Ecological Research Series
CHRONIC TOXICITY OF METHOXYCHLOR,
MALATHION, AND CARBOFURAN TO
SHEEPSHEAD MINNOWS
(Cyprinodon variegatus)
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
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RESEARCH REPORTING SERIES
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CHRONIC TOXICITY OF METHOXYCHLOR, MALATHION, AND
CARBOFURAN TO SHEEPSHEAD MINNOWS (Cyprinodon variegatus)
by
Patrick R. Parrish, Elizabeth E. Dyar, Mark A. Lindberg,
Chiara M. Shanika, and Joanna M. Enos
EG&G, Bionomics
Marine Research Laboratory
Pensacola, Florida 32507
Contract No. 68-03-0264
Project Officer
David J. Hansen
Environmental Research Laboratory
Gulf Breeze, Florida 32561
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FLORIDA 32561
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DISCLAIMER
This report has been reviewed by the Gulf Breeze Environmen-
tal Research Laboratory, 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.
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FOREWAKD
The protection of our estuarine and coastal areas from damage caused
by toxic organic pollutants requires that regulations restricting the
introduction of these compounds into the environment be formulated on a
sound scientific basis. Accurate information describing dose-response
relationships for organisms and ecosystems under varying conditions is
required. The Environmental Research Laboratory, Gulf Breeze, contributes
to this information through research programs aimed at determining:
the effects of toxic organic pollutants on individual species and
communities or organisms;
the effects of toxic organics on ecosystem processes and components;
the significance of chemical carcinogens in the estuarine and marine
environments.
This report describes effects of three insecticides in partial life-
cycle tests with an estuarine fish, the sheepshead minnow. The data will
be useful in establishing estuarine water quality criteria, and limiting
effluents containing carbofuran, malathion, or methoxychlor.
Thomas W. Duke
Director
Environmental Research Laboratory
iii
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ABSTRACT
Sheepshead minnows (Cyprinodon variegatus) were exposed to
each of three pesticides—methoxychlor, malathion, and carbofu-
ran—in flowing seawater to determine the acute and chronic (par-
tial life-cycle) effects. The calculated 96-hour LCSO's and 95%
confidence limits, based on measured concentrations, were:
methoxychlor, 49 micrograms per liter (yg/£), 37-65 yg/£; mala-
thion, 51 yg/£, 41-63 yg/£; and carbofuran, 386 yg/fc, 311-480
ygA.
Mortality of adult sheepshead minnows exposed to mean mea-
sured concentrations of methoxychlor >23 yg/£ was significantly
(P<0.05) greater than mortality of control fish during the 140-
day study- Further, hatching success of fry from eggs spawned
by fish exposed to 23 yg/£ was significantly less than hatching
success of control fry- The maximum acceptable toxicant concen-
tration (MATC) was estimated to be >12<23 yg/£ and the applica-
tion factor limits were 0.24-0.47.
Mortality of adult sheepshead minnows exposed to mean mea-
sured concentrations of malathion >18 yg/£ was significantly
greater than mortality of control fish during the 140-day study-
Mortality of fry hatched from eggs spawned by fish exposed to 9
and 18 yg/£ was significantly greater than mortality of control
fry. The MATC was estimated to be >4<9 yg/& and the application
factor limits were 0.08-0.18.
Mortality of adult sheepshead minnows exposed to mean mea-
sured concentrations of carbofuran >49 yg/£ was significantly
greater than mortality of control fish during the 131-day study.
Hatching success of fry from eggs spawned by fish exposed to 49
yg/£ was significantly less than hatching success of control fry.
Also, mortality of fry hatched from eggs spawned by fish exposed
to ?3 and 49 yg/£ was significantly greater than control fry
mortality. The MATC was estimated to >15<23 yg/£ and the appli-
cation factor limits were 0.04-0.06.
This report was submitted in fulfillment of Contract Number
68-03-0264 by EG&G, Bionomics Marine Research Laboratory, under
the sponsorship of the U.S. Environmental Protection Agency.
This report covers the period 23 May 1973 to 23 May 1975; work
was completed on 1 November 1976.
IV
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CONTENTS
Foreword iii
Abstract iv
Tables vi
Acknowledgment ix
1. Introduction 1
2. Conclusions 3
3. Recommendations 4
4. Materials and Methods
Test materials 5
Test water 5
Test animals 6
Test methods 7
Chemical analyses 9
Statistical analyses 13
5. Results and Discussion
Chemical analyses 14
Acute toxic ity 18
Chronic toxicity 19
Application factors 30
Summary 31
References 33
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TABLES
Number Page
1 Nominal and Measured Concentrations of Methoxychlor
during Acute and Chronic Exposures of Sheepshead
Minnows (Cyprinodon variegatus) in Flowing Sea-
water 14
2 Nominal and Measured Concentrations of Malathion
during Acute and Chronic Exposures of Sheeps-
head Minnows (Cyprinodon variegatus) in Flowing
Seawater 15
3 Nominal and Measured Concentrations of Carbofuran
during Acute and Chronic Exposures of Sheepshead
Minnows (Cyprinodon variegatus) in Flowing Sea-
water 16
4 Analysis of Parent and Hydrolyzed Carbofuran in Sea-
water Samples Collected from the Nominal Concen-
tration of 500 yg/& during a 131-Day Exposure of
Sheepshead Minnows (Cyprinodon variegatus) 16
5 Acute Toxicity of Three Pesticides to Sheepshead
Minnows (Cyprinodon variegatus) Exposed in Flow-
ing Seawater 18
6 Percentage Mortality of Parental Sheepshead Minnows
(Cyprinodon variegatus) Exposed to Methoxychlor
in Flowing Seawater 19
7 Growth of Sheepshead Minnows (Cyprinodon variegatus)
Exposed for 140 Days to Methoxychlor in Flowing,
Natural Seawater ..... 20
*
8 Number of Eggs Spawned by Sheepshead Minnows
(Cyprinodon variegatus) Exposed to Methoxychlor
in Flowing, Natural Seawater during Three 10-Day
Spawning Periods 21
9 Number of Eggs Spawned per Day per Female Sheeps-
head Minnow (Cyprinodon variegatus) Exposed to
Methoxychlor in Flowing, Natural Seawater during
Three 10-Day Spawning Periods 22
vi
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Number Page
10 Hatching Success of Fry from Eggs Spawned by Sheeps-
head Minnows (Cyprinodon variegatus) Exposed to
Methoxychlor in Flowing, Natural Seawater 23
11 Percentage Mortality, Average Standard Length, and
Weight (Determined In Water) of 28-Day Old Sheeps-
head Minnow (Cyprinodon variegatus) Fry Hatched
from Eggs Spawned by Fish Exposed to Methoxychlor
for 54-63 Days 23
12 Concentrations of Methoxychlor in Surviving Adult
Sheepshead Minnows (Cyprinodon variegatus) Ex-
posed for 140 Days 24
13 Accumulation of Organochlorine Pesticides by Marine
Fishes 24
14 Percentage Mortality of Sheepshead Minnows (Cyprinodon
variegatus) Exposed to Malathion in Flowing, Natural
Seawater for 140 Days 25
15 Growth of Sheepshead Minnows (Cyprinodon variegatus)
Exposed for 140 Days to Malathion in Flowing, Nat-
ural Seawater 25
16 Number of Eggs Spawned by Sheepshead Minnows
(Cyprinodon variegatus) Exposed to Malathion in
Flowing, Natural Seawater during Two 10-Day Spawn-
ing Periods 26
17 Number of Eggs Spawned per Day per Female Sheeps-
head Minnow (Cyprinodon variegatus) Exposed to
Malathion in Flowing, Natural Seawater during
Two 10-Day Spawning Periods 26
18 Hatching Success of Fry from the Eggs Spawned by
Sheepshead Minnows (Cyprinodon variegatus) Ex-
posed to Malathion in Flowing, Natural Seawater. . . 27
19 Percentage Mortality, Average Standard Length, and
Weight (Determined In Water) of 28-Day Old Sheeps-
head Minnow (Cyprinodon variegatus) Fry Hatched
from Eggs Produced by Fish Exposed to Malathion
for 87-96 Days 27
20 Percentage Mortality of Sheepshead Minnows (Cyprinodon
variegatus) Exposed to Carbofuran in Flowing, Syn-
thetic Seawater 28
VI1
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N umber Pagt
21 Growth of Sheepshead Minnows (Cyprinodon variegatus)
Exposed for 131 Days to Carbofuran in Flowing,
Synthetic Seawater . •• 28
22 Number of Eggs Spawned by Sheepshead Minnows
(Cyprinodon variegatus) Exposed to Carbofuran in
Flowing, Synthetic Seawater 29
23 Hatching Success of Fry from Eggs Spawned by Sheeps-
head Minnows (Cyprinodon variegatus) Exposed to
Carbofuran in Flowing, Synthetic Seawater 29
24 Percentage Mortality and Average Standard Length of
30-Day Old Sheepshead Minnow (Cyprinodon variegatus)
Fry which were Hatched from Eggs Spawned by Fish
Exposed to Carbofuran for 42-95 Days in Flowing,
Synthetic Seawater , . 30
25 Concentrations (yg/£) of Three Pesticides Toxic to
Sheepshead Minnows (Cyprinodon variegatus) in Acute
and Chronic Tests, and the Relationship of Acute
Toxicity to Chronic Toxicity 30
26 Comparison of Acute and Chronic Malathion Toxicity
to Two Freshwater Fishes and a Saltwater Fish. ... 31
27 Summary of Significant Effects of Methoxychlor,
Malathion, and Carbofuran on Sheepshead Minnows
(Cyprinodon variegatus) during Chronic Exposures
in Flowing Seawater. , 32
Vlll
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ACKNOWLEDGMENTS
We thank the Project Officer, Mr. David J. Hansen, for his
guidance and patience during these studies. Thanks to Mr. Terry
A. Hollister, EG&G, Bionomics Marine Research Laboratory, for
his help with statistical analyses, and thanks to Ms. Susan
Walker for typing the manuscript. The assistance of Mr. Kenneth
S. Buxton, EG&G, Bionomics Analytical Chemistry Laboratory, -is
appreciated, as is the review of the manuscript by Kenneth J.
Macek, Ph.D., and Sam R. Petrocelli, Ph.D., EG&G, Bionomics
Aquatic Toxicology Laboratory.
IX
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SECTION I
INTRODUCTION
These studies were undertaken to gain information about the
effects of three pesticides—methoxychlor, malathion, and carbo-
furan—on sheepshead minnows (Cyprinodon variegatus), a saltwater
fish. Data from these studies can be used to derive application
factors, first described by Mount and Stephan (1967). An appli-
cation factor is obtained by dividing the concentration consid-
ered to be "safe" over a long period by a short-term toxicity
value. The application factor may then be used to establish wa-
ter quality criteria for each pesticide. The use of sheepshead
minnows for long-term, life-cycle tests was proposed by Schimmel
and Hansen (1975) and a tentative method was described by Hansen
and Schimmel (1975). In our short term studies, data on 96-hour
LCSO's (the concentration of each pesticide estimated to be lethal
to 50% of the test animals after 96 hours of exposure) were ob-
tained. In our long-term studies, parental sheepshead minnows
were exposed for 28 days or more and then effects on spawning
and progeny were determined. "Safe" concentrations were esti-
mated after evaluation of data on number of eggs spawned by pa-
rental fish, hatching success of embryos, mortality of fry, and
growth of fry.
Methoxychlor is a stable, chlorinated hydrocarbon compound
used to control a wide variety of insects which attack fruits,
vegetables, field and forage crops, and livestock. It is also
used to control certain household and industrial insects. Me-
thoxychlor is a replacement for DDT in many applications (Anon-
ymous, 1972). Methoxychlor is ostensibly insoluble in water but
soluble in lipids (Gardner and Bailey, 1975) and thus might be
expected to accumulate in fish and to be cumulatively toxic dur-
ing long-term exposure.
A recent publication (Gardner and Bailey, op. cit.) provides
an excellent overview of the effects of methoxychlor on environ-
mental quality and states that although there are variances in
estimates of the acute toxicity of methoxychlor to fishes, all
reported LCSO's ranged from 5-80 micrograms per liter (pg/fc), ex-
cepting three.
We know of only one chronic (partial life-cycle) toxicity
study that has been conducted with methoxychlor and fish (Merna
and Eisele, 1973). Data from that study were insufficient for
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deriving an application factor.
Malathion is an organophosphate pesticide used throughout
the U. S. to control a variety of pests. A major use of mala-
thion is for mosquito control in both freshwater and estuarine
areas. Several studies (Parkhurst and Johnson, 1955; Westman
and Compton, 1960; Weiss, 1961; Lewallen and Wilder, 1962;
Holland and Lowe, 1966; Wellborn, 1971; and Post and Schroeder,
1971) have shown that malathion is acutely lethal to a variety
of freshwater and saltwater fishes under both field and labora-
tory test conditions. Sublethal effects (avoidance and inhibi-
tion of brain acetylcholinesterase) have also been observed in
fashes exposed to malathion (Hansen e,t al., 1972; Coppage, 1972;
and Coppage and Matthews, 1972).
Chronic studies have been conducted with malathion and two
freshwater fishes, fathead minnows (Pimephales promelas) (Mount
and Stephan, 1967), and bluegill (Lepomis macrochirus) (Eaton,
1970). The studies showed the application factor limits for the
two fishes to be very similar, ranging from 0.02-0.06.
Carbofuran is a carbamate pesticide utilized both as a con-
tact poison or as a soil-applied systemic poison. It is regis-
tered for soil-applied use on a variety of crops, including rice,
and for direct use on several insects, including mosquitoes
(Anonymous, 1971). Little research has been performed on the
effects of carbofuran on aquatic organisms.
Carbofuran was the first compound tested. Tests were con-
ducted from March-July 1974 in the EG&G, Bionomics Aquatic Toxi-
cology Laboratory, Wareham, Massachusetts. Our new laboratory
was opened in January 1975, and tests with methoxychlor and mala-
thion were conducted from June-October 1975, at EG&G, Bionomics
Marine Research Laboratory, Pensacola, Florida.
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SECTION 2
CONCLUSIONS
Sheepshead minnows (Cyprinodon variegatus) are suitable test
animals for toxicity tests which include the reproductive portion
of the life cycle and the critical life stages (embryos and fry)
of the successive generation.
Tests with these saltwater fish are practical means of de-
termining maximum acceptable toxicant concentrations and appli-
cation factors because of (a) the amenability of sheepshead
minnows to laboratory culture and (b) the relatively short peri-
od of time required to reach sexual maturity and complete the
reproductive phase of the life cycle.
The application factor limits derived for sheepshead minnows
exposed to malathion are very similar to the application factor
limits derived for two freshwater fishes, indicating that this
saltwater fish may be used effectively to obtain data on which
to base water quality criteria.
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SECTION 3
RECOMMENDATIONS
Spawning groups comprising five sheepshead minnows in the
ratio of 3 female fish:2 male fish are satisfactory to determine
spawning success.
A 10-day spawning period for a spawning group is sufficient
to monitor spawning success.
Studies should be conducted with sheepshead minnows and
other toxicants in chronic (full life-cycle) tests because tests
with this saltwater fish appear to provide accurate estimates of
MATC's in a shorter time and with less effort than do tests with
most freshwater fishes.
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SECTION 4
MATERIALS AND METHODS
TEST MATERIALS
Methoxychlor used in this study was obtained from E. I. du
Pont de Nemours & Company, Biochemicals Department, Wilmington,
Delaware. It was contained in a plastic jar labeled "Methoxy-
chlor Technical, 1 Kg." Although active ingredient was not
listed on the label, a technical data sheet dated April 1972
which accompanied the chemical stated that "...methoxychlor tech-
nical. . .contains 88% (minimum) 2,2-bis-(p-methoxyphenyl)-1, 1,
1-trichloroethane and 12% (maximum) other isomers and reaction
products.
Malathion was obtained from the American Cyanamid Company,
Agricultural Division, Princeton, New Jersey. It was contained
in a metal bottle, apparently aluminum, with a transluscent cap.
The bottle was refrigerated at all times. The material was la-
beled "MALATHION Technical, Active Ingredient: Malathion* 95%;
Inert Ingredients 5%. *0,0-dimethyl phosphorodithioate of di-
ethyl mercaptosuccinate; (1 Gallon contains 9.7 Ib of malathion)."
Carbofuran was obtained from FMC Corporation, Agricultural
Chemical Division, Middleport, New York. It was contained in a
plastic bag labeled "Carbofuran Technical (99%), FURADAN® In-
secticide, Mr L514, 2Kg. C4717-54-A, 9/14/73."
Concentrations of each pesticide are reported here as micro-
grams (yg) of the technical material described above per liter
(£) of seawater.
Stock solutions of all three pesticides were prepared on a
weight:volume basis by dissolving them in reagent grade acetone.
These I-I stock solutions were placed in amber glass bottles and
stored in the laboratory. New stock solutions were prepared as
required.
TEST WATER
Methoxychlor and Malathion
All water used for holding, acclimation, and testing was
natural seawater which was pumped from Big Lagoon into the
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laboratory- The pump intake was 85 meters (m) offshore at a
depth of approximately 3 m. Water was pumped by a #316 stain-
less steel pump through hard polyvinylchloride (PVC) pipes into
an elevated fiberglass reservoir. En route, the water passed
through a fiberglass, sand filter and a 10-micrometer (ym) poly-
propylene bag filter. From the reservoir, in which the water was
continuously and vigorously aerated, water flowed by gravity
through PVC pipes to the diluters.
No attempt was made to alter the salinity of the water, but
temperature was maintained at 30±1 degrees Celsius (°C) by heat-
ing the incoming seawater in small fiberglass-coated plywood
boxes above the diluters with electric quartz heaters and by
placing test aquaria in constant-temperature water baths.
Carbofuran
All water used for holding, acclimation, and testing was
synthetic seawater, formulated according to the methods of
LaRoche et al. (1970). Freshwater was pumped from a 120-m deep
bed-rock well at the Wareham, Massachusetts, laboratory into two
l,500-& fiberglass tanks. Ingredients were added, mixed with
freshwater, and aerated. Water was drained from one tank at the
rate of approximately 1,200 a per day until it was empty. Then,
water was drained from the second tank while fresh synthetic sea-
water was being prepared in the first tank.
All water flowed by gravity through PVC pipes to the diluter.
Temperature was maintained at 30±1°C by placing the test aquaria
in constant-temperature water baths.
TEST ANIMALS
Methoxychlor and Malathion
All sheepshead minnows used in these studies were collected
from Big Lagoon, near Bionomics Marine Research Laboratory. They-
were held in sand-banked ponds on the laboratory grounds and in
fiberglass tanks in the laboratory. All fish were acclimated to
test conditions for 14 days before testing according to the con-
ditions of U. S. Environmental Protection Agency (1975). Mor-
tality was <3% during acclimation. During holding and acclilfta-
tion, fish were fed frozen or live Artemia salina (San Francisco
Bay Brand) which contained <0.1 yg/g of chlorinated hydrocarbon
pesticides or polychlorinated biphenyls as determined by our
electron-capture gas chromatograph analyses.
Size of fish was: methoxychlor—acute test, 1.7-3.8 centi-
meters (cm) standard length (SL) and chronic test, 1.0-1.9 cm SL
and 0.15 g mean weight (determined in water); malathion—acute
test, 0.8-1.8 cm SL and chronic test, 1.0-1.8 cm SL and 0.11 g
mean weight (determined in water).
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Carbofuran
Fish were obtained from the U. S. Environmental Protection
Agency's Gulf Breeze Laboratory, Sabine Island, Gulf Breeze,
Florida, where they had spawned naturally in outside earthern
ponds. Fish for the acute and chronic tests were 1.8-4.0 cm SL.
TEST METHODS
Acute Tests
All procedures followed methods of APHA et al. (1976) and
U. S. Environmental Protection Agency (1975) , except as stated.
The 96-hour tests were conducted in an intermittent-flow system
by-using a proportional diluter (Mount and Brungs, 1967) con-
structed to deliver 1 Jl/cycle at a dilution ratio of 75%. The
average number of cycles was approximately 5/hour, providing 99%
replacement in 24 hours (Sprague, 1969) . A mechanical injector
(manufactured by George Frasier, Duluth, MN), equipped with a
50-milliliter (m£) glass syringe and stainless steel needle,
pumped methoxychlor, malathion, or carbofuran stock solution
through polyethylene tubing to the mixing cell. Test containers
were 30 X 30 X 61-cm glass aquaria. Each contained 20 fish and
approximately 28 a of water.
Chronic Tests
The tests were conducted as described above except that the
proportional diluter was constructed for 50% dilution. It de-
livered 1 Jl/cycle at a rate of approximately 5 cycles/hour. For
the methoxychlor and malathion tests, the diluter was modified
'to include a solvent control wherein the same volume of solvent/
carrier (acetone) was added to methoxychlor- or malathion-free
seawater as was added to the highest pesticide concentration.
One injector, equipped with a 30-m£ glass syringe and a stainless
steel needle, metered the respective stock solutions through
polyethylene tubing into the mixing chamber. A second injector,
equipped with a 50-m.e, glass syringe with stainless steel needle,
metered acetone to each solvent control. Maximum solvent concen-
tration was 29 y£/£ (parts per million, ppm) .
To begin each test, 20 acclimated fish were impartially se-
lected and placed in the test aquaria (a total of 40 fish per
treatment) after the toxicant delivery system had been opera-
tional for several days. Fish were daily fed flaked commercial
fish food (BiOrell® and Tetramin®) ad libitum. Salinity and dis-
solved oxygen were measured daily throughout the tests. Light
for all tests was provided by two 3.7-m fluorscent bulbs sus-
pended 46 cm above the test containers, providing approximately
1,100 lux incident to the water surface. Photoperiod was 16
hours light, 8 hours dark. Survival was monitored daily by vi-
sually inspecting each test container. Growth was monitored bi-
weekly according to the photographic method of McKim and Benoit
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(1971) and average weight was determined monthly by weighing
each group in water.
Effects of each pesticide on.-spawning were determined after
fish began to exhibit signs of sexual maturity.
Methoxychlor and Malathion—
Monitoring of spawning activity was begun on day 54 of the
methoxychlor test and on day 87 of the malathion test. Spawning
chambers were constructed by lacing pieces of 6.5-millimeter (mm)
square mesh #316 stainless steel screen together with #316 stain-
less steel wire. The chambers were 30.5-cm square X 25.5-cm
high, and were supported by 5-cm high extensions of the screen
ends. Beneath each spawning chamber, a 29.5-cm square X 4.7-cm
high egg collection tray was placed to retain the demersal eggs
that sank through the bottom of the spawning chamber. The tray
was constructed of plate glass and silicone sealant, with a 4-cm
wide strip of 480-ym square mesh nylon screen along one side of
the bottom to facilitate consolidation of eggs. Spawning groups,
which consisted of two male and three female fish, were placed in
the spawning chambers for a 10-day period. All possible 2:3
ratios in each aquarium were spawned once and extra, unspawned
fish from each replicate aquarium were combined whenever possible
to form a 2:3 spawning group. Each day, one end of each spawning
chamber was lifted slightly and the egg collection tray was re-
moved from the aquarium. The eggs spawned during the previous
24 hours were washed with seawater, transferred by large-bore
glass pipette into glass Petri dishes, counted, and separated
into groups of 50 eggs. Each 50-egg group was placed in an egg
incubator cup (a 100-m£ glass jar with the bottom cut off and
480-ym square mesh nylon screen attached with silicone sealant).
Each egg cup was then placed in the same aquarium as the spawn-
ing group which produced it. The egg incubator cups were sus-
pended from a rocker-arm apparatus (Mount, 1968) which gently
oscillated them in the test aquaria. Eggs were removed from each
egg incubator by pipette daily, counted, and the cups washed with
bursts of freshwater to clean the screens. This procedure was
repeated until all living embryos hatched. Then, 40 fry were
placed in glass chambers (14-cm wide X 20.5-cm high X 26-cm long
with 381-ym square mesh #316 stainless steel screen over one end).
Survival was monitored daily and growth (standard length anS av-
erage weight) was measured after 28 days. At least two groups
of fry per duplicate from each test concentration and controls
were monitored, except in the higher concentrations where toxi-
cant-induced mortality made it impossible to obtain spawning
groups and subsequent eggs and fry.
Carbofuran—
Effects on spawning were determined by monitoring spawning
activity of individual pairs of fish, beginning on day 28. Each
pair was placed in a 14 X 25 X 25-cm glass and #316 stainless
steel mesh spawning chamber similar to the fry chambers described
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above. Spawning was monitored for a 62-day period, during which
all possible pairs of unspawned fish in all duplicate aquaria
were spawned. Eggs that sank through a false bottom of 7-mm
square mesh #316 stainless steel screen were collected daily and
treated as described above.
CHEMICAL ANALYSES
Seawater
For each of the three acute tests, water was collected from
each aquarium at the beginning and end of the 96-hour exposure.
Water was collected from alternate duplicate aquaria weekly dur-
ing the chronic tests. Water samples were prepared and analyzed
as follows:
Methoxychlor—
Unfiltered seawater was extracted twice with two 50-m& por-
tions of Nanograde® (Mallinckrodt) dichloromethane. Volumes ex-
tracted were:
Nominal
concentration Volumes
(yg/ft) (ma)
6 500
11 300
22 300
45 100
90 100
The combined extracts were dried by elution through anhydrous
sodium sulfate (heated at 100°C for 24 hours), concentrated to
approximately 1 m£ in a Kuderna-Danish evaporator, and solvent-
exchanged with Nanograde petroleum ether. The extract volumes
were adjusted to obtain a sensitivity of 0.05 ppm (nanograms
[ng] per y£) by using a Perkin-Elmer Model 2100 gas chromatograph
equipped with a Ni63 electron-capture detector.
Operating conditions were:
Column (glass)—2 m X 4 mm ID 3% OV-101 on 80/100 mesh
Gas Chrom Q
Oven temp.—210°C Detector temp.—275°C
Injector temp.—250°C Carrier gas—Nitrogen
Malathion—
Unfiltered seawater samples of 500 m£ were extracted twice
with two 50-m£ portions of Nanograde dichloromethane. The ex-
tracts were dried by eluting through anhydrous sodium sulfate
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(heated at 100°C for 24 hours) and concentrated in a Kuderna-
Danish evaporator. The extract volumes were adjusted to obtain
a sensitivity of 1.0 ppm by using a Perkin Elmer Model 2100 gas
chromatograph equipped with a flame photometric detector oper-
ating in the phosphorus mode.
Operating conditions were:
Column (glass)—2 m X 4 mm ID 3% OV-101 on 80/100 mesh
Gas Chrom Q
Oven temp.—210°C Bead setting—500
Injector temp.—250°C Carrier gas—10% argon/methane
Carbofuran—
Unfiltered water samples (approximately 500 nu) were mea-
sured volumetrically in a graduated cylinder and placed in a l-£
separatory funnel equipped with a Teflon® stopcock. The water
was extracted three times with separate 30-m& portions of Nano-
grade dichloromethane and the combined extract was passed through
an anhydrous sodium sulfate column to remove moisture from the
solvent. The sodium sulfate was rinsed with a portion of di-
chloromethane and the extract and rinse were placed in a Kuderna-
Danish evaporator equipped with a three-ball Synder column. The
solvent was evaporated to approximately 3 nU over an 80°C water
bath, the extract was transferred to a 15-m& centrifuge tube with
a Teflon-lined cap, and evaporated to dryness at room temperature
by using a gentle stream of clean dry air. The extract was then
dissolved in an accurately known volume of Nanograde benzene and
stored in a freezer at 15°C prior to analysis by gas/liquid chro-
matography under the following conditions:
Instrument—Perkin-Elmer Model 3920 gas chromatograph
Detector—Nitrogen/phosphorus thermionic detection
Column—0.6 m X 2 mm ID glass packed with 20% SE-30 coated on 60/
80 Chromasorb W. The column was conditioned at 235°C for
two weeks prior to use. Several injections (3 X 50 y&)
of Silyl-8, a column-silanizing agent, were made ovSr the
two-week conditioning period.
Gas flows—38 cubic centimeters (cc) N2/minute (min.) carrier,
7 cc H2/min. and 100 cc air/min. to the N/P detector.
Temperatures—Injection port: 225°C
Column: 155°C
Transfer line: 245°C
Recorder—Leeds & Northrup dual pen, 0-1 mV range, 5 mm/min.
chart speed
10
-------
Response—30 ng of carbofuran and 80 ng of 3-hydroxycarbofuran
gave half-scale response with retention times of 3.4
and 6.4 min., respectively
Extraction efficiency and mean recovery for the analytical
methods were 89.9±9.8% for methoxychlor, 84.5±11.7% for mala-
thion, and 79.0±7.3% for carbofuran. Data in this report are
corrected for recovery.
Fish Tissue
Fish were collected for residue analyses as follows:
a. adults alive at the end of the respective exposure;
b. fry alive at the end of the 28-day growth period; and
c. eggs randomly collected during the spawning periods.
Methoxychlor—
Tissues were analyzed by an adaptation of the methods of
U. S. Environmental Protection Agency (1971 and 1974). Fish
tissue was weighed to the nearest 0.01 g in a beaker. The tis-
sue was transferred to a 100-m2, graduated cylinder, which had
been cut at the 80-mJi mark to reduce the height of the cylinder;
the beaker was rinsed with dichloromethane, which was then added
to the cylinder. The tissue was homogenized with approximately
30 m£ of dichloromethane for 20 seconds by using a Brinkman
Polytron Homogenizer, Model PT 10/20.
The homogenate was filtered through No. 3 Whatman paper in-
to a clean beaker, approximately 30 m£ of dichloromethane was
added to the graduated cylinder, and the Polytron probe was
rinsed for approximately 10 seconds in the solvent while the
homogenizer was operating at low speed. The probe rinse was
added to the filter and finally the filter was washed with di-
chloromethane .
The solvent was evaporated to approximately 5 m£ over a
steam bath, cooled, and evaporated to dryness at room tempera-
ture by using a gentle stream of clean air. At this point the
extract was cleaned by florisil column chromatography according
to U. S. Environmental Protection Agency (1974). Methoxychlor
eluted quantitatively from the 6% ether-in-petroleum ether frac-
tion and was sufficiently free of interfering substances to
permit analysis by electron capture detection. The 6% ethyl-
petroleum ether fraction containing methoxychlor was evaporated
to approximately 3 m£ in a Kuderna-Danish evaporator equipped
with a three-ball Snyder column, and the unit was cooled to room
temperature. The receiver was disconnected, the remainder of
the solvent was evaporated to dryness at room temperature by using
a gentle air flow, and a known volume of hexane was added to the
11
-------
receiver to dissolve the residue.
An aliquot of the extract was analyzed by gas chromatography
under the following operating conditions:
Instrument—Tracer Model MT-550 gas chromatograph
Detector—Electron capture with 15 millicuries of Ni63
Column—2 m X 2 mm ID glass packed with 3% OV-101 on 100/120
mesh HMDS-treated Supelcoport
Cashflows—30 cc N2/min. carrier, 60 cc N2/min. scavenger
Temperatures—Column: 200°C Inlet: 230°C
Transfer: 270°C Detector: 302°C
Recorder—Corning Model 841, 0-1 mV, 0.5 cm/min. chart speed
Response—2.5 ng of methoxychlor gave half-scale pen deflection
at an attenuation of 1.6 X 10~9 amperes
Three fish, weighing approximately 1 g each, were spiked
with 100 ng of methoxychlor and were analyzed by the above
method. The average precentage recovery of methoxychlor was
100±4.6%. The analytical results were not corrected for re-
covery which was considered quantitative.
Malathion—
Tissues were analyzed in the manner described for methoxy-
chlor except that malathion eluted from the florisil column
quantitatively in the 1:1 ethyl ether-in-petroleum ether frac-
tion. An aliquot of the extract was analyzed by gas chromatog-
raphy under the following operating conditions:
Instrument—Perkin-Elmer Model 3920 gas chromatograph
Detectors—Electron capture with 15 millicuries of Ni63 and
nitrogen/phosphorus thermionic detection
Effluent Splitter—10 parts to N/P and 3 parts to BCD
Column—2 m X 2 mm ID glass packed with 3% Dexsil 300 GC on 80/
100 mesh DMHS-treated Supelcoport
Gas flows—36 cc N2/min. carrier, 7 cc N2/min. and 100 cc air/
min. to the N/P detector
Temperatures—Injection port: 250°C
Column: 220°C
Transfer line and splitter: 265°C
12
-------
Recorder—Leeds & Northrup dual pen, 0-1 mV range, 1.0 cm/min.
chart speed
Response—0.30 ng of malathion gave half-scale recorder pen
deflection using the N/P detector at an attenuation
of 16 X 1. Retention time was 2.0 min.
Three whole fish, weighing approximately 1 g each, were
spiked with 100 ng of malathion and were analyzed by the above
method. The average percentage recovery of malathion was 103±
6.8%. The analytical results were not corrected for recovery
which was considered quantitative.
Carbofuran—
Results of previous research in which fish were continu-
ously exposed to radiolabeled C14 carbofuran for 28 days indi-
cated that the maximum tissue concentrations were reached within
3-10 days, after which an equilibrium concentration was observed.
A concentration factor of 5-20X was calculated (FMC, 1976). In
view of the rapid equilibrium, the low concentration factor, and
the absence of a routine gas-chromatographic analytical method
for fish tissues, fish from the carbofuran chronic test were not
analyzed for residues.
STATISTICAL ANALYSES
In the acute tests, the LCSO's and 95% confidence limits
were calculated by linear regression analysis after probit trans-
formation (Finney, 1971) .
In the chronic tests, differences between treatments were
determined by chi-square (X2) and analysis of variance (Sokol
and Rohlf, 1973). Differences were considered significant at
the 95% (P<0.05) confidence level. Post-hoc tests were con-
ducted on treatment means by using the Student-Newman-Keuls
range test (Keuls, 1952).
13
-------
SECTION 5
RESULTS AND DISCUSSION
CHEMICAL ANALYSES
Mean measured concentrations of methoxychlor in seawater
were from 57-109% of nominal during the 96-hour test and from
45-55% of nominal during the chronic test (TABLE 1). Because
this chlorinated hydrocarbon pesticide is "essentially insoluble
in water (0.10 mg/A @ 25°C)" (Anonymous, 1972) and because ex-
posure to light and the addition of particulate matter and micro-
organisms hastens its degradation (Gardner and Bailey.- 1975),,
these mean measured concentrations were within an expected and
acceptable range.
TABLE 1. NOMINAL AND MEASURED CONCENTRATIONS OF METHOXYCHLOR
DURING ACUTE AND CHRONIC EXPOSURES OF SHEEPSHEAD MIN-
NOWS (CYPRINODON VARIEGATUS) IN FLOWING SEAWATER.
Test
Concentration (yg/l.)
Nominal
Measured
0
Acute Control
22
30
40
53
70
93
125
Chronic Control
Sol. control
6
11
22
45
90
hour
<0.01
13
11
25
33
26
67
62
Mean
0.2
_a
3
5
12
23
48
96 hour
<0.01
21
29
26
35
54
86
209
S.D.
±0.5
—
±2
±4
±7
±19
±10
Mean
_
17
20
26
34
40
78
136
Range
0-2
-
1-7
2-18
4-30
9-85
34-53
% of nom.
_
77
67
65
64
57
84
109
% of nom.
_
-
50
45
55
51
53
t samples
18
-
15
15
15
15
4
JNot analyzed.
14
-------
Malathion is readily soluble in water and mean measured
concentrations during both the acute and chronic tests reflected
this characteristic; concentrations were 72-143% of nominal
(TABLE 2).
TABLE 2. NOMINAL AND MEASURED CONCENTRATIONS OF MALATHION DUR-
ING ACUTE AND CHRONIC EXPOSURES OF SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) IN FLOWING SEAWATER.
Concentration (yg/£)
Test Nominal
Acute Control
22
30
40
53
70
94
125
Chronic Control
Sol. control
4
8
15
30
60
Measured
0 hour
<0.1
16
26
25
43
62
78
108
Mean
<0.1
_a
4
9
18
37
86
96 hour
<0.1
16
27
37
50
67
99
111
S.D.
_
—
±2
±4
±6
±12
±15
Mean -
_
16
26
31
46
64
88
109
Range '
_
-
1-6
4-17
8-28
20-57
70-101
1 of nom.
_
72
87
78
89
91
95
87
'5 of nom.
_
—
100
112
120
123
143
# samples
19
-
20
20
20
10
3
aNot analyzed.
Mean measured concentrations of parent carbofuran were from
44-62% of nominal during the acute test and from 18-24% of nomi-
nal during the chronic test (TABLE 3). Evaluation of the analy-
ses of water samples collected during days 1-30 of the chronic
test shows that concentrations of parent carbofuran were approxi-
mately 40% of nominal. Thereafter, despite the use of a flowing-
water exposure system, measured concentrations of parent material
decreased to approximately 10% of nominal. Concurrently, however,
we observed a pattern of increasing concentrations of hydrolyzed
carbofuran derivitives (TABLE 4). The rapid decline of measured
concentrations of parent material in the 4-day acute test and the
stability of parent carbofuran in a stock solution over a 12-day
period (confirmed by chemical analyses) is further evidence that
carbofuran was degraded in seawater.
15
-------
TABLE 3. NOMINAL AND MEASURED CONCENTRATIONS OF CARBOFURAN DUR-
ING ACUTE AND CHRONIC EXPOSURES OF SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) IN FLOWING SEAWATER.
Concentration (yg/&)
Test Nominal
0
Acute Control
420
560
750
1,000
1,300
Chronic Control
Sol. Control
31
62
125
250
500
Measured
hour
cO.l
230
380
480
640
860
Mean
<0.1
_a
6
15
23
49
100
96 hour
<0.1
120
270
220
280
760
S.D.
-
±4
±11
±21
±44
±93
Mean '
_
175
325
350
460
810
Range '
2-12
-
2-12
1-29
1-65
2-150
20-270
£ of nom.
__
44
58
47
46
62
\ of nom.
_
-
19
24
18
20
20
# samples
7
-
7
10
12
12
10
aNot analyzed.
TABLE 4. ANALYSIS OF PARENT AND HYDROLYZED CARBOFURAN IN SEA-
WATER SAMPLES COLLECTED FROM THE NOMINAL CONCENTRATION
OF 500 yg/£ DURING A 131-DAY EXPOSURE OF SHEEPSHEAD
MINNOWS (CYPRINODON VARIEGATUS).
Test
Day
12
27
34
41
55
62
70
76
84
105
Parent
Carbofuran
(yg/£;ppb)
200
120
210
79
29
37
28
38
85
20
Hydrolyzed
Carbofurana
<1.0
<1.0
6.1
<1.0
<1.0
2.1
9.5
10.0
17.0
18.1
aThe percentage recovery of the hydrolyzed carbofuran from water
is unknown. Therefore, these values are relative to each other.
16
-------
Our justification for and methodology of determining the
hydrolyzed products of carbofuran are as follow:
Carbofuran and 3-hydroxycarbofuran were completly hydro-
lyzed within 24 hours when saturated solutions were made pH 9.2
and stored at 25°C (FMC, 1969). Similar to the base-catalyzed
degradation of Sevin® to 1-hydroxynaphthol, carbofuran was ex-
pected to eliminate the methylcarbamate group to form an hydroxy-
sub-stituent at the benzyl-oxygen. Therefore, an analytical pro-
cedure featuring derivatization of 1-naphthol was utilized to
verify any degradation products of carbofuran which possess ac-
tive hydroxy-substituents. Parent carbofuran is not detected by
this analyses since it does not contain the reactive hydroxy sub-
stituent.
Approximately 2 g of carbofuran were added to 300 m£ of nor-
mal sodium hydroxide and heated to 60°C for six hours. The solu-
tion was cooled, hydrochloric acid was added to adjust the solu-
tion to pH 4.0, and the hydrolyzed carbofuran was extracted into
methylene chloride. The solvent was evaporated and a portion of
the hydrolyzed carbofuran was weighed and dissolved in benzene
to produce the hydrolyzed carbofuran working standard solution.
An analytical procedure (U. S. Environmental Protection
Agency, 1974) designed for the gas chromatographic determination
of 1-naphthol in urine, following derivatization with chloroace-
tic anhydride, was utilized to verify the hydrolysis of carbo-
furan. Working standards of hydrolyzed carbofuran were deriva-
tized and chromatographed under operating conditions previously
described (except by using electron capture detection) with the
following results:
Weight of hydrolyzed Peak height response (mm)
Carbofuran/7 m£ benzene Retention~~\Retention
(yg) time " 3'° min' time = 4'2 min<
0 4
1 5
2 6
5 15
10 31
3
9.5
18
41
116
An aliquot of the seawater sample extracts (see TABLE 4) was
derivatized and the gas chromatograms were examined for the pre-
sence of quantity of hydrolyzed carbofuran. A graph of peak
height versus weight of hydrolyzed carbofuran was constructed,
17
-------
by using the peak eluting in 4.2 minutes, and any hydrolyzed
carbofuran found was determined with the graph. The data are
presented as relative concentrations found in the seawater since
the extraction efficiency of the hydrolyzed carbofuran moiety
from the seawater was unknown.
The concentrations of hydrolyzed carbofuran in seawater
continued to increase during the test period. It is important
to note that sample extracts were not treated with an aqueous
base, but were derivatized directly. Therefore, any hydrolysis
product of carbofuran detected was extracted from the seawater
sample. Additionally, parent carbofuran working standards sur-
vived the derivatization procedure virtually unchanged and the
peaks at 3.0 and 4.2 minutes due to hydrolyzed carbofuran were
not observed.
ACUTE TOXICITY
The acute toxicity of methoxychlor and malathion to sheeps-
head minnows was similar; carbofuran was one order of magnitude
less toxic (TABLE 5).
TABLE 5. ACUTE TOXICITY OF THEEE PESTICIDES TO SHEEPSHEAD MIN-
NOWS (CYPRINODON VARIEGATUS) EXPOSED IN FLOWING SEA-
WATER. SEAWATER FOR THE METHOXYCHLOR AND MALATHION
TESTS WAS NATURAL; THAT FOR THE CARBOFURAN TEST WAS
SYNTHETIC. CALCULATIONS WERE BASED ON MEASURED CON-
CENTRATIONS OF EACH PESTICIDE.
Compound
Methoxychlor
Malathion
Carbofuran
96-hour
LC50, \iq/H
49
51
386
95% confidence
limits, pg/£
37-65
41-63
311-480
Salinity
(°/oo)
23
20
21
Temperature
(°C)
30
29
22
The acute toxicity of methoxychlor to sheepshead minnows
tested under dynamic conditions was within the range reported
for other estuarine fishes under static conditions, where esti-
mated 96-hour LCSO's ranged from 12-150 yg/&. In static tests
with two cyprinodontid fishes, Eisler (1970) estimated 96-hour
LC50 values of 30 and 36 yg/£ for the striped killifish
(Fundulus majalis) and mummichog (F. heteroclitus), respectively.
In flowing water tests, malathion was more acutely toxic to
sheepshead minnows than to the freshwater fathead minnow (96-
hour LC50 9,000 yg/£) (Mount and Stephan, 1967) or bluegill
18
-------
(96-hour LC50 108 yg/A) (Eaton, 1970). Similarly, sheepshead
minnows were more sensitive than were all but one of the seven
estuarine fishes tested under static conditions by Eisler (1970),
including the striped killifish and mummichog, for which the 96-
hour LCSO's were 250 and 240 vg/l, respectively.
Carbofuran was of the same order of toxicity to sheepshead
minnows as to three freshwater fishes tested under static condi-
tions. Reported 96-hour TLM's (median tolerance limits; same
as LC50) for rainbow trout (Salmo gardneri) , channel catfish
(Ictalurus punctatus), and bluegill were 280, 210, and 240 yg/£,
respectively (Anonymous, 1971).
CHRONIC TOXICITY
Methoxychlor
Methoxychlor affected parental fish in the 140-day study.
Exposure to 48 yg/£ was lethal to 100% of the fish in one dupli-
cate after 10 days and after 15 days in the other. Mortality of
fish exposed to 23 yg/£ was significantly greater than mortality
of control fish (TABLE 6).
TABLE 6. PERCENTAGE MORTALITY OF PARENTAL SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) EXPOSED TO METHOXYCHLOR IN
FLOWING SEAWATER. MORTALITY IS THE AVERAGE FROM DU-
PLICATE AQUARIA AND DOES NOT INCLUDE DEATHS WHICH OC-
CURRED IN THE SPAWNING CHAMBERS.
Concentration (yg/&)
Day
1-30
31-60
61-90
91-120
121-140
Control
0
0
0
0
0
Solvent
Control
0
0
0
0
0
3
0
0
0
0
0
5
2
0
0
0
0
12
0
0
0
0
0
23.
10
8
2
0
0
48
100
-
-
-
—
Total 0 0020 20a 100a
aSignificantly different from the control.
Growth of parental fish exposed to methoxychlor was not
significantly different from growth of control fish. Although
growth was monitored biweekly, only measurements at the begin-
ning, middle, and end of the exposure are presented (TABLE 7).
19
-------
TABLE 7. GROWTH OF SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS)
EXPOSED FOR 140 DAYS TO METHOXYCHLOR IN FLOWING, NAT-
URAL SEAWATER. MEAN STANDARD LENGTH AND STANDARD DE-
VIATION ARE GIVEN IN CENTIMETERS AND WERE DETERMINED
PHOTOGRAPHICALLY. AVERAGE WEIGHT IS GIVEN IN GRAMS
AND WAS DETERMINED IN WATER.
Concentration
(ygA)
Control
Sol. control
3
5
12
23
48
Day
Length
(cm)
1.4±0.2
1.4±0.2
1.4±0.2
1.4±0.2
1.5±0.2
1.5±0.3
1.5±0.2
0
Wt.
(Sl
0.1
0.1
0.1
0.1
0.1
0.1
0.1
Day 58
Length
(cm)
2.9±0.2
2.8±0.3
2.8±0.3
2.7±0.3
2.8±0.3
2.8±0.4
^a
Wt.
(g)
0.6
0.6
0.3
0.5
0.6
0.7
—
Day
Length
(cm)
3.7±0.5
3.6±0.4
3.5±0.5
3.5±0.4
3.510.4
3.7±0.4
—
140
Wt.
. IaL
1.3
1.2
1.2
1.0
1.2
1.5
—
aAll fish had died.
Fecundity (total eggs spawned) of exposed fish was not sig-
nificantly different from that of control fish (TABLE 8). Be-
cause female fish were killed by male fish in spawning chambers
in all treatments except 23 yg/£, we calculated eggs per female
spawning day- These values were obtained by dividing the num-
ber of eggs obtained from a spawning chamber during a 24-hour
period by the number of live female fish in the spawning chamber
during the same period. There was no significant difference be-
tween eggs per female spawning day in any treatment because of
variability within the treatments, but fewer eggs were spawned
per female spawning day by fish exposed to 5, 12, and 23 yg/£
than were spawned by control fish (TABLE 9).
No female fish were killed by male fish in spawning chambers
in 23 yg/£, although 1 to 6 females were killed in spawning cham-
bers in lower concentrations and controls. Based on observations
of fish in the spawning chambers, a probable reason is that ex-
posure to 23 yg/fc of methoxychlor decreased aggressive spawning
activity of male fish.
Hatching success of fry from eggs spawned by fish exposed
to 23 yg/£ of methoxychlor was significantly less than hatching
success of control fry (TABLE 10).
Neither fry mortality to 28 days posthatch nor growth of
the fry was significantly affected by exposure to methoxychlor
(TABLE 11).
20
-------
TABLE 8. NUMBER OF EGGS SPAWNED BY SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS)
EXPOSED TO METHOXYCHLOR IN
10-DAY SPAWNING PERIODS.
WERE PLACED IN A SPAWNING
FLOWING, NATURAL SEAWATER DURING THREE
FIVE UNSPAWNED FISH (2 MALES AND 3 FEMALES)
CHAMBER IN EACH DUPLICATE AQUARIUM, A AND B.
Concentration
Day
Day
54
to
63
101
to
110
116
to
125
™?!L
TOTAL
Control
A B
443 253a
778 514
342a 413a
1,563 1,180
2,743
Solvent
Control
ABA
244a 809 746
86a 1,183 322
367a 934 528
697 2,926 1,596
3,623
3
B
921
a 991
793
2,705
4,301
(vgA)
5 12
A BAB
49 575 162 399
476 277a 94 384
563a 829 517 163a
1,088 1,681 773 946
2,769 1,719
23
A B
91 5
189 570
38 1,085
318 1,660
1,978
aDeaths occurred in spawning chamber.
-------
TABLE 9. NUMBER OF EGGS SPAWNED PER DAY PER FEMALE SHEEPSHEAD MINNOW
(CYPRINODQN VARIEGATUS) EXPOSED TO METHOXYCHLOR IN FLOWING,
to
NATURAL SEAWATER DURING THREE 10-DAY SPAWNING PERIODS.
UNSPAWNED FISH (2 MALES AND 3 FEMALES) WERE PLACED IN A
ING CHAMBER IN EACH DUPLICATE AQUARIUM, A AND B.
FIVE
SPAW
Concentration (yg/£)
Day
54-63
101-110
116-125
Mean of
duplicate
Mean of
treatment
Control
A
15
26
16
19
B
11
17
15
14
17±5
Solvent
Control
A
12
5
12
10
B
27
39
31
32
21±13
3
A
25
11
18
18
25±
5
B
31
38
26
32
9
A
2
16
20
13
B
19
11
28
19
16±9
12
A B
5 13
3 13
17 8
8 11
10±5
23
A
3
6
1
3
B
0
19
26
18
11±14
-------
TABLE 10. HATCHING SUCCESS OF FRY FROM EGGS SPAWNED BY SHEEPS-
HEAD MINNOWS (CYPRINODON VARIEGATUS) EXPOSED TO
METHOXYCHLOR IN FLOWING, NATURAL SEAWATER. MEAN PER-
CENTAGE HATCH AND STANDARD DEVIATION REPRESENTS POOLED
DATA FROM DUPLICATE AQUARIA DURING THREE 10-DAY SPAWN-
ING PERIODS.
Concentration
(ygA)
Mean percentage
hatch and S.D.
Numbers of eggs
examined
Sol
Control
. control
3
5
12
23
98±3
98±4
98±3
95±6
97±2
73±18a
1,200
2,040
2,400
1,450
700
1,055
aSignificantly different from the control.
TABLE 11. PERCENTAGE MORTALITY, AVERAGE STANDARD LENGTH, AND
WEIGHT (DETERMINED IN WATER) OF 28-DAY OLD SHEEPSHEAD
MINNOW (CYPRINODON VARIEGATUS) FRY HATCHED FROM EGGS
SPAWNED BY FISH EXPOSED TO METHOXYCHLOR FOR 54-63
DAYS.
Concentration
Sol
(yg/£)
Control
. control
3
5
12
23
Number
of fry
160
160
160
80
120
59
Mortality
Length
(%) (cm)
0
0
1
0
4
4
1.
1.
1.
1.
1.
1.
4±0.
4±0.
4±0.
3±0.
4±0.
5±0.
1
1
1
1
1
2
Weight
(g)
0.
0.
0.
0.
0.
0.
07
05
07
06
06
06
Methoxychlor was accumulated by adult fish exposed continu-
ously for 140 days. The pesticide was also accumulated in eggs
spawned by these fish. Accumulation was dependent upon water
concentration during exposure (TABLE 12). Concentration factors
(based on measured water concentrations) ranged from 113-264.
These values are much lower than concentration factors for other
chlorinated hydrocarbon pesticides and marine fishes (TABLE 13).
A maximum concentration of 1.1 yg/g was detected in eggs spawned
by fish exposed to 12
23
-------
TABLE 12. CONCENTRATIONS OF METHOXYCHLOR IN SURVIVING ADULT
SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS) EXPOSED
FOR 140 DAYS. MEAN TISSUE RESIDUES ARE WHOLE-BODY,
WET-WEIGHT. DUPLICATE ANALYSES OF EACH POOLED SAMPLE
(AT LEAST 2 FISH PER SAMPLE) WERE PERFORMED.
Concentration
Water
(pg/£)
Control
Sol. control
3
5
12
23
Tissue
(yg/g)
<0.1
<0.1
0.3410.24
1.32±0.24
1.38+0.25
3.18+0.53
Concentration
factor
—
-
113
264
115
138
Number
of samples
4
4
6
6
6
6
TABLE 13. ACCUMULATION OF ORGANOCHLORINE PESTICIDES BY MARINE
FISHES. CONCENTRATION FACTORS WERE DERIVED BY DI-
VIDING CONCENTRATIONS IN FISH (WHOLE-BODY, WET-WEIGHT)
BY CONCENTRATIONS IN TEST WATER.
Concentration
Pesticide Fish factor Exposure Source
(maximum) (days)
DDT
Dieldrin
Atlantic
croaker
Pinfish
Spot
16,300a
40,000a
6,700b
21-35
14
35
Hansen and Wilson,
1970
Parrish et al. ,
1973
Endrin
Sheepshead
minnows
4,800b
33
Schimmel et al. ,
•1975
^Based on nominal water concentration.
"Based on measured water concentration.
Malathion
Malathion affected survival of parental fish but did not
affect their growth or fecundity (TABLES 14-17). Exposure to
86 yg/& was lethal to 100% of the fish in one duplicate and 95%
24
-------
in the other after 5 days; exposure to 37 v-g/t* was lethal to 100%
in one duplicate and 80% in the other after 30 days. Exposure
to 18 yg/£ was lethal to 50% of the fish in both duplicates af-
ter 86 days. No deaths occurred in any concentration or control
after day 90 of the 140-day study.
TABLE 14. PERCENTAGE MORTALITY OF SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) EXPOSED TO MALATHION IN
FLOWING, NATURAL SEAWATER FOR 140 DAYS. MORTALITY
IS THE AVERAGE FROM DUPLICATE AQUARIA AND DOES NOT
INCLUDE DEATHS WHICH OCCURRED IN THE SPAWNING CHAM-
BERS.
Concentration (pg/£)
Day
1-30
31-60
61-90
91-120
121-140
Control
0
0
2
0
0
Solvent
Control
0
0
0
0
0
4
0
0
5
0
0
9
0
0
0
0
0
18
5
23
22
0
0
37
90
8
2
—
-
86
100
_
—
—
-
Total
0 50C
100C
100'
aSignificantly different from the control.
TABLE 15. GROWTH OF SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS)
EXPOSED FOR 140 DAYS TO MALATHION IN FLOWING, NAT-
URAL SEAWATER. MEAN STANDARD LENGTH AND STANDARD
DEVIATION ARE GIVEN IN CENTIMETERS AND WERE DETER-
MINED PHOTOGRAPHICALLY. AVERAGE WEIGHT IS GIVEN IN
GRAMS AND WAS DETERMINED IN WATER.
Concentration
Day 0
Length Wt.
Day 61
Day 140
Length
Wt.
Length Wt.
Control
Sol. control
4
9
18
37
86
1.4±0.2
1. 3±0.2
1.4±0.2
1.3±0.2
1.3+0.2
1.4±0.2
1.4±0.2
0.1
0.1
0.2
0.1
0.1
0.1
0.1
2.8±0.4
2.7±0.6
2.7±0.3
2.8±0.3
2.6±0.4
2.4±0.2
_b
0.7
0.8
0.7
0.7
0.6
0.5
-
4.1±0.4
4.1+0.5
3.9±0.4
3.9±0.4
3.7±0.3
_b
-
1.4
1.4
1.2
1.3
_a
_
-
aAll fish were used for acetylcholinesterase inhibition
analyses.
bAll fish had died.
25
-------
TABLE 16. NUMBER OF EGGS SPAWNED BY SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) EXPOSED TO MALATHION IN
FLOWING, NATURAL SEAWATER DURING TWO 10-DAY SPAWN-
ING PERIODS. FIVE UNSPAWNED FISH (2 MALES AND 3
FEMALES) WERE PLACED IN A SPAWNING CHAMBER IN EACH
DUPLICATE AQUARIUM, A AND B.
Concentration (yg/Ji)
Day
87-96
119-128
SUBTOTAL
TOTAL
Control
A
154a
538
692
1,
B
747a
258
1,005
697
Solvent
Control
A
111
364
1,141
2,
B
626
325
951
092
A
422
535
957
2,
4
B
445
729
1,174
131
A
341
738
1,079
1,
9
B
a 481a
390
871
950
18
A
299
_b
299
1,
B
353
374a
727
026
aDeath(s) occurred in spawning chamber.
spawning chamber; 50% mortality had occurred.
TABLE 17. NUMBER OF EGGS SPAWNED PER DAY PER FEMALE SHEEPSHEAD
MINNOW (CYPRINODON VARIEGATUS) EXPOSED TO MALATHION
IN FLOWING, NATURAL SEAWATER DURING TWO 10-DAY SPAWN-
ING PERIODS. FIVE UNSPAWNED FISH (2 MALES AND 3 FE-
MALES) WERE PLACED IN A SPAWNING CHAMBER IN EACH
DUPLICATE AQUARIUM, A AND B.
Concentration (yg/£)
Day
87-96
119-128
Average of
duplicate
Mean of
treatment
Control
A B
11 30
18 9
14 20
17±9
Solvent
Control
A
26
12
19
18±
B
21
11
16
7
4
A B
14 15
18 24
16 20
18±4
9
A B
16 21
25 13
20 17
19±5
18
A
10
_a
10
14±
B
21
19
16
5
aNo
spawning chamber; 50% mortality had occurred.
Malathion did not affect hatching success of fry from eggs
26
-------
spawned (TABLE 18) but did affect the fry. Mortality of fry
hatched from eggs spawned by fish exposed to 9 and 18 yg/£ of
malathion was significantly greater than mortality of control
fry. Growth of surviving fry was not affected (TABLE 19).
TABLE 18. HATCHING SUCCESS OF FRY FROM EGGS SPAWNED BY SHEEPS-
HEAD MINNOWS (CYPRINGDON VARIEGATU5) EXPOSED TO MALA-
THION IN FLOWING, NATURAL SEAWATER. MEAN PERCENTAGE
HATCH AND STANDARD DEVIATION REPRESENTS POOLED DATA
FROM DUPLICATE AQUARIA DURING TWO 10-DAY SPAWNING
PERIODS.
Concentration
(yg/£)
Percentage
hatch
Number of eggs
examined
Control
Sol. control
4
9
18
97±5
99±2
97±3
97±2
96±6
850
700
1,000
900
350
TABLE 19. PERCENTAGE MORTALITY, AVERAGE STANDARD LENGTH, AND
WEIGHT (DETERMINED IN WATER) OF 28-DAY OLD SHEEPSHEAD
MINNOW (CYPRINODON VARIEGATUS) FRY HATCHED FROM EGGS
PRODUCED BY FISH EXPOSED TO MALATHION FOR 87-96 DAYS.
Concentration
(yg/£)
Control
Sol. control
4
9
18
Number
of fry
160
160
160
160
120
Mortality
(%)
7
10
9
14b
15b
Length
(cm)
1.0±0.2
_a
1.1±0.2
1.2±0.2
1.2±0.1
Weight
(g)
0.04
0.03
0.04
0.04
0.03
^No data.
^Significantly different from the control.
Malathion was not detectable (<0.1 yg/g) in fish sampled at
the end of the 140-day study- This is not surprising because
fish readily convert malathion to the mono- and dicarboxylic
acids of malathion (Cook and Moore, 1976).
Carbofuran
Carbofuran affected survival of parental fish exposed to
the pesticide for 131 days. Exposure to 100 yg/£ was lethal to
27
-------
100% of the fish in one duplicate and 95% in the other after 14
days. Mortality of fish exposed to 49 ppb was significantly
greater than mortality of control fish after 30 days of exposure
(TABLE 20).
TABLE 20. PERCENTAGE MORTALITY OF SHEEPSHEAD MINNOWS (CYPRINODON
VARIEGATUS) EXPOSED TO CARBOFURAN IN FLOWING, SYNTHE-
TIC SEAWATER. MORTALITY IS THE AVERAGE FROM DUPLICATE
AQUARIA AND DOES NOT INCLUDE DEATHS FROM SPAWNING
ACTIVITY.
Concentration (yg/S,)
Day
1-30
31-60
61-90
91-131
Control
0
5
2
0
6
5
5
0
0
15
2
8
5
0
23
5
10
0
0
49
40
10
0
0
100
100
-
-
—
Total 7 10 15 15 50a 100a
aSignificantly different from the control.
Carbofuran did not significantly affect growth of parental
fish or number of eggs spawned in any concentration (TABLES 21-
22) .
TABLE 21. GROWTH OF SHEEPSHEAD MINNOWS (CYPRINODQN VARIEGATUS)
EXPOSED FOR 131 DAYS TO CARBOFURAN IN FLOWING, SYN-
THETIC SEAWATER. MEAN STANDARD LENGTH AND STANDARD
DEVIATION ARE GIVEN IN CENTIMETERS AND WERE DETER-
MINED PHOTOGRAPHICALLY. .AVERAGE WEIGHT IS GIVEN IN
GRAMS AND WAS DETERMINED IN WATER.
Concentration
(vgA)
Control
6
15
23
49
100
Day 2
Length
2.9±0.4
3.8±0.5
3.1±0.4
2.810.4
2.8±0.5
3.010.5
Day 60
Length
3.8+0.3
3.610.4
3.6+0.3
3.210.4
3.310.3
_a
Day
Length
4.H0.3
4.010.2
3.9+0.3
3.810.3
3.7+0.5
—
131
Weight
*
1.15
1.10
1.16
0.92
1.05
—
aAll fish had died.
28
-------
TABLE 22. NUMBER OF EGGS SPAWNED BY SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) EXPOSED TO CARBOFURAN IN
FLOWING, SYNTHETIC SEAWATER. ALL POSSIBLE PAIRS
OF FISH IN EACH DUPLICATE AQUARIUM WERE SPAWNED.
Concentration Number of eggs
(yg/&) Replicate A Replicate B Total
Control
6
15
23
49
650
854
848
475
248
449
852
1,295
820
154
1,099
1,706
2,143
1,295
402a
aFifty percent mortality had occurred.
Hatching success of fry from eggs spawned by fish exposed
to 49 yg/£ was significantly less than hatching success of con-
trol fry (TABLE 23).
TABLE 23. HATCHING SUCCESS OF FRY FROM EGGS SPAWNED BY SHEEPS-
HEAD MINNOWS (CYPRINODON VARIEGATUS) EXPOSED TO CAR-
BOFURAN IN FLOWING, SYNTHETIC SEAWATER. MEAN PER-
CENTAGE HATCH AND STANDARD DEVIATION REPRESENTS
POOLED DATA FROM SPAWNING PAIRS IN DUPLICATE AQUARIA.
Concentration Percentage Number of eggs
(yg/&) hatch examined
Control
6
15
23
49
98±3
99±3
96±10
98±2
86±10a
450
700
937
609
233
aSignificantly different from the control.
Mortality of fry hatched from eggs spawned by fish exposed
to 23 and 49 yg/2. was significantly greater than mortality of
control fry. Growth of surviving fry in all concentrations was
not affected, however (TABLE 24).
29
-------
TABLE 24. PERCENTAGE MORTALITY AND AVERAGE STANDARD LENGTH OF
30-DAY OLD SHEEPSHEAD MINNOW (CYRPINODON VARIEGATUS)
FRY WHICH WERE HATCHED FROM EGGS SPAWNED BY FISH EX-
POSED TO CARBOFURAN FOR 42-95 DAYS IN FLOWING, SYN-
THETIC SEAWATER. MORTALITY AND LENGTH ARE AVERAGES
OF FOUR GROUPS OF 40 FRY EXCEPT AS NOTED.
Concentration
(pgA)
Control
6
15
23
49
Number
of fry
160
160
160
120
80
Mortality
(%)
5
2
8
12a
41a
Length
(cm)
1.4
r. 3
1.3
1.4
1.4
aSignificantly different from the control.
APPLICATION FACTORS
Application factors were calculated from the results of the
acute and chronic toxicity tests (TABLE 25).
TABLE 25. CONCENTRATIONS (yg/£) OF THREE PESTICIDES TOXIC TO
SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS) IN ACUTE
AND CHRONIC TESTS, AND THE RELATIONSHIP OF ACUTE TOX-
ICITY TO CHRONIC TOXICITY.
96-hour LC50
(95% confidence Application
Pesticide limits) MATC limits factor limitsa
Methoxychlor
Malathion
Carbof uran
49
(37-65)
51
(41-63)
386
(311-480)
>12<23
>4<9
>15<23
0.
0.
0.
24-0.
08-0.
04-0.
47
18
06
aDerived by dividing the Maximum Acceptable Toxicant Concentra-
tion limits by the 96-hour LC50.
We find no application factors in the literature on which
to base a comparison of the sensitivity of sheepshead minnows
and any freshwater fish to methoxychlor or carbofuran. For
30
-------
malathion, however, studies by Mount and Stephan (1967) with
fathead minnows and Eaton (1970) with bluegill show that the
application factors derived for all three fishes were similar
(TABLE 26).
TABLE 26. COMPARISON OF ACUTE AND CHRONIC MALATHION TOXICITY
TO TWO FRESHWATER FISHES AND A SALTWATER FISH.
96-hour LC50
(ygA)
MATC limits
(ygA)
Application
factors limits
Fathead
minnows a
9,000
200-580
0.02-0.06
Bluegillsb
108
4-7
0.04-0.06
Sheepshead
minnows
51
4-9
0.08-0.18
aFrom Mount and Stephan, 1967.
bFrom Eaton, 1970.
SUMMARY
1. Carbofuran was less toxic to sheepshead minnows than were
methoxychlor and malathion in acute tests. Estimated 96-
hour LCSO's, based on average measured concentrations in
water, were 386, 49, and 51 yg/£, respectively.
2. All three pesticides killed parental fish in concentrations
<50 yg/£ during chronic tests, and the lowest concentrations
of the pesticides in which toxic effects were observed were
similar (TABLE 27).
3. The life stages of progeny from exposed parental fish that
were sensitive to each pesticide were: methoxychlor—embryo;
malathion—fry; and carbofuran—embryo and fry.
4. The relationship of acute toxicity and chronic toxicity for
sheepshead minnows exposed to malathion (as expressed by ap-
plication factors) was similar to that for two freshwater
fishes exposed to malathion.
5. Sheepshead minnows are a suitable estuarine fish for toxi-
city tests which include the reproductive portion of the
life cycle and the first generation.
31
-------
TABLE 27. SUMMARY OF SIGNIFICANT EFFECTS OF METHOXYCHLOR,
MALATHION, AND CARBOFURAN ON SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) DURING CHRONIC EXPOSURES
IN
Methoxychlor
Malathion
Carbofuran
FLOWING SEAWATER.
Generation Life stage
Parental Adult
FI Embryo
Parental Adult
FI Fry
Parental Adult
FI Embryo
Fry
Effect
Death
Decreased
hatch
Death
Increased
mortality
Death
Decreased
hatch
Increased
mortality
Measured-
concentration
>23
23
>18
9 and 18
>49
49
23
32
-------
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Eisler, R. 1970. Factors affecting pesticide-induced toxicity
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33
-------
FMC. 1969. Personal communication. FMC Corporation, Agricul-
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36
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TECHNICAL RCPORT DATA
(l'ii die rmJ ImLj-iii lioini u.'i tin' /ci c r\( In I"T i umi'li luii;!
REPORT NO.
ERL-GB-0010
3 RLCIPI ENT'S ACCtSSIO^* NO.
TITLE AND SUBTITLE
Chronic Toxicity of Methoxychlor, Malathion,
and Carbofuran to Sheepshead Minnows
(Cyprinodon variegatus)
6. Pi.RFORMING ORGANIZATION CODE
AUTHOHI5)
Patrick R. Parrish, Elizabeth E. Dyar, Mark
A. Lindberg, Chiara M. Shanika, and Joanna M.
Enos
0. PERFORMING ORGANIZATION RCPORT NO.
PERFORMING ORGANIZATION NAME AND ADDRESS
EG&G, Bionomics
Marine Research Laboratory
Route 6'-, Box 1002
Pensacola, Florida 32507
12..SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U,S. Environmental Protection Agency
Gulf Breeze, Florida 32561
. REPORT DAT E
April 1977 (Issuing Date)
10. PROGRAM LLEM1NT NO.
1EA615
11. CONTRACT/GRANT NO.
68-03-0264
13. TYPE OF REPORT AND PERIOD COVf RED
Final
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Sheepshead minnows (Cyprinodon variegatus) were exposed to each of
three pesticides—methoxychlor, malathion, and carbofuran--in flowing sea
water to determine the acute and chronic (partial life-cycle) effects.
Mortality of adult fish exposed to concentrations of methoxychlor
>23 yg/£ and hatching success of fry from eggs spawned by fish exposed to
23 yg/£ were significantly different from the control. The maximum ac-
ceptable toxicant concentration (MATC) was estimated to be >12<23 yg/£;
application factor (AF) limits were 0.24-0.47.
Mortality of adult fish exposed to concentrations of malathion ~>18
ygA and mortality of fry hatched from eggs spawned by fish exposed to 9
and 18 yg/£ were significantly different from the control. The MATC was
estimated to >4<9 yg/£; AF limits were 0.08-0.18.
Mortality of adult fish exposed to concentrations of carbofuran >49
yg/&, hatching success of fry from eggs spawned by fish exposed to 49
vg/^f and mortality of fry hatched from eggs spawned by fish exposed to
23 and 49 ygA were significantly different from the control. The MATJC
was estimated to >15<23 ygA; AF limits were 0.04-0.06.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Toxicity
Fish
Saltwater
Pesticides
Methoxychlor
Malathion
Carbofuran
b. IDENTIFIERS/OPEN ENDED TERMS
Chronic toxicity
Flowing seawatfer
Application factor
i1. COSATI 1 i
13. DISTRIBUTION STATEMENT
Release unlimited
10. SECURITY CLAbS / //llt'A'r/
Unclassified
NO. Of F'AGt S
36
20 SECURITY CLASS t.Y'.'i/t / dy
Unclassified
''.>. TRICE
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