PB-256 334
CHRONIC TOXICITY OF LINDANE TO SELECTED AQUATIC INVERTEBRATES AND FISHES
KENNETH J, MACEK, ET AL
BIONOMICS., EG & G, INCORPORATED
WAREHAM, MASSACHUSETTS
MAY 1976
DISTRIBUTED BY:
National Technical Information Service
U. S. DEPARTMENT OF COMMERCE
5285 Port Royal Road, Springfield Va. 22151
This document bas been approved for public release and sale.
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EPA-600/3-76-046
May 1976
CHRONIC TOXICITY OF LINDANE TO
SELECTED AQUATIC INVERTEBRATES AND FISHES
by
Kenneth J. Macek
Kenneth S. Buxton
Steven K. Derr
J. W. Dean '
Scott Sauter
Bionomics, EG§G Inc.
Wareham, Massachusetts 02571
Contract No. 68-01-0154
68-01-1841
Project Officer
i
John G. Eaton
Environmental Research Laboratory
Duluth, Minnesota 55804
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL RESEARCH LABORATORY
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. 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.
11
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ABSTRACT
Representatives of the aquatic invertebrate species of
water flea (Daphnia magna), midge (Chironomus tentans),
and scud (Gammarus fasciatus); and the fish species of
bluegill (Lepomis macrochirus), fathead minnow (Pimephales
promelas), and brook trout (Salvelinus fontinalis) were
chronically exposed to various concentrations of lindane
in separate flowing water systems.
Maximum acceptable toxicant concentrations (MATC) of lindane
for the selected species in soft water were estimated using
survival, growth, and reproduction as indicators of toxic
effects. The MATC was estimated to be between 2.2 and 5.0
jug/1 for midges, between 11 and 19 pg/1 for the water flea,
and between 4.3 and 8.6 ^ig/1 for scud. For fishes the MATC
was estimated between 9.1 and 12.5 for bluegills, between
9.1 and 23.5 ug/1 for fathead minnows, and between 8.8 and
16.6 Mg/1 for brook trout. The incipient median lethal
concentration (LC50) for fishes and the 48-hour LC50 for
invertebrates was estimated from acute exposures and used to
calculate application factors (MATC/LC50). For aquatic
invertebrates and lindane the estimated application factors
were between 0.010 and 0.024 for midges, between 0.020 and
0.029 for water flea, and between 0.11 and 0.22 for scud.
Application factors were estimated between 0.30 and 0.42
for bluegill, between 0.13 and 0.34 for fathead minnows,
and between 0.34 and 0.64 for brook trout.
This report was submitted in fulfillment of Project Number
18050 HQH, Contracts 68-01-0154 and 68-01-0841 by Bionomics,
E G & G, Inc. under the sponsorship of the Water Quality
Office, Environmental Protection Agency. Work was completed
as of February 1974.
111
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CONTENTS
SECTION PAGE
I Conclusions 1
II Recommendations 2
III Introduction 4
IV Materials and Methods 6
Chronic Exposure Systems 6
Acute Toxicity Procedures 10
Chemical Methods 10
Statistics 12
Chronic Exposure 13
Chironomus tentans 13
Daphnia magna 13
Gantmarus fasciatus 14
Lepomis macrochirus 15
Pimephales promelas 16
Salvelinus fontinalis 17
V Results 20
Acute Bioassays 20
Water Chemistry 20
Chronic Exposure 23
Chironomus tentans 23
Daphnia magna 24
Gammarus fasciatus 26
Lepomi s"macrochirus 28
Pimephales promelas 30
Salvelinus rontinaTis 33
Residue Analysis37
Calculation of Application Factors 37
VI Discussion 40
VII References 46
Preceding page blank
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TABLES
; PAGE
1, Physical characteristics of test chambers
utilized to evaluate the chronic toxicity of
lindane to fishes and fish food organism 7
2. Chemical analysis of the diluent water utilized
during chronic aqueous exposure of fishes and
aquatic invertebrates to lindane 9
3. Percent recovery of added lindane from water (ug/1)
and whole fish (ug/g) 12
4. Mean and range of measured concentrations of
hardness, alkalinity, acidity, dissolved oxygen
and pH from water samples taken periodically during
chronic exposure of aquatic invertebrates and
fishes to lindane 21
5. Nominal and measured lindane concentrations (ug/1)
in water during chronic exposure of aquatic
invertebrates and fishes to lindane 22
6. Summary of the effect of various concentrations
of lindane on hatchability, pupation and emergence
of Chironomus tentans continuously exposed for two
generations 23
7. Mean percent survival of Daphnia magna continuously
exposed to lindane for 64 days 25
8. Mean production of young per female Daphnia magna
continuously exposed to lindane for 64 days 26
9. Survival and reproductive success of Gammarus
fasciatus exposed to lindane for 17 weeks 27
10. Survival and growth during 6 and 18 months, and
results of spawning activity of bluegill (Lepomis
macrochirus) continuously exposed to lindane 29
11. Hatchability, survival and growth of bluegill
(Lepomis macrochirus) fry exposed to lindane 30
12. Survival and growth of fathead minnows (Pimephales
promelas) during 30 days, 60 days, and 43 weeks
continuous exposure to lindane 31
VI
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PAGE
13. Sexual development, spawning, hatchability of eggs,
and survival and growth of offspring after 30 and 60
days, for fathead minnow (Pimephales promelas)
continuously exposed to lindane 32
14. Growth of yearling brook trout (Salvelinus
fontinalis) during 261 days continuous exposure
to lindane 34
15. Results of spawning activity of yearling brook
trout (Salvelinus fontinalis) during continuous
exposure to lindane 35
16. Survival and growth of second generation brook
trout (Salvelinus fontinalis) during the first
90 days of development of fry continuously
exposed to lindane 36
17. Mean measured lindane concentration in water
(ug/1) and in the muscle (ug/kg) of bluegill and
brook trout and in the eviscerated carcass (>ig/kg)
of fathead minnows continuously exposed to lindane 38
18. Summary of concentrations of lindane (jig/1)
producing acute and chronic toxicity to aquatic
species, and calculated application factors
describing the relationship between acute and
chronic toxicity 39
19. Summary of application factors reported for
chemicals to various species of fish 45
vii
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ACKNOWLEDGEMENTS
During the period of time devoted to this research effort,
we were fortunate to have laboratory assistance from
Mark Lindberg and Gerald LeBlanc, who maintained exposure
systems and were significantly involved with routine aspects
of the fish chronics. Gratitude is extended to Sarah
Gnilka, Pat Costa and Karen Frey for their involvement and
expertise in conducting the invertebrate chronics. Also,
our appreciation is directed to Curtis Hutchinson for
construction of all diluter systems utilized throughout
this study, and to Patrick (Rod) Parrish for constructively
reviewing the final report.
We are grateful to Mr. Richard Griffith, Northeast Regional
Director, U.S. Bureau of Sport Fisheries, Boston, Mass,
for his assistance in obtaining disease-free brook trout
used in the chronic exposure and to Mr. Quentin Pickering,
Newtown Fish Toxicology
Cincinnati, Ohio for providing fathead minnow eggs used in
the study.
Finally, our sincere appreciation is extended to John
Eaton, Project Officer, Environmental Protection Agency,
Environmental Research Laboratory-Duluth, Duluth, Minnesota, for
his .guidance and constructive advice during the performance
of these studies.
Vlll
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SECTION I
CONCLUSIONS
Estimates (LC50) of the acute toxicity of lindane to the
species tested ranged from 26-76 jug/1 for bluegills, fathead
minnows, brook trout and gammarids, while the range of
estimated acute toxicity of lindane to daphnids and midges
was generally an order of magnitude higher (207-485 p.g/1) .
Of the six species studied during chronic bioassays, the
midges and gammarids were the most sensitive to lindane.
Estimated limits of the maximum acceptable toxicant
concentrations (MATC) of lindane for these two invertebrate
species were within the range of 2.2-8.6 jug/1.
All of the fish species studied were similar in their
susceptibility to chronic exposure to lindane. Estimated
limits of the MATC of lindane for the fishes were within
the range of 8.8-23.5 ug/1, as were those for daphnids.
The toxicity of lindane to the invertebrate species was
generally cumulative from generation to generation with
many instances of significant effects being observed at
concentrations which apparently did not affect the previous
generation.
These data suggest that populations of essential fish food
organisms may be significantly reduced in numbers by exposure
to concentrations of lindane which would apparently not
directly affect fish populations.
Calculated limits of application factors for the three
fishes were similar and were within the range of 0.13-0.64.
Comparable values for the invertebrate species were within
the range of 0.01-0.22. These data suggest that the concept
of the use of application factors for estimating the
MATC of chemicals to aquatic organisms based on the results
of acute bioassays may be valid within broad taxonomic groups.
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SECTION II
RECOMMENDATIONS
Of the species studied, midges (Chironomus tentans) were
among the most susceptible to the toxicant. This observation,
combined with the adaptability of these organisms to
laboratory systems, suggests this species as a desirable
bioassay organism for evaluating the chronic toxicity of
chemicals to aquatic organisms.
Conversely, although scud (Gammarus fasciatus) appeared to
be as sensitive as midges, it is possible this observation
is related to the inability of these organisms to adapt to
laboratory culture and handling. We believe much work needs
to be done to develop methods of handling and testing these
organisms in the laboratory which will provide better survival
and reproduction under these conditions. Such methods
would allow for more sensitive comparison between the response
of treated and control groups to toxicant exposure.
To a lesser degree, we feel the same recommendation should
be made regarding bluegill. More information regarding the
laboratory conditions necessary to induce and maintain survival
and spawning activity, and the procedures necessary to
successfully handle and feed newly hatched bluegill fry,
must be developed to enable successful completion of bluegill
chronic toxicity bioassays in a reasonable period of time.
We recommend that the fathead minnow be considered the fish
species of choice, among those tested, for chronic toxicity
bioassays. This recommendation is based on the fact that an
adequate level of spawning activity can be induced under
laboratory conditions, successful handling of eggs and
larvae is possible, and excellent survival of fry is
obtained. The combination of these factors provides
opportunities for statistically and biologically sensitive
evaluation of possible toxic effects due to chronic exposure
to chemicals. Finally, studies with this species represent
the only "true chronic" (at least one complete life history)
of the fishes studied.
Because of the cumulative nature of the toxicity of lindane
to several invertebrate species, it appears that some
information based on multi-generation studies is necessary
to adequately assess the potential long-term effects of the
pesticide to these species. We suggest that similar multi-
generation studies may be necessary with at least one fish
species to assess the possible cumulative effects of lindane
on fish.
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In view of the order of magnitude difference between
application factors calculated for fishes and those
calculated for two of the aquatic invertebrates, we suggest
that future laboratory investigations to assess the chronic
toxicity of chemicals to aquatic organisms consider at least
one fish species and one of the more sensitive invertebrate
species.
Tissue residue information on fishes, or fish fry, based
on a single interval during the life history provides
limited information on the subject of bioaccumulation.
We suggest that by including additional fish at the initiation
of a chronic exposure, and sampling periodically during the
first 60-90 days of exposure, additional significant
information regarding the uptake, accumulation and retention
of chemical residues by fish would be generated. It is
possible that this information could then be correlated with
observed biological responses to chemical exposure.
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SECTION III
INTRODUCTION
The current concern regarding the protection of aquatic
life in surface waters has prompted evaluation of the
effects of chemicals on aquatic invertebrates and fishes.
Much of the toxicological research on aquatic biota has been
limited to the development of acute toxicity values as a
measure of the biological effect of these compounds. More
recently, utilization of the chronic exposure of fishes to
chemicals has received particular attention due to the
numerous parameters that can be evaluated as indices of
toxic effects of a particular compound (Mount, 1968; Mount
and Stephan, 1967; Eaton, 1970; McKim and Benoit, 1971;
Hermanutz et al., 1973). The "Laboratory Fish Production
Index (LFPI)" as defined by Mount and Stephan (1967) reflects
toxic effects on reproduction, growth, spawning behavior,
egg mortality, and fry survival. The highest observed
toxicant concentration that has no effect on these parameters
during continuous chronic exposure is termed the maximum
acceptable toxicant concentration (MATC).
Lindane, the gamma isomer of benzene hexachloride (1,2,3,4,
5,6-hexachlorocyclohexane), is one of the few chlorinated
hydrocarbon insecticides still widely used for agricultural
purposes, particularly as a cereal seed dressing (Brooks,
1972). Current pesticide monitoring programs indicate that
lindane is present in the Mississippi River and some streams
in the Western U.S., at concentrations ranging from 2.8-
28.0 ng/1 (Matsumura, 1972). Past studies have demonstrated
that lindane is relatively persistent in natural waters and
sediment and that significant degradation occurs only under
anaerobic conditions (Brooks, 1972; Nash and Woolson, 1967).
Although the persistence of lindane could pose a significant
environmental hazard, studies to date have indicated that
the biological accumulation and persistence of lindane is
low when compared to compounds such as DDT and dieldrin
(Wilson, 1965; Gakstatter and Weiss, 1967).
Past studies with lindane have consisted mainly of low-
dose feeding experiments or short-term aqueous exposures
which generated acute toxicity values. This study was
undertaken to determine the MATC for selected aquatic
invertebrates and fishes based on continuous chronic
exposure. The organisms selected for this research effort
were:
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INVERTEBRATES
1. Chironomus tentans (Chironomidae, mdige)
2. Daphnia magna (Cladocera, water flea)
3. Gammarus fasciatus (Gammaridae, scud)
FISHES
1. Lepomis macrochirus (Centrarchidae, bluegill)
2. Pimephales promelas (Cyprinidae, fathead minnow)
3. Salvelinus fontinalis (Salmonidae, brook trout)
A reason for selecting both invertebrates and fishes is
that the susceptibility of fishes to a chemical should be
compared to the susceptibility of fish-food organisms to
that same chemical. An understanding of both these
phenomena will be invaluable in establishing realistic and
meaningful water quality criteria and standards.
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SECTION IV
MATERIALS AND METHODS
The methodology for chronic testing of fishes generally
followed the recommended bioassay procedures issued by
the Environmental Protection Agency's Environmental Research
Laboratory-Duluth, Duluth, Minnesota (Bioassay Committee, 1971a)
1971b, 1971c). Acute bioassay procedures were generally
those recommended in Standard Methods for the Examination of
Water and Wastewater (1971). Chronic testing procedures for
invertebrates were determined through communication of
Bionomics staff members with personnel at the Environmental
Research Laboratory-Duluth.
CHRONIC EXPOSURE SYSTEMS
Proportional diluters (Mount and Brungs, 1967), with a dilution
factor of 0.5 and a syringe injector, delivered the test water
and toxicant, dissolved in dimethyl sulfoxide, to the mixing
chamber, mixing cells, and ultimately to the test chambers.
Five lindane concentrations and a control flowed to mixing
containers and into separate glass delivery tubes leading to
the replicate test chambers. In the test system for Daphnia
magna, baffles were inserted in each of the quadruplicate
test chambers to minimize turbulence of influent water. The
diluter was also modified to include food cells which delivered
a measured amount of food along with the toxicant and diluent
water. All other exposure systems utilized duplicate test
chambers of varying construction and flow rates as summarized
in Table 1.
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Table 1. PHYSICAL CHARACTERISTICS OF TEST CHAMBERS UTILIZED TO EVALUATE THE CHRONIC
TOXICITY OF LINDANE TO FISHES AND FISH FOOD ORGANISMS
Species
Chironomus
tentans
Daphnia
magna
Gammarus
fasciatus
Lepomis
macrochirus
Pimephales
promelas
Salvelinus
fontinalis
Material
(shape)
glass
(rectangular)
glass
(cylindrical)
glass
(rectangular)
stainless steel
(rectangular)
glass
(rectangular)
stainless steel
(rectangular)
Dimensions
(cm)
21x26x18
17x13.5
25x40x21
40x180x30.5
30.5x90x30.5
40x90x30.5
Water
Depth
(cm)
16.0
14.0
19.0
30.5
15.0
30.5
Volume
(liters)
7.5
1.8
16.0
168.0
41.0
84.0
Flow Rate
(tank vol./
24 hours)
6
2
3
7
7
7
Dimensions are height x length x width for rectangular and height x diameter for
cylindrical chambers.
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Two growth chambers 21 x 20 x 15 cm (height x length x width)
with a water depth of 16 cm were provided for the young
Gammarus and received test water at a flow rate equal to
that in the adult test chambers. Fathead minnow test chambers
were subdivided to provide space for two growth chambers
25 x 20.5 x 12.5 cm, and test chambers for brook trout
contained a shelf which supported two growth chambers 25 x
25 x 12.5 cm with a water depth of 12.5 cm. Test water was
delivered directly to the test chamber and growth chambers
through a glass, flow-splitting chamber calibrated to keep
the flow rate equal in all chambers. The bluegill test
chambers supported two 18x28x12 cm growth chambers
with a water depth of 10.5 cm; and an air-driven system was
utilized to pump water from the test chamber to the growth
chambers at a rate of 6 chamber volumes every 24 hours. For
all fish tests, 40 mesh stainless steel screen was affixed
to one end of each growth chamber to allow water to flow
out while retaining the young fish. Bluegill and brook
trout test chambers were aerated with oil-free air in order
to maintain dissolved oxygen levels above 60 percent
saturation.
Diluent water was pumped from a 400 foot subterranean well
to a cement holding tank. Results of the chemical analysis
of the diluent water are summarized in Table 2. Diluent
water was delivered through PVC pipes to the exposure systems.
Unheated well water was mixed with heated well water from a
glass-lined heater to provide a constant temperature of 28°C
before delivery to the bluegill experimental unit. Test
chambers in the fathead minnow and brook trout units were
maintained in circulating water baths at the desired test
temperatures. Water circulating in the water baths of the
fathead minnow unit was heated by two immersion coil heaters
controlled by a mercury column thermoregulator which
maintained a temperature of 25 i l°c. Water in the baths of
the brook trout unit was cooled by recirculating through
a mechanical chiller to temperatures of 15 - 1°C from May
through August, 12 ± 1°C during September, and 9 - 1°C from
October through the end of juvenile exposure. Prior to
entering experimental units for the invertebrate species,
diluent water was delivered to a stainless steel headbox
where immersion coil heaters and thermoregulators maintained
a temperature of 19 - 1°C for Daphnia and Gammarus and
23 - 1°C for Chironomus. In addition, ultra-violet lights
(24 watt) were placed over the headbox and water cells of
each invertebrate diluter to minimize the introduction of
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Table 2. CHEMICAL ANALYSIS OF THE DILUENT WATER UTILIZED
DURING CHRONIC AQUEOUS EXPOSURE OF FISHES AND
AQUATIC INVERTEBRATES TO LINDANE
Parameter
Calcium
Magnesium
Potassium
Sulfate
Nitrate
Nitrite
Ammonia
Phenol
Chlorine
mg/liter
6.0
2.1
1.1
11.6
<0.05
<0.05
0.01
<0.001
<0.01
Parameter
Chloride
Fluoride
Cyanide
Iron
Copper
Zinc
Cadimum
Chromium
Lead
mg/liter
17.6
0.5
<0.005
<0.01
0.004
0.01
0.001
<0.001
<0.01
fungus and pathogens into the test system.
Illumination was provided by a combination of Durotest
(Optima FS) and wide spectrum Grow Lux fluorescent lights
fixed centrally above the test chambers in all experimental
units. Incandescent bulbs (100 watt) simulated a 15-minute
dawn or dusk light intensity change (Drummond and Dawson,
1970). A constant 16-hour light, 8-hour dark photoperiod was
controlled by an automatic timer in the Chironomus and Daphnia
experiments. The photoperiod for Gammarus, bluegills, and
brook trout followed the normal daylight hours of Evansville,
Indiana (representative U.S. daylength), and was adjusted
the first and fifteenth of each month. The photoperiod for
fathead minnows followed Evansville daylengths but was started
with the daylength for December 1st on day one of the
experiment (Nov. 11). All experimental units were screened
with black polyethylene curtains to prevent unnecessary
disturbance of test organisms and the influence of extraneous
lighting on the intended photoperiod.
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ACUTE TOXICITY PROCEDURES
Static acute toxicity bioassays were conducted with
invertebrates and lindane to estimate the 48-hour LC50 and
its 95% confidence interval. Five organisms in three of
four replicate containers at each concentration were exposed
at 20 - 1°C. First instar Chironomus tentans, third instar
Gammarus fasciatus, and <24 hour old Daphnia magna werfe
the organisms exposed. A linear regression equation was
calculated after converting test concentrations and
corresponding percent mortalities to logarithms and probits,
respectively, and this equation was utilized to estimate
the 48-hour LCSO's and confidence intervals.
Acute toxicity studies with fishes and lindane were conducted
in flow-through water systems using proportional diluters
(Mount and Brungs, 1967). The incipient LC50 was estimated
when no additional significant mortality (>10%) of the test
organisms was observed at any concentration for 48 hours.
At this time, the exposure was terminated and a linear
regression equation was calculated by converting lindane
concentrations and corresponding mortalities into logarithms
and probits, respectively. This equation was utilized to
estimate the incipient LC50 and 95% confidence interval.
The chronic exposure concentrations for both invertebrates
and fishes were selected by evaluating observed mortality
and no-effect concentrations from the acute studies. The
48-hour LC50 for invertebrates and the incipient LC50 for
fishes were used to estimate application factors describing
the relationship between acute and chronic toxicity.
CHEMICAL METHODS
Toxicant concentrations and basic water quality characteristics
were initially monitored in each aquarium each week to
establish that the concentrations and water quality
characteristics were constant with minimum variability.
After the determination of what these concentrations and
characteristics actually were in the experimental systems,
a minimum monitoring effort was conducted to measure
variability and detect changes from the established means.
Stock solutions of lindane (100% a.i., City Chemical Co.,
N.Y.) in nanograde dimethyl sulfoxide (DMSO) were delivered
to the dilution water from 50 ml glass syringes with
stainless steel needles. The solvent (DMSO) was not added
to the control water in any of the chronic exposures and the
amount added to the highest lindane concentration of each
chronic test ranged from 6 jig/1 during the brook trout
10
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chronic to 65 pg/1 during the Daphnia chronic. In each
case, these concentrations were determined to be less
than 1/500 of the 96-hour LC50 of the solvent for each
species. Generally, lindane concentrations were determined
once each week during each of the chronic exposures by
taking 500-ml water samples from each aquarium.
All glassware used in the analysis of samples was cleaned
according to methods described in the Analytical Handbook
for Water Quality Control in Water and Wastewater Laboratories
(U.S. EPA, 1972). Water samples (500 ml) were extracted
with three 50 ml portions of methylene chloride, dried with
anhydrous sodium sulfate (heated and purified), and
evaporated in a Kuderna-Danish evaporator, according to
the method described by U,S. EPA (1971). The resulting
extract was dissolved in hexane and an aliquot analyzed
by gas chromatography using a Beckman Model 45 gas
chromatograph. The glass column used was 180 cm x 2 mm
containing 10 percent Dexsil 300 GC on 80/100 Supelcoport
at 210° with 30 cc helium per minute at 28 psi head
pressure as the carrier. A helium-arc electron capture
detector operated at 255°C was used and received 110 cc
helium per minute. Using an electrometer attenuation of
8 x lO"-'-'-1 amps, a one-millivolt recorder produced a one
half scale deflection with 0.1 ng of lindane.
Before analysis of each set of water samples, aliquots
of hexane containing known amounts of lindane were
chromatographed and a graph of peak heights versus nanograms
of lindane was constructed as a standard curve. Nanograms
of lindane present in the sample aliquots were determined
by graphical interpolation. A mean recovery of 93 percent
obtained from analysis of known standards (Table 3) was
applied to the observed instrument response to evaluate
measured lindane concentrations in the water samples.
Lindane residues were extracted from the tissues of exposed
fishes by the method described by Hesselberg and Johnson
(1972). Exceptions to this method were the use of 1:1
petroleum and ethyl ether as the extraction solvent, and
the cleanup of the total extract rather than handling the
fats and lipids separately. After weighing, the fish tissue
samples were extracted in a Waring Blendor1* containing 1:1
petroleum and ethyl ether. The extract was filtered,
evaporated, and freed of extraneous fish oils and lipids
by methods described by EPA (1971). The clean extract
was brought to known volume with hexane and analyzed by
11
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gas chromatrography using the methods and materials described
earlier for lindane analysis of water samples. A mean
recovery of 93 percent was determined from analysis of
tissue with a known amount of lindane added (Table 3).
This percent recovery was applied to the observed instrument
response to lindane in fish_ tissue to calculate measured
lindane residues. ~ ~~
TABLE 3. PERCENT RECOVERY OF ADDED LINDANE FROM WATER (ug/1)
AND WHOLE FISH (ug/g)
Sample Type
Water
Whole Fish
Added
Cone .
(ppm)
0.025
0.025
0.025
0.125
0.125
0.125
0.101
0.164
0.118
1.27
1.12
1.77
Measured
Cone .
(ppm)
0.023
0.024
0.023
0.123
0.110
0.117
0.117
0.150
0.108
1.13
1.03
1.37
Recovery (%)
92
96
92
98
88
94
93 ± 3.5
117
91
92
89
92
77
93 ± 13
During the chronic exposures, total hardness, alkalinity,
pH and acidity were generally measured bi-weekly in the
control and one lindane concentration according to Standard
Methods for the Analysis of Water and Wastewater (APHA, 1971) ,
Temperature and dissolved oxygen concentrations were measured
in selected tanks each day using YSI dissolved oxygen meter
with a combined oxygen-temperature probe.
STATISTICS
Means of the biological parameters measured in each duplicate
container during chronic exposures were subjected to analysis
of variance according to Steel and Torrie (1960) .
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When treatment effects were indicated by Anova, the means
of these effects were subjected to Duncan's Multiple Range
Test to determine which treatments were different from the
controls. All differences were considered statistically
significant at a probability of P=.05.
CHRONIC EXPOSURE
Chironomus tentans
Stock cultures of Chironomus tentans were obtained from
Michigan State University, Eas~t Lansing, Michigan, Cultures
were Maintained in 60 1 glass aquaria utilizing a substrate
prepared by placing 50 grams of paper hand towel (Nibroc,
Brown Co.) in a blender with 5 grams of high protein
chicken feed and 1 liter of water to cover the mixture.
The mixture was homogenized for three to five minutes.
This substrate was used to cover the bottom of each
aquarium to a depth of 1.5 cm. Emergent adults were allowed
to mate within the screened aquaria and provided a ready
supply of egg masses.
Egg masses were selected from the stock cultures according
to uniformity of age, and one egg cup of 100 eggs was
suspended in each of the duplicate experimental aquaria.
Egg cups were made from 20 ml glass vials with the bottoms
cut off and replaced with 40 mesh nylon screen. After
hatching was completed in all treatments (3-5 days), the
number of surviving first instar larvae were counted and
carefully placed onto the substrate.
After 26 to 29 days, the number of emergent adults, pupal
exuvia, and dead pupa were observed and recorded. Mating
of adults was observed, and egg masses produced by mating
pairs in each replicate were collected and utilized to
conduct second generation exposures according to the above
methods. At the initiation of the second generation exposure,
new substrate was placed in the experimental aquaria which
would receive first instar larvae. The number of emergent
adults, pupal exuvia, and dead pupa was observed from each
replicate concentration and the experiment terminated.
Daphnia magna
Laboratory stocks of Daphnia magna were obtained from the
University of New Hampshire, Durham, New Hampshire, and
successfully cultured in the laboratory according to the
methods of Biesinger and Christensen (1972).
13
-------
Typically, 10 daphnids (<24 hour old) were placed into
each of four replicate experimental units, resulting in
a total of 40 animals per concentration. A food supply
consisting of trout starter and dry-powdered Cerophyl (2:1)
was prepared in an aqueous suspension (12.5 mg/ml) and
delivered from a Mariotte bottle via a volumetric delivery
system to a mixing chamber during each diluter cycle. The
diluted food suspension was subsequently transferred to
the food cells from which 25 ml (0.1 mg/ml) were delivered
to each test container during each diluter cycle.
Survival and reproduction of daphnids was recorded after
one, two, and three weeks. Reproduction was measured by
recording the number of young in each experimental chamber
weekly and discarding the progeny after weeks one and two.
At the end of the third week, the number of original animals
remaining was recorded, the specimens discarded, and 10
daphnids (<24 hours old) were randomly selected from each
chamber to begin the second generation exposure. The same
procedures were followed for the second and third generation,
after which the experiment was terminated.
Gammarus fasciatus
The test organisms were collected in October 1972 from the
raceway outlets at the National Fish Hatchery, North
Attleboro, Massachusetts. Sexually mature adults were
acclimated at 17 - 1°C in the laboratory for a period of
three weeks. After the acclimation period, females were
isolated and their progeny collected. When sufficient
1 to 22 day old gammarids were available, the chronic exposure
to lindane was initiated by placing 30 specimens into each
replicate aquarium. During the exposure, the gammardis were
fed pre-soaked maple leaves, water cress, and Elodea. Brine
shrimp (Artemia) nauplii and Daphnia were also fed to the
gammarids weekly.
Survival was recorded once each month by siphoning the
contents of each test chamber into a pan and counting the
gammarids. At the onset of reproduction, all adult chambers
were checked daily for gravid females which were isolated
singly in 400 ml beakers containing respective test solutions.
Some females were maintained for up to 28 days in beakers
with the test solution being replaced every 48 hours with
fresh test solution from the appropriate experimental
aquarium. Twenty to fifty young collected from isolated
females in the same adult chamber on the same day were
placed in the respective larval growth chamber and treatment
14
-------
continued for 30 days. At the end of 30 days, survival and
growth were recorded by the procedure of Clemens (1950).
After a period of 17 weeks, the exposure of the original
gammarids to lindane was terminated and the sex of
individual specimens was determined.
Lepomis macrochirus
In December 1971, chronic exposure of bluegills to lindane
was initiated using 7-10 cm fish obtained from a commercial
hatchery in Wisconsin. Bluegills were acclimated to the
test water for three months, after which twenty fish were
randomly distributed to each test chamber. Bluegills were
fed ad libitum the largest commercially prepared trout
pellet which they would take in two daily feedings. All
tanks were siphoned twice weekly to remove fecal material,
excess food, and detritus and were brushed when algal growth
became excessive. Total length and weight of each individual
fish was measured at the initiation of exposure, after 90
days, and at thinning (185 days) using 100 mg/1 of tricaine
methanesulfonate (MS-222) to lightly anesthetize the fish.
During the first year when secondary sex characteristics
were well developed, fish in each tank were separated into
groups of males, females, and undeveloped fish using the
shape of the urogenital opening as the main criterion
for sexual differentiation (McComish, 1968). Sexually
mature fish were randomly reduced to 3 males and 7 females
per duplicate tank and all other fish were discarded
after examination of gonadal development and measurement
of total length and weight. At this time, two spawning
substrates similar to those described by Eaton (1970) ,
were placed into each duplicate tank. Substrates measured
30.5 x 40 x 5 cm with a bowl-shaped depression 25 cm in
diameter and 4 cm deep. Although bluegills developed
secondary sex characteristica and exhibited territorial
behavior characteristic of spawning bluegill populations
under natural conditions, no spawning occurred after the
first year of exposure to lindane. Consequently, substrates
were removed and the exposure of bluegills to lindane was
continued a second year, repeating the photoperiod schedule.
During the second year of bluegill exposure, when males
demonstrated territorial behavior, the substrates were
replaced in the proper tanks and total length and weight
was determined for each fish. Substrates were checked for
eggs after 1:00 P.M. each day and eggs were brushed loose
15
-------
from the substrate under a stream of test water. Eggs were
also siphoned from the tank bottom after each spawn. Two
hundred eggs were impartially selected from each spawn and
placed in an egg cup which oscillated in the respective
test water by means of a rocker arm apparatus (Mount, 1968).
Eggs from each spawn were counted volumetrically by first
counting a subsample of exactly 1 ml and multiplying that
number by the total measured volume (ml) of spawned eggs.
Eggs were allowed to settle for two minutes in the graduated
cylinder before measuring. Hatchability was determined as
the percent of live fry hatching out of 200 eggs.
Thirty bluegill fry from the earliest two spawnings in each
tank with at least 25 percent live hatch were placed in the
growth chambers. Fry from all other spawns were discarded
after hatchability was determined. Fry in the growth
chambers were fed ad libitum three times daily with live
young zooplankton from mixed laboratory cultures of copepods,
rotifers, protozoans, and brine shrimp. Cumulative mortality
was calculated after 30, 60 and 90 days and total lengths
of fry were determined at the same time using the photographic
method of McKim and Benoit (1971). Parental bluegills were
sacrificed after spawning had ceased in all tanks for three
weeks. Total length, total weight, sex, and gonadal condition
was determined for each fish and samples of muscle were
retained for residue analysis.
Pimephales promelas
Chronic exposure of fathead minnows to lindane began in
November 1971 with 15-day old fish obtained as eggs from
the Newtown Fish Toxicology Station, Newtown, Ohio. Forty
fish were randomly distributed to each test chamber.
Cumulative mortality and total length of live fish were
determined after 30 and 60 days using the photographic method
of McKim and Benoit (1971). At 60 days, the number of fish
in each test chamber was impartially reduced to fifteen.
Fathead minnows were fed ad libitum twice daily with a
commercially prepared trout starter food which was supplemented
with daphnids and brine shrimp nauplii. All tanks were
siphoned twice weekly to remove fecal material, excess food,
and detritus and were brushed when algal growth became
excessive.
The discovery of bacteria and external parasites on a few
fish prompted the use of flush treatments of tetracycline
hydrochloride (4 mg/1 active ingredient) and a combination
of malachite green and formalin (25 ul/liter of formalin
16
-------
containing 3.7 g/liter malachite green crystals). These
treatments were concentrated between days 95 and 116 of the
experiment and were applied to all experimental units
(including controls).
Due to unequal numbers of males in each tank, some of the
male fish were removed after secondary sexual characteristics
were well developed. Five spawning sites of halved, 3 inch
transite drain tiles had been placed in each tank when
fish were released from growth chambers at 60 days. The
tiles were placed concave surface down at locations that
minimized the chance of enounters by separate egg-guarding
males. When spawning began, eggs were removed and counted
after 1:00 P.M. each day. Fifty eggs from each spawn were
oscillated in their corresponding test water by means of
an egg cup and a rocker arm apparatus (Mount, 1968). Dead
eggs were removed and counted each day until hatching was
completed (3-5 days at 25°C). Percent hatch was based on
the number of live fry from 50 eggs.
Forty fry from the earliest two spawns in each tank with at
least 80% live hatch were placed in the respective growth
chambers. Cumulative mortality and total length of live
fish were determined at 30 and 60 days by McKim and Benoit's
(1971) photographic method. Fry from all other spawns were
discarded unless a growth chamber was later made available
by termination of 60 day old fry. Finely ground starter
food and brine shrimp nauplii were fed three times daily
to fry in the growth chambers.
Parental fish were sacrificed after all spawning had ceased
for one week. Total length, weight, sex and gonadal
condition was determined for each fish and three samples
per concentration of eviscerated fish were retained for
residue analysis.
Salvelinus fontinalis
Chronic exposure of brook trout to lindane began in May 1972
with yearling fish obtained from the National Fish Hatchery,
Manchester, New Hampshire. Distribution of fish to the
test chambers was delayed by the discovery of external
parasites on a few fish. The parasites were controlled by
flush treatments of malachite green and formalin (25 pl/1 of
formalin containing 3.7 g/1 of malachite green). These
treatments were repeated shortly after the distribution of
fish to each duplicate tank to ensure control of the
parasites. Brook trout were fed twice daily with a measured
ration of the largest trout pellet they would take. The •
17
-------
feeding rate was based on a percentage of the initial
average biomass per tank and was adjusted after 90 days and
186 days when total weights of fish were again measured.
Feeding rates were from a New York State fish hatchery
feeding chart (Deuel et al., 1952). Total length and
weight of individual fish were measured at the initiation
of exposure, after 90 days, and at thinning (186 days)
utilizing 100 mg/1 of tricaine methanesulfonate (MS-222) to
quiet the fish during measuring. All tanks were siphoned
twice weekly to remove fecal material, excess food, and
detritus and were brushed when algal growth became excessive.
Secondary sexual characteristics were observed on test day
186 and males, females and undeveloped fish were identified
in each tank and the number of sexually mature fish was
randomly reduced to 2 males and 4 females per tank. All
other fish were discarded after being measured and examined
for gonadal condition. At this time, two stainless steel
spawning substrates similar to those described by Benoit
(1974) were placed in each duplicate tank. The box-like
substrates 25 x 25 x 15 cm had on their bottom a gridwork of
2.5 cm cubicles, made of 20 mesh stainless steel screen-,
to retain the eggs where they were deposited. A 25 x 25
cm square of 4 mesh stainless steel screen was placed on top
of the egg retainer and silicone adhesive was used to attach
1.3 - 2.5 cm diameter stream gravel 2 cm apart on the
screening.
Substrates were checked after 1:00 P.M. each day and eggs
were recovered by removing the gravel screen and egg
retainer, and pipetting the eggs into a pan containing the
test water. Eggs from each spawn.were counted and two egg
cups containing a group of 50 and 100 eggs were attached to
the rocker apparatus (Mount, 1968) for incubation in their
respective test water. Dead eggs were removed and recorded
daily and the number of fry hatching each day in the
incubation cups with 50 eggs was recorded. After 15 days,
the group of 100 eggs was removed and the percentage of
eggs developing a neural keel was recorded as an indication
of percent fertilization. Hatchability (percent of 50
eggs) and mean time to hatch were determined after completion
of hatching.
Twenty five fry from the earliest two spawns in each
tank with at least 50% live hatch were placed in the
respective growth chambers. Fry from all other spawns
were measured and discarded after hatchability determinations,
18
-------
Cumulative mortality and total length of live fry was
determined at the initiation of exposure and after 30,
60 and 90 days using the photographic method of McKim
and Benoit (1971). Brook trout fry were fed five times
daily ad libitum with a commercially prepared trout
starter food.
After spawning had ceased in all tanks for a period of
three weeks, parental fish were removed and examined for
gonadal condition. Total lengths and weights were determined
for each individual fish before retaining muscle samples
for residue analysis and discarding the remaining fish.
19
-------
SECTION V
RESULTS
ACUTE BIOASSAYS
The analysis of the results of acute static bioassays with
the invertebrate species indicated the 48-hour LC50 (95%
confidence interval) of lindane for Chironomus, Daphnia, and
Gammarus was 207 (157-263), 485 (301-623), and 39 (27-56)
jug/1,respectively. Lindane-induced mortality was observed
with chironomids at 180 ug/1, with daphnids at 310 ug/1, and
with gammarids at 36 ug/1. Based on these data, the highest
nominal concentrations selected for the chronic exposures
were 24 ug/1 (Chironomus) , 200 ug/1 (Daphnia) and 20 ug/1
(Gammarus).
Duplicate continuous-flow bioassays were conducted at 25 - 1 C
with 0.75 gram bluegill. The 96-hour LC50 values for lindane
in both instances were >100 ug/1. The earliest time
interval at which estimates of the LC50 were calculated was
after 8 days when the LC50 was estimated to be 52.3 (24.8-110)
and 63.7 (20.0-120) based on duplicate bioassays. After
21 d?iys, we estimated the incipient LC50 for lindane and
bluegill to be 29 (20-49) and 31 (20-49) ug/1.
Duplicate continuous-flow bioassays were conducted at
25 - 1°C with 2.0 gram fathead minnows and in both cases
the .;6-hour LC50 was >100 ug/1. After 11 days, the incipient
LC5Q- for lindane and fathead minnows was estimated to be
62.5 (19.8-119) and 75.6 (13.4-143), based on duplicate
bioassays. A single continuous-flow bioassay was conducted
at 12 * 1°C with 5.2 gram brook trout and indicated the 96-
hour LC50 for lindane was 44.3 (32.7-60.1) ug/1. After
11 days we estimated the incipient LC50 for lindane to be
26 (19-39) jag/1. Based on these data, the highest nominal
concentration selected for investigation during chronic
exposure was 12 jig/1 (bluegills) , 25 jag/1 (fathead minnows) ,
and 20 ^ig/1 (brook trout) .
WATER CHEMISTRY
The results of the chemical analyses of water samples
indicated that chemical characteristics varied minimally
within any one chronic exposure. Therefore, only means
and ranges for the various parameters are presented
(Table 4). The results of gas-chromatographic analyses
of water samples taken periodically during chronic exposures
to lindane indicated that mean measured lindane concentrations
closely approximated nominal concentrations (Table 5).
20
-------
TABLE 4. MEAN AND RANGE OF MEASURED CONCENTRATIONS OF HARDNESS, ALKALINITY, ACIDITY,
DISSOLVED OXYGEN AND pH FROM WATER SAMPLES TAKEN PERIODICALLY DURING
CHRONIC EXPOSURE OF AQUATIC INVERTEBRATES AND FISHES TO LINDANE
Species
Chironomus
tentans
Mean - S.D.
Range
# of Samples
Daphnia magna
Mean - S.D.
Range
# of Samples
Gammarus
fasciatus
Mean - S.D.
Range
# of Samples
Lepomis
macrochirus
Mean - S.D.
Range
# of Samples
Pimephales
promelas
Mean - S.D.
Range
# of Samples
Salvelinus
fontinalis
Mean - S.D.
Range
# of Samples
Hardness
(mg/1)
37.0-3.6
(32.0-43.0)
5
38.0^2.4
(33.0-39.0)
5
35.0-3.7
(27.0-46.0)
42
37.4-3.8
(29.0-42.9)
20
40.2-3.1
(32.0-46.2)
23
37.0-4.4
(31.7-44.0)
16
Alkalinity
(mg/1)
33.0-1.9
(28.0-39.0)
6
32.0^2.6
(26.0-44.0)
5
34.3-2.8
(30.0-39.0)
42
33.2^4.5
(24.8-38.5)
20
35.3-2.4
(31.0-39.5)
23
32.2-4.4
(25.6-38.4)
16
Acidity
(mg/1)
5.3-1*1
(4.0-6.8)
6
5.7^0.8
(4.7-6.8)
5
4.4-1.5
(1.9-9.6)
42
4.4-1.7
(1.9-7.7)
24
4.7-1.7
(2.9-9.6)
18
4.8-1.6
(2.9-7.7)
18
DO
(mg/1)
6.9-1.1
(5.6-8.3)
24
8.6-1.2
(6.8-8.9)
26
8.4-1.0
(6.0-9.9)
162
6.4^1.0
(3.3-8.5)
1856
7.2±1.0
(3.9-9.7)
282
8.2-0.8
(4.5-11.1)
954
pH
(6.3-7.6)
14
—
(6.1-7.3)
12
(6.4-7.2)
12
(6.4-7.4)
30
_
(6.5-7.4)
30
(6.5-7.3)
22
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TABLE 5. NOMINAL AND MEASURED LINDANE CONCENTRATIONS (ug/1)
IN WATER DURING CHRONIC EXPOSURE OF AQUATIC
INVERTEBRATES AND FISHES TO LINDANE
Species and
Nomin. Cone.
Chironomus
tentans
24
12
6
3
1.5
Daphnia
magna
200
100
50
25
12
Gamma rus
fasciatus
20
10
5
2.5
1.2
Lepomis
macrochirus
12
6
3
1.5
0.8
Pimephales
promelas
23
12
6
3
1.5
Salvelinus
fontinalis
20
10
5
2.5
1.2
Measured Concentration
Mean S.D.
28.4 ± 4.9
10.8 ± 2.4
7.3 ± 1.2
5.0 ± 1.7
2.2 ± 0.8
Range
16.0 - 42.0
7.5 - 13.0
2.6 - 8.7
1.7 - 5,1
0.6 - 2.4
200 ± 70 109 - 296
120 ± 30
42 ± 10
19 ± 7
11+6
17.7 ± 3.6
8.6 ± 2.1
4.3 ± 1.1
2.2 ± 0.5
1.2 ± 0.4
9.1 ± 4.1
4.4 ± 1.7
2.3 ± 1.0
1.1 + 0.5
0.6 ± 0.3
23.0 ±12.2
9.6 ± 5.2
5.6 ± 2.2
2.4 ± 1.0
1.3 ± 0.6
16. 6 ± 3.8
8.8 ± 2.3
4.1 ± 1.0
2.1 ± 0.7
1.0 ± 0.3
59 - 169
19 - 112
16 - 49
6-27
10.0 - 26.0
4.0 - 12.0
3.0 - 7.0
1.2 - 3.4
0.6 - 2.1
3.9 - 20.0
1.3 - 8.9
1.0 - 6.0
0.25- 2.0
o.22- 2.0
8.9 - 58.0
2.8 - 32.0
2.8 - 6.8
1.1 - 5.5
0.3 - 2.6
9.1 - 31.0
6.6 - 16.0
3.1 - 6.6
1.2 - 3.9
0.6 - 1.6
# of Samples
7
6
6
7
6
20
20
20
20
20
31
32
31
32
31
86
44
58
45
55
47
28
32
30
31
39
24
20
23
19
22
-------
CHRONIC EXPOSURE
Chironomus tentans
Continuous exposure of the original Chironomus tentans eggs
to a mean measured concentration of lindane as high as 28.4
ug/1 had no significant effect on hatchability (Table 6).
However, all first instar larvae from eggs exposed to 28.4
ug/1 died within 3 days after hatching. Eggs continuously
exposed to mean measured lindane concentrations of 10.8 and
7.3 ug/1 generally hatched successfully, but virtually none
of these larvae exceeded second instar development. Two
larvae exposed to 7.3 jug/1 did develop to fourth instar and
pupate but did not successfully emerge as adults. Continuous
exposure of chironomids to 5.0 ug/1 lindane significantly
delayed the pupation of larvae and the emergence of adults.
Larvae exposed to 5.0 pg/1 lindane completed pupation and
emergence during days 28 to 30 of exposure. All remaining
pupae from the control group, and those exposed to a mean
measured lindane concentration of 2.2 jug/1, emerged between
days 24 to 27 of exposure at which time the first generation
exposure was terminated and the second generation exposure
was initiated.
TABLE 6. SUMMARY OF THE EFFECT OF VARIOUS CONCENTRATIONS
OF LINDANE ON HATCHABILITY, PUPATION AND EMERGENCE
OF Chironomus tentans CONTINUOUSLY EXPOSED FOR TWO
GENERATIONS
Mean measured
lindane cone.
(jAg/D
Control
2.2
5.0
7.3
10.8
28.4
Repa
A
3
A
B
A
B
A
B
A
B
A
B
Generation I
% Hatch
90
94
92
76
93
86
55
81
86
47
83
76
Pupa
86
91
90
75
76
90
2
0
0
0
0
0
Adult
80
86
87
73
75
80
0
0
0
0
0
0
Generation
% Hatch
92
84
93
89
90
91
-
-
-
-
-
^
Pupa
83
76
89
78
66
38
-
-
-
-
-
^
II
Adult
80
71
85
73
62
36
-
-
-
-
-
^
One hundred eggs were initially exposed in each replicate.
23
-------
Egg masses for the second generation exposure were obtained
from treatments that contained first generation adults.
Specimens which had emerged from the 5,0 and 2.2 jig/1
treatments during the same period as had the controls (24 to
27 days) were taken, as were controls. The continuous exposure
of second generation eggs to lindane concentrations as high
as 5.0 jug/1 did not significantly affect hatchability.
Analysis of variance indicated that the number of second
generation forms pupating and emerging was not significantly
affected by continuous exposure to lindane. Also, there were
no significant differences in the numbers of pupae, numbers
of emerging adults produced, and mean time to emergence
between the first generation and the second generation of
Chironomus tentans continuously exposed to 2.2 or 5.0 pg/1
lindane.
The results of continuous exposure to lindane through two
generations of Chironomus tentans indicate that exposure
to concentrations of 7.3 jug/1 or more caused larval mortality,
severe developmental retardations, and reduction of numbers
of emerging adults. Continuous exposure to 5.0 pg/1 lindane
resulted in delayed emergence of adults. No significant
effects of continuous exposure to 2.2 jjg/1 lindane was
observed. Based on the effect of lindane on the development,
survival and emergence of Chironomus tentans, the estimated
maximum acceptable toxicant concentration for this species is
>2.2 and <5.0 jug/1.
Daphnia magna
Statistical analysis of data on survival of Daphnia continuously
exposed to lindane for 64 days indicates significant differences
due to treatment. Through the first two generations,
continuous exposure to mean measured concentrations of 210,
120 and 42 jug/1 lindane significantly reduced survival of
daphnids (Table 7). This effect on survival was cumulative,
with survival of second generation daphnids being significantly
less than that of the first generation. Survival of second
generation Daphnia continuously exposed to a mean measured
lindane concentration of 11 jug/1 was significantly lower
than controls. However, this does not appear to be toxicant-
related as survival of second generation daphnids exposed to
a mean measured lindane concentration of 19 ug/1 was comparable
to that of controls. Survival of third generation Daphnia
continuously exposed to 19 ug/1 lindane was significantly
less than controls, indicating again that the effects on
daphnids of continuous exposure to lindane are cumulative.
An untimely mechanical malfunction of the exposure system
resulting in the death of most of the third generation
24
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TABLE 7. MEAN PERCENT SURVIVAL OF Daphnia magna CONTINUOUSLY
EXPOSED TO LINDANE FOR 64 DAYS
Mean measured
cone, (ug/1)
Control
11
19
42
120
210
Generation
b I
Day
8
90
90
95
73
90
83
15
73
83
90
58
85
75
70
70
78
78
45
58
38
Generation
b II
Day
29
100
100
y7
75
20
25
36
93
48
80
58
20
12
43
86
45
70
32
13
0
Generation III
Day
50 57
93 93
20° 20
60 60
38 32
22 15
0 0
64
86
15
45
27
10
0
Each value represents the mean of four replicates.
Duration of exposure for generations, I, II, and III were
days 1-22, 22-43, 43-64, respectively.
Test containers overflowed on day 46 resulting in the death
of most test organisms at this concentration.
Daphnia exposed to 11 ug/1 lindane precludes a valid assessment
of this exposure level during this period.
Continuous exposure to 210 ug/1 lindane virtually prevented
production of young among the surviving Daphnia during the
second generation exposure (Table 8) . Statistical analysis
of data concerning production of young during the first
two generations of exposure to lindane indicates that exposure
to lindane concentrations of 120 and 42 ug/1 significantly
reduced the number of young produced per female. Assessment
of the effect of the toxicant on production of young during
the third generation exposure is hindered by the poor production
of young observed in the control group. However, it is
important to note that the number of young produced per female
among Daphnia continuously exposed to 19 ug/1 lindane was
significantly less that of Daphnia exposed to 11 ug/1, suggesting
a toxicant-related effect. It is also important to note that
the number of young produced per female among third generation
daphnids exposed to 11 ug/1 lindane was the highest observed
during the experiment suggesting that toxicant-induced inimical
effects did not occur at this level of exposure.
Based on the observed effects of lindane on survival and
reproduction of Daphnia magna continuously exposed to the
toxicant through three generations of development, the estimated
maximum acceptable toxicant concentration for this species is
and <19 ug/1.
25
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TABLE 8. MEAN PRODUCTION OF YOUNG PER FEMALE Daphnia magna
CONTINUOUSLY EXPOSED TO LINDANE FOR 64 DAYS
Mean Measured
cone, (ug/1)
Control
11
19
42
120
210
Generation I
Day
15 22
4.6 29.7
4.8 27.0
8.6 22.0
6.6 17.0
5.7 15.7
6.2 9.8
Generation II
Day
36 43
35.5 23.4
28.1 29.4
25.5 22.7
13.7 14.6
16.0 12.2
0.5 0
Generation ill
Day
57 64
6.5 8.2
37.5 35.3
12.7 7.7
20.7 7.2
9.3 6.4
0 0
Each value represents the mean of four replicates.
""Duration of exposure for generations I, II, and III were
days 1-22, 22-43, 43-64, respectively.
Gammarus fasciatus
Statistical analysis of data on survival of Gammarus fasciatus
indicated significant differences due to treatments (Table 9).
Continuous exposure to mean measured lindane concentrations
of 17.7 and 8.6 ug/1 for 30 days significantly reduced survival
of gammarids (Table 9). No effect on survival of gammarids
continuously exposed to a mean measured lindane concentration
of 4.3 pg/1 for 120 days was observed, when compared to
controls.
Pairing of males and females was first observed on days 35
and 36 among adult gammarids in the controls and in the three
lowest treatments (4.3, 2.2 and 1.2 ug/1). No significant
pairing of adult gammarids exposed to 17.7 jig/l lindane was
observed. Two gravid females were observed among this group
but no young were shed. Some pairing among gammarids exposed
to 8.6 ug/1 lindane was observed on day 40. However, none of
the young that were subsequently shed survived exposure to
this concentration of lindane. Mating pairs of Gammarus from
the controls and from the 4.3, 2.2 and 1.2 mg/1 lindane
treatments began to shed young between days 50 to 60 of
exposure (6 to 13 days after isolating the females). In all
of these groups, approximately half of all gravid females
failed to shed young.
Although there was virtually no production of young in one
replicate of gammarids exposed to 4.3 or 8.6 jug/1 lindane,
production in the other replicate was comparable to that of
the two lower treatments (2.2 and 1.2 jug/1) and of controls,
suggesting that the observed failure was not toxicant-related.
26
-------
TABLE 9. SURVIVAL AND REPRODUCTIVE SUCCESS OF Gammarus fasciatus EXPOSED TO LINDANE
FOR 17 WEEKS
Mean Measured Lindane Concentration (ug/1)
Adult Survival
Day 3 0
60
90
120
Total # Young
# young continued
in treatment
Survival after
30 days (%)
17.7
A B
60 60
27 27
7 10
0 0
-
-
8.6
A B
73 73
43 47
23 17
3 0
50 1
50 1
0 0
4.3
A B
90 90
47 77
30 30
3 10
1 38
1 38
0 35
2.2
A B
87 83
57 63
23 27
6 6
41 39
25 23
66 33
1.2
A B
93 93
70 87
27 73
10 16
54 47
54 40
65 82
Control
A B
93 83
83 77
30 17
10 16
38 48
38 48
60 4
to
-------
Data on the survival of second generation gammarids beyond
30 days are extremely variable and preclude statistical
evaluation and interpretation. Based on the effects of
lindane on survival and reproduction of Gammarus fasciatus,
the estimated maximum acceptable toxicant concentration of
lindane for this species is >4.3 and <8.6 pg/1.
Lepomis macrochirus
Survival and growth of bluegills exposed to lindane for 6 and
18 months were similar for all treatments (Table 10).
Spawning activity occurred between test days 592 and 716,
with most activity observed in tanks receiving 2.3 jug/1 of
lindane. Spawning activity was too sporadic to be conclusive
and results of spawning (number of eggs, hatchability and
fry survival) were not analyzed statistically. An attempt
was made to induce further spawning from bluegills by
administering interperitoneal injections of carp pituitary
extract on test days 714 and 715. All fish were injected
with 0.5-1.0 cc of a 200 jag/100 ml solution of carp pituitary
in physiological saline. The injections failed to induce the
desired increase in spawning and may have been administered
too late in the spawning period to be effective. Parental
fish were sacrificed on test day 735 and samples of muscle
were retained for residue analysis.
Results of egg hatchability and survival and growth of bluegill
fry are presented (Table 11). Due to the lack of spawning of
control fish, it is difficult to assess possible toxicant-
related effects on these parameters. Survival of bluegill
fry after 30 days was higher in tanks receiving 2.3 jag/liter
of lindane than in those receiving 4.4 and 1.1 jig/liter.
Survival during this period appeared to be related to an
early acceptance of available food rather than lindane
concentration. This observation is supported by the fact that
survival did not greatly change during the last two months
of exposure when feeding habits became well established and
food was more readily accepted. Total lengths at 30, 60 and
90 days did not appreciably vary for bluegill fry exposed to
4.4, 2.3 and 1.1 pg/1 of lindane.
Based on the data obtained from continuous exposure, it
appears that the maximum acceptable toxicant concentration
is greater than the highest measured lindane concentration
(9.1 ug/1) to which bluegills were exposed during the test.
Because ten percent mortality was observed among bluegills
continuously exposed to 12.5 pg/1 lindane for 23 days in a
continuous-flow acute bioassay, the maximum acceptable
toxicant concentration of lindane for this fish is estimated
to be between >9.1 and <12.5 pg/1.
28
-------
TABLE 10. SURVIVAL AND GROWTH DURING 6 AND 18 MONTHS, AND RESULTS OF SPAWNING
ACTIVITY OF BLUEGILL (Lepomis macrochirus) CONTINUOUSLY EXPOSED TO
LINDANE
Item
6 MONTHS
Survival (%)a
Total Length
(mm)b
Total weight
(g)c
18 MONTHS
Survival (%)
*/*
Total length
(mm)
-------
TABLE 11. HATCHABILITY, SURVIVAL AND GROWTH OF BLUEGILL
(Lepomis macrochirus) FRY EXPOSED TO LINDANE
Item
Hatchability (%)
# Egg groups3
# fry groups13
30 days survival
(%)
60 days survival
(%)
90 days survival
(%)
Mean total length
(mm)
30 days
60 days
90 days
Measured Lindane
4.4
70
(2)
1
16
14
14
4.
19fl.8
31^4.0
39±3.6
2.3
65
(11)
4
60
59
59
4.
19^2.3
30|3.8
39-6.1
Concentration (ug/1)
1.1
88
(1)
1
12
12
12
+
24jl.O
28^3.5
30r5.3
0.6
90
(3)
1
0
0
0
—
-
Each group contained 200 eggs.
Each fry group contained 30 one day old fry.
Pimephales promelas
Survival of fathead minnows exposed to a mean measured lindane
concentration of 23.5 jug/1 f°r 60 days appeared lower than for
all other treatments and the controls (Table 12). However,
variability in the data precluded ascribing statistical
significance to this 60-day observation.
Statistical analysis of the survival after 43 weeks exposure
to 23.5 ug/1 substantiated this trend and showed a significant
decrease in survival of fathead minnows exposed to this lindane
concentration as compared with control groups and lower
treatments. Spawning of fathead minnows in all lindane
treatments and the controls occurred between test days 124
and 271. For unknown reason(s), the number of mature females
and number of eggs produced per female were significantly
lower in controls than in all groups exposed to lindane
(Table 13). We do not believe that this observation is
indicative of some positive effect of lindane on reproduction
since the number of mature females, the number of spawnings
per female, the number of eggs per mature female, and the
number of eggs per spawn observed in groups exposed to lindane
are similar to those observed for fathead minnows from control
groups and from unaffected treatments in other chronic
studies. Conversely, the same parameters for controls in this
30
-------
TABLE 12. SURVIVAL AND GROWTH OF FATHEAD MINNOWS (Pimephales promelas) DURING 30
DAYS, 60 DAYS, AND 43 WEEKS CONTINUOUS EXPOSURE TO LINDANE
Item
30 DAYS
Survival (%)
Total length
(ram)
(S.D.)a
60 DAYS b
Survival (%)
Total length
(mm)
(S.D.)
43 WEEKS
Survival (%)
rf/S
Extra rf
removed
Total length
(mm)
-------
TABLE 13. SEXUAL DEVELOPMENT, SPAWNING, HATCHABILITY OF
GROWTH OF OFFSPRING AFTER 30 AND 60 DAYS, FOR
promelas) CONTINUOUSLY EXPOSED TO LINDANE
EGGS, AND SURVIVAL AND
FATHEAD MINNOW (Pimephales
Item
# mature 9
Spawning/ 9
Eggs/?
Eggs/spawn-
ing
Percent
Hatchb
(N)
30 DAYS
Survival (%)
Total length
(mm)
(S.D.)a
60 DAYS
Survival (%)
Total length
(mm)
(S.D.)
# fry
groups0
Mean
23.4
A B
5 7
11 9
1494 1057
136 117
86 92
(27) (29)
89.0 84.0
11.0 10.0
(2.4) (2.2)
83.0 83.0
18.0 19.0
(4.8) (3.7)
2 2
Measured Lindane Concentration (jig/liter)
9.1
A B
8 7
8 8
907 751
113 94
88 90
(31) (20)
72.0 45.0
11.0 13.0
(2.2) (2.6)
70.0 45.0
21.0 20.0
(4.6) (3.7)
2 2
5.6
A B
6 6
14 12
1504 1120
107 93
89 87
(32) (31)
71.0 75.0
12.0 12.0
(2.9) (2.1)
67.0 70.0
19.0 20.0
(4.2) (4.0)
4 4
2.4
A B
7 8
8 4
871 387
109 97
88 82
(32) (16)
71.0 45.0
12.0 12.0
(2.9) (1.8)
70.0 45.0
21.0 21.0
(4.5) (3.4)
4 2
1.4
A B
5 8
8 6
783 582
98 198
91 82
(18) (8)
88.0 42.0
11.0 12.0
(2.4) (3.2)
84.0 33.0
18.0 20.0
(4.5) (5.3)
4 2
Control
A B
2 3
4 2
147 145
37 73
87 94
(4) (5)
78.0 55.0
10.0 10.0
(2.7) (2.0)
75.0 53.0
17.0 21.0
(4.9) (5.9)
1 1
to
to
Standard Deviation.
Hatchability samples contained 50 eggs.
•*
'Each fry group contained 40 one day old fry.
-------
study were severely depressed. We have no basis on which
to speculate whether this observation is a random occurrence
or what cause-effect relationship might be applicable.
Parental fish were sacrificed on test day 304 after all
spawning had ceased for 3 weeks and samples of the edible
portion (eviscerated carcass) retained for residue analysis.
Statistical analysis indicated no significant differences
in hatchability and percent survival and growth of FI fathead
minnow fry after 30 and 60 days continuous exposure to lindane,
Based on these data derived from the continuous exposure of
fathead minnows to various concentrations of lindane for 52
weeks, the estimated maximum acceptable toxicant concentration
for this species is >9.1 and <23.5 ug/1.
Salvelinus fontinalis
Continuous exposure for 261 days to mean measured lindane
concentrations as high as 16.6 ug/1 had no significant effect
on survival of brook trout. Although continuous exposure to
lindane did not significantly effect growth of trout during
the first 90 and 1-36 days exposure, continuous exposure of
fish for 261 days to a mean measured lindane concentration of
16.6 jug/1 significantly reduced the weight and total length
of brook trout when compared to the controls and other
exposed groups (Table 14).
Spawning activity of yearling brook trout in all experimental
units occurred between days 189 and 242 of exposure. The
failure of most control fish to spawn their eggs and the
subsequent inconsistencies in data related to spawning
activity preclude valid conclusions regarding statistical
significance of these data (Table 15). Certainly, however,
it would appear that continuous exposure to 16.6 ug/1 lindane
reduced the percent of eggs developing neural keel, and
reduced the percent of eggs successfully hatching, below
those observed for all other treatments. Also, we observed
that brook trout exposed to 16.6 ug/1 lindane usually did
not spawn on the substrate provided (as did fish in all
other groups) but frequently released eggs randomly throughout
the experimental chamber making fertilization by the male
virtually impossible. Adult brook trout were sacrificed
after 261 days of exposure when no spawning had been observed
for 21 days, and samples of muscle tissue taken for residue
analysis.
No significant effects of exposure to lindane on survival
and growth of second generation brook trout were observed
(Table 16). During the first 30 days of exposure (prior
to swim-up) , survival of fry exposed to 16.6 jag/1 lindane
was 94.5% as compared to 97.0% among unexposed fish. During
the remaining 60 days observation, survival was generally
33
-------
TABLE 14. GROWTH OF YEARLING BROOK TROUT (Salvelinus fontinalis) DURING 261 DAYS
CONTINUOUS EXPOSURE TO LINDANE
Item
Mean length
(mm)
(S.D. )b
Mean weight
(g)
(S.D.)
Mean Measured Lindane Concentration (ug/liter)
16. 6
A B
251 255
(26) (10)
172 175
(47) (20)
8.8
A B
265 264
(21) (11)
219 187
(72) (28)
4.1
A B
263 270
(23) (15)
203 212
(54) (64)
2.1
A B
272 274
(23) (12)
229 219
(67) (36)
1.0
A B
270 259
(16) (13)
229 194
(58) (42)
Control
A B
281 277
(11) (19)
255 255
(53) (72)
w
Initial mean total length, 199 mm, initial mean total weight, 73 g; initial number
of fish/tank, 15.
3Standard Deviation.
-------
TABLE 15. RESULTS OF SPAWNING ACTIVITY OF YEARLING BROOK TROUT (Salvelinus
fontinalis) DURING CONTINUOUS EXPOSURE TO LINDANE
Item
No. rf/?
No. ? spawn-
ing
No spawns/?
Total # eggs
spawned
#Eggs
spawned/?
Neural Keel
developed
(%)
Incubation
time (degree
days)
Mean hatch-
ability (%)
(N)b
Mean Measured Lindane Concentration (ug/liter)
16.6
A B
1/3 2/4
3 4
3 3
1145 1461
382 365
0 28
405
28
0 (1)
8.8
A B
2/4 3/3
4 3
1 4
1210 1731
302 577
50 37
387 396
76 92
(1) (2)
4.1
A B
2/4 3/3
4 3
3 3
2630 2355
658 732
61 78
423 414
65 72
(3) (5)
2.1
A B
2/4 2/4
4 4
3 3
2260 2188
565 547
80 76
423 404
97 51
(4) (4)
1.1
A B
2/4 2/4
3 4
2 3
1747 2195
437 549
76 31
441 432
86 68
(1) (1)
Control
A B
2/4 3/
3a
2
425 101
106 33
0 7
- 36
2
- (2
CO
Ol
Unlike females from all other experimental units, females in this unit were not
spent at termination of exposure but were observed to contain mature eggs.
Indicated the number of groups of 50 eggs in which at least half successfully
developed a neural keel.
-------
TABLE 16. SURVIVAL AND GROWTH OF SECOND GENERATION BROOK TROUT (Salvelinus
fontinalis) DURING THE FIRST 90 DAYS DEVELOPMENT OF FRY CONTINUOUSLY
EXPOSED TO LINDANE
Item
No. Groups
Survival (%)
30 Days
60 Days
90 Days
Mean Length
(mm)
30 Days
60 Days
90 Days
Mean Measured Lindane Concentration (pg/liter)
16.6
A B
1
93
7
7
20
20
21
8.8 '
A B
2 2
96 94
23 4
23 4
20 20
21 21
22 21
4.1
A B
2 2
92 72
32 0
32 0
20 19
22 20
23
2.1
A B
2 2
90 100
50 46
50 46
21 21
24 23
27 28
1.1
A B
2 2
59 79
2 25
2 25
19 21
21 22
25 26
Control
A B
2a 2a
94 100
54 48
54 48
22 21
29 27
42 33
These groups were obtained from a local hatchery and were of the same age as
the remaining groups 0
-------
poor and response could not be correlated with treatments.
Except for the unexposed fish (obtained from a local hatchery)
growth of second generation fry during the final 60 days
observation was very poor and may or may not be toxicant-
related.
Based on the available evidence, derived from continuous
exposure of brook trout to lindane for 261 days, relating
to pesticide-induced reduction of growth, and apparent effects
on percent fertilization and hatchability, we estimate the
maximum acceptable toxicant concentration of lindane for
brook trout is >8.8 and <16.6 pg/1.
RESIDUE ANALYSIS
Three samples of muscle tissue from terminated adult
bluegills and brook trout were analyzed to determine lindane
residues in fish exposed to each concentration of lindane.
The small size of fathead minnows prompted the use of 3
samples of pooled eviscerated carcasses of terminated adults
exposed to each lindane concentration. Results of these
analyses (Table 17) clearly show that the amount of lindane
residue accumulated is directly proportional to the level of
exposure and that this response is essentially linear over
the range of concentrations tested.
Although the bioconcentration factors for brook trout and
bluegills are similar, those for fathead minnows appear
to be an order of magnitude higher. This may reflect the
possible inclusion of tissue other than muscle with the
fathead minnow carcass (e.g. kidney). Alternatively, these
observations may be indicative of species differences in
the tendency of fishes to accumulate and retain chemical
residues. It is interesting to note that the control fish
of each species appear to have concentrated the trace amount
of lindane in the diluent water by a factor similar to that
observed for other experimental groups.
CALCULATION OF APPLICATION FACTORS
A summary of the estimated LC50 value, the maximum acceptable
toxicant concentration, and the application factor derived
therefore, for lindane and all species studied is presented
(Table 18). The MATC for each species except bluegills is,
by definition (Mount and Stephan, 1967), an undetermined value
between the highest mean measured concentration having no
significant effect and the lowest mean measured concentration
significantly affecting the organism during chronic exposure.
The MATC for bluegills is estimated between the highest
mean measured concentration applied during chronic exposure
and the minimum mean measured concentration which produced
37
-------
TABLE 17. MEAN MEASURED LINDANE CONCENTRATION IN WATER (pg/1) AND IN THE MUSCLE
(pg/kg) OF BLUEGILL (Lepomis macrochirus) AND BROOK TROUT (Salvelinus
fontinalis) AND IN THE EVISCERATED CARCASS (pg/kg) OF FATHEAD MINNOWS
(Pimephales promelas) CONTINUOUSLY EXPOSED TO LINDANE
Item
Bluegills
735 days-water
Muscle
Bioconcentra-
tion factor (X)
Brook trout
261 days-water
Muscle
Bioconcentra-
factor (X)
Fathead minnows
304 days-water
carcass
Bioconcentra-
tion factor (X)
Treatment
1
9.1
297-38
33
16.6
1200-283
72
23.5
9533-1102
406
2
4.4
196-160
45
8.8
770-113
88
9.1
6133-2359
674
3
2.3
62-43
27
4.1
255-7
62
5.6
2867-553
512
4
1.1
25+4
23
2.1
227^86
108
2.4
1067-515
445
5
0.6
16-14
27
1.0
51-36
51
1.4
537-165
284
Control
0.05
2.8-1.6
56
0.05
2±1
40
0.05
27±6
540
CO
QO
-------
TABLE 18. SUMMARY OF CONCENTRATIONS OF LINDANE (pg/1)
PRODUCING ACUTE AND CHRONIC TOXICITY TO AQUATIC
SPECIES, AND CALCULATED APPLICATION FACTORS
DESCRIBING THE RELATIONSHIP BETWEEN ACUTE AND
CHRONIC TOXICITY
Species
Chironomus
tentans
Daphnia
magna
Gairanarus
fasciatus
Lepomis
macrochirus
Pimephales
promelas
Salvelinus
fontinalis
Common
midge
water
flea
scud
bluegill
fathead
minnow
brook
trout
LC50a
207 .
(157-273)
485
(391-623)
39
(27-56)
30
(20-49)
69
(24-112)
26
(19-39)
MATC
limits
>2.2<5.0
>11.0<19.0
>4.3<8.6
>9.1<12.5
>9.1<23.5
>8.8<16.6
Limits on
application
factor
0.01S0.02
0.02&0.03
0.11&0.22
0.30&0.42
0.13&0.34
0.34&0.64
48-hour LC50 for invertebrates, incipient LC50 for fishes.
95% confidence interval.
significant mortality during acute exposure. Application
factors describing the relationship between the acute and
chronic toxicity of lindane for the species studied are
calculated using the MATC and the 48-hour LC50 for invertebrates,
Similar application factors are calculated for fishes
utilizing the limits on the MATC and the incipient LC50 which
Eaton (1970) suggests to be a better measure of acute toxicity
for this type of calculation.
Application factors are similar for two of the three
invertebrate species tested. Application factors for the
fishes are remarkably similar. However, the factors
calculated for two of the invertebrates are an order of
magnitude smaller than those calculated for the fishes.
39
-------
SECTION VI
DISCUSSION
The estimated 48-hour LC50 value of 39 ug/1 determined for
Gammarus fasciatus is lower than the 48-hour EC50 of 88
(57-136) ug/1 which Cope (1966) obtained with G. lacustris.
This discrepancy may be attributed to either species
differences in susceptibility, or to differences in age of
the test animals between the two studies, or both. The
estimated 48-hour LC50 of 485 /ug/1 which we determined for
Daphnia magna is essentially equivalent to the previously
reported value of 460 ug/1 (FWPCA, 1968). No comparable
toxicity data are available for chironomids and lindane.
However, Karnak and Collins (1974) reported that the LC50
values for eleven insecticides to third and fourth instar
larvae of Chironomus tentans were generally within the range
of 1-10 ug/1. These data suggest either that first instar
midge larvae are generally more resistant to insecticide
poisoning than third and fourth instar forms, or that lindane
is generally 1-2 orders of magnitude less toxic to this
species than the insecticides they investigated, or a
combination of both.
As might be expected, the incipient LC50 values for lindane
to bluegills (30 jug/1) and fathead minnows (69 .ug/1) determined
in flowing bioassays were slightly lower than the 96-hour
LC50 values reported by Macek and McAllister (1970) for
bluegills (68 jug/1) and fathead minnows (87 jug/1) bioassayed
under static conditions. The incipient LC50 of lindane to
brook trout (26 jug/1) was essentially identical to 96-hour
LC50 reported by these authors for rainbow trout (27 ug/1).
The results of chronic exposure of the three invertebrate
species to lindane indicate that daphnids are the least
susceptible of the invertebrates tested, as they were during
acute toxicity bioassays. The midges and scuds were similar
in their susceptibility to chronic exposure to lindane,
and were the most sensitive of all the species tested.
The chironomids and daphnids exhibited the greatest differences
between susceptibility to acute lindane exposure and susceptibility
to chronic lindane exposure. One can speculate that this
may be related to the developmental physiology of these
organisms. For example, the midges experience four life
forms (egg, larvae (four instars), pupae and adult) which
are distinctively different in their biochemical and
physiological processes. Conceivably the chronic exposure
of Chironomus to chemicals incorporates four distinct
exposure phenomena which individually and/or cumulatively are
40
-------
responsible for increased chemical susceptibility. These
phenomena may also be cause to select a species like the
midge for evaluating the hazard to aquatic organisms
associated with the occurrence of chemicals in aquatic
ecosystems.
Except for Gammarus, little previous information is available
to provide a basis for making generalizations about the
relative susceptibility of fishes and invertebrates to
chronic exposure to chemicals in water. However, much of
the previously reported information leads one to the conclusion
that invertebrates generally are more susceptible to chronic
exposure than fishes. Pickering and Thatcher (1970) reported
the MATC of LAS for fathead minnows >0.63<1.2 mg/1, while
Arthur (1970) reported the MATC of LAS for Gammarus
pseudolimnaeus to be >0.2<0.4 indicating this invertebrate
species may be ca 3 times more susceptible to LAS than the
minnow.
Mount and Stephan (1969) reported the MATC of copper in
soft water for fathead minnows was >10.6<18.4 ug/1, while
Arthur and Leonard (1970) reported the MATC of copper in
soft water for G. pseudolimnaeus was >4.6<8.0 indicating
again that the invertebrate species may be at least 2 times
more susceptible than the minnow. More recently, Arthur
et al., (1973) has reported that gammardis may be 3 times
more susceptible to chronic exposure to NTA than were fathead
minnows. Certainly, all of our results of chronic exposure
of invertebrates to lindane support a hypothesis that
invertebrates are generally more susceptible to chronic
exposure to many chemicals than fishes and suggests they may be
very useful in evaluating chronic toxicity of chemicals to
aquatic organisms.
Only one similar chronic toxicity study with bluegills has
been previously described (Eaton, 1970). In comparing the
data from our study with those reported by Eaton, it appears
that the bluegills in our study were somewhat larger and
older. As a result, where spawning occurred, we observed
more spawns per female and greater egg production per female
than occurred in the previous work. It is extremely
unfortunate that the factors influential in stimulating or
inhibiting spawning activity in our study could not be
adequately identified. The percent hatch of bluegill eggs
which we observed (65-90%) was comparable to that observed
by Eaton (ca 72%). We experienced difficulty in maintaining
and feeding bluegill fry during the initial 90 day developmental
period. We attribute this primarily to an inability to
provide adequate amounts of suitable food forms during the
various stages of fry development. Eaton (1970) experienced
the same difficulties and suggested that lack of suitable
food was the primary factor responsible for poor fry
41
-------
survival. Before additional significant effort is expended
to investigate chronic toxicity of chemicals to bluegills,
definitive information on what are suitable food sources
for developing bluegill fry, and how adequate numbers of
these can be effectively made available to the fry, must
be developed.
The results of our investigation of the chronic toxicity
of lindane to fathead minnows, though not without its problems,
reinforces our opinion that this species provides the best
opportunity for evaluating chronic.toxicity chemicals to
fishes. Certainly more work has been reported with this
species than any other (Mount, 1968; Brungs, 1969; Mount and
Stephan, 1969; Hermanutz et al., 1973) and procedures for
utilizing this species in long-term laboratory assays are
becoming well defined. Except for the control groups, the
results of our fathead minnow chronic study in terms of
performance of the test organisms are good and indicate again
that subtle long-term effects of chemicals on fishes can be
evaluated under controlled laboratory conditions using this
species.
The number of spawnings per female, the number of eggs per
spawn, the percent hatchability, and the survival of fry
during the first 60 days development in the lindane study
were as good or better than that generally observed in
previous studies. In view of these facts, and the fact
that the fathead minnow procedure represents the only "true
chronic bioassay" of the three species tested (i.e. at least
one complete life cycle) we feel strongly that fathead
minnows should be the fish species of choice until methods
suitable for using other species are sufficiently defined.
We feel the brook trout offers significant potential for
use in "partial chronic bioassays". Although there were
inconsistencies in the spawning activity from group to
group, generally the numbers of fish participating in
spawning and the number of eggs spawned were adequate. Except
for those situations where toxicant-related effects on
spawning were indicated, the percent fertilization of eggs,
percent hatch and survival through the first 30 days (to
swim-up) were generally good. We feel that significant valid
information can be generated utilizing this species and these
procedures. However, certain areas of the methods need
refinement. One suggestion we have is that separate systems,
free from the effect of light, be provided for the egg
incubation. Leitritz (1959) suggested that both trout and
salmon eggs are sensitive to direct light. In our experiment
the light intensity at the water surface varied from 18
42
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foot-candles (fc) in most tanks to 35 fc in four tanks
randomly positioned under the center of the fluorescent
lights. The tanks receiving the highest light intensity
were both control units, duplicate A of those fish exposed
to 1.0 ug/1, and duplicate B of those fish exposed to 4.1
ug/1 lindane. These groups were among those exhibiting
the lowest percent hatchability of eggs, and percent
survival of fry (Tables 15 and 16) .
We also suspect that the decreased survival of fry observed
in all groups during the period 30-90 days after hatch may
be related to the stress associated with handling fry at 30
and 60 days to count and measure those organisms. We suggest
that a single measurement after 60 days exposure would provide
the same useful information regarding potential effects of
chemicals on second generation trout yet would minimize
possible effects of handling on survival. We have recently
conducted several similar studies with brook trout eggs and
fry and achieved excellent success hatching and fry survival
by shielding the developing eggs from light and minimizing
handling of fry.
We have suggested that continuous exposure to 16.6 ug/1
lindane for 261 days produced an inimical effect on the
spawning behavior of brook trout resulting in ineffective
fertilization of spawned eggs. Boyd (1964) reported that
15% of mosquito fish aborted their young after surviving
lindane concentrations which produced 10-40% mortality among
fish.
The accumulation of lindane residues in the muscle of
bluegills exposed for 735 days found to be 23 to 56 times the
mean lindane concentration in the water in which these fish
were exposed. Gakstatter and Weiss (1967) found that 6-9 g
bluegill exposed for 19 hours to 30 ug/1 lindane accumulated
whole body lindane residues of 1500 ug/kg, a bioconcentration
factor of SOX. These data suggest that c_a 30-50X probably
represents the equilibrium bioconcentration factor of lindane
by bluegill and that this equilibrium is rapidly established
and maintained throughout exposure. Although we did not
investigate the rate of residue elimination, Gakstatter and
Weiss reported that most of the lindane was eliminated
within two days after transfer to uncontaminated water.
During the last two years we have had the opportunity to
investigate the bioconcentration of some 40 pesticides by
bluegills during a minimum of 30 days continuous exposure
(Bionomics, unpublished data). With all but three of the
pesticides studied, we observed bioconcentration factors
43
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of at least 10X, and with more than half of those studied
we observed bioconcentration factors >50X. These data
suggest that bluegills do not bioconcentrate lindane to any
greater extent than they do most pesticides. Certainly,
this bioconcentration factor does not appear as significant
as those reported for chemicals which have recently been the
cause of great concern among regulatory agencies and
environmentalists. For example, Macek and Korn (1970) reported
brook trout continuously exposed to 3 ng/1 DDT for 120 days
concentrated the chemical ca 850OX. Dr. R. Reinert
U.S.D.I., Great Lakes Research Lab., Ann Arbor, Michigan
has found that rainbow trout bioconcentrate methylmercury
by as mush as 8000X (personal communication). Hansen et
al. (1971) reported marine fish exposed to polychlorinated
biphenyls concentrated the material 30,OOOX.
Mount and Stephan (1967) have proposed the utilization of
application factors to estimate chronic toxicity of chemicals
to fish based on acute toxicity data, and considerable infor-
mation based on chronic exposure of a variety of fishes to
pesticides and heavy metals support this hypothesis.
Certainly, the results of these investigations with lindane
provide evidence to substantiate the merits of this hypothesis.
The similar limits of the application factor for each of
the fishes tested is indeed remarkable.
Previous information has suggested that application factors
for pesticides are generally 0.1 or less. The only materials
for which application factors have been estimated to be on
the order of 0.25 or greater are LAS and NTA, materials which
are either relatively non-toxic to aquatic organisms, readily
biodegradable, or both. In view of the previously reported
application factors for chemicals and fishes it was surprising,
to say the least, to determine that the application factors
for lindane were so large (Table 19).
44
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TABLE 19. SUMMARY OF APPLICATION FACTORS REPORTED FOR
CHEMICALS TO VARIOUS SPECIES OF FISH
Chemical
Copper
Copper
Copper
Zinc
Cadmium
LAS
2,4-D(BEE)
Malathion
Malathion
NTA
Species
trout
minnow
minnow
minnow
minnow
minnow
bluegill
minnow
bluegill
minnow
Limits on
application
factors
.100-. 170
.035-. 076
.120-. 220
.003-. 020
.005-. 008
.145-. 276
.053-. 268
.022-. 066
.043-. 090
>.500
Source
McKim and Benoit, 1971
Mount, 1968
Mount & Stephan, 1969
Brungs, 1969
Pickering and Cast, 1972
Pickering & Thatcher,
1970
Mount & Stephan,
1967
Mount & Stephan,
1967
Eaton, 1970
Arthur et al. , 1973
45
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SECTION VII
REFERENCES
American Public Health Association 1971. Standard Methods
for the Examination of Water and Wastewater, 13th ed.
Am. Public Health Assoc., New York. 874 p.
Arthur, J. W. 1970. Chronic effects of linear alkylate
sulfonate detergent on Gammarus pseudolimnaeus,
Campeloma decisum and Physia Integra.Water Research
4:251-257.
Arthur, J. W., A. E. Lemke, V. R. Mattson, and B. J. Halligan
1973. Toxicity of sodium nitrilotriacetate (NTA) to the
fathead minnow and an amphipod in soft water. Water
Research 8:187-193.
Arthur, J. W. and E. N. Leonard 1970. Effects of copper on
Gammarus pseudolimnaeus, Physa Integra and Campeloma
decisum in soft water. J. Fish. Res7 Bd. Canada 27:
1277-1283.
Benoit, D. A. 1974. Artificial laboratory spawning substrate
for brook trout (Salvelinus fontinalis, Mitchill).
Trans. Am. Fish. Soc. 103:144-145.
Biesinger, K. E., and G. M. Christensen 1972. Effects of
various metals on survival, growth, reproduction, and
metabolism of Daphnia magna. J. Fish. Res. Bd. Canada
29:1691-1700.
Bioassay Committee 1971a. Recommended bioassay procedures
for fathead minnow (Pimephales^ promelas, Rafine^que)
chronic tests. National Water Quality Laboratory, U.S.
Environmental Protection Agency, Duluth, Minnesota.
13 p.
Bioassay Committee 1971b. Recommended bioassay procedures
for bluegill (Lepomis macrochirus, Rafinesque) partial
chronic tests.National Water Quality Laboratory, U.S.
Environmental Protection Agency, Duluth, Minnesota.
12 p.
Bioassay Committee 1971c. Recommended bioassay procedures
for brook trout (Salvelinus fontinalis, Mitchill) partial
chronic tests. National Water Quality Laboratory, U.S.
Environmental Protection Agency, Duluth, Minnesota.
12 p.
46
-------
Boyd, C. E. 1964. Insecticides cause mosquito fish to abort.
Progr. Fish. Cult. 26:138.
Brooks, G. T. 1972. Pesticides in Britain. In: Environmental
Toxicology of Pesticides. Academic Press, New York.
637 p.
Brungs, W. A. 1969. Chronic toxicity of zinc to the fathead
minnow Pimephales promelas, Rafinesque. Trans. Am.
Fish. Soc. 98:272-27T:
Clemens, H. P. 1950. Life cycle and ecology of Gammarus
fasciatus, Say. Hydrobio-Contrib. Vol. 12"Franz Theo.
Stone Inst., Ohio State University. 63 p.
Cope, 0. B. 1966. Contamination of the freshwater ecosystem
by pesticides. J. Appl. Ecol. 3:33-44.
Deuel, C. R., D. C. Haskell, D. R. Brockway, and 0. R.
Kingsbury 1952. New York State Fish Hatchery feeding
chart. N.Y. State Conserv. Dept. Fish. Res. Bull. No.
3, pp. 26.
Drummond, R. A. and W. F. Dawson 1970. An inexpensive method
for simulating diel patterns of lighting in the laboratory,
Trans. Am. Fish. Soc. 99:434-435.
Eaton, J. G. 1970. Chronic malathion toxicity to the bluegill
(Lepomis macrochirus, Rafinesque). Water Research 4:
673-684.
Federal Water Pollution Contracting Administration 1968. Water
Quality Criteria. Report of the National Tech. Adm.
Comm. to Seer, of the Interior. Fed. Water Poll.
Contr. Adm., D.S.D.I. 234 p.
:er. J. H. and C. M. Weiss 1967. The elimination of
>C14, dieldrin-Cl^, and lindane-C14 from fish following
Gakstatter,
DDT-
a single sublethal dose exposure in aquaria. Trans. Am.
Fish. Soc. 96:301-307.
Hansen, D. L., P. R. Parrish, L. I. Lowe, A. J. Wilson Jr.,
and P. D. Wilson 1971. Chronic toxicity uptake and
retention of Aroclor 1254 in two estuarine fishes. Bull.
Env. Contam. Toxicol. 6:113-119.
Hermanutz, R. 0., L. H. Mueller, and K. D. Kempfert 1973.
Captan toxicity to fathead minnows (Pimephales promelas),
bluegills (Lepomis macrochirus), and brook trout
47
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(Salvelinus fontinalis). J. Fish Res. Bd. Canada 30:
1811-1817.
Hesselberg, R. J. and J. L. Johnson 1972. Column extraction
of pesticide from fish food and mud. Bull. Env. Contain.
Toxicol. 7:115-120.
Karnak, R. E. and W. J. Collins 1974. The susceptibility
to selected insecticides and acetylcholinesterase
activity in a laboratory colony of midge larvae,
Chironomus tentans. Bull. Env. Contam. Toxicol. 12:62-69.
Leitritz, E. 1959. Trout and Salmon Culture. Fish Bulletin
No. 107, State of California, Dept. Fish & Game. 169 p.
Macek, K. J. and S. Korn 1970. Significance of the food
chain in DDT accumulation by fish. J. Fish. Res. Bd.
Canada 27:1496-1498.
Macek, K. J. and W. A. McAllister 1970. Insecticide
susceptibility of some common fish family representatives.
Trans. Am. Fish. Soc. 99:20-27.
Matsumura, F. 1972. Current pesticide situation in the
United States. In: Environmental Toxicology of
Pesticides. Academic Press, New York. 637 p.
McComish, T. S. 1968. Sexual differentiation of bluegills
by the urogenital opening. Prog. Fish. Cult. 29:28.
McKim, J. M. and D. A. Benoit 1971. Effects of long-term
exposures to copper on survival, growth, and reproduction
of brook trout (Salvelinus fontinalis). J. Fish. Res.
Bd. Canada 28:655-662.
Mount, D. I. 1968. Chronic toxicity of copper to fathead
minnows (Pimephales promelas, Rafinesque). Water
Research 2:215-223.
Mount, D, I. and W. A. Brungs 1967. A simplified dosing
apparatus for fish toxicological studies. Water Research
1:21-29.
Mount, D. I. and C. E. Stephen 1967. A method for establishing
acceptable toxicant limits for fish-malathion and the
butoxyethanol ester of 2,4-D. Trans. Am. Fish. Soc. 96:
185-193.
48
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Mount, D. I. and C. E. Stiephan 1969. Chronic toxicity of
copper to the fathead minnow (Pimephales promelas) in
soft water. J. Fish. Res. Bd. Canada 2b: 2449-: 2457.
Nash, R. G. and E. A. Woolson 1967. Persistence of chlorinated-
hydrocarbon insecticides in soils. Science 157:924-927.
Pickering, Q. H. and M. H. Cast 1972. Acute and chronic
toxicity of cadmium to the fathead minnow (Pitnephal
promelas). J. Fish. Res. Bd. Canada 29:109y-iibo.
Pickering, Q. H. and T. 0. Thatcher 1970. The chronic
toxicity of linear alkylate sulfonate (LAS) to Pimephales
promelas, Rafinesque. J. Water Poll. Cont. Fed. 42:
243-254.
Steel, R. G. D. and J. H. Torrle 1960. Principles and
Procedures of Statistics. McGraw Hill, New York. 481 p.
U. S. Environmental Protection Agency 1971. Methods for
Organic Pesticides in Water and Wastewater. National
Envir. Res. Center, Cincinnati, Ohio. 55 p.
U. S. Environmental Protection Agency 1972. Handbook for
Analytical Quality Control in Water and Wastewater
Laboratories. National Envir. Res. Center, Cincinnati,
Ohio. 114 p.
Wilson, A. J. 1965. Chemical assays. In: U.S. Bur. Comm.
Fish. Circ. 247, pp. 6-7. Annual Report of Bureau of
Commercial Fisheries Biological Laboratory, Gulf
Breeze, Florida. The fiscal year ending June 30, 1965.
49
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TECHNICAL REPORT DATA
(Please read Inuructions on the reverse before completing)
1 . REPORT NO.
EPA-600/3-76-046
3. RECIPIENT'S ACCESSIOWNO.
4. TITLE AND SUBTITLE
Chronic Toxicity of Lindane to Selected Aquatic
Invertebrates and Fishes
5, REPORT DATE
May 1976 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Kenneth J. Macek, Kenneth S. Buxton, Steven K. Derr,
J. W. Dean, Scott Sauter
8. PERFORMING ORGANIZATION REPORT NC
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Bionomics
EG & G Inc.
790 Main'Street
Wareham, Massachusetts 02571
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
68-01-0154 and 68-01-1841
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory-Duluth
Office of Research and Development
U.S Environmental Protection Agency
Duluth, Minnesota 55804
13. TYPE OF REPORT AND PERIOD COVERED
FinaJL
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT . . • •
Representatives of the aquatic invertebrate species of water flea (Daphnia
magna) , midge (Chironomus tentans), and scud (Gammarus fasciatus; and the fish
species of bluegill (Lepomis macrochirus), fathead minnow (Pimephales promelas),
and brook trout (Salvelinus fontinalis) were chronically exposed to various
concentrations of lindane in separate flowing water systems.
Maximum acceptable toxicant concentrations (MATC) of lindane for the
selected species in soft water were estimated using survival, growth, and
reproduction as indicators of toxic effects. The MATC was estimated to be between
2.2 and 5.0 ug/1 for midges, between 11 and 19 yg/1 for the water flea, and
between 4.3 and 8.6 Pg/1 for the scud. For fishes the MATC was estimated between
9.1 and 12.5 vg/l for bluegills, between 9.1 and 23.5 Vg/l for fathead minnows,
and between 8.8 and 16.6 yg/1 for brook trout. The incipient lethal concentration
(LC50) for fishes and the 48-hour LC50 for invertebrates was estimated from acute
exposures and used to calculate application factors (MATC/LC50). For aquatic
invertebrates and lindane the estimated application factors were between 0.010 and
0.024 for midges, between 0.020 and 0.029 for water flea, and between 0.11 and
0.22 for scud. Application factors were estimated between 0.30 and 0.42 for
bluegill, between 0.13 and 0.34 for fathead minnows, and between 0.34 and 0.64 for
brook trout.
17.' , KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
Toxicity
Fishes
Invertebrates
Pesticides
1?,. DISTRIBUTION STATLMENT
Release unlimited i •'.
b. IDENTIFIERS/OPEN ENDED TERMS
Chronic toxicity
Tissue residues
Flowing water system
Application factors
Lindane
13, SECUR'T Y CLASS (This Report)
Unclassified
20. SECURITY CLASS (This ptigc)
' Unclassified
c. COSATI Field/Group
06T
21. NO. OF PAGES
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