United States Prevention, Pesticides EPA712-C-96-120
Environmental Protection and Toxic Substances April 1996
Agency (7101)
&EPA 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.
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 850.1300 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 72-4 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 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.
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 under the carapace
in response to stress conditions in daphnids.
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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 in 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
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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.
(C) Any ephippia are produced by control animals.
(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-
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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.
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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:
(7) 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.
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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:
(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 when water supplies contain particulate 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.
(6) 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:
Chemical oxygen demand
Un-ionized ammonia
Residual chlorine
Total organophosphorus pesticides
Total organochlorine pesticides plus polychlorinated biphenyls (PCBs)
or:
Organic chlorine
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 characteristics listed above should be measured
at least twice a year or when it is suspected that these characteristics may
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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 (lohm/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 <0.1mL/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.
(C) Photoperiod of 16-h light and 8-h darkness.
(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:
(7) 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,
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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-through
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
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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.
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