United States Prevention, Pesticides EPA712-C-96-173
Environmental Protection and Toxic Substances April 1996
Agency (7101)
&EPA Ecological Effects Test
Guidelines
OPPTS 850.1925
Site-Specific Aquatic
Microcosm Test,
Laboratory
'Public Draft"
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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.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
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 202-512-0135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 850.1925 Site-specific aquatic microcosm test, laboratory.
(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 is 40 CFR 797.3100 Site-Specific Aquatic
Microcosm Test (proposed in the FEDERAL REGISTER of September 28,
1987 (52 FR 36344)).
(b) Purpose. This guideline is intended for use in developing site-
specific data on the chemical fate and ecological effects of chemical sub-
stances and mixtures ("test substances") subject to environmental effects
test regulations. This guideline prescribes methodologies to predict the po-
tential fate and/or effects of either organic or inorganic substances in a
natural aquatic ecosystem using a microcosm made of an indigenous water
column and sediment core. This test system is capable of evaluating or-
ganic chemical substances, either soluble or insoluble, which may form
either air-water surface films or aggregates which sink to bottom sedi-
ments. The EPA will use data from this test in assessing the potential
hazard of a chemical substance to a particular natural aquatic system (natu-
ral system).
(c) Definitions. The definitions in section 3 of TSCA and 40 CFR
part 792—Good Laboratory Practice Standards apply to this test guideline.
The following definitions also apply to this guideline:
Benthic community or benthos means numbers, species composition,
size range, and feeding types of organisms present in the sediment of the
natural system.
Benthic subsystem means an undisturbed core collected from the natu-
ral system and placed in the microcosm.
Bio accumulation factor or bioconcentration factor means the ratio of
the concentration of the test substance in an aquatic organism (i.e., biota)
to the associated exposure concentration of the test substance from the
food particles and the surrounding exposure medium (i.e., water or sedi-
ments).
Carrier means the organic solvent, solubilizer and/or other substance
used to disperse the test substance into microcosm water.
Chemical residues means the test substance and its transformation
products retained in the water, sediment, surface film, biota, and glass sur-
faces of the microcosm during the experiment period.
Exposure concentration means the concentration of test substance in
the water or the sediment in which the aquatic organisms live.
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Natural aquatic system or natural system means a particular geo-
graphic location consisting of a water column and its associated benthic
component.
Radioactivity budget or radioactivity mass balance means a quan-
titative relationship among the input, retention, and export of radioactivity
in a microcosm after applying a radiolabeled test substance into the micro-
cosm. The amount of radioactivity added to the microcosm during the test
usually is equal to the sum of the radioactivity remaining in the microcosm
compartments and the radioactivity exported from the microcosm with the
departed water, surface film, and exhaust air.
Ratio of benthic surface area to water volume means the ratio ob-
tained by dividing the calculated benthic surface area of the natural system
by the best estimate of water volume of the system.
Sediment means the bottom substrate existing at the mean water depth
within the natural system during the period of the test.
Site-specific aquatic microcosm means a miniaturized mimic of a spe-
cific natural aquatic system.
Slick protector means a partially submerged glass cylinder within
which surface film is removed.
Water column means the water within the natural system or the micro-
cosm tank.
Water flow rates over the sediment surface means the rate of average
water flow over the surface of the sediment as measured in the natural
system or in the microcosm tank.
Water replacement or replacement water means the natural water
added to the microcosm at specific intervals to simulate water turnover
rate.
Water turbulence means the average water motion in the water col-
umn of the natural system or the microcosm tank during the test.
Water turnover rate or residence time means the time required for
one complete water replacement or exchange within the natural system.
(d) Test procedures—(1) Summary of the test. A site-specific mi-
crocosm is constructed with an indigenous water column and the intact
sediment core associated with it. The water and sediment retain their asso-
ciated organisms in the pelagic and benthic components, respectively, of
the natural aquatic system. Environmental variables such as temperature,
water turbulence, and water turnover rate are manipulated to be similar
to the conditions found in the natural aquatic system. After the test sub-
stance is initially introduced into the microcosm, the fate of the test sub-
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stance as well as properties indicative of the structure and function of the
microcosm are monitored for at least 30 days. Effects of the test substance
on the abundance and diversity of aquatic life, and on elemental cycling
in the microcosm are determined by comparisons with microcosms that
do not contain the test substance.
(2) Administration of test substance, (i) Only test substances that
are resistant to photolysis (i.e., those having a half-life greater than or
equal to 30 days) should be tested in this microcosm system.
(ii) All the test substance added to the microcosms during the study
should be accounted for by mass balance. If the test substance is degrad-
able (not persistent), it is recommended that the test substance be
radiolabeled.
(iii) Test substances can be either gases, liquids, or solids and may
or may not be soluble in water.
(A) If the test substance is soluble in water, it should be dissolved
in distilled water to make a stock solution of known concentration. Meas-
ured portions of the stock solution should be added to the water in the
microcosms and thoroughly dispersed by adequate stirring.
(B) If the test substance is insoluble in water but soluble in a rel-
atively nontoxic, water-miscible solvent such as acetone, it should be dis-
solved in the minimum volume of carrier solvent required to form a homo-
geneous stock solution of known concentration. A measured portion of
the stock solution should be dispersed into the microcosm water at the
beginning of the test to form a homogeneous suspension. Carrier controls
should be included in the experimental design and monitored simulta-
neously with the microcosms treated with the test substance.
(C) If the test substance is a solid and is insoluble in either water
or a suitable carrier, it should be ground to a fine powder, weighed to
achieve the mass required, and added to a 1-L aliquot of the test water
contained in a 2-L separatory funnel. The separatory funnel should be
shaken vigorously to achieve as homogeneous a suspension as possible
and the suspension should be added to the microcosm water.
(D) If the test substance is a liquid, the measured portion should be
added to 1 L of the microcosm water contained in a 2-L separatory funnel,
and shaken to achieve as homogenous a suspension as possible. The sus-
pension should be mixed and added to the microcosm tanks.
(E) The amount of test substance remaining in the separatory funnel
must be determined by suitable solvent extraction and analyses to accu-
rately determine the amount added to the microcosms.
(iv) Sufficient quantities of the stock solution should be made as
needed to minimize storage time and disposal volume.
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(v) A test substance that is insoluble in both water and water-miscible
carriers should be dissolved in more than one carrier, for example, consist-
ing of a lipophilic solvent and an emulsifier, and a measured portion of
stock solution should be dispersed into the microcosm water to form a
homogeneous suspension.
(vi) The method and pattern of applying a test substance to micro-
cosms should reasonably reflect the release pattern expected in the natural
system. If the input of the test substance to the natural system is other
than a one dose application (i.e., multiple application, runoff), the test sub-
stance must be added to the microcosm tank in the same manner as the
initial dose and each time there is a microcosm water replacement, but
only in quantities sufficient to achieve the desired test concentrations in
the replacement water.
(3) Selection of treatment concentration, (i) Range-finding tests are
not recommended, but may be needed to determine treatment concentra-
tions.
(ii) Initially, the microcosms should be treated with concentrations
of the test substance that are 0.1, 1, and lOx as high as the average ambient
concentration of the test substance observed or predicted in the natural
system.
(iii) The test substance should be tested in concentrations of 1, 10,
and 100 (ig/L, if reliable data on observed or predicted average ambient
concentrations are not available.
(4) Definitive test, (i) The purpose of the definitive test is to deter-
mine the potential fate and ecological effects of a test substance in a spe-
cific aquatic ecosystem.
(ii) At least three concentrations of the test substance, exclusive of
controls, should be tested for at least 30 days. A minimum of five replicate
microcosms should be used for each concentration. All tanks within a
given airtight compartment should be treated with the same concentration
of the test substance.
(iii) A minimum of five control microcosms should be used in the
test for each water-soluble test substance. For those test substances that
require a carrier, two of the five control microcosms should be designated
carrier controls and treated with the carrier leaving the remaining micro-
cosms as carrier-free controls.
(iv) Two tests are recommended for each test substance. One should
be performed in the summer and another in the winter if the fate and
ecological effects of the test substance are expected to vary significantly
with seasons.
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(v) Microcosms should be installed and maintained in the following
manner:
(A) All microcosm tanks should be placed in a water bath maintained
within + 1 °C of the ambient water temperature in the natural system.
Water may be pumped from the natural system into the water bath to
regulate the temperature in the microcosms if the test laboratory is nearby.
(B) Water for the microcosm should be collected from the natural
system, at mid-tide for estuaries, by hand bucketing or nondestructive
pumping, e.g., diaphragm pump. If the natural water column in the natural
system is stratified, the microcosm water should contain subsamples taken
from various depths.
(C) Water samples should be transported to the test facility in glass
containers. On arrival at the test facility, water in each container should
be distributed equally among microcosms to a prescribed volume of ap-
proximately 140 L. Plankton samples must be collected from each micro-
cosm tank and analyzed to ensure homogeneous distribution.
(D) Each sediment core should be collected undisturbed from the nat-
ural system by inserting a glass cylinder into the sediment and extracting
the core from a prescribed location. The bottom of the core is sealed by
seating it in a crystallization dish slightly larger than the cylinder in the
following Figure 1. It is desirable to use scuba divers to inspect the uni-
formity of the benthic component in the natural system, to select represent-
ative cores of appropriate length to preserve intact habitats, and to collect
the cores with as little disturbance as possible.
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FIGURE i.—EXPERIMENTAL MICROCOSM (NOT DRAWN TO SCALE)
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(E) The ratio of benthic surface area to water volume in the micro-
cosm should be made equal to that ratio in the natural system being simu-
lated. Because the water volume in the microcosm is fixed, the desired
ratio is obtained by selecting benthic cylinders with the appropriate inner
diameter.
(F) The benthic cylinder housing the sediment core should be mount-
ed in the microcosm tank so that the overflow port of the box is 5 cm
above the water level in the tank (see Figure 1. in paragraph (d)(3)(v)(D)
of this guideline). Any disturbed sediment should be allowed to settle for
at least 30 minutes before starting water circulation in the benthic box
and water turbulence in the microcosm tank.
(G) The benthic pump should be mounted beside the benthic cylinder
with the outlet diffuser of the pump submerged below the surface of the
water (overflow port of the cylinder) but above the sediment surface (see
Figure 1. in paragraph (d)(3)(v)(D) of this guideline). The rate of water
flow over the sediment surface in the microcosm tank should be adjusted
to be equivalent to the average water flow rate over the sediment surface
in the natural system.
(H) The light intensity over the microcosms should be adjusted to
produce an abundance of phytoplankton statistically equivalent to that in
the natural system. Preliminary tests should be performed to establish the
proper light intensity over the microcosms and should be done with all
the microcosm equipment and facilities (i.e., water bath, tank paddle,
benthic cylinder and pump) in place. The preliminary tests should be per-
formed at several light intensities for at least 14 days. The photoperiod
in both preliminary and definitive tests should be set once every 7 days
to match the actual photoperiod within 0.5 h in the location of the natural
system.
(I) The light intensity on the surface of the sediment core in the mi-
crocosms should be adjusted to the level that is equivalent to the average
light intensity on the sediment surface in the natural system. Light intensity
can be adjusted by covering the upper portion of the benthic cylinder with
a screen, such as a nylon net, or other spectrally-neutral light filters.
(J) The speed of the stirring paddle installed in the microcosm tanks
should be adjusted to generate a water turbulence level statistically equiva-
lent to that in the natural system, as measured in the gypsum dissolution
method. This method measures the turbulence level by the average dissolu-
tion of pure gypsum. Weight loss should be at least 5 to 10 percent. This
may take several hours depending on temperature and turbulence. Dissolu-
tion rates should be measured and water turbulence adjusted in the micro-
cosms before each test.
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(K) Any resuspended sediment that settles on the bottom of a micro-
cosm tank should be collected with a tubing pump and returned to the
benthic cylinders when water turnover is simulated.
(L) Water turnover in the natural system should be simulated in the
microcosm as follows:
(7) A measured portion of the water in each microcosm tank should
be replaced at least three times every 7 days with water newly collected
from the natural system.
(2) The water replacement should match the water turnover rate ob-
served in the natural system.
(3) Water replacement should be scheduled immediately after sam-
pling of microcosm water and should occur on the same day.
(4) The volume of microcosm water to be removed each time should
be the difference between the calculated volume to be replaced and the
total volume of water samples removed to keep the water volume at
140 L.
(M) If the test substance accumulates in a thin film on the surface
of water in the microcosm tank, a portion of the film should be removed
with a filter pad or other absorbent material prior to removal of the volume
of water to be replaced. This simulates the surface film advective transport
from the natural system. The area (in squarecentimeters) of surface film
to be removed should be equal to the product of the ratio of the replace-
ment water volume to total tank volume ratio and the surface area of the
tank water, minus the area displaced by the benthic pump and cylinder.
Film area removed =
Replacement water
x [Tank water surface area - (Benthic pump area + Benthic cylinder area)]
Total tank volume
(vi) Sampling procedures for the study of chemical fate should be
performed as follows:
(A) Water samples should be taken at approximately 0, 1, 2, 3, 6,
12, and 24 h after the initial application of the test substance. Therefore,
samples should be taken before each water replacement. Water samples
should be collected through a slick protector within which the surface film
has been removed. Samples may be taken more frequently to follow the
fate of a chemical substance that is disappearing from the system at a
relatively rapid rate. The samples should be collected at a location at least
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3 cm from the side of the tank and 10 cm below the water surface while
both the stirring paddle and the benthic pump are in operation.
(B) If the test substance accumulates in a thin film on the water sur-
face, it should be sampled with a filter pad before each water replacement.
The quantity of a radiolabeled test substance absorbed onto the filter mem-
brane can be easily determined with liquid scintillation counting assuming
all radioactivity represents the original form of the test substance. If the
test substance has degraded, the percentage of the total radioactivity that
is the test substance should be determined.
(C) Samples of selected zooplankton species in the microcosm should
be collected once every 7 days to be analyzed for the test substance and,
if practical, for its transformation products.
(D) Air samples should be collected once every seven days with a
suitable sampler. For example, and inverted crystallization dish equipped
with inlet and outlet tubes on the side may be placed above the water
surface to collect air samples for chemical analysis; fresh air could be
drawn by a vacuum pump at the end of the sampling train, entering the
modified dish through the inlet tube, sweeping over the water surface, and
carrying any volatilized forms of the test substance through the outlet tube
to a suitable trap for subsequent quantification. Under the inverted dish,
air flow over the water surface should be adjusted to match the flow rate
over the rest of the water surface in the microcosm. The duration for each
sample collection should be kept as short as possible.
(E) The quantity of test substance adsorbed onto the glass surfaces
of the microcosm above and below the water surface should be sampled
and estimated as follows:
(7) For estimates of the test substance adsorbed onto the glass of
the microcosm tanks below the surface, glass rods of known surface area
should be suspended in the water column, and removed periodically from
the water and placed in a scintillation counting vial for radioassay. If a
surface film is present, glass rods should be removed through a slick pro-
tector. If possible, the estimated quantity of the radiolabeled chemical sub-
stance on the glass surfaces using the glass rod method should be verified
with extraction of the test substance from all subsurface glass surfaces
whenever a microcosm is sacrificed during the test.
(2) A portion of the interior microcosm tank wall extending from
the water surface to the lip of the tank should have an appropriate absorb-
ent material attached to it. This material should be removed and extracted
at the conclusion of the test to provide an estimate of the amount of the
test substance adsorbed to the tank walls above the water.
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(3) Any unlabeled test substance on the glass surface should be thor-
oughly extracted and quantified after the water and sediment are removed
from the microcosm.
(F) The quantities of the test substance in the benthic component
should be determined as follows:
(7) One of the five replicate microcosm tanks for each of the three
treatments should be randomly selected for sampling and samples of the
core contents should be collected on day 10; another of the remaining
replicate microcosm tanks should be selected for sampling and samples
should be collected on day 20. The three remaining treated replicates and
the controls should be sampled at the end of the test on day 30.
(2) Three sediment subcores, at least 25 cm in diameter by 7 cm
in depth, should be collected from each benthic component to determine
the vertical distribution of the test substance in the benthic component,
i.e., concentration of test substance in each centimeter of the sediment
core.
(3) Before triplicate sediment subcores are taken, the surface film (if
present) on both the microcosm tank and the benthic cylinder should be
removed with suitable tools such as a suction skimmer or a sheet of ab-
sorbent material, and the water in both the tank and the benthic cylinder
should be drained.
(4) Samples of each of the major animal species in the benthic com-
ponent should be analyzed for the test substance and its transformation
products, if possible.
(vii) Sampling procedures for ecological effects study should be per-
formed as follows:
(A) Water samples from microcosms should be taken as described
in paragraphs (d)(4)(vi)(A) and (d)(4)(vi)(B) of this guideline.
(B) When water replacement and ecological effects sampling occur
on the same day, biological samples should be taken first.
(C) Samples of at least 2 mL of water should be collected daily from
the microcosms and such samples should be analyzed for enumeration and
identification of phytoplankton.
(D) Samples of at least 2 L of water should be collected from the
microcosms at least twice each week and such samples should be analyzed
for enumeration and identification of zooplankton and transient larval
forms. The water samples should be collected at a rate sufficient to over-
come the zooplankters' avoidance reaction and should be screened through
a 20-(iM plankton net. The retained organisms should be rinsed into a
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Petri dish and preserved for subsequent determination of population den-
sity and species composition.
(E) The ammonium-nitrogen concentration in the water column of
the microcosms and the natural system should be determined once every
7 days.
(F) Population densities of phytoplankton and zooplankton in the nat-
ural system should be determined at least twice each week, and ammo-
nium-nitrogen concentration in natural water should be measured at least
once every 7 days. This can be done conveniently at the time for water
replacement.
(G) The flux rate of ammonium-nitrogen between the benthic compo-
nent and its associated water column should be determined weekly by stop-
ping the benthic pump for a period of 1 to 3 h. Ammonia concentrations
in water above the benthic component should be measured at the beginning
and end of this period. The flux rate should be expressed as the weight
of ammonium-nitrogen produced by each square meter of sediment surface
area per hour.
(H) The abundance and diversity of benthos should be determined.
Benthic animals should be captured by sieving the wet sediment through
a 0.5 mm screen. All animals retained on the screen should be identified
and counted. Similar characterization of the benthic community of the nat-
ural system should be established at the time of the experiment.
(5) Analytical measurements—(i) Instrumental methods. Atomic
absorption and gas chromatography are preferable to colorimetric methods
for quantitative analyses of metals and organic compounds, respectively.
Liquid scintillation counting is recommended for quantitative analysis of
radiolabeled test substances, and high-pressure liquid chromatography is
recommended in conjunction with liquid scintillation counting for separa-
tion and quantification of the test substance and its transformation prod-
ucts.
(ii) Chemical. (A) A stock solution of the test substance should be
prepared just before use, and its nominal concentration and purity should
be confirmed by chemical analysis. Standard analytical methods, if avail-
able, should be used to determine the chemical concentration in microcosm
samples and stock solution. The analytical methods used to measure all
environmental samples should be validated before the beginning of the
test.
(B) Concentrations of the test substance, and its transformation prod-
ucts, if possible, should be measured for the following components of the
microcosm:
(7) Air.
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(2) Surface film, if present.
(3) Water column, both particulate and dissolved fractions.
(4) Various layers of the benthic component.
(5) Representative species of zooplankton.
(6) Representative benthic organisms.
(7) Glass surfaces above and below the water surface.
(C) If a radiolabeled test substance is used, a complete budget of
all radioactivity should be calculated, including the amount of radioactivity
added to the microcosm, removed by gas transport and water replacement,
and remaining among the compartments of the microcosm.
(iii) Numerical. (A) Mean and standard deviations of biological at-
tributes should be calculated for each treatment and control. The following
information should be determined: Abundance of phytoplankton,
zooplankton, and each type of benthic fauna. If the species of plankton
can be identified, abundance should be calculated for each one.
(B) Statistical analyses should be performed to determine:
(7) Whether significant differences exist in biological attributes be-
tween:
(/) The control microcosms and the natural system.
(//) The carrier control and the carrier-free control.
(///) The control and the microcosms treated with the test substance.
(2) Whether significant differences exist in the amount, export, and
bioconcentration of the test substance among:
(/) Different compartments of the microcosms receiving the same
treatment, and
(//) The microcosms receiving different treatments.
(e) Test conditions—(1) Test species, (i) The organisms tested
should include the indigenous fauna and flora representing both the pelagic
and benthic communities of the natural system, except the macrofauna.
(ii) Neither acclimation nor supplemental food is necessary for the
test organisms.
(2) Facilities—(i) Supporting equipment. (A) The capacity of the
water bath used to maintain the water temperature and the flow rate of
the water through the water bath should be such that the water temperature
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in all microcosms will be kept within + 1 °C of the ambient water tempera-
ture in the natural system.
(B) Cool white fluorescent light should uniformly illuminate the water
surface of all microcosms. The fluorescent lights should be mounted on
a canopy above the microcosm tanks, (see Figure 2). The desired, uniform
light intensity is achieved by wrapping the fluorescent lamps with alu-
minum foil.
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FIGURE 2.—EXPERIMENTAL MICROCOSM FACILITY
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(C) In the room containing the microcosms, no light source except
that specifically for the microcosms should be allowed.
(D) To match the water turbulence in the natural system, the water
turbulence level in the microcosms should be controlled by the speed of
an electric motor that is mounted with its chain drive and drive shafts
above the canopy and controls the speed of all stirring paddles (see Figure
2 in paragraph (e)(2)(i)(B) of this guideline).
(E) The gypsum dissolution method measures the water turbulence
level by the average dissolution rate (i.e., weight loss/time) of cubes
(2.5 cm x 1.5 cm x 1.0 cm) of pure gypsum (CaSCU) suspended in the
microcosm tank or in the natural system. Gypsum cubes from the same
source and lot should be used for the entire set of dissolution tests in
the microcosms and in the natural system.
(F) The airspace between the canopy and water bath should be en-
closed and sealed with acrylic plastic sheets to facilitate containment of
the test substance transported into the gas phase (atmosphere) from the
water (see Figure 2 in paragraph (e)(2)(i)(B) of this guideline).
(7) The enclosed volume under the canopy and above the water bath
should be divided into relatively airtight compartments with Plexiglas pan-
els mounted transversely to the module and extending approximately 5
cm below the water surface of the water bath.
(2) Each airtight compartment should have its own air outlet to the
exhaust, a removable front cover to facilitate setting up and filling the
microcosm tanks, and hinged ports in the front cover to provide access
to the tanks during testing.
(G) Airflow over the water surface (microcosms and water bath) in
each compartment should be maintained by a manifold connected to an
exhaust fan which draws the air from all compartments through its outlet
tube and vents the exhaust air through a charcoal filter and a stack outside
the laboratory building (see Figure 2 in paragraph (e)(2)(i)(B) of this
guideline).
(ii) Microcosm. Each microcosm is a multitrophic level model that
combines pelagic and benthic communities similar to those existing in the
natural system.
(A) Hard glass (e.g., Pyrex) containers are preferred to soft glass or
plastic ones for the testing of organic chemicals.
(B) For each experiment, at least 20 microcosm tanks should be re-
quired. Each tank, about 140 L in capacity should hold enough water and
sediment to support the quantity of benthic invertebrates present in the
benthic subsystem, such as a medium-sized shellfish, for 30 days or more.
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(C) The benthic cylinder, up to 30 cm tall, should have an inner diam-
eter that makes the ratio of the sediment surface area to water volume
in the microcosm equal to that in the natural system.
(D) The benthic cylinder, which holds the sediment core, should be
sealed at the bottom end with a crystallization dish.
(E) The benthic pump (see Figure 1, in paragraph (d)(3)(v)(D) of this
guideline) should be an all-glass, air displacement pump. It should be large
enough to provide the appropriate water flow rate over the sediment sur-
face.
(F) To minimize disturbance of the sediment core by the discharge
from the benthic pump, a diffuser should be attached to the water outlet
tube of the benthic pump to direct the outgoing water into several hori-
zontal streams over the sediment surface.
(G) If the test substance forms a thin film covering the microcosm
water surface, a 6-cm length of glass cylinder, or surface film protector,
should be partially submerged in the water to provide a sampling port
for uncontaminated water samples after the surface film inside the cylinder
is removed.
(iii) Cleaning. Microcosm tanks, benthic cylinders, crystallization
dishes, benthic pumps, support rack, slick protectors, and glass rods should
be cleaned before use. All equipment should be washed according to stand-
ard laboratory practices to remove any residues remaining from manufac-
turing or previous use. A dichromate solution should not be used for clean-
ing glass containers. Solvents and/or high temperature (450 °C for 8 h)
combustion may be necessary to ensure the ultimate cleanliness of the
microcosms and associated glass components. If cleansing solvents are
used, disposal should conform to existing Federal regulations.
(3) Test parameters. Environmental conditions in the microcosm
should simulate the natural aquatic system as closely as possible.
(f) Reporting. The final report should include, but not necessarily
be limited to, the following information:
(1) Name and address of the facility performing the study and the
dates on which the study was initiated and was completed, terminated,
or discontinued.
(2) Objectives and procedures stated in the approved protocol, includ-
ing any changes in the original protocol.
(3) Statistical methods employed for analyzing data.
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(4) The test substance identified by name, Chemical Abstracts Service
(CAS) registry number or code number, source, lot or batch number,
strength, purity, and composition or other appropriate characteristics.
(5) Stability of the test substance under the conditions of administra-
tion.
(6) A description of the methods used, including:
(i) Description of microcosm facilities and supporting equipment; and
(ii) Description of natural system being simulated, including bound-
aries of natural system, pelagic community, benthic community, sediment
type, water quality, history of natural system, light regime, ratio of benthic
surface area to the water volume, water turbulence rate, water flow rate
over sediment surface, water turnover rate, light intensity over sediment
surface, seasonal attributes (e.g., water temperature), and ecological at-
tributes (e.g., productivity).
(7) A description of the test system used, including: microcosm tank
size, sediment core size, ratio of benthic surface area to water volume,
light intensity on water surface, light intensity on sediment surface, water
flow rate over sediment surface, and water turbulence.
(8) A description of the experimental design, treatment concentra-
tions, and methods and pattern of administration. The report results should
include:
(i) The results of the preliminary tests.
(ii) For the definitive test, various ecological effects and chemical
fate parameters may include:
(A) Ecological effects. (7) Phytoplankton abundance, in numbers per
mL, for the community or for each species.
(2) Zooplankton and transient larval forms abundances, in numbers
per liter, for the community or for each life stage of each species.
Number of organisms in the benthic community or, if known,
in each species, expressed in numbers per m3. Indicate the categories of
benthic organisms if species identification is not feasible.
(4) Concentrations of major nutrients, such as ammonium-nitrogen,
in the water column.
(5) Carrier effects when a carrier solvent is used.
(6) Assessment of microcosm realism by comparing the biological
attributes in the natural system to that in the control microcosms.
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(7) Effects of the test substance are assessed by comparing the treated
microcosms to carrier controls.
(B) Chemical fate. (1) The concentrations of test substance in rep-
resentative species of zooplankton and benthic organisms.
(2) The amount of test substance transported to the atmosphere.
(3) The amount of test substance adsorbed onto the glass surface of
the microcosm.
(4) The vertical distribution of the test substance in the sediment core
of the benthic component.
(5) The uptake and biotransformation of the test substance in biota.
(6) A mass balance consisting of the total quantity of the test sub-
stance added to the microcosm, the quantities exported from the micro-
cosm and the quantities remaining in the microcosm.
(7) Concentrations of the test substance and its transformation prod-
ucts, at steady state in the water column and sediment core, and the amount
on the glass surfaces both above and below the water surface and on the
surface film, if present.
(8) The effect of treatments on the residual concentrations of the test
substance in each ecosystem compartment.
(C) Transport of test substance and its transformation products.
(1) Amount of test substance and transformation products exported from
the microcosm through the air, water replacement, and removal of surface
film.
(2) The effect of the treatments on the export rate of test substance
and transformation products from each ecosystem compartment and on the
total amount of test substance being exported.
(D) Bioaccumulation potential of test substance in aquatic orga-
nisms. (1) The concentrations of test substance residues in aquatic orga-
nisms (mass of test substance per kilogram wet weight).
(2) The bioaccumulation factor for selected benthos as well as water
column species, such as zooplankton.
(3) The effect of the treatments concentration on the bioaccumulation
factor.
(4) A description of all circumstances that may have affected the qual-
ity or integrity of the data.
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(5) The name of the sponsor, study director, principal investigator,
names of other scientists or professionals, and the names of all supervisory
personnel involved in the study.
(6) A description of the transformations, calculations, or operations
performed on the data, a summary and analysis of the data, and a statement
of the conclusions drawn from the analysis.
(7) The signed and dated reports of each of the individual scientists
or other professionals involved in the study, including each person who,
at the request or direction of the testing facility or sponsor, conducted
an analysis or evaluation of data or specimens from the study after data
generation was completed.
The locations where all specimens, raw data, and the final report
are stored.
The statement prepared and signed by the quality assurance unit.
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