N-/EPA
United States
Environmental Protection
Agency
Office of
Solid Waste and
Emergency Response
EPA/540/2-917013B
July 1991
Guide for Conducting Treatability
Studies under CERCLA: Aerobic
Biodegradation Remedy Screening
Office of Emergency Response and Remedial Response
Hazardous Site Control Division OS-220
QUICK REFERENCE FACT SHEET
Section 121(b) of CERCLA mandates EPA to select remedies that "utilize permanent solutions and alternative treatment
technologies or resource recovery technologies to the maximum extent practicable" and to prefer remedial actions in which treatment
"permanently and significantly reduces the volume, toxicity, or mobility of hazardous substances, pollutants, and contaminants is
a principal element." Treatability studies provide data to support remedy selection and implementation. They should be performed
as soon as it becomes evident that the available information is insufficient to ensure the quality of the decision. Conducting
treatability studies early in the remedial investigation/feasibility study (RI/FS) process should reduce uncertainties associated with
selecting the remedy and should provide a sound basis for the Record of Decision (ROD). Regional planning should factor in the time
and resources required for these studies.
This fact sheet provides a summary of information to facilitate the planning and execution of aerobic biodegradation remedy
screening treatability studies in support of the RI/FS and the remedial design/remedial action (RD/RA) processes. This fact sheet
follows the organization of the "Guide for Conducting Treatability Studies Under CERCLA: Aerobic Biodegradation Remedy
Screening, Interim Guidance," EPA/540/2-91/013A, July 1991. Detailed information on designing and implementing remedy screening
and remedy selection treatability studies for aerobic biodegradation is provided in the guidance document. This guidance discusses
only screening of biological treatment. Remedy selection guidance for aerobic biodegradation is currently in the planning stages.
INTRODUCTION
There are three levels or tiers of treatability studies: remedy
screening, remedy selection and remedy design. The "Guide for
Conducting Treatability Studies Under CERCLA: Aerobic
Biodegradation Remedy Screening" discusses only the remedy
screening level.
Remedy screening studies are designed to provide a quick
and relatively inexpensive indication of whether biological
degradation is a potentially viable remedial technology. Remedy
selection and remedy design studies will also be required to
determine if bioremediation is a viable treatment alternative for
a site. The remedy screening evaluation should provide a
preliminary indication that reductions in contaminant
concentrations are due to biodegradation and not abiotic
processes such as photo decomposition or volatilization. It will
also produce the design information required for the next level of
testing, should the laboratory screening evaluation be
successful. Aerobic biological remedy screening study should
not be the only level of technology screening performed before
final remedy selection.
TECHNOLOGY DESCRIPTION AND
PRELIMINARY SCREENING
Technology Description
Bioremediation generally refers to the breakdown of organic
compounds (contaminants) by micro-organisms. In situ,
solid-phase, slurry-phase, soil heaping and composting
biological treatment techniques are available for the remediation
of contaminated soils. Aerobic biodegradation can be used as
the only treatment technology at a site or along with other
technologies in a treatment train. Use of aerobic biodegradation,
especially in situ, has been limited at CERCLA sites. However,
the technology shows promise for degrading, immobilizing or
transforming a large number of organic compounds commonly
found at CERCLA sites to environmentally acceptable
compounds.
As of fiscal year 1989 (FY89), in situ biodegradation has
been selected as a component of the remedy for 22 Superfund
sites having groundwater, soils, sludges, or sediments
contaminated with various volatile organics; phenols; creosotes;
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polynuclear aromatic hydrocarbons (PAHs); and benzene, tolu-
ene, ethyl benzene, and xylene (BTEX) compounds.
The determination of the need for and the appropriate level
of treatability studies required is dependent on the literature
information available on the technology, expert technical
judgement, and site-specific factors. Several reports and
electronic data bases exist which should be consulted to
assist in planning and conducting treatability studies as well as
help prescreen bioremediation for use at a specific site. Site-
specific technical assistance is provided to Regional Project
Managers (RPMs) and On-Scene Coordinators (OSCs) by the
Technical Support Project (TSP).
Prescreening Characteristics
One of the major parameters that influence the feasibility
of using biological processes is the biodegradability of the
compounds of concern. Prior to conducting a remedy screen-
ing of bioremediation it is important to confirm that the com-
pounds of concern are indeed amenable to biological
treatment. Consultation with experts and the TSP is critical at
this stage.
A literature search should be performed for the compounds
of wastes of interest, including compounds of similar structure.
The literature review should not be limited to a biodegradation
technology which has been chosen for preliminary
consideration. The key question to be answered is whether any
evidence of aerobic biodegradation of these compounds or
wastes exist.
The literature search should also investigate the chemical
and physical properties of the contaminants. The volatility of
the contaminants is one of the most important physical
characteristics. Knowledge of the contaminant volatility is
important in the prescreening step since highly volatile
contaminants may be volatilized, especially in stirred or highly-
aerated reactors, before biodegradation can proceed.
There is no steadfast rule which specifies when to proceed
with laboratory screening and when to eliminate aerobic
biodegradation as a treatment technology based on a
preliminary screening analysis. A literature search indicating
that biodegradation is unlikely should not automatically
eliminate aerobic biological technologies from consideration.
On the other hand, previous studies indicating that pure
chemicals will be degraded must be viewed with caution.
Chemical interactions or inhibitory effects of contaminants can
alter the biodegradability of chemicals in complex mixtures
frequently found at Superfund sites. An analysis of the existing
literature coupled with the site characterization will provide the
information required to make an "educated decision". However,
when in doubt, a laboratory screening study is recommended.
Examples of classes of compounds which are readily
amenable to bioremediation are: petroleum hydrocarbons
such as gasoline and diesel; wood treating wastes such as
creosote and pentachlorophenol; solvents such as acetone,
ketones and alcohols; and aromatic compounds such as ben-
zene, toluene, xylenes, and phenols. Several documents/review
articles which present detailed information on the biodegradability
of compounds are listed in the reference section of the complete
guidance document. However, discretion should be exercised
when using these reference materials, as micro-organisms which
can biodegrade compounds which have traditionally been
considered non-biodegradable are continually being isolated
through ongoing research and development efforts.
Technology Limitations
Many factors impact the feasibility of aerobic biodegradation
in addition to the inherent biodegradability as measured in the
screening test. These factors should be addressed prior to the
selection of aerobic biodegradation, and prior to the investment
of time and funds in further testing. A more detailed discussion
of these factors is presented in the guidance document.
THE USE OF TREATABILITY STUDIES IN
REMEDY EVALUATION
Treatability studies should be performed in a systematic
fashion to ensure that the data generated can support the remedy
evaluation and implementation process. A well-designed
treatability study can significantly reduce the overall unceratinty
associated with the decision, but cannot guarantee that the
chosen alternative will be completely successful. Care must be
exercised to ensure that the treatability study is representative of
the treatment as it will be employed (e.g., the sample is
representative of waste to be treated) to minimize the uncertainty
in the decision. The method presented below provides a
resource-effective means for evaluating one or more technologies.
There are three levels or tiers of treatability studies: remedy
screening, remedy selection and remedy design. Some or all of
the levels may be needed on a case-by-case basis. The need for
and the level of treatability testing required are management
decisions in which the time and cost necessary to perform the
testing are balanced against the risks inherent in the decision
(e.g., selection of an inappropriate treatment alternative). Figure
1 shows the relationship of three levels of treatability study to
each other and to the RI/FS process.
Remedy Screening
Remedy Screening is the first level of treatability testing for
aerobic biological technologies. It is used to establish the validity
of a technology to treat a particular contaminant. These studies
are generally low cost (e.g., $10,000-$50,000) and usually require
1 week to several months to complete. They yield data that can
be used as a preliminary indication of a technology's potential to
meet performance goals and can identify operating standards for
investigation during remedy selection testing. They generate little,
if any, design or cost data and should not be used as the sole
basis for selection of a remedy.
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Scoping
- the -
Ri/FS
Remedial investigation;
Feasibility Study (RI/FS)
Identification
of Alternatives
Record of
Decision
(ROD)
Remedy
Selection
Remedial Design.
Remedial Action
(RD/RA!
Site
Characterization
and Technology
Screening
REMEDY
SCREENING
to Determine
Technology Feasibility
Evaluation
of Alternatives
REMEDY SELECTION
to Develop Performance
and Cost Data
Implementation
of Remedy
REMEDY DESIGN
to Develop Scale-Up, Design,
and Detailed Cost Data
Figure 1. "Hie Role of Trecrtabllity Studies In the Ri/FS and iO/RA Process
Typically, laboratory-scale aerobic biological screening
studies are performed in test reactors provided with sufficient
nutrients and oxygen. These reactors may be small sacrificial
batch reactors (approximately 40 ml to one liter in size) or
larger ecosystems (1 to 10 liters) which are subsampled to
monitor the progress of biodegradation. The reactors may
contain saturated or unsaturated soil or slurries in water.
Normally, pH and contaminant loading rates are adjusted to
increase the chances of success. The microbial population
can be indigenous to the site, from another acclimated source
(i.e., wastewater treatment sludge or another area on site),
selectively cultured, a proprietary mixture provided by a
vendor, or any combination of the above. The bioreactors are
set up for replicate sampling at several time points. The test
reactors are compared to inhibited controls at each time point
to determine if aerobic biological degradation occurred. The
inhibited reactors are treated with sterilization agents in an
effort to reduce or eliminate the biological activity in the
control reactors. The mean contaminant concentration in the
inhibited control replicates is subtracted from the mean
contaminant concentration in the test reactors. The goal for a
successful treatability test is a removal rate, due to biological
processes, which is greater than the analytical error inherent
in the test design. A reduction of the contaminant
concentration over a three to six week period of 20%
(minimum) to 50% or 60% (corrected for non-biological
losses) would be typical. The goals of remedy screening are
discussed below.
REMEDY SCREENING TREATABILITY
STUDY WORK PLAN
Carefully planned treatability studies are necessary to
ensure that the data generated are useful for evaluating the
validity or performance of a technology. The Work Plan, which
is prepared by the contractor when the Work Assignment is
in place, sets forth the contractor's proposed technical
approach for completing the tasks outlined in the Work
Assignment. It also assigns responsibilities and establishes
the project schedule and costs. The Work Plan must be
approved by the RPM before initiating subsequent tasks. A
suggested organization of the Work Plan is provided in the
"Guide for Conducting Treatability Studies Under CERCLA:
Aerobic Biodegradation Remedy Screening."
Test Goals
Setting goals for the treatability study is critical to the
ultimate usefulness of the data generated. Goals must be
defined before the treatability study is performed. Each tier of
treatability study needs performance goals appropriate to that
tier.
The main goals of the remedy screening evaluation are to:
Provide an indication that reductions in contaminant
concentrations are due to biodegradation and not
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abiotic processes such as photodecomposition,
volatilization, and adsorption.
Produce the design information required for the next
level of testing, should the screening evaluation be
successful.
Normally, the average contaminant concentration should
be reduced by at least 20% during a six- to eight-week study,
as compared to an inhibited control, to conclude aerobic bio-
degradation is a potential treatment technology for the site
under investigation. The 20% contaminant reduction is a
matter of professional judgment, but is designed to maximize
the chances of success at the remedy screening tier. The
choice of a six- to eight-week study is to provide a consistent
endpoint for remedy screening studies. The choice of the
remedy screening treatability study goals (time and
contaminant reduction) will be site-specific decisions.
Experimental Design
A number of different approaches can be used to conduct
the remedy screening test. These range from simple shake
flask evaluations to soil pans or soil slurry reactors. The soil
may be either saturated or unsaturated, depending on the
goals of the study. Soil slurries will optimize mixing and will
tend to maximize biological degradation. Such studies will
maximize the chances of success at the remedy screening
level. Unsaturated soils will often limit mixing and result in
slower degradation rates. However, such systems will corre-
late better with field conditions in many cases and result in
better extrapolation to remedy selection test systems. The
object of this guidance document is not to specify a particular
remedy screening method but rather to highlight those critical
parameters which should be evaluated during the laboratory
test.
The test should include controls to measure the impact of
abiotic (non-biological) processes such as volatilization, sorp-
tion, and photodecomposition on the concentrations of con-
taminants. Inhibited controls can be established by using
formaldehyde, mercuric chloride or sodium azide to inhibit
microbiological activity. However, care should be exercised
when selecting a sterilizing agent. For example, sodium azide
can, under certain circumstances, promote spontaneous
explosive reactions. Mercuric chloride complexes certain
petroleum hydrocarbons and results in artificially low
hydrocarbon concentrations. Soil structure can also be
modified by sterilization agents.
Complete sterilization of soils can be difficult to
accomplish. Incomplete mixing of sterilization agents with
soils can result in pockets of surviving microbes in soil pores.
In some cases, microbial populations can transform and
detoxify sterilizing agents. Complete sterilization of the control
is not necessary, provided that biological activity is inhibited
sufficiently so that a statistically significant difference between
the test and control means can be determined. However, care
should be taken in interpreting remedy screening study
results. Substantial degradation In the control (e.g., 20-50%
contaminant reduction, or more) can mask the fact that
biodegradation occurred in the test reactor. If the control
reactor has the same or greater percent degradation as the
test reactor, a false negative conclusion can result.
Concluding that no biodegradation occurred, when in fact
there was some biodegradation, can lead to elimination of this
technology unnecessarily. Alternatively, closed test systems
with volatile traps can be used to monitor the volatilization of
compounds instead of using inhibited controls to estimate
abiotic losses.
A statistical experimental design should be used to
conduct the treatability study in order to support decisions
made from the treatability data. The various parameters of
interest are included as factors in the experimental design.
The treatability experiment should include monitoring the
concentration of chemicals of interest overtime. In general, at
least 3 to 4 time periods should be studied, including the
time-zero (T0) analysis. However, if the study goals are met
after a sampling period, then it is not necessary to continue
sampling at additional time periods. (For example, if 70%
reduction was achieved after one week, it would not be
necessary to continue testing if the goal was only to achieve
20% reduction.)
The test system can consist of a single large reactor or
multiple small reactors. In the case of the single reactor,
small subsamples are removed at various times and
compared to subsamples from a second reactor in which
biological activity has been inhibited. Normally, triplicate
subsamples are taken at each time point. The mean
contaminant concentration in the inhibited control subsample
is compared to that in the test subsample to determine
whether statistically significant biodegradation of contaminant
occurred at each time point. In this type of system,
heterogeneity within the soil system can lead to variability in
contaminant concentration among the various subsamples
and replicates. However, such system variability can be
overcome by thorough mixing of the soil before it is distributed
to the test and control systems. Care must be taken to
minimize the release of volatiles during mixing. Examples of
this type of system are large flasks, soil pans and other large
soil reactors. Care should be taken so that the system size
and design do not limit the availability of oxygen and moisture
and cause variability in degradation rates within the reactor.
Multiple reactors may be set up in place of a large soil
system. Triplicate reactors are established for each test
reactor and control group at each time point. Each reactor is
filled with the same amount of soil and nutrient additives. In
this case, the complete reactor contents are extracted and
analyzed for each of the triplicate test and control reactors at
each time point. Examples of such systems are serum
bottles, slurry reactors and aerated soil reactors. The
advantage of this type of experimental apparatus is that the
question of subsampling representativeness is avoided.
However, the representativeness of any one reactor is
questionable in this design. Thorough mixing of the soil,
before it is distributed among the individual reactors, is
important.
Respirometric measurements or other measures of bio-
logical activity can be used to predict the best times to take
samples. At the beginning of the experiment, activity mea-
surements should indicate minimal biological activity. Con-
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tinued monitoring can reveal either a rapid or relatively slow
onset of biological activity, and give a good indication of when
samples should be taken to monitor contaminant reductions.
However, respirometric measurements can indicate the loss
of oxygen through chemical oxidation in addition to
biodegradation.
In formulating an experimental design, the total number of
samples taken depends on the desired difference in
concentrations that the experimenter wishes to detect, the
measurement variability (the analytical coefficient of variation),
and the statistical error probabilities.
SAMPLING AND ANALYSIS PLAN
The Sampling and Analysis Plan (SAP) consists of two
parts-the Field Sampling Plan (FSP) and the Quality
Assurance Project Plan (QAPjP). A SAP is required for all
field activities conducted during the RI/FS. The purpose of the
SAP is to ensure that samples obtained for characterization
and testing are representative and that the quality of the
analytical data generated is known. The SAP addresses field
sampling, waste characterization, and sampling and analysis
of the treated wastes and residuals from the testing apparatus
or treatment unit. The SAP is usually prepared after Work
Plan approval.
Field Sampling Plan
The FSP component of the SAP describes the sampling
objectives; the type, location and number of samples to be
collected; the sample numbering system; the necessary
equipment and procedures for collecting the samples; the
sample chain-of-custody procedures; and the required
packaging, labeling and shipping procedures.
Field samples are taken to provide baseline contaminant
concentrations and material for the treatability studies. The
sampling objectives must be consistent with the treatability
test objectives. Because the primary objective of remedy
screening studies is to provide a first-cut evaluation of the
extent to which specific chemicals are removed from the soil
by biological process, the primary sampling objectives should
include, in general:
Acquisition of samples representative of conditions
typical of the entire site or defined areas within the
site. Because this is a first-cut evaluation, elaborate
statistically designed field sampling plans may not be
required. Professional judgment regarding the
sampling locations should be exercised to select
sampling sites that are typical of the area (pit,
lagoon, etc.) or appear above the average
concentration of contaminants in the area being
considered for the treatability test. This may be
difficult because reliable site characterization data
may not be available early in the remedial
investigation.
Acquisition of sufficient sample volumes necessary
for testing, analysis, and quality assurance and
quality control.
Quality Assurance Project Plan
The Quality Assurance Project Plan should be consistent
with the overall objectives of the treatability study. At the
remedy screening level the QAPjP should not be overly
detailed.
The intended purpose of this study is to determine if the
concentration of the target compounds decreases at least
20% in the biological reactor compared to the inhibited control
at an 80% confidence level. Only the relative accuracy of the
analytical measurements and the overall precision of the
experiments are important. The suggested QC approach will
consist of:
Triplicate samples of both reactor and inhibited
control at each sampling time
The analysis of surrogate spike compounds in each
sample
The extraction and analysis of a method blank with
each set of samples
The analysis of a matrix spike in approximately 10
percent of the samples.
The analysis of triplicate samples provides for the overall
precision measurements that are necessary to determine
whether the difference is significant at the 80 percent
confidence level. The analysis of the surrogate spike will
determine if the analytical method performance is consistent
(relatively accurate). The matrix spike will be used to measure
overall analytical accuracy. The method blank will show if
laboratory contamination has had an effect on the analytical
results.
Selection of appropriate surrogate compounds will depend
on the target compounds identified in the soil and the
analytical methods selected for the analysis.
TREATABILITY DATA INTERPRETATION
The information and results gathered from the remedy
screening are used to determine if bioremediation is a viable
treatment option and to determine if additional remedy
selection and remedy design studies are needed prior to the
implementation of a full-scale bioremediation process. A
threshold of greater than 20% reduction in the concentrations
of the compounds of concern, compared to the abiotic control,
indicates that bioremediation is potentially a viable cleanup
method and further testing is warranted. For some compounds
or sites, a period of time longer than the typical 6-8 weeks
may be indicative of a successful remedy screening study. An
example method for interpreting the results from a remedy
screening treatability study is provided below in Example 1.
Other specifically valid statistical methods may be used as
appropriate.
If the remedy screening indicates that bioremediation is
a potential cleanup option then remedy selection studies
should be performed.
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Example 1.
In a remedy screening treatability study for soil contaminated with a solvent, the average solvent concentrations in both
the inhibited control and in the biologically active system were 1300 ppm at T0. The average solvent concentration in the
inhibited control was reduced to 550 ppm (T3), a reduction of greater than 57 percent (Table 6-1). The average hydrocarbon
concentration in the biologically active system was reduced to 200 ppm CQ, a reduction of greater than 84 percent for the
same time period.
TABLE 6-1. Hydrocarbon Concentration (ppm) Versus Time
SAMPLE
Inhibited Control (C)
Replicate 1
Replicate 2
Replicate 3
Mean Value
Concentration Change
(Cifl-Cit) (1 = 0,1,2,3)
Bioreactor (C^)
Replicate 1
Replicate 2
Replicate 3
Mean Value
Concentration Decrease
(Cb0-Cbt) (1 = 0,1,2,3)
T0
1220
1300
1380
BOO {eg
0
1327
1320
1253
1300 (Cb0)
0
T,
1090
854
1056
1000(0,)
-300
982
865
703
850 (Cb,)
-450
T,
695
780
688
721
-579
550
674
666
630
-670
T,
575
580
495
(Cg 550 (G)
-750
225
310
^65
(Cb,) 200 (Ch.)
*; ,.*
-1100
The average contaminant concentration of the bioreactor, at each time point, is corrected by the average contaminant
concentration of the inhibited control, at the same time point, to measure the biodegradation at that time point. The
inhibited control accounts for contaminant losses due to volatilization, adsorption to soil particles, and chemical reactions.
Some contaminant loss in the control due to biodegradation may occur since total sterilization is difficult to accomplish.
However, if a statistically significant difference between the test and control means exists, then biodegradation has
occurred in the test bioreactor. The difference between the two means is tested using Analysis of Variance (ANOVA)
methods at the 80 percent confidence level for each of the test times. If the difference between the two means is significant
at T.,, no further test measurements are required. If the difference between the two means is not significant at T,, then the
remedy screening test continues until some T2. This process is repeated until a statistically significant difference between
the two means is found or the treatability study is determined to be unsuccessful and is discontinued. In this example,
a statistically significant difference between the two means occurs at T3. The data, therefore, indicate that bioremediation
is a viable treatment option and that further remedy selection studies are appropriate. The 80% confidence interval about
each mean is shown in Figure 6-1 to graphically describe the variation associated with each mean.
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E
CL
Q.
O
T"~
X
c
.2
2
"c
o>
u
c
O
U
Figure 6-1, Plot of hydrocorbon concentration versus time.
1 5-
1 4-
1.3-
1.2-
1.1-
1-
0,9-
0,8-
0.7-
0.6-
0.5-
04-
0.3-
0.2-
0.1-
0-
Time
A. inhibited control
• non-inhibited conirol
TECHNICAL ASSISTANCE
Literature information and consultation with experts are
critical factors in determining the need for and ensuring the
usefulness of treatability studies. A reference list of sources
on treatability studies is provided in the "Guide for Conducting
Treatability Studies Under CERCLA" (EPA/540/2-89-058).
It is recommended that a Technical Advisory Committee
(TAG) be used. This committee includes experts of the tech-
nology who provide technical support from the scoping phase
of the treatability study through data evaluation. Members of
the TAG may include representatives from EPA (Region and/
or ORD), other Federal Agencies, States, and consulting
firms.
OSWER/ORD operate the Technical Support Project
(TSP) which provides assistance in the planning, performance,
and/or review of treatability studies. For further information on
treatability study support or the TSP, please contact:
Groundwater Fate and Transport
Technical Support Center
Robert S. Kerr Environmental Research
Laboratory (RSKERL), Ada, OK
Contact: Don Draper
FTS 743-2200 or (405) 332-8800
Engineering Technical Support Center
Risk Reduction Engineering Laboratory
(RREL), Cincinnati, OH
Contact: Ben Blaney
FTS 684-7406 or (513) 569-7406
FOR FURTHER INFORMATION
In addition to the contacts identified above, the
appropriate Regional Coordinator for each Region located in
the Hazardous Site Control Division/Office of Emergency and
Reme dial Response or the CERCLA Enforcement
Division/Office of Waste Programs Enforcement should be
contacted for additional information or assistance.
ACKNOWLEDGEMENTS
The fact sheet and the corresponding guidance document
were prepared for the U.S. Environmental Protection Agency,
Office of Research and Development (ORD), Risk Reduction
Engineering Laboratory (RREL), Cincinnati, Ohio by Science
Applications International Corporation (SAIC) under Contract
No. 68-C8-0061. Mr. Dave Smith served as the EPA Technical
Project Monitor, Mr. Jim Rawe served as the primary technical
author and SAIC's Work Assignment Manager. Mr. Derek
Ross (ERM) served as a technical expert. The author is
especially grateful to Mr. Steve Safferman of EPA, RREL who
contributed significantly by serving as a technical consultant
during the development of this document.
Many other Agency and independent reviewers have
contributed their time and comments by participating in the
expert review meetings and/or peer reviewing the guidance
document.
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