United States
Environmental Protection
Agency
Environmental Research
Laboratory
Duluth MN 55804
Research and Development
EPA/600/S3-87/001 Aug. 1988
&EPA Project Summary
Guidelines for the Culture of
Fathead Minnows,
Pimephales pro me I as for
Use in Toxicity Tests
Jeffrey S. Denny
Fathead minnows, Pimephalesprom-
elas Rafinesque, have been cultured at
the Environmental Research Labora-
tory - Duluth (ERL-D) for use in aquatic
toxicity tests since the establishment
of the laboratory in 1967. The tech-
niques and apparatus described in this
report were developed over the years
by many researchers. This paper sets
forth the conditions and procedures
now being used to produce research
quality fathead minnow embryos,
larvae, juveniles, and adults. These
guidelines can be modified to adapt to
existing circumstances and needs.
Information on the physical system
includes water supply, construction
materials, water temperature, pho-
toperiod, and the water delivery sys-
tem. The biological section addresses
the selection of spawning fish, incuba-
tion of embryos, larval and adult
feeding, disease, and gene pool con-
siderations. This document is meant to
be a guide for those interested in
culturing fathead minnows for use in
fish toxicology research.
A fathead minnow culture facility can
provide a continuous supply of
embryos or fish of known age, raised
under known conditions, for aquatic
toxicity testing. The use of laboratory
reared animals is advantageous since
age and genetic background are
known, diet is controlled, fish are free
from disease, and are available year-
round.
The life stages of fish in greatest
demand for testing are less than 24
hour old embryos, 0-24 hour old larvae,
and 30 day old juveniles. The ERL-D
system is designed to produce these
three life stages, plus adults for future
brood stock. Ninety-six pairs of adult
spawners provide 1,000-2,000
embryos/day. These can be used
immediately for testing, or incubated
to provide larvae or juveniles for testing
at later dates.
This Project Summary was devel-
oped by EPA's Environmental
Research Laboratory, Duluth, MN, to
announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Physical System
Water Supply
Lake Superior water is supplied to the
ERL-D culture system at constant flow
volumes of about 150 ml/min per tank.
If available, springs, wells, or controlled
surface waters are recommended. De-
chlorinated tap water from a municipal
supply should be used only as a last
resort (Benoit, 1 982). The supply should
be examined for contamination by pes-
ticides, heavy metals, sulfides, disease
vectors, or any other suspected
contaminants.
The quantity of water necessary
depends on the size of the intended
culture unit. The ERL-D system of over
150 tanks consumes 15-20 liters/
minute when in full operation. Smaller
systems, or systems in areas of limited
water supply could operate on a reduced
flow. Though less desirable, static
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renewal or recirculating systems may be
effective.
Tanks
Tanks are 57 liter (15 gallon) glass
aquaria, with standpipe drains adjusted
to provide approximately 40 liters of
water. Tanks are supported on racks of
slotted angle iron and 3/4 inch plywood,
both of which are painted with epoxy
paint to resist moisture. Stainless steel
screen can be used to divide tanks into
sections for spawning pairs of fish. White
plastic dishpans, commonly available in
department stores, are used to hold
spawning tiles on which the embryos
incubate.
Water Delivery System
The ERL-D water delivery system is a
constant temperature (25°C), flow
through system. It is gravity fed, with
stainless steel mixing boxes positioned
in an open ceiling approximately 3.5 m
overhead. A standard toilet float valve
maintains water level in the headbox,
while a solenoid valve adds heated water
to maintain temperature at 25°C. The
solenoid valve responds to a temperature
probe in the headbox via a solid state
electronic controller. Air stones or some
other type of agitator must be used in
the headbox to assure complete mixing
and to prevent supersaturation.
Water flows to the tanks through a 1/2
inch PVC pipe manifold. Above each tank
is a tee with a 1 /2 to 3/8 reducer, with
a 3 ml disposable syringe barrel glued
into it. This allows the use of different
sizes of hypodermic needles to control
flow rates.
Construction Materials
Construction materials which come
into contact with the water must not
contain leachable substances. Rubber,
copper, brass, or plastics containing
fillers, additives, stabilizers, plasticizers,
etc., must not be used (Mount, 1971).
Glass, stainless steel. Teflon, and PVC
are the preferred construction materials.
All piping should be of rigid PVC. Thread-
able PVC must be used, to avoid the
danger of toxicity from PVC glue. Silicone
glue is safe to use as long as enough
curing time is allowed. Ground fault
interrupters are necessary on all elec-
trical components due to the close
proximity of electricity and water in these
systems.
Photoperiod
Photoperiod should be constant at 16
hours light/8 hours dark. The ERL-D
system uses lights that simulate the
wavelength spectra of sunlight. How-
ever, fathead minnows will spawn under
"cool white" lighting. Temperature
adjustments may be necessary to offset
the warming effects of fluorescent
lighting.
Aeration
Provide continuous gentle aeration to
the tanks to maintain dissolved oxygen
concentrations above 5.0 mg/l at all
times, but avoid vigorous aeration with
newly hatched larvae. If a level of 5.0
mg/l cannot be maintained, remove
some fish from the tank (Mount, 1971).
Check the location of air intakes and
efficient operation of laboratory air
compressors to avoid introducing con-
taminants. An oil trap or filter may be
necessary on some systems.
Spawning Substrates
Four inch diameter PVC pipe, cut into
4 inch pieces, and halved lengthwise,
provide a semicircular arch under which
fathead minnows will readily spawn. The
inside can be roughened with a wire
wheel to improve egg adhesion. Another
type of substrate can be made by halving
small clay flower pots.
Biological System
Obtaining Brood Stock
Fish that are free of disease and
adapted to laboratory conditions make
the best initial brood stock. For the least
risk of disease, and greater ease of
shipment, begin with embryos. Use of
embryos also avoids any bioaccumula-
tion of toxicants that may occur with
adults. Less desirable is the use of fish
caught in the wild, or purchased from
a bait dealer. Take care that the animals
are Pimephales promelas, and not a
related species. Examine all fish, espe-
cially wild caught or bait dealer fish, for
signs of disease.
Selection of Spawning Fish
Stock juvenile fish 3-4 months old at
a density of 35-40fish per 15 gallon tank,
and provide with approximately 4 spawn-
ing substrates. The presence of sub-
strates hastens the maturation process.
In 1 -2 weeks some males in the tank will
showsignsof maturing. Females become
gravid soon after the males exhibit
spawning color. For observation, net fish
from the tank and place individually into
a 400 ml beaker with appx 3 cm of water.
Sexual maturity can then be determined
as follows: Breeding males develop a
conspicuous gray pad of spongy tuber-
cles on the dorsal surface anterior to the
dorsal fin, and two rows of tubercles
across the snout. The sides of the body
become almost black except for two wide
vertical bars which are light colored.
Another characteristic of the breeding
male is the presence of a dark spot at
the anterior insertion of the dorsal fin.
Females remain quite drab (Eddy and
Underbill, 1974). The female fathead
minnow exhibits an ovipositor at least a
month before spawning (Flickinger,
1969). Backlighting makes the female
ovipositor easier to see.
Removal of the mature fish for
spawners will stimulate maturation of
subordinate fish to replace the dominant
fish that were removed from the hier-
archy. This method will provide a con-
tinuous source of mature fish.
Spawning
The fathead minnow is an intermittent,
multiple spawning species with an
extended breeding season, possibly
spawning intermittently all summer
(Hasler, 1946; Radcliff, 1931). Under
controlled culture conditions fathead
minnows will spawn throughout the
year.
The buoyant, adhesive embryos stick
to one another and to the undersurface
of the nesting object. After deposition is
complete, the male remains at the nest
site tending and defending the embryos
until hatching occurs (Andrews and
Flickinger, 1973). More than one female
may spawn in a male's nest, and up to
12,000 embryos have been found in one
nest (Markus, 1934), indicating that
several females contribute to the embryo
mass. Unpublished data indicate an
average of 258 eggs/spawn and an
average of 3,095 total eggs/female
during 100 days of spawning activity
(Olson, 1974).
Separation into spawning pairs redu-
ces competition between males, and
allows the fecundity of individual pairs
to be monitored. Sterile, or "spawned
out" fish can then be replaced to main-
tain egg production. Other investigators
have reported success with male to
female ratios of 2/4 (Olson, 1974), 3/6
(Benoit and Carlson, 1977), and 4/10-
15 (Mount, 1971). The males are terri-
torial, so at least as many spawning
substrates must be provided as there are
males in the tank to achieve optimum
egg production.
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-.mbryo Incubation
Spawning tiles containing embryos are
removed from the tanks daily, and new
empty tiles provided. Embryos are incu-
bated by placing tiles on edge in a
dishpan of culture water, with an air-
stone placed between two tiles. Nonvi-
able embryos must be removed daily with
a tweezers, to prevent spread of fungus.
At 22-23°C embryos will begin to hatch
in 5 days. Tiles can be disinfected
between uses by soaking in chlorine and
then neutralizing with sodium
thiosulfate.
Larval Handling
Larvae can be handled using a large
bore, 50 ml volumetric pipette. For
rearing in 15 gallon aquaria, a stocking
density of 250 larvae per tank is recom-
mended. All fathead minnow larvae less
than 30 days old are fed live brine shrimp
twice each day.-
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The EPA author, J. S. Denny is with the Environmental Research Laboratory,
Duluth. MN 55804.
R. L. Spehar is the EPA Project Officer (see below).
The complete report, entitled "Guidelines for the Culture of Fathead Minnows,
Pimephales promelas for Use in Toxicity Tests," (Order No. PB 87-165 247/
AS; Cost: $14.95. subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Research Laboratory
U.S. Environmental Protection Agency
Duluth, MN 55804
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
EPA/600/S3-87/001
000052V PS
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