United States Prevention, Pesticides EPA712-C-96-121
Environmental Protection and Toxic Substances April 1996
Agency (7101)
&EPA Ecological Effects Test
Guidelines
OPPTS 850.1400
Fish Early-Life Stage
Toxicity Test
'Public Draft"
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 850.1400 Fish early-life stage toxicity test.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both 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).
(2) Background. The source material used in developing this har-
monized OPPTS 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) EPA report 540/09-
82-024, 1982; and OECD 210 Fish Early-Life Stage Toxicity Test.
(b) Introduction. (1) Tests with the early-life stages of fish are in-
tended to define the lethal and sublethal effects of chemicals on the stages
and species tested. They yield information of value for the estimation of
the chronic lethal and sublethal effects of the substance on other fish spe-
cies.
(2) This guideline is based on a proposal from the United Kingdom
which was discussed at a meeting of OECD experts convened at
Medmenham (United Kingdom) in November 1988.
(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and the definitions in 40 CFR Part 792—Good Lab-
oratory Practice Standards apply to this test guideline. The following defi-
nitions also apply to this test guideline.
LOEC (Lowest-observed-effect-concentration) is the lowest tested
concentration of a test substance at which the substance is observed to
have a significant effect (at p<0.05) when compared with the control.
However, all test concentrations above the LOEC must have a harmful
effect equal to or greater than those observed at the LOEC.
NOEC (No-observed-effect-concentration) is the test concentration
immediately below the LOEC.
(d) Principle of the test. The early-life stages of fish are exposed
to a range of concentrations of the test substance dissolved in water, pref-
erably under flow-through conditions, or where appropriate, semistatic
conditions. The test is begun by placing fertilized eggs in the test chambers
and is continued at least until all the control fish are free-feeding. Lethal
and sublethal effects are assessed and compared with control values to
determine the LOEC and the NOEC.
(e) Information on the test substance. (1) Results of an acute tox-
icity test (see OPPTS 850.1075), preferably performed with the species
chosen for this test, should be available. This implies that the water solu-
bility and the vapor pressure of the test substance are known and a reliable
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analytical method for the quantification of the substance in the test solu-
tions with known and reported accuracy and limit of detection is available.
(2) Useful information includes the structural formula, purity of the
substance, stability in water and light, pKa, Pow, and results of a test for
ready biodegradability. (See OPPTS 835.3110 through 835.3160.)
(f) Validity of the test. For a test to be valid the following conditions
apply:
(1) The dissolved oxygen concentration must be between 60 and
100 percent of the air saturation value throughout the test.
(2) The water temperature must not differ by more than ±1.5 °C be-
tween test chambers or between successive days at any time during the
test, and should be within the temperature ranges specified for the test
species (Tables 4. and 5. under paragraphs (h)(l)(ii) and (h)(l)(iii) of this
guideline).
(3) Evidence must be available to demonstrate that the concentrations
of the test substance in solution have been satisfactorily maintained within
±20 percent of the mean measured values.
(4) Overall survival of fertilized eggs in the controls and, where rel-
evant, in the solvent-only controls must be greater than or equal to the
limits defined in Tables 4. and 5. under paragraphs (h)(l)(ii) and (h)(l)(iii)
of this guideline.
(5) When a solubilizing agent is used it must have no significant ef-
fect on survival nor produce any other adverse effects on the early-life
stages as revealed by a solvent-only control.
(g) Description of the method—(1) Test chambers. Any glass,
stainless steel, or other chemically inert vessels can be used. The dimen-
sions of the vessels should be large enough to allow compliance with load-
ing rate criteria given below. It is desirable that test chambers be randomly
positioned in the test area. A randomized block design with each treatment
being present in each block is preferable to a completely randomized de-
sign. The test chambers should be shielded from unwanted disturbance.
(2) Selection of species, (i) Recommended fish species are:
(A) Freshwater—rainbow trout, Oncorhynchus mykiss; fathead min-
now, Pimephales promelas; zebra fish, Danio rerio; ricefish, Oryzias
latipes.
(B) Salt water—sheepshead minnow, Cyprinodon variegatus.
(ii) The Office of Prevention, Pesticides and Toxic Substances prefers
rainbow trout (O. mykiss) or fathead minnow (P. promelas) as test species
for freshwater fish and sheepshead minnow (C. variegatus) or silverside
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(Menidia menidia, M. beryllina, or M. peninsulae) as test spcies for estua-
rine or marine fish. This does not preclude the use of other species. Exam-
ples of other well-documented species which have also been used are:
(A) Freshwater—coho salmon, Oncorhynchus kisutch; chinook salm-
on, Oncorhynchus tschawytscha; brown trout, Salmo trutta; Atlantic salm-
on, Salmo salar; brook trout, Salvelinus fontinalis, lake trout, Salvelinus
namaycush; northern pike, Esox lucius; white sucker, Catostomus
commersoni; bluegill, Lepomis macrochirus; channel catfish, Ictalurus
punctatus; flagfish, Jordanella floridae; three-spined stickleback,
Gasterosteus aculeatus; common carp, Cyprinus carpio.
(B) Salt water—Atlantic silverside, M menidia; Tidewater silverside,
M peninsulae.
(iii) Feeding and handling requirements of brood and test animals,
test conditions, duration, and survival criteria for these species can be
found in Tables 1. and 2. under paragraph (g)(3) of this guideline, and
Tables 4. and 5. under paragraphs (h)(l)(i) and (h)(l)(ii) of this guideline.
(iv) The test procedure might have to be adapted to provide suitable
test conditions. The rationale for the selection of the species and the exper-
imental method should be reported in this case.
(3) Holding of the brood fish, (i) Details on holding the brood stock
under satisfactory conditions may be found in the references cited under
paragraphs (j)(l), G)(2), and G)(3) of this guideline.
(ii) Conditions for recommended species are provided in the following
Table 1. Abbreviations in the table have the following meanings: BW =
body weight; FBS = frozen brine shrimp—adults Anemia sp.; BSN = brine
shrimp nauplii—newly hatched; BSN48 = brine shrimp nauplii—48 h old.
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Table 1.—Feeding and Handling Requirements of Brood and Test Animals of Recommended
Species
Species
Freshwater
Oncorhynchus
mykiss, rainbow
trout
Pimephales
promelas, fathead
minnow
Dan/o rerio, Zebra
fish
Oryzias latipes,
ricefish
Saltwater
Cyprinodon
variegatus,
Sheepshead min-
now
Food
Brood Fish
trout food
FBS
BSN48,
flake
food
flake food
FBS or
flake
food
Newly-
Hatched Lar-
vae
none 2
BSN
protozoa 4
protein 5
BSN, flake
food (or
protozoa or
rotifers)
BSN
Juveniles
Type
trout starter
BSN48
BSN48
BSN48, flake
food (or
rotifers)
BSN48
Amount
4% BW
per day
Frequency
2-4 feeds/
day
ad libitum
BSN once
daily; flake
food twice
daily or
flake food
and rotifers
once daily
2-3 feeds per
day
Post-Hatch
Transfer
Time1
14-1 6 days
post-hatch
or at swim-
up3
once hatching
is 90%
not necessary
from hatch to
swim-up
not applicable
Time to First
Feeding
19 days post-
hatch or at
swim-up
within 2 days
of hatching
6-7 days
after
spawning
within 24 h of
hatch/swim-
up
within 1 day
first hatch
1 if applicable
2 yolk-sac larvae require no food
3 not essential
4 filtered from mixed culture
5 granules from fermentation process
(iii) Conditions for other species are provided in the following Table
2.:Abbreviations in the table have the following meanings: BSN = brine
shrimp nauplii, newly hatched; BSN48 = brine shrimp nauplii, 48 hours
old; FBS = frozen brine shrimp; adult Artemia sp.
Table 2.—Feeding and Handling Requirements of Brood and Test Animals of Other Well-
Documented Species
Species
Freshwater
Oncorhynchus
kisutch, Coho
salmon
Oncorhynchus
tschawytscha, Chi-
nook salmon
Salmo trutta, brown
trout
Salmo sa/ar Atlantic
salmon
Salvelinus fontinalis,
brook trout
Food
Brood fish
trout food
trout food
trout food
trout food
trout food
Newly-
hatched lar-
vae
none 1
none
none
none
none
Juveniles
Type
trout starter
trout starter
trout starter
trout starter
trout starter
Amount
4% BW
per day
4% BW
per day
4% BW
per day
4% BW
per day
4% BW
per day
Frequency
2-4 feeds/
day
2-4 feeds/
day
5 feeds/day
5 feeds/day
5 feeds/day
Post-Hatch
Transfer Time
(if applicable)
26-36 days
post-hatch
or at swim-
up
26-36 days
post-hatch
or at swim-
up
21 days post-
hatch or at
swim-up
21 days post-
hatch or at
swim-up
21 days post-
hatch or at
swim-up
Time To First
Feeding
after swim-up
at transfer
26 days post-
hatch at
swim-up
at swim-up
at swim-up
at swim-up
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Table 2.—Feeding and Handling Requirements of Brood and Test Animals of Other Well-
Documented Species—Continued
Species
Salvelinus
namaycush, lake
trout
Esox lucius, North-
ern pike
Catostomus
commersoni, white
sucker
Lepomis
macrochirus,
bluegill
Ictalurus punctatus,
channel catfish
Jordanella floridae,
flagfish
Gasterosteus
aculeatus, three-
spined stickleback
Cyprinus carpio,
common carp
Saltwater
Menidia menidia, At-
lantic silverside
Menidia peninsulas,
Tidewater
silverside
Food
Brood fish
trout food
live min-
nows
FBS
FBS, trout
food
catfish food
FBS, flake
food,
BSN
Tetramin
FBS
Proprietary
carp
food;
freeze-
dried
tubifex or
trout
food
BSN48,
flake
food
BSN48,
flake
food
Newly-
hatched lar-
vae
none
BSN48
none
BSN
modified Or-
egon
BSN48, flake
food, or
protozoa/
rotifers3
Brachionus
rubens
(rotifer)
BSN
days 1-8
days 9-1 1
days 11 -end
days 1-8
days 9-1 1
days 11 -end
Juveniles
Type
trout starter
larval fish
BSN48
BSN48
modified Or-
egon
BSN48, flake
food
BSN48,
Tetramin
BSN48,
ground;
trout start-
er, or flake
food
rotifers3
BSN48 and
rotifers3
BSN48
rotifers3
BSN48 and
rotifers3
BSN48
Amount
4% BW
per day
Frequency
5 feeds/day
3 feeds/day
3 feeds per
day
at least 3
feeds per
day
Artemia
nauplii
once daily;
flake food
twice daily
or flake
food and
protozoa &
rotifers
once daily
BSN48, 2-3
feeds per
day;
Tetramin
once daily
3-4 feeds per
day
3 feeds per
day
2 feeds per
day
3 feeds per
day
2 feeds per
day
Post-Hatch
Transfer Time
(if applicable)
21 days post-
hatch or at
swim-up
transfer
hatched fish
daily
once all em-
bryos have
hatched
6-7 days at
26 °C 2
from hatch to
swim-up
several hours
after hatch2
once hatching
complete
not applicable
not applicable
not applicable
not applicable
not applicable
not applicable
Time To First
Feeding
at swim-up
1 week post-
hatch or
swimming
yolk-sac
stage
7-8 days
post-hatch
or at swim-
up
at swim-up
within 48 h of
swim-up
within 24 h of
hatch
within 24
hours of
hatch
36-48 h post-
hatch
within 24 h of
first hatch
within 24 h of
first hatch
within 24 h of
first hatch
within 24 h of
first hatch
within 24 h of
first hatch
within 24 h of
first hatch
1 yolk-sac larvae require no food
2 fish may be handled with a 6 mm internal diameter glass siphon tube
3 rotifers—Brachionus plicatilis
(4) Handling of embryos and larvae, (i) Initially, embryos and lar-
vae may be exposed within the main vessel in smaller glass or stainless
steel vessels, fitted with mesh sides or ends to permit a flow of test solu-
tion through the vessel. Nonturbulent flow through these small vessels may
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be induced by suspending them from an arm arranged to move the vessel
up and down but always keeping the organisms submerged. Fertilized eggs
of salmonid fishes can be supported on racks or meshes with apertures
sufficiently large to allow larvae to drop through after hatching.
(ii) Where egg containers, grids, or mesh have been used to hold
eggs within the main test vessel, these restraints should be removed after
the larvae hatch, according to the advice in Table 1. except that mesh
should be retained to prevent the escape of the fish. 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. The timing of this
transfer varies with the species and transfer may not always be necessary.
(5) Water. Any water in which the test species shows control sur-
vival, at least as good as that described in Table 4. under paragraph
(h)(l)(ii) of this guideline, and Table 5. under paragraph (h)(l)(iii) of this
guideline, is suitable as a test water. It should be of constant quality during
the period of the test. In order to ensure that the dilution water will not
unduly influence the test result (for example, by complexation of test sub-
stance) or adversely affect the performance of the brood stock, samples
should be taken at intervals for analysis. Measurements of heavy metals
(e.g. Cu, Pb, Zn, Hg, Cd, Ni), major anions and cations (e.g. Ca, Mg,
Na, K, Cl, sulfate), pesticides, total organic carbon, and suspended solids
should be made, for example, every 3 months where a dilution water is
known to be relatively constant in quality. Some chemical characteristics
of an acceptable dilution water are listed in the following Table 3:
Table 3.—Some Chemical Characteristics of an Acceptable Dilution Water
Substance
Particulate matter
Total organic carbon
Un-ionized ammonia
Residual chlorine
Total organophosphorus pesticides
Total organochlorine pesticides plus polychlorinated biphenyls
Total oraanic chlorine
Maximum Concentration
<20 mg/L
<2 mg/L
< 1 uq/L
< 1 0 ug/L
<50 ng/L
<50 ng/L
<25 na/L
(6) Test solutions, (i) For flow-through tests, a system which contin-
ually dispenses and dilutes a stock solution of the test substance (e.g. me-
tering pump, proportional diluter, saturator system) is required to deliver
a series of concentrations to the test chambers. The flow rates of stock
solutions and dilution water should be checked at intervals during the test
and should not vary by more than 10 percent throughout the test. A flow
rate equivalent to at least five test chamber volumes per 24 h has been
found suitable (see paragraph (j)(l) of this guideline).
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(ii) The use of solvents or dispersants (solubilizing agents) may be
required in some cases in order to produce a suitably concentrated stock
solution.
(iii) For the semistatic technique, two different renewal procedures
may be followed. Either new test solutions are prepared in clean vessels
and surviving eggs and larvae gently transferred into the new vessels, or
the test organisms are retained in the test vessels while a proportion (at
least two-thirds) of the test water is changed.
(h) Procedure. Useful information on the performance of fish early-
life stage tests is available in the literature, some examples of which are
included under paragraphs (j)(l), and (j)(4) through (j)(8) of this guideline.
(1) Conditions of exposure—(i) Duration. The test should start as
soon as possible after the eggs have been fertilized, the embryos preferably
being immersed in the test solutions before cleavage of the blastodisc com-
mences, or as close as possible after this stage. The test should continue
at least until all the control fish have been free-feeding. Test duration will
depend upon the species used.
(A) Data for recommended species are provided in the following
Table 4.:
Table 4.—Test Conditions, Duration, and Survival Criteria for Recommended Species
Species
Freshwater
Oncorhynchus mykiss, Rainbow
trout.
Pimephales promelas, Fathead
minnow.
Dan/o rerio Zebra fish
Oryzias latipes, ricefish
Saltwater
Cyprinodon variegatus, Sheeps-
head minnow 7.
Test Conditions
Temperature
(°C)
10±2 1
12±22 3
25±2
25±2
24 ±1 (a)
23±225
25±2
Photoperiod
(hours)
144
16
12-16 6
12-166
12-166
Recommended Duration of
Test
2 weeks after controls are
free-feeding (or 60 days
post-hatch)
32 days from start of test
(or 28 days post-hatch)
30 days post-hatch
30 days post-hatch
32 days from start of test
(or 28 days post-hatch)
Survival of Controls
(minimum percent)
Hatching
Success
>66
>66
>75
Post-Hatch
Success
70
70
70
80
80
1for embryos
2for larvae and juvenile fish
3the particular strain of rainbow trout tested may necessitate the use of other temperatures; brood stock must be held at the
same temperature as that to be used for the eggs
4darkness for larvae until one week after hatching except when they are being inspected, then subdued lighting throughout test
(12-16 h photoperiod (6))
5this supersedes the requirement for temperature control given earlier on in the test
6for any given test conditions, light regime should be constant
7salinity shall be at 15-30; for any given test this shall be performed to ±2 percent.
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(B) Data for other species are provided in the following Table 5:
Table 5.—Test Conditions, Duration and Survival Criteria for Other Well-Documented Species
Species
Freshwater
Oncorhynchus kisutch, Coho
salmon.
Oncorhynchus tschawytscha,
Chinook salmon.
Salmon trutta brown trout
Salmo salar, Atlantic salmon
Salvelinus fontinalis, brook trout .
Salvelinus namaycush, Lake trout
Esox lucius Northern pike
Catostomus commersoni, White
sucker.
Lepomis macrochirus, Bluegill
Ictalurus punctatus, Channel cat-
fish.
Jordanella floridae Flagfish
Gasterosteus aculeatus, Three-
spined stickleback.
Cyprinus carpio, common carp ...
Saltwater
Menidia menidia, Atlantic
silverside4.
Menidia peninsulas, Tidewater
silverside4.
Test Conditions
Temperature
(°C)
101, 122
101, 122
10
10
10
12-18
7
15
28
26
24-26
18-20
21-25
22-25
22-25
Photoperiod
(hours)
1 2-1 63
1 2-1 63
1 2-1 63
1 2-1 63
143
16
1 2-1 63
16
16
16
16
12-16
12-16
13
13
Recommended Duration of
Test
60 days post-hatch
60 days post-hatch
60 days post-hatch
60 days post-hatch
60 days post-hatch
60 days post-hatch
32 days from start of test
32 days from start of test
32 days from start of test
32 days from start of test
28 days
28 days post-hatch
28 days
28 days
Survival of Controls
(minimum percent)
Hatching
Success
>66
>66
>66
>66
>66
>66
>66
>66
80
>80
>80
>80
Post-Hatch
Success
70
70
70
70
70
70
70
80
75
65 (overall)
80
75
60
60
1for embryos
2for larvae and juvenile fish
3darkness for larvae until 1 week after hatching except when they are being inspected, then subdued lighting throughout test
(12-16 h photoperiod unless otherwise specified, but constant regime for a given test)
4salinity 20
(ii) Loading. The number of fertilized eggs at the start of the test
should be sufficient to meet statistical requirements. They should be ran-
domly distributed among treatments, and at least 60 eggs, divided equally
between at least two replicate test chambers, should be used per concentra-
tion. The loading rate (biomass per volume of test solution) should be
low enough in order that a dissolved oxygen concentration of at least 60
percent of the air saturation value (ASV) can be maintained without aer-
ation. For flow-through tests, a loading rate not exceeding 0.5 g/L/24 h
and not exceeding 5 g/L of solution at any time has been recommended
(see paragraph (j)(l) of this guideline).
(iii) Light and temperature. The photoperiod and water temperature
should be appropriate for the test species as given in Table 4. under para-
graph (h)(l)(ii) of this guideline.
(iv) Feeding. Food and feeding are critical, and it is essential that
the correct food for each stage should be supplied at an appropriate time
and at a level sufficient to support normal growth. Feeding should be ad
libitum while minimizing the surplus. Surplus food and feces should be
removed as necessary to avoid accumulation of waste. Detailed feeding
regimes are given in Table 1. under paragraph (g)(3)(ii) of this guideline,
8
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but, as experience is gained, food and feeding regimes are continually
being refined to improve survival and optimize growth. Effort should
therefore be made to confirm the proposed regime with acknowledged ex-
perts.
(v) Test concentrations. (A) Normally five concentrations of the test
substance spaced by a constant factor not exceeding 3.2 are required. The
curve relating LC50 to period of exposure in the acute study should be
considered when selecting the range of test concentrations. The use of
fewer than five concentrations, for example in limit tests, and a narrower
concentration interval may be appropriate in some circumstances. Justifica-
tion should be provided if fewer than five concentrations are used. Con-
centrations of the substance higher than the 96-h LC50 or 10 mg/L, which-
ever is the lower, need not be tested.
(B) Where a solubilizing agent is used, its concentration should not
be greater than 0.1 mL/L and should be the same in all test vessels. How-
ever, every effort should be made to avoid the use of such materials.
(vi) Controls. One dilution-water control and also, if relevant, one
control containing the solubilizing agent should be run in addition to the
test series.
(2) Frequency of analytical determinations and measurements, (i)
During the test, the concentrations of the test substance are determined
at regular intervals to check compliance with the validity criteria. A mini-
mum of five determinations is necessary. In studies lasting more than
1 month, determinations should be made at least once a week. Samples
may need to be filtered (e.g. using a 0.45 (im pore size) or centrifuged
to ensure that the determinations are made on the substance in true solu-
tion.
(ii) During the test, dissolved oxygen, pH, total hardness and salinity
(if relevant), and temperature should be measured in all test vessels. As
a minimum, dissolved oxygen, salinity (if relevant), and temperature
should be measured weekly, and pH and hardness should be measured
at the beginning and end of the test. Temperature should preferably be
monitored continuously in at least one test vessel.
(3) Observations—(i) Stage of embryonic development. The em-
bryonic 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 cleared.
(ii) Hatching and survival. Observations on hatching and survival
should be made at least once daily and numbers recorded. 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 the
other fish. Extreme care should be taken when removing dead individuals
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not to knock or physically damage adjacent eggs/larvae, these being ex-
tremely delicate and sensitive. Criteria for death vary according to life
stage:
(A) For eggs: Particularly in the early stages, a marked loss of trans-
lucency and change in coloration, caused by coagulation and/or precipita-
tion of protein, leading to a white opaque appearance.
(B) For embryos: Absence of body movement and/or absence of
heart-beat.
(C) For larvae and juvenile fish: Immobility and/or absence of res-
piratory movement and/or absence of heart-beat and/or white opaque color-
ation of central nervous system and/or lack of reaction to mechanical stim-
ulus.
(iii) Abnormal appearance. The number of larvae or fish showing
abnormality of body form should be recorded at adequate intervals depend-
ing on the duration of the test and the nature of the abnormality described.
It should be noted that abnormal embryos and larvae occur naturally and
can be of the order of several percent in the controls in some species.
Abnormal animals should only be removed from the test vessels on death.
(iv) Abnormal behavior. Abnormalities, e.g. hyperventilation, unco-
ordinated swimming, atypical quiescence, and atypical feeding behavior
should be recorded at adequate intervals depending on the duration of the
test. These effects, although difficult to quantify, can, when observed, aid
in the interpretation of mortality data and influence a decision to extend
the exposure period beyond the recommended duration.
(v) Weight. At the end of the test all surviving fish must be weighed.
Individual weights are preferred but, if the fish are especially small, they
may be weighed in groups by test vessel. Dry weights (24 h at 60 °C)
are preferable to wet weights (blotted dry).
(vi) Length. At the end of the test, measurement of individual lengths
is recommended: Standard, fork, or total length may be used. If however,
caudal fin rot or fin erosion occurs, standard lengths should be used.
(vii) Data for statistical analysis. These observations will result in
some or all of the following data being available for statistical analysis:
(A) Cumulative mortality.
(B) Numbers of healthy fish at end of test.
(C) Time to start of hatching and end of hatching.
(D) Numbers of larvae hatching each day.
(E) Length and weight of surviving animals.
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(F) Numbers of deformed larvae.
(G) Numbers of fish exhibiting abnormal behavior.
(i) Data and reporting—(1) Treatment of results, (i) It is rec-
ommended that a statistician be involved in both the design and analysis
of the test results since this test guideline allows for considerable variation
in experimental design as, for example, in the number of test chambers,
number of test concentrations, starting number of fertilized eggs, and num-
ber of parameters measured.
(ii) In view of the options available in test design, specific guidance
on statistical procedures is not given here. However, it will be necessary
for variations to be analyzed within each set of replicates using analysis
of variance or contingency table procedures. To make a multiple compari-
son between the results at the individual concentrations and those for the
controls, Dunnett's method might be found useful (see paragraphs G)(9)
and G)(10) of this guideline). However, care must be taken where applying
such a method to ensure that chamber-to-chamber variability is estimated
and is acceptably low. Other useful methods are also available (see para-
graphs (j)(l), G)(6), and (j)(H) of this guideline).
(2) Interpretation of results. The results should be interpreted with
caution where measured toxicant concentrations in test solutions occur at
levels near the detection limit of the analytical method.
(3) Test report. The test report must include the following informa-
tion:
(i) Test substance. (A) Physical nature and, where relevant, physico-
chemical properties.
(B) Chemical identification data.
(ii) Test species. Scientific name, strain, source and method of collec-
tion of the fertilized eggs, and subsequent handling.
(iii) Test conditions. (A) Test procedure used (e.g. semistatic or flow-
through design).
(B) Photoperiods.
(C) Test design (e.g. number of test chambers and replicates, number
of embryos per replicate).
(D) Method of preparation of stock solutions and frequency of re-
newal (the solubilizing agent and its concentration must be given, when
used).
(E) Nominal test concentrations, means of the measured values, their
standard deviations in the test vessels, and the method by which these
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were attained, and evidence that measurements refer to concentrations of
the test substance in true solution.
(F) Dilution water characteristics: pH, hardness, temperature, dis-
solved oxygen concentration, residual chlorine levels (if measured), total
organic carbon, suspended solids, salinity of the test medium (if meas-
ured), and any other measurements made.
(G) Water quality within test vessels: pH, hardness, temperature, and
dissolved oxygen concentration.
(H) Detailed information on feeding (e.g. type of feed, source, amount
given, and frequency).
(iv) Results. (A) Evidence that controls met the overall survival ac-
ceptability standard of the test species (Tables 4. and 5.).
(B) Data on mortality/survival at embryo, larval, and juvenile stages
and overall mortality/survival.
(C) Days to hatch and numbers hatched.
(D) Data for length and weight.
(E) Incidence and description of morphological abnormalities, if any.
(F) Incidence and description of behavioral effects, if any.
(G) Statistical analysis and treatment of data.
(H) NOEC for each response assessed.
(I) LOEC (at p = 0.05) for each response assessed.
(J) Any concentration-response data and curves available.
(v) Discussion of the results. [Reserved]
(j) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) American Society for Testing and Materials (ASTM). Standard
Guide for Conducting Early Life-Stage Toxicity Tests with Fishes. ASTM
E 1241-92, p. 180-207, Philadelphia, PA (1992).
(2) Brauhn, J.L. and Schoettger, R.A., Acquisition and Culture of Re-
search Fish: Rainbow trout, Fathead minnows, Channel catfish and
Bluegills. p. 54, Ecological Research Series, EPA-660/3-75-011, Duluth,
MN (1975).
(3) Brungs, W.A. and Jones, B.R., Temperature Criteria for Fresh-
water Fish: Protocol and Procedures, p. 128, Ecological Research Series
EPA-600/3-77-061, Duluth, MN (1977).
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(4) Hansen, D.J. and Parrish, P.R., 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 (1977).
(5) McKim, J.M. et al., Metal toxicity to embryos and larvae of eight
species of freshwater fish-II: Copper. Bulletin of Environmental and Con-
tamination Toxicology 19:608-616 (1978).
(6) Rand, G.M. and Petrocelli, S.R., Fundamentals of Aquatic Toxi-
cology. Hemisphere Publication Corporation, NY (1985).
(7) USEPA, Recommended Bioassay Procedure for Fathead Min-
nows, Pimephales promelas (Rafinesque), Chronic Tests, p. 13, National
Water Quality Laboratory, Duluth, MN (1972).
(8) USEPA, Recommended Bioassay Procedure for Bluegill. Lepomis
macrochirus (Rafinesque), Partial Chronic Tests, p. 11, National Water
Quality Laboratory, Duluth, MN (1972).
(9) Dunnett, C.W., A multiple comparisons procedure for comparing
several treatments with a control. Journal of the American Statistical Asso-
ciation 50: 1096-1121 (1955).
(10) Dunnett, C.W., New tables for multiple comparisons with a con-
trol. Biometrics 20:482-491 (1964).
(11) McClave, J.T. et al., Statistical Analysis of Fish Chronic Toxicity
Test Data. Proceedings of 4th Aquatic Toxicology Symposium, ASTM,
Philadelphia, PA (1980).
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