United States
Environmental Protection
Agency
Robert S. Kerr Environmental
Research Laboratory
Ada, OK 74820
Research and Development
EPA/60QVS2-89/042 Sept, 1989
c/EPA Project Summary
In Situ Bioremediation of
Spills from Underground
Storage Tanks: New
Approaches for Site
Characterization Project
Design, and Evaluation of
Performance
John T. Wilson, Lowell E. Leach, Joseph Michalowski, Steve Vendegrift, and
Randy Callaway
The full report presents a system-
atic approach for the design of In situ
bioremediation of hydrocarbon con-
tamination in ground water from the
determination of the total quantity of
hydrocarbons in the aquifer to the
utilization of that information in an
actual field bioremediation demon-
stration. The full report explains why
the total quantity of hydrocarbons in
an aquifer can only be determined by
collecting cores. A procedure to
acquire cores from a contaminated
aquifer is described. The procedures
described in the report were field-
tested in designing a demonstration
of the bioremediation of an aviation
gasoline leak. The performance of the
demonstration was consistent with
the expected performance based on
the preliminary site characterization
using the described procedures.
This Project Summary was devel-
oped by EPA's Robert S. Kerr Envi-
ronmental Research Laboratory, Ada,
OK, and the Environmental Monitoring
Systems Laboratory, Las Vegas, NV, 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 presents a systematic
approach for the design of in situ
bioremediation of hydrocarbon contami-
nation in ground water from an initial
determination of the total quantity of
hydrocarbons in the aquifer to the
utilization of that information in an actual
field bioremediation demonstration.
Bioremediation of ground water con-
taminated with hydrocarbons such as
gasoline is an on-site treatment tech-
nology that is both potentially technically
feasible and more cost-effective than
"pump and treat" technologies that
involve pumping of contaminated ground
water to the surface and removal of the
contaminant by air-stripping or carbon
adsorption. In situ bioremediation usually
consists of modifying the environment of
an aquifer by adding oxygen and other
inorganic nutrients in order to enhance
the activity of native microbial popu-
lations in degrading contaminants. Bio-
remediation is especially promising with
hydrocarbons which are potentially bio-
degradable by native subsurface bacteria
under the right environmental conditions
to harmless byproducts.
Successful bioremediation is depen-
dent upon a number of factors, including
the hydrogeology at the site and the
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availability of critical nutrients in the
aquifer. The primary limiting factor with
hydrocarbons is the aviability of oxygen.
If sufficient oxygen is not present
naturally, then oxygen must be provided
by circulating oxygenated water through
the contaminated area until degradation
is complete.
The primary factor that determines how
much oxygen and nutrients must be
supplied to a hydrocarbon leak and how
long remediation will take is the quantity
of the hydrocarbon at the site. Normally,
the amount of the leak is not known and
available methods to determine the
amount of contaminant at the site and its
location are not acceptable.
Almost all techniques that have been
applied for the analysis of oily con-
taminants in aquifers emphasize the com-
pounds of regulatory interest, and few are
appropriate for both solids and water. All
too frequently, the only information
available from a leak site is the concen-
tration of selected organic contaminants
in water from wells. Such information is
inadequate for determining the total
quantity of hydrocarbons in the aquifer.
Therefore, it is impossible to determine
how much oxygen and nutrients must be
delivered to the aquifer to support
sufficient microbial activity to degrade all
of the contaminant to harmless by-
products.
The full report explains why the total
quantity of hydrocarbons in an aquifer
can only be determined by collecting
cores. A procedure to acquire cores from
a contaminated aquifer is described.
Before the procedure was developed, it
was very difficult to recover good-quality
cores of unconsolidated sandy material
from below the water table. The report
also describes two procedures to deter-
mine how much contamination the cores
contain. Results of the two procedures
are in good agreement, even though they
are based on different principles.
The two techniques were developed
and evaluated by scientists at the Robert
S. Kerr Environmental Research Labora-
tory (RSKERL) as part of a large biore-
mediation research program. An oil-and-
grease method was adapted to estimate
total hydrocarbons in core samples. A
second method was adapted from tech-
niques for the analysis of fuels that
determines the total content of hydro-
carbons as well as the specific content of
individual compounds of interest.
Basically, the oit-and-grease method
uses infrared spectroscopy to measure
the absorbance of carbon-hydrogen
chemical bonds. Quantitation is sensitive
to the type of hydrocarbon but is
relatively insensitive to the particular
organic constituents of the fuel. In the
fuel carbon technique the hydrocarbons
are extracted into methylene chloride,
then separated and quantified by gas
chromatography. Representative peaks
are selected, and the quantity of total
hydrocarbons is calculated by comparing
the area of the representative peaks in a
standard sample of the fuel to the area of
the same peaks in the extract. The
method works well if the standard is
representative of the material being
analyzed. If the proper calibrations are
done, the concentrations of compounds
of regulatory interest, such as the alkyl-
benzenes, can be determined in the
same analytical run. The techniques for
core analysis and their performance is
discussed in Section III of the full report.
The procedures described in the report
were field-tested in designing a demon-
stration of the bioremediation of an
aviation gasoline leak. The performance
of the demonstration was consistent with
the expected performance based on the
preliminary site characterization using the
described procedures.
Site Characterization for In Situ
Bioremediation of Hydrocarbon
Leaks from Underground
Storage Tanks
The pattern of contamination from a
leak is complex. As the release drains
through the unsaturated zone, a portion is
left behind trapped by capillary forces. If
the released material is volatile, a plume
of vapors soon forms in the soil air in the
vadose zone. If the release is a light
hydrocarbon, it will drain down to the
water table, and then spread laterally.
Ground water moving through the aquifer
comes in contact with the release, and
leaches out the more water-soluble com-
ponents. As a result there are three
distinct regions or "plumes" formed at
the leak site: a plume of volatile fumes in
the soil air, a ground-water plume, and
the region primarily in the unsaturated
zone that contains the oily-phase material
which serves as a source area for both
plumes.
In practice the source area is usually
the object of remedial activities. There is
little point in treating the ground water or
vapors if the source area is left to spread
more contamination. Therefore, the first
step is to remove any leaking tanks,
transmission pipes, and the most visibly
contaminated fill-material around the
tank. Although necessary, such practices
usually do not remove all of the sou
The material trapped in the earth so
beneath the tank will remain and
serve as a continuous source of leacf
contaminants for many years.
To intelligently remediate such a
using in situ bioremediation require;
detailed understanding of the thr
dimensional distribution of the sou
area in the subsurface and good infon
tion on the quantity of contaminant in
source area.
Unless it is known how much conts
inant has escaped into the subsurfe
and where it is located, there is
sensible way to locate injection i
extraction wells, or to optimize pump
rates and concentrations of any ame
ments. Further, there is no way
determine how much time a reme<
action will take, or how much it will cos
Conventional monitoring wells t
accurately define the geometry of
ground-water plume, but often tt
cannot distinguish the source area fr
the rest of the plume. In fresh spi
differential sorption of individual cc
ponents of the plume to the aquifer sol
can result in chromatographic separal
of the components and alter the ratic
their concentrations in water from w
distant from the source area. However
older spills, whose plumes have come
sorptive equilibrium with the aquifer,
concentration of contaminants dissoh
in the ground water is similar in
source area and in the plume, althoi
the total amount of contaminant in
source area is much greater.
For example, comparisons of groi
water analyses vs. core analyses at
aviation gasoline spill site in Michic
showed that the ground water analy:
underestimated the amount of toluene
the aquifer significantly. Further analy
showed that the core contained peti
eum hydrocarbons that sorbed most
the toluene. If the data from I
monitoring well had been used to des
a remedy, the effort and exper
required to restore the aquifer would h«
been underestimated by a factor of six
Obviously, the distribution of t
source area and the extent of conta
ination can only be characterized
collecting and analyzing cores, becat
they sample the entire aquifer, not j
the ground water. Very precise inforr
tion is needed on the vertical extent
contamination, particularly for in situ t
restoration. The injected waters are v
expensive, and water injected into a cU
part of the aquifer is wasted. If injec
water moves underneath the conte
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mated interval and breaks through in a
monitoring well, it can also give the false
impression that the region of aquifer
between the two wells is clean.
Accurate techniques for analyzing
cores to determine the total quantity of
petroleum hydrocarbons in the aquifer
and the concentration of individual com-
pounds of regulatory concern are neces-
sary not only for estimating the ultimate
demand for oxygen, but also for docu-
menting at the end of the remediation
that the clean-up is complete.
Procedure for Acquiring Core
Samples
Problems with Unconsolidated
Sediments
Traditionally, unconsolidated soils or
sediments are sampled through a hollow-
stem auger with a split-spoon core barrel
or a conventional thin-walled sample
tube. The hollow-stem auger acts as a
temporary casing to keep the borehole
open until a sample can be acquired. A
borehole is drilled down to the depth to
be sampled. Then the core barrel is
inserted through the annular opening in
the auger and driven or pushed while
rotating the auger into the earth to collect
the sample. These tools work extremely
well in both unsaturated and saturated
cohesive materials. Unfortunately, they
work poorly in noncohesive aquifer
materials, such as unconsolidated sands.
There are two technical challenges to
sampling noncohesive material below the
water table. The first challenge is to keep
aquifer material out of the annular area of
the hollow stem auger. During augering,
the annular area of the hollow-stem auger
is plugged with a solid drill head that
pushes the sand out onto the auger
flights. To sample, the drill head is
removed and replaced with a core barrel.
When the drill head is pulled out of the
auger in consolidated sands, pressure on
the aquifer sediment is reduced, and
water and fluidized sand rush into the
annular area of the auger. This incon-
venient phenomenon is commonly re-
ferred to as "heaving." The core barrel
must push through (and sample) this
heaved material inside the auger before it
reaches the undisturbed sediment under-
neath. When the core is recovered, it is
usually impossible to determine how
much of the core is the fluidized material
and how much is an authentic sample of
the aquifer. Occasionally the amount of
sediment in the auger is so great that the
core barrel cannot be pushed, and no
sample can be acquired.
The second challenge is to keep the
sample in the core barrel while it is being
retrieved to the surface. When the
sampling tool is pulled out of the aquifer,
the pressure holding the sample in the
tool is reduced. Noncohesive sediment
will often fluidize and dribble out of
conventional core barrels.
Special Piston Sampling
Conventional practice to keep
sediments out of the hollow-stem of an
auger is to fill the hollow annular column
with drilling mud. As the borehole is
advanced, the weight of the mud
stabilizes the hydraulic pressure of the
aquifer. The use of drilling mud is not
acceptable in geochemical assessments
because fluids or chemicals introduced
into the borehole can drain into the
aquifer and alter the geochemistry of the
pore water or contaminate the sample
with foreign microorganisms. Such com-
promised samples cannot be used to
assess prospects for bioremediation, and
there is a strong possibility of microbial
alteration of the sample during shipment
or storage.
The staff of RSKERL have developed
and tested new tools and protocols that
consistently provide samples of the
quality needed to characterize spills from
underground storage tanks (Leach et al.,
1988). The tools and protocols are
modifications of techniques pioneered by
others, principally researchers at the
Institute for Ground Water Research,
University of Waterloo, Ontario, Canada
(Zapico et al., 1987).
Zapico et al. (1987) recently described
a sampling device that effectively retains
unconsolidated sands inside a cannister
fitted inside a core barrel. A sliding piston
inside the cannister maintains an air-tight
seal on the core. Vacuum and friction
keep the core in place. This device was
modified to meet the special require-
ments of the RSKERL protocol.
During field evaluation at Traverse City,
Michigan, the piston core barrel worked
very well, but only when a core retainer
basket was used. The piston core
sampler without a core retainer basket
often lost half or more of the sample
before it could be recovered. A conven-
tional core barrel with a core retainer
basket recovered no sample at all. The
combination of the two consistently
recovered more than 95% of the cored
interval (12 boreholes, more than 50
cores).
After the piston core barrel is brought
to the surface, the end of the sampler is
quickly covered with a plastic bag and
tightly sealed to minimize aeration of the
exposed core. The sampler is then
quickly disassembled by removing the
drive cap and manually pulling the piston
free from the top of the sample tube.
Then one end of the core barrel is
connected to a hydraulic ram mounted on
the rig, and the core is extruded. The
cores are collected in wide-mouth
canning jars. If possible, each jar is
entirely filled with sample. The seal on
the lid of the canning jar effectively
excludes oxygen and prevents loss of
volatiles.
Field Glove Box Sampling
If the cores are to be used for
treatability studies to evaluate the
prospects for bioremediation, they must
be protected from contamination by
foreign microorganisms. If naturally oc-
curring microbial processes are to be
evaluated, they must also be protected
from the atmosphere because many
anaerobic microorganisms are killed by
oxygen.
To protect from foreign microorga-
nisms, a core is collected by extruding a
small portion of the core, breaking off a
small section to reveal an uncon-
taminated face, then installing a sterile
paring device onto the end of the sample
tube. This tool peels away the outer
contaminated wall of the core as the
material is extruded.
To protect the sample from the atmos-
phere, the sample is extruded inside a
nitrogen-filled glove box. The core barrel
is introduced into the glove box through
an iris port that makes a tight seal around
the barrel.
The glove box is prepared for sample
collection by filling it with the desired
number of sterile canning jars and sterile
paring devices, sealing the box, and then
purging it with nitrogen gas. To prevent
oxygen contamination when the jars are
opened to receive the core in the field
glove box, the jars are filled with nitrogen
before they are brought to the field. They
are passed into a laboratory anaerobic
glove box, opened, then sealed air-tight.
A slight positive pressure of nitrogen is
maintained in the box during extrusion
and collection of the cores.
Procedures to Determine the
Concentration of Contaminants
The two techniques were developed
and evaluated by scientists at the
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RSKERL as part of a large biore-
mediation research program. An oil-and-
grease method was adapted to estimate
total hydrocarbons in core samples. A
second method was adapted from
techniques for the analysis of fuels that
determines the total content of hydro-
carbons as well as the specific content of
individual compounds of interest.
Basically, the oil-and-grease method
uses infrared spectroscopy to measure
the absorbance of carbon-hydrogen
chemical bonds. Quantitation is sensitive
to the type of hydrocarbon but is
relatively insensitive to the particular
organic constituents of the fuel. In the
fuel carbon technique the hydrocarbons
are extracted into methylene chloride,
then separated and quantified by gas
chromatography. Representative peaks
are selected, and the quantity of total
hydrocarbons is calculated by comparing
the area of the representative peaks in a
standard sample of the fuel to the area of
the same peaks in the extract. The
method works well if the standard is
representative of the material being
analyzed. If the proper calibrations are
done, the concentrations of compounds
of regulatory interest, such as the alkyl-
benzenes, can be determined in the
same analytical run
Comparison of the Methods
The fuel carbon method and the oil and
grease method compare favorably, even
though they are based on entirely
different principles (Powell et al., 1988).
The fuel carbon analysis is preferred at
RSKERL because it also provides infor-
mation on the concentration of alkyl-
benzenes in waste oils.
Field Demonstration of
Sampling and Analytical
Procedures in Designing a
Bioremediation
In 1969, a spill of aviation gasoline from
an underground storage tank at the U.S.
Coast Guard Air Station at Traverse City,
Michigan, contaminated a shallow, sandy,
water-table aquifer. Ground water moving
through the spill produced a large plume
that eventually moved off the base and
ruined a large number of domestic water
wells in a residential area. The spill
contained at least 25,000 gallons of
aviation gasoline, which drained to the
water table 16 feet below land surface,
then spread laterally in the capillary
fringe to contaminate a section of aquifer
about 80 yards in diameter.
Design of the Experiment
In 1988 the U.S. Coast Guard and the
U.S. EPA installed a pilot-scale study of
bioremediation in the area of the original
spill. The alkylbenzenes are the object of
the regulatory concern, and the biore-
mediation will be finished when their con-
centration is brought below 5 ug/liter, as
specified in a consent decree between
the Michigan Department of Natural
Resources and the U.S. Coast Guard.
Cores were acquired from the source
area to determine the vertical and hori-
zontal extent of contamination, the conc-
entration of total hydrocarbons in the
contaminated interval, and concentrations
of individual alkylbenzenes. The aviation
gasoline was composed primarily of
branched-chain alkanes. The material
spilled at Traverse City was 38% 2,2,4-
trimethylpentane; 15% 2,2,5-trimethyl-
hexane, 14% 2,3-dimethylpentane; 13%
2,4-dimethylhexane; 7% 2,3-dimethyl-
hexane; and 5% 2,4-dimethylpentane.
Only 10% of the original spill was
alkylbenzenes.
The gasoline was confined to a narrow
interval between 15 and 17 feet below the
land surface. This interval corresponds
closely with the seasonal high and low
water table at the site.
This information was used to identify
the most contaminated flow path through
the spill. A series of miniature monitoring
wells was installed along and below the
most contaminated flow path.
A set of infiltration wells was installed
to perfuse the contaminated area with
mineral nutrients, and oxygen or hydro-
gen peroxide.
Injection began the first week of March,
1988. The system was first acclimated to
oxygen, then switched to hydrogen per-
oxide. The concentration of hydrogen
peroxide was increased slowly, to allow
time for microbial acclimation to concen-
trations of hydrogen peroxide that are
generally toxic to most heterotrophic
bacteria.
Estimate of Oxygen Demand
Required for Remediation
The concentration of total petroleum
hydrocarbons in the most contaminated
interval near the infiltration wells was near
300 mg/kg. The highest measured con-
centration of total hydrocarbons near a
monitoring well 31 feet down gradient
from the injection wells is 8,400 mg/kg
(core 50AE4 in Figures 10 and 11 in the
full report). The highest measured con-
centration 60 feet down gradient is 6,500
mg/kg (core 50114 in Figures 19 and
in the full report). The average of co
50AE4 and 50114 (7,500 mg/kg) vi
taken as the best estimate of t
concentration of total petroleum hyd
carbons in the most contaminated inter
between the monitoring wells at 31 z
50 feet. The interval between the inject
wells and the monitoring wells could i
be cored because access was block
by a sanitary sewer line. The most ci
servative estimate would consider 1
entire interval between the injection w<
and the monitoring well at 31 feet to
contaminated at 7,500 mg/kg. The m<
liberal estimate would consider the int
val to be contaminated at 300 mg/kg.
arbitrary intermediate estimate woi
average 7,500 and 300 mg/kg. The o:
gen demand along the most conta
inated interval was calculated for all thi
estimates.
To calculate the theoretical oxyg
demand of the hydrocarbons in a se
ment of a flow path, the hydrocarb
content (mg hydrocarbon/kg aquifer) v\
multiplied by the bulk density of t
sediment (2.0 kg/liter) and divided by I
porosity of the aquifer (0.4 liter pc
space/liter total volume) to determine I
quantity of hydrocarbon exposed to ec
liter of pore water in the segment. T
quantity of hydrocarbon was multipli
by its oxygen demand to estimate 1
quantity of oxygen that must be deliver
to each liter of pore water in the segme
The interval from the injection wells
the monitoring well 31 feet down gradi<
was considered one segment. The (
mand in the flow path to the monitori
well 50 feet down gradient was estimal
as the weighted average of the dema
in the segment from the injection wells
31 feet, and in the segment from 31 to
feet.
Performance of the
Demonstration
The interval between the injecti
wells and the monitoring wells was cc
sidered remediated when detectable O)
gen broke through and alkylbenzen
disappeared. The interval to the mo
toring well at 31 feet was remediat
after 220 days (Julian Date 281), and I
interval to the monitoring well at 50 f(
was remediated after 270 days (Juli
Date 331).
The seepage velocity (as determin
by the tracer tests) was multiplied by 1
concentration of oxygen or hydrog
peroxide in the injection wells to det
mine the instantaneous flux of oxygen
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hydrogen peroxide along the flow path.
'he cumulative flux at the time of
remediation was considered the actual
oxygen demand for remediation.
The aquifer was purged of alkyl-
benzenes very quickly. Aviation gasoline
is composed primarily of branched-chain
alkanes. Only 10% of the original spill
was alkylbenzenes. The quantity of oxy-
gen and hydrogen peroxide required to
remove alkylbenzenes from the wells
agreed closely with the projected oxygen
demand of the alkylbenzenes alone.
This may, to some extent, be
fortuitous. Some of the alkylbenzenes
must have been washed from the source
area by simple physical weathering,
resulting from their relatively high water
solubility. Some of the alkylbenzenes
may have been removed by anaerobic
biological processes before the front of
oxygen swept through. Water from
anaerobic regions of the demonstration
contained significant concentrations of
volatile fatty acids and was visibly turbid
with microorganisms. In any case, the
flow paths to the monitoring wells at 31
and 50 feet from the injection wells were
remediated when a small fraction of the
oxygen demand of the spill had been
supplied.
Contribution of Water Washing
A significant fraction of the alkyl-
benzenes may simply be washed out of
the demonstration area by the flow of
water, instead of being destroyed by
biodegradation. The significance of this
physical weathering can be evaluated by
comparing the retardation factor of each
alkylbenzene in the most contaminated
interval to the number of pore volumes of
water that have been delivered to a
particular point.
After comparing the number of pore
volumes of water delivered along the
most contaminated interval to the pre-
dicted retardation ratios of individual
alkylbenzenes in the field demonstration,
it is evident that benzene could easily
have been removed by water washing,
and that a fraction of the toluene may
have been removed, but hardly any
removal of the xylenes, ethylbenzene, or
trimethylbenzene can be expected.
Confirmation of Remediation
The spill was cored in August 1987 to
provide information to design the demon-
stration, then cored again in March 1988,
just before the demonstration began, to
define the initial conditions. The propor-
tion of alkylbenzenes in the spill declined
modestly over the time interval. This was
probably due to anaerobic microbial
degradation as discussed earlier.
Shortly after the breakthrough of
oxygen in monitoring well BD 31-2, the
area near the monitoring well was cored
and analyzed for alkylbenzenes and total
fuel hydrocarbons. The aliphatic hydro-
carbons remained at their initial concen-
tration, but the alkylbenzenes were below
the analytical detection limit. It is not
surprising that the non-aromatic fraction
of the spill remained in the aquifer. A
very minor fraction of their oxygen
demand had been supplied when the
aquifer was cleansed of alkylbenzenes.
When the region near BD31-2 was
cored in March of 1 989, almost all the
petroleum hydrocarbons had been re-
moved, including the branched-chain
alkanes.
A core taken from a region in the
demonstration area where oxygen was
depleted showed an interesting pattern.
Toluene is depleted at one location even
though significant quantities of benzene
and ethylbenzene remain. It is difficult to
rationalize the selective removal of
toluene through some purely physical
mechanism.
References
Leach, Lowell E., F. P. Beck, J. T.
Wilson, and D. H. Kampbell. 1988.
Aseptic Subsurface Sampling Tech-
niques for Hollow-Stem Auger Drilling.
Proceedings of the Second National
Outdoor Action Conference on Aquifer
Restoration, Ground Water Monitoring
and Geophysical Methods, Vol. 1, pp.
31-51.
Powell, R. M., R. W. Callaway, J. T.
Michalowski, S. A. Vandegnft, M. V.
White, D. H. Kampbell, B. E. Bledsoe,
and J. T. Wilson. 1988. Comparison of
Methods to Determine Oxygen Demand
for Bioremediation of a Fuel Contam-
inated Aquifer. Intern. J. Environ. Anal.
Chem., Vol. 34, pp. 253-263.
Zapico, Michael M., S. Vales, and J. A.
Cherry. 1987. A Wireline Piston Core
Barrel for Sampling Cohesionless Sand
and Gravel Below the Water Table.
Ground Water Monitoring Review, Vol.
7, No. 3, pp. 74-82.
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The EPA authors, John T. Wilson (also the EPA Project Officer, see below) and
Lowell £ Leach are with the Robert S. Kerr Environmental Research Laboratory,
Ada, OK 74820; Joseph Michalowski, Steve Vendegrift, and Randy Callaway are
with N.S.I. Technology Services, Inc., Ada, OK 74820.
The complete report, entitled "In Situ Bioremediation of Spills from Underground
Storage Tanks: New Approaches for Site Characterization Project Design, and
Evaluation of Performance," (Order No. PB 89-219 976/AS; Cost: $15.95, 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:
Robert S. Kerr Environmental Research Laboratory
U.S. Environmental Protection Agency
Ada, OK 74820
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
EPA/600/S2-89/042
000085833
16BICf
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