EPA-600/3-77-069
June 1977
Ecological Research Series
TOXAPHENE: CHRONIC TOXICITY
TO FATHEAD MINNOWS AND
CHANNEL CATFISH
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
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ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
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This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-77-069
June 1977
TOXAPHENE: CHRONIC TOXICITY TO
FATHEAD MINNOWS AND CHANNEL CATFISH
by
Foster L. Mayer, Jr.
Paul M. Mehrle, Jr.
William P. Dwyer
Fish-Pesticide Research Laboratory
Fish & Wildlife Service
United States Department of the Interior
Columbia, Missouri 65201
Contract No. EPA-IAG-141(D)
Project Officer
Leonard H. Mueller
Environmental Research Laboratory
Duluth, Minnesota 55804
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory,
Duluth, Minnesota, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily reflect
the views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
ii
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FOREWORD
Our nation's freshwaters are vital for all animals and plants, yet our
diverse uses of water- for recreation, food, energy, transportation, and
industry physically and chemically alter lakes, rivers, and streams. Such
alterations threaten terrestrial organisms, as well as those living in water.
The Environmental Research Laboratory in Duluth, Minnesota develops methods,
conducts laboratory and field studies, and extrapolates research findings
—to determine how physical and chemical pollution affects aquatic life
—to assess the effects of ecosystems on pollutants
—to predict effects of pollutants on large lakes through the use of
models
—to measure bioaccumulation of pollutants in aquatic organisms that are
consumed by other animals, including man
This report studies the effects of the insecticide toxaphene on two
species of fish, fathead minnows and channel catfish, when continuously
exposed to five different toxaphene concentrations for periods of 8 to 10
months.
Donald I. Mount, Ph.D.
Director
Environmental Research Laboratory
Duluth, Minnesota
ill
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ABSTRACT
Fathead minnows (Plmephales promelas) and channel catfish (Ictalurus
punctatus) were continuously exposed to several toxaphene concentrations
(13-630 ng/1) in flow-through diluter systems for 8 to 10 months. Growth and
backbone quality of adult fathead minnows were decreased at 97 and 173 ng/1
exposures,but adult channel catfish were not affected by toxaphene. Effects
on reproduction were observed only in channel catfish in the 630 ng/1 con-
centration: the period from pairing to spawning was increased and the amount
of gelatinous matrix surrounding the eggs was reduced. Survival of fathead
minnows was not affected by toxaphene, but the no-effect concentration for fry
growth and bone quality was below 54 and 97 ng/1, respectively. Channel cat-
fish fry survival and growth were reduced in the 299 and 630 ng/1 exposures,
and bone quality was altered in concentrations as low as 72 ng/1. The
maximum toxaphene accumulation from water to fish was 69,000 times in fathead
minnows and 50,000 times in channel catfish. Toxaphene was excreted very
slowly in both species.
This report was submitted in partial fulfillment of Contract No. EPA-IAG-
141(D) by the Fish-Pesticide Research Laboratory, Fish and Wildlife Service
(USDI) under sponsorship of the U.S. Environmental Protection Agency. Work
was completed in May 1976.
iv
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CONTENTS
Foreword .............................................
Abs tract ............................................. iv
Figures .............................................. vi
Tables ............................................... vli
Acknowledgment ....................................... ^x
1. Introduction 1
2. Conclusions 4
3. Recommendations 5
4. Materials and Methods 6
Growth, reproduction, and mortality 6
Biochemistry 9
Residue dynamics 10
5. Results and Discussion 12
Growth, reproduction, and mortality 12
Biochemistry 19
Residue dynamics 25
General 33
References 3-*
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FIGURES
Number Page
1. Geographic distribution of fish containing detectable
toxaphene residues in the 1973 and 1974 National
Pesticide Monitoring Program 2
2. Effect of toxaphene on the proteinaceous matrix surrounding the
eggs of channel catfish 16
3. Cumulative mortality in channel catfish fry after 30, 60, and
90 days of exposure to toxaphene 18
4. X-rays and schematics of backbones of 90-day-old channel
catfish 24
vi
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TABLES
Number Page
1. Photoperiod and Temperature Regime for Partial Chronic Toxlcity
Test of Channel Catfish ...................................... 8
2. Effect of Toxaphene on Growth of Fathead Minnows as Indicated
by Weight and Length ......................................... 13
3. Spawning Activity and Mortality of Adult Female Fathead Minnows
Exposed to Toxaphene ......................................... 14
4. Spawning Acitivty and Mortality of Adult Female Channel Catfish
Exposed to Toxaphene ......................................... 15
5. Effect of Toxaphene on Growth of Channel Catfish Fry as Indi-
cated by Weight and Length .................................. 17
6. Acute Toxicity of Toxaphene to 30-day-old Fathead Minnows and
2 . 5-year-old Channel Catfish ................................ 20
7. Constituents of Backbone in Fathead Minnows Exposed to
21
Toxaphene [[[
8. Hydroxyproline in Channel Catfish Eggs and Fry as Affected by
Toxaphene [[[
9. Constituents of Backbone in 90-day-old Channel Catfish Fry
Exposed to Toxaphene ......................................... 23
10. Vitamin C Concentration in Eggs and Fry of Channel Catfish
Exposed to Toxaphene ........................................ 2"
11. Whole-body Residues of Toxaphene in Fathead Minnow Adults and
Their Eggs and Fry During Exposure to Toxaphene ............ 27
12. Extractable Lipids in Fathead Minnow Adults and Their Eggs
and Fry during Exposure to Toxaphene ...................... 28
13. Whole-body Residues of Toxaphene on a Lipid Basis in Fat-
head Minnow Adults and Their Eggs and Fry During Ex-
90
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TABLES (CONTINUED)
Number
14. Whole-body Residues of Toxaphene in Channel Catfish Adults
and Their Eggs and Fry During Exposure to Toxaphene 30
15. Extractable Lipids in Channel Catfish Adults and Their Eggs
and Fry During Exposure to Toxaphene 31
16. Whole-body Residues of Toxaphene on a Lipid Basis in Channel
Catfish Adults and their Eggs and Fry During Exposure
to Toxaphene 32
17. Concentrations of Toxaphene Producing Acute and Chronic
Toxicity to Aquatic Organisms 34
viii
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ACKNOWLEDGMENTS
Technical assistance and maintenance were provided by D. R. Buckler,
W. A. McAllister, and D. C. Zumwalt. Collagen, hydroxyproline, calcium, and
phosphorus assays were conducted by Sandra Cummins, Linda Fryer and S. J.
Hamilton and toxaphene residue analyses by J. L. Johnson. The backbone
illustrations were drawn by Becky Turk. Mayo Martin and Harry Dupree gave
assistance and offered suggestions in the channel catfish study and Leonard
Mueller assisted us throughout the project. Portions of the study were
funded by the Fish and Wildlife Service, United States Department of the
Interior.
ix
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SECTION 1
INTRODUCTION
Toxaphene, an organochlorine insecticide, has been extensively used in
the United States for over 20 years to control agronomic insect pests—
particularly in the Lower Mississippi Valley, one of the most intensively
farmed areas in the United States, where it is used for control of cotton in-
sect pests. A pesticide use survey of the Lower Mississippi Valley and Gulf
Coastal Prairie showed that toxaphene was being applied 8 to 12 times during
July through September at a rate of 2.2 to 3.4 kg/ha per application.
The distribution of toxaphene in fish, as found by the 1973 and 1974
National Pesticide Monitoring Program2, indicated that as an aquatic con-
taminant, the insecticide is mainly limited to the Southeastern United States
(Fig. 1). However, isolated occurrences of toxaphene residues were found in
rainbow trout (Salmo gairdneri) from the Kenai River, Alaska, in 1973 and in
largemouth bass (Micropterus salmoides) from the Colorado River, Arizona, in
1974. Residues in fish ranged from non-detectable (<0.05 ug/g) to 51 ug/g
overall, and of the species containing toxaphene in the important cyprinid
(minnow) and ictalurid (catfish) families, the concentrations ranged from 0.5
to 38 and 0.5 to 51 ug/g, respectively. Mayer et al.J found that whole body
residues of toxaphene of 0.6 ug/g in fry of brook trout (Salvelinus fontinalis)
were relatable to detrimental effects.
The acute effects of toxaphene on aquatic organisms and its persistence
in the aquatic environment have been summarizedJ, but data describing the
chronic effects in fish species other than brook trout, especially those im-
portant in the Southeastern United States, are absent. Fathead minnows
(Pimephales promelas) and channel catfish (Ictalurus punctatus) are indig-
enous to freshwater systems east of the Rocky Mountains and are of high
economic significance in the areas most contaminated with toxaphene. The rat-
head minnow is an important forage and commercial baitfish, and poisoning of
minnows by toxaphene and endrin in Arkansas and Louisiana has been reported .
The widespread use of channel catfish by sportsmen, commercial fishermen, and
fish farmers makes this species an important natural resource. Because ot
the continued heavy use of toxaphene in the Southeastern United States, its
presence in indigenous fish species, and the importance of fathead minnows
and channel catfish, we undertook this study to determine the effects o£
toxaphene on these two species.
The purpose of the research was to develop water quality criteria for
toxaphene which will assist the Fish and Wildlife Service and the Environmen-
tal Protection Agency to better manage and protect fishery resources.
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Geographic Distribution of Toxaphene Residues in Fish
from the National Pesticide Monitoring Program
XICO
Figure 1. Geographic distribution of fish containing detectable toxaphene residues
in the 1973 and 1974 National Pesticide Monitoring Program.
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Specific objectives of the study were: (1) to develop laboratory techniques
for testing channel catfish through their reproductive cycle in chronic
toxicity tests; (2) to determine the effect of continuous exposures of
toxaphene on the growth, reproduction, and mortality in fathead minnows and
channel catfish; (3) to further evaluate the potential use of collagen and
hydroxyproline as indicators or predictors of abnormal growth and development
in fish; and (4) to determine the degree of accumulation and elimination of
toxaphene in fathead minnows and channel catfish.
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SECTION 2
CONCLUSIONS
On the basis of growth, reproduction, mortality, and bone development,
the maximum acceptable concentration of toxaphene in water was between 25 and
54 ng/1 for fathead minnows and between 49 and 72 ng/1 for channel catfish.
Toxaphene was accumulated 10,000 to 69,000 times water concentrations
by various life stages of fathead minnows and 17,000 to 50,000 times by
channel catfish. Excretion of toxaphene was very slow in fathead minnows and
channel catfish, requiring up to 56 days for 36% elimination.
Collagen and hydroxyproline are sensitive indicators of growth and
development in fathead minnows and channel catfish.
Except for their large space requirements, channel catfish are highly
desirable test organisms for use in chronic toxicity tests.
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SECTION 3
RECOMMENDATIONS
For the protection of fathead minnows and channel catfish, toxaphene
concentrations in water should be less than 54 and 72 ng/1, respectively.
Laboratory tests show that collagen and hydroxyproline are good bio-
chemical indicators or predictors of growth and development in fishes and
should be evaluated for applicability to field monitoring of biological ef-
fects.
Channel catfish should be included among species recommended for chronic
toxicity tests, especially where there is a possibility of contamination of
water by a pesticide or other pollutant in the Southeastern United States.
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SECTION 4
MATERIALS AND METHODS
GROWTH, REPRODUCTION, AND MORTALITY
General
Seven-day-old fathead minnows obtained from the National Water Quality
Laboratory, Duluth, Minnesota, were held in proportional diluter systems at
25 C until the tests were initiated at 40 days of age. The fish were fed a
commercial trout starter (EWOS) ad libitum, which was supplemented daily with
live brine shrimp nauplii for the first 30 days. Adult channel catfish were
purchased from Schroeder's Fish Farm, Carlisle, Arkansas. Total body residues
of toxaphene averaged 1.2 ug/g, which was the lowest residue found in analysis
of fish from several sources. The catfish were held in raceways at 16 C and
fed a maintenance diet until the tests were started^. The adult fish were
fed a floating commercial catfish food (Purina); fry were fed the Modified
Oregon Test Diet° ad libitum. Eggs collected from both species during the
test were treated for 3 minutes with 60 mg/1 of malachite green for the first
3 days. Well water (characteristics previously described-') was passed
through an ultraviolet sterilizer system before it entered the diluter systems.
7 8
Diluter systems with the modification df McAllister et al. and a
dilution factor of 0.5 between the concentrations were used to deliver five
concentrations of toxaphene and a control for the chronic tests. We attempted
to select a concentration range that included a no-effect concentration,
whikh was estimated by multiplying the lethal threshold concentration
(determined when the rate of death was 10% or less of the original number of
fish in any concentration during the preceding 24 hr period') by 0.01, as
recommended in establishing water quality criteria1 . An experimental-use
sample of toxaphene (X-16189-49), furnished by Hercules Inc., was used through-
out the study. Flow-splitting chambers designed by Benoit and Puglisi
were used to thoroughly mix and divide each toxaphene concentration for
delivery to the replicated exposure tanks, and toxaphene concentrations in the
exposure water were measured every two weeks. Acetone was used as the carrier
solvent for toxaphene. The acetone concentrations in the control and highest
toxaphene concentration tested were 0.23 ml/1 in the fathead minnow study and
0.11 ml/1 in the channel catfish study.
Water temperature in the tanks was controlled by heat exchangers and
mixing valves before the water entered the systems. Artificial daylight was
provided by the method of Drummond and Dawson . The water temperature
regime and photoperiod were those recommended by EPA for fathead minnow
-------
. The temperature regime for channel catfish was developed on the
basis of data on natural water temperatures for channel catfish spawning
(Table 1), and the photoperiod was the United States average (Evansville,
Indiana), as recommended by EPA-^> .
We also conducted acute toxicity tests of toxaphene on 30-day-old fathead
minnows and 2.5-year-old channel catfish-^. The lethal threshold concentration
was determined by following the recommendations of Eaton^. A proportional
diluter system delivering seven concentrations and a control and with a
dilution factor of 0.75, was used for the fathead minnows, as described by
Johnson and Julin^-^. The test was conducted at 25 C, 20 fish were exposed to
each concentration, and mortalities were recorded daily. The channel catfish
test was run in a proportional diluter system having five concentrations
(dilution factor of 0.75 between concentrations) and a control, with five
fish per duplicate tank, at 20 C. The exposure tanks and system were the
same as those for brook trout^.
The design of the chronic toxicity studies was a randomized block de-
sign-^. Growth and biochemical data of both the adults and fry were an-
alyzed by analysis of variance, and the effects of toxaphene on mortality and
egg hatchability were determined by conducting an analysis of variance on the
arcsin transformation for proportions^ (angle = arcsin ^percentage) . A
multiple means comparison test (least significant difference) was used to
compare treatments. The LC50's for the acute toxicity test were calculated
by the method of Litchfield and Wilcoxon-'- .
Fathead Minnows
This portion of the study was conducted according to the recommended pro-
cedure for chronic tests of fathead minnows-^. Measured toxaphene concentra-
tions (standard error) were 0, 13 (2), 25 (2), 54 (4), 97 (8), and 173 (12)
ng/1. Water temperature was maintained at 25 (+0.5) C throughout the study.
The 12 glass tanks used for exposing the fish measured 30 x 91 cm, were 30
cm deep, and had a water depth of 22 cm. Each tank was divided into three
compartments. One compartment was 30 cm wide x 61 cm long and the other two
were each 15 cm wide x 30 cm long.
Forty 40-day-old fry (0.32 g, 30 mm) were randomly selected and distrib-
uted to each of the large compartments in each tank for biochemical and
residue determinations, and 10 fry were distributed to each of the smaller
compartments for growth measurements. These fish were weighed and measured
(total length) at 0, 30, 98, and 295 days of exposure. After 98 days of ex-
posure, 4 males and 10 females were randomly selected and placed in the large
compartment of each tank with five spawning tiles. All other fish were re-
moved. Fifty eggs from each spawn were used for hatchability tests, and 20
of the resulting fry were placed in a small compartment and weighed and
measured after 30 days to determine growth effects.
Channel Catfish
The toxaphene exposure was initiated 12 February 1974 and the measured
concentrations (standard error) were 0, 49 (6), 72 (9), 129 (17), 299 (22),
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TABLE 1. PHOTOPERIOD AND TEMPERATURE REGIME FOR PARTIAL CHRONIC
TOXICITY TEST OF CHANNEL CATFISH
Month
and date
February
1
15
March
1
15
April
1
15
May
1
15
June
1
15
July
1
15
August
1
15
September
1
15
Daylight
(time)
0600-1715
0600-1745
0600-1815
0600-1900
0600-1930
0600-2015
0600-2045
0600-2115
0600-2130
0600-2145
0600-2145
0600-2130
0600-2100
0600-2030
0600-2000
0600-1930
Temperature
(°0
17
17
19
22
26
26
26
26
26
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and 630 (58) ng/1. Twelve fish (1,233 g, 461 mm) were placed Into each of the
12 stainless steel test tanks (91 x 183 cm and 61 cm deep); 4 males and 4
females were used for evaluation of growth and spawning and 4 fish for
analysis of residues. The water depth of the tanks was 46 cm, giving a
volume of 765 liters. Well water was delivered to each tank at a rate of 1.6
1/min. The fish used for growth and spawning were weighed and measured
(fork length) at 0, 50, and 100 days of exposure. The males were also
weighed after day 75, and two of the four were removed for the toxaphene elim-
ination study. After 50 days of exposure, the tanks were divided into two
sections (91 x 91 cm and 46 cm deep) and the males, because of their increas-
ing pugnaciousness, were separated from the females. After the final growth
determination (100 days), dividers were inserted into one-half of the tank,
dividing it into two sections for spawning, each 46 x 91 cm and 46 cm deep.
One male and one female were placed into each of these sections. Sexual
maturity was determined by the physical appearance of the genitalia, flac-
cidness of the abdomen of females, and darkening and swelling of the heads of
males^O, 21t Females judged ready to spawn were paired with a slightly larger
ripe male. An 18.9-liter stainless steel milk can was then placed into each
section with the paired fish, and half of each section was covered with a
sheet of black plastic to reduce excitability. Cans were checked daily for
eggs.
Females that had not spawned by the end of June were injected 3 to 4
times on alternate days with human chorionic gonadotropin at 300 IU/454 g21.
Of the 15 fish injected, only 2 spawned, and the data were discarded be-
cause the hatchability of the eggs was very low. Egg masses were removed
from the milk cans daily and numbers were determined volumetrically. Three
100-egg samples from each spawn were counted and placed into plastic con-
tainers (11 cm square and 16 cm deep; water depth, 11 cm) for hatchability
determinations. The bottom was removed from each container and a rectangular
slit (3.8 x 6.4 cm) was cut in one side just below the water surface. Stain-
less steel screen (7.9 meshes/cm) was used to cover the bottom and slit. An
air stone was attached to the bottom screen and air was used to roll the eggs
and circulate water (215 ml/min, or one exchange per 6.5 min) through the
container. The fry hatched in 6 to 7 days, were counted, and returned to the
hatching containers where they were kept for 5 to 6 days until swim-up.
Fifty fry from each spawn were then weighed and placed in glass growth cham-
bers (15 x 38 cm and 14 cm deep; water depth, 10 cm) with a flow rate of
200 ml/min. The remaining fry were placed in one section of each large tank
for biochemical and residue determinations. The number of fry in each growth
chamber was reduced to 20 at 30 days and 10 at 60 days when growth deter-
minations (weight and fork length) were made. Final measurements were taken
after 90 days of exposure. Mortalities were recorded daily.
BIOCHEMISTRY
Fathead Minnows
Backbones (vertebrae) were dissected from 10 adults from each concen-
tration after 98 and 295 days of exposure and from 5 fry after 30 days of
exposure. Collagen, calcium, and phosphorus were determined for the backbone
of each adult and hydroxyproline was determined on each isolated collagen
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fraction, as described by Mayer et al.3 and Mehrle and Mayer22 Only the
amount of hydroxyproline in the dried backbone was analyzed in'fry. The
backbone was dried at 110 C for 2 hr in a forced-air oven, split into two
fractions, and weighed. Collagen was isolated from one fraction by the method
of Flanagan and Nichols^. The isolated collagen was weighed and subjected
to hydrolysis at 115 C in 5 ml of 6 N HC1 for 16 hr. Hydroxyproline was
determined in a 2-ral sample after Woessner2^. The other bone fraction was
subjected to hydrolysis at 115 C in 3 ml of 6 N HC1 for 16 hr. Calcium was
determined by atomic absorption spectrophotometry, and phosphorus was deter-
mined on the hydrolysate by the Fiske and Subbarow method . The precision of
each method varied less than 3%, and the recovery from spiked samples was
95-99%.
Channel Catfish
Backbone and blood samples were taken from female channel catfish within
24 hr after spawning. Collagen, hydroxyproline, calcium, and phosphorus
of the backbones were analyzed as described for fathead minnows. Blood was
sampled from the caudal artery and allowed to clot at room temperature; the
serum was then decanted and frozen. Calcium and phosphorus were determined
in the serum, as well as total protein and vitamin C2^. Six samples of
three eggs or fry each were sampled from each concentration, and vitamin C
analysis was performed on the eggs and on fry 15, 30, and 60 days old. Total
protein^S and hydroxyproline were determined in the eggs and in fry 15 days
after hatch. Backbones were dissected from 10 90-day-old fry from each con-
centration, and collagen, calcium, and phosphorus determined in each bone
sample and hydroxyproline was analyzed in each of the isolated collagen
fractions. In addition, eight 90-day-old fry from each concentration were
x-rayed for backbone anomalies.
RESIDUE DYNAMICS
General
Methodology for determination of toxaphene residues in water and fish
was described by Stalling and Huckins . The fish were ground and the
tissues extracted by the procedures of Benville and Tindle™ and Hesselberg
and Johnson . Initial sample cleanup was by automated gel permeation
chroma tography-^, followed by modified silicic acid chroma to graphy-*-*.
Toxaphene residues were quantified by gas liquid chromatography with Ni-
electron capture detection. A 2.1-m long x 2-mm i.d. coiled glass column
packed with 3% (w/w) OV-7 on chromosorb W-HP was used; nitrogen flow rate
was 40 ml/min and the column temperature was 200 C. The percentage of ex-
tractable lipids was determined in each residue sample^ . The minimum
detection limit of toxaphene was 10 ng/1 in water and 0.05 ug/g in fish
tissue, but values below 0.1 ug/g in tissue were difficult to quantitate. Re-
covery of toxaphene from spiked tissue samples was 97-100%, and from water
spiked at 25, 50, and 10a ng/1 was 44-57%, 76-85%, and 97-104%, respectively.
All residue values were adjusted for recovery.
10
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Fathead Minnows
A composite sample of 10 fish was collected from each concentration at
30, 98, and 295 days of exposure. The fish remaining after sampling at day
295 were placed in fresh flowing water and a combined sample of 4 fish
originating from each concentration was analyzed at 14, 28, and 56 days to
determine the elimination rate of toxaphene. All excess eggs from each spawn
within a concentration were pooled for analysis and 2 composite samples (40
to 60 fry each) of the resulting fry were collected from each concentration
at the end of the 30-day growth period.
Channel Catfish
Two adults were sampled from each concentration after 30, 50, 75, and
100 days of exposure. On day 75, the excess males were placed in fresh
flowing water for 33 days; 2 fish from each previous exposure were then
analyzed for toxaphene elimination. After spawning (137 days), 2 males were
removed from both the 72-and 299-ng/l exposures and toxaphene residues were
determined in both the fillets and the remaining carcass(offal). Samples of
eggs from each spawn were analyzed individually and two composite samples of
fry from each concentration were analyzed after 15, 30, 60, and 90 days of
exposure.
11
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SECTION 5
RESULTS AND DISCUSSION
GROWTH, REPRODUCTION, AND MORTALITY
Parent fathead minnows held in toxaphene concentrations of 97 and 173 ng/1
were significantly smaller than the controls (P<0.05) after 30 and 98 days
(before spawning) but not at 295 days, toward the end of the spawning period
(Table 2). No effects were observed on the spawns per female, eggs per
female, eggs per spawn, female survival, or percent hatch (Table 3). Fry
survival was significantly decreased only in the 97 ng/1 toxaphene concen-
tration; concentrations (ng/1) and mortalities (percent) were as follows: 0,
14; 13, 12; 25, 24; 54, 18; 97, 41; and 173, 21. Growth was significantly
reduced (P<0.05) in fry from parents that had been exposed to 54, 97, and 173
ng/1 (Table 2).
Growth of adult channel catfish was not affected by toxaphene; however,
the fish had an average weight loss of 5.7% between the initiation of the
study and the start of spawning. The fish fed poorly during the first part
of the experiment, probably because the water temperature was low; as the
temperature increased, the fish came into spawning condition and did not feed.
The only aspect of spawning activity affected by toxaphene was the number of
days between the pairing of adults and the start of spawning, which was
significantly increased (P<0.05) in the highest concentration (Table 4). Egg
hatch tended to decrease in the 129 ng/1 and higher concentrations, but the
decrease was not statistically significant. However, the proteinaceous
matrix surrounding the eggs was greatly reduced in the highest toxaphene
exposure (Fig. 2). The concentration of protein in matrix of the spawns of
control fish and fish exposed to 630 ng/1 did not differ—only the total
amount of the matrix was reduced in the test fish.
The weight of channel catfish fry was significantly reduced (P<0.05) in
the 299 and 630 ng/1 toxaphene concentrations after 30 days of exposure and
the same effect continued through 90 days (Table 5). Fry length was
significantly reduced (P<0.05) in the 49, 129, 299, and 630 ng/1 toxaphene
concentrations after 30 days, but only in the 299 and 630 ng/1 concentrations
after exposure for 60 and 90 days. Cumulative mortality continued to in-
crease throughout the 90-day exposure (Fig. 3), even though statistical
significance was limited to the two highest concentrations. It appeared that
the continuing mortality may have negated further decreases in growth by re-
moving the less resistant fish. This hypothesis is supported by the effects
of toxaphene on vitamin C (Table 10), which is essential in the detoxication
of organic compounds in the liver by microsomal hydroxylative enzymes35' 36>
12
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TABLE 2. EFFECT OF TOXAPHENE ON GROWTH OF FATHEAD MINNOWS AS INDICATED BY WEIGHT AND LENGTH
Toxaphene
concentration
(ng/1)
0
13
25
54
97
173
30
Weight (g)
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
51a
13)
52
14)
47
13)
49
15)
44*
12)
40*
11)
Length (mm)
36
(3)
36
(3)
35
(3)
35
(4)
35
(3)
34*
(3)
Days of
Weight (g)
1.02
(0.28)
1.12
(0.35)
0.95
(0.27)
1.01
(0.38)
0.86*
(0.23)
0.79*
(0.19)
exposure
98
Length (mm)
44
(4)
45
(5)
42
(5)
43
(5)
41*
(4)
40*
(3)
295
Weigh t(g)
2.
(1.
2.
(1.
2.
(0.
2.
(0.
2.
(0.
2.
(0.
62
28)
35
20)
21
92)
51
95)
30
92)
08
67)
Length (mm)
58
(5)
56
(5)
55
(5)
58
(4)
57
(6)
55
(5)
Size of fry of
2nd generation
30 days
the
at
Weight (g) Length (mm)
0
(0
0
(0
0
(0
0
(0
0
(0
0
(0
.172
.040)
.165
.035)
.174
.040)
.161*
.042)
.152*
.046)
.155*
.044)
25.3
(1.8)
24.9
(1.9)
25.1
(2.1)
24.7*
(2.5)
24.1*
(2.1)
24.4*
(2.3)
o
Mean (standard deviation).
*Significantly different from controls (P<0.05).
-------
TABLE 3. SPAWNING ACTIVITY AND MORTALITY OF ADULT FEMALE FATHEAD MINNOWS EXPOSED TO TOXAPHENE
Toxaphene
concentration
(ng/1)
0
13
25
54
97
173
Spawns
per
female
(n)
2.7
1.6
2.3
4.9
3.3
3.2
Eggs
per
female
(n)
256
125
165
604
301
258
Eggs
per
spawn
(n)
94
79
71
123
90
82
Female
mortality
(n)
1
3
1
5
2
1
Egg
hatch
(%)
79
91
78
79
89
83
20 females in each concentration.
-------
TABLE 4. SPAWNING ACTIVITY AND MORTALITY OF ADULT FEMALE CHANNEL CATFISH EXPOSED TO TOXAPHENE
Toxaphene
concentration
(ng/1)
0
49
72
129
299
630
Spawns
Potential Actual Days to spawn
number3 number" after pairing
8 6 12.4
7 5 7.4
8 4 10.8
8 6 7.0
8 4 9.2
8 4 19.0
(5
(3
(5
(2
(3
(4
.9)C
.9)
.0)
.0)
.2)
.5)*
Female
mortality
(n)
0
1
1
1
0
2
Ovaries
resorbed
(n)
2
0
2
0
0
1
Ovaries Egg
underdeveloped hatch
(n) (%)
0
1
1
1
4
1
93
95
93
90
91
75
Number of females before spawning.
One spawn was from a female injected with human chorionic gonadotropin in the control and 129 ng/1
exposures.
f*
Mean (standard deviation).
*Significantly different from controls (P<0.05)
-------
Control spawn
Spawn from 630 ng/1 exposure
Figure 2. Effect of toxaphene on the proteinaceous matrix surrounding
the eggs of channel catfish.
16
-------
TABLE 5. EFFECT OF TOXAPHENE ON GROWTH OF CHANNEL CATFISH FRY AS INDICATED BY WEIGHT AND LENGTH
Toxaphene
concentration 5
(ng/1) Weight (g)
0 0.021a
(0.004)
49 0.020
(0.003)
72 0.021
(0.003)
129 0.020
(0.002)
299 0.021
(0.003)
630 0.020
(0.004)
Days of exposure
30
Weight (g)
0.13
. (0.05)
0.11
(0.04)
0.13
(0.04)
0.11
(0.03)
0.09*
(0.03)
0.10*
(0.02)
Length (mm)
23
(3)
20*
(3)
22
(2)
21*
(3)
19*
(2)
20*
(2)
60
Weight (g)
0.65
(0.22)
0.61
(0.17)
0.63
(0.20)
0.59
(0.18)
0.46*
(0.22)
0.49*
(0.09)
90
Length (mm)
37
(4)
36
(4)
37
(4)
36
(4)
33*
(5)
34*
(6)
Weight (g)
1.56
(0.67)
1.48
(0.41)
1.48
(0.42)
1.50
(0.44)
1.00*
(0.21)
1.10*
(0.35)
Length (mm)
48
(7)
51
(5)
50
(5)
50
(4)
43*
(3)
45*
(4)
wean (standard deviation).
*Significantly different from controls (P<0.05).
-------
100
control
100
49 no/1
50
50
30 60 90
-I 1_
30 60 go
TO
t:
o
CD
CJ
100
50
72 ng/l
100
50
129 ng/l
60 90
Days
Figure 3. Cumulative mortality in channel catfish fry after 30, 60, and 90
days of exposure to toxaphene.
18
-------
37, 38. The whole-body vitamin C concentrations in fry were significantly re-
duced (P<0.05) in the 72 ng/1 and higher concentrations at 15 and 30 days of
exposure, but the effect decreased at 60 days, indicating that the remaining
fry were less susceptible to the effects of toxaphene.
Fishes were more sensitive to toxaphene than aquatic invertebrates. The
acute toxicity of toxaphene to daphnids, scuds, and midges ranged from 10 to
180 ug/1-* , whereas the lethal threshold concentrations for brook trout^,
fathead minnows, and channel catfish were 4.1, 5.3, and 15.2 ug/1, respec-
tively (Table 6). Daphnids were also more tolerant of toxaphene than fishes
under field conditions .
BIOCHEMISTRY
Collagen was significantly reduced (P<0.05) in the backbones of fathead
minnows exposed to 97 and 173 ng/1, and hydroxyproline in the isolated
collagen was reduced in the 54, 97, and 173 ng/1 toxaphene concentrations
after 98 days of exposure (Table 7). However, no effects were found in the
adult fathead minnows at the end of the spawning period. Calcium and phos-
phorus concentrations in the backbone were not affected by toxaphene as they
were in a previous study2 , but the toxaphene exposures in that study were
higher—55, 132, 288, and 621 ng/1 (or 94, 205, 399, 727, and 1,420 ng/1,
corrected for recovery). Due to the small size of the fry, only hydroxyproline
was determined; it was significantly reduced (P<0.05) in fish exposed to 97
and 173 ng/1. Since we did not begin exposure of the parent fathead minnows
to toxaphene until they were 30 days old and since the most important time of
backbone development is in the early life stages, we placed fry after 30 days
of exposure to the 0, 54, and 173 ng/1 toxaphene concentrations in fresh
water to determine if the reduction in hydroxyproline was of permanent
significance in the development of broken backs. After 66 days in fresh
water, the percentage of observable spinal curvatures was 14, 10, and 100%
in the 0, 54, and 173 ng/1 exposures, respectively.
At the concentrations tested, toxaphene had no effect on backbone
composition or serum calcium, phosphorus, or protein in adult channel cat-
fish. However, concentrations of 72, 129, 299, and 630 ng/1 significantly
reduced (P<0.05) hydroxyproline in the eggs and 15-day-old fry (Table 8).
Collagen and hydroxyproline were also significantly decreased and calcium
was increased in these concentrations in the 90-day-old fry (Table 9). Back-
bone phosphorus was reduced in the 129, 299, and 630 ng/1 exposures. The in-
cidence of backbone anomalies was not directly related to the exposure con-
centrations. Of the 8 fish x-rayed in each concentration, 0, 1, 5, 7, 5, and
5 fish showed deformed backbones in concentrations of 0, 49, 72, 129, 299,
and 630 ng/1, respectively. In many fish, portions of vertebrae were miss-
in g in the backbone, especially at the anterior and posterior regions (Fig.
4). The effects on bone composition in fathead minnows and channel catfish
were similar to those reported for toxaphene in brook trout-*' ^1 and in a
previous study with fathead minnows22 where, in general, collagen in the back-
bone decreased.
19
-------
TABLE 6. ACUTE TOXICITY OF TOXAPHENE TO 30-DAY-OLD FATHEAD MINNOWS AND 2.5'
YEAR-OLD CHANNEL CATFISH
Q
Fathead minnows
Days
1
2
3
4
5
6
7
8
9
10
LC50
(ug/1)
17.4
9.2
8.6
7.2
6.4
5.6
5.3C
4.9
4.8
4.8
95% confidence
limits
14.8-20.4
7.7-11.0
7.1-10.4
6.1-8.5
5.3-7.7
4.7-6.7
4.4-6.3
3.9-6.1
3.8-6.0
3.8-6.0
Channel catfish^
LC50
(ug/1)
34.0
22.8
17.4
16.5
15. 2C
15.2
15.2
15.0
15.0
95% confidence
limits
27.3-42.3
19.2-27.0
14.4-21.0
14.7-18.6
13.8-16.7
13.8-16.7
13.8-16.7
13.6-16.5
13.6-16.5
Mean weight, 0.32 g; length, 30 mm.
Mean weight, 767 g; length, 394 mm.
°Lethal threshold concentration according to requirements described by Eaton'
20
-------
TABLE 7. CONSTITUENTS (mg/g of dried bone) OF BACKBONE IN FATHEAD MINNOWS EXPOSED TO TOXAPHENE
tsa
Toxaphene
concentrat:
(ng/1)
0
13
25
54
97
173
Constituents and days of exposure
Lon Collagen
98
190(32)b
220(51)
200(42)
180(31)
140(26)*
150(27)*
295
290(19)
310(39)
310(31)
270(21)
270(22)
260(26)
Hydroxyproline3
98
30(3)
29(4)
29(2)
24(6)*
24(4)*
25(3)*
295
27(4)
27(4)
27(2)
28(2)
26(3)
26(5)
30 (fry)
6.
5.
6.
4.
3.
2.
0(0.6)
4(2.2)
2(1.9)
3(0.8)
9(1.0)*
2(1.1)*
Calcium
98
76
83
(8)
(16)
76(11)
85
81
79
(12)
(8)
(10)
295
120(34)
110(11)
140(5)
130(12)
110(8)
130(19)
Phosphorus
98
41(4)
41(4)
40(3)
41(4)
39(6)
39(6)
295
57(6)
59(7)
61(6)
68(5)
59(3)
75(10)
Hydroxyproline is expressed as mg/g of dried collagen in the adult fish.
Mean(standard deviation).
*Significantly different from controls (P<0.05).
-------
TABLE 8. HYDROXYPROLINE (mg/g protein) IN CHANNEL CATFISH EGGS AND FRY
AS AFFECTED BY TOXAPHENE
Toxaphene
concentration
(ng/1)
0
49
72
129
299
630
Eggs
0.37(0.07)a
0.35(0.05)
0.29(0.08)*
0.30(0.03)*
0.23(0.05)*
0.27(0.08)*
Life stage
Fry (15-day-old)
8.5(0.4)
7.8(1.1)
2.3(1.3)*
1.2(0.1)*
2.2(0.2)*
6.5(0.8)*
Mean(standard deviation).
*Significantly different from controls (P<0.05).
22
-------
TABLE 9. CONSTITUENTS (mg/g of dried bone) OF BACKBONE IN 90-DAY-OLD CHANNEL CATFISH FRY EX-
POSED TO TOXAPHENE
NJ
Ol
Toxaphene
concentration
(ng/1)
0
49
72
129
299
630
Constituents
Collagen
270 (16)b
260 (10)
240 (16)*
240 (16)*
240 (22)*
230 (30)*
Hydroxyproline
57.6 (5.0)
53.4 (6.2)
47.3 (4.6)*
51.0 (5.2)*
52.6 (6.8)*
50.6 (4.6)*
Calcium
65 (6)
81 (2)*
110 (2)*
95 (4)*
85 (9)*
81 (7)*
Phosphorus
64 (4)
66 (5)
63 (3)
60 (4)*
57 (3)*
54 (2)*
Hydroxyproline is expressed as mg/g of dried collagen.
Mean(s tandard deviation).
^Significantly different from controls (P<0.05).
-------
Figure 4. X-rays and schematics of backbones of 90-day-old channel catfish, normal (Aa) and ex-
posed to 72 ng/1 of toxaphene (Bb).
-------
It was suggested that a competition for vitamin C might exist between
detoxicatlon of toxaphene through hydroxylative enzymes and collagen formation
through hydroxylation of proline to hydroxyproline22. Vitamin C is reported
to he essential in the hydroxylation of drugs and other organic chemicals in
the liver of mammals^S, 36, 3/, 38 aruj ±n collagen formation via the
hydroxylation of proline to hydroxyproline^^, 43, 44, 45. Toxaphene signif-
icantly reduced whole-body vitamin C in channel catfish fry (Table 10). In
another study with 10-month-old channel catfish °' , vitamin C was signif-
icantly reduced (P<0.05) in bone and increased in liver by toxaphene. There-
fore, the brittle backbone and backbone anomalies observed in studies with
toxaphene appear to be caused by the reduction of vitamin C in bone, which
inhibits the formation of hydroxyproline from proline and reduces collagen
formation. Other organic chemicals which are detoxified by hydroxylative
enzymes requiring vitamin C in the liver would be expected to produce back-
bone effects similar to those produced by toxaphene.
RESIDUE DYNAMICS
After 98 days of exposure, toxaphene in water was accumulated by fathead
minnows from 69,000 times (0.9 ug/g) in the low concentration to 55,000 times
(9.6 ug/g) in the high concentration (Table 11). One reason why the effects
of toxaphene on growth and bone composition decreased from day 98 (before
spawning) to day 295 (after spawning) may have been due to the decrease in
toxaphene residues in the fish even though the exposure concentrations re-
mained constant. Many organochlorine residues, such as those of toxaphene,
are associated with lipids in animals. The amount of lipid decreased in
fathead minnows from day 98 to day 295 (Table 12), probably because of the
extra energy required for gonad development and spawning. On the basis of
concentration in lipids, toxaphene residues also decreased during spawning
(Table 13), but not as much as that observed on a wet-weight basis. Toxaphene
residues ranged from 0.1 to 1.0 ug/g in eggs and from 0.2 to 2.8 ug/g in fry.
The percentage declines in whole-body residues in adult fathead minnows 56
days after transfer to uncontaminated water were 0, 0, 25, 25, and 36% in the
13, 25, 54, 97, and 173 ng/1 exposures, respectively.
Adult channel catfish accumulated toxaphene from water after 100 days of
exposure by factors of 17,000 to 26,000 (Table 14). The factor of accumu-
lation of toxaphene by fry from water ranged from 27,000 to 50,000. Con-
centrations of toxaphene in eggs were 0.24 ug/g in the controls and 4.4 ug/g
in the highest exposure concentration, and the residues in fry continuously
exposed to toxaphene did not equal or exceed the concentrations in eggs un-
til 30 days after hatch. The lipid content of fry was also lower than that of
eggs until the fry were 30 to 60 days old (Table 15); however, residues in
eggs and fry differed little on a lipid basis, except in the two highest
toxaphene concentrations (Table 16).
Adult male catfish exposed to 72 and 299 ng/1 toxaphene were analyzed for
residue distribution in the fillet and offal after exposure for 137 days. The
toxaphene concentrations in offal were 4.4 and 4.1 times those in the fillets,
which were 0.55 and 2.4 ug/g in fish exposed to 72 and 299 ng/1, respectively.
After the adult male catfish were transferred to fresh flowing water for 33
days, the average percent decline in whole body residues was only 18% (Table 14)
25
-------
TABLE 10. VITAMIN C CONCENTRATION (ug/g) IN EGGS AND FRY OF CHANNEL CAT-
FISH EXPOSED TO TOXAPHENE
Toxaphene
Fry (exposure days)
concentration Eggs
(ng/1)
0
49
72
129
299
630
38
25
28
25
38
41
(13)a
(3)
(6)
(17)
(8)
(13)
15
47
47
32
36
27
32
(16)
(8)
(8)*
(4)*
(2)*
(9)*
30
79
68
55
60
48
45
(20)
(16)
(9)*
(8)*
(16)*
(5)*
60
60
61
47
37
44
47
(13)
(10)
(18)
(5)*
(ID*
(3)
(standard deviation).
*Significantly different from controls (P<0.05).
26
-------
to
TABLE 11. WHOLE-BODY RESIDUES (ug/g) OF TOXAPHENE IN FATHEAD MINNOW ADULTS AND THEIR EGGS AND FRY
DURING EXPOSURE TO TOXAPHENE3
Adult treatment
Toxaphene
concentrati
(ng/1)
13
25
54
97
173
on Exposure
30
0.2
0.4
1.0
3.3
6.0
98
0.9
1.3
2.7
3.3
9.6
(days)
295b
0.1
0.2
0.4
0.8
1.4
Post exposure (.days
after start of exposure) T?onc
•* - - •Ei&efa
309 323 351
0.1 0.1 0.1 0.1
0.2 0.1 0.2 0.1
0.2 0.4 0.3 0.2
0.8 0.4 0.6 0.5
1.2 1.1 0.9 1.0
Fry
(30-day exposure)
0.2
0.4
1.0
1.5
2.8
No toxaphene was detected in control samples.
All exposures were terminated on day 295, and fish were transferred to uncontaminated water for
determination of elimination rates.
-------
00
TABLE 12. EXTBACTABLE LIPIDS (% of wet weight) IN FATHEAD MINNOW ADULTS AND THEIR EGGS AND FRY
DURING EXPOSURE TO TOXAPHENEa
Adult treatment
Toxaphene
concentration
(ng/1)
0
13
25
54
97
173
Exposure
30
12
5
5
5
7
5
.6
.5
.1
.1
.1
.7
98
6.0
11.4
8.1
8.4
9.2
9.7
(days)
295a
3.1
2.3
2.0
3.2
4.1
2.3
Post exposure (days
after start of exposure) Eggs
309
3.7
4.8
3.1
2.9
3.3
5.3
323
6.2
6.4
5.7
4.8
5.4
5.8
351
6.9
8.1
7.3
6.9
8.4
7.8
Fry
(30-day
0.
0.
0.
0.
0.
0.
30
09
06
20
10
20
6
5
5
5
5
5
exposure)
.0
.0
.0
.9
.1
.2
3A11 exposures were terminated on day 295, and fish were transferred to uncontaminated water for
determination of elimination rates.
-------
r-o
vc
TABLE 13. WHOLE-BODY RESIDUES (ug/g) OF TOXAPHENE ON A LIPID BASIS IN FATHEAD MINNOW ADULTS AND
THEIR EGGS AND FRY DURING EXPOSURE TO TOXAPHENE3
Adult treatment
Toxaphene
Post exposure (days
concentration Exposure
(ng/1)
13
25
54
97
173
30
3.6
7.8
20
46
105
98
11
11
33
35
99
(days) after
295b 309
5.2 2.7
11 8.1
12 7.9
19 25
60 24
start of exposure) Eggs
323
1.1
2.6
8.3
7.0
19
351
1.2
2.7
4.3
7.1
12
133
200
125
540
510
Fry
(30-day exposure)
3.3
7.9
17
30
52
No toxaphene was detected in control samples.
All exposures were terminated on day 295 and fish were transferred to uncontaminated water for
determination of elimination rates.
-------
TABLE 14. WHOLE-BODY RESIDUES (ug/g) OP TOXAPHENE IN CHANNEL CATFISH ADULTS AND THEIR EGGS AND FRY
DURING EXPOSURE TO TOXAPHENE
Toxaphene
concentration
(ng/1)
0
49
72
129
299
630
Adult exposure (days)
30
1
1
1
1
3
3
.1
.2
.6
.8
.0
.9
50
1.4
1.6
1.3
1.3
2.0
3.3
75a
1.3
1.1
1.7
2.4
4.6
7.2
100
1.1
1.3
1.9
2.6
6.2
11
Eggs
0.24
0.74
0.90
1.7
2.6
4.4
Fry
15
0.14
0.25
0.60
0.50
1.8
1.4
exposure (days)
30
0.10
0.50
1.3
1.9
3.4
4.0
60
0.12
1.0
2.2
3.0
5.8
18
90
0.
2.
3.
4.
8.
32
17
2
0
4
1
o
Two males were removed from each exposure on day 75 and were transferred to uncontaminated water
for determination of elimination rates. After 33 days, residues were 1.0, 1.6, 1.1, 3.8, and 6.9
ug/g in the 49 through 630 ng/1 exposures.
-------
TABLE 15. EXTRACTABLE LIPIDS (% of wet weight) IN CHANNEL CATFISH ADULTS AND THEIR EGGS AND FRY
DURING EXPOSURE TO TOXAPHENE
Toxaphene
concentration
(ng/1)
0
49
72
129
299
630
Adult exposure (days)
30
10.4
8.3
8.5
7.5
10.7
9.6
50
11.0
8.6
10.3
7.8
9.0
5.9
75
7.4
6.5
6.2
7.3
8.1
7.5
100
7.8
10. 0
7.2
8.4
5.8
7.4
Eggs
1.5
1.8
1.7
1.7
2.6
2.1
Fry exposure (days)
15
1.1
0.81
0.90
0.95
0.94
0.84
30
1.6
1.5
2.5
2.0
1.8
1.3
60
3.2
1.3
3.7
3.4
2.0
4.3
90
6.2
4.8
4.4
4.6
4.3
5.5
-------
Ni
TABLE 16. WHOLE-BODY RESIDUES (ug/g) OF TOXAPHENE ON A LIPID BASIS IN CHANNEL CATFISH ADULTS AND
THEIR EGGS AND FRY DURING EXPOSURE TO TOXAPHENE
Toxaphene
concentration
(ng/1)
0
49
72
129
299
630
Adult exposure (days)
30
10
14
19
24
28
41
50
13
18
13
17
22
56
75
18
17
27
33
57
96
100
14
13
26
31
107
148
Eggs
16
41
53
100
100
209
Fry exposure (days)
15
13
31
67
52
191
167
30
6.2
33
52
92
189
308
60
3.7
77
59
88
290
418
90
2.7
46
68
95
188
582
-------
GENERAL
An application factor of 0.01 has been recommended in establishing water
quality criteria for organochlorine insecticides^. The factor is derived by
dividing the maximum acceptable toxicant concentration (MATC; the highest
continuous toxicant concentration that has no adverse effect on growth, re-
production, and mortality) by the 96-hr LC50 value^ or by the lethal thresh-
old concentration^. Mount and Stephan^° hypothesized that the application
factor for a given toxicant experimentally determined for one fish species is
applicable to other species of fish, and this hypothesis is further supported
by our data (Table 17). In both invertebrates-^ and fishes, the calculated
application factors bracketed or approached the recommended factor of 0.01.
The application factor concept is a potentially valuable aid in determin-
ing water quality criteria. It is almost impossible to experimentally deter-
mine the MATC of a contaminant for all aquatic organisms because of the long
time required for conducting chronic toxicity tests. Within this hypothesis,
however, the MATC for a species in question can be derived by conducting an
acute toxicity test and multiplying the value by an application factor that
is experimentally determined for one species only. The no effect level based
on biochemical observations was consistent with the MATC derived on the basis
of growth, reproduction, and mortality in brook trout and fathead minnows.
The no effect level on a biochemical basis for channel catfish was between 49
and 72 ng/1 but the MATC was 199 to 249 ng/1.
Mayer et al.-* recommended that collagen and hydroxyproline in backbone be
considered as early biochemical indicators of growth and developmental changes
in fishes. The studies with brook trout , fathead minnows, and channel cat-
fish show that observable effects on growth correlate well with the biochemical
measurements. However, effects on collagen and hydroxyproline in channel
catfish were significant at lower toxaphene concentrations than was growth,
and the biochemical approach may give a more adequate assessment of the
potential danger of a contaminant to fish in some situations.
33
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TABLE 17. CONCENTRATIONS OF TOXAPHENE PRODUCING ACUTE AND CHRONIC TOXICITY TO AQUATIC ORGANISMS
Species
LTCa
(ug/1)
Predicted
MATCb
(ng/1)
Observed
MATC
(ng/1)
Application
factor0
Source
(reference
no.)
Daphnids,
Daphnia magna
100
>70<120
>0.007<0.012
39
Scud,
Gannnarus pseudolimnaeus
Midge ,
Chironomus plumosus
Brook trout
Fathead minnows
Channel catfish
24e
180e
4.1
5.3
15
240
1,800
41
53
150
>130<250
>1,000<3,200
<39
>25<54
>129<299
>0.005<0.010
>0.006<0.018
<0.010
>0.005<0.010
>0.009<0.020
39
39
3
PPf
PP
lethal threshold concentration".
Predicted safe concentration, based on an arbitrary 0.01 application factor times the LTC.
Experimentally derived by application of MATC's and acute values.
d48-hr EC50.
e96-hr LC50
Present paper.
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38
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-77-069
4. TITLE AND SUBTITLE
TOXAPHKNE: CHRONIC TOXICITY TO FATHEAD MINNOWS AND
CHANNEL CATFISH
3. RECIPIENT'S ACCESSION-NO.
5. REPORT DATE
June 1977
issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Foster L. Mayer, Jr., Paul M. Mehrle, Jr., William P.
Dwyer
8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Fish-Pesticide Research Laboratory, Fish & Wildlife
Service, United States Department of the Interior,
Columbia, Missouri 65201
10. PROGRAM ELEMENT NO.
1BA021
11. CONTRACT/GRANT NO.
E. P. A. -I. A. G. -0141 fn)
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory - Duluth, MN
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Fathead minnows (Pimephales promelas) and channel catfish (Ictalurus punctatus) were
continuously exposed to several toxaphene concentrations (13-630 ng/1) in flow-through
diluter systems for 8 to 10 months. Growth and backbone quality of adult fathead
minnows were decreased at 97 and 173 ng/1 exposures, but adult channel catfish were
not affected by toxaphene. Effects on reproduction were observed only in channel
catfish in the 630 ng/1 concentration: the period from pairing to spawning was
increased and the amount of gelatinous matrix surrounding the eggs was reduced.
Survival of fathead minnows was not affected by toxaphene, but the no-effect concen-
tration for fry growth and bone quality was below 54 and 97 ng/1, respectively.
Channel catfish fry survival and growth were reduced in the 299 and 630 ng/1 exposure
and bone quality was altered in concentrations as low as 72 ng/1. The maximum
toxaphene accumulation from water to fish was 69,000 times in fathead minnows and
50,000 times in channel catfish. Toxaphene was excreted very slowly in both species.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Growth
Collagens
Reproduction (biology)
Mortality
Pesticides
Fishes
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Catfish
Fathead Minnow
Toxaphene
Chronic Effects
Continuous Exposure
Residue Dynamics
Partial Life Cycle
6C
6F
6T
3. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
49
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
39
U.S. GOVERNMENT PRINTING OFFICE 1977-757-056/6^53 Region No. 5-1 I
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