United States
Environmental Protection
Agency
National Risk Management
Research Laboratory
Ada, OK 74820
Research and Development
EPA/600/S-97/003
vvEPA
August 1997
ENVIRONMENTAL
RESEARCH BRI
Anaerobic Biodegradation of BTEX in Aquifer Material
Robert C. Borden*, Melody J. Hunt*, Michael B. Shafer* and Morton A. Barlaz*
Abstract
Laboratory and field experiments were conducted in two
petroleum-contaminated aquifers to examine the anaerobic
biodegradation of benzene, toluene, ethylbenzene and xylene
isomers (BTEX) under ambient conditions. At both sites,
destructive microcosm experiments were conducted
following the EPA protocol for estimation of anaerobic
microbiological transformation rate data (Federal Register,
Vol. 53, No. 115). Aquifer material was collected from
locations at the source, mid-plume and end-plume at both
sites, incubated under ambient conditions, and monitored
for disappearance of the test compounds. In the mid-plume
location at the second site, in-situ column experiments
were also conducted for comparison with the laboratory
microcosm and field-scale results.
In material from the first site, collected from a location in the
Sleeping Bear Dunes National Lakeshore, Michigan, toluene
biodegraded in microcosms under methanogenic conditions
after a 60- to 246-day lag period. There was no statistically
significant evidence of benzene, ethylbenzene, or xylene
biodegradation in the microcosm study.
At the second site, near Rocky Point, North Carolina, all
BTEX components biodegraded under ambient, anaerobic
conditions. In the mid-plume microcosms, m-xylene
Department of Civil Engineering, North Carolina State University,
Raleigh, North Carolina27695-7908.
biodegraded first followed by toluene, oxylene and benzene
under iron reducing conditions. None of the compounds
biodegraded in the source area microcosms. In the end-
plume microcosms, biodegradation was variable with
extensive biodegradation in some microcosms and little or
no biodegradaion in others. In all microcosm sets where
biodegradation was measured,'the compound being
investigated degraded to a low but detectable level (5 to
30 (ig/Liter), after which biodegradation slowed or stopped.
Biodegradation rates for m-xylene and benzene in in-situ
columns from the mid-plume location were similar to
microcosm rates.
Anaerobic biodegradation of individual BTEX components
often consisted of three distinct phases: (1) a lag period with
little or no biodegradation; (2) a rapid degradation period;
and (3) an asymptotic period where contaminant
concentrations remained essentially constant. This pattern
of biodegradation cannot be accurately described with a
simple first-order decay function. In contrast to the behavior
of the individual compounds, the biodegradation of total
BTEX appears to more closely approximate a first-order
decay function.
Introduction
This study focuses on the anaerobic biodegradation of benzene,
toluene, ethylbenzene and xylene isomers (BTEX) in aquifer
material from two petroleum-contaminated aquifers: Sleeping
Bear Dunes National Lakeshore (SB) in Michigan and a site near
Rocky Point, North Carolina (RP). The two sites examined were
chosen because of their differences in plume size, contaminant
Gyy Printed on Recycled Paper
-------
residence time, geochemical environment and geologic setting.
At both sites, previous field monitoring indicated that anaerobic
biodegradation of one or more BTEX components was occurring
(Bordenefa/., 1995; Wilsonefa/., 1994). Twodifferenttechniques
were used to evaluate the ability of indigenous microorganisms
to anaerobically degrade BTEX and to estimate the rate of
degradation: (1) destructively sampled laboratory microcosms;
and (2) in-situ test chambers. Both techniques consist of spiking
aquifer sediment with BTEX and monitoring compound
disappearance overtime. Experimental conditionsweredesigned
to mimic ambient conditions in the aquifer to the maximum extent
possible. Killed controls were monitored at the same time to
differentiate between biological and abiotic losses.
Procedure
Laboratory microcosms were constructed with aquifer material
from source, mid- and end-plume locations at both sites. The
microcosms were prepared in serum bottles with a high sediment
to water ratio of 1.8 dry g/mLiter. The experimental procedure
was based on the EPA protocol for estimation of anaerobic
microbiological transformation rate data (Federal Register,
Vol. 53,No.115). Multiple replicate microcosms were constructed
using blended aquifer sediment and ground water recovered
under anaerobic conditions. The aquifer sediment was collected
aseptically and anaerobically using methods developed by
U.S. EPA (Dunlap ef al., 1984). Ground water was collected
anaerobically through a 0.45 micron filter from monitoring wells
adjacent to each core location. Microcosms were spiked with
approximately 2,000 ng/Liter of benzene, toluene, ethylbenzene,
oxylene and m-xylene and incubated in anaerobic containers
stored at the ambient ground-water temperature, 16°C. The
microcosms were constructed in an anaerobic glove box using
aseptic techniques. A reducing agent was added to microcosms
constructed with SB sediment while naturally occurring Fe II (aq)
served as a reducing agent in the RP microcosms. Resazurin
was added to all microcosms and indicated anaerobic conditions
throughout the monitoring period. Triplicate microcosms and
abiotic controls were sacrificed at approximately monthly intervals
for one to two years and analyzed to determine the loss of BTEX
and changes in other electron acceptors and donors.
The in-situ columns were used at one location for comparison
with the laboratory microcosms and are similar to the system
used by Gillham et al. (1990). Each column consists of a
chamber (1.0 m long) where sediment and ground water are
isolated from the surrounding aquifer for controlled observation
(Figure 1). Columns were installed by drilling a pilot hole and then
installing a 15 cm-diameter by 3 m-Iong section of polyvinyl
chloride (PVC) casing. Stainless steel tubing and 3 m of drill rod
were attached to the equipment chamber. Argon was pumped
into the casing to displace any oxygen present. The column was
then pushed into the aquifer. Suction was applied to the stainless
steel feed line to insure that the column was completely filled with
aquifer material. The columns were then filled with anaerobic
ground water containing BTEX. Two microcosm columns and
one abiotic control were used in each experiment. The abiotic
control columns were prepared by adding an inhibitor to the
injection water (final concentration was either 0.1 N HCL or
500 mg formaldehyde/Liter). All columns were monitored for
BTEX, dissolved iron, sulfate, chloride, pH and dissolved oxygen
(DO). Analytical methods followed in the microcosm and in-situ
column experiments are described elsewhere (Hunt ef al., 1997;
Beckman, 1994).
Effective first-order removal rates for BTEX in the microcosms
and in-situ columns were estimated using the equation
C = Co exp (-Kt), where K"is the apparent first-order decay rate
(day1), f is time and Co is the initial concentration. The biological.
loss rate was calculated as the difference between the loss rates'
in the microcosms and abiotic controls over the time period in
which biological losses were observed. A two-tailed Student's
test, assuming unequal variances, was performed to determine
if the microcosm rates were statistically different from the abiotic
control rates.
Results and Discussion Sleeping Bear Site
Site Characteristics
The first site (SB), located at the Sleeping Bear Dunes National
Lakeshore, Michigan, is a highly transmissive glacial outwash
consisting of coarse sand and gravel with calcium carbonate
fragments. The soil has a relatively high organic carbon content
(up to 2.2%) that primarily occurs as coatings on sand grains
(>94%) (West ef al., 1994). The alkylbenzene plume covers a
relatively short span (25 m) before it is intercepted by the Platte
River. Contaminant residence time in the aquifer varies from 5
to 53 weeks because of fluctuations in the water table gradient.
Oxygen, nitrate and sulfate are rapidly consumed, and dissolved
iron is produced at the upgradient edge of the hydrocarbon spill.
in the source area and downgradient contaminated aquifer,
methane is produced and dissolved BTEX concentrations decline
(Table 1). Field monitoring data indicate that toluene biodegrades
Stainless Steel Tubing
Ball valve
Ground Surface
15.34 cm (61;)
PVC casing
BW casing
End plate with main screens
Test Chamber
Figure 1. Schematic of in-situ test column.
-------
TABLE 1. Geochemical Characterization of Ground Water at the SB and RP Sites
SB site
Parameter
Distance from source (m)
Well Screen (m)
Depth of Core (m)
Oxygen (mg/Liter) b
Nitrate (mg/Liter) b
Fe (aq, mg/Liter) b
Sulfate ( mg/Liter) b
Methane (mg/Liter) b
Eh (mV) c
pH =
BTEX (mg/Liter) c
Background
-23
2.7-4.7
NAa
2.4
67
3.5
20.0
0.08
NMd
NM
<0.001
Source area
0
2.7 - 3.7
2.3 - 4.2
<0.1
2.2
7.7
<0.05
0.10
-294
5.9
Mid-plume
9.1
1.2-2.1
0.5 - 2.4
<0.1
0.3
4.1
6.0
1.7
-180
6.5
3.3
End-plume
21.3
0.6-1.5
0.6 - 2.3
0.4
0.1
5.2
<0.05
3.1
-186
6.0
2.0
RP site
Parameter
Distance from source (m)
Well Screen (m)
Depth of Core (m)
Oxygen (mg/Liter) e
Nitrate (mg/Liter) e
Fe (aq, mg/Liter) e
Sulfate ( mg/Liter) e
Methane (mg/Liter) e
Eh (mV) o
pH*
Benzene (mg/Liter) e
Toluene (mg/Liter) e
Ethylbenzene (mg/Liter) e
o-Xylene (mg/Liter) e
m-, p-Xylene ( mg/Liter) e
Background
-55
0.9-3.9
NA
3.1
19.6
0.2
18.9
<0.001
196
4.6
<0.005
<0.005
<0.005
<0.005
<0.005
Source area
0
1 .0-4.0
4.3-5.0
0.6
<1.5
24.6
34.8
0.096
-132
5.8
1.33
10.4
1.82
3.88
8.18
Mid-plume
183
4.9-6.4
3.8-4.8
0.4
<1.5
52.0
4.0
0.096
-118
6.1
0.62
0.083
1.92
0.042
2.75
End-plume
327
5.0-6.5
3.8-4.6
1.4
<1.5
1.8
12.8
0.40
-187
7.1
0.62
0.028
0.042
0.010
0.34
bData are average of 2 samples taken over a 4-month period as reported by Wilson etal., 1994.
'Measuredat time of core collection.
dNM - not measured.
"Data are average of 4 to 7 measurements taken over an 18-month period as reported by Gomez, 1993.
most rapidly with slower biodegradation of ethylbenzene and the
xylene isomers (Wilson etal., 1994). There was no evidence of
anaerobic benzene degradation.
Microcosm Results
The rate and pattern of biodegradation were similar at all three
locations at the SB site. Toluene biodegraded under
methanpgenic conditions at all three locations after a lag period
that varied from 60 to 246 days. First-order decay rates during
the period of active biodegradation were 0.042, 0.023 and
0.032 d~1 for the source, mid- and end-plume microcosms,
respectively. Figure 2 shows the variation in dissolved toluene
and methane in microcosms from the mid-plume location. The
increase in methane greatly exceeded the amount that could be
expected from the measured BTEX loss, indicating that other
undefined substrates were being biotransformed. In addition,
most of the methane was produced during the period when
toluene was not biodegrading (Figure 2). Similar trends were
measured in the source and end-plume microcosms. The
aquifer material used in the microcosms contained ~0.6% organic
carbon while West etal. (1994) found up to 2.2% organic carbon
in aquifer sands 21 m upgradient of the plume. Acetate had also
been detected in ground water from the site (Wilson etal., 1994).
Thus, background organic carbon was the major electron donor
in the SB sediment from all three locations. The initiation of
methane production prior to toluene biodegradation and the long
lag times prior to the onset of toluene biodegradation may be due
to the presence of more readily degradable substrates. In a
-------
200
Days
Figure 2. Average concentration of toluene (a) and methane (b) in
microcosms (solid) and abiotic controls (open) from the mid-plume location
at the SB site. Data are the average of three destructively sampled
microcosms at each time point. Error bars represent one standard deviation.
study of methanogenic enrichment cultures derived from
contaminated aquifer sediments, Edwards and Grbic-Galic (1994)
found the presence of other organic substrates such as acetate,
amino acids and propionate inhibited anaerobic degradation of
toluene and o-xylene.
There was no evidence of o-xylene, m-xylene or benzene
biodegradation at any location or ethylbenzene biodegradation in
the source and mid-plume microcosms. However, for the end-
plume microcosms, the ethylbenzene results are ambiguous. The
first-order loss rate for ethylbenzene in the microcosms was not
significantly different from the abiotic control over the entire test
period; however, there was evidence of ethylbenzene degradation
in selected replicates.
Wilson ef a/. (1994) reported biodegradation rates for BTEX
components based on field monitoring of the SB plume coupled
with estimates of contaminant retention time prior to discharge to
the Platte River. Given the uncertainty in the calculation of both
in-situ and microcosm biodegradation rates, quantitative
comparison of these laboratory results and the field rate of Wilson
ef a/, is inappropriate. Qualitative comparison of the field and
laboratory data shows consistency with respect to: (1) the absence
of benzene biodegradation; (2) the production of methane; and
(3) the preferential biodegradation of toluene. While the
laboratory data do not support or preclude low rates of
ethylbenzene biodegradation, Wilson ef a/. (1994) reported
biodegradation rates for ethylbenzene that were 10% of the
rates reported for toluene.
Rocky Point Site
Site Characteristics
The second site (RP) is located on the coastal plain near Rocky
Point, North Carolina, and is of marine origin. The site geology
consists of dark gray and green micaceous fine sand, overlain
by 1.5 to 4.5 m of silts, clays and clayey sands. The sediment
is over 90% quartz sand with minor amounts of pyrite and
muscovite flakes in a clay matrix. X-ray diffraction has shown
that most of the iron in the aquifer solids is present as glauconite,
an iron-rich clay mineral, with smaller amounts of the clays
berthierine and possibly iron-rich illite (Becker, 1992).
Field monitoring (Table 1) indicates that toluene and o-xylene
decline rapidly during transport through the first 100 m of the
300 m-long contaminant plume (Borden ef a/., 1995). Benzene
and m-, p-xylene decline more slowly before discharging to a
small drainage ditch. Anaerobic ethylbenzene biodegradation
was not evident from the field data. At the upgradient edge of
the plume, oxygen is rapidly depleted. Sulfate is depleted
immediately downgradient of the contaminant source and
dissolved Fe (II) increases in the mid-plume area, suggesting
that biodegradation occurs using both sulfate and ferric iron as
electron acceptors. The absence of significant methane in the
monitoring wells indicates methanogenic fermentation is not a
major process at this site.
The average ground-water velocity in the aquifer is 30 m/yr,
resulting in a contaminant residence time in excess of 12 years
(Borden ef a/., 1995).
Microcosm Results
At the RP site, anaerobic biodegradation of BTEX was a
function of location within the contaminant plume. In the source
area microcosms, none of the BTEX components degraded
during 388 days of incubation. In the mid-plume microcosms,
m-xylene biodegradation began with no lag, followed by toluene,
o-xylene and benzene (Figure 3a). By day 140, both toluene
and o-xylene had degraded to between 3 and 11 u,g/Liter and
then remained constant for the duration of the experiment. With
the onset of toluene degradation after day 22, the rate of
m-xylene loss declined and did not increase until toluene and o-
xylenewerebelow22 ug/l_iterat120days. Benzene degradation
began after day 180, and by day 403 the benzene concentration
was between 8 and 12 ug/Liter in each replicate (Figure 3b).
Two isolated replicates in which BTEX degraded also exhibited
ethylbenzene biodegradation during the last 100 days of
incubation; however, the average decay rate for ethylbenzene
was minimal over the sampling period.
In the mid-plume location, the dominant electron acceptor was
ferric iron present as an iron-rich clay mineral, glauconite.
Methane was not produced in any microcosm and manganese
concentrations were below detection. While small amounts of
sulfate were present initially, the dissolved sulfate concentration
remained constant (Figure 3d) and was not sufficient to account
for the observed BTEX loss. Dissolved iron concentrations
varied but appeared to increase with time (Figure 3e). However,
this increase was relatively small in proportion to the increase
-------
Abiotic Benzene
Benzene
Toluene
V Ethylbenzene
m-Xylene
o-Xylene
O Benzene - Abiotic
• Benzene - Live
10 Ni- Live
:~H~ Abiotic
I- - - Abiotic 1st Order Rate
Live 1st Order Rate
3
1
"o
CO
CO
.15
.10
.05
.00
£
1
o
01
to
0 100 200 300 400
Days
Figures. Resultsfrommid-plumeRPmicrocosms: (a)BTEXcomponents,
(b) benzene, (c) total BTEX, (d) dissolved SO4'2, and (e) dissolved Fe. Data
in figures a, c, d and e are the average of three replicates destructively
sampled at each time point. Error bars are ± one standard deviation. Abiotic
benzene concentrations in Fgure 3a represent the behavior of the other
BTEX components. Benzenedatain FigureSbareforindividual microcosms.
in solid-phase Fe (II). At the end of the experiment, the
measured increase in total Fe (II) was 200% of the amount
predicted based on the measured loss of BTEX, assuming
conversion to CO2. This indicates that significant quantities of
non-BTEX organic carbon also biodegraded under iron reducing
conditions.
In the mid-plume microcosms, biodegradation of toluene, o-
xylene and benzene consisted of three distinct phases: (1) a lag
period with little or no biodegradation; (2) a rapid degradation
period; and (3) an asymptotic period where contaminant
concentrations remained essentially constant. This pattern of
biodegradation cannot be accurately described with a simple
first-order decay curve. However, when total BTEX is plotted
versus time (Figure 3c), the lag and asymptotic periods disappear
and the experimental results closely approximate a first-order
decay process. The first-order decay rates for individual
compounds reported in Table 2 are for the period of rapid
biodegradation and are not representative of the entire incubation
period. Use of these rates in a simple first-order decay function
will substantially underestimate the time required for
biodegradation.
Biodegradation in the end-plume RP microcosms was variable.
Some microcosms exhibited clear evidence of biodegradation by
day 106 while other microcosms did not show any evidence of
biodegradation after 327 days (Figure 4). Toluene and o-xylene
degradation appeared to proceed concurrently starting at 106
days (Figures 4b and 4c). On days 184 and 253, there was clear
evidence for toluene and o-xylene degradation in all microcosms
sampled. Yet on the last two sampling dates, the evidence was
ambiguous as toluene had degraded in three of six microcosms
sampled and o-xylene had degraded in only two of six. Benzene,
m-xylene and ethylbenzene also degraded in selected microcosms
but usually only when toluene and o-xylene were also depleted
(Figure 4). Estimation of first-order decay rates was not
appropriate at this location, since the calculated rate would be
more a function of the random order of microcosm sampling than
the actual rate of biodegradation. It was not possible to conclusively
identify the electron acceptor in the end-plume microcosms
because of the presence of high concentrations of dissolved
sulfate and interferences in the solid-phase iron analyses.
Field (Borden etal., 1995) and laboratory results indicate that at
the RP site, the various BTEX components biodegrade in a
sequential process: toluene and o-xylene are usually depleted
first, followed by benzene and/or m-xylene and finally
ethylbenzene. In the mid-plume microcosms, this pattern varied
somewhat. The m-xylene biodegradation began without a lag
but then slowed once toluene and o-xylene degradation began.
At this location in the aquifer, toluene ando-xylene concentrations
are low (~5 ug/Liter), but significant quantities of m-, p-xylene still
remain (>1000 ug/Liter). These results suggest that m-xylene
degradation was occurring/n-s/fuat the time of sediment removal.
However, toluene and o-xylene were preferentially degraded
when elevated levels of these compounds were added to the
microcosms. Benzene biodegradation did not begin until after
180 days. By this time, toluene and the xylene isomers were
depleted to less than 10 ug/Liter. In the end-plume microcosms,
the added compounds also degraded sequentially; however,
toluene and o-xylene degradation did not occur until after 106
days (Figure 4). At this time, the various factors that control the
order of biodegradation are not fully understood . Clearly, there
are some interactions between the degradation of the different
compounds. As discussed above, the presence of other organic
substrates, including acetate, amino acids and propionate, has
-------
TABLE 2. First-order Decay Rates from the RP Site
Compound
Benzene
Toluene
Ethylbenzene
o-Xylene
m-, p-Xylene
Total BTEX
Mid-Plume
Decay rate"
(d-1)
0.024
0.045
NSC
0.020d
0.0066
Microcosms
Decay Period
(days)
184-403
22-120
37-120
0-184
0-403
Mid-Plume In-Situ Columns
Decay rate3
(d-1)
Test 1 : 0.0049
Test 2: 0.023
NS°
Ns°
NSC
0.0143
0.0029
Decay Period
(days)
Test 1: 155-251
Test 2: 41 -181
121-251
121-251
Field
Decay rate"
(d-1)
0.0002
0.0021
0.0021
0.0013
0.0011
* Rates represent the difference between the microcosms and control abiotic loss rate over the period of decay.
"Reid decay rates were calculated over the entire length of the plume.
" Microcosms and control abiotic loss rates were not significantly different at the 99% level.
* Only m-xylene was present in the microcosms.
been reported to inhibit anaerobic degradation of toluene and
o-xylene. While only BTEX concentrations were quantified in the
present work, the gas chromatograms from the mid-plume
microcosmsshowthatalargenumberof unidentified compounds
were present at low concentrations at the start of the experiment.
Over the course of the experiment, these compounds also
disappeared.
The lag period prior to biodegradation varied significantly among
sampling locations at the RP site and among individual
microcosms. The exact cause of this variability is unknown but
is believed to be due to differences in prior adaptation to BTEX,
electron acceptor availability and aquifer geochemistry. In the
source area, field monitoring data indicated that concurrent
BTEX biodegradation and sulfate reduction were occurring
immediately downgradient of the gasoline spill (Borden et al.,
1995). Consequently, sediment and ground water were collected
from an area with significant concentrations of BTEX (26 mg/Liter)
and sulfate (-35 mg/Liter) in the ground water. In retrospect, it
appears that this was an area where active sulfate reduction was
not occurring, since significant concentrations of sulfate were still
present in the ground water. In the end-plume location, there
was tremendous variability in aquifer geochemistry in the
immediate area where the sediment was collected. Prior to
construction of the microcosms, the sediment from each location
was blended and passed through a No. 8 sieve to homogenize
the sediment. The highly variable response in the destructively
sampled microcosms from the end-plume location indicates that
this procedure was not sufficient to eliminate differences among
replicate microcosms.
In-Situ Column Results
Two sets of column experiments were performed at the mid-
plume location at the RP site for comparison with the microcosm
results. In each set of experiments, two microcosm columns
were operated in parallel with one abiotic control. All microcosm
and abiotic control columns exhibited an initial concentration
decrease of several hundred ng/Liter because of sorption to the
aquifer sediment (Beckman, 1994). The concentrations of
hydrocarbons in the abiotic columns remained constant or
declined slowly after the initial drop, indicating that biological
activity or short-circuiting did not occur in the control columns.
The DO concentration remained low (<0.2 jag/Liter) throughout
all experiments. Biological loss rates were calculated as the
difference between the rates in the microcosms and the abiotic
controls, when these rates were statistically different. The
microcosm decay rate was estimated by pooling the result from
the two microcosm columns over the period of active
biodegradation.
The first set of experiments was performed using ground water
from a nearby well that was depleted in toluene and o-xylene but
contained higher concentrations of benzene, ethylbenzene, m-,
p-xylene, pseudocumene (1,2,4-trimethylbenzene) and
mesitylene (1,3,5-trimethylbenzene). Samples were collected
monthly for approximately 250 days when the experiment was
terminated because of limited sample volume. The m-, p-xylene,
benzene and pseudocumene biodegraded after initial lag periods
that varied from 85 to 121 days. By day 251, m-, p-xylene had
decreased by over 90% (Figure 5), benzene had decreased by
50% (Figure 6a) and pseudocumene had decreased by 75% to
90%. In the control column, m-, p-xylene had decreased toy over
48%, benzene had decreased by 22% and pseudocumene had
decreased by 35%. There was no evidence of toluene, o-xylene,
ethyl-benzene or mesitylene biodegradation in either microcosm
column. The absence of toluene and o-xylene biodegradation
was likely due to the low initial concentration of these compounds
(<50 ng/Liter). In the microcosm columns, DO remained below
detection (<0.2 mg/Liter), sulfate declined slightly from
1.3 mg/Literto the detection limit (-0.3 mg/Liter) andpH remained
constant at 6.3. In the first microcosm column, dissolved iron
increased from 111 to over 200 mg/Liter; while in the second
microcosm column, dissolved iron increased from 99 to
140 mg/Liter.
In the second set of experiments, the columns were reloaded
with ground waterthat contained higher concentrations of benzene
(1,000 to 1,300 ng/Liter) and very low concentrations of TEX
(-20 ng/Liter) to determine if benzene biodegradation would
continue. Benzene biodegradation began after a 41-day lag
period, and by day 334 benzene had declined from over
-------
100
a 10
CD
c
I ' I •! I 'I '
2 f S
|io|
CD
N
c
CD
-^ 1
LU
n 1
!""! • s §.
•™
t t i i I i i i i I i i i i I i i i r I i i r i ^< i t i 1 i i i <
0 50 100 150 200 250 300 350
Days
Figure 4. Variation in (a) benzene, (b) toluene, (c) o-xylene, (d) m-xylene
and (e) ethyl-benzene in microcosms constructed with material from the RP
end-plume location. Microcosm (•) and control (o) results are from individual
destructively sampled microcosms. Three microcosms and threecontrols
were sampled at each time point, except the last time point, when no control
was sampled.
1,000 ug/Liter to 11 jj.g/Liter in column 1 and to 8 ng/Liter in
column 2 (Figure 6b). The ground water used to reload the in-situ
column was obtained from a nearby multilevel sampler. Previous
monitoring had indicated that the sulfate concentration of this
ground water was very low (~1 mg/Liter). However, after
reloading the columns, sulfate concentrations in microcosm
columns 1 and 2 were 85 and 65 mg/Liter, respectively. Additional
monitoring confirmed that a pulse of high sulfate ground water
had migrated past the multilevel sampler intake at the time
ground water was collected for injection into the in-situ columns.
Over the course of this experiment, sulfate remained constant in
column 2 but declined by 25% in column 1, and dissolved iron
remained constant in both columns. Data are insufficient to
positively identify the electron acceptor used for benzene
degradation in the in-situ column experiments.
First-order Biodegradation Rates
First-order biodegradation rates from the in-situ column
experiments are compared to the biodegradation rates from the
laboratory microcosms and field measurements in Table 2.
Biodegradation rates for m-, p-xylene in the in-situ columns and
m-xylene in the laboratory microcosms were similar. However,
the lag period prior to the start of biodegradation was longer in
the in-situ columns. The lag period prior to benzene biodegradation
in the laboratory microcosms and the first in-situ column
experiment was similar. However, the rate of benzene
biodegradation in the first in-situ column experiment was a factor
of five lower than the laboratory microcosm rate. During this
experiment, benzene biodegraded concurrently with m-, p-xylene.
However, in the microcosms, m-xylene was completely degraded
before the start of benzene biodegradation. In the second in-situ
experiment, only benzene was present and the benzene
biodegradation rate was similar to the laboratory microcosms.
In most cases, the measured biodegradation rates for individual
compounds are comparable in both columns and microcosms
but are one or two orders of magnitude higher than rates
estimated from field investigations (Borden et a/., 1994). One
likely cause of this difference is the procedure used to calculate
biodegradation rates. In the laboratory microcosms and the in-
situ columns, there is a definite lag period prior to the start of
biodegradation. The laboratory and in-situ column rates were
calculated during the period of active biodegradation after the lag
10
0 50 100 150 200 250 300
Days
Figure 5. Variation in m-, p-xylene in the first set of in-situ test columns.
Open symbols are abiotic controls; solid symbols are microcosm columns.
-------
10°
a
-o-
-©
•Q O
•JO I •. t i I I i i i I I I I I I I I I I I I I I I I l I I I I 1 I I I
0 50 100 150 200 250 300 350
Days
Figure 6. Variation in benzene in in-situ columns: (a) initial experiment;
and (b) after reloading with benzene only. Open symbols are abiotic
controls; solid symbols are microcosm columns.
period had ended. In the field, it is usually not possible to identify
the zones where biodegradation is most active, and the reported
degradation rates are for the travel time over the entire plume.
The large differences between laboratory microcosm, in-situ
column and field-scale biodegradation rates for individual
compounds could be reduced by using a grouped parameter
such as total BTEX. In the laboratory microcosms, one or more
BTEX components were degrading throughout the experiment,
so a simple first-order decay model closely matched the total
BTEX results for the entire experiment with no observable lag in
total BTEX biodegradation (Rgure 3c). In the field, one or more
BTEX components are biodegrading at any location, and
consequently a first-order decay function more closely matches
the field data throughout the entire length of the plume. When
laboratory microcosm, in-situ column and field rates are compared,
biodegradation rates for total BTEX are much more consistent
than the rates for individual compounds. For example, the
laboratory biodegradation rate for benzene was 120 times the
field rate, while the laboratory rate for total BTEX was only 6 times
the field rate. The highest benzene degradation rate in the in-situ
columns was 115 times the field rate, while the total BTEX
degradation rate in the columns was only 2.6 times the field rate.
A second potential cause for the observed differences between
laboratory microcosm, in-situ column and field biodegradation
rates is spatial variations in biological activity. The rates reported
in Table 2 were calculated from column and laboratory
measurements at a single location (mid-plume). At a second
location (source area) there was no evidence of BTEX
biodegradation, and in a third location (end-plume) the results
were variable. In contrast, the field degradation rates were
estimated from monitoring well data collected along the length of
the plume and should represent the large scale, spatially-
averaged rate.
Conclusions and Recommendations
Anaerobic Biodegradation of BTEX
1. Benzene, toluene, ethylbenzene and the xylene isomers are
anaerobically biodegradable under ambient subsurface
conditions using ferric iron, sulfate and/or carbon dioxide as
terminal electron acceptors.
2. A distinct order of biodegradation is often observed, with
toluene being the most rapidly biodegraded compound.
However, this order may vary from site to site. For example,
at the Rocky Point site, o-xylene was rapidly biodegraded;
while at the Sleeping Bear site, no significant biodegradation
of o-xylene was observed.
3. The more easily biodegradable compounds (toluene, o-
xylene, m-xylene) appear to anaerobically biodegrade to a
low but detectable concentration (10 to 30 ug/Liter) after
which biodegradation slows or stops. It is not clear whether
biodegradation of these compounds will continue once the
more difficult to degrade compounds are depleted.
4. Use of a simple first-order decay model does not adequately
describe the anaerobic biodegradation of individual
compounds in laboratory microcosms or in-situ columns. To
accurately simulate the anaerobic biodegradation of individual
compounds, a model that includes two variables will be
required: (1) the lag period prior to biodegradation; and (2)
the rate of biodegradation.
5. The lag period prior to the start of anaerobic BTEX
biodegradation varies from compound to compound and
from site to site. Until the source of this variability is better
understood, it will not be possible to use laboratory
microcosms or in-situ columns to accurately predict the time
required for the field biodegradation of individual compounds.
6. Destructive microcosms yield only one concentration
measurementfor each independent experiment (microcosm)
and are poorly suited to generating the data required to fit a
two-parameter model. Consequently, use of destructive
microcosms, as specified in the EPA protocol for estimation
of anaerobic microbiological transformation rate data, is not
appropriate when the lag period prior to the start of
biodegradation is significant.
7. Anaerobic biodegradation of total BTEX more closely
approximates a first-order decay curve than the
biodegradation of the individual compounds. First-order
decay rates for total BTEX estimated from laboratory
microcosms, in-situ columns and field monitoring data also
appear to be more consistent than the rates for individual
BTEX components.
Use of Laboratory Microcosms and In-situ Columns
to Evaluate Natural Attenuation of BTEX
1. At this time, biodegradation rates for individual compounds
derived from laboratory microcosms and in-situ columns
cannot be reliably used to estimate the time required for
complete biodegradation in the field.
-------
2. Laboratory microcosms are useful for: (1) demonstrating
that a compound of regulatory concern can and does
biodegrade under ambient subsurface conditions; and (2)
evaluating the effect of different environmental variables on
the rate and extent of biodegradation.
3. In-situ columns may also be used to evaluate compound
biodegradation under ambient conditions and the effect of
different amendments. In-situ columns are relatively simple
to install and operate, closely replicate ambient conditions,
and result in minimal disturbance of the aquifer material.
4. Total BTEX may be a more appropriate parameter for
describing natural attenuation than the concentration of
individual compounds. Biodegradation of total BTEX more
closely matches a first-order decay curve than the individual
compounds. The degradation rate for total BTEX also
appears to be more consistent than biodegradation rates for
individual compounds. However in some cases, it may be
necessary to model individual compounds to accurately
assess the risk to human health and the environment.
5. Estimation of field biodegradation rates from limited point
measurements (laboratory microcosms or in-situ columns)
will be difficult because of spatial variations in biological
activity.
Disclaimer
The U.S. Environmental Protection Agency through its Office of
Research and Development partially funded and collaborated in
the research described here under Cooperative Agreement
No. CR-819630 to North Carolina State University. It has been
subjected to the Agency's peer and administrative review and
has been approved for publication as an EPA document. Mention
of trade names or commercial products does not constitute
endorsement or recommendation for use.
Quality Assurance Statement
All research projects making conclusions or recommendations
based on environmentally related measurements and funded by
the Environmental Protection Agency are required to participate
in the Agency Quality Assurance Program. This project was
conducted under an approved Quality Assurance Program Plan.
The procedures specified in the plan were used without exception.
Information on the plan and documentation of the quality
assurance activities and results are available from the Principal
Investigator.
References
Becker, M. T. 1992. Iron Reduction in a Gasoline-Contaminated
Aquifer Containing Glauconite. M.S. Thesis, North Carolina
State University, Raleigh, North Carolina. 97 pages.
Beckman, M. 1994. In-Situ Measurement of Intrinsic
Bioremediation Rates, M.S. Thesis, North Carolina State
University, Raleigh, North Carolina. 121 pages.
Borden. R. C., C. A. Gomez, and M. T. Becker. 1994. Natural
Bioremediation of a Gasoline Spill, pp. 290-295. In R. E. Hinchee,
B. C. Alleman, R. E. Hoeppel, and R. N. Miller (ed.).Hydrocarbon
Bioremediation, CRC Press, Boca Raton, Florida.
Borden. R. C., C. A. Gomez, and M. T. Becker. 1995. Geochemical
Indicators of Natural Bioremediation. Ground Water 33:180-
189.
Dunlap, W. J., J. F. McNabb, M. R. Scalf, and R. L. Cosby. 1984.
Sampling for Organic Chemicals and Microorganisms in the
Subsurface. EPA/600/2-77/176, U.S. Environmental Protection
Agency, Ada, Oklahoma.
Edwards, E. A., and D. Grbic'-Galic. 1994. Anaerobic Degradation
of Toluene and o-Xylene by a Methanogenic Consortiu " Appl.
Environ. Microbiol. 60:313-322.
Gillham, R. W., R. C. Starr, and D. J. Miller. 1990. A Device for
In-situ Determination of Geochemical Transport Parameters; 2
Biochemical Reactions. Ground Water 82: 858-862.
Gomez, C. A. 1993. Characterization of a Dissolved Hydrocarbon
Plume, M.S. Thesis, North Carolina State University, Raleigh,
North Carolina. 107 pages.
Hunt, M. J., M. B. Shafer, M. A. Barlaz, and R. C. Borden. 1997.
Anaerobic Biodegradation of Alkylbenzenes in Laboratory
Microcosms Representing Ambient Conditions. Bioremediation
Journal 1(1):53-64.
West, C. C., W. G. Lyon, D. L. Ross, and L. K. Pennington. 1994.
Investigation of the Vertical Distribution and Morphology of
Indigenous Organic Matter at the Sleeping Bear Site, Michigan.
Environ. Geol. 24:176-187.
Wilson, J.T., D. H. Kampbell, and J. Armstrong. 1994. Natural
Bioreclamation of Alkylbenzenes (BTEX) from a Gasoline Spill in
Methanogenic Groundwater, pp. 201-218. In R. E. Hinchee, B.
C. Alleman, R. E. Hoeppel, R. N. Miller (ed.), Hydrocarbon
Bioremediation. CRC Press, Inc., Boca Raton, Florida.
-------
-------
-------
m
-o
5
o
o
en
o
o
CO
o o m c
o m Q.
^ en
•O 5"
I"
CO. CO
(D
CO
CD
si
iU
051
n
en 3
oo CD
3
<
3
CD
CD
Q.
CD
CO
O
oT
_ a
£§'
j] >
CD CQ
CO CD
CD 3
£U O
O
m
33
T>
O
O
&
-------
|