OPPTS Harmonized Test Guidelines Series 850 Ecological Effects Test Guidelines Volume I Guidelines 850.1000


                OPPTS HARMONIZED TEST GUIDELINES
                               Series 850




                      ECOLOGICAL EFFECTS




                         TEST GUIDELINES
                                Volume I

                        Guidelines 850.1000 - 850.1950
                                DRAFT
                                April 1996
*                 United States Environmental Protection Agency
  EPA

  740/           Office of Prevention, Pesticides, and Toxic Substances


  1996a                     Washington, D.C. 20460

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Series 850—Ecological Effects Test Guidelines
April 1996
OPPTS
Number
850.1000

850.1010
850.1020
850.1025
850.1035
850.1045
850.1055
850.1075
850.1085
850.1300
850.1350
850.1400
850.1500
850.1710
850.1730

850.1735
850.1 740
850.1 790
850.1800
850.1850
850.1900

850.1925
850.1950

850.2100
850.2200
850.2300

850.2400
850.2450
850.2500

850.3020
850.3030
850.3040

850.4000
850.4025
850.4100
850.4150
850.4200
850.4225
850.4230
850.4250
850.4300
850.4400

850.4450
850.4600
850.4800

850.5100
850.5400

850.6200
850.6800

850.7100
Name
Special consideration!! for conducting aquatic laboratory studies
Group A— Aquatic Fauna Test Guideline*.
Aquatic (nvetebrate acute toxicity. test, freshhwater daphnios
Gammand acute toxicity test
Oyster acute toxicity test (shell deposition)
Mysid acute toxicity lest
Penaeid acute toxicity test
Bivalve acute toxicity test (embryo larval)
Fish acute toxicity test, freshwater and marine
Fish acute toxicity mitigated by humic acid
Daphnid chronic toxicity test
Mysid chronic toxicity test
Fish early-life stage loxicity test
Fish life cycle toxicity
Oyster BCF
Fish BCF

Whole sediment acute toxicity invertebrates, freshwater
Whole sediment acute toxicity invertebrates, mahne
Chironomid sediment toxicity test
Tadpole/sediment subchronic toxicity test
Aquatic food chain transfer
Generic freshwater microcosm test, laboratory

Site-specific aquatic microcosm test, laboratory
Field testing for aquatic organisms

Group B— Terrestrial Wildlife Test Guidelines
Avian acute oral toxicity test
Avian dietary toxicity test
Avian reproduction test

Wild mammal acute toxicity
Terrestrial (soil-core) microcosm test
Field testing for terrestrial wildlife
Group C — Beneficial Insects and Invertebrates Test Guidelines.
Honey bee acute contact toxicity
Honey bee toxicity of residues on foliage
Field testing for pollinators
Group D — Nontarget Plants Test Guidelines.
Background — Nontarget plant testing
Target area phytoloxicity
Terrestrial plant toxicity. Tier I (seedling emergence}
Terrestrial plant toxicity. Tier I (vegetative vigor;
Seed germination/root elongation toxicity test
Seedling emergence. Tier II
Early seedling growth toxicity test
Vegetative vigor, Tier II
Terrestrial plants field study. Tier HI
Aquatic plant toxicity test using Lemna spp. Tiers I and II

Aquatic plants field study. Tier III
Rhaobium-legume loxicity
Plant uptake and translocation test
Group E — Toxicity to Microorganisms Test Guidelines.
Soil rnicrobial community loxicity test
Algai toxioly. Tiers 1 and II

Group F — Chemical-Specific Test Guidelines.
Earthworm subchronic toxicity test
Modified activated sludge, respiration inhibition test for spanngly soluble chemicals
Group G — Field Test Data Reporting Guidelines.
Data reporting for environmental chemistry methods
Existing Numbers
OTS
none

797.1300
795.120
797.1800
797.1930
797.1970
none
797.1400
797.1460
7971330
7971950
7971000
none
797.1830
797.1520

none
none
795.135
797.1995
none
797.3050,
.3100
797.3100
none

797.2175
797.2050
797.2130.
.2150
none
797.3775
none

none
none
none

none
none
none
none
797.2750
797.2750
797.2800
797.2750
none
797.1160

none
797.2900
797.2850

797.3700
797.1050

795.150
795.170

none
OPP
none

72-2
none
72-3
72-3
72-3
72-3
72-1 . 3
none
72-4
72-4
72-4
72-5
72-6
72-6,
165-4
none
none
none
none
72-6
none

none
72-7.
165-5

71-1
71-2
71-4

71-3
none
71-5

141-1
141-2
141-5

120-1
121-1
122-1
:22-i
122-1
123-1
123-1
123-1
124-1
122-2,
123-2
124-2
none
none

none
122-2.
123-2

none
none

none
OECD
none

none
none
none
none
none
none
203
none
202
none
210
none
none
305

none
none
none
none
none
none

none
none

none
205
206

none
none
none

none
none
none
none
none
none
none
none
none
none

none
none
none

none
none

207
209

none
EPA Pub.
no.
712-C-
96-113

96-114
96-130
96-115
96-136
96-137
96-100
96-118
96-117
96-120
96-166
96-121
96-122
96-127
96-129

96-354
96-355
96-313
96-132
96-133
96-134

96-173
96-135

96-139
96-140
96-141

96-142
96-143
96-144

96-147
96-148
96-150

96-151
96-152
96-153
96-163
96-154
96-363
96-347
96-364
96-155
96-156

96-157
96-158
96-159

96-161
96-164

96-167
96-168

96-348
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United Stales Prevention, Pesticides EPA712-C-96-113
Environmental Protection and Toxic Substances April 1996
Agency (7101)
SEPA Ecological Effects Test
Guidelines
OPPTS 850.1000
Special Considerations
for Conducting Aquatic
Laboratory Studies
'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 Davis1 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 850.1000 Special considerations for conducting aquatic lab-
oratory studies.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of 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) [Reserved]

(b) Introduction. (1) This guideline provides additional information
on how to design and conduct aquatic laboratory studies with emphasis
on the importance of adequate characterization of the test material and
proper understanding of how the material behaves under test conditions.
This guideline also attempts to interpret those areas that need to be defined
and set limits for designing and conducting laboratory studies.

(2) Agency guidance for performing aquatic testing sets forth a rea-
sonable position and approach for testing criteria, limits, and standards.
However, standards are set with the recognition that certain problems will
arise and provisions must be made to accommodate unavoidable problems.
This document provides for exceptions, while at the same time maintaining
a high level of scientific integrity so that testing will provide information
that is scientifically defensible and protective of the environment, while
taking into consideration the chemistry of the test material.

(c) General considerations. (1) Note that for aquatic toxicity testing,
the solubility and stability of the test material must be known for the con-
ditions under which it will be tested and chemical analysis of the batch
test material must be performed. Determining the solubility and stability
of the test material in the mixture or test solution is an important part
of these studies.

(2) The behavior of a test material should be based on experiments
which are conducted under the same conditions as those occurring during
the test. These include but are not limited to:

(i) Test solution characteristics (salt or freshwater).

(ii) Temperature, pH, conductivity, lighting.

(iii) With test organisms in place.

(iv) Use of the same test containers.

(v) Use of the same flow-through systems where appropriate.

(3) All chemistry methods used in preliminary trials, in range-finding
tests, in establishing percent purity of batches of test material, or in meas-
uring concentrations in test containers must be submitted with the study.
The documentation must include a complete description of the method so
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that a bench chemist can determine the necessary equipment and perform
the analysis. It must also include the raw data, standards, and chro-
matograms from a representative analysis using the method. This rep-
resentative analysis must be conducted with the specific media for which
it will be used during the test—i.e., analysis should be performed under
test conditions. The actual limit of detection (LOD) and limit of quantifica-
tion (LOQ) must be identified.

(d) Definitions.

EEC is the effective environmental concentration.

LOD is the limit of detection below which the qualitative presence
of the material is uncertain.

LOEC is the lowest-observable-effect-concentration.

LOQ is the limit of quantification below which the quantitative
amount of the material is uncertain relative the amount.

Measured concentration is an analytically derived measure above the
LOQ.

NOEC is the no-observed-effect-concentration.

Nominal concentration is, for aquatic tests, the nominal test level,
which is the concentration that would exist if all test material added to
the test solution was completely dissolved and did not dissipate in any
way.

Recommended means that the procedure or test is preferred in order
to avoid problems, but it is not required. If the recommended procedure
or test is not performed, the study will not necessarily be rejected.

Solubility is defined as the amount of chemical retained in the super-
natant of a conventionally centrifuged sample of test medium.

(e) Stability. (1) A test material is considered to be stable under test
conditions if, under those conditions, it does not degrade, volatilize, dis-
sipate, precipitate, sorb to test container walls, or otherwise decline to con-
centrations less than 70 percent of the day-0 measured concentration dur-
ing the study period. If it is expected to decline to less than 70 percent
of the day-0 measured concentration during the study period, either static
renewal or flow-through design is needed to try to ensure that the test
concentration is maintained at levels greater than or equal to 70 percent.
The only exception is testing with algae and diatoms, which cannot be
tested in static renewal or flow-through systems (see discussion in para-
graph (m) of this guideline on testing with algae and diatoms).

(2) Static renewal is one method to ensure relatively continuous con-
centrations when the test material is not stable under test conditions. At
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a minimum, the renewal cycle should be based on the stability of the test
material under test conditions. The time to renewal (renewal cycle) should
be shorter than the time it takes for the concentration of the test material
to decline to < 70 percent. (The renewal cycle may be shorter than required
by stability characteristics of the test material because of other factors,
such as dissolved oxygen, feeding, etc.)

(f) Sample storage. If samples of growth medium, stock solutions,
or test solutions collected for chemical analysis cannot be analyzed imme-
diately, they should be handled and stored appropriately to minimize loss
of the test material. Loss could be caused by such processes as microbial
degradation, hydrolysis, oxidation, photolysis, reduction, sorption, or vola-
tilization. Stability determination under storage conditions, whether it re-
fers to storing the test material before testing or storing samples awaiting
analysis, is required by GLP regulation.

(g) Preliminary trials. (1) The Agency recommends preliminary test-
ing for problem chemicals. The information about stability and solubility
of problem chemicals should be developed under test conditions. This in-
formation can be gained while doing the currently required range-finding
studies. A list of recommended preliminary tests is as follows:

(i) Stability trials should be conducted under test conditions. These
trials must be documented and submitted to the Agency for review with
the study to which they apply.

(ii) Solubility trials should be conducted under test conditions. These
trials must be documented and submitted with the study to the Agency
for review. Surfactants and charged polymers will be self-dispersing in
water and should be tested at or below their dispersability limits.

(iii) If solubility is a problem (<100 ppm), trials should be conducted
under test conditions using various solvents that are most likely to be ef-
fective and that are widely recognized as being nontoxic and other means
to ensure that the appropriate methods are used during the laboratory tests
to enhance solubility. Once a solvent is chosen based upon more simplistic,
comparative evaluations, the decision should be confirmed in the prelimi-
nary trials with only that solvent.

(iv) Chemical analysis methods as detailed in paragraph (1) of this
guideline.

(v) Stability of the test material in the samples to be collected for
chemical analyses should be determined during the laboratory studies. This
includes determining whether and how samples can be stored for future
analysis.
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(2) Laboratory studies must be designed taking into account this pre-
liminary information. This means the trials described are to be conducted
before the definitive laboratory studies are initiated.

(h) Toxicity tests with poorly soluble materials. (1) Existing OPP
guidelines for aquatic toxicity tests require that chemicals be tested up
to a maximum dissolved concentration of 100 ppm (milligrams per liter)
for pesticides or 1,000 ppm for industrial chemicals in an effort to obtain
an LC50 or EC50. This amount of test material is considered to represent
a conservative measure of the most bioavailable fraction, which may in-
clude some colloidal material not removed by centrimgation in addition
to the truly dissolved fraction.

(2) Applicants must demonstrate the technique used to maximize
chemical dissolution in the test media under standard conditions. Consider-
ation of the optimum technique should include use of nontoxic solvents,
saturation (solubility) columns, sonication, minor adjustments to environ-
mental conditions (i.e., temperature, pH, etc.), as appropriate. Minor ad-
justments should not extend outside the recommended range of conditions
for the specific test organism.

(3) Current policy allows chemicals that are poorly soluble (solubility
<100 ppm) or dispersible in water to be tested up to the maximum water
solubility or dispersibility limit obtainable for die given test conditions
employed, provided that certain prerequisites apply:

(i) Concentrations of test chemical in test media are measured at ap-
propriate intervals and from appropriate test chambers of all test levels
are determined from centrifuged supernatant or other appropriate separa-
tion (e.g., filtrate). Self-dispersing industrial chemicals (e.g., surfactants,
detergents, or charged [polymers) should be sampled directly.

(ii) Testing is also performed with a more soluble formulation e.g.,
emulsifiable concentrate, if one exists (in addition to testing with the tech-
nical-grade material). Testing with a more soluble formulation will not,
however, be required if it does not provide a twofold increase in solubility.

(4) Studies that involve radical changes in environmental test condi-
tions outside the recommended range of values for temperature, salinity,
pH, etc., will be considered on a case-by-case basis.

(i) Methods for solubility enhancement—(1) Saturator columns.
The use of saturation columns as an aid in the dissolution of test material
and in confirming maximum solubility is recommended but not required
for nonvolatile test chemicals with test media solubilities of 10 ppm or
less. Methods for using these columns in aquatic toxicity tests can be
adapted from the methods established for their use in determining water
solubility under OECD's Column Elution Method (see OPPTS 830.7840).
Saturator columns may be considered to generate test solutions for static
-------
studies or for flow-through studies. Furthermore, saturator columns for
these studies need only be considered if conventional techniques for dosing
the water do not result in water concentrations within twice the stated
solubility of the compound.

(2) Emulsifiers and formulation testing. Testing with a more solu-
ble formulation, if one exists and which may contain emulsifiers, dis-
persants, solubilizing agents, etc., is required for all active ingredients sub-
ject to aquatic organism testing and having a water solubility less than
100 ppm and less than an EC/LC50. A defined EC/LC50 provides a great-
ly improved basis for risk assessment.

(3) Effect of temperature. Solubility is a function of temperature
and is especially sensitive at the limits of solubility. Generally, below satu-
ration, increases of as much as 10 °C may affect the solubility up to a
factor of 2. However if test solutions are close to saturation, small changes
in temperature may result in supersaturated solutions. In addition, control
of temperature is important because of its well-known effects on the actual
toxicity of the compound.

(4) Centrifugation. Conventional centrifugation is required for all
test media where undissolved test material, precipitate, flocculant, or col-
loidal suspension except for surfactants or charged polymers) are observed
in the test chambers or where the solubility and (hence bioavailability)
are hi question. Filtration may be used instead of centrifugation if the ana-
lytical method is validated over a range of acceptable concentrations.

(j) Measurement at initiation and termination of testing—(1) Ini-
tial analysis, (i) Analysis at the 0-hour: A 30-min interval is generally
required between the addition of the test substance and the introduction
of the test organisms. 0-hour measurement should be made when test orga-
nisms are added. Industry will have to justify an exception from the 30-
min requirement for adding test organisms if the characteristics of the test
material and test system require a longer equilibration time. If preliminary
trials have been performed, this delay should be predictable.

(ii) In flow-through tests, the study should be conducted with knowl-
edge of the time it will take for the test material to reach equilibrium
or steady-state in the test container. Initiation of the test and scheduling
of the sampling times must be based on this information. In some cases,
a flow-through system may have to be run for an extended-time pretest
in an attempt to achieve equilibrium or steady-state conditions. If equi-
librium or steady-state cannot be achieved, and/or it appears that the meas-
ured concentrations will be substantially below (< 70 percent) nominal, the
study report should reflect that the laboratory was aware of this problem.
The study report should clearly identify the problem, indicate the steps
taken fo mitigate it and justify the study design and dosing levels. How-
ever, if sufficient analytical methods are available and acceptable toxicity
-------
data are produced, additional testing and evaluation with the sole objective
of obtaining initial measured concentrations greater than 70 percent of
nominal will not be required.

(2) Analysis at test termination. Where indicated, measurement at
test termination is considered necessary to determine if the test organisms
were exposed to the test material throughout the entire study and at what
levels. A significant change in test concentration during the last part of
the study may substantially alter the results. For example, if the test con-
centration dropped dramatically during the last few days of a study, the
effects that may have been caused by such exposure may not occur. The
EC50 or LC50 developed from that study would be misleading if it is
called a "96-h LC50".

(k) Replicates and concentration measurement. (1) Average con-
centrations of replicates are used in regression analysis. When replicate
test containers and measurement of test concentration are required, each
replicate in each test concentration must be analyzed separately because
the responses in each replicate are viewed as independent and it is nec-
essary to know what the concentrations were so variation can be deter-
mined. Exceptions to this occur when:

(i) Replicate treatment containers under static tests or static renewal
conditions are filled from a bulk preparation. In this case, only samples
from the bulk supply for each test level must be analyzed.

(ii) A "splitter" is used in a flow-through test to feed more than
one replicate. In this case, only samples from one replicate per treatment
level require analysis. It is recommended that samples be collected from
all replicates and be stored in case anomalous concentrations are measured
in the one that is analyzed. Analyzing the other replicates may shed light
on the cause and extent of the anomalous measurements.

(2) Replicates receiving flow from a splitter should be sampled and
analyzed alternately. In other words, if there are two replicates (A and
B), replicate A should be analyzed in the first week and replicate B in
the second week, etc.

(3) To the extent possible, variability in measured concentrations
should be minimized. The goal for limiting variability of measurements
between replicates of the same concentration, and over time in the same
concentration, is maintaining the ratio of the highest concentration to the
lowest concentration at 1.5:1 or less. Generally, variability above this
amount is not acceptable.

(4) An important factor in considering the limits of variability is the
avoidance of overlapping mean test concentrations between test levels.
High variability puts into question the reliability of the environmental
chemistry method and/or the concentrations on which to base statistical
-------
analysis and toxicological conclusions. If variability beyond the 1.5:1 ratio
occurs, an exception to it should be justified.

(5) This justification should clearly state the problem, explain why
it occurred, provide scientific justification, and identify all measures taken
to mitigate the problem. The justification also should include the rally de-
veloped chemistry method, including the documentation necessary for a
bench chemist to review and evaluate it.

(6) For cases in which variability problems are suspected, preliminary
trials are strongly recommended. If it becomes clear that high variability
cannot be avoided, an exception should be justified. Any justification
should be provided in advance. Agency scientists will decide on the valid-
ity of the rationale for the exception, and may recommend other methods
to reduce potential variability.

(1) Use of chemical analysis to confirm exposure in aquatic test-
ing—(1) Acute static tests. Except for acute aquatic algae and diatom
studies (which can only be conducted as static tests), acute static tests
may be conducted only if, among other things, the test material has been
shown to be stable under the test conditions, as defined in paragraph (e)
of this guideline. (Other factors not addressed in this guideline may pre-
clude conducting a static test even if the test material is stable under test
conditions. These include, but are not limited to, problems in maintaining
dissolved oxygen levels, feeding requirements, and concern for bacterial/
microbial contaminants.) In an acute static test with a test material that
is stable and readily soluble at the treatment levels, measurements of each
test concentration are not absolutely required. However:

(i) For static tests, the concentration of toxicant should be measured
at the beginning and end of the test in all test chambers. Further, measure-
ment of the toxicant's degradation products is desirable, but not required.

(ii) The study may be rejected if the following occurs:

(A) The test material was not stable under test conditions.

(B) Precipitates formed.

(iii) If the recommended chemical measurements were made to verify
exposure levels, the study may not be rejected. Whether the study design
was modified in a scientifically defensible attempt to accommodate these
chemical characteristics will also be considered.

(iv) If variability is expected to be a problem, it is recommended
that measurements of test concentrations be made at each test level at
0-hour, 48-h and, for tests longer than 48 h, at test termination. Replicate
-------
test containers should be measured separately, except as explained under
paragraph (k) of this guideline.

(2) Acute static renewal. Refer to the general discussion of replicates
under paragraph (k) of this guideline. If a static renewal test is conducted,
each test chamber must be sampled for chemical analysis at the 0-hour,
at the end of the first (or longest) cycle, and at test termination. It is rec-
ommended that measurements be made at the end of each renewal cycle
acute flow-through.

(3) Acute flow-through. If a flow-through test is conducted, each
test concentration must be measured at the 0-hour and at test termination.
It is recommended that for 96-h tests, an intermediate measurement be
made at 48-h to verify midtest exposure if variability is expected to be
a problem. (All acute aquatic algae and diatom tests must be conducted
as static. Flow-through and static renewal systems are not recommended
for these tests, since they are conducted with microscopic organisms that
cannot be protected from loss when renewing or draining water from the
test containers. Static tests for Lemna gibba can be conducted, regardless
of stability.)

(4) Chronic static renewal. Refer to the general discussion of rep-
licates under paragraph (k) of this guideline. Concentrations must be meas-
ured at each test level at 0-hour, at the end of the last renewal cycle
(at test termination), and at the beginning and end of an intervening cycle
at least once per week. The longest cycle in a sequence should be used
if variable-cycle periods are employed.

(5) Chronic flow-through. Refer to the general discussion of rep-
licates under paragraph (k) of this guideline. In each concentration, meas-
ure at 0-hour, every 7 days, and at test termination. At the beginning of
a study, the exact flow of the system and water output at each splitter
must be documented. In addition, system flow must be metered and mon-
itored visually or mechanically on a daily basis (every 24 h), and it is
recommended that the system flow be metered and monitored twice a day
(approximately every 12 h). Measurement of test concentration is required
each time metering fluctuation or malfunction is detected or observed. A
record of the regular inspections must be maintained and provided with
the study report.

(m) Measured concentrations versus nominal concentrations. This
section describes acceptable limits of deviation of measured from nominal
concentrations.

(1) Test endpoints are used as if the organisms were exposed to the
test material at the statistically developed value (LC50 or EC50) for the
entire test duration. One aspect of the risk assessment is to compare con-
cern levels based on the LC50 is to initial immediate concentrations. How-
ever, field conditions may exist in which concentrations that may be of

8
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acute concern may last longer or occur frequently enough to be comparable
to the 48-h, 96-h or 120-h test duration. Even though a pesticide may
degrade rapidly under one environmental condition (in water, for example),
the possibility of repeated exposure needs to be considered. Repeated ex-
posure from reservoirs of the active ingredient, occurring in environmental
compartments where persistence is greater, may occur. The Agency takes
these eventualities into account in order to generate risk assessments that
adequately address hazard to the aquatic ecosystem.

(2) Presumably, a safer chemical one that may degrade rapidly, has
low solubility, and is used at low rates. While these characteristics may
result in lower exposure levels in the field, the risk they represent can
only be determined if the actual toxicity of the pesticide is known or the
level below which the pesticide is not likely to result in 50 percent mortal-
ity (i.e., an LC50 > X-concentration situation.) When potentially low, real-
istic exposure levels are calculated and used for risk assessments, it is
imperative that the actual toxicity of the pesticide at those levels be deter-
mined. If the test is conducted using nominal concentrations, the results
could reflect a higher apparent effect concentration (e.g. LC50, EC50, or
NOEL). As a result, potential risk may be missed because the comparison
would be between a low "realistic" exposure and a high nominal test
level that was not the true toxicity level. A risk assessment based on such
a comparison and data would be faulty and could not be scientifically
defended.

(3) Pesticide chemicals that are used at very low levels tend to have
high biological activity. For this reason, it is imperative that the toxicity
data developed for these pesticides be accurate and scientifically defen-
sible.

(4) Measured concentrations are used when they are available because
they indicate what the exposure was in the test chambers. When measured
concentrations are indicated, they are considered necessary because:

(i) There are concerns that the actual concentrations to which the test
organisms are exposed may differ from "nominal." This variation may
be due to chemical characteristics, test conditions, or mechanical appara-
tus.

(ii) Measured concentrations confirm that the test system was de-
signed appropriately and is operating acceptably. Characteristics that make
testing difficult (low solubility, short half-life, high binding potential, etc.)
must be accounted for in the exposure estimates. They are not a reason
for developing misleading toxicity values from laboratory tests.

(5) Measurement of test concentrations is not performed just to deter-
mine if the technician knows how to mix the test solution once. Among
other things, it also ensures that the test solution was mixed correctly each
-------
time. It corroborates the precision of the technician or mechanics of the
test system.

(6) If test levels are not measured, the nominal values are used to
calculate the LC50, EC50, NOEC and LOEC. If the test material has de-
graded or has become unavailable because of insolubility or sorption, the
pesticide may be characterized as less toxic than it really is. For example,
if based on nominal test levels, the LC50 is 5 ppm, the pesticide would
be considered moderately toxic. No higher tier testing would be required
and that value (5 ppm) would be the basis for developing concern levels
with which to compare EECs. But if, hi reality, the concentrations to which
the organism was actually exposed were only between 0.1 and 1 ppm,
the LC50 may well be closer 0.5 ppm. For pesticides this would result
in labeling, and could trigger higher tier tests. More importantly, it would
yield substantially lower concern levels with which to compare exposure
levels.

(7) When a laboratory test design has been specifically modified to
accommodate the instability of test material or other factors likely to cause
variability in test concentrations, and the design is judged adequate based
on sufficient preliminary information, the study will not be rejected solely
on the grounds that measured concentrations varied by more than 30 per-
cent of the nominal concentration. (This assumes that the preliminary sta-
bility tests were conducted under test conditions essentially identical to
the actual test conditions.) An increase in measured test concentration of
more than 30 percent from the nominal concentration during the test will
generally not result in rejection, provided that the following conditions
are met:

(i) A reasonable and scientific explanation is given, and the variability
of results produced by the chemical analysis method is adequately charac-
terized.

(ii) All test containers exhibit a similar (but not necessarily identical)
shift. (If concentrations in some containers go up substantially (>30 per-
cent) and test concentrations in other containers go down substantially
(>30 percent), they will not be considered to have exhibited a similar shift.
The most important criterion is that test levels must not experience a shift
in "order." That is, the highest test level should remain highest, the next
should remain second, etc. If orders are shifted, the test may be rejected,
since regression analysis would not yield statistically sound median lethal
concentrations and confidence limits.)

(iii) The variability of the measured concentrations is acceptable.

(iv) A statistically valid endpoint can be derived from the measured
concentrations (either an LC50, EC50, or that the LC50 or EC50 is greater
than 100 ppm).

10
-------
(v) The preliminary stability information is provided with complete
documentation and description of methods used to derive such information.

(8) In some cases, high variability cannot be avoided because the test
concentrations are approaching the limit of detection or because of un-
avoidable binding of the test material to the chemical analysis apparatus.
When the ratio of the highest concentration to the lowest measured con-
centration is expected to vary by more than 1.5, the registrant is strongly
advised to justify an exception to this requirement in advance of conduct-
ing the aquatic laboratory studies. This exception justification should con-
sist of:

(i) Documentation of the preliminary trials indicating this problem.

(ii) The specific steps that will be taken to reduce the variation.

(iii) The fully developed chemical analysis method.

(iv) The raw data, standards, and chromatogram from a representative
analysis using the method. For each chemistry method, the actual mini-
mum detection level and level of quantification must be identified.

(9) The Agency will decide on each exception justification on a case-
by-case basis. However, if a series of aquatic tests are to be conducted
with one chemical and it is anticipated that these limits will be exceeded,
one exception justification may cover more than one study. The Agency
will then exercise judgment in evaluating studies with test materials that
are difficult to measure.

(10) Conducting flow-through or static renewal tests with aquatic
algae is not feasible with the current state of the practice. Therefore, the
following is recommended for a test material that, based on preliminary
stability testing, is expected to degrade to less than 70 percent of the nomi-
nal concentration. The study should be conducted normally, with con-
centrations measured at 0-hour and at test termination. Although it is unde-
sirable to allow the concentrations to decline throughout the study, the
problem may be unavoidable. In this case, the LC50 regression analysis
is based on the mean measured concentration. If the concentration is ex-
pected to decline to less than the minimum detection level before the end
of the study, then it is recommended that interim chemical measurements
be made to determine the decline rate.

(11) For purposes of consistency, the aquatic test with a vascular plant
(Lemna gibbet) need not be done using a flow-through or static renewal
system with the sole purpose of maintaining test concentrations. There may
be other reasons for conducting a static renewal study.
11
-------
-------
United States Prevention, Pesticides EPA712-C-96-114
Environmental Protection and Toxic Substances April 1996
Agency (7101)
&EPA Ecological Effects Test
Guidelines
OPPTS 850.1010
Aquatic Invertebrate
Acute Toxicity Test,
Freshwater Daphnids
U.S. EPA Headquarters Library
Mail pode 3201
1200 Pennsylvania Avenue NW
Washington DC 20460
"Public Draft"
-------
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.

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."
-------
OPPTS 850.1010 Aquatic invertebrate acute toxicity test, freshwater
daphnids.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 797.1300 Daphnid Acute Tox-
icity Test; OPP 72-2 Acute Toxicity Test for Freshwater Aquatic Inverte-
brates (Pesticide Assessment Guidelines, Subdivision E—Hazard Evalua-
tion; Wildlife and Aquatic Organisms) EPA report 540/09-82-024, 1982;
and OECD 202 Daphnia sp. Acute Immobilisation Test and Reproduction
Test.

(b) Purpose. This guideline is intended for use in developing data
on the acute toxicity of chemical substances and mixtures ("chemicals")
subject to environmental effects test regulations. This guideline prescribes
an acute toxicity test in which daphnids (Daphnia magna or D. pulex)
are exposed to a chemical in static and flow-through systems. The Envi-
ronmental Protection Agency will use data from this test in assessing the
hazard a chemical may present in the aquatic environment.

(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and 40 CFR Part 792—Good Laboratory Practice
Standards apply to this test guideline. In addition, the following definitions
apply to this guideline:

Brood stock means the animals which are cultured to produce test
organisms through reproduction.

EC50 means that experimentally derived concentration of test sub-
stance in dilution water that is calculated to affect 50 percent of a test
population during continuous exposure over a specified period of time.
In this guideline, the effect measured is immobilization.

Ephippium means a resting egg which develops in daphnids under
the carapace in response to stress conditions.

Flow-through means a continuous or an intermittent passage of test
solution or dilution water through a test chamber or culture tank with no
recycling.

Immobilization means the lack of movement by the test organisms.

Loading means the ratio of daphnid biomass (grams, wet weight) or
number of daphnids to the volume (liters) of test solution in a test chamber
at a point in time, or passing through the test chamber during a specific
interval.

1
-------
Static system means a test system in which the test solution and test
organisms are placed in the test chamber and kept there for the duration
of the test without renewal of the test solution.

Static-renewal system means a static test system in which the test
solution is renewed every 24 h.

(d) Test procedures—(1) Summary of the test, (i) Test chambers
are filled with appropriate volumes of dilution water. In the flow-through
test, the flow of dilution water through each chamber is adjusted to the
rate desired. The test chemical is introduced into each treatment chamber.
The addition of test chemical in the flow-through system is conducted at
a rate which is sufficient to establish and maintain the desired concentra-
tion in the test chamber. The test is started within 30 min after the test
chemical has been added and uniformly distributed in static test chambers
or after the concentration of test chemical in each flow-through test cham-
ber reaches the prescribed level and remains stable. In static-renewal test-
ing the dilution water and test chamber are renewed periodically. At the
initiation of the test, daphnids which have been cultured and acclimated
in accordance with the test design are randomly placed into the test cham-
bers. Daphnids in the test chambers are observed periodically during the
test, the immobile daphnids removed, and the findings recorded.

(ii) Dissolved oxygen concentration (DOC), pH, temperature, the con-
centration of test chemical and other water quality parameters are meas-
ured at specified intervals in selected test chambers. Data are collected
during the test to develop concentration-response curves and determine
EC50 values for the test chemical at the end of 24 and 48 h.

(2) Range-finding test, (i) A range-rinding test should be conducted
to establish test solution concentrations for the definitive test.

(ii) The daphnids should be exposed to a series of widely spaced
concentrations of the test chemical (e.g. 1, 10, 100 mg/L, etc.).

(iii) A minimum of five daphnids should be exposed to each con-
centration of test chemical for a period of 48 h. The exposure period may
be shortened if data suitable for the purpose of the range-finding test can
be obtained in less time. No replicates are required and nominal concentra-
tions of the chemical are acceptable.

(3) Definitive test, (i) The purpose of the definitive test is to deter-
mine the concentration-response curves and the 24- and 48-h EC50 val-
ues.

(ii) A minimum of 20 daphnids per concentration should be exposed
to five or more concentrations of the chemical chosen in a geometric series
in which the ratio is between 1.5 and 2.0 (e.g. 2, 4, 8, 16, 32, and
64 mg/L). An equal number of daphnids should be placed in two or more
-------
replicates. If solvents, solubilizing agents, or emulsifiers have to be used,
they should be commonly used carriers and should not possess a syner-
gistic or antagonistic effect on the toxicity of the test chemical. If carriers
are absolutely necessary, the amount used should be the minimum nec-
essary to achieve solution of the test substance. Triethylene glycol and
dimethyl formamide are preferred, but ethanol and acetone can be used
if necessary. Carrier concentrations should be kept constant at all treatment
levels. The concentration of solvent should not exceed 100 mg/L. The con-
centration ranges should be selected to determine the concentration-re-
sponse curves and EC50 values at 24 and 48 h. Concentration of test chem-
ical in test solutions should be analyzed prior to use.

(iii) Every test should include controls consisting of the same dilution
water, conditions, and procedures, and daphnids from the same population
(culture container), except that none of the test chemical is added.

(iv) The DOC, temperature, and pH should be measured at the begin-
ning and end of the test in each chamber.

(v) The test duration is 48 h. The test is unacceptable if more than
10 percent of the control organisms are immobilized during the 48-h test
period. Each test chamber should be checked for immobilized daphnids
at 24 and 48 h after the beginning of the test. Concentration-response
curves and 24-h and 48-h EC50 values for immobilization should be de-
termined along with their 95 percent confidence limits.

(vi) In addition to immobility, any abnormal behavior or appearance
should also be reported.

(vii) Test organisms should be impartially distributed among test
chambers in such a manner that test results show no significant bias from
the distributions. In addition, test chambers within the testing area should
be positioned in a random manner or in a way in which appropriate statis-
tical analyses can be used to determine the variation due to placement.

(viii) The concentration of the test chemical in the chambers should
be measured as often as is feasible during the test. In the static test the
concentration of test chemical should be measured in each test chamber
at a minimum at the beginning and at the end of the test. In the static-
renewal test, the test concentration of test chemicals should be measured
in each test chamber at a minimum at the beginning and at the end of
the renewal period. In the flow-through test the concentration of test chem-
ical should be measured in each chamber at a minimum at the beginning
of the test and at 48 h after the start of the test, and in at least one appro-
priate chamber whenever a malfunction is detected in any part of the test
substance delivery system. Among replicate test chambers of a treatment
concentration, the measured concentration of the test chemical should not
vary more than ±20 percent.
-------
(4) Analytical measurements—(i) Test chemical. Deionized water
should be used in making stock solutions of the test chemical. Standard
analytical methods should be used whenever available in performing the
analyses. The analytical method used to measure the amount of test chemi-
cal in a sample should be validated before beginning the test by appro-
priate laboratory practices. Any analytical method is not acceptable if like-
ly degradation products of the test chemical, such as hydrolysis and oxida-
tion products, give positive or negative interferences which cannot be sys-
tematically identified and mathematically corrected.

(ii) Numerical. The number of immobilized daphnids should be
counted during each definitive test. Appropriate statistical analyses should
provide a goodness-of-fit determination for the concentration-response
curves. A 24- and 48-h EC50 and corresponding 95 percent interval
should be calculated.

(e) Test conditions—(1) Test species—(i) Selection. (A) The
cladocerans, D. magna or D. pulex, are the test species to be used in this
test. Either species may be used for testing of a particular chemical. The
species identity of the test organisms should be verified using appropriate
systematic keys. First instar daphnids, <24 h old, are to be used to start
the test.

(B) Daphnids to be used in acute toxicity tests should be cultured
at the test facility. Records should be kept regarding the source of the
initial stock and culturing techniques. All organisms used for a particular
test should have originated from the same culture population.

(C) Stock daphnids may be tested periodically to determine any ge-
netic changes in the populations which may alter the sensitivity to test
chemicals.

(D) Daphnids should not be used for a test:

(1) If cultures contain ephippia.

(2) If adults in the cultures do not produce young before day 12.

(3) If more than 20 percent of the culture stock die during the 2
days preceding the test.

(4) If adults in the culture do not produce an average of at least three
young per adult per day over the 7-day period prior to the test.

(5) If daphnids have been used in any portion of a previous test, either
in a treatment or in a control.

(ii) Acclimation. (A) Brood daphnids should be maintained in
100-percent dilution water at the test temperature for at least 48 h prior
to the start of the test. This is easily accomplished by culturing them in

4
-------
the dilution water at the test temperature. During production of neonates,
daphnids should not be fed.

(B) During culturing and acclimation to the dilution water, daphnids
should be maintained in facilities with background colors and light inten-
sities similar to those of the testing area.

(iii) Care and handling. (A) Daphnids should be cultured in dilution
water under environmental conditions similar to those used in the test.
Organisms should be handled as little as possible. When handling is nec-
essary it should be done as gently, carefully, and quickly as possible. Dur-
ing culturing and acclimation, daphnids should be observed carefully for
ephippia and other signs of stress, physical damage, and mortality. Dead
and abnormal individuals should be discarded. Organisms that touch dry
surfaces or are dropped or injured in handling should be discarded.

(B) Smooth glass tubes (I.D. greater than 5 mm), equipped with rub-
ber bulbs, should be used for transferring daphnids with minimal culture
media carry-over. Care should be exercised to introduce the daphnids
below the surface of any solution to avoid trapping air under the carapace.

(iv) Feeding. A variety of foods (e.g. unicellular green algae) have
been demonstrated to be adequate for daphnid culture. Daphnids should
not be fed during testing.

(2) Facilities—(i) Apparatus. (A) Facilities needed to perform this
test include:

(7) Containers for culturing and acclimating daphnids.

(2) A mechanism for controlling and maintaining the water tempera-
ture during the culturing, acclimation, and test periods.

(3) Apparatus for straining paniculate matter, removing gas bubbles,
or aerating the water as necessary.

(4) An apparatus for providing a 16-h light and 8-h dark photoperiod
with a 15- to 30-min transition period.

(5) In addition, the flow-through system should contain appropriate
test chambers in which to expose daphnids to the test chemical and an
appropriate test substance delivery system.

(B) Facilities should be well ventilated and free of fumes and disturb-
ances that may affect the test organisms.

(C) Test chambers should be loosely covered to reduce the loss of
test solution or dilution water due to evaporation and to minimize the entry
of dust or other particulates into the solutions.
-------
(ii) Construction materials. (A) Materials and equipment that con-
tact test solutions should be chosen to minimize sorption of test chemicals
from the dilution water and should not contain substances that can be
leached into aqueous solution in quantities that can affect the test results.

(B) For static tests, daphnids can be conveniently exposed to the test
chemical in 250-mL beakers or other suitable containers.

(C) For flow-through tests, daphnids can be exposed in glass or stain-
less steel containers with stainless steel or nylon screen bottoms. The con-
tainers should be suspended in the test chamber in such a manner to ensure
that the test solution flows regularly into and out of the container and
that the daphnids are always submerged in at least 5 cm of test solution.
Test chambers can be constructed using 250-mL beakers or other suitable
containers equipped with screened overflow holes, standpipes, or V-shaped
notches.

(iii) Dilution water. (A) Surface or ground water, reconstituted water
or dechlorinated tap water are acceptable as dilution water if daphnids
will survive in it for the duration of the culturing, acclimation, and testing
periods without showing signs of stress. The quality of the dilution water
should be constant and should meet the specifications in the following
Table 1.:
Table 1.—Water Quality Parameters
Substance
Maximum concentration
Hardness as CaCOs
Particulate matter
Total organic carbon or
Chemical oxygen demand
Un-ionized ammonia
Residual chlorine
Total organophosphorus pesticides
Total organochlorine pesticides plus polychlorinated biphenyls (PCBs) or
Organic chlorine
180mg/L
20 mg/L
2mg/L
5 mg/L
20 |j.g/L
<3 ng/L
50 ng/L
50 ng/L
25 ng/L
(B) The water quality parameters should be measured at least twice
a year or whenever it is suspected that these characteristics may have
changed significantly. If dechlorinated tap water is used, daily chlorine
analysis should be performed.

(C) If the diluent water is from a ground or surface water source,
conductivity and total organic carbon (TOC) or chemical oxygen demand
(COD) should be measured. Reconstituted water can be made by adding
specific amounts of reagent-grade chemicals to deionized or distilled
water. Glass distilled or carbon-filtered deionized water with a conductiv-
ity less than 0.1 mS/m is acceptable as the diluent for making reconstituted
water.
-------
(iv) Cleaning. All test equipment and test chambers should be
cleaned before each use using standard laboratory procedures.

(v) Test substance delivery system. In flow-through tests, propor-
tional diluters, metering pump systems, or other suitable devices should
be used to deliver test chemical to the test chambers. The system should
be calibrated before each test. Calibration includes determining the flow
rate through each chamber and the concentration of the test chemical in
each chamber. The general operation of the test substance delivery system
should be checked twice during a test. The 24-h flow through a test cham-
ber should be equal to at least 5x the volume of the test chamber. During
a test, the flow rates should not vary more than 10 percent from any one
test chamber to another.

(3) Test parameters. Environmental parameters of the water con-
tained in test chambers should be maintained as specified below:

(i) The test temperature should be 20 °C. Excursions from the test
temperature should be no greater than ± 2 °C.

(ii) DOC between 60 and 105 percent saturation. Do not aerate
daphnid toxicity tests. A single air bubble can get under the carapace of
the daphnid and kill it, or float the daphnid to the surface where it will
get trapped.

(iii) The number of daphnids placed in a test chamber should not
affect test results. Loading should not exceed 40 daphnids per liter of test
solution in the static system. In the flow-through test, loading limits will
vary depending on the flow rate of dilution water. Loading should not
cause the DOC to fall below the recommended levels.

(iv) Photoperiod of 16 h light and 8 h darkness.

(f) Reporting. The sponsor must submit to the EPA all data devel-
oped by the test that are suggestive or predictive of acute toxicity and
all concomitant gross toxicological manifestations. In addition to the re-
porting requirements prescribed in 40 CFR Part 792—Good Laboratory
Practice Standards, the reporting of test data should include the following:

(1) The name of the test, sponsor, testing laboratory, study director,
principal investigator, and dates of testing.

(2) A detailed description of the test chemical including its source,
lot number, composition (identity and concentration of major ingredients
(percent active ingredient of chemical) and major impurities), known phys-
ical and chemical properties and any carriers or other additives used and
their concentrations.
-------
(3) The source of the dilution water, its chemical characteristics (e.g.
conductivity, hardness, pH, etc.), and a description of any pretreatment,
carriers and/or additives used, and their concentrations.

(4) Carriers and/or additives used and their concentrations.

(5) Detailed information about the daphnids used as brood stock, in-
cluding the scientific name and method of verification, age, source, treat-
ments, feeding history, acclimation procedures, and culture method. The
age of the daphnids used in the test should be reported.

(6) A description of the test chambers, the volume of solution in the
chambers, the way the test was begun (e.g. conditioning, test chemical
additions), number of test organisms per test chamber, number of replicates
per treatment, lighting, method of test chemical introduction or test sub-
stance delivery system, renewal schedule (in static-renewal tests), and flow
rate (in flow-through test) expressed as volume additions per 24 h.

(7) The concentration of the test chemical in each test chamber at
times designated for static and flow-through tests.

(8) The number and percentage of organisms that were immobilized
or showed any adverse effects in each test chamber at each observation
period.

(9) Utilizing the average measured test chemical concentration, con-
centration-response curves should be fitted to immobilization data at 24
and 48 h. A statistical test of goodness-of-fit should be performed and
the results reported.

(10) The 24- and 48-h EC50 values and their respective 95 percent
confidence limits using the mean measured test chemical concentration,
and the methods used to calculate both the EC50 values and their con-
fidence limits.

(11) All chemical analyses of water quality and test chemical con-
centrations, including methods, method validations, and reagent blanks.

(12) The data records of the culture, acclimation, and test tempera-
tures.

(13) Any deviation from this test guideline and anything unusual
about the test, e.g. diluter failure, temperature fluctuations, etc.

(14) If it is observed that the stability or homogeneity of the test
substance cannot be maintained, care should be taken in the interpretation
of the results, and note made that the results may not be reproducible.
8
-------
&EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-130
April 1996
Ecological Effects Test
Guidelines

OPPTS 850.1020
Gammarid Acute
Toxicity Test
"Public Draft'
-------
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.

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."
-------
OPPTS 850.1020 Gammarid acute toxicity test
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S..C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. The source material used in developing this har-
monized OPPTS test guideline is 40 CFR 795.120 Gammarid Acute Tox-
icity Test.

(a) Purpose. This guideline is intended for use in developing data
on the acute toxicity of chemical substances and mixtures subject to envi-
ronmental effects test regulations. This guideline describes a test to de-
velop data on the acute toxicity of chemicals to gammarids. The data from
this test will be used in assessing the hazard of a chemical to aquatic
organisms.

(b) Definitions. The definitions in section 3 of TSCA and in Part
792, Good Laboratory Practice Standards, apply to this test guideline. The
following definitions also apply to this guideline:

Death means the lack of reaction of a test organism to gentle prod-
ding.

Flow-through means a continuous or an intermittent passage of test
solution or dilution water through a test chamber or a holding or acclima-
tion tank, with no recycling.

LC50 means the median lethal concentration, i.e., that concentration
of a chemical in air or water killing 50 percent of the test batch of orga-
nisms within a particular period of exposure (which shall be stated).

Loading means the ratio of the biomass of gammarids (grams, wet
weight) to the volume (liters) of test solution in either a test chamber or
passing through it in a 24-hour period.

Solvent means a substance (e.g., acetone) which is combined with
the test substance to facilitate introduction of the test substance into the
dilution water.

Static system means a test chamber in which the test solution is not
renewed during the period of the test.

(c) Test procedures—(1) Summary of the test. In preparation for
the test, test chambers are filled with appropriate volumes of dilution
water. If a flow-through test is performed, the flow of dilution water
through each chamber is adjusted to the rate desired. In a static test, the
test substance is introduced into each test chamber. In a flow-through test,
the rate in which the test substance is added is adjusted to establish and
maintain the desired concentration of test substance in each test chamber.

1
-------
The test is started by randomly introducing gammarids, which have been
acclimated to the test conditions, into the test chambers. Gammarids in
the test chambers are observed periodically during the test; the dead
gammarids are removed and the findings recorded. Dissolved oxygen con-
centration, pH, temperature, and the concentration of test substance in test
chambers are measured at specified intervals. Data collected during the
test are used to develop concentration—response curves and LC50 values
for the test substance.

(2) Range-finding test, (i) A range-finding test should be conducted
to establish test substance concentrations to be used for the definitive test.

(ii) The gammarids shall be exposed to a wide-range of concentrations
of the test substance (e.g. 1, 10, 100 mg/L, etc.), usually under static condi-
tions.

(iii) A minimum of five gammarids should be exposed to each con-
centration of test substance for a period of 96 hours. The exposure period
may be shortened if data suitable for determining concentrations in the
definitive test can be obtained in less time. Nominal concentrations of the
test substance may be acceptable.

(3) Definitive test, (i) The purpose of the definitive test is to deter-
mine the 24, 48, 72, and 96—hour LC50 values and the concentration-
response curves.

(ii) A minimum of 20 gammarids per concentration shall be exposed
to five or more concentrations of the test substance chosen in a geometric
series in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32,
64 mg/L). The range and number of concentrations to which the organisms
are exposed shall be such that in 96 hours there is at least one concentra-
tion resulting in mortality greater than 50 and less than 100 percent, and
one concentration causing greater than zero and less than 50 percent mor-
tality. An equal number of gammarids may be placed in two or more rep-
licate test chambers. Solvents should be avoided, if possible. If solvents
have to be used, a solvent control, as well as a dilution control, shall be
tested at the highest solvent concentration employed in the treatments. The
solvent should not be toxic or have an effect on the toxicity of the test
substance. The concentration of solvent should not exceed 0.1 ml/L.

(iii) Every test shall include a concurrent control using gammarids
from the same population or culture container. The control group shall
be exposed to the same dilution water, conditions and procedures, except
that none of the test substance shall be is added to the chamber.

(iv) The dissolved oxygen concentration, temperature and pH of the
test solution shall be measured at the beginning of the test and at 24,
48, 72 and 96 hours in at least one replicate each of the control, and the
highest, lowest and middle test concentrations.
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(v) The test duration is 96 hours. The test is unacceptable if more
than 10 percent of the control organisms die during the test.

(vi) In addition to death, any abnormal behavior or appearance shall
also be reported.

(vii) Gammarids shall be randomly assigned to the test chambers. Test
chambers shall be positioned within the testing area in a random manner
or in a way in which appropriate statistical analyses can be used to deter-
mine whether there is any variation due to placement.

(viii) Gammarids shall be introduced into the test chambers after the
test substance has been added.

(ix) Observations on compound solubility shall be recorded. The in-
vestigator should record the appearance of surface slicks, precipitates, or
material adhering to the sides of the test chambers.

(4) Analytical measurements—(i) Water quality analysis. The
hardness, acidity, alkalinity, pH, conductivity, TOC or COD, and particu-
late matter of the dilution water shall be measured at the beginning of
each definitive test.

(ii) Collection of samples for measurement of test substance. Each
sample to be analyzed for the test substance concentrations shall be taken
at a location midway between the top, bottom, and sides of the test cham-
ber. Samples should not include any surface scum or material dislodged
from the bottom or sides. Samples shall be analyzed immediately or han-
dled and stored in a manner which minimizes loss of test substance
through microbial degradation, photogradation, chemical reaction, vola-
tilization, or sorption.

(iii) Measurement of test substance. (A) For static tests, the con-
centration of dissolved test substance (that which passes through a 0.45
micron filter) shall be measured in each test chamber at least at the begin-
ning (0-hour, before gammarids are added) and at the end of the test.
During flow-through tests, the concentration of dissolved test substance
shall be measured in each test chamber at least at 0 and 96-hours and
in at least one chamber whenever a malfunction of the test substance deliv-
ery system is observed.

(B) The analytical methods used to measure the amount of test sub-
stance in a sample shall be validated before beginning the test. This in-
volves adding a known amount of the test substance to each of three water
samples taken from a chamber containing dilution water and the same
number of gammarids as are placed in each test chamber. The nominal
concentrations of the test substance in these samples should span the con-
centration range to be used in the test. Validation of the analytical method
-------
should be performed on at least twq separate days prior to starting the
test.

(C) An analytical method is not acceptable if likely degradation prod-
ucts of the test substance give positive or negative interferences, unless
it is shown that such degradation products are not present in the test cham-
bers during the test.

(D) Among replicate test chambers, the measured concentrations shall
not vary more than 20 percent. The measured concentration of the test
substance in any chamber during the test shall not vary more than plus
or minus 30 percent from the measured concentration in that chamber at
zero time.

(E) The mean measured concentration of dissolved test substance
shall be used to calculate all LCSO's and to plot all concentration-response
curves.

(d) Test conditions for definitive test—(1) Test species—(i) Selec-
tion. (A) The amphipods, Gammarus fasciatus, G. pseudolimnaeus, and
G. lacustris are specified for this test.

(B) Gammarids can be cultured in the laboratory or collected from
natural sources. If collected, they must be held in the laboratory for at
least 14 days prior to testing.

(C) Gammarids used in a particular test shall be of similar age and/
or size and from the same source or culture population.

(ii) Acclimation. If the holding water is from the same source as
the dilution water, acclimation to the dilution water shall be done gradually
over a 48-hour period. The gammarids then shall be held at least 7 days
in the dilution water prior to testing. Any changes in water temperature
should not exceed 2 °C per day. Gammarids should be held for a minimum
of 7 days at the test temperature prior to testing.

(iii) Care and handling. Gammarids shall be cultured in dilution
water under similar environmental conditions to those used in the test.
Organisms shall be handled as little as possible. When handling is nec-
essary it should be done as gently, carefully and quickly as possible. Dur-
ing culturing and acclimation, gammarids shall be observed carefully for
signs of stress and mortality. Dead and abnormal individuals shall be dis-
carded.

(iv) Feeding. The organisms shall not be fed during testing. During
culturing, holding, and acclimation, a sufficient quantity of deciduous
leaves, such as maple, aspen, or birch, should be placed in the culture
and holding containers to cover the bottom with several layers. These
leaves should be aged for at least 30 days in a flow-through system before
-------
putting them in aquaria. As these leaves are eaten, more aged leaves should
be added. Pelleted fish food may also be added.

(2) Facilities—(i) Apparatus—(A) Facilities needed to perform this
test include:

(/) Containers for culturing, acclimating and testing gammarids;

(2) Containers for aging leaves under flow-through conditions;

(3) A mechanism for controlling and maintaining the water tempera-
ture during the culturing, acclimation and test periods;

(4) Apparatus for straining particulate matter, removing gas bubbles,
or aerating the dilution water, as necessary; and

(5) An apparatus for providing a 16-h light and 8-h dark photoperiod
with a 15- to 30-minute transition period.

(B) Facilities should be well ventilated and free of flames and disturb-
ances that may affect the test organism.

(C) Test chambers shall be covered loosely to reduce the loss of test
solution or dilution water due to evaporation and to minimize the entry
of dust or other particulates into the solutions.

(ii) Construction materials. Construction materials and equipment
that may contact the stock solution, test solution or dilution water should
not contain substances that can be leached or dissolved into aqueous solu-
tions in quantities that can alter the test results. Materials and equipment
that contact stock or test solutions should be chosen to minimize sorption
of test substances. Glass, stainless steel, and perfluorocarbon plastic should
be used wherever possible. Concrete, fiberglass, or plastic (e.g., PVC) may
be used for holding tanks, acclimation tanks, and water supply systems,
but they should be aged prior to use. Rubber, coopper, brass, galvanized
metal, and lead should not come in contact with the dilution water, stock
solution, or test solution.

(iii) Test substance delivery system. In flow-through tests, diluters,
metering pump systems or other suitable devices shall be used to deliver
the test substance to the test chambers. The system used shall be calibrated
before each test. The general operation of the test substance delivery sys-
tem shall be checked twice daily during a test. The 24-h flow shall be
equal to at least five times the volume of the test chamber. During a test,
the flow rates should not vary more than 10 percent from one test chamber
to another.

(iv) Test chambers. Test chambers shall contain at least one liter
of test solution. Test chambers made of stainless steel should be welded,
-------
not soldered. Test chambers made of glass should be glued using clear
silicone adhesive. As little adhesive as possible should be left exposed
in the interior of the chamber. A substrate, such as a bent piece of stainless
steel screen, should be placed on the bottom of each test chamber to pro-
vide cover for the gammarids.

(v) Cleaning of test system. Test substance delivery systems and test
chambers should be cleaned before each test. They should be washed with
detergent and then rinsed sequentially with clean water, pesticide-free ace-
tone, clean water, and 5 percent nitric acid, followed by two or more
changes of dilution water.

(vi) Dilution water. (A) Clean surface or ground water, reconstituted
water, or dechlorinated tap water is acceptable as dilution water if
gammarids will survive in it for the duration of the culturing, acclimating,
and testing periods without showing signs of strees. The quality of the
dilution water should be constant enough that the month-to-month vari-
ation in hardness, acidity, alkalinity, conductivity, TOC or COD, and par-
ticulate matter is not more than 10 percent. The pH should be constant
within 0.4 unit. In addition, the dilution water should meet the following
specifications measured at least twice a year:
Substance
Maximum
concentra-
tion
Paniculate matter
Total organic carbon (TOC) or
chemical oxygen demand (COD)
Boron, fluoride ,
Un-ionizsd ammonia
Aluminum, arsenic, chromium, cobalt, copper, iron. lead, nickel, zinc
Residual chlorine
Cadmium, mercury, silver
Total organophosphorus pesticides
Total organcchiorine pesticides plus:
polychlorinated biphenyls (PCBs) or
organic chlorine
20mg/L
2 mg/L
5 mg/t.
100pg/L
1 uglL
1 (ig/L
3ng/L
100 ngn.
50ng/L

50ng/L
25ng/L
(B) If the dilution water is from a ground or surface water source,
conductivity and total organic carbon (TOC) or chemical oxygen demand
(COD) shall be measured. Reconstituted water can be made by adding
specific amounts of reagent-grade chemicals to deionized or distilled
water. Glass-distilled or carbon-filtered deionized water with a conductiv-
ity less than 1 jimho/cm is acceptable as the diluent for making reconsti-
tuted water.

(C) The concentration of dissolved oxygen in the dilution water shall
be between 90 and 100 percent saturation. If necessary, the dilution water
can be aerated before the addition of the test substance. All reconstituted
water should be aerated before use.

(3) Test parameters. Environmental parameters during the test shall
be maintained as specified below:

(i) Water temperature of 18 ± 1°C.
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(ii) Dissolved oxygen concentration between 60 and 105 percent satu-
ration.

(iii) The number of gammarids placed in a test chamber shall not
be so great as to affect the results of the test. Ten gammarids per liter
is the recommended level of loading for the static test. Loading require-
ments for the flow-through test will vary depending on the flow rate of
dilution water. The loading should not cause the dissolved oxygen con-
centration to fall below the recommended levels.

(iv) Photoperiod of 16 hours light and 8 hours darkness.

(e) Reporting. The sponsor shall submit to the EPA all data devel-
oped by the test that are suggestive or predictive of toxicity. In addition,
the test report shall 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.

(2) Objectives and procedures stated in the approved protocol, includ-
ing any changes in the original protocol.

(3) Statistical methods employed for analyzing the data.

(4) The test substance identified by name, Chemical Abstracts (CAS)
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 the test.

(6) A description of the methods used, including:

(i) The source of the dilution water, its chemical characteristics (e.g.,
hardness, pH, etc.) and a description of any pretreatment.

(ii) A description of the test substance delivery system, test chambers,
the depth and volume of solution in the chamber, the way the test was
begun (e.g., test substance addition), the loading, the lighting, and the flow
rate.

(iii) Frequency and methods of measurements and observations.

(7) The scientific name, weight, length, source, and history of the
organisms used, and the acclimation procedures and food used.

(8) The concentrations tested, the number of gammarids and replicates
per test concentration. The reported results should include:

(i) The results of dissolved oxygen, pH and temperature measure-
ments.
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(ii) If solvents are used, the name and source of the solvent, the nomi-
nal concentration of the test substance in the stock solution, the highest
solvent concentration in the test solution and a description of the solubility
determination in water and solvents.

(iii) The measured concentration of the test substance in each test
chamber just before the start of the test and at all subsequent sampling
periods.

(iv) In each test chamber at each observation period, the number of
dead and live test organisms, the percentage of organisms that died, and
the number of test organisms that showed any abnormal effects in each
test chamber at each observation period.

(v) The 48, 72 and 96-h LC50's and their 95 percent confidence lim-
its. When sufficient data have been generated, the 24—h LC50 value also.
These calculations should be made using the mean measured test substance
concentrations.

(vi) The observed no-effect concentration (the highest concentration
tested at which there were no mortalities or abnormal behavioral or physio-
logical effects), if any.

(vii) Methods and data for all chemical analyses of water quality and
test substance concentrations, including method validations and reagent
blanks.

(9) A description of all circumstances that may have affected the qual-
ity or integrity of the data.

(10) The names of the sponsor, study director, principal investigator,
names of other scientists or professionals, and the names of all supervisory
personnel involved in the study.

(11) 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. Results of the analysis of data
should include the calculated LC50 value, 95 percent confidence limits,
slope of the transformed concentration-response line, and the results of
a goodness-of-fit test (e.g., X2 test).

(12) The signed and dated reports prepared by any individual scientist
or other professional 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.

(13) The locations where all specimens, raw data, and the final report
are stored.

8
-------
(14) The statement prepared and signed by the quality assurance unit.
-------
-------
&EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-115
April 1996
Ecological Effects Test
Guidelines
OPPTS 850.1025
Oyster Acute Topxicity
Test (Shell Deposition)
"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.

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 850.1025 Oyster acute toxicity test (shell deposition).
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 797.1800 Oyster Acute Tox-
icity Test and OPP 72-3 Acute Toxicity Test for Estuarine and Marine
Organisms (Pesticide Assessment Guidelines, Subdivision E—Hazard
Evaluation; Wildlife and Aquatic Organisms) EPA report 540/09-82-024,
1982.

(b) Purpose. This guideline prescribes tests to be used to develop
data on the acute toxicity of chemical substances and mixtures ("chemi-
cals") to Eastern oysters, Crassostrea virginica (Gmelin). The Environ-
mental Protection Agency will use data from these tests in assessing the
hazard of a chemical to the environment.

(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and the definitions in 40 CFR Part 792—Good Lab-
oratory Practice Standards apply to this test guideline. The following defi-
nitions also apply to this test guideline.

Acute toxicity is the discernible adverse effects induced in an orga-
nism within a short period of time (days) of exposure to a chemical. For
aquatic animals this usually refers to continuous exposure to the chemical
in water for a period of up to 4 days. The effects (lethal or sublethal)
occurring may usually be observed within the period of exposure with
aquatic organisms. In this test guideline, shell deposition is used as the
measure of toxicity.

EC50 is that experimentally derived concentration of a chemical in
water that is calculated to induce shell deposition 50 percent less than
that of the controls in a test batch of organisms during continuous exposure
within a particular exposure period which should be stated.

Shell deposition is the measured length of shell growth that occurs
between the time the shell is ground at test initiation and test termination
96 h later.

Umbo means the narrow end (apex) of the oyster shell.

Valve height means the greatest linear dimension of the oyster as
measured from the umbo to the ventral edge of the valves (the farthest
distance from the umbo).

(d) Test procedures—(1) Summary of the test, (i) The water solu-
bility and the vapor pressure of the test chemical should be known. Prior
to testing, the structural formula of the test chemical, its purity, stability
-------
in water and light, n-octanol/water partition coefficient, and pKa values
should be known prior to testing. The results of a biodegradability test
and the method of analysis for the quantification of the chemical in water
should also be known.

(ii) For chemicals with limited solubility under the test conditions,
it may not be possible to determine an EC50. If it is observed that the
stability or homogeneity of the test chemical cannot be maintained, then
care should be taken in the interpretation of the results and a note made
that these results may not be reproducible.

(iii) Test chambers are filled with appropriate volumes of dilution
water. The flow of dilution water through each chamber is adjusted to
the rate desired. The test chemical is introduced into each test chamber
and the flow-rate adjusted to establish and maintain the desired concentra-
tion in each test chamber. Test oysters, which have been acclimated and
prepared by grinding away a portion of the shell periphery, are randomly
introduced into the test and control chambers. Oysters in the test and con-
trol chambers are observed daily during the test for evidence of feeding
or unusual conditions, such as shell gaping, excessive mucus production
or formation of fungal growths in the test chambers. The observations are
recorded and dead oysters removed. At the end of 96 h the increments
of new shell growth are measured in all oysters. The concentration-re-
sponse curve and EC50 value for the test chemical are developed from
these data.

(2) Range-finding test. A range-finding test should be conducted to
establish test chemical concentrations for the definitive test. The test is
conducted in the same way as the definitive test except a widely spaced
chemical concentration series (i.e. log-interval) is used.

(3) Definitive test, (i) Oysters which meet condition criteria (age,
size, reproductive status, health) and which have been acclimated to test
conditions should have approximately 3 to 5 mm of the shell periphery,
at the rounded (ventral) end, ground away with a small electric disc grinder
or other appropriate device, taking care to remove the shell rim uniformly
to produce a smooth, rounded, blunt profile. The oyster's valves should
be held together tightly during grinding to avoid vibrating the shell and
injuring the adductor muscle. Oysters from which so much of the shell
rim has been removed that an opening into the shell cavity is visible should
not be used.

(ii) It is desirable to have shell growth values for the low and high
concentrations relatively close to, but different from, 0 and 100 percent.
Therefore, the range of concentrations to which the oysters are exposed
should be such that in 96 h relative to the controls, very little shell growth
occurs in oysters exposed to the highest concentration and shell growth
is slightly less than controls at the lowest concentration. Oysters in the
-------
remaining concentrations should have increments of shell growth such that
the concentration producing 50 percent shell growth relative to the growth
is bracketed with at least one concentration above and one below it.

(iii) The test should be carried out without adjustment of pH unless
there is evidence of marked change in the pH of the solution. In this case,
it is advised that the test be repeated with pH adjustment to that of the
dilution water and the results reported.

(iv) The test begins when at least 20 prepared oysters are placed in
each of the test chambers containing the appropriate concentrations of test
substance and controls. The steady-state flows and test chemical concentra-
tions should be documented. At least five test chemical concentrations
should be used. The dilution factor between concentrations should not ex-
ceed 1.8.

(v) Test oysters should be impartially distributed among test chambers
in such a manner that test results show no significant bias from the dis-
tributions. The oysters should be spread out equidistantly from one another
so that the entire test chamber is used. The oysters should also be placed
with the left (cupped) valve down and the open, unhinged ends all oriented
in the same direction facing the incoming flow of test solution.

(vi) The oysters are inspected at least after 24, 48, 72, and 96 h.
Oysters are considered dead if touching of the gaping shell produces no
reaction. Dead oysters are removed when observed and mortalities are re-
corded. Observations at 3 h and 6 h are also desirable.

(vii) Shell growth is the primary criterion used in this test guideline
to evaluate the toxicity of the test chemical. Shell growth increments in
all oysters should be measured after 96-h exposure. Record the length
of the longest "finger" of new shell growth to the nearest 0.1 mm. Oysters
should be handled very gently at this stage to prevent damage to the new
shell growth.

(viii) Records should be kept of visible abnormalities such as loss
of feeding activity (failure to deposit feces), excessive mucus production
(stringy material floating suspended from oysters), spawning, or appear-
ance of shell (closure or gaping).

(ix) The criteria for a valid definitive test are:

(A) The mortality in the controls should not exceed 10 percent at
the end of the test.

(B) The dissolved oxygen concentration should be at least 60 percent
of air saturation throughout the test.

(C) If evidence of spawning is observed, the test should be repeated.
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(D) There should be evidence that the concentration of the substance
being tested has been satisfactorily maintained over the test period. The
concentration of the test substance should be measured:

(7) In each chamber at time 0-h.

(2) In each chamber at 96-h; and

(3) In at least one appropriate chamber whenever a malfunction is
detected in any part of the test chemical delivery system.

(E) Dissolved oxygen, temperature, salinity, and pH measurements
should be made at the beginning and end of the test in each chamber.

(F) A minimum of 2 mm of new shell growth should be observed
in control oysters (solvent and dilution water).

(4) Test results, (i) At the end of the test, appropriate statistical anal-
ysis should be conducted on the oyster shell deposition test data. The
probit transformation should then be applied to the response variable and
then regressed, using least squares regression, on dose or log-dose. An
F Test for linearity should be conducted to determine whether the chosen
regression technique adequately describes the experimental data.

(ii) Calculate the ratio of the mean shell growth for each group of
test oysters (exposed to each of the test chemical concentrations) to the
mean shell growth of the group of control oysters. From these data the
concentration-response curve is drawn and an EC50 along with the
95 percent confidence limits on the value are determined from the curves.
The mean measured concentration of test chemical should be used to cal-
culate the EC50 and to plot the concentration-response curve.

(e) Test conditions—(1) Test species—(i) Selection. (A) The Eastern
oyster, Crassostrea virginica, should be used as the test organism.

(B) Oysters used in the same test should be 30 to 50 mm in valve
height and should be as similar in age and/or size as possible to reduce
variability. The standard deviation of the valve height should be less than
20 percent of the mean.

(D) Oysters should be in a prespawn condition of gonadal develop-
ment prior to and during the test as determined by direct or histological
observation of the gonadal tissue for the presence of gametes.

(ii) Acquisition. Oysters may be cultured in the laboratory, purchased
from culture facilities or commercial harvesters, or collected from a natural
population in an unpolluted area free from epizootic disease.
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(iii) Acclimation. (A) Oysters should be attended to immediately
upon arrival. Oyster shells should be brushed clean of fouling organisms
and the transfer of the oysters to the holding water should be gradual to
reduce stress caused by differences in water quality characteristics and
temperature. Oysters should be held for at least 12 to 15 days before test-
ing. All oysters should be maintained in dilution water at the test tempera-
ture for at least 2 days before they are used.

(B) During holding, the oysters should not be crowded, and the dis-
solved oxygen concentration should be above 60 percent saturation. The
temperature of the holding water should be the same as that used for test-
ing. Holding tanks should be kept clean and free of debris. Cultured algae
may be added to dilution water sparingly, as necessary to support life and
growth and such that test results are not affected as confirmed by previous
testing.

(C) Oysters should be handled as little as possible. When handling
is necessary, it should be done as gently, carefully, and quickly as possible.

(D) A batch of oysters is acceptable for testing if the percentage mor-
tality over the 7-day period prior to testing is less than 5 percent. If the
mortality is between 5 and 10 percent, acclimation should continue for
7 additional days. If the mortality is greater than 10 percent, the entire
batch of oysters should be rejected. Oysters which appear diseased or oth-
erwise stressed or which have cracked, chipped, bored, or gaping shells
should not be used. Oysters infested with mudworms (Polydora sp.) or
boring sponges (Cilona cellatd) should not be used.

(2) Test facilities—(i) Apparatus. (A) In addition to normal labora-
tory equipment, an oxygen meter, equipment for delivering the test chemi-
cal, adequate apparatus for temperature control, and test tanks made of
chemically inert material are needed.

(B) Constant conditions in the test facilities should be maintained as
much as possible throughout the test. The preparation and storage of the
test material, the holding of the oysters and all operations and tests should
be carried out in an environment free from harmful concentrations of dust,
vapors and gases and in such a way as to avoid cross-contamination. Any
disturbances that may change the behavior of the oysters should be avoid-
ed.

(ii) Dilution water. A constant supply of good quality unfiltered sea-
water should be available throughout the holding, acclimation, and testing
periods. Natural seawater is recommended, although artificial seawater
with food added may be used. In either case, to ensure each oyster is
provided equal amounts of food, the water should come from a thoroughly
mixed common source and should be delivered at a flowrate of at least
1 and preferably 5 L/h per oyster. The flowrate should be ±10 percent
of the nominal flow. A dilution water is acceptable if oysters will survive
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and grow normally for 14 days without exhibiting signs of stress; i.e. ex-
cessive mucus production (stringy material floating suspended from oys-
ters), lack of feeding, shell gaping, poor shell closing in response to prod-
ding, or excessive mortality. The dilution water should have a salinity in
excess of 12 ppt, and should be similar to that in the environment from
which the test oysters originated. A natural seawater should have a weekly
range in salinity of less than 10 ppt and a monthly range in pH of less
than 0.8 unit. Artificial seawater salinity should not vary more than 2 ppt
nor more than 0.5 pH unit. Oysters should be tested in dilution water from
the same origin.

(3) Test parameters—(i) Carriers. Stock solutions of substances of
low aqueous solubility may be prepared by ultrasonic dispersion or, if nec-
essary, by use of organic solvents, emulsifiers or dispersants of low tox-
icity to oysters. When such carriers are used the control oysters should
be exposed to the same concentration of the carrier as that used in the
highest concentration of the test substance. The concentration of such car-
riers should not exceed 0.1 mL/L.

(ii) Dissolved oxygen. The dissolved oxygen concentrations should
be at least 60 percent of the saturation value and should be recorded daily.

(iii) Loading. The loading rate should not crowd oysters and should
permit adequate circulation of water while avoiding physical agitation of
oysters by water current.

(iv) Temperature. The test temperature should be 20 °C. Temporary
fluctuations (less than 8 h) within ±5 °C are permissible. Temperature
should be recorded continuously.

(v) pH. The pH should be measured at the beginning and end of
the test in each test chamber.

(f) Reporting. In addition to the reporting requirements as specified
under EPA Good Laboratory Practice Standards, 40 CFR part 792, subpart
J, the following specific information should be reported:

(1) The source of the dilution water, the mean, standard deviation
and range of the salinity, pH, temperature, and dissolved oxygen during
the test period.

(2) A description of the test procedures used (e.g. the flow-through
system, test chambers, chemical delivery system, aeration, etc.).

(3) Detailed information about the oysters used, including the age
and/or size (i.e. height), source, history, method of confirmation of
prespawn condition, acclimation procedures, and food used.

(4) The number of organisms tested, the loading rate, and the
flowrate.
-------
(5) The methods of preparation of stock and test solutions, and the
test chemical concentrations used.

(6) The number of dead and live test organisms, the percentage of
organisms that died, and the number that showed any abnormal effects
in the control and in each test chamber at each observation period.

(7) The 96-h shell growth measurements of each oyster; the mean,
standard deviation and range of the measured shell growth at 96 h of oys-
ters in each concentration of test substance and control.

(8) The calculated 96-h EC50 and its 95 percent confidence limits
and the statistical methods used to calculate these values.

(9) When observed, the 96-h observed no-effect concentration (the
highest concentration tested at which there were no mortalities, abnormal
behavioral or physiological effects and at which shell growth did not differ
from controls).

(10) A graph of the concentration-response curve based on the
96-h chemical concentration and shell growth measurements upon which
the EC50 was calculated.

(11) Methods and data records of all chemical analyses of water qual-
ity parameters and test substance concentrations, including method valida-
tions and reagent blanks.

(12) Any incidents in the course of the test which might have influ-
enced the results.

(13) A statement that the test was carried out in agreement with the
prescriptions of the test guideline given above (otherwise a description
of any deviations occurring).
-------
-------
&EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-136
April 1996
Ecological Effects Test
Guidelines

OPPTS 850.1035
Mysid Acute Toxicity
Test
'Public Draft"
-------
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.

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."
-------
OPPTS 850.1035 Mysid acute toxicity test.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 797.1930 Mysid Shrimp Acute
Toxicity Test and OPP 72-3 Acute Toxicity Test for Estuarine and Marine
Organisms (Pesticide Assessment Guidelines, Subdivision E—Hazard
Evaluation; Wildlife and Aquatic Organisms) EPA report 540/09-82-024,
1982.

(b) Purpose. This guideline prescribes a test using mysids as test or-
ganisms to develop data on the acute toxicity of chemicals. The Environ-
mental Protection Agency will use data from these tests in assessing the
hazard of a chemical to the aquatic environment.

(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and in 40 CFR Part 792—Good Laboratory Practice
Standards apply to mis test guideline. The following definitions also apply
to this test guideline.

Concentration-response curve is the curve produced from toxicity
tests when percent response (e.g. mortality) values are plotted against con-
centration of test substance for a given length of exposure.

Death means the lack of reaction of a test organism to gentle prod-
ding.

Flow-through means a continuous or an intermittent passage of test
solution or dilution water through a test chamber or a holding or acclima-
tion tank, with no recycling.

LC50 means the experimentally derived concentration of test sub-
stance that is calculated to kill 50 percent of a test population during con-
tinuous exposure over a specified period of time.

Loading means the ratio of test organisms biomass (grams, wet
weight) to the volume (liters) of test solution in a test chamber.

No observed effect concentration (NOEC) is the highest tested con-
centration in an acceptable toxicity test which did not cause the occurrence
of any specified adverse effect (statistically different from the control at
95 percent level), and below which no tested concentration caused such
an occurrence.

Retention chamber means a structure within a flow-through test cham-
ber which confines the test organisms, facilitating observation of test orga-
nisms, and eliminating loss of organisms in outflow water.
-------
Static system means a test chamber in which the test solution is not
renewed during the period of the test.

(d) Test procedures—(1) Summary of the test. In preparation for
the test, test chambers are filled with appropriate volumes of dilution
water. If a flow-through test is performed, the flow of dilution water
through each chamber is adjusted to the rate desired. The test substance
is introduced into each test chamber. In a flow-through test, the rate at
which the test substance is added is adjusted to establish and maintain
the desired concentration of test substance in each test chamber. The test
is started by randomly introducing mysids acclimated in accordance with
the test design into the test chambers. Mysids in the test chambers are
observed periodically during the test, dead mysids are removed, and the
findings recorded Dissolved oxygen concentration, pH, temperature, salin-
ity, the concentration of test substance, and other water quality characteris-
tics are measured at specified intervals in test chambers. Data collected
during the test are used to develop concentration-response curves and
LC50 values for the test substance.

(2) Range-finding test, (i) A range-finding test should be conducted
to determine:

(A) Which life stage (juvenile or young adult) is to be utilized in
the definitive test.

(B) The test solution concentrations for the definitive test.

(ii) The mysids should be exposed to a series of widely spaced con-
centrations of test substance (e.g. 1,10,100 mg/L, etc.), usually under static
conditions.

(iii) This test should be conducted with both newly hatched juvenile
(<24 h old) and young adult (5 to 6 days old) mysids. For each age class
(juvenile or young adult), a minimum of 10 mysids should be exposed
to each concentration of test substance for up to 96 h. The exposure period
may be shortened if data suitable for the purpose of the range-finding test
can be obtained in less time. The age class which is most sensitive to
the test substance in the range-finding test should be utilized in the defini-
tive test. When no apparent difference in sensitivity of the two life stages
is found, juveniles should be utilized in the definitive test. No replicates
are required, and nominal concentrations of the test chemical are accept-
able.

(3) Definitive test, (i) The purpose of the definitive test is to deter-
mine the concentration-response curves and the 48- and 96-b LC50 values
with the minimum amount of testing beyond the range-finding test.
-------
(ii) The definitive test should be conducted on the mysid life stage
(juveniles or young adults) which is most sensitive to the test substance
being evaluated.

(iii) A minimum of 20 mysids per concentration should be exposed
to five or more concentrations of the test chemical chosen in a geometric
series in which the ratio is between 1.5 and 2.0 (e.g. 2, 4, 8, 16, 32, and
64 mg/L). An equal number of mysids are introduced into the test and
control chambers by stratified random assignment and should be placed
in two or more replicates. If solvents, solubilizing agents, or emulsifiers
have to be used, they should be commonly used carriers and should not
possess a synergistic or antagonistic effect on the toxicity of the test sub-
stance. Preferred carriers are dimethyl formamide, triethylene glycol, ace-
tone, or ethanol. Use of carriers should be avoided, if possible, as they
may serve as a carbon source for bacteria. The concentration of solvent
should not exceed 0.1 mL/L. The concentration ranges should be selected
to determine the concentration-response curves and LC50 values at 48 and
96 h.

(iv) Every test should include controls consisting of the same dilution
water, conditions, and procedures, and mysids from the same population
or culture container, except that none of the test chemical is added.

(v) The dissolved oxygen concentration, temperature, salinity, and pH
should be measured at the beginning and end of the test in each chamber.

(vi) The test duration is 96 h. The test is unacceptable if more than
10 percent of the control organisms die or exhibit abnormal behavior dur-
ing the 96-h test period. Each test chamber should be checked for dead
mysids at 24, 48, 72, and 96 h after the beginning of the test. Concentra-
tion-response curves and 24-, 48-, 72- and 96-h LC50 values should be
determined along with their 95 percent confidence limits.

(vii) In addition to death, any abnormal behavior or appearance
should also be reported.

(viii) Test organisms should be impartially distributed among test
chambers in such a manner that test results show no significant bias from
the distributions. In addition, test chambers within the testing area should
be positioned in a random manner or in a way in which appropriate statis-
tical analyses can be used to determine the variation due to placement.

(ix) The concentration of the test substance in the chambers should
be measured as often as is feasible during the test. During static tests,
the concentration of test substance should be measured at a minimum at
the beginning and at the end of the tests. During the flow-through test,
the concentration of test substance should be measured at the beginning
and end of the test and in at least one appropriate chamber whenever a
malfunction is detected in any part of the test substance delivery system.
-------
Equal aliquots of test solution may be removed from each replicate cham-
ber and pooled for analysis. Among replicate test chambers of a treatment
concentration, the measured concentration of the test substance should not
vary more than 20 percent.

(4) Analytical measurements—(i) Test chemical. Deionized water
should be used in making stock solutions of the test substance. Standard
analytical methods should be used whenever available in performing the
analyses. The analytical method used to measure the amount of test sub-
stance in a sample should be validated 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 and oxida-
tion products, give positive or negative interferences which cannot be sys-
tematically identified and mathematically corrected.

(ii) Numerical. The number of dead mysids should be counted during
each definitive test. Appropriate statistical analyses should provide a good-
ness-of-fit determination for the concentration-response curves. A 48- and
96-h LC50 and corresponding 95 percent interval should be calculated.
An NOEC and the slope of the dose-response curve should also be deter-
mined.

(e) Test conditions—(1) Test species—(i) Selection. (A) The mysid,
Mysidopsis bahia, is the organism specified for these tests. Either juvenile
(<24 h old) or young adult (5 to 6 days old) mysids are to be used to
start the test. It has recently been proposed, under paragraph (g)(2) of this
guideline, to place this species in a new genus, Americamysis.

(B) Mysids to be used in acute toxicity tests should originate from
laboratory cultures in order to ensure the individuals are of similar age
and experimental history. Mysids used for establishing laboratory cultures
may be purchased commercially or collected from appropriate natural
areas. Because of similarities with other mysid species, taxonomic verifica-
tion should be obtained from the commercial supplier by experienced lab-
oratory personnel or by an outside expert.

(C) Mysids used in a particular test should be of similar age and
be of normal size and appearance for their age. Mysids should not be
used for a test if they exhibit abnormal behavior or if they have been
used in a previous test, either in a treatment or in a control group.

(ii) Acclimation. (A) Any change in the temperature and chemistry
of the dilution water used for holding or culturing the test organisms to
those of the test should be gradual. Within a 24—h period, changes in water
temperature should not exceed 1 °C, while salinity changes should not
exceed 5 percent.

(B) During acclimation mysids should be maintained in facilities with
background colors and light intensities similar to those of the testing areas.
-------
(iii) Care and handling. Methods for the care and handling of mysids
such as those described under paragraph (g)(l) of this guideline can be
used during holding, culturing, and testing periods.

(iv) Feeding. Mysids should be fed daily during testing. Any food
utilized should support survival, growth, and reproduction of the mysids.
A recommended food is live Artemia spp. (48-h-old nauplii).

(2) Facilities—(i) Apparatus. (A) Facilities which may be needed
to perform this test include:

(/) Flow-through or recirculating tanks for holding and acclimating
mysids.

(2) A mechanism for controlling and maintaining the water tempera-
ture during the holding, acclimation, and test periods.

(3) Apparatus for straining particulate matter, removing gas bubbles,
or aerating the water, as necessary.

(4) An apparatus for providing a 14-h light and 10-h dark
photoperiod with a 15 to 30 min transition period. In addition, for flow-
through tests, flow-through chambers and a test substance delivery system
are required. Furthermore, it is recommended that mysids be held in reten-
tion chambers within test chambers to facilitate observations and eliminate
loss of test organisms through outflow water. For static tests, suitable
chambers for exposing test mysids to the test substance are required. Fa-
cilities should be well ventilated and free of fumes and disturbances that
may affect the test organisms.

(B) Test chambers should be loosely covered to reduce the loss of
test solution or dilution water due to evaporation and to minimize the entry
of dust or other particulates into the solutions.

(ii) Cleaning. Test substance delivery systems and test chambers
should be cleaned before each test following standard laboratory practices.

(iii) Construction materials. (A) Materials and equipment that con-
tact test solutions should be chosen to minimize sorption of test chemicals
from dilution water and should not contain substances that can be leached
into aqueous solution in quantities that can affect test results.

(B) For use in the flow-through test, retention chambers utilized for
confinement of test organisms can be constructed with netting material
of appropriate mesh size.

(iv) Dilution water. (A) Natural or artificial seawater is acceptable
as dilution water if mysids will survive and successfully reproduce in it
for the duration of the holding, acclimating, and testing periods without
-------
showing signs of stress, such as reduced growth and fecundity. Mysids
should be cultured and tested in dilution water from the same origin.

(B) Natural seawater should be filtered through a filter with a pore
size of <20 |Ltm prior to use in a test.

(C) Artificial seawater can be prepared by adding commercially avail-
able formulations or specific amounts of reagent-grade chemicals to
deionized water. Deionized water with a conductivity less than
1 ^iohm/cm at 12 °C is acceptable for making artificial seawater. When
deionized water is prepared from a ground or surface water source, con-
ductivity and total organic carbon (or chemical oxygen demand) should
be measured on each batch.

(v) Test substance delivery system. In flow-through tests, propor-
tional diluters, metering pumps, or other suitable systems should be used
to deliver test substance to the test chambers. The system to be used should
be calibrated before each test. Calibration includes determining the flow
rate through each chamber and the concentration of the test substance in
each chamber. The general operation of the test substance delivery system
should be checked twice daily during a test. The 24-h flow through a
test chamber should be equal to at least 5x the volume of the test chamber.
During a test, the flow rates should not vary more than 10 percent among
test chambers or across time.

(3) Test parameters. Environmental parameters of the water con-
tained in test chambers should be maintained as specified below:

(i) The test temperature should be 25 °C. Excursions from the test
temperature should be not greater than ±2 °C.

(ii) Dissolved oxygen concentration between 60 and 105 percent satu-
ration. Aeration, if needed to achieve this level, should be done before
the addition of the test substance. All treatment and control chambers
should be given the same aeration treatment.

(iii) The number of mysids placed in a test solution should not be
so great as to affect results of the test. Loading should not exceed 30
mysids per liter for a static test. Loading requirements for the flow-through
test will vary depending on the flow rate of dilution water. The loading
should not cause the dissolved oxygen concentration to fall below the rec-
ommended levels.

(iv) Photoperiod of 14 h light and 10 h darkness, with a 15 to
30 min transition period.

(v) Salinity of 20 ±3 ppt.

(f) Reporting. The sponsor should submit to the EPA all data devel-
oped during the test that are suggestive or predictive of acute toxicity and
-------
all concomitant toxicologic manifestations. In addition to the reporting re-
quirements as specified under Good Laboratory Practice Standards, 40
CFR part 792, subpart J, the following specific information should be re-
ported:

(1) The nature of the test, laboratory, name of the investigator, test
substance, and dates of test should be supplied.

(2) A detailed description of the test substances should be provided.
This information should include the source, lot number, composition, phys-
ical and chemical properties, shelf life and storage conditions, and any
carrier or additives used.

(3) Detailed information about the shrimp should be provided: Com-
mon and scientific names, source of supply, age, history, weight, acclima-
tion procedure, and feeding history should be reported.

(4) A description of the experimental design including the number
of test solution concentrations, number of replicates, and number of shrimp
per replicate should be provided.

(5) The source of the dilution water, its chemical characteristics (e.g.
salinity), and a description of any pretreatment.

(6) A description of the test chambers, the depth and volume of solu-
tion in the chamber, the number of organisms per treatment, the number
of replicates, the loading, the lighting, the test substance delivery system
and flow rate expressed as volume additions per 24 h.

(7) The concentration of the test substance in each test chamber be-
fore the start of the test and at the end.

(8) The number of dead shrimp and measurements of water tempera-
ture, salinity, and dissolved oxygen concentration in each test chamber
should be recorded at the protocol-designated times.

(9) Methods and data records of all chemical analyses of water quality
and test substance concentrations, including method validations and rea-
gent blanks.

(10) Recorded data for the holding and acclimation period (tempera-
ture, salinity, etc.).

(11) Concentration-response curves should be fitted to mortality data
collected at 24, 48, 72, and 96 h. A statistical test of goodness-of-fit should
be performed.

(12) For each set of mortality data, the 48- and 96-h LC50 and 95
percent confidence limits should be calculated on the basis of the average
measured concentration of the test substance. When data permits, LC50
values with 95 percent confidence limits should be computed for 24— and
-------
72-h observations. The NOEC and slope of the dose-response curves
should also be calculated.

(13) The methods used in calculating the concentration-response
curves and the LC50 values should be fully described.

(g) References. The following references should be consulted for ad-
ditional background material on this test guideline.

(1) Environmental Protection Agency, Bioassay Procedures for the
Ocean Disposal Permit Program, EPA Report No. 600-9-78-010 (Gulf
Breeze, Florida, 1978).

(2) Price, E.W. et al. Observations on the genus Mysidopsis Sars,
1864 with the designation of a new genus, Americamysis, and the descrip-
tions of Americamysis alleni and A. stucki (Pericarda: Mysidacea:
Mysidae), from the Gulf of Mexico. Proceedings of the Biological Society
of Washington 107:680-698 (1994).
8
-------
&EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-137
April 1996
Ecological Effects Test
Guidelines

OPPTS 850.1045
Penaeld Acute Toxicity
Test
"Public Draft"
-------
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.

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."
-------
OPPTS 850.1045 Penaeid acute toxicity test.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 797.1970 Penaid Shrimp Acute
Toxicity Test and OPP 72-3 Acute Toxicity Test for Estuarine and Marine
Organisms (Pesticide Assessment Guidelines, Subdivision E—Hazard
Evaluation; Wildlife and Aquatic Organisms) EPA report 540/09-82-024,
1982.

(b) Purpose. This guideline prescribes tests using penaeid shrimp as
test organisms to develop data on the acute toxicity of chemicals. The
Environmental Protection Agency will use data from these tests in assess-
ing the hazard of a chemical to the aquatic environment.

(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and 40 CFR Part 792—Good Laboratory Practice
Standards apply to this test guideline. The following definitions also apply
to this guideline:

Concentration-response curve is the curve produced from toxicity test
data when percent response (e.g. mortality) values are plotted against con-
centration of test substance for a given length of exposure.

Death is the lack of reaction of a test organism to gentle prodding.

Flow-through is a continuous passage of test solution or dilution
water through a test chamber, holding, or acclimation tank with no recy-
cling.

LC50 is the experimentally derived concentration of test substance
that is calculated to have killed 50 percent of a test population during
continuous exposure over a specified period of time.

Loading is the ratio of test organism biomass (grams, wet weight)
to the volume (liters) of test solution in a test chamber.

No-observed-effect-concentration (NOEC) is the highest tested con-
centration in an acceptable toxicity test which did not cause the occurrence
of any specified adverse effect (statistically different from the control at
the 95 percent level), and below which no tested concentration caused such
an occurrence.

ppt is parts per thousand (salinity units).

(d) Test procedures—(1) Summary of the test. Prior to testing, the
bottoms of the test chambers are covered with 2 to 3 cm of sand and
-------
then filled with appropriate volumes of dilution water. The flow is adjusted
to the rate desired to achieve loading requirements. Penaeids are intro-
duced into the test chambers according to the experimental design. The
shrimp are acclimated by maintaining them in the test chambers for a pe-
riod of 3 to 7 days prior to the beginning of the test. The test begins
when the test substance is introduced into the test chambers. The rate of
flow is adjusted to maintain the desired test substance concentration in
each chamber. The shrimp are observed during the test; dead shrimp are
counted, removed, and the findings recorded. Dissolved oxygen concentra-
tion (DOC), pH, temperature, salinity, test substance concentration, and
other water quality characteristics are measured at specified intervals in
selected test chambers. The concentration of test substances with low water
solubility may have to be determined with more frequency. Data collected
during the test are used to develop concentration-response curves and
LC50 values for the test substance.

(2) Range-finding test, (i) A range-finding test should be conducted
to determine the test substance concentrations to be used for the definitive
test. Substances which have low solubility and/or unusual adsorbance char-
acteristics may require special handling procedures (physical procedures
or the use of carrier substances) and attention to the type of materials
used in the testing chambers to enhance solubility or decrease adsorption.

(ii) The shrimp should be exposed to a series of widely spaced con-
centrations of test substance (e.g. 1, 10, 100 mg/L, etc.).

(iii) A minimum of five penaeid shrimp should be exposed to each
concentration of test substance for up to 96 h. No replicates are required
and nominal concentrations of the chemical are acceptable.

(3) Definitive test, (i) The purpose of the definitive test is to deter-
mine the concentration-response curves and the 48- and 96-h LC50 values
with the minimum amount of testing beyond the range-finding test.

(ii) A minimum of 20 shrimp per concentration should be exposed
to five or more concentrations of the chemical chosen in a geometric series
in which the ratio is between 1.5 and 2.0 (e.g. 2, 4, 8, 16, 32 and
64 mg/L). An equal number of shrimp are introduced into the test and
control chambers by stratified random assignment and should be placed
in two or more replicates. If solvents, solubilizing agents, or emulsifiers
have to be used, they should be commonly used carriers and should not
possess a synergistic or antagonistic effect on the toxicity of the test sub-
stance. Preferred carriers are dimethyl formamide, triethylene glycol, ace-
tone, or ethanol. Use of carriers should be avoided, if possible, as they
may serve as a carbon source for bacteria. The concentration of solvent
should not exceed 0.1 mL/L. The concentration ranges should be selected
to determine the requested concentration-response curves and LC50 values.
The concentration of test substance in test solutions should be determined
-------
prior to use and at designated times. Abnormal or unexpected observations
should trigger chemical analysis of the test water. If a specific test chamber
seems to be affected, its water should be analyzed.

(iii) Every test should include controls consisting of the same dilution
water, conditions, procedures, and shrimp from the same population or
culture container, except that none of the chemical is added. If carriers
are used, a separate carrier control should also be included.

(iv) The DOC, temperature, salinity, and pH should be measured at
the beginning of the test and at 24, 48, 72, and 96 h in each test chamber.

(v) The test duration is 96 h. The test is unacceptable if more than
10 percent of the control organisms die or appear to be stressed or diseased
during the 96-h test period. Each test chamber should be checked for dead
shrimp at 3, 6, 12, 24, 48, 72, and 96 h after the beginning of the test.
Concentration-response curves and 48- and 96-h LC50 values should be
determined along with their 95 percent confidence limits.

(vi) In addition to death, any abnormal behavior or appearance should
also be reported.

(vii) Distribution of shrimp among test chambers should be random-
ized. In addition, test chambers within the testing area should be positioned
in a random manner or in a way in which appropriate statistical analyses
can be used to determine the variation due to placement.

(viii) The concentration of dissolved test substance (that which passes
through a 0.45 Jim filter) in the test chambers should be measured as often
as is feasible during the test. The concentration of test substance should
be measured:

(A) In each chamber at the beginning of the test and at 48 and
96 h after the start of the test.

(B) In at least one chamber containing the next to the lowest test
substance concentration at least once every 24 h during the test.

(C) In at least one appropriate chamber whenever a malfunction is
detected in any part of the test substance delivery system. Among replicate
test chambers of a treatment concentration, the measured concentration of
the test substance should not vary more than 20 percent.

(ix) Observations on compound solubility should be recorded. The
investigator should report the appearance of surface slicks, precipitates,
or material adhering to the sides of the test chambers.

(4) Analytical measurements—(i) Test chemical. Deionized water
should be used in making stock solutions of the test substance. Standard
analytical methods should be used whenever available in performing the
-------
analyses. The analytical method used to measure the amount of test sub-
stance in a sample should be validated before beginning the test by appro-
priate laboratory practices. An analytical method is not acceptable if likely
degradation products of the test substance, such as hydrolysis and oxida-
tion products, give positive or negative interferences which cannot be sys-
tematically identified and corrected mathematically.

(ii) Numerical. The number of dead shrimp should be counted during
each definitive test. Appropriate statistical analyses should provide a good-
ness-of-fit determination for the concentration-response curves. A 48- and
96-h LC50 and corresponding 95 percent intervals should be calculated.
An NOEC and the slope of the dose response curve should also be deter-
mined.

(e) Test conditions—(1) Test species—(i) Selection. This test should
be conducted using one of three species of penaeid: Penaeus aztecus
(brown shrimp), Penaeus duorarum (pink shrimp), or Penaeus setiferus
(white shrimp). Post-larval juvenile shrimp should be utilized. Shrimp may
be reared from eggs in the laboratory or obtained directly as juveniles
or adults. Shrimp used in a particular test should have been obtained from
the same source, be of similar age, and be of normal size and appearance.
Shrimp should not be used for a test if they exhibit abnormal behavior
or if they have been used in a previous test, either in a treatment or control
group.

(ii) Acclimation. During acclimation, shrimp should be maintained
in facilities with background colors and light intensities similar to those
of the testing areas. In addition, any change in the temperature and chem-
istry of the dilution water used for holding and acclimating the test orga-
nisms to those of the test should be gradual. Within a 24-h period, changes
in water temperature should not exceed 1 °C, while salinity changes should
not exceed 2 percent.

(iii) Care and handling. Upon arrival at the test facility, the shrimp
should be transferred to water closely matching the temperature and salin-
ity of the transporting medium. Shrimp should be held in glass tanks of
30 L capacity or larger. No more than 22 to 24 shrimp should be placed
in a 30 L tank unless the flow-through apparatus can maintain dissolved
oxygen levels above 60 percent of saturation. With species of the genus
Penaeus, a minimum flow rate of 7.5 L/g body weight day should be
provided. Larger flows, up to 22 L/g body weight day, may be desirable
to ensure dissolve oxygen concentrations above 60 percent of saturation
and the removal of metabolic products. The period of acclimation to ambi-
ent laboratory conditions should be at least 4 to 7 days.

(iv) Feeding. Penaeid shrimp should not be fed during testing. Every
2 or 3 days during the acclimation period, shrimp should be fed fish pieces
approximately 1 cm2. Uneaten food should be removed daily.
-------
(2) Facilities—(i) Apparatus. (A) Facilities which may be needed
to perform this test include: Flow-through tanks for holding and
acclimating penaeid shrimp; a mechanism for controlling and maintaining
the water temperature and salinity during the holding period; apparatus
for straining paniculate matter, removing air bubbles, or aerating water
when necessitated by water quality requirements; and an apparatus provid-
ing a 12-h light and 12-h dark photoperiod with a 15- to 30-min transi-
tion period. Facilities should be well ventilated, free of fumes, and free
of all other disturbances that may affect test organisms.

(B) Acid-washed sand, free of excess organic matter, should be placed
in the bottom of test chambers to a depth of 2-3 cm.

(C) Test chambers should be loosely covered to reduce the loss of
test solution or dilution water due to evaporation, minimize entry of dust
and other particles, and prevent escape of the shrimp.

(ii) Cleaning. Test substance delivery systems and test chambers
should be cleaned before each test following standard laboratory practices.

(iii) Construction materials. Materials and equipment that contact
test solutions should be chosen to minimize sorption of test chemicals from
dilution water and should not contain substances that can be leached into
aqueous solution in quantities that can affect test results.

(iv) Dilution water. (A) Natural or artificial seawater is acceptable
as dilution water if shrimp will survive in it without signs of stress, such
as unusual behavior or discoloration. Shrimp should be acclimated and
tested in dilution water from the same origin.

(B) Natural seawater should be filtered through a 5 um filter with
a pore size <20 u.m prior to use in a test.

(C) Artificial seawater can be prepared by adding commercially avail-
able formulations or specific amounts of reagent-grade chemicals to
deionized water. Deionized water with a conductivity less than
0.1 mS/m at 12 °C is acceptable for making artificial seawater. When
deionized water is prepared from a ground or surface water source, con-
ductivity and total organic carbon (or chemical oxygen demand) should
be measured on each batch.

(v) Test substance delivery system. Proportional diluters, metering
pumps, or other suitable systems should be used to deliver test substance
to the test chambers. The system used should be calibrated before each
test. Calibration includes determining the flow rate through each chamber
and the concentration of the test substance in each chamber. The general
operation of the test substance delivery system should be checked twice
daily during a test. The 94-h flow through a test chamber should be equal
-------
to a least 5x the volume of the test chamber. During a test, the flow rates
should not vary more than 10 percent among test chambers or across time.

(3) Test parameters. Environmental parameters of the water con-
tained in test chambers should be as specified below:

(i) Temperature of 23 ± 1 °C.

(ii) DOC between 60 and 105 percent saturation. Aeration, if needed
to achieve this level, should be done before the addition of the test sub-
stance. All treatment and control chambers should be given the same aer-
ation treatment.

(iii) The number of shrimp placed in a test solution should not be
so great as to affect results of the test. Loading requirements will vary
depending on the flow rate of dilution water. The loading should not cause
the DOC to fall below the recommended levels.

(iv) Photoperiod of 12-h light and 12-h darkness, with a 15- to
30-min transition period.

(v) Salinity of 20 ± 3 ppt.

(f) Reporting. The sponsor should submit to the EPA all data devel-
oped by the test that are suggestive or predictive of acute toxicity and
all other lexicological manifestations. In addition to the general reporting
requirements prescribed under Good Laboratory Practice Standards, 40
CFR part 792, subpart J, the reporting of test data should include the fol-
lowing:

(1) The nature of the test, laboratory, name of the investigator, test
substance, and dates of the test should be supplied.

(2) A detailed description of the test substances should be provided.
This information should include the source, lot number, composition, phys-
ical and chemical properties, shelf life, storage conditions, and any carrier
or additives used.

(3) Detailed information about the shrimp should be provided: Com-
mon and scientific names, source of supply, age, history, weight, acclima-
tion procedure, and feeding history should be reported.

(4) A description of the experimental design including the number
of test solution concentrations, number of replicates, and number of shrimp
per replicate should be provided.

(5) The source of the dilution water, its chemical characteristics (e.g.
salinity), and a description of any pretreatment.

(6) A description of the test chambers, the depth and volume of solu-
tion in the chamber, the number of organisms per treatment, the number
-------
of replicates, the loading, the lighting, the test substance delivery system,
and flow rate expressed as volume additions per 24 h.

(7) The concentration of the test substance in each test chamber be-
fore the start of the test and at the end.

(8) The number of dead shrimp and measurements of water tempera-
ture, salinity, and DOC in each test chamber should be recorded at the
protocol-designated times.

(9) Methods and data records of all chemical analyses of water quality
and test substance concentrations, including method validations and rea-
gent blanks.

(10) Recorded data for the holding and acclimation period (tempera-
ture, salinity, etc.).

(11) Concentration-response curves should be fitted to mortality data
collected at 24,48, 72, and 96 h. A statistical test of goodness-of-fit should
be performed.

(12) For each set of mortality data, the 48- and 96-h LC50 and 95
percent confidence limits should be calculated on the basis of the average
measured concentration of the test substance. When data permits, LC50
values with 95 percent confidence limits should be computed for 24- and
72-h observations. The NOEC and slope of the dose-response curves
should also be calculated.

(13) The methods used in calculating the concentration-response
curves and the LC50 values should be fully described.

(g) References. The following references should be consulted for ad-
ditional background material on this test guideline.

(1) Environmental Protection Agency, Bioassay Procedures for the
Ocean Disposal Permit Program. EPA Report No. 600-9-78-010 (Gulf
Breeze, FL 1978).

(2) [Reserved]
-------
-------
&EPA
United States
Environmental Protection
Agency
Prevention. Pesticides
and Toxic Substances
(7101)
EPA712-C-96-160
April 1996
Ecological Effects Test
Guidelines

OPPTS 850.1055
Bivalve Acute Toxicity
Test (Embryo-Larval)
"Public Draft"
-------
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.

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."
-------
OPPTS 850.1055 Bivalve acute toxicity test (embryo-larval).
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.).

(2) Background. The source material used in developing this har-
monized OPPTS test guideline is OPP 72-3 Acute Toxicity Test for Estua-
rine and Marine Organisms (Pesticide Assessment Guidelines, Subdivision
E—Hazard Evaluation; Wildlife and Aquatic Organisms) EPA report 5407
09-82-024, 1982.

(b) Purpose. This guideline prescribes tests to be used to develop
data on the acute toxicity of chemical substances and mixtures ("chemi-
cals") to Eastern oysters (Crassostrea virginica), Pacific oysters
(Cmssostrea gigas), quahogs (Mercenaria mercenarid), or bay mussels
(Mytilus edulis). The Environmental Protection Agency will use data from
these tests in assessing the hazard of a chemical to the environment.

(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and the definitions in 40 CFR Part 792—Good Lab-
oratory Practice Standards apply to this guideline. The following defini-
tions also apply to this test guideline.

Acute toxicity is the discernible adverse effects induced in an orga-
nism within a short period of time (days) of exposure to a chemical. The
effects (lethal or sublethal) occurring may usually be observed within the
period of exposure with aquatic organisms. In this test guideline, abnormal
development or death is used as the measure of toxicity.

48-h EC50 (Effective Median Concentration) is that experimentally
derived concentration of a chemical in water in which 50 percent of the
larvae exposed to test material are dead or abnormally developed compared
to larvae in the controls (not exposed to test material) after a 48-h expo-
sure.

Embryo is the stage between the fertilization of the egg and the
trochophore (2 to 8 cell stage).

Larva includes the trochophore and the straight hinge stage.

LOEC is the lowest observed effect concentration.

NOEC is the no observed effect concentration.

Veliger is the larval stage in which the ciliated velum (swimming
organ) is present.

(d) Test procedures—(1) Summary of the test, (i) The water solu-
bility and the vapor pressure of the test chemical should be known. Prior
to testing, the structural formula of the test chemical, its purity, stability
-------
in water and light, «-octanol/water partition coefficient, and pK values
should be known. The results of a biodegradability test and the method
of analysis for the quantification of the chemical in water is also desirable.

(ii) It may be possible to determine an EC50 for a chemical with
limited solubility under the test conditions. If the stability or homogeneity
of the test chemical cannot be maintained, care should be taken in the
interpretation of the results and a note made that these results may not
be reproducible.

(iii) This study consists of a static 48-h exposure that is used to evalu-
ate the proportion of living and normal D-shaped veligers exposed to the
test material compared to the proportion of the same in controls not ex-
posed to test material. The concentration-response curve and EC50 value
for the test chemical are developed from these data.

(3) Definitive test, (i) The test is started about 4 h after fertilization
while die embryos are in the 2- to 4-cell stage (determined microscopi-
cally). At this stage embryos (15-30 embryos/rnL/replicate) are added to
the test solution. The endpoint for this test is the determination of a
48-h EC50. This will be based on the proportion of normal larvae (those
that are alive with completely developed shells containing meat) exposed
to test solution as compared to normal larvae in controls. An LOEC and
an NOEC are also to be calculated. Constant conditions should be main-
tained in the test facilities as much as possible throughout the test. The
preparation and storage of the test material, the holding of the oysters,
and all operations and tests should be carried out in an environmental free
from harmful concentrations of dust, vapors, and gases and in such a way
to avoid cross-contamination. Any disturbances that may change the be-
havior of the test organisms should be avoided.

(ii) The test chemical concentrations are to be documented in all tests.
At least five test concentrations are to be used with a dose separation
factor not to exceed 1.8 between concentrations.

(iii) Test organisms are to be impartially distributed among test cham-
bers in such a manner that the test results show no significant bias from
the distributions.

(iv) Test organisms are inspected at regular intervals. Dead bivalves
are removed when observed.

(v) The criteria for a valid definitive test are:
-------
(A) Mortality or aberrant development in the controls are not exceed
30 percent percent for oysters or 40 percent for clams at the end of each
test.

(B) The dissolved oxygen concentration should be at least 60 percent
of air saturation throughout all tests.

(D) The difference between the time-weighted-average (TWA) meas-
ured temperatures for any two test chambers from the beginning to the
end of the test should not be greater than 1 °C. No single measured tem-
perature in any test chamber should be more than 3 °C different from the
mean of the TWA measured temperatures for the individual test chambers.
The difference between the measured temperatures hi any two test cham-
bers should not be more than 2 °C at any one time.

(e) Test conditions—(1) Test species—(i) Selection. (A) Eastern
oysters (C. virginica) are the preferred test species, but Pacific oysters
(C. gigas), quahogs (A/, mercenaria), or bay mussels (M. edulis) may also
be used.

(B) The test must begin with embryos within 4-h of fertilization when
embryos are in the 2- to 4-, and 8-cell stages.

(C) Embryos used to start a test should be obtained from females
and males that have been maintained for at least 2 weeks in the dilution
water in the laboratory before they are stimulated to spawn.

(D) The spawning of bivalve test organisms is induced by rapidly
elevating the temperature 5-10°C above the conditioning temperature. An
added stimulus of heat-killed bivalve sperm may be used. To fertilize the
eggs, sufficient sperm suspension should be added to the egg suspension
to yield I05 to 107 sperrn/rnL in the final mixture. Additional guidance
may be found in paragraph (g)(l) of this guideline.

(ii) Acquisition. Bivalves may be cultured in the laboratory, pur-
chased from culture facilities or commercial harvesters, or collected from
a natural population in an unpolluted area free from epizootic disease.

(2) Test facilities—(i) Apparatus. (A) Test vessels, equipment and
facilities that contact stock solutions, test solutions, or any water into
which any brood stock or test organisms will be placed should not contain
substances that can be leached or dissolved by aqueous solutions in
amounts that adversely affect test organisms.

(B) Test chambers are defined as the smallest physical units between
which there are no water connections. Tests are usually conducted in glass
chambers that are 1- to 2-L in capacity.
-------
(ii) Dilution water. A constant supply of good quality unfiltered sea-
water should be available throughout the holding, acclimation, and testing
periods. The dilution water should be acceptable to adult bivalve molluscs
and their embryos and larvae. For oysters, at least 70 percent of the em-
bryos resulting from eggs and sperm of appropriately conditioned adults
result in normal larvae while being maintained in the dilution water for
48 h. For clams, this should be 60 percent of the embryos resulting in
normal larvae. Also, a dilution water is acceptable if adult oysters or clams
will survive and grow normally for 14 days without exhibiting signs of
stress, i.e., excessive mucus production (stringy material floating sus-
pended from oysters) lack of feeding, shell gaping, poor shell closing in
response to prodding, or excessive mortality. Natural seawater is rec-
ommended, although artificial seawater with food added may be used. The
dilution water is to have a salinity in excess of 12 ppt. A natural seawater
should have a weekly range in salinity of less than 10 ppt and a monthly
range in pH of less than 0.8 unit. Artificial seawater salinity should not
vary more than 2 ppt nor more than 0.5 pH unit. Oysters are to be tested
in dilution water from the same origin.

(3) Test parameters—(i) Carriers. Stock solutions of substances of
low aqueous solubility may be prepared by ultrasonic dispersion or, if nec-
essary, by use of organic solvents, emulsifiers or dispersant of low toxicity
to oysters. When such carriers are used the control oysters are to be ex-
posed to the same concentration of the carrier as that used in the highest
concentration of the test substance. The concentration of such carriers
should not exceed 0.1 mL/L.

(ii) Dissolved oxygen. The dissolved oxygen concentrations are to
be at least 60 percent of the saturation value and should be recorded daily.

(iii) Loading. The loading rate should not crowd oysters and should
permit adequate circulation of water while avoiding physical agitation of
oysters by water current.

(iv) Temperature. Tests with C. gigas should be conducted at 20 °C,
with C. virginica and M. mercenaria at 25 °C, and with M. edulis at 16 °C.
The temperature for C. gigas, C. virginica, and M. mercenaria should
never exceed 32 °C, nor 20 °C for M. edulis (even during spawning induc-
tion). Temperature should be recorded continuously.

(v) pH. The pH is to be measured at the beginning and end of the
test in each test chamber.

(f) Reporting. In addition to the reporting requirements prescribed
in 40 CFR Part 792—Good Laboratory Practice Standards, the report is
to contain the following:
-------
(1) The source of the dilution water, the mean, standard deviation
and range of the salinity, pH, temperature, and dissolved oxygen during
the test period.

(2) A description of the test procedures used (e.g., the flow-through
system, test chambers, chemical delivery system, aeration, etc.).

(3) Detailed information about the oysters used, including the age
and/or size (i.e., height), source, history, method of confirmation of
prespawn condition, acclimation procedures, and food used.

(4) The number of organisms tested, the loading rate, and the
flowrate.

(5) The methods of preparation of stock and test solutions, and the
test chemical concentrations used.

(7) The calculated 48-h EC50 and its 95 percent confidence limits
and the statistical methods used to calculate these values.

(8) The calculated LOEC and a NOEC must also be developed.

(9) Methods and data records of all chemical analyses of water quality
parameters and test substance concentrations, including method validations
and reagent blanks.

(10) Any incidents in the course of the test which might have influ-
enced the results.

(11) A statement that the test was carried out in agreement with the
prescriptions of the test guideline given above (otherwise a description
of any deviations occurring).

(g) References. The following references should be consulted for ad-
ditional background material on this test guideline.

(1) ASTM. Standard Guide for Conducting Static Acute Toxicity
Tests Starting with Embryos of Four Apecies of Saltwater Bivalve
Molluscs. E 724-89. American Society for Testing and Materials, Philadel-
phia, PA. 18 pp (1989).

(2) [Reserved]
-------
-------
&EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-118
April 1996
Ecological Effects Test
Guidelines
OPPTS 850.1075
Fish Acute Toxicity Test,
Freshwater and Marine
"Public Draft"
-------
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.

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."
-------
OPPTS 850.1075 Fish acute toxicity test, freshwater and marine.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 797.1400 Fish Acute Toxicity
Test; OPP 72-1 Acute Toxicity Test for Freshwater Fish and 72-3 Acute
Toxicity Test for Estuarine and Marine Organisms (Pesticide Assessment
Guidelines, Subdivision E—Hazard Evaluation; Wildlife and Aquatic Or-
ganisms) EPA report 540/09-82-024, 1982; and OECD 203 Fish Acute
Toxicity Test.

(b) Purpose. The purpose of the acute toxicity test with fish species
is to help in the assessment of possible risk to similar species in natural
environments, as an aid in determination of possible water quality criteria
for regulatory purposes, and for use in correlation with acute testing of
other species for comparative purposes. Data on a cold and warm fresh-
water species are generally required. The rainbow trout, Oncorhynchus
myfass, and bluegill sunfish, Lepomis macrochirus, are preferred species
to meet this requirement since they are sensitive indicator species and a
large data base which characterizes the response to environmental contami-
nants is available. Other species as identified in paragraph (e)(4)(i)(A) of
this guideline may be used. However, under certain circumstances, when
potential environmental exposures may lead to significant risks, data on
the preferred species may be required for risk assessment purposes so that
the Agency can conduct comparative analyses with alternative chemical
substances. Historically, it appears that many chemical classes are subject
to comparative analyses. Development of a good data base could ulti-
mately result in the use of other species in comparative analyses. In any
case, the results of such a study should not be construed to represent be-
havior of the test material in the natural environment where other factors
may come into play, but rather as a indicator of effects which might occur
under comparable conditions as those utilized in the study.

(c) Principle of the test—(1) Definitive test The goal of the defini-
tive test is to determine concentration-response curves for fish mortality,
the LCSO's, and the 95 percent confidence intervals for each species tested
at 24, 48, 72, and 96 h in a static, static-renewal, or flow-through system.

(2) Range-finding or limit testing. Definitive testing may be waived
if limit testing with at least 30 organisms shows LC50 levels to be greater
than 1,000 mg/L based on 100 percent active ingredients (AI), or the limits
of water solubility or dispersibility. For pesticides, a lower level of 100
mg AI/L may be tested when estimated environmental concentrations are
not expected to exceed 100 mg/L (ppm) as might occur with pesticide
use. Prior to selection of definitive test concentrations it may be advisable
-------
to conduct a range-finding test. Results of any range-finding and limit tests
should be reported with results of the definitive test.

(3) Information on the test substance. The material to be tested
should be technical grade unless the test is designed to test a specific for-
mulation, mixture, or effluent. The degree of purity must be recorded for
technical ingredients and mixtures. The percentage of each impurity should
be reported and percentages should total 100 percent. A complete descrip-
tion of physicochemical characteristics (i.e. solubility, vapor pressure, hy-
drolysis in pH 5, 7, and 9) should be included with description of the
AI used in specific chemical testing. A reliable analytical method for quan-
tification of test substance concentrations must be available.

(d) Validity of the test. (1) Maximum-allowable control or solvent
control mortality is 10 percent (or 1 mortality if 7 to 10 control fish are
used) for a 96-h period of testing. If the test is continued past 96 h, the
maximum-allowable additional mortality is 10 percent.

(2) Constant conditions must be maintained throughout the test pe-
riod. Flow-through procedures are preferred over static-renewal or
semistatic procedures and static-renewal procedures are preferred over a
static test procedure.

(3) In static tests, the dissolved oxygen (DO) in each replicate should
at all times be greater than 60 percent saturation. In flow-through tests,
the DO should be maintained above 75 percent saturation.

(4) Measured concentrations are required if the test chemical is unsta-
ble or a flow-through diluter system is employed. Exception may be made
in cases where hydrolysis studies indicate chemical to be stable (<5 per-
cent degradation) in 96 h at a pH comparable to test dilution water. In
any case there must be evidence that test concentrations remained at least
80 percent of the nominal concentrations throughout the test or that mean
measured concentrations are an accurate representation of exposure levels
maintained throughout the test period.

(e) Description of the method—(1) Apparatus. Normal laboratory
equipment and especially the following is necessary:

(i) Equipment for determination of water hardness, etc.

(ii) Adequate apparatus for temperature control.

(iii) Tanks constructed of chemically inert material and of suitable
capacity to allow recommended loading levels.

(2) Water, (i) Clean surface or ground water, seawater (for estuarine
or marine species), and reconstituted water are acceptable as dilution
water. Dechlorinated water should not be used because some forms of
chlorination are difficult to remove adequately. If dechlorinated tap water
-------
is used, then daily chlorine analysis should be performed. Reconstituted
or natural water is preferred.

(ii) Chemical analysis of water used in testing should include the fol-
lowing elements and limitations on maximum concentrations based on at
least biannual testing:
Maximum con-
centration

20.0 mg/L
5.0 mg/L
2.0 mg/L
< 100.0 mg/L
0.003 mg/L
0.020 mg/L
0.001 mg/L
<0:100
0.050 \igfL
0.050 ng/L
<1.0 jiohms
Substance

Participate matter
Chemical oxygen demand (COD)
Total organic carbon (TOC)
Boron and fluoride
Residual chlorine
Un-ionized ammonia
Aluminum, arsenic, chromium, cobalt, copper, iron, lead, nickel, and
zinc.
Cadmium, mercury, and silver
Total organophosphorus pesticides
Total organochlprine pesticides + PCBs or organic chlorine
Specific conductivity

(iii) Salinity should be 20 ± 5 ppt for estuarine species.

(iv) Hardness should range between 40 and 180 mg/L as CaCO3 for
freshwater species.

(v) Water hardness or salinity, as appropriate, should be measured
at the beginning of each test.

(vi) In marine flow-through tests, salinity should be recorded at the
beginning of the test, on day 4, and if extended, on days 7 and 14.

(3) Solutions of test water, (i) Distilled water should be used in mak-
ing stock solutions of the test substance. If the stock volume is more than
10 percent of the test solution volume, dilution water should be used. If
a carrier, i.e. a solvent and/or dispersant, is absolutely necessary to dissolve
the test substance, the amount used should not exceed the minimum vol-
ume necessary to dissolve or suspend the test substance in the dilution
water. If the test substance is a mixture, formulation, or commercial prod-
uct, none of the ingredients is considered a carrier unless an extra amount
is used to prepare the stock solution.

(ii) Solvent concentration may not exceed 0.5 mL/L in static-renewal
or static testing, and 0.1 mL/L in flow-through testing.

(iii) Preferred solvents are dimethyl formamide, triethylene glycol,
methanol, acetone, or ethanol. Solvent use should be avoided if possible.

(iv) Solvent concentrations selected should be kept constant in the
solvent control and all test solutions. The concentration of solvent in high-
est treatment level should be used in the solvent control.
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(v) The use of a solubility (saturation) column is permitted in the
preparation of stock solutions. This may help to ensure the aqueous solu-
bility limit is attained for poorly soluble test materials.

(vi) The pH should not be adjusted after the addition of the test chem-
ical or stock solution into dilution water.

(vii) The pH should be measured in each replicate at the beginning
of the test and every 24 h thereafter.

(viii) The pH must be monitored in low, medium, and high test con-
centrations and must remain > 6.0 and < 8.0 for freshwater testing and > 7.5
and < 8.5 for marine testing.

(ix) The pH may be adjusted in stock solutions to match the pH of
dilution water if pH change does not affect stability of compound in water.
HC1 and NaOH may be used for this adjustment if warranted.

(4) Selection of test species—(i) Test species. One or more of the
following species may be used:

(A) Freshwater species—Atlantic salmon, Salmo salar; bluegill sun-
fish, Lepomis macrochirus; brook trout, Salvelinus fontinalis; channel cat-
fish, Ictalurus punctatus\ coho salmon, Oncorhynchus kisutch; common
carp, Cyprinus carpio; fathead minnnow, Pimephales promelas; guppy,
Poecilia reticulata; rainbow trout, Oncorhynchus mykiss; red killifish,
Oryzias latipes; threespine stickleback, Gasterosteus aculeatus; and zebra-
fish, Brachydanio rerio.

(B) Saltwater species—Atlantic silverside, Menidia menidia; sheeps-
head minnow, Cyprinodon variegatus; and tidewater silverside, Menidia
penisulae.

(C) Data on both a warm and a cold freshwater species are generally
required. The preferred warm water species is the bluegill sunfish. The
rainbow trout is the preferred cold water species. When data on a marine
or estuarine species is desired, the Atlantic silversides is preferred.

(ii) Acclimation. (A) A minimum 12-day acclimation period is re-
quired with 14 days recommended. A minimum of 7 days of the acclima-
tion period must be performed in test dilution water.

(B) Holding water should come from the same source as the test dilu-
tion water, if not, acclimation to the dilution water should be done gradu-
ally over a 48-h period.

(D) No feeding is permitted within 48 h of test initiation.
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(E) Pretest mortality must be < 5 percent during acclimation. If pretest
mortality is >10 percent, then the entire batch must be rejected and a
new batch begun in acclimation.

(F) Any changes in water temperature should not exceed 3 °C per
day. Fish should be held for a minimum of 7 days at the test temperature
prior to testing.

(G) During the final 48 h of acclimation fish should be maintained
in facilities with background colors and light intensities similar to those
of testing area.

(iii) Age and size of test fish. (A) Juvenile fish must be tested. Juve-
nile fish <3.0 g should be used and the longest should not be more than
twice the length of the shortest. The fish should be of normal size and
appearance for their age. All fish must be of the same age.

(B) Wild caught fish may be used to satisfy testing guidelines if size,
age, and source requirements are satisfied. Wild caught fish should be
quarantined 7 days before acclimation procedures begin.

(C) Fish must originate from the same source and population. Records
should be kept regarding the source of the initial stock and/or culturing
techniques.

(D) Fish should not be used for a test if they appear stressed, or
if more than 5 percent die during the 48 h immediately prior to the test,
or if they were used in previous tests for treatments or controls.

(iv) Temperature. The recommended test temperatures are:
Species
Atlantic salmon
Atlantic silverside
Bluegill sunfish
Brook trout
Channel catfish
Coho salmon
Common carp
Fathead minnnow
Guppy
Rainbow trout
Red killifish
Sheepshead minnow ...
Threespine stickleback
Tidewater silverside
Zebra-fish
Temperature, °C

12 ±2.0
22 ±2.0
22±2.0
12 + 2.0
22 ±2.0
12 + 2.0
22 + 2.0
23 + 2.0
23 + 2.0
12 + 2.0
23 ±2.0
22 + 2.0
10+2.0
22±2.0
23+2.0
(v) Feeding. Feeding of test fish daily until 48 h prior to test initiation
is suggested.
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(f) Performance of the test—(1) Test design—(i) Test duration.
Acute testing must be performed for a minimum of 96 h.

(ii) Controls. Every test should include controls consisting of the
same dilution water, conditions, procedures, and test population, except
that no test substance is added. Solvent (carrier) controls are also required
if a solvent was used.

(iii) Introduction of fish. Fish should be added to test chambers with-
in 30 min of addition of the test material to dilution water. Fish may be
added prior to addition of test material. Fish should be introduced ran-
domly to individual replicates.

(iv) Number of test organisms. A minimum of seven fish per rep-
licate is required. The use of 10 fish per replicate is preferred to obtain
a more statistically accurate representation of the dose-response curve, to
allow for mortality which may occur, yet be unrelated to chemical effect,
and to avoid unnecessary repetitions of the test due to excessive control
mortality.

(v) Replicates. (A) Two replicates per test concentration are preferred
to avoid test repetition due to system failures, and to provide a stronger
statistical baseline.

(B) Each test chamber should contain an equal volume of test solution
and equal numbers of test fish. Replicate test chambers should be phys-
ically separated.

(vi) Loading. (A) The number of fish placed in each replicate should
not be so great as to affect the test results.

(B) In static or static-renewal tests, loading should not exceed
0.8 g (fresh weight) of fish per liter of test solution in a replicate at any
one time.

(C) In flow-through tests, loading should not exceed 0.5 g fresh
weight of fish (FWF) per liter of test solution passing through a replicate
within 24 h.

(vii) Test chambers and support equipment. (A) Construction ma-
terials and equipment that contact the stock solution, test solution, or dilu-
tion water should not contain substances that can be leached or dissolved
into aqueous solutions in quantities that can affect the test results. Mate-
rials and equipment that contact stock or test solutions should be chosen
to minimize sorption of test chemicals. Glass, no. 316 stainless steel, nylon
screen, and perfluorocarbon plastic (e.g. Teflon) are acceptable materials
and should be used whenever possible. Concrete, fiberglass, or plastic (e.g.
PVC) may be used for holding tanks, acclimation tanks, and water supply
systems, but they should be thoroughly conditioned before use. Rubber,
copper, brass, galvanized metal, epoxy glues, lead, and flexible tubing
-------
should not come in contact with the dilution water, stock solution, or test
solution.

(B) Test chambers should be loosely covered to reduce evaporation
and to minimize the entry of dust or other particulates into solutions and
to prevent loss of test fish.

(C) Size. Many different sizes of test chambers have been used suc-
cessfully. The size, shape, and depth of the test chamber is acceptable
if the specified flow rate and loading requirements can be achieved. Test
vessels must be of adequate size to maintain a load rate of
FWF > 0.8 g FWF/L for static or static-renewal tests, or
FWF > 0.5 g FWF/L for flow-through tests.

(D) Test substance delivery system. (1) In flow-through tests, propor-
tional diluters, metering pump systems, or other suitable systems should
be used to deliver the test chemical to the test chambers. The choice of
a specific delivery system depends on the specific properties and require-
ments of the test substance.

(2) The system should be calibrated before and after each test. Cali-
bration includes determining the flow rate and test concentration in each
replicate. The apparatus used should accurately and precisely deliver the
appropriate amount of stock solution and dilution water to each replicate.

(3) A closed flow-through system may be used to test volatile com-
pounds when more than 20 percent of the test substance would be lost
through volatility or the test substance would cause oxygen levels may
fall below 60 percent of the saturation level. A design description of this
type of system should be included in the study report.

(E) Aeration. Gentle aeration of test vessels used in static systems
during the exposure period is permitted only in cases where oxygen levels
are in danger of dropping below 60 percent saturation due to chemical
characteristics of the test material. Test concentrations must be measured
during the test if aeration is used. No aeration of actual test vessels may
be utilized in flow-through tests.

(viii) Light (A) The photoperiod with 15 to 30 min transition periods
is suggested. Photoperiods may range from 12D/12N to 16D/8N, where
D - day, and N = night.

(B) Light intensity should range from 30 to 100 1m at the water sur-
face; the intensity selected should be duplicated as closely as possible in
all replicates.

(ix) Temperature. (A) Temperatures must be recorded in all rep-
licates at the beginning of the test and every 24 h thereafter. The tempera-
ture should be recorded at least hourly in one replicate throughout the
-------
test. Temperature should vary no more then 1.0 °C in any given 24-h
period.

(B) The test system should be equipped with an automatic alarm sys-
tem to alert staff of temperature changes in excess of 2.0 °C.

(x) Dissolved oxygen. DO concentrations should be measured in each
replicate at the beginning of the test and every 24 h thereafter.

(xi) Feeding. Fish may not be fed during the treatment period.

(xii) Disturbances. Any disturbance which might change the behavior
of the test fish should be avoided.

(2) Test concentrations, (i) A minimum of five test concentrations
must be employed.

(ii) Five or more concentrations in a geometric series should be tested.
Test concentrations must be at least 50 percent greater than the next lowest
test concentration (not to exceed 120 percent). Range-finding studies prior
to testing may allow more accurate selection of test concentrations.

(iii) No more than 25 percent variation is allowed between test con-
centrations within the same treatment during the test.

(iv) Concentration selection. (A) Test concentrations should be se-
lected to produce a no-observable-effect concentration (NOEC) and, pref-
erably, at least two partial mortalities, i.e. one greater than and the other
less than 50 percent, after 96 h. The highest test concentration should not
exceed the chemical's aqueous solubility limit if the chemical is not a
surfactant or the chemical's self-dispersibility limit if the chemical is a
surfactant or a charged polymer.

(B) Exceptions may be required in testing certain pesticide AIs as
products. Product formulations may increase the solubility of the AI be-
yond its aqueous solubility limit.

(v) Concentration analysis. (A) Concentration analysis must be per-
formed at initiation and every 48 h of the study thereafter.

(B) In static tests, the test substance concentration should be measured
in each replicate minimally at the beginning (0-hour, before test organisms
are added), at 48 h, and at the end of the test.

(C) In static-renewal tests, the test substance should be measured in
each replicate at the beginning and end of test and just before and after
each renewal.

8
-------
(D) In flow-through tests, the test substance should be measured as
follows:

(/) In each replicate at 0, 48, and 96 h, and every 96 h thereafter,
as long as the test is continued.

(2) In at least one appropriate chamber whenever a malfunction is
detected in any part of the test substance delivery system.

(3) Collection of samples for measurement, (i) Water samples must
be removed from a central point within the test vessel, not from inflow
or outflow points.

(ii) These samples should not contain any surface particulates or ma-
terial dislodged from the bottom or sides. Samples should be analyzed
immediately, or handled and stored in a manner which minimizes loss of
test substance through microbial degradation, photodegradation, chemical
reaction, volatilization, or sorption.

(iii) The test solution volume should not be reduced during the test
by more than 10 percent as a result of sampling.

(iv) Samples from each test concentration replicate should not be
pooled for analyses.

(v) Diluter systems must be monitored for proper adjustment, and
operation every 24 h, and should be monitored during the first hour of
operation.

(vi) Surface films and precipitates must be reported should they occur.

(vii) The flow rate to each replicate should be measured at the begin-
ning and end of each test.

(viii) During a test, the flow rates should not vary more than a factor
of 10 from any one replicate to another.

(ix) Minimum number of test vessel replacements should be 6 to 10
per 24-h period for flow-through testing.

(4) Observations, (i) Mortality observations should be recorded at
6, 24,48, 72, and 96 h.

(ii) If the test is continued past 96 h, additional observations should
be made every 24 h until termination.

(iii) In addition to mortality, any abnormal behavior should be re-
corded, such as, but not limited to, erratic swimming, loss of reflex, in-
creased excitability, lethargy, and changes in appearance or physiology
such as discoloration, excessive mucous production, hyperventilation,
opaque eyes, curved spine, or hemorrhaging.
-------
(g) Data and reporting—(1) Treatment of results. The cumulative
percentage mortality for each exposure period is plotted against concentra-
tion on logarithmic paper. Normal statistical procedures are then employed
to calculate the LC50 for the appropriate exposure period. Confidence lim-
its (CI) with p = 0.95 for the calculated LC50 values are to be included.

(2) Test report, (i) The test report must include the following:

(ii) Test facilities, test dates, and personnel must be reported.

(iii) Identification of the test substance and purity.

(iv) Water quality characteristics as reported in the laboratory records
for the study. These must include 24-h records of DO, pH, and tempera-
ture.

(v) Methods of stock solution preparation and the concentrations used
in definitive testing.

(vi) All test concentrations measured during the test and at termi-
nation.

(vii) The number of test organisms in each replicate and/or test con-
centration.

(viii) The LC50 concentration-response curves, LC50 values, and as-
sociated 95 percent CI should be determined for 24, 48, 72 and 96 h,
whenever sufficient data exist.

(ix) A graph of the concentration-mortality curve at test termination.
Any control mortality observed during the acclimation or study period.

(x) An NOEL for the 96-h test should also be reported.

(xi) If no LC50 value is determined, but it can be demonstrated that
the concentrations tested were the highest possible due to the test chemi-
cal's aqueous solubility limit, self-dispersibility limit, or other physico-
chemical limitations, then the data will be considered for acceptance. Ex-
planation should include details of the solvents which were tried prior to
initiation of the final study.

(xii) Any abnormal behavior displayed by the test fish.

(xiii) Any protocol deviations or occurrences which may have influ-
enced the final results of the test.

(xiv) A quality control methods and quality assurance statement
should accompany all final study reports.

(xv) Raw data must be available to support study author's conclusions
and should be presented with the study report.

10
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(xvi) Methods of statistical analysis should be reported.

(xvii) Methods used in analysis of test concentrations of the test
chemical should be described. The accuracy of the method (i.e. detection
limit and quantification limit) should be given.

(h) References. The following references should be consulted for ad-
ditional background material on this test guideline.

(1) Standard Guide for Conducting Acute Toxicity Tests with Fishes,
Macroinvertebrates, and Amphibians, E 729-88a. American Society Test-
ing Materials, Philadelphia, PA. Approved Nov. 21, 1988.

(2) Organization of Economic Cooperation and Development, Guide-
lines for Testing of Chemicals, Guideline 203 "Fish Acute Toxicity Test."
Adopted July 17, 1992.

(3) Test Guideline EG-9, Fish Acute Toxicity Test, Office of Pollu-
tion Prevention and Toxics, Office of Prevention, Pesticides and Toxic
Substances, U.S. Environmental Protection Agency, Washington DC.

(4) Standard Evaluation Procedure Acute Toxicity Test for Freshwater
Fish, EPA-540/9-85-006, Office of Pesticide Programs, Office of Preven-
tion Pesticides and Toxic Substances, U.S. Environmental Protection
Agency, Washington DC. Revised June 1985.

(5) Acute Toxicity Test for Estuarine and Marine Organisms (Estua-
rine Fish 96-Hour Acute Toxicity Test), EPA 540/9-85-009, Office of
Pesticide Programs, Office of Prevention, Pesticides, and Toxic Sub-
stances, U.S. Environmental Protection Agency, Washington DC. Revised
June 1985.

(6) Federal Insecticide, Fungicide, Rodenticide Act, Subdivision E,
Hazard Evaluation, Wildlife and Aquatic Organisms, U.S. Environmental
Protection Agency. October 1982.

(7) Finney, D.J., Probit Analysis. 3rd Edition. Cambridge University
Press: London and New York (1971).

(8) Stephen, C.E., "Methods for Calculating an LC50" Aquatic Toxi-
cology and Hazard Evaluation, ASTM STP 634, American Society of Test-
ing and Materials, Philadelphia, PA (1977).

(9) Canada, Environment Canada. Biological test method: acute
lethality test using threespine stickleback {Gasterosteus aculeatus). Envi-
ronmental Protection, Conservation and Protection, Environment Canada,
Report EPS l/RM/10 (1990).
11
-------
-------
&EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-9&-117
April 1996
Ecological Effects Test
Guidelines

OPPTS 850.1085
Fish Acute Toxicity
Mitigated by Humic Acid
"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.

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 850.1085 Fish acute toxicity mitigated by humic acid.
(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 797.1460 Fish Acute Toxicity
Mitigated by Humic Acid.

(b) Purpose. This guideline may be used to develop data on the acute
toxicity of chemical substances and mixtures under static or static renewal
conditions, subject to environmental effects testing. This guideline pre-
scribes procedures to be used to develop data on the acute toxicity of
chemicals to fish with and without the presence of naturally occurring dis-
solved organic substances (.e.g., humic acids and their salts). EPA will
use data from these tests in assessing the hazard of a chemical to the envi-
ronment. For additional background information on this test guideline see
OPPTS 850.1075.

(c) Definitions. In addition to the definitions in section 3 of the Toxic
Substances Control Act (TSCA), and the definitions in 40 CFR Part 792—
Good Laboratory Practice Standards, the following definitions also apply
to this test guideline:

Acclimation means the physiological compensation by test organisms
to new environmental conditions (e.g. temperature, hardness, pH).

Acute toxicity test means a method used to determine the concentra-
tion of a substance that produces a toxic effect on a specified percentage
of test organisms in a short period of time (e.g. 96 h). In this guideline,
death is used as the measure of toxicity under static or static renewal con-
ditions only.

Carrier means a solvent used to dissolve a test substance prior to
delivery to the test chamber.

Death means the lack of opercular movement by a test fish.

Dissolved organic carbon (DOC) means various organic molecules
occurring in lotic and lentic ecosystems, which in this test are restricted
to a heterogeneous group of humic substances.

Total organic carbon (TOC) means the sum of all organic carbon
molecules, which are dissolved, particulate, and suspended, occurring in
test dilution waters.

Humic substances means humic acids (HAs), fulvic acids, and humin
fractions, and their various salts, resulting from chemical fractionation of
this heterogeneous naturally-occurring organic substance. For purposes of
-------
this test, HA, sodium salt (e.g. Aldrich Catalog No. HI,675-2; mention
of a commercial company or product does not constitute approval or en-
dorsement by the Agency) may be used as the source of DOC.

LC50 means that the test substance concentration calculated from ex-
perimentally-derived mortality data is lethal to 50 percent of a test popu-
lation during continuous exposure over a specified period of time.

Loading means the ratio of fish biomass (in grams, wet weight) to
the volume (in liters) of test solution in a test chamber or passing through
it in a 24-h period.

Static means the test solution is not renewed during the period of
the test.

Test solution means the dilution water containing the dissolved test
substance to which test organisms are exposed.

(d) Test procedures—(1) Summary of the test, (i) This test is de-
signed to determine the acute effects of the test substance on one of three
species offish with HA. Test chambers are filled with appropriate volumes
of dilution water.

(ii) The test substance is introduced into each test chamber. Some
test chambers contain only dilution water; other contain a concentration
of spiked HA.

(iii) Test fish which have been acclimated in accordance with the
test design are introduced into the test and control chambers by stratified
random assignment.

(iv) Fish in the test and control chambers are observed periodically
during the test; dead fish are removed at least twice each day and the
findings are recorded.

(v) The dissolved oxygen (DO) concentration, pH, and temperature
are measured at intervals in selected test chambers.

(vi) A concentration-response curve and LC50 value for the test sub-
stance in dilution water spiked with a known amount of HA are developed
from the mortality data collected during the test.

(2) Range-finding test, (i) If the toxicity of the test substance in HA
is not already known, a range-finding test should be performed to deter-
mine the range of concentrations to be used in the definitive test. The
highest concentration of test substance for use in the range-finding test
should not exceed its solubility in water or the permissible amount of car-
rier used.

(ii) Initially, two fish test is performed at 20 mg/L of HA. In some
cases, the 20 mg HA/L concentration may be so high that no toxicity will
-------
be present due to the formation of viscous, colloidal complexes. If this
occurs, the 20 mg HA/L concentration should be decreased to 15 mg/L,
or an appropriately lower concentration.

(3) Definitive test, (i) A minimum of 20 fish should be exposed to
each of five or more test substance concentrations in dilution water spiked
with a known amount of HA. The range of test substance concentrations
to which the fish are exposed should be such that in 96 h there are at
least two partial mortality exposures bracketing 50 percent survival.

(ii) For exposure to each concentration of a test substance in dilution
water spiked with a known amount of HA, an equal number of test fish
should be placed in two or more replicate test chambers. Test fish should
be impartially distributed among test chambers in such a manner that test
results show no significant bias from the distributions.

(iii) Every test should include a control consisting of the same dilution
water, conditions, procedures, and fish from the same group used in the
test, except that none of the test substance is added. Every test should
also include negative controls consisting of dilution water with HA alone.

(iv) Mortality data collected during the test are used to calculate a
96-h LC50 value. The 24-, 48-, and 72-h values should be calculated
whenever there is sufficient mortality data to determine such values.

(v) Test fish should not be fed while they are being exposed to the
test substance under static conditions.

(4) Test results, (i) Death is the primary criterion used in this test
guideline to evaluate the toxicity of the test substances on the presence
of a known amount of HA.

(ii) In addition to death, any abnormal behavior such as, but not lim-
ited to, erratic swimming, loss of reflex, increased excitability, lethargy,
or any changes in appearance of physiology, such as discoloration, exces-
sive mucous production, hyperventilation, opaque eyes, curved spine, or
hemorrhaging should be recorded.

(iii) Observations on compound solubility and/or dispersibility should
be recorded. The investigator should report the appearance of surface
slicks, precipitates, or material adhering to the sides of the test chamber.

(iv) Each test and control chamber should be checked for dead fish
and observations recorded at 24, 48, 72, and 96 h after the beginning of
the test or within 1 h of the designated times. If the test is continued
past 96 h, additional observations should be made every 24 h until termi-
nation.

(v) The mortality data are used to calculate LC50 values and their
95 percent confidence limits, and to plot concentration-response curves
-------
for each time interval whenever sufficient data exists. The methods rec-
ommended for use in calculating LC50 values include probit, logit, bino-
mial, and moving average angle.

(vi) A test is be unacceptable if more than 10 percent of the control
fish die or exhibit abnormal behavior during a 96-h test.

(5) Analytical measurements—(i) Water quality analysis. (A) The
hardness, acidity, alkalinity, pH, conductivity, TOC, or chemical oxygen
demand (COD), and total suspended solids (TSS) of the dilution water
should be measured at the beginning of each static test. The month-to-
month variation of the above values should be less than 10 percent and
the pH should vary less than 0.4 units.

(B) During static tests, the DO concentration, temperature, and pH
should be measured in each test chamber at the beginning and end of
the test. The test solution volume should not be reduced by more than
10 percent as a result of these measurements.

(ti) Dissolved organic carbon. The naturally-occurring DOC selected
for this test should be HA, which is available from the Aldrich catalog,
(No. HI,675-2).

(iii) Collection of samples for measurement of TOC. Samples to
be analyzed for TOC should be taken from the control chambers midway
between the top, bottom, and sides of the test chamber. These samples
should not include any surface scum or material dislodged from the bottom
or sides.

(iv) Measurement of TOC. (A) For static tests, DOC should be
measured (as TOC) at a minimum in each test chamber at the beginning
(time 0, before fish are added) of the test. Three TOC measurements
should be made and the average reported.

(B) The analytical methods used to measure the TOC in a sample
should be validated before beginning the test. The accuracy of a method
should be verified by a method such as using known additions. This in-
volves adding a known amount of the dissolved organic carbon source
to three water samples taken from a chamber containing dilution water
to be used in the test. The normal concentration of dissolved organic car-
bon in those samples should span the TOC concentration range to be used
in the test.

(C) The nominal concentration of test substance based on 100 percent
active ingredient (AI) should be used to calculate all LC50 values and
to plot all concentration-response curves.

(e) Test conditions—(1) Test species— (i) Selection. The test spe-
cies for this test are the rainbow trout (Oncorhynchus mykiss = Salmo
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gairdneri), bluegill (Lepomis macrochirus), and fathead minnow
(Pimephales promelas).

(ii) Age and condition of fish. Juvenile fish should be used. Fish
used in a particular test should be the same age and be of normal size
and appearance for their age. The longest fish should not be more than
twice the length of the shortest. All newly acquired fish should be quar-
antine and observed for at least 14 days prior to use in a test. Fish should
not be used for a test if they appear stressed or if more than 5 percent
die during the 48 h immediately prior to the test.

(iii) Acclimation of test fish. If the holding water is not from the
same source as the test dilution water, acclimation to the dilution water
should be done gradually over a 48-h period. The fish should be held
an additional 14 days in the dilution water prior to testing. Any changes
in water temperature should not exceed 3 °C per day. Fish should be held
for a minimum of 7 days at the test temperature prior to testing. During
the final 48-h of acclimation, fish should be maintained in facilities with
background colors and light intensities similar to those of the testing area
and should not be fed.

(2) Facilities—(i) General. Facilities needed to perform this test in-
clude:

(A) Flow-through tanks for holding and acclimating fish.

(B) A mechanism for controlling and maintaining the water tempera-
ture during the holding, acclimation, and test periods.

(D) Apparatus for providing a 16-h light and 8-h dark photoperiod
with a 15- to 30-min transition period.

(E) Chambers for exposing test fish to the test substance.

(ii) Construction materials. Construction materials and commer-
cially purchased equipment that may contact the stock solution, test solu-
tion, or dilution water should not contain substances that can be leached
or dissolved into aqueous solutions in quantities that can alter the test re-
sults. Materials and equipment that contact stock or test solutions should
be chosen to minimize sorption of test chemicals. Glass, stainless steel,
and perfluorocarbon plastic should be used whenever possible. Concrete,
fiberglass, or plastic (e.g. PVC) may be used for holding tanks, acclimation
tanks, and water supply systems, but they should be thoroughly condi-
tioned before use. If cast iron pipe is used in freshwater supply systems,
colloidal iron may leach into the dilution water and strainers or filters
should be used to remove rust particles. Rubber, copper, brass, galvanized
-------
metal, epoxy glues, and lead should not come in contact with the dilution
water, stock solution, or test solution.

(iii) Test chambers. Test chambers made of stainless steel should
be welded, not soldered. Test chambers made of glass should be fused
or bonded using clear silicone adhesive. As little adhesive as possible
should be left exposed in the interior of the chamber.

(iv) Cleaning of test system. Test chambers should be cleaned before
each test. They should be washed with detergent and rinsed in sequence
with clean water, pesticide-free acetone, clean water, and 5 percent nitric
acid, followed by two or more changes of dilution water.

(v) Dilution water. (A) Clean surface or ground water, reconstituted
water, or dechlorinated tap water is acceptable as dilution water if the
the test fish will survive in it for the duration of the holding, acclimating,
and testing periods without showing signs of stress, such as discoloration,
hemorrhaging, disorientation, or other unusual behavior. The quality of
the clean dilution water (without spiked HA) should be constant and
should meet the specifications in the following Table 1., measured at least
twice a year:
Table 1.—Specifications for Dilution Water
Substance
Maximum Concentration
Total suspended solids
Total organic carbon (TOC), or chemical oxygen demand
(COD).
Un-ionized ammonia
Residual chlorine
Total organophosphorus pesticides
Total organochlorine pesticides plus polychlorinated biphenyls
(PCBs).or organic chlorine.
Hardness (as CaCO3 during testing)
20 mg/L
2 mg/L, or 5 mg/L, respectively

20fig/L
1 ^g/L
50 ng/L
50 ng/L, or 25 ng/L, respectively

180 mg/L
The quality of the dilution water after spiking with HA should meet all
the previous specifications except for TOC or COD.

(B) The DO concentration in the dilution water should be between
90 and 100 percent saturation; 9.8 to 10.9 mg/L for tests with trout, and
8.0 to 8.9 mg/L for tests with bluegill or fathead minnow at sea level.
If necessary, the dilution water can be aerated before the addition of the
test substance. All reconstituted water should be aerated before use.
Buffered soft water should be aerated before but not after the addition
of buffers.

(C) Diseased organisms present in the dilution water in sufficient
number to cause infection of the fish should be killed or removed by suit-
able equipment.
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(D) Glass-distilled or carbon-filtered deionized water with a con-
ductivity less than 1 fiS/cm is acceptable for use in making reconstituted
water. If the reconstituted water is prepared from a ground or surface water
source, conductivity and TOC should be measured on each batch.

(vi) Carriers. Only distilled water should be used in making stock
solutions of the test substance. However, if the stock volume is more than
10 percent of the test solution volume, dilution water should be used. Car-
bon-based carriers cannot be used in this test. If necessary, stock solution
pH should be adjusted to pH 7.

(3) Test parameters—(i) Loading. The number of fish placed in a
test chamber should not be so great as to affect the results of the test.
The loading should not be so great that the test substance concentrations
are decreased by more than 20 percent due to uptake by the fish. Loading
should not exceed 0.5 g of fish/L of solution in the test chamber at any
one time. These loading rates should be sufficient to maintain the DO
concentration above the recommended levels and the ammonia concentra-
tion below 20 Hg/L.

(ii) Dissolved oxygen concentration. During static tests with rainbow
trout, the DO should be maintained above 5.5 mg/L in each test chamber.
In tests with bluegill and fathead minnow, the DO should be greater than
4.5 mg/L in each test chamber.

(iii) Temperature. The test temperature should be 22 °C for bluegill
and fathead minnow, and 12 °C for rainbow trout. Deviations from the
test temperature should be no greater than ±2 °C. The temperature should
be measured at least hourly in one test chamber.

(iv) Light. A 16-h light and 8-h dark photoperiod should be main-
tained.

(f) Reporting. The sponsor should submit to the EPA all data devel-
oped by the test that are suggestive or predictive of toxicity. In addition
to the reporting requirements prescribed in 40 CFR Part 792—Good Lab-
oratory Practice Standards, the reported test data should include the follow-
ing:

(1) The source of the dilution water, a description of any pretreatment,
and the measured hardness, acidity, alkalinity, pH, conductivity, TOC,
COD, and total suspended solids.

(2) The source of the HA (e.g., batch number), as well as a complete
description and chemical characterization.

(3) A description of the test chambers, the depth and volume of solu-
tion in the chamber, and the specific way the test was begun (e.g., condi-
tioning and test substance additions).
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(4) Detailed information about the test fish, including the scientific
name and method of verification, average weight (grams, wet weight),
standard length, age, source, history, observed diseases, treatments and
mortalities, acclimation procedures, and food use.

(5) The number of replicates used, the number of organisms per rep-
licate, and the loading rate.

(6) The measured DO, pH, and temperature and the lighting regime.

(7) A description of preparation of the stock solution. If the pH of
the stock solution was adjusted, describe the adjustment.

(8) The concentrations of the dissolved organic carbon as TOC from
the HA control just before the start of the test, all triplicate measurements,
and average TOC values.

(9) Results from any range-finding tests performed at 20 mg/L of
HA.

(10) The number of dead and live tests organisms, the percentage
of organisms that died, and the number that showed any abnormal effects
in the control and in each test chamber at each observation period.

(11) The 96-h LC50, and when sufficient data have been generated,
the 24—, 48-, 72-h LC50 values, then- 95 percent confidence limits, and
the methods used to calculate the LC50 values and their confidence limits.

(12) When observed, the no-observed-effect-concentration (the high-
est concentration tested at which there were no mortalities, abnormal be-
havioral, or physiological effects) in treatments.

(13) The concentration-response curve at each observation period for
which LC50 values are calculated.

(14) Methods and data records of all chemical analyses of water qual-
ity parameters, TOC, including method validations and reagent blanks.
8
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oEPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-120
April 1996
Ecological Effects Test
Guidelines
OPPTS 850.1300
Daphnid Chronic
Toxicity Test
'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.

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 850.1300 Daphnid chronic toxicity test.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 797.1330 Daphnid Chronic
Toxicity Test; OPP 72^1 Fish Early Life-Stage and Aquatic Invertebrate
Life-Cycle Studies (Pesticide Assessment Guidelines, Subdivision E—-
Hazard Evaluation; Wildlife and Aquatic Organisms) EPA report 540/09-
82-024, 1982; and OECD 202, Daphnia sp. Acute Immobilisation Test
and Reproduction Test.

(a) Purpose. This guideline prescribes a chronic toxicity test in which
daphnids are exposed to a chemical either in a static-renewal or a flow-
through system. The Environmental Protection Agency will use data from
this test in assessing the hazard a chemical may present to the aquatic
environment. No preference is given in this guideline on the type of test
system to be used, either static-renewal or flow-through. However, the
former works well if individual daphnids need to be monitored during the
test. The latter works well with chemicals that are volatile, have low water
solubilities, and an oxygen demand, and for those that degrade, hydrolyze,
or photolyze easily. Flow-through systems allow maintenance of near con-
stant chemical concentrations throughout the test.

(b) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and the definitions in 40 CFR Part 792—Good Lab-
oratory Practice Standards apply to this test guideline. The following defi-
nitions also apply to this test guideline.

Brood stock means the animals which are cultured to produce test
organisms through reproduction.

Chronic toxicity test means a method used to determine the concentra-
tion of a substance in water that produces an adverse effect on a test orga-
nism over an extended period of time. In this test guideline, mortality and
reproduction (and optionally, growth) are the criteria of toxicity.

Ephippium means a resting egg which develops under the carapace
in response to stress conditions in daphnids.

1
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Flow-through means a continuous or intermittent passage of test solu-
tion or dilution water through a test chamber or culture tank with no recy-
cling.

Immobilization means the lack of movement by daphnids except for
minor activity of the appendages.

Loading means the ratio of daphnid biomass (grams, wet weight) to
the volume (liters) of test solution in a test chamber at a point in time
or passing through the test chamber during a specific interval.

LOEC (lowest observed effect concentration) means the lowest con-
centration of a material used hi this test that has an adverse effect on
the test organisms and is the test concentration immediately above the
NOEC.

MATC (maximum acceptable toxicant concentration) means the maxi-
mum concentration at which a chemical can be present and not be toxic
to the test organism.

NOEC (no observed effect concentration) means the highest con-
centration of a material used in this test that does not have an adverse
effect on the test organisms and is the test concentration immediately
below the LOEC.

Static-renewal system means the technique in which test organisms
are periodically transferred to fresh test solution of the same composition.

(c) Test procedures—(1) Summary of the test, (i) Test chambers
are filled with appropriate volumes of dilution water. In the flow-through
test the flow of dilution water through each chamber is then adjusted to
the rate desired. The test substance is introduced into each test chamber.
The addition of test substance in the flow-through system is done at a
rate which is sufficient to establish and maintain the desired concentration
of test substance in the test chamber.

(ii) The test is started within 30 min after the test substance has been
added and uniformly distributed in the test chambers in the static-renewal
test or after the concentration of test substance in each test chamber of
the flow-through test system reaches the prescribed level and remains sta-
ble. At the initiation of the test, daphnids which have been cultured or
acclimated in accordance with the test design, are randomly placed into
the test chambers. Daphnids in the test chambers are observed periodically
during the test, immobile adults and offspring produced are counted and
removed, and the findings are recorded. Dissolved oxygen concentration,
pH, temperature, the concentration of test substance, and other water qual-
ity parameters are measured at specified intervals in selected test cham-
bers. Data are collected during the test to determine any significant dif-
ferences (p < 0.05) in immobilization and reproduction as compared to the
-------
control. At the end of the test, the growth of surviving adults is measured
as the total body length or dry weight or both.

(2) Range-finding test, (i) A range-finding test should be conducted
to establish test solution concentrations for the definitive test.

(ii) The daphnids should be exposed to a series of widely spaced
concentrations of the test substance (e.g. 1, 10, 100 mg/L), usually under
static conditions.

(iii) A minimum of five daphnids should be exposed to each con-
centration of test substance for a period of time which allows estimation
of appropriate chronic test concentrations. No replicates are required and
nominal concentrations of the chemical are acceptable.

(3) Definitive test, (i) The purpose of the definitive test is to deter-
mine concentration-response curves, EC50 values, and effects of a chemi-
cal on immobilization and reproduction during chronic exposure.

(ii) A minimum of 10 daphnids per concentration should be exposed
to five or more concentrations of the chemical chosen in a geometric series
in which the ratio is between 1.5 and 2.0 (e.g. 2, 4, 8, 16, 32,
64 mg/L). In flow-through testing, an equal number of daphnids (minimum
of 20 per concentration) should be placed in two or more replicates or
test chambers, e.g. four replicates each with five daphnids, for each con-
centration. In static-renewal tests, 10 or more replicates of one daphnid
each, for each concentration, should be used. The concentration ranges
should be selected to determine the concentration-response curves, EC50
values, and MATC. Solutions should be analyzed for chemical concentra-
tion at designated times during the test.

(iii) Every test should include controls consisting of the same dilution
water, conditions, procedures and daphnids from the same population (cul-
ture container), except that none of the chemical is added.

(iv) The test duration is 21 days. The test is invalid and unacceptable
if any of the following occur:

(A) More than 20 percent of the control organisms appear to be im-
mobilized, stressed, or diseased during the test.

(B) Each control daphnid living the full 21 days produces an average
of less than 60 young.

(v) The number of immobilized daphnids in each chamber should be
recorded on day 21 of the test. After offspring are produced, they should
be counted and removed from the test chambers every 2 or 3 days. Con-
centration-response curves, EC50 values, and associated 95 percent con-
-------
fidence limits for adult immobilization should be determined for day 21.
An MATC should be determined for the most sensitive test criteria meas-
ured (number of adult animals immobilized, number of young per adult,
and number of immobilized young per adult).

(vi) Growth of daphnids is determined by measuring total body length
or dry weight, or both, of each surviving adult. It is preferred that both
measures be taken.

(vii) In addition, any abnormal behavior or appearance should also
be reported.

(viii) Test organisms should be impartially distributed among test
chambers in such a manner that test results show no significant bias from
the distributions. In addition, test chambers within the testing area should
be positioned in a random manner as in a way in which appropriate statis-
tical analyses can be used to determine the variation due to placement.

(4) Analytical measurements—(i) Test chemical. Deionized water
should be used in making stock solutions of the test substance. Standard
analytical methods should be used whenever available in performing the
analyses. The analytical method used to measure the amount of test sub-
stance in a sample should be validated before beginning the test by appro-
priate laboratory practices. An analytical method is not acceptable if likely
degradation products of the test substance, such as hydrolysis and oxida-
tion products, give positive or negative interferences which cannot be sys-
tematically identified and corrected mathematically.

(ii) Numerical. The number of immobilized adults, total offspring
per adult, and immobilized offspring per adult should be counted during
each test. Appropriate statistical analyses should provide a goodness-of-
fit determination for the adult immobilization concentration-response
curves calculated on day 21. A 21-day EC50 based on adult immobiliza-
tion and corresponding 95 percent confidence intervals should also be cal-
culated. Appropriate statistical tests (e.g. analysis of variance, mean sepa-
ration test) should be used to test for significant chemical effects on chron-
ic test criteria (cumulative number of immobilized adults, cumulative num-
ber of offspring per adult and cumulative number of immobilized offspring
per adult) on day 21. An MATC should be calculated using these chronic
test criteria.

(d) Test conditions—(1) Test species—(i) Selection. (A) The
cladocerans, Daphnia magna or D. pulex, are the species to be used in
this test. Either species can be utilized for testing of a particular chemical.
The species identity of the test organisms should be verified using appro-
priate systematic keys.

(B) First instar daphnids, <24 h old, are to be used to start the test.

4
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(ii) Acquisition. (A) Daphnids to be used in chronic toxicity tests
should be cultured at the test facility. Records should be kept regarding
the source of the initial stock and culturing techniques. All organisms used
for a particular test should have originated from the same culture popu-
lation.

(B) Daphnids should not be used for a test if:

(1) Cultures contain ephippia.

(2) Adults in the cultures do not produce young before day 12.

(3) More than 20 percent of the culture stock die in the 2 days preced-
ing the test.

(4) Adults in the culture do not produce an average of at least three
young per adult per day over the 7-day period prior to the test.

(5) Daphnids have been used in any portion of a previous test either
in a treatment or in a control.

(iii) Feeding. (A) During the test the daphnids should be fed the same
diet and with the same frequency as that used for culturing and acclima-
tion. All treatments and controls should receive, as near as reasonably pos-
sible, the same ration of food on a per-animal basis.

(B) The food concentration depends on the type used. Food con-
centrations should be sufficient to support normal growth and development
and to allow for asexual (parthenogenic) reproduction. For automatic feed-
ing devices, a suggested rate is 5 to 7 mg food (either solids or algal
cells, dry weight) per liter of dilution water or test solution. For manual
once-a-day feeding, a suggested rate is 15 mg food (dry weight) per liter
of dilution water or test solution.

(iv) Loading. The number of test organisms placed in a test chamber
should not affect test results. Loading should not exceed 40 daphnids per
liter in the static-renewal system. In the flow-through test, loading limits
will vary depending on the flow rate of the dilution water. Loading should
not cause the dissolved oxygen concentration to fall below the rec-
ommended level.

(v) Care and handling of test organisms. (A) Daphnids should be
cultured in dilution water under similar environmental conditions to those
used in the test. A variety of foods has been demonstrated to be adequate
for daphnid culture. They include algae, yeasts, and a variety of mixtures.

(B) Organisms should be handled as little as possible. When handling
is necessary it should be done as gently, carefully, and quickly as possible.
During culturing and acclimation, daphnids should be observed carefully
for ephippia and other signs of stress, physical damage, and mortality.
-------
Dead and abnormal individuals should be discarded. Organisms that touch
dry surfaces or are dropped or injured during handling should be discarded.

(C) Smooth glass tubes (I.D.>5 mm) equipped with a rubber bulb
can be used for transferring daphnids with minimal culture media carry-
over.

(D) Care should be exercised to introduce the daphnids below the
surface of any solution in order not to trap air under the carapace.

(vi) Acclimation. (A) Brood daphnids should be maintained in 100
percent dilution water at the test temperature for at least 48 h prior to
the start of the test. This is easily accomplished by culturing them in dilu-
tion water at the test temperature. During acclimation, daphnids should
be fed the same food as will be used for the definitive test.

(B) During culturing and acclimation to the dilution water, daphnids
should be maintained in facilities with background colors and light inten-
sities similar to those of the testing area.

(2) Facilities—(i) General. (A) Facilities needed to perform this test
include:

(2) Containers for culturing and acclimating daphnids.

(2) A mechanism for controlling and maintaining the water tempera-
ture during the culturing, acclimation, and test periods.

(3) Apparatus for straining paniculate matter, removing gas bubbles,
or aerating the water when water supplies contain paniculate matter, gas
bubbles, or insufficient dissolved oxygen, respectively.

(4) An apparatus for providing a 16-h light and 8-h dark photoperiod.

(5) An apparatus to introduce food if continuous or intermittent feed-
ing is used.

((5) In addition, the flow-through test should contain appropriate test
chambers in which to expose daphnids to the test substance and an appro-
priate test substance delivery system.

(B) Facilities should be well ventilated and free of fumes and other
disturbances that may affect the test organisms.

(ii) Test chambers. (A) Materials and equipment that contact test
solutions should be chosen to minimize sorption of test chemicals from
the dilution water and should not contain substances that can be leached
into aqueous solution in quantities that can affect test results.

(B) For static-renewal tests, daphnids can be conveniently exposed
to the test solution in 250-mL beakers or other suitable containers.
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(C) For flow-through tests daphnids can be exposed in glass or stain-
less steel containers with stainless steel or nylon screen bottoms. Such
containers should be suspended in the test chamber in such a manner to
ensure that the test solution flows regularly into and out of the container
and that the daphnids are always submerged in at least 5 cm of test solu-
tion. Test chambers can be constructed using 250-mL beakers or other
suitable containers equipped with screened overflow holes, standpipes, or
V-shaped notches.

(D) Test chambers should be loosely covered to reduce the loss of
test solution or dilution water due to evaporation and to minimize the entry
of dust or other particulates into the solutions.

(iii) Test substance delivery system. (A) In the flow-through test,
proportional diluters, metering pump systems, or other suitable systems
should be used to deliver the test substance to the test chambers.

(B) The test substance delivery system should be calibrated before
each test. Calibration includes determining the flow rate through each
chamber and the concentration of the test substance in each chamber. The
general operation of the test substance delivery system should be checked
twice daily during a test. The 24-h flow rate through a test chamber should
be equal to at least 5x the volume of the test chamber. During a test,
the flow rates should not vary more than 10 percent from any one test
chamber to another. For the static-renewal test, test substance dilution
water should be completely replaced at least once every 3 days.

(iv) Dilution water. (A) Surface or ground water, reconstituted water,
or dechlorinated tap water are acceptable as dilution water if daphnids
will survive in it for the duration of the culturing, acclimation, and testing
periods without showing signs of stress. The quality of the dilution water
should be constant and should meet the specifications in the following
Table 1.:
Table 1.—Specifications for Dilution Water
Substance
Particulate matter
Total organic carbon or:
Un-ionized ammonia
Residual chlorine
Chemical oxygen demand
Total organophosphorus pesticides
Total organochlorine pesticides plus polychlorinated biphenyls (PCBs)
or:
Organic chlorine
20 mg/L
2mg/L
5 mg/L
20 ng/L
< 3 \ig/L
50 ng/L

50 ng/L
25 ng/L
(B) The water quality characteristics listed above should be measured
at least twice a year or when it is suspected that these characteristics may
-------
have changed significantly. If dechlorinated tap water is used, daily chlo-
rine analysis should be performed.

(C) If the diluent water is from a ground or surface water source,
conductivity and total organic carbon (TOC) or chemical oxygen demand
(COD) should be measured. Reconstituted water can be made by adding
specific amounts of reagent-grade chemicals to deionized or distilled
water. Glass-distilled or carbon-filtered deionized water with a conductiv-
ity of less than 1 |iohm/cm is acceptable as the diluent for making reconsti-
tuted water.

(D) If the test substance is not soluble in water, an appropriate carrier
should be used at a concentration ^O.lmL/L. Triethylene glycol and di-
methyl formamide are preferred solvents, but ethanol or acetone can be
used if necessary.

(v) Cleaning of test system. All test equipment and test chambers
should be cleaned before each use following standard laboratory proce-
dures. Cleaning of test chambers may be necessary during the testing pe-
riod.

(3) Test parameters, (i) Environmental conditions of the water con-
tained in test chambers should be maintained as specified in this paragraph:

(A) The test temperature should be 20 °C. Excursions from the test
temperature should be no greater than ± 1 °C.

(B) Dissolved oxygen concentration between 60 and 105 percent satu-
ration. Aeration, if needed to achieve this level, should be done before
the addition of the test substance. All treatment and control chambers
should be given the same aeration treatment.

(ii) Additional measurements include:

(A) The concentration of the test substance in the chambers should
be measured during the test.

(B) At a minimum, the concentration of test substance should be
measured as follows:

(1) In each chamber before the test.

(2) In each chamber on days 7, 14, and 21 of the test.

(3) In at least one appropriate chamber whenever a malfunction is
detected in any part of the test substance delivery system. Equal aliquots
of test solution may be removed from each replicate chamber and pooled
for analysis. Among replicate test chambers of a treatment concentration,

8
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the measured concentration of the test substance should not vary more
than 20 percent.

(C) The dissolved oxygen concentration, temperature, and pH should
be measured at the beginning of the test and on days 7, 14, and 21 in
at least two chambers of the high, middle, low, and control test concentra-
tions.

(e) Reporting. The sponsor should submit to the EPA all data devel-
oped by the test that are suggestive or predictive of chronic toxicity and
all associated toxicologic manifestations. In addition to the reporting re-
quirements prescribed under Good Laboratory Practice Standards, 40 CFR
part 792, subpart J, the reporting of test data should include the following:

(1) The name of the test, sponsor, testing laboratory, study director,
principal investigator, and dates of testing.

(2) A detailed description of the test substance including its source,
lot number, composition (identity and concentration of major ingredients,
percent active ingredient, and major impurities), known physical and
chemical properties, and any carriers or other additives used and their con-
centrations.

(3) The source of the dilution water, its chemical characteristics (e.g.
conductivity, hardness, pH), and a description of any pretreatment.

(4) Detailed information about the daphnids used as brood stock, in-
cluding the scientific name and method of verification, age, source, treat-
ments, feeding history, acclimation procedures, and culture methods. The
age of the daphnids used in the test should be reported.

(5) A description of the test chambers, the volume of solution in the
chambers, the way the test was begun (e.g. conditioning, test substance
additions), the number of test organisms per test chamber, the number of
replicates per treatment, the lighting, the static-renewal process and sched-
ule for the static-renewal chronic test, the test substance delivery system
and flow rate expressed as volume additions per 24 h for the flow-dirough
chronic test, and the method of feeding (manual or continuous), and type
of food.

(6) The concentration of the test substance in test chambers at times
designated for static-renewal and flow-through tests.

(7) The number and percentage of organisms that show any adverse
effect in each test chamber at each observation period.

(8) The cumulative adult and offspring immobilization values and the
progeny produced at designated observation times, the time (days) to first
brood, the number of offspring per adult in the control replicates and in
-------
each treatment replicate, and the growth of surviving adults measured as
total length or dry weight or both.

(9) All chemical analyses of water quality and test substance con-
centrations, including methods, method validations, and reagent blanks.

(10) The data records of the culture, acclimation, and test tempera-
tures.

(11) Any deviation from this test guideline, and anything unusual
about the test, e.g. dilution failure, temperature fluctuations.

(12) The MATC to be reported is calculated as the geometric mean
between the lowest measured test substance concentration that had a sig-
nificant (p < 0.05) effect (LOEC) and the highest measured test substance
concentration that had no significant (p<0.05) effect (NOEC) on day 21
of the test. The most sensitive of the test criteria (number of adult animals
immobilized, the number of young per surviving female, the number of
immobilized young per female, and the growth of surviving females) is
used to calculate the MATC. The criterion selected for MATC computa-
tion is the one which exhibits an effect (a statistically significant difference
between treatment and control groups; p < 0.05) at the lowest test substance
concentration for the shortest period of exposure. Appropriate statistical
tests (analysis of variance, mean separation test) should be used to test
for significant test substance effects. The statistical tests employed and
the results of these tests should be reported.

(13) Concentration-response curves utilizing the average measured
test substance concentration should be fitted to cumulative adult immo-
bilization data at 21 days. A statistical test of goodness-of-fit should be
performed and the results reported.

(14) An EC50 value based on adult immobilization with correspond-
ing 95 percent confidence limits when sufficient data are present for day
21. These calculations should be made using the average measured con-
centration of the test substance.
10
-------
&EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-120
April 1996
Ecological Effects Test
Guidelines

OPPTS 850.1350
Mysid Chronic Toxicity
Test
"Public Draft"
-------
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.

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."
-------
OPPTS 850.1350 Mysid chronic toxicity test.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 797.1950 Mysid Shrimp
Chronic Toxicity Test; OPP 72-4 Fish Early Life-Stage and Aquatic Inver-
tebrate Life-Cycle Studies (Pesticide Assessment Guidelines, Subdivision
E—Hazard Evaluation; Wildlife and Aquatic Organisms) EPA report 5407
09-82-024, 1982; and OECD 202 Daphnia sp. Acute Immobilisation Test
and Reproduction Test.

(b) Purpose. This guideline prescribes tests using mysids as test orga-
nisms to develop data on the chronic toxicity of chemicals. The Environ-
mental Protection Agency will use data from these tests in assessing the
hazard of a chemical to the aquatic environment.

(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and in 40 CFR Part 792—Good Laboratory Practice
Standards apply to this test guideline. The following definitions also apply
to this guideline:

Chronic toxicity test is a method used to determine the concentration
of a substance that produces an adverse effect from prolonged exposure
of an organism to that substance. In this test, mortality, number of young
per female, and growth are used as measures of chronic toxicity.

Death is the lack of reaction of a test organism to gentle prodding.

Flow-through is a continuous or an intermittent passage of test solu-
tion or dilution water through a test chamber or a holding or acclimation
tank, with no recycling.

Gl (Generation 1) are those mysids which are used to begin the test,
also referred to as adults; G2 (Generation 2) are the young produced by
Gl.

LC50 is the experimentally derived concentration of test substance
that is calculated to kill 50 percent of a test population during continuous
exposure over a specified period of time.

Loading is the ratio of test organism biomass (gram, wet weight) to
the volume (liters) of test solution in a test chamber.

MATC (maximum-acceptable-toxicant-concentration) is the maximum
concentration at which a chemical can be present and not be toxic to the
test organism.

1
-------
Retention chamber is a structure within a flow-through test chamber
which confines the test organisms, facilitating observation of test orga-
nisms and eliminating washout from test chambers.

(d) Test procedures—(1) Summary of the test, (i) In preparation
for the test, the flow of test solution through each chamber is adjusted
to the rate desired. The test substance is introduced into each test chamber.
The rate at which the test substance is added is adjusted to establish and
maintain the desired concentration of test substance in each test chamber.
The test is started by randomly introducing mysids acclimated in accord-
ance with the test design into retention chambers within the test and the
control chambers. Mysids in the test and control chambers are observed
periodically during the test, the dead mysids removed, and the findings
reported.

(ii) Dissolved oxygen concentration (DOC), pH, temperature, salinity,
the concentration of test substance, and other water quality characteristics
are measured and recorded at specified intervals in selected test chambers.

(iii) Data collected during the test are used to develop an MATC
and to quantify effects on specific chronic parameters.

(2) Range-finding test, (i) A range-finding test should be conducted
to establish test solution concentrations for the definitive test.

(ii) The mysids should be exposed to a series of widely spaced con-
centrations of the test substance (e.g. 1, 10, 100 mg/L), usually under static
conditions.

(iii) A minimum of 10 mysids should be exposed to each concentra-
tion of test substance for a period of time which allows estimation of ap-
propriate chronic test concentrations. No replicates are required and nomi-
nal concentrations of the test substance are acceptable.

(3) Definitive test, (i) The purpose of the definitive test is to deter-
mine concentration-response curves, LC50 values, and effects of a chemi-
cal on growth and reproduction during chronic exposure.

(ii) A minimum of 40 mysids per concentration should be exposed
to five or more concentrations of the test chemical chosen in a geometric
series in which the ratio is between 1.5 and 2.0 (e.g. 2, 4, 8, 16, 32, and
64 mg/L). Mysids should be physically separated into replicate groups of
no more than eight individuals when most of the mysids reach sexual ma-
turity (usually 10-14 days after the beginning of the test). If solvents, solu-
bilizing agents, or emulsifiers have to be used, they should be commonly
used carriers and should not possess a synergistic or antagonistic effect
on the toxicity of the test substance. The concentration of solvent should
not exceed 0.1 mL/L. The concentration ranges should be selected to deter-
mine the concentration response curves, LC50 values, and MATC. Con-
-------
centration of test substance in test solutions should be determined prior
to use.

(iii) Every test should include controls consisting of the same dilution
water, conditions, procedures, and mysids from the same population or
culture container, except that none of the test chemical is added.

(iv) The DOC, temperature, salinity, and pH should be measured
weekly in each chamber.

(v) The test duration is 28 days. The test is unacceptable if more
than 25 percent of first generation females in the control groups fail to
produce young or if the average number of young produced per female
in the controls is less than three per day. The number of dead mysids
in each chamber should be recorded on days 7, 14, 21, and 28 of the
test. The number of male and female mysids in each test chamber should
be recorded at the time when sexual characteristics become discernible.
This generally occurs after 10-12 days in the control, but may be delayed
in those mysids exposed to the test substance. Females are identified by
the presence of a ventral brood pouch. Body length (as measured by total
midline body length, from the anterior tip of the carapace to the posterior
margin of the uropod) should be recorded for males and females at the
time when sex can be determined simultaneously for all mysids in control
and treatment groups. This time cannot be specified because of possible
delays in sexual maturation of mysids exposed to test substances. A second
observation of male and female body lengths should be conducted on day
28 of the test. To reduce stress on the mysids, body lengths can be re-
corded by photography through a stereomicroscope with appropriate scal-
ing information. As offspring are produced by the Gl mysids (approxi-
mately 13 to 16 days in controls), die young should be counted and sepa-
rated into retention chambers at the same test substance concentration as
the chambers where they originated. If available prior to termination of
the test, observations on the mortality, number of males and females and
male and female body length should be recorded for the G2 mysids. Con-
centration-response curves, LC50 values and associated 95 percent con-
fidence limits for the number of dead mysids (Gl) should be determined
for days 7, 14, 21, and 28. An MATC should be determined for the most
sensitive test criteria measured (cumulative mortality of adult mysids,
number of young per female, and body lengths of adult males and fe-
males).

(vi) In addition to death, any abnormal behavior or appearance should
also be reported.

(vii) Test organisms should be impartially distributed among test
chambers in such a manner that test results show no significant bias from
the distributions. In addition, test chambers within the testing area should
-------
be positioned in a random manner or in a way in which appropriate statis-
tical analyses can be used to determined the variation due to placement.

(viii) The concentration of the test substance in the chambers should
be measured as often as is feasible during the test. The measured con-
centration of the test substance should not vary more than 20 percent
among replicate test chambers of a treatment concentration. The concentra-
tion of test substance should be measured:

(A) At each test concentration at the beginning of the test and on
days?, 14, 21, and 28.

(B) In at least one appropriate chamber whenever a malfunction is
detected in any part of the test substance delivery system.

(4) Analytical measurements—(i) Test chemical. Deionized water
should be used in making stock solutions of the test substance. Standard
analytical methods should be employed whenever available in performing
the analyses. The analytical method used to measure the amount of test
substance in a sample should be validated before beginning the test by
appropriate laboratory practices. An analytical method is not acceptable
if likely degradation products of the test substance, such as hydrolysis and
oxidation products, give positive or negative interferences which cannot
be systematically identified and corrected mathematically.

(ii) Numerical. (A) The number of dead mysids, cumulative young
per female, and body lengths of male and female mysids should be re-
corded during each definitive test. Appropriate statistical analyses should
provide a goodness-of-fit determination for the day-7, -14, -21, and -28
adult (Gl) death concentration-response curves.

(B) A 7- 14-, 21- and 28-day LC50, based on adult (Gl) death,
and corresponding 95 percent confidence intervals should be calculated.
Appropriate statistical tests (e.g. analysis of variance, mean separation test)
should be used to test for significant chemical effects on chronic test cri-
teria (cumulative mortality of adults, cumulative number of young per fe-
male, and body lengths of adult male and females) on designated days.
An MATC should be calculated using these chronic tests criteria.

(e) Test conditions—(1) Test species—(i) Selection. (A) The mysid
Mysidopsis bahia, is the organism specified for these tests. Juvenile
mysids, <24-h old, are to be used to start the test. It has recently been
proposed, under paragraph (g)(2) of this guideline, to place this species
in a new genus, Americamysis.

(B) Mysids to be used in chronic toxicity tests should originate from
laboratory cultures in order to ensure the individuals are of similar age
and experimental history. Mysids used for establishing laboratory cultures
may be purchased commercially or collected from appropriate natural
-------
areas. Because of similarities with other mysid species, taxonomic verifica-
tion should be obtained from the commercial supplier, by experienced lab-
oratory personnel, or by an outside expert.

(D) Mysids should not be used for a test if they exhibit abnormal
behavior, or if they have been used in a previous test, either in a treatment
or in a control group.

(ii) Acclimation. (A) Any change in the temperature and chemistry
of the water used for holding or culturing the test organisms to those of
the test should be gradual. Within a 24-h period, changes in water tem-
perature should not exceed 1 °C, while salinity changes should not exceed
5 percent.

(B) During acclimation mysids should be maintained in facilities with
background colors and light intensities similar to those of the testing areas.

(iii) Care and handling. Methods for the care and handling of mysids
such as those described in paragraph (g)(l) of this guideline can be used
during holding, culturing, and testing periods.

(iv) Feeding. Mysids should be fed during testing. Any food utilized
should support survival, growth, and reproduction of the mysids. A rec-
ommended food is live Artemia spp. nauplii (approximately 48-h old).

(2) Facilities—(i) Apparatus. (A) Facilities which may be needed
to perform this test include:

(/) Flow-through or recirculating tanks for holding and acclimating
mysids.

(2) A mechanism for controlling and maintaining the water tempera-
ture during the holding, acclimation, and test periods.

(3) Apparatus for straining particulate matter, removing gas bubbles,
or aerating the water, as necessary.

(4) An apparatus for providing a 14-h light and 10-h dark
photoperiod with a 15- to 30-min transition period. In addition, flow-
through chambers and a test substance delivery system are required. It
is recommended that mysids be held in retention chambers within test
chambers to facilitate observations and eliminate loss through outflow
water.

(B) Facilities should be well ventilated and free of fumes and disturb-
ances that may affect test organisms.
-------
(C) Test chambers should be loosely covered to reduce the loss of
test solution or dilution water due to evaporation and to minimize the entry
of dust or other particulates into the solutions.

(ii) Cleaning. Test substance delivery systems and test chambers
should be cleaned before each use following standard laboratory practices.

(iii) Construction materials. (A) Materials and equipment that con-
tact test solutions should be chosen to minimize sorption of test chemicals
from the dilution water and should not contain substances that can be
leached into aqueous solution in quantities that can affect the test results.

(B) Retention chambers utilized for confinement of test organisms
can be constructed with netting material of appropriate mesh size.

(iv) Dilution water. (A) Natural or artificial seawater is acceptable
as dilution water if mysids will survive and successfully reproduce in it
for the duration of the holding, acclimating, and testing periods without
showing signs of stress, such as reduced growth and fecundity. Mysids
should be cultured and tested in dilution water from the same origin.

(B) Natural seawater should be filtered through a filter with a pore
size of > 20 |U.m prior to use in a test.

(C) Artificial seawater can be prepared by adding commercially avail-
able formulations or by adding specific amounts of reagent-grade chemi-
cals to deionized or glass-distilled water. Deionized water with a con-
ductivity less than 0.1 mS/m at 12 °C is acceptable as the diluent for
making artificial seawater. When deionized water is prepared from a
ground or surface water source, conductivity and total organic carbon (or
chemical oxygen demand) should be measured on each batch.

(v) Test substance delivery system. Proportional diluters, metering
pumps, or other suitable systems should be used to deliver test substance
to the test chambers. The system used should be calibrated before each
test. Calibration includes determining the flow rate and the concentration
of the test substance in each chamber. The general operation of the test
substance delivery system should be checked twice daily during a test.
The 24-h flow rate through a chamber should be equal to at least 5x the
volume of the chamber. The flow rates should not vary more than 10 per-
cent among chambers or across time.

(3) Test parameters. Environmental parameters of the water con-
tained in test chambers should be maintained as specified below:

(i) The test temperature should be 25 °C. Excursions from the test
temperature should be no greater than ± 2 °C.

(ii) DOC between 60 and 105 percent saturation. Aeration, if needed
to achieve this level, should be done before the addition of the test sub-
-------
stance. All treatment and control chambers should be given the same aer-
ation treatment.

(iii) The number of mysids placed in a test solution should not be
so great as to affect results of the test. Loading requirements for the test
will vary depending on the flow rate of dilution water. The loading should
not cause the DOC to fall below the recommended levels.

(iv) Photoperiod of 14 h light and 10 h darkness, with a 15-30 min
transition period.

(v) Salinity of 20 ±3 ppt.

(f) Reporting. The sponsor should submit to the EPA all data devel-
oped by the test that are suggestive or predictive of chronic toxicity and
all concomitant toxicologic manifestations. In addition to the general re-
porting requirements prescribed under Good Laboratory Practice Stand-
ards, 40 CFR part 792, subpart J, the reporting of test data should include
the following:

(1) The source of the dilution water, its chemical characteristics (e.g.
salinity, pH, etc.) and a description of any pretreatment.

(2) Detailed information about the test organisms, including the sci-
entific name and method of verification, average length, age, source, his-
tory, observed diseases, treatments, acclimation procedures, and food used.

(3) A description of the test chambers, the depth and volume of solu-
tion in the chamber, the way the test was begun (e.g. conditioning, test
substance additions, etc.), the number of organisms per treatment, the num-
ber of replicates, the loading, the lighting, the test substance delivery sys-
tem, and the flow rate expressed as volume additions per 24 hours.

(4) The measured concentration of test substance in test chambers
at the times designated.

(5) The first time (day) that sexual characteristics can be observed
in controls and in each test substance concentration.

(6) The length of time for the appearance of the first brood for each
concentration.

(7) The means (average of replicates) and respective 95 percent con-
fidence intervals for:

(i) Body length of males and females at the first observation day (de-
pending on time of sexual maturation) and on day 28.

(ii) Cumulative number of young produced per female on day 28.

(iii) Cumulative number of dead adults on day 7, 14, 21, and 28.
-------
(iv) If available prior to test termination (day 28), effects on G2
mysids (number of males and females, body length of males and females,
and cumulative mortality).

(8) The MATC is calculated as the geometric mean between the low-
est measured test substance concentration that had a significant (p<0.05)
effect and the highest measured test substance concentration that had no
significant (p<0.05) effect in the chronic test. The most sensitive of the
test criteria for adult (Gl) mysids (cumulative number of dead mysids,
body lengths of males and females, or the number of young per female)
is used to calculate the MATC. The criterion selected for MATC computa-
tion is the one which exhibits an effect (a statistically significant difference
between treatment and control groups (p<0.05)) at the lowest test sub-
stance concentration for the shortest period of exposure. Appropriate statis-
tical tests (analysis of variance, mean separation test) should be used to
test for significant chemical effects. The statistical tests employed and the
results of these tests should be reported.

(9) Concentration-response curves should be fitted to the cumulative
number of adult dead for days 7, 14, 21, and 28. A statistical test of good-
ness-of-fit should be performed and the results reported.

(10) An LC50 value based on the number of dead adults with cor-
responding 95 percent confidence intervals for days 7, 14, 21, and 28.
These calculations should be made using the average measured concentra-
tion of the test substance.

(11) Methods and data records of all chemical analyses of water qual-
ity and test substance concentrations, including method validations and re-
agent blanks.

(12) The data records of the holding, acclimation and test temperature
and salinity.

(g) References. The following references should be consulted for ad-
ditional background information on this test guideline:

(1) Environmental Protection Agency, Bioassay Procedures for the
Ocean Disposal Permit Program, EPA Report No. 600/9-78-010 (Gulf
Breeze, Florida, 1978).

(2) Price, W.W. et al. Observations on the genus Mysidopsis Sars,
1864 with the designation of a new genus, Americamysis, and the descrip-
tions of Americamysis alleni and A. stucki (Peracarida: Mysidacea:
Mysidae), from the Gulf of Mexico. Proceedings of the Biological Society
of Washington 107:680-698 (1994).
8
-------
&EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-121
April 1996
Ecological Effects Test
Guidelines

OPPTS 850.1400
Fish Early-Life Stage
Toxicity Test
'Public Draft'
-------
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.

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."
-------
OPPTS 850.1400 Fish early-life stage toxicity test.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 797.1600 Fish Early Life Stage
Toxicity Test; OPP 72-4 Fish Early Life-Stage and Aquatic Invertebrate
Life-Cycle Studies (Pesticide Assessment Guidelines, Subdivision E—
Hazard Evaluation; Wildlife and Aquatic Organisms) EPA report 540/09-
82-024, 1982; and OECD 210 Fish Early-Life Stage Toxicity Test.

(b) Introduction. (1) Tests with the early-life stages of fish are in-
tended to define the lethal and sublethal effects of chemicals on the stages
and species tested. They yield information of value for the estimation of
the chronic lethal and sublethal effects of the substance on other fish spe-
cies.

(2) This guideline is based on a proposal from the United Kingdom
which was discussed at a meeting of OECD experts convened at
Medmenham (United Kingdom) in November 1988.

LOEC (Lowest-observed-effect-concentration) is the lowest tested
concentration of a test substance at which the substance is observed to
have a significant effect (at p<0.05) when compared with the control.
However, all test concentrations above the LOEC must have a harmful
effect equal to or greater than those observed at the LOEC.

NOEC (No-observed-effect-concentration) is the test concentration
immediately below the LOEC.

(d) Principle of the test. The early-life stages of fish are exposed
to a range of concentrations of the test substance dissolved in water, pref-
erably under flow-through conditions, or where appropriate, semistatic
conditions. The test is begun by placing fertilized eggs in the test chambers
and is continued at least until all the control fish are free-feeding. Lethal
and sublethal effects are assessed and compared with control values to
determine the LOEC and the NOEC.

(e) Information on the test substance. (1) Results of an acute tox-
icity test (see OPPTS 850.1075), preferably performed with the species
chosen for this test, should be available. This implies that the water solu-
bility and the vapor pressure of the test substance are known and a reliable

1
-------
analytical method for the quantification of the substance in the test solu-
tions with known and reported accuracy and limit of detection is available.

(2) Useful information includes the structural formula, purity of the
substance, stability in water and light, pKa, Pow, and results of a test for
ready biodegradability. (See OPPTS 835.3110 through 835.3160.)

(f) Validity of the test. For a test to be valid the following conditions
apply:

(1) The dissolved oxygen concentration must be between 60 and
100 percent of the air saturation value throughout the test.

(2) The water temperature must not differ by more than ±1.5 °C be-
tween test chambers or between successive days at any time during the
test, and should be within the temperature ranges specified for the test
species (Tables 4. and 5. under paragraphs (h)(l)(ii) and (h)(l)(iii) of this
guideline).

(3) Evidence must be available to demonstrate that the concentrations
of the test substance in solution have been satisfactorily maintained within
±20 percent of the mean measured values.

(4) Overall survival of fertilized eggs in the controls and, where rel-
evant, in the solvent-only controls must be greater than or equal to the
limits defined in Tables 4. and 5. under paragraphs (h)(l)(ii) and (h)(l)(iii)
of this guideline.

(5) When a solubilizing agent is used it must have no significant ef-
fect on survival nor produce any other adverse effects on the early-life
stages as revealed by a solvent-only control.

(g) Description of the method—(1) Test chambers. Any glass,
stainless steel, or other chemically inert vessels can be used. The dimen-
sions of the vessels should be large enough to allow compliance with load-
ing rate criteria given below. It is desirable that test chambers be randomly
positioned in the test area. A randomized block design with each treatment
being present in each block is preferable to a completely randomized de-
sign. The test chambers should be shielded from unwanted disturbance.

(2) Selection of species, (i) Recommended fish species are:

(A) Freshwater—rainbow trout, Oncorhynchus mykiss; fathead min-
now, Pimephales promelas; zebra fish, Danio rerio; ricefish, Oryzias
latipes.

(B) Salt water—sheepshead minnow, Cyprinodon variegatus.

(ii) The Office of Prevention, Pesticides and Toxic Substances prefers
rainbow trout (O. mykiss) or fathead minnow (P. promelas) as test species
for freshwater fish and sheepshead minnow (C variegatus) or silverside
-------
(Menidia menidia., M. beryllina, or M. peninsulae) as test spcies for estua-
rine or marine fish. This does not preclude the use of other species. Exam-
ples of other well-documented species which have also been used are:

(A) Freshwater — coho salmon, Oncorhynchus kisutch', Chinook salm-
on, Oncorhynchus tschawytscha', brown trout, Salmo trutta; Atlantic salm-
on, Salmo salar; brook trout, Salvelinus fontinalis, lake trout, Salvelinus
namaycush; northern pike, Esox lucius; white sucker, Catostomus
commersoni; bluegill, Lepomis macrochirus; channel catfish, Ictalurus
punctatus; flagfish, Jordanella floridae; three-spined stickleback,
Gasterosteus aculeatus; common carp, Cyprinus carpio.

(B) Salt water — Atlantic silverside, M. menidia; Tidewater silverside,
M peninsulae.

(iii) Feeding and handling requirements of brood and test animals,
test conditions, duration, and survival criteria for these species can be
found in Tables 1. and 2. under paragraph (g)(3) of this guideline, and
Tables 4. and 5. under paragraphs (h)(l)(i) and (h)(l)(ii) of this guideline.

(iv) The test procedure might have to be adapted to provide suitable
test conditions. The rationale for the selection of the species and the exper-
imental method should be reported in this case.

(3) Holding of the brood fish, (i) Details on holding the brood stock
under satisfactory conditions may be found in the references cited under
paragraphs (j)(l), G)(2)> ^d (j)0) of this guideline.
(ii) Conditions for recommended species are provided in the following
Table 1. Abbreviations in the table have the following meanings: BW =
body weight; FBS = frozen brine shrimp — adults Artemia sp.; BSN = brine
shrimp nauplii — newly hatched; BSN48 = brine shrimp nauplii — 48 h old.
-------
Table 1.—Feeding and Handling Requirements of Brood and Test Animals of Recommended
Species
Species
Freshwater
Oncorhynchus
rnykiss, rainbow
trout
Pimephales
promelas, fathead
minnow
Danio rerio. Zebra
fish

Oryzias latipes,
ricefish

Saltwater
Cyprinodon
variegatus.
Sheepshead min-
now
Food
Brood Fish

trout food

FBS

BSN48,
flake
food
flake food

FBS or
flake
food

Newly-
Hatched Lar-
vae

none2

BSN

protozoa *
protein 5

BSN, flake
food (or
protozoa or
rotifers)

BSN

Juveniles
Type

trout starter

BSN48

BSN48. flake
food (or
rotifers)

BSN48

Amount

4%BW
per day

Frequency

2-4 feeds/
day

ad libitum

BSN once
daily; flake
food twice
daily or
flake food
and rotifers
once daily

2-3 feeds per
day

Post-Hatch
Transfer
Time1

14-16 days
post-hatch
or at swim-
once hatching
is 90%

not necessary

from hatch to
swim-up

not applicable

Time to First
Feeding

19 days post-
hatch or at
swim-up
within 2 days
of hatching

6-7 days
after
spawning
within 24 h of
hatch/swim-
up

within 1 day
first hatch

1 if applicable
2 yolk-sac larvae require no food
3 not essential
4 filtered from mixed culture
5 granules from fermentation process
(iii) Conditions for other species are provided in the following Table
2.:Abbreviations in the table have the following meanings: BSN = brine
shrimp nauplii, newly hatched; BSN48 = brine shrimp nauplii, 48 hours
old; FBS = frozen brine shrimp; adult Anemia sp.
Table 2.—Feeding and Handling Requirements of Brood and Test Animals of Other Well-
Documented Species
Species
Freshwater
Oncorhynchus
kisutch, Coho
salmon

Oncorhynchus
tschawytscha, Chi-
nook salmon

Salmo trutta, brown
trout

Salmo safer Atlantic
salmon

Salvelinus fontinalis.
brook trout
Food
Brood fish

trout food

Newly-
hatched lar-
vae

none '

none

Juveniles
Type

trout starter

Amount

4%BW
per day

4% BW
per day

4% BW
per day
Frequency

2-4 feeds/
day

5 feeds/day

Post-Hatch
Transfer Time
(if applicable)

26-36 days
post-hatch
or at swim-
up
26-36 days
post-hatch
or at swim-
up
21 days post-
hatch or at
swim-up
21 days post-
hatch or at
swim-up
21 days post-
hatch or at
swim-uo
Time To First
Feeding

after swim-up
at transfer

26 days post-
hatch at
swim-up

at swim-up

-------
Table 2.—Feeding and Handling Requirements of Brood and Test Animals of Other Well-
Documented Species—Continued
Species
Saivelinus
namaycush, lake
trout
Esox iucius, North-
em pike

Catostomus
comm&soni, white
sucker

Lepomis
macrochirus,
bluegill
Ictalurus punctaius.
channel catfish

Jordanella fioridae,
flagfish

Gasterosteus
aculeatus, three-
spined stickleback

Cyphnus carpio,
common carp

Saltwater
Menidia menidia. At-
lantic silverside

Menidia peninsulas.
Tidewater
silverside

Food
Brood fish
trout food

live min-
nows

FBS

FBS, trout
food

catfish food

FBS, flake
food,
BSN

Tetramin
FBS

Proprietary
carp
food:
freeze-
dried
tubifex or
trout
food

BSN48,
flake
food

Newly-
hatched lar-
vae
none

BSN48

none

BSN

modified Or-
egon

BSN48, flake
food, or
protozoa/
rotifers3

Brachionus
rubens
(rotifer)

BSN

days 1-8

days 9-1 1

days 11-end

days 1-8

days 9-11

days 11-end

Juveniles
Type
trout starter

larval fish

BSN48

modified Or-
egon

BSN48, flake
food

BSN48,
Tetramin

BSN48.
ground;
trout start-
er, or flake
food

rotifers3

BSN4B and
rotifers3
BSN48

rotifers3

BSN48 and
rotifers3
BSN48

Amount
4% BW
per day

Frequency
5 feeds/day

3 feeds/day

3 feeds per
day

at least 3
feeds per
day
Anemia
nauplii
once daily;
flake food
twice daily
or flake
food and
protozoa &
rotifers
once daily
BSN48, 2-3
feeds per
day;
Tetramin
once daily
3-4 feeds per
day

3 feeds per
day

2 feeds per
day

3 feeds per
day

2 feeds per
day

Post-Hatch
Transfer Time
(if applicable)
21 days post-
hatch or at
swim-up
transfer
hatched fish
daily

once all em-
bryos have
hatched

6-7 days at
26 "C *

from hatch to
swim-up

several hours
after hatch2

once hatching
complete

not applicable

Time To First
Feeding
at swim-up

1 week post-
hatch or
swimming
yolk-sac
stage
7-8 days
post-hatch
or at swim-
up
at swim-up

within 48 h of
swim-up

within 24 h of
hatch

within 24
hours of
hatch

36-48 h post-
hatch

within 24 h of
first hatch

within 24 h of
first hatch
within 24 h of
first hatch
within 24 h of
first hatch

within 24 h of
first hatch
within 24 h of
first hatch
1 yolk-sac larvae require no food
2 fish may be handled with a 6 mm internal diameter glass siphon tube
3 rotifers—Brachionus plieatilis
(4) Handling of embryos and larvae, (i) Initially, embryos and lar-
vae may be exposed within the main vessel in smaller glass or stainless
steel vessels, fitted with mesh sides or ends to permit a flow of test solu-
tion through the vessel. Nonturbulent flow through these small vessels may

-------
be induced by suspending them from an arm arranged to move the vessel
up and down but always keeping the organisms submerged. Fertilized eggs
of salmonid fishes can be supported on racks or meshes with apertures
sufficiently large to allow larvae to drop through after hatching.

(ii) Where egg containers, grids, or mesh have been used to hold
eggs within the main test vessel, these restraints should be removed after
the larvae hatch, according to the advice in Table 1. except that mesh
should be retained to prevent the escape of the fish. If there is a need
to transfer the larvae, they should not be exposed to the air, and nets
should not be used to release fish from egg containers. The timing of this
transfer varies with the species and transfer may not always be necessary.

(5) Water. Any water in which the test species shows control sur-
vival, at least as good as that described in Table 4. under paragraph
(h)(l)(ii) of this guideline, and Table 5. under paragraph (h)(l)(iii) of this
guideline, is suitable as a test water. It should be of constant quality during
the period of the test. In order to ensure that the dilution water will not
unduly influence the test result (for example, by complexation of test sub-
stance) or adversely affect the performance of the brood stock, samples
should be taken at intervals for analysis. Measurements of heavy metals
(e.g. Cu, Pb, Zn, Hg, Cd, Ni), major anions and cations (e.g. Ca, Mg,
Na, K, Cl, sulfate), pesticides, total organic carbon, and suspended solids
should be made, for example, every 3 months where a dilution water is
known to be relatively constant in quality. Some chemical characteristics
of an acceptable dilution water are listed in the following Table 3:
Table 3.—Some Chemical Characteristics of an Acceptable Dilution Water
Substance
Participate matter ,
Total organic carbon
Un-ionized ammonia
Residual chlorine
Total organophosphorus pesticides
Total organochlorine pesticides plus polychlorinated biphenyls
Total oraanic chlorine
Maximum Concentration
< 20 mg/L
<2 mg/L
<1 ua/L
< 1 0 ua/L
<50 ng/L
< 50 ng/L
< 25 na/L
(6) Test solutions, (i) For flow-through tests, a system which contin-
ually dispenses and dilutes a stock solution of the test substance (e.g. me-
tering pump, proportional diluter, saturator system) is required to deliver
a series of concentrations to the test chambers. The flow rates of stock
solutions and dilution water should be checked at intervals during the test
and should not vary by more than 10 percent throughout the test. A flow
rate equivalent to at least five test chamber volumes per 24 h has been
found suitable (see paragraph (j)(l) of this guideline).
-------
(ii) The use of solvents or dispersants (solubilizing agents) may be
required in some cases in order to produce a suitably concentrated stock
solution.

(iii) For the semistatic technique, two different renewal procedures
may be followed. Either new test solutions are prepared in clean vessels
and surviving eggs and larvae gently transferred into the new vessels, or
the test organisms are retained in the test vessels while a proportion (at
least two-thirds) of the test water is changed.

(h) Procedure. Useful information on the performance of fish early-
life stage tests is available in the literature, some examples of which are
included under paragraphs (j)(l)> aii^ G)(4) through (j)(8) of this guideline.
(1) Conditions of exposure — (i) Duration. The test should start as
soon as possible after the eggs have been fertilized, the embryos preferably
being immersed in the test solutions before cleavage of the blastodisc com-
mences, or as close as possible after this stage. The test should continue
at least until all the control fish have been free-feeding. Test duration will
depend upon the species used.

(A) Data for recommended species are provided in the following
Table 4.:

Table 4.— Test Conditions, Duration, and Survival Criteria for Recommended Species
Species
Freshwater
Oncortiynchus mytoss. Rainbow
trout.
Pimephales promelas, fathead
minnow.
Danio rerio Zebra fish
Oryzias latipes ricefish
Saltwater
Cyprinodon variegatus, Sheeps-
head minnow ?.
Test Conditions
Temperature
rc)
10±21
12±223
25±2
25+2
24 ±1 (a)
23±2 2 *
25±2
Photoperiod
(hours)
14"
16
12-16 6
12-16 «
12-16 6
Recommended Duration of
Test
2 weeks after controls are
free-feeding (or 60 days
post-hatch)
32 days from start of test
(or 28 days post-hatch)
30 days post-hatch
30 days post-hatch
32 days from start of test
(or 28 days post-hatch)
Survival of Controls
(minimum percent)
Hatching
Success
>66
>66
>75
Post-Hatch
Success
70
70
70
SO
60
1for embryos
2for larvae and juvenile ftsh
3the particular strain of rainbow trout tested may necessitate the use of other temperatures; brood stock must be held at the
same temperature as that to be used for the eggs
"darkness for larvae until one week after hatching except when they are being inspected, then subdued lighting throughout test
(12-16 h photoperiod (6))
5this supersedes the requirement for temperature control given earlier on in the test
sfor any given test conditions, light regime should be constant
7salinity shall be at 15-30; for any given test this shall be performed to ±2 percent.
-------
(B) Data for other species are provided in the following Table 5:
Table 5.—Test Conditions, Duration and Survival Criteria for Other Well-Documented Species
Species
Freshwater
Oncorhynchus kisutch. Coho
salmon.
Oncorhynchus tschawytscha,
Chinook salmon.
Salmon trutta, brown trout
Salmo salar Atlantic salmon
Satvelinus fontinalis, brook trout .
Salvelinus namaycush. Lake trout
£sox lucius. Northern pike
Catostomus commersoni. White
sucker.
Lepomis macnxhirus, Bluegitl
tctalurus punctatus. Channel cat-
fish.
Jordanella fioridae, Flagfish
Gasterosteus aculeatus, Three-
spined stickleback.
Cyprinus carpio, common carp ...
Saltwater
Menidia menidia. Atlantic
silverside4.
Menidia peninsutae. Tidewater
silverside4.
Test Conditions
Temperature
<°c)

101, 122

101. 122

10
10
10
12-18
7
15

28
26

24-26
18-20

21-25

22-25

Photoperiod
(hours)

12-163

12-1 63
12-163
143
16
12-163
16

16
16

16
12-16

12-16

Recommended Duration of
Test

60 days post-hatch

60 days post-hatch
60 days post-hatch
60 days post-hatch
60 days post-hatch
32 days from start of test
32 days from start of test

32 days from start of test
32 days from start of test

28 days

28 days post-hatch

28 days

Survival of Controls
(minimum percent)
Hatching
Success

>66

>66
>66
>66
>66
>66
>66

>80

Post-Hatch
Success

70
70
70
70
70
80

75
65 (overall)

1for embryos
2for larvae and juvenile fish
^darkness for larvae until 1 week after hatching except when they are being inspected, then subdued lighting throughout test
(12-16 h photoperiod unless otherwise specified, but constant regime for a given test)

-------
but, as experience is gained, food and feeding regimes are continually
being refined to improve survival and optimize growth. Effort should
therefore be made to confirm the proposed regime with acknowledged ex-
perts.

(v) Test concentrations. (A) Normally five concentrations of the test
substance spaced by a constant factor not exceeding 3.2 are required. The
curve relating LC50 to period of exposure in the acute study should be
considered when selecting the range of test concentrations. The use of
fewer than five concentrations, for example in limit tests, and a narrower
concentration interval may be appropriate in some circumstances. Justifica-
tion should be provided if fewer than five concentrations are used. Con-
centrations of the substance higher than the 96-h LC50 or 10 mg/L, which-
ever is the lower, need not be tested.

(B) Where a solubilizing agent is used, its concentration should not
be greater than 0.1 mL/L and should be the same in all test vessels. How-
ever, every effort should be made to avoid the use of such materials.

(vi) Controls. One dilution-water control and also, if relevant, one
control containing the solubilizing agent should be run in addition to the
test series.

(2) Frequency of analytical determinations and measurements, (i)
During the test, the concentrations of the test substance are determined
at regular intervals to check compliance with the validity criteria. A mini-
mum of five determinations is necessary. In studies lasting more than
1 month, determinations should be made at least once a week. Samples
may need to be filtered (e.g. using a 0.45 fim pore size) or centrifuged
to ensure that the determinations are made on the substance in true solu-
tion.

(ii) During the test, dissolved oxygen, pH, total hardness and salinity
(if relevant), and temperature should be measured in all test vessels. As
a minimum, dissolved oxygen, salinity (if relevant), and temperature
should be measured weekly, and pH and hardness should be measured
at the beginning and end of the test. Temperature should preferably be
monitored continuously in at least one test vessel.

(3) Observations—(i) Stage of embryonic development. The em-
bryonic stage at the beginning of exposure to the test substance should
be verified as precisely as possible. This can be done using a representative
sample of eggs suitably preserved and cleared.

(ii) Hatching and survival. Observations on hatching and survival
should be made at least once daily and numbers recorded. Dead embryos,
larvae, and juvenile fish should be removed as soon as observed since
they can decompose rapidly and may be broken up by the actions of the
other fish. Extreme care should be taken when removing dead individuals
-------
not to knock or physically damage adjacent eggs/larvae, these being ex-
tremely delicate and sensitive. Criteria for death vary according to life
stage:

(A) For eggs: Particularly in the early stages, a marked loss of trans-
lucency and change in coloration, caused by coagulation and/or precipita-
tion of protein, leading to a white opaque appearance.

(B) For embryos: Absence of body movement and/or absence of
heart-beat.

(C) For larvae and juvenile fish: Immobility and/or absence of res-
piratory movement and/or absence of heart-beat and/or white opaque color-
ation of central nervous system and/or lack of reaction to mechanical stim-
ulus.

(iii) Abnormal appearance. The number of larvae or fish showing
abnormality of body form should be recorded at adequate intervals depend-
ing on the duration of the test and the nature of the abnormality described.
It should be noted that abnormal embryos and larvae occur naturally and
can be of the order of several percent in the controls in some species.
Abnormal animals should only be removed from the test vessels on death.

(iv) Abnormal behavior. Abnormalities, e.g. hyperventilation, unco-
ordinated swimming, atypical quiescence, and atypical feeding behavior
should be recorded at adequate intervals depending on the duration of the
test. These effects, although difficult to quantify, can, when observed, aid
in the interpretation of mortality data and influence a decision to extend
the exposure period beyond the recommended duration.

(v) Weight. At the end of the test all surviving fish must be weighed.
Individual weights are preferred but, if the fish are especially small, they
may be weighed in groups by test vessel. Dry weights (24 h at 60 °C)
are preferable to wet weights (blotted dry).

(vi) Length. At the end of the test, measurement of individual lengths
is recommended: Standard, fork, or total length may be used. If however,
caudal fin rot or fin erosion occurs, standard lengths should be used.

(vii) Data for statistical analysis. These observations will result in
some or all of the following data being available for statistical analysis:

(A) Cumulative mortality.

(B) Numbers of healthy fish at end of test.

(D) Numbers of larvae hatching each day.

(E) Length and weight of surviving animals.

10
-------
(F) Numbers of deformed larvae.

(G) Numbers of fish exhibiting abnormal behavior.

(i) Data and reporting—(1) Treatment of results, (i) It is rec-
ommended that a statistician be involved in both the design and analysis
of the test results since this test guideline allows for considerable variation
in experimental design as, for example, in the number of test chambers,
number of test concentrations, starting number of fertilized eggs, and num-
ber of parameters measured.

(ii) In view of the options available in test design, specific guidance
on statistical procedures is not given here. However, it will be necessary
for variations to be analyzed within each set of replicates using analysis
of variance or contingency table procedures. To make a multiple compari-
son between the results at the individual concentrations and those for the
controls, Dunnett's method might be found useful (see paragraphs Q)(9)
and (j)(10) of this guideline). However, care must be taken where applying
such a method to ensure that chamber-to-chamber variability is estimated
and is acceptably low. Other useful methods are also available (see para-
graphs (j)0), 0X6), and (j)(ll) of this guideline).

(2) Interpretation of results. The results should be interpreted with
caution where measured toxicant concentrations in test solutions occur at
levels near the detection limit of the analytical method.

(3) Test report. The test report must include the following informa-
tion:

(i) Test substance. (A) Physical nature and, where relevant, physico-
chemical properties.

(B) Chemical identification data.

(ii) Test species. Scientific name, strain, source and method of collec-
tion of the fertilized eggs, and subsequent handling.

(iii) Test conditions. (A) Test procedure used (e.g. semistatic or flow-
through design).

(B) Photoperiods.

(D) Method of preparation of stock solutions and frequency of re-
newal (the solubilizing agent and its concentration must be given, when
used).

(E) Nominal test concentrations, means of the measured values, their
standard deviations in the test vessels, and the method by which these

11
-------
were attained, and evidence that measurements refer to concentrations of
the test substance in true solution.

(F) Dilution water characteristics: pH, hardness, temperature, dis-
solved oxygen concentration, residual chlorine levels (if measured), total
organic carbon, suspended solids, salinity of the test medium (if meas-
ured), and any other measurements made.

(G) Water quality within test vessels: pH, hardness, temperature, and
dissolved oxygen concentration.

(H) Detailed information on feeding (e.g. type of feed, source, amount
given, and frequency).

(iv) Results. (A) Evidence that controls met the overall survival ac-
ceptability standard of the test species (Tables 4. and 5.).

(B) Data on mortality/survival at embryo, larval, and juvenile stages
and overall mortality/survival.

(D) Data for length and weight.

(E) Incidence and description of morphological abnormalities, if any.

(F) Incidence and description of behavioral effects, if any.

(G) Statistical analysis and treatment of data.

(H) NOEC for each response assessed.

(I) LOEC (at p = 0.05) for each response assessed.

(J) Any concentration-response data and curves available.

(v) Discussion of the results. [Reserved]

0) References. The following references should be consulted for ad-
ditional background material on this test guideline.

(1) American Society for Testing and Materials (ASTM). Standard
Guide for Conducting Early Life-Stage Toxicity Tests with Fishes. ASTM
E 1241-92, p. 180-207, Philadelphia, PA (1992).

(2) Brauhn, J.L. and Schoettger, R.A., Acquisition and Culture of Re-
search Fish: Rainbow trout, Fathead minnows, Channel catfish and
Bluegills. p. 54, Ecological Research Series, EPA-660/3-75-011, Duluth,
MN (1975).

(3) Brungs, W.A. and Jones, B.R., Temperature Criteria for Fresh-
water Fish: Protocol and Procedures, p. 128, Ecological Research Series
EPA-600/3-77-061, Duluth, MN (1977).

12
-------
(4) Hansen, D.J. and Parrish, P.R., Suitability of sheepshead minnows
(Cyprinodon variegatus) for life-cycle toxicity tests. In Aquatic Toxicology
and Hazard Evaluation (edited by F.L. Mayer and J.L. Hamelink), pp.
117-126, ASTM STP 634 (1977).

(5) McKim, J.M. et al., Metal toxicity to embryos and larvae of eight
species of freshwater fish-II: Copper. Bulletin of Environmental and Con-
tamination Toxicology 19:608-616 (1978).

(6) Rand, G.M. and Petrocelli, S.R., Fundamentals of Aquatic Toxi-
cology. Hemisphere Publication Corporation, NY (1985).

(7) USEPA, Recommended Bioassay Procedure for Fathead Min-
nows, Pimephales promelas (Rafinesque), Chronic Tests, p. 13, National
Water Quality Laboratory, Duluth, MN (1972).

(8) USEPA, Recommended Bioassay Procedure for Bluegill. Lepomis
macrochirus (Rafinesque), Partial Chronic Tests, p. 11, National Water
Quality Laboratory, Duluth, MN (1972).

(9) Dunnett, C.W., A multiple comparisons procedure for comparing
several treatments with a control. Journal of the American Statistical Asso-
ciation 50: 1096-1121(1955).

(10) Dunnett, C.W., New tables for multiple comparisons with a con-
trol. Biometrics 20:482^91 (1964).

(11) McClave, J.T. et al., Statistical Analysis of Fish Chronic Toxicity
Test Data. Proceedings of 4th Aquatic Toxicology Symposium, ASTM,
Philadelphia, PA (1980).
13
-------
-------
vvEPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-122
April 1996
Ecological Effects Test
Guidelines
OPPTS 850.1500
Fish life cycle toxicity
'Public Draft'
-------
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.

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."
-------
OPPTS 850.1500 Fish life cycle toxicity.
(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 OPP 72-5 Life-Cycle Tests of Fish (Pes-
ticide Assessment Guidelines, Subdivision E—Hazard Evaluation; Wildlife
and Aquatic Organisms) EPA report 540/09-82-024, 1982.

(b) When required. (1) Data obtained from a life-cycle test of fish
are required by 40 CFR 158.145 to support the registration of an end-
use product intended to be applied directly to water or expected to trans-
port to water from the intended use site, and when any of the following
conditions apply:

(i) If the estimated environmental concentration is equal to or greater
than one-tenth of the no-effect level in the fish early life-stage or inverte-
brate life-cycle test; or

(ii) If studies of other organisms indicate the reproductive physiology
of fish may be affected.

(2) See 40 CFR 158.50, "Formulators' exemption", to determine
whether these data must be submitted. Section II-A of this Subdivision
provides an additional discussion on this subject.

(c) Test standards. Data sufficient to satisfy the requirements in 40
CFR 158.145 should be derived from tests which comply with the general
test standards in § 70-3 and the following test standards:

(1) Test substance. Data shall be derived from testing conducted with
the technical grade of each active ingredient in the product.

(2) Duration of tests. Fish should be cultured in the presence of the
test substance from one stage of the life cycle to at least the same stage
of the next generation (e.g. egg to egg).

(3) Species. Testing should be performed on a freshwater fish (e.g.
fathead minnow). An estuarine species (e.g. sheepshead minnow) may be
used if the pesticide is expected to enter the estuarine environment.

(4) Concentration analysis. The concentration of the test substance
in the water should be determined at the start of the study and periodically
throughout the study to verify concentrations.

(d) Reporting and evaluation of data. In addition to the basic infor-
mation provided in §70-4, the test report should contain the following
information (where appropriate):
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(1) Reproductive effects;

(2) Detailed records of spawning, egg numbers, fertility, and fecun-
dity;

(3) No-effect level, and mortality data;

(4) Statistical evaluation of effects;

(5) Locomotion, behavioral, physiological, and pathological effects;

(6) Definition of the criteria used to determine effects;

(7) Summary of general observation of signs of intoxication or other
effects;

(8) Stage of life cycle in which organisms were tested;

(9) Duration of the test; and

(10) Concentration analysis.

(e) Acceptable protocol—(1) Freshwater fish life-cycle test. An ex-
ample of an acceptable protocol is found in the following reference:

National Water Quality Laboratory Committee on Aquatic Bioassays.
1971. Recommended bioassay procedure for fathead minnow Pimephales
promelas (Rafinesque) chronic tests. (Revised January, 1972). Pp. 15-24
in Biological Field and Laboratory Methods. U. S. Environmental Protec-
tion Agency, Office of Res. and Dev. EPA-670/4-73-001.

(2) Estuarine fish life-cycle test. Examples of acceptable protocols
are found in the following references:

(i) Schimmel, S.C., and D.J. Hansen. 1974. Sheepshead minnow
Cyprinodon variegatus: an estuarine fish suitable for chronic (entire
lifecycle) bioassays. Proc. 28th Ann. Cong. S.E. Assoc. Game-Fish Comm.
Pp. 392-398.

(ii) Hansen, D.J., P.R. Parrish, S.C. Schimmel, and L.R. Goodman.
1978. Life-cycle toxicity test using sheepshead minnows (Cyprinodon
variegatus). Pp. 109-116 in Bioassay Procedures for the Ocean Disposal
Permit Program. U.S. Environmental Protection Agency, Office of Re-
search and Development. EPA-600/9-78-010.
-------
&EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-127
April 1996
Ecological Effects Test
Guidelines
OPPTS 850.1710
Oyster BCF
"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.

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 850.1710 Oyster BCF.
(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 materials used in developing this har-
monized OPPTS test guideline are 40 CFR 797.1830 Oyster
Bioconcentration Test and OPP 72-6 Aquatic Organism Accumulation
Tests (Pesticide Assessment Guidelines, Subdivision E—Hazard Evalua-
tion; Wildlife and Aquatic Organisms) EPA report 540/09-82-024, 1982.

(b) Purpose. This guideline is to be used for assessing the propensity
of chemical substances to bioconcentrate in tissues of estuarine and marine
molluscs. This guideline describes a bioconcentration test procedure for
the continuous exposure of Eastern oysters (Crassostrea virginicd) to a
test substance in a flow-through system. EPA will use data from this test
in assessing the hazard a chemical or pesticide may present to the environ-
ment.

(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and in 40 CFR Part 792—Good Laboratory Practice
Standards are applicable to this test guideline. The following definitions
also apply:

(1) Acclimation is the physiological compensation by test organisms
to new environmental conditions (e.g. temperature, salinity, pH).

(2) Bioconcentration is the net accumulation of a chemical directly
from water into and onto aquatic organisms.

(3) Bioconcentration factor (BCF) is the quotient of the concentration
of a test chemical in tissues of aquatic organisms at or over a discrete
time period of exposure divided by the concentration of test chemical in
the test water at or during the same time period.

(4) Depuration is the elimination of a test chemical from a test orga-
nism.

(5) Depuration phase is the portion of a bioconcentration test after
the uptake phase during which the organisms are in flowing water to which
no test chemical is added.

(6) EC50 is that experimentally derived concentration of a chemical
in water that is calculated to induce shell deposition 50 percent less than
that of the controls in a test batch of organisms during continuous exposure
within a particular period of exposure (which should be stated).

(7) Loading is the ratio of the number of oysters to the volume (liters)
of test solution passing through the test chamber per hour.
-------
(8) Organic chlorine is the chlorine associated with all chlorine-con-
taining compounds that elute just before lindane to just after mirex during
gas chromatographic analysis using a halogen detector.

(9) Organochlorine pesticides are those pesticides which contain car-
bon and chlorine, such as aldrin, ODD, DDE, DDT, dieldrin, endrin, and
heptachlor.

(10) Steady-state is the time period during which the amounts of test
chemical being taken up and depurated by the test oysters are equal, i.e.
equilibrium.

(11) Steady-state bioconcentration factor is the mean concentration
of the test chemical in test organisms during steady-state divided by the
mean concentration of the test chemical in the test solution during the
same period.

(12) Stock solution is the concentrated solution of the test substance
which is dissolved and introduced into the dilution water.

(13) Test chamber is the container in which the test oysters are main-
tained during the test period.

(14) Test solution is dilution water containing the dissolved test sub-
stance to which test organisms are exposed.

(15) Umbo is the narrow end (apex) of the oyster shell.

(16) Uptake is the sorption of a test chemical into and onto aquatic
organisms during exposure.

(17) Uptake phase is the initial portion of a bioconcentration test dur-
ing which the organisms are exposed to the test solution.

(18) Valve height is the greatest linear dimension of the oyster as
measured from the umbo to the ventral edge of the valves (the farthest
distance from the umbo).

(d) Test procedures—(1) Summary of the test. Oysters are continu-
ously exposed to a minimum of one constant, sublethal concentration of
a test chemical under flow-through conditions for a maximum of 28 days.
During this time, test solution and oysters are periodically sampled and
analyzed using appropriate methods to quantify the test chemical con-
centration. If, prior to day 28, the tissue concentrations of the chemical
sampled over three consecutive sampling periods have been shown to be
statistically similar (i.e. steady-state has been reached), the uptake phase
of the test is terminated, and the remaining oysters are transferred to un-
treated flowing water until 95 percent of the accumulated chemical resi-
dues have been eliminated, or for a maximum depuration period of 14
days. The mean test chemical concentration in the oysters at steady-state
-------
is divided by the mean test solution concentration at the same time to
determine the bioconcentration factor (BCF), If steady-state is not reached
during 28 days of uptake, the steady-state BCF should be calculated using
non-linear parameter estimation methods.

(2) [Reserved]

(3) Range-finding test. The oyster acute toxicity test is used to deter-
mine the concentration levels to be used in the oyster bioconcentration
test.

(4) Definitive test, (i) The following data on the test chemical should
be known prior to testing:

(A) Solubility in water.

(B) Stability in water.

(D) Acute toxicity (e.g. propensity to inhibit shell deposition) to oys-
ters.

(E) The validity, accuracy, minimum detection, and minimum quan-
tification limits of selected analytical methods.

(ii) At least two concentrations should be tested to assess the propen-
sity of the compound to bioconcentrate. The concentrations selected should
not stress or adversely affect the oysters and should be less than one-
tenth the EC50 or
-------
estimate should also be used to designate a sampling schedule. The uptake
phase should continue until steady-state has been reached. The uptake
phase should continue for at least 4 days, but need not be longer than
28 days.
(A) The time to steady-state (S in hours) can be estimated from the
water solubility of the octanol-water partition coefficient for chemicals
whose uptake and depuration follow a two-compartment, two-parameter
model (ASTM, 1986, under paragraph (g)(l) of this guideline). The fol-
lowing equations were developed from data on fish but are considered
useful in this test as well:
S = 3.0/antilog(0.431 log W - 2.11)
or
S = 3.0/antilog(-0.414 log P + 0.122)
where
W = water solubility (mg/L)
P = octanol-water partition coefffient
For example, S for a chemical of log P 4.0 would be estimated as
3.0/antilog(-0.414(4.0) + 0.122) = 3.0/0.029 = 103.4 h.
Bioconcentration kinetic studies have also been performed specifically for
molluscs, e.g. as investigated by Hawker and Connell (under paragraph
(g)(2) of this guideline) and these may also be consulted.
(B) The depuration phase should continue until at least 95 percent
of the accumulated test substance and metabolites have been eliminated,
but no longer than 14 days.
(C) Based on the estimate of the time to steady-state, one of the fol-
lowing sampling schemes may be used to generate the appropriate data.
Table—Time to Steady-State in Days

Test Period
Exposure2

S<4
Sampling
days
11
61
1
2
3
4

S>4<14
Sampling
days
41
1
3
7
10
12
14
S>15<21
Sampling
days
1
3
7
10
14
18
22
S>21
Sampling
days
1
3
7
10
14
21
28
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Table—Time to Steady-State in Days—Continued

Test Period
Depuration2

S<4
Sampling
days
11
61
121
1

S>4<14
Sampling
days
1
2
4
6

S>15<21
Sampling
days
1
3
7
10

S>21
Sampling
days
1
3
7
10
14
1 Hours
2 Additional sampling times may be needed to confirm that steady-state has been
attained
(v) The following criteria should be met in order for the test to be
valid:

(A) If it is observed that the stability or homogeneity of the test chem-
ical cannot be maintained in the test solution, care should be taken in
the interpretation of the results and a note should be made that these results
may not be reproducible.

(B) The mortality in the controls should not exceed 10 percent at
the end of the test.

(D) There should be evidence (using measured test chemical con-
centrations) that the concentration of the chemical being tested has been
satisfactorily maintained over the test period.

(E) If evidence of spawning is observed, the test should be discon-
tinued and later repeated.

(F) Temperature variations from 20 °C should be held to a minimum,
preferably ±2 °C.

(vi) The following methodology should be followed:

(A) The test should not be started until the test chemical delivery
system has been observed to be functioning properly and the test chemical
concentrations have equilibrated (i.e. the concentration does not vary more
than 20 percent). Analyses of two sets of test solution samples taken prior
to test initiation should document this equilibrium. At initiation (time 0),
test solution samples should be collected immediately prior to the addition
of oysters to the test chambers.
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(B) The appropriate number of oysters (see paragraph (d)(4)(vii)(A)
of this guideline) should be brushed clean and should be impartially dis-
tributed among test chambers in such a manner that test results show no
significant bias from the distributions. The number of oysters used in ths
test will depend on the length of the test, number of replicate test chambers
used, and if, in addition to a nonsolvent control, a solvent-control is used.
Also important are the size of each oyster and the size of the test chamber.
For example, in a 28-day test, a minimum of 28 oysters in the uptake
(exposure) phase and an additional 20 oysters in the depuration phase per
test chemical concentration would be needed. These oysters could be dis-
tributed among two or more replicates at each concentration. A minimum
of 48 oysters would be required for each control. The oysters should be
spread out equidistant from one another and placed with the left (cupped)
valve down and the unhinged ends (opposite from umbo) all oriented in
the same direction facing the incoming flow.

(C) Oysters should be exposed to the test chemical during the uptake
phase until steady state has been reached or for a maximum of 28 days.
The uptake phase should be a minimum of at least 4 days. Then the re-
maining oysters should be transferred to untreated flowing water and sam-
pled periodically to determine if depuration of the test chemical occurs.
Every test should include a control consisting of the same dilution water,
conditions, procedures, and oysters from the same group used in the test,
except that none of the test chemical is added. If a carrier is present in
the test chamber, a separate carrier control is required.

(D) Oysters should be observed (and data recorded) at least daily for
feeding activity (deposition of feces) or any unusual conditions such as
excessive mucus production (stringy material floating suspended from oys-
ters), spawning, or appearance of shell (closure or gaping). If gaping is
noted, the oyster(s) should be prodded. Oysters which fail to make any
shell movements when prodded are to be considered dead, and should be
removed promptly with as little disturbance as possible to the test
chamber(s) and remaining live oysters.

(E) For oysters sampled, careful examination of all the tissues should
be made at the time of shucking for any unusual conditions, such as a
watery appearance or differences in color from the controls.

(F) Observations on compound solubility should also be recorded.
These include the appearance of surface slicks, precipitates, or material
adsorbing to the test chamber.

(vii) Sampling. (A) At each of the designated sampling times, trip-
licate water samples and enough oysters should be collected from the test
chambers) to allow for tissue analyses of at least four oysters. The con-
centration of test chemical should be determined in a minimum of four
oysters analyzed individually at each sampling period. If individual analy-
-------
sis is not possible, due to limitations of the sensitivity of the analytical
methods, then pairs, triplicates or more oysters may be pooled to constitute
a sample for measurement. A similar number of control oysters should
also be collected at each sample point, but only those collected at the
first sampling period and weekly thereafter, should be analyzed. Triplicate
control water samples should be collected at the time of test initiation
and weekly thereafter. Test solution samples should be removed from the
approximate center of the water column.

(B) At each sampling period the appropriate numbers of oysters are
removed and treated as follows:

(/) The valve height of each oyster should be measured.

(2) Oysters should be shucked as soon as practical after removal and
should never be refrigerated or frozen in the shell. The shell should be
opened at the hinge, the adductor muscle severed and the top valve re-
moved. The remaining adductor muscle should be severed where it at-
taches to the lower valve and the entire oyster removed.

(5) The shucked oysters should then be drained 3 min, blotted dry,
weighed and analyzed immediately for the test chemical. If analyses are
delayed, the shucked oysters should be wrapped individually in aluminum
foil (for organic analysis) or placed in plastic or glass containers (for metal
analysis) and frozen.

(C) If a radiolabeled test compound is used, a sufficient number of
oysters should also be sampled at termination to permit identification and
quantitation of any major (greater than 10 percent of parent) metabolites
present. It is crucial to determine how much of the activity present in
the oyster is directly attributable to the parent compound, and to correct
the bioconcentration factor appropriately.

(5) Test results (i) Steady-state has been reached when the mean
concentrations of test chemical in whole oyster tissue for three consecutive
sampling periods are statistically similar (F test, P = 0.05). A BCF is then
calculated by dividing the mean tissue residue concentration during steady-
state by the mean test solution concentration during the same period. A
95 percent confidence interval should also be derived from the BCF. This
should be done by calculating the mean oyster tissue concentration at
steady-state (Xo ) and its 97.5 percent confidence interval Xo ± t (S.E.)
where t is the t statistic at P = 0.025 and S.E. is the one standard error
of the mean. This calculation would yield lower and upper confidence lim-
its (Lo and Uo). The same procedure should be used to calculate the mean
and 97.5 percent confidence interval for the test solution concentrations
at steady-state, XS ± t (S.E.), and the resulting upper and lower confidence
limits (Ls and Us). The 95 percent confidence interval of the BCF would
then be between Lo/Us and Uo/Ls. If steady-state was not reached during
the maximum 28-day uptake period, the maximum BCF should be cal-
-------
culated using the mean tissue concentration from that and all the previous
sampling days. An uptake rate constant should then be calculated using
appropriate techniques. This rate constant is used to estimate the steady-
state BCF and the time to steady-state.

(ii) If 95 percent elimination has not been observed after 14 days
depuration then a depuration rate constant should also be calculated. This
rate constant should be based on the elimination of the parent compound.

(iii) Oysters used in the same test should be 30 to 50 mm in valve
height and should be as similar in age/size as possible to reduce variability.
The standard deviation of the height should be less than 20 percent of
the mean (N = 30).

(6) Analytical measurements, (i) All samples should be analyzed
using EPA methods and guidelines whenever feasible. The specific meth-
odology used should be validated before the test is initiated. The accuracy
of the method should be measured by the method of known additions.
This involves adding a known amount of the test chemical to three water
samples taken from an aquarium containing dilution water and a number
of oysters equal to that to be used in the test. The nominal concentration
of these samples should be the same as the concentration to be used in
the test. Samples taken on two separate days should be analyzed. The accu-
racy and precision of the analytical method should be checked using ref-
erence or split samples or suitable corroborative methods of analysis. The
accuracy of standard solutions should be checked against other standard
solutions whenever possible.

(ii) An analytical method should not be used if likely degradation
products of the test chemical, such as hydrolysis and oxidation products,
give positive or negative interferences, unless it is shown that such deg-
radation products are not present in the test chambers during the test.
Atomic absorption spectrophotometric methods for metal and gas
chromatographic methods for organic compounds are preferable to colon-
metric methods. Spectrophotometry is also acceptable provided Beer's law
is followed and an acceptable extinction coefficient can be determined.

(iii) In addition to analyzing samples of test solution at least one rea-
gent blank should also be analyzed when a reagent is used in the analysis.

(iv) When radiolabelled test compounds are used, total radioactivity
should be measured in all samples. At the end of the uptake phase, water
and tissue samples should be analyzed using appropriate methodology to
identify and estimate the amount of any major (at least 10 percent of the
parent compound) degradation products or metabolites that may be present.

(e) Test conditions—(1) Test species, (i) The Eastern oyster,
Crassostrea virginica, should be used as the test organism.

8
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(ii) Oysters used in the same test should be 30 to 50 mm in valve
height and should be as similar in age and/or size as possible to reduce
variability. The standard deviation of the valve height should be less than
20 percent of the mean.

(iii) Oysters used in the same test should be from the same source
and from the same holding and acclimation tank(s).

(iv) Oysters should be in a prespawn condition of gonadal develop-
ment prior to and during the test as determined by direct or histological
observation of the gonadal tissue for the presence of gametes.

(v) Oysters may be cultured in the laboratory, purchased from culture
facilities or commercial harvesters, or collected from a natural population
in an unpolluted area free from epizootic disease.

(vi) The holding and acclimation of the oysters should be as follows:

(A) Oysters should be attended to immediately upon arrival. Oyster
shells should be brushed clean of fouling organisms, and the transfer of
the oysters to the holding water should be gradual to reduce stress caused
by differences in water quality characteristics and temperature. Oysters
should be held for at least 12 days before testing. All oysters should be
maintained in dilution water at the test temperature for at least 2 days
before they are used.

(B) During holding, the oysters should not be crowded, and the dis-
solved oxygen concentration should be above 60-percent saturation. The
temperature of the holding waters should be the same as that used for
testing. Holding tanks should be kept clean and free of debris. Cultured
algae may be added to dilution water sparingly, as necessary to support
life and growth, such that test results are not affected, as confirmed by
previous testing. Oysters should be handled as little as possible. When
handling is necessary, it should be done as gently, carefully, and quickly
as possible.

(C) A batch of oysters is acceptable for testing if the percentage mor-
tality over the 7-day period prior to testing is less than 5 percent. If the
mortality is between 5 and 10 percent, acclimation should continue for
7 additional days. If the mortality is greater than 10 percent, the entire
batch of oysters should be rejected. Oysters which appear diseased or oth-
erwise stressed or which have cracked, chipped, bared, or gaping shells
should not be used. Oysters infested with mudworms (Polydora sp.) or
boring sponges (Cilona cellata) should not be used.

(2) Facilities—(i) Apparatus. (A) An oxygen meter, dosing equip-
ment for delivering the test chemical, adequate apparatus for temperature
control, test tanks made of chemically inert material and other normal lab-
oratory equipment are needed.
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(B) Constant conditions in the test facilities should be maintained as
much as possible throughout the test. The preparation and storage of the
test material, the holding of the oysters and all operations and tests should
be carried out in an environment free from harmful concentrations of dust,
vapors and gases and in such a way as to avoid cross-contamination. Any
disturbances that may change the behavior of the oysters should be avoid-
ed.

(C) The test chambers should be made from materials that will not
absorb the test substance. Delivery systems and test chambers should be
cleaned before and after each use. If absorption of the test substance oc-
curs, those applicable parts of the delivery system should be discarded.

(D) The test substance delivery system used should accommodate the
physical and chemical properties of the test substance and the selected
exposure concentrations. The apparatus used should accurately and pre-
cisely deliver the appropriate amount of stock solution and dilution (sea)
water to the test chambers. The introduction of the test substance should
be done in such a way as to maximize the homogeneous distribution of
the test substance throughout the test chamber.

(ii) Dilution water. A constant supply of good quality unfiltered sea-
water should be available throughout the holding, acclimation, and testing
periods. Natural seawater is recommended, although artificial seawater
with food (algae) added may be used. In either case, to ensure each oyster
is provided equal amounts of food, the water should come from a thor-
oughly mixed common source and should be delivered at a flow rate of
at least one, and preferably 5 L/h per oyster. The flowrate should be
± 10 percent of the nominal flow. A dilution water is acceptable if oysters
will survive and grow normally over the period in which the test is con-
ducted without exhibiting signs of stress, i.e. excessive mucus production
(stringy material floating suspended from oysters), lack of feeding, shell
gaping, poor shell closing in response to prodding, or excessive mortality.
The dilution water should have a salinity in excess of 12 ppt, and should
be sirm'liar to that in the environment from which the test oysters origi-
nated. A natural seawater should have a weekly range in salinity of less
than 10 ppt and a monthly range in pH of less than 0.8 units. Artificial
seawater should not vary more than 2 ppt nor more than 0.5 pH units.
Oysters should be tested in dilution water from the same origin. If natural
sea water is used, it should meet the following specifications, measured
at least twice a year.
Substance
Concentration
Suspended sotids
Un-ionized ammonia
<20 mg/L
<20 mg/L
10
-------
Substance Concentration

Residual chlorine <3 ug/L
Total organophosphorus pesticides <50 ug/L
Total organophosphorus pesticides <50 ug/L
plus PCB's
(3) Test parameters—(i) Carriers. Stock solutions of substances of
low aqueous solubility may be prepared by ultrasonic dispersion or, if nec-
essary, by use of organic solvents, emulsifiers or dispersants of low tox-
icity to oysters. When such carriers are used, the control oysters should
be exposed to the same concentration of the carrier as that used in the
highest concentration of the test substance. The concentration of such car-
riers should not exceed 0.1 mL/L (100 mg/L).

(ii) Dissolved oxygen. This dissolved oxygen concentration should
be at least 60 percent of the air saturation value and should be measured
daily in each chamber.

(iii) Loading. The loading rate should not crowd oysters and should
permit adequate circulation of water while avoiding physical agitation of
oysters by water current.

(iv) Temperature. The test temperature should be 20 °C. Temporary
excursions (less than 8 h) within ± 5 °C are permissible. Temperature
should be recorded continually.

(v) pH. The pH should be measured daily in each test chamber.

(vi) The amount of total organic carbon (TOG) in the dilution water
can affect the bioavailability of some chemicals. Thus, TOC should be
measured daily.

(i) Reporting. In addition to the reporting requirements prescribed
in 40 CFR Part 792—Good Laboratory Practice Standards, the report
should contain the following:

(1) The source of the dilution water, the mean, standard deviation
and range of the salinity, pH, TOC, temperature, and dissolved oxygen
during the test period.

(2) A description of the test procedures used (e.g. the flow-through
system, test chambers, chemical delivery system, aeration, etc.).

(3) Detailed information about the oysters used, including age, size
(i.e. height), weight (blotted dry), source, history, method of confirmation
of prespawn condition, acclimation procedures, and food used.

11
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(4) The number of organisms tested, sampling schedule, loading rate
and flowrate.

(5) The methods of preparation of stock and test solutions and the
test chemical concentrations used.

(6) The number of dead and live organisms, the percentage of oysters
that died and the number that showed any abnormal effects in the control
and in each test chamber at each observation period.

(7) Methods and data records of all chemical analyses of water quality
parameters and test chemical concentrations, including method validations
and reagent blanks.

(8) Description of sampling, sample storage (if required) and analyt-
ical methods of water and tissue analyses for the test chemical.

(9) The mean, standard deviation and range of the concentration of
test chemical hi the test solution and oyster tissue at each sampling period.

(10) The time to steady-state.

(11) The steady-state or maximum BCF and the 95 percent confidence
limits.

(12) The time to 95 percent elimination of accumulated residues of
the test chemical from test oysters.

(13) Any incidents in the course of the test which might have influ-
enced the results.

(14) If the test was not done in accordance with the prescribed condi-
tions and procedures, all deviations should be described in full.

(g) References.

(1) American Society for Testing and Materials. ASTM E 1022-84.
Standard practice for conducting bioconcentration tests with fishes and
saltwater bivalve molluscs. In 1986 Annual Book of ASTM Standards,
vol. 11.04: Pesticides; resource recovery; hazardous substances and oil
spill response; waste disposal; biological effects, pp. 702—725 (1986).

(2) Hawker, D.W. and D.W. Connell, Bioconcentration of lipophilic
compounds by some aquatic organisms, Ecotoxicology and Environmental
Safety 11:184-197 (1986).

(3) Schimmel, S.C. and R.L. Gamas, Interlaboratory comparison of
the ASTM bioconcentration test method using the eastern oyster, pp. 277-
287. In R.C. Banner and R.T. Hansen (eds.), Aquatic Toxicology and Haz-
ard Assessment: Eighth Symposium, ASTM STP 891, American Society
for Testing and Materials, Philadelphia, PA (1985).

12
-------
vvEPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-129
April 1996
Ecological Effects Test
Guidelines

OPPTS 850.1730
Fish BCF
"Public Draft"
-------
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.

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."
-------
OPPTS 850.1730 Fish BCF
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.\

(2) Background. The source material used in developing this har-
monized OPPTS test guideline is 40 CFR 797.1520 Fish Bioconcentration
Test; OPP guideline 72-6 Aquatic Organism Bioavailability/
Biomagnification/Toxicity Tests (Pesticide Assessment Guidelines, Sub-
division E—Hazard Evaluation; Wildlife and Aquatic Organisms) EPA re-
port 540/09-82-024, 1982 and OPP 165-4 Laboratory Studies of Pesticide
Accumulation in Fish (Pesticide Assessment Guidelines, Subdivision N—
Environmental Fate) EPA report 540/09-82-031, 1982; and OECD 305E
Bioaccumulation: Flow-Through Fish Test.

(b) Introduction—(1) Purpose. The purpose of the study is to deter-
mine uptake and depuration rate constants and bioconcentration factors
(BCFs) for fish exposed to a test chemical in aqueous solution. Another
purpose is to identify and quantify major degradates at steady state. BCF
values for the test chemical should always be based on concentrations of
the chemical in fish tissue and exposure water, and not on total
radiolabeled residues. BCFs may be used to help assess risks to the fish
and to nontarget organisms (including humans) above them in the food
chain.

(2) Criteria for performing test. The test is most commonly required
for chemicals that are relatively persistent (stable) in water and have a
relatively high potential for bioaccumulation as indicated by log Pow (log
of the octanol/water partition coefficient) values less than or equal to 1.0.

(3) Criteria for degradate characterization. BCFs based on total
radiolabeled residues in fish tissue and exposure water can be used to help
determine whether major degradates should be identified and quantified.
If the BCF in terms of total radiolabeled residues is greater than or equal
to 1,000, OPP requires that an attempt be made to identify and quantify
pesticide degradates representing greater than or equal to 10 percent of
total residues in fish tissues at steady state. If degradates representing
greater than or equal to 10 percent of total radiolabeled residues in the
fish tissue are identified and quantified, then degradates in the test water
should also be identified and quantified.

(4) Desired information on the test chemical. To determine whether
a BCF test is warranted (see paragraph (b)(2) of this guideline), it is nec-
essary to know aqueous fate characteristics of the test chemical that deter-
mine its persistence in water and its octanol/water partition coefficient.
Aqueous fate characteristics include rates of abiotic hydrolysis,
biodegradation, direct photolysis in natural sunlight, and volatilization
from water. Henry's law constant (approximated by the ratio of the chemi-
-------
cal's vapor pressure to its solubility in water) is a good indicator of vola-
tilization potential. It is necessary to know the test chemical's solubility
in water to ensure that exposure concentrations do not exceed it. It is also
necessary to know the toxicity of the chemical to test fish to ensure expo-
sure concentrations do not adversely effect them (see paragraph (g)(2) of
this guideline). The purity of the test chemical should be known as well
as its radiopuriry if radiolabeled. The structure and radiolabeled positions
should be known. An appropriate analytical method, of known accuracy,
precision, and sensitivity, for the quantification of the substance in the
test solutions and in biological material must be available, together with
details of sample preparation and storage. Analytical detection limit of test
substance in both water and fish tissues should also be known.

(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR
Part 792—Good Laboratory Practice Standards (GLP) apply to this test
guideline. The following definitions also apply to this test guideline.

Bioconcentration/bioaccumulation is the increase in concentration of
the test substance in or on an organism (specified tissues thereof) relative
to the concentration of test substance in the surrounding medium.

The bioconcentration factor (BCF or KB) at any time during the up-
take phase of this accumulation test is the concentration of test substance
(expressed in milligrams per gram or parts per million) in/on the fish or
specified tissues thereof, divided by the concentration of the chemical in
the surrounding medium (BCF = Cj/Cw).

The depuration (loss) rate constant (k2) is the numerical value defin-
ing the rate of reduction in the concentration of the test substance in the
test fish (or specified tissues thereof) following the transfer of the test
fish from a medium containing the test substance to a medium free of
that substance (k2 is expressed in day1)-

The exposure or uptake phase is the time during which fish are ex-
posed to the test chemical.

Kinetic concentration factors (BCFK) are bioconcentration factors
calculated directly from kinetic rate constants (ki/k2).

The octanol-water partition coefficient (Pow) is the ratio of the solu-
bility of a chemical in n-octanol and water at equilibrium and can also
be expressed as KQW. Log Pow is used as an indication of a chemical's
potential for bioconcentration by aquatic organisms.

A plateau or steady-state is reached when the the plot of yhe con-
centration of test substance in fish (Cf) against time becomes parallel to
the time axis and three successive analyses of Cf made on samples taken
at intervals of at least 2 days are within ± 20 percent of each other, and
there are no significant differences among the three sampling periods. At
-------
least four successive analyses are required when pooled samples are ana-
lyzed. For test substances which are taken up slowly, the intervals would
more appropriately be 7 days.

The postexposure or depuration (loss) phase is the time, following
the transfer of the test fish from a medium containing test substance to
a medium free of that substance, during which the depuration (or the net
loss) of the substance from the test fish (or specified tissue thereof) is
studied.

The steady state bioconcentration factor is found when the BCF does
not change significantly over a prolonged period of time, the concentration
of the test substance in the surrounding medium being constant during
this period of time.

The uptake rate constant (k\) is the numerical value defining the rate
of increase in the concentration of test substance in/on test fish (or speci-
fied tissues thereof) when the fish are exposed to that chemical (kj is
expressed in day-1).

(d) Principle of test—(1) Uptake and depuration phase. The test
consists of two phases—the exposure (uptake) and postexposure
(depuration) phases. During the uptake phase, separate groups of fish of
one species are exposed to at least two concentrations of the test substance
until steady state is achieved or to a maximum of 28-60 days (see para-
graph (g)(3) of this guideline). They are then transferred to a medium free
of the test substance for a depuration phase of adequate duration (see para-
graph (g)(4) of this guideline). The concentration of the test substance in/
on the fish (or specified tissue thereof) and in water is followed through
both phases of the test.

(2) Determination of rate constants and BCFs. (i) Concentrations
of the test chemical in fish tissue and water as a function of time through-
out the uptake and depuration phases are used to determine the uptake
(ki) and depuration (k2) rate constants (see paragraph (i)(l) of this guide-
line.

(ii) Both the steady state and kinetic bioconcentration factors should
be calculated (see paragraph (i)(2) of this guideline). The steady state
bioconcentration factor (BCFs) is calculated as the ratio of the concentra-
tion in the fish (Cf) and to that in the water (Cw) at apparent steady-state.
The kinetic bioconcentration factor (BCFK) is calculated as the ratio of
the uptake rate constant (ki) to the depuration rate constant (ka) assuming
first-order kinetics

(iii) At a minimum, BCFs should be computed for the whole fish.
Whenever possible, they should also be calculated for edible and nonedible
tissue. BCFs should be related to both the weight and lipid content of
-------
the fish. If first-order kinetics are obviously not obeyed, more complex
models should be employed under paragraph (d)(2)(iv) of this guideline.

(iv) Mode! discrimination. Most bioconcentration data have been as-
sumed to be reasonably well described by a simple two-compartment/two-
parameter model, as indicated by the rectilinear curve which approximates
to the points for concentrations in fish, during the depuration phase, when
these are plotted on semilog paper. (Where these points cannot be de-
scribed by a rectilinear curve then more complex models should be em-
ployed, see paragraph (k)(20) of this guideline.)

(A) Graphical method for determination of depuration (loss) rate con-
stant k2-

Plot the concentration of the test substance found in each sample of
fish against sampling time on semilog paper. The slope of that line is
k2.

k2 = ln(Cfl/Cf2)/(t2-t,)

Note that deviations from a straight line may indicate a more complex
depuration pattern than first order kinetics. A graphical method may be
applied for resolving types of depuration deviating from first order kinet-
ics.

(B) Graphical method for determination of uptake rate constant ki.

Given k2, calculate ki as follows:

Equation 1

ki=Cfk2/CwTlx(l-e-*2t)

The value of Cf is read from the midpoint of the smooth uptake curve
produced by the data when log concentration is plotted versus time (on
an arithmetical scale).

The preferred means for obtaining the bioconcentration factor and ki
and k2 rate constants is to use nonlinear parameter estimation methods
on a computer. These programs find values for ki and k2 given a set of
sequential time concentration data and the model:

Equation 2

Cf=Cwxki/k2x(l -e-k2t) 0
-------
Equation 3

Of = Ow X
- ek-2<
t>tc
where tc = time at the end of the uptake phase.

This approach provides standard deviation estimates of ki and ka.

(D) As ka in most cases can be estimated from the depuration curve
with relatively high precision, and because a strong correlation exists be-
tween the two parameters ki and k2 if estimated simultaneously, it may
be advisable first to calculate k2 from the depuration data only, and subse-
quently calculate kj from the uptake data using nonlinear regression.

(e) Materials—(1) Exposure tanks and tubes. Care should be taken
to avoid the use of materials, for all parts of the equipment, that can dis-
solve, sorb or leach and have an adverse effect on the fish. Standard rec-
tangular or cylindrical tanks, made of chemically inert material and of a
suitable capacity in compliance with loading rate (see paragraph (e)(7) of
this guideline), can be used. The use of soft plastic tubing should be mini-
mized. Use Teflon, stainless steel and/or glass tubing. Experience has
shown that for substances with high adsorption coefficients, such as the
synthetic pyrethroids, silanized glass may be required. In these situations
the equipment will have to be discarded after use.

(2) Diluter. For flow-through tests, a system which continuously dis-
penses and dilutes a stock solution of the test substance (e.g. metering
pump, proportional diluter, saturator system) is required to deliver the test
concentrations to the test chambers. Preferably allow at least five volume
replacements through each test chamber per day. The flow rates of stock
solutions and dilution water should be checked both 48 hours before and
then at least daily during the test. Include in this check the determination
of the flow-rate through each test chamber and ensure that it does not
vary by more than 20 percent either within or between chambers. The
flow-through mode is to be preferred, but where this is not possible (e.g.
when the test organisms are adversely affected) a semi-static technique
may be used provided that the validity criteria are satisfied (see paragraph
(g)(ll) of this guideline).

(3) Dilution water, (i) Natural water is generally used in the test
and should be obtained from uncontaminated and uniform quality source.
The dilution water must be of a quality that will allow the survival of
the chosen fish species for the duration of the acclimation and test periods
without them showing any abnormal appearance or behavior. Ideally, it
should be demonstrated that the test species can survive, grow and repro-
duce in the dilution water (e.g. in laboratory culture or a life-cycle toxicity
test). The water should be characterized at least by pH, hardness, total
solids, total organic carbon and, preferably also ammonium, nitrite and
alkalinity and, for marine species, salinity. Although the parameters which
-------
are important for optimal fish well-being are not fully known, the follow-
ing Table 1. gives recommended maximum concentrations of a number
of parameters for fresh and marine test waters.

Table 1.—Some chemical characteristics of an acceptable dilution water
Substance
Limit con-
centration
Particulate matter 5 mg/L
Total organic carbon 2 mg/L
Un-ionized ammonia 1 mg/L
Residual chlorine 10 mg/L
Total organophosphorus pesticides 50 ng/L
Total organochlorine pesticides 50 ng/L
plus polychlorinated biphenyls 25 ng/L
Total organic chlorine 1 ug/L
Aluminium 1 ug/L
Arsenic 1 ug/L
Chromium 1 ug/L
Cobalt 1 ug/L
Copper 1 ng/L
Iron 1 ug/L
Lead 1
Nickel 1
Zinc 1
Cadmium 100 ng/L
Mercury 100 ng/L
Silver 100 ng/L
(ii) The water should be of constant quality during the period of a
test. The pH value should be within the range 6.0 to 8.5, but during a
given test it should be within a range of ±0.5 pH units. In order to ensure
that the dilution water will not unduly influence the test result (for exam-
ple, by complexation of the test substance) or adversely affect the perform-
ance of the stock of fish, samples should be taken at intervals for analysis.
Determination of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd, Ni), major anions
and cations (e.g. Ca, Mg, Na, K, Cl, 564), pesticides (e.g. total
organophosphorus and total organochlorine pesticides), total organic car-
bon and suspended solids should be made, for example, every 3 months
where a dilution water is known to be relatively constant in quality. If
water quality has been demonstrated to be constant over at least I year,
determinations can be less frequent and intervals extended (e.g. every 6
months).

(iii) The natural particle content as well as the total organic carbon
(TOC) of the dilution water should be as low as possible to avoid adsorp-
tion of the test substance to organic matter which may reduce its
bioavailability. The maximum acceptable value is 5 mg/L for particulate
matter (dry matter, not passing a 0.45 u.m filter) and 2 mg/L for total
-------
organic carbon. If necessary, the water should be filtered before use. The
contribution to the organic carbon content in water from the test fish (ex-
creta) and from the food residues should be as low as possible. Throughout
the test, the concentration of organic carbon in the test vessels should not
exceed the concentration of organic carbon originating from the test sub-
stance and, if used, the solubilizing agent by more than 10 mg/L
(±20 percent).

(4) Test chemical. Whether radiolabeled or not, the chemical purity
of the test chemical should be as high as practical (preferably greater than
or equal to 98 percent). If radiolabeled, the radiopurity should be greater
than or equal to 95 percent.

(5) Test chemical stock solutions. Prepare a stock solution of the
test substance at a suitable concentration. The stock solution should pref-
erably be prepared by simply mixing or agitating the test substance in
the dilution water. The use of solvents or dispersant (solubilizing agents)
is not recommended; however this may occur in some cases in order to
produce a suitably concentrated stock solution. Solvents which may be
used are, ethanol, methanol, ethylene glycol monomethyl ether, ethylene
glycol dimethyl ether, dimethylformamide and triethylene glycol. Dispers-
ant which may be used are Cremophor RH40, Tween 80, methylcellulose
0.01 percent and HCO-40. Care should be taken when using readily bio-
degradable agents as these can cause problems with bacterial growth in
flow-through tests.

(6) Test species, (i) Important criteria in the selection of species are
that they are readily available, can be obtained in convenient sizes and
can be satisfactorily maintained in the laboratory. Other criteria for select-
ing fish species include recreational, commercial, ecological importance
as well as comparable sensitivity, past successful use etc. Recommended
test species and test conditions are given in the following Table 2. Other
species may be used but the test procedure may have to be adapted to
provide suitable test conditions. The rationale for the selection of the spe-
cies and the experimental method should be reported in this case.
-------
Table 2.—Fish species recommended for testing

O(JcUlco

Danio rerio 1 (Teleostei, Cyprinidae) (Hamilton-Bu-
chanan) Zebra-fish
Pimephales promelas (Teleostei, Cyprinidae)
(Rafinesque) Fathead minnow
Cyprinus carpio (Teleostei, Cyprinidae) (Linnaeus) Com-
mon carp
Oryzias latipes (Teleostei, Poecilliidae) (Temminck and
Schlegel) Ricefish
Poecilia reticulata (Teleostei, Poeciliidae) (Peters) Guppy
Lepomis macrochirus (Teleostei Centrarchidae)
(Rafinesque) Bluegill
Oncorhynchus mykiss (Teleostei Salmonidae (Walbaum)
Rainbow trout
Gasterosteus aculeatus (Teleostei, (Gasterosteidae) (Lin-
naeus) Three-SDined stickleback
Test tem-
perature

(°C)
20-25
20-25
20-25
20-25
20-25
20-25
13-17
18-20
Total length
of test ani-
meal
(cm)
3.0+0.5
5.0 + 2.0
5.0+3.0
4.0+1.0
3.0 ±1.0
5.0+2.0
8.0+4.0
3.0+1.0
1 Meyer A. and G. Orti. Proceedings of the Royal Society of London 252 (Series
B):231 (1993).
(ii) Various estuarine and marine species have been used in different
countries, for example: Spot (Leiostomus xanthurus); Sheepshead minnow
(Cyprinodon variegatus); Silverside (Menidia beryllina); Shiner perch
(Cymatogaster aggregata); English sole (Parophrys vetulus); Staghorn
sculpin (Leptocottus armatus); Three-spined stickleback (Gasterosteus
aculeatus)', Sea bass (Dicentracus labrax); Bleak (Alburnus alburnus)

(iii) The fresh water fish listed are easy to rear and/or are widely
available throughout the year, whereas the availability of marine and estua-
rine species is partially confined to the respective countries. They are capa-
ble of being bred and cultivated either in fish farms or in the laboratory,
under disease-and parasite-controlled conditions, so that the test animal
will be healthy and of known parentage. These fish are available in many
parts of the world.

(7) Reference chemicals. The use of reference compounds of known
bioconcentration potential would be useful in checking the experimental
procedure, when required. However, specific substances cannot yet be rec-
ommended.

(f) Fish care and health—(1) Acclimation. Acclimate the stock pop-
ulation of fish for at least 2 weeks in water at the test temperature and
8
-------
feed throughout on a sufficient diet (see paragraph (f)(3) of this guideline)
and of the same type to be used during the test.

(2) Pretest mortality and health, (i) Following a 48-h settling-in
period (during acclimation), mortalities are recorded and the following cri-
teria applied:

(A) Mortalities of greater than 10 percent of population in 7 days,
reject the entire batch.

(B) Mortalities of between 5 and 10 percent of population in 7 days,
acclimate for 7 additional days.

(C) Mortalities of less than 5 percent of population in 7 days, accept
the batch. If more than 5 percent mortality during the second 7 days, reject
the entire batch.

(ii) Ensure that fish used in tests are free from observable diseases
and abnormalities. Discard any diseased fish. Fish should not receive treat-
ment for disease in the two weeks preceding the test, or during the test.

(3) Feeding, (i) During the acclimation and test periods, feed an ap-
propriate diet of known lipid and total protein content to the fish in an
amount sufficient to keep them in a healthy condition and to maintain
body weight. Feed daily throughout the acclimation and test periods at
a level of approximately 1 to 2 percent of body weight per day; this keeps
the lipid concentration in most species of fish at a relatively constant level
during the test. The amount of feed should be recalculated, for example,
once per week, in order to maintain consistent body weight and lipid con-
tent. For this calculation, the weight of the fish in each test chamber can
be estimated from the weight of the fish sampled most recently in that
chamber. Do not weigh the fish remaining in the chamber.

(ii) Siphon uneaten food and faeces daily from the test chambers
shortly after feeding (30 min to 1 h). Keep the chambers as clean as pos-
sible throughout the test so that the concentration of organic matter is kept
as low as possible (see paragraph (e)(3), since the presence of organic
carbon may limit the bioavailability of the test substance under paragraph
(k)(6) of this guideline.

(iii) Since many feeds are derived from fishmeal, the feed should be
analyzed for the test substance. It is also desirable to analyze the feed
for pesticides and heavy metals.

(g) Exposure conditions during test—(1) Optional preliminary
test to determine optimal conditions. It may be useful to conduct a pre-
liminary experiment in order to optimize the test conditions of the defini-
tive test, e.g. selection of test substance concentrations, duration of the
uptake and depuration phases.
-------
(2) Exposure concentrations of test chemical, (i) During the uptake
phase, expose fish under flow-through conditions to at least two concentra-
tions of the test substance in water. Normally, select the higher (or highest)
concentration of the test substance to be about 1 percent of its acute as-
ymptotic LC50, and to be at least tenfold higher than its detection limit
in water by the analytical method used. The highest test concentration can
also be determined by dividing the acute 96-h LC50 by an appropriate
acute/chronic ratio (e.g. appropriate ratios for some chemicals are about
3, but a few are above 100). If possible, choose the other concentrations
such that it differs from the one above by a factor of 10. If this is not
possible because of the 1 percent of LC50 criterion and the analytical limit,
a lower factor than 10 can be used or the use of 14C labeled test substance
should be considered.

(ii) No exposure concentration used should be above the solubility
in water of the test substance.

(iii) Where a solubilizing agent is used in the stock solution, its di-
luted concentration in the exposure water should not be greater than
0.1 mL/L and should be the same in all test vessels. Its contribution (to-
gether with the test substance) to the overall content of organic carbon
in the test water should be known. However, every effort should be made
to avoid the use of such materials.

(iv) Minimize results reported as "not detected at the limit of detec-
tion" by pretest method development and experimental design, since such
results cannot be used for rate constant calculations. Pretest results can
be used to determine the exposure concentrations necessary to ensure that
concentrations in fish tissue are generally above method detection limits.

(3) Duration of uptake phase, (i) A prediction of the duration of
the uptake phase and time required to reach steady state can be obtained
from practical experience (e.g. from a previous study or an accumulation
study on a structurally related chemical) or from certain empirical relation-
ships utilizing knowledge of either the solubility in water or the octanol/
water partition coefficient of the test substance (see paragraph (g)(5) of
this guideline).

(ii) The uptake phase should be run for 28 days unless it can be
demonstrated that equilibrium has been reached earlier. If the steady-state
has not been reached by 28 days, the uptake phase should be extended,
taking further measurements, until steady-state is reached or 60 days,
whichever is shorter. The depuration phase is then begun.

(4) Duration of depuration phase, (i) The depuration period is
begun by transferring the fish to the same medium but without the test
substance in another clean vessel. A depuration phase is always necessary
unless uptake of the substance during the uptake phase has been insignifi-
cant (e.g. the BCF is less than 10).

10
-------
(ii) A period of half the duration of the uptake phase is usually suffi-
cient for an appropriate (e.g. 95 percent) reduction in the body burden
of the substance to occur (see paragraph (g)(5) of this guideline for an
explanation of the estimation). If the time required to reach 95 percent
loss is unpractically long, exceeding for example twice the normal duration
of the uptake phase (i.e. more than 56 days) a shorter period may be used
(e.g. until the concentration of test substance is less than 10 percent of
steady-state concentration). However, for substances having more complex
patterns of uptake and depuration than are represented by a one-compart-
ment fish model, yielding first order kinetics, allow longer depuration
phases for determination of loss rate constants. The period may, however,
be governed by the period over which the concentration of test substance
in the fish remains above the analytical detection limit.

(5) Prediction of the duration of the uptake and depuration
phases — (i) Prediction of the duration of the uptake phase. (A) Before
performing the test, an estimate of k2 and hence some percentage of the
time needed to reach steady-state may be obtained from empirical relation-
ships between k2 and the n-octanol/water partition coefficient (Pow) or k2
and the aqueous solubilities.

(B) (1) An estimate of k2 (day-1) may be obtained from the following
empirical relationship (see paragraph (k)(20) of this guideline):

Equation 1

log k2 = -0.414 log Pow + 1.47(r2 = 0.95)

For other relationships see see paragraph (k)(14) of this guideline.

(2) If the partition coefficient (Pow) is not known, an estimate can
be made (see paragraph (k)(4) of this guideline) from a knowledge of the
aqueous solubility (s) of the substance using:

Equation 2

log Pow = 0.862 log(s) + 0.710(r2 = 0.994)

where s = solubility expressed as moles per liter: (n=36)
(3) These relationships apply only to chemicals with log Po
between 2 and 6.5 (see paragraph (k)(12) of this guideline).
values
The time to reach some percentage of steady-state may be obtained
by applying the k2-estimate, from the general kinetic equation describing
uptake and depuration (first-order kinetics):
or, if Cw is constant:
11
-------
Equation 3

Cf=k1/k2-Cw(l-(exp)-^«

When steady-state is approached (as t approaches infinity), equation
3 may be reduced (see paragraphs (k)(3) and (k)(9) of this guideline) to:

Cf=ki/k2Cw

Cf/Cw = ki/k2 = BCF

Then ki/k2-Cw is an approach to the concentration in the fish at steady-
state (Cf,s). Equation 3 may be transcribed to:
or

Equation 4

Cf/Cf(S = 1 - e-**

Applying equation 4, the time to reach some percentage of steady-state
may be predicted when ki is preestimated using equation 1 or 2.

As a guideline, the statistically optimal duration of the uptake phase
for the production of statistically acceptable data (BCFK) is that period
which is required for the curve of the logarithm of the concentration of
the test substance in fish plotted against linear time to reach its midpoint,
or 1.6/k2, or 80 percent of steady-state but not more than 3.0/k2 or 95
percent of steady-state (see paragraph (k)(19) of this guideline).

The time to reach 80 percent of steady-state is (equation 4):

0.8 - 1 - e-k2'

Equation 5

t8o= 1.6/k2

Similarly 95 percent of steady-state is:

Equation 6

t95 - 3.0/k2

For example, the duration of the uptake phase (up) for a test substance
with log Pow = 4 would be (using equations 1,5, and 6):

12
-------
:2 = -0.414.(4) + 1.47
k2 = 0.652 days-'
up (80 pct)= 1.6/0.652 i.e. 2.45 days (59 h)
or
up (95 pct)= 3.0/0.652 i.e. 4.60 days (110 h)
Similarly, for a test substance with s = 10~5 mol/L, (log(s) = - 5.0),
the duration of up would be (using equations 1, 2 and 5, 6):
log (Pow) = -0.862 (-5.0) + 0.710 = 5.02
log k2 =-0.414 (5.02)+1.47
k2 = 0.246 days-'
up (80 pet) = 1.6/0.246, i.e. 6.5 days (156 hours)
or
up (95 pet) = 3.0/0.246, i.e. 12.2 days (293 hours)
Alternatively, the expression:
teq = 6.54 x 10-3POW + 55.31 (hours)
may be used to calculate the time for effective steady-state to be reached
(see paragraph (k)(12) of this guideline).
(ii) Prediction of the duration of the depuration phase. (A) A pre-
diction of the time needed to reduce the body burden to some percentage
of the initial concentration may also be obtained from the general equation
describing uptake and depuration (first order kinetics) (see paragraphs
(k)(13) and (k)(20) of this guideline.
For the depuration phase, Cw is assumed to be zero. The equation
may then be reduced to:
dCf/dt - -k2Cf
or
where Cf,o is the concentration at the start of the depuration period.
50 percent depuration will then be reached at the time (tso):
or
Similarly 95 percent depuration will be reached at:
13
-------
t95« 3.0/k2

If 80 percent uptake is used for the first period (1.6/k2) and 95 percent
loss in the depuration phase (3.0/k2), then depuration phase is approxi-
mately twice the duration of the uptake phase.

It is important to note, however, that the estimations are based on
the assumption that uptake and depuration patterns will follow first order
kinetics. If first order kinetics are obviously not obeyed, more complex
models should be employed (e.g. paragraph (k)(16) of this guideline).

(6) Numbers and characteristics of test fish, (i) Select the numbers
of fish per test concentration such that a minimum of four fish per sample
are available at each sampling. If greater statistical power is required, more
fish per sample will be necessary.

(ii) If adult fish are used, report whether male or female, or both
are used in the experiment. If both sexes are used, differences in lipid
content between sexes should be documented to be nonsignificant before
the start of the exposure; pooling all male and all female fish may be
necessary.

(iii) In any one test, select fish of similar weight such that the smallest
are no smaller than two-thirds of the weight of the largest. All should
be of the same year-class and come from the same source. Since weight
and age of a fish appear sometimes to have a significant effect on BCF
values (see paragraph (k)(6) of this guideline) record these details accu-
rately. It is recommended that a sub-sample of the stock of fish is weighed
before the test in order to estimate the mean weight (see paragraph (h)(2)
of this guideline).

(7) Loading of fish, (i) Use high water-to-fish ratios in order to mini-
mize the reduction in Cw caused by the addition of the fish at the start
of the test and also to avoid decreases in dissolved oxygen concentration.
It is important that the loading rate is appropriate for the test species used.
In any case, a loading rate of 0.1-1.0 g of fish (wet weight) per liter
of water per day is normally recommended. High loading rates can be
used if it is shown that the required concentration of test substance can
be maintained within ±20 percent limits, and that the concentration of
dissolved oxygen does not fall below 60 percent saturation.

(ii) In choosing appropriate loading regimes, take account of the nor-
mal habitat of the fish species. For example, bottom-living fish may de-
mand a larger bottom area of the aquarium for the same volume of water
than pelagic fish species.

(8) Light and temperature. The photoperiod is usually 12 to 16 h
and the temperature (±2 °C) should be appropriate for the test species
(see Table 3. under paragraph (e)(6)(i) of this guideline). The type and

14
-------
characteristics of illumination should be known. Caution should be given
to the possible phototransformation of the test substance under the irradia-
tion conditions of the study. Appropriate illumination should be used
avoiding exposure of fish to unnatural photoproducts. In some cases it
may be appropriate to use a filter to screen out UV irradiation below
290 nm.

(9) Water quality measurements. During the test, dissolved oxygen,
TOC, pH and temperature should be measured in all vessels. Total hard-
ness and salinity (if relevant) should be measured in the controls and one
vessel at the higher (or highest) concentration. As a minimum, dissolved
oxygen and salinity (if relevant) should be measured 3 times—at the begin-
ning, around the middle, and end of the uptake period—and once a week
in the depuration period. TOC should be measured at the beginning of
the test (24 h and 48 h prior to test initiation of uptake phase) before
addition of the fish and, at least once a week, during both uptake and
depuration phases. Temperature should be measured daily, pH at the begin-
ning and end of each period and hardness once each test. Temperature
should preferably be monitored continuously in at least one vessel.

(10) Controls. In addition to the two test concentrations, a control
group of fish is held under identical conditions except for the absence
of the test substance, to relate possible adverse effects observed in the
bioconcentration test to a matching control group and to obtain background
concentrations of test substance. One dilution water control and if relevant,
one control containing the solubilizing agent should be run.

(11) Validity of test. For a test to be valid the following conditions
apply:

(i) The temperature variation is less than ± 2 °C.

(ii) The concentration of dissolved oxygen does not fall below
60 percent saturation.

(iii) The concentration of the test substance in the chambers is main-
tained within ± 20 percent of the mean of the measured values during the
uptake phase.

(iv) The mortality or other adverse effects/disease in both control and
treated fish is less than 10 percent at the end of the test; where the test
is extended over several weeks or months, death or other adverse effects
in both sets of fish should be less than 5 percent per month and not exceed
30 percent in all.

(h) Sampling and analysis of fish and water—(1) Fish and water
sampling schedule, (i) Sample water from the test chambers for the deter-
mination of test substance concentration before addition of the fish and
during both uptake and depuration phases. As a minimum, sample the

15
-------
water at the same time as the fish and before feeding. During the uptake
phase, the concentrations of test substance are determined in order to check
compliance with the validity criteria (see paragraph (g)(ll) of this guide-
line).

(ii) Sample fish on at least five occasions during the uptake phase
and at least on four occasions during the depuration phase. Since on some
occasions it will be difficult to calculate a reasonably precise estimate of
the BCF value based on this number of samples (especially when other
than simple first-order depuration kinetics are indicated), it may be advis-
able to take samples at a higher frequency in both periods (see the follow-
ing Table 3.) Store the extra samples as described in paragraph (h)(3) and
analyze them only if the results of the first round of analyses prove inad-
equate for the calculation of the BCF with the desired precision.

(iii) An example of an acceptable sampling schedule is given in the
following Table 3.
Table 3.—Theoretical example of sampling schedule for bioconcentration tests of substances
with log Row = 4
Fish Sampling
Uptake phase

1st

2nd

3rd

4th

5th
Depuration phase
6th

7th

8th

9th

Sample time schedule
Minimal re-
quired fre-
quency
(days)
-1
0
0.3
0.3
0.6
1.2
2.4
4.7
5.0
5.9
9.3
14.0
Additional
sampling
(days)
0.9
1.7
3.3
5.3
7.0
11.2
17.5
No. of water
samples**
2*
2
2
(2)
2
(2)
2
(2)
2
(2)
2
No. of fish per sample**
Add 45-80 fish
4
(4)
4
(4)
4
(4)
4
(4)
6
Transfer fish to water
free of test chemical
4
(4)
4
(4)
4
(4)
6
f4)
*Sample water after minimum of 3 "chamber-volumes" have been deliv-
ered.

**Values in parentheses are numbers of samples (water, fish) to be taken
if additional sampling is carried out.
16
-------
Note: Pretest estimate of k2 for log Pow of 4.0 is 0.652 days-1. The total
duration of the experiment is set to 3 x up — 3 x 4.6 days = 14 days.
For the estimation of up see paragraph (g)(5) of this guideline.

Other schedules can readily be calculated using other assumed values of
POW to calculate the exposure time for 95 percent uptake.

(iv) Continue sampling during the uptake phase until a steady-state
has been established or for 28 days, whichever is the shorter. If the steady-
state has not been reached within 28 days continue until a steady-state
has been attained or 60 days, whichever is shorter. Before beginning the
depuration phase transfer the fish to clean tanks.

(2) Sampling methodology, (i) Obtain water samples for analysis by
siphoning through inert tubing from a central point in the test chamber.
Since neither filtration nor centrifuging appears always to separate the
nonbioavailable fraction of the test substance from that which is
bioavailable (especially for superlipophilic chemicals, those chemicals with
a log Pow greater than or equal to 5) (see paragraphs (k)(6) and (k)(8)
of this guideline), samples may not be subjected to those treatments. In-
stead, measures should be taken to keep the tanks as clean as possible
and the content of total organic carbon should be monitored during both
the uptake and depuration phases (see paragraph (g)(9) of this guideline).

(ii) Remove an appropriate number of fish (normally a minimum of
four) from the test chambers at each sampling time. Rinse the sampled
fish quickly with water, blot dry, kill instantly, using the most appropriate
and humane method, and then weigh.

(3) Sample storage, (i) It is preferable to analyze fish and water im-
mediately after sampling in order to prevent degradation or other losses
and to calculate approximate uptake and depuration rates as the test pro-
ceeds. Immediate analysis also avoids delay in determining when a plateau
has been reached.

(ii) Failing immediate analysis, store the samples by an appropriate
method. Obtain information on the proper method of storage for the par-
ticular test substance before the beginning of the study—for example,
deep-freezing, holding at 4 °C, duration of storage, extraction, etc.

(4) Analysis of fish samples, (i) Radiolabeled test substances can
facilitate the analysis of water and fish samples, and may be used to deter-
mine whether degradate identification and quantification should be made.
BFCs based on total radiolabeled residues (e.g. by combustion or tissue
solubilization) can serve as one of the criteria for determining if degrades
identification and quantification is necessary. However, BCF determina-
tions for the parent compound should be based upon the concentration
of the parent compound in fish and water, not upon total radiolabeled resi-

U.S. EPA Headquarters Library
17 Mail code 3201
1' 1200 Pennsylvania Avenue NW
Washington DC 20460
-------
(ii) If the BCF in terms of total radiolabeled residues is greater than
or equal to 1,000, it may be advisable, and for certain categories of chemi-
cals such as pesticides strongly recommended, to identify and quantify
degradates representing greater than or equal to 10 percent of total residues
in fish tissues at steady state. If degradates representing greater than or
equal to 10 percent of total radiolabeled residues in the fish tissue are
identified and quantified, then it is also recommended to identify and quan-
tify degradates in the test water. The major metabolites may be character-
ized at steady-state or at the end of the uptake phase, whichever is the
sooner. It is possible to combine a fish metabolism study with a
bioconcentration study to identify and quantify residues in tissues.

(iii) The concentration of the test substance should usually be deter-
mined for each weighed individual fish. If this is not possible, pooling
of the samples on each sampling occasion may be done but pooling does
restrict the statistical procedures which can be applied to the data. If a
specific statistical procedure and power are important considerations, then
an adequate number of fish to accommodate the desired pooling, procedure
and power, should be included in the test. See paragraphs (k)(7) and
(k)(10) of this guideline for an introduction to relevant pooling procedures.

(5) Determination of lipid content. BCF should be expressed both
as a function of total wet weight and, for high lipophilic substances, as
a function of the lipid content. Determine the lipid content of the fish
on each sampling occasion if possible. Suitable methods should be used
for determination of lipid content (see paragraphs (k)(5) and (k)(15) of
this guideline). Chloroform/methanol extraction technique may be rec-
ommended as standard method (see paragraph (k)(ll) of this guideline).
The various methods do not give identical values (see paragraph (k)(18)
of this guideline), so it is important to give details of the method used.
When possible, the analysis for lipid should be made on the same extract
as that produced for analysis for the test substance, since the lipids often
have to be removed from the extract before it can be analyzed
chromatographically. The lipid content of the fish (as mg/kg wet weight)
at the end of the experiment should not differ from that at the start by
more ±25 percent. The tissue percent solids should also be reported to
allow conversion of lipid concentration from a wet to a dry basis.

(6) Quality of analytical method. Since the whole procedure is gov-
erned essentially by the accuracy, precision, and sensitivity of the analyt-
ical method used for the test substance, check the precision and reproduc-
ibility of the chemical analysis experimentally, as well as recovery of the
test substance from both water and fish to ensure that they are satisfactory
for the particular method. Also, check that the test substance is not detect-
able in the dilution water used. If necessary, correct the values of Cw and
Cf obtained from the test for the recoveries and background values of con-
trols. Handle the fish and water samples throughout in such a manner as

18
-------
to minimize contamination and loss (e.g. resulting from adsorption by the
sampling device).

(i) Data analysis—(1) Determination of uptake and depuration
rate constants, (i) Obtain the uptake and depuration curves of the test
substance by plotting its concentration in/on fish (or specified tissues) in
the uptake and in the depuration phase against time on arithmetic scales.
The depuration rate constant (k2) is usually determined from the depuration
curve (i.e. a plot of the decrease in test substance concentration in the
fish with time). The uptake rate constant (ki) is then calculated given ka
and a value of Cf which is derived from the uptake curve. See paragraph
(d)(2)(iv) of this guideline for a description of these methods. The pre-
ferred method for obtaining BCFK and the rate constants, ki and k2, is
to use nonlinear parameter estimation methods on a computer (see para-
graph (k)(15) of this guideline). Otherwise, graphical methods may be used
to calculate ki and k2- If the depuration curve is obviously not first-order,
then more complex models should be employed (see paragraphs (k)(3),
(k)(4), (k)(9), (k)(12), (k)(13), (k)(14), (k)(19, and (k)(20) of this guide-
line) and advice sought from a biostatistician.

(ii) The uptake rate constant, the depuration (loss) rate constant (or
constants, where more complex models are involved), the bioconcentration
factor, and where possible, the confidence limits of each of these param-
eters are calculated from the model that best describes the measured con-
centrations of test substance in fish and water.

(iii) The results should be interpreted with caution where measured
concentrations of test solutions occur at levels near the detection limit of
the analytical method. Clearly defined uptake and loss curves are an indi-
cation of good quality bioconcentration data. The variation in uptake/
depuration constants between the two test concentrations should be less
than 20 percent. Observed significant differences in uptake/depuration
rates between the two applied test concentrations should be recorded and
possible explanations given. Generally the confidence limit of BCFs from
well-designed studies approach ± 20 percent.

(2) Determination of the steady state and kinetic BCFs. (i) Obtain
the uptake curve of the test substance by plotting its concentration in/on
fish (or specified tissues) in the uptake phase against time on arithmetic
scales. If the curve has reached a plateau, that is, become approximately
asymptotic to the time axis, calculate the steady state BCFs from the fol-
lowing relationship:

Cf at steady state (mean)/Cw at steady state (mean)

(ii) When no steady state is reached, it may be possible to calculate
a BCFs of sufficient precision for hazard assessment from a steady-state
at 80 percent (1.6/k2) or 95 percent (3.0/k2) of equilibrium.

19
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(iii) Determine the concentration factor (BCFK) as the ratio
the two first-order kinetic constants.

(iv) The BCF is expressed as a function of the total wet weight of
the fish. However, for special purposes, specified tissues or organs (e.g.
muscle, liver), may be used if the fish are sufficiently large or the fish
may be divided into edible (fillet) and nonedible (viscera) fractions. Since,
for many organic substances, there is a clear relationship between the po-
tential for bioconcentration and lipophilicity, there is also a corresponding
relationship between the lipid content of the test fish and the observed
bioconcentration of such substances. Thus, to reduce this source of varia-
bility in test results for those substances with high lipophilicity (i.e. with
log POW greater than or equal to 3), bioconcentration should be expressed
in relation to lipid content in addition to whole body weight. The lipid
content should be determined on the same biological material as is used
to determine the concentration of the test substance, when feasible.

(j) Test report. The test report must include the following informa-
tion.

(1) Summary. Test chemical and test species, uptake and depuration
rate constants, and steady state and kinetic BCFs

(2) Materials, (i) Exposure tanks and tubes-material and size of
tanks.

(ii) Diluter-type and description.

(iii) Dilution water. Source, description of any pretreatment, and
water characteristics including pH, hardness, temperature, dissolved oxy-
gen concentration, residual chlorine levels (if measured), total organic car-
bon, suspended solids, salinity of the test medium (if appropriate) and any
other measurements made.

(iv) Test substance. Physical nature and, where relevant, physico-
chemical properties; chemical identification data (including the organic
carbon content, if appropriate); if radio-labeled, the precise position of the
labeled atoms and the percentage of radioactivity associated with impuri-
ties.

(v) Stock solutions. Method of preparation of stock solutions and fre-
quency of renewal (the solubilizing agent, its concentration and its con-
tribution to the organic carbon content of test water must be given, when
used).

(vi) Test species. Scientific name, strain, source, any pretreatment,
age, size-range, etc.

20
-------
(vii) Care of fish. Acclimation, pretest mortality and health, feeding
(e.g. type of foods, source, composition—at least lipid and protein content
if possible, amount given and frequency).

(3) Test conditions— (i) Test design. Number and size of test cham-
bers, water volume replacement rate, number of replicates, number of fish
per replicate tank, number of test concentrations, controls,

(ii) Exposure concentrations. The nominal concentrations, the means
of the measured values and their standard deviations in the test vessels.

(iii) Length of uptake and depuration phases. Give the lengths of
the uptake and depuration phases and the rationale behind them

(iv) Light. Type and characteristics of illumination used and
photoperiods.

(v) Water quality within test vessels. pH, hardness, TOC, tempera-
ture and dissolved oxygen concentration.

(4) Sampling and analysis, (i) Sampling frequency for fish and water
samples.

(ii) Sample storage.

(iii) Sample extraction and analysis.

(iv) Detection and quantification limits.

(v) Accuracy and precision—results of spike and replicate analyses

(5) Results, (i) Data obtained in any preliminary test.

(ii) Validity of the test. Fish mortality and/or abnormal behavior for
exposed and control, variations in exposure concentrations, variations in
temperature, and minimum dissolved oxygen with respect to test validity
criteria.

(iii) Lipid content of the test fish.

(iv) Uptake and depuration curves of the test chemical in fish; graphi-
cal representation of data.

(v) Concentrations of parent in fish tissue and exposure water. Tab-
ular representation of data; Cf and Cw (with standard deviation and range,
if appropriate) for all sampling times (Cf expressed in milligrams per gam
of wet weight (parts per million) of whole body or specified tissues thereof
e.g. lipid, and Cw expressed in milligrams per gam of wet weight (parts
per million). Cw values for the control series (background should also be
reported).

21
-------
(vi) Uptake and depuration rate constants. Give values and 95 percent
confidence limits for the uptake and depuration (loss) rate constants, de-
scribe the computation.

(vii) Steady state and kinetic BCFs. The BCFs and the BCFK (both
expressed in relation to the whole body and the total lipid content, if meas-
ured, of the animal or specified tissues thereof), confidence limits and
standard deviation (as available).

(viii) Degradate concentrations. Where radiolabeled substances are
used, and when required, the accumulation of any major metabolites at
steady state or at the end of the uptake phase.

(ix) Deviations and/or unusual observations. Report anything unusual
about the test, any deviation from these procedures, and any other relevant
information.

(k) References. The following references should be consulted for ad-
ditional background material on this test guideline.

(1) American Society for Testing and Materials. ASTM E-1022-84.
Standard Practice for conducting Bioconcentration Tests with Fishes and
Saltwater Bivalve Molluscs (1988).

(2) Bintein, S. et al. Nonlinear dependance of fish bioconcentration
on w-octanol/water partition coefficient. Environmental Research 1:29—390
(1993).

(3) Branson, D.R. et al. Transactions of the American Fisheries Soci-
ety 104:785-792 (1975).

(4) Chiou, C.T. and Schmedding D.W. Partitioning of organic com-
pounds in octanol-water systems. Environmental Science and Technology
16:4-10 (1982).

(5) Compaan, H. Chapter 2.3, Part II in The determination of the
possible effects of chemicals and wastes on the aquatic environment: deg-
radation, toxiciiy, bioaccumulation. Government Publishing Office, The
Hague, The Netherlands (1980).

(6) Connell, D.W. Bioaccumulation behavior of persistent chemicals
with aquatic organisms. Reviews of Environmental Contaminant Toxi-
cology W2:l 11-156 (19SZ).

(7) Environmental Protection Agency. Section 5, A(l) Analysis of
Human or Animal Adipose Tissue in Analysis of Pesticide Residues in
Human and Environmental Samples. Thompson J.F. (ed). Research Tri-
angle Park, NC 27711 (1974).

22
-------
(8) Environmental Protection Agency. 822-R-94-002. Great Lake
Water Quality Initiative Technical Support Document for the Procedure
to Determine Bioaccumulation Factors (1994).

(9) Ernst W. Accumulation in Aquatic Organisms. In: Appraisal of
tests to predict the environmental behavior of chemicals. Ed. by Sheehman
P., Korte F., Klein W. and Bourdeau P.M. Part 4.4 pp 243-255. 1985
SCOPE, John Wiley & Sons Ltd., New York (1985).

(10) Food and Drug Administration. Pesticide analytical manual. Vol.
1. 5600 Fisher's Lane, Rockville, MD 20852, (1975).

(11) Gardner et al. Limnology and Oceanography 30:1099-1105
(1995).

(12) Hawker, D.W. and D.W. Cornell D.W. Influence of partition
coefficient of lipophilic compounds on bioconcentration kinetics with fish.
Water Research 22: 701-707.

(13) Konemann, H. and K. Van Leeuwen Toxicokinetics in Fish: Ac-
cumulation and Elimination of Six Chlorobenzenes by Guppies. Chemo-
sphere 9:3-19 (1980).

(14) Kristensen P. (1991) Bioconcentration in fish: comparison of
bioconcentration factors derived from OECD and ASTM testing methods;
influence of paniculate organic matter to the bioavailability of chemicals.
Water Quality Institute, Denmark.

(15) Kristensen, P. and N. Nyholm. CEC. Bioaccumulation of chemi-
cal substances in fish: the flow-through method—Ring Test Programme,
1984-1985 Final report, March 1987.

(16) Organization for Economic Cooperation and Development.
Guidelines for testing of chemicals. Paris (1993).

(17) OECD, Paris (1995). Direct Phototransformation of chemicals
in water. Guidance Document. February 1996.

(18) Randall R.C., Lee H., Ozretich R.J., Lake J.L. and Pruell
R.J.(1991). Evaluation of selected lipid methods for normalizing pollutant
bioaccumulation. Environ. Toxicol. Chem. Vol.10, pp. 1431-1436.

(19) Reilly P.M. et al. Guidelines for the optimal design of experi-
ments to estimate parameters in first order kinetic models. Canadian Jour-
nal of Chemical Engineering 55:614-622 (1977).

(20) Spacie, A. and J.L. Hamelink Alternative models for describing
the bioconcentration of organics in fish. Environmental Toxicology and
Chemistry 1:309-320 (1982).

23
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-------
&EPA
United States
Environmental Protection
Agency
Prevention. Pesticides
and Toxic Substances
(7101)
EPA712-C-96-354
April 1996
Ecological Effects Test
Guidelines
OPPTS 850.1735
Whole Sediment Acute
Toxicity Invertebrates,
Freshwater
"Public Draft"
-------
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.

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."
-------
OPPTS 850.1735 Whole sediment acute toxicity invertebrates,
freshwater.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.}.

(2) [Reserved]

(b) Objective. This guideline may be used to determine the toxicity
and bioaccumulation potential of chemicals in sediments in freshwater in-
vertebrates. Natural sediment is spiked with different concentrations of test
chemical and the results from the sediment toxicity tests can be used to
determine causal relationships between the chemical and biological re-
sponse. Reported endpoints from whole sediment toxicity tests may in-
clude the LC50 (median lethal concentration), EC50 (median effective
concentration), NOEC (no-observable-effect-concentration), or the LOEC
(lowest-observable-effect-concentration).

Clean. Clean denotes a sediment or water that does not contain con-
centrations of test materials which cause apparent stress to the test orga-
nisms or reduce their survival.

Concentration. Concentration is the ratio of weight or volume of test
material(s) to the weight or volume of sediment.

Contaminated sediment. Contaminated sediment is sediment contain-
ing chemical substances at concentrations that pose a known or suspected
threat to environmental or human health.

Control sediment. Control sediment is sediment that is essentially free
of contaminants and is used routinely to assess the acceptability of a test.
Any contaminants in control sediment may originate from the global
spread of pollutants and does not reflect any substantial input from local
or non-point sources. Comparing test sediments to control sediments is
a measure of the toxicity of a test sediment beyond inevitable background
contamination.

Effect concentration (EC). Effect concentration is the toxicant con-
centration that would cause an effect in a given percent of the test popu-
lation. Identical to LC when the observable adverse effect is death. For
example, the EC50 is the concentration of toxicant that would cause death
in 50% of the test population.

Inhibition concentration (1C). Inhibition concentration is the toxicant
concentration that would cause a given percent reduction in a non-quantal
measurement for the test population. For example, the IC25 is the con-
centration of toxicant that would cause a 25% reduction in growth for
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the test population and the IC50 is the concentration of toxicant that would
cause a 50% reduction.

Interstitial water or pore water. Interstitial water or pore water is
water occupying space between sediment or soil particles.

Lethal concentration (LC). Lethal concentration is the toxicant con-
centration that would cause death in a given percent of the test population.
Identical to EC when the observable adverse effect is death. For example,
the LC50 is the concentration of toxicant that would cause death in 50%
of the test population.

Lowest observable effect concentration (LOEC), Lowest observable
effect concentration is the lowest concentration of a toxicant to which or-
ganisms are exposed in a test which causes an adverse effect on the test
organisms (i.e., where the value for the observed response is statistically
significant different from the controls).

No observable effect concentration (NOEC). No observable effect
concentration is the highest concentration of a toxicant to which organisms
are exposed in a test that causes no observable adverse effect on the test
organisms (i.e., the highest concentration of a toxicant in which the value
for the observed response is not statistically significant different from the
controls).

Overlying water. Overlying water is the water placed over sediment
hi a test chamber during a test.

ppt. ppt is parts per thousand.

Reference sediment. Reference sediment is a whole sediment near an
area of concern used to assess sediment conditions exclusive of material(s)
of interest. The reference sediment may be used as an indicator of local-
ized sediment conditions exclusive of the specific pollutant input of con-
cern. Such sediment would be collected near the site of concern and would
represent the background conditions resulting from any localized pollutant
inputs as well as global pollutant input. This is the manner in which ref-
erence sediment is used in dredge material evaluations.

Reference-toxicity test. Reference-toxicity test is a test conducted in
conjunction with sediment tests to determine possible changes in condition
of the test organisms. Deviations outside an established normal range indi-
cate a change in the condition of the test organism population. Reference-
toxicity tests are most often performed in the absence of sediment.

Sediment. Sediment is paniculate material that usually lies below
water. Formulated particulate material that is intended to lie below water
in a test.
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Spiked sediment. Spiked sediment is a sediment to which a material
has been added for experimental purposes.

Whole sediment. Whole sediment is sediment and associated pore
water which have had minimal manipulation. The term bulk sediment has
been used synonymously with whole sediment.

(d) Test method. (1) Whole sediment toxicity tests are outlined for
the amphipod, Hyalella azteca and the midge, Chironomus tentans. Dura-
tion of whole sediment tests is 10 to 28 days and is accomplished in 300-
mL test chambers containing 100 mL of sediment and 175 mL of overlying
water. The overlying water may be renewed daily or a flow-through sys-
tem may be used. Test organisms are fed during the toxicity test. The
endpoint for H. azteca is survival, and for C. tentans, survival, growth
and/or emergence.

(2) A range-finding test to establish a suitable range of test concentra-
tions is recommended. A definitive test will not be required if no toxicity
is observed at concentrations of 100 mg/kg dry weight of sediment.

(e) Water, formulated sediment, reagents, and standards—(1)
Water, (i) Testing and culture water must be of uniform quality, and is
acceptable if it allows satisfactory survival, growth, and reproduction of
the test organisms. Disease or apparent stress (e.g. discoloration, unusual
behavior) should not be prevalent. If problems occur during testing or cul-
turing, water characteristics should be analyzed.

(ii) Natural water is considered to be of uniform quality if the ranges
of hardness, alkalinity, and specific conductance are within 10 percent of
the respective averages. The monthly pH range should be <0.4 units.
Sources of natural water should be uncontaminated well or spring or sur-
face water. Special considerations for surface water include minimizing
quality and contamination variables, maximizing the levels of DO, and
confirming that sulfides and iron levels are low. Chlorinated water should
not be used for testing or culturing because chlorine-produced oxidants
and residual chlorine are toxic to aquatic organisms. Tap water is accept-
able if it is dechlorinated, deionized, and carbon filtered, but its use is
not encouraged.

(iii) If source water is contaminated with facultative pathogens, it
should be UV-irradiated using intensity meters and flow-controls, or fil-
tered through 0.45 pm pore size.

(iv) The DO concentration of source water should be between 90 and
100 percent saturation. In some cases aeration may be required using air
stones, surface aerators, or column aerators.

(v) High-purity distilled or deionized water may be reconstituted by
adding specified amounts of reagent grade chemicals. The deionization
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system should produce water with a resistance of 1 MH. For each batch
of reconstituted water, the following parameters should be measured: Con-
ductivity, pH, hardness, DO, and alkalinity. Aeration should be employed
to maintain acceptable levels of pH and DO.

(vi) The preparation of 100 L of reconstituted water was developed
at the USEPA EMSL-Cincinnati and has been tested with H. azteca, C.
tertians, and Chironomus riparius in round-robin tests and is given as fol-
lows:

(A) Add approximately 75 L of deionized water to a properly cleaned
container capable of holding 100 L.

(B) Add 5 g of CaSO4 and 5 g of CaCl2 to a 2-L aliquot of deionized
water and mix (e.g., on a stir plate) for 30 min or until the salts dissolve.

(C) Add 3 g of MgSO4, 9.6 g NaHCO3, and 0.4 g KC1 to a second
2-L aliquot of deionized water and mix on a stir plate for 30 min.

(D) Pour the two 2-L aliquots containing the dissolved salts into the
75 L of deionized water and fill the carboy to 100 L with deionized water.

(E) Aerate the mixture for at least 24 h before use.

(F) The water quality of the reconstituted water should be approxi-
mately the following: Hardness, 90 to 100 mg/L as CaCOa, alkalinity 50
to 60 mg/L as CaCO3> conductivity 330 to 360 ^.S/crn, and pH 7.8 to
8.2.

(vii) Synthetic seawater may be prepared by adding commercial sea
salts to deionized water. H. azteca may be cultured or tested at salinities
up to 15 ppt.

(2) Artificial sediment. Artificial sediments consist of mixtures of
materials designed to mimic natural sediments. Because artificial sedi-
ments have not been used routinely to assess the toxicity of contaminants
in sediment, the use of uncontaminated natural sediment is recommended.
If the use of artificial sediment is necessary, detailed information may be
found in paragraph (1)(1) of this guideline.

(3) Reagents. All reagents and chemicals purchased from supply
houses should be accompanied by appropriate data sheets. All test mate-
rials should be reagent grade. However, if specified as necessary, commer-
cial product, technical-grade, or use-grade materials may be used. Dates
for receipt, opening, and shelf-life should be logged and maintained for
all chemicals and reagents. Do not use reagents beyond shelf-life dates.

(4) Standards. Acceptable standard methods for chemical and phys-
ical analyses should be used. When appropriate standard methods are not
-------
available or lack the required sensitivity, other sources should be consulted
for reliable methods.

(f) Sample collection, storage, manipulation, and characteriza-
tion—(1) Sample collection, (i) Procedures for handling natural sediments
should be established prior to collection. Pertinent data such as location,
time, core depth, water depth, and collection equipment should be re-
corded.

(ii) Replicate sampling should be used for the collection of natural
sediment to determine the variance in sediment characteristics. While some
disruption of the sediment is inevitable regardless of the sampling equip-
ment used, disruption of sediment should be kept to a minimum. Several
devices are available for collecting sediment, but benthic grab or core sam-
plers are recommended. The depth of sediment collected should reflect
the expected exposure. During sediment collection, exposure to direct sun-
light should be kept to a minimum. Cooling of sediment to 4 °C is rec-
ommended.

(2) Storage. Storage of sediment may affect bioavailability and tox-
icity. Although nonionic and nonvolatile organic contaminants in sediment
may not result in substantive changes, metals and metalloids may affect
redox, oxidation, or microbial metabolism in sediment. It is best to hold
sediments at 4 °C in the dark and test within 2 to 8 weeks after collection.
Long storage may result in changes of sediment properties. Sediment tests,
and especially pore water tests, should be performed within 2 weeks of
collection to minimize property changes in the sediment.

(3) Manipulation, (i) During homogenization, water above sediment
that may have settled during shipment should be mixed back into the sedi-
ment. Sieving should not be used to remove indigenous microorganisms,
unless an excessive number of oligochaetes are present. Because
oligochaetes may inhibit the growth of the test organisms, it may be advan-
tageous to remove them as well as other macroorganisms, rocks, wood,
and the like by sieving. If sieving is used, sediment samples should be
analyzed before and after sieving to document the influence of sieving
on sediment characteristics. Sediments collected from multiple locations
or sites may be pooled and mixed using suitable apparatus (e.g. stirring,
rolling mill, feed mixer, etc.).

(ii) The preparation of test sediment may be accomplished by the
spiking of natural or artificial sediments. Additional research is needed
before artificial sediments may be used routinely. The responses of spiked
sediment may be affected by mixing time and aging. Spiked sediment may
be aged for at least 1 month to achieve equilibrium with the spiked chemi-
cals, if the chemical is known to be persistent. Sediments spiked with in-
dustrial chemicals should be used as soon as possible. Point estimates of
toxicity or minimum concentrations at which toxic effects are observed
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may be determined by spiking natural sediments with a range of chemical
concentrations. The test material should be reagent grade unless there is
a specific need-to-use commercial product, technical-grade, or use-grade
material. Specific information required for all test materials includes but
is not limited to the following:

(A) Identity and concentration of major ingredients and impurities.

(B) Solubility in test water.

(D) When measured test concentrations are required, the precision
and bias of the analytical method at the planned concentrations of test
material.

(£) Recommended handling and disposal procedures.

(iii) Organic solvents should not be added to the sediment mixture
because they may affect the concentration of dissolved organic carbon in
pore water, and should not be used.

(4) Characterization, (i) The characteristics of all sediment should
be determined, and at a minimum, the following factors should be meas-
ured: pH and ammonia concentration of pore water, organic carbon content
(total organic carbon (TOC)), particle size distribution (percent sand, silt,
clay), and percent water content. Additional analyses are suggested and
include biological oxygen demand, chemical oxygen demand, cation ex-
change capacity, Eh, total inorganic carbon, total volatile solids, acid vola-
tile sulfides, metals, synthetic organic compounds, oil and grease, and pe-
troleum hydrocarbons. Various physicochemical parameters should also be
determined for interstitial water. Sediment characterization should also in-
clude qualitative parameters such as color, texture, and the presence of
macrophytes or animals.

(ii) Standard analytical methods should be used to determine chemical
and physical data. Precision, accuracy, and bias should be determined in
sediment, water, and tissue for each analytical method. Analysis should
include analytical standards and reagent blanks as well as recovery calcula-
tions.

(iii) Concentrations of spiked chemicals may be measured in sedi-
ment, interstitial water, and overlying water at the beginning and at the
end of the test if so required. Measurement of degradation products may
also be required. Sediment chemistry should be monitored during and at
the end of a test. Separate replicates resembling the biological replicates
and containing organisms should be specified for chemical sampling. The
concentration of test material in water is measured by pipetting water sam-
ples from 1 to 2 cm above the sediment surface. Caution should be used
to eliminate the presence of any surface debris, material from the sides
-------
of the chamber, or sediment in the overlying water sample. At the end
of the test, the test material may be removed for chemical analysis by
siphoning (without disturbing sediment) the overlying water. Appropriate
samples of sediment can then be removed for chemical analysis. The sug-
gested method for isolation of interstitial water is by centrifugation without
filtration.

(g) Collection, culture, and maintainence of test organisms—(1)
Hyalella azteca—(i) Life history. (A) H. azteca are found throughout
North and South America in permanent lakes, ponds, and streams. They
are commonly found in mesotrophic or eutrophic lakes that are capable
of supporting aquatic plants and that remain warm (20 to 30 °C) for most
of the summer months. Densities may exceed 10,000 M2 in optimal habi-
tats. H. azteca are epibenthic detritivores that burrow into the sediment.
They may be found in saline waters up to 29 percent, but are sensitive
to hardness (e.g. they are not found in waters with calcium at
< 7 mg/L and DO at < 2 mg/L).

(B) H. azteca reproduce sexually, averaging 18 eggs per brood and
approximately 15 broods every 152 days. Hatching occurs approximately
5 to 10 days after fertilization at 24 to 28 °C. They proceed through a
minimum of 9 instars, which are separated into 5 to 8 prereproductive
instars and an indefinite number of postreproductive instars. Instars 1
through 5 form the juvenile life stage, instars 6 and 7 form the adolescent
stage of development, instar 8 is the nuptial life stage, and later instars
form the adult stages of the amphipod.

(C) H. azteca may be cultured under illumination of 500-1,000 Ix.
They feed during daylight and avoid bright light by hiding under litter.

(D) H. azteca is tolerable of a wide range of temperatures (0-33 °C),
but are immobile at temperatures <10 °C and die at temperatures
>33 °C. Reproduction can occur at temperatures of 10-18 °C, but the
highest rate of reproduction occurs at temperatures between 26 and
28 °C.

(E) H. azteca can tolerate a wide range of substrates. Survival and
growth of have not been shown to be negatively affected by either particle
size (>90 percent silt and clay particles to 100 percent sand-sized particles)
or grain size and organic matter in 10-day tests. In tests where organisms
were not fed, survival decreased.

(ii) Culturing procedures. (A) To start a sediment test, 7- to
14-day-old amphipods must be produced. If growth is an endpoint, a nar-
rower range, such as 1- to 2-day-old amphipods should be used. Details
and further discussion of acceptable culture procedures for H. azteca are
presented in paragraph (1)(1) of this guideline.
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(B) H. azteca should be held and fed under the same conditions as
the mass culture for at least 2 days prior to test initiation.

(2) Chironomus tentans—(i) Life history. (A) C. tentans are found
in eutrophic ponds and lakes. In soft bottoms, approximately 95 percent
of chironomid larvae are found in the upper 10 cm. Chironomid larvae
are generally not found in sediments with hydrogen sulfide concentrations
>0.3mg/L.

(B) The aquatic phases of C. tentans include the larval and pupal
stages. Female chironomids can oviposit eggs within 24 h of emergence,
releasing a single gelatinous egg mass containing roughly 2,300 eggs.
Hatch occurs in 2 to 4 days at 23 °C. The emergence of pupae as adults
occurs after 21 days at 23 °C.

(C) C. tentans are able to tolerate a wide range of grain sizes and
percentage organic matter. However, low percentage organic matter in con-
junction with no feeding may result in decreased survival. Survival is best
above pH 6.5. Poor control survival occurs at pH <6.5. Growth may also
be impacted by coarser sediment.

(ii) Culturing procedures. (A) The third instar chironomids must be
used to start a sediment test. Larvae should develop to the third instar
within 9 to 11 days at a temperature of 23 °C. The instar stage of midges
must be confirmed by head capsule width (-0.38 mm). Weight and height
of midges should be monitored at the beginning of a sediment test. Details
and further discussion of acceptable culture procedures are presented in
paragraph (1)(1) of this guideline.

(B) The time to first emergence and the success of emergence should
be recorded for all culture chambers. Growth may be monitored by peri-
odically measuring the midge head capsule width.

(h) Test method: Hyalella azteca 10- to 28-day sediment toxicity
test—-(1) Test conditions. General test conditions required for a 10-day
sediment toxicity test with H. azteca are presented in the following table
XX. The 10-day sediment toxicity test must be conducted at 23 °C with
a 161ight:8dar photoperiod. Illumination should be approximately 500 to
1,000 Ix. The recommended test chambers are 300-mL high-form beakers
without lips containing 100 mL of sediment and 175 mL of overlying
water. The test is started using 10 7- to 14-day-old amphipods. Eight
replicates/treatment are recommended for routine testing. Because of po-
tential impacts on study results, feed added to the test chamber should
be kept to a minimum. Thoroughly mix food prior to removing aliquots.
In order to prevent bacterial and fungal growth, feeding should be sus-
pended for 1 to 2 days if food collects on sediment. Feeding should also
be suspended if DO falls below 40 percent of saturation. When feeding
is suspended in one treatment it should be suspended in all treatments.
Feeding rates and appearance of sediment surface should be observed daily

8
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and detailed records maintained. Each chamber should receive 2 volume
additions per day or flow-through of overlying water. Sources of overlying
water can be culture water, well water, surface water, site water, or recon-
stituted water.
Table XX.—General Test Conditions for 10-day Sediment Toxicity with H. azteca
Parameter
Conditions
1. Test type
2. Temperature
3. Light quality
4. Illuminance
5. Photoperiod
6. Test chamber
7. Sediment volume
8. Overlying water volume
9. Renewal of overlying water
10. Age of organisms
11. Number of organisms/chamber
12. Number or replicate chambers/
treatment.
13. Feeding
14. Aeration
15. Overlying water
16. Test chamber cleaning
17. Overlying water quality
18. Test duration
19. Endpoints
20. Test acceptability
Whole-sediment toxicity test with renewal of overlying water
23± 1 °C
Wide-spectrum fluorescent lights
500 to 1000 Lux
16L8D
300-mL high-form lipiess beaker
100mL
175mL
2 volume additions/d
7- to 14- d old at start of test
10
8

Feed 1.5 mL daily to each test chamber
None (unless D.O. drops below 40% of saturation)
Culture water, well water, surface water, site water or reconstituted water
Gently brush outside of screen when clogged
Hardness, alkalinity, conductivity, pH, and ammonia at beginning and end of test; tem-
perature and D.O. daily
10-28d
Survival (growth optional)
Minimum mean control survival of 80% and above conditions
(2) Sediment into test chambers, (i) Sediment should be thoroughly
mixed and added to test chambers the day before (day—1) the start of
the test. The degree of homogeneity should be inspected visually. Homo-
geneity may be quantified by taking replicate subsamples and analyzing
for TOC, chemical concentration, and particle size.

(ii) Equal amounts of sediments should be added to each test chamber
on the basis of volume or dry weight. To minimize disturbance of sedi-
ment, overlying water should be poured gently along the sides of the test
chambers or poured over a Teflon baffle (with handle) positioned above
the sediment. The renewal of overlying water should commence on day -
1. The test begins once organisms are added to the test chambers (day-
0).
(3) Renewal of overlying water. Renewal or flow-through of over-
lying water is recommended during a test. Flow rates through any two
test chambers should not differ by more than 10 percent at any time during
the test. Each water-delivery system should be calibrated prior to test initi-
ation to verify that the system is functioning properly. Renewal of over-
lying water is started on day--l before the addition of test organisms
or food on day - 0.
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(4) Acclimation. Test organisms must be cultured and tested at
23 °C. The same water used for culture should be used for testing. Accli-
mation of test organisms to the test water is not required.

(5) Placement of organisms in test chambers. Handle test organisms
as little as possible. Amphipods may be placed into test chambers by
pipetting the organisms directly into the overlying water just below the
air-water interface or by placing the organisms into 30-mL counting cups
and floating them in the test chamber for 15 min prior to placement into
the overlying water. Measurements of length or weight should be made
on a subset of 20 organisms prior to test initiation.

(6) Monitoring a test. All test chambers should be checked daily.
Test organisms should be observed for abnormal behavior, such as sedi-
ment avoidance. The exposure system should also be monitored daily to
assure proper operation.

(7) Measurement of overlying water-quality characteristics, (i)
Conductivity, hardness, pH, alkalinity, and ammonia should be measured
in all treatments at the beginning and end of a test, and during any test
should not vary more than 50 percent. Samples should be removed with
a pipet from 1 to 2 cm above the sediment surface without disturbance.
Caution is required to avoid removing test organisms when sampling.

(ii) DO should be measured daily, and should be maintained between
40 percent and 100 percent saturation. Both DO and pH may be measured
in overlying water using a probe.

(iii) Temperature should be measured daily in one test chamber from
each treatment. The mean and instantaneous temperatures should not vary
from the desired temperature by more than 1 °C and 3 °C, respectively.

(8) Feeding. H. azteca may be fed with a mixture of yeast, Cerophyl,
and trout chow (YCT) at a rate of 1.5 mL daily per test chamber. Food
is required for proper maintenance of the test organisms but should be
kept to a minimum to prevent alteration of contaminant availability or the
growth of microbials such as fungus and bacteria. Collection of food on
the bottom of the test chamber or reduced concentration of DO are indica-
tors of possible overfeeding. Should either of the above conditions occur,
feeding should be suspended in all test chambers until conditions have
readjusted. Detailed records and observations should be made daily.

(9) Ending a test. Surviving amphipods may be pipetted from the
test chamber prior to sieving the sediment. Immobile organisms isolated
from either sediment or sieved material are considered dead. Sediment may
be sieved by pouring one-half of the overlying water volume followed
by one-half of the sediment through a #50 sieve (300 |j.m) into an examina-
tion pan. The coarser sediment remaining in the test chamber should be
washed through a #40 (425 fim) sieve into a second examination pan.

10
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Surviving organisms should be isolated and preserved (e.g. 8 percent sugar
formalin) and measured for growth. The amount of time taken to recover
test organisms should be consistent (e.g. 10 min per replicate). A recovery
rate of 90 percent of organisms from the sediment is acceptable.

(10) Test data, (i) The primary endpoint for 10-day sediment toxicity
test with H, azteca is survival.

(ii) Amphipod body length should be measured from the base of the
first of antenna to the tip of the third uropod along the curve of the dorsal
surface.

(iii) To determine dry weight of surviving amphipods:

(A) Pool all surviving organisms from a replicate.

(B) Dry the sample to constant weight at 60 to 90 °C.

(D) Weigh the sample of organisms to the nearest 0.01 mg. This
measure will give the mean weight of surviving organisms per replicate.

(11) Interpretation of results—(i) Age sensitivity. The relative sen-
sitivity of H. azteca is comparable up to 24- to 26-day-old organisms.
Amphipods 7- to 14-day-old represent sensitivity of H. azteca up to adult
life stage.

(ii) Grain size. H. azteca tolerate a wide range of substrates. Neither
grain size nor TOC correlate with the toxic response in sediment toxicity
tests.

(iii) Isolating organisms at the end of a test. Quantitative recovery
of amphipods <7-days-old is difficult. Starting testing with
7-day-old amphipods facilitates recovery.

(iv) Influence of indigenous organisms. The presence of
oligochaetes does not reduce the survivability of amphipods in 28-day
sediment tests. However, high density of oligochaetes does reduce the
growth of amphipods. The number of oligochaetes and presence of preda-
tors in test sediment should be determined to improve the interpretation
of growth data.

(i) Interferences. (1) Interferences are defined as those characteristics
of sediment or sediment test systems that are unrelated to sediment-associ-
ated contaminants, but have the potential to affect the survival of test orga-
nisms. Interferences may lead to both Type I (false-positive) and Type
II (false-negative) errors.

(2) Interferences may result from sediment characteristics that affect
survival independently of chemical concentration, altered bioavailability

11
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(e.g. sediment manipulation, storage, etc.), or when indigenous species are
present.

(3) Test procedures and organism selection criteria were designed to
minimize impacts due to interferences, and are suitable for providing direct
measure of contaminant effects on benthic organisms.

(4) Several noncontaminant factors have the potential to affect sedi-
ment toxicity. These factors include but are not limited to avoidance, light-
ing, and geomorphological and physicochemical characteristics. Although
laboratory sediment toxicity tests results may be used to predict effects
in the field, extrapolations to the field may not prove valid in cases where
motile organisms are able to avoid exposure.

(5) Toxicological responses of some chemicals may be altered by UV
radiation contained in natural sunlight. Sediment testing with some chemi-
cals, which are photoinduced by UV light, may not provide results useful
for predicting field effects, because typical lighting (i.e. fluorescent) does
not emit UV radiation.

(6) Natural geomorphological and physicochemical characteristics of
sediment should be within the tolerance limits of the test organism. Factors
such as texture, grain size, and organic carbon may influence the toxic
response of the test organism.

(7) Sediment toxicity tests were designed to predict anticipated con-
taminant-related effects in the field or under natural conditions. However,
sediment toxicity is related to bioavailability, which can be altered by
physical manipulation, temperature, adjuncts, and organism uptake.

(8) In some cases bioavailability may differ between the laboratory
and in situ. Sediment collection, handling, and storage are critical to pre-
serving the integrity of contaminant equilibrium. The manipulation of sedi-
ment may disrupt the equilibrium with organic carbon and the pore water/
particle system, resulting in the increased availability of organic com-
pounds.

(9) The testing temperature is important to bioavailability. Tempera-
ture affects contaminant solubility, the partitioning coefficient, as well as
the physical and chemical characteristics of sediment. Bioavailability may
also be altered by interactions between sediment and overlying water.

(10) Adjuncts such as food, water, or solvents may alter
bioavailability and promote the growth of microorganisms. While food ad-
dition is necessary, the quantity and composition of food added must be
carefully considered.

(11) Uptake of contaminants by the test organisms or test chambers
may influence bioavailability. Test organisms are sinks for contaminants,
but to a lesser degree than sediments.

12
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(12) The routes of exposure for sediment contaminants are not always
known. In some cases, it may desirable to normalize sediment concentra-
tions of contaminants to factors other than dry weight, such as organic-
carbon for nonionic organic compounds or acid volatile sulfides for certain
metals.

(13) The Agency recommends using natural sediments for spiking in
sediment toxicity tests. However, indigenous species sometimes exist in
field-collected sediments and their presence could negatively effect the
growth rates of test organisms. Biological activity may be inhibited by
gamma radiation, heat, sieving, mercuric chloride, or antibiotics, and their
impact on sediment characteristics must be determined prior to the com-
mencement of testing.

(j) Test method—Chironomus tentans 10-day survival and
growth test for sediments—(1) Test conditions. The 10-day sediment
toxicity test with C tentans should be conducted at a temperature of 23
°C and photoperiod of 16 h light:8 h dark at 500 to 1,000 Ix. The rec-
ommended test chambers are 300-mL high-form beakers without lips con-
taining 100 mL of sediment and 175 mL of overlying water. Each test
chamber is filled with 10 third-instar midges to begin the test. All orga-
nisms must be third-instar (50 percent of organisms) or younger. For rou-
tine testing, eight replicates are recommended. Midges should be fed 1.5
mL of a 4 g/L suspension of Tetrafm daily. Overlying water in each test
chamber should receive two volume changes per day and can be culture
water, well water, surface water, site water, or reconstituted water.

(2) Sediment into test chambers. Test sediment should be mixed
thoroughly and placed into test chambers one day (day—1) before com-
mencement of the test. Sediment should be checked for homogeneity vis-
ually and quantitatively by analyzing TOC, chemical concentrations, and
particle size. Equal volumes of sediment should be added to each test
chamber, and on day-1 overlying water should be added by pouring water
along a baffle to avoid any disturbance of the sediment. The test begins
once the test organisms are added to the test chambers (day-0).

(3) Renewal of overlaying water. The renewal of overlying water
is required and should be conducted on day—1 prior to the addition of
test organisms or food on day-0. Flow rates should not vary by more
than 10 percent between any two test chambers at any time during the
test. Proper system operation should be verified by calibration prior to
initiation of the test.

(4) Acclimation. The required culture and testing temperature is
23 °C. The test organisms should be cultured in the same water to be
used for testing. Acclimation of the test organisms to the test water is
not required.

13
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(5) Placing organisms in test chambers. Handle test organisms as
little as possible. Midges may be placed into test chambers by pipetting
the organisms directly into the overlying water just below the air-water
interface or by placing the organisms into 30-mL counting cups and float-
ing them in the test chamber for 15 min prior to placement into the over-
lying water. Measurements of length or weight should be made on a subset
of 20 organisms prior to test initiation. Head capsule widths should be
measured on midges to determine the instar used at test initiation.

(7) Measurement of overlying water-quality characteristics, (i)
Conductivity, hardness, pH, alkalinity, and ammonia concentration should
be measured in all treatments at the beginning and end of a test, and during
any test should not vary more than 50 percent. Samples should be removed
with a pipet from 1 to 2 cm above the sediment surface without disturb-
ance. Caution is required to prevent removing test organisms when sam-
pling-

(ii) DO should be measured daily, and should be maintained between
40 and 100 percent saturation. Both DO and pH may be measured in over-
lying water using a probe.

(iii) Temperature should be measured in one test chamber from each
treatment daily. The mean and instantaneous temperatures should not vary
from the desired temperature by more than 1 and 3 °C, respectively.

(8) Feeding. Food is required for proper maintenance of the test orga-
nisms but should be kept to a minimum to prevent alteration of contami-
nant availability or the growth of microbials such as fungus and bacteria.
Collection of food on the bottom of the test chamber or reduced concentra-
tion of DO are indicators of possible overfeeding. Should either of the
these conditions occur, feeding should be suspended in all test chambers
until conditions have readjusted. Detailed records and observations should
be made daily.

(9) Ending a test. Surviving amphipods may be pipetted from the
test chamber prior to sieving the sediment. Immobile organisms isolated
from either sediment or sieved material are considered dead. Surviving
organisms should be preserved (e.g. 8 percent sugar-formalin) and meas-
ured for growth. Specific sieving instruction may be found in paragraph
(1)(1) of this guideline.

(10) Test data, (i) The endpoints measured in 10-day sediment tests
with C. tentans are dry weight and survival. At the end of the test, C.
tentans in control sediment should have an average size of 0.6 mg. Head

14
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capsule width should be measured prior to dry weight. To determine dry
weight of surviving midges:

(A) pool all surviving organisms from a replicate.

(B) Dry the sample at 60 to 90 °C to constant weight.

(D) Weigh the sample of organisms to the nearest 0.01 mg. This
measure will give the mean weight of surviving organisms per replicate.

(iv) Pupae and adults should be excluded from dry weight determina-
tions. Length measurement is optional, but measurements should be from
the anterior of the labrum to the posterior of the last abdominal segment.

(11) Interpretation of results—(i) Age sensitivity. First and second
instar midges are more sensitive than third and fourth instar midges. Sedi-
ment tests should be initiated with midges of uniform size and age to
avoid changes in sensitivity. Sediment tests are conducted with the third-
instar midges because the greater size facilitates handling and isolation
from sediment at test termination.

(ii) Grain size. C. tentans are tolerant of a wide range of substrates.
The sensitivity of midges does not correlate with TOC or grain size. How-
ever, sensitivity may be influenced by artificial sediment when test orga-
nisms are not fed during the test.

(iii) Isolating organisms at the end of a test. Isolation and recovery
of midges at the end of the test is not difficult. The midges are typically
red and greater 5-mm in length.

(iv) Influence of indigenous organisms. There are no reports on the
influence of indigenous organisms on C. tentans survival and response
in sediment toxicity tests. However, survival of a congener, Chironomus
riparius, was not reduced in the presence of oligochaetes, but growth was
reduced in the presence of high numbers of oligochaetes. The number of
oligochaetes and presence of predators in test sediment should be deter-
mined to improve the interpretation of growth data.

(k) Reporting. In addition to information meeting general reporting
requirements, a report of the results of a whole sediment toxicity test
should also include the following:

(1) Name of test and investigators, name and location of laboratory,
and dates of start and end of test.

(2) Source of control or test sediment, method for collection, han-
dling, shipping, storage and disposal of sediment.

15
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(3) Source of test material, lot number if applicable, composition
(identities and concentrations of major ingredient and impurities if known),
known chemical and physical properties, and the identity and concentra-
tions of any solvent used.

(4) Source and characteristics of overlying water, description of any
pretreatment, and results of any demonstration of the ability of an orga-
nism to survive or grow in the water.

(5) Source, history, and age of test organisms: Source, history, and
age of brood stock, culture procedures and source and date of collection
of the test organisms, scientific name, name of person who identified the
organisms and the taxonomic key used, age or life stage, means and ranges
of weight or length, observed diseases or unusual appearance, treatments
holding procedures.

(6) Source and composition of food, concentrations of test material
and other contaminants, procedure used to prepare food, feeding methods,
frequency and ration.

(7) Description of the experimental design and test chambers, the
depth and volume of sediment and overlying water in the chambers, light-
ing, number of test chambers and number of test organisms/treatment, date
and time test started and ended, temperature measurements, DO concentra-
tion (as percent saturation) and any aeration used before starting a test
and during the conduct of a test.

(8) Methods used for physical and chemical characterization of sedi-
ment.

(9) Definitions of the effects used to calculate LC50 or ECSOs, bio-
logical endpoints for tests, and a summary of general observations of other
effects.

(10) A table of the biological data for each test chamber for each
treatment including the controls in sufficient detail to allow independent
statistical analysis.

(11) Methods used for statistical analyses of data.

(12) Summary of general observations on other effects or symptoms.

(13) Anything unusual about the test, any deviation from these proce-
dures, and any other relevant information.

(14) Published reports should contain enough information to clearly
identify the methodology used and the quality of the results.

(1) References. The following references should be consulted for ad-
ditional background material on this test guideline.

16
-------
(1) U.S. Environmental Protection Agency. Methods for Measuring
the Toxicity and Bioaccumulation of Sediment-Associated Contaminants
with Freshwater Invertebrates. EPA 600/R-94/024 (1994).

(2) [Reserved]
17
-------
-------
&EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-355
April 1996
Ecological Effects Test
Guidelines
OPPTS 850.1740
Whole Sediment Acute
Toxicity Invertebrates,
Marine
'Public Draft"
-------
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.

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."
-------
OPPTS 850.1740 Whole sediment acute toxicity invertebrates, ma-
rine.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.).

(2) [Reserved]

(b) Objective. This guideline may be used to determine the toxicity
and bioaccumulation potential of chemicals in estuarine or marine sedi-
ments in marine invertebrates. Natural sediment is spiked with different
concentrations of pesticide or contaminant and the results from the sedi-
ment toxicity tests can be used to determine causal relationships between
the chemical and biological response. Reported endpoints from whole sedi-
ment toxicity tests include the LC50 (median lethal concentration), EC50
(median effective concentration), NOEC (no-observable-effect-concentra-
tion), or the LOEC (lowest-observable-effect-concentration).

Clean. Clean denotes a sediment or water that does not contain con-
centrations of test materials which cause apparent stress to the test orga-
nisms or reduce their survival.

Concentration. Concentration is the ratio of weight or volume of test
material(s) to the weight or volume of sediment.

Contaminated sediment. Contaminated sediment is sediment contain-
ing chemical substances at concentrations that pose a known or suspected
threat to environmental or human health.

1
-------
the test population and the IC50 is the concentration of toxicant that would
cause a 50% reduction.

Interstitial water or pore water. Interstitial water or pore water is
water occupying space between sediment or soil particles.

Lowest observable effect concentration (LOEC). Lowest observable
effect concentration is the lowest concentration of a toxicant to which or-
ganisms are exposed in a test which causes an adverse effect on the test
organisms (i.e., where the value for the observed response is statistically
significant different from the controls).

Overlying water. Overlying water is the water placed over sediment
in a test chamber during a test.

Sediment. Sediment is particulate material that usually lies below
water. Formulated particulate material that is intended to lie below water
in a test.

Spiked sediment. Spiked sediment is a sediment to which a material
has been added for experimental purposes.
-------
Whole sediment. Whole sediment is sediment and associated pore
water which have had minimal manipulation. The term bulk sediment has
been used synonymously with whole sediment.

(d) Test method. (1) Whole sediment toxicity tests are outlined for
four species of estuarine/marine amphipods, Ampelisca abdita,
Eohaustorius estuarius, Rhepoxynius abronius, and Leptocheirus
plumulosus. Whole sediment tests last 10 or more days, and are conducted
in in 1-L test chambers containing 175 mL (2 cm) of sediment and
800 mL of overlying water. The overlying water does not have to be re-
newed and test organisms do not have to be fed during the toxicity test,
The endpoint is survival, but reburial for E. estuarius, L. plumulosus, and
R. abronius is optional.

(2) A range-finding test to establish a suitable range of test concentra-
tions is recommended. If no toxicity is observed at concentrations of
100 mg/kg dry weight of sediment, a definitive test is not required.

(e) Water, reagents, and standards—(1) Water, (i) Sea water used
in sediment toxicity test should be of uniform quality and allow satisfac-
tory survival, growth, or reproduction of the test organisms. Organisms
cultured and tested in the selected sea water should not show signs of
disease or stress.

(ii) Natural sea water should be from uncontaminated surface-water
upstream of known discharges. Sea water should be collected at slack high
tide or within 1 h of high tide. Full strength sea water should be collected
from areas with salinities of 28 ppt. Sea water for estuarine test may be
collected from areas with salinities close to the test salinity or diluted with
freshwater. Water prepared from natural sea water should be covered,
maintained at 4 °C, and used with 2 days.

(iii) Although natural sea water is preferable, reconstituted water is
acceptable. Reagent grade chemicals should be added to high-purity dis-
tilled or deionized water (1 MQ). Each batch of reconstituted water should
be measured for salinity, pH, and dissolved oxygen (DO). Suspended par-
ticles should be removed by filtration (<5 urn) from reconstituted water
at least 24 h before use.

(2) Reagents. All reagents and chemicals purchased from supply
houses should be accompanied by appropriate data sheets. All test mate-
rials should be reagent grade. However, if specified as necessary, commer-
cial product, technical-grade, or use-grade materials may be used. Dates
for receipt, opening, and shelf-life should be logged and maintained for
all chemicals and reagents. Do not use reagents beyond shelf-life dates.

(3) Standards. Acceptable standard methods for chemical and phys-
ical analyses should be used. When appropriate standard methods are not
-------
available or lack the required sensitivity, other sources should be consulted
for reliable methods.

(2) Storage. Storage of sediment may affect bioavailability and tox-
icity. Although nonionic and nonvolatile organic contaminates in sediment
may not result in substantive changes, metals and metalloids may affect
redox, oxidation, or microbial metabolism in sediment. It is best to hold
sediments at 4 °C in the dark and test within 2 to 8 weeks after collection.
Long storage may result in changes of sediment properties. Sediment tests,
and especially pore water tests, should be performed within 2 weeks of
collection to minimize property changes in the sediment.

(3) Manipulation, (i) Dining homogenization, water above sediment
that may have settled during shipment should be mixed back into the sedi-
ment. Sieving should not be used to remove indigenous organisms, unless
an excessive number of oligochaetes are present. Because oligochaetes
may inhibit the growth of the test organisms, it may be advantageous to
remove them by sieving. If sieving is used, sediment samples should be
analyzed before and after sieving to document the influence of sieving
on sediment characteristics. Sediments collected from multiple locations
or sites may be pooled and mixed using suitable apparatus (e.g. stirring,
rolling mill, feed mixer, etc.).

(ii) The preparation of test sediment may be accomplished by the
spiking of natural or formulated sediments. Additional research is needed
before formulated sediments may be used routinely. The responses of
spiked sediment may be affected by mixing time and aging. Spiked sedi-
ment should be used immediately. Point estimates of toxicity or minimum
concentrations at which toxic effects are observed may be determined by
spiking natural sediments with a range of chemical concentrations. The
test material should be reagent grade unless there is a specific need to
use commercial product, technical-grade, or use-grade material. Specific
-------
information required for all test materials include but is not limited to
the following:

(A) Identity and concentration of major ingredients and impurities.

(B) Solubility in test water.

(D) When measured test concentrations are required, the precision
and bias of analytical method at the planned concentrations of test mate-
rial.

(E) Recommended handling and disposal procedures.

(iii) Organic solvents should not be added to the sediment mixture
because they may affect the concentration of dissolved organic carbon in
pore water.

(4) Characterization, (i) The characteristics of all sediment should
be determined, and at a minimum the following factors should be meas-
ured: pH and ammonia concentration of pore water, organic carbon content
(total organic carbon, TOC), particle size distribution (percent sand, silt,
clay), and percent water content. Additional analyses are suggested and
include biological oxygen demand, chemical oxygen demand, cation ex-
change capacity, Eh, total inorganic carbon, total volatile solids, acid vola-
tile sulfides, metals, synthetic organic compounds, oil and grease, and pe-
troleum hydrocarbons. Various physicochemical parameters should also be
determined for interstitial water. Sediment characterization should also in-
clude qualitative parameters such as color, texture, and the presence of
macrophytes or animals.

(ii) Standard analytical methods should be used to determine chemical
and physical data. For each analytical method, precision, accuracy, and
bias should be determined in sediment, water, and tissue. Analysis should
include analytical standards and reagent blanks as well as recovery calcula-
tions.

(iii) Concentrations of spiked chemicals should be measured in sedi-
ment, interstitial water, and overlying water at the beginning and at the
end of the test. Degradation products should also be measured where ap-
propriate. Sediment chemistry should be monitored during and at the end
of a test. Separate replicates resembling the biological replicates and con-
taining organisms should be specified for chemical sampling. The con-
centration of test material in water is measured by pipetting water samples
from 1 to 2 cm above the sediment surface. Caution should be used to
eliminate the presence of any surface debris, material from the sides of
the chamber, or sediment in the overlying water sample. At the end of
the test, the test material may be removed for chemical analysis by siphon-
ing (without disturbing sediment) the overlying water. Appropriate samples
-------
of sediment can then be removed for chemical analysis. The suggested
method for isolation of interstitial water is by centrifugation without filtra-
tion.

(g) Collection and maintainence of test organisms. (1) The methods
for collection of test organisms are species-specific. Subtidal species, such
as A. abdita and L. plumulosus, may be collected with a small dredge
or grab and may also be collected by skimming the sediment surface with
a long-handled, fine-mesh net. E. estuarius and R. abronius are found both
subtidally and intertidally. The aforementioned methods are suitable for
subtidal populations. Intertidal populations may be collected using a shov-
el. Approximately one-third more organisms should be collected than are
required for testing.

(2) All collecting, sieving, and transporting equipment should be clean
and constructed of nontoxic material and clearly marked "live only". All
apparatus should be cleaned and rinsed with an appropriate water source
before use.

(3) Collected organisms should be handled gently, carefully, quickly,
and no more than necessary. The sieving operation should be conducted
by slowly immersing the sieve into collection site water. Sieved test orga-
nisms should be kept submerged in ambient collection water at all times.
Direct exposure to sunlight of amphipods out of sediment must be avoided.

(4) A. abdita and L. plumulosus should be isolated from collection
site sediment by using a 0.5 mm mesh sieve. A. abdita which remain in
tubes must be left undisturbed for 20 to 30 min to allow for natural exit
of the organisms. A 1.0 mm sieve should be used to isolate E. estuarius
and R. abronius.

(5) A. abdita and L. plumulosus may be collected with a small dredge
or grab apparatus (e.g. PONAR, Van Veen, etc.) and E. estuarius and
R. abronius may be collected with a shovel. Collected amphipods should
be sieved in the field by slow immersion in collection site water. Sieved
amphipods should be separated from detritus and predators and transferred
gently to transport containers containing 2 cm of collection site sediment.
Mesh sizes of 0.5 to 1.0 mm should be utilized. Salinity and temperature
of collection site sediment should be recorded surface and bottom loca-
tions. Amphipods should be transported in coolers with ice packs and held
in the collection-site sediment at or below the temperature at the collection
site. Aeration is recommended for transport times exceeding 1 h. Collec-
tion site sediment should be used as holding sediment in the laboratory
and as control test sediment.

(h) Ampelisca abdita, Eohaustorius estuarius, Leptocheirus
plumulosus, or Rhepoxynius abronius 10- to 28-Day Survival Test For
Sediments—(1) Recommended test method. The recommended test tem-
peratures for conducting sediment toxicity tests with E. estuarius and R.
-------
abronius, A. abdita, and L. plumulosus are 15, 20, and 25 °C, respectively.
E. estuarius and L. plumulosus should be tested at a salinity of 20 ppt
and A. abdita and R. abronius at a salinity of 28 ppt. The recommended
photoperiod is 24 h of light with illumination of 500 to 1,000 Ix. Sediment
(175 mL) and 800 mL of overlying seawater are placed in 1-L glass test
chambers. Twenty organisms are placed in each test chamber to begin the
test. Five replicates per treatment are recommended, however, this number
may vary depending upon need. The size and life stage of amphipod re-
quired for testing varies from 2-4 mm for L. plumulosus to 3-5 mm for
the three remaining species. Additionally, no mature male or female A.
abdita or L plumulosus should be used for testing. Overlying water does
not have to be renewed and test organisms do not have to be fed during
the test.

(2) General procedure—(i) Introduction of sediment. The test sedi-
ment should be homogenized one day before the test is to commence
(day—1) using a rolling mill, feed mixer, or other suitable apparatus. The
sediment should be observed for homogeneity visually and quantitatively
by measuring TOC, chemical concentrations, and particle size. A
175-mL aliquot of homogenized sediment should be added to each test
chamber, and settled with the use of a nylon, fluorocarbon, or polyethylene
spatula.

(ii) Addition of overlying water. A turbulence reducer (a disk cut
from polyethylene, nylon, or Teflon, or a glass Petri dish attached to a
glass pipet) should be used when adding overlying water. Turbulence re-
ducers should be rinsed with seawater between replicates, and individual
turbulence reducers used between treatments. Test chambers should be
covered, immersed in a temperature bath, and gently aerated. The test com-
mences once the test organisms are added to the test chambers (day-0).

(iii) Addition of amphipods. Twenty amphipods are randomly added
to each test chamber in batches of 5 to 10 on day—0 following the addi-
tion of sediment and overlying water. One-third more amphipods than nec-
essary are sieved from culture or control sediment and transferred to sort-
ing trays. Recommended sieve sizes are 0.5 mm for A. abdita and L.
plumulosus and 1.0 mm for £. estuarius and R. abronius. Isolated
amphipods are transferred from the sorting tray to 150 mL of test sea
water using pipets. The test organisms are observed for injury or stress
after addition. If any E. estuarius, L. plumulosus, and R. abronius have
not burrowed within 5 to 10 min, they should be replaced. A. abdita that
have not burrowed within 1 h should also be replaced. Organisms express-
ing sediment avoidance, should be removed, recorded, but not replaced.

(3) Test conditions—(i) Aeration. Overlying sea water should be
continuously aerated from day-1 to day- 10 except when test organisms
are being added. DO should be maintained at approximately 90 percent
U.S. EPA Headquarters Library
Mail code 3201
1200 Pennsylvania Avenue NW
Washington DC 20460
-------
saturation using gentle aeration without disturbing the sediment. Results
are unacceptable if DO falls to below 60 percent saturation.

(ii) Lighting. Lights must be left on for the duration of the 10-day
testing period. The constant light assures that the test organisms to remain
burrowed during the test.

(iii) Feeding. None of the four test species need to be fed during
the 10-day testing period.

(iv) Water temperature. The respective selected test temperatures
are representative of the summertime thermal maximum for each species.
E. estuarius and R. abronius (Pacific Coast amphipods) must be tested
at 15 °C. A. abdita and L. plumulosus must be tested at 20 °C and
25 °C, respectively.

(v) Salinity. Overlying water salinity should be 28 ppt for A. abdita
and R. abronius and 20 ppt for E. estuarius and L. plumulosus. Pore water
salinity must be measured prior to the start of the test.

(4) Measurements and observations, (i) Temperature should be
measured daily from at least one replicate from each treatment. Tempera-
ture of the water bath or exposure chamber must be monitored continu-
ously.

(ii) Salinity, DO, and pH should be measured in overlying water daily
in one test chamber in each treatment. These parameters should be meas-
ured in all test chambers at the beginning of the test and at termination.

(iii) Ammonia concentration should be measured near day-2 and
day-8 during the 10-day test period. Ammonia concentration measure-
ments should be accompanied by pH and temperature measurements. pH,
temperature, and ammonia concentration should be measured in pore water
at the beginning of the test.

(iv) Air-flow to overlying sea water must be monitored daily. Any
test organisms trapped in air-water interface must be gently pushed back
down using a glass rod or pipet.

(5) Ending a test, (i) Recovery of organisms from control sediment
should equal or exceed 90 percent in a 10-day test or 80 percent in a
28-day test.

(ii) Test animals are isolated from the test chambers by sieving with
sea water. Sieves should not exceed 0.5 mm. Test organisms should be
washed into sorting trays containing sea water. Caution should be taken
that no tube-dwelling organisms remain trapped on the sieve. Slapping
the sieve forcefully against the surface of the water should successfully
dislodge all A. abdita. The remaining species should be easily separated
by the sieving process.

8
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(iii) Small portions of material should be washed into sorting trays
and should be examined carefully. The tubes of A. abdita should be teased
apart under a dissecting microscope to ensure that all organisms are ac-
counted for. The numbers of living, missing, or dead amphipods should
be observed and recorded for all test chambers. Missing animals and all
observed animals failing to respond to gentle prodding (i.e. neuromuscular
twitch of pleopods or antennae) are recorded as dead.

(6) Test data. The primary endpoint for the 10-day sediment test
is survival. Effective mortality (the sum of dead animals plus survivors
that fail to rebury) may also be determined. To determine reburial, E.
estuarius, L. plumulosus, and R. abronius should be transferred to a
2-cm layer of 0.5 mm sieved control sediment and overlying test sea water
(2 cm).

(7) Interpretation of results—(i) Influence of indigenous orga-
nisms. Because test sediments collected from the field may contain indige-
nous species, data interpretation may be complicated by the presence of
organisms similar to the test organism or predatory organisms.

(ii) Effect of grain size. While the four estuarine/marine test species
are generally tolerable of a wide range of sediment types, grain size may
adversely affect some species of amphipod. When this possibility exists,
a clean control/reference sediment should be incorporated into the test de-
sign to facilitate distinction of contaminant effects versus particle size ef-
fects. Species-specific ranges of grain sizes are as follows.

(A) A. abdita: Survival may be impacted in sediments containing 95
percent or more sand. Test sediment should contain less than 95 percent
sand.

(B) L. plumulosus: Survival should not be impacted in clean sedi-
ments containing 100 percent sand to 100 percent sand + clay.

(C) E. estuarius: Survival is unaffected by clean sediments containing
0.6 to 100 percent sand. However increased mortality may be associated
with increased proportions of fine-grained sediment. In these cases an ap-
propriate control/reference should be included.

(D) R. abronius: Very fine grains, particularly silts and clays, may
reduce survival of this species. When test sediments contain silts and clays,
the use of control/reference groups with particle sizes characteristic of the
test sediment is recommended.

(iii) Effects of pore water salinity. The range of salinity tolerance
is variable for the four amphipod species. For sediment testing, two sce-
narios for test salinity are acceptable given that appropriate conditions are
met:
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(A) Salinity tolerance range is the range of salinity in which a given
species can survive for 10 days when the overlying water salinity is
matched to that of the pore water salinity. In laboratory sediment testing,
the overlying water salinity can be based on the standard salinity for each
test species, or adjusted to match the salinity of pore water. It is critical
that pore water salinity be measured prior to test initiation and that the
appropriate species be used. Salinity tolerance ranges for A. abdita, E.
estuarius, L. plumulosus, and R. abronius are 20-32 ppt, 2-34 ppt, 1.5-
32 ppt and 25-32 ppt, respectively.

(B) Salinity application range is the range of pore water salinities
in which a given species can survive for 10 days when using the species-
specific overlying water salinity. Salinity application ranges for A. abdita
with overlying water salinity of 28 to 32 ppt, E. estuarius with overlying
water salinity of 20 ppt, L. plumulosus with overlying salinity of 20 ppt,
and R. abronius with overlying water salinity of 28 to 32 ppt are 0 to
34, <2 to 34, <1.5 to 32, and 25 to 34, respectively.

(iv) Effects of sediment-associated ammonia. Ammonia concentra-
tions in field-collected sediments may be toxic to amphipods. When am-
monia concentration exceeds the water column no-effect levels, any mor-
tality observed during the 10-day sediment test may be due to the ammo-
nia. Ammonia levels should be measured approximately 1 cm above the
sediment surface on day-0 and, if necessary, reduced prior to the addition
of test organisms by flushing the overlying water for up to two consecutive
24-h periods (six volume replacements per hour). Following flushing, the
overlaying water ammonia concentration should be remeasured. If ammo-
nia is at acceptable levels testing may be initiated but flushing at a rate
of sic volume changes per 24-h period must be maintained throughout
the test. Ammonia concentrations in overlaying water should be measured
again on day-10. If ammonia is not at acceptable levels, 24-h flushings
must continue at the six-volume change per 24 h rate and ammonia con-
centration measured every 24 h.

(i) Interferences. (1) Interferences are characteristics of sediment or
the sediment test system with potential to affect survival of test organisms
independent of sediment-associated contaminant affects. Interferences are
categorized into three categories: Noncontaminant factors causing reduced
survival, changes in bioavailability due to manipulation or storage, and
the presence of indigenous species. Noncontaminant factors can make ex-
trapolation of laboratory test results to the field difficult. Specifically, the
motility of organisms (i.e. escapism) and photoinduced toxicity due to UV
light in from the sun (e.g. UV light absent from fluorescent light) may
be markedly different between laboratory conditions and the natural envi-
ronment. Other noncontaminant factors include sediment particle size, pore
water salinity, and pore water ammonia concentration. The test conditions
must be matched appropriately with the tolerance limits of the four
amphipod test species (see paragraph (k)(l) of this guideline).

10
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(2) Bioavailability of sediment-associated contaminants can be altered
by collection, handling, and storage. The handling, storage, and preparation
of test sediment should be as consistent as possible. Test sediments should
be presieved and rehomogenized prior to introduction into the test cham-
bers. Bioavailability may also be affected by temperature, salinity, the ratio
of sediment to overlying water, and me depletion of contaminant due to
organismal uptake. In some cases it is advantageous to normalize sediment
concentrations to dry weight, organic-carbon content, or acid volatile sul-
fides.

(3) Test sediment collected from the field may contain indigenous
organisms, and can potentially make interpretation of treatment effects dif-
ficult. If the presence of indigenous or predatory organisms is suspected,
the test sediment should be press-sieved prior to test initiation.

(j) Reporting. In addition to information meeting the general report-
ing requirements of a toxicity test, a report of the results of a sediment
toxicity test for estuarine and marine amphipods should include the follow-
ing:

(1) Name of test and investigators, name and location of laboratory,
and dates of start and end of test.

(2) Source of control or test sediment, method for collection, han-
dling, shipping, storage, and disposal of sediment.

(3) Source of test material, lot number if applicable, composition
(identities and concentrations of major ingredients and impurities if
known), known chemical and physical properties, and the identity and con-
centrations of any solvent used.

(4) Source and characteristics of overlying water, description of any
pretreatment, and results of any demonstration of the ability of an orga-
nism to survive or grow in the water.

(5) Source, history, and age of test organisms; source, history and
age of brood stock, culture procedures; and source and date of collection
of the test organisms, scientific name, name of person who identified the
organisms and the taxonomic key used, age or life stage, means and ranges
of weight or length, observed diseases or unusual appearance, treatments,
holding procedures.

(6) Source and composition of food, concentrations of test material
and other contaminants, procedure used to prepare food, feeding methods,
frequency and ration.

(7) Description of the experimental design and test chambers, the
depth and volume of sediment and overlying water in the chambers, light-
ing, number of test chambers an number of test organisms/treatment, date
and time test starts and ends, temperature measurements, DO concentration

11
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(as percent saturation) and any aeration used before starting a test and
during the conduct of a test.

(8) Methods used for physical and chemical characterization of sedi-
ment.

(9) Definitions of the effects used to calculate LCSOs or ECSOs, bio-
logical endpoints for tests, and a summary of general observations of other
effects.

(10) A table of the biological data for each test chamber for each
treatment including the controls in sufficient detail to allow independent
statistical analysis.

(11) Methods used for statistical analyses of data.

(12) Summary of general observations on other effects or symptoms.

(13) Anything unusual about the test, any deviation from these proce-
dures, and any other relevant information.

(k) References. The following references should be consulted for ad-
ditional background material on this test guideline.

(1) U.S. Environmental Protection Agency. Methods for Assessing
the Toxicity of Sediment-Associated Contaminants with Estuarine and Ma-
rine Amphipods. EPA 600/R-94/025 (1994).

(2) [Reserved]
12
-------
&EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-313
April 1996
Ecological Effects Test
Guidelines
OPPTS 850.1790
Chironomid Sediment
Toxicity Test
'Public Draft"
-------
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.

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."
-------
OPPTS 850.1790 Chironomid sediment toxicity test.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. The source material used in developing this har-
monized OPPTS test guideline is 40 CFR 795.135 Chironomid Sediment
Toxicity Test (proposed in the FEDERAL REGISTER of June 25, 1991 (56
FR 29149)).

(b) Purpose. This guideline may be used to develop data on the tox-
icity and bioavailability of chemical substances and mixtures ("chemi-
cals") in sediments subject to environmental effects test regulations under
TSCA. This guideline prescribes tests to be used to develop data on the
toxicity of chemicals present in sediments to Chironomid larvae (midges).
The EPA will use data from these tests in assessing the hazard of a chemi-
cal to the environment.

(c) Definitions. The definitions in section 3 of TSCA and 40 CFR
part 792, Good Laboratory Practice Standards (GLPS), apply to this test
guideline. In addition, the following definitions also apply:

Bioconcentration factor (BCF) is the quotient of the concentration
of a test substance in tissues of the chironomids at or over a specific time
period of exposure divided by the concentration of test substance in the
overlying water, interstitial water, or in the sediments at or during the
same time period.

Cation exchange capacity (CEC) is the sum total of exchangeable
cations that a sediment can absorb. The CEC is expressed in
milliequivalents of negative charge per 100 g or milliequivalents of nega-
tive charge per gram of sediment (dry weight).

COD is chemical oxygen demand.

EC50 is an experimentally-derived concentration of test substance in
the sediment that is calculated to affect 50 percent of a test population
during continuous exposure over a specified period of time.

Flow-through is a continuous or intermittent passage of dilution water
through a test chamber or culture tank with no recycling of water.

Geometric mean is the calculated mean between the highest test con-
centration with no statistically significant effects and the lowest concentra-
tion showing significant effects.

Interstitial water is liquid which is found in or directly adjacent to
sediments and can be extracted from these sediments by several processes.

1
-------
Loading is the ratio of chironomid biomass (grams wet weight) to
the volume (liters) of test solution in a test chamber at a specified time
or passing through the test chamber during a specific interval.

Lowest-observed-effect-concentration (LOEC) is the lowest treatment
(i.e., test concentration) of a test substance that is statistically different
in adverse effect on a specific population of test organisms from that ob-
served in controls.

MATC (maximum acceptable toxicant concentration) is the maximum
concentration at which a chemical may be present and not be toxic to
the test organism.

No-observed-effect-concentration (NOEC) is the highest treatment
(i.e., test concentration) of a test substance that shows no statistical dif-
ference in adverse effect on a specific population of test organisms from
that observed in controls.

Overlying water is liquid which is found above or placed over sedi-
ments. For purposes of this guideline, overlying water is equivalent to the
term water column.

Partial life-cycle toxicity test is one which uses a sensitive portion
of the life of a test organism (second instar of midges) to assess the effects
of test substances.

Redox potential (Eh) means the oxidizing or reducing intensity or
condition of a solution expressed as a current, referenced against a hydro-
gen electrode. Zero or negative Eh values may be exist due to reducing
conditions within wet sediments.

Sediment is matter that settles to the bottom of a liquid in natural
situations or a substrate prepared from a combination of natural sediments
and artificial components. Sediment is equivalent to the term solid-phase
sediments in this guideline.

Sediment partition coefficient is the ratio of the concentration of test
substance on the sediment to the concentration in the overlying water. For
the purposes of this guideline, this term is identical to soil-water partition
coefficient.

Spiking is the addition of a test substance to a negative control and/
or reference sediment so that the toxicity of a known quantity of test sub-
stance can be determined in a known nontoxic sediment. Often a solvent
carrier is needed for low-water soluble test substances.

Subchronic toxicity test is a method used to determine the concentra-
tion of a test substance in water and for sediment which produces an ad-
verse effect on chironomids over a partially extended period of time. In
-------
this guideline, mortality and growth (expressed as change in wet weight
of midges) are the criteria of toxicity.
TOC is total organic carbon.
(d) Test procedures—(1) Summary of test, (i) This flow-through
test consists of three parts. Part 1. is a 14-day aqueous exposure test, with
minimal sediments, with food, and with the test substance added to the
overlying water. Part 2. is a 14-day sediment exposure test, with one or
more sediments (4 to 6 cm in thickness) which may have varying amounts
of organic carbon, with food, and with the test substance added to sedi-
ments. Part 3. is a 14-day interstitial exposure test, with one or more sedi-
ments (4 to 6 cm in thickness) which may have varying amounts of organic
carbon, with food, and with the test substance added to overlying water.
The flow-through test is illustrated in the following Table 1.

Table 1.—Experimental Design for the Chironomid Sediment Flow-Through Toxicity Test

Test system
Parti
14— Day Aqueous Exposure
Control (2 reps)
Solvent Control (2 reps)
Part 2
14— Day Sediment Exposure
Control (2 reps)
Solvent Control (2 reps)
Part 3
14-Day Interstitial Water/Sediment Expo-
sure ..
Control (2 reps)
Solvent Control (2 reosl
Test sub-
centrations
(2 replicates
each) '
5(10)
na
na
5(10)
na
na
5 no)
na
na
Number of
sediments
(2 replicates
each)
na*
na
na
1-3 s (2-6)
1 (2)
1 (2)
1 -3 5 (2-6)
1 (2)
1 2)
Number ol
Overlying
water P/C *
5 (10)
1(2)
2
5 (10)
1 (2)
1 (2)
5 (10)
1 (2)
1 (2)
Samples Anal
Interstitial
water P/C 2
na
na
na
na
na
na
5 (10)
1 (2)
1 2)
yzed (2 replica
Sediments
na
na
na
5(10)
1 (2)
12
5(10)
1 (2)
1(2)
tes each)
Midges 3
5(10)
1 (2)
1 (2)
5(10)
1 (2)
1(2)

1 (2)
1(2
1 Test substance concentration in all replicates measured at days 0 and 14 (reps - replicates)
2 P/C = physical/chemical measurements (dissolved oxygen, temperature (In °C), and pH) on days 0, 4, 7, 10 and 14.
3 Midges are observed throughout the test, dead chironomids recorded, removed, and weighed on days 4, 7, and 10. At end of
each test, remaining midges from each replicate are removed, counted, and weighed.
4 na = not applicable
5 Number of sediment types tested will depend on range of TOC content tested; 1 to 3 types (low, medium, and high TOC lev-
els) are recommended.

(ii) The day before the test is to be started, sediments (in treatments,
and reference and negative controls) should be screened to remove large
particles and endemic animals (especially midge predators) and added to
the test chambers. The amount of sediments to be added to each test cham-
ber will depend on the experimental design and test species. Only a mini-
mum amount (to a depth of 2 mm) should be added in the aqueous expo-
sure portion of the test. Each replicate test chamber should contain the
same amount of sediments. Overlying water should be added to each test
chamber.

(iii) In this flow-through test, the flow of dilution water through each
chamber is begun and adjusted to the rate desired. The test substance
should be introduced into each test chamber. The addition of test substance
in the flow-through system should be done at a rate which is sufficient
-------
to establish and maintain the desired concentration of test substance in
the test chamber.

(iv) At the initiation of the test, chironomids which have been cul-
tured or acclimated in accordance with the test design are randomly placed
into the test chambers. Midges in the test chambers are observed periodi-
cally during the test. Immobile or dead larvae should be counted, removed,
and weighed, and the findings recorded. "Floating" larvae are nonviable
and should be replaced. Dissolved oxygen (DO) concentration, pH, tem-
perature, the concentration (measured) of test substance, and other water
quality parameters should be measured at specified intervals in selected
test chambers, during all three parts of this test. (See Table 1 under para-
graph (d)(i)(i) of this guideline.) Data should be collected during the test
to determine any significant differences (P< 0.05) in mortality and growth
as compared to the controls. BCFs should be calculated at the end of the
test based on route of exposure.

(2) Range-finding test, (i) A range-finding test should be conducted
prior to beginning each of the three parts of the test to establish test solu-
tion concentrations for the three definitive parts of the test.

(ii) The chironomids should be exposed to a series of widely spaced
concentrations of the test substance (e.g., 1, 10, 100 mg/L).

(iii) A minimum of 10 chironomids should be exposed to each con-
centration of test substance for a period of time which allows estimation
of appropriate test concentrations. No replicates are required and nominal
concentrations of the chemical are acceptable.

(3) Definitive test, (i) The purpose of the definitive portion of the
test is to determine concentration-response curves, EC50 values, effects
of a chemical on mortality and growth, and the determination of BCFs
during subchronic exposure.

(ii) A minimum of 30 midges per concentration (15 midges per rep-
licate test chamber) should be exposed in each part of the test to five
or more concentrations of the test substance chosen in a geometric series
in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, 64 mg/
L). An equal number of chironomids should be placed in two replicates.
The concentration ranges should be selected to determine the concentra-
tion-response curves, EC50 values, and MATC. Solutions should be ana-
lyzed for chemical concentration prior to use and at designated times dur-
ing the test.

(iii) Each test should include controls consisting of the same dilution
water, sediments, conditions, procedures, and midges from the same popu-
lation (same egg mass in culture container), except that none of the test
substance is added.
-------
(iv) The test duration is 14 days for each of the three parts of the
test. The test is unacceptable if more than 20 percent of the control orga-
nisms die or are stressed or diseased during the test. A test period longer
than 14 days may be necessary for high log KOW chemicals.

(v) The number of dead chironomids in each test chamber should
be recorded on days 4, 7, 10, and 14 of the test. At the end of the test,
surviving midges are removed from the test chambers and weighed after
being blotted dry. Concentration-response curves, EC50 values, and associ-
ated 95 percent confidence limits for mortality should be determined for
days 4, 7, 10, and 14 in the aqueous exposure portion of the test. MATC,
NOEC, and LOEC values should be determined for midge survival and
growth.

(vi) In addition to survival and growth, any abnormal behavior or
appearance of the chironomids should be reported.

(vii) Distribution of midges among the test chambers should be ran-
domized. In addition, test chambers within the testing area should be posi-
tioned in a random manner or in a way that appropriate statistical analyses
can be used to determine variation due to placement.

(viii) A control sediment and/or a reference sediment should be used
in each part of this test. Use of these controls/references will help deter-
mine if the test is acceptable, serve to monitor the health of the
chironomids used in the testing and the quality and suitability of test condi-
tions, parameters and procedures, and aid in analyzing data obtained from
this test. A negative control should be run in the test, using a sediment
known to be nontoxic to the midges. A reference sediment can be run
in the test in addition to or in place of the negative control. The reference
sediment should be obtained from an area that is known to have low levels
of chemical contamination and which is similar to or identical to the test
sediments in physical and chemical characteristics.

(ix) In the first part of this test, the aqueous exposure, a minimal
amount of sediment (<2mm) is placed in the test chambers. The presence
of sediment is necessary to allow the midges to construct tubes, to reduce
stress to the chironomids, and to reduce cannibalism.

(x) BCFs should be calculated at the end of each part of the test.

(4) Analytical measurements—(i) Water quality analysis. (A) The
hardness, acidity, alkalinity, conductivity, TOC or COD, and paniculate
matter of the dilution water serving as the source of overlying water should
be measured on days 0 and 14. The month-to-month variation of these
values should be less than 10 percent and the pH should vary less than
0.4 units.
-------
(B) During all three parts of the flow-through test, DO, temperature,
and pH should be measured in each chamber on days 0, 4, 7, 10, and
14.

(ii) Analysis of test substance. (A) Deionized water should be used
in making stock solutions of the test substance. Standard analytical meth-
ods should be used whenever available in performing the analyses of water
and sediments. Radiolabeling of the test substance (e.g., by use of 14C)
may be necessary to measure quantities present in the sediments accu-
rately. The analytical method used to measure the amount of test substance
in the sample should be validated by appropriate laboratory practices be-
fore beginning the test. An analytical method is not acceptable if likely
degradation products of the test substance, such as hydrolysis and oxida-
tion products, give positive or negative interference which cannot be sys-
tematically identified and corrected mathematically. When radiolabeled
test substances are used, total radioactivity should be measured in all sam-
ples. At the end of the test, water, sediments, and tissue samples should
be analyzed using appropriate methodology to identify and estimate any
major (at least 10 percent of the parent compound) degradation products
or metabolites that may be present.

(B) The overlying water from each test chamber should be sampled
for the test substance on days 0, 7, and 14 for all three aqueous exposure
parts of this test.

(C) For the nonaqueous exposure parts of the test, the interstitial
water from each test chamber should be analyzed for the test substance
on days 0, 7, and 14. Interstitial water can be sampled by using a variety
of methods, such as removal of overlying water and centrifugation, filtra-
tion of sediments, pressing the sediments, or using an interstitial water
sample. Care should be taken during these measurements to prevent the
biodegradation, transformation, or volatilization of the test substance.

(D) For the nonaqueous exposure portion of the test, the sediments
from each test chamber should be analyzed for the test substance on days
0, 7, and 14.

(E) The sediment partition coefficient or soil-water partition coeffi-
cient is determined by dividing the average test substance concentration
in sediment by the respective average concentration in the water column.
Concentrations of test substance in the sediments to be used in this test
can be chosen by measuring these partition coefficients. This sediment par-
tition coefficient should be determined in triplicate by placing a quantity
of a sediment with a known TOC content and spiked with the radiolabeled
test substance into a quantity of dilution water. The ratio of sediment to
dilution water should simulate the ratio present in the test. The sediment/
dilution water mixture is shaken periodically, and the radiolabeled test sub-
-------
stance measured. This shaking and sampling procedure is repeated until
equilibrium is reached, as defined by the stage on the desorption curve.

(F) Overlying water samples should be filtered through a 0.45 ^.m
filter to determine the concentration of dissolved test substance.

(G) BCFs should be calculated by determining the amount of test
substance in the midge tissue and dividing by the concentration of test
substance in the water column, interstitial water, and sediments. At test
termination, the midges remaining in each test concentration are analyzed
for test substance. Suitable methods are available, such as radiolabeling
(I4C) the test substance, combusting the midges, trapping and counting
the resulting radioactivity and the BCF calculated. If insufficient
chironomid biomass is present at the conclusion of the test replicates may
be pooled. BCFs cannot be calculated if after pooling there is insufficient
biomass or if the accumulated test substance concentration is lower than
the detection limit for the test substance.

(iii) Numerical. (A) The number of dead midge second instars should
be counted during each definitive test. Appropriate statistical analyses
should provide a goodness-of-fit determination for mortality concentration-
response curves calculated on days 4, 7, 10, and 14. A 4-, 7-, 10-, and
14-day LC50 value based on second instar mortality, and with correspond-
ing 95 percent confidence intervals, should be calculated. The methods
recommended for calculating ECSOs include probit, logit, binomial, and
moving average.

(B) Appropriate statistical tests (e.g., analysis of variance and mean
separation tests) should be used to test for significant chemical effects on
growth (measured as wet weights) on days 4, 7, and 14. An MATC should
be calculated using these test criteria.

(C) In no case should any analytical measurements be pooled except
when calculating BCFs when there is insufficient biomass available for
individual measurements as described under paragraph (d)(4)(ii)(G) of this
guideline.

(e) Test conditions—(1) Test species—(i) Selection. (A) The midge,
Chironomus tentans or C. riparius, should be used in this test. Both spe-
cies are widely distributed throughout the United States, and the larvae
and adult flies can be cultured in the laboratory. The larval portion life
cycles of both species is spent in a tunnel or case within the upper layers
of benthic sediments of lakes, rivers, and estuaries. Feeding habits of both
species include both filter feeding and ingesting sediment particles.

(B) Second instar chironomids (< 10 days) of the same age and size
are to be used in this test. Third and fourth instar are less desirable, as
some evidence indicates they are less sensitive, at least to copper. Each
instar is 4 to 7 days in duration.
-------
(ii) Acquisition. (A) Chironomids to be used in this test should be
cultured at the test facility. Adult flies are collected from the chironomid
cultures and allowed to mate and lay egg masses. Two egg masses are
collected and allowed to hatch. The larvae are fed daily. When the second
instar stage (about 10 days after hatching) is reached, larvae are removed
and placed in the test chambers. Records should be kept regarding the
source of the initial stock and culturing techniques. All organisms used
for a particular test should have originated from the same population (cul-
ture container) and be the same age and size.

(B) Chironomids should not be used in a test if:

(7) During the final 48 hours of midge holding, obvious mortality
is observed.

(2) The larvae are not in the second instar.

(iii) Feeding. (A) During the test, the Chironomids should be fed the
same diet at the same frequency as that used for culturing and acclimation.
All treatments and controls should receive, as near as reasonably possible,
the same amount of food on a per-animal basis.

(B) The food concentration depends on the type used and the nutri-
tional requirements of the midges. The latter in turn is dependent upon
their developmental stage.

(iv) Loading. The number of test organisms placed in a test chamber
should not affect the test results. Loading should not exceed 30
Chironomids per liter per 24 hours in the flow-through test. Loading should
not affect test concentrations or cause the DO concentration to fall below
the recommended level.

(v) Care and handling of test organisms. (A) Chironomids should
be cultured in dilution water under similar environmental conditions as
those in the test. Food such as Tetra Conditioning Food has been dem-
onstrated to be adequate for chironomid cultures.

(B) Organisms should be handled as little as possible. When handling
is necessary, it should be done as gently, carefully, and as quickly as pos-
sible. During culturing and acclimation, midges should be observed for
any signs of stress, physical damage, and mortality. Dead and abnormal
individuals should be discarded. Organisms that are damaged or dropped
during handling should be discarded.

(vi) Acclimation. (A) Midges should be maintained in 100 percent
dilution water at the test temperature for at least 4 days prior to the start
of the test. This is easily accomplished by culturing them in the dilution

8
-------
water at the test temperature. Chironomids should be fed the same food
during the test as is used for culturing and acclimation.

(B) Midges should be maintained in facilities similar to those of the
testing area during culturing and acclimation to the dilution water.

(2) Test system—(i) General. (A) Facilities needed to perform this
test include:

(/) Containers for culturing and acclimating the chironomids.

(2) A mechanism for controlling and maintaining the water tempera-
ture during the culturing, acclimation, and test periods.

(3) Apparatus for straining paniculate matter, removing gas bubbles,
or aerating the water as necessary to ensure that the test solution flows
regularly into and out of the test chamber.

(4) Test chambers can be small aquaria capable of holding 3 L of
water or test solution, 5.7-L clear glass battery jars, or 1-L beakers made
of borosilicate glass. Each chamber should be equipped with screened
overflow holes, standpipes, or U-shaped notches covered with Nitex
screen.

(B) Construction materials and commercially purchased equipment
that may contact dilution water should not contain substances that can be
leaked or dissolved into aqueous solutions in quantities that can alter the
test results. Materials and equipment that contact test solutions should be
chosen to minimize sorption of test substances.

(C) Test chambers should be loosely covered to reduce the loss of
test solution or dilution water by evaporation, and to minimize the entry
of dust or other particulates into the solutions.

(ii) Test substance delivery. (A) In the flow-through test, propor-
tional diluters, metering pump systems, or other suitable systems should
be used to deliver the test substance to the test chambers.

(B) The delivery system should be calibrated before and after each
test. Calibration includes determining the flow rate through each chamber
and the concentration of the test substance in each chamber. The general
operation of the test substance delivery system should be checked twice
daily during the test. The 24-h flow rate through a test chamber should
be equal to at least 5x the volume of the test chamber. During a test,
the flow rates should not vary more than 10 percent from any one test
chamber to another or from one time to any other.

(iii) Cleaning of test system. AH test equipment and test chambers
should be cleaned before each test following standard laboratory proce-
-------
dures. Cleaning of test chambers may be necessary during the testing pe-
riod.

(iv) Dilution water. (A) Surface or ground water, reconstituted water,
or dechlorinated tap water are acceptable as dilution water if chironomids
will survive in it for the duration of the culturing, acclimation, and testing
periods without showing signs of stress. The quality of the dilution water
should be constant and should meet the specifications in the following
Table 2.:

Table 2.—Specifications For Dilution Water

Substance Maximum Concentration
Particulate matter 20 mg/L
TOC or COD 2 mg/L or 5 mg/L, respectively
Boron, fluoride 100 u.g/L
Un-ionized ammonia 10 ng/L
Aluminum, arsenic, chromium, cobalt, copper, 1 ug/L
iron, lead, nickel, zinc..
Residual chlorine 3 ug/L
Cadmium, mercury, silver 100 ng/L
Total organophosphorus pesticides 50 ng/L
Total organochlorine pesticides and poly- 50 ng/L or 25 ng/L respectively
chlorinated biphenyls (PCBs) or organic chlo-
rine..

(B) The water quality characteristics listed in Table 2. should be
measured at least twice a year or when it is suspected that these character-
istics may have changed significantly. If dechlorinated tap water is used,
daily chlorine analysis should be performed.

(C) If the diluent water is from a ground or surface water source,
conductivity, hardness, alkalinity, pH, acidity, particulate matter, TOC or
COD, and particulate matter should be measured. Reconstituted water can
be made by adding specific amounts of reagent-grade chemicals to
deionized or distilled water. Glass distilled or carbon filtered deionized
water with conductivity of less than 1 ^lohm/cm is acceptable as the diluent
for making reconstituted water.

(D) If the test substance is not soluble in water, an appropriate carrier
such as triethylene glycol (CAS No. 112-27-6), dimethylformamide (CAS
No. 68-12-2), or acetone (CAS No. 67-64-1) should be used. The con-
centration of such carriers should not exceed 0.1 mL/L.

(v) Sediments. (A) Preparation and source. (7) Sediments used in
this test may contain low (<1 percent) to high (> 15 percent) amounts of
organic carbon because they are derived from variable natural sediments.
Prior to use, the sediments should be sieved to remove larger particles.
The should be characterized for particle size distribution (sand, silt, clay
percentages), percent water holding capacity, total organic and inorganic
carbon, total volatile solids, COD, BOD, cation exchange capacity, redox

10
-------
potential (Eh), oils and greases, petroleum hydrocarbons, organophosphate
pesticide concentrations, organochlorine pesticide and polychlorinted
biphenyl (PCB) concentrations, toxic metal concentrations, and pH.

(2) The source of the sediments used in this test should be known
and the characteristics listed above should be measured every time addi-
tional sediments are obtained. The sediments should not contain any en-
demic organisms, as these may be chironomid predators.

(5) Sediments should not be resuspended during the test.

(3) Test parameters, (i) Environmental conditions of the water con-
tained in test chambers should be maintained as specified below:

(A) Temperature of 20 ±1 °C for C. tentans and 22 ±1 °C for C.
riparius.

(B) DO concentration of the dilution water should be 90 percent of
saturation or greater. The DO concentrations of the test solutions should
be 60 percent or greater of saturation throughout the test. Aeration may
be necessary, and if this is done, all treatment and control chambers should
be given the same aeration treatment.

(ii) Additional measurements include:

(A) The concentration of dissolved test substance (that which passes
through a 0.45 urn filter) in the chambers should be measured during the
test.

(B) At a minimum, the concentration of test substance should be
measured as follows:

(7) In each chamber before the test.

(2) In each chamber on days 7 and 14 of the test.

(3) In at least one appropriate chamber whenever a malfunction is
detected in any part of the test substance delivery system.

(C) Among replicate test chambers of a treatment concentration, the
measured concentration of the test substance should not vary by more than
20 percent at any time or 30 percent during the test.

(D) The dissolved oxygen concentration, temperature, and pH should
be measured at the beginning of the test and on days 7 and 14 in each
chamber.

(f) Calculated values—(1) Sediment partition coefficient. (A) The
sediment or soil-water partition coefficient (Kp) is defined as the ratio of

11
-------
the concentration of the test substance in the sediment (Cs) to the con-
centration in the water or interstitial water (Cw) as given in the follwing
expression:
The resultant Kp values for the sediment or sediments tested are used to
select test substance concentrations for the sediment test.

(B) The Kp value is equivalent or related to the sediment organic
carbon sorption coefficient multiplied by the percent organic carbon con-
tent of the sediment.

(C) The sediment partition coefficient should be determined in trip-
licate for each sediment type at equilibrium by spiking with the
radiolabeled test substance and shaking. The test substance concentration
in the water is measured radiometrically at intervals and the data used
to create a desorption curve. The process is repeated until an equilibrium
is reached, as defined by the shape of the curve.

(2) BCFs. BCFs should be calculated for each part of the test. These
values are computed as the amount of test substance present in the midge
tissues divided by test substance concentrations in the water column, inter-
stitial water, and sediments. At test termination, the chironomids remaining
in each test concentration are analyzed for radiolabeled test substance.

(g) Reporting. The sponsor should submit all data developed by the
test that are suggestive and predictive of toxicity and all associated
toxicologic manifestations to the Agency. In addition to the reporting re-
quirements prescribed in the GLPS, the reporting of test data should in-
clude the following:

(1) The name of the test, sponsor, testing laboratory, study director,
principal investigator, and dates of testing.

(2) A detailed description of the test substance including its source,
lot number, composition (identity and concentration of major ingredients
and major impurities), known physical and chemical properties, and any
carriers or other additives used and their concentrations.

(3) The source of the dilution water, its chemical characteristics (e.g.,
conductivity, hardness, pH, TOC or COD, and particulate matter) and a
description of any pretreatment.

(4) The source of the sediment, its physical and chemical characteris-
tics (e.g., particle size distribution, TOC, pesticide and metal concentra-
tions), and a description of any pretreatment.

(5) Detailed information about the chironomids used as a stock, in-
cluding the scientific name and method of verification, age, source, treat-

12
-------
merits, feeding history, acclimation procedures, and culture methods. The
age (in days) and instar stage of the midges used in the test should be
reported.

(6) A description of the test chambers, the volume of solution in the
chambers, and the way the test was begun (e.g., conditioning and test sub-
stance additions). The number of test organisms per test chamber, the num-
ber of replicates per treatment, the lighting, the test substance delivery
system, flow rates expressed as volume additions per 24 hours for the
flow-through subchronic test, the method of feeding (manual or continu-
ous), and type and amount of food.

(7) The concentration of the test substance in the water, interstitial
water, and sediments in test chambers at times designated in the flow-
through tests.

(8) The number and percentage of organisms that show any adverse
effect in each test chamber at each observation period, and wet weights
of midges in each test chamber at days 7 and 14.

(9) BCFs for all three parts of the test (i.e., overlying water or water
column, sediment, and interstitial water modes of exposure).

(10) All chemical analyses of water quality and test substance con-
centrations, including methods, method validations, and reagent blanks.

(11) The data records of the culture, acclimation, and test tempera-
tures. Information relating to calculation of sediment (or soil-water) parti-
tion coefficients (Kp).

(12) Any deviation from this test guideline, and anything unusual
about the test (e.g., diluter failure and temperature fluctuations).

(13) An LC50 value based on mortality and an EC50 value based
on adverse effects on growth (wet weights), with corresponding 95 percent
confidence limits, when sufficient data are present for days 4, 7, and 14.
These calculations should be made using the average measured concentra-
tion of the test substance.

(14) Concentration-response curves utilizing the average measured
test substance concentration should be fitted to both number of midges
that show adverse effects (mortality) and effects on growth or wet weights
of midges at days 4, 7, and 14. A statistical test of goodness-of-fit should
be performed and the results reported.

(15) The MATC to be reported is calculated as the geometric mean
between the lowest measured test substance concentrationthat had signifi-
cant (P < 0.05) effect and the highest measured test substance concentration
that had no significant (P>0.05) effect on days 4, 7, and 14 of the test.
The criterion selected for MATC computation is the one which exhibits

13
-------
an effect (a statistically significant difference between treatment and con-
trol groups (P<0.05) at the lowest test substance concentration for the
shortest period of exposure. Appropriate statistical tests (analysis of vari-
ance and mean separation tests should be used to test for significant test
substance effects. The statistical tests employed and the results of these
tests should be reported.

(h) References. The following references should be consulted for fur-
ther background information on this test guideline.

(1) Adams, W.J. et al. Aquatic safety assessment of chemicals sorbed
to sediments. R.D. Cardwell, R. Purdy, and R.C. Banner, eds. In: Aquatic
Toxicology and Hazard Assessment. ASTM STP 854. American Society
for Testing and Materials, Philadephia, PA (1985).

(2) Nebeker, A.V. et al. Relative sensitivity of Chironomus tentans
life stages to copper. Environmental Toxicology and Chemistry 3:151-158.
(1984).

(3) Nebeker, A.V. et al. Biological methods for determining toxicity
of contaminated freshwater sediments to invertebrates. Environmental
Toxicology and Chemistry 3:617-630. (1984).
14
-------
dEPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-132
April 1996
Ecological Effects Test
Guidelines
OPPTS 850.1800
Tadpole/Sediment
Subchronic Toxicity Test
'Public Draft"
-------
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.

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."
-------
OPPTS 850.1800 Tadpole/sediment subchronic toxicity test.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.} and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. The source material used in developing this har-
monized OPPTS test guideline is 40 CFR 797.1995 Tadpole/sediment
subchronic toxicity test.

(b) Purpose. This guideline may be used to develop data on the
subchronic toxicity of chemical substances and mixtures subject to envi-
ronmental effects testing. This guideline prescribes tests to be used to de-
velop data on the subchronic toxicity of chemicals sorbed to natural sedi-
ments to bullfrog tadpoles. The EPA will use data from these tests in as-
sessing the hazard of a chemical to the environment.

(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA), and the definitions in 40 CFR part 792—Good Lab-
oratory Practice Standards for physical, chemical, persistence, and ecologi-
cal effects testing apply to this test guideline. The following definitions
also apply:

Acclimation means the physiological compensation by test organisms
to new environmental conditions (e.g., temperature, hardness, pH).

Carrier means a solvent or dispersant used to dissolve a test sub-
stance.

Cation exchange capacity (CEC) means the sum total of exchangeable
cations that a sediment can absorb. The CEC is expressed in
milliequivalents of negative charge per 100 g or milliequivalents of nega-
tive charge per gram of sediment (dry weight).

Clay mineral analysis means the estimation or determination of the
kinds of clay-size minerals and the amount present in a sediment.

Conditioning means the exposure of construction materials, test cham-
bers, and testing apparatus to dilution water or to test solutions prior to
the start of a test in order to minimize the sorption of the test substance
onto the test facilities or the leaching of substances from the test facilities
into the dilution water or test solution.

Control means the exposure of test organisms to uncontaminated sedi-
ments.

Death means the total lack of movement by a test tadpole.

EC50 means that test substance concentration calculated from experi-
mentally-derived growth or sublethal effects data that has affected 50 per-
-------
cent of a test population during continuous exposure over a specified pe-
riod of time.

Flow-through means a continuous or an intermittent passage of dilu-
tion water through a test chamber, or a holding or acclimation tank with
no recycling.

LC50 means the test substance concentration calculated from experi-
mentally-derived mortality data that is lethal to 50 percent of a test popu-
lation during continuous exposure over a specified period of time.

Loading means the ratio of tadpole biomass (in grams, wet weight)
to the volume (in liters) of test solution in a test chamber or passing
through it in a 24-h period.

Lowest-observed-effect-concentration (LOEC) means the lowest treat-
ment (i.e., test concentration) of a test substance that is statistically dif-
ferent in adverse effect on a specific population of test organisms from
that observed in controls.

No-observed-effect-concentration (NOEC) means the highest treat-
ment (i.e., test concentration) of a test substance that shows no statistical
difference in adverse effect on a specific population of test organisms from
that observed in controls.

Organic matter is the organic fraction of the sediment; it includes
plant and animal residues at various stages of decomposition, cells and
tissues of sediment-based organisms, and substances synthesized by the
microbial community.

Particle size analysis is the determination of various amounts of dif-
ferent particle sizes in a sample (i.e., sand, silt, and clay), usually by sedi-
mentation, sieving, micrometry, or combinations of these methods. The
names and diameter ranges commonly used in the United States are pro-
vided in the following Table 1.:
Table 1.—Particle Size
Name
Diameter range
Very coarse sand
Coarse sand
Medium sand
Fine sand ,
Very fine sand ....
Silt
Clay
2.0 to 1.0 mm
1.0 to 0.5 mm
0.5 to 0.25 mm
0.25 to 0.125 mm
0.125 to 0.052 mm
0.052 to 0.002 mm
< 0.002 mm
Sediment is the unconsolidated inorganic and organic material that
is suspended in and being transported by surface water, or has settled out
and has deposited into beds.
-------
Static means the test solution is not renewed during the period of
the test.

Subchronic toxicity test means a method used to determine the con-
centration of a substance that produces adverse effects on a specified per-
centage of test organisms in a specified period of time (e.g., 30 days)
which is a significant portion of the organism's life cycle. In this guideline,
survival (i.e., death) and growth is used as the measure of toxicity.

Test slurry means the test substance and the natural sediment on
which the test substance is sorbed. This sediment/test substance slurry is
dosed directly into the tadpole.

(d) Test procedures—(1) Summary of the test, (i) Test chambers
are filled with appropriate volumes of dilution water, or appropriate
amounts of contaminated natural sediments and dilution water. If a flow-
through test is performed, the flow of dilution water through each chamber
is adjusted to the rate desired.

(ii) This toxicity test may be performed by either of two methods:

(A) Dosing the tadpole directly with a sediment/test substance slurry
and maintaining tadpoles in test chambers with only clean dilution water.

(B) Maintaining tadpoles in test chambers containing contaminated
sediments and allowing tadpoles to ingest contaminated sediments ad lib-
itum.

(iii) Tadpoles which have been acclimated in accordance with the test
design are introduced into the test and control chambers by stratified ran-
dom assignment.

(iv) Tadpoles in the test and control chambers should be observed
daily during the test. Dead tadpoles should be removed at least twice each
day and the findings recorded.

(v) Live tadpoles in the test and control chambers should be weighed
at least every 3 days.

(vi) The dissolved oxygen (DO) concentration, pH, temperature, and
the concentration of test substance in contaminated sediments and/or water
should be measured at intervals in selected test chambers.

(vii) Concentration-response curves, LC50, EC50, LOEC, and NOEC
values for the test substance are developed from the survival and growth
data collected during the test.

(2) Range finding test. If the toxicity of the test substance is not
already known, a range-finding test should be performed to determine the
range of concentrations to be used in the definitive test.
-------
(3) Definitive test, (i) This toxicity test may be conducted by either
of two methods:

(A) Dosing the tadpole directly with a sediment/test substance slurry
and maintaining tadpoles in test chambers with only clean dilution water.

(B) Maintaining tadpoles in test chambers containing sediments and
allowing tadpoles to ingest contaminated sediments ad libitum.

(ii) If this test is to be performed by dosing the tadpoles directly,
the sediment/test chemical slurry should be placed directly into the buccal
cavity of the tadpole with a pipet. The slurry should be shaken or mixed
and 50 uL of the slurry should be placed directly into the posterior portion
of the buccal cavity. The dosed tadpole should be held out of the water
for about 1 minute after dosing to ensure ingestion and then returned to
the test chamber. The test slurry should be prepared by adding 5 mL of
distilled water to 1 g of dry sediment; the test chemical is added and the
final volume is brought to 10 mL. This test slurry should be mixed on
a mechanical shaker for at least 8 h before dosing.

(iii) If this test is to be conducted by maintaining tadpoles in test
chambers containing contaminated sediments and allowing tadpoles to in-
gest contaminated sediments ad libitum, appropriate amounts of contami-
nated sediments sufficient to cover the bottom of each test chamber with
about 3 to 5 cm of the contaminated sediment should be prepared. An
appropriate amount of clean dilution water (i.e., about 10 to 20 cm above
the sediment) should be added carefully to each chamber followed by tad-
poles.

(iv) It is recommended that this test be performed three times, each
time with a different natural sediment depending on the organic carbon
content: Low (0.1 to 0.2 percent), medium (0.5 to 1.0 percent), and high
(2.0 to 3.0 percent) organic carbon content (refer to OPPTS guideline
835.1220). However, natural sediments with a medium organic carbon
content should be used if this test is to be done only once. Sediments
selected for testing should be characterized by sampling location, general
clay fraction mineralogy, percent sand, silt, and clay (particle size analy-
sis), percent organic matter, percent organic carbon, pH (1:1 solidsrwater),
and CEC.

(v) A minimum of 20 tadpoles should be exposed to each of five
or more test substance concentrations (i.e., treatments) and a control. Test
concentrations should be chosen in a geometric series in which the ratio
is between 1.5 and 2.0 mg/kg (e.g., 2, 4, 8, 16, 32, and 64 mg/kg). All
test concentrations should be based on milligrams of test chemical (100
percent active ingredient (AI)) per kilogram of sediment (dry weight). The
concentration range should be selected to determine the concentration-re-
sponse relationship, EC50 values, LOEC, and NOEC values for survival,
sublethal effects, and growth.
-------
(vi) An equal number of tadpoles should be placed in two or more
replicates. The distribution of individual tadpoles among the test chambers
should be randomized. Test concentrations in sediment and/or dilution
water should be analyzed for test chemical concentrations prior to the start
of the test and at designated times during the test.

(vii) Every test should include a control consisting of uncontaminated
sediments, the same dilution water, conditions, procedures, and tadpoles
from the same group used in the test, except that none of the test substance
is added.

(viii) The test duration is 30 days.

(ix) It is recommended that this test be performed under flow-through
conditions.

(x) The number of dead tadpoles should be recorded daily. In addi-
tion, the number of tadpoles showing sublethal effects and the type of
effect (e.g., any abnormal behavior or appearance) should also be recorded
daily. Each tadpole should be weighted every 3 days. Data on survival,
sublethal effects, and growth which are collected during the test are used
to calculate the LC50 value for survival, the EC50 value for sublethal
effects, the EC50 value for growth, and to determine the LOEC and NOEC
values on days 10, 20, and 30.

(xi) Tadpoles should be fed a suitable food every day. Food which
sinks to the bottom should be used; food which floats on the water surface
should not be used. In tests in which the tadpoles are dosed with a sedi-
ment/test chemical slurry and held in dilution water without sediments,
any excess food or fecal material should be removed when observed. In
tests in which tadpoles are allowed to feed ad libitum on contaminated
sediments, excess food should not be given.

(4) Test results, (i) Survival and growth should be the primary cri-
teria used in this test guideline to evaluate the toxicity of the test sub-
stance.

(ii) In addition to death, any abnormal behavior such as, but not lim-
ited to, erratic swimming, loss of reflex, increased excitability, lethargy,
or any changes in appearance or physiology, such as discoloration (e.g.,
reddened leg), excessive mucous production, opaque eyes, curved spine,
or hemorrhaging should be recorded.

(iii) Each test and control chamber should be checked for dead or
effected tadpoles and observations recorded every 24 h after the beginning
of the test or within 1 h of the designated times. Dead tadpoles should
be removed at least twice a day.

(iv) Live tadpoles in the test and control chambers should be weighted
at least every 3 days and fresh weights recorded.
-------
(v) The mortality data should be used to calculate LC50 values and
their 95 percent confidence limits, and to plot concentration-response
curves at 10, 20, and 30 days. The statistical methods recommended for
use in calculating LC50 values include probit, logit, moving average angle,
and binomial.

(vi) The sublethal effects and growth (i.e., fresh weight) data should
be used to plot concentration-response curves, calculate EC50 values, and
determine LOEC and NOEC values. The statistical methods recommended
for use in calculating the EC50 values include probit, logit, moving aver-
age angle, and binomial. Appropriate statistical methods (e.g., analysis of
variance and multiple comparison test) should be used to test for signifi-
cant differences between treatment means and determine LOEC and NOEC
values.

(vii) A test is unacceptable if:

(A) More than 20 percent of the control tadpoles die or appear to
be stressed, or are seen to be diseased during the test.

(B) The tadpoles in the control lose a significant amount of weight
during the test, i.e. 30 percent.

(5) Analytical measurements—(i) Water quality analysis. (A) The
hardness, acidity, alkalinity, pH, conductivity, total organic carbon (TOC)
or chemical oxygen demand (COD), and particulate matter of the dilution
water should be measured in the control test chambers at the beginning
of each static test and at the beginning and end of each flow-through test.
The month-to-month variation of the above values should be less than 10
percent and the pH should vary less than 0.4 units.

(B) During static tests, the DO concentration, temperature, and pH
should be measured in each test chamber at the beginning of the test, and
as often as needed thereafter, to document changes from the initial levels.
The dilution water volume should not be reduced by more than 10 percent
as a result of these measurements.

(C) During flow-through tests, the DO, temperature, and pH measure-
ments should be made in each chamber at the beginning of the test and
every 48 hours thereafter until the end of the test. It is recommended that
this test be done under flow-through conditions.

(ii) Collection of samples for measurement of test substance. Sam-
ples of sediment to be analyzed for the test substance should be taken
with a coring device. Samples of dilution water to be analyzed for
desorbed test substance should be taken midway between the top, bottom,
and sides of the test chamber. These samples should not include any sur-
face scum or material dislodged from the bottom or sides. Samples should
be analyzed immediately or handled and stored in a manner which mini-
-------
mizes loss of test substance through microbial degradation,
photodegradation, chemical reaction, volatilization, or sorption.

(iii) Measurement of test substance. (A) The concentration of test
substance in sediment and/or dilution water should be measured at a mini-
mum in each test chamber at the beginning (zero-hour, before tadpoles
are added) and every 10 days thereafter.

(B) The analytical methods used to measure the amount of test sub-
stance in a sample should be validated before beginning the test. The accu-
racy of a method should be verified by a method such as using known
additions. This involves adding a known amount of the test substance to
three samples of dilution water or sediment taken from a chamber contain-
ing dilution water and the same number of tadpoles as are used in the
test. The nominal concentration of the test substance in those samples
should span the concentration range to be used in the test. Validation of
the analytical method should be performed on at least 2 separate days
prior to starting the test.

(D) In addition to analyzing samples of dilution water and sediment,
at least one reagent blank, containing all reagents used, should also be
analyzed.

(E) Among replicate test chambers, the measured concentrations in
sediment should not vary more than 20 percent. The measured concentra-
tion of the test substance in sediment in any chamber during the test should
not vary more than 30 percent from the measured concentration prior to
initiation of the test.

(F) The mean measured concentration of test substance in sediment
(dry weight) should be used to plot all concentration-response curves and
to calculate all LC50, EC50, LOEC, and NOEC values.

(e) Test conditions—(1) Test species—(i) Selection. The test species
for this test is the bullfrog tadpole (Rana catesbeiana).

(ii) Age and condition of tadpoles. (A) Tadpoles having the mor-
phological characteristics of premetamorphic stages VI through IX as de-
scribed by Taylor and Kollros (1946) under paragraph (g)(3) of this guide-
line, characterized by the emergence of hind paddles and respiration by
gills, should be used. Tadpoles used in a test should be the same age,
weight (i.e., 2 to 5 g), and be of normal size and appearance for their
age. The longest tadpole should not be more than twice the length of the
shortest tadpole.
-------
(B) All newly acquired tadpoles should be quarantined and observed
for at least 14 days prior to use in a test.

(C) Tadpoles should not be used for a test if they appear stressed
or if more than 5 percent die during the 48 h immediately prior to the
test.

(iii) Acclimation of test tadpoles. (A) If the holding water is not
from the same source as the test dilution water, acclimation to the dilution
water should be done gradually over a 48-h period and tadpoles should
be held an additional 14 days in the dilution water prior to testing. Any
changes in water temperature should not exceed about 1 °C per hour or
3 °C per day. Tadpoles should be held for a minimum of 7 days at the
test temperature prior to testing.

(B) During the final 48 h of acclimation, tadpoles should be main-
tained in facilities with background colors and light intensities similar to
those of the testing area.

(2) Facilities—(i) General. Facilities needed to perform this test in-
clude:

(A) Flow-through tanks for holding and acclimating tadpoles.

(B) A mechanism for controlling and maintaining the water tempera-
ture during the holding, acclimation, and test periods.

(D) Apparatus for providing a 16-h light/8-h dark photoperiod with
a 15- to 30-min transition period.

(E) Chambers for exposing test tadpoles to the test substance.

(F) A dilution water delivery system for flow-through tests.

(ii) Construction materials. Construction materials and commer-
cially purchased equipment that may contact the stock solution or dilution
water should not contain substances that can be leached or dissolved into
aqueous solutions in quantities that can alter the test results. Materials and
equipment that contact stock or test solutions should be chosen to mini-
mize sorption of test chemicals. Glass, no. 316 stainless steel, and
perfiuorocarbon plastic should be used whenever possible. Concrete, fiber-
glass, or plastic (e.g., PVC) may be used for holding tanks, acclimation
tanks, and water supply systems, but they should be thoroughly condi-
tioned before use. If cast iron pipe is used in freshwater supply systems,
colloidal iron may leach into the dilution water and strainers should be
used to remove rust particles. Rubber, copper, brass, galvanized metal,

8
-------
epoxy glues, and lead should not come in contact with the dilution water
or stock solution.

(iii) Dilution water delivery system. In flow-through tests, the sys-
tem used should be calibrated before each test. Calibration includes deter-
mining the flow rate of dilution water through each chamber. The general
operation of the dilution water delivery system should be checked twice
daily during a test. The 24-h flow rate through a test chamber should
be a minimum of six tank volumes. During a test, the flow rates should
not vary more than 10 percent from one test chamber to another or from
one time to any other.

(iv) Test chambers. Test chambers made of stainless steel should
be welded, not soldered. Test chambers made of glass should be fused
or bonded using clear silicone adhesive. As little adhesive as possible
should be left exposed in the interior of the chamber.

(v) Cleaning of test system. Dilution water delivery systems and test
chambers should be cleaned before each test. They should be washed with
detergent and rinsed in sequence with clean water, pesticide-free acetone,
clean water, and 5 percent nitric acid, followed by two or more changes
of dilution water.

(vi) Dilution water. (A) Clean surface or ground water, reconstituted
water, or dechlorinated tap water is acceptable as dilution water if the
test tadpoles will survive in it for the duration of the holding, acclimating,
and testing periods without showing signs of stress, such as discoloration
(i.e., reddened leg), hemorrhaging, disorientation, or other unusual behav-
ior. The quality of the dilution water should be constant and should meet
the specifications in the following Table 2. when analyzed (at least twice
a year).
Table 2.—Specifications for Dilution Water
Substance
Particulate matter ...
Total organic carbon (TOC)
Chemical oxygen demand (COD) .
Un-ionized ammonia
Residual chlorine
Total organochlorine pesticides
Total organochlorine pesticides.
plus poly chlorinated biphenyls (PCBs)
Organic chlorine
Maximum
Concentra-
tion
20.0 mg/L
20 mg/L
50 mg/L
1.0 uq/L
1.0 ng/L
50.0 ng/L
50.0 ng/L
25.0 nq/L
(B) The concentration of DO in the dilution water should be between
90 and 100 percent saturation, or >5 mg/L at sea level. If necessary, the
dilution water can be aerated before the addition of the test substance.
-------
All reconstituted water should be aerated before use. Hardness should be
<180 mg/L as CaCO3; pH should be 6.5 to 8.5.

(C) If disease organisms (e.g., pathogenic bacteria) are present in the
dilution water in sufficient numbers to cause infection, they should be
killed or removed by suitable equipment.

(D) Glass distilled or carbon filtered deionized water with a con-
ductivity less than 1 jiS/cm is acceptable for use in making reconstituted
water. If the reconstituted water is prepared from a ground or surface water
source, conductivity, and TOC or COD should be measured on each batch.

(vii) Carriers. (A) Distilled water should be used in making stock
solutions of the test substance. If a carrier is absolutely necessary to dis-
solve the test substance, the volume used should be minimal. If the test
substance is a mixture, formulation, or commercial product, none of the
ingredients is considered a carrier unless an extra amount is used to pre-
pare the stock solution. Concentrations of stock solution should be based
on 100 percent AI of the test chemical.

(B) Triethylene glycol and dimethyl formamide are the preferred car-
riers, but acetone can also be used.

(3) Test parameters—(i) Loading. The number of tadpoles placed
in a test chamber should not be so great as to affect the results of the
test. The loading should not be so great that the test substance concentra-
tions in treated sediments are decreased by more than 20 percent due to
uptake by the tadpoles. Loading should not exceed one tadpole per liter
of dilution water in the test chamber at any time. Loading rates should
be adjusted to maintain the DO concentration above the recommended lev-
els and the ammonia concentration below 20 (J,g/L.

(ii) Dissolved oxygen concentration. The DO in each test chamber
should be greater than 5.0 mg/L.

(iii) Temperature. The test temperature should be about 18 °C. The
temperature should be measured at least hourly in one test chamber.

(iv) Light. A 16-h light/8-h dark photoperiod with a 15- to 30-
minute transition period should be maintained.

(e) Reporting. (1) The final report should include, but not necessarily
be limited to, the following information.

(i) Name and address of the facility performing the study, and the
dates on which the study was initiated and was completed, terminated,
or discontinued.

(ii) Objectives and procedures stated in the approved protocol, includ-
ing any changes in the original protocol.

10
-------
(iii) Statistical methods used for analyzing the data. 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.

(iv) The test substance identified by name, Chemical Abstracts Serv-
ice (CAS) registry number or code number, source, lot or batch number,
strength, purity, and composition, or other appropriate characteristics.

(v) Stability of the test and, if used, control substances under the con-
ditions of administration.

(vi) A description of the methods used, which should include the fol-
lowing:

(A) Description of the test chambers, the depth and volume of solu-
tion in the chamber, the specific way the test was begun (e.g., conditioning
and test substance additions), and for flow-through tests, a description of
the dilution water delivery system including a diagram if the design is
complex.

(B) The source of the dilution water, a description of any
pretreatrnent, and the measured hardness, acidity, alkalinity, pH, con-
ductivity, TOC or COD, and paniculate matter.

(C) The source of the natural sediment (i.e., sampling location), sedi-
ment physical-chemical properties, percent sand, silt, and clay (particle
size analysis), percent organic matter, percent organic carbon, pH (1:1 sol-
ids:water), CEC, general clay fraction mineralogy, and procedures used
to determine the above properties.

(D) Methods used to determine the placement of test chambers and
the assignment of treatment concentrations to particular test chambers to
ensure randomization of exposure.

(E) Frequency, duration, and methods of observations.

(F) Detailed information about the test tadpoles, including the sci-
entific name and method of verification, source of test species, histories
of the species, average fresh weight (grams), average size, age, observed
diseases, treatments and mortalities, acclimation procedures, and food
used.

(G) The number of treatments and replicates used, the number of or-
ganisms per replicate, the loading rate, and the flow rate of dilution water
for flow-through tests.

(H) A description of the preparation of the sediment/test substance
slurry or the treated sediments. A description of the dosing procedures
if tadpoles were dosed directly.

11
-------
(I) The concentration of the test substance in the test slimy or in
sediments and/or dilution water in each test chamber just before the start
of the test and at all subsequent sampling periods. The concentration of
the test substance in the stock solution, if used, and the type and concentra-
tion of carrier solvent, if used.

(vii) The measured DO, pH, and temperature and the lighting regime.

(viii) The reported results should include:

(A) The results of the preliminary test and measurements. The number
of tadpoles and concentrations of test substance used and observed effects
on tadpoles should be stated.

(B) For the definitive test, in each untreated control and for each treat-
ment concentration used:

(/) The number of dead and live tadpoles.

(2) The percentages of tadpoles that died or showed adverse sublethal
effects.

(3) The number that showed any abnormal effects.

(4) The fresh weights of live tadpoles.

(5) The LC50, EC50, LOEC, and NOEC values at days 10, 20, and
30.

Results of the data analysis should include the concentration-response
curves with 95 percent confidence limits and the results of a goodness-
of-fit (e.g., X2-square test).

(ix) A description of all circumstances that may have affected the
quality or integrity of the data.

(x) Methods and data records of all chemical analyses of water quality
parameters and test substance concentrations, including method validation
and reagent blanks.

(xi) 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.

(xii) 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.

(xiii) The locations where all specimens, raw data, and the final report
are stored.

12
-------
(xiv) The quality control statement prepared and signed by the quality
assurance unit.

(g) References. The following references should be consulted for ad-
ditional background information on this test guideline.

(1) National Research Council. Amphibians: Guidelines for the
Breeding, Care, and Management of Laboratory Animals. National Acad-
emy of Sciences, Washington, DC (1974).

(2) Perkins, K. W. et al. Reptiles and Amphibians: Care and Culture.
Carolina Biological Supply Co., Burlington, NC (1981).

(3) Taylor, A. C. and Kollros, J. J. Stages in the Normal Development
of Rana pipiens. Anatomy Records 94:2 (1946).
13
-------
-------
&EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-133
April 1996
Ecological Effects Test
Guidelines

OPPTS 850.1850
Aquatic Food Chain
Transfer
"Public Draft"
-------
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.

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."
-------
OPPTS 850.1850 Aquatic Food Chain Transfer
(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 OPP 72-6 Aquatic Organism Accumula-
tion Tests (Pesticide Assessment Guidelines, Subdivision E—Hazard Eval-
uation: Wildlife and Aquatic Organisms) EPA report 540/09-82-025
(1982).

(b) Test standards. Data sufficient to satisfy the requirements in 40
CFR 158.145 should be derived from tests which comply with the follow-
ing test standards:

(1) Test substance. Data should be derived from testing conducted
with the technical grade of each active ingredient in the product (studies
using radioisotopes require analytical grade) or the purest available form
of the principal degradatioc products, whichever has a water solubility of
less than 0.5 mg/L, an octanol/water partition coefficient greater than
1,000, and is persistent in water (i.e., a half-life greater than 4 days).

(2) Test organisms, (i) Consultation with the Agency is advised be-
fore selection of species is made. One or more of the following species
may be used in accumulation testing:

(A) A typical bottom-feeding fish (e.g., catfish or carp).

(B) A cold-water fish, a warm-water fish, or marine fish (e.g. brrok
trout, rainbow trout, bass, bluegill, northern pike, walleye, or sheepshead
minnow).

(D) Crustaceans (e.g., Daphnia spp., shrimp, or crayfish).

(E) Insect nymphs (e.g., mayfly).

(ii) The following factors should be considered in selecting species:

(A) The use pattern of the formulated product.

(B) The relative sensitivity of the different species to toxic effects.

(c) Reporting and evaluation of data. Specific data reporting and
evaluation guidance should be determined by consultation with the Agen-
cy.

1
-------
(d) References. The following references can provide usefiill back-
ground information on developing protocols. The conditions under which
an accelerated aquatic organism test may be an acceptable substitute for
a full-length test should be determined by consulting with the Agency.

(1) Johnson, B.T. and R.A. Schoettger. A biological model for esti-
mating the uptake, transfer, and degradation of xenobiotics in a food chain.
FEDERAL REGISTER 40(123):26906-26909. (June 25, 1975).

(2) Macek, K.J. et al. Bioconcentration of 14C pesticides by bluegill
sunflsh during continuous exposure. Pp. 119-142 in Structure-activity cor-
relations of studies of toxicity and bioconcentration with aquatic orga-
nisms. Proceedings of a symposium held at Burlington, Ontario, March
11-13, 1975. G.D. Veith and D.E. Konasewich, eds. Sponsored by the
Standing Committee on Scientific Basis for Water Quality Criteria of the
International Joint Commission's Research Advisory Board. (1975).

(3) Schimmel, S.C. et al. Acute toxicity to and bioconcentration of
endosulfan by estuarine animals. Pp. 241-252 in Aquatic Toxicology and
Hazard Evaluation. F.L. Mayer and J.L. Hamelink, eds. STP no. 634,
American Society for Testing and Materials, Philadelphia, PA (1977).

(4) Branson, D.R. et al. Bioconcentration of 2,2',4,4'-
tetrachlorobiophenyl in rainbow trout as measured by an accelerated test.
Transactions of the American Fish Society. 104:785-792 (1975).
-------
&EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-134
April 1996
Ecological Effects Test
Guidelines
OPPTS 850.1900
Generic Freshwater
Microcosm Test,
Laboratory
"Public Draft"
U.S. EPA Headquarters Library
Mail code 3201
1200 Pennsylvania Avenue NW
Washington DC 20460
-------
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.

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."
-------
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.
-------
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.
-------
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.
-------
(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 ECSOs 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-
-------
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.

(7) One day 28, the following criteria should be met in the static
microcosms:

(/) At least 90 percent reduction in nitrate (NOa) concentration.

O'O Algal biomass in each mL of medium has exceeded
2,000 x 104 (um)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 p.M 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.
-------
(2) From day 28 to the conclusion of the test, the performance of
control microcosms should always meet the following criteria:

(/) Algal biomass exceeds 100 x 104 (jim)3/mL per mL.

(if) Positive oxygen gain in daytime.

(Hi) Daphnid population density exceeds 15 Daphnia/100 mL.

(/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 gam 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:

(/) 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 macroinvertebrates to be stocked into each microcosm include:

(0 Sixteen daphnids consisting of 3 adults with embryos, 3 adults
without embryos, and 10 juveniles.

(«') Six ostracods.

(Hi) Twelve amphipods consisting of three mating pairs (if possible)
and six juveniles.
-------
(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:

(j) Dissolved oxygen gain in the daytime.

(K) pH value (pre-light).

(iii) Abundance of daphnids and the presence of ostracods and
amphipods.

(iv) 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:

(/) 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.

(if) 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.

(Hi) 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.

(iv) 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)(^) 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:

(f) 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.
-------
(H) 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.

(Hi) 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:

(/) 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.

(P) The test substance (and carrier, if needed) should be introduced
into appropriate microcosms 6 weeks following initial inoculation of the
system.

8
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(/) 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.

(#) 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.
-------
(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.

(//) 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.

(12) 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:

(1) 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-ujn 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.

10
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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:

(j) 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.

(M) Dissolved inorganic 14C should be kept sterile before the test.
For example, it may be kept in a sealed ampule and autoclaved.

(Hi) 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.

(/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 |lCi of 14C-labeled NaHCO3 (specific activity about 1.0
(O.Ci/1.0 jig) 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.

(v/) About 1 |iCi of NaHI4CO3 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.

(v//) After incubation, the phytoplankton culture in each rube should
be filtered through a 0.45 Jim filter membrane over a vacuum flask. The

11
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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 paniculate 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 NaHCOs) in
the incubation tube, should be measured simultaneously with measurement
of 14C uptake rate. Total CC>2 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:

(/) 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.

(if) Sieved microcosm water should be filtered under suction through
a 0.45 urn filter pad, which is covered with a fine powder of MgCCb
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.

(MI) 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.

(iv) 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 fluorornetric or spectrophotometric method.

(VH) 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] = mlI.64(A663 - A750) - 2.16(AM5 - A750) + 0.10(A630 - A75o) %
(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 photosynthetic activity.

13
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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 urn 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 l4C-g!ucose de-
composition should be performed as follows:

(/) A 15 mL water sample should be collected in a 50-mL flask.

(H) A glucose solution that contains 0.15 jiCi radioactivity in 0.3 mL
of distilled water should be added to the flask.

(HI) 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.

(/v) 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 CC^.

(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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(/5) 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.

(/ 7) Water-borne bacteria can be counted weekly.

(B) Chemical fate. Sampling should be performed according to the
following procedures:

(1) 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 man 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:

(/) Macrophytes, subdivided into roots, shoots, and leaves.

(K) Phytoplankton.

(m) Zooplankton.

(iv) Benthic fauna.

(v) Sediment core, sectioned into 1-cm subcores.

(v/) 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.
(6) Water pH.
(7) Nutrients (at least nitrate) in water.
(B) The following data should be obtained from the mixed-flask, mi-
crocosm test:
(/) Abundance of phytoplankton and zooplankton.
(2) Net daytime production (DO gain).
(3) Net nighttime respiration (DO loss).
(4} Chlorophyll a concentration.
(J) Water pH.
(6) 14C glucose decomposition rate.
(C) The following data should be obtained for the pond microcosm:
(1) 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.

(/) 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 rum 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.

18
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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).

(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. (J) 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
NaNO3
MgSO47H2O
KH2PO4
NaOH
CaCI22H2O
NaCI
Al2{SO4)s 18H2O
Na2SiC>3-9H2O
sand
Trace Metals:
FeSO47H2O
H3BO3
ZnSO4-7H2O
MnCI24H2O
Na2MoO42H2O
CuSO4-5H20
Co(NC-3)26H2O
EDTA
Vitamins:
Calcium pantothenate
Cyanocobalamin (Bi2)
Thiamin (Bi)
Riboflavin (B2)
Nicotinamide
Folic Acid
Biotin . .
Putrescine
Choline
Inositol
Pyrtdoxine monohydrochloride

Molecular
weight
85.0
246.5
136.0
40.0
147.0
58.5
666.5
284.0
278.0
61.8
287.5
197.9
242.0
249.7
291.0
292.0
476.5
1,355.4
337.3
376.4
122.1
441.4
244.3
161.1
181.7
216.2
205.7
Concentration
Units
mM
0.5
0.1
0.04
0.099
1.0
1.5
0.0048
0.80
\M
1.12
0.75
0.025
0.25
0.025
0.005
0.0025
1.42
1.47
0.000022
0.18
0.11
1.06
0.75
0.12
0.19
2.75
5.09
2.43
Element of
concern
N
Mg
P
Na
Ca
Na
Al
Na
Si
Fe
B
Zn
Mn
Mo
Cu
Co
EDTA
mg/L
7.0
2.43
1.23
2.27
40.0
34.5
0.26
36.8
22.4
0.0625
0.008
0.0015
0.0135
0.0024
0.00032
0.00015
0.4145
0.70
0.00003
0.06
0.04
0.13
0.33
0.03
0.03
0.50
1.10
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)(l) of this guideline used in the early protocol
development is also adequate.

(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.

21
-------
(C) Standard deviation of light intensities among the microcosms
within ±10 percent of the mean and a light intensity of 150 (j,Enr2sec
for this test.

(e) Reporting. (1) The final report should include, but not necessarily
be limited to, the following information:

(i) 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.
test.
(xi) The materials, the methods, and the results of any range-finding
(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
-------
(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.

(i) Absorbance density at 440 nm, as an index of the paniculate mate-
rials, including phytoplankton.

(it) Content of chlorophyll a.

(Hi) 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 photosynthetic 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.

(11) 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:

(1) 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.

23
-------
(6) EC5Q 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.

(1) 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).

24
-------
(10) Element cycling such as ammonium-nitrogen content, in
micrograms per liter.

(//) Maximum and minimum diel DO concentration on sampling
date.

(12) 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 paniculate and dis-
solved fractions of the water column.

(/5) Concentration of test substance in representative species of
zooplankton and benthos.

(16) Concentration of test substance in periphyton.

(17) 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

25
-------
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.
26
-------
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"
-------
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.

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."
-------
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.

Bioaccumulation 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.
-------
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-
-------
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.
-------
(v) A test substance that is insoluble in both water and water-miscible
earners 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 (Xg/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.
-------
(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.
-------
FIGURE i.—EXPERIMENTAL MICROCOSM (NOT DRAWN TO SCALE)
-------
(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.
-------
(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:

(/) 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

8
-------
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:

(/) 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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(5) 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.

(5) 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-jiM plankton net. The retained organisms should be rinsed into a

10
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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.

11
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(2) Surface film, if present.
(5) 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:
(/) Whether significant differences exist in biological attributes be-
tween:
(/) The control microcosms and the natural system.
(«) The carrier control and the carrier-free control.
(iii) 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:
(0 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
12
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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.
13
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FIGURE 2.—EXPERIMENTAL MICROCOSM FACILITY
r -o 0>
14
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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).

(1) 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.

15
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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.

16
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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.

(3) 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.

17
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(7) Effects of the test substance are assessed by comparing the treated
microcosms to carrier controls.

(B) Chemical fate. (/) 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.
(/) 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.

18
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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.

(5) The locations where all specimens, raw data, and the final report
are stored.

(P) The statement prepared and signed by the quality assurance unit.
19
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United States Prevention. Pesticides EPA712-C-96-135
Environmental Protection and Toxic Substances April 1996
Agency (7101)
vvEPA Ecological Effects Test
Guidelines
OPPTS 850.1950
Field Testing for Aquatic
Organisms
'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.

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 850.1950 Field testing for aquatic organisms.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.} and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. The source material used in developing this har-
monized OPPTS test guideline are OPP 72-7 Simulated or Actual Field
Testing for Aquatic Organisms (Pesticide Assessment Guidelines, Subdivi-
sion E—Hazard Evaluation; Wildlife and Aquatic Organisms) EPA report
540/09-82-024, 1982 and subsequent guidance on aquatic mesocosm tests
under paragraph (e)(3) of this guideline.

(b) Test standards—(1) Test substance. Unless specified otherwise,
data should be derived from testing conducted with an end-use product.
An end-use product may be the applicant's own product or a typical end-
use product.

(2) Concentration analysis. The concentration of the test substance
in the water should be determined at the start of the study and samples
should be collected periodically for analysis to verify concentrations.

(3) Test conditions. The test conditions for conducting field tests
should resemble the conditions likely to be encountered under actual use.
Specifically, the pesticide should be applied according to the rate, fre-
quency, and method specified on the label.

(4) Endangered species. Studies should not be conducted in critical
habitats or areas containing, or suspected to contain, endangered or threat-
ened plants or animals which may be threatened by the tests to be con-
ducted.

(5) Residue levels. When the test substance is applied under simu-
lated or actual field condition testing, residues should be determined in
appropriate vegetation, soil, water, sediments, and other environmental
components, and in selected tissues of test organisms.

(6) Other standards. Any additional standards for conducting these
tests will be provided by the Agency in writing following consultation
between the applicant and the Agency, and will take into account the
mechanisms by which a pesticide may enter the environment, and the food
sources and habitats that may be affected.

(c) Simulated field studies (mesocosm)—(1) Physical description—
(i) Experimental design. (A) One acceptable design is a minimum of four
experimental treatments consisting of a control which receives no test
compound, an X treatment level representing expected exposures, an X+
treatment level representing an upper bound, and an X- treatment level
representing a lower bound. At least three replicates per treatment level
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are needed to provide the requisite resolution of effects and probability
of their occurrence. However, it is recommended that the number of rep-
licates be dictated as a function of the parameters of interest and the sen-
sitivity of their analysis.

(B) Alternative designs which emphasize regression analysis and uti-
lize more treatment levels with fewer or no replicates may also be appro-
priate. Regression designs are most useful for determining maximum expo-
sure conditions which provide no significant impacts or a specified level
of effect in test systems.

(ii) Mesocosm number. A minimum of 12 mesocosms is required,
with additional mesocosms added as replicates or treatments when needed
to increase the sensitivity of analysis for specific parameters.

(iii) Mesocosm size. Dimensions of a mesocosm must be large
enough to accommodate a viable finfish population. Depth should be suffi-
cient to provide a representative open water area, and sloped sides should
provide a littoral area for macrophyte growth and finfish reproduction. An
acceptable design would occupy approximately 0.1 acre surface area with
a volume of at least 300 m3 and a maximum depth of 2 m. Sides of the
mesocosm should be sloped approximately 1 unit of drop for every 2-
3 units of linear distance.

(iv) Mesocosm features. (A) Mesocosms can be constructed as dug-
out ponds or enclosures of existing impoundments. The mesocosms should
be lined with an impervious material of known adsorption for the test
compound. The sediment used should be well-defined and representative
in composition (percent clay, silt and sand, organic carbon, and organic
nitrogen and ion exchange capacity) to pond sediments in the intended
use area of the pesticide. The sediment depth at the bottom of the systems
should be a minimum of 15 cm. Sediments may consist of natural pond
sediment or top soil. If top soil is used, the complete mesocosm should
be seasoned for 1 year prior to experimental use. This time is necessary
to develop benthic biota. If pond sediments are used, a shorter seasoning
period (e.g. 6 mon) is adequate. Organic content of the top soil should
be at least 2 percent.

(B) A means of interchange (circulation, fill-drain-refill, etc.) of the
water between the systems during initial establishment is desirable to en-
sure even distribution of biota among the mesocosms. Once the systems
have become established or at initiation of a test the circulation should
be stopped and each system kept separate from all other systems. The
required precautions to ensure no cross contamination from pond overflow
during rainstorms, leakage in the circulation system, etc., should be taken
from the outset.

(v) Mesocosm biota. (A) The mesocosms must contain a representa-
tive pond biota. It is recommended that an established pond with diverse
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biota act as a parent pond. The water in the mesocosrn should be equiva-
lent to the water of the parent pond and biota collected from the parent
pond should be evenly distributed to each mesocosm to act as a starter
base. Biota from other sources may be used to augment a natural assem-
blage to ensure adequate representation of important taxa.

(B) Phytoplankton are expected to reach a concentration consistent
with the nutrient levels of the system prior to introduction of
macroinvertebrates. Nutrient levels should be within a mesotrophic classi-
fication. The macroinvertebrate fauna should include representatives of the
rotifers, annelids, copepods, cladocerans, amphipods, aquatic insects, and
gastropods. Introduced macroinvertebrates, if necessary to augment natu-
rally colonized populations, should not exceed 10 g wet-weight/m3 and
finfish should not be introduced at more than 2 g wet-weight/m3. Fish
species used in the test must be of known sensitivity to the test compound
(determined from acute toxicity tests) and appropriate to small pond enclo-
sures. Finfish species used must be native North American species
(bluegill sunfish alone or in combination with largemouth bass are rec-
ommended).

(vi) Mesocosm treatment. Treatment levels of the mesocosms should
be based on exposure models and residue monitoring data if available.
In a three-replicate by four-treatment design, the three experimental treat-
ments should be separated into a low, intermediate, and high treatment
(dosed) and a control treatment (undosed). The intermediate treatment
should approximate the estimated environmental concentration determined
through modeling and experiential data for the intended pesticide use. It
is recommended that the low treatment be 1/10 and the high treatment
lOx the intermediate concentration. Regression designs should bracket ex-
pected exposures and expected response concentrations. Loading of pes-
ticide into the mesocosms is to be by direct overspray to simulate drift
and aerial deposition and with a sediment/water slurry channeled into the
system at predetermined points to simulate runoff. Model predictions with
available monitoring data will dictate the timing, frequency, and mode of
introduction of the test material.

(2) Measured parameters—(i) Chemical/physical properties. (A)
Mesocosm water should be monitored for pH, temperature, transparency
(turbidity), dissolved oxygen, alkalinity, total nitrogen, total phosphorus,
conductivity (total hardness), and particulate and dissolved organic carbon
at appropriate intervals (e.g., biweekly). Observations are to be made at
several locations throughout the mesocosm (which will be dictated by the
physical design of the mesocosm) and at appropriate depths to allow quan-
tification of vertical and horizontal variations. A complete water analysis
should be conducted at test initiation and termination, and at significant
periods during the test (i.e., pesticide inputs, substantial changes in other
observed parameters, etc.). Temperature, pH, and dissolved oxygen should
be monitored on a continuous basis for 24 h on a biweekly schedule and
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at significant periods during the test to provide an estimate of gross pro-
duction and community respiration.

(B) Mesocosm sediment must be analyzed for pesticide content, par-
ticle size, cation exchange capacity, organic content, and pH at the initi-
ation of the test.

(ii) Biological structure. (A) Biota will be identified to species or
lowest taxonomic unit practical. The schedule for sampling and collection
of biological samples will depend on the design and composition of the
mesocosm and must be determined prior to the initiation of the test. Col-
lections should not be so frequent as to disrupt the system.

(B) Phytoplankton are to be collected from the water column, domi-
nant species identified, and biomass determined by measuring chlorophyll
a and phaeophytin. All samples should be preserved for archival reference.
Periphyton are to be collected from glass slide substrates placed in the
mesocosm and exposed for a minimum of 2 weeks. Periphyton should
be analyzed for chlorophyll and ash-free weight. Macrophytes are to be
identified to species, biomass determined by dry weight, and percent cover
of the mesocosm determined.

(C) Zooplankton will be collected weekly with tube cores of the water
column and vertical net tows. All samples are to be archived for future
reference. Zooplankton samples will be analyzed biweekly by enumerating
and identifying dominant species. Cladocerans should be identified to
genus and differentiated by size (e.g., measured for length of muon).
Macroinvertebrates, at a minimum, should be collected from emergent in-
sect traps and artificial substrates. Sampling of sediment directly (e.g.,
Ekman dredge), should be employed cautiously, if necessary for tracking
benthic community parameters, to minimize disruption to the benthic com-
munity. Samples should be enumerated, identified to lowest practical
taxon, and archived.

(D) Finfish will be identified to species, enumerated, sexed (when
possible) and measured in length and weight (wet) at introduction into
the mesocosms and at test termination. Also at test termination, females
will be assessed for fecundity and all collected fish will be examined for
gross pathology. Spawning substrates will be placed in the systems and
periodically surveyed for number of deposited eggs.

(E) Toxicity testing and bioassays with indigenous fauna on-site and
in the laboratory may be used to assist in confirming cause and effect
relationships.

(iii) Residue analysis. Residues of the test material and major
degradates/metabolites will be analyzed at appropriate intervals to the envi-
ronmental properties of the compound in the water, sediments, and biota
at a sensitivity consistent with concentrations of concern.
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(iv) Meteorological conditions. Continuous monitoring of air tem-
perature, wind velocity, precipitation, evaporation, and solar radiation are
required within 1 mile of the mesocosm test facility.

(d) Actual field studies. Data from an actual field study are required
on a case-by-case basis to support registration of an end-use product in-
tended for outdoor application. Consultation with the Agency is advised
before undertaking these tests. Whenever data are required, the determina-
tion will be made in writing by the Agency and will state which properties
and use patterns of the product were used in the determination.

(e) References. The following references can provide useful back-
ground information for conducting a simulated or actual field study for
aquatic organisms.

(1) Graney, R.L. et al. (Eds.). Aquatic Mesocosm Studies in Ecologi-
cal Risk Assessment, Lewis, Boca Raton, FL (1994).

(2) Hill, I.R. et al. (Eds). Freshwater Field Tests for Hazard Assess-
ment of Chemicals, Lewis, Boca Raton, FL (1994).

(3) Touart, L.W. Aquatic Mesocosm Tests to Support Pesticide Reg-
istrations. U.S. Environmental Protection Agency, Hazard Evaluation Divi-
sion; Technical Guidance Document. National Technical Information Serv-
ice, Springfield, VA) (1988).

(4) Voshell, Jr., J.R. (Ed.). Using Mesocosms to Assess the Aquatic
Ecological Risk of Pesticides: Theory and Practice. MPPEAL 75 (1989).
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