Investigations in fish control

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

UNITED STATES DEPARTMENT OF THE INTERIOR
FISH AND WILDLIFE SERVICE

Investigations in Fish Control, published by the Fish and Wildlife Service, include reports on the results of work
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See inside back cover for list of current issues.

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ISSN 0565-0704 = Investigations in fish control

1. Fishes—Control—Collected works. I. U.S. Fish and
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[DNLM: W2 A B895]]

SH157.7.158 C3997 a, 75-643274
AACR 2 MARC-S

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

UNITED STATES DEPARTMENT OF THE INTERIOR
FISH AND WILDLIFE SERVICE
Washington, D.C. © 1988

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Acute and Chronic Toxicity of Rotenone to Daphnia magna

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 by the 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 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 ug/L in a 21-day chronic toxicity test, the EC50 was 2.1 ug/L. The no-observed-effect concen-

tration was 1.25 pg/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, inverte-
brates, 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 CaCO3, and alkalinity
110-120 mg/L as CaCO3 (ASTM Committee E-35 on
Pesticides 1980). Water samples were analyzed accord-
ing to methods of the American Public Health Associa-
tion 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.

Exposure concentrations of rotenone used were 0.5,
2.5, 5.0, 7.5, and 10.0 g/L in the acute toxicity test and
0.312, 0.625, 1.25, 2.5, and 5.0 pg/L in the chronic tox-
icity test. For both types of tests, rotenone stock solu-
tions 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 col-
lected samples from the stock solutions before algal cells
and food supplement were added. Rotenone was concen-
trated 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 ug/L concentration and
800 mL for the 2.5- and 1.25-ug/L concentrations. Water
samples were filtered, buffered to pH 5.0, and passed
through a disposable Baker C;g 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 spectro-
photometer, and a Micro-Pak (30 cm x4 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 (wave-
length 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 photo-
period 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 con-
sidered 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 daph-
nids. 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 trans-
ferred 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 daph-
nids, were required for each experimental treatment (con-
trol, 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 ECSO values (toxicant concentration
resulting in immobilization or death of 50% of the ex-
posed 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 solu-
tion 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 through-
out the test.)

Dissolved
Date Alkalinity Hardness oxygen
(1986) pH (mg/L) (mg/L) (mg/L)
16 April 8.34 112 166 9.0
23 April 8.18 108 164 a
5 May 8.02 94 168 9.4

4No 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 ECS0 values
and the 95% C.I. were determined by the method of Litch-
field 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 com-
ponents 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 con-
taining 1.25, 2.5, and 5.0 ug/L rotenone were analyzed
(Table 3). Measured rotenone concentrations (mean +
SD) were 1.38+0.078 ug/L for the 1.25-ug/L concen-
tration, 2.63 +0.137 ug/L for the 2.5-ug/L concentration,
and 5.27 wg/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 ug/L (Table 4). The calculated 48-h EC50 value
for rotenone was 3.7 ug/L (95% C.I1., 2.5-5.5 ug/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 ug/L (Table 5). The calculated
EC50 value for rotenone in the chronic 21-day test was
2.1 ug/L (95% C.I., 0.9-4.9 ug/L). Each surviving
daphnid produced 40 or more young in the rotenone con-
centrations of 0.312-1.25 pg/L, but only 19 in the
2.5-ug/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 con-
ducted 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 of rotenone 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
Alkalinity Hardness oxygen

Test pH (mg/L) (mg/L) (mg/L)

concentration

(ug/L) Fresh 3 days Fresh 3 days Fresh 3 days Fresh 3 days
0.0 (control) 8.02 8.16 94 106 168 166 9.4 8.7
0.312 8.29 8.44 94 97 164 164 9.4 8.8
0.625 8.30 8.41 105 100 168 166 2) 8.7
175) 8.31 8.43 104 98 166 164 9.4 8.6
DES 8.26 8.45 111 103 164 166 9.4 8.8

Table 3. Rotenone concentrations (ug/L)? determined by
high pressure liquid chromatography during the 21-day
chronic toxicity test.

Calculated rotenone

concentrations
(ug/L)
Date

Day (1986) 125 DES 5.0°

0 16 April 1k33 2.53 a1)

7 23 April 1.49 2.55 —

14 30 April 1.33 2.61 —

19 5 May - 1.36 2.83 —

4Rotenone was not detectable at calculated concentrations of 0.312 or
0.625. Detection level was 1 pg/L.
Dall organisms died within 5 days.

Table 4. Survival of daphnids (n = 20) exposed to rote-
none in a 48-h acute toxicity test.

Concentration Survival

(ug/L) (%)*
0.0 (water control) 100
0.0 (acetone control) 100
0.5 100
D5) 80
5.0 25
VS 15

10.0 5

4EC50 = 3.7 ug/L; range, 2.5-5.5 ug/L.

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

Survival of

Concentration test organisms

(ug/L) (%)*
0.0 (water control) 90
0.0 (acetone control) 80
0.312 70
0.625 100
1.25 80
DS) 40
5.0 0

4EC50 = 2.1 pg/L; range, 0.9-4.9 pg/L.

Table 6. Mean lengths (mm) of daphnids in chronic ex-
posure groups.

Length (mm)
Concentration SS SSS
Mean SE

(ug/L)
0.0 (water control) 3.82 0.17
0.0 (acetone control) 3.94 0.08
0.312 3.83 0.17
0.625 3.88 0.04
25) 3.56 0.08
DES 3).30 0.28

Number of
young produced by
test organisms

Average number of
young produced per
‘surviving adult

483 53.7
456 57.0
459 65.6
578 57.8
351 43.9

78 19.5

standard toxicity testing (Committee on Methods for Tox-
icity 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 ECS0 value for Daphnia
was 3.7 g/L, whereas Marking and Bills (1976) showed
that, for fish, the LC50 ranged from 21.5 to 389 ug/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 waste-
water, 16th ed. American Public Health Association, Wash-
ington, D.C. 1268 pp.

ASTM Committee E-35 on Pesticides. 1980. Standard practice
for conducting acute toxicity tests with fishes, macroinverte-
brates, 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 Organ-
isms. 1975. Methods for acute toxicity tests with fish, macro-
invertebrates, 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. Allen.
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 macroinverte-
brates 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, Ist ed. W. H.
Freeman and Company, San Francisco, Calif. 776 pp.

U.S. Environmental Protection Agency. 1984. Interim pro-
cedures for conducting the Daphnia magna toxicity assay.
Environmental Research Laboratory, Duluth, Minn., and
Environmental Monitoring Systems Laboratory, Las Vegas,
Nev., February 1984. 28 pp. + appendixes (Unpubl.
manuscr.).

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Toxicity of Rotenone to Developing Rainbow Trout

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 (Salmo gairdnert)
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 ug/L did not kill the eyed eggs, which hatched
normally after 5 or 6 days of exposure. Mortality of young larvae increased as rotenone concentra-
tions increased. After 32 days of exposure, and after all larvae had transformed to the swim-up
stage, the LCS0 was 2.08 ug/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 pg/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 hatch-
ing 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 sensi-
tive 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 con-
ducted with a proportional diluter (Mount and Brungs
1967) that provided about 12 turnovers of test solution
daily. Test organisms were exposed to rotenone continu-
ously for 28 days at the following concentrations (mean
aS D) Ose OME OL095 2 210665) 2e7 a= 02424"
4.37+40.092, 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
CaCO3, and total alkalinity was 105 mg/L as CaCO3.

l

Table 1. Cumulative mortality (%) in duplicated exposures (100 each) of eyed eggs and early larval stages of rain-
bow trout to rotenone in water at 12°C.

Exposure concentration (ug/L)

Day 0.00 1.01 2.21
y> 0.0 0.0 0.0
5¢ 0.0 0.5 0.0
10 3.5 2.0 6.0
15 4.5 2.0 33.0

20 6.0 OS 47.5
25 6.5 3.0 53.0
324 7.0 4.0 58.0

2.75 4.37 5.32 ISD 10.04
0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 6.0

17.0 97.5 99.0 100.0 100.0

91.0 100.0 100.0 100.0 100.0

94.0 100.0 100.0 100.0 100.0

96.5 100.0 100.0 100.0 100.0

96.5 100.0 100.0 100.0 100.0

41 ethal concentrations (ug/L) and 95% confidence at 32 days: LC50, 2.08 (1.98-2.18); and LCO1, 1.00 (0.89-1.12).

Pall eggs were eyed.
“Eggs began hatching at all concentrations.

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

Eyed rainbow trout eggs obtained from Trout Lodge, Inc.,
McMillan, Washington, were handled according to pro-
cedures 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). Dupli-
cate groups of 100 organisms were exposed to each test
concentration. The LCO1 and LCSO (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 one-
way analysis of variance, and the significance of differ-
ences 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 pg/L. Dissolved
oxygen remained at 8.1 mg/L or higher and water tem-
perature 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 yg/L or higher died
within 5 days after hatching, and 90% of those exposed
to 2.75 ug/L died within 15 days. The 32-day LCSO (and
95% C.1.) was 2.08 yg/L (1.98-2.18) and the 32-day
LCOI was 1.00 ug/L (0.894-1.12).

Growth of fry that survived exposure to 2.21 or
2.75 ug/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 dose-
related 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 fingerlings in rainbow trout
in static exposures (Marking and Bills 1976). The 96-h
LCS50 for newly fertilized eggs ranged from 2.5 mg/L in
very hard water to 5.6 mg/L in very soft water; com-
mensurate values for fingerlings were 53.0 g/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

d Length (mm)
concentration
(ug/L) Mean SD
QO (control) 26.7 i 527/
1.01 26.8 1.65
2.21 21.73 1.35
QaI5 23.63 0.53

Significantly shorter (P < 0.05) than the controls or the fish exposed
to 1.01 pg/L.

eyed eggs, and both egg stages were far more resistant
than fingerlings.

There is little likelihood of chronic exposures of fish
or fish eggs to rotenone because the approved use pat-
terns 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 waste-
water, 16th ed. American Public Health Association, Wash-
ington, D.C.

ASTM Committee E-35 on Pesticides. 1980. Standard practice
for conducting acute toxicity tests with fishes, macroinverte-
brates, 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 Organ-
isms. 1975. Methods for acute toxicity tests with fish, macro-

invertebrates, 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. Allen.
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. Environ-
mental Monitoring and Support Laboratory, U.S. Environ-
mental 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, Liter-
ature Review 74-15. NTIS (National Technical Information
Service) PB-235 454/AS, 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.

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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 sum-
marized 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 con-
centrations 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 dur-
ing 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
environmental effects of pesticides, particularly their tox-
icity to nontarget organisms. The use of many pesticides
has been curtailed or terminated because of suspected
hazards of parent compounds or degradation products.
Attention has been directed even at compounds that long
were “‘generally regarded as safe.’’

Rotenone has been used as a fish toxicant for more than
50 years (Schnick 1974) and also has a long history of
safe use as an insecticide. However, its continued registra-
tion for this use requires the development of information
on its toxicity to nontarget organisms in accordance with

regulations of the U.S. Environmental Protection Agency
(EPA).

Numerous formulations of rotenone have been used as
fish toxicants (Schnick et al. 1986). The present registra-
tion carries two specific restrictions: Fish killed by rote-
none cannot be used as food for humans or animals; and
the toxicant can be applied only by, or after consultation
with, State or Federal agencies. Rotenone is now the com-
pound most widely used for the control or eradication of
unwanted fish populations, and fish managers hope that
the registration of rotenone for this purpose will be main-
tained. Current EPA regulatory guidelines indicate that

l

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 Prac-
tice 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 veri-
fied 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 toxicity 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 Boule-
vard, 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 micro-
scopic study.

The incidence of clinical signs did not differ signif-
icantly 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, correspond-
ingly, 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. Informa-
tion was collected from two litters (Fj, and F7,) produced
by two generations of rats (Fo and F;,) through the wean-
ing of F), litters and terminal necropsy of Fj, adults.
Immature Charles River CD(SD)BR rats (4 weeks old
upon arrival) were later mated to provide Fo generation
animals.

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

Dietary rotenone concentration (ppm)

Sex and 0 eS 37.5 75.0

WeCiOlt = |),

study Mean SD Number Mean SD Number Mean SD Number Mean SD Number
Males

0 118.3 6.40 40 IN — Dedd7/ 40 HIS029 95524 40 S59 5.81 40
20 348.5 24.03 40 343.3 21.30 40 342.1 21.07 40 342.7 16.03 40
40 390.4 26.68 39 385.5 26.25 40 380.4 24.46 40 380.0 18.81 40
60 412.0 32.04 39 410.7 28.73 37/ 401.6 27.54 39 402.0 20.21 39
80 401.2 28.59 36 399.0 32.46 36 383.07 24.59 37 365.17 24.16 39
104 384.2 26.46 25 382.8 33.43 24 354.1% 21.30 34 627/282 any enn Bil
Females

0 90.1 3.61 40 90.5 3.06 40 89.1 3.87 40 89.8 2.67 40
20 209.0 9.95 40 210.8 8.42 40 198.07 8.63 40 Sele 8.02 40
40 233.8 12.24 40 231.4 9.59 40 211.1% 9.50 38 180.9? 8.14 39
60 ZAM Neds) 40 266.9 19.41 40 235.27 11.95 37) 190.5? 8.22 38
80 292.8 22.01 39 284.6 21.21 39 229.07 12.48 35 185.0? 7.98 38
104 306.5 31.94 29 309M 15295 32 239). NOY 27 187.77 —- 10.58 35

4Group 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 Fo generation animals
received selected concentrations of rotenone in the diet
continuously from 6 weeks of age through weaning of
Fj, litters and until they were necropsied during week 33
on test. The Fj, 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 Fo and
Fj, 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 treat-
ment group than in the controls for the F,, and F, 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 appar-
ent 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. Tox-
icity was ascertained by monitoring for toxic, pharm-
acologic, and behavioral changes; for feed consumption
and body weight changes; for hematologic and blood
chemistry changes; for gross necropsy and histopatho-
logic 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 pre-
liminary 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.

Litter size Pup weight
Generation and (number) Survival (g)

dietary rotenone Number of Sassen SS SSS SS

concentration (ppm) litters Mean SD Mean SD Mean SD
Generation F),

0 23 S71) 0.56 98.7 3.44 47.5 4.56

eS) 18 9.6 0.77 98.4 5.01 45.9 3.53

7/0) 22 9.1 1.57 96.9 8.17 31 5.03

75.0 21 8.6 1.56 96.5 7.44 24.4* 4.08
Generation F>,

0 20 95 1.32 100 — 45.6 5.97

7.5 18 8.5 2.37 100 — 43.2 6.24

37.5 21 9.3 1.63 97.1 7.17 36.3* 6.58

75.0 21 8.0 2.65 95.1 11.12 24.6* 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 gastrointes-
tinal 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 con-
sumption, 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
LDS0 to be 305 pg/kg of rotenone in rainbow trout (Salmo
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 con-
centrations 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 imposi-
tion 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 (Salmo gairdneri). Aquat. Toxizol. 9:263-274.

Gosalvez, M., and J. Merchan. 1973. Induction of rat mam-
mary tumors with the respiratory inhivitor 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 tumor-
igenicity in mice: a preliminary note. J. Natl. Cancer Inst.
42:1101-1114.

Khera, K. S., C. Whalen, and G. Angers. 1982. Teratogen-
icity 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 asso-
ciation 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, Liter-
ature 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.

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(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. Cumming, 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 Chironomus
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-Trifluoromethy]-4-nitrophenol
(TFM), by W. P. Dwyer, F. L. Mayer, J. L. Allen, 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.

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