INVESTIGATIONS IN FISH CONTROL

32. Toxicity of Hyamine 35OO to Fish

33. Voidance Time for 23 Species of Fish

34. Laboratory Studies on

Possible Fish-Collecting Aids

With Some Toxicities for the 

Isomers of Cresol

United States Department of the Interior

Fish and Wildlife Service 

Bureau of Sport Fisheries and Wildlife
INVESTIGATIONS IN FISH CONTROL

Investigations in Fish Control, published by the Bureau of Sport Fisheries and Wildlife, include reports on the results of work at the Bureau's Fish Control Laboratories at La Crosse, 

Wis., and Warm Springs, Ga., and reports of other studies related to that work. Though each 

report is regarded as a separate publication, several may be issued under a single cover, for 

economy.

Current reports in this series are 

(Reports 1 and 2 are in one cover.)

1. Laboratories and Methods for Screening Fish-Control Chemicals, by Robert E. Lennon 

and Charles R. Walker. 1964. 15 p.

2. 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. 1964. 18 p.

(Reports 3 through 8 are in one cover.)

3. Minimum Lethal Levels of Toxaphene as a Piscicide in North Dakota Lakes, by Dale L. 

Henegar. 1966. 16 p.

4. Effects of Toxaphene on Plankton and Aquatic Invertebrates in North Dakota Lakes, by 

Robert G. Needham. 1966. 16 p.

5. Growth Rates of Yellow Perch in Two North Dakota Lakes After Population Reduction with 

Toxaphene, by Donald C. Warnick. 1966. 9 p.

6. Mortality of Some Species of Fish to Toxaphene at Three Temperatures, by Mahmoud 

AhmedMahdi. 1966. 10 p.

7. Treatment of East Bay, Alger County, Michigan, with Toxaphene for Control of Seal Lampreys, by William E. Gaylord and Bernard R. Smith. 1966. 7 p.

8. Effects of Toxaphene on Fishes and Bottom Fauna of Big Kitoi Creek, Afognak Island, 

Alaska, by William R. Meehan and William L. Sheridan. 1966. 9 p.

(Reports 9 through 11 are in one cover.)

9. Relation of Chemical Structure to Fish Toxicity in Nitrosalicylanilides and Related Compounds, by Charles R. Walker, Roland J. Starkey, and Leif L. Marking. 1966. 12 p.

10. Evaluation of p,p'-DDT as a Reference Toxicant in Bioassays, by Leif L. Marking.

1966. 10 p.

11. Evaluation of an Electronic Method of Measuring Hematocrits of Fish, by Richard A. 

Schoettger and Arnold M. Julin. 1966. lip.

(Reports 12 through 17 are in one cover.)

12. Toxicity of MS-222 to Selected Fishes, by Leif L. Marking. 1967. 10 p.

13. Efficacy of MS-222 as an Anesthetic on Four Salmonids, by Richard A. Schoettger and 

Arnold M. Julin. 1967. 15 p.

14. Method for Determining MS-222 Residues in Fish, by Charles R. Walker and Richard A. 

Schoettger. 1967. 10 p.

15. Residues of MS-222 in Four Salmonids Following Anesthesia, by Charles R. Walker and 

Richard A. Schoettger. 1967. 11 p.

16. Annotated Bibliography on MS-222, by Richard A. Schoettger. 1967. 15 p.

17. MS-222 as an Anesthetic for Channel Catfish: Its Toxicity, Efficacy, and Muscle Residues, 

by Richard A. Schoettger, Charles R. Walker, Leif L. Marking, and Arnold M. Julin.

1967. 14 p.

Fish Control Laboratories

Bureau of Sport Fisheries and Wildlife

U.S. Department of the Interior

P.O. Box 862

La Crosse, Wisconsin 54602

Continued on inside back cover 
INVESTIGATIONS IN FISH CONTROL

32. Toxicity of Hyamine 3500 to Fish

By James W. Hogan

330 Voidance Time for 23 Species of Fish

By Thomas H. Lane and Howard M. Jackson

34. Laboratory Studies on Possible Fish-Collecting Aids 

With Some Toxicities for the Isomers of Cresol 

By Robert M. Howland

United States Department of the Interior, Walter J. Hickel, Secretary

Leslie L. Glasgow, Assistant Secretary

for Fish and Wildlife, Parks, and Marine Resources

Fish and Wildlife Service, Charles H. Meacham, Commissioner

Bureau of Sport Fisheries and Wildlife, John S. Gottschalk, Director

Washington, D.C. October 1969

For sale by the Superintendent of Documents, U.S. Government Printing Office 

Washington, D.C. 20402 - Price 35 cents
IN THIS COVER

Pages 

Toxicity of Hyamine 3500 to Fish, by James W. Hogan. ........ 1-9

Voidance Time for 23 Species of Fish, by Thomas H. Lane and

Howard M. Jackson .............................. 1-9

Laboratory Studies on Possible Fish-Collecting Aids, With Some

Toxicities for the Isomers of Cresol, by Robert M. Howland ... 1-10

ii
INVESTIGATIONS IN FISH CONTROL

32. Toxicity of Hyamine 35OO to Fish

By James W. Hogan

United States Department of the Interior, Walter J. Hickel, Secretary

Leslie L. Glasgow, Assistant Secretary

for Fish and Wildlife, Parks, and Marine Resources

Fish and Wildlife Service, Charles H. Meacham, Commissioner

Bureau of Sport Fisheries and Wildlife, John S. Gottschalk, Director

Washington, D.C. October 1969
CONTENTS

Page 

Abstract................................... 3

Materials and methods ......................... 3

Results. ................................... 4

Trout. ................................. 4

Warm water fish........................... 5

Effects of temperature ...................... 5

Effects of water quality...................... 5

Discussion ................................. 5

Conclusions. ................................ 7

References ................................. 9
TOXICITY OF HYAMINE 3500 TO FISH

By James W. Hogan, Chemist

Bureau of Sport Fisheries and Wildlife

Southeastern Fish Control Laboratory

Warm Springs, Georgia

ABSTRACT. Toxicity of Hyamine 3500 to three species of trout and 

11 species of warmwater fish was determined in static bioassays. Twentynine lots of fish from nine sources were used in water at various levels of 

pH, temperature, and total hardness. Hyamine 3500 is more toxic in alkaline or acidic water than in water with a pH of 7. In general, toxicity varies 

directly with temperature and inversely with total hardness. Dilute solutions 

of Hyamine 3500 appear to degrade rapidly in open vessels.

Because of its efficacy as a microbicide 

and its relatively low toxicity to mammals 

(Rohm and Haas Co., 1965), Hyamine 3500 

is being considered as a disinfectant in fish 

culture (Warren, 1964). Before it can be 

considered for general use, its toxicity to 

fish must be known. The purpose of this 

study was to determine the toxicity of Hyamine 

3500 to several species of fish in waters of 

various qualities.

MATERIALS AND METHODS

The active ingredients in Hyamine 3500 

are a selected blend of alkyl (C 14 , 50 percent; 

C12 , 40 percent; and C16 ,10 percent) dimethyl 

benzyl ammonium chlorides having an average molecular weight of 358 g /mole. Two 

forms of Hyamine 3500 are available: an 

80-percent concentrate in ethanol, and a 50- 

percent aqueous solution. We used the latter. 

The other ingredients in the aqueous solution include 40 percent of water and 10 percent of ethanol. This solution is pale yellow, 

has a mild odor, congeals at low temperatures, and is miscible in all proportions 

with water, lower alcohols, and ketones.

The species, lot numbers, weights, and 

sources of fish are listed in table 1. Most

of the lots were from National Fish Hatcheries, but two lots of goldfish were from a private producer, and one lot of channel catfish 

was from a Georgia State Fish Hatchery, and 

one lot of trout was from a Wisconsin State Fish 

Hatchery. The term "lot" refers to a particular group of fish of a species received from a 

hatchery in a single shipment.

Routine bioassays were conducted in accordance with the methods outlined by Lennon 

and Walker (1964), with slight modifications. 

In general, the formulation of water of various 

levels of total hardness, alkalinity, and pH 

was accomplished by the methods outlined by 

Marking (1966, 1967). Water temperatures in 

vessels were maintained at 12°± 1°, 17°± 2°, or 

220+ 1° c.

Immediately before each bioassay, concentrated stock solutions of Hyamine 3500 

were prepared with a deionized water-acetone 

(50/50, v/v) solvent. Concentrations were 

calculated on the basis of product rather than 

active ingredient. Aliquots of these stock 

solutions were measured directly into bioassay 

vessels to produce desired concentrations. The 

amount of acetone per bioassay vessel never 

was in excess of the volume tolerated by the 

test fish.

The data from each bioassay were analyzed 

by the method outlined by Litchfield and
Investigations in Fish Control 32: Bureau of Sport Fisheries and Wildlife

Wilcoxon (1949). By this method, LC50 values 

(concentrations producing 50-percent mortality) and their 95-percent confidence intervals were calculated.

RESULTS

Trout. The 24-hour LC50 values at 12° C. 

indicate that rainbow trout were less sensitive

to Hyamine 3500 than lake trout and brown 

trout (table 2). The 96-hour LC 50 values for 

the three species ranged from 1.90 to 2.45 

p p m ; rainbow trout were still the least 

sensitive. The 95-percent confidence intervals of lake trout and brown trout overlap, 

indicating no statistically significant differences in the sensitivity of these two 

species.

TABLE 1. The 14 species of fish used in toxicity tests of famine 3500

Species

Brown trout, Salmo trutta. .........................

Lake trout , Salvelinus namaycush. ..................

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

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

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

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

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

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

Smallmouth buffalo , Ictiobus bubalus ...............

Brown bullhead, Ictalurus nebulosus ................

Channel catfish, Ictalurus punctatus ...............

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

Bluegill, Lepomis macrochirus ......................

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

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

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

Smallmouth bass, Micropterus dolomieui. ............

Largemouth bass, Micropterus salmoides. ............

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

Lot

368

309

346

W107

W116

W133

Iffl 53

W162

WHO

W113

W194

W139

W163

W148

W115B

W157

W164

W109

Will

W119

W128

W112

W117 

W146

W140

W141

Average weight 

and range (grams)

0.8(0.5 - 1.1)

1.2(0.7 - 1.9)

1.9(1.5 - 2.2)

1.3(1.1 - 2.2)

1.3(0.8 - 2.1)

1.4(1.0 - 1.8)

1.1(0.7 - 1.5)

1.0(0.9 - 2.1)

0.7(0.6 - 1.0)

1.0(0.5 - 2.0)

0.8(0.6 - 1.4)

0.8(0.6 - 1.4)

1.1(0.5 - 1.4)

1.1(0.5 - 1.2)

2.2(1.6 - 3.3)

0.9(0.5 - 2.1)

0.9(0.6 - 1.1)

0.8(0.7 - 1.0)

1.1(0.7 - 1.4)

1.1(0.7 - 1.4)

1.2(0.8 - 1.6)

1.4(1.1 - 2.4)

1.1(0.8 - 1.5) 

0.7(0.5 - 0.9)

0.8(0.6 - 1.2)

0.6(0.4 - 0.8)

Source

Manchester NFH, la.

McNenny NFH, S. Dak.

St. Croix Falls SFH, Wis.

Welaka NFH, Fla.

Tallassee, Ala.

Do.

Marion NFH, Ala.

Do.

Do.

Do.

Do.

Do.

Do.

Do.

Cordele SFH, Ga.

Marion NFH, Ala.

Do.

Do.

Do.

Do.

Do.

Do.

Do. 

Mammoth Springs NFH, Ark.

Welaka NFH, Fla.

Warm Springs NFH, Ga.

TABLE 2.--Toxici-cy of Hyamine 3500 to three species of trout at 12° C. at 24, 48, and 96 hours of exposure

Species Lot

368 

309 

346

LCjo and y5-percent confidence interval (ppm) at 

24 hours

3.65 (3.38 - 3.94) 

2.61 (2.14 - 3.18) 

2.74 (2.51 - 2.99)

48 hours

2.80 (2.59 - 3.02) 

2.13 (1.79 - 2.53) 

2.35 (2.12 - 2.61)

96 hours

2.45 (2.19 - 2 

1.95 (1.65 - 2 

1.90 (1.71 - 2

.74) 

.30) 

.11)
James W. Hogan: Toxicity of Hyamine 3500 to Fish

Warmwater fish. The 96-hour LC^ *s of 

Hyamine 3500 for 10 species of warmwater 

fish range from 0.32 to 2.25 p p m at 17° C. 

(table 3). The response of the fish varied 

somewhat for different exposure periods. For 

instance, 3-hour LC M values show that smallmouth bass were the most sensitive, while 

96-hour data indicate that bluegill were the 

most sensitive. Similarly, 3-hour LC50 *s show 

that brown bullhead were the most resistant, 

but 96-hour data indicate that green sunfish 

were the most resistant. Thus, some species 

having a relatively high resistance in short 

exposures may have a comparatively low resistance in longer exposures, and for other 

species the converse is true.

The toxicity varied among lots of a given 

species, and within the same lot. For 

example, the 96-hour LCso of lot Will of 

bluegills was twice that of lot W109. Two 

tests on goldfish from lot W116 gave 3-hour 

LCSO values of 6.55 and 10 p p m , but results 

generally were consistent and predictable.

Effects of temperature. Usually, the toxicity of Hyamine 3500 increases with temperature as indicated by the LC 60 values for lake 

trout (table 4), fathead minnow, and channel 

catfish (table 5). At 24 and 48 hours, the 

LCSO *s for lake trout at 17° are less than 

half those at 7° C. All of the 24-, 48-, and 

96-hour LC w *s for fathead minnow and channel 

catfish indicate increasing toxicity with an 

increase in temperature from 12° to 22° C. 

When both the 95-percent confidence interval 

and the LC 50 value are examined, it is apparent that some of the differences in toxicity 

indicated by the LC ^ fs alone are not statistically significant. Temperature changes appeared to have less effect on the toxicity of 

Hyamine 3500 to goldfish and bluegill than 

to the other two species tested. Some LC 60 

values indicate that toxicity increased with 

temperature, but other values show the opposite to be true. For instance, the 96-hour 

LC60 value for goldfish at 12° is 2.09 p p m 

and at 22° is 2.56 p p m , while the 3-hour 

LC 60 at 12° is 13.30 p p m and at 22° it is 

8.50.

Effects of water quality. The toxicity of 

Hyamine 3500 to lake trout, fathead minnow, 

and bluegill is greater in soft water than in

hard water (tables 6 and 7). The 96-hour 

LCso's in hard water are almost twice those 

in soft water.

The toxicity to goldfish and fathead minnow 

is greater at both acidic and alkaline pH 

levels than at neutrality (table 8). The effect 

of pH on the toxicity of Hyamine 3500 does 

not appear to be as great as the effect of 

water hardness.

DISCUSSION

In general, for at least the initial 24 hours, 

the toxicity of Hyamine 3500 to the 14 species 

of fish was dependent on the concentration of 

chemical and duration of exposure. Toxicity 

increased only slightly when exposures were 

continued from 24 to 96 hours. For instance, 

green sunfish from lotW164had3- and 6-hour 

LCSO *s of 8.00 and 4.50 p p m , respectively, 

while the 24-, 48-, and 96-hour LCSO values 

were 2.32, 2.25, and 2.25 p p m , respectively. For many species the 24- and 48-hour 

LC 50 *s or the 48- or 96-hour LCso *s were 

identical, or when slight differences existed 

they were not statistically significant.

There are many possible causes for the 

comparatively slow increase in toxicity when 

exposures were continued from 24 to 96 

hours. Marking (1966) indicated that this 

slowness may be caused by absorption and 

metabolism of the toxicant by the test fish, 

and by natural degradation of the chemical in 

solution. The latter would seem to be especially true of Hyamine 3500 since Nicholes 

and Burton (1961) found that solutions of 

Hyamine 3500 deteriorated when stored in 

open vessels. Furthermore, they indicated 

that both the chemically titratable activity and 

the germicidal efficiency of Hyamine 3500 

solutions decreased upon standing. This deterioration was found to increase rapidly if 

the storage temperature was higher than 

22° C.

Bioassays showed that not only fish from 

different sources but fish from the same 

source and even fish from the same lot differ 

in their sensitivity to Hyamine 3500. Marking 

(1966) found that geographic location, water 

quality, pond fertilization, herbicide application, and feeding and handling during rearing
Investigations in Fish Control 32: Bureau of Sport Fisheries and Wildlife

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James W. Hogan: Toxicity of Hyamine 3500 to Fish 7

TABLE 4-. Toxicity of famine 3500 to lake trout at three temperatures at 24, 48, and 96 hours of exposure.

Temperature

(°c.)

7 .............................

12 .............................

17 .............................

Lot

34-6 

34-6 

34-6

LC5 o and 95-percent confidence interval (ppm) at 

24 hours

4-. 00 (3.31 - 4-. 84-) 

2.74- (2.51 - 2.99) 

1.36 (0.91 - 2.04)

48 hours

2.45 (2.15 - 2.79) 

2.35 (2.12 - 2.61) 

1.22 (0.86 - 1.73)

96 hours

1.80 (1.58 - 2.05) 

1.90 (1.71 - 2.11) 

1.00 (0.74 - 1.36)

may influence the relative tolerance of specimens to a toxicant. Also, water quality and 

feeding and handling during the holding time 

immediately before testing may have a direct 

influence on the physiological condition of the 

test fish. The 24-, 48-, and 96-hour LC50 values 

obtained with lots of goldfish from Welaka 

National Fish Hatchery and Tallassee, Ala., 

indicated that the fish had statistically significant differences in their tolerance to Hyamine 

3500. The 24-hour LC50 values from separate 

tests conducted on different occasions with 

brown bullheads from lot W148 and with smallmouth bass from lot W146 show that there 

were statistically significant differences in 

resistance among fish from the same lot.

It is interesting that the 24-hour LC & values 

did not vary over a wide range among the 14 

species tested. The range for warmwater 

fishes was from 0.72 p p m for bluegill to 

2.71 p p m for goldfish at 17° C. As a group 

the three species of trout were more resistant 

than warmwater fishes to Hyamine 3500 at 

12° C. Of the four species of warmwater 

fish tested at 12° C., only goldfish were as 

resistant as the trout.

In the only field data currently available, 

Drake 1 reported on the use of Hyamine 3500 

for control of gill disease at Pendills Creek 

National Fish Hatchery in Michigan. In these 

tests, lake trout held in lentic water were 

exposed to Hyamine 3500 for 1 hour and observed for 24 hours following exposure to 

determine mortality. The tests were conducted in two similar types of water having 

the following physical and chemical characteristics: (1) raceway water having temperatures ranging from 2° to 5° C., a pH of 

7.2, and a total hardness of 51 p p m , and

1 Letter from Peter G. Drake, Manager, Pendills 

Creek NFH, Brimley, Mich0 1966.

(2) tap water with temperatures ranging from 

6° to 10° C., a pHof 7.5, and a total hardness 

of 51 p p m. Under the conditions listed, a 

1-hour exposure to 2 to 5 p p m of Hyamine 

3500 killed all fish within 45 minutes following the test. Similar exposures to concentrations of 1.50 to 1.75 ppm killed half the 

fish, while exposure to concentrations ranging 

from 1.10 to 1.40 ppm resulted in 0- to 

80-percent mortality. Drake stated that mortality was greater when water temperature 

exceeded 4° C. He concluded that Hyamine 

3500 was not suitable for treatment of gill 

disease in lake trout at Pendills Creek National Fish Hatchery.

Considering Drake's report, and the fact 

that warmwater fish are as sensitive as trout 

to Hyamine 3500, it appears that more field 

data are required before any recommendations 

can be made regarding Hyamine 3500 for control of fish diseases.

CONCLUSIONS

1. Decreasing mortality among fish indicates 

rapid degradation of dilute solutions of 

Hyamine 3500 in open vessels.

2. Toxicity of Hyamine 3500 to fish decreases 

as water hardness increases.

3. In general, toxicity of Hyamine 3500 to 

fish decreases as water temperature decreases.

4. Hyamine 3500 is more toxic at acid and 

alkaline pH than at pH 7.

5. The three species of trout tested are as 

resistant to Hyamine 3500 as are warmwater fish.
8 Investigations in Fish Control 32: Bureau of Sport Fisheries and Wildlife

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James W. Hogan: Toxicity of Hyamine 3500 to Fish 9

TABLE 6. Toxicity of Ifyamine 3500 to lake trout (lot 34-6) at various levels of total hardness at two temperatures

Temperature

<°c.)

12...................

12...................

12..............

17 ...................

17 ...................

17 ...................

Total hardness 

as CaC03 (ppm)

10 

42 

170

10 

42 

170

LC 50 and 95-percent confidence interval (ppm) at --

24 hours

2.85 (2.57 - 3.16) 

2.74 (2.51 - 2.99) 

4.18 (3.67 - 4.77)

0.59 (0.43 - 0.81) 

1.36 (0.91 - 2.04) 

0.96 (0.74 - 1.25)

48 hours

1.82 (1.69 - 1.92) 

2.35 (2.12 - 2.61) 

3.15 (2.89 - 3.43)

0.50 (0.36 - 0.69) 

1.22 (0.86 - 1.73) 

0.70 (0.59 - 0.84)

96 hours

1.49 (1.37 - 1.62) 

1.90 (1.71 - 2.11) 

2.83 (2.53 - 3.17)

0.42 (0.31 - 0.59) 

1.00 (0.74 - 1.36) 

0.70 (0.59 - 0.84)

TABLE 7. Toxicity of Ifyamine 3500 to fish in waters of various qualities at 17° C.

Species

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

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

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

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

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

Lot

W113

W113

W113

W119

W119

W119

Average 

PH

6.99

7.54

8 T ^

6.99

7 54

8 1 ^

Total hardness 

as CaC0 3 (ppm)

11.0

41.3

150.0

11.0

J 1 O

150.0

96-hour LC50 and 95-percent 

confidence interval 

(ppm)

0.82 (0.73 - 0.92)

1 OQ ( T n& T *^ ^

1.67 (1.52 - 1.84)

0.43 (0.33 - 0.56)

0.50 (0.38 - 0.65)

0.82 (0.65 - 1.03)

TABLE 8. Toxicity of famine 3500 to fish at three pH levels at 17° C.

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

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

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

Fathead minnow 

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

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

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

Lot

W153

W153

W153

W139 

W139

W139

W139

pH

5.0 + 0.1

7.0 + 0.1

9.0 i 0.1

5.0 i 0.1 

5-0 + o.l

7.0 - 0.1

9.0 i 0.1

LCso and 95 perc

24 - hours

1.89 (1.60 - 2.23)

1.85 (1.62 - 2.11)

0.74 (0.67 - 0.81)

1.35 (1.24 - 1.47)

1.04 (0.96 - 1.12)

ent confidence interval

48 - hours

1.80 (1.56 - 2.07)

2.00 (1.74 - 2.30)

1.73 (1.57 - 1.90)

0.73 (0.64 - 0.84) 

0.62 (0.55 - 0.69)

1.18 (1.07 - 1.30)

0.93 (0.85 - 1.01)

(ppm) at -

96 - hours

1.83 (1.59 - 2.10)

0.60 (0.^.9 - 0.74) 

0.37 (0.27 - 0.50)

1.10 (1.00 - 1.21)

0.93 (0.85 - 1.00)

REFERENCES

Lennon, Robert Ee, and Charles R, Walker. 

1964. Investigations in Fish Control: 1. Laboratories and methods for screening fish-control 

chemicals. Bureau of Sport Fisheries and Wildlife, Circular 185. 15 p.

Litchfield, J. T0 Jr.. and F. Wilcoxon. 

1949. A simplified method of evaluating dose-effect 

experiments. Journal of Pharmacology and Experimental Therapeutics, vol. 96, no. 2, p. 99-113.

Marking, Leif L.

1966. Investigations in Fish Control: 10. Evaluation 

of ptp1 -EOT as a reference toxicant in bioassays. 

U,S. Bureau of Sport Fisheries and Wildlife, Resource Publication 14. 10 p.

1967. Investigations in Fish Control: 12. Toxicity of 

MS-222 to selected fishes. U.S. Bureau of Sport

Fisheries and Wildlife, Resource Publication 18. 

10 p.

Nicholes, Paul S., and Francis C. Burton.

1961. An examination of the effectiveness of various 

sanitizing agents against yeasts isolated from liquid 

sugar and sucrose-corn syrup blends. Journal of 

the American Society of Sugar Beet Technologists, 

vol. 11, no. 5, p. 399-405.

Rohm and Haas Company.

1965. Hyamine 3500. Agricultural and Sanitary 

Chemicals Department, Technical Bulletin SAN 

170-8. 7 p.

Warren, James W.

1964. Laboratory tests of the bactericidal action and 

fish toxicity of six quaternary disinfectants. Abstract. Proceedings of the Northwest Fish Culture 

Conference, Oregon State University, Corvallis, 

December 2-3, p. 22-23.
INVESTIGATIONS IN FISH CONTROL

33. Voidance Time for 23 Species of Fish

By Thomas H. Lane and Howard M. Jackson

United States Department of the Interior, Walter J. Hickel, Secretary

Leslie L. Glasgow, Assistant Secretary

for Fish and Wildlife, Parks, and Marine Resources

Fish and Wildlife Service, Charles H. Meacham, Commissioner

Bureau of Sport Fisheries and Wildlife, John S. Gottschalk, Director

Washington, D.C. October 1969
CONTENTS

Page

Abstract. ....................................... 3

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

Results ........................................ 5

Discussion ...................................... 7

Conclusions. ..................................... 8

References ...................................... 8
VOIDANCE TIME FOR 23 SPECIES OF FISH

By Thomas H. Lane and Howard M. Jackson, Fishery Biologists

Bureau of Sport Fisheries and Wildlife 

Southeastern Fish Control Laboratory, Warm Springs, Georgia 31830

ABSTRACT. Observations on 23 species of fingerling-size bioassay 

fish indicated that voidance time (time required for food residues to pass 

through the alimentary canal) ranged from 12 to 108 hours.

Many species of fish are used in bioassays 

of pesticides, pollutants, and other chemicals 

(Douglas, 1960; Ward and Irwin, 1961; and 

Gould and Irwin, 1965). Guidelines for the 

acquisition, acclimatization, and evaluation 

of bioassay fish were defined by Hart, 

Doudoroff, and Greenbank (1945) and Doudoroff 

et al. (1951). They recommended feeding 

specimens during the 10-day pretest holding 

period, but they advised that food be withheld 

for 1 to 2 days before bioassays and during 

bioassays. Lennon and Walker (1964) advocated withholding food from test fish for as 

long as 4 days before bioassay, depending on 

the life stage and species of fish. The objective of the withholding is to empty the digestive tract of food wastes before the fish is 

introduced into bioassay.

Ward and Irwin (1961) stated that fish in 

good condition may not be greatly affected by 

short periods without food, but they cited 

evidence that the resistance of fish to chemicals may decrease with longer periods of 

starvation. In any case, the period off feed 

should be held to a minimum. Accordingly, 

the purposes of this investigation were to 

measure voidance times for various freshwater fishes and to determine whether voidance time is constant for a given species at 

one or two temperatures.

Some information concerning digestion 

rates, food passage rates, nutrition, and 

feeding behavior of fish is scattered throughout the literature. Results are diverse for a

species, even though size of fish, water 

temperature, and other variables are similar. 

Many of the variables characteristically found 

in this type of investigation are either referred to or briefly discussed by Darnell and 

Meierotto (1962) in their study on digestion 

rate in a population of small black bullheads. 

According to these authors, digestion rate and 

rate of food passage through the digestive 

tracts of fishes have been shown to vary with 

temperature, age of experimental animal, and 

type and amount of food. They also point out 

that digestion rates are affected by the general 

activity of the animals which in turn may be 

influenced by the light regimen.

Phillips et al. (1960) in a test using brook 

trout averaging 6 grams observed no significant change in rate of food passage which 

could be ascribed to a temperature reduction 

of 2.8° C., from 11.1° to 8.3°. Earlier studies 

showed a deceleration in food passage in 

fingerling brook trout when the temperature 

was reduced 8.3° C., from 10.5° to 2.2° 

(Phillips et al., 1956). Markus (1932) and 

Baldwin (1957) demonstrated in experiments 

with warm- and cold-water fish that a 

temperature change of 4° C. can appreciably 

affect digestion rate.

Phillips et al. (1956) observed that 6-gram 

brook trout showed a significant change in 

food passage rate when fed different types of 

food, that is, meat and a mixture of meat and 

dry meal.
Investigations in Fish Control 33: Bureau of Sport Fisheries and Wildlife

METHODS AND MATERIALS

The observations on voidance time were 

made at the Fish Control Laboratory at La 

Crosse, Wis. and the Southeastern Fish Control Laboratory at Warm Springs, Ga. The 

water in the indoor holding facilities at La 

Crosse is supplied from a deep well. It is 

hard (220 to 330 parts per million as CaCOs), 

and its temperature during the test was 

12°t 1° C. The holding and test temperatures 

were so similar that no acclimation was 

necessary when transferring fish into test 

facilities.

The water at Warm Springs for outdoor 

holding facilities and indoor testing is supplied by a spring and is hardened with lime to 

35 to 45 ppm as CaCO3 . Its temperature is 

17° C. Test temperatures ranged from 12° to 

25°, and experimental fish were carefully 

acclimated to them.

Most of the fish were acquired from Federal 

and State hatcheries. Golden shiners and 

brown bullheads were obtained from private 

ponds. The list of species is given in table 1.

The fish were fed routinely during the 

holding period, with special care taken within 

the last 24 hours before trials to provide as

TABLE 1. Fish used in voidance tests

Rainbow trout, Salmo gairdneri, 

Lake trout, Salvelinus namaycush. 

Northern pike, Bsox lucius. 

Goldfish, Carassius auratus.

Carp, Qyprinus carpio. 

Golden shiner, Notemigonus crysoleucas. 

Fathead minnow, RLmephales promelas. 

White sucker, Catostomus commersoni.

Bigmouth buffalo, Ictiobus cyprinellus. 

White catfish, Ictalurus catus. 

Black bullhead, Ictalurus melas. 

Yellow bullhead, Ictalurus natalis.

Brown bullhead, Ictalurus nebulosus. 

Channel catfish, Ictalurus punctatus. 

Green sunfish, Lepomis cyanellus. 

Pumpkinseed, Lepomis gibbosus.

Bluegill, Lepomis macrochirus. 

Longear sunfish, Lepomis megalotis. 

Smallmouth bass, Micropterus dolomieui. 

Largemouth bass, Micropterus salmoides.

White crappie, Pomoxis annularis. 

Yellow perch, Perca flavescens. 

Walleye, Stizostedion v. vitreum.

much food as the fish would eat. Carnivorous 

species were furnished with live foods such 

as daphnia and fish fry. Some lots of fish 

were supplied with dry food, but most lots 

were given a synthetic food originally formulated at the Southeastern Fish Cultural 

Laboratory, Marion, Ala., for use in studies 

on the nutrition of channel catfish (Harry K. 

Dupree and Kermit E. Sneed. Purified diets 

for channel catfish nutritional research. 

Manuscript.)

All voidance observations at La Crosse and 

initial tests at Warm Springs were conducted 

in 1-gallon wide-mouth jars. Later experiments were accomplished in funnel-type 

aquariums which proved more useful (fig. 1). 

Cover screens were necessary on the funnel 

aquariums to prevent escape of some species 

and to minimize the effect of the observer's 

movements. Bottom screens of epoxy-coated 

hardware cloth were installed to prevent 

coprophagy.

Each test vessel in a battery of four vessels per species contained 2.5 liters of reconstituted, deionized water which was continuously aerated (Lennon and Walker, 1964). 

The fish were loaded into vessels at a rate of 

about 8 grams per liter of water. Observations were made twice daily on the fish and 

feces; any dead fish were removed; and the 

presence or absence of fecal material was 

noted. At each observation, test vessels containing significant amounts of solid feces 

were scored with a plus (+). Those containing 

only one or two small deposits or none were 

scored with a minus (-).

Needle Valve 

Screen

 Air Stone

Glass Funnel ( 4 liter )

Screen

One-Hole Stopper 

Rubber Tube 

Pinchcock

Figure 1. Funnel-type aquarium used in voidance tests.
T. H. Lane and H. M. Jackson: Voidance Time for 23 Species of Fish

The tests of most species continued for 4 or 

5 days. Voiding was judged to be complete at 

the end of the first interval during which two 

of the four test vessels were free of fecal 

deposits, that is, when a 50-percent reduction 

of + observations occurred. If three vessels 

were free of feces at the end of a period, 

voidance was listed as complete at the previous observation period, 12 hours earlier.

At La Crosse, the fish were transferred to 

clean jars at each observation period. At 

Warm Springs, about 10 percent of the water 

was removed from each jar each 12 hours 

and replaced with fresh reconstituted 

water.

Three techniques were used in identifying 

and enumerating feces: (1) Discrete fecal deposits were observed and counted; (2) Sudan 

111, a red dye insoluble in water but soluble 

in oil, was added to the Marion catfish diet at 

1 part per thousand to permit differentiation 

of food-waste feces from non-food feces which 

were found after last meals were completely 

voided; (3) A colorimetric method of detecting 

Sudan 111 in food-waste feces was employed.

The colorimetric method involved placing 

fecal samples in a Squibb separatory funnel 

after excess water was decanted. Glass beads 

and 20 milliters of chloroform were added to 

the funnel and shaken vigorously until the dye 

was extracted. The bottom layer of chloroform 

with dye was drawn off and read in a DB spectrophotometer at a wavelength of 510 mn. as 

percent transmittance. The peak absorbance 

of this wavelength was determined previously 

for Sudan 111 which was extracted from fecal 

material with chloroform. Readouts for each 

of four replications were averaged and plotted 

for each observation period (fig. 2).

During preliminary colorimetric experiments, reference blanks of sol vent-extracted 

feces from fish fed the same synthetic diet 

without dye and blanks of 100-percent solvent 

were compared. The transmittance of the 

blanks was not significantly different, and the 

chloroform blank was used, therefore, in tests 

plotted in figure 2.

lOO-i

UJ

o

I

CO

z

80-

6CH

h- 40-

H

UJ

UJ 

Q.

bigmouth buffalo 

green sunfish 

bluegill 

fathead minnow

12 24 36 48 60

OBSERVATION INTERVAL (hours)

Figure 2. Relative concentrations of Sudan 111 in 

fecal voidings of four fishes.

RESULTS

At La Crosse, 30 of 32 groups of fish voided 

within 36 to 96 hours at 11° to 13° C. (table 2). 

Metabolism is lower at these temperatures, 

and fish generally require more time to complete voiding. One group of lake trout did not 

complete voiding for 108 hours. One lot of 

channel catfish voided in less than 36 hours 

but it had appeared to feed poorly during pretrial holding. Another lot of channel catfish, 

a lot of yellow perch, and one of bluegill fed 

only lightly during holding, and the intestines 

of several specimens from each lot were 

examined during the tests. Because of the 

comparatively small amounts of food found in 

them, the voiding results probably should be 

regarded as minimal values (table 2).

In tests at Warm Springs in warm water 

(19° to 25°), 13 of 17 groups of fish completed 

voiding within 36 to 60 hours (tables 3 and 4). 

One group each of goldfish and yellow bullheads required 72 hours to complete voiding, 

and one group each of fathead minnows and 

bigmouth buffalo completed voiding in less 

than 36 hours.

Three groups of bluegills from lot number 

17 (table 4) were tested at 12°, 17°, and 22°C. 

to ascertain the effects of temperature on 

voiding. The voidance times were 84, 48, and 

36 hours, respectively. The expected
Investigations in Fish Control 33: Bureau of Sport Fisheries and Wildlife

TABLE 2. Voidance time for fish held at 12°+1° C. at La Crosse, Wisconsin

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

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

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

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

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

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

Channel catfish.... 

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

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

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

Longear sunfish.... 

Smallmouth bass .... 

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

Largemouth bass .... 

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

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

Wn 1 1 p VA

Total leng

Average

1.0

2.8

2.8

3.7

1.5

2.9

1.4

1.8

2.0

2.1

2.2

3.5

1.6

1.7

1.8

1.6 

1.3 

1.9

1.4 

1.8

1.4

1.9

1.6

th (inches)

Range

1.6-2.4

2 5-2 9

2 4-3 2

3.4-4.1

1.3-1.7

2.6-3.1

1.2-1.9

1.5-2.7

1.6-2.5

1.7-2.5

3.5-3.9

1.6-2.5

3.0-4.1

1.3-2.0

1.7-2.5

1.5-2.1

1.2-2.0

1.4-2.0

1.4-2.0 

1.1-1.6 

1.5-2.1

1.2-1.4 

1.4-2.0

1.2-1.7

1.8-2.2

1.2-2.0

Weight

Average

0.2

1.2

3.1

2.5

5.6

0.4

1.9

O

rt

2.5

2.5

1.7

6.0

2.1

2.1 

5.2

1.4

2.6

1.1

1.2

1.3

1.0 

0.5 

1.3

0.5 

00

0.5

0.8

0.5

(grams)

Range

__

__

__

4 4-6 5

0 0 n &

0.6-1.5

2.0-3.6

0.9-3.1

0.9-3.2

1.4-3.7 

3.5-8.0

0.5-1.7

0.8-2.1

O fJ o n

0.7-1.8 

0.3-0.7

~

0.9-1.1

Number

of fish

Qn

30

24

32

12

40

On

100

74

80

48

12

36

36 

16

72

45

sn

60

60

125 

40 

120

160 

96

160

100

360

Number of

replicates

3

3

4

4

4

4

8

4

7

8

4

3

4

4 

4

4

3

4

3

4

7 

4 

8

4 

4

4

4

12

Time to

void (hours)

36

60

60

108

60

1 72

72

48

36

60

36

60

84

2 24 

2 36

60

84

2 60

84

36

72 

1 72 

48

84 

48

60 2 36

60

Observations made at 24-hour intervals. 

2 Fish may not have consumed sufficient food before being placed in test vessels.

TABLE 3. Voidance time for fish held at 20 ±1° C. at Warm Springs. Ga.

Golden shiner. .....

Fathead minnow. ....

Bigmouth buffalo. . .

White catfish......

Brown bullhead. ....

Green sunfish. .....

Largemouth bass ....

White crappie......

Weight

Average

1.4

0.9

1.5

1.5

3.2

0.6

1.2

1.2

1.6

1.2

0.4

(grams)

Range

0.5-3.5

0.6-1.9

1.2-3.0

1.2-3.0

2.0-5.1

0.6-0.9

0.7-2.0

0.5-2.0

0.2-0.7

Number 

of

fish

80

40

40

40

40

40

40

40

40

80

40

40

Number 

of

cates

8

4

4

4

4

4

4

4

8

4

4

Time to 

void

(hours)

36

12 x (24)

24 1 (24)

48

48

60

48 x (48)

36 i (48)

48

60

60

TABLE 4.--Voidance tine for fish held at various temperatures at 

Warm Springs, Ga.

Species

Yellow bullhead

Lot 

number

13 

14 

14 

15

16 

17 

17 

17

Weight (grams)

Average

0.8 

3.0 

3.6 

1.5

0.5 

0.6 

0.6 

0.6

Range

0.6-0.9 

1.8-4.2 

2.4-5.6 

0.8-2.0

0.1-0.9 

0.4-1.5 

0.4-1.5 

0.4-1.5

Number

of 

fish

40

40 

40 

40

40 

20 

20 

20

Number 

of 

replicates

4 

4 

4 

4

4 

2 

2 

2

Average

temp.

25.0 

23.5 

22.5 

24.0

25.0 

22.0 

17.0 

12.0

Time 

to 

void 

(hours)

60 

48 

48 

72

36 

36 

48 

84

1 Values obtained by colormetric method.
T. H. Lane and H. M. Jackson: Voidance Time for 23 Species of Fish

physiological effects of increased temperature were apparent, especially in tests made 

at 12° and 17°.

Visual assessments of voidance time for 

four species were made concurrently on 

samples which were evaluated by using the 

transmittance-time curve of the colorimetric 

method (fig. 2), and they were well corroborated by the latter.

DISCUSSION

The 1-gallon test jars used at La Crosse 

and initially at Warm Springs had several disadvantages. They made it necessary to handle 

the fish at each 12-hour period, which resulted 

in unnecessary stress and stress-induced defecation. Several types of tests vessels were 

tried, but those which were funnel-like in design proved to be the most satisfactory. The 

design shown in figure 1 minimized handling 

of fish and permitted easy and efficient recovery of solid wastes which were concentrated in the neck of the funnel. According 

to Brockway (1950) excretory products in solution also tend to accumulate on the bottom in 

ponds and raceways because of their greater 

density. We presumed, therefore, that a large 

portion of liquid wastes at the bottom of an 

aquarium would be drawn off with the 250- 

milliliter sample of excrement and water.

The later feeding with the synthetic diet 

eliminated the early variance due to different 

types of foods. Some of the early variations in 

results with a species tested both at La Crosse 

and Warm Springs were due more to different 

types of food than to different temperatures. 

The synthetic diet also facilitated the use of 

a dye, Sudan 111, to detect the end point of 

voidance. It was acceptable, either with or 

without the dye, to all of the species tested 

except largemouth bass. Some species preferred it over several types of dry food. The 

gastric and intestinal mucosae of fish which 

were fasted for several days after eating dyed 

food were examined for evidence of dye 

absorption, but none was found.

The frequency of taking food varied with 

each test lot so that no pattern could be followed in feeding the fish. Food was offered 

frequently for 24 or more hours prior to

testing, and each time to the point of satiation.

The fish used in the voiding experiments 

were exposed to a change in water hardness. 

The reconstituted deionized water has a calcium hardness of about 16 ppm as CaCO3 . 

Holding water at La Crosse has a calcium 

hardness range of 150 to 232 ppm, and that 

at Warm Springs ranges from 35 to 45 ppm. 

Phillips et al. (1954) observed that a diminution in water calcium was accompanied by an 

increase in metabolic activity in brook trout. 

A variable such as this may have influenced 

voidance times for fish at both laboratories.

Of the factors shown to affect digestion 

rates and food passage rates in fish (Darnell 

and Meierotto, 1962), temperature and those 

factors associated with diets and feeding 

probably are the most important contributors 

to the variance in voidance times. Large differences were found between voidance times 

of fish of the same species and size from different hatchery lots which were tested at the 

same temperature. This observation leads us 

to believe that interpopulation differences may 

be significant in causing variation in voidance 

time. The test results for different lots of fish 

of the same species and size were not averaged 

so that the differences would remain apparent. 

Even so, sufficient data are not available to 

evaluate effects which might result from 

population differences, and the question remains unanswered.

The problem of distinguishing between fecal 

deposits which contain food residues and those 

which do not became evident in early experiments with green sunfish, carp, goldfish, and 

bigmouth buffalo. According to Hawk, Oser, 

and Summerson (1954), feces resulting from 

food digestion and absorption are composed 

of (1) food residues, (2) the remains of intestinal and digestive secretions, (3) substances secreted into the intestinal tract, 

principally salts, (4) bacterial flora and their 

metabolic end products, (5) cellular elements, 

and (6) abnormal elements. During fasting, 

some species apparently void large quantities 

of any one, or possibly all, of the elements 

mentioned above except food residues. In one 

test not included in the tables, bigmouth buffalo voided for 156 hours without any noticeable decrease in the quantity or change in the
8 Investigations in Fish Control 33: Bureau of Sport Fisheries and Wildlife

appearance of the fecal deposits. However, 

two tests with dyed food were performed with 

fish from the same lot. The results showed 

that voidance of food residues was complete 

in 24 and 48 hours (table 3) even though deposition of fecal material continued as before. 

Thus, the value of the dye method is clearly 

demonstrated.

The colorimetric method, although timeconsuming, was used as a check on the visual 

method. It is considered more accurate since 

all of the excreted dye can be extracted and 

measured.

CONCLUSIONS

1. All but 2 of the 32 groups of fish tested at 

La Crosse at 11° to 13° C. completed voiding in 36 to 96 hours. One group of lake 

trout required 108 hours, and one group of 

channel catfish, which fed poorly, completed voiding in less than 36 hours.

REFERENCES

Baldwin, N. S.

1957. Food consumption and growth of brook trout at 

different temperatures. Transactions of the 

American Fisheries Society, vol. 86, p. 323-328.

Brockway, Donald R.

1950. Metabolic products and their effects. Progressive Fish-Culturist, vol. 12, no. 3, p. 127-129.

Darnell, Rezneat M., and Richard R. Meierotto.

1962. Determination of feeding chronology in fishes. 

Transactions of the American Fisheries Society, 

vol. 91, no. 3, p. 313-320.

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

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

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.

1960. A study of the comparative use of different 

species of fish in the toxicity bioassay of petroleum 

refinery effluent. Proceedings of the Fourteenth 

Annual Conference of the Southeastern Association 

of Game and Fish Commissioners, p. 215-222.

2. All but 4 of 17 groups of fish tested at 

Warm Springs at 19° to 25° C. completed 

voiding in periods of 36 to 60 hours. One 

group each of goldfish and yellow bullheads 

required 72 hours, and one group each of 

fathead minnows and bigmouth buffalo completed voiding in less than 36 hours.

3. Solid feces formed as a result of feeding 

and those formed during fasting could not 

be discriminated in green sunfish, carp, 

goldfish, and bigmouth buffalo without use 

of a dye marker. Voidance times determined using the dye, Sudan 111, were 

corroborated by the colorimetric 

method.

4. The period of time that fingerling-size fish 

should be held off feed before use in bioassays depends principally on the species, 

type of food, and temperature. If contamination of bioassays by food-waste feces is to be 

minimized, food should be withheld from 

most of the 23 species studied for 2 to 3 

days before bioassays.

Gould, W. R. Ill, and W. H. Irwin.

1965. The suitabilities and relative resistances of 

twelve species of fish as bioassay animals for oilrefinery effluents. Proceedings of the Sixteenth 

Annual Conference of the Southeastern Association 

of Game and Fish Commissioners (1962), p. 333- 

348.

Hart, W. Bregy, Peter Doudoroff, and John Greenbank. 

1945. The evaluation of the toxicity of industrial 

wastes, chemicals and other substances to freshwater fishes. Waste Control Laboratory, The 

Atlantic Refining Company, Philadelphia. 317 p.

Hawk, Phillip B., Bernard L. Oser, and William H. Summers on.

1954. Practical physiological chemistry. 13th 

edition, McGraw-Hill Book Company, Inc., New 

York. 1439 p.

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

1964. Laboratories and methods for screening fishcontrol chemicals. U.S. Bureau of Sport Fisheries 

and Wildlife, Investigations in Fish Control No. 1. 

(Bureau Circular 185). 15 p.

Markus, Henry C.

1932. The extent to which temperature changes influence food consumption inlargemouthbass (Huro 

floridana). Transactions of the American Fisheries 

Society, vol. 62, p. 202-210.
T. H. Lane and H. M. Jackson: Voidance Time for 23 Species of Fish 9

Phillips, Arthur M., Jr., Floyd E. Lovelace, Henry A. 

Podoliak, Donald R. Brockway, and George C. Balzer, 

Jr.

1954. The nutrition of trout. Cortland Hatchery Report No. 23, New York Conservation Department, 

Fisheries Research Bulletin 18. 52 p.

Phillips, Arthur M., Jr., Henry A. Podoliak, Donald R. 

Brockway, and George C. Balzer, Jr.

1956. The nutrition of trout. Cortland Hatchery Report No. 25, New York Conservation Department, 

Fisheries Research Bulletin 20, 61 p.

Phillips, Arthur M., Jr., Henry A. Podoliak, Donald L. 

Livingston, Richard F. Dumas, and Glenn L. Hammer.

1960. The nutrition of trout. Cortland Hatchery Report No. 29, New York Conservation Department, 

Fisheries Research Bulletin 24, 76 p.

Ward, Claud M., and W. M. Irwin.

1961. The relative resistance of thirteen species of 

fishes to petroleum refinery effluent. Proceedings 

of the Fifteenth Annual Conference of the Southeastern Association of Game and Fish Commissioners, p. 255-276.
INVESTIGATIONS IN FISH CONTROL

34. Laboratory Studies on

Possible Fish-Collecting Aids 

With Some Toxicities for the 

Isomers of Cresol

By Robert M. Howland

United States Department of the Interior, Walter J. Hickel, Secretary

Leslie L. Glasgow, Assistant Secretary

for Fish and Wildlife, Parks, and Marine Resources

Fish and Wildlife Service, Charles H. Meacham, Commissioner

Bureau of Sport Fisheries and Wildlife, John S. Gottschalk, Director

Washington, D.C. October 1969
CONTENTS

Page

Abstract. ...................................... 3

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

Chemicals. .................................. 4

Toxicity.................................... 4

Efficacy.................................... 4

Results. ....................................... 5

Toxicity.................................... 5

Observations during toxicity tests................... 6

Efficacy .................................... 7

Discussion ..................................... 8

Conclusions. .................................... 9

References ..................................... 9
LABORATORY STUDIES ON POSSIBLE FISH-COLLECTING AIDS, 

WITH SOME TOXICITIES FOR THE ISOMERS OF CRESOL

By Robert M. Rowland, Fishery Biologist

Bureau of Sport Fisheries and Wildlife 

Fish Control Laboratory, La Crosse, Wisconsin 54601

ABSTRACT. The relative merits of quinaldine (2 methylquinoline), 

McNeil-JR-7464 (dl-l-(l-phenyl-ethyl)-5-(propoxy-carbonyl)-imidazole 

hydrochloride), and three isomers of cresol (p-methylphenol, o-methylphenol, and m-methylphenol) as collecting agents were determined in a 

lotic system under laboratory conditions at 12°C. The toxicity of the three 

cresol isomers to rainbow trout, brown trout, and brook trout also was 

measured in bioassays conducted in standard constituted water, and LC50 

values were calculated for exposures of 6, 24, 48, and 96 hours. The 

toxicity of para-cresol was also established for carp, fathead minnow, 

black bullhead, channel catfish, bluegill, and yellow perch. Conclusions of 

these toxicity and efficacy tests were as follows: (1) Para-cresol is the 

most active of the three isomers of cresol in inducing total incapacitation 

of rainbow, brown, and brook trout; (2) The cresols are too harsh physiologically to warrant further development as fish-collecting agents; (3) 

MS-222 is not well suited as a fish-collecting tool; it does not induce 

surfacing and a high concentration is necessary for total incapacitation;

(4) Quinaldine may have some usefulness for collecting fish in small 

streams with rapid flow but its physiological effects need elucidation;

(5) McNeil-JR-7464 does not induce surfacing but is effective at low 

concentrations and appears to have desirable characteristics as a fishcollecting agent which merits further investigation.

Chemical compounds are needed for collecting 

fish without harming them. Such compounds as 

rotenone, cresol, quinaldine, aqualin, and 

tranquilizers have come under scrutiny 

(Wilkins, 1955; Louder and McCoy, 1962; 

Blanchard, 1965; Penfold, 1965; Tate, Moen, 

and Severson, 1965). Chemically induced 

surfacing of fish has been considered a 

desirable characteristic by some investigators (Loeb, 1962; Blanchard, 1965). Commercial cresol evoked a degree of surfacing, 

according to Wilkins (1955), when used as a 

stream sampling aid by the Tennessee Game 

and Fish Commission.

Because of the performance of cresol in 

past trials (Embody, 1940; Wilkins, 1955), we 

decided on further laboratory studies on the 

chemical. We used static bioassay to determine the toxicities of three isomers of cresol 

to rainbow trout, brown trout, and brook trout. 

The toxicity of par a-ere sol was also determined on carp, fathead minnows, black bullheads, channel catfish, bluegill sunfish, and 

yellow perch. The efficacies of ortho-cresol, 

meta-cresol, and para-cresol as collecting 

aids were measured with rainbow trout. 

Subsequently, quinaldine, MS-222, and McNeilJR-7464 were investigated along with the
4 Investigations in Fish Control 34: Bureau of Sport Fisheries and Wildlife

most effective isomer of cresol, against 

rainbow trout, to compare their relative 

merits as collecting agents in a lotic system.

METHODS AND MATERIALS

Chemicals

The isomers of cresol were a practical 

grade of para-cresol (p-methylphenol) and a 

purified grade of ortho-cresol (o-methylphenol) and meta-cresol (m-methylphenol). 

The quinaldine (2-methylquinoline) was 

practical grade. The McNeil-JR-7464 is 

dl-l-(l-phenyl-ethyl)-5-(propoxy-carbonyl)- 

imidazole hydrochloride.

Toxicity

Static bioassays with the isomers of cresol 

were with acclimated 2-inch fish in 5-gallon 

glass jars (Lennon and Walker, 1964). We 

exposed 10 fish to each concentration, and 20 

served as controls. The statistical methods of 

Litchfield and Wilcoxon (1949) were used for 

determining LC50 values, slope functions, and 

95-percent confidence intervals. Observations 

were made on surfacing and signs of physiological stress.

Efficacy

The lotic system for constant-flow experimentation was a modified alumium hatchery 

through 14 feet long (fig.l). A head-box

3 25967

14 15

Figure 1. A lotic system for constant-flow experimentation: (1) standard aluminum hatchery 

trough, (2) head-box, (3) water supply, (4) overflow tube, (5) spout, (6) 1-gallon poultry 

waterer, (7) inverted-plastic-pail cover to reduce evaporation and noxious fumes, (8) 

admixture of inflowing water and chemical solution, (9) siphon tube, (10) enlargement of 

siphon tube, (11) modified teflon stopcock, notched at each hole with a shallow tapering 

v-groove to increase flow adjustability, (12j diffusion screen, (13 and 14) test chamber 

screens, (15) outlet standpipe with adjustable rubber sleeve for depth regulation.
Robert M. Rowland: Possible Fish-Collecting Aids

provided a uniform flow of water. Chemicalstock solution was held in a 1-gallon poultry 

waterer positioned below the head-box to allow 

admixture of chemical and inflowing water. 

Metering-in of the stock solution was accomplished with a glass siphon tube, equipped 

with an adjustable stopcock, modified from a 

standard burette. With this arrangement we 

could maintain a flow of 20 milliliters per 

minute with solutions of different viscosities, 

for the short periods involved in the experiments.

The trough was divided into compartments 

with screens 1 foot, 3.5 feet, and 5.5 feet 

from the point of inflow. The first screen 

served to enhance mixing and the following 

screens formed a test compartment for the 

fish. The inflow from the head-box was 

a djusted by using spouts of different diameters. 

The required amounts of chemicals and stock 

solutions were determined by the method 

described by Burrows (1949).

The stocks for quinaldine and the cresols 

were prepared with a 50:50 acetone:water 

solution. MS-222 and McNeil-JR-7464 dissolved readily in water. The progress of 

chemicals through the trough was simulated 

with 5 ppm (parts per million) of malachite 

green. Samples taken 1 minute apart at the 

head-screen of the fish compartment and 

analyzed by spectrophotometer provided a 

chronology of the increase toward maximum 

concentration. Thus, parameters were 

established for the subsequent exposure of 

test fish.

To appraise the value of these chemicals 

for collecting purposes, criteria of response 

were established in preliminary trials with 

cresol (mixture) and 3-inch rainbow trout:

1. Repellency minimal to reduce evasion in 

areas of weaJc concentration; surfacing desirable.

2. Total incapacitation, i.e. loss of equilibrium with only weak fin and directional 

movements remaining, within 10 minutes of 

contact.

3. Recovery not so rapid as to interfere 

with collecting.

4. No sign of physiological damage. We 

used twenty 3-inch rainbow trout to test each 

concentration for determining the most active 

isomer of cresol. Ten 9-inch rainbow trout 

were used at each concentration in the comparative testing of four compounds. Fish used 

in the lotic system were acclimated overnight. 

We kept a chronological record of fish 

responses for each trial. The flow of chemical 

was halted when all fish were in total incapacitation or when this stage was not attained 

within 15 minutes.

All tests were carried out at 12°C. in reconstituted water (Lennon and Walker, 1964). 

Table 1 lists the sources and sizes for test 

fish.

TABLE 1. Species, sizes, and sources of test fish

Species

Rainbow trout, Salmo gairdneri ......

Brown trout, Salrao trutta. ..........

Brook trout, Salvelimis f ontinalis . .

Carp, Cyprinus carpio ...............

Fathead minnow, Pimephales promelas. 

Black bullhead, Ictalurus melas.....

Channel catfish, Ictalurus punctatus

Yellow perch, Perca flavescens. . . . . .

Average 

length 

( inches )

2

2

2

2

2 

2

2

2

2

Source

Nevin, Wis., SFH. 1 

Manchester, Iowa, NFH. 2

Soap Lake, Wash.

Do.

Osceola, Wis., SFH.

Lake Mills, lifts., NFH.

Bud's Marina, La Crosse, 

Wis.; collected from 

Minnesota lakes

Necedah, Wis., NWR. 3

Fairport, Iowa, NFH.

Lake Mills, Wis., NFH.

Do.

1 State Fish Hatchery.

2 National Fish Hatchery.

3 National Wildlife Refuge.

RESULTS

Toxicity

The three isomers of cresol were toxic to 

trout (table 2). The 24-hour LCSO's reveal the 

differences in toxicity most clearly: against 

rainbow trout the value for para-cresol was 

9.2 ppm, for qrtho-cresol 9.9 ppm, and for 

meta-cresol 10.4 ppm. Similarly, against 

brown trout, para-cresol produced a value of 

4.4 ppm, ortho-cresol 7.2 ppm, and metacresol 8.6 ppm. The values against brook
6 Investigations in Fish Control 34: Bureau of Sport Fisheries and Wildlife

TABLE 2. Toxicities of the isomers of cresol to fish at 12° C.

ortho-cresol .......

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

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

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

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

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

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

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

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

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

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

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

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

Species

of fish

Rainbow trout

Rainbow trout

Bluegill

LC50 (ppn

6 hours

11.0

9.6-12.7

14.1

10.4-19.0 

14.9

13.4-16.6 

11.0

9.7-10.6 

11.4

9.7-13.4 

11.4

10.2-12.8

4

rj

4.1-5.3

8

C

7.3-9.9

£LC r\

54.0-78.0

19.5

14.7-26.0

) and 95-percent

24 hours

9.9

8.8-11.1 

7.2

6.7-7.8 

7.9

5.3-11.8 

10. -4

9.3-11.6 

8.6

7.5-9.8

8

0

7.7-8.8 

9.2

8.4-10.1 

-4.4

3.9-4.9 

6.3

5.4-7.4 

22.0

21.8-22.2 

60.3

50.0-72.9 

120.0

112.1-128.4

KSi Ci

47.0-71.2 

7.9

6.5-9.3 

12.3

10.5-14.4

confidence inte

48 hours

8.6

7.6-9.8 

7.2

6.7-7.8 

n si

5.9-10.3 

10.2

9.3-11.2 

81

7.4-9.6

7

s

7.0-8.2 

8.4

7.2-9.8 

4.4

3.9-4.9 

<=. si

5.1-6.6 

15.0

14.9-15.1

cn tj

37.9-67.9 

94.0

85.5-103.5 

50.0

39.1-64.0 

7.1

6.0-8.4 

10.0

8.1-12.3

rval at

96 hours

7.0

6.1-8.0

6

O

5.2-7.4 

7.2

6.7-7.7

8

r

7.7-9.6 

81

7.8-9.6

7

s

7.0-8.2 

7.4

6.4-8.5 

4.4

3.9-4.9 

<=. si

5.1-6.6 

13.3

13.2-13.4 

. 15.5

13.8-17.4 

57.5

46.0-72.0 

39.7

28.3-55.6 

7.1

6.0-8.4 

10.0

8.9-11.2

Mean

function

1.400

1.253

2.135

1.228

1.288

1.208

1.203

1.230

1.250

1.182

1.450

1.215

1.635

1.473

1.505

trout were 6.3 for para-cresol, 7.9 for orthocresol, and 8.2 for meta-cresol. The data 

show that the isomers are only slightly more 

toxic to the salmonids at 96 hours than they 

are at 24 hours. Regarding all LCSO's for the 

salmonids, a definite order oftoxicity emerges 

for the three isomers: para-cresol is most 

toxic, followed by ortho-cresol. and finally 

meta-cresol.

Among the warm-water fish in bioassays 

with para-cresol, fathead minnows, black 

bullheads, and channel catfish proved to be 

most resistant. For these species as well as 

carp, the toxicity increased markedly in 

relation to time of exposure.

Observation during toxicity tests

During the first 10 minutes of the bioassays 

against brook trout at 6 to 20 ppm, the 

approximate incidences of surfacing were 

meta-cresol 20 percent, ortho-cresol 30 

percent, and para-cresol 90 percent. The 

incidences for warm-water species exposed 

to para-cresol were carp 80 percent at 15 

to 23 ppm, fathead minnows 30 percent at 30 

to 150 ppm, bluegills 30 percent at 14 to 16 

ppm, and yellow perch 50 percent at 12 to 18 

ppm. Black bullheads and channel catfish did 

not surface at any concentration. Hemorrhages 

of the gills and at the base of the pectoral 

and/or pelvic fins were common in all bioassays.
Robert M. Rowland; Possible Fish-Collecting Aids

Efficacy

Tests conducted at 10 ppm with the isomers 

of cresol in the lotic system confirmed the 

order of potency revealed in the static bioassays 

(fig. 2). Meta-cresol caused total incapacitation in 15 of 20 rainbow trout within 11.5 

minutes, after which recovery to a higher 

level of activity occurred despite the continuing 

flow of chemical. Ortho-cresol induced total 

incapacitation in 17 of 20 fish within 15 

minutes, with 3 fish remaining partially active. 

Para-cresol totally incapacitated 20 fish 

within 8.5 minutes.

15'

10 

 cresol 

^ cresol 

 para cresol

..X'

K>

NUMBER OF FISH

20

Figure 2. Induction times and numbers of fish in total 

incapacitation from exposure to the isomers of cresol 

at 10 ppm.

Rainbow trout reacted violently upon contact 

with the isomers, as did all species tested in 

the static bioassays. Random lunging and 

thrashing occurred among the rainbows. 

Surfacing appeared during the throes preceding stupefaction but was not sustained. As the 

fish lost equilibruim, they became darker, 

the gular area reddened in some, and their 

bellies bloated as swimming activity 

diminished. Most fish settled to the bottom 

where blanched gills and several fin-base 

hemorrhages were noted. These responses 

were evoked quite uniformly by all three 

isomers.

The results of the flowing trials and static 

bioassays make it apparent that para-cresol 

is the prominent isomer for the incapacitation 

of rainbow, brown, and brook trouts. Thus it 

was decided to evaluate para-cresol in 

conjunction with certain compounds which are 

known to immobilize trout (Muench, 1958; 

Bove', 1962; Thienpoint and Niemegeers, 1965).

The concentrations of MS-222, quinaldine, 

and McNeil-JR-7464 which induce total 

incapacitation in 10 fish within specified times 

are presented in table 3. Flows and depths 

differed between these trials and those involving the isomers of cresol shown in figure 2. 

Therefore, the induction times of the two 

trials are not comparable. At least 7 minutes 

were required to attain the maximum concentration of a chemical in the test chamber, 

using malachite green as a tracer. Therefore, 

in some tests the fish never were exposed to 

the desired concentration, and in others they 

received the full dosage for only a portion of 

the time stated in the table. The calculated, 

ultimate concentrations achieved the desired 

effect in the times listed through buildup 

toward or to maximum level, as would be the 

case in a practical, stream application.

TABLE 3. Concentration of four compounds necessary to induce total 

incapacitation in 9-inch rainbow trout within specified times

Time

(minutes)

Number

fish para-cresol

40.0

3oncentration (pj

McNeil-JR-7464

3.0

2.5

2.3

2.2

1.8

1.7

m) of 

Quinaldine

37.5

35.0

25.0

20.0

15.0

MS-222

110.0

95.0

80.0

65.0

56.3

Concentrations of para-cresol below 14.0 

ppm required more than 10 minutes to induce 

total incapacitation; concentrations of 35 and 

40 ppm did not improve on the induction time 

at 30 ppm (table 3). Repellency was strong at 

all concentrations. The incidence of surfacing 

was about 50 percent during partial incapacitation, but transitory with each fish. They 

settled to the bottom at the onset of total 

incapacitation.
8 Investigations in Fish Control 34: Bureau of Sport Fisheries and Wildlife

The symptoms of stress, observed in the 

flowing trials of the isomers of cresol, were 

manifested again in these trials. Nine of 70 

rainbow trout exposed to para-cresol developed 

hemorrhages at the base of pectoral and/or 

pelvic fins. Eight of 40 fish died as a result 

of the trials above 25 ppm. Recovery from 

incapacitation required 5.5 to 26.5 minutes, 

the longer times being associated with higher 

concentrations.

MS-222 was less efficacious than paracresol and did not induce total incapacitation 

within 10 minutes at levels below 56.3 ppm 

(table 3). However, higher concentration were 

efficacious, although there was no improvement in reaction time at concentrations 

greater than 110 ppm. Neither repellency nor 

surfacing was observed, and no external signs 

of stress were visible. The time for recovery 

ranged between 4 and 10 minutes, and there 

were no deaths among 60 fish.

Quinaldine was effective in causing total 

incapacitation within 10 minutes at levels 

from 15.0to 37.5 ppm (table 3). Concentrations 

below 15.0 ppm were not efficacious within 10 

minutes; doses above 37.5 ppm did not improve 

significantly on reaction time. Moderate 

repellency was noted, and the rainbow trout 

darted about, shaking their heads and bodies. 

Only incidental surfacing occurred prior to 

total incapacitation. The only unusual manifestation in the stupefied fish was gyrating of 

the eyes. As recovery commenced, tremoring 

of the bodies was general. No deaths were 

recorded among 80 fish, and recovery required 

5 to 14 minutes.

McNeil-JR-7464 was most effective between 

1.7 and 3.0 ppm (table 3). There was no repellency and surfacing was only incidental before 

total incapacitation. Most of the fish assumed 

an inverted position while stupefied at the 

bottom. No signs of stress were evident at 

any stage of testing. The time for recovery 

ranged from 12 to 22 minutes, and no losses 

resulted among 50 fish.

DISCUSSION

Of the compounds tested, para-cresol fared 

poorly according to the criteria established 

for testing potential collecting compounds. It

caused violent repellency before rapid 

incapacitation and deaths of more than 10 

percent. The active surfacing induced by 

para-cresol is not conducive to collecting fish 

as would a passive surfacing. Cresol-induced 

surfacing is a constantly shifting phenomenon 

which appears to be a vague effort to escape 

the irritating solution during the paroxysms 

which precede stupefaction.

There was considerable evidence of physiological disturbance from contact with the 

cresols at effective concentrations. Violent 

lunging and gulping was followed by bloating 

of the abdomen in most cases. Some fish 

reddened in the gular area, and others 

developed hemorrhages at the bases of the 

pectoral and/or pelvic fins. When the fish 

settled to the bottom, opercula and mouths 

were agape, and the gills were considerably 

blanched. They retained this appearance until 

death. These symptoms are similar to those 

described by Jones (1964) for phenol and 

cresols against perch.

As for the physiological damage which 

occurs, a Polish investigator has provided 

detailed accounts of phenol-induced changes 

in the blood and tissues of the bream Abramis 

brama (L) (Waluga, 1966a, 1966b). Among his 

findings were quantitative changes in the blood, 

circulatory impairment, hemorrhages, cell 

necrosis leading to disintegration, and damage 

to the central nervous system; in summary, a 

general poisoning of the fish, damaging systems and organs essential to life.

The cresols are alkyl derivatives of phenol, 

and their systemic actions are so identical as 

not to warrant separation (Goodman and 

Oilman, 1965). It is apparent that the search 

for fish-collecting agents shoul be directed 

toward substances less harsh than the cresols.

MS-222 met the test criteria satisfactorily, 

although it did not induce surfacing. The 

relatively high concentrations required would 

make practical employment of this expensive 

drug unfeasible.

Quinaldine caused moderate irritation and 

repellency and no significant surfacing. This 

compound probably would be satisfactory as a 

collecting aid in small swiftly flowing streams,
Robert M. Rowland: Possible Fish-Collecting Aids

where current action would wash the immobilized fish into a blocking seine. However, 

further data on the physiological effects of 

this compound are necessary.

McNeil-JR-7464 produced most of the 

desired effects on fish at very low concentrations. It should be effective for collecting in 

swift waters, and possibly in slower sections 

as well. The long recovery period and the 

inverted position of stupefied trout, exposing 

their light undersides, should enhance collection in nonturbid slow waters. This compound 

lacks only surfacing action to have excellent 

potential as a collecting tool. Its combination 

with a compound which induces only active 

surfacing might yield a valuable collecting 

tool.

CONCLUSIONS

1. Para-cresol is the most active of the three 

isomers of cresol in inducing total 

incapacitation of rainbow, brown, and 

brook trout.

2. The cresols are too harsh physiologically 

to warrant further development as fishcollecting agents.

3. MS-222 is not well suited as a fishcollecting tool; it does not induce surfacing, and a high concentration is necessary 

for total incapacitation

4. Quinaldine may have some usefulness for 

collecting fish in small streams with rapid 

flow. Its physiological effects need elucidation.

5. McNeil-JR-7464 does not induce surfacing 

but is effective at low concentrations and 

appears to have desirable characteristics 

as a fish-collecting agent. It merits 

further investigation.

REFERENCES

Blanchard, J. H.

1966. Preliminary report. The use of tranquilizers as a possible sampling tool. Proceedings 

of the Nineteenth Annual Conference of the 

Southeastern Association of Game and Fish 

Commissioners, October 10-13, 1965, Tulsa, 

Oklahoma, p. 394-396.

Bove, Frank J.

1962. MS-222 Sandoz the anaesthetic of choice 

for fish and other coldblooded organisms. 

Sandoz News, No. 3. 12 p.

Burrows, Roger E.

1949. Prophylactic treatment for control of fungus 

(Saprolegnia parasitica) on salmon eggs. 

Progressive Fish-Culturist, vol. 11, no. 2, 

p. 97-103.

Embody, Daniel R.

1940. A method of estimating the number of fish 

in a given section of stream. Transactions of 

the American Fisheries Society, vol. 69, 1939, 

p. 231-236.

Goodman, Louis S., and Alfred Oilman.

1965. The pharmacological basis of therapeutics. 

Third edition. MacMillan Co., New York. 

1,785 p.

Jones, J. R. Erichsen.

1964. Fish and river pollution. Butterworth & 

Co., London, England. 203 p.

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

1964. Laboratories and methods for screening 

fish-control chemicals. U.S. Bureau of Sport 

Fisheries and Wildlife, Investigations in Fish 

Control No. 1 (Bureau Circular 185). 15 p.

Litchfield, J. T., Jr., and F. Wilcoxon.

1949. A simplified method of evaluating doseeffect experiments. Journal of Pharmacology 

and Experimental Therapeutics, vol. 96, no. 2, 

p. 99-113.

Loeb, Howard A.

1962. Effect of lysergic acid diethylamide (LSD25) on the surfacing behavior of laboratory 

carp. New York Fish and Game Journal, vol. 

9, no. 2, p. 127-132.

Louder, Darrell E., and Edward G. McCoy.

1962. Preliminary investigations of the use of 

aqualin for collecting fishes. Proceedings of 

the Sixteenth Annual Conference of the Southeastern Association of Game and Fish Commissioners, October 14-17, 1962, Charleston, 

South Carolina, p. 240-242.

Muench, Bruce.

1958. Quinaldine, a new anesthetic for fish. 

Progressive Fish-Culturist, vol. 20, no. 1, 

p. 42-44.

Penfold, Vincent F.

1965. An anesthetic fish collecting chemical. 

Vancouver Public Aquarium, Vancouver, 

British Columbia, Canada. 1 p.
10 Investigations in Fish Control 34 : Bureau of Sport Fisheries and Wildlife

Tate, Bill, Tom Moen, and B. I. Severson.

1965. The use of rotenone for recovery of live 

fish. Progressive Fish-Culturist, vol. 27, no. 

3, p. 158-160.

Thienpoint, D., and C. J. E. Niemegeers.

1963. R7464 - a new potent anaesthetic in fish. 

International Zoo Yearbook, 5, p. 202-205.

Waluga, Danuta.

I966a. Phenol effects on the anatomicohistopathological changes in bream Abramis 

brama (L.). (Zmiany anatomo-histologiczne

u leszcza pod wplywem fenolu). Acta Hydrobiologica, Krakow, vol. 8, no. 1, p. 55-78.

1966b. Phenol induced changes in the peripheral 

blood of the breams Abramis brama (L.). 

(Zmiany we krivi obwodowej leszczy pod 

wplywem fenolu). Acta Hydrobiologica, 

Krakow, vol. 8, no. 2, p. 87-95.

Wilkins, L, Price.

1955. Observations on the field use of cresol as 

a stream survey method. Progressive FishCulturist, vol. 17, no. 2, p. 85-87.

* U. S. GOVERNMENT PRINTING OFFICE : 1969 O - 369-964
(Reports 18 through 21 are in one cover.)

18. Toxicity of 22 Therapeutic Compounds to Six Fishes, by Wayne A. Willford. 1967. 10 p.

19. Toxicity of Bayer 73 to Fish, by Leif L. Marking and James W. Hogan. 1967. 13 p.

20. Toxicity of Dimethyl Sulfoxide (DMSO) to Fish, by Wayne A. Willford. 1967. 8 p.

21. Labor-Saving Devices for Bioassy Laboratories, by Robert J. Hesselberg and Ralph M. 

Burress. 1967. 8 p.

(Reports 22 through 24 are in one cover.)

22. Efficacy of Quinaldine as an Anesthetic for Seven Species of Fish, by Richard A. Schoettger 

and Arnold M. Julin. 1969 . 10 p.

23. Toxicity of Quinaldine to Selected Fishes, by Leif L. Marking. 1969 . 10 p.

24. Quinaldine as an Anesthetic for Brook Trout, Lake Trout, and Atlantic Salmon, by 

David O. Locke. 1969. 5 p.

(Reports 25 through 28 are in one cover.)

25. Field Trials of Antimycin as a Selective Toxicant in Channel Catfish Ponds, by Ralph M. 

Burress and Charles W. Luhning. 1969. 12 p.

26. Laboratory Studies on Antimycin A as a Fish Toxicant, by Bernard L. Berger, 

Robert E. Lennon, and James W. Hogan. 1969. 19 p.

27. Field Trials of Antimycin A as a Fish Toxicant, by Philip A. Gilderhus and Robert E. 

Lennon. 1969. 21 p.

28. Use of Antimycin for Selective Thinning of Sunfish Populations in Ponds, by Ralph M, 

Burress and Charles W. Luhning. 1969. 10 p.

(Reports 29 through 31 are in one cover.)

29. Efficacy of Methylpentynol as an Anesthetic on Four Salmonids, by Robert M. Howland 

and Richard A. Schoettger. 1969. lip.

30. Toxicity of Methylpentynol to Selected Fishes, by Leif L. Marking. 1969. 7 p.

31. Annotated Bibliography on Methylpentynol, by Gerald E. Svendsen. 1969. 7 p.
As the Nation's principal conservation agency, the Department of 

the Interior has basic responsibilities for water, fish, wildlife, 

mineral, land, park, and recreational resources. Indian and Territorial affairs are other major concerns of this department of 

natural resources.

The Department works to assure the wisest choice in managing 

all our resources so that each shall make its full contribution to a 

better United States now and in the future.

UNITED STATES 

DEPARTMENT OF THE INTERIOR

FISH AND WILDLIFE SERVICE

BUREAU OF SPORT FISHERIES AND WILDLIFE 

WASHINGTON, D.C. 20240