vvEPA
United States
Environmental Protection
Agency
Environmental Research
Laboratory
Duluth MN 55804
EPA/600/3-87/001
January 1987
Research and Development
Guidelines
for the Culture of
Fathead Minnows
Pimephales
Prom el as for Use in
Toxicity Tests
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EPA/600/3-87/001
January 1937
GUIDELINES FOR THE CULTURE OF FATHEAD MINNOWS
PIMEPHALES PROMELAS FOR USE IN TOXICITY TESTS
by
Jeffrey S. Denny
Environmental Research Laboratory - Duluth
Duluth, Minnesota 55804
ENVIRONMENTAL RESEARCH LABORATORY - DULUTH
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804
U.S. Environments! Faction Agency
D^^'"i.. P-, i ;ur. - • f<~>i ion
77 V'i'\i Jackson Buu c.ard, 12th Floor
Chicago, IL 60604-3590
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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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FOREWORD
Fathead minnows, Pimephales promelas Rafinesque, have been
cultured at the Environmental Research Laboratory - Duluth for
use in aquatic toxicity tests since the establishment of the
laboratory in 1967. The techniques 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 different circumstances and needs.
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ABSTRACT
This paper describes the mechanical apparatus and
biological techniques now in use to culture fathead minnows
the us Environmental Protection
Agency's Environmental Research Laboratory in Duluth, Minnesota.
Physical system information includes water supply, construction
materials, water temperature, photoperiod, and the water delivery
system. The biological section addresses the selection of
spawning fish, incubation of embryos, larval and adult feeding,
disease, and gene pool considerations. This document is meant to
be a guide for those interested in culturing fathead minnows for
use in fish toxicology research.
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ACKNOWLEDGEMENTS
For continual help throughout the development of this guide
I would like to thank Larry Herman, Barb Halligan, Jodi Collins,
and Andy Peterson. For expert advice on the culture of fatheads
I would like to thank Barb Halligan, Armond Lemke, Duane Benoit,
Dick Carlson, and Don Mount. For manuscript review I am indebted
to all of the above mentioned people, as well as to Richard
Siefert, Quentin Pickering, J. Howard McCormick, Teresa Norberg-
King, Scott Heinritz, John Brazner, and Jim Taraldsen. I extend
special thanks to Jodi Collins for illustrations and Eric
Peterson for photographic support.
Much of the credit for this document is due to present and
former staff members of the Environmental Research Laboratory -
Duluth, who have, over the years, developed this system of
fathead minnow culture.
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CONTENTS
Foreword ii
Abstract iii
Acknowledgements iv
1. Purpose 1
2. Design 1
3. Physical System
3.1 Tanks 3
3.2 Water supply 4
3.3 Water delivery system 6
3.4 Construction materials 9
3.5 Temperature 10
3.6 Aeration 10
3.7 Photoperiod 11
3.8 Spawning substrates 11
3.9 Fail safe devices 12
3.10 Tank cleaning 12
3.11 Disturbance 14
3.12 Brine shrimp hatchery 14
4. Biological System
4.1 Obtaining brood stock 16
4.2 Selection of spawning fish 17
4.3 Spawning 21
4.4 Embryo incubation 23
4.5 Larval feeding 27
4.6 Adult feeding 29
4.7 Disease 29
4.8 Gene pool 31
4.9 Record keeping 32
5. Literature cited 33
6. Additional reading 35
7. List of suppliers 41
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PURPOSE
A fathead minnow culture facility provides 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.
This document describes in detail fathead minnow culture
techniques used at the Environmental Protection Agency's
Environmental Research Laboratory in Duluth, Minnesota. Present
practices are based on years of expertise developed by staff
scientists. Local conditions and specific needs of researchers
may require modification of these guidelines.
DESIGN
The life stages of fish in greatest demand for testing are
less than 24 hr old embryos, 0 - 24 hr old larvae, and 30 day old
juveniles. This system is designed to best produce these three
life stages.
The fathead culture unit at ERL-D has 54 m2 of floor space
in two adjacent rooms. The main room (36 m2) contains 24
spawning tanks, 60 juvenile rearing tanks, 20 brood stock tanks,
6 hatching trays for eggs, and 20 more tanks for
experimental/emergency use (See Photograph #1). This provides
ample space for 96 pairs of adult spawners, 400 - 500 maturing
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fish for use as future spawning stock, and about 15,000 juveniles
being reared to 30 days of age. This system produces from 1000
- 2000 eggs/day, and provides 400 - 500 30 day old juveniles
each day for toxicity testing. The adjacent room (18 m2)
provides space to quarantine and breed incoming wild fish used to
intermittently diversify the gene pool.
Production now supplies about 15 - 20 researchers engaged in
full time toxicity testing , and fills occasional requests from
other scientists. A slight over - production is necessary to
allow for variations in egg production and for recycling spare
fish into new brood stock.
PHYSICAL SYSTEM
3.1 Tanks
The tanks are 57 liter (15 gallon) glass aquaria, 31 cm x 61
cm x 32 cm deep, with standpipe drains adjusted to provide 20 cm
of water depth. These contain approximately 40 liters of water
per tank. The tanks, supported on racks made of slotted angle
iron and 1.9 cm (3/4 inch) plywood, are arranged in 2 tiers, in
rows of 12 tanks. Two rows placed back to back form a bank of 48
tanks. Three such banks make up the entire culture system.
When starting a culture program, new tanks must be acid (10%
nitric), and acetone rinsed before using. Used tanks must be
disinfected with hypochlorite. Allow culture water to flow
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through the tanks for a day or two before adding fish.
For spawning fish, divide the tanks into four sections
with stainless steel screen (eg: 5 mm mesh, 0.89 mm wire), glued
in place with silicon glue. The screen material must be acid
leached before use to remove machine oil. A single water inlet,
drain, and air stone can service each tank.
White plastic dishpans, commonly available in department
stores (eg: 53 cm x 40 cm x 12 cm deep) make good hatching trays
for embryo incubation. White trays provide the best background
color for seeing the newly hatched larvae. Place six hatching
trays in a temperature controlled water bath (eg: 130 cm x 125 cm
x 8 cm deep, constructed from 1.9 cm (3/4 ijich) plywood and
sealed on the inside with epoxy paint). If this bath is placed in
a rack below a bank of fish tanks, drain water from the fish
tanks will provide an inexpensive heating source for embryo
hatching. Fish tanks at 25°C will warm hatching pans to 23°C.
3.2 Water Supply
Unfiltered Lake Superior water is the water supply for the
ERL - D fathead culture unit. It has a pH range of 7.4 - 8.2,
alkalinity (as CaCO3) of 42 mg/1, and total hardness of 45 mg/1.
For more detailed chemical characterization of Lake Superior
water, see Glass (1977).
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Water supply must be the most important consideration in
establishing a fathead culture facility. Use a natural supply
such as a spring, well, or controlled surface water with
consistent water quality, if possible. Culture water should be
similar to water used in testing. Examine the source for
contamination by pesticides, heavy metals, sulfides, disease
vectors, or any other suspected contaminants. Make checks of
water quality periodically. Filtration may be necessary if well
water is used (Mount, 1971). Dechlorinated tap water from a
municipal water supply should be used only as a last resort
(Benoit, 1982). If the water supply is contaminated with fish
pathogens, pass the water through an ultraviolet or similar
sterilizer immediately before it enters the system (Allison and
Hermanutz, 1977).
Water quality parameters such as hardness, alkalinity, and
anions should fall within the following limits: hardness - 40 -
300 mg/1 (as CaCO3), and alkalinity slightly less than the
hardness. The anions should be those found in a normal stream or
lake. Avoid well or spring waters which have high iron, silica,
sulfides, or chlorides not found in surface waters.
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 renewal
or recirculating systems may be effective.
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3.3 Water Delivery System
The ERL - D water delivery system is a constant temperature,
flow-through system. It is gravity fed, with custom welded
stainless steel mixing boxes (46 cm x 28 cm x 40 cm deep)
positioned in an open ceiling approximately 3.5 m overhead. Lake
Superior water warmed to 20°C flows through a toilet tank valve1
to maintain water level in the headbox. Water heated to 30°C
flows through a solenoid valve2 positioned over the headbox. To
achieve the desired constant temperature, a temperature probe3
suspended in the headbox controls a solid state temperature
controller (Syrett and Dawson, 1975). This controller opens and
closes the solenoid valve when resistance of the temperature
probe changes due to change in temperature. When the headbox
temperature falls below 25°, the solenoid valve activates,
adding 30° water until the headbox temperature reaches 25°, when
the probe/relay system closes the solenoid valve. Headbox
temperature must be maintained at 26- 27° to provide 25° at the
tanks. This is due to heat loss to ambient temperature. This
basic water delivery system can supply most experimental water
^ Numbers refer to suppliers that manufacture or distribute
the type of product described. A list of suppliers is appended.
Mention of trade names or commercial products does not constitute
endorsement or recommendation for use by the U.S. Environmental
Protection Agency.
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temperatures, depending only on the temperature of the water
supply to the float and solenoid valves. (See Figure #1).
Air stones or some other type of agitator4 must be used in
the headbox to assure complete mixing and oxygenation, and to
prevent supersaturation of gases caused by heating water. Water
flows from the headbox through a 1.27 cm (1/2 inch) threadable,
polyvinyl chloride (PVC) pipe (schedule 80, available from
plumbing supply houses). The outlet is on the side of the
headbox, about 2 -3 cm up from the bottom. Detailed instructions
on the design of the mixing boxes, electronic relays, etc., are
available in Syrett and Dawson, 1972, 1975, and McCormick and
Syrett, ms, 1970.
Water flows into the tanks through a 1.27 cm (1/2 inch) PVC
pipe manifold. Above each tank there is a T with a 1.27 cm (1/2
inch) to .95 cm (3/8 inch) reducer attached. Into the reducer a
3 ml disposable syringe barrel5 is glued with silicone glue.
This allows the use of different sizes of hypodermic needles5 to
control flow rates. Seventeen gauge needles provide 100 - 150
ml/min with 1 - 2 m head pressure.
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3.4 Construction Materials
Construction materials which come in 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. Schedule 80,
threadab^e pvc must be used to avoid the danger of toxicity
from PVC glue. Silicone6 glue is safe to use as long as enough
curing time is allowed. (Follow manufacturers instructions for
complete curing.) Check all batches of neoprene stoppers for
toxicity prior to use. Recent static tests at ERL - D show that
certain types of neoprene stoppers are acutely toxic to fathead
minnow larvae. A simple 24 hr static test exposing fathead
larvae to the stoppers in a beaker of culture water will indicate
whether the stoppers are safe.
Ground fault interrupters7 are necessary on all electrical
components because of the close proximity of electricity and
water in these systems. Electrical equipment must be three
pronged and well grounded. Avoid hanging extension cords, or
extension cords on the floor, where tank overflow could cause
them to get wet.
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3.5 Temperature
Water temperature in all tanks in the ERL - D fathead
culture unit is maintained at 25°C + 1°C for maximum egg
production and growth. Brungs (1971a) found that temperatures
below 22°C or above 26°C reduced fathead minnow reproduction.
The described temperature control system will maintain 25 + i°c.
Culture systems will function down to 22°C.
3.6 Aeration
Provide continuous gentle aeration to the tanks to maintain
dissolved oxygen concentrations above 5.0 mg/1 at all times, but
avoid vigorous aeration, especially with newly hatched larvae.
If a level of 5.0 mg/1 cannot be maintained, remove some fish
from the tank (Mount, 1971). Brungs (1971b), found that larval
growth was the most sensitive indicator of sublethal effects of
lowered dissolved oxygen, and significant effects were seen at. 5
mg/1. Provide an oil free air supply of at least 150 cc/min per
tank. An oil trap or filter may be necessary on some systems.
Check the location of air intakes and efficient operation of
laboratory air compressors to avoid introducing contaminants.
Arrange air tubing, brass air valves, and air stones,
(obtainable in aquarium stores) to provide an airline to each
tank. Incorporate a pressure regulator into the system and feed
each group of 6 or 12 tanks from a central, larger diameter
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manifold. A pressure regulator on the main manifold will allow
use of valves with slip-on tubing, without blowing the tubing
off.
3.7 Photoperiod
Use lights that simulate the wavelength spectra of sunlight.
A combination of Durotest Optima FS8 and wide spectrum Sylvania
Gro - Lux9 fluorescent tubes has proven satisfactory in the ERL -
D system. Light intensities at the water surface should average
400 - 500 lux. The photoperiod should be constant at 16 hours
light/8 hours dark (Mount, 1971). Gradual changes in light
intensity at dawn and dusk may be included within the photoperiod
if desired (see Drummond and Dawson, 1970). Paragon Model #4001
timers10 control photoperiod in the ERL - D system. Lighting
warms the culture water, and this effect must be considered as
part of the overall design.
3.8 Spawning Substrates
Since fathead minnows deposit their adhesive eggs on the
underside of submerged or floating objects, the spawning
substrate provided must yield a similar surface. The ERL - D
culture system uses fiber cement water pipe, 7.6 to 10.2 cm in
diameter, cut into 7-10 cm long sections. When halved
lengthwise and inverted, these pipe sections form a semicircular
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arch. Benoit (1982), used stainless steel (16 - 18 gauge), cut
and bent into the same size and shape with a thin layer of quartz
sand glued on the underside with silicone glue. This type of
substrate can come unglued after prolonged use, however. Gale
and Bunyak (1982) , successfully used PVC pipe in the same
configuration. (See Photograph #2 for general view of spawning
and egg incubation apparatus).
3.9 Fail Safe Devices
An alarm system or fail-safe device will protect against
temperature control or water flow failure. Temperature
recorders11 monitored hourly by security personnel are in use at
ERL - D. Alternatively, use high - low alarm systems, or solenoid
valves to shut off water to the tanks if the temperature rises
above 28° or falls below 20°C.
3.10 Tank Cleaning
ERL - D personnel scrape tanks and siphon residue on a
biweekly, rotating schedule. Some tanks are siphoned between
cleanings if necessary. Most culturists allow light growths of
algae, rotifers, etc, to remain, for these provide a dietary
supplement for fish. Excessive growths of blue-green algae or
fungus must be removed. Test cleaning tools for possible toxicity
before use.
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3.11 Disturbance
*
Shield the fish from continual or drastic disturbance. Avoid
construction noises, continual human presence, and extraneous
lights that might alter the photoperiod.
3.12 Brine Shrimp (Artemia spp.) Hatchery
The brine shrimp hatchery at ERL - D consists of a
rectangular fiberglass tank, 2.1 m x 0.6 m x 0.5 m deep, that
serves as a water bath. (See Photograph #3). Rails mounted on
the inside, 9 cm down from the top, support 10 to 12 movable
plywood racks (53 cm x 15 cm). Each of these racks has three
11.5 cm diameter holes that hold brine shrimp hatching jars., A
manifold of airline tubing provides an air supply to each
hatching jar.
Brine shrimp will hatch in any container with a conical
bottom, as long as air bubbled at the bottom of the cone keeps
the eggs in constant motion. Some culturists use separatory
funnels, and others use inverted 1 liter plastic soda bottles
with the bottoms cut out.
The hatching jars in use at ERL - D are made from round
bottom glass light fixtures12, about 11 cm in diameter and 24 cm
long. A ring of airline tubing glued around the jar 5 cm from
the open end suspends each jar through a hole in the plywood
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rack, partially submerged in the water bath. The capacity of each
jar is about 1.5 liters. This system provides for up to 36
hatching jars. A temperature probe and controller system similar
to the headboxes feeding the fish tanks maintains the bath at
25°C.
BIOLOGICAL SYSTEM
4.l 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 bioaccumulation of toxicants that
may occur with adults. Less desirable is the use of fish
captured in the wild or purchased from a bait dealer. Take care
to verify that the animals are PAniejDliaJ.es^ p_romej.as^ and not a
related species. Examine all fish, and especially wild caught
or bait dealer fish, for signs of disease. Use prophylactic
treatment (described in section 4.7) to insure good health.
These fish should then be bred through one full generation to
determine vigor, fecundity, and freedom from disease before use
in toxicity testing.
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4.2 Selection of spawning fish
Determination of the sex of immature fathead minnows is
nearly impossible, which makes it difficult to select mating
pairs before the fish actually begin breeding. Breeding males
develop a conspicuous gray pad of spongy tubercles on the dorsal
surface anterior to the dorsal fin, and two rows of strong
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). (See Photographs #4 & 5, and Figure #2).
Breeder fish are selected as follows: juvenile fish 3-4
months old are stocked at a density of 30 - 40 fish per 15 gallon
T
tank, and provided with 2 or 3 spawning substrates as previously
described. The presence of spawning substrates hastens the
maturation process. In 1 - 2 weeks some males in the tank will
show signs of maturing. Females will begin to 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 approximately 3 cm of water. Sexual maturity can
then be determined as previously described. Backlighting makes
the female ovipositor easier to see.
Removal of the mature fish for service as spawners will
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stimulate the subordinate juveniles in the brood stock tanks to
ripen, and take the places of the dominant fish that were removed
from the hierarchy. This method will provide a continuous source
of mature fish. Some of each month's leftover larvae or
juveniles not used in testing must be set up in brood stock
tanks to begin the maturation process. The ERL - D system uses
16 brood tanks, along with 8 larger holding tanks (208 liters)
for bringing fish to maturity.
4.3 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.
Fathead minnows usually spawn beneath objects, even under
such transient objects as old maple and oak leaves (Isaak, 1961).
Males clean the underside of the object selected for the nest
site using the head tubercles in a scraping action and pulling
pieces of algae and associated debris off the nest surface with
the mouth (Andrews and Flickinger, 1973). The male and female
swim back and forth beneath the prepared overhead site and roll
on their sides to emit the sex products. Close lateral contact
and body vibration characterize the act of spawning. McMillan
(1972) describes the spawning act as follows: "Finally, when a
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sufficient degree of vibratory stimulation has been reached, the
male lifts and presses the female's ventral surface against the
object's underside. In doing so, he turns so that he is beneath
the female and can use the posterior part of his body to
manipulate her upward. The tubercules on his large pectoral fins
also help him to grip the female tightly. As the fishes' bodies
are taut and strained in this position, the female emits one or
perhaps several eggs, and the male probably releases sperm at
this instant. Then they abruptly separate, although a new bout
of vibrating may begin only seconds later."
The buoyant, adhesive embryos stick to each other 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 the 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.
Egg counts in wild mature females have ranged from 800 to
9,000 eggs per female, depending on size of female, water
temperature, geographic strain, etc. Unpublished data indicate
an average of 258 eggs/spawn and an average of 3095 total
eggs/female during 100 days of spawning activity (Olson, 1974).
For spawning tanks, a standard tank is divided into 4
chambers as previously described. Each spawning chamber is then
approximately 15 cm x 30 cm. A spawning substrate, and a pair of
sexually mature fish is placed in each section, for a total of
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four pairs per spawning tank. There are 24 spawning tanks in the
ERL - D system, for a total of 96 spawning pairs.
Separation into spawning pairs is not essential. 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 territorial, so at least as many
spawning substrates must be provided as there are males in the
tank to achieve optimum egg production.
Paired spawning reduces fighting and competition between
males. When ERL - D began to use the paired method, the number
of embryos produced in the culture unit almost doubled (Benoit,
1982). Paired spawning also allows the culturist to follow the
fecundity of individual fish. Sterile or "spawned out" fish
identified through daily spawning records can then be replaced to
maintain egg production. A good procedure is to review daily
spawning records every three weeks. If a pair has not spawned
during that period, discard both male and female and replace
with new spawners.
4.4 Embryo Incubation
Each morning at approximately 10:30 am (7 days/week), all
spawning tiles are checked for the presence of embryos. It is
not necessary to remove the tile from the tank to see the
embryos. Gently running the fingertips on the undersurface of
the spawning substrates will reveal their presence. If no
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embryos are present, the spawning substrate is left in the tank.
This creates less disturbance than daily removal, inspection, and
replacement of each substrate (See Photograph #6).
When checking for embryos, estimate and record the number
deposited by each spawning pair. Provide fresh tiles for each
chamber from which embryos were removed. Researchers requiring
embryos less than 24 hrs old for testing can take charge of the
embryos at this point. If the embryos are to be incubated, leave
them on the spawning tiles, and place the tiles in the dated
hatching tray. Wipe the tops of the tiles clean with a sponge
to reduce transfer of sediment to the hatching pan. Place tiles
on end, with two tiles pushed together to form a circle, and
place an airstone between them to circulate water. (See
Photograph #7). Inspect embryos daily, and remove any dead ones
with tweez€srs to decrease the spread of fungus. (Nonviable
embryos are opaque or clear with a white "dot" inside where the
yolk has precipitated. Viable embryos will be clear for the
first 36-48 hrs until they reach the eyed stage). If fungus
has attacked more than 50% of the embryos on a particular tile,
discard the entire spawn on that tile.
At 22°C embryos will begin to hatch in 5 days. If
embryos have been removed from spawning chambers each day, so
that the age of each group is known to within 24 hours, then 95%
should hatch within a 24 hour period. If a toxicity test is
planned that calls for less than 24 hr. old larvae, provide the
fish to the researchers at this point. If larvae are to be
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Photograph 6. Fathead minnow embryos on the underside of a
spawning substrate.
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-5
3
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reared to 30 days old for testing as juveniles, or to adulthood
for future brood stock, remove them from the hatching tray using
a large bore, 50 ml volumetric pipette. Count larvae into lots
of 250, and stock into rearing tanks (15 gal tanks with appx 20
cm water depth). Label each rearing tank with the date of hatch.
Thirty day old juveniles that weigh approximately 150 mg each
(wet weight) are the desired goal, although fish in the range of
100 to 300 mg per fish are acceptable (EPA, 1982).
Clean spawning tiles after each use by soaking them in a
mild HTH13 bath (sodium hypochlorite, appx 12 g HTH/liter) for 1
hour. Follow with a tap water rinse, and then soak tiles in
appx. 5 x 10~3 M sodium thiosulfate for at least 10 minutes to
remove residual chlorine. After a final rinse in culture water,
set tiles aside to dry and store for reuse.
4.5 Larval Feeding
All fathead minnow larvae less than 30 days old are fed live
brine shrimp twice each day. Since larvae begin feeding during
the first day of hatch, feeding must start immediately. It is
important that the brine shrimp nauplii be small enough for the
fathead larvae to ingest (Norberg and Mount, 1985). Gape size of
larval fathead minnows is in the range of .24 - .28 mm, so the
"width" of the nauplii when offered must be slightly less than
this for the nauplii to be ingested. Growth rates depend on
the brine shrimp nauplii used, since different strains of the
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cysts vary in nutritional adequacy (ASTM Draft No. 2, 1984).
The brand now in use by the ERL - D fathead culture unit is
Biomarine1 which has yielded more uniform growth than some
others. Any strain that offers a good hatching percentage, small
size of nauplii upon hatch, nutritional adequacy, and freedom
from contaminants will yield good results. Some culturists also
supplement the larval diet with powdered or flake foods to insure
a more rounded diet. Opinions vary as to the efficacy of this
supplementary feeding. Some trout chow formulations have proven
to be inadequate fathead minnow diets in experiments in our
laboratories.
Conditions for hatching each strain of cysts are usually
provided by the supplier. Live nauplii are harvested from 24 -
48 hrs after set-up at 25°C. To harvest, remove the air tube from
the hatching jars approximately 15 minutes before feeding, to
allow the live shrimp to settle. Unhatched cysts will form a
brown layer at the bottom of the hatching jar, while the live
shrimp will form a bright orange layer just above the unhatched
cysts. The layer of live shrimp is then siphoned from the
hatching jars into a 1 liter beaker. Rinsing the brine shrimp is
not necessary in flow-through systems with large tanks. In
static systems rinsing is desirable to avoid salt build up.
Exact quantification of shrimp fed to each tank is not necessary
as long as all fish receive an adequate amount. Inspect tanks 10
- 15 minutes after feeding. If all shrimp have been eaten in
this short time, provide more. It is important that larvae be
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feed ad libitum, especially during the first few weeks of life.
Approximately 50 ml of the concentrate is fed to each tank of
250 larvae, twice per day. Larvae less than 1 week old require
slightly less. Nine 1.5 liter jars started with approximately 25
cc of eggs each, provide two feedings per day for 60 tanks of 250
larvae each. Different brands, feeding schedules, and stocking
densities may alter the amount required.
Adult Feeding
Feed spawning pairs, replacement brood stock, and any other
T ^
fish over 30 days old frozen brine shrimp-1- twice per day.
Allow frozen brine shrimp to thaw slightly (not fully) for ease
of handling. Feed fish ad libitum, with each spawning pair
receiving 1/8 - 1/4 teaspoon, and other tanks according to number
of fish. A rule of thumb is that the right amount of food will
be consumed in about 10 minutes.
4.7 Disease
Discard any diseased lots of fish and disinfect the tanks
with hypochlorite. To minimize the risk of spreading disease
between tanks, disinfect all nets, siphons, brushes, and other
tools if disease is thought to be present. Adding fifty ml
formalin16, and 10 ml Roccal II17 to 50 liters of water in a
plastic garbage can makes a handy disinfectant bath.
29
-------
Fungus on eggs is not considered a disease (Mount, 1971).
Fungus is ubiquitous in the aquatic environment, and its presence
should not be considered a valid reason to discard all
associated embryos unless the labor involved in picking and
sorting out the bad embryos is not worth the return at hatch.
Examine fins, skin, and gills of fish from random tanks
monthly for parasites. Perform other disease checks sufficient to
assure that disease is not a problem. If present, it is best to
destroy fish from infected tanks. Treatment is in order only if
the fish cannot be sacrificed.
Treated fish are rarely, if ever, used in bioassays.
Disease treatment does have a place, however, for fish brought in
from the wild. When bringing in wild brood stock the following
prophylactic treatments may be useful. First, bring fish to
25°C from their arrival temperature, at a rate not to exceed 2°C
per day. After an extra day at 25° for acclimation, disease
treatments begin, following the methods of ORSANCO (1974).
Perform disease treatments on incoming fish in a quarantined
area, away from the main culture unit, if possible.
Treat fish with potassium permanganate for external
parasites. Using a 1% KMnO4 solution, add 1.0 ml per liter of
water. The water will turn dark purple, and must be aerated
vigorously during the treatment. After 1/2 hour, neutralize the
KMn04 with .01 ml/liter of 0.1N sodium thiosulfate. Within 15 -
20 minutes the solution will turn yellow - brown. Siphon the
tank or drain it down as far as possible without stressing the
30
-------
fish, and then refill. Give this treatment for two consecutive
days, and curtail feedings during this time.
Once a day for the next four consecutive days use a
tetracycline bath, at the rate of 25 mg/liter, for bacterial
control. After a one hour static bath turn the water on, so that
the initial dose is being slowly diluted. Do not drain the tanks
after this treatment, and feed the fish during the treatment.
Tetracycline can also be added to the feed as an antibacterial
drug against Aeromonas, Hemophi].us, and Pseudomonas. The
recommended dose is 2.5 - 3.75 g/45 kg fish/day for 10 days, in
feed (Schnick, et al, 1986).
4 .8 Gene pool
The Environmental Research Laboratory - Duluth periodically
(every 2 years) mixes existing brood stock with healthy wild
minnows to eliminate the risk of developing a homogenous strain
(Benoit, 1982). Homogenous genetic stock may provide smaller
variance in test results, but it correspondingly reduces the
strength of possible inferences that can be made. Excessive
inbreeding leads to increased rate of genetic deformities. When
bringing in fish for the infusion of new genes make every attempt
to obtain the healthiest fish possible. Fish that show any sign
of disease, or that have a known history of exposure to disease,
dissolved oxygen stress, or exposure to other environmental
perturbations must be rejected. Fish that are selected for the
31
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outbreeding process must be subjected to the previously described
temperature acclimation and disease treatment measures.
After acclimation and treatment, set up the new brood stock
in an area separate from the main culture unit. Maintain these
fish in quarantine, and breed through the second generation using
the same procedures as in the main culture unit. When second
generation fish are sexually mature, mix in spawning pairs with
original brood stock, in combinations of new male/existing female
and new female/existing male. The generation resulting from these
crossings will be the new brood stock for the main culture unit.
Replace one fourth of the spawning pairs in the culture unit
every three weeks until all pairs have been changed. Consult
researchers using the fish in toxicity tests so that they are
aware that new genes will be entering the pool.
4.9 Record keeping
Maintain records on the estimated number of eggs spawned
each day by each spawning pair. Also record the date that each
spawning pair was introduced, and the dates on which each holding
tank was stocked with larvae. Measure and record routine water
chemistries (pH and D.O.) at least twice weekly on random tanks.
Records on source of new brood stock, date of arrival, disease
treatments, and procedures used for mixing into the gene pool are
also important. Record all mortalities in the culture unit, and
observe fish daily for abnormal appearance or behavior.
32
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LITERATURE CITED
Allison, D.T., and R.O. Hermanutz. 1977. Toxicity of diazinon to
brook trout and fathead minnows. U.S. Environmental Protection
Agency. EPA - 600/3 - 77 - 060.
American Society for Testing and Materials (ASTM). 1984. Proposed
new standard practice for the use of brine shrimp nauplii as food
for test animals in aquatic toxicology. Draft No. 2, August, ASTM
Subcommittee E47.01 on Aquatic Toxicology.
Andrews, A.K., and S.A. Flickinger. 1973. Spawning requirements
and characteristics of the fathead minnow. Proc. 27th. Ann. Conf.
SE Assoc. Game and Fish Comm. pp 759 - 766.
Benoit, D.A., and R.W.Carlson. 1977. Spawning success of fathead
minnows on selected artificial substrates. Prog. Fish. Cult.
39(2).67 -69.
Benoit, D.A. 1982. User's guide for conducting life cycle chronic
toxicity tests with fathead minnows (Pimephales promelas). U.S.
Environmental Protection Agency. EPA - 600/8 - 81 - Oil. 17pp.
Brungs, W.A. 1971a. Chronic effects of elevated temperature on
the fathead minnow (Pimephales promelas Rafinesque). Trans. Am.
Fish. SOC. 100(4). 659 - 664.
Brungs, W.A. 1971b. Chronic effects of low dissolved oxygen
concentrations on the fathead minnow, (Pimephales promelas). J.
Fish. Res. Bd. Canada. 28. 1119 - 1123.
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.
Eddy, S., and J.C. Underbill. 1974. Northern fishes. Univ. Minn.
Press, Mpls., MN. 414pp.
EPA, 1982. Culture of 30 day old fathead minnows for use in acute
tests at ERL - D. Standard operating procedure #CR - 01 - 01.
U.S. Environmental Protection Agency, Duluth, MN 55804. 2pp.
Flickinger, S.A. 1969. Determination of sexes in the fathead
minnow. Trans. Am. Fish. Soc. 3. 526 - 527.
Gale, W.F., and G.L.Bunyak. 1982. Fecundity and spawning
frequency of the fathead minnow - a fractional spawner. Trans.
Am. Fish. Soc. 111. 35 - 40.
33
-------
Glass, G.E. 1977. Identification and distribution of inorganic
components in water: what to measure? Ann. NY Acad. Sci. 298. 31
-46.
Hasler, A.D., H.P. Thomsen, and J.C.Neess. 1946. Facts and
comments on raising two common bait minnows. Wis. Cons. Bull.
#210 - A - 46. 12 pp.
Isaak, D. 1961. The ecological life history of the fathead
minnow, P!roejoha].es P_rome_las (Raf inesque). Ph.D. Thesis, Univ.
Minn. 150 pp.
McCormick, J.H., and R.F. Syrett. 1970. A controlled temperature
apparatus for fish egg incubation and fry rearing.
manuscript.U.S. Environmental Protection Agency, Duluth, MN
McMillan, V. 1972. Mating of the fathead. Nat. Hist. 81(5).73 -
78.
Mount, D.I. 1971. Tentative plans for the design and of operation
of a fathead minnow stock culture unit. U.S. Environmental
Protection Agency, Duluth, MN 55804. 7pp.
Norberg, T.J., and D.I. Mount. 1985. A new subchronic fathead
minnow (PiraepJiaJ^s P_rome,lasJ toxicity test. Env. Tox. Chem. 4.
711 -718.
Olson, D.T. 1974. Fathead minnow egg production as a function of
the number of females per spawning chamber. Interim report of the
fathead minnow methods study, Part I. 15pp. U.S. Environmental
Protection Agency, Duluth, MN 55804.
ORSANCO. 1974. 24 hour bioassay. Ohio River Valley Sanitary
Commission. Cincinnati, OH 45202. 21pp.
Radcliff, L. 1931. Propagation of minnows. Trans. Amer. Fish.
Soc. 61. 131 - 137.
Schnick, R.A., F.P. Meyer, and D.F. Walsh. 1986. Status of
fishery chemicals in 1985. Prog. Fish. Cult. 48(1). l - 17.
Syrett, R.F., and W.F. Dawson. 1972. An inexpensive electronic
relay for precise water temperature control. Prog. Fish. Cult.
34(4). 241 - 242.
Syrett, R.F., and W.F. Dawson. 1975. An inexpensive solid state
temperature controller. Prog. Fish. Cult. 37(3). 171 - 172.
34
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ADDITIONAL READING
Adelman, I.R., and L.L. Smith, Jr. 1976. Fathead minnows
(Piro^Ekf*.!6.^ E££3l®.ll:fL) and goldfish (Carass_ius auratus) as
standard fish in bioassays and their reaction to potential
reference toxicants. J. Fish. Res. Board Can. 33. 209-214.
Andreasen, J.K. 1975. Occurrence of the fathead minnow,
Pimephales promelas, in Oregon. Calif. Fish and Game. 61(3). 155-
156.
Andrews, A.K. 1970. The distribution and life history of the
fathead minnow (Pimejohaleis promelas Rafinesque) in Colorado.
Ph.D. Thesis. Colorado State Univ. Fort Collins. 131pp.
Andrews, A.K. 1970. Squamation chronology of the fathead minnow,
Pimephales promelas. Trans. Am. Fish. Soc. 99(2). 429-432.
Andrews, A.K. 1971. Altitudinal range extension for the fathead
minnow (Pimephales promelas). Copeia. 1. 169.
Andrews, A.K. 1972. Survival and mark retention of a small
cyprinid marked with flourescent pigments. Trans. Am. Fish. Soc.
101(1). 128-133.
Bernstein, J.W., and R.J.F. Smith. 1983. Alarm substance cells in
fathead minnows do not affect the feeding preference of rainbow
trout. Environ. Biol. Fish. 9(3-4). 307-311.
Brauhn, J.L., and R.A. Schoettger. 1975. Acquisition and culture
of research fish: rainbow trout, fathead minnows, channel
catfish, and bluegills. US Env. Prot. Agcy. EPA-660/3-75-011.
45pp.
Brown, B.E. 1970. Exponential decrease in a population of fathead
minnows. Trans. Am. Fish. Soc. 99(4). 807-809.
Chiasson, A.G., and J.H. Gee. 1983. Swim bladder gas composition
and control of buoyancy by fathead minnows (Pimephales promelas)
during exposure to hypoxia. Can. J. Zool. 61(10). pp 2213-2218.
Coble, D.W. 1970. Vulnerability of fathead minnows infected with
yellow grub to largemouth bass predation. J. Parasit. 56(2). 395-
396.
Coyle, E.E. 1930. The algal food of PiinejDha3.es promejjas. Ohio J.
Sci. 30(1). 23-35.
Dixon, R.D. 1971. Predation of mosquito larvae by the fathead
minnow, Pimephales promelas Rafinesque. Manit. Entomol. 5. 68-70.
35
-------
Flickinger, S.A. 1973. Investigation of pond spawning methods for
fathead minnows. Proc. 26th. Ann. Conf. SE Assoc. Game and Fish
Comm. 376-391.
Cast, M.H., and W.A. Brungs. 1973. A procedure for separating
eggs of the fathead minnow. Prog. Fish Cult. 35. 54.
Gee, J.H. 1977. Effects of size of fish, water temperature, and
water velocity on buoyancy alteration by fathead minnows,
Pimephales promelas. Comp. Biochem. Physiol. A56(4). 503-508.
Gee, J.H., R.F. Tallman, and H.J. Smart. 1978. Reactions of some
great plains fishes to progressive hypoxia. Can. J. Zool. 56.
1962-1966.
Grabar, F., and R.A. Flickinger. 1977. Replicon number and growth
rate in fathead minnow cells cultured at 14 and 34°C. Cell Tissue
Kinet. 10(5). 505-507.
Gravell, M., and R.G. Malsberger. 1965. A permanent (epithelial)
cell line from the fathead minnow (Pi.m ep_ha 3. es
promelas)(cultivation method, temperature control, chromosome
analysis, virus susceptibility). Ann NY Acad Sci. 126(1). 555-
565.
Guest, W.C. 1977. Technique for collecting and incubating eggs of
the fathead minnow. Prog. Fish. Cult. 39(4). 188.
Hart, J.S. 1947. Lethal temperature relations of certain fish of
the Toronto region. Trans. Roy. Soc. Can. 91(3). 57-71.
Held, J.W. 1971. Some ecological aspects of the fathead minnow,
Piro^Eli^i6-3- PJ-!P.S!®.l3ts Rafinesque, in North Dakota saline lakes.
Ph.D. Thesis. North Dakota State Univ. Fargo, ND. 80pp.
Held, J.W., and J.J. Peterka. 1974. Age, growth, and food habits
of the fathead minnow, P_imej3haJ.es p_romeJLa£, in North Dakota
saline lakes. Trans. Am. Fish. Soc. 103(4). 743-756.
Hendrickson, G.L. 1979. Ornj-thodi-p^ostomum ptychocheilus;
migration to the brain of the fish intermediate host, Pimephales
promelas. Exp. Parasit. 48. 245-258.
Hoffman, G.L. 1958. Studies on the life eye], e of
2£Hi^ho^i£l.2st°lH51 ptychocheilus (Faust) (TrematodarStrigeoidea)
and the "self cure" of infected fish. J. Parasit. 44(4). 416-421.
Ingram, R., and W.D. Wares,II. 1979. Oxygen consumption in the
fathead minnow (Pimep_hales p_romelas Rafinesque) II: Effects of
pH, osmotic pressure, and light level. Comp. Biochem. Physiol.
62A. 895-897.
36
-------
Kelly, R.K., B.W. Souter, and H.R. Miller. 1978. Fish cell lines:
comparisons of CHSE-214, FHM, and RTG-2 in assaying IHN and IPN
viruses. J. Fish. Res. Board Can. 35(7). 1009-1011.
Klak, G.E. 194Q.Neascus infestation of blackhead, blunt nosed,
and other forage minnows. Trans. Amer. Fish. Soc. 69. 273-278.
Klinger, S.A., J.J. Magnuson, and G.W. Gallepp. 1982. Survival
mechanisms of the central mudminnow (Umbra limi), fathead minnow
(Pimephales promelas), and brook stickleback (Culea inconstans)
for low oxygen in winter. Environ. Biol. Fish. 7(2). 113-120.
Konefes, J.L., and R.W. Bachmann. 1972. Growth of the fathead
minnow (Pimephales promelas) in tertiary treatment ponds. Proc.
Iowa Acad. Sci. 77. 104-111.
Lemke, A.E., W.A. Brungs, and B.J. Halligan. 1978. Manual for
construction and operation of toxicity testing proportional
diluters. US Env. Prot. Agcy. EPA-600/3-78-072.
Lewis, D.H., and N.L. Savage. 1972. Detection of antibodies to
Aeromonas iiquefjici.ens in fish by an indirect flourescent
antibody technique. J. Fish. Res. Board Can. 29(2). 211-212.
Lord, R.F., Jr. 1927. Notes on the use of the blackhead minnow,
PimephjQes P_romel.asj, as a forage fish. Trans. Am. Fish. Soc. 57.
92-99.
Luoma, M.E., and J.H. Gee. 1980. Seasonal factors affecting
buoyancy attained in still water and current by fathead minnows,
Pimephales promelas. Can. J. Fish. Aquat. Sci. 37. 670-678.
Manner, H.W., and C.M. Casimira. 1974. Early embryology of the
fathead minnow Pimephales promelas Rafinesque. Anat. Rec. 180(1).
99-109.
Manner, H.W., and C. Muehlman. 1975. Permeability and uptake of
3H-uridine during teleost embryogenesis. Sci. Biol. J. 1(3). 81-
82.
Manner, H.W., M. VanCura, and C. Muehlman. 1977. The
ultrastructure of the chorion of the fathead minnow, Pimephales
promelas. Trans. Amer. Fish. Soc. 106(1). 110-114.
Marcus, L.F., and J.H. Vandermeer. 1966. Regional trends in
geographic variation. Syst. Zool. 15(1). 1-13.
Markus, H.C. 1931. Life history of the blackhead minnow,
Pimephales promelas. Copeia. 3. 116-122.
Mayer, F.L. 1976. 2,4-D reduces SajDro_legji.ia on fathead minnow
eggs. Prog. Fish. Cult. 38(1). 19.
37
-------
Merritt, R.B. 1972. Geographic distribution and enzymatic
properties of lactate dehydrogenase allozymes in the fathead
minnow, Pimephales promelas. Am. Nat. 106(948). 173-184.
Meyer, F.P. 1958. Helminths of fishes from Trumbull Lake, Clay
County, Iowa. Proc. Iowa Acad. Sci. 65. 477-516.
Minchew, C.D. 1981. Unicauda magna sp.n. (Protozoa: Myxozoa): A
new Myxozoan from the fin tissues of the fathead minnow,
Pimephales promelas Rafinesque. J. Fish. Dis. 4(6). 513-518.
Ming, F.W, and D.L.G. Noakes. 1984. Spawning site selection and
competition in minnows (P_imepjia_les notatus and P^ prgmejlas)
(Pisces, Cyprinidae). Biol. Behav. 9(3). 227-234.
Mitchell, A.J., C.E. Smith, and G.L. Hoffman. 1982. Pathogenicity
and histopathology of an unusually intense infection of white
grubs (Posthodi.p_lostomum m. m_in_imum) in the fathead minnow
(Pimephales promelas). J. Wildl. Dis. 18(1). 51-57.
Mitchell, L.G., C.L. Seymour, and J.M. Gamble. 1985. Light and
electron microscopy of Myxobolus hendri.cksoni (Myxozoa,
Myxobolidae) infecting the brain of the fathead minnow,
Pimephales promelas Rafinesque. J. Fish. Dis. 8(1). 75-89.
McCarraher, D.B., and R. Thomas. 1968. Some ecological
observations on the fathead minnow, Pimephales promelas, in the
alkaline waters of Nebraska. Trans. Am. Fish. Soc. 97(1). 52-55.
McMillan, V.E. 1972. An ethological study of agonistic and
reproductive behaviour in the fathead minnow (Pimephales promelas
Rafinesque). Ph.D. Thesis. Univ. Saskatchewan, Saskatoon, Sask.,
Canada. 147 pp.
Nagel, T. 1976. Technique for collecting newly hatched fathead
minnow fry. Prog. Fish. Cult. 38(3). 137.
Niazi, A.D. 1964. The development of the Weberian system and
early embryology of Pimeghal.es P_rome_las (Osteichthyes:
Cyprinidae). Diss. Abstr. 25(4). 2674.
Peterson, D.L., and F.A. Hennagir. 1980. Minnesota live bait
industry assessment study. Proj. #30261-R. Minn. Dept. Natl.
Res., St.Paul, MN. 100pp.
Prather, E.E. 1958. Preliminary experiments on winter feeding
small fathead minnows. Proc. llth. Ann. Conf. SE Assoc. Game and
Fish Comm. 249-253.
Prather, E.E. 1959. Further experiments on feeds for fathead
minnows. Proc. 12th. Ann. Conf. SE Assoc. Game and Fish Comm.
176-178.
38
-------
Radabaugh/ D.C. 1980. Changes in minnow, P^me^
Rafinesque, schooling behaviour associated with infections of
brain-encysted larvae of the fluke, Orni.thodi^jDn.ostoinuin
ptychocheilus . J. Fish. Biol. 16(6). 621-628.
Richardson, L.R. 1937. Observations on the mating and spawning of
Pimephales promelas (Rafinesque). Can. Field-Natur. 51(1). 1-4.
Scott, W.B., and E.J. Grossman. 1973. Freshwater fishes of
Canada. Bulletin 184. Fish. Res. Board Can. Ottowa. 966pp.
Smith, H.T., C.B. Schreck, and O.E. Maughan. 1978. Effect of
population density and feeding rate on the fathead minnow
(Pimephales promelas) . J. Fish. Biol. 12. 449-455.
Smith, R.J.F. 1970. 1970. A technique for marking small fish with
injected flourescent dies. J. Fish. Res. Board Can. 27(10). 1889-
1891.
Smith, R.J.F. 1973. Testosterone eliminates alarm substance in
male fathead minnows. Can. J. Zool. 51(8). 875-876.
Smith, R.J.F. 1974. Effects of 17-methyltestosterone on the
dorsal pad and tubercles of fathead minnows (Pimegha^les
promelas) . Can. J. Zool. 52(8). 1031-1038.
Smith, R.J.F. and B.D. Murphy. 1974. Functional morphology of the
dorsal pad in fathead minnows (PimejohaJ.es Erome^as) . Trans. Am.
Fish. Soc. 103(1). 65-72.
Smith, R.J.F. 1976. Male fathead minnows (Pimejoh^less ...
Rafinesque) retain their fright reaction to alarm substance
during the breeding season. Can. J. Zool. 54. 2230-2231.
Smith, R.J.F. 1978. Seasonal changes in the histology of the
gonads and dorsal skin of the fathead minnow, P_imepha.,le£
promelas. Can. J. Zool. 56. 2103-2109.
Snyder, D.E., M.B.M. Snyder, and S.C. Douglas. 1977.
Identification of golden shiner, Notemigonus crysoleucas, spotfin
shiner, NotroEis. j^i-koj^t6.^3.' anc* fathead minnow,
promelas, larvae. J. Fish. Res. Board. Can. 34(9). 1397-1409.
Solis, J. de lac. 1969. Propagation and characterization of
animal viruses in a poikilothermic cell line from the fathead
minnow, (Pimephales promelas) . Diss. Abstr. Intl. 30B. 312-313.
Sullivan, J.F., and G.J.Atchison. 1978. Predator - prey behaviour
of fathead minnows, PAieEhales P_romeJL.a£, and largemouth bass,
Micropterus sa:L5!oJ:
-------
Sykora, J.L., E.J. Smith, and M. Synak. 1972. An inexpensive fish
egg incubator. Prog. Fish. Cult. 34(3). 183-184.
Tallman, R.F., K.H.Mills, and R.G. Rotter. 1984. The comparative
ecology of pearl dace (Se?notd.l.us margarrta) and fathead minnow
(Pimephales promelas) in lake 114, the Experimental Lakes Area,
northwestern Ontario, with an appended key to the cyprinids of
the Experimental Lakes Area. Can. Manuscr. Rep. Fish. Aquat. Sci.
1756.
Till, J.E. 1977. Laboratory technique for obtaining fathead
minnow eggs for use in toxicity experiments. Prog. Fish. Cult.
39(1). 24-27.
Tsai, C. 1971. Occurrence of the fathead minnow, Pimegha^es
promelas (Pisces: Cyprinidae), in the Chesapeake Atlantic slope
drainages. Chesapeake Science. 12(4). 274-275.
Unger, L.M. 1983. Nest defense by deceit in the fathead minnow,
Pimephales promelas. Behav. Ecol. Sociobiol. 13(2). 125-130.
Vandermeer, J.H. 1966. Statistical analysis of geographic
variation of the fathead minnow, Pimephales promelas. Copeia. 3.
457-466.
Vaughan, G.E., and D.W. Coble. 1975. Sublethal effects of three
ectoparisites on fish. J. Fish. Biol. 7(3). 283-294.
Wabuke-Bunoti, M.A.N., and C.E. Firling. 1983. The prehatching
development of the thyroid gland of the fathead minnow,
Pimephales promelas (Rafinesque). Gen. Comp. Endocrinol. 49. 320-
331.
Walker, E.R., S.F. Fidler, and D.E. Hinton. 1981. Morphology of
the buccopharyngeal portion of the gill in the fathead minnow
Pimephales promelas (Rafinesque). Anat. Rec. 200. 67-81.
Wares, W.D.II., and R. Ingram. 1979. Oxygen consumption in the
fathead minnow, (Pimephales promelas Rafinesque)- I: Effects of
weight, temperature, group size, oxygen level, and opercular
movement rate as a function of temperature. Comp. Biochem.
Physiol. 62A. 351-356.
Wynne-Edwards, V.C. 1932. The breeding habits of the black-headed
minnow (P_imej3haj.es PJZP.E^i3.3. Rafinesque). Trans. Am. Fish. Soc.
62. 382-383.
Yoakim, E.G., and J.M. Grizzle. 1980. Histological,
histochemical, and ultrastructural studies on the interrenal and
chromaffin cells of the fathead minnow, PAniepJia_les £rome].as
Rafinesque. J. Fish. Biol. 17(5). 477-494.
40
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P.O. Box 632
Cibolo, TX 78108
512/658 - 3503
16. HUMCO Laboratory Formalin
P.O. Box 2550
Texarkana, TX 75501
214/793 - 3174
17. National Laboratories
Lehri and Fink Industrial Prod. Div. Roccal II
Sterling Drug, Inc.
Montvale, NJ 07645
201/391 - 8500
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•&U.S. GOVERNMENT PRINTING OFFICE:1987/748-121/40684
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