analyzed in various common components of fish diets (Table 2). In the
past, we used the Cortland No. 7 diet (Phillips, personal communi-
cation)* that was formulated from. selected ingredients shown in Table
2. The reference research diet used presently at FPRL is outlined in
DIETS FOR EXPERIMENTAL FISH.

SCHEDULING

Even though hatchery fish are easily acquired, good planning is
necessary for the best sequence of availability, shipment, receipt,
quarantine, acclimation and final experimental use. Obtaining
immature or adult fish may require more than a year of advance sched-
uling with the supplying hatchery. Therefore, maintaining a continuous
flow of fish to the laboratory requires communication between the
researcher, laboratory culturist (if present), and the supplying
hatchery so that scheduling is periodically revised in light of

current research needs.

Availability of any particular life stage of fish varies with
latitude. For example, fingerling bluegills in southern states can
be obtained approximately 60 days earlier than those at more
northerly latitudes. Thus, with proper scheduling between southern
and northern hatcheries, bluegills of a particular size would be
available over 120 days.

Fish availability can also be extended by selecting strains with
different spawning times, or by manipulating normal reproductive cycles.
The scheduled acquisition of different strains insures adequate

* Dr. A. M. Phillips, Sr., U.S. Bureau of Sport Fisheries and Wildlife,
Tunison Laboratory of Fish Nutrition, Cortland, New York. 1967.

12


Rainbow trout for use in static, acute toxicity tests (0.5-1.5 g)
should be fed regularly for proper maintenance. In general, small
fish consume more of their daily ration if portions are fed several
times per day. Larger fish may be fed less often, down to once per
day as the trout approach sexual maturity (Buterbaugh and Willoughby,
1967; Phillips, 1970). Bumgarner (1971) controlled trout growth by
starvation. However, this practice must be used with caution because
disease resistance may be lowered by starvation. Nevertheless, the
technique may have some use in maintenance of rainbow trout, providing

an optimum physiological state is reached before research use.

RAINBOW TROUT EGGS

On occasion, it is more convenient to obtain rainbow trout by shipping
eggs, and specific studies may require eggs or alevins. Rainbow trout
eggs may be shipped 24-36 hours after fertilization, but then they
become more sensitive to disturbances during later development (Hayes,
1949). After eyes appear in the embryo, the eggs may be air-shipped
great distances with minimal losses.

Shipment is accomplished by gradually reducing egg temperature to 1°C,
packing the eggs in cheesecloth, placing an ample ice supply above the
eggs and then putting both eggs and ice in a well-insulated box. When
eggs are received, we reverse this procedure until the eggs reach 12°C,
the appropriate incubation temperature. First, the eggs are gently
removed from the shipping container and placed in an egg pan containing
water at the same temperature as the eggs. Optimal water quality is
similar to that for rainbow trout fingerlings. Then, clean water at
12°C is gradually added to limit the egg temperature increase to 2°C
per hour. The egg pan is also floated in a tank of 12°C water to

33


the Bureau of Sport Fisheries and Wildlife maintain well-qualified
disease diagnosticians who may be consulted. At the other extreme,
our investigators have experienced problems among fish that were
grossly over-treated by culturists with good intentions. Indeed,
compatible host-parasite relationships may be more representative of

fish found in natural ecosystems.

Unless seriously diseased fish are irreplaceable, they are immediately
discarded or quarantined to prevent transfer of the disease to healthy
fish. In exceptional situations, appropriate therapeutic measures are
initiated when the disease is diagnosed and are continued until symp-
toms disappear. Post-treatment observation for 10 days is advisable

before they are used for research.

Some therapeutants, particularly the antibiotics, may accumulate in
fish tissues (Herman et al., 1969). The residues may interact with
other compounds or cause side effects that will affect research

results. For this reason, the use of therapeutants on research fish
should be avoided whenever possible. If therapeutants are used,

records should be maintained and the investigator should be informed
of the disease, type of therapeutant, and its method of application.

FISH BEHAVIOR

Observations of fish behavior are a valuable tool to the culturist in
assessing the day to day condition of fish. However, the natural
behavior patterns of pond-reared fish are altered by placing them in
restricted areas. Culturists and researchers should be aware of the
behavior change. Experienced culturists know that abnormal behavior
may be the first indication of stress-producing factors.

29


SECTION VIII

REFERENCES

Allison, R. 1957. A preliminary note on the use of di-n-butyl tin
oxide to remove tapeworms from fish. Prog. Fish-Cult. 19(3):
128-129.

American Public Health Association. 1971. Standard Methods for the
Examination of Water and Wastewater. 13th ed. American Public
Health Assoc., Inc., New York. 874 p.

Beamish, F. W. H., and P. S. Mookherjii. 1964. Respiration of
fishes with special emphasis on standard oxygen consumption.

I. Influence of weight and temperature on respiration of
goldfish, Carassius auratus L. Can. J. Zoo. 42:161-175.

Brauhn, J. L. 1971. Fall spawning of channel catfish. Prog. Fish-
Cult. 33(3):150-152.

Brauhn, J. L., D. Holz, and R. O. Anderson. 1972. August spawning
of largemouth bass. Prog. Fish-Cult. 34(4):207-210.

Breder, C. M., Jr., and D. E. Rosen. 1966. The Modes of Reproduction
in Fish. The Natural History Press, Garden City, New York. 941 p.

Bumgarner, D. H. 1971. A technique for controlling trout growth.
Prog. Fish-Cult,, 53(1):41.

Buss, K., and J. E. Wright. 1956. Results of species hybridization
within the family Salmonidae. Prog. Fish-Cult. 18(4):149-158.

Buss, K., and D. Waite. 1961. Research units for egg incubation
and fingerling rearing in fish hatcheries and laboratories.
Prog. Fish-Cult. 23(2) :83-86.

Buterbaugh, G. C., and H. Willoughby. 1967. A feeding guide for brook,
brown, and rainbow trout. Prog. Fish-Cult. 29(4):210-215.

38


aquifer and used once in a flow-through system. We think this source
is superior to most surface waters, shallow wells, or springs that
could be intermittently contaminated. Water from deep wells may
require aeration to increase dissolved oxygen, or to eliminate

supersaturation of dissolved gases.

Water of excellent quality and quantity may be rendered useless for
fish if pipes and valves that release heavy metals such as zinc
(Pickering and Vigor, 1965: Eisler, 1967) or other contaminants are
used. Vinyl plastic pipes, particularly the softer plastic, should
not be used because these materials continually contribute plasti-
cizers such as di-2-ethylhexyl and di-n-butyl phthalates to water
(Mayer et al., 1972; Mayer and Sanders, 1973; Sanders et al., 1972;
Stalling et al., 1973). Black iron, high density polyethylene and
polypropylene piping do not contaminate water. Teflon (polytetra-

fluorethylene) and some types of nylon pipe are suitable but high
cost limits their use.


TECHNICAL REPORT DATA .
(Please read Instructions on the reverse before completing)

7 REPORT NO.
EPA-660/3-75-011

2

3. RECIPIENT'S ACCESSION NO.

4. TITLE AND SUBTITLE
Acquisition and Culture of Research Fish:

Bluegills

Trout, Fathead Minnows, Channel Catfish, and

5. REPORT D,

Rainbow 1/30/75 tDate of approval)

6. PERFORMING ORGANIZATION CODE

7. AUTHOR(S)
James L. Brauhn and Richard A. Schoettger

Ig. PERFORMING ORGANIZATION REPORT NO.

[9. PERFORMING ORGANIZATION NAME AND ADDRESS
Fesh-Pesticide Research Laboratory
Bureau of Sport Fisheries and Wildlife
United States Department of the Interior
Columbia MO 65201

10. PROGRAM ELEMENT NO.
1BA021

11, CONTRACT/GRANT NO.
EPA~IAG-0153 (D)

12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency

National Water Quality Laboratory
6201 Congdon Blvd.
Duluth MN 55804

13, FYPE OF REPORT AND PERIOD COVERED
Final

14, SPONSORING AGENCY CODE

15. SUPPLEMENTARY NOTES

16. ABSTRACT

widely for toxicological research.

parative or confirmatory research.

variable status.
pagation techniques provide the basis for

contaminants in diets and fish.

Rainbow trout (Salmo gairdneri), channel catfish (Ictalurus punctatus), fathead
minnows (Pimephales promelas), and bluegills (Lepomis macrochirus) are cultured
However, vacillant or extreme cultural conditions|
are sometimes suspected of compromising the
Because
maintenance and culture of the four species
standardized practices that are intended to
Water quality, nutrition,
handling, gross behavior, and required facilities are discussed.

emphasis is given to diets, diet preparation, and residues of pesticides or other

test animals and, thus, results of com-
exact optimum conditions for indoor
are not well defined, we have adopted
reduce cultural conditions to a common
genetic variation, diseases, fish

Well known pro-
the intensive care methods used. Special

KEY WORDS AND DOCUMENT ANALYSIS

DESCRIPTORS

b.IDENTIFIERS/OPEN ENDED TERMS |c. COSATI Field/Group

*freshwater fish; *quality control;
food; feeding rates; water quality;
rainbow trout; channel catfish; fish
diseases; pesticide residues

*sources; fathead
minnows; bluegill sun-
fish; genetic stocks

18. DISTRIBUTION STATEMENT

19, SECURITY CLASS (This Report) 21. NO, OF PAGES

Release unlimited

unclassified 52
20. SECURITY CLASS (This page) ~/22. PRICE
unclassified

EPA Form 2220-1 (9-73)

‘# U.S. GOVERNMENT PRINTING OFFICE: 1975-698-446 1140 REGION 10


Sanders, H. 0., F. L. Mayer, Jr., and D. F. Walsh. 1972. Toxicity,
residue dynamics, and reproductive effects of phthalate esters
in aquatic invertebrates. Environ. Res. 6(1):84-90.

Shell, E. W. 1966. Comparative evaluation of plastic and concrete
pools and earthen ponds in fish-cultural research. Prog. Fish-
Cult. 28(3):201-205.

Snow, J. R. 1962. A comparison of rearing methods for channel
catfish fingerlings. Prog. Fish-Cult. 24(3):112-118.

Spotte, S. H. 1970. Fish and Invertebrate Culture. Wiley-
Interscience, New York. 145 p.

Stalling, D. L., J. W. Hogan, and J. L. Johnson. 1973. Phthalic
ester residues - their metabolism and analysis in fish.
Environ. Health Perspec. 3:159-173.

Subcommittee on Fish Nutrition, National Research Council. 1973.
Nutrient requirements of domestic animals. No. 11, Nutrient
requirements of trout, salmon and catfish. National Academy
of Sciences, Washington, D. C. 57 p.

Tiemeier, 0. W., C. W. Deyoe, and C. Suppes. 1967. Production and
growth of channel catfish fry. Trans. Kans. Acad. Sci. 70(2):
164-170.

U. S. Bureau of Sport Fisheries and Wildlife. 1970a. List of state
fish hatcheries and rearing stations. Div. Hatcheries, Fish
Hatchery Leaflet No. 41. 20 p.

—___ 1970b. Dealers in trout and pond fishes. Div. Hatcheries,
Fish Hatchery Leaflet No. 46. 121 p.

—__— 1970c. Report to the fish farmers. Resource Publ. No. 83.
124 p.

U. S. Environmental Protection Agency. 1972. Recommended bioassay
procedure for fathead minnows, Pimephales promelas Rafinesque---
chronic tests. National Water Quality Laboratory, Duluth,
Minn. 13 p.

44


numbers of rainbow trout fingerlings for testing throughout the year.
However, strains spawning at different times are not developed for
most species, and availability can be extended by altering normal
reproductive cycles (Hazard and Eddy, 1950; Carlson and Hale, 1972).
For example, channel catfish normally spawn in early summer, with
fingerlings available for research in the fall. But, spawning can be
controlled by holding adults at relatively cool temperatures and
then gradvally increasing temperature to stimulate ripening (Yamamoto
et al., 1966; Brauhn, 1971). Similar techniques have been used to
induce fall spawning in largemouth bass (Micropterus salmoides)

(Brauhn et al., 1972; Carlson, 1973) and other teleosts (DeVlaming,
1972).

The importance of communication between the culturist and a proposed

supplying hatchery cannot be overemphasized since hatchery rearing
conditions may vary daily.

TRANSPORT

Truck transport is a reliable and economical method of transporting

large numbers of adult or immature fish for short distances (Davis, 1967),
but air transport is probably the most efficient and least stressful
method of securing small numbers of fingerling fish (Nemoto, 1957;

Hulsey, 1962; Gebhards, 1965). Air shipment is the only practical

means of transporting fish eggs great distances.

The primary hazards in transport of research fish are 0, deficiency,
excess co, and NH; pressure changes, and adverse temperatures
(Leitritz, 1960; Moss and Scott, 1961; Doudoroff and Shumway, 1970).
Of these, the first three are due to metabolism of fish and other

16


prevent rapid warming from the air. When the incubation temperature
is reached, the eggs are gently removed from the pan and placed in a
Heath* incubator tray supplied with 5-10 liter per minute clean water.
Davis (1967) described techniques for removing dead eggs and prophy-
lactic treatment for fungal infections.

FATHEAD MINNOWS

Fathead minnows are commonly propagated in ponds (Dobie, et al., 1956)
for use as forage or bait fish. They are fed supplementally to aid
growth (Prather, 1957). Thus, before newly-acquired fathead minnows
are used for research, they must be conditioned to accept a new diet,
and they must be acclimated to their new surroundings. The length of

"training" will vary with the feeding history of each group of minnows,
but generally is not long.

We have found that water temperatures of 21-24°C apparently increase
metabolism sufficiently to stimulate food intake and, therefore, make
conditioning to artificial diets easier. Portions of the daily ration
should be offered several. times per day. Excess food and fecal material
should be siphoned out of the tank because the common practice of
partially draining the tank and brushing it may injure the fish or
interfere with their conditioning to artificial feeds. Acceptance of
food and a positive response to the culturist indicates that the fish
are ready for long-term holding or research use. Temperature may then
be varied gradually at 1°C per day to the desired experimental tempera-
ture and feeding rates are reduced to 1-2 percent of total body weight
per day.

*
Heath-Techna Plastics Corp., Tacoma, Washington.

34


CONTENTS

Abstract

List of Tables

Acknowledgements

Sections

I Recommendations

II Introduction

III Fish Holding Facilities

Iv Acquisition

Vv Receipt of Fish and Acclimation
vi Maintenance

VII Specific Care and Problems
VIII References

iii

19

23

32

38


prior to, or during the experiment. Fish intended for toxicologic
research should be maintained throughout their life-span on the ration
to be used during research. For example, Mehrle et al. (in press)
have shown ithat the susceptibility of rainbow trout to chlordane can
be altered by the type of diet fed before testing.

Natural food organisms from lakes, streams, or ponds should not be
considered for routine maintenance of experimental fish because of
gross contamination by industrial pollutants and pesticides, variation
in seasonal supply, diversity of weight and size, and species variation
in protein, carbohydrate, and fat. However, rearing of small swim-up
fry of species such as bluegills is difficult without live food.

Eaton (personal communication) * recommends feeding very early instars
of daphnia or copepods, or a small~size variety of brine shrimp from

the West Coast.
GRADING

Hatchery fish may be delivered as a group of relatively mixed sizes,
and certain research may require greater uniformity. The mechanical
fish grader devised by Morton (1956) is useful for separating fish
into groups of even size. The spaced-bar type of grader used by
Pruginin and Shell (1962) is used at FPRL because it is efficient,
accurate, and inflicts minimal injury to the fish.

Fish grading should be done only when necessary and then, very
carefully. Only a few fish should be passed through the grader at
one time and the transfer must be made with fine meshed nets of soft

*
Mr. J. G. Eaton, U. S. Environmental Protection Agency, National
Water Quality Laboratory, Duluth, Minn. 1974.

27


€T

Table 2. SELECTED CONTAMINANTS DETECTED IN DIET COMPONENTS ANALYZED
INTERMITTENTLY AT FPRL FROM 1970 - 1973
Residues (ug/g)
Total
Sample Hexachloro- organo-
a/ b/ benzene Diel-] Chlor- Lin- | chlorine Phthalate /
DpT—"_| PcB— (HCB) drin | dane Endrin} dane | content esters—
Protein sources
Fish protein -/ |_| 0.0002 - - - - | 0.0002 -
concentrate
Herring meal 0.005- | 0.10- - - - - - 0.015- -
0.05 0.20 | 0.0003 0.250
Menhaden meal 0.84 1.6 - 0.30 - 0.01 - 2.75 -
Peruvian fish meal|0.73 1.8 - 0.01 - - - 2.54 -
Rough fish meal 0.20 2.8 - 0.02 - - - 3.02 -
Casein 0.005 - - - - - - 0.005 0.6
Gelatin 0.028 | 0.1 - 0.06 - - - 0.188 7.0
Skim milk - - - - - - - - -
Soybean meal - 0.1 - 0.03 - - - 0.130 0.6



SECTION V

RECEIPT OF FISH AND ACCLIMATION

Constructing adequate facilities, finding a suitable water supply
system and acceptable source of fish, and arranging transport of
fish to the laboratory is wasted if attention is not given to
the critical period immediately after receipt of fish at the
laboratory. Water quality, stress, disease, or physical damage
in this period may determine whether fish are fit for research.
Therefore, we have established a fish receipt protocol that is

followed whenever possible.
WATER QUALITY

Changes in water quality during shipment and differences between
that of the supplying hatchery and receiving laboratory are common
causes of stress and they should be given first consideration when
fish arrive. Leitritz (1960) and Davis (1967) summarized water
quality factors influencing trout culture. Because temperature,
alkalinity, dissolved solids, and dissolved gases of a hatchery's
water may differ appreciably from that of a laboratory, the
researcher must allow incoming fish to adjust to the laboratory.
Transfer of fish to water differing radically in temperature, hard-
ness, pH, and 0 may result in severe shock or death (Holmes and
Donaldson, 1969). If possible, the researcher should consider
acquiring fish from hatcheries with water qualities similar to those
of the laboratory. However, when water quality differences are
extreme, we modify a sufficient volume of receiving water in pH,

alkalinity, hardness, and temperature to approximate that of the

19


ST

Table 2 (continued).

SELECIED CONTAMINANTS DETECTED IN DIET COMPONENTS ANALYZED
INTERMITTENTLY AT FPRL FROM 1970 - 1973

Residues (ug/g)

Total
Sample Hexachloro- brgano-
a b benzene Diel-| Chlor- Lin- (chlorine Phthalate
por’! pope! (HcB) drin_ | dane Endrin | dane content esters—

Carbohydrates a/ 7 . _ . _ _ _ _
Dextrin -
Wheat middlings 0.05 - - 0.01} 0.03 - 0.14 0.23 0.2
Cornstarch 0.005 | 0.10 - - - - - 0.105 -
Vitamins, minerals,

and binders
Bone meal 0.03 | - - 0.01} - » - - | 0.04 0.9
Distillers 0.005 | 0.1- - 0.01} - - - 0.115 -

solubles 0.3 0.30
Brewers yeast | - - - - - 0.03 - 0.03 -
Carboxymethy1 - - - - 0.72 - - 0.72 -

cellulose
Alphacel - - - - - - - - -
Mineral mix - - - = - - - - -
Minimum detection {0.005 | 0.01 0.0001 0.01] 0.1 0.005 | 0.001) 0.1-

limits 0.5

al All DDT analogs

b/ ALL polychlorinated biphenyls, but usually Aroclor® 1254 and 1260

c/ di-n~butyl phthalate and di-2-ethylhexyl phthalate

a/ None detected

NOTE: In addition to the above residues, 2.08 ag/g of methoxychlor and 0.5 ~g/g

were found in wheat middlings

of malathion


material. Overloading nets or graders may cause scale loss, skin
puncture, eye damage, severe shock, or even mortality of some fish.
Injuries promote infection by pathogenic bacteria which may later
spread to healthy fish. We watch for delayed appearance of injuries

for at least one week after grading before the fish are used in
research,

DISEASES AND PROPHYLAXIS

Hygenic measures are required to prevent the initiation and spread

of disease among experimental fish. Nets, buckets, fish graders, and
hands are disinfected with 200 mg/l of Hyamine 1622 or a similar
bactericide before they are placed in holding waters. Also, a 1-hour
treatment with 2.0 mg/l Hyamine 1622 is applied to all fish at 14-day
intervals. Routine maintenance schedules should include the recording
of daily mortality and assessment of possible causes of death.

Most states now require that fish and eggs transferred, particularly
of salmonids, across their boundaries be certified disease-free by
a qualified fish pathologist. These regulations have helped to
minimize disease problems among FPRL research fish.

When diseases occur, references of Davis (1967), Leitritz (1960), and
the Fish Disease Leaflet series* published by the U.S. Bureau of Sport
Fisheries and Wildlife can be used, among others, to identify common
diseases encountered in fish culture and the methods for treatment.
Disease diagnosis is difficult at times and success frequently depends

on the biologist's experience. Many state conservation departments and

*¥
Available from the Superintendent of Documents, U.S . Government
Printing Office, Washington, D. C.

28


RESIDUES

Hatchery fish may also contain undesirable residues if concentrations
of contaminants in their diets and water supplies are sufficiently
high. In 46 samples composed of eggs, fingerlings, and adults of

the four species from 11 hatcheries, we found polychlorinated biphenyls
(PCB) residues in 83 percent (0.01-2.7 ug/g), residues of the DDT
complex (o,p'- and p,p'-DDT, DDE, and DDD) in 76 percent (0.01-0.29
mg/g), and phthalate acid ester plasticizers in 21 percent (0.16-1.0
ug/g). Other contaminants analyzed included chlordane in 11 percent
(0.02-0.6 g/g); dieldrin in 17 percent (0.003-0.37 mg/g); endrin in

6 percent (0.05-0.88 wg/g); toxaphene in 2 percent (9-20.0 ag/g in fat);
and heptachlor, benzene hexachloride (BHC), and hexachlorobenzene (HCB)
in 2 percent of the fish (0.002-0.03 mg/g). Accordingly, we now analyze
a small sample of fish before accepting the main shipments. The
analyses are made approximately 2 weeks before the requested fish reach
the desired size at the supplying hatchery. The pre-shipment analyses
for residues permit the researcher to decide whether or not contaminants
are present in sufficient quantity to interfere with his research. The
procedure minimizes the risk of invalidating research results and
eliminates cost of transporting fish that cannot be used.

Hatchery diets can be the source of residues in research fish. Although
commercial dry diets may provide adequate nutrition for rainbow trout,
fathead minnows, channel catfish, and bluegills, special consideration
should be required when one feeds fish destined for toxicologic
research. Residues of pesticides and industrial contaminants in
several fish diets have been analyzed by FPRL for several years.
Analyses for 1972 and 1973 are shown in Table 1. Residues are also

10


or air. Incoming fish are quarantined for 2 weeks. During this time,
we examine the fish for diseases and observe behavior. However, the
best disease preventative measure is avoidance,or destruction of fish
known to be diseased. Microscopic examination of gill lamellae and
the skin is used during the quarantine period to identify possible
disease agents, or to determine the severity of infestation. Some
diseases that are extremely pathogenic to rainbow trout and channel

catfish are discussed and control measures for these diseases are
outlined.

To maintain the 4 species for extended intervals, acceptable loading
capacities should not be exceeded. The capacity for rainbow trout can
be used because their maintenance requirements are generally more
restrictive than those of the other three species. Other recommenda-
tions for maintaining research fish include minimal handling or
grading, observation of behavior, and routine feeding of the proper
food. Bluegills and fathead minnows may require a brief period of
temperature elevation to stimulate their conversion from natural to
synthetic diets. This period, however, may not require conditioning
if they have been fed artificial diets before. In general, conditioned
behavioral patterns can reflect fishes' physical condition before more
observable disease symptoms appear. Therefore, consistent feeding and
other good cultural practices can be used to condition these behavioral
patterns and provide an excellent diagnostic tool to the fish culturist.


Subcommittee on Fish Nutrition, National Research Council (1973). We
have modified the diet (Table 3) as suggested by the Bureau of Sport
Fisheries and Wildlife's Committee on Fish Nutrition Research and
Development. This Committee recommends that the diet be termed and
used as a reference research diet. If fish meal is substituted for
casein, we suggest that the meal and whole dry mixture contain less
than 0.1 g/g of organochlorine residues. Because casein is derived
from milk, it is monitored closely by the U.S. Department of Agriculture
for contaminating residues and discarded if residues exceed tolerance
or action levels. Also, we require a pre-shipment analysis of all
other components to permit selection of those relatively free of
contamination. We prefer that fish oils contain no more than 2 wg/g
of organochlorine residues. By purchasing a 6-month supply of compo-
nents, the costly replication of residue analysis is eliminated and
variations in background residues are minimized. Rainbow trout and
channel catfish grow well on the diet with no apparent abnormalities.
Its suitability for bluegill and fathead minnows remains to be demon-
strated. We are using this diet for the four species but encourage
future nutrition research to clearly define the requirements of

bluegills and fathead minnows maintained solely on artificial rations.

The reference research diet can be made wet or dry, but we prefer the
latter because it has longer shelf life and requires less refrigerated
storage area. The feed is prepared at present as a rolled pellet in
any selected size with a Dravo pelletizer.*

Since the nutrition of experimental fish may influence results, diets
should be selected carefully by investigators. Unfortunately, many
research fish are initially fed one diet, then offered another ration

*
Dravo Corporation, Neville Island, Pittsburg, Pennsylvania.
25


ACCLIMATION

Each of the four species under discussion must be acclimated to their
new surroundings. Fish accustomed to unrestricted swimming in a
rearing pond undergo intense competition for food and swimming space
when placed in holding facilities. They may be frightened of nearby
movements, and also respond to variations in background color and
light intensity. The skin abrasions resulting from collisions with
solid objects can lead to bacterial or fungal infections and, ulti-
mately, the fish may have to be discarded. To prevent jumping, the
facilities should be covered with screens, which may also reduce

the light intensity. Channel catfish avoid light and appear to
acclimate faster to new surroundings if the tank is covered with
black polyethylene plastic. However, the cover should later be
removed a section at a time over 2 days until normal lighting is
reached. In general, fish should be maintained in facilities with
color backgrounds and light intensities similar to those of research
units to prevent a second acclimation period and lost research time.
Rainbow trout seem less affected by restriction of swimming space

and changes in background colors than are the other three species.

22


ACKNOWLEDGMENTS

We wish to acknowledge the following individuals and organizations

whose contributions, suggestions, and/or critical reviews made

publication of this manuscript possible:

Mr.

Mr.

Dr.

Dr.

Dr.

Ronald Henry and the staff, Fish Control Laboratory,
Bureau of Sport Fisheries and Wildlife, La Crosse, Wisc.

James L. Johnson, Dr. Foster L. Mayer and Dr. Paul M. Mehrle,
Fish-Pesticide Research Laboratory, Bureau of Sport Fisheries
and Wildlife, Columbia, Mo.

John E. Halver, Western Fish Nutrition Laboratory, Bureau
of Sport Fisheries and Wildlife, Cook, Wash.

Gary Rumsey, Tunison Laboratory of Fish Nutrition, Bureau
of Sport Fisheries and Wildlife, Cortland, New York

Harry K. Dupree, Southeastern Fish Cultural Laboratory,
Bureau of Sport Fisheries and Wildlife, Marion, Ala.

John Eaton, and the staff, National Water Quality Laboratory,
Environmental Protection Agency, Duluth, Minn.

*Information contained herein and/or mention or products by commercial
or trade names does not constitute endorsement by the U.S. Department

of Interior, Fish and Wildlife Service, Fish-Pesticide Research

Laboratory.


TABLES

Selected contaminants detected in diets analyzed
intermittently at FPRL from August, 1972 - August,
1973.

Selected contaminants detected in diet components
analyzed intermittently at FPRL from 1970 - 1973.

Reference research diets for cold and warm water
fish,

Observations of abnormal fish behavior and some
common causes for this behavior.

iv:

13

26

31


Tt

Table 1.

SELECTED CONTAMINANTS DETECTED IN DIETS ANALYZED INTERMITTENTLY
AT FPRL FROM AUGUST, 1972 - AUGUST, 1973

Residues (ag/

Du Total
lets Hexachloro- organo- c/
/ b/ benzene chlorine | Phthalate~
ppt PCB— (HCB) Dieldrin Endrin | content esters
Oregon Moist 0.06 0.30 x - - 0.36 -
Glenco 0.11 0.30 - - - 0.41 -
Clark 0.11 0.20 - - - 0.31 -
Silver Cup 0.08- 0.20- - - - 0.37 -
(2 samples) 0.17 0.32 0.06 0.01 - 0.555 -
EWOS 0.15~ 0.20- 0.008- 0.01- - 0.361- -

(3 samples) 0.39 0.30 0.046 0.02 0.01 0.766 3.0
Colorado State Diets 0.11- 0.10- - 0.01- - 0.213- -

(9 samples) 0.84 2.80 0.003 0.30 0.01 3.953 -
BSFW Hatchery Diets 0.12- 0.20- - - - 0.32- -

(2 samples) 0.19 0.30 0.01 0.50 -
Purina Catfish Chow - - - 0.01 - 0.01 -
Reference research diet| - <0.1 - - - <0.1 -

(4 samples)

Minimum detection 0.1-

limits 0.005 0.1 0.0001 0.01 0.01 0.5

a/ 411 DDT analogs

b/ al polychlorinated biphenyls, but usually Aroclor® 1254 and 1260

c/ di-n-butyl phthalate and di-2-ethylhexyl phthalate

4/ None detected


SECTION I

RECOMMENDATIONS

Holding facilities for research fish should be adequate for the
required size, number, and species of fish. A flow-through type of
holding system is superior to closed, water recirculation systems.
Round fiberglass tanks are generally superior to permanent concrete
tanks for holding fish.

Water of uniform high quality should be supplied to holding facilities
through chemically inert piping. Generally, a deep well is superior

to surface water sources, shallow wells, or springs.

Fish to be used for toxicological research should be morphologically
typical of the species and genetically consistent. Hatchery fish,
rather than wild fish, are used because their history is relatively
well known with regard to genetic strain, disease, diet, spawning
times, and rearing water.

Contamination of fish diets and hatchery water supplies has resulted
in the accumulation, to different degrees, of industrial compounds and
pesticides by fish intended for research. Pre-shipment analyses of
"candidate" test fish, and feeding a relatively uncontaminated diet
assist in minimizing foreign residue accumulations in research speci-

mens.

Stress producing factors should be minimized during transport of fish
to the laboratory. After arrival at the laboratory, special considera-
tions must be given to acclimating fish to laboratory water quality
and temperature, depending on whether the fish were shipped by truck


organisms such as bacteria that live on feces, mucous, and other
organic material in transport water. Therefore, fish should be held
without food for at least 48 hours prior to transport. Lane and
Jackson (1969) determined the fecal voidance time for 23 species of
fish including rainbow trout, fathead minnows, channel catfish and
bluegills. They found that voidance was temperature dependent, but
usually complete in 2 to 3 days. Haskell and Davies (1958) and
Leitritz (1960) suggest the loading should not exceed 454 g (1 1b)
per 3.8 1 (1 gal).

Some hatcheries reduce bacteria in transport water by adding acri-
flayine or other bacteriostatic compounds. Since these compounds

may be accumulated by the fish while in transport and later interfere
with research, we discourage use of such chemicals. Healthy fish,
minimal fecal accumulation, and proper loading rates make bacteriostats

unnecessary.

All-season truck transport of fish requires special equipment including
an oxygenation system, properly designed insulated tanks, a water
recirculation system, and, in some instances, refrigeration equipment.
It is generally impractical for research laboratories to invest in

this expensive equipment. However, researchers should be aware that
this kind of equipment is often necessary to transport rainbow trout

during summer.

Because of the distances between supplying hatcheries and the laboratory
(>500 miles), we ship many experimental fish by commercial air freight.
Before air-shipment, fingerlings are not fed for 48 hours and then the
water temperature is gradually lowered to 3°C during the second 24
hours. Approximately 450 g of fish are placed into a clear polyethylene

17




Eller, L. 1970. Histopathology. p. 27-28. Annual report of the
Fish-Pesticide Research Laboratory. In: Progress in Sport
Fishery Research, 1970. Bur. Sport Fish. and Wildl. Resource
Publ. 106. 318 p.

Ferguson, D. E., and C. R. Bingham. 1966. The effects of combinations
of insecticides on susceptible and resistant mosquitofish.

Bull. of Environ. Contam. Toxicol. 1(3):97-103.

Fry, F. E. J. 1957. The aquatic respiration of fish. p. 1-63. In:
The Physiology of Fishes, Brown, M. E. (ed.). Vol. 1. Academic
Press, New York. 525 p.

Gebhards, S. V. 1965. Transport of juvenile trout in sealed
containers. Prog. Fish-Cult. 27(1):31-36.

Giudice, J. J. 1966a. An inexpensive recirculating water system.
Prog. Fish-Cult. 28(1):28.

1966b. Growth of a blue X channel catfish hybrid as
compared to its parent species. Prog. Fish-Cult. 28(3):142-145.

Grant, B. F., and P. M. Mehrle. 1970a. Chronic endrin poisoning in
goldfish, Carassius auratus. J. Fish. Res. Bd. Can. 27(12):
2225-2232.

1970b. Pesticide effects on fish endocrine systems.
p. 20-24. Annual report of the Fish-Pesticide Research Labora-
tory. In: Progress in Sport Fishery Research, 1970. Bur.
Sport Fish. and Wildl. Resource Publ. 106. 318 p.

1973. Endrin toxicosis in rainbow trout (Salmo
gairdneri). J. Fish. Res. Bd. Can. 30(1):31-40.

Grant, B. F., and R. A. Schoettger. 1972. The impact of organochlorine
contaminants on physiologic functions in fish. pp. 245-250. In:
Proc, Tech, Sessions, 18th Ann, Mtg., Inst. Environ, Sci., New
York City.

Haskell, D. C. 1955. Weight of fish per cubic foot of water in
hatchery troughs and ponds. Prog. Fish-Cult. 17(3):117-118.

40


SECTION III

FISH HOLDING FACILITIES

Inadequate, poorly designed facilities hamper research progress
because they cannot sustain the required number, sizes, or species of
fish for the duration of an experiment, or, more importantly,
concurrent experiments. Potential water sources and supply systems

in conjunction with special considerations, such as contaminants from
construction materials, water additives (chlorine, fluoride), lighting,
space limitations, and facility size and location should influence
facility design and construction.

Many types of fish holding facilities are used to maintain research
fish. Concrete tanks used at FPRL and FCL were designed to hold small
numbers of various species for several months but they can be used to
rear fingerlings to sexual maturity. Whereas concrete tanks are
permanent and require little maintenance, tanks of redwood or fiber-
glass are moveable, durable, and versatile. Concrete tanks do not
permit reallocation of space when fish holding requirements are reduced.
Circular fiberglass tanks appear to meet space and versatility
requirements and if inflows and outflows are properly designed, they
are "self-cleaning" and require less water flow than rectangular tanks
(Davis, 1967). However, Mairs (1961) noted fish death due to
improperly "cured" epoxy resin cements and low water flows. The
intensive culture concept of Buss and Waite (1961) is incorporated
into some circular tank designs.


SECTION II

INTRODUCTION

Use of fish for research in toxicology, physiology, and medicine has
increased greatly in recent years. Lennon (1967) reviewed research
needs for selected strains of fish and emphasized the importance of
information on sources, care, handling, feeding, and testing various
experimental fishes. Such information is essential to provide research
animals that yield reproducible and comparable bioassay results
(Henderson and Tarzwell, 1957; Lennon and Walker, 1964; American
Public Health Association, 1971).

The need for high quality research fish is recognized, but methods for
assessing the quality of prospective fish and procedures for their
acquisition and maintenance are not well established. Most fish
cultural techniques are directed at mass propagation of fishes and do
not consider the intensive care necessary for obtaining and maintaining
suitable research fish. Relative resistance of test fish to reference
toxicants such as DDT or antimycin were used to estimate quality by
Marking (1966) and Hunn et al. (1968). The latter authors also
presented methods of handling and maintaining bioassay fish, but
pointed out that the most appropriate methods are still in the develop-
mental stage. They hoped to stimulate further discussion and exchange
of information on this topic. In response, this paper presents culture
methods currently in use at the Bureau of Sport Fisheries and Wildlife's
Fish~Pesticide Research (FPRL) and the Fish Control (FCL) Laboratories
for rainbow trout (Salmo gairdneri), channel catfish (Ictalurus
punctatus), fathead minnows (Pimephales promelas), and bluegills
(Lepomis macrochirus). We recognize that some of these methods are
not yet supported by research data, but they do represent our best
opinions based ‘on empirical evidence.

3


Another serious disease of channel catfish is columaris (Davis, 1967).
Infected fish may be dipped into a 1:100,000 malachite green solution
for 10 seconds to successfully control this disease. This treatment
is repeated daily for 4 days. Also, daily applications of 25 mg/1
Combiotic or Dystrillin for one hour under static conditions and
repeated for 10 days is an effective control for this disease (see
DISEASE AND PROPHYLAXIS).

During routine handling, the barbed pectoral spines of channel catfish
frequently catch in nets, and soft fin tissue is easily damaged. Like
other mechanical injuries, the surrounding tissues can then become
infected with bacteria or fungus. This problem can be minimized by
coating nets used for channel catfish with asphalt varnish.

BLUEGILLS

Since bluegills are reared in ponds on natural foods (Davis, 1967),
their physiological state is tied closely with seasonal variations

in light, temperature, and food. Thus, we have experienced difficulty
in changing the reproductive cycle of bluegills with temperature alone.
Previous experience shows that adult males establish a territory
within indoor holding tanks when temperature is elevated but females

appear unresponsive.

Bluegills also. establish a "peck order}’ or sequence of dominant indi-
viduals (Breder and Rosen, 1966). Some fish, at the lower end of an
established peck order, refuse to eat, become emaciated, and die. This
dominance is usually established in the first 3 weeks after the fish
are brought indoors, but is is disrupted each time they are handled or

36


Loading capacities have not, however, been adequately investigated for
warm water species maintained in laboratory facilities. The minimum
dissolved oxygen (D.0.) requirements of warm water species are generally
lower than those for rainbow trout (Doudoroff and Shumway, 1970; Moss
and Scott, 1961). So we utilize carrying capacities for rainbow trout.
First, the permissible weight of fish is established empirically for

a given water inflow rate (Piper, 1971). Permissible weight of fish

is established by placing fish into a holding unit, varying inflow rates
and monitoring behavior, 0,» and NH, at each rate. Safe limits of
dissolved gasses for rainbow trout are: D.0., above 5 mg/l and NH

3?
below 0.5 mg/1. Then loading is determined using the formula:

where F = loading factor
_ W L = length of fish (cm or in) @)
LxI I = 1 or gal ‘per min water inflow
W = permissible weight of fish

(kg or 1b)

Subsequently, with additional growth, F can be substituted back into
the equation to establish new holding requirements. Previously estab-
lished feeding rates are used, when known, and as the fish grow, or as
additional fish are placed into the unit, equation 2) is used to
estimate the correct water inflow. This method is checked frequently
according to the safe limits for rainbow trout. If a hazardous level

of these gasses is noted, a new F is determined.

DIETS FOR EXPERIMENTAL FISH

The problem of residue accumulation in test fish from contaminated
food is circumvented by preparing our diet from "non-contaminated"
constituents. The diet is basically the H440 test diet reported by the

24


Haskell, D. C., and R. 0. Davies. 1958. co, as a limiting factor in
trout transportation. N. Y. Fish and Game J. 5(2):175-183.

Hayes, F. R. 1949. The growth, general chemistry, and temperature
relations of salmonid eggs. Quart. Rev. Biol. 24(4):281-308.

Hazard, T. D., and R. E. Eddy. 1950. Modification of the sexual
cycle in brook trout (Salvelinus fontinalis) by control of light.
Trans. Amer. Fish. Soc. 80:158-162.

Henderson, C., and C. M. Tarzwell. 1957. Bioassays for control of
industrial effluents. Sewage Ind. Wastes 29(9):1002-1017.

Henderson, C., W. L. Johnson, and A. Inglis. 1969. Organochlorine
insecticide residues in fish (National Pesticide Monitoring
Program). Pestic. Monit. J. 3(3):145-171.

Herman, R. L., D. Collis, and G. L. Bullock. 1969. Ocytetracycline
residues in different tissues of trout. Bur. Sport Fish. and
Wildl. Tech. Paper No. 37. 4 p.

Hoffman, G. L. 1962. Whirling disease of trout. U. S. Dep. Interior,
Fishery Leaflet No. 508. 3 p.

Holmes, W. N., and E. N. Donaldson. 1969. The body compartments and
the distribution of electrolytes. p. 50-79. In: Fish Physiology.
Hoar, W. S.,and D. J. Randall (ed.). Vol. 1. Academic Press,
New York and London. 465 p.

Hulsey, A. H. 1962. Transportation of channel catfish fry in plastic
bags. p. 354-356. In: Proc, 16th Ann, Conf., Southeast Assn,
Game and Fish Comm,

Hunn, J. B., R. A. Schoettger, and E. W. Whealdon. 1968. Observations
on the handling and maintenance of experimental fish. Prog.
Fish-Cult. 30(3):164-168.

Lane, T. H., and H. M. Jackson. 1969. Voidance time for 23 species
of fish. Bur. Sport Fish. and Wildl., Invest. Fish Contr. No.
33. 9 p.

41


SECTION VI

MAINTENANCE

Fish often'must be maintained for extended periods in laboratory
facilities before they can be used, making capacity of facilities,
diet, size, grading, disease, and behavior critical.

CAPACITY OF FACILITIES

Estimates of the proper number and weight of fish that can be held
satisfactorily in the laboratory depend upon the size of fish, dis-
solved oxygen, feeding rate, temperature, and rate of water exchange
in the holding unit. In general, the fish's oxygen requirement at
constant temperature is associated with its caloric intake (Fry, 1957).

An acceptable loading capacity (weight of fish per liter of inflow)
of holding facilities is estimated for salmonids by the method of

Haskell (1955).

Weight (g) of feed per liter of

Weight (g) of fish per _ inflow ql)
liter of inflow Fractional percent of body weight
fed

Haskell's equation must be used in conjunction with the feeding

tables of Deuel et al. (1952). This basic relationship was modified
by Willoughby (1968), Piper (1971), and Liao (1971). Other investi-
gators (Buterbaugh and Willoughby, 1967) have also developed
correlations between trout length, weight, and the percent of body
weight to feed for maximal growth. Examples and further discussion of
these methods are presented by Phillips (1970).

23


**When reporting uses of, or research on drugs and pesticides, the
authors do not imply that the drug or pesticide uses discussed have
been cleared or registered. Clearance of drugs and registration of
pesticides are necessary before recommendation. Appropriate state
and federal regulatory agencies should be consulted to determine if
the chemical in question can be used and under what conditions the
uses are permitted.

vi


4T

Table 2 (continued).

INTERMITTENTLY AT FPRL FROM 1970 - 1973

SELECTED CONTAMINANTS DETECTED IN DIET COMPONENTS ANALYZED

Residues (ug/g)

Total
Sample Hexachloro- organo-
a/ b/ benzene Diel- | Chlor- Lin-| chlorine} Phthalat
ppt="'|_ PcB~ (HCB) drin | dane | Endrin| dane| content esters®
Oils
Tuna oil 6.99- | 2.0- 0.052- 0.05-| = - - 9.09- -
(2 samples) 7.45 2.6 0.07 0.06 - - - 10.18
Menhaden oil 1.09 4.7 0.11 - - - - 5.90 -
Redfish oil 2.14 1.6 0.07 0.06 - - - 3.87 -
Herring oil 0.69- | 1.0- - - - - - 1.69- - -
(3 samples) 3.40 2.3 0.07 0.46 6.23
Cod oil 0.87- | 3.0- 0.06- 0.07-| - - - 4.00- -
(3 samples) 0.96 3.5 0.38 1.10 5.94
Pollock oil 3.0 32.0 - - - - - 35.0 -
Salmon oil 0.23- | 0.20- - - - - - 0.43- -
(3 samples) 0.90 1.60 - 0.03 2.53
Linseed oil - - - - - - - - -
Corn oil - 0.1 - - - - - 0.1 1.1

Corn oil



Te

Table 4. OBSERVATIONS OF ABNORMAL FISH BEHAVIOR AND SOME COMMON CAUSES FOR THIS BEHAVIOR.
SEVERAL OBSERVATIONS MAY BE MADE SIMULTANEOUSLY AND MAY BE CAUSED BY A
COMBINATION OF TWO OR MORE PROBLEM AREAS.

Observation

Possible problem

1. Surfacing and swimming with mouth half emerged - | a. insufficient oxygen; toxic chemicals;
a, b, c, de high NH,
2. Scraping against sides of tanks - al b. parasitism of skin or gills.
b, d, f
c. rapid temperature changes; parasitism
3. Erratic swimming - of nervous system; virus disease
a, b,c
d. parasitism of intestine; bacterial
4. Crowding around water inflow - disease
a, b
e. diet consistency
5. Distended abdomen and difficulty in swimming -
d, e, f £. nutrition deficiency
6. Refusal to feed actively -
a, c, d, e, f
7. Listlessness, emaciation -
b, c, d, e, £
a/

Normal fathead minnow courtship behavior includes coloration change and display near tank sides.


SECTION VII

SPECIFIC CARE AND PROBLEMS

Whereas the preceeding subjects dealt with a broad spectrum of
essentials for adequate fish maintenance, this section delineates
specific cultural practices for each of the four species under
discussion. The information presented here was determined empirically
over 4 years by working with several hundreds of thousands of fish.

RAINBOW TROUT

Water that contains 5-10 mg/1 of D.O.(but is not supersaturated), has
a pH of 7-7.5, and is 9-13°C produces optimum vigor in rainbow trout
(Leitritz, 1960). Dissolved oxygen and temperature are probably most
critical since each limits the weight of fish held per unit of water
volume (see CAPACITY OF FACILITIES). Water temperatures between

0° to 9°C cause sluggishness and reduction of appetite, whereas
temperatures above 13°C stimulate appetite and growth. Temperatures
near 25°C approximate the upper lethal temperature (Fry, 1957). In
general, temperatures above 17°C to 20°C should be avoided.

Rainbow trout are susceptible to infectious viral diseases, such as
pancreatic necrosis (Wolf, 1966a) and hemorrhagic septicemia (Wolf,
1966b). ‘They are also susceptible to whirling disease (Hoffman, 1962),
a highly contagious protozoan-caused disease. Since these diseases
are not detected by routine microscopic analysis, a qualified fish
pathologist should be consulted when appropriate symptoms appear. At
present, since there are no therapeutic measures effective against
these diseases, only complete sterilization of holding facilities will
control them and permit introduction of new stocks.

32


hatchery. We then add laboratory water at a rate sufficient to give
a complete exchange of water every 24 hours. If necessary, we adjust
the rate of temperature change to approximately 3 to 4°C per day. :
Beamish and Mookherjii (1964) recommended a temperature change rate
of 1°C per day when acclimating goldfish (Carassius auratus) to a

research temperature, which we follow after quarantine.

Receiving air shipments requires a slightly different procedure.
Except when water quality between the supplying hatchery and the
laboratory differ greatly, it is less important than temperature
difference. Dissolved co, and NH, will accumulate during shipment
and influence the pH. Also, oy concentration in the water will
gradually decrease as the time of shipment increases. Therefore,
the fish may be in water radically changed from that in which they
were reared. Fish should be changed to a new water quality over’

2 or more hours to prevent lethal stress. Temperature acclimation
should receive primary consideration, because the shipments should
be at a reduced temperature (see TRANSPORT). This is done by
allowing the shipping bags to float on the surface of the tank in
which they are to be placed. When temperatures are equalized, water
may be added gradually at a rate of approximately 50 ml/min to the
shipping bag. When a volume of water approximately equal to that
in the shipping bag has been added, the fish may then be eased into
their holding tank.

QUARANTINE

We assume that all incoming fish may be diseased. To prevent the
spread of disease and to protect healthy resident fish, we quarantine
incoming fish for 2 weeks while their behavior is observed and they
are examined at least twice for ectoparasites (see DISEASE AND
PROPHYLAXIS) .

20


ABSTRACT

Rainbow trout (Salmo gairdneri), channel catfish (Ictalurus
punctatus), fathead minnows (Pimephales promelas), and bluegills
(Lepomis macrochirus) are cultured widely for toxicological research.
However, cultural conditions are sometimes suspected of compromising
the test animals and, thus, results of research findings. Because
optimum conditions for indoor maintenance and culture of the four
species are not well defined, we have adopted standardized practices
that are intended to reduce cultural conditions to a common variable
status. Water quality, nutrition, genetic variation, diseases, fish
handling, gross behavior, and required facilities are discussed. Well
known propagation techniques provide the basis for the intensive care
methods used. Special emphasis is given to diets, diet preparation,
and residues of pesticides or other contaminants in diets and fish.

This report was submitted in fulfillment of Contract EPA-IAG-141(D)
by the U. S. Department of the Interior, Bureau of Sport Fisheries and
Wildlife, Columbia, Missouri, under the partial sponsorship of the

Environmental Protection Agency, Work was completed as of June, 1974.

Ai


Carlson, A. R., and J. G. Hale. 1972. Successful spawning of large-
mouth bass, Micropterus salmoides under laboratory conditions.
Trans. Amer. Fish. Soc. 101(3):534-542.

Carlson, A. R. 1973. Induced spawning of largemouth bass [Micropterus
salmoides (Lacepede)]. Trans. Amer. Fish. Soc. 102(2):442-444.

Childers, W. F., and G. W. Bennet. 1961. Hybridization between three
species of sunfish (Lepomis). I11. Nat. Hist. Sur., Biol. Notes
No. 46. 15 p.

Clark, J. R., and R. L. Clark. 1964. Sea-water systems for experi-
mental aquariums. U.S. Fish and Wildl. Serv., Res. Rep. No. 63.
192 p.

Davis, H. S. 1967. Culture and diseases of game fishes. 1953 ed.
University of California Press, Berkeley and Los Angeles. 332 p.

DeVlaming, V. L. 1972. Environmental control of teleost reproductive
cycles: A brief review. J. Fish Biol. 4(1):131-140.

Deuel, C. R., D. C. Haskell, D. R. Brockway, and O. R. Kingsbury.
1952. The New York State fish hatchery feeding chart. 3rd ed.
Fish. Res. Bull. No. 3. New York State Conservat. Dep., Albany,
N. Y. 61 p.

Dobie, J., 0. L. Meehean, S. F. Snieszko, and G. N. Washburn. 1956.
Raising bait fishes. U.S. Fish Wildl. Serv., Cir. No. 35. 124 p.

Dollar, A. M., and M. Katz. 1964. Rainbow trout brood stocks and
strains in American hatcheries as factors in the occurrence of
hepatoma. Prog. Fish-Cult. 26(4):167-174.

Doudoroff, P. and D. L. Shumway. 1970. Dissolved oxygen requirements
of freshwater fishes. FAO Fish. Tech. Paper No. 86, Food and
Agr. Organ., U. N., Rome, Italy. 291 p.

Drummond, R. A., and W. F. Dawson. 1970. An inexpensive method for
simulating diel patterns of lighting in the laboratory. Trans.
Amer. Fish. Soc. 99(2):434-435.

Eisler, R. 1967. Acute toxicity of zinc to killifish, Fundulus
heteroclitus. Chesapeake Sci. 8(4):262-264.

39


graded. The dominance order, more frequent in adults than fingerlings,
causes uneven growth and may be promoted by the change from natural

to artificial diets. That is, some fish may adapt to the artificial
diet sooner than others. Feeding the same quantity of food less
frequently, may ameliorate this problem.

Pond-reared bluegills are accustomed to natural food and the change
‘to an artificial diet requires a conditioning period similar to that
used for fathead minnows. This period is critical to bluegill

survival and may extend over 2 weeks, with food offered at approxi-

mately the same time each day.

Bluegills are more susceptible to physical injury during handling
than rainbow trout or channel catfish, therefore we use a 3-percent
NaCl dip for 1-2 minutes (Hunn, et al., 1968) after their handling
or grading. This reduces the possibility of external infection
resulting from loss of mucous or injury to the skin.

37


SECTION IV

ACQUISITION

Fish used for research should be healthy, relatively free of
pollutants, of known age, and "physiologically representative" of
the species as a,whole. Factors such as hybridization, disease,

"

injury, nutritional state, and exposure to pollution may "compromise"
the usefulness of wild fish in toxicologic and physiologic research.

SOURCES OF FISH

Fish are obtained primarily from national and state fish hatcheries
(U.S. Bureau of Sport Fisheries and Wildlife, 1970a). Snow (personal
communication)* selected "reference" strains of bluegills and channel
catfish that are propagated at two national fish hatcheries. These
strains are used for toxicologic research whenever possible.

However, Eller (1970) noted an abnormally high incidence of ovotestis
in third-generation bluegills of the reference strain. This finding
May eventually preclude our using these bluegills for certain types
of research. It also exemplifies the need for monitoring potential
ill effects of inbreeding. Fish for experimental use are also
available from commercial sources (U.S. Bureau of Sport Fisheries and
Wildlife, 1970b).

The significance of research fishes' genetic background is not always
clear, but many investigators believe that variation in results is
reduced by using selected strains. Some types of research may require
fish from polymorphic gene pools, but for reproducible laboratory
results from a reasonably small sample size, a selected population

*
Mr. J. R. Snow, U.S. Bureau of Sport Fisheries and Wildlife, National
Fish Hatchery, Marion, Ala. 1972.

7


in commercial production (Dollar and Katz, 1964). These strains are
geographically separate and spawn at various times throughout the year,
and several research installations may use different strains of trout
for similar research. The advantage of year-around availability of
test fish from different strains may outweigh the potential disadvan-
tage of response variability between strains.

In general, we discourage the use of wild fish because of the high
potential for genetic variability, widespread pollution, and disease.
Wild fish may be carriers of various parasites, bacteria, and viruses
that, with crowding in the laboratory, may spread to healthy groups

of research fish. The resulting changes in physiological stages of
these fish may alter their susceptibility to toxicants as well as
interfere with biochemistry and pathology observations (Lennon and
Walker, 1964). Unavoidable epidermal and gill damage or sublethal
stress induced in wild fish during netting, trapping, shocking, or
transport will generally render these fish unfit for research use.
Injuries and stress make the fish more susceptible to bacterial and
fungal infections. Nutritional state may be unimportant except when
stunted or starved wild fish are collected from overpopulated waters.
The ubiquity of pesticides (Henderson et al., 1969) and other residues
(Mayer, 1970) in wild fish may limit their utility in certain research
because of variations in dose-mortality responses (Ferguson and
Bingham, 1966), interference in studies of chemical kinetics (Mayer,
1970), and untoward embryological or other physiological effects (Grant
and Mehrle, 1970a, 1970b, 1973; Grant and Schoettger, 1972).


EPA-660/3-75-011
MAY 1975

ACQUISITION AND CULTURE OF RESEARCH FISH:

RAINBOW TROUT, FATHEAD MINNOWS, CHANNEL CATFISH, AND BLUEGILLS

by

James L. Brauhn
Richard A. Schoettger
Fish-Pesticide Research Laboratory
Bureau of Sport Fisheries and Wildlife
United States Department of the Interior
Columbia, Missouri 65201

EPA-IAG~141(D)
Program Element 1BA021
ROAP/Task No, 16AAK/017

Project Officer

Leonard H. Mueller
National Water Quality Laboratory
National Environmental Research Center
6201 Congdon Boulevard
Duluth, Minnesota 55804

NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330


Hunn et al. (1968) observed behavior patterns of individual groups of
fish for 10 days prior to their use as research animals. The culturist
should observe the fish's response to food, their swimming posture,
location in the tank, and unusual or erratic movements (Table 4). In
addition, they are observed for visible injuries or changes in body
conformation. Precision in the observations can be enhanced by in-
ducing conditioned responses to scheduled, routine cultural practices.
For example, Phillips (1970) suggested that small trout be fed small
amounts of feed at hourly intervals in an 8-hour day and waste food
and fecal material should be removed daily by siphoning or by partially
draining the tanks. Deviations in the usual responses to these
activities at appointed times may portend serious cultural problems.

Also, each species responds differently to laboratory space restric-
tions. It is normal for bluegills to disperse throughout the holding
unit and maintain that posture, but it is abnormal for a schooling
fish such as channel catfish fingerlings to do so, except when seeking
food. Fathead minnows also exhibit a schooling tendency, and may
exhibit a strong migration urge upon receipt. Thus, observations of
fish behavior depend on the time of day that the observations are made,
the species of fish, feeding schedule, the length of time in holding
facilities, and the size of fish.

30


bag containing 7.5 liters of water at 3°C. Frequently, two bags are

used, one inside the other, to minimize punctures or other leaks.
Another bag containing 1.5 kg of ice is placed in the bottom of a
0.5-m> styrofoam box and the bag containing fish is set on the ice.
The fish bag is partially inflated (to avoid bursting in unpressurized
aircraft compartments) with pure 0, and sealed tightly with rubber

bands. Fingerling rainbow trout packed in this manner have survived

12 hours with less than 5 percent loss.

18


RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development,

U.S. Environmental Protection Agency, have been grouped into
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:

- Environmental Health Effects Research
Environmental Protection Technology
Ecological Research

- Environmental Monitoring
Socioeconomic Environmental Studies

aPwn—

This report has been assigned to the ECOLOGICAL RESEARCH STUDIES
series. This series describes research on the effects of pollution

on humans, plant and animal species, and materials. Problems

are assessed for their long- and short-term influences. Investigations
include formation, transport, and pathway studies to determine

the fate of pollutants and their effects. This work provides

the technical basis for setting standards to minimize undesirable
changes in living organisms in the aquatic, terrestrial and atmospheric
environments.

This report has been reviewed by the Office of Research and
Development, EPA, and approved for publication. Approval does
not signify that the contents necessarily reflect the views and
policies of the Environmental Protection Agency, nor does mention
of trade names or commercial products constitute endorsement or
recommendation for use.


Leitritz, E. 1960. Trout and salmon culture. Calif. Dept. of Fish
and Game, Fish. Bull. No. 107. 169 p.

Lennon, R. E. 1967. Selected strains of fish as bioassay animals.
Prog. Fish-Cult. 29(3):129-133.

Lennon, R. E., and C. R. Walker. 1964. Laboratories and methods for
screening fish-control chemicals. Bur. Sport Fish. and Wildl. ~
Invest. Fish Contr. No. 1 (Cir. No. 185). 15 p.

Leteux, F., and F. P. Meyer. 1972. Mixtures of malachite green and
formalin for controlling Ichthyophthirius and other protozoan
parasites of fish. Prog. Fish-Cult. 34(1):21-27.

Lewis, W. M. 1962. Maintaining Fishes for Experimental and Instruc-
tional Purposes. Southern Illinois University Press, Carbondale.
100 p.

Liao, P. B. 1971. Water requirements of salmonids. Prog. Fish-Cult.
33(4):210-216.

Mairs, D. F. 1961. Toxicity of epoxy cement to fishes. Prog.
Fish-Cult. 23(4):178.

Marking L. 1966. Evaluation of p,p'-DDT as a reference toxicant in
bioassays. Bur. Sport Fish. and Wildl. Invest. Fish Contr.

No. 10, Resource Publ. No. 14. 10 p.

Mayer, F. L., Jr. 1970. Chronic toxicity of pesticides to fish.

p- 10-15. Annual Report of the Fish-Pesticide Research
Laboratory. In: Progress in Sport Fishery Research, 1970.
Bur. Sport Fish. and Wildl., Resource Publ. 106.

Mayer, F. L., Jr., D. L. Stalling, and J. L. Johnson. 1972.
Phthalate esters: An environmental contaminant. Nature 258:
411-413.

Mayer, F. L., Jr., and H. 0. Sanders. 1973. Toxicology of phthalic
acid esters in aquatic organisms. Environ. Health Perspect.
3:153-158.

42


a/

Table 3. REFERENCE RESEARCH DIETS— FOR
COLD AND WARM WATER FISH
Component Percent of dry diet for
Cold water fish Warm water fish

Casein 35.0 28.0
Gelatin 15.0 12.0
Dextrin 28.0 28.0
a-cellulose mie!

a-cellulose 8.0 18.0

Vitamin mix 1.0 1.0
Mineral mixt/ 4.0 4.0
Corn oil 6.0 6.0
Fish oil 3.0 3.0

al Recommended by the Committee on Fish Nutrition Research and

Development, Bureau of Sport Fisheries and Wildlife.

b/ Subcommittee on Fish Nutrition, National Research Council (1973).

26


Altering the natural temperature regime of fathead minnows frequently
alters their reproductive cycle. Water temperature elevation stimu-
lates breeding behavior and spawning before and after the normal
spawning period. Detailed recommendations for culturing this species
are presented by the U.S. Environmental Protection Agency (1972).

CHANNEL CATFISH

Methods are available for economical pond propagation of channel
catfish for sport fishing and for food (Snow, 1962; Tiemeier, et al.,
1967; U.S. Bureau of Sport Fisheries and Wildlife, 1970c). The same
methods are used to rear fingerlings for research use, hence, the
fingerlings should be accustomed to competitive feeding on artificial
rations. At FPRL they are fed a ration similar to that for trout,
but, in general, their protein requirement is somewhat less (Phillips
et al., 1957; Nail, 1962).

Channel catfish are more susceptible to several diseases than the

other three species. One of these is the protozoan, Icthyophthirius
multifilius, or ich, which is well known among culturists (Meyer, 1969).
We assume that all newly-acquired channel catfish are carrying one or
more life stages of ich. Probably the most effective control for ich
in catfish is to place them in flowing water and elevate the temperature
to 32°C for one week. This procedure hastens the life cycle of ich and
is lethal to its infective stages (Meyer, 1969). The treatment is most
successful with fingerling channel catfish, but it must be judiciously
applied to adults. Adult channel catfish are sexually stimulated by
this increase in water temperature (Brauhn, 1971) and they may initiate
courtship behavior. This behavior includes competitive biting among
males. The resulting lesions become infected rapidly with bacteria
and/or fungus, and the fish cannot be used until a formalin treatment

is applied. .
35


We designate a semi-isolated portion of the fish holding facilities

as a permanent quarantine area. The quarantine procedure is initiated
before fish arrive by sterilizing a tank with about 200 mg/1 HTH
(calcium hypochlorite) for 24 hours (See DISEASE AND PROPHYLAXIS).
During this time, the tank walls and plumbing are scrubbed with this
solution. Then, the HTH is flushed from the tank and at least three
exchanges of water are passed through the tank in the next 24 hours.
All nets, screens, and buckets used in the tanks are treated simi-

larly, then thoroughly rinsed with clean water.

Immediately after receipt, fresh preparations are made from the

skin, gills, and gastro-intestinal (GI) tract of six fish and examined
microscopically for the presence of ecto- and endoparasites, and
physical damage to the epidermis or fins is noted. For ectoparasites,
we initially apply a 1l-hour flow-through treatment of one part
formalin to 6,000 parts incoming water. Then, a mixture of formalin
and malachite green is applied to give concentrations of 25 mg/1 and
0.1 mg/1, respectively, in three, 1~hour treaments on alternating

days (Leteux and Meyer, 1972). Parasitism of the GI tract is treated
with one dose of di-n-butyl tin oxide incorporated into the diet at

a rate of 250 mg/kg of fish (Allison, 1957). We repeat the microscopic
examination of skin, gills, and GI tract of fish on the 10th day of
quarantine. Then, if there are no untoward side effects of treatment,
and if no ecto- or endoparasites are present, the fish are moved from
the quarantine section to a long-term holding tank or to experimental
tanks.

Bacterial infections in newly-arrived fish warrant consultation with
a qualified fish disease diagnostician as to the seriousness of
infection, the causative organism, and possible therapeutic measures.
(See DISEASE AND PROPHYLAXIS).

21


Concrete tanks are widely used in salmonid culture (Davis, 1967),

but studies to determine the suitability of raceway troughs for
culturing warmwater species were initiated only recently. Shell (1966)
found that managed earthen ponds were more conducive to warmwater fish
production, and thus they were preferred for culturing research fish
over concrete ponds and plastic pools. We have found that the
plasticizers (phthalic acid esters) in polyvinyl plastic pools
contaminate fish and make this type of container undesirable for
research use (Mayer and Sanders, 1973).

Outdoor culture provides natural light and at least some natural food
(see DIETS FOR EXPERIMENTAL FISH), but fish may be exposed to broad
temperature extremes and ponds may be covered with ice in winter. For
this reason, our fish holding facilities are indoors. If a programmed
photoperiod is desired, the apparatus designed by Drummond and Dawson
(1970), or that by Wickham et al. (1971), may be incorporated into
indoor lighting systems.

Many laboratories have only one water source, and if they must hold
numerous species, optimal systems are often difficult to replicate.
Aquarists have long recognized problems associated with maintaining
optimal environments, and they have devised a number of water filtra-
tion and recycling systems (Lewis, 1962; Spotte, 1970; Clark and
Clark, 1964). Partial recirculation or closed systems are desirable
for holding research fish when optimal water quality is not possible.
These systems may have a specific use, e.g., the channel catfish egg
incubation system designed by Giudice (1966a). If only water requiring
dechlorination or decontamination is available, recycling with small
volume addition may be desirable. The probability of water contamina-
tion at FPRL is low because water is pumped from a deep (1,100 ft)


Mehrle, P. M., W. W. Johnson, and F. L. Mayer, Jr. Nutritional
effects on chlordane toxicity in rainbow trout. Bull. Environ.
Contam. Toxicol. (in press).

Meyer, F. Ps 1969. Parasites of freshwater fish: Icthyophthirius
multifilius. U.S. Dep. Interior, Fish Disease Leaflet No. 2.
4p.

Morton, K. E. 1956. A new mechanically adjustable multi-size fish-
grader. Prog. Fish-Cult. 18(2):62-67.

Moss, D. D., and D. C. Scott. 1961. Dissolved oxygen requirements
for three species of fish. Trans. Amer. Fish. Soc. 90(4):377-
393.

Nail, M. L. 1962. The protein requirement of channel catfish,
Ictalurus punctatus (Rafinesque). p. 307-316, In: Proc.
16th Ann, Conf., Southeast Assn, Game and Fish Comm,

Nemoto, C. M. 1957. Experiments with methods for air transport of
live fish. Prog. Fish-Cult. 19(4):147-157.

Phillips, A. M., Sr. 1970. Trout feeds and feeding. In: Manual
of fish culture. U. S. Dep. Interior. 49 p.

Phillips, A. M., Sr., H. A. Podoliak, D. R. Brockway, and R. R. Vaughn.
1957. Cortland Hatchery Report No. 26 for the year 1956. Fish.
Res. Bull. No. 21, N. Y. State Conservat. Dep., Albany, 93 p.

Pickering, Q. H. and W. M. Vigor. 1965. Acute toxicity of zinc to
eggs and fry of the fathead minnow. Prog. Fish-Cult. 27(3):153-
157.

Piper, R. G. 1971. Know the proper carrying capacities of your farn.
Amer. Fish. U. S. Trout News. 15(1):4-6.

Prather, E. E. 1957. Preliminary experiments on winter feeding small
fathead minnows. p. 249-253. In: Proc, 1lth Ann, Conf,, Southeast
Assn, Game and Fish Comm,

Pruginin, Y. and E. W. Shell. 1962. Separation of the sexes of
Tilapia nilotica with a mechanical grader. Prog. Fish-Cult.

24(1):37-40.
43


Wickham, D. A., J. B. Cagle, and F. Hightower, Jr. 1971. Apparatus
for controlling ambient light cycles in experimental environments.
Trans. Amer. Fish. Soc. 100(1):128~136.

Willoughby, H. A. 1968. A method for calculating carrying capacities
of hatchery troughs and ponds. Prog. Fish-Cult. 30(3):173-175.

Wolf, K. 1966a. Infectious pancreatic necrosis of salmonid fishes.
U.S. Dep. Interior, Fishery Leaflet No. 1. 4 p.

1966b. Viral hemorrhagic septicemia of rainbow trout. U.S.,
Dep. Interior, Fishery Leaflet No. 6. 4 p.

Yamamoto, K., Y. Nagahama, and F. Yamazaki. 1966. A method to induce
artificial spawning of goldfish all through the year. Bull.
Jap. Soc. Sci. Fish. 32:977-983.

45


having a restricted gene pool is desired. In most instances, minimal
variation in animal responses due to homogeneity is essential for
interpretation of experimental results. However, in other cases,
heterogeneity may be desirable to estimate the range of responses in
natural populations (Ferguson and Bingham, 1966). Lennon (1967)
stressed the importance of establishing standardized reference strains
of fish for research. Marking (1966) reported variability of bioassay
results when fish from two sources were used. Also, bluegill response
to standard toxicants was found to vary with the history of stress
prior to bioassay use (Lennon and Walker, 1964).

Hatchery-reared fish are the alternative to wild fish, though some

may argue that the former do not truly represent the species. However,
for laboratory research, this factor is probably less important than
the ease of acquisition and quality of hatchery fish reared under
relatively controlled conditions.

Certain hatcheries promote inbreeding to maintain parent stock,
whereas others replenish brood fish from wild stocks. In the past,
wild fathead minnows, channel catfish, and bluegills were collected
for brood stock. However, hybridization is common in wild populations
of centrarchids (Childers and Bennet, 1961), and Giudice (1966b)
reported different growth qualities of hybrid catfish. In contrast,
Buss and Wright (1956) report that salmonid hybrids have poor survival
rates and are less likely to be encountered than hybrids of other
species. Therefore, to minimize experimental variation and insure
known lineage, only hatcheries maintaining stocks that are several
generations removed from wild populations should be considered as
reliable sources of research fathead minnows, channel catfish, and
bluegills. At least 12 established strains of rainbow trout are used