INVESTIGATIONS IN FISH CONTROL

67. Method for Assessment of Toxicity or Efficacy 

of Mixtures of Chemicals

United States Department of the Interior

Fish and Wildlife Service
INVESTIGATIONS IN FISH CONTROL

67. Method for Assessment of Toxicity or Efficacy 

of Mixtures of Chemicals

United States Department of the Interior

Fish and Wildlife Service
INVESTIGATIONS IN FISH CONTROL

67. Method for Assessment of Toxicity or 

Efficacy of Mixtures of Chemicals

By Leif L. Marking and Verdel K. Dawson

United States Department of the Interior

Fish and Wildlife Service 

Washington, B.C. 1975
CONTENTS

Page 

Abstract.................................................................... 1

Introduction................................................................ 1

Materials and methods ..................................................... 2

Applications and discussion .............................................. 3

Literature cited ..........,.................................................. 7
METHOD FOR ASSESSMENT OF TOXICITY OR EFFICACY 

OF MIXTURES OF CHEMICALS

by

Leif L. Marking and Verdel K. Dawson 

Fish Control Laboratory, La Crosse, Wisconsin

ABSTRACT

The individual toxic contributions of poisons were summed, and the additive 

toxicity was defined by a linear index for two chemicals in combination. This index 

expresses the toxicity quantitatively: zero indicates additive toxicity, negative 

values indicate less than additive toxicity, and positive values indicate greater than 

additive toxicity. We selected examples from the literature and conducted tests in the 

laboratory to assess the additive toxicity of selected chemical mixtures to fish. The 

values ranged from -1.37 for zinc and cyanide to 7.20 for malathion and Delnav (R) . 

The method quantifies additive toxicity or efficacy, and assists in evaluating the 

advantages as well as environmental hazards resulting from chemical mixtures.

INTRODUCTION

The effect of mixtures of two or more 

chemicals is commonly referred to as additive, 

synergistic, or antagonistic depending on the 

relation of the toxicity of the mixtures to that of 

the individual components. Because these terms 

are abiguous and nonquantitative (Fingl and 

Woodbury 1965), a better system of terminology 

and quantification is needed.

British researchers have investigated 

methods of predicting the toxic effects of 

chemical mixtures in water by adding up "toxic 

units" of individual toxic materials (Lloyd 1961; 

Herbert and Shurben 1964; Herbert and Vandyke 1964; Brown et al. 1968; Brown et al. 1969; 

and Brown and Dalton 1970). Brown (1968) 

stated his reservations about the use of this 

technique and cautioned that actual bioassays 

of polluted water are to be preferred. Sprague 

and Ramsey (1965) used the "toxic unit" method

to predict the toxicity of copper and zinc 

mixtures to Atlantic salmon (Salmo salar).

Other techniques for evaluating the toxicity of 

mixtures of chemicals to mammals have been 

advanced (Keplinger and Deichmann 1967; 

Smyth et al. 1969); most of which follow the 

mathematical model for additive joint toxicity 

that yields the harmonic mean of the LDSO's for 

the components (Finney 1952). This model tests 

the hypothesis that the toxicity of chemical 

mixtures is simply additive. Smyth et al. (1969) 

normalized the values obtained from Finney's 

equation with a frequency distribution curve 

and adjusted the values to indicate additive 

toxicity with zero. Smyth et al. (1970) derived 

values in terms of adjusted ratios for mixtures of 

industrial organic chemicals fed to rats.

The objective of this paper is to adapt current 

methods and terminology to quantitatively 

describe additive toxicity of chemicals in water 

and to assign significance to the additive 

toxicity index.
MATERIALS AND METHODS

For toxicity tests, hatchery-reared rainbow 

trout (Salmo gairdneri) and bluegill (Lepomis 

macrochirus) were maintained as described by 

Hunn et al. (1968) and acclimated to test waters. 

The toxicities of individual chemicals to fish 

were determined according to standard 

laboratory procedures (Lennon and Walker 

1964; Marking 1969a). The toxicities of 

chemicals in mixtures were determined similarly except that two chemicals were added in a 

fixed ratio to the test vessels. Toxicity was 

defined by the LCSO's (concentrations producing 50% mortality) and their 95% confidence 

intervals (Litchfield and Wilcoxon 1949).

The additive indices of mixtures of chemicals 

were derived as follows. The effective contributions of each chemical (A and B) in a 

mixture are represented by the forA B

mula: _ + _!!!=S, where A and B are chemicals,

A, B,

i and m are the toxicities (LC50's) of the individual chemicals and the mixtures, respectively, and S is the sum of the biological activity. If 

the sum of toxicity of the chemicals is simply 

additive, S = 1.0; sums that are less than 1.0 

indicate greater than additive toxicity, and 

sums greater than 1.0 indicate less than 

additive toxicity (Fig. 1). This sum alone could 

function as a quantitative indication of additive 

toxicity, except that values greater than 1.0 are 

not linear with values less than 1.0.

Am

AI

GREATER THAN

JJm

B?

LESS THAN ADDITIVE TOXICITY

ADDITIVE TOXICITY

1.0 2.0 3.0

SUM OF TOXIC CONTRIBUTIONS

4.0

S(-l) + I = 

LESS THAN ADDITIVE TOXICITY GREATER THAN ADDITIVE TOXICITY

2.0 -1.0 0 +1.0 +2.0 

CORRECTED SUM OF TOXIC CONTRIBUTIONS

Figure 1. Sums (S) of toxic contributions for a chemical mixture, -which are nonlinear for less than additive 

and greater than additive toxicity (upper illustration) were corrected for linearity and direction of plus and 

minus values (lower illustration).
A system in which the index represents simple 

additive, greater than additive, and less than 

additive effects by zero, positive, and negative 

values, respectively, would be desirable. Such a 

system can be developed by establishing linearity and by assigning a reference point of zero for 

simple additive toxicity (Fig. 1). We established 

linearity by using the reciprocal of the values of 

S which were less than 1.0, and a zero reference 

point was achieved by subtracting 1.0 (the 

expected sum for simple additive toxicity) from

the reciprocal !"(_)-11. Thus greater than L S J 

additive toxicity is represented by index values

greater than zero. Index values representing 

less than additive toxicity were obtained by 

multiplying the values of S which were greater 

than 1.0 by -1 to make them negative, and a zero 

reference point was achieved by adding 1.0 to 

this negative value [S(-l)+l]. Thus, less than 

additive toxicity is represented by negative 

index values. The sum of effective contributions 

(S) is modified by one of two procedures: either

the additive index = £ -! for S< 1.0 (greater than 

o

additive toxicity) or S (-1) + 1 for S>1.0 (less 

than additive toxicity). A sum (S) of 1 yields an 

index value of zero by either modifying 

procedure and represents simple additive toxicity. A summary of the procedure follows.

Am Bm

 + =S, the sum of biological effects

A, B,

Additive index = -1.0 for S<1.0 and

S

Additive index = S(-l) + 1.0 for S>1.0

We assessed the significance of additive 

indices close to zero in our data by substituting 

values from the 95% confidence intervals into 

the formula to determine whether the range for 

additive indices overlapped zero (simple additive toxicity). The range was derived by 

selecting values of the 95% confidence interval 

yielding the greatest deviation from the additive 

index. The lower limits of the individual 

toxicants (At and Bi) and the upper limits of the 

mixtures (An-, and Bm ) were substituted for 

LC50's to determine the lower limit of the index. 

Correspondingly, the upper limits of the individual toxicants (At and Bt ) and the lower 

limits of the mixtures (Am and Bm ) were

substituted into the formula to determine the 

upper limit of the index.

Applications and Discussion

To test the method on existing information, 

we selected toxicity data from published papers 

in which the author had provided the toxicity for 

the individual components and for the mixtures. 

In some instances observed mortality was 

reported and used in place of statistically 

derived LCSO's. The significance of additive 

indices derived from data in the literature was 

not assigned because 95% confidence intervals 

were usually not reported.

Doudoroff (1952) studied the toxic activity of 

mixtures of zinc and copper, two suspected 

metal ion contaminants in some effluents. He 

showed extraordinary toxic activity against 

fathead minnows (Pimephales promelas) in 8-h 

tests in which survival was recorded rather than 

median lethal concentrations (LCSO's). The 

additive index is

1.0 0.025

( ) ~ l = 3'

020 - 0250 

a value which definitely supports Doudoroff s 

conclusions (Table 1). Lloyd (1961) and Sprague 

and Ramsey (1965) found less or no potentiation 

in zinc and copper mixtures, but they defined the 

toxicity with LCSO's for longer exposures; 

however, they did report that lethal mixtures of 

those metal ions act 2 to 3 times as fast as the 

metals singly.

Cairns and Scheier (1968) reported "slight 

antagonistic interaction" of zinc and cyanide to 

fathead minnows, in contrast to the opposite 

activity of zinc and copper. They attributed the 

effect to complexation of these ions. The 

additive index (- 1 .37) as computed in the present 

study indicates considerable antagonism. Chen 

and Selleck (1969) also considered the zinccyanide combination to be very antagonistic, 

but they did not quantify the toxicity.

Rowland (1969) reported additive effects for 

mixtures of two fish toxicants, antimycin and 

rotenone, whereas our index is -0.39. Although 

our value suggests slightly less than additive 

toxicity, the significance of the value should be 

defined before the results are interpreted.

Antimycin could be applied to water with a 

fluorescent tracer, rhodamine B. The tracer
Table 1. Toxicity or efficacy of chemicals applied individually and in combination against fishes 

and the calculated additive index

Chemical 

mixtures

Zinc a 

and

Copper3

Zinc 

and

Cyanide

Antimycin 

and

Rotenone

Antimycin 

and 

Rhodamine B

MS-222 

and 

QdSO4

Malachite green 

and 

Formalin'3

Toxic 

unit

mg/l

mg/l

mg/l

mg/l

M9/I

/&9/I

^ig/l 

mg/l

mg/l 

mg/l

mg/l

96-h LC50 or

Individually

8.0

0.2

4.2

0.18

0.032

57.0

0.048 

217

80 

25

0.2 

50

EC50 of chemical /y,Mitiv« Reference

In combination mdex

1.0 

3.00 Doudoroff 1952

0.025

3.90 

-1.37 Cairns and Scheier 1968

0.26

0.027 

-0.39 Howland 1969

31.0

0.047 

0.00 Marking 1969b 

5.0

30 

0.29 Berger 1969 

10

0.05 

0.83 Leteux and Meyer 1972 

15

a An 8-h time response, based on survival rather than LC50. 

Concentrations effective against parasites.

interacts little, if any, with the toxicant (additive index = 0.00), and the suitability of this 

tracer was supported (Marking 1969b).

The additive index method can be used for 

characteristics other than toxicity. For instance, the efficacy of MS-222 (tricaine 

methanesulfonate) and QdSO4 (quinaldine 

sulfate), two fish anesthetics, shows the rapid 

and sustaining anesthetic qualities, respectively, and an improved safety factor when the two 

chemicals are mixed rather than applied 

separately. The index of 0.29 suggests greater 

than additive efficacy and agrees with Berger's 

(1969) interpretation that the interaction was 

synergic. Schoettger and Steiicke (1970) discussed advantages of the anesthetic mixture, including a cost reduction of 60 to 80%. Mixtures of 

some fish therapeutants are more effective than 

individual disease treatment chemicals. For

example, the additive index for the efficacy of 

malachite green and formalin is 0.8 (Leteux and 

Meyer 1972).

Several pairs of toxic chemicals were chosen 

for determining additive toxicity in our 

laboratory (Table 2). When the fish toxicants 

antimycin and TFM (lampricide) were tested 

individually and in combination against 

bluegills, the additive index was 0.343 and the 

range computed from the 95% confidence 

interval was -0.189 to 1.17. Since the range 

overlaps zero, the toxicity for these two 

chemicals in this particular test was merely 

additive.

The index for mixtures of antimycin and 

Dibrom(R) against rainbow trout was -0.574 

(range, -1.12 to -0.173). Since the range did not 

overlap zero, the toxicity of the mixture was less
Table 2. Toxicity of toxicants applied individually and in combination to fish in 96-h, 

standardized tests at 12 C

Species, toxicants, 

and toxic unit

LC50 and 95% confidence interval

Individually In combination

Additive 

index

Bluegill

Antimycin ( /u.g/1)

and 

TFM (mg/l)

Rainbow trout 

Antimycin ( /u.g/1)

and 

Dibrom (mg/l)

0.0710 

0.0574-0.0879

4.96 

4.10-5.99

0.0312 

0.0266-0.0366

0.0490 

0.0279-0.0633

0.0390 

0.0296-0.0506

0.970 

0.744-1.26

0.0300 

0.0272-0.0331

0.0300 

0.0272-0.0331

0.340 

-0.189 to 1.17

-0.574 

-1.12 to 0.173

Antimycin

and 

KMnO4 (mg/l)

0.0412 

0.0371-0.0457

1.22 

1.08-1.38

3.79 

2.78-5.16

1.0

-91.8

Malathion (/u.g/1)

and 

Delnav ( /xg/l)

70.0 

59.2-82.7

47.2 

42.4-52.6

3.44 

2.92-4.06

3.44 

2.92-4.06

7.20 

5.09 to 10.0

than additive. Berger (1971) reported that 

mixtures of those toxicants are synergistically 

toxic to black bullhead (Ictalurus melas), 

largemouth bass (Micropterus salmoides), and 

yellow perch (Perca flavescens). These data 

suggest that additive toxicity may vary for 

different species of fish, ratios of toxicants, or 

test conditions. For example, Hoff and Westman 

(1965) reported that a 3:2 ratio of Dibrom<R> and 

malathion showed promising selectivity toward 

bluegills and pumpkinseeds (Lepomis gibbosus) 

in the presence of largemouth bass. For these 

reasons, the additive index for toxicants should 

be determined for nontarget as well as target 

species.

Antimycin is readily detoxified by oxidizing 

agents such as potassium permanganate 

(Walker 1967). Both chemicals were toxic to fish, 

but the counteraction through oxidation greatly 

decreased the toxicity of the mixture. Potassium 

permanganate was added at 1.0 mg/l to each 

concentration of antimycin because that concentration of permanganate has been effective 

in fish management applications. As expected, 

the combination produced extreme antagonistic 

activity against rainbow trout; the additive 

index was -91.8 in standardized tests at pH 7.5 

(Table 2). This procedure could be used to assess 

the effectiveness of permanganate for detoxifying antimycin under other conditions of
different pH or temperature.

(R) Mixtures of malathion and Delnav "', two 

organic phosphates reported to be synergistic 

against insects, are extremely toxic to fish. The 

additive index is 7.20 for rainbow trout. That 

value is the highest for any toxicant mixture 

evaluated by our method and further 

emphasizes the potency of certain pesticide 

mixtures.

The 95% confidence interval influences the 

significance of the additive index, and 

significance becomes more difficult to show as 

the range widens because of the greater 

likelihood that the range will overlap zero. The 

95% confidence interval is influenced by the 

number of concentrations and the number of 

test organisms per concentration. Therefore, 

well planned toxicity tests which result in 

narrow confidence intervals are the most useful 

in the assignment of effects of chemical mixtures.

The study of mixtures of toxic chemicals in 

water and the resultant benefits or hazards is 

fairly new, and only a few methods have been 

investigated. Conceivably, the additive toxicity 

of more than two chemicals could be evaluated 

by simply adding the contributions of additional chemicals according to the following

formula,

Am Bm Cm

 + + + - - - = S.

Ai B, Ci

This additive toxicity index method could be 

useful for assessing the economics of mixtures 

of chemicals, for determining toxicity advantages against target brganisms, for determining 

hazards or disadvantages against nontarget 

organisms, for assessing the additive toxicity of 

different ratios of chemicals in a mixture, and 

for assessing physical influences of the environment on additive toxicity. Advantages of this 

method over existing methods include linearity 

for all index values and a procedure for 

determining the significance of indices.
LITERATURE CITED

Berger, B. L. 1969. A synergic mixture of MS-222 and

quinaldine sulfate as an anesthetic. Presented at the 31st

Midwest Fish and Wildlife Conference, St. Paul,

Minnesota. 7 pp. mimeo. 

Berger, B. L. 1971. Fish toxicant compositions and method

of using them. U.S. Patent 3,608,072. Filed March 21,1969,

patented September 21, 1971. 2 pp. 

Brown, V. M. 1968. The calculation of the acute toxicity of

mixtures of poisons to rainbow trout. Water Res..

2:723-733. 

Brown, V. M., and R. A. Dalton. 1970. The acute lethal

toxicity to rainbow trout of mixtures of copper, phenol,

zinc and nickel. J. Fish Biol. 2:211-216. 

Brown, V. M., D. H. M. Jordon, and B. A. Tiller. 1969. The

acute toxicity to rainbow trout of fluctuating concentrations and mixtures of ammonia, phenol and zinc. J.

Fish Biol. 1:1-9. 

Brown, V. M., V. V. Milrovic, and G. T. C. Stark. 1968.

Effects of chronic exposure to zinc on toxicity of a mixture

of a detergent and zinc. Water Res. 2:255-263. 

Cairns, J., Jr., and A. Scheier. 1968. A comparison of toxicity

of some common industrial waste components tested

individually and combined. Prog. Fish-Cult. 30(l):3-8. 

Chen, C. W., and R. E. Selleck. 1969. A kinetic model offish

toxicity threshold. J. Water Pollut. Control Fed. 41 (part

2):R294-R308. 

Doudoroff, P. 1952. Some recent developments in study of

toxic industrial wastes. Proc. Annu. Pac. Northwest Ind.

Waste Conf. 4. 21 pp. 

Fingl, E., and D. M. Woodbury. 1965. General principles.

Pages 1-36 in L. S. Goodman and A. Gilman, eds.

Pharmacological basis of therapeutics. 3rd ed. The

Macmillan Co., New York. 

Finney, D. J. 1962. Probit analysis, 2nd ed. Cambridge Univ.

Press, London and New York. 318 pp. 

Herbert, D. W. M., and D. S. Shurben. 1964. The toxicity to

fish of mixtures of poisons. I. Salts of ammonia and zinc.

Ann. Appl. Biol. 53:33-41. 

Herbert, D. W. M., and J. M. Vandyke. 1964. The toxicity to

fish of mixtures of poisons. II. Copper-ammonia and zincphenol mixtures. Ann. Appl. Biol. 53:415-421. 

Hoff, J. B., and J. R. Westman. 1965. Experiments with

Dibrom-malathion formulation as a selective piscicide.

N.Y. Fish Game J. 12:99-107. 

Howland, R. M. 1969. Interaction of antimycin A and

rotenone in fish bioassays. Prog. Fish-Cult. 31(l):33-34.

Hunn, J. B., R. A. Schoettger, and E. W. Whealdon. 1968.

Observations on the handling and maintenance of

bioassay fish. Prog. Fish-Cult. 30:164-167. 

Keplinger, M. L., and W. B. Deichmann. 1967. Acute toxicity

of combinations of pesticides. Toxicol. Appl. Pharmacol.

10:586-595. 

Lennon, R. E., and C. R. Walker. 1964. Laboratories and

methods for screening fish control chemicals. U.S. Bur.

Sport Fish. Wildl. Invest. Fish Control. No. 1 (Circ. 185). 15

pp. 

Leteux, F., and F. P. Meyer. 1972. Mixtures of malachite

green and formalin for controlling Ichthyophthirius and

other protozoan parasites of fish. Prog. Fish-Cult.

34(l):21-26. 

Litchfield, J. T., Jr., and F. Wilcoxon. 1949. A simplified

method of evaluating dose-effect experiments. J. Pharm.

Exp. Therap. 96:99-113. 

Lloyd, R. 1961. The toxicity of mixtures of zinc and copper

sulfates to rainbow trout (Salmo gairdnerii Richardson).

Ann. Appl. Biol. 49:535-538. 

Marking, L. L. 1969a. Toxicological assays with fish. Bull.

Wildl. Dis. Assoc. 5:291-294. 

Marking, L. L. 1969b. Toxicity of rhodamine B and

fluorescein sodium to fish and their compatibility with

antimycin A. Prog. Fish-Cult. 31(3):139-142. 

Schoettger, R. A., and E. W. Steucke, Jr. 1970. Synergic

mixtures of MS-222 and quinaldine as anesthetics for

rainbow trout and northern pike. Prog. Fish-Cult.

34(4):202-205. 

Smyth, H. F., Jr., C. S. Weil, J. S. West, and C. P. Carpenter.

1969. An exploration of joint toxic action: Twenty-seven 

industrial chemicals intubated in rats in all possible pairs. 

Toxicol. Appl. Pharmacol. 14:340-347. 

Smyth, H. F., Jr., C. S. Weil, J. S. West, and C. P. Carpenter.

1970. An exploration of joint toxic action. II. Equitoxic

versus equivolume mixtures. Toxicol. Appl. Pharmacol.

17:498-503. 

Sprague, J. B., and B. A. Ramsey. 1965. Lethal levels of

mixed copper-zinc solutions for juvenile salmon. J. Fish.

Res. Board Can. 22(2):425-432. 

Walker, C. R. 1967. Deactivation of antimycin. Pages

173-194 in Progress in sport fishery research 1966. U.S.

Bur. Sport Fish. Wildl., Resour. Publ. No. 39. 21 pp.
(Reports 53 through 55 are in one cover.)

53. Toxicity of Mixtures of Quinaldine Sulfate and MS-222 to Fish, by Verdel K. Dawson and Leif L. Marking 

1973. 11 pp.

54. The Efficacy of Quinaldine Sulfate: MS-222 Mixtures for the Anesthetization of Freshwater Fish, by Philip 

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

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

Sulfate:MS-222, by Joe B. Sills, John L. Alien, Paul D. Harman, and Charles W. Luhning. 1973. 12 pp.

(Reports 56 through 59 are in one cover.)

56. Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) to 10 Species of Algae, by A. A. Maki, L. 

D. Geissel, and H. E. Johnson. 1975. 17 pp.

57. Acute Toxicities of 3-Trifluoromethyl-4-nitrophenol (TFM) and 2',5-Dichloro-4'-nitrosalicylanilide (Bayer 

73) to Larvae of the Midge Chironomus tentans, by J. A. Kawatski, M. M. Ledvina, and C. R. Hansen. 1975.7 

pp.

58. Acute Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) to Nymphs of Mayflies 

(Hexagenia spj, by C. R. Fremling. 1975. 8 pp.

59. Toxicity and Residue Dynamics of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) in Aquatic 

Invertebrates, by H. O. Sanders and D. F. Walsh. 1975. 9 pp.

(Reports 60 through 62 are in one cover.)

60. Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) to Nontarget Fish in Static Tests, by Leif 

L. Marking and Lee E. Olson. 1975. 27 pp.

61. Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) to Nontarget Fish in Flow-Through 

Tests, by Leif L. Marking, Terry D. Bills, and Jack H. Chandler. 1975. 9 pp.

62. Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) to Selected Aquatic Invertebrates and 

Frog Larvae, by Jack H. Chandler and Leif L. Marking. 1975. 7 pp.

(Reports 63 through 66 are in one cover.)

63. Laboratory Efficacy of 3-Trifluoromethyl-4-nitrophenol (TFM) as a Lampricide, by Verdel K. Dawson, 

Kenneth B. Gumming, and Philip A. Gilderhus. 1975. 13 pp.

64. Effects of 3-Trifluoromethyl-4-nitrophenol (TFM) on Developmental States of the Sea Lamprey, by George 

V. Piavis and John H. Ho well. 1975. 8 pp.

65. Accumulation and Loss of Residues of 3-Trifluoromethyl-4-nitrophenol (TFM) in Fish Muscle Tissue: 

Laboratory Studies, by Joe B. Sills and John L. Alien. 1975. 10 pp.

66. Residues of 3-Trifluoromethyl-4-nitrophenol (TFM) in a Stream Ecosystem after Treatment for Control of 

Sea Lampreys, by Philip A. Gilderhus, Joe B. Sills, and John L. Alien. 1975. 7 pp.

Fish Control Laboratories

Fish and Wildlife Service

U.S. Department of the Interior

P.O. Box 862 

La Crosse, Wisconsin 54601

GPO 859 -739



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