United States Prevention, Pesticides EPA712-C-98-297
Environmental Protection and Toxic Substances January 1998
Agency (7101)
&EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.3170
Shake Flask Die-Away
Test
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0132 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines."
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OPPTS 835.3170 Shake flask die-away test.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline are the articles referenced under paragraphs
(j)(2) through (j)(9) of this guideline.
(b) General. (1) This guideline describes procedures for assessing
the biodegradation of chemicals in natural surface water samples, and pro-
vides an opportunity to evaluate rates of biodegradation in the presence
and absence of natural sediment materials. It also may provide limited
information on the abiotic degradation rate, and sorption to sediment and
vessel walls. The method allows for the development of a first-order rate
constant, based on the disappearance of the test compound with time, and
a second-order rate constant, normalized for changes in microbial biomass.
(2) A compound-specific analytical method is required and the con-
centrations of test compound employed depend on the sensitivity of the
analytical method. The test method is designed to be applicable to com-
pounds that are not inhibitory to bacteria at the concentrations used in
the test, that do not rapidly volatilize from water, that are soluble at the
initial test concentration, and that do not degrade rapidly by abiotic proc-
esses, such as hydrolysis.
(3) This guideline may involve hazardous materials, operations, and
equipment, but does not purport to address all of the safety problems asso-
ciated with its use. It is the responsibility of the user to establish appro-
priate safety and health practices and determine the applicability of regu-
latory limitations prior to use.
(c) Summary of test method. The shake-flask die-away biodegrada-
tion method is similar to river water die-away tests described by Degens
et al., Eichelberger and Lichtenberg, Paris et al., and Saeger and Tucker
under paragraphs (j)(6) through (j)(9) of this guideline. It is based on the
Chemical/Toxicity Abatement Test (CTA Test) described by Cripe et al.
under paragraph (j)(5) of this guideline, and is essentially identical to
ASTM Standard Test Method E 1279-89 under paragraph (j)(3) of this
guideline. It differs from most die-away methods by providing for an eval-
uation of the effects of natural sediments on the transformation of the test
compound and by the use of shaking to ensure a dissolved oxygen supply.
Each test compound is dissolved in water collected from a field site, with
and without added natural sediment, and with and without sterilization.
Initial test compound concentrations typically are relatively low
(micrograms per liter), analytical capabilities permitting. Loss of test
compound with time is followed by an appropriate compound-specific ana-
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lytical technique. Changes in microbial biomass may also be followed by
the use of an appropriate technique such as bacterial plate counts. Data
obtained from this test are used to provide the abiotic degradation rate
in the presence and absence of sediment, and the combined biotic and
abiotic degradation rate in the presence and absence of sediment.
(d) Significance and use. (1) Most of the simpler methods used to
screen chemicals for biodegradation potential employ measures of biodeg-
radation that are not specific to the test compound, such as loss of dis-
solved organic carbon (DOC), evolution of respiratory CC>2, or uptake of
dissolved oxygen (biochemical oxygen demand (BOD)). Such methods are
used to evaluate ultimate biodegradability. They require the use of rel-
atively high initial concentrations of the test compound, generally 10 mg/
L or higher, unless the tests are conducted using radiolabeled
(14C-labeled) test compounds. Biodegradation tests measuring 14C-CO2
evolution, for example, can be conducted using initial concentrations of
test compound in parts per billion. However, these tests require specialized
equipment, and the custom preparation of appropriately labeled compounds
is often very expensive.
(2) Die-away biodegradation methods are simple simulation methods
that employ water collected from natural water sources and follow the
disappearance of an added amount of the test compound resulting from
the activity of microorganisms in the water sample. The compound-spe-
cific analytical techniques used to follow the disappearance of the test
compound typically are employed using relatively low initial concentra-
tions of the test compound. Most environmental pollutants are present in
the environment at relatively low concentrations (less than 1 mg/L), and
it has been observed that biodegradation rates obtained using high test
compound concentrations may be quite different from those observed at
lower concentrations (refer to paragraph (j)(4) of this guideline).
(3) The transformation of the test compound to an extent sufficient
to remove some characteristic property of the molecule, resulting in the
loss of detection by the compound-specific analytical technique, is referred
to as primary biodegradation. For many purposes, evidence of primary bio-
degradation is sufficient, especially when it is known or can be shown
that toxicity or some other undesirable feature associated with the test
compound is removed or significantly reduced as a result of the primary
biodegradation. A determination of ultimate biodegradation, on the other
hand, is usually required only when treatability or organic loading are is-
sues of concern, or when assurance is needed that potentially toxic or bio-
accumulative degradation products are not formed. Nonspecific measures
of biodegradation such as DOC and BOD (see paragraph (d)(l) of this
guideline) do not directly reflect primary degradation.
(4) Because the use of low test compound concentrations enhances
the probability of observing first-order or pseudo first-order kinetics, a rate
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constant for the primary biodegradation reaction and a half-life for the
test compound can be derived under defined incubation conditions. Rate
constants are required in many environmental fate mathematical models.
(e) Materials and apparatus. The following materials and apparatus
are needed to perform the test.
(1) Carefully cleaned glass or plastic carboys, required for the collec-
tion and transport of field water samples.
(2) Field sediment samples, obtained using scoop, beaker, or box sam-
pler, as appropriate.
(3) A rotary shaker, capable of holding 2-L Erlenmeyer flasks and
shaking at 140 to 150 r/min is required for the incubation of test flasks.
Temperature control ( ±2 °C) may be incorporated in an incubator/shaker
unit or may be attained by placing the shaker in a temperature-controlled
space. The flasks should be constructed of material that minimizes sorption
of test or reference compound to the walls of the flasks. In general, glass
is the best choice.
(4) A gas chromatograph, or other suitable instrument equipped with
a detector sensitive to the test and reference compounds, is required for
compound-specific analysis.
(5) The use of reference compounds is desirable to evaluate the bio-
degradation potential of the microbial population. A suitable reference
compound will be biodegradable under the test conditions but not so read-
ily biodegradable that it is completely degraded within a small fraction
of the normal test period.
(f) Procedure—(1) Field sampling, (i) Collect water and sediment
from a selected field site (for example, river, lake, or estuary) the day
before test initiation. Measure the salinity (when appropriate), water tem-
perature, and pH at the time of sampling. Collect water from approxi-
mately 60 mm below the air/water surface in clean glass or plastic carboys.
Remove floating or suspended particulates, preferably by filtering the
water through a 3-(im pore-size membrane filter. Collect the upper 5 to
10 mm of underlying sediment by skimming with a beaker, scoop, or box
sampler. Screen the sediment through a sieve with 2 mm-openings to re-
move larger particles and biota. Remove sand by resuspending detritus
and fine particles and decanting the suspension from the sand. (This is
necessary because sand cannot be quantitatively transferred from a slurry
with a pipet.) Adjust the volume of field water by adding it to or decanting
it from the sieved sediment until there is approximately a 1:1 ratio between
sediment and water volumes. Transport the water sample and the sediment
slurry to the laboratory in closed containers.
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(ii) If there is no sediment layer at the field site (for example, the
stream or lake bed is all rock), omit the sediment collection and use proce-
dures.
(2) Handling of field samples, (i) Stir the sediment slurry and site
water continuously at room temperature until they are used in the test.
(ii) Measure the concentration of sediment in the slurry by filtering
5-mL samples of well-mixed slurry through predried (at 105 °C for
1 h) 0.45-(j,m pore-size membrane filters. The slurry must be stirred vigor-
ously during sampling to ensure homogeneity. Rinse the slurry sampling
pipet, sediment, and filter with 2 to 3 mL of deionized water, and dry
the filter and sediment at 105 °C for 1 h. Weigh the sediment after the
dried filter and sediment have cooled to room temperature in a desiccator.
Use the weight of sediment per milliliter of slurry to calculate the volume
of slurry to be used in test flasks.
(3) Preparation of flasks—(i) Test compound concentration. Initial
test compound concentration in the method typically is 200 (ig/L. This
concentration is generally high enough for analytical sensitivity yet low
enough to be environmentally realistic. Choose other concentrations as ap-
propriate.
(ii) Control water (CW) flasks. Add 1 L of site water to each of
two 2-L Erlenmeyer flasks.
(iii) Control sediment (CS) flasks. Add 900 to 950 mL of site water
to each of two 2-L Erlenmeyer flasks, then add sufficient sediment slurry
to each flask to achieve a final (following a second addition of site water)
suspended sediment concentration of 500 mg/L based on dry weight of
sediment. Add additional site water to achieve a final volume of site water
plus sediment equal to 1 L.
(iv) Amended site water. Add sufficient test or reference compound
to 9 to 10 L of site water to produce the desired initial concentration.
Generally, analytical sensitivity permitting, the desired initial concentration
is 200 (ig/L, so that 2.0 mg of test compound is added to 10 L of site
water. Test compound may be added as a stock solution in a volatile sol-
vent (for example, acetone). Add the solution to a clean, empty vessel,
remove the volatile solvent by flushing with a stream of clean air or nitro-
gen, and add 10 L of site water. Analyze the final solution to determine
the concentration of test compound. To compensate for the volume of sedi-
ment slurry and formalin added later (see paragraphs (f)(vi) through
(f)(viii) of this guideline), an excess of test compound may be added to
yield a concentration greater than 200 (ig test compound per liter. The
amount of amended site water added to the active water, active sediment,
sterile water, and sterile sediment flasks is adjusted to yield a final con-
centration of 200 (ig test compound per liter. Unamended site water is
used, as necessary, to produce a final volume of 1 L in each flask.
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(v) Active water flasks. Add 1 L of amended water to each of two
2-L Erlenmeyer flasks.
(vi) Active sediment flasks. Add 900 to 950 mL of amended water
to each of two 2-L Erlenmeyer flasks and then add sufficient sediment
slurry to each flask to achieve a final (following a second addition of
amended site water) suspended sediment concentration of 500 mg/L. Add
additional amended site water to achieve a final volume of water plus
sediment equal to 1 L.
(vii) Sterile water flasks. Add 900 to 950 mL of amended water
to each of two 2-L Erlenmeyer flasks. Add 20 mL of 37 percent formalde-
hyde solution (formalin) to each flask to act as a sterilizing agent. Add
additional amended site water to each flask to achieve a volume of 1 L.
If any interaction between formalin and the test or reference compound
is likely or suspected, another sterilization procedure (for example, use
of phenylmercuric acetate or autoclaving) may be required.
(viii) Sterile sediment flasks. Add 900 to 950 mL of amended water
to each of two 2-L Erlenmeyer flasks and then add sufficient sediment
slurry to each flask to achieve a final (following a second addition of
amended water) suspended sediment concentration of 500 mg/L, then add
20 mL of formalin to each flask to act as a sterilizing agent. Add additional
amended site water to each flask to achieve a final volume of site water,
sediment, and formalin equal to 1 L.
(ix) Flask incubation. Close the flasks with polyurethane foam plugs
and place them on a rotary shaker at 140 to 150 r/min and 25+2 °C.
If a closer simulation of site conditions is desired, the incubation may
be held at a temperature representative of the collection site ±2 °C. Deter-
mine the pH of the water in each flask on day-0 and at least every other
day for the remainder of the test. Maintain the pH at the value observed
at the time of collection (+ 0.2 pH units) throughout the test by adding
a few drops of 1 N HC1 or 1 N NaOH as required.
(4) Preliminary check. This test method is not suitable for biodeg-
radation rate determinations of compounds that are rapidly lost (50 percent
or greater decrease in 24 h) from solution due to chemical instability, vola-
tility, or photolysis. To determine suitability, a preliminary test may be
set up using a 2-L flask containing 1.0 L of reagent water, with a purity
equal to or better than Type II of Specification D 1193 under paragraphs
(j)(l) and (j)(2) of this guideline, to which 20 mL of formalin is added.
Amend the flask with test compound to a concentration of about 200 (ig/
L. Close the amended flask with a stopper and provide with laboratory
lighting of the same type and intensity provided to the test shaker flasks.
This flask serves as a check for abiotic losses (for example, by photolysis,
hydrolysis, or volatilization). Sample the flask for test compound con-
centration at time zero (to) and after 24 h. If one-half or less of the test
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compound is present at 24 h, no further work is carried out and the method
is considered unsuitable for testing the compound.
(5) Total organic carbon (TOC) analysis. Analyze well-mixed sam-
ples from the CS and CW flasks for TOC content using a suitable method,
such as that described in ASTM Test Method D 4129 under paragraph
(j)(2) of this guideline. This value is used in calculating the equilibrium
sorption coefficient.
(6) Equilibrium sorption coefficient. Sample the sterile sediment
flasks at half-hour intervals until the test compound concentrations are rel-
atively constant at each of two sequential sampling times, indicating no
more sorption to sediment and vessel walls. This generally occurs within
the first 6 h. Place duplicate 25-mL samples from each flask in centrifuge
tubes and centrifuge to remove suspended particles before analysis to de-
termine test or reference compound concentration. Concentration at to (Co)
is the concentration observed in samples obtained immediately following
the preparation of the sterile sediment flasks.
(g) Sampling. (1) Collect samples from each flask according to a
schedule appropriate to the rate of biodegradation of the test and reference
compounds. Sampling should be sufficiently frequent to establish plots of
degradation versus time and to permit the determination of rate constants.
Collect a minimum of six samples from to until completion of the test.
A nominal test duration of 28 days allows a reasonable period for observa-
tions with slowly degraded compounds. The test period may be extended
beyond 28 d if necessary to calculate a half-life. Tests may be terminated
prior to 28 days when more than 50 percent of the starting material has
disappeared from solution due to biodegradation.
(2) Remove duplicate samples of a sufficient size from each flask
at appropriate intervals from day 1 (t = 24 h) until completion of the test.
Centrifuge each sample to remove suspended particles. Analyze the super-
natant (or a suitable extract of the supernatant) to determine the concentra-
tion of test or reference compound. Maintain a record of compound con-
centration versus time for each flask. If sorption to sediment solids is a
significant factor, extract the sediment plug and analyze the extract to ac-
count for untransformed test compound.
(3) If microbial adaptation (a lag phase with little or no loss of test
compound followed by relatively rapid loss) is suspected, add additional
test compound to that flask and the corresponding control flask, at or near
the normal end of the test period. Adaptation is indicated if the microorga-
nisms in the test flask degrade the added compound without a lag period
and the control flask, to which test compound has just been added for
the first time, exhibits a lag prior to degradation. Do not use the lag period
in the calculation of the biodegradation rate. If there is a lag period due
to adaptation, use the end of the lag period as to when calculating the
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first-order rate constant (see paragraph (h)(2)(i) of this guideline). For ex-
ample, see Cripe et al. under paragraph (j)(5) of this guideline.
(4) If desired, samples may also be taken for biomass determinations.
Sampling times should coincide with the times of sampling for test and
reference compound concentration.
(h) Calculations — (1) Equilibrium sorption coefficient. Calculate
the equilibrium sorption coefficient (Koc) using the following equation:
= 1000 (Co - Ce)/Ce (CS - CW)
where
Co = test compound concentration at to ((ig/mL)
Ce = test compound concentration at equilibrium ((ig/mL)
CS = TOC in control sediment sample (g/L)
CW = TOC in control water (CW) sample (g/L)
(2) Biodegradation rates and half-lives — (i) First-order rate con-
stants. (A) First-order rate constants (Ki) are determined using a regres-
sion equation of the type
C = a + Kit
where
C = concentration of test compound (|ig/L)
a = Y-axis intercept
KI = slope (first-order rate constant)
t = time
(B) See paragraph (g)(3) of this guideline for information on calculat-
ing KI if there is microbial adaptation resulting in a lag period.
(ii) Half-life. The half-life of the test compound, based on the first-
order rate constant, is given by ti/2 = 0.693/Ki. Calculate the half-life for
the test compound in each flask and calculate an average value for rep-
licate flasks.
(3) Second-order rate constants, (i) Second-order rate constants are
of interest because some mathematical fate models use a second-order rate
expression to describe the biotransformation of chemical compounds in
environmental waters. In such models, the disappearance rates for com-
pounds are calculated from the concentration of indigenous bacteria, the
concentration of the compound, and the second-order rate constant.
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(ii) A second-order rate constant (K2) can be obtained by dividing
KI by the average bacterial concentration (B). If plate count methods are
used, (B) is expressed in colony forming units per mililiter. Bacterial con-
centrations normally do not change significantly during these tests, since
concentrations of growth substrates are normally too low to support signifi-
cant microbial growth. Therefore, measured bacterial concentrations are
averaged to obtain (B). K2 is calculated as follows:
K2 = Ki/B
(i) Report. Report the following data and information:
(1) Test and reference compound identities.
(2) Site, date, and time of field water and sediment collection.
(3) Temperature, pH, and salinity (when appropriate) of site water
at the time of collection.
(4) Concentration of sediment (dry weight) per milliliter of slurry.
(5) TOC in the CS and CW samples expressed as grams per liter.
(6) Measured concentrations of test and reference compounds at each
sampling time during the preliminary check, sorption coefficient deter-
mination, and test sampling steps.
(7) Equilibrium sorption coefficient (Koc) calculations and results.
(8) The average first-order rate constants for each replicate pair of
flasks. If microbial adaptation was observed (with a lag period following
test startup), describe the lag period and how it was evaluated.
(9) The average half-life for the compound in each replicate pair of
flasks.
(10) The plate count or other biomass data, if applicable.
(11) The average second-order rate constants for each replicate pair
of flasks, if applicable.
(j) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) American Chemical Society (ACS). Reagent Chemicals: American
Chemical Society Specifications. ACS, Washington, DC (1994).
(2) American Society for Testing and Materials (ASTM). Annual
Book of Standards ASTM, Philadelphia, PA (1993).
8
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(3) American Society for Testing and Materials (ASTM). Standard
Test Method for Biodegradation by a Shake-flask Die-Away Method,
ASTM E 1279-89. ASTM, Philadelphia, PA (1993).
(4) Boethling, R. S. and M. Alexander. Effect of Concentration of
Organic Chemicals on Their Biodegradation by Natural Microbial Commu-
nities. Applied and Environmental Microbiology 37:1211-1216 (1979).
(5) Cripe, C.R. et al. A Shake-Flask Test for the Biodegradability
of Toxic Organic Substances in the Aquatic Environment. Ecotoxicology
and Environmental Safety 14:239-251 (1987).
(6) Degens, P.N., Jr. et al. Influence of Anionic Detergents on the
Diffused Air Activated Sludge Process. Sewage and Industrial Wastes
27:10-25(1955).
(7) Eichelberger, J.W. and J.J. Lichtenberg. Persistence of Pesticides
in River Water. Environmental Science and Technology 5:541-544 (1971).
(8) Paris, D.F. et al. Second-Order Model to Predict Microbial Deg-
radation of Organic Compounds in Natural Waters. Applied and Environ-
mental Microbiology 41:603-609 (1981).
(9) Saeger, V.W. and E.S. Tucker. Biodegradation of Phthalic Acid
Esters in River Water and Activated Sludge. Applied and Environmental
Microbiology 31:29-34 (1976).
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