INVESTIGATIONS IN FISH CONTROL

41. Identification of MS-222 Residues 

in Selected Fish Tissues 

by Thin Layer Chromatography

42. Dynamics of MS-222 in the

Blood and Brain of Freshwater Fishes 

During Anesthesia

43. Effect of MS-222 on

Electrolyte and Water Content 

In the Brain of Rainbow Trout

United States Department of the Interior

Fish and Wildlife Service 

Bureau of Sport Fisheries and Wildlife
INVESTIGATIONS IN FISH CONTROL

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

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

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

economy.

Current reports in this series are 

(Reports 1 and 2 are in one cover.)

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

Charles R. Walker. 1964. 15 p.

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

Animals, by Charles R. Walker, Robert E. Lennon, and Bernard L. Berger. 1964. 18 p.

(Reports 3 through 8 are in one cover.)

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

Henegar. 1966. 16 p.

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

Robert G. Needham. 1966. 16 p.

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

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

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

Mahdi. 1966. 10 p.

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

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

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

(Reports 9 through 11 are in one cover.)

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

10. Evaluation of j),F>'-DDT as a Reference Toxicant in Bioassays, by Leif L. Marking.

1966. 10 p.

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

Schoettger and Arnold M. Julin. 1966. lip.

(Reports 12 through 17 are in one cover.)

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

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

Arnold M. Julin. 1967. 15 p.

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

Schoettger. 1967. 10 p.

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

Richard A. Schoettger. 1967. lip.

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

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

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

1967. 14 p.

Fish Control Laboratories

Bureau of Sport Fisheries and Wildlife

U.S. Department of the Interior

P.O. Box 862

La Crosse, Wisconsin 54601

Continued on inside back cover 
INVESTIGATIONS IN FISH CONTROL

41. Identification of MS-222 Residues 

in Selected Fish Tissues 

by Thin Layer Chromatography

By John L. Alien, Charles W. Luhning, and Paul D. Harman

42. Dynamics of MS-222 in the Blood and Brain 

of Freshwater Fishes During Anesthesia

By Joseph B. Hunn

43. Effect of MS-222 on Electrolye and Water Content 

in the Brain of Rainbow Trout

By Wayne A. Willford

UNITED STATES DEPARTMENT OF THE INTERIOR, WALTER J. HICKEL, SECRETARY

Leslie L. Glasgow, Assistant Secretary for Fish and Wildlife and Parks

Fish and Wildlife Service, Charles H. Meacham, Commissioner

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

Washington, D.C. July 1970

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

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

Pages

Identification of MS-222 Residues in Selected Fish 

Tissues by Thin Layer Chromatography, by John L. 

Alien, Charles W. Luhning, and Paul D. Harman .......... 1-7

Dynamics of MS-222 in the Blood and Brain of Freshwater Fishes During Anesthesia, by Joseph B. Hunn. ....... 1-8

Effect of MS-222 on Electrolyte and Water Content

in the Brain of Rainbow Trout, by Wayne A. Willford. ...... 1-7

ii
INVESTIGATIONS IN FISH CONTROL

41. Identification of MS-222 Residues 

in Selected Fish Tissues 

by Thin Layer Chromatography

By John L. Alien, Charles W. Luhning, and Paul D. Harman

UNITED STATES DEPARTMENT OF THE INTERIOR, WALTER J. HICKEL, SECRETARY

Leslie L. Glasgow, Assistant Secretary for Fish and Wildlife and Parks

Fish and Wildlife Service, Charles H. Meacham, Commissioner

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

Washington, D.C. July 1970
CONTENTS

Page

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

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

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

Reagents and apparatus ........................... 4

Tissue collection ............................... 4

Sample preparation and extraction. ................... 4

Cleanup of extracts. ............................. 5

Thin layer chromatography ........................ 5

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

References ...................................... 7
IDENTIFICATION OF MS-222 RESIDUES IN SELECTED 

FISH TISSUES BY THIN LAYER CHROMATOGRAPHY

By John L. Alien, Chemist, 

Charles W. Luhning and Paul D. Harman, Physical Science Technicians

Bureau of Sport Fisheries and Wildlife 

Southeastern Fish Control Laboratory, Warm Springs, Georgia

ABSTRACT. MS-222, a commonly used fish anesthetic, reacts with the 

Bratton-Marshall reagents to form a wine-red dye. Residues of MS-222 

determined by this reaction are not distinguished from other primary 

aromatic amines. Thin layer chromatography was used to identify MS-222 

in the presence of background primary aromatic amines in fish muscle, 

brain, and blood. This method, in which the Bratton-Marshall reaction is 

used to visualize the spots, gave both the specificity of the Bratton-Marshall 

reaction for primary aromatic amines and the Rf of MS-222 as tools for 

identification of the residues. Recoveries of 25 to 60 percent were obtained 

in muscle samples spiked with 2 to 10 ppm of MS-222. Quantitative estimation was difficult in samples spiked with 2 ppm or less, but presence 

of MS-222 residues could be confirmed in samples spiked with as little as 

0.2 ppm. Since the meta-aminobenzoate ester can be identified at these 

concentrations, this should be a useful ancillary or confirmatory method 

for determining the rate of disappearance of drug residues in fish flesh 

and obtaining data for clearance and registration of the anesthetic with the 

Food and Drug Administration.

MS-222 (methanesulfonate of meta- to investigate the application of thin layer 

aminobenzoic acid ethyl ester) is used exten- chromatography, using modified Brattonsively as an anesthetic for fish. Walker and Marshall reagents, to visualize MS-222 resiSchoettger (1967) described a method for quan- dues in fish tissue. This system offers both 

titative determination of MS-222 residues in the specificity of the Bratton-Marshall reaction 

fish tissues using a modification of the Bratton- for primary aromatic amines and the Rf of 

Marshall (1939) method for sulfonamides. MS- MS-222 as tools for the identification of the 

222, a primary aromatic amine, gives a compound, 

wine-red color when reacted with the BrattonMarshall reagents. Since other primary aromatic

amines give the same color, a method for specific METHODS AND MATERIALS 

identification of MS-222 residues is needed.

Experiments on operating parameters

Thin layer chromatography has been useful "

in identifying primary aromatic amines. This Various solvent systems were investigated 

technique was used by Bican-Fister and for possible use as developers of the chroKajganovic (1963) to visualize sulfonamides on matograms on silica gel chromatography 

thin layer plates by spraying the plates with sheets without fluorescent dye. When a solvent 

modified Bratton-Marshall reagents. We chose system of 2-percent methanol in benzene was
Investigations in Fish Control 41: Bureau of Sport Fisheries and Wildlife

used, MS-222 gave an Rf of approximately 0.5. 

However, a substance occurring in certain untreated carp (Cyprinus carpio) and goldfish 

(Carassius auratus) tissues gave the same red 

color with about the same Rf as MS-222 when 

this system was employed. This problem was 

overcome by using a solvent system of 91 percent benzene, 4 percent acetic acid, and 5 percent ethyl ether. With this system MS-222 

gave an Rf of about 0.45, while the interfering 

substance in carp and goldfish tissue did not 

migrate above the spotting line.

We used the diazo coupling agent of Bratton 

and Marshall (1939) to develop a red-colored 

azo dye in the MS-222 spot on the chromatogram. This diazotization reaction with MS-222 

was described in detail by Walker and Schoettger (1967). We found that this coupling reagent in spray solution gave the darkest spot 

when 0.2-percent sodium nitrite in 1.5-percent 

hydrochloric acid was used. After the plates 

were sprayed with the acidic nitrite solution, 

they were dried in a stream of hot air to destroy the excess nitrous acid. A coupling 

reagent of 0.1-percent N-1-naphthylethylenediamine dihydrochloride in water was satisfactory.

The minimum time required for the extraction of MS-222 residues from fish muscle in a 

Soxhlet extractor was determined. Fish muscle 

samples fortified with MS-222 were analyzed 

after 4, 8, 12, 16, and 24 cycles. Extraction 

was essentially complete after 8 cycles.

Reagents and apparatus

Reagents and apparatus were as follows 1 :

1. Methanol, reagent grade.

2. Petroleum ether, reagent grade.

3. Florisil, 100-200 mesh.

4. Alumina, neutral, Brockman activity 1, 

80-200 mesh.

5. Developing solution: 91-percent benzene 

(pesticide grade), 4-percent acetic acid 

(reagent grade), and 5-percent ethyl ether 

(U.S.F.).

Reference to a company or product does not imply 

recommendation to the exclusion of others that may be 

suitable.

6. 0.2-percent sodium nitrite in 1.5-percent hydrochloric acid: Dissolve 0.20 g 

of sodium nitrite in 50 ml of water, add 

1.5 ml of concentrated hydrochloric acid 

and dilute to 100 ml with distilled water. 

Make fresh daily.

7. 0.1-percent N-1-naphthylethylenediamine 

dihydrochloride: Dissolve 0.10 g in 100 

ml of distilled water. Store in a dark container, refrigerate, and make fresh 

weekly.

8. Standard solution of MS-222: Dissolve 

10.0 mg of MS-222 in 100 ml of methanol.

9. Tissue homogenizer.

10. Silica gel thin layer plates, Eastman 

Chromogram Sheet without fluorescent 

dye, 20 x 20 crn.

11. Micropipettes, 1, 5, and 10 gl.

12. Chromatography tank, 4 by 8 by 9 inches, 

lined with absorbent paper.

13. Chromatographic column, 400 x 24 mm 

I.D. with sealed in, coarse fritted disk.

14. Soxhlet extraction apparatus, I.D. of extraction tube 30 mm ; 80 x 25 mm 

thimbles.

15. Teflon coated muffin pan.

Tissue collection

Blood samples are drawn from specimens by 

cardiac puncture. (Walker and Schoettger, 1967) 

with a heparinized syringe fitted with an 18- or 

20-gage needle.

Other tissue samples are collected from fish 

after killing them by a blow on the head or by 

pithing. Brain samples are dissected out of the 

immobilized fish, and muscle samples are collected by filleting the fish.

Sample preparation and extraction

Blood samples are extracted by adding 0.5ml 

blood to 9.5 ml methanol, mixed thoroughly and 

applied to the chromatographic column for cleanup.

Brain samples are extracted by homogenizing 

1 g of brain, or the entire brain if it weighs less 

than 1 g with 5 ml of methanol. The homogenate 

is then placed in the chromatographic column 

for cleanup.
Alien, Luhning, and Harman: Identification of MS-222 Residues by Chromatography

Muscle samples (5 g) are taken from a homogenate of the entire fillet, spread thin in the 

bottom of the muffin pan, and dried at 80°C. 

for 6 hours. Grind the dried tissue to a powder 

with a mortar and pestle, transfer to a Soxhlet 

extraction thimble, and wash with three 15-ml 

portions of petroleum ether (discard the washings). Air-dry the washed sample and extract 

with 70 ml of methanol in a Soxhlet for a minimum of 8 reflux cycles. After the final cycle, 

allow the upper portion of the extractor to fill, 

but not to siphon over. The extract and the remaining methanol in the boiling flask is ready 

for column chromatographic cleanup.

Cleanup of extracts

Prepare a 400-mm by 24-mm column by 

adding 2.5 cm of alumina followed by 7.5 cm 

of Florisil. Tap the column gently to pack the 

adsorbent. Prewash the column with 50 ml of 

methanol. When the methanol wash just sinks 

into the surface of the column, add the sample 

extract to the column and begin collecting the 

eluate in a 100-ml beaker. Discard the methanol prewash. Rinse the flask which contained 

the extract 3 times with 2-ml portions of 

methanol, adding each consecutive rinse to the 

column just as the previous rinse disappears 

into the surface of the column. As the last of 

the methanol rinse sinks into the column 

surface, add 50 ml of methanol and collect the 

eluate only until the last of the methanol has 

disappeared into the surface of the column.

Use a hot water bath and a stream of dry air 

to concentrate the eluate to 3 to 5 ml. Quantitatively transfer the eluate to a 15-ml graduated centrifuge tube with methanol. Place the 

centrifuge tube in a hot water bath and concentrate the eluate to 0.5 ml under a stream 

of dry air.

as determined by the method of Walker and 

Schoettger (1967), spot a volume of extract 

equivalent to 0.5^/g of MS-222. To compensate 

for interferences inherent in the colorimetric 

procedure, spot 100 £.1 of extract from samples containing 2.0 ppm or less of MS-222 

residue.

When screening unknown samples which 

may contain MS-222 residues, a maximum of 

lOO^/1 of sample extract is spotted on the 

spotting line. On the same line, spot 50, 10, 

and 5 g 1 of extract along with a series of 

MS-222 standards in the range of 0.1 fig to 

1.0 £g.

Thirty minutes before developing the thin 

layer plate, pour 200 ml of developing solution into a chromatographic tank lined with 

absorbent paper. Place the thin layer plate in 

the tank and allow the developing solution to 

rise to the previously marked solvent-front 

line. Remove the plate from the tank, mark 

any deviations of the solvent front, and allow 

to air-dry in a horizontal position.

Spray the plate with the acidic nitrite solution until the plate is uniformly damp, wait 

3 to 5 minutes, and dry in a stream of hot air. 

When the plate is completely dry, spray with 

0.1 -percent ^N- 1-naphthy lethylenediamine 

dihydrochloride solution until damp and dry 

immediately with hot air.

MS-222 is seen as a red spot. The amount 

of MS-222 can be estimated by comparing the 

intensity and size of the sample spot to the 

MS-222 standard spots. MS-222 standards 

must be run simultaneously with the samples 

so direct comparison of the Rf of standard and 

sample can be made on the same plate. After 

quantitiative estimation is complete, store 

plate in a dark dry container.

Thin layer Chromatography

Mark a spotting line 2.5 cm, and a solventfront line 12.5 cm, from the bottom of an 8- 

by 8-inch thin layer plate.

When thin layer Chromatography is employed 

to confirm the presence of MS-222 residues,

RESULTS AND DISCUSSION

The Bratton-Marshall color reaction is 

specific for primary aromatic amines. Therefore, any naturally occurring primary aromatic amine or drug containing a primary 

aromatic amine group develops a color when 

treated with the Bratton-Marshall reagents.
Investigation in Fish Control 41: Bureau of Sport Fisheries and Wildlife

The presence of low levels of MS-222 residue 

is difficult to ascertain by the modified Bratton-Marshall method of Walker and Schoettger 

(1967) because of the background readings 

obtained from the tissues being analyzed.

Thin layer chromatography separates MS222 from nine other chemicals containing the 

primary aromatic amine group. The Rf values 

for 0.5-/*g spots of MS-222 and nine other 

compounds containing the primary aromatic 

amine group are shown in table 1. The comparison of Rf values must be made on the 

same plate since these values may vary between determinations.

The minimum level at which quantitative 

estimations can be made from MS-222 standard spots was found to be 0.1//g. The maximum amount of muscle extract that can be 

spotted on the thin layer plate was found to be 

approximately 100 gl, which is equivalent to 

1 g of tissue.

The efficiency of the method was evaluated 

by analyzing muscle samples from channel 

catfish (Ictalurus punctatus) spiked with 0.2 

to 10.0 ppm of MS-222 (table 2). Recoveries 

of MS-222 from samples spiked with 2.0 to 

10.0 ppm ranged from 25 to 60 percent. Quantitative estimation becomes difficult at residue levels of 2.0 ppm or less owing to the 

large amount of sample which must be spotted. 

When large amounts of samples are spotted, 

accurate quantitation is prevented by spreading of the spot caused by interfering fats and 

other extraneous materials. However, presence of MS-222 in muscle tissue was confirmed 

in samples spiked with as little as 0.2 ppm of 

MS-222. The samples were spiked by injecting 

a methanol solution of MS-222 standard into 

the samples before they were oven-dried.

The method was effective for eliminating 

background interferences in the analysis of 

muscle tissue from 8 species of fish (table 3). 

A red spot was noted only in goldfish and carp, 

but it did not migrate above the spotting line.

Brain and blood samples from three channel 

catfish treated with 100 ppm of MS-222 to deep 

anesthesia were analyzed by thin layer chromatography after 0-hour, 1/2-hour, and 1-hour

Table 1. Rf values for MS-222 and nine other compounds 

whicn produce a red color by the MS-222 thin layer 

chromatographic method when the 91 percent benzene, 4 

percent acetic acid, and 5 percent ethyl ether developing solution was used and 5 01 of a 100-ppm solution 

of each amine was spotted

Compound R value MS-2221

MS-222 (tricaine methanesulf onate ) . 0.41 

0.35 

0.27 

0.16 

0.07 

0.07 

0.00 

0.00 

0.00

1.00 

0.85 

0.66 

0.39 

0.17 

0.17 

0.00 

0.00 

0.00

of sample

Rf of MS-222 standard 

2 Penicillin G, procaine, 300,000 u/cc.

Table 2. Estimated recovery of MS-222 spiked into 5-g samples of 

channel catfish muscle as determined by the thin layer chromatographic method

Tr trace

Concentration 

of spike

Number 

of 

fish

Equivalent

amount of 

muscle 

spotted 1

(g)

Estimated

amount of 

MS-222 

detected

(ftg)

Estimated

concentration of 

MS-222

(ppm)

cent 

reControl. .....-- ^ i _nn n.n n.n

0.2 ppm.

0.5 ppm.

1.0 ppm.

2.0 ppm.

5.0 ppm.

10.0 ppm.

4 1.00 Tr Tr Tr

4 1.00 Tr-0.3 Tr-0.3 Tr-60

3 0.50-0.75 Tr-0.6 Tr-0.3 Tr-80

4 0.50-1.00 0.5-0.6 0.5-0.6 25-30

4 0.10 0.2-0.3 2.0-3.0 40-60

4 -0.05 0.2-0.3 4.0-6.0 40-60

1 High and low amounts of tissue spotted do not necessarily coincide with high and low amounts of MS-222 detected.

Table 3. Analyses of eight species of untreated fish for the presence of naturally occuring interferences by the thin layer cnromatographic method for MS-222

Species

Northern pike, Es_ox lucius««-

Muskellunge, Esox masquinongy

Goldfish, Carassius auratus..

Channel catfish, Ictalurus

Bluegill, Lepomis

Largemouth bass, Micropterus

Walleye, Stizostedion

o_ _ Rf of sample

Equivalent

amount of 

muscle 

spotted

(g)

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

Red spot Rf 

detected spc

No

No

Yes 0.

No

No

No

of RMS-222 

t

..

00 1 0.00

__

__

0.00

"""" R- of MS-222 standard 0.41 ~ "'^

withdrawals in fresh water. The cleanup on 

these samples was satisfactory for thin layer 

chromatography. No recoveries were run on 

these tissues as the cleanup procedure for 

blood and brain is identical to that of muscle
Alien, Luhning, and Harman: Identification of MS-222 Residues by Chromatography

tissue. Residues of MS-222 were shown to be 

present in each sample of blood and brain by a 

red spot having the same Rf as the MS-222 

standards.

Kidney and liver samples could not be analyzed for MS-222 residues by this method, because our cleanup procedure was not effective 

for these tissues.

Since we were able to effectively isolate, 

recover, and identify trace concentrations of 

meta-aminobenzoate ester, this should be a 

useful ancillary or confirmatory method for 

determining the rate of disappearance of residues in fish flesh. To obtain clearance and 

registration of MS-222 as an anesthetic with 

the Food and Drug Administration, we must

generate residue data by analytical methods of 

sufficient sensitivity and reliability with confirmation by an ancillary method.

REFERENCES

Bican-Fister, T., and V. Kajganovic.

1963. Separation and identification of sulfonamides 

by thin layer Chromatography. Journal of Chromatography, vol. 11, no. 4, p. 492.

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.

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

1967. Method for determining MS-222 residues in 

fish. U.S. Bureau of Sport Fisheries and Wildlife, 

Investigations in Fish Control, No. 14. 10 p.
INVESTIGATIONS IN FISH CONTROL

42. Dynamics of MS-222 in the

Blood and Brain of Freshwater Fishes 

During Anesthesia

By Joseph B. Hunn

UNITED STATES DEPARTMENT OF THE INTERIOR, WALTER J. HICKEL, SECRETARY

Leslie L. Glasgow, Assistant Secretary for Fish and Wildlife and Parks

Fish and Wildlife Service, Charles H. Meacham, Commissioner

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

Washington, D.C. July 1970
CONTENTS

Page

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

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

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

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

Summary ...................................... 7

References ..................................... 7
DYNAMICS OF MS-222 IN THE BLOOD AND BRAIN OF 

FRESHWATER FISHES DURING ANESTHESIA

By Joseph B. Hunn, Fishery Biologist

Bureau of Sport Fisheries and Wildlife

Fish Control Laboratory, La Crosse, Wisconsin

ABSTRACT. Eleven species of freshwater fishes were rapidly anesthetized in solutions of MS-222 containing from 100 to 1,000 milligrams 

of MS-222 per liter. MS-222 concentrations in blood and brain after 1 

minute of exposure indicate that MS-222 rapidly diffuses across the gill 

and passes the blood-brain barrier. Evidence of metabolism of the drug 

was seen in the presence of acetylated MS-222 in the blood of all species 

studied. The concentration of free MS-222 in the brain increased with depth 

of anesthesia to loss of reflex and then either increased or declined 

slightly as the fish approached medullary collapse.

MS-222 (methanesulfonate of metaaminobenzoic acid ethyl ester) is an effective 

fish anesthetic when administered by immersing fish in a solution or by spraying it on 

their gills (Schoettger, 1967). In either case, 

the route of entry is the gills. MS-222 is a 

lipid-soluble compound which is only 0.01- 

percent ionized at body pH (Maren, Embry, 

and Broder, 1968). This lipid solubility most 

likely accounts for its rapid diffusion across 

the gills.

Once the drug enters the bloodstream, it is 

distributed throughout the body. Although 

the site of action of MS-222 has not been 

established, it is thought to be in the brain. 

The blood-brain barrier in fish is known to 

exclude certain dyes, such as sulfonilic acid, 

from the cerebrospinal fluid (Rail, 1967). 

Preliminary investigations by Stenger and 

Maren (1968) indicate that MS-222 effectively 

crosses this barrier in the dogfish shark 

(Squalus acanthias). My studies were designed to extend this observation by measuring the rate of uptake of MS-222 in blood and 

brain of freshwater fish during the induction 

of anesthesia.

METHODS AND MATERIALS

Eleven species of fish were obtained from 

several sources (table 1). All specimens were 

maintained according to the methods of Hunn, 

Schoettger, and Whealdon (1968), except carp

Table 1. Sources and sizes of fish used in the MS-222 uptake studies

. Length 

SP60168 (inches)

Shortnose gar 

Lepisosteus platostomis 21.5-27.5

Longnose gar 

Lepisosteus osseus 27.0-33.5

Bowfin 

Ami a calva 23.0-31.0

Rainbow trout 

Sulmo gairdneri 11.0-16.0

Northern pike 

Esax lucius 10.0-18.0

Carp 

Cyprinus carpio 8.5-10.8

Spotted sucker 

Miavtrema melanops 11.8-14.3

Black bullhead

Ictalurus melas 7.0-9,6

Channel catfish 

Ictalurus punctatus 9.8-15.8

White bass 

Roccus chrysops 12.2-14.0

Bluegill 

Leponds macrochirus 7.5-9.3

, Wei^ Source (grams)

Mississippi River 

Guttenberg, Iowa

Mississippi River 

Guttenberg, Iowa

Mississippi River 

Guttenberg, Iowa

National Fish Hatchery 

296-720 Manchester, Iowa

Mead Wildlife Area 

Marshfield, Wis.

Mississippi River 

140-350 Genoa, Wis,

Mississippi River 

Guttenberg, Iowa

Mead Wildlife Area

84-220 Marshfield, Wis.

Mississippi River 

104-540 Lansing, Iowa

Mississippi River 

435-620 Guttenberg, Iowa

National Fish Hatchery 

197-345 Fairport, Iowa
Investigations in Fish Control 42; Bureau of Sport Fisheries and Wildlife

and black bullheads, which were held at 17° C. 

The anesthetic solution of MS-222 in well 

water was made up fresh daily. The MS-222 

was technical grade (99.4 percent) methanesulfonate of m-aminobenzoic acid ethyl ester 

obtained from Sandoz Pharmaceuticals. Desired concentrations of the drug were achieved 

by adding the crystaline material to measured 

volumes of well water in 5-gallon stainlesssteel pails or in 45- or 100-liter polyethlene 

tanks. Individual fish were immersed in the 

anesthetic solution for periods of 1, 3, 5, 8, 

or 11 minutes. All fish were anesthetized to 

loss of reflex, and most were nearing medullary collapse in 8 to 11 minutes of exposure.

Blood samples were taken by caudal puncture (Steucke and Schoettger, 1967). The 

spinal cord of the fish was then severed and 

the brain removed. Concentrations of MS-222 

and background primary aromatic amines in 

whole blood and brain were determined by the 

Bratton-Marshall method as modified by 

Walker and Schoettger (1967). The average 

concentration of background amines was subtracted from total aromatic amines to determine the concentration of MS-222.

RESULTS

MS-222 moves rapidly across the gills and 

enters the bloodstream of fishes (table 2). 

Within 1 minute of exposure, the drug concentration greatly exceeds the background 

level of primary aromatic amines. The ratio 

of the highest average concentration of MS222 in whole blood to that of the anesthetic 

solution ranged from 0.14 in shortnose gar to 

0.83 in rainbow trout.

Background primary aromatic amines in 

whole blood ranged from 0.6 to 5.4 milligrams per liter (mg/1) as free amines, and 

0.0 to 4.0 mg/1 as acetylated amines.

In seven of the eleven species, the brain 

concentration of MS-222 exceeded that of the 

whole blood after the first minute of exposure (table 2). The brains of all species 

contained amounts of MS-222 in excess of 

those in the blood after 3 minutes.

Table 2. Concentration of MS-222 in whole blood and train of 11 species of fish during the induction of anesthesia

[Condition of anesthesia for each species listed in table 3]

Species and 

exposure 

time

Concentration in ppm

Whole blood

Free MS-222

n Mean Range

Acetylated MS-222

n Mean Range

Brain

Free MS-222

n Mean Range

Brain - 

blood 

ratio

Shortnose gar:

0 min. 1 ...........

1 min. ............

3 min.............

8 min .............

Longnose gar:

0 min. 1 . ..........

1 min. ............

3 min .............

5 min. ............

8 min. ............

Bowf in :

0 min. 1 ...........

1 min. ............

3 min. ............

5 min .............

8 min .............

...... 2

...... 2

...... 2

...... 2

...... 2

...... 2

...... 1

...... 2

...... 2

...... 2

...... 2

...... 2

...... 2

...... 2

...... 2

0.6

143. 2

135.0

108.2

122.8

2.1

74.7

135.3

114.3

127.1

3.9

202.2

178.5

186.0

117.3

0.6

93.8-192

135.0

105.4-111

95.0-150

1.6-2.6

_

126.4-144

110.0-118

124.8-129

3.2-4,6

163.6-240

166.6-190

184.6-187

100.0-134

.6

.0

.6

.2

.6

.4

.8

.4

.4

.6

2

2

2

2

2

2

1

2

2

2

2

2

2

2

2

1.8

10.3

8.2

8.8

0.9

1.2

2.8

8.5

1.7

8.0

0.2

105.1

64.0

37.0

0.0

1.0-2.6

0.0-20.6

7.8-8.6

8.6-9.0

0.0-1.8

0.6-1.8

_

0.0-17.0

0.0-3.4

5.8-10.2

0.0-0.4

75.0-135.2

6.6-121.4

34.2-39.8

-

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

3-6

135.6

342.8

269.6

230.4

2.9

88.3

265.1

277.5

228.7

8.6

58.3

217.3

188.8

222.8

3.2-4.0

128.8-142.4

301.6-384.0

238.4-300.8

190.8-270.0

2.4-3.4

58.8-117.8

221.6-308.6

262.4-292.6

225.6-231.8

7.2-10.0

45.6-71.0

157.8-276.8

184.8-192.8

192.8-252.8

6.0

0.95

2.54

2.49

1.88

1.38

1.18

1.96

2.43

1.80

2.21

0.29

1.22

1.02

1.90

See footnotes at end of table.
Joseph B. Hunn: Dynamics of MS-222 in Freshwater Fishes

Table 2. Concentration of MS-222 in whole blood and brain of 11 species of fish during the induction

of anesthesia Continued

Species and 

exposure 

time

Concentration in ppm

Whole blood

Free MS-222

n Mean Range

Acetylated MS-222

n Mean Range

Brain

n

Free MS-222

Mean Range

Brainblood 

ratio

Rainbow trout:

0 min1 . .............

1 min. ..............

2 min. ..............

4 min. ..............

6 min ...............

10 min...............

Northern pike:

0 min1 .............

3 min. ..............

5 min ..............

8 min. .............

Carp:

0 min1 .............

1 min. .............

3 min ..............

5 min. .............

8 min. .............

11 min ..............

Spotted sucker:

0 min. 1 ............

3 min ..............

5 min ..............

8 min. .............

11 min. .............

Black bullhead:

0 min. 1 ............

3 min ..............

5 min. .............

8 min ..............

11 min. .............

Channel catfish:

0 min. 1 . ...........

1 min ..............

3 min. .............

5 min. .............

8 min..............

11 min ..............

White bass:

0 min. 1 ...........

1 min. ............

3 man. ............

5 min. ............

8 min. ............

Bluegill:

0 min. 1 ...........

1 min. ............

3 min. ............

5 min. ............

8 min. ............

11 min. ............

..... 8

..... 4

..... 5

..... 5

..... 4

..... 5

..... 6

..... 6

..... 6

..... 6

..... 6

..... 6

..... 6

..... 6

..... 6

..... 6

..... 6

..... 6

..... 2

..... 2

..... 2

..... 2

..... 2

..... 8

..... 6

..... 5

..... 6

..... 6

..... 6

..... 16

..... 4

..... 4

..... 4

..... 4

..... 4

..... 3

..... 2

..... 5

..... 2

..... 2

..... 6

..... 6

..... 6

..... 12

..... 6

..... 6

1.7

69.2

51.9

68.6

68.5

83.1

1.9

35.9

81.9

86.0

95.7

1.4

63.9

96.0

90.2

100.8

89.5

1.8

113.4

100.3

108.9

121.0

102.7

2.3

68.8

121.5

145.5

146.8

127.9

1.2

114.4

137.3

116.4

106.7

115.2

4.1

42.8

86.7

90.3

88.9

1.9

62.7

104.3

121.8

98.5

95.5

1.3-2.8

43.3-104.2

42.7-66.8

49.9-82.2

60.9-72.6

66.3-94.2

1.0-2.4

23.0-57.6

60.4-111.6

73.0-100.4

83.0-105.6

1.2-2.0

34.4-82.0

78.4-114.0

82.8-97.8

88.8-115.6

72.4-105.0

1.6-2.2

86.2-140.6

66.0-134.6

58.8-159.0

103.8-138.2

83.2-122.2

1.6-3.0

42.0-148.0

102.6-174.2

109.0-197.0

130.4-165.8

110.0-156.2

0.6-1.8

104.4-120.6

123.8-164.0

99.8-120.4

94.0-111.0

104.2-140.8

3.4-5.4

39.7-45.9

44.3-102.5

84.7-95.9

86.3-91.5

1.4-2.6

27.6-93.4

87.2-123.0

87.7-134.1

91.0-104.6

74.2-117.0

8

4

5

5

4

5

5

6

5

5

6

6

6

6

6

5

6

6

2

2

2

2

2

8

6

4

5

6

6

16

4

4

4

4

4

3

2

5

2

2

6

6

5

11

6

6

0.7

3.8

3.1

1.7

2.0

2.9

0.7

1.5

3.4

13.1

7.7

0.7

3.8

2.5

17.2

1.7

3.5

0.0

0.0

10.7

6.6

0.7

1.5

0.2

0.4

23.1

19.3

10.9

11.1

1.2

3.2

11.7

11.4

7.6

9.2

0.4

3.8

10.1

21.2

13.8

0.9

2.9

5.1

12.8

17.5

8.7

0.4-1.1

0.6-7.9

0.6-6.9

0.0-5.3

0.4-3.1

0.0-4.9

0.4-4.0

0.0-3.9

1.3-5.3

4.0-16.8

2.4-11.2

0.0-2.8

0.0-17.2

1.4-2.8

3.4-29.8

0.0-2.6

0.0-6.3

-

_

7.0-14.4

6.4-6.8

0.0-1.4

1.0-2.0

0.0-0.6

0.0-2.8

0.0-62.2

12.0-27.4

1.2-15.4

8.0-15.4

0.6-1.6

0.0-8.2

6.2-14.2

0.8-19.6

2.2-9.8

5.6-13.2

0.0-1-2

3.6-4.0

2.8-14.4

14.8-27.6

12.8-14.8

0.0-2.2

0.0-8.6

0.0-9.2

0.0-63.9

12.0-20.6

0.0-14.6

12

8

8

8

5

5

^3

23

23

^3

23

^3

23

23

23

23

23

^3

^P

^P

P

P

P

^4

2 3

23

23

23

23

8

4

4

4

3

4

3

2

5

2

2

^3

23

^3

^6

^3

23

3.2

116.0

145.1

165.4

156.8

154.1

1.8

66.9

152.9

204.6

248.1

1.1

54.6

164.1

165.8

190.0

156.5

4.9

35.5

143.7

190.0

200.3

184.1

4.8

111.3

162.8

227.1

266.9

240.5

3.9

188.6

260.3

235.6

182.3

217.9

2.7

47.7

109.7

115.1

130.9

3.1

89.0

168.2

174.2

196.5

174.2

2.3-5.6

107.7-125.7

136.8-150.7

159.1-169.2

146.1-172.0

144.9-159.6

1.6-2.2

60.6-78.4

140.8-158.6

183 . 6-223 . 8

230.4-257.8

1.0-1.4

44.2-66.6

155.8-171.4

151.4-192.6

187.0-192.6

134.6-176.2

4.0-6.6

-

-

-

-

-

4.0-5.5

102.8-119.6

140.3-185.7

194.4-259.7

252.0-284.5

220.0-250.5

2.6-4.6

178.1-198.1

239.7-290.5

227.7-243.7

150.7-240.2

184.5-258.6

2.0-3.8

45.1-50.3

77.1-132.9

104. 9- 125. 3

125.3-136.5

2.8-3.2

67.6-109.6

152.4-189.2

329.3-206.5

180.0-208.0

163.6-179.6

1.83

1.68

2.80

2.41

2.29

1.85

0.95

1.86

1.87

2.38

2.59

0.79

0.85

1.71

1.84

1.88

1.75

2.7

0.3

1.4

1.7

1.6

1.8

2.09

1.62

1.34

1.56

1.82

1.88

3.25

1.65

1.89

2.02

1.71

1.89

0.65

1.11

1.27

1.27

1.47

1.61

1.42

1.61

1.43

1.99

1.82

-" Background level of primary aromatic amines. 

2P = pooled sample, 2 brains per sample.
6 Investigations in Fish Control 42: Bureau of Sport Fisheries and Wildlife

Table 3. Concentrations of MS-222 in blood and brain of 11 species of fish at loss of reflex stage of anesthesia

Shortnos 

Longnose 

Bowf in. .

Rainbow 

Northern

Spotted 

Black bu 

Channel 

White ba 

Bluegill

m . Anesthetic Temperature Species 0/1 concentratic

G' (us/1)

e fTa-r* i o i nnn

gar. ........................ 12 800

............................. 12 1,000

trout........................ 12 100

pike........................ 12 150

............................. 17 200

sucker....................... 12 200

llhead. ...................... 17 200

catfish. ..................... 12 200

ss........................... 12 150

............................. 12 200

Time in Average concentration 

anesthetic of free MS-2221

of reflex In whole blood In brain 

(minutes) (mg/1) (mg/kg)

2-3 135.0 342.8 

2-3 135.0 265.1 

2-3 178.0 217.3 

3-4 68.6 165.4 

2-3 81.9 152.9 

3-4 96.0 164.1 

2-3 100.3 143.7 

5-6 145.5 227.1 

2-3 137.3 260.3 

2-3 83.6 107.1 

2-3 104.3 168.2

Average concentrations of free MS-222 compiled from table 2.

A minimum concentration of 100 milligrams per kilogram (mg/kg) of free MS222 appears to be necessary for anesthesia 

to loss of reflex judging from the average 

concentrations measured in the brain of 11 

species (table 3).

DISCUSSION

Diffusion of MS-222, a highly lipid-soluble 

nonpolar drug, across the gills of fish is 

quite rapid. Movement of the drug may be in 

either direction depending on the concentration gradient. This study has shown concentrations of MS-222 in both blood and brain 

greatly in excess of background amines after 

1 minute of exposure to the anesthetic solution. As shown by Maren, Embry, and Broder 

(1968) in their study on the dogfish shark, 

the gill is quite efficient in clearing the blood 

of MS-222 during recovery from anesthesia. 

Hunn, Schoettger, and Willford (1968) have 

indirectly measured the same phenomenon in 

rainbow trout. Preliminary investigations by 

Maren, Broder, and Stenger (1968) showed 

that the nonpolar ethyl m-aminobenzoate and 

its N-acetyl derivative are both excreted 

across the gill while the polar m-aminobenzoic 

acid and its N-acetyl derivative are excreted 

via the kidney. Most of the MS-222 and its 

congeners are excreted via the gills during 

recovery; 95 percent in the dogfish shark 

(Maren, Embry, and Broder, 1968) and 79 to 

85 percent in the rainbow trout (Hunn, 

Schoettger, and Willford, 1968).

Concentrations of MS-222 in whole blood 

(table 2) drawn via caudal puncture did not 

reach the levels in the anesthetic solutions 

during exposures as long as 11 minutes (fish 

approaching medullary collapse). This is 

probably due to the fact that blood drawn by 

this method is usually venous blood which 

would contain a lesser concentration of the 

drug than arterial blood until the drug is in 

equilibrium between the fish and the anesthetic solution.

The appearance of acetylated MS-222 in 

most blood samples indicates that all 11 

species are able to metabolize it. Highest 

concentrations of acetylated drug were 

usually detected after 3 to 5 minutes of exposure. The bowfin had the greatest blood 

concentration of acetylated MS-222 of any of 

the 11 species studied, 34 percent after a 1- 

minute exposure. Concentrations in the 10 

species were usually less than 20 percent. 

Maren, Broder, and Stenger (1968) found the 

same level of acetylated drug in the plasma 

of the dogfish shark during recovery from 

anesthesia.

Stenger and Maren (1968) reported that during MS-222 anesthesia of the dogfish shark, 

the drug rapidly reaches the cerebrospinal 

fluid and the brain. My observations confirm 

this finding. In all 11 species, the concentration of free MS-222 in the brain was significantly above background after 1 minute of exposure. The concentration of drug in the brain
Joseph B. Hunn: Dynamics of MS-222 in Freshwater Fishes

increased with depth of anesthesia to loss of 

reflex. With deeper anesthesia, the concentration of free MS-222 either increased 

slightly or declined in comparison with the 

concentration at loss of reflex. A concentration of at least 100 mg/kg is necessary for 

anesthesia to loss of reflex in susceptible 

species like rainbow trout, whereas the more 

resistant species like black bullhead require 

approximately 200 mg/kg of the free drug for 

a similar level of anesthesia.

In a previous paper (Hunn, 1968) I noted that 

rapid recovery in fresh water is associated 

with the declining concentration of free MS-222 

in the brain of channel catfish. The brain concentration of free drug was 91.6 mg/kg when 

the catfish righted themselves whereas it was 

260.3 mg/kg when they exhibited loss of reflex. Indeed, in all studies published to date 

anesthesia and recovery in fresh water have 

been strictly associated with the concentration 

of free drug in the blood and brain (Schoettger 

et al., 1967; Walker and Schoettger, 1967b).

SUMMARY

Eleven species of freshwater fish were 

rapidly anesthetized in solutions of MS-222 

containing from 100 to 1,000 mg/1 of drug. 

MS-222 (free and acetylated) concentrations 

in whole blood and brain after 1 minute of exposure indicate that MS-222 rapidly diffuses 

across the gill and passes the blood-brain 

barrier. Blood samples drawn by caudal puncture contained lower concentrations of MS-222 

than those of the anesthetic solutions.

The presence of acetylated MS-222 in the 

blood of all species studied is evidence that 

fish metabolize the drug. Concentratins of 

acetylated drug were usually less than 20 percent of the total MS-222 except those in bowfin 

which had 34-percent acetylation after a 1- 

minute exposure.

MS-2 22 rapidly enters the brain from the 

blood. The concentration in the brain increases with depth of anesthesia to loss of 

reflex. As fish enter more deeply into anesthesia, the drug concentration either increases 

slightly or declines in ratio to the levels at 

loss of reflex. Anesthesia and recovery in 

fresh water appears to be associated with the 

concentration of free MS-222 in the blood and 

brain.

REFERENCES

Hunn, Joseph B.

1968. Anesthetics physiology. In Progress in Sport 

Fisheries Research 1967, p. 119-120.U.S. Bureau 

of Sport Fisheries and Wildlife, Resource Publication 64.

Hunn, Joseph B., Richard A. Schoettger, and Everett W. 

Whealdon.

1968. Observations on handling and maintenance of 

bioassay fish. Progressive Fish-Culturist, vol. 

30, no. 3, p. 164-167.

Hunn, Joseph B., Richard A. Schoettger, and Wayne A. 

Willford.

1968. Turnover and urinary excretion of free and 

acetylated M.S. 222 by rainbow trout, Salmo 

gairdneri. Journal of the Fisheries Research 

Board of Canada, vol. 25, no. 1, p. 25-31. 

Maren, Thomas H., Lawrence E. Broder, and Vincent G. 

Stenger.

1968. Metabolism of ethyl m-aminobenzoate(MS 222) 

in the dogfish, Squalus acanthias. Bulletin Mount 

Desert Island Biological Laboratory, vol. 8, p. 39. 

Maren, Thomas H., Rebecca Embry, and Lawrence E. 

Broder.

1968. The excretion of drugs across the gill of the 

dogfish, Squalus acanthias. Comparative Biochemistry and Physiology, vol. 26, no. 3, p. 853- 

864. 

Schoettger, Richard A.

1967. Annotated bibliography on MS-222. U.S. 

Bureau of Sport Fisheries and Wildlife, Investigations in Fish Control, No. 16. 15 p.

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

Marking, and Arnold M. Julin.

1967. MS-222 as an anesthetic for channel catfish: 

Its toxicity, efficacy, and muscle residues. 

U.S. Bureau of Sport Fisheries and Wildlife, Investigations in Fish Control, No. 17. 14 p.

Stenger, Vincent G., and Thomas H. Maren.

1968. The pharmacology of ethyl m-aminobenzoate 

(MS 222) in the dogfish, Squalus acanthias. Bulletin Mount Desert Island Biological Laboratory, 

vol. 7, p. 51.

Steucke, Erwin W., Jr., and Richard A. Schoettger. 

1967. Comparison of three methods of sampling 

trout blood for measurements of hematocrit. 

Progressive Fish-Culturist, vol. 29, no. 2, p. 98- 

101.
8 Investigations in Fish Control 42* Bureau of Sport Fisheries and Wildlife

Walker* Charles R., aftd Richard A. Schoettger. 1967b. Residues of MS-222 in four salmonids

1967a. Method for determining MS-422 residues in following anesthesia. U.S. Bureau of Sport Fishfish. U.S. Bureau Of Sport Fisheries and Wildlife, eries and Wildlife, Investigations in Fish Control, 

Investigations in Fish Control, No, 14. 10 p. No. 15. lip.
INVESTIGATIONS IN FISH CONTROL

43. Effect of MS-222 on

Electrolyte and Water Content 

in the Brain of Rainbow Trout

By Wayne A. Willford

UNITED STATES DEPARTMENT OF THE INTERIOR, WALTER J. HICKEL, SECRETARY

Leslie L. Glasgow, Assistant Secretary for Fish and Wildlife and Parks

Fish and Wildlife Service, Charles H. Meacham, Conmissioner

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

Washington, D.C. July 1970
CONTENTS

Page 

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

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

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

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

Summary ........................................ 5

References ....................................... 6
EFFECT OF MS-222 ON ELECTROLYTE AND WATER CONTENT 

IN THE BRAIN OF RAINBOW TROUT

By Wayne A. Willford, Chemist 

Bureau of Sport Fisheries and Wildlife 

Fish Control Laboratory, La Crosse, Wisconsin

ABSTRACT. Rainbow trout (Salmo gairdneri) were exposed to 100-milligrams-per-liter solutions of MS-222 for 1-, 2-, 4-, and 10-minute intervals and their brains were analyzed for sodium, potassium, calcium, 

magnesium, zinc, iron, and water content. The mean potassium content 

decreased 17.7 percent and iron increased 56.2 percent during 2-minute 

exposures. Sodium and calcium increased slightly (7.4 and 9.4 percent); 

magnesium, zinc, and water content remained relatively constant. All of 

the affected electrolytes returned toward control values with 4- and 10- 

minute exposures. These shifts in electrolytes appear to be related to depth 

of anesthesia and to the concentrations of free MS-222 in the brain.

MS-222 (methanesulfonate of meta-aminobenzoic acid ethyl ester) has been used extensively as an anesthetic for coldblooded animals 

including fish and amphibians (Schoettger, 1967). 

However, little is known about its mode of action.

It is generally believed that anesthetics act 

by absorption or combination with lipid groups 

in the cell membrane and somehow alter the 

cell membrane's function of establishing ionic 

gradients and regulating respiratory rates in 

cells (Skou, 1961; Quastel, 1963). The result is 

disruption of ionic differential, the biopotential 

and ratios by which nerve impulses are propagated. In addition, upsetting the ionic equilibriums may further affect the rates of reactions which restore standing biopotentials 

(Hillman, 1966).

Walsh and Schopp (1966) demonstrated that 

MS-222 and related compounds reduce the 

frequency of electric organ discharges in the 

electric fish (Gnathonemus moori). They concluded that these agents apparently inhibit 

pacemaker cells in the mesencephalic command nucleus. Stenger and Maren (1968) and 

Hunn (1970) have further shown that MS-222

rapidly crosses the gill of fish and is concentrated in the brain. The depth of anesthesia was 

associated with the concentration of MS-222 in 

the brain.

The objective of this investigation was to 

detect and measure changes in brain electrolytes which are associated with MS-222 

anesthesia. The electrolytes chosen for study 

were those which appear essential to the production and maintenance of nerve potential and 

metabolic activity.

MATERIALS AND METHODS

Rainbow trout (Salmo gairdneri) were obtained from the National Fish Hatchery, Manchester, Iowa. The fish ranged in weight from 

390 to 720 grams and were delivered in two shipments during the 6 months of testing. Each 

group of fish was held in flowing well water at 

12° C. and was fed on a diet of commercial 

trout pellets supplemented with liver.

The test fish were placed individually into 5 

liters of well water containing 100 mg/1 (milligrams per liter) of MS-222 for 1, 2, 4 or 10
Investigations in Fish Control 43: Bureau of Sport Fisheries and Wildlife

minutes. This concentration of anesthetic produces deep anesthesia in rainbow trout within 

3 minutes and medullary collapse in approximately 10 minutes (Schoettger and Julin, 1967).

After exposure, the fish were removed from 

the anesthetic and decapitated. Whole brains 

were removed carefully to avoid contamination and were blotted dry and placed in tared 

porcelain crucibles. After determination of wet 

weight, the samples were dried to constant 

weight at 95° C. Brains of control fish were 

excised and processed in the same manner.

All samples were dry-ashed according to the 

approved method for lead using 1 milliliter of 

15.5 N nitric acid as the "ash-aid" (Horwitz, 

1960). After ashing, 2 ml of 12.1 N hydrochloric acid were added to each crucible, and 

the resulting solution was concentrated to approximately 1 ml. The concentrate was quantitatively transferred by multiple rinses with 

distilled, deionized water into a 10-ml volumetric flask containing 1 ml of a 5-percent 

lanthanum solution in 25-percent (V/V) hydrochloric acid. The lanthanum chloride reduces 

chemical interference during analysis (Elwell 

and Gidley, 1966).

The samples were analyzed for sodium 

(Na+), potassium (K+), calcium (Ca2+), magnesium (Mg24), zinc (Zn 24), and iron (Fe34) 

on an atomic absorption spectrometer. Standard curves were prepared from composite 

standard solutions of all six elements in the 

presence of 0.5-percent lanthanum and 10- 

percent (V/V) hydrochloric acid.

The experiment was performed six times 

over a 6- month period using three fish for 

each of the four exposure intervals, and three 

fish for controls. The data for each brain constituent were analyzed statistically using a 

two-way analysis of variance to determine the 

significance (p=0.05) of observed changes 

(Snedecor, 1956).

RESULTS

Anesthesia of rainbow trout in 100 mg/1 of 

MS-222 at 12° C. resulted in significant shifts 

(p<0.005) of K+ and Fe3+ concentrations in the

brain (table 1). There was a 17.7-percent reduction in K+ and a 56.2-percent increase in 

Fe 3+ during the initial 2 minutes of exposure 

as determined by comparison with the controls. With longer exposures, the concentrations of K 4" and Fe34" returned toward control values.

Minor, nonsignificant (p>0.25), increases of 

7.4 and 9.4 percent were observed in the concentrations of Na+ and Ca 2.+ respecitvely in 

the brains of MS-222 anesthetized trout. Though 

these shifts were not significant, they do suggest an effect of the anesthetic which is similar 

to that observed in Fe34". The significance of 

these measurements may have been masked 

by indeterminant variation.

The Mg 24", Zn2+, and water contents of the 

brains remained relatively constant over the 

entire range of exposures.

In addition to the changes observed during 

anethesia, significant variation (p=0.025 to 

<£ 0.005) of all the cations and the water content 

occurred between monthly replicates. The 

monthly variation in control fish appeared to 

be random, and resulted in diverse levels of

Table 1. Brain cation and water content of rainbow trout 

exposed to 100 mg/1 of MS-222 at 12° C. for selected 

intervals of time

Constituent

Exposed for 

Uhexposed 1 

minute

2

minutes

4 

minutes

10 

minutes

Na+ 

rag/kg1

K+ * 

mg/kg

Ca2+ 

mg/kg

Mg 2+ 

mg/kg

Zn 2+ 

mg/kg

Fe3+ * 

mg/kg

H,0 

g/100 g

2 8,978

+1, 160

14,260 

+1,890

612.6 

+68.2

570.0 

+37.3

47.82 

±5.39

79.65 

+13.45

81.43 

+0.57

9,370 

+1,589

13,140 

+1,850

656.9 

+115.1

593.4 

±55.9

47.00 

+7.63

104.06 

+29.98

81.26 

+0.82

9,646 

+1, 119

11,740 

+2,330

670.2 

+120.4

584.2 

+32.4

49.48 

+14.03

124.38 

+40.46

81.14 

+0.73

9,650 

+1,092

13,720 

+2, 140

649.7 

+94.4

578.6 

+33.1

48.97 

+9.35

119.13 

+43.40

81.28 

+0.77

9,514 

+1,147

13,920 

+2,310

625.3 

+99.3

576.7 

+47.4

47.48 

+9.10

103.24 

+33.61

81.41 

+0.62

1 Concentration of all cations based on dry tissue 

weight.

2 Mean + standard deviation (n=l8). 

^Significant variance attributable to exposure 

(p<0.005).
Wayne A. Willford: Effect of MS-222 in the Brain of Rainbow Trout

Table 2. Monthly variation of brain cation and water content of 

rainbow trout not exposed to US-222

Constituent

Na+ 

mg/kg1

K* 

mg/kg

Ca2+ 

ng/kg

X

Zn2+ 

mg/kg

Fe3+ 

ng/kg

H20

g/ioo g

Sampled on 

11/16/67

2 8,333

±1,032

12,380 

±3, 110

617.3 

±61.7

571.0 

±32.2

51.30 

±3.81

75.59 

±17.41

80.99 

±0.67

12/11/67

7,839 

±128

1A,990 

±460

549.2 

±31.4

512.8 

±11.9

47.43 

±3.88

80.04 

±3.80

81.35 

±0.54

1/16/68

8,451 

±901

14,260 

±680

581.9 

±100.1

566.3 

±29.4

52.39 

±9.29

69.06 

±10.94

81.45 

±0.26

2/12/68

10,353 

±370

15,570 

±880

641.5 

±32.7

596.3 

±12.1

43.61 

±1.87

78.22 

±10.00

81.58 

±0.75

3/21/68

9,945 

±1,001

12,950 

±1,800

596.0 

±51.1

561.8 

±26.6

44.27 

±0.44

82.43 

±6.12

82.08 

±0.42

5/6/68

8,952 

±1,081

15,410 

±1,690

690.3 

±53.8

612.3 

±12.1

47.92 

±6.02

92.57 

±22.66

81.13 

±0.39

1 Concentration of all cations based on dry tissue weight.

2 Mean ± standard deviation (N=3).

ions and water in the brain from replicate to 

replicate (table 2). However, the shift in ions 

attributable to MS-222 exposure (table 1) was 

similar within each replicate.

DISCUSSION

The results of this study show that MS-222 

disrupts, directly or indirectly, specific cationic equilibrums in the brain of rainbow trout 

during anesthesia. Disruption of ionic differentials, such as K+/Ca 2+ and K+/Na+ ratios, has 

a profound effect on nerve potentials and 

respiration, this being the basis of the general 

theory of anesthetic action (Quastel, 1963; 

Hillman, 1966).

A major decrease in brain K+ , however, is 

not peculiar to anesthesia. Systemic stress 

such as anoxia, heat, and cold also produce 

similar cationic imbalance in fish and mammals in vivo (Benjamin, Anastasi, and Helvey, 

1961a; Hickman et al., 1964; Van Harreveld, 

1966; Bandurski, Bradstreet, and Scholander, 

1968). Benjamin, Anastasi, and Helvey, (1961b) 

have further shown by in vitro studies with rat 

brain that temperature does not directly effect K+ release but both anoxia and lack of 

glucose do. Since MS-222 reduces the respiratory rate of fish (Campbell and Davis, 1963; 

Randall, Smith, and Brett, (1965), an associated 

anoxia or hypoglycemia may have contributed 

to the K + depression which I observed.

Unlike the progressive K + loss that reaches 

a plateau during systemic stress, MS-222-induced anesthesia causes an initial K+ decrease 

during 1- and 2-minute exposures followed by 

a return toward control levels up to the time 

when the fish is approaching death at 10 

minutes. The Fe3+ change is the reverse of 

this pattern.

Hunn (1970) showed that a similar pattern 

occurs in the concentration of free MS-222 in 

the brain of rainbow trout during anesthesia. 

The concentration of MS-222 increases rapidly 

during the first 2 to 4 minutes of exposure and 

then slowly declines with longer exposures. 

The exposure period at which the cation and 

MS-222 concentrations reverse direction of 

change coincides with the approximate exposure which produces loss of reflex in the 

fish. Electrolyte content of the brain 

appears to be associated with the MS-222 concentration in the brain and the depth of anesthesia.

Hillman (1966) stated that in nearly all conditions examined in mammals, changes in the 

K+ content of brain are mirrored in opposite 

changes in the Na+ content. In this study, as 

previously mentioned, the K4" decrease is 

mirrored by a marked increase in the Fe3 + 

content and only a slight increase in the Na+ 

content. Possibly this phenomenon can be explained in light of the work by Germain and 

Gagnon (1968). They demonstrated that the 

blood of hagfish (Myxine glutinosa L.) accumulates in subcutaneous sinuses during MS-222 

anesthesia. They postulated that this accumulation was due to reduced respiratory movements and profound changes in hemodynamics 

during narcosis.

Since the brain is a relatively vascular 

tissue (Zwehl, 1961; Heisey, 1968), it is possible that a pooling or plasma skimming which 

concentrates erythrocytes, may occur in the 

brain during anesthesia. This would tend to 

mask changes in Na+ and K + concentrations 

and cause an increase in the Fe + content of 

the brain.

SUMMARY

1. Anesthetizing rainbow trout in 100 mg/1 

of MS-222 at 12° C. for 2 minutes causes a
6 Investigations in Fish Control 43: Bureau of Sport Fisheries and Wildlife

significant reduction of K + and increase of 

Fe 3+ in the brain. A concurrent minor increase in the Na+ and Ca2+ of the brain is 

observed.

2. Exposure of the fish to MS-222 for periods 

longer than 2 minutes results in a return 

of K+, Fe 3 "1" , Na+, and Ca 2+ towards control values. This pattern of change appears 

to be associated with the concentration of 

free MS-222 in the brain and with the depth 

of anesthesia.

3. MS-222 anesthesia of rainbow trout does 

not affect the concentrations of Mg 2+, 

Zn2+, or water in the brain.

4. Whereas shifts in ions appear to be closely 

associated with anesthesia, it is possible 

that the large increase in Fe 3+ may be a 

secondary effect due to erythrocyte pooling 

in the brain.

5. Significant monthly variation in the electrolyte and water content of brain occurs 

in rainbow trout held under the conditions 

described.

REFERENCES

Benjamin, F. B.t Joan N. Anastasi, and W. M. Helvey.

196la. Effect of stress on potassium content of rat 

brain. Proceedings of the Society for Experimental Biology and Medicine, vol. 107, no. 4, 

p. 972-973.

196lb. Effect of stress on potassium release from 

surviving rat brain. Proceedings of the Society 

for Experimental Biology and Medicine, vol. 107, 

no. 4, p. 973/974.

Bandurski, Robert S., Edda Bradstreet, and 

P. F. Scholander.

19680 Metabolic changes in the mud-skipper during asphyxia or exercise. Comparative Biochemistry and Physiology, vol. 24, p. 271-274.

Campbell, G. D., and D. H. Davies.

1963. Effect of ethyl m-aminobenzoate (MS-222) 

on the elasmobranch electrocardiograph. 

Nature (London), vol. 198, p. 302.

Elwell, W. T.f and J. A. F. Gidley.

1966. Atomic - absorption spectrophotometry. 

Second edition, Pergamon Press Ltd., London. 

139 p.

Germain, Paul, and Andre Gagnon.

1968. A simple and reliable method for obtaining 

large blood samples from the anaesthetized 

hagfish. Comparative Biochemistry and 

Physiology, vol. 26, p. 371-375.

Heisey, Richard S.

1968. Brain and choriod plexus blood volumes in 

vertebrates. Comparative Biochemistry and 

Physiology, vol. 26, p. 489-498.

Hickman, C. P., Jr., R. A. McNabb, J. S. Nelson, 

E. D. Van Breeman, and D. Comfort.

1964. Effect of cold acclimation on electrolyte 

distribution in rainbow trout (Salmo gairdnerii). 

Canadian Journal of Zoology, vol. 42, p. 577- 

597.

Hillman, H.

1966. The role of potassium and sodium ions as 

studied in mammalian brain. International 

Review of Cytology, vol. 20, p. 125-137.

Horwitz, William. (Editor)

1960. Official methods of analysis of the Association of Official Agricultural Chemists. 

Ninth edition, Association of Official Agricultural Chemists. Washington, D.C. 832 p.

Hunn, Joseph B.

1970. Dynamics of MS-222 in the blood and brain 

of freshwater fishes during anesthesia. 

Bureau of Sport Fisheries and Wildlife, Investigations in Fish Control. No. 42. 

Quastel, J. H.

1963. Effects of anesthetics, depressants, and 

tranquilizers on cerebral metabolism, 

p. 517-538. In Metabolic inhibitors; A comprehensive treatise. Volume 11. Academic Press, 

New York.

Randall, D. J., L. S. Smith, and J. R. Brett.

1965. Dorasl aortic blood pressures recorded 

from the rainbow trout (Salmo gairdneri). 

Canadian Journal of Zoology, vol. 43, p. 863- 

872.

Schoettger, Richard A.

1967. Annotated bibliography on MS-222.

Bureau of Sport Fisheries and Wildlife, Investigations in Fish Control, No. 16. 15 p.

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

1967. Efficacy of MS-222 as an anesthetic on four 

salmonids. Bureau of Sport Fisheries and 

Wildlife, Investigations in Fish Control, No. 13. 

15 p.

Skou, Jens Chr.

1961. The effect of drugs on cell membranes with 

special reference to local anaesthetics. Journal 

of Pharmacy and Pharmacology, vol. 13, 

p. 204-217.
Wayne A. Willford: Effect of MS-222 in the Brain of Rainbow Trout

Snedecor, George W.

1956. Statistical methods. Fifth edition, Iowa State 

University Press, Ames, Iowa. 534 p.

Stenger, Vincent G., and Thomas H. Maren.

1968. The pharmacology of ethyl m-aminobenzoate 

(MS-222) in the dogfish, Squalus acanthias. 

Bulletin Mount Desert Island Biological Laboratory, vol. 7, p. 51.

Van Harreveld, A.

1966. Brain tissue electrolytes. Butterworth Inc., 

Washington, D.C. 171 p.

Walsh, Raymond R., and Robert T. Schopp.

1966. Frequency-depressing action of ethylaminobenzoate isomers on momyrid electric fish. 

American Journal of Physiology, vol. 211, no. 1, 

P. 1-5.

Zwehl, Vera von.

1961. Uber die Blutgefassversorgung des Gehirns 

bei einigen Teleostiern. Zoologische 

Jahrbucher, Abteilung Fur Anatomie und 

Ontogenie der Tiere, vol. 79, p. 371-438.

* U. S. GOVERNMENT PRINTING OFFICE ; 1970 O - 404-780
(Reports 18 through 21 are in one cover.)

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

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

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

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

Burress. 1967. 8 p.

(Reports 22 through 24 are in one cover.)

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

and Arnold M. Julin. 1969. 10 p.

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

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

David O. Locke. 1969. 5 p.

(Reports 25 through 28 are in one cover.)

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

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

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

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

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

Lennon. 1969. 21 p.

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

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

(Reports 29 through 31 are in one cover.)

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

and Richard A. Schoettger. 1969. lip.

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

31. Annotated Bibliography on Methylpentynol, by Gerald E. Svendsen. 1969. 7 p.

(Reports 32 through 34 are in one cover.)

32. Toxicity of Hyamine 3500 to Fish, by James W. Hogan. 1969. 9 p.

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

Jackson. 1969. 9 p.

34. Laboratory Studies on Possible Fish-Collecting Aids, WithSomeToxicities for the Isomers 

of Cresol, by Robert M. Howland. 1969. 10 p.

35. Toxicology of Thiodan in Several Fish and Aquatic Invertebrates, by Richard A. Schoettger. 

^ 1970. 31 p.

(Reports 36 through 38 are in one cover.)

36. A Method for Rating Chemicals for Potency Against Fish and Other Organisms, by Leif L. 

Marking. 1970. 8 p.

37. Comparative Toxicity of 29 Nitrosalicylanilides and Related Compounds to Eight Species of 

Fish, by Leif L. Marking and Wayne A. Willford. 1970. 11 p.

38. Toxicity of 33NCS (3'-chloro-3-nitrosalicylanilide) to Freshwater Fish and Sea Lampreys, 

by Leif L. Marking, Everett L. King, Charles R. Walker, and John H. Howell. 1970. 16 p.

(Reports 39 and 40 are in one cover.)

39. Effects of Antimycin A on Tissue Respiration of Rainbow Trout and Channel Catfish, by 

Richard A. Schoettger and Gerald E. Svendsen. 19 70. 10 p.

40. A Resume on Field Applications of Antimycin A to Control Fish, by Robert E. Lennon and 

Bernard L. Berger. 1970. 19 p.
As the Nation's principal conservation agency, the Department 

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

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

natural resources.

The Department works to assure the wisest choice in managing 

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

a better United States now and in the future.

UNITED STATES DEPARTMENT OF THE INTERIOR 

BUREAU OF SPORT FISHERIES AND WILDLIFE

FISH CONTROL LABORATORIES 

LA CROSSE, WISCONSIN 5^601

POSTAGE AND FEES PAID 

U.S. DEPARTMENT OF THE INTERIOR