1

* Department of Civil Engineering, North Carolina State University,

Raleigh, North Carolina 27695-7908.

National Risk Management

Research Laboratory

Ada, OK 74820

United States

Environmental Protection

Agency

Research and Development

ENVIRONMENTAL

RESEARCH BRIEF

EPA/600/S-97/003 August 1997

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

Anaerobic Biodegradation of BTEX in Aquifer Material

Robert C. Borden*, Melody J. Hunt*, Michael B. Shafer* and Morton A. Barlaz*

biodegraded first followed by toluene, o-xylene and benzene

under iron reducing conditions. None of the compounds

biodegraded in the source area microcosms. In the endplume 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 μg/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
2

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

(Borden et al., 1995; Wilson et al., 1994). Two different techniques

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 over time. Experimental conditions were designed

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 et 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 μg/Liter of benzene, toluene, ethylbenzene,

o-xylene and m-xylene and incubated in anaerobic containers

stored at the ambient ground-water temperature, 16 o

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-long 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 0.1N 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 et 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

(day-1), t 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 et 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

Figure 1. Schematic of in-situ test column.
3

TABLE 1. Geochemical Characterization of Ground Water at the SB and RP Sites

SB site

Parameter Background Source area Mid-plume End-plume

Distance from source (m) -23 0 9.1 21.3

Well Screen (m) 2.7-4.7 2.7 - 3.7 1.2 - 2.1 0.6 - 1.5

Depth of Core (m) NAa 2.3 - 4.2 0.5 - 2.4 0.6 - 2.3

Oxygen (mg/Liter) b 2.4 <0.1 <0.1 0.4

Nitrate (mg/Liter) b 67 2.2 0.3 0.1

Fe (aq, mg/Liter) b 3.5 7.7 4.1 5.2

Sulfate ( mg/Liter) b 20.0 <0.05 6.0 <0.05

Methane (mg/Liter) b 0.08 0.10 1.7 3.1

Eh (mV) c NMd -294 -180 -186

pH c NM 5.9 6.5 6.0

BTEX (mg/Liter) c <0.001 3.3 2.0

RP site

Parameter Background Source area Mid-plume End-plume

Distance from source (m) -55 0 183 327

Well Screen (m) 0.9-3.9 1.0-4.0 4.9-6.4 5.0-6.5

Depth of Core (m) NA 4.3-5.0 3.8-4.8 3.8-4.6

Oxygen (mg/Liter) e 3.1 0.6 0.4 1.4

Nitrate (mg/Liter) e 19.6 <1.5 <1.5 <1.5

Fe (aq, mg/Liter) e 0.2 24.6 52.0 1.8

Sulfate ( mg/Liter) e 18.9 34.8 4.0 12.8

Methane (mg/Liter) e <0.001 0.096 0.096 0.40

Eh (mV) e 196 -132 -118 -187

pH e 4.6 5.8 6.1 7.1

Benzene (mg/Liter) e <0.005 1.33 0.62 0.62

Toluene (mg/Liter) e <0.005 10.4 0.083 0.028

Ethylbenzene (mg/Liter) e <0.005 1.82 1.92 0.042

o-Xylene (mg/Liter) e <0.005 3.88 0.042 0.010

m-, p-Xylene ( mg/Liter) e <0.005 8.18 2.75 0.34

aNA - not applicable

bData are average of 2 samples taken over a 4-month period as reported by Wilson et al., 1994.

cMeasured at time of core collection.

dNM - not measured.

eData 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 et al., 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

methanogenic 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 et al. (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 et al., 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
4

0

1

2

3

4

5

6

7

Toluene (μM)

0 100 200 300 400

Days

0

200

400

600

800

1000

Methane (

μM)

b

a

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 endplume 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 et al. (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

et al. 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 et al. (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 et al., 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 et al., 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 μg/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 oxylene were below 22 μg/Liter at 120 days. Benzene degradation

began after day 180, and by day 403 the benzene concentration

was between 8 and 12 μg/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
5

0.1

1

10

100

Concentration (μM)

Abiotic Benzene

Benzene

Toluene

Ethylbenzene

m-Xylene

o-Xylene

0.1

1

10

Benzene (μM)

Benzene - Abiotic

Benzene - Live

0 100 200 300 400

Days

.0

.2

.4

.6

.8

Dissolved Fe (mM)

Fe- Abiotic

Fe - Live

.00

.05

.10

.15

Dissolved SO 4 (mM)

SO4 - Abiotic

SO4-Live

e

d

b

a

1

10

100

Total BTEX (μM)

Live

Abiotic

Abiotic 1st Order Rate

Live 1st Order Rate

c

Figure 3. Results from mid-plume RP microcosms: (a) BTEX components,

(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. Benzene data in Figure 3b are for individual 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, oxylene 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 et al., 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 and o-xylene concentrations

are low (~5 μg/Liter), but significant quantities of m-, p-xylene still

remain (>1000 μg/Liter). These results suggest that m-xylene

degradation was occurring in-situ at 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 μg/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
6

TABLE 2. First-order Decay Rates from the RP Site

Mid-Plume Microcosms Mid-Plume In-Situ Columns Field

Compound Decay ratea Decay Period Decay ratea Decay Period Decay rateb

(d -1) (days) (d -1) (days) (d -1)

Benzene 0.024 184-403 Test 1: 0.0049 Test 1: 155-251 0.0002

Test 2: 0.023 Test 2: 41-181

Toluene 0.045 22-120 NSc 0.0021

Ethylbenzene NSc Nsc

o-Xylene 37-120 NSc 0.0021

m-, p-Xylene 0.020d 0-184 0.0143 121-251 0.0013

Total BTEX 0.0066 0-403 0.0029 121-251 0.0011

a Rates represent the difference between the microcosms and control abiotic loss rate over the period of decay.

b Field decay rates were calculated over the entire length of the plume.

c Microcosms and control abiotic loss rates were not significantly different at the 99% level.

d 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

microcosms show that a large number of 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 midplume 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 μg/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 μg/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 by 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 μg/Liter). In the microcosm columns, DO remained below

detection (<0.2 mg/Liter), sulfate declined slightly from

1.3 mg/Liter to the detection limit (~0.3 mg/Liter) and pH 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 water that contained higher concentrations of benzene

(1,000 to 1,300 μg/Liter) and very low concentrations of TEX

(~ 20 μg/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
7

0.1

1

10

100

Toluene (μM)

0.1

1

10

100

Benzene (μM)

0 50 100 150 200 250 300 350

Days

0.1

1

10

Ethylbenzene (μM)

0.1

1

10

o-Xylene (μM)

0.1

1

10

m-Xylene (μM)

a

b

c

d

e

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 three controls

were sampled at each time point, except the last time point, when no control

was sampled.

1,000 μg/Liter to 11 μg/Liter in column 1 and to 8 μg/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 withm-, 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 al., 1994). One

likely cause of this difference is the procedure used to calculate

biodegradation rates. In the laboratory microcosms and the insitu 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

0 50 100 150 200 250 300

Days

10

1

10

2

103

m-,p-Xylene (

μg/L)

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.
8

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 (Figure 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

10

2

103

Benzene (

μg/L)

0 50 100 150 200 250 300 350

Days

10

1

10

2

103

Benzene (

μg/L)

b

a

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.

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, spatiallyaveraged 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, oxylene, m-xylene) appear to anaerobically biodegrade to a

low but detectable concentration (10 to 30 μg/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

measurement for 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.
9

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 Consortium. 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.

 ́  ́