INVESTIGATIONS IN FISH CONTROL

53. Toxicity of Mixtures of Quinaldine 

Sulf ate and MS-222 to Fish

54. The Efficacy of Quinaldine Sulfate:MS-222 Mixtures 

for the Anesthetization of Freshwater Fish

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

Anesthesia with Mixtures of Quinaldine Sulfate:MS-222

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 47-50 (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.

(Reports 51 and 52 are in one cover.)

51. Methods for Simultaneous Determination and Identification of MS-222 and Metabolites in 

Fish Tissues, by Charles W. Luhning. 1973. 10 p.

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

Anesthesia, by Charles W. Luhning. 1973. 11 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

53. Toxicity of Mixtures of Quinaldine Sulfate 

and MS-222 to Fish 

By Verdel K. Dawson and Leif L. Marking

54. The Efficacy of Quinaldine Sulfate:MS-222 Mixtures 

for the Anesthetization of Freshwater Fish 

By Philip A. Gilderhus, Bernard L. Berger, 

Joe B. Sills, and Paul D. Harman

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

Anesthesia with Mixtures of Quinaldine Sulfate:MS-222 

By Joe B. Sills, John L. Alien, Paul D. Harman, 

and Charles W. Luhning

United States Department of the Interior

Fish and Wildlife Service 

Bureau of Sport Fisheries and Wildlife
IN THIS COVER

Pages

Toxicity of Mixtures of Quinaldine Sulfate and MS-222 to Fish,

by Verdel K. Dawson and Leif L. Marking ............... 1-11

The Efficacy of Quinaldine Sulfate: MS-222 Mixtures for the 

Anesthetization of Freshwater Fish, by Philip A. Gilderhus, 

Bernard L. Berger, Joe B. Sills, and Paul D. Harman ....... 1-9

Residues of Quinaldine and MS-222 in Fish Following Anesthesia 

with Mixtures of Quinaldine Sulfate:MS-222, by Joe B. Sills, 

John L. Alien, Paul D. Harman, and Charles W. Luhning ..... 1-12

ii
FOREWORD

All chemicals, including anesthetics, which are to be used on food or 

game fishes must be approved and registered by the Food and Drug Administration. Research to support petitions for registration of such compounds is an integral part of the program of the Fish Control Laboratories. Studies involving anesthetics have centered on quinaldine and tricaine 

methanesulfonate (MS-222, Finquel^), the two most commonly used 

anesthetics for fish.

While each compound is effective in itself, studies have shown that 

when MS-222 and quinaldine are used together, desired anesthesia is 

achieved without undesirable side effects noted when the compounds are 

used singly.

Registration-oriented research on MS-222 was reported in Investigations in Fish Control (IFC). numbers 12-17. The development of a 

water-soluble salt of quinaldine and related studies to support a petition 

for its registration are found in IFC, numbers 47-50.

The papers which follow are concerned with research on the toxicity, 

efficacy, and residues associated with the use of mixtures of quinaldine 

sulfate and MS-222 as an anesthetic for selected coldwater and warmwater fishes. The data presented will be used to support a petition for 

registration to permit the use of such mixtures on fish.

Fred P. Meyer, Director 

Fish Control Laboratories

iii
INVESTIGATIONS IN FISH CONTROL

53. Toxicity of Mixtures of Quinaldine Sulfate 

and MS-222 to Fish

By Verdel K. Dawson and Leif L. Marking

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.............................. 4

Results ........................................ 5

Effects of QdSO^ :MS-222 combinations on test solutions. .... 5

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

Effects of temperature and water hardness ............. 6

Effects of pH ................................. 6

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

Quantification of additive toxicity.................... 7

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

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

References ..................................... 10
TOXICITY OF MIXTURES OF QUINALDINE SULFATE AND MS-222 TO FISH

By Verdel K. Dawson and Leif L. Marking 

Fish Control Laboratory, La Crosse, Wisconsin

ABSTRACT.~The acute toxicities of mixtures of two fish anesthetics 

(quinaldine sulfate and MS-222) to coho salmon, rainbow trout, brown 

trout, brook trout, lake trout, carp, channel catfish, bluegill, and largemouth bass of various sizes were determined in 15-, 30-, and 60-minute 

and 24-, 48-, and 96-hour static toxicity tests. The effects of various 

temperatures, water hardnesses, and pH's on the mixture's toxicity were 

evaluated. The 96-hour LCSO's of QdSO4:MS-222 in ratios of 1 : 4 ranged 

from 4.23:16.9 mg/1 for lake trout to 8.63:34.5 mg/1 for carp in standard reconstituted water at 12° C. Temperature changes had little influence on the effect of the drugs. In very soft water, solutions of the 

combination are acidic and considerably less toxic than in harder water. 

The toxicity of the mixture decreases with decreasing pH, especially 

below pH 6.5. Safety indices (lethal concentration/effective concentration) indicate that the safety margin is greater at shorter exposures.

INTRODUCTION

An anesthetic is an important tool for handling fish during operations such as artificial 

spawning, marking, weighing, measuring, 

transporting, and others. MS-222 has been 

shown to be an effective anesthetic for fish 

and other coldblooded organisms. The action 

of MS-222 is characterized by rapid and deep 

anesthetization, but concentrations that render 

fish immobile are not tolerated for extended 

periods (McFarland, 1959; Bove, n.d.; 

Schoettger and Julin, 1967). Bove'(n.d.)identifiedMS-222as the methanesulphonateof metaaminobenzoic acid ethyl ester. The compound 

is a fine, white crystalline powder which is soluble to 11 percent in water and forms a clear, 

colorless, acidic, and relatively stable solution.

The anesthetic effect of quinaldine (2- 

methylquinoline) on fish was first reported by 

Muench (1958). Schoettger and Julin (1969) 

further investigated the use of quinaldine as 

an anesthetic for several species of hatcheryreared fish under a variety of temperature and 

water quality conditions. The action of 

quinaldine in fish is characterized by long, 

safe exposure times, but it does not entirely

block reflex movements. Quinaldine occurs in 

coal tar and is made from aniline, acetaldehyde, and hydrochloric acid. It is a colorless, oily liquid that turns reddish-brown upon 

exposure to air. Quinaldine is soluble in 

alcohol, ether, chloroform, and acetone but is 

insoluble in water (Stecher, 1968).

Schoettger and Steucke (1970) reported mixtures of quinaldine and MS-222 to be synergic 

for anesthetizing fish. The combination of anesthetics exhibits the rapid sedation and lack of 

reflex response typical of MS-222 and the long, 

safe exposure time typical of quinaldine.

Amendments to the Federal Food, Drug, and 

Cosmetic Act require that chemicals used on 

fish be registered for their specific uses (Lennon, 1967). The registration of MS-222 has 

been supported by information on its toxicity 

or maximum safe exposure to fish (Marking, 

1967) and the persistence of residues in fish 

tissues (Walker and Schoettger, 1967).

Recently, Alien and Sills (1973) synthesized 

quinaldine sulfate (QdSCv;), a salt of quinaldine, 

which is water soluble and has a less pungent 

odor than quinaldine. Gilderhus, et al. (1973)
4 Investigations in Fish Control 53: Bureau of Sport Fisheries and Wildlife

evaluated the efficacy of QdSO4 to 15 species 

of fish and found it to be as effective, on the 

basis of active ingredient, as that of quinaldine. The toxicity of QdSO^ to fish under a 

variety of conditions was determined by Marking and Dawson (1973). Sills, et al. (1973) 

measured QdSO^ residues in 10 species of 

fish.

Because QdSO^ is more convenient to use 

than quinaldine, tests of mixtures of QdSO^ and 

MS-222 were devised. The purpose of this investigation was to define concentrations of 

three ratios of the combination which are toxic 

to various species and sizes of freshwater 

fish at selected exposure periods in water at 

three temperatures, four water hardnesses, 

and four pH's. In addition, the safety must be 

determined for use pattern concentrations and 

exposures.

METHODS AND MATERIALS

The QdSO.; (quinaldine sulfate) was synthesized at the Southeastern Fish Control 

Laboratory, Warm Springs, Ga. The MS-222 

(methane sulfonate of meta-aminobenzoic acid 

ethyl ester) was Finquel^ , marketed by 

Ay erst Laboratories, Inc.

Static toxicity tests of mixtures of QdSO^ 

and MS-222 were conducted with 3- to 6-cm 

fish in glass jars containing 15 1 of water according to the methods of Lennon and Walker 

(1964). Larger fish were exposed to the anesthetics in 45-1 polyethylene tanks. The two 

drugs were tested for toxicity against coho 

salmon (Oncorhynchus kisutch), rainbow trout 

(Salmo gairdneri), brown trout (Salmo trutta), 

brook trout (Salvelinus fontinalis), lake trout 

(Salvelinus namaycush), carp (Cyprinus 

carpio), channel catfish (Ictalurus punctatus), 

bluegill (Lepomis macrochirus), and largemouth bass (Micropterus salmoides). The fish 

were obtained from fish hatcheries, maintained under a fish culturist's care (Hunn, 

et al., 1968), acclimated to the test water 

before the chemical was added, and incinerated after death. Ten fish were exposed 

to each concentration of the anesthetics, and 

mortalities were recorded periodically the

first day and daily thereafter during the 

96-hour tests.

The hardness of the test water was altered 

by adding selected amounts of reconstituting 

salts to deionized water, and the pH in certain tests (ranging from 6.5 to 9.5) was adjusted and maintained with chemical buffers 

(Marking and Dawson, 1973). Temperatures of 

7°, 12°, and 17° C were controlled by water 

baths.

Stock solutions of the anesthetics dissolved 

in water were added to the bioassays to obtain 

the desired concentrations. QdSO^ and MS222 were tested in a ratio of 1:4 against the 

nine available species, while additional ratios 

of 1:6 and 1:2 were tested against representative coldwater and warmwater species.

The mortality data were analyzed according 

to the method of Litchf ield and Wilcoxon (1949) 

to determine LCSO's (concentration causing 50 

percent mortality), variations, slope functions, 

and 95-percent confidence intervals.

Finger ling rainbow trout (1.1 g) were exposed to mixtures of QdSO4 and MS-222 (1 : 2) 

to determine safety indices (Marking, 1967). 

A safety index refers to the margin between 

efficacy and mortality and is expressed by the 

quotient of a lethal concentration (LC50) and 

an effective concentration (EC50). The EC50 

defines the concentration of drugs which produces total loss of equilibrium (stage 2) in 

half the organisms (Schoettger and Julin, 

1967). The maximum safety index (LC1/EC99) 

is lower than the safety index and is biased in 

favor of greater safety.

The toxicity of mixtures of the anesthetics 

was defined by an additive index1 developed 

at the Fish Control Laboratory. The index expresses the toxicity quantitatively with zero 

indicating strictly additive toxicity. Negative 

values indicate less than additive toxicity and 

positive values indicate greater than additive 

toxicity.

1 Lelf L. Marking and Verdel K, Dawson0 A method 

to assess the toxic or other effects of mixtures of 

chemicals. (Manuscript)
Dawson and Marking: Toxicity of Mixtures of Quinaldine Sulfate and MS-222 to Fish

A B

 + = S, the sum of biological effects

A. ID. 

1 1

Additive index = - -1.0 for S<1.0 and 

Additive index = [ S(-l) ] +1.0forS>1.0

where A and B represent concentrations of 

chemicals. Individual concentrations are 

designated by i and mixtures of A and B are 

designated by m.

RESULTS

Effects of QdSO4 ;MS-222 Combinations on 

Test Solutions

QdSO4 :MS-222 solutions are acidic and influence the pH of bioassay water, especially 

softer waters. A stock solution containing 30 

g of QdSO4 and 60 g of MS-222 in a liter of 

deionized water has a pH of 1.25. Each chemical decreases the pH of bioassay waters significantly (Marking and Dawson, 1973; Alien 

and Harman, 1970). The extent of the reduction of pH in waters of various hardnesses by 

QdSO4 :MS-222 solutions in the ratio of 1 : 2 is 

given in table 1. Very soft water is poorly 

buffered, and the pH is lowered more than 

40 percent by a QdSO4 :MS-222 concentration 

of 35:70 mg/1. In harder waters the pH is

more stable, and in very hard water the pH 

drops only 12.6 percent at this concentration 

of the drugs.

The extent to which the pH is decreased appears to be independent of the ratio of the anesthetics. The percentage reduction of the pH 

was very nearly the same at ratios of 1:2, 1 : 4, 

and 1:6 where the total concentration of the 

two anesthetics was 75 mg/1 (table 2).

Species and Sizes of Fish

The toxicity of the mixture of QdSO^ and 

MS-222 to nine species of fish is presented in 

table 3. We selected one ratio of the combination (1:4) to scrutinize the effects of the 

drugs on various species tested. Lake trout 

are the most sensitive at all exposures to the 

1:4 ratio of QdSO4 and MS-222 (LC50 = 7.25: 29.0 

mg/1 at 1 hour and 4.23:16.9 mg/1 at 96 

hours), and coho salmon are the most resistant (LC50 = 11.3:45.0 mg/1 at 1 hour and 

6.53:26.1 mg/1 at 96 hours) (table 3).

Among the warmwater species tested, 

largemouth bass are the most sensitive to the 

QdSO4:MS-222 combination with 1- and 96- 

hour LCSO's of 7.75:31.0 and 5.38: 21.5 mg/1, 

respectively. At a 1-hour exposure to the anesthetics, channel catfish are the most resistant (LC50 = 14.0:56.0 mg/1), but at 96 hours 

of exposure carp are the most resistant 

(LC50 = 8.63:34.5 mg/1).

Table 1. Influence of combinations of Q£S04 :MS-222 (1:2) on the pH of test

solutions of various hardnesses

Water 

hardness

Initial 

pH

Reduction of pH at Q1S04 : MS-222 mixtures (mg/l) of

15:30

Final 

pH

Percentage 

reduction

25:50

Final 

pH

Percentage 

reduction

35:70

Final 

pH

Percentage 

reduction

very soft, 

soft, 

hard, 

very hard,

6.48

7.36

7.86

8.20

4.53

6.57

7.19

7.57

30.1

10.7

8.52

7.68

3.95

5.98

6.95

7.34

39.0

18.8

11.6

10.5

3.74

5.60

6.75

7.17

42.3

23.9

14.1

12.6
6 Investigations in Fish Control 53: Bureau of Sport Fisheries and Wildlife

Table 2. Influence of three ratios of QdS04 :MS-222 (75.0 mg/1 total) on the 

pH of test solutions of various hardnesses

Water 

hardness

Initial 

pH

Reduction of pH at QdS04 : MS-222 ratios (mg/1) of

25:50

Final 

pH

Percentage 

reduction

15:60

Final 

PH

Percentage 

reduction

10.7:64.3

Final 

pH

Percentage 

reduction

very soft .... 

soft .........

hard .........

very hard ....

6.4-8 

7.36

7.86

8.20

3.95 

5.98

6.95

7.34

39.0

~\ ft ft

n

£L

10.5

3.94 

6.03

6.94 ,

7.35

39.2 

18.1

~\ ~i "7

10.4

3.96 

6.02

6.96

7.38

38.9 

18.2

11.5

10.0

Larger sizes of coho salmon and brook 

trout were exposed to the 1:4 ratio of QdSQ; 

and MS-222. The 13.2-g coho salmon and 

11.6-g brook trout were more resistant than 

smaller fish of the same species (table 3).

Effects of Temperature and Water Hardness

The toxicity of the drugs to fingerling rainbow trout in soft reconstituted water was not 

significantly different (P= 0.05) at temperatures of 7°, 12°, and 17° C (table 4). The lack 

of influence by temperature was evident at all 

exposure periods and concentration ratios 

tested.

Water hardness apparently influences the 

toxicity of mixtures of the two anesthetics. 

The 96-hour LCSO's for rainbow trout in very 

soft water (12 mg/1 of total hardness) were 

significantly (P = 0.05) greater than in harder 

waters (table 4). The decreased activity of the 

combination in very soft water can possibly be 

attributed to a decrease in pH as indicated in 

table 2. At a total of 75 mg/1 of QdSO4 and 

MS-222 in ratios of 1 : 2, 1 : 4, or 1:6, the pH of 

the test solution dropped to about 4.0. This is 

below the pKa value of 5.42 for quinaldine 

(Knight et al., 1955; Sober, 1968) and is very 

near the pKa value of 3.5 for MS-222 (Maren 

et al., 1968). The equilibrium for both chemicals, therefore, is shifted in favor of the 

ionized form which is relatively unavailable 

to the fish (Sills and Alien, 1971).

In soft water (44 mg/1 of total hardness), 

the combination is more toxic than in very 

soft water (12 mg/1 of total hardness), and 

the 96-hour LCSO's for the 1:4 ratio against 

rainbow trout are 5.50:22.0 mg/1 and 

7.63:30.5 mg/1, respectively. At 96 hours the 

toxicity of the drugs is insignificantly 

(P= 0.05) different in soft, hard (170 mg/1 of 

total hardness) and very hard (300 mg/1 of 

total hardness) water (table 4).

Effects of pH

Although solutions were chemically buffered 

to specific pH's, the acidic nature of the stock 

solution of the anesthetics caused a reduction 

in the pH of the bioassay water. Various 

amounts of 1 N NaOH were added to each test 

vessel, depending upon the concentration of 

the anesthetics, to readjust the pH to the 

original value. A linear regression of the ml 

of 1 N NaOH required to readjust the pH to its 

original value versus the total concentration 

of the combination of anesthetics produced 

a slope, intercept, and correlation coefficient 

of 0.08, 0.0, and 0.9995, respectively. The 

slope indicates that regardless of the initial 

pH of the solution, 0.08 ml of 1 N NaOH is 

required for each mg/1 of the combination in 

order to readjust the pH to its original value.

Tests of the combination of QdSO^ :MS-222 in 

the ratio of 1:2 at pH 6.5, 7.5, 8.5, and 9.5 indicate the activity is greater at the higher
Dawson and Marking: Toxicity of Mixtures of Quinaldine Sulfate and MS-222 to Fish 7

Table 3. Toxicity of combinations of QjiSO,; a 1 MS-222 to fish in soft reconstituted water at 12° C

Species

Average 

weight 

(g)

Average 

length 

(cm)

Ratio of 

QiSO*: 

MS-222

LC50 of QiSO,;: MS-222 combinations (mg/l) at

1 hour 3 hour's 6 hours 24 hours 96 hours

Coho salmon....

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

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

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

Rainbow trout.. 

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

Brown trout....

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

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

Brook trout....

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

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

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

Lake trout.

Carp. 

Do. 

Do.

Channel catfish 

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

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

Bluegill. 

Do..... 

Do.....

Largemouth bass

0.7

0.7

0.7

13.2

0.6 

0.6 

0.6

1.2 

1.2

1.2 

11.6

0.5

1.3 

1.3 

1.3

1.8 

1.8

1.5 

1.5 

1.5

4.1

4.1

4.1

10.7

3.1 

3.8

3.8 

3.8 

3.8

4.8

4.8

4.8

10.2

4.1

4.3 

4.3 

4.3

4.3 

4.3 

4.3

1:6 

1:4 

1:2 

1:4

1:4 

1:2

1:6 

1:4 

1:2

1:6 

1:4 

1:2 

1:4

1:4

1:6 

1:4 

1:2

1:6 

1:4 

1:2

1:6 

1:4 

1:2

1:4

7.67:46.0 

11.3:45.0 

16.6:33.1 

11.2:44.8

6.98:41.9 

11.0:43.9 

16.6:33.1 

11.2:44.8

6.85:41.1 

10.6:42.4 

16.6:33.1 

11.1:44.2

5.21:31.3 

7.70:30.8 

12.8:25.5 

9.58:38.3

4.93:29.6 

6.53:26.1 

10.3:20.5 

8.55:34.2

10.5:42.0 

16.0:31.9

6.83:41.0 

10.5:42.0 

16.5:33.0

9.40:37.6 

14.5:29.0

6.50:39.0 

9.05:36.2 

14.9:29.7

8.05:32.2 

13.5:27.0

6.28:37.7 

8.73:34.9 

13.7:27.3

6.35:25.4 

9.05:18.1

4.45:26.7 

6.68:26.7 

10.0:20.0

5.50:22.0 

9.05:18.1

4.45:26.7 

5.73:22.9 

9.15:18.3

8.58:51.5 

11.0:44.0 

18.9:37.8 

13.2:52.7

8.42:50.5 

10.7:42.7 

18.7:37.3 

11.7:46.6

7.85:47.1 

10.3:41.2 

16.8:33.6 

10.3:41.0

5.95:35.7 

7.23:28.9 

10.9:21.8 

8.70:34.8

4.70:28.2 

6.15:24.6 

9.85:19.7 

7.63:30.5

7.25:29.0 6.53:26.1 5.95:23.8 4.25:17.0 4.23:16.9

8.83:53.0 

11.0:44.1 

22.3:44.5

7.22:43.3 

10.2:40.9 

18.1:36.1

6.90:41.4 

9.78:39.1 

16.9:33.8

6.90:41.4 

8.78:35.1 

16.0:32.0

6.68:40.1 

8.63:34.5 

14.7:29.3

9.25:55.5

14.0:56.0 

23.4:46.7

8.50:51.0 

9.25:37.0 

16.1:32.1

9.25:55.5 

11.9:47.7 

20.4:40.7

5.53:33.2 

7.08:28.3 

13.8:27.5

7.67:46.0 

11.6:46.2 

20.0:40.0

4.87:29.2 

7.08:28.3 

12.3:24.6

7.23:43.4 

9.25:37.0 

17.2:34.3

4.87:29.2 

6.93:27.7 

11.0:22.0

5.48:32.9 

7.70:30.8 

11.7:23.3

4.80:28.8 

6.93:27.7 

11.0:22.0

7.75:31.0 6.75:27.0 6.25:25.0 5.50:22.0 5.38:21.5

pH's, especially in longer exposures (table 5) 

However, the influence of pH in this range is 

relatively small. The 96-hour LCSO's for the 

combination at pH 6.5 and 9.5 are 8.35:16.7 

and 5.90:11.8 mg/l, respectively.

Safety Indices

Safety indices (LC50/EC50) and maximum 

safety indices (LC1/EC99) were determined 

for mixtures of QdSO4 :MS-222 (1 : 2)against 

rainbow trout at 5-f 10-, 15-, and 30-minute 

exposures (table 6). The safety indices ranged 

from 4.23 to 2.60 at 5 and 30 minutes, respectively. The safety index values averaged 

1.6 times the corresponding maximum safety 

indices. As the exposure time increased, the 

safety margin decreased. Therefore, increased safety is achieved by using concentrations that are effective at shorter exposures.

Quantification of Additive Toxicity

Schoettger and Steucke (1970) indicated 

that the combination of MS-222 and quinaldine 

was synergic for anesthetizing fish. The extent of the synergism, or the effect of changing the ratio of the two materials, was not 

fully defined. The additive index was determined for the data to quantitate the extent of 

synergism at selected ratios of the two chemicals. If the index is greater than zero, 

synergism or greater than additive effect 

is indicated.

Table 7 presents the index values for selected 

species, exposure periods, and ratios of the two 

anesthetics. In all cases the index values are 

greater than zero, and the average of all values 

is 0.26 indicating that the effect of the two chemicals is greater than additive. Statistical analysis failed to show any significant difference 

in the additive toxicity between any of the 

ratios tested (P = 0.05).
8 Investigations in Fish Control 53: Bureau of Sport Fisheries and Wildlife

Table 4. Toxicity of combinations of QdSCU and MS-222 to fingerling rainbow trout at various

temperatures and water hardnesses

Temp, (°c) Water 

hardness

Ratio of 

QdS04 :MS-222

LC50 of QdS04 :MS-222 combinations (mg/l) at

0.25 hour 0.5 hour 1 hour 3 hours 6 hours 24- hours 96 hours

7........

Do.....

12.......

Do.....

17.......

Do.....

12 .......

Do. . . . .

Do.....

Do. . . . .

Do.....

Do.....

1:2

1:4

1:2

1:4

1:2

1:4

1:2

1:4

1:2

1:4

1:2

1:4

25.3:50.6 16.5:32.9 15.6

9.80

27.5:55.0 16.2:32.3 16.0

10.5

26.5:52.9 15.4:30.8 14.6

10.4

14.8

15.6:31.1 14.9

9.00

15.0:30.0 14.1

8.63

:31.2

:39.2

:31.9

:42.0

:41.5

:59.0

:36.0

:34.5

13.8:27.6

9.10:36.4

14.5:29.0

9.40:37.6

13.5:27.0

8.80:35.2

34.1:68.2

12.8:51.3

14.1:28.1

8.38:33.5

14.1:28.2

8.18:32.7

11.0

8.05

13.5

8.05

12.5

7.50

28.0

12.1

12.1

:21.9

:32.2

:27.0

:32.2

:25.0

:29.0

:56.0

:24.1

:24.1

9.25:18.5

6.35:25.4

9.05:18.1

6.35:25.4

8.50:17.0

5.73:22.9

16.8:33.6

9.75:39.0

8.75:17.5

6.00:24.0

9.90:19.8

5.80:23.2

8

9

8

5

7

8

5

9

5

.40

.50

.05

^o

,35

.40

,63

.75

.73

.05

,55

:16.8

-.22.0

:18.1

:22.0

:16.7

:21.6

:30.5

:17.5

 22.9

:18.1

:22.2

Table 5.--Toxicity of combinations of QdS04 and MS-222 (1:2) to fingerling 

rainbow trout in soft reconstituted water at 12° C buffered to selected pH's

Time 

( hours)

0.25.........

0.50.........

1.0..........

3.0..........

6.0..........

24.0.........

96.0.........

LC50 of QdS04 :MS-222 combinations (mg/l)

6.5

20.0:40.0 

15.3:30.5 

13.7:27.4 

12.5:25.0

9.60:19.2 

8.35:16.7

7.5

18.0:36.0 

15.0:30.0 

13.3:26.6 

11.5:23.0

8.10:16.2 

8 . 10 : 16 . 2

8.5

24.1:48.1 

15.0:30.0 

12.5:25.0 

9.55:19.1 

9.55:19.1 

6.20:12.4 

6.20:12.4

at pH

9.

17.2 

15.5 

10.6 

9.05 

9.05 

6.15 

5.90

5

:34.4 

:31.0 

:21.1 

:18.1 

:18.1 

:12.3 

-.11.8

Table 6. Safety indices for mixtures of QdSO^.: MS-222 (1:2) using rainbow 

trout in soft reconstituted water at 12° C

Exposure

( min) L

5.........

10.........

15 .........

30.........

Concentration of

C50 EC50

56.7 13.4

42.2 11.5

34.5 10.8

27.3 10.5

anesthetics

LC1

37.5

i£. n

25.1

21.8

(nig/1)

EC99

K

ry

-IQ rt

13.1

1?.8

Safety

LC50/EC50

4 P3

3.67

3.19

P.60

indices

LC1/EC99

2.55

2.61

1.92

1.70
Dawson and Marking: Toxicity of Mixtures of Quinaldine Sulfate and MS-222 to Fish 9

Table 7.--Toxicity of QdS04 and MS-222 (LC50's in mg/l) individually and in 

combination and their additive indices at selected exposure periods

Species Exposure 

(hours)

Individually

CflSO*1 MS-222 2

In combination

QdS04 MS-222 Ratio Additive 

index

Rainbow trout. . .

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

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

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

Brown trout .....

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

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

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

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

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

Brook trout. ....

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

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

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

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

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

Lake trout. .....

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

Bluegill. .......

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

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

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

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

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

Largemouth bass.

24

24-

96

96

24

24

24

96

96

96

24

24

24-

96

96

96

24

96

24

24-

24

96

96

96

24

37.0

37.0

31.8

31.8

32.7

32.7

32.7

28.3

28.3

28.3

27.2

27.2

27.2

22.2

22.2

22.2

16.3

15.5

36.8

36.8

36.8

32.0

32.0

32.0

16.0

39.0

39.0

38.4

38.4

38.5

38.5

38.5

43.8

43.8

43.8

50.7

50.7

50.7

50.0

50.0

50.0

33.8

32.0

45.7

45.7

45.7

45.7

45.7

45.7

47.0

6.35

9.05

5.50

9.05

4.45

6.68

10.0

4.45

5.73

9.15

5.95

7.23

10.9

4.70

6.15

9.85

4.25

4.23

4.87

6.93

11.0

4.80

6.93

11.0

5.50

25.4

18.1

22.0

18.1

26.7

26.7

20.0

26.7

22.9

18.3

35.7

28.9

21.8

28.2

24.6

19.7

17.0

16.9

29.2

27.7

22.0

28.8

27.7

22.0

22.0

1:4

1:2

1:4

1:2

1:6

1:4

1:2

1:6

1:4

1:2

1:6

1:4

1:2

1:6

1:4

1:2

1:4

1:4

1:6

1:4

1:2

1:6

1:4

1:2

1:4

0.22

0.41

0.34

0.32

0.21

0.11

0.21

0.30

0.38

0.35

0.08

0.20

0.20

0.29

0.30

0.19

0.31

0.25

0.30

0.26

0.28

0.28

0.22

0.21

0.23

1 From (Marking and Dawson, 1973)

2 From (Marking, 1967)

DISCUSSION

The pattern of toxic response of the mixture of QdSC>4:MS-222 among various species 

and sizes of fish is similar to that of each 

component when tested individually (Marking, 1967; Marking and Dawson, 1973). In 

both cases lake trout were the most sensitive of the coldwater species tested and largemouth bass were the most sensitive of the 

warmwater species tested. Also, larger fish

of the same species were more resistant than 

smaller ones.

The effect of temperature, however, does 

not show a similar pattern when tested individually. Temperature had very little effect on the toxicity of the mixture of the anesthetics to rainbow trout. However, when 

MS-222 was tested individually the trout were 

more resistant at lower temperatures. This
10 Investigations in Fish Control 53: Bureau of Sport Fisheries and Wildlife

was true also of the QdSO4 for longer exposures, but the trend was reversed in 1- to 

6-hour exposures.

Tests of the anesthetic mixture at adjusted 

pH's of 6.5, 7.5, 8.5, and 9.5 indicated the pH 

had only a slight influence on the toxicity of 

the anesthetics in this range. This is not surprising considering the pKa value of each of 

the components is more than one pH unit 

below the lowest pH tested. According to the 

Henderson-Hasselbach equation, even at pH 

6.5, 92.3 percent of the QdSCX; and 99.9 percent of the MS-222 would be un-ionized. The 

un-ionized forms of both molecules are lipidsoluble, thereby making both anesthetics potentially available to the fish (Sills and Alien, 

1971).

On the other hand, there was a singificant 

decrease in the pH of poorly buffered solutions. This is because QdSO4 is a watersoluble salt of quinaldine, and MS-222 is a 

water-soluble salt of m-aminobenzoic acid 

ethyl ester. Being water-soluble, the salt 

forms are easier to handle, but the sulfuric 

acid from QdSC>4 an<^ the methane sulfonic 

acid from MS-222 are strong acids. If the 

anesthetic mixture were used in soft, unbuffered water, the pH may go below 6.5, and 

there would be a substantial decrease in both 

toxicity and efficacy.

The toxicity of the anesthetics is increased 

when they are combined as indicated by an 

average additive index of 0.26. The increased 

toxicity of the combination would be hazardous 

when the desired effect is sedation and not 

mortality. However, when the additive index 

formula is applied to information presented 

by Berger (1969) on the efficacy of the mixture as an anesthetic, a value of 0.29 is obtained. The index for toxicity and the index 

for anesthesia are both greater than one, thus 

indicating that although the mixture is more 

toxic it also is more effective as an anesthetic. The important advantage in using the 

mixture is in combining the rapid, deep anesthetization of MS-222 and the long, safe exposure time of QdSO,;.

CONCLUSIONS

1. Ninety-six hour LCSO's for the 1:4 ratio 

of QdSO4 :MS-222 among nine species of 

fish ranged from 4.23:16.9 mg/1 for lake 

trout to 8.63:34.5 mg/1 for carp in soft 

reconstituted water at 12° C.

2. Larger fish are generally more resistant 

to the combined anesthetics than smaller 

fish.

3. The toxic effect of the combination is 

greater than additive as indicated by an 

average additive index of 0.26. The additive toxicity of QdSO4 :MS-222 ratios of 

1:2, 1 : 4, and 1:6 were insignificantly different (P = 0.05).

4. The toxicity of the drugs to fingerling 

rainbow trout was not influenced by temperature changes from 7° to 17° C.

5. The combination of anesthetics is slightly 

less toxic in solutions adjusted to pH 6.5 

than in solutions adjusted to pH 9.5. The 

lower pH probably reduces the concentration of the active, un-ionized form of the 

molecules.

6. The mixture is less toxic in very soft 

water than in harder water, but the decreased pH in very soft water is considered responsible for the reduced activity.

7. Safety indices indicate that the safety 

margin is greater at shorter exposures.

REFERENCES

Alien, John L.* and Joe B. Sills.

1973. Preparation and properties of quinaldine sulfate, 

an improved fish anesthetic,, U.S. Bureau of Sport 

Fisheries and Wildlife. Investigations in Fish Control, No. 47, 7 p.
Dawson and Marking: Toxicity of Mixtures of Quinaldine Sulfate and MS-222 to Fish 11

Alien, John L., and Paul D. Harman. 

1970. Control of pH in MS-222 anesthetic solutions. 

The Progressive Fish-Culturist, vol. 32, noa 2, 

P. 100.

Berger, Bernard L.

1969. A synergic mixture of MS-222 and quinaldine 

sulfateasan anesthetic. Presented at the 31st Midwest Fish and Wildlife Conference. St. Paul, 

Minnesota. Mimeo. 7 p.

Bove, Frank J.

n.d. MS-222 Sandoz the anaesthetic and tranquilizer 

of choice for fish and other cold-blooded organisms. 

Sandoz, Ltd. Pharmaceutical Chemicals Division. 

Basle, Switzerland. 20 p.

Gilderhus, Philip A^, Bernard L. Berger, Joe B. Sills, 

and Paul D. Harman.

1973. The efficacy of quinaldine sulfate (QdSO^.) as an 

anesthetic for freshwater fish. U.S. Bureau of Sport 

Fisheries and Wildlife, Investigations in Fish Control, 

No. 49, 9 p.

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

Whealdon.

1968. Observations on the handling and maintenance of 

bioassay fish. The Progressive Fish-Culturist, 

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

Knight, Samuel B0 R, H. Wallick, and Carolyn Bolch. 

1955. The ultraviolet spectra and the dissociation constants of the monochloroquinolines and the 

monomethylquinolines. Journal of the American 

Chemical Society, vol. 17, p. 2577-2579.

Lennon, Robert E.

1967. Clearance and registration of chemical tools 

for fisheries,, The Progressive Fish-Culturist, 

vol. 29, no, 4, p. 187-193.

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

1964. Laboratories and methods for screening fishcontrol chemicals. U.S. Bureau of Sport Fisheries 

and Wildlife, Investigations in Fish Control, No. 1, 

Circular 185, 15 p.

Litchfield, J0 T0 JrM and F. Wilcoxon, 

1949. A simplified method of evaluating dose-effect 

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

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, p. 853-864.

Marking, Leif Le

1967. Toxicity of MS-222 to selected fishes. U.S. Bureau of Sport Fisheries and Wildlife, Investigations 

in Fish Control, No. 12, 10 p.

Marking, Leif L., and Verdel K. Dawson. 

1973. Toxicity of quinaldine sulfate to fish. U.S. Bureau of Sport Fisheries and Wildlife, Investigations 

in Fish Control, No. 48, 8 p.

McFarland, William N.

1959. A study of the effects of anesthetics on the be. 

haviorand physiology of fishes. Publications of the 

Institute of Marine Science, University of Texas, 

vol. 6, p. 23-55.

Muench, Bruce.

1958. Quinaldine, a new anesthetic for fish. The Progressive Fish-Culturist, vol. 20, no. 1, p. 42-44.

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., and Arnold M. Julin.

1969. Efficacy of quinaldine as an anesthetic for seven 

species of fish. U.S. Bureau of Sport Fisheries and 

Wildlife, Investigations in Fish Control, No. 22, 

10 p.

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

1970. Synergic mixtures of MS-222 and quinaldine as 

anesthetics for rainbow trout and northern pike. 

Progressive Fish-Culturist, vol. 32, no. 4, p. 202- 

205.

Sills, Joe B., and John L. Alien.

1971. The influence of pH on the efficacy and residues 

of quinaldine. Transactions of the American Fisheries Society, vol. 100, no. 3, p. 544-545.

Sills, Joe B., John L. Alien, Paul D. Harman, and 

Charles W. Luhning.

1973. Residue of quinaldine in ten species offish following anesthesia with quinaldine sulfate. U.S. Bureau of Sport Fisheries and Wildlife, Investigations 

in Fish Control, No. 50, 9 p.

Sober, Herbert A. (ed.)

1968. Physical and chemical data. Section J, p. J1-J256 

In Handbook of biochemistry. Selected data for molecular biology. The Chemical Rubber Company, 

Cleveland, Ohio.

Stecher, Paul G. (ed.)

1968. The Merck Index. Eighth edition. Merck and Co., 

Inc., Rahway, New Jersey, 1713 p.

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

1967. Residues of MS-222 in four salmonids following 

anesthesia. U.S. Bureau of Sport Fisheries and 

Wildlife, Investigations in Fish Control, No. 15, 

Up.
INVESTIGATIONS IN FISH CONTROL

54. The Efficacy of Quinaldine Sulfate: MS-222 Mixtures 

for the Anesthetization of Freshwater Fish

By Philip A. Gilderhus, Bernard L. Berger, 

Joe B. Sills, and Paul D. Harman

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

Results. ........................................ 5

Behavior of the fish ............................. 5

Efficacy of the anesthetics......................... 6

Water quality. ................................. 8

Water temperature.............................. 8

Repeated exposure .............................. 8

Repeated use of solutions. ......................... 8

Discussion ...................................... 8

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

References ...................................... 9
THE EFFICACY OF QUINALDINE SULFATE: MS-222 MIXTURES FOR THE 

ANESTHETIZATION OF FRESHWATER FISH

By Philip A. Gilderhus, Bernard L. Berger, Joe B. Sills, 

and Paul D. Harman

ABSTRACT. Combinations of quinaldine sulfate (QdSO^) and MS-222 

were tested for their efficacy in anesthetizing 14 species of freshwater fish. 

The combinations induced rapid and deep anesthesia as does MS-222 and 

permitted long safe holding times as does QdSCX;. The concentrations of 

the combined anesthetics needed were considerably lower than those 

needed when MS-222 is used alone. Most salmonids tested required concentrations of 10: 20 to 10:40 mg/1 (QdSO4 :MS-222) for effective anesthetization. Warmwater species generally required higher concentrations 

of 10:40 to 20:75 mg/1. Large adult fish usually required higher concentrations than smaller fish.

Both compounds lower the pH of the solution, and at pH's approaching 

6.0 or below the combinations were much less effective. In soft waters 

where the pH was lowered to that point, buffering the pH back to 6.5 or 

higher restored the activity of the anesthetics.

INTRODUCTION

The individual attributes and use patterns 

of quinaldine and MS-222 as anesthetics for 

fish have been well documented (Schoettger 

and Julin, 1967, 1969). Schoettger and Steucke 

(1970) tested mixtures of quinaldine and MS222 against rainbow trout1 and northern pike 

and found the combinations to possess most 

of the attributes of both anesthetics. Furthermore, substantially less of each component 

was necessary when they were used in combination. The combination in concentrations 

from 5 : 20 mg/1 (quinaldine: MS-222) for 

rainbow trout to 20;60 mg/1 for northern pike, 

rapidly anesthetized the fish and permitted 

them to be held safely in the chemical solution 

for at least 60 minutes.

Most recently Alien and Sills (1973) synthesized quinaldine sulfate (QdSO^), a salt of 

quinaldine which is more convient to use than

The common and scientific names of the fish used 

in the present study are given in table 1.

quinaldine, because it is water soluble. The 

efficacy of QdSO4 was found to be essentially 

the same, on an active ingredient basis, as 

that of quinaldine (Gilderhus et al. 1973).

Since QdSCX; is a crystalline material, it 

appeared to be ideal for use in combination 

with MS-222. The two compounds could be 

blended together and stored or marketed as a 

ready-to-use mixture. Using the data of 

Schoettger and Steucke (1970) as a starting 

point, our objectives were to determine the 

effective concentrations and ratios of concentrations of the combined anesthetics for 14 

species of fish, and evaluate the influences of 

water quality and temperature on the efficacy 

of the anesthetics.

METHODS AND MATERIALS

The quinaldine sulfate (QdSO^) used in these 

tests was synthesized at the Southeastern Fish 

Control Laboratory, Warm Springs, Ga. The 

MS-222 (methane sulfonate of meta-aminobenzoic acid ethyl ester) was Finquel(R) , marketed by Ayerst Laboratories, Inc.
Investigations in Fish Control 54: Bureau of Sport Fisheries and Wildlife

The anesthetics for each combination were 

weighed individually and either introduced directly into the test vessel or mixed into water 

solution in a flask and then introduced into the 

test vessel. The same procedures were used 

for both laboratory and field tests.

Tests were conducted at the Fish Control 

Laboratories, La Crosse, Wis., and Warm 

Springs, Ga., depending on the availability of 

test fish. Fingerling-size fish were exposed 

to the anesthetics in 15-1 glass jars, and 45- 

and 100-1 polyethylene tanks were used for 

tests with larger fish. The temperature was 

maintained by placing the test vessel in a circulating water bath equipped with heating or 

cooling equipment.

The efficacy of the combined anesthetics was 

evaluated against five species of salmonids 

and nine species of warmwater fish (table 1). 

The fish for laboratory tests were obtained 

from federal or state fish hatcheries except

Table 1.--Species of fish used in tests of the 

efficacy of QdSO,; :MS-222 mixtures as anesthetics for fish

Common name Scientific name

Coho salmon .......

Rainbow trout .....

Brown trout .......

Brook trout .......

Lake trout ........

Northern pike ....

Muskellunge 

Carp .......

White amur ........

White sucker ......

Black bullhead ....

Channel catfish . . 

Bluegill ..........

Largemouth bass ... 

Walleye ...........

Oncorhynchus kisutch 

Salmo gairdneri 

Salmo trutta 

Salvelinus fontinalis

Salvelinus namaycush 

Esox lucius

Esox Masquinongy 

Cyprinus carpio

Ctenopharyngodon idellus

Catostomus commersoni

Ictalurus melas

Ictalurus punctatus

Lepomis macrochirus

Micropterus salmoides 

Stizostedion vitreum

for small coho salmon and rainbow trout which 

were hatched and reared at the La Crosse 

laboratory. All fish used in laboratory tests 

were maintained as described by Hunn et al. 

(1968). They were acclimated to the test conditions for 16 to 24 hours before the anesthetics were added. Tests were also conducted 

against the five species of salmonids, northern 

pike, and walleyes at fish hatcheries during 

their spawning and marking operations.

The laboratory tests were conducted in well, 

city, and reconstituted waters at La Crosse 

and in limed spring water at Warm Springs 

(table 2). The efficacy of tbe anesthetics at 

selected pH's was assessed in reconstituted 

waters in which the pH was adjusted with a 

KHgPO^-NaOH buffer system (Marking, 1969). 

In some tests where the anesthetic chemicals 

lowered the pH of the water below the point 

where they were effective, the pH was raised 

by adding NaHCOs. For example, to raise the 

pH of 45 1 of water to 7.0, 1.9 and 11.0 g of 

NaHCOs were added to waters of pH 5.3 and 

3.8, respectively.

Laboratory tests were conducted at 7 , 12°, 

and 27° C at La Crosse and at 19° C at Warm 

Springs. Field tests were conducted at the

Tatle 2.--Characteristics of waters used for 

laboratory tests of QdSO«v :MS-222 mixtures 

as anesthetics for fish

Water 

type pH

Total

Alkalinity

(mgA)

Hardness 

(mg/1)

well .........

city .........

spring 1 ......

Reconstituted

very soft 

soft......

very hard.

7.5-8.0 232-262 238-371

7.4-8.2 209-250 289-340

6.8-7.0 ( 2 ) 20

6.4-6.8 10-13 10-13

7.2-7.6 30-35 40-48

8.0-8.4 225-245 280-320

1 CaO added to water to prevent osmotic shock in 

the fish.

2 Not analyzed.
Gilderhus et al. : Efficacy of Quinaldine Sulfate:MS-222 Mixtures for Anesthetization 5

existing water temperatures of the hatchery 

water supplies (table 3).

Schoettger and Julin (1967) defined loss of 

equilibrium, stage 2, as the degree of anesthesia 

at which locomotion ceases, and opercular rate 

slows, but there is still some reflex response 

to pressure on the caudal peduncle. We found 

that fish anesthetized by mixtures of QdSCU: 

MS-222 were easily handled when in loss of 

equilibrium, stage 2. Therefore, the tests were 

designed to determine the concentrations and

ratios of QdSO4:MS-222 which would anesthetize the fish to loss of equilibrium, stage 2, 

in approximately 4 min or less.

RESULTS

Behavior of the Fish

Fish exposed to the combination of anesthetics 

generally go through a period of 20 to 30 seconds of 

normal swimming before becoming sedated. The 

progression of anesthesia is rapid from sedation 

to loss of equilibrium, stage 2, at which point it

Table 3. Characteristics of hatchery water supplies used in tests of 

QdS04:MS-222 mixtures as anesthetics for fish

Location

Platte River SFH1

Mlchigan

Manchester NFH2

Iowa

Manchester NFH

Iowa

Osceola SFH

Wisconsin

Crystal Springs SFH 

Minnesota

Jordan River NFH 

Michigan

Lansing SFH 

Iowa

Valley City NFH 

North Dakota

Lansing SFH 

Iowa

Water

feff temp, tested /oJ\

Coho salmon 6

Rainbow trout 9

Brown trout 8

Brook trout 9

Lake trout 8 

(adult)

Lake trout 7 

(fingerling)

Northern pike 5

Muskellunge 18

Walleye 10

Total

pH Alkalinity 

(mg/1)

7.8 150

7.5 172

(3)

8.1 171

7.5 257

7.6 120

9.2 141

7.9 179

9.2 133

Hardness

168

215

.._

208

280

120

144

213

164

1 State Fish Hatchery.

2 National Fish Hatchery.

3 Same water supply as used for rainbow trout. Analysis not done.
Investigations in Fish Control 54: Bureau of Sport Fisheries and Wildlife

either slows or stops. As with quinaldine sulfate alone (Gilderhus et al. 1973), the combination of chemicals rarely induces total loss 

of reflex. However, the fish are easily handled 

while in loss of equilibrium, stage 2, and the reflexes which are retained are usually weak and 

of little consequence to the handler.

Efficacy of the Anesthetics

Combinations of QdSO4 :MS-222 proved to be 

effective anesthetics for all species of fish on 

which they were tested. Concentrations of 

10:20 mg/1 (QdSO4 :MS-222) were the lowest 

which were effective for four species of trout 

in laboratory tests. Coho salmon were slightly 

more sensitive, requiring a combination of 

5;20 mg/1. The salmonids recovered rapidly 

in fresh water, and they recovered faster in 

warmer water, requiring up to 4.7 min at 

17° C and 20 min at 7° C (table 4).

The larger salmonids exposed to the anesthetics at field stations were somewhat more 

resistant than the smaller fish exposed in the 

laboratory, requiring concentrations of 10:20 

to 10:40 mg/1 (table 5). Brook and lake trout

required the highest concentrations under field 

conditions to subdue them to a handleable condition for artificial spawning. This agrees with 

Schoettger and Julin (1967) who found brook and 

lake trout to be more resistant than other 

trouts to MS-222 alone.

The combination of drugs was less active on 

most of the species of warmwater fish than on 

salmonids, and all but small walleyes required 

higher concentrations. Black bullheads were the 

most resistant in laboratory tests requiring 

concentrations of 20:75 mg/1 (table 6). Northern 

pike, carp, and white suckers were the next 

most resistant requiring 20:50 mg/1 to 20:75 

mg/1. Small walleyes, the most sensitive fish, 

were anesthetized by a combination of 5:15 

mg/l(QdSO4:MS-222).

Higher concentrations of the combined anesthetics also were required for larger specimens of the warmwater species. Large northern 

pike required 20:75 mg/1 and small northern 

pike 20:50 mg/1; large channel catfish required 

40:60 mg/1 and small channel catfish 20:50 mg/1; 

large walleyes required 10:30 mg/1 and small 

walleyes 5:15 mg/1.

Table 4. Efficacy of QdS04:MS-222 combinations as anesthetics for salmonids in laboratory tests

Species

Mean 

weight 

(g)

No. 

of 

fish

Temp. (°c) Water 

type

Concentration 

(mg/1)

Loss of 

equilibrium 

stage 2 

(min)

Length of 

exposure 

(min)

Recovery in 

fresh water 

(min)

Coho salmon. . . 

Do. .........

Do..........

Rainbow trout. 

Do..........

Do..........

Do. .........

Do..........

Do..........

Do..........

Brown trout. . . 

Do..........

Do. .........

Brook trout. . . 

Do..........

Do. .........

Lake trout. . . . 

Do..........

Do..........

19 

16

19

25 

1.4

1.5

18

0.9

0.9

25

18 

28

16

27 

1.1

27

25

30

25

15 

15

10

15 

15

10

10

15

15

15

10 

15

15

15 

15

15

10 

15

15

7 

12

12

7 

12

12

12

17

17

17

7 

12

17

7 

12

12

7 

12

17

well 

soft

 oral 1

well 

soft

txr&~\ "1

soft

1 pH-7.6 

soft

1 pH-8.5

well

woT 1

w^T "1

well 

soft

well

well

5:20 

10:20

5:20

10:20 

10 '40

10:20

10:20

10:20

10:20

10:20

10:20 

10:20

10:20

10:20 

10:20

10:20

10:20 

10:20

10:20

1.5-2.6 

1.4-3.0

1.3-1.8

0.9-1.5 

1 3-4 5

0.5-0.6

0.9-1.5

1.4-3.3

1.8-2.9

0.8-0.9

1.1-1.5 

0.9-1.5

1 0-1 3

0.9-1.4 

1.0-3.5

0.9-1.4

1.6-2.6 

0.9-1.7

1.0-2.5

15-60 

15-60

15-30

15-60 

15-60

5.5-15

15-30

15-60

15-60

15-60

15-30 

15-60

15-60

15-60 

5.5-15

15-60

15-30 

15-60

15-60

3.2-5.5 

3.1-5.3

2.0-3.0

3.0-7.8 

2.7-10.0

2.0-3.2

2.0-3.8

1.5-3.0

2.0-4.5

1.0-4.0

3.0-6.3 

3.0-7.0

2.0-3.8

3.5-9.4 

1.5-2.6

3.0-5.0

7.0-20.0 

3.0-11.0

pH adjusted with buffers.
Gilderhus et al. : Efficacy of Quinaldine Sulfate:MS-222 Mixtures for Anesthetization 7

Table 5. Efficacy of QdS(X;:MS-222 combinations as anesthetics for fish at field stations

Species 

and 

location

Platte River SFH1

Manchester NFH2 

Brown trout ......................

Manchester NFH

Osceola SFH

Crystal Springs SFH

Jordan River NFH

Lansing SFH

Valley City NFH 

Walleye ..........................

Lansing SFH

Mean No. Effect 

weight of concentr 

(kg) fish (mg/1

Time (min) to

ation Loss of RecOvery in 

) equilibrium ,, , , 

 , « iresh water

3.5 15 5:10 0. 

3.2 74 10:30 1. 

2.6 62 5:30 2. 

1.0 57 10:40 1. 

2.5 55 10:40 3. 

0.01 2,700 10:20 1. 

1.1 12 20:50 2. 

2.3 8 20:50 1. 

0.9 10 10:30 2.

8-2.3 3.0-4.0 

2-3.0 3.2-5.0 

0-3.2 1.0-4.5 

2-1.5 2.2-5.5 

0-4.0 4.0-5.0 

2-2.0 4.0-5.5 

5-8.0 8.8-22.0 

2-1.5 3.0-6.6 

5-3.5 6.0-10.0

1 State Fish Hatchery. 

2 National Fish Hatchery.

Table 6. Efficacy of QdS04:MS-222 combinations as anesthetics for warmwater fish in laboratory tests

Mean No 

Species weight of 

(g) fist

Northern pike......... 10 15

Do.................. 1791 18

Do. ................. 115 10

Do. ................. 10 15

Do. ................. 115 15

Carp.................. 387 20

Do.................. 387 10

White amor. ........... 227 3

White sucker.......... 138 10

Black bullhead ........ 208 10

Do.................. 208 10

Do.................. 129 10

Channel catfish. ...... 1.8 10

Do.................. 1.5 10

Do.................. 1.8 10

Do.................. 1316 30

Bluegill. ............. 77 14

Do.................. 135 49

Do.................. 80 15

Do.................. 15 10

Do. ................. 1044 30

Walleye............... 1.1 15

i as- ( c > »r tyPe £S£ (mg/1)

12 well 10:40 

12 well 20:75 

17 very soft 20.50 

17 well 10:40 

22 very soft 20:50

12 well 20:50 

27 well 20:50

19 spring 20 :40 

12 well 20:50

12 well 20:75 

17 well 20:75 

27 well 20:50

12 well 30:30 

17 well 20:50 

17 very hard 20:50 

19 spring 40 : 60

17 well 10:40 

19 spring 10 :40 

27 well 10:40

17 well 20:50 

17 very hard 20:50 

19 spring 20:40

12 well 5:15

Loss of 

equilibrium 

Stage 2 

(min)

2.4-3.1

3.0-5.0 

1.7-2.5 

1.8-2.0 

0.9-2.0

2.1-3.3 

1.6-2.3

1.5-1.8 

1.7-2.0

3.3-4.1 

3.5-4.1 

2.5-3.1

1.5-2.3 

0.6-1.5 

1.5-2.5 

2.0-3.0

1.4-2.1 

2.5-3.5 

1.1-1.2

1.0-1.1 

0.8-1.5 

1.5-3.1

1.5-3.3

Length of Recovery in 

exposure fresh water 

(min) (min)

15-60 5.5-11.0 

30 19.0-26.0 

5.5-15 3.5-5.9 

15-60 4.5-5.0 

5.5-30 3.2-8.5

5.5-15 4.5-9.0 

5.5-15 2.5-6.5

30 4.0-5.0 

5.5 5.1-7.0

5.5-15 10.0-22.0 

15 4.5-16.0 

5.5-15 4.2-6.5

5.5-15 2.2-14.0 

15-30 1 1.8-3.5 

5.5-15 2.0-2.8 

30 3.5-7.5

15-60 5.0-8.2 

30 1.5-3.0 

5.5-15 1.7-3.0

15 5.0-6.5 

5.5-15 2.7-10.0 

30 2.5-9.5

15-60 5.0-26.0

Some fish killed by 30-minute exposure.
8 Investigations in Fish Control 54: Bureau of Sport Fisheries and Wildlife

The time required for the fish to recover in 

fresh water was inversely related to the water 

temperature with longer times being necessary 

in colder water. Most fish recovered in less 

than 10 min, but some needed up to 26 min 

(table 6).

Water Quality

The efficacy of the combined anesthetics 

was affected by the chemical characteristics 

of the water. Both of the compounds are acidic, 

lowering the pH of the water to which they are 

added (Alien and Barman, 1970; Marking and 

Dawson, 1973). We found that in anesthetic 

solutions of about pH 6 or below, the anesthetics were diminished in efficacy, depending on 

the concentration. The lowering of the pH was 

critical only when the anesthetics were placed 

in soft or very soft, unbuffered water. With 

rainbow trout, 10:20 mg/1 were effective in 

well water and not effective in very soft water 

at 12° C. The combination anesthetized northern 

pike at 10: 30 mg/1 in well water; whereas 

20:75 mg/1 were ineffective in soft water at 

12°C. Increasing the concentration sometimes 

compensated for the loss of activity in soft 

waters, but adding NaHCO^ until the pH of the 

solution was 6.5 or higher, assured satisfactory activity.

Water Temperature

The water temperature did not decisively or 

consistently affect the efficacy of the combined 

anesthetics. The concentrations needed for effective anesthetization of salmonids were the 

same over a wide range of water temperatures. 

There was some indication that northern pike 

and black bullheads might be anesthetized by 

lower concentrations at higher temperatures 

but the results were not conclusive. Inconsistent results related to temperature were 

not surprising. The efficacy of MS-222 apparently is affected by temperature (Schoettger 

and Julin, 1967), whereas the efficacy of 

quinaldine is not (Schoettger and Julin, 1969). 

The recovery time for fish was more consistently related to temperature with recovery 

being more rapid at higher temperatures.

Repeated Exposure

Repeated anesthetization of the same fish 

does not appear to affect the sensitivity of the

fish to the combined anesthetics. A group of 

ten 20-cm rainbow trout was anesthetized 11 

times in 15 days by a 20:50 mg/1 solution and 

anesthetization and recovery times were unaffected.

Repeated Use of Solutions

The repeated use of solutions of QdSO4:MS222 was evaluated during the fin clipping of 

lake trout at Jordan River NFH. We found that 

1,800 fish (a total of 25 kg) could be anesthetized in 8 1 of a 10: 20-mg/l solution before the 

solution had to be spiked or replaced.

Apparently, raising the concentration slightly 

from that normally used will help ensure continued effectiveness for a period of several 

days. Fifty 1 of a 20:100-mg/1 solution were 

used for 3 days to anesthetize 2.2-3.6 kg northern pike. A total of 135 pike was anesthetized 

the first day, 125 the second, and 120 the third 

without noticeable loss of activity of the solution.

DISCUSSION

The combinations of QdSO4 a"d MS-222 combined the attributes of the individual anesthetics and induced anesthesia more effectively 

than QdSG>4 al°ne and more safely than MS-222 

alone. Fish can be safely held for 1 hour or 

more in concentrations which effectively 

anesthetize the respective species. This is in 

contrast to the 5.5- to 12-minute safe holding 

times for salmonids in MS-222 given by 

Schoettger and Julin (1967). An exception was 

channel catfish which suffered mortalities 

after 30 min of exposure to 20 : 50 mg/1, the 

lowest effective concentration. The long safe 

holding time afforded by the combination is a 

distinct advantage because more fish can be 

anesthetized at one time without danger to the 

last ones handled. Whereas the combinations 

do not consistently induce total loss of reflex 

as does MS-222, they do make fish more handleable than does QdSO,; alone.

The concentrations of the drugs used in 

combination represent a substantial saving of 

chemicals over the concentrations necessary 

when they are used alone. For example, when 

used alone for salmonids, the concentrations
Gilderhus et al. : Efficacy of Quinaldine Sulfate:MS-222 Mixtures for Anesthetization 9

needed are 80 to 100 mg/1 of MS-222 or 25 

mg/1 of QdSO4. When used in combination, the 

concentrations necessary to anesthetize 

salmonids are 10: 20 mg/1 (QdSO 4:MS-222).

The water chemistry appears to be the only 

factor which consistently influences the efficacy of the combined anesthetics. Apparently, 

the two chemicals are affected differently, but 

both have reduced activity in very soft water. 

Both compounds lower the pH of the water, 

contributing to the ionization and inactivation 

of QdSO4 at pH's below 6. MS-222 is less effective in soft waters, apparently because the 

lack of calcium ions induces osmotic stress 

in the fish which interferes with the activity 

of the anesthetic (Schoettger and Julin, 1967).

CONCLUSIONS

1. Combinations of QdSO^ and MS-222 effectively anesthetize a wide variety of fishes.

2. The combinations possess the attributes of 

both anesthetics that is, the long safe 

holding time with QdSCU a"d the rapid anesthetization with MS-222.

3. Combining the anesthetics greatly reduces 

the concentrations over those necessary 

when they are used alone.

4. Higher concentrations of the combination 

are generally needed for large adult fish 

than for small, immature fish.

5. The combination is relatively ineffective if 

it lowers the pH of the water to 6 or below. 

This is more prone to occur in soft or unbuffered water.

6. If the combined anesthetics lower the pH of 

the solution to near 6 or below, the pH 

should be raised to 6.5 or higher with 

NaHCCb or another satisfactory buffer.

REFERENCES

Alien, John L., and Paul D. Harman.

1970. Control of pH in MS-222 anesthetic solutions. 

The Progressive Fish-Culturist, vol. 32, no. 2, 

p. 100.

Alien, John L., and Joe B. Sills.

1973. Preparation and properties of quinaldine sulfate, 

an improved fish anesthetic. U.S. Bureau of Sport 

Fisheries and Wildlife, Investigations in Fish Control, No. 47, 7 p.

Gilderhus, Philip A., Bernard L. Berger, Joe B. Sills, 

and Paul D. Harman.

1973. The efficacy of quinaldine sulfate as an anes= 

thetic for freshwater fish. U.S. Bureau of Sport 

Fisheries and Wildlife, Investigations in Fish Control, No. 49, 9 p.

Hunn, Joseph B., Richard A. Schoettger, andEverettW, 

Whealdon.

1968. Observations on handling and maintenance of 

bioassay fish. The Progressive Fish-Culturist, 

vol. 30, no0 3, p. 164-167.

Marking, Leif L.

1969. Toxicological assays with fish. Bulletin of the 

Wildlife Disease Association, vol. 5, p. 291»2940

Marking, Leif Lc, and Verdel K. Dawson.

1973. Toxicity of quinaldine sulfate to fish. U.S. Bureau of Sport Fisheries and Wildlife, Investigations 

in Fish Control, No. 48, 8 p.

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

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

salmonids. U.S0 Bureau of Sport Fisheries and Wildlife, Investigations in Fish Control, No. 13, 15 p.

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

1969. Efficacy of quinaldine as an anesthetic for seven 

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

Wildlife, Investigations in Fish Control, No. 22, 8 p.

Schoettger, Richard A., and Erwin W. Steucke, Jr0 

19700 Synergic mixtures of MS-222 and quinaldine as 

anesthetics for rainbow trout and northern pike. The 

Progressive Fish-Culturist, vol. 32, no. 4, p. 202- 

2050
INVESTIGATIONS IN FISH CONTROL

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

Anesthesia with Mixtures of Quinaldine 

Sulfate:MS-222

By Joe B. Sills, John L. Alien, Paul D. Harman, 

and Charles W. Luhning

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 .............................. 4

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

Discussion ...................................... 10

Conclusions. ..................................... \2

References ...................................... 12
RESIDUES OF QUINALDINE AND MS-222 IN FISH FOLLOWING 

ANESTHESIA WITH MIXTURES OF QUINALDINE SULFATE: MS-222

By Joe B. Sills, John L. Alien, Paul D. Harman, 

and Charles W. Luhning

ABSTRACT.---Residues of quinaldine and MS-222 in 10 species of fish 

exposed to mixtures of quinaldine sulfate and MS-222 were determined 

using gas chromatography and spectrophotometry for quinaldine and colorimetry for MS-222. The residue concentrations of quinaldine and MS-222 

decreased rapidly following withdrawal from the anesthetics. The mean 

concentration of 0-hour residues of quinaldine ranged from 0.15 to 6.92 

Mg/g depending on concentration, temperature, length of exposure, and 

species. The mean concentration of 0-hour residues of MS-222 ranged 

from 1.9 to 27.3 Mg/g and decreased to near the background reading of the 

controls after 24 hours of withdrawal. The fish exposed to the same concentration of the mixture of anesthetics for 15 minutes generally contained higher concentrations of residues than those exposed for 5.5 

minutes. Two weight groups of brook trout were exposed to the same concentrations of the mixed anesthetics for the same length of time. The 

smaller fish contained 1.22 times higher concentration of quinaldine residue and 1.43 times higher concentration of MS-222 residue than the larger 

fish.

INTRODUCTION

MS-222 (methanesulfonate of meta-aminobenzoic acid ethyl ester) and quinaldine (2-methylquinoline) are effective and widely-used fish 

anesthetics (Schoettger and Julin, 1967, 1969). 

The choice of which anesthetic to use depends 

upon the specific properties that are desired. 

MS-222 causes rapid immobility, but fish are 

unable to tolerate long exposures. On the other 

hand, quinaldine is tolerated for long periods, 

but does not completely block reflex movement. 

Schoettger and Steucke (1970) found that mixtures of these drugs offer advantages over the 

use of each separately. The mixture produced 

rapid immobility and prolonged toleration at 

slightly reduced concentrations of each drug.

Alien and Sills (1973) prepared a watersoluble form of quinaldine by forming its 

sulfate salt. The toxicity of quinaldine sulfate 

(QdSO4) to fish was determined by Marking 

and Dawson (1973), and its efficacy as a fish

anesthetic was determined by Gilderhus et al. 

(1973a). The toxicity of the anesthetic mixture 

(quinaldine sulfate;MS-222) was determined by 

Dawson and Marking (1973). Gilderhus et al. 

(1973b) determined the efficacy of the combination anesthetic in the laboratory and under field 

conditions.

Residue data on four salmonids1 and channel 

catfish anesthetized with MS-222 have been reported (Walker and Schoettger, 1967; Schoettger 

et al., 1967). Alien et al. (1972) determined 

MS-222 residues in northern pike, muskellunge, 

and walleye anesthetized with MS-222. Sills 

et al. (1973) determined residues of quinaldine 

in five species each of coldwater and warmwater fish following anesthesia with quinaldine 

sulfate. Sills and Harman (1970) determined 

quinaldine residues in striped bass (Morone 

saxatilis) following anesthesia with quinaldine 

sulfate.

common and scientific names of fish used in the 

present study are given in table 1.
4 Investigations in Fish Control 55: Bureau of Sport Fisheries and Wildlife

Before the mixture of quinaldine sulfate and 

MS-222 can be registered for general use, 

more information is needed about the fate of 

quinaldine and MS-222 in fish tissues. Therefore, this study was undertaken to measure 

the concentration and persistence of quinaldine 

and MS-222 residues in five species each of 

coldwater and warmwater fish following anesthesia with efficacious concentrations of the 

mixture.

METHODS AND MATERIALS

Ten species of fish (table 1) were exposed 

quinaldine sulfate and MS-222 (Gilderhus 

et al., 1973b). Temperatures of treatment 

ranged from 7° to 19° C t and exposure times 

ranged from 5.5 to 30 minutes. A wide range 

of concentrations was necessary, because of 

the variety of species and temperatures involved.

Withdrawal times began when exposed fish 

were placed in fresh, flowing water for recovery. At least three fish were collected for 

residue analysis at 0, 1, 2, 4, either 6 or 8, 

and 24 hours. Samples of muscle tissue were 

collected and held frozen until analyzed. Whole 

fillets were homogenized after thawing to obtain representative samples of edible tissue.

Table 1. Species of fish analyzed for quinaldine 

and MS-222 residues following anesthesia with 

mixtures of quinaldine sulfate and MS-222

Common Name Scientific Name

Coho salmon .......

Brown trout .......

Rainbow trout .....

Lake trout ........

Brook trout .......

Northern pike .....

Channel catfish ... 

Largemouth bass ... 

Bluegill ..........

Walleye ...........

Oncorhynchus kisutch 

Salmo trutta 

Salmo gairdneri 

Salvelinus namaycush 

Salvelinus fontinalis 

Esox lucius 

Ictalurus punctatus 

Micropterus salmoides 

Lepomis macrochirus 

Stizostidion vitreum

The samples were analyzed by the colorimetric method of Walker and Schoettger (1967) 

for MS-222 residue and by the gas chromatographic and U.V. spectrophotometric methods of Alien and Sills (1970a and 1970b) 

for quinaldine residue. The minimum detectable concentration of the quinaldine methods 

is 0.01 Mg/g and the minimum detectable concentration of the MS-222 method is 0.1 Mg/g. 

Residues of quinaldine less than 0.01 Mg/g 

are reported as zero. The minimum detectable 

concentration of the MS-222 method is limited 

also by the background aromatic amines, and 

all MS-222 results include these.

RESULTS

Coho salmon

Spawning-migrant coho salmon from Lake 

Michigan were exposed to a mixture of 5 mg 

of quinaldine sulfate and 10 mg of MS-222 per 

liter of water at 12° C for 5.5 and 15 minutes 

(table 2). MS-222 residues ranged from mean 

concentrations of 1.9 to 3.3Mg/g at the 0-hour 

interval and decreased to a background level 

of the controls or slightly above at the 24-hour 

withdrawal interval. Quinaldine residues ranged 

from mean concentrations of 0.15 to 0.51 Mg/g 

at the 0-hour interval to zero after 4 to 8 hours 

of withdrawal. The coho salmon were the 

largest fish tested.

Brown trout

Brown trout (table 2) were exposed to a mixture of 5 mg of quinaldine sulfate and 30 mg 

of MS-222 per liter of water at 12° C for 5.5 

and 15 minutes. MS-222 residues ranged from 

mean concentrations of 7.2 to 14.6Mg/g at the 

0-hour withdrawal and were within background 

levels after 8 to 24 hours. Quinaldine residues 

ranged from mean concentrations of 0.33 to 

0.63Mg/g at the 0-hour withdrawal, and were 

down to zero after 8 hours of withdrawal.

Rainbow trout

Hatchery-reared rainbow trout were tested 

the most extensively (table 3). Those exposed 

at 7° C to a mixture of 5 mg of quinaldine sulfate and 30 mg of MS-222 per liter of water 

for 15 minutes contained a mean concentration
Table 2. Residues of anesthetics in muscle tissue of coho salmon and brown trout t

quinaldine sulfate (QjdS04 ) and MS-222 at 12° C for 5.5 and 15 mi

Treatment

QdSO* 

(mg/1)

MS-222 

(mg/1)

Exposure 

(min)

Mean 

weight 

(kg)

Residu

Quinaldine

Mean Range

Coho salmon

Control ............

0-hour .............

1-Jiour .............

2-Jaour .............

4-hour .............

8-Jaour .............

24-hour .............

0-hour .............

24-hour .............

Brown trout

Control ............

0-hour .............

2-hour .............

0 Jaour .............

2-Jaour ............

4-hour .............

8-hour .............

24-hour .............

..... 0

..... 5

..... 5

..... 5

..... 5

..... 5

5

..... 5

..... 5

..... 0

..... 5

..... 5

5

..... 5

5

..... 5

..... 5

..... 5

0

10

10

10

10

10

10

10

10

0

30

30

30

30

30

30

30

30

0

5.5

5.5

5.5

5.5

5.5

5.5

15

15

0

5.5

5.5

5.5

15

15

15

15

15

2.03

3.82

1.53

3.20

3.91

2.71

1.57

3.91

4.37

0.58

0.44

0.56

0.50

0.41

0.48

0.56

0.53

0.51

0.00

0.15

0.10

0.02

0.00

0.00

0.00

0.51

0.00

0.00

0.33

0.01

0.00

0.63

0.03

0.01

0.00

0.00

0.00-0.00

0.09-0.1

0.07-0.14

0.01-0.0

0.00-0.00

0.00-0.00

0.00-0.00

0.34-0.65

0.00-0.00

0.00-0.00

0.23-0.40

0.01-0.0

0.00-0.00

0.55-0.75

0.02-0.04

0.01-0.0

0.00-0.00

0.00-0.0

1 Each mean value represents the average of three analyses.
Table 3. Residues of anesthetics in muscle tissue of rainbow trout anestheti

quinaldine sulfate (QdSO^) and MS-222 at 7°, 12°, and 17° C for 5.5 an

Withdrawal 

interval

Treatment

(mg/1)

MS- 222 

(mg/1)

Exposure 

(min)

Temp.

Mean 

weight

(g)

Re

Quinaldine

Mean Rang

0-hour.

1-hour.

2 -hour.

4-hour.

Control ........

0-hour.

1-hour.

2 hour

4-hour.

8-hour.

24-hour.

0-hour.

8-hour.

24-hour.

Control ........

0

_Vi r\\ T -r»

1-hour.

2-hour.

4-hour.

1-hour.

2 -hour.

4-hour.

8-hour ......

24-hour. . . . . .

5

5

5

5

0

10

10

10

10

10

10

10

10

10

0

5

5

5

5

5

5

5

5

5

5

30

30

30

30

0

40

40

40

40

40

40

40

40

40

0

30

30

30

30

30

30

30

30

30

30

15

15

15

15

0

5.5

5.5

5.5

5.5

5.5

5.5

15

15

15

0

5.5

5.5

5.5

5.5

15

15

15

15

15

15

7

7

7

7

12

12

12

12

12

12

12

12

12

12

17

17

17

17

17

17

17

17

17

17

17

496

532

483

445

135

116

148

168

172

155

100

T ^)£.

152

i / n

135

J on

j ~\ o

467

390

T O£l

-i i rt

145

1 -I!}

152

155

0.29

0.24

0.13

0.02

0.00

0.72

0.22

0.03

0.02

0.02

0.00

1 44

0.01

0.00

0.00

0.53

0 23

0.12

0.03

1.05

O oy

0.06

0.01

0.01

0.00

0.25-

0.20-

0.07-

0.01-

0.00-

0.64-

0.14-

0.02-

0.01-

0.01-

0.00-

O

rt/~\ 

0.01-

0.00-

0.00-

O y rt 

0.20-

0.08-

0.02-

0.72-

0

~\ £. 

0.04-

0.01-

0.01-

0.00-

Each mean value represents the average of three analyses.
Sills, Alien, Harman, and Luhning: Residues of Quinaldine and MS-222 in Fish 7

of quinaldine residue of 0.29 Mg/g and a mean 

concentration of MS-222 residue of 5.2/ug/g at 

0-hour withdrawal. After 4 hours of withdrawal 

the MS-222 residue approached the background 

of the controls and the quinaldine residue had 

decreased to a mean concentration of 0.02 Mg/g.

Rainbow trout exposed at 12° C to a mixture 

of 10 mg of quinaldine sulfate and 40 mg of 

MS-222 per liter of water for 5.5 minutes contained a mean concentration of quinaldine residue of 0.72Mg/g and a mean concentration of 

MS-222 residueof 11.9Mg/g at the 0-hour withdrawal. After 8 hours of withdrawal the MS-222 

residue was equal to the background of the 

controls (0.5 Mg/g); however, the 24-hour withdrawal samples showed a residue of 1.0Mg/g of 

MS-222. The 24-hour withdrawal samples 

showed no quinaldine residue. Those exposed 

at 12° C to a mixture of 10 mg of quinaldine 

sulfate and 40 mg of MS-222 per liter of water 

for 15 minutes contained a mean concentration 

of quinaldine residue of 1.44 Mg/g and a mean 

concentration of MS-222 residue of 17.0 Mg/g. 

After 24 hours of withdrawal the MS-222 residue (including background aromatic amines) 

was still slightly higher (1.0 Mg/g) thanthebackground of the controls (0.5 Mg/g). The 24-hour 

withdrawals contained no detectable quinaldine 

residue.

Rainbow trout exposed at 17° C to a mixture 

of 5 mg of quinaldine sulfate and 30 mg of 

MS-222 per liter of water for 5.5 minutes contained a mean concentration of quinaldine 

residue of 0.53 Mg/g and a mean concentration 

of MS-222 residue of 6.9 Mg/g at the 0-hour 

withdrawal. After 4 hours of withdrawal the 

MS-222 residue had decreased to within the 

range of the background in the controls, and 

the mean concentration of quinaldine residue 

had decreased to 0.03 Mg/g. Rainbow trout exposed at 17 C to a mixture of 5 mg of quinaldine sulfate and 30 mg of MS-222 per liter of 

water for 15 minutes contained a mean concentration of quinaldine residue of 1.05Mg/g 

and a mean concentration of MS-222 residue of 

16.5 Mg/g. After 24 hours of withdrawal the 

MS-222 residue had decreased to the background of the controls and no quinaldine residue was detected.

Lake trout

Hatchery-reared lake trout were exposed to 

a mixture of 10 mg of quinaldine sulfate and 

40 mg of MS-222 per liter of water at 12° C for 

5.5 and 15 minutes (table 4). Those exposed to 

this combination for 5.5 minutes contained mean 

concentrations of residues of 0.74 Mg/g and 

14.1 Mg/g of quinaldine and MS-222, respectively. 

After 24 hours of withdrawal the MS-222 residue had decreased to less than the background 

of the controls and no quinaldine residue was 

detected. Those exposed to this combination 

for 15 minutes contained mean concentrations 

of residues of 1.26 Mg/g and 17.9 Mg/g of 

quinaldine and MS-222, respectively. The 24- 

hour withdrawal samples showed no residue 

above the background of the control.

Brook trout

Hatchery-reared brook trout were exposed 

at 9° C to a mixture of 10 mg of quinaldine 

sulfate and 40 mg of MS-222 for 5.5 minutes 

(table 4). Two weight groups were tested. 

Brook trout weighing approximately 0.3 kg 

contained mean concentrations of residues of 

1.17 Mg/g and 9.2 Mg/g °f quinaldine and MS222, respectively; those weighing approximately 0.8 kg contained 0.96Mg/g and 6.4 

Mg/g of quinaldine and MS-222, respectively. 

Both groups showed no detectable residue 

above the background of the controls of either 

anesthetic after the last withdrawal interval.

Northern pike

Spawning adult northern pike from the Mississippi River were exposed to a mixture of 20 

mg of quinaldine sulfate and 50 mg of MS-222 

per liter of water at 7° C and to a mixture of 

20 mg of quinaldine sulfate and 75 mg of MS222 per liter of water at 12° C for 30 minutes 

(table 5). The fish treated at 7° C contained 

mean concentrations of 1.60 Mg/g and 9.6 Mg/g 

residues of quinaldine and MS-222, respectively. After 24 hours of withdrawal no residue 

of quinaldine or MS-222 was detected in this 

group. Those treated at 12° C contained a mean 

concentration of quinaldine residue of 1.80 

Mg/g at the 0-hour withdrawal and no quinaldine residue was detected after 24 hours of
Table 4. Residues of anesthetics in muscle tissue of lake trout treated with mixtu

sulfate (QdSO*) and MS-222 at 12° C for 5.5 and 15 minutes and brook trout treate

quinaldine sulfate (QdSCU) and MS-222 at 9° C for 5.5 minutes

Withdrawal interval

Treatment

QdS04

(me/D

MS-222 

(mg/1)

Exposure 

(min)

Mean 

weight 

(kg)

Residue

Quinaldine

Mean Range

Lake trout

0-hour ..........

1-hour ..........

4-hour ..........

8-hour ..........

24- hour ..........

0-hour ..........

8-hour ..........

24- hour ..........

Brook trout

Control ..........

0-hour ..........

1-hour ..........

2-hour ..........

4-hour ..........

8-hour ..........

24-hour

Control

0-hour ..........

15-hour ..........

....... 0

....... 10

....... 10

....... 10

....... 10

....... 10

....... 10

........ 10

,.......- 10

........ o

........ 10

........ 10

........ 10

........ 10

........ 10

........ 10

0

........ 10

........ 10

0

40

40

40

40

40

40

40

40

0

40

40

40

40

40

40

0

40

40

0

5.5

5.5

5.5

5.5

5.5

15

15

15

0

5.5

5.5

5.5

5.5

5.5

5.5

0

5.5

5.5

1.63

1.40

1.47

1.27

1.78

1.35

1.82

1.25

1.57

0.30

0.30

0.31

0.33

0.30

0.31

0.30

0.80

0.88

0.68

0.00

0.74

0.24

0.02

0.01

0.00

1.26

0.01

0.00

0.00

1.17

0.20

0.03

0.02

0.01

0.00

0.00

0.96

0.00

0.00-0.00

0.55-1.02

0.19-0.27

0.02-0.03

0.00-0.02

0.00-0.00

1.05-1.60

0.00-0.01

0.00-0.00

0.00-0.00

0.71-1.80

0.17-0.24

0.02-0.04

0.02-0.02

0.01-0.01

0.00-0.00

0.00-0.01

0.46-1.31

0.00-0.00

Each mean value represents the average of three analyses.
Table 5. Residues of anesthetics in muscle tissue of northern pike treated with m

sulfate (QdSCU) and MS-222 for 30 minutes at 7° and 12° C and in muscle tissue 

treated with the combination anesthetic for 30 minutes at 19° C

Withdrawal interval

Treatment

QdS04 

(mg/1)

MS-222 

(ing/D

Temp. (°c)

Mean 

weight 

(kg)

Resi

Quinaldine

Mean Range

Northern pike 

Control...,

0-hour.

1-hour.

2-hour,

4-hour.

24-hour.

0

20

20

20

20

20

0

50

50

50

50

50

1.43 0.00 0.00-0.

1.21

1.02

1.13

1.20

1.21

1

0

0

0

0

.60

.53

.33

.06

.00

1

0

0

0

0

.40-1

.48-0

.22-0

.05-0

.00-0

.

.

.

.

.

Control,

0-hour.

1-hour.

2-hour,

4-hour,

6-hour,

24-hour.

Channel catfish

0

20

20

20

20

20

20

0

75

75

75

75

75

75

12

12

12

12

12

12

12

1.48 0.00 0.00-0.

1.67

2.08

1.53

1.55

1.94

1.65

1.80

0.34

0.07

0.04

0.02

0.00

1.50-1.

0.24-0.

0.06-0.

0.02-0.

0.01-0.

0.00-0.

1-hour. ...........

2-hour. ...........

4- hour. ...........

6-hour. ...........

24-hour. ...........

...... 40

...... 40

...... 40

. . . . . 40

...... 40

...... 40

W

60

60

60

60

60

60

JLS'

19

19

19

19

19

19

-L. <C 1

1.50

1.27

1.36

T P7

T 3P

1.14

6

00

.92 

3.19

1.79

0.56

0.30

0.00

\j . uu u. 

6 

or? 1-7 

. J /-/ . 

I

/TO o 

0.44-0.

0.10-0.

0.00-0.

Each mean value represents the average of three analyses.
10 Investigations in Fish Control 55: Bureau of Sport Fisheries and Wildlife

withdrawal. The 0-hour and 24-hour withdrawal 

samples of this group for MS-222 analysis were 

lost; however, the 1-hour withdrawal sample 

contained a mean MS-222 residue of 3.0 Mg/g 

which is very close to the concentration of 

MS-222 residue found in the 1-hour withdrawal 

samples treated at 7° C (2.8 Mg/g). The residue of MS-222 in the 6-hour withdrawal samples 

was within the background of the controls.

Channel catfish

Hatchery-reared channel catfish were exposed to a mixture of 40 mg of quinaldine 

sulfate and 60 mg of MS-222 per liter of water 

at 19°C for 30 minutes (table 5). They contained mean concentrations of residues of 

6.92 Mg/g and 13.7 Mg/g of quinaldine and MS222, respectively. After 24 hours of withdrawal 

the MS-222 residue was less than the background of the controls and no quinaldine residue was detected.

Largemouth bass

Hatchery-reared largemouth bass were exposed to a mixture of 20 mg of quinaldine sulfate and 40 mg of MS-222 per liter of water at 

19° C for 30 minutes (table 6). The mean concentrations of residues of quinaldine and MS222 at 0-hour were 4.07Mg/g and 15.1 Mg/g, 

respectively. After 24 hours of withdrawal no 

residues of quinaldine or MS-222 were detected.

Bluegill

Hatchery-reared bluegills were exposed to 

a mixture of 10 mg of quinaldine sulfate and 

40 mg of MS-222 per liter of water at 19 °C 

for 30 minutes (table 6). The mean concentrations of residues of quinaldine and MS-222 at 

0-hour were 3.13 Mg/g and 27.3 Mg/g, respectively. After 24 hours of withdrawal no 

residues of quinaldine or MS-222 were detected.

Walleye

Spawning adult walleyes from the Mississippi River were exposed to a mixture of 10 

mg of quinaldine sulfate and 30 mg of MS-222 

per liter of water at 7° C for 30 minutes 

(table 6). The mean concentrations of residues 

of quinaldine and MS-222 at 0-hour were 2.20

Mg/g and 14.1 Mg/g, respectively. After 6 hours 

of withdrawal from the mixture, the quinaldine 

residue had decreased to a mean of 0.27Mg/g 

and the MS-222 residue had decreased to 2.3 

Mg/g. Only enough fish were available for 6 

hours of withdrawal.

DISCUSSION

The decrease in concentration of quinaldine 

and MS-222 residues during withdrawal of the 

fish from the mixed anesthetic follow a pattern 

similar to that of the individual anesthetics 

(Walker and Schoettger, 1967; Schoettger et al., 

1967; Alien et al., 1972; Sills and Harman, 

1970; and Sills et al., 1973). After 24 hours of 

withdrawal residues of both anesthetics decreased to near the background reading of the 

controls for MS-222 and to less than 0.01 Mg/g 

for quinaldine.

More residues of the two anesthetics were 

accumulated in smaller brook trout (0.3 kg) 

than in larger fish (0.8 kg). At the 0-hour withdrawal period 1.22 times more quinaldine residue and 1.43 times more MS-222 were found 

in the smaller fish than in the larger fish.

MS-222 appears to be taken up by both coldwater and warmwater fish more readily than 

quinaldine. The mixed anesthetic solutions contained from 1.5 to 6 times higher concentrations of MS-222 than quinaldine sulfate and 

muscle residues at the 0-hour withdrawal interval contained from 2.0 to 23 times higher 

concentrations of MS-222 than quinaldine residue.

The warmwater species were exposed to the 

highest concentrations of the anesthetics at the 

highest temperature. This group of fish showed 

slightly higher concentrations of anesthetic 

residues at the 0-hour withdrawal than the 

coldwater fish.

The length of exposure influenced the concentration of anesthetic residues as found by 

the earlier investigators. Fish exposed to the 

same concentrations of the mixture of anesthetics for 15 minutes contained from 1.2 to 

3.4 times the concentration of quinaldine residue and from 1.3 to 2.4 times the MS-222 residue as those exposed for 5.5 minutes, with the 

exception of coho salmon which showed higher 

MS-222 residues in the 5.5-minute exposure.
Table 6. Residues of anesthetics in muscle tissue of largemouth bass and bluegills 

of quinaldine sulfate (QdSO^) and MS-222 for 30 minutes at 19° C and walleyes trea

tion anesthetic for 30 minutes at 7° C

Withdrawal interval

Treatment

QdS04 

(mg/1)

MS-222 

(fflg/1)

Mean 

weight 

(kg)

Residues 

Quinaldine

Mean Range

Largemouth bass

Control ..........

0-hour ...........

1-hour ...........

2-hour ...........

4-hour ...........

6-hour ...........

24-hour ...........

Bluegill

Control ..........

0-hour ...........

1-hour ...........

2-hour ...........

4- hour ... . ....

6- hour

24-hour ...........

Walleye

Control ..........

0-hour ...........

1-hour . .

2-hour ...........

6-hour ...........

..... 0

, . . . . 20

, . . . . 20

, . . . . 20

. . . . . 20

, . . . . 20

20

..... 0

..... 10

..... 10

..... 10

..... 10

10

10

..... 0

.... 10

... 10

.... 10

.... 10

0

40

40

40

40

40

40

0

40

40

40

40

40

40

0

30

30

30

30

1.09

1.18

1.04

1.09

1.22

0.91

1.27

0.12

0.14

0.15

0.14

0.15

0.17

0.15

0.81

0.89

1.14

0.76

1.02

0.00

4.07

0.56

0.49

0.17

0.06

0.00

0.00

3.13

0.30

0.14

0.02

0.02

0.00

0.00

2.20

0.86

0.38

0.27

0.00-0.00

3.10-4.60

0.46-0.62

0.43-0.54

0.14-0.22

0.06-0.07

0.00-0.00

0.00-0.00

2 . 80-3 . 80

0.27-0.34

0.12-0.17

0.02-0.03

0.01-0.04

0.00-0.00

0.00-0.00

2.00-2.40

0.64-1.10

0.17-0.64

0.04-0.53

Each mean value represents the average of three analyses,
12 Investigations in Fish Control 55: Bureau of Sport Fisheries and Wildlife

CONCLUSIONS

1. The residues of quinaldine and MS-222 in 

the species tested varied considerably depending on concentration of the anesthetic, 

temperature, and length of exposure. As 

any of these parameters was increased, the 

residue concentrations at 0-hour withdrawal 

increased.

2. The residue concentrations of quinaldine 

decreased to less than 0.01 Mg/g and those 

of MS-222 decreased to near the range of 

the background of the controls after 24 hours 

of withdrawal.

3. MS-222 is taken up more readily from the 

mixed anesthetic solutions than quinaldine.

REFERENCES

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

1972. Residues of MS-222 in northern pike, muskellunge, and walleye. U.S. Bureau of Sport Fisheries 

and Wildlife, Investigations in Fish Control, No. 45, 

8 p.

Alien, John L., and Joe B. Sills.

I970a. GLC determination of quinaldine residue in 

fish. Journal of the Association of Official Analytical Chemists, vol. 53, p. 20-23.

Alien, John L., and Joe B. Sills.

I970b. U.V. identification and quantative measurement 

of quinaldine residues in fish. Journal of the Association of Official Analytical Chemists, vol. 53, 

p. 1170-1171.

Alien, John U, and Joe B. Sills.

1973. Preparation and properties of quinaldine sulfate, 

an improved fish anesthetic. U.S. Bureau of Sport 

Fisheries and Wildlife, Investigations in Fish Control, No. 47, 7 p.

Daws on, Verdel K., and Leif L. Marking. 

1973. Toxicity of mixtures of quinaldine sulfate and 

MS-222 to fish. U.S. Bureau of Sport Fisheries and 

Wildlife, Investigations in Fish Control, No. 53, 

Up.

Gilderhus, Philip A., Bernard L. Berger, Joe B. Sills, 

and Paul D. Harman.

I973a. The efficacy of quinaldine sulfate as an anesthetic for freshwater fish. U.S. Bureau of Sport Fisheries and Wildlife, Investigations in Fish Control, 

No. 49, 9 p.

Gilderhus, Philip A., Bernard L. Berger, Joe B. Sills, 

and Paul De Harman0

1973b. The efficacy of quinaldine sulfate:MS-222 mixtures for the anesthetization of freshwater fish. 

U.S. Bureau of Sport Fisheries and Wildlife, Investigations in Fish Control, No0 54, 9 p.

Marking, Leif L., and Verdel K. Daws on.

1973. Toxicity of quinaldine sulfate to fish. U.S. Bureau of Sport Fisheries and Wildlife, Investigations 

in Fish Control, No. 48 8 p.

Schoettger, Richard A., and A. 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.

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

1969. Efficacy of quinaldine as an anesthetic for seven 

species of fish. U.S. Bureau of Sport Fisheries and 

Wildlife, Investigations in Fish Control, No. 22,10 p.

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

1970. Synergic mixtures of MS-222 and quinaldine as 

anesthetics for rainbow trout and northern pike. 

Progressive Fish-Culturist, vol. 32, no. 4, p. 202- 

205.

Sills, Joe B., John L. Alien, Paul D. Harman, and 

Charles W. Luhning.

1973. Residue of quinaldine in ten species of fish following anesthesia with quinaldine sulfate. U.S. Bureau of Sport Fisheries and Wildlife, Investigations 

in Fish Control, No. 50, 9 p.

Sills, Joe B., and Paul D. Harman, 

1970. Efficacy and residues of quinaldine sulfate, an 

anesthetic for striped bass (Roccus saxatilis). 

Proceeding of 24th Annual Conference of the Southeastern Association of Game and Fish Commissioners, p. 546-549.

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. 15, 10 p.

U. S. GOVERNMENT PRINTING OFFICE : 1973 O - 526-823
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