INVESTIGATIONS IN FISH CONTROL

9. Relation of Chemical Structure to Fish Toxicity 

in Nitrosalicylanilides and Related Compounds

10. Evaluation of p, p'-DDT

as a Reference Toxicant in Bioassays

11. Evaluation of an Electronic Method of 

Measuring Hematocrits of Fish

J

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. (Circular 185.)

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

(Circular 186.)

(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. (Resource Publication 7.)

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

Robert G. Needham. 1966. 16 p. (Resource Publication 8.)

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

Toxaphene, by Donald C. War nick. 1966. 9 p. (Resource Publication 9.)

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

Ahmed Mahdi. 1966. 10 p. (Resource Publication 10.)

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. (Resource Publication 

11.)

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. (Resource Publication 12.)

(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. 

(Resource Publication 13.)

10. Evaluation of p,p'-DDT as a Reference Toxicant in Bioassays, by Leif L. Marking. 1966. 

10 p. (Resource Publication 14.)

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

Schoettger and Arnold M. Julin. 1966. lip. (Resource Publication 15.)

Fish Control Laboratories

Bureau of Sport Fisheries and Wildlife

U.S. Department of the Interior

P. O. Box 862

La Crosse, Wisconsin 54602
INVESTIGATIONS IN FISH CONTROL

9. Relation of Chemical Structure to Fish Toxicity 

in Nitrosalicylanilides and Related Compounds

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

(Resource Publication 13, p. 1-12)

10. Evaluation of p, pr -DDT as a Reference Toxicant in

Bioassays

By Leif L. Marking (Resource Publication 14, p. 1-10)

11. Evaluation of an Electronic Method of Measuring 

Hematocrits of Fish

By Richard A. Schoettger and Arnold M. Julin 

(Resource Publication 15, p. 1-11)

Washington, D.C. 

February 1966

United States Department of the Interior, Stewart L. Udall, Secretary

Stanley A. Cain, Assistant Secretary for Fish and Wildlife and Parks

Fish and Wildlife Service, Clarence F. Pautzke, Commissioner

Bureau of Sport Fisheries and Wildlife, John S. Gottschalk, Director
INVEST/GA77ONS IN FISH CONTROL

9. Relation of Chemical Structure to Fish Toxicity 

in Nitrosalicylanilides and Related Compounds

By Charles R. Walker, Chemist 

Fish Control Laboratory 

Bureau of Sport Fisheries and Wildlife 

La Crosse, Wisconsin

Roland J. Starkey, Microbiologist 

Ben Venue Laboratories, Inc. 

Bedford, Ohio

Leif L. Marking, Chemist

Fish Control Laboratory

Bureau of Sport Fisheries and Wildlife

La Crosse, Wisconsin

U.S. DEPARTMENT OF THE INTERIOR

Fish and Wildlife Service 

Bureau of Sport Fisheries and Wildlife 

Resource Publication 13 

Washington . February 1966
CONTENTS

Page

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

Methods and materials .............................. 4

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

Nitrosalicylanilides ............................. 5

Mono-substituted nitrosalicylanilides. .............. 5

Poly-substituted nitrosalicylanilides ............... 8

Benzanilides .................................. 9

Discussion ...................................... 11

Conclusions. ..................................... 12

Summary ....................................... 12

Literature cited................................... 13
RELATION OF CHEMICAL STRUCTURE TO FISH TOXICITY 

IN NITROSALICYLANHIDES AND RELATED COMPOUNDS

By Charles R. Walker, Chemist 

Fish Control Laboratory, La Crosse, Wis.

Roland J. Starkey, Microbiologist 

Ben Venue Laboratories, Bedford, Ohio

and Leif L. Marking, Chemist 

Fish Control Laboratory, La Crosse, Wis.

ABSTRACT. Relations between chemical structures of salicylanilides 

and benzanilides and their toxicity to rainbow trout and goldfish were 

evaluated in standard, static bioassays. Single and multiple substitutions 

of alkyl-, nitro-, and halo- groups were tested. Substitution of hydroxyat ortho (2) accompanied by nitro- at meta (3) or sub-meta (5) on the 

benzoic acid moiety are basic requirements for toxic activity against 

fish. Halogenation at para (41 ) markedly affects the selective toxicity of 

the compounds to either rainbow trout or goldfish.

A search for potent piscicides is a primary 

function of the Fish Control Laboratory at La 

Crosse, Wis., and the Southeastern Fish Control Laboratory at Warm Springs, Ga. Candidate compounds are bioassayed to determine 

their potentials as selective or general fish 

toxicants. Among the more toxic compounds 

studied to date is antimycin A (Walker, 

Lennon, and Berger, 1964). Strong (1958) 

described the capacity of antimycin A to inhibit the electron-transport system in higher 

animals, and Derse and Strong (1963) suggested that the antibiotic may be useful as a 

toxicant highly selective to fish. Since antimycin A is a 3-formamidosalicylic lactone, 

interest was stimulated in the chemical and 

biological activities of N-substituted nitrosalicylates and formidosalicylates. Dickie 

et al. (1963) demonstrated that certain nitroand formido-substitutions were significantly 

more active biologically than others. We 

elected to investigate certain salicylic acid derivatives to detect relations between chemical 

structure and biological activity against fish.

Investigations with other test organisms have 

shown some relation between chemical structure of salicylanilides and toxicity. Taborsky 

et al. (1959, 1962, 1963) described the importance of specific substitutions to antimicrobial 

activity. Baichwal et al. (1960a) described fungicidal activities, and Schraufstatter (1962) 

demonstrated relations between structure and 

molluscicidal properties. Recently, Starkey and 

Howell (1965) pointed out the importance of 

molecular structure of salicylanilides to selective toxicity to the sea lamprey (Petromyzon 

marinus).

The object of this study was to ascertain the 

structural requirements of compounds to produce toxicity in two fishes. The compounds included certain substituted nitrosalicylanilides 

and benzanilides with consideration given to 

single and multiple substituents, and in particular to the locus of the nitro and halogen 

groups. The fish were rainbow trout (Salmo 

gairdneri) and goldfish (Carassius 

auratus).
METHODS AND MATERIALS

The bioassay methods used were described 

by Lennon and Walker (1964) for the evaluation of fish control agents. All bioassays were 

conducted in reconstituted water at 12°C. The 

rainbow trout were acquired from the Manchester, Iowa, National Fish Hatchery and 

acclimated to the laboratory water supply. 

The mean LC 50 of p,p'-DDT for them was 

6.8 parts per billion, with a range from 1.9 to 

14.0 p.p.b. (table 1). The goldfish were obtained from the Lake Mills, Wis., National 

Fish Hatchery and weighed from 0.7 to 2.5 

grams. The mean LC 50 of p,p'-DDT for them 

was 41.2 p.p.b., with a range from 27.0 to 

76.0 p.p.b. (table 1).

TABLE 1. Comparative sensitivities of different lots of fish 

reference standard p,p'-DDT expressed as the LC5 o value in p.

to the 

p.b.

Species and lot

Rainbow trout:

Goldfish:

Average 

weight 

(grams)

0.92

2.50

Date 

bioassayed

Mar. t,, 1964

LC 5 o 

(p.p.b.)

H.O

76.0

Toxicity of the compounds was determined 

at three concentrations (0.1, 1.0, and 10 parts 

per million) on rainbow trout and goldfish in 

2.5- or 15-liter glass containers. Ten fish 

were bioassayed at each concentration, and 

the effects of the chemicals upon the fish were 

recorded at 0.25, 0.5, 1, 3, 24, 48, 72, and 96 

hours. The data presented in this paper are 

confined to observations made at the end of 

3 and 48 hours, which appeared to be the critical times for differentiating the gross acute 

toxic effects.

Ben Venue Laboratories, Inc., Bedford, 

Ohio, furnished substituted salicylanilides and 

benzanilides for evaluation of relations between structure and piscicidal activity (fig. 1 

and table 2). Substituted nitrosalicylanilides 

of the general formula depicted in figure 1 

have been documented (Taborsky et al., 1959; 

Taborsky and Starkey, 1963; and an unpublished 

manuscript "Some substituted salicylanilides"

R6. R5.

SALICYLANILIDES

R2 = OH accompanied by one or more

of the following: 

R3 s N02,and/or Rg = N02,CI,Br, or

acetylamine 

R2«-R6« * N02,F,CI,Br,I,CH3,OCH3,

C 2 H 5' and/or phenylazo in 

one or more positions.

BENZANILIDES

R 2 -R5 = N02,CI, and/or OH with the

following: 

R2.-R6. = N02,CI,Br,I,CH3,OCH3,

and C 2 H 

positions.

in one or more

Figure 1. --General structure of substituted salicyianilides and benzanilides.

by Roland J. Starkey). They can be arbitrarily 

divided into five groups based upon substituents 

in the aniline moiety: (1) halonitrosalicylanilides; (2) halonitrosalicylotoluidides; 

(3) nitrosalicyloxylidides; (4) nitrosalicylanisidides; and (5) a miscellaneous group including 4f-phenylazo-3-nitrosalicylanilide and 

2'-ethyl-3-nitrosalicylanilide.

Salicylanilides are synthesized by the condensation of salicylic acid or a derivative with 

aniline or a derivative thereof. This produces 

a biphenyl molecule with a CONH bridge and 

is characterized by the presence of an ortho 

phenolic hydroxyl group (OH) in the acid 

moiety. Benzanilides, also biphenyl in structure with the CONH bridge but devoid of the 

hydroxyl group, are prepared in the same 

manner with a derivative of benzoic acid.

3-nitrosalicylanilides, in addition to a 

hydroxyl group, possess a nitro (NO 2 ) group 

at the 3 (meta) position of the acid moiety
TABLE 2. Compounds used in evaluating the relation of chemical structure to fish toxicity

Compound

1.

2.

3.

4.

5.

6. 

7. 

8. 

9. 

10.

11. 

12.

13. 

14. 

15.

16. 

17. 

18. 

19. 

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

Chemical name 1

3-nitrosalicylanilide

2 -iodo-3-nitrosalicylanilide

2 -ethyl-3-nitrosalicylanilide

3 -chloro-3-nitrosalicylanilide

3 -bromo-3-nitrosalicylanilide

3 -iodo-3-nitrosalicylanilide 

4 -chloro-3-nitrosalicylanilide 

4 -bromo-3-nitrosalicylanilide 

4 -iodo-3-nitrosalicylanilide 

4 -phenylazo-3-nitrosalicylanilide

2 -chloro-5-nitrosalicylanilide 

3 -fluoro-5-nitrosalicylardlide 

4 -chloro-5-nitrosalicylanilide 

4 -iodo-5-nitrosalicylanilide 

3 -chloro-5-acetamidosalicylanilide

2 ,4 ' , 6 ' -trichloro-3-nitrosalicylanillde 

3,4' -dichloro-3-nitrosalicylanilide 

2,5' -dibromo-3-nitrosalicylanilide 

4 -chloro-5-bromo-3-nitrosalicylanilide 

4 ,5-dibromo-3-nitrosalicylanilide

3-nitro-2',3'-salicyloxylidide

3-nitro-2 ' ,4 ' -salicyloxylidide

3-nitro-2 ' , 5 ' -salicyloxylidide

3-nitro-2 ' , 6 ' -salicyloxylidide

3' -chloro-3 -nitro-o_-salicylotoluidide

4 ' -chloro-3-nitro-o-salicylotoluidide

2 ' -chloro-3-nitro-p-salicylotoluidide

6 ' -chloro-3-nitro-2-sa:1- icy:1-0'toluid:i- de

3, 5 ' -dinitro-o-salicylotoluidide

2 ' ,3-dinitro-p-salicylotoluidide

4 ' -bromo-3-nitro-o-salicylotoluidide

5'-chloro-3-nitro-o-salicylanisidide

4 ' -chloro-2 ' , 5 ' -dimethoxy-3-nitrosalicylanilide

5 ' -methyl-o-salicylanisidide

4 ' -nitro-o-salicylanisidide

Compound

36.

37.

38.

39.

40.

41. 

42. 

43. 

44. 

45.

46. 

47. 

48. 

49. 

50.

51. 

52. 

53. 

54. 

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

Chemical name 1

2 '-nitro-p-salicylanisidide

2 ' ,3-dinitro-m-salicylanisidide

2'-chloro-3,4'-dinitrosalicylanilide

3-nitrosalicylanilide

benzanilide

3 ' -chloro-3-hydroxybenzanillde 

4 '-chloro-3-hydroxybenzaniUde 

2 '-chloro-2-nitrobenzanilide 

3 '-chloro-2-nitrobenzanilide 

4 ' -chloro-2-nitrobenzanilide

4 ' -bromo-2-nitroben2anilide 

3'-chloro-2-nitro-o-benzotoluidide 

3-nitrobenzanilide 

2 '-chloro-3-nitrobenzanilide 

3 ' -chloro-3-nitrobenzanilide

3 ' -chloro-3-nitro-p-benzotoluidide 

2 ' -chloro-4-nitrobenzanilide 

3 '-chloro-4-nitrobenzanilide 

5 ' -chloro-4-nitrobenzanilide 

p-chlorobenzanilide

3, 5-dinitrobenzanilide

2 '-chloro-3, 5-dinitrobenzanilide

3 ' -chloro-3, 5-dinitrobenzanilide

3 ' -bromo-3, 5-dinitrobenzanilide

4 '-bromo-3, 5-dinitrobenzanilide

4 ' -iodo-3, 5-dinitrobenzanilide

2 '-fluoro-3, 5-dinitrobenzanilide

3, 5-dinitro-o-benzotoluidide

3 , 5-dinitro-2 ' , 3 ' -benzoxylidide

3 ' -chloro-3, 5-dinitro-p-benzotoluidide

5 '-chloro-3, 5-dinitro-o-benzotoluidide

3 '-chloro-3, 5-dinitro-o-benzotoluidide

3,3' , 5-trinitrobenzanilide

2 ' ,3,4 ' j'S-tetranitrobenzanilide

5 ' -chloro-3, 5-dinitro-o-benzanisidide

2 ' , 5 ' -diethyl-3, 5-dinitrobenzanilide

1 A.C.S. or I.U.C. nomenclature

(fig. 1 and table 2). 5-nitrosalicylanilides differ with the locus of the nitro group being 

oriented to the five position. In several instances halogens have also been substituted 

at the 5 position to produce 5-chloro-3- 

nitrosalicylanilides or 5-bromo-3-nitrosalicylanilides. Variations in these compounds 

have been achieved by introducing single 

(mono) or multiple (poly) substituents in the 

anilide portion of the molecule. Diversity was 

attained with isomers by changing the locus 

of one or all of the substituents. In one case 

(compound 15, table 2) the 5-substituted nitro 

group has been reduced to an acetamido group.

RESULTS

NITROSALICYLANILIDES

Mono-substituted nitrosalicylanilides. 

This series, with the exception of 2l-ethyl-3- 

nitrosalicylanilides, 2 f-chloro-5-nitrosalicylanilide, and the 5-acetamldo- derivative 

of 3*-chloro-5-nitrosalicylanilide (compounds 3, 11, 15, table 3), effected a complete kill of rainbow trout during a 48-hour

test period at 1 p.p.m. Most of the substituted 3-nitrosalicylanilides at this concentration were lethal within 3 hours. 3-nitrosalicylanilide per se (compound 1, table 3) 

did not share this property of producing death 

quickly. Substituted 5-nitrosalicylanilides did 

not kill fish quickly at less than 10 p.p.m. 

4'-phenylazo-3-nitrosalicylanilide and 4'- 

iodo-3-nitrosalicylanilide (compounds 9 and 

10, table 3) were the only mono-halonitrosalicylanilides lethal to trout at 0.1 p.p.m.

Seven of the mono-substituted nitrosalicylanilides (compounds 6-10 and 13-14, table 3) 

killed all rainbow trout and goldfish at 

10 p.p.m. Two of them, 4'-chloro-3-nitrosalicylanilide and 4f-iodo-3-nitrosalicylanilide, were effective within 3 hours. 4fchloro-3-nitrosalicylanilide was lethal to 

goldfish at 0.1 p.p.m. but not to rainbow trout 

during the 48-hour test. Seven others (Nos. 1, 

2, 4, 5, 11, 12, and 15) were lethal to trout at 

1 p.p.m. but varied in toxicity to goldfish.

The toxicity of mono-substituted nitrosalicylanilides to goldfish increased as the locus 

of the substituent in the aniline moiety was
TABLE 3. Relation of chemical structure to piscicidal activity of mono-substituted nitrosalicylanilides to two species of fish at 12°C.

56 o

Compound

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

Locus of substituent on the structure

2

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

3

N02

N02

N02 

N0 2

N02

N02

N02

N02

N02

N0 2

_

_

4

_

~

_

_

~

_

_

5

_

_

._

--

_

N02

M02

N02

N02

AcAra3

6

_.

_

_

_

_

_

--

--

2'

_

I

C 2 H5

_

_

Cl

_

--

3'

_

_

Cl

Br

I

__

_

--

_

F

Cl

4'

_

_

~

Cl

Br

I

PA2

_

_

Cl

I

5'

_

 .

_

~

_

_

6'

_

--

_

_

--

__

--

Toxicity of the compound to 

Rainbow trout at 

0.1

p. p.m.

0

0

0

0

0

0

0

0

9

10

0

0

0

0

1

1.0

p. p.m.

10

10

0

10

10

10

10

10

10

10

8

10

10

10

9

10.0

p. p.m.

10

10

_

10

10

10

10

10

10

10

10

10

10

10

10

Goldfish at 

0.1

p. p.m.

0

0

0

0

0

0

10

0

0

0

1

0

1

1

0

1.0

p. p.m.

0

0

0

6

9

10

1 10

10

1 10

10

1

1

10

10

1

10.0

p.p.m.

10

1 10

__

1 10

1 10

1 10

1 10

1 10

1 10

10

1 10

10

1 10

1 10

10

1 All dead at 3 hours.

2 Phenyl-azo.

3 Acetamido.

oriented from ortho (2 ! ) to para (41 ). For example, although iodo-3-nitrosalicylanilides 

were evaluated at all three positions, only 

2'-iodo-3-nitrosalicylanilide was nontoxic to 

goldfish at 1 p.p.m. S'-iodo- and 4'-iodo-3- 

nitrosalicylanilide are lethal to both species 

at this concentration, but 4'-iodo-3-nitrosalicylanilide was lethal to both species within 

3 hours. In addition the latter compound 

killed 9 out of 10 rainbow trout at 0.1 p.p.m.,

but the ortho (2 1 ) and meta (31 ) isomers were 

ineffective. Likewise, 3'-chloro-3-nitrosalicylanilide required 48 hours to kill 6 out of 

10 goldfish and all of the rainbow trout at 

1 p.p.m. 4l-chloro-3-nitrosalicylanilide produced total kills of both species within 3 hours. 

Further trials revealed that 4 l-chloro-3- 

nitrosalicylanilide is far more toxic to goldfish than to rainbow trout at 0.1 p.p.m. 

(tables 4 and 5).

TABLE 4. Relation of chemical structure of nitrosalicylanilides to selective piscicidal activity for rainbow trout and goldfish at 12°C.

Compound

10.

19.

20.

9.

31.

17.

8.

6.

13.

14.

33.

7.

Locus of substituent on the structure

2

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

3

N0 2

N02

N02

N02

N02

N02

N02

N0 2

_

N02

N02

4

_

--

_

5

_

Br

Br

--

_

N02

N02

6

, _

--

-_

_

_

_

--

2'

..

CH3

_

_

_

CH3 0

y

..

Cl

i

4'

PA2

Cl

Br

I

Br

Cl

Br

Cl

I

Cl

Cl

5'

_

_

~

_

CH3 0

6'

_

--

--

__

_

_

Toxicity of the compound to 

Hainbow trout at 

0.1 

p.p.m.

10

10

10

9

2

1

0

0

0

0

0

0

1.0 

p.p.m.

10

10

10

10

10

10

10 1 10

10

10 1 10

1 10

10.0 

p.p.m.

10

10

10

10

10

10

10

10

10

10

10

10

Goldfish at 

0.1 

p.p.m.

0

3

2

0

0

1

0

0

1

1

1

10

1.0 

p.p.m.

10

10

10

10

10

10

1 10

10

10

10

10

1 10

10.0 

p.p.m.

10

10

10

10

10

10

10

10

10

10

10

10

1 All dead at 3 hours.

2 C 6H5-N=N-(phenyl-azo).
TABLE 5. Obligatory structural requirements for 4'-chloro-3-nitrosalicylanilide as a selective piscicidal agent for goldfish and rainbow trout

5 6

Compound

7.

17.

4.

41.

44.

50.

58.

42.

54.

2

OH

OH

OH

_

NOj

"

3

N02

N02

N02

OH

--

N02

N02

OH

"

4

..

--

..

--

--

N02

5

..

--

..

N02

--

6

--

_

--

-

--

2'

..

"

_

"

"

--

3'

Cl

Cl

Cl

Cl

Cl

Cl

--

4'

Cl

Cl

--

_

--

"

Cl

5'

--

--

_

"

--

--

6'

--

--

_

--

--

--

Toxicity of the compound to 

Rainbow trout at 

p.p.m.

0

1

0

0

0

0

0

0

0

p.p.m.

1 10

1 10

10

0

0

2

0

0

0

p.p.m.

1 10

1 10

1 10

10

10 1 10

7

10

10

Goldfish at--

p.p.m.

10

1

0

0

0

0

0

0

0

p.p.m.

1 10

10

6

0

0

0

0

0

0

p.p.m.

1 10

1 10

10

7

6

10

0

2

6

1 All dead at 3 hours.

Poly-substituted nitrosalicylanilides. 

Tests were made to determine whether toxicity and selectivity could be enhanced with 

poly-substituted derivatives of 3-nitrosalicylanilide. This series includes compounds 

with multiple substituents, some containing 

a halogen and a methyl group (CH 3 ) in the 

aniline moiety (halo-3-nitrosalicylotoluidides),

some with two methyl groups (3'-nitrosalicyloxylidides), and others with halogenatedmethoxy (CH 3O) derivatives (halo-3- 

nitrosalicylanisidides) (table 6).

3',4l-dichloro-3-nitrosalicylanilide (compound 17, tables 2, 4, and 6) killed all goldfish and rainbow trout at 1 p.p.m., but 9 out

TABLE 6. Relation of chemical structure to piscicidal activity of poly-substituted nitrosalicylanilides to two species of fish at 12 C.

2' 3'

Compound

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27. 

28 .

29.

30.

31.

32.

33.

34. 

35.

36.

37.

38.

39,

Locus of substituent on the structure

2

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH 

OH

OH

OH

OH

OH

OH

OH 

OH

OH

OH

OH

OH

3

N02

N02

N02

N0 2

N02

N02

N02

N02

N02

N02

N02

N02

N0 2 

N02

N02

N02

N0 2

NOj

N02

N02 

N02

N02

N02

N0 2

4

--

_

--

_

--

--

_

"

..

~

--

--

--

5

--

Br

Br

_

--

_

-.

--

..

--

6

_

._

--

_

--

_

--

-

..

--

--

--

2'

Cl

_

Br

CH3

CH3

CH3

CH3

CH3

CH3

Cl

CH3

N02

CH3

CH3 0

CH3 0

CH3 0

N02

N02

Cl

3'

Cl

--

..

CH3

_

--

Cl

"

-_

--

--

--

--

4'

Cl

Cl

Cl

Br

_

CH3

~

_

Cl

CH3

CH3

Br

..

Cl

N02 

CH3 0

N02

--

5'

_

Br

_

CH3

~

_

-

N0 2

,.

--

Cl

CH3 0

CH3

CH3 0

6'

Cl

_

..

..

CH3

_

Cl

_

"

..

--

Toxicity of the compound to 

Rainbow trout at 

0.1 

p.p.m.

0

1

0

10

10

0

0

0

0

1

0

1

0

0

0

2

0

0

0 

0

0

0

0

0

1.0 

p.p.m.

0

1 10

10

1 10

1 10

10

10

9

1

10

1 10

1 10 

0

0

10

1 10

1 10

1 10

9 

2

8

10 1 10

0

10.0 

p.p.m.

1 10

!. 10

1 10

1 10

1 10

1 10

1 10

1 10

10

1 10

1 10

1 10 

10

1 10

1 10

1 10

1 10

1 10

10 

1 10

1 10

1 10

1 10

10

Goldfish at--

0.1 

p.p.m.

0

1

0

3

2

2

0

0

0

0

0

1 

0

0

0

0

0

1

1 

1

0

0

2

0

1.0 

p.p.m.

0

10

3

10

10

0

1

0

0

2

10

1 10 

0

0

0

10

1

10

0 

0

4

0

10

0

10.0 

p.p.m.

10

1 10

1 10

1 10

1 10

10

1 10

10

10

10

1 10

1 10 

0

9

10

1 10

1 10

1 10

10

10

1 10

0

1 10

10

1 All dead at 3 hours.
of 10 of each species survived at 0.1 p.p.m. 

Compared with 4'-chloro-3-nitrosalicylanilide, this compound was less toxic to goldfish but more toxic to rainbow trout. Also, 

2 t ,4',6f-trichloro-3-nitrosalicylanilide (compound 16, table 6) was toxic to goldfish and 

rainbow trout at 10 p.p.m. but not at 1 p.p.m. 

It thus appears that the receptors of goldfish, 

sensitive to 4f-chloro-3-nitrosalicylanilide, 

cannot easily accommodate bulkier molecules 

irrespective of a common para (4') substituent. A similar observation on the sea lamprey was made by Starkey and Howell (1965).

A change in locus of the nitro- group from 

meta (3) to sub-meta (5) on the salicylic acid 

moiety of para (4') halogenated anilides resulted in slightly more toxicity to goldfish 

than rainbow trout at 0.1 p.p.m. (compounds 13 

and 14, table 6). The toxicity of 5-nitrosalicylanilides to rainbow trout and goldfish was reduced when the halogen was moved to the 

meta (31 ) position. The ortho (2 f ) substitution 

was even less toxic.

Among the poly-substituted 3-nitrosalicylanilides, 4',5-dibromo-3-nitrosalicylanilide 

and 4f-bromo- 5- chloro- 3- nitrosalicylanilide 

(compounds 19-20, tables 4 and 6) were lethal 

to rainbow trout at 0.1 p.p.m. but only slightly 

toxic to goldfish. At 1 p.p.m. both compounds 

were lethal to trout within 3 hours whereas 

goldfish succumbed later during the 48-hour 

test.

The toxicity of 3-nitrosalicyloxylidides 

(compounds 21-24, table 6) to rainbow trout 

varies with the loci of the methyl (CH 3 ) substituents in the aniline moiety. Each compound in this series has an ortho (21 ) methyl 

group in common but differs with the 

ring position of the second substituent either 

at the meta (3'), para (4'), sub-meta (5*), or 

sub-ortho (6*) positions. The 2 I,3I - and 2 f ,4fisomers are completely lethal to rainbow trout 

at 1 p.p.m. In comparison, the 2',5f- and 

2',6f- isomers produce 90- and 10-percent 

mortality, respectively. None of the isomers 

was toxic to goldfish except at ,10 p.p.m. A 

similar variation in the toxicity of 3-nitrosalicyloxylidides of the sea lamprey was reported by Starkey and Howell (1965).

Halo-3-nitrosalicylotoludides were tested to 

ascertain if the activity of 3-nitrosalicyloxylidides is significantly altered by substituting a halogen, nitro, or methoxy group 

for a methyl group (table 7). 3-nitro-2,3- 

salicyloxylidide and 3-nitro-2,4-salicyloxylidide (compounds 21 and 22) were not 

toxic to goldfish at 1 p.p.m. Substitution of 

the meta (3 1 ) and para (41 ) with chlorine 

atoms, however, produced compounds (3fchloro-3-nitro-o-salicylotoluidide and 4fchloro- 3-nitro-o- salicylotoluidide, compounds 25 and 26) which were lethal at 

1 p.p.m. but not at 0. 1 p.p.m. Substitution of 

the ortho (2 f ) methyl group with a nitro group, 

as in the case of 2 f ,3-dinitro-p-salicylotoluidide (compound 30, table 7), resulted in a 

compound which was nontoxic to goldfish at 

1 p.p.m.

Other compounds in which one of the methyl 

groups was replaced by a methoxy group (3- 

nitrosalicylanisidides) were relatively nontoxic to goldfish at 1 p.p.m. The only exception was 2',3-dinitro-p-salicylanisidide (compound 36) which killed 4 of 10 goldfish. Its 

isomer, 2l ,3-dinitro-5-salicylanisidide (compound 37), was nontoxic to goldfish at 

10 p.p.m. but killed all rainbow trout at 

1 p.p.m. Altering the loci of chloro and 

methyl groups, as in the case of 2'-chloro-3- 

nitro-6-salicylanisidide (compound 28) eliminated toxicity to goldfish at 10 p.p.m. In no 

instance did the halo-3-nitrosalicylotoludides, 3-nitrosalicyloxylidides, and 3- 

nitrosalicylanisidides exhibit selective toxicity to goldfish over rainbow trout. Only one 

compound, a 2',5f-dimethoxy-3-nitrosalicylanilide, demonstrated a slight toxicity to goldfish at 0. 1 p.p.m. and did not affect rainbow 

trout.

BENZANILIDES

Benzanilides in this series (table 8), differing from salicylanilides by the absence of 

an ortho hydroxyl substituent, include benzanilide (compound 40), meta (3) hydroxybenzanilides (compounds 41-42), ortho (2) nitrobenzanilides (compounds 43-47), meta

(3) nitrobenzanilides (compounds 48-51), para

(4) nitrobenzanilides (compounds 52-54), para

8
TABLE 7. Relation of chemical structure to piscieidal activity of 3-nitrosalicyloxylidides, 3-nitrosalieylanisidides, and 3-nitrosalicylotoluidides

to two species of fish at 12°C.

21.

25.

22.

26.

27.

38.

30.

36.

35.

29.

32.

34.

37.

28.

Locus of substituent on the structure

2

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

3

N02

N02

N02

N02

N02

N02

N02

N02

N02

N02

N02

N0 2

N02

N02

4

._

5

..

~

_

"

6

..

_

2'

CH3

CH3

CH3

CH3

Cl

Cl

N02

N02

CH3 0

CH3

CH30

CH30

N02

Cl

3'

CH3

Cl

_

~

_

~

_

~

4'

CH3

Cl

CH3

N02

CH3

CH30

N02

_

__

__

5'

~

_

_

_

--

_

N02

Cl

CH3

CH3 0

~

6'

_

_

_

_

_

"

_

_

_

._

CH3

Toxieity of the compound to 

Rainbow trout at 

0.1 

p. p.m.

0

1

0

0

1

0

0

0

0

0

0

0

0

0

1.0 

p. p.m.

10

10

10 1 10

1 10

1 10

10

8

2

0

1 10

9

10

0

10.0 

p. p.m.

10

1 10

10

1 10

1 10

1 10

1 10

1 10

1 10

1 10

1 10

10

1 10

10

Goldfish at 

0.1 

p. p.m.

2

0

0

1

1

2

0

0

1

0

1

0

0

0

1.0 

p. p.m.

0

10

1

1 10

1 10

10

0

4

0

0

10

0

0

0

10.0 

p. p.m.

10

1 10

10

1 10

1 10

1 10

10

1 10

10

9

1 10

0

0

0

1 All dead at 3 hours.

TABLE 8. Relation of chemical structure to piscieidal activity of mono- and poly-substituted benzanilides to two species of fish at 12°C.

560 2' y

V C - HH

Compound

40.

41.

42.

43.

44.

45.

46.

2 47.

2 48.

49.

50.

2 51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

2 61.

62.

2 &3.

2 64.

65.

66.

2 67.

68.

69.

70.

71.

Locus of substituent on the structure

2

N02

N02

N02

N02

N02

_

_

_

_-

_

-_

..

_.

3 1

OH

OH

_

--

"

N02

N02

N02

N02

-.

--

N02

N02

N02

N02

N02

N02

N02

N02

N02

N02

N02

N02

N02

N02

N02

N02

4

_

_

N02

N02

N02

Cl

_

_

_

_

"

5

_

"

_

N02

N02

N02

N02

N02

N02

N02

N02

N02

N02

N02

N02

N02

N0 2

N02

N02

6

_

_

--

_

~

_

~

_

"

_

2'

"

Cl

CH3

_

Cl

Cl

_

Cl

_

F

CH3

CH3

CH3

CH3

_

N02

CH3 0

C 2H5

3'

Cl

_

Cl

Cl

_

Cl

Cl

Cl

_

Cl

Br

_

--

_

Cl

Cl

N02

~

4'

__

Cl

_

__

Cl

Br

_

CH3

Cl

_

Br

I

_

CH3

CH3

_

N02

"

5'

_

_

_

__

_

"

_

--

_

_

Cl

"

_

Cl

6'

__

~

_

"

._

_

_

_

_

C 2H5

Toxieity of the compound to 

Rainbow trout at 

9.1 

p. p.m.

0

0

0

0

0

0

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

0

3 0

1.0 

p.p.m.

0

0

0

0

0

0

0

0

0

0

2

0

0

0

0

0

0

0

0

4

0

0

0

0

0

0

0

0

0

0

0

3 0

10. 0 

p.p.m.

0

10

10

0

10

10

10

0

10

10

1 10

0

0

0

10

0

0

0

7

0

0

0

10

10

1

2

1 10

1 10

1 10

10

0

0

Goldfish at 

0.1 

p.p.m.

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

0

0

0

0

0

0

1.0 

p.p.m.

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

1

0

10.0 

p.p.m.

0

7

2

0

6

5

2

0

10

1 1 10

0

0

0

6

0

0

0

0

0

0

0

10

8

0

0

0

0

0

0

0

1

1 All dead at 3 hours.

2 Precipitation at 10 p.p.m.

3 Sounding.
(4) chlorobenzanilides (compound 55), and 

3,5-dinitrobenzanilides (compounds 57-71). 

As in the substituted salicylanilides, the substituents (mono or poly) are either halogen, 

methyl, or nitro groups on the anilide portion 

of the molecule (fig. 1, table 2).

In general, the benzanilides are relatively 

nontoxic to rainbow trout and goldfish (tables 

5 and 8). Only three (compounds 48, 50, and 

62) were lethal to goldfish at 10 p.p.m. It 

appears that substituted benzanilides have 

minimal toxicity in comparison with salicylanilides. This is attributed to the lack of an 

ortho hydroxyl substituent. These findings 

agree with those of Baichwal et al. (1960a) 

and Starkey and Howell (1965). For example, 

3'-chloro-2-nitrobenzanilide and 4'-chloro2-nitrobenzanilide (compounds 44 and 45, 

tables 5 and 8) were the most toxic to both 

species. The speed of activity was increased 

markedly by moving the nitro group to the 

meta (3) position in the acid moiety. Substitution of the nitro group at the para (4) 

position produced compounds intermediate in 

toxicity.

The 3,5-dinitrobenzanilides were relatively 

insoluble at 10 p.p.m. and were therefore 

difficult to bioassay. They were, with several 

exceptions, selective to rainbow trout at 

10 p.p.m. (table 5). Only two (compounds 62 

and 63) were significantly toxic to goldfish at 

this concentration. 3,3',5-trinitrobenzanilide was more toxic than 2l,3,4',5-tetranitrobenzanilide (compounds 68 and 69) and may 

reflect the effects of saturation on both toxicity and selectivity.

Another group of benzanilides (compounds 

41, 44, 50, 53, and 58) possess a meta (31 ) 

chloro-substituent and vary only in the locus 

of a substituent in the acid moiety (table 8). 

Of them, compounds 44, 50, 53, and 58 are 

various substituted nitrobenzanilides and 

compound 41 is a substituted 3-hydroxybenzanilide. None has the ortho hydroxyl substituent characteristic of salicylanilides. Compound 53 which has the nitro substituted in 

the para (4) position was nontoxic to both 

fishes. The other compounds (41, 44, 50, and 

58) were lethal to rainbow trout at 10 p.p.m., 

but only compound 50 was toxic at 1 p.p.m.

Compounds 41, 44, and 50 varied in their 

toxicity to goldfish, and only compound 50 

killed all specimens at 10 p.p.m. None was 

toxic to the species at 1 p.p.m. Compound 58 

was nontoxic at either concentration.

2l ,6'-diethyl-3,5-dinitrobenzanilide (compound 71, table 8) was nontoxic to rainbow 

trout and goldfish but caused sounding reaction among the rainbows at 0.1 and 1 p.p.m.

DISCUSSION

Chemical structures which exhibit the 

greater toxicity to rainbow trout and goldfish 

were nitrosalicylanilides. We attribute this tc 

the ortho (2) hydroxy substitution on the acid 

moiety. Baichwal et al. (1960a) and Starkey ar 

Howell (1965) made similar findings with respect to fungi and sea lampreys respectively. 

In contrast, benzanilides which do not contain the hydroxy substitution in the ortho 

position are relatively nontoxic to the test 

fishes.

The ortho (2) hydroxy substitution also 

appears to accelerate the biological activity 

of a compound, because the rainbow trout and 

goldfish often succumbed in tests within 3 

hours.

The substitution of meta (3 1 ) nitro on the 

benzoic acid moiety of benzanilides produced 

greater toxicity to rainbow trout and goldfish 

than other nitro substitutions. A nitro substitution in the para (4) position had the least 

effect of any nitro substitution.

Substitutions of halogens in the para (4*) 

position on the aniline moiety of salicylanilides markedly increased toxicities to fish. 

They also increase the speed of toxic action. 

Furthermore, the size of halogen atom has a 

dramatic effect on selective activity between 

rainbow trout and goldfish. A para substitution of iodine (atomic weight 127) is most 

toxic to rainbow trout, whereas bromine 

(atomic weight 80) is equally toxic to the two 

species. Chlorine (atomic weight 35) in the 

para position, however, is selectively toxic 

to goldfish.

10
Of the halogen substitutions, those in the 

3rtho (2 1 ) position on the aniline moiety of 

either benzanilides or salicylanilides had 

teast effect. One noteworthy exception occurred when an additional substitution of a 

litro was made in the para (4 1 ) position. The 

result was greater toxicity to goldfish over 

rainbow trout. Similarly, the chloro substitution in the para (41 ) position in 4f-chloro-3- 

litrosalicylanilide caused selective toxicity 

to goldfish. This particular change in the 

structural configuration strengthens the concept that selective toxicity occurs when the 

molecular geometry of a toxicant fits the 

receptor geometry of the fish. Baichwal et al. 

(1960b) investigated the fungicidal properties 

3f salicylanilides and derivatives and indicated that activity may be attributed to interference to an enzyme or enzyme system(s) by 

hydrogen bonding or chelation via the phenol 

(ortho (41 ) hydroxy-) and amidic oxygen 

atoms. Furthermore, according to Ariens 

(1964), the introduction of an electron spending group, such as the hydroxy (-OH) in the 

ortho or jpara_ position, increases the electron 

density in a conjugated system. Conversely, 

he nitro group decreases the electron density 

of this system.

Our further substitution of a bromine in the 

sub-meta (5) position on the salicylic acid 

moiety of the para (41 ) halo-3-nitrosalicylanilides gave even greater toxic action to fish in 

general. Baichwal et al. (1960a) noted a similar increase in toxicity by 5-chloro substitutions on fungicides.

CONCLUSIONS

The chemical structure of salicylanilides 

and benzanilides was related to toxicity and 

selectivity to rainbow trout and goldfish. 

Salicylanilides were more toxic than benzanilides to the fishes. The ortho (2) hydroxy 

substitution of salicylanilide accelerates biological activity against fish. The meta (3) 

nitro substitution on the salicylanilides and 

benzanilides increases toxicity to fish. The 

para (4') halogen substitution on nitrosalicylanilides markedly increased toxicities to fish 

and enhanced the speed of toxic action. The

size of the halogen atom substituted on 3- 

nitrosalicylanilide affected selective toxicity 

between rainbow trout and goldfish. The ortho 

(2 1 ) halogen substitution on 3-nitrosalicylanilide became selectively toxic to goldfish by 

adding a para (41 ) nitro on the aniline moiety. 

The para (4 1 ) halogen substitution on 5-nitrosalicylanilides caused slight selective toxicity 

to goldfish. A para (41 ) chloro substitution on 

the 2 f ,5f-dimethoxy-3-nitrosalicylanilide resulted in selective toxicity to goldfish. A 

bromine substituted in the sub-meta (5) position on para (41 ) halo-3-nitrosalicylanilides 

increased toxicity to fish.

SUMMARY

Relations between the chemical structures 

of salicylanilides and benzanilides and their 

toxicity to rainbow trout and goldfish were 

investigated. Single and multiple substitutions 

of alkyl, nitro, and halo groups on the salicylanilides and benzanilides were tested in 

standard, static bioassays with fingerling fish 

at the Fish Control Laboratory.

The nitro salicylanilides were more toxic to 

the fish than benzanilides. A hydroxy substitution at ortho (2) appears to accelerate toxic 

action. The nitro substitution at meta (3) or 

sub-meta (5) on the benzoic acid moiety 

caused greater toxicity to the fish than other 

nitro substitutions.

Halogens in the para (41 ) position on the 

aniline moiety of salicylanilides markedly increased toxicities to fish and accelerated 

toxic action. The size of the halogen atom was 

found to have a great effect on selective activity between the two fishes. Iodine in the 

para (41 ) position produced greater toxicity 

to rainbow trout, whereas chlorine in the 

same position brought about an increase in 

toxicity to goldfish.

Halogens in the meta position were less 

toxic than para, and least toxic in the ortho 

position. An exception occurs when the chloro 

in the ortho (21 ) position was accompanied by 

a nitro in the para (41 ) position. The resulting 

compound was selectively toxic to goldfish. 

Halogenation in the sub-meta (5) position on 

the salicylic acid moiety produced even 

greater toxicity to both species.

11
The results of changes in structural configuration of salicylanilides support the concept 

that general and selective toxicity occurs 

when molecular geometry of a toxicant conforms to the receptor geometry of the fish.

LITERATURE CITED

Ariens, E. J.

1964. Molecular Pharmacology, the mode 

of action of biologically active compounds. Vol. 1. Academic Press, New 

York. 503 p.

Baichwal, R. S., R. M. Baxter, S. I. Kandel, 

and G. C. Walker.

1960a. Antifugal action of salicylanilide. 

II. Canadian Journal of Biochemistry and 

Physiology, vol. 38, p. 245-251.

Baichwal, R. S., R. M. Baxter, and G. C. 

Walker.

1960b. The lack of a reversing effect of 

certain metallic ions on the inhibitory 

action of salicylanilide. Canadian Journal 

of Microbiology, vol. 6, p. 595-597.

Derse, Phillip H., and F. M. Strong.

1963. Toxicity of antimycin to fish. Nature, 

vol. 200, No. 4906, p. 600-601.

Dickie, J. P., M. E. Loomans, T. M. Parley, 

and F. M. Strong.

1963. The chemistry of antimycin A. XI. 

N-substituted 3-formamidosalicylic 

amides. Journal of Medical Chemistry, 

vol. 6, p. 424-427.

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

1964. Investigations in fish control. 1. Laboratories and methods for screening fishcontrol chemicals. U.S. Bureau of Sport 

Fisheries and Wildlife, Circular 185. 

15 p.

Schraufstatter, E.

1962. Chemical development of Bayluscide 

Pflanzenschutz-Nachrichten - Bayer, 

vol. 15, p. 25-33.

Starkey, R. J., and J. H. Howell.

1965. Substituted nitrosalicylanilides: a 

new class of larvicides selectively toxic 

toward sea lamprey, Petromyzon 

marinus. (In press).

Strong, F. M.

1958. Topics in microbial chemistry. 

John Wiley and Sons, New York. 166 p.

Tabor sky, Robert G., Grant D. Darker, and 

Saul Kaye.

1959. Substituted salicylanilides with antimicrobial activity. Journal of the Amer< 

ican Pharmaceutical Association, vol. 4f 

No. 9, p. 503-507.

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

1962. Substituted salicylanilides. II. Further antimicrobial and other studies witl 

halonitrosalicylanilides. Journal of 

Pharmaceutical Sciences, vol. 51, No. 1! 

p. 1152-1155.

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

1963. Substituted salicylanilides. III. New 

salicylanilides and related compounds 

with antimicrobial activity. Journal of 

Pharmaceutical Sciences, vol. 52, No. 6, 

p. 542-545.

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

Bernard L. Berger.

1964. Investigations in fish control. 2. 

Preliminary observations on the toxicity of antimycin A to fish and other 

aquatic animals. U.S. Bureau of Sport 

Fisheries and Wildlife, Circular 186. 

18 p.

12
INVESTIGATIONS IN FISH CONTROL

1O. Evaluation of p,p-DOT

as a Reference Toxicant in Bioassays

By Leif L. Marking, Chemist 

Fish Control Laboratory 

Bureau of Sport Fisheries and Wildlife 

La Crosse, Wisconsin

U.S. Department of the Interior

Fish and Wildlife Service 

Bureau of Sport Fisheries and Wildlife 

Resource Publication 14 

Washington . February 1966
CONTENTS

Page

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

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

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

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

Conclusions. ..................................... 8

Summary ....................................... 9

Literature cited................................... 9
EVALUATION OF p,p'-DDT AS A REFERENCE TOXICANT IN BIOASSAYS

By Leif L. Marking, Chemist 

Fish Control Laboratory, La Crosse, Wis.

ABSTRACT. p,p'-DDT was tested as a reference standard toxicant 

against 19 species of freshwater fish, including 39 lots from 10 sources. 

In particular, the rapidity, nonselectivity, and consistency of its toxicity 

to fish were evaluated in 96-hour static bioassays. The chemical was 

rapidly and consistently toxic to lake trout, carp, green sunfish, bluegill, 

and yellow perch. It lacked either rapid or consistent toxicity to rainbow 

trout, brook trout, goldfish, fathead minnows, and longear sunfish in 96- 

hour tests. Thus, p,p'-DDT is of limited usefulness as a reference standard toxicant in large bioassays with many species of fish.

Necessity for standardization of facilities 

and techniques in bioassay,andreproducibility 

and comparability of results from test to test 

and investigation to investigation was recognized early in the fish control program. Others 

have also emphasized this necessity. Hart, 

Doudoroff, and Greenbank (1945) stated the 

need for a reference evaluation in bioassays 

to serve as a link between work of different 

investigators and to compare the relative 

toxicities of substances. Warren and Doudoroff

(1958) observed that living organisms are 

highly variable and in order to obtain reproducible results in bioassays, uniformly tolerant test organisms must be found. Cutting et al.

(1959) pointed out the unique advantages of 

fish as indicator organisms, but noted the persistent difficulties in making needed comparisons of bioassays because of the variety of 

fishes used. Cope (1961) suggested that it is 

essential to have some measure of the condition of test fish which are used in bioassays. 

Douglas and Irwin (1962) stressed the fact that 

results of independent bioassays often cannot 

be related because the comparative resistance 

of many test fishes has not been established. 

They pointed out the need for knowledge on the 

reactions of different species of fish when exposed to a particular toxicant.

Standardization of bioassay facilities and 

methods at the Fish Control Laboratories,

La Crosse, Wis., and Warm Springs, Ga., 

were reported by Lennon and Walker (1964). 

The reproducibility and comparability of results in bioassays may be achieved best by 

employing a standardized test fish, but there 

is none at present. In lieu of a standard test 

fish, the potentials of p,£'-DDT as a standard 

reference chemical in bioassays were investigated.

A standard reference chemical should have 

the following qualifications:

1. A rapid, nonselective, and consistent 

toxicity to fish.

2. Uncommon in nature, thus reducing 

risks of previous exposure among fish.

3. A known mode of action on fish.

The purpose of this study was to determine 

whether j^p'-DDT has the first qualification. 

Accordingly, we evaluated the toxicity of the 

chemical against 19 species of freshwater 

fish, including 39 separate lots of fish from 10 

different sources.

METHODS AND MATERIALS

The bioassays of p,p'-DDT [1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethanol] at 

La Crosse and Warm Springs were static tests
in 5-gallon glass jars as described by Lennon 

and Walker (1964). Stock solutions of the reagent grade chemical were made in acetone, 

and aliquots were added directly to bioassay 

vessels. The quantity of acetone in bioassays 

never exceeded 1 ml. per 1. The 96-hour 

LC 50 values (concentrations producing 50- 

percent mortality) were calculated according to the method of Litchfield and Wilcoxon 

(1949). This method gives a 95-percent confidence interval, the range in which 50 percent of the fish die 95 percent of the time.

The species and sources of fish are listed 

in table 1. Most of the lots were received 

from National Fish Hatcheries, but one lot of 

brook trout was received from a Wisconsin 

State Hatchery and two species were collected 

at the Necedah National Wildlife Refuge in 

Wisconsin. A "lot" is a particular group of 

fish of a species received from a hatchery in 

a single shipment.

TABLE 1. Species and source of fish used in bioassays of p,p'-DDT

Common nane

Rainboiv trout. .....

Brown trout ........

Brook trout. .......

Lake trout .........

Northern pike ......

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

Northern redbelly 

dace. ............

Carp. ..............

Fathead minnow. . .

Black bullhead...

Channel catfish. .

Brook stickleback

Green sunf ish. . . .

Pumpkinseed. .......

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

Longear sunf ish. . . .

Largemouth bass. . . .

Yellow perch. ......

Fresh water drum. . .

Technical name

Salmo gairdneri

Salmo trutta

Salvelinus fontinalis

Salvelinus fontinalis

Salvelinus namaycush

Esox lucius

Carassius auratus

Cyprinus carpio

Pimephales promelas

Ictalurus melas

Ictalurus punctatus

Eucalia inconstans

Lepomis cyanellus

Lepomis gibbosus

Lepomis macrochirus

Lepomis megalotis

Micropterus salmoides

Perca flavescens

Aplodinotus grunniens

Source

Manchester NFH, la.

Lake Mills NFH, Wis.

St. Croix Falls SFH, Wis.

Jordan River NFH, Men.

Yankton NFH, S.D.

Lake Mills NFH, tiis.

Necedah NWR Wis

Lake Mills NFH, Wis.

Lake Mills NFH, Wis.

Guttenberg NFH, la.

Fairport NFH, la.

Necedah NWR, Wis.

Lake Mills NFH, Wis.

Lake Mills NFH, Wis.

Fairport NFH, la.

Genoa NFH, Wis.

Lake Mills NFH, Wis.

Lake Mills NFH, Wis.

Upon delivery, the fish were maintained in 

a fish holding house and fed commercial pellets. The lengths and weights of representative samples in each lot were measured routinely. The fish at La Crosse and Warm 

Springs were held and tested at 12° and 17° 

C., respectively. Before testing, each lot was 

in quarantine for 10 days; food was withheld 

the last 3 days. If, after quarantine, a lot of 

fish was judged acceptable for bioassay, it 

was used in tests of £,£'-DDT and candidate 

fish controls. The tests with p,p'-DDT were

repeated biweekly for as long as a lot remained on hand in usable condition. Subsequent acquisitions of the same species were 

assigned different lot numbers even though 

they may have come from the same hatchery 

stock as a previous lot.

Mortalities of the test fish were recorded 

throughout each 96-hour bioassay. Preliminary investigations disclosed that p,p'-DDT 

produces similar effects on all species of 

fish, although there are slight variations in 

the behavior of different species. Reactions 

in catfishes, for instance, are less obvious 

than in trout. Generally, irritation and excitation are readily noticeable in the earlier 

stages of response. Swimming becomes erratic, upside down, on a side, with surfacing 

and colliding with walls of the container. The 

swimming is continuous until the final stages 

of response. Total loss of equilibrium, loss of 

reflex reactivity, and cessation of locomotion 

immediately precede death. Moribund fish 

often turn light in color. The operculums are 

usually distended and the spine is sometimes 

curved, indicating convulsive reactions at the 

time of death.

RESULTS

The toxicity of p,p'-DDT differed greatly 

among the 19 species of fish. The compound 

was consistently very toxic to some fishes but 

less and inconsistently so to others (table 2). 

The toxicity also varied among lots of a species, and details are given in tables 3 to 7.

TABLE 2. Order of toxicity of p,p'-DDT to various species of fish

Species Number 

of lots

2

1

3

2

1

Number 

of tests

3

1

Mean LC50 

in p.p.b.

0.9
TABLE 3. Toxicity of p,p'-DDT to trout and northern pike in 96 hours 

~ ~ at 12°C.

TABLE 6.--Toxicity of p,p'-DDT to centrarchids in 96 hours at 12°C.

Species and lot

Rainbow trout:

Brook trout:

Lake trout:

Average 

weight 

(grams)

0.9

0.9

1.2

1.6

4.0

Date 

tested

3-24-64

4-24-64

5-15-64

6- 5-64

LC50 

(p.p.b. )

14.0

4.6

-1 rt

95-percent confidence interval

12.8-15.4

29-74

12.0-19.0

9.6-12.3

TABLE 4. Toxicity of p,p_'-DDT to cyprinids in 96 hours at 12°C.

Species ar.d lot

Goldfish:

Northern redbelly dace:

Carp:

Average

weight 

(grams)

Date 

tested

LC 50 

(p.p.b.)

6.0

95-percent confidence interval

1.3- 12.0

TABLE 5. The toxicity of p,p'-DDT to catfishes and brook stickleback in 

~ ~96 hours at 12°C.

Species and lot

Black bullhead:

Channel catfish:

Brook stickleback:

Average 

weight 

(grams)

Date 

tested

LC 50 

(p.p.b.)

95-percent confidence interval

54.5-82.4

Species and lot

Green sunfish:

Pumpkinseed :

Bluegill:

Longear sunfish;

Largemouth bass: Lot 57 . . 

Average 

weight 

(grams)

0.7

1.0

0.7

0.8

1.3

0.6

0.5

Date 

tested

5-22-64

6- 5-64

6-20-64

10- 9-64

10-29-64

11-20-64

12-31-64

1-22-65

10-29-64

11-13-64

12-11-64

12-31-64

1- 8-65

4-24-64

5- 8-64

9-18-64

10-16-64

10-29-64

12- 4-64

1- 5-65

12- 3-64

12-14-64

12-28-64

11-20-64

12- 4-64

7-17-64

LC 50 

(p.p.b.)

2.8

3.0

-3.9

6.7

4.4

3.6

5.0

6.7

2.8

3.6

1.8

4.3

1.7

3.0

7.0

3.6

4.6

2.8

0.8

95-percent confidence interval

2.3- 3.4

2.5- 3.6

3.1- 4.9

5.0- 9.0

5.6- 7.3

3.6- 5.4

2.7- 4.8

4.1- 6.1

6.4- 8.8

5.7- 7.8

2.3- 3.4

2.7- 4.8

1.2- 2.6

3.5- 5.3

1.3- 2.1

2.3- 4.0

6.6- 7.4

6.4- 7.7

2.6- 5.0

0.9- 1.6

3.7- 5.8

6.4-13.9

1.9- 4.0

4.0- 6.0

7.6-20.6

0.7- 0.9

TABLE 7. Toxicity of p,p'-DDT to yellow perch and freshwater drum in 

~ ~~ 96 hours at 12°C.

Species and lot

Yellow perch:

Freshwater drum: Lot 117...

Average 

weight 

(grams)

1.1

0.8

1.2

3.3

Date 

tested

10-30-64

LC 50 

(p.p.b.)

0.8

0.6

1.5

10.0

95-percent confidence interval

0.6- 1.0

0.4- 0.8

1.1- 2.0

5.6-18.0

The jvp'-DDT was most toxic to largemouth bass, yellow perch, and northern pike, 

in that order; mean LCgo values were 0.8, 

0.9, and 1.7p.p.b. The fish were small fingerlings, however, and they did not appear to 

hold up well in the 2 to 4 days of starvation 

during the test period. Had live food been 

available in quantity before and during the 

tests, the fish might have shown greater resistance to the toxicant. Furthermore, the 

holding and test temperature of 12° C. may 

have been below optimum for fingerlings of 

these species.

The mean LC ^ of the toxicant was 4.5 

p.p.b. for green sunfish, pumpkinseed, and 

bluegill (table 2). The toxicity ranged from 2.8 

to 6.7 p.p.b. for green sunfish, 1.8 to 7.5 p.p.b. 

for pumpkinseed, and 1.2 to 9.4 p.p.b. for
bluegill, and the higher values for them occurred in October (table 6). Green sunfish in 

lot 25 doubled their average weight, 0.7 to 

1.4 g. in a month, and the LC50 increased 

from 2.8 to 3.9 p.p.b. In contrast, green sunfish in lot 95 lost in average weight from 1.1 

to 0.7 g. and LC 50 from 6.7 to 4.4 p.p.b. The 

LC 50 values for lots of the three species 

which merely maintained their average 

weights over 2-week to 1-month periods varied up and down, but the trend was generally 

downward.

The p,p'-DDT produced consistent results 

among bluegills tested at La Crosse and Warm 

Springs. The biweekly LC50 values for one lot 

of longear sunfish increased from 4.9 to 12.5 

p.p.b. Since the confidence intervals did not 

overlap, the difference was significant (table 6).

The chemical was less toxic to carp than to 

most of the centrarchids but more toxic than 

to trouts (table 2). It was tested three times 

against carp in lot 83 which fed, grew farily 

rapidly, and became more resistant.

The toxicity of the compound was intermediate to trouts (table 2). Of the four species, it was most toxic to lake trout and least 

to brook trout. The data suggest selectivity 

to some degree among these closely related 

species. Hatch (1957) related an interesting 

example of different responses among salmonids to DOT. An aerial application of the insecticide to a hatchery watershed was sublethal to rainbow and brook trout but killed all 

landlocked Atlantic salmon. His further observations confirmed that the trouts could 

survive concentrations deadly to the salmon.

The bioassays included four lots of rainbow 

trout, and the LC 50 values for them were inconsistent. Generally, the larger fish were 

more resistant to the chemical. The mean 

LCg, for trout over 1.5 g. was approximately 

14. p.p.b. while the value dropped to 7 p.p.b. 

for fish less than this weight.

The candidate chemical was less toxic to 

channel catfish and black bullhead (table 2). 

There was only one lot of channel catfish 

available, however, and the fish showed identical tolerances in tests 2 weeks apart. The

LC50 for black bullhead declined from 42 to 

17 p.p.b. over a 7-week period (table 5).

The p,£'-DDT was one-fifth as toxic to 

goldfish as to trouts. Moreover, the interlot 

and intralot differences in responses to the 

toxicant were great. The LC^ values for lots 

ranged from 21 to 180 p.p.b., and confidence 

intervals were extremely wide (table 4). 

These intervals do not overlap in all instances 

and suggest that the tolerance differs.

The p,p'-DDT was least toxic to brook 

stickleback (table 5) and northern redbelly 

dace (table 4), and their resistances exceeded 

the mean tolerance of goldfish. The LC50 values are six times as great as the mean for 

trouts (table 2).

Tests of the toxicant against fathead minnows presented unique problems. The results 

were neither rapid nor consistent. The toxicity did not increase uniformly with increased 

concentrations up to 1,000 p.p.b. within 96 

hours. A reliable LC^ could not be obtained 

in repeated trials. Furthermore, a marked 

abdominal distention occurred in exposed fish. 

It began at 24 hours and became more pronounced at 96 hours. Examination of the peritoneal cavity revealed an enlarged air bladder. 

It is not known at present what relation this 

distention has to the toxicant. Henderson, 

Pickering, and Tarzwell (1959) noted the phenomenon among fathead minnows which had 

been exposed to chlorinated hydrocarbons. 

They indicated, however, that the LC^ of 

JVP'-DDT to fatheads was 32 p.p.b. in 24 

hours and 26 p.p.b. in 96 hours. We could not 

confirm their results.

DISCUSSION

The p,p*-DDT was rapidly and consistently 

toxic to lake trout, carp, channel catfish, 

green sunfish, bluegill and yellow perch, but 

not so to rainbow trout, brook trout, and black 

bullhead. Results were unsatisfactory with 

goldfish and fathead minnows in 96-hour bioassays. The regression curves, plotted on logarithmic coordinates, in figure 1 illustrate the 

consistency of toxic responses in rainbow 

trout and goldfish. The calculated slope function
99

>- 90

»-

< 80

»- 

<* 70

^ 60 

50 

40

>- 30

" 20 u

OL 

LU

o. 10

10 

CONCEN TRATION-PPB

100

Figure 1. --Kesponses ot rainbow trout (x) and goldfish (o) to p, p' -DDT.

for rainbow trout is 1.16 whereas that for 

goldfish is 6.02.

The regression curve for rainbow trout 

(lot 16a) indicates toxicity at very low levels. 

The slope of the curve reveals a narrow margin between survival and mortality. The confidence intervals are relatively narrow, thus 

indicating that toxicity is more accurately 

defined.

The regression curve for goldfish (lot 1) 

contrasts sharply with that for rainbow trout. 

The curve is flat rather than steep, and it 

shows that increased concentrations in the 

survival and mortality range produce little 

effect within a 96-hour bioassay. The confidence intervals are wide, and the toxicity of 

p,pf-DDT to goldfish is not defined accurately.

The results with p,£'-DDT further demonstrated that lots of fish within a single species 

may vary significantly in responses. The variations may be due to several factors. Centrarchids appeared to have seasonal cycles in 

relative resistance to the toxicant. Green sunfish, pumpkinseed, and bluegill displayed 

higher tolerances in autumn (table 6). It was 

impossible, however, to evaluate total cyclic 

effects since the fish were not available for 

more than a few months of the year. Shell 

(1961) found systematic and seasonal variations in blood components of smallmouth bass. 

He noted that most of the changes took place 

in August and suggested that changes in the 

anabolic-catabolic balance were responsible 

for the cyclic concentrations of the various 

blood components. This may explain why fish 

are at times more vulnerable to toxicants or 

other stresses.
Hoi den (1962) determined that fish in poor 

condition or with a low reserve of fatty tissue , 

are more sensitive to DOT. This was noticeable in some of our tests with pumpkinseed, 

bluegill, and yellow perch. In contrast, relatively healthy fish which were feeding and 

gaining in weight tended to become more resistant to the toxicant. Examples of increasing 

resistance were afforded by rainbow trout (lot 

17), brook trout (lot 18), goldfish (lot 19), 

carp (lot 83), and green sunfish (lot 25). 

Phillips, Livingston, and Dumas (1960) reported that starved brook trout have a tendency to increase in water content and decrease in body fat and protein. Such changes 

in body chemistry may contribute to decreased 

resistances of the fish to the reference compound.

The bioassays of p,p'-DDT showed that fish 

obtained from different sources display different tolerances. Geographic location, water 

quality, pond fertilization, herbicide applications, and feeding and handling during the 

rearing period probably influence the relative 

tolerance of specimens to a toxicant. Lots of 

brook trout from Lake Mills National Fish 

Hatchery and St. Croix Falls State Fish 

Hatchery varied considerably in resistance. 

On the other hand, the bluegills from Lake 

Mills and Warm Springs National Fish Hatcheries reacted quite similarly.

A good reference standard chemical should 

be sufficiently uncommon in nature that there 

is little risk that bioassay fishes have i>een 

exposed 10 it or its closely related compounds. 

The j>,£f-DDT hardly meets this qualification, 

since bioassay fishes from various hatcheries 

may have been exposed previously to DOT. It 

is very widely used as a pesticide and resists 

decomposition. A hatchery in an agricultural 

watershed, for example, may very well be 

contaminated to some extent. Also, the natural 

or prepared diets of the fish are likely to 

contain small quantities of DOT. Such exposures to the insecticide may have influenced 

the tolerances of fish in our tests. Many of 

the trials were unsuccessful because all specimens either lived or died at concentrations 

of jvp'-DDT which had previously resulted in 

partial kills.

Cope (1960) reported that trout and whitefish retained DOT in considerable quantities 

almost a year after an aerial application for 

spruce budworm in a western watershed. King 

(1962) found that guppies became more tolerant to DOT in repeated exposures. Vinson, 

Boyd, and Ferguson (1963) observed that 

mosquitofish (Gambusia affinis) preexposed to 

DOT near treated cotton fields were considerably more tolerant than fish from untreated 

areas. Boyd and Ferguson (1964) observed 

that tolerance to DOT persisted through one 

to three generations of mosquitofish which 

were removed from an environment contaminated by the insecticide.

CONCLUSIONS

Thej>,p'-DDT PartiaHy fulfilled the qualification that a reference standard toxicant be 

rapidly, nonselectively, and consistently toxic. 

It was rapidly and consistently toxic to lake 

trout, carp, channel catfish, green sunfish, 

bluegill, and yellow perch. It was sufficiently 

toxic to brown trout, northern pike, northern 

redbelly dace, black bullhead, brook stickleback, largemouth bass, and freshwater drum, 

but there were not enough lots of each species 

to determine the consistence of toxicity. It 

lacked either rapid toxicity or consistent 

toxicity to rainbow trout, brook trout, goldfish, fathead minnows, and longear sunfish in 

96-hour bioassays. The p,p'-DDT exhibited 

a selective toxicity among the 19 fishes 

instead of the desired nonselectivity. For 

example, the mean LC^ for largemouth bass 

was 0.8 p.p.b. whereas the LCso for northern redbelly dace was 68 p.p.b. Moreover, a 

reliable LC50 for fathead minnows could not 

be obtained within a 96-hour bioassay.

There were intraspecific as well as interspecific differences in the sensitivity of fish 

to the toxicant. For example, the range of 

LCso values for four lots of goldfish was 21 

to 180 p.p.b. The £,£'-DDT was generally 

more toxic to small fish than large ones within a species. Thus, p,p'-DDT is limited in 

usefulness as a reference standard in largescale bioassays with many species of freshwater fish. The tests against 19 species demonstrate the continuing need for an adequate

8
standard reference compound and/or a standard reference fish. In spite of the limitations, 

it is useful in the selection, testing, and evaluation of other candidate reference compounds.

SUMMARY

A standard reference compound is needed 

in the fish control program to facilitate reproducibility and comparisons in bioassays in 

which many species, sizes, conditions, and 

sources of fish, and many kinds and concentrations of chemicals are involved. The 

p-p^-DDT was tested for rapid, nonselective, 

and consistent toxicity against 19 fishes, including different lots of the same species and 

repeated samples within lots. The trials were 

accomplished in static bioassays in the laboratory at 12° C. We established the LC5e of 

p,p'-DDT for each lot of fish received from 

hatcheries or the field, but only after 10 days 

of quarantine in a fish-holding house. The tests 

were repeated biweekly for as long as a lot 

remained on hand in testworthy condition.

The p,p'-DDT was rapidly and consistently 

toxic in 96-hour bioassays to 6 of the 19 

species. It was sufficiently toxic to 8 others, 

but there were insufficient lots to evaluate 

consistency. It lacked either rapid toxicity or 

consistent toxicity to five species. The mean 

LC50 for largemouth bass was 0.8 p.p.b., and 

the mean for northern redbelly dace was 

68 p.p.b., thus it was more selective in its 

action than would be desirable.

There were intraspecific as well as interspecific differences in sensitivity among the 

fish to the toxicant. The chemical was more 

toxic to small fish than large ones within a 

species.

Since the toxicity of jvp'-DDT varied in 

rapidity, selectivity, and consistency against 

19 species of freshwater fish, the compound 

is of limited usefulness as a standard reference in large-scale bioassays. The results 

are helpful, however, in the selection, testing, 

and evaluation of other candidate reference 

compounds.

LITERATURE CITED

Boyd, Claude E., and Denzel E. Ferguson. 

1964. Susceptibility and resistance of 

mosquito fish to several insecticides. 

Journal of Economic Entomology, vol. 

57, No. 41, p. 430-431.

Cope, Oliver B.

1960. The retention of D.D.T. by trout 

and whitefish. Biological problems 

in water pollution, U.S. Department of 

Health, Education, and Welfare, Transactions of the 1959 seminar, Technical 

Report W60-3, p. 72-76.

Cope, Oliver B.

1961. Standards for reporting fish 

toxicity tests. Progressive FishCulturist, vol. 23, No. 4, p. 187-190.

Cutting, Windsor, Morris Baslow, Dorothy 

Read, and Arthur Furst.

1959. The use of fish in the evaluation 

of drugs affecting the central nervous 

system. Journal of Clinical and Experimental Psychopathology, vol. 20, 

No. 1, p. 26-32.

Douglas, Neil H., and W. H. Erwin.

1962. Evaluation and relative resistance 

of sixteen species of fish as test 

animals in toxicity bioassays of petroleum refinery effluents. (Mimeo) 

Contribution No. 351, Department of 

Zoology, Oklahoma State University. 

40 p.

Hart, W. B., P. Doudoroff, and J. Greenbank. 

1945. The evaluation of the toxicity of 

industrial wastes, chemicals and other 

substances to freshwater fishes. Waste 

Control Laboratory, Atlantic Refining 

Co., Philadelphia. 332 p.

Hatch, Richard W.

1957. Relative sensitivity of salmonids to 

DOT. Progressive Fish-Culturist, 

vol. 19. No. 2, p. 89-92.

Henderson, C., Q. H. Pickering, and C. M. 

Tarzwell.

1959. Relative toxicity of ten chlorinated 

hydrocarbon insecticides to four
species of fish. Transactions of the 

American Fisheries Society, vol. 88, 

No. 1, p. 23-32.

Holden, A. V.

1962. A study of the absortion of

14 C-labelled DOT from water by fish. 

Annals of Applied Biology, vol. 50, 

p. 467-477.

King, Susan Frances.

1962. Some effects of DOT on the guppy 

and the brown trout. U.S. Fish and 

Wildlife Service, Special Scientific 

Report Fisheries No. 399. 21 p.

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

1964. Investigations in fish control. 

1. Laboratories and methods for 

screening fish-control chemicals. U.S. 

Bureau of Sport Fisheries and Wildlife, 

Circular 185. 15 p.

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

1949. A simplified method of evaluating 

dose-effect experiments. Journal of 

Pharmacology and Experimental 

Therapeutics, vol. 96, No. 2, p. 99-113.

Phillips, Arthur M., Jr., Donald L. Livingston, 

and Richard F. Dumas.

1960. Effect of starvation and feeding on 

the chemical composition of brook 

trout. Progressive Fish-Culturist, 

vol. 22, No. 4, p. 147-155.

Shell, Eddie Wayne.

1961. Chemical composition of blood of 

smallmouth bass. U.S. Bureau of Sport 

Fisheries and Wildlife, Research 

Report 57. 36 p.

Vinson, S. B., Claude E. Boyd, and Denzel E. 

Ferguson.

1963. Resistance to DDT in mosquito 

fish, Gambusia affinis. Science, 

vol. 139, No. 3551, p. 217-218.

Warren, Charles E., and Peter Doudoroff. 

1958. The developments of methods for 

using bioassays in the control of pulp 

mill waste disposal. Technical Association of Pulp and Paper Industry, 

vol. 41, No. 8, p. 211A-216A.

10
INVESTIGATIONS IN FISH CONTROL

11. Evaluation of an Electronic Method of 

Measuring Hematocrits of Fish

By Richard A. Schoettger and Arnold M. Julin, Fishery Biologists 

Fish Control Laboratory 

Bureau of Sport Fisheries and Wildlife 

La Crosse, Wisconsin

U.S. Department of the Interior

Fish and Wildlife Service 

Bureau of Sport Fisheries and Wildlife 

Resource Publication 15 

Washington . February 1966
CONTENTS

Page

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

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

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

Reproducibility of hematocrits ...................... 5

Comparability of hematocrits....................... 5

Effects of saline and protein on electronic hematocrit ...... 7

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

Summary ....................................... 9

Literature cited................................... 10
EVALUATION OF AN ELECTRONIC METHOD OF 

MEASURING HEMATOCRITS OF FISH

By Richard A. Schoettger and Arnold M. Julin, Fishery Biologists 

Fish Control Laboratory, La Crosse, Wis.

ABSTRACT. Conductivity measurements of mammalian blood are considered satisfactory estimates of hematocrit. The YSI Electronic Hematocrit gives rapid and reproducible results with fish blood, and the readings 

can be corrected to microhematocrit values. In the low hematocrit range, 

however, readings do not change proportionately with microhematocrits; 

they are difficult to correct; but they may prove useful as a gross, rapid 

method for detecting anemia. Electrolyte and protein concentrations in 

fish blood influence the electronic hematorcrit; the relative size of 

erythrocytes may also.

Hematological parameters are used frequently to estimate the physiological condition 

of fish. Among them, the hematocrit is one of 

the easier values to secure. Procedures, 

problems, and modifications associated with 

microhematocrit determinations in fish were 

discussed by Hesser (1960), Snieszko (1960), 

Larsen and Snieszko (1"961), and Mairs and 

Kennedy (1962).

The microhematocrit, or centrifuge, technique is better suited to laboratory routine 

than to measurements of hematocrit in the 

field. Electric line power is needed for the 

centrifuge, and transportation of blood 

samples to the laboratory is time consuming. 

There is need for a method which complements the centrifuge technique in the laboratory and is convenient for studies in the field.

An electrical device for relating the conductivity of mammalian blood to the hematocrit was described by Okada and Schwan

(1960). The pointed out that its validity is affected by changes in serum conductivity or 

the average size of erythrocytes. Kernen et al.

(1961) investigated the reproducibility of results obtained with the instrument on human 

blood and studied the effect of anticoagulants

and blood protein levels on conductivity. 

Further tests were conducted by the Yellow 

Springs Instrument Company, which manufactures a similar instrument called an Electronic Hematocrit.

Our investigation was designed to study the 

comparability of the YSI electronic and 

centrifuge methods for measuring hematocrits in fish, the reproducibility of the electronic hematocrits, and some physiological 

variables in the blood of fish which could influence conductivity.

METHODS AND MATERIALS

Hematocrits were determined as described 

by Larsen and Snieszko (1961) for the 

centrifuge method and according to the manufacturer's instructions for the electronic 

method.

Briefly, the YSI Model 30 Electronic 

Hematocrit is portable, weighs 2.8 pounds, 

is powered by a 6-volt mercury cell, and is 

compensated for use at 50° to 105° F. A 

blood sample of at least 0.02 cc. is drawn 

and placed between two electrodes imbedded
in a glass cell. The cell is placed between the 

circuit contacts, a button is pressed, and the 

hematocrit is read directly from the instrument. The reproducibility of readings is reported by the manufacturer to be 0.1 unit with 

a standard deviation of ± 0.3 unit. Anticoagulants other than heparin and abnormal levels 

of serum proteins in human blood influence 

conductivity.

The operating principle of the electronic 

hematocrit is based on the insulating characteristics of the erythrocyte membrane 

which separates the conductive interior from 

the conductive serum outside the cell. The 

YSI instrument contains a bridge circuit. Two 

arms of the bridge consist of a precision, 

center-tapped transformer. The third is a 

thermistor-resistor which automatically 

compensates for ambient and glass cell 

temperatures. A fixed volume of blood within 

a glass cell constitutes the unknown fourth 

resistance. Unbalance of the bridge is a 

measure of the concentration of erythrocytes 

(manufacturer's specifications; and Okada 

and Schwan, 1960).

Blood samples were collected from fish by 

cardiac puncture with a syringe or from 

severed caudal vessels directly into capillary 

tubes. The syringes, tubes, and scalpels were 

coated with heparin.

The reproducibility of the two hematocrit 

methods was estimated by repeated measurements on relatively large samples of blood. 

The samples were secured from one each 

shovelnose sturgeon, spotted sucker, northern redhorse, brown bullhead, white bass, 

largemouth bass, and walleye. During the 

measurement of hematocrits a magnetic 

mixer was used to maintain uniform distribution of red blood cells within a sample, as 

described by Snieszko (1963).

Tests of the comparability of the hematocrit methods were conducted with fingerling 

and larger-size fish including: rainbow trout, 

brown trout, brook trout, lake trout, and 

goldfish. Two capillary tubes were filled

from each specimen. One was spun in an International Microcapillary Centrifuge, Model 

MB, and the hematocrit determined with a 

plastic reader. The second sample was utilized 

for electrical measurements.

The effects of sodium chloride and protein 

concentrations on electronic hematocrits of 

rainbow trout and carp were determined in an 

experimental design similar to that of Kernen 

et al. (1961) except that the tests were conducted with salines containing 0.65-, 0.75-, 

0.85-, and 1.0-percent sodium chloride. According to Wolf (1963) this range of salines 

has been used in physiological studies of fish. 

Powdered bovine albumin was added to the 

salines to obtain protein levels of 1 to 9 grams 

per 100 cc. This albumin was selected because it was used by Kernen et al. (1961). The 

erythrocytes were separated from heparinized 

blood by centrifuging at 850 r.p.m. for 15 

minutes. The cells were washed twice in the 

appropriate saline at 20° C. and resuspended 

in various protein-saline solutions to give 

approximate hematocrits of 25 to 30 percent. 

A third saline rinse occasionally resulted in 

hemolysis of the erythrocytes in the final resuspension. Wolf (1959) found that red cells of 

salmonids coagulated in various sodium 

chloride solutions which did not contain anticoagulant. In our tests, residual heparin 

probably served a similar function. Ionic 

coagulants such as the oxalates alter the 

conductivity of samples (Kernen et al., 

1961).

Throughout the tests, capillary tubes were 

examined for clotting and hemolysis. The 

electronic cells were checked for clots. It 

was essential to conduct electrical measurements rapidly to minimize the effect of 

erythrocyte sedimentation within the glass 

cell.

The trout and goldfish were obtained from 

State and National fish hatcheries. The other 

species were collected in the Mississippi 

River. They were maintained in flowing well 

water at the Fish Control Laboratory for at 

least 2 months before use.
RESULTS AND DISCUSSION

REPRODUCIBILITY OF HEMATOCRITS

The tests with seven species of fish to 

evaluate the relative reproducibility of the 

two hematocrit methods indicated that the 

centrifuge technique was somewhat superior 

(table 1). The standard deviations, for example, were smaller except in the case of the

hematocrits. Statistical tests showed that the 

differences between the means of the two 

methods were significant at the 0.01 level.

The electronic hematocrit can be corrected 

to compare with centrifuge value by using 

the regressions in figure 1 or correcting 

with mean differences between the methods 

(table 2). The second readings in the table will 

be discussed later.

TABLE 1. Comparative reprodueibility of electronic and centrifuge hematocrits

Species 

of 

fish

Shovelnose sturgeon. .

Northern redhorse ....

Hematocrit

Electronic

Number of 

hematocrits

12 

12

20 

12 

12 

8 

9

Mean

15.5 

20.0 

31.0 

16.0 

14.0 

14.5 

28.0

Range

14.0-17.0 

17.0-23.0 

28.5-32.5 

14.0-18.0 

12.0-18.0 

12.0-16.0 

25.0-29.0

Standard 

deviation

0.8 

1.4 

1.2 

0.9 

1.5 

1.4 

1.4

Centrifuge

Number of 

hematocrits

12 

10 

24 

12 

11 

12 

12

Mean

22.0 

33.5 

33.0 

22.0 

31.0 

26.0 

40.0

Range

20.5-23.0

32.0-34.0 

31.0-34.5 

21.0-22.5 

30.0-32.5 

24.0-27.0 

39.0-41.5

Standard 

deviation

1.0

0.7 

0.7 

0.5 

0.7 

1.0 

0.7

Centrifuge 

minus 

electronic

6.5 

13.5 

2.0 

6.0 

17.0 

11.5 

12.0

shovelnose sturgeon. In contrast, the electronic hematocrits were consistently low and 

standard deviations were greater. Statistical 

tests for each species of fish demonstrated a 

significant difference between the results of 

the two hematocrit methods at the 0.01 level.

COMPARABILITY OF HEMATOCRITS

The electronic hematocrit was considered 

sufficiently reproducible. The differences 

between the means of the two methods suggested a need to correct electronic readings 

for individual and species variation. The 

regressions of electronic hematocrit on 

centrifuge hematocrit were determined for 

five species (fig. 1). The mean centrifuge 

values for the various species were as follows: rainbow trout, 40.4; brown trout, 33.0; 

brook trout, 37.1; lake trout, 35.7; and goldfish, 42.8. The mean electronic hematocrits, 

depending on species, were generally 20 to 60 

percent lower than the means of the centrifuge

The regression technique is preferable because differences between hematocrit methods 

vary with the magnitude of the hematocrit. 

For example, an electronic hematocrit of 10 

for rainbow trout corresponds to a centrifuge 

value of 25 and a reading of 30 to 49.5 when 

corrected by the regression. Using the mean 

difference as a correction, these values would 

be 27.5 and 47.5.

The regressions and the differences between means for rainbow and brown trout are 

similar (fig. 1, table 2). The 95-percent confidence intervals indicate that the same 

factor could be used to correct the electronic 

hematocrits of either salmonid. Separate corrections are required for other species.

The standard deviations of the differences 

between electronic and centrifuge hematocrits indicate a considerable variation about 

their means (table 2). Since the electronic 

hematocrit is relatively reproducible, and the 

measurements were made on the same
50-

40-

o

E 30-

o

X

10-

X Erythrocyte dilutions 

__ __ _ Extrapolation

S.E.I.

 - Goldfish

 Brook trout

 Rainbow trout

S _ Brown trout

x-Lake trout

i

10 20

l

30

4*0 50

Centrifuge Hematocrit

Figure 1. Plot of electronic hematocrit against centrifuge hematocrit. Straight-line 

regression equations were computed for the data. S.E.I, and S.E.M. correspond to 

standard errors of individual and mean estimates respectively.

TABLE 2.--lfean differences between hematocrit methods for five species of fish

Species and electronic 

reading

Rainbow trout:

Brown trout :

Brook trout :

Lake trout:

Goldfish:

Second reading .............................

Number 

of 

measurements

19

Ifean 

difference; 

centrifuge 

minus 

electronic

14.3

1.4

Range of 

differences

9-24

5-19

1 4- 5

Standard 

deviation

4.0

3.3

3.1

4.6

2.6

Standard 

error

0.84

0.80

0.70

0.65

0.61

0.43

0.62

0.60

95-percent 

confidence 

interval

15.6-19.1

6.6- 9.7

12.9-15.9

5.0- 7.7

20.2-22.6

10.0-11.8

7.8-10.4

0.1- 2.6

1 All centrifuge nematocrits exceeded electronic readings except for three values in this group. The range extended from four units above to five units 

below the centrifuge hematocrit.
samples, the standard deviations suggest 

variations in sample conductivity due to 

factors other than percentage of cells. For 

this reason, the corrected electronic hematocrit should only be considered an estimate of 

the centrifuge hematocrit.

The extrapolations of regressions in figure 1 

converge toward a common intercept as the 

hematocrit decreases. Unfortunately, anemic 

fish were unavailable to test the extrapolations, 

but plasma dilutions of rainbow erythrocytes 

were used to simulate anemia. The measurements of simulated samples were comparable 

to the predicted values at centrifuge hematocrits above 25; between 10 and 25, the readings 

ranged from 4 to 6 and increased with hematocrits less than 10. Thus the correction of electronic hematocrits of anemic fish may give 

misleading estimates of the centrifuge hematocrit. Readings of 4 to 6, however, might be 

used as gross, but rapid, indicators of anemia. 

Though severe anemia may give higher electronic values, the condition is usually detected 

by visual examination of the blood.

The conductivities of solutions containing 

0.45- to 1.20-percent sodium chloride were 

tested on the YSI Electronic Hematocrit. A 

plot of the readings against the salt concentrations formed a curve similar to that shown 

in figure 1 for plasma dilutions of red blood 

cells. Salines of 0.60 to 0.75 percent sodium 

chloride gave electronic readins of 5 to 6. 

The similarity of the two curves suggests that 

the number of erythrocytes in blood affect 

the electronic hematocrit like changes in the 

electrolyte concentration. The electronic 

hematocrit of the plasma used to dilute the 

erythrocytes corresponded to that for a 0.86- 

percent solution of salt. A 0.86-percent saline 

has a freezing point similar to that for the 

bloods of most freshwater teleosts (Black, 

1957). The data indicate that electronic 

measurements of plasma, or serum, might be 

used to estimate the electrolyte levels in fish 

blood.

Changes in the conductivity of a blood 

sample were observed upon re-reading. The 

value rose shortly after the first reading, increased to a peak, and then declined. The 

rate of change varied with individuals and

groups of fish. The apex was usually attained 

in 3 to 7 minutes and occasionally approximated the value obtained by the centrifuge 

method. An examination of blood within the 

glass cell indicated that the change in conductivity may be related to sedimentation 

and an unequal distribution of erythrocytes 

between the electrodes. This factor may be 

significant when measurements are delayed 

or when several samples are collected before 

reading.

Limited studies were conducted on the possibility of using the second electronic reading to estimate hematocrit. The mean differences between hematocrit methods, using 

the second reading, are shown in table 2. A 

correction factor for the second reading, like 

the first, is needed to estimate hematocrit. 

The factors are considerably smaller and 

have lower standard deviations, and standard 

errors. The second reading is time consuming and requires the use of heparinized blood. 

We found that blood could be drawn directly 

into the glass cell, without heparinization, 

and the first reading made within approximately 30 seconds. The blood began to clot 

shortly afterward, and a second reading was 

not usually possible. The alternative for 

using the second electronic reading is the 

preliminary collection of blood in heparinized 

capillary tubes; however, the technique detracts from the ease and speed of the electronic hematocrit.

Although the electronic determination of 

hematocrit is relatively rapid, time is lost in 

cleaning the glass cells. Several rinses in 

distilled water and drying with acetone were 

usually adequate. If blood dried or clotted in 

the cell, it was difficult to remove. If it remained, it changed sample volumes.

EFFECTS OF SALINE AND PROTEIN ON 

ELECTRONIC HEMATOCRIT

Experiments were conducted with rainbow 

trout and carp to determine whether variations 

in the electrolyte and protein concentration of 

fish blood influence electronic hematocrit 

(table 3 and fig. 2). The electronic hematocrit 

was inversely proportional to the concentration
TABLE 3. Effect of various protein and sodium chloride concentrations on the deviation of electronic from centrifuge hematocrits

Species and concentration of 

sodium chloride

Rainbow trout:

Carpi

Protein concentrations of (grains per 100 cc.) 

1

-1.5 

-1.0

-11.5 

-14.5 

-27.5

-26.0

0.0 

-1.0 

-14.0 

-15.5

2

-12.0 

-14.0 

-24.5 

-24.5

-12.5 

-11-5

3

-0.5 

0.0 

-6.0 

-7.0 

-12.5 

-11.0 

-24.5 

-23.5

+1.0 

+1.5 

-11.5 

-12.5

5

+1.0 

+0.5 

-2.5 

-6.0 

-9.5 

-9.0 

-22.0 

-21.5

+2.5 

+0.5 

-11.0 

-9.0

7

-7.5 

-7.5 

-20.5 

-19.5

-7.5 

-7.5

9

+2.0 

+1.5 

-3.0 

-4.0 

-5.0 

-8.0 

-18.0 

-19.0

+4.0 

+4.5 

-5.0 

-8.0

30

±20"

10-

0-

.f20-

E

30-

Rainbow trout 

Carp

4 5 

grams per 100 c c

Figure 2. Plot of hematocrit deviation against protein concentration for various levels of sodium 

chloride (as listed in table 3), Straight-line regression equations were computed for the data.

of sodium chloride and directly proportional 

to the level of protein. Suspensions of erythrocytes in salines containing 0.65- to 1.00- 

percent sodium chloride gave electrical

readings from 4.5 units above to 27.5 units 

below their corresponding centrifuge values. 

The concentration of protein had less effect 

on electronic hematocrits as the level of

8
sodium chloride declined. The readings increased with protein concentration by 7.0 to 

9.5 units at 1-percent and by 2.5 to 5.5 units 

at 0.65-percent sodium chloride. The difference in conductivity between erythrocytes of 

rainbow trout and carp were relatively small.

The deviation of electronic readings due to 

variations in the electrolyte content of fish 

blood can be estimated from Black's (1957) 

review of freezing points of bloods for several 

teleosts. The values are related to species 

and osmotic concentration of the external environment. The bloods of most fish in freshwater froze between -0.49 and -0.67° C. According to Hodgman, Weast, and Selby (1960), 

salines containing approximately 0.80- to 1.15- 

percent sodium chloride have similar freezing 

points. Phillips et al. (1956) suggested that 

nutritional conditions may influence the range 

of variability in the concentration of inorganic 

elements in brown trout blood. Seasonal 

changes in these constituents were observed 

in the blood of smallmouth bass by Shell 

(1961). Considering these data it is possible 

that the electronic hematocrit readings in 

tables 1 and 2 were affected by blood electrolytes.

The quantities of serum proteins of fish 

vary according to sex, reproductive condition, 

food, disease, oxygen depletion, and other 

factors (Booke, 1964). Some concentrations of 

protein reported per 100 cc. of blood include 

1.8 to 4.5 grams for smallmouth bass (Shell, 

1961), 2.01 to 2.04 grams for brown and 

brook trout (Phillips et al., 1957), 3.33 to 4.70 

grams for rainbow trout (Meisner and Hickman, 1962), and 3.18 to 7.96 grams for brook 

trout (Brooke, 1964).

Assuming a physiologic level of electrolytes 

in the blood, electronic hematocrits could 

deviate 4 or 5 units depending on protein concentration. This may not be a comparatively 

large error, but the degree to which bovine 

albumin simulates blood proteins of fish with 

respect to conductivity is not known.

Though the regressions in figure 2 demonstrate the effect of electrolyte and protein 

concentration on conductivity, they do not 

correspond to results obtained by Kernen, 

Wurzel, and Okada (1961) on human blood.

They found that electronic values corresponded to centrifuge hematocrits at a 

protein concentration of 7.4 grams per 100 cc. 

(bovine albumin dissolved in physiological 

saline). Their regression extended from -9.9 

hematocrit units at 2.5 grams of protein per 

100 cc. to +6.6 units at 11.0 grams per 100 cc. 

The inconsistency between data on humans 

and on fish may be related to the preparation 

of cellular suspensions or, perhaps, to an additional variable such as the relative size of 

fish erythrocytes. The red blood cells of fish 

are generally larger than and shaped differently from those of mammals, and it is 

conceivable that the comparatively low electronic hematocrits are partially correlated 

with differences in their insulatory characteristics (Smith, 1952; Hawk, Oser, and Summerson, 1954; and Mott, 1957). Larger cells may 

have less surface area per volume and have 

an effect on the electronic hematocrit which 

is analogous to that of fewer cells.

CONCLUSIONS

The YSI Electronic Hematocrit gives relatively rapid and reproducible results with 

fish blood. Centrifuge hematocrits greater 

than 25 can be estimated by correcting the 

electronic readings. Electronic readings 

were not correlated linearly with centrifuge 

values which were less than 25. They may be 

used, however, as gross but rapid indicators 

of anemia. Observations on the conductivities of plasma and various-strength 

salines suggest the instrument could be used 

to measure electrolyte levels in fish blood. 

The conductivities of blood samples changed 

with time and became closer to centrifuge 

values than the initial readings. The estimations of hematocrits from the later readings were time consuming and required 

heparinized blood. The electrical measurement may be influenced by variations in blood 

electrolytes and proteins, and by the average 

size of erythrocytes.

SUMMARY

Experiments with a YSI, Model 30 Electronic 

Hematocrit indicate that measurements of 

the conductivity of fish blood can be used to 

estimate hematocrit. The method is relatively
rapid and reproducible, but readings must be 

corrected to estimate centrifuge hematocrits 

of approximately 25 or more. The individual 

differences between the two methods varied 

widely and the differences between means of 

the methods deviated according to species. 

The electronic hematocrits do not change 

proportionately with centrifuge values less 

than 25, and are not easily corrected. The 

disproportionate change of electronic readings 

in the low hematocrit range may prove useful as a gross but rapid method for detecting 

anemia. Variations in serum electrolytes and 

proteins of fish, and in the average size of 

erythrocytes may interfere seriously with 

electrical measurements of hematocrits.

LITERATURE CITED

Black, Virginia Safford.

1957. Excretion and osmoregulation. In 

The physiology of fishes, Vol. 1, p. 163- 

265. Edited by Margaret E. Brown. 

Academic Press, New York.

Booke, Henry E.

1964. A review of variations found in fish 

serum proteins. New York Fish and 

Game Journal, vol. 11, No, 1, p. 47-57.

1964. Blood serum protein and calcium 

levels in yearling brook trout. Progressive Fish-Culturist, vol. 26, No. 3, 

p. 107-111.

Hawk, Philip B., Bernard L. Oser, and 

William H. Summerson.

1954. Practical physiological chemistry, 

13th ed. McGraw-Hill Book Co., New 

York. 1439 p.

Hesser, E. F.

1960. Methods for routine fish hematology. Progressive Fish-Culturist, 

vol. 22, No. 4, p. 164-171.

Hodgman, Charles D., Robert C. Weast, and 

Samuel M. Selby.

1960. Handbook of chemistry and physics, 

42nd ed. Chemical Rubber Publishing 

Co., Cleveland, Ohio. 3481 p.

Kernen, Jules A., Harold Wurzel, and Robert 

Okada.

1961. New electronic method for measuring hematocrit, clinical evaluation. 

Journal of Laboratory and Clinical 

Medicine, vol. 57, No. 4, p. 635-641.

Larsen, Howard N., and S. F. Snieszko.

1961. Modification of the microhematocrit technique with trout blood. Transactions of the American Fisheries 

Society, vol. 90, No. 2, p. 139-142.

Mairs, Donald F., and Theresa E. Kennedy.

1962. Evaluation of some heparinized 

capillaries for micro-hematocrit 

determinations of warm-water fishes. 

Progressive Fish-Culturist, vol. 24, 

No. 4, p. 188-189.

Meisner, Herman M., and C. P. Hickman. 

1962. Effect of temperature and photoperiod on the serum proteins of the 

rainbow trout, Salmo gairdneri. 

Canadian Journal of Zoology, vol. 40, 

p. 127- 130.

Mott, J. C.

1957. The cardiovascular system. In 

The physiology of fishes, vol. 1, 

p. 81-108. Edited by Margaret E. 

Brown. Academic Press, New York.

Okada, R. H., and H. P. Schwan.

1960. An electrical method to determine 

hematocrits. IRE Transactions on 

Medical Electronics, vol. ME-7, 

p. 188-192.

Phillips, Arthur M., Jr., Henry A. Podoliak, 

Donald R. Brockway, and Ray R. Vaughn.

1956. The nutrition of trout. Cortland 

Hatchery Report No. 25, New York 

Conservation Department, Fisheries 

Research Bulletin 20. 61 p.

1957. The nutrition of trout. Cortland 

T atchery Report No. 26, New York Cont. 3rvation Department, Fisheries Research Bulletin 21. 93 p.

Shell, Eddie Wayne.

1961. Chemical composition of blood of 

smallmouth bass. U.S. Fish and Wildlife Service, Research Report 57. 36 p.

10
Smith, Charles G., William M. Lewis, and 

Harold M. Kaplan.

1952. A comparative morphologic and 

physiologic study of fish blood. Progressive Fish-Culturist, vol. 14, No. 4, 

p. 169-172.

Snieszko, S. F.

1960. Microhematocrit as a tool in 

fishery research and management. 

U.S. Fish and Wildlife Service, Special 

Scientific Report Fisheries No. 341. 

15 p.

1963. An aid in the preparation of blood 

samples from fish. Progressive FishCulturist, vol. 25, No. 4. P. 174.

Wolf, Ken.

1959. Plasmoptysis and gelation of 

erythrocytes in coagulation of blood 

of freshwater bony fishes. Blood, 

vol. 14, No. 12, p. 1339-1344.

1963. Physiological salines for freshwater teleosts Progressive. FishCulturist, vol. 25, No. 3. p. 135-140.

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