EPA-600/3-77-099
August 1977
Ecological Research Series
A RAPID ASSESSMENT OF THE TOXICITY OF
THREE CHLORINATED CYCLODIENE
INSECTICIDE INTERMEDIATES TO
FATHEAD MINNOWS
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
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The nine series are:
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This report has been assigned to the ECOLOGICAL RESEARCH series. This series
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This document is available to the public through the National Technical Informa-
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EPA-600/3-77-099
August 1977
A RAPID ASSESSMENT OF THE TOXICITY OF THREE CHLORINATED
CYCLODIENE INSECTICIDE INTERMEDIATES TO FATHEAD MINNOWS
by
Robert L. Spehar
Oilman D. Veith
David L. DeFoe
Barbara A. Bergstedt
Environmental Research Laboratory-Duluth
Duluth, Minnesota 55804
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
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, U.S. Environmental Protection Agency, and approved for publication.
Mention of trade names or commercial products does not constitute endorsement
or recommendation for use.
ii
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FOREWORD
This report describes the toxic!ty of three organochlorine compounds used
as intermediates in the manufacture of pesticides. The U.S. Food and Drug
Administration noted residues in fish samples collected below a pesticide
manufacturing plant and alerted the Environmental Protection Agency for
necessary action. This research demonstrates the application of current state
of the art methods to measure chronic toxicity and residue forming potential
in a 30-day test.
A broad base of data accumulated over the past 10 years suggests
that the results of embryo-larval and early juvenile tests with associated
residue measurements will provide results within a factor of two of the values
that would be obtained in full life-cycle chronic tests.
Donald I. Mount, Ph.D.
Director
Environmental Research Laboratory
Duluth, Minnesota
iii
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ABSTRACT
A rapid assessment study to determine the toxicity and bioaccumulation of
three chlorinated cyclodiene insecticide intermediates; hexachlorocyclo-
pentadiene, hexachloronorbornadiene, and heptachloronorhoraene to fathead
minnow larvae and early juveniles was conducted for 30 days under flow-through
conditions.
A concentration of 7.3 yg/liter of hexachlorocyclopentadiene caused
significant decreases in survival after 4 days. Growth of fish exposed for
30 days was not significantly decreased at any of the concentrations tested.
The highest concentration of hexachlorocyclopentadiene having no adverse effect
was 3.7 yg/liter.
Concentrations of 122 and 226 yg/liter of hexachloronorbornadiene caused
significant decreases in survival after 4 days. Growth of 30 day-old larvae
was significantly decreased at 38.4 yg/liter and was the most sensitive
indicator of toxicity. The highest concentration having no adverse effect was
20.0 yg/liter'. The average bioconcentration factor for fish exposed to less
than 38.4 yg/liter of this compound was 6400.
Survival of fathead minnows exposed to heptachloronorbornene was
significantly decreased at 83.5 yg/liter after 4 days. Growth was significantly
reduced at 40 yg/liter after 30 days and was the most sensitive indicator of
toxicity. The highest concentration having no adverse effect was 25.9 yg/liter.
The average bioconcentration factor for fish exposed to less than 40 yg/liter
of this compound in water was 11,200.
iv
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CONTENTS
Foreword ill
Abstract ' iv
Figures ..... vi
Tables vii
Acknowledgments viii
1. Introduction 1
2. Conclusions 5
3. Recommendations 6
4. Materials and Methods 7
Water Characteristics 7
Exposure System 7
Chemical Conditions 7
Biological Methods 8
Residue Analysis 12
Statistical Analysis 12
5. Results 13
Hexachlorocyclopentadiene ("hex") 13
Toxicity 13
Hexachloronorbornadiene ("X") 13
Toxicity 13
Accumulation 13
Heptachloronorbornene ("Y") 16
Toxicity 16
Accumulation 16
6. Discussion 17
References 19
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FIGURES
Number Page
1 The manufacture of endrin from hexachlorocyclopentadiene
("hex"), reproduced from Barthel £t al. (1969) 3
2 Relationship between LC50 (log scale) of hexachlorocyclo-
pentadiene ("hex"), hexachloronorbornadiene ("X"), and
heptachloronorbornene ("Y") to exposure time (log scale)
for fathead minnows 14
3 Residue accumulation (log scale) of. hexachloronorbornadiene
("X") and heptachloronorbomene ("Y") measured in fathead
minnows exposed for 30 days 15
vi
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TABLES
Number Page
1 Survival and growth of fathead minnows exposed to various
concentrations of hexachlorocyclopentadiene ("hex") 9
2 Survival and growth of fathead minnows exposed to various
concentrations of hexachloronorbornadiene ("X") 10
3 Survival and growth of fathead minnows exposed to various
concentrations of heptachloronorbornene ("Y") 11
vii
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ACKNOWLEDGMENTS
The authors wish to thank Mr. D. Tanner for making routine water analysis,
constructing test equipment, and for daily assistance; and the Environmental
Research Laboratory-Duluth's staff for technical assistance, advice and
manuscript review.
viii
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SECTION 1
INTRODUCTION
The purpose of this study was to make a rapid assessment of the toxiclty
and bioaccumulation of three chlorinated cyclodiene insecticide intermediates;
hexachlorocyclopentadiene (."hex"), hexachloronorbornadiene C"X"), and
heptachloronorbornene ("Y") using larval and early juvenile stages of the
fathead minnow. The latter two compounds have been referred to as "X" and
"Y", respectively, in the literature (Barthel ejt al., 1969}.
The study was Initiated upon request for assistance from the Regional
Administrator, Region IV, to aid the U.S. Environmental Protection Agency
(EPA) in carrying out its responsibilities under the Federal Water Pollution
Control Act of 1972 concerning the discharge of these chemicals Into the
Mississippi River near Memphis, Tennessee.
Results of a study by the U.S. Food and Drug Administration C1976} in 1972-
1974 indicate that significant amounts of "X" and "Y" along with an epoxy
derivative (a metabolite of "X") were found in edible fish from the Mississippi
River near Memphis. The data showed that the most frequent occurrence and
highest residues of these chemicals were found In fish from the Memphis area
near a primary manufacturer of endrin and heptachlor, in contrast to low
amounts found in fish from other parts of the United States. This agrees with
work by Barthel jst al. (1966, 1969) who found that pesticide manufacturing
operations near Memphis was a source of significant pesticide contamination
in the sediments and water during studies conducted in 1964, 1966, and 1967.
These results showed that high concentrations (parts per thousand) of several
insecticide residues and residues of "X" and "Y" occurred In the bottom
sediments, spoils, and flood plain deposits.
In December, 1975, "hex11 was qualitatively identified as a contaminant in
the discharge of a pesticide production plant In Memphis (U.S. EPA, 1977). In
May, 1977, "hex" was also qualitatively identified at a pesticide production
plant in Michigan. Concentrations of "hex" were identified in the air, in the
plant's aqueous discharge and in fish tissue in the receiving stream.
Hexachlorocyclopentadiene is the key Intermediate in the synthesis of
stable chlorinated cyclodiene insecticides including aldrln, dleldrln, endrin,
endosulfan, heptachlor, chlordane, isodrin and mlrex (Brooks, 1974). The
accumulation of residues of these insecticides In higher trophic levels has
been demonstrated by several authors and is presently being used as a guideline
for water quality criteria (National Academy of Sciences and National Academy
of Engineering, 1973). Some other products derived from "hex" are nonflammable
resins, fungicides, heat resistant and shock proof plastics, acids, esters,
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ketones and fluorocarbons. It is a clear, yellow, slightly soluble liquid that
is produced by two companies in the United States; Hooker Chemical Corporation
(at Montague, Michigan and Niagara Falls, New York) and Velsicol Chemical
Corporation (at Memphis, Tennessee). Using the Diels-Alder reaction,
hexachlorocyclopentadiene can be transformed by the addition of vinyl chloride
to heptachloronorbornene and further converted by dehydro-chlorination to
hexachloronorbornadiene, a key intermediate in the synthesis of isodrin and
endrin (Brooks, 1974) (Figure 1). The estimated production of hexachloro-
cyclopentadiene in the U.S. is approximately 7 to 15 million pounds per year
(U.S. EPA, 1977).
The toxicity of chlorinated cyclodiene insecticides to aquatic life has
been studied by several authors: Henderson e£ al. (1959), Mount (1962), Mount
et al. (1966), Mount and Putnicki (1966), Johnson (1967), Reinert (JL967), Brungs
and Mount (1967), Macek et al. (1969), Grant (1976), Cardwell Q977) and
numerous others. These compounds and their metabolites have been shown to
accumulate in aquatic systems and are directly toxic to various aquatic
organisms at water concentrations of less than 1 pg/liter. However, only
limited information is available in the literature concerning the toxicity of
these three chemical intermediates. Cole (1954) investigated the germicidal
effects of a commercial "hex" preparation called P-162 in sewage effluents and
found that this chemical was more toxic to bacteria, coliform and Salmonella
typhasa than was chlorine. Results showed that 10 mg/liter of "hex" reduced
bacteria counts by at least 90% in 2-hr while chlorine over the same time
period and concentration reduced the total count by about 45%. Similar results
were observed with coliform and Salmonella at 5 and 10 mg/liter. Davis and
Hardcastle (1959) determined the 24, 48, and 96-hr median tolerance limit
(TL ) of "hex" for bluegills (Lepomis macrochirus) and largemouth bass
(Micropterus salmoides). The results of static bioassays for these time
periods were respectively, for bluegills, <500, 30, and 25 mg/liter and for bass
<500, 35, and 20 mg/liter (average water hardness, 77 mg/liter). The toxicity
of "hex" to fathead minnows was tested by the U.S. Department of Health,
Education and Welfare (1956) using two dilution waters and two formulations
for preparing test concentrations. The results demonstrated that (lhex" was
slightly less toxic in an acetone solution than in a water emulsion and more
toxic in hard water. Recorded 24, 48, and 96-hr TI^ values in hard water
(emulsion) were 0.075, 0.059 and 0.059 mg/liter, respectively. A model
ecosystem study by Lu et al. (1975) showed that "hex" has considerable
ecological stability and moderate biomagnification potential in algae
(Oedogonium cardiacum), snails (Physa sp.), mosquito larvae (Culex pipiens
quinquefasciatus) and fish (Gambusia affinis). Studies reported by Mount and
Putnicki (1966) showed that the chemical intermediates "X" and "Y" and endrin
accumulated in all samples of fish dying in the Mississippi River in 1963.
Laboratory studies by these authors indicated that these two intermediates
were approximately 1,000 to 10,000 times less toxic than endrin to guppies
(Poecilia reticulata).
Studies by Ingle (1953), Treon e* al. (1955) and Naishstein and Lisovskaya
(1965) have shown that hexachlorocyclopentadiene can produce toxicity in
mammals via ingestion, inhalation, or dermal exposures. Degenerative changes, in
the brain, heart, adrenals, liver, kidney and lungs were observed in severly
poisoned animals by all routes of administration.
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CH,-CHCL
VINYL CHLORIDE
"Y"OR HEPTdCHLORONORBORNENE
CYCLDPENTADIENE
*X" OR HEXACHLORONORBORNADIENE
BOOR IN
Figure 1. The manufacture of endrin from hexachlorocyclopentadiene C"hex''l, reproduced
ftom Barthel et al, Q9691.
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No specific studies on human toxicity of these three chemical intermediates
were shown in the literature. However, several inferences of the effects of
"hex" to researchers using these chemicals, such as eye irritation, headaches
and skin irritations were made by Ingle (1953), Treon e£ al* (1955), and Hooker
Chemical and Plastics Corporation (1969). Taste and odor threshold concentrations
of "hex" in water were reported as 1.4 and 1.6 yg/liter, respectively,
(Naishstein and Lisovskaya, 1965).
The study herein describes the toxicity and bioaccumulation of the above
named chlorinated cyclodiene insecticide intermediates in 30 day tests with
early life stages of the fathead minnow. The use of short term toxicity tests
involving the early developmental stages of fish to predict chronic toxicity
has been proposed by several investigators (Pickering and Thatcher, 1970;
Pickering and Cast, 1972; McKim e£ al., 1975; Eaton et al., 1977; and McKim et
al.', 1977). An extensive review of the literature (McKim, 1977) on life-cycle
toxicity tests with fish showed that embryo-larval and early-juvenile stages were
the most or among the most sensitive to chemical pollutants. It was concluded
that tests utilizing these stages can be used to estimate the maximum acceptable
toxicant concentration (MATC) within a factor of two and should be useful in
screening large numbers of chemicals.
The ability to predict the bioconcentratlon potential and steady state
concentrations of chemicals from relatively short exposure periods was discussed
by Blau et^al., 1975. Hansen et_ al^. (1971) demonstrated that chlorinated
compounds such as PCB's were rapidly stored in fish with maximum residue
concentrations being attained in 14 to 28 days. Thereafter, the concentrations
in the tissues remained constant with continued exposure. Consequently, the
bioconcentration factors reported in this study can be considered estimates
of what would be determined from longer exposures.
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SECTION 2
CONCLUSIONS
1. Thirty day exposures with larval and early juvenile fathead minnows shoved
that concentrations of 7.3, 38.4, and 40.0 pg/liter and above of hexachloro-
cyclopentadlene ("hex"), hexachloronorbornadiene ("X"), and heptachloro-
norbornene ("Y"), respectively, would be deleterious to this species.
2. Hexachloronorbornadiene and heptachloronorbornene decreased growth at 40
pg/liter, 2 to 3 times lower than concentrations affecting survival.
3. Larval and early juvenile stages of fathead minnows under the present test
conditions were more sensitive to all three intermediate compounds than
this same species and other species tested for similar time periods in
earlier studies.
4. The toxlcity curve for "hex" showed that this compound was a non-cumulative
poison. A median lethal threshold was attained within 4 days. Toxicity
curves for "X" and "Y" indicate that these compounds had a cumulative action.
5. Residue concentrations of "X" and "Y" Increased with increased exposure
concentrations up to a concentration causing decreases in growth of the
fish. The average bioconcentration factors for fish exposed to "X" and
"Y" were 6,400 and 11,200, respectively. Residue concentrations of "hex"
in fathead minnows were not obtained.
6. A rapid assessment of the toxlcity of hexachlorocyclopentadlene, hexachloro-
norbornadiene, and heptacbloronorbomene utilizing 30 day tests with larval
and early juvenile fathead minnows indicate that these compounds may present
a potential hazard to aquatic systems. The bioaccumulation of hexachloro-
norbornadiene and heptachloronorbornene in these fish suggest their
possible biomagnlfication in higher food chain organisms.
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SECTION 3
RECOMMENDATIONS
1. Concentrations of hexachlorocyclopentadiene, hexachloronorbornadiene, and
heptachloronorbornene not exceeding 3.7, 20, and 25.9 yg/liter, respectively,
appear "safe" for fathead minnows under the conditions tested and may be
used as first approximations of non-toxic concentrations. Concentrations
may need to be lowered to comply with residue limits once acceptable residue
concentrations are established.
2. An assessment of the toxicity of these compounds to other aquatic life
should be completed.
3. Studies involving the toxicity of mixtures of these chemical intermediates
along with mixtures of other toxicants, particularly the organochlorine
insecticides for which they are used to synthesize, are needed to evaluate
interactions.
4. Since these intermediate compounds have been found in edible fish,
investigations concerning what concentrations are safe for human consumption
should be made.
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SECTION 4
MATERIALS AND METHODS
WATER CHARACTERISTICS
Unfiltered Lake Superior water was heated and used in all tests at 25+2
C. Chemical characteristics of the test water were determined weekly according
to methods described by the American Public Health Association et al^. (1975).
Ranges for these measurements were (in milligrams per liter): dissolved
oxygen, 7.2-8.6; hardness, 45-47 as CaCOa; alkalinity, 42-43 as CaC03; and
acidity, 1.5-2.5 as CaCOa. The pH ranged from 7.2 to 7.7.
EXPOSURE SYSTEM
The three tests were conducted with intermittent-flow exposure systems
consisting of a multi-toxicant injection system (DeFoe, 1975) which delivered
five toxicant concentrations with equal amounts of acetone (4 mg/liter) and a
control to duplicate exposure chambers. The test chambers were glass aquaria,
45 x 16 x 18 cm, with a water volume of 8.9 liters. Water depth was 13.5 cm.
Flow rate to each chamber was 500 ml every 3 min providing a 93% replacement
of the test water every 2.7-hr (Sprague, 1969).
Flourescent bulbs provided a light intensity of 18-28 lumens at the water
surface. An automatically controlled 16-hr photoperiod was used.
CHEMICAL CONDITIONS
Chemicals used in this study were supplied by Velsicol Chemical Corporation,
Memphis, Tennessee. Water samples were collected daily from the test chambers
by siphoning each sample through a glass tube directly into volumetric flasks
containing hexane. Due to the differences in test concentrations, 500 ml flasks
containing 50 ml hexane were used in the test with "hex" and 250 ml flasks
containing 50 ml hexane were used in the tests with "X" and "Y". All samples
were stirred for 1.5 hr at a speed great enough to cause rigorous vortex mixing.
The phases were allowed to separate for 1 hr and an aliquot of hexane was
transferred to a vial for gas-liquid chromatograph (GLC) analysis. The samples
were analyzed on a Hewlett Packard 5730 H gas chromatograph equipped with an
auto sampler and Ni-63 electron capture cell. The column was 1.8 m x 2 mm (ID)
glass coil filled with 4% SE-30 and 6% OV-210 on 80/100 mesh Gas Chrom Q. The
carrier gas was argon containing 5% methane and all chromatograms were produced
at a column temperature of 150 C.
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To estimate the precision of the GLC injection procedure, six aliquots
of extracts from the next to the lowest concentration in each test were
transferred to vials for replicate analysis. The results showed that the GC
reproducibility gave relative standard deviations of 9.8% for the hexachloro-
cyclopentadiene at a concentration of 0.67 yg/liter, 1.5% for hexachloronor-
bornadiene at 36.7 yg/liter, and 0.6% for heptachloronorbornene at 45 yg/liter.
It is generally accepted that GLC analyses should have a precision of approxi-
mately 5%. Consequently, the precision of hexachlorocyclopentadiene analysis
is slightly less than many pesticide analyses and the precision of the other
analyses are well within the anticipated variability. The decrease in precision
in the former case is likely due to the much greater volatility of hexachloro-
cyclopentadiene and the fact that the concentrations were a order of
magnitude lower than the other chemicals.
The precision of the sampling and extraction procedure was examined
by analyzing six replicate water samples from a single tank in each diluter.
The results showed that the measured concentration could be estimated with
a relative standard deviation of 10.3%, 1.4%, and 0.9% for "hex", "X", and
"Y", respectively. Since the variation of water analyses is typically less than
10%, these measurements are within acceptable limits. Moreover, the variability
due to sample collection and extraction does not significantly increase the
variability of the overall analysis arising from the GLC injection.
Measured concentrations of each chemical are included in Tables 1, 2, and
3. One tank representing each concentration in each diluter system was sampled
18 times during this 30 day study, except those tanks in which all fish died
before the end of the test. On 11 and 12 of these sampling days, the
appropriate volume of Lake Superior water from the respective control tanks was
spiked to a concentration of 10 yg/liter of each chemical in acetone and the
sample was extracted and analyzed to determine the recovery of the extraction
procedure. The recovery of hexachlorocyclopentadiene from 12 spiked samples
was 93.7 + 6.6%, of hexachloronorbomadiene from 11 spiked samples was
102 + 4%, and of heptachloronorbornene from 11 spiked samples was 101 + 3%.
BIOLOGICAL METHODS
All three 30-day tests were conducted simultaneously beginning in April
and ending in May, 1977. To begin each test, 25 one-day-old fathead minnow
larvae were randomly selected and distributed to each duplicate exposure
chamber. All fish were fed brine shrimp nauplii 3 to 4 times a day. Mortalities
were recorded after the fourth day and then once a week for the remainder of
the test. Death was defined as complete immobilization and failureof the animals
to respond to probing.
After the 30 day period all surviving fish were killed in ice water and
immediately measured for total length, blotted and weighed. Whole fish were
then frozen in stainless steel weighing dishes for residue analysis.
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TABLE 1. SURVIVAL AND GROWTH OF FATHEAD MINNOWS EXPOSED TO VARIOUS CONCENTRATIONS OF
HEXACHLOROCYCLOPENTADIENE ("HEX"). ASTERISK (*) DENOTES VALUES SIGNIFICANTLY LESS
THAN CONTROLS (ANALYSIS OF VARIANCE, DUNNETT'S TEST, P=0.05).
VO
Item
Measured concentration (yg/liter)
9.1a ± 1.8 7.3 ± 4.7 3.7 ± 1.2 1.7 ±0.78 0.78 ± 0.31 <0.04 (control)
ABb ABABAB AB A B
4-day
Survival (%) 4*0 64 * 76 100 88 100 96 96 96 100 96
Survival (%)
Length (mm)
Weight (g)
30-day
* 0 60 * 72 96 84 100 92
25.6 ± 2.9 24.6 ± 2.9 24.7 ± 2.3
0.13 ± 0.04 0.11 ± 0.04 0.11 ± 0.03
92 96
25.1 ± 2.3
0.12 ± 0.03
92 96
24.8 ± 2.5
0.12 ± 0.04
Mean ± S.D. of duplicate chambers.
Duplicate chamber.
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TABLE 2. SURVIVAL AND GROWTH OF FATHEAD MINNO'WS EXPOSED TO VARIOUS CONCENTRATIONS OF
HEXACHLORONORBORNADIENE ("X"). ASTERISK (*) DENOTES VALUES SIGNIFICANTLY LESS THAN
CONTROLS (ANALYSIS OF VARIANCE, DUNNETT'S TEST, P=0.05).
Item
Survival (%)
Survival (%)
Length (mm)
Weight (g)
Measured
226a ± 26.3 122 ± 8.8 56.9
A B b A B A
28 * 48 84 * 72 96
0*0 72 * 60 96
19. 0 ± 2.3* 24.5
0.06 ± 0.02 0.11
concentration
± 10.2 38.4 ±
B A
4-day
96 96
30-day
96 92
± 1.6* 24.6 ±
± 0.03* 0.11 ±
(yg/liter)
3.1 20.0 ± 3.9
BAB
96 100 100
92 100 100
2.3* 25.0 ± 2.5
0.03 0.12 ± 0.03
<0.04 (control)
A B
100 100
96 92
25.9 ± 1.7
0.13 ± 0.03
Mean ± S.D. of duplicate chambers.
Duplicate chamber.
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TABLE 3. SURVIVAL AND GROWTH OF FATHEAD MINNOWS EXPOSED TO VARIOUS CONCENTRATIONS OF
HEPTACHLORONORBORNENE ("Y"). ASTERISK (*) DENOTES VALUES SIGNIFICANTLY LESS THAN
CONTROLS (ANALYSIS OF VARIANCE, DUNNETT'S TEST, P=>0.05).
Item
Survival (%)
Survival (%)
Length (mm)
Weight (g)
Measured concentration (yg/liter)
180. 3a + 14.8 164.9 ± 36.6 83.5 ± 7.1 40.0 ± 10.8 25.9 ± 3.4 <0.04 (control)
A Bb A BABABABA B
4-day
0*0 0*0 44 * 76 100 76 84 96 100 100
30-day
0*0 0*0 8 * 36 92 76 84 96 96 96
19.2 ± 3.3* 24.2 ± 2.2* 24.7 ± 2.8 25.4 ± 1.9
0.07 ± 0.03 0.11 ± 0.03 0.12 ± 0.03 0.12 ± 0.04
Mean ± S.D. of duplicate chambers.
Duplicate chamber.
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ANALYSIS
Whole fish were analyzed on a wet weight basis using the methods described
fry ffeith and Lee (1971). Composite samples of all surviving fish in each
ifttyfticete tank were homogenized with anhydrous Waj»S
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SECTION 5
RESULTS
HEXACBLOROCYCLOPENTADIENE ("hex")
Toxicity
Survival of fathead minnow larvae was significantly decreased at 7.3
pg/liter and above after 4 days of exposure (Table 1). A concentration of
9.1 yg/liter killed all but one fish after 4 days and all fish by 30 days.
Growth of fish surviving at concentrations of 7.3 yg/liter and below was not
significantly decreased from that in the controls (Table 1).
The toxicity curve relating median lethal concentration (LC50) for "hex"
to exposure time is illustrated in Figure 2.
HEXACHLORONORBORNADIENE ("X")
Toxicity
Concentrations of 122 and 226 tig/liter of "X" caused significant decreases
in survival of fathead minnow larvae after 4 days of exposure (Table 2). By
the end of the test (30 days) all fish were dead at 226 yg/liter and the mean
survival at 122 pg/liter was 66%. Significant decreases in survival did not
occur in concentrations lower than 122 yg/liter. Growth of 30 day-old larvae,
however, was significantly decreased at concentrations of 38.4 yg/liter and above.
The toxicity curve relating LC50 for "X" to exposure time is included in
Figure 2.
Accumulation
The concentration of "X" accumulated by fathead minnows was directly
proportional to the mean exposure concentration up to a concentration of 38.4
yg/liter which decreased the growth of the fish. As illustrated in Figure 3,
residues in 30 day-old fish exposed to 20 yg/liter contained an average of
129 yg/g which resulted in a bioconcentration factor of 6450. A similar
bioconcentration factor of 6350 was obtained when fish were exposed to 38.4
pg/liter. However, the bioconcentration factor at 56.9 yg/liter decreased to
4500, and further decreased to 4000 at 122 yg/liter. Consequently, the
average bioconcentration factor for hexachloronorbornadiene in fish exposed to
concentrations less than 38.4 yg/liter was approximately 6400.
13
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300
200
100
50
Q
IO
10
2O
3O
EXPOSURE TIME (days)
Figure 2. Relationship between LC50 (log scale) of hexachlorocyclopentadiene
("hex"), hexachloronorbornadiene ("X"), and heptachloronorbornene
("Y") to exposure time (log scale) for fathead minnows. Symbol •
indicate mean value of "hex", • of "X", and o of "Y". Bars indicate
95% confidence limits.
14
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lOOOr
EXPOSURE CONCENTRATION (jig/1)
Figure 3. Residue accumulation (log scale) of "X" (•! and "Y" (pi measured
in fathead minnows exposed for 30 -days. Symbols and bars indicate
the mean of duplicate pooled samples and S.D., respectively.
Symbol with no bar indicates one pooled sample.
15
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HEPTACHLORQNORBORNENE ("Y")
Toxicity
Survival of fathead minnow larvae exposed to "Y" was significantly
decreased at 83.5 yg/liter and above after 4 days. Higher concentrations of
164.9 and 180.3 yg/liter, killed all the fish within this period. Additional
mortality occurred at 83.5 yg/liter after 30 days resulting in 22% survival.
Decreases in survival were not observed at the two lower concentrations of
40 and 25.9 yg/liter. Lengths of fish, however, were significantly reduced
at 40 yg/liter and above after 30 days of exposure.
The toxicity curve relating LC50 for "Y" to exposure time is included in
Figure 2.
Accumulation
The concentration "Y" accumulated by fathead minnows was directly
proportional to the mean exposure concentration up to the "effect" concentration
of 40 yg/liter (Figure 3). Fish exposed to 25.9 yg/liter "Y" accumulated an
average of 296 yg/g which resulted in a bioconcentration factor of 11,400,
Fish exposed to 40.0 yg/liter contained residues of 438 yg/g, which resulted
in a bioconcentration factor of approximately 11,000. However, at 83.5 yg/liter,
588 yg/g were accumulated and the bioconcentration factor was reduced to 7000.
Consequently, the average bioconcentration factor for fish exposed to "Y" at
less than 40 yg/liter was 11,200.
16
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SECTION 6
DISCUSSION
Hexachlorocyclopentadiene (."hex") was more toxic to fathead minnows than
hexachloronorbornadiene ("X") or heptachloronorbornene ("Y"). A concentration
of "hex" causing adverse effects was approximately 5 times lower than concen-
trations of "X" and "Y" causing adverse effects to fathead minnows after the
same time period. Hexachloronorbornadiene and heptachloronorbornene
significantly decreased growth at approximately the same concentrations.
Concentrations that decreased growth were 2 to 3 times lower than those
affecting survival. This effect was different from that caused by "hex" since
this compound caused significant decreases in survival but not in growth.
Comparison of the results of this test to earlier studies show that all
three chemical intermediates were more toxic to fathead minnows in this study
than to this same species and others exposed to these compounds for similar
time periods. The 96-hr LC50 value of "hex" reported for fathead minnows in
this test was 8 times lower than the value reported for this species by the
U.S. Department of Health, Education and Welfare (1956). The 96-hr LC50
values of "hex" in static bioassays reported for bluegills and bass (Davis and
Hardcastle, 1959) were approximately 2800 to 3600 times higher than 96-hr
values reported in this test. Mount and Futnicki (1966) indicated that com-
pounds of "X" and "Y" would be 1,000 to 10,000 times less toxic than endrin
to guppies. An earlier study by Mount (1962) showed that 0.4 to 0.5 yg/liter
of endrin caused mortality to guppies and bluntnose minnows and that little
mortality was due to endrin at 0.25 yg/liter. These results indicate that "X"
and "Y" would be toxic to guppies at concentrations of 500 yg/liter and above
and that little effect would occur at concentrations below this. However, in
the present test, approximately 40 yg/liter of "X" and "Y" caused significant
reductions in growth and 122 and 83.5 yg/liter, respectively, caused
significant decreases in survival of fathead minnows. The lower values
obtained in this test for all three compounds are probably due to the
utilization of intermittent-flow exposure systems and/or the use of the most
sensitive life stages of development for testing (Mount, 1962; McKim, 1977).
The toxicity curve for hexachlorocyclopentadiene showed that a median
lethal threshold (the concentration at which acute toxicity of 50% of the
test animal ceases) was attained within 4 days. The presence of a threshold
level for this exposure time would indicate that this compound was non-cumulative.
Toxicity curves for hexachloronorbornadiene and heptachloronorbornene showed
that these compounds may have had a cumulative action. This is also
suggested because adverse effects on growth were observed at concentrations
much lower than those which decreased survival and by their high bioaccumulation
in whole body tissue.
17
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The accumulation of hexachloronorbornadiene and heptachloronorbornene in
fathead minnows was substantial at low exposure concentrations after 30 days.
The ability of these compounds to accumulate in fish is similar to that of the
persistent organochlorine insecticides including endrin, one of the most toxic
of all economic poisons to fish (Grant, 1976). Their accumulation in fathead
minnows indicate both a real and potential hazard to higher food chain organisms.
Both "X" and "Y" have been found in edible fish such as catfish, carp, and
others (U.S. Food and Drug Administration, 1976).
Residue concentrations of "hex" have not been found in edible fish and
its accumulation in fathead minnows was not demonstratable in this test because
of losses by vaporization during extraction. Additional studies are needed
to determine the bioconcentration factor of "hex" since it is found in water
bodies together with sewage water from manufacturers of poisonous chemicals
and plastics (Naishstein and Lisovskaya, 1965).
No information was found in the literature on the toxicity of hexachloro-
norbornadiene and heptachloronorbornene to mammals or humans. Studies by
Ingle (1953), Treon et jil. (1955), and Hooker Chemicals and Plastics Corpora-
tion (1969), however, have shown that hexachlorocyclopentadiene is toxic to
mammals (guinea pigs, rats, mice and rabbits) via inhalation, ingestion or
dermal exposure. Other effects caused by "hex" on mammals include degenerative
changes in several body organs, pulmonary edema, bronchitis, pneumonia, tremors,
irritation of mucous and respiratory membranes, increased breathing and others.
All three studies indicated that this compound can cause noxious effects to
humans such as skin burns and discomfort, eye irritation and headaches.
18
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Barthel, W. F., J. C. Hawthorne, J. H. Ford, 6. C. Bolton, L. L. McDowell,
E. H. Grissinger, and D. A. Parsons. Pesticides in Water. Pest.
Monit. J., Vol. 3, No. 1:8-67. 1969.
Barthel, W, F., D. A. Parsons, L. L. McDowell, and E. H. Grissinger. Surface
Hydrology and Pesticides - A Preliminary Report on the Analysis of
Sediments of the Lower Mississippi River. In: Pesticides and Their
Effects on Soil and Water. ASA Special Publication No. 8:128-144. 1966.
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Blau, 6. E., W. B. Neely, and D. R. Branson. Ecokinetics: A study of the Fate
and Distribution of Chemicals in Laboratory Ecosystems. AIChE Journal,
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Brooks, G. T. Chlorinated Insecticides. Vol. I. Technology and Application.
CRC Press, Inc., Cleveland, Ohio. 249 p. 1974.
Brungs, W. A., and D. I. Mount. Lethal Endrin Concentration in the Blood of
Gizzard Shad. J. Fish. Res. Board Can. 24:429-432. 1967.
Cardwell, R. D., D. G. Forman, T. R. Payne, and D. J. Wilbur. Acute and
Chronic Toxicity of Chlordane to Fish and Invertebrates. EPA-600/3-77-019.
U.S. Environmental Protection Agency, Duluth, Minnesota. 126 p. 1977.
Cole, E. J. Treatment of Sewage with Hexachlorocyclopentadiene. Appl. Micro-
biol. 2:198-199. 1954.
Davis, J. T., and W. S. Hardcastle. Biological Assay of Herbicides for Fish
Toxieity, Weeds, 7:397-404. 1959.
Defoe, D. L. Multichannel Toxicant Injection System for Flow-Through Bioassays.
J. Fish. Res. Board Can. 32:544-546. 1975.
Eaton, J. G., J. M. McKim, and G. W. Holcombe. Metal Toxicity to Embryos and
Larvae of Seven Freshwater Fish Species - I Cadmium. Bull. Environ.
Contam. Toxicol. In Press, 1977.
19
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Grant, B. F. Endrin Toxicity and Distribution in Freshwater. A Review.
Bull. Environ. Contam. Toxicol. 15(3):283-290. 1976.
Hamilton, M. A., R. C. Russo, and R. V. Thurston. The Trimmed Spearman-Karber
Method for Estimating Median Lethal Concentrations in Toxicity Bioassaya.
Environ. Sci. and Technol. 11(3):714-719. 1977.
Hansen, D. J., P. R. Parrish, J. I. Lowe, A. J. Wilson Jr., and P. D. Wilson.
Chronic Toxicity, Uptake and Retention of AroclorR 1254 in Two Estuarine
Fishes. Bull. Environ. Contam. Toxicol. 6(2):113-119. 1971.
Henderson, C., Q. H. Pickering, and C. M. Tarzwell. Relative Toxicity of Ten
Chlorinated Hydrocarbon Insecticides to Four Species of Fish. Trans.
Am. Fish. Soc. 88:23-32. 1959.
Hooker Chemicals and Plastics Corp., Industrial Chemicals Division.
Hexachlorocyclopentadiene. Data Sheet No. 815A. 1969.
Ingle, L. Toxicity of Chlordane Vapors. Sci. 118:213-214. 1953.
Johnson, H. E. The Effects of Endrin on the Reproduction of a Freshwater
Fish (Oryzias latipes). Ph.D. Thesis, University of Washington. 136 p.
1967.
Lu, P. Y., R. L. Metcalf, A. S. Hirwe, and J. W. Williams. Evaluation of
Environmental Distribution and Fate of Hexachlorocyclopentadiene, Chlordane,
Heptachlor, and Heptachlor Epoxide in a Laboratory Model Ecosystem.
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Macek, K. J., C. Hutchinson, and 0. B. Cope. The Effects of Temperature on the
Susceptibility of Bluegills and Rainbow Trout to Selected Pesticides. Bull.
Environ. Contam. Toxicol. 4:174. 1969.
McKim, J. M. Evaluation of Tests With Early Life Stages of Fish for Predicting
Long-Term Toxicity. J. Fish. Res. Board Can. 34(8):1148-1154. 1977.
McKim, J. M., J. W. Arthur, and T. W. Thorslund. Toxicity of a Linear Alkylate
Sulfonate Detergent to Larvae of Four Species of Freshwater Fish. Bull.
Environ. Contam. Toxicol. 14:1-7. 1975.
McKim, J. M., J. G. Eaton, and G. W. Holcombe. Metal Toxicity to Embryos and
Larvae - Early Juveniles of Eight Species of Freshwater Fish II. Copper.
Bull. Environ. Contam. Toxicol. In Press, 1977.
Mount, D. I. Chronic Effects of Endrin on Bluntnose Minnows and Guppies. U.S.
Fish & Wildl. Serv. Res. Rep. 58. 1962.
Mount, D. I., and G. J. Putnicki. Summary Report of the 1963 Mississippi Fish
Kill. Trans. 31st No. Amer. Wildl. and Nat. Res. Conf. p. 177. 1966.
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Mount, D. I., L. W. Vigor, and M. L. Schafer. Endrin: Use of Concentration
in Blood to Diagnose Acute Toxicity to Fish. Sci. 152:1388-1390. 1966.
Naishstein, S. Ya., and E. V. Lisovskaya. Maximum Permissible Concentration of
Hexachlorocyclopentadiene in Water Bodies. Gig. Sanit., 30:177-181. 1965.
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Environmental Studies Board, Washington, D.C. 594 p. 1973.
Pickering, Q. H., and M, H. Cast. Acute and Chronic Toxicity of Cadmium to
the Fathead Minnow, Pimephales promelas. J. Fish. Res. Board Can.
29:1099-1106. 1972,
Pickering, Q. H., and T. 0. Thatcher. The Chronic Toxicity of Linear Alkylate
Sulfonate (LAS) to Pimephales promelas Rafinesque. J, Water Poll. Cont.
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Reinert, R. E. The Accumulation of Dieldrin in an Alga (Scenedesmus obliguus),
Daphnia (Daphnja magna), Guppy (Lebistes reticulatus) Food Chain. Ph.D.
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Sprague, J. B. Measurements of Pollutant Toxicity to Fish. 1. Bioassay
Methods for Acute Toxicity. Water Res., 3:793-821. 1969.
Steel, R. G. D., and J, H. Torrie. Principles and Procedures of Statistics
with Special Reference to the Biological Sciences. McGraw-Hill, Inc.
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Treon, C,, F. Cleveland, and J, Cappel. The Toxicity of Hexachlorocyclopentadiene.
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Environmental Health for Hooker Chemical Company, Niagara Falls, New
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Chemicals in Edible Fish, 1,2,3,4,7,7-hexachloronorbornadiene, 1,2,3,4,5,
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21
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
,1. REPORT NO.
! fl»ft*"600/3-77-099
3. RECIPIENT'S ACCESSION NO.
4i "Plflie-AtilO SUBTITLE
& R&pld Assessment of the Toxicity of Three Chlorinate<
CT^lodiene Insecticide Intermediates to Fathead
W&swows
5. REPORT DATE
August 1977 issuing date
6. PERFORMING ORGANIZATION CODE
L. Spehar, Oilman D. Veith, David L. DeFoe,
Barbara A. Bergstedt
8. PERFORMING ORGANIZATION REPORT NO.
ORGANIZATION NAME AND ADDRESS
ffittXironmental Research Laboratory - Duluth, MN
(Office of Research and Development
$.S. Environmental Protection Agency
Duluth, Minnesota 55804
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
In-house
12. SPONSORING AGENCY NAME AND ADDRESS
SAME AS ABOVE
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A rapid assessment study to determine the toxicity and bioaccumulation of three
chlorinated cyclodiene insecticide intermediates; hexachlorocyclopentadiene,
hexachloronorbornadiene, and heptachloronorbornene to fathead minnow larvae and early
juveniles was conducted for 30 days under flow-through conditions.
A concentration of 7.3 yg/liter of hexachlorocyclopentadiene caused significant
decreases in survival after 4 days. Growth of fish exposed for 30 days was not
significantly decreased at any of the concentrations tested. The highest concen-
tration of hexachlorocyclopentadiene having no adverse effect was 3.7 yg/liter.
Concentrations of 122 and 226 yg/liter of hexachloronorbornadiene caused
significant decreases in survival after 4 days. Growth of 30 day-old larvae was
significantly decreased at 38.4 yg/liter and was the most sensitive indicator of
toxicity. The highest concentration having no adverse effect was 20.0 yg/liter. The
average bioconcentration factor for fish exposed to less than 38.4 yg/liter of this
compound was 6400.
Survival of fathead minnows exposed to heptachloronorbornene was significantly
decreased at 83.5 yg/liter after 4 days. Growth was significantly reduced at 40
yg/liter after 30 days and was the most sensitive indicator of toxicity. The highest
concentration having no adverse effect was 25.9 yg/liter. The average bioconcen-
tration factor for fish exposed to less than 40 us/liter of this compound in water was
,7.11,200.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Bioassay
Freshwater
fishes
Residues
Survival
Growth
Minnows
Toxicity
Fathead minnows
Acute toxicity tests
Chronic toxicity tests
Early developmental
stages
Bioaccumulation
Organochlorine
insecticides
06 F
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS {ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
30
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
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