INVESTIGATIONS IN FISH CONTROL

45. Residues of MS-222

in Northern Pike, Muskellunge, and Walleye

46. Methods of Estimating the Half-Life

of Biological Activity of Toxic Chemicals 

in Water

United States Department of the Interior

Fish and Wildlife Service 

Bureau of Sport Fisheries and Wildlife
INVESTIGATIONS IN FISH CONTROL

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

La Crosse, Wis., and Warm Springs, Ga., and reports of other studies related to that work. 

Though each report is regarded as a separate publication, several may be issued under a 

single cover, for economy.

Current reports in this series are 

(Reports 1 and 2 are in one cover.)

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

Charles R. Walker. 1964. 15 p.

2. Preliminary Observations on the Toxicity of Antimycin A to Fish and Other Aquatic Animals, by Charles R. Walker, Robert E. Lennon, and Bernard L. Berger. 1964. 18 p.

(Reports 3 through 8 are in one cover.)

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

Henegar. 1966. 16 p.

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

Robert G. Needham. 1966. 16 p.

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

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

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

Mahdi. 1966. 10 p.

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

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

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

(Reports 9 through 11 are in one cover.)

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

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

1966. 10 p.

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

Schoettger and Arnold M. Julin. 1966. 11 p.

(Reports 12 through 17 are in one cover.)

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

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

Arnold M. Julin. 1967. 15 p.

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

Schoettger. 1967. 10 p.

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

Richard A. Schoettger. 1967. 11 p.

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

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

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

1967. 14 p.

(Reports 18 through 21 are in one cover.)

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

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

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

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

Burress. 1967. 8 p.

(Reports 22 through 24 are in one cover.)

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

Schoettger and Arnold M. Julin. 1969. 10 p.

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

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

David 0. Locke. 1969. 5 p.

Continued on inside bac^ cover- 
INVESTIGATIONS IN FISH CONTROL

45. Residues of MS-222

in Northern Pike, Muskellunge, and Walleye

By John L. Alien, Charles W. Luhning, 

and Paul D. Harman

46. Methods of Estimating the Half-Life

of Biological Activity of Toxic Chemicals 

in Water

By Leif L. Marking

United States Department of the Interior

Fish and Wildlife Service 

Bureau of Sport Fisheries and Wildlife

Washington, B.C. July 1972

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

Washington, D.C. 20402 - Price 30 cents

Stock Number 2410-00336
IN THIS COVER

Pages

Residues of MS-222 in Northern Pike, Muskellunge, 

and Walleye, by John L. Alien, Charles W. Luhning, 

and Paul D. Harman ............................... 1-8

Methods of Estimating the Half-Life of Biological 

Activity of Toxic Chemicals in Water, by Leif L. 

Marking .......................................... 1-9

ii
INVESTIGATIONS IN FISH CONTROL

45. Residues of MS-222

in Northern Pike, Muskellunge, and Walleye

By John L. Alien, Charles W. Luhning, 

and Paul D. Harman

United States Department of the Interior

Fish and Wildlife Service 

Bureau of Sport Fisheries and Wildlife

Washington, B.C. July 1972
CONTENTS

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

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

Methods and Materials .................. 3

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

Analysis ........................ 4

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

Northern pike ..................... 4

Muskellunge ...................... 6

Walleye ........................ 6

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

Summary ......................... 8

References ........ , ................ 8
RESIDUES OF MS-222 

IN NORTHERN PIKE, MUSKELLUNGE, AND WALLEYE

By John L. Alien, Chemist 

Charles W. Luhning, Technician 

and Paul D. Harman, Technician 

Southeastern Fish Control Laboratory 

Bureau of Sport Fisheries and Wildlife 

Warm Springs, Georgia 31830

ABSTRACT.--Residues of MS-222 (tricaine me thane sulfonate) 

in muscle tissue of northern pike, muskellunge, and walleye 

following anesthesia were measured by a modified BrattonMarshall colorimetric method and confirmed by thin-layer chromatography. The residues dissipate rapidly from the muscle when 

fish are withdrawn from the anesthetic and are near the background 

readings of the controls within 24 hours.

The U. S. Food and Drug Administration 

requires that certain disinfectants, antimicrobials, and anesthetics be cleared for 

their continued use on fish subject to human 

consumption. A part of the information 

necessary for clearance of MS-222 (tricaine methanesulfonate) as an anesthetic 

is its residues in treated fish. MS-222 has 

gained wide acceptance as an anesthetic for 

fish. The drug has been used to facilitate 

handling of fish during marking, spawn 

taking, and transporting operations.

The demand for popular species of fish 

such as northern pike (Esox lucius), muskellunge (Esox masquinongy), and walleye 

(Stizostedion vitreum vitreum) has become 

so great that they are extensively propagated in hatcheries. These fish are difficult to handle during such hatchery operations as spawning and marking unless 

they have been anesthetized. The release 

or use of fish treated with an anesthetic 

without prior knowledge of the persistance 

of the compound could result in consumption of an adulterated product.

Walker and Schoettger (1967a) measured MS-222 residues in four salmonids

following anesthesia, and Schoettger et al. 

(1967) measured MS-222 residues in muscle of channel catfish (Ictalurus punctatus).

This study was undertaken to measure 

residues of MS-222 in the muscle of northern 

pike, muskellunge, and walleye.

METHODS AND MATERIALS

Fish

The inve'stigation of MS-222 residues in 

northern pike, muskellunge, and walleye was 

performed on fish treated with MS-222 in the 

springs of 1967 and 1968 (table 1). The fish 

were treated as outlined by Schoettger and 

Julin (1967) and Schoettger and Steucke (1970). 

Samples of muscle tissue collected by personnel of the Fish Control Laboratory in 

La Crosse, Wis.} were individually wrapped, 

frozen, and shipped on dry ice to the Southeastern Fish Control Laboratory, Warm 

Springs, Ga., for analysis. Tissue samples of all species collected in 1967 were 

taken by removing a portion of muscle adjacent to the dorsal fin as described by Walker 

and Schoettger (1967b). Northern pike muscle tissue samples collected in 1968 were
4 Investigations in Fish Control 45: Bureau of Sport Fisheries and Wildlife 

Table 1.--Species and sources of fish

Common 

name

Northern pike

Scientific 

name

Esox lucius

Average weight 

(grams )

1,180 

690 

690 

1,178

Year

1967 

1967 

1967 

1968

Site of treatment I/

NFH, Valley City, N. D. 

NFH, Genoa, Wis. 

FCL, La Crosse, Wis. 

NFH, Valley City, N. D.

Muskellunge 

Wa1leye

Esox masquinongy

770

Stizostedion vitreum vitreum 1,286

1,286

1967 NFH, New London, Minn.

1967 NFH, Valley City, N. D,

1967 NFH, Genoa, Wis.

1967 FCL, La Crosse, Wis.

]_/NFH ^National Fish Hatchery; FCL = Fish Control Laboratory.

taken by filleting the fish and homogenizing 

the entire fillet before analysis.

Analysis

The samples from northern pike, muskellunge, and walleye treated in 1967 were 

analyzed by the modified Bratton-Mar shall 

colorimetric method of Walker and 

Schoettger (1967b). In addition, at least 

one fish from each withdrawal interval was 

analyzed according to the thin-layer chroma- Northern pike 

tographic method of Alien, Luhning, and 

Harman (1970), except that a developing 

solution of 2-per cent methanol in benzene 

was used.

indicate when the residues had decreased 

to a concentration below the sensitivity of 

the procedure, which is approximately 

0. 2 ppm. Residues of less than 2. 0 ppm 

are reported as a trace. The Rf of the 

sample spot was compared with the Rf of an 

MS-222 standard on the same plate to identify the residues as MS-222.

RESULTS

The samples of northern pike collected 

in 1968 were taken from homogenized 

fillets and were analyzed by the colorimetric method of Walker and Schoettger 

(1967a). Each sample was analyzed also 

by the ancillary thin-layer chromatographic 

(TLC) method of Alien, Luhning, and 

Harman (1970).

Although the colorimetric method provides the accuracy of a photometric measurement, background readings from 

naturally occurring primary aromatic 

amines prevent the detection of MS-222 

residues less than background. The thin;- 

layer chromatographic procedure was used 

to identify the residues of MS-222 and to

Residues of free MS-222 in northern 

pike treated in 1967 were measured by 

colorimetric analysis and averaged 8. 4 ppm 

in the 0-hour withdrawal at 12° C to within 

the background readings of the controls at 

24-hour withdrawal. The fish in the colder 

water accumulated higher residue concentrations at 0-hour than those in the warmer 

water. At all temperatures the 24-hour 

withdrawal samples were within the background readings of the controls. No residues of MS-222 could be detected even by 

the more selective thin-layer chromatographic analysis after 24 hours withdrawal 

(table 2). Residues of MS-222 were confirmed by thin-layer chromatography only in 

samples showing colorimetric readings 

higher than the control samples. One control sample showed a trace of.MS-222 by thinlayer chromatography, but this sample may 

have been contaminated with MS-222 during
Table 2: Residues of MS-222 including background aromatic amines in muscle tissue of 

selected temperatures and withdrawal intervals following deep anesthesia wit

of MS-222

Temperature 

and Site and 

withdrawal year of 

interval treatment

8° C

Control

0-hour

2-hour

24-hour

12° C

Control

0-hour

2-hour

24-hour

17° C

Control

0-hour

2-hour

6-hour

24-hour

12° C

Control

0-hour

6-hour

24-hour

17° C

Control

0-hour

6-hour

24-hour

Valley City NFH

Do

Do

Do

Genoa NFH

Do

Do

Do

La Crosse FCL

Do

Do

Do

Do

La Crosse FCL

Do

Do

Do

La Crosse FCL

Do

Do

Do

Number of 

fish 

analyzed

1967

1967

1967

1967

1967

1967

1967

1967

1967

1967

1967

1967

1967

1968

1968

1968

1968

1968

1968

1968

1968

3

3

3

3

3

3

3

3

3

3

3

3

2

3

3

3

3

3

3

3

3

(1)

(D

(1)

(1)

(1)

(D

(1)

(D

(3)

(0

(D

(2)

(2)

(3)

(3)

(3)

(3)

(3)

(3)

(3)

(3)

Bratton-Marshal 1 method-- 

residues in ppm

I/ 6e

if Mean

0.7

10.9

5.3

0.8

1.2

8.4

2.9

0.8

1.0

7.3

0.7

0.3

1.1

0.1

19.5

0.1

0.0

0.1

9.4

1.6

0.7

MS-222

Range

0.1- 1.8

5.4-16.0

2.4- 9.0

0.0- 1.2

0.4- 2.8

3.2-17.0

1.6- 4.6

0.5- 1.0

0.0- 2.4

3.0-10.6

0.0- 2.0

0.0- 1.0

0.0- 2.2

0.0- 0.2

12.0-30.0

0.0- 0.2

0.0- 0.0

0.0- 0.2

6.0-12.0

1.0- 2.4

0.2- 1.0

Free and

Mean

2.0

11.3

6.2

1.3

2.6

9.1

5.7

2.0

2.1

8.3

0.5

0.5

1.0

1.4

22.0

0.7

0.7

1.8

12.2

2.0

1.1

acetylated

1

5

4

0

2

5

4

1

1

2

Range

.5- 2.

.4-18.

.8- 8.

.8- 2.

.0- 3.

.4-15.

.0- 7-

.5- 2.

.5- 2.

.8-13.

0.0- 1.

0

1

1

16

0

0

1

9

1

1

.1- 1.

.0- 1.

.0- 1.

.8-28.

.2- 1.

.6- 1.

.4- 2.

.0-15.

.6- 2.

.0- 1.

MS-222

4

0

4

0

0

0

0

4

8

0

0

0

0

6

0

0

0

4

0

8

4

\J Number in parentheses indicates the number of fish analyzed by the TLC method. 

2/ ND = None detected; Trace = less, than 2.0 ppm.

3/ Two of the three fish in this group showed a trace of MS-222 residue, but no resid

third fish.
6 Investigations in Fish Control 45: Bureau of Sport Fisheries and Wildlife

analysis. There was not enough sample 

remaining to repeat the analysis.

In 1968, northern pike were anesthetized to medulary collapse with 150 ppm of 

MS-222 at 12° and 17° C at the Fish Control Laboratory at La Crosse, Wis. Residues of free MS-222 measured by colorimetric analysis averaged 19. 5 ppm at 

0-hour withdrawal to within the background 

readings of the controls at 6-hour withdrawal in fish treated at 12° C. Thin-layer 

chromatographic analysis of each fish from 

each withdrawal interval confirmed the 

presence of MS-222 residues only at the 

0-hour withdrawal interval (table 2). At 

17° C, residues of free MS-222 determined 

by colorimetric analysis averaged 9. 4 ppm 

at 0-hour to trace after 24 hours withdrawal from the anesthetic. Thin-layer 

chromatography confirmed the presence of 

MS-222 residues in all the fish treated at 

this temperature with the exception of one 

fish from the 24-hour withdrawal interval.

Muskellunge

Residues of free MS-222 in muskellunge 

anesthetized to medullary collapse with 

150 ppm of MS-222 at 10° C at the New 

London National Fish Hatchery in Minnesota averaged 7. 9 ppm at 0-hour withdrawal, decreasing to background during 

24 hours of recovery in fresh water 

(table 3). Thin-layer chromatographic 

analysis of two fish from each withdrawal 

period confirmed the presence of MS-222 

residues at the 0-hour and 2-hour withdrawals. No residues were detected at the 

24-hour withdrawals by TLC.

Walleye

In walleye treated at 17° C with 100 ppm 

of MS-222, residues of free MS-222 

averaged 2. 4 ppm at 0-hour withdrawal, 

and had decreased to within the background 

readings of the control samples during 24 

hours of recovery in fresh water (table 4). 

Thin-layer chromatography of one fish

from each withdrawal interval confirmed 

the presence of MS-222 residues in fish 

from all withdrawal intervals except those 

at 24 hours. The 6-hour withdrawal sample showed only a trace of MS-222 residue 

remaining.

Walleye exposed to 120 ppm of MS-222 

at 10° C at the Genoa National Fish Hatchery 

in Wisconsin showed residues of free 

MS-222 at 0-hour that averaged 4. 3 ppm 

and decreased to within the background 

levels of the controls after 24 hours in 

fresh water (table 4). Thin-layer chromatography of one fish from each withdrawal 

interval confirmed the presence of MS-222 

at each withdrawal interval except at 24 

hours.

DISCUSSION

MS-222 residues remaining after 24 

hours withdrawal from the drug were shown 

to vary little from the background amines of 

the controls as detected by colorimetric 

analysis. The colorimetric analysis for 

total free plus acetylated aromatic amines 

generally showed only slightly higher concentrations than the analysis for free aromatic amines only. Certain samples showed 

slightly higher readings for free MS-222 

than for both free and acetylated aromatic 

amines. It is possible that small amounts 

of some of these amines could have been 

lost during the digestion step.

The identification of the residues was 

made by comparing the Rj? of the spot obtained from the sample to the Rf of the spot 

given by the MS-222 standard. The semiquantitation of the residues by thin-layer 

chromatography was accomplished by comparing the size and intensity of the sample 

spot with a series of standard spots of known 

concentrations. The quantitative estimates 

made by thin-layer chromatography were in 

agreement with the results obtained by 

colorimetric analysis. This indicates that 

most of the aromatic amine above the background reading of the controls detected by the 

colorimetric method was MS-222.
Alien, Luhning, and Harman: MS-222 Residues in Pike, Muskellunge, and Walleye 7

Table 3t--Residues of MS-222 including background aromatic amines in muscle tissue of

muskellunge at selected withdrawal intervals following deep anesthesia with a 

150 ppm solution of MS-222 at 10° C

Site 

Withdrawal of 

interval treatment

Control

0-hour

2-hour

24-hour

New London NFH

Do

Do

Do

Bratton-Marshal 1 

residues in

Free

Mean

0.3

7.9

0.8

0.4

MS-222

Range

0.1- 1.0

3.4-10.5

0.2-

0.0-

1.0

1.0

method-- 

ppm

Free and acetylated MS-222

Mean

1.2

10.7

2.2

2.0

Range

1

7

1

1

.0- 1

.6-13

.5- 2

.0- 2

.5

.0

.8

.8

(3)

(3)

(3)

(3)

TLC method -- 

estimated free 

MS-222 residues in 

ppm (mean) I/ 2/

ND

4.3

Trace

ND

(2)

(2)

(2)

(2)

\J Number of fish in parentheses,

2/ ND = None detected; Trace = less than 2.0 ppm.

Table 4: Residues of MS-222 including background aromatic amines in muscle of walleye 

at selected concentrations, temperatures, and withdrawal intervals following 

deep anesthesia

Temperature, 

concentration, 

and

Sites 

of

withdrawal interval treatment MeanRange

TLC method--

_____________________________ estimated free 

Free MS-222Free and acetylated MS^2l2 MS-222 residues

Bratton-Marshal1 method 

residues in ppm_______

Mean Range in ppm I/ 2/

10° C, 120 ppm

Control

0-hour

2-hour

6-hour

24-hour

17° C, 100 ppm

Control

0-hour

2 -hour

6-hour

24-hour

Genoa NFH

Do

Do

Do

Do

La Crosse

Do

Do

Do

Do

Do

1.0

4.3

1.8

1.6

0.4

FCL

1.7

2.4

2.8

2.0

1.3

0.6-2.0

1.0-7.6

0.4-4.0

0.4-4.0

0.0-1.0

0.4-2.8

0.4-3.6

1.0-5.0

1.4-2.8

1.0-2.0

1.1

3.6

1.0

0.4

1.0

2.3

2.5

3.3

2.5

2.3

0.3-2.0

0.5-7.6

1.0-1.0

0.0-0.8

0.5-2.2

2.0-2.8

1.0-3.6

2.0-5.4

2.0-2.8

1.2-3.4

(3)

(3)

(3)

(3)

(3)

(3)

(3)

(3)

(3)

(3)

ND

8.0

Trace

Trace

ND

ND

4.0

Trace

Trace

ND

(1)

(D

(D

(D

(D

(D

(D

(D

(D

(D

\J Number of fish in parentheses.

2_/ ND = None detected; Trace = less than 2.0 ppm.

With the exception of two samples of 

northern pike (table 2), no MS-222 residues were detected by thin-layer chromatography in fish withdrawn from the drug 

for 24 hours. Two of the three northern 

pike treated at 17° C and withdrawn from 

the anesthetic for 24 hours in 1968 showed

a trace of MS-222 by thin-layer chromatography, but no residue was detected in 

the third sample. The lower limit of sensitivity of the thin-layer chromatographic 

procedure is 0. 2 jig of MS-222, but 

quantitation generally is not possible in 

samples spiked at less than 2. 0 ppm.
8 Investigations in Fish Control 45: Bureau of Sport Fisheries and Wildlife

Thus a trace of MS-222 as detected by TLC 

indicates the presence of MS-222 at concentrations of 2.0 ppm or less. The concentration of free MS-222 including background amines for the two 24-hour withdrawal samples treated at 17° C in 1968 

was 1. 0 ppm by colorimetric analysis. 

Both the thin-layer chromatographic and 

colorimetric method showed that these two 

samples contained less than 2. 0 ppm of 

MS-222 residue.

Walleye were treated at a lower concentration of MS-222 and had the lowest 

concentration of MS-222 residues at 0- 

hour withdrawal. Northern pike had the 

highest concentration of MS-222 residues 

at 0-hour withdrawal. At the end of 24 

hours, however, there were no significant 

differences in residue concentrations in 

any of the samples.

SUMMARY

Residues of MS-222 in muscle of northern pike, muskellunge, and walleye 

following anesthesia at selected temperatures were measured by the modified 

Bratton-Mar shall colorimetric method 

(Walker and Schoettger, 1967b) and confirmed by thin-layer chromatography 

(Alien, Luhning, and Harman, 1970).

The mean concentration of MS-222 residues at the 0-hour withdrawal interval 

ranged from 2. 4 to 19. 5 ppm. The level 

of MS-222 residues at 0-hour withdrawal 

was highest in northern pike and lowest in 

walleye. After 24 hours withdrawal from 

the anesthetic, residues were detected inonly 2 of the 12 northern pike analyzed by 

thin-layer chromatography, and these two 

samples were shown to contain less than 

2. 0 ppm.

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.

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 Erwin W.

Steucke, Jr.

1970. Quinaldine and MS-2 22 as spawning aids for northern pike, muskellunge, 

and walleyes. Progressive FishCulturist, Vol. 32, No. 4, p. 199-201.

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.

l967a. Residues of MS-222 in four

salmonids following anesthesia. U. S. 

Bureau of Sport Fisheries and Wildlife, Investigations in Fish Control, 

No. 15, 11 p.

1967b. Method for determining MS-222 

residues in fish. U. S. Bureau of 

Sport Fisheries and Wildlife, Investigations in Fish Control, No. 14, 10 p.
NVESTIGATIONS IN FISH CONTROL

46. Methods of Estimating the Half-Life

of Biological Activity of Toxic Chemicals 

in Water

By Leif L. Marking

United States Department of the Interior

Fish and Wildlife Service 

Bureau of Sport Fisheries and Wildlife

Washington, B.C. July 1972
CONTENTS

Page 

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

Methods ......................... 4

Method A .................... 4

Example .................... 5

Method B .................... 5

Example .................... 7

Discussion and Conclusions .............. 7

References ....................... 9
METHODS OF ESTIMATING THE HALF-LIFE 

OF BIOLOGICAL ACTIVITY OF TOXIC CHEMICALS IN WATER

By Leif L. Marking, Chemist 

Bureau of Sport Fisheries and Wildlife 

Fish Control Laboratory, La Crosse, Wisconsin

ABSTRACT. --In the absence of analytical methods, the half-life 

of biological activity of a chemical (the time required to decrease its 

toxic activity by one-half) can be estimated by bioassays. The methods 

.presented require the determination of LC50 values (concentration producing 50% mortality) for organisms in aged solutions containing unknown residual concentrations, and concurrent tests of solutions containing known concentrations. The half-life of biological activity is 

determined by plotting (1) the percent concentrations remaining in 

aged solutions or (2) the deactivation indices against aging time on 

cyclic semilogarithmic graph paper. By the first method, a 5-day 

half-life for antimycin, a fish toxicant, was determined using resistant channel catfish (Ictalurus punctatus) and goldfish (Carassius 

auratus) and for sensitive green sunfish (Lepomis cyanellus) and 

rainbow trout (Salmo gairdneri) in soft water at pH 7. 5. The second 

method, using only rainbow trout, also estimated a 5-day half-life for 

antimycin in soft water at pH 7. 5. These methods could be used also 

to determine the half-life of antimycin at different pH's, temperatures, 

and light intensities, the factors thought to influence the toxicant's 

efficacy. The methods are not limited to fish and fish toxicants.

The biological activity of a toxicant is ample, antimycin, a fish toxicant, kills

the killing power, which decreases as the rainbow trout at 30 parts per trillion

toxicant is deactivated biologically and (Berger et al., 1969), but analytical

chemically with time. The half-life of a methods aren't available to quantitate this

toxicant's biological activity is the time concentration. Walker et al. (1964) obnecessary for that activity to be reduced served that antimycin degrades rapidly in

by one-half. Regulatory agencies now re- water, especially under alkaline conditions,

quire data on the persistence of toxicants and recommended further definition of the

in the environment, and the half-life or detoxification rate, 

rate of disappearance is a good measure

of persistence. In lieu of inadequate analytical methods,

	I have outlined two laboratory bioassay

The half-life of most fish toxicants in methods, free of analytical instrumentation,

water is influenced by pH, temperature, to estimate the half-life of biological activity

sunlight, metabolism by aquatic organisms, for toxicants used in water. The first

and other environmental variables. method requires test animals that are

	differentially sensitive to a toxicant in

Analytical methods to detect and order to quantitate the decreased concenmeasure very small concentrations of some trations resulting from degradation. The

fish toxicants are unavailable. For ex- second method requires one test species
4 Investigations in Fish Control 46: Bureau of Sport Fisheries and Wildlife

and greater than lethal concentrations of 

a chemical that are aged to levels commensurate with the tolerance of the selected species.

Mortalities in the bioassays with degrading concentrations are compared with 

those in concurrent reference tests on the 

same organism using known concentrations 

of the toxicant. The experiments with 

antimycin included sensitive and resistant 

species of fish, but other aquatic organisms such as mollusks, aquatic insects, and plants may be used in the bioassays for the half-life determination. The 

intra-species tolerance differences are of 

little consequence in the procedure since 

a reference bioassay is conducted concurrently with the degrading solutions.

The half-life of biological activity of 

a toxicant can be determined for various 

environmental parameters such as pH and 

temperature. Such information' would be 

invaluable to fishery biologists in selecting minimum lethal concentrations of fish 

toxicants or aquatic herbicides for target 

species and in avoiding hazards to nontarget species. Field biologists using antimycin have reported incomplete kills of 

fish that probably resulted more from 

rapid deactivation of the antibiotic in 

water of a particular quality than from 

applying a nontoxic concentration. For 

example, 1 ppb of antimycin may kill all 

of the target fish in water of pH 8, but fail 

to kill any in water of pH 9. Knowledge of 

chemical persistence of toxicants in the 

aquatic environment is essential for efficacious, safe, and economical control of 

target organisms.

METHODS

The fish toxicant antimycin was chosen 

as an example to determine the half-life of 

biological activity according to the methods 

described. Previous reports indicate that 

the toxicity of antimycin is influenced by 

chemical and biological properties of the

water, especially those controlling pH, but 

no one has been able to quantify the rate of 

deactivation. The static bioassay routine 

and maintenance of test fish were essentially those described by Lennon and 

Walker (1964) and Hunn et al. (1968). All 

the tests were conducted in soft water at 

pH 7. 5 and 12° C.

Method A

The concentration of biologically active chemicals remaining in solutions permitted to age for a selected period of time 

can be estimated by introducing fish or 

other susceptible aquatic organisms for 

bioassay. More resistant species are exposed to lethal solutions of toxicants that 

have been aged slightly, whereas more 

sensitive species are exposed to older 

solutions that have become deactivated to 

lower concentrations. Concurrently with 

tests in aged media, identical organisms 

must be employed in bioassays of fresh 

solutions under identical test conditions 

and exposure periods at concentrations expected to produce mortality. The mortality 

data from the tests in aged and fresh solutions are analyzed to determine the statistical LC50 (concentration producing 50 percent 

mortality) according to the method of 

Litchfield and Wilcoxon (1949). The LCSO's 

are calculated for both tests on the basis 

of applied concentrations even though the 

concentration has decreased in the aging 

tests.

To determine the approximate concentration remaining in the aged bioassays, a 

formula was developed using hypothetical 

data for ideal disappearance of biological 

activity presented in table 1. The hypothetical data are characteristic for a toxicant with a half-life of 1 day. LCSO's for 

all of the tests in aged solutions (Cj) are 

identical at 50 ppb in this hypothetical 

case. In actuality, the statistical LC50 

would not be consistent for each test, but 

I have kept it constant for simplicity. The 

actual concentrations remaining in the aged
Marking: Estimating Half-Life of Biological Activities of Toxic Chemicals in ^A ater 5

solutions are unknown after aging begins, 

yet the LC50's are calculated from the 

concentrations originally applied to the 

test water. In reference tests with fresh 

solutions (C2 ), the concentrations required decrease proportionately with the 

deactivation of aged solutions, and therefore more sensitive species are required 

to conform to these lower concentrations. 

The percent concentration remaining is 

calculated according to the following 

equation:

Percent concentration 

remaining = 100 -

- C2 )

c

100

Where Cj = LC50 of aged solutions 

C2 = LC50 of fresh solutions

This computation is repeated for each 

aging time interval.

A plot of the percent concentration remaining and aging time (table 1) on semilogarithmic graph paper reveals a typical 

first-order decay curve with a half-life of 

1 day. The unit of days was chosen for the 

hypothetical situation, but hours or weeks 

are appropriate when toxicants are deactivated slower or faster.

Example

Four series of uniform concentrations 

of antimycin were set up and permitted to 

age prior to introducing the test species. 

Each series included concentrations of 20, 

40, 60, 80, and 100 ppb of antimycin and 

a control with no toxicant. After 7 days of 

aging, 10 channel catfish were introduced 

into each concentration and the control 

vessel of a single series. On the same 

day, antimycin was added to five vessels 

containing 10 channel catfish each at concentrations ranging from 1 to 40 ppb. The 

96-hour LC50 of the 7-day-old solutions 

was calculated to be 47. 300 (C1 ), and that 

value for the fresh solutions is 20. 400 ppb

By substituting the values of C^ and 

C2 into the formula, the percent concentration remaining after 7 days is calculated to be 43. 13 (table 2 ).

Goldfish were added to the second 

series of antimycin concentrations of 20, 

40, 60, 80, and 100 ppb after the solutions 

had aged for 30 days. Concurrently, goldfish were bioassayed in fresh solutions of 

antimycin, and the 96-hour LC50's were 

computed for both tests. The resulting 

percent concentration remaining after 30 

days is 1. 10 (table 2).

Green sunfish were added to another 

series of aged solutions after 35 days of 

deactivation, and rainbow trout after 44 

days of deactivation. The percent concentration remaining was computed for both 

species (table 2).

The reference LCSO's (C^) show 

different sensitivity among the four species, 

and the 96-hour LCSO's range from 20.400 

ppb for the more resistant catfish to 0. 049 

ppb for the more sensitive trout. The 

LCSO's are characteristic for these species 

in this water quality, but change in tests 

using different water.

The percent concentration remaining 

and aging time from table 2 are plotted on 

cyclic semilogarithmic graph paper (fig. 1). 

The curve approximates a first-order decay 

curve and describes the rate of disappearance 

for antimycin in soft water of pH 7. 5. The 

percent concentration remaining is 50 

after 5 days and 25 after 10 days; therefore the half-life of biological activity is 

5 days.

Method B

The half-life of biological activity can 

be estimated by using only one species of 

test organism. The procedure requires 

aged solutions of toxicant, but stronger 

solutions are established and allowed to 

age for longer periods, such as 2, 4, 8,
6 Investigations in Fish Control 46: Bureau of Sport Fisheries and Wildlife

Table 1: Percent of concentrations remaining as calculated from hypothetical data on aged 

and fresh solutions of toxicant

Aging time 

for C] 

(days)

0

1

2

3

k

5

6

7

8

9

10

LC50 I/

Aged 

solutions

50.000

50.000

50.000

50.000

50.000

50.000

50.000

50.000

50.000

50.000

50.000

(ppb) of

Fresh 

(Ci) solutions ( 2)

50.000

25.000

12.500

6.250

3.125

1.563

0.782

0.391

0.196

0.098

0.0^9

Percent 

reduction 

of LC50

0.00

50.00

75.00

87.50

93.75

96.87

98.^3

99.21

99.60

99.80

99.90

Percent 

concentration 

remaining

100.00

50.00

25.00

12.50

6.25

3.13

1.57

0.79

O.^fO

0.20

0.10

j/Calculated from original concentrations of aged and fresh solutions bioassayed concurrently.

Table 2: Toxicity of antimycin to fish'in aged and fresh solutions at pH 7*5 and 12° C.

Species

Channel catfish 

(Ictalurus punctatus)

Goldfish 

(Carassius auratus)

Green sunfish 

(Lepomis cyanellus)

Rainbow trout 

(Salmo gairdneri )

Age in days 

of aged test 

solutions

7

30 

35

Mf

96-hour LC50

aged (Cj) 

sol uti ons

if7-300

60.500 

52.000

3^.^00

(ppb) of

fresh (C-2) 

solutions

20.^00

0.650 

0.308

0.0^9

Percent 

concentration 

remaining

^3.13

1.10 

0.59

0.20

16, 32, or more days. As the solutions 

deactivate to a level of toxicity commensurate with the tolerance of the test organism, they are bioassayed against the

test species. Concurrently with the bioassay of aged solutions, fresh solutions are 

bioassayed against individuals of the same 

species to provide reference data.
Marking: Estimating Half-Life of Biological Activities of Toxic Chemicals in Water 7

100-

50-

O

z

z

10- 

,0-

1.0-

z

uj 0-51

U

oc

0.1-

i i

10 20 

TIME

Figure 1 

at pH 7.

30 40 

IN DAYS 

:--The inactivation of antimycin 

5 and 12° C by method A.

50

The results obtained by bioassaying 

aged and fresh solutions against a single 

species are used to compute a deactivation 

index. The deactivation index is derived 

by dividing the LC50 of an aged solution by 

the LC50 of a fresh reference solution. 

The index has a value greater than 1 if the 

chemical deactivates at all. A value of 2 

indicates that the concentration of an aged 

solution has diminished by one-half. The 

aging time required for a deactivation index of 2, calculated from the LCSO's, is 

equivalent to the half-life of biological 

activity of the toxicant.

The deactivation index invariably does 

not equal exactly 2 for any particular test. 

Therefore, the deactivation index is computed for each aging period, and the results are plotted on semilogarithmic 

coordinates. The slope of the curve

influences the half-life value and is derived from a line fitting the plotted deactivation indices.

Example

Rainbow trout were selected to assess 

this alternate method for half-life of 

biological activity. Five series of different 

concentrations of antimycin were prepared 

on different days during a 16-day period. 

The first series of solutions contained 

higher concentrations than subsequent 

series because it would be deactivating for 

16 days prior to introducing the fish. The 

next series aged for 8 days, and the initial 

concentrations were lower than in the first 

series. Another series of concentrations 

was aged 4 days, and another for 2 days 

prior to introducing the trout. All of the 

aged solutions were bioassayed on the 

same day. Concurrently, a reference 

bioassay with rainbow trout was conducted 

using known concentrations in fresh solutions. The 96-hour LCSO's for solutions 

aged for different time periods were used 

to compute the deactivation indices 

(table 3).

The deactivation indices range from 

1. 25 to 7. 5 and signify the deactivation 

occurring over the test period. They are 

plotted against aging time on semilogarithmic graph paper (fig. 2K From this graph, 

the half-life of biological activity for antimycin in soft water at pH 7. 5 and 12° C 

is about 5 days. This value agrees with 

that determined by method A.

DISCUSSION AND CONCLUSIONS

The model (table 1) conveniently presents data for each 1-day interval of the 

aging process. Quite frequently, as in the 

example of antimycin, species are unavailable for consistent 1-day increments and 

consequently the aging times before introducing the species do not coincide with the 

half-lifes. This does not prevent the halflife determination, however, since the
8 Investigations in Fish Control 46: Bureau of Sport Fisheries and Wildlife

Table 3: Toxicity of antimycin (ppb) to rainbow trout in aged (C-\ ) and fresh ( 2) 

solutions in 96-hour tests at pH 7.5 and 12° C.

Aged solutions

Age i n 

days

LC50

Fresh 

solut ions 

LC50 (C 2 )

Deactivation 

index

8

16

0.0^98 

0.0787 

0.130 

0.300

0.0^0 

Q.OkO 

0.040 

0.0^0

1.25

1.97

3.25

7.50

z 

o

2-

1 -

0 4 8 12 16

TIME IN DAYS

Figure 2:--The inactivation of antimycin 

at pH 7.5 and 12° C by method B..

percent reduction can be plotted at any 

time a species can be found which is sensitive to the aged solutions.

In field applications, target organisms 

must be exposed to a minimum lethal concentration or greater for a certain period

of time for successful eradications. Knowledge of the half-life of biological activity 

permits selection of concentrations which 

will retain sufficient toxicity throughout an 

exposure period to kill the target organism. 

In addition, the concentration remaining in 

the treated water can be calculated for any 

reasonable length of time. For instance, 

highly sensitive species may be introduced 

into waters in which highly resistant species 

were eradicated. The time required for 

the toxicant to become sublethal to the sensitive species can be extrapolated from the 

half-life curve.

In the event that a chemical detoxifies 

too slowly and bioassay space and equipment cannot be tied up for long periods, the 

procedure for method A may be divided into 

segments which can be accomplished concurrently. For example, resistant species 

may be tested in aged (C}) and fresh (C2 ) 

solutions while at the same time sensitive 

species may be tested in lower concentrations of aged (C^) and fresh (C2 ) solutions. The percent reduction in concentrations for each time segment is plotted 

on a single graph and the half-life is obtained by averaging the rates for individual 

species. This practice reduces the time 

that single solutions must age, thereby 

reducing problems caused by evaporation 

and decreasing the chance of contamination 

in the test solutions. It also detects changes 

in rates of detoxification for different concentrations of toxicants.
Marking: Estimating Half-Life of Biological Activities of Toxic Chemicals in Water 9

A difficulty sometimes arises in determining the proper time to bioassay the 

aged solutions, especially without prior 

knowledge of the toxicant's persistence. 

After the initial test in method A, however, 

the data can be plotted using the single percent concentration remaining and assuming 

100 percent biological activity at the beginning of the test. The slope of the curve 

may be influenced in subsequent tests, but 

through a quick half-life approximation the 

activity can be predicted for longer aging 

periods and more sensitive species. 

Following the tests of more sensitive 

species, the curve is redrawn for best fit 

to all of the calculated values.

Method A is limited by the number of 

differentially sensitive organisms available. In fact, to more accurately derive 

the half-life by this method, three or more 

values are desirable to establish the 

slope of the curve. Several of these values 

may be obtained using one species, although more than one species should be 

used in deriving the half-life curve. The 

model presents a reduction in toxicity by 

a factor of 1, 000 using 10 different aging 

periods, but experimental chemicals frequently are not that differentially toxic nor 

are that many species available.

Advantages of method B are that only 

one species is required, fewer reference 

tests are necessary, and the total time and 

facility requirements are less. Both 

methods are estimates because the values 

derived are taken from a curve representing statistical calculations on the 

tolerance of biological organisms. The 

plotted values are experimental and do not 

necessarily occur on the curve. In method 

A the variation is among different species, 

whereas in method B the variation is 

among different tests of the same species. 

For better results, several species could 

be tested in method B and values could be 

averaged.

Neither method is intended to account 

for absorption and adsorption of test chemicals to different kinds of bioassay vessels 

or for complexing to organic matter found 

in different waters. Also, they do not 

differentiate between chemical degradation 

and biological inactivation. The methods 

estimate the available total activity of a 

chemical and its degradation products under 

defined test media conditions.

REFERENCES

Berger, Bernard L., Robert E. Lennon,

and James W. Hogan.

1969. Laboratory studies on antimycin A 

as a fish toxicant. U. S. Bureau of 

Sport Fisheries and Wildlife, Investigations in Fish Control, No. 26, 19 p.

Hunn, Joseph B., Richard A. Schoettger,

and Everett W. Whealdon.

1968. Obervations on the handling and 

maintenance of bioassay fish. The 

Progressive Fish-Culturist, 30(3): 

164-167.

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

1964. Laboratories and methods for

screening fish-control chemicals. U. S. 

Bureau of Sport Fisheries and Wildlife, 

Investigations in Fish Control, No. 1 

(Circular 185). 15 p.

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

1949. A simplified method for evaluating 

dose-effect experiments. Journal of 

Pharmacology and Experimental 

Therapeutics, 96:99-113.

Walker, Charles R., Robert E. Lennon,

and Bernard L. Berger.

1964. Preliminary observations on the 

toxicity of antimycin A to fish and 

other aquatic animals. U. S. Bureau 

of Sport Fisheries and Wildlife, 

Investigations in Fish Control, No. 2 

(Circular 186). 18 p.

if U.S. GOVERNMENT PRINTING OFFICE : 1972 O-475-000
(Reports 25 through 28 are in one cover.)

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

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

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

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

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

Lennon. 1969. 21 p.

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

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

(Reports 29 through 31 are in one cover.)

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

and Richard A. Schoettger. 1969. 11 p.

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

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

(Reports 32 through 34 are in one cover.)

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

33. Voidance Time for 23 Species of Fish, by Thomas H. Lane and Howard M. Jackson. 1969. 9 p.

34. Laboratory Studies on Possible Fish-Collecting Aids With Some Toxicities for the Isomers of Cresol, by Robert M. Rowland. 1969. 10 p.

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

Schoettger. 1970. 31 p.

(Reports 36 through 38 are in one cover. In press.)

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

> Leif L. Marking.

V3*/. Comparative Toxicity of 29 Nitrosalicylanilides and Related Compounds to Eight Species 

of Fish, by Leif L. Marking and Wayne A. Willford.

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.

(Reports 39 and 40 are in one cover.)

39. Effects of Antimycin A on Tissue Respiration of Ralnhow 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.

Fish Control Laboratories

Bureau of Sport Fisheries and Wildlife

U.S. Department of the Interior

P.O. Box 862

La Crosse, Wisconsin 54601
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

U.S. DEPARTMENT OF THE INTERIOR

I NT ^23