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/A newsletter about soil, sediment, and ground-water characterization and remediation technologies
Issue 41
Trtw mt/e o/Technology News and Trends features site-specific approaches for applying, opti-
mizing, and evaluating the potential for or efficacy of bioremediation approaches such as sub-
surface injection ofmicrobial cultures or emulsified vegetable oil.
March 2009
DOD Evaluates Bioaugmentation for Remediating Low-pH Aquifers
The U.S. Department of Defense (DOD) has
completed pilot-scale work on the "MAG-1" site
at Fort Dix, NJ, to test suitability of anaerobic
bioremediation for an aquifer with low pH.
Project results will be used to design full-scale
ground-water treatment at MAG-1. In addition,
technical criteria gained from the demonstration
will be integrated into DOD's remediation
strategy for its many facilities situated above
low-pH Coastal Plain aquifers.
As part of recent DOD facility realignment, Fort
Dix now operates as an Army Reserve and
National Guard training reservation. The facility
is located in central New Jersey's Pine Barrens
region, within the recharge area of the Kirkwood-
Cohansey Aquifer that serves as the primary
source for the area's domestic wells. As a result
of past discharge of chlorinated solvents during
motor pool operations, the MAG-1 site contains
a significant and cumulative trichloroethene
(TCE) plume within a Class 1 -A aquifer situated
wholly beneath the property. The aquifer
comprises primarily silty fine sand, with
chlorinated volatile organic compound (CVOC)
contamination primarily in the upper part of
the formation in a saturated thickness of
approximately 10 feet.
Baseline studies of the aquifer indicated a
pH of 4.2 to 5.3 and a positive oxidation-
reduction (redox) potential ranging from 40
to 145 mV. Sulfate concentrations were
relatively low (27-61 mg/L), and dissolved iron
concentrations ranged from 3,500 to 7,500
|Ig/L. Primary contaminants are TCE andc/'s-
1,2-dichloroethene (c/'s-DCE), at pre-
treatment concentrations of 30-1500 (Ig/L
and 190-1300 (Ig/L, respectively; vinyl
chloride (VC) concentrations were below
detection limits. An initial qPCR (quantitative
polymerase chain reaction) analysis of site
samples for the dechlorinating organisms
Dehalococcoides spp. (DHC) revealed that
native DHC were present in relatively low
numbers (below 105 DHC/L).
Although anaerobic bioremediation has been
used widely for treating ground water
contaminated with chlorinated ethenes
(particularly tetrachloroethene [PCE], TCE,
and cw-DCE), past applications have been
ineffective in low-pH aquifers due to minimal
natural biodegradation activity. In addition, PCE
and TCE degradation that does occur tends to
accumulate a's-DCE as a terminal end product,
without proceeding to ethene. Formation of
large contaminant plumes in low-pH
environments over time reduces utility of
common remedial alternatives such as in situ
chemical oxidation and decreases practicality
of adjusting pH for the purpose of increasing
natural biodegradation.
Bioaugmentation suitability tests for MAG-1
began with laboratory microcosm tests. Results
confirmed that dechlorination was extremely
limited at the low natural pH; even pH adjustment
resulted in limited dechlorination by indigenous
microbial populations. Testing also suggested
that native DHC were unable to efficiently
degrade cis-DCE and a "cw-DCE stall" was
occurring in microcosms. In contrast, microcosms
inoculated with a bioaugmentation culture (SDC-
9™) and adjusted to pH levels greater than 6
demonstrated progression of TCE and cis-DCE
dechlorination and production of stoichiometric
[continued on page 2]
Contents
DOD Evaluates
Bioaugmentation
for Remediating
Low-pH Aquifers page 1
Optimization of
EVO Delivery
Improves Enhanced
Reductive
Dechlorination of
Ground Water page 3
Bioremediation/
Natural Attenuation
Continues after
ISCO Treatment page 4
Information
Resources for
Bioremediation
of Sites with
Contaminated
DNAPL
Upcoming
Nanotechnology
Conference
page6
page6
Online Resources
The "Technologies:
Remediation" focus area of
the U.S. EPA's CLU-IN
website provides additional
project profiles of
bioremediation strategies
such as bioreactors, biowalls,
bioventing, bioaugmentation,
and biostimulation. The area
also provides links to key
references including AFCEE
protocols, ITRC guidance, and
interagency training sessions.
To learn more, visit:
www.clu-in.org/remediation/.
Recycled/Recy cl abl e
Printed with Soy/Canola Ink on paper that
contains at least 50% recycled fiber
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[continued from page 1]
amounts of ethene. Microcosm results were
verified and an extended laboratory column
study was conducted to gather additional
data on dechlorination rates, bacterial
transport, and aquifer buffering.
Bioaugmentation evaluation in the field
involved construction of a recirculation
system to capture ground water at an
extraction well and re-inject it at an
injection well approximately 40 feet
upgradient. The design provided a re-
circulating treatment loop approximately
15 feet wide and 10 feet below ground
surface (bgs). In addition, two monitoring
wells were installed between the
extraction and injection wells. All wells
were screened across the entire
contaminated saturated zone.
Laboratory tests and ground-water flow
models were used to estimate the
amount of sodium bicarbonate
required to raise ground water pH to
above 6 and to determine the amount
of electron donor (sodium lactate),
nutrients (diammonium phosphate), and
bacteria needed to facilitate in situ
bioremediation. The ground-water
circulation system initially operated at
a flow rate of 0.5 L/min to create a 30-
day hydraulic flow time between the
injection and extraction wells.
Start-up operations in late 2007 included
characterization of the flow system and
efforts to reduce redox potential. The first
bio augmentation event involved injection
of 10 liters of SDC-9 culture (containing a
DHC titer of 10" DHC/L) directly into the
recirculation loop. Upon observation of
a pH spike to above pH 9 and loss of
bioaugmentation substrate, a second,
identical injection was conducted
approximately 100 days later. Each
culture cost approximately $1,000.
DHC numbers in the aquifer subsequently
increased to approximately 108 DHC/L in
a row of monitoring wells located 20 feet
from the injection wells, which suggested
significant in situ growth and distribution
100 200
Days
Figure 1. Performance monitoring of aquifer bioaugmentation during the
MAG-1 demonstration showed significant production of vinyl chloride, as a
TCE degradation end-product, beginning approximately 140 days after the
second inoculation.
of organisms concomitant with CVOC
dechlorination. SDC-9 bioaugmentation in
the re-circulation loop reduced the CVOC
concentration to approximately 20 ppb
within one year (Figure 1), from a combined
TCE and cw-DCE concentration of 1,500
ppb prior to treatment.
It was difficult to achieve and maintain an
acceptable pH in the treatment zone. During
the first three months of operation, more
than 1,700 Ibs of sodium bicarbonate
were injected into each recirculation
loop. To reduce the excessive labor or
automated loading costs associated with
administering this volume, which was
significantly higher than estimates derived
from earlier testing and the column study,
sodium carbonate was selected as an
alternate buffering agent.
The sodium carbonate additions were
found to cause the pH spike observed
approximately three months after the first
injection. Buffering in the form of
carbonate also led to excess fouling of the
injection wells, which necessitated
periodic well redevelopment and a reduced
volume of buffering agent. To maintain a
suitable pH during the remainder of the
demonstration, a total of 2,400 pounds of
sodium carbonate was used.
Analysis of post-injection data indicated
that DHC growth could not be stimulated in
the aquifer without pH adjustment, despite
existence of the organisms at measurable
levels, and that pH adjustment alone could
not facilitate biological degradation of cis-
DCE and VC. Results demonstrated that
complete dechlorination could be
achieved by concurrently adjusting the
pH to a level greater than 6 and
inoculating the formation with
bioaugmentation cultures.
Full-scale bioaugmentation at Fort Dix
is expected to begin this summer, in
accordance with final cleanup goals
set by the New Jersey Department
of Environmental Protection. The
demonstration was conducted under
DOD's Environmental Security and
Technology Certification Program, in
conjunction with development of
bioaugmentation guidance for DOD and
the U.S. Department of Energy.
Contributed by Nancy Ruiz, Naval
Facilities Engineering Service Center
(nancy.ruiz&navy.mil or 805-982-1155)
and Robert Steffan, Ph.D., Shaw
Environmental and Infrastructure, Inc.
(rob, steffan&shawgrp. com or
609-895-5350)
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Optimization of EVO Delivery Improves Enhanced Reductive Dechlorination of Ground Water
Optimization of an emulsified vegetable oil
(EVO) delivery system led to uniform
distribution and ongoing biological treatment
of a PCE source area last summer at the former
Naval Training Center (NTC) in Orlando, FL.
Remedial efforts targeted contaminated
ground water at a former laundry and dry
cleaning facility at NTC's Operable Unit 4
(OU4), where earlier injections of potassium
permanganate (KMnO4) failed to reduce
contaminant concentrations in the source
area. Performance monitoring last fall
indicated that the EVO system was operating
successfully and aquifer conditions were
favorable for enhanced reductive
dechlorination (ERD), with total organic
carbon (TOC) concentrations approaching
200 mg/L, in contrast to a pre-treatment
average of approximately 10 mg/L.
Contamination of OU4 ground water was
attributed to past spills and leaks in drain
lines, floor drains, and other components of
the dry cleaning facility's wastewater
collection and conveyance system.
Subsurface investigations detected PCE
concentrations reaching 22,600 j-ig/L, which
suggested the presence of dense non-
aqueous phase liquid (DNAPL). From
March through October 2003, a KMnO4
recirculation delivery system was
implemented. Subsequent field studies
indicated that the elevated natural oxidant
demand of the aquifer material had caused
formation of manganese dioxide solids, which
in turn plugged the KMnO4 injection wells
and limited oxidant distribution.
In 2006, an optimization study was conducted
to evaluate viable remedial alternatives for
addressing the remaining DNAPL source area.
Enhanced in situ biodegradation using EVO
was selected as a cost-effective strategy to
treat source-zone contamination, reduce mass
flux, and control plume migration over time.
Recirculation initially was selected as the
delivery method most likely to uniformly
distribute EVO in the 60- by 80-foot source
area. The target treatment zone was defined
by a PCE concentration of 2,000 (-ig/L,
approximately 1% of the compound's aqueous
solubility. Target depths extended through a
shallow source area above a 5-foot cemented
sand unit located 20-25 feet bgs. The delivery
system comprised a central recovery well
surrounded by six equally-spaced injection
wells. Based on short-duration aquifer
performance tests, an average extraction rate
of 2.5 gpm was expected in the shallow zone
(5-20 feet bgs) (Figure 2).
EVO application began in September 2007
with ground-water recovery from the single
extraction well. Based on the design flow rate,
Recovery Well .
(Recirculation, 1 of 1)
Recovery Well _
(Recirculation, 1 of 1)
Injection Well Pair -
(Recirculation, 1 of 6)
the anticipated duration of recirculation
(one pore volume) was 14 days. Performance
monitoring indicated that the maximum
sustained extraction rate of 1 gpm was well
belowthe design flow rate of 2.5 gpm, and
that two monitoring wells (MW1 and MW2)
within the recirculation footprint had not
been impacted by EVO recirculation.
Inadequate distribution of the EVO was
attributed to poor extraction from the
central recovery well. As a result, the
recirculation effort was abandoned after
approximately 10% of the design volume
was injected (approximately 8,200 gallons
of a 1% EVO solution).
Following the initial EVO recirculation event,
analysis of ground water in MW1 indicated
PCE concentrations had increased from 1,800
to 3,500 ng/L, and daughter products TCE
and cis-DCE also increased an order of
magnitude. In MW2, PCE and TCE
concentrations remained unchanged, but
cis-DCE decreased an order of magnitude.
Vinyl chloride was not detected in samples
from either monitoring well, and no changes
in TOC were observed. A significant
decrease in redox potential, from -70 to -252
mV, was observed in MW1 after two months
of recirculation. In addition, geochemical
indicators suggested that sulfate and iron
reduction were occurring, but methane
concentrations were unchanged.
Injection Well Pair
(Direct, 3 of 14)
100
Evaluation of the initial attempt
to recirculate EVO in the
shallow zone led to several
"lessons learned." A longer
aquifer test would have
evaluated sustainable injection
and extraction rates better,
thereby optimizing design of
[continued on page 4]
Figure 2. Treatment of ground
water at NTC OU4 involved
optimized delivery of EVO
through a system of shallow-
zone recirculation recovery
wells and injection wells.
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[continued from page 3]
the recirculation system array. Distances
between the injection and extraction wells
may have been too large to achieve
adequate control of the injected EVO. In
addition, injection of EVO at a higher
concentration may have resulted in better
substrate distribution in the subsurface.
Subsequent optimization activities involved:
(1) bench-scale tests to determine the
adsorptive oil capacity of the soil and optimal
EVO dose; (2) an evaluation of EVO delivery
options; and (3) a pilot-scale study to
determine optimal injection spacing. Bench-
scale tests were performed on two shallow
(fine sand) and two deep (fine to medium
sand) samples collected from outside the
target treatment zone. Results indicated a
median adsorptive capacity of 0.00384
(pounds of EVO per pound of soil), which
was greater than three times the adsorptive
capacity assumed during EVO recirculation.
Direct-push technology (DPT) points
rather than permanent injection wells were
selected for EVO delivery due to the high
cost and extensive field resources
associated with installing numerous
permanent wells. In addition, deployment
of temporary DPT points could focus EVO
injection in a discrete vertical interval.
A March 2008 pilot study evaluated 5- and
7.5-foot radii of influence for injection point
spacing. The 7.5-foot radius was selected
for full-scale delivery due to an increase in
TOC and positive results from a bromide
tracer test in the monitoring wells. Full-scale
injection of EVO, using 22 DPT points
spaced 15 feet apart, was conducted in July
2008. A 4-foot screen was used to inject
66,888 gallons of a 2.1-6.3% EVO solution
over an interval of 8-20 feet bgs.
Ground-water samples from MW1 and MW2
in November 2008 indicated that TOC
concentrations had risen from 10 mg/L to
160-180mg/L. InMW1, PCE concentrations
decreased from 1,800 ug/L to 756 ug/L, while
TCE increased from 64 ug/L to 914 ug/L, cis-
DCE increased from 87 ug/L to 11,700 ug/L,
and vinyl chloride increased from non-detect
levels to 92.9 ug/L. Similarly, concentrations
of PCE in MW2 decreased from 18,000 ug/L
to 184 ug/L, while TCE decreased from 1,200
ug/L to 38 ug/L, cis-DCE increased from 1,500
ug/L to 9,210 ug/L, and vinyl chloride
increased from non-detect concentrations
to 2,800 ug/L. Ethene was detected in MW2
at concentrations as high as 19 |Ig/L.
Methanogenesis within the treatment zone
was evidenced by an increase in methane
concentrations of up to 7 mg/L. In addition,
Dehalococcoides ethenogenes was detected
within the treatment zone in populations
reaching 7.61E+03 cells/mL. Overall results
based on formation of TCE daughter products
and the shift in aquifer geochemical
conditions (from moderately reducing to
highly reducing methanogenic conditions)
indicate that EVO optimization has improved
enhanced reductive dechlorination of OU4
ground water. Further optimization of the
ERD system will be conducted based on
future monitoring results.
Contributed by Michael A. Singletary,
NAVFAC Southeast
(michael.a.singletary(q)navy.mil or
904-542-6303) and Casey E. Hudson,
CH2MHILL, Inc. (770-604-9095 or
casev.hudson(a)ch2m. com)
Bioremediation/Natural Attenuation Continues after ISCO Treatment
Scientists and engineers from the private
sector, and EPA are evaluating the
potential for in situ bioremediation and/
or natural attenuation to successfully
treat residual soil and ground-water
contamination after source-area in situ
chemical oxidation (ISCO) treatment. The
studies were prompted by concerns that
the two treatment technologies may be
incompatible due to detrimental impacts
on microbial communities as well as
decreases in aquifer permeability resulting
from ISCO. Recent field evaluation at a
former manufacturing site in Framingham,
MA, and other investigations indicated
that adverse impacts from ISCO were not
permanent and did not affect subsequent
enhanced in situ bioremediation or
natural attenuation.
An approximate 2,000-ft2 TCE plume existed
at the Framingham site as a result of past
activities involved with electronics
manufacturing. The plume contained TCE in
high concentrations of approximately 22,000
ppb and 1,000 ppb of cis-DCE and vinyl
chloride, respectively, and was situated 20-
35 feet bgs within layered fine sands and silts.
The water table ranges from 8 to 12 feet bgs.
The presence of daughter products indicated
that reductive dechlorination was active.
In December 2001, injection of 450 gallons of a
20% solution of sodium permanganate
(NaMnO4) was performed through a single
injection well screened at 24-34 feet bgs within
the suspected source area. Atotal of 750 pounds
(dry weight) of NaMnO4 was injected over
two days. Permanganate was detected at least
30 feet downgradient of the injection point.
Analyses of ground water at downgradient
monitoring wells showed that
permanganate persisted in the aquifer for
approximately six months following
injection. The wells also showed an
immediate reduction in TCE followed by
temporary rebound attributed to slow
mass transfer and mass transport
mechanisms. Over the following 1.5
years, TCE concentrations steadily
declined to 5,000 ppb, and a small
increase in daughter product cw-DCE
was observed, showing that reductive
dechlorination activity had rebounded.
Bioremediation efforts were initiated in
2003, 30 months after the ISCO treatment,
to accelerate degradation of the
contaminant plume through bacterial
[continued on page 5]
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[continued from page 4]
processes. A total of 1,026 pounds of
sodium lactate was administered over 1.5
years during five injection events in two
of the monitoring wells located in the
I SCO treatment area and downgradient
of the ISCO injection well. Laboratory
analysis of ground-water samples
collected from treatment wells two years
after the start of sodium lactate additions
indicated TCE concentrations had
decreased to approximately 2,500 ppb.
Cw-DCE and vinyl chloride levels also
increased following lactate injection.
NaMnO4 and other ISCO oxidants such as
hydrogen peroxide, permanganate, and
ozone exhibit antiseptic properties with
potential for inhibiting or killing microbial
organisms involved in subsurface biotic
processes, particularly under anaerobic
conditions. Oxidant injection results in a
significant increase in redox potentia that
can interfere with reducing conditions
needed under anaerobic conditions, and
consequently inhibit microbial activity
and contaminant transformations. To
evaluate these impacts more closely,
microbial communities were assessed at
another site, where approximately 60,000
pounds of NaMnO4 and potassium
permanganate (KMnO4) were injected
into a two-acre area over one year. Bio-
Trap® devices were deployed in
monitoring wells for passive, in-situ
collection of microbes in ground water
over an extended time.
Microbial sampling and PLFA analysis of
three wells (a few months after and four
years after ISCO) indicated an increase in
post-oxidation biomass levels in
monitoring wells impacted by
permanganate, when compared to
upgradient ground water representative
of background conditions (Figure 3).
PLFA data from permanganate-impacted
wells also indicated a complex
consortium of microbes including
aerobes, anaerobes, and metal- and
sulfate-reducing bacteria. Collectively,
this consortium is capable of degrading
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hydrocarbons and enhancing reductive
dechlorination of CVOCs.
No loss in permeability was observed at the
Massachusetts facility, in contrast to
common assertions that reduction of
permanganate ion consistently leads to
manganese oxide (MnO2) accumulation in
porous media and associated reduction in
permeability and treatment efficacy. A
critical analysis of MnO2(s) accumulation
in porous media indicated that MnO2(s)
precipitation filled approximately 8% of the
void volume in a porous medium with an
oxidant demand of 50-60 g/kg. Other
mechanisms such as MnO2(s) particle
transport and filtration, mechanical
straining, electrostatic interactions,
chemical bridging, or specific adsorption
may cause immobilization of MnO2(s)
particles within porous media and lead to
changes in permeability.
Study of ISCO applications at multiple sites
showed that KMnO4 injections typically
involve concentrations of 2-3g/L, which are
below the solubility (6.5 g/L at 20° C), but
are sensitive to temperatures. For example,
differences in temperature between the
KMnO4 solution in a mixing tank and the
(cooler) aquifer can result in precipitation
of KMnO4 in the aquifer, causing rapid but
temporary permeability reduction. In
addition, insufficient pre-injection mixing
duration or methods can produce an
injection solution containing a significant
quantity of KMnO4 particulates. Study
findings indicated that although
accumulation of KMnO4 particulates in the
Figure 3.
Significant
changes were
observed
between pre-
and post-
oxidation
microbial
biomass in wells
impacted by
permanganate at
one study site.
aquifer can decrease permeability, the effect
typically is temporary due to dissolving of
KMnO4 over time. Similarly, generation of
carbon dioxide gas caused by microbial
activity or oxidation of organics can
temporarily decrease permeability before
the gas dissolves in water.
Results at the Massachusetts site and two
other sites gave no indication that ISCO
resulted in sterilization of aquifer material
or permanent inhibition of microbial
activity. In subsurface systems, contact
between oxidant and microbial
populations can be limited due to
preferential pathways and microniches,
allowing microbiota to survive rigorous
applications of oxidant. Spatial
separation between oxidant injection into
source areas and downgradient
microbially active areas also diminishes
the impact of the oxidant. Potential for
successful bioremediation and/or natural
attenuation following ISCO applications
may vary with oxidant, physical and
chemical characteristics of subsurface
materials, or varying contaminant
concentrations and characteristics.
Contributed by Scott Hu ling, U.S. EPA's
Robert S. Kerr Laboratory
(huling. scott(q),epa. gov or
580-436-8610, Dick Brown, ERM
(dickbrown(a)erm.com or
609-403-7530), and Robert Luhrs,
Raytheon robert_c_luhrs(q)raytheon. com
or 781-768-3995)
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Solid Waste and
Emergency Response
(5203P)
EPA 542-N-09-002
March 2009
Issue No. 41
United States
Environmental Protection Agency
National Service Center for Environmental Publications
P.O. Box 42419
Cincinnati, OH 45242
Presorted Standard
Postage and Fees Paid
EPA "
Permit No. G-35
Official Business
Penalty for Private Use $300
Information Resources for Bioremediation of
Sites with Contaminated DNAPL
EPAcontinues to collaborate with federal and state agencies to develop tools for using in
situ bioremediation as a treatment option for subsurface DNAPLs, particularly those
associated with chlorinated ethenes. In June 2008, the Interstate Technology and Regulatory
Council (ITRC) issued a guidance entitled/« Situ Bioremediation of Chlorinated Ethene:
DNAPL Source Zones. The document covers technical and regulatory issues related to
bioremediation technology selection, applicability, design, and operation and monitoring.
The full guidance is available on the ITRC website (www. itrc web . org/Do cuments/
bioDNPL_Docs/BioDNAPL3 .pdf).
The ITRC also delivered a recent internet seminar on in situ bioremediation of chlorinated
summarized general steps available to practitioners and regulators for objective
decisionmaking on bioremediation technology assessment, monitoring, and optimization
in DNAPL areas. The seminar presentations may be viewed in EPA's CLU-IN online
archives (www. clu-in. org/conf/itrc/bioDNAPL 090908). This seminar will be offered
again on June 1 1 , 2009.
CLU-DSTs contaminant focus area on DNAPLs offers a range of bioremediation information
applying to DNAPL treatment. Topics include overviews of chemical and behavioral
aspects, environmental occurrence, toxicology, detection or characterization, and treatment
technologies such as soil vapor extraction, solidification, and thermal processes. The
DNAPL focus area is at: www.clu-in.org/contaminantfocus/default.focus/sec/
Dense Nonaqueous Phase Liquids (DNAPLsVcat/Overview.
Contact Us
Technology News and Trends
is on the NET! View, download,
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Contributions may be submitted to:
John Quander
Office of Superfund Remediation
and Technology Innovation
U.S. Environmental Protection Agency
Phone:703-603-7198
quander.john@.epa.qov
Upcoming Conference
The University of Massachusetts'
Environmental Institute and the U.S.
EPA Office of Superfund Remediation
and Technology Innovation will host the
International Conference on the
Environmental Implications and
Applications of Nanotechnology on
June 9-11, 2009, in Amherst, MA
EPA is publishing this newsletter as a means of disseminating useful information regarding innovative and alternative treatment techniques and
technologies. The Agency does not endorse specific technology vendors.
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