INVESTIGATIONS IN FISH CONTROL

51. Methods for Simultaneous Determination

and Identification of MS-222 and Metabolites 

in Fish Tissues

52. Residues of MS-222, Benzocaine, and Their Metabolites 

in Striped Bass Following Anesthesia

United States Department of the Interior

Fish and Wildlife Service 

Bureau of Sport Fisheries and Wildlife
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. [See Investigations in Fish Control 45-46 (in one cover) for 

list of issues published prior to 1970]

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. 1970. 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.

(Reports 41 through 43 are in one cover.)

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. 1970. 7 p.

42. Dynamics of MS-222 in the Blood and Brain of Freshwater Fishes During Anesthesia, by 

Joseph B. Hunn. 1970. 8 p.

43. Effect of MS-222 on Electrolyte and Water Content in the Brain of Rainbow Trout, by 

Wayne A. Willford. 1970. 7 p.

44. A Review of Literature on TFM (3-trifluormethyl-4-nitrophenol) as a Lamprey Larvicide, 

by Rosalie A. Schnick. 1972. 31 p.

(Reports 45 and 46 are in one cover.)

45. Residues of MS-222 in Northern Pike, Muskellunge, and Walleye, by John L. Alien, 

Charles W. Luhning, and Paul D. Harman. 1972. 8 p.

46. Methods of Estimating the Half-Life of Biological Activity of Toxic Chemicals in Water, 

by Leif L. Marking. 1972. 9 p.

(Reports 47 through 50 are in one cover.)

47. Preparation and Properties of Quinaldine Sulfate, an Improved Fish Anesthetic, by 

John L. Alien and Joe B. Sills. 1973. 7 p.

48. Toxicity of Quinaldine Sulfate to Fish, by Leif L. Marking and Verdel K. Dawson. 

1973. 8 p.

49. The Efficacy of Quinaldine Sulfate as an Anesthetic for Freshwater Fish, by Philip A. 

Gilderhus, Bernard L. Berger, Joe B. Sills, and Paul D. Harman. 1973. 9 p.

50. Residue of Quinaldine in Ten Species of Fish Following Anesthesia with Quinaldine 

Sulfate, by Joe B. Sills, John L. Alien, Paul D. Harman, and Charles W. Luhning. 

1973. 9 p.

Fish Control Laboratories

Bureau of Sport Fisheries and Wildlife

U.S. Department of the Interior

P.O. Box 862

La Crosse, Wisconsin 54601
INVESTIGATIONS IN FISH CONTROL

51. Methods for Simultaneous Determination and Identification 

of MS-222 and Metabolites in Fish Tissues 

By Charles W. Luhning

52. Residues of MS-222, Benzocaine, and Their Metabolites 

in Striped Bass Following Anesthesia

By Charles W. Luhning

United States Department of the Interior

Fish and Wildlife Service 

Bureau of Sport Fisheries and Wildlife

DEC 1973
IN THIS COVER

Pages

Methods for Simultaneous Determination and Identification of

MS-222 and Metabolites in Fish Tissues, by Charles W.Luhning . . 1-10

Residues of MS-222, Benzocaine, and Their Metabolites in Striped

Bass Following Anesthesia, by Charles W. Luhning ......... 1-11

For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price 60 cents

Stock Number 2410-00374

ii
INVESTIGATIONS IN FISH CONTROL

51. Methods for Simultaneous Determination

and Identification of MS-222 and Metabolites 

in Fish Tissues

By Charles W. Luhning

United States Department of the Interior

Fish and Wildlife Service 

Bureau of Sport Fisheries and Wildlife
CONTENTS

Page 

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

Introduction. ..................................... 3

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

3 Reagents and apparatus...........................

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

Analytical procedures............................ 5

Results. ........................................ 7

Discussion ...................................... 9

Conclusions. ..................................... 10

References ...................................... 10
METHODS FOR SIMULTANEOUS DETERMINATION AND IDENTIFICATION 

OF MS-222 AND METABOLITES IN FISH TISSUES

By Charles W. Luhning

Southeastern Fish Control Laboratory

Warm Springs, Georgia

ABSTRACT. MS-222 (methanesulfonate of meta-aminobenzoic acid 

ethyl ester) is a primary aromatic amine commonly used to anesthetize 

fish. Like all primary aromatic amines, its diazonium salt reacts with 

J^-1-naphthylethylenediamine dihydrochloride to form a wine-red azo dye 

with a maximum absorbance at 545 nm. Basic carbon tetrachloride extraction separates the azo dyes of MS-222 and its acid metabolite, 

m-aminobenzoic acid, and quantitative determination of each compound is 

made colorimetrically. By this method, recoveries of 82 to 110 percent 

for MS-222 and 84 to 117 percent for m-aminobenzoic acid were obtained 

from largemouth bass muscle and liver tissues spiked with 1 to 10 Mg/g 

of each compound. Confirmation of MS-222 and m-aminobenzoic acid residues in fish tissue was made by thin-layer chromatography. Quantitative 

estimation and identification of each compound were possible from samples 

spiked with as little as 1 Mg/g of MS-222 or m-aminobenzoic acid.

INTRODUCTION

MS-222 (methanesulfonate of meta-aminobenzoic acid ethyl ester) is a commonly used 

fish anesthetic. When any chemical or drug is 

used on a food commodity item, the U.S. Food 

and Drug Administration requires data on 

residual amounts of the drug and its metabolites left in the commodity after treatment. 

Previous methods of analysis for MS-222 

residues in fish tissue did not identify 

m-aminobenzoic acid, a metabolite of 

MS-222. The method described by Walker and 

Schoettger (1967) included the residue of 

m-aminobenzoic acid, but the method did not 

separate it from MS-222 residues. The thinlayer chromatographic method of Alien, 

Luhning, and Harman (1970) only confirmed 

residues of MS-222 determined by the first 

method.

The analytical method described here will 

detect residues of MS-222 and its metabolites, 

namely, acetylated MS-222, m-aminobenzoic

acid and m-acetylaminobenzoic acid. The thinlayer chromatographic procedures are done 

simultaneously with the analytical procedures, 

which save time and materials.

METHODS AND MATERIALS

Reagents and apparatus

Reference to a company or product does not 

imply recommendation to the exclusion of 

others that may be suitable. Reagents and apparatus used were as follows:

1. Carbon tetrachloride, reagent grade.

2. Trichloroacetic acid: Dissolve 60 g 

crystalline TCA in distilled water. 

Transfer to a 100-ml volumetric flask 

and bring to volume with distilled water. 

Dilute 50 ml to 200 ml to obtain 15- 

percent TCA, and dilute 5 ml of the 

original stock to 100 ml to obtain 3- 

percent TCA.
Investigations in Fish Control 51: Bureau of Sport Fisheries and Wildlife

3. 0.2-percent sodium nitrite: Transfer 

0.2 g NaNC>2 into a 100-ml volumetric 

flask, dissolve, and bring to volume with 

distilled water. Make fresh daily.

4. 0.5-percent ammonium sulfamate: 

Transfer 0.5 g NH 4SO3 NH 2 into a 

100-ml volumetric flask and bring to 

volume with distilled water.

5. 0.1 -percent _N-1 -naphthylethylenediamine 

dihydrochloride: Transfer 0.1 g C 12^14 

N2-2HC1 into a 100-ml volumetric flask, 

dissolve, and bring to volume with distilled water. Refrigerate and make up 

fresh weekly.

6. 4N hydrochloric acid: Dilute 40 ml ION 

HC1 to 100 ml with distilled water.

7. 10-percent hydrochloric acid (V/V).

8. Concentrated ammonium hydroxide.

9. Standard solutions: Weigh 0.01 g of 

MS-222 and quantitatively transfer to a 

100-ml volumetric flask, add 20 ml of 

15-percent TCA and bring to volume 

with distilled water. One ml of this 

stock solution diluted to 100 ml with 3- 

percent TCA will make a 1 Mg/ml solution. Make fresh daily. Standards of 

m-aminobenzoic acid are made up 

weekly in like manner.

10. Ethyl acetate, reagent grade.

11. Iso-butanol, reagent grade.

12. Developing solutions: The first solution 

contains 180 ml ethyl ether, 10 ml acetone, and 10 ml concentrated glacial 

acetic acid. The second solution contains 

120 ml chloroform, 38 ml ethyl alcohol, 

38 ml methanol, and 4 ml concentrated 

ammonium hydroxide. All solvents are 

reagent grade or better.

13. Waring blendor.

14. Tissue homogenizer, Virtis "45".

15. Orbital centrifuge.

16. Chromatography tank 10.2 by 20.3 by 

22.9 cm, lined with absorbent paper.

17. Silica gel thin-layer plates, Eastman 

chromagram sheets without flourescent 

dye, 20 by 20 cm.

18. Chromatography spray bottle.

19. Micropipettes, 1, 5, 10, and 25 /d.

20. Centrifuge tubes, 15 and 40 ml.

21. Spectrophotometer cuvettes, round, 13 

by 100 mm.

22. Spectrophotometer, Bausch and Lomb 

"Spectronic 20".

23. Whatman #40 filter paper, 9.0 cm diameter.

24. Funnels, 5 cm diameter.

Tissue collection and treatment

Blood samples are taken by caudal puncture 

(Steucke and Schoettger, 1967) with a heparinized syringe fitted with a 20-gauge needle. 

Place 1.0 ml of blood in a 40-ml graduated 

centrifuge tube, and add 15 ml of distilled 

water and 4 ml of 15-percent TCA. Mix thoroughly and centrifuge at 2,500 RPM for 30 

minutes. Filter the supernatant through #40 

Whatman filter paper, and the sample is 

ready for analysis. If a 1.0-ml sample of 

blood cannot be obtained, use the amount obtained and proportional amounts of water and 

TCA.

Fillet each fish after the other tissues have 

been removed, and homogenize the entire 

fillet in a Waring blendor to obtain a homogeneous sample (Luhning and Harman, 1971). 

Weigh out 1.0 g of homogenized tissue in a 

tared homogenizing flask, add 10 ml of distilled water and homogenize for 1 minute. 

Quantitatively transfer to a 40-ml graduated 

centrifuge tube and bring to a volume of 16 

ml with distilled water. Then add 4 ml of 15- 

percent TCA to coagulate the protein, mix 

thoroughly, and centrifuge at 2,500 RPM for 

20 minutes. Filter the supernatant through 

#40 Whatman filter paper, and the sample is 

ready for analysis. Kidney, liver, and brain 

are dissected from the fish and prepared the 

same as muscle tissue, except that these 

samples are not homogenized before weighing 

out a 1.0-g sample.
Luhning: Determination and Identification of MS-222 and Metabolites

Analytical procedures

The first seven steps are basically the same 

as those described by Walker and Shoettger 

(1967). The procedure is as follows:

1. Pipette 5 ml of 3-percent TCA into a 

clean 50-ml Erlenmeyer flask for a 

reagent blank.

2. Pipette 5 ml of 1-Mg/ml standard of 

MS-222 and m-aminobenzoic acid into 

Erlenmeyer flasks. Also pipette 5 ml of 

mixed MS-222 and acid standards, each 

at a 1-wg/ml concentration, into an 

Erlenmeyer flask.

3. Pipette 5 ml of filtrate from each sample 

into separate 50-ml Erlenmeyer flasks.

4. Add 0.5 ml of 0.2-percent sodium nitrite 

to each flask, swirl, and let stand for 15 

minutes. Keep samples at room temperature and out of direct sunlight.

5. Add 0.5 ml of 0.5-percent ammonium 

sulfamate to each flask, swirl, and let 

stand for 3 minutes.

6. Add 0.5 ml of 0.1-percent ^J-1-naphthylethylenediamine dihydrochloride to each 

flask, swirl vigorously, and let stand for 

10 minutes.

7. Pour samples into a clean cuvette. Zero 

spectrophotometer at 100-percent transmittance using the reagent blank. Read 

percent transmittance of each sample in 

the spectrophotometer at 545 nm.

8. Transfer the azo dyestuff to a labeled 

15-ml centrifuge tube containing 0.5 ml 

of 7.5 M ammonium hydroxide and 2 ml 

of carbon tetrachloride. Shake vigorously, and centrifuge at 1,500 RPM for 3 

minutes.

9. Rinse each cuvette with distilled water 

and shake dry. Be sure to keep cuvettes 

in order. Add 1 ml of 60-percent TCA to 

each cuvette.

10. After centrifugation (step 8), transfer 5 

ml of the aqueous layer back into the 

original cuvette and mix gently.

11. Zero the spectrophotometer at 100- 

percent transmittance using the extracted reagent blank, and again read 

percent transmittance of each sample 

with a spectrophotometer at 545 nm.

12. Convert all percent transmittance readings to absorbance, and calculate 

residues as follows;

A 2

S,x - S 2

Mg/gfree MS-222 = x 20

HM

Mg/g free jm-aminobenzoic acid =_Ji x 20

A 2

51 = absorbance of sample before extraction.

5 2 = absorbance of sample after extraction.

M-L = absorbance of mixed standard (1 

Mg/ml MS-222 and 1 Mg/ml rnaminobenzoic acid) before extraction.

M 2 = absorbance of mixed standard 

after extraction.

A -L = absorbance of rn-aminobenzoic 

acid standard (1-Mg/ml) before 

extraction.

A 2 = absorbance of m-aminobenzoic 

acid after extraction.

13. Possible acetylated derivatives can be 

analyzed as follows:

A. Place 5 ml of filtrate from each

sample into separate 15-ml graduated 

centrifuge tubes and add 0.5 ml of 

4N HC1.

B. Place tubes in a boiling water bath 

for 1 hour, cool and adjust volume to 

5 ml with distilled water.
Investigations in Fish Control 51: Bureau of Sport Fisheries and Wildlife

C. Transfer to a 50-ml Erlenmeyer 

flask and analyze as before, starting 

with step 4.

D. The amount of acetylated residues is 

obtained by subtracting the concentration of the first analysis (free 

residues) from the concentration of 

the second analysis.

14. For the identification and confirmation 

of MS-222 and m-aminobenzoic acid 

residues by thin-layer chromatography, 

proceed as follows;

A. React another 5 ml of filtrate from 

each sample and standards with the 

Bratton-Marshall reagents (steps 1 

through 6).

B. Transfer the azo dyestuff to a stoppered test tube containing 1 ml,of 

iso-butanol-ethyl acetate (50/50, 

V/V). Shake vigorously, and let stand 

for 30 minutes in a dark place.

C. Mark a spotting line 2.5 cm and a 

solvent-front line 12.5 cm from the 

bottom of a 20 by 20 cm silica gel 

thin-layer plate.

D. With a calibrated micropipette, spot 

enough iso-butanol-ethyl acetate on 

spotting line to give a visible, colored spot for each sample. On the 

same plate, spot 1, 2, and 5 Ml of the 

mixed standard (1- Mg/ml concentration of each).

E. Thirty minutes before developing the 

plate, mix 180 ml of ethyl ether, 10 

ml of acetone, and 10 ml of concentrated acetic acid (90:5:5) and pour 

into a chromatography tank lined with 

absorbent paper. Place the plate in 

the tank and allow the solvent to rise 

to the top of the plate, then remove 

and dry under an electric hair dryer 

in a fume hood.

F. Develop the plate again in 120 ml of 

chloroform, 38 ml of ethanol, 38 ml

of methanol, and 4 ml of concentrated 

ammonium hydroxide (60:19:19:2). 

Allow the solvent front to migrate 

up to the previously marked line 

(12.5 cm from the bottom), then remove the plate, dry, and spray with 

10-percent HC1 under a fume hood.

G. Compare the Rf values of the standards to those of the samples. In general, the Rf values will be 0.83 for 

MS-222 and 0.18 for m.-aminobenzoic 

acid. An estimate of the concentration present in the samples can be 

made by comparing the color intensity and size of the sample spot to 

that of a spot obtained from a known 

amount of standard.

A flow chart has been prepared to diagram 

the sequence of the above procedures (fig. 1). 

The chemical structures of MS-222 and its 

metabolites are illustrated in figure 2.

_________ HOMOGENIZE ^ SAMPLE ________ WITH WATER

ADD TCA

CENTRIFUGE

FILTER

ACETYLATED RESIDUES

STANDARDS 

(I JUG. /ML.)

FREE RESIDUES

ADD HCL. 

HEAT FOR I HOUR

-£a^b .x

ADD BRATTON-MARSHALL REAGENTS

CONFIRMATORY METHOD ANALYTICAL METHOD

ADD ISO-BUTANOL-ETHYL ACETATE READ PERCENT TRANSMITTANCE 

J (SAMPLES-S|, STANDARDS-M| AND A|) 

SPOT SOLVENT ON TLC PLATE ^

J ADD NH4 OH AND CCL4 

DEVELOP J 

J CENTRIFUGE 

COMPARE RF VALUES

USE AQUEOUS LAYER______y DISCARD CCL 4

ADD TCA

I 

READ PERCENT TRANSMITTANCE

(SAMPLES'S2 , STANDARDS- M2 AND

CONVERT PERCENT TRANSMITTANCE TO ABSORBANCE

I 

CALCULATE JUU3./G. RESIDUES

Figure 1. Flow chart of procedures for the determination and identification of MS-222 and metabolites.
Luhning: Determination and Identification of MS-222 and Metabolites

Methanesulfonate of 

nj-aminobenzoic acid 

ethyl ester (MS-222)

m-Aminobenzoic acid

Oj-Acetylaminobenzoic 

acid ethyl ester

NHCOCH3

m-Acetylaminobenzoic 

acid

Figure 2.~Chemical structures of MS-222 

and metabolites.

RESULTS

The efficiency of the extraction procedure 

was evaluated by analyzing a series of pure 

and mixed standards of MS-222 and m-aminobenzoic acid before and after extraction with 

basic carbon tetrachloride (fig. 3). The concentration of the standard did not influence the 

amount extracted, for the regression of absorbance values was relatively linear with increasing concentration of the standards. The 

absorbance value for the pure m-aminobenzoic 

acid standard before extraction, divided by the 

absorbance value after extraction (A2 /Ax ) 

does not equal the known dilution factor of the 

procedure (D). The difference is attributed to 

a small loss of material left in the cuvette 

after transferring the material to a centrifuge 

tube. Thus, in figure 3, the slope of the absorbance curve for MS-222 obtained from the 

mixed standards (M x X A2 /A! - M 2 ) is 

slightly different from one calculated by the 

known dilution factor (MS X D).

.050

0 0.2 0.4 0.6 0.8 1.0 

CONCENTRATIONS(wg/ml )

Figure 3, Absorbance curves at 545 nm for: (A2) 

standards of jn-aminobenzoic acid extracted with carbon tetrachloride: (M2 ) standards of MS-222 and 

jm-aminobenzoic acid mixed and extracted with carbon 

tetrachloride; (M-L X ^ - M 2) the calculated absorbance of MS-222 after extracting the acid from the 

mixed standards; and (MS X D) standard curve of 

MS-222 multiplied by the known dilution factor resulting from the extraction of the acid.

When a fresh standard of MS-222 is diazotized and extracted with basic carbon tetrachloride, the aqueous fraction has zero absorbance. Thus, the amount of MS-222 present 

in a sample has to be calculated from the 

information obtained by extracting a 1-Mg/ml 

standard of acid and the absorbance values 

of a mixed 1-Mg/ml standard of MS-222 and 

m-aminobenzoic acid before and after extraction.

The amount of recoverable MS-222 and 

m-aminobenzoic acid was determined by spiking samples of largemouth bass (Micropterus 

salmoides) and liver tissue with 1 to 10 Mg/g 

amounts of the two compounds (table 1). Recovery studies were done only on muscle and 

liver tissues because interfering substances
8 Investigations in Fish Control 51: Bureau of Sport Fisheries and Wildlife

Table 1. Recovery of MS-222 and m-aminobenzoic acid spiked into 1-g samples 

of largemouth bass muscle and liver tissue as determined by the colorimetric method and confirmed by the thin-layer chromatographic method

Tissue

Number 

of 

analyses

Concentration 

added (jug)

MS -222 Acid

Concentration 

recovered (/ug)

MS-222 Acid

Percent 

recovery

MS -222 Acid

Confirmed by 

TLC

MS -222 Acid

3 

3 

3 

3 

3 

Muscle 3 

3

3

3

3

3

3

1

1

liver 1

1

1

1

1. 

5. 

7. 

10. 

0. 

0. 

0.

0.

1.

2.

5.

7.

5.

10.

0.

0.

2.

7.

0 

0 

5 

0 

0 

0 

0

0

0

5

0

5

0

0

0

0

5

5

0.0 

0.0 

0.0 

0.0 

1.0 

5.0 

7.5

10.0

1.0

7.5

5.0

2.5

0.0

0.0

5.0

10.0

7.5

2.5

0 

4 

6 

10

0

2

4

7

4

10

2

8

.827 

.740 

.750 

.680

__

.870

.730

.790

.720

.880

.230

--

--

.650

.270

0.860 

4.970 

7.470

9.950

0.910

7.340

4.870

2.180

__

--

4.780

10.500

8.770

2.100

82.7 

94.8 

90.0 

106.8

_-

87.0

109.2

95.8

102.9

97.6

102.3

--

--

106.0

110.3

86 

99 

99

99

91

97

97

87

_

-

95

105

116

84

.0 

.6 

.6

.5

.0

.9

.4

.2

_

_

.6

.0

.9

.0

Yes 

Yes 

Yes 

Yes 

No 

No 

No

No

Yes

Yes

Yes

Yes

Yes

Yes

No

No

Yes

Yes

No 

No 

No 

No 

Yes 

Yes 

Yes

Yes

Yes

Yes

Yes

Yes

No

No

Yes

Yes

Yes

Yes

are most prevalent in liver and least prevalent in muscle tissue. Recoveries ranged from 

82.7 to 110.3 percent for MS-222 and from 

84.0 to 116.9 percent for m-aminobenzoic 

acid.

Diazotization of compounds mentioned in 

this study was accomplished by modification 

of the Bratton and Marshall (1939) procedure 

described by Walker and Schoettger (1967). 

The reaction of primary aromatic amines with 

nitrous acid yields a diazonium salt, which 

will couple with certain aromatic amines to 

yield strongly colored azo compounds. Naturally occurring primary aromatic amines in 

certain fish tissues, especially liver, react 

with the Bratton-Marshall reagents and cause 

serious quantitative errors in the determination of MS-222 and rn-aminobenzoic acid residues. Thus, background levels of these 

amines are determined in control samples

and substracted from levels obtained in treated 

samples. This is an accepted procedure in any 

analytical method by spectrophotometry.

Another standard procedure should be a 

confirmatory method of analysis. The thinlayer chromatographic method described previously will separate azo dyestuffs of naturally 

occurring primary aromatic amines from MS222 and rn-aminobenzoic acid. The following 

Rf values were obtained: 0.83 for the azo 

dyestuff of MS-222, 0.70 for m-aminophenol, 

0.34 for sulfanilic acid, 0.26 for j>-aminobenzoic acid, 0.18 for JTI-aminobenzole acid, and 

0.00 for liver extract. The comparison of Rf 

values must be made on the same thin-layer 

plate since these values may vary between 

determinations.

The minimum amount of azo dyestuff from 

a MS-222 standard that can be visualized on a
Luhning: Determination and Identification of MS-222 and Metabolites

thin-layer plate was found to be 5 ng. The 

maximum amount of azo dyestuff from muscle 

extract that can be spotted on a thin-layer 

plate is 100 /ul, which is equivalent to 25 mg 

of tissue. Thus, the sensitivity of the method 

is 0.2 ng/mg. Quantitative estimations of residues in samples can be accomplished by 

spotting a series of MS-222 and m-aminobenzoic acid standards in the range of 5 to 25 ng 

amounts on the same plate along with the 

samples.

DISCUSSION

The analytical and confirmatory methods 

described here will effectively determine and 

positively confirm which metabolites of MS222 are present. Different species of fish 

seem to eliminate residues of MS-222 by 

various means of excretion and deactivation,

which in turn will influence the amount of 

metabolites present in the muscle (table 2).

Excellent recoveries of MS-222 and maminobenzoic acid were obtained from spiked 

samples of muscle tissue. Recoveries were 

higher from spiked liver samples because of 

inconsistent background interference from 

naturally occurring primary aromatic amines, 

which amounted to a mean of 89 percent 

transmittance in the control liver samples 

after extraction. By thin-layer chromatography, the control liver samples contained no 

spot with the same Rf value as MS-222 or jnaminobenzoic acid.

The accuracy of the analytical method is 

governed by the tissue being analyzed and by 

the techniques of extraction and dilution. 

The dilution factor attributed to extraction

Table 2. Average concentrations of MS-222, m-aminobenzoic acid, and their 

N-acetyl derivatives in muscle tissue of four species of fish after a 

15-minute exposure to a 100-mg/l aqueous solution of MS-222 at 18° C.

Number 

of 

fish

Mean concentration of residues by 

colorimetric method

Free

MS-222

(Mg/g)

Acetylated 

MS-222 

(xg/g)

Free 

acid 

(^g/g)

Acetylated 

acid 

(^g/g)

Free residues 

confirmed 

by TLC

MS -222 Acid

Channel catfish 5 67.9 4.5 3.4 1.7 Yes Yes

(Ictalurus punc- 1 (0.7) (0.5) 

tatus)

Striped bass 5 23.0 0.0 14.7 3.3 Yes Yes

(Morone saxatilis) (0.5) (1.2)

Bluegill 5 23.8 0.6 0.0 0.0 Yes No

(Lepomis macroch- (l.O) (0.0)

Largemouth bass 

(Micropterus 

salmoides)

34.4 

(1.9)

0.0 1.4 

(0.0)

0.7 Yes Yes

1 Residues after 6 hours recovery in fresh water at 18° C; not analyzed 

by TLC.
10 Investigations in Fish Control 51: Bureau of Sport Fisheries and Wildlife

= °. 725) as determined by the extraction of a l-/ug/ml standard of m-aminobenzoic 

acid was quite consistent. Particular care 

must be taken to remove all gas bubbles that 

form on the sides of the cuvettes. Generally, 

these bubbles will float to the top when the 

sides of the cuvette are tapped lightly with a 

soft object. The maximum absorbance for the 

azo dyes tuff of rn-aminobenzoic acid is identical to that of MS-222, which is 545 nm.

CONCLUSIONS

1. The colorimetric method presented will 

effectively determine MS-222 and rnaminobenzoic acid residues in fish tissues.

2. Recovery of the compounds from spiked 

samples of muscle and liver tissues 

ranged from 82.7 to 110.3 for MS-222 

and from #4.0 to 116.9 percent for maminobenzoic acid.

3. The thin-layer chromatographic method 

presented will positively identify and 

separate MS-222 and m-aminobenzoic 

acid residues in fish tissues.

4. The Rf values for the azo dyestuff of 

MS-222 and rn-aminobenzoic acid are 

near 0.83 and 0.18, respectively.

5. Quantitative estimation and identification 

by the thin-layer chromatographic method 

were possible from samples spiked with 

as little as 1 *tg/g of MS-222 or maminobenzoic acid.

REFERENCES

Alien, John LM Charles W. Luhning, and Paul D. Harman. 

1970. Identification of MS-222 residues in selected 

fish tissues by thin-layer chromatography. U.S. 

Bureau of Sport Fisheries and Wildlife, Investigations in Fish Control, No. 41, 7 p.

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

1939. A new coupling component for sulfanilamide determination. Journal of Biological Chemistry, vol. 

28. p. 537-550.

Luhning, Charles WM and Paul D0 Harman.

1971. Sampling of fish muscle for MS-222 and quinaldine residues. Journal of the Fisheries Research 

Board of Canada, vol. 28, no. 1, p. 113-115.

Steucke, Erwin WM Jr0, 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.

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

52. Residues of MS-222, Benzocaine, and Their

Metabolites in Striped Bass Following Anesthesia

By Charles W. Luhning

United States Department of the Interior

Fish and Wildlife Service 

Bureau of Sport Fisheries and Wildlife
CONTENTS

Page

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

Introduction. ..................................... 3

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

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

Anesthetization of fish. ........................... 4

Analyses of samples ............................. 4

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

MS-222 treated fish ............................. 4

Benzocaine treated fish........................... 7

Discussion ...................................... 9

Conclusions. ..................................... 10

References ...................................... 10
RESIDUES OF MS-222, BENZOCAINE, AND THEIR METABOLITES IN 

STRIPED BASS FOLLOWING ANESTHESIA

By Charles W. Luhning 

Southeastern Fish Control Laboratory 

Warm Springs, Georgia

ABSTRACT. Striped bass (Morone saxatilis) anesthetized in a 

100-mg/l solution of MS-222 at 17.5° C contained an average of 57.9 

Mg/g of MS-222 and 23.3 /zg/g of m-aminobenzoic acid residues in the 

muscle tissue immediately after a 30-minute exposure to the drug (0-hour 

withdrawal samples). After this tissue was homogenized and stored in a 

refrigerator for 1 week at 1.7° C, residues were 100 percent m-aminobenzoic acid. Fish anesthetized with benzocaine and treated in like manner 

still contained residues of benzocaine and a small amount of jp-aminobenzoic 

acid (3.4 percent) after storage. The ester and acid residues of both 

anesthetics decreased steadily with length of recovery time. The residues 

were measured by a modified Bratton-Marshall colorimetric method and 

confirmed by thin-layer chromatography.

INTRODUCTION

MS-222 (methanesulfonate of meta-aminobenzoic acid ethyl ester) is an effective and 

widely used fish anesthetic (Schoettger, 1967; 

Schoettger and Julin, 1967; Schoettger, Walker, 

Marking, and Julin, 1967). Residues of this 

drug occur in various tissues of anesthetized 

fish, but decline to background levels within 

9-24 hours after fish are placed into fresh 

water (Walker and Schoettger, 1967a). Other 

than the fact that residues do disappear, little 

is known about the metabolic fate of MS-222. 

In striped bass muscle tissue analyzed by the 

modified method of Walker and Schoettger 

(1967b), MS-222 residues were found in 0-hour 

through 8-hour withdrawal samples. When 

these same samples were analyzed by the 

thin-layer chromatographic method (Alien, 

Luhning, and Harman, 1970), only the 0-hour 

withdrawal samples contained MS-222 residues. 

These results implied that the former method 

did not distinguish between MS-222 and some 

metabolite. The metabolite was identified as 

m-aminobenzoic acid and was analyzed by the 

method of Luhning (1973).

The objective of this study was to determine 

the amount of MS-222 and rn-aminobenzoic 

acid residue in muscle tissue of striped bass 

anesthetized with MS-222. Analyses of benzocaine, a structural analogue of MS-222, and 

j>-aminobenzoic acid residues in muscle tissue 

of striped bass, bluegill, and largemouth bass 

anesthetized with benzocaine were included 

for comparative purposes.

METHODS AND MATERIALS

Fish

Striped bass (Morone saxatilis), ranging 

from 12.7 to 20.3 cm long, were obtained from 

the National Fish Hatcheries at Edenton, N.C., 

and Welaka, Fla. Bluegill (Lepomis macrochirus), ranging from 16.5 to 21.6 cm long, and 

largemouth bass (Micropterus salmoides). 

ranging from 16.5 to 25.4 cm long, were obtained from the National Fish Hatchery at 

Marion, Ala. All fish were held in limed, 

spring water at the Warm Springs Laboratory 

and were maintained according to the methods 

of Hunn, Schoettger, and Whealdon (1968).
Investigations in Fish Control 52: Bureau of Sport Fisheries and Wildlife

Anesthetization of fish

I used technical-grade (99.4-percent) MS222, the methane sulfonate of m-aminobenzoic 

acid ethyl ester, marketed for experimental 

purposes by Ay erst Laboratories, Inc., New 

York, N.Y., under the trade name of FINQUEL(R) . The m- and_p-aminobenzoic acid 

and_p-aminobenzoic acid ethyl ester were purchased from Eastman Kodak Company. 1

Striped bass, bluegill, and largemouth bass 

were anesthetized for 30 minutes in a 100-mg/l 

aqueous solution of MS-222 buffered to pH 7.0 

with sodium bicarbonate. Also, these three 

species of fish were anesthetized for 15 minutes in a 63.216-mg/l aqueous solution of benzocaine (63.216 mg/1 para-aminobenzoic acid 

ethyl ester is equal on a mole basis to 100 

mg/1 methanesulfonate salt of meta-aminobenzoic acid ethyl ester) buffered to pH 6.5 

with sodium bicarbonate. All fish were exposed 

to 75 1 of anesthetic solution at a temperature 

of 18 + 0.5° C.

Benzocaine is only slightly soluble in water. 

To obtain the concentration of anesthetic 

equivalent to that of MS-222, the benzocaine 

was dissolved in ethanol containing a small 

amount of methanesulfonic acid. Thus, 4.7412 g 

of benzocaine dissolved in 50 ml of ethanol 

were added to 75 1 of limed, spring water to 

obtain a concentration of anesthetic equivalent 

to 100 mg/1 of MS-222.

After the specified exposure time, the fish 

were withdrawn from the anesthetic solution, 

and placed in fresh, flowing water for recovery. This action marked the beginning of 

withdrawal time. Fish killed immediately after 

withdrawal from the anesthetic solution were 

labeled as 0-hour withdrawal samples. Five 

fish were used for residue analyses at each 

withdawal interval.

Analyses of samples

The head, scales, fins, and viscera were 

removed from each fish, and the remaining

Reference to a company or product does not imply 

recommendation to the exclusion of others that may be 

suitable.

tissues were homogenized in a Waring blender. 

One-gram samples of these homogenates were 

analyzed for residues of MS-222, benzocaine, 

and their acid metabolites on the same day the 

fish were killed. The remainder of each 

homogenate was stored in a refrigerator at 

1.7° C, and a sample was analyzed 1 week 

later. One-gram samples of liver and 1 ml of 

blood from some fish also were analyzed on 

the same day the fish were killed.

Samples of homogenized tissue were initially 

analyzed by the colorimetric method described 

by Walker and Schoettger (1967b). In addition 

to this method, the azo dyestuff of each sample 

was partitioned with basic carbon tetrachloride, 

centrifuged, and the aqueous layer separated, 

and made acidic (Luhning, 1973). This aqueous 

fraction contained only the azo dyestuff of the 

acid (m- or j>-aminobenzoic acid).

Standard solutions of mixed ester and acid 

at a concentration of 1 jug/ml each, also were 

partitioned. The amounts of ester and acid 

present in the samples were calculated on a 

ratio basis with standards of the ester and 

acid partitioned in the same manner as the 

samples.

RESULTS

MS-222 treated fish

Twenty striped bass were anesthetized for 

30 minutes in a 100-mg/l solution of MS-222 

at 17.5° C. Deep anesthesia in striped bass 

occurred about the same time as it did in the 

largest specimens among the largemouth bass. 

No mortalities occurred with bluegill or largemouth bass, whereas one striped bass died 

during the 24-hour recovery period.

Striped bass rapidly hydrolyzed the m-aminobenzoic acid ethyl ester to m-aminobenzoic 

acid during exposure and withdrawal (table 1). 

During the first 4 hours of recovery, residues 

of m-aminobenzoic acid dissipated from the 

muscle tissue at a slower rate than did the 

MS-222 residues. For comparison, five bluegill and five largemouth bass anesthetized and 

analyzed in the same way as the striped bass 

contained a small percentage of total residue 

as acid at the 1-hour withdrawal interval.
Luhning: Residues of MS-222, Benzocaine, and Their Metabolites in Striped Bass 5

Table 1. Average concentrations of MS-222 and m-aminobenzoic acid residues 

in muscle tissue of five striped bass, bluegill, and largemouth bass following deep anesthesia in a 100-mg/l aqueous solution of MS-222 buffered to 

pH 7.0 at 1.7.5° G

Species

Exposure 

time 

(minutes)

Withdrawal 

time 

(hours)

Free MS -222 residues

(Mg/g)

Meant s__

X

Range

Free m-aminobenzoic 

acid residues (Mg/g)

Mean± s_

X

Range

Striped 0 

bass2

30

Bluegill4 30

Largemouth 30

bass 5

0

0

1

4

24

1

1

0.0

57.

6.

0.

3 0.

4.

8.

9±

5±

8±

5±

8±

1±

5.59

1.06

0.23

0.16

0.27

0.61

36.1-66.

2.6-

0.2-

0.2-

3.7-

5.8-

8.

1.

0.

5.

9.

5

9

5

8

2

5

0.3±

23. 3±

16. 5±

7.5±

0.2±

0.5±

0.3±

0.09

2.63

1.94

0.79

0.06

0.13

0.10

0.

15.

11.

5.

0.

0.

0.

0- 0.5,

3-30.6

1-21.1

7-10.0

0- 0.2

2- 0.9

0- 0.6

1 Standard error of the mean for five fish.

2 Average length of 15.2 cm and average weight of 46.9 g.

3 Mean of four fish.

4 Average length of 20.1 cm and average weight of 179.0 g.

5 Average length of 20.3 cm and average weight of 154.0 g.

A limited number of striped bass were 

available to test the rate of hydrolysis during 

a 50-minute exposure to a 100-mg/l aqueous 

solution of MS-222 and at intervals during recovery from the drug in fresh, flowing water 

(table 2). The amount of residue accumulated 

after 30 minutes of exposure and no recovery 

time was 44.6 Mg/g, of which 49.6 percent was 

free MS-222. This amount is not in agreement 

with the data found in tables 1 and 4. This discernible difference could have been due to fish 

size, number of fish analyzed, temperature 

difference, source of fish difference, or experimental error.

The added information gained from this 

test was the fact that m-aminobenzoic acid 

residues continued to increase during 50 

minutes of anesthesia and MS-222 residues 

started to decrease after 30 minutes of

anesthesia (fig. 1). Analyses of acetylated 

MS-222 and m-aminobenzoic acid also were 

performed on these samples, and only a small 

amount of the residue was found to be acetylated, mostly as jn-acetylaminobenzoic acid 

(table 2).

Hydrolysis of MS-222 also occurred in 

homogenized striped bass tissue during storage (table 3). Samples of homogenized muscle 

tissue from striped bass, bluegill, and largemouth bass were previously analyzed for MS222 and m-aminobenzoic acid residues (table 1). 

Additional samples were analyzed after storage 

for 1 week at 1.7° C. The 0-hour withdrawal 

samples of striped bass contained an average 

of 51.6 Mg/g acid residues, but no residues of 

MS-222 could be detected at the end of the 

storage period. A reduction in the MS-222 

concentration occurred during storage of
Investigations in Fish Control 52 : Bureau of Sport Fisheries and Wildlife

Table 2. Residues of MS-222, m-aminobenzoic acid and their N-acetyl 

derivatives in muscle tissue of striped bass during different times 

of exposure to and recovery from a 100-mg/l aqueous solution of 

MS-222 buffered to pH 7.0 at 18.5° C

Exposure 

time

(minutes)

Withdrawal 

time 

(minutes )

Average free 1 

residues (Mg/g)

MS -222 Acid

Average acetylated2 

residues (Mg/g)

MS-222 Acid

Total 

residues 

(*g/g)

Percent 

free 

MS-222

15

30

50

30

30

30

30

0

0

0

15

30

45

60

19.0

22.1

15.8

13.2

12.3

7.8

3 5.6

11.9

19.4

24.3

17.8

11.6

11.6

9.4

0.0

0.0

0.0

0.0

0.0

0.5

0.0

3.3

3.1

2.5

2.2

2.0

1.5

1.6

34.2

44.6

42.6

33.2

25.9

21.4

' 16.6

55.6

49.6

37.1

39.8

47.5

36.4

33.7

1 Average of two fish having an average length and weight of 17.8 cm and 

67.6 g., respectively.

2 Obtained by subtracting the free residue and background concentration of 

control (untreated) fish from the amount measured in the samples after acid 

hydrolysis and extraction.

3 Average of two analyses from one fish.

0 = rn-Aminobenzoic acid residues 

X-MS-222 residues

- During exposure

 During recovery

Figure 1. Uptake and hydrolysis of MS-222 residues in 

striped bass muscle tissue during different lengths of 

exposure to a 100-mg/l solution of MS-222 buffered 

to pH 7.0 at 18.5°C, and elimination of MS-222 and 

jn-aminobenzoic acid residues during 1 hour of recovery time in fresh water.

MINUTES
Luhning: Residues of MS-222, Benzocaine, and Their Metabolites in Striped Bass 7

Table 3. Residues of free MS-222, benzocaine, and their acid metabolites in 

homogenized muscle tissue of three species of treated fish stored for 1 

week at 1.7°± 0.2° C

Species

Withdrawal 

time 

(hours)

MS -222 treated 1

MS-222

(Mg/g)

Mean 1 s__3

X

m-aminob enzoic acid 

(*g/g)

Mean± s

X

Benzocaine treated2

Benzocaine

(Mg/g)

Mean± s

X

p-aminobenzoic acid 

(Mg/g)

Mean± s

X

Striped bass

Bluegill

Largemouth bass

0 

1 

4

1

1

0. 

0.

3.

5.

0.0 

1± 0.07 

5± 0.17

8± 0.61

4 (o.o)

0± 0.99 

(0.0)

51 

16 

5

0

0

.6± 

.5± 

.8±

.6± 

(0.

.4± 

(0.

5.73 

2.44 

0.81

0.08 

6)

0.02 

6)

25. 

3. 

0.

1.

2.

8± 

3± 

6±

5± 

(0.

1+ 

(0.

1.83 

0.19 

0.05

0.50 

0)

0.32 

0)

0. 

0. 

0.

0.

9± 0.10 

2± 0.10 

1± 0.04

0.0 

(0.6)

06± 0.04 

(0.4)

1 Anesthetized for 30 minutes in a 100-mg/l solution of MS-222 buffered to 

pH 7.0 at 17.5° C.

2 Anesthetized for 15 minutes in a 63.216-mg/l solution of benzocaine 

(equivalent to 100-mg/l of MS-222) buffered to pH 6.5 at 17.8° C.

3 Standard error of the mean for five samples.

4 Acetylated fraction obtained by subtracting the free and background concentrations of control (untreated) fish from the amount measured in the 

samples after acid hydrolysis and extraction.

homogenized muscle tissue from bluegill and 

largemouth bass, but no significant change occurred in the acid concentration (tables 1 and 

3).

In another test using 0-hour withdrawal 

samples of striped bass tissue, the temperature at which samples were stored greatly influenced the rate at which MS-222 residues 

were hydrolyzed to the acid (tables 4 and 5). 

Homogenized tissue stored at -12.2° C for 3 

weeks contained an average of 21.7 /ug/g MS222 residues. However, after this tissue was 

stored for an additional 8 days at 4.4° C, it 

contained no residues of MS-222.

Residues of MS-222 and m-aminobenzoic 

acid were confirmed in all the 0- and 1-hour 

withdrawal samples of striped bass by the 

thin-layer chromatographic method described 

by Luhning (1973).

Benzocaine treated fish

Striped bass, bluegill, and largemouth bass 

were anesthetized in a 63.216-mg/l aqueous 

solution of benzocaine for 15 minutes at 17.8° C. 

Deep anesthesia occurred in about the same 

time as it did when MS-222 was used. No mortalities of largemouth bass or bluegill occurred before the withdrawal time of 1 hour, 

but one striped bass died during the 24-hour 

recovery period.

Benzocaine residues comprised from 93.5 

to 100 percent of the total residues found in 

these three species of fish (table 6). The mean 

residues of p-aminobenzoic acid in striped 

bass and largemouth bass were about the same 

concentration at the 1-hour withdrawal interval, whereas residues of m-amino benzole acid 

differed greatly between these two species at 

the same withdrawal interval (tables 1 and 6).
8 Investigations in Fish Control 52: Bureau of Sport Fisheries and Wildlife

Table 4. Residues of MS-222 and m-aminobenzoic acid in 0-hour withdrawal 

samples of striped bass muscle tissue during storage at -12.2° C

Storage time

"1 Yl TRTO O IfCS

I

Average /ug/g

MS -222

i/Iean Range

0 35. A- 30.4-44.9

1 28.4 22.3-36.8

2 24.5 15.9-31.9

3 21.7 12.3-31.9

4 18.2 9.9-26.4

5 16.3 8.1-20.9

6 15.3 6.9-20.3

8 15.0 6.8-20.8

free residues 1

m-aminobenzoic acid

Mean

6.9

11.2

12.1

12.7

13.2

14.3

15.7

16.0

Range

5.6- 8.0

10.0-12.2

10.0-14.0

10.0-15.3

11.7-16.0

12.6-16.9

14.1-17.9

14.1-17.9

Percent 

MS -222

83.7

71.7

66.9

62.8

58.0

53.3

49.4

48.4

1 Average of three fish, which were exposed for 30 minutes in a 100-mg/l 

aqueous solution of MS-222 at 20° C.

Table 5. Residues of MS-222 and m-aminobenzoic acid in 0-hour withdrawal 

samples of striped bass muscle tissue kept at -12.2° C for 3 weeks, then 

analyzed during storage at 4.4° C

Storage time 

in days

Average Mg/g free residues -1

MS-222

Mean Range

m-aminobenzoic acid

Mean Range

Percent 

MS-222

0 21.7

1 13.2

3 5.2

6 0.3

8 0.0

12.3-31.9

5.8-19.6

0.4- 8.2

0.0- 0.9

12.7

17.0

20.7

23.7

24.9

10.0-15.3

16.3-17.6

19.7-22.5

21.8-26.4

22.5-28.2

62.8

43.7

20.1

1.3

0.0

1 Average of three fish, which were exposed for 30 minutes in a 100-mg/l 

aqueous solution of MS-222 at 20° C.
Luhning: Residues of MS-222, Benzocaine, and Their Metabolites in Striped Bass 9

Table 6. Average concentrations of benzocaine and p_-aminobenzoic acid residues in muscle 

tissue of five striped bass, bluegill, and largemouth bass following deep anesthesia in 

a 63.216-mg/l aqueous solution of benzocaine buffered to pH 6.5 at 17.8° C

Species

Exposure 

time 

(minutes )

Withdrawal 

time 

(hours)

Free benzocaine 

residues (^g/g)

Meant s_1

X

Range

Free p_-aminobenzoic 

acid residues (Mg/g)

Mean± s__

X

Range

Striped bass2 0.0 0.3± 0.09 0.0-0.5

15

Bluegill3 15

Largemouth bass* 15

0

1

4

24

1

1

37.

7.

0.

0.

1.

2.

9±

3±

7±

2±

9±

9±

2.43

0.99

0.21

0.07

0.51

0.57

28

3

0

0

0

2

.6-41.

.4- 8.

.1- 1.

.0- 0.

.9- 3.

.0- 4.

4

8

2

5

8

8

1.5±

0.2±

0.1±

0.

0.

0.2±

0.46

0.07

0.04

0

0

0.00

0.

0.

0.

8-3.3

0-0.4

0-0.2

--

--

--

1 Standard error of the mean for five fish.

2 Average length of 16.0 cm and average weight of 54.5 g.

3 Average length of 18.0 cm and average weight of 154.0 g.

4 Average length of 19.1 cm and average weight of 106.8 g.

Both the ester and acid residues decreased 

during storage in a refrigerator at 1.7°± 0.2° C 

for 1 week (table 3). This decrease was due 

mainly to acetylation of the free amine. Residues of benzocaine and_p-aminobenzoic acid 

were confirmed in all of the 0-hour withdrawal 

samples of striped bass by thin-layer chromatography.

DISCUSSION

Striped bass muscle tissue contains an 

esterase or a substance capable of cleaving 

the ester of MS-222. Muscle tissue of striped 

bass spiked with MS-222 and analyzed 1 week 

later contained both MS-222 and m-aminobenzoic acid. It is not known whether the muscle 

tissue is completely responsible for the ester 

cleavage while the fish is still alive. Preliminary investigations showed that rn-aminobenzoic acid residues amounted to 27.1 percent 

of the total free residues in striped bass 

muscle tissue, 20.6 percent in blood, and 15.8 

percent in liver after a 30-minute exposure in 

a 100-mg/l solution of MS-222. In the 1-hour 

withdrawal samples, muscle tissue had the

lowest concentration of free acid when compared to blood and liver. No residues of 

acetylated MS-222 were detected in the 0-hour 

withdrawal samples of blood, liver, and muscle. 

In the 0-hour withdrawal samples, residues of 

m-acetylaminobenzoic acid were highest in 

the blood (22.5 Mg/g) and lowest in the muscle 

(1.7 ,ug/g).

A limited amount of information is available 

on the acid metabolite of MS-222, ni-aminobenzoic acid, in freshwater fishes. On the other 

hand, a considerable amount of data is available on the N-acetyl derivative of MS-222, but 

no differentiation is made between the acetylated ester and the acid (Schoettger, Walker, 

Marking, and Julin, 1967; Hunn, Schoettger, 

and Willford, 1968). Presumably MS-222 and 

acetylated MS-222 are eliminated by the gills 

during recovery, whereas m-aminobenzoic 

acid and m-acetylaminobenzoic acid are excreted renally (Hunn, 1970). Investigations by 

Maren, Broder, and Stenger (1968) on the 

metabolism of ethyl-m-aminobenzoate (MS222) in the dogfish shark (Squalus acanthias), 

revealed that 2 hours after dogfish sharks were
10 Investigations in Fish Control 52: Bureau of Sport Fisheries and Wildlife

injected with MS-222, the predominant metabolites in the urine were m-aminobenzoic acid 

and jn-acetylaminobenzoic acid. Hunn, Schoettger, and Willford (1968) reported that acetylated MS-222 was found in much higher concentrations in the urine than in the blood of 

rainbow trout (Salmo gairdneri). No analyses 

were done on urine of striped bass.

In this study, residues of MS-222 and rn_- 

aminobenzoic acid steadily decreased with 

length of recovery time. On a percentage basis 

of all residues determined, m-aminobenzoic 

acid residues increased with recovery time in 

striped bass muscle tissue, but not in muscle 

tissue of bluegill and largemouth bass. Residue 

concentrations of m-aminobenzoic acid increased with an increase in sample storage 

time only in striped bass muscle tissue, whereas residue concentrations of j>-aminobenzoic 

acid remained relatively low in fresh and stored 

muscle tissue of all fish analyzed. The decrease in the concentration of free benzocaine 

residues which occurred in all stored fish 

samples was due mainly to acetylation of the 

free amine. Thus, samples should be analyzed 

as soon as possible to obtain data pertinent to 

each withdrawal interval.

Residues of acetylated MS-222 (rn_-acetylaminobenzoic acid ethyl ester) cannot be determined accurately by the acetylation test of 

Walker and Schoettger (1967b). In this test the 

acetylated fraction is obtained by subtracting 

the free MS-222 and background concentrations 

from the total aromatic amines measured in 

the sample after acid hydrolysis. When the 

ester and acid fractions are separated before 

and after acid hydrolysis, the MS-222 fraction 

obtained after hydrolysis is generally less than 

the amount obtained before hydrolysis. This 

indicates that some free MS-222 is converted 

to free m-aminobenzoic acid during the acid 

hydrolysis. Thus, the esterified compounds 

should be quantified before and after acid hydrolysis, to determine the amount of free MS222 and acetylated MS-222 that might be converted to free m-aminobenzoic acid during 

hydrolysis.

CONCLUSIONS

1. Among the species of fish analyzed to date, 

only the striped bass effectively hydrolyzes

the ester of MS-222 to m-aminobenzoic 

acid in vivo.

2. Hydrolysis of MS-222 also occurs in frozen, 

homogenized muscle tissue from treated 

striped bass.

3. A reduction in the concentration of MS-222 

residues after 1 week storage at 1.7°C in 

bluegill and largemouth bass muscle tissue 

was due mainly to the acetylation of the 

free amine.

4. Analysis of striped bass muscle tissue for 

MS-222 residues must be done soon after 

the fish are killed to determine the amount 

of parent drug rather than its acid metabolite, because hydrolysis occurs during 

storage.

REFERENCES

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

1970. Identification of MS-222 residues in selected 

fish tissues by thin-layer chromatography. U.S. 

Bureau of Sport Fisheries and Wildlife, Investigations in Fish Control No. 41, 7 p.

Hunn, Joseph B.

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

freshwater fishes during anesthesia. U.S. Bureau of 

Sport Fisheries and Wildlife, Investigations in Fish 

Control No. 42, 8 p.

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 MS-222 by rainbow trout, Salmo gairdneri. Journal of the Fisheries Research Board of 

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

Luhning, Charles W.

1973. Methods for simultaneous determination and 

identification of MS-222 and metabolites in fish 

tissues. U.S. Bureau of Sport Fisheries and Wildlife, Investigations in Fish Control No. 51, 10 p.

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

Stenger.

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

in the dogfish, Squalus acanthias. Bulletin Mount 

Desert Island Biological Laboratory, vol. 8, p. 39.
Luhning: Residues of MS-222, Benzocaine, and Their Metabolites in Striped Bass 11

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., and Arnold M. Julin.

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

salmonids. U.S. Bureau of Sport Fisheries and Wildlife, Investigations in Fish Control No. 13, 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.

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

I967a. Residues of MS-222 in four salmonids following anesthesia. U.S. Bureau of Sport Fisheries 

and Wildlife, Investigations in Fish Control No. 

15. lip.

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U.S. GOVERNMENT PRINTING OFFICE : 1974 O-527-811
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

FISH AND WILDLIFE SERVICE

BUREAU OF SPORT FISHERIES AND WILDLIFE 

WASHINGTON. D. C. 2O24O

POSTAGE AND FEES PAID 

U.S. DEPARTMENT OF THE INTERIOR

INT 423