EPA/600/3-91/063
December 1991
GUIDELINES FOR CONDUCTING EARLY LIFE STAGE TOXICITY TESTS
WITH JAPANESE MEDAKA (ORYZIAS LATIPES)
BY
DUANE A. BENOIT, GARY W. HOLCOMBE, AND ROBERT L. SPEHAR
U.S. ENVIRONMENTAL PROTECTION AGENCY
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
DULUTH, MINNESOTA 55804
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804
Printed on Recycled Paper
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NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
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CONTENTS
Page
Foreword ^
Abstract 5
1. Physical System
1.1 Diluter 6
1.2 Toxicant mixing and flow splitting 6
1.3 Test tanks 6
1.4 Embryo incubation cups 7
1.5 Dilution water 8
1. 6 Loading and flow rate 8
1.7 Aeration 8
1.8 Test water temperature 9
1.9 Photoperiod and lighting 9
1.10 Cleaning 9
1.11 Disturbance 10
1.12 Construction materials 10
2. Chemical System
2.1 Preparing a stock solution 11
2.2 Measurement of toxicant concentration 11
2.3 Measurement of other variables 12
2.4 Residue analysis 12
3. Biological System
3.1 Source of test fish 13
3.2 Preliminary tests 13
3.3 Obtaining embryos . 14
3.4 Embryo filament removal 16
3.5 Embryo exposure 17
3.6 Embryo hatch 18
3.7 Larval exposure 19
3.8 Calculation of results 20
References 22
3
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FOREWORD
In recent years the Japanese Meclaka, Oryzias latipes. has become an
important test animal for use in evaluating carcinogenesis. A review of the
literature by Couch and Harshbarger (1.) revealed that the medaka are known to
be quite susceptible to some classes of chemical carcinogens. Hawkins, et al.
(2) have also demonstrated the development of chemically induced tumors in
several medaka organs such as the liver, kidney, eye and muscle. Because of
the medaka's apparent sensitivity to chemically induced tumor development, its
short life cycle time, rapid growth and well known culture techniques, the
U.S. Environmental Protection Agency's Environmental Research Laboratory in
Duluth, Minnesota recently began a medaka chemical carcinogen screening
program. The USEPA at Duluth is also currently evaluating the medaka for use
in developing freshwater aquatic life criteria documents.
This procedural guide is based upon evaluations of published papers and
recent methods development: work conducted at the U.S. EPA laboratory in
Duluth, Minnesota. The purpose of this report is to provide information and
basic guidelines for conducting an early life stage toxicity and/or carcinogen
test with medaka. This test starts with 24 hour old embryos and ends 4 weeks
after hatch. The guideline also describes recently developed methods for
shortening the hatch period and methods for incubating embryos without the use
of chemical treatments to control fungus. It is also the intention of the
authors to make these procedures compatible with ASTM's Annual Book of ASTM
Standards: Guide for Conducting Early Life-Stage Toxicity Tests with Fishes,
(Volume 11.04; designation E1241-88). The report has been reviewed by the
Environmental Research Laboratory-Duluth, U.S. Environmental Protection
Agency, and approved for publication. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
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ABSTRACT
This manual represents a procedural guide for conducting embryo-larval early
life stage (ELS) toxicity tests with Japanese medaka (Oryzias latipes). These
procedures are based upon evaluation of published papers and recent methods
development work conducted at our laboratory in Duluth. The published papers
are referenced in the appropriate places throughout the text of this report.
If more detailed information on test: apparatus or specific biological and
chemical methods is desired, the reader is encouraged to study the reference
material or contact one of the authors of this manual. All routine methods
not covered in this procedure (physical and chemical determinations), should
be followed as described in Standard Methods for the Examination of Water and
Wastewater (3). Those routines dealing with handling of fish not covered in
this procedure should be followed as described by ASTM (4).
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CHAPTER 1
PHYSICAL SYSTEM
1.1 Diluter
Intermittent-flow proportional diluters (5, 6) or continuous-flow serial
diluters (7, 8) should be used. The operation of the diluter should be
checked daily, either directly or through measurement of toxicant
concentrations. A minimum of five toxicant concentrations with a dilution
factor not greater than 0.50 and a control should be used for each test.
1.2 Toxicant Mixing and Flow Splitting
If a proportional diluter is used, a container to promote mixing toxicant and
diluent water should be used between the diluter and test tanks for each
concentration (9). Separate flow splitter delivery tubes should run from this
container to each replicate tank. If a continuous-flow serial diluter is
used, additional mixing containers are not: needed, but separate flow splitter
delivery tubes must run from the diluter to all test tanks. Delivery tubes
are allocated to tanks by stratified random assignment (random assignment of
one delivery tube for each, level of concentration in a row followed by random
assignment of the duplicate delivery tube for each level of concentration in
the other row). Flow splitting accuracy should be within 10% and should be
checked periodically to see that the intended amount of test water reaches
each tank.
1.3 Test Tanks
All test tanks should be made either of glass or stainless steel. Glass is
usually preferred because it is less expensive. Typically, duplicate test
tanks have been arranged in two rows back to back with test concentrations
assigned by stratified random assignment. Many sizes of test tanks have been
used successfully. Any tank size, shape and depth is acceptable if the flow
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rates and loading requirements can be achieved. One small test tank which has
been used routinely for medaka ELS tests is described as follows:
Up to sixty medaka (hatch-1 month old) have been tested in each tank
(duplicate tanks per treatment) that measured 133 x 178 x 127 mm high
containing 2000 mL of test water with a water depth of 85 mm and a flow rate
of 33 ml/minute. Water levels were controlled by a side drain in the end of
each tank. Side drains were covered with a removable 40 mesh (40 openings per
inch) cylindrical stainless steel screen to prevent loss of 1-10 day old fish
after which a 20 mesh cylindrical stainless steel screen was used to prevent
loss of 11-28 day old fish. It is also recommended that a circulating water
bath system be used with the test tanks so that temperature differences
between tanks can be held to within + 0.5 °C.
1.4 Embrvo Incubation Cups
Embryo incubation cups (Figure 1) are made from 20 ml glass scintillation
vials with the bottom portion cut away leaving a 4.5 cm upper portion to form
the cup. Sixty mesh Nitex screen is glued over the 1.5 cm ID mouth of the
vial. The bottomless vial is then inverted and a # 5 1/2 neoprene rubber
stopper with a 1 cm hole bored through it is inserted into the cut end of the
vial (2.5 cm ID). A 20 gauge stainless steel wire loop handle about 3.5 cm
high is shaped and inserted into the upper end of the stopper. During embryo
incubation the small cups are oscillated vertically 2.5-4.0 cm in the test
water by means of a rocker arm apparatus driven by a 10 rpm motor (Figure 2).
The combination of this size motor and cup configuration causes each embryo to
move up and down in the vial with a cascading rolling motion. Recent studies
at our laboratory have shown that this method of embryo incubation for medaka
greatly shortens the time to hatch period (mean test days to complete hatch @
28°C - 7.5; mean hatch - 96%). The method also significantly reduces the
number of fungused embryos throughout the incubation period as compared to the
embryo incubation system used for fathead minnows which utilizes a 2 rpm motor
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B
Figure 1. Medaka embryo incubation cup made from a 20 ml glass
scintillation vial (A) with 60 mesh Nitex screen (B) #5.5 rubber
stopper (C) with a 1 cm hole (D) and wire handle (E).
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drive (10). These studies have also shown no increase in larval mortality
and/or deformities at hatch.
1.5 Dilution Water
The water used should be from a well or spring, if at all possible, or from a
surface water source (4) Only as a last resort should dechlorinated water
from a municipal water supply be used.
1.6 Loading and Flow Rate
The loading in test tanks should not exceed 0.1 gram of fish per liter of test
water passing through the tanks in 24 hours (4). Flow rates to larger test
tanks (10-20 liter) should be at least 6-10 water volumes per 24 hours. Flow
rates, to the smaller test tanks (0.5 - 2.0 liter) should be at least 20-24
volumes per 24 hours. During a test, flow rates should not vary more than 10%
between any tanks. Flow rates should be great enough so that dissolved oxygen
does not drop below 60% of saturation (4) or toxicant concentrations drop by
more than 20% when fish are in the test tanks. Flow can be increased above
those specified rates to maintain proper dissolved oxygen or toxicant
concentrations. With a continuous flow diluter system delivering one tank
volume every hour, oxygen levels can be maintained above 75% in 2 liter volume
tanks containing 60 four week old medaka.
1.7 Aeration
Diluent water should be aerated vigorously (with oil-free air) or passed over
a screen column with a recirculating pump before flowing through the diluter.
Aeration of diluent water will eliminate supersaturation of dissolved gases
and also ensure that dissolved oxygen concentrations will be at or near
saturation (90-100%). However, the test tanks themselves should not be
aerated (aeration can alter toxicity by driving of volatile chemicals and it
is also difficult to aerate all tanks at exactly the same rate).
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1.8 Test Water Temperature
Test water temperature should not deviate from 28°C by more than +1°C and
should not remain outside the range of 26 to 30°C for more than 48 hours at a
time. At a minimum, temperature should be measured in one tank at each
toxicant concentration once a week, alternating between duplicate tanks from
week to week. Temperature should also be recorded daily in one control tank.
1.9 Photoperiod and Lighting
A 16-hour light and 8-hour dark photoperiod controlled by an automatic timer
should be used throughout the test in order to provide long daylight feeding
hours and to promote normal maturation. A 15-30 minute transition period
between light and dark is optional (11). Cool white fluorescent tubes have
been used successfully for embryo-larval tests and light intensities at the
water surface have ranged from 20-60 lumens (12). The intensity selected
should be duplicated as closely as possible for all test tanks. Another
option for lighting is a. 50:50 combination of Durotest (Optima FS) and wide
spectrum Grow-Lux fluorescent tubes.
1.10 Cleaning
Incubation cup screen bottoms should be cleaned periodically after embryos are
removed. Screens can usually be cleaned with tap water pressure, and/or a
small brush. After hatch is complete and larvae swim-up all tanks should be
carefully siphoned at least three times a week and scraped if algal or fungal
growth becomes noticeable. Siphoning should be done just before the last
feeding of the day.
Siphoning can be done safely with a large pipette (50 ml) fitted with a
squeeze bulb or with a siphon hose leading to a white dishpan. Fish which are
siphoned accidentally can be observed easily in the pipette or white pan and
returned carefully to the tank without harm.
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1.11 Disturbance
Fish should be shielded from excessive disturbances such as people walking
past the tanks or extraneous lights that might alter the intended photoperiod,
1.12 Construction Materials
Construction materials which contact the diluent water should not contain
leachable substances and should not absorb significant amounts of substances
from the water (4). Stainless steel and glass are the preferred construction
materials. Rubber and plastics containing fillers, additives, stabilizers,
plasticizers, etc., should not be used. Teflon, nylon, and their equivalents
are not known to contain leachable materials, nor do they absorb significant
amounts of test substances. All batches of neoprene stoppers should be
checked for toxicity prior to use in the diluter and exposure tanks. Static
tests at the U.S. EPA, Environmental Research Laboratory-Duluth, have shown
that certain lots of neoprene stoppers are acutely toxic to fathead minnow
larvae (S.J. Broderius, personal communication, U.S. Environmental Protection
Agency, Duluth MN 55804).
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CHAPTER 2
CHEMICAL SYSTEM
2.1 Preparing a Stock Solution
Distilled or diluent water should be used in making up the test stock
solutions. The development of several chemical saturators for use with
hydrophobic chemicals has eliminated the need to use carrier solvents with
most test chemicals (13-17).
If carrier solvents other than water are absolutely necessary, they should be
of reagent grade or better but the amounts used should be kept to a minimum.
Triethylene glycol (TEG) and dimethyl formamide (DMF) are preferred, but
methanol, ethanol or acetone can also be used. The calculated solvent
concentration to which any test organisms are exposed should never exceed 0.1
ml/liter.
When a carrier is used, use two sets of duplicate controls. One set should
contain no solvent and one set should contain the highest concentration of
solvent to which any organism in tha test is exposed (4).
2.2 Measurement of Toxicant Concentration
At a minimum, the concentration of toxicant in the test water should be
measured in one replicate at each toxicant; concentration every week,
alternating weekly between replicates. Water samples should be taken about
midway between the top and bottom and the sides of the tank and should not
include surface film or material stirred up from the bottom or sides of the
tank.
Methods described in Methods for Chemical Analysis of Water and Wastes (18)
and Manual of Analytical Methods for the Analysis of Pesticides in Human and
Environmental Samples (19) should be used when possible. Accuracy should be
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measured using the method of known additions for all analytical methods for
toxicants. Reference samples should be analyzed periodically for each
analytical method.
2.3 Measurement of Other Variables
Dissolved oxygen and pH should be measured at each concentration at least once
a week, alternating weekly between replicates.
A control, high and median test concentration should be analyzed at least
initially for alkalinity, hardness, conductance and turbidity to show the
variability in the test water. If any of these characteristics is affected by
the toxicant, that characteristic should be measured at each concentration at
least once a week and alternated between replicates. Alkalinity, hardness,
conductance and turbidity should be measured at least once a week in alternate
control replicates for the duration of the test.
2.4 Residue Analysis
After the ELS test is complete all surviving fish may be analyzed for toxicant
residues. Additional samples for analyzing toxicant residues can be measured
during the test, if desired, to determine the toxicant uptake curve. The
frequency and amount of samples taken will depend on the test objectives.
Since medaka usually are consumed whole by predators, whole body residues
should be reported.
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CHAPTER 3
BIOLOGICAL SYSTEM
3.1 Source of Test Fish
Sufficient numbers of embryos can be transported by express from a brood stock
culture unit to other laboratories or field sites to initiate an early life
stage test. These embryos should be shipped in well-oxygenated water in
insulated containers.
It is, however, recommended that a laboratory brood stock culture unit be
started at your facility by obtaining embryos from another well-established
culture unit such as is maintained at the Environmental Research Laboratory in
Duluth MN. Guidelines for the culture of several life stages of the medaka
can be obtained from the Environmental Research Laboratory in Duluth, MN (20).
At 27-28°C and a constant 16-hour day light photoperiod, fish fed unrestricted
quantities of live brine shrimp nauplii will mature in 3-4 months. With
proper care and maintenance these adult fish will produce quality embryos for
6-8 months (20).
3.2 Preliminary Tests
Selection of ELS test concentrations, a critical part of the experiment,
should be made using the utmost judgment and care. All embryos or larvae used
in preliminary and ELS tests should be taken from the same adult stock. As a
general rule, one should conduct a small scale 96-hour static range finder
test (five concentrations plus control) with ten 1-2 day old larvae in each of
six 250 ml beakers with 200 ml of test water in each beaker. Test beakers
should be covered (i.e., with parafilm) to slow evaporation and to reduce the
loss of volatile chemicals. Larvae need not be fed during the 96-hour test.
The selection of range finder concentrations (based upon previous toxicity
information and/or saturation concentration) should be spread out by as much
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as a factor of 10 depending on how much is known about the test chemical. If
the chemical in question is highly volatile and/or more than 20% is lost
during the range finder, then the test: may have to be conducted as a renewal
test or possibly even as a flow-through test. Throughout the 96-hour test,
all fish should be carefully observed so that not only mortality is noted but
also any behavioral effects such as abnormal swimming, surfacing, nervous
movements, or lethargy. Concentrations which produce definite behavioral
effects during the 96-hour larval test will usually cause significant survival
and/or growth effects during the longer ELS test.
Depending on how distinct the effects are from the range finder test, the
investigator may then either set up another static preliminary test with a
dilution factor of 0.5 or conduct a small scale 10-12 day flow-through
exploratory test (0.5 dilution factor, five concentrations plus control) with
ten 1-2 day old larvae (fed live brine shrimp). Either test should be based
upon the most sensitive indicator of stress observed during the range finder
test.
If mortality was the only observed effect during preliminary testing, then the
highest test concentration selected for the ELS test should be no less than
the 96-hour LC20 and no more than the 96-hour LC50 calculated from the
preliminary tests. If distinct behavioral effects were the most sensitive
indicators of stress then the highest test concentration selected for the ELS
test should be equal to that concentration.
3.3 Obtaining Embryos
It is desirable to have a medaka culture unit located on site so that the
cultures can more easily be managed to provide adequate embryos at the proper
time. Two days before the ELS test is to be started all spawning sponges (20)
in the culture unit are removed and cleared of embryos. Since medaka nearly
always spawn during the early daylight hours (21), these sponges should be
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cleared in the afternoon. Some females may still be carrying their egg clutch
from the morning spawning but to prevent injury to the brood stock it is not
recommended that these clutches be removed.
Maximum numbers of similar age medaka embryos (<24 hours old) can be obtained
for testing if the sponges are not replaced in the spawning tanks until the
very late evening hours (shortly before photoperiod controlled lights go out)
on the day before embryos are to be collected. By that time all of the
females carrying clutches from the previous day will have dropped them and
most of the females spawning during the early morning hours of that day will
also have dropped them. By using this technique, about 85-95% of the embryos
collected the following afternoon are less than 24 hours old.
The following afternoon all sponges with embryos are removed from the spawning
tank and suspended in a shallow white dishpan in flowing water (-50 ml/min @
27-28°C) (20). A rough estimate of total number of embryos is also made at
this time to determine if there are enough to start an ELS test (20). The
embryos are left to develop on the sponges overnight, and are removed the next
morning to start the test. This important step helps to reduce fungus growth
during embryo incubation. With the aid of a dissecting scope, 24 hour old
viable embryos (neurula stage) can easily be staged and counted. Nonviable
eggs and fungused, opaque or abnormal appearing embryos, as well as 48 hour
old embryos (optic lens and cup stage) laid by females which were still
carrying egg clutches when the spawning sponges were replaced in the tanks,
can also be identified and discarded. It has been our experience that medaka
embryo viability can be expected to be 95-98%.
Prior to staging each group of embryos, the long sticky filaments are removed
from the chorion of each embryo as described in the following section. If
filaments are not removed, embryos will be difficult to separate when staging
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and will clump together in the embryo incubation cup which will cause fungus
to grow on them before hatch.
3.4 Embryo Filament Removal
Recent methods development work conducted at our laboratory has provided a
simple apparatus and technique for the fast and safe removal of sticky
filaments from each embryo. The "embryo roller" apparatus is made from a wide
mouth 270 ml glass jar (72 mm OD) with four evenly spaced 5 mm deep notches
cut into the top, and the bottom cut off and replaced with soft Nitex screen
(approximately 90 mesh) attached with silicone glue (Figure 3). The modified
jar is then inverted with the notched mouth side down and placed in the bottom
portion of a glass petri dish (100 x 20 mm).
Embryos (200-300) from about 10-12 sponges at a time are gently picked off by
hand (index finger and thumb) and placed in a small round-bottomed glass or
stainless steel bowl containing diluent water maintained at ~28°C. Embryos
are then gently swirled around in the bowl. When the swirling motion slows
down, embryos can easily be siphoned up from the center of the bowl with a 50
ml pipette and squeeze bulb (pipette tip cut off and a 7 mm OD glass tube
inserted and glued to the inside of the pipette tube). Embryos are then
carefully transferred from the pipette to the 90 mesh screen on the embryo
rolling apparatus. Water transferred with the embryos will drain through the
screen and into the petri dish below. Embryos are then rolled around on the
screen for about two minutes with a gentle circular motion of the index finger
while pressing on them lightly. The motion and pressure of the finger is
almost identical to that described by Cast and Brungs (22) for removing
fathead minnow embryos from spawning substrates. The rolling action of the
embryos on the screen causes the filaments to begin sticking to one another
which in turn causes the filaments to begin breaking off from each rolling
embryo. Loose filaments form a sticky ball which quickly adheres to other
attached filaments and breaks them off, This method, developed for filament
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B
Figure 3. Medaka embryo roller for removal of long sticky filaments.
Embryo roller is made from a wide mouth 270 ml glass jar (A)
with 90 mesh Nitex screen (B) drainage notches (C) and a glass
petri dish (D) placed underneath.
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removal, has been shown to have no adverse effects on either embryo
development or hatch.
Embryos with filaments removed are then transferred for staging by simply
turning the embryo roller over and immersing embryos on the screen into a
water filled petri dish (100 x 20 mm). Filament balls can be easily picked
out of the petri dish with tweezers and discarded. After each group of 200-
300 embryos is staged, they should be pooled in a small round-bottomed bowl
containing 28°C diluent water.
3.5 Embryo Exposure (test day 0-9)
The test is started by counting and distributing a minimum of 40 embryos (-24
hours old) with a large bore eye dropper to one incubation cup standing in 27-
28°C diluent water and then randomly assigning that cup to a replicate test
tank. This process is repeated until all replicates have one incubation cup
suspended on the moving rocker arm apparatus.
Because of the medaka embryo's tendency to attract fungus rapidly, it is
necessary to gently flush out each group daily from the incubation cup into a
glass petri dish. Embryos are then examined with a dissecting scope and dead
or fungused embryos counted and discarded. Embryos can easily be flushed out
by inverting the cup at a sharp angle over the petri dish and slowly pouring
27-28°C diluent water through the bottom of the cup screen. The cup should
then be examined under a magnifier viewer light to make sure that all embryos
are out of the cup. Early fungus development can be more easily observed on
the embryos if an index card is placed over the stage light of the dissecting
scope so that the embryos can be viewed over the light along the edge of the
index card. The detracted light helps to make the early stages of fungus
development more visible.
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On test day 4 the embryos will be well developed and eyed-up (23); therefore,
on this day each group should be randomly reduced to a minimum of 20 per cup
after they are flushed out for their daily examination. This number may vary
depending upon the number of fish needed for examination at the end of the
test.
3.6 Embryo Hatch (test day 5-9).
With an incubation temperature of 28°C, embryos (with sticky filaments
removed) will normally begin to hatch on the fifth day of the test. It has
been our experience that most embryos that are incubated as described in
section 1.4 will hatch on days 6 and 7 of the test.
Hatched larvae from each cup should be counted daily after they are flushed
into the petri dish. Live fish that appear lethargic or show abnormal
swimming behavior or physical appearance should also be noted at this time,
but should not be discarded. All live larvae are then released into the test
tank, dead larvae are discarded and unhatched embryos returned to the cups
after inspection under the dissecting scope for fungal growth. Embryos not
hatched after ten days of exposure should be counted as dead and discarded.
It might, however, be necessary to extend the incubation period for a few days
if it is suspected that the toxicant is causing a delayed hatch. The average
exposure days-to-hatch (to the nearest day) in each cup should also be
recorded.
Recent medaka experiments at our laboratory, using the above embryo handling
techniques, have demonstrated that one can expect the normal mean percent
hatch to be about 95% (minimum 80%; maximum 100%); and the average normal test
days to complete hatch to be about 7.5 days (minimum 7; maximum 9). It has
also been determined upon close examination that those embryos which have not
hatched by test day 10 are either dead or in such a weakened condition that
they are unable to hatch. Further studies using the above methods by the
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authors have also shown that normal larval mortality and/or deformities at
hatch were consistently low, (mean = 0.3%, minimum 0%; maximum 2%).
3.7 Larval Exposure (mean hatch test day 7 - final test day 35).
Feeding methods for the medaka are generally the same as those used for the
fathead minnow (24). Larvae should be fed equal quantities of live brine
shrimp nauplii (e.g., Bio-Marine, San Francisco Bay, or Jungle Brand) within
1-2 days after hatching. Measured volumes of the nauplii in a concentrated
freshwater slurry are dispensed to each replicate with a small pipette (3-5
ml) fitted with a squeeze bulb. Quantities fed should be increased weekly as
the fish grow larger in order to insure that adequate food is available
throughout the daylight hours. The slurry must be mixed well between
replicate feedings to insure uniformity of volumes fed. Feeding schedules are
generally either twice a day about six hours apart or three times a day at
least four hours apart. Fish may, however, be fed double rations once a day
on weekends and holidays. Studies completed by the authors have shown that
brine shrimp are quite hardy and can remain alive for up to 5-6 hours in fresh
water. Each new lot of brine shrimp eggs should be analyzed for presence of
pesticides, before use.
Dead fish should be counted and removed daily throughout the exposure period.
Sometimes larvae which die (especially during the first 2 weeks -after hatch)
deteriorate so rapidly in the warm water that they cannot be easily observed
in the test tank; therefore, the final count of live fish at the end of the
test is used to calculate survival.
On exposure day 35 (28 days after mean hatch day), surviving fish in each
replicate are killed by placing them on ice for several minutes. The wet
weight of each individually blotted fish is then determined to the nearest
milligram. Total length also may be reported if desired. Make note of any
lethargic or any fish exhibiting abnormal movements, swimming behavior or
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physical appearance. Fish should not be fed during the last 24 hours of the
exposure period.
If fish are to be transferred to clean water for further studies such as
residue half-live studies or tumor development research, live fish can be
weighed as follows. Individual fish are blotted on paper toweling through a
small net and then are carefully dropped from the net into a container of
water which has been tared to zero on a balance. The balance is tared to zero
after each fish is weighed. If desired, total lengths of live fish can be
determined photographically as described by McKim and Benoit (25). An early
life-stage test with medaka is generally considered to be unacceptable when
the overall average survival in any control tank is less than 70%.
Recent medaka experiments at our laboratory using the above larval handling
techniques have demonstrated that one can expect the normal mean survival from
embryo thinning (test day 4) to 28 days after hatch to be about 90% (minimum
80%; maximum 98%). Results from these experiments also demonstrated that the
normal mean weight measured 28 days after hatch was 55 mg per fish (minimum -
47 mg; maximum 72 mg) with a standard deviation of + 7 mg.
3.8 Calculation of Results
The primary data to be analyzed from the medaka early life-stage test are
those on (a) percent survival in each replicate (may be analyzed as embryo
survival, larval survival and/or overall survival), (b) mean weight of
individual survivors in each replicate, and (c) mean chemical concentration of
the test solution in each treatment. Depending on the individual test
objectives and results, additional analyses may also be done to determine
toxicant effects on embryos, hatchability, deformities, total lengths and
other chemical factors such as residue uptake and measurements on temperature,
oxygen, pH, etc.
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The variety of procedures that can be used to calculate results of a medaka
early-life stage test can be divided into two categories: those which test
hypotheses, and those which provide point estimates (4). The calculation
procedures and interpretation of results must be appropriate to the
experimental design of the test. Alternative methods and points to be
considered when selecting and using procedures for calculating results of
early-life stage toxicity tests are discussed by ASTM (4).
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REFERENCES
1. Couch, J.A. and J.C. Harshbarger. 1985. Effects of caracinogenic agents
on aquatic animals: An environmental and experimental overview. Environ.
Carcinogenesis. Revs. 3(1). 63-105.
2. Hawkins, W.E., et. al. 1985. Development of aquarium fish models for
environmental carcinogenesis: Tumor induction in seven species. Journal
of Applied Toxicology. 5(4): 261-264.
3. American Public Health Association, American Water Works Association, and
Water Pollution Control Federation. 1985. Standard methods for the
examination of water and wastewater. 16th ed., APHA, Washington, DC
20005.
4. ASTM. 1990. Annual book of ASTM Standards, Water and Enviromental
Technology: Pesticides, resource recovery, hazardous substances and oil
spill responses, waste disposal, biological effects. Volume 11. 04:827
designation E1241-88, ASTM Philadelphia, PA 19103-1187.
5. Mount, D.I. and W.A. Brungs. 1967. A simplified dosing apparatus for
fish toxicology studies. Water Res. 1:21-29.
6. Lemke, A.E., W.A. Brungs, and B.J. Halligan. 1978. Manual for
construction and operation of toxicity-testing proportional diluters.
EPA-600/3-78-072. National Technical Information Service, Springfield, VA
22161.
7. Benoit, D.A., V.R. Mattson, and D.L. Olson. 1982. A continuous-flow
mini-diluter system for toxicity testing. Water Res. 16:457-464.
22
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8. Carton, R.R. 1980. A simple continuous-flow toxicant delivery system.
Water Res. 14:227-230.
9. Benoit, D.A. and F.A. Puglisi. 1973. A simplified flow-splitting chamber
and siphon for proportional diluters. Water Res. 7:1915-1916.
10. Benoit, D.A., 1982. Users guide for conducting life-cycle chronic
toxicity tests with fathead minnows (Pimephales promelas). U.S. EPA
600/8-81-011, Environmental Research Laboratory, Duluth, MN 55804.
11. Drummond, R.A. and W.F. Dawson. 1970. An inexpensive method for
simulating diel patterns of lighting in the laboratory. Trans. Am. Fish.
Soc. 99:434-435.
12. Holcombe, G.W., D.A. Benoit, D.E. Hammermeister, E.N. Leonard, andR.D.
Johnson. 1991. Acute and long-term effects of ten chemicals on the
Japanese Medaka (Orvzias latipes). (Draft: U.S. EPA, Environmental
Research Laboratory, Duluth, MN 55804).
13. Chadwick, G.C. and U. Kiigemagi. 1968. Toxicity evaluation of a
technique for introducing dieldrin into water. J. Fish. Res. Board Can.
40:76-82.
14. Veith, G.D. and V.M. Comstock. 1975. Apparatus for continuously
saturating water with hydrophobic organic chemicals. J. Fish. Res. Board
Can. 32:1849-1851.
15. Gingerich, W.H., W.K. Seim, and R.D. Schonbrod. 1979. An apparatus for
the continuous generation of stock solutions of hydrophobic chemicals.
Bull. Environ. Contam. Toxicol. 23:685-689.
23
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16. Phipps, G.L., G.W. Holcombe, and J.T. Fiandt. 1982. Saturator system
for generating toxic water solutions for aquatic bioassays. Prog. Fish-
Cult. 44:115-116.
17. Kahl, M.D., C.L. Russom, D.L. DeFoe, and D.E. Hammermeister. 1991.
Liquid/liquid and glass wool column equilibration units. (Draft: AScI
Corp. and U.S. EPA, Environmental Research Laboratory, Duluth, MN 55804).
18. U.S. Environmental Protection Agency. 1979. Methods for chemical
analysis of water and wastes. EPA-600/4-79-020. National Technical
Information Service, Springfield, VA 22161.
19. U.S. Environmental Protection Agency. 1980. Manual of analytical
methods for the analysis of pesticides in human and environmental
samples. EPA-600/8-80-038. National Technical Information Service,
Springfield, VA 22161.
20. Denny, J., R. Spehar, K. Mead, and S. Yousuff. 1991. Guidelines for the
culture of the medaka Oryzias latipes. (Draft: U.S. EPA, Environmental
Research Laboratory, Duluth, MN 55804).
21. Yamamoto, Toki-0. 1975. Medaka (killifish) biology and strains.
Keigaku Publishing Company. Tokyo, Japan.
22. Cast, M.H. and W.A. Brungs. 1973. A procedure for separating eggs of
the fathead minnow. Prog. Fish-Cult. 35:54.
23. Kirchen, R.V. and W.R. West. 1976. The Japanese Medaka: Its care and
development. Developmental Biology and Photography Departments, Carolina
Biological Supply Company, Burlington, NC 27215.
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24. Benoit, D.A., F.A. Puglisi, and D.L. Olson. 1982. A fathead minnow
(Pimephales promelas) early life stage toxicity test method evaluation
and exposure to four organic chemicals. Environ. Pollut. (Series A).
28:189-197.
25. McKim, J.M. and D.A. Benoit. 1971. Effect of long-term exposures to
copper on survival, reproduction, and growth of brook trout (Salvelinus
fontinalis). J. Fish. Res. Board can. 28:655-662.
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