United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA 712–C–96–121

April 1996

Ecological Effects Test

Guidelines

OPPTS 850.1400

Fish Early-Life Stage

Toxicity Test

‘‘Public Draft’’
i

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 published 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. Environmental Protection Agency under the Toxic Substances Control Act (15

U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act

(7 U.S.C. 136, et seq.).

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 ‘‘Environmental 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 comments. 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, Arlington, 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 Bulletin 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.’’
1

OPPTS 850.1400 Fish early-life stage toxicity test.

(a) Scope—(1) Applicability. This guideline is intended to meet testing 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 harmonized 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 intended 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 species.

(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 Laboratory Practice Standards apply to this test guideline. The following definitions 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, preferably 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 toxicity test (see OPPTS 850.1075), preferably performed with the species

chosen for this test, should be available. This implies that the water solubility and the vapor pressure of the test substance are known and a reliable
2

analytical method for the quantification of the substance in the test solutions 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 between 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)(1)(ii) and (h)(1)(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 relevant, in the solvent-only controls must be greater than or equal to the

limits defined in Tables 4. and 5. under paragraphs (h)(1)(ii) and (h)(1)(iii)

of this guideline.

(5) When a solubilizing agent is used it must have no significant effect 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 dimensions of the vessels should be large enough to allow compliance with loading 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 design. 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 minnow, 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 estuarine or marine fish. This does not preclude the use of other species. Examples of other well-documented species which have also been used are:

(A) Freshwater—coho salmon, Oncorhynchus kisutch; chinook salmon, Oncorhynchus tschawytscha; brown trout, Salmo trutta; Atlantic salmon, 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)(1)(i) and (h)(1)(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 experimental 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)(1), (j)(2), and (j)(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 Artemia sp.; BSN = brine

shrimp nauplii—newly hatched; BSN48 = brine shrimp nauplii—48 h old.
4

Table 1.—Feeding and Handling Requirements of Brood and Test Animals of Recommended

Species

Species

Food

Post-Hatch

Transfer

Time1

Time to First

Feeding Brood Fish

NewlyHatched LarvaeJuveniles

Type Amount Frequency

Freshwater

Oncorhynchus

mykiss, rainbow

trout

trout food none 2 trout starter 4% BW

per day

2–4 feeds/

day

14–16 days

post-hatch

or at swimup3

19 days posthatch or at

swim-up

Pimephales

promelas, fathead

minnow

FBS BSN BSN48 ad libitum once hatching

is 90%

within 2 days

of hatching

Danio rerio, Zebra

fish

BSN48,

flake

food

protozoa 4

protein 5

BSN48 not necessary 6–7 days

after

spawning

Oryzias latipes,

ricefish

flake food BSN, flake

food (or

protozoa or

rotifers)

BSN48, flake

food (or

rotifers)

BSN once

daily; flake

food twice

daily or

flake food

and rotifers

once daily

from hatch to

swim-up

within 24 h of

hatch/swimup

Saltwater

Cyprinodon

variegatus,

Sheepshead minnow

FBS or

flake

food

BSN BSN48 2–3 feeds per

day

not applicable 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 WellDocumented Species

Species

Food

Post-Hatch

Transfer Time

(if applicable)

Time To First

Feeding Brood fish

Newlyhatched larvaeJuveniles

Type Amount Frequency

Freshwater

Oncorhynchus

kisutch, Coho

salmon

trout food none 1 trout starter 4% BW

per day

2–4 feeds/

day

26–36 days

post-hatch

or at swimup

after swim-up

at transfer

Oncorhynchus

tschawytscha, Chinook salmon

trout food none trout starter 4% BW

per day

2–4 feeds/

day

26–36 days

post-hatch

or at swimup

26 days posthatch at

swim-up

Salmo trutta, brown

trout

trout food none trout starter 4% BW

per day

5 feeds/day 21 days posthatch or at

swim-up

at swim-up

Salmo salar Atlantic

salmon

trout food none trout starter 4% BW

per day

5 feeds/day 21 days posthatch or at

swim-up

at swim-up

Salvelinus fontinalis,

brook trout

trout food none trout starter 4% BW

per day

5 feeds/day 21 days posthatch or at

swim-up

at swim-up
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Table 2.—Feeding and Handling Requirements of Brood and Test Animals of Other WellDocumented Species—Continued

Species

Food

Post-Hatch

Transfer Time

(if applicable)

Time To First

Feeding Brood fish

Newlyhatched larvaeJuveniles

Type Amount Frequency

Salvelinus

namaycush, lake

trout

trout food none trout starter 4% BW

per day

5 feeds/day 21 days posthatch or at

swim-up

at swim-up

Esox lucius, Northern pikelive minnowsBSN48 larval fish transfer

hatched fish

daily

1 week posthatch or

swimming

yolk-sac

stage

Catostomus

commersoni, white

sucker

FBS none BSN48 3 feeds/day once all embryos have

hatched

7–8 days

post-hatch

or at swimup

Lepomis

macrochirus,

bluegill

FBS, trout

food

BSN BSN48 3 feeds per

day

at swim-up

Ictalurus punctatus,

channel catfish

catfish food modified Oregonmodified Oregonat least 3

feeds per

day

6–7 days at

26 °C 2

within 48 h of

swim-up

Jordanella floridae,

flagfish

FBS, flake

food,

BSN

BSN48, flake

food, or

protozoa/

rotifers3

BSN48, flake

food

Artemia

nauplii

once daily;

flake food

twice daily

or flake

food and

protozoa &

rotifers

once daily

from hatch to

swim-up

within 24 h of

hatch

Gasterosteus

aculeatus, threespined stickleback

Tetramin

FBS

Brachionus

rubens

(rotifer)

BSN48,

Tetramin

BSN48, 2–3

feeds per

day;

Tetramin

once daily

several hours

after hatch2

within 24

hours of

hatch

Cyprinus carpio,

common carp

Proprietary

carp

food;

freezedried

tubifex or

trout

food

BSN BSN48,

ground;

trout starter, or flake

food

3–4 feeds per

day

once hatching

complete

36–48 h posthatch

Saltwater

Menidia menidia, Atlantic silversideBSN48,

flake

food

days 1–8 rotifers3 3 feeds per

day

not applicable within 24 h of

first hatch

days 9–11 BSN48 and

rotifers3

2 feeds per

day

not applicable within 24 h of

first hatch

days 11–end BSN48 not applicable within 24 h of

first hatch

Menidia peninsulae,

Tidewater

silverside

BSN48,

flake

food

days 1–8 rotifers3 3 feeds per

day

not applicable within 24 h of

first hatch

days 9–11 BSN48 and

rotifers3

2 feeds per

day

not applicable within 24 h of

first hatch

days 11–end BSN48 not applicable 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 larvae 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 solution through the vessel. Nonturbulent flow through these small vessels may
6

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 survival, at least as good as that described in Table 4. under paragraph

(h)(1)(ii) of this guideline, and Table 5. under paragraph (h)(1)(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 substance) 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 Maximum Concentration

Particulate matter .................................................................................... < 20 mg/L

Total organic carbon ................................................................................ < 2 mg/L

Un-ionized ammonia ................................................................................ < 1 μg/L

Residual chlorine ..................................................................................... < 10 μg/L

Total organophosphorus pesticides ........................................................ < 50 ng/L

Total organochlorine pesticides plus polychlorinated biphenyls ............. < 50 ng/L

Total organic chlorine .............................................................................. < 25 ng/L

(6) Test solutions. (i) For flow-through tests, a system which continually dispenses and dilutes a stock solution of the test substance (e.g. metering 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)(1) of this guideline).
7

(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 earlylife stage tests is available in the literature, some examples of which are

included under paragraphs (j)(1), 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 commences, 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

Test Conditions

Recommended Duration of

Test

Survival of Controls

(minimum percent)

Temperature

(°C)

Photoperiod

(hours) Hatching

Success

Post-Hatch

Success

Freshwater

Oncorhynchus mykiss, Rainbow

trout.

10 ± 2 1

12 ± 2 2 3

14 4 2 weeks after controls are

free-feeding (or 60 days

post-hatch)

> 66 70

Pimephales promelas, Fathead

minnow.

25 ± 2 16 32 days from start of test

(or 28 days post-hatch)

> 66 70

Danio rerio, Zebra fish ................. 25 ± 2 12–16 6 30 days post-hatch 70

Oryzias latipes, ricefish ............... 24 ± 1 (a)

23 ± 2 2 5

12–16 6 30 days post-hatch 80

Saltwater

Cyprinodon variegatus, Sheepshead minnow 7.25 ± 2 12–16 6 32 days from start of test

(or 28 days post-hatch)

> 75 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.
8

(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

Test Conditions

Recommended Duration of

Test

Survival of Controls

(minimum percent)

Temperature

(°C)

Photoperiod

(hours) Hatching

Success

Post-Hatch

Success

Freshwater

Oncorhynchus kisutch, Coho

salmon.

101, 122 12–163 60 days post-hatch > 66 70

Oncorhynchus tschawytscha,

Chinook salmon.

101, 122 12–163 60 days post-hatch > 66 70

Salmon trutta, brown trout ........... 10 12–163 60 days post-hatch > 66 70

Salmo salar, Atlantic salmon ....... 10 12–163 60 days post-hatch > 66 70

Salvelinus fontinalis, brook trout . 10 143 60 days post-hatch > 66 70

Salvelinus namaycush, Lake trout 12–18 16 60 days post-hatch > 66 70

Esox lucius, Northern pike .......... 7 12–163 32 days from start of test > 66 70

Catostomus commersoni, White

sucker.

15 16 32 days from start of test > 66 80

Lepomis macrochirus, Bluegill ..... 28 16 32 days from start of test 75

Ictalurus punctatus, Channel catfish.26 16 32 days from start of test 65 (overall)

Jordanella floridae, Flagfish ........ 24–26 16

Gasterosteus aculeatus, Threespined stickleback.18–20 12–16 28 days 80 80

Cyprinus carpio, common carp ... 21–25 12–16 28 days post-hatch > 80 75

Saltwater

Menidia menidia, Atlantic

silverside4.

22–25 13 28 days > 80 60

Menidia peninsulae, Tidewater

silverside4.

22–25 13 28 days > 80 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 randomly distributed among treatments, and at least 60 eggs, divided equally

between at least two replicate test chambers, should be used per concentration. 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 aeration. 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)(1) 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 paragraph (h)(1)(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,
9

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 experts.

(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. Justification should be provided if fewer than five concentrations are used. Concentrations of the substance higher than the 96–h LC50 or 10 mg/L, whichever 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. However, 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 minimum 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 μm pore size) or centrifuged

to ensure that the determinations are made on the substance in true solution.

(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 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 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
10

not to knock or physically damage adjacent eggs/larvae, these being extremely delicate and sensitive. Criteria for death vary according to life

stage:

(A) For 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.

(B) For embryos: Absence of body movement and/or absence of

heart-beat.

(C) For 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.

(iii) Abnormal appearance. The number of larvae or fish showing

abnormality of body form should be recorded at adequate intervals depending 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, uncoordinated 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.
11

(F) Numbers of deformed larvae.

(G) Numbers of fish exhibiting abnormal behavior.

(i) Data and reporting—(1) Treatment of results. (i) It is recommended 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 number 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 comparison between the results at the individual concentrations and those for the

controls, Dunnett’s method might be found useful (see paragraphs (j)(9)

and (j)(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 paragraphs (j)(1), (j)(6), and (j)(11) 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 information:

(i) Test substance. (A) Physical nature and, where relevant, physicochemical properties.

(B) Chemical identification data.

(ii) Test species. Scientific name, strain, source and method of collection of the fertilized eggs, and subsequent handling.

(iii) Test conditions. (A) Test procedure used (e.g. semistatic or flowthrough 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 renewal (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
12

were attained, and evidence that measurements refer to concentrations of

the test substance in true solution.

(F) Dilution water characteristics: pH, hardness, temperature, dissolved oxygen concentration, residual chlorine levels (if measured), total

organic carbon, suspended solids, salinity of the test medium (if measured), 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 acceptability 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 additional 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 Research 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 Freshwater 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 Contamination Toxicology 19:608–616 (1978).

(6) Rand, G.M. and Petrocelli, S.R., Fundamentals of Aquatic Toxicology. Hemisphere Publication Corporation, NY (1985).

(7) USEPA, Recommended Bioassay Procedure for Fathead Minnows, 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 Association 50: 1096–1121 (1955).

(10) Dunnett, C.W., New tables for multiple comparisons with a control. 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).