;
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EPA/505/8-89/002b
CULTURING OF FATHEAD MINNOWS (PIMEPHALES PROMELAS):
SUPPLEMENTAL REPORT FOR VIDEO TRAINING TAPE
Teresa Norberg-King
Jeff Denny
U.S. Environmental Protection Agency
Environmental Research Laboratory
6201 Congdon Blvd.
Duluth, MN 55804
U.S. Environmental Protection Agency
Office of Water Enforcement and Permits
Permits Division
401 M Street, SW
Washington, DC 20460
EPA Contract No. 68-C8-0015
1989
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NOTICE
This report has been funded wholly or in part by the Environmental Protection Agency
under Contract 68-C8-0015. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
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FOREWORD
This report serves as a supplement to the video tape "Culturing of Fathead Minnows
(Pimephales promelas)(EPA, 1989A)". The methods illustrated in the tape and described in this
report support the methods published in the U.S. Environmental Protection Agency's (EPA's)
Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms,
Third Edition (1985) and Short-term Methods for Estimating the Chronic Toxicity of Effluents
and Receiving Waters to Freshwater Organisms, Second Edition (1989B), referred to as the acute
and chronic manuals, respectively. The video tape and this report provide details on setting up
and maintaining cultures based on the expertise of the personnel at the EPA's Environmental
Research Laboratory - Duluth (ERL-D), Minnesota.
This report and its accompanying video tape are part of a series of training tapes produced
by EPA's Office of Water Enforcement and Permits. The tape entitled "Fathead Minnow Larval
Survival and Growth Toxicity Test" (EPA, 1989C) complements the material in this video by
explaining the 7-day subchronic test method. These tapes are available through the National
Audiovisual Center, Capitol Heights, MD, 20743. Other available tapes include "Culturing of
Ceriodaphnia dubia" (EPA, 1989D), and its companion tape "Ceriodaphnia Survival and
Reproduction Toxicity Tests" (EPA, 1989E).
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ACKNOWLEDGEMENTS
This report was produced for the Office of Water Enforcement and Permits (Laura Phillips,
Work Assignment Manager). Technical direction of the culturing methods presented in this
report was provided by Jeff Denny, Teresa Norberg-King, and staff at EPA's Environmental
Research Laboratory-Duluth (ERL-D), Minnesota.
The methods described in this report are based on the laboratory experience of the
following people: Jeff Denny, Teresa Norberg-King, and Craig Wilson (ERL-D), Jim Gordon
(University of Minnesota-Duluth, Natural Resources Research Institute), Barb Halligan, Larry
Herman, Don Mount, Jodi Collins, Andy Peterson, Armond Lemke, Duane Benoit, Dick Carlson,
Dan Tanner, and Ron Carlson (ERL-D). This list is by no means inclusive, as many more of
ERL-D's present and former staff have contributed to the present system of fathead minnow
culturing.
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INTRODUCTION
Fathead minnows (Pimephales promelas) have been cultured for use in aquatic toxicity tests
for over 30 years, and are the most common fish species used to determine sublethal toxicity of
chemicals and complex effluents. The fathead minnow has a widespread distribution and is an
important forage fish. It is also readily cultured in captivity. A large data base on the effects of
single chemicals has been developed using the fathead minnow for acute partial and life-cycle
tests.
Recent modifications to the 32-day early life stages (ELS) test have produced a 7-day larval
growth and survival toxicity test. Norberg and Mount (1985) describe this rapid method to assess
the chronic toxicity of effluents using fathead minnows.
Healthy animals are the most important aspect for a good toxicity test. Emphasis should be
placed on determining the quality of the organisms used for producing the test organisms. This
report and the video "Culturing of Fathead Minnows (Pimephales promelas)" were produced by
EPA to clarify and expand on culturing methods explained in the acute manual. To ensure
successful toxicity testing, laboratory personnel should be familiar with the handling and
culturing procedures detailed below.
The fathead minnow is an adaptable organism and can be cultured in the laboratory under a
variety of conditions. Factors that must not be overlooked are the types of food to use for larvae
and adults, the stocking rates to grow testing and breeding stock, and the water to use. For
example, use of the brine shrimp (Artemia) as a basic food source has been essential at ERL-D.
Dried or processed fish foods such as the trout chow can be very good as a dietary supplement,
but few cultures have been successful using those diets alone. For cultures that require constant
reproduction, the use of the fresh or frozen Artemia has been essential.
The density of the fish in the culture is also very important. While fish can survive at high
stocking densities, the rapid growth, uniformity of size, sexual maturation, high reproduction
rates, and limiting of the spread of disease can be achieved with lower densities. With good diets,
space, constant temperature, and photoperiod, the fathead minnow can be cultured with ease.
Keen observation and regular maintenance of the culture animals and conditions are essential for
year-round organism production.
1
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The first section of this report covers the selection of the culture water, and explains
procedures for natural, dechlorinated, and synthetic culture water preparation. The second
section discusses the food requirements and preparation. The third section explains procedures
for initiating and maintaining fathead minnow cultures. The methods described in this report
cover culturing requirements for both acute and chronic tests, although the emphasis is primarily
on generating animals for sublethal tests.
2
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CULTURE WATER PREPARATION AND DELIVERY
The waters to be used for culturing fathead minnows are any toxicity-free water including
natural water, drinking water, or reconstituted water. The water source chosen for culturing may
not necessarily be the same type of water used for testing. However, whichever water is chosen
for culturing or testing, it must be tested to ensure that good survival and reproduction of the
organisms are possible and that consistency is achievable. Before any water is used, it should also
be tested for possible contamination by pesticides, metals, sulfides, or any other suspected
contaminants (OECD, 1989). The quality of the water must meet the acceptable levels described
in Table 1. In any case, the water quality should ensure adequate survival, growth, and
reproduction and it should be from a consistent source to provide constant quality during any
given test period.
Natural Water
Natural water can be from a variety of sources such as a surface water (e.g., river, lake,
pond), well water, or spring water. Natural waters should be carbon and/or sand filtered, along
with a fine filter as well (~ 5^m). When using a natural water that has resident fish populations,
an ultraviolet sterilizer or ultrafilter may need to be added after the roughing filters, to remove
any potential fish pathogens.
Dechlorinated Water
Drinking water (i.e., city or tap water) may be used provided that it has received adequate
treatment, but it may require dechlorination which can be accomplished either by aeration for
24 hours or by using a carbon filter to remove residual chlorine. Sodium thiosulfate may also be
used but it may act as a reducing/chelating agent in the water. The addition of 1.0 mg/1 of
anhydrous sodium thiosulfate will reduce 1.5 mg/1 of chlorine. For fathead minnows, the
96-hour LCS0 of sodium thiosulfate is 7.3 g/1 (EPA, 1988).
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Reconstituted Waters
Two types of synthetic water are frequently used in testing, but the volumes required for
culturing fish have limited their application for culture. This is not to say that they are not
suitable, but that their preparation is labor intensive, and the body of knowledge based on
culturing in synthetic waters is small. Two types of synthetic fresh dilution water can be
prepared. One is made using reagent grade chemicals and the other using a commercial mineral
water. Both recipes are described in the chronic manual (EPA, 1989). Recipes for preparing 20
liters of moderately hard water are given below.
The deionized water may be obtained from a Millipore Milli-Q® system, Nanipore®, or
equivalent. Acceptable ranges for the physical/chemical characteristics of the dilution water are
given in Table 2. In order to extend the life of the Milli-Q® cartridges, use a preconditioned
(deionized) feed water by using a Culligan®, Continental®, or equivalent system in front of the
Milli-Q® system. In a four-cartridge Milli-Q® system place the cartridges in the order of (1) ion
exchange, (2) carbon, (3) an organic column such as Organex-Q®, and (4) a final bacteria filter
(0.22-fim fine filter). For a five cartridge system, add an additional carbon cartridge. The order
of the filter heads may need to be re-plumbed so that the water flows over the cartridges
correctly. Conductivity of this filtered water should be zero /imhos/cm. All filters should be
changed at least every six months, but more frequent changes may be needed. The frequency of
change is totally dependent on the source water.
The synthetic water made with reagent grade chemicals (Table 3) can be prepared in batches
of 20 liters using the following recipe:
1. Place 19 liters of Milli-Q® or equivalent water in a properly cleaned plastic carboy.
2. Add 1.20 g of MgS04, 1.92 g of NaHC03, and 0.080 g of KC1.
3. Add 1.20 g of CaS04«2H20 to 1 liter of Milli-Q® or equivalent water in a separate
flask. Stir on a magnetic stirrer until the CaS04 is dissolved, and add it to the 19 liters
and mix well.
4. Aerate vigorously for 24 hours to dissolve the added chemicals and stabilize the medium.
5. The measured pH, hardness, and alkalinity should be as listed in Table 2.
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The synthetic water prepared using commercially available mineral water can also be
prepared in large batches (Table 3). The instructions in this report are specifically for Perrier®
water (EPA, 1989). While other commercial waters have been tested, the properties of other
waters have not been evaluated extensively; therefore no other commercial water source is
provided. To prepare 20 liters of water:
Place 16 liters of Milli-Q® or equivalent water in a properly cleaned carboy.
Add 4 liters of Perrier® water.
Aerate vigorously for 24 hours to stabilize the medium.
The measured pH, hardness, and alkalinity of the aerated water should be as listed in
Table 2.
This synthetic water prepared with Perrier® water is referred to as 20% diluted mineral
water (20% DMW) in toxicity test methods.
To aerate the water, use air free of oils and fumes. Organic vapors and oils can be removed
using an in-line activated carbon filter such as Balston® C-l (Balston, Inc., Lexington, MA.).
Particles are removed using another in-line filter such as the Balston® Grade RX filter, used
frequently in combination with the carbon filter.
Store both types of water in the carboys in which they were prepared and use each batch for
only 14 days. Water should be stored away from direct light, and should be kept covered.
Bacterial growth may occur in the water as it ages, which can cause problems for the culture
organism.
Water Delivery
If possible, a flow-through system should be used for culturing. The water delivery
system should provide at least three to four turnovers per day. Threaded polyvinyl chloride
(PVC) pipe is the most widely used construction material but glass, stainless steel, or Teflon® can
be used. Rubber, copper, brass, or plastics containing fillers, additives, stabilizers, or plasticizers
may cause toxicity, and therefore, should not be used. Glued PVC should be used for drainage
only, as the glue can be toxic.
1.
2.
3.
4.
5.
5
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Recirculating systems often consist of aquaria at table top level for the fish, and an
aquarium on the floor that acts as a trickling filter for the drain water from the fish tanks. This
filter can be made of any non-toxic, high surface area material like crushed coral, pea gravel, or
specially designed plastic media for trickling filters. These are available through filtration
suppliers. Nitrifying bacteria in these filters convert ammonia to nitrate when the system is in
balance. Ammonia levels must be monitored closely in this type of system. Water can be
pumped from a pump at the bottom of the filter up to a headbox above the fish tanks, where it
flows back into the tanks.
Culturing fathead minnows is also possible using a static system. Each tank should have
either an under-the-gravel or external filtration system. Supplies for these types of systems are
available at hobby shops and aquarium supply houses. Every two to three days, renew the water
by siphoning down at least 25% of the volume and adding new water. Use distilled water when
replacing water lost due to evaporation to avoid concentrating the dissolved salts. An additional
consideration is that larvae must be protected from being captured in the filtering system. A
container made of fine mesh will allow water to flow through, while protecting the larvae.
For breeding, the aquariums can be divided into four chambers with stainless steel mesh. In
flow-through and recirculating systems, each tank can be serviced by one water source, air stone,
and drain.
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Table 1. Chemical characteristics of an acceptable dilution water (OECD, 1989)
Substance Concentration
Particulate matter <20 mg/l
Total organic carbon < 2 mg/l
Un-ionized ammonia ¦ _ <1 /xg/1
Residual chlorine <10 /xg/1
Total organophosphorus pesticides <50 ng/1
Total organochlorine pesticides plus
Polychlorinated biphenyls <50 ng/1
Total organic chlorine <25 ng/1
Table 2. Water quality parameters for reconstituted waters (EPA, 1989)
Water Type pH Hardness3 Alkalinity1
Very Soft 6.4 - 6.8 10 - 13 10 - 13
Soft 7.2 - 7.6 40 - 48 30 - 35
Moderately Hard 7.4 - 7.8 80 - 100 60 - 70
Hard 7.6 - 8.0 160 - 180 110 - 120
Very Hard 8.0 - 8.4 280 - 320 225 - 245
a Expressed as mg/l as CaC03.
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Table 3. Preparation of synthetic fresh water (procedures taken from EPA,1989)a
Reagent Recipes
Reagent Added fms/H
Type
NaHCO,
CaSO4.2H20
MgS04
KC1
Mineral Water Recipes
Volume of Proportion
Mineral Water Mineral
Added Water
(ml/1) (%)
Very Soft
12.0
7.5
7.5
0.5
50
2.5
Soft
48.0
30.0
30.0
2.0
100
10.0
Moderately Hard
96.0
60.0
60.0
4.0
200
20.0
Hard
192.0
120.0
120.0
8.0
400
40.0
Very Hard
384.0
240.0
240.0
16.0
—
—
a Add reagent grade chemicals and/or mineral water to Milli-Q® or equivalent water.
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FOOD PREPARATION
Fathead minnows are fed different forms of food during their development. From hatching
to approximately 30 days old their main diet is live brine shrimp (Artemia salina), and generally
after one month old they are weaned over to partially frozen brine shrimp. The amount fed to
each tank should be adjusted for the number and size of the fish.
Feeding the Larvae
Feed the fish live brine shrimp twice each day, Monday through Friday, and once each day
on weekends. Larvae require very small amounts of food during the first few days (days 1-5),
but require increasing amounts on days 6-10. Feed the larvae two times a day Monday through
Friday, and once a day on Saturday and Sunday. After 10 days, the feeding rate must increase
substantially each day and is proportional to the number of fish maintained. Care must be taken
not to overfeed during the first few days, and not to underfeed in the later stages. Careful
observation is critical and waste food on the bottom of the tank indicates overfeeding. This
decaying food will cause the dissolved oxygen (DO) levels to drop. Rapid consumption of all
food right after feeding (i.e., all Artemia is consumed 5-10 minutes post feeding) indicates
underfeeding. Shortage of food is also evident by wide size variability in 30-day old juvenile
fish. The rotifers, Brachionus spp., have recently received attention as an alternative food for the
first feedings of larval fathead minnows.
Observe the amount of food left at the end of the day and adjust the feeding rate
accordingly. Each day, siphon out any excess food as waste food will grow fungus that can trap
the larvae.
Live Brine Shrimp
The brine shrimp used for feeding the larvae and juveniles is Artemia salina. Some sources
of Artemia cysts are
Aquarium Products Biomarine Research
180 L. Penrod Ct. c/o Aquafauna
Glen Burnie, MD 21061 P.O. Box 5
Hawthorne, CA 90250
and other commercial suppliers.
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Upon receiving brine shrimp cysts, date the containers and store them in a freezer to
prolong their shelf life.
Brine shrimp cysts are most easily hatched in containers with conical shaped bottoms or in
separatory funnels. Typically hatching instructions are provided with the cysts. A common
procedure is to make a 15-ppt salinity medium using un-iodized salt, and bubbling the water
with filtered air from the bottom to keep the cysts circulating.
At 25<>-280C, the Artemia begin to hatch in 24 hours. Larval fathead minnow must be fed
less than 24-hour post-hatch Artemia nauplii so that they are small enough for the larvae to
ingest. At this age, the nauplii also have their highest nutritional value as their yolk sack has not
yet been depleted. This nauplii size requirement makes it necessary to start new Artemia cultures
daily.
To collect Artemia for feeding, remove the air supply and allow the hatched cysts to settle to
the bottom of the hatching jar (~ 5 minutes). The live shrimp settle forming an orange layer at
the bottom of the container with a brown layer of unhatched cysts below it. The empty shells of
the hatched cysts will rise to the top. The live shrimp can be removed using a large-bore pipette
or a siphon. Either a 50-ml or 100-ml pipette (inverted) works well. Before feeding in static
systems, or for static tests, rinse the Artemia with distilled, deionized, or culture water to prevent
salt buildup in the tanks.
Frozen Brine Shrimp
Fish that are over 30 days old are fed partially frozen brine shrimp twice daily, Monday
through Friday, and once a day on weekends. Some sources for the brine shrimp are
Jungle Laboratories Corp. San Francisco Bay Brand, Inc.
P.O. Box 632 Newark, CA 94560
Cibolo, TX 78108
or other commercial suppliers.
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For easier handling, allow the brine shrimp to thaw slightly at room temperature (not
completely). Each spawning pair should receive approximately 1/8-1/4 teaspoon of the brine
shrimp. As a general guide, feed each tank of fish the amount of food that can be consumed in
about 10-20 minutes.
Nutritional quality and contaminant levels vary widely between strain, year of harvest,
location of harvest, and supplier of Arlemia. It is useful to get as much information as possible
from the supplier concerning the nutritional quality and contamination or have the Artemia
checked for these parameters as new batches are ordered.
Supplements
The periphyton that grows naturally in the tanks provides a good dietary supplement for the
fish. In addition, flake food such as commercially available Tetramin® or trout chow may be
used as supplements. Trout chow is available from several sources such as
Ziegler Bros., Inc. Glencoe Mills Murray Elevators
P.O. Box 95 Glencoe, MN 55416 118 West 4800 South
Gardners, PA 17324 Murray, UT 84107
Again, contaminant levels and nutritional content can vary widely, and screening and analysis
may be required to ascertain the suitability of the foods.
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CULTURES
Initiating Cultures
Fathead minnow cultures should be started with fish from a reliable source such as a
commercial supplier or a research laboratory. Both ERL-D and EMSL-Newton, Ohio provide
starter cultures, typically embryos. The embryos or fish should be shipped by overnight mail in
an oxygenated container that is packed in a cooler to minimize temperature fluctuations. Upon
receipt, allow the water in the shipping container to acclimate in a water bath or use aquarium
heaters for the temperature adjustment. Once acclimated, empty the container containing the
starter cultures into a pan, aerate, and maintain the temperature at 25°C. In 4-5 days the
embryos will hatch, at which time the larvae should be moved to rearing tanks using a large-bore
pipette. For an 8- to 10-gallon tank, the recommended stocking density is 200-250.
Once embryos hatch, feed them Artemia salina nauplii that are less than 24 hours old. Feed
the fish 2 to 3 times a day, 5 days a week, for at least the first 2 weeks. On weekends, feeding
only once each day has proven to be adequate.
Breeding
When the fish are 30 days old, at ERL-D the fish are either used for acute toxicity testing
or grown out as brood stock. In a 50- to 70-gallon tank, 300-400 fish can be grown to maturity.
For the brood stock fish, wean them over to frozen brine shrimp. To hasten the maturation
process, thin the 3-4 month old fish to 30-35 per 10-gallon tank, and the addition of spawning
tiles may speed up the maturation.
Fathead minnows begin to show signs of maturity at three to four months of age (Figure 1).
The male will develop an enlarged head with rows of tubercles across the snout. These are used
to clean the underside of the spawning substrate on which the eggs are deposited. The male will
also develop black coloration on his sides. The female is smaller than the male and will not have
tubercles. She is an olivaceous color and when mature, exhibits an ovipositor.
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Tubercles
Female
Ovipositor
Figure 1. Male Fathead Minnow (top) and Female Fathead Minnow (below)
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Spawning tiles can be made of clay tiles or 10 cm-diameter PVC pipes cut into 7-10 cm
long sections, then cut in half lengthwise to create a semicircular arch. The inner side, where the
eggs will be deposited, is then roughened with sandpaper. When placed in the aquariums, the
fish use the underside to deposit and fertilize the eggs. The rough surface will help the eggs to
adhere to the substrate. In the wild, fathead minnows use the underside of submerged or floating
objects.
Two options can be used for separating the fish for spawning. The first separates the fish
into spawning pairs. This is easily accomplished by dividing a 10- or 15-gallon aquarium into
quarters using stainless steel screens. Paired spawning reduces competition which allows for
greater production and makes it possible to monitor the fecundity of each pair. Daily records of
reproduction can be used to identify sterile or spawned-out fish, which can be replaced by a new
pair to maintain a high production rate.
A second breeding option is to place groups of mature adult fish in tanks with a female:
male ratio of 8 : 3. Use four spawning substrates in an 8- to 10-gallon tank with approximately
20 fish. When the egg production rate in these tanks drops, replace the entire group of fish.
Spawning
The male cleans the underside of the spawning tile with his tubercles and draws the female
underneath. The male directs her toward the tile where she releases the eggs. Fertilization of the
eggs is external and the buoyant eggs stick to one another and adhere to the underside of the tile.
Females release an average of 100-200 eggs per spawn, with larger females releasing 200-400.
Fathead minnows spawn approximately every 4-5 days, but can spawn as often as every 2 days.
Monitor the reproduction rate of each brood pair or group of adults. If no embryos are
produced in a 3-week period, replace the pair or the entire group. Younger fish can be allotted
longer periods of time if they are just beginning to spawn.
Collecting the Embryos
The substrates in each tank should be checked daily for embryos. In order not to disrupt
the early morning spawning, check the tanks midmorning. To retrieve the tiles, use tongs that
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are dipped in boiling water between each tank to minimize any possible transfer of disease from
one tank to another.
If needed for toxicity testing, the embryos can be removed from the tile with a gentle,
circular, rubbing motion while keeping the tile underwater to prevent premature hatching caused
by the disruption. If the embryos are to be hatched directly from the tiles, transfer the tiles
immediately to the hatching system.
Hatching
Two options for hatching the fathead minnow embryos are to remove the embryos from the
tiles to aerated water in separatory funnels or to keep them on the tiles and hatch them in aerated
water in larger pans. The first option requires that the embryos be rolled off of the tiles and
pipetted into a separatory funnel containing aerating culture water. After 2 days in this system
the embryos are placed in a pan containing aerated culture water where they will hatch in another
2-3 days.
The second option is to place the tiles directly into a pan containing aerating culture water
in a holding pan. A white pan allows the larvae to be seen more easily. The tiles are placed on
their sides covered with culture water, and aerated. At a temperature of 25°C, the embryos
should hatch in 4-5 days.
The aeration of the water in the separatory funnel and around the tiles provides circulation
and helps keep sediment and fungal spores from settling on the embryos. Check the embryos on
the tiles on days 1 and 2 for fungus or lack of viability and remove any such embryos with
tweezers. On days 3, 4, and 5, check the tiles, but minimize any disturbances as it may cause
early hatching of the larvae. Embryos that appear cloudy should be removed and discard all of
the eggs from any tile on which 50% or more die. Figure 2 is a check list used at ERL-D to
track the daily tasks required for fathead minnow culturing.
After each use, the tiles are disinfected in a chlorine bath for 1 hour, rinsed with tap water,
neutralized with sodium thiosulfate for at least 10 minutes to remove residual chlorine, and
finally, rinsed in culture water and allowed to air dry.
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DAILY CHECK-LIST FOR FATHEAD CULTURE UNIT
(initial when done)
DATE
MORNING
Notes
Check temperatures
Check water flow
to all tanks
Feed adult fish
frozen brine shrimp
Feed fry live
brine shrimp
Check tiles in pans
for bad eggs
Pull spawning tiles
&est. no. eggs (10:30)
AFTERNOON
Set up fry
Feed adult fish
frozen brine shrimp
Feed fry live
brine shrimp
Set up new live
brine shrimp
Check temperatures
before leaving
Figure 2. Suggested Daily Tasks for Fathead Culturing
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Larvae for future brood stock should be the progeny of as many adult spawning fish as
possible. A few larvae collected each month from many different spawning pairs will provide a
broader gene pool than hundreds of larvae from one or two spawning pairs in one week.
Tracking the Fish
If the fathead minnows are used for toxicity testing there will be a need to anticipate the
demand for the eggs or larvae. The number of eggs that are needed for testing will determine the
number left to hatch for larval testing or to grow out for future brood stock. Figure 3 is an
example of a request form used to anticipate the demand on the cultures. Figure 4 is a tracking
form that ERL-D uses to monitor the performance of their broods that are used for toxicity tests.
It represents one method used to track the health of the cultures.
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EGG/LARVAE REQUEST FORM
NAME
DATE
NEED
(eggs or larvae)
HOW
MANY
WHEN
REMARKS 1
Figure 3. Testing Request Form
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FATHEAD CULTURE UNIT
Fish Tracking Form
Date:
Researcher:
Age of Fish (Embryos, Larvae?):
Number Taken:
Type of Test (Embryo-Larval, 7-Day Etc.):
Test Conditions (Static, Renewal,
Flow-Through?):
Temperature:
Dilution Water:
Test Chemical:
Hatching Percentage (Embryos):
Control Survival (%) (Reps Different?):
Abnormal Survival In Low Cones?:
Deformities In Control Fish?:
Observations (ie: Condition of Fish,
Test Conditions):
RETURN FORM TO FATHEAD CULTURE UNIT BEFORE INITIATING NEXT TEST!
Figure 4. Performance Tracking Form
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REFERENCES
EPA, 1989A. Culturing of Fathead Minnows {Pimephales promelas). Video Tape (17:10 min).
EPA, 1989B. Short-term Methods for Estimating the Chronic Toxicity of Effluents and
Receiving Waters to Freshwater Organisms. Second Edition. EPA/600/4-89/001.
Cincinnati, OH.
EPA, 1989C. Fathead Minnow Larval Survival and Growth Toxicity Tests. Video Tape and
Supplemental Report. EPA/505/8-89/001b. Washington, DC.
EPA, 1989D. Culturing of Ceriodaphnia dubia. Video Tape and Supplement Report.
EPA/505/8-89-002a. Washington, DC.
EPA, 1989E. Ceriodaphnia Survival and Reproduction Toxicity Tests. Video Tape and
Supplemental Report. EPA/505/8-89-001a. Washington, DC.
EPA, 1987. Guidelines for the Culture of Fathead Minnows (Pimephales promelas) for Use in
Toxicity Tests. EPA/600/3-87/001. Duluth, MN.
EPA, 1985. Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine
Organisms. Third Edition. EPA/600/4-85/013. Cincinnati, OH.
Norberg, T.J. and D.I. Mount, 1985. A new fathead minnow (Pimephales promelas) subchronic
toxicity test. Environ. Toxicol. Chem. 4:1-711-718.
Organization for Economic Cooperation and Development (OECD), 1989. Draft Standard of the
Fathead minnow (Pimephales promelas) Larval Survival and Growth Test. OECD, Italy.
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GLOSSARY
Artemia. Common brine shrimp (Order Anostraca) found in the Great Salt Lake and similar
saline lakes and ponds scattered throughout the world; 4-10 mm long.
Larvae. General term for any independent, active, immature stage of an animal which is
morphologically quite unlike the adult; grows into an adult by a complicated metamorphosis
in most cases. For fathead minnows, young fish are considered larvae for the first days
post-hatch.
Nauplii. Free-swimming microscopic larvae stage characteristic of copepods, ostracods,
branacles, etc. typically only with three pairs of appendages.
Ovipositor. The tubular extension of the female pore in certain fishes used to assist in depositing
eggs.
Pimephales promelas. Scientific name for the fathead minnow, a common minnow of the family
Cyprinidae which is widely distributed east of the Rockies.
Tubercles. Spongy protrusions on the dorsal surface of the male fish, anterior to the dorsal fin;
these are used by the male to clean the debris from spawning substrate and fertilized
embryos.
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