INVESTIGATIONS IN FISH CONTROL

87. Ethyl-p-aminobenzoate (Benzocaine): Efficacy as an 

Anesthetic for Five Species of Freshwater Fish

88. Influences of Selected Environmental Factors on the 

Activity of a Prospective Fish Toxicant, 2-(Digeranylamino)-ethanol, in Laboratory Tests

89. Toxicities of the Lampricides

3-Trifluoromethyl-4-nitrophenol (TFM) 

and the 2-Aminoethanol Salt of 

2',5-Dichloro-4'-nitrosalicylanilide (Bayer 73) 

to Four Bird Species

UNITED STATES DEPARTMENT OF THE INTERIOR

FISH AND WILDLIFE SERVICE
Investigations in Fish Control, published by the Fish and Wildlife Service, include reports 

on the results of work at the National Fishery Research Laboratory at La Crosse, Wis., and 

the Southeastern Fish Control Laboratory at 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. [See Investigations in Fish Control 

47-50 (in one cover) for list of issues published prior to 1973.]

(Reports 51 and 52 are in one cover.)

51. Methods for Simultaneous Determination and Identification of MS-222 and Metabolites in Fish Tissues, by Charles W. Luhning. 1973.10 pp.

52. Residues of MS-222, Benzocaine, and Their Metabolites in Striped Bass Following 

Anesthesia, by Charles W. Luhning. 1973.11 pp.

(Reports 53 through 55 are in one cover.)

53. Toxicity of Mixtures of Quinaldine Sulfate and MS-222 to Fish, by Verdel K. Dawson 

and Leif L. Marking. 1973.11 pp.

54. The Efficacy of Quinaldine Sulfate:MS-222 Mixtures for the Anesthetization of Freshwater Fish, by Philip A. Gilderhus, Bernard L. Berger, Joe B. Sills, and Paul D. Harman. 1973. 9 pp.

55. Residues of Quinaldine and MS-222 in Fish Following Anesthesia with Mixtures of 

Quinaldine Sulfate:MS-222, by Joe B. Sills, John L. Alien, Paul D. Harman, and 

Charles W. Luhning. 1973.12 pp.

(Reports 56 through 59 are in one cover.)

56. Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) to 10 Species of 

Algae, by A. W. Maki, L. D. Geissel, and H. E. Johnson. 1975.17 pp.

57. Acute Toxicities of 3-Trifluoromethyl-4-nitrophenol (TFM) and 2',5-Dichloro-4'-nitrosalicylanilide (Bayer 73) to Larvae of the Midge Chironomus tentans, by J. A. Kawatski, M. M. Ledvina, and C. R. Hansen. 1975. 7 pp.

58. Acute Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) to Nymphs of 

Mayflies (Hexagenia sp.), by C. R. Fremling. 1975. 8 pp.

59. Toxicity and Residue Dynamics of the Lampricide 3-Trifluoromethyl-4-nitrophenol 

(TFM) in Aquatic Invertebrates, by H. O. Sanders and D. F. Walsh. 1975. 9 pp.

(Reports 60 through 62 are in one cover.)

60. Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) to Nontarget Fish in 

Static Tests, by L. L. Marking and L. E. Olson. 1975. 27 pp.

61. Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) to Nontarget Fish in 

Flow-Through Tests, by L. L. Marking, T. D. Bills, and J. H. Chandler, Jr. 1975. 9 pp.

62. Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) to Selected Aquatic 

Invertebrates and Frog Larvae, by J. H. Chandler, Jr. and L. L. Marking. 1975. 7 pp.

(Reports 63 through 66 are in one cover.)

63. Laboratory Efficacy of 3-Trifluoromethyl-4-nitrophenol (TFM) as a Lampricide, by 

V. K. Dawson, K. B. Cumming, and P. A. Gilderhus. 1975. 13 pp.

64. Effects of 3-Trifluoromethyl-4-nitrophenol (TFM) on Developmental Stages of the Sea 

Lamprey, by G. W. Piavis and J. H. Howell. 1975. 8 pp.

65. Accumulation and Loss of Residues of 3-Trifluoromethyl-4-nitrophenol (TFM) in Fish 

Muscle Tissue: Laboratory Studies, by J. B. Sills and J. L. Alien. 1975.10 pp.

66. Residues of 3-Trifluoromethyl-4-nitrophenol (TFM) in a Stream Ecosystem after Treatment for Control of Sea Lampreys, by P. A. Gilderhus, J. B. Sills, and J. L. Alien. 1975. 

7pp.

67. Method for Assessment of Toxicity or Efficacy of Mixtures of Chemicals, by L. L. 

Marking and V. K. Dawson. 1975. 7 pp.

68. Development and Evaluation of On-site Toxicity Test Procedures for Fishery Investigations, by R. M. Burress. 1975. 8 pp.
INVESTIGATIONS IN FISH CONTROL

87. Ethyl-p-aminobenzoate (Benzocaine): Efficacy as an 

Anesthetic for Five Species of Freshwater Fish

By V. K. Dawson and P. A. Gilderhus

88. Influences of Selected Environmental Factors on the 

Activity of a Prospective Fish Toxicant, 2-(Digeranylamino)-ethanol, in Laboratory Tests

By C. A. Launer and T. D. Bills

89. Toxicities of the Lampricides

3-Trifluoromethyl-4-nitrophenol(TFM) 

and the 2-Aminoethanol Salt of 

2',5-Dichloro-4'-nitrosalicylanilide (Bayer 73) 

to Four Bird Species

By R. H. Hudson

UNITED STATES DEPARTMENT OF THE INTERIOR

FISH AND WILDLIFE SERVICE

Washington, D.C. 1979

Ethyl-p-aminobenzoate (Benzocaine): Efficacy as an Anesthetic for

Five Species of Freshwater Fish

by

V. K. Dawson and P. A. Gilderhus

U.S. Fish and Wildlife Service

National Fishery Research Laboratory

La Crosse, Wisconsin 54601

Abstract

Ethyl-/j-aminobenzoate (benzocaine) was tested for its efficacy as an anesthetic for rainbow 

trout (Salmo gairdneri), brown trout (Salmo trutta), northern pike (Esox lucius), carp (Cyprinus 

carpio), and largemouth bass (Micropterus salmoides). Since benzocaine is not water soluble, it 

was applied with acetone as a carrier. Concentrations of 100 to 200 mg/1 were required for large 

adult northern pike, compared with 50 to 100 mg/1 for small fish. Rates of sedation and recovery 

were slower in cold water than in warm water. Water hardness had little influence on the activity 

of benzocaine. Fish were anesthetized faster and recovered more slowly in acid than in alkaline 

water. Benzocaine produced deep anesthesia, but concentrations that rendered the fish handleable within 5 min were generally not safe for exposures longer than 15 min. Concentrations of 

benzocaine efficacious for fish were not acutely toxic to eggs of coho salmon (Oncorhynchus 

kisutch), chinook salmon (Oncorhynchus tshaivytscha), rainbow trout, brown trout, or lake trout 

(Saluelinus namaycush). Benzocaine is not registered for fishery use and is neither more effective 

nor safer than the registered anesthetic, tricaine methanesulfonate (MS-222).

Many substances have been tested for their ability 

to anesthetize fish (McFarland 1959; Bell 1967; 

McErlean 1967; Schoettger and Julin 1967; Schoettger 

and Julin 1969; Rowland and Schoettger 1969; Gilderhus et al. 1973). Few of these have been used extensively as anesthetics, however, and only one tricaine 

methanesulfonate (MS-222) is registered for fishery 

use by the U.S. Food and Drug Administration (Office 

of the Federal Register 1976).

McErlean (1967) suggested the use of ethyl-paminobenzoate (benzocaine) as a possible alternative 

to MS-222, and McErlean and Kennedy (1968) compared some anesthetic properties of the two compounds. Wedemeyer (1970) and Soivio et al. (1977) 

evaluated physiological effects of anesthesia with MS222 and benzocaine on rainbow trout (Salmo gairdneri). Both studies revealed similarities between the 

two compounds at pH 7.0.

Benzocaine and MS-222 are similar in structure but 

differ in the addition of a methanesulfonate group to 

MS-222, to make it water soluble; the amine group is 

meta-substituted in MS-222 and para-substituted in 

benzocaine.

Although benzocaine is not registered for fishery 

use, it has been used extensively in veterinary 

(Windholz 1976) and human medicine (Baker 1976).

The present study was conducted to evaluate the 

potential of benzocaine as a fish anesthetic, under 

selected water conditions, for several species and sizes 

of fish. The investigation involved a laboratory study 

with subadult fish and a field study with adult northern pike (Esox lucius). The toxicity of benzocaine to 

fish eggs which might be exposed during hatchery 

spawning operations was also evaluated.

Materials and Methods

Benzocaine (90%) used in the tests was obtained 

from Aldrich Chemical Co., Milwaukee, Wisconsin. 

Stock solutions were made up in acetone and added to 

the test vessels in the quantities needed to produce the 

desired concentrations. Fish eggs were exposed in 

glass jars containing 2.5 liters of soft reconstituted 

water. Subadult fish were exposed in polyethylene 

tanks containing 45 liters of water and adults in tanks 

containing 1QO liters.

Hardness of water used in the tests was produced by 

adding selected amounts of salts to aerated, deionized 

water as described by Marking and Dawson (1973). 

Selected pH values (from 6.5 to 9.5) were produced and 

maintained with chemical buffers according to the 

method of Dawson et al. (1975). Temperatures of 7,12,
Table 1. Efficacy ofbenzocaine as an anesthetic against five species offish in standard reconstituted water.

Species and 

average weight 

(g)

Rainbow trout

65

Brown trout

1

90

90

90

Carp

50

Largemouth bass

50

Northern pike

6

6

6

6

1,543

1,543

Temp

12

12

7

12

17

12

12

7

12

17

12

3

12

Concn

(mg/1)

50

50

50b

50

50

100

50b

60b

60b

60b

100

200

200

Time (min) to loss of:

Equilibrium3

2.8

2.1

3.9

2.4

2.5

2.6

9.3

7.0

4.0

2.8

2.5

5.0

3.0

Reflex

5.0

2.5

8.8

4.5

4.3

3.7

9.7

13.3

8.0

7.5

3.5

5.0

3.0

Exposure 

time 

(min)

10

10

10

10

10

15

15

15

15

15

15

15

15

Recovery 

time 

(min)

12.0

3.4

6.7

7.4

7.5

18.0

11.5

23.5

20.0

8.5

15.0

35.0

35.0

Survival

60

5

100

100

10

100

100

100

100

100

100

100

100

aLoss of equilibrium, stage 2 of Schoettger and Julin (1967). 

bLoss of reflex did not occur within the desired period of 5 min.

and 17 C were maintained with water baths.

We tested the anesthetic against rainbow trout, 

brown trout (Salmo trutta), northern pike, carp (Cyprinus carpio), and largemouth bass (Micropterus 

salmoides), and evaluated its toxicity to green eggs of 

coho salmon (Oncorhynchus kisutch), chinook salmon 

(Oncorhynchus tshawytscha), rainbow trout, brown 

trout, and lake trout (Salvelinus namaycush). Eggs 

were exposed to the anesthetic within 24 h after fertilization.

Fish used in the laboratory tests were obtained from 

Federal or State fish hatcheries, maintained under a 

fish culturist's care, and acclimated to test waters 2 

days before the benzocaine was added. Ten fish or 25 

fish eggs were exposed to each concentration of anesthetic.

Field tests were conducted with adult northern pike 

collected from the Mississippi River with fyke nets in 

April. Most were females (average length, 61 cm, and 

weight, 1,543 g) that had been artificially spawned 3 

days previously. Most tests were conducted at the 

State Fish Hatchery, Lansing, Iowa, in water from the 

Mississippi River (temperature, 3 C; pH, 7.8; total 

alkalinity, 135 mg/1 as CaCO3 ; and total hardness, 192 

mg/1 as CaC03 ). Tests at the National Fishery 

Research Laboratory were conducted in well water 

(temperature, 12 C; pH, 7.8; total alkalinity, 206 mg/1 

as CaC03 ; and total hardness, 224 mg/1 as CaC03 ). 

Three fish were exposed to each concentration.

The tests were designed to establish the effective 

concentration that would result in loss of reflex and

thus render the fish handleable within 5 min and still 

permit survival of all fish after at least 10 min of exposure. We determined the various stages of anesthesia 

by using characteristics defined by Schoettger and 

Julin (1967): sedation (decreased reactivity to stimuli); 

partial loss of equilibrium (swimming ability disrupted); total loss of equilibrium, stage 1 (usually the 

fish turn over but swimming ability persists); total 

loss of equilibrium, stage 2 (locomotion ceases but fish 

respond to pressure on the caudal peduncle); loss of 

reflex (failure to respond to external stimuli); and 

medullary collapse (opercular activity ceases).

We analyzed mortality data according to the method 

of Litchfield and Wilcoxon (1949) to determine LC50 's 

(concentration causing 50% mortality) and 95% confidence intervals. A safe exposure index was obtained 

by dividing the time required for the first fish to reach 

medullary collapse by the time required for fish to 

reach loss of reflex (Schoettger and Julin 1967). An 

index value of less than 1.0 indicates that the anesthetic causes mortality before it produces the desired 

level of anesthesia.

Results

Efficacy to Subadult Fish

At 12 C, rainbow trout and brown trout exposed to 

50 mg/1 of benzocaine showed a total loss of equilibrium (stage 2) within less than 3 min and loss of
reflex within 5 min (Table 1). A 10-min exposure at 50 

mg/1 caused a mortality of 40% in rainbow trout and 

95% in brown trout fry. Brown trout exposed to the 

50-mg/l concentration at 7 C did not experience loss of 

reflex within the desired period of 5 min, and at 17 C, 

90% died.

Warmwater fishes consistently withstood longer 

exposures and higher concentrations than did coldwater species. For example, largemouth bass and 

northern pike fingerlings exposed to concentrations of 

50 and 60 mg/1, respectively, at 12 C did not reach loss 

of reflex within 5 min; at 100 mg/1, however, the 

northern pike as well as carp were completely anesthetized. However, exposure to the 100-mg/l concentration for 30 min killed subadults of all five species 

(data not shown). The safe exposure indices for the different species ranged from 1.5 to 2.5.

Efficacy to Adult Northern Pike

Adult northern pike did not progress through the 

stages of anesthesia shown by small fish. Most 

remained active until they turned on their sides. They 

had then lost reflex, having bypassed total loss of equilibrium, stages 1 and 2. Several fish were hyperactive 

for 20 to 30 s immediately before they turned over. 

Northern pike exposed to 200 and 300 mg/1 in 12 C well 

water at the Laboratory exhibited some headshaking 

immediately after being placed in the solution, suggesting that the solution might be mildly irritating.

Anesthetization of adult northern pike at 3 C 

required exposure for 7 to 8 min to concentrations of 

100 to 160 mg/1 of benzocaine. A concentration of 200 

mg/1 rendered them motionless in 4.0 to 6.5 min at 3 C 

and in 2.5 to 3.5 min at 12 C. Anesthesia was induced 

in 3.5 to 4.8 min at 3 C and in 2.0 to 3.5 min at 12 C at a 

concentration of 300 mg/1 of benzocaine; however, 

some mortality occurred at the higher concentration. 

The length of time required for fish to recover in fresh 

water varied from 35 to 100 min after 15 min of exposure to 200 mg/1 at 3 C.

In the 3 C water at the Lansing (Iowa) tests, there 

were no mortalities among fish exposed to 200 or 300 

mg/1 for 30 min. In the 12 C water at the Laboratory, 

northern pike suffered 67% mortality (two of three 

fish) after 60 min of exposure to 200 mg/1 and after 30 

min of exposure to 300 mg/1.

Effect of Temperature

There was almost no difference in the rates of 

anesthesia at 12 and 17 C for brown trout, and the 

times to loss of equilibrium and reflex were only 

slightly longer for subadult northern pike at 12 C than 

at 17 C. At 7 C, however, the times to loss of equilibrium and reflex were significantly longer for both

species. Little difference was noted in recovery time 

for brown trout at any of the test temperatures, but 

northern pike recovered considerably faster at 17 C 

than at 7 or 12 C (Table 1).

Effect of Water Hardness andpH

Water hardness had little influence on the average 

time to loss of reflex in subadult brown trout; the rate 

of anesthesia, however, appeared to be somewhat 

slower at higher pH's (Table 2). The rate of recovery 

after anesthesia (10-min exposure to 50 mg/1) was not 

influenced by water hardness or pH except that recovery tended to be slower at pH 6.5 than at pH 8.0 or 9.5 

(Table 3).

Table 2. Average time (min) for 10 subadult brown 

trout to reach loss of reflex in 50 mg/l of benzocaine at 12 C in waters of different total hardness 

andpH.

Water hardness

pH

6.5

8.0

9.5

Soft

3.3

4.9

5.3

Hard

3.1

3.7

4.3

Very hard

4.2

5.0

5.2

Table 3. Average recovery time (min) for 10 subadult 

brown trout after a 10-min exposure to 50 mg/l of 

benzocaine at 12 C in waters of different total hardness and pH.

Water hardness

pH

6.5

8.0

9.5

Soft

11.2

4.6

4.8

Hard

6.3

5.7

6.4

Very hard

9.9

5.7

8.2

Safety to Fish Eggs

The toxicity of benzocaine to green eggs ranged 

from an LC50 of 88.0 mg/l for rainbow trout to 43.0 mg/l 

for coho salmon after 1 day of exposure (Table 4). The 

5-day LC50 's for eggs of these species were 47.0 and 

38.4 mg/l, respectively. The most sensitive eggs after 5 

days of exposure were those of brown trout (LC50 = 

17.8 mg/l). Toxicity to eggs of all species tested was 

not substantially increased by exposures as long as 10 

days.

Eggs of rainbow trout were resistant to benzocaine 

exposures that might be encountered during hatchery 

spawning operations. No mortalities occurred after a
Table 4. Toxicity (LCM and 95% confidence interval, mg/l) of benzocaine to fish eggs in standard 

reconstituted water at 12 C.

Exposure period (days)

1

88.0

75.3-103

62.0

53.9-71.4

65.0

56.9-74.3

43.0

34.0-54.4

64.1

54.6-75.3

5

47.0

36.2-61.0

17.8

14.1-22.4

36.5

30.1-44.2

38.4

32.2-45.8

46.0

37.7-56.1

10

42.5

33.0-54.3

14.5

11.9-17.7

29.2

23.1-37.0

32.0

28.1-36.5

44.0

33.2-58.4

Table 5. Mortalities (%) of green eggs of rainbow 

trout after exposures to different concentrations 

of benzocaine for 1 to 3 h and transfer to fresh 

water for 96 h.

Species 

Rainbow trout

Brown trout 

Lake trout 

Coho salmon 

Chinook salmon

1-h exposure to 500 mg/1 of benzocaine or to a 3-h exposure to 100 mg/1 (Table 5). Exposure for 3 h to 200 and 

500 mg/1 of benzocaine resulted in mortalities of 24 and 

100%, respectively.

Discussion

The observed progression of adult northern pike 

exposed to the anesthetic, from partial loss of equilibrum directly to loss of reflex, would present no use 

problem, since fish in the loss of reflex stage are easier 

to handle.

The activity of many chemicals is influenced by the 

pH of the test solution, often as a result of an ionization equilibrium where only the un-ionized form of the 

molecule is able to penetrate the gill membrane (Sills 

and Alien 1971; Dawson and Marking 1973; Hunn and 

Alien 1974; Dawson et al. 1977). However, the ionization constant (pKa) for benzocaine (2.38; Fasman 1976) 

is so low that little ionization occurs at the pH's of 6.5 

to 9.5 used in our tests. Therefore most benzocaine 

molecules should have been in the lipid-soluble, unionized form. The apparent increase in activity at 

lower pH's may be related to increased stress on the 

fish in acidic water.

The safe exposure indices for benzocaine (1.5-2.5) are 

closely similar to those of its structural homologue, 

MS-222, as reported by Schoettger and Julin (1967).

Benzocaine does not depress the pH of poorly buffered solutions as do the fish anesthetics MS-222 and 

quinaldine sulfate (Dawson and Marking 1973). Benzocaine, however, is not water soluble and is not registered for fishery use. Furthermore, by comparison 

with the reported efficacy (Schoettger and Julin 1967) 

and toxicity (Marking 1967) of the registered anesthetic, MS-222, benzocaine is neither more effective 

nor safer.

Concentration 

(mg/1)

100 

200 

500

Exposure period (h) 

0.5 1

0 

0 

0

0 

0 

0

0

24

100

Acknowledgment

We thank K. J. Horn for her assistance in the study 

while she was working on a Junior Academy of Science 

project.

References

Baker, C. E., Jr., publisher. 1976. Physician's desk reference, 

30th ed. Litton Industries, Inc., Oradell, N.J. 2046 pp.

Bell, G. R. 1967. A guide to the properties, characteristics, 

and uses of some general anaesthetics for fish, 2nd ed. Bull. 

Fish. Res. Board Can. 148. 9 pp.

Dawson, V. K., K. B. Cumming, and P. A. Gilderhus. 1975. 

Laboratory efficacy of 3-trifluoromethyl-4-nitrophenol 

(TFM) as a lampricide. U.S. Fish Wildl. Serv., Invest. Fish 

Control 63. 13 pp.

Dawson, V. K., K. B. Cumming, and P. A. Gilderhus. 1977. 

Efficacy of 3-trifluoromethyl-4-nitrophenol (TFM), 2',5- 

dichloro-4'-nitrosalicylanilide (Bayer 73), and a 98:2 mixture as lampricides in laboratory studies. U.S. Fish Wildl. 

Serv., Invest. Fish Control 77. 11 pp.

Dawson, V. K., and L. L. Marking. 1973. Toxicity of mixtures 

of quinaldine sulfate and MS-222 to fish. U.S. Fish Wildl. 

Serv., Invest. Fish Control 53.11 pp.

Fasman, G. D., editor. 1976. Handbook of biochemistry and 

molecular biology, Section D Physical and Chemical 

Data, 3rd ed., vol. 1. CRC Handbook, Chemical Rubber Co., 

Cleveland, Ohio. 576 pp.

Gilderhus, P. A., B. L. Berger, J. B. Sills, and P. D. Harman. 

1973. The efficacy of quinaldine sulfate:MS-222 mixtures 

for the anesthetization of freshwater fish. U.S. Fish Wildl. 

Serv., Invest. Fish Control 54. 9 pp.

Howland, R. M., and R. A. Schoettger. 1969. Efficacy of 

methylpentynol as an anesthetic on four salmonids. U.S. 

Fish Wildl. Serv., Invest. Fish Control 29. 11 pp.

Hunn, J. B., and J. L. Alien. 1974. Movement of drugs across 

the gills of fishes. Annu. Rev. Pharmacol. 14:47-55.

Litchfield, J. T., Jr., and F. Wilcoxon. 1949. A simplified 

method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96(2):99-113.

Marking, L. L. 1967. Toxicity of MS-222 to selected fishes. 

U.S. Fish Wildl. Serv., Invest. Fish Control 12. 10 pp.

Marking, L. L., and V. K. Dawson. 1973. Toxicity of quinaldine sulfate to fish. U.S. Fish Wildl. Serv., Invest. Fish 

Control 48. 8 pp.

McErlean, A. J. 1967. Ethyl p-aminobenzoate: an anesthetic 

for cold-blooded vertebrates. Copeia 1967(1):239-240.
McErlean, A. J., and V. S. Kennedy. 1968. Comparison of

some anesthetic properties of benzocaine and MS-222.

Trans. Am. Fish. Soc. 97(4):496-498. 

McFarland, W. N. 1959. A study of the effects of anesthetics

on the behavior and physiology of fishes. Publ. Inst. Mar.

Sci., Univ. Tex. 6:23-55. 

Office of the Federal Register. 1976. Tricaine methanesulfonate. Page 157 in Code of Federal regulations, title 21,

parts 500-599. Office of the Federal Register, Washington,

B.C. 

Schoettger, R. A., and A. M. Julin. 1967. Efficacy of MS-222

as an anesthetic on four salmonids. U.S. Fish Wildl. Serv.,

Invest. Fish Control 13. 15 pp. 

Schoettger, R. A., and A. M. Julin. 1969. Efficacy of quinaldine as an anesthetic for seven species of fish. U.S. Fish

Wildl. Serv., Invest. Fish Control 22.10 pp. 

Sills, J. B., and J. L. Alien. 1971. The influence of pH on the

efficacy and residues of quinaldine. Trans. Am. Fish. Soc.

100(3):544-545.

Soivio, A., K. Nyholm, and M. Huhti. 1977. Effects of anesthesia with MS 222, neutralized MS 222 and benzocaine on

the blood constituents of rainbow trout, Salmo gairdneri. J.

FishBiol. 10(1):91-101. 

Wedemeyer, G. 1970. Stress of anesthesia with M.S. 222 and

benzocaine in rainbow trout (Salmo gairdneri}. J. Fish. Res.

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Windholz, M., ed. 1976. The Merck index, 9th ed. Merck &

Co., Inc., Rahway, N.J. 1313 pp. + indices.

Influences of Selected Environmental Factors on the Activity of a 

Prospective Fish Toxicant, 2-(Digeranylamino)-ethanol, in Laboratory

Tests

by

C. A. Launer and T. D. Bills

U.S. Fish and Wildlife Service

National Fishery Research Laboratory

La Crosse, Wisconsin 54601

Abstract

Fathead minnows (Pimephales promelas), brown trout (Salmo trutta), and rainbow trout (S. 

gairdneri) were used to assess the influences of temperature, pH, turbidity^ ultraviolet light, and 

aquatic vegetation on the toxicity of an experimental fish toxicant, 2-(digeranylamino)-ethanol 

(GD-174). Also examined was the feasibility of chemical counteraction by oxidation or reduction. 

The activity of the compound was reduced in cold water, in acid water, and in turbid water where 

the turbidities were produced by bentonite or Crowley silt loam, and in water containing waterweed (Elodea sp.K The activity was not affected by exposure to ultraviolet light, by the presence 

of duckweed (Lemna sp.), or by turbidities produced by kaolin or barium sulfate. GD-174 can be 

counteracted by either oxidation or reduction; oxidation by potassium permanganate was the 

most effective procedure tested.

Rotenone and antimycin are currently the only two 

piscicides registered for use in the United States. Both 

are general toxicants. Through an intensive screening 

program seeking selective chemicals for the control of 

undesirable fishes, Marking (1974) determined that a 

terpine derivative, 2-(digeranylamino)-ethanol (GD174), was more toxic to carp than to other warmwater 

species under laboratory conditions. Although the 

chemical has shown great promise in laboratory 

studies as a general fish toxicant and potential for the 

selective control of carp, results in small-scale field 

applications have been inconsistent. We conducted 

several laboratory studies to assess the effects of 

water temperature, pH, turbidity, aquatic vegetation, 

and ultraviolet radiation on the activity of GD-174. 

We also examined the effectiveness of selected oxidizing and reducing agents for counteracting the 

chemical.

Materials and Methods

Stock solutions of technical grade GD-174 obtained 

from Glidden Durkee, Division of SCM Corporation, 

Jacksonville, Florida, were prepared by solubilizing 

the chemical in an equal volume of 20% glacial acetic 

acid and then diluting to the desired volume with 

water. To prepare test solutions of desired concentrations, we pipetted portions of the stock solutions 

into test vessels and then stirred the mixture to ensure 

homogeneity.

Fish species tested were brown trout, Salmo trutta 

(average weight 7.5 g); rainbow trout, S. gairdneri 

(2.5 g); and fathead minnows, Pimephales promelas 

(1.1 g). Fish were either obtained from Federal fish 

hatcheries or cultured at the National Fishery 

Research Laboratory, La Crosse, Wisconsin.

Acute toxicity tests for the effects of water temperature and pH on the activity of GD-174 were conducted 

according to methods described by the Committee on 

Methods for Acute Toxicity Tests with Aquatic 

Organisms (1975).

In photodegradation studies, stock solutions of GD174 were exposed to UV light (21 /iW/cm2 X 100) from 

a GE-275W sunlamp at a distance of 25 cm from the 

surface for periods of 6, 12, or 24 h. These solutions 

were maintained in a temperature-controlled water 

bath and continuously stirred to ensure homogeneity. 

Portions of these solutions were then added to test 

vessels and their toxicities compared with those of reference solutions aged in darkness for the same period.

In studies on turbidity, GD-174 was added to a 

series of solutions containing either barium sulfate, 

kaolin, bentonite, or Crowley silt loam (classified as a 

Typic Albaqualf; consists of 6% sand, 83% silt, 11% 

clay) and having turbidities of 25, 50, 75, and 100 

nephelometric turbidity units (NTU). Turbidity was 

measured with an HF DRT-100 model turbidimeter. 

The solutions were compared to a reference series of 

clear water (0.53 NTU), and the toxicities compared.

In studies to determine the effects of aquatic vege-
tation on the toxicity of GD-174, waterweed (Elodea 

sp.) and duckweed (Lemna sp.) collected from the wild 

were used. In outdoor tests, GD-174 was added to 

polyethylene tanks containing reconstituted water 

and either 0, 5, 10, or 15 g/1 of waterweed or 0 or 5 g/1 of 

duckweed. In outdoor tests, 0, 2, 4, 8, or 12 g/1 of 

waterweed were used. After 24 h, the vegetation was 

removed, fish were introduced, and the toxicities were 

compared with those of reference solutions which had 

contained no vegetation.

In counteraction studies, potassium permanganate 

(KMnO4 ) at 2 mg/1, chlorine at 0.05 mg/1 (active C12 

from commercial grade calcium hypochlorite), or 

sodium thiosulfate (Na2S2O3 ) at 10 mg/1 was introduced into a series of GD-174 solutions 6 h before 

the introduction of fish. The toxicities of these solutions were then compared with that of reference 

solutions.

The method of Litchfield and Wilcoxon (1949) was 

used to calculate LC50 's and 95% confidence intervals. 

Significant differences in all data were based on P = 

0.05. All reported data met the chi-square test for 

acceptability.

Table 1. Toxicity of 2-(digeranylamino)-ethanol to fathead minnows in water of selected temperatures and 

pH's.

Temp

ra

7

12

17

22

12

12

12

12

LC50 and 95% confidence interval (jtl/1)

pH

7.5

7.5

7.5

7.5

6.5

7.5

8.5

9.5

24 h

>1.0

0.467

0.377-0.578

0.196

0.169-0.228

0.100

0.0803-0.125

0.820

0.712-0.943

0.386

0.329-0.452

0.243

0.225-0.263

0.178

0.146-0.216

96 h

0.547

0.461-0.649

0.200

0.164-0.244

0.150

0.126-0.179

0.0750

0.0573-0.0982

0.324

0.274-0.383

0.137

0.113-0.166

0.100

0.0788-0.126

0.0730

0.0653-0.0815

Results

Water Temperature

Toxicity of GD-174 was greater at higher than at 

lower water temperatures (Table 1). At 96 h, the LC50 's 

for fathead minnows were significantly higher at 12 C 

than at 7 C and at 22 C than at 17 C. Furthermore, the 

toxic action was more rapid at the higher temperatures: there were no significant differences between 

the 24- and 96-h LC50 's at either 17 or 22 C, whereas at 

7 and 12 C the LC50 's for 96 h equaled only about half 

those for 24 h (Table 1).

Turbidity

The activity of GD-174 was severely reduced in 

turbid water where the primary source of turbidity 

was produced by either bentonite or Crowley silt loam. 

In fact, no mortality of fathead minnows was observed 

in solutions where bentonite was the source of turbidity. A slight reduction in toxicity was observed in 

kaolin turbidities, and barium sulfate turbidities had 

no significant effect on toxicity (Table 3).

pH

The GD-174 was more toxic to fathead minnows in 

alkaline than in acid waters (Table 1). At 24 h, there 

were significant differences between the LC50 's at each 

pH tested (6.5, 7.5, 8.5, and 9.5), and a fourfold difference between pH 6.5 and pH 9.5. At 96 h, there were 

no significant differences between pH's 7.5 and 8.5 or 

between pH's 8.5 and 9.5.

Ultraviolet Light

In tests in which stock solutions of GD-174 exposed 

to UV light were compared with unexposed reference 

solutions, no significant differences were found in the 

toxicities to fathead minnows, based on 96-h LC50 's 

(Table 2).

Table 2. Toxicity of 2-(digeranylamino)-ethanol solutions exposed to ultraviolet light (21 pW/cm2 X 100) 

to fathead minnows in soft water at 12 C.

Exposure3 or

LC50 and 95% confidence intervals (/J/l)

aging time (h) Unexposed solutions15 Exposed solutions

6

12

24

0.268

0.241-0.298

0.200

0.172-0.232

0.172

0.127-0.232

0.250

0.219-0.285

0.250

0.220-0.283

0.150

0.121-0.185

aTime the solutions were irradiated with ultraviolet light. 

bUnexposed solutions were shielded with aluminum foil.
Table 3. Effects of selected turbidities on the toxicity of 2-(digeranylamino)-ethanol to fathead minnows. 

Source of turbidity3 , exposure time, and LC50 's and 95% confidence intervals (^1/1)

Turbidity

(NTU)

0.53b

25

50

75

100

Bentonite

24 h

0.410

0.345-0.487

>2.0

>2.0

>2.0

>2.0

96 h

0.142

0.119-0.169

>2.0

>2.0

>2.0

>2.0

Crowley silt loam

24 h

0.353

0.285-0.438

0.353

0.285-0.438

0.690

0.552-0.862

0.790

0.681-0.916

0.750

0.582-0.967

96 h

0.128

0.110-0.149

0.247

0.234-0.357

0.289

0.234-0.357

0.290

0.249-0.338

0.292

0.250-0.341

Kaolin

24 h

0.500

0.413-0.605

0.542

0.435-0.675

0.560

0.477-0.658

0.557

0.446-0.696

0.640

0.549-0.745

96 h

0.128

0.108-0.152

0.194

0.165-0.227

0.209

0.187-0.234

0.196

0.165-0.232

0.221

0.181-0.270

Barium sulfate

24 h

0.560

0.475-0.660

0.448

0.320-0.627

0.531

0.400-0.704

0.474

0.394-0.570

0.532

0.430-0.658

96 h

0.141

0.118-0.168

0.123

0.108-0.139

0.140

0.109-0.179

0.184

0.155-0.218

0.143

0.120-0.171

aGrams of substance per 15 liters of solution used to produce turbidities of 25, 50, 75, and 100 NTU's were: bentonite 1.5, 3.75, 

7.5, and 15.0; Crowley silt loam-1.8, 3.45, 5.1, and 6.75; kaolin 0.375, 0.75, 1.125, and 1.5; and barium sulfate 0.27, 0.615, 

0.99, and 1.35.

 Turbidity of standard reconstituted water.

Table 4. Toxicity of 2-(digeranylamino)-ethanol to three 

species offish after 24-h exposure of test solutions to 

duckweed or waterweed.

Species of fish,

and type and amount

(g/1) of vegetation

Brown trout 

Duckweed 

0

Rainbow trout 

Waterweed 

0

5

10

15

LC50 and 95% confidence interval

24 h

0.347 

0.278-0.433

0.323 

0.275-0.378

0.298 

0.248-0.358

0.562 

0.495-0.638

0.800 

0.694-0.921

1.07 

0.931-1.23

96 h

0.323 

0.270-0.386

0.263 

0.198-0.347

0.264 

0.222-0.314

0.562 

0.495-0.638

0.800 

0.694-0.921

1.07 

0.931-1.23

Fathead minnows

Waterweed

0

2

4

8

12

0.728

0.688-0.771

0.800

0.736-0.869

1.06

0.889-1.26

1.00

0.871-1.15

1.40

1.05-1.88

0.218

0.183-0.259

0.390

0.344-0.440

0.363

0.300-0.439

0.618

0.560-0.681

0.720

0.657-0.789

Aquatic Vegetation

Duckweed appeared to have no effect on the toxicity 

of GD-174, whereas waterweed reduced the toxicity 

significantly (Table 4). The reduction was directly 

proportional to the amount of waterweed in the solutions. In rainbow trout, toxicity decreased 20.1% for 

each 1 g increase of waterweed per liter of solution, 

and results with fathead minnows were similar.

Counteraction

Our study indicated that GD-174 was subject to 

detoxification by both oxidation and reduction. 

However, sodium thiosulfate and chlorine were considerably less effective than potassium permanganate 

(Table 5).

Table 5. Toxicity of 2-(digeranylamino)-ethanol 

solutions containing selected oxidizing or reducing 

agents to fathead minnows in soft water at 12 C.

Agent and 

concentration

Reference

solution

KMnO4

(2 mg/1)

(0.05 mg/1) 

Na2S203 

(10 mg/1)

LC50 and 95% confidence interval (pll\)

24 h

0.250

0.192-0.324

2.45 

2.04-2.97 

0.970

0.844-1.12 

0.635 

0.560-0.720

96 h

0.0895

0.0688-0.116

1.42 

1.14-1.77 

0.450

0.382-0.529 

0.263 

0.197-0.351

aCommercial grade calcium hypochlorite (65% available C12 )
Discussion and Conclusions

The compound GD-174, like rotenone (Gersdorff 

1943; Spitler 1970) and antimycin (Berger et al. 1969), 

was more toxic at high than at low temperatures. 

However, unlike rotenone (Brooks 1961; Spitler 1970) 

and antimycin (Berger et al. 1969), GD-174 was more 

toxic in alkaline than in acid waters. Toxic action of 

GD-174 did not appear to be altered by ultraviolet 

light, whereas that of both antimycin and rotenone is 

decreased (Dawson 1973; Cheng et al. 1972).

In tests with plants, the toxicity was reduced by the 

submerged waterweed, whereas the floating duckweed 

had no effect. Although it is difficult to assess the 

cause of the differences, it could be related to differences in metabolism, or to differential sorption of 

the toxicant onto the surface of the plants. Because 

submerged vegetation has much more area in contact 

with the solution than the floating variety, it would 

reduce activity by a greater degree.

In field situations, the inhibiting effects of aquatic 

vegetation and turbidity on the activity of GD-174 are 

likely to be additive. Furthermore, interactions between those factors and pH and temperature, which 

may themselves either antagonize or enhance the 

activity, will make difficult the choice of the proper 

concentration for selective carp control. Therefore, a 

precise on-site toxicity test will be necessary to prescribe the proper concentration for field application.

The counteraction studies showed that the toxicant

could be deactivated rapidly with strong oxidizing or 

reducing agents. Potassium permanganate was the 

most effective and would be the chemical of choice for 

field application. However, no chemicals are currently 

registered for aquatic use of this type.

References

Berger, B. L., R. E. Lennon, and J. W. Hogan. 1969. Laboratory studies on antimycin A as a fish toxicant. U.S. Fish

Wildl. Serv., Invest. Fish Control 26. 21 pp. 

Brooks, I. C. 1961. Research methods and findings on fish

toxicants and their application. Res. Dep., S. B. Penick and

Co., New York. 10 pp. 

Cheng, H., I. Yamamoto, and J. E. Casida. 1972. Rotenone

photodecomposition. J. Agric. Food Chem. 20(4):850-856. 

Committee on Methods for Acute Toxicity Tests with

Aquatic Organisms. 1975. Methods for acute toxicity tests

with fish, macroinvertebrates, and amphibians. Ecol. Res.

Ser. EPA [Environ. Prot. Agency]-660/3-75-009. 61 pp. 

Dawson, V. K. 1973. Photodecomposition of the piscicides

TFM (3-trifluoromethyl-4-nitrophenol) and antimycin.

M. S. Thesis. University of Wisconsin, La Crosse. 65 pp. 

Gersdorff, W. A. 1943. Effect of change of temperature on

relative toxicity of rotenone and phenol. J. Agric. Res.

67(2):65-80. 

Litchfield, J. T., Jr., and F. Wilcoxon. 1949. A simplified

method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96(2):99-113. 

Marking, L. L. 1974. Toxicity of 2-(digeranylamino)-ethanol,

a candidate selective fish toxicant. Trans. Am. Fish. Soc.

103(4):736-742. 

Spitler, R. J. 1970. An analysis of rotenone treatments for

elimination of fish populations in southern Michigan lakes,

1957-1967. Mich. Acad. 3(l):77-82.
Toxicities of the Lampricides 3-Trifluoromethyl-4-nitrophenol

(TFM) and the 2-Aminoethanol Salt of 2',5-Dichloro-4'-nitrosalicylanilide (Bayer 73) to Four Bird Species 1

by

Rick H. Hudson2

U.S. Fish and Wildlife Service

Patuxent Wildlife Research Center

Laurel, Maryland 20811

Abstract

The acute oral toxicities of the lampricides 3-trifluoromethyl-4-nitrophenol (TFM) and the 2- 

aminoethanol salt of 2',5-dichloro-4'-nitrosalicylanilide (Bayer 73, Bayluscide) were determined in 

mallards (Anas platyrhynchos), ring-billed gulls (Larus delawarensis), bobwhites (Colinus virginianus), and California quail (Lophortyx californicus). Ring-billed gulls were the most sensitive 

to both chemicals, alone and in combination. Field grade TFM (35% TFM in N,N-dimethylformamide) was toxic; median lethal dosages (LD^'s) ranged from 250 mg/kg in gulls to 546 mg/kg in 

California quail. Toxicity of Bayer 73 (70%) was lower; LDM, values ranged from 500 mg/kg in 

gulls to more than 2,000 mg/kg in mallards and bobwhites. Field grade TFM was more toxic than 

purified TFM (> 96% pure) to mallards. Toxicity of a mixture of Bayer 73 with field grade TFM 

was additive in gulls and possibly additive in mallards. Mallards exposed for 48 h to drinking and 

swimming water containing the lampricides showed mild signs of intoxication but no birds 

became severely ill. On the basis of these studies, use of TFM or Bayer 73, alone or in combination, presents little toxic hazard to birds when applied in the amounts routinely used for 

lamprey control.

The toxicant 3-trifluoromethyl-4-nitrophenol (TFM) 

has been used extensively in the Great Lakes region 

for controlling the sea lamprey (Petromyzon marinus). 

Since Howell et al. (1964) reported that the 2-aminoethanol salt of 2',5-dichloro-4'-nitrosalicylanilide 

(Bayer 73, Bayluscide) acts synergistically to increase 

the toxicity of TFM to sea lamprey larvae, Bayer 73 

has sometimes been used in combination with TFM at 

concentrations corresponding to 2% or less of the 

TFM concentrations, based on active ingredients of 

each.

Under ideal conditions TFM is 2 to 10 times more 

toxic to sea lamprey larvae than to most fish, but it is 

also toxic to many other organisms (Schnick 1972). 

Bayer 73, on the other hand, is extremely toxic to a 

wide variety of aquatic organisms, but appears to have

This study was funded in part by the Great Lakes Fishery 

Commission, through the National Fishery Research 

Laboratory, La Crosse, Wisconsin. The work was 

conducted at the Denver Wildlife Research Center before 

March 1976, when the Environmental Contaminants Evaluation Program was consolidated at the Patuxent Wildlife 

Research Center.

Present address: Department of Zoology, University of 

Washington, Seattle, Washington 98195.

little effect on mammals (Marking and Hogan 1967). 

Although the toxicity of TFM to sea lampreys is 

increased by the addition of Bayer 73, its selectivity is 

decreased; consequently fish kills occur more frequently when the combination of the two chemicals is 

used (Schnick 1972). The present study was conducted 

to gather basic data on the toxicity of TFM, Bayer 73, 

and a mixture of the two chemicals to four bird 

species mallards (Anas platyrhynchos), ring-billed 

gulls (Larus delawarensis), bobwhites (Colinus virginianus), and California quail (Lophortyx californicus) and to assess the toxic hazard of aqueous 

solutions of these compounds to mallards.

Materials and Methods

The lampricides used in these studies, obtained from 

the National Fishery Research Laboratory, La Crosse, 

Wisconsin, consisted of purified TFM (TFM[A]; > 96% 

pure, Aldrich Chemical Co., Lot No. 060217); field 

grade TFM (TFM[FG]; 35% TFM in N,N-dimethylformamide); and Bayer 73 (70% pure, Chemagro Corp., 

Lot No. 8059410). Reagent grade N,N-dimethylformamide (DMF), obtained locally, was tested alone and in 

combination with TFM(A) to assess its contribution to

1
toxicity in the field grade formulation.

Mallards used in this study were raised at the 

Denver Wildlife Research Center, either from stock 

lines (for the studies with purified and field grade 

TFM, and DMF) or from 1-day-old ducklings obtained 

from the Max McGraw Wildlife Foundation, Dundee, 

Illinois (for the studies on Bayer 73 and the mixture of 

Bayer 73 with field grade TFM). Ring-billed gulls were 

obtained from a captive breeding colony maintained at 

the Denver Wildlife Research Center, and California 

quail were raised at the Center, from stock lines. Bobwhites came from Highland Game Bird Farms, Franktown, Colorado. Age and sex of test birds varied, 

depending on availability. Previous studies have indicated that, unlike mammals, nonbreeding birds show 

only minor sex-dependent differences in sensitivity to 

oral administration of pesticides in acute toxicity tests 

(Tucker and Crabtree 1970; Tucker and Haegele 1971), 

and differences between age groups are generally small 

(Hudson et al. 1972). However, these differences occasionally are significant and should not be ignored. The 

age and sex of the birds used in the present tests are 

shown in Table 1.

Table 1. Acute oral toxicity (LD50)a of lampricides to 

four species of birds.

Lampricide0

TFM(FG) + 

Speciesb TFM(A) TFM(FG) Bayer 73 Bayer 73

Mallard 458 308 > 2,000 472

(324-649) (237-400) (408-546) 

Ring-billed 250 500 154

gull (170-368) (77.8-3,210) (118-200) 

Bobwhite > 2,000 435

(335-565)

California 546 

quail___________(313-950)_______________

aLD50 values are expressed as milligrams of toxicant per 

kilogram of body weight; 95% confidence limits are shown in 

parentheses.

bAge and sex of birds exposed to the different chemicals 

follow. Mallards: TFM(A) and TFM(FG), 1-year-old drakes; 

Bayer 73, 1-year-old hens; TFM(FG) + Bayer 73, 14- to 17- 

week-old drakes. Ring-billed gulls: TFM(FG), immature 

males and females; Bayer 73, adult females; TFM(FG) + 

Bayer 73, immature and adult males and females. Bobwhites: all tests, 18- to 24-week-old cocks. California quail: 

TFM(FG), 1-year-old hens.

eLampricides: TFM(A) = purified, > 96% active 3-trifluoromethyl-4-nitrophenol; TFM(FG) = field grade, 35% active 

TFM in the carrier N,N-dimethylformamide (DMF); and 

Bayer 73 = 70% active 2-aminoethanol salt of 2',5-dichloro4'-nitrosalicylanilide. The LD50 's of DMF alone (not shown) 

were > 2,000 mg/kg in mallards, > 185 in ring-billed gulls, 

and > 460 in bobwhites.

Mallards and ring-billed gulls were exposed in 

groups of 2 to 16 birds in unheated, concrete-floored 

pens (2.4 X 3.0 m, 2.4 m high), which were enclosed and 

covered, and contained a 37-liter swimming pond with 

about 2,450 cm2 of water surface. Quail were held individually in indoor cages (30 X 56 cm, 23 cm high) provided with a water trough. All birds were held in the 

pens for at least 1 day before they were exposed to 

lampricides. Temperature in the indoor test room was 

maintained at 20 to 24 C. Mallards and quail were fed a 

commercially prepared game bird diet, and gulls a 

commercially prepared dry dog food. Feed was available ad libitum, except during a 20-h fasting period 

before oral administration of toxicants for the acute 

toxicity tests. Water was available ad libitum, and 

fresh water was constantly available in the mallard 

and gull cages, except during 48-h exposures of 

mallards to lampricides in the water under static 

conditions.

For determination of the acute oral median lethal 

dosage (LD50 ), the test compounds were administered 

either by stomach tube or in gelatin capsules inserted 

through glass tubing. Each compound was delivered 

to the level of the proventriculus in mallards and gulls 

and to the crop in both species of quail. Gelatin capsules were used for the administration of TFM(A) to 

mallards; of Bayer 73 to mallards, gulls, and bobwhites; and of DMF to gulls and bobwhites. A 

stomach tube was used to deliver TFM(FG) to 

mallards, gulls, and California quail, and DMF to 

mallards.

In tests to assess the toxicity of a mixture of the 

lampricides, TFM(FG) and Bayer 73 were administered sequentially (because the chemicals could not 

be successfully administered simultaneously) to 

mallards, gulls, and bobwhites as follows: (1) Bayer 73 

in gelatin capsules was administered through glass 

tubing; (2) a small amount of pure DMF was administered by stomach tube; and (3) within 5 to 18 min, 

TFM(FG) was administered by stomach tube. Dosages 

of the individual lampricides administered to each 

animal provided the active ingredients in the ratio of 

98 parts TFM to 2 parts Bayer 73 (34.3% active TFM 

and 0.7% active Bayer 73 in the mixture).

Three to six birds were treated at each of two to four 

geometrically spaced dosage levels. Acute oral LD50 

values were computed by the methods of Thompson 

(1947) and Weil (1952).

The indices used to describe the toxicity of lampricides administered in combination were derived by 

Marking and Dawson (1975). An additive index 

greater than 0 indicates greater than additive toxicity 

(synergism), an index less than 0 indicates less than 

additive toxicity (antagonism), and an index of 0 indicates simple additive toxicity of the toxicants in a 

mixture. If the confidence interval for the additive
index overlaps 0, simple additive toxicity is indicated.

Mallards were exposed to lampricide-contaminated 

water in their swimming ponds for 48 h under static 

conditions. Experimental groups in these tests were 

composed of five males and five females. The ages of 

the ducks exposed to the different chemicals were as 

follows: TFM(A), 6-8 weeks; TFM(FG) and Bayer 73, 1 

year; and the mixture of TFM(FG) with Bayer 73, 17 

weeks. Appropriate quantities of TFM(A) were 

dissolved in 59.2 ml ethanol and then added to the tap 

water in the swimming ponds to make up treatment 

levels of 5, 15.8, 50, and 500 mg/1 of active TFM. Only 

the solvent (59.2 ml of ethanol) was added to the swimming pond of the control group. In other swimming 

ponds TFM(FG) was at concentrations of 57.1 and 286 

mg/1 (20 and 100 mg/1 active TFM); Bayer 73 at concentrations of 0.01, 0.10, and 1.0 mg/1 (0.007, 0.07, and 

0.70 mg/1 of active Bayer 73); and the mixture of 

TFM(FG) and Bayer 73 (of the same composition as 

that administered orally) at concentrations of 11.7, 

58.4, and 292 mg/1 (4.01 and 0.082, 20.0 and 0.409, and 

100 and 2.04 mg/1 of active TFM and Bayer 73, respectively). At the end of the 48-h exposure, the lampricidecontaminated water was flushed out and replaced with 

tap water, augmented by a small continuous flow. The 

swimming ponds of the controls for all tests other than 

TFM( A) contained untreated tap water.

Mortality, signs of intoxication, and body weight 

changes were observed for 14 to 21 days after treatment. (In general, the weights of the survivors of all 

studies were normal by the end of the observation 

periods.)

Results

Acute Oral Toxicity Tests

Of the species exposed, ring-billed gulls were the 

most sensitive to all of the compounds (TFM[A] not 

tested), alone or in combination (Table 1). Median 

lethal dosages for TFM(FG) ranged from 250 mg/kg in 

gulls to 546 mg/kg in California quail. Bayer 73 was 

less toxic than TFM; LD50 values ranged from 500 

mg/kg in gulls to > 2,000 mg/kg in mallards and 

bobwhites.

The additive toxicity indices of combinations of 

lampricides and solvent (DMF) are presented in 

Table 2. TFM(A) and DMF produced an additive index 

of 1.98 (greater than additive toxicity) with mallards. 

TFM(FG) and Bayer 73 produced an additive index of 

-0.506, which suggests less than additive toxicity, 

although the range for that index could not be calculated because there was no 95% confidence interval for 

Bayer 73 tested individually. Since the index is close to 

zero, however, the toxicity possibly was additive. The 

combination of TFM(FG) and Bayer 73 was additive in

toxicity to ring-billed gulls (the interval for the additive index, -0.179 to 2.17, overlapped zero).

Mallards treated with oral doses of TFM(FG) drank 

excessive amounts of water and showed a number of 

other responses: regurgitation, stumbling, excessive 

sitting, weakness, incoordination, imbalance, labored 

breathing, immobility, and terminal wing-beat convulsions accompanied by arching of the head and neck 

over the back. Mallards treated with TFM(A) also displayed these signs of intoxication (except for weakness), in addition to social withdrawal, imperturbability, convulsions, and temporary whole-body rigidity. Signs of intoxication were produced more rapidly 

by TFM(FG) than by TFM(A): signs of TFM(FG) 

intoxication appeared as soon as 1 min after treatment, and deaths from 4 to 30 min after treatment, 

whereas the effects from TFM(A) did not appear for 13 

min and deaths occurred 20 to 50 min after treatment. 

Recovery (when it occurred) usually was complete 

within 4 h after treatment with TFM(FG), but was 

prolonged for up to 7 or 8 days after treatment with 

TFM(A).

The signs of intoxication observed in ring-billed 

gulls dosed with TFM(FG) were similar to those observed in mallards with the addition of rapid breathing, imperturbability, wing-droop, outspread wings, 

and temporary whole-body rigidity, but the gulls did 

not display stumbling, weakness, imbalance, immobility, or terminal wing-beat convulsions. The timing 

of the appearance of signs and deaths was usually 

similar to that in mallards; however, one gull died 11 

days after treatment. There was no apparent sexrelated difference in response among the gulls.

After treatment with TFM(FG), California quail displayed signs of intoxication similar to those observed 

in gulls and mallards, except that regurgitation did 

not occur. The timing of the appearance of signs and 

deaths was also similar.

Bayer 73 produced intoxication in all of the species 

tested at all of the dosage levels administered. Signs 

observed in mallards included tenseness of the masseter, regurgitation, high carriage, excessive drinking 

and sitting, imperturbability, incoordination, imbalance, rapid and labored breathing, tremors, and 

temporary whole-body rigidity followed by death. 

Signs observed in bobwhites and gulls were similar; 

however, regurgitation in gulls was more profuse, 

especially at the higher dosage levels, and probably 

accounts for the wide confidence interval (Table 1) 

calculated for this test (regurgitation did not occur in 

bobwhites). Deaths occurred at 500, 1,000, and 2,000 

mg/kg in mallards (one of the six mallards treated at 

each of these levels died), and one of three bobwhites 

treated at 2,000 mg/kg died. Toxic signs in all three 

species appeared as soon as 15 min after treatment 

and persisted for up to 24 h; deaths occurred from
30 min to 3 h after treatment.

After treatment with the mixture of TFM(FG) and 

Bayer 73, signs observed in mallards included most of 

those observed after treatment with the individual 

chemicals. The timing of the appearance of effects, 

deaths, and recovery in mallards was similar to that 

after treatment with TFM(FG) alone. Toxic signs 

observed after treatment of ring-billed gulls with the 

mixture were similar to those observed in mallards, 

although the appearance of signs, deaths, and recovery was more rapid: deaths usually occurred in 2 to 12 

min and recovery within 1 h. Signs of intoxication 

observed after treatment of bobwhites with the 

mixture were similar to those observed in mallards, 

except that regurgitation did not occur. Timing of the 

appearance of signs, deaths, and recovery was also 

similar to that observed in mallards.

Signs of intoxication in mallards (1-year-old drakes) 

given DMF at dosages up to 2,000 mg/kg were limited 

to slowness, incoordination, and falling, which developed within 10 min after treatment. All ducks used in 

the tests had recovered within about 24 h. Bobwhites 

(19- to 20-week-old cocks) treated at dosages up to 505 

mg/kg and gulls (immature and adult males and 

females) treated at dosages up to 249 mg/kg showed no 

signs of intoxication after DMF treatment.

Water Exposure Toxicity Tests

No mortalities occurred in mallards exposed to 

lampricides in the ponds in their enclosures, and body 

weights by the end of the observation periods were 

normal. However, various clinical signs of poisoning 

were observed.

All of the 6- to 8-week-old mallards exposed to 5 mg/1 

TFM(A) appeared normal throughout the 48-h exposure and subsequent 12-day observation period. In the 

group treated with 15.8 mg/1, several birds appeared 

wobbly and showed slight masseter tenseness about 

7 h after exposure began. About 22 h later most of the 

mallards in this test group may have had slight 

degrees of incoordination, and 49 h after exposure the 

group was underactive and all birds tended to sit; one 

drake showed a moderate degree of incoordination and 

jerky movements. All birds in the 50-mg/l group were 

strongly affected after 3 h of exposure. They displayed 

excessive sitting, underactivity, and weakness of the 

leg muscles, and some ran and fell with moderate to 

extreme degrees of incoordination. Although one bird 

was extremely incoordinated 22 h after exposure 

began, most birds had begun to recover. All were 

normal 24 to 48 h after exposure ended. The group 

exposed to 500 mg/1 apparently avoided consumption 

of the solution after initial exposure. This group sat 

huddled together more than controls and stumbled 

and showed slight to moderate degrees of incoordination; they appeared normal 24 h after exposure 

ended.

The 1-year-old mallards exposed to 57.1 mg/1 

TFM(FG) appeared normal throughout the study. 

Those exposed to 286 mg/1 displayed incoordination, 

slowness, and stumbling; however, recovery occurred 

within 24 h after the end of treatment.

During the water exposure studies of Bayer 73 and 

the mixture of Bayer 73 with TFM(FG), only mild 

signs of intoxication were observed. One mallard 

exposed to 0.10 mg/1 Bayer 73 showed slight incoordination 24 to 48 h after the beginning of treatment, 

and one exposed to 292 mg/1 of the mixture showed 

slight incoordination on the day treatment was begun.

Discussion

The present tests showed that TFM is moderately 

toxic to birds exposed by acute oral administration, 

and that it has about the same magnitude of toxicity 

in birds as in mammals. Bayer 73 appears to have a 

low order of toxicity in birds, although birds may be 

more susceptible than mammals to acute oral administration. The analysis for additive toxicity revealed 

that the mixture of TFM(A) and DMF worked synergistically in mallards (Table 2). Maki et al. (1975), who 

studied the toxicity of purified and field grade TFM to 

several species of algae, noted that DMF in the field 

grade material may augment the toxicity of TFM; 

Sanders and Walsh (1975) found a synergistic effect of 

the field grade formulation in crayfish (Orconectes 

raw's); and Marking and Olson (1975) noted that the 

field grade TFM was more toxic to several species of 

fish. Apparently the carrier (DMF) increases dispersion, reduces particle size, or enhances the ionic state 

of the TFM molecule (Marking and Olson 1975). The 

increased activity of TFM(FG) in mallards can probably be attributed to increased absorption of TFM in 

the gastointestinal tract as a result of the presence of 

DMF. Whether DMF acts by altering the epithelial 

lining, or merely by changing the state of the TFM 

(solution vs. solid) is not known, but DMF is known to 

increase the epidermal absorption of many substances 

(Wurster 1972).

In these studies it appears that the mixture of 

TFM(FG) with Bayer 73 was additive in ring-billed 

gulls and possibly additive in mallards. Kawatski et al. 

(1975) noted that the toxicity of TFM and Bayer 73 to 

larvae of a midge (Chironomus teutons) also was 

usually additive.

Concentrations of the lampricide TFM routinely 

used for sea lamprey control range from 1 to 15 mg/1 

(active ingredient), and exposures generally last 12 to 

15 h. Concentrations of Bayer 73 routinely used are 

2% or less of the TFM concentration. Exposures of 

mallards to drinking and swimming water containing
Table 2. Additive toxicity indices of lampricides administered individually and in combination to birds 

in acute oral tests.

Species and 

lampricide3

Mallard

TFM(A)

and

DMF

Mallard

TFM(FG)

and

Bayer 73

Ring-billed gull

TFM(FG)

and

Bayer 73

LD60b and95% 

confidence limits \AAH--

Individually

458

(324-649)

> 2,000

308

(237-400)

> 2,000

250

(170-368)

500

(77.8-3,210)

 ^iciciiuivcr

In combination index0

108

(83.0-140)

1.98

200

(154-260)

463

(400-535)

-0.506

4.72

(4.08-5.46)

151

(116-196)

0.647

1.54 (-0.179-2.17)

(1.18-2.00)

aLampricides: TFM(A) = purified, > 96% active 3-trifluoromethyl-4-nitrophenol; DMF = reagent grade N,N-dimethylformamide; TFM(FG) = field grade, 35% active TFM in 

DMF; and Bayer 73 = 70% active 2-aminoethanol salt of 

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

bLD5n values are expressed as milligrams of toxicant per 

kilogram of body weight.

c For discussion of derivation, see Marking and Dawson 

(1975).

lampricides lasted longer than those used in field treatments; no mortalities occurred at any of the concentrations tested; and toxic responses were lacking or 

mild at the concentrations used in sea lamprey control.

Conclusions

 The lampricide TFM was toxic to birds in acute oral 

tests, and apparently has about the same magnitude 

of toxicity in birds as in mammals.

 Bayer 73 appeared to have a low order of toxicity in 

birds, but may be more toxic to birds than to 

mammals.

 Field grade TFM appeared to be more toxic than 

purified TFM to mallards.

 Mixtures of Bayer 73 and TFM (2:98) appeared to 

have additive toxic action in ring-billed gulls and 

possibly additive action in mallards.

 Among mallards exposed to lampricides in the drinking and swimming water, none died at the concentrations tested, and toxic responses to use-pattern 

concentrations were lacking or mild.

The addition of TFM or Bayer 73, alone or in combination, to the environment in the amounts routinely 

used for lamprey control appears to present little 

toxic hazard to birds.

Acknowledgments

I thank the Max McGraw Wildlife Foundation for 

providing the mallards used for the tests on Bayer 73 

and the mixture of Bayer 73 with TFM(FG), and the 

various individuals especially Richard K. Tucker and 

M. A. Haegele who assisted in the studies.

References

Howell, J. H., E. L. King, Jr., A. J. Smith, and L. H. Hanson. 

1964. Synergism of 5,2'-dichloro-4'-nitro-salicylanilide and 

3-trifluormethyl-4-nitrophenol in a selective lamprey larvicide. Great Lakes Fish. Comm., Tech. Rep. 8. 21 pp.

Hudson, R. H., R. K. Tucker, and M. A. Haegele. 1972. Effects of age on sensitivity: acute oral toxicity of 14 pesticides to mallard ducks of several ages. Toxicol. Appl. Pharmacol. 22:556-561.

Kawatski, J. A., M. M. Ledvina, and C. R. Hansen, Jr. 1975. 

Acute toxicities of 3-trifluoromethyl-4-nitrophenol (TFM) 

and 2',5-dichloro-4'-nitrosalicylanilide (Bayer 73) to larvae 

of the midge (Chironomus tentans). U.S. Fish Wildl. Serv., 

Invest. Fish Control 57. 7 pp.

Maki, A. W., L. D. Geissel, and H. E. Johnson. 1975. Toxicity 

of the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) to 

10 species of algae. U.S. Fish Wildl. Serv., Invest. Fish 

Control 56. 17 pp.

Marking, L. L., and V. K. Dawson. 1975. Method for assessment of toxicity or efficacy of mixtures of chemicals. U.S. 

Fish Wildl. Serv., Invest. Fish Control 67. 8 pp.

Marking, L. L., and J. W. Hogan. 1967. Toxicity of Bayer 73 

to fish. U.S. Fish Wildl. Serv., Invest. Fish Control 19. 

13pp.

Marking, L. L., and L. E. Olson. 1975. Toxicity of the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) to nontarget 

fish in static tests. U.S. Fish Wildl. Serv., Invest. Fish 

Control 60. 27 pp.

Sanders, H. O., and D. F. Walsh. 1975. Toxicity and residue 

dynamics of the lampricide 3-trifluoromethyl-4-nitropheriol 

(TFM) in aquatic invertebrates. U.S. Fish Wildl. Serv., 

Invest. Fish Control 59. 9 pp.

Schnick, R. A. 1972. A review of literature on TFM (3-trifluoromethyl-4-nitrophenol) as a lamprey larvicide. U.S. 

Fish Wildl. Serv., Invest. Fish Control 44. 31 pp.

Thompson, W. R. 1947. Use of moving averages and interpolation to estimate median-effective dose. I. Fundamental 

formulas, estimation of error, and relation to other 

methods. Bacteriol. Rev. 11:115-145.

Tucker, R. K., and D. G. Crabtree. 1970. Handbook of toxicity of pesticides to wildlife. U.S. Fish Wildl. Serv., 

Resour. Publ. 84. 131 pp.

Tucker, R. K., and M. A. Haegele. 1971. Comparative acute 

oral toxicity of pesticides to six species of birds. Toxicol. 

Appl. Pharmacol. 20:57-65.

Well, C. S. 1952. Tables for convenient calculation of medianeffective dose (LD5n or ED,,,) and instruction in their use. 

Biometrics 8:249-263.

Wurster, D. E. 1972. Some practical applications of percutaneous absorption theory. Adv. Biol. Skin 12:153-168.

GPO 850-3 130



(Reports 69 and 70 are in one cover.)

69. Toxicity of 3-trifluoromethyl-4-nitrophenol (TFM), 2',5-dichloro-4'-nitrosalicylanilide 

(Bayer 73), and a 98:2 Mixture to Fingerlings of Seven Fish Species and to Eggs and 

Fry of Coho Salmon, by T. D. Bills and L. L. Marking. 1976. 9 pp.

70. The Freshwater Mussel (Anodonta sp.) as an Indicator of Environmental Levels of 3- 

trifluoromethyl-4-nitrophenol (TFM), by A. W. Maki and H. E. Johnson. 1976. 5 pp.

71. Field Tests of Isobornyl Thiocyanoacetate (Thanite) for Live Collection of Fishes, by 

R. M. Burress, P. A. Gilderhus, and K. B. Gumming. 1976.13 pp.

72. Toxicity of Rotenone to Fish in Standardized Laboratory Tests, by L.L. Marking and 

T.D.Bills. 1976. llpp.

(Reports 73 through 76 are in one cover.)

73. Formalin: Its Toxicity to Nontarget Aquatic Organisms, Persistence, and Counteraction, by T. D. Bills, L. L. Marking, and J. H. Chandler, Jr. 1977. 7 pp.

74. Chlorine: Its Toxicity to Fish and Detoxification of Antimycin, by L. L. Marking and 

T. D. Bills. 1977. 5 pp.

75. Malachite Green: Its Toxicity to Aquatic Organisms, Persistence, and Removal with 

Activated Carbon, by T. D. Bills, L. L. Marking, and J. H. Chandler, Jr. 1977. 6 pp.

76. Toxicity of Furanace to Fish, Aquatic Invertebrates, and Frog Eggs and Larvae, by 

L. L. Marking, T. D. Bills, and J. H. Chandler, Jr. 1977. 6 pp.

(Reports 77 through 79 are in one cover.)

77. Efficacy of 3-Trifluoromethyl-4-nitrophenol (TFM), 2',5-Dichloro-4'-nitrosalicylanilide 

(Bayer 73), and a 98:2 Mixture as Lampricides in Laboratory Studies, by V. K. Dawson, 

K. B. Cumming, and P. A. Gilderhus. 1977.11 pp.

78. Toxicity of the Molluscicide Bayer 73 and Residue Dynamics of Bayer 2353 in Aquatic 

Invertebrates, by H. O. Sanders. 1977. 7 pp.

79. Accumulation, Elimination, and Biotransformation of the Lampricide 2',5-Dichloro-4'- 

nitrosalicylanilide by Chironomus teutons, by J. A. Kawatski and A. E. Zittel. 1977. 

8pp.

(Reports 80 and 81 are in one cover.)

80. Effects of Antimycin A and Rotenone on Macrobenthos in Ponds, by L. J. Houf and 

R. S. Campbell. 1977. 29 pp.

81. Aquatic Macroinyertebrates in a Small Wisconsin Trout Stream Before, During, and 

Two Years After Treatment with the Fish Toxicant Antimycin, by G. Z. Jacobi and 

D. J. Degan. 1977. 24 pp.

82. Investigations in Fish Control: Index to Numbers 1-72, 1964-76, by R. A. Schnick and 

K. A. Graves. 1977.19 pp.

(Reports 83 through 85 are in one cover.)

83. Survival of Two Species of Freshwater Clams, Corbicula leana and Magnonaias boykiniana, After Exposure to Antimycin, by L. L. Marking and J. H. Chandler, Jr. 1978. 

5pp.

84. Chronic and Simulated Use-Pattern Exposures of Brook Trout (Salvelinus fontinalis] to 

3-Trifluoromethyl-4-nitrophenol (TFM), by W. P. Dwyer, F. L. Mayer, J. L. Alien, and 

D. R. Buckler. 1978. 6 pp.

85. Hydrolysis and Photolysis of the Lampricide 2',5-Dichloro-4'-nitrosalicylanilide (Bayer 

73), by D. P. Schultz and P. D. Harman. 1978. 5 pp.

86. Registration of Thirty-three Fishery Chemicals: Status of Research and Estimated 

Costs of Required Contract Studies, by R. A. Schnick and F. P. Meyer. 1978.19 pp.

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