs (Dill and Saunders, 1974). Furthermore, several investigations revealed that its best-known a dverse effects are those on reproduction. For other wildlife, DDT has an endocrine disrupting eff ect. And female herring gu ll (Colborn et al., 1996) in the Great Lakes was observed abnormal in ma ternal behavior under the stress of DDT. DDT,

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16 especially its metabolite DDE, also alters the ratios of male and female birds by feminizing their male sexual organs (Fox, 1992). Another aspect of significant adverse impact on reproductive success is the eggshell thinning. Similar ad verse effects on repr oduction and estrogenic development such as advanced puberty and persistent vaginal estrus ha ve been observed in laboratory animals. Because many developing count ries still apply DDT to control insects and malaria, the chronic nervous-system effects of long-term occupational exposure to DDT was evaluated and showed significan t correlations between time period of exposure and worse neurobehavioural functions and an increase of neuropsychological and psychiatric symptoms, even though the mechanism of action for DDT to affect neurological function was not elucidated (de Joode et al., 2001). Probable car cinogenicity of organochlorine pe sticides has also been paid much attention. DDT and its persistent metabo lites cause cancer problems in experimental animals. Zheng (Zheng et al., 1999) investigated the relation be tween DDE and DDT exposure and breast cancer risk and did not find any significance relationship. However, the study is limited by lack of exposure data and a relatively small number of deaths A possible carcinogenic role in human for DDT is not impossible consider ing the similarity of structure and chemical properties shared by halogenated aromatic hydrocarbons. Efforts of demonstrating the toxicological mechanisms of DDT provide a few ways to understand how the organisms responded to this insecticide and its metabolites. Some researchers believed that o,p or p, p isomers interact with estrogen or androgen receptors of rats (Heinrich et al., 1971) and then cause a series of abnormal effects, as mentioned above. Blocking androgen action at its receptor and altering the expression of androgen-dependent genes (Kelce et al., 1995; Kelce et al., 1997) were considered another likely mechanism for DDEs changing the sex differentiation in male rats. Additionally, the decrease in serum

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17 testosterone levels in rats (Krause, 1977) a nd alligators in Lake Apopka (Guillette et al., 1994) exposed to DDT is another avenue to affect androgen homeostasis. The understanding of the toxicological mode of action and the metabolism of toxaphene in various organisms has slow due to its complicat ed structure and the difficulties in analysis. Generally, toxaphene affects through the central nervous system, liver mixed function oxidases, and other metabolic pathways. Some receptors bi nding to specific sites in mammal and insect brains were shown to be inhibited competitiv ely by toxaphene and its purified components (Lawrence and Casida, 1984; Matsumura and Tanaka, 1984). Additionally, toxaphene has an effect on the central nervous systems by increasing K+ and Ca2+ and inhibiting ATPase activities in cockroaches (Whitson and Crowder, 1979) and mice (Trottman and Desaiah, 1979). In addition to central nervous system, the process of glycolysis and other energy metabolisms can be inhibited by toxaphene (Trosko et al., 1987). Environmental Fate of Toxaphene and DDT W ith the increasing concerns about environm ental health problems resulting from these pesticides, a large number of studies have invest igated the environmental fate and environmental characteristics of chlorinated pesticides such as toxaphene and DDT. The physical and chemical characteristics of these pesticides such as low water solubility, high stability and semi-volatility contribute to its relatively high pe rsistence in the environment. The percentage of pesticides remaining in the soil depends on the applicati on rate before the monitoring investigation. The higher concentration applied, the higher percentage remained in the soil. The environmental conditions have a very important effect on the persiste nce of organochlorine pesticides (OCPs). Organic matter is the most important factor am ong those affecting the persistence of OCPs, OCPs persisting longer in soils rich in organic matters. For example, in the muck with high organic matter ranging from 27 to 56 %, mean concentration of DDTr was as high as 4685 ppb

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18 dry weight and residues of DDT were still present there, abou t 70 % decreasing after 16 years (Oloffs et al., 1971; Szeto and Price, 1991). In the silt loam farms with organic matter ranging from 3.7 to 6.5 %, only 433 ppb of DDTr were detect ed. The dissipation of DDTr in the loamy sand soils, with less than 2% organic matter, was almost complete after 16 years. Organic matters adsorbed DDT as a result of its high lipophilicity so as to increase the persistence by removing DDT from water solution in soils and preventing leaching throu gh soil. Soil acidity, another chemical property of soil, may cause pesticides more persis tent (Gong et al., 2004). When the pH was closer to acidity, the changes of structure of the hum ic acid would lead to bigger cavities in its polymer, resulting in more organochlorine pesticides trapped into these cavities (Alawi et al., 1995). B ecause of high solar radiation and microorganism diversity in tropical region, degradation of degr adative pesticides was remarkable in this kind of area than in temperature regions (Khan, 1994). Additionally, high temperature can also accelerate the volatilization of organochlor ines in spite of their low vapor pressure (Farmer et al., 1972; Nash, 1983; Singh and Agarwal, 1995). Both toxaphene and DDT are the persistent pollu tants in the environments, long half-life in soils, sediment and water (Table 1-2). One st udy revealed the large amount of residue of toxaphene in the soil (Willis and McDowell, 1987) It was estimated that only between 9 and 19% of toxaphene applied to cotton fields by aircra ft was partitioned in plants, while most of the toxaphene went to the soil (Willis and McDowell 1987). Then how did the toxaphene in the soil translocate? Compared with ot her chlorinated hydrocarbon insect icides, toxaphene exhibited a relatively long half-life of 11 years in a Congaree sandy loam so il, while the half-life for purified aldrin, technical aldrin, dieldrin, chlordane, he ptachlor, Dilan, BHC and DDT were respectively 5, 9, 7, 8, 2 to 4, 4, 2, and 10.5 yrs (Table 1-2) (Nash and Woolson, 1967). Even after 20 years,

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19 45.1% of applied toxaphene was detected (Nash et al., 1973) because of the rather slow degradation rate. The extent of chlorina tion may explain its long half-life though, the chlorination rendering the much difficulties in the biodegradation. Major components of toxaphene detected in sediment, soils, sewage sludge and various mammals (Stern et al., 1996; Buser et al., 2000) come from the reductive dechlorination of toxaphene. The study on the distribution and movement of toxaphene in an aerobic saline marsh soils revealed that the concentration of toxaphene decreased with dept h (Gallagher et al., 1979 ). The translocated toxaphene, therefore, became the pollution source of groundwater. Volat ilization is of great importance accounting for the loss of toxaphene from soil. Glotfelty report ed (Glotfelty et al., 1989) substantial losses (31 %) of surface-applied toxaphene resi dues within a 21-day period and a decrease in the volatilization when the soil surface became dry. On the other hand, DDT has also been detect ed in the global, most heavily in humid temperate and tropical zones. In general, DDT residues disappear from three primary mechanisms (Woodwell et al., 1971) including vo latilization, water runoff, and degradation. Because DDT can be degraded to DDE with the existence of oxygen, DDE maked up the major proportion of the total DDT residues in the superf icial or well-aerated soils (Spencer, 1975; Aislabie et al., 1997). Greater vapor pressure (Table 1-1) leads DDE to be the major component found in the atmosphere over a field which was previously applied t echnical DDT (Spencer, 1975; Aislabie et al., 1997). For the other, in the flooding cond itions, a higher percentage of DDD constituted the residues (Spenc er, 1975; Aislabie et al., 1997). Extraction of Toxaphene and DDT from Soils To date, a variety of extraction m ethods have been developed for organic compounds. In 1879, a German chemist pioneered the extraction technology with the solid-liquid extraction apparatus that bears his name, Soxhlet (SOX). The Soxhlet extraction process can ensure the

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20 intimate contact of the sample matrix with the extraction solvent. As a traditional method, it cannot overcome the limitation at the point of process period that requires the use of long time cycles e.g. 8 to 24 hours. Alterna tive methods such as solvent-sh ake extraction require intensive labor for the complicated steps. In addition, a large quantity of solven t is used in those extractions, thereby increasing the cost and producing much waste to be disposed. In 1995, accelerated solvent ex traction (ASE) was introduced for analysis of organics because it requires only a small amount of solven t and less time than Soxhlet, which has sparked increasing interest in it use in environmental studi es (Richter et al., 1996) The solvent is first pumped into the extraction cell c ontaining the sample, and then th e temperature and pressure are elevated quickly, respectively, to 100 C and 2000 p.s.i. (Fitzpat rick et al., 2000). Increased temperature and pressure allow the solvent to penetrate the sample matrix by decreasing the viscosity of organic solvents (Montgomery et al., 1995). In addition, increased temperature and pressure can keep the solvent liquefied above th e boiling point and disrupt the strong interactions between the solute molecules and active site of the matrix, and hence improve extraction. After a few minutes, the extract is purged from the cell to a collection vial for further processing, such as additional cleanup if required or direct analysis. Due to the reduced sample preparation time and the lower amount of solvent required, ASE has been increasingly used to extract organic contaminan ts from various environments, including soils and sediments (Montgomery et al., 1995; Fisher et al., 1997; Schantz et al., 1997; Saim et al., 1998). And comparisons between ASE and some traditional techniques such as Soxhlet extraction and fluid extract ion for extracting pesticides s how an agreement that ASE has similar recoveries to those from traditional ones (Conte et al., 1997; Frost et al., 1997; Fitzpatrick et al., 2000). Additionally, ASE has also been ev aluated when applied to extract DDT and its

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21 metabolites from environmental reference mate rials and good agreement for extracting these organic pollutants was achieved between SOX and ASE (Fitzpatrick et al., 2000). For choosing a proper extraction te chnique, it is important to realize that various factors affect the performance of the specific extraction methods to some extent. ASE is sensitive to the soil characteristics; an increasing amount of car bon in the sediments caused chlorinated organic compounds less accessible to the extraction solv ent (Josefsson et al., 2006). The choice of extraction solvent has been demonstrated as one of the most significant variables for polycyclic aromatic hydrocarbons (Parr and Smith, 1974). As an important factor, water content was shown critical to obtain high extraction efficiencies fo r organic pollutants from low organic carbon soils in many investigations (Wall and Stratton, 1991 ; Heemken et al., 1997; Spack et al., 1998; Berglof et al., 2000). Microbiology of Toxaphene and DDT Degradation Reductive Dechlorination of Organochlorine Contaminants Despite their persistence, long half-life in the environm ent, and resistance to any chemical, physical and biological degrada tion, higher halogenated compounds may be dehalogenated when the soil is subjected to a re ducing environment. Lower haloge nated pollutants may be oxidized aerobically by microbial enzymatic systems, while aerobic microorganisms may not be capable of oxidizing higher halogenated ones. Reductive dechlorination is considered as the most important reaction of the biodegradation of complicated chlorinated compounds, and the participation of transition metal-containing co enzymes (Esaac and Matsumura, 1980; Mohn and Tiedje, 1992) has been documented for this process of an electron transfer with the release of a chlorine and its replacement by hydrogen (Fingerling et al., 1996). Using this alternative electron acceptor benefits the dehalogenating bacteria si nce the standard free energy change for the

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22 oxidation of hydrogen under this situation can yield more en ergy than some more common electron acceptors such as methane and sulfate in anaerobic soils. When hydrogen is used as electron donor, the amount of energy available fr om general dechlorination of halogenated compounds results in G values between 101 and 159 kJ/mol of chlorine removed according to a conservative estimate. This is at least 3 times more than the amount of energy available from sulfate reduction or methanogenesis per mole of hydrogen consumed (Dolfing and Harrison, 1992). In general, sulfate reduc ing bacteria (SRB) widely di stribute in the anaerobic environments and relate to such process whic h is coupled with energy conservation with the haloorganic compounds serving as the terminal electron acceptor instead of sulfate. Some SRB directly dehalogenate the chemi cals (Utkin et al., 1994; Boucha rd et al., 1996; Dennie et al., 1998; Boyle et al., 1999; Gerritse et al., 1999; Wiegel et al., 1999; Drzyzga and Gottschal, 2002; Zanaroli et al., 2006) or indirec tly participate in th e dehalogenation by providing dehalogenating bacteria with hydrogen, an electron donor (de Best et al., 1997; Drzyzga et al., 2002; Drzyzga and Gottschal, 2002). However, dehalogenating bact eria were reported to compete with SRB for electron donors under sulfate redu cing conditions in some cases (Hoelen and Reinhard, 2004). With the diversities of SRBs role in the deha logenation of complex organic contaminants, their microbiology and biochemistry related to halore spiration still remain to be elucidated. Based on our previous study, SRB is present in the NSRA soils (Cas tro and Ogram, data not shown) so that we assumed that this group of versatile bacteria might be involved in the potential biodegradation of organochl orine pesticides (OCPs) in NSRA. Microbial Dechlorination and Degradat ion of toxaphene, DDT, DDD, and DDE Reductive dechlorination of DDT in anoxic condition to DDD is considered to be the dominant reaction of two major routes (Johnsen, 1976; Esaac and Matsumura, 1980; Lal and Saxena, 1982; Boul, 1996; Aislabie et al., 1997). In most cases, DDT dechlorination is not an

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23 energy-yielding process as DDT does not serv e as the terminal electron accepter in a bioenergetic pathway (Foght et al., 2001). U nder anaerobic conditions, DDD can be further metabolized (Fig. 1-2). Similar to the first reduc tive dechlorination reac tion, all steps after the first reductive transformation of DDT to DDD un til the formation of benzhydrol were found to require co-metabolic substrates (Foght et al., 2001). For another, dehydrochlorination of DDT under aerobic conditions to DDE is the other possi ble pathway despite not occurring often (Fig. 1-3). Degradation of DDT by microorganisms in pure culture has been studied for a few decades. As early as 1980, a pure strain, Pseudomonas aeruginosa 640X (Golovleva et al., 1980), was isolated to degrade DDT from soils w ith a series conditions including aeration and alternating co-substrates after th e fist step of reductive dechlorination. Till today, many pure cultures and consortia have been reported to play important roles in destroying DDT under aerobic or anaerobic conditions. The physiology a nd biochemistry of biodegradation of DDT have been investigated on a labor atory scale and reveal many importa nt strategies or processes to control the metabolism process. Some studies showed the toxicity of DDT to microbial degraders. For a DDT-degrading consortium co mposed of four bacterial strains, the Serratia marcescens DT-1P (Bidlan and Manonmani, 2002) was shown to be capable of degrading DDT up to 15 ppm DDT; however, 50 ppm DDT inhibi ted degradation. DDT and polychlorinated biphenyls (PCBs) share similar aspects of their chem ical structures, and some researchers studied degradation of DDT by PCB degr ading bacteria. For example, Alcaligenes eutrophus A5 was originally enriched from PCB-contaminated sediment and was found to degrade DDT via 4-CB aerobically (Nadeau et al., 1994). With Alcaligenes denitrificans ITRC-4 (Ahuja and Kumar, 2003) degrading DDT, sodium acetate and sodium succinate inhibit aerobic metabolism by 38%

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24 and 47%, respectively. And also, biphenyl vapo rs and 2,2-bipyridyl inhibit degradation completely by competing with structural anal og to DDT to bind at th e active site of the metabolizing enzyme and making it unavailable for DDT metabolism or inhibiting the ironcontaining enzyme in the metabolic pathway. Nevertheless, glucose shows no inhibition of aerobic dechlorination of DDT, and even enhances the transformation of DDT to DDD by 50%. Since microorganisms in soil co-metabolize DDTr the amendment of suitable carbon source will successfully improve the DDT degradation rate To date, plenty metabolism explorations resulted in a basically complete degradation pa thway under either aerobic or anaerobic condition (Fig. 1-2 and Fig. 1-3). Because of its complex structure, the degrad ation pathways of toxaphene have not been studied as much as those of DD T, DDD and DDE, its major products. Most of the investigations about the biodegradation of toxaphene revealed that the major pathway was carried on under anaerobic conditions (Murthy et al ., 1984) and follows first-order kinetics, which implicates that the degradation rate depends on the concentratio n of toxaphene remained in the system. Except for the well-established anaerobic metabolism, the oxidative process plays an important role in the degradation of toxaphene (Chandurkar and Ma tsumura, 1979; Maiorino et al., 1984). A strain of Pseudomonas putida was shown to decompose toxaphene aerobically and anaerobically in that study. Recently, the white-rot fungus Bjerkandera sp. strain BOL13 (Romero et al., 2006) was isolated to aerobically degrade toxaphene, while Dehalospirillum multivorans (Ruppe et al., 2002) and Enterobacter cloacae (Lacayo-Romero et al., 2005) anaerobically degrade toxaphene. Nevertheless, biochemistry of de gradation by these microorganisms has not been reported yet. Two pathways are proposed for biodegra dation of toxaphene including reductive dechlorination, a major process, (Fingerling et al., 1996) and dehydrochlorination, a minor

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25 process, (Ruppe et al., 2004) and then lower chlorinated complex are taken over by aerobes. Dechlorination of toxaphene is reported to be ster eoselective. The first loss of chlorine atom usually occurs in the C-2endo position, the most labile position in the carbon ring, from each geminal dichloro group, which two chlorines are attached to the same carbon in the ring. However, reasons for anaerobes favoring 2-e ndo chloro substitution remain unclear. The dechlorination rate, in most of cases, depends on the chlorination stage and follows the order of nonachlorobornanes > octachlororbornanes > heptach lorobornanes (Fig. 1-4) (Fingerling et al., 1996; Buser et al., 2000). Generally, a hexachlo robornane (Hx-Sed) and a heptachlorobornane (Hp-Sed) are the predominant end metabolites of t oxaphene in treated environments (Stern et al., 1996; Buser et al., 2000). Redox Potential and Biodegradation of Organochlorine Compounds Redox potential (Eh) is a m easure of the free electron activity of a system. Redox conditions in vadose zones and sa turated environments are relate d to promoted microorganisms growth and efficient biodegrada tion of organic contaminants (Szeto and Price, 1991; Jin and Englande, 1996; Crawford et al., 2000; Davis et al., 2004). A chemical with more positive Eh will have a more affinity for an electron and te ndency to be reduced. The poly-chlorination of organic chemicals results in the increase in thei r electrophilicity and oxidation status (Jin and Englande, 1996), which leads to the instability and biodegrad ation under the reduction condition and explained why they can be easily broken do wn under the anaerobic conditions. As shown in Table 1-4, higher halogenated compounds have more positive potentials than lower halogenated chemicals have and the reduction of the haloge nated contaminants produce more energy than sulfate reduction or even denitrification in some cases, making these organic compounds the favorable electron acceptor. A high Eh indicate s the environment that allows the oxidation reaction with a relatively high tendency, while the reduction reaction more possibly occurs when

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26 the system has a low Eh. It is therefore evid ent that redox potential in the environments represents a rather important fact or that may have an effect on the efficiency of organochlorine compounds such as toxaphene, DDT, DDD and DDE. To date, many investigations have revealed that various redox poten tials influenced the biodegradation efficiency and le d to different degradation pa thways (Esaac and Matsumura, 1980; Doong and Wu, 1992). Detailed information a bout the influence of redox potentials on the degradation of chlorinated com pounds has been reported in a study by Jin and Englande (Jin and Englande, 1996). Under the differe nt redox potentials the degradati on rate of carbon tetrachloride (CT) was investigated, the lowest minimum redox potential corresponding to highest degradation rate, K (Table 1-3), the over all rate constants depending on cell mass and k, specific rate constants independent of cell mass. Redox potentials can be made negative by the ad dition of organic matter (Szeto and Price, 1991), carbon source (Davis et al., 2004), electron acceptors, and microbial activities (Crawford et al., 2000). In general, as the organic matter in the soil itself is small and not readily available, the Eh is more positive in th e environment without any amen dment (Ugwuegbu et al., 2001). A flooded soil can limit the diffusion of oxygen into the soil and the oxygen in both water phase and soils can be removed by microbial respiratio n. The addition of acetate, glucose, or other energy source is capable of accelerating the proc ess of reducing oxygen and driving the Eh to drop. Bioremediation of Toxa phene, DDT, DDD an d DDE The management of toxaphene, DDT and its meta bolites in the soil, water and air has been concerned especially with remediation of cont aminated environments. Conventional techniques for organochlorine contaminated soils involve physical and chemical strategies including excavation and incineration, thermal desorption (Norris et al., 1997), microwave-enhanced

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27 thermal decontamination (Kawala and Atamancz uk, 1998), leaching with surfactant (Kile and Chiou, 1989; Ganeshalingam et al., 1994; Parfitt et al., 1995), and supercr itical fluid extraction (Sahle-Demessie and Richardson, 2000). Even th ough the above technologies are more rapid than bioremediation, they do not change the chemi cals themselves but tran sfer the contamination from one place to another and disturb the original landscape. Bioremediation overcomes these disadvantages and may be more cost-effective than chemical and physical remediation. It utilizes the metabolic diversity of bacteria, fungi, and pl ants. These powerful cleaning creatures degrade the pollutants, produce less toxic or nontoxic ch emicals and allow them to enter the clean geochemical cycles. Therefore, in the recent years, bioremediation has gained a lot of interest in research and application. As early as the 1970s, the biodegradation of DDT partially succeeded in an Everglades muck (Parr and Smith, 1974) and flooded soil (Guenzi and Beard, 1976). Regardless of the extreme complexity of structure of toxaphene, a few publications have documented toxaphene biodegradation in anaerobic soils (Parr and Smith, 1974; Lacayo-Romero et al., 2006), sewage sludge (Buser et al., 2000), and water (LacayoR et al., 2004). Alt hough the biological process for degradation of toxicants is se lective in general, toxaphene degradation doesnt show the specificity (Buser et al., 2000). At this point, toxaphene offers sp ecial convenience for microbial attack and thereby improving the degradation rate For such a recalcitra nt pollutant, microbial remedy is probably the most promising soluti on to the toxaphene environmental topic. Because highly chlorinated contaminants prefer anaerobic metabolisms and less chlorinated species favor aerobic metabolisms, complete mineralization of these compounds can be achieved by anaerobic treatment followe d with aerobic oxidation. More efficient bioremediation of organochlorine compounds has b een reported in alternating anaerobic/aerobic

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28 mode. Promising results have been obtained from the complete investiga tions (Anid et al., 1991) on the destruction of PCBs in river sediment under sequential anaerobic-aerobic processes. During the anaerobic period, reductive dechlorination of PCBs occurr ed, and tri-, tetra-, penta-, and hexachlorobiphenyl concentrations decrease d, whereas monoand dichlorobiphenyl were accumulated. Under the following aerobic phase, significant degradation of the anaerobic products occurred. By the end of the anaerobicaerobic treatment, only 43% of the 300 mg of PCB/kg of soil remained. Despite the lack of th e quantification of DDT degradation under the sequential anaerobic-aerobic proces s, prospects of alternating anaerobic and aerobic incubation were indicated in an in vitro study of cell free extracts of Pseudomonas sp. extensively degrading DDT (Pfaender and Alexander, 1972). Pseudomonas sp. can not dechlorinate DDT under the strict aerobic condition but can anaerobically co-metaboli ze DDT. Under the anaerobic conditions, DDT can be metabolized to 4,4-dichlorobenzophenone (DBP) as the end product through DDD, 1-chloro-2,2-bis(4-chlo rophenyl)ethane (DDMS), 2,2-bis(4chlorophenyl)ethylene (DDNU) by a Pseudomonas sp. (Pfaender and Alexander, 1972). Nevertheless, subsequently ae robic incubation with fresh Pseudomonas sp. produced a pchlorophenylacetic acid (PCPA), which was not found in the strict anaer obic conditions. This product was readily further degraded by an Arthrobacter sp., producing pchlorophenylglycoaldehyde. It implies that th e ring cleavage of DDT n eeds the existence of oxygen and alternating anaerobic and aerobic incuba tion conditions can promote more complete DDTr biodegradation. Although various microorganisms are at the la boratory capable of biologically degrading toxaphene, DDT and its residues, DDTr, their mineralization in soil is very slow. In environments unlike in laboratory, carbon sour ce and electron donors may not be sufficient;

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29 degraders may compete with other strains; bioavailability of contaminants may be low or concentrations of contaminants may be too high as to be toxic for degrading microbes, etc. These difficulties can not overshadow the prospective of bioremediation of toxaphene and DDTr and biodegradation has the potential to be enhanced by some strategies. For example, the addition of co-substrates such as lime (Parr and Smith, 1974) and alfalfa (Parr and Smith, 1974) seemed to increase the bacterial popul ation, thereby increasing the microbial degradation. Among environmental factors, temperature was very im portant for enhancing th e conversion of DDT to DDD (Guenzi and Beard, 1976), while aerating with N2 was capable of activating the anaerobes responsible for toxaphene degradation (Pa rr and Smith, 1974). In order to improve the bioavailability of contaminants, surfactant addition is the most common approach (You et al., 1996). Fortunately, all these enhancements can be used in the field and aid in bioremediation during large scale cleanup processes. Site Characteristics Lake Apopka is located in north west Orange and southeast Lake counties (Fig. 1-5), and is one of the most polluted lakes in Florida. Farm ing operations, wastewater discharged from shoreline communities and citrus processing plan ts were the main pollution sources. Excessive phosphorus in Lake Apopka resulted in the dec line of the vegetation, food for the fish and wildlife under the water. As a result, the State of Florida cl osed this area to farming in 1998 and is now restoring the lake and surrounding la nd. During the winter of 1998-1999, over 600 birds, primarily white pelicans, associated with the lake died. The reasons for this large bird kill are still unclear, although the tissues of some of the pelicans cont ained high concentrations of OCPs. The major pesticides in this area are toxaphe ne, DDT, DDD, and DDE. According to En Chem, in 1999, Lake Apopka NSRA had 16 ppm of toxaphene, 0.35 ppm of DDT, 2 ppm of DDE, and 0.54 ppm DDD in the moderately contaminated ar eas. In the highly contaminated sites, the

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30 contaminated pesticides and metabolites had a pproximate recovery of 48 ppm of toxaphene, 1.2 ppm of DDT, 2.4 ppm of DDE, and 1.6 ppm DDD. The concentra tions of these pollutants in the extremely highly contaminated sites were as hi gh as 150 ppm of toxaphene, 2.4 ppm of DDT, 2.8 ppm of DDE, and 1.8 ppm DDD. Obviously, the contamination le vel of toxaphene exceeds the maximum contaminant level (MCL) of 5 ppb by EP A. The removal of these OCP is of great concern. The data also give a clue that DDT has probably converted to DDD and DDE through intrinsic degradation. We could be confident that the bioremediation of the pesticides in this restoration area is feasible. Research Hypotheses Published studies dem onstrated that the biodegradation of toxaphene, DDT and its major metabolites under appropriate conditions could occu r. As a promising technique for restoring these OCP-contaminated soils, bioremediation has not been studied extens ively. Specifically, the microbiology at this site of th eir biodegradation remains unknown. The high organic matter in NSRA soils result s in the difficulty in the extraction and remediation of OCPs. Our overall objective was to establish the optimal extraction methods of pollutants, the reasonability and applicability of bioremediation in the upper scale and the most efficient conditions for restor ing the NSRA. Specific hypotheses were proposed to gain the information for the extraction of OCPs and their biodegradation in both microcosm and mesocosm scales of the NSRA soils. Hypothesis 1: Compared with SOX, a traditiona l extraction method, ASE simultaneously extracts toxaphene and DDT family from c ontaminated soils from NSRA with highest efficiency, and repeatability. Solvent system, moisture content and organic matter impact the performance of the extraction methods. Hypothesis 2: Among six most commonly used elec tron donors under sulfate reducing conditions, introduced H2 will be directly utilized by dehalogenaters. Or dehalogenaters may be stimulated by hydrogen transfor med from special donors through syntrophy.

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31 Toxaphene, DDT and its major metabolites can be used as the sole energy source or electron acceptors by stimulated de graders under these conditions. Hypothesis 3: A pure culture or consortium native in the contaminated soils reductively dechlorinates toxaphene by utilizing toxaphene as electron acceptor when provided with the electron donor that shows th e potential effect on stimulati ng biodegraders in study of Hypothesis 2. Hypothesis 4: Mesocosms, a scale up study, will extend the concepts about relationship between electron donors and biodegradation deve loped in the bench scale experiments; readily available plant materials might be fermented and produce nutrients, especially electron donors, which has the equivalent effects on stimulating OCP degraders to technical electron donor (lactate). Study Overview Our study was conducted to: 1) optim ize th e extraction methods for toxaphene, DDT, DDD, and DDE from soils with high organic matter contents; 2) more im portantly, establish the feasibility and further the valuable information of bioremediation of OCPs contaminated soils in the bench scale by testing various electron donors and attempting to characterize the responsible organisms; 3) extend the concepts in the micr ocosm studies to the scale up of mesocosm. Chapter 2 investigates the most efficient methods for extracting toxaphene, DDT and its major metabolites from NSRA soils. Conditions for the simultaneous determination of these pollutants from different matric es are optimized using both ASE and SOX. The influence of water content in samples on the extraction efficien cy is examined. Efficiency of isolating these chemicals using solvents methylene chloride and hexane is compared. Chapter 3 focuses on whether sulfate reducing conditions would promote the dechlorination of toxaphene and DDT. And also, th is chapter compares degradation of the OCPs with important electron donors in dechlorination to find the best combination for degradation of the target OCPs. Finally, this Chapter tries to identify the biodegraders and investigate whether pure culture or a consortium of bact eria cometabolizes toxaphene or DDT.

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32 Chapter 4 tests the concepts developed fr om the microcosm studies by extending the microcosm studies to a mesocosm scale. Furthermore, this Chapter evaluates the efficacy of produced lactate and hydrogen from the fermenta tion of readily available plant materials as electron donors in order to decrease the cost and make the process safely or practically handled. Finally, Chapter 5 summarizes implications of this research toward greater understanding of biodegradation of toxaphene, DDT, DDD and DDE, and factors co ntrolling this process in NSRA soils.

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33 Table 1-1. Physical and chemical properties of toxaphene, p,p -DDT, DDD and DDE Toxaphene p,p' -DDT p,p' -DDD p,p' -DDE Chemical formula C10H10Cl8 C14H9Cl5 C14H10Cl4 C14H8Cl4 Molecular weight 413.82 (avg) 354.49 320.04 318.02 Melting point (C) 65 90 106.5 109.5 89 Boiling point (C) decomposes decomposes 350 336 Density (g/cm) 1.65 1.6 1.39 No data Vapor pressure (mm Hg at 20 C) 0.2 0.4 1.5 107 1.35 106 6 106 Partition coefficients: Log Kow 3.30 6.91 6.02 6.51 Log Koc 2.47 5.18 5.18 4.70 Water solubility (mg/L at 25 C) 3 0.023 0.09 0.12

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34 Table 1-2. Half-life of toxaphene, DDT, DDD and DDE in different matrices Toxaphene DDT DDD DDE Soil 11a yr 10.5a yr No data 5.7e yr Sediment No data 12c yr No data No data Surface water No data 350b day No data No data Water 20d yr 3.3-3.7f day Ground water 31b yr No data No data a: Nash 1967 b: Howard 1991 c: Van Metre 2005 d: Miller 1999 e: Beyer 1989 f: ATSDR 2002 Table 1-3. Effect of Eh on CT biodegradation by Providencia stuartii Min. Eh (mv) Overall rate C. K (r) (hr-1) Specific rate C. k (r) [hr-1/(mg/l cell mass)] Control -4.2 1.29 10-3 (0.963) 6.91 10-6 (0.959) Treatment1 -181.7 2.31 10-2 (0.900) 1.26 10-4 (0.897) Treatment2 -239.5 2.57 10-2 (0.952) 1.47 10-4 (0.984) Treatment3 -208.5 2.29 10-2 (0.931) 6.02 10-5 (0.939) Data were taken from Jin and Englande (1996). C: concentration of CT (ppb); r: correlation coefficients.

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35 Table 1-4. Gibbs free energy values and redox potentials of selected chlorinated compounds and inorganic redox couples Electron acceptor Half-reactio n of reductive conversion Go' (kJ/electr on) Eo' (mV) O2 O2 + 4H+ + 4e 2H2O -78.7 816 Fe3+ Fe3+ + e Fe2+ -74.4 771 CT CCl4 + H+ + 2e CHCl3 + Cl -65.0 673 PCE C2Cl4 + H+ + 2e C2HCl3 + Cl -55.4 574 CF CHCl3 + H+ + 2e CH2Cl2 + Cl -54.0 560 TCE C2HCl3 + H+ + 2e C2H2Cl2 + Cl -53.0 to -50.8 550 to 527 hexachlorobenzene C6Cl6 + H+ + 2e C6HCl5 + Cl -171.4 478 pentachlorobenzene C6HCl5 + H+ + 2e C6H2Cl4 + Cl -163.4 to -167.7 455 to 421 pentachlorophenol C6HCl5O + H+ + 2e C6H2Cl4O + Cl -167.8 to -156.9 455 to 399 NO3 NO3 + 2H+ + 2 e NO2 + H2O -41.7 432 DCE C2H2Cl2 + H+ + 2e C2H3Cl + Cl -40.6 to -38.3 420 to 397 tetrachlorophenol C6H2Cl4O + H+ + 2e C6H3Cl3O + Cl -157.1 to -132.2 400 to 271 trichlorobenzoate C6H3Cl3O2 + H+ + 2e C6H4Cl2O2 + Cl -163.1 to -147.4 397 to 331 chlorobenzoate C6H5ClO2 + H+ + 2e C6H6O2 + Cl -145.4 to -137.3 339 to 297 Fe(OH)3 Fe(OH)3 + 3H+ + e Fe2+ + 3H2O -11.4 118 SO4 2 SO4 2 + 9H+ + 8e HS + 4H2O +20.9 -217 HCO3 HCO3 + 9H+ + 8e CH4 + 3H2O +23.0 -238 H+ 2H+ + 2e H2 +40.5 -420 Fe2+ Fe2+ + 2e Fe(0) +42.5 -440 The Go' and Eo' values were taken from De Wildeman and Verstraete (2003) and Dolfing and Harrison (1992).

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36 A B C D Figure 1-1. A: Toxaphene: C10H10Cln (n=5-12); B: DDT: C14H9Cl5; C: DDD: C14H10Cl4; D: DDE: C14H8Cl4.

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37 Figure 1-2. Anaerobic degradation pathway of DD T (A, D, E and F are multiple steps whose intermediates are not identified yet.). (http://umbbd.ahc.umn.edu/ddt2/ddt2_map.html)

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38 Figure 1-3. Aerobic degradation pathway of DDT (A, B, C, D, and E are multiple steps whose intermediates are not identified yet.). (http://umbbd.ahc.umn.edu/ddt/ddt_map.html)

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39 Figure 1-4. Six environmentally relevant to xaphene components; I: 2,2,5-endo,6-exo,8c,9b,10aheptachlorobornane (Tox B); IIa: 2, 2,5-endo,6-exo,8b,8c,9c,10a-octachlorobornane (Tox A1); IIb: 2,2,5-endo,6-exo,8c,9b,9c,10a -octachlorobornane (Tox A2); III: 2,2,5endo,6-exo,8c,9b,10a,10b-octachlorobornane ; IV: 2,2,5-endo,6-exo,8b,8c,9c,10a,10cnonachlorobornane; V: 2,2,5-endo,6-e xo,8c,9b,9c,10a,10b-nonachlorobornane.

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40 Figure 1-5. The North Shore Restora tion area of Lake Apopka in FL

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41 CHAPTER 2 EVALUATION OF EXTRACTION OF TOXAPHE NE, DDT, DDD, AND DDE F ROM NSRA SOILS WITH HIGH OR GANIC CARBON CONTENT Toxaphene (a complex mixture of polych lorinated monoterpenes), 1,1,1-trichloro-2,2bis( p-chlorophenyl)ethane (DDT), 1,1-dichloro-2,2-bis( p -chlorophenyl)ethylene (DDE), and 1,1dichloro-2,2-bis( p -chlorophenyl)ethane (DDD) are organochlorine pesticides (OCP) which have been of environmental concern due to their carcinogenic toxicity, adverse effects on human development, and long environmental persiste nce (Hill and McCarty, 1967; Nash and Woolson, 1967; Nash et al., 1973; Zeiger, 1987; Saleh, 1991; Sunyer et al., 2006). Adsorption of hydrophobic molecules to organic materials in so ils and sediments as a result of low water solubility decreased availability for uptake and transformation by soil microorganisms and limited degradation rates in soils (Alexander, 2000; Ghosh et al., 2003) Adsorption of these contaminants to soils with high organic carbon contents may also make extraction difficult for subsequent quantification. The extr action efficiency of contaminants can be affected by a variety of factors, including soil properties, extraction solvent, and sample moisture (Bandh et al., 2000; Josefsson et al., 2006). A variety of approaches for extracting organic chemicals from soils have been used over the years, including Soxhlet (SOX) and Accelerat ed Solvent Extraction (ASE). SOX is based on hot solvent extractions, and has relatively good extrac tion efficiencies from most soils (Spack et al., 1998). SOX is time consuming (12 to 24 hours) however, and requires at least 100 ml of solvent. ASE is a relatively new approach, a nd is intended to bypass so me of the limitations associated with SOX (Richter et al., 1995). In ASE, approximate ly about 50 ml of solvent is pumped into an extraction cell c ontaining the sample, at which tim e the temperature and pressure are elevated. After a few minutes, the extract is purged from the cell to a collection vial for further cleanup or direct analysis ASE is faster (5 to 15 minut es) and requires less extraction

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42 solvent than SOX, making it a pr omising alternative for extraction of analytes such as OCPs from soil. Information available comparing extraction efficiencies of hydrophobic OCPs by SOX and ASE from organic soils is limited, however. We are primarily interested in the fate of OC Ps in the organic soils from the northern shore of Lake Apopka, FL. The marshes of the norther n shore were drained in 1941 for muck farming (2000). Nutrient runoff from the highly productive farms resulted in the lake becoming highly eutrophic. As a result, the Stat e of Florida closed this area to farming in 1998 and is now restoring the lake and surr ounding land. During the winter of 1998-1999, over 600 birds, primarily white pelicans, associated with the lake died over this period. The reasons for this large bird kill are still unclear, although the tissues of some of the pelicans contained high concentrations of OCPs. Toxaphe ne and DDT concentrations from the northern shore of Lake Apopka have been reported to range from 21 ppb to 150 ppm, and removal of these OCPs is important for the complete restoration of Lake A popka. In order to evaluate the effectiveness of potential bioremediation strategi es in the organic soils of La ke Apopka, effective extraction methods are required for use with es tablished analytical strategies. In this study, ASE was compared with S OX to extract the toxaphene, DDT and its metabolites from Lake Apopka North Shore Re storation Area (NSRA). Conditions for the simultaneous determination of these pollutants fro m different matrices were investigated using both ASE and SOX. The influence of water conten t in samples on the extraction efficiency was examined. Efficiency of extracting these chem icals using solvents methylene chloride and hexane were compared. Except for the effect of soil characteristics on the recovery rate, all the evaluations of the extraction methods and parameters were investigated for extraction of aged residues of toxaphene, DDT, DDD a nd DDE.USING THE FORMATTING TEMPLATE

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43 Materials and Methods Samples: Collection and Preparation Three different levels of contamination, low, moderate, and high based on data reported by En Chem (Green Bay, WI) in 1999 (Table 2-1) we re used in this study. The low contaminated soil sample was collected from Lake Apopka NSRA (Lat 28 42.439 N, Long 81 38 59.113 W). The moderately contaminated sample was from Lat 28 42.213N, Long 81 36 24.836 W. The highly contaminated soil was initially excavat ed near a former airstrip approximately 18 months prior to collection for this study, and kept in a large vat under shelter. The definition of contamination levels was based on the concentra tion of toxaphene in the soil. Upon collection, the samples were placed in an ice cooler and transported to University of Florida campus in Gainesville. Immediately upon arrival in Gainesville, the soils were stored at 4C until use. Total carbon and nitrogen content was determined in th e Teaching Analytical Chemistry Laboratory of the Soil and Water Science Department, the Un iversity of Florida, using a TruSpec CN Elemental Determinator (LECO, St. Joseph, MI). Soil samples were ground and passed through a 20 mesh sieve (850 micrometer) after allowing to air dry for three days at room te mperature. Following drying, the soil volumetric water content was adjusted to intermediate or sa turated, and allowed to equilibrate for three days in sealed containers. The prepared samples were extracted within seven days after moisture adjustment. A surrogates solution (Decachlorobi phenyl (DCB) and tetrachloromethyl xylene (TCMX), AccuStandard, Inc., USA) and intern al standards (Toxaphene, DDD, DDE, and DDT, AccuStandard, Inc., USA) were added to sample s prior to extraction. 50 ppm of technical DDD, DDE, DDT, and 100 ppm of technical toxaphene we re spiked to low and highly contaminated soils, respectively, to test the recoveries.

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44 Extraction Procedures SOX Four gram s of soils in triplicate we re mixed in cellulose thimbles (25 80mm, Ahlstrom Mt. Holly Springs, LLC, PA, USA) with four grams of anhydrous sodium sulfate, which had been purified at 400 C for four hours. Using methylene chloride/acetone (4:1, v/v) or hexane/acetone (1:1, v/v) as solv ent, OCPs were extracted at 5 to 6 cycles per hour for 12 hours. ASE. A Dionex ASE 100 Accelerated Solven t Extractor (Dionex, Sunnyvale, CA) was used to perform the extractions. Triplicates of soil samples were mixed with hydromatrix (Varian, Palo Alto, CA) in 2:1 proportion and extracted in a 34 ml stainless vessel using methylene chloride/acetone (4:1, v/v) or hexane/ acetone (1:1, v/v) as solvent. The cell was filled with the solvent and heated to 100 C. The stat ic extraction was performed for 5 min, followed by flushing the vessel with 60% of the 34 ml cell of fresh solvent. Finally, the vessel was purged with nitrogen for 100 sec at 150 ps i. The total amount of extracti on solvent was about 50 ml, for carrying out one static cycle. Cleanup and Analysis All extracts were concen trated to n ear dryness by a gentle stream of N2. Repeated this process twice to remove acetone and methylen e chloride by adding hexane. The volume of extracts was measured in a glass graduated volumet ric cylinder. At this point, the samples were split for analysis of toxaphene and DDT and its metabolites. For analysis of DDT and its metabolites, one ml of extracts was passed through a florisil cartridge packed with ap proximately 500 mg of 200 m Florisil (Varian, Palo Alto, CA). Nine ml of hexane:acetone (9:1, v/v) were used to rinse the column. The resulting eluent was concentrated to 1 ml by an N2 stream and then transferred to a glass GC vial (2 ml, Fisherbrand, Atlanta, GA) and crimped with a Teflon lined cap for subsequent analysis by gas chromatography.

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45 Another one ml extract was pur ified by sulfuric acid treatmen t for further analysis of toxaphene. Equal amounts of concen trated sulfuric acid and sample were mixed in a 10 mL vial. The vial was capped and vortexed for 1 min. Phas es were allowed to separate, and the upper (organic) phase transferred to a GC vial for analysis. Extracts were analyzed with a gas chroma tograph (Shimadzu GC-17A) equipped with a 63Ni electron capture detector (ECD). The co lumn was a RTX-5 (0.25 mm ID 15 m long; Restek Corporation, Bellefonte, PA, USA). In most cases of DDT analyses, 250 C oven temperature was used, however toxaphene was cons idered as the first concern such that we applied the program which Paan and Chen deve loped (Pearson et al., 1997; Paan et al., 2006). The temperature was programmed as follows: 60 C, 3.0 C/min to 70 C for 1 min, 10 C/min to 130 C for 3 min, 10 C/min to 160 C, 20 C/min to 200 C. A calibration curves with 6 points bracketing the experimental concentrations wa s used for both analyses. The concentration calculation followed EPA 8000B and methods used by Pearson and Paan (Pearson et al., 1997; Paan et al., 2006). Statistical Analysis One-way ANOVA was used to test the di fference am ong treatment groups. Pairwise comparisons for differences in concentration m eans between different soil characteristics, extraction methods, and extraction solvents we re done by Tukeys Studentized test, while correlations were tested by Spearman and Pearso n using SAS version 9.1 (SAS Institute, Cary, NC). The null hypothesis was rejected at p < 0.05, thus the probability of observing no difference among the pair wise is less than 5%.

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46 Results and Discussion Correlation of ASE and Soxhlet Recoveries to Soil Characteristics Since true concentrations of toxaphene, DDT DDD and DDE in the soils of the NSRA soils are not known, the true extrac tion recovery is difficult to dete rm ine. Recoveries here refer to the rate at which the spiked technical chemicals of these OCPs were extracted. The recoveries of OCPs from the two sample sites were compared to the site characteristics in Fig. 2-1 (organochlorine pesticides con centration, total of carbon, and tota l of nitrogen, all data from Table 2-1) to examine whether a correlation exists between the soil proper ties and the recoveries. Table 2-2 gave the Pearson correlation and p values. Table 2-2 indicated that the re covery for toxaphene in ASE wa s correlated to the site. The correlation between recovery and total of organic carbon (TOC), total of nitrogen (TON) for toxaphene was further illustrated in Table 22 by a high correlation (-0.98) and the very low associated p value (<0.0001). High TOC and low C/N of sampling site seemed to decrease the toxaphene recovery in ASE by 36. 4%. This tendency is consistent with the investigations performed where an increasing am ount of carbon in the sediments caused chlorinated organic compounds less accessible to the extraction solven t in ASE (Bandh et al., 2000). However, some studies showed the opposite trend, in which th e TOC did not influence pressurized liquid extractions or the recoveries decreased as the C/ N ratio increased (Josefsson et al., 2006). In that study, the sediments used for examination had a relatively limited range of low organic carbon contents, ranging from 1.4 to 6.3%. Recoveries of the two different sites for DDD, DDE, and DDT were similar in both ASE and Soxhlet, while toxaphene in Soxhlet, ca n be observed from Fig. 2-1. The low Pearson correlations and high p-values indicated the lack of correla tion between the soil characteristics and the recoveries for the DDT family. Th ese findings agree with a published study on

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47 pendimethalin (Spack et al., 1998). Even though the so il properties varied gr eatly with regard to soil pH, soil structure, total C, total N, the re covery was not significantl y different when using Soxhlet extraction. Even though nega tive correlations were observed between recoveries of DDT and its metabolites, and water insoluble organic carbon (WIOC) were shown in another study (Tao et al., 2004), the variation in the WIOC was produced by amending the reagent to the soil while the soil without any amendment was used as the control. On the other hand, other major matrix characteristics, such as the C/N ratio, we re shown to correlate with decreased recoveries. In a report by Josefsson et al. (2006), the C/N ratio was significantly corr elated with extraction efficiency of polychlorinated biphenyls (PCB) by ASE (Josef sson et al., 2006). The higher C/N ratio indicates the possible aging of matrix, which causes the contaminants to intertwine with the matrix affecting the extractability. Nevertheless, the C/N ratio of soils in that study ranged from 15.6 to 32.3, which allowed for observation of the si gnificant correlation of the recovery to the C/N ratio. The C/N ratios in the present study only extended across a 1.2 fold range, such that more research might be of value in revealing a potential correlation between C/N and extractability in ASE and Soxhlet. Effect of Extraction Solvents Extraction s olvent has been demonstrated as one of the most signifi cant variables for PAH (Parr and Smith, 1974). Methylene chloride/acetone and hexane/acetone are the most commonly used solvent systems for the pesticide resi due analysis. Pairwise comparisons of OCP concentrations extracted by thes e two solvent systems with ASE and Soxhlet are shown in Table 2-3. Using ASE, methylene chlori de/acetone (4:1, v/v) provided significantly higher extraction efficiency for toxaphene, DDE, and DDT than did hexane/acetone (1:1, v/v); however, extraction of DDD did not differ between the two solvent systems. Significantly more toxaphene, DDT and DDE were extracted by ASE with methylene chlori de/acetone than with hexane/acetone (Table

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48 2-3). The boiling point of the solvents and the extraction temperature of the compared two procedures can explain our findings. For one thin g, boiling point for methyl ene chloride (40 C) is much lower than hexane (68 ~ 70 C), 30 C. Therefore, boiling point for methylene chloride/acetone (4:1, v/v) would be lower th an hexane/acetone (1:1, v/v), which acetone has a boiling point of 56 C. This fact indicates that hexane/acetone should be better than methylene chloride/acetone since the boiling point of the solvent system is the extraction temperature of SOX. In ASE, extraction temperature is fixed as 100 C for both solvent systems. Our study revealed that methylene chloride/acetone is a better solvent than hexane/acetone at the same extraction temperature. However, methylene chlori de as the only solvent in the extractions did not extract significantly higher am ounts of OCP than hexane/acetone (1:1, v/v) when evaluated pressurized fluid extraction (PFL ) and Soxhlet for the extracti on of DDT, DDE, and DDD from various environmental matrices, such as urban dust, sediment, and fish (S chantz et al., 1997). On the contrary, they were comparable with PFL fo r all the tested matrices. The difference may be ascribed to aging and other characteristics of NS RA soil. It suggests th at the decision of the appropriate extraction solvent de pends on the environmental matri x. Interestingly, the extraction of DDD was rather easily influenced by SOX but not affected much by ASE (Table 2-3). Additionally, the tendency of ef fects of solvent on other chem icals except for DDT using SOX showed to be opposite to that using ASE (Table 2-3). Pairwise comparisons revealed that extraction efficiencies of both DDE and DDD using hexane/acetone (1 :1, v/v) were significantly higher than using methylene chlori de/acetone (4:1, v/v) at 95% confidence level. And the effect of hexane/acetone on toxaphene and DDT was significant at 90% c onfidence level. The means of DDE and DDD concentrations using hexane/acet one as extractant were nearly double those using methylene chloride/acetone. Additionally, the extraction efficiency for toxaphene using

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49 Soxhlet with hexane/acetone is comparable to th at using ASE with methylene chloride/acetone. We can conclude, therefore, that the optimum extractant depends on the procedure. Hence, the appropriate combination extraction method with the extraction solvent system is a very important factor to gain optimum extraction efficiency. Comparison of ASE and Soxhlet Since m ethylene chloride/acetone (4:1, v/v) was evaluated for ASE, extraction methods were further compared with this solvent syst em. The ASE extractions for extracting from the highly contaminated sample generally showed highe r extraction efficiencies than SOX, with the exception of DDT (Table 2-4). Means for extr acted toxaphene, DDE, and DDD using ASE were 31.69%, 55%, 52.4% higher, respectively, than for SOX. The rather low p-values further confirm that ASE extracted these organic compounds more efficiently than S oxhlet at the statistical level ( p <0.05). Although ASE did not extract DDT with significantly higher efficiency at the statistical, it was comparable to SOX. Large re lative standard deviation (RSD) values indicated SOX extraction was less precise than ASE (Table 2-4). For the moderate contaminated soil, ASE ex tractions performed well in comparison with SOX (Table 2-5). ASE was 55.14% more effici ent than SOX for toxaphene extraction. A pairwise analysis of extracti on methods indicated ASE was significantly more efficient than SOX. Similar to DDT measured from the highly c ontaminated soil, the means were very similar. Statistical tests did no t indicate significant difference in means of DDE and DDD, unlike the observation from the highly contaminated soil. RSD values of means for extracting with ASE were also satisfactory in general. To date, most, if not all, extraction studies conducted for comparison of ASE with SOX conducted with polyaromatic hydrocarbons, PCBs and other organic compounds (Williams et al., 1993; Heemken et al., 1997; Schantz et al., 199 7; Brumley et al., 1998; Bandh et al., 2000)

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50 showed that ASE performed at least as well as S OX. For the first time, the efficiency of ASE for extracting the residue of pesticid es from the high organic carbon soils was compared with that of SOX. Our investigation agreed with the previous studies by showing the comparable or better efficiencies. Effect of Volumetric Water Content To investigate the influence of water content on extraction efficiency, OCPs were extracted from aged samples by ASE and Soxhlet with methyl ene chloride/ace tone (4:1, v/v) with varying water contents (Table 2-6; Fig. 2-1). As reported in other inves tigations of water effect on the extraction efficiency for orga nic pollutants (Wall and Stra tton, 1991; Heemken et al., 1997; Spack et al., 1998; Berglof et al., 2000), water co ntent was shown to be an important factor to obtain high extraction efficien cies from low organic carbon soils. Water content was significantly correlated to extraction efficien cy for DDT by SOX, and for DDE, DDT, and toxaphene by ASE. The correlation of water c ontent to DDE extraction efficiency was not significant. In both ASE and SOX, intermediate water content (Fig. 2-1) yielded the greatest overall efficiencies for extracting toxaphene, DDT and its metabolites simultaneously from the aged contaminated soils w ith high organic carbon. Two explanations have been proposed to e xplain the effects of water content on the extraction efficiency (Wall and Stratton, 1991; Sp ack et al., 1998). Firs t, water content may change some properties that are responsible for the adsorption of pesticides to soil. A certain amount of moisture may swell the organic matter in soil, allowing ex traction solvents to come in contact with the OCP, and is especially important for NSRA soils that contain high organic carbon contents. Such effect of the swelling or deactivating function of water was proposed to render pendimethalin sorption sites more accessible to supercritical fluid extraction (Spack et al., 1998). When water was not available, soil particle s or organic matter collapsed and adsorbed the

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51 OCPs strongly so as to keep them unavailable fo r solvents. Excessive wate r still led to extraction difficulties because the soil particles are su rrounded by water, which potentially blocks extraction solvent accessed to the OCPs. In a ddition, water soluble organic carbon (WSOC) may be another factor that influenced the adsorption of pollutants. Th e increase in the water content reduces the WSOC by partitioning it into the water matrix, which will combine with organic pollutants, thereby improving the extrac tion efficiency (Tao et al., 2004). This study compared the traditional and alte rnative extraction methods for extracting toxaphene, DDT and its major metabolites from the high organic soils. The parameters of interest were evaluated. When using ASE, the recovery of toxaphene was highly correlated to TOC and TON, while SOX was not affected. Both ASE a nd SOX extracted DDT and its metabolites with similar recoveries despite the different sample s. As an important factor in the extraction efficiency, intermediate water content yielded the greatest overall efficiencies for extracting OCP in both ASE and SOX. Significantly more toxa phene, DDT, and DDE were extracted by ASE using methylene chloride/acetone (4:1, v/v) than those with hexa ne/acetone (1:1, v/v). However, the hexane/acetone system show ed statistically higher effici encies for DDE and DDD at 95% confidence level and toxaphene at 90% confiden ce level using SOX. Statistical evaluations showed that equivalent results (moderate cont aminated soils) or higher efficiencies (highly contaminated soils) were achieved for the simu ltaneous extraction of residues of toxaphene, DDT and its metabolites by ASE as compared to SOX. Considering much less solvent consumption and extraction time and comparable efficiency, ASE is the preferable extraction technique for the investigation of high organic carbon contaminated soils.

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52 Table 2-1. Soil characteristics for low, m oderately, and highly contaminated soils Low Moderate High Total organic carbon (TOC %) 26.07(1.14) 41.34(2.30) 18.07(1.59) Total organic nitrogen (TON %) 2.70(0.26) 3.71(0.10) 1.59(0.12) C/N 9.66 11.14 11.36 Toxaphene (ppm) 5.1 16 48 DDE (ppb) 160 2000 2400 DDD (ppb) 34 540 1600 DDT (ppb) 21 350 1200 Note: number in the parentheses re presents the standard deviation. Table 2-2. Correlation of soil properties to recovery rate in Soxhlet and ASE Soxhlet ASE toxaphene DDT DDE DDD toxapheneDDT DDE DDD a 0.11 -0.49 0.33 0.39 -0.98 0.07 0.11 0.62 pb 0.7862 0.1681 0.3912 0.2954 <0.0001 0.8671 0.7808 0.0501 rc-Low 0.635 1.398 0.845 0.560 0.517 1.396 0.762 0.481 rc-High 0.612 1.496 0.814 0.534 0.812 1.377 0.747 0.431 a Pearson correlation coefficient. b Probability of significant correla tion; statistical significance set at p <0.05. c Recovery rates for low and highly contaminated soils respectively.

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53 Table 2-3. Comparison of extraction solvents for extracting toxaphene, DDT and its metabolites using Soxhlet and ASE from highly contaminated soil ASE Soxhlet Concentration (ppm) Concentration (ppm) methyleneCl:acetone hexane: acetone p-value methyleneCl:acetone hexane: acetone p-value Toxaphene 40.10 30.03 0.0214 30.45 41.47 0.0680 DDE 5.58 3.41 0.0076 3.60 6.65 0.0039 DDD 3.49 3.41 0.8610 2.29 4.03 0.0096 DDT 9.25 5.12 0.0039 8.73 6.07 0.0640 Table 2-4. Comparison of ASE and Soxhlet for extracting toxaphene, DDT and its metabolites from highly contaminated soil. Methylene chlo ride/acetone (4:1, v/v) was used as the solvent. ASE Soxhlet Concentration Concentration (ppm) RSDf (ppm) RSD p -value Toxaphene 40.10 0.095 30.45 0.052 0.0153 DDE 5.58 0.071 3.60 0.086 0.0024 DDD 3.49 0.094 2.29 0.178 0.0168 DDT 9.25 0.107 8.73 0.190 0.6618

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54 Table 2-5. Comparison of ASE and Soxhlet for extracting toxaphene, DDT and its metabolites from moderately contaminated soil. Methyl ene chloride/acetone (4:1, v/v) was used as the solvent. ASE Soxhlet Concentration Concentration (ppm) RSD (ppm) RSD p -value Toxaphene 2.87 0.114 1.85 0.052 0.0241 DDE 3.88 0.131 4.93 0.086 0.0810 DDD 2.65 0.018 3.48 0.178 0.1048 DDT 10.23 0.013 10.46 0.190 0.8763 Table 2-6. Correlation of water content in the so il to extraction efficiency by Soxhlet and ASE. Methylene chloride/acetone (4:1, v/v) was used as the solvent. Soxhlet ASE toxaphene DDT DDE DDD toxapheneDDT DDE DDD g -0.47 0.90 0.53 0.37 0.79 0.9 -0.47 -0.05 pb 0.0820 <0.0001 0.0524 0.1396 <0.0001 <0.0001 0.0326 0.4506 g Spearman correlation coefficient. b Probability of significant co rrelation; statistical significance for one-side test set at p <0.05.

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55 Figure 2-1. Effect of water content on the av erage means of extracted pesticides in the intermediate level of contaminated soils. Methylene chloride/acetone (4:1, v/v) was used as the solvent. Soxhlet 0 5 10 15 20 25 DDEDDDDDTtoxaphene pollutantsconcentration of pollutants (ppm) airdry intermediate saturated ASE 0 5 10 15 20 DDEDDDDDTtoxaphene pollutantsconcentration of pollutants (ppm) airdry intermediate saturated

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56 CHAPTER 3 BIODEGRADATION OF TOXAPHENE, DD T, AND ITS METABOLITES IN MICROCOSMS Toxaphene is a low volatile organochlorin e pesticide consisting of hundreds of chlorobornanes and other bicyc lic compounds. Following the ban of toxic 1, 1, 1-trichloro -2,2bis(4-chlorophenyl)ethane (DDT), toxaphene be came one of the worlds most commonly used pesticides in agriculture, used on crops such as cotton, grains, fr uits, and vegetables (Howdeshell and Hites, 1996). The global use of toxaphene reached as high as 1,330,000 tons (Voldner and Li, 1993). The United States Environmental Prot ection Agency (EPA) banned toxaphene in 1982 due to its carcinogenic toxicity (Hill and Mc Carty, 1967; Nash and Woolson, 1967; Nash et al., 1973; Zeiger, 1987; Saleh, 1991), and high persiste nce under natural envi ronmental conditions (10-14 yrs) (Nash and Woolson, 196 7; Menzie, 1972). As a result of agricultural practices in the second half of the 20th century, Lake Apopka has become Floridas most polluted lake. During the winter of 1998-1999, over 600 birds associated with the lake died (2000). Most of them were white pelicans, wood storks, and great blue hero ns. Even though the reasons for the death of these birds are still unclear, high concentrations of organochlorine pesticides (OCPs) detected in the tissues of some of the pelicans are the primar y factor or the cause of the death, these birds probably accumulated OCP by consuming contaminated fish. In general, techniques for cleaning up OCPs contaminated environments include physical, chemical, and biological methods. Chemical remediation of toxa phene has been investigated (Clark et al., 2005); in particular, physico-c hemical treatments by Fe(II)/Fe(III) redox coupling (Khalifa et al., 1976; Williams and Bidleman, 1978) have been applied to clean up toxaphene contamination. In situ bioremedia tion is an attractive st rategy for cleaning contamination of toxic OCP in soils. The metabolic versatility of indigenous microorganisms to metabolize and detoxify

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57 toxic compounds, the low cost, and avoidance of secondary pollution, are attractive properties compared with chemical or physical remedi ation strategies. Bior emediation of DDT by anaerobic and/or aerobic micr obes has been given a great d eal of interest (Subbarao and Alexander, 1985; Bidlan and Manonmani, 2002; Chiu et al., 2004). Despite the complex structure (consisting of at le ast 670), toxaphene has been re ported to be degraded under anaerobic conditions (Williams and Bidleman, 1978; Fingerling et al., 19 96; Buser et al., 2000; Lacayo-Romero et al., 2006). Reductive dechlorination is the first and lim iting step in the metabolism of many OCP by anaerobic bacteria. Sulfate-reducing bacteria (SRB) are a group of microorganisms reportedly involved in reductive dechlorination of chlori nated pollutants, whic h may be either a cometabolic process (de Best et al., 1997; Drzy zga et al., 2001; Drzyzg a et al., 2002) with the appropriate electron donor/a cceptor (sulfate) or a metabolic pr ocess (Zwiernik et al., 1998; Fava et al., 2003; Zanaroli et al., 2006) using chlorinated chemicals as the alternative electron acceptor upon the depletion of sulfate. The objective of this investigation was to in vestigate whether sulfate reducing conditions would promote the dechlorination of toxaphe ne and DDT by setting up two batches of experiments: one with low concen tration of sulfate; a nd the other with a hi gher concentration of sulfate. At the same time, we compared degrad ation of the OCPs with important electron donors in dechlorination (de Best et al ., 1997; Villemur et al., 2006), such as butyrate, propionate, formate, acetate, lactate, and hydrogen to find the best combination for degradation of the target OCPs. Batch enrichment culture followed by isolation of pure culture is a widely used method to isolate biodegraders despite some limitations. Additional objectives of this study were to identify

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58 the biodegraders and investigate whether pure cultur e or a bacterial consortium could be isolated that would metabolize toxaphene or DDT. Informa tion gained from this study will be used to design large-scale mesocosm experiments that will pr ovide a valuable test of principles for field scale treatment of contaminated areas of the north shore restoration area (NSRA). Materials and Methods Sampling Two soil samples with different levels of contam ination, termed moderate and high as described in Table 3-1, were used in this study. The moderately contaminated sample was from Lat 28 42.213N, Long 81 36 24.836 W. The highly contam inated soil was initially excavated near the former Lust airstrip approximately 18 months prior to collection for this study, and kept in a large vat under shelter. Th ese soils were placed in an ice cooler and transported to University of Florida campus in Gainesville. Immediately upon arrival, the soils were stored at 4C until use. Total carbon and nitrogen content was determined in the Teaching Analytical Chemistry Laboratory of the Soil and Water Science Department, the University of Florida, using a TruSpec CN Elemental Determinator (LECO, St. Joseph, MI). Microcosm Microcosm s were constructed from well-mixe d highly contaminated soil. Five g of soil were added to each culture tube and mixed with 45 ml of an autoclaved mineral salts solution consisting of (g/L of distilled water): K2HPO4, 4.8; KH2PO4, 1.2; NH4NO3, 1; CaCl2 .2H2O, 0.025; MgSO4 .7H2O, 0.2; Fe2(SO4)3, 0.001; resazurin, 0.001; pH was adjusted to 7.5. The medium was boiled for 5 minutes and then cooled under nitrogen gas. 200 mM anaerobic stock solution of acetate, lactate, formate, butyrat e, and propionate was pr epared by gassing the solution and then the headspace with nitrogen ga s. Then, 2% cysteine-sodium sulfide and 5mL electron donor solution were added in to serum vials using sterile syringes, plus a 100 kPa H2

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59 and one control with only 100 kPa N2 gas in the headspace. A sterile control microcosm was included for each electron donor microcosm to test the importance of each electron donor, and consisted of soil sterilized by three successive autoclavings and all components for each experimental replicate described above. Each microcosm was supplemented with a final of 20 ppm toxaphene (technical mix; Accustandard, New Haven, CT, USA) which was solved in hexane. The hexane wa s blown off with N2 before the medium was added. Serum tubes were incubated in the dark at 30 C without shaking. A second batch of enrichments similar to those described above was established, in additon 20 mg MgSO4 .7H2O per liter was added to in the mineral salt solution. Microcosms we re sacrificed after 25 days. In order to establish OCP biodegradation and increase statistical power, three replicates were constructed using the moderately contamin ated soil incubated for two months. The added study only tested three potentiall y promising electron donors, hydrogen, lactate, and acetate, plus one control. The medium components and procedure remained same. OCP Determination Extraction Procedure. OCP concentrations were determ ined in both soil and solution phases. OCPs in the aqueous phase and the por tion adsorbed to the walls and lids of the microcosms were determined via extraction with hexane. Soils were separated from liquid via centrifugation at 8,000 xg for 30 mi nutes. All the soils were gri nded and then passed through a 20 mesh sieve (nominal pore size, 850 micrometers) after three-day air dry. Surrogates solution of 50 ppb (Decachlorobiphenyl and TCMX, AccuSta ndard, Inc., USA) was spiked to samples prior to extraction. A Dionex ASE 100 Accelerated Solvent Extract or (Sunnyvale, CA) was used to perform the extractions. Triplicates of soil samples were mixed with hydr omatrix (Varian, Palo Alto, CA) in 2:1 proportion and extracted in a 34 ml stainl ess vessel using methylene chloride/acetone (4:1,

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60 v/v) or hexane/acetone (1:1, v/v) as solvent. The cell was filled with the solvent and heated to 100 C. The static extraction wa s performed for 5 min, followed by flushing the vessel with 60% of the 34 ml cell of fresh solvent. Finally, the vessel was purged with nitrogen for 100 sec at 150 psi. The total amount of extrac tion solvent was about 50 ml, for carrying out one static cycle. Cleanup and Analysis. All extracts were concentrated to near dryness by a gentle stream of N2. Repeated this process twice to remove acetone and methylene chloride by adding hexane. The volume of extracts was measured in a glass gr aduated volumetric cylinde r. At this point, the samples were split for analysis of to xaphene and DDT and its metabolites. For analysis of DDT and its metabolites, one ml of extracts was passed through a florisil cartridge packed with ap proximately 500 mg of 200 m Florisil (Varian, Palo Alto, CA). Nine ml of hexane:acetone (9:1, v/v) were used to rinse the column. The resulting eluent was concentrated to 1ml by an N2 stream and then transferred to a glass GC vial (2 ml, Fisherbrand, Atlanta, GA) and crimped with a Teflon lined cap for subsequent analysis by gas chromatography. Another one ml extract was pur ified by sulfuric acid treatmen t for further analysis of toxaphene. Equal amounts of concen trated sulfuric acid and sample were mixed in a 10 mL vial. The vial was capped and vortexed for 1 min. Phas es were allowed to separate, and the upper (organic) phase transferred to a GC vial for analysis. Extracts were analyzed with a gas chroma tograph (Shimadzu GC-17A) equipped with a 63Ni electron capture detector (ECD). The co lumn was a RTX-5 (0.25 mm ID 15 m long; Restek Corporation, Bellefonte, PA, USA). In most cases of DDT analyses, 250 C over temperature was used, however toxaphene was consid ered as first concern such that we applied the program which Paan and Chen developed (Pearson et al., 1997; P aan et al., 2006). The

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61 temperature was programmed as follows: 60 C, 3.0 C/min to 70 C for 1 min, 10 C/min to 130 C for 3 min, 10 C/min to 160 C, 20 C/min to 200 C. A calibration curves with 6 points bracketing the experimental concentrations wa s used for both analyses. The concentration calculation followed EPA 8000B and methods used by Pearson and Paan (Pearson et al., 1997; Paan et al., 2006). Isolation of Biodegraders The m ineral basal medium used for enrichment (MBME) of degraders consisted of (per L of distilled water) 5.455 g Na2HPO4; 0.675 g KH2PO4; 0.25 g NH4NO3; 0.029 g Ca(NO3)2 .4H2O; 0.2 g MgSO4 .7H2O; 0.001g resazurin; and 1mL of trace mineral solution. The trace mineral solution contained (g/L of distilled water): 1 g Fe2(SO4)3; 1 g MnSO4 .H2O; 0.366 g CuSO4 .5H2O; 0.25 g Na2MoO4 .2H2O; 0.1 g H3BO3 and 5 ml of concentrated H2SO4. pH was adjusted to 7.5. Sodium sulfide (0.5%) was used as the anaerobic reductant instead of cysteine-sodium sulfide to avoid the interference of Cl in measurement of dechlorination. Technical toxaphene or DDT (AccuStandard, New Haven, CT, USA) was absorbed to the bottom of the serums to a final concentration of 20 ppm of each. The medium was prepared anaer obically as described above. Dechlorination of Toxaphene and DDT. Five g moderately and highly contaminated soils were added to 45 ml of MBME using lact ate as the electron donor. After one month, 5 ml of the enriched medium were transferred to fresh MBME (45 ml). A total of 5 transfers after the first transfer were made every 14 days before th e genetic analysis and ch loride ion measurement were conducted. Ten ml of the solution in th e serum was centrifuged and filtered through a 0.45 m syringe filter (Nalge, Roches ter, NY) after 5, 8, 11, 14, and 17 days respectively. Chloride ion concentrations were measured by ion ch romatography using a Dionex Model LC20 equipped with an AS14 column and an ECD50 conductivit y detector (Dionex, Sunnyvale, CA), using 3.5 mM sodium carbonate/1.0 mM s odium bicarbonate as eluent.

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62 Roll Tube Experiment. A modified Hungate roll tube technique was used to isolate potential OCP degraders. Serum tubes, rather than screw capped tubes, of MBME with 1.6% agar were utilized, plus 100kPa N2 gas. Solid medium was inocul ated with 0.2 ml suspension of enriched medium, and was then incubated at 30 C in the dark until well-formed colonies were observed. Colonies were picked up taking into a ccount morphological differences and transferred to the liquid fresh medium. This process was repe ated several times to make sure that only one single strain tolerating or toxaphene had been picked. DNA Sequencing Analysis Nucleic acids were extracted with UltraCle an Soil DNA kits (Mobio, Solana Beach, CA). Following the m anufacturers instruction, 1 ml of culture was used. PCR for 16S rRNA genes was firstly conducted with SRB primers designed by Wagner et al. (Wagner et al., 1998), which amplifies a 1.9-kb fragment of the DSR gene. This set consists of primers DSR1F (5'ACSCACTGGAAGCACG-3') a nd DSR4R (5'-GTGTAGCAGTTACCGCA-3'). And then the universal primer set 27F/1492r (Lane, 1991) was used. One l of diluted DNA solution was mixed with 10 l of HotStarTaq Master Mix (Qiagen, Valencia, CA), 7 l of H2O provided with Master Mix, 1 l of each primer (10 pmol/ l). PCR amplification was carried out in a GeneAmp PCR system 2400 (Perkin-Elmer Applied Biosystems, Norwalk, CT). The initial enzyme activation and DNA denatura tion was performed at 95 C, followed by 35 cycles of 30s at 95 C, 30s annealing at 58 C, and 30s extension at 72C for amplification of 16S rRNA genes. A 7 min final extension was performed at 72 C. The amplicons were elec trophoresed through 1% agarose gels in TAE buffer. Fresh PCR amplicons were pretreated and transformed into chemically competent E. coli TOP 10F cells following the manufacturers inst ructions (Invitrogen, Ca rlsbad, CA). Twenty

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63 individual colonies of E. coli were picked up for screening by direct PCR amplification using T7 and SP6 primers and electrophoresed as descri bed before. Restriction Fragment Length Polymoprhism analyses performe d with the restriction enzyme Hha 1 were used to classify clones in order to reduce the number of clones seque nced. Digests were electrophoresed through 2% agarose. Selected clones were sequenced at the DNA Sequencing Core Laboratory at the University of Florida using the 27F primer. Phylogenetic analysis based on 16S rDNA sequences was performed with the aid of PH YLIP (version 3.6a2; J.L. Felsen stein, Department of Genetics, University of Washington, Seattle, WA). Statistical Analysis One-way ANOVA was used to test the di fference am ong treatment groups. Pairwise comparisons for differences in concentration means between different soil characteristics, extraction methods, and extraction solvents we re done by Tukeys Studentized test, while correlations were tested by Spearman and Pearso n using SAS version 9.1 (SAS Institute, Cary, NC). Contrast for different treatments for stimul ating degradation betwee n sulfate concentration, sterile control, and electron donors were tested using SAS version 9.1 (SAS Institute, Cary, NC). The null hypothesis was rejected at p <0.05, thus the probability of observing no difference among the pair wise is less than 5%. Results and Discussion Degradation of Toxaphene, DDT and its Metabolites Our initial h ypothesis was that toxaphene, and DDT and its metabolites at NSRA may be anaerobically degraded under sulf ate reducing conditions. A preliminary screening of soils from a flooded area of the NSRA indicated sulfate re duction occurred, confirming the presence of active SRB (data not shown).

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64 Under anaerobic conditions, soils containing high levels of recalcitrant contaminants are typically enriched with those microorganisms capable of utilizing the contaminant as either nutrient or terminal electron accep tor, such that higher levels of biodegradation may be expected in these soils than in moderately contaminated soils. Microcosms were established with a range of electron donors, including acetate, lactat e, formate, butyrate, propionate, and H2. These electron donors are major fermenta tion products that are used by a number of anaerobic bacteria, including those SRB (Villemur et al., 2006) that may dechlorinate organic compounds. Acetate and lactate are used by relative ly few anaerobic bacteria, and almost exclusively by SRB when sulfate is in abundance (de Best et al., 1997; Villemur et al., 2006). H2 is a very electron rich donor that controls a variety of anaerobic processes, although SRB are among the primary utilizers under sulfate reducing conditions. Experimental controls included live soil in which no exogenous electron donors were added to test the importance of individual electron donors, and a sterile autoclaved control for each of the el ectron donors. The sterile control was used as reference for degradation. Two concentrations of sulfate were tested, including 200 mg MgSO4 .7H2O per L medium and 20 mg MgSO4 .7H2O per L medium. The higher sulfat e concentration was considered sufficient to maintain an actively growing popula tion of SRB with the concentration of electron donors provided. The lower level of sulfate was inte nded to provide sufficient sulfate to enrich SRB, but not to sustain the growth of SRB w ith the level of electr on donors provided. If SRB capable of utilizing OCP as terminal electron acce ptors are present, they might be expected to switch to OCP once sulfate became limiting. The highest levels of degradat ion relative to sterile controls were observed for toxaphene (Fig. 3-1), with relatively little degradati on observed for DDT, DDE, and DDD (Fig. 3-2 Fig.

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65 3-4). Very little toxaphene was observed in medium solution or in hexane extracts of the bottles (data not shown), suggesting that hexane was not an efficient extr actant of toxaphene from the glass microcosm bottles used in this study. Statistically significant differences were detected in the concentrations of toxaphene for the 200 mg/L MgSO4 .7H2O ( p<0.001) amended and 20 mg/L MgSO4 .7H2O ( p<0.0001) amended treatments compared to the autoclaved soil control, using the least squared means test adjusted to Tukey. Over all, there was no significant di fference between the two levels of MgSO4, which means we do not have the evidence that SRB were responsible for the significant decrease in the concentrations of the tested OCP. Among the two different levels of sulfate supplies, however, the choice of electron donors re sulted in statistically signifi cant difference in promoting biodegradation. Four electron donors among the six tested ones significantly ( p<0.05) stimulated the degradation of toxaphene. With 20 mg/L MgSO4 .7H2O supplied media, toxaphene was degraded in the systems with hydr ogen, acetate, and lactate as the electron donors, in contrast to the sterile soil. Butyrate had a significant ( p <0.05) role in the decr ease in the toxaphene concentration with the s upplement of 200 mg/L MgSO4 .7H2O. Formate and propionate were less effective. This observation may be explained because acetate, butyrate, and lactate may be utilized more efficiently by some secondary fermenters than formate and propionate through syntrophy to produce hydrogen, lead ing to halorespiration with hydrogen as the electron donor. Furthermore, hydrogen amended to the headspace of the microcosm system was directly used as the electron donor for halorespir ation. The lack of significan t evidence between the amended electron donors and live soil cont rol showed that the natural a ttenuation of toxaphene occurred by the fact that 51% and 39% of toxaphene was re duced even in the control sets with low and high level of sulfate media. The effect of sulfate was particular ly important with lactate and H2,

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66 in which degradation was higher in low sulfate th an in high sulfate. Seemingly, sulfate depletion is necessary for observing the degradation. This ob servation is consistent to microbial reductive dechlorination of polychlorinated biphenyls (P CBs) by SRB according to Zanaroli (Zanaroli et al., 2006). However, in the presence of butyrate, degradation was higher in high sulfate than in low. Degradation of toxaphene under limiting sulfate conditions with H2 and lactate raised the possibilities that sulfate inhibited toxaphene degradation because, as initially hypothesized, sulfate was used preferentially as the terminal electron acceptor, or that SRB competed with another group responsible for toxaphene degradation. However, th e duplicates exhi bited a large relative standard deviati on (RSD). For example, even though acetate, lactate, and butyrate were observed to stimulate some microorganisms to degrade toxaphene, the experimental system exhibited RSDs as high as 0.905, 1.315, and 0.632, respectively (Fig. 3-1). Significant ( p<0.05) degradation of DDE was also observed in the 20 mg/L MgSO4 .7H2O group compared to the high sulfate set and st erile control. Among th e six electron donors, formate was the only effective compound to be utili zed to reduce DDE. In contrast to the control, 44% of DDE disappeared with the existence of fo rmate. Seemingly adequate sulfate did not help reduce DDE (Fig. 3-3) since the average concentr ation of DDE for all six electron donors plus control was similar to that in the sterile soil set. Some bacteria favored formate and propionate as electron donors to a ttack DDT. Formate in the 20 mg/L MgSO4 .7H2O medium most efficiently stimulated bacteria that can de stroy DDT in comparison with ot her electron donors when treated by low level of sulfate (Fig. 3-2), which the concentration of DDT decreased 19%. In 200 ppm sulfate treated group propionate played a role in degrading DDT by 20%. There was no statistical evidence to show the degradati on of DDD (Fig. 3-4) in all thr ee-treatment groups with various donors after 25-day incubation. No t surprisingly, the high RSD effected the confidence in

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67 determination of DDD and DDT. RSD for DDT ranged from 0.001 to 0.494, while RSD for DDD ranged from 0.006 to 0.244. A limitation of the experimental design was that only two replicates were included for each electron donor and control, and the time of incubation was relatively short for an anaerobic incubation of this type. Two, rather than three, replicates were used in order to reduce the number of analyses, and hence time required for gas chromatography. The limited number of replicates made statistically si gnificant differences between treat ments difficult to observe (Figs. 3-1 3-4). Microcosm studies were repeated with three repli cates and a two-month incubation period for those treatments that appeared promising. Another potential limitation of this experime nt was that the soil used was excavated approximately 18 months prior to our collecti on, during which time the soil became air dried. The numbers of non-spore forming microorgani sms may have been reduced, thereby likely reducing initial degradation rate s. Repeat of this study should use fresh soil that has high endogenous levels of OCP. Establishment of Degradation of Toxaphene, DDT and its Metabolites Degradation of toxaphene, DDT and its m eta bolites was observed to some extent in previous microcosms with H2, lactate, or butyrate as electron donors. However, the degradation needs to be established by using moderately co ntaminated soils, the aged OCP contaminated soils with fresh indigenous microorganisms. Additionally, the anaerobic microcosms were stopped after incubating two month for three replicates, thereby gaining greater statistical power. In the second study, anaerobic degradation of OCPs in soils was established (Fig. 3-5 Fig. 3-8). After two months of incubati on, statistically significant ( p<0.01) degradation of toxaphene was observed in the non sterile groups (Fig. 3-5) In agreement with the exploratory study,

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68 overall toxaphene degradation was not distingui shable between the low and high levels of sulfate. Lactate and butyrate we re capable of stimulating toxaphene degradation at the statistical level p <0.10 when the sulfate concentration was relatively high (200 mg/L MgSO4 .7H2O). On the contrary, another study of aged and freshl y toxaphene contaminated soils in anaerobic bioreactors indicated that lactic acid interfered with the biode gradation (Lacayo-Romero et al., 2006). They reported that toxaphene congeners showed a signifi cantly lower degradation rate after the addition of lactic acid. The structure of microbial commun ity or soil conditions in their investigation and our soils may contribute to such a difference. As discussed above, lactate and other electron donors such as butyrate can not be used by some dehalogenating bacteria such that they have to be fermented to hydrogen through syntrophy. Generally, SRB or other microorganisms participate in such fermentation when sulfate is not sufficient (Drzyzga and Gottschal, 2002). Hydrogen is the preferred electron donors by dechlorinaters. Once dechlorinaters obtain hydrogen, replacement of the chloride of OCP with hydrogen is accomplished. High concentration of sulfate, for the other, will make SRB outnumber dehalogenating bacteria and stop the dehalogena tion. In the present study, abundant sulfate salt (200 mg/L MgSO4 .7H2O) allowed the consumption of lactate or butyrate to hydrogen, thereby promoting the disappearance of toxaphene. Po ssibly, other groups of microorganisms used lactate or butyrate as the el ectron donor and dehalogenating mi crobes utilized the produced hydrogen to yield energy by prefer ring dehalogenation, more effi cient energy yielding approach, to sulfate reduction (Dolfing and Harrison, 1992). When hydrogen is provided artificially, dehalogenating bacteria would show higher degradation. In our st udy, we supplied with hydrogen gas, therefore, more degradation was detected with highly statistical significance ( p<0.05), for each contrast of 200 mg/L MgSO4 .7H2O group vs sterilized 200 mg/L

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69 MgSO4 .7H2O group and 20 mg/L MgSO4 .7H2O group vs sterilized 200 mg/L MgSO4 .7H2O group. Intrinsic disappearance of toxaphene was c onfirmed in the control systems of both high and low sulfate medium with the statistical evidence ( p<0.05). With three replicates, RSDs were appreciably uniform and acceptable (within 0.2). In the second study, RSDs were relatively lo w and the degradation was confirmed more precisely for DDT and its metabolites. DDT si gnificantly disappeared in non sterile groups. Compared with the sterile group, the concentrati ons of DDT in the groups treated with 20 ppm and 200 ppm sulfate differed from the sterile control with strong st atistical evidence ( p<0.0001). In both non sterile groups, DDT was degraded ( p <0.05) notably in all the three chosen electron donors. With limited supplement of sulfate (20 mg/L MgSO4 .7H2O), bacteria degraded DDT with the highest efficiency (Fig. 3-8), 47% re duction utilizing hydrogen as the electron donor in contrast to other electron donor s. This finding supported our cont ention that the dechlorinating process can be stimulated under sulfate reducin g condition (Zwiernik et al., 1998; Fava et al., 2003; Zanaroli et al., 2006). Additionally, DDT in moderately contaminated soil without any electron donor was degraded intrinsically, an average of 16% and 29% of decrease in the concentration in comparison with the sterile gr oup. Unlike in the highly contaminated soil, DDD was observed degraded in the moderately contaminated soil after incubated for two month. The treatment factors such as the le vel of sulfate salt, different el ectron donors, and sterilization had statistically significant effects ( p<0.0001) on DDD biodegradation. Within 200 ppm sulfate salt group, all these three electron donors participat ed in the statisti cally significant ( p <0.05) disappearance of DDD in comparison with sterile control; degradation even happened greatly in control set. Although intrinsic degradation of DDD was observed in treatments of 20 mg/L MgSO4 .7H2O, the applied electron donors significantly promoted the degradation (Table 3-2).

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70 Microcosms in this system seemed to be activat ed to the highest extent, which degraded 45% of DDD. In contrast to the first set, we didnt obser ve a significant decrease but an increase in the concentration of DDE in some serum bottles. Because DDT was considered dechlorinated to DDE, accumulation of DDE indicated the degradation of DDT. Isolation of Toxaphene Degraders Dechlorination of Toxaphene In order to characterize th e OCPs biodegrader(s) in the NSRA conta minated soils, a batch of enri chment experiments were established. After three months with five transfers, chloride ion was determined to confirm the occurrence of biodegradation in the enrichment media. During the test period of experiment, the concentration of chloride ion increased in both serum bottles en riched from highly and moderate contaminated NSRA samples (Figs. 3-9 and 3-10). But the dyna mics of released chloride differed in two different levels of contamination. In both systems, 5 ppm of chloride ion was found following five days after the transfer, wher eas bacteria from moderate soils were more active to decompose the organochlorine chemical. Until the 11th day, 15 ppm of chloride ion was produced in the serum bottles originated from the highly contaminated sample. But 18 ppm of chloride was determined only after 8 days, and 28 ppm chloride appeared at the point of 11 days in moderate soil. In contrast to moderately contaminated so il, only 25 ppm of chloride was released after 17day incubation in high soil. The production of Claccompanying the growth of the bacteria strongly suggested the mechanism of reductive de chlorination of toxaphene (Khalifa et al., 1976; Fingerling et al., 1996 ; Buser et al., 2000). Although DDT showed a degradable potential in the previous microcosm study, there was no detectable Cl-. Possibly, the DDT degrading consortium wa s lost somewhere in the process of those five transfers. Furthermore, the turbidity of the medium that re vealed the growth of bacteria suggested that lact ate was the preferred carbon and energy source to DDT for the

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71 bacteria in the medium. We, thereby, drew a conc lusion that the strains which would be isolated from the solution might not degrade DDT with the presence of lactate. Roll Tube, 16S rDNA and Sequencing In this study, anaerobi c roll tube experiments were set up to separate the col onies and transferred to the limite d medium with t oxaphene as the sole substrate and lactate as the electron donor. Each of very small colonies that developed were picked and cultured in the limited liquid medi um. The turbidity in the limited medium was observed and 16S rDNA was sequenced. However, the sequencing analysis still revealed that different species existed in the medium. In additi on, the second or third transfer of the culture to the fresh liquid medium with toxaphene and lact ate did not show any grow th. It seemed that a consortium participated in the degradation of toxaphene. As a powerful tool to isolate the pure strains roll tube is there are a few limitations. Problems coming with this met hod include: first, real environmental conditions are very different from those in th e enrichment experiments, such as the redox potential; second, th e concentration used as the source of carbon and energy to isolate the degraders is usually higher than in the environment; third, many pesticides are shown to be attacked by consortia instead of pure cultures. In order to identify the bacteria who were capable to grow in the presence of toxaphene and dechlorinate toxaphene, their 16S rRNA genes were sequenced and compared to others in GenBank. As specific primers for SRB were tried to isolate the degrader s, no DNA was detected with this type of primers. A range of strains of bacteria was characterized from enrichment cultures when using the universal primers. Phyl ogenetical analysis demonstrated that some sequences related to the genus Citrobacter with 99.9% sequence similarity were most abundant in the clone libraries (Fig. 3-11); others were closely related to the gram positive genus of Sporomusa, with 98% similarity (Fig. 3-12) Citrobacter species have been previously reported

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72 to tolerate or accumulate heavy metals such as lead (Levinson and Mahler, 1998), cadmium (Montgomery et al., 1995), copper (Williams et al ., 1993; Pathak and Gopal, 1994; Pickup et al., 1997) and nickel (Pickup et al., 1997). Additionally, anaerobic degradation a range of organic pollutants such as phenol (Kanekar et al., 1999) biphenyl (Grishchenkov et al., 2002), TNT (Li et al., 1989), 2,4-dichlorophenol (DCP), 2,4,6-trichlorophenol (TCP) and pentachlorophenol (PCP) (Martinez et al., 2000) has been reported to abscribe to Citrobacter species. For the first time, this work demonstrated the potential biodegr adation of toxaphene at the microbial level and related Citrobacter to toxaphene degradation. And also, Sporomusa ovata was reported to reductively dechlorinate perchloroethene (PCE ) to trichloroethylene (TCE) (Terzenbach and Blaut, 1994) as the sole product with methanol as an electron donor. The re versible inhibition with propyl iodide sugges ted that Corrinoids in S. ovata were involved in the dechlorination reaction of PCE to TCE. The mech anisms that the bacteria natu rally occurred in the studied enrichment culture used to dechlorinate toxaphe ne are attempted to demonstrate. Recently, a white-rot fungus, Bjerkandera sp., (Romero et al., 2006) has been reported to degrade toxaphene when supplied with wood chips, wheat husk or cane molasses as cosubstrates in batch culture experiments. Another strain which has been isolat ed from the aged contaminated soil to degrade toxaphene was analyzed to be related to Enterobacter cloacae (Lacayo-Romero et al., 2005) and characterized to be a facultative anaerobic, Gram-negative hete rotrophic bacterium. However, both investigations did not reve al the real mechanisms for these microorganisms to use for toxaphene degradation.

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73 Table 3-1. Soil characteristics for mode rately and highly contaminated soils Moderate High Total organic carbon (TOC %) 41.34 18.07 Total organic nitrogen (TON %) 3.71 1.59 C/N 11.14 11.36 Toxaphene (ppm) 16 48 DDE (ppb) 2000 2400 DDD (ppb) 540 1600 DDT (ppb) 350 1200

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74 Table 3-2. Statistical contrast analyses of roles of various el ectron donors in biodegradation of OCPs in microcosms after two-month incubation OCPs Electron donors TestEstimate p-value 200 ppma vs S200 ppmb-1.640.0872 Lactate 20 ppmc vs S200 ppmb-1.150.2027 200 ppma vs S200 ppmb-2.150.0317 H2 20 ppmc vs S200 ppmb-2.320.0239 200 ppma vs S200 ppmb-2.580.0537 Toxaphene Butyrate 20 ppmc vs S200 ppmb-0.250.8077 200 ppma vs S200 ppmb-1.670.0014 Lactate 20 ppmc vs S200 ppmb-1.700.0013 200 ppma vs S200 ppmb-1.580.0002 H2 20 ppmc vs S200 ppmb-2.23<0.0001 200 ppma vs S200 ppmb-1.950.0059 DDT Butyrate 20 ppmc vs S200 ppmb-1.120.0534 200 ppma vs S200 ppmb-2.200.0008 Lactate 20 ppmc vs S200 ppmb-2.260.0007 200 ppma vs S200 ppmb-1.65<0.0001 H2 20 ppmc vs S200 ppmb-2.67<0.0001 200 ppma vs S200 ppmb-2.690.0019 DDD Butyrate 20 ppmc vs S200 ppmb-1.570.0215 200 ppma vs S200 ppmb0.210.5083 Lactate 20 ppmc vs S200 ppmb-0.490.1495 200 ppma vs S200 ppmb-0.100.6091 H2 20 ppmc vs S200 ppmb-1.290.0004 200 ppma vs S200 ppmb0.500.2085 DDE Butyrate 20 ppmc vs S200 ppmb0.600.1441 a: medium with 200 mg/L MgSO4.7H2O b: medium with 200 mg/L MgSO4.7H2O and sterile soil c: medium with 20 mg/L MgSO4.7H2O

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75 Figure 3-1. Degradation of toxaphene in microc osms containing differe nt electron donors and sulfate concentrations. Note: MgSO4 was added as the form of MgSO4.7H2O. Error bars represent +/one standard deviation. 0 10 20 30 40 50 60cont r o l a cet at e lactat e f or m at e butyrat e propionat e hydrog e n concentration (ppm) 200 ppm MgSO4 20 ppm MgSO4 sterile control

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76 Figure 3-2. Degradation of DDT in microcosms containing different el ectron donors and sulfate concentrations Note: MgSO4 was added as the form of MgSO4.7H2O. Error bars represent +/one standard deviation. 4 8 12 16 20 24control ac et at e l actat e f or m at e b ut yr at e p ropionat e hydr og e n concentration (ppm) 200 ppm MgSO4 20 ppm MgSO4 sterile control

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77 Figure 3-3. Degradation of DDE in microcosms containing different el ectron donors and sulfate concentrations Note: MgSO4 was added as the form of MgSO4.7H2O. Error bars represent +/one standard deviation. 4 8 12 16 20 24contro l a c e ta t e l acta t e fo rmat e b u tyrat e p ro pi o n a t e hydroge n concentration (ppm) 200 ppm MgSO4 20 ppm MgSO4 sterile control

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78 Figure 3-4. Degradation in DDD in microcosms containing different el ectron donors and sulfate concentrations Note: MgSO4 was added as the form of MgSO4.7H2O. Error bars represent +/one standard deviation. 4 8 12 16 20 24con t rol acetate l a ct a t e form a t e b utyrat e pr o pi o na t e hy d rog e nconcentration (ppm) 200 ppm MgSO4 20 ppm MgSO4 sterile control

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79 Figure 3-5. Degradation of toxaphene in microc osms containing differe nt electron donors and sulfate concentrations Note: MgSO4 was added as the form of MgSO4.7H2O. Error bars represent +/one standard deviation. 0 3 6 9 12 15control hydro ge n lactat e b ut y rat e concentration (ppm) 20 ppm MgSO4 200 ppm MgSO4 sterile control

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80 Figure 3-6. Degradation of DDE in microcosms containing different el ectron donors and sulfate concentrations Note: MgSO4 was added as the form of MgSO4.7H2O. Error bars represent +/one standard deviation. 0 1 2 3 4 5 6 7control hydr o gen lact a t e bu t yrat e concentration (ppm) 20 ppm MgSO4 200 ppm MgSO4 sterile control

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81 Figure 3-7. Degradation of DDD in microcosms containing different el ectron donors and sulfate concentrations Note: MgSO4 was added as the form of MgSO4.7H2O. Error bars represent +/one standard deviation. 0 2 4 6 8c ontrol hy dr oge n lactate b u tyra teconcentration (ppm) 20 ppm MgSO4 200 ppm MgSO4 sterile control

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82 Figure 3-8. Degradation in DDT in microcosms containing different el ectron donors and sulfate concentrations Note: MgSO4 was added as the form of MgSO4.7H2O. Error bars represent +/one standard deviation. 0 1 2 3 4 5 6contro l hydrogen l a ctate bu t y r a t econcentration (ppm) 20 ppm MgSO4 200 ppm MgSO4 sterile control

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83 Figure 3-9. Dechlorination of toxaphene with lactate as the electron donors enriched from moderately contaminated soils Note: Error bars represent +/one standard deviation. 0 10 20 30 40 05101520 incubation time (day)concentration of Cl ion (ppm ) control lactate

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84 Figure 3-10. Dechlorination of toxaphene with lactate as the electron donors enriched from highly contaminated soils Note: Error bars represent +/one standard deviation. 0 10 20 30 40 50 05101520 incubation time (day)concentration of Cl ion (ppm ) control lactate

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85 Figure 3-11. Phylogenetic tree of dominant se quences (K13s, L12, L1s, K10, K19, L13s, L19, L19s, K1s, K19s, L13) enriched from NSRA soils 0.1 Escherichi Citrobacter werkmanii CDC 0876-58 (AF025373) Citrobacter sp. TSA-1 (AF463533) Citrobacter freundii CDC 621-64 (AF025365) Citrobacter freundii WA1 (AY259630) Citrobacter freundii DSM 30039 (AJ233408) L13s K19 K10 L1s L12 K13s Citrobacter sp. CDC 4693-86 (AF025367) L19 Citrobacter freundii IRB3 (AY870315) Citrobacter freundii HPC255 (AY346317) Citrobacter sp. HPC369 (AY999033) Citrobacter freundii MBRG 7.3 (AJ514240) L19s Citrobacter freundii SSCT56 (AB210978) Citrobacter youngae CECT 5335 (AJ564736) Citrobacter freundii 07F (AB182001) K19s K1s L13 Citrobacter freundii A1 (AY163805) 100 52 60 55 50 55 78 53 59

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86Figure 3-12. Phylogenetic tree of Gram-positive speci es (K4, K5, K6, L5) enri ched from NSRA soils 0.1 S elenomonas sp. oral clone JI021 (AY349409) A naerospora hon g kon g ensis HKU15 (AY372051) S poromusa acidovorans DSM 3132 (AJ279798) S poromusa sphaeroides DSM 2875 (AJ279801) S poromusa paucivorans DSM 3637 (M59117) Uncultured bacterium TCE33 (AF349762) D esul f osporomusa pol y tropa STP11 (AJ006606) D esul f osporomusa pol y tropa STP13 (AJ006608) S poromusa sp. DR5 (Y17761) S .silvacetica DG-1 (Y09976) S poromusa sp. DR6 (Y17760) S poromusa sp. DR15 (Y17762) S poromusa ovata DSM 2662 (AJ279800) S poromusa termitida JSN-2 (M61920) S poromusa malonica DSM 5090 (AJ279799) S poromusa sp. DR1/8 (Y17763) S poromusa aerovorans TMAO3 (AJ506191) S poromusa aerovorans TMAM3 (AJ506192) K4 K5 K6 L5 A naeromusa acidaminophila DSM 3853 (AF071415) A naerovibrio burkinabensis DSM 6283(T) (AJ010961) Clostridium quercicolum ATCC 25974 (M59110) Clostridium quercicolum DSM 1736(T) (AJ010962) 100 72 100 51 74 83 96 69 82 100 100 93 100 69 100 100 100 100 100 100

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87 CHAPTER 4 BIOLOGICAL BREAKDOWN OF PESTICID E S IN LAKE APOPKA NORTH SHORE RESTORATION AREA SOIL IN A MESOCOSM EXPERIMENT Residues of toxaphene, DDT (1,1,1-trichloro-2,2-bis( p -chlorophenyl)-ethane), DDE (1,1dichloro-2,2-bis( p -chlorophenyl)ethylene), and DDD (1,1-dichloro-2,2-bis( pchlorophenyl)ethane) are frequently detected in soils, surface water supplies, and aquatic sediments (Szeto and Price, 1991; Hargrave et al ., 1993; Paasivirta et al., 1993; Pearson et al., 1997; Xie et al., 1997; Kelderman et al., 2000; Shiv aramaiah et al., 2002). Since their toxicities and potential for bioaccumulation are significant, removing them from environmental matrices via an efficient and economical strategy is of interest. In situ bioremediation of organochlorine pesticides (OCPs) is a particularly attrac tive option because of its cost efficiency. The marshes of the northern shore of Lake Apopka, FL were drained in 1941 for muck farming. Nutrient runoff from the highly producti ve farms resulted in the lake becoming highly eutrophic. The State of Florida closed this area to farming in 1998 and is now restoring the lake and surrounding land. During the winter of 19981999, over 600 birds, primarily white pelicans, associated with the lake died ove r this period. The reasons for this large bird kill are still unclear, although the tissues of some of the pelicans cont ained high concentrations of OCPs. Toxaphene and DDT concentrations from the northern shore of Lake Apopka have been reported to range from 21 ppb to 150 ppm, and removal of these OCPs is important for the co mplete restoration of Lake Apopka. Studies about biodegradation of contamin ants usually start from the bench scale microcosm. In this scale, conditions for s timulating the biodegrader s responsible for the degradation are easily controlled. Once the feasibility of the bioremediation strategy in the microcosm system is confirmed, a larger scale investigation is preferred to represent the conditions in the field for evaluating such a stra tegy for cleaning up the contaminated site at both

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88 cost and efficiency aspects as findings in the be nch scale can not necessar ily occur as exactly as in the field. To evaluate the feasibility of bioremediation of OCPs, we tested a set of six different electron donors under sulfate redu cing conditions and demonstrat ed that appropriate electron donors stimulated the bacteria wh ich can utilize OCP as a term inal electron acceptor or energy source after incubating one month. In order to test the concepts developed from the microcosm studies, we extended the microcosm studies to a series of replic ated tanks containing soil from the NSRA. Furthermore, since hydrogen can not be safely or practically handled, and pure chemicals of lactate applied in the microcosms are relatively expensive, we tested the efficacy of produced lactate and hydrogen from the fermentation of some readily available plant materials as electron donors. DeBusk and Reddy reported that th e cellulose content of cattail is approximately 40% (DeBusk and Reddy, 1998), which means 1 kg cattail would contain approximately 400 grams cellulose and therefore would be theoreti cally transformed to approximately 5 moles of lactate through the fermentation pathway. In addi tion to cellulose, other fermentable substrates are also present in cattail to be the potential candidates for forming more fermentation products. Cycling between anoxic and oxic systems is believ ed to be an efficient way to completely detoxificate persistent OCPs (Gavrilescu, 2005) because complicated pollutants are resistant to the oxidative metabolism such that anoxic process, e.g. dechlorination reactions, has to degrade the chemicals to smaller molecular for the aer obes to take over. Since hydrology at NSRA is simply controlled, such cycling is allowed; one cycle of anoxic/oxic incu bations was included in these studies to gain the most possible degradation.

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89 Materials and Methods Site Prior to d etermining the site for soil collection for the mesocosm studies, soil samples were taken from five separate sites at NSRA. Candidate sites were se lected, marked, and cleared of vegetation by district personnel. Samples from the individual sites were composited from five randomly selected spots at each site. The soil from each site was initially mixed on site, transported to the laboratory in Gainesvill e, where soil was thoroughly mixed by hand in the laboratory. Approximately half the soil in each sa mple was shipped to Pace Analytical Services, Inc. for analysis of OCP residues, and the other half was analyzed at the University of Florida using accelerated solvent extraction (A SE) methods as described below. The site chosen for soil collection for use in the mesocosms was ZSS0750, located in a field south of Lust Rd (UTM coordinates, X 44411, Y 3171355), and was selected on the basis of OCP concentration and ease of access (Fig. 4-1). The OCP values reported by Pace for this site were: toxaphene, 9200 g/kg; DDT, 130 g/kg; and DDXs, 1620 g/kg (including DDT, DDE, and DDD). Approximately 6 cubic yards of soil were collect ed from a 13 ft x 13 ft plot at this site on December 12, 2005. The soil was mixed at the s ite using a trackhoe by a hazardous materials contractor under the direction of District personnel (Fig. 4-2). The soil was loaded onto a dump truck and transported to the Soil and Water Scie nce Departments greenho uses on the University of Florida campus in Gainesville. Mesocosm Once at the University of Florida greenhous es, soil was initially m anually mixed with shovels, and added to the tanks sequentially, one wheel barrow at a time. Mixing was deemed to

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90 be satisfactory, as indicated by th e relatively low variability with in OCP concentrations within and between tanks, as reported by Pace Analytical Services. Mesocosms were based on a design used by the Wetland Biogeochemistry Laboratory at the University of Florida. Mesocosms were constructed from 150 gallon Rubbermaid "Farm Tough" stock tanks; these are constructed of hi gh density polyethylene an d are approximately 35 inches (88.9 cm) wide at the top, 54 inches ( 137.16 cm) long at top, and 23 inches (58.42 cm) deep, with drains built into the sides of the ta nks. Prior to addition of soil, a layer of gravel approximately five inches (12.5 cm) thick wa s added to the bottom of each mesocosm to facilitate drainage. Nine mesocosm tanks (Table 4-1) were set on tables reinforced with cement block supports within the greenhouse (Fig. 4-3). The order of the mesocosms, from north to south, is Control-1, Cattail-1, Lactate-1; Control-2, Ca ttail-2, Lactate-2; Control-3, Cattail-3, Lactate-3. This design is intended to randomize any effects that lo cation in the greenhouse might have, such as temperature. Soil was added to the mesocosm tanks to a depth of approximately 26 cm. The top 10 cm soil was removed from each of the cattail tanks and mixed with 1 kg dried, ground cattail in a small electric cement mixer. Cattail for this study was harvested from Lake Alice on the University of Florida campus. This level of plant mate rial is not related to standing crop on site (which has little or no cattail), but rather th at amount of cattail that could be easily obtained and plowed into the soil. The mixture wa s then added back to th e appropriate tanks. Ten samples were randomly collected from each tank at soil depths of 5 cm, and mixed to form a single composite sample for each mesocosm. On January 10, 2006, water was added to a heig ht of 10 cm above soil surface. For the lactate mesocosms, lactic acid was mixed with tap water in 20 L containers to form a final

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91 concentration of 10 mM lactate. Approximately 160 L lactate solution was required to fill each tank to 10 cm above soil surface. Ten soil samples were collected randomly from each mesocosm at a depth of 5 cm, mixed to form one composite sample per tank, and frozen at C. The redox potentials in all tanks stabilized by February 21, 2006, which was considered the starting point for analyses. Anoxic incubatio n continued until June 8, 2006, at which time the water was removed from the tanks to begin the aerobic phase. Water was removed by siphon from the top of the tanks, and allowed to drain from the bottom through the drain. Drainage was very slow to ensure that wa ter above soil was siphoned off. DNA Extraction Procedure Soils were collected fro m five sites ra ndomly selected from each tank and then composited. Nucleic acids were extracted with UltraClean Soil DNA kits (Mobio, Solana Beach, CA). Following the manufacturers instruction, one gram of soil was used. PCR for 16S rRNA genes was conducted with the universal primer set 27F/1492r (Lane, 1991) and DHC primers, Fp DHC 587/Rp DHC 1090 (Hendrickson et al., 2002), for Dehalococcoides One l of diluted DNA solution was mixed with 10 l of HotStarTaq Master Mix (Qiagen, Valencia, CA), 7 l of H2O provided with Master Mix, 1 l of each primer (10 pmol/ l). PCR amplification was carried out in a GeneAmp PCR system 2400 (Perkin-Elmer Applied Biosystems, Norwalk, CT). The initial enzyme activation and DNA denaturation was performed at 95 C, followed by 35 cycles of 30s at 95C, 30s annealing at 58 C, and 30s extension at 72 C for amplification of 16S rRNA genes. A 7 min final extension was performed at 72 C. The amplicons were electrophoresed through 1% agarose gels in TAE buffer.

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92 Redox Potential Measurement Three redox probes were perm anently inst alled in each tank (o ne at each end approximately 30 cm from the wall, one in the ce nter) at a soil depth of 5 cm. Redox potential (Eh) was determined at weekly intervals duri ng the anoxic treatment. Eh was measured with permanently installed platinum electrodes; pl atinum wire (2 mm diameter, 15 mm length) was welded on a copper wire protected with insulati on material. Eh was read using ORION SA 230, (Orion Research Inc., Boston). A photograph of permanently installed re dox probes is presented in Fig. 4-4. Temperature Measurement Tem perature was determined every two weeks at the time of sampling at a depth of 5 cm. A glass thermometer was inserted in selected mesocosms clos e to the two sides of window installed with fans in summer or the middle of the green house where the temperature was relatively high. Water Content Measurement The water content of the m esocosms in th e aerobic phase was measured at randomly chosen ten locations at 12cm depth in the soil by TDR 300 soil moisture meter (Spectrum Technologies, Inc. East Plainfield, Illinois). Sample Collection When redox potentials in the m esocosms stab ilized, ten samples were taken every two weeks from each mesocosm to a depth of approxi mately 5 cm using a 50 ml disposable syringe with the end cut off. The use of a syringe was ne cessary because the soils exhibited a slurry-like consistency that precluded the use of more trad itional core samplers. The ten samples from each mesocosm were mixed and centrifuged in the laboratory to form composite samples. The separated solution and so ils were stored at oC until analysis.

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93 Organic Acid Analysis Acetate is a n important metabolite of cellulose fermentation; thus, acetate concentrations provide information on the functioning of the system. Acetate and lactate concentrations in pore water were determined by high pressure liqui d chromatography equipped with a UV detector (Waters, Co), with Aminex HP 87H column as the separating column (300 X 7.5 mm) and sulfuric acid (5mM) as eluent. OCP Analysis Toxaphene, DDT, DDE and DDD co ncentrations were determined by extraction with accelerated solvent extraction (A SE) and gas chromatography. Preliminary tests conducted to investigate the influence of soil moisture co ntent on OCP extraction by ASE indicated that medium water content yielded the greatest effici encies of recovery. Samples were air dried, ground, and sieved prior to analysis. Deionized wa ter was added to sieved soils and allowed to equilibrate for three days in a closed dessicato r. Water content was adjusted to 50% before extraction. A surrogates solution (50 ppb) (Decach lorobiphenyl and TCMX, AccuStandard, Inc., USA) was added to samples prior to extraction. ASE A Dionex ASE 100 Accelerated Solvent Extractor (Dionex, PA, USA) was used to perform the extractions. Triplicates of soil samp les were mixed with hydromatrix (Varian, Palo Alto, CA) in 2:1 proportion and extracted in a 34 mL stainless ve ssel using methylene chloride/acetone (4:1, v/v) or he xane/acetone (1:1, v/v) as solven t. The cell was filled with the solvent and heated to 100C. Th e static extraction was perfor med for 5 min, followed by flushing the vessel with 60% of the cell of fresh solvent. Finally, the vessel was purged with nitrogen for 100 sec at 150 psi. The total amount of extraction solvent was about 50 mL, and one static cycle was conducted.

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94 Cleanup and Analysis All extracted volumes were reduced and then hexane was added by an N2 stream. Repeated this process twice to remove acetone and methylene chloride. The volume of extracts was measured in a glass grad uated volumetric cylinder. At this point, the samples were split for analysis of toxaphene and DDT and metabolites. For analysis of DDT and its metabolites, one mL of extracts was passed through a florisil cartridge packed with approximately 500 mg of 200 m Florisil (Varian, Palo Alto, CA). Nine mLs of hexane:acetone (9:1, v/v) were used to rinse the column. Th e resulting eluent was concentrated to 1mL by an N2 stream and then transferred to a glass vial (2 mL, Fisherbrand, Atlanta, GA) and crimped with a Teflon lined cap for subsequent analysis by gas chromatography. Another one mL extract was purified by sulfur ic acid treatment for further analysis of toxaphene. Equal amounts of concen trated sulfuric acid and sample were mixed in a 10 mL vial. The vial was capped and vortexed for 1 min. Phas es were allowed to separate, and the upper (organic) phase transferred to a GC vial for analysis. Extracts were analyzed with a gas chroma tograph (Shimadzu GC-17A) equipped with a 63Ni electron capture detector (ECD). The co lumn was a RTX-5 (0.25mm ID 15m; Restek Corporation, Bellefonte, PA, USA). The temperature was programmed as follows: 60C, 3.0C/min to 70C for 1 min, 10C/min to 130C for 3 min, 10C/min to 160C, 20C/min to 200C. A calibration curves with 6 points bracketing the experimental concentrations was used for both analyses. The concen tration calculation followed EP A 8000B and methods used by Pearson and Paan (Pearson et al., 1997; Paan et al., 2006). Statistical Analysis One-way ANOVA was used to test the difference am ong tr eatment groups. Comparisons for differences in concentration least square means between sulfat e concentration, st erile control,

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95 and electron donors were done by Tukeys adjustment using SAS version 9.1 (SAS Institute, Cary, NC). The null hypothesis was rejected at p <0.05, thus the probability of observing no difference among the pair wise is less than 5%. Results and Discussion Physical Conditions Related to OCPs Degradation w ithin Mesocosms Temperature Temperature was monitored throughout anoxic and oxic phases of the investigation. In treatment of Cattail-1, the incr ease in temperature from 22C in February to above 30C after May (Fig. 4-5) was observed. The similar scenario occurred with other monitored mesocosms. It was not difficult to understand the fluctuation in all monitored mesocosms from the overall low temperature to the overall high temperature from the very beginning until the end of the experiment. It likely resulted from the poor cooling system in the greenhouse that could not keep temperature in the ho tter days of summer as same as in the cold days of winter. Furthermore, temperature in th e mesocosm located in the middle of the row of nine tanks was higher than that in the mesocosms in the two ends of the row. For example, the average temperature in Cattail-2 in summer was ge nerally higher (Fig. 4-5) than that in Control1. Such variability may be explained by the fact th at fans were only installed on the two sides of the window wall so as to cool the air around the ends of row closed to the windows. Redox Potential Redox potentials were monitored in all tanks following flooding and throughout the anoxic phase by averaging three re adings from each mesocosm (Fig. 4-6). The fastest drop of redox potentials to ca. -250 mV, which was re ported as the desirable redox potentials for reductive dechlorination (Jin a nd Englande, 1996), happened in the tanks with added carbon amendments approximately one m onth after flooding. This indicated that the cattails were decomposed and released the ca rbon, probably other nutr ients, quickly after amended, which were comparable with the pure ch emical, lactate, as far as their effect on the

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96 change in the soil conditions was concerned. In contrast, re dox potentials in the control mesocosms kept falling throughout the flooded phase. It was not unexpected that the applications of carbon source in the treatments helped reduce th e redox potentials in the system and maintain a more reductive environment very efficien tly (Ugwuegbu et al., 2001 ; Davis et al., 2004; Lookman et al., 2005), which would suggest pot ential for anaerobic biodegradation of OCPs. While in controls limited in carbon and electron energy redox potentials could not decrease rapidly as in treatments. Following drainage of water, the aerobic rang e of the redox potential was approached within two weeks (Fig. 4-7). Water Content Volumetric water content was monitored in all mesocosms following draining on June 8, 2006 by averaging 10 readings by TDR 300 from each mesocosm. The surface soils were rather wet after draining a nd remained water was continually evaporated thanks to the hot summer. However, volumetric wa ter content in some tanks (Lactate-1, Cattail2, and Lactate-2) was still high, around 50%, (Fig. 4-8) until the end of July. Other mesocosms showed a relatively constant water content. Degradation of Toxaphene in Mesocosm Table 4-2 shows sim ilar toxaphene concentratio ns at the beginning of the flooded phase in both controls and treatments. However, statisti cal significance of toxaphene concentrations between treatments and controls (Table 4-2) in the end of flooding was revealed by pair-wise comparison as would be expected. Throughout th e anoxic phase, toxaphene was continually degraded when supplied with an appropriate electron donor and toxaphene decreased until May 31 (Fig. 4-9). Toxaphene concentrations in contro l mesocosms did not decrease until May 3 (Fig. 4-9), which seems to correspond with much more reductive redox potential (-200 mv). This phenomenon indicated the metabolism mechanism that highly chlorinated compounds can only be degraded under the anaerobic conditions. On the other hand, intrinsic degradation was

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97 observed in the non sterile group in our previous laboratory studies, explai ning why insignificant degradation of toxaphene in controls was found until an anaerobic condition. However, toxaphene concentrations in control mesocosms di d not continue decreasing after this time point. At the end of anoxic period, toxaphene concentr ations in lactate a nd ground cattail reduced to 13.20 ppm and 12.63 ppm respectively, while control mesocosms still had 18.38 ppm of toxaphene. This observation reflect ed the results of the microcosm studies. In microcosms, the appropriate electron donors incl uding lactate and hydrogen were capable of stimulating the toxaphene feeders during the two months of anaer obic incubation. As expected that cattail can be consumed and produce some electron donors including lactate and then syntrophs utilized lactate to produce hydrogen (Drzyzga et al., 2002), halogenated chemi cals such as toxaphene are believed to release the chlorine ion with the replacement by hydrogen accompanied by a twoelectron transfer (Fingerling et al., 1996). Fermentation process in all lactate and cattail mesocosms was also confirmed by both gas pr oduction and the rapid decrease in the redox potential soon after flooding in comparison with those in control systems. These phenomena demonstrated that the microbial community quickly responded to the amendment of the exogenous carbon source and this became the stimulus for the degradation, although others reported an inhibition of toxaphene biodegradation with the additi on of lactic acid as the carbon source (Lacayo-Romero et al., 2006). No obvious production of sulfide c onfirmed that sulfate reducing bacteria might not take a role in a ttacking toxaphene in the anoxic phase. Probably, some other group of bacteria were feeding upon toxaphene as the carbon and energy source and electron acceptor. Up to now, two strains of anaerobes, Enterobacter sp. strain D1 (LacayoRomero et al., 2005) and Dehalosprillum multivorans (Ruppe et al., 2002), were isolated to degrade toxaphene. Dehalococcoides was detected and in NSRA soils (personal

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98 communication). In fact, reductive dechlorination of certain compounds by Dehalococcoides sp., has been shown by Krajmalnik-Brown (Krajmalni k-Brown et al., 2004) to dechlorinate some compounds using lactate as elec tron donor in consortia, and H2 as electron donor in pure culture (He et al., 2003). Lactate was thought to be fermented to H2 in consortia in that study, which then served as the primary electron donor fo r reduction of trichloroethylene by Dehalococcoides Although both lactate and H2 were identified as efficient el ectron donors for OCP degradation in our previous microcosm studies, microorganisms en riched as capable of utilizing toxaphene as electron acceptor did not incl ude any strain with 16S rDNA sequence similarity to Dehalococcoides sp. Amplicons could not be obtained using Dehalococcoides sp. specific PCR primers from soils used in mesocosms before flooding (data not presented). This evidence was not strong enough to exclude the degrading role of Dehalococcoides in NSRA, which probably resulted from the procedures we followed, altho ugh it was not detected in the microcosm studies. For most samplings from the beginning to the end of anoxic period, gr ound cattail and lactate treatments could not be distingu ished at the statistical level. This study demonstrated that the cheap and readily available plant materials as amendments can achieve a very high efficacy of degrading toxaphene under anae robic conditions. During the oxic phase, continued degradation of toxaphene was not observed. On the contra ry, all the treatment mesocosms showed the tendency to be constant and no significant loss of toxaphe ne was observed under the oxic condition. Statistical analyses yielded significantly lower conc entration of toxaphene in treatment systems than that determined in the control mesocosms (Table 4-2). No significant difference between the cattail and lactate treatmen ts can be found from the pair-wise comparison analyses. Dehalogenation of toxaphene in our study did not seem to prefer the aerobic incubation. Even if we could not determine the structure of components, products of

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99 dechlorination of toxaphene in the treatment mesocosms, the fact that aerobic soil microorganisms generally fail to metabolize higher halogenated pesticides might be thought to be the reason of no further de gradation during the oxic phase. Degradation of DDT and its Meta bolites in Mesocosm At the beginning of the anaerobic phase, c oncentrations of DDT and DDD and DDE did not show any statistically significant difference among the tanks (Table 4-2). In comparison with controls, degradation of DDT was observed in the cattail mesocosms, concentrations of DDT declining constantly throughout the anoxic pha se (Fig. 4-10). Even though concentrations of DDT in the lactate mesocosms in the starting of the anaerobic period were not sensitive to the treatmen t, a decrease was noted after May 3, pending the end of anoxic phase. It is likely that the mi crobial community in the NSRA so ils took some time to respond to the addition of the applied carbon and energy source. By the end of the anoxic phase, concentrations of DDT were significantly lower in the lactate and cattail mesocosms than in the control mesocosms (Table 4-2) The observations of such de gradation supported and extended our bench scale investigations. In our previous study, we obser ved that lactate and hydrogen stimulated the significant disappearance of DDT from NSRA soils under both low and high concentrations of sulfate salt. Concentrations of DDD and DDE, the major produc ts of transformation of DDT, were also determined. An increase in DDD concentrations in the cattail mesocosms was noted in the first weeks of anaerobic phase (Fig. 411), corresponding to a decrease in DDT concentrations despite the big variation (Fig. 4-10), wh ile the concentrations of DDD in the lactate mesocosms kept unchanged at the same period. As flooding condition ended, a general increase in DDD concentrations was observed in th e lactate and cattail mesocosms, consistent with the relatively large decrease observed in those tanks for DDT Such findings led the confidence that the

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100 observed degradation of DDT in the treated me socosms was real. At the end of the flooded phase, DDD concentrations were significantly higher in the lactate mesocosms than in the control mesocosms. Although the effect of cattail could not be distinguish ed from controls, the tendency of increase of DDD was not negligible and probably masked by the variabilities of the DDD measurement. DDE concentrations revealed little informa tion of degradation under anaerobic conditions. No loss of DDE was observed in any treatment during the anoxic phase (Fig. 4-12, Table 4-2), nevertheless not surprising according to the de gradation pathway of DDT under the anaerobic condition (Fig. 1-2). Generally, DDE can not be degraded further to any other compounds in the anaerobic phase as would be expected, whic h was already observed in our bench scale microcosms. Our results indicated that added electron donor s in the form of ground cattail and lactate promoted DDT degradation relative to contro l mesocosms, and stimulated production of DDD under anoxic conditions. The fact that no lactate or acetate was dete cted in porewaters of any of the mesocosms (data not shown) confirmed that specific bacteria utilized lactate direct from the chemicals or the fermentation of the plant re sidues as the electron donors. As well known, many contaminants were degraded under methanogeni c conditions or sulfate reducing conditions. However, relatively few microorganisms are capable of directly utiliz ing lactate as electron donor and largely restricted to secondary fermenters (syntrophs) in consortia with methanogens (Sekiguchi et al., 2006). Sulfat e reducing bacteria, a major group of bacteria involved in the anaerobic degradation of hal ogenated compounds, were possibly not the microorganisms that were capable of utilizing lactate or a fermentation product as electron donor, either directly or indirectly, and using OCP as electron accepto r (van Pee and Unversucht, 2003) based on the

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101 evidence that no noticeable sulfide was produced during the experiments period. In our study, another valuable finding was that mesocosms with lactate and ground cattail were approximately equivalent in stimulating degradation of DDT. The plant residues from the cattail treatments were capable of being transformed to H2 and lactate, indicated by ga s production and then drove DDT degradation as expected. Th is observation suggested that ch eap and easily available plants materials made the cost efficien t remediation strategy possible. At the end of the oxic phase, no significant difference was observed for DDT and DDE, as with toxaphene. On the other hand, the same modes of higher concentrations of DDD in mesocosms treated by lactate and ca ttail and rather lower in contro ls were observed at the end of anoxic phase as the end of the oxic phase. The lack of degradation du ring the oxic phase was somewhat surprising. Potential nitrogen and phos phorus limitations at later stages of the incubation were possible reason responsible for no further degradation throughout the oxic phase. These investigations have shown that much of the observed degradation of OCPs occurred prior to draining, while continue d degradation was not observed dur ing the oxic phase. In this case, the primary and most efficient approach for these OCPs degradation suggested was anaerobic and, therefore, future efforts for NSRA remediation should be attempted for anaerobic process. Another important factor in degradation is th e bioavailability of the contaminants for the organisms responsible for metabolism of the compounds. These degraders in NSRA soils are likely limited to access to the OCPs as a resu lt of their hydrophobic properties and the high organic maters in the soils. Increasing the availa bility of OCPs to micr obial attack may provide an optimal strategy for accelerating their degrad ation (Walters and Aitken, 2001). Other reported

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102 strategies for increasing availabi lity included the application of surfactants (Kommalapati et al., 1997) (Gao et al., 2000; Lunney et al., 2004). In our project of cl ean-up of NSRA contaminated soils, application of surfactants with incorporation of plant residue prior to flooding might be desirable by resulting in increased degradation.

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103 Table 4-1. Experimental de sign of mesocosm studies Treatment/Control Description No. of Tanks Treatment Lactate 10 mM lactate 3 Treatment Cattail 0.04 g ground cattail per gram soil 3 ControlNo electron donor soil and water only 3 Total Mesocosms 9 Table 4-2. Pair-wise Comparison of the treat ments on the degradation of pesticides ToxapheneDDT DDD DDE Control 22.22(A) 3.17(A) 1.54(A) 3.16(A) Cattail 24.52(A) 3.34(A) 1.83(A) 3.53(A) Starting concentrations (Feb. 21, 2006) Lactate 25.21(A) 3.14(A) 1.47(A) 3.44(A) Control 18.38(A) 3.16(A) 1.42(B) 3.92(A) Cattail 12.63(B) 2.08(B) 1.90(B) 4.18(A) Anaerobic period (Ending May 31, 2006) Lactate 13.20(B) 2.55(AB) 4.10(A) 4.11(A) Control 18.60(A) 3.07(A) 1.87(B) 3.93(A) Cattail 12.49(B) 2.74(A) 4.02(A) 4.24(A) Aerobic period (Ending Aug. 21, 2006) Lactate 14.42(B) 2.23(A) 4.34(A) 3.98(A) Note: Concentrations are pres ented in mg/Kg and concentrati ons within a given time point sharing the same letter did not s how significant difference at the p<0.05 level.

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104 Figure 4-1. Soil collection site ZSS0750 in the North Shore Restoration Area at Lake Apopka, Orange County, Florida.

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105 Figure 4-2. Soil mixed in a 13 ft x 13 ft plot at site ZSS0750 by a trackhoe prior to collection and transport to the greenhouses at the Departme nt of Soil and Water Science, University of Florida, Gainesville.

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106 Figure 4-3. Mesocosm tanks in the greenhouse at the Department of Soil and Water Science, University of Florida, Gainesville.

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107 Figure 4-4. Permanently installed re dox electrodes in mesocosm tank.

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108 Figure 4-5. Temperatures within mesocosms at a depth of 5 cm. Note: Vertical black arrow indicat es date of draining (June 8). 15 20 25 30 35 40 2/2/20063/24/20065/13/20067/2/20068/21/2006 Datetemperature (centidegree) Control-1 Cattail-1 Lactate-1 Cattail-2 Control-3 Lactate-3

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109 Figure 4-6. Redox potentials with meso cosm tanks during flooded phase. -400 -300 -200 -100 0 100 200 3002/1 0/ 2 0 06 2/17/2006 2/2 4/2006 3/3/2006 3/1 0/ 2006 3/17/2006 3/2 4/20 06 3/3 1/ 2006 4 / 7/200 6 4/1 4/20 06 4/21 / 2006 4/ 2 8/2006 5/5/2 0 06 5/12/2006 5/19/2006 5 / 2 6/2006 6/2/2 00 6Dateredox potential (mv ) Control-1 Cattail-1 Lactate-1 Control-2 Cattail-2 Lactate-2 Control-3 Cattail-3 Lactate-3

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110 Figure 4-7. Redox potentials following draining. -400 -300 -200 -100 0 100 200 300 400 500 6006 / 1 6 / 2 00 6 6 / 1 9 / 2 00 6 6/2 2 / 2 006 6/25/2006 6/28/2006 7/1/2006 7/4/2006 7/7/ 2 00 6 7 / 1 0/20 0 6Dateredox potential (mv Control-1 Cattail-1 Lactate-1 Control-2 Cattail-2 Lactate-2 Control-3 Cattail-3 Lactate-3

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111 Figure 4-8. Volumetric water co ntents following draining. 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.007/31/2006 8/2/2006 8 /4 / 20 0 6 8 /6 /2 0 06 8/8/2006 8/ 1 0/ 2 00 6 8/12/2006 8 /1 4 /2 0 06 8/ 1 6/ 2 00 6 8/18/2006 8 /2 0 /2 0 06Date volumetric water content (%) Control-1 Cattail-1 Lactate-1 Control-2 Cattail-2 Lactate-2 Control-3 Cattail-3 Lactate-3

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112 Figure 4-9. Concentrations of toxaphene during study. Note: Error bars represent +/1 standard deviation. Vertical black arrow indicates date of draining (June 8). 10 15 20 25 302/21/2006 3/7/2006 3/21/2006 4 / 4/2006 4 / 18/200 6 5 / 2 / 2 0 06 5 / 1 6 / 2 00 6 5 / 3 0 / 2 00 6 6 / 1 3 / 2 00 6 6 / 2 7 / 2 00 6 7 / 1 1 / 2 00 6 7/25 / 2 0 06 8/8/20 0 6 8/22 / 2 0 06DateConcentration of Toxaphene (ppm) control cattail lactate

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113 Figure 4-10. Concentrati ons of DDT during study. Note: Error bars represent +/1 standard deviation. Vertical black arrow indicates date of draining (June 8). 1.5 2 2.5 3 3.5 4 4.5 52 / 2 1 / 2 00 6 3 / 7 / 2 0 06 3 / 2 1 / 2 00 6 4 / 4 / 2 0 06 4 / 1 8 / 2 00 6 5 / 2 / 2 0 06 5 / 1 6 / 2 00 6 5 / 3 0 / 2 00 6 6 / 1 3 / 2 00 6 6 / 2 7 / 2 00 6 7 / 1 1 / 2 00 6 7 / 2 5 / 2 00 6 8 / 8 / 2 0 06 8 / 2 2 / 2 00 6DateConcentration of DDT (ppm) control cattail lactate

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114 Figure 4-11. Concentrati ons of DDD during study. Note: Error bars represent +/1 standard deviation. Vertical black arrow indicates date of draining (June 8). 0 1 2 3 4 5 62/ 2 1/ 20 06 3/ 7 /2 00 6 3/ 2 1/ 20 06 4/ 4 /2 00 6 4/ 1 8/ 20 06 5/ 2 /2 00 6 5/ 1 6/ 20 06 5/ 3 0/ 20 06 6/ 1 3/ 20 06 6/ 2 7/ 20 06 7/ 1 1/ 20 06 7/ 2 5/ 20 06 8/ 8 /2 00 6 8/ 2 2/ 20 06DateConcentration of DDD (ppm) control cattail lactate

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115 Figure 4-12. Concentrati ons of DDE during study. Note: Error bars represent +/1 standard deviation. Vertical black arrow indicates date of draining (June 8). 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.002 / 2 1 / 2 00 6 3 / 7 / 2 0 06 3 / 2 1 / 2 00 6 4 / 4 / 2 0 06 4 / 1 8 / 2 00 6 5/2 / 2 0 06 5/1 6 / 2 006 5/30/2006 6/13/2006 6/27/2006 7/11/2006 7/25/2006 8/8/ 2 00 6 8/22/20 0 6DateConcentration of DDE (ppm) control cattail lactate

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116 CHAPTER 5 SUMMARY AND CONLUSIONS OCPs have endangered wildlife and hum ans as a result of widespread use and their carcinogenic toxicities. Removal of OCPs from contaminated environments has become an environmental priority. Cost effi ciency leads bioremediation to be a desirable strategy compared with physical and chemical techniques. The NS RA is such a widely contaminated site by toxaphene and DDTr that the presence of the OCPs constitutes a risk to wildlife, and toxaphene and DDTr may have contributed to the deaths of several hundred white pe licans. The large area of contamination makes the NSRA to be a good candidate for researching environmental behavior and remediation of OCPs. This research was conducted to understand the microbiological proce sses of toxaphene and DDTr degradation under appropr iate conditions, with special emphasis on anaerobic biodegradation at the bench and mesocosm scales. Chapter 2 addressed an elementary probl em for studying biodegradation of OCPs by evaluating conventional and altern ative extraction methods and optim izing factors that affected the extraction efficiency. The correlation of the recovery of toxaphene using ASE with TOC and TON was very negatively high, -0.95, while SOX was not affected much by the sample characteristics. The recovery of DDTr was not correlated to TOC and T ON using either ASE or SOX. These results confirmed previous work (Bandh et al., 2000) that indicated that high organic carbon contents more st rongly adsorbed toxaphene, making it difficult to remove the toxicant from the soil matrix using ASE. Theref ore, choosing ASE for gaining high recovery of toxaphene should consider the soil characteristic s, especially amounts of organic matter. Among other soil properties, the water content influenced the extraction efficiency, with intermediate water content yielding the greatest overall effi ciencies for extracting OCPs in both ASE and

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117 SOX. Our finding indicated that suitable wet condition, intermediate wa ter content, swelled organic soil structure allowing the OCPs in the soil pores available for solvent to carry out into the bulk extraction liquid, especi ally important for NSRA soils that contained high organic carbon. When water was not available, soil partic les collapsed and adsorbed the OCPs strongly so as to keep then unavailable for solvents. Ex cessive water still led to extraction difficulties because the soil particle s are surrounded by water, which potentially blocks extraction solvent accessed to the OCPs. Water soluble organic car bon could be partitioned into water matrix instead of combining with OCPs, thereby improvi ng the extraction efficiency (Tao et al., 2004), which would be another reason to indicate the effect. Significantly more toxaphene, DDT, and DDE were extracted by ASE using methylene chloride/acetone (4:1, v/v) than using hexane/acetone (1:1, v/v). However, pairwise comparisons revealed that using SOX, the hexane/acetone system showed statistically higher efficienci es for DDE and DDD, at 95% confidence level and toxaphene at 90% confidence level. Reasons responsib le for performance of extractant have not been well elucidated based on previous publications. Differences in polarity and boiling point might contri bute tbo explain the phenomena. The present study strongly suggested that the choice of extraction solvent syst em must be dependent on the extraction procedure. Statistical evaluations showed that equivale nt results (moderate contaminated soils) or higher efficiencies (highly contaminated soils) were achieved for the simultaneous extraction of residues of toxaphene, DDT and its metabolites by ASE in contrast to SOX. Considering the solvent consumption and extraction time, ASE is the preferable extraction technique for the investigation of contaminated high organic car bon soils. Although many studies investigated the extraction of similar PAH, none have reported the comparison of toxaphene and DDTr extraction

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118 procedures and evaluated the e ffects of environmental characte ristics and extraction solvents. The present study provided a guideline in practice to gain effici ent extraction of toxaphene and DDTr given all parameters of interest. Chapter 3 offered a biological concept to tr eating OCP-contaminated soils by making use of OCP-degrading bacteria. In our microcosm st udy, statistically significant degradation of toxaphene was observed in the non sterile groups. This finding confirmed the feasibility of biodegradation of toxaphene. Among three pr omising electron donors which showed possible roles in the metabolism of OCP, lactate and bu tyrate were capable of stimulating toxaphene degradation at the marginal statistical level ( p<0.10) when the sulfate concentration was relatively high (200 mg/L MgSO4 .7H2O). As well known, anaerobic dechlorination is the first step of complex organochlorine pesticides and may be achieved by different pathways. One possibility occurs when lactate or other electron donors except for hydrogen are present in the system. Dechlorination may not be processed wit hout hydrogen such that the lactate or other donors have to be fermented by syntrophs to hydrogen. Generally, SRB or other microorganisms participate in such fermentation when sulfate sa lt is not sufficient (Drz yzga and Gottschal, 2002). Once dechlorinaters obtain hydr ogen, replacement of a chlori de of OCP with hydrogen is accomplished. Alternatively, high concentratio ns of sulfate will make SRB outnumber dehalogenating bacteria and stop the dehaloge nation. In our study, abundant sulfate salt (200 mg/L MgSO4 .7H2O) allowed the consumption of lactat e or butyrate to produce hydrogen, thereby promoting the disappearance of toxaphene. Since no amplicons were detected related to sulfate reduction in enrichment culture, we conc luded that other groups of microorganisms used lactate or butyrate as the el ectron donor and degraders utili zed the produced hydrogen to yield energy by preferring dehalogenation, a more ef ficient energy yielding approach, to sulfate

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119 reduction (Dolfing and Harrison, 1992). In my ex periments, when hydrogen gas was supplied in serum bottles, statistically significant degrad ation of toxaphene, DDT and DDD was detected between high level of sulfate group vs sterilized group, and low level group vs sterilized group. With limited supplement of sulfate (20 mg/L MgSO4 .7H2O), the responsible bacteria degraded DDT with the highest efficiency in hydrogen. Th is finding further indicat ed that the biological degradation of OCPs was not influenced by the c oncentration of sulfate only if hydrogen existed in the anaerobic system. Further, dechlorina ting microorganisms were the major group of degrading the OCPs based on the dechlorination noticeable observed in enrichment medium. A second mechanism attributed to dechlorina tion of OCP is that some dehalogenaters utilize halogenated compounds as energy source and degrade OCP through halorespiration when hydrogen is supplied as the electr on donor. The effect of hydrogen added to the serum bottles in the present work demonstrated the presence of dehalogenating bacteria and their metabolizing roles. Additionally, DDT and DDD in non sterile moderate soil without added electron donor were not degraded significantly in compar ison with the sterile group after two months incubation. In contrast to the first set, a significant decrease was not observed, but an insignificant increase in the concentration of DDE in some serum bottles, which indicated anaerobic transformation of DDT to DDE. Alth ough degradation of toxaphene, DDT, and DDD were observed in the present work, the biodegradation products are not k nown at this moment. The limitation of this present study resulted from the lack of instruments such as GC-MS. Further work should confirm the structure and to xicities of metabolic products and conversion rate of parent chemicals to metabolites. The production of Clduring the growth of the bacteria in enrichment cultures strongly suggested the mechanism of reductive dechlorina tion of toxaphene. The different dynamics of

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120 released chloride confirmed our speculation for microcosms study that microbial inactivity resulted in limited degradation of OCPs in highly contaminated soils. These findings suggested that biodegradation of OCPs should take the mi crobial activities into account for gaining the largest degradation. Phylogenetic analysis demonstrated that some sequences most abundant in the clone libraries were related to the genus Citrobacter (99.9% similarity), some closely related to Gram positive Sporomusa, (98% similarity) Sporomusa are homoacetogens capable of producing acetate with CO2 as the electron acceptor and hydrogen as the electron donor. Both species are involved in the degradation of organic contaminants. Sporomusa ovata is capable of reductive dechlorinating PCE to TCE with methanol as an electron donor (Terze nbach and Blaut, 1994). On the other hand, facultative anaerobic Citrobacter belongs to Enterobacteriaceae and can ferment glucose, glycerol, or other carbon source to acetate, lactate, etc. It has been reported to be capable of utilizing chlorophenol or biphenyl as sole carbon and energy source (Martinez et al., 2000; Grishchenkov et al., 2002). Even though Citrobacter can tolerate or accumulate a variety of toxicants and Sporomusa was involved in dechlorina tion, it was not possible to conclude how Citrobacter and Sporomusa are involved in toxaphene dechlorination in our enrichment experiments. Further work should co ntribute to isolation of the pure culture or determine the members of consortium which can degrade toxaphene. Chapter 4 supported and extended the bench scale investigations that concluded that added electron donors promote OCPs degradation under anoxic conditions. By the end of the anoxic phase, both lactate and cattail trea tments significantly promoted the degradation of toxaphene and DDT as expected based on the microcosm studies. As flooding condition ended, a general increase in concentrations of DDD, the major products of reductive dechlorination of DDT, was

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121 observed in the lactate and cattail mesocosms, cons istent with the relatively large decrease in those tanks for DDT. Such findings led to the co nfidence that the observe d degradation of DDT in the treated mesocosms was real The fact that no lactate or acet ate was detected in porewaters of any of the mesocosms indicated that bacteria immediately metabolized lactat e in the systems by aerobic (in the water phase and the soil surface) and anaerobic (responsible for degradation) bacteria. This was not unexpected since our micr ocosm studies already indicated the significant role of acetate and lactate in degrading toxaphene. This stud y demonstrated that use of inexpensive and readily available plant material s cattail as amendments can promote degradation of toxaphene under anaerobic conditions at the NSRA. Alternating anaerobic with aerobi c conditions is considered to be an effective strategy for OCP decontamination, but in this case, the primar y and most efficient approach for these OCPs degradation suggested was anaerobic and therefor e future efforts for NSRA remediation should be attempted for anaerobic process. The fact that aerobic soil microorganisms generally fail to metabolize higher halogenated pesticides might be the reason for no further degradation during the oxic phase. Potential nitrogen and phosphorus limitations at later stag es of the incubation may have been a possible reason as well. Finally, degraders in NSRA soils are likely limited to access to the OCPs as a result of their hydr ophobic properties and the high organic matter content of the soils. In future, increasing the availability of OCPs to microbial attack, e.g. application of surfactants may provide an optimal strategy for accelerating their degradation.

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135 BIOGRAPHICAL SKETCH Yun Cheng was born in Yingkou, P.R. China. She received her B.S. in Biology in 1999 f rom Shenyang Normal University, P.R. China. At the same year, she was admitted to the graduate school in Chinese Academy of Scie nces to pursue her M.S. in Ecology after the nationally competitive exam. In 2002, she completed her M.S. degree and joined the Soil and Water Science Department at the University of Florida to start her Ph.D. studies. In 2003, she joined Dr. Andrew Ograms research team Her project focused on bioremediation of contaminated soils in Lake Apopka, one of the most polluted lakes in Florida.