	Office of Chemical Safety and Pollution Prevention

(7101)	EPA 712-C-16-005

October 2016

	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  HYPERLINK
"http://www.epa.gov/ocspp" http://www.epa.gov/ocspp  and select “Test
Methods & Guidelines” on the navigation menu. You may also access the
guidelines in  HYPERLINK "http://www.regulations.gov"
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.

  SEQ CHAPTER \h \r 1 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.

― 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 Reregistration 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 E1193-97, Standard Guide for Conducting
Daphnia magna Life-Cycle Toxicity Tests (see paragraph (j)(1) 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 Daphnia pulex) 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. 

F0 (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.

F1 (Filial generation, first) are the offspring produced by the parental
generation (F0). 

(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 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 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 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)(1) 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)(1) of
this guideline). For D. 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 (HCl) 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. 

(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, 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)(1). 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,
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)(1) 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.

(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 to1080 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 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) F0 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 (F0) 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 (F1) 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. 

. 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.

(f) Treatment of results.

(1) Response variable calculations. Response variables calculated from
primary data include proportion of surviving F0 adults, F0 production
rate of first brood release, reproductive output as total number of live
offspring per surviving F0 adult and number of live offspring per F0
adult per reproductive day, and mean surviving F0 adult dry wieght
and/or length and are discussed in paragraphs (f)(1)(i) through
(f)(1)(v) of this guideline.

(i) Proportion of surviving F0 adults. The proportion of surviving F0
adults at test termination for a given replicate is calculated using
Equation 1 of this guideline.

 	Equation 1

where:

 = proportion of surviving F0 adults at test termination in the
replicate;

 = number of surviving F0 adults at test termination in the replicate;
and

N = number of F0 adults at test initiation in the replicate.

(ii) F0 production rate of first brood. The time or rate of F0
production of the first brood (rb) for a given replicate is calculated
using Equation 2 of this guideline.

 	Equation 2

where:

  = F0 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 (F1) were observed in the replicate; and

li = length of observation interval i, 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 F0 adult (see paragraph
(f)(1)(iii)(A) of this guideline) and the number of live offspring per
F0 adult per reproductive day (see paragraph (f)(1)(iii)(B) of this
guideline).

 ) for a given replicate is calculated using Equation 3. 

 	Equation 3

where:

 = total number of live offspring per surviving F0 adult;

 ; and

 = number of surviving F0 adults at test termination in the replicate.

(B) Number of live offspring per F0 adult per reproductive day. The
number of live offspring per F0 adult per reproductive day (Rb) for a
given replicate is calculated using Equation 4. The number of
reproductive days is the total number of days an F0 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 F0 adult was observed alive and the
first observation day it was observed dead. When the test design
consists of a single F0 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 F0 adults in a replicate and one F0 adult (or more)
dies before the end of the test, the number of days an F0 adult
reproduced from the first day offspring were observed in the test is the
average of the last observation day the F0 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 F0 adults alive in the replicate for
each reproduction day. These data are available from the daily survival
data. An F0 adult is considered dead for the whole 24 hours preceding
observed death. For example, if an F0 adult is observed dead on day 21,
then that replicate would have one less F0 adult reproduction day. 

 	Equation 4

where:

 = number of live offspring per F0 adult per reproductive day;

y = total number of live offspring produced in the replicate; and

f = total number of reproductive days at test termination for the
replicate calculated using Equation 5.

 	Equation 5

where:

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 F0 adults at observation event i; and

li = length of observation interval i, in days (if inspected daily, the
length of the inspection interval is 1 day).

  is the mean surviving F0 adult dry weight.

 	Equation 6

where:

  = mean surviving F0 adult dry weight;

w = dry weight of surviving F0 adult or pooled surviving F0 adults, if
replicate consists of multiple F0 adults, for the replicate (measured as
described in paragraph (e)(9)(iii)(E) of this guideline); and

  = number of surviving F0 adults that were weighed at test termination
in the replicate.

  is the mean surviving F0 adult body length. 

 	Equation 7

where:

 = mean surviving F0 adult body length.

 ;

Lk = body length of individual k in the replicate (measured as described
in paragraph (e)(9)(iii)(E) of this guideline); and

 = number of surviving F0 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 F0 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. 

(3) Percent inhibition. For all response variables in Table 1, the
percent inhibition (%I) as compared to the control(s) for each test
substance treatment level is calculated using Equation 8.

 	Equation 8

where:

%I = percent inhibition as compared to the control(s);

C = mean control response value (e.g., mean F0 production rate of first
brood); and

X = mean response value (e.g., mean F0 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. 

―Test Response Variables to Calculate and Evaluate

 )



(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

≤2 mg/L

Renewal of test solutions	Static-renewal test: minimum of 3 times per
week with a maximum renewal period of 3 days; 

≤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.

(1) Background information. Paragraph (k)(1) 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.

(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.

(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 F0 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 F0 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.

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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 E1193-97, 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.

(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
Reregistration Rejection Rate Analysis: Ecological Effects, EPA
738-R-94-035, Office of Prevention, Pesticides and Toxic Substances,
December, 1994. 

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