United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA 712-C-96-134
April 1996
&EPA Ecological Effects Test
Guidelines
OPPTS 850.1900
Generic Freshwater
Microcosm Test,
Laboratory
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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
(7 U.S.C. 136, 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 805 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 850.1900 Generic freshwater 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 797.3050 Generic freshwater
microcosm test (proposed in the Federal Register of September 28,
1987 (52 FR 36344)). This guideline may be used with OPPTS 850.7100.
(b)	Purpose. This guideline is intended for use in developing data
on the chemical fate and/or ecological effects of chemical substances and
mixtures ("test substances") subject to environmental effects testing regu-
lations under the Toxic Substances Control Act (TSCA) (Pub. L. 94-469,
90 Stat. 2003, 15 U.S.C. 2601 et seq.) This guideline prescribes meth-
odologies to predict the potential fate and/or effects of a chemical sub-
stance in freshwater ecosystems using various types of microcosms, i.e.,
standardized aquatic microcosm, naturally derived mixed-flask culture mi-
crocosm, or naturally derived pond microcosm, with and without sediment.
The microcosms contain freshwater algae and zooplankton with an assort-
ment of unidentified bacteria and fungi. The United States Environmental
Protection Agency (EPA) will use data from this test in assessing the po-
tential hazard of a chemical substance to freshwater ecosystems.
(c)	Definitions. The definitions in second 3 of TSCA and the defini-
tions in Part 792—Good Laboratory Practice Standards apply to this guide-
line. The following definitions also apply to this guideline:
Aseptic means free from contaminating organisms, e.g., aseptic trans-
fer of an algal culture into a sterilized tube via a sterile inoculating loop.
Axenic means free from other living organisms. An axenic culture
(pure culture) of algae contains only one species of algae, no bacteria,
and no fungi.
Batch culture means a culture of organisms that use only the initial
supply of nutrients in the culture medium. Without replenishment of nutri-
ents, concentrations of nutrients decline and waste products accumulate
in the culture medium with the increase in numbers of organisms.
Bioconcentration factor (BCF) means the ratio of the concentration
of the test substance in an organism or tissue (i.e., the biota) to the con-
centration in microcosm water or sediment, as specified.
Carrier means the organic solvent, solubilizer and/or other substance
used to disperse the test substance into microcosm water.
Detritivore means an organism (e.g., ostracod) that feeds on detritus,
i.e., dead organic matter.
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Ecosystem means a community of organisms and its interrelated phys-
ical and chemical environment functioning as a unit.
ECX means the experimentally derived test substance concentration,
in the aqueous phase, that is calculated to affect X percent of the test
species.
Generic microcosm means a general representation of an aquatic eco-
system in which a microcosm is maintained under constant laboratory con-
ditions and no attempt is made to simulate the physical/chemical environ-
ment of the natural system.
Gnotobiotic means a culture or community containing only known
species or organisms.
Grazer means an animal that grazes or feeds on growing plants, e.g.,
daphnids, rotifers, and some protozoa.
Herbivore means an animal that feeds on plants, synonymous with
grazer.
Linear contrast means the statistical comparison of the means of two
treatment groups, e.g., the control and another treatment group.
Medium means the chemically-defined culture solution used in the
microcosms.
Microcosm means a miniaturized model of a natural ecosystem.
Naturally-derived means using an assortment of organisms and/or
water and sediment collected from one or more natural freshwater
ecosystems.
Net daytime production means the increase in dissolved oxygen (DO)
concentration in microcosm water during the light phase of the
photoperiod.
Nighttime respiration means the decline in DO concentration during
the dark phase.
Semicontinuous culture means an algae culture that is periodically
harvested by partial draining and replenished with an equal volume of
fresh nutrient solution.
Standardized aquatic microcosm (SAM) means a culture of a commu-
nity containing known species of algae and aquatic invertebrates, but con-
taining uncharacterized species of protozoa and microorganisms.
Treatment group means the replicate microcosms that receive the
same amount (if any) of the test substance; controls are treatment groups
that receive none of the test substance.
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Unialgal culture means the cultivation or growth of a single species
of algae; each species of algae is established and maintained in a separate
culture.
Xenic means a culture or community containing one or more kinds
of unidentified organisms.
(d) Test procedures—(1) Summary of the test, (i) In preparation
for the test, a sufficient number of containers for the test plus an appro-
priate number of extra containers should be filled with appropriate volumes
of nutrient medium or natural water, numbers and types of organisms, and,
in some cases, natural or artificial sediment. Microcosm components
should be allowed to interact and adjust to one another for a specified
period of time. After culling microcosms which deviate most from the
group as a whole, microcosms should be randomly assigned to treatment
groups and to specific locations in the test area.
(ii)	The test should be started by applying the test substance to the
microcosms. Appropriate control groups should be established. Micro-
cosms should be sampled and/or monitored for changes in one or more
attributes at specified intervals during the exposure period or the recovery
period or both. The means of the attributes should be compared using suit-
able statistical methods to assess the fate or effects of the test substance.
Dose-response curves should be plotted for appropriate attributes.
(iii)	Microcosms should be monitored for at least 6 weeks after the
test substance is applied. Monitoring may be terminated earlier if all test
parameters in the treatment microcosms treated with the test substance re-
main the same as the control microcosms for 2 weeks after the application
of test substance (the last application in the case of multiple applications).
(2) Administration of test substance, (i) When possible, it is pre-
ferred that a test substance be radiolabeled so that its residues can be rap-
idly and accurately measured by radioassay.
(ii)	A test substance that is soluble in water should be dissolved in
distilled water to make a stock solution of known concentration; a nominal
concentration of test substance could be established in the microcosm by
adding a measured volume of stock solution and thoroughly dispersing
it by adequate stirring.
(iii)	A test substance that is insoluble in water, but that is soluble
in relatively non-toxic, water-miscible solvents, such as acetone, should
be dissolved in the minimum volume of carrier solvent required to form
a homogenous stock solution of known concentration. At the beginning
of the test, a measured portion of stock solution should be added to micro-
cosm water and dispersed to form a homogeneous suspension. Carrier con-
trols should be included in the experimental design and monitored simulta-
neously with microcosms treated with test substance.
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(iv)	A test substance that is insoluble in both water and water-miscible
solvents 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 microcosm water to form a homo-
geneous suspension.
(v)	In the pond microcosm, where stirring is hampered by the
macrophyte vegetation and the potential siltation of natural sediment, the
stock solution of test substance may be mixed thoroughly with 1 or 2
L of water taken from the microcosm, and poured slowly back into the
microcosm while the microcosm water is gently stirred.
(vi)	Sufficient quantities of stock solution should be made as needed
to minimize storage time and disposal volume.
(vii)	If the test substance is a formulated preparation, the strength
of the active ingredient (AI) in the preparation and the concentration of
the test substance in microcosm water should be specified in terms of per-
cent AI.
(viii)	The nominal concentration of test substance in both stock solu-
tion and microcosm water should be confirmed by chemical analyses at
the beginning of the exposure period.
(3) Range-finding test, (i) A range-finding test may be conducted
to establish if definitive testing is necessary and, if it is necessary, to estab-
lish concentrations of the test substance for the definitive test.
(ii)	Culled, old control, or newly established microcosms should be
exposed for 2 weeks to a series of test substance concentrations (e.g., 0.1,
1.0, 10, and 100 mg/L). Controls should also be used. The exposure period
may be shortened if sufficient data are gathered in a shorter time.
(iii)	The lowest test substance concentration in a test series, exclusive
of controls, should be the lowest concentration which can be analytically
quantified. The highest concentration should be 100 mg/L or the maximum
water solubility of the test substance at ambient temperature. Replicates
are not needed, and nominal concentrations of the test substance are ac-
ceptable for range-finding. If all calculated EC50s for all species are great-
er than 100 mg/L or less than the analytical detection limit, definitive test-
ing is not necessary. However, replicates and measured concentrations of
the appropriate dose are needed to substantiate this result.
(iv)	A range-finding test is not necessary if data on environmental
concentrations of the test substance are available from monitoring studies,
or environmental releases of the test substance are known or can be pre-
dicted from models, and the objective of the test is to bracket environ-
mental concentrations which result from the releases. Otherwise, a range-
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finding test is advisable since microcosm response can differ significantly
from single species tests.
(4) Definitive test—(i) Purpose. The purpose of the definitive test
is to determine the potential ecological effects and/or fate of a test sub-
stance released into the freshwater environment.
(ii)	Concentration. At least three concentrations of test substance,
exclusive of controls, should be tested. The concentration range selected
should define the dose-response curves for major microcosm species be-
tween EC 10 and EC90, unless a known environmental or release con-
centration is being bracketed. A minimum of six replicate microcosms
should be used for each concentration.
(iii)	Controls—(A) General requirements. Each test should include
controls consisting of the same nutrient medium or natural water, types
of biological groups, kind and amount of sediment (if present), and other-
wise should be treated the same as exposed groups, except that none of
the test substance is added. If a carrier is used to dissolve or suspend
the test substance, additional controls containing the carrier should also
be included in the test to determine any effect of the carrier on the micro-
cosms.
(B) Standardized aquatic microcosm. To demonstrate the health of
standardized microcosms in use, untreated controls should meet the criteria
specified below; otherwise, test data may be rejected by EPA, unless ade-
quately justified.
(1) One day 28, the following criteria should be met in the static
microcosms:
(z) At least 90 percent reduction in nitrate (NO3) concentration.
(ii)	Algal biomass in each mL of medium has exceeded
2,000 x 104 (|im)3.
(iii)	Oxygen gain has exceeded 4 mg/L (ppm).
(iv)	Population density of daphnids, including members of all size
groups, has exceeded 85 Daphnia per 100 mL.
(v)	Coefficient of variation for each microcosm attribute within ± 0.5
more than 50 percent of the time except as noted; coefficient of variation
should not be calculated for any nitrate concentration below 2 |iM or for
oxygen gain below 1 mg/L (ppm).
(vi)	pH values in late-afternoon between 6 and 10; coefficient of vari-
ation among replicate microcosms within ± 0.05 more than 50 percent
of the time.
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(2)	From day 28 to the conclusion of the test, the performance of
control microcosms should always meet the following criteria:
(z) Algal biomass exceeds 100 x 104 (|im)3/mL per mL.
(zz) Positive oxygen gain in daytime.
(in) Daphnid population density exceeds 15 Daphnia/100 mL.
(z'v) More than 50 percent of the time, the coefficient of variation
is within ± 0.5 among replicates of control microcosms for algal biomass,
daphnid population density, and for oxygen gain above 1.00 mg/L (ppm).
(v) pH values in late-afternoon between 6 and 9, and coefficient of
variation for pH values among control replicates within ± 0.05 more than
50 percent of the time.
(3)	When control microcosms fail to meet the above criteria, adequate
statistical justification is required for EPA acceptance of test data.
(iv) Initiation and maintenance of microcosms—(A) Standardized
aquatic microcosm. The standardized microcosm should be initiated and
maintained as follows:
(7) At least 36 glass jars (or more if extra controls are needed) should
be filled with 3 L of culture medium, 200 g of acid-washed silica sand,
0.5 g of rinsed chitin, and 0.5 g of cellulose powder, and sterilized in
an autoclave as specified in paragraph (e)(2)(ii)(A)(2) of this guideline.
(2)	On day 0, at least 30 of the 36 autoclaved jars containing sterilized
culture media should be inoculated with 10 species of algae at 103 cells/
mL for each species. Algal cultures are covered and incubated on a white
table under adequate illumination.
(3)	On day 4, algae cultures should be examined for algal abundance,
pH, oxygen gain, and other variables and each jar of algal culture should
be stocked with five species of animals, which include both grazers and
detritivores. The numbers of microinvertebrates to be added to each liter
of algal culture are 110 Hypotrich protozoans and 30 Philodina rotifers.
The volume of media with protozoa and rotifers should not exceed 5 mL.
The macro invertebrates to be stocked into each microcosm include:
(z) Sixteen daphnids consisting of 3 adults with embryos, 3 adults
without embryos, and 10 juveniles.
(zz) Six ostracods.
(z/z) Twelve amphipods consisting of three mating pairs (if possible)
and six juveniles.
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(4)	On day 7, the 30 microcosms should be reexamined and any
outliers should be culled. At least 24 microcosms should be selected for
the test. The following attributes of microcosms should be used in the
selection of the 24 microcosms:
(z) Dissolved oxygen gain in the daytime.
(z'z) pH value (pre-light).
(z/z) Abundance of daphnids and the presence of ostracods and
amphipods.
(zv) Abundance of Selenastrum and Chlamydomonas.
(5)	Selected microcosms should be randomly assigned to one of the
treatment groups including the controls, and located on the support table
in a six-block design as follows:
(z) Each of the 24 selected microcosms (the number of microcosms
for a typical test) should be randomly assigned to one of the four treatment
groups (including the control), appropriately labeled, and treated with ap-
propriate concentrations of the test substance except that the control micro-
cosm does not receive the test substance.
(zz) Each of the six microcosms in each of the four treatment groups
should be randomly assigned to one of the six block groups on the table;
therefore, each block group has four microcosms, one from each treatment
group.
(z/z) Finally, each of the four microcosms in each block group should
be randomly assigned to one of the four specific locations within that block
on the table.
(zv) To facilitate the handling of microcosms during the test, a series
of new numbers should be assigned to the microcosms according to their
ordered locations on the table.
(6)	The test substance should be added after sampling on experiment
day 7 (see paragraph (c)(4)(iv)(A)(7) of this guideline).
(7)	The standardized microcosm should be sampled and examined at
least once every 7 days after the test substance is added and reinoculated
as follows:
(z) After sampling and enumeration on each Friday, any microcosm
that is underpopulated (less than three individuals) with mature
macroinvertebrates should be reinoculated with reproductive age adults so
that each microcosm contains at least three individual amphipods,
daphnids, and ostracods.
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(zz) About 0.05 mL (1 drop) of dense Hypotrich protozoan culture
and the same volume of dense Philodina rotifer culture should be added
to each microcosm after each examination.
(z/z) Each microcosm should be reinoculated every 7 days with about
0.05 mL of an algal mixture that is prepared by pooling equal volumes
of monoculture from each of the 10 algal species.
(B)	Naturally-derived mixed-flask microcosm. The mixed-flask mi-
crocosm should be initiated and maintained as follows:
(7) A culture medium should be prepared from fresh refrigerated
stock solution (warmed to ambient temperature before measuring) in suffi-
cient volume to fill each container with 950 mL of culture medium from
the same stock solution.
(2)	Stock cultures, which are derived from biotic samples collected
from a variety of ecosystems, should be at least 3 months old before they
are inoculated into the microcosms.
(3)	Each microcosm should contain 50 mL of inoculum, 950 mL of
culture medium, and 50 mL of acid-washed sand, and should be randomly
assigned to one of the four treatment groups, including controls.
(4)	Inoculum in each 50-mL beaker should be placed under micro-
cosm water with the beaker and decanted into the microcosm water to
avoid exposing the zooplankton to the air during inoculation and cross-
seeding.
(5)	Microcosms are placed in the environmental chamber according
to a randomized block design.
(6)	All microcosms should be cross-seeded at least twice per week
for 3 weeks following inoculation. Cross-seeding should be performed by
collecting a 50-mL aliquot of a homogeneous suspension from each micro-
cosm, carefully pooling and mixing them together and returning a 50-
mL aliquot of the mixture to each microcosm.
(7)	Each microcosm should be reinoculated weekly with a 50-mL
inoculum.
(8)	Following weekly reinoculation, distilled water should be added
to each microcosm to return the volume to 1 L to compensate for loss
of water through evaporation.
(9)	The test substance (and carrier, if needed) should be introduced
into appropriate microcosms 6 weeks following initial inoculation of the
system.
(C)	Naturally-derived pond microcosm. The pond microcosms
should be initiated and maintained as follows:
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(7) All microcosm components, including water, sediment and biota,
should be collected from a single ecosystem, preferably on the same day
they are to be used. A shouldow pond is the best source of material for
pond microcosms, but littoral zones of lakes, or slow-moving rivers, may
be acceptable alternatives.
(2)	Water should be collected before sediment. At least 60 L of water
should be collected from the pond for each microcosm.
(3)	Sediment should be collected from the upper 26 cm of the pond
bottom and placed in appropriate containers for transportation. Stones,
twigs, and other large debris should be removed before the sediment is
placed in microcosm containers. At least 12 L of sediment are required
for each microcosm.
(4)	If a macrophyte community is present in the pond, a portion
should be collected from the bottom and placed in an appropriate con-
tainer. All organisms naturally associated with the macrophyte community
may be included in the samples except crayfish. At least 100 gm of the
macrophytes is needed for each microcosm. If macrophyte communities
are unavailable in the pond, filamentous algae communities may be col-
lected instead if present.
(5)	Water, sediment, and biota should be protected from bright sun-
light and extreme temperatures, and placed, as soon as possible, in an envi-
ronmental chamber that is set at a temperature equal to that of the pond
water.
(6)	The glass aquaria should be positioned in the environmental cham-
ber before filling.
(7)	Approximately 12 L of sieved sediment should be placed in each
aquarium, resulting in a layer of sediment about 6.7 cm thick. Sediment
in each transportation container should be equally divided among all mi-
crocosms.
(8)	If interstitial water sampling is planned, two suitable water collec-
tors, such as a glass diffuser, should be embedded in the sediment of each
microcosm. The fritted-glass disk of the air diffusers should be positioned
4 cm below the sediment surface which is leveled and smoothed.
(9)	Approximately 55 L of pond water should be added slowly to
each aquarium. Pond water in each transportation container should be
equally divided among all microcosms. To minimize resuspension of sedi-
ment during water filling, a plastic film may be used to cover the sediment
layer and a simple diffuser should be used to dissipate the kinetic force
of the water flow. The diffuser may be made of the bottom half of a
4-L polyethylene jug with holes punched around the perimeter.
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(10) One hundred grams of drained macrophytes or filamentous algae
from the source, such as Elodea canadensis, should be planted in the sedi-
ment in each microcosm.
(77) After macrophytes are planted, 1 to 2 L of water remaining in
the macrophyte collection container should be added to each microcosm
as an additional source of biota.
(72) The microcosm should be incubated in the environmental cham-
ber for at least 4 weeks before the test substance is applied.
(13) Distilled water should be added to the microcosms periodically
to compensate for the loss of water through evaporation. If a significant
volume of microcosm water is removed in sampling, it should be replaced
with an equal volume of dechlorinated tap water or well water.
(v) Sampling procedures—(A) Ecological effects. Sampling of mi-
crocosms for routine monitoring and final sampling can be performed as
follows:
(7) Each species of macroinvertebrates, including daphnids, ostracods,
and amphipods, in the microcosm can be counted visually if the numbers
of animals are less than 20 and the water is clear enough for counting.
When a dense population or turbid water hampers direct counting of all
macroinvertebrates in the microcosm, a series of 100-mL subsamples
should be taken out of the standardized microcosm for enumeration of
each macroinvertebrate species until 20 of each invertebrate are counted
or 6 subsamples are removed, whichever occurs first. Water samples
should be quickly captured and confined in a wide-mouth sampler before
removal. Periphyton should be scraped from the glass surface and thor-
oughly dispersed into the culture media preceding sampling of the water
column. Zooplankton should be counted in the mixed-flask microcosm by
removing a series of 25 mL subsamples. Four such samples are usually
sufficient. In the pond microcosm, zooplankton population should be meas-
ured twice per week. They are captured with a 2-L beaker that is sub-
merged rapidly into the microcosm water, concentrated on a 80-(im mesh
plankton bucket, stained, and preserved. Population density for three
groups of zooplankton, (i.e., cladocera, copepod, and rotifers) should be
counted in the pond microcosm: major groups of zooplankton should be
identified according to genus, or species if possible.
(2) The population density of protozoa and rotifiers should be deter-
mined in standardized aquatic microcosms, a water sample of up to 2 mL
should be dispersed in a 0.01-, 0.1- or 0.2-mL aliquot on counting plates
(e.g., Palmer cell with water depth of 4 mm) at 12x magnification under
a stereomicroscope. The total volume of aliquots examined should contain
at least 50 individuals per species.
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(3)	The population density of each algal species can be counted twice
per week. In the standardized aquatic microcosm, at least 50 cells should
be counted for each known algal species from a series of up to 35 fields
on the counting chamber under the microscope. If species cannot be identi-
fied, the major genus of the phytoplankton and periphyton should be iden-
tified for the following groups of algae: diatoms, green algae, euglenoid,
and blue-green algae.
(4)	Filamentous algae in the algal mat should be examined every 7
days with a microscope to detect the potential extinction of any inoculated
algae and the possible presence of contaminant algal species.
(5)	The biomass of primary producers should be estimated twice per
week with in vivo fluorescence or optical density of chlorophyll a in ace-
tone solution.
(6)	The rate of uptake of dissolved inorganic carbon-14 by
phytoplankton should be measured every 7 days as follows:
(z) Primary productivity in each microcosm should be measured in
duplicate bottles under the same light intensity as that intensity over the
microcosm, with a set of two duplicate bottles placed in the dark as con-
trols.
(z'z) Dissolved inorganic 14C should be kept sterile before the test.
For example, it may be kept in a sealed ampule and autoclaved.
(z/z) About 100 mL of water should be taken from each microcosm,
sieved through a 440-(im nylon screen and placed in a 125-mL conical
flask.
(z'v) The sieved phytoplankton suspension in each flask should be
shaken vigorously and poured into a set of four 16.5 mL test tubes until
water rises to the rim of each tube, which are then sealed with a serum
stopper.
(v) About 10 (iCi of 14C-labeled NaHCC>3 (specific activity about 1.0
(iCi/1.0 |ig) per milliliter of alkaline aqueous solution should be main-
tained at pH 9.5, packed in a glass ampule, and sterilized after the ampule
is sealed.
(vz) About 1 (iCi of NaH14CC>3 in 0.1 mL aqueous solution should
be injected into each of the four 16.5-mL test tubes. Two of the tubes
should be immediately placed in the dark inside a light-tight box while
the other two should be exposed to the same level of light intensity as
that prevailing over the microcosms. All tubes should be vigorously shaken
during the 2-h incubation.
(vz'z) After incubation, the phytoplankton culture in each tube should
be filtered through a 0.45 (iin filter membrane over a vacuum flask. The
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membrane filter and the phytoplankton retained on its surface should be
dried and stored in a desiccator over silica gel before the radioassay.
(viii)	Immediately before liquid scintillation counting, each filter
membrane with the phytoplankton materials should be fumed over con-
centrated hydrochloric acid for at least 90 seconds to remove remaining
inorganic 14C, and placed in a counting vial for radioassay.
(ix)	The counting rate for each liquid scintillation counting vial that
holds the particulate matter from one of the incubation tubes should be
properly calibrated for quenching effects.
(x)	If the absolute rate of carbon assimilation (besides the relative
14C uptake) is desired, the total dissolved inorganic carbon should be de-
termined. The total content of dissolved inorganic carbon in the micro-
cosm, which affects the specific activity of 14C (added as NaHCO.i) in
the incubation tube, should be measured simultaneously with measurement
of 14C uptake rate. Total CO2 content is usually calculated from measured
values of total carbonate alkalinity and pH in the microcosm water. It can
also be measured by gas chromatography if the buffering capacity of the
microcosm medium interferes with the alkalinity-pH method.
(7) The content of chlorophyll a in microcosm water should be meas-
ured weekly as follows:
(z) A sample of microcosm water, from 30- to 60-mL depending on
the standing crop of algae, should be sieved through a 0.3-mm nylon
screen to remove any macroinvertebrates among the phytoplankton.
(zz) Sieved microcosm water should be filtered under suction through
a 0.45 (im filter pad, which is covered with a fine powder of MgCC>3
at about 10 mg/cm2 of filter area. Following filtration, phytoplankton on
the filter pad should be immediately extracted for chlorophyll a or tempo-
rarily stored at -30 °C.
(z/z) Retained on the filter pad, the phytoplankton and magnesium car-
bonate should be placed in a glass, pestle-type homogenizer with
3 to 5 mL of 90 percent acetone and homogenized at 500 rpm for about
1	min.
(z'v) After each homogenate is transferred to a graduated centrifuge
tube equipped with a cap, the homogenizer and its pestle should be rinsed
2	to 3x with 90 percent acetone before its next use. The final volume
of pooled homogenate and washes should be adjusted to 15.0 mL.
(v) After the cap is fastened, the centrifuge tube with its contents
should be allowed to stand in a dark, cold (below 10 °C) place for at
least 1 h, and centrifuged at 4,000-5,000 g for approximately 10 minutes.
Any turbid supernatant should be recentrifuged if its absorbance at 750
nm exceeds 0.005 at 1 cm of light path.
12

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(vz) Without disturbing the precipitate, the supernatant in the cen-
trifuge tube should be poured or pipetted into a tube, capped, placed in
a dark place, and warmed to room temperature before quantification of
chlorophyll a by a fluorometric or spectrophotometry method.
(vzz) In spectrophotometry, the band-width of each monochromatic
light should be 3 nm or less. The absorbance (A) of the acetone extract
should be measured at 750, 663, 645, and 630 nm against a 90 percent
acetone blank. The concentration of chlorophyll a (x) in the acetone extract
(in micrograms per milliliter) should be calculated from the length of the
optical path (in centimeters) and the absorbance at each of the four wave
lengths using the formula:
[x] = mll.64(A663 - A750) - 2.16(A645 - A750) + 0.10(A63o - A750) %
(light path).
(viii) The concentration of chlorophyll a in a water sample (in
micrograms per milliliter) is calculated by multiplying the concentration
in the extract by the volume of the extract (in milliliters), and dividing
the product by the total volume of the water sample (in liters).
(8)	At least twice each week, the peak and troughs on the diel curve
of DO in microcosm water can be measured to estimate oxygen gain and
loss resulting from daytime photosynthesis and nighttime respiration, re-
spectively. The morning measurement of DO should be taken immediately
before the light is turned on, while the afternoon measurement should be
taken in the late afternoon or evening after the DO concentration in each
microcosm has reached the peak in its diel cycle. At least once during
the early part of the study, DO readings should be taken hourly during
the light cycle to determine when the peak occurs. The net daytime com-
munity production, which is the gain in DO during the 12-hour
photophase, should be calculated as the difference between the DO con-
centration at the end of the photophase and the DO concentration at the
end of the preceding dark phase. The net nighttime community respiration,
which is the loss of DO in the microcosm during the dark phase, should
be calculated as the difference between the DO concentration at the end
of the photophase and the DO concentration at the end of the following
dark phase.
(9)	The pH values of microcosm water should be read to 0.01 unit
after the pH meter is calibrated with standard buffers of pH 7 and pH
10, and the pH probe should be rinsed very thoroughly between readings.
The pH value should be taken at the same time day on scheduled sampling
dates after addition of the test substance to the microcosm as, for example,
0, 1, 2, 3, 5, 7, 10, 14, 21, 28, 35, and 42 days after addition of the
test substance. It is preferable to take the pH reading at the end of the
dark phase to reflect community respiration or at the end of the photophase
to reflect photo synthetic activity.
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(10) Dissolved nutrients in the microcosms should be monitored at
least twice each week for the first 4 weeks and at least once every 7 days
thereafter; the samples should be filtered through a 0.45 (iin membrane
and kept frozen before they are analyzed by standard analytical methods
for soluble reactive phosphorus, ammonia, nitrite, and nitrate.
(77) Net daytime community production and net nighttime community
respiration should be measured on scheduled sampling dates as, for exam-
ple, days 0, 1, 2, 3, 5, 7, 10, 14, 21, 28, 35, and 42 after addition of
the test substance.
(12)	Biomass decomposition rate, represented by the decomposition
rate of 14C-glucose in 15 mL of microcosm suspension, can be measured
on scheduled sampling dates as, for example, days 0, 1, 2, 3, 5, 7, 10,
14, 21, 28, and 35, after addition of the test substance to the microcosms.
Sampling for biomass decomposition (14C-glucose decomposition) should
precede reinoculation if both occur on the same day. The 14C-glucose de-
composition should be performed as follows:
(z) A 15 mL water sample should be collected in a 50-mL flask.
(zz) A glucose solution that contains 0.15 (iCi radioactivity in 0.3 mL
of distilled water should be added to the flask.
(z/z) The flask should be immediately sealed with a specially designed
serum stopper, fitted with a plastic center well containing a 2 x 5 cm
strip of chromatographic paper, and shaken gently for approximately 15
min in the dark.
(iv)	The heterotrophic activity should be stopped by injecting 1.0 mL
of 2N H2SO4 into the flask. A CO2 trapping agent, such as carbosorb,
should be immediately injected onto each filter paper under the stopper
after the acidification to collect the evolving CO2.
(v)	The flask should be gently shaken for at least 2 h, and the 14C
activity in the filter paper should be counted with a liquid scintillation
counter.
(vi)	The percentage deviation in the counts per minute (CPM) of the
treatment from the control should be calculated.
(13)	Total alkalinity, dissolved organic carbon, and specific con-
ductivity of microcosm water can be measured weekly.
(14)	Interstitial water in the sediment, if present, can be collected
weekly to be analyzed for ammonium-nitrogen content. The first 5-mL
water sample from the embedded gas diffuser, as specified in the pond
microcosm, should be discarded, and the second sample of 10 mL should
be filtered before chemical analysis.
14

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(75) Any extinction of macrophytes, such as Elodea canadensis in
the pond microcosm, in treated microcosms can be noted during the test,
and biomass of macrophytes should be determined at the end of the test.
(16) The extinction and reappearance of benthic fauna, such as in-
sects, snails, and oligochaetes, can be observed weekly in those micro-
cosms containing natural sediments.
(77) Water-borne bacteria can be counted weekly.
(B) Chemical fate. Sampling should be performed according to the
following procedures:
(7) The initial concentration of test substance in microcosm water
should be determined by chemical analysis of samples that are taken im-
mediately after the test substance is thoroughly dispersed in microcosm
water.
(2)	The dissolved test substance, its total residue, or both should be
measured in the filtrate of microcosm water semiweekly immediately after
the test substance is applied and at least once more during the first week,
measured at least once during the second week, and measured biweekly
until the end of the test. The filtrate may be substituted with unfiltered
microcosm water if the test substance is partitioned into the particulate
fraction in such a high proportion that the chemical concentration in the
filtrate fraction falls below the analytical detection limit for the test sub-
stance using the most practical analytical method.
(3)	The concentration of test substance in macrophyte shoots, if
present, can be measured biweekly if the sample is less than 5 percent
of biomass.
(4)	Distribution of the test substance among compartments of micro-
cosms can be determined at the end of the test; the components may in-
clude:
(z) Macrophytes, subdivided into roots, shoots, and leaves.
(zz) Phytoplankton.
(z/z) Zooplankton.
(z'v) Benthic fauna.
(v) Sediment core, sectioned into 1-cm subcores.
(vz) Periphyton, if any.
(5)	Analytical measurements—(i) Chemical. Standard analytical
methods should be used in performing analyses. The analytical method
used to measure the amount of test substance in a sample should be vali-
15

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dated by appropriate laboratory practices before beginning the test. An
analytical method is not acceptable if likely degradation products of the
test substance, such as hydrolysis or oxidation products, give positive or
negative interference which cannot be systematically identified and mathe-
matically corrected.
(ii) Numerical. (A) The following data should be obtained from the
standardized microcosm test:
(7) Abundance of each species of alga and macroinvertebrate.
(2)	Abundance of each type of microscopic animal (i.e., protozoa and
rotifers).
(3)	Net daytime production.
(4)	Net nighttime respiration.
(5)	Chlorophyll a concentration.
(<5) Water pH.
(7) Nutrients (at least nitrate) in water.
(B)	The following data should be obtained from the mixed-flask, mi-
crocosm test:
(7) Abundance of phytoplankton and zooplankton.
(2)	Net daytime production (DO gain).
(3)	Net nighttime respiration (DO loss).
(4)	Chlorophyll a concentration.
(5)	Water pH.
(6)	14C glucose decomposition rate.
(C)	The following data should be obtained for the pond microcosm:
(7)	Abundance of phytoplankton and zooplankton.
(2)	Abundance of each type of benthic fauna.
(3)	Net daytime production.
(4)	Net nighttime respiration.
(5)	Chlorophyll a concentration.
(6)	Water pH, alkalinity, conductivity, and dissolved oxygen.
16

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(7)	Concentrations of the test substance in each compartment of the
microcosm.
(8)	Bioconcentration factor.
(D)	Means and standard deviations of each chemical and biological
attribute specified in this test rule should be calculated for the replicates
of each treatment and control groups.
(E)	EC50 values and their 95-percent confidence limits should be
calculated for each of the appropriate attributes for the time between appli-
cation of the test substance and recovery from test substance treatment,
if data are adequate for statistical analysis. Otherwise, ECX should be cal-
culated as the percent deviation of an attribute in a treatment group from
that in the control.
(F)	Appropriate statistical analyses (e.g., Dunnett's procedure) should
be performed to determine whether significant differences in attributes
exist between the carrier (if appropriate) and carrier-free controls and be-
tween the control and treated groups, and between microcosms receiving
different concentrations of test substance.
(G)	For the pond microcosm, appropriate statistical analyses should
be performed to determine whether significant differences in the amount
and in the bioconcentration factor of the test substance exist between treat-
ed different compartments within treated microcosms and between treated
microcosms receiving different treatments.
(e) Test conditions—(1) Test species—(i) Selection. (A) The orga-
nisms inoculated into the standardized microcosm should include 10 algal
species; 1 each of protozoa, rotifer, daphnid, ostracod, and amphipod spe-
cies; and an uninvited assortment of unidentified heterotrophs, such as bac-
teria and fungi.
(7) The following 10 species of algae should be included: Anabaena
cylindrica, Ankistrodesumus sp., Chlamydomonas reinhardi, Chlorella
vulgaris, Lyngbya sp., Nitzschia kutzigiana, Scenedesmus obliquus,
Selenastrum capricornutum, Stigeeclonium sp., Udotheric sp.,
(2)	Daphnia magna should be included. Species identity of the test
daphnids should be verified using appropriate systematic keys.
(3)	Amphipods, Hyaletia azteca, also named H. knickerbockeri,
should be used in the test. Mating pairs and the young are inoculated into
the microcosm.
(4)	Ostracods chosen should be either Cypridopsis or Cyprinotus sp.
Only adults should be used.
17

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(5)	Protozoa should belong to the order Hypotrichida, and the culture
should be 72-h-old when it is inoculated into the microcosm.
(6)	Rotifers should belong to the Philodina sp.
(B)	Inoculum for the mixed-flask microcosm test should at least con-
tain the following:
(7)	Two species of single-celled green algae or diatoms.
(2)	One species of filamentous green alga.
(3)	One species of nitrogen-fixing blue-green alga.
(4)	One species of grazing macroinvertebrate.
(5)	One species of benthic, detritus-feeding macroinvertebrate.
(6)	Bacteria and protozoa.
(C)	The following broad groups of biota should be included in the
pond microcosm: Macrophyte, phytoplankton, periphyton, zooplankton,
and benthic animals.
(ii) Source. (A) Each unialgal culture that is a part of the 10-species
composite inoculum for all standardized microcosms in a test should be
of the same batch that in turn is subcultured to the exponential growth
phase from a single source. Before the test, at least two successive
subcultures outside the microcosm are required to acclimate the algal
monoculture from agar slant to microcosm medium. A semicontinuous cul-
ture system is recommended for culture of unicellular algaeAnabaena,
Ankistrodesumus, Selenastrum, and Lyngbya should be cultured in batch
culture before they are inoculated into microcosms. Recommended proce-
dures for culturing algae as well as the other organisms used in this test
are described by Taub and Read under paragraph (g)(2) of this guideline.
(B)	The original stock culture for the mixed-flask microcosm should
be collected from a variety of natural ecosystems. New stock culture
should be added to the old stock cultures at least twice each year. To
prepare the inoculum for microcosms, samples from several different aged
stock cultures should be mixed together. Stock cultures should be at least
3 mon old to be used as a source of microcosm inoculum. Distilled water
should be added to the stock cultures in the open aquaria as needed to
replace losses by evaporation. Aquatic organisms collected from a variety
of natural ecosystems should be inoculated into culture medium to start
stock cultures.
(C)	Organisms for the pond microcosm should be obtained from the
same natural ponds that supply the water and sediment used in the micro-
cosm.
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(2) Facilities—(i) Apparatus. (A) The environmental chambers or
room housing the microcosms should provide adequate environmental con-
trols to meet temperature, irradiation, photoperiod, and air circulation re-
quirements. Chambers should be designed to prevent escape of contami-
nated internal air into the external environment by using appropriate filter-
ing devices to prevent contamination of the external environment with the
test substance.
(B)	Laboratory facilities where the test substance is handled should
have nonporous floor covering, absorbent bench covering with imper-
meable backing, and adequate disposal facilities to accommodate liquid
waste (e.g., test and waste solutions containing the test substance at the
end of each test), and solid wastes (e.g., bench covering, lab clothing,
disposable glassware, or other contaminated materials).
(C)	The test substance should be stored in a room separate from stock
cultures and microcosms.
(D)	A large autoclave capable of sterilizing several 1-gal microcosm
containers should be used. An autoclave large enough for sterilizing cul-
ture medium in a 20-L (5-gal) carboy is desirable.
(E)	The dimensions of the bench space for the gnotobiotic micro-
cosms should be at least 2.6 x 0.85 m and should have a white top or
white covering.
(F)	Standard laboratory equipment and, if the test substance is
radiolabeled, a liquid scintillation counter for radioassays is required.
(G)	For the standardized and mixed-flask microcosm tests, a special
sampler should be used to capture macroinvertebrates from the microcosm.
The sampler should be taller than the microcosm to reach the bottom of
the jar, have a large diameter for free passage of water into the sampler,
and a rubber stopper attached to a long glass rod to stir the water before
sampling and to seal the bottom of the sampler for transferring water out
of the microcosm after the sample is taken.
(ii) Containers and media—(A) Standardized microcosm. (7) The
containers used in each standardized microcosm test should be new glass
jars with the capacity of at least 1 gal (3.8 L). The jars should be at least
25 cm in height and 16.0 cm in diameter, with an opening 10.6 cm in
diameter. The new jars should be washed with diluted (1:10) HC1, flushed
with tap water, and rinsed with distilled water before use.
(2) Each standardized microcosm should contain at least 3 L of a
medium, such as Taub's T82MV, in addition to an artificial sediment made
of silica sand (200 g) enriched with chitin (0.5 g) and cellulose (0.5 g).
Before use, the sand should be washed with diluted (1:10) HC1 for 2 h,
repeatedly rinsed with clean water until the pH rises to 7, and dried in
19

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an oven at 120 °C. The crude chitin from commercial sources should be
rinsed with distilled water, air-dried, ground in a blender, and sifted
through a 0.4 mm sieve. The cellulose powder, which is also packing ma-
terial for chromatographic columns, is commercially available.
(B)	Naturally derived mixed-flask microcosm. Hard-glass contain-
ers (e.g., 1-L Pyrex beakers), should be selected for testing organic sub-
stances in mixed-flask microcosms. Polypropylene beakers may be used
for testing inorganic substances.
(C)	Naturally derived pond microcosm. For the pond microcosm
test, 72-L glass aquaria (60 cm long by 30 cm wide by 40 cm deep)
should be used as containers. About 12 L of sieved sediment and 55 L
of pond water should be added to each aquarium.
(D)	Materials and equipment. Materials and equipment that contact
test solutions should be selected to minimize sorption of test substances
from the microcosm and should not contain substances that can be leached
into aqueous solution in quantities that can affect test results.
(iii)	Cleaning and sterilization. Microcosm containers, stock culture
containers, nutrient storage containers, and all other glassware should be
cleaned before use. All glassware and equipment should be washed accord-
ing to good standard laboratory procedures to remove any residues remain-
ing from manufacturing or previous use. Dichromate solution should not
be used for cleaning glassware. In the standardized microcosm, all glass
containers and equipment for culturing and testing organisms should be
sterilized by autoclave where possible. DO and pH probes may be cleaned
with ethanol and thoroughly rinsed with distilled water before use. All
sampling devices should be sterilized before each test; sampling devices
in contact with lake water or sediment should be sterilized after each use.
(iv)	Nutrient media. (A) Taub's T82MV (see paragraph (g)(2) of
this guideline) medium is recommended for use in the standardized micro-
cosm. Its composition is given in the following Table 1.
20

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Table 1.—Nutrient Medium, Taub T82MV
[pH adjusted to 7.0 with dilute HCI (1:10)]
Compound
Molecular
weight
Concentration
Units
Element of
concern
mg/L


mM


NaN03 	
85.0
0.5
N
7.0
MgS047H20 	
246.5
0.1
Mg
2.43
KH2P04	
136.0
0.04
P
1.23
NaOH 	
40.0
0.099
Na
2.27
CaCI22H20 	
147.0
1.0
Ca
40.0
NaCI 	
58.5
1.5
Na
34.5
AI2(S04)318H20	
666.5
0.0048
Al
0.26
Na2Si03 9H20 	
284.0
0.80
Na
36.8
sand 	


Si
22.4
Trace Metals:

|^M


FeS047H20 	
278.0
1.12
Fe
0.0625
H3BO3 	
61.8
0.75
B
0.008
ZnS04 7H20 	
287.5
0.025
Zn
0.0015
MnCI24H20 	
197.9
0.25
Mn
0.0135
Na2Mo042H20 	
242.0
0.025
Mo
0.0024
CuS04 5H20 	
249.7
0.005
Cu
0.00032
Co(N03)26H20 	
291.0
0.0025
Co
0.00015
EDTA	
292.0
1.42
EDTA
0.4145
Vitamins:




Calcium pantothenate 	
476.5
1.47
—
0.70
Cyanocobalamin (Bi2)	
1,355.4
0.000022
-
0.00003
Thiamin (Bi) 	
337.3
0.18
—
0.06
Riboflavin (B2) 	
376.4
0.11
—
0.04
Nicotinamide 	
122.1
1.06
—
0.13
Folic Acid 	
441.4
0.75
—
0.33
Biotin 	
244.3
0.12
—
0.03
Putrescine 	
161.1
0.19
—
0.03
Choline 	
181.7
2.75
—
0.50
Inositol 	
216.2
5.09
—
1.10
Pyridoxine monohydrochloride	
205.7
2.43
-
0.50
(B)	The recommended medium for growth and establishment of stock
cultures for the mixed-flask microcosm is Taub's T82, which is the same
as T82MV without vitamins. The modified Taub's no. 36 medium (Leffler
1981) under paragraph (g)(1) of this guideline used in the early protocol
development is also adequate.
(C)	There is no need to add nutrients to pond microcosms.
(3) Test parameters. Environmental conditions for the microcosms
should be maintained as follows:
(A)	Temperature within 21 to 25 °C (23 ±2 °C).
(B)	Photoperiod of 12 h light/12 h darkness.
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(C) Standard deviation of light intensities among the microcosms
within ± 10 percent of the mean and a light intensity of 150 (iEm 2sec
for this test.
(e) Reporting. (1) 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 completed, terminated, or dis-
continued.
(ii)	Objectives and procedures stated in the approved protocol, includ-
ing any changes in the original protocol.
(iii)	Statistical methods used for analyzing the data.
(iv)	The test substance identified by name, Chemical Abstract Service
(CAS) Registry number or code number, source, lot or batch number,
strength, purity, and composition, or other appropriate characteristics.
(v)	Stability of the test substance under the conditions of administra-
tion.
(vi)	A description of the methods used, including the facilities and
supporting equipment.
(vii)	A description of the test system used, including: Microcosm di-
mensions and water volume, sediment type and volume if used, tempera-
ture, photoperiod, and light intensity over the water surface.
(viii)	A description of the organisms included in the microcosms rep-
resenting various functional groups that are essential for the maintenance
of a healthy microcosm.
(ix)	A description of the nutrient media, if applicable.
(x)	A description of the experimental design, treatment concentrations
and media, and pattern of administration.
(xi)	The materials, the methods, and the results of any range-finding
test.
(xii)	For the definitive test, reported results should include:
(A) For the standardized microcosm, a description of the following
ecological effects and the fate of the test substance in the biota:
(7) Phytoplankton abundance, in numbers per milliliter, for each spe-
cies.
(2)	Population density of rotifers and protozoans, in numbers per mil-
liliter, for each species.
22

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(3)	Abundance of daphnids, in numbers per liter, for each size group
(small, medium, and large).
(4)	Abundance of amphipods, in numbers per microcosm, for each
size group (small and large).
(5)	Abundance of ostracods, in numbers per microcosm.
(6)	Relative abundance of phytoplankton in microcosms.
(z) Absorbance density at 440 nm, as an index of the particulate mate-
rials, including phytoplankton.
(zz) Content of chlorophyll a.
(z/z) In vivo fluorescence.
(7)	Concentrations of major mineral nutrients, such as orthophosphate,
ammonia, nitrite, and nitrate in the filtrate of microcosm water.
(8)	Primary productivity, as measured by 14C-uptake methods.
(9)	Community production and respiration, measured by the three-
point methods (the net gain in dissolved oxygen during the photophase
is the photo synthetic production of phytoplankton, while the loss of DO
during the dark phase is an indicator of community respiration).
(10)	Carrier effects when a carrier is used. Assessed by comparing
biological variables in carrier controls to those in plain-water controls.
(77) Chemical effects assessed by comparing biological data in treated
microcosms to that in plain-water controls or in combined controls for
both the carrier and plain water.
(B) For the mixed-flask microcosm, a description of the following
ecological effects and the fate of the test substance in biota:
(7) Phytoplankton abundance for the entire community or standing
crop for each of the major species, in number of plants per milliliter.
(2)	Zooplankton abundance for the community or standing crop for
each life stage of the major species, in numbers of animals per liter.
(3)	Type and total number of the benthic organisms, or the standing
crop for each species of benthic organism, in numbers of organisms per
square meter.
(4)	Carrier effects when carrier is used.
(5)	Chemical effects assessed by comparing treated microcosms to
controls.
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(<5) EC50 values for the test substance expressed in terms of pH
change, net daytime community production, net nighttime community res-
piration, and decomposition rate of organic matter.
(7)	Concentration of test substance residues in aquatic organisms or
in specific tissues.
(8)	The bioconcentration factors of the test substance or its total resi-
dues.
(9)	Effect of the initial concentration of the test substance on its
bioconcentration factor.
(C) For the pond microcosm, a description of the following ecological
effects and fate of the test substance in biota:
(7) Phytoplankton abundance for the entire community or standing
crop for each of the major species, number of phytoplankton per milliliter
or chlorophyll a concentration.
(2)	Chlorophyll a content of periphyton and the major groups of
periphytons, such as diatoms, green algae, blue-green algae, and euglenoid,
if possible, genus or species names.
(3)	Abundance of macrophytes in the microcosm calculated by esti-
mating the volume of microcosm water occupied by the macrophytes and
determining the standing crop of the macrophytes, including tops and
roots.
(4)	Zooplankton abundance for the community or standing crop for
each life stage of the major species, in number of animals per liter.
(5)	Type and total number of benthic organisms, or standing crop
for each species of benthic organism, in number of organisms per square
meter.
(6)	Concentration of major dissolved nutrients, such as ammonium-
nitrogen, nitrate and nitrite, and orthophosphate, in microcosm water.
(7)	Carrier effects when carrier solvent is used.
(8)	Chemical effects assessed by comparing treated microcosms to
controls.
(9)	The median effect concentration (EC50) and its 95-percent con-
fidence limit if the concentration of test substance causes partial reduction
in any biological attribute in enough treatment groups. If the partial reduc-
tion occurs in only a few treatment groups, indicate the percentage reduc-
tion of biological abundance caused by the concentration of test substance
(ECX).
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(10) Element cycling such as ammonium-nitrogen content, in
micrograms per liter.
(77) Maximum and minimum diel DO concentration on sampling
date.
(72) Net daytime production and net nighttime respiration, in milli-
grams per liter of DO change.
(13)	Ratio of production to respiration (P/R ratio).
(14)	Concentrations of the test substance in both particulate and dis-
solved fractions of the water column.
(15)	Concentration of test substance in representative species of
zooplankton and benthos.
(16)	Concentration of test substance in periphyton.
(77) Vertical distribution of the test substance in the sediment core.
(18)	Concentrations of the test substance in total biota.
(19)	Concentrations of the test substance which may include its trans-
formation products, at steady state in the water column and sediment pro-
file, and the amount in the periphyton on the glass surface.
(20)	Effect of the test substance concentration applied to the micro-
cosm on the residual concentration of the test substance in each compart-
ment.
(21)	Bioconcentration factors of the test substance or its total residues.
(22)	Effect of the initial concentration of test substance on its
bioconcentration factors.
(D) A description of any circumstance that may have affected the
quality or integrity of the data, including reporting and explaining any sig-
nificant excursions from normal for microcosm parameters during the test.
(xiii)	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.
(xiv)	A description of the transformations, calculations, or operations
performed on the data, and a statement of the conclusion drawn from the
analysis.
(xv)	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
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an analysis or evaluation of data or specimens from the study after data
generation was completed.
(xvi)	The locations where all specimens, raw data, and the final report
are stored.
(xvii)	The statement prepared and signed by the quality assurance
unit.
(g) References. The following references should be consulted for ad-
ditional background information on this guideline, :
(1)	Leffler, J.W. Tentative protocol of an aquatic microcosm screen-
ing test for evaluating ecosystem-level effects of chemicals. Final report,
EPA Contract No. 68-01-5043 (Subcontract No. T6411(7197)025 with
EPA Office of Toxic Substances, Washington, DC (1981)). Available from
J.V. Nabholz, 7403, Environmental Effects Branch, Health and Environ-
mental Review Division, Office of Pollution Prevention and Toxics, U.S.
Environmental Protection Agency, 401 M St., SW., Washington, DC
20460-0001.
(2)	Taub, F.B., and Read, P.L. Standardized aquatic microcosm pro-
tocol. Draft final report, U.S. Food and Drug Administration Contract No.
223-83-7000 with FDA, Washington, DC 20005 (1986). Available from
Dr. B.L. Hoffmann, U.S. FDA, HFF-304, Environmental Impact Staff,
1110 Vermont Ave., NW., Suite 710, Washington, DC 20005.
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