United States
Environmental Protection
Agency
Office of Pesticides Prograrrs
Washington, DC
EPA £40/9-86-133
July 198S
Hazard Evaluation Division
Standard Evaluation Procedure
Fish Early Life-Stage Support Document 5o
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HA2AR0 EVALUATION DIVISION
STANDARD EVALUATION PROCEDURE
FISH EARLY LIFE-STAGE TEST
Prepared by
Miachel Rexrode, M.S.
and
Thomas M. Armitage, Ph.D.
Standard Evaluation Procedures Project Manager
Stephen L. Johnson
Hazard Evaluation Division
Office of Pesticide Programs
United States Environmental protection Agency
Office of Pesticide Programs
Washington, B.C. 20460
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STANDARD EVALUATION PROCEDURE
PREAMBLE
This Standard Evaluation Procedure (SEP) is one of a set of
guidance documents which explain the procedures used to evaluate
environmental and human health effects data submitted to the
Office of Pesticide Programs. The SEPs' are designed to ensure
comprehensive and consistent treatment of major scientific topics
in these reviews and to provide interpretive policy guidance
where appropriate. The Standard Evaluation Procedures will be
used in conjunction with the appropriate Pesticide Assessment
Guidelines and other Agency Guidelines, While the documents were
developed to explain specifically the principles of scientific
evaluation within the Office of Pesticide Programs, they may also
be used by other offices in the Agency in the evaluation of
studies and scientific data. The Standard Evaluation Procedures
will also serve as valuable internal reference documents and will
inform the public and regulated community'of important consider-
ations in the evaluation of test data for determining chemical
hazards. I believe the SEPs will improve both the quality of
science within EPA and, in conjunction with the Pesticide Assess-
ment Guidelines, will lead to more effective use of both public
and private resources..
W. Melonef Director
Hazard Evaluation Division
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TABLE OF CONTENTS
I. INTRODUCTION
A, When Required I
B. Purpose . 1
C. Test Material . ... 2
D. Acceptable Protocols ..........P....,,.....,. • 2
II. MATERIALS, METHODS, AND REPORTING REQUIREMENTS
A. Biological System 2
1. Acceptable Species 2
2 . Source * 2
3. Eggs from Adult Fish 3
4. Embryo Exposure (Test Begins) » 3
5. Post Hatch, Larval Fish 4
6. Controls 5
7. Data Endpoints ........................... 5
B. Physical System 5
1. Test Water .. . .' 5
a. Saltwater Pish 5
b. Freshwater Fish 6
2. Temperature 6
3, Photo period 6
4 . Dosing Apparatus 6
.5. Toxicant Mixing 6
6. Test Vessels .....,,.» 6
7 . Embryo Cups ,.,«... 6
8. Flow Rate 7
i . Aeration 7
C. Chemical System ..»..»....,.... 7
1. Concentrations 7
2. Measurement of Other Variables .......... 7
3. Solvents 7
D. Calculations * 8
III. REVIEWER'S EVALUATION
A. Verification of Statistical Analysis ......... 8
B. Conclusions .................................. 8
1. Categorization of Results 8
2 . Rationale .'...... 9
3. Reparability 9
4. Descriptive Conclusions ................. 9
REFERENCES . 10
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llJ[§H_gARLY LIFE-STAGE
I. INTRODUCTION
A. When Required
The fish early life-stage test1/ is required to support an
end-use product is intended to be applied directly to water or
is expected to transport to water from the intended use site,
and when any one of the following conditions apply:
0 If the pesticide is. intended for use such that its
presence in water is likely to be continuous or recurrent
regardless of toxieity, as revealed by studies required
by 40 CFR §158.130 "'~ ~
0 If any LC^Q or £€50 value determined in the testing
required by 40 CFR §158.145 [§§ 72-1, -2, or ~3J is
less than 1 mg/1;
'° If the estimated environmental concentration in water
is equal to or greater than 0.01 of any £€59 or *LC$Q
determined in acute testing required by 40 CFR §158.145;
or
0 If the actual or estimated environmental concentration
in water resulting from use is less than 0.01 of any
EC50 or L^5Q determined in testing required by 40 CFS
§158.145 and any of the following conditions exists:
Studies of other organisms indicate the repro-
ductive physiology of fish and/or invertebrates
may be affected;
Physicochenucal properties indicate cumulative
effects? or
The pesticide is persistent in water (e.g., half-
life in water greater than four days),
B. Purpose
0 To establish chronic toxieity levels of the active
ingredient to fish;
In cases where risk criteria for both fish and invertebrates
are exceeded, the more sensitive organism must be tested in
a fish early life-stage or invertebrate life cycle study.
Both studies may,, however, may be required to complete a
risk assessment.
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0 To compare toxicity information with measured or
estimated pesticide residues in art aquatic environment
in order to assess potential impact to fish;
8 To provide support for precautionary label statements;
and
0 To indicate the need for further laboratory testing or
field testing.
C. Test Material
Testing must be conducted with the technical grade of the
active ingredient (a,i.)« If more than one active ingredient
constitutes a technical product, the technical grade of each
active ingredient must be tested separately.
D, acceptable Prg_tgcols
The Ecological Effects Branch (EEB) does not endorse any
one protocol. It is sometimes necessary and desirable to alter
the -procedures presented in published protocols to meet the
needs of the chemical or test organisms used. However, EEB
does recommend some protocols as guidance for performing a fish
early life-stage toxicity test. These protocols include;
American Public Health Association, American Water Works
Association and Water Pollution Control Federation (1985)
Standard Methods for the Examination of Water and Wa&tewater
Sixteenth Edition. Publication Office: American Public
Health Association, -1015 18th Street NW, Washington,
DC 20036. 854 pp.
Goodman, L.R. '(1985} Comparative Toxicological
Relationship Demonstrated in Early Life. Stage Tests with
Marine Fish, Environ. Res. Lab., Gulf Breeze, PL.
EPA/60Q/*85/135,
Middaugh, D.P., M.J. Hemmer, and L.R. Goodman, 1987,
Methods for Spawning, Culturing and Conducting Toxicity
Tests with Early Life Stages of Four Atherinid Fishes: The
inland silverslde, Menj.dj_a beryllina, Atlantic sllverstde,
M. menidia, tidewater silverside, M. peninsulas, and
California" grunion, Leuresthes tenuis. Office of Research
and Development, U.S. Environmental Protection Agency,
Environmental Research Laboratory at Gulf Breeze Florida,
EPA/600/8-87/D04 (Januraryp'1987) » 56 pp.
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II. MATERIALS, METHODS, AND REPORTING REQUIREMENTS
A. Biologic aJLjS y s t e m
I. AcceptableSpecies
The selected species should have a demonstrated sensitivity
to known toxicants. If possible, they should be species that
occur in the area of exposure or be related to exposed species.
The acceptable freshwater species,(1, 2) are rainbow trout
(Saj.mg g_a_irdrier i) , brook trout [Salve jLinus fontinalis) , coho
salmon (Onco_rKyiichus kisutch) , Chinook (O. tshawytscha) , bluegill
(Lepomis macrochirus) , brown trout (S. ;t£u_tta) , lake trout (S_.
namaycush) /northern pike (Esox lucius), fatKead minnow
{Pimephaj-es pj^melas) , white sucker ("Catostomus commersuni) ,
and channel catfish (Ictalurus punctatus). The silverside
species (Menidia roenidia , menldjji bejcyll_i_na, and Mg£ULd_i_a
peninsulas) and the sheepshead minnow (Cyprinodon variegatus)
are acceptable estuarine species.{1, 2)
2. Source
Gametes used for conducting a fish early life-stage test
roay be obtained: 1) directly from hatcheries or commercial
sources; 2) from wild populations of adult fish collected in
the field; oc 3) from brood fish cultured in the laboratory*
Whenever salmon or trout are used, they should be obtained from
a hatchery that has been certified disease-free.
3. Eggs from Adult Fish
Eggs can be obtained either by stripping ripe females or
by collecting eggs deposited directly on substrata. Manual
removal is usually conducted on salmon, pike, trout, bluegill,
and silversides. This procedure is usually preceded by killing
or by anesthetizing ripe females in MS-222 ( » iQO-ppm) or
quinaldine { « 10 ppm). Eggs are forced from the vent by
manual or air pressure techniques. If fish are sacrificed, an
incision is made along the median ventral line from the vent to
the pectoral fins of ripe females. Care should be taken to
keep eggs free from mucous and blood. Eggs still adhering to
the ovaries ace not taken, Eggs from at least three females
should be fertilized with sperm from at least three males.
Male specimens (e.g., salmon, trout) anesthesized with • 100
ppm of MS-222 can be collected by gently pressing the sides of
'the abdomen.(1) Sperra collection from pike is best accomplished
by sacrificing the fish.(l) The fish are killed and the testes
removed through an incision in the abdomen. Testes are placed
in clean cheesecloth and squeezed to extrude milt.(l)
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Fertilized eggs may be shipped but can be damaged by rough
handling. Eggs should be water hardened .for one-hour prior to
shipping and kept cool (< 10 °C) . (1) Preferably eggs and sperm
should be shipped separately with plenty of ice and a blanket
of pure oxygen on the sperm. Unfertilized eggs and semen can
be transported for a period of 24 hours after stripping if they
are kept in an air-tight container . (1) Using a 0.75 percent
saline solution when mixing eggs and semen may facilitate
fertilization,
Eggs can 'be obtained from channel catfish, fathead minnow,
sheepshead minnow, and bj.uegi.ll by facilitating natural spawning
either in the laboratory or a brood pond.(lf 3, 4, 5, 6}
4 . Embryo Exposure (Test B_eg_i_ns_)_
can be either fertilized prior to toxicant exposure
or fertilized in the test solution. Verification of the precise
embryonic stage at the beginning of the exposure should be
attempted if possible.
to/initiate Jrfie study shouldx&e at the &ysd st^ge,
and E&ficted from a group^of which 70% ate fertilizer;
A minimum of 20 embryos are randomly selected per replicate
cup with four replicates per concentration (80 embryos total) . (1)
Cups containing embryos are placed into the exposure chambers,
Water may flow directly over the embryos in the cup or the cups
may be oscillated in the test solution by means of rocker arm
apparatus driven by a low speed electric motor,
Embryos should be 2 to 24 hours old at the beginning of
tfce_te.|5t:;L> Twenty-four hours alter being placed in the incuba-
/tTon c~ups they should be counted and examined for dead or
j heavily fungused individuals, which should be discarded without
disturbing the viable embryos . ( 2} This counting and examination
is repeated on a daily basis. The range of time-to-hatch in
each .cup . is .species specific as noted in table 1.
Cu-e_. pLF ^ -y- xjn U.^ 44, a.
HAS, old, Sc^orvic) eVu-yCxS j^ay JJKL,
'^ ^ JD daa
h,
- -W,- -te ••
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c
Table 1. Average Time-to-Hatch for Several Species of Fish,
Relative to Temperature(1)
Species
Rainbow trout (Salmo gairdneri)
Brook trout (Salvelinus fontinalis)
Coho salmon (Oncorhynchus k_isutch)
Brown trout (Salvelinus trutta)
Lake trout (Salvelinus namaycush)
Chinook (Oncorhynchus tshawytscha)
Northern pike (Esox lucius)
Bluegill (Lepomis macrochirus)
Fathead minnow (Pimephales promelas)
Channel catfish (Ictaluras punctatus)
Sheepshead (Cyprinodon variegatus)
Silverside (Menidia menidia)
Temperature Days-to-Hatch
10 °C
10 °C
10 °C
10 °C
7 °C
10 °C
15 °C
28 °C
25 °C
26 °C
30 °C
25 °C
25 °C
31
44
55
41
72
56
6
6
5
6-7
4
7
8
5., Post Hatch, Larval Fish
When hatching is about 90 percent completed or 48 hours
after first hatch, live young fish should be counted.(1) All
of the normal and abnormal live fish should be released into
the test chambers.(1) Fish numbers can be thinned to at least
30 per treatment.(1) A test should be terminated if the average
percent of embryos (based on the number of embryos after
thinning) that produce live fry for release into the test
chambers in any control treatment is less than 50 percent or if
the percent hatch in any control embryo cup is more than 1.6
times that in another control embryo cup.(l, 3}
Test fish over two days old (post hatch swim-ups) must be
fed live newly hatched brine shrimp.
Fish should
feedings will be
be fed at least
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meaningful endpoint.
twice daily. Time between
species specific, and must be based on a
Control and treatment
of food if growth
teedings will be species specitic,
reliable hatchery feeding schedule.
fish must receive equal amrmni-c- r\f
«.*
amounts
is to be a
Dead fish should be removed and recorded when/observed.
At a" minimum, the live fish should be counted (including those
which are lethargic or grossly abnormal in either swimming
behavior or physical appearance) 11, 18, 25, and 32 days after
hatching.(1)
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Fisb should not be fed for at least 24 hours prior to
termination on day 32. At termination, all live fish should be
weighed (wet, blotted dry).
6. Controls
A test is not acceptable if the average survival of the
controls at the end of the test is less than 80 percent or if
survival in any control chamber is less than 70 percent.fi)
The relative standard deviation -(RSD = 100 x standard deviation
divided by mean) of. weights of the fish that were alive at the
end of the test in any control test chamber must not be greater
than 40 percent. (1) , _
A negative control (no toxicant or carrier) and a carrier.
control (when applicable) are required, Regardless of the
carrier used, the carrier concentration should be equal in each
exposure concentration and carrier control. If they are not,
the carrier concentration in the control (carrier) must be at
least as high as that in any toxicant test chamber.
7, Data Endpojnts
A record of the results of an acceptable test must include
the number of erobryos hatched, -tine to hatch, mortality of
embryos, larvae, and juveniles, time to swim-up, measurement of
growth, incidence of pathological "or histological effects, and
observations of other effects or clinical signs in each treat-
ment, Endpoints defined in terms of statistically significant
differences and biologically significant differences are based
on contingency table, or other hypothesis testing procedures
and regression analysis, concentration-effect curve analysis,
and other estimation procedures. (1} Tests for chamber to chamber
heterogeneity within treatments are generally based on analysis
of variance or contingency table procedures , (1)
B . Ph y s i c a 1 S y sj: en?
1 . Te s t_ Wa t e r_
. a . Saltvater
1) "*est water may be natural (sterilized ancl filtered to
remove particles 15 mierons and- larger) or a commercial mixture
(provided that there are no adverse affects to test organisms
or alterations in test material toxicity) ; 2} Natural seawater
is considered to be of constant quality if the weekly range of
salinity is .less than six percent, and if monthly pH range. is
less than 0.8 of a pH unit; 3)* Salinity should -be >_ 15 parts
per thousand; 4) Water must be free of pollutants. (7} Use of
ultraviolet light irradiation is recommended to sterilize the
test water.
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b. Freshwater Fish .
1) Test water can be supplied from a well or-spring provided
that the source is not polluted; 2) Water should be sterilized
with ultraviolet irridiation and tested for pesticides, heavy
metals, and other possible contaminants; 3) Hardness of 40 to
4-8 mg/L as CaCO3 and pH of 7.2 -to 7.6 is recommended;
4) Reconstituted water can be used. Detailed descriptions of
acceptable procedures for preparing diluent are found in the
protocols by the American Society of Testing Materials (1980).(2)
2. Temperature . . .
x. ~-—~~
Test temperature depends upon the test species and should
not deviate by more than 2°C from the appropriate temperature
(refer to section A, 4).
3-. Photoperiod '
A photoperiod of 16L/8D can be used with a light intensity
of 400 to 800 Lux at the surface 'of the test'solution. However,
in -general salmonid eggs should be incubated under dim lighting
(< 20 ft-candles) or total darkness. (1)
4 . Dosing Apparatus. -
Intermittent-flow proportional diluters as described by
Mount and Brungs (8) or continuous-flow serial diluters, as
described by Carton (7) should be employed. A minimum of five
toxicant concentrations with a dilution factor not greater than
0.50 and controls should be used.
5. Toxicant Mixing -
A mixing chamber is recommended to assure adequate mixing
of test material. Aeration should not be used for mixing.
Separate flow splitter delivery tubes should run -from this
container to each replicate larval tank.(3) Depending upon the
apparatus used, a mixing chamber may not be required/ but it
must be demonstrated that the -test solution is completely mixed
before introduction into the test system. Flow splitting
accuracy must be within 10 percent and should be checked
periodically for accurate distribution of test water to each
tank.(1)
6. - Test' Vessels- - *
All test tanks should be of either all glass' or glass with
a stainless steel frame. Exposure vessels will vary in size
according to the species under test. Generally, it is desirable
to have a depth of water of at least 15 to 30 cm.(1)
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7. Embryo Cups
Embryo incubation cups should be made from 120 mL glass
jars with the bottoms replaced with 40 mesh stainless steel or
nylon screen. Cups can be oscillated vertically (2.5 to 4.0
cm) in the test water (rocker arm apparatus, 2 rpro motor) or
placed in separate chambers with self-starting siphons. Both
methods should insure adequate exchange of water and test
material.
8. Flow Rate
Plow rates to larval cups should provide 90 percent
"replacement in 8 to 12 hours, (3) Flow rate must be capable of
maintaining dissolved oxygen at above /5 percent of saturation
and maintain the toxicant level (concentration cannot drop
below 20 percent with fish in the tank).
9, Aeration
Dilution water should be aerated vigorously insuring that
dissolved oxygen concentration will be at or near 90 to 100
percent saturation. Test tanks and embryo cups should not be
aerated.
C. Chemical System
1. Concentrations
A minimum of five concentrations of toxicant and a
control, (all replicated) are used in this chronic test. A
solvent control is added if a solvent is utilized. At a
minimum, the concentration of toxicant must be measured in
one tank at each toxicant level every week. Water samples
should be taken about midway between top and bottom and -the
sides of the tank. One concentration selected must adversely
affect a life-stage and one concentration must not affect any
life-stage.
2. Measurement of Other variables
Dissolved oxygen must be measured at each'concentration
at least once a week. Freshwater parameters in a control and
one concentration must be analyzed once a week. These
parameters should include pH, alkalinity, hardness, and
conductance. Natural seawater must maintain a constant
salinity and not fluctuate more than six percent weekly or a
monthly pH range of less than 0.8 of a pH unit.
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3. Solvents
If solvents other than water are necessary, they should be
used sparingly and not to exceed 0.1 mL/L in a flow-through
system. The following solvents are acceptable:(2)
dime thy Iforntamide
triethylene glycol
methanol
acetone
ethanol
The development of chemical saturators for use with hydro-
phobic chemicals may be used with most test chemicals.(6, 9)
D.'. Calculations • •
Data from these toxicity studies are of two types,
continuous (i.e., length, weight) and discrete (i.e., number of
fish hatching or surviving). In general, continuous data
should be analyzed with the appropriate analysis of variance
(ANOVA) technique followed by an appropriate multiple comparison
test. Dichotomous data should be analyzed using some form of a
2x2 contingency table.
As a part of the ANOVA, it is desirable to plot the
residuals versus concentration and determine whether there have
been any obvious violations of homoscedasticity on the assumption
of normality. All test results must be accompanied by copies
of the original (raw) data for the reviewer's evaluation.
Transcripts of the original raw data may be submitted if they
provide all of the information available in the original,
including comments or notes of the investigator.
III. REVIEWER'S EVALUATION
The reviewer should identify each aspect of the reported
procedures and determine if there is any inconsistency with
recommended methodologies. The number of deviations and their
severity will determine the validity of the study and the
interpretation of the results.
A. Verification of Statistical Analysis '
Reviewer should ensure that a maximum allowable toxic
concentration (MATC) has been properly derived by recalculating
the reported "results. If the recalculated results differ
substantially from the submitted results, the reviewer should
no.te this and attempt to explain the differences.
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B. Conclusions
1. Categorization of Results
The significance of inconsistencies in the test procedures
must be determined by the reviewer so that the results of the
test can be categorized as to whether they fulfill Part 158
regulations and are useful in performing a risk assessment.
Categories are described as:
° Core; All essential information was reported and the
study was performed according to recommended protocols.
Minor inconsis.tencies with standard methodologies may
be apparent; however, the deviations do not detract
from the study's soundness or intent. Studies within
this category fulfill the basic requirements of current
guidelines and are acceptable for use in a risk
assessment.
0 Supplemental: Studies in this category are scientifi-
cally sound; however, they were performed under condi-
tions that deviated substantially from recommended
protocols. Results do not meet guideline requirements;
however, the information may be useful in a risk
assessment.
Some of the conditions that may place a study in a
supplemental category include:
— Unacceptable test species;
inappropriate test material; or
Deviations from recommended test solution charac-
teristics (variations in DO, temperature, hardness,
and pH can affect toxicological response),
0 Invalid; These studies .provide no useful information.
They may be scientifically unsound, or they were
performed under conditions that deviated so significantly
from recommended protocols that the results will not be
useful in a risk assessment.
Examples of studies placed in this category commonly
include those where the test system was aerated, test
vessels were constructed from materials other than
glass, or there were problems of solubility or volatility
of the test material. Unless acceptable chemical.
analyses of actual toxicant concentrations were performed
in studies such as these, the reviewer cannot be sure
that test organisms were actually exposed to nominally
designated concentrations.
A study where the test material was not properly
identified can also be'invalidated,
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2. Rationale
Identify what makes the study supplemental or invalid.
While all- deviations from recommended protocol should be noted,
the reviewer is expected to exercise judgment in the area of
study categorization.
'3. Reparability , ,. .
Indicate whether the study may be upgraded or given a
higher validation category if certain conditions are met.
Usually this would involve the registrant submitting more data
about the study. . v
' ^* Descriptive Conclusions ' . '
The reviewer should indicate what' the results were and
how much, information can be drawn from them. - These results are
useful in a risk assessment. • • . .
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REFERENCES
(I) ASTM Standard E 729-80, Practice n,r Conducting Acute
Toxicity Tests with Fishes, Mac* " ! iw^r tebrates and
Amphibians. American Society fo«- Tent-inn Br1j L .. . ,
1916 Rac* street, Philadelphia, PA 19103? ^ Materials,
(2} Benoit, D.A. (1981) User's
Chronic Toxicity Tests with
promelas). Environ. Res. Lab.-m, j „,.,
600/8-81-011,
(3) Hansen, D.J.; Parrish, P.R. ; Sch iltltflfl,
(1978) Toxlclty Test Using Sheet,,hq-
var j.egatus) . Bioassay Procedure^ r0r I-HB noo3» ni^^L^ii
Permit Program. EPA-600/9-78-OK,. °r the °Cean DlsP°sal
(4) Smith, W.E. (1976) Larval Feed 1 ny and Maturation
of Bluegill in the Laboratory. fro,. ri=h-riii*- TR-
95-97 pp. cult. j».
(5) Sneed, K.E, , H.P. Clemens (1960) ,,8ft f pi h PituitarieB
to induce Spawning in Channel CatfUh" u S Pish and
Wildlife Service, Washington, DC, %«cial scientific
Report - Fisheries No. 329. 12 pp.
(6) Garten, R.R. (1980) A Simple Cont Inuou.-Plow Toxicant
Delivery System. Water Res. i^-y,Ji7-y^
(7) Mount, D.I , Brungs, w.A. (1967) A gimplified Dosi
Apparatus for Fish Toxicology Stu
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