INVESTIGATIONS IN FISH CONTROL

12. Toxicity of MS-222 to Selected Fishes

13. Efficacy of MS-222 as an Anesthetic 

on Four Salmonids

14. Method for Determining MS-222 Residues 

in Fish

15. Residues of MS-222 in Four Salmonids 

Following Anesthesia

16. Annotated Bibliography on MS-222

17. MS-222 as an Anesthetic for Channel Catfish: 

Its Toxicity, Efficacy, and Muscle Residues

United States Department of the Interior

Fish and Wildlife Service 

Bureau of Sport Fisheries and Wildlife
INVESTIGATIONS IN FISH CONTROL

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

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

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

economy.

Current reports in this series are~

(Reports 1 and 2 are in one cover.)

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

and Charles R. Walker. (Bureau Circular 185.) 1964. 15 p.

2. Preliminary Observations on the Toxicity of Antimycin A to Fish and Other Aquatic 

Animals, by Charles R. Walker, Robert E. Lennon, and Bernard L. Berger. (Bureau 

Circular 186.) 1964. 18 p.

(Reports 3 through 8 are in one cover.)

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

Henegar. (Resource Publication 7.) 1966. 16 p.

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

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

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

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

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

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

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

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

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

1966. 9 p.

(Reports 9 through 11 are in one cover.)

9. Relation of Chemical Structure to Fish Toxicity in Nitrosalicylanilides and Related Compounds, by Charles R. Walker, Roland J. Starkey, and Leif L. Marking. (Resource Publication 13.) 1966. 12p.

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

(Resource Publication 14.) 1966. lOp.

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

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

(Reports 12 through 17 are in one cover.)

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

1967. lOp.

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

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

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

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

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

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

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

1967. 15p.

17. MS-222 as an Anesthetic for Channel Catfish: Its Toxicity, Efficacy, and Muscle Residues, by Richard A. Schoettger, Charles R. Walker, Leif L. Marking, and Arnold M. 

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

Fish Control Laboratories

Bureau of Sport Fisheries and Wildlife

U.S. Department of the Interior

P.O.Box 862

La Crosse, Wisconsin 54602
INVESTIGATIONS IN FISH CONTROL

12. Toxicity of MS-222 to Selected Fishes

By Leif L. Marking (Resource Publication 18, p. 1-10)

13. Efficacy of MS-222 as an Anesthetic on Four Salmonids

By Richard A. Schoettger and Arnold M. Julin 

(Resource Publication 19, p. 1-15)

14. Method for Determining MS-222 Residues in Fish

By Charles R. Walker and Richard A. Schoettger 

(Resource Publication 20, p. 1-10)

15. Residues of MS-222 in Four Salmonids Following Anesthesia

By Charles R. Walker and Richard A. Schoettger 

(Resource Publication 21, p. 1-11)

16. Annotated Bibliography on MS-222

By Richard A. Schoettger (Resource Publication 22, p. 1-15)

17. MS-222 as an Anesthetic for Channel Catfish: 

Its Toxicity, Efficacy, and Muscle Residues

By Richard A. Schoettger, Charles R. Walker, Leif L. Marking, 

and Arnold M. Julin (Resource Publication 33, p. 1-14)

United States Department of the Interior, Ste\vart L. Udall, Secretary

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

Fish and Wildlife Service, Clarence F. Pautzke, Commissioner

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

Washington, D.C. January 1967
FOREWORD

The Food and Drug Administration of the U.S. Department of Health, 

Education, and Welfare advised the Bureau of Sport Fisheries and Wildlife that certain drugs and chemicals used in the culture and management of game fishes must be cleared for continued use. We must demonstrate to the PDA that fish exposed to the compounds pose no danger, 

because of residues, to people who might eat them.

MS-222 is among the drugs mentioned because it is commonly used to 

anesthetize fish during marking or tagging, spawning by stripping, and 

transportation. The information necessary for clearance includes the 

toxicity of the drug to fish, its efficacy as an anesthetic, and its residues 

in fish. PDA also requires that fish of various sizes be represented in 

tests of the drug at three or more'temperatures in waters of different 

qualities. Accordingly, we investigated the toxicity of MS-222 to eight 

species of game fish, its efficacy on four trouts, and its residues in the 

same trouts (emphasis was placed on residues in muscle because muscle 

is the principal tissue eaten by people).

MS-222 was synthesized by Maurice Sandoz (Sandoz, Ltd., Basle, 

Switzerland, and Hanover, N.J.) during his search about 45 years ago 

for a synthetic substitute for cocaine. It has been used as a local anesthetic in preparations for humans, but its general anesthetic activity 

against cold-blooded animals was recognized early. Within the past 

decade, its use as an anesthetic in fish culture and fish management has 

become widespread in the United States and abroad. For example, the 

millions of lake trout stocked in the upper Great Lakes in the past several years were first anesthetized with MS-222 and fin-clipped.

The considerable variety of common and chemical names applied to 

MS-222 in the literature causes some confusion. The names include:

MS-222. Metacaine methanesulphonate.

MS-222 Sandoz. m-amino ethyl benzoate.

M. S.-222 Sandoz. Ethyl m-aminobenzoate.

TS-222. Methane sulfonic acid salt of

TS-222 Sandoz. meta-amino ethyl benzoate.

Tricaine. Methanesulphonate of

Tricaine - Sandoz. meta-aminobenzoic acid. 

Tricaine methanesulfonate. Methanesulphonate meta-aminoMetacaine. benzoic acid ethyl ester.

Of these names, we used MS-222, tricaine methanesulfonate, and 

methanesulphonate of meta-aminobenzoic acid ethyl ester.

Robert E. Lennon, Director 

Fish Control Laboratories
/NVfST/GAT/ONS IN FISH CONTROL

12. Toxicity of MS-222 to Selected Fishes

By Leif L. Marking

United States Department of the Interior, Stewart L. Udal!, Secretary 

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

Fish and Wildlife Service, Clarence F. Pautzke, Commissioner 

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

Resource Publication 18 January 1967 Washington, D.C.
CONTENTS

Page

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

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

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

Species and sizes of fish ..................... 5

Effect of temperature ....................... 6

Effect of water quality. ...................... 6

Recovery ............................... 6

Safety indices ............................ 7

Discussion ................................. g

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

Summary .................................. 9

Literature cited.............................. 10
TOXICITY OF MS-222 TO SELECTED FISHES

By Leif L. Marking, Chemist,

Bureau of Sport Fisheries and Wildlife

La Crosse, Wis.

Abstract. Toxicity of MS-222 to rainbow trout, brown trout, brook 

trout, lake trout, northern pike, bluegill, largemouth bass, and walleye 

of various sizes was determined in 15-, 30-, and 60-minute and 24-, 48-, 

and 96-hour static bioassays at selected temperatures. Twenty-fourhour LC 50 values for the eight species ranged from 33.8 to 63.0 p.p.m. 

Exposures longer than 24 hours had little effect on toxicity. Small fish of 

a species were more sensitive to the drug than large ones, and trout were 

more sensitive at warmer temperatures. Safety indexes were calculated 

on the basis of the brief exposures.

Bove (1962) identified MS-222 as the 

methanesulphonate of meta-aminobenzoic acid 

ethyl ester. The compound is a fine, white 

crystalline powder with a molecular weight of 

261.3 and a melting point of 145° to 150° C. It 

leaves only minimum traces of ash upon ignition and is free from chlorides, sulfates, alkaloids, and heavy metals. Less than 0.5 percent 

of its weight is lost upon heating to 103° C. It 

is soluble to 11 percent in water and forms a 

clear, colorless, acid, and relatively stable 

solution. He also reported that a 10-percent 

solution retained its potency during 3 days' 

storage, but decreased 5 percent in activity 

after 10 days. Exposure of a solution to light 

caused a change in color to yellow or brown, 

but its activity was not affected.

The molecular structure of MS-222, although not defined in literature, possibly conforms to figure 1. Since it is highly soluble 

(up to 22.5 percent in our laboratory) a proton bond probably exists between the amine 

group on the benzoate structure and the hydrogen on the methane sulfonante. In the ionization of the compound, the hydrogen probably 

splits off the sulfonate group, being attracted 

to the nitrogen of the amine group.

COOC 2 H 5

Figure 1. Structure of MS-222.

MS-222 is an effective anesthetic for fish 

and other coldblooded animals (McFarland, 

1959; Eisler and Backiel, 1960; Bove*, 1962; 

Schoettger and Julin, 1966). Rothlin (1932) 

stated that the drug is three times less toxic 

to coldblooded animals than novocaine and ten 

times less toxic than cocaine.

There are reports, however, that under certain circumstances MS-222 is toxic to certain 

species or strains of fish. Marvin Smith 

(Regional Fishery Management Biologist at 

Atlanta, in a memorandum report to the 

Branch of Game Fish and Hatcheries, June 12, 

1956) recommended that MS-222 be bioassayed for toxicity to rainbow trout under
Investigations in Fish Control 12 : Bureau of Sport Fisheries and Wildlife

various water qualities. Thompson (1959) observed a 95-percent mortality among cutthroat trout exposed to 50 p.p.m. of MS-222 

for 5 hours. Others have noted that excessive 

exposures cause mortalities among fish 

(Nelson, 1953; Parkhurst and Smith, 1957; and 

Eisler and Backiel, 1960).

The purpose of this investigation was to define the concentrations of MS-222 which are 

toxic to eight species of freshwater, game 

fish of various sizes. The experimental conditions included different water qualities and 

durations of exposure at four temperatures. 

Safety indices for rainbow trout were established.

METHODS AND MATERIALS

Eight species of fish were obtained from 

various fish hatcheries (table 1). The three 

size groups included small fingerlings 1 to 3 

inches long, an intermediate group of 3 to 5 

inches, and a group 6 to 9 inches long. All fish 

were quarantined for 10 days and if judged 

acceptable, were then acclimated to the temperatures and conditions of the tests of 

MS-222.

The static bioassays of the anesthetic 

against 2-inch fish were conducted in 5- 

gallon glass jars (Lennon and Walker, 1964). 

At least 120 fish of this size were included 

in each test of 10 concentrations of drug. 

Ten fish were exposed to each concentration, 

and 20 served as controls. The tests against

TABLE 1. Fishes used in toxioity tests of MS-222 

Source

Rainbow trout (Salmo gairdnerl)....

Brown trout (Salmo trutta)

Brook trout (Salvelinus fontlnalls). 

Lake trout (Salvelinus namaycush)..

Northern pike (Esox luelus).

Bluegill (Lepomis maerochirus).....

Largemouth bass (Mlcropterus 

salmoides).................

Walleye (Stlzostedlon vitreum).

Manchester National Fish Hatchery, 

Iowa.

Manchester National Fish Hatchery, 

Iowa.

State Fish Hatchery at Osoeola, Wis.

Jordan River National Fish Hatchery, 

Moh., and St. Croix Falls, Wis.

Garrison Dam National Fish Hatchery, 

N. Dakota and Yankton National 

Fish Hatchery, S. Dakota.

Lake Mills National Fish Hatchery,

Genoa National Fish Hatchery, Wis.

Garrison Dam National Fish Hatchery, 

N. Dakota.

4- and 9-inch fish were made in aerated, 

polyethylene tanks which contained 45 liters 

of anesthetic solution. Ten of the larger fish 

were exposed to each of 5 concentrations per 

test, and 10 served as controls.

Water temperatures were controlled by 

placing the bioassay vessels in water baths at 

7°, 12°, 170, and 22° C. Water hardnesses of 

10, 42, and 176 p.p.m. were arranged by adding different amounts of the mixture of reconstituting salts to deionized water (table 2).

TABLE 2. Water qualities obtained with various amounts of reconstituting 

salts in deionized water

Water 

quality

Soft. ..... 

Standard. . 

Hard......

Salts in mg./l.

NaHC03

12 

48 

192

CaSO^

7.5 

30 

120

MgSO^

7.5 

30 

120

KC1

.75 

3 

12

Total 

hardness

(p.p.m.)

10 

42 

176

Total 

alkalinity 

(p.p.m.)

10 

30 

116

PH

6.72 

6.75 

7.70

Concentrated stock solutions of MS-222 

were prepared daily in deionized water. Aliquots of the stock were added to bioassay 

vessels following the introduction of fish.

The responses of fish to MS-222 were recorded for several hours after exposure began 

and daily thereafter throughout the bioassay. 

It was possible during the tests to differentiate between dead and anesthetized fish. Dead 

specimens were removed immediately. Fish 

which remained in anesthesia throughout a 

test were placed in fresh water until they 

recovered. The times for recovery and survival were noted.

The data on survival and death of fish exposed to selected amounts of MS-222 were 

analyzed according to the method of Litchfield and Wilcoxon (1949) to define concentrations which produce 50-percent mortaility 

(LC5Q). In addition, the variance and the 95- 

percent confidence intervals were determined.

RESULTS

MS-222 demonstrated a definite and consistent toxicity to the eight species of fish. 

Furthermore, the concentrations permitting 

survival or causing mortality can be predicted with a high degree of confidence.
Leif L. Marking: Toxicity of MS-222 to Selected Fishes

Species and sizes of fish

Among the four trouts, the toxicity of MS222 at a given temperature is dependent on 

variables such as species, size, and duration 

of exposure. Lake trout are the most sensitive and brook trout the least (table 3). Brown 

and rainbow trout are equal in their sensitivity 

and intermediate to lake and brook trout.

Larger fish of a species are usually more 

resistant to MS-222 than smaller fish. This is 

apparent with rainbow trout as the 24-hour 

I_,C 50 increases from 39 p.p.m. for 2-inch 

fish to 52 p.p.m. for 9-inch fish. The trend is 

similar in 48- and 96-hour assays. The 

larger lake trout and brown trout also showed 

greater resistance than smaller fish at all 

periods of observation.

There was little difference in toxicity among 

brook trout of different sizes, but 6-inch

specimens appeared to be more sensitive 

than 4-inch fish. It turned out, however, that 

the larger fish had become infected with 

furunculosis just prior to the bioassay, and 

quite possibly the disease lowered their resistance. A repeat trial with healthy brook 

trout was not possible.

Exposures beyond 24 hours do not have a 

pronounced effect on toxicity. In several instances the toxicity at 96 hours was identical 

to the 24-hour LC 50 . Among the trouts, only 

2-inch brown trout exhibited a significant difference, with a 20-percent drop in resistance 

between 24 and 96 hours.

Other species in the toxicity trials included 

northern pike, bluegill, largemouth bass, and 

walleye (table 4). Two-inch bluegill, largemouth bass, and walleye were only slightly 

less resistant than small brook trout. The 

larger specimens of bluegill and largemouth

TABLE 3. Toxicity of MS-222 to salmonids at 12° C.

Species and lot

Rainbow trout:

Brown trout:

Brook trout:

Lake trout:

Lot 162........................

weight 

(grams)

35.0

Approxilength 

(inches)

7

LC50

TO e

 3-3 0

35.8

39.8

At 24 hours

95-percent 

confidence interval

39.0-40.6

LC 5 o

 3C 0

39.2

At 48 hours

95-percent 

confidence interval

37.8-40.2

49 7-54 3

31 7-34 3

38.0-40.4

LC 50

43.8

43.0

"3c ft

38.5

At 96 hours

95-percent 

confidence interval

37.0-39.7

44 3-49 8

27 7-34 2

40.2-46.0

37.4-39.7

TABLE 4. Toxioity of MS-222 to warm-water fish at 12° C.

Species and lot

Northern pike:

Bluegill:

Largemouth bass:

Walleye: 

Lot 55.........................

weight 

(grams)

1

0

2

et

0.7

Approximate

length 

(inches)

2

LC 50

49.0

At 24 hours

95-percent 

confidence interval

46.2-51.9

LC50

48.5

At 48 hours

95-percent 

confidence interval

45.8-51.4

LC50

48.5

At 96 hours

95-percent 

confidence interval

44 7-48 4

45.8-51.4
6 Investigations in Fish Control 12: Bureau of Sport Fisheries and Wildlife

bass were more resistant than smaller ones. 

For example, the LC 50 for 7-inch largemouth 

bass was 50 percent greater than for 1-inch 

bass. Also, all sizes of bluegill, largemouth 

bass, and walleye were affected very little by 

exposures longer than 24 hours.

The northern pike were of two sizes, but the 

data were insufficient for separate treatment 

and they were combined to facilitate statistical 

evaluation. Both sizes were healthy, but it is 

difficult to bioassay pike because they are 

voracious and turn to cannibalism unless other 

live food is available. Their possible hunger 

stress was demonstrated by decreased resistance at 96 hours.

Effects of temperature

The effects of temperature on the toxicity 

of MS-222 to rainbow trout which averaged 

0.6 g. and 1.6 in. were evaluated (table 5). The 

trout were slightly more resistant at 7° than 

at 12° and 17° C. There was little difference 

between the LCso values at 24 and 96 hours 

at a given temperature.

TABLE 5. Toxici-ty of MS-222 to rainbow trout at selected temperatures

Temperature

riOfi

12°C........

 \rfOri

At 24 hours

LCSO

40.0 

37.2 

37.5

95-percent 

confidence 

interval

36.6-43.7 

36.3-38.1 

35.6-39.6

At 48 hours

LC50

39.5 

37.2 

37.2

95-percent 

confidence 

interval

37.6-41.6 

36.3-38.1 

36.7-37.7

At 96 hours

^50

39.5 

37.2 

36.5

95-percent 

confidence 

interval

37.6-41.6 

36.3-38.1 

35.9-37.1

MS-222 was more toxic to small bluegills at 

12° or 22° C. than at 17° (table 6). The differences, however, between LCso values obtained at 3 temperatures in 24-, 48-, and 96- 

hour tests were not large. The fish tested at 

22° were slightly smaller than the others, 

which may account for their greater sensitivity.

Effects of water quality

The effects of water hardness on the toxicity of MS-222 were established using rainbow trout which averaged 1.1 grams and 1.9 

inches (table 7). The fish were from the 

same lot, and they were exposed concurrently to the anesthetic at three hardnesses. 

Results were consistent and accurate at 42 

and 176 p.p.m. total hardness, but variable at 

10 p.p.m. which necessitated several repeats 

for reliability. The inconsistent mortalities 

at lower concentrations indicated an erratic 

action of the drug in soft water. Partial mortality occurred at 33 and 34 p.p.m. in 24 

hours, but no mortality occurred at 36 p.p.m. 

A previous, 24-hour test indicated considerably higher mortalities at 30 and 33 p.p.m. 

than at 38 p.p.m.

Recovery

The recovery of fish after 96-hour exposures was observed. At relatively low concentrations of MS-222, all fish recovered to 

some extent during the bioassay and completely during the recovery period immediately following the 96-hour exposure. At 

intermediate concentrations, which resulted 

in a partial kill during a 96-hour exposure, 

some of the survivors recovered partially 

previous to terminating the bioassay. Very 

few of the surviving fish died during the recovery period following exposure. The numbers of fish and the rates at which they recovered from anesthesia and survived 

exposures to the intermediate concentrations are listed in table 8.

The trout species recovered faster from 

partial-kill concentrations of MS-222 than the 

other fishes. Recovery was considered complete when specimens regained equilibrium 

and the ability to swim against a current.

TABLE 6.--Toxicity of US-222 to bluegills at selected temperatures

Temperature

12<V

17°C. ........

22°C.........

Lot 

No.

152

weight 

grams

0.8

length 

inches

1.6

^50

39.8

At 24 hours

95 -percent confidence 

interval

41.7-44.3

/ill, 1-45 ,9

39.0-40.6

LXso

/i/n 9

39.8

At 48 hours

95-percent 

confidence 

interval

41.7-44.3

39.0-40.6

M»o

39.8

At 96 hours

95 -percent 

confidence 

interval

41.7-44.3

42.7-45.3

39.0-40.6
Leif L. Marking: Toxicity of MS-222 to Selected Fishes

TABLE 7. Toxlcity of IB-222 to rainbow trout in selected water qualities at 12° C.

Total 

hardness alkalinity 

(p. p.m.)

pH K«

At 24 hours

95-peroent 

confidence 

interval

LC 50

 36 A

At 48 hours

95 -percent 

confidence 

interval

">«

At 96 hours

95-peroent 

confidence 

interval

TABLE 8. Recovery from anesthesia among fish exposed to concentrations of IB-222 causing partial Idlls within % hours at 12° c.

Species and length

Rainbow trout:

Brown trout:

Brook trout:

Lake trout:

Northern Pike: 

Bluegill :

Largemouth bass:

Walleye:

Partial kill 

concentration 

(p.p.m.)

34-44 

46-48 

48-50

20-40 

40-46 

44-46

44-50 

46-52 

46-54

32-36 

33-38 

36-40

47-54 

44-46 

45-47

44-56 

46-56

40-50

Number of fish

Surviving 

at 96 hours

37 

10 

15

25 

6 

6

31 

29 

25

9 

29 

24

14 

13 

29

10 

45

34

Recovering 

after % hours

34 

10 

15

25 

6 

6

31 

29 

25

8 

23 

24

13 

18 

29

10 

45

32

Minutes to recover 

in fresh water

2- 5 

1- 2 

3- 4

2- 8 

2- 4 

3- 13

2- 9 

2- 20 

4- 15

1- 2 

4- 12 

10- 30

2- 30 

10- 30 

2- 14

5- 60 

5-120

60-180

Rainbow trout exposed to 34 to 50 p.p.m. for 

96 hours recovered within I to 5 minutes when 

removed to fresh water. In contrast, walleye 

exposed to 40 to 50 p.p.m. for 96 hours required 60 to 180 minutes to recover. Of the 

356 fish removed to and recovering in fresh 

water, only 8 subsequently died. Among the 

survivors, the 2-inch rainbow trout exposed 

to 34 to 44 p.p.m. of the drug for 96 hours and 

removsf to fresh water were observed for 7 

days. There was no mortality; they fed readily 

and behaved similarly to controls.

Safety indexes

Rainbow trout averaging 1.4 grams and 2 

inches were exposed to MS-222 in brief bioassays to determine safety indexes. A safety 

index refers to the margin between efficacy 

and mortality. It is expressed by the quotient 

of a lethal concentration and an effective concentration.

Safety indexes for rainbow trout in table 9 

were derived from LCso and ECso values. 

The EC 50 here relates to the concentration 

of drug which produces total loss of equilibrium in half of the specimens (Schoettger 

and Julin, 1966). The best results were obtained in 15-, 30-, and 60-minute exposures. 

Shorter exposures may be desirable from the 

point of view of field practice, but they give 

less accurate results. The fish do not die 

uniformly or quickly enough. Longer exposures of 1 to 6 hours were not meaningful 

because some fish recovered from anesthesia; 

others died at concentrations close to the effective concentration.

The maximum safety index (M.S.I.) is the 

quotient of the LC^ and the £699 . The values 

were extrapolated from the regressions used 

in determining the LC50 and EC 50 . The 

M.S.I, is lower than the safety index and is 

biased in favor of greater safety.
8 Investigation in Fish Control 12: Bureau of Sport Fisheries and Wildlife

TABLE 9. Safety and maximum safety indexes of M3-222 against 2-inoh rainbow trout in brief exposures at 12° C.

Exposure

Water 

hardness 

(p. p.m.)

42 

42 

42

10 

10 

10

Safety index

LC 

(p. p.m.)

64.6 

56.8 

55.5 

64.0 

58.0 

54.2

EC50

(p. p.m.)

32.0 

31.8 

29.2 

34.0 

31.5 

31.2

Index 

LC /EC 

50 50

2.0 

1.8 

1.9 

1.9 

1.8 

1.7

Maximum safety index

LCi 

(p.p.m.)

54.0 

49.5 

46.0 

50.0 

46.5 

44.0

EC,, 

(p.p.m.)

38.2 

38.5 

36.8 

47.5 

45.0 

40.4

Index 

LCi/EC99

1.4 

1.3 

1.3 

1.1 

1.0 

1.1

The S.I. and M.S.I, derived for MS-222 

against small rainbow trout demonstrate that 

the shorter exposures are safer for the fish 

than longer exposures (table 9). This was true 

in waters with hardnesses of 10 and 42 p.p.m. 

The data also show that the drug is not as 

safe in soft water as in harder water.

DISCUSSION

Toxicity of MS-222 to the eight species of 

fish depended on the concentration of drug 

and the duration of exposure. There was a 

straight-line relation between concentration 

and time of exposure within the initial 24 

hours. Beyond 24 and up to 96 hours a given 

concentration showed little or no difference in 

toxicity. For example, the 15- and 30-minute 

and 24- and 96-hour LC 5o values for 2-inch 

rainbow trout were 64.6, 55.5, 39.0, and 

38.4 p.p.m. respectively. The 24- and 96-hour 

LC 50 values for some species were identical, 

such as 47 p.p.m. for 6-inch rainbow trout.

The reasons for identical LC so values at 

24 and 96 hours may include a decrease of 

concentration by absorption and metabolism 

in the fish, some natural degradation of the 

drug in solution, and greater activity of the 

chemical on fish within the early hours of 

exposure.

It is noteworth that the LCso values for the 

eight species did not differ greatly. The range 

of 24-hour LCso values for the more susceptible lake trout to the more tolerant largemouth bass was 33.8 to 63.0 p.p.m.

Among the salmonids, brook trout were the 

more resistant to MS-222. Interestingly, 

Walker and Schoettger (1966), detected lower 

residues of the drug in brook trout than in

rainbow, brown, and lake trout upon withdrawal from effective anesthesia. This may 

explain, in part, the higher resistance of the 

species to the anesthetic.

Water hardness did not influence the toxicity of MS-222 to rainbow trout, except that 

results at 10 p.p.m. were erratic. Parkhurst 

and Smith (1957) observed that rainbows react differently in various water supplies to the 

same concentrations of the drug. These inconsistencies suggest that the fish may not 

be exposed to MS-222 as safely in soft water 

as in hard water.

Sandoz Pharmaceuticals (in an undated leaflet "Toxicity of MS-222 to fish and frogs", 

2 p.) stated that the therapeutic index (LCj / 

EC 99 ) of MS-222 for rainbow trout was 1.57. 

The LC i, however, was defined in a 15- 

minute exposure of fish whereas the ECgg 

was defined in 3- to 4-minute exposures. 

Since the exposures for LC and EC differ, 

their index is not comparable to our safety 

indexes.

The S.I. (LCso /EC50 ) and the M.S.I. 

(LCi/EC99 ) displayed similar trends throughout the bioassays of MS-222 with one significant exception. There was little difference between the S. I. for rainbow trout in hard and 

soft water, but there was a significant difference between the M.S.I. The index was higher 

in the hard water. The slope of the regressions for LC and EC in soft water was less 

than in hard water, and the LCj was therefore smaller and the EC99 greater. These 

relations suggest that MS-222 is safer to 

rainbow trout in hard water than in soft. This 

was confirmed by Schoettger and Julin (1966) 

in their studies on the efficacy of the drug to 

rainbows in waters of various qualities. It is 

advisable to calculate both the S.I. and the
Leif L. Marking: Toxicity of MS-222 to Selected Fishes 9

M.S.I, because one may suggest a possible 

hazard when the other doesn't.

The speed and degree of recovery of fish 

from anesthesia are important. Complete recovery from brief exposures is rapid, and it 

is proportional to the concentration of MS222 and the duration of exposure according to 

Klontz (1964). This is not wholly the case in 

long exposures up to 96 hours. The concentrations of drug in long exposures did not 

hold all fish at a certain stage of narcosis for 

96 hours. Some fish were recovering from 

anesthesia each day after the first 24 hours.

The trout recovered from anesthesia more 

rapidly than the other species. In fact, most 

fish recovered rapidly, and the extended recovery times for some species reflect the 

very slow recoveries of a few individuals.

Sublethal exposures to MS-222 did not 

seem to harm fish. Rainbow trout which 

survived partial-kill concentrations were 

observed for 7 days. They fed and behaved 

as well as controls.

CONCLUSIONS

MS-222 demonstrated a definite and consistent toxicity to eight species of fish in 24- 

to 96-hour bioassays. The range of 24-hour 

LCso values for the more susceptible lake 

trout to the more tolerant largemouth bass 

was 33.8 to 63.0 p.p.m. The toxicity was not 

much greater in exposures of more than 24 

hours.

Larger specimens of a species are usually 

more resistant to MS-222 than small ones.

The trout were slightly more resistant to 

the anesthetic at colder temperatures. Bluegills were more resistant at 17° than at 12° 

to 22° C.

Toxicity of MS-22 2 to rainbow trout was 

influenced very little by water hardness, but 

the fish reacted inconsistently in very soft 

water.

Among the survivors in partial-kill concentrations of MS-222 in 96-hour exposures,

the trout recovered from anesthesia more 

rapidly than other species.

Safety indexes based on the quotient of 

lethal and effective concentrations can be 

used to distinguish the margin between 

toxicity and efficacy.

SUMMARY

Toxicity of MS-2 22 to rainbow trout, brown 

trout, brook trout, lake trout, northern pike, 

bluegill, largemouth bass, and walleye of 

various sizes was determined in 15-, 30-, 

and 60-minute and 24-, 48- and 96-hour bioassays at selected temperatures. The 24- 

hour LC 50 values for the more sensitive 

lake trout and more tolerant largemouth 

bass ranged from 33.8 to 63.0 p.p.m. and 

mortalities were not much greater in exposures which exceeded 24 hours.

Larger fish of a species are more resistant to the drug than small ones. Also, 

the trout were more tolerant at colder 

temperatures. Bluegills, on the other hand, 

were more tolerant at 17° than at 12° and 

22° C.

The drug was tested against rainbow trout 

in waters with hardnesses of 10, 42, and 176 

p.p.m. The fish reacted inconsistently in the 

softer water.

The recovery from anesthesia was evaluated in fish which survived partial-kill concentrations of MS-222 in 96-hour exposures. 

The trout recovered more rapidly than the 

other species, and posttreatment survival was 

good.

Safety indexes (LC so/EC50) and maximum 

safety indexes (LQ /ECgg) were calculated 

from data obtained in 15-, 30-, and 60- 

minute exposures of rainbow trout to MS-222. 

They can be used to estimate the margin 

of safety between toxicity and efficacy. In 

one instance, the maximum safety index more 

clearly demonstrated the possible hazard of 

exposing small rainbow trout to the drug in 

soft water than did the safety index.
10 Investigations in Fish Control 12: Bureau of Sport Fisheries and Wildlife

REFERENCES

Bove, Frank J.

1962. MS-222 Sandoz the anaesthetic of 

choice for coldblooded organisms. Sandoz 

News, No. 3, 12p.

Eisler, Ronald, and Tadeusz Backiel. 

1960. Narcotization of chinook salmon 

fingerlings with tricaine methanesulfonate 

(M.S. 222). Transactions of the American 

Fisheries Society, vol. 89, no. 2, p. 164- 

167.

Klontz, George W.

1964. Anesthesia of fishes. Proceedings of 

the Symposium on Experimental Animal 

Anesthesiology, Brooks Air Force Base, 

December 14-16. 13 p.

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

1964. Investigations in Fish Control: 

1. Laboratories and methods for screening fish-control chemicals. U.S. Bureau 

of Sport Fisheries and Wildlife, Circular 

185. 15 p.

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

1949. A simplified method of evaluating 

dose-effect experiments. Journal of 

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

McFarland, William N.

1959. A study of the effects of anesthetics 

on the behavior and physiology of fishes. 

Publications of the Institute of Marine 

Science, University of Texas, vol. 6, 

p. 23-55.

Nelson, P. R.

1953. Use of three anesthetics on juvenile 

salmon and trout. Progressive FishCulturist, vol. 15, no. 2, p. 74.

Parkhurst, Z. E., and M. A. Smith.

1957. Various drugs as aids in spawning 

rainbow trout. Progressive Fish-Culturist, vol. 19, no. 1, p. 39.

Rothlin, E.

1932. M.S. 222 (losliches Anaesthesin), ein 

Narkotikim fur Kaltbliiter. Schweizerische 

Medizinische Wochenschrift, vol. 62, no. 

45, p. 1042-1043.

Schoettger, Richard A., and Arnold M. Julin. 

1966. Investigations in Fish Control: 

13. Efficacy of MS-222 as an anesthetic 

on four salmonids. U.S. Bureau of Sport 

Fisheries and Wildlife, Resource Publication 19.

Thompson, R. B.

1959. Tricaine methanesulfonate (M.S. 222) 

in transport of cutthroat trout. Progressive Fish-Culturist, vol. 21, no. 2, p. 96.

Walker, Charles R., and Richard A. 

Schoettger.

1966. Investigations in Fish Control: 

15. Residues of MS-222 in four salmonids following anesthesia. U.S. Bureau 

of Sport Fisheries and Wildlife, Resource 

Publication 21.
INVESTIGATIONS IN FISH CONTROL

13. Efficacy of MS-222 as an Anesthetic 

on Four Salmonids

By Richard A. Schoettger and Arnold M. Julin

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

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

Fish and Wildlife Service, Clarence F. Pautzke, Commissioner 

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

Resource Publication 19 January 1967 Washington, D.C.
CONTENTS

Page

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

Method and materials ......................... 4

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

Rapid anesthesia ......................... 6

Effects of water hardness ................... 7

Effects of repeated anesthetizations ............ 8

Moderately rapid anesthesia. ................. 8

Sedation ............................... 9

Use of MS-222 as a spawning aidr .............. 11

Other observations on MS-222 ................ 12

Conclusions. ............................... 12

Summary . ................................ 13

Literature cited. ............................ 13
EFFICACY OF MS-222 AS AN ANESTHETIC ON FOUR SALMONIDS

By Richard A. Schoettger and Arnold M. Julin, Fishery Biologists

Bureau of Sport Fisheries and Wildlife

La Crosse, Wis.

Abstract. MS-222 was tested for its efficacy as an anesthetic for 

rainbow trout, brown trout, brook trout, and lake trout. Eighty to 135 

p.p.m. of MS-222 anesthetized fish within 3 minutes at 7° to 17° C. The 

fish could be exposed for a total time of 4 to 12 minutes. Fifty to 60 

p.p.m. induced a moderate rate of anesthesia which could be maintained 

for approximately 30 minutes. Sedation was produced within 15 minutes 

and maintaine for 5 to 6 hours at 15 to 30 p.p.m. The efficacy of sedating 

concentrations appeared to decrease with time at 17° C. Lake trout required larger doses than the other salmonids for complete anesthesia 

but tolerated only short exposures. There was no relation between size 

of fish and efficacy of MS-222. Smaller fish occasionally had shorter 

exposure times. The drug was equally efficaceous at pH values of 5.0, 

7.0, and 8.5. Anesthetic solutions with a total hardness of 10 p.p.m. 

were less effective in anesthetizing rainbow trout than those containing 35 and 180 p.p.m. Individuals which were anesthetized in soft water 

recovered sooner.

MS-222 (tricaine methanesulfonate) was 

discovered by Maurice Sandoz during investigations to develop a synthetic substitute for 

cocaine. It was classified as a local anesthetic 

for use in human medicine. During the late 

1920's, the drug also came into use as an 

anesthetic for poikilotherms including fish, 

salamanders and frogs (Bove, 1962).

The potential usefulness of MS-222 was not 

recognized widely in fisheries until Wood 

(1956) and Ball and Cowen (1959) reported on 

the carcinogenic properties of urethane, then 

a commonly used anesthetic for fish. An editorial comment accompanying Wood's report 

indicated that MS-222 might be a suitable substitute for urethane. Since then, the anesthetization of fish with MS-222 has become routine 

to facilitate the handling of both marine and 

freshwater species. The toxicity of the drug to 

fish was determined by Marking (1966). Walker 

and Schoettger (1966) measured its residues 

in various tissues of salmonids.

General guidelines for effective and safe 

use of MS-222 in fisheries are frequently 

misleading or lacking. The concentrations 

employed in fish culture, fisheries management and research were reviewed by Bove 

(1962), the manufacturer 1 f Eisler and Backiel 

(1960), and Schoettger (1966). They differ 

widely depending on factors such as species 

and temperature. Unfortunately, potentially 

lethal concentrations may be required to 

achieve desired rates or depths of anesthesia. 

Thus, the durations of exposure are critical. 

If many fish are anesthetized at one time, 

some may become over exposed and die. Concentrations for selected rates of anesthesia, 

exposure times tolerated by the fish and factors affecting the drug's efficacy therefore

1 Sandoz Pharmaceuticals: (No date) M.S. 222-Sandoz, 

the anesthetic of choice in work with cold-blooded animals, Sandoz Pharmaceutical s, Hanover, N.J.,'Technical 

Bulletin. 10 p.
4 Investigations in Fish Control 13: Bureau of Sport Fisheries and Wildlife

required definition under controlled conditions.

Because of the widespread use of MS-222 

in the culture and management of salmonids, 

we selected four representatives of the group 

for this study. The influences of temperature, 

water hardness, pH, repeated exposure and 

size of fish on the action of the anesthetic 

were evaluated.

METHODS AND MATERIALS

The efficacy of MS-222 was tested against 

rainbow trout, brown trout, brook trout, and 

lake trout of 2 to 6 inches and 7 to 12 inches 

(table 1). The test fish were held in well 

water at 12° C. and starved for two days 

prior to their placement in the anesthetic 

solutions. Twenty-four hours before an experiment, the fish were temperature conditioned in reconstituted water which was prepared according to methods described by 

Lennon and Walker (1964). Fish less than 4 

inches long were tested in 15 liters of reconstituted water. Larger individuals were tested 

in 45 liters. Since preliminary tests failed to 

show that variable loading had any effect on 

efficacy, the loading levels were approximately 

10 g./l. for the smaller fish, and up to 40 g./l. 

for the larger fish.

TABLE 1. Trouts used in tests of KB-222 

Source

TABLE 2. General behaviorial response of fish which characterize 

progressive levels of anesthesia

Sedation..................... Decreased reactivity to visual and

vibrational stimuli; opereular and locomotor activity reduced slightly; darker 

in color.

Partial loss of equilibrium.. Loss of equilibrium in water current; 

increased opercular rate; swimming 

ability disrupted.

Total loss of equilibrium:

Stage 1.................... Usually turn over; swimming ability persists; opercular rate rapid; react to 

vibrational stimuli.

Stage 2.................... Locomotion ceases; fin movement may continue; tactile response only to pressure 

on caudel fin or peduncle; opercular rate 

slowed.

Loss of reflex activity...... Failure to respond to external stimuli,

particularly pressure on caudal fin or 

peduncle; opercular rate slow and erratic.

Medullary collapse........... Opercular activity ceases.

responses were reported by McFarland (1959 

and 1960) and Klontz (1964). Recognition of 

these stages of anesthesia is essential to the 

selection of dosage rates, and to reduce the 

risk of excessive exposures.

The criteria established to determine the 

efficacy of MS-222, though somewhat arbitrary, were based on general considerations 

reported in the literature (table 3). It is difficult to predict concentrations of MS-222 

which satisfy all requirements of an anesthetic; however, the levels associated with these 

criteria provide reference points which are 

adaptable to the specific needs for an anesthetic. The periods that fish tolerated exposure 

to MS-222 were determined by entry of the 

first and the last individual into medullary

Rainbow trout (Salmo gairdneri)..... Manchester National Fish Hatchery,

Manchester, Iowa.

Brown trout (Salmo trutta).......... Manchester National Fish Hatchery,

Manchester, Iowa, and State Fish 

Hatchery at Lanesboro, Ittrm.

Brook trout (Salvelinus fontlnalis). State Fish Hatcheries at Osceola,

Wis., St. Croix Falls, Wis., and 

Lanesboro, Ittnn.

Lake trout (Salvelinus namaycush)... Jordan River National Fish Hatchery,

Charlevoix, tfioh., and State Fish 

Hatchery at St. Croix Falls, Wis.

Initial trials with MS-222 demonstrated the 

need to establish: (1) general behavioral responses of fish to the anesthetic, and (2) criteria to evaluate effective concentrations.

The behavioral responses of fish treated 

with MS-222 are shown in table 2. Similar

Table 3. Criteria for testing anesthetics

Handling: Fin clipping, 

stripping, etc.......

rransportation of 

fish............

Desired effect

A. Rapid immobility 

Rapid recoveryBrief immersion 

time.

B. Itoderately rapid and 

sustained anesthesia.

Reduced activity. Reduced oxygen consumption. Dispersal of 

fish in containers. 

Rapid recovery.

Criteria for effective concentration

A. Concentration producing loss of reflex 

in all fish within 

3 minutes. Total 

immersion time dependent on the onset 

of medullary collapse.

B. Concentration producing total loss of 

equilibrium, stage 

11, in all fish 

within- 15 minutes. 

Fish recover after 

15 to 30 minutes of 

immersion.

Concentration producing 

sedation within minutes 

and maintaining it for 

5 to 6 hours.
Schoettger, Julin: Efficacy of MS-222 as an Anesthetic on Four Salmonids

collapse. The time of response is useful in 

estimating the numbers of fish which can be 

handled safely in a batch. In actual practice 

the more susceptible fish respond first to the 

anesthetic and are removed.

A series of concentrations of MS-222 were 

tested at a temperature of 12° C. and at pH 

7.0. The levels giving reasonable anesthetization, holding and recovery times, and which 

produced minimum mortality were considered 

for further trials at 7°, 12° and 17° C., and at 

pH 5.0, 7.0, and 8.5. Temperature control was 

maintained by placing the test containers in 

water baths. The pH of some solutions was 

varied by the addition of sodium bicarbonate, 

or potassium acid phthalate and sodium hydroxide. The oxygen concentrations of the solutions were maintained close to saturation by 

aeration before, or during the experiments.

The metabolic rates of fish sedated with 

MS-222 were compared with those of unanesthetized fish. The studies were conducted in 

open and closed systems, and were designed 

to estimate the usefulness of the drug for suppressing the metabolism of fish in tanks, and 

in plastic bags.

Lake trout averaging 4 inches in length and 

6.3 grams in weight were used in the opensystem tests. Five individuals were placed in 

each of sixteen 1-gallon jars, each containing 

2.5 liters of reconstituted water and the volume 

adjusted slightly to achieve a constant loading 

of 15 g./l. Eight test solutions at 12° C. contained sufficient MS-222 to produce sedation 

(20 p.p.m.), and were saturated with oxyben. At 

intervals, one container of fish from each experimental and control group was selected randomly and the oxygen was measured by the 

modified Winkler method. The tests continued 

until the fish showed signs of distress because 

of low oxygen.

In the closed-system trials, one or two 6- 

inch rainbow trout were weighed and sealed in 

1-gallon jars that were completely filled with 

reconstituted water at 12° C. The solutions 

were saturated with oxygen at the beginning of 

the tests. The fish were sedated with 20 to 30 

p.p.m. of MS-222 and the quantities of oxygen 

consumed by them were compared to controls

after 30 to 60 minutes. The values were corrected for the loading level in each jar.

The influence of water hardness on the 

efficacy of MS-222 was measured with 4- 

inch and 9-inch rainbow trout. The hardnesses of the various solutions were altered 

by adding different amounts of the mixed reconstituting salts. Preliminary tests were 

conducted in solutions of MS-222 which had 

hardnesses ranging from 17 to 350 p.p.m. as 

calcium carbonate. In subsequent trials a concentration of 100 p.p.m. of the drug was tested 

for efficacy in solutions with hardnesses of 10 

and 180 p.p.m. These contained 5 and 75 p.p.m. 

of calcium ion, 1.5 and 7.0 p.p.m. of chloride 

ion, 9.5 and 116 p.p.m. total alkalinity, and had 

pH's of 7.14 and 8.20 respectively. They will 

later be referred to as relatively soft and 

hard waters. The trout were acclimated in 

both types of water for 24 to 48 hours preceeding the experiments and were placed in 

well water for recovery.

The effect of repeated exposure of fish to 

MS-222 was tested by anesthetizing 12-inch 

rainbow trout and 6-inch lake trout daily in a 

concentration of 100 p.p.m. The time preceeding the onset of medullary collapse, recovery time and survial were used as indices 

of sensitivity. The trials with rainbow trout 

continued for 21 days and were conducted in 

reconstituted water at 12° C. The fish were 

placed in well water for recovery. In similar 

experiments lake trout received 12 treatments. Their sensitivity to the anesthetic was 

compared periodically with untreated individuals. The fish were anesthetized and recovered in well water. Both species were 

tested in groups of 10 and fed their normal 

ration throughout the trials.

RESULTS AND DISCUSSION

The efficacy of MS-222 for inducing rapid 

and moderately rapid anesthesia and sedation 

in two size groups of salmonids was tested at 

7° to 17° C. and pH 5.0 to 8.5. The efficacy of 

the drug appeared unaltered by pH and these 

data were grouped, without differentiation, 

with those for size of fish and temperature.
6 Investigations in Fish Control 13: Bureau of Sport Fisheries and Wildlife

Rapid anesthesia

Rainbow trout were anesthetized to loss of 

reflex within 3 minutes in solutions containing 

100 p.p.m. of MS-222 (table 4). Fish in both 

size ranges were equally sensitive to the drug 

and in contrast to other salmonids, there appeared to be less effect of temperature on 

efficacy. The fish withstood exposure for approximately 5.5 to 12 minutes. The durations 

were somewhat shorter at higher temperatures and with smaller fish.

A concentration of 80 p.p.m. was 100-percent effective on brown trout at 17°, however, 

the concentration was raised to 100 p.p.m. at 

7° and 12° C. to achieve efficacies of 86.6 to 

.100 percent (table 4). Though a weaker solution was used at 17°, the fish reached medullary collapse and recovered sooner than at 

7° and 12°. This suggests a greater metabolism of MS-222 at higher temperatures.

Brook trout and lake trout were effectively 

anesthetized by 100 p.p.m. of MS-222 at 17°

TABLE 4. Concentrations of MS-222 producing rapid anesthesia in four salmonids at three temperatures.

Species and concentration

Rainbow trout:

Brown trout:

Brook trout:

Lake trout:

Temperature 

(C.)

70 

70 

12° 

12° 

17°

12° 

17°

^ 7° 

120

17° 

70

7° 

12° 

12°

120 

17° 

12° 

7°

7° 

12° 

17° 

12° 

7°

Size of 

fish 

inches

2-6

7-12 

2-6 

7-12 

7-12

2-6 

2-6 

7-12 

2-6 

2-6 

7-12 

2-6 

7-12 

2-6 

7-12

2-6

7-12 

7-12 

7-12

2-6 

2-6

7-12 

7-12 

7-12

Fish in loss of 

' Equilibrium, stage 

11, with 2 minutes

Number

60/60 

20/20 

173/173 

95/95 

20/20

20/20 

5/5 

20/20 

13/15 

40/40 

8/8 

30/30 

34/35 

60/60 

55/55

70/70 

20/20 

30/30 

24/25

38/120 

lOJ/120 

25/25 

30/30 

18/25

Percent

100.0 

100.0 

100.0 

100.0 

100.0

100.0 

100.0 

100.0 

86.6 

100.0 

100.0 

100.0 

97.1 

100.0 

100.0

100.0 

100.0 

100.0 

96.0

31.6 

84.1 

100.0 

100.0 

72.0

Reflex within 

3 minutes

Number

60/60 

20/20 

170/173 

95/95 

20/20

13/20 

5/5 

20/20 

9/15 

39/40 

8/8 

26/30 

32/35 

60/60 

53/55

43/60 

20/20 

30/30 

24/25

10/120 

73/120 

25/25 

29/30 

20/25

Percent

100.0 

100.0 

98.3 

100.0 

100.0

65.0 

100.0 

100.0 

60.0 

97.5 

100.0 

86.6 

91.4 

100.0 

96.3

71.6 

100.0 

100.0 

96.0

8.3 

59.2 

100.0 

96.7 

80.0

Ifean range of 

exposure times 

(minutes)

First 

fish

6.6 

6.8 

5.5 

8.0 

5.5

6.9 

5.3 

4.6 

9.4 

5.9 

4.5 

7.9 

9.5 

6.1 

7.9

5.0 

4.2 

6.5 

8.8

6.9 

4.7 

3.9 

5.0 

5.6

Last 

fish

8.1 

11.4 

7.0 

11.9 

6.9

13.3 

7.3 

7.0 

10.4 

8.3 

6.1 

9.4 

12.1 

8.1 

11.0

5.4 

8.7

11.4

8.5 

5.4 

4.7 

6.3 

6.5

exposure 

index 1

2.2

2.3 

1.8 

2.7 

1.8

2.3 

1.8 

1.5 

3.1 

2.0 

1.5 

2.6 

3.2 

2.0 

2.6

1.7 

1.4 

2.2 

2.9

2.3 

1.6 

1.3 

1.7 

1.9

Recovery

Ifean 

time range 

minutes

4.0-11.0 

5.1-11.8 

2.8- 8.2 

4.2- 8.6 

4.3- 9.0

3.3- 6.6 

6.0- 6.3 

3.9- 8.2 

5.8-10.1 

3.8- 7.6 

4.8-17.0 

6.4-18.5 

7.3-11.0 

4.1- 9.6 

4.9-13.3

2.0- 6.4 

5.1- 8.9 

4.4- 7.6 

4.1- 7.7

5.5-19.2 

4.1-15.8 

3.4- 5.3 

3.5- 9.1 

4.4- 7.1

Survival 

(percent)

98.4 

100.0 

93.1 

100.0 

93.3

100.0 

40.0 

86.6 

100.0 

100.0 

75.0 

100.0 

96.6 

100.0 

98.1

100.0 

93.3 

100.0 

100.0

85.8 

91.7 

100.0 

100.0 

100.0

1 Index obtained by dividing the time for the first fish to reach medullary collapse by the time (3 minutes) for fish to reach lose of reflex.

and by 110 p.p.m. at 12° (table 4). The dosage 

was raised to 120 p.p.m. for brook trout, and 

135 p.p.m. for lake trout at 7°. The concentrations for lake trout are similar to those 

used at a national fish hatchery 2 in Michigan.

In general, the chars were more resistant 

to MS-222 at 7° and 12° than rainbow trout or 

brown trout. This may indicate a lower, and 

possibly narrower, range of optimum ternPersonal communication from George Drake, 

Hatchery Manager, Pendills Creek National Fish Hatchery, Brimley, Mich.

peratures for the former species. The expoposures for brook trout ranged from 4.2 

minutes at 17° to 11.4 minutes at 7°. The 

values for lake trout, at the same temperatures, were 3.9 and 6.5, and they reflect a 

greater sensitivity than the other salmonids 

to prolonged anesthetization. Also, the lake 

trout died quickly when they were not removed from the ariesthetic soon after the 

cessation of respiration. This was not as 

critical with the other species and suggests 

that the respiratory and cardiac centers of 

lake trout are inhibited at a similar rate by 

the drug.
Schoettger, Julin: Efficacy of MS-222 as an Anesthetic on Four Salmonids

The progress of anesthesia in the various 

salmonids slowed, and exposure times lengthened with decreasing temperature. Although 

lower dosages were used at 17° C., with the 

exception of rainbow trout, it was necessary 

to remove the fish from the anesthetic solution sooner than at 7°. The reduced efficacy 

at relatively low temperatures is in accord 

with the results of Meister and Ritzi (1958). 

We concluded that rapid anesthesia is more 

safely induced and maintained at lower temperatures.

The drug appeared to be similarily effective 

on both size ranges of rainbow trout and brown 

trout; however, larger fish withstood longer 

anesthesia. Brook trout and lake trout of 

smaller sizes were not tested at the concentrations used for larger specimens. The trouts 

usually recovered from deep anesthesia within 3 to 15 minutes. There were no consistent 

relations between recovery time and temperature, size of fish, or total exposure.

MS-222 is capable of inducing rapid anesthesia which makes it useful in spawning, 

marking, and measuring fish, and in immobilizing specimens for various physiological 

investigations (Nelson, 1953; Butler, 

1.957 Parkhurst and Smith, 1957; Schiffman 

and Fromm, 1959; Crawford and Hulsey, 1963; 

Black and Connor, 1964). Its relatively high 

narcotic potency to fish is related to its 

molecular weight (McFarland, 1959).

The rate of anesthetization with MS-222 can 

be controlled by the dosage. According to the 

literature, rapid results have been achieved 

with concentrations ranging from approximately 80 to 1,000 p.p.m. The action of the 

drug is reversible at these concentrations 

provided exposure time is manipulated to 

minimize mortality. We observed that anesthetized trout always recovered when they 

were transferred to fresh water before cessation of respiratory activity. Rodman (1963) 

found that anesthesia of white sturgeon was 

not reversible when the fish were exposed to 

a concentration of 1:40,000 for 30 to 48 hours. 

Marking (1966) maintained rainbow trout 

under anesthesia with MS-222 for 96 hours. 

They recovered quickly in fresh water and 

appeared to be healthy seven days later.

Our results, and those of other investigators, demonstrate that relatively high concentrations of MS-222 are effective and 

safe for the rapid anesthetization of trout. 

The dependence of survival on exposure time 

suggests than an index for the safe exposure 

of fish to MS-222 can be obtained from a 

quotient of the time for medullary collapse 

and that for anesthesia. Indexes for the variour species and sizes of fish were calculated 

for temperatures of 7°, 12°, and 17° C. (table 

4). They are biased in favor of greater safety. 

High indexes may be calculated from times 

for shallower anesthesia and longer exposure. 

Usually, the indexes were greater than 1.5. 

The values of 12° and 17° were relatively 

lower than those at 7° and indicated a greater 

hazard in anesthetizing fish with MS-222 at 

higher temperatures. Brook trout and lake 

trout, for example, had respective indices of 

1.4 and 1.3 at 17°, whereas the values were 

2.9 and 1.9 at 7° C.

Effects of water hardness. In preliminary 

studies, we found that the water harnesses 

from 17 to 350 p.p.m. had little influence on 

the anesthetic properties of MS-222 against 

rainbow trout.

Phillips et al. (1955) established the importance of the calcium ion in the metabolism 

and osmoregulation of brook trout. Calcium 

concentrations of 5 p.p.m. increased metabolism, presumably in active opposition to the 

osmotic inflow of water. We anticipated that 

solutions with a hardness of 10 p.p.m. and 

a calcium ion level of 5 p.p.m. might introduce an osmotic stress which would alter the 

anesthetic action of MS-222 against rainbow 

trout. The results of trials in solutions with 

hardnesses of 10 p.p.m. and 180 p.p.m. are 

shown in table 5. In soft water, the times to

TABLE 5. Effect of water hardness on the efficacy of 100 p.p.m. of 

MS-222 against 9-inch rainbow trout at 12° C.

Mean time in minutes to 

Six tests (80 fish)......

Differences between means

Loss of reflex 

in water with 

hardness of 

10 

p.p.m.

7.98 

1.98

180 

p.p.m.

2.43 

0.29

5.55 

17.56*

Medullary 

collapse in 

water with 

hardness of 

10 

p.p.m.

16.80 

3.61

180 

p.p.m.

8.10 

1.94

8.70 

13.41*

Recovery in 

water with 

hardness of 

10 

p.p.m.

4.68 

1.57

180 

p.p.m.

6.67 

2.22

1.99 

4.98*

"Significant at the 0.01 level.
8 Investigations in Fish Control 13: Bureau of Sport Fisheries and Wildlife

reach complete anesthesia and durations of 

exposure were double those for hard water, 

and recovery times were shorter. The mean 

differences between soft and hard water were 

statistically significant at the 0.01 level. 

Onkst et al. (1957) observed that induction of 

anesthesia in guppies with pentobarbital was 

inhibited when the fish were acclimated and 

tested in a calcium dificient medium. Marking 

(1966) observed that higher concentrations of 

MS-222 were required to anesthetize rainbow 

trout in soft water.

The results in hard water agree closely 

with those in table 4 which were determined 

in waters with a hardness of 35 p.p.m. The 

data in tables 4 and 5 indicate that there is a 

threshold of ions below which the efficacy of 

MS-222 is partially inhibited. Ions other than 

calcium may also contribute to this effect.

The reduced effectiveness of MS-222 in 

soft water is not due to lower uptake of the 

drug. Greater amounts of MS-222 were found 

in the muscle tissues of fish anesthetized in 

soft water than in those treated in hard water 

(Walker and Schoettger, 1966). The longer 

exposures of trout in soft water, and increases in their metabolism to maintain 

osmotic equilibrium, may account for the 

higher levels of MS-222 in these fish.

We observed that the rates of anesthetization of fish to total loss of equilibrium, stage 

1, were similar in both soft and hard water. 

The later stages of anesthesia, however, occurred slowly in soft water. Using McFarland's (1959) suggested classification of 

anesthesia in fishes, there appeared to be no 

change in the depressive action of MS-222 on 

the telencephalon, sensory areas of the tectum 

and tegmentum, and midbrain and thalamus of 

fish treated in soft water. Depressions of the 

motor nuciei in the medulla, the spinal reflex 

arcs, and of respiratory and cardiac centers 

are responsible for deeper anesthesia. It is 

possible that in these areas the activity of the 

anesthetic was antagonized by osmotic disturbances of the potassium-calcium balance 

in the nerve cells. Also, considering the results of Phillips et al. (1955) and Walker and 

Schoettger (1966), the rate of absorption and 

deactivation of MS-222 may have increased

along with higher metabolic rates of fish in 

soft water.

Phillips et al. (1957) showed that the narcotization of brook trout with MS-222 increased their absorption of radioactive cobalt. They suggested that there may be an 

adjustment of the osmotic processes of narcotized fish. According to Quastel (1963) 

anesthetics have a twofold mechanism of 

action on the respiration and metabolism of 

brain tissue: (1) effects on the cationic equilibrium of the cell membrane, and (2) the 

anesthetic may affect the oxidation of diphosphopyridine nucleotide within the cell. The 

first mechanism may occur without the 

second. Changes in ionic balance such as an 

increase in cellular potassium or a decrease 

in calcium stimulate respiration of the 

nerve cell. An anesthetic may deactivate carriers at the cell membrane which are responsible for cation movements, and have an 

effect similar to that of a respiratory inhibitor 

acting on the mitochondria. It is not known 

whether the action of MS-222 on the central 

nervous system of fish is in accord with this 

scheme for other anesthetics.

Effects of repeated anesthetization. The 

daily anesthetization of 10 rainbow trout over 

21 days, and 10 lake trout over 12 days with 

100 p.p.m. of MS-222 produced no progressive 

increase, or decrease in their tolerance to the 

drug. The tolerances of lake trout were compared periodically with those of previously 

untreated individuals. The repeatedly exposed 

fish withstood anesthesia for approximately 

1.0 to 1.5 minutes longer than the control 

group. The higher tolerance apparently originated after the first treatment. Recovery 

time and survival were not affected by repeated exposure. Klontz (1964) reported a 

slight increase in tolerance of fish anesthetized repeatedly with MS-222 which was 

compensated by increasing the concentration.

Moderately rapid anesthesia

A moderate rate of anesthesia is useful in 

situations where fish must be handled over a 

longer time, but where rapid immobilization 

is not essential. For example, MS-222 is used 

to anesthetize fish during surgery and hematological studies (Robertson, 1958; Smith and 

Bell, 1964).
Schoettger, Julin: Efficacy of MS-222 as an Anesthetic on Four Salmonids 9

We determined that concentrations of 50 to 

60 p.p.m. anesthetized the four salmonids to 

total loss of equilibrium, stage 2, within 15 

minutes (table 6). The fish usually could remain in the anesthetic for 30 minutes.

A concentration of 60 p.p.m. was effective 

in anesthetizing rainbow trout within 15 minutes, but attempts to maintain them under 

anesthesia for 30 minutes resulted in mortalities of approximately 8 to 25 percent. More 

individuals died at 12° than at 7° C. The anesthetic was almost as effective at 50 to 55 

p.p.m., and mortality was less.

MS-222 was effective on brown trout at 50 

p.p.m., and only 1 fish of 170 failed to recover 

(table 6).

The chars were relatively less sensitive to 

MS-222 than the other fish and a concentration 

of 60 p.p.m. was required to induce anesthesia 

within 15 minutes. The data indicate that there 

was a slight reduction in efficacy of the drug 

at 12° C., which suggests some deterioration, 

or increased metabolism, of MS-222 in higher 

temperatures. Although lake trout appeared 

to be resistant, some did not tolerate exposures to 60 p.p.m. longer than 15 minutes. 

A concentration of 50 p.p.m. lengthened the 

immersion time, but reduced the initial speed 

of anesthetization.

All of the brook trout recovered from anesthesia, but mortalities of lake trout ranged

from 15 to 35 percent and were greater at 

12° than at 7° C. The mortalities were probably related to our inability to pinpoint the 

onset of medullary collapse. The duration of 

the loss of reflex in lake trout is very brief.

The recovery of fish in these tests usually 

occurred within 1 to 15 minutes.

Sedation

MS-222 can be used to produce sedation in 

fish. Solutions containing 20 p.p.m. of the drug 

were, with several exceptions. 80- to 100-percent effective in sedating rainbow trout (table 

7). Brown trout were sedated by 15 p.p.m. 

Thirty p.p.m. were required for larger rainbow trout at 17° and for brook trout at 7° C. 

The trout usually recovered within 1 minute 

after they were transferred to fresh water. 

Concentrations similar to these have been 

used to sedate white sturgeon, sockeye 

salmon, cutthroat trout, rainbow trout, and 

bluegill (Rodman, 1963; Meehan and Revet, 

1962; Thompson, 1959: Dollar, 1963; Gebhard, 

1965; Webb, 1958). Lamarque (1964) recommended dosages of 20 to 100 p.p.m., depending on temperature, for the preliminary anesthesia of fish before they are placed in plastic 

bags for shipment. He suggested 10 p.p.m. for 

tranquilization of fish in hauling tanks.

McFarland (1959) stated that pH may influence the absorption of anesthetics into the 

gills of fish. Our results, however, showed no

TABLE 6. Concentrations of MS-22? producing moderately rapid anesthesia in fish to total loss of equilibrium, stage 2, within 15 minutes

Species and concentration

Rainbow trout:

Brown trout:

Brook trout:

Lake trout:

Temperature 

(C.)

J?

12°

1 

J°

* 

?12°

Size of fish 

( inches )

2-6 

2-6 

2-6 

2-6 

2-6

2-6 

7-12 

2-6

2-6 

2-6

2-6 

2-6 

2-6 

2-6 

2-6

Fish in anesthesia

Number

43/45 

182/210 

20/20 

134/135 

85/85

30/30 

10/10 

130/130

40/40 

50/60

0/10 

72/120 

47/140 

133/140 

84/120

Percent

95.5 

86.7 

100.0 

99.2 

100.0

100.0 

100.0 

100.0

100.0 

83.3

0.0 

60.0 

33.5 

95.0 

74.0

Exposure time 

(minutes)

15-> 30 

>30 

30 

15->30 

15- 30

30 

30 

30

20- 30 

30

30 

30 

15- 30 

15- 30 

15- 30

Recovery

Mean time range 

(minutes)

1.40- 4.00 

1.59-17.54 

2.75-11.25 

3.40- 9.20 

2.20-16.60

4.40- 6.-80 

6.20-52.00 

2.30- 4.60

3.20- 8.00 

3.20- 5.70

1.00- 1.50 

3.41-13.57 

2.23-15.23 

3.70-13.10 

1.60-21.80

Survival 

(percent)

100.0 

98.9 

100.0 

91.8 

75.2

100.0 

90.0 

100.0

100.0 

100.0

100.0 

83.3 

65.8 

85.0 

65.0
10 Investigations in Fish Control 13: Bureau of Sport Fisheries and Wildlife

TABLE 7. Concentrations of MS-222 producing sedation in four salmonlds

Species and concentration

Rainbow trout:

Brown trout:

Brook trout:

Lake trout:

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

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

Temperature

(C.)

7° 

7° 

12° 

12° 

17° 

12° 

17°

7° 

7° 

12° 

17° 

7° 

12° 

12°

7° 

12° 

17°

S S17°

12° $

12° 

17°

Size range 

(Inches )

2-6 

7-12 

2-6 

7-12 

7-12 

7-12 

7-12

2-6 

7-12 

2-6 

7-12 

2-6 

2-6 

7-12

2-6 

7-12 

7-12 

2-6 

7-12 

2-6 

7-12 

7-12

2-6

7-12 

2-6 

7-12 

7-12

Fish in sedation at 

15 minutes

Number

132/135 

80/120 

175/177 

81/90 

32/40 

20/20 

14/20

20/35 

45/50 

28/30 

20/20 

9/36 

21/72 

17/20

10/10 

20/20 

20/20 

57/60 

20/20 

29/30 

9/20 

12/20

179/180 

17/20 

280/280 

20/20 

18/20

Percent

97.8 

66.6 

98.8 

90.0 

80.0 

100.0 

70.0

57.1 

90.0 

93.3 

100.0 

25.0 

29.2 

85.0

100.0 

100.0 

100.0 

95.0 

100.0 

96.7 

45.0 

60.0

99.5 

85.0 

100.0 

100.0 

90.0

5-6 hours

Number

129/135 

78/120 

177/177 

84/90 

22/40 

16/20 

16/20

33/35 

49/50 

30/30 

20/20 

23/36 

70/72 

20/20

5/10 

20/20 

20/20 

50/60 

19/20 

24/30 

20/20 

15/20

146/150 

17/20 

185/220 

20/20 

16/20

Percent

95.6 

65.0 

100.0 

93.3 

55.0 

80.0 

80.0

94.3 

98.0 

100.0 

100.0 

63.9 

97.2 

100.0

50.0 

100.0 

100.0 

83.3 

95.0 

80.0 

100.0 

75.0

97.3 

85.0 

84.0 

100.0 

80.0

Behavior1 of fish 

not in sedation at 

15 minutes

> 

> 

> 

> 

<

>

> 

> 

>

> 

> 

>

>

> 

> 

>

> 

<

<

5-6 hours

>

>

> 

< 

> 

>

> 

>

> 

>

<

> 

> 

>

< >

> 

< 

<

<

>= deeper anesthesia; < = similar to controls.

important effects of pH 5.0 to 8.5 on the sedating properties of MS-222.

Sedation is not a static stage of anesthesia. 

The stage shifts toward recovery, or deeper 

anesthesia, according to the concentration of 

anesthetic, temperature, and, in some instances, the size of the fish. The individuals 

in nearly 70 percent of the trials conducted 

at 17° C. showed signs of recovery after 6 

hours. The trend was never toward deeper 

anesthesia in tests at this temperature. The 

shift at 7° and 12° was toward recovery in 40 

percent of the tests, and toward deeper anesthesia in 20 percent. Sedation persisted in the 

remaining 40 percent. The reduced effectiveness of the drug at relatively higher temperatures is probably related to its deterioration, 

increased metabolism by the fish, or both. 

More conclusive evidence regarding the temperature dependence of MS-222 was obtained 

by McFarland (1959, 1960). His observations 

on the behavior and metabolism of anesthetized California killifish demonstrate that 

the drug was an effective sedative at 18° to 

21° C., but not at 24° to 27° c. Sedation at 

the lower temperature range was not maintained beyond 6 to 8 hours.

One of the principal uses of anesthetics in 

the transportation of fish is to control metabolism. Baudin (1932a, 1932b), McFarland 

(1959), and Blahm (1961) reported that MS-222 

reduces the rate of oxygen consumption in 

fish. We observed that the drug reduced the 

oxygen consumption of rainbow trout in a 

closed system by approximately 30 percent 

(table 8). Statistical analyses indicated that 

the reduction was significant at the 0.05 but 

not at the 0.01 level. In contrast, the anesthetic failed to reduce significantly the oxygen 

consumption rate of lake trout in open-system 

tests (table 9). This may have been the result 

of the differential diffusion of oxygen into 

those solutions with greater oxygen gradients. 

Thus, there is little advantage in using MS222 in open systems to reduce oxygen consumption. On the other hand, the fish in these 

experiments were not purposely stimulated to 

higher levels of activity. Fry (1957) demonstrated that the oxygen uptake of stimulated 

fish may be four to five times the basal 

metabolic rate. The drug may be efficacious 

in open systems by lowering metabolic rates 

of this magnitude. McFarland (I960) suggested 

the pre-sedation of fish for several hours to 

lower their metabolic rates before transport.
Schoettger, Julin: Efficacy of MS-222 as an Anesthetic on Four Salmonids 11

TABLE 8. Oxygen consumption of 6-inch rainbow trout sedated with MS-222 

in closed systems at 12° C.

Test.

Difference between mean rates....

Bates of oxygen consumption 

(mg.02/kg. tody wt./hr.)

Sedated fish

164.4 

103.7 

142.6 

138.7 

83.2 

272.0 

137.9 

178.0 

62.2

142.5 

57.7

Control fish

162.0 

230.4 

242.'9 

212.9 

182.6 

338.0 

118.1 

216.0 

188.5

210.2 

57.6

67.7 

2.63*

^Significant at the 0.05, tut not at the 0.01 level.

The reports on the benefits of sedating fish 

with MS-222 during transport are contradictory (Webb, 1958; Martin and Scott, 1959; 

Thompson, i959; Meehan and Revet, 1962; 

Dollar, 1963; Rodman, 1963; Gebhards, 1965). 

The drug has been used more successfully at 

low, than at high temperatures, and it may be 

instrumental in reducing injuries to fish because of hyperactivity (Gossington, 1957; 

McFarland, 1960; Collins and Hulsey, 1963).

Use of MS-222 as a spawning aid

Anesthetization of gravid rainbow trout and 

brook trout with MS-222 was observed at 

three hatcheries. The rainbow trout were 

treated at national fish hatcheries at Genoa, 

Wis., and Manchester, Iowa, and the brook 

trout were exposed at the state fish hatchery 

at Osceola, Wis. The concentrations of drug 

were those normally used at the hatcheries

during the collection of eggs and sperm. The 

observations are listed in table 10.

The amounts of MS-222 used to anesthetize 

the sexually mature, adult rainbow trout were 

similar to those producing rapid and moderately rapid anesthesia in the efficacy tests 

(table 4 and 6). A concentration of 58 p.p.m. 

produced loss of reflex in about 6 minutes, 

and it was as effective on fish weighing 8 

pounds as on smaller individuals. The fish 

remained in this concentration for 6 to 28 

minutes, and all recovered in 3 to 5 minutes. 

The desirable feature of this dosage was 

that a greater number of fish could be 

anesthetized over a longer period than 

would have been possible using higher 

dosages. Twenty fish were exposed in one 

trial and some remained in the anesthetic 

for nearly 30 minutes. They were not 

completely anesthetized and reacted 

slightly when handled. The more susceptible 

individuals were spawned within 5 minutes 

after contact with the anesthetic.

The fish exposed to 100 p.p.m. reached loss 

of reflex in 1.25 and 3.25 minutes. The duration of exposure was 2 to 7 minutes, and all 

recovered in 1 to 3 minutes.

The 166 p.p.m. of MS-222 used for male 

brook trout were higher than those employed 

in our-efficacy tests (table 4). They were exposed for 4.25 to 7.5 minutes and all recovered in 7 to 9 minutes. The hatchery personnel reported that as many as 150 fish are 

anesthetized in one solution of MS-222 before

TABLE 9. The oxygen consumption of 4-inch lake trout sedated with MS-222 in open systems at 12° C.

Elapsed time

Difference between mean rates......

Sedated fish

Concentration of 

oxygen 

(p.p.m.)

9.8 

8.3

7.0

6.5 

5.6 

5.2

5.0 

4.2 

3.6

Consumption of oxygen

p.p.m./hr.

1.2 

1.9

1.7 

1,7 

1.5

1.4 

1.4 

1.4

1.5

mg.02/kg. 

body wt./hr.

80.0 

126.7

113.3 

113.3 

100.0

93.3 

93.3 

93.3

101.7 

15.0

Control fish

Concentration of

 oxygen 

(p.p.m.)

9.4 

6.3 

6.4

6.2 

4.7 

4.6

4.4 

4.0 

3.0

Consumption of oxygen

p,p.m./hr.

2.1

2.0

1.6 

1.9 

1.6

1.4 

1.4 

1.4

1.7

ng.02/kg. 

body wt./nr.

140.0 

133.3

106.7 

126.7 

106.7

93.3 

93.3 

93.3

111.6 

19.1

9.9

1.1
12 Investigations in Fish Control 13: Bureau of Sport Fisheries and Wildlife

TABLE 10. Observations on use of MS-222 in anesthetizing rainbow trout and brook trout during spawning operations

Species and hatchery

Rainbow trout:

Genoa, Wis.

Brook trout:

Concentration 

(p. p.m.)

58 

58 

58 

58 

58 

100 

100

110 

110 

166 

166

Temperature 

(F.)

54° 

54° 

54° 

54° 

54° 

51.5° 

51.5°

48° 

48° 

48° 

48°

Mean 

weight 

per fish 

(pounds)

7.0 

7.0 

7.0 

1.0 

1.0 

0.9 

1.0

1.6 

1.6 

1.4 

1.4

Number of 

fish per 

test

10 

6 

11 

20 

15 

2 

2

2 

7 

2

7

Number 

of

tests

1 

1 

1 

1 

1 

3 

6

2 

2 

2 

2

Sex

F 

F 

F 

M 

M 

F 

M

F 

K 

M 

M

Time (minutes) to reach 

loss of 

Equilibrium, 

stage 2

4.00 

5.00 

5.50 

3.25 

5.50 

1.00-1.50 

1.00-2.00

0.75 

1.00-1.50 

1.00 

1.00

Reflex

N.R. 1 

N.R. 1 

N.R. 1 

6.00 

N.R. 1 

1.50-3.25 

1.25-3.00

1.25-1.50 

2.25-3.25 

1.25-2.00 

2.00-2.25

Range of 

exposures 

(minutes )

6.00- 8.00 

6.00- 8.00 

6.00- 8.00 

7.00-28.00 

N.R. 1 

2.00- 5.00 

3.50- 7.00

2.00- 2.50 

3.75- 5.75 

4.25- 5.00 

4.00- 7.50

Recovery in 

fresh water

Percent

100 

100 

100 

100 

100 

100 

100

100 

100 

100 

100

Time 

(minutes)

3.00 

4.00 

3.00 

3.00-5.00 

N.R. 1 

2.50-4.50 

1.00-3.00

2.00 

8.00-9.75 

7.00-8.00 

8.00-9.00

1 Not recorded.

its effectiveness is greatly reduced. Meister 

and Ritzi (1958) reported that from 22 to 35 

pounds of brook trout, 36 to 86 pounds of 

Atlantic salmon, or 93 pounds of Jake trout 

could be anesthetized per gram of MS-222.

The use of MS-222 during spawning speeds 

the operation and reduces the mortality of 

fish due to forceful handling. The anesthetic 

apparently has no adverse effects on the survival of eggs or parents (Parkhurst and Smith, 

1957; Crawford and Hulsey, 1963). The exposed fish, however, are usually dipped into 

clean water before stripping to prevent the 

anesthetic from contacting reproductive 

products (Allison, 1961).

Other observations on MS-222

Minor deviations in the efficacy of MS-222 

were observed from time to time. We believe 

that they were partially related to variations 

in the susceptibilities of individuals and 

groups of fish, or combinations of unknown 

factors. In any case, it is advisable to conduct preliminary bioassays of anesthetic solutions with several individuals from the stocks 

of fish which are to be narcotized. The precaution was recommended also by other investigators including Klontz (1964), Lamarque (1964) and Bell (1964). Observations on 

the depths of anesthesia and exposure times 

can be used to determine the numbers of fish 

which can be anesthetized safely.

CONCLUSIONS

Anesthetization of rainbow trout, brown 

trout, brook trout, and lake trout with MS-222

produced behavioral responses which were 

similar to those reported for other anesthetics and species of fish.

Concentrations of MS-222 which produced 

rapid anesthesia in the four salmonids ranged 

from 80 to 135 p.p.m. Levels of 50 to 60 p.p.m. 

induced moderately rapid anesthesia, and 15 to 

30 p.p.m. were effective for sedation.

The action of MS-222 was reversible when 

the fish were removed from anesthetic solutions 

prior to the cessation of respiratory activity 

(medullary collapse). The retention of fish in 

solutions which are effective for rapid and 

moderately rapid anesthesia may result in 

mortality depending on the duration of exposure.

Brook trout and lake trout were more resistant to MS-222 at 7° and 12° C. than the 

other salmonids. Lake trout could withstand 

only short exposures and they died quickly 

after entering medullary collapse.

Effective concentrations and durations of 

exposure, particularly for rapid anesthesia, 

were inversely related to temperature. The 

rapid anesthetization of fish is more safely 

induced at lower temperatures.

There was little relation between size of 

fish and the efficacy of MS-222; however, the 

smaller fish occasionally had shorter exposure 

times.

The anesthetic solutions which contained 10 

p.p.m. total hardness were less effective in 

narcotizing rainbow trout than those with hardnesses of 35 and 180 p.p.m. The fish anesthetized in soft water recovered sooner.
Schoettger, Julin: Efficacy of MS-222 as an Anesthetic on Four Salmonids 13

The trout which were repeatedly anesthetized 

with MS-222 were slightly more tolerant than 

previously unexposed individuals. The tolerance, however, did not increase with repeated 

treatments.

Sedation was not a static stage of anesthesia, 

but shifted toward recovery, or deeper anesthesia depending on the concentration of anesthetic and temperature.

MS-222 did not reduce the rate of oxygen consumption of lake trout in open systems. The 

consumption rate of rainbow trout in closed 

systems was reduced by approximately 30 

percent. The efficacy of MS-222 was not altered to a measurable degree by pH 5.0 to 

8.5.

Our results and those of other investigators 

demonstrate that MS-222 is effective and safe 

for inducing various stages of anesthesia in 

the four salmonids.

SUMMARY

Investigations were made to determine the 

concentrations of MS-222 which produce rapid, 

moderately rapid, and sedating anesthesia in 

rainbow trout, brown trout, brook trout, and 

lake trout. Eighty to 135 p.p.m. of MS-222 

rapidly anesthetized the fish within 3 minutes 

at temperatures of 7° to 17° C. The fish 

could be exposed to the anesthetic for approximately 4 to 12 minutes. The effective concentrations and exposure times varied with 

species and temperature. Fifty to 60 p.p.m. 

induced a moderate rate of anesthesia which 

could be maintained for approximately 30 

minutes. Sedation was produced within 15 

minutes and maintained for 5 to 6 hours at 

15 to 30 p.p.m. The efficacy of sedating concentrations appeared to decrease with time 

at 17° C. Lake trout required higher dosages 

of MS-222 than other salmonids for complete anesthesia, but they tolerated only short 

exposures.

There was no relation between size of fish 

and efficacy of MS-222. Smaller fish, however, 

occasionally had shorter exposure times. The 

efficacy of the drug was not influenced by pH 

5.0 to 8.5.

Anesthetic solutions with a total hardness 

of 10 p.p.m. were less effective in anesthetizing rainbow trout than those containing 35 to 

180 p.p.m. Individuals which were anesthetized in soft water recovered sooner.

Repeatedly anesthetized trout were slightly 

more tolerant than previously unexposed fish. 

Tolerance did not increase with repeated 

treatments.

Sedating concentrations of MS-222 did not 

lower the rate of oxygen consumption of lake 

trout in open systems. In closed systems, the 

consumption rate of rainbow trout was reduced approximately 30 percent.

We concluded that MS-222 is an effective 

and safe anesthetic for inducing anesthesia in 

the four salmonids. The relative safety of the 

drug was greater at lower temperatures. Our 

results and those of other investigators indicate that preliminary bioassays of anesthetic 

solutions are advisable to determine the desired rates of anesthesia and exposure times 

for specific conditions and lots of fish. Close 

observance on stages of anesthesia is essential to prevent excessive exposure of the fish 

at hi&h concentrations.

REFERENCES

Allison, Leonard N.

1961. The effect of tricaine methanesulfonate (M.S. 

222) on the motility of brook trout sperm. Progressive Hsh-Culturist, vol. 23, no. 1, p. 46-48.

Ball, J. N., and P. N. Cowen.

1959. Urethane as a carinogen and as an anasthetic 

for fishes. Nature, vol. 184, p. 370.

Baudin, Louis.

1932a. Action de la tricaine sur la consummation 

d'oxygene de Carassiusauratus, Comptes rendus 

des Seances de la Soci6te de biologie, vol. 109, 

p. 731-733.

1932b. Perte de la sensibilite a la depression chez 

les poissons anesthesies a la tricaine. Comptes 

rendus des Seances de la Societe de biologie, vol. 

110, p. 151-153.

Bell, Gordon R.

1964. A guide to the properties, characteristics, and 

uses of some general anesthetics for fish. Fisheries Research board of Canada, Bulletin 148. 4 p.
14 Investigations in Fish Control 13: Bureau of Sport Fisheries and Wildlife

Black, Edgar Cmt and Anne R. Connor. 

1964. Effects of MS 222 on glycogen and lactate 

levels in rainbow trout (Salrno gairdneri). Journal of the Fisheries Research Board of Canada, 

vol. 21, no. 6, p. 1539-1542.

Blahm, T. H.

1961. Effects of tricaine methanesulfonate on oxygen consumption of juvenile sockeye salmon. 

Transactions of American Fisheries Society, vol. 

90, no. 2, p. 226-227.

Bove, Frank J.

1962. MS-222 Sandoz the anaesthetic of choice for 

fish and other coldblooded organisms. Sandoz 

News, No. 3, 12p.

Butler, Robert L.

1957. The development of a vinyl plastic subcutaneous tag for trout. California Fish and Game, vol. 

43, no. 3, p. 201-212.

Collins, James L., and Andrew H. Hulsey.

1963. Hauling mortality of threadfin shad reduced 

with M.S. 222 and salt. Progressive Fish-Culturist, vol. 25, no. 2, p. 105-106.

Crawford, Bruce, and Andrew Hulsey.

1963. Effects of M.S. 222 on the spawning of channel catfish. Progressive Fish-Culturist, vol. 25, 

no. 4, p. 214.

Dollar, Alexander M.

1963. Air transportation of living rainbow trout. 

Progressive Fish-Culturist, vol. 25, no. 3, p. 167- 

168.

Eisler, Ronald, and Tadeusz Backiel.

1960. Narcotization of chinook salmon fingerlings 

with tricaine methanesulfonate (M.S. 222). Transactions of the American Fisheries Society, vol. 

89, no. 2, p. 164-167.

Fry, F.E.J.

1957. The aquatic respiration of fish. In: The Physiology of Fishes. Volume 1. Edited by Margaret 

E. Brown, Academic Press, New York, p. 1-63.

Gebhards, Stacy V.

1965. Transport of juvenile trout in sealed containers. Progressive Fish-Culturist, vol. 27, 

no. 1, p. 31-36.

Gossington, Robert.

1957. An aid to fish handling-tricaine. Aquarium 

Journal, vol. 28, no. 9, p. 318-321.

Klontz, George W.

1964. Anesthesia of fishes. From: Proceedings of 

the Symposium on Experimental Animal Anesthesiology, Brooks Air Force Base, December 14- 

16. 13 p.

Lamarque, Pierre.

1964. Anesthesie et transport. Bull. Inf. Cons. Sup. 

Pgche, vol. 55, p. 5-9.

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

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

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

Marking, Leif L.

1966. Investigations in Fish Control: 12. Toxicity 

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

Sport Fisheries and Wildlife, Resource Publication 18.

Martin, N. V., and D. C. Scott.

1959. Use of tricaine methanesulfonate (M.S. 222) 

in the transport of live fish without water, Progressive Fish-Culturist, vol. 21, no. 4, p. 183- 

184.

McFarland, William N.

1959. A study of the effects of anesthetics on the 

behavior and physiology of fishes. Publications 

of the Institute of Marine Science, University of 

Texas, vol. 6, p. 23-55.

1960. The use of anesthetics for the handling and 

the transport of fishes. California Fish and Game, 

vol. 46, no. 4, p. 407-431.

Meehan, William R., and L. Revet.

1962. The effect of tricaine methanesulfonate (M.S. 

222) and/or chilled water on oxygen consumption 

of sockeye salmon fry. Progressive Fish-Culturist, vol. 24, no. 4, p. 185-187.

Meister, Alfred L., and Charles F. Ritzi.

1958. Effects of chloretone and M.S. 222 on eastern 

brook trout. Progressive Fish-Culturist, vol. 

20, no. 3, pe 104-110.

Nelson, P. R.

1953. Use of three anesthetics on juvenile salmon 

and trout. Progressive Fish-Culturist, vol. 15, 

no. 2, p. 74.

Onkst, H., J. Jacoby, and D. G. Scarpelli.

1957. Effect of calcium binding substances on rate 

and duration of narcosis. Proceedings of Society 

for Experimental Biology and Medicine, vol. 96, 

no. 2, p. 397-399.

Parkhurst, Z. E., and M. A. Smith.

1957. Various drugs as aids in spawning rainbow 

trout. Progressive Fish-Culturist, vol. 19, no. 1, 

P. 39.

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

Podoliak, Donald R. Brockway, and George C. 

Balzer, Jr.

1955. The nutrition of trout. Cortland Hatchery 

Report 24, New York Conservation Department, 

Fisheries Research Bulletin 19. 56 p.
Schoettger, Julin.- Efficacy of MS-222 as an Anesthetic on Four Salmonids 15

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

R. Brockway, and Ray R. Vaughn. 

1957. The nutrition of trout. Cortland Hatchery 

Report 26, New York Conservation Department, 

Fisheries Research Bulletin 21. 93p.

Quastel, J. H.

1963. Effects of anesthetics, depressants, and 

tranquilizers on cerebral metabolism. In: 

Metabolic inhibitors; A comprehensive treatise. 

Volume II. Edited by R. M. Hochster and J. H. 

Quastel. Academic Press, New York, p. 517-538.

Robert son, O. H.

1958. Accelerated development of testis after unilateral gonadectomy, with observations on normal 

testis of rainbow trout. U.S. Fish and Wildlife 

Service, Fishery Bulletin, No. 127, vol. 58, p. 9-30.

Rodman, Duane T.

1963. Anesthetizing and air-transporting young 

white sturgeons. Progressive Fish-Culturist, 

vol. 25, no. 2, p. 71-78.

Schiffman, R. H., and P. O. Fromm.

1959. Measurement of some physiological parameters in rainbow trout (Salmo gairdnerii). Canadian Journal of Zoology, vol. 37, p. 25-32.

Schoettger, Richard A.

1966. Investigations in Fish Control: 16. Annotated 

bibliography on MS-222. U.S. Bureau of Sport 

Fisheries and Wildlife, Resource Publication 22.

Smith, Lynwood S., and Gordon R. Bell.

1964. A technique for prolonged blood sampling in 

free-swimming salmon. Journal of the Fisheries 

Research Board of Canada, vol. 21, no. 4, p. 711- 

717.

Thompson R. B.

1959. Tricaine methanesulfonate (M.S. 222) in 

transport of cutthroat trout. Progressive FishCulturist, vol. 21, no. 2, p. 96.

Walker, Charles R., and Richard A. Schoettger. 

1966. Investigations in Fish Control: 15. Residues 

of MS-222 in four species of salmonids following 

anesthesia. U.S. Bureau of Sport Fisheries and 

Wildlife, Resource Publication 21.

Webb, Robert T.

1958. Distribution of bluegill treated with tricaine 

methane sulfonate (M.S. 222). Progressive FishCulturist, vol. 20, no. 2, p. 69-72.

Wood, E. M.

1956. Urethane as a carcinogen. Progressive FishCulturist, vol. 18, no. 3, p. 135-136.
INVESTIGATIONS IN FISH CONTROL

14. Method for Determining MS-222 Residues in Fish

By Charles R. Walker and Richard A. Schoettger

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

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

Fish and Wildlife Service, Clarence F. Pautzke, Commissioner 

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

Resource Publication 20 January 1967 Washington, D.C.
CONTENTS

Page

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

Methods and materials ......................... 3

Experiments on operating parameters ............ 3

Fish. .................................. 4

Tissue collection and treatment ................ 4

Analysis of residues ......................... 5

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

Chemistry of the colorimetric reaction ........... 6

Measuring MS-222 in fish .................... 8

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

Summary .................................. 9

Literature cited.............................. 10
METHOD FOR DETERMINING MS-222 RESIDUES IN FISH

By Charles R. Walker, Chemist, and Richard A. Schoettger, Fishery Biologist

Bureau of Sport Fisheries and Wildlife

La Crosse, Wisconsin

Abstract. MS-222 is a primary aromatic amine. Its diazonium salt 

reacts with the Bratton-Marshall reagent (N-1-naphthylethylenediamine 

dihydrochloride) to form a wine-red azo dye with a maximum absorbance 

at 545 millimicrons. The diazotization reagent had to be modified and the 

reaction time extended to obtain a measurable yield of the diazonium salt. 

The regression of absorbance values versus concentrations of MS-222 

which ranged from 0 to 7.5 p.p.m. had a slope of 1 and adhered to Beer's 

law. MS-222 was spiked into samples of blood, muscle, liver, and kidney 

from rainbow trout, and excellent recoveries were measured by the analytical method. The residues had to be distinguished from a background of 

primary aromatic amines which varied in each tissue, between individual 

specimens and with each lot of fish. Backgrounds were higher in liver 

and kidney, and residues of MS-222 were more difficult to differentiate. 

The method was particularly effective in determining residues of the drug 

in muscle and blood of trout.

MS-222 (tricaine methanesulfonate) is 

widely used as a fish anesthetic. Residues 

which might occur in fish flesh following use 

of the drug must be ascertained for safety of 

human consumers. Development of adequate 

information on residues required a method 

capable of measuring finite concentrations in 

the tissues.

A primary consideration in the acceptance 

of any analytical method for residues is the 

capacity to detect them at levels related to 

their toxicity to mammals. A contract study 

done for us by the Wisconsin alumni Research 

Foundation in March 1965 indicated that the 

oral LD 50 of MS-222 for Sprague-Dawley 

strain laboratory rats is between 5 and 10 

grams per kilogram of body. Thus, the drug 

is relatively nontoxic to mammals according 

to the standards of the American Industrial 

Hygiene Association (Spector, 1956). An analytical procedure capable of defining residues 

in parts per million should be adequate for 

all practical purposes.

Friddle and Snieszko (1950), using the 

Bratton-Marshall (1939) method, found that 

MS-222 caused an error in their evaluation 

of sulfa residues in anesthetized brook trout. 

We believe this can be explained by the fact 

that MS-222 is a primary aromatic amine and 

reacts readily in the colorimetric procedure. 

We chose to investigate the applicability of 

this colorimetric-reaction to determinations 

of MS-222 in fish tissues.

METHODS AND MATERIALS

Experiments on operating parameters

Diazotization of the m-aminoethylbenzoate 

constituent of the MS-222 salt was accomplished by modification of the procedure described by Bratton and Marshall (1939). The 

conditions for formation of the diazonium ion 

and the subsequent reaction with the coupling 

reagent were established in a series of exr 

periments.
4 Investigations in Fish Control 14: Bureau of Sport Fisheries and Wildlife

The diazonium salt was obtained in a 5-ml. 

sample containing MS-222, m-aminoethyLbenzoate, by adding 0.5 ml. of 0.2-percent 

sodium nitrite. The excess nitrous acid was 

destroyed by ammonium sulfamate. The diazonium salt was reacted with the BrattonMarshall reagent, N-1-naphthylethylenediamine dihydrochloride, to form the azo 

dyestuff.

The effect of diazotization time upon the 

concentration of azo dye formed was measured in 40 tests involving 160 reactions made 

with 0, 1, 3, 5, or 7.5 p.p.m. of MS-222 standards. Diazotization reactions were stopped 

at 3, 5, 7 to 10, and 15 to 30 minutes. The 

percent transmittance values were plotted 

for each concentration and the regression 

was calculated for each time interval.

The speed of azo dye formation upon addition of the coupling reagent was measured 

at 0, 1, 3, and 5 p.p.m. of MS-222 in standards reacted at room temperature following 

diazotization. The transmittance values were 

recorded at 30-second intervals for the first 

5 minutes, 1-minute intervals for the next 10 

minutes, and at 5-minute intervals up to 1 

hour to measure the effect of development 

time.

The effect of temperature on the diazotization reaction and the resultant concentration 

of azo dye formed was tested at 15°, 22.5°, 

and 30° C. Twenty tests were conducted at 

the longer diazotization times at each 1-minute interval from 10 minutes through 30 

minutes for each concentration of 1, 3, and 

5 p.p.m. of MS-222. The mean transmittance 

value for each concentration and temperature 

was calculated for 5 measurements taken at 

10-15 minutes, for 5 at 15-20 minutes, and 

for 10 between 20 and 30 minutes.

The maximum limit for the reaction of the 

diazonium ion was between 5 and 7.5 p.p.m. 

of MS-222 without causing precipitation on 

the sides of cuvettes and or optical density 

less than 20 percent. Thus, dilutions were 

designed to keep the concentration in samples 

in the working range of the instrumentation.

Measurements of the maximum absorbance value of the azo dye at 420 to 650

millimicrons were made with a Beckman DB 

spectrophotometer and a Sargent SRL recorder. Mechanical Adjustments for slit 

width for the maximum absorbance band 

were established on standard solutions of 

MS-222 at concentrations up to 7.5 p.p.m. 

with a Perkin-Elmer 139 spectrophotometer 

equipped with a photomultiplier.

Fish

We used rainbow trout in the tests of the 

residue method. The 8- to 14-inch fish were 

furnished by the Manchester, Iowa, National 

Fish Hatchery and were held in facilities of 

the Fish Control Laboratory. They were fed 

a pelleted, open-formula diet. It is important 

to point out that several ingredients of the 

diet were fortified with primary aromatic 

amines or vitamins such as folic acid, 

vitamin B, 2 , p-aminobenzoic acid plus protein sources such as brewer's yeast.

Tissue collection and treatment

Blood samples were drawn from specimens 

at specified intervals by cardiac puncture with 

a heparinized syringe and 18- or 20-gage 

needle. Within 15 minutes after the blood was 

taken, the samples were mixed with trichloroacetic acid (TCA) for coagulation of 

protein.

Each fish was immobilized by a blow on 

the head or pithing and the other tissues were 

dissected out. Skin was removed to expose 

the thick muscular area adjacent to the 

dorsal fin. A sample about 1 inch square was 

removed, minus bones, and placed in a tared 

beaker and weighed. It was homogenized and 

diluted 1:20 with TCA and distilled water to 

make up a 3-percent TCA solution.

The entire liver, excluding the gall bladder, 

was removed. Kidney tissue was dissected 

posterior to the isthmus. The tissues were 

weighed in tared beakers and those not 

processed immediately were quick-frozen 

with dry ice. The tissues were processed 

for chemical analysis in the same manner 

as muscle.
Walker, Schoettger: Method for Determining MS-222 Residues in Fish

Analysis of residues

The concentration of aromatic amines and 

other masking or interfering substances 

were determined for blood, muscle, kidney 

and liver in 9 specimens of rainbow trout and 

expressed in terms of parts per million of 

MS-222. The recovery of MS-222 from each 

tissue was evaluated by spiking at rates of 

0, 20 and 100 p.p.m. in each lot of 3 fish by 

using the outlined procedure.

The colorimetric reaction described by 

Bratton and Marshall (1939) was used, with 

modifications, for detection of MS-222 in 

fish tissues. The process was as follows:

Reagents:

1. 15-percent trichloroacetic acid: Dissolve 30 g. crystalline TCA in distilled 

water. Transfer to a 200-ml. volumetric flask and bring to volume with 

distilled water. Dilute 20 ml. to 100 to 

get 3-percent TCA.

2. 0.2-percent sodium nitrite: Dissolve 

0.5 g. NaNC>2 in water. Transfer to a 

100-ml. volumetric flask and bring to 

volume. Transfer 40 ml. to a second 

100-ml. volumetric flask and bring to 

volume. Make fresh daily.

3. 0.5-percent ammonium sulfamate: Dissolve 0.5 g. in distilled water and dilute 

to 100 ml.

4. 0.1-percent N-1-naphthylethylenediamine dihydrochloride: Dissolve 0.5 g. 

in distilled water and bring to 100 ml. 

Transfer 20 ml. to 100-ml. volumetric 

flask and bring to volume. Refrigerate 

and make up fresh weekly.

5. 4N hydrochloric acid: Dilute 40 ml. 

concentrated HC1 to 100 ml. Mix and 

titrate a 5 ml. portion with standard 

IN NaOH. Adjust to exactly 4N.

6. 10-percent HC1.

Standards:

1. Stock solution: Weigh 0.1000 g. of 

MS-222 and dissolve in distilled water.

Quantitatively transfer to 100-ml. 

volumetric flask, add 20 ml. of 15-percent TCA and dilute to volume with distilled water. Stock equals 1,000 p.p.m. 

of MS-222 in 3-percent TCA.

2. Dilute 2.0 ml. stock to 200 ml. with 3- 

percent TCA. This stock contains 10 

p.p.m. of MS-222.

3. Volume of 10 p.p.m. stock is diluted to 

50 ml. with 3-percent TCA:

Desired p.p.m.

9.0 

7.5 

5.0 

3.0 

1.0 

0.5 

0.25

ml. of 

10-p.p.m. stock

45.0

37.5

25.0

15.0

5.0

2.5

1.25

Procedure:

1. Sample preparation blood and tissue.

A. Blood: Use an anticoagulant, and 

draw blood with care to avoid contamination with body fluids or water. 

Place 0.5 ml. in 7.5 ml. of distilled 

water and let stand 10 minutes or 

until other samples are ready. Add 

2 ml. of 15-percent TCA. Mix 

thoroughly and let set 5 minutes. 

Centrifuge at 2,500 RPM for 30 

minutes. Filter to remove fat or 

other materials not spun down. Go 

to Part 2, Analysis.

B. Other tissue (muscle, kidney and 

liver): Carefully dissect out the 

sample, weigh, homogenize and 

dilute in the following manner: One 

gram of tissue is homogenized and 

diluted to 16 ml. with distilled water 

in a graduated centrifuge tube. A 

1:20 dilution of the sample is 

achieved by adding 4 ml. of 15-percent TCA. The precipitate of protein 

is removed by the centrifugation and 

filtration outlined for blood as above. 

The supernatant is then analyzed 

according to part 2, Analysis.
Investigations in Fish Control 14; Bureau of Sport Fisheries and Wildlife

2. Analysis.

A. Pipette 5 ml. of filtrate into a clean 

25-ml. Erlenmeyer flask.

B. Pipette 5 ml. of 3-percent TCA into 

another flask as a reagent blank.

C. Add 0.5 ml. of 0.2-percent NaNO 2 

to each flask, swirl, and let stand 

15 minutes. Keep out of direct sunlight.

D. Add 0.5 ml. of 0.5-percent ammonium sulfamate to each flask, 

swirl and let stand 2 to 3 minutes.

E. Add 0.5 ml. of 0.1-percent N-lnaphthylethylenediamine dihydrochloriue to each tube or flask. 

Swirl for 30 to 60 seconds and let 

stand 5 or 10 minutes. Keep out of 

direct sunlight.

F. Pipette or pour into cuvettes and 

read in spectrophotometer at 545 

millimicrons. A Model 139, Perkin 

and Elmer spectrophotometer with 

a slit width of 0.05 mm. and sensitivity of XI was used. Sensitivity of 

the photomultiplier was set at 5. 

Obtain p.p.m. or microgram from 

precalibration curve or use a standard. Correct for dilution and quantity of tissue. The concentration 

will be in terms of free MS-222.

G. Possible acetylated derivatives: 

Place 10 ml. of filtrate into a 

graduated centrifuge tube. Add 

0.5 ml. of 4N HC1. Place in a boiling water bath for 1 hour, cool, and 

adjust volume to 10 ml. with water. 

Transfer 5 ml. to a 25-ml. Erlenmeyer flask and treat as above for 

free MS-222.

3. Other considerations.

A. Bubble formation: Occasionally 

bubbles form in cuvettes. The 

cuvettes should be capped and 

slowly tipped. Most of the bubbles 

on the transparent surface should be

absorbed by the large air bubble in 

the cuvette. The use of larger Erlenmeyer flasks may also reduce bubble 

formation.

B. Glassware cleanliness: Flasks and 

cuvettes which have been used in 

steps E and F should be rinsed, first 

in 10-percent HC1 and then in distilled water. A colored residue may 

adhere to the glassware and produce 

errors in subsequent analyses. Several rinsings with acid are usually 

sufficient to remove the residue.

RESULTS

Chemistry of the colorimetric reaction

The majority of applications of the Bratton-Marshall reaction employing the N-lnaphthylethylenediamine coupling reagent 

have involved par a-substituted compounds 

such as sulfonamides, procaine hydrochloride, p-aminobenzoic acid, folic acid, and 

chloramphenicol (Meites, 1963; Welcher, 

1963; V,arley, 1963). However, coupling of 

diazonium salts of both para- and orthoprimary aromatic amines are widely illustrated in the chemistry of azo dyestuffs 

(Brewster and McEwer, 1961). In our application, we are diazotizing a primary aromatic amine in the meta position to-a carboxyl group. The preparation of the diazonium 

salt of MS-222 would probably follow the flow 

scheme illustrated by Natelson (1961) (fig. 1).

N=NOH

Aryl amine Aryl diazonium ion

NH-CH2 -CH2 -NH2

Colored Azo Dye

N-l-naph1hylethylenediamine

Figure 1. Probable flow scheme of the diazotization 

of the ethyl ester of m-aminobenzoate (MS-222) and 

the coupling reaction with I^-1-naphthylethylenediamine after Natelson (1961). The HC1 maintains the 

pH at 1 to 2.
Walker, Schoettger: Method for Determining MS-222 Residues in Fish 7

Apparently, this reaction proceeds much 

slower with the meta isomer than with ortho 

and para isomers. The suggested time for 

diazotization of par a-substituted sulfonamides 

was 3 minutes with 0.1-percent sodium nitrite reagent (Bratton and Marshall, 1939). 

The diazotization of MS-222, however, required at least 0.2-percent sodium nitrite. 

A more consistent production of diazonium 

salt occurred at 15° to 30° C. in 15 to 30 

minutes (table 1). Holbourn and Pattle (1943) 

observed the necessity for longer diazotization time in their work with sulfanilamide 

and the Bratton-Marshall reagent. They also 

noted the effect of light on the reaction and 

coupling with the reagent.

According to Marshall and Litchfield (1938) 

and Bratton and Marshall (1939), the ammonium sulfamate effectively destroys the 

excess nitrous acid. Compared to the reaction time indicated for the sulfa drugs, the 

diazonium salt of MS-222 appears to undergo 

nitroso formation at a relatively slow rate, 

thus providing more latitude of time prior to 

the termination of diazotization. Upon addition of the Bratton-Marshall reagent, N-lnaphthylethylenediamine dihydrochloride solution, the azo dyestuff formed rapidly and 

attained a maximum intensity within a few 

minutes (fig. 2). The azo dye coupling 

reaction proceeds immediately to form a 

wine-red colored azo dyestuff with a maximum absorbance in the spectral range of 545 

millimicrons (fig. 3). This absorbance curve 

is very similar to that illustrated for sulfa 

drugs by Natelson (1961). The absorbance 

values or percent transmittance were plotted

TABLE 1. Effect of diazotization times at three temperatures on the 

percentage of transmittance values observed for MS-222 in the BrattonMarshall reaction

Diazotization time 

and 

temperature

10-15 minutes: 

15.0° C.. ........

22 5® C

15-20 minutes : 

i s cfi r

30.0° C.... ......

20-30 minutes: 

15.0° C.... ......

22 5° C

30.0° C. .........

Percentage of transmittance at 

1 p.p.m.

Mean

78.3 

77.8 

77.0

77.2 

76.8 

76.3

76.1 

76.6

75.3

Range

77.8-81.5 

76.5-80.4 

76.5-78.8

76.8-79.1 

76.8-77.6 

75.7-77.0

75.8-76.5 

75.0-76.1 

74.9-75.8

3 p.p.m.

Mean

48.4 

47.7 

46.9

47.0 

46.6 

46.0

44.6 

44.0 

43.5

Range

46.9-50.3 

45.0-48.4 

45.0-47.6

46.3-50.0 

45.5-47.0 

45.0-46.7

43.9-45.0 

43.3-44.4 

43.0-44.0

5 p.p.m.

Mean

29.7 

28.7 

28.2

28.1 

27.5 

27.0

26.2 

25.8 

25.3

Range

29.5-30.6 

27.5-30.2 

27.4-29.3

27.6-29.5 

26.5-28.9 

26.3-27.9

25.8-26.6 

25.2-26.2 

24.7-25.8

100

5 10 

DEVELOPMENT TIME (min.)

60

Figure 2. Speed of development of the azo dyestuff 

in the coupling reaction of N-l -naphthylethylenediamine with three concentrations of diazotized 

MS-222.

lOO-i

(O

(C

60-

40-

z

UJ 

O

400 500 600 

WAVE LENGTH (mjj)

Figure 3.--Absorption spectrum for the azo dyestuff 

developed in the reaction of diazotized MS-222 and 

N-l -naphthylethylenediamine.
8 Investigations in Fish Control 14: Bureau of Sport Fisheries and Wildlife

on the logarithmic scale versus the concentration in p.p.m. as MS-222 on an 

arithmetic scale with 1-cycle logarithmic paper (fig. 4). A straight line 

relation exists between means of points 

established at 1, 3, and 5 p.p.m., and it 

adheres to Beer's law over a wide range of 

diazotizatlon times. Greater sensitivity was 

obtained at 15 to 30 minutes, and the values 

did not vary as greatly as those at shorter 

diazotizatlon periods.

The accuracy of the determination is 

greatest in the middle section of the curve. 

Hawk et al. (1954) mention that higher concentrations produce colors too deep for precise 

measurement. The dyestuff appears to precipitate at the liquid-air interface at the 

higher concentration of MS-222, and it adheres to the sides of glassware. It is exceedingly important that cuvettes be rinsed with 

10-percent HC1 prior to introducing the developed sample. We also found it expedient 

to rinse the cuvettes at least once with the

lOOi

80

2*60

H 

2

CO

o 

oc

20

 3 min.

 5 min.

 7-10 min.

 15-30 min.

MS-222

3 

in p.p.m.

Figure 4. Transmittance curves of MS-222 showing 

the effects of different diazotization times on the 

intensity of color development at 545 millimicrons 

and the range of values observed at 5 p.p.m.

developed sample solution to avoid a negative 

dilution error. Contaminants and/or gas 

bubbles on the sides of cuvettes in the light 

path may produce serious quantitative errors 

at high transmittance. Particular care must 

be taken to remove detergent film from glassware by a thorough rinsing with dilute HC1 

and deionized water. Detergents cause yellow or green-brown turbidity in extreme 

cases of contamination. This turbidity is 

distinguishable from the nitroso formation 

of azoxy compounds of MS-222 and natural 

aromatic amines which are usually reddishbrown in color.

Measuring MS-222 in fish

Since the Bratton-Marshall color reaction 

is specific to aromatic amines, the quantltation of MS-222 has to be differentiated from 

natural occurring amines such as p-aminobenzoic acid (Hawk et al., 1954). Thus, we 

determined background levels of amines and 

other masking substances in blood, muscle, 

kidney, and liver (table 2). Further, the 

amounts of recoverable MS-222 were determined by spiking samples of two concentrations (table 3). This determined its effectiveness in measuring different concentrations 

in the various tissues over and above background interferences. Recoveries ranged 

from 89 to 112 percent for all samples.

TABLE 2. Concentrations of background at 545 millimicrons in rainbow trout

Tissue

Liver

Number 

of 

fish

9 

9 

9 

9

Background

Mean 

(p.p.m.)

0.91 

0.87 

5.42 

18.13

Range 

(p.p.m.)

0.0- 2.0 

0.0- 2.0 

2.8- 9.0 

11.6-20.0

Standard 

error

0.19 

0.29 

0.58 

0.%

95-percent 

confidence 

interval

0.46- 1.36 

0.21- 1.53 

4.08- 6.76 

15.92-20.34

TABLE 3. Recovery of MS-222 spiked into various tissues of rainbow trout

Tissue

Number

of 

fish

3 

3

3

3

3

Concenof spikes 

(p.p.m.)

100 

20

100

100

100

lu

Mean 

(p.p.m.)

99.47 

21.00

101.87

88.93

103.30

B-222 in tissue

Range 

(p.p.m.)

96. 6-101.0 

20.6- 21.4

94.2-106.2

86.2- 91.6

95.2-109.6

Percent 

recovery

99.5 

105.0

101.9

88.9

103.3
Walker, Schoettger: Method for Determining MS-222 Residues in Fish

Mooney and Pasarela (1964) refined the 

cleanup of samples to reduce contamination 

by procaine penicillin and other interfering 

substances in the background. They employed a Dow-50W-X2 (H+) ion exchange 

resin to extract sulfonamides selectively in 

acid phase and to elute them in alkaline 

phase. This method was not applicable to the 

cleanup of fish tissues because we could not 

elute MS-222 from the column.

Liver gave the most interference by producing a true red, azo dye color reaction, 

and the levels were quite consistent with a 

small deviation and narrow confidence limits. 

Kidney samples were characterized by a yellow color. This pigment introduced a negative interference with the azo dye reaction, 

but gave a consistent recovery of spiked 

MS-222. Blood and muscle gave relatively 

little color reaction or interference. The 

volumes of blood samples, however, were 

small, and contributed to positive dilution 

errors. The errors were particularly significant in the recovery of lower concentrations of MS-222. In general, the overall 

accuracy of the method is governed by the 

techniques of extraction and dilution.

CONCLUSIONS

MS-222 can be detected in fish tissues by 

means of a modified Bratton-Marshall 

method. Interferring substances were more 

prevalent in liver and kidney than in blood 

and muscle. The recovery of MS-222 from 

spiked samples ranged from 89 to 112 percent. The method was more accurate for 

measuring MS-222 in blood and muscle than 

in kidney and liver.

SUMMARY

Use of MS-222 as a fish anesthetic necessitated an investigation into the residues in 

fish following exposure. Fortunately, the 

toxicity of MS-222 to mammals was found to 

be quite low with an acute oral LD50 of approximately 5-10 g./kg. to laboratory rats. 

Thus, an analytical procedure which would

define concentrations in p.p.m. was 

considered adequate.

MS-222 has the chemical structure of a 

primary aromatic amine and it reacts colorimetrically with the Bratton-Marshall reagent. We modified the method commonly 

used for determination of sulfa drugs.

MS-222 forms a diazonium salt much more 

slowly than the para-substituted aromatic 

amines and thus required a higher concentration of nitrous acid and longer time for 

development. The azo dye coupling reaction 

proceeds almost immediately with N-lnaphthylethylenediamine dihydrochloride to 

form a wine-red dye complex with a maximum absorbance in the spectral range of 

545 millimicrons. The regression of absorbance values was almost linear with a 

slope of 1.0, and it bore evidence of adherence to Beer's law.

The fish tissues were extracted in distilled water, and the protein was precipitated 

in a 3-percent TCA solution. The solution 

was centrifuged and then filtered to remove 

the lighter fatty fraction. A l-to-20 dilution 

was necessary to adjust the concentration 

within the limits of the transmission curve 

(0 to 7.5 p.p.m.).

We obtained excellent recoveries of MS222 from all spiked samples of fish tissue. 

They ranged from 89 to 112 percent which 

excluded the aromatic amines of natural 

origin. The background of primary aromatic 

amines varied with each tissue and between 

individual specimens. The backgrounds were 

higher in liver and kidney, and residues of 

MS-222 were more difficult to differentiate. 

In liver, it was due, presumably, to the 

diazotized amines such as vitamin B12 , 

p-aminobenzoic acid, folic acid, and the 

many proteins in fortified diets for trout. 

A yellow pigment persisted in extracts from 

kidneys which introduced a negative interference with the azo dye reaction. The modified Bratton and Marshall method was, however, particularly effective in determining 

residues of MS-222 in muscle and blood of 

trout.
10 Investigations in Fish Control 14: Bureau of Sport Fisheries and Wildlife

REFERENCES

Bratton, A. Calvin, and E. K. Marshall, Jr.

1939. A new coupling component for sulfanilamide 

determination. Journal of Biological Chemistry, 

vol. 128, p. 537-550.

Brewster, Ray Q., and William E. McEwen.

1961. Organic chemistry. 3d. ed. Prentice-Hall, 

Englewood Cliffs, N.J. 854 p.

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

1950, Effects of tricaine methanesulfonate on the 

determination of sulfonamides. Science, vol. 112, 

no. 2902, p. 181-182.

Hawk, Philip B., Bernard L. Oser, and William H. 

Summerson.

1954. Practical physiological chemistry. 13th ed., 

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

1439 p.

Holbourn, A. H. S., and R. E. Pattle.

1943. Sources of error in sulfanilamide determinations. Journal of Laboratory and Clinical Medicine, vol. 28, p. 1028-1033.

Marshall, E. K., Jr., and J. T. Litchfield, Jr.

1938. The determination of sulfanilamide. Science, 

vol. 88, no. 2273, p. 85-86.

Meites, Louis.

1963. Handbook of analytical chemistry. 1st ed., 

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

1805 p.

Mooney, R. P., and N. R. Pasarela.

1964. A colorimetric procedure for the determination of sulfonamides in animal tissues. Journal 

of Agriculture and Food Chemistry, vol. 12, no. 2, 

p. 123-127.

Natelson, Samuel.

1961. Microtechniques of Clinical Chemistry. 2d ed. 

Charles Ce Thomas, Springfield, 111. 578 p.

Spector, William S.

1956. Handbook of toxicology. Volume 1, Acute 

toxicities of solids, liquids, and gases to laboratory animals. W. B. Saunders Company, Philadelphia. 408 p.

Varley, Harold.

1963. Practical clinical biochemistry. William 

Heinemann Medical Books and Interscience 

Books, New York. 689 p.

Welcher, Frank J.

1963. Standard methods of chemical analysis. 

Volume 2, Industrial and natural products and 

noninstrumental methods. 6th ed., D. Van 

Nostrand Co., Princeton, N.J. 2613 p.
INVESTIGATIONS IN FISH CONTROL

15. Residues of MS-222 in Four Salmonids 

Following Anesthesia

By Charles R. Walker and Richard A. Schoettger

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

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

Fish and Wildlife Service, Clarence F. Pautzke, Commissioner 

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

Resource Publication 21 January 1967 Washington, D.C.
CONTENTS

Page

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

Methods and materials ......................... 3

Fish. .................................. 3

Anesthetization ........................... 4

Collection and analyses of tissue. ............... 4

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

Preliminary analyses ....................... 4

Intensive analyses on four species. .............. 6

Discussion ................................. g

Conclusions. ................................ IQ

Summary .................................. IQ
RESIDUES OF MS-222 IN FOUR SALMONIDS FOLLOWING ANESTHESIA

By Charles R. Walker, Chemist and Richard A. Schoettger, Fishery Biologist

Bureau of Sport Fisheries and Wildlife

La Crosse, Wisconsin

Abstract. Residues of MS-222 (tricaine methanesulfonate) in the 

blood, muscle, liver, and kidney of rainbow trout and in the muscle of 

brown trout, brook trout, and lake trout were measured by a modified 

Bratton-Mar shall colorimetric method. Temperatures were 7°, 12°, and 

17° C. in waters with total hardnesses of 10 to 180 p.p.m. The residues 

were easily detected and measured in blood and muscle, but they were 

masked in liver and kidney by background substances. The anesthetic 

dissipated rapidly in the muscle of the four species within 1 to 6 hours, 

and the residues approached the background levels of controls within 9 

to 24 hours after withdrawal from exposure. The differences in residue 

levels in the muscle at 0-hour withdrawal between species, at three 

temperatures, and in soft and hard water largely disappeared within 24 

hours.

MS-222 (tricaine methanesulfonate) has 

gained wide acceptance as a fish anesthetic 

and transportation sedative. There has been 

no investigation, however, on its residues in 

fish following treatment. This information is 

especially important in situations where fish 

exposed to anesthetic or sedating solutions 

are eaten soon after by people.

An extensive review of the literature on 

the many uses of MS-222 was made by 

Schoettger (1966). Schoettger and Julin 

(1966) defined the criteria for desired 

stages of anesthesia, and the efficacy of 

MS-222 at various concentrations and 

times of exposure to rainbow trout, 

brown trout, brook trout, and lake trout. 

The analyses of residues of the anesthetic 

in these species were performed concurrently with the investigations of efficacy.

We selected the extremes of concentration 

and depth of anesthesia to ascertain the existence and persistence of residues in the 

four trouts. The major emphasis was placed 

on the determination of residues in muscle

since it is more important than other tissues 

in human consumption. The rainbow trout, 

the species more widely propagated for 

sport and food in the United States, was 

used for evaluation of residues in other tissues: blood, liver and kidney. We also concentrated observations at 12° and to lesser 

extent at 7° and 17° C. This range of temperature is considered to be the most suitable for trout production (Davis, 1956).

METHODS AND MATERIALS

Fish

The rainbow trout, brown trout, brook 

trout, and lake trout ranged from 7 to 14 

inches long, and were obtained from State 

fish hatcheries at Lanesboro, Minn. and St. 

Croix, Wis., and from national fish hatcheries at Jordan River, Mich., Lake Mills, 

Wis. and Manchester, la. They were held in 

well water and facilities described by Lennon and Walker (1964) and taken off feed for
Investigations in Fish Control 15: Bureau of Sport Fisheries and Wildlife

at least 24 hours before treatment with 

MS-222.

Anesthetization

Schoettger and Julin (1966) established the 

efficacy of MS-222 as an anesthetic for several species of trout at various temperatures 

and water qualities. Concurrent with these 

studies, we sampled tissues from the fish 

which were anesthetized at 7, 12 and 17° C. 

The concentrations of anesthetic solutions 

ranged from 80 to 135 p.p.m., and the mean 

time to reach the desired stage of anesthesia 

ranged from 4 to 12 minutes depending on 

temperature and species. After specimens 

reached deep anesthesia, they were withdrawn from the drug solution and placed in 

fresh water for recovery. This action 

marked the beginning of withdrawal time.

Collection and analyses of tissues

The methods used for collecting and analyzing the blood, muscle, liver, and kidney 

of trout were described by Walker and 

Schoettger (1966).

RESULTS

Preliminary analyses

Initially we sought to detect and measure 

residues of MS-222 in blood, muscle, liver, 

and kidney of rainbow trout at intervals of 

0, 5, 10, 20, and 30 minutes after withdrawal 

from 9 minutes of exposure to 100 p.p.m. of 

drug at 12° C. The 12- to 14-inch fish were 

in advanced loss of reflex and some had 

reached medullary collapse (table 1). Residues occurred in all tissues and the higher 

concentrations were found in blood, liver, 

and kidney at 0-minute withdrawal. The 

higher concentration of residue in muscle 

was detected at the 10-minute withdrawal. 

The results were variable. It was apparent 

that withdrawal time should be extended 

beyond 30 minutes, more samples should be 

used, and all specimens should be in the 

same stage of anesthesia.

TABLE 1. Residues of MS-222 including background amines recovered from 

adult rainbow trout following withdrawal from anesthesia

Fish, 

sample

No. 12.......

Interval

after 

withdrawal 

(minutes)

0 

0 

5 

5

10 

10 

15 

15

20 

20 

30 

30

Residues in p.p.m.

Blood

61.6

17.6 

20.2

13.0 

9.1 

6.9 

9.5

7.1 

6.3 

5.9 

5.1

Miscle

7.3 

6.7 

1.3 

9.8

18.9

1.1 

1.1 

7.3

9.2 

4.2

4.0 

0.7

liver

49.5 

53.7 

21.3 

14.6

25.4 

8.3

37.4 

11.5

41.3 

23.6

11.5 

11.5

Kidney

91.5 

52.3 

29.2 

27.8

23.9 

9.4 

9.6 

10.6

14.9 

5.9 

8.7 

2.3

Another group of 15 rainbow trout, 12 to 

14 inches long, was anesthetized in 100 

p.p.m. of MS-222 at 12° C. to a state of 

medullary collapse. Tissues were sampled 

for residues at 0, 1, and 2 hours following 

withdrawal (table 2). The concentrations of 

the drug were highest in the blood of fish at 

0-hour withdrawal. They dissipated rapidly 

,as shown in a regression (fig. 1).

A similar pattern was noted in the muscle, 

although the initial concentration of residue 

was somewhat lower (table 2 and fig. 2). The 

regressions indicate that the amounts of 

MS-222 in blood and muscle approach background quantities of aromatic amines after 

2 hours of withdrawal from the anesthetic 

solution. The 95-percent confidence intervals overlap at the 2-hour withdrawal.

The livers of untreated fish had a wide 

range in concentration of background amines, 

and it was difficult to distinguish between 

them and actual residues of MS-222 (table 2). 

The apparent concentration of the drug in 

liver was highest at the 1-hour reading and 

fell off in 2 hours to values only slightly 

above controls. An extrapolation of the 

regression indicates that residues of the 

drug persist beyond 5 hours (fig. 1).

The residues of MS-222 in kidney were 

somewhat more persistent than those in 

muscle but also declined during the 2-hour 

period following withdrawal (table 2). A 

projection of the regression makes an interception very close to the 2-hour reading 

(fig. 1). The broad confidence intervals at 

the 1- and 2-hour withdrawals overlap
Walker, Schoettger: Residues of MS-222 in Four Salmonids Following Anesthesia

TABLE 2. Residues of MS-222 including background amines recovered from adult rainbow trout following withdrawal from anesthesia

Tissue and withdrawal

interval

Blood:

Misole:

Liver:

Kidney:

Nunher

fish

Mean

Length (inches)

1 O ft

 } o ft

13.1

lot*

size

Weight (grams)

 5T e f>

Residue

Mean

2 60

1 ^ ftSt

5

Q£

eo fiSt

B in p. p.m.

Range

31 0 50 2

Standard

error

0 50

O

rtrt

95-percent

interval

60 T7 78 03

60'

Q. 

Q.

CN 

CN 

CN

40-

Q 20H

CO

0-

012345 

HOURS FOLLOWING WITHDRAWAL

Figure 1. Regressions of the residues of MS-222 in 

blood, muscle, liver, and kidney of rainbow trout 

on withdrawal time. The residues are expressed as 

concentration above the mean background value for 

control fish.

considerably. We also found that the high 

levels of background in untreated controls 

differed slightly between these time intervals.

The uptake of MS-222 was higher in individuals which were anesthetized in soft water

30-

E

Q. 

Q.

CN 20-1

CN

CN

I

to

RESIDUE

10-

7°C

 170C

CONTROL imiTflflNt 

6 1 

HOURS

I l

3 6 

FOLLOWING

9 24

WITHDRAWAL

Figure 2. Regression of the residues of MS-222 in 

muscle of four species of trout on withdrawal time 

following deep anesthesia at three temperatures. 

The lines were drawn through mean values for 

residues at each withdrawal.

than those treated in hard water (table 3). The 

greater uptake of drug in soft water appeared 

to be related to longer exposures. The times
Investigations in Fish Control 15: Bureau of Sport Fisheries and Wildlife

TABLE 3. Residues of MS-222 including bacltground in muscle of rainbow trout following deep anesthesia in hard and soft water at 12° C.

Hardness and withdrawal 

interval

Hard water (180 p.p.m.):

Soft water (10 p.p.m.):

Number 

of 

fish

3 

3

3 

3

Mean size

Length 

(inches)

11.5 

10.6

11.5 

11.1

Weight 

(grams)

233.3 

188.3

233.3 

216.3

Residues in p.p.m.

Mean

9.60 

2.37

30.73 

2.4?

Range

3.4-16.0 

2.1- 2.6

27.6-35.6 

2.2- 2.8

Standard 

error

2.10 

0.09

1.43 

0.10

95-percent 

confidence 

interval

0.99-18.21 

2.01- 2.73

24.39-36.57 

2.03- 2.90

for anesthetization of fish in soft water were 

approximately double those in hard water, but 

recoveries were more rapid (Schoettger and 

Julin, 1966). The concentrations of acetylated 

MS-222 in fish anesthetized in soft and hard 

water were approximately the same. However, 

they amounted to 20 percent of the total aromatic amines which were measured in fish 

treated in soft water as compared with 40 

percent for fish in hard water at the 0-hour 

withdrawal. We were unable to show any 

differences in concentrations of free MS-222 

at 24 hours, and approximately 30 to 35 percent of the total aromatic amines were 

acetylated.

Intensive analyses on four species

Rainbow trout. Seventy-five 8- to 12-inch 

rainbow trout were anesthetized to medullary 

collapse in 100 p.p.m. of MS-222 at 7°, 12°, 

and 17° C. (table 4). The induction of anesthesia ranged from 6.8 to 11.4 minutes at 7°, 

8.0 to 11.9 minutes at 12°, and 5.5 to 6.9 

minutes at 17° C.

The residues including background in blood 

at 0-hour withdrawal ranged from 39.8 to 

65.8 p.p.m., with the least variation at the 

higher temperature (table 4). They decreased 

rapidly at 7° and 12° compared with 17° C. 

The levels of background in these tests were 

0.8 to 1.0 p.p.m. (table 5). Thus, the residues 

of MS-222 approach the background levels 

after 24 hours withdrawal.

The highest concentrations of residues of 

MS-222 including background in muscle occurred at 0-hour withdrawal at all water 

temperatures (table 6). They varied greatly 

between individual fish, thereby causing large 

standard errors and making the calculation 

of 95-percent confidence intervals impractical. The standard errors decreased as the

TABLE 4. Residues of 16-222 including background in blood of rainbow 

trout following deep anesthesia at selected time intervals and water 

temperatures

Temperature and 

withdrawal 

interval

7° C.:

12° C.:

17° C. :

Number 

of 

fish

2

2 

2

1

2

2 

2

1 

2

2 

2 

2 

2

Ifean size

Length 

(inches)

10.00 

9.70 

9.95 

10.00

9.85 

10.00 

9.80 

10.10 

9.25

10.55 

10.15 

10.25 

10.40

Weight 

(gram)

154.0 

131.5 

1A1.0 

154.0

1A1.5 

150.5 

150.0 

155.0 

118.0

187.0 

159.5 

166.5 

168.0

Residues in 

p.p.m.

Mean

52.8 

2.5

1.6 

1.0

60.6 

6.2 

2.1 

2.6 

1.9

45.8 

5.1 

4.6 

1.4

Range

39.8-65.8 

2.0- 3.0 

1.4- 1.8

57.2-64.0 

5.6- 6.8 

2.0- 2.2

1.8- 2.0

43.8-47.8 

4.8- 5.4 

4.6- 4.6 

1.0- 1.8

Standard 

error

13.00 

0.50 

0.20

3.40 

0.60 

0.10

0.10

2.00 

0.30 

0.00 

0.40

time of withdrawal increased. Furthermore, 

the residues of 9.4 to 72.0 p.p.m. at 0-hour 

withdrawal declined rapidly to a range of 0.8 

to 3.8 p.p.m. at 6 hours.

The mean residues in muscle at the 9-hour 

withdrawal were equal to or less than background values, and the confidence intervals 

were similar (table 5). It appears that no significant residues of MS-222 remain in muscle 

at 9 hours after withdrawal.

The residues of MS-222 in livers exceeded 

the mean background by 17 to 59 p.p.m. at 

0-hour withdrawal (table 7). Thereafter, the 

residues were masked by the high and variable levels of background amines (table 5).

The residues in kidneys at 0-hour withdrawal exceeded the mean background by 

21 to 90 p.p.m. (table 8). At 7° and 17°, the 

mean residues at 9- to 24-hour withdrawals 

were below the means of background in untreated controls (tables 5 and 7). At 12°, the 

mean residues beyond 3 hours of withdrawal 

fell within the range of background values.
Walker, Schoettger: Residues of MS-222 in Four Salmonids Following Anesthesia

TABLE 5. Background residues of aromatic amines in tissues of fish which were used as untreated controls In the study of MS-222

Species and tissue

Rainbow trout:

Number

fish

3

Mean

Length 

(inches)

7.60

size

Weight 

(grams)

45.67

Residue

Mean

1 44

0.40

s in p. p.m.

Range

0.8- 1.0

11.6-16.0

2.6-10.4

0.2- 0.6

0.2- 0.6

Standard

error

0.22

0.26

0.72

1.36

0.12

0.07

0.11

95-percent

interval

0.72- 2.16

9.60-18.00

4.34-10.90

0.00- 0.92

0.23- 0.83

0.00- 0.87

TABLE 6. Residues of MS-222 including background in muscle of rainbow trout following deep anesthesia at selected water temperatures

Temperature and 

withdrawal interval

7° C.:

12° C. :

17° C. :

Number 

of 

fish

5 

5 

5 

5

5 

5 

5 

5 

5 

5

5 

5 

5 

5

Mean size

Length 

(inches)

9.50 

9.26 

9.82 

9.78

8.14 

9.64 

8.64 

8.94 

8.32 

8.76

10.22 

9.62 

9.82 

10.02

Weight 

(grams)

127.2 

119.6 

135.8 

141.2

86.2 

129.4 

102.6 

116.0 

87.6 

95.8

167.4 

133.4 

143.8 

149.8

Residues in p. P.M.

Mean

41.92 

1.48 

1.00 

0.80

26.40 

9.72 

2.64 

1.04 

1.00 

1.44

18.04 

3.44 

1.52 

1.24

Range

21.4-72.0 

1.0- 2.2 

0.8- 1.2 

0.6- 1.0

10.2-50.8 

7.4-17.2 

1.6- 4.4 

0.8- 1.4 

0.8- 1.4 

1.0- 2.0

9.4-23.8 

2.8- 3.8 

0.8- 2.8 

1.0- 1.6

Standard 

error

7.13 

0.21 

0.06 

0.06

7.20 

1.92 

0.48 

0.10 

0.14 

0.20

2.58 

0.17 

0.34 

0.11

95-percent 

confidence 

interval

0.58- 2.38 

0.74- 1.26 

0.54- 1.06

0.46-17.98 

0.57- 4.71 

0.61- 1.47 

0.60- 1.40 

0.58- 2.30

2.71- 4.17 

0.06- 2.98 

0.77- 1.71

TABLE 7. Residues of MS-222 including background (grams) in livers of 

rainbow trout following deep anesthesia at selected time intervals and 

water temperatures

TABLE 8. Residues of MS-222 including background (grams) in kidneys of 

rainbow trout following deep anesthesia at selected time intervals and 

water temperatures

Temperature and 

withdrawal 

interval

7° C.:

12° C.:

17° C.:

Number 

of 

fish

2 

2 

2 

2

2 

2 

2 

2 

2

2 

2 

2 

2

Mean size

Length 

(inches)

10.00 

9.70 

9.95 

10.00

9.85 

10.00 

9.80 

9.50 

9.25

10.55 

10.15 

10.25 

10.40

weight 

(grams)

154.0 

131.5 

141.0 

146.5

141.5 

150.5 

150.0 

132.0 

118.0

187.0 

159.5 

166.5 

168.0

Residues in 

p.p.m.

Mean

59.6 

17.5 

20.0 

15.0

60.6 

19.5 

17.9 

11.1 

13.4

49.3 

20.5 

17.5 

13.7

Range

46.2-73.0 

15.8-18.2 

18.2-21.8 

14.8-15.2

57.2-64.0 

18.4-20.6 

16.0-19.8 

11.0-11.2 

12.0-14.8

31.0-67.6 

19.6-21.4 

17.0-18.0 

13.0-14.4

Standard 

error

13.39 

1.20 

1.80 

0.06

3.40 

1.10 

1.90 

0.06 

1.39

18.30 

0.90 

0.50 

0.70

Temperature and 

withdrawal 

interval

7° C.:

12° C.:

17° C.:

Number 

of 

fish

2 

2 

2 

2

2 

2 

2 

2 

2

2

1 

2 

2

Mean size

Length 

(inches)

10.00 

9.70 

9.95 

10.00

9.85 

10.00 

9.80 

9.50 

9.25

10.55 

10.30 

10.25 

10.40

weight 

(grams)

154.0 

131.5 

141.0 

146.5

141.5 

150.5 

150.0 

132.0 

118.0

187.0 

170.0 

166.5 

168.0

Residues in 

p.p.m.

Mean

84.2 

9.4 

6.6 

6.4

38.0 

15.5 

10.8 

8.9 

9.1

62.8

11.2 

8.0 

5.4

Range

71.4-97.0 

9.2- 9.6 

6.0- 7.2 

6.0- 6.8

28.2-47.8 

11.6-19.4 

7.8-13.8 

8.4- 9.4 

7.0-11.2

58.8-66.8

5.8-10.2 

5.2- 5.6

Standard 

error

12.79

0.20 

0.60 

0.40

9.80 

3.90 

3.00 

0.50 

2.10

4.00

2.20 

0.20
8 Investigations in Fish Control 15: Bureau of Sport Fisheries and Wildlife

The reliability of the modified BrattonMarshall colorimetric reaction for detection 

and measurement of MS-222 in liver and kidney is comromised by the high and variable 

concentrations of background amines. Therefore, residues in these tissues were measurable only at 0-hour withdrawal. The detection of residues in these organs is of little 

importance other than to indicate the ability 

of fish to deactivate or metabolize the anesthetic. The liver and kidney are rarely consumed by people, and they comprise only a 

small fraction of the total weight of a fish.

Brown trout. Groups of 6- to 9-inch 

brown trout were brought to medullary collapse by 80 p.p.m. of MS-2-22 at 17° within 

4.6 to 7.0 minutes. Because the species is 

less sensitive to the drug at 7° and 12°, 

100-p.p.m. solution was used to achieve 

medullary collapse within 9.5 to 12.1 minutes at 7° and within 7.9 to 11.0 minutes at 

120.

The residues of MS-222 in brown trout 

were measured only in muscle (table 9). The 

concentrations, including background, ranged 

from 9.8 to 60.8 p.p.m. at 0-hour withdrawal 

and declined 80 to 99 percent within 6 hours. 

They were 0.4 to 5.4 p.p.m. at 6 hours and 

0.6 to 1.4 p.p.m. at 24 hours. Also, they were 

least variable at 17° and persisted longer 

at 7°. After 24 hours, the mean residues of 

MS-222 were only a fraction of a part per 

million over background.

Brook trout. Brook trout 7 to 10 inches 

long were anesthetized to medullary collapse. 

The concentrations of drug were 100 p.p.m. 

at 17°, 110 p.p.m. at 12°, and 120 p.p.m. at 

7°. Anesthesia was induced within 4.2 to 5.4 

minutes at 17°, 6.5 to 8.7 minutes at 12°, and 

8.8 to 11.4 minutes at 7°.

The residues, including background, in 

muscle ranged from 6.0 to 34.8 p.p.m. at 0- 

hour withdrawal at the three temperatures 

(table 10). This range was lower than in any 

of the other trout, and it is noteworthy because the brook trout required higher concentrations of the drug for anesthesia. Within 9 to 24 hours after withdrawal, the residues 

including background were 0.6 to 2.8 p.p.m. 

At 24 hours, the mean residues of MS-222 

exceeded the backgrounds by 1.1 p.p.m. at 

17°, 0.3 p.p.m. at 12°, and 1.4 p.p.m. at 7°, 

but their 95-percent confidence intervals 

overlapped.

Lake trout. Lake trout 6 to 8 inches long 

were brought to medullary collapse by 100 

p.p.m. of MS-222 at 17°. 110 p.p.m. at 12°, 

and 135 p.p.m. at 7° within 3.9 to 6.5 minutes 

overall. The highest concentrations of residues, including background, were measured 

in muscle from fish at 7°. These fish had 

longer exposures to higher concentrations 

of drug than the others.

The mean values for residues of anesthetic 

at 6-, 9-, and 24-hour withdrawals at the

TABLE 9. Residues of MS-222 including background in muscle of brown trout following deep anesthesia at selected time intervals and water temperatures

interval

7° C.:

12° C. :

17° C. :

Number

fish

*$

3

3

3

Mean

Length (inches)

8.03

7

j»q

7 on

6

crt

size

Weight (grams)

83.3

88.0

Residues

Mean

27.53

5.07

4.67

1.13

0.60

12.93

in p.p.m.

Range

9.8-43.4

4.8- 5.4

4.4- 4.6

1.0- 1.4

20.8-60.8

4.6- 7.4

0.4- 0.8

0.7- 1.4

12.2-23.2

error

9.75

0.18

0.18

0.23

0.88

0.11

0.20

0.37

0.11

0.11

95-percent

confidence 

interval

4.30-5.84

3.90-5.44

0.14-2.12

1.88-9.46

0.90-2.44

0.13-1.37

0.14-1.89

0.33-1.27

1.33-2.27
Walker, Schoettger: Residues of MS-222 in Four Salmonids Following Anesthesia

TABLl 10. Residues of MS-222 including background in muscle of brook trout following deep anesthesia at selected time intervals and water temperatures

Temperature -and withdrawal 

interval

7 C. :

12° C. :

17° C. :

Number 

of 

fish

3 

3 

3 

3

3 

3 

3 

3 

3 

3

3 

3 

3 

3

Mean size

Length finches)

8.00 

8.83 

9.00 

8.43

9.23

8.77 

9.63 

8.20 

8.70 

8.60

8.93 

9.73 

8.33 

8.83

Weight (grams)

138.3 

123.0 

118.3 

94.7

118.0 

110.3 

148.3 

81.7 

106.7 

90.7

112.0 

143.0 

110.0 

102.0

Residues in p. p.m.

Mean

16.67 

3.53 

2.60 

1.93

28.07 

3.73 

1.37 

0.83 

1.03 

0.80

15.47 

3.33 

2.33 

1.67

Range

6.0-22.6 

3.2- 3.8 

2.4- 2.8 

1.8- 2.2

17.8-34.8 

3.2- 4.6 

0.7- 1.8 

0.8- 0.9 

0.9- 1.2 

0.6- 1.0

13.0-19.0 

3.0- 3.6 

2.2- 2.4 

1.2- 2.0

Standard 

error

5.34 

0.18 

0.11 

0.23

5.22 

0.44 

0.34 

0.03 

0.10 

0.11

1.86 

0.18 

0.07 

0.24

95-percent 

confidence 

interval

2.76-4.30 

2.13-3.07 

0.94-2.92

1.84-5.62 

0.00-2.83 

0.70-0.96 

0.60-1.46 

0.33-1.27

2.56-4.10 

2.03-2.63 

0.64-2.70

TABLE 11. Residues of MS-222 including background in muscle of lake trout following deep anesthesia at selected time intervals and water temperatures

Temperature and 

withdrawal interval

7° C.:

12° C. :

17° C. :

Number 

of 

fish

3 

3

3 

3

3 

3 

3 

3 

3 

3

3 

3 

3 

3

Mean size

Length 

( inches }

7.43 

7.17 

7.33 

6.73

7.13 

7.30 

6.67 

7.73 

7.18 

6.93

7.83 

7.57 

7.40 

7.46

Weight 

(grams)

46.3 

42.3 

43.3 

35.3

43.3 

40.7 

30.7 

51.7 

40.3 

45.0

53.3 

48.7 

41.7 

43.7

Residues in p. p.m.

Mean

32.00 

3.40 

4.53 

2.87

31.67 

6.80 

1.00 

0.93 

1.40 

0.87

14.87 

2.27 

1.80 

1.40

flange

20.0-55.6 

2.8- 4.4 

3.8- 5.6 

2.6- 3.2

22.8-37.4 

6.0- 8.2 

0.8- 1.2 

0.8- 1.0 

1.0- 1.8 

0.8- 1.0

14.0-16.6 

2.0- 2.6 

1.6- 2.0 

1.2- 1.6

Standard 

error

11.82 

0.50 

0.54 

0.18

4.50 

0.70 

0.11 

0.07 

0.23 

0.05

0.96

0.18 

0.11 

0.11

95-percent 

confidence 

interval

1.25-5.55 

2.21-6.85 

2.10-3.64

3.79-9.81 

0.53-1.47 

0.63-1.23 

0.41-2.39 

0.65-1.09

1.50-3.04 

1.33-3.27 

0.93-1.87

three temperatures were 0 to 4 p.p.m. above 

backgrounds, but the overlap of confidence 

intervals suggests that little, if any, residue 

remains beyond 9 hours (table 11). The residues dissipated more rapidly at 12°, and no 

significant amount persisted after 3 hours. 

The dissipation was intermediate and gradual 

at 17° and slowest at 7°.

DISCUSSION

Residues of MS-222 dissipated rapidly in 

rainbow trout, brown trout, brook trout, and 

lake trout within 1 to 6 hours after withdrawal 

from deep anesthesia (fig. 2). Regardless of 

species and temperature, there was very 

little difference in muscle between the residues of MS-222 and quantities of background 

amines at 9 to 24 hours. The higher concentrations of residues in all fish were measured 

at 0-hour withdrawal, and it was here that

differences were greater among individuals 

and species.

Rainbow trout and brown trout had higher 

initial residues than brook trout and lake 

trout despite the fact that the latter species 

were exposed to higher concentrations of 

MS-222. The chars, however, had the briefer 

exposures.

The chars may have oral and gill membranes which are less permeable than those 

of trouts to MS-222. The lake trout appear 

to be less able than brook trout to metabolize 

or detoxify the drug because they showed less 

tolerance and higher residues. Also, the 

anesthetic is more toxic to lake trout than 

brook trout (Marking, 1966).

The mineral content of water affected the 

mean time to anesthetize fish and the amount 

of residue in them. The 0-hour residues in
10 Investigations in Fish Control 15: Bureau of Sport Fisheries and Wildlife

fish exposed in soft water (10 p.p.m. total 

hardness) were triple those in fish in hard 

water (180 p.p.m. total hardness). The fish 

in soft water, however, were exposed twice 

as long as in harder water. At the end of 24 

hours, there were no significant differences 

in residues.

Acetylated residues in fish were not unexpected when one considers the similarity of 

MS-222 to p-aminobenzoic acid and the sulfonamides. These aromatic amines were 

probably acetylated in the liver or kidney 

and excreted (Hawk et al., 1954; Jessop, 1961). 

We do not know whether the conjugation of 

MS-222 with acetic acid in fish is a significant mechanism of detoxification or deactivation.

In general, the residues of MS-2 22 in fish 

at 7° and 12° were greater at 0-hour withdrawal than at 17° (figure 2). They persisted 

longer at 7°, but the concentrations of anesthetic solution and the durations of anesthesia 

also were greater at this temperature. The 

shorter exposures to lower concentrations of 

drug at 17° resulted in less but more consistent residues in all species at 0-hour withdrawal.

CONCLUSIONS

Residues of MS-222 occurred in the blood, 

muscle, liver, and kidney of deeply anesthetized rainbow trout. They dissipated 

rapidly within 1 to 6 hours, but those in liver 

and kidney were masked by background substances which interfered with the modified 

Bratton-Marshall colorimetric method.

The residues of MS-2 22 in muscle varied 

greatly at 0-hour withdrawal among anesthetized rainbow trout, brown trout, brook 

trout, and lake trout. They were higher in the 

trouts than in the chars. Regardless of species and temperature, there was very little 

difference between the residues of MS-222 and 

background amines in muscle at 9 to 24 hours 

after withdrawal. The residues of anesthetic 

in muscle of fish at 7° and 12° C. were greater

at 0-hour withdrawal than at 17°. Residues in 

muscle persisted longer at 7° than at warmer 

temperatures.

The 0-hour residues of drug in muscle of 

fish exposed in soft water were triple those of 

fish in hard water. At the end of 24 hours, 

however, there were no significant differences 

in residues.

SUMMARY

The residues of MS-222 in selected tissues 

of rainbow trout, brown trout, brook trout, 

and lake trout at 7°, 12°, and 17° C. and in 

waters of various hardnesses were measured 

by means of the modified Bratton-Marshall 

colorimetric method. The concentrations of 

drug in the blood, muscle, liver, and kidney 

of deeply anesthetized rainbow trout dissipated rapidly within 1 to 6 hours. Those in 

the liver and kidney were masked by background substances which interfered with the 

colorimetric method.

The amounts of MS-222 including background in muscle of the four species at 0-hour 

withdrawal were 6 to 72 p.p.m. The mean concentrations at 7°, 12°, and 17° C. were 18 to 

42 p.p.m. in rainbow trout, 13 to 44 p.p.m. in 

brown trout, 15 to 28 p.p.m. in brook trout, 

and 15 to 32 p.p.m. in lake trout. At 24 hours 

after withdrawal from the anesthetic, the 

mean concentrations including background 

were 0.8 to 1.2 p.p.m. in rainbows, 0.8 to 1.4 

in browns, 0.8 to 1.9 in brook trout, and 0.9 

to 2.9 p.p.m. in lake trout. Actually, there 

was little difference between the residues of 

drug and background amines at 9 to 24 hours 

after withdrawal.

At 7° and 12° the concentrations of residue 

including background were generally above 20 

p.p.m. at 0-hour withdrawal, whereas at 17° 

they were below 20 p.p.m. The residues 

persisted longer at 7° than at the warmer 

temperatures. At 9 to 24 hours the concentrations of drug at the three temperatures 

approached those of background amines in 

controls.
Walker, Schoettger: Residues of MS-222 in Four Salmonids Following Anesthesia 11

REFERENCES

Davis, H. S.

1956. Culture and diseases of game fishes. University of California Press, Berkeley and Los 

Angeles. 332 p.

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

Summerson.

1954. Practical physiological chemistry. 13th ed. 

McGraw-Hill Book Co., New York. 1439 p.

Jessop, William John Edward.

1961. Fearon's introduction to biochemistry. 4th ed. 

Academic Press, New York. 473 p.

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

1964. Investigations in Fish Control: 1. Laboratories 

and methods for screening fish-control chemicals. 

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

Marking, Leif L.

1966. Investigations in Fish Control. 12. The toxicity of MS-222 to selected fishes: U.S. Bureau of 

Sport Fisheries and Wildlife, Resource Publication 18.

Schoettger, Richard A.

1966. Investigations in Fish Control: 16. Annotated 

bibliography on MS-222. U.S. Bureau of Sport 

Fisheries and Wildlife, Resource Publication 22.

Schoettger, Richard A., and Arnold M. Julin.

1966. Investigations in Fish Control: 13. Efficacy of 

MS-222 as an anesthetic on four salmonids. U.S. 

Bureau of Sport Fisheries and Wildlife, Resource 

Publication 19.

Walker, Charles R., and Richard A. Schoettger. 

1966. Investigations in Fish Control: 14. Method 

for determining MS-222 residues in fish. U.S. 

Bureau of Sport Fisheries and Wildlife, Resource 

Publication 20.
INVESTIGATIONS IN FISH CONTROL

16. Annotated Bibliography on MS-222

By Richard A. Schoettger

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

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

Fish and Wildlife Service, Clarence F. Pautzke, Commissioner 

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

Resource Publication 22 January 1967 Washington, D.C.
ANNOTATED BIBLIOGRAPHY ON MS-222

By Richard A. Schoettger, Fishery Biologist

Bureau of Sport Fisheries and Wildlife

La Crosse, Wisconsin

Abstract. This bibliography contains 86 selected references on uses 

of MS-222 on cold-blooded animals including fish and amphibians. Most 

of the references are annotated.

The Fish Control Laboratories at La 

Crosse, Wis., and Warm Springs, Ga., initiated studies in 1964 on the toxicity, efficacy, 

and residues of MS-222 in fish. The data were 

required by the U.S. Food and Drug Administration in order to clear the drug for continued use. In connection with the studies, a 

substantial bibliography on uses of MS-222 

was compiled. A considerable number of the 

more useful references were annotated.

Some recognized references on MS-222 

were not available conveniently for review 

and they have been included by title only.

BIBLIOGRAPHY

Allison, Leonard N.

1961. The effect of tricaine methanesulfonate (M.S. 222) on motility of brook 

trout sperm. Progressive Fish-Culturist, vol. 23, no. 1, p. 46-48. 

The spermatozoa of brook trout remained 

motile less than 10 seconds in a concentration of 18.9 p.p.m. of MS-222. Allison recommended that the drug should not contact reproductive products during spawn-taking.

Bailey, Merryll M.

1965. Lake trout fin-clipping rates at two 

national fish hatcherie'fe. Progressive 

Fish-Culturist, vol. 27, no. 3, p. 169- 

170.

MS-222 was used as an anesthetic during 

the fin-clipping of more than 3.5 million lake

trout. The fish ranged from 4.1 to 7.9 inches 

long and were young-of-the-year to 3-yearolds. One fin each was removed from 2 million fish, and 2 fins each were clipped on 

1.5 million. Postmarking mortality usually 

was 0.1 to 0.2 percent; it increased with 

temperatures over 65° F. Cooling the solutions and reducing the concentration of 

MS-222 lowered mortality.

Ball, J. N., and P. N. Cowen.

1959. Urethane as a carcinogen and as an 

anaesthetic for fishes. Nature, vol. 184, 

p. 370.

Urethane is carcinogenic in the lung and 

other tissues of mice and rats. It does not 

produce tumors in rabbits, chickens, and 

guinea-pigs, but carcinogencity may vary 

with species (and strain). It has a leucopenic 

effect on humans and can be absorbed from 

the skin.

MS-222 has not been reported to be carcinogenic. Its effectiveness on fish and 

amphibians varies with species, size of the 

animal, and temperature. Suitable concentrations are determined empirically for every 

species and situation.

Baudin, Louis.

1932a. Action de la tricaine sur la consommation d'oxygene de Carassius 

auratus. Comptes rendus des Seances 

de la Societe* de biologie, vol. 109, 

p. 731-733.

A 1:1,000 solution of MS-222 anesthetized 

goldfish within 1 to 2 minutes. Observations

3
Investigations in Fish Control 16: Bureau of Sport Fisheries and Wildlife

on the circulation of blood in the fins revealed a slower than normal rate with 

prolonged exposure to the anesthetic. A concentration of 1:20,000 produced a level of 

anesthesia in 30 to 60 minutes at 16.5° C. 

which could be maintained for 24 hours without apparent damage. The rate of oxygen 

consumption was depressed to 50 percent of 

normal after 1 hour and to 25 percent after 

11 hours of exposure. The respiratory rate 

returned to normal within 2 hours after the 

fish were placed in fresh water. Above 16° 

C., narcosis was incomplete and the reduction of oxygen consumption was less. Below 

this temperature, a longer period was required for anesthesia and, for a fixed period, 

the rate of oxygen consumption was higher 

than at 16.5° C.

1932b. Action de la tricaine sur le quotient resperatoire de Carassius auratus. 

Ibid., p. 1081-1083.

1932c. Perte de la sensibilite a la depression chez les poissons anesthesies a la 

tricaine. Ibid., vol. 110, p. 151-153. 

Oxygen consumption by goldfish was increased at lower atmospheric pressures. The 

rates for fish which were anesthetized with 

1:20,000 of MS-222 were relatively low under 

conditions of both normal and reduced pressure. The sensitivity of the fish to reduced 

pressure was not completely suppressed by 

concentrations of 1:30,000 and 1:40,000.

1932d. Respiration du poisson (Carassius 

auratus) anesthesie a la tricaine et 

soumes a une elevation brusque de 

temperature. Ibid., p. 235-237.

1934. Action de la tricaine sur le sang 

des poissons. Ibid., vol. 115, p. 510-512. 

A 1-percent solution of MS-222 anesthetized several species of fish, including 

goldfish and perch, within 1 minute. There 

were no effects on the erythrocytes, erythrocyte count, or oxygen capacity of the blood. 

The oxygen saturation of the blood was near 

zero, and the carbon dioxide level was 

slightly in excess. Lower concentrations of 

the drug and longer exposures induced exaggerated respiratory movement which increased the oxygen saturation and lowered

the carbon dioxide level. The author concluded 

that MS-222 may excite respiratory centers 

of the brain, or interfere with exchanges of 

gases, thereby causing anoxia and hyperventilation. With complete narcosis, the blood 

showed signs of asphyxia and there was a 

slowing of the circulation which contributed 

to reductions in erythocyte numbers and oxygen capacity of the blood.

Bell, Gordon R.

1964. A guide to the properties, characteristics, and uses of some general 

anaesthetics for fish. Fisheries Research Board of Canada, Bulletin 148,

4 P.

Properties of eleven chemicals used as 

fish anesthetics are summarized. The chemicals are carbon dioxide, chloral hydrate, 

chloretone, ether, methyl pentynol, MS-222, 

phenoxyethanol, quinaldine, sodium amytal, 

tribromoethanol, and tertiary amyl alcohol. 

The common and chemical names of the chemicals, manufacturers, costs, solubilities, 

stabilities, hazards, toxicities, emergency 

treatments, approximate dosages, precautions, 

behavioral effects, uses, and modes of action 

are included. Concentrations of MS-222 from 

1:9,000 to 1:4,500 were recommended for 

brief exposures. The generally useful range 

is between 1:25,000 to 1:12,500.

Black, Edgar C., and Anne R. Connor. 

1964. Effects of MS 222 on glycogen and 

lactate levels in rainbow trout (Salmo 

gairdneri). Journal of the Fisheries 

Research Board of Canada, vol. 21, no. 6, 

p. 1539-1542.

A concentration of 0.5 g. of MS-222 per 

gal. anesthetized trout within 60 seconds. The 

fish showed signs of initial stimulation. The 

dosage might have been fatal with prolonged 

exposure. The hemoglobin, blood and muscle 

lactate, and muscle glycogen levels were 

similar in anesthetized and control fish.

Blahm, T. H.

1961. Effect of tricaine methanesulfonate 

on oxygen consumption of juvenile sockeye salmon. Transactions of the American Fisheries Society, vol. 90, no. 2, 

p. 226-227.

MS-222 uniformly reduced the oxygen 

consumption of both large (160-180 mm. fork
Richard A. Schoettger: Annotated Bibliography on MS-222

length) and small (60-80 mm. fork length) 

sockeye salmon. The fish were anesthetized 

by 1:20,000 in a constant flow respirometer.

Hove1 , Frank J.

1962. MS-222 Sandoz the anaesthetic of 

choice for fish and other cold-blooded 

organisms. Sandoz News, no. 3, 12 p.

The author summarizes the history of 

MS-222 from its use as a local anesthetic in 

human medicine to its current status as a 

general anesthetic for fish, amphibians, and 

other cold-blooded forms. His personal communications with various investigators reveal that concentrations ranging from 1:3,785 

to 1:17,500 have been used in measuring and 

weighing, marking, and spawning of various 

salmonids. A concentration of 1:12,000 

seemed to be most popular. Solutions containing approximately 1:3,000 of MS-222 were 

effective on tropical fish, goldfish, bluegills, 

largemouth bass, and bullheads.

Effective dosages for amphibians ranged 

from U250 to 1:20,000, but 1:1,000 to 1:3,000 

were preferred. Specimens with gills and 

those undergoing metamorphosis were more 

sensitive to the anesthetic.

Butler, Robert L.

1957. The development of a vinyl plastic 

subcutaneous tag for trout. California 

Fish and Game, vol. 43, no. 3, p. 201- 

212.

MS-222 at 0.5 g./gal. prepared fish for 

the operation within 30 seconds at 50° F. 

The fish were exposed no longer than 3 minutes. As temperature increases, it is necessary to reduce the exposure time or the concentration. The drug had no effect on feeding 

by the fish after they recovered from anesthetization.

Campbell, G. D., and D. H. Davies.

1963. Effect of ethyl m-aminobenzoate 

(MS-222) on the elasmobranch electrocardiograph. Nature, vol. 198, no. 4877, 

p. 302.

MS-222 caused coincidental decreases in 

the pulse and respiratory rates of stingrays 

indicating a neurological relationship between 

the cardiac and respiratory centers in the 

brain. The drug did not affect the ECG complex.

Christensen, K.

1931. Effect of castration on the secondary 

sex characters of males and females of 

Rana pipiens. Anatomical Record, vol. 

48, p. 241.

Collins, James L., and Andrew H. Hulsey. 

1963. Hauling mortality of threadfin shad 

reduced with M.S. 222 and salt. Progressive Fish-Culturist, vol. 25, no. 2, 

p. 105-106.

A combination of 0.5-percent salt and 

MS-222 equivalent to 1 g./12 gal. facilitated 

hauling of more than 200,000 fish with a survival of 95 percent.

Copenhaver, W. M.

1939. Initiation of beat and intrinsic contraction rates in the different parts of 

the Amblystoma heart. Experimental 

Zoology, vol. 80, p. 139.

Crawford, Bruce, and Andrew Hulsey.

1963. Effects of M.S. 222 on the spawning 

of channel catfish. Progressive FishCulturist, vol. 25, no. 4, p. 214. 

The anesthetization of adult channel catfish with MS-222 at a rate of 4 g./8 gal. did 

not affect the success of spawning nor the 

viability of fry. The fish were narcotized for 

sexing and placement in spawning pens.

Dollar, Alexander M.

1963. Air transportation of living rainbow

trout. Progressive Fish-Culturist, vol.

25, no. 3, p. 167-168. 

The fish were placed in lake water containing MS-222 at a concentration of 1:100,000, 

The water was cooled gradually over a 3-hour 

period at 33° F. The fish and water were 

transferred to a plastic bag and packed in 

ice. Oxygen was added to the bags. The fish 

survived in the sealed containers for 24 

hours.

Eisler, Ronald, and Tadeusz Backiel. 

1960. Narcotization of chinook salmon 

finger lings with tricaine methanesulfonate (M.S. 222). Transactions of the 

American Fisheries Society, vol. 89, 

no. 2, p. 164-167.

A concentration of 1:33,000 effectively 

anesthetized this species within 5 minutes.
Investigations in Fish Control 16: Bureau of Sport Fisheries and Wildlife

The rate of narcotization was dependent on 

concentration. Recovery time increased with 

exposures up to an hour, but declined with 

longer exposure. The drug was equally active 

in fresh and salt water. The authors reviewed 

concentrations of MS-222 which have been 

used to anesthetize various species of fish.

Ellis, Robert J.

1964. The effect of confinement on blood 

lactate levels in chinook and coho salmon. 

Research Briefs, Fish Commission of 

Oregon, vol. 10, no. 1, p. 28-34 

The lactic acid levels in the blood of trollcaught salmon declined more rapidly in those 

individuals which were held in solutions containing MS-222 at a concentration of 1:150,000.

Ewing, Ann.

1965. Current U.S. patents. A method for 

marking fish by which scales are transplanted painlessly from one part of the 

body to another has been patented. 

Science News Letter, vol. 87, no. 15, 

p. 228.

Dr. Louis Levy and Miss Carol A. De 

Fusco (1965) have patented a method of marking fish by replacing scales of one color from 

one part of the body with those of contrasting 

color from a different area. The fish are 

anesthetized during the operation with 50 to 

100 p.p.m. of MS-222.

The technique was successful on goldfish, 

carp, blue acara, blackspot barb, and guppies, and has been used to measure the 

effects of drugs on the rejection time of 

scales from different fish.

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

1950. Effect of tricaine methanesulfonate

on the determination of sulfonamides.

Science, vol. 112, no. 2902, p. 181-182. 

Concentrations of 2 to 4 mg.%, as sulfamerazine, were detected in the tissues of 

trout which should not have contained the 

sulfa drug. The fish had been anesthetized 

for 1 minute in a 1:5,000 solution of MS-222. 

This anesthetic, or others with similar molecular structures, should not be used whenever they may interfere with the colorimetric test for sulfonamides.

Fromm, Paul O.

1958. A method for measuring the oxygen 

consumption of fish. Progressive FishCulturist, vol. 20, no. 3, p. 137-139. 

MS-222 is used to immobilize fish for 

length and weight measurements before they 

are placed in a respirometer. A concentration of 0.03 percent produces anesthesia 

within 30 to 45 seconds. The drug apparently 

has no lasting effect on the general metabolic 

rate.

Gebhards, Stacy V.

1965. Transport of juvenile trout in sealed 

containers. Progressive Fish-Culturist, 

vol. 27, no. 1, p. 31-36. 

The use of sedating levels of MS-222 did 

not increase the loading density or survival of 

rainbow trout in sealed containers. Greater 

survival was associated with the starvation 

period prior to loading.

Gilbert, P. W., and F. G. Wood.

1957. Methods of anaesthetizing large

sharks and rays safely and rapidly.

Science, vol. 126, p. 212. 

A 1:1,000 solution of MS-222 is sprayed 

into mouth, spiracles, or gill exits by 

means of a hand sprayer, such as a water 

pistol. Sharks as large as 400 pounds are 

anesthetized in a minute or less and may be 

handled, out of water, for 5 to 30 minutes. 

The volume of anesthetic solution required to 

anesthetize fish varies with the size of the 

individuals.

Glucksohn, Salome.

1932. Aussere Entwicklung der Extremitaten und Stadieneinteilung der Larvenperiode von Triton taeniatus Leyd. und 

Triton cristatus Laur. W. Roux* Archiv 

fur Entwicklungsmechanik der Organismen, vol. 125, p. 341-405. 

Metamorphosing salamanders were anesthetized with 1:3,000 of MS-222 for 30 minutes 

a day. The growth of treated specimens was 

somewhat less than controls, but the former 

were proportioned normally.

Goodrich, H. B., and R. Nichols.

1931. The development and regeneration 

of color pattern in Brachydanio rerio. 

Anatomical Record, vol. 51, p. 513.
Richard A. Schoettger: Annotated Bibliography on MS-222

Gossington, Robert.

1957. An aid to fish handling tricaine.

Aquarium Journal, vol. 28, no. 9, p. 318-

321.

Fish could be anesthetized safely with 

MS-222 in concentrations of 0.24 to 0.32 

g./gal. Larger specimens required somewhat higher concentrations. Live-bearers 

were generally more resistant than egglayers. The drug reduced the chances of 

injury to fish from thrashing during shipment. Under anesthesia, Siamese fighting 

fish could be shipped together in a single 

container.

Hadian, Z., and M. S. Dunn.

1938. Localisation in the occulomotor 

nuclei of the goldfish. Journal of Comparative Neurology, vol. 68, p. 191.

Hublou, Wallace F.

1957. A method of using an anesthetic in 

marking fins. Progressive Fish-Culturist, vol. 19, no. 1, p. 40-43. 

MS-222 was used in a recirculating system 

to fin-clip fingerling salmon and steelhead 

trout. The marking rate was improved by 

49.5 percent with the use of an anesthetic. 

Advantages of the system included better 

marks, lower rate of injury, reduction of 

worker fatigue, and saving in time and 

money.

Johnson, Harlan E., and J. M. Shelton.

1958. Marking chinook salmon fry.

Progressive Fish-Culturist, vol. 20,

no. 4, p. 183-185.

Groups of 10 to 20 individuals were caught 

in a small net and placed in a 1:7,500 solution of MS-222. Fin-clipping began when all 

the fish were on their sides and after the net 

had been placed in fresh water. The last fish 

was marked just before it recovered. Approximately 487,000 fry were marked at a 

cost of $50.

Karczmar, Alexander G., and Theodore 

Koppanyi.

1948. Action of central nervous system 

depressants at different growth periods 

of salamander (Amblystoma punctatum) 

larvae. Federation Proceedings, vol. 7, 

p. 231-232.

Larvae of different ages were immersed 

in a 1:7,500 solution of MS-222, and the times 

for anesthesia were recorded. Paraldehyde, 

ethyl alcohol, sodium barbital, nembutal, 

chloral hydrate, and chloretone were also 

tested. Anesthesia with MS-222 was more 

rapid in older and larger individuals.

Klontz, George W.

1964. Anesthesia of fishes. From: Proceedings of the Symposium on Experimental Animal Anesthesiology, Brooks 

Air Force Base, December 14-16, 13 p. 

The efficacy and characteristics of 14 

methods which are used to anesthetize fish 

were discussed.

Concentrations of 25 to 35 p.p.m. of MS222 are recommended for transporting fish; 

50 to 100 p.p.m. are used to induce deep 

anesthesia. In general, induction time requires 1 to 3 minutes of exposure and the 

fish recover, in fresh water, within 3 to 15 

minutes. Fish which are repeatedly exposed 

to MS-222 showed a slight increase in tolerance which is corrected by raising tht concentration slightly. The drug appears to be 

toxic to those fish which are treated in salt 

water and in direct sunlight.

Knight, Alexis E.

1964. Intracellular hemoglobin crystallization in two centrarchids, the^ largemouth bass and the bluegill. Progressive Fish-Culturist, vol. 26, no. 3, 

p. 115-117.

A 1:5,000 solution of MS-222 was used to 

narcotize fish during the collection of blood 

samples.

Koppanyi, Theodore, and Alexander G. 

Karczmar.

1948. Comparison of anesthetic action of 

acetanilid, tricaine (MS-222) and aliphatic depressants. Federation Proceedings, vol. 17, p. 234. 

The action of acetanilid on salamander 

larvae was independent of larval stage. Subanesthetic dosages of the drug acted additively with subanesthetic levels of MS-222, 

nembutal, and chloretone. The anesthetic 

effects of chloretone, MS-222, alcohol, 

paraldehyde, and acetanilid were reversed 

rapidly.
8 Investigations in Fish Control 16: Bureau of Sport Fisheries and Wildlife

Lemarque, Pierre.

1964. Anesthesie et transport. Bull. Inf.

Cons. Sup. P£che, vol. 55, p. 5-9. 

MS-222 may be used in concentrations of 

1:10,000 to 1:50,000 to anesthetize fish before they are placed in plastic bags at a loading level of 1 kilogram of fish per 1 to 2 

liters of water. The bags were filled with 

oxygen. Dosages of 1:100,000 were recommended for the tranquilization of fish in 

transportation tanks.

Larsen, Howard N.

1964. Comparison of various methods of 

hemoglobin determination of catfish 

blood. Progressive Fish-Culturist, 

vol. 26, no. 1, p. 11-15. 

The fish were anesthetized in a 1:5,000 

solution of MS-222 to facilitate the collection 

of blood samples.

Levy, Louis Encino, and Carol A. DeFusco.

1965. Identification of scaly teleosts. 

U.S. Patent Office, Patent No. 3,174,458. 

3 p.

Lumb, William V.

1963. Small animal anesthesia. Chapter: 

Anesthesia of laboratory and zoo animals, p. 269-310. Lea and Febiger, 

Philadelphia. 420 p.

MS-222 is used to immobilize fish and 

other cold-blooded animals by completely 

bathing small subjects, by gill spraying in 

large fish, or by injection in large animals. 

Concentrations of 0.5 to 1.0 grams of drug 

per gallon are used for most teleosts and 

the temperature is maintained at 40° to 60° 

F. Repeated use of anesthetic solutions reduces their efficacy. Longer anesthesia or 

sedation can be maintained with lower concentrations. The drug can also be used in 

treatment of fungus infections and other 

localized diseases on pet or ornamental 

fish.

The uses of ether, sodium amytal, carbon dioxide, urethane, and cresol in anesthetizing fish are discussed.

Marking, Leif L.

1966. Investigations in Fish Control. 12. 

Toxicity of MS-222 to selected fishes. 

U.S. Bureau of Sport Fisheries and 

Wildlife, Resource Publication 18.

The 24-hour LC 59 concentrations of MS222 for various species of fish, at 12° C., 

were found to be: rainbow trout, 39.0 to 52.0 

p.p.m.; brown trout, 38.5 to 45.6 p.p.m.; 

brook trout, 50.7 to 52.2 p.p.m.; lake trout, 

33.8 to 39.8 p.p.m.; northern pike, 56.0 p.p.m.; 

bluegill, 45.7 to 46.9 p.p.m.; largemouth bass, 

42.0 to 61.5 p.p.m.; and walleye, 49.0 p.p.m. 

The results indicated that exposures of MS222 for 24 to 96 hours had no significant effect on the toxicity of the chemical.

In general, MS-222 was more toxic to 

smaller individuals, and at higher temperatures. Water hardness had little effect on 

toxicity.

The safety index of MS-222 for rainbow 

trout was determined by comparisons of the 

LCso and EC 50 concentrations after 15, 30, 

and 60 minutes of exposure in relatively soft 

and hard water. The indexes ranged from 1.7 

to 2.0 and decreased slightly with exposure 

time. A comparison of the LCi and EC 99 

for hard water gave an index of about 1.3 and 

1,0 to 1.1 in soft water.

Maintenance of anesthesia in fish for 96 

hours was not harmful. After the fish had 

been placed in fresh water and recovered, 

they fed as well as controls.

Martin, N. V., and D. C. Scott.

1959. Use of tricaine methanesulfonate 

(M.S. 222) in the transport ctf live fish 

without water. Progressive Fish-Culturist, vol. 21, no. 4, p. 183-184. 

Hybrid trout were anesthetized in 60 

p.p.m. of MS-222 and packed in layers of 

chipped ice and sphagnum moss. The fish 

were maintained under these conditions for 

4 to 4.5 hours with little mortality.

McFarland, William N.

1959. A study of the effects of anesthetics

on the behavior and physiology of fishes.

Publications of the Institute of Marine

Science, University of Texas, vol. 6,

p. 23-55.

The anesthetic effects of 21 chemicals, 

including MS-222, were tested against 

Fundulus parvipinnis, Gambusia affinis, 

Paralabrax clathratus, and Girella nigricans. 

The behavioral changes induced in fish by 

anesthetics were classified into the following 

levels of anesthesia: sedation, loss of
Richard A. Schoettger: Annotated Bibliography on MS-222

equilibrium, loss of reflex reactivity and 

medullary collapse. Anesthesia in fish was 

compared to that in humans and was found to 

be a similar process involving sequential 

suppression of higher to lower central nervous centers.

The narcotic potencies of the various 

compounds increased with their molecular 

weights. MS-222 was rated as highly potent. 

The ratio of the dosages necessary to induce 

sedation and medullary collapse during a 

12-hour period was 7.1.

Anesthesia with MS-222 was more rapid 

at 27° than at 12° C., but anesthesia did not 

progress as deeply at the lower temperature. 

Metabolic studies indicated that MS-222 was 

depleted, with time, at a greater rate than 

other anesthetics.

McFarland, William N.

1960. The use of anesthetics for the handling and the transport of fishes. California Fish and Game, vol. 46, no. 4, 

p. 407-431.

MS-222, tertiary amyl alcohol, and 

methylparafynol were suggested as beneficial for the induction of deep anesthesia 

because they act quickly and recovery is 

rapid. Recovery from anesthesia was complete provided respiratory movements had 

not ceased for more than a few minutes. 

MS-222 at 0.03 g./gal. induced loss of reflex in Fundulus parvipinnis within 1 hour. 

Higher concentrations were recommended 

for more rapid anesthesia; however, fish 

must be removed from the anesthetic after 

the desired stage of anesthesia has been induced.

MS-222 was not recommended for transporting fish. The drug failed to maintain a 

lowered rate of metabolism at higher temperatures.

It is advisable to pretreat fish in an anesthetic before transporting to reduce metabolic 

rates which may be stimulated due to handling.

McGovern, Beulah H., and Roberts Rugh. 

1944. Efficacy of m-amino ethyl benzoate 

as an anesthetic for amphibian embryos. 

Proceedings of the Society for Experimental Biology and Medicine, vol. 57, 

p. 127-130.

Dosages of 1:3,000 did not affect the 

motility or fertility of frog spermatozoa. The 

eggs which were fertilized in this solution 

developed normally when the exposure did 

not exceed 1 hour. Longer exposures produced decreasing numbers of abnormal embryos as development progressed through 

gastrulation and neurulation. The older embryos withstood anesthesia for 24 hours; however, mortality of the embryos undergoing 

transition from external to internal gill 

respiration increased with immersions in 

MS-222 longer than 2 hours. The drug inhibited muscular action, but not ciliary 

activity. MS-222 was considered to be nontoxic to frog embryos within the exposure 

times adequate for surgical operations.

Meehan, William R., and L. Revet.

1962. The effect of tricaine methanesulfonate (M.S. 222) and/or chilled water 

on oxygen consumption of sockeye salmon fry. Progressive Fish-Culturist, 

vol. 24, no. 4, p. 185-187. 

The most favorable conditions for survival of fish during transportation appeared 

to be uncrowded numbers of fish in water 

colder than that from which they were removed. The fish also survived well when 

uncrowded in their normal environmental 

water to which was added 0.1 g. of MS-222 

per 4,000 ml. Unsatisfactory results were 

obtained with crowding, or when the fish were 

placed in solutions of MS-222 which were 

colder than their environmental water.

Meister, Alfred L., and Charles F. Ritzi. 

1958. Effect of chloretone and MS-222 on 

eastern brook trout. Progressive FishCulturist, vol. 20, no. 3, p. 104-110. 

MS-222 was considered superior to chloretone as an anesthetic for fishery use. The 

former had a wider range of practical field 

concentrations, lesser inhibitory effect on 

respiration, and was easier and more predictable for use in the field. Both drugs produced more rapid anesthetization when temperatures were increased. Anesthesia with 

MS-222 was induced within approximately 10 

minutes by concentrations ranging from 

1:5,000 to 1:15,000 at 37° to 39° F., and 

1:5,000 to 1:25,000 at 48° to 51° F. A concentration of 1:1,000 produced respiratory
10 Investigations in Fish Control 16: Bureau of Sport Fisheries and Wildlife

arrest in brook trout after 5 minutes, but 

continued exposure for 6 minutes was not 

fatal.

They observed that 22 to 35 pounds of 

brook trout, 36 to 86 pounds of salmon or 93 

pounds of lake trout could be anesthetized 

per gram of MS-222.

Moss, D. D., and D. C. Scott.

1964. Respiratory metabolism of fat and 

lean channel catfish. Progressive FishCulturist, vol. 26, no. 1, p. 16-20. 

MS-222 was used at the rate of 1 g./3.8 1. 

to anesthetize channel catfish for measurements of lengths and weights. The fish were 

placed in a respirometer and recovered from 

the anesthetic within 1 to 2 minutes. The oxygen consumption of fat fish was greater than 

that of thin fish at 25° C. At 30° C. the respiratory rates for both groups were similar.

Nelson, P. R.

1953. Use of three anesthetics on juvenile 

salmon and trout. Progressive FishCulturist, vol. 15, no. 2, p. 74. 

MS-222, chlorobutanol, and urethane were 

used as aids in weighing and measuring coho 

salmon, red salmon, and Dolly Varden trout. 

MS-222 was used at the rate of 1:12,500 at 

12° to 17° C. and effectively anesthetized the 

fish within several minutes. The concentration was increased slightly for fingerlings. 

A dosage of 1:10,000 caused 100 percent 

mortality.

Normandeau, Donald A.

1962. Microhematocrit values for some

salmonids reared in New Hampshire.

Progressive Fish-Culturist, vol. 24,

no. 4, p. 172-176.

A solution containing 1:10,000 of MS-222 

was used to anesthetize landlocked salmon, 

rainbow, brook, lake, and splake trout before 

collection of microhematocrits. The fish 

were anesthetized sufficiently within 1 minute. There was a significant relation of 

mean hematocrits to sampling date, but not 

to water temperature.

Parkhurst, Z. E., and M. A. Smith.

1957. Various drugs as aids in spawning 

rainbow trout. Progressive Fish-Culturist, vol. 19, no. 1, p. 39.

MS-222, sodium amytal, methyl pentynol, 

urethane, and chloretone were used to anesthetize rainbow trout.

A concentration of 264 p.p.m. of MS-222 

induced complete anesthesia within 30 to 45 

seconds. Longer exposures resulted in some 

mortality. The fish were in good condition 

75 days after treatment. The hatching success of eggs from anesthetized and control 

fish was similar.

Methyl pentynol at 2,400 p.p.m. was effective within 3.5 minutes, a 0.5-percent solution of urethane in 2 minutes, and 400 p.p.m. 

of choretone in 1.0 to 1.5 minutes. Sodium 

amytal, because of its slow action, had no 

practical value.

The experiments were conducted at a 

temperature of 43° F.

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

Donald R. Brockway, and Ray R. Vaughn. 

1957. The nutrition of trout. Cortland 

Hatchery Report 26, New York Conservation Department, Fisheries Research 

Bulletin 21. 93 p.

The absorption of radioactive cobalt was 

elevated in brook trout which were narcotized 

with MS-222. It was suggested that there may 

be an adjustment of the osmotic processes of 

narcotized fish.

Pickford, Grace E.

1953. A study of the hypophysectomized 

male killifish, Fundulus heteroclitus 

(Linn.) Bulletin of the Bingham Oceanographic College, vol. 14, no. 2, p. 5-41.

1957. Methods of hypophysectomy in 

fishes. Appendix to: The physiology of 

the pituitary gland of fishes, by Grace E. 

Pickford and James W. Atz, p. 485-487. 

New York Zoological Society, New York. 

MS-222 is recommended over several 

other techniques and agents for anesthetizing 

fish during hypophysectomy. The drug gave 

excellent results with Fundulus- heteroclitus, 

but suitable strengths must be determined 

for each species.

Piper, Robert G., and Robert F. Stephens. 

1962. A comparative study of the blood of 

wild and hatchery reared lake trout.
Richard A. Schoettger: Annotated Bibliography on MS-222 11

Progressive Fish-Culturist, vol. 24,

no. 2, p. 81-84.

Blood samples were collected by heart 

puncture after anesthetizing the trout in a 

1:1,000 solution of MS-222. The hemoglobin 

levels and erythrocyte counts of the hatcheryreared and wild lake trout were similar.

Pulford, Earl F., and L. M. Woodall.

1963. An operculum marking experiment 

on juvenile chinook salmon. Research 

Briefs, Fish Commission of Oregon, 

vol. 9, no. 1, p. 30-36. 

MS-222 was used as an anesthetic during 

marking of 1.5-inch salmon. There was little 

mortality of the fish which were maintained 

under observation for 112 days.

Randall, D. J.

1962. Effect of an anaesthetic on the heart 

and respiration of telost fish. Nature, 

vol. 195, no. 4840, p. 506.

Heart and respiratory rates were measured in tench exposed to 25 to 200 mg./l. of 

MS-222 at 17° C. The heart rate in undisturbed, control fish was 15 to 30 beats per 

minute. At a concentration of 33 p.p.m. of 

MS-222, the rate exceeded 50 per minute, 

and increased at higher dosages. The respiratory rate and amplitude also increased, but in 

a variable manner.

MS-222 probably acts on the heart via the 

parasympathetic nervous system since the 

direct effect of the drug on isolated and perfused hearts of tench, trout and roach decreased the beat frequency. Bilateral sectioning of the vagi of the fish, which were 

exposed to MS-222, resulted in a reduced 

heart rate.

Respiratory collapse occurred at 100 to 

200 p.p.m. of MS-222.

Robinson, Clay.

1965. Those chasing-rainbows. U.S. Trout

News, January-February, p. 5. 

The anesthetic action of MS-222 varies 

according to water temperature and hardness.

Robertson, O. H.

1958. Accelerated development of testis 

after unilateral gonadectomy, with observations on normal testis of rainbow 

trout. U.S. Fish and Wildlife Service,

Fishery Bulletin, No. 127, vol. 158, 

p. 9-30.

MS-222 was used to anesthetize fish for 

gonadectomy and for length and weight measurements. A concentration of 1:20,000 induced adequate anesthesia in 2 to 3 minutes. 

The fish recovered rapidly, and no harmful 

effects were observed even after daily use 

for a number of weeks.

The operational technique included starvation of the fish for 48 hours and initial anesthesia in 1:20,000 of MS-222. The fish were 

placed on an operating board and then dipped 

into a 1:25,000 solution of the anesthetic.

The sequence of histological changes in 

gonadectomized fish and those in which 

laparotomy only was performed were the same 

as in normally maturing gonads.

Rodman, Duane T.

1963. Anesthetizing and air-transporting 

young white sturgeons. Progressive 

Fish-Culturist, vol. 25, no. 2, p. 71-78. 

MS-222, tertiary amyl alcohol, and reduced temperature were employed in airtransporting young sturgeon. Shipments 

following use of two drugs were not successful. Fish exposed to a 1:40,000 solution of 

MS-222 survived for 30 to 48 hours but later 

died. A temperature of 40° F. provided adequate cold sedation which could be maintained 

during transport for approximately 30 hours 

with dry ice. The fish were shipped successfully by this method.

Rothlin, E.

1932. M.S. 222 (losliches Anaesthesin), 

ein Narkotikum fur Kaltbluter. Schweizerische Medizinische Wochenschrift, 

vol. 62, no. 45, p. 1042-1043. 

MS-222 is a third as toxic to cold-blooded 

animals as novocaine and a tenth as toxic as 

cocaine. MS-222 was more efficacious in 

comparison with novocaine, strovain, alypin, 

tutokain, panthesin, kokaine, barokain, and 

eukain. A frog was completely anesthetized 

within 5 to 7 minutes in solutions of 1:1,000 

to 1:2,000 of MS-222. Narcosis lasted several hours, and when the animal was placed 

in fresh water it recovered in 30 to 60 minutes. Studies with homologs of MS-222 allyl, 

isopropyl, n-butyl ester gave no better results than the ethyl ester.
12 Investigations in Fish Control 16: Bureau of Sport Fisheries and Wildlife

Rotmann, Eckhard.

1931. Die Rolle des Ektoderms und 

Mesoderms bei der Formbildung der 

Kiemen und Extremitaten von Triton. 

1. Operation in Gastrulastadium. 

W. Roux Archiv fur Entwicklungsmechanik, vol. 124, p. 747-794. 

There were no side or after effects of 

MS-222 on salamanders which were anesthetized by concentrations of 1:3,000 for 

1 hour. Repeated anesthetization was not 

harmful.

Ryder, R. A.

1960. Comparative tagging returns employing three different anesthetics. 

Canadian Fish Culturist, no. 26, p. 23- 

25.

Ether, urethane, and MS-222 were used 

to anesthetize Stizostedion v. vitreum for 

tagging. After 2 years, approximately twice 

as many fish tagged with the help of MS-222 

had been recovered as those tagged with the 

help of either of the other anesthetics.

Sakano, Ei-ichi.

1961. Anaesthetizing experiments of chum 

salmon fry with tricaine methanesulfonate (M.S. 222). Scientific Reports of 

the Hokkaido Salmon Hatchery, No. 16, 

p. 103-106.

Sandoz, M.

1920. Recherches experimentales sur les 

anesthesiques locaux. 1. Preparations 

et propriet6s physiologiques de la 

tricaine et de quelques-un de ses 

derives, Bull. Soc. Vaud. Sc. Nat., 

vol. 53, p. 263-302.

Sandoz Pharmaceuticals

(No date) The toxicity of MS-222 to fish 

and frogs. Sandoz Pharmaceuticals, 

Hanover, N.J. (Mimeo) 2 p. 

The 30-minute LCso of MS-222 for frogs 

was 1:160.

A 1:12,200 concentration produced 50- 

percent mortality of ypung trout in 15 minutes. The maximal tolerated concentration 

(LCi ) for trout was 1:15,900 and a 1:25,000 

solution induced anesthesia in 99 percent of 

the individuals (EC 99 ) within 3 to 4 minutes. 

These data gave a therapeutic index for MS222 of 1.57.

A 10-percent solution of MS-222 which 

was stored at room temperature showed no 

loss in activity after 3 days. After 10 days 

there was a reduction in activity of about 5 

percent. No difference was noted between 

solutions protected or not protected against 

light.

Sandoz Pharmaceuticals

(No date) M.S. 222-Sandoz, the anesthetic 

of choice in work with cold-blooded animals. Sandoz Pharmaceuticals, Hanover, 

N.J., Technical Bulletin. 10 p. 

Concentrations of 0.5 to 1.0 g./gal. anesthetic silver salmon, sockeye salmon, lake 

trout, brown trout, and largemouth and smallmouth bass within 2 to 4 minutes at 40° to 

60° F. A dosage of 0.25 to 1.0 g./gal. is 

recommended for rainbow trout.

A concentration of 0.14 g./gal. is recommended for tranquilizing bait fish during 

transport. Levels up to 0.32 g./gal. are 

effective for various tropical species.

Sato, T.

1930. Beitrage zur Analyse de Wolff'schen 

Linsen regeneration. Wilh. Roux Archiv 

flir Entwicklungs mechanik d. Oragnismen, vol. 122, p. 451.

Schiffman, R. H., and P. O. Fromm.

1959. Measurement of some physiological 

parameters in rainbow trout (Salrno 

gairdnerii). Canadian Journal of Zoology, vol. 37, p. 25-32. 

Rainbow trout were anesthetized in 300 

p.p.m. of MS-222, placed on their backs and 

their hearts exposed. Blood samples were 

collected by cardiac puncture. The samples 

were divided for measurements of hematocrit, hemoglobin, and erythrocyte count 

and size. In addition, weights of organs and 

body water and volumes of blood and plasma 

were determined.

Schoettger, Richard A., and Arnold M. Julin. 

1966. Investigations in Fish Control: 13. 

Efficacy of MS-222 as an anesthetic on 

four salmonids. U.S. Bureau of Sport 

Fisheries and Wildlife, Resource Publication 19.

Concentrations of MS-222 ranging from 80 

to 135 p.p.m. effectively anesthetized rainbow,
Richard A. Schoettger: Annotated Bibliography on MS-222 13

brown, brook and lake trout within 3 minutes 

at 7° to 17° C. The fish were exposed safely 

to these concentrations for 4 to 12 minutes. 

Dosages of 50 to 60 p.p.m. induced anesthesia .within 15 minutes, and 15 to 30 p.p.m. 

were effective for sedation. The effective 

concentrations and exposure times were inversely related to temperature; however, 

the efficacy of sedating concentrations declined with time at 17° C. The narcotic action 

of the drug was reversible, provided the fish 

were removed from the anesthetic prior to the 

cessation of respiratory activity.

The efficacy of MS-222 was not affected 

significantly by size of fish or pH of the solution. Anesthetic solutions with hardnesses of 

10 p.p.m. were less effective than those containing 35 to 180 p.p.m. total hardness, but the 

fish treated in soft water recovered sooner.

The exposure tolerances of fish which 

were repeatedly anesthetized with MS-222 

were slightly greater than those which were 

unexposed previously.

Trout sedated in closed systems at 12° C. 

reduced their rate of oxygen consumption by 

30 percent; the rate was not depressed significantly in open systems.

Serfaty, A., R. Labat, and R. Quillier. 

1959. Les reactions cardiaques chez la 

carpe (Cyprinus carpio) au cours d'une 

anesthesie prolongee. Hydrobiologia, 

vol. 13, p. 144-151. 

Anesthetizing carp with 100 p.p.m. of 

MS-222 caused a primary and secondary 

tachycardia. The first was believed to be 

correlated with encephalic penetration of the 

drug, and the second with a reduction of vagal 

tonus and lack of oxygen. Eventually, auriculoventricular dissociation occurred which 

was attributed to damage of the intracardiac 

system.

Shelton, G., and D. J. Randall.

1962. The relationship between heart beat 

and respiration in teleost fish. Comparative Biochemistry and Physiology, 

vol. 7, p. 237-250.

The heart and respiratory rates of tench 

were increased by anesthetizing them in 80 

to 200 p.p.m. of MS-222. After 12 minutes 

in a 200 p.p.m. solution, the fish stopped 

breathing and the heart rate fell to a level

similar to that of unanesthetized individuals. 

The heart beat and breathing became absolutely synchronized in animals lightly anesthetized in MS-222. The direct effect of MS222 on isolated hearts was to decrease the 

beat rate at 15 p.p.m. and decrease beat 

amplitude above 30 to 60 p.p.m. Since fish 

have no sympathetic innervation of the heart, 

the authors suggested the presence of some 

cardioaccelerator fibers in the vagus nerve.

Smith, Lloyd L., Jr., Robert H. Kramer, and 

J. Cameron MacLeod. 

1965. Effects of pulpwood fibers on fathead 

minnows and walleye finger lings. Journal, Water Pollution Control Federation, 

vol. 37, no. 1, p. 130-140. 

A concentration of 1,000 p.p.m. of MS-222 

rapidly anesthetized fathead minnows for the 

measurement of hematocrits. The hematocrits 

of treated and control fish were not significantly different. The quantity of blood obtained 

from anesthetized individuals was 17 percent 

less than controls and probably indicates a 

reduced rate of circulation.

Smith, Lynwood S., and Gordon R. Bell. 

1964. A technique for prolonged blood

sampling in free-swimming salmon.

Journal of the Fisheries Research

Board of Canada, vol. 21, no. 4, p. 711-

717.

The dorsal aortas of pink and sockeye 

salmon were cannulated to permit the 

sampling of blood over extended periods. 

The fish were anesthetized during the operation by irrigating the gills in a 1:15,000 solution of MS-222 using a pump recycling system. 

The cannula was attached to a length of polyethylene tubing which extended dorsal from 

the roof of the mouth to above the snout.

Snieszko, S. F.

1960. Microhematocrit as a tool in fishery 

research and management. U.S. Fish and 

Wildlife Service, Special Scientific 

Report Fisheries No. 341. 15 p. 

A technique for measuring the hematocrit 

of fish was described. The fish were anesthetized for approximately 1 minute in a 1:2,000 

solution of MS-222.
14 Investigations in Fish Control 16: Bureau of Sport Fisheries and Wildlife

Steinbrecht, Karl.

1957. Narkose von Fischen. Die Aquarienund Terrarien-Zeitschrfit, vol. 10, 

no. 11, p. 305-306.

There were no abnormalities of offspring 

from guppies which were frequently anesthetized in a 1:2,000 solution of MS-222 

following fertilization.

Steucke, Erwin, W., Jr., and Charles R. 

Atherton.

1965. Use of microhematocrit values to 

sex largemouth bass. Progressive FishCulturist, vol. 27, no. 2, p. 87-90. 

MS-222 at 1:3,000 was used to anesthetize 

largemouth bass. They were narcotized in 

about 2 minutes.

Thompson, R. B.

1959. Tricaine methanesulfonate (M.S. 

222) in transport of cutthroat trout. 

Progressive Fish-Culturist, vol. 21, 

no. 2, p. 96.

Young cutthroat trout were transported 

successfully in plastic bags which contained 

oxygen and a 1:40,000 solution of MS-222. 

The temperature was reduced by packing 

the bags in ice.

One-quart, plastic food boxes were tested 

in place of plastic bags. Approximately 500 

individuals were added to each box which 

was half filled with the 1:40,000 solution of 

anesthetic. Oxygen was not used in these 

tests, but the containers were packed in ice. 

The fish were released after 3 hours, and 

only 19 out of 2,000 failed to recover from 

anesthesia.

Villwock, W.

1958. Narkose bei Fischen. AquarienTerrarien-Zeitschrift, vol. 11, p. 28.

Walker, Charles R., and Richard A. 

Schoettger.

1966. Investigations in Fish Control: 15. 

Residues of MS-222 in four salmonids 

following anesthesia. U.S. Bureau of 

Sport Fisheries and Wildlife, Resource 

Publication 21.

Residues of MS-222 were measured in 

rainbow, brown, brook, and lake trout. The 

fish were anesthetized to medullary collapse 

in concentrations of 80 to 135 p.p.m., depending on species and temperature. Residues 

ranged from 6 to 72 p.p.m. in the muscle 

tissues of fish at the time of medullary collapse. The levels in fish which had recovered 

in freshwater at 12° C. declined rapidly within 3 hours and approached background values 

after 6 to 9 hours. A slower dissipation of 

residues occurred in tests at 7° and 17° C. 

The total residue including background did 

not exceed 5 p.p.m. at the end of 9 hours, or 

3 p.p.m. after 24 hours for all species at all 

temperatures.

Residues of MS-222 in the blood, liver, 

and kidney of rainbow trout declined in a pattern similar to that for muscle.

1966. Investigations in Fish Control: 14. 

Method for determining MS-222 residues 

in fish. U.S. Bureau of Sport Fisheries 

and Wildlife, Resource Publication 20. 

The Bratton-Marshall method is sensitive 

for primary aromatic amines such as sulfa 

drugs. It was modified to detect MS-222.

Selected tissues from anesthetized trout 

were processed to extract the drug. The recovery of known amounts in muscle, blood, 

kidney, and liver ranged from 90 to 112 percent. The concentrations of interferring 

amines were evaluated.

The acute oral LD 50 of MS-222 to laboratory rats is between 5 and 10 g./k. This indicates a relatively low toxicity of MS-222 to 

mammals.

Watson, John E.

1961. Tricaine methanesulfonate as an 

anesthetic for herring. Progressive 

Fish-Culturist, vol. 23, no. 4, p. 174. 

MS-222 was tested in sea water as an 

anesthetic for herring. The fish were anesthetized within 8 minutes by a solution of 

1:20,000 at 8° C. They recovered in approximately 8 minutes after removal to fresh seawater. Their prolonged exposure to MS-222 

for 3 to 4 minutes after complete anesthesia 

was usually lethal.

Webb, Robert T.

1954. Tricaine methanesulfonate (M.S. 

222) as an anesthetic for some common 

pond fishes. Unpublished thesis. Alabama Polytechnic Institute, Auburn.
Richard A. Schoettger: Annotated Bibliography on MS-222 15

Webb, Robert T.

1958. Distribution of bluegill treated with

tricaine methanesulfonate (M.S. 222).

Progressive Fish-Culturist, vol. 20,

no. 2, p. 69-72.

Tests were conducted to determine 

whether the application of MS-222 would increase the number or pounds of bluegills 

which could be carried in a distribution truck. 

A concentration of 0.1 g./gal. appeared to be 

the most promising. The results of the tests 

were inconclusive; successful tests could not 

be duplicated; and at times the control fish 

hauled as well or better than the drugged 

ones.

Witschi, E.

1927. Testis grafting in tadpoles of Rana 

temporaria L. and its bearing on the hormone theory of sex determination. Journal of Experimental Zoology, vol. 47, 

p. 269.

Wood, E. M.

1956. Urethane as a carcinogen. Progressive Fish-Culturist, vol. 18, no. 3, 

p. 135-136.

Urethane induces lung tumors in mice 

which develop whether the drug is administered by injection, in drinking water, by nasal 

instillation, or from painting the skin.

An editorial comment accompanying this 

report indicated that MS-222 might be a suitable substitute for urethane.
INVESTIGATIONS IN FISH CONTROL

17. MS-222 as an Anesthetic for Channel Catfish 

Its Toxicity, Efficacy, and Muscle Residues

By Richard A. Schoettger, Charles R. Walker, 

Leif L. Marking, and Arnold M. Julin

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

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

Fish and Wildlife Service, Clarence F. Pautzke, Commissioner 

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

Resource Publication 33 January 1967 Washington, D.C.
CONTENTS

Page

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

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

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

Efficacy................................ 5

Residues. ............................... 5

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

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

Effects of size......................... 5

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

Effects of water quality................... 6

Safety indexes ......................... 6

Efficacy ................................ 6

Rapid anesthesia ....................... 6

Moderately rapid anesthesia. ............... 8

Sedation ............................. 9

Residues. ............................... 9

Discussion ................................. 11

Toxicity ................................ 11

Efficacy ................................ 12

Residues. ............................... 13

Summary .................................. 13

References ................................. 14
MS-222 AS AN ANESTHETIC FOR CHANNEL CATFISH: 

ITS TOXICITY, EFFICACY, AND MUSCLE RESIDUES

By Richard A. Schoettger, Fishery Biologist

Charles R. Walker and Leif L. Marking, Chemists

and Arnold M. Julin, Fishery Biologist

Bureau of Sport Fisheries and Wildlife

La Crosse, Wisconsin

Abstract. MS-222 was tested as an anesthetic on channel catfish. Its 

acute toxicity is approximately 65 to 50 p.p.m. over periods of 24 to 96 

hours. Anesthesia is induced within 2 minutes by concentrations above 

100 to 140 p.p.m., and within 15 minutes by 70 p.p.m. Concentrations of 

20 to 40 p.p.m. maintain sedation for 6 hours. Residues of MS-222 occur 

in muscles of anesthetized catfish, but decrease about 90 to 95 percent at 

1 hour of withdrawal from the drug. Nine to 24 hours after withdrawal the 

residues decline to within the statistical variations of the background 

aromatic amines. The influences of duration of exposure, size of fish, 

temperature, and water quality on toxicity, efficacy, and residues are 

discussed.

MS-222 (tricaine methanesulfonate) is commonly employed to anesthetize fish during 

marking, spawning, and transporting. The drug 

has been used less commonly on channel catfish than on salmonids or centrarchids. In 

recent years the channel catfish, Ictalurus 

punctatus, has become highly regarded in 

many areas of the United States as a commercial and sport fish; it is being reared on 

rice-fish farms, in commercial hatcheries, 

and in farm ponds. We anticipate that a 

greater use of MS-222 will accompany increased effort to advance the culture and 

management of this species.

The U.S. Food and Drug Administration requires that certain disinfectants, antimicrobials, and anesthetics be cleared for their 

continued use on fish which may be consumed 

by people.

The information necessary for clearance of 

MS-222 as an anesthetic for channel catfish

Arnold M. Julin is now at the Fish Genetics Laboratory, Bureau of Sport Fisheries and Wildlife, Beulah, Wyo.

includes its toxicity, its efficacy as an anesthetic, and its residues in tissues of treated 

fish. Similar studies were conducted on 

salmonids at the Fish Control Laboratories 

(Marking, 1966; Schoettger and Julin, 1966; 

and Walker and Schoettger, 1966a, 1966b). 

The history and development of M-222 as a 

fish anesthetic were reviewed by Bove (1962), 

the manufacturer (Sandoz Pharmaceuticals), 

Eisler and Backiel (I960), and Schoettger 

(1966).

The scope of the investigation on channel 

catfish was somewhat narrower than that of 

our earlier study on salmonids. We tested 

the influences of temperature or size of fish 

on toxicity and efficacy. The effects of 

water hardness on toxicity and of pH on efficacy were also measured. Determinations 

of MS-222 residues in catfish were confined 

to those in muscle because residues in blood, 

liver, and kidney of salmonids decreased with 

withdrawal time at a rate similar to that in 

muscle; because background substances of 

natural origin in the livers and kidneys of
Investigations in Fish Control 17: Bureau of Sport Fisheries and Wildlife

salmonids interfered with analyses of low 

residues; and because muscle is the principal 

edible tissue in catfish.

METHODS AND MATERIALS

The channel catfish used in the toxicity, efficacy, and residue experiments were obtained 

from the national fish hatcheries at Fairport 

and Guttenberg, Iowa, and from the Mississippi River (by the Iowa Conservation Department). They were held in well water at 12° C. 

and without feed for 2 days before placement 

in the anesthetic solutions. Twenty-four hours 

before an experiment, the fish were acclimated to temperature in reconstituted 

water prepared according to methods described by Lennon and Walker (1964).

TOXICITY

The static bioassays of MS-222 were conducted with 1.9-, 2.6-, and 3.5-inch fish according to the methods of Lennon and Walker 

(1964). They were tested in 5-gallon glass 

jars containing 15 liters of test solution, 

with aeration applied to solutions containing 

the 3.5-inch fish. Water temperatures were 

maintained at 12°, 17°, and 22° C. by placing 

the bioassay vessels in thermostatically 

controlled water baths.

The data were analyzed statistically according to the method of Litchfield and Wilcoxon 

(1949) to define concentrations which produced 

50 percent mortality (LC.so). In addition, the 

variances, slope functions, and 95 percentconfidence intervals were determined.

Different water qualities were obtained by 

varying the amounts of reconstituting salts 

added to deionized water (table 1).

Safety indexes (S.I.) were determined for 

the anesthesia of channel catfish with MS-222. 

We define the indexes as the margin between 

concentrations effective for anesthesia and 

those which cause mortality, expressed as a 

number obtained by dividing the lethal concentration (LC50) by the effective concentration (EC50). An effective level anesthetizes 

the catfish to loss of equilibrium. This stage 

of anesthesia is defined later in methods for 

measuring the efficacy of MS-222.

The S.I. values were calculated for 15-, 30-, 

and 60-minute exposures. These exposures 

were selected to give consistent results because shorter exposures required extremely 

high concentrations. In longer exposures, the 

anesthetized fish occasionally recovered in 

the test solutions.

Ten fish were exposed to each concentration, 

and 10 or 20 served as controls. Ten concentrations were selected to yield survival 

and mortality of 1.9- and 2.6-inch fish; 5 

concentrations were selected for the 3.5-inch 

fish.

The maximum safety indexes (M.S.I.) were 

calculated from the LC j and ECgg values obtained by extrapolating the regressions used 

in determining LCso and ECso values. The 

maximum safety index is lower than the safety 

index and is biased in favor of greater safety.

Table 1. Water qualities obtained with various amounts of reconstituting salts in deionized water

Water quality

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

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

NaHC03

12

48

192

Salts i

CaS04

7.5

30.0

120.0

n nig. /I.

MgS04

7.5

30.0

120.0

KC1

0.75

3.00

12.00

Total 

hardness

as CaCOs 

(p. p.m. )

10-13

40-48

160-180

Total 

alkalinity

as CaC03 

(p. p.m. )

10-15

30-35

110-120

PH

6.4-6.8

7.2-7,6

7.6-8.0
Schoettger, Walker, Marking, and Julin: MS-222 as an Anesthetic for Channel Catfish

EFFICACY

Channel catfish of 2 to 6 inches and 7 to 12 

inches were used to determine the efficacy of 

MS-222. Fish less than 4 inches were tested 

in 15 liters of reconstituted water. Larger 

individuals were exposed in 45 liters. Since 

preliminary trials failed to show that variable 

loading had any effect on efficacy, the loading 

levels were approximately 10 g./l. for the 

smaller fish, and up to 40 g./l. for the larger 

fish.

The methods of evaluating efficacy of 

MS-222 against channel catfish were similar 

to those reported in our trials with salmonids. 

A series of concentrations of MS-222 were 

tested at a temperature of 12 C. and at pH 

7.0. The levels giving reasonable anesthetization, holding and recovery times, and which 

produced miminum mortality were considered 

for further trials at 7°, 12° 17°, 22°, and 27° 

C., and at pH 5.0, 7.0, and 8.5.

RESIDUES

Residues of MS-222 were measured in 

muscles of 6- to 13-inch channel catfish which 

had been treated during the efficacy trials. 

Groups, each composed of three individuals, 

were anesthetized to medullary collapse by 

270 p.p.m. of MS-222 at 7°, 12 , 17°, 22°, and 

27° C. In this series of tests, the drug was 

less effective at 7° and some fish tolerated an 

exposure exceeding 1 hour; at higher temperatures the exposures were approximately 5 to 

10 minutes long.

Five or six groups of catfish were anesthetized at each temperature. One narcotized 

group was selected for each temperature for 

analyses of residues at 0-hour withdrawal. 

The remaining groups were placed in fresh 

water for recovery. Their muscles were 

analyzed 1, 3, 6, 9, and 24 hours after withdrawal. We also analyzed control fish at each 

temperature.

The criteria for assessing efficacy of MS222 against channel catfish were changed 

slightly from those used for salmonids; therefore, a portion of the results which governed 

methodology are included in this section. The 

behavioral responses of catfish in deep 

anesthesia differed from those of trout. In 

catfish, at high concentrations of MS-222, we 

were unable to distinguish clearly the transition from total loss of equilibrium, stage n, 

into loss of reflex. The reflex response to 

constant pressure on the caudal fin or peduncle is frequently delayed for 1 to 10 

seconds. Also, loss of reflex is not always 

distinct from medullary collapse. Opercular 

movements occasionally cease before reflex 

activity stops. A concentration of MS-222 

was considered effective for rapid or moderately rapid anesthesia when it induced total 

loss of equilibrium, stage II, within 2 and 15 

minutes respectively. This stage of anesthesia 

is more safely induced and maintained than 

loss of reflex, and the fish appear to be almost as easily handled. An effective concentration for sedation induces the response 

within 15 minutes and maintains it for 6 

hours.

The residues of MS-222 in fish flesh were 

determined by the modified Bratton-Marshall 

method developed by Walker and Schoettger 

(1966a).

RESULTS

TOXICITY

Effects of size. The toxicity of MS-222 to 

channel catfish is dependent, in part, on their 

size. The larger fish appear more resistant 

to the anesthetic than smaller individuals 

(table 2). The 24-hour LCso values for 1.9-, 

2.6-, and 3.5-inch specimens are 58.0, 64.0, 

and 66.2 p.p.m., respectively. This sizetoxicity relationship was also observed in the 

48- and 96-hour bioassays.

Effects of temperature. The toxicity of 

MS-222 to channel catfish is influenced by 

temperature. The data indicate a higher resistance of 1.9-inch fish at 17 than at 12 or 

22° C. (table 2). The 24-hour1 LCso values for 

the latter two temperatures are similar, but
Investigations in Fish Control 17: Bureau of Sport Fisheries and Wildlife

Table 2. Toxicity of MS-222 to channel catfish at three temperatures

Temperature 

and lot

At 12° C.: 

Lot 276......

Lot 92.......

Lot 122......

At 17° C. : 

Lot 276......

At 22° .C. : 

Lot 276......

Averag

Length 

(in.)

1.9

2.6

3.5

1.9

1.9

e size

Weight 

(g.)

0.9

1.9

5.2

0.9

0.9

24

LC50 

(p. p.m. )

«^O A

64.0

SS o

60.5

CQ a

lours

95-percent 

confidence 

interval

55.6-61.5

Crt o r~ir\ >

63.0-69.5

ert -i s*-\ Q

58.0-61.5

48

LC50 

(p. p.m. )

55.0

55.0

/1O -I

60.0

Kft c}

hours

95-percent 

confidence 

interval

53.3-56.8

52.9-57.2

60.9-63.3

58.3-61.7

57.0-60.6

96

LC50 

(p. p.m.)

51.1

55.0

s O T

60.0

Crt ft

hours

9 5 -per cent 

confidence 

interval

49.0-53.5 

53.9-56.1

60.9-63.3

«^{J 1 £T r?

57.0-60.6

Table 3. Toxicity of MS-222 in selected water qualities at 12° C.

Total 

hardness

12 p. p.m...

46 p. p.m. . .

170 p. p.m..

Total

alkalinity 

(p. p.m. )

15

30

114

24 hours

PH

6.6

7.5

7.8

LC50 

(p. p.m. )

64.0

58.0

54.0

95-percent 

confidence 

interval

58.7-69.8

55.6-61.5

51.0-57.4

48 hours

LC 50 

(p. p.m.)

60.0

55.0

54.0

95-percent 

confidence 

interval

57.1-63.0

53.3-56.8

51.0-57.4

96 hours

LC 50 

(p. p.m. )

50.0

51.1

54.0

95-percent 

confidence 

interval

46.7-53.5

49.0-53.5

52.6-55.5

the 96-hour LCso was lower at 12 than at 

22° C. In general, the relative change in 

toxicity of the anesthetic with exposure is less 

at 17° and 22° than at 12° C.

Effect of water quality. The toxicity of 

MS-222 to catfish was evaluated in relatively 

soft, medium, and hard water (table 3). The 

LCso's range from 50 to 64 p.p.m., and the 

drug is more toxic at 24 hours in hard water 

than in soft water. After 96 hours of exposure 

the drug appears more toxic in soft water. 

The toxicities of MS- 222 to catfish in medium 

and hard waters are similar, except at 96 

hours of exposure. Here, the L-C^Q values for 

medium and soft water are much alike, and 

the 95-percent confidence limits overlap considerably.

The greatest change in toxicity of MS-222 

with exposure occurred in tests in soft water. 

These trials also gave the most variable results as shown by the relative widths of the 

95-percent confidence intervals (table 3).

Safety indexes. The S.I. values in table 4 

range from 2.4 to 3.0 and indicate that 15- 

minute exposures are safer than those lasting 

30 or 60 minutes. The M.S.I, values, on the 

other hand, show greater safety with longer 

exposures. This is probably due to the large 

variance which is associated with the broad 

range of concentrations tolerated by channel 

catfish.

EFFICACY

The efficacy of MS-222 for inducing rapid 

and moderately rapid anesthesia and sedation 

in channel catfish appears to be unaltered by 

pH 5.0 to 8.5. These data were pooled, without 

differentiation, with those for size of fish and 

temperature.

Rapid anesthesia. Concentrations of 140 to 

270 p.p.m. are all equally effective for the 

rapid anesthesia of 7- to 12-inch catfish at 

12° to 27° C. (table 5). Levels above 100 to 

120 p.p.m. anesthetize 2- to 6-inch fish.
Schoettger, Walker, Marking, and Julin: MS-222 as an Anesthetic for Channel Catfish

Table 4. Safety indexes and maximum safety indexes for anethesia of channel catfish with

MS-222 at 12° C.

Exposure

30 minutes ...............

Safety index

LC 50 

(p. p.m. )

139.0 

118.0 

110.0

EC50 

(p. p.m. )

46.5 

45.0 

46.4

Index 

LC50/EC50

3.0 

2.6 

2.4

Maximum safety index

LC-L 

(p. p.m. )

76.0 

74.0 

80.0

EC 99 

(p. p.m. )

71.9 

62.0 

62.8

Index 

LCi/ EC 9 9

1.1 

1.2 

1.3

Table 5. Concentrations of MS-222 producing rapid anesthesia in two sizes of channel catfish at

five temperatures

Temperature and 

concentration

At 7° C.:

160-270 p. p.m.

160-270 p. p.m.

200-270 p. p.m.

At 12° C.:

80 p. p.m.

100 p. p.m.

120-260 p. p.m.

270 p. p.m.

At 17° C.:

80 p. p.m.

100-220 p. p.m.

140-270 p. p.m.

At 22° C.:

270 p. p.m.

At 27° C.:

100-130 p. p.m.

140-270 p. p.m.

Size 

of 

fish

(in.)

2- 6

7-12

7-12

2- 6

2- 6

2- 6

7-12

2- 6

2- 6

7-12

7-12

7-12

7-12

Lot 

No.

122

114

199 & 203

122

122

122

203

122

122

114 & 203

203

203

203

Fish in loss

of equilibrium, 

stage II, within 

2 minutes

Number

39/ 40

24/ 30

2/ 48

O/ 40

10/ 20

331/345

18/ 18

O/ 10

90/ 90

63/ 80

24/ 24

12/ 25

53/ 55

Percent

98

80

4

0

50

95

100

0

100

79

100

48

96

Mean range

of exposure times 

(min. )

First 

fish

2 6

2 6

25

2 30

2 11

2 7

6

2 20

2 6

8

4

7

4

Last 

fish

--

--

48

7

11

5

9

5

Safe 

exposure 

index1

--

3.0

4.0

2.0

2.0

Recovery 

mean

time

range 

(min.)

6-24

6-13

19-33

2- 5

2- 9

7-18

7-10

8-15

3- 7

4- 8

5- 7

3- 9

5-10

Survival 

(percent)

100

100

98

100

100

97

100

100

100

100

100

100

80

1 Index obtained by dividing the time for the first fish to reach medullary collapse by the 

time (2 min.) for all fish to reach loss of equilibrium, stage 11.

2 Fish removed from the anesthetic before all reach medullary collapse.

Catfish to tolerate exposures to MS-222 for approximately 6 to 11 minutes at 12 and 17 , and 

4 to 5 minutes at 22 and 27 C. They usually 

recover in fresh water in less than 15 

minutes; at 27 , however, 20 percent of the 

test fish died when they were not removed 

quickly enough to prevent overexposures.

The results of tests at 7 C. were variable 

(table 5). Concentrations of 160 to 270 p.p.m. 

were 80 to 98 percent effective on lots 114 

and 122, but 200 to 270 p.p.m. were ineffective on lots 199 and 203. Although individuals 

in lots 114 and 122 were effectively anesthetized and tolerated exposure for about 6
Investigations in Fish Control 17: Bureau of Sport Fisheries and Wildlife

minutes, lots 199 and 203 required 3 to 5 

minutes for anesthesia, and some individuals 

tolerated exposures for more than an hour.

Fish in lot 199 were used for residue 

analyses, and the numbers remaining were 

insufficient for comparative tests to determine whether they were also more resistant 

than lot 114 at higher temperatures. The 

sensitivities of catfish in lot 203 were similar 

to those in lot 199 at 7 and those in lot 114 

at 17 C., and the results were combined at 

the respective temperatures. The purpose in 

comparing sensitivities of the different lots 

is to point out that there was some variable 

in the tests at 7 which increased the 

resistance of catfish to MS-222 in one instance but not in another. Analysis of the 

sources of the fish and their general conditions indicated that these were probably not 

causes of the inconsistencies. On the other

hand, fish in lot 114 were tested in the fall, 

and those in lots 199 and 203 in the spring. It 

is conveivable that the natural acclimation 

of the latter fish to low temperatures during 

the winter may have contributed to their 

resistance to MS-222 at 7° C.

We were unable to calculate safe exposure 

indexes for all of the trials reported in table 

5. In most instances the fish were placed in 

fresh water before they all entered medullary 

collapse. The indexes in the remaining trials 

range from 2 to 4. They indicate that catfish 

can be safely exposed to MS-222 for about 2 

to 4 times longer than required to induce loss 

of equilibrium, stage II.

Moderately rapid anesthesia. Seventy 

p.p.m. of MS-222 induce loss of equilibrium 

in channel catfish within 15 minutes (table 6). 

They tolerate exposure to this concentration

Table 6.--Concentrations of MS-222 producing moderately rapid anesthesia in two sizes of channel

catfish at four temperatures

Concentration, temperature and 

size of fish

At 70 p.p.m.:

At 7° C.:

2- 6 in. ........................

7-12 in.........................

At 12° C.:

2- 6 in. ........................

7-12 in.........................

At 17° C.:

2- 6 in.........................

At 60 p.p.m. :

At 27° C.:

7-12 in. ........................

At 70 p.p.m.:

At 27° C.:

7-12 in.........................

Lot 

No.

122

114

122

114

122

203

203

Fish in loss

of equilibrium, 

stage II, 

within 15 minutes

Number

10/10

6/ 6

70/70

33/35

10/10

4/10

8/8

Percent

100

100

100

94

100

40

100

Exposure 

time 

(min. )

30

30

30

30

30

1 24

180

Recovery

Mean 

time

T»QTicro

(min. )

2- 4

2- 6

3- 5

2-10

1- 2

1- 2

Survival 

(percent)

100

100

100

100

100

100

75

Hours
Schoettger, Walker, Marking, and Julin: MS-222 as an Anesthetic for Channel Catfish

for at least 30 minutes and many can be exposed longer. In one test, at 27 C., the 

majority of the fish survived at 3-hour exposure.

Water temperature and size of fish appear 

to have no influence on the efficacy of a concentration inducing a moderate rate of 

anesthesia. The fish recover faster, however, at 22 and 27° than at 7°, 12°, or 17°. 

The sensitivities of the different lots of test 

fish at various temperatures were only partially compared.

A moderate rate of anesthesia appears most 

applicable to handling operations such as 

measuring and weighing, or when large numbers of individuals are involved. A level of 70 

p.p.m. may also be useful for long-term surgical operations, provided sufficient time is 

allowed for reflex responses to decline.

Sedation. Channel catfish are sedated in 

20 p.p.m. of MS-222 at 7°, 12°, and 17° C. 

(table 7). This level was not effective at 27° C.,

and the concentration was increased to 40 

p.p.m. to maintain sedation for 6 hours. These 

data suggest that sedating concentrations of 

MS-222 are metabolized or deactivated at 

higher temperatures. This finding is supported by the results of McFarland (1959 and 

1960), Schoettger and Julin (1966), and our 

toxicity trials with catfish.

RESIDUES

Residues of MS-222 occur in the muscles of 

anesthetized channel catfish and appear to 

vary with temperature. Seventeen to 147.2 

p.p.m. of the drug, including background 

aromatic amines, were measured in fish 

narcotized to medullary collapse in 270 

p.p.m. of MS-222 (table 8). The mean concentrations of residue at 0-hour withdrawal 

are: 31.3 p.p.m. at 7° C., 69.9 p.p.m. at 12°, 

125.5 p.p.m. at 17°, 97.5 p.p.m. at 22°, and 

65.7 p.p.m. at 27 . The levels in fish treated 

at 12°, 17°, 22°, and 27° are two to four times 

greater than in those exposed at 7 C.

Table 7. Concentrations of MS-222 producing sedation in two sizes of channel catfish at four

temperatures

Concentration, 

temperature , 

and size of fish

At 20 p.p.m: 

At 7°C. : 

2- -6 in. ..............

7-12 in...............

At 12° C. : 

2- 6 in. ..............

At 17° C: 

2- 6 in. ..............

7-12 in. ..............

At 27° C. : 

7-12 in. ..............

At 30 p.p.m: 

At 27° C. : 

7-12 in. ..............

At 40 p.p.m. : 

At 27° C. : 

7-12 in. ..............

Lot 

No.

122 

114

122

122 

114

203 

203 

203

Fish in sedation at--

15 minutes

Number

10/ 10 

6/ 6

138/140

50/ 50 

6/ 6

12/ 12 

13/ 13 

6/ 6

Percent

100 

100

99

100 

100

100 

100 

100

6 hours

Number

10/ 10 

6/ 6

132/140

50/ 50 

6/ 6

O/ 12 

8/ 13 

2 6/ 6

Percent

100 

100

94

100 

100

0 

62 

100

Behavior1 of fish 

not in 

sedation at 

15 min.

<>

6 hrs.

>

<

<

1 > = deeper anesthesia; < = similar to controls.

2 Fish in sedation at 3 and 19 hours.
10 Investigations in Fish Control 17: Bureau of Sport Fisheries and Wildlife

Table 8. Residues of MS-222 including background in muscle of channel catfish for various

withdrawal times at selected temperatures

T*O1TTT^OT»Q "l"l 1T»^ aTif!

withdrawal, time

At 7° C. : 

0 hour. .......................

6 hours .......................

24 hours .......................

At 12° C. :

At 17° C. :

At 22° C. :

1 hour. .......................

At 27° C. : 

0 hour. .......................

1 hour. .......................

6 hours .......................

24 hours .......................

Number

of 

fish

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

Mean

Length 

(in.)

12.13

12.53

12.53

12.03

12.77

10.57

rr cjrr

8 07

7.73

8

nrr

7

c»r7

9.00

8

CJ-3

9.20

9.33

9.00

8.90

7.70

8 #7

7.53

8

T "7

8

^ "7

8 on

9.13

9.23

S rt'j

8 Q^

8

r7 r'

size

Weight 

(g.)

199.7

223.0

214 3

223.3

oy t> n

140.7

-47.3

61.7

-45.0

51.7

-49.3

77.0

x-r; r»

GO "7

8-4.3

"70 1

67.3

J K. <->

65.3

-41.0

cs rj

57.3

71.7

71.0

80.0

64 3

6-4.0

f o n

Residue;

Mean

31.30

26.70

8. S3

5.07

-4.53

69.93

6.57

1.20

1.10

0.83

0.90

125.53

6.57

1.40

2.23

1.00

97. -47

6.57

1.47

1.20

1.27

0.60

65.67

4.00

4.23

~\ jn

1.27

0.77

3 in p.p.m.

Range

17.0- 38.5

22.5- 34 9

7.6- 11.2

4.0- 5.8

2.5- 7.6

56.8- 77.2

4.9- 9.3

0.5- 1.2

0.8- 1.3

0.6- 1.0

0.8- 1.0

88.6-147.2

4.8- 8.7

1.3- 1.5

1.7- 2.8

0.7- 1.4

87.6-105.4

4.5- 9.3

1.2- 1.9

0.9- 1.7

0.8- 1.8

0.5- 0.7

~V7 ? ftn n

2.0- 6.4

1.9- 6.8

0.9- 1.9

0.8- 1.6

0.6- 1.0

error

7.26

4.10

1.18

0.55

0.91

6.58

1.40

0.47

0.15

0.18

0.06

18.55

1.15

n 0*5

0.32

0.21

5.23

1.43

0.22

0 ?5

0.29

JD.06

14 23

1.29

0.59

0.30

0.24

0.12

95 -percent

confidence 

interval

ft 5ft_AZ. 3<4

1.08-16.58

2.70- 7 44

0.61- 8.45

0.55-12.59

0.00- 3.22

0.45- 1.75

0.06- 1.60

0 6A- 1 "l£

1 62-11 52

1.19- 1.62

0.85- 3.61

n i 0- i QO

O y o no r?o

0.52- 2.42

0

~\ O "> O0

n ro ? *i?

0.34- 0.86

O nn Q ^'i

1

£jrj x- 1-71-7

O

n rt o rjx:

O oy o in

0.25- 1.29

As in salmonids, a background of aromatic 

amines is also present in the muscles of unanesthetized catfish. The background in catfish ranges from 0.5 to 3.6 p.p.m. (table 9). 

Both the upper and the lower limits of the 

range occur in samples from fish (lot 199, 

table 5) maintained at 7 . The mean background at this temperature is 1.7 p.p.m. with 

a standard error of 0.96. The 95-percent confidence interval around the means is 0 to 5.33 

p.p.m. The mean concentrations in flesh at 

other temperatures range from 0.8 to 1.5 

p.p.m. with standard errors from 0.05 to 0.18.

Their 95-percent confidence intervals overlap 

between 0.10 and 1.72 p.p.m.

The residues of MS-222 in muscles of catfish decline approximately 90 to 95 percent 

after 1 hour of withdrawal at 12 , 17 , 22 , 

and 27° C. (table 8). They decrease further 

after 6 to 24 hours of withdrawal and are 

withdrawal and are within the 95-percent 

confidence intervals around the mean backgrounds of controls. The residues in catfish 

at 7° decline more slowly than at higher 

temperatures, but are within the background
Schoettger, Walker, Marking, and Julin: MS-222 as an Anesthetic for Channel Catfish 11

Table 9.--Background residues of aromatic amines in muscle of channel catfish used as

controls in the study of MS-222

Temperature

7° r

ipo r

17° C....................

OpO n

27° C....................

Number 

of 

fish

3 

5 

3 

3 

3

Mean size

Length 

(in.)

12.20 

9.80 

9.20 

9.43 

8.37

Weight 

(g-)

220.7 

108.2 

80.7 

80.7 

51.7

Concentration 

in p. p.m.

Mean

1.70 

0.80 

1.07 

1.50 

0.87

Range

0.5-3.6 

0.6-1.0 

0.9-1.3 

1.4-1.5 

0.6-1.2

Standard 

error

0.96 

0.07 

0.12 

0.05 

0.18

95 -percent 

confidence 

interval

0.00-5.83 

0.61-0.99 

0.55-1.59 

1.28-1.72 

0.10-1.65

Table 10. Acetylated aromatic amines in muscle of channel catfish for various withdrawal

times at 7° C.

Withdrawal 

time

0 hour .......

6 hours ......

24 hours ......

Number

of 

fish

3

3

3

3

3

Mean

Length 

(in.)

12.1

12.5

12.5

12.0

1 *D ft

size

Weight 

(g.)

199.7

223.0

11 j t

223.3

ov cJ n

Residues

Mean

1.07

3

rjf

5.50

6.00

3

ft i

in p.p.m.

Range

0.0-2.4

? A- 5 1

J O £. ">

5.3-7.0

2.4-4.9

error

0.71

O ijft

0.40

0.56

0.75

95 -percent

confidence 

interval

O J r\ r? 10

3

r7/r r» *-)>

3

£.l C> OQ

0

£ o r? c\J

Percent 

of total

aromatic 

amines

o &

12 4

 30 j

c*j -~>

47.9

confidence interval for this temperature after 

9 to 24 hours of withdrawal.

Channel catfish which had been anethetized 

at 7 were also analyzed to detect possible 

acetylated derivatives of MS-222 (table 10). 

The presence of these derivatives may indicate a mechanism within the fish for deactivation of the drug. The acetylated fraction is 

obtained by subtracting the free MS-222 and 

background concentrations (table 8) from the 

total aromatic amines measured in the sample 

after acid hydrolysis. At 0-hour withdrawal 

the muscles contain an average of 1.07 p.p.m. 

of acetylated aromatic amines, or 2.8 percent 

of the total aromatic amines. When the fish 

are transferred to fresh water, the concentrations of acetylated substances increase to a 

mean high of 6 p.p.m. after 9 hours of withdrawal, and then decline. Within this period, 

free residues decrease so that the relative 

amount of acetylated amines increase to 54.2 

percent. The concentrations of both fractions 

decline at 24 hours of withdrawal, but their 

proportions are similar.

DISCUSSION

TOXICITY

The channel catfish is one of the species 

more resistant to MS-222. The 24- to 96-hour 

LCso's range from 66.2 to 51.1 p.p.m. Marking (1966b) reported LCso's for similar exposures of 52.2 to 31.0 p.p.m. for various 

salmonids and 61.5 to 39.4 p.p.m. for northern 

pike, bluegill, largemouth bass, and walleye. 

This greater resistance of catfish to certain 

chemicals has been noted in other investigations at this laboratory (Walker et al., 1964; 

Marking, 1966a). Also, large catfish are more 

resistant to the drug than small ones, and this 

finding agrees with the size-sensitivity relation between other species and MS-222 noted 

by Marking (1966b).

Catfish are somewhat more resistant to 

MS-222 at 17° than at 12° or 22° C. Marking 

(1966b) made a similar observation on the 

toxicity of the anesthetic to bluegill. Whether 

the resistance of 1.9-inch catfish at 17 C. is
12 Investigations in Fish Control 17: Bureau of Sport Fisheries and Wildlife

significant seems doubtful. The confidence 

interval for the 24-hour LCso at this temperature includes the LCso for 1.9-inch fish 

at 22 and almost includes the value at 12 .

The interaction of temperature and exposure 

seems to have a greater effect on toxicity of 

MS-222 than temperature alone. The fact that 

the drug increases in toxicity with time at 

12°, but not significantly at 17° and 22° suggests that it degrades at the higher temperatures.

We found that catfish are relatively more 

resistant when the anesthetic is dissolved in 

soft water. The resistance declines with exposure, and after 96 hours the soft water solutions are slightly more toxic. Schoettger and 

Julin (1966) reported that MS-222 is less effective against rainbow trout in very soft 

water. They suggested that the rate of absorption and deactivation of the drug may increase 

along with higher metabolic rates of trout in 

soft water. The decreased resistance of catfish with time, however, may reflect the effects of osmotic stress.

The safety indexes for anesthesia of channel 

catfish with MS-222 show that the lethal concentrations of drug are approximately 2.5 to 

3.0 times greater than the levels which induce 

narcosis. In actual practice this method of 

evaluating safety of fish anesthetics is not 

entirely applicable. The fish may be anesthetized at similar or slightly higher concentrations than those used to calculate safety indexes, but exposure may determine lethality. 

For this reason some indexes based on safe 

exposure were calculated in the efficacy experiments.

EFFICACY

MS-222 is effective and safe for the rapid 

and moderately rapid anesthesia of channel 

catfish. The concentrations for the former 

rate of narcotization, 100 to 270 p.p.m., exceed the 80 to 135 p.p.m. which were effective 

for salmonids (Schoettger and Julin, 1966) and 

suggest that catfish tolerate a much broader 

range of concentrations. The levels for moderate rates in salmonids and catfish are similar. The low end of the range of concentrations 

inducing rapid anesthesia in catfish probably 

represents a threshold, or minimum effective 

level, since concentrations up to 270 p.p.m. 

are essentially no more effective. Concentrations within this range have been used to 

anesthetize channel catfish for sexing and 

physiological investigations (Crawford and 

Hulsey, 1963; Larsen, 1964; Moss and Scott, 

1964).

The inconsistent efficacy of MS-222 on three 

lots of fish at 7 C. may reflect differential 

sensitivities of the lots. The differences may 

be related to prior acclimation to environmental temperatures. Residues of MS-22 were 

later measured in muscles of fish in a resistant lot and, as discussed elsewhere, the 

residues differ markedly from those in fish 

treated at higher temperatures.

In general, temperature has little influence 

on the efficacy of MS-222 against catfish, 

but they tolerate less exposure at 22 and 27 

than at 7 , 12 , or 17 C. It was necessary to 

increase the concentrations of the drug for 

salmonids at lower temperatures, but a similar influence of temperature on the resistance 

of catfish may have been masked by the broad 

range of effective concentrations.

Although channel catfish appear to be more 

resistant to MS-222 than salmonids, the durations of exposure tolerated by both are similar. The behavioral responses of the catfish 

must be observed, however, to minimize the 

risk of overexposures. Medullary collapse 

occurs almost simultaneously widi loss of reflex, and therefore, anesthesia to the latter 

stage may be unsafe and unnecessary. The 

delayed reflex response which occurs late in 

loss of equilibrium does not usually preclude 

the handling of fish. The cessation of opercular activity in catfish, as in trout, is a good 

criterion for transferring them to fresh water, 

although many individuals may recover completely from brief exposures beyond this point.

Sedation can be induced and maintained in 

channel catfish by MS-222 at 20 to 40 p.p.m., 

depending on temperature. Schoettger and 

Julin (1966) cited contradictory reports on
Schoettger, Walker, Marking, and Julin: MS-222 as an Anesthetic for Channel Catfish 13

the benefits of sedating various species of fish 

with the drug for transport. We are unable 

to locate published reports on the use of MS222 for transport of channel catfish.

RESIDUES

The mean residues of MS-222 in the flesh of 

anesthetized channel catfish at 12° and 17° C. 

are approximately two to eight times as great 

as mean residues measured by Walker and 

Schoettger (1966a) in salmonids at the same 

temperatures. The mean values at 7 are 

similar. This indicates that relatively more 

MS-222 is taken up by salmonids at colder 

temperatures than at warmer temperatures, 

whereas the reverse applied to catfish. One 

explanation for the differential deposition of 

MS-222 in salmonids and catfish may be the 

influence of temperature on the efficacy of 

MS-222 in salmonids, and a relative lack of 

its effect in catfish. The concentrations of 

MS-222 -were adjusted upward to maintain efficacy against salmonids at lower temperatures, but only one level, two to three times 

that for salmonids, was used to anesthetize 

catfish in the residue experiments. On the 

other hand, the differential may represent the 

influence of theoretical temperature optimums 

or acclimation on drug metabolism.

Residues of MS-222 in salmonids and 

channel catfish decline rapidly during the first 

hour of withdrawal, except those in catfish at 

7 . The latter are initially low and decrease 

gradually. The acclimation of these individuals 

to winter temperatures may have contributed 

to their low uptake and turnover of MS-222, 

and to their resistance to anesthetization at 

7 . The background of aromatic amines in untreated catfish is slightly higher at 7 and 

suggests a temperature-related reduction in 

metabolism of these substances. Residues of 

the drug, at all temperatures, cannot be distinguished from the background in controls 

when the fish are held in fresh water for 9 to 

24 hours after anesthesia.

Interestingly, the mean concentrations of 

residues at 0-hour withdrawal form a parabolic curve when plotted against temperature, 

and peak at 17 C. We have no explanation for 

this, unless it is caused by a temperaturedependent, uptake-and-metabolism relation. 

The pattern does not persist after withdrawal.

The acetylation of MS-222 may be an effective mechanism for its deactivation in catfish. 

The concentrations of acetylated derivatives 

in muscles of fish treated at 7° increase during withdrawal times up to 9 hours, and as the 

amount of free MS-222 decreases. They decline after 24 hours of withdrawal, when the 

concentration of the free fraction is lowest. 

The inverse change in concentrations of free 

and acetylated derivatives during withdrawal 

suggests metabolism of MS-222. The absolute 

amounts of acetylated substances are relatively small, however, and may simply reflect the turnover of natural aromatic amines.

SUMMARY

MS-222 was tested against channel catfish 

to determine its toxicity, efficacy, and residues 

in muscle. Several size groups of fish were 

exposed to solutions with various temperatures, pH, and water qualities.

The LCso values of MS-222 for catfish 

range from 66.2 to 51.1 p.p.m. in 24- to 96- 

hour bioassays. The variations in toxicity are 

associated with exposure, size of fish, temperature, and water quality. Channel catfish 

are more resistant to the drug than other 

species we have tested. The safety indexes 

for anesthesia of catfish decrease from 3.0 

to 2.4 with exposures of 15 to 60 minutes.

Concentrations of drug above 100 to 140 

p.p.m. are effective for inducing rapid loss of 

equilibrium in catfish within 2 minutes. They 

tolerate exposure for 4 to 11 minutes. A level 

of 70 p.p.m. produces moderately rapid 

anesthesia, and 20 to 40 p.p.m. are effective 

for sedation. The fish tolerate concentrations 

for moderately rapid anesthesia for at least 

30 minutes. They can be maintained in sedation for 6 hours by 20 to 40 p.p.m. The efficacy of MS-222 is not influenced greatly by 

temperature, pH, or size of fish. At 22° or 

27° C., the fish tolerate less exposure to high 

drug concentrations, and at 27° the sedation 

concentration has to be increased to compensate for an apparent degradation of MS-222.
14 Investigations in Fish Control 17: Bureau of Sport Fisheries and Wildlife

The deep stages of MS-222-induced narcosis, 

beyond loss of equilibrium, appear less distinct in catfish than in salmonids. The 

behavior of the fish must be observed to 

minimize the risk of overexposures, and 

cessation of opercular activity is a good 

criterion for transferring them to fresh 

water.

Residues of MS-222 occur in the muscles of 

anesthetized catfish. They vary, according to 

temperature, from 17.0 to 147.2 p.p.m. including background aromatic amines. The 

background, in control fish, ranges from 0.5 

to 3.6 p.p.m. The lowest concentrations of 

residue at 0-hour withdrawal occur in fish 

treated at 7° C., but after 1 hour of withdrawal from the drug the levels in individuals 

exposed at this temperature are highest. The 

residues in muscles after 9 to 24 hours of 

withdrawal at all temperatures decline to 

within the statistical variations in the background concentrations.

The concentrations of acetylated aromatic 

amines in muscles of catfish anesthetized at 

7 C. increase as MS-222 residues decrease 

during 9 hours of withdrawal. The inverse 

relation suggests metabolism of MS-222 by 

acetylation, but the small amounts of acetylated derivatives may be aromatic amines of 

natural origin.

REFERENCES

Bove, Frank J.

1962. MS-222 Sandoz, the anaesthetic of choice 

for fish and other cold-blooded organisms. 

Sandoz News, no. 3f 12 p.

Crawf ord, Bruce, and Andrew Hulsey.

1963. Effects of M.S.-222 on the spawning of 

channel catfish. Progressive Fish-Culturist, 

vol. 25, no. 4, p. 214.

Eisler, Ronald, and Tadeusz Backiel.

1960. Narcotization of chinook salmon fingerlings 

with tricaine methanes^ulfonate (M.S. 222). 

Transactions of the American Fisheries Society, 

vol. 89, no. 2, p. 164-167.

Larsen, Howard N.

1964. Comparison of various methods of hemoglobin determination of catfish blood. 

Progressive Fish-Culturist, vol. 26, no. 1, 

p. 11-15.

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

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

chemicals. U.S. Bureau of Sport Fisheries and 

Wildlife, Circular 185, p. 1-15.

Marking, Leif L.

1966a. Investigations in Fish Control: 10. Evaluation of p,p'-DDT as a reference toxicant in 

bioassays. U.S. Bureau of Sport Fisheries and 

Wildlife, Resource Publication 14, p. 1-10. 

1966b. Investigations in Fish Control: 12. Toxicity of MS-222 to selected fishes. U.S. Bureau of 

Sport Fisheries and Wildlife, Resource Publication 18.

McFarland, William N.

1959. A study of the effects of anesthetics on the 

behavior and physiology of fishes. Publications 

of the Institute of Marine Science, University of 

Texas, vol. 6, p. 23-55.

1960. The use of anesthetics for the handling and 

transport of fishes. California Fish and Game, 

vol. 46, no. 4, p. 407-431.

Moss, D. D., and D. C. Scott.

1964. Respiratory metabolism of fat and lean 

channel catfish. Progressive Fish-Culturist, 

vol. 26, no. 1, p. 16-20.

Sandoz Pharmaceuticals.

(no date) M.S. 222 Sandoz, the anesthetic of 

choice in work with cold-blooded animals. 

Sandoz Pharmaceuticals, Hanover, N.J., Technical Bulletin, 10 p.

Schoettger, Richard A.

1966. Investigations in Fish Control: 16. Annotated bibliography on MS-222. U.S. Bureau of 

Sport Fisheries and Wildlife, Resource Publication 22.

Schoettger, Richard A., and Arnold M. Julin.

1966. Investigations in Fish Control: 13. Efficacy 

of MS-222 as an anesthetic on four salmonids. 

U.S. Bureau of Sport Fisheries and Wildlife, 

Resource Publication 19.

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

Berger.

1964. Investigations in Fish Control: 2. Preliminary observations on the toxicity of 

antimycin A to fish and other aquatic animals. 

U.S. Bureau of Sport Fisheries and Wildlife, 

Circular 186, p. 1-18.

Walker, Charles R., and Richard A. Schoettger.

1966a. Investigations in Fish Control: 15. Residues of MS-222 in four salmonids following 

anesthesia. U.S. Bureau of Sport Fisheries and 

Wildlife, Resource Publication 21.

1966b. Investigations in Fish Control:

14. Method for determination of MS-222 residues in fish. U.S. Bureau of Sport Fisheries 

and Wildlife, Resource Publication 20.

U. S. GOVERNMENT PRINTING OFFICE ! 1972 O - 456-687