United States
Environmental Protection
Iml M m Agency
Office of Chemical Safety EPA 712-C-16-008
and Pollution Prevention October 2016
(7101)
Ecological Effects
Test Guidelines
OCSPP 850.1400:
Fish Early Life Stage
Toxicity Test
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NOTICE
This guideline is one of a series of test guidelines established by the United States
Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention (OCSPP) for
use in testing pesticides and chemical substances to develop data for submission to the Agency under
the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide,
Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food,
Drug and Cosmetic Act (FFDCA) (21 U.S.C. 346a). Prior to April 22, 2010, OCSPP was known as
the Office of Prevention, Pesticides and Toxic Substances (OPPTS). To distinguish these guidelines
from guidelines issued by other organizations, the numbering convention adopted in 1994 specifically
included OPPTS as part of the guideline's number. Any test guidelines developed after April 22, 2010
will use the new acronym (OCSPP) in their title.
The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions under
TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the test, and is also
used by EPA, the public, and the companies that are subject to data submission requirements under
TSCA, FIFRA, and/or the FFDCA. As a guidance document, these guidelines are not binding on
either EPA or any outside parties, and the EPA may depart from the guidelines where circumstances
warrant and without prior notice. At places in this guidance, the Agency uses the word "should." In
this guidance, the use of "should" with regard to an action means that the action is recommended
rather than mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline, but EPA recognizes that departures may be
appropriate in specific situations. You may propose alternatives to the recommendations described in
these guidelines, and the Agency will assess them for appropriateness on a case-by-case basis.
For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.gov/ocspp and select "Test Methods & Guidelines" on the
navigation menu. You may also access the guidelines in http://www.regulations.gov grouped by
Series under Docket ID #s: EPA-HQ-OPPT-2009-0150 through EPA-HQ-OPPT-2009-0159, and
EPA-HQ-OPPT-2009-0576.
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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.
(2) Background. The source materials used in developing this harmonized OCSPP test
guideline are 40 CFR 797.1600 Fish Early Life Stage Toxicity Test; OPP 72-4 Fish Early
Life-Stage and Aquatic Invertebrate Life-Cycle Studies (Pesticide Assessment
Guidelines, Subdivision E — Hazard Evaluation: Wildlife and Aquatic Organisms; see
paragraph (j)(14) of this guideline); EPA Pesticide Reregi strati on Rejection Rate
Analysis: Ecological Effects (see paragraph (j)(l5) 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)(l) 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.
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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
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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.
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(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 salary 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
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in paragraph (j)(l) 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)(l) 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.
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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)(l) 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)(l), 0X2),
0X3), (j)(4), G)(8), 0X12), 0)(13), 0)(16), 0X17), and 0X18) 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
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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)(l) 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.
(v) Diet and feeding. Food type and feeding quantity and frequency are critical,
and supplying the correct food for each stage at an appropriate time and at a level
sufficient to support optimal growth and survival is essential. The amount of food
should be based upon the number and size of individuals present and should be
adjusted over the course of the test to account for changes in number and size.
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One method for accomplishing this is to measure the amount of food consumed
by all fish in a control replicate vessel within a 10-minute feeding duration. The
amount of food consumed is divided by the number of fish present to derive a
feed ration per individual. This amount is then used to calculate the food ration
per test vessel based on the number of surviving fish. Surplus food and feces
should be removed once daily and as necessary to avoid accumulation of waste.
Suggested feeding regimes for brood and test animals can be found in Tables 1
and 2. Keeping the amount of food constant between control(s) and test
treatments is important to avoid bacterial development and to observe the amount
of food consumption. Fish should not fed during the 24 hours prior to termination
of the test.
Table 1.—Recommended Diet and Feeding Frequency for Freshwater Brood and Test
Fish Species 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
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
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Species
Food1
Post-Hatch
Transfer Time3
Time to First
Feeding
Brood
Fish
Newly-
Hatched
Larvae2
Juveniles
Type
Frequency
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
Saimo trutta,
Brown trout
trout food
none4
trout starter5
5 feeds per day
ca. 21 days
post-hatch or at
swim-up
at swim-up
Saiveiinus
fontinaiis,
Brook trout
trout food
none4
trout starter5
5 feeds per day
ca. 21 days
post-hatch or at
swim-up
at swim-up
Saiveiinus
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).
Table 2.—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 beryiiina
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 peninsuiae,
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.
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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 (HC1)
and sodium hydroxide (NaOH) may be used for this adjustment if warranted.
See additional information about pH during testing in (e)(8)(ii).
(ii) Exposure technique. The test should be conducted using 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.
10
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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.
(ii) Introduction of test organisms. The test should be started by introducing
fertilized eggs, preferably at a stage before cleavage of the blastodisc commences,
into the test vessels after the test substance has been added. Tests should be begun
with embryos less than or equal to (<) 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
11
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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.
12
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Natural seawater should be filtered through a filter with a pore size of <20
micrometers (|im) 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
13
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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.
Table 3.—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
CO
CM
I
O
T
(O
CM
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 peninsuiae,
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
Pimephaies promeias,
Fathead minnow
23-27
16
32 days from test initiation
(or 28 days post-hatch)
66
70
Oryzias iatipes, 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 minnow3
23-27
12-16
32 days from test initiation
(or 28 days post-hatch)
75
80
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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
12-16®
60 days post-hatch
66
70
Oncorhynchus
tschawytscha, Chinook
salmon
8-124, 10-145
12-16®
60 days post-hatch
66
70
Salmon trutta, Brown
trout
10
12-16®
60 days post-hatch
66
70
Salvelinus fontinalis,
Brook trout
10
14®
60 days post-hatch
66
70
Salvelinus namaycush,
Lake trout
7
12-16®
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
15
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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.
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(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.
17
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(1) Response variable calculations.
(i) Mean time to hatch. The mean time to hatch for a given test vessel represents
the mean time span, in days, between the introduction of fertilized embryos (day 0
of the test) and hatch within the test vessel. The mean time to hatch, in days, for
replicate j (h ,) is calculated using Equation 1.
where:
hj = mean time to hatch, in days, for replicate /;
/ = 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;
hj = number of eggs that hatch between observation event i-1 and observation
event / (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
Hj = total number of eggs that hatch by the end of the hatch period in replicate j.
(ii) Mean time to swim-up. For species with a swim-up life stage (e.g., salmon
and trout), the mean time to swim-up represents the mean time span, in days,
between the introduction of fertilized embryos (day 0 of the test) and swim-up
within the test vessel. The mean time to swim-up, in days, for a replicate j (su,)
is calculated using Equation 2.
suj = mean time to swim-up, in days, for replicate /;
i = index of observation events from 1 (first observation event after introduction
of fertilized embryos (day 0 of the test)) through m\
Equation 1
Equation 2
where:
18
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m = maximum number of observation events;
stj = 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.
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.
where:
phj = post-hatch success for replicate /;
Sj = number of post-hatch fish surviving to test termination in replicate /;
Equation 3
Equation 4
and
19
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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.
where:
phj = post-hatch success for replicate /;
ns0 = number of surviving post-hatch fish at start of the first thinning
event in replicate y;
Hj = total number of eggs that hatched by the end of the hatch period in
replicate y;
nsXj= 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 y;
nfx = total number of fish kept or transferred at the first thinning event in
replicate y;
ns2.= 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
nf2 = 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 5
20
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Equation 6
where:
Oj = overall survival for replicate /;
Sj = number of surviving fish at test termination in replicate /;
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)(l)(iii) of this guideline) by
post-hatch success (see paragraph (f)(l)(iv) of this guideline) as described
in Equation 7.
where:
Oj = overall survival for replicate /;
hsj = hatching success for replicate j (see paragraph (f)(l)(iii) of this
guideline); and
phj = post-hatch success for replicate j (see paragraph (f)(l)(iv) of this
guideline);
(vi) Mean body length of surviving fish. The response measure for growth based
on body length is the mean body length of surviving fish at test termination in a
replicate. The mean body length of surviving fish for replicate j (Lj) is calculated
using Equation 8.
where:
Lj = mean body length for replicate /;
k = index number from 1 to Sj of length measurement for surviving fish at test
termination in replicate j\
Oj = {hsj\phj)
Equation 7
Equation 8
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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.
(vii) Mean body weight of surviving fish. The response measure for growth
based on weight (dry weight preferred) is the mean weight of surviving fish at test
termination in a replicate. The mean body weight of surviving fish for replicate j
(wj) is calculated using Equation 9.
where:
wj = mean body weight for replicate /;
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 9
%/ = (C-*X'0Q)
Equation 10
C
22
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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
Mean time to hatch (hj)
Mean time to swim-up (for those species with a swim-up stage) (suj)
Hatching success (proportion of eggs that hatch) (hsj)
Post-hatch success (proportion of post-hatched fish surviving to the end of the test) (phj)
Overall survival (proportion of fertilized eggs that hatch and survive to the end of the test) (o,)
Mean body length of surviving fish (Lj)
Mean body weight of surviving fish (dry weight preferred) (^)
(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
23
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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.
Table 6.—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 CaCCb
(freshwater tests)
<250 mg/L (preferably <180 mg/L); 40-50 mg/L for testing with metals
Salinity (saltwater tests)
20±5 ppt (constant during test within ±2 ppt for selected salinity)
Total organic carbon (TOC)
<2 mg/L
Test vessel size/volume
Adequate to maintain loading and dependent upon species
Age of test organisms
Preferably before cleavage of blastodisc (generally <24 hours post
fertilization)
Number of test organisms per
concentration
80 minimum
Number of replicate test
vessels per concentration
4 minimum
Loading
<0.5 g/L/day or 5 g/L at any time
Feeding regime
Dependent upon species tested (see Tables 1 and 2)
Test vessel aeration
Not recommended; gentle aeration of test vessels may only be used
in cases where the dissolved oxygen levels are in danger of dropping
below 60% saturation. In such cases, assurances should be made
that the use of aeration does not stress the test organisms; test
substance concentrations should be measured during the test; and all
treatment and control vessels should be given the same aeration
treatment.
Vehicle concentration, if used
<0.1 mL/L for recommended solvents (see OCSPP 850.1000)
Test concentrations
Definitive test: minimum of 5 test concentrations chosen in a
geometric series plus a dilution water control and a vehicle (solvent)
control, if a vehicle is used
Measures of effect or
measurement endpoints
Definitive test: NOEC/LOEC for 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
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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)(l) of OCSPP 850.1000 describes the
minimum background information to be supplied in the report.
(2) Guideline deviations. Provide a statement of the guideline or protocol followed.
Include a description of any deviations from the test guideline or any occurrences which
may have influenced the results of the test, the reasons for these changes, and any
resulting effects on test endpoints noted and discussed.
(3) Test substance.
(i) Identification of the test substance: common name, IUPAC and CAS names,
CAS number, structural formula, source, lot or batch number, chemical state or
form of the test substance, purity {i.e., for pesticides, the identity and
concentration of active ingredient(s)), and radiolabeling, if any, including the
location of label(s) and radiopurity.
(ii) Storage conditions of the test chemical or test substance and stability of the
test chemical or test substance under storage conditions if stored prior to use.
(iii) Methods of preparation of the test substance and the treatment concentrations
used in the range-finding and definitive tests, or limit test. Identify whether the
nominal concentrations are corrected or uncorrected for purity of the test
substance.
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(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
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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.
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(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).
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(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.
(1) American Society for Testing and Materials. ASTM E1241-05, 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
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(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 Reregi strati on 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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