the binding and availability of 2,4,6-trinitrotoluene (tnt) from sand, clay minerals, and soils the effects of aging and the implications for biotreatment

THE BINDING AND AVAILABILITY OF 2,4,6-TRINITROTOLUENE TNT FROM SAND, CLAY MINERALS, AND SOILS — THE EFFECTS OF AGING AND THE IMPLICATIONS FOR BIOTREATMENT A Dissertation Submitted to the

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THE BINDING AND AVAILABILITY OF 2,4,6-TRINITROTOLUENE (TNT) FROM SAND, CLAY MINERALS, AND SOILS — THE EFFECTS OF AGING

AND THE IMPLICATIONS FOR BIOTREATMENT

A Dissertation

Submitted to the Graduate School

of the University of Notre Dame

in Partial Fulfillment of the Requirements

for the Degree of

Doctor of Philosophy

by

Kara M Young, B.S.C.E., M.S.C.E

/ 1 WM 7® Director Graduate Program in Civil Engineering and Geological Sciences

Notre Dame, Indiana

April 2006

UMI Number: 3207141

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THE BINDING AND AVAILABILITY OF 2,4,6-TRINITROTOLUENE (TNT) FROM SAND, CLAY MINERALS, AND SOILS —- THE EFFECTS OF AGING AND THE

IMPLICATIONS FOR BIOTREATMENT

Abstract

by

Kara M Young

For many years, the ultimate fate of the most important explosive, 2,4,6-

trinitrotoluene (TNT), has been of interest to the scientific community Despite its

importance, the fate of TNT in the environment remains unknown The objective of this research was to better understand the binding and physical availability of TNT from geosorbents including sand, clay minerals, and soils — the effects of aging and the

implications for biotreatability The physical availability of freshly-spiked TNT from geosorbents was investigated using sorption isotherm and thermal programmed

desorption-mass spectrometry (TPD-MS) experiments TPD-MS experiments

demonstrated for the first time that TNT release is dependent upon the character of the geosorbent and that release energy values increase with increasing complexity of the geosorbent (i.e., montmorillonite > kaolinite > sand) Similarly, sorption coefficients

Kara M Young

indicated a stronger binding affinity and thus a decreased availability with increasing

geosorbent complexity The geosorbents were then aged to assess the effects of aging on

the physical availability of TNT Chemical extractions revealed that a small fraction of

TNT was bound to the soils after only 30-60 days while TNT remained fully extractable

from the clay minerals In contrast, aqueous desorption experiments demonstrated a reduced availability of TNT with only 30 days of aging for all geosorbents, suggesting that sequestration was the dominant mechanism of interaction

The biotreatability of TNT was investigated by composting Anaerobically

digested biosolids were used for the first time as a sole amendment by

blending/composting with the geosorbents TNT was effectively removed in systems

amended with only 2.5% biosolids Microbial growth corresponded to TNT degradation

The organic matter and the active microbial community in the soils were factors for the increased TNT removal in comparison to the clay minerals; thus, the type of geosorbent does affect the availability of TNT for treatment Geosorbents were then aged for 30 days and subjected to composting The soils did bind a fraction of the TNT in 30 days, but the remaining TNT was available for biotreatment In contrast, the individual clay minerals did not bind TNT after aging for 30 days, but the TNT was less available for biotreatment from the aged than freshly-spiked clay minerals due to reversible sorption processes

To Michael, Kira, Emma and Buck my family

1.1 Problem Definition ee eeeseessesesecsssesessessessessesescnsersssrassssesseesesersesees 1

1.2 Research Objectrve and Hypotheses - - HH HH ưệt 3

2.1 Why Are We Concerned with TÌN T”? - «cv HH HH HH HH Hưện 7 2.1.1 Toxicity and Health Effects of TNT - - án nen, 7

2.1.2 A History of TNT Contaminated SŠ1tes - Ăn 8 2.1.3 Environmental Transformations of TÌNT - sen eeg 9

2.1.4 Soil Constituents Involved in TNT Binding - «c2 se 10

2.1.4.1 Quartzite Sand and Clay MineraÌs 7s cseeesee 10 2.1.4.2 Natural Organic Maf€T ng HH nhưm 12 2.2 How Do We Assess Binding and Avallab1]Ity? -sc sccnnHie, 13 2.2.1 Bound Residues - - SH ng nh ng nu ngư nh 13

2.2.2 Availability nh 14

"No 6h 16

2.3 How Amenable is TNT to Biotreatment? 00.0 eee eeeeeeeseeneeeeeeeeneeeseneeenee 17 2.3.1 Biodegradation Of TNT .cccsccscesseesessecneeseceeenetsasesecseeserseeeaessenssensens 17 2.3.2 Bioremediation Strategies for TNT .ccccsccssccesesecceeceeseeseeeeeeeeeeaeens 20 2.3.2.1 Phytoremediation 0.0.0.0 eeeeeeeeenseneessceseseseesseeesnesssesaseseseaesenee 20 2.3.2.2 Bioslurry 'TreatInenif - sư 21

°ˆ Y6 §®uu 7 22 2.4 What Are the Mechanisms of Aging that Influence the Availability and

5)10i4721101)1A 0088016 012002222.55 23

2.4.1 Covalent Binding - - - cm TH 0 TH 25

CHAPTER 3: ASSESSING THE BINDING AND PHYSICAL AVAILABILITY

OE 2,4,6-TRINITROTOLUENE (TNT) EROM GEOSORBENTS 28

Kể án 28 3.2 Experimental SecfIOn ch ng TH ng HH HH He 29 3.2.1 Mat€rlaÌS - - HH TH Họ nh và 29 3.2.2 TPD-MS BackgTrOUnd - - + sgk 32 3.2.3 TPD-MS InstrumenfatiOn - - s Lt.vk ng g ng nrg 32

3.2.4 TPD-MS Spiking ProcedUre Ác ch HH ng ng 34

° W0 S0, ôn 34

EVI.N ¿PO 09.4 35

3.2.7 TPD-MS Analysis Method - óc HH HH re 36 3.2.8 Sorption Isotherm EXxperlmenS - HH HH He 37

3.2.9 TNT Extraction and ÀnaÌyS1S - HH HH ng gkc 39 3.2.10 Brunauer, Emmett, and Teller (BET) Specific Surface Area 40 3.2.11 Atomic Force Microscopy (AFM) ccccsccssccssssssesssessscssseesessseeneesseeees 40 3.2.12 Powder X-Ray Diffraction (XRD) - Án ren 41

3.3 Results and ID1SCUSSIOTI 0 HH HT TH TH TH HH nh 41

3.3.1 TPD-MS Initial Data Processing ác treo 41 3.3.2 TPD-MS Ramp Rate DIÍÍerences ng net 46 3.3.3 TPD-MS Sorbate DIÍferences óc ng HH HH ngà, 47 3.3.4 TPD-MS Sorbent DIfferences cà kg 1 0 xen 48 3.3.5 Release Energy from TPD-MS, ác vn HH g0 01 1 ke 53 3.3.6 Sorption IsOtl€rTnS sgk 34

CHAPTER 4: THE AVAILABILITY OF 2,4,6-TRINITROTOLUENE (TNT)

FROM GEOSORBENTS FOR BIOREMEDIATION USING

LAN Go 59 4.2 Materials and Methods «HH gọn TH HH Hà ng 62

“AC on 62

lý V0 an 62 8c 0 nh 63 4.2.4 Microbial Population AnaÌYS1S - ch HH ng ng 64

ch 0008x011 84 909/0 on ố 87

CHAPTER 5: A COMPARISON OF AQUEOUS AND THERMAL

DESORPTION FOR ASSESSING THE PHYSICAL AVAILABITTY OF AGED

bo 89 5.2 Materials and Methods - . óc HH HH HT nh 90 5.2.1 Chemical Reagents and GeosorbenIS - - sn Hi, 90

5.2.2 SpIking and Aging of TÌNT - sxnttnHg nHnHHHHHkưy 91

5.2.3 Aqueous Desorption of Aged TNT-Spiked Geosorbens 91 5.2.4 Thermal Desorption of Aged TNT-Spiked Geosorbents 93 5.2.5 TNT Extraction and ÀnaÌy§1S - co HH cư 94 6c an 94 5.3.1 Effect of Aging Time on Aqueous TT Desorption 94

5.3.2 Effect of Geosorbent Type on Aqueous TNT Desorption 96

5.3.3 Kinetic Modeling Results 2.0.0.0 cccccssecesscesecseeeeeneessecseeeeenetaceneeeeens 102

5.3.4 Effect of Aging Time on TNT Thermal Desorption 109

=8 1 119 6.3.1 Composting via Bloaugmentation of Aged Geosorbents 119 6.3.2 Degradation Rates of TÌNT, - kg ng kg 125 6.3.3 Microbial Population - - - sgk, 127

FIGURES

Figure 2.1 — TNT transformation pathways (taken from Spain and Nishino, 2002) 10 Figure 2.2 — Processes Inherent to aging (taken from Reid et al., 2000) 24

Figure 3.1 — Schematic of TPD-MS instrument showing the direct insertion probe and

sample crucible (an exploded VI€W) sgk 33

Figure 3.2 — Raw thermogram for TNT spiked on sand with individual thermogram

for TNT (m/z 210) shown in the inset 0.0.0 eee eseceseenesesseereeeeseerscesessascnsseresesees 42 Figure 3.4 — Comparison of a) the initial TNT thermogram and b) the TNT

thermogram smoothed using the Gaussian smoothing function - 44 Figure 3.5 — Variation in triplicate thermogram runs for TNT spiked on sand

600990) 45 Figure 3.6 - TNT desorption thermograms illustrating the peak changes with

1ncreasing ramp rate for sand (Š500ppim) - «ngàng 46 Figure 3.7 - NAC desorption from glass beads, at 10°/min, illustrating a thermogram shift to the right with increasing molecular welghi( - sen, 47

Figure 3.8 — Normalized TNT desorption thermograms, at 10°/min, from glass vials, glass beads, sand, kaolinite, and soil-c, illustrating a shift in peak temperature

Figure 3.9 — TNT thermograms for a) glass vials, glass beads, sand, b) kaolinite and

soil-c, illustrating the decreasing intensity of desorption with increasing

Figure 3.10 — AFM images of a) kaolinite and b) montmorillonite surfaces 52 Figure 3.11 — Yong and Lang plot for glass, sand, kaolinite, and soil-C 33

Figure 3.12 — a) Linear and b) Freundlich adsorption isotherms of TNT on kaolinite,

montmorillonite, and soil-C (I = 10mM KC|Ì) ó5 St rseeee 55

Figure 4.1 — Schematic diagram of the experimental design for composting with

it) 66

Figure 4.3 -Experiment #1 - TNT degradation for 0%, 2.5%, 5%, 10%, and 20%

biosolids/sand amended r€aCfOTS - - ng HH HH rệp 73

Figure 4.4 — Experiment #1 - Formation of ADNTs for the 2.5%, 5%, 10%, and 20% biosolids/sand amended r€äCfOTS G9 nH gn ng 74 Figure 4.5 — Experiment #2 - TNT removal for 0%, 0.25%, 0.5%, 1%, and 2.5%

biosolids/sand amended reactors 0.0 cceeccsseecesceseceereesceescessncessecssceseeeseeeasesseveseesaee 75

Figure 4.6 — Experiment #3 - TNT removal for 0%, 2.5%, and 5% biosolids amended

41181010201 76

Figure 4.7 - TNT degradation pattern in 2.5% amended soil-A in comparison to

controls without amendments and autoclaved-bioso]1ds «-«-ss«cceee 78

Figure 4.8 — TNT degradation pattern in 2.5% amended soil-C in comparison to

controls without amendments and autoclaved-biosoÌ1ds - - «sec 78 Figure 4.9 - TNT degradation pattern in 2.5% amended kaolinite in comparison to

controls without amendments and autoclaved-biosoÌ1iđs -. -s«ecesee 80 Figure 4.10 — TNT degradation pattern in 2.5% amended montmorillonite in

comparison to controls without amendments and autoclaved-biosolids 80

Figure 4.11 — Total viable microbial population for kaolinite amended with 0%, 2.5%,

Figure 4.12 — Total viable microbial population for montmorillonite amended with

0%, 2.5%, and 2.5%A blosoÌ1ds HH HH TH TH ng HH nọ HH 85 Figure 4.13 — Total viable microbial population for soil-A amended with 0%, 2.5%, and 2.5%A Đ1OSỌ1ỞS - 0 HH HT TH TH TH TH TH HH 85 Figure 4.14 — Total viable microbial population for soil-C amended with 0%, 2.5%,

Figure 5.1 — (a) TNT desorption from sand after 0, 30, 60, 90, and 180 days of aging; (b) TNT desorption from kaolinite after 0, 30, 60, 90, and 180 days of aging; (c) TNT desorption from montmorillonite after 0, 30, 60, 90, and 180 days of aging;

(d) TNT desorption from soil-A after 0, 30, 60, 90, and 180 days of aging; and

(e) TNT desorption from soil-C after 0, 30, 60, 90, and 180 days of aging 97 Figure 5.2 — (a) TNT desorption from sand, kaolinite, montmorillonite, soil-A, and

soil-C without aging; (b) TNT desorption from sand, kaolinite, montmorillonite,

soil-A, and soil-C following 30 days of aging; (c) TNT desorption from sand,

kaolinite, montmorillonite, soil-A, and soil-C following 60 days of aging; (d)

TNTdesorption from sand, kaolinite, montmorillonite, soil-A, and soil-C

following 90 days of aging; and (e) TNT desorption from sand, kaolinite,

montmorillonite, soil-A, and soil-C following 180 days of aging 99 Figure 5.3 — (a) TNT desorption from sand, kaolinite, montmorillonite, soil-A, and

soil-C without aging and normalized to BET SA; (b) TNT desorption from sand, kaolinite, montmorillonite, soil-A, and soil-C following 30 days of aging and

normalized to BET SA; (c) TNT desorption from sand, kaolinite,

montmorillonite, soil-A, and soil-C following 60 days of aging and normalized

to BET SA; (d) TNT desorption from sand, kaolinite, montmorillonite, soil-A, and soil-C following 90 days of aging and normalized to BET SA; and (e) TNT desorption from sand, kaolinite, montmorillonite, soil-A, and soil-C following

180 days of aging and normalized to BET ŠA - Ă Gv 101 Figure 5.4 — Fraction of TNT remaining with sand after 0, 30, 60, 90, and 180 days of aging The model fit is plotted with the experimental data for each aging period .104

Figure 5.5 — Fraction of TNT remaining with kaolinite after 0, 30, 60, 90, and 180

days of aging The model fit is plotted with the experimental data for each aging POTION, 105 Figure 5.6 — Fraction of TNT remaining with montmorillonite after 0, 30, 60, 90, and

180 days of aging The model fit is plotted with the experimental data for each 4113806 e 106

Figure 5.7 — Fraction of TNT remaining with soil-A after 0, 30, 60, 90, and 180 days

of aging The model fit is plotted with the experimental data for each aging

POTION, 00 107 Figure 5.8 — Fraction of TNT remaining with soil-C after 0, 30, 60, 90, and 180 days

of aging The model fit is plotted with the experimental data for each aging

POTION, 0.0 — 108 Figure 5.9 — (a) Thermograms for desorption of TNT from sand after aging at 30, 60,

90, and 180 days The thermogram without aging is not shown (b) Thermogram

for TNT desorption after 180 days OŸ aØ1ng HH gen 110 Figure 5.10 -Thermograms for desorption of TNT from kaolinite after aging at 30,

60, 90, and 180 days The thermogram without aging is not shown 111 Figure 5.11 -Thermograms for desorption of TNT from soil-A after aging at 30, 60,

90, and 180 days The thermogram without aging 1s not shown - 111 Figure 5.12 -Thermograms for desorption of TNT from soil-C after aging at 30, 60,

90, and 180 days The thermogram without aging 1s not shown -« 112 Figure 5.13 — TNT extraction data for the five geosorbents after aging for 0, 30, 60, L8 1 114 Figure 6.1 - TNT degradation pattern in 2.5%-amended sand, aged for 30 days, in

comparison to controls without amendments and autoclaved-biosolids 121 Figure 6.2 — TNT degradation pattern in 2.5%-amended kaolinite, aged for 30 days,

in comparison to controls without amendments and autoclaved-biosolids 121

Figure 6.3 — TNT degradation pattern in 2.5%-amended montmorillonite, aged for 30

days, in comparison to controls without amendments and autoclaved-biosolids 122 Figure 6.4 — TNT degradation pattern in 2.5%-amended soil-A, aged for 30 days, in comparison to controls without amendments and autoclaved-biosolids 124 Figure 6.5 - TNT degradation pattern in 2.5%-amended soil-C, aged for 30 days, in comparison to controls without amendments and autoclaved-biosolids 124 Figure 6.6 — Total viable microbial population for aged sand amended with 0%, 2.5%, and 2.5%A DiOSOLS 128 Figure 6.7 — Total viable microbial population for aged kaolinite amended with 0%, 2.5%, and 2.5%%A_ b1OSỌ1ỞS - Q00 1011191119 ng KH v03 E6 129 Figure 6.8 — Total viable microbial population for aged montmorillonite amended

with 0%, 2.5%, and 2.5%%A blosỌ1S Ăn ng ng nh cr 129

Figure 6.9 — Total viable microbial population for aged soil-A amended with 0%,

2.5%, ANd 2.5%A DIOSOLIS .cccessesessccecscssscccvscescecessececscsessceccsceesersecesevsteesevessases 130 Figure 6.10 — Total viable microbial population for aged soil-C amended with 0%,

TABLES

TABLE 3.1 - PROPERTIES OF GEOSORBENTS nen 30 TABLE 3.2 - CHEMICAL PROPERTIES OF TNT AND TETRYL - 31 TABLE 3.3 - RELEASE ENERGY VALUES FOR GLASS, SAND, KAOLINITE, AND SOIL-C càng go TH TH TH HT HH HH HA 54 TABLE 3.4 - SORPTION PARAMETERS FIT TO LINEAR AND FREUNDLICH

TABLE 4.1 - REACTOR SCHEMES USED FOR THE BIOSOLIDS

9)380/0/2.99/9)6.409 90115 67 TABLE 4.2 - PROPERTIES OF GEOSORBENTS FOR COMPOSTTING 69 TABLE 4.3 - CHARACTERIZATION OF ANAEROBICALLY DIGESTED

BIOSOLIDS nà nàng HH HH Tà tàn TH TH TH Tà Tà TH ni 70 TABLE 4.4 - SEQUENCE RESULTS TAKEN FROM THE DGGE PROFILES OF WET, DRIED, AND DRIED-AUTOCLAVED BIOSOLIDS .-.- 72 TABLE 4.5 - INITIAL AND FINAL TNT CONCENTRATIONS IN CONTROL,

BIOSOLIDS, AND AUTOCLAVED-BIOSOLIDS AMENDED

TABLE 4.6 - CORRELATION COEFFICIENTS FOR ZERO-, FIRST-, AND

SECOND-ORDER LINEAR MODELS - Án niên 82 TABLE 4.7 - FIRST-ORDER REACTION RATE CONSTANTS FOR TNT

DEGRADATION IN SOILS AMENDED WITH 0%, 2.5%, AND 2.5%A

TABLE 5.1 - SUMMARY OF THE EXTENT OF DESORPTION AND %

DESORBED FOR EACH GEOSORBENT AFTER EACH AGING PERIOD .100

TABLE 5.2 - SUMMARY OF KINETIC DESORPTION PARAMETERS FOR

EACH GEOSORBENT AFTER EACH AGING PERIOD 103 TABLE 5.3 - SUMMARY OF THERMAL DESORPTION PARAMETERS FOR

EACH GEOSORBENT AFTER EACH AGING PERIOD 113 TABLE 6.1 - PERCENT RECOVERY OF TNT AFTER 0 AND 30 DAYS OF

, 610 6 120 TABLE 6.2 - INITIAL AND FINAL TNT CONCENTRATIONS IN CONTROL,

BIOSOLIDS, AND AUTOCLAVED-BIOSOLIDS AGED GEOSORBENTS 123 TABLE 6.3 - CORRELATION COEFFICIENTS FOR ZERO-, FIRST-, AND

SECOND-ORDER LINEAR MODELS FOR AGED GEOSORBENTS 126

TABLE 6.4 - FIRST-ORDER REACTION RATE CONSTANTS FOR TNT

DEGRADATION IN GEOSORBENTS AGED FOR 0 AND 30 DAYS,

AMENDED WITH 0%, 2.5%, AND 2.5%A BIOSOLIDS 127

ACKNOWLEDGMENTS

I would like to acknowledge the United States Army Corps of Engineers —

Baltimore District and the United States Army Engineer Research and Development

Center — Waterways Experiment Station for their support of this work I would also like

to acknowledge funding from the Bayer Predoctoral Research Fellowship Lastly, I

would like to acknowledge the American Association of University Women for awarding

me an Engineering Dissertation Fellowship I am grateful to my advisor, Dr Jeffrey W Talley, and the members of my committee — Dr Patricia Maurice for her laboratory guidance and classroom interactions, Dr Jennifer Woertz for more laboratory guidance and frequent discussions, and Dr Robert Nerenberg for allowing me to present to his research group and engage in thoughtful discussions related to my research I thank both the current and former members of my research group — Sara Nicholl, Erica Pirie,

Guojing Liu, Tim Ruggaber, Caitlyn Shea and Dr Xiangru Zhang I would like to make

special acknowledgements to Tina Mitchell, Maria Avila, Joshua Hunn, and Dennis Birdsell for the many hours spent with me in the laboratory I also thank the Center for Environmental Science and Technology for the use of their facility to conduct many of the analytical parts of this research My thanks also go to Dr Maciej Manecki for his help with the Atomic Force Microscopy I especially thank my Parents, Grandparents and many Friends for their love, support, and encouragement over the past years

CHAPTER 1

INTRODUCTION

1.1 Problem Definition

There is a great need to understand soil interactions with nitroaromatic

compounds (NACs) and the effects of these interactions on both availability and

treatability of NACs in the environment This is especially true for the compound 2,4,6- trinitrotoluene (TNT) due to its extensive usage and connection with military bases and

former munitions manufacturing plants (Eriksson et al., 2004) Much of the soil

contaminated with TNT today is a result of the wide scale use of explosives dating back

to World Wars I and II The adherence and transformation of TNT in soils over such an extended period of time creates an obstacle for remediation (Lewis et al., 2004) and

therefore challenges our understanding of associated risks and accepted cleanup standards

(Alexander, 1995) Whether or not these contaminants are truly available for uptake by

plants and organisms and whether they pose a threat to human health and the

environment (Tang et al., 1998) are questions of current interest This dissertation

research examines the physical availability and biotreatability of TNT in contaminated sand, clays, and soils It also looks at the effects of aging; i.e., the length of time during

which the contaminated matrix has been impacted with TNT before any means of

remediation is attempted

In the aging process, compounds can become bound via numerous mechanisms such that their bioavailability is significantly decreased (Kottler and Alexander, 2001) The time-dependent decline in availability may result from compounds becoming bound

by mechanisms that are still not well understood (Chung and Alexander, 2002) Several

mechanisms for aging are described in the literature The mechanisms of greatest

importance for the aging of chemicals in soils and sediments include increasing sorption and partitioning into the soil organic matter For example, aging of herbicide residues

leads to increased sorption and, thus, to decrease in leaching (Walker et al., 2005) The aging process entails prolonged contact with soil organic matter during which time the molecule of interest becomes progressively more tightly bound to the organic matrix and

correspondingly less bioavailable (Barraclough et al., 2005) While the influence of soil

organic matter is strong, it may not be the sole contributor to the process (Chung and

Alexander, 1998, 2002; Bogan and Sullivan, 2003) and the literature seeks to identify

other parameters of interest (i.e., clay minerals, porosity, etc.) Despite work conducted to better understand the importance of aging on the environmental fate of organic

compounds, information about TNT remains limited

Time-dependent changes in availability are characteristic of both hydrophobic and hydrophilic compounds (Hatzinger and Alexander, 1995) However, with the exception

of Hatzinger and Alexander’s work (1995) on 4-nitrophenol, previous research on aging focused on mainly hydrophobic organic compounds (HOCs) such as polycyclic aromatic hydrocarbons (PAHs), and did not examine in detail the effects of aging on NACs,

compounds that are typically only slightly hydrophobic and sparingly water soluble A

comprehensive study that examines the influence of aging of TNT in a fundamental way

by isolating the effects of individual soil components (i.e., clay minerals and organic matter) is needed to expand our knowledge of TNT’s binding and availability

With an improved understanding of the binding and availability of TNT, we can better understand the potential effects of contaminant aging on our current risk-

assessment methods Most research on the fate of TNT has been site-specific, focusing

only on soils This research will bring forth new information on the aging of TNT in laboratory-aged sand, clay minerals, and soils It will examine how mostly mineral or organic carbon environments influence TNT binding and availability This information has great implications for the biotreatability of TNT with respect to both natural

attenuation systems and in-situ bioremediation schemes that are typically less energy intensive and less expensive than are more aggressive ex-situ treatment strategies

1.2 Research Objective and Hypotheses

The primary research objective of this dissertation was to better understand the

binding and availability of TNT in contaminated sand, clays, and soils; the effects of aging; and the implications for biotreatment To accomplish this goal, a review of the literature on TNT, availability, biotreatability, and aging was performed (Chapter 2) The

physical availability of TNT was investigated by using model geosorbents in sorption

isotherm experiments and thermal programmed desorption — mass spectrometry (TPD- MS) experiments (Chapter 3) Experiments were conducted that examined the availability

and biotreatability of TNT from model geosorbents in laboratory-scale composting

systems (Chapter 4) Anaerobically digested biosolids were used as the sole additive, and

for the first time, to investigate the biotreatability of TNT in the presence of different contaminated matrix type (i.e., sand, clay minerals, and soils) Aging experiments were

conducted to assess the effects of aging on the binding and availability of TNT as

determined via chemical extraction, aqueous desorption experiments, and TPD-MS

experiments (Chapter 5) The biotreatability of TNT after aging in contaminated sand, clays, and soils was investigated (Chapter 6) Finally, the binding and availability of TNT

in sand, clay minerals, and soils was summarized with respect to its environmental fate, the effects of aging, and the implications for biotreatment (Chapter 7)

Specifically, the following hypotheses were investigated for this research:

Hypothesis #1 (Chapter 3) — It is hypothesized that the physical availability, as

determined via aqueous sorption and thermal desorption experiments, of TNT

from the studied model geosorbents will be greatest in the order sand > kaolinite >

montmorillonite > soil-C (2.4% organic matter)

Hypothesis #2 (Chapter 4) — It is hypothesized that TNT and its reduced transformation

products can be degraded by the biota present in anaerobically digested biosolids Additionally, the biosolids can reduce the TNT concentrations by incorporating

TNT and its reduced products into the organic matter I hypothesize that the bioaugmented systems, those receiving biosolids, will perform better than control systems with no biosolids and systems receiving autoclaved-biosolids; thus,

confirming that the added biota are critical to the treatment process.

Hypothesis #3 (Chapter 5) — It is hypothesized that there will be differences in the rate

and extent of sequestration, the rate and extent of desorption, and the release energy of TNT from geosorbents that are characterized as mostly mineral, having low organic carbon, and having high organic carbon as these geosorbents undergo

aging in the lab As the aging period increases and the percent organic carbon

increases, the rate and extent of both sequestration and desorption will decrease while the estimated release energy will increase

Hypothesis #4 (Chapter 6) — It is hypothesized that differences in the concentration of

organic carbon (zero, low, high) for aged contaminated geosorbents will affect both the rate and extent of biotreatability and the percent recovery of TNT With

increasing organic carbon, the percent recovery and the rate and extent of

biotreatability will decrease

CHAPTER 2

LITERATURE REVIEW

The objective of this research was to investigate the binding, availability, and biotreatability of 2,4,6-trinitrotoluene (TNT) in quartz sand, clay minerals (specifically kaolinite and montmorillonite), and soils A major emphasis was placed on how aging affects both the availability and biotreatability as well as how these vary with different components found in TNT-contaminated soils It is important to understand how changes

in the availability of TNT over time could influence its mobility through soils, and its potential to contaminate surface waters or groundwater resources, and the potential for its

biotreatment This literature review provides background information needed to

understand this research, while simultaneously supporting the need for more fundamental research concerning the effects of aging on the binding and availability of TNT in soils

To do so, it answers the following four questions:

1) Why are we concerned with TNT?

2) How do we assess the binding and availability of TNT?

3) How amenable is TNT to biotreatment?

4) What are the important mechanisms of aging that influence the availability and biotreatability of TNT?

The answer to the first question discusses TNT in general, providing motivation

for why this research is important In answering the second question, definitions of bound

residues and availability are given and assessment of TNT binding and availability are

discussed The answer to the third question reviews the current status of TNT

bioremediation efforts The answer to the final question reviews aging mechanisms

previously proposed to explain decreased availability of organic contaminants due to aging

2.1 Why Are We Concerned with TNT?

2.1.1 Toxicity and Health Effects of TNT

The toxicity of TNT is well documented (Harvey et al., 1990; Kaplan and Kaplan, 1982; and Won et al., 1976) TNT is known to be toxic towards bacteria, fish, plants, and mammalian cells (Drzyzga et al., 1995; Layton et al., 1987; and Honeycutt, 1996) TNT

is also on the list of USEPA Priority Pollutants, is a known mutagen, and can cause pancytopenia as a result of nitrenium ions formed by enzymatic oxidation (Rodgers and

Bunce, 2001) There have been numerous cases of munitions workers developing liver

damage and anemia due to TNT exposure (Sax, 1963; Bridge et al., 1942; Hamilton,

1921; Voegtlin et al., 1920) Soils contaminated with TNT pose a significant risk to

human health via numerous exposure routes, and TNT transformation products may be equally if not more toxic than the parent TNT molecule

2.1.2 A History of TNT Contaminated Sites

Soils contaminated with TNT exist at former ordinance production plants as well

as existing military ranges throughout the world (Spain and Nishino, 2002; Thiboutot et al., 2002) Much of the contamination at the production sites resulted from wastewaters

discharged into unlined lagoons or directly onto soil (Pennington and Patrick, 1990;

Selim et al., 1995) Although these practices are no longer employed, localized areas of residual contamination still exist (Harvey et al., 1990) TNT is recalcitrant to degradation

in aerated and unsaturated soils, and thus contributes to numerous long-term

contamination problems (Fuller et al., 2003) It is well understood that contamination of the environment by explosives like TNT is a major consequence of munitions

development and testing There nevertheless remains an operational need for testing and training with conventional weapons in order to maintain combat readiness in our armed services (Thiboutot et al., 2002) The environmental impact of meeting this need presents

a serious environmental challenge

The estimated cost for the cleanup of Department of Defense (DOD) sites

contaminated with TNT in the U.S alone is $2.66 billion, and may include up to 750,000 cubic yards of soil and 530 billion gallons of groundwater (Spain and Nishino, 2002) Although a number of TNT (see Rodgers and Bunce, 2001 for a review) treatment

technologies have been developed at the bench-scale, these techniques do not result in the

mineralization of the TNT molecule (Hughes et al., 1998; Boopathy et al., 1998; Bruns- Nagel et al., 2000) Furthermore, when applied to the full-scale, these remediation

strategies are often not capable of achieving soil cleanup goals

2.1.3 Environmental Transformations of TNT

TNT can undergo transformation via photochemical and microbiological

processes in soil (Walsh et al., 1995) Environmental transformation of TNT is significant for two reasons First, the transformation products may be as undesirable as TNT because

of their toxicity (Griest et al., 1993; Gibbs et al., 1996) and second, they may have the

potential for immobilization reactions with soil components (Daun et al., 1998; Drzyzga

et al., 1998; and Bruns-Nagel et al., 2000)

It is well established in the literature that the three nitro groups of TNT reduce the electron density of the aromatic ring and impede electrophilic attack (Rieger and

Knackmuss, 1995) Consequently, catabolic pathways initiated by oxygenation are

unknown for TNT The electron deficiency of the ring system favors initial reductive reactions so that even aerobic microorganisms harbor the ability to transfer reduction equivalents to TNT Reductive pathways can lead to reduction of the nitro group to hydroxylamino- (HADNTs), amino- (ADNTs), azo-, and azoxy-dinitrotoluenes (AZTs) Simultaneous oxidation and reduction was reported by Bruns-Nagel et al (1999), who found that the major product from TNT was 2-amino-4,6-dinitrobenzoic acid, in which one of the nitro groups had been reduced and the methyl group had been oxidized

Hydrogenation of the aromatic m-electron system, that give rise to the formation of hydride Meisenheimer complexes, has been identified as a novel initial catabolic

reaction Figure 2.1 illustrates the transformation pathways for TNT

2-Amino-4,6-dinitro- 4-Amino-2,6-dinitrotoluenc H’-Meisenheimer

Figure 2.1 — TNT transformation pathways (taken from Spain and Nishino, 2002)

2.1.4 Soil Constituents Involved in TNT Binding

Since the mechanisms controlling TNT availability in soils are likely similar to

the mechanisms fundamentally developed for nonpolar organic compounds, a brief

review of the soil constituents that are important for controlling TNT fate processes (1.e., sorption, desorption, and partitioning) is provided

2.1.4.1 Quartzite Sand and Clay Minerals

In typical soils, minerals make up over 90% of the soil matrix and are composed

of various arrangements of silica, aluminum, oxygen, and iron (Sparks, 1995) The

surface of most of these minerals is charged The mineral matrix itself is impenetrable and rigid; hence, sorption to mineral components is a surface or near surface interaction and takes place in a fixed pore system (Pignatello, 1998)

Quartz, the second most abundant mineral in the Earth’s crust, is important to

TNT binding because it dominates in sands, many soils, and some sediments Quartzite

sand is essentially pure S102, but it may contain trace amounts of other elements It consists of a three-dimensional framework of SiO, tetrahedra, with all oxygens shared by two tetrahedra A large number of nitroaromatic compounds (NACs), such as TNT, have minimal interaction with the surface of quartz For example, Haderlein and

Schwarzenbach (1993) showed that NACs were weakly adsorbed to silica The use of quartz sand as a geosorbent in this research provided a good control for demonstrating

minimal TNT binding to more homogeneous surface sites The sorption of TNT to clay

minerals, on the other hand, is of greater significance

Clay minerals are a group of sheet silicates with related atomic structures Most

are hydrated aluminum or magnesium silicates that are products of weathering Clays are usually very fine grained, often less than 1 um in size, which results in the presence of large surface areas available for the exchange of ions and molecules between the solids

and surrounding solutions Two of the most common clay minerals are kaolinite and montmorillonite

Kaolinite is a common secondary mineral, forming after aluminous silicates, and

is a component of many soils Kaolinite clays are two-layer structures with a chemical

formula of Al¿(S14O¡o)(OH)s; The atomic structure of kaolinite is based on layers of S104

tetrahedra with OH’ and A!** in between, which leads to two distinctly different potential

adsorption surfaces: an alumina surface with surface hydroxyl groups and a silica surface with oxygen-bridged silica atoms (van Duin and Larter, 2001) The hydrophobic siloxane surfaces (exterior oxygen plane and siloxane cavities) are the preferred sites for the

adsorption of non-polar organic molecules via van der Waals type interactions (Yariv and

Cross, 2002) Given the presence of these relatively hydrophobic surfaces as well as the high surface area of kaolinite, nonpolar organic molecules, such as TNT, are bound more

tightly to kaolinite as compared to quartz sand

Montmorillonite, the other most common clay mineral, dominates modern clay-

rich sediments Montmorillonite is basically a three-layer structure that can take up extra water or other fluids between the layers of its atomic structure In the process it expands;

thus, we sometimes refer to it as an expandable or swelling clay The structure is based

on groups of three layers, where single sheets of (Al,Mg)(O,OH)¢ octahedra are

sandwiched between two sheets of SiO, tetrahedra Uzgiris et al (1995) attributed a

recalcitrant fraction of polychlorinated biphenyls (PCBs) in montmorillonite to PCBs trapped in the interstitial layers of the clay

2.1.4.2 Natural Organic Matter

Natural organic matter (NOM) is an assemblage of organic compounds derived

from plants and animals and found in practically every terrestrial environment NOM may include recognizable biopolymers like proteins, lignin, and cellulose, but also a menagerie of macromolecules from the partial degradation and cross-linking of organic residues remaining from organisms or photochemical reactions (Schwarzenbach et al., 1993) NOM is predominantly made of carbon, but can also consist of almost as many

oxygens as carbon in their structure Naturally, the structure of the material will depend

on the ingredients supplied for a particular water or soil

Soil organic matter (SOM) is decomposed plant and microbial material, with the

bulk of SOM consisting of humic substances (HSs) Substances are typically referred to

as humic substances (HSs) if they are soluble or extractable in aqueous base, and humin

or kerogen if they are not The humic substances are further subdivided into fulvic acids (FAs) if they are soluble at all pHs and humic acids (HAs) if they are not soluble in acidic

conditions (< pH 2) but are soluble at higher pHs HSs are a refractory mixture or

macromolecules with molecular weights ranging from a few hundred to many tens of

thousands of grams per mole (or daltons) Partitioning into humic matter may be

important in the sorption of nonionic organic chemicals, like TNT, and could be a

mechanism of its aging in soils and sediments

2.2 How Do We Assess Binding and Availability?

2.2.1 Bound Residues

Bound residues were first defined in 1975 by the American Institute of Biological Sciences — Environmental Task Group as “‘unextractable and chemically unidentifiable.”

Fuhr et al (1998) proposed the following definition for bound residues: “bound residues

represent compounds in soil, plant, or animal which persist in the matrix in the form of the parent substances or its metabolites after extractions in general; the formation of bound residues reduces the accessibility and the bioavailability significantly.” Weller et

al (1998) proposed the following set of definitions:

e Covalently bound residue — the parent compound or a major metabolite that is covalently bound to the substrate;

e Soluble covalently bound residues — those that are extracted along with the matrix

by a specific procedure where the covalent bond is preserved (i.e., humic acids extractions);

e Adsorbed residues — parent compounds or main metabolites which are bound to

the matrix by reversible non-covalent interactions; and

e Entrapped residues — parent compounds or main metabolites which are retained within the matrix by steric effects; entrapped residues are essentially bound unless the matrix structure is modified

The term “bound residue” usually corresponds to the sum of covalently bound residues, adsorbed residues, and entrapped residues The binding of a chemical in a fashion that reduces availability may be linked with another constituent or soil property or several acting together, e.g., amount and type of organic matter, amount and type of clay mineral, surface area, nanoporosity, or cation-exchange capacity (CEC) (Alexander and

Alexander, 2000), and the loss of availability is a characteristic of chemicals with many

dissimilar properties (Kottler and Alexander, 2001)

2.2.2 Availability

How we define availability is critical The available portion of a compound in soil

is generally described to be the portion of a compound that can be extracted from soil, typically by an organic solvent and without alteration to the chemical structure of the compound as well as the non-extractable portion (Northcott and Jones, 2000) The

bioavailable fraction is further defined as the quantity of a chemical in soil or another

environmental medium that is actually accessible to an organism (Alexander and

Alexander, 2000) Bioavailability, the term as used here and as commonly used among

soil and environmental scientists, does not relate to toxicity (Alexander and Alexander, 2000) Decreasing bioavailability with increasing contaminant-soil contact time has been shown for microorganisms (Kelsey et al., 1997; White et al., 1997), earthworms (Kelsey

et al., 1997; White et al., 1997), and other organisms (Leppanen and Kukkonen, 1998) However, in many cases, the fraction of a compound that is not bioavailable does not

inversely relate to the fraction of the compound that is considered as bound residue

(Edwards, 1974) Decreased bioavailability of contaminants in soils may result from chemical oxidation reactions incorporating contaminants into organic matter, slow

diffusion into very small pores, and absorption into organic matter — all related to the aging process

It is generally agreed that one necessary aspect to bioavailability is the release of a solid-bound contaminant (Ehlers and Luthy, 2003) Therefore, before a compound can be

bioavailable, it must actually be physically available This release or physical availability

is defined as the capability of a chemical for transport from its bound state on or within a

geosorbent to a new environmental medium Physical availability can be linked to the extent to which a contaminant can partition into the aqueous phase compared to how

much is sequestered (Chung and Alexander, 1998) and more recently, the energy required

to release the chemical from its sorbent matrix Thus physical availability can be assessed using more traditional aqueous desorption methods and the novel thermal programmed desorption-mass spectrometry (TPD-MS) experiment

2.2.3 TPD-MS

The theory behind TPD-MS is simple, yet complex The physical availability, as controlled by sorption and diffusion, of a compound can be assessed semi-quantitatively using release energy, with higher energy values indicating a more tightly bound

compound (Talley, 2000; Talley et al., 2004) Release energy is not to be used

synonymously with the term activation energy from the surface science literature because

a release energy actually represents the total energy required to release a compound from its sorbent, including the energy required for desorption from and diffusion through the

sorbent matrix Release energy values are a function of the type of sorbate and sorbent

involved Release energy values can be calculated using both integral and differential approaches These calculations result in values for the pre-exponential factor (v) and

release energy (E) from desorption rate curves that model thermograms produced during

a TPD-MS experiment Nicholl et al (2004) used the analytical solution to the simplified Polanyi-Wigner equation to calculate energy values of polycyclic aromatic hydrocarbons (PAHs) from several geosorbents The results of this method indicate that, as suggested

in other reviews of TPD methods (Talley et al., 2004), the ‘coverage dependence’ of both

E and v must be addressed in order to determine a release energy value However, this type of modeling may be possible using a differential approach

To date, TPD-MS using direct insertion probes has been used for the study of pyrolysis (Yun and Meuselaar, 1991); as a means for inserting a sample into a mass spectrometer for compound identification; and to study desorption of organic substrates from solids (Yang et al., 1999; Talley, 2000; Ghosh et al., 2001; Talley et al., 2002; Xi et al., 2003; Talley et al., 2004; Nicholl et al., 2004) The work to date regarding desorption

of organic substrates has centered on hydrophobic organic compounds (HOCs) such as

PAHs, PCBs, and dioxins In previous work, it was found that TPD-MS could be used to

assess the availability of PAHs from sediments (Talley et al., 2002, Ghosh et al., 2001) Specifically, it was discovered that a PAH-impacted material had accessible pollutant fractions and a bound residue that was much more biologically unavailable and

immobile The available (treatable) fraction was associated with a low organic carbon environment and lower release energies, while the bound (untreatable) residue was found

to be present primarily in a high organic carbon environment and associated with higher

release energies

2.3 How Amenable is TNT to Biotreatment?

Research regarding the biodegradation of TNT dates back to the 1970s Since

TNT was the most widely produced and used military explosive in the world and it is now the primary explosive contaminant of concern to the DOD, tremendous work was and continues to be done in an attempt to achieve mineralization of this compound

However, numerous investigations show that the TNT molecule is very recalcitrant

against microbial mineralization (Gorontzy et al., 1994; Rieger and Knackmuss, 1995; Hughes et al., 1998; Boopathy et al., 1998)

2.3.1 Biodegradation of TNT

In general, bioremediation’s greatest successes involve technologies that exploit the use of aerobic processes (USEPA, 2002) The electron-withdrawing nature of the nitro groups, however, causes the TNT molecule to be resistant to oxidation (Hundal et al., 1997) The most common biological transformation of TNT is reduction of one or

more nitro groups (Spain, 1995) via nitroso and hydroxylamino intermediates to form

monoamines and diamines However, reduction of TNT in the literature occurs under both anaerobic (Lewis et al., 1996; Funk et al., 1995; Boopathy and Kulpa, 1994;

Boopathy et al., 1993; Funk et al., 1993; Preuss et al., 1993) and aerobic conditions (Kalafut et al., 1998; French et al., 1998; Vorbeck et al., 1998; Bruns-Nagel et al., 1996; Bradley et al., 1994; Vorbeck et al., 1994; Boopathy et al., 1994a; Boopathy et al., 1994b; Boopathy et al., 1994c; Duque et al., 1993) via bacteria and fungi Under anaerobic conditions, the reductions occur more rapidly than in the presence of oxygen, and the unstable hydroxylamino intermediates do not accumulate or form linkages In contrast, under aerobic conditions, the production of aminodinitrotoluenes and azoxy compounds

can lead to the formation of azo or azoxy linkages and to dimerization or polymerization

(Carpenter et al., 1978)

In the last decade, aerobic pure cultures that degrade TNT were identified

Vorbeck et al (1994) demonstrated that a Mycobacterium sp strain was able to

metabolize TNT under aerobic conditions producing a Meisenheimer complex as its

initial metabolite Montpas et al (1997) isolated a strain of Serratia marcescans from a TNT-contaminated site that could degrade TNT as the sole source of carbon and energy Vorbeck et al (1998) enriched two bacterial strains with TNT as a sole source of nitrogen under aerobic conditions, TN7-8 and TNT-32, which could carry out nitro-group

reduction reactions Kalafut et al (1998) reported on three aerobic bacterial strains

(Pseudomonas aeruginosa, Bacillus sp., and Staphylococcus sp.) that can co-metabolize TNT, with varying metabolic preferences, if the cell concentration is sufficiently high

Enterobacter cloacae PB2 was found by French et al (1998) to be capable of slow

aerobic growth with TNT as the sole nitrogen source

The anaerobic metabolism of TNT to reduced intermediates was investigated by Funk et al (1993), using starch as a supplemental carbon substrate and incubation under static conditions Clostridium bifermentans was shown to reductively transform TNT under anaerobic conditions (Lewis et al 1996) Anaerobic biotransformation of TNT was carried out by a methanogenic mixed culture and stimulated by the addition of electron donors, such as Hp (Adrian et al 2003) Recently, Fleischmann et al (2004) investigated the degradation of TNT by bovine rumen fluid, a novel source of anaerobic microbes, and found that it reduced TNT via similar pathways already described in the literature;

however, it may have also resulted in metabolites of TNT that were further transformed than previously reported

Not only are bacteria effective in the bioremediation of TNT, so too are fungi

(Barr and Aust, 1994; Bumpus and Tatarko, 1994; Fernando et al., 1990) Several studies using Phanerochaete chrysosporium discuss the mineralization of TNT Fernando et al (1990) investigated the biodegradation of TNT with Phanerochaete chrysosporium and found 18.4% and 19.6% mineralization in water and soil, respectively Hawari et al (1999) investigated the biotransformation of TNT with Phanerochaete chrysosporium, and found an unusually high number of intermediates, but found that mineralization in this case did not exceed 1% Thus, microbial transformation of TNT to carbon dioxide and water has been observed in several studies, but concentrations were generally too low

to simulate the actual conditions in polluted sites, where TNT concentrations can reach 1% or 10,000 mg TNT/kg soil (Comfort et al., 1995)

2.3.2 Bioremediation Strategies for TNT

The greatest advantages of bioremediation are that 1) contaminants can be

degraded to nontoxic byproducts and some can be mineralized, potentially resulting in the complete removal of the contaminant from the environment; 2) bioremediation uses

natural means to achieve contaminant removal; and 3) bioremediation is perceived as

relatively inexpensive and environmentally friendly (USEPA, 2002) Recommended bioremediation strategies for TNT-contaminated soils include phytoremediation,

bioslurry reactors, and composting

2.3.2.1 Phytoremediation

Phytoremediation, the use of green plants to remediate contaminated soil or water (Cole, 1997), is a promising method for the treatment of explosive-contaminated sites

(Hannink et al., 2002) Phytoremediation is considered an inexpensive, low maintenance

biotechnology that can tolerate high concentrations of contaminants better than some

microorganisms (Rodgers and Bunce, 2001) Hughes et al (1997) was the first to confirm the intrinsic ability of the plants Myriophyllum and C roseus to transform TNT

Thompson et al (1998) studied the translocation and fate of TNT in a poplar tree hybrid (Populus sp deltoidesXnigra, DN34) Bhadra et al (1999) examined processes in plants for the formation of fate products of TNT beyond its aminated reduction products,

2ADNT and 4ADNT Bhadra et al (1999) also isolated and characterized products of oxidative metabolism of TNT in plants Dramatically enhanced detoxification of TNT has been demonstrated in transgenic plants expressing the bacterial nitroreductase (Hannik et al., 2001) Burken et al (2002) indicated that the removal of NACs by plants corresponds

to a “green liver” model in which contaminants are transformed and then conjugated into plant molecules

2.3.2.2 Bioslurry Treatment

Bioslurry treatment involves mixing TNT-contaminated soil with water and treating it in a reactor (e.g., tank or lagoon) either anaerobically, aerobically, or

anaerobically-aerobically (Rieger and Knackmuss 1995 and Lenke et al 1997) The

advantage of the bioslurry system is the high degree of mixing and effective contact time which leads to often faster and more complete degradation of TNT and formation of

bound residues Bioslurry treatment for TNT is considered to be an effective method of TNT treatment by means of immobilization (e.g., fixation by irreversible binding to the organic soil matrix)

Drzyzga et al (1998) estimated the actual fate of '*C-TNT in a molasses-

supplemented soil bioreactor after a sequential anaerobic-aerobic treatment process that

transformed 97% of the initially added TNT This was the first study to show data that demonstrated the incorporation of nearly 84% of the originally applied radioactivity into the organic soil matrix following the sequential anaerobic-aerobic bioremediation

process Based on alkaline hydrolyses of the solvent extracted soil samples taken after nine weeks of treatment, the major part of the radioactivity was found to be strongly bound to the humin fraction At about that same time, Daun et al (1998) investigated the

interaction of TNT and metabolites with soil and concluded that, without the use of 4c

TNT, their studies indicated that the irreversible binding of TNT reduction products with soil components was inevitable In their companion paper (Lenke et al 1998), the same

researchers conducted laboratory-scale and full-scale experiments to test for biologically

induced immobilization of TNT, but still could not address the relative stability of the covalent binding of reduced TNT metabolites to soil organic matter

2.3.2.3 Composting

Composting, which involves mixing TNT-contaminated soil with bulking agents (e.g., straw, bark, sawdust, and wood chips) and organic amendments (e.g., manure or fruit and vegetable processing waste), is an attractive and economical bioremediation technology The process has been studied extensively (Kaplan and Kaplan 1982, Griest et

al 1991, Williams and Marks 1991) With suitable porosity, temperature, oxygen levels,

pH and moisture content, TNT can be effectively biotransformed and incorporated into

the soil organic matter Toxicity studies have shown that the composting of TNT-

contaminated soils reduced its concentration as well as mutagenicity

Pennington et al (1995) investigated the fate of TNT in a simulated compost system in an attempt to complete an overall mass balance on '*C-TNT At the time, the best method for tracing TNT and its metabolites was the use of a radiolabeled tracer that

could be compartmentalized into the different fractions of soil organic matter This study

indicated that TNT was not mineralized, no volatile organic compounds were produced, and more than half of the radioactivity was recovered in the cellulose plus humin

fractions of the compost mixture The long-term stability of this binding was still unclear

The use of a radiolabeled tracer is really only indicative of immobilization taking place and not of the actual chemical structures of the immobilized residues that are formed during biotreatment More recently, the application of '"N NMR experiments

proved a viable technique, offering direct spectroscopic evidence for the formation of

covalent linkages to the organic matter In a two part study, Knicker et al (1999) and Bruns-Nagel et al (2000) applied solid-state 'SN-NMR spectroscopy to examine the nature of nitrogen-containing products that were formed and immobilized during TNT

degradation via composting Thorn and Kennedy (2002) analyzed the reacted products of

TNT and its major reductive degradation products with soil humic acid using °N NMR

and confirmed the ability of the products to form covalent bonds with the soil humic acid

These highly reactive TNT reduction products (amino groups), once bound to the soil organic matter, can significantly influence the availability and risk of TNT residues in

soil

2.4 What Are the Mechanisms of Aging that Influence the Availability and

Biotreatability of TNT?

Several mechanisms have been described to explain the aging of chemicals in

soils (Chiou et al., 1983; Steinberg et al., 1987; Brusseau et al., 1991; Brusseau and Rao, 1991) Aging is believed to be strongly affected by sorption, sequestration, partitioning and/or entrapment in organic matter, and covalent bonding, with each process being

dominant and influential at different times during aging The literature presents two opposing opinions on what the term ‘aging’ should include Some authors believe that aging includes the formation of covalent bonds of parent compounds or their degradation

products (Gevao et al., 2000), while others believe aging does not include reactions that alter the structure of the molecule (Hatzinger and Alexander, 1995) The authors do agree

that the passive processes, such as partitioning into organic matter and entrapment within

small pores, do occur with aging in soil (Alexander, 1995; Pignatello and Xing, 1996;

Hatzinger and Alexander, 1995; Kelsey et al., 1997; Luthy et al., 1997; Mader et al.,

1997; White et al., 1997; Chung and Alexander, 1998; Nam and Alexander, 1998; Piatt

and Brusseau, 1998) Figure 2.2 displays the processes that are inherent to the aging of organic compounds in the presence of both minerals and organic matter (Reid et al.,