United States Prevention, Pesticides EPA 712-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
'Public Draft"
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 850.1010 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.
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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-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 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
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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.
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(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:
(7) 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
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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.
Apparatus for straining particulate 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.
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(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 CaCO3
Particulate matter ....
Total organic carbon or
Chemical oxygen demand
Un-ipnized ammonia
Residual chlorine
Total organophosphorus pesticides
Total organochlorine pesticides plus polychlorinated biphenyls (PCBs) or
Organic chlorine
180 mg/L
20 mg/L
2 mg/L
5 mg/L
20 ug/L
<3 u,g/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.
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(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.
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(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.
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