United States
Environmental Protection
Agency
Environmental
Research Laboratory
Athens GA 30613-7799
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Research and Development
EPA/600/S3-88/048 June 1989
&EPA Project Summary
Effects of Temperature and
Redox Conditions on
Degradation of Chlorinated
Phenols in Freshwater
Sediments
John E. Rogers, Gert-Wieland Kohring, and Juergen Wiegel
The physical and chemical proper-
ties of the sediment environment can
vary in time as well as in location.
Sediment temperatures change over
the seasons, and sediments directly
exposed to the sun in summer can
reach temperatures in excess of
50°C. Freshwater sediments are
primarily methanogenic, whereas
marine sediments are dominated by
sulfate reducing conditions. These
fluctuations and differences can
influence chemical as well as
microbiological processes involved
in the transformation or degradation
of hazardous organic chemicals.
Therefore, during the development of
mathematical models to predict the
fate of toxic organic compounds in
the environment, it is important to
include parameters that describe the
influence of temperature and chem-
ical characteristics on anaerobic bio-
degradation.
The effect of temperature and
redox conditions on the anaerobic
degradation of 2,4-dichlorophenol
(2,4-DCP) was investigated in
anaerobic sediment slurries prepared
from local freshwater pond
sediments. Under methanogenic
conditions, 2,4-DCP dechlorination
occurred in the temperature range
between 5 and 50°C. Although
dechlorination was not observed
above 50°C, anaerobic bacterial
activity was Indicated by methane
formation up to 60 °C. In sediment
samples from two sites and at all
temperatures from 5 to 50°C, 2,4-DCP
was transformed to 4-chlorophenol
(4-CP). The 4-CP was further
degraded after several weeks.
Adaptation periods decreased
between 5 and 25 "C, were essentially
constant between 25 and 35°C, and
increased between 35 and 40 °C.
Degradation rates increased expo-
nentially between 15 and 30°C, had a
second peak at 35 °C and decreased
to about 5% of the peak activity by
40 "C. In one sediment sample, an
increase in degradation rates was
observed following the minimum at
40 "C, suggesting that at least two
different organisms were involved in
the 2,4-DCP dechlorination.
The addition of sulfate at a level of
0.25% resulted in a marked reduction
in the rate of methane formation, a
marked reduction in the rate of
reductive dechlorination, and an
increase in the length of the
adaptation period before reductive
dechlorination could be detected.
The addition of nitrate at a level of
0.15% resulted in the complete
inhibition of the reductive
dechlorination reaction for at least
127 days.
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Storage of the original sediment
slurries for 2 months at 12°C resulted
in increased adaptation times, but did
not affect the degradation rates.
This Project Summary was devel-
oped by EPA's Environmental
Research Laboratory, Athens, GA, to
announce key findings of the research
project that Is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
The full report explains the effect of
temperature and redox conditions on the
reductive dechlorination of 2,4-dichloro-
phenol (2,4-DCP) and 4-chlorophenol (4-
CP) sediment slurries. It also shows that,
for a restricted temperature (e.g.,from 15
to 30°C), dechlorination rates under
methanogenic conditions can be
described by an Arrhenius function.
Both 2,4-DCP and 4-CP are released to
the environment by direct disposal or as
break-down products of higher
chlorinated compounds such as the
extensively used wood preservative pen-
tachlorophenol (PCP) or the herbicides
2,4-dichlorophenoxyacetic acid (2,4-D)
and 2,4,5-trichlorophenoxyacetic acid
(2,4,5-T). These compounds are highly
toxic and 4-chlorophenol is a suspected
carcinogen. Thus, there is a clear need
for modeling the fate of these compounds
in order to evaluate their potential risk to
man and his environment.
The reductive dechlorination of chloro-
phenols has been reported in a variety of
anaerobic sediments such as sewage
sludge, sediment, and subsurface aquifer
environments, all of which were methano-
genic. Only recently has the reductive
dechlorination of 2,4-DCP been reported
to occur under sulfate reducing
conditions and, to our knowledge,
reductive dechlorination under denitri-
fying conditions has not been reported.
The proposed pathway for the anaerobic
degradation of 2,4-DCP is the pro-
gressive replacement of the chlorines by
hydrogen (reductive dechlorination) lead-
ing to phenol, which is then further
degraded to methane and carbon dioxide.
Similar reductive pathways have been
reported for chlorinated benzoates and
other chlorinated phenols.
The addition of sulfate to methanogenic
aquifer materials has resulted in the
inhibition of the dechlorination reaction.
Interestingly, however, the degradation
intermediate phenol was readily de-
graded under sulfate reducing conditions.
Adding sulfate to methanogenic aquifer
material has also led to increased
degradation of cresols. Unfortunately,
little else is known about the effects of
environmental factors on these reactions.
An extensive study of the effects of redox
couples or temperature has not been
reported. Our work was undertaken to
learn more of these effects to provide a
better base for the development of
environmental fate and effects models.
Procedures
Freshwater lake sediments from two
different sites near Athens, GA, were
used in this investigation. Individual
reaction flasks consisted of anaerobic
tubes that were filled with 20 ml of
sediment slurry and crimp sealed with
butyl rubber stoppers. All procedures
were conducted under flowing nitrogen
that had been purged of oxygen.
Methanogenic slurries were prepared by
combining sediment with site water to
give 0.2 to 0.4 mg dry wt. sediment/ml of
slurry, adding sufficient 1 M K2HP04/
KH2P04 to yield a buffer concentration of
20 mM, and then adjusting the pH to 6.9
by the addition of small quantities of 2 N
NaOH. The slurries were prepared in a
blender and were periodically mixed to
obtain a homogeneous solution. Sulfate
reducing or denitrifying slurries were
prepared by adding sulfate (0.25%) or
nitrate (0.15%), respectively.
Degradation experiments were initiated
by adding 0.7 ml of an aqueous 2,4-DCP
stock solution to give a final
concentration of about 125 iimol/l (20
ppm). Duplicate samples were incubated
in waterbaths at different temperatures.
Sediment slurries were removed
periodically and combined with an equal
volume of acetonitrile to terminate the
reaction and to facilitate recovery of
compounds bound to the sediment
matrix. These mixtures were subseq-
uently analyzed by high performance
liquid chromatography (HPLC) for 2,4-
DCP, 4-CP, phenol, and benzoate. Abiotic
controls were prepared by autoclaving
the sediment samples on 3 consecutive
days for 45 min each before 2,4-DCP
was added.
For methane formation experiments, 10
ml of the slurry without 2,4-DCP were
placed in Hungate tubes and the filled
tubes were then incubated in a
temperature gradient incubator (7 to
80°C) with continuous shaking. Period-
ically, 100-jil samples were taken from
the headspace with a gas lock syringe
and the methane concentration deter-
mined by gas chromatography.
Conclusions
The authors observed a sequential
dechlorination of 2,4-DCP in the
temperature range between 5 and 52 °C
for unacclimated sediment dominated by
methanogenic activity. At all
temperatures, the first product was 4-CP,
and although at least two different groups
of microorganisms were involved in 2,4-
DCP degradation at the different
temperatures, they all used the same
reaction to initiate degradation.
The authors also found that, although
anaerobic microbial dechlorination of 2,4-
DCP occurred from 5 to 52°C, only a
small portion of the data (15 to 35°C]
could be fit to an Arrhenius equation
Above and below the linear range the
temperature dependence varied marked-
ly. Although this range appears limited, i'
does indicate the temperatures ovei
which microorganisms will significantly
influence the fate of chlorinated aromatic
compounds in anaerobic environments
and does cover a significant fraction o
environments where these micro'
organisms would be active. Furthermore
it indicates that laboratory tests could be
performed at the faster rates (e.g. a
28°C) and then extrapolated to environ
mental temperatures (e.g. at 20 °C).
Unlike a chemical reaction where
temperature may only affect the reaction
in a microbiologically mediated process
temperature affects both the compositioi
of the active community and the enzyme
catalyzed reaction. The effects o
population become even more pro
nounced when more than one organism
as in the case of reductive dechlorinatior
may be required for activity and differen
organisms may catalyze the rate
determining step at different tempera
tures. In this study, the major peaks c
activity observed above and below 40 °<
probably resulted from the contribution
of thermophilic and mesophilic orgar
isms, respectively. Interestingly, becaus
thermophilic activity was only observed i
half of the samples from one of th
sediments, the key thermophilic orgar
isms appeared to be present at signi
icantly lower numbers.
Storing sediment samples before us
appeared to affect adaptation more tha
the rate of dechlorination. For exampli
adaptation periods, determined over th
temperature range of 5 to 25°
decreased from a range of 140 to 8 dav
for the fresh sediments to a range of 27
to 12 days for the sediments stored for
months at 12°C. The corresponding 2,
DCP transformation rates were m
significantly affected. Therefore the ui
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kof stored samples could lead to
srroneous estimates of the adaptation
'period.
Under sulfate reducing conditions, the
rate of reductive dechlorination activity
was observed only between 18 and 40°C
after 365 days incubation. More than
30% of the sulfate remained following
complete degradation of the 2,4-DCP and
methanogenic activity was inhibited for
the duration of the experiment. The rates
varied from 2 to 19% of the rates
observed under methanogenic condi-
tions. No reductive dechlorination was
observed under denitrifying conditions
after 127 days of incubation.
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The EPA author, John £. Rogers (also the EPA Project Officer, see below), is with
the Environmental Research Laboratory, Athens, GA 30613-7799; Gert-Weiland
Kohring and Juergen Wiegel are with the University of Georgia, Athens, GA.
The complete report, entitled "Effects of Temperature and Redox Conditions on
Degradation of Chlorinated Phenols in Freshwater Sediments," (Order No. PB
89-729 US/AS; Cost: $73.95, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Research Laboratory
U.S. Environmental Protection Agency
Athens, GA 30613-7799
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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