United States Prevention, Pesticides EPA712-C-98-083
Environmental Protection and Toxic Substances January 1998
Agency (TS-788)
&EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.3180
Sediment/Water
Microcosm
Biodegradation 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. I36etseq.).
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 835.3180 Sediment/water microcosm biodegradation 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
(e)(3) through (e)(9) and (e)(ll) through (e)(16) of this guideline.
(b) Introductory information—(1) Prerequisites. Sediment and
water containing a representative sample of the natural microbial commu-
nity from a test site of interest are required.
(2) Guidance information. A preliminary study such as a shake-flask
test with site water and sediment (under paragraph (e)(l) of this guideline)
is recommended to provide preliminary information about the fate of a
test compound. For example, a preliminary study may help identify those
fate processes such as volatilization that should receive close attention dur-
ing a microcosm study, and can provide guidance on sampling frequency
and dosing patterns. In addition, an activated sludge respiration inhibition
test (under paragraph (e)(10) of this guideline) is recommended to evaluate
potential adverse effects of a test substance on the natural microbial com-
munity in the receiving environment. This test measures respiration rate
under controlled conditions, and can help to obtain information on micro-
bial toxicity that may be important to microcosm design and set up.
(3) Qualifying statements, (i) This guideline establishes criteria of
minimum acceptability for the development of sediment/water microcosms
for use in biodegradation studies. If the nature of a receiving environment
requires testing under strictly anaeraobic conditions, other test methods
may be more appropriate (e.g., the test guideline for anaerobic
biodegradability referenced under paragraph (e)(17) of this guideline).
(ii) This test guideline does not require any specific microcosm design
because design and operation are compound and site specific.
(iii) Performance of procedures discussed in this guideline may in-
volve contact with hazardous materials and operation of potentially hazard-
ous equipment. This guideline does not purport to address all of the safety
concerns associated with its use. It is the responsibility of the user of this
guideline to establish appropriate safety and health practices and determine
the applicability of regulatory limitations prior to using this guideline.
(4) Recommendations, (i) The use of 14C-labeled test substances is
recommended.
(ii) The use of a mass-balance approach is recommended.
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(c) Method — (1) Introduction, purpose, scope, relevance, applica-
tion, and limits of test — (i) Background. (A) The fate of chemicals re-
leased to the environment may be evaluated in the field or in laboratory
studies. Field studies can be used to obtain important information about
the fate of chemicals in a particular ecosystem, but have many disadvan-
tages. In field studies, environmental variables generally cannot be con-
trolled and the study may be subject to wide fluctuations in variables such
as temperature, rainfall, or sunlight. It is also difficult in many cases to
distinguish whether an observed effect is a result of one fate process vs.
another, or a result of the interaction of more than one fate process. Micro-
cosms may be used to replicate many of the processes affecting the fate
of a chemical in complex ecosystems. These model systems provide an
opportunity to manipulate various test conditions and to observe the effects
of these alterations and their interactions on fate processes. Because micro-
cosms are more easily replicated, effects of environmental variability are
more easily studied than under field conditions. Finally, microcosms can
be used to examine the significance of various fate processes (e.g. hydroly-
sis and biodegradation). This makes it possible to focus on critical proc-
esses and consider site-specific environmental situations.
(B) This guideline provides guidance on the development, use and
evaluation of microcosms containing intact benthic sediment and overlying
water for laboratory evaluations of the fate of chemical substances in the
aerobic aquatic environment.
(C) Several examples of information on the fate of chemicals that
might be obtained from microcosm studies are:
(7) The ability of a chemical substance to persist in its original form
in a particular aquatic environment.
(2) Relative importance of abiotic and biotic processes in determining
the fate of a chemical.
The effect that partitioning of a chemical substance into benthic
sediment has on the ultimate fate of the compound.
(ii) Definitions. (A) Within this guideline, a microcosm is defined
as an intact, minimally disturbed portion of an ecosystem brought into
the laboratory for study under controlled experimental conditions.
(B) Ultimate biodegradation is any biologically mediated conversion
of an organic compound to inorganic compounds (e.g. CCh, H2o, etc.),
other products associated with normal metabolic processes, and microbial
biomass.
(C) Primary biodegradation is any biologically mediated trans-
formation that changes the molecular structure of the parent chemical
compound.
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(D) Within this guideline, a mass balance approach is one in which
the test substance's transport to or from and appearance in all applicable
media of the microcosm, including sediment, overlying water, interstitial
water, off-gases, and test equipment washings is determined, and formation
of 14C-labeled CCh is determined if radiolabeled parent compound is used.
(iii) Reference substances. If required, methyl parathion or linear
alkylbenzene sulfonate (LAS) may be used as an appropriate reference
standard with the shake flask and microcosm tests.
(iv) Principle of the test method. Microcosms are laboratory test
systems designed to study fate processes such as biodegradation of chemi-
cal substances by natural microbial communities. Microcosms contain
sediment and water that have been collected from test sites in a manner
that maintains the physical and biological integrity of the ecosystem under
study. Physical parameters such as lighting, mixing, and temperature may
be controlled to simulate the environmental conditions of the site from
which water and sediment samples are collected. Test compounds are in-
troduced into the system either as a single dose or by continuous dosing
over the duration of testing. The microcosms are sampled on a periodic
basis, and the water, sediment and off-gases (latter for volatile test com-
pounds) are analyzed for disappearance of the parent compound (i.e. pri-
mary biodegradation) and, if feasible, appearance of metabolites. Measure-
ments of ultimate biodegradation may also be made.
(v) Quality criteria—(A) Reproducibility. Microcosms containing
sediment and water samples collected simultaneously from the same site
should provide for adequate reproducibility between replicate microcosms,
provided that standard testing conditions are strictly observed. Variability
may be greater when sediment and water samples are collected at different
test sites or at the same sites over different time intervals. The presence
of organisms other than microorganisms (e.g., amphipods) in sediment/
water samples may also cause significant changes to occur in the sedi-
ments, and therefore complicate test results.
(B) Sensitivity. The sensitivity of the test is dependent upon the dos-
ing protocol, adequate control of specific testing conditions, and the analyt-
ical methods used.
(C) Specificity. This method is applicable to various classes of inor-
ganic and organic compounds. The specific type of test compound used
should be considered in selecting an appropriate microcosm design, testing
protocol, and analytical technique.
(D) Possibility of standardization. Standardization of all aspects of
microcosm testing is difficult, but minimum criteria for acceptability are
provided by this guideline. Specific microcosm designs and testing proto-
cols should be selected based on the type of test compound used and the
environmental conditions at the test site.
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(E) Possibility of automation. Not foreseen.
(2) Description of test procedure— (i) Physical/chemical site char-
acterization—(A) Sediment/water. To ensure that aerobic, sediment/
water microcosms adequately replicate the environmental habitats from
which they are derived and to identify important environmental factors
that could potentially affect the rate or extent of biodegradation of a chemi-
cal substance, physical/chemical characteristics of the site of collection
should be determined. These include:
(7) Physical characteristics. Light intensity and photoperiod, tem-
perature, total suspended solids (TSS), etc.
(2) Chemical characteristics. Dissolved oxygen (DO), total organic
carbon (TOC), dissolved organic carbon (DOC), alkalinity, conductivity,
pH, redox gradient, etc.
(B) Site contamination. Because certain contaminants can adversely
affect the fate (e.g. rate or extent of biodegradation) of a chemical sub-
stance, it is recommended that to the extent possible collection sites be
screened for the presence, identity, and extent of contamination by the
test chemical or close chemical analogs and the following chemical sub-
stances: Pesticides, PCBs and other hazardous substances, heavy metals.
(ii) Design features—(A) Size. Microcosms vary in size from a frac-
tion of a liter to several hundred liters. A microcosm should be sufficiently
large to permit removal of water and sediment samples without signifi-
cantly affecting surface area to volume ratios over the course of an experi-
ment, and to readily accommodate monitoring probes and mixing appara-
tus where necessary. Alternatively, smaller microcosms may be used in
sufficient numbers to allow for destructive sampling. Small microcosms
(i.e. containing several hundred milliliters or less of site water and sedi-
ment) may be the most appropriate for studies of chemical fate processes
such as biodegradation and sorption.
(B) Alternative methods for establishing microcosms. (7) One
method of establishing a sediment/water microcosm is to use a sediment
coring device which becomes the microcosm vessel. A glass tube of suit-
able size (e.g. approximately 3.5 cm in diameter and 40 cm in length)
is inserted into the sediment to a depth equal to or greater than the depth
of biological activity. The top of the tube is closed with a silicone stopper.
The tube is removed from the sediment and closed with another silicone
stopper. After the cores are transported back to the laboratory, additional
site water (i.e. collected separately) may be added to the microcosm as
necessary to achieve the desired sediment surface area to water volume
ratio.
(2) Alternatively, an intact core may be obtained in the field and ex-
truded into a microcosm vessel in the laboratory. A simple and effective
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coring device can be made from clear, acrylic pipe. The coring device
is inserted into the sediment to a depth equal to or greater than the depth
of biological activity. The coring device is closed with stoppers as noted
above and the core is returned to the laboratory. The bottom stopper is
removed and the core is inserted into the microcosm vessel. The top stop-
per is removed and the coring device is lifted out of the microcosm leaving
the sediment core intact. For large water volume to sediment surface area
ratios, the coring device may be placed in a glass dish (e.g. a crystallization
dish) having a diameter slightly larger than the corer, and the entire assem-
bly (core, corer, and dish) placed into the microcosms.
(C) Diagrams of representative microcosms. Figures 1, 2 and 3
present a representative selection of microcosm designs that are appro-
priate for conducting sediment/water microcosm bio degradation tests.
Figure 1. Ecocore Microcosm (under paragraph (e)(12) of this guide-
line)
Figure 2. Sediment/Water System (under paragraph (e)(13) of this
guideline)
Figure 3. Flow-Through Microcosm (under paragraph (e)(14) of this
guideline)
(iii) Preparation—(A) Reagents—(7) Water. Water for the micro-
cosm shall be collected from above or nearly above the site of sediment
core collection. Water may be collected by hand bucketing, grab sample,
or pumping. Water samples shall be transported to the test facility with
minimum delay. If water must be held in the laboratory overnight, it may
be kept at room temperature and gently stirred. Where necessary (e.g.
flow-through microcosm test), larger quantities of site water may be held
for longer periods of time at 4 °C. Prior to use in an aerobic, sediment/
water microcosm test, water should be brought to the test temperature ±2
°C and gently stirred.
(2) Sediment cores. Sediment cores shall be collected in such a man-
ner as to preserve to the extent possible the structural integrity of the sam-
ple, including the redox gradient and the benthic community.
(B) Materials—(7) Sampling containers. In order to minimize
leaching of plasticizers and other contaminants into the water, sampling
containers shall be composed of materials such as glass or fluorocarbon
plastics (e.g. Teflon®).
(2) Microcosm construction materials. Microcosms shall be com-
posed of inert fluorocarbon plastics (e.g. Teflon®) and/or glass. If rubber
stoppers are used, they shall be composed of silicone rubber.
(iv) Procedure for setting up and maintaining microcosms—(A)
Control microcosms—(7) Sterile control microcosms. Use of sterile
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control microcosms permits determination of the relative importance of
biotic and abiotic processes in the fate of a test compound.
(2) Other control microcosms. If necessary, solvent control micro-
cosms, glucose-amended controls, and controls containing other standard
reference compounds such as aniline should also be used.
(B) Dosing microcosms. (7) To the extent possible, the method and
pattern of applying a test substance to a microcosm should reflect the re-
lease pattern expected in the natural environment.
(2) Microcosms may be maintained in either flow-through or static-
renewal modes. For the latter, a fixed percentage of microcosm water is
replaced with fresh site water at appropriate time intervals. A single (i.e.
pulse) dose of test compound may be applied in conjunction with either
of these modes. For a pulse dose in a flowing system, the relationship
between molecular turnover (partial replacement) time and the flow rate
and volume of the microcosm chamber has been described by Sprague
(under paragraph (e)(15) of this guideline)
For flow-through systems employing relatively large concentra-
tions of test compounds, a pump or headbox/siphon arrangement is rec-
ommended. All parts of the pump and delivery tubing that come into con-
tact with either the test compound or diluent water should be composed
of inert materials to minimize sorption of the test substance.
(4) The test substance may be dissolved in a carrier and the resulting
stock solution metered into flowing diluent water. Ideally, the carrier
should be dissolved in sterile diluent water. Peristaltic pumps using sili-
cone rubber tubing may be used for adding diluent water. However, it
is not recommended that these be used to deliver stock solutions because
sorption of test compound to the tubing may be significant. A syringe
pump with glass syringes and inert plastic (fluorocarbon) plungers and tub-
ing is more desirable for introducing a test compound and carrier into
flowing diluent water.
(5) If the test compound is insoluble in water, but soluble in a
nontoxic, water-miscible solvent, it should be dissolved in the minimum
volume of carrier or solvent required to form a homogeneous stock solu-
tion of known concentration. Carriers other than water that are acceptable
in aquatic toxicity testing may be used, including acetone, ethanol, meth-
anol, diethyl sulfoxide, ethylene glycol monomethyl ether,
dimethylformamide, and triethylene glycol (under paragraph (e)(2) of this
guideline). Care should be taken that an increased organic carbon load
due to the addition of a carrier does not significantly affect the test results.
In addition, if a carrier or solvent is used, a control microcosm should
be established to assess the effect of the solvent on microbial activity.
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(6} If the test compound is added continuously, the stock solution
and delivery lines should be kept free of microbial contamination to avoid
degradation of the test compound before it reaches the microcosm.
(C) Test compound concentration. The test compound concentra-
tion^) shall approximate the expected ambient environmental concentra-
tion. Solubility in water of the test compound, analytical detection limit,
and toxic effects on microbiota in the microcosm should be considered
in the selection of the test compound concentration. A preliminary shake-
flask study (under paragraph (e)(l) of this guideline) and/or a microbial
respiration inhibition test (under paragraph (e)(10) of this guideline) may
aid in identifying an appropriate concentration.
(D) Temperature. It is recommended that microcosms be maintained
at field temperature ±2 °C. However, specific circumstances may suggest
that another temperature may be more appropriate. The microcosms should
be placed in a water bath to maintain a constant temperature.
(E) Lighting. (7) It may be desirable to control the quantity and qual-
ity of light entering the microcosms. Light intensity may be adjusted to
a level that is equivalent to the average light intensity on the sediment
surface in the natural system from which the core was obtained. The pre-
ferred source of artificial light is a xenon lamp since its spectrum is closest
to that of sunlight.
(2) Photoperiod may be controlled by the use of simple timers.
Photoperiod is usually fixed at some arbitrary ratio (e.g. 12 h of light
and 12 h of dark), or is maintained at ambient field conditions.
(F) Mixing. Mixing of the microcosm water shall be adequate to uni-
formly distribute the test chemical in the water column but not so great
as to resuspended sediment. Mixing can be accomplished with pumps, aer-
ation, or stirrers. Use of glass or fluorocarbon plastic (e.g. Teflon®) stirrers
attached to small motors is recommended. Simple aeration of microcosm
water is often unsatisfactory because it may cause significant losses of
volatile test substance and may result in uneven mixing.
(v) Microcosm replication techniques. A detailed discussion of tech-
niques for determining adequate microcosm replication and appropriate
statistical treatment of the relevant test data is beyond the scope of this
guideline. However, treatment microcosms must be established in triplicate
for each identified dose (e.g. 1 ppm, 10 ppm, 1,000 ppm), and control
microcosms in duplicate for each identified type of control (e.g. sterile
control, solvent control, glucose-amended control), to be minimally accept-
able for this test guideline.
(vi) Microcosm sampling techniques—(A) Monitoring physical/
chemical characteristics. To ensure the functional capability of the sedi-
ment/water microcosms and to maintain environmental conditions that ade-
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quately represent those of the receiving environment, it is recommended
that to the extent that they are applicable, certain physical/chemical charac-
teristics (listed in paragraph (c)(2)(i)(A) of this guideline) be monitored
over the course of the test.
(B) Water samples. (7) Two replicate water samples shall be col-
lected from each microcosm at dosing time (after an appropriate mixing
period) and periodically thereafter. Sampling regimes for both static and
flow-through systems should be designed according to the expected dis-
appearance rate of the parent compound.
(2) To characterize sorption and/or volatilization of the chemical sub-
stance during the start-up and initial phase of the microcosm test, it may
be appropriate to collect water samples at frequent intervals initially (e.g.
at 0, 1, 3, 6, 12, and 24 h) and at less frequent intervals (e.g. 1, 4, 7,
14, 24, 36 days) thereafter.
(3) The duration of microcosm testing should be limited to 60 days
or less, unless specific circumstances and microcosm function warrant
longer operation. During this time, the microcosm should be monitored
to ensure its viability and stability (under paragraph (e)(ll) of this guide-
line).
(4) A preliminary study such as a shake-flask test (under paragraph
(e)(l) of this guideline), using site water and sediment, is recommended
to help identify a sampling protocol that is appropriate for the specific
site and test parameters.
(C) Sediment samples. Sediment samples shall be collected periodi-
cally during the test. To determine the importance of partitioning to sedi-
ment, it is recommended that sediment samples be collected initially at
frequent intervals (e.g. 0, 12 h), and at less frequent intervals (e.g. 1, 4,
7, 14, 24, 36 days) thereafter. Sediment samples shall be collected in trip-
licate from each treatment microcosm, and in duplicate from each control
microcosm, for each time interval and test concentration. If the diameter
of the sediment core is sufficiently large for repeated sampling without
disturbing the sediment/water interface, this may be accomplished at a
minimum by collecting one sample from each of the triplicate treatment
microcosms for each concentration of test substance. However, it should
be noted that such a sampling design may not be statistically optimal, since
the extent of sample variability for each treatment microcosm is not
known. If the nature of the sediment or the diameter of the core is such
that disturbance of the sediment is a likely consequence of sampling, it
is recommended that the study design include three times as many treat-
ment microcosms as there are sampling times, such that treatment micro-
cosms are destructively sampled in triplicate.
(D) Additional sampling. All of the test substance added to the mi-
crocosm during the study should be accounted for by mass balance. The
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use of radiolabeled test compound is therefore recommended. Potential
losses of test substance and transformation products to the atmosphere may
be evaluated by trapping and sampling off-gases using sampling techniques
described by Bourquin et al. (under paragraph (e)(3) of this guideline),
and in the U.S. Environmental Protection Agency Test Guideline for a
site-specific microcosm test (under paragraph (e)(16) of this guideline).
The significance of volatilization and sorption can also be evaluated based
on preliminary tests and sterile controls, and should be accounted for in
the design of the microcosm.
(vii) Analytical methods. Detailed discussion of compound-specific
analytical methods is beyond the scope of this guideline. Gas chroma-
tography (GC) and high performance liquid chromatography (HPLC) are
suitable for the quantification of many test compounds. Use of appro-
priately radiolabeled test substances is recommended, especially when
quantifying mineralization or identifying degradation products that need
further characterization by conventional analysis.
(d) Data and reporting. (7) A mass balance shall be determined for
the test substance describing its fate, including its transport to or from
and appearance in all applicable media of the microcosm. Where appro-
priate, the media should include at a minimum the following: sediment,
overlying water, sediment core washings, resin traps for volatile com-
pounds, KOH or other standard solvent used as a trap for 14C-labeled CCh
from parent compound, and test equipment washings. Analysis of these
components should account for >80 percent of the initial added concentra-
tion of 14C-labeled substrate or parent compound (under paragraph (e)(4),
(5), (6), (7), (8), (9), (12), and (13) of this guideline). A spreadsheet format
may be useful for reporting data.
(2) The rate constant for the loss of the test compound from the water
column can be determined, assuming first-order kinetics, from a plot of
In C vs. t:
In C = kit + a
where C is the test compound concentration, a is the Y-axis intercept,
and t is time. The rate constant is ki, which is determined as the best-
fit slope of a liner regression of In C vs. t. The half-life (ti/2) can then
be determined by use of the following equation:
ti/2 = 0.693/ki
(3) Reports should account for any unusual observations as well as
include where applicable the following information:
(/) Date the study began and ended.
(//) Name and address of testing laboratory.
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(///) Principal investigator(s).
(/v) Staff members actually conducting the test.
(v) Full description of the experimental design and procedures, includ-
ing a description of the test equipment.
(v/) Identity of the test substance, percentage of active ingredient, mo-
lecular structure, and radiolabel placement.
(v//) Manufacture and lot number of the test substance.
(v///) Physical and chemical properties of the site.
(ix) Results of all plate counts.
(jc) Principal mathematic equations.
(xi) Residue data or contamination profile of sediment and water.
(xii) Results of trapped volatiles and CCh analyses.
(xiif) Residue decline curves (i.e. loss of parent compound).
(jc/v) Metabolite characteristics as determined by TLC, HPLC or other
analytical technique suitable for identifying metabolites; identity of each
metabolite with >10 percent yield.
(xv) Incubation temperature; photoperiod and lighting conditions.
(xvi) Materials balance, rate constants, half-lives.
(xvii) Concentration of dissolved 14C-labeled CCh in water.
(xviii) Dates and results of QA/QC audits.
(xix) Location of raw data.
(xx) Complete description of any deviation from the established test
guideline.
(xxi) Experimental test system monitoring data including TOC, pH,
lighting, temperature, etc.
(e) References. (1) American Society for Testing and Materials
(ASTM), ASTM Method E1279-89: Standard test method for biodegrada-
tion by a shake-flask die-away method. Philadelphia, PA (1989).
(2) American Society for Testing and Materials (ASTM), ASTM
Method E729-88a: Standard guide for conducting acute toxicity tests with
fishes, macroinvertebrates, and amphibians. Philadelphia, PA (1989).
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(3) Bourquin A.W. et al., An artificial microbial ecosystem for deter-
mining effects and fate of toxicants in a salt-marsh environment. Devel-
opmental Industrial Microbiology 10: 185-191 (1977).
(4) Clark J.M. and Matsumura F., Metabolism of toxaphene by aquat-
ic sediment and a camphor-degrading pseudomonad. Archives of Environ-
mental Contamination and Toxicology 8: 285-298 (1979).
(5) Giddings J.M. et al., Transport and fate of anthracene in aquatic
microcosms. In: Microbial Degradation of Pollutants in Marine Sediments
(Bourquin A.W. and Pritchard P.H., eds.). Gulf Breeze, FL: U.S. EPA
Office of Research and Development, Environmental Research Laboratory,
pp. 312-320. EPA-600-979-012 (1979).
(6) Heitkamp M.A. and Cerniglia C.E., Effects of chemical structure
and exposure on the microbial degradation of polycyclic aromatic hydro-
carbons in freshwater and estuarine ecosystems. Environmental and Toxi-
cological Chemistry 6: 535-546 (1987).
(7) Heitkamp M.A. et al., Fate and metabolism of isopropylphenyl
diphenyl phosphate in fresh water. Environmental Sciience and Technology
18: 434-439 (1984).
(8) Jensen K. et al., The use of ecocores to evaluate biodegradation
in marine sediments. Water Air Soil Pollution 39: 89-99 (1988).
(9) O'Neill E.J. et al., Effects of lugworms and seagrass on kepone
(chlordecone) distribution in sediment/water laboratory systems. Environ-
mental and Toxicological Chemistry 4: 453-458 (1985).
(10) Organization for Economic Cooperation and Development
(OECD), Activated sludge, respiration inhibition test. OECD Guidelines
for Testing of Chemicals, no. 209. Paris (1981).
(11) Perez K.T. et al., Environmental assessment of phthalate ester,
di(2-ethylhexyl) phthalate (DEHP), derived from a marine microcosm. In:
Aquatic Toxicology and Hazard Assessment: Sixth Symposium (Bishop
W.E., Cardwell R.D. and Heidolph B.B., eds). Philadelphia, PA: American
Society for Testing and Materials, pp. 180-191 (1983).
(12) Pritchard P.H. et al., System design factors affecting environ-
mental fate studies in microcosms. In: Microbial Degradation of Pollutants
in Marine Sediments (Bourquin A.W. and Pritchard P.H., eds). Gulf
Breeze, FL: U.S.EPA Office of Research and Development, Environmental
Research Laboratory, pp. 251-271. EPA-600-979-012 (1979).
(13) Pritchard P.H. et al., Movement of kepone (chlordecone) across
an undisturbed sediment-water interface in laboratory systems. Environ-
mental and Toxicological Chemistry 5: 647-657 (1986).
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(14) Rodgers J.H. et al., Use of microcosms to study transport, trans-
formation, and fate in aquatic systems. Environmental and Toxicological
Chemistry 2: 155-167 (1983).
(15) Sprague J.B., Measurement of pollutant toxicity in fish: bioassay
methods for acute toxicity. Water Research 3: 793-821 (1969).
(16) U.S. Environmental Protection Agency, Office of Toxic Sub-
stances. Toxic Substances Control Act test guidelines. Proposed rule: site-
specific aquatic microcosm test. Federal Register 52:36352 (1987).
(17) U.S. Environmental Protection Agency. Anaerobic
biodegradability of organic chemicals. 40 CFR 796.3140 (1992).
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