INVESTIGATIONS IN FISH CONTROL

100. Observations on the Effects of Irrigation Water Containing 

3-Trifluoromethyl-4-Nitrophenol (TFM) on Plants

101. Residues of Malachite Green in Muscle, Eggs, and Fry of Treated 

Atlantic Salmon and Chinook Salmon

102. Effects of Water Temperature, Hardness, and pH on the Toxicity of 

Benzocaine to Eleven Freshwater Fishes

OCH 3

CH 2

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 Fisheries Research Center at La Crosse, Wisconsin, and reports of other studies related to fish control and acute 

toxicity of piscicides. Though each report is regarded as a separate publication, several may be issued under a single 

cover, for economy. See inside back cover for list of current issues.

Copies of this publication may be obtained from the Publications Unit, U.S. Fish and Wildlife 

Service, 1849 C Street, N.W, Mail Stop 130 ARLSQ, Washington, DC 20240, or may be purchased 

from the National Technical Information Service (NTIS), 5285 Port Royal Road, Springfield, VA 

22161.

ISSN 0565-0704
INVESTIGATIONS IN FISH CONTROL

100. Observations on the Effects of Irrigation Water Containing 

3-Trifluoromethyl-4-Nitrophenol (TFM) on Plants

By Philip A. Gilderhus

101. Residues of Malachite Green in Muscle, Eggs, and Fry of Treated 

Atlantic Salmon and Chinook Salmon

By J. L. Alien

102. Effects of Water Temperature, Hardness, and pH on the Toxicity of 

Benzocaine to Eleven Freshwater Fishes

By Terry D. Bills, George E. Howe, and Leif L. Marking

U.S. Fish and Wildlife Service 

National Fisheries Research Center La Crosse

P.O. Box 818 

La Crosse, Wisconsin 54602

1990

Observations on the Effects of Irrigation Water 

Containing 3-Trifluoromethyl-4-Nitrophenol (TFM)

on Plants

by

Philip A. Gilderhus

U.S. Fish and Wildlife Service 

National Fisheries Research Center La Crosse

P.O. Box 818 

La Crosse, Wisconsin 54602

ABSTRACT. Because concerns have been expressed about the effects of irrigating truck-garden 

crops with water from a stream treated with the lampricide 3-trifluoromethyl-4-nitrophenol (TFM), I 

conducted studies on the effects of TFM on young plants of common vegetables and fruits. Plants 

established in horticultural flats were irrigated for 12 h with water containing 10 mg/L of TFM and 

later compared with plants irrigated for a similar period with untreated water. Lettuce, radish, sweet 

corn, and potato plants were virtually unaffected. Green bean and tomato plants developed brown or 

dead spots on many leaves but growth rates and survival were not affected. Cucumber and cantaloupe 

plants were severely damaged; some were killed and about 40% of the leaves on surviving plants were 

dead or dying. Two weeks after treatment, the mean weight of surviving treated cantaloupe plants was 

significantly less than that of control plants.

The parasitic sea lamprey (Petromyzon marinus) is controlled in the Great Lakes by treating more than 400 nursery streams with the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) to kill the larvae. Some of these tributaries 

flow through agricultural land, where the stream water is 

used to irrigate truck-garden crops. Farmers have been 

reluctant to forego irrigation while a stream is being 

treated, without evidence that TFM would damage their 

crop. Lack of irrigation for even 1 day reportedly can 

affect crop growth, delay marketing, and prevent a farmer 

from getting full market price for a product. To date, sea 

lamprey control agencies have had no specific information to give to farmers about potential damage to their 

crops from using TFM-treated irrigation water.

It is known that TFM has adverse effects on aquatic 

plants ranging from reduced production after exposure to 

10 mg/L for 1 h to plants becoming limp and cyanotic after 

exposure to 20 mg/L for 3 h (National Research Council of 

Canada 1985). No studies of the effects of TFM on terrestrial plants have been published. In response to requests 

from control agents for information on how TFM affects 

terrestrial plants, I conducted studies at the National Fisheries Research Center, La Crosse, Wisconsin, to determine

if irrigation with water containing TFM is deleterious to 

selected truck-garden plants.

Methods and Materials

Plants of eight common vegetables and fruits were 

raised in horticultural flats filled with sandy loam soil. 

Lettuce, radish, green bean, cucumber, and cantaloupe 

plants were started from seed in flats 53 X 27 cm and 6 cm 

deep. Corn and potatoes, which need deeper soil, were 

planted in flats 36 X 33 cm and 15 cm deep. Seedling 

tomato plants were transplanted from a garden plot into 

flats that were divided into sections 8 cm square and 6 cm 

deep. The plants were allowed to grow until they were 

well established (25 to 35 days after planting). Their sizes 

and the numbers used are listed in the Table.

The studies were conducted in a series of verticalwalled 0.005-ha concrete ponds (Figure). Two ponds held 

treatment water and the plants were kept in two unfilled 

ponds. For treatment, the plants were placed on racks with 

an oscillating sprinkler between the racks at the same 

height. A submersible pump transferred fresh water or

1
INVESTIGATIOS IN FISH CONTROL 100

Table. Size and number of plants used and effects of irrigating them with water containing TFM.

Plant

Lettuce

Radish

Green bean

Sweet corn

Potato

Tomato

Cucumber

Cantaloupe

"At time of treatment.

bSee text for details.

Height of

plants (cm)a

8-10

8-10

8-10 '

25-30

25-30

15-18

13-18

13-18

Plants

per pan

15

15

15

16

4

6

9

15

Pans per

treatment

3

3

2

3

3

3

3

2

Damage from

TFM exposureb

None

None

Leaf spotting

None

None

Leaf spotting

Severe

Severe

water containing TFM (field grade; 39.9% active ingredient) from the sump of the reservoir pond to the sprinkler 

(Figure). The spray crossed each rack twice per minute. 

Treatments were designed to simulate the maximum exposure likely to occur; the plants were sprayed with water 

containing 10 mg/L of TFM (calculated as the sodium salt) 

for 12 h. Controls were sprayed with clear water during the 

same period. Except during treatment, the plant flats were 

kept on the floor of the pond and covered with perforated 

aluminum screen to partly shield them from direct sunlight. Plants were watered daily before and after treatment, 

except when there was adequate rain. The water was applied from above the plants with a fine-spray watering can. 

Mean weights of treated and control plants of green 

beans and cantaloupe were compared by use of the Student's t test.

Results

Lettuce, radish, sweet corn, and potato plants showed 

no adverse effects from the application of TFM. Green 

bean and tomato plants showed some damage.

At the time of treatment, the green bean plants were 8 to 

10 cm high, each with two large unifoliate leaves. By 6 

days after the TFM treatment, 70% of the leaves had 

brown spots of dead tissue that covered as much as 50% of 

individual leaves (average, about 15%). After 12 days, the 

dead spots on treated leaves were about the same as after 6 

days, and all treated plants were growing and adding new 

trifoliate leaves at the same rate as the controls. Fifteen 

days after the first treatment, the bean plants were subjected to a second treatment to provide data on more 

advanced plants. The results were about the same as in the 

first treatment; about 90% of the new leaves sustained

some damage (brown, dead spots) but all plants continued 

to grow. Two weeks after the second treatment, the plants 

were cut off at the ground and weighed to the nearest 0.1 g. 

Mean weights of treated and control plants (N = 16) did not 

differ significantly.

Two days after treatment, 50% of the leaves on treated 

tomato plants had numerous small brown spots; 4 to 5 days 

after treatment, the tomato plants began to look healthier 

and began developing new leaves; and 8 days after treatment, the treated plants were growing and blooming at 

about the same rate as the controls.

Cucumber and cantaloupe plants sustained severe damage from irrigation with water containing TFM. Some 

damage to cucumber plants was apparent 20 h after treatment, as evidenced by brown spots on some of the leaves. 

Two days after treatment, 60-70% of the leaves had large 

brown spots of dead tissue. At 4 days after treatment, five 

plants were dead and the brown areas on live plants were 

drying out. By 8 days after treatment, 40% of the leaves on 

surviving treated plants were dead or dying, but the plants 

continued to grow at their tips and produced blossoms. 

Overall, cucumbers sustained major damage; 20% of the 

plants were killed and 40% of the leaves were severely 

damaged on surviving plants.

Cantaloupe plants were vigorous and healthy at the time 

of treatment. At 6 days after treatment, 75% of the leaves 

on treated plants showed damage (brown spots), two 

plants were dead, and two were nearly dead. After 2 weeks 

all of the treated plants looked unhealthy; 40% of the 

leaves showed damage and new leaves were small and 

pale. The control plants were growing well, appeared 

healthier, and had added large, dark green leaves. At 2 

weeks after treatment, the mean weight (N= 16) of treated 

plants (10.8 g) was 33% less than that of controls (16.2 g). 

The difference was highly significant (P < 0.01).
OBSERVATIONS ON THE EFFECTS OF IRRIGATION WATER

SUMP

TFM RESERVOIR

RACKS

REGULAR POSITION 

OF PLANTS

DRY POND

SPRINKLER

DRY POND

WATER RESERVOIR

Figure. Schematic diagram of 0.005-ha ponds used for irrigating 

plants with water containing TFM.

Discussion

The treatment regimen used in these studies represented 

the maximum exposure that might occur when plants are 

irrigated with water from streams treated with TFM, and 

the length of treatment probably exceeded normal irrigation periods. Irrigation for a shorter period or at a lower 

concentration of TFM would probably show proportionately less effect on plants.

Although green beans and tomatoes showed no apparent growth inhibition, there was sufficient damage to individual leaves to indicate that exposure of these plants to 

TFM should be avoided. The severe damage sustained by 

cucumber and cantaloupe plants indicated that these 

plants and perhaps all plants of the family Cucurbitaceae 

should not be irrigated with water containing TFM. Because there is a wide variation in effects between types of 

plants, the best policy for types that have not been tested is 

to avoid irrigating them with water containing TFM.

Reference

National Research Council of Canada, Panel on TFM and Bayer 

73. 1985. TFM and Bayer 73: Lampricides in the aquatic 

environment. National Research Council of Canada, Ottawa. 

Publication NRCC-22488. 184pp.

Gilderhus, Philip A. 1990. Observations on the Effects of Irrigation Water 

Containing 3-Trifluoromethyl-4-Nitrophenol (TFM) on Plants. U.S. Fish 

Wildl. Serv., Invest. Fish Control 100. 3 pp.

The effects of 3-trifluoromethyl-4-nitrophenol (TFM) on young plants of common vegetables and fruits were studied. Plants established in horticultural flats 

were irrigated for 12 h with water containing 10 mg/L of TFM and later compared 

with plants irrigated for a similar period with untreated water. Lettuce, radish, 

sweet corn, and potato plants were virtually unaffected. Green bean and tomato 

plants developed brown or dead spots on many leaves but growth rates and 

survival were not affected. Cucumber and cantaloupe plants were severely 

damaged.

Key words: Lampricides, TFM, irrigation, terrestrial plants.

_

Gilderhus, Philip A. 1990. Observations o

Containing 3-Trifluoromethyl-4-Nitrop

Wildl. Serv., Invest. Fish Control 100. 3 

The effects of 3-trifluoromethyl-4-nitroph

mon vegetables and fruits were studied. Pla

were irrigated for 12 h with water containing 

with plants irrigated for a similar period w

sweet corn, and potato plants were virtually 

plants developed brown or dead spots on 

survival were not affected. Cucumber an

damaged.

Key words: Lampricides, TFM, irrigation, 

Gilderhus, Philip A. 1990. Observations on the Effects of Irrigation Water Containing 3-Trifluoromethyl-4-Nitrophenol (TFM) on Plants. U.S. Fish Wildl. Serv., Invest. Fish Control 100. 3 pp.

The effects of 3-trifluoromethyl-4-nitrophenol (TFM) on young plants of common vegetables and 

fruits were studied. Plants established in horticultural flats were irrigated for 12 h with water containing 

10 mg/L of TFM and later compared with plants irrigated for a similar period with untreated water. 

Lettuce, radish, sweet corn, and potato plants were virtually unaffected. Green bean and tomato plants 

developed brown or dead spots on many leaves but growth rates and survival were not affected. Cucumber 

and cantaloupe plants were severely damaged.

Key words: Lampricides, TFM, irrigation, terrestrial plants.

Gilderhus, Philip A. 1990. Observations on the Eff

fluoromethyl-4-Nitrophenol (TFM) on Plants. U.S. 

The effects of 3-trifluoromethyl-4-nitrophenol (TFM

fruits were studied. Plants established in horticultural fla

10 mg/L of TFM and later compared with plants irriga

Lettuce, radish, sweet corn, and potato plants were virtu

developed brown or dead spots on many leaves but growth 

and cantaloupe plants were severely damaged.

Key words: Lampricides, TFM, irrigation, terrestrial pl

Residues of Malachite Green in Muscle, Eggs, and 

Fry of Treated Atlantic Salmon and Chinook Salmon

by

J. L. Alien

U.S. Fish and Wildlife Service 

National Fisheries Research Center La Crosse

P.O. Box 818 

La Crosse, Wisconsin 54602

ABSTRACT. Residues of malachite green in muscle, eggs, and fry of Atlantic salmon (Salmo salar) 

and chinook salmon (Oncorhynchus tshawytscha) were determined by colorimetric analysis after the 

fish had been routinely treated with the chemical at fish hatcheries (10-47 times, 1 ppm for 1 h). The 

concentration of residues in fish muscle generally depended on the elapsed time since the last 

treatment; concentrations were usually highest (about 1.0 to 2.5 ug/g) in fish sampled 1 or 2 days after 

the last treatment and had declined somewhat to values as low as 0.33 ug/g after 18-41 days. Residues 

in eggs taken from adults that had been treated with malachite green were about 0.1 to 4.2 ug/g in 

Atlantic salmon and 0.1 to 1.0 ug/g in chinook salmon; there was little relation between the residue 

concentrations in the eggs and the elapsed time since the last treatment. Residues of malachite green in 

fry newly hatched from eggs of treated chinook salmon ranged from 0.14 to 1.16 ug/g.

Malachite green has been used extensively in fish culture (usually in the zinc-free oxalate form) for the control 

of fungal infections and external parasites (Nelson 1974; 

Alderman 1985). It is the most effective antifungal treatment used in aquaculture (Meyer and Hoffman 1976; Bailey 1983; Alderman 1985). It has never been registered for 

use on food fish (such use was banned in 1978) because of 

potential health risks; use is currently limited to the treatment of nonfood fish (e.g., salmon brood stock) under an 

Investigational New Animal Drug Application issued by 

the U.S. Food and Drug Administration.

The potential threat of malachite green to human health 

was first pointed out by Werth and Boiteux (1958), who 

described marked increases in the incidence of internal 

tumors in the progeny of laboratory rats fed malachite 

green. Meyer and Jorgenson (1983) reported a significant 

increase in gross abnormalities in rainbow trout (Oncorhynchus mykiss) hatched from eggs treated with malachite green at concentrations of 1 mg/L for 1 h daily (30 

applications); 3 mg/L for 1 h every other day (15 applications), and 5 mg/L for 1 h weekly (5 applications). They 

also reported that malachite green produced significant 

teratological effects at all levels of treatment when it was 

administered orally to New Zealand white rabbits at doses

as low as 5 mg/kg body weight. Although a different 

delivery system and a higher dose rate were used in the 

rabbits than are normally used in fish culture, the existence 

of a serious potential hazard is obvious.

Information on residues of malachite green that might 

occur in fish flesh must be determined as part of the 

evaluation of its use. Poe and Wilson (1983), who reported the presence of malachite green in channel catfish (Ictalurus punctatus) after treatment, wrote that 

a greenish color appeared on the surface of the catfish 

muscle tissue after frozen storage for 13 to 60 days; and 

T. D. Bills (personal communication) indicated that a 

solution of 85% ethyl alcohol, 10% formalin, and 5% 

acetic acid (AFA) that was used to preserve fish exposed 

to malachite green developed a bluish color. Thus, the 

presence of malachite green can be determined by colorimetric analysis.

Alien and Hunn (1986) reported preliminary results of 

malachite green analysis after residues were extracted 

with AFA from muscle tissue of rainbow trout and muscle 

tissue and eggs of Pacific salmon (Oncorhynchus sp.). 

Colorimetric analysis showed that muscle of rainbow 

trout exposed to 0.1 mg/L of malachite green for 24 h at 

14° C contained 1.87 |lg/g of malachite green; the residue

1
INVESTIGATIONS IN FISH CONTROL 101

declined to 0.22 [ig/g in muscle after 6 days of withdrawal 

in fresh water. Residues in eggs from these fish ranged 

from 0.1 to 4.1 ppm. In the present study, I used AFA 

extraction and colorimetric analysis to examine adult 

salmonids treated with malachite green to obtain further 

data on residues of malachite green in the muscle tissue 

and eggs and, especially, newly hatched fry. The analyses 

were done on fish from national fish hatcheries treated and 

sampled under hatchery conditions.

Materials and Methods

Adult Atlantic salmon (Salmo salar} at the Berkshire 

(Massachusetts) National Fish Hatchery (NFH) were 

treated on 5 consecutive days each week 20 to 47 

times with 1 ppm of malachite green for 1 h. Samples of 

muscle tissue and eggs were collected from 1 to 41 days 

after the last treatment of these fish, as well as from untreated adults (to determine background readings of malachite green).

Adult chinook salmon (Oncorhynchus tshawytscha) 

from the Warm Springs (Oregon) NFH and Winthrop 

(Washington) NFH were also treated on 5 consecutive 

days each week with 1 ppm of malachite green for 1 h. 

The fish at Warm Springs NFH were treated 27 to 30 

times and samples of muscle tissue were collected 1, 6, 

12, or 18 days after the last treatment; those at Winthrop 

NFH received 10 treatments at various intervals and were 

sampled 8 days after the last treatment. At both hatcheries, eggs were collected at fertilization (0 h) and 24 h 

after fertilization, and fry were sampled 24 h after hatching. Muscle tissue, eggs, and fry from untreated adults 

were collected at Warm Springs NFH to provide background values.

Samples of fish tissue, eggs, and fry from the several 

hatcheries were frozen and shipped to the La Crosse (Wisconsin) National Fisheries Research Center for analysis. I 

extracted 10-g samples (wet weight) with 25 mL of AFA at 

room temperature in the dark for 24 h, centrifuged samples 

at about 2,000 rpm, and filtered them through a glass fiber 

filter. The absorbance of sample extracts at 615 nm was 

determined on a Beckman DU6 spectrophotometer and 

compared to the absorbance of malachite green oxalate 

standards in AFA. Residue concentrations are reported as 

fig/g malachite green oxalate.

Results and Discussion

The presence of malachite green residues in the fish 

eggs and fry was readily apparent from the blue-green

color of the tissue extracts. Because fish and eggs have a 

naturally occurring yellowish color, malachite green residues are seen as green by the human eye. The presence of 

naturally occurring colors also produced a low background reading in the untreated fish, eggs, and fry, but too 

few untreated samples were tested to establish a useful 

mean background reading for subtraction from the sample 

residue concentrations. Background color readings in the 

muscle from two untreated adult Atlantic salmon were 

0.09 to 0.15 |j,g/g (malachite green equivalents) and 

0.10 \ig/g in the eggs of one (Table 1). The residue concentrations reported here include the background values.

Residues reported as malachite green in Atlantic salmon showed no relation between the number of treatments 

received and the concentration of residue in the muscle 

(Table 1). Residues ranged from 0.33 to 2.54 jig/g. The 

residue concentrations were lowest (0.33, 0.35 fig/g) in 

muscle from two adults that received the fewest treatments 

(20) and were sampled 24 and 33 days after the last treatment; however, the residue concentration in the muscle of 

one other adult was 2.54 [ig/g after 20 treatments. Eggs 

from treated fish contained 0.21 to 4.16 |J,g/g of residual 

malachite green.

Chinook salmon from Warm Springs NFH tested for 

malachite green residues had received 27 to 30 treatments 

of 1.0 ppm for 1 h and were sampled at 1, 6, 12, and 18 

days after the last treatment. Background color readings 

were 0.32 and 0.35 |j,g/g in muscle tissue, 0.03 to 

0.08 fig/g in eggs, and 0.22 [ig/g in fry (Table 2). The mean 

concentration of malachite green in muscle of the treated 

fish ranged from 0.473 to 1.08 fig/g. Residues in the muscle were slightly higher in fish sampled at 1 day after the 

last treatment than in those sampled after 6-18 days.

The mean concentration of malachite green in eggs 

taken from adults after different frequencies of treatment 

ranged from 1.16 to 1.86 |J,g/g. In eggs sampled 24 h after 

fertilization, the average levels ranged from 0.84 to 

1.54 \ig/g. In fry from treated adults, average residues 

ranged from 0.97 to 1.16 |U.g/g.

The mean concentration of malachite green in muscle of 

adult salmon from Winthrop NFH that had been treated 10 

times at 1.0 ppm for 1 h was 0.43 [ig/g (Table 2). The mean 

concentration in eggs was 0.173 \ig/g at collection and 

0.113 \ig/g in 24 h; the average concentration in the fry 

was 0.14 [ig/g.

In general, malachite green seemed to be readily taken 

up by adult salmon treated with it and was incorporated 

into their eggs before spawning. Residues in the fry 

hatched from eggs of treated fish indicated that little of the 

chemical was lost during incubation.
RESIDUES OF MALACHITE GREEN

Table 1. Residues of malachite green oxalate (\ig/g) in muscle and eggs of Atlantic salmon subjected to 20 to 47 1-h 

treatments with 1 ppm of malachite green at the Berkshire National Fish Hatchery. Each row of data represents one 

fish.

Time after last 

treatment (days)

No treatment

No treatment

1

1

1

2

3

5

6

7

24

33

41

Number of 

treatments 

at 1 ppm

0

0

42

45

47

20

45

20

47

47

20

20

45

Sample

Muscle

0.15

0.09

1.66

2.13

2.54

1.11

0.64

2.22

0.33

0.35

0.91

Eggs

0.10

3.15

3.26

4.16

2.51

3.60

0.72

2.67

3.28

2.29

0.21

Table 2. Mean residues (SD in parentheses) of malachite green ([Lg/g) in muscle, eggs, and fry from treated (1 ppmfor 

1 h) and untreated chinook salmon at two national fish hatcheries (NFH).

Time from 

last 

treatment 

(days)

Warm Springs NFH

1

1

6

6

12

18

No treatment

Winthrop NFH

8

Number 

of 

fish

3

3

3

3

3

3

2

3

Number 

of 

treatments 

at 1 ppm

27

30

29

30

30

30

0

10

Sample

Muscle

0.997

(0.197)

1.08

(0.159)

0.75

(0.14)

0.473

(0.186)

0.577

(0.228)

0.743

(0.104)

0.335a

0.43

(0.044)

Eggs 

(Oh)

1.34

(0.835)

1.82

(0.300)

1.86

(0.681)

1.47

(0.941)

1.16a

1.69

(0.662)

0.08a

0.173

(0.055)

Eggs 

(24 h)

0.843

(0.401)

1.40

(0.332)

1.45

(0.561)

0.973

(0.601)

1.24

(1.06)

1.54

(0.611)

0.03a

0.113

(0.04)

Fry

(24 h)

0.970

(0.423)

1.04

(0.091)

1.00

(0.246)

1.16s

1.01

(0.946)

1.03

(0.375)

0.22"

0.14

(0.01)

"Average for two fish. 

bRepresents one fish.
INVESTIGATIONS IN FISH CONTROL 101

References

Alderman, D. J. 1985. Malachite green: a review. J. Fish Dis.

8:289-298. 

Alien, J. L., and J. B. Hunn. 1986. Fate and distribution studies of

some drugs used in aquaculture. Vet. Hum. Toxicol. 28:21-24. 

Bailey, T. A. 1983. Method for in vitro screening of aquatic

fungicides. J. Fish Dis. 6:91-100. 

Meyer, F. P., and G. L. Hoffman. 1976. Parasites and diseases of

warrnwater fishes. U.S. Fish Wildl. Serv., Resour. Publ. 127.

20pp.

Meyer, F. P., and T. A. Jorgenson. 1983. Teratological and other 

effects of malachite green on development of rainbow trout 

and rabbits. Trans. Am. Fish. Soc. 112:818-824.

Nelson, N. C. 1974. A review of the literature on the use of 

malachite green in fisheries. National Technical Information 

Service, Springfield, Va. PB-235-450/AS. 79 pp.

Poe, W. E., and R. P. Wilson. 1983. Adsorption of malachite 

green by channel catfish. Prog. Fish-Cult. 45:228-229.

Werth, V. G., and A. Boiteux. 1958. Disturbances of the hereditary pattern and production of tumors by experimental tissue 

anoxia. Arzneim.-Forsch. 8:735-744.
Alien, J. L. 1990. Residues of Malachite Green in Muscle, Eggs, and Fry from 

Treated Atlantic Salmon and Chinook Salmon. U.S. Fish Wildl. Serv., 

Invest. Fish Control 101. 4 pp.

Residues of malachite green in muscle, eggs, and fry of Atlantic salmon (Salmo 

salar) and Chinook salmon (Oncorhynchus tshawytscha) were determined by 

colorimetric analysis after the fish had been routinely treated with the chemical at 

fish hatcheries (10--47 times, 1 ppm for 1 h). The concentration of residues in fish 

muscle generally depended on the elapsed time since the last treatment; concentrations were usually highest (about 1 .0 to 2.5 (ig/g) in fish sampled 1 or 2 days 

after the last treatment and had declined somewhat to values as low as 0.33 |J.g/g 

after 18-41 days. Residues in eggs taken from adults that had been treated with 

malachite green were about 0.1 to 4.2 [ig/g in Altantic salmon and 0.1 to 1 .0 (ig/g 

in chinook salmon; there was little relation between the residue concentrations in 

the eggs and the elapsed time since the last treatment. Residues of malachite green 

in newly hatched fry ranged from 0.14 to 1.1

Key words: Malachite green, residues, Atlantic salmon, Chinook salmon, salmon 

eggs, fry, colorimetric analysis.

Alien, J. L. 1990. Residues of Malachite 

Treated Atlantic Salmon and Chin

Invest. Fish Control 101. 4 pp.

Residues of malachite green in muscle, 

salar) and chinook salmon (Oncorhynch

colorimetric analysis after the fish had bee

fish hatcheries (10--47 times, 1 ppm for 1 h

muscle generally depended on the elapsed 

trations were usually highest (about 1.0 t

after the last treatment and had declined s

after 18-41 days. Residues in eggs taken 

malachite green were about 0.1 to 4.2 [lg/g 

in chinook salmon; there was little relation 

the eggs and the elapsed time since the last 

in newly hatched fry ranged from 0.14 to 

Key words: Malachite green, residues, At

eggs, fry, colorimetric analysis.

Alien, J. L. 1990. Residues of Malachite Green in Muscle, Eggs, and Fry from Treated Atlantic 

Salmon and Chinook Salmon. U.S. Fish Wildl. Serv., Invest. Fish Control 101.4 pp.

Residues of malachite green in muscle, eggs, and fry of Atlantic salmon (Salmo salar) and chinook 

salmon (Oncorhynchus tshawytscha) were determined by colorimetric analysis after the fish had been 

routinely treated with the chemical at fish hatcheries (10-47 times, 1 ppm for 1 h). The concentration of 

residues in fish muscle generally depended on the elapsed time since the last treatment; concentrations 

were usually highest (about 1 .0 to 2.5 ^g/g) in fish sampled 1 or 2 days after the last treatment and had 

declined somewhat to values as low as 0.33 ^.g/g after 18-41 days. Residues in eggs taken from adults that 

had been treated with malachite green were about 0. 1 to 4.2 ^.g/g in Altantic salmon and 0. 1 to 1 .0 ^.g/g in 

chinook salmon; there was little relation between the residue concentrations in the eggs and the elapsed 

time since the last treatment. Residues of malachite green in newly hatched fry ranged from 0.14 to

Alien, J. L. 1990. Residues of Malachite Green in 

Salmon and Chinook Salmon. U.S. Fish Wildl. S

Residues of malachite green in muscle, eggs, and 

salmon (Oncorhynchus tshawytscha) were determine

routinely treated with the chemical at fish hatcheries (

residues in fish muscle generally depended on the ela

were usually highest (about 1 .0 to 2.5 Hg/g) in fish sa

declined somewhat to values as low as 0.33 ^.g/g after 

had been treated with malachite green were about 0. 1 t

chinook salmon; there was little relation between the 

time since the last treatment. Residues of malachite 

Key words: Malachite green, residues, Atlantic salmon, Chinook salmon, salmon eggs, fry, colorimetric 

analysis.

Key words: Malachite green, residues, Atlantic salmo

analysis.

Effects of Water Temperature, Hardness, and pH on the 

Toxicity of Benzocaine to Eleven Freshwater Fishes

by

Terry D. Bills, George E. Howe, and Leif L. Marking

U.S. Fish and Wildlife Service 

National Fisheries Research Center La Crosse

P.O. Box 818 

La Crosse, Wisconsin 54602

ABSTRACT. The toxicity of benzocaine (ethyl 4-aminobenzoate; 98%) to eleven freshwater fishes 

was evaluated under various physical and chemical conditions. Under standard test conditions (pH 7.8, 

12° C, soft water), the 24-h LC50 (mg/L) was 17.2 for lake sturgeon (Acipenserfulvescens), 22.5 for 

rainbow trout (Oncorhynchus mykiss), 34.0 for muskellunge (Esox masquinongy), 24.0 for northern 

pike (Esox lucius), 19.0 for common carp (Cyprinus carpio), 25.9 for fathead minnow (Pimephales 

promelas), 28.0 for channel catfish (Ictaluruspunctatus), 28.1 for striped bass (Morone saxatilis), 21.9 

for green sunfish (Lepomis cyanellus), 21.9 for bluegill (L. macrochirus), and 22.0 for walleye 

(Stizostedion vitreum). Tests conducted at three water temperatures, four hardnesses, and three pH's 

showed that none of these variables influenced toxicity. The higher temperatures tested (to 22° C) did 

not increase the rate of toxicosis. Rainbow trout survived exposure to the recommended use concentration of 25 mg/L for 15 min, but did not survive exposure to 75 or 125 mg/L. Users of benzocaine should 

be cautioned that overdoses may lead to undue stress or mortality in exposed fish. Comparison of 

benzocaine concentrations by high performance liquid chromatography at 0 and 96 h showed no 

degradation of the compound during the exposure period.

Anesthetics are used for a number of fishery applications, ranging from mild sedation for transport to total 

anesthetization for marking, tagging, spawn-taking, and 

surgical procedures. Although many chemicals have been 

used to anesthetize fish (McFarland 1959; Bell 1967), 

tricaine methanesulfonate (MS-222) is the only chemical 

registered for use on fish in the United States. The label for 

MS-222 forbids anesthetized fish to be released to the wild 

or used as food until after a 21-day withdrawal. Salmon 

anesthetized with MS-222 and killed during spawning 

must be discarded rather than used as human or animal 

food (Gilderhus 1989). Because of the withdrawal period 

for MS-222, there is a need for an alternate anesthetic.

Many substances have been tested as potential anesthetics for fish (McFarland 1959; Bell 1967; Gilderhus et al. 

1973; Dawson and Gilderhus 1979). McErlean (1967) 

first suggested that ethyl-p-aminobenzoate be used as an 

anesthetic for cold-blooded vertebrates. The 183 fishery 

workers who responded to a survey conducted by Marking 

and Meyer (1985) used a total of 11 different chemicals to 

anesthetize fish. Gilderhus and Marking (1987) identified

benzocaine, from a group of 16 anesthetics, as a possible 

candidate for use in fisheries. Chemically, it is similar to 

MS-222, differing only by the position of a single substituent: the amino group is in the meta position in MS-222 

and in the para position on benzocaine. Also, because 

benzocaine is widely used in human over-the-counter drug 

preparations, its registration for fishery use may be easier 

and less costly than for other candidate anesthetics (Gilderhus 1989).

Previous studies of benzocaine (Dawson and Gilderhus 

1979; Gilderhus 1989) determined the compound's effectiveness as a fish anesthetic but provided little information 

on its toxicity to fish. Standardized toxicity information is 

required to satisfy requirements of the U.S. Food and Drug 

Administration for minor use of animal drugs. The purpose of our study was to determine the toxicity of benzocaine to representative coldwater and warmwater fish in 

laboratory tests; to evaluate the effects of water temperature, hardness, and pH on toxicity; and to determine the 

safety to fish of treatment concentrations that were 3 and 5 

times the effective rate for trout.

1
INVESTIGATIONS IN FISH CONTROL 102

Materials and Methods

Static test procedures used in this study followed those 

prescribed by the Committee on Methods for Acute Toxicity Tests with Aquatic Organisms (1975), ASTM Committee E-35 on Pesticides (1980), and the U.S. Department 

of Agriculture (1986). We exposed 20 fish to each concentration of benzocaine in glass jars containing 15 L of 

oxygen-saturated test water. Reconstituted test waters 

were prepared according to standardized procedures to 

produce the desired water quality.

The solutions were adjusted to a selected pH (± 0.2 

unit) with chemical buffers (Committee on Methods for 

Acute Toxicity Tests with Aquatic Organisms 1975), 

before each test and at 24-h intervals, as needed. Temperatures were regulated by immersing the test jars in 

constant-temperature water baths. To assess the effects of 

water hardness, we buffered the test solutions to a constant 

pH with sodium bicarbonate, using the procedure of 

Marking (1975).

The test species were lake sturgeon (Acipenser fulvescens), rainbow trout (Oncorhynchus my kiss), muskellunge (Esox masquinongy), northern pike (Esox lucius\ common carp (Cyprinus carpio), fathead minnow 

(Pimephales promelas), channel catfish (Ictalurus punctatus), striped bass (Morone saxatilis), green sunfish (Lepomis cyanellus), bluegill (L. macrochirus), and walleye 

(Stizostedion vitreum). They were obtained from a State or 

Federal fish hatchery or produced at the National Fisheries 

Research Center, La Crosse, Wisconsin, and were maintained according to the standard procedures for handling 

experimental fish. The fish were acclimated to the desired 

water chemistries and temperatures for 24 h before each 

test. Mortalities were recorded at 1,3, 6, and 12 h on the 

first day of exposure and daily thereafter for 96 h. The 

methods of Litchfield and Wilcoxon (1949) were used to 

compute the LC50's (concentration causing 50% mortality) and 95% confidence intervals.

Three species (rainbow trout, channel catfish, and bluegill) were used in tests to determine the effects of water 

temperature, hardness, and pH on the toxicity of benzocaine. In tests to determine safe use pattern levels, we 

exposed groups of 300 rainbow trout to benzocaine at the 

prescribed effective treatment concentration of 25 mg/L 

for 15 min (Gilderhus 1989) and to 3 and 5 times this 

effective level (U.S. Department of Agriculture 1986). We 

observed the fish for a 14-day postexposure period for 

unusual behavior or delayed mortality, using the criteria 

set forth by Lennon and Walker (1964).

Benzocaine (ethyl 4-aminobenzoate, 98%; Aldrich 

Chemical Company, Milwaukee, Wisconsin) was dissolved in ethanol to make stock solutions, and aliquots of

these solutions were pipetted into test vessels to reach the 

desired test concentrations.

Benzocaine concentrations in water samples were 

quantified at 0 and 96 h by high performance liquid chromatography (HPLC). Quantification equipment and conditions were as follows: Waters HPLC system, consisting 

of a 71 OB WISP autosampler, a 510 variable speed pump, 

a 481 spectrophotometer detector, and a 730 data module 

integrator. A Micro Pak, C 18 , reverse-phase column 

(30 cm X 4 mm) was used with a mobile phase consisting 

of 70% HPLC methanol, 26% HPLC water, and 4% acetic 

acid. Flow rate for the mobile phase was 2.0 mL/min and 

the detector was set at a wavelength of 286 nm. This 

method resulted in a retention time of about 3.6 min. To 

quantify peak areas, we used the Waters External Standard 

Quantification program.

Results

Toxicity to Eleven Species of Fish

Benzocaine was toxic to all species exposed in soft 

water at 12° C; the 24-h LC50's ranged from 17.2 mg/L for 

lake sturgeon to 34.0 mg/L for muskellunge (Table 1). 

Coldwater and warmwater species responded similarly; 

the 24-h LC50 was 22.5 mg/L for rainbow trout and 

21.9 mg/L for bluegills. The toxicity of benzocaine did not 

increase significantly with longer exposures; the LC50's 

changed little between 1 and 24 h for the species exposed.

Influence of Temperature, Water Hardness, 

andpH

The toxicity of benzocaine was not significantly 

affected by any of the water characteristics tested. Increased water temperature, which increases the metabolic 

rate of poikilotherms, usually increases the rate of uptake 

of toxic chemicals and results in greater mortality. However, the toxicity of benzocaine was not affected by 

changes in water temperature in these exposures. For example, with rainbow trout, the 24-h LC50 was 17.0 mg/L 

in water at 7° C and 20.5 mg/L in water at 17° C (Tables 2, 

3, and 4). The 24-h LC50's for channel catfish were 

28.0 mg/L at 12° C and 27.9 mg/L at 22° C.

Likewise, neither water hardness nor pH affected toxicity. For example, in channel catfish, the 24-h LC50 was 

30.2 mg/L in very soft water (10-12 mg/L, total hardness 

as milligrams per liter CaCO3 ) and 30.0 mg/L in very hard 

water (300-320 mg/L total hardness). The 24-h LC50's 

for channel catfish were 30.1 mg/L in acidic water (pH 

6.5) and 29.0 mg/L in alkaline water (pH 9.5).

The HPLC analysis of benzocaine concentrations in 

water from randomly selected test vessels agreed closely
EFFECTS OF WATER TEMPERATURE, HARDNESS, AND pH

Table 1. Toxicity (LC50 in mgIL, and 95% confidence interval) ofbenzocaine to eleven species offish in soft water

at 12°C.

Species

Lake sturgeon3

Rainbow trout

Northern pike

Muskellunge3

Fathead minnow

Common carp

Channel catfish

Striped bassa

Bluegill

Green sunfish

Walleye3

1

28.0

25.3-31.0

27.0

26.7-28.4

35.0

28.0-43.7

34.0

29.5-39.1

35.0

33.0-37.2

22.6

21.2-24.1

36.0

33.1-39.1

32.9

27.6-39.2

26.5

22.6-31.0

26.5

25.0-28.1

37.7

32.4-43.9

3

24.2

20.0-29.2

24.2

23.3-25.1

35.0

28.0-43.7

34.0

29.5-39.5

29.0

27.2-30.9

21.9

20.7-23.2

35.0

32.5-37.7

28.1

22.7-34.8

22.8

20.4-25.4

25.0

22.8-27.4

25.9

22.1-30.4

Duration of exposure (hours)

6 12

20.5 

18.4-22.8 

23.0 22.5

21.8-24.2 21.3-23.8

35.0 27.5

28.0-43.7 26.1-29.0

34.0 34.0

29.5-39.1 29.5-39.1

26.0 25.9

24.8-27.4 24.9-26.9

21.9 19.3

20.7-23.2 18.6-20.0

29.5 29.0

27.8-31.3 27.2-30.9

28.1 28.1

22.7-34.8 22.7-34.8

22.8 21.0

20.4-25.4 19.5-22.6

23.0 22.0

21.8-24.3 20.8-23.3

22.0 22.0

19.7-24.6 19.7-24.6

24

17.2

15.5-19.1

22.5

21.3-23.8

24.0

21.6-26.6

34.0

29.5-39.1

25.9

24.9-26.9

19.0

17.9-20.1

28.0

26.7-29.3

28.1

22.7-34.8

21.9

20.3-23.6

21.9

20.6-23.2

22.0

19.7-24.6

96

17.2

15.5-19.1

11.0

10.2-11.8

20.0

18.1-22.0

30.0

26.7-33.7

25.9

24.9-26.9

19.0

17.9-20.1

18.5

12.3-14.8

28.1

22.7-24.8

17.0

15.8-18.3

20.2

19.0-21.4

 

"Ten fish per test concentration; 20 for other species.

Table 2. Toxicity (LC50 in mg/L, and 95% confidence interval) of benzocaine to rainbow trout in water of different

temperatures, hardnesses, and pH's.

Temperature

(°C)

7

12

17

12

12

12

12

12

12

12

Hardness pH

Soft 7.8

Soft 7.8

Soft 7.8

Very soft 8.2

Soft 8.2

Hard 8.2

Very hard 8.2

Soft 6.5

Soft 8.5

Soft 9.5

1

23.0

21.8-24.3

27.0

25.7-28.4

26.0

23.6-26.4

31.0

28.4-33.8

28.0

26.2-29.9

24.0

21.5-26.8

30.0

27.6-32.6

27.0

24.9-29.2

35.5

32.5-38.8

31.0

28.5-33.7

Duration of exposure (hours)

3 6 12

19.8 19.0 17.0

18.8-20.9 18.0-20.1 15.7-18.4

24.2 23.0 22.5

23.3-25.1 21.8-24.2 21.3-23.8

23.8 22.0 22.0

22.7-24.9 20.7-23.3 20.7-23.3

26.8 24.0 24.0

24.7-29.1 21.4-26.9 21.4-26.9

26.0 24.0 24.0

23.7-28.5 21.4-26.9 21.4-26.9

24.0 24.0 24.0

21.5-26.8 21.5-26.8 21.5-26.8

27.0 27.0 27.0

24.9-29.2 24.9-29.2 24.9-29.2

24.0 24.0 24.0

21.4-26.9 21.4-26.9 21.4-26.9

30.4 29.0 24.0

27.9-33.1 26.8-31.4 21.4-26.9

24.2 24.2 21.0

21.5-27.2 21.5-27.2 19.7-22.4

24

17.0

15.7-18.4

22.5

21.3-25.8

20.5

19.2-21.9

22.0

20.3-23.9

24.0

21.4-26.9

24.0

21.5-26.8

23.2

21.1-25.5

21.0

19.5-22.6

22.0

20.4-23.8

19.0

17.3-20.8

96

13.3

12.3-14.3

11.0

10.2-11.8

7.20

5.92-9.76

8.60

7.98-9.26

7.50

6.53-8.62

9.70

8.90-10.6

23.2

21.1-25.5

 

 

 
INVESTIGATIONS IN FISH CONTROL 102

Table 3. Toxicity (LC50 in mg/L, and 95% confidence interval) of benzocaine to blue gills in water of different

temperatures, hardnesses, andpH's.

Temperature

(°Q

12

17

22

12

12

12

12

12

12

12

Table 4.

Hardness

Soft

Soft

Soft

Very soft

Soft

Hard

Very hard

Soft

Soft

Soft

Toxicity (LC50 in

PH

7.8

7.8

7.8

8.2

8.2

8.2

8.2

6.5

8.5

9.5

Duration of exposure (hours)

1

26.5

22.6-31.0

27.0

20.7-35.2

27.0

24.8-29.4

29.0

27.4-30.7

28.5

26.6-30.5

29.5

26.7-32.5

29.0

27.2-30.9

25.5

23.4-27.8

29.0

26.1-32.2

25.0

22.2-28.1

3

22.8

20.4-25.4

25.0

22.4-27.9

22.0

19.5-24.8

27.0

25.4-28.7

25.0

23.8-26.2

24.5

22.6-26.5

28.0

26.7-29.4

22.0

20.2-24.0

25.0

23.0-27.1

23.5

21.2-26.1

6

22.8

20.4-25.4

25.0

22.4-27.9

21.0

19.5-22.6

25.0

22.7-27.6

23.0

21.8-24.3

23.0

21.8-24.3

25.6

24.3-27.0

21.2

20.1-22.3

23.0

21.3-24.8

23.0

20.8-25.4

mg/L, and 95% confidence interval) ofbenzocaine

12

21.0

19.5-22.6

25.0

22.4-27.9

18.6

16.9-20.4

22.3

21.1-23.6

22.9

21.7-24.2

23.0

21.6-24.5

24.3

23.4-25.3

21.2

20.1-22.3

21.5

19.9-23.2

22.5

20.8-24.3

24

21.9

20.3-23.6

23.0

21.1-25.1

17.8

16.5-19.2

22.3

21.1-23.6

22.0

20.8-23.3

23.0

21.6-24.5

21.3

19.8-22.9

21.2

20.1-22.3

20.6

19.3-22.0

22.5

20.8-24.3

96

17.0

15.8-18.3

18.0

16.2-20.0

9.20

7.48-11.3

16.0

14.6-17.5

17.0

15.8-18.3

18.0

16.9-19.2

15.1

13.6-16.7

19.0

17.4-20.8

19.5

18.6-20.4

22.0

20.7-23.3

to channel catfish in water of, different

temperatures, hardnesses, andpH's.

Duration of exposure (hours)

Temperature

(°C)

12

17

22

12

12

12

12

12

12

12

Hardness

Soft

Soft

Soft

Very soft

Soft

Hard

Very hard

Soft

Soft

Soft

pH

7.8

7.8

7.8

8.2

8.2

8.2

8.2

6.5

8.5

9.5

1

36.0

33.1-39.1

34.5

32.4-36.7

34.0

31.3-36.9

49.0

44.8-53.4

48.0

43.9-52.3

49.0

43.4-55.3

49.0

44.8-53.5

49.0

44.8-53.5

48.0

43.9-52.5

42.0

39.2-45.0

3

35.0

32.5-37.7

33.0

31.2-34.9

33.0

31.2-34.9

35.0

32.9-37.3

34.7

32.6-37.0

33.0

31.2-34.9

34.7

32.6-36.9

38.0

34.8-41.5

35.5

33.0-38.2

35.1

32.6-37.8

6

29.5

27.8-31.3

33.0

31.2-34.9

30.0

28.3-31.2

34.0

31.9-36.2

34.7

32.6-37.0

33.0

31.2-34.9

34.7

32.6-36.9

32.0

30.5-33.6

35.0

32.9-37.3

33.0

31.2-34.9

12

29.0

27.2-30.9

31.0

29.4-32.7

28.0

26.7-29.4

31.0

29.5-32.5

30.2

28.7-31.8

30.0

28.4-31.7

30.0

28.2-31.9

31.0

29.5-32.6

31.5

30.0-33.1

29.0

27.2-30.9

24

28.0

26.7-29.3

29.0

26.9-31.2

27.9

26.5-29.3

30.0

28.2-31.9

30.2

28.7-31.8

30.0

28.2-31.9

29.4

27.6-31.3

30.1

28.5-31.8

31.0

29.3-32.7

29.0

27.2-30.9

96

13.5

12.3-14.8

16.0

14.1-18.2

13.8

11.3-16.8

20.0

17.9-22.5

15.8

13.7-18.2

28.0

26.6-29.4

15.0

13.8-16.3

27.5

26.1-29.0

28.0

26.2-30.0

26.0

23.7-28.5
EFFECTS OF WATER TEMPERATURE, HARDNESS, AND pH

with the calculated concentrations (Table 5). At the beginning of the exposures, concentrations in the water samples 

were within 5% of the calculated values. Analysis of the 

same vessels 96 h later showed little, if any, change in the 

benzocaine concentration. Also, water temperature, hardness, and pH did not affect the rate of degradation of 

benzocaine over the 96-h period.

Use Pattern Exposure

Rainbow trout exposed to benzocaine at the use pattern 

concentration of 25 mg/L for 15 min recovered from the 

anesthesia within 15 min or less and responded much like 

control fish during the 14-day postexposure period. However, all fish exposed to 3 or 5 times the effective concentration died within 30 min.

Discussion

The use pattern concentration of 25 mg/L was chosen to 

represent the level that was effective for anesthetizing 

small salmonids (Gilderhus 1989). At that level, fish were 

effectively anesthetized in 3 min or less, recovered from 

anesthesia in less than 5 min, and survived 15 min of 

exposure. In our use pattern exposures, the fish were also 

exposed for 15 min, which is about 5 times the duration 

necessary for effective anesthesia. Although the X3 and 

X5 concentrations were lethal in 15-min exposures, they 

would be much less toxic in 3-min exposures. However, 

the margin of safety to treated fish is not high, and users

should be aware that overdosing could result in undue 

stress or mortality.

Benzocaine is uniformly toxic to different species of 

fish and at various water temperatures, pH's, and hardnesses. This consistency is an advantage because users 

need not be concerned about alterations in safety to the 

fish. The toxicity of many other fishery chemicals is influenced by water characteristics, especially pH (Hunn and 

Alien 1974). Fasman (1976) reported the ionization constant (pka) for benzocaine to be 2.38. At this pH, half the 

benzocaine would be in the ionized form and half in the 

un-ionized form (Dawson and Gilderhus 1979). At the 

pH's of 6.5-9.5, there would be little change in the concentration of the un-ionized, lipid soluble form of benzocaine 

available for uptake; thus, little, if any, change in toxicity 

at these pH's would be expected.

The 96-h exposures were of little relevance for evaluating safety, but the values generated demonstrate that exposure time beyond 1 h does not critically increase the toxicity. The concentrations of benzocaine in the water 

solutions remained nearly constant for 96 h, indicating 

that the chemical was not degraded and the fish did not 

remove a significant portion of the chemical at this loading 

rate. In some of the exposures for 96 h, dissolved oxygen 

fell below 50% saturation. As a result, toxicity increased 

in those tests. The ethanol solvent seemingly provided 

bacteria with a nutrient source that allowed them to proliferate in some test vessels. Variability in the 96-h results is 

probably due to these bacterial growths.

Toxicity of benzocaine to fish compares closely to that 

of MS-222. Marking (1967) reported that for MS-222, the

Table 5. HPLC analysis of benzocaine concentrations of 7.5, 10, and 30 mg/L (calculated) in water at selected

temperatures, hardness, andpH's at 0 and 96 h.

Benzocaine concentration (mg/L)

Temperature

7

12

17

12

12

12

12

12

12

12

Hardness

Soft

Soft

Soft

Very soft

Soft

Hard

Very hard

Soft

Soft

Soft

pH

7.8

7.8

7.8

8.2

8.2

8.2

8.2

6.5

8.5

9.5

Calculated

10

10

10

7.5

7.5

7.5

7.5

7.5

7.5

7.5

Measured

0 ha, 96 ha

9.5, 9.4

9.4, 9.3

9.6, 9.0b

7.1,6.4

7.3, 6.7

7.3, 6.9

7.3b, 6.8

6.8, 6.6

7.1 b, 6.8

6.9, 6.7

Calculated

30

30

30

30

30

30

30

30

30

30

Measured

0 ha, 96 ha

28.5, 29.4

28.4, 29.4

29.2, 29.3

28.5, 28.1

28.2, 29.1

28.4, 29.4

27.3, 27.6

27.0, 27.8

28.3,29.1

26.8, 26.9

"Duplicate samples. 

bAverage of triplicate analysis.
INVESTIGATIONS IN FISH CONTROL 102

24-h LCSO's ranged from 34 to 64 mg/L. Although the 

values for MS-222 are slightly higher than those for benzocaine, MS-222 was formulated as a methanesulfonate 

salt. The higher molecular weight of the formulation 

would be expected to yield an anesthetic less active than 

the benzocaine formulation. The toxicity of MS-222 also 

was affected little by water hardness or temperatures, and 

safety for rainbow trout in 15-min exposures to MS-222 

was similar to that for benzocaine.

References

ASTM Committee E-35 on Pesticides. 1980. Standard practice 

for conducting acute toxicity tests with fishes, macroinvertebrates, and amphibians E729-80. Pages 1-25 in Annual book 

of ASTM standards. Part 46. End use and consumer products. 

American Society for Testing and Materials, Philadelphia, Pa.

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

uses of some general anesthetics for fish. 2nd ed. Bull. Fish. 

Res. Board Can. 148. 9 pp.

Committee on Methods for Acute Toxicity Tests with Aquatic 

Organisms. 1975. Methods for acute toxicity tests with fish, 

macroinvertebrates, and amphibians. U.S. Environ. Prot. 

Agency, Ecol. Res. Serv., EPA 660/3-75-09. 61 pp.

Dawson, V. K., and P. A. Gilderhus. 1979. Ethyl-p-aminobenzoate (benzocaine): efficacy as an anesthetic for five species of 

freshwater fish. U.S. Fish Wildl. Serv., Invest. Fish Control 87. 

5pp.

Fasman, G. D., editor. 1976. CRC handbook of biochemistry and 

molecular biology, section D Physical and chemical data. 

3rd ed. Vol. 1. CRC Press, Cleveland, Ohio. 576 pp.

Gilderhus, P. A. 1989. Efficacy of benzocaine as an anesthetic for

salmonid fishes. N. Am. J. Fish. Manage. 9:150-153.

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

Gilderhus, P. A., and L. L. Marking. 1987. Comparative efficacy 

of 16 anesthetic chemicals on rainbow trout. N. Am. J. Fish. 

Manage. 7:288-292.

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

gills of fishes. Pages 47-55 in H. W. Elliot, ed. Annual review 

of pharmacology. Vol. 14. Annual Reviews, Inc., Palo Alto, 

Calif.

Lennon, R. E., and C. R. Walker. 1964. Laboratories and methods 

for screening fish-control chemicals. U.S. Fish Wildl. Serv., 

Invest. Fish Control 1 (Circ. 185). 15 pp.

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

of evaluating dose-effect experiments. J. Pharmacol. Exp. 

Ther. 96:99-113.

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. 1975. Toxicological protocol for the development 

of piscicides. Pages 26-31 in P. H. Eschmeyer, ed. Rehabilitation of fish populations with toxicants: A symposium. Am. 

Fish. Soc. Spec. Publ. 4.

Marking, L. L., and F. P. Meyer. 1985. Are better anesthetics 

needed in fisheries? Fisheries (Bethesda) 10(6):2-5.

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

cold-blooded vertebrates. Copeia 1967:239-240.

McFarland, W. H. 1959. A study of the effects of anesthetics on 

the behavior and physiology of fishes. Publ. Inst. Mar. Sci., 

Univ. Tex. 6:23-55.

U.S. Department of Agriculture. 1986. Interregional Research 

Project No. 4. Guidelines for IR-4 investigations. New Jersey 

Agricultural Experiment Station, Cook College, Rutgers University, New Brunswick. 6 pp.
Bills, Terry D., George E. Howe, and Leif L. Marking. 1990. Effects of Water 

Temperature, Hardness, and pH on the Toxicity of Benzocaine to Eleven 

Freshwater Fishes. U.S. Fish. Wildl. Serv., Invest. Fish Control 102. 6 pp.

The toxicity of benzocaine (ethyl 4-aminobenzoate; 98%) to eleven freshwater 

fishes was evaluated under various physical and chemical conditions. Under 

standard test conditions (pH 7.8,12° C, soft water), the 24-h LC50 (mg/L) 17.2 for 

lake sturgeon (Acipenserfulvescens), was 22.5 for rainbow trout (Oncorhynchus 

mykiss), 34.0 for muskellunge (Esox masquinongy), 24.0 for northern pike (Esox 

lucius), 19.0 for common carp (Cyprinus carpio), 25.9 for fathead minnow 

(Pimephales promelas), 28.0 for channel catfish (Ictalurus punctatus), 28.1 for 

striped bass (Morone saxatilis), 21.9 for green sunfish (Lepomis cyanellus), 21.9 

for bluegill (L. macrochirus), and 22.0 for walleye (Stizostedion vitreum). Tests 

conducted at three water temperatures, four hardnesses, and three pH's showed 

that none of these variables influenced toxicity. The higher temperatures tested (to 

22° C) did not increase the rate of toxicosis. Rainbow trout survived exposure to 

the recommended use concentration of 25 mg/L for 15 min but did not survive 

exposure to 75 or 125 mg/L. Users of benzocaine should be cautioned that 

overdoses may lead to undue stress or mortality in exposed fish. Comparison of 

benzocaine concentrations by high performance liquid chromatography at 0 and 

96 h showed no degradation of the compound during the exposure period.

Key words: Toxicity, benzocaine, anesthetic, environmental factors, fish.

_

Bills, Terry D., George E. Howe, and Leif 

Temperature, Hardness, and pH on th

Freshwater Fishes. U.S. Fish. Wildl. S

The toxicity of benzocaine (ethyl 4-amino

fishes was evaluated under various physi

standard test conditions (pH 7.8,12° C, soft 

lake sturgeon (Acipenserfulvescens), was 2

mykiss), 34.0 for muskellunge (Esox masqu

lucius), 19.0 for common carp (Cyprinus 

(Pimephales promelas), 28.0 for channel c

striped bass (Morone saxatilis), 21.9 for gre

for bluegill (L. macrochirus), and 22.0 for 

conducted at three water temperatures, fou

that none of these variables influenced toxici

22° C) did not increase the rate of toxicosis

the recommended use concentration of 25 

exposure to 75 or 125 mg/L. Users of b

overdoses may lead to undue stress or mort

benzocaine concentrations by high perform

96 h showed no degradation of the compou

Key words: Toxicity, benzocaine, anesthet

Bills, Terry D., George E. Howe, and Leif L. Marking. 1990. Effects of Water Temperature, Hardness, 

and pH on the Toxicity of Benzocaine to Eleven Freshwater Fishes. U.S. Fish. Wildl. Serv., Invest. 

Fish Control 102. 6 pp.

The toxicity of benzocaine (ethyl 4-aminobenzoate; 98%) to eleven freshwater fishes was evaluated 

under various physical and chemical conditions. Under standard test conditions (pH 7.8, 12° C, soft 

water), the 24-h LC50 (mg/L) 17.2 for lake sturgeon (Acipenserfulvescens), was 22.5 for rainbow trout 

(Oncorhynchus mykiss), 34.0 for muskellunge (Esox masquinongy), 24.0 for northern pike (Esox lucius), 

19.0 for common carp (Cyprinus carpio), 25.9 for fathead minnow (Pimephales promelas), 28.0 for 

channel catfish (Ictalurus punctatus), 28.1 for striped bass (Morone saxatilis), 2 1.9 for green sunfish 

(Lepomis cyanellus), 21.9 for bluegill (L. macrochirus), and 22.0 for walleye (Stizostedion vitreum). Tests 

conducted at three water temperatures, four hardnesses, and three pH's showed that none of these 

variables influenced toxicity. The higher temperatures tested (to 22° C) did not increase the rate of 

toxicosis. Rainbow trout survived exposure to the recommended use concentration of 25 mg/L for 15 min 

but did not survive exposure to 75 or 125 mg/L. Users of benzocaine should be cautioned that overdoses 

may lead to undue stress or mortality in exposed fish. Comparison of benzocaine concentrations by high 

performance liquid chromatography at 0 and 96 h showed no degradation of the compound during the 

exposure period.

Key words: Toxicity, benzocaine, anesthetic, environmental factors, fish.

Bills, Terry D., George E. Howe, and Leif L. Marking. 19

and pH on the Toxicity of Benzocaine to Eleven Fr

Fish Control 102. 6 pp.

The toxicity of benzocaine (ethyl 4-aminobenzoate; 

under various physical and chemical conditions. Unde

water), the 24-h LC50 (mg/L) 17.2 for lake sturgeon (A

(Oncorhynchus mykiss), 34.0 for muskellunge (Esox ma

19.0 for common carp (Cyprinus carpio), 25.9 for fat

channel catfish (Ictalurus punctatus), 28.1 for striped 

(Lepomis cyanellus), 21.9 for bluegill (L. macrochirus), a

conducted at three water temperatures, four hardness

variables influenced toxicity. The higher temperatures 

toxicosis. Rainbow trout survived exposure to the recom

but did not survive exposure to 75 or 125 mg/L. Users o

may lead to undue stress or mortality in exposed fish. Co

performance liquid chromatography at 0 and 96 h show

exposure period.

Key words: Toxicity, benzocaine, anesthetic, environm



(Reports 87 through 89 are in one cover.)

87. Ethyl-p-aminobenzoate (Benzocaine): Efficacy as an Anesthetic for Five Species of Freshwater Fish, by 

V. K. Dawson and P. A. Gilderhus. 1979. 5 pp.

88. Influences of Selected Environmental Factors on the Activity of a Prospective Fish Toxicant, 2-(Digeranyl-amino)- 

ethanol, in Laboratory Tests, by C. A. Launer and T D. Bills. 1979. 4 pp.

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. 1979. 5 pp.

(Reports 90 and 91 are in one cover.)

90. Accumulation and Loss of 2',5-Dichloro-4'-nitrosalicylanilide (Bayer 73) by Fish: Laboratory Studies, by 

V. K. Dawson. J. B. Sills, and Charles W. Luhning. 1982. 5 pp.

91. Effects of Synergized Rotenone on Nontarget Organisms in Ponds, by R. M. Burress. 1982. 7 pp.

(Reports 92 through 94 are in one cover.)

92. Acute and Chronic Toxicity of Rotenone to Daphnia magna, by J. J. Rach, T. D. Bills, and L. L. Marking. 1988.5 pp.

93. Toxicity of Rotenone to Developing Rainbow Trout, by T. D. Bills, J. J. Rach, and L. L. Marking. 1988. 3 pp.

94. Oral Toxicity of Rotenone to Mammals, by L. L. Marking. 1988. 5 pp.

95. Deposition and Persistence of Rotenone in Shallow Ponds During Cold and Warm Seasons, by P. A. Gilderhus, 

V. K. Dawson, and J. L. Alien. 1988. 7 pp.

(Reports 96 and 97 are in one cover.)

96. Effects of Environmental Factors on the Toxicity of Chloramine-T to Fish, by T. D. Bills, L. L. Marking, 

V. K. Dawson, and J. J. Rach. 1988. 6 pp.

97. Effects of Organic Matter and Loading Rates of Fish on the Toxicity of Chloramine-T, by T. D. Bills, L. L. Marking, 

V. K. Dawson, and G. E. Howe. 1988. 4 pp.

98. History of Acute Toxicity Tests with Fish, 1963-1987, by J. B. Hunn. 1989. 16 pp.

99. Evaluation of 215 Candidate Fungicides for Use in Fish Culture, by T. A. Bailey and S. M. Jeffrey. 1989. 9 pp.

NOTE: Use of trade names does not imply U. S. Government endorsement of commercial products.
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