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THESiS
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LIBRARY
Michigan State
University

 

 

 

This is to certify that the

thesis entitled

Microbial Transformation of DDT in Pine River Sediments

Date

0-7639

presented by

Michael Gene Roberts

has been accepted towards fulfillment
of the requirements for

Master of Science Crop and Soil Sciences

degree in

Major professor

 

 

/7

  

 

October S, 2001

 

MS U i: an Affirmative Action/Equal Opportunity Institution

 

 

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MICROBIAL TRANSFORMATION OF DDT IN PINE RIVER SEDIMENTS

By

Michael Gene Roberts

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Crop and Soil Sciences

2001

ABSTRACT
MICROBIAL TRANSFORMATION OF DDT IN PINE RIVER SEDIMENTS
By

Michael Gene Roberts

This study examined the fate of DDT in sediments at five sampling locations near
the Pine River Superfimd site. River sediments were analyzed for DDT and its
dechlorination products, DDD, DDE, and DDMU (collectively referred to as DDX).
Both p, p'- and o, p'-isomers for each compound were detected at all sites at
concentrations ranging from 0.05 ppm to 20,000 ppm. Compositional analysis revealed
an increase in p, p'- vs 0, p'— isomers and higher proportions of DDD relative to technical
grade DDT at 3 of the sites. In addition to analyzing for DDX compounds, we conducted
microcosms studies to determine the abilities of the sediments to support anaerobic
dechlorination and to clarify specific degradation pathways. We compared the extent of
anaerobic transformations of 14C- radio-labeled DDT, DDD, and DDE amended to live
and autoclaved anaerobic sediments slurries. Although DDT was converted primarily to
DDD in both live and autoclaved treatments, no transformation of DDD or DDE
occurred. Our results indicate that the majority of DDD formation in the Pine River
sediment microcosms was due to abiotic processes, but there was also a substantial biotic

contribution (19 % to 41 %) during the 40 week incubations.

This work is dedicated to my mother, Annie Mae Roberts, who always sacrificed for my
education, and to my wife, Jennifer, whose support and patience has made this goal
achievable.

iii

ACKNOWLEDGEMENTS

I would like to express my appreciation to my major professor Dr. Stephen Boyd for his
guidance and support for my study. I also would like to extend my appreciation to Dr.
James Tiedj e and Dr. Matthew Zabik for serving on my M.S. Guidance Committee and
their assistance with my research. A special thanks is given to Dr. John Quensen for his
sincere friendship and valuable assistance with all aspects of my research.

I want to acknowledge the College of Agriculture and Natural Resources and Crop and
Soil Sciences for their financial support. I give thanks to Dr. James Jay for his friendship
and assistance.

Finally, appreciation is extended to my wife, family, friends and professors for their

support and patience throughout this program.

iv

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................ vii
LIST OF FIGURES ......................................................................................................... viii
CHAPTER 1
INTRODUCTION ............................................................................................................... 1
Historical Information .............................................................................................. 1
Environmental Fate .................................................................................................. 3
Dechlorination Reactions ......................................................................................... 3
Oxidative Reactions ................................................................................................. 8
Compounds detected at DDT contaminated sites .................................................. 10
Specific Transformations ....................................................................................... 11
DDT to DDD .............................................................................................. ll
DDT to DDE .............................................................................................. 12
DDD to DDMU .......................................................................................... 13
DDE to DDMU .......................................................................................... 14
DDMU and beyond .................................................................................... 14
REFERENCES .................................................................................................................. 15
CHAPTER 2
INTRODUCTION ............................................................................................................. 21
Sampling and Chemical Analysis of Pine River sediments ................................... 21
MATERIALS AND METHODS ....................................................................................... 24
Sampling and Chemical Analysis .......................................................................... 24
RESULTS AND DISSCUSION ........................................................................................ 26
Site Description ...................................................................................................... 26
REFERENCES .................................................................................................................. 34
CHAPTER 3
INTRODUCTION ............................................................................................................. 35
OBJECTIVE ...................................................................................................................... 35
MATERIALS AND METHODS ....................................................................................... 36
Materials ................................................................................................................ 36
Sediments Sampling and Analyses ........................................................................ 36
Methods ........................................................................................................ ’ .......... 37
Sediment Slurry Preparation .................................................................................. 37
Microcosms Setup .................................................................................................. 37
Methane Analysis ................................................................................................... 38
Dechlorination Assays (Extraction and Analysis) ................................................. 38
RESULTS AND DISSCUSION ........................................................................................ 4O
DDT Transformations ............................................................................................ 4O
Transformations in live microcosms .......................................................... 41

V

Transformations in abiotic microcosms ..................................................... 45

Estimated (calculated) biotic transformation ............................................. 51

Abiotic and biotic formation of DDD ........................................................ 52

14C Total Recoveries .............................................................................................. 54

DDD and DDE Transformation in sediment microcosms ......................... 56

SUMMARY ....................................................................................................................... 58
REFERENCES .................................................................................................................. 59
APPENDIX ........................................................................................................................ 60

vi

LIST OF TABLES

Table 1.1. Abbreviations, chemical formulae and names of DDT and its degradation
products given in the text ..................................................................................................... 7

Table 2.1. Mean concentrations (ug/ g) of DDT and metabolites and total DDX in
sediments collected from the Pine River. Values, in ugrams/ grams (calculated from
measurements of estimated concentration in sample extract), are the average of two
replicates. The component percentage of the total DDX concentration is shown in
parentheses ......................................................................................................................... 28

Table 2.2. Percent of Total DDT and metabolites in Technical DDT (Adapted from West
and Campbell, 1950) .......................................................................................................... 30

Table 2.3. Mass concentrations (ug / g) and percentage of DDT and its derivatives in
sediments collected from the Pine River. Values, in ugrams/ grams (calculated fi'om
measurements of estimated concentration in sample extract), are means of two replicates
except for three replicates from site 4 sediments. The component percentage of the total
concentration is shown in parentheses ............................................................................... 32

Table 3.1. Comparison of observed percent l4C-radioactivity recovered as various
analytes from Pine River sediments (5 sites) incubated under anaerobic conditions. The
standard deviation is reported in parentheses. The t-Test was applied to determine the
statistical significance (P<0.05) for differences in DDT transformations among the
biologically active and abiotic controls ............................................................................. 43

Table 3.2. Total 14C activity recovered at O and 40 weeks for various analytes with live
and autoclaved microcosms prepared with biologically active (live) and autoclaved
sediments amended with 14C-DDT .................................................................................... 55

Table 3. 3. Total 14C activity recovered after 40 weeks for various analytes with live and

autoclaved microcosms prepared with biologically active (live) and autoclaved sediments
amended with either 4C- DDD or l4C-DDE ...................................................................... 57

vii

LIST OF FIGURES

Figure 1.1. Metabolic pathway of DDT biotransformation (Adapted from Rochkind and
Blackburn, 1986) with additions of (Jensen et al.1971; Marei et al., 1978; Quensen et a1.
1998). The steps observed most common in DDT the degradation pathway are indicated
by underlined letters, and the step most commonly found in Pine River sediment
microcosms are indicated by a thick arrow. In addition to DDD, DDE and DDMU, there
are several other degradation products that are less commonly observed ........................... 6

Figure 1.2. Cometabolic, meta-fission products of DDE as proposed for biphenyl-grown
Pseudomonas acidovorans M3GY (Adapted from Hay and Focht, 1998).

(1) DDE; (2) 1,l-dichloro-2-(dihydroxy-4- chlorophenyl )-2—(4-chlorophenyl)ethylene;
(4) 4, 6-oxo-2-hydrooxy-7-(4-chlorophenyl)-4,8,8-trichloroocta-2,4-dienoic acid; (5) 5,
2-(4- chlorophenyl)-3,3-dichloropropenoic acid; (6) 6, 4-chlorophenylacetic acid; (7) 7,
4-chloroacetophenone; (8) 8, 4-chlorobenzaldehyde; (9) 9, 4-chlorobenzoic acid ............. 9

Figure 1.3 Proposed pathway for reductive dechlorination reaction of DDT
(Schwarzenbach et al., 1993) in anaerobic systems. The oxidation state for aliphatic
carbon atom for each compound is shown in parenthesis .................................................. 12

Figure 1.4. Proposed pathway for dehydrochlorination of DDT in aerobic environments

(Schwarzenbach et al.,
1 993) ........................................................................................................ 1 3

Figure 2.1. Map of sampling locations at the Pine River .................................................. 27

Figure 2.2. Percentage of DDX component distributions among sites, the components
were grouped according to p, p'- versus 0, p'- substitution ................................................ 33

Figure 3.1. Dechlorination of DDT to DDD in sediment microcosms by microorganisms
under anaerobic conditions during 40 weeks of incubation. There were no significant
differences in DDD formation among sites at the 8 and 40 week sample intervals .......... 44

Figure 3.2. Microbial anaerobic dechlorination of DDT to DDD in sediment microcosms
prepared from site 1 sediments (refer to Figure 2.1 for sampling location) during 40
weeks incubations. Dashed line indicates Recovery Total added as DDT. These results
were obtained by liquid scintillation counting of scrapings fiom the TLC plates.
Recovery Total represents the amount of 14C-labeled radioactivity DDT recovered in the
solvent and aqueous extracts. Percent of Methane produce at time of analysis by GC....46

Figure 3.3. Microbial anaerobic dechlorination of DDT to DDD in sediment microcosms
prepared fiom site 2 sediments (refer to Figure 2.1 for sampling location) during 40
weeks incubations. Dashed line indicates Recovery Total added as DDT. These results
were obtained by liquid scintillation counting of scrapings from the TLC plates.

viii

Recovery Total represents the amount of l4C-labeled radioactivity DDT recovered in the
solvent and aqueous extracts. Percent of Methane produce at time of analysis by GC....47

Figure 3.4. Microbial anaerobic dechlorination of DDT to DDD in sediment microcosms
prepared from site 3 sediments (refer to Figure 2.1for sampling location) during 40 weeks
incubations. Dashed line indicates Recovery Total added as DDT. These results were
obtained by liquid scintillation counting of scrapings fiom the TLC plates. Recovery
Total represents the amount of l4C-labelcd radioactivity DDT recovered in the solvent
and aqueous extracts. Percent of Methane produce at time of analysis by GC ................ 48

Figure 3.5. Microbial anaerobic dechlorination of DDT to DDD in sediment microcosms
prepared from site 4 sediments (refer to Figure 2.1 for sampling location) during 40
weeks incubations. Dashed line indicates Recovery Total added as DDT. These results
were obtained by liquid scintillation counting of scrapings fiom the TLC plates.
Recovery Total represents the amount of 14C-labeled radioactivity DDT recovered in the
solvent and aqueous extracts. Percent of Methane produce at time of analysis by GC....49

Figure 3.6. Microbial anaerobic dechlorination of DDT to DDD in sediment microcosms
prepared from site 5 sediments (refer to Figure 2.1 for sampling location) during 40
weeks incubations. Dashed line indicates Recovery Total added as DDT. These results
were obtained by liquid scintillation counting of scrapings from the TLC plates.
Recovery Total represents the amount of ”C-labeled radioactivity DDT recovered in the
solvent and aqueous extracts. Percent of Methane produce at time of analysis by GC....50

Figure 3.7. The fraction of DDD recovered resulting from biotic and abiotic
transformations of DDT in Pine River sediment microcosms. The abiotic fraction was
determined by dividing the DDD recovered in the autoclaved treatments by the DDD
recovered in the live treatments (X 100 %). Similarly the biotic fraction was determined
by dividing the difference of the live and autoclaved treatments by the live treatment (X
100 %). Only sites that had a significant difference between live and autoclaved are
shown ................................................................................................................................. 53

Figure 3.8. Summary of metabolites detected in Pine River sediments and proposed
reactions leading to formation. Transformation marked with X were not observed in
laboratory microcosms containing river sediments and added ”C-DDT, -DDD or DDE.
............................................................................................................................................ 58

ix

CHAPTER 1

INTRODUCTION

Historical Information

In 1874, Othmar Zeidler, a young German chemist at the University of
Strassburg, was the first to synthesize 1,1,1-trichloro-2, 2-bis (p-chlorophenyl) ethane,
commonly known as DDT, and examine its chemical and physical properties. While
working on the synthesis of aromatic compounds through dehydration, he published a
report on a series of compounds resulting from the condensation of chloral with mono-
chlorobenzene (Zimmerman et al., 1946). Unknown to Zeidler, DDT would later save
millions of lives by killing disease carrying insects (Zimmerman et al., 1946);
unfortunately, due to its persistence and toxicity, it would also become a global
environmental problem.

In 1936, a Swiss chemist, Paul Muller, discovered the insecticidal properties of
DDT (Cruz, 1946). DDT was patented as an insecticide by the Geigy Company, Inc. for
commercial production in 1943 in Switzerland, and later by Du Pont, Merck, and
Hercules Powder Co. in the United States (Zimmerman et al., 1946). By the early
months of 1944, the United States Army was using DDT to protect troops against pests.
Specifically, DDT was first used to stop the epidemic of typhus fever in Naples, Italy.
DDT also proved effective for curbing vectors of other diseases such as malaria, yellow

fever and bubonic plague.

Shortly after DDT was used by the military in 1944, the general public began
using it extensively to control many pests. Aside from transmitting diseases, insects
destroy property and cause discomfort. To reduce diseases and to maintain relatively
pest-free homes, DDT was sprayed directly onto beds, walls, and floors, and dusted onto
the body and hairs of humans, livestock and pets.

Between 1946, when the toxicity of DDT to humans was first reported
(Zimmerman et al., 1946), and the 1960s, evidence started surfacing that DDT and its
metabolites were persistent in the environment and 'bioaccumulate in higher animals.
During this period it began to be realized that exposure to DDT threatened the survival of
many species (Heberer and Dunnbier, 1999). Some studies (Stine, et al., 1996) pointed
out that fish and birds of prey suffered most from effects of DDT. DDT and its
metabolites are responsible for causing a decrease in the eggshell thickness of birds
(Korte and Stuttgart, 1992). A direct negative correlation has been found between 2,2-bis
(p-chlorophenyl) 1,1—dichloroethylene (DDE) levels in Alaskan hawks and eggshell
thickness (Cade et al., 1971; Peakall, 1974).

It has been estimated that 1.2 billion pounds of DDT were used in the United
States between 1945 and 1972 (Chem. Eng. News, 1988a). The US. Environmental
Protection Agency (EPA) (EPA, 1988; Stine, etal., 1996) banned DDT in US for
agricultural purposes in 1972 and for all other purposes in 1988. Today, in the United
States, DDT is no longer produced; however, Mexico, China, Russia, and India, the last
known producers of DDT, continue to manufacture it for use against mosquitoes (C&EN,

1998). The World Health Organization (WHO) reports that roughly two million people

die annually from malaria, and therefore, DDT remains in use for controlling mosquito-
bome malaria in many nations in Africa, Asia and Latin America.

DDT is inexpensive to produce and has been accepted as among the best man-
made compounds for controlling potential outbreak of insect-bome diseases. However,
because of its toxicity and environmental effects, many environmental groups have

advocated that DDT be phased out of use worldwide by 2007.

Environmental Fate

Pathways for the degradation of DDT suggest sequential dechlorination of the
three aliphatic chlorine atoms through a combination of two types of dechlorination
reactions: reductive dechlorination and dehydrochlorination. Additionally, the carbon
skeleton of DDT can be degraded through oxidative reactions, presumable during any

stage of dechlorination. The following sections will discuss these general reactions.

Dechlorination Reactions

Reductive dechlorination (or reductive dehalogenation) proceeds by replacing a
chlorine (or halogen) atom with a hydrogen atom (Hill and McCarty, 1967; Morrill et al.,
1982). For DDT and analogues this occurs on the non-aromatic chlorine bearing carbon.
Reductive dechlorination is a reduction reaction as the two-electron transfer reduces the
oxidation-state of the carbon atom by 2 and also adds H to the molecule. This is a
thermodynamically favorable reaction that has been shown to support the growth of a
variety of anaerobic microorganisms through the process of halorespiration (Mohn and

Tiedje, 1992; Wohlfarth and Diekert, 1997; J anssen, Oppentocht and Peolarends, 2001).

Reductive dechlorination typically occurs under anaerobic conditions and is commonly
observed in the degradation pathway of DDT.

Dehydrochlorination involves the simultaneous removal of hydrogen and chlorine
producing a double bond. This reaction typically takes place between a saturated
chlorinated carbon and the adjacent hydrogen on the neighboring carbon (Lal and Saxena
1982). Overall, this is a redox-neutral reaction as the carbon losing the hydrogen atom is
oxidized and the carbon atom losing the chlorine is reduced. Dehydrochlorination is a
common abiotic transformation and also occurs frequently as an aerobic microbial
reaction. The details of this process for abiotic dehydrochlorination of
chlorohydrocarbons are found in Lal and Saxena (1982).

DDT and its partial transformation products are persistent and toxic compounds
that threaten ecosystems and humans. Many studies have examined the fate of DDT in
the environment (Monill et al., 1982; Gibson, 1984; Bitton and Gerba, 1984; Ward et al.,
1985). DDT can be transformed in soils and sediments by reductive dechlorination,
dehydrochlorination, and oxidation through both abiotic and biotic processes. The
relative contribution from the different reactions varies among soils and microorganisms.
The types of transformations of DDT in aerobic and anaerobic environments are
summarized in Figure 1.1.

Microorganisms play a major role in the breakdown of a wide array of pesticides,
including DDT, in environments such as soils, river and lake sediments, and in sewage
sludges. Studies with a variety of pure cultures have shown the microbially mediated
transformation of DDT into several different products (Lal and Saxena 1982). In a

laboratory experiment with sewage sludge, evidence of dechlorination of DDT was also

obtained (Marci et al., 1978 and Ware et al., 1980). Miskus et a1. (1965), and Essac and
Matsumura (1980) also have established that degradation of DDT can be mediated by
iron porphyrins under anaerobic conditions. Despite the potential of microorganism to
transform DDT, it is evident from the literature that DDT often persists in soil and water
for long periods of time. Thus, studies on transformation of DDT in specific well defined
environments remain important in order to understand why DDT persists.

Laboratory studies have shown that anaerobic reductive dechlorination by
microorganism is an important step in DDT degradation. Dechlorination of DDT may
result in products with different biological activities than the parent compound. For
example, the p, p'-isomer of DDT was found to be weakly anti-androgenic while its
major metabolite p, p'-DDE was a strong anti- androgen (Kelce et al., 1995). In the case
of other persistent chlorinated aromatic compounds, anaerobic dechlorination may reduce
toxicity. For example, microbially dechlorinated polychlorinated biphenyls (PCB)
mixtures (Aroclors) showed substantially lower “dioxin-like” activity as compared to
unaltered Aroclors (Quensen et al., 1998). Generally, dechlorination makes the resultant
compounds more easily degradable in aerobic environments. Recently, considerable
interest has developed in utilization of anaerobic reductive dechlorination for
environmental remediation because it has the potential to reduce toxicity and increase the
likelihood of further microbial degradation. The use of sequential anaerobic/ aerobic
microbial processes to degrade PCBs, DDT and their metabolites has often been

considered for contaminated sites.

We (30:0 200 mac-reg; mu
0;? .0951?”
1 11:1" Nmkm Rwy p__1)MU
mm “W on 0W”)

H—C—Cl

”f...

DDNC

H H o
.Q._©..._.010M2.O 0
OJ-.." I ll
_ 3" f— —H H—C— H

DDA 0“
l DDOH DDNU
‘1‘ ’3 i‘
00$O~ moor} -—~ {mom
I
H on o
DPM DBH DBP

Figure 1.1. Metabolic pathway of DDT biotransformation (Adapted from Rochkind and
Blackburn, 1986) with additions of (Jensen et al.1971; Marei et al., 1978; Quensen et a1.
1998). The steps observed most common in DDT the degradation pathway are indicated
by underlined letters, and the step most commonly found in Pine River sediment
microcosms are indicated by a thick arrow. In addition to DDD, DDE and DDMU, there
are several other degradation products that are less commonly observed.

Table 1.1. Abbreviations, chemical formulae and names of DDT and its degradation

products given in the text.

DDT= 1,1,1-trichloro-2, 2-bis (p-chlorophenyl) ethane;
DDD= 1,1-dichloro-2, 2-bis (p-chlorophenyl) ethane
DDMU= 1-chloro-2, 2-bis (p-chlorophenyl) ethylene;
DDMS= 1—chloro-2, 2-bis (p-chlorophenyl) ethane;
DDNU= 1,1-bis (p-chlorophenyl) ethylene;

DDOH= 2,2-bis (p-chlorophenyl) ethanol;

DDA= dichlorodiphenylacetate, or bis (p-chlorophenyl) acetic acid;
DBH= dichlorobenzhydrol;

DBP= dichlorobenzophenone;

DDE= 2,2-bis (p-chlorophenyl) 1,1-dichloroethylene
DDNC= bis (p-chlorophenyl)-acetonit1i1e

DPM= p, p'-dichlordiphenylmethane

Oxidative Reactions

In addition to dechlorination reactions, the carbon structure of DDT and its
metabolites can be degraded through oxidative reactions. DDT is degraded by a variety
of soil microorganisms in aerobic environments. Focht and Alexander (1970) reported
that certain microorganisms have the ability to cometabolize DDT and its metabolites.
The term “cometabolism” refers to the ability of a microorganism to metabolize a
substrate that it cannot use as a source of energy (Alexander et al., 1967). Currently,
there is no evidence that DDT can be used as a carbon source for growth by microbes in
the degradation of DDT.

In pure culture studies, Hay and Focht (1998) have proposed that Pseudomonas
acidovorans M3GY cometabolically transformed DDE and its unchlorinated analog, 1,1-
diphenylethylene (DPE) (Figure 1.2). DDE was cometabolized by a dioxygenase at the
ortho and meta positions. Megharaj et al. (1997) reported transformation of DDT by pure
cultures of 4,4'-dichlorobiphenyl and DPE-degrading bacteria through oxidative
cometabolism. DDE is resistant to reductive cometabolism by pure obligately anaerobic
microorganism (Strompl and Thiele, 1997; Megharaj et al., 1997). In general,
cometabolic processes are slow, and the transformations of DDT are controlled by

environmental conditions (Alexander et al., 1967).

com (IO-cw
6

Figure 1.2. Cometabolic, meta-fission products of DDE as proposed for biphenyl-grown
Pseudomonas acidovorans M3 GY (Adapted from Hay and Focht, 1998).

(1) DDE; (2) 1,l-dichloro-2-(dihydroxy-4- chlorophenyl )-2-(4-chlorophenyl)ethylene;
(4) 4, 6-oxo-2-hydrooxy-7-(4-chlorophenyl)-4,8,8-trichloroocta-2,4-dienoic acid; (5) 5,
2-(4- chlorophenyl)-3,3-dichloropropenoic acid; (6) 6, 4-chlorophenylacetic acid; (7) 7,
4-chloroacetophenone; (8) 8, 4-chlorobenzaldehyde; (9) 9, 4-chlorobenzoic acid.

Compounds detected at DDT contaminated sites

Several studies have proposed pathways for the degradation of DDT, and Figure
1.1 represents a generalized scheme (Rochkind and Black 1986). The degradation
reactions are reductive dechlorination, dehydrochlorination and oxidation. DDT can be
degraded under both aerobic and anaerobic conditions and it degradation can be catalyzed
by either microbial or abiotic reactions. The sequential order of these reactions
determines the degradation pathway.

An initial reductive dechlorination of DDT produces 1,1-dichloro-2, 2-bis (p-
chlorophenyl) ethane (DDD). This is a commonly observed reaction by both abiotic and
anaerobic microbial systems. An initial dehydrochlorination of DDT produces DDE.
This is most commonly observed as an aerobic microbially mediated process.
Traditionally DDE has been considered a dead-end product. Recently, however, Quensen
et al. (1998) showed that DDE is further converted to 1-chloro-2, 2—bis (p-chlorophenyl)
ethylene (DDMU) by a reductive dechlorination reaction. DDD has been shown to also
be converted to DDMU by a dehydrochlorination reaction (Wedemeyer et al., 1967).
Further reduction of DDMU has not been studied in as much detail, but is believed to
form 1,1-bis (p-chlorophenyl) ethylene (DDNU) either directly from a reductive
dechlorination reaction, or by a dehydrochlorination reaction following a hydrogenation
reaction. DDNU has a completely dechlorinated aliphatic carbon and further degradation
proceeds through sequential oxidation of this carbon to form 2,2-bis (p-chlorophenyl)
ethanol (DDOH), then bis (p-chlorophenyl) acetic acid (DDA), before decarboxylation to
form p, p'-dichlordiphenyhnethane (DPM). There is evidence that DPM may also be

oxidized to dichlorobenzhydrol (DBH) and then dichlorobenzophenone (DBP) (Perry et

10

al., 1963 and Pinto et al., 1965). The complete mineralization of DDT beyond this point
has also been suggested (Hay and F ocht, 1998).

Specific Transformations

DDT to DDD There have been two central studies describing research on DDT
transformations in sediments (Matsumura etal., 1971; Jensen etal., 1972). Matsumura et
al. (1971) examined the fate of DDT in Lake Michigan sediments, and observed that
DDT was reductively dechlorinated to DDD (Figure 1.3). This is consistent with other
studies of isolated bacteria (and other microorganisms), as well as in anaerobic soil
conditions, where the major pathway of DDT involves reductive dechlorination (Guenzi,
and Beard 1967; Johnsen, 1976; Essac and Matsummura, 1980; Lal and Sexena, 1982;
Rochkind and Blackburn, 1986). Jensen et al. (1972) showed that DDT was rapidly
transformed to DDD within several hours of incubation in anaerobic sewage sludge and
lake sediments. Often DDD is the first and sole transformation product of DDT under
anaerobic conditions (Figure 1.3). The extent of this transformation can be significant;
Wedemeyer (1967) and Guenzi et al. (1967) showed that less than 1% of original DDT
remained in the soil afier 12 weeks of incubation. In the reductive dechlorination of DDT
to DDD, DDT accepts 2 electrons and the oxidation state of the aliphatic carbon atom
changes from +3 to +1, while the oxidation state of all other atoms remain the same

(Guenzi and Beard, 1968; Parr et al., 1970; Parr and Smith, 1974).

11

+Cl

 

Figure 1.3 Proposed pathway for reductive dechlorination reaction of DDT
(Schwarzenbach et al., 1993) in anaerobic systems. The oxidation state for aliphatic
carbon atom for each compound is shown in parenthesis.

In addition to microbially mediated reactions, the addition of some chemical
agents (e. g., Fe (II) porphyrin, heamatin, detergent) in anaerobic systems may accelerate
the transformation of DDT to DDD (Quirke et al., 1979). It has been shown that the
extent of DDT degradation to DDD is negatively correlated with redox potential (Burge,
1971; Guenzi et al., 1971; Parr and Smith, 1974). Furthermore, Glass (1972) showed that
DDD formation is related to the levels of ferrous (Fey) iron present in the anaerobic
environment.

DDT to DDE DDT can be transformed to DDE by microorganisms through
dehydrochlorination (Figure 1.4). Dehydrochlorination is the dominant pathway by
which microorganisms degrade DDT in aerobic environments. Aerobic
dehydrochlorination of DDT is a process whereby hydrogen and chlorine are removed
simultaneously fi'om the aliphatic side-chain portion of the molecule, reducing the

number of chlorines and generating a carbon-carbon double bond.

12

(+2)
C1 C1

+ 110‘ ——+ ‘0’ + H20 + Cl
CI Cl Cl
DDE

 

DDT

Figure 1.4. Proposed pathway for dehydrochlorination of DDT in aerobic environments
(Schwarzenbach et al., 1993).

A number of studies have shown that microorganisms can dehydrochlorinate
DDT to DDE (Ott and Gunther, 1965; O’Brien, 1967; Menzie, 1969; and Metcalf, 1971).
This is consistent with other studies in aerobic soil conditions, where DDE is the primary
metabolite of DDT transformations (Wedemeyer,1967), but other compounds such as
dichlorobenzophenone (DBP) and DDMU have been reported (Marci et al.,l97 8). Quirke
et al., (1979) observed that the transformation of DDT to DDE involves a reduction and
subsequently oxidative step via a free radical mechanism. As discussed below, DDT can

be transformed to DDE, and subsequently to DDMU (Quensen et al., 1998).

DDD to DDMU Several studies examined the fate of DDD and proposed a pathway for
degadation of DDD (Quirke et al., 1979; Quensen et al., 1998). DDD can be
transformed to DDMU by microbes in aerobic and anaerobic environments. Aerobic
dehydrochlorination of aliphatic side carbon is the main reaction for the degradation of
DDD, however, this reaction is typically slow (Engst and Kujawa, 1967; Quirke et al.,
1979). Quirke et al. (1979) have proposed that DDD can be dehydrochlorinated to

DDMU via a free radical process involving a hydrogen radical. Pereira et al (1996) also

13

described finding DDMU in environmental samples. Evidence of anaerobic degradation
of DDD has previously been established for microbial cultures (Wedemeyer, 1967). The
findings indicated that DDD was converted to p, p' dichlorobenzophenone (DBP) via

DDMU and bis (p-chlorophenyl) acetic acid (DDA).

DDE to DDMU Traditionally, DDE, a toxic breakdown product, has been viewed a
dead-end metabolite in the environment (Spencer et al., 1996; Hay and Focht, 1998).
DDE is extremely persistent in the environment, and degradation is largely controlled by
the environmental conditions. Despite the persistence of DDE in soils, studies with a
variety of pure cultures have shown degradation of DDE by microbes (Hay and F ocht,
1998). In sediments, the presence of DDMU among the anaerobic degradation products
of DDT has been attributed to reductive dechlorination of DDE (Quensen et al., 1998).
Laboratory assays have shown that DDE can be reductively dechlorinated to DDMU by

microbes under methanogenic and sulfidogenic conditions (Quensen et al., 1998).

DDMU and beyond In addition to DDD and DDE, intermediate compounds such as
DDMU, l-chloro-2, 2-bis (p-chlorophenyl) ethane (DDMS), DDNU, DDA, and DBP can
occur from anaerobic degradation of DDT. There is an evidence for the degradation of
DDT beyond DDMU. Jensen et a1. (1971) proposed that bis (p-chlorophenyl)-
acetonitrile (DDNC) is formed fiom DDT by reduced hematin and ammonia.
Furthermore, it has been shown that DDNC can be oxidized to DBP in aerobic

environments by dehydrochlorination reactions (Marei et al., 1978; Quirke et al., 1979).

14

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18

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20

CHAPTER 2

INTRODUCTION

Sampling and Chemical Analysis of Pine River sediments

Between 1936 and 1978, Velsicol Chemical Corporation, formerly Michigan
Chemical, manufactured a wide array of compounds such as 1,1,1-trichloro-2, 2-bis (p'-
chlorophenyl) ethane (DDT), polybrominated biphenyls (PBBs), hexabromobenzene
(HBBs), and tris (2,3-dibromopropy1) phosphate (Tris). These synthetic compounds
entered Pine River sediments through the discharge of wastewater and sludge. The
Velsicol Chemical manufacturing site (“Site”) is an approximately 50-acre parcel located
on the Pine River. The Site has been viewed as a threat to general public health, welfare,
and the environment. The Site, including the adjoining Pine River, has been subject to
considerable attention from regulatory agencies and ultimately did not withstand
regulatory scrutiny for PBBs and DDT. The State of Michigan and US. Environmental
Protection Agency (U .8. EPA) closed the Velsicol Chemical facility in 1976 after forty
years of intense activities with production of 11 million pounds of PBBs. This action
resulted from contamination of dairy cattle throughout Michigan by PBB- tainted feed
and operating violations of the company’s landfill in 1974. The former incident occurred
when Velsicol mistakenly shipped PBB rather then the feed additive magnesium oxide
(MgO) to operators.

As part of a five-year remediation plan, the Velsicol Chemical Company and the

State of Michigan began work to treat the contaminated site by dismantling the

21

manufacturing facility, and placing a slurry wall around it and a five-foot thick clay cap
over it to stabilize contaminants. However, in negotiations between Velsicol and the
State of Michigan regarding clean up, a no action alternative regarding the contaminated
sediments of the Pine River was agreed to. The idea was that with time the contaminated
sediments would become physically isolated by the deposition of non-contaminated
sediments. A no consumption advisory was established for fish in the Pine River (St.
Louis) to its confluence with the Chippewa River (a 20 mile stretch) in 1977 and by
1982, the site was identified as a Superfimd site at the National Level (F orba et al., 1982)

For the past three decades EPA has monitored contamination of the river by DDT,
PBB and other chemicals. This began in the early 1980’s when Forba et al. (1980 and
1982) reported that sediments in Pine River were heavily contaminated with DDT,
petroleum products and brominated compounds such as HBBs and PBBs. Analysis of
sediments in the Pine River reservoir adjoining the Velsicol manufacturing site indicated
that the average concentration of DDT was 3,500 ug/g (ppm) and the maximum
concentration detected was 44,000 ug/g (F orba et al., 1982).

Analysis of fish in Pine River showed that DDT levels were extremely high in
1985 and exceeded the Federal Department of Agriculture (FDA) tolerance level of 5
ppm for DDT (Forba et al., 1982). Subsequent analysis of DDT levels in fish (e.g. carp
and black crappie) taken in 1989, 1994, 1995 and 1997 from the Pine River within and
below the St. Louis impoundment show that concentrations have remained high. During
this period the average DDT concentrations in fish appear to have increased about 2X but
the statistical significance of this is difficult to establish because of the wide variability of

concentrations and limited number of fish taken. The average DDT concentration in the

22

1995 samples was highest at 15.6 ug/g, but concentrations of up to 200 ug/g were
observed in individual fish. These findings indicate that the no action alternative taken
for sediment remediation in this region of the river had failed. The high levels of DDT in
fish tissue indicate continual exposure of the fish to contaminated sediments within the
St. Louis impoundment. Concern for human exposure to DDT via consumption of
contaminated fish has driven a $ 40,000,000 Superfund clean-up which is currently
underway.

In 1998 the U.S.EPA approved a dredging plan for the removal of the
contaminated sediments fi'om the impoundment. The first stage of this intensive cleanup
program was to install sheet piling around the most contaminated sediments near the old
plant outfall. The overlying water was then pumped out and the sediments were
solidified then excavated down to the underlying peat. The excavated sediments were
dewatered before they were landfilled offsite. The water from the sediments was treated
on site before being returned to the river. The process has now been expanded to the rest
of the impoundment and should be complete by the end of 2001.

In this study, we measured the total concentration of DDT and related compounds
in sediments from Pine River Reservoir. Similar to previous analyses, we found that
sediments of the Pine River in close vicinity to former manufacturing site contain high
concentrations of p, p'-DDT, o, p'-DDT, p, p'-DDE, o, p'-DDE, p, p'-DDD, o, p'-DDD,
and p, p'-DDMU (collectively referred to as DDX). Of the DDX compounds present in
Pine River sediments, the components comprising the greatest amounts of DDX were

DDT and DDD while DDE and DDMU represented smaller portions.

23

MATERIALS AND METHODS
Sampling and Chemical Analysis

DDT contaminated sediments were collected in September 1998 from the Pine
River (PR) at Velsicol Chemical Company Superfilnd Site in St. Louis, Michigan.
Sections of polyvinyl chloride pipes with inner diameter of 7.62 cm and approximately
90 cm in length were used as sampling devices for withdrawing sediments. Pre-drilled
rubber stoppers fitted with a rubber flapper were inserted at one end of each pipe for
releasing water and gas as the pipes were pushed into sediments to a depth of
approximately 90 cm. The pipes were withdrawn, capped and sealed with tape to reduce
exposure to oxygen, and the cores were then transported to the laboratory and stored at
4°C. Samples were taken in triplicate from five locations.

Prior to analysis, the sediments were extruded from the PVC pipes, air-dried at
room temperature for 3 to 5 days, and passed through a 2 mm sieve. Sediments from
each core were homogenized over depth and duplicate ten-gram samples were extracted
with 250 ml of hexane and acetone (1:1, vol: vol) in a Soxhlet apparatus for 24 hours.
The solvent extract was then transferred to a separatory firnnel and back-extracted with
approximately 50 mls of 2% aqueous NaCl. After draining off the aqueous portion, 2 to
4 mls of concentrated sulfuric acid (HzSO4) was added to the remaining hexane extract,
and the whole was shaken by hand then allowed to separate. The acid was drained off
and the hexane residues were then washed 2-3 times with 25 mls of 2% NaCl solution to
remove residual acidity. The hexane extract was then filtered through anhydrous Na2804

to remove water. To remove polar compounds and sulfur, the sample was eluted through

24

a 160 mm sample clean-up column (Supelco, Bellefonte, PA Cat. No. 58099) packed in
layers with 5 mm anhydrous sodium sulfate, 60mm of a F lorisil-acid rinsed copper
powder mixture (4:1 ratio, vol: vol), and 5 mm more of sodium sulfate.

The solvent extracts were then analyzed for DDT analogs (p, p'-DDT, o, p'-DDT,
p, p'-DDE, o, p'-DDE, p, p'-DDD, o, p'-DDD, and p, p'-DDMU) by high-resolution gas
chromatography. The retention times of authentic standards were matched to the DDX
components in the extracts. An internal standard (octachloronapthalene) method was
used to quantify the amount of DDX in each sample. The analysis was performed on a
HP 5890 gas chromatograph equipped with an electron capture detector and a capillary
column (HP Ultra 2, 50m long 0.22 mm id and 0.33 um film thickness). The inlet
temperature was 220° C, and the detector temperature was 325° C. The column
temperature program began at 140 ° C for 1min and was increased to 240 ° C at the rate of
2 ° C/min, then held at 240 ° C for 4 min. A Sul sample was injected using a split ratio of

approximately 10:1.

25

RESULTS AND DISSCUSION

Site Description

Sediments from five locations in the Pine River between the Washington Street
and Mill Street bridges (Figure 2.1) were analyzed for DDX components. Sediments at
the five sites contained varying concentrations of DDX. The highest concentrations of
DDX were found in the middle basin (at Site 2) between the industrial site jetty and the
Mill Street bridge (Figure 2.1), with the total concentration of DDX in sediments being
~20,000 ug/g (Table 2.1). The high levels of DDX were not surprising in this region of
the river because it lies adjacent to and slightly downstream from the former Vesicol
Chemical Company manufacturing site. Previously, Forba (1980) reported a maximum
DDX concentration of 44,000 ug/g in sediments collected from this region of the Pine
River. Similar results by National Enforcement Investigations Center (NEIC) and the
EPA (1997) indicated that maximum concentrations of total DDX were 26,000 ug/g and

44,000 ug/ g, respectively for this portion of reservoir, consistent with our results.

26

 

 

Pine River
St. Louis, Michigan

 

 

 

 

 
 

E'
E
Jetty
Impoundment .
Site
\ - Dam
Former Velsicol i
Plant Site ‘

Xshington St.

Ly State St.

Figure 2.1. Map of sampling locations at the Pine River.

 

 

 

 

The five sites sampled grouped naturally into one site with high (site 2), two sites
with medium (sites 1 and 5), and two sites with low (sites 3, 4) levels of DDX (Table
2.1). The total concentration of DDX was obtained by summing the concentrations of o,
p' and p, p'-isomers of all DDX compounds for each sediment. Total concentrations of

DDX for sediment collected near the Mill Street bridge and across from the Velsicol

Plant site (site 1 and site 5, Figure 2.1) were intermediate among the samples taken with
DDX concentrations of approximately 13 ug/g for site 5 and 5.5 ug/g for site 1. In the
upper basin (sites 3 and 4), located upstream of the Velsicol Plant site, the DDX levels
were relatively low at approximately 0.5 ug/g. DDX levels were high (approximately
20,000 ug/g) at site 2, located adjacent to the former Velsicol manufacturing site. The
field study indicated differences in total concentrations of DDX as well as the relative
distribution of p, p'- and o, p' - DDX isomers at the five sediment sampling location
(Table 2.2). p, p'-DDT was the predominant isomer at site 1 whereas for other sites a
more even distribution of the four DDT and DDD isomers was observed. In general,

concentrations of DDE and DDMU were considerably less that those of DDT and DDD.

Table 2.1. Mean concentrations (pg/g) of DDT and metabolites and total DDX in
sediments collected from the Pine River. Values, in ugrams/grams (calculated from
measurements of estimated concentration in sample extract), are the average of two

 

 

 

replicates. The component percentage of the total DDX concentration is shown in
parentheses.
Mean Concentration (pg/g)
Analyte . Site 1 Site 2 Site 3 Site 4 Site 5
p, p'-DDT 4.29 (77.4) 7020 (35.1) 0.04 (27.8) 0.10 (25.9) 5.43 (42.0)
o, p'-DDT 0.20 (3.61) 5380 (26.9) 0.04 (27.1) 0.06 (15.06) 0.59 (4.54)
p, p'-DDD 0.05 (0.96) 2910 (14.5) 0.03 (18.2) 0.11 (27.8) 2.89 (22.4)
o, p'-DDD 0.01 (0.12) 3930 (19.7) 0.03 (18.4) 0.10 (26.01) 1.99 (15.43)
p, p'-DDE 0.42 (7.52) 301 (1.51) 0.01 (5.5) 0.01 (1.83) 1.0 (4.75)
o, p'-DDE 0.06 (1.17) 200 (1.0) 0.001 (0.7) 0.001 (0.43) 0.39 (3.05)
p, p'-DDMU 0.51 (9.23) 252 (1.3) 0.003 (2.4) 0.01 (2.95) 1.01 (7.82)
Total DDX 5.54 19993 0.154 0.391 13.3

 

 

28

In general technical grade DDT is composed of several compounds listed in
(Table 2.2) and the mixture typically contains from 73% to 77% p, p'- DDT and up to
20% o, p'-DDT (West and Campbell, 1950). The exact composition of DDT products
that were originally discharged into the Pine River reservoir is unknown. It is likely,
however, that the DDT composition was relatively similar to technical grade DDT. The
DDT analogs p, p'-DDT, o, p'-DDT, p, p'-DDE, o, p'-DDE, p, p'-DDD, o, p'-DDD, and p,
p'-DDMU were detected in all sediments sampled. The presence of these compounds
may be due to the original mixtures of DDT that were discharged into the river or the
result of microbial and chemical transformation of DDT. Of the DDX compounds found
in PR sediments, DDT and DDD were consistently higher than other components. DDE
and DDMU were detected in sediments at comparatively low levels; these compounds are
not normally detectable in technical DDT mixtures (West and Campbell, 1950). Because
it is unknown what exact DDT mixtures were discharged into the PR, and because the
composition of the DDT products may vary over time, it remains uncertain whether the
presence of DDE and DDMU at low concentrations is an indication of DDT
transformation. However, these are known environmental transformation products of

DDT so this is their likely origin in the Pine River Sediments.

29

Table 2.2. Percent of Total DDT and metabolites in Technical DDT (Adapted

from West and Campbell, 1950).

 

Composition of Technical DDT

 

 

 

Compound Constitution (%) of sample of given setting point

886°C 91 .2°C 91 .4°C
p, p'-DDT 70.5 66.7 72.7
o, p'-DDT 20.9 19.0 11.9
o, o'-DDT - - 0.01
p, p'-DDD 4.0 0.3 0.17
0, P'-DDD - - 0.04
2,2,2,-Trichloro-1-o- 1.8 0.4 0.57
chlorophenyl ethyl-p-

chlorobenzenesulfonate

l-p-Chlorophenyl-Z, 2, 2- - 0.2 -
trichloroethanol

1 ,1 ,1 ,2-tetrachloro-2-p- - _ _
chlorophenylethane

Bis (p-chlorophenyl)- 0.1 0.6 0.03
sulfone

o-chlorophenyl 0.007 - -
chloroacetamide

p-chlorophenyl 0.01 - 0.006
chloroacetamide

Sodium p- - 0.02 -
chlorobenzenesulfonate

Ammonum p- - - 0.005
chlorobenzenesulfonate

 

30

In examining the DDX component distributions (Table 2.1), it is apparent that site
1 has a much higher percent component of p, p'-DDT as compared to the other four sites.
Interestingly, the p, p'-DDT component percentage (of total DDX) at sitel (77.4%) is
similar to the typical composition of technical grade DDT. Thus, among the sites
evaluated, the DDX composition at site 1 may be most representative (1. e. least
transformed) of what was originally discharged. However, the component percentage of
o, p'-DDT at site 1 (3.6%) is considerably lower than that typically found in technical
grade DDT. In the other four sites the p, p'-DDT component percentage ranges from 26
to 42% of total DDX. This is considerably lower than would typically be found in
technical DDT indicating significant transfonnation of p, p'-DDT. Transformation of p,
p'-DDT presumably yields predominately p, p'-DDD. In fact, for sites 2-5 the component
percentage of p, p'-DDD are significantly elevated compared to that found in technical
DDT. For sites 2-4 the component percentage of 0, p'-DDT are similar to those found in
technical DDT whereas for sites 1 and 5 they are significantly lower. The component
percentages of o, p'-DDD appear to be elevated, especially for sites 2-5, as compared to
technical DDT. Overall, this presents somewhat confusing picture regarding the
composition of technical DDT originally discharged into the river, and regarding what
environmental transformations may have occurred. It does seem clear however that a
significant amount of p, p'-DDT has been transformed into p, p'-DDD in four of the five
sites assessed.

In a further attempt to identify differences in DDX component distributions
among sites, the components were grouped according to p, p'- versus 0, p'- substitution

(Table 2.3 and Figure 2.2). Again, the major component at site 1 is DDT and there is

31

surprisingly little DDD. This is true for both p, p'- and 0, p'-substitution. At the other
four sites, the component percentages of DDT are lower and those of DDD are higher.
This indicates significant environmental transformation of DDT to DDD for both p, p'-
and o, p'-substitution. Significant component percentages of DDE are also observed at
each site. Since DDE is not typically found in technical DDT products, its presence
likely originates from in-situ transformation of DDT. Both p, p'-DDE and o, p'-DDE are
present at somewhat similar component percentages “indicating no obvious preference for

transformation of the p, p'- versus 0, p'-isomers.

Table 2.3. Mass concentrations (11g /g) and percentage of DDT and its derivatives in
sediments collected from the Pine River. Values, in ugrams/grarns (calculated from
measurements of estimated concentration in sample extract), are means of two replicates
except for three replicates from site 4 sediments. The component percentage of the total
concentration is shown in parentheses.

 

p, p' - Substituted Compound, Mean Concentration (pg/g)

 

 

 

Compound Site 1 Site 2 Site 3 Site 4. Site 5
DDT 4.29 (81.40) 7020 (66.97) 0.04 (48.19) 0.10 (43.48) 5.43 (54.63)
DDD 0.05 (0.95) 2910 (27.76) 0.03 (36.14) 0.11 (47.83) 2.89 (29.07)
DDE 0.42 (7.97) 301 (2.87) 0.01 (12.05) 0.01 (4.35) 1.00 (6.14)
DDMU 0.51 (9.70) 252 (2.40) 0.003 (3.61) 0.01 (4.35) 1.01 (10.16)
Total 5.27 10483 0.083 0.23 10.33

 

o, p' - Substituted Compound, Mean Concentration (pg/g)

 

 

DDT 0.20 (74.07) 5380 (56.57) 0.04 (56.34) 0.06 (37.27) 0.59 (19.87)
DDD 0.01 (3.70) 3930 (41.32) 0.03 (42.25) 0.10 (62.11) 1.99 (67.00)
DDE 0.06 (22.22) 200 (2.10) 0.001 (1.41) 0.001 (0.62) 0.39 (13.13)
Total 0.27 9510 0.071 0.161 2.97

 

32

% of DDX
Components

 

; 100.
l E 80.
l X
n “’ .
t D g 60
"6 E 40.
,\° 6
o 20.

 

 

[.Lrggmmnr.

Figure 2.2. Percentage of DDX component distributions among sites, the
components were grouped according to p, p'- versus 0, p'- substitution.

33

REFERENCES

EPA. 1997. Ecological Risk Assessment Guidance for Superfund: Process for Designing
and Conducting Ecological Risk Assessments, Interim Final. Environmental Response
Team, Edison, NJ, EPA 540-R-97-006.

Forba, R. W. US. EPA Pine River Contamination Survey, St. Louis, MI, EPA-330/2-80-
031, 1980.

F orba, R. W. US. EPA Summary of Pine River Reservoir Sediment Sampling Survey, St.
Louis, MI, EPA-330/2-82-001, 1982.

Morris, P.J.; Quensen, J.F., III; Tiedje, J.M., and Boyd, SA. 1993. An Assessment of
the Reductive Debromination of Polybrominated Biphenyls in the Pine River Reservoir.
Environ. Sci. Technol. 27: 1 580-1586.

Quensen, J. F ., III, J. M. Tiedje, and S. A. Boyd. 1988. Reductive dechlorination of
polychlorinated biphenyls by anaerobic microorganisms fiom sediments. Science 242:
752-754.

Quensen, J. F., 111, S. A. Boyd and J. M. Tiedje. 1990. Dechlorination of four
commercial Aroclors by anaerobic microorganisms from sediments. Appl. Environ.
Microbiol. 56:2360-2369.

Quensen, J. F., III, Mueller, S. A.; Jain, M. K.; Tiedje, J. M. 1998. Science. 280: 722-
724.

Quirke, J. M. E.; Marei, A. S. M.; Eglinton, G. 1979. The Degradation of DDT and its
degradative products by reduced iron (1H) porphyrins and ammonia. 3:151-155.

Rochkind, M. L., and Blackburn. 1986. Microbial decomposition of chlorinated aromatic
compounds. EPA/600/2-86/090 p.138-145.

Shelton, D. R., and Tiedje, J. M. 1984. General method for determining anaerobic
biodegradation potential. Appl. Environ. Microbiol. 47:850-857.

West, T.F. and Campbell, G.A., DDT and Newer Persistent Insecticides, revised 2“d ed.,
Chapman and Hall, London, 1950.

34

CHAPTER 3
INTRODUCTION

From analysis of DDX concentrations in environmental samples, such as data
presented in chapter 2, it is ofien difficult to decipher degradation pathways
unequivocally. Laboratory studies using proposed intermediates, preferably radio-
labeled, as substrates are needed to clarify specific degradation pathways. In chapter 2,
several DDT analogs were quantified in Pine River sediments, mainly DDD, DDE and to
a lesser extent DDMU. Although these intermediates are clearly present, several
questions remain including whether DDD is produced chemically or biologically, and
whether DDMU is being formed from DDT via DDD, as is traditionally believed, or via
DDE as was recently shown by Quensen et a1. (1998) in marine sediments. It is also
unclear whether DDMU, or any other metabolites, are dead-end products.

OBJECTIVE

In this study we examined the fate of DDX in Pine River sediments contaminated
with DDT ranging from 0.05 to 20,000ug/g sediment. We compared the extent of
transformations over a 40 week period. The objective of this study was to determine the

pathway of DDT degradation and distinguish between abiotic and microbial processes.

35

MATERIALS AND METHODS
Materials
Chemicals 1,1,1-trichloro-2, 2-bis (p-chlorophenyl) ethane (99% purity); 1,1-dichloro-
2, 2-bis (p-chlorophenyl) ethane (99%pu1ity) ; 2,2-bis (p-chlorophenyl) 1,1-
dichloroethylene (99.9% purity) were obtained fi'om Ultra Scientific, Co., North

Kingstown, RI. ”(z—DDT (12.7 mCi/mmol, 97% radiochemical purity) ”(z-DDD (2.6
mCi/mmol, 100 % radiochemical purity) and MC-DDE (13 mCi/mmol, 97.7%

radiochemical purity) were ring labelled and provided by the Sigma Chemical Co. St.

Louis, MO. The chemical purity for l4c—DDT, DDD and DDE were 97, 99.4 and 98.2%

respectively.
Sediments Sampling and Analyses

Three sediment cores from each of five sites were collected in September 1998
from the Pine River (PR) at the Velsicol Chemical Company Superfund Site in St. Louis,
Michigan (Figure 2.1). Sections of polyvinyl chloride pipe with inner diameter of 7.62
cm and approximately 90 cm in length were used as sampling devices for withdrawing
sediments. Pre-drilled rubber stoppers with an attached rubber flapper were placed at one
end of the pipes for releasing water and gas as the pipes were inserted (by hand) into the
sediment as deep as possible (up to 90 cm). After withdrawal, the pipes were capped and
sealed with tape to reduce the exposure of oxygen, and cores (in vertical positions) were
then transported to the laboratory. The samples were stored at 4°C until they were used

in this study.

36

Methods

Sediment Slurry Preparation

Five sediment samples (one fiom each site) were selected from the fifteen
sediment cores for further study. Sediments were extruded from the PVC pipes then
homogenized by mixing with an equal volume of Reduced Anaerobic Mineral Medium
(RAMM) as described by Quensen et al. (1998). The RAMM was prepared as previously
described in the general method for determining anaerobic biodegradation potential
(Shelton and Tiedje, 1984). The autoclaved RAMM (600 ml) was added to a 2000 ml
Erlenmeyer flask that had been pre-flushed with oxygen-flee nitrogen and carbon dioxide
(80:20, vol/vol). While continuously sparging with the N2/COz gas mixture, an equal
amount of contaminated sediments was then added to the flask with a sterile spatula and
mixed on a magnetic stirrer.

Microcosms Setup

l4C-radiolabelled-DDT, DDD and DDE were used as substrates separate

dehalogenation assays. A mixture containing 2.8, 12.3 or 2.4 mg of '4C- DDT, DDD or
DDE, respectively, and also including 54.2, 44.7 or 54.6 mg of unlabelled-DDT, DDD or
DDE was diluted with acetone to 2 ml. This gave a solution of each compound with a
concentration of 28.5 rig/111 and an activity of approximately 1.07X105 (1an 111, as
verified by liquid scintillation counting (Beclonan LS 6500). A volume of 7 mls of a
sediment slurry (containing about 2 g of sediments on a dry weight basis) was placed in

each sample vial (20ml) with a sterile Pasteur pipette. The entire contents of each vial

were spiked with 7111 14C-labeled DDT, DDD or DDE. This gave an activity of

37

approximately 7.5X105 dpm per microcosm and a DDX concentration of approximately

100ug/ g sediment (dry weight). The microcosms were then stored in the dark at room
temperature, approximately 25°C. Both biologically active and sterile (abiotic) samples

were set up in parallel. The abiotic treatments were autoclaved on three consecutive days
for one hour prior to addition of l4C-labeled compounds to vials. Four replicate samples

were sacrificed and evaluated after 0, 4, 8, 12, 16, 24 and 40 week intervals for live, and

0, 8, 16, and 40 week intervals for abiotic treatments.
Methane Analysis

After predetermined periods of incubation, sediment microcosms were analyzed
for methane using a gas chromatograph fitted with a flame ionization detector. Methane
production in headspace of anaerobic sediment microcosms was used to establish the
existence of anaerobic conditions, and as a relative index or the biologically activity of
each microcosm. Methane activity was determined as the percent of headspace gas in
microcosms. The mean of three or four samples at each time interval was considered as
one observation.

Dechlorination Assays (Extraction and Analysis)

After incubation for various time intervals, the entire contents of each sediment
microcosm were extracted three times by shaking for 10 minutes with 7 mls of a
petroleum ether-acetone mixture (5:2, vol: vol). The solvent extracts were combined and
evaporated to a final volume of 0.5 mls under a stream of dry nitrogen. Samples of the

reduced volume extracts (20111) were spotted on activated silica gel plates that were

38

subsequently developed to 15 cm with a petroleum ether-hexane mixture (5:95) in a lined
thin layer chromatography (TLC) chamber at room temperature. Autoradiography was
used to determine the locations of the parent compound and metabolites on the TLC
plates. Kodak Scientific Imaging Film (X-OMAT AR) was exposed to the TLC plates
for 7 days at minus 20° C and then developed. Autoradiography films and TLC plates
were aligned on a back-lighted box, and the parent compound and metabolite zones were
visualized and marked for scraping. These zones corresponded to DDT, DDE, DDD,
DDMU, and the origin where polar metabolites would remain. The l4C-labeled activity
in the scraping was determined by liquid scintillation counting.

The results for DDT degradation and dechlorination are presented as the percent

of 14C recovered. The results for DDT degradation were determined by dividing the

amount of 14C-DDT recovered by the amount of l4C-DDT added (X100) to get the

percent of total recovery. The percent of radioactivity recovered for each compound,

thus, was not normalized to total radioactivity recovered.

39

RESULTS AND DISSCUSION

DDT Transformations

The Pine River sediments in St. Louis, Michigan are heavily contaminated with
DDT, polybrominated biphenyls (PBB), hexabromobenze (I-IBB), petroleum, chlordane,
and heavy metals. Previous studies on Pine River sediments sought to determine the
degradation potential for PBBs at this site. Morris et al. (1993) found evidence for
limited in situ anaerobic biodegradation of PBBs based on the distribution of congener
patterns in sediment. Morris et a1. (1993) indicated that the Pine River sediments
partially debrominated the commercial PBB mixture F iremaster BP6 by removal of meta
and para bromide. Although microorganisms capable of PBB debromination were found
in the sediments, high concentrations of co-contaminants were believed to inhibit in situ
debromination.

Previous studies on the Pine River Reservoir revealed the presence of DDT in
high concentrations (up to 40,000 ppm) (Forba et a1. 1980; 1982). In addition to
detecting up to 20,000 ppm DDT, our sediment analysis also identified the proposed
DDT metabolites DDD, DDE, and DDMU (Chapter 2). Quensen et al. (1998) recently
demonstrated DDMU formation from the reductive dechlorination of DDE. This
conclusion differed from previous studies that have reported DDE as a dead end product
from DDT biodegradation, and DDMU formation from DDD via dehydrochlorination
(Quirke et al., 1979; Spencer et al., 1996; Hay and Focht, 1998). The present study was
undertaken to examine the degradation pathway of DDT in Pine River sediments in order
to determine the origin of DDMU detected in the sediments as well as the origin and fate

of DDD and DDE under anaerobic conditions.

40

Sediments from five locations in the Pine River Reservoir were used to study their
ability to support anaerobic dechlorination. Thin layer chromatography and
autoradiography of solvent extracts were used to analyze transformations of DDT, DDE
and DDD in sediment microcosms under anaerobic conditions. In addition to live
treatments, replicate samples were autoclaved to account for abiotic transformations.
Biologically mediated transformations were estimated by examining the difference
between live and autoclaved samples. When statistically valid, comparisons were made
between the contribution of biotic and abiotic transformations at a given site. The Pine
River sediments supported microbial dechlorination of DDT, but did not support
anaerobic reductive dechlorination of DDD or DDE. Therefore, this discussion will
focus on the transformations of DDT to DDD in microcosms.

Transformations in live microcosms

In the active microcosms, the anaerobic sediments supported anaerobic reductive
dechlorination of DDT to DDD. As the counts for DDT declined over time, there were
comparable increases for DDD (Table 3.1), but not for DDE or DDMU (data not shown).
Also, there was no significant temporal increase of counts at the origin (putative polar
metabolite) (Table 3.1). Thus dechlorination of DDT to DDD was only significant
reaction observed in the live microcosms. The greatest dechlorination of DDT to DDD
occurred by 8 weeks, and gradually increased thereafter (Table 3.1). For the live samples
at 8 weeks DDT recovery ranged from 38 to 53%. Furthermore, most DDT
dechlorination had occurred in microcosms by 16 weeks of incubation. After 40 weeks
of incubation, the average recovery of DDT for four sites (1,3,4 and 5) was 28% of the

applied DDT and the average recovery of DDT at site 2 was 39% (Table 3.1). A low

41

percentage of radioactivity in microcosms was recovered at time zero as DDD and polar
metabolites (Table 3.1); DDD amounts ranged fiom 3 to 14% (of added l4C-DDT) among
the five sampling sites. Two possible explanations for this are: (l) DDT was transformed
rapidly within several hours before they were transferred to the freezer, or (2) DDT may
have been transformed to DDD while microcosms were stored in the freezer. For the live
treatments, the average of radioactivity as DDD at weeks 8 and 16 in live samples was
33% and 36%, respectively, among all sites (Table 3.1). After 24 weeks average DDD
was 41% of the added DDT (data not shown), and after 40 weeks of incubation DDD
ranged from 40% to 44%. The general trends observed in Pine River sediments for

conversion of DDT to DDD under anaerobic conditions are shown in (Figure 3.1).

42

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43

Of the 14C-DDT added in microcosms, between 2 and 5 % was recovered as polar

metabolites; similar amounts occurred in all treatments (live and sterile) and at all
sampling points. Because they represented a small stable fraction of analytes in all

treatments, no attempt was made to identify the polar metabolites.

 

 

 

 

 

% of 14C recovered as DDD

 

 

 

 

 

 

 

0 I T I T T I
0 8 16 24 32 40 48
Incubation Time (Weeks)

Figure 3.1. Dechlorination of DDT to DDD in sediment microcosms
by microorganisms under anaerobic conditions during 40 weeks

of incubation. There were no significant differences in DDD
formation among sites at the 8 and 40 week sample intervals.

Sediments from site 2, which had the highest of DDX levels, were collected close
to sites that were reported to contain high PBB levels (~125ug/g) and other co-
contaminants (Morris et al., 1993). Morris et al. (1993) suggested that the presence of

organic co-contaminants, petroleum products, and heavy metals in sediments such as

44

those found at site 2 can inhibit in situ dehalogenation. Furthermore, Morris et al. (1993)
found that Pine River sediments containing the highest PBB did not support microbial
dechlorination of PCBs during a 32-week incubation. High sulfate levels in sediments
from this region of the Pine River probable may also inhibit dehalogenation (Mon-is et
al., 1993), but Quensen et a1. (1998) showed biodegradation of DDE to DDMU in
sediments under sulfidogenic conditions. Our results show even the sediments from site

2 which contained the highest concentration DDX compounds supported dechlorination

of added 14c-DDT.

Transformations in abiotic microcosms

Since methanogens are ubiquitous in anaerobic inhabitants, methane production
was used as an indicator microbial activity in the microcosms. At zero time, methane
production was below detection limits. In the live sediment microcosms, methane was
detected within 4 weeks of incubation and gradually increased during the 40 weeks
incubation (Figures 3.2, 3.3, 3.4, 3.5, 3.6). There was no correlation between the extent
of DDT dechlorination and the initial or cumulative methane production in the sediments.
Unlike live treatments that contained up to 25% methane content in the headspace by 40
weeks, methane in the autoclaved samples was less than 0.01% or not detected. This
suggests that there was no methanogensis in autoclaved microcosms, and the

transformations in the autoclaved treatments are likely due to abiotic processes.

45

% of 14C recovered

100

 

Total DDT _ 80

 

 

 

 

 

 

60 ~ — 60
4o « _, a 4% ~ 40
20 1 ~ 20
V —A
0 ""9 r r l I 1 I 0
0 8 16 24 32 40 48
Incubation Time (Weeks) _x_ Total DDT
—l— DDD
+ DDT

+ Polar Metabolites
—e— CH4

 

 

 

Figure 3.2. Microbial anaerobic dechlorination of DDT to DDD in sediment
microcosms prepared from site 1 sediments (refer to Figure 2.1 for sampling
location) during 40 weeks incubations. Dashed line indicates Recovery Total
added as DDT. These results were obtained by liquid scintillation counting
of scrapings from the TLC plates. Recovery Total represents the amount of

14C-labeled radioactivity DDT recovered in the solvent and aqueous extracts.
Percent of Methane produce at time of analysis by GC.

46

% of Methane Gas in Headspace

% of 14C recovered

 

100 ‘00
Total oor
so P _—}_‘”"‘}' —————— I — 80
+ \
\
60 ~ \ — so

 

L40

~20

°/o of Methane Gas in Headspace

 

 

 

 

 

w 0
0 8 16 24 32 40 48
Incubation Time (Weeks) __‘_ Total DDT
-I— DDD
-O— DDT
+ Polar Metabolites
-e— CH4

 

 

 

Figure 3.3. Microbial anaerobic dechlorination of DDT to DDD in sediment
microcosms prepared from site 2 sediments (refer to Figure 2.1 for sampling
location) during 40 weeks incubations. Dashed line indicates Recovery Total
added as DDT. These results were obtained by liquid scintillation counting
of scrapings from the TLC plates. Recovery Total represents the amount of

14C-labeled radioactivity DDT recovered in the solth and aqueous extracts.
Percent of Methane produce at time of analysis by GC. *

47

% of 14C recovered

 

 

 

 

 

 

 

 

 

 

100 100
Total DDT
80 % I — 80
1 ‘1
60 — — 60
4o - Ti e 40
.—
20 - i — 20
A
o 'g’ F I r F I I 0
0 8 16 24 32 40 48
Incubation Time (Weeks)
—u— Total DDT
—I— DDD
+ DDT

 

-0- CH4

 

 

Figure 3.4. Microbial anaerobic dechlorination of DDT to DDD in sediment
microcosms prepared from site 3 sediments (refer to Figure 2.1for sampling
location) during 40 weeks incubations. Dashed line indicates Recovery Total
added as DDT. These results were obtained by liquid scintillation counting
of scrapings from the TLC plates. Recovery Total represents the amount of

14C-labeled radioactivity DDT recovered in the solvent and aqueous extracts.
Percent of Methane produce at time of analysis by GC.

48

% of Methane Gas in Headspace

% of 14C recovered

100 '

100

 

 

Total DDT
r 80

—60

~40

~20

 

 

 

 

 

 

a 0
48
Incubation Time (Weeks) _*_ Tow DDT
-l- DDD
—0— DOT

 

+ Polar Metabolites
‘9‘ CH4

 

 

Figure 3.5. Microbial anaerobic dechlorination of DDT to DDD in sediment
microcosms prepared from site 4 sediments (refer to Figure 2.1 for sampling
location) during 40 weeks incubations. Dashed line indicates Recovery Total
added as DDT. These results were obtained by liquid scintillation counting
of scrapings from the TLC plates. Recovery Total represents the amount of

14C-labeled radioactivity DDT recovered in the solvent and aqueous extracts.
Percent of Methane produce at time of analysis by GC.

49

% of Methane Gas in Headspace

% of 14C recovered

100

 

100 *

     

Total DDT
— 80

604 -60

 

—40

«b
O
L

 

~20

N
O

A

T T *T 0

O 8 1 6 24 32 4O 48

Incubation Time (Weeks) —«— Total DDT

—|— DDD

+ DDT

+ Polar Metabolites

—e— CH4

 

 

 

 

 

 

Figure 3.6. Microbial anaerobic dechlorination of DDT to DDD in sediment
microcosms prepared from site 5 sediments (refer to Figure 2.1 for sampling
location) during 40 weeks incubations. Dashed line indicates Recovery Total
added as DDT. These results were obtained by liquid scintillation counting
of scrapings from the TLC plates. Recovery Total represents the amount of

14C--labeled radioactivity DDT recovered in the solvent and aqueous extracts.

Percent of Methane produce at time of analysis by GC.

50

°/o of Methane Gas in Headspace

Transformation of DDT to DDD was observed in both live and autoclaved
treatments, however it was higher for the live treatments than for autoclaved treatments.
After 8 weeks of incubations, the recovery of DDT ranged from 39 to 61% for autoclaved
samples (Table 3.1). In general there was a more gradual decline in DDT levels between
8 and 40 weeks, similar to the trends observed in the biologically active incubations
(Table 3.1). After 40 weeks of incubation under sterile conditions, DDT recovered
ranged from 34 to 51% for the autoclaved samples. As in the live samples, DDD was
recovered in autoclaved samples at week zero with concentrations ranging from 3 to 9 %.
By 40 weeks of incubation DDD formation ranged from 26 to 34 %.

Estimated (calculated) biotic transformation

Since abiotic transfonnations were likely to also occur in live treatments, the
difference between autoclaved and live treatments was used to estimate biologically
mediated (biotic) transformations. Before computing differences, however, the t-Test
was performed to compare the statistical significance (P<0.05) for differences in DDT
transformations between biologically active and corresponding abiotic controls (Table
3.1). In general, the relatively large standard deviations for each time point resulted in
varied levels of significance. There were, however, significant differences between DDD
recovery in live and autoclaved treatments at sites 4 and 5 at week 16 and at sites 1, 2, 3,
and 5 at 40 weeks. The estimated recovered radioactivity that resulted from biotic
transformation of DDT to DDD at 16 weeks was 12.88 % at site 4 and 16.98 % at site 5.
At 40 weeks, the biotic contribution was 14.62 % at site 1, 10.79 % at site 2, 17.78 % at

site 3, and 7.90 % at site 5.

51

Abiotic and biotic formation of DDD

The results discussed above demonstrate DDD was formed from DDT by both
abiotic and biotic transformations. The relative contributions of each of these were
calculated for each statistically valid time point (Figure 3.7). At 16 weeks, 67 % of DDD
formation was abiotic and 33 % was biotic at site 4; whereas, 59% was abiotic and 41%
was biotic at site 5. At 40 weeks the abiotic fraction of DDD formation was 64, 73, 59,
and 81 % for sites 1, 2, 3, and 5 respectively. The biotic contribution was 36, 27, 41, and
19% for sites 1, 2, 3, and 5 respectively. These results demonstrate that the majority of
DDD formation in the Pine River sediment microcosms is due to abiotic processes, but

there is also a substantial biotic contribution.

52

IAbiotic

 

0
IB' t'
70 IO lo 16 weeks
C
.9.
‘5
E
.2
o
o
‘2
.\
site 4 site 5
Site Location
90 IAbiotic 40 weeks
IBiotic
C
.2
u
I!
E
o
u.
D
D
0
.\°

 

Site 1 site 2 site 3 site 5
Site Location

Figure 3.7. The fraction of DDD recovered resulting from biotic and abiotic
transformations of DDT in Pine River sediment microcosms. The abiotic fraction was
determined by dividing the DDD recovered in the autoclaved treatments by the DDD
recovered in the live treatments (X 100 %). Similarly the biotic fraction was determined
by dividing the difference of the live and autoclaved treafinents by the live treatment (X
100 %). Only sites that had a significant difference between live and autoclaved are

shown.

53

/—

14C Total Recoveries

There was DDT degradation in both live and autoclaved samples during 40 weeks

of incubation. At time zero, the 14C recovered as total DDT ranged from 84% to 93 % in

biologically active microcosms, and similar recoveries of total DDT ranged between 87%
and 93% for autoclaved microcosms (Table 3.2). The total recovery decreased at 8
weeks in live treatments and remained stable thereafter. A similar trend was observed
with the autoclaved treatments, however with the live samples higher amounts of

radioactive materials were recovered at intermediate time points. After 40 weeks of
incubation, the 14C recovered as total DDT ranged from 73% to 84% for live treatments,

and similar recoveries of total DDT were detected for the sterile samples across the sites
1-5 (Table 3.2). In both live and autoclaved treatments, it is clear that a portion of
radioactive materials were unaccounted for. The reason for failure to recover a portion of
the added radioactivity DDT is not clear. Guenzi and Beard (1967) have shown DDT
recoveries ~ 79% for soils incubated for 12 weeks under anaerobic conditions. This is
similar to recoveries of total DDT in our experiment for sediments incubated for 40
weeks. They also concluded that between 15% and 20% of the added l4C-DDT was not
accounted for in autoclaved treatments. The observed loss of DDT in microcosms may
be due to several factors including losses during extraction, cleanup and concentrating

steps as well as volatilization.

54

 

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DDD and DDE Transformation in sediment microcosms

After 40 weeks of incubation, DDD and DDE were not degraded in sediment
samples that were sterilized prior to incubation, and no degradation occurred for any of
the non-sterile sediment microcosms. After completion of the incubations the average of
radioactivity recovered as DDD in live treatments was 87% of the added DDD. Trace
amounts of unidentified polar metabolites or impurities that always were recovered also
occurred in controls. For the sterile control microcosms the recovery ranged from 83.5 to
93.07 % as DDD across the five sites (Table 3.3). The results for DDE amended
sediment samples closely resemble those of DDD. By 40 weeks of incubation, the
radioactivity recovered as DDE ranged from 88% to 94% for biologically active
microcosms across all five sites sampling (Table 3.3). These results indicate that the Pine
River sediments do not support dechlorination of DDD or DDE in laboratory microcosms

after 40 weeks incubation.

56

 

 

 

 

 

 

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57

SUMMARY

DDT was converted primarily to DDD in both live and autoclaved treatments.
Microbial dechlorination accounted for 19 to 41 % of DDD formation during 40 weeks of
incubation in live treatments (Figure 3.7). There was no evidence for the transformation
of DDT to DDE in laboratory microcosms. This was not surprising since DDE is a major
degradation product of aerobic DDT degradation and considering that our conditions
were strictly anaerobic. No dechlorination of DDD or DDE was observed in either live
or autoclaved treatments. No significant differences were found among sites despite the
fact that endogenous DDT concentrations ranged from 0.05 ug/g to 20,000ug/g sediment.
The results from this laboratory experiment provide no evidence for DDMU formation,

although DDMU was observed at sites 2, 3 and 4.

noise
/ x

DDD

' i0“ “Oi-O“

Cl—C—Cl H— C —Cl

.5; \x. X .—
00; -O-“

Cl— C —Cl

DDE

Figure 3.8. Summary of metabolites detected in Pine River sediments and proposed
reactions leading to formation. Transformation marked with X were not observed in
laboratory microcosms containing river sediments and added l4C-DDT, -DDD or DDE.

58

 

REFERENCES

Forba, R. W. US EPA Pine River Contamination Survey, St. Louis, MI, EPA-330/2-80-
031, 1980.

Forba, R. W. U.S. EPA Summary of Pine River Reservoir Sediment Sampling Survey, St.
Louis, MI, EPA-330/2-82-001, 1982.

Guenzi, W. D., and Beard, W. E. 1967. Anerobic biodegradation of DDT to DDD in
soil. Science. 156: 1116-1117.

Hay, A. G., and Focht, D. D. 1998. Cometabolism of 1,1-Dichloro—2, 2-Bis (4-
Chlorophenyl) Ethylene by Pseudomonas acidovorans M3GY Grown on Biphenyl. Appl.
Environ. Microbiol. 64:2141-2146.

Morris, P.J.; Quensen, J.F., IH; Tiedje, J.M., and Boyd, SA. 1993. An Assessment of
the Reductive Debromination of Polybrominated Biphenyls in the Pine River Reservoir.
Environ. Sci. Technol. 27:1580-1586.

Quensen, J. F., III, Mueller, S. A.; Jain, M. K.; Tiedje, J. M. 1998. Science. 280: 722-
724.

Quirke, J. M. E.; Marei, A. S. M.; Eglinton, G. 1979. The Degradation of DDT and its
degradative products by reduced iron (III) porphyrins and ammonia. 3:151-155.

Spencer, W. F ., Singh, G. C., Taylor, D., LeMert, R. A. M., Cliath, M.; and Farmer, W. J.

1996. DDT persistence and volatility as affected by management practices after 23 years.
J. Environ. Qual. 25:815-821.

59

APPENDIX

Figure A. 1. Dechlorination of DDT to DDD by non-biological processes in sediment
microcosms prepared from site 1 sediments (refer to Figure 2.1 for sampling location)
during 40 weeks incubations. Dashed line indicates Recovery Total added as DDT.
These results were obtained by liquid scintillation counting of scrapings from the TLC
plates. Recovery Total represents the amount of l4C-labeled radioactivity DDT recovered
in the solvent and aqueous extracts .................................................................................... 61

Figure A.2. Dechlorination of DDT to DDD by non-biological processes in sediment
microcosms prepared from site 2 sediments (refer to Figure 2.1 for sampling location)
during 40 weeks incubations. Dashed line indicates Recovery Total added as DDT.
These results were obtained by liquid scintillation counting of scrapings from the TLC
plates. Recovery Total represents the amount of MC-labeled radioactivity DDT recovered
in the solvent and aqueous extracts .................................................................................... 62

Figure A.3. Dechlorination of DDT to DDD by non-biological processes in sediment
microcosms prepared from site 3 sediments (refer to Figure 2.1 for sampling location)
during 40 weeks incubations. Dashed line indicates Recovery Total added as DDT.
These results were obtained by liquid scintillation counting of scrapings from the TLC
plates. Recovery Total represents the amount of l4C-labeled radioactivity DDT recovered
in the solvent and aqueous extracts .................................................................................... 63

Figure A.4. Dechlorination of DDT to DDD by non-biological processes in sediment
microcosms prepared from site 4 sediments (refer to Figure 2.1 for sampling location)
during 40 weeks incubations. Dashed line indicates Recovery Total added as DDT.
These results were obtained by liquid scintillation counting of scrapings from the TLC
plates. Recovery Total represents the amount of l4C-labeled radioactivity DDT recovered
in the solvent and aqueous extracts .................................................................................... 64

Figure A.5. Dechlorination of DDT to DDD by non-biological processes in sediment
microcosms prepared from site 5 sediments (refer to Figure 2.1 for sampling location)
during 40 weeks incubations. Dashed line indicates Recovery Total added as DDT.
These results were obtained by liquid scintillation counting of scrapings from the TLC
plates. Recovery Total represents the amount of l4C--labeled radioactivity DDT recovered
in the solvent and aqueous extracts .................................................................................... 65

60

% of 14C recovered

 

100

80 \ /

 

 

_‘
~
‘
‘
~
‘
~.
~‘
x

Total DDT

 

 

 

0 8 1 6 24 32

Incubation Time (Weeks)

 

 

 

40 48
—x— Total DDT
—a— DDD
+ Polar Metabolites

 

 

Figure A.1. Dechlorination of DDT to DDD by non-biological processes

in sediment microcosms prepared from site 1 sediments (refer to Figure 2.1
for sampling location) during 40 weeks incubations. Dashed line indicates
Recovery Total added as DDT. These results were obtained by liquid
scintillation counting of scrapings from the TLC plates. Recovery Total

represents the amount of 14C-labeled radioactivity DDT recovered in the

solvent and aqueous extracts

61

% of 14C recovered

100

80 ..

60

40

20

 

 

 

 

 

 

 

Total DDT
\ \ \
%—~~r ——————————— i
L A
0 8 16 24 32 4O 48
Incubation Time (Weeks)
—x— Total DDT
—a— DDD
—9— DOT

+ Polar Metabolites

 

 

 

Figure A.2. Dechlorination of DDT to DDD by non-biological processes

in sediment microcosms prepared from site 2 sediments (refer to Figure 2.1
for sampling location) during 40 weeks incubations. Dashed line indicates
Recovery Total added as DDT. These results were obtained by liquid
scintillation counting of scrapings from the TLC plates. Recovery Total

represents the amount of 14C-labeled radioactivity DDT recovered in the
solvent and aqueous extracts

62

% of 14C recovered

 

100

   
 
 

Total DDT

\

80 \

\E___¥ ........ I

60‘

 

40~

#é

 

 

 

 

 

 

20 ~
A
0 T I fi' I I T T
8 16 24 32 40 48
Incubation Time (Weeks)

—x- Total DDT
—a— DDD
—e— DDT
+ Polar Metabolites

 

 

 

Figure A.3. Dechlorination of DDT to DDD by non-biological processes

in sediment microcosms prepared from site 3 sediments (refer to Figure 2.1
for sampling location) during 40 weeks incubations. Dashed line indicates
Recovery Total added as DDT. These results were obtained by liquid
scintillation counting of scrapings from the TLC plates. Recovery Total

represents the amount of 14C-labeled radioactivity DDT recovered in the
solvent and aqueous extracts

63

% of 1“C recovered

100

 

Total DDT

 

 

 

 

 

 

It
20 ~ T
A

0 I I j f

 

 

0 8 16 24 32 40 48
Incubation Time (Weeks)
—x— Total DDT
-B— DDD
_9_ DDT

+ Polar Metabolites

 

 

 

Figure A.4. Dechlorination of DDT to DDD by non-biological processes

in sediment microcosms prepared from site 4 sediments (refer to Figure 2.1
for sampling location) during 40 weeks incubations. Dashed line indicates
Recovery Total added as DDT. These results were obtained by liquid
scintillation counting of scrapings from the TLC plates. Recovery Total

represents the amount of 14C-labeled radioactivity DDT recovered in the
solvent and aqueous extracts

64

% of 14C recovered

100

 

Total DDT

60*

4o-

20‘

 

 

———A

 

 

 

 

0 l T— l I
0 8 16 24 32 40 48
Incubation Time Meeks)
—x— Total DDT
—a— DDD
—O—- DDT

-A— Polar Metabolites

 

 

 

Figure A.5. Dechlorination of DDT to DDD by non-biological processes

in sediment microcosms prepared from site 5 sediments (refer to Figure 2.1
for sampling location) during 40 weeks incubations. Dashed line indicates
Recovery Total added as DDT. These results were obtained by liquid
scintillation counting of scrapings from the TLC plates. Recovery Total

represents the amount of 14C-labeled radioactivity DDT recovered in the
solvent and aqueous extracts

65

IIIIllliiiliiijliiilllilflllI