INVESTIGATIONS IN FISH CONTROL

18. Toxicity of 22 Therapeutic Compounds 

to Six Fishes

19. Toxicity of Bayer 73 to Fish

20. Toxicity of Dimethyl Sulfoxide (DMSO) to Fish

21. Labor-Saving Devices for Bioassay Laboratories

United States Department of the Interior

Fish and Wildlife Service 

A, ^ 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. (Bureau Circular 185.) 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. (Bureau 

Circular 186.) 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. (Resource Publication 7.) 1966. 16 p.

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

Robert G. Needham. (Resource Publication 8.) 1966. 16p.

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

Toxaphene, by Donald C. Warnick. (Resource Publication 9.) 1966. 9 p.

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

Ahmed Mahdi. (Resource Publication 10.) 1966. 10 p.

7. Treatment of East Bay, Alger County, Michigan, with Toxaphene for Control of Sea Lampreys, by William E. Gaylord and Bernard R. Smith. (Resource Publication 11.) 1966. 7p.

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

Alaska, by William R. Meehan and William L. Sheridan. (Resource Publication 12.)

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. (Resource Publication 13.) 1966. 12p.,

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

(Resource Publication 14.) 1966. lOp.

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

Schoettger and Arnold M. Julin. (Resource Publication 15.) 1966. lip.

(Reports 12 through 17 are in one cover.)

12. Toxicity of MS-222 to Selected Fishes, by Leif L. Marking. (Resource Publication 18.)

1967. lOp.

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

Arnold M. Julin. (Resource Publication 19.) 1967. 15p.

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

Schoettger. (Resource Publication 20.) 1967. lOp.

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

Richard A. Schoettger. (Resource Publication 21.) 1967. lip.

16. Annotated Bibliography on MS-222, by Richard A. Schoettger. (Resource Publication 22.) 

1967. 15p.

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. (Resource Publication 33.) 1967. 14p.

Continued on inside back cover 
INVESTIGATIONS IN FISH CONTROL

18. Toxicity of 22 Therapeutic Compounds to Six Fishes 

By Wayne A. Willford (Resource Publication 35, p. 1-10)

19. Toxicity of Bayer 73 to Fish

By Leif L. Marking and James W. Hogan 

(Resource Publication 36, p. 1-13)

20. Toxicity of Dimethyl Sulfoxide (DMSO) to Fish 

By Wayne A. Willford (Resource Publication 37, p. 1-8)

21. Labor-Saving Devices for Bioassay Laboratories 

By Robert J. Hesselberg and Ralph M. Burr ess 

(Resource Publication 38, p. l-8>

ERRATA 

- INVESTIGATIONS IN FISH

Col. Line item

l* ll 2 44 0.081 instead of 0.81 

21 4 2 11 3-1/2 " » 1-1/2

United Stales Department of the Interior, Slewart L. Udall, Secretary

Stanley A. Cain, Assistant Secretary for Fish and Wildlife and Parks

Fish and Wildlife Service, Clarence F. Pautzke, Commissioner

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

Washington, B.C. April 1967

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

Washington, D.C., 20402 - Price 30 cents
INVEST/CATIONS IN FISH CONTROL

18. Toxicity of 22 Therapeutic Compounds to Six Fishes

By Wayne A. Willford, Chemist 

Fish Control Laboratory 

Bureau of Sport Fisheries and Wildlife 

La Crosse, Wisconsin

United States Department of the Interior, Stewart L. Udall, Secretary

Stanley A. Cain, Assistant Secretary for Fish and Wildlife and Parks

Fish and Wildlife Service, Clarence F. Pautzke, Commissioner

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

Resource Publication 35 Washington, D.C. December 1966
CONTENTS

Page

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

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

Results. .............................. 6

Discussion ............................ 6

Conclusions and summary .................. 9

References ............................ 9
TOXICITY OF 22 THERAPEUTIC COMPOUNDS 

TO SIX FISHES

By Wayne A. Willford, Chemist 

Fish Control Laboratory, La Crosse, Wis.

ABSTRACT. Of 22 therapeutic chemicals (18 parasiticides and 4 oral 

bacteriostats) tested by bioassays, 16 were toxic to fish and 6 were not. 

Tests were in 24- and 48-hour static bioassays on rainbow, brown, brook, 

and lake trout and bluegills at 12° C. and channel catfish at 17° C. The 

16 toxic chemicals, in descending order, were malachite green, Trolene, 

CoRal, Tiguvon, Roccal, P.M. A., Acriflavine, amopyroquin dihydrochloride, 

merthiolate, methylene blue, Neguvon, Ruelene, TV-1096, nickel sulfate, 

formalin, and quinacrine hydrochloride; the 6 that did not appear to be 

toxic were erythromycin thiocyanate, quinine hydrochloride, Flagyl, 

sulfamerazine, sulfamethazine, and sulfisoxazole.

An objective of the Fish Control Laboratories is to develop chemical tools to prevent 

and control fish diseases. Although efficacious 

concentrations of many drugs have been determined, a thorough examination of their 

toxicity has not been reported. Prior to clearance of drugs, the Food and Drug Administration requires data on their toxicity. The purpose of this study was to define the toxicity of 

22 therapeutic chemicals to six species of 

fish before further research is undertaken on 

their efficacy and residues.

Eighteen parasiticides of known or possible 

value as external treatments for fish were selected for investigation upon recommendations 

by other investigators. Four oral bacteriostats 

were tested to determine whether any toxicity 

to fish would result through leaching, or excretion, of the compounds into water.

MATERIALS AND METHODS

Six species of fish were obtained from various fish hatcheries (table 1). All were quarantined for 10 days, and those judged acceptable for bioassays were acclimated to 

conditions of the tests.

The static bioassays were made in facilities 

described by Lennon and Walker (1964). We 

used 5-gallon glass jars which contained 15 

liters of reconstituted, deionized water at 

total hardness of 42 p.p.m., and a maximum 

of 1 gram of fish per liter of water. Each test 

included 10 concentrations of a chemical. Ten 

fish were exposed to each concentration, and 

20 fish served as controls.

The 22 therapeutic compounds were tested 

at 12 °C. against five species of fish at La 

Crosse, Wis. (table 2). Tests against channel 

catfish at 17° were made at the Southeastern 

Fish Control Laboratory, Warm Springs, Ga.

A concentrated stock solution of each compound, using acetone or deionized water or 

both as solvents, was usually prepared for addition to the test vessels immediately before 

each test. When solubility of the compound 

prevented preparation of concentrated stocks, 

the compound was added directly and allowed 

to dissolve in the test vessel.

Observations on survival and mortality 

were recorded at 24 and 48 hours. The data 

were then analyzed by plotting concentration 

against mortality on logarithmic normal
Investigations in Fish Control 18: Bureau of Sport Fisheries and Wildlife

TABLE 1. Fishes used in toxicity trials

Species

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

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

Brook trout, Salvelinus f ontinallis. . . .

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

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

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

Channel catfish, Ictalurus punctatus. . .

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

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

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

Lot

159

159

177

177

161

161

78

78

rjQ

W-70

W-74

115

131

131

Average 

length 

( inches)

1.5

1.8

1.7

1.9

1.5

1.6

4.0

4.0

4.1

2.1

2.2

1.6

1.4

1.7

Average 

weight 

(grains)

0.'5

0.9

0.8

1.2

0.4

0.6

2.5

2.8

3.2

1.2

1.5

0.8

0.7

1.1

Grading 

date

1-21-65

2-15-65

3-16-65

4- 1-65

1-21-65

2-15-65

8-L4-64

8-28-64

10- 7-64

7-21-65 '

8- 4-65

11- 5-64

11-17-64

12- 1-64,,

Source

National Fish Hatchery,

National Fish Hatchery,

Lake Mils, Wis.

State Fish Hatchery,

St. Croix Falls, Wis.

National Fish Hatchery,

National Fish Hatchery,

Marion, Ala.

National Fish Hatchery,

Lake Mills, Wis.

TABLE 2. Common names and active ingredients of compounds tested

Common name

«

Quinacrine hydrochloride

Roocal. ..............................

TV- 1096 (Parke, Davis & Company) . . . . .

Grade or 

formulation

80-percent soluble

50-percent concentrate. .......

technical. ....................

Active ingredient

3,6-diamino-lO-methyl acridinium chloride and 3,6-diaminoacridine 

4-(7-chloro-4-quinolylamino) -«-l-pyrrolidyl-£-cresol dihydrochloride

0,0-diethyl 0-3-chloro-4-methyl-2-ojro-2H-l-benzopyran-7-yl-phosphorothioate 

erytoromycin thiocyanate

1- ( 2-hydroxyethyl) -2-methyl-5-nitroimidazole 

37-percent formaldehyde gas in water

bis-(p-dimethylaminophenyl) phenylmethane treated with HCL 

sodium ethylmereurithiosalicylate

3,7-bis(dimethylamino) phenazathionium chloride 

0, 0-dimethyl 2,2,2-trichloro-l-hydroxyethyl phospbonate

NiS0 4 . 6H,0 

pyridylmer curio acetate

3-chloro-7-methoxy-9- (l-methyl-4-diethylaminobutylamino) acridine diliydrochloride 

quinine hydrochloride 

aliyl dimethylbenzylammonium chlorides 

4-tert-butyl-2-chlorophenyl methyl methylphosphoramidate

y/1 -(4-methyl-2-pyrimdyl) sulfanilamide 

y/1 .(4,6-dimethyl-2-pyrijnidinyl) sulfanilamide

y^1 -(3,4-dimethyl-5-isoxazolyl) sulfanilamide 

0,0-dimethyl 0-[4-(methylthio)-m-tolyl] phosphorothioate

0, 0-dimethyl 0-2,4, 5-trichlorophenyl phosphorothioate 

Lg-threo-2- ( 5-nitro-2-furyl) -5-( p- nitrophenyl) -2-oxazoline-4-methanol

(probability) graph paper to define the concentration that produced 50-percent mortality 

(LCso) as described by Litchfield and Wilcoxon (1949). Variance and the 95-percent 

confidence interval (C.I.) were also determined.

Most of the compounds tested were technical or U.S.P. materials, and the rest were 

formulated materials. To eliminate confusion, 

all results are reported in terms of p.p.m. of

total material (formulated or technical) instead of active ingredient.

RESULTS

Of the 22 compounds, 16 were toxic to the 

six species of fish, and the LC so values were 

determined (tables 3 to 8).

The most toxic compound, malachite green, 

is relatively uniform in toxicity to the six
Wayne A. Willford: Toxicity of 22 Therapeutic Compounds to Six Fishes

TABLE 3. Toxicity of 16 compounds to rainbow trout at 12° C. TABLE 6.--Toxicity of 16 compounds to lake trout at 12° C.

Compound

TV- 1096.............. '......

At 24 hours

LC50 

(p.p.m.)

30.1 

47.0

2.60 

207

0.50 

60.5

25.0 

32.5

320 

5.00

3.24

35.0 

5.30

1.17 

24.2

95-percent

C.I.

26.2-34.6 

43.5-50.8

2.28-2.96 

182-236

0.36-0.69 

53.5-68.4

20.5-30.5 

29.3-36.1

302-339 

4.35-5.75

2.92-3.60

31.5-38.8 

4.82-5.83

0.89-1.53 

22.8-25.7

At 48 hours

LC50 

(p.p.m.)

19.9

35.3

0.55 

168

0.39 

21.2

16.0 

12.2

160 

3.75

172 

2.57

32.0 

4.35

0.74 

16.1

95-percent 

C.I.

17.0-23.3 

33.3-37.4

0.51-0.59 

154-183

0.33-0.46 

18.6-24.2

13.8-18.7 

10.6-14.0

150-171 

3.02-4.65

159-186 

2.16-3.06

30.5-33.6 

3.62-5.22

0.64-0.86 

14.9-17.4

Compound

crif ine

P M A

At 24 hours

LC 50 

(p. p.m.)

37.5 

15.5

6.80 

220

0.57 

13.0

35.0 

41.0

170 

12.5

28.0 

2.70

27.0 

6.50

0.73 

32.0

95 -per cent 

C.I.

34.7-40.5 

12.1-19.8

4.00-11.56 

200-242

0.49-0.66 

9.6-17.6

29.4-41.6 

38.7-46.5

139-209 

11.8-13.2

18.8-42.0 

2.41-3.02

25.0-29.2 

6.08-6.96

0.62-0.86 

28.6-35.8

At 48 hours

LC 50 

(p.p.m.)

28.0 

U.O

4.00 

167

0.40 

2.13

34.0 

9.00

75.0 

7.60

21.0 

1.95

27.0 

5.30

0.62 

16.5

95 -per cent 

C.I.

24.4-32.2 

10.8-18.1

1.25-12.80

160-174

0.33-0.49 

1.06-4.26

29.3-39.4 

7.20-11.25

55.6-101.2 

6.33-9.12

12.4-35.7 

1.68-2.26

23.9-30.5 

4.91-5.72

0.53-0.72 

13.2-20.6

TABLE 4.--Toxicity of 16 compounds to brown trout at 12° C. TABLE 7.--Toxicity of 16 compounds to channel catfish at 17° C.

Compound

At 24 hours

LC50 

(p.p.m.)

40.0 

42.0

0.92 

325

0.45 

110

54.0 

54.0

400 

9.30

390 

2.95

2b.2

4.^0

0.53

95 -percent

C.I.

36.4-44.0 

37.5-47.0

0.84-1.00 

3CK-348

0.42-0.49 

75-160

46.2-63.2 

48.2-60.5

345-464 

8.30-10.42

361-421 

2.46-3.54

24.7-27.8 

4.09-4.95

0.38-0.74

At 48 hours

LC50 

(p.p.m.)

27.0 

36.0

0.73 

185

0.34 

54.0

32.8 

16.5

270 

6.22

230 

2.05

25.7 

3.62

0.39

95 -percent 

C.I.

25.0-29.2 

33.3-38.9

0.62-0.86 

165-208

0.30-0.38 

47.8-61.0

28.8-37.4 

11.8-23.1

241-302 

5.71-6.78

184-288 

1.74-2.42

24.2-27.2 

2.78-4.71

0.30-0.51

Compound

At 24 hours

LC50 

(p.p.m.)

43.5 

19.8

6.80 

137

0.21 

7.50

120 

80.0

368 

3.22

198 

1.28

39.5 

5.90

1.76 

27.0

95-percent

C.I.

39.9-47.4 

17.7-22.2

5.81-7.96 

129-145

0.17-0.27 

6.41-8.73

110-131 

72.7-88.0

334-405 

2.66-3.90

169-232 

1.16-1.41

37.6-41.5 

4.50-7.73

1.54-2.01 

24.8-29.4

At 48 hours

LC50 

(p.p.m.)

33.2

12.5

96.0

0.20 

5.65

104 

32.0

165

2.89

70.0 

1.12

34.8 

5.90

1.26

20.3

95-percent 

C.I.

31.0-35.5 

11.8-13.2

90.6-101.8

0.16-0.26 

4.79-6.67

93-116

24.8-41.3

129-211 

2.60-3.21

59.3-82.6 

1.03-1.22

32.5-37.2 

4.50-7.73

1.09-1.46 

19.3-21.3

TABLE 5. Toxicity of 16 compounds to brook trout at 12° C. TABLE 8. Toxicity of 16 compounds to bluegills at 12° C.

Compound

P M. A

TV- 1096....................

At 24 hours

LCso 

(p.p.m.)

48.0 

52.0

1.06 

196

0.30 

89.5

49.8 

34.0

15.5

4.13

36.8 

6.15

0.59 

29.3

95-percent 

C.I.

43.2-53.3 

44.8-60.3

0.87-1.29 

187-206

0.22-0.40 

85.2-94.0

41.2-60.3 

23.4-49.3

12.9-18.6

3.79-4.50

34.4-39.4 

5.21-7.26

0.44-0.78 

26.4-32.5

At 48 hours

LCso 

(p.p.m.)

1A.8 

40.0

0.80 

157

0.26 

74.5

22.9 

16.8

242 

10.7

230 

3.40

35.0 

5.50

0.39 

19.0

95 -percent 

C.I.

H. 0-15. 7 

38.1-42.0

0.70-0.91 

143-173

0.22-0.31 

71.0-78.2

17.2-30.5 

14.1-20.0

224-261 

9.8-11.7

177-299 

3.09-3.74

31.5-38.8 

5.1A-5.88

0.26-0.59 

16.8-21.5

Compound

TV- 1096....................

At 24 hours

LC50 

(p.p.m.)

18.0 

33.0

10.5 

185

0.26 

110

51.0 

78.0

20.0

120 

2.10

36.0 

15.7

2.50 

37.0

95-percent

C.I.

16.8-19.3 

23.6-42.2

8.1-13.6 

156-220

0.22-0.31 

87-139

40.2-64.8 

64.5-94.4

18.0-22.2

73-198 

1.94-2.27

34.3-37.8 

13.2-18.7

2.25-2.78 

33.3-41.1

At 48 hours

LC50 

(p.p.m.)

13.5 

18.5

8.00 

140

0.11 

64.5

33.0 

71.0

495 

16.0

79.0 

1.68

35.0 

8.90

1.00 

28.2

95 -per cent 

C.I.

12.6-U.4 

16.7-20.5

6.11-10.48 

127-154

0.09-O.U 

57.6-72.2

26.2-41.6 

55.9-90.2

450-544 

13.4-19.0

54.1-115.3 

1.56-1.81

33.0-37.1 

7.67-10.32

0.67-1.50 

25.9-30.7
Investigations in Fish Control 18: Bureau of Sport Fisheries and Wildlife

species, and LC 50 values range from 0.11 to 

0.40 p.p.m. at 48 hours. Clemens and Sneed 

(1958a) reported its LC « to channel catfish 

as 0.14 p.p.m. in 24 and 48 hours at 25°C. 

Our results show the LC ^ values to be 0.21 

and 0.20 p.p.m. in 24 and 48 hours respectively at 17°. This variation between results 

may be due to differences in test temperatures.

Following malachite green in decreasing 

order of toxicity are Trolene, CoRal, and 

Tiguvon, all of which have tjhe basic structure 

of phosphorothioate. In the same general 

range of toxicity are Roccal and P.M.A., with 

Roccal the more toxic of the two. Roccal, like 

malachite green, exhibits relatively uniform 

toxicity, and LC» values range from 1.12 to 

3.40 p.p.m. at 48 hours for all species.

P.M. A. exhibits a much wider range of toxicity with LC W values of 2.9 to 16.0 p.p.m. at 

48 hours. Clemens and Sneed (1958a) reported 

its LCgo to channel catfish as 3.8 p.p.m. in 

24 hours at 24 ° C. In a later publication, these 

authors (1958b) reported the LC 50 of P.M. A. 

to channel catfish as 2.96 and 2.81 p.p.m. in 

24 and 48 hours, respectively, at 16.5°. Both 

reports compare favorably with our LC^ 

values of 3.22 and 2.89 p.p.m. for 24 and 48 

hours at 17°.

Acriflavine, amopyroquin dihydrochloride, 

merthiolate, methylene blue, Neguvon, Ruelene, and TV-1096 fall into an intermediate 

toxicity range with LC^ of 10 to 100 p.p.m. 

Only Ruelene exhibits a uniform LC^, range 

of 25.7 to 35.0 p.p.m. for six species in 48 

hours. The other compounds of this group 

demonstrate a relatively wide range of toxicity to the different species.

TV-1096 is soluble only to approximately 

30 p.p.m. in water. Amounts above this level 

produce a saturated solution with a precipitate on the bottom of the test vessel. Brown 

trout fail to succumb to concentrations below 

30 p.p.m. and for this reason, LC» values 

could not be derived for the species. Also, 

amounts of TV-1096 in excess of a saturated 

solution are nontoxic to brown trout. In contrast, LCM values of TV-1096 for the other 

species range from 16.1 to 28.2 p.p.m. at 48 

hours.

Nickel sulfate, formalin, and quinacrine hydrochloride are the least toxic of the compounds analyzed. Formalin exhibits a fairly 

uniform LCso range of 96 to 185 p.p.m. at 48 

hours. The other two have a much wider 

range.

Clemens and Sneed (1958a) reported the 

LC 50 values of formalin on channel catfish to 

be 87 and 69 p.p.m. in 24 and 48 hours, respectively, at 25o whereas we found them to 

be 137 and 96 p.p.m. in 24 and 48 hours, respectively, at 17 °C. This variation in results 

seems to indicate that the toxicity of formalin 

may be increased by an increase in temperature. The observation is supported by our results which show that formalin is more toxic 

to channel catfish at 17° than it is to four 

species of trout and to bluegills at 12°.

Erythromycin thiocyanate and quinine hydrochloride were tested at an arbitrary level 

of 100 p.p.m. Their solubility would have permitted higher concentrations but preliminary 

tests indicated little toxicity. At 100 p.p.m., 

the substances were not toxic to the fish.

The poor solubility of Flagyl, sulfamerazine, sulfamethazine, and sulfisoxazole prevented the determination of LC^ values. 

Solutions were saturated before lethal levels 

could be reached. The arbitrary concentration 

of 100 p.p.m. was selected then for tests. This 

resulted in saturated solutions with excess 

chemical remaining on the bottom of the bioassay vessels. None of them was toxic to the 

six species of fish.

DISCUSSION

Malachite green has been in use for many 

years as a fungicidal dip for fish (Foster and 

Woodbury, 1936). Recently, Amlacher (1961) 

recommended it for prolonged treatment of 

fish in ponds to combat Ichthyophthirius, 

Chilodenella, and Costia. He applied 0.15 

p.p.m. and allowed it to dissipate in the water. 

Concentrations of 0.11 to 0.40 p.p.m. in our 

bioassays proved toxic within 48 hours to the 

six species of fish tested. Thus, there is a 

risk with concentrations over 0.11 p.p.m. in 

long-term treatments.
Wayne A. Willford: Toxicity of 22 Therapeutic Compounds to Six Fishes

Trolene, CoRal, and Tiguvon are under consideration as prolonged treatments for control 

of Ichthyophthirius. Tiguvon is the least toxic 

of these organophosphates to fish. This indicates that it may prove the most valuable of 

the group if minimum concentrations required 

for control of "Ich" are approximately the 

same for all three.

Roccal has been in use as a bactericide for 

many years (Fish, 1947). Putz (1964) reported 

its possible value in prolonged, indefinite 

treatments at 0.25 to 0.50 p.p.m. for Ichthyophthirius. In treatments such as this, the 

chemical attacks the free-living stages of 

"Ich". He did not say which formulation of the 

chemical he used, but 10-percent active is the 

formulation commonly used in hatcheries 

(Davis, 1956). We used 50-percent active, and 

upon converting from 10-percent active to 

50-percent active, the treatment levels could 

be reduced to 0.05 and 0.10 p.p.m. This permits a comparison between treatment levels 

and toxicity which shows a 10-fold difference 

in concentrations.

P.M. A. has been of considerable value in 

combating bacterial and protozoan diseases 

(Davis, 1956). Evidence of its greater toxicity 

to rainbow trout than other trouts has been 

reported over the years (Foster and Olson, 

1951; Rodgers et al., 1951; Wolf, 1951; 

Hammer, 1960). Allison (1957) reported variations in the toxicity of P.M. A. from lot to 

lot of chemical. We used only one lot of 

P.M. A. in this study, and the results support 

the earlier findings that it has greater toxicity 

to rainbow trout. For example, it was up to 

three times as toxic to rainbow trout as to 

brook trout. Channel catfish appear to be sensitive to the compound at 17 °C.

Snieszko and Friddle (1948) used merthiolate (sulfo) as a disinfectant for rainbow trout 

eggs. Van Duijn (1956) cautioned against use 

of merthiolate as a fish bath since the compound is a mercurial and is certain to be toxic 

to fish in contact with it for some period. We 

find an extreme variation in its toxicity to 

different species. This is especially true at 

24 hours where LC W values range from 7.5 

p.p.m. for channel catfish to 110.0 p.p.m. for 

brown trout. This variation diminishes somewhat at 48 hours, and lake trout become the 

most sensitive to the chemical. The LC 50 at 

48 hours for lake trout is 2.1 p.p.m. in contrast with 74.5 p.p.m. for brown trout. Variations in resistance such as this may make 

merthiolate extremely difficult to work with 

in routine treatments of several species.

Acriflavine, amopyroquin dihydrochloride, 

methylene blue, Neguvon, Ruelene, and TV1096 are under consideration as prolonged, 

indefinite treatments for control of Ichthyophthirius. Putz (1964) reported that 3 p.p.m. 

of acriflavine shows promise against the parasite. Our results indicate that bluegills are 

the most sensitive to the compound with LC 50 

values of 18.0 and 13.5 p.p.m. at 24 and 48 

hours respectively. Channel catfish are the 

most resistant with LC 50 values of 43.5 and 

33.2 p.p.m. at 24 and 48 hours.

Clemens and Sneed (1958a) reported the 

LC 50 values of acriflavine on channel catfish 

at 24 and 48 hours to be 11.5 and 6.8 p.p.m., 

respectively, at 20 ° C. Our finding, is that it 

is only about one-fourth as toxic as that. Possible causes for the discrepancy are many. 

Among them are differences in water quality 

and temperature, differences in the condition 

of fish, and purity of the compound used. In 

addition to this unexplained variation in the 

toxicity of acriflavine, another factor warrants serious consideration in its use. Van 

Duijn (1956) reported sterility in both egglaying and live-bearing aquarium fish. This 

is a temporary situation and normal fertility 

is restored after several months.

Amopyroquin dihydrochloride also shows 

promise as a prolonged, indefinite treatment 

at 0.05 to 0.10 p.p.m. for control of Ichthyophthirius (Putz, 1964). Our results show that 

its toxicity to all trout tested, with the exception of lake trout, is between 35 and 40 p.p.m. 

for 48 hours. Also, bluegills at 12 OC. and 

channel catfish at 17° are approximately as 

sensitive as lake trout. A treatment level of 

0.1 p.p.m. would include a safety margin in 

use of more than a hundredfold even against 

these three species.

Van Duijn (1956) recommended methylene 

blue as a satisfactory control for Ichthyophthirius in aquariums. He used 2 to 4 p.p.m.
8 Investigations in Fish Control 18: Bureau of Sport Fisheries and Wildlife

of it in a permanent bath at temperatures between 21 and 27° C. We found that rainbow 

trout are the most sensitive to the dye, and the 

LC M is 16 p.p.m. at 48 hours. The most resistant species is channel catfish with an 

LC 50 of 104 p.p.m. at 48 hours. The remaining species are intermediate in sensitivity 

with a 48-hour LC 50 range of 22.9 to 34.0 

p.p.m. Comparison of the use levels with toxicity levels indicates a good safety margin.

Neguvon and Ruelene are of approximately 

the same toxicity except in one very important 

respect. Neguvon has a marked difference between the 24-hour and 48-hour LC 5,,. The 

most striking example of this involves lake 

trout with LC 50 values of 41 p.p.m. at 24 

hours and 9 p.p.m. at 48 hours. Differences 

between the 24- and 48-hour LC 50 values by a 

factor of at least two are common except with 

bluegills. For some unknown reason the difference with bluegills is only 78 to 71 p.p.m.

Ruelene provides a contrast with Neguvon 

because it exhibits approximately the same 

toxicity at 24 and 48 hours. In the case of lake 

trout, the 24- and 48-hour LC ^ values are 

identical at 27 p.p.m. It is possible that Ruelene degrades very rapidly in the test vessel 

to a nontoxic level.

TV-1096 has toxicity comparable to that of 

Neguvon and R"uelene. Like Neguvon, it does 

not appear to degrade as rapidly as Ruelene.

Nickel sulfate is under consideration as a 

prolonged, indefinite treatment for control of 

Ichthyophthirius. Our results show that it is 

relatively low in toxicity when compared with 

the other compounds tested, but it has a fairly 

wide range of toxicity among the species 

tested. The LC 50 values at 48 hours range 

from 75 p.p.m. for lake trout to 495 p.p.m. for 

bluegills. Twenty-four-hour tests of 50 to 275 

p.p.m. on brook trout and 200 to 500 p.p.m. on 

bluegills did not cause death.

Allison (1957) reported use of formalin as a 

parasiticide in long-term treatments in ponds. 

He suggested 5 p.p.m. for Gyrodactylus and 

15 p.p.m. for Trichodina and Ichthyophthirius. 

Our results show that formalin is relatively 

and uniformly low in toxicity when compared

with the other compounds tested. It does appear to increase in toxicity as temperatures 

rise from 17° to 25° C. Even with this increase in toxicity, the compound retains a 

safety margin of at least sixfold at recommended use levels.

Van Duijn (1956) recommended use of quinacrine hydrochloride in treatment of stubborn 

cases of "Ich" in aquarium fish. The treatment consists of three applications of 1 p.p.m. 

at 48-hour intervals. This totals 3 p.p.m. if 

no degradation of the compound occurs. He 

also stated that this treatment should not be 

extended over long periods and that 8 to 10 

days should be sufficient.

Our results show that lake trout are approximately 10 times as sensitive to quinacrine hydrochloride as the other trout and 3 

or 4 times as sensitive as bluegills and channel catfish. The sensitivity is complicated by 

the fact that the toxicity to lake trout is quite 

erratic and some deaths occur over a wide 

range of concentrations. The 48-hour LC 0 of 

quinacrine hydrochloride for lake trout is 

approximately 10 p.p.m., the LC 50 is 21 

p.p.m., and the LC 100 is 110 p.p.m. Some fish 

succumb to the chemical quickly and at comparatively low concentrations whereas the 

rest survive for long periods. Further evidence of this lingering is shown by the slight 

difference between the 24-hour LC » of 28 

p.p.m. and the 48-hour LCso of 21 p.p.m. In 

contrast, there is a considerable difference 

between the 24- and 48-hour LC 50 values obtained for the other species. A possible explanation is that there is considerable variation in resistance among lake trout 

individuals.

Van Duijn (1956) recommended use of quinine hydrochloride for treatment of Ichthyophthirius in aquarium fish. The treatment 

consists in adding 1 p.p.m. on 3 successive 

days, a final treatment level of 3 p.p.m. He 

cautioned against use of the treatment for long 

periods because of possible fertility problems. 

Our results show that 100 p.p.m. of the chemical in water are not toxic within 48 hours to . 

the species tested.

Erythromycin thiocyanate has been used as 

a food additive for control of kidney disease in 

rainbow trout at 4.5 grams per 100 pounds of
Wayne A. Willford: Toxicity of 22 Therapeutic Compounds to Six Fishes

fish per day for 21 days (Piper, 1961). Warren 

(1963) reported that it is toxic to rainbow 

trout at 500 mg. per kg. Our results show that 

100 p.p.m. of the antibiotic in water is not 

toxic to the species tested within 48 hours.

Flagyl has been used in medicine as an 

antiprotozoal agent (Cutting, 1962). Putz 

(1964) reported experimental use of it at 1.5 

p.p.m. for control of Ichthyophthirius. Our 

results show that Flagyl is nontoxic at 100 

p.p.m. The finding is qualified somewhat since 

the compound is not immediately soluble at 

100 p.p.m. It dissolves slowly, however, and 

is completely in solution within 48 hours.

Snieszko and Bullock (1957) reported use of 

sulfamerazine, sulfamethazine, and sulfisoxazole as food additives in the treatment of 

furunculosis at 8 to 10 grams per 100 pounds 

of fish per day for 10 to 20 days. Van Duijn 

(1956) recommended use of the sodium salt of 

sulfamerazine at 1 part per thousand as an 

effective cure for worm cataract in aquarium 

fish. In our water, sulfamerazine, sulfamethazine, and sulfisoxazole are not soluble at 100 

p.p.m., and saturated solutions are not toxic 

to the six species within 48 hours.

All of our results were obtained with fish 

which were, to the best of our knowledge, 

healthy. They showed no signs of disease or 

physical injuries. The toxicity of these compounds to fish which are sick or in poor condition might be significantly different.

None of the compounds reported herein are 

cleared by the Food and Drug Administration 

and the Department of Agriculture for use on 

fish destined for human consumption. The 

data and discussion presented in this paper 

should not be construed as recommendations 

for use.

CONCLUSIONS AND SUMMARY

The toxicities of 22 therapeutic compounds 

to rainbow trout, brown trout, brook trout, 

lake trout, and bluegills at 12 °C. and channel 

catfish at 17° were determined in 24- and 48- 

hour static bioassays.

LC so values for malachite green, the most 

toxic compound tested, range from 0.1 to 0.4 

p.p.m. for all species tested. CoRal, P.M.A., 

Roccal, Tiguvon, and Trolene are less toxic 

than malachite green, but still rank relatively 

high in toxicity. Their LCg,, values range 

from approximately 0.5 to 10 p.p.m. for all 

species.

Acriflavine, amopyroquin dihydrochloride, 

merthiolate, methylene blue, Neguvon, Ruelene, and TV-1096 are intermediate in toxicity. The LC 50 values range from approximately 10 to 100 p.p.m. for all species. 

Merthiolate has wide variations in toxicity to 

various species.

Formalin, nickel sulfate, and quinacrine 

hydrochloride have relatively low toxicities. 

The LC M values are usually above 100 p.p.m. 

Quinacrine hydrochloride is substantially 

more toxic to lake trout than to the other 

species.

No tests of erythromycin thiocyanate and 

quinine hydrochloride were made at concentrations above 100 p.p.m. They are not toxic 

within 48 hours at this concentration. Saturated solutions of Flagyl, sulfamerazine, 

sulfamethazine, and sulfisoxazole are also not 

toxic within 48 hours.

REFERENCES

Allison, Leonard N.

1957. Variation in strength of pyridylmercuric acetate technical and its effect on rainbow trout. Progressive 

Fish-Culturist, vol. 19, no. 3, p. 108- 

111.

Allison, Ray.

1957. Some new results in the treatment 

of ponds to control some external 

parasites of fish. Progressive FishCulturist, vol. 19, no. 2, p. 58-63.

Amlacher, Erwin.

1961. Taschenbuch der Frischkrankheiten. VEB Gustav Fischer Verlag, 

Jena, Germany. 286 p.
10 Investigations in Fish Control 18: Bureau of Sport Fisheries and Wildlife

Clemens, Howard P., and Kermit E. Sneed.

1958a. The chemical control of some 

diseases and parasites of channel catfish. Progressive Fish-Culturist, vol. 

20, no. 1, p. 8-15.

1958b. Effect of temperature and physiological condition on tolerance of channel catfish to pyridylmercuric acetate 

(PMA). Progressive Fish-Culturist, 

vol. 20, no. 4, p. 147-150.

Cutting, Windsor C.

1962. Handbook of pharmacology. Second 

edition. Appleton-Century-Crofts, New 

York. 647 p.

Davis, H. S.

1956. Culture and diseases of game 

fishes. University of California Press. 

332 p.

Fish, Frederic F.

1947. A technique for controlling infectious disease in hatchery fish. Transactions of the American Fisheries Society, vol. 74 (1944), p. 209-222.

Foster, Fred J., and Lowell Woodbury.

1936. The use of malachite green as a 

fish fungicide and antiseptic. Progressive Fish-Culturist, no. 18, p. 7-9.

Foster, R. F., and P. A. Olson.

1951. An incident of high mortality 

among large rainbow trout after treatment with pyridylmercuric acetate. 

Progressive Fish-Culturist, vol. 13, 

no. 3, p. 129-130.

Hammer, Glen L.

1960. Toxicity of pyridylmercuric acetate to cutthroat trout. Progressive 

Fish-Culturist, vol. 22, no. 1, p. 14.

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

1964. Investigations in fish control. 

1. Laboratories and methods for 

screening fish-control chemicals. U.S. 

Bureau of Sport Fisheries and Wildlife, 

Circular 185. 15 p.

Litchfield, J. T., 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.

Piper, Robert G.

1961. Toxic effects of erythromycin 

thiocyanate on rainbow trout. Progressive Fish-Culturist, vol. 23, no. 3, p. 

134-135.

Putz, Robert E.

1964. Recent studies on Ichthyophthirius 

disease of fish. Presented at the Northeastern Fish and Wildlife Conference, 

Hartford, Conn., January 19-22. 

Mimeo. 14 p.

Rodgers, E. O., B. H. Hazen, S. B. Friddle, 

and S. F. Snieszko.

1951. The toxicity of pyridylmercuric 

acetate technical (PMA) to rainbow 

trout (Salmo gairdnerii). Progressive 

Fish-Culturist, vol. 13, no. 2, p. 71-73.

Snieszko, S. F., and G. L. Bullock.

1957. Treatment of sulfonamide-resistant furunculosis in trout and determination of drug sensitivity. U.S. Fish 

and Wildlife Service, Fishery Bulletin, 

No. 125, vol. 57, p. 555-564.

Snieszko, S. F., and S. B. Friddle.

1948. Disinfection of rainbow trout eggs 

with sulfomerthiolate. Progressive 

Fish-Culturist, vol. 10, no. 3, p. 143- 

149.

Van Duijn, C., Jr.

1956. Diseases of fishes. First edition. 

Water Life, Dorset House, London. 

174 p.

Warren, James W.

1963. Toxicity tests of erythromycin 

thiocyanate in rainbow trout. Progressive Fish-Culturist, vol. 25, no. 2, 

p. 88-92.

Wolf, Louis E.

1951. Comments: Letter to the editor of 

the Progressive Fish-Culturist. Progressive Fish-Culturist, vol. 13, no. 1, 

p. 42.
/INVESTIGATIONS IN FISH CONTROL

19. Toxicity of Bayer 73 to Fish

By Leif L. Marking and James W. Hogan, Chemists

Bureau of Sport Fisheries and Wildlife

Fish Control Laboratories

La Crosse, Wis., and Warm Springs, Ga.

United States Department of the Interior, Stewart L. Udall. Secretary

Stanley A. Cain, Assistant Secretary for Fish and Wildlife and Parks

Fish and Wildlife Service, Clarence F. Pautzke, Commissioner

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

Resource Publication 36 Washington, D.C. April 1967
CONTENTS

Page

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

Materials and methods .............................. 4

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

Preliminary screening ........................... 5

Delineative screening ............................ 5

General toxicity. ............................ 5

Comparative toxicity. ......................... 7

Short exposures............................. 7

Effects of temperature ........................ 9

Effects of water quality........................ 9

Effects of pH............................... 9

Discussion ...................................... 10

Conclusions. ..................................... n

References ...................................... 12
TOXICITY OF BAYER 73 TO FISH

Leif L. Marking and James W. Hogan, Chemists

/

Fish Control Laboratories 

La Crosse, Wis., and Warm Springs, Ga.

ABSTRACT. Bayer 73, a molluscicide sold commercially as Bayluscide, 

is highly toxic to 18 species of freshwater fish. Various temperatures and 

water qualities in static bioassays do not influence the toxicity greatly, but 

pH variations in chemically buffered solutions do. The biodegradability, efficacy, and relative safety of Bayer 73 in aquatic situations indicate possible 

usefulness as a general fish toxicant.

Nitrosalicylanilides display intense biological activity against a broad spectrum of organisms. Taborsky et al. (1959) and Taborsky 

and Starkey (1962 and 1963) reported antimicrobial activity of salicylanilides. They 

found various levels of activity of nitro- and 

halo-substituted salicylanilides against fungi 

and bacteria, and concluded that certain positions on the salicylanilide molecule are 

more active than others.

Taborsky and Starkey (1962) also reported 

antitumoral activity of substituted salicylanilides. Molnar and Baron (1964) and Vinson 

et al. (1961) stated that salicylanilides possess 

germicidal properties and are efficacious as 

hospital sanitizers.

The molluscicidal activity of Bayer 73, 2', 

5-dichloro-4f-nitrosalicylanilide, was first 

recognized by Gb'nnert and Schraufstatter 

(1958). Since then it has been tested thoroughly 

in the laboratory and field against snails which 

are intermediate hosts for schistosomiasis 

(Gonnert, 1961; Schraufstatter et al., 1961; 

Schraufstatter, 1962;Strufe and Gonnert, 1962; 

Foster, 1962; and Webbe, 1963). In addition, 

Gillet and Braux (1962) determined in laboratory tests with snails that Bayer 73 is 

ovicidal and cercaricidal, which enhances its 

effectiveness in breaking the cycle of schistosomes. Meyling et al. (1962) found that it 

killed 100 percent of the snail eggs at 1.0 

p.p.m. in 4 to 5 hours.

Bayer 73 is relatively nontoxic to mammals. 

Hecht and Gloxhuber (1962) reported that dogs 

tolerated 0.1 g./kg. orally. Duhm (1963) indicated that humans and animals are not harmed 

by drinking water containing molluscicidal 

concentrations of Bayer 73. Foster (1962) and 

Holz and Hwa (1963) reported survival of 

plants exposed to molluscicidal concentrations. Shiff and Garnett (1961) reported reduced planktonic life immediately after application of 1.0 p.p.m. of Bayer 73, but the effect 

was short lived, and 32 days later populations 

were back to normal.

Gonnert (1962) and Webbe (1963) reported 

fish kills with Bayer 73 at a concentration of 

0.3 p.p.m. Howell et al. (1964) found that 

Bayer 73 is highly toxic to sea lamprey larvae 

and rainbow trout. They further discovered 

that Bayer 73 synergized 3-trifluoromethyl-4- 

nitrophenol, and potentiated the compound as 

a selective lamprey larvicide.

In research for fish control chemicals, 

Walker et al. (1966) described the structureactivity relation of substituted salicylanilides 

to fish. Starkey and Howell (1966) showed that 

a number of substituted salicylanilides are 

more toxic to larval sea lampreys than rainbow trout. Marking et al. (in press) defined 

the toxicity of 3 f -chloro-3-nitrosalicylanilide 

to larval sea lamprey and many species of
Investigations in Fish Control 19: Bureau of Sport Fisheries and Wildlife

fish in the laboratory under standard test 

conditions and also in simulated stream environments. The compound is highly toxic to 

all species but most toxic to larval lamprey 

and several species of rough fish.

In view of the possible wide use of Bayer 73 

in acquatic environments for controlling schistosomiasis and the piscicidal activity previously reported, there is a need to define its 

toxicity to fish and evaluate its potential in 

controlling fish populations. Accordingly, 

Bayer 73 was bioassayed at the Fish Control 

Laboratories at La Crosse, Wis., and Warm 

Springs, Ga., to determine its effects on fish 

in various laboratory environments.

MATERIALS AND METHODS

The sample (control No. C910) was obtained 

from Chemagro Corporation, Kansas City, 

Mo. as a technical formulation containing 99 

percent 2-aminoethanol salt of 2 1 , 5-dichloro4-nitrosalicylanilide. Its structure is illustrated in figure 1. The material is a bright 

yellow, crystalline powder.

Test fish were obtained from National, 

State, and private fish hatcheries (table 1) 

and were acclimated to test conditions ac-

-N02 -NH2-(CH2)2-OH

OH

Figure 1. Structure of 2-aminoethanol salt of 

2', 5-dichloro-4'-nitrosalicylanilide.

cording to procedures described by Lennon 

and Walker (1964).

Preliminary screening was in 15 liters of 

bioassay medium at 12° C. at La Crosse and 

at 17° C. at Warm Springs. The fish were 

exposed to 0.1, 1.0, and 10.0 p.p.m. Bayer 73 

to determine the general range of toxicity and 

the rate of reaction at the higher concentrations.

Delineative tests, including variations in 

test media such as temperature, water quality, 

and pH were conducted using 10 fish per concentration and at least 9 concentrations per 

test, plus a control. Temperature variations 

were maintained mechanically, and water 

quality variations were obtained by adding selected quantities of reconsitution salts to deionized water (table 2).

TAELE 1. Sizes and sources of test fish

Species

Brook trout, Salvelinus fontinalis. ...........

Goldfish, Carassius aura t us. ..................

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

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

TVi

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

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

TVi

Tilapia, Tilapia mossambica. ..................

Lot

279 

295 

W116 

W96A 

W78 

W86 

W89 

367 

W113 

381 

W79 

393 

WU8 

W74A 

W87 

W154 

W75 

W119 

340 

W91 

W146 

WUO 

382 

W156

Average 

length 

( inches)

1.9 

1.8 

1.7 

1.6 

1.7 

2.0 

2.3 

1.5 

1.8 

2.0 

2.0 

1.9 

1.8 

3.0 

2.8 

2.2 

1.6 

1.6 

1.5 

1.6 

1.6 

1.6 

1.7 

1.5

Average 

weight 

(grams)

1.0 

1.0 

1.3 

1.3 

1.1 

1.9 

2.2 

0.8 

1.0 

1.2 

1.5 

1.3 

1.1 

2.8 

3.0 

1.1 

1.4 

1.1 

0.6 

0.9 

0.7 

0.8 

0.6 

0.8

Source

Nevin SFH, Wis. 

Osceola SFH, Wis. 

Tallassee PFH, Ala. 

Marion NFH, Ala. 

Marion NFH, Ala. 

Marion NFH, Ala. 

Marion NFH, Ala. 

Genoa NFH, Wis. 

Marion NFH, Ala. 

Lake Mills NFH, Wis. 

Marion NFH, Ala. 

Guttenberg NFH, Iowa 

Marion NFH, Ala. 

Marion NFH, Ala. 

Marion NFH, Ala. 

Marion NFH, Ala. 

Marion NFH, Ala. 

Marion NFH, Ala. 

Lake Mills, NFH, Wis. 

Marion NFH, Ala. 

Mammoth Springs NFH, Ark. 

Welaka NFH, Fla. 

Lake Mills NFH, Wis. 

Marion NFH, Ala.
L. L. Marking and J. W. Hogan: Toxicity of Bayer 73 to Fish 5

TABLE 2. Quality and composition of reconstituted water used at the Fish Control Laboratories

Classification 

of 

water

Soft..................

Medium1 ...............

Hard. .................

Salt added in mg. per liter

NaHC03

12 

48 

192

CaS04

7.5 

30.0 

120.0

MgS04

7.5 

30.0 

120.0

KC1

0.50 

1 2.00 

8.00

pH 

range

6.4-6.8 

7.2-7.6 

7.6-8.0

Concentration in 

p. p.m. CaCOs as total

hardness

10- 13 

40- 48 

160-180

alkalinity

10- 13 

30- 35 

110-120

1 Standard reconstituted water used in routine bioassays.

Different buffers to control the pH of test 

media were prepared from reagent grade 

chemicals. The normal and molar solutions 

and corresponding dilutions are listed in table 

3. The pH's of solutions were checked daily 

and adjusted as necessary to yield approximately the desired pH level.

The delineative data were analyzed according to the methods of Litchfield and Wilcoxon 

(1949) to determine LC 50 values, variation, 

slope functions, and 95-percent confidence 

intervals.

TABLE 3. Volumes of buffer reagents (ml.) necessary to 

yield desired pH levels in 15 liters of test solution

Reagent and strength

Potassium acid phthalate 

(KHC8 H404 )...................

Boric acid ( HsBOs) .............

...1.0 N

...0.1 M

...1.0 M

...1.0 M

Volume to yield 

pH of 

approximately 

5

10 

510

7

10 

30

10

20 

21

RESULTS

PRELIMINARY SCREENING

Initial tests with Bayer 73 indicated a high 

level of biological activity in fish. In a concentration of 0.1 p.p.m., all rainbow trout 

succumbed in 15 minutes, flathead catfish died 

within an hour, and yellow perch died within 

24 hours, but not all white suckers, black 

bullheads and bluegills died within the 96-hour 

bioassay. Exposure to a 1.0 p.p.m. concentration for 15 minutes caused deaths of all 

white suckers, flathead catfish, green sunfish,

and yellow perch. Carp, black bullheads, and 

bluegills were more resistant, but died during 

a 15-minute exposure to a concentration of 

10.0 p.p.m.

The response of fish to the chemical is noticeable immediately after exposure to the 

higher concentrations. They appear irritated, 

and become excited when exposed to sound or 

external movements. Their early reactions 

include rapid, irregular respiration. Swimming 

is aimless, and some fish skitter on the surface while losing equilibrium. Just before 

death, respiration and swimming actions 

gradually slow and become irregular, and 

finally are spastic. Often the opercula are 

extended on the dead fish.

DELINEATIVE SCREENING

General toxicity. Bayer 73 is toxic to the 

18 species of fish tested. The LC^ values, 

95-percent confidence intervals, and mean 

slope functions for routine tests are given in 

table 4 for the 24- to 96-hour exposure periods. Exposure beyond 24 hours produces little 

additional mortality. In fact, the figures indicate no difference in the 24- and 48-hour LCso 

values because the confidence intervals overlap for all species at the two exposure periods. 

The LC 50 values for brook trout, one lot of 

carp, and brown bullheads remained the same 

from 24- to 96-hour exposures.

The 95-percent confidence intervals of 24- 

to 96-hour LC 50 's overlap for all species except brown bullhead, redear sunfish, smallmouth bass, largemouth bass and tilapia. 

Intervals are narrow in most cases, indicating 

consistent toxic effects with little range between 

concentrations resulting in survival and death.
6 Investigations in Fish Control 19: Bureau of Sport Fisheries and Wildlife

TABLE 4.--Toxicity of Bayer 73 to selected species of fish

Species Temp. 

C.

LC50 (p.p.m.) and 95-percent confidence interval

24 hours 48 hours 96 hours

Mean slope 

function

Rainbow trout.................. 12°

Brook trout.................... 12°

Goldfish....................... 17°

Carp........................... 12°

Do........................... 17°

Do........................... 17°

Fathead minnow................. 17°

White sucker................... 12°

Bigmouth buffalo............... 17°

Black bullhead................. 12°

Brown bullhead................. 17°

Channel catfish................ 17°

Flathead catfish............... 17°

Green sunfish.................. 17°

Bluegill....................... 12°

Do........................... 17°

Redear sunfish................. 17°

Smallmouth bass................ 17°

Largemouth bass................ 17°

Yellow perch................... 12°

Tilapia........................ 17°

0.052 

0.049-0.056

0.061 

0.058-0.065

0.279 

0.243-0.321

0.143 

0.133-0.154

0.148 

0.142-0.159

0.245 

0.227-0.265

0.106 

0.097-0.116

0.084 

0.073-0.097

0.080 

0.059-0.108

0.104 

0.084-0.123

0.071 

0.065-0.077

0.084 

0.079-0.089

0.043 

0.040-0.046

0.158 

0.145-0.172

0.105 

0.095-0.116

0.082 

0.072-0.092

0.157 

0.148-0.167

0.089 

0.085-0.099

0.111 

0.099-0.124

0.082 

0.076-0.088

0.180 

0.159-0.203

0.052 

0.049-0.056

0.061 

O.C58-0.065

0.279 

0.243-0.321

0.139 

0.134-0.145

0.148 

0.142-0.159

0.235 

0.214-0.258

0.103 

0.096-0.111

0.081 

0.075-0.088

0.064 

0.056-0.073

0.096 

0.087-0.106

0.071 

0.066-0.077

0.084 

0.079-0.089

0.043 

0.040-0.046

0.115 

0.101-0.130

0.098 

0.085-0.112

0.082 

0.070-0.096

0.153 

0.142-0.165

0.089 

0.085-0.099

0.097 

0.087-0.109

0.081 

0.069-0.087

0.150 

0.133-0.170

0.050 

0.047-0.054

0.061 

0.058-0.065

0.230 

0.201-0.263

0.139 

0.134-0.145

0.148 

0.142-0.159

0.225 

0.208-0.243

0.102 

0.089-0.117

0.081 

0.076-0.086

0.088 

0.078-0.098

0.056 

0.049-0.064

0.082 

0.077-0.088

0.043 

0.040-0.046

0.100 

0.094-0.107

0.094 

0.083-0.107

0.068 

0.057-0.080

0.088 

0.086-0.090

0.060 

0.048-0.074

0.062 

0.050-0.076

0.081 

0.069-0.087

0.109 

0.090-0.132

1.133

1.070

1.147

1.053

1.060

1.106

1.106

1.084

1.245

1.156

1.107

1.063

1.060

1.097

1.131

1.158

1.069

1.223

1.157

1.077

1.157
L. L. Marking and J. W. Hogan: Toxicity of Bayer 73 to Fish 7

The slope functions were consistent in exposures of 24, 48, and 96 hours, and they were 

averaged to yield the figures given in table 4. 

The values, ranging from 1.060 to 1.245, are 

low, the regression is nearly vertical, and 

there is little difference between concentration permitting complete survival and that 

producing complete mortality. In contrast, the 

slope function of p,p'-DDT for goldfish is 6.02 

(Marking, 1966).

Comparative toxicity. Bayer 73 is highly 

toxic to all species, with 96-hour LC50 values 

ranging from 0.043 to 0.230 p.p.m. Flathead 

catfish, rainbow trout, brown bullheads, smallmouth bass, brook trout, and largemouth bass 

in that order are the most sensitive species to 

Bayer 73 (fig. 2). Goldfish are the most resistant, followed by carp. Intermediate in 

sensitivity are white suckers, yellow perch, 

channel catfish, black bullheads, redear sunfish, bluegills, green sunfish, tilapia, and fathead minnows. Bayer 73 does not appear specific to any undesirable species of fish but 

rather is generally toxic to game and rough 

fish.

Short exposures. Among the six species 

selected to determine effects of short-term . 

exposures to Bayer 73, rainbow trout were 

most sensitive and carp most resistant 

(table 5). Rainbow trout, brook trout, and 

yellow perch respond rather quickly; LC M 

values are only slightly greater at 1 hour than 

at 6 hours. The 95-percent confidence intervals overlap for each species in the 2-, 3-, 

and 6-hour exposures. Also, the values at 6 

hours and 24 hours for rainbow trout and 

brook trout do not differ significantly (tables 

5 and 6).

Carp, white suckers, and black bullheads 

respond somewhat more slowly at 1 hour, although their resistance is greater initially and 

the increments in LCso values from 1- to 96- 

hour exposures are small and fairly uniform.

Rainbow trout 

Brook trout 

Goldfish 

Carp

Fathead minnow 

White sucker 

Black bullhead 

Brown bullhead 

Channel catfish 

Flathead catfish 

Green sunfish 

Bluegill

Redear sunfish 

Smallmouth bass 

Largemouth bass 

Yellow perch 

Tilapia

0.1 

C oncentrat ion

0.2

in p.p.m.

0.3

Figure 2. 95-percent confidence intervals for LCjo values of Bayer 73 to 17 species of fish. The data was

taken from table 4 at 96-hour exposures.
8 Investigations in Fish Control 19: Bureau of Sport Fisheries and Wildlife

TABLE 5.--Toxieity of Bayer 73 in short exposures at 12° C.

Species

Black bullhead. . . .

LCgo (p. p.m. ) and 95-percent confidence interval at 

1 hour

0.063 

0.060-0.066

0.077 

0.069-0.086

0.300 

0.267-0.338

0.180 

0.177-0.183

0.275 

0.230-0.329

0.120 

0.103-0.140

2 hours

0.056 

0.053-0.059

0.069 

0.063-0.076

0.250 

0.232-0.264

0.108 

0.092-0.127

0.210 

0.181-0.243

0.116 

0.103-0.131

3 hours

0.052 

0.049-0.056

0.064 

0.060-0.068

0.200 

0.188-0.214

0.105 

0.091-0.122

0.156 

0.138-0.177

0.116 

0.101-0.133

6 hours

0.052 

0.049-0.056

0.064 

0.060-0.068

0.188 

0.173-0.204

0.100 

0.088-0.114

0.156 

0.138-0.177

0.100 

0.089-0.112

Me en 

slope 

function

1.064 

1.089 

1.080 

1.126 

1.156 

1.143

TABLE 6. Effects of temperature on tcxioity of Bayer 73 to fish

Species

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

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

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

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

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

Channel catfish. . . . 

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

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

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

Do

Temp. 

C.

7° 

12° 

17° 

12° 

17° 

22° 

12° 

17° 

22° 

12° 

17° 

22°

LC50 (p. p.m. ) and 95-percent confidence interval at 

3 hours

0.059 

0.053-0.066

0.052 

0. 049-0. 05b

0.054 

0.048-0.059

0.340 

0.315-0.367

0.350 

0.311-0.394

0.290 

0.260-0.320

0.065 

0.060-0.070

0.058 

0.055-0.061

0.141 

0.128-0.155

0.120 

0.107-0.135

0.100 

0.093-0.108

6 hours

0.050 

0.044-0.057

0.052 

0.049-0.056

0.053 

0.048-0.058

0.270 

0.250-0.290

0.283 

0.258-0.311

0.268 

0.237-0.302

0.056 

0.053-0.059

0.058 

0.055-0.061

0.115 

0.103-0.129

0.096 

0.082-0.110

0.092 

0.080-0.106

24 hours

0.048 

0.042-0.055

0.052 

O.U49-0.056

0.052 

0.048-0.057

0.228 

0.207-0.251

0.279 

0.243-0.321

0.252 

0.221-0.287

0.052 

0.048-0.056

0.084 

0.079-0.089

0.055 

0.052-0.058

0.106 

0.094-0.120

0.082 

0.072-0.092

0.082 

0.065-0.103

43 hours

0.048 

0.042-0.055

0.052 

0.049-0.056

0.052 

0.048-0.057

0.228 

0.207-0.251

0.279 

0.243-0.321

0.206 

0.181-0.235

0.050 

0.046-0.054

0.084 

0.079-0.089

0.055 

0.052-0.058

0.092 

0.085-0.099

0.082 

0.070-0.096

0.066 

0.058-0.076

96 hours

0.047 

0.043-0.052

0.050 

0.047-0.054

0.050 

0.048-0.054

0.228 

0.207-0.251

0.230 

0.201-0.263

0.223 

0.179-0.279

0.049 

0.045-0.053

0.082 

0.077-0.088

0.042 

0.035-0.051

0.092 

0.085-0.099

0.068 

0.057-0.080

0.054 

0.046-0.063
L. L. Marking and J. W. Hogan: Toxicity of Bayer 73 to Fish

The values for all 6 species vary from 0.063 

to 0.300 p.p.m. in 1 hour to 0.050 to 0.139 in 

96 hours. The figures indicate that fish die 

rather quickly at concentrations only slightly 

greater than those required to kill at 96-hour 

exposures.

The average slope functions for 1-, 2-, 3-, 

and 6-hour exposures are low and similar to 

those for longer exposures (table 5). The figures indicate a steep regression where small 

increases in concentration produce considerable additional mortality.

Effects of temperature. Bayer 73 was 

tested with rainbow trout, goldfish, channel 

catfish, and bluegills at 7°, 12°, and 17° C. in 

3-, 6-, 24-, 48-, and 96-hour exposures (table 

6). These changes in temperature had no significant effect on the toxicity of Bayer 73 to 

rainbow trout.

Goldfish, although more resistant than rainbow trout, reacted similarly at 12°, 17°, and 

22° C. The difference is not significant at any 

one temperature, but the data indicate greater 

toxicity at 22° than at 12° or 17° C. The confidence intervals overlap for the 6-, 24-, 48-, 

and 96-hour exposures but not for the 3-hour 

exposure.

The channel catfish tested at 17° were different from the lot tested at 12° and 22° C. 

The results indicate greater resistance at 

17°, but the difference may be attributable to 

the source and physical condition of the fish. 

Another lot of channel catfish tested in 3- and 

6-hour bioassays indicated little or no difference at 17°. The responses at 12° and 22° obtained from the same lot of catfish show no 

significant differences.

Bluegills respond to increases in temperature more than the other species (table 6). 

Bayer 73 is more toxic at 22° than at 12Q or 

17°. The confidence intervals do not overlap 

at 12° and 22° at exposures of 3, 48, and 96 

hours. The increase in toxicity at longer exposures also is more noticeable with bluegills 

than with the other three species.

Effects of water quality. Bayer 73 was 

tested in soft, medium, and hard water prepared according to table 2. At all exposure 

periods, the chemical is more toxic to rainbow trout in soft water and less toxic in hard 

water (table 7). The greater decrease in toxicity is between medium and hard water and 

suggests degradation of Bayer 73 at higher pH 

values and higher alkalinities.

Carp respond much the same as rainbow 

trout, although the confidence intervals overlap at the extreme water quality variations 

(table 7). The carp tested in water of medium 

hardness were from a different lot of fish. The 

data indicate greater sensitivity in water of 

medium hardness than in soft water, but the 

difference is not significant.

The toxicity of Bayer 73 to channel catfish 

decreases significantly in hard water but 

changes very little from soft to medium hard 

water. The toxic effects are uniform within 

each hardness, and do not increase significantly from the 3-hour to the 96-hour exposure (table 7).

Bluegills also are more susceptible to 

Bayer 73 in softer waters (table 7). The LC50 

values in hard water are 2 or more times 

those in medium water. Confidence intervals 

for medium and hard water do not overlap at 

any exposure.

Effects of pH. Variations in pH were accomplished by adding buffering agents to 

standard test water according to table 3. 

There were significant differences in the toxicity of Bayer 73 to goldfish at pH 5, 7, and 10 

(table 8). The tests at pH 7 conform somewhat 

to tests in standard bioassays although these 

goldfish are from a different lot and appear 

more sensitive to Bayer 73 than the goldfish 

tested in standard water.
10 Investigations in Fish Control 19: Bureau of Sport Fisheries and Wildlife

TABLE 7.--Effects of water quality on toxicity of Bayer 73 to fish

Species

Rainbow trout. . . .

F)n

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

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

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

Channel catfish. . 

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

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

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

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

Water

quality

soft

soft

3 hours

0.046 

0.043-0.049

0.054

0.050-0.059 

0.082

0.075-0.090

0.082 

0.075-0.090

0.064

0.058-0.071 

0.079

0.091-0.104 

0.092

0.084-0.101 

0.120

0.106-0.135 

0.200

0.183-0.219

LC50 (p. p.m. ) and

6 hours

0.046 

0.043-0.049

0.053

0.049-0.055 

0.082

0.075-0.090

0.342

0.309-0.374

0.083

0.074-0.093 

0.096

0.082-0.110 

0.190

0.170-0.212

95-percent confid

24 hours

0.046 

0.043-0.049

0.053

0.049-0.055 

0.082

0.075-0.090 

0.197

0.168-0.230 

0.169

0.163-0.178 

0.229

0.210-0.250

0.070 

0.066-0.074

0.062

0.056-0.068 

0.093

0.085-0.101 

0.083

0.074-0.093 

0.082

0.072-0.092 

0.178

0.152-0.209

ence interval at--

48 hours

0.046 

0.043-0.049

0.053

0.049-0.055 

0.082

0.075-0.090 

0.188

0.181-0.196 

0.169

0.163-0.178 

0.229

0.207-0.242

0.070 

0.066-0.074

0.062

0.056-0.068 

0.093

0.085-0.101 

0.073

0.060-0.088 

0.082

0.070-0.096 

0.154

0.136-0.175

96 hours

0.046 

0.043-0.049

0.053

0.049-0.055 

0.070

0.066-0.074 

0.191

0.170-0.214 

0.169

0.163-0.178 

0.224

0.207-0.242 

0.060

0.054-0.067 

0.093

0.085-0.101 

0.064

0.055-0.074 

0.068

0.057-0.080 

0.117

0.100-0.137

TABLE 8. Effects of pH on the toxicity of Bayer 73 to 

goldfish at 12° C.

PH

5...

7...

10...

LCjo (p. p.m. ) and 95-percent confidence 

interval at--

3 hours

4.600 

3.880-5.460

-

-

24 hours

3.520 

3.120-3.980

0.180 

0.163-0.198

6.420 

5.780-7.130

48 hours

3.500 

3.100-4.000

0.180 

0.163-0.198

6.150 

5.690-6.640

96 hours

3.500 

3.100-4.000

-

3.190 

2.850-3.580

DISCUSSION

Bayer 73 is toxic to fish in very brief exposures and does not become proportionately 

more toxic with increased exposure time. In 

several cases the LC50 differed only little between 1 and 96 hours. This suggests rapid 

degradation of the toxicant either by the water 

or the fish. Duhm et al. (1963) found that rats

fed 50 mg./kg. 5,2'-dichloro-4l-nitro-salicicanilide excrete most of the material as 5,2'- 

dichloro-4-aminosalicylic-anilide in a conjugated form. The amino-structures excreted 

are very unstable and decompose when the excrement is left to stand for several hours. 

They concluded that the chemical is reduced 

and detoxified by metabolism in rats. Quite 

possibly, fish are able to detoxify Bayer 73 by 

a reduction process similar to that in rats. 

This may explain to some extent why the comparatively high initial activity of the compound 

is followed by minimum time-concentration 

effects.

Strufe and Gonnert (1962) reported that 

water temperature influences the efficacy of 

Bayer 73 in destroying snails. They found that 

0.3 p.p.m. in 24-hour exposures is sufficient 

to kill all the snails at 20°, but 0.5 p.p.m. is 

necessary to yield 100-percent mortality at 

5° C. They also indicated that solutions of 

Bayer 73 exposed to diffused daylight at 20°
L. L. Marking and J. W. Hogkn: Toxicity of Bayer 73 to Fish 11

to 50° C. for 9 hours show no significant loss 

of active ingredient. Our data indicate greater 

biological activity at higher temperatures, and 

the results probably would be more significant 

at extreme temperatures.

Gonnert (1962) reported that environmental 

factors such as salt content and pH of the 

water do not decisively influence activity of 

Bayer 73 against snails during the required 

exposure time. Strufe and Gonnert (1962) 

stated that the biological action of Bayer 73 

against snails is not influenced to any measurable degree by salty waters containing up to 

500 p.p.m. of calcium and magnesium. The 

laboratory tests with fish indicate less activity 

of the compound in harder water but, as other 

scientists report on snails, the difference is 

not considerable.

 iMI"*

The pH variations in different water qualities which ranged from 6.4 to 8.0, did not influence toxicity drastically; the buffered pH 5 

and 10 solutions did. Data from the latter test 

indicate less toxicity at low and high pH values 

and little difference at pH 7. The probable 

cause of decreased toxicity at extreme pH's 

may be a solubility or ionization factor in 

which less active chemical is available to the 

fish. Recently, Meyling and Pitchford (1966) 

found that as the pH of Bayer 73 solutions increase from 6.0 to 8.0 the solubility increases 

from less than 1.0 to 5.0 p.p.m. They did not 

define the solubility at pH 10. Strufe and Gonnert (1962) concluded from irradiation tests 

that up to 15 percent of the molluscicide is 

inactivated in neutral and weakly alkaline conditions, and up to 30 percent in the acid range 

of pH 5 to 6. Our tests at the extreme pH 

values of 5 and 10 confirm their findings.

-^ ~,

Fox et al. (1963) reported that at pH 9.7 to 

9.9 about 4 times as much Bayer 73 is required to kill snails as at pH 7.0 to 9.2. They 

suggest that snails require continuous stimulation by chlorine in order to bring into play 

their detoxifying mechanisms.

Meyling et al. (1962) reported that concentrations of Bayer 73 in hard water exposed to 

sunlight were reduced from 1.0 to 0.57 p.p.m. 

in 16 hours whereas in darkness the concentrations did not change after 312 hours. Apparentl^ sunlight, hard water, high pH, and 

alkalini|ty contribute to the reduction of Bayer 

73.

Bayer 73 is highly toxic to certain fish 

species resistant to other chemicals. Catfishes, for example, are readily susceptible 

to it but are resistant to such other toxicants 

as antimycin A (Walker et al., 1964) and rotenone (Henegar, 1966).

Gonnert (1961) summarizes the results of 

laboratory and field trials with Bayer 73 in a 

number of countries. Minimum lethal concentration!? for snails and snail ova were found to 

be between 0.2 and 0.5 p.p.m. Applications of 

1.0 p.p, m. of Bayer 73 destroyed snails in 

standing and flowing water. He also indicated 

that the toxicity of Bayer 73 to fish is very 

similar to that for snails.

Our tests in the laboratory show that Bayer 

73 produces 50-percent mortality in 18 species

at concentrations of 0.043 to 0.230 

in standard 96-hour bioassays. More 

0 p.p.m. was required to kill fish only

of fish

p.p.m.

than 1.

when the bioassays were chemically buffered

to pH 5 and 10. Thus, in the laboratory, molluscicidal concentrations of Bayer 73 are

toxic tp all species of fish tested. Applications

of 1.0 p.p.m. are probably toxic to fish in

natural environments provided that extreme

pH values of 5 or 10 do not prevail.

CONCLUSIONS I Bay^r 73, a molluscicide, is highly toxic to 

18 spebies of fish in static bioassays. Concentrations lethal to snails in the laboratory are 

also tdxic to fish.

Bay^r 73 kills 50 percent of the rainbow 

trout, JDrook trout, brown bullheads, flathead 

catfistj, smallmouth bass, and largemouth 

bass at 0.062 p.p.m. or less in 96-hour exposures. Fathead minnows, white suckers, 

black bullheads, channel catfish, green sunfish, biuegills, redear sunfish, yellow perch, 

and tilapia are intermediate in sensitivity, and 

LC50 values range from 0.81 to 0.109 p.p.m. 

in 96 hours. Goldfish and carp are more resistani: species, with LCso values of 0.230 and 

0.148 p.p.m.
12 Investigations in Fish Control 19: Bureau of Sport Fisheries and Wildlife

The toxicity of Bayer 73 to bluegills increased with temperature, but the toxicity to 

trout, goldfish, and catfish was not significantly affected. Various water qualities also

greatlvT ^ijtfgfflg

slgnif icantly reduce the^tpxicity 01' ' **^

Bayer 73 to figfc* This effect 'may be ,.-» « W»WW*fll I«.|J,H«««*W»"«WI»«1«*»WI**UW' ' " "****-'5-1«*''^fc^St»«Mpr. l.-,,w,,. _ ,--lx., , - ,__

decreased ionization at extreme pH values or 

to a metabolic reduction 6T"fM^5ornpquiid 

through stimulation of thej^teixlFying system 

in fish. " ' '

Because of its biodegradability and safety 

in water, Bayer 73 deserves consideration as 

a general fish toxicant.

REFERENCE

Duhm, B.t W. Maul, H. Medenwald, K. Patzschke, and 

L. A. Wagner. 1963. Radioactive studies on a new 

molluscicide. (from: Z. Naturforschg. 16b, 509-515 

(1961)). Pflanzenschutz-Nachrichten-Bayer, vol. 16, 

no. 5, p. 258-268.

Foster, R.

1962. Further observations on trials with 

BAYLUSCIDE(R) against certain East African 

snails. Pflanzenschutz-Nachrichten-Bayer, vol. 

15, no. 1, p. 75-82.

Fox, I., L. S. Ritchie, and L. P. Frick.

1963. Effect of pH on molluscicidal activity of Bayer 

73 vs. Australorbis glabratus of Puerto Rico under 

laboratory conditions, (from: Experiment Parasitology 13, 167-172 (1963)). Pflanzenschutz-Nachrichten-Bayer, vol. 16, no. 5, p. 269-276.

Gillet, J., and P. Bruaux.

1962. Laboratory and field testing of BAYLUSCIDE(R) 

("Bayer 73"). Pflanzenschutz-Nachrichten-Bayer, 

vol. 15, no. 1, p. 70-74.

Gohnert, Rudolf, and Ernst Schraufstatter.

1958. A new molluscicide: Molluscicide Bayer 73. 

(from: Proceedings of the Sixth International Congresses on Tropical Medicine and Malaria, vol. 11, 

p. 197-202. (1958).) Chemotherapeutiches Laboratorium und Pharmazeutisch - Wissenschaftliches 

Laboratorium der Farbenfabriken Bayer A G., 

Wuppertal - Elberfeld, Germany.

Gonnert, Rudolf.

1961. Results of laboratory and field trials with the 

molluscicide Bayer 73. Bulletin of the World Health 

Organization, vol. 25, p. 483-501.

Gonnert, Rudolf.

1962. BAYLUSCIDE(R) a new compound for controlling medically important freshwater snails. 

Pflanzenschutz-Nachrichten-Bayer, vol. 15, no. 1, 

P. 4-25.

Hecht, G., and Chr. Gloxhuber.

1962. Studies on the tolerance of 5,2'-dichloro-4- 

nitro-salicylic-anilide-ethanolamine. Pflanzenschutz-Nachrichten-Bayer, vol. 15, no. 1, p. 34-41.

Henegar, Dale L.

1966. Investigations in fish control. 3. Minimum 

lethal levels of toxaphene as a piscicide in North 

Dakota lakes. U.S. Bureau of Sport Fisheries and 

Wildlife, Resource Publication 7, 16 p.

Holz, J., and Ong Bie Hwa.

1963. Influence of the molluscicide ( R) Bayluscide on 

plankton in Indonesian fish ponds and rice fields. 

Pflanzenschutz-Nachrichten-Bayer, vol. 16, no. 5, 

p. 253-257.

Howell, John H., Everett L. King, Jr., Alien J. Smith, 

and Lee H. Hanson.

1964. Synergism of 5,2'-dichloro-nitro-salicylanilide 

and 3-trifluormethyl-4-nitrophenol in a selective 

lamprey larvicide. Great Lakes Fishery Commission, Technical Report No. 8, 21 p.

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

1964. Investigations in fish control. 1. Laboratories 

and methods for screening fish-control chemicals. 

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

Litchfield, J. T., 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 

£,p'-DDT as a reference toxicant in bioassays. U.S. 

Bureau of Sport Fisheries and Wildlife, Resource 

Publication 14, 10 p.

Marking, Leif L., Everett L. King, Charles R. Walker, 

and John H. Howell.

In press. Investigations in fish control. Acute toxicity 

of 3'-chloro-3-nitrosalicylanilide (33 NCS) to freshwater fish and sea lamprey. U.S. Bureau of Sport 

Fisheries and Wildlife, Resource Publication.

Meyling, A. H., C. H. J. Schutte, and R. J. Pitchford. 

1962. Some laboratory investigations on Bayer 73 and 

IC1 24223 as molluscicides. Bulletin of the World 

Health Organization, vol. 27, p. 95-98.

Meyling, A. H., and R. J. Pitchford.

1966. Physico-chemical properties of substances 

used as molluscicides. Precipitation and Adsorption. Bulletin of the World Health Organization, vol. 

34, p. 141-146.
L. L. Marking and J. W. Hogan: Toxicity of Bayer 73 to Fish 13

Molnar, N. M., and Seymour Baron.

1964. Two new stable polybrominated salicylanilides 

for antibacterial use in soap and detergent products. 

The Journal of the American Oil Chemists' Society, 

vol. 41, no. 7, p. 478-480.

Sniff, C. J., and Bridget Garnett.

1961. The short-term effects of three molluscicides 

on the microflora and microfauna of small, biologically stable ponds in Southern Rhodesia. Bulletin of the World Health Organization, vol. 25, 

p. 543-547.

Schraufstatter, Ernst Von, Werner Meiser, and Rudolf 

Gbnnert.

1961. Untersuchungen uber ein neues Molluscicid. 

I. Mitt.: Boziehungen zwischen Struktur und 

Wirkung. Zietschrift fur Naturforschung, Band 

16b, Heft 2, p. 95-108.

Schraufstatter, E.

1962. Chemical development of Bayluscide (R). 

Pflanzenschutz-Nachrichten-Bayer, vol. 15, no. 1, 

p. 25-33.

Starkey, Roland J., and John H. Howell.

1961. Substituted nitrosalicylanilides: a new class 

of selectively toxic sea lamprey (Petromyzon 

marinus) larvicides. Great Lakes Fishery Commission, Technical Report, 11, p. 21-29.

Strufe, R., and R. Gonnert.

1962. Comparative studies on the influence of environmental factors upon the efficiency of 

BAYLUSCIDE< R\ Pf lanzenschutz-NachrichtenBayer, vol. 15, no. 1, p. 50-70.

Taborsky, Robert G., Grant D. Darker, and Saul Kaye. 

1959. Substituted salicylanilides with antimicrobial 

activity. Journal of the American Pharmaceutical 

Association, vol. 48, no. 9, p. 503-507.

Taborsky, Robert G., and Roland J. Starkey.

1962. Subsituted salicylanilides. II. Further antimicrobial and other studies with halo-nitrosalicylanilides. Journal of Pharmaceutical Sciences, 

vol. 51, no. 12, p. 1152-1155.

Taborsky, Robert G., and Roland J. Starkey.

1963. Substituted salicylanilides. III. New salicylanilides and related compounds with antimicrobial 

activity. Journal of Pharmaceutical Sciences, vol. 

52, no. 6, p. 542-545.

Vinson, L. J., P. Dineer, and W. Schneider.

1961. Evaluation of four hospital detergent-sanitizers. 

Soap and Chemical Specialities, October and November. 11 p.

Walker, Charles R., Roland J. Starkey, and Leif L. 

Marking.

1966. Investigations in fish control. 9. Relation of 

chemical structure to fish toxicity in nitrosalicylanilides and related compounds. U.S. Bureau of 

Sport Fisheries and Wildlife, Resource Publication 

13, p. 1-12.

Webbe, G.

1963. The application of a molluscicide to the Mironga 

River in Mwanza, Tanganyika. PflanzenschutzNachrichten-Bayer, vol. 16, no. 5, p. 244-252.
INVESTIGATIONS IN FISH CONTROL

2O. Toxicity of Dimethyl Sulfoxide (DMSO) to Fish

By Wayne A. Willford, Chemist

Bureau of Sport Fisheries and Wildlife 

Fish Control Laboratory, La Crosse, Wis.

United Stales Department of the Interior, Stewart L. Udall. Secretary

Stanley A. Cain, Assistant Secretary for Fish and Wildlife and Parks

Fish and Wildlife Service, Clarence F. Pautzke, Commissioner

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

Resource Publication 37 Washington, D.C. April 1967
CONTENTS

Page

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

Materials and methods ............................... 4

Results and discussion ............................... 5

Preliminary testing .............................. 5

General toxicity................................. 5

Effect of water quality and temperature on toxicity.......... 5

Effect of DMSO on toxicity of antimycin ................. g

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

References ....................................... 7
TOXICITY OF DIMETHYL SULFOXIDE (DMSO) TO FISH

By Wayne A. Willford, Chemist 

Fish Control Laboratory, La Crosse, Wis.

ABSTRACT. Toxicities of dimethyl sulfoxide (DMSO) to rainbow trout, 

brook trout, lake trout, carp, black bullhead, channel catfish, green sunfish, 

bluegill, and yellow perch were determined in 24-, 48-, and 96-hour static 

bioassays at 12° C. Toxicity was of low order, around 30 p.p.t. Water quality had little effect, but increased temperature increased the toxicity to 

rainbow trout. A preliminary test indicated that DMSO has little effect on 

the toxicity of antimycin to bluegill.

Dimethyl sulf oxide (DMSO) is the simplest 

of the homologous series of organic sulf oxides; 

it is prepared by oxidation of dimethyl sulfide. 

Its formula is (CH^ ) 2 SO, and the pure form is 

a hygroscopic, colorless, odorless liquid, 

melting at 18.45° and boiling at 189° C., with 

a specific gravity of 1.100 (Stecher, I960).

DMSO was first synthesized in 1867 but remained a laboratory curiosity until the 1940's 

when it was successfully used as a solvent for 

the spinning of polyacrylonitrile fibers (Block, 

1964). The first physiochemical data on the 

compound appeared in 1948, and since that 

time DMSO has been used extensively in several fields.

DMSO has been shown to possess remarkable potential as a solvent for many types of 

inorganic and organic compounds including 

gases (Willson et al., 1965). It has also been 

used as a preservative during freeze storage 

of red blood cells (Lovelock and Bishop, 

1959), platelets (Geisler et al., 1964), spermatozoa (Sherman, 1964), mitochondria 

(Greiff, 1961), protozoa (Hwang, 1964), bone 

marrow (Ashwood-Smith, 196 la; Persidsky 

and Richards, 1963), cardiac muscle (Levy 

et al., 1962), and tissue culture cells 

(Dougherty, 1962; Porterfield and AshwoodSmith, 1962). DMSO also exhibited radioprotective action against lethal doses of Xirradiation in mice (Ashwood-Smith, 196 Ib).

In the medical field, the compound is under 

investigation as a penetrant carrier, local 

analgesic, anti-inflammatory adjunct, bacteriostatic agent, diuretic, tranquilizer, and 

potentiator (Jacob et al., 1964a). It is even 

reported to be good for headaches (Jacob, 

1965). The most promising of these areas appears to be the ability of DMSO to penetrate 

biological membranes and act as a carrier for 

other drugs (Horita and Weber, 1964; Jacob 

et al., 1964b; Stoughton and Fritsch, 1964; 

Stoughton, 1965).

In the agricultural field, DMSO has been 

shown to be of value as a solvent carrier for 

certain compounds used in the control of 

plant diseases (Bean, 1965; Keil et al., 1965). 

It also has some herbicidal activity when 

used by itself for the control of purple nutsedge (Anderson and Dunford, 1966).

Additional information and references on 

DMSO can be obtained from articles written 

by Rosenkrantz et al. (1963), and Kligman 

(1965a and 1965b).

These interesting properties of DMSO plus 

the fact that it is miscible in all proportions 

with water suggested that it might be useful
4 Investigations in Fish Control 20: Bureau of Sport Fisheries and Wildlife

in fisheries as a nontoxic solvent in toxicity 

studies and for the administration of nonwater-soluble drugs to fish. In addition, if 

DMSO enhances the absorption by fish of 

compounds dissolved in it, a major breakthrough in the fields of fish control and fish 

disease control would result. It is for these 

purposes that the following toxicity studies 

were undertaken.

MATERIALS AND METHODSr

Samples of DMSO were obtained from 

Ayerst Laboratories, New York, N.Y. The 

formulation was 90 percent DMSO and 10 percent water.

The fish were obtained from several fish 

hatcheries (table 1), and were introduced to 

the static bioassays after routine acclimation 

as described by Lennon and Walker (1964).

Preliminary bioassays were conducted in 

1-gallon glass jars each containing 3 liters 

of bioassay media and two fish. After the approximate level of toxicity had been established, delineative bioassays were conducted 

in 5-gallon glass jars each containing 15

liters of reconstituted deionized water and 

10 fish. The proper volume of bioassay 

media was maintained by removal of a quantity of water equal to the aliquot of DMSO 

which was to be added. Each test included 

5 to 9 concentrations of chemical and 50 to 

90 test fish plus 10 fish for controls.

Various water qualities were obtained by 

adding selected concentrations of reconstituting salts to deionized water (table 2). Tests 

were maintained at 7°, 12°, or 17° C. by 

water baths.

Survival and mortality were recorded at 

24, 48, and 96 hours. The data were analyzed 

by plotting concentration versus mortality on 

logarithmic normal (probability) graph paper 

to define the concentration which produced 

50-percent mortality (LCw ), slope function, 

variation, and 95-percent confidence intervals 

(C.I.) as described by Litchfield and Wilcoxon 

(1949).

All results are reported in parts per thousand (p.p.t.), by volume, of total material 

added to the test vessel instead of active ingredient.

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

Species

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

Brook trout, Salvelinus f ontinales. ................

Average 

length 

(inches )

1.8

1.8

1.5

1.9

1.6

2.0

2.0

1.5

1.3

2.6

Average 

weight 

(grams)

0.9

1.0

0.5

0.7

1.0

1.7

1.1

0.8

0.5

1.7

Source1

SFH, Osceola, Wis.

SFH, St. Croix Falls, Wis.

NFH, Lake Falls, Wis.

NFH, New London, Minn.

NFH, Fairport, Iowa.

NFH, Lake Mills, Wis.

Do.

Do.

NFH = National Fish Hatchery; SFH = State Fish Hatchery.

TABLE 2. Composition and analysis of reconstituted, deionized water used in bioassays

Classification 

of 

water

Soft. ............

Medium1 ..........

Hard.............

Amount of salts added (mg./l. )

NaHC03

12.0 

48.0 

192.0

CaS04

7.5 

30.0 

120.0

MgS04

7.5 

30.0 

120.0

KCL

0.5 

2.0 

8.0

pH 

range

6.4 - 6.8 

7.2 - 7.6 

7.6 - 8.0

Range of 

total hardness 

as p. p.m. CaCOa

10-13 

40-48 

160-180

Range of 

total alkalinity 

as p. p.m. CaCOa

10-13 

30-35 

110-120

Standard reconstituted water used in routine bioassays.
Wayne A. Willford: Toxicity of DMSO to Fish

RESULTS AND DISCUSSION

PRELIMINARY TESTING

Preliminary tests to determine the approximate level of DMSO toxicity were carried out with yellow perch as the test species. 

In order to preserve the supply of test material it was necessary to prepare a 3-liter 

solution containing 500 p.p.t. of DMSO. Subsequent concentrations were obtained by dilution of this solution.

Results in terms of the approximate time 

to death at each concentration were as follows:

500 p.p.t. - 10 minutes

250 p.p.t. - 30 minutes

125 p.p.t. - 1.5 hours

62 p.p.t. - 24 hours

31 p.p.t. - no mortality within 96 hours

From these data, the 24-, 48-, and 96-hour 

LC 50 values appear to lie between 30 and 60 

p.p.t., and concentrations were selected accordingly for the routine toxicity bioassays.

GENERAL TOXICITY

DMSO exhibited a consistent and nonselective toxicity of very low order to the nine 

species tested (table 3).

Probit analysis yielded slope functions on 

logarithmic paper which ranged from 1.06 to 

1.20 with a mean of 1.11 on all tests. This

indicates that as the level of acute toxicity 

is approached a minimal change in concentration is required to produce either 0- or 

100-percent mortality.

The comparative resistance of the nine 

species was extremely close. The 96-hour 

LC 50 T s ranged from only 32.3 to 43.0 p.p.t. 

for the various species.

The order of susceptibility of the nine 

species varied with the observation period. 

At 24 and 48 hours, for example, channel catfish were the most susceptible, with bluegill 

the most resistant at 24 hours and yellow 

perch the most resistant at 48 hours. By 

96 hours, rainbow trout became the most 

susceptible and green sunfish the most resistant. This fluctuation in the comparative 

order of sensitivity further exemplifies the 

extremely nonselective toxicity to all fish 

tested.

Recent studies at the Western Fish Nutrition Laboratory with yearling coho salmon 

(Oncorhynchus kisutch) determined the median tolerance limit (TLm) to be 72, 55, and 

46 p.p.t. at 24. 48, and 96 hours respectively.1 

Ball (1966) reported that the 48-hour LC 50 of 

DMSO to goldfish (Carassius auratus) is 43 

p.p.t. at 15° C. These results are in close 

agreement with the results at this laboratory 

and serve to further substantiate the consistent and nonselective toxicity of DMSO.

1 Personal communication from Pete Benville, Jr., 

Chemist, Western Fish Nutrition Laboratory, Bureau 

of Sport Fisheries and Wildlife, Cook, Wash., 1966.

TABLE 3. Toxicity of 90-percent DMSO to nine species of fish at 12° C.

Species

At

LC5 o 

(p.p.t.)

53.0

54.5

47.8

44.0

j o e:

39.0

65.0

72.0

65.0

24 hours

95-percent

C.I.

48.6 - 57.8

50.9 - 58.3

42 3 - 54 0

39.3 - 49.3

37.9 - 47.6

36.1 - 42.1

61.3 - 68.9

£-0 o Oo n

61.3 - 68.9

At

LC 5 o 

(p.p.t.)

41.7

46.0

38.2

44 0

39.2

34.5

52.5

56.0

57.0

48 hours

95-percent 

C.I.

39 3 _ 44 2

j o o ^n i

35.4 41.3

39.3 - 49.3

35.3 - 43.5

31.7 - 37.6

47.7 - 57.8

51.9 - 60.5

52.3 - 62.1

At 9

LC 5 o 

(p.p.t.)

32.3

36.5

37.3

41.7

36.5

32.5

43.0

33.5

37.0

6 hours

95-percent 

C.I.

30.2 - 34.6

33.2 - 40.2

35.2 - 39.5

36.3 - 48.0

33.8 - 39.4

29.8 - 35.4

35.8 - 51.6

29.9 - 37.5

33.9 - 40.3
Investigations in Fish Control 20: Bureau of Sport Fisheries and Wildlife

Rabinowitz and Myerson (1966) stated that 

a concentration of 19 p.p.t. of DMSO produced 

an approximate 48-hour LD so with neon 

tetras (Paracheirodon innesi), platys (Xiphosphorus rnaculatus), mollies (Pescilia 

latipinna), and guppies (Poecilia reticulata). 

The LD^s for zebras (Brachydanio rerio) 

and catfish (Corydoras paleatus) were somewhere in excess of 25 p.p.t. These results 

were obtained in distilled water at 24° to 

25° C. and the increased toxicity indicated 

may be the result of osmotic stress induced 

by the test media.

EFFECT OF WATER QUALITY AND 

TEMPERATURE ON TOXICITY

Changes in water quality at 12° C. had 

little or no effect upon the toxicity of DMSO 

(table 4). The LC 50 confidence interval in 

any particular water quality overlaps the 

LC 50's in other water qualities within the 

same observation period in all cases except 

one. This exception was in hard water at 

96 hours. In general, it appears that DMSO 

is slightly less toxic in hard water than it is 

in waters of soft or medium hardness.

Changes in temperature at medium hardness 

exhibited a substantial influence on toxicity 

(table 4). An increase in toxicity in excess of 

10 p.p.t. as the temperature increases from 

7° to 17° C. was observed at all observation 

periods.

This increase in toxicity at warmer temperature is in agreement with observations 

made at the Western Fish Nutrition Laboratory.

EFFECT OF DMSO ON TOXICITY 

OF ANTIMYCIN

A preliminary test was performed to determine what, if any, influence DMSO has 

on the toxicity of antimycin to bluegill. Various concentrations of antimycin were added 

in combination with enough DMSO to produce 

1.0 p.p.t. of DMSO in the test vessel. A comparison test was run using only acetone as 

solvent for the antimycin.

The 96-hour LC 50 of antimycin and acetone 

alone was 0.089 parts per billion (p.p.b.), 

while antimycin in combination with 1.0 p.p.t. 

of DMSO produced a 96-hour LC 50 of 0.084 

p.p.b. These results reflect biological variation and indicate that DMSO has no effect on 

the toxicity of antimycin at 96 hours. It is 

possible that in a bioassay designed to yield 

toxicity with shorter exposures, DMSO could 

enhance the absorption of antimycin sufficiently to affect toxicity.

Ball (1966) compared the relative toxicity of 

0.05 p.p.m. j>,p'-DDT to goldfish when used in 

combination with 6 and 18 p.p.t. of either 

DMSO or acetone. His results indicated that 

DMSO does not significantly affect the median 

survival time of goldfish when compared to 

acetone. He further suggested that DMSO may 

be a better solvent than acetone for pesticide 

toxicity studies.

Rabinowitz and Myerson (1966) were unable 

to show a significant difference in the uptake 

by aquarium fish of radioactive labeled dyes 

when used in combination with 1.0 p.p.t. of 

DMSO.

TABLE 4. Effect of water quality and temperature on toxicity of DMSO (90-percent) to rainbow trout

Temperature 

C.

7°..............

12°..............

17°..............

12°. .............

12°..............

Water 

quality

At

LCso 

(p.p.t.)

65.5

53.0

41.5

53.5

57.0

24 hours

95-percent 

C.I.

57.0-75.3

/ fj f. cry £j

-31-1 rj ) c f

49.1-58.3

c-i jj /lO rj

At

LC50 

(p-p.t.)

46.0

41 7

35.0

42 3

44 8

48 hours

95-percent

C.I.

41.8-50.6

39.3-44.2

31.8-38.5

38.4-46.5

41.5-48.4

A

LC50 

(p.p-t.)

41.5

32.3

27.7

33.5

38.0

t 96 hours

95-percent 

C.I.

37.7-45.6

30.2-34.6

25.0-30.7

30.7-36.5

35.8-40.3
Wayne A. Willford: Toxicity of DMSO to Fish

All of these results indicate that DMSO, 

when used as a diluted constituent, does not 

affect the absorption of some chemicals by 

fish. It has been shown to be an excellent 

solvent, and is worthy of further investigation as such for certain chemicals used in 

fisheries. In addition, the potential of DMSO 

as a penetrant carrier of certain drugs used 

in human medicine suggests investigation of 

similar potential in the treatment of fish 

disease.

CONCLUSIONS

1. The acute toxicity of DMSO to fish is of a 

very low order.

2. When the level of acute toxicity is reached, 

DMSO is abruptly and nonselectively toxic 

to the nine species tested.

3. Various water qualities at 12° C. have 

little effect upon the toxicity of DMSO to 

rainbow trout.

4. Increases in temperature cause a definite 

increase in the toxicity of DMSO to rainbow 

trout.

5. Preliminary results indicate that 1.0 p.p.t. 

of DMSO has no effect on the toxicity of 

antimycin to bluegill at 96 hours.

REFERENCES

Anderson, W. Powell, and Max P. Dunford.

1966. The herbicidal activity of dimethyl sulfoxide on 

purple nutsedge. Weeds, vol. 14, no. 3, p. 195-197.

Ashwood-Smith, M. J.

1961a. Preservation of mouse bone marrow at -79° C, 

with dimethyl sulphoxide. Nature (London), vol. 

190, no. 4782, p. 1204-1205.

1961b. The radioprotective action of dimethyl sulphoxide and various other sulphoxides. International 

Journal of Radiation Biology (London), vol. 3, no. 

1, p. 41-48.

Ball, lan R.

1966. Toxicity of dimethyl sulphoxide to the goldfish 

Carassius auratus. Nature (London), vol. 210, no. 

5036, p. 639-640.

Bean, George A.

1965. The use of dimethyl sulfoxide (DMSO) with 

certain fungicides for controlling Helminthosporium 

disease of Kentucky bluegrass. Plant Disease Reporter, vol. 49, no. 10, p. 810-811. 

Block, Lawrence H.

1964. Diiaethyl sulfoxide. Drug and Cosmetic Industry, vcl. 95, no. 3, p. 342, 345-346, 462-465.

Dougherty, Robert M.

1962. Use of dimethyl sulphoxide for preservation of 

tissue culture cells by freezing. Nature (London), 

vol. 193, no. 4815, p. 550-552.

Geisler, Phillip H., loulious A. lossifides, and Mary F. 

Eichman.

1964. Preservation of lactic dehydrogenase activity 

of platelets by freezing in dimethylsulfoxide and 

plasma. Blood; Journal of Hematology, vol. 24, 

no. 6, p. 761-764.

Greiff, D., and M. Myers.

1961. Effect of dimethyl sulphoxide on the cryotolerance of mitochondria. Nature (London), vol. 

190, no. 4782, p. 1202-1204.

Horita, A., and L. J. Weber.

1964. Skin penetrating property of drugs dissolved 

in dimethylsulfoxide (DMSO) and other vehicles. 

Life Sciences, vol. 3, no. 12, p. 1389-1395.

Hwang, Shuh-Wei, E. E. Davis, and M. T. Alexander.

1964. Freezing and viability of Tetrahymena 

pyriformis in dimethylsulfoxide. Science, vol. 144, 

no. 3614, p. 64-65.

Jacob, Stanley W.

1965. Dimethyl sulfoxide, its basic pharmacology 

and usefulness in the therapy of headache. Headache, vol. 5, no. 3, p. 78-81.

Jacob, Stanley W., Margaret Bischel, and Robert J. 

Herschler.

1964a. Dimethyl sulfoxide (DMSO): A new concept 

in pharmacotherapy. Current Therapeutic Research, vol. 6, no. 2, p. 134.

1964b. Dimethyl sulfoxide: Effects on the permeability of biologic membranes (preliminary report). Current Therapeutic Research, vol. 6, no. 3, 

p. 193-198.

Keil, H. L., B. C. Smale, and B. A. Wilson.

1965. Control of peach bacteria leaf spot with sprays 

of oxytetracycline plus dimethyl sulfoxide. Phytopathology, vol. 55, no. 5, p. 505.

Kligman, Albert M.

1965a. Topical pharmacology and toxicology of 

dimethyl sulfoxide, Part I. Journal of the American Medical Association, vol. 193, no. 10, p. 140- 

148.

1965b. Dimethyl sulfoxide, Part II. Journal of the 

American Medical Association, vol. 193, no. 11, 

p. 923-928.
8 Investigations in Fish Control 20: Bureau of Sport Fisheries and Wildlife

Lennon, Robert E., 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, p. 1-15.

Levy, Joseph V., Victor Richards, and Maxim Persidsky. 

1962. Effect of dimethyl sulfoxide and gly -erol on 

cation content of freeze-thawed cardiac muscle. 

Proceedings of the Society for Experimental 

Biology and Medicine, vol. 110, no. 4, p. 789-791.

Litchfield, J. T., 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.

Lovelock, J. E., and M. W. H. Bishop.

1959. Prevention of freezing damage to living cells 

by dimethyl sulfoxide. Nature (London) vol. 183, 

no. 4672, p. 1394-1395.

Persidsky, Maxim, and Victor Richards.

1963. Optimal conditions and comparative effectiveness of dimethyl sulphoxide and polyvinylpyrrolidone 

in preservation of bone marrow. Nature (London), 

vol. 197, no. 4871, p. 1010-1012.

Porterfield, J. S., and M. J. Ashwood-Smith. 

1962. Preservation of cells in tissue culture by 

glycerol and dimethyl sulphoxide. Nature (London), 

vol. 193, no. 4815, p. 548-550.

Rabinowitz, Joseph L., and Ralph M. Myerson. 

1966. Exposure of aquarium fish to dimethyl sulfoxide (DMSO) with special reference to toxicity and

effects on uptake of radioactive dyes. Proceedings 

of the Society for Experimental Biology and Medicine, vol. 121, no. 4, p. 1065-1067.

Rosenkrantz, Harris, Zareh Hadidian, Heather Seay, 

and Marcus M. Mason.

1963. Dimethyl sulfoxide: Its steroid solubility and 

endocrinologic and pharmacologic-toxicologic 

characteristics. Cancer Chemotherapy Reports, 

no. 31, p. 7-24.

Sherman, J. K.

1964. Dimethyl sulfoxide as a protective agent during freezing and thawing of human spermatozoa. 

Proceedings of the Society for Experimental 

Biology and Medicine, vol. 117, no. 1, p. 261-264.

Stecher, Paul G.

1960. The Merck index of chemicals and drugs. 

Seventh edition, Merck and Co., Rahway, N.J. 

1641 p.

Stoughton, Richard B.

1965. Percutaneous absorption. Toxicology and 

Applied Pharmacology, supplement 2, p. 1-6.

Stoughton, R. B., and William Fritsch.

1964. Influence of dimethyl sulfoxida (DMSO) on 

human percutaneous absorption. Archives of 

Dermatology, vol. 90, no. 5, p. 512-517.

Willson, J. E., D. E. Brown, and E. K. Timmens.

1965. A toxicologic study of dimethyl sulfoxide. 

Toxicology and Applied Pharmacology, vol. 7, no. 1, 

p. 104-112.
INVESTIGATIONS IN FISH CONTROL

21. Labor-saving Devices for Bioassay Laboratories

By Robert J. Hesselberg, Chemist and 

Ralph M. Burress, Fishery Biologist

Southeastern Fish Control Laboratory 

Bureau of Sport Fisheries and Wildlife 

Warm Springs, Georgia

United Stales Department of the Interior, Slewarl L. Udall. Secretary

Stanley A. Cain, Assistant Secretary for Fish and Wildlife and Parks

Fish and Wildlife Service, Clarence F. Pautzke, Commissioner

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

Resource Publication 38 Washington, D.C. April 1967
CONTENTS

Page

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

Jar rinsing rack. .................................. 3

Description. .................................. 3

Use ........................................ 3

Automatic liquid-measuring vessel ...................... 4

Description. .................................. 4

Use ........................................ 6

Jar emptier. ..................................... 7

Description. .................................. 7

Use ........................................ 7

Reference. ...................................... 8
LABOR-SAVING DEVICES FOR BIOASSAY LABORATORIES

By Robert J. Hesselberg, Chemist 

and Ralph M. Burress, Fishery Biologist 

Southeastern Fish Control Laboratory 

Warm Springs, Ga.

ABSTRACT. Three inexpensive pieces of labor-saving apparatus for 

bioassay laboratory use are described and illustrated. Construction features, material costs, and use of a jar rinser, automatic liquid measuring vessel, and jar emptier are discussed.

Nearly all of the indoor bioassays at the 

Fish Control Laboratories require the use of 

5-gallon glass jars. Considerable work is involved in handling the scores of jars used 

daily, and much thought and effort have been 

given to developing methods to reduce the 

labor.

In recent months three labor-saving devices 

were developed and put into use: a rack for 

simultaneously rinsing 26 jars, a vessel for 

automatically measuring 15 liters of water 

into each jar, and a simple apparatus for 

emptying jars. Hundreds of man-hours of 

work are saved, and the hazards in handling 

heavy, slippery jars are reduced. The devices 

are not expensive and may be adapted to meet 

needs which differ from our own.

JAR RINSING RACK

DESCRIPTION

The essential features of the rack are shown 

in figure 1. It is about 15 feet 6 inches long, 

28 inches wide, and 18 inches high, but dimensions may be varied according to needs. Materials are not of critical importance either, 

unless highly corrosive wastes will be encountered. In our case, availability of materials and the ease with which they could be

worked were the chief factors considered. 

Two-inch lumber and large nails are used to 

construct load-bearing structures (sides, 

ends, and cross bracing), while the pipe supports and grate-retaining rim are of 1-inch 

material. All wooden parts were given two 

coats of high-grade enamel. The sections of 

aluminum grating which hold the jars were 

part of the covering for the floor trench over 

which the jar-rinsing rack is supported and 

into which waste water drains. Wooden crosspieces could be substituted for the metal 

gratings at considerable savings in material 

costs. Plumbing components include 3/4-inch 

plastic pipe and fittings (1/2-inch pipe would 

be adequate for a small rack), 3/4-inch 

valves (one for each pipe), 1/4-inch brass 

spray nozzles, and 1/4- by 1 1/8-inch allthread nipples for attaching nozzles to the 

pipe. Total cost of materials exclusive of the 

gratings was $54.20.

USE

After the jars are emptied and removed 

from bioassay troughs, they are cleaned according to a procedure like that outlined by 

Lennon and Walker (1964). The rack not only 

provides jar support at a convenient working 

height during washing, but also cuts the time 

for washing and rinsing to less than half that 

required when jars were cleaned individually.
Investigations in Fish Control 21: Bureau of Sport Fisheries and Wildlife

Figure 1. Jar-rinsing rack with sections of grating removed to show construction detail.

When fewer than 26 jars are rinsed, a board 

is laid over the unused nozzles to deflect 

water into the floor trench.

AUTOMATIC LIQUID 

MEASURING VESSEL

DESCRIPTION

Most of the custom-made shell of the vessel 

was constructed of sections of polyethylene 

pipe having a wall thickness of one-fourth inch 

and inside diameters of 3, 4, and 8 inches

(fig. 2). The main body of the vessel was made 

by heat-fusing a specially shaped top and bottom on a 17-inch-long section of 8-inch pipe. 

The convex top plate was made from a polyethylene block machined to a thickness of onefourth inch, and the weight-bearing, concave 

lower plate was similarly machined to a 

thickness of one-half inch. An 8-inch-long 

piece of the 4-inch pipe was fused to the top 

plate to form the neck of the vessel, and a 

1 1/2-inch-long piece of 3-inch pipe was 

fused to the bottom plate to serve as a drain 

(it also holds the vessel in place on the jar).
R. J. Hesselberg and R. M. Burress: Labor-saving Devices for Bioassay Laboratories 5

aluminum rod

1"adjustable 

coupling

height adjusted 

for fifteen liters

1" 

4

l"plastic overflow

tube

2 aluminum rod

4 grooves l"w by 1 d

 13 rubber stopper^ 

aluminum nut stopper, i"rod and overflow not shown in this view

Top View Cross section view

Figure 2.--Schematic illustration of automatic measuring vessel.

Special shaping of the upper and lower plates 

prevents air bubbles from being trapped 

under the upper plate, and results in rapid, 

complete drainage from the lower plate. The 

underside of the lower plate was made with 

four radial grooves one-half inch wide and 

one-fourth inch deep to permit rapid escape 

of air from the jar as it fills.

In filling the vessel, water enters through 

a 1/2-inch hard-rubber stopcock near the 

bottom, and any excess overflows through a 

1-inch plastic pipe inserted through the side 

of the vessel near the top. The internal upper

section of the overflow pipe extends upward 

into the neck to the required level, and the 

external lower section extends down to the 

bottom of the vessel where it is rigidly attached. A threaded plastic coupling at the top 

of the overflow allows for calibration of the 

volume measured.

The vessel is emptied through a drain hole 

in the bottom plate, the sides of which are 

tapered at an angle of 11°. A No. 13 rubber 

stopper and a 34-inch-long piece of aluminum 

rod serve as valve and handle, respectively. 

The lower 6 inches of the rod are threaded.
6 Investigation in Fish Control 21: Bureau of Sport Fisheries and Wildlife

The stopper, which is located about 5 inches 

from the lower end of the rod, is held securely 

in place by means of aluminum washers and 

nuts above and below it. The lower end of the 

rod extends through a hole in a 3-inch-long 

piece of 1/2-inch aluminum rod which is held 

in place across the inside of the drain by 

small metal screws in each end. This guides 

the stopper into place, and when the vessel is 

emptied, upward travel of the rod is limited 

by the aluminum nut on its lower end. The 

midsection of the handle is bent to form a 2- 

inch offset which gives clearance around the 

upper end of the overflow pipe. The upper end 

of the rod extends slightly above the top of the 

vessel and is bent at a right angle to form a 

hand grip.

In the last vessel constructed, a stainlesssteel rod was used for the handle, and a 

toilet-tank ball of soft, flexible rubber was 

used for a valve. The dimensions of the vessel 

could be changed easily to permit measurement of a wide range of volumes. The total 

cost of materials for the vessel was $37.20.

USE

To facilitate rapid filling of jars, two vessels are used concurrently. Water is supplied 

to each through sections of 5/8-inch plastic 

tubing connected to a single hose by a "Y" 

connector (fig. 3). Under our conditions it 

takes about 25 seconds to fill the measuring

Figure 3. Automatic measuring vessels ready for use.
R. J. Hesselberg and R. M. Burress: Labor-saving Devices for Bioassay Laboratories

vessel with 15 liters of water, and less than 

half that long to empty it. Thus, a full vessel 

can be emptied and moved to another jar 

while the other vessel is filling. The plastic 

vessels are light, safe to handle, and quite 

durable.

JAR EMPTIER

DESCRIPTION

This tool consists of a U-shaped tube made 

of 1 1/2-inch plastic pipe, about a foot of 

which is inserted through a rubber seal (fig. 

4). The seal is fashioned from the thickwalled upper section of a 2-way force cup

(plumber's helper). A short section of 3/8- 

inch cooper tubing having a flared inner end 

also is inserted through the rubber cup to 

pass compressed air into the jar. The intake 

end of the 1 1/2-inch pipe is covered with a 

1/8-inch mesh screen to prevent loss of fish 

as water is removed. The cost of materials 

was $2.35.

USE

The safe, low-pressure (5 p.s.i.) air supply 

used for aerating fish tanks provides the 

power to empty water from the bioassay jars. 

Once the screened end of the jar emptier and 

the rubber seal are inserted in the jar, compressed air seats the seal firmly against the

Figure 4. Jar emptier partially inserted into jar; compressed air is supplied through the small tube while waste

water is expelled through the 1 1/2-inch pipe.
8 Investigations in Fish Control 21: Bureau of Sport Fisheries and Wildlife

neck. Nearly 15 liters of water are expelled strating that the necessary margin of safety

in 11 seconds, and the resultant 30-pound was provided.

reduction in weight makes the task of lifting

jars from bioassay troughs much easier and REFERENCE

safer. Tests were conducted to learn what

would happen when the outlet screen v/as de- Le11^1 ' ?obert E".^Charles R. Walker

, , 1964. Investigations In Fish Control. 1. Laboratories hberately plugged and pressure was allowed flnd methods for screening fish_control chemicals,

to build up. In each case the seal was harm- Bureau of Sport Fisheries and Wildlife. Circular 185,

lessly forced out the neck of the jar, demon- 15 p.

* U.S. GOVERNMENT PRINTING OFFICE : 1967 O 271-162
(Reports 18 through 21 are in one cover.)

18. Toxicity of 22 Therapeutic Compounds to Six Fishes, by Wayne A. Willford. (Resource 

Publication 35.) 1967. 10p.

19. Toxicity of Bayer 73 to Fish, by Leif L. Marking and James W. Hogan. (Resource Publication 36.) 1967. 13p.

20. Toxicity of Dimethyl Sulfoxide (DMSO) to Fish, by Wayne A. Willford. (Resource Publication 37.) 1967. 8p.

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

Burress. (Resource Publication 38.) 1967. 8p.

Fish Control Laboratories

Bureau of Sport Fisheries and Wildlife

U.S. Department of the Interior

P.O. Box 862

La Crosse, Wisconsin 54602
The Department of the Interior, created in 1849, is a Department of 

Conservation, concerned with management, 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.

UNITED STATES 

DEPARTMENT OF THE INTERIOR

FISH AND WILDLIFE SERVICE

BUREAU OF SPORT FISHERIES AND WILDLIFE 

WASHINGTON. D. C. 2O24O