reproductiF	Office of Chemical Safety and Pollution Prevention

(7101)	EPA 712-C-16-008

October 2016

	Ecological Effects

Test Guidelines

OCSPP 850.1400:

	Fish Early Life Stage 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 850.1400: Fish early life stage 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)(14) of this guideline); EPA Pesticide Reregistration Rejection Rate
Analysis: Ecological Effects (see paragraph (j)(15) of this guideline);
OECD 210 Fish Early-Life Stage Toxicity Test, 2013 (see paragraph
(j)(12) of this guideline); and ASTM E1241-05 Standard Guide for
Conducting Early Life-Stage Toxicity Tests with Fishes (see paragraph
(j)(1) of this guideline).

(b) Purpose. This guideline is intended for use in developing data on
the toxicity of chemical substances and mixtures (“test chemicals”
or “test substances”) subject to environmental effects test
regulations. This guideline describes a subchronic toxicity test in
which early life stages of fish (embryonic through larval and early
juvenile development) are exposed to a test substance, preferably in a
flow-through system. The Environmental Protection Agency will use data
from this test to assess the hazards and risks a chemical 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 guideline:

Blastodisc is a dome of cytoplasm (which is disc-like in larger teleost
eggs such as Fundulus and Salmo) that segregates from the yolk towards
the animal pole during and after the one-cell stage and undergoes
cleavage.

Days from post-hatch is the number of days from the mean hatch day in
the control(s).

Embryo or incubation cup refers to a small glass jar or similar
container with a screened bottom in which the embryos of some species
(i.e., minnow) are placed during the incubation period and which is
normally oscillated to ensure a flow of water through the cup.

Juvenile, as used in this guidance, is a young fish not yet sexually
mature.

Larval, as used in this guidance, refers to the life-stage of the fish
between the time of hatching and the end of the test.

Swim-up is the life stage that begins when larval fish, such as
salmonids, have absorbed or almost completely absorbed their yolk-sac
and swim up from the bottom of the test vessel toward the water surface.

(d) General considerations.

(1) Summary of the test. Organisms 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) from the fertilized egg stage
through embryonic, larval, and early juvenile development. The test
lasts approximately 28 to 32 days for warm water fish and approximately
70 to 90 days for cold water fish. Observations are made on when fish
hatch and on embryo, larval, and juvenile survival periodically during
the test. For those species with a swim-up life stage, observations are
made on the time taken to reach the swim-up stage. In addition,
observations are made on the occurrence of any abnormal behavior or
appearance in the test organisms. Body weight and length of surviving
fish are determined at test termination. The test is designed to
determine the relationship between aqueous concentrations of the test
substance and effects on time to hatch, time to swim-up, hatching
success, post-hatch success, overall survival, and early juvenile
growth. 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. The range-finding test may be a flow-through acute
toxicity test (see OCSPP 850.1075) using the same dilution water, test
substance, and species as the early life stage test. In the absence of
such a test, an acute range-finding test may be conducted in which 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)). The details of the acute range-finding test do not have
to be the same as those of definitive acute 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. The relevance of the
acute toxicity test may be increased by extending the test duration
beyond 96 hours (up to 10 days) and by using fish from an earlier life
stage. The highest concentration of test substance in the early life
stage test may then be selected to be equal to the lowest concentration
that caused adverse effects in either the definitive or range-finding
acute tests. Alternatively, if an acute-to-chronic ratio has been
determined for the test substance with a species of comparable
sensitivity, the concentrations of test substance in the early life
stage test may then be selected based upon the acute test result divided
by the acute-to-chronic ratio of the other species. The intent of any of
these methods is to identify an appropriate range of test concentrations
for establishing a test NOEC and LOEC. The relationship relating LC50 to
period of exposure in the acute test should also be considered when
selecting the range of test concentrations. If a range-finding test is
conducted, its results should be reported along with the results of the
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 survival and early growth and development of fish
early life stages 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 6 of this guideline. Test
validity elements are listed in Table 7.

(5) Limit test. In some situations, it is only necessary to ascertain
that no effects on fish early life stages occur at a certain limit
concentration (i.e., NOEC greater than or equal to (≥) limit
concentration). In a limit test, at least 80 fertilized eggs, divided
into 4 replicates of 20 eggs each, are exposed to a single “limit
concentration,” with the same number of organisms (and replicates) in
appropriate controls. 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 6 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 5 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 will depend on the species and the
test temperature (see Tables 3 and 4) and ranges from approximately 28
to 32 days for warm water species to over 70 days for cold water
species. The post-hatch duration at the designated test temperatures
provides for fish to begin actively feeding, followed by a growth period
of sufficient duration to allow detection of effects on growth. The
difference in test duration between fish species reflects differences in
metabolism, in when fish begin actively feeding, and in physiologic
time-dependence for growth at the given test temperatures.

	(3) Test organism.

		(i) Species.

(A) The preferred test species are:

(1) Freshwater. Rainbow trout, Oncorhynchus mykiss, for a cold water
species and bluegill sunfish, Lepomis macrochirus, for a warm water
species.

(2) Saltwater. Atlantic silverside, Menidia menidia; inland silverside,
M. beryllina; or tidewater silverside, M. peninsulae.

(B) The listing of preferred species does not preclude the use of other
species. Alternative well-documented species that have been used in an
early life stage test include:

(1) Freshwater. Fathead minnow, Pimephales promelas; medaka, Oryzias
latipes; zebra fish, Danio rerio

(2) Saltwater. Sheepshead minnow, Cyprinodon variegatus

(C) Other freshwater species that have also been referenced as possible
test species include: Atlantic salmon, Salmo salar; brook trout,
Salvelinus fontinalis; brown trout, Salmo trutta; channel catfish,
Ictalurus punctatus; chinook salmon, Oncorhynchus tshawytscha; coho
salmon, Oncorhynchus kisutch; common carp, Cyprinus carpio; flagfish,
Jordanella floridae; lake trout, Salvelinus namaycush; northern pike,
Esox lucius; three-spined stickleback, Gasterosteus aculeatus; white
sucker, Catostomus commersoni. Additional information regarding these
species can be found in paragraph (j)(1) of this guideline. If one of
these species or another species is used, the rationale for the
selection of the species and the experimental method should be reported
along with documentation of appropriate husbandry conditions and control
performance.

(D) The test should start as soon as possible after the eggs have been
fertilized with the embryos preferably being immersed in the test
solutions before cleavage of the blastodisc commences or as close as
possible after this stage. The embryonic stage at the beginning of
exposure to the test chemical should be verified as precisely as
possible. This can be done using a representative sample of eggs
suitably preserved and cleaned.

Gametes may be obtained directly from hatcheries or commercial sources,
from wild populations of adult fish collected in the field, or from
brood fish cultured in the laboratory. Wild caught fish should be
quarantined 14 days in addition to the defined minimum holding and
acclimation periods (see paragraph (e)(3)(ii)). Fish captured by
electroshocking, chemical treatment, or gill nets should not be used.
Whenever salmonids are used (as gametes or a brood stock source), they
should be obtained from a hatchery that has been certified disease-free
(e.g., free of infectious pancreatic necrosis, furunculosis, kidney
disease, enteric redmouth, and whirling disease). Fish and gametes used
for a particular test should originate from the same adult brood stock
source and population. Records should be kept regarding the source of
the gametes, initial stock, and/or culturing techniques. Guidance for
obtaining gametes from many fish species can be found in paragraph
(j)(1) and for select species in paragraphs (j)(7) and (j)(17) of this
guideline.

(ii) Holding and acclimation. When gametes are received from an outside
culture source at a temperature that differs from the recommended test
temperature, they should be acclimated to the test temperature. When
eggs are received, they should be immediately unpacked, and the
temperature of the surrounding water should be determined. Sudden
temperature changes should be avoided. Acclimation to the appropriate
test temperature should be accomplished within a period of 6 hours and
should incorporate the use of dilution water. Holding water for brood
stock should come from the same source as the test dilution water; if
not, acclimation to the dilution water should be done gradually over a
48-hour settling-in period. To maintain organisms in good condition and
avoid unnecessary stress, brood fish should not be crowded or subjected
to rapid changes in temperature or water quality. During culturing,
holding, and acclimation, organisms should be observed carefully for
mortality, disease, and other signs of stress and physical damage. Dead
and abnormal individuals should be discarded. If the fish appear
severely diseased, destroying or discarding the entire lot immediately
is recommended. Brood fish should be fed as necessary to optimize
survival, growth, and reproduction.

Following a 48-hour settling-in period, mortalities in the brood fish
should be recorded, and the following guidelines should be applied:

(A) Mortalities of greater than 10% of the population in the 7 days
directly preceding the test: rejection of entire batch;

(B) Mortalities of between 5 and 10% of the population during the 7 days
of acclimation: holding continued for additional 7 days;

(C) Mortalities of less than 5% of the population during the 7 days of
acclimation: acceptance of batch.

 (iii) Health status and condition.

(A) Brood fish. Adult brood stock should not be used for a test:

(1) If they appear stressed, diseased, have physical damage (including
if injured during handling), or abnormalities;

(2) If they have been used in a previous test, either in a treatment or
in a control group.

(B) Embryos. Embryos should not be obtained from fish treated for
disease for at least 14 days after treatment, and no disease treatments
should be administered during testing. Embryos should be visually
inspected prior to placement in the embryo cups or screen trays. During
visual inspection, empty shells, opaque embryos, and embryos with fungus
or partial shells attached should be removed and discarded. If less than
50% of the eggs to be used appears healthy, all embryos in the lot
should be discarded (see paragraph (j)(1) of this guideline).

(iv) Care and handling.

(A) Brood fish. The brood fish should be handled as little as possible,
but when necessary, it should be done as carefully and quickly as
possible. Any disturbance that may change the outcome of the test should
be avoided. Organisms that touch dry surfaces or are dropped or injured
during handling should be discarded. Detailed instructions for the care
and handling of fish, such as those described under paragraphs (j)(1),
(j)(2), (j)(3), (j)(4), (j)(8), (j)(12), (j)(13), (j)(16), (j)(17), and
(j)(18) of this guideline, can be used during the culturing, holding,
acclimating, and testing periods.

(B) Gametes, embryos, larvae, and juveniles. Embryos and fish should be
handled as little as possible, but when necessary, it should be done as
carefully and quickly as possible. Any disturbance that may change the
outcome of the test should be avoided. Organisms that that touch dry
surfaces or are dropped or injured during handling should be noted and
discarded. Fertilized eggs may be distributed to test vessels using a
pipette with a large bore or a similar apparatus.

Guidance on inducing spawning, collection of eggs and sperm, and
fertilization methods is provided for a number of fish species in
paragraph (j)(1) of this guideline.

Initially, fertilized eggs may be placed within the test vessels in
smaller glass or stainless steel compartments fitted with mesh sides or
ends to permit a flow of test solution through the compartment.
Non-turbulent flow through these smaller compartments may be induced by
suspending them from an arm arranged to move the compartment up and down
within the test vessel but always keeping the organisms submerged.
Fertilized eggs of salmonid fishes can be supported on racks or meshes
placed in the test vessels with apertures sufficiently large to allow
larvae to drop through after hatching.

When placed in a test vessel, salmonid eggs should be separated so that
they are not stacked upon each other. For silverside species, the
chorionic fibrils should be cut before indiscriminately placing
fertilized eggs in test vessels. Additionally, silverside fry are
injured easily and are susceptible to impingement on the mesh of egg
containers. Consequently, water flow into and out of the egg containers
when counting fry should be at a slow rate; a minimum water depth of 5
centimeters (cm) should always be maintained in the container.

When egg containers, grids, or mesh have been used to hold eggs within
the test vessels, these restraints should be removed after the larvae
hatch, according to the time schedule recommended in Tables 1 and 2. The
timing of this transfer varies with the species, and transfer may not
always be necessary. If there is a need to transfer the larvae, they
should not be exposed to the air, and nets should not be used to release
fish from egg containers. Late hatching embryos may be left in the egg
containers to determine if they will eventually hatch or not.

When embryos begin to hatch, they should not be handled. Newly-hatched
silverside are very sensitive to handling and should not be handled at
all during the first 5 days after hatching begins. The counting of
surviving fry is not recommended until six days post-hatch. All of the
normal and abnormal fry should be gently released by allowing the fry to
swim out of each embryo cup; nets should not be used.

―Recommended Diet and Feeding Frequency for Freshwater Brood and Test
Fish Species and Schedule for Larval Transfer and First Feeding

  SEQ CHAPTER \h \r 1 Species	Food1	Post-Hatch Transfer Time3	Time to
First Feeding

	Brood

Fish	Newly-Hatched Larvae2	Juveniles

	Type	Frequency

Preferred Test Species

Oncorhynchus mykiss, rainbow trout	trout food	none4	trout starter5	2-4
feeds per day	ca. 14-16 days post-hatch or at swim-up	ca. 19 days
post-hatch or at swim-up

Lepomis macrochirus, bluegill	FBS, trout food	BSN	BSN, BSN48	3 feeds per
day	once hatching is complete or 48 hours after first hatch	at swim-up

Other Well Documented Test Species

Pimephales promelas, Fathead minnow	FBS, flake food	BSN	BSN	3 feeds per
day at ca. 4 hours apart 5 days a week and twice per day the other two
days of the week	once hatching is complete or within 48 hours of first
hatch	within 2 days of hatching

Oryzias latipes, medaka	flake food	BSN, flake food, or protozoa and
rotifers	BSN48, flake food or rotifers	BSN once daily; flake food twice
daily or flake food and rotifers once daily	once hatching is complete
ca. within 2 days of hatching

Danio rerio, Zebrafish	BSN48, flake food	Commercial larvae food,
protozoa6

Protein7	BSN48, flake food	BSN once daily; flake food twice daily	once
hatching is complete 	within 2 days of hatching

Other Documented Test Species

Catostomus commersoni, white sucker	FBS	none4	BSN	3 feeds per day	once
hatching is complete	7-8 days post-hatch

Esox lucius, Northern pike	live minnows	BSN	BSN, larval fish	3 feeds per
day	at day 10	ca. day 12 or at swim-up stage

Ictalurus punctatus,

channel catfish	catfish food	modified Oregon8 or BSN	modified Oregon8 or
BSN	At least 3 feeds per day	once hatching is complete	at swim-up

Oncorhynchus kisutch, Coho salmon	trout food	none4	trout starter5	2-4
feeds per day	ca. 26-36 days post-hatch or at swim-up	after swim-up at
transfer

Oncorhynchus tschawytscha,

Chinook salmon	trout food	none4	trout starter5	2-4 feeds per day
ca.26-36 days post-hatch or at swim-up	ca. 23 days post-hatch at swim-up

Salmo trutta,

Brown trout	trout food	none4	trout starter5	5 feeds per day	ca.21 days
post-hatch or at swim-up	at swim-up

Salvelinus fontinalis,

Brook trout	trout food	none4	trout starter5	5 feeds per day	ca.21 days
post-hatch or at swim-up	at swim-up

Salvelinus namaycush,

Lake trout	trout food	none4	trout starter5	5 feeds per day	ca. 21 days
post-hatch or at swim-up	at swim-up

1 Abbreviations: BSN = brine shrimp nauplii, newly hatched (i.e., less
than or equal to 24 hours old); BSN48 = brine shrimp nauplii, 48 hours
old; FBS = frozen brine shrimp, adult Artemia sp.

2 Feed ad libitum if feeding is applicable.

3 If applicable, timing (based on each treatment level) of transfer of
hatched fish out of egg cups. Complete hatching means at least 90%
hatched (based on control(s)).

4 Yolk-sac larvae require no food.

5 Recommended amount of feed is 4% of body weight per day.

6 Filtered from mixed culture.

7 Granules from fermentation process.

8 Oregon moist pellet formulations (see paragraph (j)(5) of this
guideline) or similar formulated feed that meets nutritional
requirements of the species (see paragraphs (j)(6), (j)(10), and (j)(11)
of this guideline).

―Recommended Diet and Feeding Frequency for Saltwater Brood and Test
Fish and Schedule for Larval Transfer and First Feeding

Species	Food1	Post-Hatch Transfer Time3	Time to First Feeding

	Brood Fish	Newly-Hatched Larvae2	Juveniles

	Type	Frequency

Preferred Test Species

Menidia beryllina

inland silverside	BSN48, flake food	days 1-8: rotifers4

days 9-11: BSN5 and rotifers4

days 11-test end: BSN6	3 feeds per day

2 feeds per day

2 feeds per day	six days after initial hatching	within 24 hours of first
hatch

Menidia menidia, Atlantic silverside	BSN48, flake food	days 1-8:
rotifers4

days 9-11: BSN5 and rotifers4

days 11-test end: BSN6	3 feeds per day

2 feeds per day

2 feeds per day	six days after initial hatching	within 24 hours of first
hatch

Menidia peninsulae, Tidewater silverside	BSN48, flake food	days 1-8:
rotifers4

days 9-11: BSN5 and rotifers4

days 11-test end: BSN6	3 feeds per day

2 feeds per day

2 feeds per day	six days after initial hatching	within 24 hours of first
hatch

Other Well Documented Test Species

Cyprinodon variegatus, Sheepshead minnow	FBS or flake food	BSN	BSN	2-3
feeds per day	once hatching is 90% complete in the control(s) or within
48 hours of first hatch	within 24 hours of first hatch

1 Abbreviations: BSN = brine shrimp nauplii, newly hatched (i.e., less
than or equal to 24 hours old); BSN48 = brine shrimp nauplii, 48 hours
old; and FBS = frozen brine shrimp, adult Artemia sp.

2 Feed ad libitum if feeding is applicable.

3 If applicable, timing (based on each treatment level) of transfer of
hatched fish out of egg cups.

4 Rotifers—Brachionus plicatilis; recommend 5,000 to 10,000 organisms
per egg container (based on 15 fish per container which is equivalent to
about 330 to 670 organisms per fish) per feeding period for days 1-11.

5 Recommend 2,500 organisms per test vessel (based on 15 fish per
container) which is equivalent to about 170 organisms per fish.

6 Recommend number of organisms are gradually increased to approximately
5,000 organisms per test vessel (based on 15 fish per container), which
is equivalent to about 330 organisms per fish, by test day 28.

	(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 described 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 the 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)(9) 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 the
flow-through exposure technique; the static-renewal exposure technique
could possibly be used but is not recommended. Guidance on the selection
of the appropriate exposure technique is provided in OCSPP 850.1000.

(iii) Treatment concentrations. At least 5 test solution concentrations
should be used for definitive testing, plus the appropriate control(s).
A range-finding test is recommended 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 occurred 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 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 survival and hatching success in any control
do not meet the performance standards in Tables 3 and 4.

 (6) Number of test organisms and replicates. There should be a minimum
of 4 replicate test vessels, each with at least 20 fertilized eggs, for
each test concentration and control. Each test vessel should contain an
equal volume of test solution and an equal number of eggs. Replicate
test vessels should be physically separated, since the test vessel is
the experimental unit.

(ii) Loading. The number of embryos or fish 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
flow-through tests, loading requirements will vary depending upon the
flow rate of dilution water, but should not exceed 0.5 gram wet weight
of organism per liter (g/L) of test solution passing through a test
vessel in 24 hours or 5 g/L at any time.

≤) 24 hours after fertilization. It is noted that for some test
species, e.g., salmonids, use of <24 hour old embryos may not be
possible under all circumstances; however, it is encouraged that tests
begin with embryos ≤ 48 hours after fertilization. The embryos should
be should be randomly or indiscriminately distributed among treatments.
For example, a representative sample of the test embryos could be
indiscriminately distributed by adding that no more than 20% of the
total number of embryos to each cup or screen tray and repeating the
process until each cup or screen tray contains the specified number of
embryos. Test vessels for treatment levels should be randomly or
indiscriminately located within the testing area, and the embryo cups or
screen trays should be randomly or indiscriminately distributed into the
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 that are well ventilated and free of fumes and disturbances
which may affect the test organisms. There should be flow-through tanks
for culturing, holding, and acclimating brood stock; a system for
providing a continuous supply of live brine shrimp nauplii (Artemia
salina) as well as a system for providing a supply of rotifers
(Brachionus plicatilis) for approximately 11 days during the test for
silversides. A suitable magnifying viewer for examination of embryos, if
appropriate, should be available as well as 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 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, test
vessels, egg containers, and screens should be cleaned before each test.
See OCSPP 850.1000 for further information. Test vessels should be
cleaned during the test as needed to maintain the dissolved oxygen
concentration, remove uneaten food, and prevent bacterial development
and clogging of the egg container mesh and screens.

(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 suitable 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, to minimize the entry of dust or other particulates
into the solutions, and to prevent the loss of test fish. Flow-through
systems should contain an appropriate test substance delivery system.

Egg containers or screens should be constructed of inert material (e.g.,
glass, polyethylene tubes, glass petri dishes, stainless steel or nylon
mesh of various sizes, silicon adhesive). There should be mechanisms,
like a rocker arm apparatus, for oscillating or moving egg containers up
and down within test vessels.

(vi) Dilution water. Clean surface water, ground water, reconstituted
water, or natural or artificial seawater (for saltwater species) are
acceptable as dilution water if the test species will survive in it for
the duration of the culturing, holding, acclimation, and testing periods
without showing signs of stress.

Natural seawater should be filtered through a filter with a pore size of
<20 micrometers (µm) prior to use in a test. Artificial seawater can be
prepared by adding commercially available formulations or specific
amounts of reagent-grade chemicals to reagent water (deionized,
distilled, or reverse osmosis water), surface water, or ground water.
For saltwater species, a salinity should be selected from a range of 15
and 25 parts per thousand (ppt). For artificial seawater or natural
seawater that is diluted with freshwater, salinity should be
maintainable within a weekly range of 2 ppt.

Dechlorinated tap water is not recommended (either as the freshwater
source, preparation of artificial seawater, or dilution of natural
seawater) 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 are in OCSPP 850.1000.

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.

For freshwater testing, hardness, alkalinity, and conductivity should be
measured in the dilution water at the beginning of the test. For
saltwater testing, salinity should be measured in the dilution water at
the beginning of the test.

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, pH, and salinity) in test solutions during the test
are described in detail in OCSPP 850.1000.

(i) Temperature. Recommended test temperatures are provided in Table 3
for the preferred test species and in Table 4 for other documented test
species. If the tabulated temperature is given as a range, the selected
test temperature within that range should be constant within plus or
minus (±) 1°C during the test. Additionally, the water temperature
should not differ by more than 1°C between test vessels at any one time
during the test.

(ii) pH and salinity. The pH should be between 6.0 and 8.5 for
freshwater species and between 7.5 and 8.5 for saltwater species and
should vary less than 1 pH unit within a test vessel and between test
concentrations (including control(s)). During a given test, the salinity
(selected from a range of 15 to 25 ppt) should be constant within ± 2
ppt.

(iii) Lighting and photoperiod. Recommended photoperiods are provided in
Table 3 for the preferred test species and in Table 4 for other
documented test species. 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.

―Test Environmental Conditions, Duration, and Control Survival
Standards for Preferred Test Species

Species	Recommended Test Conditions	Recommended Duration of Test
Survival of Control(s)

(minimum percent)

	Temperature

(°C)1	Photoperiod (hours)2

Hatching Success	Post-Hatch Success

Freshwater

Oncorhynchus mykiss, Rainbow trout	10 – 123	12 - 164	2 weeks after
control(s) are free-feeding (or 60 days post-hatch)	66	70

Lepomis macrochirus, Bluegill	28	16	32 days from test initiation	75
(overall)

Saltwater

Menidia beryllina,

Inland silverside5	25	13-16	28 days post-hatch	80	60

Menidia menidia,

Atlantic silverside5	22-25	13-16	28 days post-hatch	80	60

Menidia peninsulae,

Tidewater silverside5	22-25	13-16	28 days post-hatch	80	60

1 The temperature should be constant within ±1 °C of the selected
temperature during the test.

2 Hours of light is listed. For any given test, the light regime should
be constant.

3 The particular strain of rainbow trout tested may necessitate the use
of other temperatures; brood stock should be held at the same
temperature as that to be used for the fertilized eggs.

4 Except for when they are being inspected, larvae are kept in darkness
until 1 week after hatching; subdued lighting is then used throughout
the remainder of the test.

5 Salinity 20±5 ppt.

Table 4.―Test Environmental Conditions, Duration, and Control Survival
Standards for Other Freshwater and Saltwater Test Species

Species	Recommended Test Conditions	Recommended Duration of Test
Survival of Control(s)

(minimum percent)

	Temperature (°C)1	Photoperiod (hours)2

Hatching Success	Post-Hatch Success

Other Well Documented Test Species

Freshwater

Pimephales promelas, Fathead minnow	23 - 27	16	32 days from test
initiation (or 28 days post-hatch)	66	70

Oryzias latipes, Medaka	23 - 25	12-16 	30 days post-hatch	80 (overall)

Danio rerio, Zebrafish	23 -27	12-16 	30 days post-hatch	70 (overall)

Saltwater

Cyprinodon variegatus, Sheepshead minnow 3	23 - 27	12-16	32 days from
test initiation (or 28 days post-hatch)	75	80

Other Documented Test Species

Freshwater

Catostomus commersoni,

White sucker	15	16	32 days from test initiation	66	80

Esox lucius,

Northern pike	15	16	32 days from test initiation	66	70

Ictalurus punctatus, Channel catfish	25	16	32 days from test initiation
65 (overall)

Oncorhynchus kisutch, Coho salmon	8-124, 10-145	12-166	60 days
post-hatch	66	70

Oncorhynchus tschawytscha, Chinook salmon	8-124, 10-145	12-166	60 days
post-hatch	66	70

Salmon trutta, Brown trout	10	12-166	60 days post-hatch	66	70

Salvelinus fontinalis, Brook trout	10	146	60 days post-hatch	66	70

Salvelinus namaycush, Lake trout	7	12-166	60 days post-hatch	66	70

1 The temperature should be constant during the test within ±1 °C of
the selected test temperature.

2 Hours of light is listed. For any given test, the light regime should
be constant.

3 Salinity 20±5 ppt.

4 For embryos.

5 For larvae and juvenile fish.

6 Except for when they are being inspected, larvae are kept in darkness
until 1 week after hatching; subdued lighting is then used throughout
the remainder of the test.

(iv) Dissolved oxygen. The dissolved oxygen concentration should be
between 60 and 100% saturation during the test. Dissolved oxygen
concentrations should be maintained by the use of appropriate loading
and flow rates as well as removal of uneaten food and regular cleaning.
If aeration is needed to achieve an appropriate dissolved oxygen level,
it should be done before the addition of the test substance. 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 not recommended. 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.

(iv) 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; however, more volume replacements per 24-hour period may
be necessary to maintain environmental conditions for some test species.
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) Stage of embryonic development at test initiation. The embryonic
stage at the beginning of exposure to the test substance should be
verified as precisely as possible. This can be done using a
representative sample of eggs suitably preserved and cleaned.

(iv) Measures of effect.

(A) Hatching and survival. The number of hatched embryos and the number
of dead embryos, larvae, and juveniles (as appropriate) should be
counted and recorded daily, except for the silverside (see paragraph
(e)(3)(iv)(B) of this guideline). Unfertilized eggs should not be
included as part of the test population when determining
hatchability/viability.

Dead embryos, larvae, and juvenile fish should be removed as soon as
observed since they can decompose rapidly and may be broken up by the
actions of other fish. Within the first 2 days of the exposure, eggs
that are heavily fungus-infected should be counted and discarded to help
prevent further infection. Extreme care should be taken not to knock or
physically damage adjacent eggs/larvae when removing dead or
fungus-infected individuals as eggs/larvae are extremely delicate and
sensitive.

			Test organisms should be declared dead under the following
conditions:

(1) Eggs. Particularly in the early stages, a marked loss of
translucency and change in coloration caused by coagulation and/or
precipitation of protein, leading to a white opaque appearance.

(2) Embryos. Absence of body movement and/or absence of heart-beat.

(3) Larvae and juvenile fish. Immobility and/or absence of respiratory
movement and/or absence of heart-beat and/or white opaque coloration of
central nervous system and/or lack of reaction to mechanical stimulus.

(B) Time to swim-up. For salmon, trout, and char, the number of fry that
swim-up during an observation period should be recorded to the nearest
day.

(C) Body length. At test termination, all surviving fish should be
measured (to the nearest 0.1 millimeters (mm)); standard, fork, or total
length may be used to measure individual length. If caudal fin rot or
fin erosion occurs, standard lengths should be used.

(D) Body weight. At test termination, all surviving fish should be
weighed. Individual weights (to the nearest 0.01 grams for minnows and
similar small-sized fish and 0.1 grams for salmon, trout, and char) are
preferred, but if the fish are especially small, they may be weighed in
groups by test vessel. Dry weights (dried at 60 °C for 24 to 48 hours
or to constant weight) are preferable to wet weights (blotted dry). If
the fish exposed to the test substance appear to be edematous compared
to control fish, determination of dry weight rather than wet weight is
recommended.

(E) Abnormal appearance. The number of larvae or fish showing
abnormality of body form (e.g., hemorrhaging, discoloration, excessive
mucous, scoliosis, stunted bodies, etc.) should be recorded daily and
described in detail. It should be noted that deformed embryos and larvae
occur naturally and can be of the order of several percent in the
control(s) in some species. Abnormal appearing animals should only be
removed from the test vessels upon death. A photographic record of the
physical abnormalities may be made. Deformed fish that die, or are
sacrificed at the termination of the test, may be preserved for possible
future pathological examination.

(F) Abnormal behavior. Hyperventilation, uncoordinated swimming,
atypical quiescence, atypical feeding behavior, etc. should be recorded
daily and described in detail. These effects, although difficult to
quantify, can aid in the interpretation of mortality data and influence
a decision to extend the exposure period beyond the recommended
duration. Abnormally behaving animals should only be removed from the
test vessels upon death.

(f) Treatment of results.

	(1) Response variable calculations.

 ) is calculated using Equation 1.

 	Equation 1

where:

 = mean time to hatch, in days, for replicate j;

i = index of observation events from 1 (first observation event after
introduction of fertilized embryos (day 0 of the test)) through m;

m = maximum number of observation events;

hij = number of eggs that hatch between observation event i-1 and
observation event i (for i = 1, the number of eggs that hatch between
introduction of fertilized embryos (day 0 of the test) and observation
event 1) in replicate j;

ti = time span, in days, between the introduction of fertilized embryos
(day 0 of the test) and observation event i;

Hj = total number of eggs that hatch by the end of the hatch period in
replicate j.

 ) is calculated using Equation 2.

 	Equation 2

where:

 = mean time to swim-up, in days, for replicate j;

i = index of observation events from 1 (first observation event after
introduction of fertilized embryos (day 0 of the test)) through m;

m = maximum number of observation events;

sij = number of fry that swim-up between observation event i-1 and
observation event i (for i = 1, the number of fry that swim-up between
introduction of fertilized embryos (day 0 of the test) and observation
event 1) in replicate j;

ti = time span, in days, between the introduction of fertilized embryos
(day 0 of the test) and observation event i;

SUj = total number of fry that swim-up in replicate j.

(iii) Hatching success. Hatching success is the proportion of eggs that
hatched for a given replicate. The hatching success for replicate j
(hsj) is calculated using Equation 3.

 	Equation 3

where:

hsj = hatching success for replicate j;

Hj = total number of eggs that hatch by the end of the hatch period in
replicate j; and

ej = number of fertilized eggs at test initiation in replicate j.

(iv) Post-hatch success. Post-hatch success is the proportion of
post-hatch fish that survive to the end of the test. Tests in which the
number of exposed fish is reduced or thinned during the post-hatch
exposure phase by removal of live individuals prior to test termination
may be conducted. In some tests, one or more thinning events may occur.
Alternatively, the test may be conducted with no thinning event(s).

(A) Without any thinning events. Equation 4 is used to calculate
post-hatch success for a given replicate when there is no thinning event
of post-hatch fish during the test.

 	Equation 4

where:

phj = post-hatch success for replicate j;

sj = number of post-hatch fish surviving to test termination in
replicate j; and

Hj = total number of eggs that hatched by the end of the hatch period in
replicate j.

	(B) With thinning event(s). The proportion of post-hatch fish that
survive to the end of the test when one or more thinning events occur is
calculated by multiplying the portion surviving each event. Equation 5
is used to calculate post-hatch success for a given replicate when there
are 2 thinning events of post-hatch fish during the test; if there is
only one thinning event, the equation reduces down.

 	Equation 5

where:

phj = post-hatch success for replicate j;

 = number of surviving post-hatch fish at start of the first thinning
event in replicate j;

Hj = total number of eggs that hatched by the end of the hatch period in
replicate j;

 = number of fish that were kept at the first thinning event that
survived to the start of the second thinning event (or to the end of the
test, if this is the only thinning event) in replicate j;

 = total number of fish kept or transferred at the first thinning event
in replicate j;

 = number of fish that were kept at the second thinning event that
survived to test termination in replicate j (if there was only one
thinning event, this value is not applicable and is dropped); and

 = total number of fish kept at the second thinning event in replicate j
(if there was only one thinning event, this value is not applicable and
is dropped).

(v) Overall survival. Overall survival is the proportion of fertilized
eggs that successfully hatch and survive to the end of the test.

(A) Without any thinning events. Equation 6 is used to calculate overall
survival for a replicate when there is no thinning event during the
test.

 	Equation 6

where:

oj = overall survival for replicate j;

Sj = number of surviving fish at test termination in replicate j;

ej = number of fertilized eggs at test initiation in replicate j.

(B) With post-hatch thinning event(s). Overall survival for a replicate
when one or more thinning events occur post-hatch is calculated by
multiplying hatching success (see paragraph (f)(1)(iii) of this
guideline) by post-hatch success (see paragraph (f)(1)(iv) of this
guideline) as described in Equation 7.

 	Equation 7

where:

oj = overall survival for replicate j;

hsj = hatching success for replicate j (see paragraph (f)(1)(iii) of
this guideline); and

phj = post-hatch success for replicate j (see paragraph (f)(1)(iv) of
this guideline);

 ) is calculated using Equation 8.

 	Equation 8

where:

  = mean body length for replicate j;

k = index number from 1 to Sj of length measurement for surviving fish
at test termination in replicate j;

Lk = body length of individual k in replicate j (measured as described
in paragraph (e)(9)(iv)(C) of this guideline); and

Sj = number of surviving fish at test termination that were measured in
replicate j.

 ) is calculated using Equation 9.

 	Equation 9

where:

  = mean body weight for replicate j;

wj = total weight (dry weight, preferred) for all surviving fish at test
termination in replicate j (measured as described in paragraph
(e)(9)(iv)(D) of this guideline); and

Sj = number of surviving fish at test termination that were weighed in
replicate j.

	(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 5 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 number of embryos or fish showing
abnormality of body form should be summarized by type of abnormality,
time of observation, treatment group, and replicate.

(iii) Abnormal behavior. Behavioral abnormalities (e.g.,
hyperventilation, uncoordinated or erratic swimming, atypical
quiescence, and atypical feeding behavior) should be summarized by type
of behavior, time of observation, treatment group, and replicate.

(3) Percent inhibition. For all response variables in Table 5, the
percent inhibition (%I) as compared to the control(s) at each test
substance treatment level is calculated using Equation 10.

 	Equation 10

where:

%I = percent inhibition as compared to the control(s) for test substance
treatment level;

C = mean control response value (e.g., mean body length of surviving
fish); and

X = mean response value (e.g., mean body length of surviving fish) 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, if there is a
statistically significant inhibition in any of the response variables in
Table 5 at the limit concentration as compared to the control(s) are
observed at the limit concentration, a multiple-concentration definitive
test should be conducted.

	(5) Definitive test.

(i) NOEC. A NOEC and a LOEC should be determined for each of the
response variables in Table 5 using appropriate statistical methods. 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 (hatch,
post-hatch, or overall) and the 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.

Table 5.―Early Life Stage Primary Response Variables to Calculate and
Evaluate

 )

(g) Tabular summary of test conditions. Table 6 lists the important
conditions that should prevail during the multiple-concentration
definitive test. The same conditions are recommended for a limit test
except for a difference in the number of test concentrations. Meeting
these test conditions will greatly increase the likelihood that the
completed test will be acceptable or valid.

―Summary of Test Conditions for Fish Early Life Stage Toxicity Test

Test type	Flow-through preferred

Test species	See paragraph (e)(3)(i) of this guideline

Test duration	Dependent upon species tested (see Tables 3 and 4)

Temperature	Dependent upon species tested (see Tables 3 and 4)

Light quality	Ambient laboratory illumination

Light intensity	540-1080 lux (approximately 50-100 ft-c)

Photoperiod	Dependent upon species tested (see Tables 3 and 4)

pH	Between 6.0 and 8.5 for freshwater species; between 7.5 and 8.5 for
saltwater species (constant during testing within ±1 pH unit)

Water hardness, as CaCO3 (freshwater tests)	<250 mg/L (preferably <180
mg/L); 40-50 mg/L for testing with metals

≤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 each response parameter in Table 5

(h) Test validity elements. This test would be considered to be
unacceptable or invalid if one or more of the conditions in Table 7
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 7.―Test Validity Elements for the Fish Early Life Stage
Toxicity Test

1. All test vessels and compartments 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 or compartments.

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 embryos long after first cleavage of the
blastodisc (see paragraph (e)(6)(ii) of this guideline).

5. The test was terminated before the appropriate duration given for the
test species in Tables 3 and 4.

6. Control survival and hatching success did not meet the performance
standards in Tables 3 and 4.

7. 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 at 20(C, 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) Source of fertilized eggs, and a description of the methods,
handling, and any transport used to obtain fertilized eggs for the test.
The description should include the number of females and males from
which gametes were collected.

(iv) Information about the parents: source, culture practices, and
holding and acclimation procedures and conditions, including acclimation
period, water used, feeding history, and health status (mortality of
parents before test initiation and any preventative or disease
treatments).

(v) Embryo age in terms of time post-fertilization at test initiation
and the embryonic stage of the test organisms from a representative
sample (range and distribution) 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 vessel: 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 rate 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; salinity (saltwater); pH; hardness and
alkalinity (freshwater); dissolved oxygen; un-ionized ammonia; total
organic carbon or chemical oxygen demand; particulate matter;
conductivity; metals, pesticides, and residual chlorine (mean, standard
deviation, range). Describe the frequency and sample date(s) for
documenting dilution water quality.

(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) Description of the number of fertilized eggs exposed at test
initiation in each test group and replicate, the occurrence of any
thinning event, and the number of fish in each test group and replicate
kept after a thinning event.

(vii) Number of test vessels (replicates) per treatment level and
control(s).

(viii) Methods used for treatment randomization and assignment of test
organism 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, salinity (if applicable) and light intensity.

(xiii) Methods and frequency of measuring dissolved test substance to
confirm exposure concentrations.

(xiv) Methods and frequency of observing or measuring dead embryos and
fish, time of hatch, time of swim-up (if applicable), body length and
weight of surviving fish, and 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, the 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, salinity (if applicable), 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 dead embryos and dead
fish removed, the number of hatched eggs, the number of fish reaching
the swim-up stage (for applicable species with a swim-up life stage),
and the number of surviving post-hatch fish 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 dead embryos and
dead fish removed, the number of hatched eggs, the number of fish
reaching the swim-up stage (if swim-up life stage is applicable), and
the number of surviving post-hatch fish in each test vessel, for all
treatment levels and control(s), at each observation time (provide the
raw data) and descriptive statistics (mean, standard deviation, minimum,
maximum).

(vi) For limit and definitive tests, provide the first day of the test
that hatch is observed in each replicate and the first day of the test
that swim-up is observed in each replicate (if swim-up life stage is
applicable).

(vii) For limit and definitive tests, a tabulation by treatment and
replicate of mean time to hatch, mean time to swim-up (if swim-up life
stage is applicable), hatching success, post-hatch success, overall
survival, mean body length of surviving fish, and mean body weight of
surviving fish. 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 %I 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
the number of embryos or post-hatch fish displaying abnormal appearance
or behavioral signs of toxicity by test vessel, treatment, and
observation time (provide raw data).

(x) For limit test, provide findings of hypothesis tests for each of the
primary endpoints and for abnormal behavior and appearance as compared
to the control(s).

(xi) 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.

(xii) When appropriate, a description of statistical method(s) used for
point estimates, including the software package for determining LC50 and
IC50 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.

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1241-05, Standard Guide for Conducting Early Life-Stage Toxicity Tests
with Fishes. In Annual Book of ASTM Standards, Vol. 11.06, ASTM, West
Conshohocken, PA. Current edition approved 2005.

(2) A.P.H.A., A.W.W.A, W.E.F., 1998. Standard Methods for the
Examination of Water and Wastewater, 20th edition. Section 8921, Fathead
Minnow.

(3) Brauhn, J.L. and R.A. Schoettger, 1975. Acquisition and Culture of
Research Fish: Rainbow trout, Fathead minnows, Channel catfish and
Bluegills. p. 54, Ecological Research Series, EPA-660/3-75-011, Duluth,
MN.

(4) Brungs, W.A. and B.R. Jones, 1977. Temperature Criteria for
Freshwater Fish: Protocol and Procedures. p. 128, Ecological Research
Series EPA-600/3-77-061, Duluth, MN.

(5) Crawford, D.L., D.K. Law, T.B. McKee and J.W. Westgate, 1973.
Storage and nutritional characteristics of modified Oregon moist rations
as an intermediate-moisture product. The Progressive Fish-Culturist
35(1): 32-38.

(6) Conklin, D.E. 2000. Chapter 3: Diet In The Laboratory Fish. The
Handbook of Experimental Animals. G.K. Ostrander (ed). Academic Press,
San Diego, CA.

(7) Environment of Canada. 1998. Biological Test Method: Toxicity Tests
Using Early Life Stage of Salmonid Fish (Rainbow Trout). Environmental
Protection Series, Method Development and Application Section,
Environmental Technology Centre, EPS1/RM/28 Second Edition

(8) Hansen, D.J. and P.R. Parrish, 1977. Suitability of sheepshead
minnows (Cyprinodon variegatus) for life-cycle toxicity tests. In
Aquatic Toxicology and Hazard Evaluation (edited by F.L. Mayer and J.L.
Hamelink), pp. 117-126, ASTM STP 634.

(9) 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.

(10) National Research Council. 1993. Nutrient Requirements of Fish.
Subcommittee on Fish Nutrition, Committee on Animal Nutrition, Board on
Agriculture, National Academy Press, Washington, DC.

(11) National Research Council. 2011. Nutrient Requirements of Fish and
Shrimp. Subcommittee on Nutrient Requirements of Fish and Shrimp,
Committee on Animal Nutrition, Board on Agriculture, National Academy
Press, Washington, DC. (consult latest version)

(12) Organization for Economic Co-operation and Development. 2013. OECD
Guidelines for Testing of Chemicals, Guideline 210, Fish Early
Life-Stage Toxicity Test.

(13) U.S. EPA, 1972. Recommended Bioassay Procedure for Fathead Minnows,
Pimephales promelas (Rafinesque), Chronic Tests. p. 13, National Water
Quality Laboratory, Duluth, MN.

(14) 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.

(15) 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.

(16) U.S. Environmental Protection Agency, 2002. Short-term methods for
estimating the chronic toxicity of effluents and receiving water to
freshwater organisms, Fourth Edition. EPA-821-R-02-013. October, 2002.

(17) U.S. Environmental Protection Agency, 2002. Short-term methods for
estimating the chronic toxicity of effluents and receiving water to
marine and estuarine organisms, Third Edition. EPA-821-R-02-014.
October, 2002.

(18) U.S. Environmental Protection Agency, 2015. Endocrine Disruptor
Screening Program Test Guidelines, OCSPP 890.2200: Medaka extended one
generation reproduction test (MEOGRT), EPA 740-C-15-002, Office of
Chemical Safety and Pollution Prevention, July, 2015.

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