INTERIM PROTOCOL
for determining the
AEROBIC DEGRADATION POTENTIAL
of
HAZARDOUS ORGANIC CONSTITUENTS IN SOIL
Developed by
BIOSYSTEMS TECHNOLOGY DEVELOPMENT PROGRAM
SCIENTIFIC STEERING COMMITTEE
with assistance from
THE SOIL TREATMENT PROCESSSES COMMITTEE
ROBERT S. KERR ENVIRONMENTAL RESEARCH LABORATORY
SEPTEMBER 1988

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TABLE OF CONTENTS
1.0 Scope and Approach
2.0 Summary of Method
3.0 Collection and Sampling of Site Materials
3.1	Sample Selection
3.2	Sample Collection
3.3	Sample Moisture Characterization
3.4	Sample Transportation
3.5	Sample Preservation
3.6	Sample Holding Times
4.0 Apparatus and Materials
4.1	Reactor Components
4.2	Reactor Design
5.0 Procedure
5.1	Reactor Set-Up
5.2	Reactor Operation
5.3	Analysis of Reactor Contents
6.0 Data Recording and Analysis
6.1	Data to Be Reported
6.2	Determination of Degradation Rates
7.0 References
7.1	General
7.2	Chemical Analysis
7.3	-Sampling
U.S. EPA Region III
Regional Center for Environmental
Information
1650 Arch Street (3PM52)
Philadelphia, PA 19103

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Appendices
A.	Chemical Analysis of Test Chemicals and/or Waste Chemicals
B.	Gas Flow Measurements and Analysis
C.	Soil Moisture Determinations
D.	Evaluation of Samples from Treatability Test for
Toxicological Hazard

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1.0 SCOPE AND APPROACH
The selection of technologies for the cleanup of
National Priority List Sites using biological treatment is
often based, in part, on information obtained from treat-
ability tests. In bioremediation, an individual or company
(a vendor) frequently uses treatability information to
substantiate their proposed technology and strategy for the
biological cleanup of a hazardous waste site. If not pro-
vided with guidance, however, a vendor will use a variety of
methods and techniques to obtain the treatability information.
This can lead to interpretational problems and relevancy
concerns by the third party (usually an EPA regional office or
state agency representative responsible for the coordination
of remedial actions to clean up a hazardous waste site)
evaluating the proposed technology and strategy. Thus
protocols are required to provide consistency in the develop-
ment of treatability information and interpretation of the
resulting data.
This particular protocol provides a vendor with a
standard method for comparing aerobic degradation rates of
hazardous organic chemicals in contaminated surface soils. -
The protocol can be used as a standard guideline for the
submission of data in support of their claims of aerobic
treatability. Use of the protocol by a third party to
evaluate a vendor's technology provides the possibility of an
unbiased assessment.
Contaminated surface soils that are appropriate for the
treatability protocol include any soils that can be
mechanically perfused with water as part of the treatment
technology or any subsurface soils that can be excavated and
treated in the surface environment.
Data collected from the treatability tests specified in
the protocol;
a.)	provide a first cut evaluation of the rate and
extent by which specific chemicals, chemical mixtures and /or
toxicity of a waste are removed in conjunction with a
proposed bioremediation strategy.
b.)	insure, through the use of a mass balance principle,
that the removal or loss of the hazardous organic chemical(s)
is the result of degradation and not some other process such
as chemical decay, volatilization, stripping or sorption.
c). are not intended for predicting the rate or extent
of biodegradation at field scale.
d.) and cannot be used in predicting the cost of full
scale implementation, or.the time required to bring the site
to closure.

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The protocol provides optional applications. It can ;
a.)	compare the rates of degradation under different
environmental conditions [pH, oxygen status, moisture, depth
zone of contamination, presence of co-occurring contaminants]
and/or under different treatment conditions (aeration,
supplements, slurries,etc.).
b.)	compare rates to determine if the selected
environmental condition(s) or the selected treatment
conditions affect the degradation rates significantly in
unaltered or untreated conditions.
2.0 SUMMARY OF METHOD
The protocol is based on the use of small scale
experimental reactors designed to mimic the conditions
proposed by the vendor for an aerobic biological treatment
(bioremediation) of contaminated soils. Four basic reactor
designs are considered:
1.	no tillage,
2.	periodic tillage,
3.	forced ventilation and
4.	slurried reactor.
A minimum of two^tests are required for evaluation of a
proposed bioremediation strategy. One, called the complete
treatment test uses the reactor design which most closely
mimics the proposed bioremediation strategy and includes any
proposed biological inocula, nutrient amendments, adjustments
or other procedures to stimulate biological activity. The
second, called a no treatment test. is standard against which
the vendor's proposed strategy is compared. It consists of
the reactor with no tillage and involves no additions,
adjustments or manipulations.
Other tests can also be set up to determine the
necessity of all or some of the adjustments to of the soil
proposed in the bioremediation strategy. For example, one
may want to evaluate the effectiveness of nutrient additions.
In this case, a third test is included in which the nutrient
addition is eliminated but all other proposed additions and
manipulations are maintained. Or the contaminants to be
treated may be very volatile (as judged, for example, from
changes in contaminant concentration during handling) and thus
a specialized reactor design might be included.
The basic framework of the protocol is to establish
treatability efficacy of the contaminants in the soil by
determining the rate and extent of disappearance of specific
chemicals over time. In addition, a method for the use of a
bioassay to follow the loss of toxicity over time is provided

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in the protocol to compare with the chemical disappearance
data.
Procedures for dealing with hazardous wastes and
ensuring the health and safety of laboratory personnel are
not addressed in this protocol. Any laboratory using the
protocol must comply with the appropriate procedures for
handling and disposal of the wastes and the appropriate Good
Laboratory Practices.

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1.0 COLLECTION AND HANDLING OF SITE MATERIALS
5.1	SAMPLE SELECTION
Samples of contaminated soils will be collected from
ireas where the vendor has proposed to use bioremediation
strategies. Sampling areas should be selected that are
representative of conditions most typical of the site. That
Ls, sampling areas should be based on the site
characterization data and the treatment strategy. In
general, areas with the highest concentrations of
contaminants should be selected. If, however, there are any
compounds present that are suspected to be inhibitory to the
biological treatment process, their concentration and
toxicity should be included in the sampling design. For
example, the microbes can tolerate concentrations of lead and
zinc that are an order of magnitude higher than cadmium or
nickel. Therefore sampling should be based on the
concentrations of cadmium or nickel even though the
concentrations may be considerably smaller than other less
toxic metals.
Sampling areas for any particular treatability study,
should not differ substantially in terms of soil type and
chemical composition (for example, the presence or absence of
heavy metals). Sampling plans should be developed in
accordance with the recommendations given in the USEPA
compendium of methods (SW - 846, 3rd Edition, November 1986).
3.2	Sample Collection
Enough soil must be collected from each sampling area
for a minimum of two tests (gpmplete treatment and no
treatment) and any additional test proposed. All the soil
collected from a sampling area must be composited and
thoroughly mixed at the point of collection. Subsamples used
in the treatability tests are then taken from this mixture.
Three replicate 200 gm (dry weight) samples of
composited soil should also be collected for chemical
analysis. These samples should be wrapped tightly in heavy
gauge aluminum foil and quick frozen with dry ice.
Additional samples should be taken for moisture
characterization. The quantities of soil collected should be
recorded.
If possible, the soil should be drained at the time of
collection. Sampling should not occur after a major climatic
event, such as rain, abnormal droughts, or seasonal changes.

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3.3 SAMPLE MOISTURE CHARACTERIZATION
A soil moisture curve should be determined on the
composited samples from each area of the field site
immediately following sample collection. This curve, which
relates the capillary tension on water in the soil to the
water content on a mass basis, should be determined using the
procedures outlined in Appendix C. The moisture information
is necessary for properly adjusting the moisture content of
the soils in the reactors.
3.4	SAMPLE TRANSPORTATION
Soil samples must be transported to the laboratory in
containers capable of;
a.)	preventing loss of volatile organic compounds,
b.)	protecting the soil sample from light, and
c.)	minimizing adsorption of chemicals to container and
cap surfaces.
Screw-capped, wide mouth, glass bottles having a lid
with a Teflon TR liner are recommended (cf. Chapter Four - ¦
Organic Analyates, Section 4.1.2 SW-846). Collected samples
will be maintained on wet ice or in a refrigerator at less
than 10 C during transport and until the soil is used in the
reactors. The soil samples should be kept at a moisture
content representative of the field or specified by the
vendor. Caution should be exercised to prevent wet soils
from becoming anaerobic during shipment. If a sample
container is broken or opened before the soil is to be used
in the reactors, the sample must be discarded and if
necessary, the area resampled.
3.5	Sample Preservation
The addition of chemical preservatives is prohibited.
The sample must be iced down prior to any shipment.
3.6	Sample Holding Times
The soil samples to be evaluated in the treatability
test can be held for a maximum of 14 days if the samples are
refrigerated (not frozen). Soil samples that are to be
chemically analyzed, can be held indefinitely as long as they
are frozen. No holding time is allowed for soils that are
used to determine moisture content.

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4.0 APPARATUS AND MATERIALS
4.1	REACTOR COMPONENTS
The basic reactors, which are drawn from the soil flask
system of USEPA (1984, 1986a, 1986b, 1988) and Sims et al.
(1982, 1986), are shown in Fig. 1. The reactor is designed to
determine both the rate of loss by volatilization and
biodegradation. The reactor consists of a 500 ml Erlenmeyer
flask (Kontes cat. No. K-617000-0624 or equivalent) with a
standard taper ground glass joint that accepts an aeration cap
(Inlet Adapter, Kontes cat. No. K-1881000-2440 or equivalent)
modified to deliver air to the flask as depicted in the
figure. The joint is protected with a Teflon TR sleeve. The
aeration cap admits chemically clean air through Teflon TM
tubing. The soil flask can be supplied with breathing quality
compressed air, or laboratory air can be cleaned by pumping it
through an appropriate filter trap. The purge air flows over .
the surface or through the soil-waste mixture within the flask
and exits the aeration cap through an effluent tube close to
the top of the flask. Split stream sampling is conducted
through glass tees in the flask effluent line. An air
splitter can be used to divert the appropriate flow to the •
sorbent tube, or the tube can be connected to a constant flow
vacuum pump.
4.2	Reactor Design
The different treatment types are mimicked as follows:
a.)	Treatment without tillage is obtained by incubating
reactors in a static fashion with uniform air flow above the
soil surface.
b.)	Treatment with tillage is obtained by turning the
reactors on their side and thumping them gently to stir the
soil. This is performed periodically following a schedule
supplied by the vendor.
c.)	Treatment with forced ventilation is obtained by
layering the soil in the reactor on top of a layer of pea
gravel or sand and then delivering humidified air to the
gravel or sand to purge the air through the soil. This
modification is shown in Fig. 2.
d.)	A stirred reactor is obtained by adding water to the
soil, to bring it to the soil-to-water ratio specified by the
vendor, and then adding the slurry to the reactor. The
slurry is aerated by delivering the air to the bottom of the
reactor; if the slurry is not to be aerated, the air should
be delivered above the slurry. The slurry can be stirred
with a magnetic spin bar. This arrangement is depicted in
Fig. 3. Other mechanical mixing devices can be selected by
the vendor depending on soil and slurry texture.

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Influent
Purge Gee
Oat Cleaning
Trap
Pump
~ Effluent Purge Gee-v
Sorbent
TuDe

*»0-
Capillary Flow
Controller
)
Soll/Waete siow Meter
Mixture
Flow Meter
Conetent Flow
Vocuum Pufnp
Effluent Purge Gas
LABORATORY FLASK APPARATUS USED FOR MASS BALANCE
MEASUREMENTS

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influent
Purge Got
Teflon™
Sleeve -*
||hmz
o
effluent Purge Gas
Soil
Gravel or
Course Send
FIGURE 2. LABORATORY FLASK APPARATUS USED FOR SOIL VENTING
EVALUATION

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Influmt
Puryi Gat
T#fionTH
Sittvi->
Jut-Hi
O
Effluinl Purge Cos
6011
Slurry
Mcgnstfe Spin
Bar
Magnetic
SUrr$r
FIGURE 3. LABORATORY FLASK APPARATUS USED FOR SLURRY TAPE
MATERIAL

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5.0 PROCEDURE
5.1 REACTOR SET-UP
The composite field sample is brought to room
temperature in the laboratory and thoroughly mixed. This
step is extremely important if the composite soil sample was
wrought to the laboratory in more than one container. The
nixed soil is then divided into portions equalling the number
3f tests to be performed. Portions will be required for a
minimum of two treatability tests; a.) a no treatment test
consisting of a reactor mimicking treatment without tillage
and containing an unamended/unprepared subsample of the
composited field sample and b.) a complete treatment test
consisting of a reactor most closely mimicking the treatment
strategy proposed by the vendor for cleanup of the
contaminated site and containing a subsample of the composite
field sample treated according to the inoculations,
amendments, adjustments, and/or manipulations proposed by the
vendor. Further portions of soil may be required depending
on other tests designated by the site manager or suggested by
the vendor. The intent of the protocol is to provide enough
flexibility to mimic the proposed treatment as closely as
possible.
Treatment of the soils should be accomplished after
addition of the soil to the reactor. For example, if tillage
of material(s) (i.e., inocula of bacteria, nutrients,
chemicals, etc.) into the soil is part of the treatment
strategy, then the material(s) should be till into the
composited soil samples before it is added to the reactors.
If, on the other hand, the proposed treatment strategy
requires that material(s) be applied only to the soil surface
without mixing, then the material(s) should be applied to the
surface of the soil in the reactor without mixing. Similar
considerations should be given for other types of the
treatment strategies.
The recommended quantity of soil for each reactor is
equivalent to 200 gm (dry wt), however, as little as 50 gm
can be used and larger amounts are also appropriate if the
size of the reactor is also increased. All weights of soil
added to the reactors should be recorded. Moisture content
of the soils should be adjusted before addition of soil to
the reactors.
The reactors are arranged in triplicate sampling sets
for analysis at a minimum of four sampling times,
geometrically spaced (the first time being zero). Additional
sampling times will be negotiated between the vendor and the
third party, depending on such factors as the compounds of
interest, the soil used, and the environmental
conditions,etc. With a minimum of four sampling intervals in
triplicate, a minimum of two treatability tests required

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(complete treatment test and no treatment test), and complete
sampling of the contents of a reactor with each sampling
time, a minimum of 24 reactors will be have to be set up for
each treatability study.
5.2 REACTOR OPERATION
An experimental test is initiated when a reactor is
filled with the proper amount of contaminated soil, capped
and the purge gas flow begun at approximately 200 ml/min.
Time zero analysis of concentration of contaminants in
the soil (initial reactor concentration) is performed by
sacrificing triplicate reactors immediately after the soil is
placed in the reactor and the reactor capped, but prior to
commencement of the purge gas flow.
The frozen samples collected in the field for chemical
analysis (field concentration) should also be analyzed at
this time.
The addition of extraction solvent directly to the
reactor at any sampling time, will terminate any biological
activity.
Gas flow measurements and analysis should be initiated
at this time, according to instructions given in Appendix B.
The reactors should be incubated in the dark (or
protected from the light with aluminum foil) and at a
constant temperature that reflects the average temperature of
the field site when it is to be biologically remediated.
The test soil in the reactors should be maintained at a
soil moisture tension between -0.3 and -1.0 bar unless soil
moisture is a variable to be evaluated (not applicable to
slurry reactors). The reactors will be weighed on a daily
basis, and water added to the reactors as needed to keep them
within the specified moisture range (see Appendix C). The
moisture tension in all the reactors in the protocol may vary
from -0.3 to -1.0 bar, but all the replicates in all the
experimental treatments may not vary from each other by more
than +/- 0.2 bar.
5.4 Analysis of Reactor Contents
Reactors will be sampled by sacrificing the contents of
an entire reactor. All soil in the reactor will be extracted
and analyzed according to chemical procedures given in
Appendix A, If the reactor contains a slurry, the soil and
water can be extracted together if extraction efficiency of
the contaminated soil is not affected by the presence of
water. Otherwise, the water and soil should be extracted
separately.

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6.0 DATA REPORTING AND ANALYSIS
6.1 DATA TO BE REPORTED
The following data will be reported for each of the
treatability tests performed.
-	A record of all sampling transactions including
sampling procedure, map of the site showing
sampling areas (identified on a plan map to within
+/- 2 m and vertical depth of sample to +/- 2 cm),
time of sampling, sample size, and storage of
samples.
-	Concentration of chemicals in the frozen samples at
the time of sampling (field concentration) and
before the samples are added to the reactors (time
zero reactor concentration).
-	Amount of soil used in the reactors and a description
of all modifications to the reactors.
-	Quantity of chemical(s) in samples taken in the field
and in the same samples at the time of their
preparation for addition to the reactors
-	Quantity of residual chemical(s) in each of the
reactors at each sampling time.
-	Quantity of chemical(s) in the traps for volatile
organics at each air sampling time.
-	Quantity of the chemical(s) in the solvent
washings of the reactor glassware, tubing, and
other associated equipment at each sampling time.
-	Information on the presence of toxic materials, such
as heavy metals, in the samples taken from the
field site.
-	The soil moisture curve for the soils sampled for the
tests.
-	Written log (indicating type, extent and time of any
action) of the temperature profile over the entire
experiment.
-	Written log (indicating type, extent and time of any
action) of the sample pump rate and purge gas flow
rates and time interval that the trap is on line
for each reactor at each sampling time.

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-	Written log (indicating type, extent and
action) of all the additions of water
reactors, reactor weights as measured
loss.
-	Concentration of chemical(s) in the reactor head space
at equilibrium from the volatility test.
-	Written log (indicating type, extent and time of any
action) of any other additions, removals, changes,
manipulations, or mishaps which occur during the
course of the experiment.
-	Written log of all cited analytical procedures (see
Appendix A).
-	Hard copies of all GC/HPLC recorder tracings.
6.2 INTERPRETATION OF REPORTED DATA
The change in concentration of the test chemical(s) over
time will determine the degradation rate. The chemical(s)
concentration can change as a result of either;
a.)	decreased extraction efficiency during chemical
analysis,
b.)	volatilization,
c.)	chemical decomposition, and/or
d.)	biodegradation.
Changes in extraction efficiencies over the course of
the experiment and chemical decomposition are both unlikely
but their contribution to the disappearance of the
contaminants can not be directly determined with the
information given in this protocol. Volatilization is
readily determined by the amount of chemical detected in the
volatilization traps assuming that a good mass balance
(greater than 90%) is obtained. Biodegradation is the most
likely process affecting the contaminants if the addition of
inorganic or organic nutrients and/or oxygen results in a
substantially faster decrease in concentration of the
contaminants than without the additions. If the additions do
not affect the disappearance rates, then some combination of
biological, chemical and physical may be controlling the
rates.
time of any
to the
for moisture

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Time
No
Treatment
Complete
Treatment
Partial
Treatment
RVDFVDRV
D
T(0)
T (1)
T(2)
T(3)
t (n)
R - The fraction remaining in the reactor is determined by
dividing amount of chemical remaining in the reactor
(including chemical found on reactor walls and tubing) at
(0,l,2,3,n) by the amount in the reactor at T(0).
V - The fraction of the chemical lost from volitilization and
stripping is determined by dividing the amount of chemical in
the reactor at time T(0) by the amount of chemical accumulated
in the volatile organic traps over incubation period T(0) to
D - The fraction of chemical lost to degradative processes is
then assumed to be the fraction not accounted for in R and V.
D is determined by combining R and V and subtracting this
value from 1.0.
T(n)
FIGURE 4. DATA TABLE FOR RECORDING DEGRADATION RATE
INFORMATION

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The relative importance of biodegradation and
volitilization to the loss of hazardous chemicals from the
reaction vessels can be determined from a pseudo mass balance
illustrated by the table in Figure 4. The data from the
treatability test showing changes in chemicals concentration
over time, is first transformed^ to determine the fraction of
chemical (R) remaining in the reactor at any time (T), the
total amount of chemical lost by volitilization and stripping
(V) over the time period from T(0) to T(n), and the amount of
lost from apparent degradation processes (D). Methods for
determining R, V, and D are described in the footnote of
Figure 4.	|
The relative importance of volitilization and stripping
over degradation is determined by comparing mean values in the
V and D columns within each treatment (No Treatment, Complete
Treatment, Partial Treatment, etc.)* The relative rates of
loss due to degradation and volitility can be determined by
comparing the rates with either the V or D columns between
treatments (No and Complete, No and Partial, Partial and
Complete, etc.)
An indication of degradation can be determined by
comparing the mean of three replicates at any given sampling
time to any other mean with a t-test for the differences of
two means. If the data follow a normal distribution, they
may be subjected to statistical analysis as collected. If
the data follow a log-normal distribution, as is frequently
the case with concentrations of organic chemicals in soils
and geological materials, a log transformation may be taken
before the data are subjected to statistical analysis.
Biodegradation is assumed if removal of the
contaminant(s) of interest in the experimental test mimicking
the treatment proposed by the vendor, is greater than 2 0%
per month at 95% confidence, after correcting for removal in
the controls. If an adaptation or acclimation process is
expected, then the test should be incubated for a long enough
period to see greater than a 20% change; i.e., to cover the
acclimation period.
In some instances, the concentration of test chemical(s)
at the beginning and end of a treatability study, as depicted
in the actual chromatographs, can be compared for a quick
estimate of the effectiveness of the biological treatment. In
other cases, a decrease in concentration of a component(s)
within a chemical mixture may be observed and eventually
quantified.

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7.0 REFERENCES
Sims, R. C. 1982. Larid application design criteria for
recalcitrant and toxic organic compounds in fossil fuel
wastes. PhD dissertation. North Carolina State
University, Raleigh, NC.
Sims, R.C., D.L. Sorensen, W.J. Doucette, and L.. Hastings.
1986. Waste/soil treatability studies for hazardous
wastes: Methodologies and results. Vols. 1 and 2. U.S.
Environmental Protection Agency, Robert S. Kerr
Environmental Research Laboratory, Ada, Ok.
EPA/6—/6-86/003a And b. NTIS No.
U.S. EPA. 1984. Review of in-place treatment techniques for
contaminated surface soils. Volume 2: background
information for in situ treatment. EPA-540/2-84-003b.
U.S. Environmental Protection Agency, Cincinnati, OH.
U.S. EPA. 1986a. Permit guidance manual on hazardous waste
land treatment demonstrations. Final Version.
EPA-530/SW-860932.
U.S. EPA. 1986b. Evaluation of volatilization of hazardous
constituents at hazardous waste land treatment sites.
EPA/600/2-86/071. NTIS No. PB86-233939, U.S.
Environmental Protection Agency, RESKERL, Ada, OK.
U.S. EPA. 1988. Treatment potential for 56 listed hazardous
chemicals in soil. EPA/600/6-88/001. NTIS No.
PB88-17446. U.S. Environmental Protection Agency,
RSKERL, Ada, OK.

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APPENDIX A
CHEMICAL ANALYSIS OF TEST, CHEMICALS AND/OR WASTE SAMPLES
¦ •'
¦ ] \
The selection of;a suitable extraction procedure for a
given combination of analyte(s) and soil matrix generally
requires some method development (Coover et al. 1987). For
example methods that successfully recover a compound from one
medium may not adequately recover the same chemical from
similar media (Albro 1979). Also, extration recoveries from a
given set of structually similar media may vary (Albro 1979).
Where possible we recommend that the existing and
established analytical methods described in Test Methods for
Evaluating Solid Waste (USEPA SW-846 3rd Edition November
1986) be used.
The recommended SW-84 6 methodology for selected analytes
are:
Gas Phase Volatiles
Modified Method 5 Sampling Train
Source Assessment Sampling System
(SSAS)
Volatile Organic Sampling Train (VOST)
Protocol for Analysis of Sorbent
Cartridges from Volatile Organic
Sampling Train
Soil Phase Volatiles
Method 0010
Method 0020
Method 003 0
Method 5040
Method 5030
ffiethod 8010
Method 8015
Method 8020
Method 8030
Purge and Trap
Halogenated Volatile Organics
Non-Halogenated Volatile Organics
Aromatic Volatile Organics
Acrolein, Acrylonitrile, Acetonitrile
Selected Non-Volatiles
Method 8040	Phenols
Method 8060	Phythalate Esters
Method 8080	Organic Pesticides and PCB's
Method 8090	Nitroaromatics
Method 8100	Polynuclear Aromatic Hydrocarbons
Method 8120	Chlorinated Hydrocarbons
Method 8140	Organophosphorous Pesticides
Method 8150	Chlorinated Herbicides
GC/MS analytical methods are not recommended for this
protocol due to the reltively high cost. Analytical

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methodology using gas chromatographic and liquid
chromatographic analysis is sufficient for the use of this
protocol. For a select few analytes GC/MS, or other
specialized techniques, may be the only means to correctly
identify and analyze tor their presence.
Recommended extraiztion/conpentraiton techniques (soils
and sediments) are: \
i
I
Method 3540 Soxhlet Extraction
Method 3550 Sonication Extraction
Other published methods for Soxhlet extraction (Anderson
et al. 1985, Bossert et'al. 1984, Coover et al. 1987, Eiceman
et al. 1986, Kjolholt 1985, Grimalt et al. 1986), sonication
extraction (de Leevw et al. 1986, Sims 1982) and
homogenization and extraction(Coover et al. 1987, Fowlia and
Bulman 1986, Lopez-Avila et al. 1983, Sims 1982, Stott and
Tabatabai 1983, and U.S. EPA 1982a, and extraction of
materials from treatability studies (Brunner et al. 1985,
Russell and McDuffle 1983) are available for reference and
special applications.
Soil spiking and recovery studies should be conducted to
determine the effects of soil, test substance(s), and soil
test substance(s) matrix on chemical extraction and recovery
efficiency. Soil samples should be sterilized using a method
such as mercuric chloride, causing minimal change in soil
physical and chemical properties. (Fowlie and Bulman 1986) .
The sterile soil should be spiked with the test substance(s)
to achieve a range of initial oil concentrations (Coover et
al. 1987). The range of concentration should include the
highest concentration and less than one-half of the lowest
initial concentration to be used in degradation evaluations.
Extractions of the soil/test-substance(s) mixtures using the
selected procedure will allow the evaluation of the effect of
test substance(s) soil concentrations on recovery efficiency.
The effect of soil concentration on recovery efficiency was
evaluated and found to be significant for anthracene and
benzo[a]pyrene by Fowlie and Bulman (1986).
Extracts of the soil and complex wastes should be spiked
with test substance(s) of interest to evaluate the effect of
these matrices on chemical identification and quantification.
Interferences due to the extract matrix may be identified.
Extraction procedures or instrumentation used for
identification and quantification may then be changed if
necessary.
Standard curves should be prepared using primary
standards of the test substance(s), or chemicals in the test
substance, dissolved in a suitable solvent that does not
interfere with chemical identification and quantification.
Standard curves should be generated using at least six points

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ranging from the highest concentration anticipated to the
detection limit for the chemical.

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REFERENCES
Albro, P.W. 1979. Problems in analytical methodology:
Sample handling, extraction,and cleanup. Ann. N.Y.
Acad. Sci. 320:1^9-27.
R. Theilen, and A.F. Weston,
for determination of
Anderson, J.W., G.H. Herman, D.]
1985. Method verification
tetrachlorodibenztadioxin in soil. Chemosphere 14:
1115-1126.	\
Bossert, I., W.M. Kachel, and R. Bartha. 1984. Fate of
hydrocarbons duririg oil sludge disposal in soil.
Applied and Environ. Micro. 47:763-767.
Brunner, W., F.H. Sutherland, and d.d. Focht. 1985.
Enhanced biodegradation of polychlorinated biphyenyls in
soil by analog enrichment and bacterial inoculation. J.
Environ. Qual. 14:324-328.
Coover, M.P., R.C. Sims, and W.J. Doucette. 1987.
Extraction of polycyclic aromtic hydrocarbons from
spiked soil. J. Assoc. Off. anal. Chem.
70(6):1018-1020.
de Leeuw, J.W.E., W.B. de Leer, J.S. S. damste, and P. J. W.
Schuyl. 1986. Screening of anthropogenic compounds in
polluted sediments and soils by flash
evaporation/pyrolysis gas chromatography-mass
spectrometry. Anal. Chem. 58:1852-1857.
Eiceman, G.A., B. Davani, and J. Ingram. 1986. Depth
profiles for hydrocarbons and polycyclic aromatic
hydrocarbons in soil beneath waste disposal pits from
natural gas production. J. Environ. Sci. Technol.
20:500-514. Federal Register. 1979. 44(53):
16727-16280 (Friday, March 16).
Fowlie, P.J.A., and T.L. Bulman. 1986. Extraction of
anthracene and benzo(a)pyrene from soil. Anal. Chem.
58:721-723.
Grimalt, J., C. Marfil, and J. Albaiges. 1986. Analysis of
hydrocarbons in aquatic sediments. Int. J. Environ.
Anal. Chem. 18:183-194.
Kjolholt, J. 1985. Determination o trace amounts of
organophorous pesticides and related compounds in soils
and sediments using capillary gas chromatography and a
nitrogen-phosphorus detector. Journal of Chrom.
325:231-238.
Lopez-Avila, V. , R. Northcutt, J. Onstot, M. Wickham, and S.
billets. 1983. Determination of 51 priority organic
compounds after extraction from standard reference
materials. Anal. Chem.
55:881-889.
Russell, D.J., and B. McDuffie. 1983. Analysis for
phthalate esters in environmental samples: Separation
from PCSs and pesticides using dual column liquid
chromatography. Int. J. Environ, anal. Chem.
15:165-183.
Sims, R. C. 1982. Land application design criteria for
recalcitrant and toxic organic compounds in fossil fuel

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wastes. PhD dissertation. North Carolina State
University, Raleigh, NC.
Sims, R.C., D.L. Sorensen, W.J., Doucette, and L.. Hastings.
1986. Waste/soil%treatability studies for hazardous
wastes: Methodologies and results. Vols. 1 and 2. U.S.
Environmental Protection Agency, Robert S. Kerr
Environmental Research Laboratory, Ada, Ok.
EPA/6—/6-86/003a and b. NTIS No. PB87-111738.
Stott, D.E. and M.A. Tabatabai. 1985. Identification of
phospholipids in soils and sewage sludges by
high-performance liquid chromatography. J. Environ.
Qual. 14:107-110.1

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APPENDIX B
z'
GAS FLpW MEASUREMENTS AND ANALYSIS
For reactors containing tilled and untilled soil, or
stirred reactors that are not aerated, the purge gas should
flow at a rate between 1200 and 20 ml/minute. This range of
flow rates turns the heladspace of the reactor over every one
to ten minutes. The flpw rate is selected at the
convenience of the laboratory doing the testing, based on the
performance of the trapping system; but one selected the flow
rate must be rigidly controlled (within 2%). The flow rate
through reactors with forced ventilation or aerated slurried
reactors is specified by the vendor.
The concentration of volatile organics should be
determined in the headspace of the sealed samples brought
back to the laboratory to fill the reactors. This is most
easily done by fitting a septum into the lid of the sample
jar(s). The headspace gas can be sampled with a syringe and
injected directly into a gas chromatograph. The measured
concentration of volatile organic compounds multiplied by the
flow rate of purge gas and the total time of incubation of
the reactors sets an upper boundary on the quantity of each
volatile organic compound that can be released from the soil
sample. This calculated upper bound must be compared to the
total amount of the volatile organic compounds in the samples
frozen in the field to determine the initial concentration of
contaminants. If the calculated upper bound for volatile
loss is more that 20% of the total amount present for any
compound of regulatory concern, the loss of volatiles from
the reactors must be determined as described below.
Emissions of volatile organic compounds should be
monitored in the three replicate reactors that are to be
incubated for the longest time increment. The suggested
sampling interval for the contents of the reactors is time
zero, one month, two months, and a final sampling time
selected by the site manager. If this interval is to be
followed, a system of traps and flow splitters should be
devised that can contain (without breakthrough) the
calculated upper bound for volatile loss in a one-day period
for any of the compounds of regulatory interest.
The system of traps and splitters should be able to
detect (MDL) a quantity of each organic compound of
regulatory concern equal to 1% of the amount of each compound
originally present, divided by 0.01. This will ensure that
error in the estimated contribution of volatilization to the
mass balance will be no more than 5% of the amount originally

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As Sampled
10
20
30
to
so
gms ^0/200 gm dry soil
FIGURE 5. SOIL MOISTURE CHARACTERISTIC CURVE

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The volatile emissions should he trapped and quantified
in the 24 hour period representing the first day of the
second week in incubation, the last day corresponding of the
one-month incubation,\the last day of the final incubation.
If an alternate sampling schedule is used, the sampling of
volatile emission should be compressed accordingly? however,
the entire first 24 hours must be monitored.
\
The total loss of each volatile compound should be
estimated by fitting a burve through the data points (zero-
order or first-order on|the time of incubation, depending on
which is the best statistical fit), then determining the area
under the curve.

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APPENDIX C,
SOIL MQISTURE DETERMINATIONS
Prior to setting iip the bibreactors, the soil moisture
characteristic curve should be determined on a sample of the
material that will be subjected to the protocol. This
procedure requires several days and should be started as soon
as possible after the sample arrives in the laboratory. A
portion of the sample should be air-dried at 150°C for 24
hours to determine its water content. Then the moisture
characteristic curve should be consulted to determine the
moisture tension in the sample. If the tension is greater
than -0.3 bar, water should be added to the bioreactors. The
moisture characteristic curve should be consulted to
determine the amount of water in the soil at -0.3 bar. The
difference between the amount of water in the soil and the
amount there at -0.3 is the amount that should be added.
The soil moisture characteristic curve should be
consulted to determine the weight of water that can be lost
before the moisture tension drops below -0.1 bar. The
reactors should be weighed on a daily basis, and water added
to the reactors as needed to keep them within the specified
range. The moisture tension in all the reactors in the test
may vary from -0.3 to -0.1 bar, but all the replicates in the
experimental treatments may not vary from each other by more
that 0.2 bar at any one time.
The figure shows a hypothetical soil moisture
characteristic curve. As sampled the soil contained 2 3 gm
water in 23 + 200 grams of wet soil. After air drying, 100
gm of wet soil weighed 81.3 grams. To attain 200 gm of air-
dried soil in the reactors, 223 gm of the soil sample should
be added to each reactor.
x	100
200	81.3
The soil in the bioreactor contains 23 gm water. From the
moisture characteristic curve, the soil at -0.3 bar contains
42 gm water, so 20 ml of water should be added to each
reactor. From the curve the soil at -0.1 bar contains 8 gm
water. The reactors can loose up to 43 -8=35 gm of water
before more water needs to be added.•

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APPENDIX D
EVALUATION OF SAMPLES FROM TREATABILITY TEST FOR
?OXICOLOGlCAL HAZARD
I. INTRODUCTION
At the time site Coordinators consider using the Aerobic
Soil Treatability Protocol, available options for remedial
action will have been identified and biodegradation should be
among those options. If this is the case, two primary
questions in risk assessment must be answered;
a.	Does the bioremediation process eliminate or
significantly decrease the hazard potential of the treated
soil?
b.	How does the reduction in hazard compare with other
remediation options?
The risk assessment procedures described within the
Public Health Evaluation Manual (SPHEM) rely upon a review of
the toxicity of individual indicator chemicals identified at
the site. This manual provides risk assessment managers with
appropriate guidance for risk assessments in five areas:
carcinogenicity, mutagenicity, reproductive effects, exposure
assessments, and assessments of chemical mixtures (U.S.
Environmental Protection Agency, 1986a,b,c,d,e). Key within
this process is the ability to compare public health risks.
Comparisons are not made in necessarily absolute terms but in
a comparative manner among the remedial actions developed in
other parts of the Remedial Investigation and Feasibility
Study process.
The SPHEM guideline, using data and information
generated from treatability protocols can be used to help
answer both of the above questions. However in its present
state, it best applied to situations involving only one or a
few pollutant chemicals with known level of toxicity.
Inherent within the SPHEM guideline is a health assessment
process which is designed to compare results, relative to
risk, from bioremediation to other treatment alternatives,
including the no action alternative.
Because quantitative genotoxicity (mutagenicity/
carcinogenicity) data is available for only a few of the
pollutants at a hazardous waste site, a simple surrogate
method for determining the absence or presence of
genotoxicants is useful. This appendix provides information
concerning such a system.

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II. DEVELOPING GENETIC TOXICOLOGY DATA USING THE SALMONELLA
BIOASSAY FOR MUTAGENICITY
Since genotoxicity (carcinogenicity and mutagenicity)
information is often critical in a risk assessment, the risk
assessor may want to uke a rapicl, relatively inexpensive
short-term bioassay. Such an assay should help him/her
establish whether a particular treatment process, as
initially assessed throtagh the use of a treatability
protocol, eliminate, reduces, or increases the genotoxicity
of the pollutant organics found in the soil. The bioassay
will only be a qualitative indicator of the effect a
particular treatment process will have on the hazardous
compounds found in the soil samples. In addition, these
bioassays can be conducted and the results interpreted
without knowledge of the specific chemicals within the
mixture and without knowing the mutagenicity of each
constituent chemical. Because the bioassay most likely used
in conjunction with treatability tests is the Salmonella
bioassay, this appendix limits its discussion to this assay.
Mutagenicity tests using bacteria have been available ¦
for approximately 3 0 years. In 1951, Demarec et al. used an
Escherichia coli (E. coli) bioassay to test 31 chemicals. In
1971, Ames and Yamasaki published a mutagen detection system
using histidine requiring mutants of Salmonella typhimurium
(S. tvnhimurium). This test later gained the pseudonym "Ames
test." Dr. H. V. Mailing (1971) made a significant
improvement to in vitro tests, including the Ames test, by
incorporating a mammalian metabolizing system into in vitro
tests. This allows these assays to detect promutagens
(substances that can be metabolized to mutagens) directly.
The term Ames test usually refers to the plate
incorporation technique. With this protocol, the
investigator mixes the bacteria, the substance under test,
and any metabolic activating system in a melted, soft-agar
overlay and pours the mixture onto the minimal media plate.
After incubation, one examines for toxicity, contamination,
and numbers of colonies. Because each substance is tested
with 5 to 7 doses using 2 or 3 replicate plates per dose and
2 or more bacterial strains and metabolic activation systems,
a single experiment can contain over 200 plates for each
substance tested. Although the data can be summarized in a
number of ways, a modeled slope value taken from dose-
responsive data provides a quantitative method for
summarizing results. Generally, the statistical model used
is either that of Bernstein et al (1982) or Stead, et al
(1981).
The bioassay procedure, developed by Ames et al (1975)
and later refined (Maron and Ames, 1983), detects reverse
mutations that occur in histidine-requiring strains of S.

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tvphimurium. The indicator strains used were developed from
either spontaneous or induced mutants of the parental strain
LT-2 that will not grow on histidine-free medium. When these
histidine-requiring strains undergo a reverse mutation to
prototropy (normal wild type), Ithe mutants will form
countable colonies on a minimal nutrient media deficient in
histidine. Since more\ than one type of gene mutation is
inducible in DNA, several indicator strains were developed.
Besides the histidine-locus mutation, the most commonly used
strains also have additional mutations incorporated. These
additional mutations enhance the sensitivity of the assay
(Maron and Ames, 1983).
While the plate incorporation test protocol is
sufficient to screen most substances, other types of
specialized protocols do exist. Maron and Ames (1983)
described most of these alternative methods and their uses.
Guidance for the performance, quality assurance criteria,
data gathering and interpretation of the Salmonella assays
are available (Claxton, et al, 1987). In general, an expert
in these types of assays should be consulted before the use
of bioassays is initiated. The consultant should be made
aware of the type of samples to be tested, the types of
pollutants that are likely to be present, and how the
bioassay information will be used.
Although this assay is not designed to detect all types
of genetic damage, it does detect those chemicals that cause
small gene mutations. Since gene mutation appears to be one
of several possible steps in the process of carcinogenicity,
compounds which cause gene mutation and are detected with the
Ames test have and increased•likelihood of being carcinogens
(Clayton, et al, 1988). Since cancer can be induced by non-
genetic mechanisms, this bioassay does not detect all
carcinogens. An increase in the mutagenicity of a
bioremediation sample indicates to the risk assessor that the
process being investigated produces additional hazardous
substances. On the other hand, a decrease in mutagenicity
helps to confirm that the bioremediation process is effective
in reducing the genotoxicity of organic compounds in the soil
sample. Chemical and bioassay information should supply
complementary information and will strengthen the risk
assessors evaluation.

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III. PROCEDURE FOR EVALUATING BIODEGRADATION PRODUCTS
/'
The purpose of bioassay testing is to determine whether
or not an apparently efficacious treatment (the contaminant
of concern is removed) decreasejs, does not change, or
increases the overall genotoxicity of the soil sample. This
section provides an outline of how to incorporate and
interpret the bioassay 'information.
i
Selecting Samples for Bioassay. In order to minimize
cost, samples are selected after the third (two-month) time-
interval of the degradation potential protocol is completed.
After the two-month time interval, reactors representing an
untreated condition and the most efficacious condition
(greatest removal of contaminant) are selected for bioassay.
Using this approach, not all reactors need to be bioassayed.
These two sets of reactors will represent the two extreme
conditions for bioremediation.
Extraction. Concentration, and Solvent Exchange. In
many cases, the extracts of the reaction flasks can be
aliquated for both chemical analysis and bioassay. If
separate reactors are used in preparation for the bioassay,
the selected extraction method should be the same as that
used for chemical analysis. The extraction solvent for
bioassay should also be the same as the chemical analysis
extraction solvent unless it prohibits solvent exchange of
the extracted mass into dimethylsulfoxide (DMSO) or some
other solvent compatible with bioassay. If the same solvent
will not allow this, a separate solvent that will allow the
appropriate solvent exchange should be used to extract a
separate aliquot. After extraction , a small aliquot is used
to determine gravimetric mass so the concentration (mass per
ml solvent) can be determined. If sufficient mass is
available, the remaining sample is solvent exchanged into
DMSO at 10 mg/ml concentration. If a precipitate forms at 10
mg/ml, a lower concentration is used.
Bioassay. The chosen samples are tested concurrently at
a minimum of 5 doses using two plates per dose with and
without a mammalian metabolizing system. In order to
conserve sample, an initial range finding test using one
strain is performed using 2 mg per plate as the highest dose
followed by 4 other doses spaced at half-log intervals. If
enough sample is available, definitive testing is done with
strains TA98 and TA100. If excess sample is available, other
indicator strains also are used. All testing is replicated.
If the amount of sample prohibits using at least two strains,
the strain chosen should be one that will detect at least some
of the known genotoxicants. If it is not known which strain
is most appropriate, TA98 with mammalian metabolizing enzymes
is used.

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It is recommended that the protocol of Maron and Ames
(1983) be followed according to. the guidelines given by
Clayton et al (1987).% If either this protocol and/or
guidelines are not followed, justification should be given.
Data Analysis and! Evaluation. In order to determine
whether or not a sample is mutagenic, existing guidelines
(Clayton, et al.# 1987)' should be followed. When the samples
are positive and testedl concurrently, they can be
quantitatively compared using the slope of the dose response
curve. If there is less than a two fold difference in slope
values, the difference is generally not significant. Slope
values can effectively be determined by using either the
models of Stead et al (1981) or Bernstein et al (1982) or by
doing a least squares linear regression of the linear portion
of the dose response curve.
The purpose of bioassay testing is to determine whether
or not an apparently efficacious treatment (the contaminant
of concern is removed) decreases, does not change, or
increases the genotoxicity of the soil sample. If the
genotoxicity of the sample after aerobic degradation is
decreased, as one would expect with the removal of toxic
components, the regulator can have increased confidence that
this type of treatment has potential utility. If there is no
mutagenicity associated with the samples before_or after
treatment, the regulator can rely upon the results of the
chemical assay for the best available assessment of
genotoxicity. If the mutagenicity of the treated samples is
greater than the mutagenicity of the original sample (whether
or not the chemistry indicates destruction of the
contaminant), the biotreatment has produced or made
available additional mutagenic compounds. If it cannot be
determined in this final situation that the increased
mutagenicity is due to artifactual causes, the regulator
should assume that the biotreatment is ineffective or
detrimental. If the mutagenicity of a sample decreases, the
biotreatment is effective. The degree of its effectiveness
can be assessed by comparing the slope values (potency) of
the untreated and treated samples.

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REFERENCES
Ames, B.N. McCann, J.(and Yamasaki, E. (1975) Methods for
detecting carcinogens,and mutagens with the
Salmonella/mammalian-microsome mutagenicity test. Mutation
Res. 31:347-364.	j
Ames, B.N. and Yamasaki, E. (1971) The detection of chemical
mutagens with enteric bacteria (Chapter 9) in: "Chemical
Mutagens: Principles arifJ Methods for their Detection, Vol I,"
A. Hollander, ed., Plenum Press, New York.
Bernstien, L, Kaldor, J., McCann, J., and Pike, M.C. (1982)
An empirical approach to the statistical analysis of
mutagenesis data from the Salmonella test. Mutation Res.
97:267-281.
Clayton, L.D., Allen, J., Auletta, A., Mortelmans, K.,
Nestmann, E., and Zeiger, E. (1987) Guide for the Salmonella
typhimurium/ mammalian microsome tests for bact6rial
mutagenicity, Mutation Res. 189:83-91.
Clayton, L.D., Stead, A.G., and Walsh, D. (1988) An Analysis
by chemical class of Salmonella mutagenicity as predictors of
animal carcinogenicity. Mutation Res. 205:197-225.
Demarec, M., Bertani, G., and Flint, J. (1951) A survey of
chemicals for mutagenic action of £. coli. American
Naturalist 85:119-136.
Mailing, H.V. (1971) Dimethylnitrosamine: formation of
mutagenic compounds by interaction of mouse liver microsomes,
Mutation Res. 13:425-429.
Maron, D., and Ames, B.N. (1983) Revised methods for the
Salmonella mutagenicity test, Mutation Res. 134:1-47.
Stead, A., Hasselblad, V., Creason, J., and Clayton, L.
(1981) Modeling the Ames test, Mutation Res. 85:13-27.
U.S. Environmental Protection Agency (1986a) Guidelines for
Carcinogen Risk Assessment. Federal Register 51:33992-34003.
U.S. Environmental Protection Agency (1986b) Guidelines for
Exposure Assessment. Federal Register 51:34042-34054.
U.S. Environmental Protection Agency (1986c) Guidelines for
Mutagenicity Risk Assessment. Federal Register 51:34006-
34012.
U.S. Environmental Protection Agency (1986d) Guidelines for
Health Assessment of Suspect Developmental Toxicants.
Federal Register 51:34028-34040.

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U.S. Environmental Protection Agency (1986e) Guidelines for
Health Risk Assessment of Chemical Mixtures. Federal
Register 51:34014-3402^

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