United States
Environmental Protection
Iml M m Agency
Office of Chemical Safety EPA 712-C-16-005
and Pollution Prevention October 2016
(7101)
Ecological Effects
Test Guidelines
OCSPP 850.1300:
Daphnid Chronic
Toxicity Test
-------
NOTICE
This guideline is one of a series of test guidelines established by the United States
Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention (OCSPP) for
use in testing pesticides and chemical substances to develop data for submission to the Agency under
the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide,
Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food,
Drug and Cosmetic Act (FFDCA) (21 U.S.C. 346a). Prior to April 22, 2010, OCSPP was known as
the Office of Prevention, Pesticides and Toxic Substances (OPPTS). To distinguish these guidelines
from guidelines issued by other organizations, the numbering convention adopted in 1994 specifically
included OPPTS as part of the guideline's number. Any test guidelines developed after April 22, 2010
will use the new acronym (OCSPP) in their title.
The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions under
TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the test, and is also
used by EPA, the public, and the companies that are subject to data submission requirements under
TSCA, FIFRA, and/or the FFDCA. As a guidance document, these guidelines are not binding on
either EPA or any outside parties, and the EPA may depart from the guidelines where circumstances
warrant and without prior notice. At places in this guidance, the Agency uses the word "should." In
this guidance, the use of "should" with regard to an action means that the action is recommended
rather than mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline, but EPA recognizes that departures may be
appropriate in specific situations. You may propose alternatives to the recommendations described in
these guidelines, and the Agency will assess them for appropriateness on a case-by-case basis.
For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.gov/ocspp and select "Test Methods & Guidelines" on the
navigation menu. You may also access the guidelines in http://www.regulations.gov grouped by
Series under Docket ID #s: EPA-HQ-OPPT-2009-0150 through EPA-HQ-OPPT-2009-0159, and
EPA-HQ-OPPT-2009-0576.
l
-------
OCSPP 850.1300: Daphnid chronic toxicity test
(a) Scope.
(1) Applicability. This guideline is intended for use in meeting testing 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, et seq.). It describes
procedures that, if followed, would result in data that would generally be of scientific
merit for the purposes described in paragraph (b) of this guideline.
(2) Background. The source materials used in developing this harmonized OCSPP 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; see
paragraph (j)(4) of this guideline); Standard Evaluation Procedure: Daphnia magna Life-
Cycle (21-Day Renewal) Chronic Toxicity Test (see paragraph (j)(5) of this guideline);
EPA Pesticide Reregi strati on Rejection Rate Analysis: Ecological Effects (see paragraph
(j)(6) of this guideline); OECD 211 Daphnia magna Reproduction Test, 2012 (see
paragraph (j)(3) of this guideline); and ASTM El 193-97, Standard Guide for Conducting
Daphnia magna Life-Cycle Toxicity Tests (see paragraph (j)(l) of this guideline).
(b) Purpose. This guideline is intended for use in developing data on the chronic toxicity of
chemical substances and mixtures ("test chemicals" or "test substances") subject to
environmental effects test regulations. This guideline describes a chronic toxicity test in which
daphnids {Daphnia magna or Daphniapulex) are exposed to a test substance in static-renewal or
flow-through systems. The Environmental Protection Agency will use data from this test to
assess the hazards and risks a test substance may present in the aquatic environment.
(c) Definitions. The definitions in OCSPP 850.1000 apply to this test guideline. In addition, the
following more specific definitions apply to this test guideline:
Effective concentration, median (EC50) is the experimentally derived concentration of test
substance in dilution water that would be expected to cause a defined adverse effect in 50
percent (%) of a group of test organisms under specified exposure conditions. In this
guideline, the effect measured is immobilization as a surrogate for death.
Ephippium is a resting egg that develops in daphnids under the carapace in response to
stress conditions.
Immobilization refers to a lack of movement except for minor spontaneous, random
activity of appendages; those animals that are not able to swim within 15 seconds after
gentle agitation of the test vessel are considered to be immobilized even if they can still
move their antennae. Immobilization, as defined for this guideline, is used as a surrogate
for death. Dead animals are counted as immobilized.
Fo (Parental generation) are those daphnids (less than (<) 24 hours old) that are used to
begin the test and subsequently develop into adults and produce offspring during the test.
1
-------
Fi (Filial generation, first) are the offspring produced by the parental generation (Fo).
(d) General considerations.
(1) Summary of the test. Daphnids are exposed to the test substance and to appropriate
controls {i.e., dilution water control and a vehicle (solvent) control, if a vehicle is used)
for 21 days, during which observations are made on survival, growth, and reproduction.
The results of the test are used to calculate, at a minimum, the hypothesis-based no
observed effect concentration (NOEC) and the lowest observed effect concentration
(LOEC) for these endpoints. Concentration-response regression-based models may also
be fit, and point estimates {e.g., inhibition concentration (ICX) and effect concentration
(ECX)) may be calculated ancillary to NOEC and LOEC determinations where there is
sufficient coverage of the concentration-response curve.
(2) General test guidance. The general guidance in OCSPP 850.1000 applies to this
guideline except as specifically noted herein.
(3) Range-finding test. A range-finding test is usually conducted to establish the
appropriate test solution concentrations to be used in the definitive test. In the range-
finding test, the test organisms are generally exposed to a series of widely-spaced
concentrations of the test substance {e.g., 1, 10, 100 milligrams per liter (mg/L)) for a
period of time that allows estimation of the appropriate chronic test concentrations. Often
chronic range-finding test concentrations are selected based upon the results of an acute
toxicity test. It should be noted, however, that the presence of food in the chronic test can
affect the results and consequently the predictive ability of the acute test. The details of
the range-finding test do not have to be the same as those of definitive testing in that the
number of replicates, the number of test organisms, and duration of exposure may be less
than that used in definitive testing. In addition, the types of observations made on test
organisms may not be as detailed or as frequently observed as that of a definitive test.
Results of range-finding tests should be reported along with the results of the definitive
test, if range-finding tests are conducted.
(4) Definitive test.
(i) The goal of the definitive test is to determine the effects of the test substance
on immobilization, growth, and reproduction during chronic exposure. The test
endpoints are expressed in terms of the results of hypothesis-based testing {i.e.,
NOEC and LOEC). Concentration-response regression-based models may also be
fit, and point estimates {e.g., inhibition concentration (ICX) and effect
concentration (ECX)) may be calculated ancillary to NOEC and LOEC
determinations where there is sufficient coverage of the concentration-response
curve.
(ii) A minimum of 5 concentrations of the test substance, plus appropriate
controls, should be tested. For hypothesis-based testing, there should be 1
concentration level where no adverse effects occur as compared to the control(s)
and a second, higher concentration with an adverse effect. For regression-based
2
-------
endpoints, if calculated, the selected test concentrations, at a minimum, should
bracket the desired median inhibition/effect concentration (ICX/ECX) of the most
sensitive endpoint. Analytical confirmation of dissolved test concentrations
should be performed as described in OCSPP 850.1000. Summaries of the test
conditions are presented in Table 2 of this guideline. Test validity elements are
listed in Table 3.
(5) Limit test. In some situations, it is only necessary to ascertain that no effects on
daphnid survival, growth, or reproduction occur at a certain limit concentration (i.e.,
NOEC greater than or equal to (>) limit concentration). For guidance on the number of
replicates and organisms to use at the limit concentration, refer to paragraph (e)(6) in this
guideline. For most industrial chemicals, the lower of 10 mg/L or the limits of water
solubility or dispersion is considered appropriate as the limit concentration. For
pesticides, the lower of 10 milligrams active ingredient per liter (mg a.i./L), when
estimated environmental concentrations are not expected to exceed 10 mg/L, or the limit
of water solubility may be used as the limit concentration. Except for the number of test
concentrations, limit tests should follow the same test procedures, have the same duration
as the multiple-concentration definitive test (see Table 2 of this guideline), and have both
a dilution water control and a vehicle (solvent) control, if a vehicle is used. Limit tests,
like definitive tests, should include analytical confirmation of the dissolved concentration
of the test substance. If there is a statistically significant inhibition in any of the response
variables in Table 1 at the limit concentration as compared to the control(s) (i.e., no
observed effect concentration (NOEC) less than (<) limit concentration), a multiple-
concentration definitive test should be conducted.
(e) Test standards.
(1) Test substance. The substance to be tested should be technical or reagent grade
unless the test is designed to test a specific formulation, mixture, or end-use product. For
pesticides, if more than one active ingredient constitutes a technical product, then the
technical grade of each active ingredient should be tested separately, in addition to the
combination, if applicable. OCSPP 850.1000 lists the type of information that should be
known about the test substance before testing and discusses methods for preparation of
test solutions.
(2) Test duration. The test duration is 21 days.
(3) Test organism.
(i) Species. The test species are Daphnia magna or Daphnia pulex. First instar
daphnids, <24 hours old, should be used to start the test.
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 originate from the
same culture population that should have been maintained for at least 21 days in
3
-------
the same conditions and in the same dilution water source as those used for
testing.
(ii) Holding and acclimation. During culturing and acclimation to the dilution
water, stock animals should be maintained in culture conditions (e.g.,
temperature, light intensity) similar to those to be used in the test. Any changes in
the temperature and chemistry of the dilution water used for culturing the test
organisms to those of the test should be gradual, and a group of brood daphnids
should be maintained in dilution water at the test temperature for at least 48 hours
prior to the start of the test. Within a 24-hour period, changes in water
temperature should not exceed 1 degree Celsius (°C).
(iii) Health status and condition. Daphnids should not be used for a test:
(A) If the culture contains ephippia;
(B) If more than 5% of the culture stock dies or shows signs of stress (e.g.,
exhibits abnormal behavior) during the 48 hours preceding the test;
(C) If adults in the culture do not produce offspring before day 12;
(D) If adults in the culture do not produce an average of at least 3
offspring per adult per day over the 7-day period prior to the test;
(E) If they are first-brood progeny;
(F) If they have been used in a previous test, either in a treatment or in a
control group.
(iv) Care and handling. Organisms should be handled as little as possible, but
when necessary, it should be done as carefully and quickly as possible. During
culturing, holding, 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 also be discarded. Smooth glass
tubes (inner diameter greater than (>) 5 millimeters (mm)) equipped with rubber
bulbs have been used in 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.
(v) Diet and feeding. During the test, the daphnids should be fed the same diet as
that used for culturing, holding, and acclimation. All treatments and controls
should receive, as near as possible, the same ration of food on a per-animal basis.
The feeding regime should provide for an acceptable level of reproduction (see
Table 3 of this guideline).
Feeding should occur once per day in static-renewal tests, typically after the test
solutions are renewed (on renewal days) and at the same time on non-renewal
4
-------
days. At least 2 feedings, and preferably 3 feedings, per day are recommended in
flow-through tests. Continuous feeding devices may also be used.
Various combinations of trout chow, yeast, alfalfa, and algae have been
successfully used for culturing and testing daphnids; however, research has
demonstrated that it is best to incorporate algae into the diet to maintain
consistently healthy daphnids (see paragraph (j)(l) of this guideline). The
concentration of food provided 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 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. Other recommended
feeding regimes include 1 mg/L trout chow suspension (optional) or 1 x 108 algal
cells/L, or both (see paragraph (j)(l) of this guideline). For I). magna, ration
levels between 0.1 and 0.2 mg organic carbon per daphnid per day should be
sufficient for achieving the number of offspring to meet the test validity elements
(see paragraph (j)(2) of this guideline).
(4) Administration of test substance.
(i) Preparation of test solutions. Preparation of test solutions depends on the
solubility and stability of the test substance. Guidance for preparation of test
solutions, especially for difficult or low solubility test substances, is provided in
OCSPP 850.1000. Dilution water source and quality used in the test are described
in OCSPP 850.1000 and paragraph (e)(7)(vi) of this guideline.
The concentration of vehicle solvent should not exceed 0.1 milliliters per liter
(mL/L). A previous review recommends that solvent concentrations as low as 0.02
mL/L of dilution water be used (see paragraph (j)(2) of this guideline).
The pH of stock solutions may be adjusted to match the pH of dilution water or to
a neutral pH if pH change does not affect the stability of the test substance in
water. The pH of test solutions may be adjusted after the addition of the test
substance or stock solution into the dilution water. However, all pH adjustments
need to be made prior to the addition of test organisms. Hydrochloric acid (HC1)
and sodium hydroxide (NaOH) may be used for this adjustment if warranted.
See additional information about pH during testing in (e)(8)(ii).
(ii) Exposure technique. The test should be conducted using one of two basic
exposure techniques: static-renewal or flow-through. Guidance on the selection of
the appropriate exposure technique is provided in OCSPP 850.1000.
For a static-renewal test, the frequency of medium renewal will depend on the
stability of the test substance, but should be at least three times per week with a
maximum renewal period of 3 days.
5
-------
(iii) Treatment concentrations. At least 5 test solution concentrations should be
used for definitive testing, plus the appropriate control(s). A range-finding test, as
well as the EC50 curve in an acute test, may be used to establish the appropriate
test solution concentrations for the definitive test (see paragraph (d)(3) of this
guideline). Justification should be provided if fewer than 5 concentrations are
used. OCSPP 850.1000 provides guidance on selection of test concentrations.
For hypothesis-based testing, there should be 1 concentration level where no
adverse effects occur as compared to the control(s) and a second, higher
concentration with an adverse effect. For regression-based endpoints, if
calculated, the selected test concentrations, at a minimum, should bracket the
desired median inhibition/effect concentration (ICX/ECX) of the most sensitive
endpoint.
For a limit test, there is a single treatment concentration, plus the appropriate
control(s). Guidance on the limit concentration is provided in paragraph (d)(5) of
this guideline.
(5) Controls. Every test includes a dilution water control and a vehicle (solvent) control,
if a vehicle is used. Controls consist of the same dilution water, conditions, procedures,
and test population as the test solutions, except that no test substance is added.
A test is not acceptable if:
(i) More than 20% of the organisms in any control showed signs of disease, stress
(e.g., discoloration, unusual behavior, immobilization), and/or death; or
(ii) Ephippia were produced in any control; or
(iii) Daphnids in any control that lived for 21 days did not produce, on average, at
least 60 offspring in the 21 days of the test.
(6) Number of test organisms and replicates. There are 2 test designs that are
commonly used. The first design, which is often used with the static-renewal exposure
technique, employs 10 replicate test vessels, each with a single daphnid, for each test
concentration and control. The second design, which is often used with the flow-through
exposure technique, employs 4 replicate test vessels, each with 5 daphnids, for each test
concentration and control resulting in a total of 20 daphnids per test concentration and
control. Regardless of the design, each test vessel should contain an equal volume of test
solution and an equal number of daphnids. Replicate test vessels should be physically
separated, since the test vessel is the experimental unit.
(i) Loading. The number of daphnids placed in a test vessel should not be so large
as to cause the dissolved oxygen concentration to fall below the recommended
levels, the un-ionized ammonia concentration to exceed the recommended levels,
or affect the results of the test. In static-renewal tests, loading should not exceed 1
daphnid per 40 mL of test solution (25 daphnids per liter). In flow-through tests,
loading requirements will vary depending upon the flow rate of the dilution water,
6
-------
but should not exceed 0.5 grams wet weight of organism per liter (g/L) of solution
passing through the test vessel in 24 hours or 5 g/L at any time.
(ii) Introduction of test organisms. The test should be started by introducing
<24-hour old daphnids, from acclimated parents, into the test vessels after the test
substance has been added. Test vessels for treatment levels should be randomly or
indiscriminately located within the test area, and test organisms should be
randomly or indiscriminately distributed among test vessels. Further guidance is
provided in OCSPP 850.1000.
(7) Facilities, apparatuses, and supplies. Normal laboratory equipment should be used,
especially the following:
(i) Facilities. Facilities for culturing, holding, acclimating, and testing daphnids
that are well ventilated and free of fumes and disturbances which may affect the
test organisms. Equipment for culturing and/or handling food sources for
daphnids. Drying ovens, aluminum weighing pans, and an analytical balance
capable of accurately weighing to 0.01 mg.
(ii) Environmental control equipment. Mechanisms for controlling and
maintaining the water temperature and lighting during the culturing, holding,
acclimation, and test periods. Apparatus for aerating the dilution water and
removing gas bubbles as necessary. For flow-through tests, apparatus for aerating
the dilution water in the head box before mixing with the test substance or
delivery to test vessels. An apparatus providing a 30-minute lighting transition
period may be needed.
(iii) Water quality testing instruments. Equipment for determination of water
quality characteristics (pH, hardness, temperature, etc)
(iv) Cleaning of test system. Test substance delivery systems and test vessels
should be cleaned before each test. See OCSPP 850.1000 for further information.
Cleaning of test vessels may be necessary during the testing period and can be
accomplished as described in paragraph (j)(l). Daily brushing of the outside of
the screens (during flow-through tests) is recommended.
(v) Test containers and delivery system. 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 solutions in
quantities that can affect the test results. Construction materials and equipment
that contact stock or test solutions should be chosen to minimize sorption of test
substances. Refer to OCSPP 850.1000 for additional information on appropriate
construction materials. Test vessels, which should be constructed of chemically
inert material, should be of a capacity to maintain the loading rate and
environmental conditions. Test vessels 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. The flow-through system, if used,
7
-------
should contain an appropriate test substance delivery system. In addition, there
should be an apparatus to introduce food if continuous or intermittent feeding is
used.
For flow-through tests, daphnids can be retained in glass or stainless steel
containers {i.e., test compartments) with stainless steel or nylon screen bottoms
within the test vessel. The containers should be suspended in the test vessel in
such a manner as 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
(cm) of test solution.
Test vessels can be constructed using 250-mL beakers or other suitable containers
equipped with screened overflow holes, standpipes, or V-shaped notches.
(iv) Dilution water. Clean surface water, ground water, or reconstituted water are
acceptable as dilution water if daphnids will survive in it for the duration of the
culturing, holding, acclimation, and testing periods without showing signs of
stress. Dechlorinated tap water is not recommended because some forms of
chlorination are difficult to remove adequately. If dechlorinated tap water is used,
recommended maximum chlorine levels as well as other ways to demonstrate
suitability as a dilution water source can be found in OCSPP 850.1000. The
addition of selenium and vitamin B12 may also be desirable in reconstituted water
(see paragraph (j)(l) of this guideline). Daphnids may also be cultured and tested
in a defined medium (see paragraph (j)(3) of this guideline).
Dissolved oxygen in the dilution water (prior to use in a test) should be between
90 and 100% saturation. If necessary, the dilution water can be aerated before the
addition of the test substance.
Hardness, alkalinity, and conductivity should be measured in the dilution water at
the beginning of the test. When using soft water (40-50 mg/L hardness), the test
species should be D. pulex, as D. magna is intolerant of soft water.
Measurement of total organic carbon (TOC) or chemical oxygen demand (COD)
in the dilution water at the beginning of the test is recommended, but at a
minimum, TOC and COD should be analyzed periodically in the dilution water
source to document and characterize their magnitude and variability. For tests
with cationic substances, TOC or COD should be measured at the beginning of
the test.
Specifications for dilution water quality and constancy are described in OCSPP
850.1000.
(8) Environmental conditions. Environmental parameters during the test should be
maintained as specified below. The number and frequency of measurements
recommended for documenting and confirming the magnitude and variability of water
quality parameters {e.g., temperature, dissolved oxygen, and pH) in test solutions during
the test are described in detail in OCSPP 850.1000.
8
-------
(i) Temperature. The water temperature should be 20 °C and should be constant
within plus or minus (±) 2 °C during the test.
(ii) pH. The pH should be between 6.0 and 8.5 and vary less than 1 pH unit
during the test within a test vessel and between test concentrations (including
control (s)).
(iii) Lighting and photoperiod. A photoperiod should be selected from regimes
of 12 hours light: 12 hours dark to 16 hours light: 8 hours dark. For any given test,
the light regime should be constant. Light intensity should range from 540 to 1080
lux (approximately 50-100 foot-candles (ft-c)). A 15- to 30-minute transition
period between light and dark is suggested.
(iv) Dissolved oxygen. The dissolved oxygen concentration should be between 60
and 100% saturation during the test. If aeration is needed to achieve an
appropriate dissolved oxygen level, it should be done before the addition of the
test substance. The use of more frequent renewal of test solutions or a higher flow
rate should be used to maintain adequate dissolved oxygen levels. For flow-
through exposures, the dilution water may be aerated vigorously prior to delivery
to the test vessels (e.g., in the diluter head box) such that the dissolved oxygen
concentration is at or near 90 to 100% saturation. If the water is heated,
precautions should be taken to ensure that supersaturation of dissolved gases is
avoided. Aeration of test solutions during the test is strongly discouraged because
a single air bubble can become caught under the carapace of a daphnid and kill it
or float the daphnid to the surface where it will become trapped. Therefore, gentle
aeration of test vessels during the exposure period may only be utilized in cases
where the dissolved oxygen levels are in danger of dropping below 60%
saturation. In such cases, assurances should be made that the use of aeration does
not stress the test organisms; test substance concentrations should be measured
during the test to ensure that they are not affected by the use of aeration; and all
treatment and control vessels should be given the same aeration treatment.
(v) Flow in a flow-through system. During a test, the flow rates should not vary
more than 10% between any one replicate and another. The minimum number of
test vessel volume replacements should be five per 24-hour period. It is
recommended that diluter systems be monitored for proper adjustment and
operation at least twice daily throughout the test period to better ensure that the
target test concentrations are achieved and maintained. The flow rate to each test
vessel should be measured weekly and at the beginning and end of the test.
(9) Observations.
(i) Measurement of test substance. OCSPP 850.1000 describes the
recommended sampling methods, frequency of sampling, and sample processing
(especially of low solubility test substances) for analytical confirmation of
dissolved test concentrations and characterization of test substance stability
throughout the test. The analytical methods used to measure the amount of
9
-------
dissolved test substance in a sample should be validated before beginning the test,
as described in OCSPP 850.1000, and the relevant method detection limit(s) and
limit(s) of quantification should be reported.
(ii) Test solution appearance. Observations on test solution appearance and test
substance solubility should be made daily and at the beginning and end of the test.
The appearance of surface slicks, precipitates, or material adhering to the sides of
the test vessels or in any part of the mixing and delivery system should be
recorded at a minimum at the beginning and end of the test and during the test
when the test solution appearance changes.
(iii) Measures of effect.
(A) Fo survival. During the test, daphnids in the test vessels should be
observed periodically, preferably daily, but at least 3 times per week. The
number of surviving, immobilized, and dead parental (Fo) daphnids should
be counted and recorded. Immobilized daphnids should be removed from
test vessels at the time of observation.
(B) Time to release of first brood. The time of appearance of the first
brood in each test vessel should be recorded.
(C) Live offspring produced. Live offspring (Fi) should be counted and
removed, preferably daily, but at least 3 times per week, beginning with
the appearance of the first brood.
(D) Number of aborted eggs or immobilized offspring. Only the
number of living offspring is used to calculate the reproductive output for
the test, but the presence of aborted eggs and immobilized or dead
offspring should also be counted and recorded.
(E) Fo growth. At test termination, the growth of the Fo adults should be
determined. This can be accomplished by measuring a constant dry weight
or length of each surviving adult. Both measurements (weight and length)
are preferred. For experimental designs where a replicate consists of
multiple test animals, the total dry weight of the pooled surviving animals
in the replicate can be measured rather than each individual animal.
Constant dry weight can be achieved by exposing daphnids to 60 °C for 72
hours or 100 °C for 24 hours. Length is defined as the distance from the
apex of the helmet to the base of the spine or is extrapolated from a
standard curve of dry weight of a test animal to body length. Since
measuring dry weight is a destructive process, lengths should be taken
before the animals are dried.
(F) Appearance and behavior. Observation of any abnormal behavior or
appearance including the presence of males, ephippia, or aborted broods
should be recorded, preferably daily, but at least 3 times per week.
10
-------
(f) Treatment of results.
(1) Response variable calculations. Response variables calculated from primary data
include proportion of surviving Fo adults, Fo production rate of first brood release,
reproductive output as total number of live offspring per surviving Fo adult and number
of live offspring per Fo adult per reproductive day, and mean surviving Fo adult dry
wieght and/or length and are discussed in paragraphs (f)(l)(i) through (f)(l)(v) of this
guideline.
(i) Proportion of surviving Fo adults. The proportion of surviving Fo adults at
test termination for a given replicate is calculated using Equation 1 of this
guideline.
ps = proportion of surviving Fo adults at test termination in the replicate;
ns = number of surviving Fo adults at test termination in the replicate; and
N= number of Fo adults at test initiation in the replicate.
(ii) Fo production rate of first brood. The time or rate of Fo production of the
first brood (n) for a given replicate is calculated using Equation 2 of this
guideline.
where:
rb = Fo production rate of first brood;
i = index of observation events from 1 (first observation event after test initiation)
through m\
ti = time, in days, from test initiation to the first observation event offspring (Fi)
were observed in the replicate; and
k = length of observation interval /, in days (if inspected daily, the length of the
inspection interval is 1 day).
(iii) Reproductive output. Reproductive output is expressed in terms of the total
number of live offspring per surviving Fo adult (see paragraph (f)(l )(iii)(A) of this
Equation 1
where:
Equation 2
11
-------
guideline) and the number of live offspring per Fo adult per reproductive day (see
paragraph (f)(l)(iii)(B) of this guideline).
(A) Total number of live offspring per surviving Fo adult. The total
number of live offspring per surviving Fo adult (Rs ) for a given replicate
is calculated using Equation 3.
Rs = total number of live offspring per surviving Fo adult;
y = total number of live offspring produced in the replicate, which is the
sum of the live offspring counted at each observation event i (= ^ yt);
and
ns = number of surviving Fo adults at test termination in the replicate.
(B) Number of live offspring per Fo adult per reproductive day. The
number of live offspring per Fo adult per reproductive day (Rh) for a given
replicate is calculated using Equation 4. The number of reproductive days
is the total number of days an Fo adult was observed alive in a replicate
starting from the first day offspring were observed in the test, irrespective
of treatment or replicate. When observations are not made daily, the
number of reproductive days is the average between the last observation
day the Fo adult was observed alive and the first observation day it was
observed dead. When the test design consists of a single Fo adult per
replicate and observations are made daily, Rb is calculated as the total
number of live offspring produced divided by the total number of
reproductive days. When the test design consists of multiple Fo adults in a
replicate and one Fo adult (or more) dies before the end of the test, the
number of days an Fo adult reproduced from the first day offspring were
observed in the test is the average of the last observation day the Fo adult
was observed alive and the first observation day it was observed dead (see
Equation 5 of this guideline). The value for reproduction days for a given
replicate is calculated by summing the number of Fo adults alive in the
replicate for each reproduction day. These data are available from the
daily survival data. An Fo adult is considered dead for the whole 24 hours
preceding observed death. For example, if an Fo adult is observed dead on
day 21, then that replicate would have one less Fo adult reproduction day.
Equation 3
where:
Equation 4
12
-------
where:
Rb = number of live offspring per Fo adult per reproductive day;
y = total number of live offspring produced in the replicate; and
/= total number of reproductive days at test termination for the replicate
calculated using Equation 5.
i = index of observation events from FBR through m;
FBR = observation event that the first brood release occurred in the test,
irrespective of treatment or replicate;
m = maximum number of observation events;
fi = number of live Fo adults at observation event z; and
li = length of observation interval z, in days (if inspected daily, the length
of the inspection interval is 1 day).
(iv) Mean surviving Fo adult dry weight. The response measure for growth
based dry weight is the mean surviving Fo adult dry weight for a given replicate
(w) and is calculated using Equation 6. When the test design consists of a single
Fo adult per replicate, w is the dry weight of the surviving Fo adult. When the test
design consists of multiple Fo adults in a replicate, w is the mean surviving Fo
adult dry weight.
where:
w = mean surviving Fo adult dry weight;
w = dry weight of surviving Fo adult or pooled surviving Fo adults, if replicate
consists of multiple Fo adults, for the replicate (measured as described in
paragraph (e)(9)(iii)(E) of this guideline); and
ns = number of surviving Fo adults that were weighed at test termination in the
replicate.
Equation 5
where:
Equation 6
13
-------
(v) Mean surviving Fo adult body length. The response measure for growth
based on body length is the mean surviving Fo adult body length for a given
replicate (L) and is calculated using Equation 7. When the test design consists of
a single Fo adult per replicate, L is the length of the surviving Fo adult. When the
test design consists of multiple Fo adults in a replicate, L is the mean surviving Fo
adult body length.
where:
L = mean surviving Fo adult body length.
k = index number of length measurement for surviving Fo adult in the replicate
from 1 to ns;
Lk = body length of individual k in the replicate (measured as described in
paragraph (e)(9)(iii)(E) of this guideline); and
ns = number of surviving Fo adults that were measured at test termination in the
replicate.
(2) Summary statistics.
(i) Response variables. For each test group, including the control(s), summary
statistics (mean, median, minimum, maximum, and the first and third quartiles)
for each response variable in Table 1 should be calculated and plotted.
Additionally, the standard deviation, coefficient of variation, standard error of the
mean, and 95% confidence interval of the mean for each test group, including the
control(s), should be calculated.
(ii) Abnormal appearance. The occurrence (number or proportion is desired) in
Fo adults or offspring (alive or dead) of abnormality of body form, males, and
ephippia should be summarized by type of abnormality, time of observation,
treatment group, and replicate.
(iii) Abnormal behavior. Behavioral abnormalities (e.g., erratic swimming,
atypical quiescence, atypical feeding behavior, immobilized offspring) should be
summarized by type of behavior, time of observation, treatment group, and
replicate.
Equation 7
14
-------
(3) Percent inhibition. For all response variables in Table 1, the percent inhibition
as compared to the control(s) for each test substance treatment level is calculated using
Equation 8.
%/ = (C~^X100) Equation 8
where:
%I= percent inhibition as compared to the control(s);
C = mean control response value (e.g., mean Fo production rate of first brood); and
X= mean response value (e.g., mean Fo production rate of first brood) for test substance
treatment level.
Stimulation or a greater response in the test substance treatment than the control(s) is
reported as negative %I.
(4) Evaluation of limit test results. At test termination (21 days), if there is a
statistically significant inhibition in any of the response variables in Table 1 at the limit
concentration as compared to the control(s), a multiple-concentration definitive test
should be conducted.
(5) Evaluation of multiple-concentration definitive test.
(i) NOEC. A NOEC and a LOEC should be determined for each of the response
variables in Table 1 using appropriate statistical methods. Involvement of a
statistician in both the design and analysis of the test results is recommended
since OCSPP 850.1300 allows for variation in experimental design (e.g., number
of test animals per replicate, number of replicates). The overall NOEC and LOEC
values for the test are the lowest values (i.e., most sensitive) of all the response
variables considered.
(ii) Regression-based endpoints. The test should be conducted to obtain
hypothesis-based endpoints (see paragraph (f)(5)(i) of this guideline). Ancillary to
NOEC and LOEC determinations, the LCX for survival and the ECX/ICX for
growth (length and weight) may be estimated when there is sufficient coverage of
the concentration-response curve.
(iii) Statistical methods. All methods used for statistical analysis should be
described completely. Experimental units (replicates) are the individual test
vessels within each treatment level. Additional discussion about endpoints and
statistical procedures can be found in OCSPP 850.1000.
15
-------
Table 1.—Test Response Variables to Calculate and Evaluate
Proportion of surviving Fo adults (Ps)
Fo production rate of first brood (rt)
Total number of live offspring per surviving Fo adult (Rs)
Number of live offspring per Fo adult per reproductive day (Rb)
Mean surviving Fo adult dry weight per replicate (w) and/or
Mean surviving Fo adult body length per replicate (L)
(g) Tabular summary of test conditions. Table 2 lists the important conditions that should
prevail during the multiple-concentration definitive test. The same conditions are recommended
for a limit test, except for the difference in the number of test concentrations. Meeting these test
conditions will help ensure the satisfactory performance of the test.
Table 2.—Summary of Test Conditions for Daphnid Chronic Toxicity Test
Test type
Static-renewal or flow-through
Test species
Daphnia magna or Daphnia pulex
Test duration
21 days
Temperature
20° C (constant during test within ±2 °C)
Light quality
Ambient laboratory illumination
Light intensity
540-1080 lux (approximately 50-100 ft-c)
Photoperiod
Selected from among 12 hours light: 12 hours dark to 16 hours
light:8 hours dark schemes
PH
Between 6.0 and 8.5 (constant during test within ±1 pH unit)
Water hardness (as CaCCb)
<250 mg/L (preferably <180 mg/L); 40-50 mg/L for testing with
metals
TOC
mg/L
Renewal of test solutions
Static-renewal test: minimum of 3 times per week with a maximum
renewal period of 3 days;
Flow-through test: minimum of volume changes per 24 hours
Age of test organisms at test initiation
<24 hours old
Number of organisms per test vessel
The minimum ranges from 1 to 5 depending upon test design
Number of replicate test vessels per
concentration
The minimum ranges from 4 to 10 depending on test design
Number of organisms per test
concentration
10 (minimum)
Loading
Static-renewal test: should not exceed 1 daphnid per 40 mL;
Flow-through test: <0.5 g/L per 24 hours and <5 g/L at any time
Feeding regime
To allow sufficient growth and reproduction, typically at least on a
daily basis
16
-------
Test vessel aeration
Strongly discouraged because a single air bubble can become
caught under the carapace of a daphnid and kill it or float the
daphnid to the surface where it will become trapped. Gentle
aeration of test vessels may only be used in cases where the
dissolved oxygen levels are in danger of dropping below 60%
saturation. In such cases, assurances should be made that the use
of aeration does not stress the test organisms; test substance
concentrations should be measured during the test; and all
treatment and control vessels should be given the same aeration
treatment.
Vehicle concentration, if used
<0.1 mL/L for recommended solvents (see OCSPP 850.1000)
Test concentrations
Definitive test: minimum of 5 test concentrations chosen in a
geometric series plus a dilution water control and a vehicle (solvent)
control, if a vehicle is used
Measures of effect or measurement
endpoints
Definitive test: NOEC/LOEC for response parameters in Table 1
(h) Test validity elements. This test would be considered to be unacceptable or invalid if one or
more of the conditions in Table 3 occurred. These parameters are not the only elements
considered when evaluating the acceptability of a test, and it is possible that a test could be found
unacceptable or invalid based on other considerations. However, except for the conditions listed
in Table 2 and in OCSPP 850.1000, it is unlikely that a test will be rejected when there are only
slight variations from guideline environmental conditions and test design unless the control
organisms are significantly affected and/or significant biases are introduced in defining the
magnitude of effect on measurement endpoints as compared to guideline conditions. Before
departing significantly from this guideline, the investigator should contact the Agency to discuss
the reason for the departure and the effect the change(s) may have on test acceptability. In the
test report, all departures from the guideline should be identified, reasons for the changes given,
and any resulting effects on test endpoints noted and discussed.
Table 3.—Test Validity Elements for the Daphnid Chronic Toxicity Test
1. All test vessels were not identical.
2. Treatments were not randomly or indiscriminately assigned to individual test vessel locations, or
individual test organisms were not randomly or indiscriminately assigned to test vessels.
3. A dilution water control (and vehicle (solvent) control, if a vehicle was used) was not included in the
test.
4. The test was begun with daphnids more than 24 hours old.
5. More than 20% of the organisms in either the dilution water or vehicle (solvent) controls showed signs
of disease, stress (e.g., discoloration, unusual behavior, immobilization), and/or death.
6. Daphnids that lived for 21 days in any control did not produce, on average, at least 60 offspring in the
21 days.
7. Ephippia were produced in the control(s).
8. A surfactant or dispersant was used in the preparation of a stock or test solution. (However, adjuvants
may be used when testing pesticide typical end-use products.)
(i) Reporting.
17
-------
(1) Background information. Paragraph (k)(l) of OCSPP 850.1000 describes the
minimum background information to be supplied in the report.
(2) Guideline deviations. Provide a statement of the guideline or protocol followed.
Include a description of any deviations from the test guideline or any occurrences which
may have influenced the results of the test, the reasons for these changes, and any
resulting effects on test endpoints noted and discussed.
(3) Test substance.
(i) Identification of the test substance: common name, IUPAC and CAS names,
CAS number, structural formula, source, lot or batch number, chemical state or
form of the test substance, purity (i.e., for pesticides, the identity and
concentration of active ingredient(s)), and radiolabeling, if any, including the
location of label(s) and radiopurity.
(ii) Storage conditions of the test chemical or test substance and stability of the
test chemical or test substance under storage conditions if stored prior to use.
(iii) Methods of preparation of the test substance and the treatment concentrations
used in the range-finding and definitive tests, or limit test. Identify whether the
nominal concentrations are corrected or uncorrected for purity of the test
substance.
(iv) Physicochemical properties of the test substance such as water solubility,
vapor pressure, UV absorption, pKa, and Kow.
(v) If a vehicle (solvent) is used to prepare stock or test substance, provide the
name and source of the vehicle, the nominal concentration(s) of the test substance
in the vehicle in stock solutions or mixtures, and the vehicle concentration(s) used
in the treatments and vehicle control. If different vehicle concentrations are used
at different treatment levels, the report should, at a minimum, identify the
maximum vehicle concentration used. It is helpful to support the vehicle choice
by including a description of any measures that were taken to identify an
appropriate vehicle for use in the test, such as the types and concentrations of
vehicles used and their corresponding effects on solubility during any preliminary
work.
(vi) If a positive control is used, provide the name and source of positive control
and the nominal concentration(s) of the positive control material in stock
solutions or mixtures.
(4) Test organisms.
(i) Scientific name and common name.
(ii) Method for verifying the species.
18
-------
(iii) Information about the daphnids used as brood stock in the test: source, culture
practices, and holding and acclimation procedures and conditions, including
acclimation period, water used, feeding history, and health status (mortality of
brood stock before test initiation and any preventative or disease treatments).
(iv) Age of test organisms at test initiation.
(5) Test system and conditions. Provide a description of the test system and conditions
used in the definitive or limit test and any preliminary range-finding tests.
(i) Description of range-finding test, if any: test concentrations, other relevant
conditions, and results from test that were used to determine conditions for the
definitive test.
(ii) Description of the test vessels: size, type, material, and fill volume.
(iii) Description of the exposure technique: static-renewal, flow-through, open or
closed system. If static-renewal, the frequency of test solution renewal, and if
flow-through, a description of the flow-through system, including flow rates and
test vessel turnover rate. For closed systems, a description of the closed system
design. For all systems, a description of the calibration and validation methods.
(iv) Description of the dilution water and any water pretreatment: source/type;
temperature; pH; hardness and alkalinity; dissolved oxygen; total organic carbon
or chemical oxygen demand; particulate matter; conductivity; metals, pesticides,
and residual chlorine concentrations (mean, standard deviation, range). If natural
water, also provide the un-ionized ammonia concentration. Describe the
frequency and sample date(s) for documenting dilution water quality and
consistency.
(v) Use of aeration, if any, and location of aeration within exposure system (e.g.,
test solution or dilution water prior to test substance addition).
(vi) Number of test organisms added to each test vessel at test initiation.
(vii) Number of test vessels (replicates) per treatment level and control(s).
(viii) Methods used for treatment randomization and assignment of test organisms
to test vessels.
(ix) Date of introduction of test organisms to test solutions and test duration.
(x) Loading rate.
(xi) Photoperiod and light source.
19
-------
(xii) Methods and frequency of environmental monitoring performed during the
definitive or limit test for test solution temperature, dissolved oxygen, pH, light
intensity, and ammonia.
(xiii) Methods and frequency of measuring dissolved test substance to confirm
exposure concentrations.
(xiv) Methods and frequency of counting number of immobilized daphnids and
measuring any other toxic symptoms.
(xv) Detailed information on feeding (e.g., type of feed, source, amount given,
and frequency). Feed should be analyzed periodically to identify background
contaminants such as heavy metals (e.g., arsenic, cadmium, lead, mercury, and
selenium) and persistent pesticides, especially chlorinated insecticides.
(xvi) For definitive and limit tests, description of all analytical procedures,
accuracy of the method, method detection limit, and limit of quantification.
(6) Results.
(i) Nominal exposure concentrations and a tabulation of test substance analytical
results by treatment group and test vessel (provide raw data), descriptive statistics
(mean, standard deviation, minimum, maximum, coefficient of variation), and
percent of nominal.
(ii) Environmental monitoring data results (test solution temperature, dissolved
oxygen, pH, light intensity, and ammonia) in tabular form (provide raw data for
measurements not made on a continuous basis) and descriptive statistics (mean,
standard deviation, minimum, maximum).
(iii) For preliminary range-finding test, if conducted, a tabulation of the effects
monitored by treatment level. Describe findings and use in setting definitive test
exposure concentrations. If information from other tests such as acute test results
was used to set definitive test exposure concentrations, provide a description.
(iv) For limit test, a tabulation of the number of surviving and immobilized Fo
adults and the number of live offspring counted and removed in each test vessel,
for the limit concentration and control(s), at each observation time (provide the
raw data) and descriptive statistics (mean, standard deviation, minimum,
maximum).
(v) For definitive test, a tabulation of the number of surviving and immobilized Fo
adults and the number of live offspring counted and removed in each test vessels,
for all treatment levels and control(s), at each observation time (provide the raw
data) and descriptive statistics (mean, standard deviation, minimum, maximum).
(vi) Time (days) to first brood release.
20
-------
(vii) For limit and definitive tests, a tabulation by treatment level and replicate of
the proportion of surviving Fo adults at test termination, Fo production rate of the
first brood, total number of live offspring per surviving Fo adult, number of live
offspring per Fo adult per reproductive day, mean surviving Fo adult dry weight at
test termination, and/or mean surviving Fo adult body length at test termination.
Descriptive statistics (mean, standard deviation, standard error, 95% confidence
interval, median, first and third quartiles, minimum, maximum) and a plot of these
effects by treatment level. Tabulation of the % inhibition calculated as compared
to control(s). Provide sufficient raw data for performance of an independent
statistical analysis.
(viii) For limit and definitive tests, a description and tabulation of abnormal
appearance and behavioral signs of toxicity by test vessel, treatment, and
observation time (provide raw data).
(ix) For limit test, provide findings of hypothesis tests for each response variable
and for abnormal behavior and appearance as compared to the control(s).
(x) For definitive test, a tabulation of NOEC and LOEC determinations for each
response variable and a description of statistical method(s) used for the NOEC
and LOEC determinations, including the software package, and the basis for the
choice of method.
(xi) When appropriate, a description of statistical method(s) used for point
estimates, including the software package for determining EC50 values and fitting
the concentration-response model, and the basis for the choice of method. Provide
results of any goodness-of-fit tests.
(j) References. The following references should be consulted for additional background material
on this test guideline.
(1) American Society for Testing and Materials. ASTM El 193-97, Standard Guide for
Conducting Daphnia magna Life-Cycle Toxicity Tests. In Annual Book of ASTM
Standards, Vol. 11.06, ASTM, West Conshohocken, PA. Current edition approved April
1, 2004.
(2) Hutchinson, T.H., N. Shillabeer, M.J. Winter and D.B. Pickford, 2006. Acute and
chronic effects of carrier solvents in aquatic organisms: A critical review. Aquatic
Toxicology, 76, 69-92.
(3) Organization for Economic Cooperation and Development, 2012. OECD Guidelines
for Testing of Chemicals, Guideline 211, Daphnia magna Reproduction Test.
(4) U.S. Environmental Protection Agency, 1982. Pesticide Assessment Guidelines,
Subdivision E, Hazard Evaluation, Wildlife and Aquatic Organisms, EPA 540/09-82-024,
U.S. Environmental Protection Agency, Washington, DC.
21
-------
(5) U.S. Environmental Protection Agency, 1986. Hazard Evaluation Division Standard
Evaluation Procedure: Daphnia magna Life-Cycle (21-Day Renewal) Chronic Toxicity
Test, EPA-540/9-86-144, Office of Pesticide Programs, Office of Prevention, Pesticides
and Toxic Substances, U.S. Environmental Protection Agency, Washington DC.
(6) U.S. Environmental Protection Agency, 1994. Pesticides Reregi strati on Rejection
Rate Analysis: Ecological Effects, EPA 738-R-94-035, Office of Prevention, Pesticides
and Toxic Substances, December, 1994.
22
------- |