[Code of Federal Regulations]
[Title 40, Volume 30]
[Revised as of July 1, 2004]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR797.1330]
[Page 110-115]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 797_ENVIRONMENTAL EFFECTS TESTING GUIDELINES--Table of Contents
Subpart B_Aquatic Guidelines
Sec. 797.1330 Daphnid chronic toxicity test.
(a) Purpose. This guideline is intended for use in developing data
on the chronic toxicity of chemical substances and mixtures
(``chemicals'') subject to environmental effects test regulations under
the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003,
15 U.S.C. 2601 et seq.). This guideline prescribes a chronic toxicity
test in which daphnids are exposed to a chemical in a renewal or a flow-
through system. The United States Environmental Protection Agency will
use data from this test in assessing the hazard a chemical may present
to the aquatic environment.
(b) Definitions. The definitions in section 3 of the Toxic
Substances Control Act (TSCA), and the definitions in part 792 Good
Laboratory Practice Standards of this chapter apply to this test
guideline. In addition, the following definitions apply to this
guideline:
(1) Brood stock means the animals which are cultured to produce test
organisms through reproduction.
(2) Chronic toxicity test means a method used to determine the
concentration of a substance in water that produces an adverse effect on
a test organism over an extended period of time. In this test guideline,
mortality and reproduction (and optionally, growth) are the criteria of
toxicity.
(3) EC50 means that experimentally derived concentration
of test substance 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.
(4) Ephippium means a resting egg which develops under the carapace
in response to stress conditions in daphnids.
(5) Flow-through means a continuous or intermittent passage of test
solution or dilution water through a test chamber or culture tank with
no recycling.
(6) Immobilization means the lack of movement by daphnids except for
minor activity of the appendages.
(7) 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.
(8) MATC (Maximum Acceptable Toxicant Concentration) means the
maximum concentration at which a chemical can be present and not be
toxic to the test organism.
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(9) 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 minutes after the test substance
has been added and uniformly distributed in the test chambers in the
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 stable. 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 quality parameters are measured at
specified intervals in selected test chambers. Data are collected during
the test to determine any significant differences (p[lE]0.05) in
immobilization and reproduction as compared to the control.
(2) [Reserved]
(3) 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
concentration 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.
(4) Definitive test. (i) The purpose of the definitive test is to
determine concentration-response curves, EC50 values and
effects of a chemical on immobilization and reproduction during chronic
exposure.
(ii) A minimum of 20 daphnids per concentration shall 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). An equal number of daphnids shall be placed in two or more
replicates. The concentration ranges shall be selected to determine the
concentration-response curves, EC50 values and MATC.
Solutions shall be analyzed for chemical concentration at designated
times during the test.
(iii) Every test shall include controls consisting of the same
dilution water, conditions, procedures and daphnids from the same
population (culture container), except that none of the chemical is
added.
(iv) The test duration is 21 days. The test is unacceptable if:
(A) More than 20 percent of the control organisms appear to be
immobilized, 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 shall be
recorded on day 21 of the test. After offspring are produced, they shall
be counted and removed from the test chambers every 2 or 3 days.
Concentration-response curves, EC50 values and associated 95
percent confidence limits for adult immobilization shall be determined
for day 21. An MATC shall be determined for the most sensitive test
criteria measured (number of adult animals immobilized, number of young
per adult, and number of immobilized young per adult).
(vi) In addition to immobility, any abnormal behavior or appearance
shall also be reported.
(vii) Test organisms shall be impartially distributed among test
chambers in such a manner that test results show
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no significant bias from the distributions. In addition, test chambers
within the testing area shall be positioned in a random manner as in a
way in which appropriate statistical analyses can be used to determine
the variation due to placement.
(5) [Reserved]
(6) 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
substance in a sample shall 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. The number of immobilized adults, total offspring
per adult, and immobilized offspring per adult shall 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
immobilization and corresponding 95 percent confidence intervals shall
also 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 criteria (cumulative number of
immobilized adults, cumulative number of offspring per adult and
cumulative number of immobilized offspring per adult) on day 21. An MATC
shall 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 appropriate systematic keys.
(B) First instar daphnids, [lE]24 hours old, are to be used to start
the test.
(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 shall have originated from the same
culture population.
(B) Daphnids shall 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
preceding the test.
(4) Adults in the culture do not produce an average of at least 3
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 shall be fed the
same diet and with the same frequency as that used for culturing and
acclimation. All treatments and control(s) shall receive, as near as
reasonably possible, the same ration of food on a per-animal basis.
(B) The food concentration depends on the type used. Food
concentrations should be sufficient to support normal growth and
development and to allow for asexual (parthenogenic) reproduction. For
automatic feeding devices, a suggested rate is 5 to 7 mg food (either
solids or algal cells, dry weight) per liter dilution water or test
solution. For manual once-a-day feeding, a suggested rate is 15 mg food
(dry weight) per liter dilution water or test solution.
(iv) Loading. The number of test organisms placed in a test chamber
shall not affect test results. Loading shall not exceed 40 daphnids per
liter in the renewal system. In the flow-through test, loading limits
will vary depending on the flow rate of the dilution water. Loading
shall not cause the dissolved oxygen concentration to fall below the
recommended 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 have been demonstrated to be
adequate for daphnid
[[Page 113]]
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 shall be discarded. Organisms
that touch dry surfaces or are dropped or injured during handling shall
be discarded.
(C) Smooth glass tubes (I.D. greater than 5mm) 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 so as not to trap air under the carapace.
(vi) Acclimation. (A) Brood daphnids shall be maintained in 100
percent dilution water at the test temperature for at least 48 hours
prior to the start of the test. This is easily accomplished by culturing
them in dilution water at the test temperature. During acclimation,
daphnids shall 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
intensities similar to those of the testing area.
(2) Facilities--(i) General. (A) Facilities needed to perform this
test include:
(1) Containers for culturing and acclimating daphnids.
(2) A mechanism for controlling and maintaining the water
temperature during the culturing, acclimation and test periods.
(3) 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-hour light and 8-hour dark photo
period.
(5) An apparatus to introduce food if continuous or intermittent
feeding is used.
(6) In addition, the flow-through test shall contain appropriate
test chambers in which to expose daphnids to the test substance and an
appropriate 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 renewal tests, daphnids can be conveniently exposed to the
test solution in 250 ml beakers or other suitable containers.
(C) For flow-through tests daphnids can be exposed in glass or
stainless steel containers with stainless steel or nylon screen bottoms.
Such containers shall 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
centimeters 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.
(D) Test chambers shall 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 shall 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-hour flow rate through a test
chamber shall be equal to at least five times the volume
[[Page 114]]
of the test chamber. During a test, the flow rates shall not vary more
than 10 percent from any one test chamber to another. For the renewal
test, test substance dilution water shall 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
following specificiations:
------------------------------------------------------------------------
Substance Maximum concentration
------------------------------------------------------------------------
Particulate matter......................... 20 mg/l.
Total organic carbon or.................... 2 mg/l.
Chemical oxygen demand............... 5 mg/l.
Un-ionized ammonia......................... 20 [mu]g/l.
Residual chlorine.......................... <3 [mu]g/l.
Total organophosphorus pesticides.......... 50 ng/l.
Total organochlorine pesticides plus 50 ng/l.
polychlorinated biphenyls (PCBs).
or organic chlorine.................. 25 ng/l.
------------------------------------------------------------------------
(B) The water quality characteristics listed above shall 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 chlorine analysis shall 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) 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
conductivity of less than 1 microohm/cm is acceptable as the diluent for
making reconstituted water.
(D) If the test substance is not soluble in water an appropriate
carrier should be used.
(v) Cleaning of test system. All test equipment and test chambers
shall be cleaned before each use following standard laboratory
procedures. Cleaning of test chambers may be necessary during the
testing period.
(3) Test parameters. (i) Environmental conditions of the water
contained in test chambers should be maintained as specified in this
paragraph:
(A) The test temperature shall be 20 [deg]C. Excursions from the
test temperature shall be no greater than 2
[deg]C.
(B) Dissolved oxygen concentration between 60 and 105 percent
saturation. Aeration, if needed to achieve this level, shall be done
before the addition of the test substance. All treatment and control
chambers shall be given the same aeration treatment.
(C) Photoperiod of 16-hours light and 8-hours darkness.
(ii) Additional measurements include:
(A) The concentration of the test substance in the chambers shall 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, the measured concentration of the test substance should
not vary more than 20 percent.
(4) An apparatus for providing a 16-hour light and 8-hour dark
photoperiod.
(C) The dissolved oxygen concentration, temperature and pH shall 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
concentrations.
(e) Reporting. The sponsor shall submit to the U.S. Environmental
Protection Agency all data developed by the test that are suggestive or
predictive of chronic toxicity and all associated toxicologic
manifestations. In addition to the reporting requirements prescribed in
the part 792--Good Laboratory Practice Standards of this chapter the
reporting of test data shall include the following:
(1) The name of the test, sponsor, testing laboratory, study
director, principal investigator, and dates of testing.
[[Page 115]]
(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), and a description of any
pretreatment.
(4) Detailed information about the daphnids used as brood stock,
including the scientific name and method of verification, age, source,
treatments, feeding history, acclimation procedures, and culture
methods. The age of the daphnids used in the test shall 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 renewal process
and schedule for the renewal chronic test, the test substance delivery
system and flow rate expressed as volume additions per 24 hours 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 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 and the number of offspring per adult in the control
replicates and in each treatment replicate.
(9) All chemical analyses of water quality and test substance
concentrations, including methods, method validations and reagent
blanks.
(10) The data records of the culture, acclimation, and test
temperatures.
(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
significant (p[lE]0.05) effect and the highest measured test substance
concentration that had no significant (p[lE]0.05) effect on day 21 of
the test. The most sensitive of the test criteria (number of adult
animals immobilized, the number of young per female and the number of
immobilized young per female) is used to calculate the MATC. The
criterion selected for MATC computation is the one which exhibits an
effect (a statistically significant difference between treatment and
control groups; p[lE]0.05) at the lowest test substance concentration
for the shortest period of exposure. Appropriate statistical tests
(analysis of variance, mean separation test) shall be used to test for
significant test substance effects. The statistical tests employed and
the results of these tests shall be reported.
(13) Concentration-response curves utilizing the average measured
test substance concentration shall be fitted to cumulative adult
immobilization data at 21 days. A statistical test of goodness-of-fit
shall be performed and the results reported.
(14) An EC50 value based on adult immobilization with
corresponding 95 percent confidence limits when sufficient data are
present for day 21. These calculations shall be made using the average
measured concentration of the test substance.
[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19060, May 20, 1987]