INVESTIGATIONS IN FISH CONTROL

1. Laboratories and methods

for screening fish-control chemicals

2. Preliminary observations 

on the toxicity of antimycin A 

to fish and other aquatic animals

United States Department of the Interior

Fish and Wildlife Service 

Bureau of Sport Fisheries and Wildlife
UNITED STATES DEPARTMENT OF THE INTERIOR

Stewart L. Udall, Secretary 

Frank P. Brings, Assistant Secretary for Fink and Wildlife

FISH AND WILDLIFE SERVICE

Clarence F. Pautzke, Commissioner

BUREAU OF SPORT FISHERIES AND WILDLIFE

Daniel H. Janzen, Director

The United States Department of the Interior, created in 

1849, is concerned with nanagement, conservation, and development of the Nation's water, wildlife, fish, mineral, 

forest, and park and recreational resources. It has major 

responsibilities also for Indian and Territorial affairs.

As America's principal conservation agency, the Department 

works to assure that nonrenewable resources are developed 

and used wisely, that park and recreational resources are 

conserved for the future, and that renewable resources make 

their full contribution to the progress, prosperity, and 

security of the United States, now and in the future.
INVESTIGATIONS IN FISH CONTROL

1. Laboratories and methods

for screening fish-control chemicals

Bureau Circular 185

2. Preliminary observations

on the toxicity of antimycin A 

to fish and other aquatic animals

Bureau Circular 186

Washington, D.C. June 1964
Investigations in Fish Control are reports on the results 

of work at the Bureau's Fish Control Laboratories at 

La Crosse, Wis., and Warm Springs, Ga. The two reports 

presented here are the first of several reports that are 

planned on work now under way.

CONTENTS

Pages

Laboratories and methods for screening fish-control chemicals,

by Robert E. Lennon and Charles R. Walker ............... 1-15

Preliminary observations on the toxicity of antimycin A to fish

and other aquatic animals, by Charles R. Walker, Robert E.

Lennon, and Bernard L. Berger ....................... 1-18
INVESTIGATIONS IN FISH CONTROL

I. Laboratories and methods

for screening fish-control chemicals

By Robert E. Lennon, Fishery Research Biologist 

Charles R. Walker, Chemist 

Bureau of Sport Fisheries and Wildlife 

La Crosse, Wis.

Bureau of Sport Fisheries and Wildlife

Circular 185 

Washington, D.C. June 1964
CONTENTS

Page

Abstract. ....................................... 1

Facilities ....................................... 3

Bioassay laboratories............................ 3

Other laboratory facilities......................... 4

Fish-holding facilities. ........................... 5

Outdoor bioassay pools ........................... 5

Methods ........................................ 6

Test chemicals ................................ 8

Test fishes ................................... 8

Responses of test fish. ........................... 10

Screening.................................... 12

Recording and reporting results ..................... 14

Summary ....................................... 14

Literature cited................................... 15

ii
Laboratories and methods for screening fish-control chemicals

By Robert E. Lennon, Fishery Research Biologist

* and Charles R. Walker, Chemist 

Bureau of Sport Fisheries and Wildlife 

La Crosse, Wis.

Abstract. This report describes the physical and technical facilities 

and the procedures of the Fish Control Laboratories at La Crosse, Wis., 

and Warm Springs, Ga. The laboratories emphasize screening of chemicals to find a variety of fishery management tools. Preliminary Screening 

ascertains whether a chemical in three concentrations has a desirable 

biological activity on eight species of fish in reconstituted water at 12° 

and 17o c. Delineative Screening ascertains effective concentrations 

(EC 100) on eight species in reconstituted water (the method for which is 

described) at 12Qf 17°, 22°, and 27°. Intensive Screening of promising 

fish-control agents ascertains effects on 24 species of fish and on other 

aquatic organisms, at different temperatures and in waters of various 

qualities, in the laboratory and in the field.

Fish Control Laboratories were established 

by the Bureau of Sport Fisheries and Wildlife 

at La Crosse, Wis., in 1959 and at Warm 

Springs, Ga., in 1963. The mission of the Laboratories is the development of means for 

efficient manipulation of fresh-water fish. 

In particular, safe and economical controls 

chemical, biological, electrical, or mechanical are sought for undesirable populations in 

standing and flowing waters. The objectives 

are sufficiently broad to encompass investigation and development of any new tools that 

may be useful in fishery management, fish 

culture, or fishery research.

At La Crosse, the buildings of the National 

Fish Hatchery were remodeled and expanded 

in 1960-62 to provide a large research facility 

(figs. 1 and 2). The subsidiary Laboratory at 

Warm Springs is new construction on the 

grounds of the National Fish Hatchery (fig. 3). 

Their locations offer contrasting advantages 

to be exploited through close coordination in 

the research on fish control:

La Crosse

Northern fishes. 

Cold climate. 

Cold water. 

Hard water.

Warm Springs

Southern fishes. 

Warm climate. 

Warm water. 

Soft water.

In equipping and staffing the laboratories, 

early recognition was given to the potentials of 

chemical control agents. The bioassay (wet), 

chemistry, and physiology laboratories are 

concerned with general and selective toxicants, 

attractants, repellants, anesthetics, sterilants, 

spawning inducers, osmoregulators, marking 

dyes, medications for diseases, and sedatives 

and decontaminants for fish distribution. Emphasis is on finding selective toxicants for 

longnose and shortnose gars, gizzard shad, 

goldfish, carp, squawfishes, white sucker, black 

bullhead, rock bass, green sunfish, pumpkinseed, yellow perch, and freshwater drum.

Ample justification for research on selective 

piscicides is contained in fishery literature.
Figure 1. The Fish Control Laboratory at La Crosse, Wis,

Figure 2. The fish holding house at La Crosse, Wis.
Figure 3. The Fish Control Laboratory (foreground) and wet laboratory holding house at Warm Springs, Ga. 

Construction and grading were incomplete when tfie photograph was taken.

LesVeaux (1959) listed 30 States that need control of certain troublesome fishes. Among the 

qualities desired in selective toxicants are 

specificity to certain life stages or to certain 

fish, low cost, ease and safety of application, 

rapid degradation to nontoxic residues, harmlessness to warm-blooded animals, and effectiveness at low temperatures. Applegate et al. 

(1961) reported on an effective and selective 

sea lamprey larvicide. Loosanoff, MacKenzie, 

and Shearer (1960) showed the possibilities of 

controlling certain shellfish predators with 

chemicals in marine environments. These and 

other studies stimulated interest in investigations to find selective toxicants for various 

fresh-water fish. The American Fisheries Society, for example, resolved at its 88th annual 

meeting in 1958 to recommend, to the Secretary of the Interior, an expansion of research 

in fish control. Congress in the same year 

made the first appropriation for establishment 

of the Fish Control Laboratory at La Crosse.

FACILITIES

Bioassay laboratories

At La Crosse and Warm Springs there are 

wet laboratories for large-scale screening of

chemicals against fish. Fiberglass or aluminum troughs serve as water baths for bioassay 

vessels, and fiberglass or concrete tanks hold 

selected fish for experiments (figs. 4 and 5). 

Ground water is used for the water baths and 

fish holding, and temperatures are adjusted 

by means of thermostatically controlled immersion heaters or refrigeration units.

Deionized water of at least 1 million ohms 

resistivity is reconstituted according to a 

formula developed at the Bureau's FishPesticide Research Laboratory and is employed as a test medium in the bioassay 

vessels. The following chemicals are added 

per liter of deionized water: 30 mg. of calcium 

sulfate, 30 mg. of magnesium sulfate, 48 mg. 

of sodium bicarbonate, and 3 mg. of potassium 

chloride.

Glass vessels are preferred for bioassays 

in the laboratory. Most of the screening is 

done in economical 1-gallon pickle jars which 

are used once and discarded. Five-gallon water 

buckets of glass are employed in advanced 

screening. They are reused after thorough decontamination and washing which include several steps;

a. Rinse jar with tap water.
Figure 4. View of wet laboratory at La Crosse showing batteries of bioassay vessels in concrete, aluminum, and

fiberglass tanks.

b. Add 6.3 grams of activated charcoal 

(1 gram/3 liters), fill jar with deionized water, and let stand over night.

c. Empty and rinse; wash with strong 

detergent in hot tap water and rinse 

thoroughly.

d. Sponge entire jar with 10- to 14-percent hydrochloric acid and rinse twice 

with deionized water.

Whenever residual contamination is detected 

or suspected, the jar is discarded. All discarded bioassay glassware is smashed to prevent further use.

All test solutions are discarded into a floor 

drain which continually carries at least 300 

g.p.m. of waste water from the fish holding 

tanks. The dilution has been found to be more 

than sufficient to eliminate hazards.

All fish used in the bioassays are disposed 

of in gas-fired incinerators of complete-combustion type.

Other laboratory facilities

Each Fish Control Laboratory has chemistry, biochemistry, and physiology laboratories as adjuncts to the bioassay facilities. 

Chemicals for testing are received in the 

chemistry laboratories, stored in fire- and 

explosion-resistant vaults, and prepared in 

proper solutions and dilutions for bioassay. 

Compounds showing promise as fish-control 

agents are investigated in the biochemistry 

laboratories to evolve methods for application, effective and economical formulations, 

possibilities for potentiation, means for minimizing side effects and hazards, and techniques 

for detoxification. In the final stages of development, a control agent is studied in the
Figure 5. View of a wet laboratory at Warm Springs showing a battery of bioassay vessels in a fiberglass tank.

physiology laboratories to define its mode of 

action on fishes and other organisms, any 

chronic effects, the fate of residues in live 

animals, and the risks, if any, to consumers 

of treated fish.

for long periods in usable, disease-free condition. A holding house and outside pools are 

provided at each Laboratory for the maintenance, feeding, sorting, and grading of the 

experimental fish (fig. 3 and 6).

Fish-holding facilities

Large quantities of fish are required for 

the chemical screening programs. At La 

Crosse, for example, 498,000 fish of 34 

species were used in 1963. Most are obtained 

from Federal, State, or private hatcheries 

and rearing stations; it is more satisfactory 

to arrange small, frequent deliveries than 

attempt to maintain large quantities on hand

Outdoor bioassay pools

An intermediate step between laboratory 

testing and field trials of promising fishcontrol agents is essential to detect and 

evaluate some of the physical or chemical 

factors that influence the performance of 

a candidate agent in natural waters. Raceways and portable plastic pools are located 

at each Laboratory for this purpose.
Figure 6. Interior view of fish holding house at La Crosse.

Inexpensive vinyl wading pools, 9 and 10 

feet in diameter, 2.5 feet deep, and about 

1,000 gallons in capacity, are set up as described by Lawrence and Blackburn (1962) in 

outdoor testing areas (figs. 7 and 8). Bottom 

soils of various types, pond or ground waters, 

aquatic plants, invertebrates, fish, and amphibians are used in them as needed during 

chemical trials. Contaminated vinyl liners 

are economically replaced. The pools at La 

Crosse are employed only during the warm 

season, but those at Warm Springs are in 

operation all year.

Ten- and 20-foot concrete raceways are 

used for extraordinary tests in running or 

standing water. Disposable vinyl liners are 

used when necessary to avoid harmful contamination of the raceways.

METHODS

The static bioassay is the first approach 

in screening chemicals for control agents. 

Constant-flow bioassays are reserved for 

advanced stages of testing.

Bliss (1957) defined a bioassay as a determination of the potency of a physical, chemical, or biological agent by means of a biological indicator. Noting its development 

during the past 20 or 30 years by scientists 

from many and diverse fields, he listed 

principles which characterize the modern 

bioassay: (1) Potency is a property of the 

drug, not of the response; (2) potency is 

relative, not absolute; (3) the assayed 

potency of an unknown is only an estimate 

of its true value; and (4) both the reliability
Figure 7. Vinyl bioassay pools on levee at La Crosse.

Figure 8. Vinyl bioassay pools at Warm Springs. 

7
and efficiency of an assay are linked inseparably with its design. Observance of these 

principles overcomes some major disadvantages of the bioassay as a research tool.

Fish have long been employed as biological 

indicators in bioassays of water pollutants, 

insecticides, herbicides, detergents, and 

other substances. The standards recommended by Doudoroff et al. (1951), Henderson 

and Tarzwell (1957), and Henderson (1960) 

for such tests have been widely accepted and 

applied, although difficulties in comparing 

studies have arisen because of the many 

kinds of fish involved. Douglas and Irwin 

(1962) pointed out that the results of independent bioassays often cannot be related 

because the comparative resistance of the 

many test fishes has not been established. 

They noted that certain species have been 

more useful in toxicity bioassays than others, 

and they emphasized the need for knowledge 

about the reactions of different species of 

fish when exposed to a particular toxicant.

The large body of literature on methods 

and results of bioassays with fish has been 

helpful in defining the screening programs of 

the Fish Control Laboratories. In general, 

the practical methods proposed by the investigators cited above are followed but with 

some modifications since we seek more in 

biological activities than acute toxicity only.

Test chemicals

The test chemicals are selected by staff 

chemists and biologists and are contributed by 

industry. Preference is given to compounds 

that have demonstrated biological activity or 

are suspected of possessing a useful activity 

against fish. It is also desirable to have as 

much information as possible before screening on the nature and porperties of each 

chemical, its shelf life and stability, its 

solubility, and its potential hazards to investigators. Security is respected, and precautions are taken with compounds and test data 

to protect the rights of contributors.

The chemicals obtained for screening are 

arbitrarily classified as follows in order to

facilitate scheduling of tests, observation of 

responses, and reporting of results:

1. Natural organic products:

a. Animal extracts (steroids, proteins,

etc.). 

b. Plant extracts (rotenoids, alkaloids,

phenols, etc.). 

c. Fermentation products (antibiotics,

etc.).

2. Synthetic organic products:

a. Halogenated hydrocarbons.

b. Nitrogen-bearing hydrocarbons 

(salicylanilides, carbamates, 

triazines, etc.).

c. Phosphorus-bearing hydrocarbons.

d. Sulfur-bearing hydrocarbons

(mercapto, thioates, thiozines, 

etc.).

e. Miscellaneous compounds and combinations.

3. Inorganic products.

The progress of a chemical through testing 

and development is depicted in figure 9. A 

compound is introduced into Preliminary 

Screening to detect whether it has activity 

against fish. If it has, it is advanced into 

Delineative Screening, where the effective 

concentrations are defined and the possible 

usefulness of the substance is declared. If 

results are favorable, the candidate is referred to Intensive Screening, where it is fully 

evaluated in the laboratory and in the field as 

a fishery tool. Compounds which fail to meet 

requirements at any stage of screening are 

shelved or discarded immediately.

Test fishes

It is of utmost importance that the validity 

and comparability of the bioassays be assured 

by using only fish of selected species, of 

certain sizes, and in good condition as biological indicators. The criteria are defined 

and strictly observed for each stage of screening. Therefore, the rate of progress of a 

chemical through screening and development 

depends largely on the availability of the prescribed fishes.
PRELIMINARY SCREENING

Time and 

Chemical

6 to 12 mo. 

10 groms

rejection

DELI NE ATI VE SCREENING 6 to l£ mo. 

50 grams

rejection . evaluation

rejection Consultation with supplier

rejection

INTENSIVE SCREENING I2to2,4mo.

I kilogram

Laboratory Physiology Outdoor pools Field testing

rejection

\/

rejection

1

rejection

FINAL EVALUATION

rejection acceptance

Figure 9.-eSchematic on the progress of chemicals through screening.

9
TABLE 1. Time required by certain fish held at 12° C. to empty the 

digestive tracts after food is withheld

Number

Tested

of fish

Per pound

Voiding

time (hours)

36

Lots of hatchery fish are requested for 

delivery at least 2 weeks prior to use in bioassays. They are placed in the care of fish 

culturists, and everything possible is done to 

minimize stresses. During the first 10 days 

they are fed, prophylactically or therapeutically treated as necessary, and observed 

to evaluate them as test animals. Lots in 

which the mortalities exceed 10 percent 

within the period are not moved into the 

bioassay program.

The fish for experiments are carefully 

graded to desired and uniform size and transferred into the wet laboratories or outside 

pools 3 or 4 days before use. Food is withheld 

for as long as 96 hours before screening, 

depending on the life stage and species of 

fish. Generally, young fish and certain species require less time to empty the intestinal 

tract than others (table 1). The fish spend at 

least 24 hours in water similar to the test 

medium before being introduced into the 

bioassay vessels.

A help in maintaining comparable results 

in bioassays is use of a recognized reference 

toxicant against a sample of fish from each 

test lot. We employ para-, para f -DDT 

(1,1, l-trichloro-2,2-bis(p-chlorophenyl) 

ethane), and the test is made coincidentally 

with the introduction of a lot of fish into the 

screening program; the test is repeated 

biweekly if the lot remains on hand. The 

sensitivity (EC50) of the sample is determined for comparison with regression curves 

established for the species from experience 

or literature. Although costly, the test provides the only measure of relative sensitivity 

of fish used in the bioassays.

The value of reference tests was demonstrated during shakedown trials of facilities

and methods at La Crosse. For example, goldfish from Missouri and Wisconsin had such 

widely divergent sensitivities that they 

bracketed those of other species. Also, t^ie 

sensitivities within a lot of fish differ sijmificantly if some specimens are exposed to presumably harmless electric shocks like ti ose 

experienced in electrofishing. Moreover, the 

stress of disease, malnutrition, temperature 

change, or altered water quality may influence 

sensitivity to a toxicant. Holding time is 

another factor, and we observed that the 

sensitivities changed greatly in a lot of g'oldfish which was retained for 2 months. Saila 

(1953) emphasized the significance of this 

and showed that the resistance of his mo^quitofish to rotenone decreased in rough proportion 

to the length of time they were held.

Responses of test fish

We recognize that the observer may be the 

greatest source of error in bioassays with fish. 

The criteria of response are arbitrary and 

subject to individual interpretation. Moreover, 

they are complicated at the Fish Control Laboratories because effects in addition to and 

more subtle than acute toxicity are sought. To 

achieve individual and mutual consistency, the 

observers are trained to use definite criteria 

of response.

Chemicals may have either short-term or 

long-term effects on fishes. Those of short 

term are typically identified with the following:

1. Acute toxicity:

a. Selective toxicant, 

b. General toxicant.

2. Movement: 

a. Attractant. 

b. Repellant.

3. Facilitating capture, handling, or 

transport: 

a. Anesthetic, 

b. Sedative, 

c. Osmoregulator.

4. Marking live fish: 

a. Immersion stain, 

b. Internal stain.

10
5. Therapy or prevention of disease: 

a. Bactericide. 

b. Parasiticide, 

c. Fungicide, 

d. Prophylactic, 

e. Antiseptic.

The long-term activities of potential control chemicals may be difficult to detect and 

evaluate. They are associated with 

1. Control of reproduction:

a. Hormonal spawning inducer. 

b. Hormonal spawning inhibitor, 

c. Sterilant.

2. Control of growth and development: 

a. Growth stimulant, 

b. Growth inhibitor.

c. Inverted.

d. On side.

e. Against tank sides on bottom.

Integument;

1. Pigmentation:

a. No observable change, 

b. Light discoloration, 

c. Dark discoloration, 

d. Varidiscoloration.

2. External mucosa:

a. No observable change, 

b. Shedding; patchy, 

c. Copious exudate. 

d. Coagulation.

3. Hemorrhagic.

The criteria employed to evaluate the responses of fish are rather refined, and are 

applicable to short-term responses. They are 

used as necessary at each observation period 

to identify fully the reactions of the fish. 

They are 

General behaviour;

1. No observable difference from control.

2. Quiescent.

3. Excitable.

4. Irritated.

5. Surfacing.

6. Sounding.

7. Twitching.

8. Motionless: 

a. Tetany. 

b. Flaccidity.

9. Swimming:

a. Erratic; convulsive, 

b. Gyrating; skittering.

Respiration;

1. Respiratory rate:

a. No observable change.

b. Rapid.

c. Slow.

d. Irregular.

e. Ceased.

2. Gulping air.

3. Structures, organs:

a. No observable change, 

b. Mouth gaping, 

c. Hemorrhage in gills, 

d. Irritative response, 

e. Copious mucus in gills.

Alimentary responses;

1. No observable change.

2. Egurgitating mucus or other material.

3. Defecating mucus or other material.

Nervous responses;

1. No observable change.

11
Positive Negative

2. Sensitivity to stimuli:

a. Exterior movement.,

b. Light.....................

c. Sound....................

d. Touch....................

e. Electric probe........

Moribundity;

1. No motion.

2. No respiration.

3. Distended operculum.

4. Opaque eyes.

5. Death.

Recovery;

1. Complete.

2. Incomplete.

Additional and more definitive observations 

are made of fish at the in-test, recovery, or 

postmortem stages in the biochemistry and 

physiology laboratories. Their objectives 

may be, for example, the modes of action and 

side effects of control agents.

Screening

Preliminary Screening. Preliminary 

Screening is designed to detect whether a 

selected chemical at 0.1, 1, and 10 p.p.m. 

has a biological activity (see list of criteria 

above) against eight species of fish within 48 

hours. It is a static bioassay in 1-gallon jars 

containing 2.5 liters of pre-aerated, reconstituted water. No artificial aerating is done 

during the test. The temperatures of water

baths are 12° C. at La Crosse and 17° C. at 

Warm Springs.

The fish include the following species:

La Crosse

Rainbow trout. 

Goldfish. 

White sucker. 

Yellow perch.

Warm Springs

Carp.

Black bullhead.

Green sunfish.

Bluegill.

Goldfish from the same gene pool and from 

the same source are employed for occasional 

toxicity checks between the two laboratories.

At least 10 fish of each species are 

with each concentration of chemical and as 

controls. They weigh between half a gram and 

2 grams each, but the weight range of the fish 

in each test does not exceed 15 percent. They 

are distributed at one species per jar about 

16 hours before the bioassay at a loading of 

1 gram or less of fish per liter of test medium 

Thus, there may be but one or two fish per 

jar, and up to 10 jars may be needed for each 

concentration of chemical.

A stock solution of a test compound is prepared within an hour of the bioassay. The 

solvents preferred are water, acetone, ard 

ethanol, in that order. Because of their tcxicity to fish, precautions are taken that the 

concentrations of acetone and ethanol do not 

exceed 4 parts per thousand in the bioassay 

media. Furthermore, whenever these solvents 

are used, equal volumes of them are applied 

in control vessels. Thus the volume in controls is equal to the highest volume used in a 

dosage series. Small aliquots of a stock solution are thoroughly mixed into bioassay media 

to avoid improper dilution or stratification.

The responses of the fish in bioassays and 

controls are observed routinely at 0.75, 1.5, 3, 

6, 24, and 48 hours, but so far as possible they 

are recorded on the first day and thereaf -^r 

as often as the nature of a candidate compound 

warrants.

If a compound has a desirable activity against 

the fish, it is held for further screenings, which 

are discussed briefly below.

12
Delineative Screening. The effective concentrations (EC 100) of a chemical against 

fish are determined in Delineative Screening. 

The vessels may be jars or troughs for static 

or flowing water trials. The water, aeration, 

species and size of fish, duration of tests, and 

controls are the same as in Preliminary 

Screening. A compound is first bioassayed at 

12° C. to define concentrations which evoke 

all-or-none responses from the fish. The 

approach to these concentrations may be 

direct by bracketing and interpolation, or it 

may be indirect by probit analysis, whichever 

is the shorter. The EC 100, if seemingly 

practical, is confirmed by seven replicate 

trials.

A chemical which yields favorable results 

at 12° C. is retested at 17°, 22°, and 27° to 

evaluate the effects of temperature on the 

effective concentrations. At 22° and 27°, rainbow trout are omitted from the bioassays, 

and the loading rates for other species are 

half a gram or less per liter of test medium.

If a compound succeeds in these trials, its 

potential as a fish-control agent is estimated. 

Information is sought on its possible application; on the source and manufacturing costs; 

on possible hazards, conflicts, or limitations 

in use; and on the size of the market. Only if 

it continues to appear promising is the chemical promoted into the more elaborate and 

expensive Intensive Screening.

Intensive Screening. Intensive Screening 

is directed toward development of a promising 

chemical as a fish-control agent and the definition of its advantages and limitations. These 

are broad objectives, and some subdivision 

of approaches is in order.

A. Stages: The advanced tests of a compound are accomplished in the wet laboratories, in outside pools, and finally in the 

field. They may include static or flowing 

water bioassays with durations of 24, 48, and 

96 hours or longer. The vessels may include 

1-gallon jars with 2.5 liters of test medium, 

5-gallon jars with 15 liters of medium, and 

fibergalss troughs of 4-, 8-, and 16-foot 

lengths. The outdoor pools include 1,000- 

gallon vinyl units and 10- and 20-foot concrete

raceways. Proportioning pumps ar? used in 

the flowing bioassays to achieve consistent 

concentrations of chemical.

The initial trials of a compound in the 

field are conducted in waters closed to public 

use. These waters are found, for example, on 

the grounds of Federal and State fish hatcheries, on wildlife refuges, or on military reservations. Trials are manned or immediately 

supervised by the Laboratory staff. Subsequent 

experiments in the field may be accomplished 

by selected cooperators in State ard Federal 

agencies.

B. Varieties of fish: Sixteen species in 

addition to those included in Preliminary and 

Delineative Screening are employed. They are:

1. Brook trout.

2. Northern pike.

3. Fathead minnow.

4. Brook stickleback.

5. Pumpkinseed.

6. Longear sunfish.

7. Smallmouth bass.

8. Walleye.

9. Gizzard shad.

10. Golden shiner.

11. Bigmouth buffalo.

12. Brown bullhead.

13. Channel catfish.

14. Redear sunfish.

15. Largemouth bass.

16. White crappie.

Other species may be used on occasion in 

advanced tests.

C. Life stages: Various life rfages of 

fish, from egg to mature adult, may be involved 

in the more advanced bioassays to detect the 

presence, absence, or variation of response to 

a promising chemical. It may be a great advantage or disadvantage if a potential control 

agent is selective for one life stage and not 

for another.

D. Other organisms: The effects of a 

potential chemical tool on other aquatic life 

must be assessed at least minimally in the 

laboratory before it is tested in the field. Some 

forms such as water fleas, snails, and plants

13
are cultured for this purpose. Others, such as 

freshwater scuds, damselfly and mayfly 

nymphs, and tadpoles, are collected in the 

field. Static bioassays in glass jars are conducted with chemical concentrations and experimental conditions similar to those of the 

fish assays.

E. Temperature; The influences of 

water temperature on the biological activity 

of a candidate chemical are defined for all 

species and different life stages from bioassays under ice and at 2°, 7°, 12°, 170, 22Q, 

and 27° C. It is especially desirable to develop compounds that may be applied effectively during cold seasons when recreational 

use of public waters is low.

F. Water quality: It is recognized that 

water quality may exert tremendous influence 

on the activity of an introduced chemical. Accordingly, the effectiveness of a candidate 

control is observed in hard and soft waters, 

in alkaline and acid waters, and in waters of 

low and high organic content, in the laboratory and field. Initially, the formula for reconstituting deionized water is altered to detect trends.

G. Formulations: Formulation is often 

the key to an effective biological activity or 

efficient application of a compound. Density 

and solubility, for example, may be extremely 

important factors in a fish-control agent. Various formulations with carriers, wetting agents, 

dispersing agents, potentiators, or inhibitors 

may be prepared or acquired for testing. 

Combinations of biologically active chemicals 

are tested to ascertain their potentials as 

multiple controls for fish and aquatic weeds, 

fish and parasites, or fish and amphibians.

Moreover, the practicality of a control 

agent might depend on or be enhanced by an 

applicator's ability to modify or arrest its 

activity efficiently at a given point. Hence, 

the Intensive Screening includes experiments 

with possible deactivators, detoxifiers, or 

decontaminants for potential controls.

Unsatisfactory results or hazardous 

side effects, and new information on manufacturing, marketing, or competitive control

chemicals may contribute at any point in Intensive Screening to the abandonment of further 

trials and development.

Recording and reporting results

The results obtained with each chemical 

are analyzed soon after completion of the 

Preliminary, Delineative, and Intensive 

Screenings. They are furnished to the contributor of the chemical immediately. After 

screening, we evaluate the compound as a 

potential fishery management tool, to determine patent positions, to refer it for studies 

of residues and chronic effects, and to investigate requirements for clearance and labeling.

For compounds with negative results the 

screening data are segregated accordiric to 

the classes of chemicals involved and vill be 

published as soon as the accumulation of data 

warrants. The results with chemicals vhich 

succeed as fish-control agents will be reported 

individually and addressed primarily to fish 

managers or fish culturists. Thus, we plan to 

define the range of effective concentrations for 

certain target fishes in waters of different 

temperatures, different types, and of various 

qualities.

SUMMARY

The Fish Control Laboratories at La Crosse, 

Wis., and Warm Springs, Ga., are equipped to 

find and develop chemicals which may be 

used as fishery management tools. Objectives 

of the research are general and selective toxicants, attractants, repellents, anesthet'^s, 

sterilants, spawning inducers, marking dyes, 

medications for diseases, and sedatives for 

fish distribution. The facilities include chemistry, physiology, and wet laboratories; fish 

holding structures; and outdoor pools and raceways. The program involves three stages of 

chemical screening.

Preliminary Screening detects whether a 

selected chemical at 0.1, 1, and 10 p.p.m. has 

an activity in static bioassays on eight species 

of fish in reconstituted water at 12° and 

17° C.

14
Delineative Screening defines the effective 

concentrations (EC 100) of a chemical in static 

or flowing bioassays on eight species of fish 

in reconstituted water at 12°, 17°, 22°, and 

27° C.

Intensive Screening is reserved for chemicals which show great promise as fish-control 

agents. Effective concentrations are determined in the laboratory, in outdoor pools, and 

in the field against 24 or more species of 

fish in various life stages from egg to adult, 

against selected aquatic organisms, and in 

waters of different temperatures and various 

qualities. The modes of action, side effects, 

chronic effects, and deactivators also are 

studied, as necessary.

The results of screening are reported 

direct to contributors. They are also published by the Bureau. The effective concentrations of fishery tools on target fishes under 

certain conditions are given.

LITERATURE CITED

Applegate, Vernon C.t John H. Howell, James W. 

Moffett, B. Ge He Johnson, and Manning A. Smith. 

1961. Use of 3-Trifluormethyl-4-nitrophenol as 

a selective sea lamprey larvidde. Great Lakes 

Fishery Commission, Technical Report 1, 35 p.

Bliss, C. L.

1957. Some principles of bioassay. American 

Scientists, vol. 45, No. 5, p. 449-466.

Doudoroff, P., B. G. Anderson, G. E. Burdick, P. S. 

Galtsoff, W. B. Hart, R. Patrick, E. R. Strong, E. W. 

Surber, and W. M. Van Horn. 

1951. Bio-assay methods for the evaluation of 

acute toxicity of industrial wastes to fish.

Sewage and Industrial Wastes, vol, 23, No. 11, 

p. 1380-1397.

Douglas, Neil H., and W. H. Irwin.

1962. Evaluation and relative resistance of sixteen species of fish as test animals in toxicity 

bioassays of petroleum refinery effluents. 

Department of Zoology, Oklahoma State University, Contribution No. 351. Mimeo. 40 p.

Mender son, Croswell.

1960. Bioassay procedures, aims, and equipment. 

Biological Problems in Water Pollution, Technical Report W60-3, The Robert A. Taft Sanitary Engineering Center, U.S. Public Health 

Service, p. 246-248.

Henderson, Croswell, and Clarence M. Tarzwell. 

1957. Bio-assays for control of ind "Strial effluents. Sewage and Industrial Wastes, vol. 29, 

No. 9, p. 1002-1017.

Lawrence, J. M., and R. D. Blackburn.

1962. Evaluating herbicidal activity of chemicals 

to aquatic plants and their toxicity to fish in the 

laboratory and in plastic pools. Auburn University. Mimeo. 23 p. (In press, Proceedings of 16th 

Annual Conference of Southeastern Association 

of Game and Fish Commissioner?, Columbus, 

S.C.).

LesVeaux, John F.

1959. Summary report of survey to evaluate the 

need for specific fish toxicants in sport fishing 

waters. Progressive Fish-Culturist, vol. 21, 

No. 3, p. 99-110.

Loosanoff, V. L., C. L. MacKenzie, Jr., and L. W. 

Shearer.

1960. Use of chemicals to control shellfish predators. Science, vol. 131. No. 3412, p. 1522- 

1523.

Saila, Saul B.

1953. Bio-assay procedures for the evaluation 

of fish toxicants with particular reference to 

rotenone. Transactions of the American 

Fisheries Society, vol. 83, p. 104-114.

15
INVESTIGATIONS IN FISH CONTROL

2. Preliminary observations 

on the toxicity of antimycin A 

to fish and other aquatic animals

By Charles R. Walker, Chemist

Robert E. Lennon, Fishery Research Biologist

Bernard L. Berger, Chemist

Bureau of Sport Fisheries and Wildlife

Bureau of Sport Fisheries and Wildlife

1 Circular 186 

Washington, D.C. June 1964
CONTENTS

Page

Abstract. ......................................... 1

Antimycin. ........................................ 1

Sources and uses ................................. 1

Composition and structure........................... 2

Physical and chemical properties ...................... 2

Biological activity ................................ 2

Methods and materials ................................ 4

Laboratory tests ................................. 4

Field tests ..................................... 4

Results of laboratory studies ............................ 6

Trouts ........................................ 7

Herrings. ...................................... 7

Perches ....................................... 8

Pikes. ........................................ 8

Suckers ....................................... 9

Sunfishes ...................................... 9

Sticklebacks .................................... 9

Minnows and carps................................ 9

Fresh-water catfishes. ............................. 10

Other animals ................................... 11

Results of field studies ................................ 11

Tests in wading pools .............................. 11

Tests in hatchery ponds. ............................ 14

Discussion of field studies ........................... 15

Conclusions. ....................................... 16

Literature cited. .................................... 17

ii
Preliminary observations on the toxicity of antimycin A to fish and other aquatfc animals

By Charles R. Walker, Chemist 

Robert E. Lennon, Fishery Research Biologist

and Bernard L. Berger, Chemist 

Bureau of Sport Fisheries and Wildlife

Abstract. Antimycin A, an antifungal antibiotic, has been suggested 

for use as a fish toxicant. Preliminary tests were made to evaluate its 

effects at concentrations of 0.01 to 120 p.p.b. on 24 species of freshwater fish in the laboratory and 25 species in outdoor pools. Responses 

of a select group of other animals and aquatic plants are discussed. The 

antibiotic is a powerful fish toxicant. Carp and other rough fish were 

killed by small concentrations in short exposures at cool and warm temperatures. Longnose gar, bowfin, black bullheads, and yellow bullheads 

were relatively resistant to the quantities tested. Plankton, aquatic plants, 

bottom fauna, salamanders, tadpoles, and turtles were not harmed by 

pisicicidal concentrations. Antimycin A degrades rapidly in water, 

especially in the presence of free hydroxide. Detoxification occurred 

within 24 to 96 hours. Further studies are planned on the performance 

of antimycin A against various life stages of fish, on other aquatic animals, and in waters of differing qualities and temperatures. The process 

of detoxification and the fate of residues deserve further attention.

An objective of the Fish Control Laboratories is the development of new fish toxicants 

that can be used safely and economically in 

the management of fish populations. Antimycin 

A exhibits properties desired in a candidate 

fish toxicant. It is lethal to certain target 

fishes in low concentration and on short exposure; it works in cool and warm water and 

in the presence of aquatic plants; it degrades 

rapidly in water and appears to leave no harmful residue.

This report summarizes data obtained on 

antimycin A in the laboratory and small outdoor pools and larger hatchery ponds. Development and efficacy of the compound as a 

fishery tool is to be further investigated.

ANTIMYCIN

Sources and uses

Antimycin is an antifungal antibiotic isolated 

from the bacteria Streptomyces sp, and identified by Dunshee, Leben, Keitt, and Strong 

(1949) at the University of Wisconsin. Following this discovery, at least seven st>ecies of 

Streptomyces were found to be producers of 

antimycin. Burger, Teitel, and Grunberg 

crystalized the antibiotic from two species of 

Streptomyces (Strong, 1956). Later at the 

University of Wisconsin, another culture produced an antimycin-like product wlich showed 

promise as an antibiotic for plant pathogens 

(Lockwood et al., 1954).
Harada and associates (Nakayama et al., 

1956) in Japan discovered an antimycinproducing culture Of Streptomyces kitazau-aensis 

which differed from the first culture at the 

University of Wisconsin, but both produce an 

antitumor substance (carzinomyceticus). Research at the University of Tokyo by Watanebe 

et al. (1957) on S. blastmyceticus yielded an 

antibiotic called blastmycin which consists 

largely of antimycin A3. Harada et al. (1959) 

devoted special attention to the antifungal 

property of blastmycin as a control for rice 

blast disease (Piricularia oryzae) in Japan.

Derse and Strong (1963) related that antimycin is an antibiotic of unusual chemical 

structure which is toxic to yeasts, other fungi, 

insects, and mammals, but not to bacteria. 

They also reported that it is extremely toxic 

to goldfish at 1 p.p.b. On the basis of this 

observation, on the rapid degradation of the 

chemical, and its much lower toxicity to 

higher animals, they suggested that antimycin 

may be useful in fish management.

Composition and structure

The complex structure of antimycin was 

elucidated by Dunshee et al. (1949), Tener 

et al. (1953), Strong (1956) and Strong et al. 

(I960), van Tamelen et al. (1959 and 1961), 

and Dickie et al. (1963). It is illustrated in 

figure 1.

Lockwood et al. (1954) described antimycin 

as a complex made up of several active fractions which they identified from paper 

chromatograms as AI, A2, A3, and A^ according to increasing Rp values. Liu and Strong 

(1959) determined that one or more of these 

Rp values were represented in antimycin 

A-35, antimycin A-102, blastmycin, and 

virosin, and they investigated them. Further 

study by Dickie and his associates (1963) 

established that the fractions differ only in 

the alkyl side chain (R) in figure 1. The antimycin AI and A^ fractions are probably isomeric with R = n-hexyl, and calculations of 

the elemental composition indicate that the 

emperical formula is 028^40^2^9. The A2 

and A3 isomers bear the ri-butyl side chain, 

and the emperical formula is perhaps 

^26^36^209. The percentage composition

of fractions or isomers is very important 

to the biological activity of the antimycin 

complex.

Physical and chemical properties

The fermentation extracts of antimycin are 

dark, tarry substances which upon further 

purification yield a fine crystalline material. 

This nitrogenous, phenolic complex is characterized by solubility in polar organic solvents 

including ethanol, acetone, and chloroform; 

slight solubility in nonpolar solvents including 

petroleum ether, benzene, and carbon tetrachloride; and relative insolubility in water and 

5-percent solutions of hydrochloric acid, 

sodium bicarbonate, and sodium carbonate 

(Keitt, Leben, and Strong, 1953).

The infrared absorption spectrum of antimycin has been identified in isolates from 

several cultures, although the crystalline 

products appear to have different properties. 

These differences are attributed to the intricate composition of the antibiotic and the 

presence of impurities associated with samples (Strong, 1956). For example, blastmycin 

has almost the duplicate IR spectrum of antimycin A-35 isolate, but the melting points are 

166°-167° and 140.5°-141.5° C. respectively. 

Blastmycin is composed primarily of the antimycin A3 fraction with a trace of A4 ir contrast to antimycin A-35, antimycin A-102, and 

virosin, which contain additional subcompounds 

A! and A 2 (Strong, 1956; Liu and Strong, 1959).

Antimycin is susceptible to alkaline degradation as indicated in figure 1. Hydrolytic 

cleavage occurs at the lactone carbonyl sites 

on the cyclic diester and leads to the formation of antimycic acid or blastmycic ard the 

neutral fragment (van Tamelen et al., 1961; 

Liu et al., I960; and Tener et al., 1953). The 

degradation is rapid in water, and detoxification of 10 p.p.b. is accomplished withir 7 days 

according to Derse and Strong (1963); it is 

accelerated in the presence of light, high 

alkalinity, and warm temperatures.

Biological activity

Antimycin is a powerful and highly selective 

inhibitor of the electron transport in oxidative
CONH

NHCHO

0

fl 

0 C CH2CH(CH3)2

6 '* 

Antimycin A, ; R* n-hexyl : C 28H4QN209

Antimycin A3 , R » n - butyl :

I COOH

I -CONH CH

L^w JL » ^^ \r fi

^XN>H | 

NHC-HO CH3

Blastmycic acid C^H^OS

^ COOH ^ 

^^^>.CONH CH HCOOH

f^ir * Formic acid 1 H COH V^

<r 

0" L\f n-» 

NH 2 3

Antimycic acid 

C M H .4N 2°5

3-omino- Glycine 

salicylic acid 

C,H.NO, C 2 H5 N02

R' X R 

Antimycin 1 acton r

R ^ V O C VflMV rl i%^O«fTM

fm « <^C

' n 

R»OOCCH2CH(CHr)2

 ^ Hydroxy 1 act one

R=OH 

R'*!!- C 6 H I3

 >-<><- n-hexyl - 

r levulinic acid 

C| | H2003

 >.Methylethylactetic acid

Ffeure 1. Structure of antimycin and the assumed process of breakdown under alkaline conditions

in the laboratory.
phosphorylation systems (Strong, 1956). It 

retards the respiration of cells, and the selective action, in the electron transport chain at 

the cytochrome b - (Coenzyme Q) - cytochrome 

c has made antimycin an indispensable reagent 

for enzyme studies. Its effects on the succinic-oxidase system have been described 

as the "antimycin-A-blocked factor." Gottlieb 

and Ramachandran (1961) illustrated the site 

of action of antimycin and ascosin as follows:

Substrate p> Pyridine nucleotide ^> Flavoprotein ^> 

cytochrome b,

__ __ "s- S

s* **.

^coenzyme Q^ 

cytochrome c ^> cytochrome a ^> oxygen

N--^>"AS" site >>

Because of its extreme potency as an 

inhibitor of electron transport, Derse and 

Strong (1963) surmised that antimycin is 

absorbed into the gills and interferes with 

respiration in fishes.

METHODS AND MATERIALS

Crystalline antimycin A was supplied by the 

Wisconsin Alumni Research Foundation from 

Kyowa Fermentation Company, Ltd., in Tokyo, 

Japan. This material was isolated from the 

Culture of Streptomyces kitazawaensis and had 

the following fractions by weight: Aj, 40 percent; A2, 20 percent; A3, 20 percent; and A4, 

10 percent. Although the fraction A 3 amounts 

to only 20 percent, it accounts for about 60 

percent of the biological activity.

Stock solutions were prepared with 100 miligrams of crystalline antimycin A dissolved in 

1 liter of acetone. They were renewed with 

each series of bioassays, although tests indicated that solutions in acetone are relatively 

stable up to 24 days. Crystalline material 

stored at room temperature for 2 years also 

remained stable.

Laboratory tests

The methods and facilities employed for 

evaluation of potential fish-control agents were 

described by Lennon and Walker (1964). The

bioassays of antimycin A were conducted in 

slightly alkaline and medium hard, reconstituted water at 12°, 17°, and 22° C. Twentyfour species of fish, representing nine families, were included (table 1). They were, 

supplied by national fish hatcheries, the Wisconsin Conservation Department, and O^ark 

Fisheries, Inc., and each lot was graded to 

a desired size before use.

Aliquots of the stock solution of antimycin 

A were diluted and stirred into the 1- or 5- 

gallon bioassay vessels in the presence of 

fish. The responses of the fish to the toxicant 

were observed at 24, 48, 72, and 96 hours.

Other animals included in bioassays were 

water fleas (Daphnia magna), crayfish (Cambants 

sp.), damselfly nymphs (Ischnura sp.), t'ger 

salamander (Ambystoma tigrinum), and bullfrog 

tadpoles (Rana catesbiana). They were Stocked 

in bioassay vessels as follow: 10 water fleas 

or 2 damselfly nymphs in each 16-ounce jar, 

1 crayfish or 2 bullfrog tadpoles in each 1- 

gallon jar, and 1 adult tiger salamander in 

each 5-gallon jar.

Field tests

Vinyl wading pools. Only a few outdoor 

bioassays were made in 1962 and 1963 because 

only small quantities of toxicant were available.

TABLE 1. Hie 24 fishes used in laboratory tests of antimycin A

Common name

Brown trout ..............

Stone roller. ............. 

Goldfish. ................

Golden shiner. ........... 

Fathead minnow. ..........

Black bullhead........... 

Channel catfish. .........

Green sunf ish. ........... 

Pumpkinseed. .............

Bluegill.................

Longear sunf ish. .........

Iowa darter. .............

Yellow perch. ............ 

Walleye ..................

technical name

Dorosoma cepedianum

Salmo trutta

Campostoma anomalum 

Carassius auratus

Notemigonus erysoleucas

Pimephales promelas 

Catostomus conmersoni

lotalurus melas

IctaluruB natalis 

Ictalurus punotatus

Lepomis cyanellus 

Lepomis gibbosus

Lepomis maerochirus

Lepomis megalotis

Pomoxis annularis 

Etheostoma exile

Perca f lavescene 

Stizostedion vitreum

Size range 

(grams)

12.0-15.0 

1.0- 1.6

1.2- 1.4 

0.5- 0.6

3.0- 4.0 

1.5- 2.4

1.0- 2.2

0.9- 1.8 

1.3- 2.8

0.7- 2.3

1.2- 2.2 

1.5-^1.8

0.8- 2.5 

1.0- 2.3

1.2- 2.4

1.0- 2.5

1.5- 3.0 

0.6- 1.2

0.6- 3.0 

0.4- 0.8
The test vessels were 1,000-gallon wading 

pools similar to those described by Lawrence 

and Blackburn (1962). Some physical, chemical, and biological conditions characteristic 

of ponds were simulated or intrinsic. The 

physical aspects included bottom soils of sand 

and loam, naturally varying temperatures, 

turbidity, and natural light. The chemistry of 

the well water in the pools was modified by 

physical and biological factors.

Of the 18 pools, 9 had 3 inches of sand on 

the bottom, and 9 had 3 inches of silt loam. 

After the pools were filled, the following were 

introduced: Sagittaria latifolia, Elodea canadensis, 

Myriophyllum heterophyllum, Potamogeton nodosus, 

P. pectinatus, Spirogyra spp., and phytoplankton. 

They were established, and the water chemistry was stabilized, during the 4- to 8-week 

periods before fish were added. Fingerling 

and adult fish were stocked 1 to 2 weeks before applications of the toxicant.

The rate of detoxification of the antimycin 

was observed, and some of the killed fish were 

shipped to the Wisconsin Alumni Research 

Foundation for mammalian toxicity tests. 

Bottom fauna were sampled and quantitated. 

Data were obtained on water chemistry during

POND NO. 2

the course of tests according to standard 

methods (American Public Health Association 

et al., 1960).

Hatchery ponds. The Wisconsin Conservation Department provided two pondr for tests 

at the Delafield Warmwater Fisher:'es Research Station in September 1963. 7"he surface areas of ponds No. 2 and No. 5 are 0.47 

and 0.78 acre respectively (fig. 2).

Pond No. 2 was stocked with 18 soecies of 

fish at the rate of 240 pounds per acre, and 

pond No. 5 with 19 species at 225 pounds per 

acre, 1 week before antimycin was applied. 

Samples of water, plankton, and bottom fauna 

were taken from each pond soon aft^r the 

fish were stocked and again just before the 

ponds were drained (table 2).

TABLE 2. Concentrations of antiaycin A which caused ajj-or-none survival 

among rainbow trout and brown trout at selected water temperatures in 

24 and 96 hours

Species

Rainbow trout. . . .

Brown trout. .....

Number 

of 

fish

1,829

120

348

120

Tenperature

(° c)

12 

17

12 

17

Concentrations (p.p.b.) and survival

At 24 hours

All

0.10 

0.02

0.10 

0.02

None

0.60 

0.08

0.40 

0.06

At 96 hours

All

0.02 

<0.02

<0.06 

<0.04

None

0.08 

0.04

0.08 

0.06

POND NO. 5

YY Charo

* + Spirogyra

0 o Potamogeton

CONTOUR

0 - I ft. 

I -2ft. 

Z + ft.

ACRE-FEET

0.44

0.33

0.01

TOTAL 0.78

CONTOUR

0 - I ft.

1 - 2 ft.

2 - 3 ft. 

3+ ft.

ACRE-FEET

0.68

0.63

0.42

0.17

TOTAL 1.90

Figure 2. Sketch of ponds No. 2 and No. 5 at the Delafield Warmwater Fisheries Research Station.

5
Two formulations of antimycin A were prepared for application at 10 p.p.b. Pond No. 2 

received 9.72 grams of technical material in 

a carrier formulated by the S. B. Penick Company to make up a total volume of 300 ml. 

Pond No. 5 received 23.37 grams of technical 

material dissolved in 300 ml. of acetone as a 

carrier. Each aliquot was mixed with 2 gallons of water and applied to a pond surface 

with a hand-powered garden sprayer. The 

applications were made from a rowboat in 

late afternoon, and frequent observations 

were made during the next 8 hours. Observations and recovery of dead fish were made 

daily in the following 4 days.

RESULTS OF LABORATORY STUDIES

We found that antimycin A is toxic to the 

24 species of fish tested. The toxicity varies 

among species and is correlated with water 

temperature and time. Trends in sensitivity 

reflect taxonomic relationships of the fishes,

and variations in susceptibility amonir individuals was more pronounced in som^ species 

than others. The following remarks pertain 

principally to the concentrations which delineate the all-or-none survival EC0 to EC 100 

ranges, of fish at 24 or 96 hours in b^oassays 

at 12°, 17°, or 22° C. Data are shown graphically in figures 3 and 4.

Among the 24 species, the group of fish 

most sensitive to antimycin A includes gizzard shad, rainbow trout, brown trou*:, white 

sucker, Iowa darter, yellow perch, and walleye. All survived exposure to 0.08 p.p.b. for 

24 hours at 12° C; all perished at 0.8 p.p.b.

The group intermediate in sensitiv'ty included northern pike, stoneroller, carp, 

golden shiner, fathead minnow, bigmouth 

buffalo, brook stickleback, green sun*ish, 

pumpkinseed, bluegill, longear sunfish, 

largemouth bass, and white crappie (*ig. 5). 

Concentrations of 0.1 and 1.6 p.p.b. c^fined 

their all-or-none survival in 24 hours at 12° C.

GIZZARD SHAD-----^__ C

RAINBOW TROUT - - 

BROWN TROUT - -

NORTHERN PIKE 

STONEROLLER - - - 

GOLDFISH 

CARP - - - - - -

GOLDEN SHINER 

FATHEAD MINNOWWHITE SUCKER - 

BIGMOUTH BUFFALO 

BLACK BULLHEAD

YELLOW BULLHEAD

CHANNEL CATFISH -

BROOK STICKLEBACK

GREEN SUNFISH - - -_^^£

PUMPKINSEED- - - -_......... P

TEMPERATURE

BLUEGILL- - _________f * YffffffffffffZZ2Etr mm |20C

LONGEAR SUNFISH ----- i ^^MM^^^^» -j -, ,Toc

LARGEMOUTH BASS - 

_____________________________ iffjrj^

WHITE CRAPPIE 

IOWA DARTER 

YELLOW PERCH--

WALLEYE - 

O.I 1.0 10 too

Figure 3. The 24-hour responses of 24 fishes in the laboratory to antimycin A in pj>.b. The solid, plain, 

and cross hatched bars span the ranges between the ECQ and ECjoo at 12°- 17°» and 22° c»
GIZZARD SHADRAINBOW TROUT - - - - 

GOLDFISH

CARP

FATHEAD MINNOW

WHITE SUCKER -----

BLACK BULLHEAD - - - 

YELLOW BULLHEADCHANNEL CATFISH

GREEN SUNFISH 

PUMPKINSEED - - - - 

BLUEGILL

LONGEAR SUNFISH - - -

LARGEMOUTH BASS _____--- 

IOWA DARTER ------

YELLOW PERCH--- -g^

-ezzzi

TEMPERATURE

 I2°C

<=3 |7t

B2Z3 22°C

.01 O.I 1.0 10 100

Figure 4. The 96-hour responses of 16 fishes in the laboratory to antimycin A in p.p.b. The solid, plain, 

and crosshatched bars span the ranges between the ECQ and ECjoo at 12°, 17°, and 22° C.

The more resistant group of fish was 

represented by goldfish, black bullhead, 

yellow bullhead, and channel catfish. The 

concentrations required for kills in 24 hours 

at 12° C were 20 p.p.b. for channel catfish, 

80 p.p.b. for yellow bullhead, 100 p.p.b. for 

goldfish, and 120 p.p.b. for black bullhead.

Increases in water temperature or duration 

of exposure made significant differences in 

the toxicity of antimycin A to fish in the three 

groups. For example, the toxicity to goldfish 

was increased tenfold at the higher temperature of 17° C. Among catfishes, the toxicity 

was enhanced about twofold at 17°. At the 

maximum temperature of 22°, the black and 

yellow bullheads were about 10 times as 

tolerant to antimycin A as goldfish, but 

channel catfish were only slightly more 

resistant.

For more detailed discussion on the toxicity 

of antimycin A, the species are grouped 

according to their respective families. The

families, in turn, are presented in order of 

their sensitivity to the toxicant.

T routs

Rainbow trout and brown trout were extremely sensitive to antimycin A (trble 2). At 

12°, the rainbow trout succumbed tc 0.6 p.p.b. 

in 24 hours and to 0.08 p.p.b. in 96 hours. At 

the same temperature, brown trout were killed 

by 0.4 p.p.b. in 24 hours and by O.OF p.p.b. in 

96 hours. Both species tolerated concentrations of 0.1 p.p.b. for 24 hours. In 9< -hour 

tests, the rainbow trout survived 0.02 p.p.b. 

whereas brown trout withstood 0.06 p.p.b.

Herrings

At 12° C., all gizzard shad died within 24 

hours upon exposure to 0.8 p.p.b. ard within 

96 hours at 0.1 p.p.b. (table 3). They were 

especially sensitive to the toxicant at 22°:
MOST SENSITIVE INTERMEDIATE LEAST SENSITIVE

TROUTS PIKES FRESHWATER CATFISHS

PERCHES SUNFISHES GARS

HERRINGS SUCKERS BOWFINS

STICKLEBACKS

A A* i

CARPS

> . GOLDFISH <..

**" .^*- * 

Figure 5. The order of sensitivity of 11 families of fish to antimycin A in the laboratory and field.

concentrations of 0.04 p.p.b. caused complete 

kills within 24 hours, and partial kills occured 

at 0.02 p.p.b. or more. It was noted that a 

narrow range of concentrations yielded all-ornone survival, particularly at the higher temperature and longer exposure.

Perches

The Iowa darter, yellow perch, and walleye 

were also very sensitive to antimycin A 

(table 4). All specimens in 0.08 p.p.b. at 12O 

for 24 hours survived, but those in 0.66 p.p.b. 

died. The narrow range in concentrations 

which caused all-or-none survival was more 

apparent at 22° and 96-hour exposures. Yellow 

perch, for example, survived 0.02 p.p.b. for 

24 hours and 0.01 p.p.b. for 96 hours; they

died at 0.08 p.p.b. within 24 hours and at 

0.06 p.p.b. within 96 hours.

Pikes

The fry and fingerlings of northern pike 

were difficult to use in bioassays beca use of 

cannibalism and rapid growth. Nevertl ^less, 

they exhibited great susceptibility to entimycin A. Complete kills were obtained in 24

TABLE 3. Concentrations of antimycin A which caused all-or-none survival 

among gizzard shad at selected water temperatures in 24 and 96 hours

Number

of

120

60

60

ature f °n "i

12

17

22

Concentrations (p.p.b.) and survival

At 24 hours

All

0.10

0.08

0.02

None

0.80

0.40

0.04

At 96 hours

All

0.06

0.04

0.02

None

0.10

0.08

0.04

8
hours by 0.8 p.p.b. at 12°, 0.2 p.p.b. at 17°, 

and 0.1 p.p.b. at 22°. In contrast, all specimens survived 0.4 p.p.b. at 12°, 0.08 p.p.b. 

at 17°, and 0.06 p.p.b. at 22°. Greater toxicity 

was detected in 48-hour exposures, but the 

concentrations related to all-or-none survival 

were not defined.

Suckers

The white sucker and bigmouth buffalo 

differed in their sensitivities to the toxicant, 

and the former was among the most susceptible fishes tested (table 5). Concentrations greater than 0.06 p.p.b. produced partial kills of white suckers at 12° in 96 hours, 

and 0.22 p.p.b. caused complete kills. Even 

greater sensitivity was observed at 22°. The 

bigmouth buffalo, on the other hand, required 

concentrations of antimycin A in excess of 

0.4 p.p.b. for complete kills in 96 hours at 12°.

Sunfishes

Green sunfish, pumpkinseed, bluegill, longear sunfish, largemouth bass, and white crappie 

were moderately sensitive to antimycin A

TABLE 4. Concentrations of antimyoin A which caused all-or-none survival 

among Iowa daters, yellow perch, and walleye at selected water temperatures in 24 and 96 hours

Species

Iowa darters.... 

Yellow perch. . . .

Number 

of 

fish

275

560 

504 

60

20

20

Temperature

(°c.)

12

12 

17 

22

12

17

Concentrations (p.p.b.) and survival

At 24 hours

All

0.10

0.10 

0.08 

0.02

0.08 

<0.08

Hone

0.66

0.40 

0.40 

0.08

0.60 

0.10

At 96 hours

All

0.08

0.06 

0.02 

0.01

None

0.14

0.20 

0.08

0.06

TABLE 5. Concentrations of antimycin A which caused all-or-none survival 

among white sucker and bigmouth buffalo at selected temperatures in 24 

and 96 hours

Species

White sucker ....

Bigmouth buffalo

Number 

of 

fish

810 

36

72

430

Temperature

C°c.)

12 

17 

22

12

Concentrations fp.p.b.) and survival

At 24 hours

All

0.22 

0.06 

<0.04

0.20

None

0.64 

0.40 

0.20

2.00

At 96 hours

All

0.06 

0.04 

<0.06

<0.10

Hone

0.22 

0.10

0.10

0.40

(table 6). The concentrations required to cause 

complete kills of them at 12° ranged from 1 to 

6 p.p.b. in 24 hours and from 0.2 tc 0.8 p.p.b. 

in 96 hours. At 22°, killing concentrations 

ranged from 0.2 to 0.8 p.p.b. in 24 hours and 

from 0.08 to 0.4 p.p.b. in 96 hours.

The pumpkinseed and bluegill were the more 

sensitive of the six species, and they were 

followed in order of decreasing sersitivity by 

longear sunfish, largemouth bass, v'hite crappie, 

and green sunfish.

Sticklebacks

Brook sticklebacks were moderately sensitive to antimycin A at 12°. Concentrations of 

5 p.p.b. killed all specimens within 24 hours, 

and partial kills occurred at concentrations 

greater than 0.5 p.p.b. The exposures beyond 

24 hours failed to give consistent results. The 

condition of the fish was suspect because of 

difficulty in maintaining them without feeding 

during the longer test periods.

Minnows and carps

Stoneroller, goldfish, carp, golden shiner, 

and fathead minnow responded over a wide 

range of concentrations in an interesting 

pattern of susceptibility. In contract to other 

families, the minnows exhibited greater variation in response between species as well as 

between individual speciments (table 7).

TABLE 6. Concentrations of antimycin A which caused all-or-none survival 

among green sunfish, pumpkinseed, bluegill, longear sunfish, largemouth 

bass, and white orappie at selected water temperatures in 24 and 96 hours

Species

Green sunfish. . . .

Pumpkinseed. .....

Longear sunfish..

Largemouth bass.. 

Write crappie....

Number 

of 

fish

396 

216 

30

480 

120 

180

1,053 

360 

200

240 

48 

48

800 

180

Temperature

(° c)

12 

17 

22

12 

17 

22

12 

17 

22

12 

17 

22

12 

12

Concentrations (p.p.b.) and survival

At 24 hours

All

0.80 

0.20 

0.08

0.40 

0.06 

0.01

0.20 

0.10 

0.04

0.20 

0.08 

0.08

0.20 

0.60

Hone

6.00 

2.00 

0.80

2.00 

1.00 

0.20

1.00 

0.60 

0.20

2.00 

0.40 

0.40

<6.00 

>2.00

At 96 hours

All

0.04 

0.08 

0.08

0.08 

0.04 

0.01

0.06 

0.01 

0.02

0.04 

0.04 

0.02

0.10

None

0.80 

0.60 

0.40

0.20 

0.10 

0.08

0.40 

0.10 

0.08

0.40 

0.20 

0.20

0.80
TABLE 7. Concentrations of antimycin A which caused all-or-none survival 

among stoneroller, goldfish, carp, golden shiner, and fathead minnow at 

selected water temperatures in 24 and 96 hours

Species

Golden shiner. . . .

Fathead minnow. . .

Number 

of 

fish

531

1,469 

312 

200

240

84

60 

60 

40

816 

96 

78

Temperature 

(° C)

12

12 

17 

22

12 

17

12 

17 

22

12 

17 

22

Concentrations (p.p.b.) and survival

At 24 hours

All

0.08

4.00 

1.00 

0.30

0.60 

0.40

0.20 

0.10 

0.05

0.10 

<0.80 

<0.10

None

1.00

100.00 

8.00 

4.00

2.00 

1.00

2.00 

0.40 

0.50

2.00 

2.00 

0.80

At 96 hours

All

0.40 

0.20 

0.08

0.08 

0.08

0.05

0.08 

<0.06 

<0.10

None

2.00 

1.00 

0.60

0.60 

0.40

0.60

0.40 

0.10 

0.10

bility were observed; a concentration which 

killed on one occasion failed on the next.

The golden shiner and fathead minniw were 

somewhat similar to the stoneroller in sensitivity, but all-or-none effects were delineated within a narrow range of concertrations. 

The golden shiners succumbed to 0.6 p.p.b. 

within 96 hours at 12°, and survival vas noted 

at 0.05 p.p.b. Fathead minnows died at 0.4 

p.p.b. and survived at 0.08 p.p.b.

Fresh-water catfishes

An outstanding highlight of the screening 

program was the discovery that carp are 

vulnerable to small concentrations of antimycin A. This prolific exotic is widely considered a most undesirable species in gamefish waters and is difficult to control with 

existing means.

At 12° all test carp were killed by 2 p.p.b. 

of antimycin in 24 hours and by 0.6 p.p.b. in 

96 hours; at 17° all were killed by 1 p.p.b. in 

24 hours and by 0.4 p.p.b. in 96 hours. Temperatures had less effect on toxicity to carp than 

to most species. There were only slight differences due to temperature in 24-hour exposures and even less at 96 hours. All carp 

survived 0.08 p.p.b.

The results on goldfish contrasted sharply 

with those on carp. In fact, the goldfish was 

the most tolerant of the minows tested against 

antimycin A. It required 100 p.p.b. for complete kills within 24 hours at 12°, but only 2 

p.p.b. were needed for kills within 96 hours. 

Higher temperatures contributed to greater 

toxicities, and all goldfish perished within 

96 hours when exposed to 1 p.p.b. at 17° and 

0.6 p.p.b. at 22°.

Stonerollers were among the more sensitive minnows. Concentrations of toxicant as 

low as 1 p.p.b. killed all specimens within 

24 hours at 12°, but variations in susceptiThe catfishes were significantly lers sensitive to antimycin A than other families 

(table 8). Channel catfish were more susceptible than bullheads. They survived 24-hour 

exposures at 12° to 2 p.p.b. but perished at 

20 p.p.b. All specimens died at 6 p.p.b. in 

96-hour tests at 22°.

The black bullhead was the more tc^erant 

to the toxicant, and the yellow bullhea d was 

only slightly less so. Concentrations of 120 

and 100 p.p.b. respectively were required 

for complete kills in 24 hours at 12 . These 

concentrations are more than 100 tiires 

greater than those needed to kill fish of the 

most sensitive families.

The bullheads were affected by somewhat 

smaller quantities of chemical at 17 . Nevertheless, black bullheads tolerated 4 p.p.b. for 

24 hours at 22°, and all died at 40. p.j\b.

TABLE 8. Concentrations of antimyein A which caused all-or-none survival 

among black bullhead, yellow bullhead, and channel catfish at selected 

water temperatures in 24 hours and 96 hours

Species

Black bullhead...

Yellow bullhead..

Channel catfish. .

Number 

of 

fish

848

120 

120

192 

84

120 

120 

180

Teraperature (°C)

12 

17 

22

12 

17

12 

17 

22

Concentrations (p.p.b.) and survival

At 24 hours

All

10.0 

6.0 

4.0

20.0 

<10.0

2.0 

1.0 

0.8

None

120.0 

60.0 

40.0

80.0 

60.0

20.0 

10.0 

6.0

At 96 hours

ATJL

10.0 

4.0 

2.0

20.0

2.0 

1.0 

0.8

None

80.0 

40.0 

40.0

80.0

20.0 

10.0 

6.0

10
Other animals

Four hundred water fleas were used in 

trials with antimycin A. At 12° C., specimens 

survived 1 and 0.5 p.p.b., but died in 100 p.p.b. 

in 24 hours and in 10 p.p.b. in 48 hours. Their 

susceptibility increased with temperature. At 

22°, they survived 0.1 p.p.b., but died in 10 

p.p.b. in 24 hours and in 0.5 p.p.b. in 48 hours.

There were no mortalities among 20 crayfish exposed to 10 p.p.b. of toxicant at 12° for 

96 hours.

Tests with 120 damselfly nymphs disclosed 

that the insects were relatively tolerant to 

antimycin A. At 12°, specimens survived 100 

and 50 p.p.b. for 24 and 48 hours respectively, 

and 1,000 and 500 p.p.b. were required to kill 

them in the same time periods. At 22°, they 

survived 50 and 10 p.p.b., but died at 500 and 

100 p.p.b. in 24 and 48 hours. The observations 

were not continued to 96 hours because high 

mortalities began to occur among controls.

Ninety-six tiger salamanders were exposed 

to antimycin A at 12°. Specimens survived 80 

p.p.b. for 96 hours, but were killed by 600 

p.p.b.

Among the 40 bullfrog tadpoles tested for 

24 hours at 12°, the individuals exposed to 20 

p.p.b. of toxicant survived whereas those 

subjected to 40 p.p.b. perished.

RESULTS OF FIELD STUDIES

Tests in wading pools

Results in 1962. Some preliminary bioassays were conducted in 18 pools in July and 

October, to determine the utility of the pools 

as bioassay vessels and to yield information 

on the performance of antimycin A outdoors. 

A shortage of toxicant limited the scope of the 

trials, and a scarcity of fish of desirable 

species, sizes, and condition affected their 

validity. A number of the species were wild 

fish which later proved to be unsatisfactory 

test animals because of variable sizes, heavy 

parasitism, and poor condition.

The wading pools worked well as bioassay 

vessels. Fish, invertebrates, and plants did 

well in the test units and controls. "Tiere were 

some differences in the quantity of plankton 

and aquatic vegetation in the sand- and loambottom units because the latter were more fertile. The abundance of plants, we believe, contributed to increases in pH and alterations of 

alkalinity, and these in turn influenced the 

efficacy of antimycin A.

Goldfish, golden shiner, black bullhead, bluegill, largemouth bass, and yellow perch were 

exposed to 5 and 10 p.p.b. of toxicart in July. 

Most of them survived in the sand pools. The 

mortality was greater in the loam pools, especially at 10 p.p.b., but in no instance did it 

reach 100 percent. The black bullheads exhibited high tolerance to the toxicant in all 

pools.

Another series of tests was made in October 

with higher concentrations against rainbow 

trout, goldfish, golden shiner, bluntiose minnow, yellow bullhead, green sunfish, and yellow 

perch (table 9). The pH values in th^ pools at 

the time ranged from 7.5 to 9.9. Ninety to 100 

percent of the trout, golden shiner, bluntnose 

minnow, green sunfish, and perch, and 60 percent of the goldfish were killed by 20 p.p.b. 

over sand and loam bottoms. At 40 p.p.b., there 

was very low survival among the tr->ut, goldfish, and sunfish, but nearly complete survival 

of bullheads.

There appeared to be rapid degradation and 

detoxification of antimycin in the pc ols within 

24 to 96 hours, depending on the initial concentration and the pH. Small numbers of goldfish, golden shiner, bluntnose minnow, bluegill, and largemouth bass were stocVed later 

in pools in which antimycin A had b^en present 

for 24 to 72 hours. No more than h^lf of the 

golden shiners and bluegills perished within 

the following 2 days.

Results In 1963. The plants, plankton, and 

bottom fauna were permitted to develop in the 

pools for 2 months before toxicity trials. In 

July, acetone solutions of antimycir A were 

tested at 10, 20, 40, and 80 p.p.b. against eight 

species of fish of various sizes (talles 10, 11, 

12, and 13). Golden shiners, bluegills, largemouth bass, and yellow perch were the more

11
TABLE 9. Toxicity of antimycin A at 20 and 40 p.p.b. on adult and fingerling fish in sand- and loam-bottom pools 

[Mortalities are cumulative by observation periods]

Species 

of

fish

Adults:

Finger lings:

Do. ............................

Type 

of 

bottom

sand 

loam

sand 

loam

sand 

loam

sand 

loam

sand 

loam

sand 

loam

sand 

loam

Antimycin A at 20 p.p.b.

Number 

of fish

20 

20

40 

40

40 

40

40 

40

40 

40

40 

40

40 

40

Number dead in (hours) --

24

16 

8

0

0

0 

0

1 

1

35 

37

33 

31

40 

40

48

16 

12

0 

0

0

0

11

7

39 

40

39 

39

96

17 

15

0 

0

7

5

16 

40

40 

40

336

19 

18

1 

3

37 

39

25

Antimycin A at 40 p.p.t.

Number

of fish

20 

20

40 

40

40 

40

40 

40

40 

40

40 

40

40 

40

Number dead in (hours) 

24

14

0

0

1 

2

3 

14

40 

40

40 

40

40 

40

48

17 

16

0 

0

1 

2

21 

40

96

18

17

0 

0

12

11

37

336

20

19

1 

0

40 

39

37

TABLE 10. Numbers and sizes of fish exposed to antimycin A in wading pools 

in July 1963

Species

Black bullhead

Total 

number

T fln

1/1 4

UJ\

Average 

weight (grams)

sensitive, and they were killed by 1C p.p.b. 

within 48 hours over sand and loam bottoms. 

Carp and green sunfish perished at 10 p.p.b. 

within 24 hours over loam bottoms and at 20 

p.p.b. in the same time over sand scU. Some 

lots of goldfish died at 20 p.p.b., but all succumbed at 40 p.p.b. A concentration of 80 

p.p.b. killed all fingerling and adult black 

bullheads within 48 hours over sand bottom 

but only one-half of them over loam.

The green sunfish and yellow perch appeared at the surface of pools withir 2 hours

TABLE 11. Toxicity of antimyoin A at 10 and 20 p.p.b. on adult and fingerling fish in sand- and loam-bottom wading pools 

[Mortalities are cumulative by observation period]

Species

Adults:

Do. ...........................

Finger lings:

Type 

of 

bottom

sand 

loam

sand 

loam

sand 

loam

sand 

loam

sand 

loam

sand 

loam

sand 

loam

sand 

loam

sand 

loam

sand 

loam

Antimycin A at 10 p.p.b.

Number 

of fish

20 

20

20 

20

20 

20

20 

20

14 

14

20

20

16 

16

40 

40

16 

16

20 

20

Number dead in (hours) 

24

0 

0

17 

20

6 

7

14 

20

14 

14

0 

0

15 

16

40 

40

16 

16

20 

18

48

0

0

20

6 

7

14

0 

0

15 

20

96

0 

0

6 

7

14

0 

0

15

480

2

1

6 

7

14

0 

0

15

Antimycin A at 20 p.p.b.

Number 

of fish

20 

20

20 

20

20 

20

20 

20

W 

14

20 

20

16 

16

40 

40

16 

16

20 

20

Number dead in (hours) ~

24

0 

0

20 

20

20 

10

20 

14

14 

14

0 

0

16 

16

40 

40

16 

16

20 

20

48

0 

0

10 

14

0 

0

96

0 

0

10 

14

0 

0

480

0

3

10 

14

0 

0

12
TABLE 12. Toxici-ty of antimycin A at 40 and 80 p.p.b. on adult and fingerling fish In sand- and loam-bottom wading pools 

[Mortalities are cumulative by observation period]

Species

Adults:

Finger lings:

Type 

of 

bottom

sand 

loam

sand 

loam

sand 

loam

sand 

loam

sand 

loam

sand 

loam

sand 

loam

sand 

loam

sand 

loam

sand 

loam

Antimycin A at 40 p.p.b.

Number 

of fish

20 

20

20 

20

20 

20

20 

20

14 

14

20 

20

16 

16

40 

40

16 

16

20 

20

Number dead in (hours) 

24

0 

0

20 

20

20 

20

20 

20

14 

14

0 

0

16 

16

40 

40

16

16

20 

18

48

0 

0

0 

0

20

96

0 

0

0 

0

480

2

1

0 

0

Antimycin A at 80 p.p.b.

Number 

of fish

20 

20

20 

20

20 

20

20 

20

14 

14

20

20

16

16

40 

40

16 

16

20 

20

Number dead in (hours) 

24

6

1

20 

20

20 

20

20 

20

14 

14

20

11

16 

16

40 

40

16 

16

20 

14

48

20 

2

11 

20

96

2

11

480

3

11

TABLE 13. Average values of analyses made on water from sand- and loambottom wading pools before and after applications of antimycin A in 

July 1963

Item

Dissolved oxygen. .....

Total alkalinity......

Calcium hardness ......

Total phosphorus ...... 

Ammonia nitrogen. ..... 

Hltrite nitrogen. ..... 

Hitrate nitrogen. .....

Unit of

measurement

°C

at 20^C

p.p.m.02

pH

p.p.m.CaCOj

p.p.m.CaC03

p.p.m.PO^ 

p.p.m.NH3 

p.p.m.N02 

p.p.m.H03

Sa

Before

8.7

204.4 

( 10.7)

53.6

*yK ft

0.059 

0.399 

0.006 

0.117

nd

After

9.1

181.2 

( 11.5

47.9

0.071 

0.730 

0.013 

0.191

Lo

Before

9.7

8

ft

198.2 

( L4.6)

60.0

1 d o

0.082 

0.710 

0.005 

0.154

am

After

9.7

183.7 

( W.3)

53.6

0.106 

1.100 

0.018 

0.220

after exposure to the toxicant, and they exhibited a narcosislike condition. They showed 

little response to motion stimulus or handling 

with a dip net. Some of the larger bullheads 

behaved as if in distress and were subject to 

development of an unidentified funguslike 

condition on the body prior to death.

The trials in October included two formulations of antimycin. One was a solution in acetone, and the other an emulsifiable concentrate, applied to pools at 1, 5, 10, and 100 

p.p.b. against 10 species of fish. T^e pH 

values at the time in all pools were about 10, 

and the antimycin A degraded so r?oidly that 

most fish escaped toxic effects (tal^e 14). The

TABLE 14. Average values of analyses made on water from sand- and loambottom wading pools before and after applications of antimycin A in 

October 1963

Dissolved oxygen. .....

Total alkalinity......

Calcium hardness ......

Total phosphorus ...... 

Ammonia nitrogen. ..... 

Nitrite nitrogen. ..... 

Nitrate nitrogen. .....

Unit of

measurement

°C

p.p.m.02

pH

p.p.m.CaC03

p.p.m.CaC03

p.p.m.P04 

p.p.m.NH3 

p.p.m.N02 

p.p.m.N03

San

Before

10.0

10.0

114.0

(85.0)

27.4

17.8

0.090 

0.25 

0.0 

0.0

d

After

16

3431

9.6

0.0

10.0

107.0 

(27.5)

148.0

33.0 

0.026

15.3

0.084 

0.270 

0.0 

0.0 

15.25

Lo

Before

16

3439

10.0 

0.0

10.0

127.0 

(36.0)

(91.0)

155.0

38.0 

0.036

18.0

0.043 

0.000 

0.0 

0.0 

11.0

am

After

15

3396

9.5 

0.0

9.8

121.0 

(34.0)

(88.0)

154.0

35.0 

0.028

14.0

0.035 

0.550 

0.0 

0.0 

13.6

13
exceptions were those individuals exposed to 

100 p.p.b. It appeared that the acetone solution of toxicant deteriorated sooner than the 

other preparation.

Of the 10 species of fish, 7 species succumbed totally to 100 p.p.b. of acetoneantimycin A, and 9 species to the emulsifiable 

formulation, within 24 hours over sand bottoms; only carp, fathead minnow, bluegill, 

longear sunfish, and yellow perch died over 

loam bottoms. The black bullhead was the 

sole survivor of 100 p.p.b. over both bottom 

types. Neither preparation of toxicant caused 

100-percent kills of any species within 96 

hours at 5 or 10 p.p.b.

In general, most of the susceptible fish 

showed signs of distress within a short time 

after exposure, and many came to the surface 

of the pools. The length of time which elapsed 

before death varied with the species and 

water temperature, and ranged from a few 

hours to several days. It is significant that all 

specimens which displayed symptoms of distress eventually died. This suggests that the 

action of the toxicant on fish is irreversible.

There were no grossly toxic effects by antimycin A on the plankton, bottom fauna, or 

aquatic plants during the course of the July 

and October trials. For example, in the four 

pools which received 20 p.p.b. of aritimycin A 

in July, the average quantity of plankton was 

0.0036 cc./l. (range- 0.0020 to 0.0044) before 

treatment and 0.0040 cc./l. (range: 0.0033 to 

0.0061) at 20 days after treatment. The quantities in two control pools were 0.0047 and 

0.0089 cc./l. during pretreatment sampling 

and 0.0022 and 0.0044 cc./l. during posttreatment sampling.

Tests in hatchery ponds

There appeared to be a more rapid response of fish to the antimycin A which was 

formulated with an emulsifiable concentrate 

than with acetone. With the former preparation in pond No. 2, fish surfaced within 4 to 

6 hours after application, whereas in pond 

No. 5 there were no comparable effects for 

another 10 hours. By the end of the first full

day, we saw no significant differences in the 

effects produced by the two formulations. 

Table 15 gives before and after water analyses 

for the two ponds.

Northern pike were the first fish tc exhibit 

distress. They surfaced and appeared to be in 

a state of narcosis which was followed by complete locomotor ataxia. The rainbow trout, 

white suckers, carp, walleye, and surfishes 

followed in order with similar symptoms. The 

great majority of specimens were dead within 

48 hours (tables 16 and 17). It is noteworthy 

that goldfish a species which was relatively

TABLE 15. Analyses of water from ponds No. 2 and No. 5 at Delafield 

Warrawater Fisheries Research Station before and after applications 

of antimycin A in September 1963

Dissolved oxygen. .....

Total alkalinity. .....

Calcium hardness. .....

Total phosphorus ...... 

Ammonia nitrogen. ..... 

Nitrite nitrogen. ..... 

Nitrate nitrogen. .....

Unit of

measurement

°r

p.p.m.02

pH

p. p.m. CaC0 3

p.p.m.CaC0 3

p.p.m.Fe 0

p.p.m.PO^ 

p.p.m.NHj 

p.p.m.N02 

p.p.m.N03

Pond

Before

210.0 

( 0.0)

77.0

M 3

1.40 

0.20 

0.0 

0.07

No. 2

After

6.9

202.0 

( 0.0)

82.0

0.10 

0.19 

0.0 

0.50

Pond

Before

7.5

201.1 

( 8.8)

80.0

0.50 

0.18 

0.0 

0.07

to. 5

After

8.2

189.5 

( 0.0)

75.0

0.15 

0.38 

0.0 

0.43

TABLE 16. Effects of 10 p.p.b. of antimycin A In emulsifiable concentrate 

on 18 species of fish in pond No. 2

Species

Longnose gar. .....

Rainbow trout. ....

Northern pike. ....

White sucker......

Black bullhead. . . .

Yellow bullhead...

Brown bullhead. . . .

Green sunfish. ....

Black crappie. ....

Largemouth bass. . .

Hybrid sunfish....

Total 

fish 

stocked

3

312

7

4

600

4

1

3

27 

7

4-

1,400

1

Average

length 

(inches)

25.6

4.0

17.8

15.1

3.8

8.3

4.2

3.8

6.1 

8.3

15.4

1.7

13.5

Average 

weight 

(grams)

658

82

713

554

18

168

50

14

68 

95

795

9

318

Number of fish dead at 

(hours) 

24

0

312

5

3

0

0

0

0

21

5

1

1,400

1

49

0

5

3

0

0

0

0

21

5

4

_

96

0

5

3

0

0

0

1

22 

5

--

_

480

0

7

4

0

0

0

3

27 

7

--

_

--

14
TABLE 17. Effects of 10 p.p.b. of antimycin A in acetone solution on 19 

species of fish in pond No. 5

Species

Rainbow trout.....

Northern pike. ....

Goldfish. .........

Black bullhead. . . .

Yellow bullhead. . .

Brown bullhead. . . .

Green sunf ish. ....

Black erappie.....

Largemouth bass...

Hybrid sunfish. . . .

Total 

fish 

stocked

470

8

1,400

27

875

1

6

4

43 

9

5

2,055

1

Average 

length 

(inches)

->T fj

4.1

19.1

15.3

3.7

5.7

11.4

4.2

6.4 

8.8

12.8

1.7

11.6

Average 

weight 

(grams)

86

976

1,112

14

59

377

23

86 

136

477

9

272

Number of fish dead at 

(hours ) 

24

470

6

0

0

- 0

0

19 

1

0

2,055

0

48

6

27

?

0

0

1

28 

4

2

_

0

96

8

3

0

0

2

36 

4

4

_

1

480

157

0

1

4

43 

9

5

_

--

TABLE 18. Abundance of bottom fauna In ponds No. 2 and No. 5 before and 

after treatment with 10 p.p.b. of antlmyolr A

[Each collection consisted of 16 one-square foot samples

tolerant to antimycin A in the laboratory died 

in both ponds within 24 hours.

The longnose gar, bowfin, black bullhead, 

yellow bullhead, and brown bullhead were the 

only species which were not affected greatly 

by the toxicant at 10 p.p.b. Seventy percent 

of them were recaptured alive when the ponds 

were drained after 20 days.

The detoxification of antimycin A was monitored throughout the first 96 hours. It occurred 

within 72 hours after application, and fish 

placed in live cages after this time survived 

until the ponds were drained.

Plankton was sampled in both ponds during 

the experimental period. In pond No. 2, the 

pretreatment quantity was 0.018 cc./l and the 

posttreatment quantity was 0.047 cc./l. Pond 

No. 5 had pretreatment and posttreatment 

quantities of 0.0035 and 0.039 cc./l. None of 

the relatively minor changes was attributed 

to the toxicant. Also, there were no observable 

changes in the aquatic plants in the ponds.

Pretreatment and posttreatment samples of 

bottom fauna were taken. We concluded that 

antimycin A was nontoxic to the 15 taxonomic 

groups which were represented in both ponds 

because there were no significant changes in 

species composition or numerical abundance 

(table 18). The midges were the more numerous in all samples, and they increased by 55 

to 65 percent during the experimental period. 

The nymphs of mayflies, dragonflies, and

Organism

Horsehair worm

Aquatic earthworm 

Leech 

Scuds

Mayflies 

Damselflies 

Dragonflies 

ffaterbugs

Caddisflies 

Water beetles 

Mosquitoes 

Midges

Biting midges 

Soldierflies 

Snails

(Nematomorpha) .... 

(Oligocheata) .....

(Ephemeroptera. . . .

(Trichoptera) .....

(Tendipedidae ) . . . .

(Ceratopogonidae) . . 

(Stratiomyiidae)...

Average number per square foot

Pond No. 2

Sept. 23

10.7

0.0 

0.0 

4.7

9.0 

1.7 

0.0 

0.0

0.7 

2.7 

0.0 

209.7

1.3 

0.3 

4.0

244.8

Oct. 15

0.7

0.0 

1.3 

2.0

145.3 

2.0 

0.7 

2.7

2.0 

18.0 

0.0 

388.0

0.0 

0.0 

30.7

593.4

Pond No. 5

Sert. 17

1.0

34.5 

5.2

17.5

6.8 

6.8 

0.5 

1.0

0.2 

5.0 

0.8 

269.5

2.8

6.0

72.8

-^24.4

Oct. 1A

1.0

3.0 

1.0 

39.0

6.5 

7.8 

0.5 

1.5

0.0 

3.5 

0.0 

422.0

1.0 

0.0 

30.5

517.3

damselflies were also more abundant in the 

posttreatment samples.

Care was taken to note any gross effects of 

the toxicant on frogs, salamanders, and turtles, but there were none.

Discussion of field studies

There was a lack of consistency in the performance of antimycin A in sand- and loambottom pools in July and October, 1962 and 

1963, and in the hatchery ponds. The cause, 

we believe, was the chemistry of the waters 

and particularly the presence of the hydroxyl 

ion.

An alkaline shift occurred in the vading 

pools as the plant biomass increased. The 

relatively hard, well water which wa s used 

to fill the pools was gradually softened because of the decrease in calcium. Tl ^re was 

a shift from bicarbonate (methyl orange alkalinity) to free hydroxide (phenolphthalein 

alkalinity). The measure of the acid-base 

shift was pH which rose from 7.5 upward to 

10 or more. Diurnal fluctuations of several 

pH units are not uncommon in ponds, and the 

pH in wading pools ranged accordingly between 

morning and afternoon.

The highest pH values were observed in 

late afternoon in the presence of abundant 

plants and sunshine. In this situatior, the

15
hydroxyl ions appear, and often they are not 

checked by buffering salts. Magnesium prevails as calcium ions are removed from 

solution, and the result is the sort of alkaline shift observed in softer waters.

We assume that the relative success of the 

toxicity trials in hatchery ponds was due in 

large part to the fact that the water had high 

buffer capacity and little reserve alkalinity 

in the form of hydroxide. Thus, the antimycin 

A was not subject to immediate detoxification 

by action of free hydroxide, and the 10 p.p.b. 

were effective in killing fish.

In contrast, the poorer results obtained in 

the wading pools reflected the greater concentrations of free hydroxide present. In 

July 1963, the pools had approximately the 

same pH and total alkalinity as the hatchery 

ponds, but there was more free hydroxide 

present. Therefore, the degradation of the 

toxicant was more rapid, and 20 to 40 p.p.b. 

were needed to kill fish.

The contrast was heightened by results in 

-October 1963. The water was much softer and 

lower in buffer capacity, and there was even 

more free hydroxide present. The pH ranged 

up to 10. Under these conditions, there was 

almost immediate detoxification of the antimycin, and only partial fish kills were obtained at 100 p.p.b.

CONCLUSIONS

Antimycin A is a powerful toxicant to 

fresh-water fish. We observed the responses 

of many specimens to concentrations which 

ranged from 0.01 to 120 p.p.b. Among them, 

the carp a most undesirable fish in many 

waters proved vulnerable to small concentrations and short exposures at cool and warm 

temperatures. Other fishes which at times 

may be undesirable, such as goldfish, white 

suckers, green sunfish, and pumpkinseeds, 

were also killed.

The sensitivities to antimycin A varied 

among species, and they were correlated with 

temperature and duration of exposure. The 

tests in the laboratory at 12°, 17°, and 22° C.

indicated that smaller quantities of toricant or 

shorter exposures produced kills of fish in 

warmer waters, but the results at 12° were 

nonetheless satisfactory.

There were three general degrees cf sensitivity detected among the 24 species o* fish in 

the" laboratory and a similar order arrong the 

25 species used in outdoor trials. Indicative 

of the extremes in response, gizzard shad 

perished at 0.04 p.p.b. of toxicant whereas 

black bullheads survived 100 p.p.b. Trsre also 

appeared to be a tendency for sensitivities to 

follow family lines, but species in the nine 

families tested exhibited great variations in 

susceptibility. For example, fingerling carp 

in the laboratory died within 24 hours upon 

exposure to 0.6 p.p.b. at 12°, but up to 100 

p.p.b. were required for complete kills of 

goldfish.

Observations in the laboratory and field 

demonstrated that antimycin A was less toxic 

to other animals. Water fleas were killed by 

100 p.p.b. in 24 hours at 12°, but their susceptibility increased at warmer temperatures 

or longer exposures. Crayfish were not harmed 

by 10 p.p.b. over 96 hours, and damselfly 

nymphs survived 50 p.p.b. for 48 hours. Tiger 

salamanders survived 80 p.p.b. for 96 hours 

at 12°, and bullfrog tadpoles were unharmed 

by 20 p.p.b. for 24 hours.

The plankton in wading pools and hatchery 

ponds was not significantly affected during 

experiments, and there was no gross evidence 

of toxicity to filamentous algae, and submersed 

and emergent plants. No deleterious effects 

were detected on the composition, numbers, 

and growth of bottom fauna in hatcher;" ponds.

Antimycin A degraded rapidly in water, and 

detoxification was complete within 24 to 96 

hours under field conditions. The rate of 

molecular breakdown was accelerated sharply 

in the presence of free hydroxide, and this 

suggests a possibility for artificial detoxification. Bioassays with fish following the degradation of the toxicant revealed an absence 

of harmful residues in water.

Further investigation on antimycin A as a 

fish toxicant is warranted in the laboratory

16
and field. Studies are contemplated or in 

progress at the Fish Control Laboratories 

on its performance against various life 

stages of fish from egg to adult; against 

additional species; on minimum killing concentrations and exposures; in waters of various chemistries; and at cold and warm temperatures. Appropriate formulations for 

standing and flowing waters are desirable. 

Further attention must also be given to the 

effects of the toxicant on other aquatic organisms. The factors in water which contribute to degradation of the toxicant deserve 

study, and the nature and fate of residues 

require definition. Also and depending on 

adequate supplies of toxicant many, and more 

comprehensive, trials in the field are needed 

for full and fair evaluation of this material 

which has potential value in fishery management and research.

LITERATURE CITED

American Public Health Association. American Water 

Works Association, and Water Pollution Control 

Federation.

I960. Standard methods for the examination of 

water and waste-water, llth. ed. American 

Public Health Association, New York. 626 p.

Derse, P. H., and F. M. Strong.

1963. Toxicity of antimycin to fish. Nature, vol. 

200. No. 4906, p. 600-601.

Dickie, J. P., M. E. Loomans, T. M. Parley, and 

F. M. Strong.

1963. The chemistry of antimycin A. XI. Nsubstituted 3-formamidosalicylic amides. 

Journal of Medicinal Chemistry, vol. 6. p. 424- 

427.

Dunshee, B. R., C. Leben. G. W. Keitt, and F. M. 

Strong.

1949. Isolation and properties of antimycin A. 

Journal of the American Chemical Society, vol. 

71, p. 2436-2437.

Gottlieb, D., and S. Ramachandran.

1961. Mode of action of antibiotics. 1. Site of 

action of ascosin. p. 391-396.

Harada. Y., K. Nakayama, and F. Okamoto.

1959. Antimycin A, an antibiotic substance useful 

in prevention and treatment of imochibyo, a 

disease of rice. Japan, 2200, (Chemical Abstracts, vol. 53 (1959), p. 19286i).

Keitt, George W., Curt Leben, and Frank M. Strong. 

1953. Antimycin and process for production. U.S. 

Patent Office. Patent No. 2,657,170, 10 p.

Lawrence, J. M., and R. D. Blackburn.

1962. Evaluating herbicidal activity of chemicals 

to aquatic plants and their toxicity tc fish in the 

laboratory and in plastic pools. Auhnrn University, mimeo: p. 1-23, (In press, Proceedings 

of 16th Annual Conference of Southeastern 

Association of Game and Fish Commissioners, 

Columbia, S.C.).

Lennon, Robert E., and Charles R. Walker.

1964. Investigations in Fish Control; 1 Laboratories 

and methods for screening fish-confol chemicals. Bureau of Sport Fisheries ancJ Wildlife, 

Circular 185.

Liu, Wen-chih, and F. M. Strong.

1959. The chemistry of antimycin A. VI. Separation and properties of antimycin A subcomponents. Journal of the American Chemical Society, vol. 81, p. 4387-4390.

Liu, Wen-chih, E. E. van Tamelen, and F. M. Strong.

1960. The chemistry of antimycin A . VIII. Degradation of antimycin A. Journal of the American Chemical Society, vol. 82, p. 16f 2-1654.

Lockwood, J. L., C. Leben, and G. W. Keitt,

1954. Production and properties of antimycin A 

from a new Streptomyces isolate. Phtopathology, vol. 44, p. 438-446.

Nakayama, K., F. Okamoto, and Y. Harada.

1956. Antimycin A: Isolation from Streptomyces 

kitazawaensis and activity against rice plant 

blast fungi. Journal of Antibiotics (Japan) Ser. 

A 9,p. 63-66(Chemical Abstracts, voV, 53 (1959), 

p. 19030h).

Strong, F. M.

1956. Topics in microbial chemistry. John Wiley 

and Sons, Inc., New York, 166 p.

Strong, F. M., J. P. Dickie, M. E. Loomanr, E. E. van 

Tamelen, and R. S. Dewey.

1960. The chemistry of antimycin A. IX. Structure of the antimycins. Journal of tl ^ American 

Chemical Society, vol. 82, p. 1513.

Tener, G. M., F. Merlin Bumpus, Bryant R. Dunshee, 

and F. M. Strong.

1953. The chemistry of antimycin A. II. Degradation studies. Journal of the American Chemical Society, vol. 75, p. 1100-1104.

Tener, G. M., E. E. van Tamelen, and F. M. Strong. 

1953. The chemistry of antimycin A. in. The 

structure of antimycic acid. Journal of the 

American Chemical Society, vol. 75, p. 3623- 

3625.

17
van Tamelen, E. E., F. M. Strong, and U. Carol Quarck. ture of the antimycins. Journal of the. A merican 

1959. The chemistry of antimycin A. IV. Studies Chemical Society, vol. 83, p. 1639-1646. 

on the structure of antimycin lactone. Journal

of the American Chemical Society, vol. 81, p. Watanabe, K., T. Tanaka, K. Fukuhara, N. Miyairi, 

750-751. H. Yonehara, and H. Umezawa.

1957, Blastmycin, a new antibiotic from

van Tamelen, E. E., J. P. Dickie, M. E. Loomans, R. Streptomyces. Journal Antibiotics (Jaoan), vol. 

S. Dewey, and F. M. Strong. A10, p. 39-45. (Chemical Abstracts, vol. 53 

1961. The chemistry of antimycin A. X. Struc- (1959), p. 2222Ig).

18
FISH CONTROL

PURPOSE

INDUCE SPAWNING

PREVENT SPAWNINGIMPROVE GROWTH, 

VIGOR, FECUNDITY, 

DISEASE RESISTANCE

TRANSPORT

PREVENT 

OSMOTIC SHOCK

PREVENT ENTRY

RESTRICT MOVEMENT

SELECTIVE REDUCTION

FACILITATE CAPTURE

ERADICATION

METHODS

RADIANT ENERGY - LIGHT, HEAT, ELECTRICITY, SOUND

Electric shock 

Controlled light 

Sound stimulant

Electric shock 

Radiation 

Controlled light Sound depressant

Ligh* control

Electro-narcosis

Electrical barrier

Electrical array

Pulsed current

Sonic attractant 

Electrical guiding array

Lethal current

PHYSICAL AND 

MECHANICAL

Hydraulic control

Aeration 

Hydraulic control 

Temperature control

Acclimatization

Barrier 

Hydraulic manipulation

Hydraulic manipulation

Water level manipulation 

Gear development

Water level manipulation 

Gear development

Gear development

CHEMICAL AND 

BIOCHEMICAL

Hormones

Sterilants 

Hormones

Anesthetics 

Sedatives Decontaminants 

Diet manipulation

Osmoregulatory compounds

Repellents

Attractants 

Repellents

Selective toxicants

Attractants

Lethal compounds

GENETICS

Select for season and 

duration of spawning

Select for low fertility

Brood stock selection 

Select for wildness 

Select for rapid growth 

Select for disease resistance

Select for adaptability

Select for 

non-migratory strain

Select for catchability

BIOLOGICAL AGENTS

Plant introductions

Plant introduction or 

removal

Selective infectious disease 

Selective parasites 

Predator introduction 

Competitor introduction
POSTAGE AND FEES PAID 

U.S. DEPARTMENT OF THE INTERIOR

UNITED STATES 

DEPARTMENT OF THE INTERIOR

FISH AND WILDLIFE SERVICE

BUREAU OF SPORT FISHERIES AND WILDLIFE

WASHINGTON, D.C. 20240