INVESTIGATIONS IN FISH CONTROL

36. A Method for Rating Chemicals 

for Potency Against Fish 

and Other Organisms

37. Comparative Toxicity of 

29 Nitrosalicylanilides 

and Related Compounds 

to Eight Species of Fish

38. Toxicity of 33NCS to

Freshwater Fish and Sea Lampreys

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 L0 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 _p,p'-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. lip.

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

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

by Richard A. Schoettger, Charles R. Walker, Leif L. Marking, and Arnold M. Julin. 1967. 

14 p.

Fish Control Laboratories

Bureau of Sport Fisheries and Wildlife

U.S. Department of the Interior

P.O. Box 862

La Crosse, Wisconsin 54601

Continued on inside back cover 
INVESTIGATIONS IN FISH CONTROL

36. A Method for Rating Chemicals 

for Potency Against Fish 

and Other Organisms

By Leif L. Marking

37. 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

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

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

Fish and Wildlife Service, Charles H. Meachaih, Commissioner

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

Washington, D.C. July 1970

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

Washington, D.C. 20402 - Price 45 cents
IN THIS COVER

Pages

A Method for Rating Chemicals for Potency Against Fish and

Other Organisms, by Leif L. Marking ................. 1-8

Comparative Toxicity of 29 Nitrosalicylanilides and Related 

Compounds to Eight Species of Fish, by Leif L. Marking and 

Wayne A. Willford .............................. 1-11

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 ................ 1-16

ii
INVESTIGATIONS IN FISH CONTROL

36. A Method for Rating Chemicals 

for Potency Against Fish 

and Other Organisms

By Leif L. Marking

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

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

Fish and Wildlife Service, Charles H. Meacham, Commissioner

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

Washington, D.C. July 1970
CONTENTS

Page

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

Potency rating.................................... 3

Discussion and conclusions ........................... 6

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

References ...................................... 7

Appendix. ....................................... 8
A METHOD FOR RATING CHEMICALS FOR POTENCY 

AGAINST FISH AND OTHER ORGANISMS

By Leif L. Marking, Chemist

Bureau of Sport Fisheries and Wildlife

Fish Control Laboratory, La Crosse, Wisconsin

ABSTRACT. A potency rating is presented by which the toxicity of 

chemicals to organisms can be assessed with a minimum of data from 

preliminary bioassays. This method permits effective and rapid evaluation 

of toxicity when data from preliminary tests are inadequate for statistical 

analysis. In bioassays of chemicals against fish, the mortality is recorded 

in 3- to 96-hour tests when exposed to three concentrations. The potency 

rating of a bioassayed chemical, when compared with the maximum value 

on the rating scale, indicates the relative toxicity of the chemical. Concentrations and exposure times can be changed to fit a specific bioassay 

program. Results of 24- or 48-hour bioassays can be compared with 

96-hour bioassays because the maximum values of the rating scales are 

approximately equal at all time intervals. The potency rating is more 

applicable to toxicants which have similar dose-effect curves.

Data from preliminary bioassays are usually tedious and difficult to analyze since the 

toxicity is not defined by statistical parameters such as those described by Litchfield 

and Wilcoxon (1949). A minimum number of 

organisms and concentrations limit statistical 

evaluations for median tolerance limits. Preliminary bioassay data usually indicate toxic 

ranges of concentrations and are followed by 

more definitive tests. However, a greater 

number of chemicals can be tested in preliminary trials such as those described by 

Lennon and Walker (1964), but a mathematical 

method is needed for analyzing and summarizing these data in a form that is easily stored 

and retrieved. In programs in which many 

chemicals are not tested beyond preliminary 

screening, the potency rating of a large number of compounds is useful in making comparisons and selecting those having potential. 

It also reduces the bulk of data to easily 

comprehended terms.

POTENCY RATING

Potency is necessarily stated in absolute 

units for therapeutic applications, but for 

comparison of drugs relative potency is a 

more convenient term, as Fingl and Woodbury 

(1965) point out. They also state that doseeffect curves which are similar in slope 

permit comparisons of relative potencies, 

while those with dissimilar dose-effect 

curves do not. Relative potency is more applicable to bioassay data derived from related 

chemical structures or from chemicals producing similar biological effects on the test 

organisms.

Relative potency is described by Finney 

(1962) and is calculated as a constant difference between the dosage-response regressions of two stimuli. This method does not 

apply to preliminary type data which cannot 

be analyzed by probit regression methods.
Investigations in Fish Control 36: Bureau of Sport Fisheries and Wildlife

I have therefore derived an equation expressing relative activities of chemicals to fish or 

other organisms. This equation defines potency rating.

The potency rating effectively separates 

toxic from nontoxic chemicals. It also indicates the degree of general toxicity and the 

differential toxicity among species tested. 

The values obtained are compared with a 

maximum potency rating calculated by assuming complete mortality at all concentrations 

and time intervals.

Preliminary screening yields data on numbers of organisms killed at selected concentrations in multiples of 10 that are easily 

analyzed by comparing their potency ratings. 

Mortality is dependent upon the concentration 

and exposure time. Potency rating (Pr) is 

dependent on the mortality at each concentration and each exposure time. In addition, 

the number of tests must be considered, since 

Pr is the accumulation of values throughout 

a bioassay period. The relation of Pr to the 

variables involved is suggested by formula 1 

where mortality is a function of concentration 

and time (table 1). In essence, the mortality 

(M) is multiplied by the reciprocal of the concentration (C) times the reciprocal of the log 

of time (T) at each concentration and time

interval. The sum of these factors is divided 

by the log of the number of observations (N). 

The arrangement of the values used in deriving this index of activity does not follow any 

particular mathematical relations, and the 

values do not express units of measurement. 

The Pr merely indicates the activity of one 

test substance in comparison with others.

Reciprocals for concentration and the log 

of time are necessary to emphasize the 

effects of lower concentrations and shorter 

exposure periods (figs. 1 and 2). If the reciprocal relations are neglected, the accumulated 

potency rating increases drastically in longer 

exposures even though none of the organisms 

die during this period, and the effects of 

higher concentrations are weighted more 

heavily. The effects of the reciprocals are 

best seen in table 2, where maximum potencies (Pmax) are calculated for five time 

intervals and three concentrations. I have assumed that 10 organisms die among 10 tested 

in each case. The contributions of potency 

ratings are listed at each time interval under 

each concentration of 0.1, 1.0, and 10 ppm. 

Pmax contribution at 3 hours at 0.1 ppm is 

209.60, whereas that for 96 hours at 0.1 

ppm is 50.45, and subsequent contributions at 

higher concentrations are less by factors 0.1 

and 0.01. Therefore, chemicals which are

TABLE 1. Formulas for potency rating

j = 1 i = 1

Where M = mortality among the total number tested 

C = concentration

T = time of exposures in hours, T > 1 

N = number of observations, N > 1

j = finite variables of T: j^ = 3, j 2 = 6, J 3 = 24, j 4 = 48, j 5 = 96 

i = finite variables of C: ±± = 0.1, i 2 = 1.0, 13 = ID

[

,M x 1 x 1 > ,M x .1 x 1 .. ,M x _1 x 1 v 1 

* 0.1 log Tj' k 1.0 log Tj' ^ ID log Tj'J

When the individual concentrations are considered, formula 1 expands to: 

5

(2) Pr = / .

3=1______________________________________

log N 

When the observation times are considered also, the formula expands further to:

log N

x 1 

log 96
Leif L. Marking: Rating Chemicals for Potency Against Fish

ouu 

500

§400

Of t 

Z

0 300

Z

o 

o

\

\

\

\

\

\

TABLE 2. Maximum potencies at three concentrations and five time intervals

200

100

\

\

0.1 IjO 

CONCENTRATION

10

Figure l.~-The potency rating is larger at lower 

concentrations through the reciprocal relationship.

300

250

200

150

a. 

£

100

50

V

24 48 72 

EXPOSURE HOURS

96

Figure 2. The potency rating is larger at shorter 

exposure periods through the reciprocal relationship.

3 hours .

6 hours .

24 hours .

48 hours .

96 hours .

Total....

0.4771

0.7782

1.3802

1.6812

1.9823

--

log T

2.0960

1.2850

0.7245

0.5948

0.5045

--

3

6

9

12

15

--

0.4771

0.7782

0.9542

1.0792

1.1761

--

Concentration (

0.1 1.0

209.60 20.96

128.50 12.85

72.45 7.25

59.48 5.95

50.45 5.04

520.48 52.05

10.0

2.10

1.29

0.72

0.59

0.50

5.20

Sum TT_ at T faa

232.66 487.65

142.64 482.27

80.42 467.11

66.02 483.45

55.99 491.23

577.73

highly toxic in short exposure periods indicate greater potency than chemicals which are 

effective only in longer exposures. Chemicals 

which are toxic only at higher concentrations 

will definitely yield lower potency ratings 

regardless of exposure time. The values at 

1.0 and 10 ppm are rounded off to two decimal 

places.

The divisor in the formula, log N, accounts 

for the number of observations being analyzed. 

This arbitrary factor also reduces the additive 

effect of potency once the fish are recorded 

dead. Pmax in table 2 indicates little change 

at any time interval, and data from 24- or 

48-hour tests could be compared with data 

from 96-hour tests. The maximum potency in 

a 96-hour bioassay is 491.23; thus it becomes 

the standard for comparison of the calculated 

experimental potencies. If the potencies were 

merely^ summed at the various time intervals, the maximum potency would be 577.73 

(table 2), but these effects are cumulative, 

and the sum depends on the number of time 

intervals in the testing program. This cumulative potency increases even though no more 

fish die.

The size of the number describing Pr and 

the number of digits the Pr contains adequately indicate the toxicity with respect to 

time. Since Pmax is 467.11 to 491.23 depending on T, and the contribution at 0.1 ppm is 

a 3-digit whole number, I have assumed that 

3-digit whole-number Pr's indicate mortality 

at 0.1 ppm. Corresponding, 2-digit whole 

numbers indicate mortality at 1.0 ppm (maximum contribution = 52.05) and values less 

than 10 indicate mortality at 10 ppm (maximum 

contribution = 5.20). If none of the specimens 

die at the highest concentration, the value 

becomes zero. The Pr values can vary from
Investigations in Fish Control 36: Bureau of Sport Fisheries and Wildlife

0 to 491.23, and their proportion of the maximum potency with respect to T defines the 

relative activity. Values greater than 300 indicate mortality of 10 organisms at 0.1 ppm in 

6 hours or less.

The virtues of the potency rating concept 

are more comprehensible through example. 

Table 3 lists results of a preliminary fish 

bioassay and defines the individual potencies 

at each concentration and time interval. The 

value at 1.0 ppm is found by using the part of 

formula 2 pertaining to 1.0 ppm and 3 hours 

exposure time, or the value may be obtained 

from the appendix.

Pr = M x -- x 

1.0 log T

= 5x 1

0.4771 = 10.48

The sum of the individual values, 284.46, 

is divided by log N (1.1761) according to formula 3, and the Pr of this chemical to fish is 

241.87. The Pr of 241.87 is then compared 

with Pmax of 491.23 in table 2, and since this 

3-digit whole number is a large proportion of 

Pmax but not 300, mortality was assumed to 

have occurred at 0.1 ppm in exposures less 

than 24 hours.

If the chemical produced no mortality at 

0.1 ppm, Pr becomes the sum of potencies at 

1.0 and 10 ppm divided by log N, or 45.07/ 

1.1761, which equals 38.32. This 2-digit 

whole-number Pr indicates mortality at 

1.0 ppm in the shorter exposures but not 

complete mortality in 3 hours, since the 

maximum in this case is 57.25/1.1761 or 

48.68. The 2-digit whole-number value also 

indicates no mortality at 0.1 ppm.

TABLE 3. Potency rating determined from mortality among 

10 fish

Exposure

3 hours.

6 hours .

24 hours.

48 hours.

96 hours.

Mortality at 

concentrations 

(ppm) of 

0.1

0

5

9

10

10

1.0

5

9

10

10

10

10.0

Contributions 

to Pr at 

concentrations 

(ppm) of--

0.1

8 0.00

10 64.25

10 65.21

10 59.48

10 50.45

1.0

10.48

11.57

7.25

5.95

5.04

10.0

Sum

1.68 12.16

1.29 77.11

0.72 73.18

0.59 66.02

0.50 55.99

Since the concentrations are in multiples of 

10, the contribution to Pr can be calculated 

for one concentration, such as 0.1 ppm, for 

total mortality among the 10 fish. These figures can then be used for the contributions to 

Pr at 1.0 and 10 ppm by multiplying them by 

0.1 and 0.01 and rounding off to two decimals. 

Once the values for mortalities are established, the procedure becomes simple and 

efficient.

DISCUSSION AND CONCLUSIONS

The potency rating concept is more applicable to data derived from toxicants similar 

in nature or ones with similar modes of 

action. The concept is therefore limited to 

materials producing dose-effect curves similar in slope. Chemicals which produce rapid 

effects cannot be compared accurately with 

ones which require longer time for results.

The potency rating concept is especially 

applicable to data such as those reported by 

Walker, Starkey, and Marking (1966) and 

Marking and Willford (1970). The compounds 

in these investigations are related to structure and activity against fish and produce 

similar slopes on dose-effect curves.

The formula and concept are general and 

can be used to include different concentrations 

and time exposures depending on the program. 

In each case, the maximum potency must be 

calculated and the Pr values compared with it. 

If larger or smaller figures are sought, the 

values can be multiplied by a constant to 

yield the desired number of digits.

Pmax is uniform at all exposure times 

(table 2), but Pr changes to some extent with 

exposure time. Pr increases with exposure 

time when the mortality is delayed, but the 

increase is much less than the additive 

effects of the potencies at the different exposures. If mortality occurs at 3 hours, the Pr 

values approximate more closely the uniformity of Pmax found in table 2.

Equal numbers of test animals must be used 

in trials in order to compare Pr values with 

a single maximum potency. Data containing
Leif L. Marking: Rating Chemicals for Potency Against Fish 7

different numbers of test animals must be 

grouped and compared with a maximum 

potency for each group.

SUMMARY

Potency ratings effectively estimate the 

toxicity of chemicals to organisms from preliminary data which otherwise are often considered inadequate. The concept is especially 

useful in comparing results of numerous 

toxins tested against a wide variety of organisms. The values derived indicate the general 

and differential toxicity to the species tested.

The formula is versatile in that different 

concentrations and exposures can be selected 

to better fit a specific testing program. Mortality is a function of concentration and time, 

and the potency rating does not change in 

relation to the maximum potency if different 

concentrations or time intervals are selected. 

Data from 24- or 48-hour tests can be compared with those from 96-hour tests since 

the maximum potency is approximately equal 

for the three exposure times.

Shorter exposures and lower concentrations 

are emphasized in the formula by reciprocating the concentrations and the log of the time 

exposures. The effect is greater Pr's for 

highly toxic materials which kill organisms 

rapidly.

The potency rating concept is more applicable to data obtained from chemicals producing similar dose-effect curves. Chemicals 

of similar structure and chemicals which

produce similar response to organisms can 

be readily evaluated and compared.

REFERENCES

Fingl, Edward, and Dixon M. Woodbury.

1965. General Principles. In The pharmacological 

basis of therapeutics, edited by Louis S. Goodman 

and Alfred Oilman, p. 1-36, Macmillan Company, 

New York.

Finney, D. J.

1962. Probit analysis. A statistical treatment of 

the sigmoid response curve. Second edition. 

University Press, Cambridge. 318 p.

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 (Bureau Circular 185). 15 p.

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

1949. A simplified method of evaluating doseeffect experiments. Journal of Pharmacology 

and Experiment Therapeutics, vol. 96, no. 2, 

p. 99-113.

Marking, Leif L., and Wayne A. Willf ord. 

1970. Comparative toxicity of 29 nitrosalicylanilides and related compounds to eight 

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

and Wildlife, Investigations in Fish Control, 

No. 37. 11 p.

Walker, Charles R., Roland J. Starkey, and Leif L. 

Marking.

1966. Relation of chemical structure to fish 

toxicity in nitrosalicylanilides and related compounds. U.S. Bureau of Sport Fisheries and 

Wildlife, Investigations in Fish Control, No. 9. 

12 p.
Pr contributions at 0.1, 1.0, and 10.0 ppm for 10 test organisms

Exposure 

and 

concentration

3 hours: 

0.1 ppm. ......

1.0 ppm. ......

10.0 ppm.......

6 hours:

1.0 ppm. ......

10.0 ppm.......

24 hours: 

0.1 ppm. ......

10.0 ppm.......

48 hours: 

0 . 1 ppm .......

1.0 ppm. ......

10.0 ppm.......

96 hours: 

0 . 1 ppm .......

1 . 0 ppm .......

10.0 ppm.......

1

20.96 

2.10 

0.21

12.85 

1.29 

0.13

7.25 

0.73 

0.07

5.95 

0.60 

0.06

5.05 

0.51 

0.05

2

42.92 

4.29 

0.43

25.70 

2.57 

0.26

14.49 

1.45 

0.15

11.90 

1.19 

0.12

10.09 

1.01 

0.10

Pr

3

62.88 

6.29 

0.63

38.55 

3.86 

0.39

21.74 

2.17 

0.22

17.84 

1.78 

0.18

15.14 

1.51 

0.15

contributions calculated from

4

83.84 

8.38 

0.84

51.40 

5.14 

0.51

28.98 

2.90 

0.29

23.79 

2.38 

0.24

20.18 

2.02 

0.20

5

104.80 

10.48 

1.05

64.25 

6.43 

0.64

36.23 

3.62 

0.36

29.74 

2.97 

0.30

25.23 

2.52 

0.25

6

125.76 

12.58 

1.26

77.10 

7.71 

0.77

43.47 

4.35 

0.44

35.69 

3.57 

0.36

30.27 

3.03 

0.30

death of 

7

146.72 

14.67 

1.47

89.95 

9.00 

0.90

50.72 

5.07 

0.51

41.64 

4.16 

0.42

35.32 

3.53 

0.35

8

167.68 

16.77 

1.68

102.80 

10.28 

1.03

57.96 

5.80 

0.58

47.58 

4.76 

0.48

40.36 

4.04 

0.40
INVESTIGATIONS IN FISH CONTROL

37. Comparative Toxicity of 

29 Nitrosalicylanilides 

and Related Compounds 

to Eight Species of Fish

By Leif L. Marking and Wayne A. Willford

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

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

Fish and Wildlife Service, Charles H. Meacham, Commissioner

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

Washington, D.C. July 1970
CONTENTS

Page

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

Materials and methods .............................. 4

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

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

Conclusions and summary ............................ 8

References ...................................... 9

Appendix. ....................................... 10
COMPARATIVE TOXICITY OF 29 NITROSALICYLANILIDES 

AND RELATED COMPOUNDS TO EIGHT SPECIES OF FISH

By Leif L. Marking and Wayne A. Willford, Chemists

Bureau of Sport Fisheries and Wildlife 

Fish Control Laboratory, La Crosse, Wisconsin

ABSTRACT. The relative potencies of 29 nitrosalicylanilides and 

related structures against rainbow trout, goldfish, carp, fathead minnows, black bullheads, green sunfish, bluegills, and yellow perch were 

determined in 96-hour static bioassays. They varied from zero to the 

maximum potency of the system, depending on the type and position 

of substitutions. The 4'- substitutions on 3-nitrosalicylanilide are 

essential in producing high toxicity and selectivity to fish. The toxic 

activity of nitrosalicylanilides increases as halogen substitutions 

are shifted from the 2' to the 3* and to the 4' positions on the aniline 

moiety. Activity increases as molecular weight of the substituent 

increases. 4'-azophenyl-3-nitrosalicylanilide and 4'-iodo-3-nitrosalicylanilide are more toxic than 4'-bromo- or 4'-chloro-3-nitrosalicylanilide. The dihalo-substituted 3-nitrosalicylanilides are 

more toxic than monohalo-substituted 3-nitrosalicylanilides provided one of the halo- substitutions is at the 4 ! - position on the 

aniline moiety. 4'-bromo-3-nitrosalicylanilide is more toxic to 

carp, fathead minnows, bluegills, and yellow perch than to rainbow 

trout. Several compounds are selective to yellow perch. Goldfish 

are the most resistant species to the salicylanilides.

The nitrosalicylanilides are among the 

more biologically active chemicals and are 

under intensive investigation by scientists in 

many fields. Gb'nnert (1962) and Schraufstatter 

(1962) defined the molluscicidal activity of 

salicylanilides and reported reduction of 

Schistosomiasis through control of the vector 

snail. Vinson, Dineen, and Schneider (1961) 

point out that salicylanilides are efficacious 

as hospital detergent sanitizers. Molnar and 

Baron (1964) stated that certain polybrominated salicylanilides possess the desirable 

properties of germicides to the maximum 

extent. Molnar (1965) reported synergistic 

activity against bacteria when two polybrominated salicylanilides were combined.

The salicylanilide structure is also of 

interest in sea lamprey control. Bayluscide, 

chemically known as 2',5-dichloro-4f-nitrosalicylanilide and better known as Bayer 73, 

is presently used to synergize 3-trifluoromethyl-4-nitrophenol (TFM) (Howell et al., 

1964). Bayer 73 is highly toxic to fish, and 

Marking and Hogan (1967) reported 96-hour 

LCSO's of 0.043 to 0.230 ppm. They found that 

the toxicity of the compound is reduced in 

waters of extremely high or low pH, but it is 

not reduced or enhanced greatly by water 

temperatures between 7° and 22° c.

Starkey and Howell (1965) investigated the 

activity of a number of salicylanilides and
Investigations in Fish Control 37: Bureau of Sport Fisheries and Wildlife

related compounds against rainbow trout and 

larval sea lampreys and reported greater 

toxicity to larval lampreys in many instances.

The biological activity of salicylanilides 

may be enhanced or diminished by specific 

substitution on the molecule. Taborsky, 

Darker, and Kaye (1959) and Taborsky and 

Starkey (1962, 1963) demonstrated various 

antimicrobial activities of salicylanilides with 

selected halogens at certain positions.

Walker, Starkey, and Marking (1966) showed 

that the piscicidal activity of salicylanilides 

and related compounds is governed by the 

nature and location of substitutions on the 

molecule. The halo- substitutions on the nitrosalicylanilides, in particular, had greater 

influence on toxicities to fish than other 

substituents.

Twenty-nine nitrosalicylanilides and related 

structures, many having halo- substitutions at 

selected positions, were obtained for preliminary bioassays against eight species of 

fish. These tests, conducted at the Fish 

Control Laboratories at La Crosse, Wis. and 

Warm Springs, Ga., in facilities described by 

Lennon and Walker (1964), provide comparative data on the toxicity of the chemicals to 

selected freshwater fish.

MATERIALS AND METHODS

The nitrosalicylanilides and related structures were furnished by Ben Venue Laboratories, Bedford, Ohio. Each structure was 

chosen according to its toxicity determined 

previously on rainbow trout and goldfish. The 

general structure of the compounds is illustrated in figure 1.

The fish were obtained from National Fish 

Hatcheries (table 1). They were introduced to 

the bioassay following a routine procedure of 

10-day quarantine, removal from feed, and 

acclimation to water chemistry and temperature as outlined by Lennon and Walker (1964).

The tests were conducted in 15 liters of 

standard bioassay medium at 12° C. at 

La Crosse and at 17° C. at Warm Springs.

OH

Figure 1. Basic structure of salicylanilides. Benzanilides lack the OH ~ at the 2 position.

TABLE 1. Sizes and sources of test fishes

Species and lot

Rainbow trout ( Salmo gairdneri) :

Goldfish ( Carassius auratus) :

Carp ( Cyprinus carpio) :

Fathead minnow (Pimephales 

promelas) :

Black "bullhead (Ictalurus melas):

Green sunfish (Lepomis cyanellus) :

Bluegill (Lepomis macrochirus) :

Lot W84

Yellow perch ( Perca flavescens) :

Lot 267.........................

Average 

length 

( inches)

2.0 

1.2 

1.9 

1.9

1.6 

1.7

1.3 

1.5 

1.7 

1.6

1.6 

1.8

2.0 

1.3 

2.1 

2.2

1.4 

1.7 

1.5 

1.5

1.5 

1.2 

1.5

2.2 

2.3

Average 

weight 

(grams)

1.2 

0.2 

1.1 

1.1

2.4 

1.1 

2.3

0.7 

0.6 

1.0 

0.8

0.7 

1.0

1.6

0.5 

2.0 

2.2

0.8 

1.7 

1.1 

0.8

0.9 

0.4 

0.9

1.6 

1.9

Source

Manchester NFH, Iowa 

La Crosse FCL, Wis. 

Manchester NFH, Iowa 

Do.

Lake Mills NFH, Wis. 

Marion NFH, Ala. 

Lake Mills NFH, Wis.

Marion NFH, Ala. 

Do. 

Lake Mills NFH, Wis. 

Do.

Do. 

Do.

Necedah NWR, Wis. 

Genoa NFH, Wis. 

Guttenberg NFH, Iowa 

Do.

Lake Mills NFH, Wis. 

Do. 

Do. 

Do.

Marion NFH, Ala. 

Lake Mills NFH, Wis. 

Do.

Do. 

Do.

Ten specimens were exposed to each concentration of 0.1, 1.0, and 10.0 ppm of the 

candidate compounds. A stock solution of 

each compound, using acetone as the solvent, 

was prepared immediately before each test. 

Test concentrations of 0.1, 1.0, and 10.0 mg/Z 

were obtained by adding an appropriate aliquot of a stock to 15 liters of reconstituted 

water. Observations on the effects of the 

compounds were recorded at 15 and 30 minutes and at 1, 3, 24, 48, 72, and 96 hours.

The data were analyzed according to the 

method of Marking (1970) in which the potency 

rating (Pr) is defined for each chemical to
Marking and Willford; Toxicity of 29 Nitrosalicylanilides to Fish

each species of fish. The values derived indicate the toxicity as a proportion of the predetermined maximum potency with respect to 

concentrations and observation periods. Since 

the observations were taken at 3, 24, 48 and 

96 hours in these experiments, the Pr is found 

as follows:

TABLE 2. Maximum potency at four time intervals

48,96

Pr =

1.0,10.0

L log

j = 3,24 i = 0.1,

logN

where M = mortality among 10 fish. 

C = concentration in ppm. 

T = time of exposure in hours, T>1. 

N = number of observations, N >1. 

j = finite variables of T. 

i = finite variables of C.

When the individual concentrations of 0.1, 

1.0, and 10.0 ppm at each observation period 

are considered, the formula becomes

M x 1 x 1

Pr =

logN

M x J._ x 1 _ _ _ M x 1 x 1

* i f^ i ~ *>' ' "* "* \ -i f\ i___ r\£.i 10 log V 10 log 96'

logN

Pmax is obtained by assuming that all of 

the fish died at each concentration and observation interval, and the values are given in 

table 2. Pmax is 403.16 in a 96-hour bioassay, and Pr values for experimental chemicals are compared with it. The number of 

digits in the Pr and size of the number effectively define the potency. Three-digit whole 

numbers indicate mortality at 0.1 ppm. Threedigit whole numbers over 200 indicate mortality at 0.1 ppm in 3 hours, and as Pr approaches the value of Pmax a greater number 

of fish die at this concentration and time 

interval.

Two-digit whole numbers indicate mortality at 1.0 ppm, and 1-digit whole numbers

Time log T

1

N log N

At concentration

(I

0.1

3 hours 0.4771 2.0960 3 0.4771 209.60

24 hours 1.3802 0.7245 6 0.7782 72.45

48 hours 1.6812 0.5948 9 0.9542 59.48

96 hours 1.9823 0.5045 12 1.0792 50.45

)pm) of 

1.0

20.96

7.25

5.95

5.04

ID

Sum Pmax

2.10 232.66 487.65

0.72 80.42 402.31

0.59 66.02 397.30

0.50 55.99 403.16

indicate mortality at 10 ppm. If none of the 

fish die at any concentration and time interval, Pr becomes zero.

RESULTS

The behavioral response of fish on contact 

with the nitrosalicylanilides is immediate at 

10 ppm, and with some at 1.0 ppm. The response usually involves irritation, which is 

revealed by intermittent opercular flipping 

that simulates a coughing action. Swimming 

becomes irregular and is characterized by 

surfacing and diving. Loss of equilibrium 

and orientation are usually followed by a 

period of slow movement and quiescence. 

The opercular flipping becomes infrequent 

just before death.

The gill covers of dead fish are often distended, and a proliferation of mucuous is 

noticeable about the gill area. In some cases, 

long strings of mucous were observed in the 

anal area and on the ends of the ventral fins. 

Microscopic examination indicates that the 

bodies of the dead fish are devoid of mucous. 

Hemorrhage of the gills occurs frequently at 

the higher concentrations.

The iodo- substitutions on the nitrosalicylanilide structure (table 3, compounds 

1, 2, 3, and 4) are toxic to all the species 

tested. The degree of toxicity is progressively 

enhanced for most species by moving the 

iodo- from the 2' to the 3', and from the 3 1 to 

the 4' positions. Also, the rate of toxic action 

is progressive. Survival is almost complete 

at 1.0 ppm in 3 hours with the 2'-iodo- substitution, whereas complete mortality occurs 

at this concentration and exposure with the 

4f-iodo- substitution. By shifting the nitrogroup from the 3 position, as in compounds 

1 to 3, to the 5 position (compound 4) the 

general activity decreases.
Investigations in Fish Control 37: Bureau of Sport Fisheries and Wildlife

TABLE 3. The potency rating of 29 chemicals to fish in bioassays at 12° C. 

[Rating calculated from data in the appendix]

Chemical

(1) 2'-iodo-3-nitrosalieylanilide. . . 

(2) 3'-iodo-3-nitrosalicylanilide. . . 

(3) 4'-iodo-3-nitrosalieylanilide. . . 

(4) 4'-iodo-5- nitrosalieylanilide. . . 

(5) 2'-bromo-3-nitrosalieylaiiilide. . 

(6) 3 ' -bromo-3-nitrosalicylanilide . . 

(7) 4'-bromo-3-nitrosalieylanilide. . 

(8) 4',5-dibromo-3-nitrosalieyl-

( 9 ) 4 ' -bromo -2 ' -methyl-3 -nitro -

(10) 2 ' ,5-dibromo-3-nitrosalieyl-

(11) 4 ' -chloro-3 -nitrosalieylanilide . 

(12) 2 ' -ehloro-4 ' -me thy 1-3 -nitro -

(13) 4 '-chloro-2 1 -methyl-3 -nitro-

(14) 2 ' -ehloro-4 ' -nitro -3 -nitro -

(15) 5 ' -chloro-2 ' -methoxy-3 -nitro-

(16) 2 ' ,5'-dimethoxy-4 ' -ehloro-3-

(17) 2 ' -chloro-5 ' -trif luoromethyl-3 -

(18) 3 ' ,4 ' -diehloro-3-nitrosalieyl-

(19) 4 ' -chloro -5 -bromo -3 -nitro -

(20) 2 ' -chloro-5 -nitrosalieylanilide . 

(21) 3'-chloro-5-nitrosalicylanilide. 

(22) 4' -ehloro-5-nitrosalieylanilide. 

(23) 2 ' -methoxy-4 ' -nitro-3-nitro-

(24) 4 ' -methoxy-2 ' -nitro-3-nitro-

(25) 2 ' ,4 ' -dimethyl-3 -nitrosalieylanilide. ........................

(26) 4 '-azophenyl -3 -nitrosalieylanilide. ........................

(27) 3' -ehloro-3-nitrobenzanilide. . . .

(28) 2',6'-diethyl-3,5-dinitrobenzanilide. ........................

(29) 5-bromo-3-nitrosalieylie acid...

Rainbow 

trout

39.95 

39.37 

241.08 

21.80 

39.95 

39.95 

39.37

208.94

67.03

39.95

48.18 

53.11

39.37

39.37

56.85

39.37

175.14

53.11

39.95 

15.94 

21.80

7.01

20.52

39.95

228.36

7.47

0 

39.95

Goldfish

1.68 

19.61 

39.95 

19.84 

2.26 

10.22 

20.52

38.30

26.19

6.07

41.53 

53.11

19.61

67.03

52.37

39.95

£+£.*£.£.

2.84 

1.68 

20.60

-\ £.3

-> rt -i r

3.62

39.37

3.62

0 

20.52

Carp

22.47 

4.09 

208.94 

19.18 

20.52 

38.00 

403.16

/*r7 or;

190.00

19.85

44.30

 a cJ r\c\

133.39

37.33

r4. 7«i

175. 1/4

jo.UO

173.27 

11.40 

10.67 

15.26

5

rtr

4.56

19.85

176.58

5.30

0 

20.52

Fathead 

minnow

20.52 

26.35 

208.94 

20.52 

39.95 

39.95 

128.51

186.69

85.46

36.06

104.90 

39.95

45.41

r rt *\O

r)f oc

26.35

165.20

148.53

16.94

20.52 

6.60

15.25

15.39

208.94

3.62

1.01 

20.52

Black 

bullhead

20.52 

39.95 

208.94 

20.52 

20.52 

39.95 

87.26

-i ccj co

39.95

28.29

58.65 

90.65

39.95

cl fjn

20.52

168.66

20o . y4

s O o 1

17.84 

3.62 

32.65

3

y *a

20.52

182.09

3.23

0 

20.52

Green 

sunfish

6.79 

20.52 

112.82 

13.81 

6.33 

13.73 

39.95

Cjo CJ/-1

110.18

26.35

44.62 

ocj nn

19.85

20.52

17.53

JO.UU

^\J £.*+ 

-i -i s rtr;

18.77 

5.02 

20.52

3.35

/, /, /,

4.56

137.50

3.62

0 

27.14

Bluegill

7.01 

20.52 

202.23 

20.52 

9.65 

20.52 

99.87

-\ /-Q OCJ

25. 19

8.40

31.82 

39.95

20.20

25.19

29.87

114.49

W9.72

152.79 

3.62 

3.62 

20.52

3

/-VCJ

3.50

4.56

208.94

3.43

0 

85.48

Yellow 

perch

20.52 

73.75 

247.79 

92.23 

20.52 

56.85 

228.36

208 94

202.23

39.95

73.75 

235.56

175.14

52.97

 3O -» fj

95.70

127.92

209.42

OOEJ *3£L

11.10 

19.18 

46.25

7

r\s

22.47

208.94

5.30

0 

205.06

The 2 1 -, 3'-, and 4'-bromo- substitutions 

on the 3-nitrosalicylanilide structures (compounds 5, 6, and 7) are slightly less toxic than 

the equivalent iodo- substitutions with the 

exception of compound 7 to carp. Of the 

mono-bromo- substitutions, 4t-bromo-3-nitrosalicylanilide is the most toxic, killing all

species at 1.0 ppm and all carp at 0.1 ppm in 

3 hours. The Pr of this chemical to carp is 

403.16 and equals the Pmax of the system. 

Its Pr values suggest specificity for carp. 

All carp and yellow perch and 20 percent of 

the fathead minnows were killed at 0.1 ppm 

within 24 hours, but no mortality occurred
Marking and Willford: Toxicity of 29 Nitrosalicylanilides to Fish

among the other species. At 96 hours, 90 percent of the fathead minnows and bluegills and 

70 percent of the black bullheads died.

In most instances, the toxicity of the 

4'-bromo- substitution is further enhanced 

by addition of a second bromo- on the acid 

portion of the molecule at the 5 position 

(compound 8). This congener produced 

100-percent mortality in all species at 

0.1 ppm within 96 hours. The rate of toxic 

action, however, decreases. This is evidenced 

by less mortality of fish at 1.0 ppm after 

3 hours, and longer survival of carp at 

0.1 ppm than with the mono- 4'-bromosubstitution (appendix).

Substitution of a methyl group in 2' position on 4'-bromo-3-nitrosalicylanilide does 

not increase the activity of the compound 

(compound 9). All carp and yellow perch, 

and 60 percent of the green sunfish died when 

exposed to 0.1 ppm for 96 hours (appendix). 

The 2',5'-dibromo- substitution (compound 10) 

produces approximately the same toxicity as 

the mono-, 2'-bromo- substitution (compound 5).

Corresponding chloro- substitutions on 

the 3-nitrosalicylanilide are usually less 

toxic than the bromo- or iodo- substitutions 

(compounds 11-19). This is illustrated with 

4'- substitutions of the three halogens. Toxicity increases as the substitution is changed 

from chloro- to bromo- to iodo- (compounds 

3, 7, and 11).

Methyl-, nitro-, methoxy-, or dimethoxysubstitutions on mono-chloro-3-nitrosalicylanilide fail to exhibit outstanding toxicity (compounds 11-16). However, the 2'- 

chloro-4'-methyl and 4'-chloro-2'-methyl 

structures are relatively toxic to yellow 

perch (compounds 12 and 13) and may have 

potential as selective toxicants. The Pr of 

cpd 12 is 235.56 and is 2 to 6 times as great 

as the Pr values for other species.

The S'-trifluoromethyl substitution (compound 17) on 2'-chloro-3-nitrosalicylanilide 

is more toxic than mono-substituted 4'- 

chloro-3-nitrosalicylanilide to six of the 

eight species.

The 3'-chloro- or 5-bromo- substitutions 

(compounds 18 and 19) on 4'-chloro-3-nitrosalicylanilide also enhance the general toxicity of the molecule. The dichloro- substitution, 

however, failed to kill carp or green sunfish 

at 0.1 ppm, while the bromo- substitution 

killed all carp and green sunfish at 0.1 ppm 

in 96 hours. Both compounds killed all of the 

fathead minnows, bluegills, and yellow perch 

at 0.1 ppm in 96 hours (appendix).

The chloro-5-nitrosalicylanilides (compounds 20-22) are less toxic than the chloro3-nitrosalicylanilides. Activity increases by 

relocating the chloro- from the 2' or the 3' to 

the 4' positions in much the same manner as 

demonstrated by the iodo- and bromo-3- 

nitrosalicylanilides.

Nonhalo- substitutions on the 3-nitrosalicylanilide produce variable results. The 

2'-methoxy-4'-nitro- substitution (compound 

23) appears less toxic than the 4'-methoxy-2 tnitro- and the 2',4'-dimethyl- substitutions 

(compounds 24 and 25). None of these are as 

toxic as mono- 4'-halo- substitutions.

Substitution of the larger azophenyl- radical at the 4' position (compound 26) produces 

greater toxicity than any of the nonhalo-, and 

in most cases greater than the halo- substitutions. All fish tested at 0.1 ppm died within 

96 hours (appendix).

The 3'-chloro-3-nitrobenzanilide (compound 

27) is far less toxic than the halo- substitutions on 3- or 5-nitrosalicylanilides. The 3'- 

chloro-3-nitrobenzanilide produces little 

mortality of any species at 1.0 ppm in 96 

hours, and Pr values range from 3.23 to 7.47 

for all species.

The toxicity of 2 t ,6'-diethyl-3,5-dinitrobenzanilide (compound 28) is considerably 

less than that of 3'-chloro-3-nitrobenzanilide. 

This compound, containing multiple substitutions, fails to kill any species at 10.0 ppm 

in 96 hours, and consequently the Pr values 

are zero. One fathead minnow died unnaturally at 1.0 ppm in 48 hours, and this produces a Pr of 1.01 at 96 hours.
8 Investigations in Fish Control 37: Bureau of Sport Fisheries and Wildlife

The 5-bromo-3-nitrosalicylic acid (compound 29), although composed of only a portion of the nitrosalicylanilide molecule, contains the vital hydroxy- in the 2 position and 

the nitro- in the 3 position. Its toxicity is 

approximately equal to or greater than that of 

the 2' and 3' bromo- or iodo- on 3-nitrosalicylanilides (compounds 1, 2, 5, and 6) 

but less than that of the 4' bromo- or iodo- 

(compounds 3 and 7).

DISCUSSION

The highly toxic nitrosalicylanilides are 

active in shorter periods than the tables 

indicate. In 15 to 30 minutes, 10 ppm of the 

4 l-halo-3-nitrosalicylanilides kill rainbow 

trout, fathead minnows, and yellow perch. 

The more potent 4'-azophenyl-3-nitrosalicylanilide kills all species tested including 

the relatively resistant carp, black bullheads, 

green sunfish, and bluegills in 30 minutes at 

10 ppm. Several of these species are killed in 

one hour at 1.0 ppm. All of the nitrosalicylanilides at 10 ppm produce stress in trout 

and yellow perch within 15 minutes.

We found that the activity of the halo- substitutions increases as the atomic weight of 

the substituted halogen increases. This agrees 

with the results of Walker, Starkey, and Marking (1966). However, this order of activity 

does not correspond to that for fungi and bacteria. Tabor sky and Starkey (1962) reported 

that 4'-chloro- and 4t-bromo- substitutions on 

the 3- and 5-nitrosalicylanilides are among 

the more active antimicrobials. They also 

observed that 4'-bromo-5-nitrosalicylanilide was a more potent antimicrobial than 

4'-chloro-3-nitrosalicylanilide. Our data 

indicate that the 3-nitrosalicylanilides are 

more toxic to fish than 5-nitrosalicylanilides. 

The inverse antimicrobial activity of these 

compounds suggests their possible use in 

treating fish diseases of bacterial and fungal 

origin.

The 4'- position appears to be a key position 

on the molecule. The halo- substitutions at 

this position are toxic to all species tested. 

The azophenyl- substitution in this position

enhances the compound even further and suggests that bulkier unsaturated substitutions 

may increase the activity.

The potency ratings indicate that several 

chemicals are selective to target species of 

fish. Compound 7 (4'-bromo-3-nitrosalicylanilide) appears more toxic to carp, fathead minnows, black bullheads, bluegills, 

and yellow perch than to rainbow trout. Compound 12 (2 ! -chloro-4f -methyl-3-nitrosalicylanilide) and compound 29 (5-bromo-3-nitrosalicylic acid) are considerably more toxic to 

yellow perch than to any of the other species 

tested.

CONCLUSIONS AND SUMMARY

Nitrosalicylanilides continue to be of interest in fish control. The versatility of the parent structure permits a wide variety of substitutions enabling a critical review of the 

structure-activity relationships. The potency 

ratings on the three chemicals tested against 

eight species of fish indicate desirable selective toxicities. Of the 29 chemicals, 28 were 

toxic to fish at concentrations of 10 ppm or 

less.

Nitrosalicylanilides are more toxic to 

the eight species of fish tested than nitrobenzanilides with respective substitutions. 

This may be attributed to the hydroxy- at the 

2 position on the benzole acid moiety of the 

nitrosalicylanilides. Also, the activity of 

nitrosalicylanilides increases as the nitrois shifted from the 5 to the 3 position.

The activity of a structure increases as the 

halogen is shifted from the 2' to the 3 1 and to 

the 4' positions on the aniline moiety. In addition, activity increases as the molecular 

weight of the substituent increases. Greater 

toxicity is observed with the heavier azophenyl- substitution, followed by iodo-, 

bromo-, and chloro- in that order. The activity of 4'-bromo-3-nitrosalicylanilide is 

enhanced further with the addition of another 

bromo-, providing that a bromo- remains at 

the 4 1 position.

The 5-bromo-3-nitrosalicylic acid produces toxicity which is approximately equal to
Marking and Willford: Toxicity of 29 Nitrosalicylanilides to Fish

or greater than that of 2 1 and 3 1 bromo- or 

iodo- substitutions on the 3-nitrosalicylanilide. 

This molecule, although smaller and simpler, 

contains the hydroxy- at the 2 position which 

is necessary to produce maximum biological 

activity.

The results indicate that 4' halo- and 

azophenyl- substitutions on 3-nitrosalicylanilide produce the greatest toxicity 

against fish. These compounds deserve further consideration as fish control agents.

The relative potency concept expedited the 

critical analysis of these preliminary data. 

It permitted rapid comparison to distinguish 

highly toxic from less toxic chemicals and 

aided in distinguishing selectivity.

REFERENCES

Gonnert, R.

1962. BayluscideW a new compound for controlling medically important freshwater snails. 

Bayer, A. E4, Pflanzenschutz-Nachrichten, 

vol. 15, no. 1, p. 4-24.

Howell, John H.t Everett L. King, Jr., Alien J. Smith, 

and Lee H. Hanson.

1964. Synergism of 5,2'-dichloro-4'-nitro-salicylanilide and 3-trifluormethyl-4-nitrophenol in a 

selective lamprey larvicide. Great Lakes Fishery Commission, Technical Report 8. 21 p.

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. (Bureau Circular 185). 15 p.

Marking, Leif L.

1970. A method for rating chemicals for potency 

against fish and other organisms. U.S. Bureau of 

Sport Fisheries and Wildlife, Investigations in 

Fish Control, No. 36. 8 p.

Marking, Leif L., and James W. Hogan.

1967. Toxicity of Bayer 73 to fish. U.S. Bureau of 

Sport Fisheries and Wildlife, Investigations in 

Fish Control, No. 19. 13 p.

Molnar, Nicholas M.

1965. New soap/detergent bacteriostat. Soap and 

Chemical Specialties, February. 6 p.

Molnar, N. M., and Seymour Baron.

1964. Two new stable polybrominated salicylanilides for antibacterial use in soap and detergent products. Journal of the American Oil 

Chemists' Society, vol. 41, no. 7, p. 478-480.

Schraufstatter, E.

1962. Chemical development of BayluscideW. 

Bayer, A. G., Pflanzenschutz-Nachrichten, 

vol. 15, no. 1, p. 25-33.

Starkey, Roland J., and John H. Howell.

1966. Substituted nitrosalicylanilides: A new class 

of selectively toxic sea lamprey larvicides. 

Great Lakes Fishery Commission, Technical 

Report 11, p. 21-29.

Taborsky, Robert G., Grant D. Darker, and Saul Kaye. 

1959. Substituted salicylanilides with antimicrobial activity. Journal of the American Pharmaceutical Association, vol. 48, no. 9, p. 503-507.

Taborsky, Robert G., and Roland J. Starkey.

1962. Substituted salicylanilides: II. Further antimicrobial and other studies withhalonitrosalicylanilides. Journal of Pharmaceutical Sciences, 

vol. 51, no. 12, p. 1152-1155.

1963. Substituted salicylanilides: III. New salicylanilides and related compounds with antimicrobial activity. Journal of Pharmaceutical 

Sciences, vol. 52, no. 6, p. 542-545.

Vinson, L. J., P. Dineen, and W. Schneider.

1961. Evaluation of four hospital detergent - 

sanitizers. Soap and Chemical Specialties, 

October and November. 11 p.

Walker, Charles R., Roland J. Starkey, and Leif L. 

Marking.

1966. Relation of chemical structure to fish toxicity in nitrosalicylanilides and related compounds. 

U.S. Bureau of Sport Fisheries and Wildlife, 

Investigations in Fish Control, No. 9. 12 p.
10 Investigations in Fish Control 37: Bureau of Sport Fisheries and Wildlife

APPENDIX

Toxicity of 0.1, 1.0, and ID ppm of experimental chemicals to fish at 12° C. 

[Numbers indicate mortalities among ID fish in each bioassay]

Chemical and exposure time

( 1) 2 ' - iodo-3-nitrosalicylanilide:

(2) 3' -iodo-3-nitrosalicylanilide:

(3) 4' -iodo-3-nitrosalicylanilide:

(4) 4'-iodo-5-nitrosalicylanilide:

(5) 2'-bromo-3-nitrosalicylanilide:

(6) 3'-bromo-3-nitrosalicylanilide:

(7) 4'-bromo-3-nitrosalicylanilide:

(8) 4'-bromo-5-bromo-3-nitrosalicylanilide:

(9) 4 ' -bromo-2 ' -methyl -3-nitrosalicylanilide:

(10) 2',5 I -dibromo-3-nitrosalicylanilide:

(11) 4 ' -chloro-3-nitrosalicylanilide:

(12) 2'-chloro-4'-methyl-3-nitrosalioylanilide:

(13) 4 1 -chloro-2'-methyl-3-nitrosalioylanilide:

(14) 2'-chloro-4' -nitro-3-nitrosalioylanilide:

Rainbow trout

O.ll 1.0

0 ID 

0 ID 

0 10 

0 10

0 10 

0 10 

0 10

2 ID 

9 10 

10 ID 

ID 10

0 2 

0 ID 

0 ID

0 10 

0 10 

0 10 

0 10

0 10 

0 ID 

0 ID 

0 10

0 ID 

0 10 

0 10

0 10 

10 ID 

ID 10 

10 10

0 ID 

2 ID 

2 ID

0 ID 

0 10 

0 10 

0 10

0 9

0 10 

1 10 

1 10

0 10 

1 10 

1 ID

0 ID 

0 ID 

0 10

0 10 

0 10 

0 ID

ID

10 

10

10 

10

10 

10 

10

ID 

ID 

ID 

10

ID 

10 

ID

ID 

ID

10 

10

10 

ID 

10 

ID

10 

ID 

10

10 

10 

10 

ID

ID 

ID 

ID

10 

10 

ID 

ID

ID 

ID 

ID 

10

ID 

ID 

10

ID 

ID 

ID

ID 

ID 

ID

Goldfish

0.1

0 

0 

0

0

0 

0 

0

0 

0 

0 

0

0 

0

1

0 

0 

0 

0

0 

0 

0 

0

0 

0 

0 

0

0 

0 

3

0 

0 

0

0 

0 

0 

0

0 

0 

0 

2

0

1 

1

0 

0 

0

0 

2 

2

1.0

0 

0 

0 

0

1

ID 

10

10 

ID 

ID 

ID

0 

5 

ID

0 

0 

0 

0

1 

1 

3 

5

0 

ID 

ID 

ID

1 

10 

ID

4 

ID

10

0 

2 

3 

3

6 

10 

10 

10

10 

ID 

10

1 

10 

10

0 

ID 

ID

ID

0 

10

10

10

ID 

ID

10

10 

ID 

ID 

ID

10 

ID 

ID

3

ID 

ID

10

10 

10 

10 

10

10 

ID 

10 

10

10 

10 

10

ID 

ID 

ID

0 

10 

10

10

10 

10 

ID 

10

10 

ID 

ID

ID 

ID 

ID

10 

ID 

10

Carp

0.1

0 

0 

0 

0

0 

0 

0 

0

0 

ID 

10 

ID

0 

0 

0 

0

0 

0 

0 

0

0 

0 

0 

0

10 

ID 

10 

10

0 

0 

0 

ID

0 

8 

9 

ID

0 

0 

0 

0

0 

0

1 

1

0 

0 

0 

0

0 

4 

6 

8

0 

0

1

2

1.0

I 

10 

10 

10

0 

0 

0

1

10 

10 

ID 

10

0 

8

10 

10

0 

10

10 

10

9 

10 

ID 

10

10 

10 

10 

ID

0 

ID

10 

ID

10 

10 

10 

ID

0 

9 

ID

10

7 

10 

10 

10

9 

10 

10 

10

8

ID 

10 

ID

1 

10 

10 

ID

ID

10 

ID 

ID 

10

10 

10 

10 

10

10 

10 

ID 

ID

10 

ID 

10 

ID

10 

10

10 

10

10 

10 

10 

10

10 

10 

10 

ID

10 

10 

10 

10

10 

10 

10 

ID

10 

ID 

ID 

10

10 

10 

ID 

10

10 

10 

10 

10

ID 

10 

10 

ID

10 

10 

10 

10

Fathead minnow

0.1

0 

0 

0 

0

0 

0 

0 

0

0 

ID

10 

10

0 

0 

0 

0

0 

0 

0 

0

0 

0 

0 

0

0 

2 

6 

9

0 

9 

ID

10

0

1

4 

4

0 

0 

0 

0

0 

0 

5 

8

0 

0 

0 

0

0 

0 

0 

2

0 

0 

2 

5

1.0 10

0 ID 

10 ID 

ID ID 

10 ID

3 ID 

ID ID 

10 ID 

ID ID

ID 10 

ID 10 

ID 10 

ID 10

0 10 

10 10 

10 ID 

10 ID

10 ID 

10 10 

10 10 

ID ID

10 ID 

10 ID 

10 10 

10 ID

10 ID 

10 10 

10 ID 

10 ID

2 ID 

10 ID 

ID ID

10 10

9 ID 

ID 10 

ID 10 

ID ID

8 10 

10 10 

10 10 

10 ID

ID ID 

ID ID 

ID ID 

ID 10

ID 10 

ID 10 

10 ID 

10 10

8 ID 

ID 10 

ID ID 

10 10

7 ID 

10 ID 

10 ID 

10 10

Black bullhead

0.1 1.0

0 0 

0 10 

0 10 

0 ID

0 ID 

0 10 

0 10 

0 ID

0 10 

ID ID 

10 ID 

ID ID

0 0 

0 ID 

0 ID 

0 ID

0 0 

0 10 

0 10 

0 10

0 ID 

0 ID 

0 ID 

0 10

0 9 

0 ID 

3 10

7 10

0 7 

5 ID 

8 ID 

ID 10

0 ID 

0 10 

0 10 

0 10

0 4 

0 ID 

0 ID 

0 ID

0 10 

0 10 

0 10 

4 ID

0 ID 

3 10 

3 ID 

3 10

0 ID 

0 10 

0 10 

0 ID

0 10 

0 10 

1 10 

2 10

ID

ID 

ID 

ID 

ID

ID 

ID 

ID 

ID

10 

ID 

10 

10

10 

10 

10 

10

10 

10 

10 

10

ID

13 

10 

10

ID 

10 

10 

13

ID 

10 

ID 

10

ID 

ID 

10 

10

10 

10 

ID 

10

ID 

ID 

ID 

ID

ID 

ID

10 

10

10 

10 

10 

10

ID 

10 

ID

in

Green sunfish

O.ll 1.0

0 0 

0 1 

0 2 

0 3

0 0 

0 ID 

0 10 

0 10

0 ID 

2 10 

4 10 

8 10

0 0 

0 0 

0 ID 

0 ID

0 0 

0 1 

0 2 

0 2

0 0 

0 4 

0 6 

0 9

0 10 

0 ID 

0 10 

0 ID

0 0 

0 ID 

3 ID 

ID ID

0 10 

3 10 

4 10 

6 10

0 3 

0 10 

0 ID 

0 ID

0 10 

0 10 

0 10 

1 10

0 9

0 10 

0 10 

0 13

0 1 

0 10 

0 10 

0 ID

0 0 

0 9 

0 10 

0 13

10

10 

ID 

10 

10

ID 

10 

ID 

10

10 

ID 

10

10

10

10 

10 

10

10 

10 

10 

10

10 

ID 

10 

10

10 

10 

10 

10

10 

13 

ID 

10

ID 

ID 

ID 

10

10 

10 

13 

10

ID 

ID 

10 

ID

ID 

10 

10 

10

10 

ID

10 

10

10 

10 

10

in

Bluegill

0.1

0 

0 

0 

0

0 

0 

0 

0

0 

9 

10 

ID

0 

0 

0 

0

0 

0 

0 

0

0 

0 

0 

0

0 

0 

5 

9

0 

7 

ID 

ID

0 

0 

0

1

0 

0

0 

0

0 

0 

0 

2

0 

0 

0 

0

0 

0 

0 

0

0 

0 

0

1

1.0 ID

0 ID 

2 ID 

2 ID 

2 ID

0 ID 

10 ID 

ID ID 

ID 10

ID 10 

ID ID 

10 10 

10 10

0 ID 

ID ID 

ID ID 

ID ID

0 10 

0 ID 

5 10 

7 10

0 10 

10 10 

ID 10 

10 ID

5 10 

ID 10 

ID 10 

10 10

0 10 

10 10 

10 ID 

10 ID

0 10 

10 ID 

10 10 

ID 10

0 10 

2 10 

2 ID 

5 ID

1 ID 

10 10 

ID ID 

10 ID

10 ID 

10 ID 

10 ID 

ID ID

0 

10 

10 

ID 

0 ID 

10 ID 

10 10 

10 10

Yellow perch

O.ll 1.0 1 ID

0 0 ID 

0 10 ID 

0 10 ID 

0 ID ID

0 10 ID 

2 10 10 

2 ID ID 

2 10 ID

2 10 ID 

ID ID ID 

10 10 ID 

10 10 10

0 6 10 

3 ID 10 

3 10 10 

5 10 10

0 0 ID 

0 10 10 

0 10 10 

0 10 10

0 10 ID 

1 10 ID 

1 10 ID 

1 10 10

1 10 ID 

10 ID 10 

10 ID 10 

10 ID 10

0 ID 10 

10 10 10 

ID 10 10 

10 ID 13

0 10 ID 

9 10 ID 

ID 10 ID 

10 10 ID

0 ID 10 

0 ID 10 

0 ID ID 

0 10 10

0 10 10 

2 ID 10 

2 ID 10 

2 10 ID

2 ID ID 

9 ID ID 

9 10 ID 

ID 10 ID

0 10 10 

8 ID 10 

8 10 10 

8 10 ID

0 8 ID 

1 ID ID 

1 10 10 

1 10 ID
Marking and Willford: Toxicity of 29 Nitrosalicylanilides to Fish 11

Toxicity of 0.1, 1.0, and 10 ppm of experimental chemicals to fish at 12° C. Continued

( 15 ) 5' -chloro-2 ' -methoxy-3-nitrosalicylanilide:

(16) 2',5'-dimethoxy-4'-chloro-3- 

nitrosalicylanilide:

(17) 2 ' -chloro-5 ' -trif luoromethyl3 -nitrosalicylanilide:

(18) 3 ' ,4 ' -dichloro-3-nitrosalicylanilide:

(19) 4 ' -chloro -5 -bronio -3 -nitrosalicylanilide:

(20) 2'-chloro-5-nitrosalicylanilide:

(21) 3 '-chloro-5 -nitrosalicylanilide:

(22) 4'-chloro-5-nitrosalicylanilide:

(23) 2 1 -methoxy-4'-nitro-3-nitrosalicylanilide:

(24) 4 l -methoxy-2'-nitro-3-nitrosalicylanilide:

(25) 2',4 1 -dimethyl-3-nitrosalicylanilide:

(26) 4 '-azophenyl-3 -nitrosalicylanilide:

(27) 3'-chloro-3-nitrobenzanilide:

(.28) 2',6'-diethyl-3,5-dinitrobenzanilide:

(29) 5-bromo-3-nitrosalicylic acid:

Rainbow trout

O.l| 1.0 | 10

0 10 ID

Goldfish

0.1 | 1.0 | 10

0 10 ID

Carp

0.1 | 1.0 | 10

0 10 ID

Fathead minnow

0.1 |l.O | 10

0 10 ID

Black bullhead

0.1 | 1.0 | 10

0 ID 10

Green sunfish

O.lj 1.0 | 10

1 0 10

1 0 10

1 10 10

Bluegill

0.1 | 1.0 | 10

0 10 ID

0 ID ID

0 ID 10

0 0 10

0 0 10

0 0 10

0 0 10

0 0 10

0 0 10

0 0 10

0 1 ID

0 0 ID

0 2 10

0 ID 10

10 ID ID

8 ID ID

Yellow perch

0.1 | 1.0 | 10

0 6 ID

4 ID ID

0 ID ID

6 ID 10

0 1 ID

0 3 ID

0 3 10

0 4 10

0 0 10

0 8 10

0 10 10

0 1 10

0 ID 10

0 ID 10

10 ID 10

0 1 ID

ID ID 10
INVESTIGATIONS IN FISH CONTROL

38. Toxicity of 33NCS to

Freshwater Fish and Sea Lampreys

By Leif L. Marking, Everett L. King, 

Charles R. Walker, and John H. Howell

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

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

Fish and Wildlife Service, Charles H. Meacham, Commissioner

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

Washington, D.C. July 1970
CONTENTS

Page

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

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

Fish Control Laboratories......................... 4

Hammond Bay Biological Station. .................... 5

Closed system bioassays....................... 5

Flowing water bioassay........................ 5

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

Fish Control Laboratories......................... 6

Preliminary investigations. ..................... 6

Delineative investigations ...................... 7

Effects of temperature ........................ 8

Effects of water quality........................ 8

Effects of preexposure ........................ 10

Hammond Bay Biological Station. .................... 10

Preliminary testing .......................... 10

Effects of temperature ........................ 11

Effects of water quality........................ 12

Flowing water bioassay........................ 12

Discussion ...................................... 14

Conclusions. ..................................... 15

References ...................................... 16
TOXICITY OF 33NCS (3'-CHLORO-3-NITROSALICYLANILIDE) 

TO FRESHWATER FISH AND SEA LAMPREY

By Leif L. Marking, Everett L. King, 

Charles R. Walker, and John H. Howell

ABSTRACT. The chemical 33NCS (3'-chloro-3-nitrosalicylanilide) 

was evaluated as a fish control agent and as a larvicide for sea lampreys 

at the Fish Control Laboratories of the Bureau of Sport Fisheries and 

Wildlife and the Hammond Bay Biological Station of the Bureau of 

Commercial Fisheries. The chemical is rapidly toxic to many species. 

Sea lampreys, bowfin, and channel catfish are the most sensitive species. 

Carp are more sensitive than trouts or sunfishes. Use of 33NCS in 

selective control of freshwater fishes or sea lampreys requires precise control because its toxicity is strongly influenced by variations in 

water quality.

The salicylanilides are of great interest in 

fishery research because of their promise as 

selective controls for freshwater fish and 

sea lamprey larvae (Walker, Starkey, and 

Marking, 1966; Starkey and Howell, 1966). 

Among them, 33NCS (3'-chloro-3-nitrosalicylanilide) showed the greatest potential 

for development as a sea lamprey larvicide 

during early testing at the Hammond Bay 

Biological Station. It also showed properties 

of interest during preliminary screening at 

the Fish Control Laboratories. A cooperative 

investigation was initiated to evaluate 33NCS 

as a selective toxicant under a variety of 

conditions.

The importance of studying the biological 

activity of a compound in different environments was demonstrated repeatedly during 

the development of the sea lamprey larvicide 

TFM (3-trifluoromethyl-4-nitrophenol) and 

its synergist, Bayer 73 (2 t ,5-dichloro-4lnitrosalicylanilide) (Applegate et al., 1961;

Leif L. Marking and Charles R. Walker are Chemists 

at the Fish Control Laboratory of the Bureau of Sport 

Fisheries and Wildlife, La Crosse, Wis.

Everett L. King and John H. Howell are Fishery Biologists at the Hammond Bay Biological Station of the Bureau of Commercial Fisheries, Millersburg, Mich.

Howell et al., 1964). Walker, Lennon, and 

Berger (1964) found that environmental conditions strongly influence the toxicity of 

antimycin. Strufe and Gb'nnert (1962) observed 

that the molluscicidal activity of Bayer 73 is 

affected by chemical and physical factors. 

Later, Fox, Ritchie, and Frick (1963) determined the effect of pH on the efficacy of 

Bayer 73 and postulated that the inconsistencies in published lethal concentrations are 

the result of differences in water quality.

Recognizing that the biological activity of 

33NCS may be influenced by chemical or 

physical factors, our bioassays were conducted in reconstituted and natural waters of 

different qualities. A variety of organisms 

including invertebrates and fish were exposed 

to the toxicant in laboratory or simulated 

field tests.

MATERIALS AND METHODS

Pharmaceutical grade 33NCS was used in 

all tests. Organic solvents employed in formulating the toxicant were not in excess of 

amounts tolerated by the test animals.
Investigations in Fish Control 38: Bureau of Sport Fisheries and Wildlife

The common and scientific names of the 

six kinds of invertebrates and 30 species of 

fish used in the bioassays are given in table 1,

Fish Control Laboratories

The facilities and procedures for routine 

bioassays were described by Lennon and 

Walker (1964).

Reconstituted deionized water was used in 

most of the bioassays of 33NCS, and hard and 

soft water was formulated by varying the

TABLE.1.--Common and scientific names of test organisms

Common name

Mollusks :

Snail....................

Crustaceans :

Crayfish .................

Insects :

Stonefly nymph. ..........

Mayfly nymph .............

Fish: 

Sea lamprey. .............

Bowfin. ..................

Rainbow trout ............

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

Brook trout ..............

Lake trout ...............

Northern pike ............

Goldfish. ................

Lake chub ................

Sand shiner ..............

Longnose dace ............

Longnose sucker ..........

Black bullhead. ..........

Yellow bullhead. .........

Trout-perch. .............

Rock bass ................

Pumpkinseed. .............

Bluegill.................

Largemouth bass ..........

Yellow perch .............

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

Scientific name

Physa spp.

Cambarus spp.

Togoperia spp.

Hexagenia spp.

Ophiogpmphus spp.

Petromyzon marinus

Amia calva

Salmo gairdneri

Salmo trutta

Salvelinus fontinalis

Salvelinus namaycush

Esox lucius

Carassius auratus

Hybopsis plumbea

Notropis cornutus

Notropis stramineus

Pimephales promelas

Rhinichthys cataractae

Catostomus catostomus

Catostomus commersoni

Ictalurus melas

Ictalurus natalis

Ictalurus nebulosus

Ictalurus punctatus

Percopsis omiscomaycus

Ambloplites rupestris

Lepomis cyanellus

Lepomis macrochirus

Micropterus dolomieui

Micropterus salmoides

Perca flavescens

Percina caprodes

Stizostedion vitreum

quantity of salts added (table 2). Desired pH 

levels were obtained with buffer systems of 

potassium hydrogen phthalate (KHCgH^^), 

boric acid (HsBQs), and sodium hydroxide 

(NaOH). The bioassay solutions were aerated 

prior to introduction of fish.

Most of the bioassays were carried out in 

5-gallon glass jars containing 15 liters of 

water each. Bioassays with larger fish were 

made in plastic tanks which held up to 45 

liters of water. The toxicant, 33NCS, was 

dissolved in acetone, and aliquots of stock 

solutions were pipetted directly into bioassay 

vessels to produce desired concentrations.

The fish in routine bioassays ranged from 

1.0 to 2.5 inches in length and were obtained 

from National, State, and private fish hatcheries in the vicinity of La Crosse, Wis., and 

Warm Springs, Ga. Larger fish used in special 

tests were also obtained from these sources. 

All fish were held in quarantine for at least 

10 days. Three days before testing, the fish 

were taken off feed and acclimated to the 

water chemistry and temperatures of the 

bioassay.

The preliminary bioassays took place in 

standard reconstituted water at concentrations of 0.1, 1.0, and 10 ppm of 33NCS and 

at 12° and l?o c. Ten fish of each species 

were exposed to each concentration and their 

responses were observed at 0.5, 1, 3, 6, 24, 

48, 72, and 96 hours.

Delineative bioassays were performed at 

7°, 12°, 17°, and 22O c. in waters of various 

qualities. Ten fish were exposed to each of 

ten concentrations. The results were used to 

delineate concentrations which produced 

50-percent mortality (LC50 values) at selected 

observation periods. The LCSO's, confidence

TABLE 2. Quality and composition of reconstituted water used at the Fish 

Control Laboratories

Classification of 

water

Jfedium2 ......

Salt added in sag/1

NaHC03

12 

48 

192

CaSCU

7.5 

30.0 

120.0

MgSO*

7.5 

30.0 

120.0

KC1

pH 

range

Total 

hardness1

Total 

alialinity1

0.75 6.4-6.8 10- 13 10- 13 

3.00 7.2-7.6 40- 48 30- 35 

12.00 7.6-8.0 160-180 110-120

 ' As ppm CaCOs2Standard reconstituted water used in routine bioassays.
Marking, King, Walker, Howell: Toxicity of 33NCS to Freshwater Fish and Lampreys

intervals (C.I.), and slope functions were 

derived according to methods of Litchfield 

and Wilcoxon (1949).

Hammond Bay Biological Station

Closed system bioassays. The water used 

in most tests and for holding test specimens 

was pumped directly from Lake Huron at 

Hammond Bay. The chemical and physical 

characteristics of the lake water fluctuate 

seasonally but are considered to be generally 

intermediate to the extremes found among 

tributaries of the upper Great Lakes: total 

alkalinity (expressed as ppm CaCO3) is 85 to 

118, pH is 7.4 to 8.3, and specific conductance 

is 148 to 203 micromhos at ISO c.

Special tests were made to evaluate the 

effect of water quality on the toxic action of 

33NCS, with natural water from representative 

tributaries in which sea lampreys spawn.

In standard bioassays, 5 liters of water 

were added to 10-liter glass battery jars 

which were then placed in constant-temperature troughs maintained at 12.8° c. The 

troughs were similar to those described by 

Lagler (1953). The effect of temperature was 

determined by tests at 1.7°, 7.2°, 12.8°, 18.3°, 

and 23.9° C.

Two larval sea lampreys 3.5 to 5.0 inches 

long and two rainbow trout 3.0 to 5.0 inches 

long were placed in each jar and were acclimated for 1 to 2 hours before introduction of 

the toxicant. At least four replications were 

made simultaneously at each concentration.

Aliquots of a 5-percent stock solution of 

33NCS in dimethyl formamide (DMF) were 

diluted to 1 liter and added to the test jars to 

establish the final 6-liter test volume and 

desired concentrations. All test solutions 

were aerated to maintain oxygen at or near 

saturation.

Bioassays provided data on minimum lethal 

(MLC100) and maximum allowable (MAC 25) 

concentrations (Howell and Marquette, 1962). 

The MLC100 is the lowest concentration which 

produces a complete kill of larval lampreys. 

The highest concentration causing no greater

than a 25-percent kill among other test fish 

is the MAC25. The working range of 33NCS 

falls between these values. The ratio of working range to the MLC100 gives the permissible 

additional flow (PAF) and represents the 

amount of dilution before the MAC 25 will be 

reduced to the MLC100. In sea lamprey control applications, a PAF less than 1 is considered to limit utility (Howell and Marquette, 

1962).

Hourly observations on specimens were 

recorded through the first 12 hours and at 

the end of each assay period, usually 21 to 

24 hours. The bioassays at Hammond Bay 

were not conducted for longer periods, since 

the period of exposure in sea lamprey control 

is seldom greater than 24 hours.

Flowing water bioassay. Experiments 

with 33NCS were conducted in raceways with 

flowing water to simulate natural stream conditions. These also permitted the simultaneous 

exposure of a variety of aquatic species and 

larger fish than could be accommodated in 

the laboratory. Simulated stream tests also 

provided information on the physical properties of 33NCS when applied to flowing water, 

the type of formulations required, and the 

adequacy of the chemical metering devices 

for stream application.

Raceway tests were made in the early fall 

of 1964 and again in the late spring of 1965. 

Chemical and physical analyses of the water 

were made daily during the two test periods. 

Ranges of values for the 1964 tests were as 

follows: pH, 7.8 to 8.3; conductivity, 176 to 

197 micromhos; total alkalinity, 97 to 106 

ppm; and. water temperature 5.5° to 11.1° 

(mean 8.2° C.). The 1965 test characteristics 

were: pH, 7.6 to 8.2; conductivity, 127 to 180 

micromhos; total alkalinity, 96 to 108 ppm, 

and water temperature, between 11.1° and 

19.4° C. (mean 15.6°).

The concrete raceways were 65 feet long, 

6 feet wide and 30 inches deep. Water from 

Lake Huron was delivered to them from a 

surge tank which stabilized the flow. The flow 

was held constant at 0.34 cfs by a "V" weir. 

Water depth throughout the length of the raceway was maintained at 14 inches by a stopboard.
6 Investigations in Fish Control 38: Bureau of Sport Fisheries and Wildlife

Trout were provided by State Fish Hatcheries (Michigan Department of Conservation) 

at Grayling and Oden, Mich., except for one 

lot of brown trout which was obtained in the 

fall of 1964 from the Rifle River, Ogemaw 

County, Mich. Most of the other specimens 

used in the raceway tests were collected from 

the Ocqueoc River and Bullhead and Lost 

lakes, Presque Isle County, Mich. Several 

species were from the Cheboygan River, 

Cheboygan County, and from a commercial 

bait dealer at Traverse City, Mich. All specimens were held in flowing Lake Huron water 

for at least 7 days before exposure to 33NCS.

Larger fish were allowed to move freely 

within the raceway. Smaller specimens were 

confined in cylindrical screen cages. Larval 

lampreys were placed in cages containing 3 to 

5 inches of clean beach sand at least 24 hours 

before each test to allow sufficient time for 

them to establish burrows. The confinement 

of larval lampreys and the other smaller 

specimens in cages facilitated rapid and 

accurate observations of mortality and prevented predation by larger fish.

Stock solutions of 33NCS were prepared 

using DMF as the solvent. An electric fuel 

pump was used to apply solutions of various 

strengths depending on the concentration 

desired (Anderson, 1962).

RESULTS

Fish Control Laboratories

Preliminary investigations. The results of 

early trials with 33NCS indicate a high degree 

of toxicity to the fish at 12° and l?o c. 

(table 3). The fish respond to the toxicant 

immediately after its introduction into the 

water. At lower concentrations they become 

mottled in appearance, are quiescent, and 

swim slowly to the surface or bottom. At 

higher concentrations they become disoriented, irritated, and excited. Erratic 

swimming and surfacing occurs within 15 minutes at less than 1 ppm. Close examination 

shows that the entire integument is devoid of 

mucous. Respiration is usually rapid and

TABLE 3.--Toxicity of 0.1, 1.0, and 10.0 ppm of 33NCS to fish at two temperatures 

[expressed as the number of mortalities in a bioassay of 10 fish]

Temperature 

and species

At 12° C.

Bowf in. ..............

Goldfish. ............

Black bullhead. ......

Bluegill. ............

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

At 17° C.

Goldfish. ............

Average 

length 

(in.)

2.2 

1.2 

1.9

2.1 

2.1

1.8

1.5 

1.6 

1.3

1.7 

1.5 

1.0

2.0 

1.3

1.6 

1.2 

2.1

1.9 

2.0

Mortality at 

3 hours

0.1

0 

0 

0

0 

0 

0

0 

0 

0

0 

0 

1

0 

0

1 

0 

0

0 

0

1.0

10 

10 

3D

9 

10 

0

9 

2 

8

30 

0 

1

3D 

3D

0 

2

0

3 

9

10

10 

10 

30

10 

10 

10

10 

10 

10

10 

10 

10

10 

10

10 

10 

10

10 

3D

24 hours

0.1

10 

0 

0

0 

0 

0

7

0 

0

10 

0 

1

8 

5

1 

0 

0

0 

0

1.0

3D 

10 

10

10 

10 

9

10 

10 

30

10 

10

ID

10 

10

3 

9 

3D

9

10

10

3D 

10 

10

ID 

10

10

]D 

10 

10

]D 

10 

]D

10 

ID

10 

10 

10

ID 

ID

48 hours

0.1

10 

0 

0

0

0

7

0 

0

]D 

0 

1

8 

8

1 

0 

0

0 

0

1.0

10 

10 

10

10

9

3D 

3D 

10

3D 

10 

3D

3D 

3D

7 

3D

3D

3D 

10

3D

3D 

10 

10

3D

10

3D 

3D

10

10 

10 

3D

3D 

3D

3D 

3D 

10

3D 

3D

96 hours

0.1

10 

0 

0

0

0 

8 

0 

0

3D 

1 

3

9 

3D

1 

0 

0

0 

0

1.0

10 

10 

3D

3D

3D

3D 

3D 

10

3D 

3D 

10

3D 

3D

7 

10 

3D

3D

3D

10

10 

10 

10

10

30

3D 

10 

10

3D 

ID 

10

10 

10

10 

3D

10

3D

30
Marking, King, Walker, Howell; Toxicity of 33NCS to Freshwater Fish and Lampreys

irregular, and the operculums are distended 

and retracted abruptly in a coughlike action 

in all species. The respiratory rate slows 

preceding death, accompanied by spastic 

gulps, twitching, and tetany. The response to 

touch and sound progressively decreases and 

the fish soon lose equilibrium. Death is accompanied also by the occurrence of mucous 

about the gills and anus. The fish have open 

mouths and extended operculums at the time 

of death.

33NCS is rapidly as well as highly toxic to 

fish. Ten ppm kills all species in 3 hours. 

One ppm kills all bowfin, rainbow trout, brown 

trout, northern pike, channel catfish, yellow 

perch, and walleye within 3 hours, and it produces partial mortality among lake trout, 

carp, fathead minnow, black bullhead, green 

sunfish, bluegill, and smallmouth bass within 

the same time (table 3). Goldfish, carp, and 

green sunfish are the only species among 

which mortality was not complete at 1.0 ppm 

in 24 hours. Even 0.1 ppm was highly toxic, 

and bowfin, channel catfish, and walleye died 

within 96 hours.

Delineative investigations. The LCSO's of 

33NCS for 11 species of fish range from 

0.068 to 0.830 ppm in 96 hours at 12° c.

(table 4). Channel catfish are by far the most 

sensitive species. Carp, yellow perch, brown 

trout, lake trout, rainbow trout, fathead minnow, and black bullhead are intermediate in 

sensitivity to 33NCS. Goldfish, green sunfish, 

and bluegill are more resistant.

- 33NCS is unique because it kills most species in a matter of minutes. Statistical expressions of LC50 are possible in 1 to 6 hours 

at concentrations only slightly higher than 

those required to kill all fish in 24 hours. 

Also, concentrations necessary to produce 

mortality are within a very narrow range at 

each time interval. Minute increments in 

concentrations delineate all-or-none effects 

as denoted by extremely low slope functions. 

This is illustrated by a very low mean slope 

function which ranges from 1.13 to 1.67 

(table 4). In contrast, Marking (1966) found 

that the slope function for goldfish exposed to 

j),p^-DDT was 6.02.

The trout respond considerably faster to 

33NCS than warmwater species, and LC50 

values were established in a 1-hour exposure 

(fig. 1). Higher concentrations are required to 

kill warmwater fish in short-term exposures. 

For instance, it requires less than twice as 

much 33NCS to kill rainbow trout in 3 hours

TABLE 4. Toxicity of 33NCS for selected fishes at 12° C.

Species

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

Goldfish. ..........

Black tnillhead. ....

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

Channel catfish. . . .

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

Bluegill. ..........

Average

length 

(inches)

1.9

1.9

3.3

2 1

I

et

1.5

1.6

2.4

1.3

1.7

1.5

1.7

1.3

2.2

A1

LC 50 

(ppm)

0.233

n 3on

0.173

0.325

1.400

0.155

0.385

0 440

O OC>r\

0.085 

0.707

0.840

0.625

0.195

j 24 hours

95-percent 

C.I.

0.275-0.327

0.142-0. 210

0.304-0.348

1.240-1.580

0.119-0.202

0.324-0.458

0.350-0.550

0.339-0.426

0.075-0.096 

0.637-0.785

0 750-0 900

0.521-0.750

0.156-0.244

At

LC 5 o 

(ppm)

0.203

0.260

0.167

1.030

0.130

0.250

0.255

0

-2OCJ

0.068 

0.600

0.500

0.530

0.172

48 hours

95-percent 

C.I.

O n on n oT7

0.245-0.276

0.190-0.226

O &j n T or?r\

0.104-0.162

0.172-0.362

0.203-0.307

O oc>^ n "yin

0.050-0.071

0

1 &i r\ rior)

0.465-0.537

0.465-0.604

O.L46-0.203

At

LC 50 

(ppm)

0.203

0.167

0.200

0.830

0.123

0.245

0.255

0.260

0.068 

0.490

0.320

0

} O£j

0.155

96 hours

95-percent 

C.I.

0 190-0 217

0.214-0.269

0.176-0.228

0.690-1.000

0.098-0.154

0.194-0.309

0.203-0.307

0.050-0.071 

0.422-0.568

0.270-0.380

O otjo r\ J r?o

0.135-0.178

Mean

slope 

function

1.33

1.15

1.25

1.39

I

t rt

1.67

1 40

1.39

1.19

1.27

1 O&

1.16

1

1 o

1.39
8 Investigations in Fish Control 38 : Bureau of Sport Fisheries and Wildlife

96-

48-

£24.

o

x

12-

3-

'. \\ ' v\ \ \\ \ \\ \ x\

1 \\ * * \

'. \ \

\ \ ^ \ \

\ \ \

 .Rainbow trout \

limn Brown trout \

 Fathead minnow *

- Black bullhead *,

 « Green sunfish

 

\

*

\ \* 

*

\ \

V

\

V

o.i .2 .3 .4 .5 .6 

LC 5Q in p.p.m.

.8 1.0 1.2 1.4

Figure 1. Rate of response for several species of 

fish to 33NCS.

17- \

12-

7-

0

;\» *

\

-- 1-HOUR

 -24-HOURS

......,,...n....96-HOURS

1 .2

L

\

\ 

\

\ \

\ \

\ \

\ \

 i

.3 .4

C 50 in P-P- m -

1

.5 .<

i

\1

5 .7 .8 .<

\

? 1.0

Figure 2. Effects of temperature on the toxicity of 

33NCS to rainbow trout.

as in 96 hours whereas it takes three times 

as much or more for carp, fathead minnow, 

black bullhead, and channel catfish.

Effects of temperature. The 1-hour LC50 

for rainbow trout at 7° is 2.5 times that at 

17° C. (fig. 2 and table 5); at 24 and 96 hours, 

the LC50's at 7° are less than twice those at 

17°. Although the same trend prevails with 

green sunfish, the difference in LC50 values

for 12°, 17°, and 22° is not as great. There is, 

however, a very similar decline in the toxic 

concentrations calculated at 1 to 5 hours as 

opposed to the 24- and 96-hour values. The 

data also indicate that 33NCS is more effective 

at warmer temperatures, and much less 

material is required to produce lethal effects. 

Examination of the slope functions at all temperatures reveals consistent values, and the 

narrow range of concentrations necessary to 

permit survival and produce mortality (allor-none effect) prevails at all temperatures.

Effects of water quality. The toxicity of 

33NCS to rainbow trout is much greater in 

soft water than in hard water (table 6). The 

LCSO's in hard water are approximately 

5 times as great as those for soft water at 6, 

24, 48, and 96 hours. The LCSO's in water of 

medium hardness are about twice those for 

soft water. The range of pH is 6.4-6.8 for 

soft water and 7.6-8.0 for the hard water. 

Thus, pH and water hardness influence toxicity of 33NCS.

The toxicity of 33NCS to rainbow trout at 

all water hardnesses manifests itself within 

a short period of time. The LCSO's at 96 hours 

are only slightly less than those at 6 hours. 

Decreases in LC50 values of 28, 19, and 22 

percent are noted for soft, medium, and hard 

water respectively.

Recognizing the variable responses in 

waters of different pH and hardness, we sought 

to identify the constituents of the reconstituted 

water which may affect toxicity. The toxicity 

of 33NCS to brook trout is considerably less 

when four times as much sodium bicarbonate 

is added to the reconstituted formula (table 7). 

Four times the regular amounts of potassium 

chloride, magnesium sulfate, or calcium sulfate had no appreciable effect on toxicity of 

the chemical. The addition of more sodium 

bicarbonate increases the pH from 7.2 to 8.2, 

whereas adding more of the other constituents 

has less effect on pH. Further, the slight 

increase in pH from 7.7 to 8.2 accounts for 

complete survival of fish at 0.5 ppm.

The specific effect of pH is demonstrated 

by using buffer systems at pH 5.8 and 8.5 with 

the standard reconstituted water (table 8). We
Marking, King, Walker, Howell: Toxicity of 33NCS to Freshwater Fish and Lampreys

TABLE 5. The toxicity of 33NCS to fish at selected temperatures

Species and

exposure 

time

Rainbow trout:

Green sunfish:

A

LC 50 

(ppm)

1 080

0.331

0.281

0.275

t 7° C.

95- percent 

C.I.

0.302-0.360

0.264-0.296

0.262-0.288

J

LC 50 

(ppm)

0 642

0.300

0.260

0 240

1.220

0.730

0.610

0.511

it 12° C.

95-percent 

C.I.

0.500-0.686

0.275-0.327

0.245-0.276

0.2U-0.269

0.665-0.800

0.565-0.660

0.464-0.552

1

LC 50 

(ppm)

0.380

0.195

0.180

0.142

0.870

0.710

0.500

0.361

Vt 17° C.

95-percent 

C.I.

0.331-0.437

n 177-0 ^>~\L

0.172-0. 187

0.123-0.163

0.820-0.920

0.635-0.795

0.437-0.634

0 302-0 432

A

LCso 

(ppm)

0.715

0.600

0.445

0.271

b 22° C.

95-percent 

C.I.

0.660-0.775

0.550-0.655

0.364-0.542

0.208-0.352

TABLE 6. The toxicity of 33NCS to rainbow trout in various water qualities at 12° C.

Water

hardness

Soft. .......

Hard. .......

At

LC 50 

(ppm)

0.118

0.250

0.580

6 hours

95-percent 

C.I.

0.092-0.152

0.242-0.258

0.560-0.603

At

LC50 

(ppm)

0.104

0.233

0.520

24 hours

95-percent 

C.I.

0.078-0.138

0.216-0.252

0.481-0.562

At

LC 50 

(ppm)

0.102

0.203

'0.500

48 hours

95-percent 

C.I.

0.083-0.125

0.190-0.217 .

0.463-0.540

At

LC 50 

(ppm)

0.092

0.203

0.420

96 hours

95-percent 

C.I.

0.069-0.123

0.190-0.217

0.385-0.458

TABLE 7. The toxicity of 0.5 ppm of 33NCS to 7.5-inch brook 

trout in selected water qualities

[Ten fish per test in 45-liter tanks with aeration]

TABLE 8. Mortality of 2.5-inch rainbow trout resulting 

from exposure to 33NCS at selected pH values

Observation 

time

3 hours. ....

6 hours. ....

24 hours. . . .

96 hours. . . .

Percent mortality

pH 7.2 

( standard 

bioassay 

water)

pH 7.5 

(4 times 

KC1)

pH 7.6 

(4 times

pH 7.7 

(4 times 

CaSO^ )

pH 8.2 

(4 times 

NaHC03 )

30 10 0 20 0

30 20 20 30 0

40 40 40 60 0

40 40 40 60 0

40 40 40 60 . 0

observed that 0.35 ppm of 33NCS killed all 

fish within 15 minutes at pH 5.8; it killed all 

fish in 24 hours at pH 7.4; but none died in 

96 hours at pH 8.5. Thus, pH governs effectiveness and rate at which the chemical kills 

fish.

Brook trout which averaged 7.2 inches long 

were exposed in aerated vessels containing 

45 liters of test solution. Erratic results such 

as mortality at lower concentrations and survival at higher concentrations suggested inconsistent test media. At 0.5 ppm all of the 

trout were dead in 1 hour, whereas 30 percent 

of the fish survived exposure to 0.6 ppm for 

96 hours. At 0.7 ppm, 40 percent of the fish 

survived 1 hour, and 10 percent survived 

3 hours. The swift and complete mortality at

pH

5.8......

5

0-

7.4......

8.5......

8.5......

Buffer 

applied

KHP - NaOH

KHP - NaOH

Noxis

NaOH - H3 B03

NaOH - H3 B03

33NCS 

(ppm)

0.33

0.35

0.35

0.35

0.37

Percent mortality 

at given time

1DO at 1 to 24 hours

0.5 ppm cannot be explained. However, in a 

subsequent bioassay using 2.5-inch rainbow 

trout in 15 liters of aerated test solution, 

the pH changed slightly owing to aeration 

rates. Here, 0.34 and 0.36 ppm produced 

100-percent mortality in 3 hours, while 0.4 

ppm produced no mortality in 48 hours and 

only 10 percent mortality in 96 hours. A measure of pH revealed that the values for the 

lower concentrations were 7.6 and 7.5 but that 

of the bigger concentration was 8.0. Thus, a 

small increment in pH of only 0.4 or 0.5 unit 

was responsible for the decreased activity of 

33NCS.

The effects of pH and alkalinity were differentiated in bioassays conducted with 2.5-inch 

rainbow trout. The LCSO for 96 hours using standard reconstituted water was 0.24 ppm (C.I.= 

0.21-0.27). An increase of pH from 7.2 to 8.2 

through the use of buffers increases the LCSO
10 Investigations in Fish Control 38: Bureau of Sport Fisheries and Wildlife

value to 0.35 ppm (C.I.=0.32-0.37). The toxicant in hard water, pH 8.2, had an LC50 value 

of 0.44 ppm (C.I.=0.41r0.47), whereas in hard 

water buffered to pH 7.2 the value was 0.32 

ppm (C.I.=0.29-0.35) in 96 hours. These results indicate that pH apparently has slightly 

greater effect on the toxicity of 33NCS than 

does hardness.

Larger fish appear slightly more resistant 

to 33NCS than smaller fish. Although LC50 

values are not available, 8-inch brown trout 

and 7.2-inch brook trout survived 0.4 ppm for 

96 hours in an aerated bioassay. Smaller trout 

tested under regular bioassay conditions are 

more sensitive, and LCSO's range from 0.20 to 

0.24 ppm in 96 hours. Aeration increases pH,

Hammond Bay Biological Station

Preliminary testing. The toxicity of 33NCS 

to larval lampreys varied little from April 28, 

1964, to April 22, 1965 (table 10). The MLCIOO 

values ranged from 0.7 to 0.3 ppm (average 

0.4 ppm) and exceeded 0.5 ppm only three 

times in the 42 assays performed.

Toxicity to finger ling rainbow trout was 

more variable. The MAC25 ranged from 0.8 

to 2.1 ppm (average 1.3 ppm). Again the

TABLE 10. Biological activity of a 5-percent-by-weight formulation 

of 33NCS in Lake Huron water in 21-hour assays at 12.8° C.

Date

Lamprey

larvae

MIC 100 

(ppm)

Rainbow

trout

MAC 25 

(ppm)

PAF 1

Properties of ttst water

Conductivity 

(gmhos/180 C.) alkalinity 

(ppm CaC03 )

pH

ferences.

Several of the larger fish which had completely lost equilibrium were placed in fresh

water to see whether they would recover. "

Two brown trout exposed to 0.6 ppm for

15 minutes regained equilibrium in 45 minutes. One of the fish died, and the other

apparently recovered fully only after 24 hours

in fresh water. Brook trout exposed to 0.5

ppm for 1 hour died within 10 minutes after

being placed in fresh water. Thus, it appears

that fish do not recover after critical exposure

periods.

Effects of preexposure. The rate of toxic

action is reduced by preexposure to a sublethal level of 33NCS (table 9). Rainbow trout

preexposed to 0.2 ppm of 33 NCS for 15 minutes are more resistant at 3 hours than unexposed trout. The difference in resistances

at 24 hours is reduced considerably and at

48 hours it is insignificant.

TABLE 9. The response of rainbow trout to 33NCS after preexposure to

sublethal concentration

At 3 hours At 24 hours At 48 hours

LC5 o 95-percent I£ 50 95-percent LC 50 " 95-percent 

(ppm) C.I. (ppm) C.I. r ppm) C.I.

Unexposed. . . . . . 0.368 0.350-0.390 0.290 0.245-0.342 0.237 0.214-0.257

Preexposed1 .... 0.625 ( 2) 0.325 0.290-0.364 0.239 0.204-0.283

1964

Apr. 28

June 8

15

July 20

21 

22

23

24

27

Aug. 4 

5

6

17

18

19 

20

Sept. 1

Oct. 12

19 

20

Nov. 9

10

Dec. 2

7

14

15 

17

1965

Jan. 4

8

11

12

13

14

18

Feb. 26

Mar. 5

12

19

29

Apr. 6

13

22

Average

value

.

0.3

0.4

0.4

0.3

0.4 

0.5

0.4

0.5

0.3

0.6 

0.7

0.3

0.3

0.4

0.5 

0.3

0.5

0.3

0.4 

0.4

0.3

0.3

0.4

0.4

0.5

0.5 

0.6

0.4

0.4

0.4

0.4

0.4

0.4

0.5

0.3

0.5

0.3

0.4

0.5

0.4

0.5

0.4

0.4

0.9

1.4

1.0

1.6

1.4 

1.5

1.7

2.1

2.0

1.6 

1.8

1.8

1.6

1.4

1.6 

1.4

1.2

1.2

1.0 

1.0

1.0

1.0

1.0

0.8

1.2

0.9 

1.2

0.9

0.8

1.4

1.2

1.2

1.4

1.4

1.2

0.9

1.4

1.0

1.0

0.9

1.0

0.8

1.3

2.0

2.5

1.5

4.3

2.5 

2.0

3.3

3.0

5.7

1.7 

1.6

5.0

4.3

2.5

2.2

3.7

1.4

3.0

1.5 

1.5

2.3

2.3

1.5

1.0

1.4

0.8 

1.0

1.3

1.0

2.5

2.0

2.0

2.5

1.8

3.0

0.8

3.7

1.5

1.0

1.3

1.0

1.0

2.2

169.0

184.0

163.0

171.0

173.0 

174.0

174.0

160.0

158.0

149.0 

172.0

172.0

186.0

181.0

181.0 

177.0

175.0

-

_

192.0

185.0

179.0

156.0

179.0

159.0 

203.0

180.0

170.0

172.0

186.0

173.0

183.0

201.0

190.0

186.0

179.0

179.0

177.0

175.0

I £?p Q

148.0

175.7

90.0

104.0

99.0

94.0

93.0 

93.0

96.0

86.0

93.0

93.0 

96.0

96.0

103.0

96.0

96.0 

98.0

97.0

-

:

105.0

100.0

96.0

85.0

103.0

96.0 

118.0

106.0

91.0

94.0

100.0

94 0

101.0

110.0

98.0

94.0

94.0

99.0

94.0

95.0

106.0

85.0

97.1

8.0

8.1

8.0

8.0

8.1 

8.1

8.0

8.0

8.1

8.2 

8.3

8.1

8

f\ U

8.3

8.3 

8.3

8.2

-

_

8.2

8.2

8.1

7.6

8.0

7.9 

7.8

7.8

7.7

7.4

7.8

7^8

7.9

7.9

7.8

7.8

7.8

7.8

7.8

7

tJ

 0

7.9

8.0

 " Preexposed to 0.2 ppm for 15 minutes. 

295-percent confidence intervals not available.

second is the ratio of working range to MLCioo (see text for 

explanation).
Marking, King, Walker, Howell: Toxicity of 33NCS to Freshwater Fish and Lampreys 11

higher values occurred infrequently; only 

twice did the MLC25 exceed 1.8 ppm.

The PAF values were highly variable, 

ranging from 0.8 to 5.0 and averaging 2.2. 

A PAF of less than 1.0, which is considered 

below usefulness for stream treatment, 

occurred only twice.

The data from all 42 assays show that 

there is some seasonal variation in toxicity 

of 33NCS in Lake Huron water, at least for 

rainbow trout. The results are therefore 

divided into two groups, June through October (summer) and November through April 

(winter).

The average ML 100 of 0.42 ppm for summer 

and 0.41 ppm for winter assays did not differ 

significantly (P>0.04), but the average MAC25 

levels for rainbow trout of 1.49 ppm for 

summer and 1.07 ppm for winter assays were 

highly significant (P<0.001). Because PAF 

values reflect the relation between MLC100 

and MAC25, they also differed significantly 

(P<0.005) between the two seasons (averages 

were 2.80 in the summer and 1.68 in the 

winter).

Only pH showed a significant variation 

(P<0.001) between the two seasons (fig. 3).

TFM was more selective than 33NCS in 

6 out of 9 tests in Lake Huron water (table 11), 

but 33NCS was 5 to 10 times as potent on 

larval lamprey (MLC100) and 6 to 12 times as 

potent on rainbow trout (MAC25) as TFM. The 

average PAF value was 3.0 for TFM and 2.2 

for 33NCS; the MAC25's of TFM and 33NCS 

for rainbow trout were 2.5 to 6 times the 

MLClOO's for lamprey larvae.

Effects of temperature. The toxicity of 

33NCS, expressed as LC50, becomes greater 

with each increment of test temperature 

(table 12). But, an examination of the 95- 

percent confidence limits on those values 

indicates that the variations are slight between 

7.2° and 18.3Q c. The difference observed 

between 1.7° and 7.2O is greater but even 

more so between 18.3° and 23.9° C. The slope 

function for each species is essentially the 

same and did not vary with temperature. 

These slope functions (1.20-1.25 for rainbow

1.8-

1.6

1.4-

1.2-

1.0-

CO

O 0.8-

Z

CO 

CO

0.6

0.4-

0.2-

-8.4

8.2

8.0

7.8

7.6

J FMAMJ JASOND 

Months of the year

Figure 3. Seasonal variations in pH (solid line) and 

concentrations of 33NCS (broken line) required to 

kill 25 percent (MAC25) of the rainbow trout.

TABLE 11. Relative toxicity and selectivity of TFM and 33NCS in 

Lake Huron water conducted at 12.8° C. for 21 hours

Lamprey larvae

MCL100 (Ppm) 

TFM 33NCS

Potency 

ratio 

(TFM/33NCS)

Rainbow

MAC 25 (ppm) 

TFM 33NCS

trout

Potency 

ratio 

(TFM/33NCS)

PAF

TFM 33NCS

3.0

3.0

2.0

3.0

3.0

3.0

4.0

4.0

4.0

Average

3.2

0.3

0.3

0.4

0.5

0.5

0.5

0.5

0.6

0.7

values

0.5

10

10

5

6

6

6

8

7

6

7

12.0

12.0

12.0

18.0

12.0

10.0

12.0

10.0

14.0

12.4

1.8

1.4

1.0

1.6

1.2

1.4

1.0

1.6

1.8

1.4

7

9

12

11

10

7

12

6

8

9

3.0

3.0

5.0

5.0

3.0

2.3

2.0

1.5

2.5

3.0

5.0

3.7

1.5

2.2

1.4

1.8

1.0

1.7

1.6

2.2

TABLE 12. Effect of temperature on toxicity of 33NCS to larval 

lamprey and rainbow trout in 21-hour bioassays

Temperature 

(C.)

Lamprey larvae

LC50 

(ppm)

95-percent

C.I.

Rainbow trout

LC 50 

(ppm)

95 -percent 

C.I.

Slope function

Lamprey 

larvae

Rainbow 

trout

1.7°.....

7.20.....

12.8°.....

18.3°.....

23.9°.....

0.80

0.28

0.26

0.25

0.17

0.70-0.92

0.25-0.32

0.22-0.30

0.21-0.29

0.14-0.12

1.72

1.36

1.20

1.20

0.25

1.47-2.01

1.17-1.58

1.07-1.34

1.07-1.34

0.21-0.30

1.20

1.13

1.25

1.28

1.20

1.25

1.25

1.22

1.22

1.20
12 Investigations in Fish Control 38 : Bureau of Sport Fisheries and Wildlife

TABLE 13. Effect of temperature on toxicity, 

selectivity, and utility of 33NCS in 21-hour Moassays

Temperature

(c.)

1.7°.........

7.2°.........

12.8°.........

18.3°.........

23.9°.........

Lamprey larvae 

MLCioo

(ppm)

1.2

0.4-

0.4.

0.4-

0.3

Rainbow trout 

MAC 25

(ppm)

1.4

1.0

1.0

1.0

0.2

PAF

0.2

1.5

1.5

1.5

-0.3

trout and 1.13-1.28 for larval lampreys) indicate that the narrow range of concentrations 

required to produce an "all-or-none" response 

held throughout the series of temperatures 

used.

The MLCIOO, MAC25, and PAF values obtained between 7.2° and 18.3° C. are the 

same and indicate a usable degree of selectivity (table 13). The complete loss of selectivity observed at 23.9° C. is probably a 

result of unnatural stress placed on the rainbow trout rather than a true reflection of 

toxicity, since this temperature is near the 

lethal threshold for the species.

Effects of water quality. The toxicity of 

33NCS and TFM decreases with an increase 

in alkalinity and conductivity (table 14). The

MAC25's increase from 0.6 to 3.5 ppm for 

33NCS and from 5.0 to 26.0 ppm for TFM in 

waters of increasing alkalinity. MLCIOO values 

show a parallel increase as best illustrated 

in figure 4. The potency ratios of 33NCS to 

TFM range from 4.0 to 8.5 ppm for MLCIOO 

and 7.1 to 10.0 ppm for MAC25in waters of 

various alkalinities. Therefore, alkalinity, 

conductivity, and pH and their interactions 

profoundly influence the toxicity of 33NCS 

and TFM to larval lamprey and rainbow trout.

Flowing water bioassay. Of the fish used, 

the sea lamprey was by far the most sensitive 

species to 33NCS in "simulated" stream tests 

in 1964 and 1965 (tables 15 and 16). In contrast, the trouts are among the most resistant. 

The LCSO's for various life stages of the sea 

lamprey range from 0.37 to 0.61 ppm. The 

calculated LCSO's for other fish ranged from 

0.66 to 1.75 ppm. Consistent with the laboratory tests, the narrow range of concentrations 

which delineate "all-or-none effects" are demonstrated by these low slope function values. 

Equally important is the narrow range of the 

confidence intervals about the LC50 values.

Data on several species of fish were insufficient to calculate the LC50 values, but 

we were able to determine the highest concentration which did not kill at least 25 percent of the test fish (table 17).

TABLE 14.--Variation in the toxicity of TFM and 33NCS in waters of various alkalinities in Michingan

[Tests conducted for 21 hours at 12.8° C]

Source of test water

Little Billies Creek, Cheboygan

Ocequeoc River, Presque Isle County. . 

Pere Marquette River, Mason County. . .

Trout River, Presque Isle County.....

Properties

Alkalinity 

(ppm CaC03 )

43.0

97.0

148.0

163.0

175.0 

184.0

189.0

200.0

of test water

Conductivity P 

(//mhos/l8°C.)

74.0 7

175.0 8

oo^; n 3

250.0 8 

274.0 7 

345.0 8

304.0 8

310.0 8

H Toxicant

.6 TFM

33NCS 

.2 TFM

33NCS

1

T»T?Tijr

33NCS

.3 TFM 

33NCS

.9 TFM 

33NCS

.2 TFM

33NCS

3

TT?TUf

33NCS

.1 TFM 

33NCS

Lamprey

MLCioo(PPm)

2.0

0.3

3.0

0.5 

7.0

0.8

5.0 

0.8

5.0 

1.3

7.0

1.3

8

r\

1.6

8.0 

1.1

Rainbow

MA.C 25 (ppm)

5.0

0.6 

12.0

1.2 

-i a n

2.2

16.0 

2.2

18.0 

2.5

25.0

3.5 

oy n

2.8

26.0 

3.0

PAF

1.5

1.0 

3.0

1.4 

1.6

1.8

2.2

1.8

2.6

1.0

2

/*

1.8 

2.0

0.8

2.3

1.8
Marking, King, Walker, Howell: Toxicity of 33NCS to Freshwater Fish and Lampreys 13

30-1 

20-

lOu

Z 2.0-

00 

CO

E 1.0-

O .3-

o 

U

I I

0 100 200 

Total Alkalinity in p.p.m. CaCO3

Figure 4. The effect of alkalinity on the toxicity of 

33NCS and TFM to rainbow trout and larval 

lamprey.

TABIE 16. Order of toxicity of 33NCS to mature fish in 21 hours 

(unless otherwise specified) in simulated stream tests in 

spring, 1965

Test organism

White sucker ( immature) ....

LC30 (ppm) 95 -percent C.I

0.38 0.37-0.40 

0.61 0.55-0.68 

0.85 

0.88 0.80-0.97 

0.90 0.80-1.02 

1.14 0.94-1.38 

1.19 1.02-1.39 

1.20 1.09-1.32 

1.24 1.08-1.43 

1.25 0.96-1.60 

1.30 1.15-1.47 

1.31 1.16-1.48 

1.50 1.32-1.71 

1.75 1.59-1.94

Slope 

., function

1.22 

1.08 

1.29 

1.27 

1.22 

1.20 

1.35 

1.35 

1.08 

1.08 

1.25 

1.15 

1.23 

1.20

TABLE 17. Highest concentrations of 33NCS in ppm which 

did not kill more than 25 percent of the fish in 

simulated stream tests

Species

Rainbow trout (immature 

Lake c hub

)........

Rock bass

Pumpkins eed sunfish (immature)..

Bluegill. .......................

Largemouth bass (immatu r*p ^

LotTDerch

1964

1.80 

1.80 

1.80 

1.80 

1.60 

1.80 

1.80

1.20

1965

1.59 

0.40 

1.59 

1.59 

1.59 

1.59 

1.59 

1.59 

1.59 

1.00 

C.50

TABI£ 15. Order of toxicity of 33NCS to mature fish in 21 hours 

(unless otherwise specified) in simulated stream tests in fall, 

1964

Test organism

Sea lamprey (transformer).

White sucker (immature)...

Rainbow trout (immature)..

LC30 (ppm)

0.37 

0.43

0.66

O f}/

0.89

1.00

1.05

1.13

1.18

' 1.65

95 -percent C.I.

0.32-0.43

O

rtQ r\ &f"

0.79-1.00

0.83-1.20

0.96-1.16

1.02-1.37

1.37-1.60

1.35-1.67

-i yon >7Q

1.29-2.11

Slope 

function

1.11 

Iort

1-35 

1.03

1.15

-i r\c>

1.43

1.35

1.35 

1.25

1 O&

1.06

1.22

1.14 .

-1 I f>

1 Stream-dwelling fish collected from the Rifle River, Ogemaw 

County, Mien.

33NCS is toxic to invertebrates, and 

LCSO's range from 0.78 to 1.8 ppm. The LC50 

value for 33NCS on stonefly nymphs was 

0.78 ppm (0.64 to 0.95) in 1964 with a slope 

function of 1.25. The LC50 for burrowing mayfly nymphs in 1965 was 1.26 ppm (1.07 to 1.49) 

with a slope function of 1.34. Estimates of the

LC50 values in 1964 and 1965 tests for other 

organisms were 1.4 and 1.26 ppm for snails, 

1.4 and 1.59 for clams, and 1.8 and 1.59 for 

crayfish. We also estimated the LC50 to be 

1.0 ppm for stoneflies and 1.59 for dragonfly 

nymphs in 1965.

Although the LC50 values of 33NCS varied 

among some species of fish from one raceway 

test to the other, a general pattern is evident. 

33NCS is almost uniformly nontoxic to centrarchids at the levels tested. In descending 

order of resistance, after the Centrarchidae, 

are the Salmonidae, Ictaluridae, Catostomidae, 

Cyprindae, Percidae, Percopsidae, and Petromyzonidae (tables 18 and 19). This listing of 

relative resistance to 33NCS is similar to one 

given by Howell (1966) for TFM.

Among the many species tested, both vertebrates and invertebrates, none is as sensitive to the toxic effect of 33NCS as the larval 

lamprey (LC50 = 0.38 to 0.43 ppm). The compound is also highly toxic to the lamprey at
14 Investigations in Fish Control 38: Bureau of Sport Fisheries and Wildlife

TABLE 18. Order of selectivity of 33NCS calculated from 

simulated stream tests in 1964 based on curves plotted 

using the data from table 15

Species

Yellow "Derch

Brown trout (stream dwelling)..

MA.C25 (ppm)

0.54

0.70

0.82

0.85

0.92

0.96

0.98

1.02

1.09

1.14

1.25 

1.30

1.40

1.47

PAF 1

-0.10

0.17

0.37

0.42

0.53

0.60

0.63

0.70

0.82

0.90

1.08 

1.17

1.33

1.45

Based on a MCLioo of 0.60 ppm.

TABLE 19. Order of selectivity of 33NCS calculated from 

simulated stream tests in 1965 based on curves plotted 

using the data from table 16

Species

Yellow Derch

MAC 2 g (ppm)

0.72

0.76

0.79

0.96

0.98

1.00

1.12

1.16

1.20

1.20

1.30

1.55

PAF 1

0.14

0.21

0.25

0.52

0.56

0.59

O

rirt

0 84

0.90

0.90

1.06

1.46

1 Based on a MLCioo of 0.63 ppm.

other stages in the life cycle (LC50 = 0.37 to 

0.61 ppm). The trout-perch may be as susceptible as the adult lamprey to 33NCS, but 

the data on trout-p$rch are limited, and we 

merely state that the LC50 is less than 0.50 

ppm.

The LC50 varied somewhat among the 

trouts in the two tests. The LC50 for immature (3- to 5-inch) rainbow trout was 1.18 

in 1964 and greater than 1.59 in 1965. The 

reason for this difference is unknown, but 

both values are within the limits observed 

during laboratory studies. In the 1964 raceway series, mature brown trout (7- to 14-inch) 

from two sources were compared. The LC50 

was 1.30 for hatchery stocks, whereas it was 

1.65 for fish collected from a stream. The

two values, noted during the same test, illustrate that toxicity may vary among fish from 

different sources. In both tests, brook trout 

were more sensitive to 33NCS than brown or 

rainbow trout.

The brown bullhead was one of the most 

resistant species during the 1964 test 

(LC50 = 1.60). In the second series, brown and 

yellow bullhead were slightly less tolerant 

than the trouts. the LC50 values for these two 

species are in close agreement (1.14 for 

brown bullhead and 1.24 for yellow bullhead 

during the 1965 tests).

There were some differences in the toxicity 

of 33NCS to suckers and minnows in the two 

series of tests. These differences were small 

except for the common shiner. The LC50 value 

in 1964 was 0.84 ppm, whereas the value in 

1965 was 1.75. It is difficult to account for 

these differences on the basis of water quality, temperature, or source of test animals, 

and it seems more likely to be a reflection of 

some basic physiological difference between 

the specimens used in each test.

DISCUSSION

At Hammond Bay, the 21-hour LC50 of 

33NCS to rainbow trout in Lake Huron water 

was 1.2 ppm. The 24-hour LC50 in standard 

reconstituted water at the Fish Control Laboratories was 0.3 ppm, four times the toxicity 

at approximately the same temperature and 

exposure time. The contrast is even greater 

in softer waters.

Bioassays on rainbow trout in harder water 

at La Crosse yielded an LC50 of 0.52 ppm in 

24 hours. Water of similar alkalinities from 

Pendills Creek and Lake Huron produced 

LC50 values of 0.74 and 1.42 ppm, respectively, in aerated bioassays in 21 hours.

The apparent differences in results of 

bioassays at Hammond Bay and the Fish 

Control Laboratories are attributable to 

variations in the quality of aerated and nonaerated waters. Data in table 9 indicate that 

an increase in the bicarbonate of bioassay 

water, with corresponding increase in pH and
Marking, King, Walker, Howell: Toxicity of 33NCS to Freshwater Fish and Sea Lampreys 15

alkalinity, reduces the toxicity of 33NCS 

considerably. Seasonal variations in pH and 

alkalinity in Lake Huron water show a similar 

effect (table 10). For a specific concentration 

of toxicant, the pH alone can effect either 

100 percent mortality or survival (table 8).

Aeration is a major influence on the toxicity 

of 33NCS; trout survive 0.5 ppm of 33NCS in 

reconstituted water only when the bioassay is 

aerated (table 7). This observation is substantiated further by the decreased toxicity in 

aerated bioassays at Hammond Bay.

Another factor influencing the toxicity of 

33NCS is the volume of fish per volume of 

test solution. Heavier loads of fish in test 

vessels are possible at Hammond Bay because 

of aeration. At the heavier loadings, the ratio 

of toxicant per unit of biomass is considerably 

less than with lighter loadings. The size of 

test fish also is a factor. In general, larger 

fish are more tolerant to 33NCS.

The toxicity of 33NCS at temperatures 

between 7° and 18° c. showed little variation 

at Hammond Bay and at the Fish Control 

Laboratories. Toxicity to larval lamprey, 

rainbow trout, and green sunfish tends to 

increase at the higher temperatures. The 

extreme temperatures of 1.7° and 23.9° C. 

produce more drastic variations in toxicity, 

and such variations are more noticeable in 

rainbow trout than in lamprey larvae.

PAF values for 33NCS were usually less 

than those calculated for TFM on rainbow 

trout in laboratory and simulated stream 

trials. Lower PAF values for other species 

in simulated stream trials were in the descending order of resistance to 33NCS: Centrarchidae, Salmonidae, Ictaluridae, Catastomidae, Cyprinidae, and Percopsidae. 

These PAF values suggest that 33NCS may be 

limited as a selective larvicide to situations 

involving only trouts and sunfishes.

CONCLUSIONS

1. 33NCS is rapidly toxic to a wide variety 

of fishes and invertebrates.

2. The toxicity and selectivity of 33NCS are 

comparable under different test condi10.

11,

tions even though absolute values may 

vary. Sea lamprey, bowfin, carp, and 

channel catfish are more sensitive than 

trouts or sunfishes.

33NCS was more toxic to fish in bioassays 

at the Fish Control Laboratories than at 

Hammond Bay. Water quality, aeration, 

loading, and size and source of test fish 

contributed to the differences in results 

in flowing and static bioassays.

The toxicity of 33NCS decreases with 

increasing pH, alkalinity, and hardness 

of water. A change in pH from 7.8 to 

8.2 is very critical, and it may cause 

100-percent mortality or permit survival 

of fish exposed to a given concentration.

The toxicity of 33NCS increases with 

temperature. At extreme temperatures 

the effect is more profound.

The regressions for 33NCS are nearly 

parallel for all species tested, even 

under different test conditions. Minute 

increments in concentrations delineate 

all-or-none effects as denoted by extremely low slope functions.

The toxicity of 33NCS varies with seasons. The bioassays indicate greater 

toxicity to rainbow trout from November 

through April.

Preexposure does not affect the ultimate 

lethal level but does appear to retard the 

rate of acute toxic action.

Trout did not recover after critical 

exposures to 33NCS.

33NCS is approximately 6 times as toxic 

to larval lamprey as TFM in most waters 

tested, but 33NCS does not yield as high 

PAF values as TFM under the same 

conditions.

PAF values are greater than 1.0 for 

trouts and sunfishes in simulated stream 

trials. However, MLC100 values for sea 

lamprey and MAC25 values for other 

species of fish in these studies produce 

PAF values which may seriously limit
16 Investigations in Fish Control 38; Bureau of Sport Fisheries and Wildlife

use of 33NCS for selective control of 

freshwater fish and sea lamprey larvae.

12. In field use, 33NCS would require rigid 

control since its performance is highly 

dependent on water quality.

13. The results of this study form a base 

for further investigation of the selective 

activities of 33NCS and closely related 

nitrosalicylanilides

REFERENCES

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1962. Three portable feeders for metering chemical into streams for control of sea lamprey. 

Progressive Fish-Culturist, vol. 24, no. 4, 

p. 190-192.

Applegate, Vernon C., John H. Howell, James W. 

Moffett, B. G. H. Johnson, and Manning A. Smith. 

1961. Use of 3-triflurornethyl-4-nitrophenol as a 

selective sea lamprey larvicide. Great Lakes 

Fishery Commission, Technical Report 1. 35 p.

Fox, I., L. S. Ritchie, and L. P. Frick.

1963. Effect of pH on molluscicidal activity of 

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Rico under laboratory conditions. Bayer A. G., 

Pflanzenschutz-Nachrichten, vol. 16, no. 5, 

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Howell, John H.

1966. The life cycle of the sea lamprey and a 

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and Lee H. Hanson.

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Kanayama, Richard K.

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Marking, Leif L.

1966. jvpJ-DDT as a reference toxicant in bioassays. U.S. Bureau of Sport Fisheries and 

Wildlife, Investigations in Fish Control, No. 10. 

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1962. Comparative studies on the influence of 

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U.S. GOVERNMENT PRINTING OFFICE : 1970 O - 404-V81