INVESTIGATIONS IN FISH CONTROL

92. Acute and Chronic Toxicity of Rotenone to Daphnia magna

93. Toxicity of Rotenone to Developing Rainbow Trout

94. Oral Toxicity of Rotenone to Mammals

OCH 3

UNITED STATES DEPARTMENT OF THE INTERIOR 

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Library of Congress 75[8802r83]rev
INVESTIGATIONS IN FISH CONTROL

92. Acute and Chronic Toxicity of Rotenone to Daphnia magna

By J. J. Rach, T. D. Bills, and L. L. Marking

93. Toxicity of Rotenone to Developing Rainbow Trout

By T. D. Bills, J. J. Rach, and L. L. Marking

94. Oral Toxicity of Rotenone to Mammals

By L. L. Marking

I .S.

KISII&XVII 1)1 IKK 

SKKVU'K

UNITED STATES DEPARTMENT OF THE INTERIOR 

FISH AND WILDLIFE SERVICE

Washington, D.C. 1988

Acute and Chronic Toxicity of Rotenone to Daphnia magna

by

J. J. Rach, T. D. Bills, and L. L. Marking

National Fisheries Research Center 

U.S. Fish and Wildlife Service

P.O. Box 818 

La Crosse, Wisconsin 54602

Abstract

The continued use of rotenone as a fish toxicant depends on the development of information 

requested bj ' e U.S. Environmental Protection Agency for reregistration. To meet one of the 

requirements, we exposed Daphnia magna to rotenone in toxicity tests. In exposures of 0.5 to 

10.0 ptg/L rotenone in a 48-h acute toxicity test, the EC50 was 3.7 ptg/L; in exposures of 0.312 to 

5.0 ptg/L in a 21-day chronic toxicity test, the EC50 was 2.1 ^g/L. The no-observed-effect concentration was 1.25 ptg/L.

Rotenone is widely used in fishery management for the 

removal of unwanted fish populations. Its usefulness stems 

from its high toxicity to fish, low toxicity to mammals, 

and rapid decomposition in the environment (Lennon et al. 

1970; Haley 1978). Its use has been questioned, however, 

by environmental groups and others who are concerned 

about the use of chemicals in the environment.

The U.S. Environmental Protection Agency (EPA) has 

requested data needed for the reregistration of rotenone. 

During the last several years, studies of its toxicity, 

accumulation, and depuration in fish, mammals, invertebrates, and plants have been completed as part of the EPA 

requirements.

One of the studies required consisted of acute and 

chronic toxicity tests on Daphnia magna, the organism 

chosen because it is sensitive to toxic substances, is small, 

can be easily identified, is available from laboratories and 

commercial suppliers, and has been used extensively in 

toxicity testing (EPA 1984). The tests we conducted to 

meet this requirement are described here.

Materials

Analytical grade rotenone (96.47% pure) used for this 

study was obtained from the S. B. Penick Corporation, 

Lyndhurst, New Jersey. Purity was determined by high 

pressure liquid chromatography (HPLC) as described by 

Dawson et al. (1983).

Reconstituted water, prepared by adding selected salts 

to deionized water used for all exposures and culture 

waters, had the following characteristics: pH 7.6-8.0, 

hardness 160-180 mg/L as CaCOs, and alkalinity 

110-120 mg/L as CaCO3 (ASTM Committee E-35 on 

Pesticides 1980). Water samples were analyzed according to methods of the American Public Health Association et al. (1985).

Controls for the acute exposures included a blank water 

control, and a blank acetone control that contained acetone 

at a concentration equal to that in the highest exposure 

concentration. Controls for the chronic test were similar, 

except that algal food was added.

1
Exposure concentrations of rotenone used were 0.5, 

2.5, 5.0, 7.5, and 10.0 /-tg/L in the acute toxicity test and 

0.312,0.625, 1.25, 2.5, and 5.0 j^g/L in the chronic toxicity test. For both types of tests, rotenone stock solutions were prepared in 2-L volumetric flasks containing 

diluted water and rotenone; for the chronic toxicity tests, 

predetermined amounts of algal cells and food supplement 

were added. Rotenone concentrations in all tests were 

verified analytically by HPLC.

For the analysis of rotenone concentrations, we collected samples from the stock solutions before algal cells 

and food supplement were added. Rotenone was concentrated from the samples by the method of Dawson et al. 

(1983). The volume of water extracted increased with 

decreases in the theoretical rotenone concentration: 

400 mL were extracted for the 5.0 ^g/L concentration and 

800 mL for the 2.5- and 1.25-^g/L concentrations. Water 

samples were filtered, buffered to pH 5.0, and passed 

through a disposable Baker Cjg column with the aid of 

a Baker-10 vacuum manifold. Extracted rotenone was then 

eluted from the columns with 2 mL of methanol.

For the direct analysis of eluted methanol samples, we 

used a Waters WISP710B autoinjector in conjunction with 

a Waters 510 HPLC pump, Waters 481 LC spectrophotometer, and a Micro-Pak (30 cmx4 mm) MCH-10 

reverse-phase column. A mobile phase of methanol: water 

(78:22; volume:volume) was used at a flow rate of 

1.0 mL/min. An ultraviolet spectrophotometer (wavelength 295 nm) enabled detection of rotenone in samples; 

attenuation was 0.01. Rotenone concentrations in the 

samples were calculated on the basis of peak area.

Cultures of Daphnia magna were maintained at 

20+1 °C in a constant temperature water bath, at a photoperiod of 16 h light and 8 h darkness. We transferred adult 

brood stock daphnids to fresh water weekly in fire-polished 

pipettes with an inside diameter of 5-8 mm or at least 

1.5 times the diameter of the daphnids. We introduced 

the organisms beneath the surface of the new medium to 

avoid trapping air under the carapace. Cultures were considered healthy and suitable for use when survival in each 

culture was >90% over a 2-week period, no air-locked 

daphnids were present, no ephippia were produced, and 

large numbers of young were present (EPA 1984).

Adult daphnids that were about to have their second to 

sixth broods were cultured under conditions similar to 

those described. Young daphnids produced from these 

adults provided brood stock for later testing. These young 

brood stock daphnids were reared in 2-L glass containers, 

each having 20 daphnids per 1,600 mL of water. The 

young produced by these brood daphnids were used for 

acute and chronic tests (EPA 1984).

The alga Selenastrum capricornutum, obtained from the 

EPA Laboratory, Duluth, Minnesota, was cultured at 

20+2°C in supplemented Woods Hole Marine Biological 

Laboratory medium (EPA 1984), as food for the daphnids. It was inoculated into three sterile culture vessels 

and allowed to bloom. The resulting cultures were used 

to maintain the Daphnia magna brood stock and to feed 

the experimental animals during the chronic toxicity study. 

The daphnids were provided algae at densities of 108 

cells/L of test water, as recommended by EPA (1984). 

A liquefied solution of a commercial trout food, used to 

supplement the Selenastrum culture (EPA 1984), was 

added every other day to the exposure beakers and to 

Daphnia cultures to yield a concentration of 5 mg/L.

Procedures in Acute and Chronic Tests

A routine procedure was used to ensure that the young 

daphnids used in the tests were <24 h old. One day before 

the test, we removed 15 adult brood daphnids and transferred them into individual 100-mL beakers containing 

80 mL of water and food. The next day we transferred 

the offspring to a 1-L beaker containing 800 mL of water. 

The young daphnids were then randomly pipetted into the 

exposure beakers used for the acute and chronic tests. 

Photoperiod in all tests was 16 h light and 8 h darkness.

In the acute test, daphnids were transferred into test 

vessels, which were 100-mL beakers containing 80 mL 

of test solution. Four beakers, each containing five daphnids, were required for each experimental treatment (control, acetone control, and each test concentration). The 

beakers were labeled, covered with watch glasses, and 

randomly placed in a water bath at 20± 1°C. After 48 h, 

the beakers were removed and mortality was recorded. 

We determined EC50 values (toxicant concentration 

resulting in immobilization or death of 50% of the exposed organisms) and estimated 95 % confidence interval 

(C.I.) by the methods of Litchfield and Wilcoxon (1949).

In the chronic test, one daphnid was transferred into 

each of 10 100-mL beakers containing 80 mL of test solution with food supplement. As in the acute test, the beakers 

were labeled, covered with watch glasses, and randomly 

placed in a water bath at 20±1°C.

Daphnids for the chronic exposures were transferred 

to clean beakers with fresh test solutions and observations 

on mortalities and molting were made three times a week 

(Monday, Wednesday, Friday). Rotenone concentrations 

were verified by HPLC on days 0, 7, 14, and 19. 

Chemical characteristics of the reconstituted water used 

to prepare rotenone test solutions were checked on days 

0, 7, and 19. The number of young produced and water
Table 1. Analysis of the reconstituted water for 21-day 

chronic toxicity test. (Temperature was 20°C throughout the test.)

Date

(1986)

16 April

23 April

5 May

pH

8.34

8.18

8.02

Alkalinity

(mg/L)

112

108

94

Hardness

(mg/L)

166

164

168

Dissolved

oxygen

(mg/L)

9.0

a

9.4

aNo determination.

bath temperature were recorded daily. Surviving daphnids 

on day 21 were measured under a microscope equipped 

with an ocular micrometer. Lengths of the daphnids (top 

of the head to the base of the spine) in each concentration 

were statistically compared by analysis of variance and 

application of the Student-Newman-Keuls multiple range 

test (Sokal and Rohlf 1969).

Criteria for a valid Daphnia chronic toxicity test require 

that control animals produce an average of at least 40 

young, and that survival in the control units be 80% or 

higher over the 3-week test (EPA 1984). The EC50 values 

and the 95 % C.I. were determined by the method of Litchfield and Wilcoxon (1949).

Results

Chemical characteristics of the reconstituted water for 

the chronic study (Table 1) remained within acceptable 

limits established by the ASTM Committee E-35 on 

Pesticides (1980). Chemical characteristics of the water

in the rotenone exposures were analyzed at the start 

and end of batch replacements. Concentrations of components remained within the established acceptable ranges, 

except that alkalinity levels were slightly below normal 

(Table 2).

Rotenone concentrations of 0.312 and 0.625 pg/L were 

below minimum detection levels; only the solutions containing 1.25, 2.5, and 5.0 ftg/L rotenone were analyzed 

(Table 3). Measured rotenone concentrations (mean + 

SD) were 1.38 ±0.078 ftg/L for the 1.25-jug/L concentration, 2.63 ±0.137 jug/L for the 2.5-ptg/L concentration, 

and 5.27 |ug/L for the 5.0-/ug/L concentration.

In the acute toxicity test, all daphnids survived in the 

controls and at 0.5 /ug/L rotenone, but only 5% survived 

at 10.0 Mg/L (Table 4). The calculated 48-h EC50 value 

for rotenone was 3.7 Mg/L (95% C.I., 2.5-5.5 ftg/L).

In the 21-day chronic toxicity test, the survival rate in 

the acetone control was 80%, and the no-observed-effect 

concentration was 1.25 Mg/L (Table 5). The calculated 

EC50 value for rotenone in the chronic 21-day test was 

2.1 Mg/L (95% C.I., 0.9-4.9 Mg/L). Each surviving 

daphnid produced 40 or more young in the rotenone concentrations of 0.312-1.25 Mg/L, but only 19 in the 

2.5-Mg/L concentration. The size (lengths) of daphnids 

in the control group and the rotenone-treated groups 

(Table 6) did not differ significantly.

Discussion

The standard toxicity tests described here were conducted to update the data base on rotenone toxicity to 

crustaceans. Daphnia magna was used as the test organism 

because of its importance in the aquatic environment 

and because it has been recommended by the EPA for

Table 2. Chemical characteristics ofrotenone exposure water used for a batch replacement during the 21-day chronic 

toxicity test, freshly prepared and after 3 days of exposure. (Temperature was 20°C throughout the test.)

Dissolved

Test 

concentration

(Mg/L)

0.0 (control) 

0.312

0.625

1.25

2.5

pH

Fresh

8.02 

8.29

8.30

8.31

8.26

3 days

8.16 

8.44

8.41

8.43

8.45

Alkalinity 

(mg/L)

Fresh

94 

94

105

104

111

3 days

106

97

100

98

103

Hardness

(mg/L)

Fresh

168 

164

168

166

164

3 days

166 

164

166

164

166

oxygen

(mg/L)

Fresh

9.4 

9.4

9.2

9,4

9.4

3 days

8.7 

8.8

8.7

8.6

8.8
Table 3. Rotenone concentrations (\ag/L} a determined by 

high pressure liquid chromatography during the 21-day 

chronic toxicity test.

Calculated rotenone 

concentrations 

fag/L)

Day

0

7

14

19

T^ot<a

(1986)

16 April

23 April

30 April

5 May

1.25

1.33

1.49

1.33

1.36

2.5

2.53

2.55

2.61

2.83

5.0b

5.27

a Rotenone was not detectable at calculated concentrations of 0.312 or 

0.625. Detection level was 1 Mg/L. 

bAll organisms died within 5 days.

Table 4. Survival of daphnids fn = 20) exposed to rotenone in a 48-h acute toxicity test.

(Mg/L)

0.0 (water control)

0.0 (acetone control)

0.5

2.5

5.0

7.5

10.0

Survival

100

100

100

80

25

15

5

aEC50 = 3.7 ng/L; range, 2.5-5.5

Table 5. Survival of daphnids fn = 10) exposed to rotenone during a 21-day chronic toxicity test.

Concentration

(ng/L)

0.0 (water control)

0.0 (acetone control)

0.312

0.625

1.25

2.5

5.0

Survival of 

test organisms

90

80

70

100

80

40

0

Number of 

young produced by 

test organisms

483

456

459

578

351

78

Average number of 

young produced per 

surviving adult

53.7

57.0

65.6

57.8

43.9

19.5

aEC50 = 2.1 Mg/L; range, 0.9-4.9

Table 6. Mean lengths (mm) of daphnids in chronic exposure groups.

(Mg/U

0.0 (water control)

0.0 (acetone control)

0.312

0.625

1.25

2.5

Length (mm)

Mean

3.82

3.94

3.83

3.88

3.56

3.32

SE

0.17

0.08

0.17

0.04

0.08

0.28

standard toxicity testing (Committee on Methods for Toxicity Tests with Aquatic Organisms 1975).

The toxicity data indicated that daphnids were much 

more sensitive than fish to rotenone, as shown earlier by 

Kemp et al. (1971). The 48-h EC50 value for Daphnia 

was 3.7 jug/L, whereas Marking and Bills (1976) showed 

that, for fish, the LC50 ranged from 21.5 to 389 jug/L 

of the formulated material. Even though the toxicity data 

for fish and daphnids were developed in a laboratory 

environment, it is likely that daphnid numbers would be 

reduced after rotenone was used to remove nuisance fish 

populations. However, the organisms are prolific and have
been shown to recover quickly from chemical treatments 

and adverse conditions (Jacobi and Degan 1977).

References

American Public Health Association, American Water Works 

Association, and Water Pollution Control Federation. 1985. 

Standard methods for the examination of water and wastewater, 16th ed. American Public Health Association, Washington, D.C. 1268 pp.

ASTM Committee E-35 on Pesticides. 1980. Standard practice 

for conducting acute toxicity tests with fishes, macroinvertebrates, and amphibians. E729080. Pages 1-25 in Annual book 

of ASTM standards, Part 46. American Society for Testing 

and Materials, Philadelphia, Pa.

Committee on Methods for Toxicity Tests with Aquatic Organisms. 1975. Methods for acute toxicity tests with fish, macroinvertebrates, and amphibians. U.S. Environ. Prot. Agency, 

Ecol. Res. Ser. EPA-660/3-75-009. 61 pp.

Dawson, V. K., P. D. Harman, D. P. Schultz, and J. L. Alien. 

1983. Rapid method for measuring rotenone in water at 

piscicidal concentrations. Trans. Am. Fish. Soc. 112:725-727.

Haley, T. J. 1978. A review of the literature of rotenone. 

J. Environ. Pathol. Toxicol. 1:315-339.

Jacobi, G. Z., and D. J. Degan. 1977. Aquatic macroinvertebrates in a small Wisconsin trout stream before, during, and 

two years after treatment with the fish toxicant antimycin. U.S. 

Fish Wildl. Serv., Invest. Fish Control 81. 24 pp.

Kemp, H. T., J. P. Abrams, and R. C. Overbeck. 1971. Water 

quality criteria data book. Vol. 3. Effects of chemicals on 

aquatic life, selected data from the literature through 1968. 

EPA Water Pollution Center Research Serial 18050GWV05/ 

71, U.S. Government Printing Office, Washington, D.C.

Lennon, R. E., J. B. Hunn, R. A. Schnick, and R. M. Burress. 

1970. Reclamation of ponds, lakes, and streams with fish 

toxicants: a review. FAO Fish Tech. Pap. 100. 99 pp.

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

for evaluating dose-effect experiments. J. Pharmacol. Exp. 

Ther. 96:99-113.

Marking, L. L., and T. D. Bills. 1976. Toxicity of rotenone 

to fish in standardized laboratory tests. U.S. Fish Wildl. Serv., 

Invest. Fish Control 72. 11 pp.

Sokal, R. R., and F. J. Rohlf. 1969. Biometry, 1st ed. W. H. 

Freeman and Company, San Francisco, Calif. 776 pp.

U.S. Environmental Protection Agency. 1984. Interim procedures for conducting the Daphnia magna toxicity assay. 

Environmental Research Laboratory, Duluth, Minn., and 

Environmental Monitoring Systems Laboratory, Las Vegas, 

Nev., February 1984. 28 pp. -I- appendixes (Unpubl. 

manuscr.).

Rach, J. J., T. D. Bills, and L. L. Marking. 1988. Acute and chronic toxicity 

of rotenone to Daphnia magna. U.S. Fish Wildl. Serv., Invest. Fish Control 

92. 5 pp.

The continued use of rotenone as a fish toxicant depends on the development 

of information requested by the U.S. Environmental Protection Agency for reregistration. To meet one of the requirements, we exposed Daphnia magna to 

rotenone in acute and chronic toxicity tests. In exposures of 0.5 to 10.0 ^g/L 

rotenone in a 48-h acute toxicity test, the EC50 was 3.7 ^g/L; in exposures of 

0.312 to 5.0 ftg/L in a 21-day chronic toxicity test, the EC50 was 2.1 ^g/L. 

The no-observed-effect concentration of rotenone to daphnids was 1.25

Key words: Rotenone, acute toxicity, chronic toxicity, Daphnia magna, noobserved-effect concentration.

Rach, J. J., T. D. Bills, and L. L. Marking

of rotenone to Daphnia magna. U.S. Fi

92. 5 pp.

The continued use of rotenone as a fish t

of information requested by the U.S. Envir

registration. To meet one of the requireme

rotenone in acute and chronic toxicity test

rotenone in a 48-h acute toxicity test, the E

0.312 to 5.0 \iglL in a 21-day chronic tox

The no-observed-effect concentration of ro

Key words: Rotenone, acute toxicity, ch

observed-effect concentration.

Rach, J. J., T. D. Bills, and L. L. Marking. 1988. Acute and chronic toxicity of rotenone to Daphnia 

magna. U.S. Fish Wildl. Serv., Invest. Fish Control 92. 5 pp.

The continued use of rotenone as a fish toxicant depends on the development of information requested 

by the U.S. Environmental Protection Agency for reregistration. To meet one of the requirements, 

we exposed Daphnia magna to rotenone in acute and chronic toxicity tests. In exposures of 0.5 to 

10.0 ng/L rotenone in a 48-h acute toxicity test, the EC50 was 3.7 ng/L; in exposures of 0.312 to 

5.0 /jg/L in a 21-day chronic toxicity test, the EC50 was 2.1 ng/L. The no-observed-effect concentration of rotenone to daphnids was 1.25 ^g/L.

Key words: Rotenone, acute toxicity, chronic toxicity, Daphnia magna, no-observed-effect 

concentration.

Rach, J. J., T. D. Bills, and L. L. Marking. 1988. Acute 

magna. U.S. Fish Wildl. Serv., Invest. Fish Contr

The continued use of rotenone as a fish toxicant depen

by the U.S. Environmental Protection Agency for rere

we exposed Daphnia magna to rotenone in acute and 

10.0 ng/L rotenone in a 48-h acute toxicity test, the E

5.0 /ig/L in a 21-day chronic toxicity test, the EC50 wa

tion of rotenone to daphnids was 1.25 ^g/L.

Key words: Rotenone, acute toxicity, chronic tox

concentration.

Toxicity of Rotenone to Developing Rainbow Trout

by

T. D. Bills, J. J. Rach, and L. L. Marking

U.S. Fish and Wildlife Service 

National Fisheries Research Center

P. O. Box 818 

La Crosse, Wisconsin 54602

Abstract

In a flow-through toxicity test, we exposed eyed eggs and larvae of rainbow trout (Solmo gairdnerf) 

for 32 days to crystalline technical grade rotenone to determine its effects on survival, hatching, 

and growth. Rotenone concentrations of 1.0 to 10.0 /ng/L did not kill the eyed eggs, which hatched 

normally after 5 or 6 days of exposure. Mortality of young larvae increased as rotenone concentrations increased. After 32 days of exposure, and after all larvae had transformed to the swim-up 

stage, the LC50 was 2.08 /ng/L. Hatching time was unaffected by exposures to rotenone, but growth 

was significantly decreased in fish that survived concentrations of 2.21 and 2.75 ^g/L.

Rotenone has been used extensively as an insecticide, 

and has been used by fishery managers since the 1930's 

to remove unwanted fish populations from lakes and 

streams (Schnick 1974). The toxicity of rotenone to 

aquatic organisms is well documented (Marking and Bills 

1976); however, owing to changes in regulations of the 

U.S. Environmental Protection Agency regarding the 

toxic effects of contaminants on aquatic organisms, the 

U.S. Fish and Wildlife Service undertook additional 

studies to determine whether rotenone can be safely used 

as a piscicide.

The present study (funded by the Division of Federal 

Aid of the U.S. Fish and Wildlife Service) was designed 

to evaluate the effects of rotenone on the eyed eggs and 

young of rainbow trout (Salmo gairdneri) under chronic 

exposure conditions. We observed the effects on hatching and on growth and mortality of larvae during a 32-day 

exposure. This period was chosen because embryos or 

larvae have been shown to be the life stages most sensitive to toxicants (Woltering 1984).

Materials and Methods

Crystalline rotenone (96.47% pure) for the study was 

supplied by S. B. Penick and Company. Tests were conducted with a proportional diluter (Mount and Brungs 

1967) that provided about 12 turnovers of test solution 

daily. Test organisms were exposed to rotenone continuously for 28 days at the following concentrations (mean 

±SD): 0, 1.01±0.09, 2.21±0.266, 2.75±0.424, 

4.37±0.092, 5.32±0.197. 7.52+0.577. and 10.0±0.436. We 

measured concentrations of rotenone in water by high 

-pressure liquid chromatography (HPLC), using the 

method of Dawson et al. (1983). Samples for analysis 

were collected once every 5 days. Dissolved oxygen and 

temperature in test solutions were monitored daily.

Chemical characteristics of water used in the tests were 

determined in accordance with the methods outlined by 

the American Public Health Association et al. (1985); 

average pH was 8.0, total hardness was 138 mg/L as 

CaCO}. and total alkalinity was 105 mg/L as
Table 1. Cumulative mortality (%) in duplicated exposures (100 each) of eyed eggs and early larval stages of rainbow trout to rotenone in water at 72°C. a

Day 0.00

Exposure concentration (jug/L)

1.01 2.21 2.75 4.37 5.32 7.52

aLethal concentrations (yg/L) and 95% confidence at 32 days: LC50, 2.08 (1.98-2.18); and LC01, 1.00 (0.89-1.12).

All eggs were eyed. 

cEggs began hatching at all concentrations.

All surviving larvae reached swim-up stage and appeared to be searching for food.

10.04

l b

5C

10

15

20

25

32d

0.0

0.0

3.5

4.5

6.0

6.5

7.0

0.0

0.5

2.0

2.0

2.5

3.0

4.0

0.0

0.0

6.0

33.0

47.5

53.0

58.0

0.0

0.0

17.0

91.0

94.0

96.5

96.5

0.0

0.0

97.5

100.0

100.0

100.0

100.0

0.0

0.0

99.0

100.0

100.0

100.0

100.0

0.0

0.0

100.0

100.0

100.0

100.0

100.0

0.0

6.0

100.0

100.0

100.0

100.0

100.0

Eyed rainbow trout eggs obtained from Trout Lodge, Inc., 

McMillan, Washington, were handled according to procedures outlined by Hunn et al. (1968). Mortality was 

determined daily and dead eggs or larvae were removed. 

Testing was done in accordance with methods outlined 

by the Committee on Methods for Toxicity Tests with 

Aquatic Organisms (1975), ASTM Committee E-35 on 

Pesticides (1980), and Horning and Weber (1985). Duplicate groups of 100 organisms were exposed to each test 

concentration. The LC01 and LC50 (concentrations 

producing 1 and 50% mortality) and 95% confidence 

intervals (C.I.) were calculated according to the method 

of Litchfield and Wilcoxon (1949). Data on growth in 

length after 32 days of exposure were analyzed by oneway analysis of variance, and the significance of differences in length among treatment means was determined 

by use of the Student-Newman-Keuls multiple range test.

Results and Discussion

Measured concentrations of rotenone in the exposure 

solutions ranged from 1.01 to 10.04 pig/L. Dissolved 

oxygen remained at 8.1 mg/L or higher and water temperature was held at 12.0°C throughout the tests.

No rainbow trout eggs died as a result of exposure to 

rotenone (Table 1). Hatching was not affected; all eggs 

hatched on the fifth or sixth day of exposure. All larvae 

exposed to concentrations of 4.37 /-ig/L or higher died 

within 5 days after hatching, and 90% of those exposed 

to 2.75 pjg/L died within 15 days. The 32-day LC50 (and 

95% C.I.) was 2.08 p<g/L (1.98-2.18) and the 32-day 

LC01 was 1.00 ng/L (0.894-1.12).

Growth of fry that survived exposure to 2.21 or 

2.75 /^g/L rotenone was significantly less (P < 0.05) than 

that of the controls (Table 2). Studies of mammals 

chronically exposed to rotenone have also shown doserelated responses: In rats, pup weights and weight gains 

were both reduced after exposure to 37.5 and 75.0 mg/L 

diet concentrations of rotenone (however, at these high 

concentrations the reduced food intake may have caused 

the weight differences); rotenone exposure had no effect 

on the reproductive performance of either sex in two 

successive generations (MacKenzie and Kehoe 1983).

Noxfish, a 5% emulsifiable formulation of rotenone, 

was less toxic to eggs than to fmgerlmgs in rainbow trout 

in static exposures (Marking and Bills 1976). The 96-h 

LC50 for newly fertilized eggs ranged from 2.5 mg/L in 

very hard water to 5.6 mg/L in very soft water; commensurate values for fmgerlings were 53.0 /-ig/L in very 

hard water and 54.4 /^g/L in very soft water. Accordingly, 

green eggs of rainbow trout were more resistant than

Table 2. Mean length of surviving rainbow trout after 32 

days of exposure to rotenone at three concentrations.

Exposure 

concentration

(Mg/L)

0 (control)

1.01

2.21

2.75

Length (mm)

Mean

26.7

26.8

21. 7a

23. 6a

SD

1.27

1.65

1.35

0.53

aSignificantly shorter (P < 0.05) than the controls or the fish exposed 

to 1.01
eyed eggs, and both egg stages were far more resistant 

than fmgerlings.

There is little likelihood of chronic exposures of fish 

or fish eggs to rotenone because the approved use patterns and the label restrictions specify application periods 

of less than 12 h in streams and single applications in 

ponds or lakes. Rotenone dissipates rapidly and degrades 

in the natural environment (Schnick 1974); only in cold 

water does it remain toxic for more than 4 days. P. A. 

Gilderhus (personal communication) demonstrated that 

residues persisted for as long as 57 days in water when 

ice was present, but that treated waters were no longer 

toxic to fathead minnows (Pimephales promelas) after 

30 days.

References

American Public Health Association, American Water Works 

Association, and Water Pollution Control Federation. 1985. 

Standard methods for the examination of water and wastewater, 16th ed. American Public Health Association, Washington, D.C.

ASTM Committee E-35 on Pesticides. 1980. Standard practice 

for conducting acute toxicity tests with fishes, macro invertebrates, and amphibians. E729080. Pages 1-25 in Annual book 

of ASTM standards, Part 46. End use and consumer products, 

American Society for Testing and Materials, Philadelphia, Pa.

Committee on Methods for Toxicity Tests with Aquatic Organisms. 1975. Methods for acute toxicity tests with fish, macroinvertebrates, and amphibians. U.S. Environ. Prot. Agency, 

Ecol. Res. Ser. EPA-660/3-75-009. 61 pp.

Dawson, V. K., P. D. Harman, D. P. Schultz, and J. L. Alien. 

1983. Rapid method for measuring rotenone in water at 

piscicidal concentrations. Trans. Am. Fish. Soc. 112:725-727.

Horning, W. B., and C. I. Weber. 1985. Short-term methods 

for estimating the chronic toxicity of effluents and receiving 

waters to freshwater organisms. EPA/600/4-85/014. Environmental Monitoring and Support Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio. 162 pp.

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

Observations on handling and maintenance of bioassay fish. 

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

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

for evaluating dose-effect experiments. J. Pharmacol. Exp. 

Ther. 96:99-113.

MacKenzie, K. M., and D. F. Kehoe. 1983. Reproduction study 

for safety evaluation of rotenone using rats. Contract 14-16- 

009-79-097 by U.S. Fish and Wildlife Service. Hazleton 

Raltech, Inc., Madison, Wis. Vol. 1. 254 pp.

Marking, L. L., and T. D. Bills. 1976. Toxicity of rotenone 

to fish in standardized laboratory tests. U.S. Fish Wildl. Serv., 

Invest. Fish Control 72. 11 pp.

Mount, D. I., and W. A. Brungs. 1967. A simplified dosing 

apparatus for fish toxicology studies. Water Res. 1:21-29.

Schnick, R. A. 1974. A review of the literature on use of 

rotenone in fisheries. U.S. Fish and Wildlife Service, Literature Review 74-15. NTIS (National Technical Information 

Service) PB-235 4547AS, Springfield, Va. 130 pp.

Woltering, D. M. 1984. The growth response in fish chronic 

and early life stage toxicity tests: a critical review. Aquat. 

Toxicol. 5:1-21.

Bills, T. D., J. J. Rach, and L. L. Marking. 1988. Toxicity of rotenone to 

developing rainbow trout. U.S. Fish Wildl. Serv., Invest. Fish Control 93. 

3 pp.

Eyed eggs and resulting larvae of rainbow trout (Salmo gairdneri) were exposed 

for 32 days to technical grade rotenone to determine its effects on survival, 

hatching, and growth. Rotenone concentrations of 1.0 to 10.0 /ug/L did not kill 

the eyed eggs, which hatched normally after 5 or 6 days of exposure. After 

32 days of exposure, and after all larvae had transformed to the swim-up stage, 

the LC50 was 2.08 /Jg/L. Growth was significantly decreased in fish that survived concentrations of 2.21 and 2.75

Key words: Rotenone, embryo, larvae, swim-up, LC50, growth, hatching.

Bills, T. D., J. J. Rach, and L. L. Ma

developing rainbow trout. U.S. Fish 

3 pp.

Eyed eggs and resulting larvae of rainbo

for 32 days to technical grade rotenon

hatching, and growth. Rotenone concen

the eyed eggs, which hatched normally 

32 days of exposure, and after all larvae 

the LC50 was 2.08 /Jg/L. Growth was s

vived concentrations of 2.21 and 2.75

Key words: Rotenone, embryo, larvae

-h

Bills, T. D., ]. J. Rach, and L. L. Marking. 1988. Toxicity of rotenone to developing rainbow trout. 

U.S. Fish Wildl. Serv., Invest. Fish Control 93. 3 pp.

Eyed eggs and resulting larvae of rainbow trout (Salmo gairdneri) were exposed for 32 days to 

technical grade rotenone to determine its effects on survival, hatching, and growth. Rotenone concentrations of 1.0 to 10.0 ng/L did not kill the eyed eggs, which hatched normally after 5 or 6 days of 

exposure. After 32 days of exposure, and after all larvae had transformed to the swim-up stage, the 

LC50 was 2.08 f*g/L. Growth was significantly decreased in fish that survived concentrations of 2.21 and 

2.75 Mg/L.

Key words: Rotenone, embryo, larvae, swim-up, LC50, growth, hatching.

Bills, T. D.,i. i. Rach, and L. L. Marking. 1988. T

U.S. Fish Wildl. Serv., Invest. Fish Control 93

Eyed eggs and resulting larvae of rainbow trout 

technical grade rotenone to determine its effects on 

trations of 1.0 to 10.0 fig/L did not kill the eyed e

exposure. After 32 days of exposure, and after all 

LC50 was 2.08 f*g/L. Growth was significantly decre

2.75 ^g/L.

Key words: Rotenone, embryo, larvae, swim-up, 

Oral Toxicity of Rotenone to Mammals

by

Leif L. Marking

U.S. Fish and Wildlife Service 

National Fisheries Research Center

P.O. Box 818 

La Crosse, Wisconsin 54602

Abstract

Information required by the U.S. Environmental Protection Agency to support the continued use 

of rotenone as a fish toxicant includes data on toxicity to nontarget organisms. Information summarized here was developed in three mammalian safety studies of rotenone: chronic oral toxicity 

in rats, effects on reproduction in rats, and subchronic oral toxicity in dogs. The no-observed-effect 

level (NOEL) of rotenone, determined in rats in a 24-month exposure, was 7.5 mg per kilogram 

of diet. The only difference related to exposure was the lower weight of rats exposed to the higher 

concentrations (37.5 and 75.0 mg/kg) probably because the animals ate less food as a result of 

taste avoidance. Rotenone administered continuously to two successive generations of rats at concentrations of 7.5, 37.5, and 75.0 mg/kg in the diet had no effect on the reproductive performance 

of either sex. The NOEL for toxicity again was 7.5 mg rotenone per kilogram of diet. In beagles 

that received daily doses in gelatin capsules, animals that received the highest daily dose rate of 

10 mg/kg showed the most obvious effects: diarrhea; decreased feed consumption; weight loss during the first 2 months of exposure; mild anemia; and consistent decreases in blood glucose, total 

lipids, and cholesterol. A daily oral dose of 2 mg/kg produced only mild signs of these disorders, 

and the low dose of 0.4 mg/kg was considered the NOEL in dogs. Results of studies reported here 

and in the literature show that even unusually high treatment rates of rotenone do not cause tumors 

or reproductive problems in mammals.

In recent years, there has been increasing concern about regulations of the U.S. Environmental Protection Agency

environmental effects of pesticides, particularly their tox- (EPA).

icity to nontarget organisms. The use of many pesticides Numerous formulations of rotenone have been used as 

has been curtailed or terminated because of suspected fish toxicants (Schnicket al. 1986). The present registrahazards of parent compounds or degradation products, tion carries two specific restrictions: Fish killed by roteAttention has been directed even at compounds that long none cannot be used as food for humans or animals; and 

were "generally regarded as safe." the toxicant can be applied only by, or after consultation

Rotenone has been used as a fish toxicant for more than with, State or Federal agencies. Rotenone is now the com50 years (Schnick 1974) and also has a long history of pound most widely used for the control or eradication of

safe use as an insecticide. However, its continued registra- unwanted fish populations, and fish managers hope that

tion for this use requires the development of information the registration of rotenone for this purpose will be mainon its toxicity to nontarget organisms in accordance with tained. Current EPA regulatory guidelines indicate that

	1
additional information on safety to nontarget aquatic 

organisms and mammals is needed.

Methods

Contract research on rats and dogs for determination 

of mammalian safety is summarized here. The studies 

were done in accordance with the Good Laboratory Practice Regulations as required in the Code of Federal 

Regulations [21 CFR 58.35(b)(6)(7)]. Authorized methods 

and standard procedures were used in each study and a 

Quality Assurance Unit performed inspections and verified the accuracy of the results. The data from each study 

have been submitted to the EPA for inclusion in the 

rotenone file.

Three studies are summarized:

1. Chronic toxiciry study of rotenone in rats. Study 6115- 

100, Volumes 1-4, Hazleton Laboratories America, Inc., 

3301 Kinsman Boulevard, Madison, Wisconsin. (U.S. 

Fish and Wildlife Service contract 14-16-009-81-043.) 

2. Reproduction study for safety evaluation of rotenone 

using rats. Study 81007, Volumes 1-3, Hazleton Raltech, 

Inc., a subsidiary of Hazleton Laboratories America, Inc., 

3301 Kinsman Boulevard, Madison, Wisconsin. (U.S. 

Fish and Wildlife Service contract 14-16-009-79-097.) 

3. Subchronic oral dosing study for safety evaluation of 

rotenone using dogs. Midwest Research Institute Project 

4853-B, Midwest Research Institute, 425 Volker Boulevard, Kansas City, Missouri. (U.S. Fish and Wildlife 

Service contract 14-16-0009-79-115.)

Details of the studies and records of all the original data 

are in bound volumes on file at the National Fisheries 

Research Center, U.S. Fish and Wildlife Service, P.O. 

Box 818, La Crosse, Wisconsin 54602.

Chronic Toxicity of Rotenone 

in Rats

Chronic effects of rotenone in rats were determined by 

submitting the animals to continuous dietary exposure for 

24 months. A total of 320 Fischer 344 rats, 6 weeks old, 

were assigned at random to groups (40 animals of each 

sex per group) and fed 0, 7.5, 37.5, or 75.0 mg of 

rotenone per kilogram of feed. Doses were based on 

the results of a 15-day subchronic study in which doses 

of 600 and 1,200 mg/kg caused death in rats of both 

sexes. The lower dose sequence for the present study 

was designed to establish a no-observed-effect level 

(NOEL).

The exposure that began when the rats were about 

6 weeks old continued for 2 years. Body weights were 

recorded weekly for 12 weeks and every fourth week 

thereafter. Food consumption was recorded weekly 

through 12 weeks and then for weeks 26, 39, 52, 

65, 78, 92, and 104. Urinalyses and hematology and 

blood chemistry tests were done at 3, 6, 12, 18, and 

24 months of exposure. At terminal sacrifice, all animals 

were examined macroscopically, selected organs were 

weighed, and selected tissues were prepared for microscopic study.

The incidence of clinical signs did not differ significantly among groups. Males and females treated with 

37.5 and 75.0 mg/kg had significantly lower mean body 

weights and cumulative dose-related body weight gains 

than did those of their respective control groups (Table 1). 

However, the feed consumption of females treated with 

37.5 and 75.0 mg/kg was lower than that of control 

females, probably because of taste avoidance.

Lower total protein and albumin in high-dose-group 

females and higher serum urea nitrogen in females of the 

middle and high dose group seemed to be treatment 

related. However, no histopathology was noted that could 

be correlated with these changes.

Treatment-related findings were minor or lacking. 

The only macroscopic finding was the lower average 

body weight of animals treated at the high doses. In 

both sexes, terminal body weights and, correspondingly, organ weights and ratios of organ weight to body 

weight, were significantly lower in animals treated with 

37.5 and 75.0 mg/kg of rotenone than in controls or 

animals treated at 7.5 mg/kg. No pathological evidence 

was observed in the hematology, blood chemistry, 

urinalysis, or histology of exposed individuals. The NOEL 

of rotenone for rats in a 24-month exposure was 7.5 mg/kg 

in the diet.

Effects of Rotenone* on 

Reproduction in Rats

The present study was conducted to determine the 

effects of rotenone on reproductive function and fetal 

development in two successive generations of rats that had 

been exposed continuously through their diet. Information was collected from two litters (Fia and F2a) produced 

by two generations of rats (Fo and Fia) through the weaning of F2a litters and terminal necropsy of Fia adults. 

Immature Charles River CD(SD)BR rats (4 weeks old 

upon arrival) were later mated to provide FQ generation 

animals.
Table 1. Summary of weights (g) of rats fed diets containing different concentrations of rotenone.

Dietary rotenone concentration (ppm)

Sex and 

week of 

study

Males

0

20

40

60

80

104

Females

0

20

40

60

80

104

Mean

118.3

348.5

390.4

412.0

401.2

384.2

90.1

209.0

233.8

271.7

292.8

306.5

0

SD

6.40

24.03

26.68

32.04

28.59

26.46

3.61

9.95

12.24

17.35

22.01

31.94

Number

40

40

39

39

36

25

40

40

40

40

39

29

Mean

117.8

343.3

385.5

410.7

399.0

382.8

90.5

210.8

231.4

266.9

284.6

309.1

7.5

SD

5.57

21.30

26.25

28.73

32.46

33.43

3.06

8.42

9.59

19.41

21.21

15.95

Number

40

40

40

37

36

24

40

40

40

40

39

32

Mean

115.03

342.1

380.4

401.6

383. Oa

354. l a

89.1

198. Oa

211. l a

235. 2a

229.03

239. 2a

37.5

SD

5.24

21.07

24.46

27.54

24.59

21.30

3.87

8.63

9.50

11.95

12.48

19.29

Number

40

40

40

39

37

34

40

40

38

37

35

27

Mean

115.9

342.7

380.0

402.0

365. l a

327. 8a

89.8

175. l a

180.93

190.53

185.03

187. 7a

75.0

SD

5.81

16.03

18.81

20.21

24.16

17.71

2.67

8.02

8.14

8.22

7.98

10.58

Number

40

40

40

39

39

31

40

40

39

38

38

35

aGroup mean is significantly different from the mean of the control group at P < 0.05 (Dunnett's test).

The rotenone used in this study, which was 97-98% 

pure, was incorporated into the diet at rates of 0, 7.5, 

37.5, and 75.0 mg/kg. For each treatment group, 15 males 

and 25 females were selected at random and continued 

on the rotenone-treated diet. The FQ generation animals 

received selected concentrations of rotenone in the diet 

continuously from 6 weeks of age through weaning of 

Fi a litters and until they were necropsied during week 33 

on test. The Fi a generation animals selected for the 

reproduction study were exposed to rotenone while in 

utero, through weaning, and in their diet continuously 

through termination of the study during week 32 of the 

test.

A dose-related decrease in the average body weight of 

parental male and female animals began in week 13 and 

continued throughout the study. Body weights of FQ and 

Fla generation male rats exposed to 37.5 and 75.0 mg/kg 

were significantly lower than those of control animals. 

However, reduced organ weights were detected only at 

the highest treatment level and seemed to be related to 

the reduced body weights.

Mean litter size at birth was smaller in the high treatment group than in the controls for the F la and p2a litters 

(Table 2). Treatment-related reductions in pup weights 

and in weight gains were detected throughout lactation. 

There were no other significant differences in litter data, 

and no physical or behavioral abnormalities were apparent in any of the offspring.

It was concluded that rotenone administered to two 

successive generations of rats at concentrations as high 

as 75.0 mg/kg in the diet had no effect on reproductive 

performance of either sex or on fetal development. The 

NOEL for toxicity was determined to be 7.5 mg/kg of 

rotenone in the diet.

Subchronic Toxicity of 

Rotenone to Dogs

The subchronic toxicity of rotenone to dogs was 

determined by administering the compound in gelatin 

capsules daily at one of three treatment rates. Toxicity was ascertained by monitoring for toxic, pharmacologic, and behavioral changes; for feed consumption 

and body weight changes; for hematologic and blood 

chemistry changes; for gross necropsy and histopathologic examinations; and for the survival of treated 

animals. The intent was to define a toxic dose and a safe 

dose.

A total of 60 AKC-registerable beagles (30 of each sex), 

4 to 5 months old, were used in the studies. In a preliminary 28-day study, the dogs were exposed to daily 

doses of 0.08 to 50 mg/kg to establish treatment levels 

to be used for the main study. Six males and six females 

were then given daily doses of 0.0, 0.4, 2.0, or 10 mg/kg 

of encapsulated rotenone for 26 weeks.
Table 2. Numbers and weights of rats in litters at day 21 of exposure to different concentrations of rotenone.

Generation and 

dietary rotenone

concentration (ppm)

Generation Fi a

0

7.5

37.5

75.0

Generation p2a

0

7.5

37.5

75.0

Number of

litters

23

18

22

21

20

18

21

21

Litter size 

(number)

Mean

9.7

9.6

9.1

8.6

9.5

8.5

9.3

8.0

SD

0.56

0.77

1.57

1.56

1.32

2.37

1.63

2.65

Survival

Mean

98.7

98.4

96.9

96.5

100

100

97.1

95.1

SD

3.44

5.01

8.17

7.44

7.17

11.12

Pup weight 

(g)

Mean

47.5

45.9

37.7*

24.4*

45.6

43.2

36.3*

24.6*

SD

4.56

3.53

5.03

4.08

5.97

6.24

6.58

8.37

^Significantly different from control (P < 0.01).

The major effect caused by rotenone was on the 

gastrointestinal tract; diarrhea or soft stools occurred 

throughout the study and were more common in the 

males. The dogs became tolerant of the gastrointestinal effects and survived the entire test period, even 

at the highest dose rate. No treatment-related effects 

were seen in the urinalyses or in the histopathologic 

evaluations.

The high daily dose of 10 mg/kg adversely affected 

gastrointestinal functions: diarrhea, decreased feed consumption, and weight loss were observed during the first 

2 months of exposure. In addition, dogs in this group 

showed a mild anemia and consistent decreases in blood 

glucose, total lipids, and cholesterol.

The daily dose of 2 mg/kg was less toxic and caused 

a milder form of the gastrointestinal effects. No decrease 

in body weight was evident until the later months of 

the study. The low daily dose of 0.4 mg/kg produced 

no observed effects and was considered the NOEL in 

dogs.

Discussion

Most studies on the toxicity of rotenone to mammals 

have been on the rat. Lehman (1952) found an increased 

incidence of nodules and tumors in the livers of rats fed 

rotenone; however, a later similar study on rats by Hansen 

et al. (1965) showed no tumors, and a mouse bioassay 

by Innes et al. (1969) revealed no tumorigenicity.

The toxicity of chemicals to mammals and fish is 

difficult to compare because the routes of exposure are

usually different. Rotenone is purported to be extremely 

toxic to fish and much less toxic to mammals; however, 

Erickson and Gingerich (1986) reported the intravenous 

LD50 to be 305 pig/kg of rotenone in rainbow trout (Sabno 

gairdneri) indicating that intravenous toxicity in fish is 

similar to that in mammals when rotenone is administered 

in defined doses by the same route.

In a study with dogs, Hansen et al. (1965) reported that 

groups of four beagles fed a diet containing fixed concentrations of rotenone for 28 months developed no 

unusual symptoms. The highest daily dose rate was 

0.52 mg/kg of rotenone. In a simultaneous study with rats, 

Hansen et al. (1965) reported that 50 mg/kg had no effect, 

but that doses as high as 1,000 mg/kg caused reduction 

in weight gains. In another study, Khera et al. (1982) 

reported that exposure of rats to daily doses of 5 and 

10 mg/kg of rotenone was associated with reductions in 

maternal body weight gain, fetal weight, and skeletal 

ossification.

Gosalvez and Merchan (1973) reported that 6 weeks 

or more of daily intraperitoneal dosing of rats with 

rotenone caused transplantable mammary tumors to 

develop some months later. This information placed 

suspicion on the safety of rotenone and led to the imposition by EPA of a temporary Rebuttable Presumption 

Against Registration. However, when other scientists 

were unable to reproduce the results of Gosalvez and 

Merchan, the restriction was removed. Results of 

the studies summarized here and in the literature 

show that even high doses of rotenone do not cause 

tumors or reproductive failure, nor adversely affect fetal 

development.
References

Erickson, D. A., and W. H. Gingerich. 1986. Effect of injected 

rotenone on the production and composition of urine from the 

rainbow trout (Salmogairdneri). Aquat. Toxicol. 9:263-274.

Gosalvez, M., and J. Merchan. 1973. Induction of rat mammary tumors with the respiratory inhibitor rotenone. Cancer 

Res. 33:3047-3050.

Hansen, W. H., K. J. Davis, and O. G. Fitzhugh. 1965. 

Chronic toxicity of cube. Toxicol. Appl. Pharmacol. 

7:535-542.

Innes, J. R. M., B. M. Ulland, M. G. Valerio, L. Petrucelli, 

L. Fisbein, E. R. Hart, A. J. Pallotta, R. R. Bates, H. L. 

Falk, J. J. Gart, M. Klein, I. Mitchell, and J. Peters. 1969. 

Bioassay of pesticides and industrial chemicals for tumorigenicity in mice: a preliminary note. J. Natl. Cancer Inst. 

42:1101-1114.

Khera, K. S., C. Whalen, and G. Angers. 1982. Teratogenicity study on pyrethrum and rotenone (natural origin) and 

Ronnel in pregnant rats. J. Toxicol. Environ. Health 

10:111-119.

Lehman, A. J. 1952. Chemicals in food: a report to the association of food and drug officials on current developments. 

Part II. Pesticides. Section V. Pathology. Association Food 

Drug Office, U.S. Q. Bull. 16:126-132.

Schnick, R. A. 1974. A review of the literature on use of 

rotenone in fisheries. U.S. Fish and Wildlife Service, Literature Review 74-15. NTIS (National Technical Information 

Service) PB-235 454/AS, Springfield, Va. 130 pp.

Schnick, R. A., F. P. Meyer, and D. L. Gray. 1986. A guide 

to approved chemicals in fish production and fishery resource 

management. U.S. Fish and Wildlife Service and University 

of Arkansas Cooperative Extension Service, Little Rock. 

MP 241-11M-1-86. 24 pp.

Marking, L. L. 1988. Oral toxicity of rotenone to mammals. U.S. Fish Wildl. 

Serv., Invest. Fish Control 94. 5 pp.

Information summarized was developed from three mammalian safety studies 

of rotenone: chronic oral toxicity in rats, effects on reproduction in rats, and 

subchronic oral toxicity in dogs. The no-observed-effect level (NOEL) of 

rotenone, determined in rats in a 24-month exposure, was 7.5 mg per kilogram 

of diet. Rotenone administered continuously to two successive generations of 

rats at concentrations of 7.5 to 75.0 mg/kg in the diet had no effect on reproductive performance. The NOEL for rotenone in beagles that received daily doses 

in gelatin capsules was 0.4 mg/kg. Even these unusually high treatment rates 

of rotenone did not cause tumors or reproductive problems in mammals.

Key words: Rotenone, oral toxicity, no-observed-effect level, rats, beagles.

Marking, L. L. 1988. Oral toxicity of rot

Serv., Invest. Fish Control 94. 5 pp.

Information summarized was developed 

of rotenone: chronic oral toxicity in rats, 

subchronic oral toxicity in dogs. The n

rotenone, determined in rats in a 24-month 

of diet. Rotenone administered continuou

rats at concentrations of 7.5 to 75.0 mg/kg 

tive performance. The NOEL for rotenone 

in gelatin capsules was 0.4 mg/kg. Even 

of rotenone did not cause tumors or repr

Key words: Rotenone, oral toxicity, no-

Marking, L. L. 1988. Oral toxicity of rotenone to mammals. U.S. Fish Wildl. Serv., Invest. Fish 

Control 94. 5 pp.

Information summarized was developed from three mammalian safety studies of rotenone: chronic 

oral toxicity in rats, effects on reproduction in rats, and subchronic oral toxicity in dogs. The noobserved-effect level (NOEL) of rotenone, determined in rats in a 24-month exposure, was 7.5 mg 

per kilogram of diet. Rotenone administered continuously to two successive generations of rats at concentrations of 7.5 to 75.0 mg/kg in the diet had no effect on reproductive performance. The NOEL 

for rotenone in beagles that received daily doses in gelatin capsules was 0.4 mg/kg. Even these unusually 

high treatment rates of rotenone did not cause tumors or reproductive problems in mammals.

Key words: Rotenone, oral toxicity, no-observed-effect level, rats, beagles.

Marking, L. L. 1988. Oral toxicity of rotenone to m

Control 94. 5 pp.

Information summarized was developed from three 

oral toxicity in rats, effects on reproduction in rats, 

observed-effect level (NOEL) of rotenone, determine

per kilogram of diet. Rotenone administered continuou

centrations of 7.5 to 75.0 mg/kg in the diet had no e

for rotenone in beagles that received daily doses in gelat

high treatment rates of rotenone did not cause tumor

Key words: Rotenone, oral toxicity, no-observed-ef

 & U.S. Government Printing Office: 1988 573-205/80032

(Reports 73 through 76 are in one cover.)

73. Formalin: Its Toxicity to Nontarget Aquatic Organisms, Persistence, and Counteraction, by T. D. Bills, L. L. Marking, 

and J. H. Chandler, Jr. 1977. 7 pp.

74. Chlorine: Its Toxicity to Fish and Detoxification of Antimycin, by L. L. Marking and T. D. Bills. 1977. 5 pp.

75. Malachite Green: Its Toxicity to Aquatic Organisms, Persistence, and Removal with Activated Carbon, by T. D. Bills, 

L. L. Marking, and J. H. Chandler, Jr. 1977. 6 pp.

76. Toxicity of Furanace to Fish, Aquatic Invertebrates, and Frog Eggs and Larvae, by L. L. Marking, T. D. Bills, and J. H. 

Chandler, Jr. 1977. 6 pp.

(Reports 77 through 79 are in one cover.)

77. Efficacy of 3-Trifluoromethyl-4-nitrophenol (TFM), 2',5-Dichloro-4'-nitrosalicylanilide (Bayer 73), and a 98:2 Mixture 

as Lampricides in Laboratory Studies, by V. K. Dawson, K. B. Gumming, and P. A. Gilderhus. 1977. 11 pp.

78. Toxicity of the Molluscicide Bayer 73 and Residue Dynamics of Bayer 2353 in Aquatic Invertebrates, by H. O. Sanders. 

1977. 7 pp.

79. Accumulation, Elimination, and Biotransformation of the Lampricide 2',5-Dichloro-4'-nitrosalicylanilide by Chlronomus 

tentans, by J. A. Kawatski and A. E. Zittel. 1977. 8 pp.

(Reports 80 and 81 are in one cover.)

80. Effects of Antimycin A and Rotenone on Macrobenthos in Ponds, by L. J. Houf and R. S. Campbell. 1977. 29 pp.

81. Aquatic Macroinvertebrates in a Small Wisconsin Trout Stream Before, During, and Two Years After Treatment with the 

Fish Toxicant Antimycin, by G. Z. Jacobi and D. J. Degan. 1977. 24 pp.

82. Investigations in Fish Control: Index to Numbers 1-72, 1964-1976, by R. A. Schnick and K. A. Graves. 1977. 19 pp.

(Reports 83 through 85 are in one cover.)

83. Survival of Two Species of Freshwater Clams, Corbicula leana and Magnonaias boykiniana, After Exposure to Antimycin, 

by L. L. Marking and J. H. Chandler, Jr. 1978. 5 pp.

84. Chronic and Simulated Use-Pattern Exposures of Brook Trout (Salvelinus fontinalis) to 3-Trifluoromethyl-4-nitrophenol 

(TFM), by W. P. Dwyer, F. L. Mayer, J. L. Alien, and D. R. Buckler. 1978. 6 pp.

85. Hydrolysis and Photolysis of the Lampricide 2',5-Dichloro-4'-nitrosalicylanilide (Bayer 73), by D. P. Schultz and P. D. 

Harman. 1978. 5 pp.

86. Registration of Thirty-three Fishery Chemicals: Status of Research and Estimated Costs of Required Contract Studies, by 

R. A. Schnick and F. P. Meyer. 1978. 19 pp.

(Reports 87 through 89 are in one cover.)

87. Ethyl-p-aminobenzoate (Benzocaine): Efficacy as an Anesthetic for Five Species of Freshwater Fish, by V. K. Dawson 

and P. A. Gilderhus. 1979. 5 pp.

88. Influences of Selected Environmental Factors on the Activity of a Prospective Fish Toxicant, 2-(Digeranyl-amino)-ethanol, 

in Laboratory Tests, by C. A. Launer and T. D. Bills. 1979. 4 pp.

89. Toxicities of the Lampricides 3-Trifluoromethyl-4-nitrophenol (TFM) and the 2-Aminoethanol Salt of 2',5-Dichloro-4'- 

nitrosalicylanilide (Bayer 73) to Four Bird Species, by R. H. Hudson. 1979. 5 pp.

(Reports 90 and 91 are in one cover.)

90. Accumulation and Loss of 2',5-Dichloro-4'-nitrosalicylanilide (Bayer 73) by Fish: Laboratory Studies, by V. K. Dawson, 

J. B. Sills, and Charles W. Luhning. 1982. 5 pp.

91. Effects of Synergized Rotenone on Nontarget Organisms in Ponds, by R. M. Burress. 1982. 7 pp.

Use of trade names does not imply U.S. Government endorsement of commercial products.
As the Nation's principal conservation agency, the Department of the Interior 

has responsibility for most of our nationally owned public lands and natural 

resources. This includes fostering the wisest use of our land and water resources, 

protecting our fish and wildlife, preserving the environmental and cultural values 

of our national parks and historical places, and providing for the enjoyment of 

life through outdoor recreation. The Department assesses our energy and mineral 

resources and works to assure that their development is in the best interests of 

all our people. The Department also has a major responsibility for American 

Indian reservation communities and for people who live in island territories under 

U.S. administration.

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