United States Office of Pesticides Programs EP/W540/9-86-137
Environmental Protection Washington, DC 20460 ju|y igg0
Agency
vvEPA Hazard Evaluation Division
Standard Evaluation Procedure
Fish Life-Cycle Toxicity Tests
-------
HAZARD EVALUATION DIVISION
STANDARD EVALUATION PROCEDURE
FISH LIFE-CYCLE TOXICITY TESTS
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, D.C. 20460
-------
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.
tfohn W. Melone, Director
Hazard Evaluation Division
-------
TAELE OF CONTENTS
Pa9e
I. INTRODUCTION
A. When Required 1
B. Purpose 1
C. Test Material 1
D. Acceptable Protocols 1
II. MATERIALS, METHODS, AND REPORTING REQUIREMENTS
A. Eiological System 2
1. Acceptable Species 2
2. Source and Acclimation of Fish 2
3. Eggs from Adult Fish 2
4. Feeding 3
5. Embryo Removal 3
6. Embryo Exposure (Four-Five Days) 3
7. Larval-Juvenile Exposure (Eight Weeks) . 4
8. Juvenile-Adult Exposure (32-40 Weeks) .. 4
9. Second Generation Embryo Exposure
(Four-Five Days) 4
10. Second Generation Larval-Juvenile
Exposure (Four-Eight Weeks) 5
B. Physical System 5
1. Test Water 5
a. Sheepshead Minnow 5
b. Fathead Minnow 5
2. Temperature 5
a. Fathead Minnow 5
b. Sheepshead Minnow 6
3. Photoperiod 6
a. Sheepshead Minnow 6
b. Fathead Minnow 6
4. Dosing Apparatus 8
5. Toxicant Mixing 8
6. Test Tanks 8
a. Fathead Minnow 8
b. Sheepshead Minnow 8
7. Embryo and Fry Chambers 8
8. Flow Rate 9
• 9. Aeration 9
C.. Chemical System 9
1. Concentrations 9
2. Measurement of Other Variables 9
3. Solvents 9
D. Calculations 10
-------
TABLE OF CONTENTS (Continued)
Page
III. REVIEWER'S EVALUATION
A. Verification of Statistical Analysis 10
B. Conclusions 10
1. Categorization of Results 10
2. Rationale 11
3. Reparability 11
4. Descriptive Conclusions 11
REFERENCES 12
-------
FISH LIFE-CYCLE TOXICITY TESTS
I. INTRODUCTION
A. When Required
The fish life-cycle test is designed to evaluate risk from
chronic pesticide exposure to fish reproduction and other life
stages. This study is required when an end-use product is
intended to be applied directly to water or is expected to
transport to water from the intended use site, when any of the
following conditions apply:
° If the estimated environmental concentration is equal
to or greater than one-tenth of the no-effect-level in
the fish early life-stage or invertebrate life-cycle
test; or
° If studies of other organisms indicate the reproductive
physiology of fish may be affected.
B. Purpose
° To establish chronic toxicity levels of the active
ingredient to non-target fish;
° To compare toxicity information with measured or estimated
pesticide residues in an aquatic environment to assess
...potential impact "to fish;
0 To provide support for precautionary label statements;
and
° 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 Protocols
^Ecological Effects Branch (EEB) does not endorse any one
prptocol. It is sometimes necessary and desirable to alter the
procedures presented in published protocols to meet the needs
ofJ.the chemical or test organism used.. However, 1 EEB dbes
recommend some protocols as guidance for performing a fish
life-cycle toxicity test. These protocols include:
-------
-2-
Benoit, D.A. (1981) User's Guide for Conducting Life-Cycle
Chronic Toxicity Tests with Fathead Minnows (Pimephales
promelas). Environ. Res. Lab.-Duluth, Duluth, MN. EPA
600/8-81-011.
Hansen, D.J., Parrish, P.R., Schimmel, S.C., and Goodman, L.R.
(1978) Toxicity Test Using Sheepshead Minnows (Cyprinodon
variegatus). Bioassay Procedures for the Ocean Disposal
Permit Program. EPA-600/9-78-010.
American Public Health Association, American Water Works
Association and Water Pollution Control Federation (1985)
Standard Methods for the Examination of Water and Wastewater.
Sixteenth Edition. Publication Office: American Public
Health Association, 1015 18th Street NW, Washington, DC
20036. 854 pp.
These reference protocols are presented as flexible guidance to
help researchers design scientific protocols and to help the
reviewer validate studies.
II. MATERIALS, METHODS, AND REPORTING REQUIREMENTS
A. Biological System
1. Acceptable Species
The preferred test species are fathead minnow (Pimephales
promelas) and sheepshead minnow (Cyprinodon variegatus).
2. Source and Acclimation of Fish
Adult fish are obtained from either wild populations or
suitable culture laboratories. Sheepshead minnows.are to be held
in a flowing 30°C seawater of 15 percent salinity for at least
two weeks prior to breeding.(1) Fathead minnows should be main-
tained at 25°C and a constant 16-hour day-light photoperiod
(embryos will mature in five to .six months under ..these-conditions) .
Neither species of fish or eggs (embryos obtained from well-esta-
blished culture units as found at the Environmental Research es
Laboratory in Duluth, MN) should exhibit excess mortality.(2)
3. Eggs from Adult Fish
^ ^Artificial;inducement and natural spawning are.the:two methods
for obtaining a sufficient number jof eggs ; for ;a.;chrohic-exposure.
Artificial inducement. entails the stimulation.: of >egg production
by injection of human gonadotrophic hormone. -Sheepshead minnow
females-can be injected intraper itoneally with; five .IU-.HCG. pn3two
consecutive days. Two days following the second.injection, ova
from females are stripped and mixed with sperm derived from
excised macerated testes. Usually ten females and five males
should be used.(l)
-------
-3-
Natural spawning is possible with a few considerations for
each fish species. Sheepshead minnow embryos are obtained by
combining five or more females and three males in spawning
chambers measuring 20 x 35 x 22 cm. Mature adults should attain
a minimum standard length of 2 > mm and display courtship charac-
teristics (sexual dimorphism, territoriality, and aggressive
behavior by the male). Fish from each spawning group are left in
chambers for a minimum of 14 days. (2) Fathead minnows require
paired spawning in order to eliminate fighting and competition.
Culture units for this fish can consist of one tank measuring
30.5 x 30.5 x 61 cm with a water depth of 18 cm and four individual
spawning chambers (15.2 x 30.5 cm) formed by stainless steel
screen dividers (5 mesh, 0.89 mm wire).(2)
Adult deaths during spawning should be noted; dead animals
are removed, but not replaced. At termination of each spawning
group, lengths and weights of individual fish are measured.
4. Feeding
Fry of both fish species should be fed equal portions of live
brine shrimp nauplii at least two times daily about six hours
apart for three weeks (frozen nauplii are not to be used).
Juveniles (four weeks posthatch) and adults can be fed twice
daily on equal portions of dry food (e.g., Tetramin® or BiOrell)
supplemented with frozen adult brine shrimp. Each batch of food
should be checked for pesticides and metals.
5. Embryo Removal
A record of numbers and egg fertility must be maintained
daily. All embryos are examined daily with a dissecting scope.or
magnifying viewer to remove empty shells and opaque, or abnormal
appearing embryos. If less than 50 percent of the embryos from
a spawn appear to be healthy and fertile, all embryos from that
spawn should be discarded. (2) Embryos should be removed at a
fixed time each day so spawning activity is not disturbed
unnecessarily.
6. Embryo Exposure (Four-Five Days)
The life-cycle chronic toxicity test must begin with embryos
from at least three separate spawnings that are < 24 hours old
and have soaked in dilution water for at least two hours. (2)
Testing begins by randomly distributing 50 embryos to each of the
four replicate larval growth chambers.(2) Ten embryos are trans-
ferred with a large bore eye dropper to successive incubation
cups which are standing in dilution water." This is repeated
until 50 embryos are in each cup. The incubation cups are then
distributed to each replicate larval chamber.
-------
-4-
Survival of embryos, time required to hatch, hatching
success, and survival of fry for four weeks are determined and
recorded. Dead embryos usually turn opaque and must be counted
and removed each day until hatching is complete. Live fungused
embryos must be removed daily and counted as dead.(2)
7. Larval-Juvenile Exposure (Eight Weeks)
After hatching, each group of larvae is randomly reduced
to 25, and released in replicate larval growth chambers.(2)
This random selection must include any fish that are lethargic
or deformed. Survival should be determined in each replicate
growth chamber at least once a week. Survival during this
period is determined by counting the number of live fish, since
dead larvae deteriorate rapidly.
At four and eight weeks after hatching, total lengths (mm)
of all fish must be recorded.(2) Techniques suggested for
measuring fish include direct measurement and a photographic
method outlined by McKim and Benoit (1971).(3) In order to
treat growth as a valid endpoint, the amount of food given to
the control and treated fish must be kept constant between
exposures.
8. Juvenile-Adult Exposure (32-40 Weeks)
All fish are transferred to the adult spawning tank (samei:
concentration) eight weeks after hatching.(2) Each tank should
have 25 randomly selected fish (deformed individuals included).
When secondary sexual characteristics are well-developed,
fathead minnow (20-24 week post hatch) males will exhibit
tubercles, pads and body color, while females will exhibit
extended transparent and canals (urogenital papilla) . At this.£&
time, mature fish should be placed in spawning tank, separate;"
f.rom undeveloped fish. (2) The spawning tank will be divided-
into four individual spawning chambers with appropriate spawning
substrates. Four males and four females are randomly chosen
and assigned to spawning chambers. Substrates are examined
daily and embryos removed, counted, and recorded separately for
each pair.
. The:adult exposure (fathead minnow) should be terminated
when, during the decreasing day-length photoperiod, a one-weekne
period passes in which no spawning occurs.(2) Testing usingJ?sT
sheepshead minnows should terminate after spawning is observecLH
for two weeks because this fish spawns readily and almost dailyi
unless immature or affected by a-pollutant.(1) "
-------
-5-
9. Second Generation Embryo Exposure (Four-Five Days)
Fifty embryos from each concentration level are randomly
selected and transferred to incubation cups for hatch. Those
embryos not selected are discarded. Test procedures used during
embryo removal and embryo exposure (sections A, 5 and A, 6,
respectively) are repeated with second generation embryo
exposure.(1, 2)
10. Second Generation Larval-Juvenile Exposure
(Four-Eight Weeks)
Eight week exposure begins with the release of two groups
of 25 larvae in replicate growth chambers. These larvae should
have been produced from different breeding pairs in each spawning
tank. Selection of each group should be from early spawnings.
(1, 2) Testing procedures are the same as those described in
section A, 7.
Each group of second generation fish is terminated eight
weeks after hatching. Fish are blotted, weighed, and measured
before being discarded.(1, 2)
B. Physical System
1. Test Water
a. Sheepshead Minnow
1) Test water may be natural (sterilized and filtered to
remove particles 15 microns 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 sterilized and free of
pollutants. (1) Irradiation with ultraviolet light is recommended
to sterilize test water.
b. Fathead Minnow
1) Test water can be supplied from a well or spring
provided that the source is not polluted; 2) Water should be!
sterilized with ultra violet irradiation and tested for pesti-
• - ~ on
cides, heavy metals, and other possible contaminants; 3) Hardness
of 40 to 48 mg/L as CaC03 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) '. (4)
-------
-6-
2. Temperature
A continuous record of temperature of test water must be
kept.
a. Fathead Minnow
Temperature should be maintained at 25°C and should not remain
outside the range of 24 to 26°C for more than 48 hours.(2)
b. Sheepshead Minnow
Temperature should be maintained at 30°C.(1)
3. Photoper iod
Lighting above each replicate must be balanced and must
simulate the wavelength spectra of sunlight. Light intensities
at the water surface should range from 10 to 100 lumens. One
lumen per square meter is equal to one lux.
a. Sheepshead Minnow
A 16-hour light/8-hour dark cycle is maintained throughout
the test. (1)
b. Fathead Minnow
A 16-hour light/8-hour dark cycle is maintained throughout
the test.
4. Dosing Apparatus
Intermittent-flow proportional diluters as described by
Mount and Brungs(5) or continuous-flow serial diluters, as
described by Garton(6) should be employed. A minimum of five
toxicant concentrations with a dilution factor not greater than
0.50 and one control 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 and adult tank.(2) Depending-
upon' the apparatus used a mixing chamber may not be required ;;^0
It must, however, be demonstrated that the test solution is "-10
completely mixed before introduction into the test systenf.-
Flow splitting accuracy must be within 10 percent and should" be
checked periodically for accurate distribution of test water'to
each tank.(2)
-------
-7-
6. Test Tanks
All test tanks should be of either all glass or glass with
a plastic or stainless steel frame.
a. Fathead Minnow
Adult spawning tanks should measure 30.5 x 30.5 x 91.4 cm
or 30.5 x 30.5 x 61 cm long with a screened-off or separate
larval tank. (2) Each larval section is divided in half allowing
for two larval growth chambers for each adult spawning tank.
Larval chambers should be designed with glass bottoms and
drains that allow water to be drawn down to 3 cm. (2) Test
water must be delivered separately to each adult tank and
larval section, with one-third of the water volume going to the
latter. Larval tanks can also be conveniently located directly
above spawning tanks containing test solutions of the same
concentrations so they can be drained directly into the spawning
tank. Test water depth in adult tanks and larval chambers
should be a minimum of 15 cm.(2)
b. Sheepshead Minnow
Tanks 45 x 90 x 26 cm with a water depth of 19 cm have
been successful. Larval chamber design and test water divided
are the same as described for fathead minnow.(1)
7. Embryo and Fry Chambers
Embryo incubation chambers should be made from 120 ml
glass jars with the bottoms replaced with 40 mesh stainless
steel or nylon screen. Chambers can be oscillated vertically
(2.5 to 4.0 cm) in the test water (rocker arm apparatus, 2 rpm
motor) or placed in separate chambers with self-starting siphons.
Both methods should insure adequate exchange of water and test
material.(1,2)
8. Flow Rate
Flow rates to adult tanks or larval chambers should provide
90 percent replacement in 8 to 12 hours. (2) Flow rate must be
capable of maintaining dissolved oxygen at above 75 percent of
saturation and maintain the toxicant level (concentration
cannot drop below 20% 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 chambers should not
be aerated. (1, 2)
-------
-8-
C. Chemical System
1. Concentrations
A minimum of five concentrations of toxicant and a control
(all duplicated) are used in this chronic test. A solvent
control is added if a solvent is utilized. As 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. (2)
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:(4)
dimethylformamide
triethylene glycol
methanol
acetone
ethanol
The development of chemical saturators for use with
hydrophobic chemicals may be used with most test chemicals.(7,
8, 9)
D. Calculations
Data from these toxicity studies are of two types,
continuous (i.e.-, length, weight) and discrete ..(i .e ., number _o.f
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
2 x :2^contingency table.
-------
-9-
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 the
original (raw) data for the reviewer's evaluation.
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 c
oncentration {MATC) has been properly derived by recalculating
the reported results. If the recalculated results differ
substantially from the submitted results, the reviewer should
note this and attempt to explain the differences.
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 inconsistencies 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.
° Supplemental: Studies in this category are scienti-
fically sound; however, they were performed under
conditions that deviated substantially from recommended
protocols. Results do not meet guideline requirements;n
however, the information may be useful in a risk
assessment.
-------
-10-
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).
° 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."
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.
4. 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.
-------
-11-
REFERENCES
(1) Hansen, D.J.; Parrish, P.R.; Schimmel, S .C. ; Goodman, L.R.
(1978) Toxicity Test Using Sheepshead Minnows (Cyprinodon
variegatus). Bioassay Procedures for the Ocean Disposal
Permit Program. EPA-600/9-78-010.
(2) Benoit, D.A. (1981) User's Guide for Conducting Life
Cycle Chronic Toxicity Tests with Fathead Minnows
(Pimephales promelas). Environ. Res. Lab.-Duluth, Duluth,
MN. EPA-600/8-81-011.
(3) McKim, J.M.; Benoit, D.A. (1971) Effect of long-term
exposures to copper and survival, reproduction and growth
of rainbow trout (Salvelinus fontinalis). J. Fish. Res.
Board Can. 28:655-662.
(4) ASTM Stnadard E 729-80, Practice for Conducting Acute
Toxicity Tests with Fishes, Macroinvertebrates, and
Amphibians. American Society for Testing and Materials,
1916 Race street, Philadelphia, PA 19103.
(5) Mount, D.I.; Brungs, W.A. (1967) A simplified dosing
apparatus for fish toxicology studies. Water Res. 1:21-
29.
(6) Garton, R.R. (1980) A simple continuous-flow toxicant
delivery system. Water Res. 14:227-230 pp.
(7) Chadwick, G.C.; Kiigemagi, U. (1968) Toxicity evaluation
of a technique for introducing dieldrin into water.
J. Fish. Res. Board Can. 40:76-82.
(8) Gingerich, W.H.; Seim, W.K.; Schonbrod, R.D. (1979) An
apparatus for the continuous generation of stock solutions
of hydrophobic chemicals. Bull. Environ. Contam. Toxicol.
23:685-689.
(9) Veith, G.D.; Comstock, V.M. (1975) Apparatus for
continuously saturating water with hydrophobic organic
chemicals. J. Fish. Res. Board Can. 32:1849-1851.
------- |