660/3-75-015
MAY 1975
Ecological Research Series
Effects of Mirex and Methoxychlor
on Striped Mullet, Mug// cephalus L
national Environmental Research Center
Office of Research and Development
U.S. Environmental Protection Agency
CorvaNis, Oregon 97330
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EPA-660/3-75-015
MAY 1975
EFFECTS OF MIREX AND METHOXYCHLOR ON
STRIPED MULLET, Mugil cephalus L.
by
Jong H. Lee, Colin E. Nash and Joseph R. Sylvester
Project Officer
David J. Hans en
Gulf Breeze Environmental Research Laboratory
National Environmental Research Center
Sabine Island
Gulf Breeze, Florida 32561
Grant No. R 802348
Program Element No. 1EA077
ROAP/TaskNo. 10 AKC/040
NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
For Sale by the National Technical Information Service
U.S. Department of Commerce, Springfield, VA 22151
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ABSTRACT
The effects of two chlorinated insecticides, mirex and methoxychlor, on eggs,
larvae, juveniles, and adults of the striped mullet, Mugil cephalus L., were
studied. Test concentrations of both insecticides used were 0. 01, 0.1, 1. 0 and
10.1 mg/1 in dynamic bioassays of juveniles and adults and static bioassays of
eggs and larvae.
Young juveniles, standard length 20-43 mm, were apparently more susceptible
to mirex exposure than older juveniles, standard length 70 - 150 mm, and
adults, standard length 260 - 380 mm. No mortalities occurred in older juve-
niles and adults exposed to mirex for 96 hours. Young juveniles exposed to 0.1
and 1. 0 mg/1 of mirex had higher mortality rates (27 and 32 percent) than con-
trol fish, but mortalities of 0. 01- and 10. 0-mg/l exposed fish were not different
from controls. Significant amounts of mirex residues were accumulated in the
body tissue of the test fish. Juveniles and adults exposed to 0.01 mg/1 mirex
were found to accumulate 0.2 and 0.4 ptg/g respectively during the bioassays.
The concentrations in fish increased with increasing mirex concentrations in
test water. The highest accumulation was found in the adult fish exposed to
10.0 mg/l(37.3pg/g).
Methoxychlor was more toxic to mullet than mirex. Mortalities were greater
than 90 percent over a 96-hour period for juvenile and adult fish at concentra-
tions of 0.1, 1. 0 and 10. 0 mg/1. Methoxychlor residues accumulated in the tis-
sues of test fish in smaller amounts than mirex in mirex-exposed fish. Young
adults and juveniles exposed to 0. 01 mg/1 methoxychlor accumulated 0.1 and
0.2 ng/g respectively. The accumulated amounts in 10. 0 mg/1 concentration
were 1.7 jug/g for young juveniles and 11.1 # g/g for adult fish.
Results of the experiments on eggs and larvae were inconclusive because of the
natural high mortality of both stages in culture conditions. Egg and larval
survival was generally better in mirex than in methoxychlor over a 96-hour
period.
This report was submitted in fulfillment of Grant Number R 802348 by the
Oceanic Foundation under the sponsorship of the Environmental Protection
Agency. Work was completed as of April 30, 1974.
11
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CONTENTS
Section Page
I Conclusions 1
II Introduction 2
in Effects of mirex and methoxychlor on juvenile and adult 3
striped mullet, Mugil cephalus L.
IV Effects of mirex and methoxychlor on the eggs and larvae 13
of striped mullet, Mugil cephalus L.
V References 16
iii
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TABLES
No.
Analysis of seawater used in the continous-flow bioassay 4
for mirex and methoxychlor.
Percent mortality of striped mullet exposed to mirex for 7
96 hours in a continous-flow bioassay.
Concentration of mirex accumulated by striped mullet 8
exposed to mirex and measured concentration in water
for 96 hours in a continuous-flow bioassay.
Concentration of mirex accumulated by adult striped 8
mullet (278 - 310 mm) exposed to 0. 5 mg/1 mirex and
measured concentration in water for 96 hours in a
continuous-flow bioassay.
Percent mortality of striped mullet exposed to methoxy- 9
chlor for 96 hours in a continuous-flow bioassay.
Concentration of methoxychlor accumulated by striped n
mullet exposed to methoxychlor and measured concentra-
tion in water for 96 hours in a continuous-flow bioassay.
Concentration of methoxychlor accumulated by juvenile 11
striped mullet exposed to 10 fig/1 methoxychlor and
measured concentration in water for 96 hours in a
continuous-flow bioassay.
Concentration of methoxychlor accumulated by adult striped 12
mullet exposed to IQug/l methoxychlor and measured
concentration in water for 96 hours in a continuous-flow
bioassay.
Percent survival of striped mullet eggs in mirex and 14
methoxychlor and measured concentrations of both
insecticides in test water for 48 hours in a static bioassay.
iv
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TABLES (cont.)
No. Page
10 Percent survival of striped mullet larvae in mirex 15
and methoxychlor and measured concentration of both
insecticides for 96 hours in a static bioassay.
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SECTION I
CONCLUSIONS
1. Young juvenile mullet (20-43 mm standard length) were apparently more
susceptible to mirex exposure than older juveniles (70-150 mm standard length)
or adults (260-380 mm standard length).
2. Mirex was accumulated by mullet, and body concentrations in whole fish
(wet weight) increased with increased concentration in test water, which indi-
cates that the insecticide is stable in striped mullet.
3. Mirex was accumulated most (21.5 jug/g) in visceral organs and least
(1.4 jug/g) in skin and muscle of adult mullet for a 96-hour exposure period.
Gills and hearts accumulated 2.0 /zg/g for the same exposure time.
4. Methoxychlor was more toxic to mullet than mirex at concentrations of 0.1,
1. 0 and 10.1 mg/1 over a 96-hour period. However, mortalities of young juve-
niles in 0.1- and 1.0-mg/l mirex-treated water were 27 and 32 percent respec-
tively, by far the highest among mirex-exposed fish or controls. No mortality
occurred among older juveniles and adults exposed to mirex over the same
experimental period.
5. Relative to mirex, small amounts of methoxychlor were accumulated in
whole juvenile and adult mullet. Test fish exposed to 10. 0 mg/1 methoxychlor
contained 1.7 pig/g for juveniles and 11.1 /ug/g for adults over a 96-hour period.
6. Results with eggs and larvae were inconclusive because of the natural high
mortality of both stages in culture conditions. Eggs and larvae from the same
broodstock had a better survival rate in mirex than in methoxychlor.
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SECTION n
INTRODUCTION
Mirex, apolycyclic chlorocarbon insecticide (dodecachloroocta-hydro-1,3,4-
metheno-2H-cyclobuta[ cd] pentalene), has been used many years for control of
the imported fire ant (Solenopsis savissima richteri) in various southeastern
states (Coon and Fleet, 1970). Accumulation of mirex residues by various
species of aquatic organisms (Butler, 1969; Wolfe and Norment, 1973;
Borthwick et al., 1973) is indicative of the widespread occurrence and extent
of environmental contamination by this insecticide. The toxicity of mirex to
non-target organisms has been reported by a number of workers on selected
freshwater fishes (Van Valin et al., 1968), estuarine and freshwater inverte-
brates (Lowe et al., 1971; Naqyi et al., 1973), larval crabs (Bookout et al.,
1972), and freshwater crayfish (Ludke et al., 1971).
Methoxychlor (1,1, l-trichloro-2,2-bis[p-methoxyphenyl] ethane) has been a
commercial insecticide since discovery of its insecticidal properties (Lauger
et al., 1944), and has replaced DDT, especially for the control of blackfly
larvae in streams, elm bark beetles, and fruit and garden pests (Burdick et
al., 1968; Reinbold et al., 1971). The metabolism of the insecticide by warm-
blooded animals is rapid and its toxicity to many insects is similar to that of
DDT (Menzie, 1969). The physico-chemical properties of methoxychlor and
its toxicology to higher animals indicate that methoxychlor may be one of the
safest of all insecticides (Negherbon, 1959).
However, methoxychlor could be hazardous to aquatic life because it accumu-
lates in the environment like other chlorinated insecticides. The toxicity of
methoxychlor has been reported for decapod marine crustaceans (Eisler, 1969),
selected freshwater fish species, such as fathead minnow, goldfish, guppies,
and mummichog (Henderson et al., 1959; Eisler, 1970), bluegills and certain
freshwater invertebrates (Kennedy et al., 1970), and tadpoles (Sanders, 1970).
The purpose of this investigation was to determine the acute toxicity and accu-
mulation rates of mirex and methoxychlor to juvenile and adult striped mullet
(Mugil cephalus L.) using 96-hour continuous flow bioassays; and to eggs and
larvae in 48-hour and 96-hour static bioassays respectively. Striped mullet
have an extensive geographic distribution and can tolerate a wide range of
physical parameters in both fresh and saltwater environments (Sylvester et al.,
1974; Thompson, 1966). The striped mullet are predominantly herbivores and
detritivores and a highly desirable food fish in many parts of the world.
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SECTION HI
EFFECTS OF MIREX AND METHOXYCHLOR ON JUVENILE AND
ADULT STRIPED MULLET, Mugil cephalus L.
MATERIALS AND METHODS
Juvenile mullet used in this study were seined from coastal streams and bays
around the island of Oahu, Hawaii. Young juveniles ranged in standard length
from 20 to 43 mm and older juveniles ranged in standard length from 70 to 150
mm. Adult mullet, standard length 260 to 380 mm, were collected from the
island of Hawaii. All fish were transported to the laboratory in aerated tanks
and acclimated from ten days to two weeks in a 4,000-gallon vat or in ponds.
Mirex was obtained from the Allied Chemical Corporation and contained a mini-
mum of 95 percent active mirex. Methoxychlor contained a minimum of 88
percent active ingredient and was obtained from the E. I. du Pont de Nemour
Company.
BIOASSAY TECHNIQUES
Stock solutions of mirex and methoxychlor were prepared with acetone and
stored in two-gallon glass jars. Insecticide stock solutions were siphoned at
0.9 ml/min. into baffled mixing chambers where they mixed with 5-/n filtered
seawater (Table 1) flowing at 650 ml/min. The seawater solvent and insecti-
cide in the mixing chamber then flowed into a delivery chamber which supplied
the desired concentrations to each test aquarium. Acetone and seawater con-
trols were supplied with the same delivery system. The 20-gallon glass
aquaria maintained a constant volume of 55 liters of fluid.
The dosing apparatus and procedures of the test were similar to those described
by Burke and Ferguson (1968) and Sprague (1969). Four different concentra-
tions of one insecticide—0.01, 0.1, 1.0 and 10.1 mg/1—were run simultane-
ously with four replicates of each concentration, four controls containing sea-
water, and four with seawater and acetone. Total flow rate through the test
aquaria was 260 ml/min. of insecticide and seawater. The number of mullet
used in each test was 25 for young juveniles, 10 for older juveniles, and two
for adults. The test fish were transferred into the aquaria approximately 20
hours after the toxicant started flowing.
Mortalities were recorded daily during a 96-hour exposure period and percent
mortality adjusted for control mortality according to the method of Ludke et al.
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(1971):
= Sc-StxlOO
where M^. = percent mortality in insecticides
S_ = percent survival in controls
c
Sj. = percent survival in insecticides.
In accumulation tests of both mi rex and methoxychlor on juveniles and adults,
identical experimental apparatus were used. Desired concentrations were 0.5
mg/1 for mirex and 0. 01 mg/1 for methoxychlor. A set of five aquaria for one
insecticide was used in the continuous-flow tests for 96 hours. The number of
mullet used in each aquarium was ten for juveniles and two for adults. A
sample of fish was taken from the acclimation tank at the start of the experiment
before the rest were put under test (0-hour sample). Further samples were
taken at 8, 24, 48, and 96 hours with replicates at each time. Samples for
adults were taken 24, 48, 72, and 96 hours after they were transferred to the
test aquaria.
Table 1. ANALYSIS OF SEAWATER USED IN
THE CONTINUOUS-FLOW BIOASSAY
FOR MIREX AND METHOXYCHLOR.
Analysis Range
Temperature (°C)
Salinity (°/oo)
PH
Dissolved oxygen (ppm)
Phosphate (JJL mole)
Nitrate (fi mole)
Ammonia (fj. mole)
Mirex (ng/1)
Methoxychlor (fig/1)
23.0
32.5
7.6
4.5
3.5
40
1.4
- 25.6
- 32.9
-8.3
-6.5
-4.0
- 50
-2.1
ND*
ND*
*none detected: less than 0.1 /ig/1 mirex,
0.8 ng/1 methoxychlor.
Dissolved oxygen, temperature and salinity were monitored daily with Model 54
YSI oxygen meter and Bissett-Berman salinometer (Model 6263).
RESIDUE ANALYSIS
At the end of 96 hours, live fish were killed in acetone, then rinsed with ace-
tone and frozen for residue analysis. Dead fish in mirex-treated tanks were
-------
removed as soon as they were observed, then rinsed in acetone and kept in the
freezer. Methoxychlor was measured from fish killed by the insecticide and
pooled at the end of the bioassays, except those in 0. 01 mg/1 methoxychlor tanks
and controls (1-10 ppm). Frozen samples were minced in a laboratory blender.
Approximately 5 grams of the minced sample (duplicates from individual test
aquaria) were ground thoroughly with additions of anhydrous sodium sulfate in a
mortar and pestle. For fish tested for accumulation rates, skin and muscle,
gills and heart, and viscera were separately taken from individuals (10 for
juveniles, 2 for adults) and blended samples for each part were used for insecti-
cide analyses.
Insecticide was extracted from the mixture with four portions of 50 ml petroleum
ether in a water bath at 40°C and concentrated in 50-ml boiling flasks using a
rotary evaporator. The extract was then dissolved in 10 ml of petroleum ether
saturated with acetonitrile, transferred into a separatory funnel, and extracted
three times with 25-ml portions of acetonitrile saturated with petroleum ether.
The acetonitrile extract was then combined in a 500 ml separatory funnel con-
taining 300 ml of 2 percent sodium chloride solution and 50 ml of petroleum
ether. The ether extract was filtered through a 2-inch column of anhydrous
sodium sulfate in a 150 x 24 mm filter tube and condensed to ca. 5 ml using the
evaporator. The residue was transferred into a 30 cm x 15 mm i.d. chromato-
graphic column containing 4 inches of Florisil (60/100 mesh, activated at 130°C)
topped with 1/2-inch anhydrous sulfate, eluted with 6 percent diethyl ether in
petroleum ether, and reduced to an appropriate volume.
Water samples were collected during tests from aquaria for insecticide analysis.
The insecticides in the water samples were extracted with 6 percent diethyl
ether/hexane mixture. The extract was then dried with anhydrous sodium sul-
fate and reduced to an appropriate volume.
Identification and quantitative determination of the insecticides were made by
gas chromatography equipped with Ni-63-electron capture detector. Opera-
tional conditions of the two U-type glass columns (6 ft x 1/4 in. o. d.) were as
follows:
Column I Column H
Liquid phase 1.5% SP 2250 / 1.95% SP 2401 4% SE-30 / 6% SP 2401
Solid phase 100/120 mesh Supelcon AWDMCS 80/100 mesh Supelcon AWDMCS
Temperature, °C
Met 215 215
Column 200 200
Detector 275 275
N gas flow, cc/min.
2 Carrier 80 80
Purge 20 20
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All the organic solvents used were nanograde quality. Sodium chloride, sodium
sulfate and glass wool were pre-washed with hexane for the removal of any
possible contamination.
RESULTS AND DISCUSSION
The results indicate that young striped mullet juveniles are more susceptible to
mirex poisoning than either older juveniles or adults (Table 2). No mortalities
occurred among older juveniles or adults in the 96-hour bioassay. Among young
juveniles highest mortalities of 32.1 and 26.9 percent (adjusted mortality)
occurred in mirex concentrations of 1. 0 and 0.1 mg/1. However, mortalities in
10. 0 and 0. 01 mg/1 (9. 0 and 6.4 percent) did not much differ from those in con-
trols. The causes of the anomaly in mortality between extreme concentrations
of 10. 0 and 0. 01, and the intermediate ones of 1.0 and 0.1, are obscure. The
behavior of the young juveniles before death was similar in all test tanks and
control tanks.
The anomaly in mortality of juvenile striped mullet could result from a specific
action of mirex on the species and the physical parameters of the experimental
conditions. Inconsistencies in the effects of mirex on other fishes have been
reported elsewhere. In a study of the effects of mirex on bluegill, Lepomis
machrochirus, and goldfish, Carrasius auratus, Van Valin et al« (1968)
reported that bluegill exposed to 0. 0013 mg/1 and 1.0 mg/1 showed no relation-
ship between mortalities and mirex exposure. However, they observed 68.8
percent mortality in goldfish in 0.1 mg/1, and 85.5 percent mortality in
1.0 mg/1 during a 308-day experimental period.
The results of the mirex residue analyses and the measured concentrations of
mirex in water are presented in Table 3. The largest amounts of mirex were
found in the adults, possibly because adults may have a higher proportion of
body fat than juveniles. Amounts of mirex in fish increased with increasing
insecticide concentration in test water. An intensive study of the mirex toxicity
to estuarine organisms by Lowe et al. (1971) showed that juvenile pinfish,
Lagodon rhomboides, concentrated up to 40 mg/kg of mirex in their tissues when
fed food having 20 mg/kg mirex with the weekly addition of fire ant bait to the
aquaria over a period of five months.
Adult mullet, 278-310 mm in standard length, were exposed to 0.5 mg/1 of
mirex in water for 96 hours in a continuous-flow system. Organs from various
parts of the test mullet were analyzed for mirex accumulation (Table 4). The
results indicate that mirex was concentrated most in visceral organs and least
in skin and muscle parts. Amounts of mirex found in 96-hour samples were
21.5 ng/g from viscera, 2.0 jxg/g from gills and heart, and 1.4 ng/g from skin
and muscle.
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Table 2. PERCENT MORTALITY OF STRIPED MULLET EXPOSED TO MIREX
FOR 96 HOURS IN A CONTINUOUS-FLOW BIOASSAY. FOUR REPLI-
CATE AQUARIA WERE USED FOR EACH CONCENTRATION AND
CONTROL. NUMBER OF FISH TESTED PER REPLICATE
AQUARIUM: 25 YOUNG JUVENILES, 10 JUVENILES, 2 ADULTS.
Age Group
Young
juvenile
20-43 mm
Juvenile
70-150 mm
V
Adult
260-380 mm
\
Mirex
(mg/1)
acetone
control
seawater
control
0.01
0.1
1.0
10.0
acetone
control
seawater
control
0.01
0.1
1.0
10.0
acetone
control
seawater
control
0.01
0.1
1.0
10.0
Mortality £
Observed
Mean S. D.
22.0 8.5
22.0 2.8
27.0 10.5
43.0 5.0
47.0 5.0
29.0 2.0
0
0
0
0
0
0
0
0
0
0
0
0
6)
Adjusted
Range
16 - 28
20- 24
16-36 6.4
36-48 26.9
40 - 52 32. 1
28-32 9.0
0
0
0
0
0
0
0
0
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Table 3. CONCENTRATION OF MIREX ACCUMULATED BY
STRIPED MULLET EXPOSED TO MIREX AND
MEASURED CONCENTRATION IN WATER FOR 96
HOURS IN A CONTINUOUS-FLOW BIOASSAY. CON-
CENTRATION IN MULLET EXPRESSED AS WHOLE
BODY WET WEIGHT. NUMBER OF FISH USED FOR
ANALYSIS: 10 JUVENILES, 2 ADULTS PER REPLI-
CATE AQUARIUM.
Age group Mirex cone, (mg/1) Mirex residue Qng/g)*
Juvenile
Adult
added
0.01
0.1
1.0
10.0
0.01
0.1
1.0
10.0
measured
0.010
0.104
1.108
5.180
0.012
0.107
1.033
9.540
mean
0.17
0.85
3.90
17.81
0.38
1.02
6.15
37.30
S.D.
0.02
0.31
0.16
2.47
0.07
0.17
1.91
4.83
*Duplicate samples per replicate were taken for analysis. Mean
values were obtained with the data from eight separate analyses
for each concentration.
Table 4. CONCENTRATION OF MIREX ACCUMULATED BY ADULT
STRIPED MULLET (278-310 mm) EXPOSED TO 0. 5 mg/1
MIREX AND MEASURED CONCENTRATION IN WATER FOR
96 HOURS IN A CONTINUOUS-FLOW BIOASSAY. CONCEN-
TRATIONS IN ORGANS EXPRESSED AS WET WEIGHT AND
ARE FROM EIGHT ADULT FISH.
Exposure time Measured cone. Residue Qag/g)
(hr) (mg/1) skin, muscle gills, heart viscera
24
48
72
96
0.49
0.46
0.45
0.45
1.75
1.44
1.62
1.38
1.27
2.36
2.37
2.03
5.86
11.63
17.25
21.50
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Methoxychlor was highly toxic to striped mullet. Young mullet were more sus-
ceptible than adults, and both age groups were more sensitive to methoxychlor
than to mirex. Young juveniles exposed to 10.0 mg/1 sustained 100 percent
mortality within three hours of initial exposure while all adults exposed to the
same concentration died within six hours. In 1. 0 mg/1 all juveniles were killed
within nine hours and adults within 15 hours. At a concentration of 0.1 mg/1,
about 95 percent mortality in the juveniles and 63 percent mortality in the adults
occurred within 48 hours (Table 5). After 96 hours of exposure, mortality of
adults and juveniles was nearly identical.
Table 5. PERCENT MORTALITY OF STRIPED MULLET EXPOSED TO METH-
OXYCHLOR FOR 96 HOURS IN A CONTINUOUS-FLOW BIOASSAY.
FOUR REPLICATE AQUARIA WERE USED FOR EACH CONCENTRA-
TION AND CONTROL. NUMBER OF FISH TESTED PER REPLICATE
AQUARIUM: 25 YOUNG JUVENILES AND 2 ADULTS.
Mortality (%)
Age group Methoxychlor cone. Observed, mean Adjusted
(mg/1) 48-hr 96-hr 48-hr 96-hr
Young
juvenile
20-43 -m-m
acetone control
seawater control
23.0
21.0
25.0
24.0
Adult
260-380 mm
0.01
0.1
1.0
10.0
acetone control
seawater control
26.0
96.0
100.0
100.0
0
0
29.0
98.0
-
-
0
0
4.0
94.8
100.0
100.0
5.1
97.4
-
-
0.01 0 0 0 0
0.1 ! 62.5 100.0 62.5 100.0
1.0 100.0 - 100.0
10.0 100.0 - 100.0
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During the experiments, affected mullet in the experimental tank showed
behavior apparently caused by insecticide poisoning. This behavior included
sudden random movements, attempts to jump out of the test tanks, gradual loss
of equilibrium, and cessation of respiratory movements.
The concentrations of methoxychlor at which 50 percent of the test fish sur-
vived (TLr0) were estimated by the graphical method outlined by the American
Public Health Association (1971). For young juvenile mullet, 48-hr and 96-hr
TLso values were 32 pg/l and 31 pg/1; for adult mullet the 48-hr and 96-hr
values were 65 peg/1 and 32 pg/1.
The toxicity study of methoxychlor on other fishes was made by Merna et al.
(1972). They reported 96-hr continuous-flow TL50 of 7.5 ptg/1 for fathead
minnow, Pimephales promelas, and 20.0/zg/l for yellow perch, Perca
flavescens. A TL50 value of 40. 0 ptg/1 for the yellow perch was found by Merna
et al. (1972) for static toxicity tests. They attributed this higher value to the
relatively more rapid breakdown of methoxychlor in static bioassay conditions.
Eisler (1970) reported a 96-hr TL50 value of 63 ptg/1 for juvenile mullet, Mugil
cephalus, from the east coast of the United States using a static bioassay
technique.
Relatively small amounts of methoxychlor accumulated in the mullet (Table 6).
Mullet exposed to 0. 01 mg/1 of methoxychlor for 96 hours concentrated about
0.1 jLtg/g in juveniles and 0.2 jitg/g in adults. Accumulations of methoxychlor by
the mullet exposed to the other concentrations were not significantly different
for the same age groups. Juvenile and adult mullet exposed to 0.01 mg/1 of
methoxychlor in the accumulation test showed that skin and muscle, and gills
and heart did not, in general, concentrate the insecticide as a function of expo-
sure time while viscera did, even though the rates were very slow compared
with those of mirex (Tables 7 and 8). Amounts of methoxychlor accumulated in
visceral organs were approximately 0.3 jtg/g for juveniles and 0.4 pg/g for
adults after 96-hr exposure. However, the methoxychlor amounts found in
other organs were not significantly different among the samples of each age,
especially adults collected at 24-, 48-, 72- and 96-hr intervals after the initial
exposure (0.01 ng/g for all skin and muscle and 0.12 pg/g or less for all gill
and heart samples).
10
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Table 6. CONCENTRATION OF METHOXYCHLOR ACCUMULATED BY
STRIPED MULLET EXPOSED TO METHOXYCHLOR AND MEASURED
CONCENTRATION IN WATER FOR 96 HOURS IN A CONTINUOUS-
FLOW BIOASSAY. CONCENTRATION IN MULLET EXPRESSED AS
WHOLE BODY WET WEIGHT. NUMBER OF FISH USED FOR ANALY-
SIS: 16 YOUNG JUVENILES, 2 ADULTS PER AQUARIUM.
Age group Methoxychlor cone, (mg/1) Methoxychlor residue Qug/g)*
Young juvenile
Adult
added
0.01
0.1
1.0
10.0
0.01
0.1
1.0
10.0
measured
0.009
0.104
1. 018
9.361
0.010
0.108
1.022
9.690
mean
0.06+
1.64
2.40
1.69
0.20+
11.92
11.83
11.12
S.D.
0.02
0.57
0.59
0.30
0.04
2.00
2.40
2.72
* Duplicate samples per replicate were taken for analysis. Mean values were
obtained with the data from eight separate analyses for each concentration.
+ Residue amounts in fish not killed by the insecticide.
Table 7. CONCENTRATION OF METHOXYCHLOR ACCUMULATED
BY JUVENILE STRIPED MULLET EXPOSED TO 10 jug/1
METHOXYCHLOR AND MEASURED CONCENTRATION IN
WATER FOR 96 HOURS IN A CONTINUOUS-FLOW BIO-
ASSAY. CONCENTRATION IN ORGANS EXPRESSED AS
WET WEIGHT. NUMBER OF FISH USED FOR EACH
ANALYSIS: 10 (75 - 134 mm STANDARD LENGTH).
Exposure time Measured cone,. Residue Qng/g)
(hr)
0
8
24
48
96
(mg/1)
9.7
9.9
9.5
9.1
skin, muscle
ND*
0.01
0.03
0.01
0.02
gills, heart
ND
0.02..
0.10
0.03
0.11
viscera
ND
0.06
0.10
0.15
0.27
*ND—none detected: less than 0. Ol^ig/g.
11
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Table 8. CONCENTRATION OF METHOXYCHLOR ACCUMULATED
BY ADULT STRIPED MULLET EXPOSED TO 10jug/l
METHOXYCHLOR AND MEASURED CONCENTRATION IN
WATER FOR 96 HOURS IN A CONTINUOUS-FLOW BIO-
ASSAY. CONCENTRATION IN ORGANS EXPRESSED AS
WET WEIGHT. NUMBER OF FISH USED FOR ANALYSIS:
10 IN EACH AQUARIUM (310-381 mm STANDARD LENGTH).
Exposure time Measured cone. Residue Qng/g)
(hr)
24
48
72
96
(mg/1)
11
14
11
12
skin, muscle
0.01
0.01
0.01
0.01
gills, heart
0.12
0.10
0.10
0.12
viscera
0.24
0.11
0.17
0.39
Reinbold et al. (1971) in a study of uptake and metabolism of methoxychlor by
some aquatic organisms found that the insecticide is readily metabolized in
tilapia, Tilapia mossambica, and green sunfish, Lepomis cyanellus. The
biodegradability of methoxychlor in aquatic organisms such as Gambusia
affinls fish and mosquito larvae was also observed by Kapoor et al. (1970) and
Metcalf et al. (1971).
12
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SECTION IV
EFFECTS OF MIREX AND METHOXYCHLOR ON THE EGGS AND LARVAE
OF STRIPED MULLET, Mugil cephalus L.
MATERIALS AND METHODS
Fertilized eggs and pro-larvae were available for experimentation following
induced breeding of the adults in February 1974. Artificial spawning was
induced in the laboratory by injecting salmon gonadotropin into the female
mullet. After fertilization the eggs are usually incubated at 22 °C in well-
aerated and specially constructed seawater tanks.
In static acute bioassays (48 hrs for eggs, 96 hrs for larvae), eggs and larvae
from the same female and male mullet were used. The test container adapted
for the study was an 8" x 8" x 10" nylon net of Nitex #351 mesh immersed into
a 20-gallon glass aquarium containing 55 liters of seawater. Desired amounts
of the insecticide, previously dissolved in acetone (10 ml portion per tank),
were added two hours before the eggs and larvae were introduced. Average
temperature of the seawater measured throughout the bioassays was 22°C, with
the range of 21.2 to 23.5°C; average salinity 32.5 %0; and average dissolved
oxygen 6.4 ppm (range 4.5 - 7.2 ppm).
Once spawning occurred and fertilization was confirmed (95%), approximately
300 eggs (0.9 mm diameter) were transferred into each test container.
Desired concentrations of mirex and methoxychlor were 0. 01, 0.1, 1.0 and
10. 0 Mg/l» together with acetone and seawater control tanks. Three groups of
50 eggs each were taken daily from the individual test containers to estimate
the percentage of dead embryos during 48-hour incubation bioassay. Viability
was determined by microscopic examination and recorded on an Aristo hand
tally counter. Developmental stages of live eggs were determined from illustra-
tion by Kuo et al. (1973).
Using an identical experimental system, a total of 300 pro-larvae (3 mm in
length) was introduced into each container for 96-hour bioassay. Dead larvae
were counted daily by three independent observers and the totals combined and
averaged. Test water was sampled daily for insecticide analyses.
RESULTS
During the initial 12 hours of the egg bioassay, heavy mortalities occurred from
all the test tanks including controls (Table 9). The mortality rates were
13
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apparently higher in methoxychlor than in mirex throughout the test period.
None of the eggs hatched during the period. Optimum hatching time has been
determined by Kuo et al. (1973) to be 48 to 56 hours after fertilization at
20 - 24°C.
Table 9. PERCENT SURVIVAL OF STRIPED MULLET EGGS IN MIREX AND
METHOXYCHLOR AND MEASURED CONCENTRATIONS OF BOTH
INSECTICIDES IN TEST WATER FOR 48 HOURS IN A STATIC BIO-
ASSAY. TESTS RUN SIMULTANEOUSLY FOR BOTH INSECTICIDES.
Concentration (mg/1)
Measured
Mean survival
Insecticide
Added
24-hr 48-hr
12-hr 24-hr
48-hr
Mirex
Methoxychlor
acetone
control
seawater
control
0.01
0.1
1.0
10.0
0.01
0.1
1.0
10.0
0.008
0.091
0.584
4.277
0.009
0.083
0.848
8.273
N.D.*
N.D.
0.006
0.062
0.435
3.366
0.007
0.070
0.612
7.683
73
80
43
25
18
29
14
19
9
14
58
80
24
23
14
24
11
12
8
9
50
64
23
23
13
14
10
7
7
7
*N. D. —none detected: less than 0.1 /ng/1 mirex, 0.08 (j.g/1 methoxychlor.
The measured insecticide concentrations in the test water (Tables 6 and 7)
varied. Concentrations measured from the fourth-day samples collected from
the larval bioassay tanks decreased rapidly compared with those from the first-
day samples for all levels; and the higher the added concentrations, the greater
the decreasing rate observed in both mirex and methoxychlor test water. This
was probably due to the low solubility, adsorption by apparatus, uptake by test
organisms, volatilization, rapid breakdown, etc., of the insecticides in the
standing seawater.
It was apparent that methoxychlor was more toxic to mullet larvae than mirex
14
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(Table 10). Larval mortalities in 10 mg/1 methoxychlor test water on the first
and second day were 95 and 100 percent, respectively, compared with 24 and
52 percent in the same concentration of mirex test water. Representative
tolerance limit values of both insecticides for mullet larvae were not obtained
from this study. The unexpectedly severe mortalities observed from the ace-
tone control tanks prevented application of statistical testing.
Table 10. PERCENT SURVIVAL OF STRIPED MULLET LARVAE IN MIREX AND METHOXY-
CHLOR AND MEASURED CONCENTRATION OF BOTH INSECTICIDES FOR 96
HOURS IN A STATIC BIOASSAY. BIOASSAY WAS MADE SIMULTANEOUSLY FOR
BOTH INSECTICIDES.
Insecticide
Concentration (mg/1)
Mean survival (%)
Added
Measured
24-hr 48-hr 72-hr 96-hr 24-hr 48-hr 72-hr 96-hr
acetone
control
seawater
N.D.*
N.D.
87
96
70
92
56
82
55
77
control
Mirex
Methoxychlor
0.01
0.1
1.0
10.0
0.01
0.1
1.0
10.0
0.009
0.158
0.835
6.811
0.011
0.133
0.736
8.268
0.008
0.102
0.867
5.489
0.007
0.163
0.823
8.386
0.007
0.080
0.569
3.322
0.008
0.133
0.531
5.210
0.006
0.062
0.389
2.238
0.007
0.266
0.238
1.260
94
92
85
76
93
70
49
5
89
85
84
48
69
53
1
0
86
83
81
46
60
50
0
-
77
75
69
45
41
38
—
-
*N. D. —none detectable.- less than 0.1 pg/1 mirex, 0.08 us/1 methoxychlor.
The culture of marine and brackishwater organisms is still very much at a
developmental stage. Incubation of eggs and rearing the larvae in static and/or
non-aerated conditions, in general, increases mortality. Furthermore, differ-
ences in the quality of the eggs from individual broodstock are being demon-
strated by biochemical analyses. Consequently there are inherent differences
in the viability of each larval population. A fertilized egg produced by induced
spawning methods as yet does not guarantee a viable larva. In order to evalu-
ate effectively the susceptibility of eggs and larvae to any pesticide, several
tests are necessary with the progeny of different broodstock, and preferably
with the same broodstock in subsequent breeding seasons.
15
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SECTION V
REFERENCES
American Public Health Association. 1971. Standard methods for the examina-
tion of water and wastewater. 13th ed. APHA, Washington, D. C. 874 pp.
Bookhout, C.G., A.J. Wilson, Jr., T.W. Duke and J.I. Lowe. 1972. Effects
of mirex on the larval development of two crabs. Water, Air, Soil Pollut.
1:165-180.
Borthwick, P.W., T.W. Duke, A.J. Wilson, J.I. Lowe, J.M. Patrick, Jr.
and J. C. Oberhen. 1973. Accumulation and movement of mirex in
selected estuaries of South Carolina. Pestic. Monit. J. 7:6-14.
Burdick, G. E., H. J. Dean, E.J. Harris, J. Skea, C. Frisa and C. Sweeney.
1968. Methoxychlor as a blackfly larvicide, persistence of its residues in
fish and its effect on stream arthropods. J. N. Y. Fish Game 15:121-142.
Burke, W.D. and D. E. Ferguson. 1968. A simplified flow-through apparatus
for maintaining fixed concentrations of toxicants in water. Trans. Am.
Fish. Soc. 97:498-502.
Butler, P. A. 1969. Monitoring pesticide pollution. BioScience 19:889-891.
Coon, D.W. andR.R. Fleet. 1970. The ant war. Environment 12:38.
Eisler, R. 1969. Acute toxicities of insecticides to marine decapod crusta-
ceans. Crustaceana 16:302-310.
Eisler, R. 1970. Acute toxicities of organochlorine and organophosphorus
insecticides to estuarine fishes. U.S. Bur. Sport Fish. Wildl. Technical
Paper No. 46. 12 pp.
Henderson, C., Q.H. Pickering and C. M. TarzwelL 1959. Relative toxicity
of ten chlorinated hydrocarbon insecticides to four species of fish. Trans.
Amer. Fish. Soc. 88:23-32.
Kapoor, I.P., R.L. Metcalf, R.F. Nystrom and G.K. Sangha. 1970. Compar-
ative metabolism of methoxychlor, methiochlor and DDT in mouse, insects,
and in a model ecosystem. J. Agr. Food Chem. 18:1145-1152.
16
-------
Kennedy, H. D., L. L. Eller and D. F. Walsh. 1970. Chronic effects of meth-
oxychlor on bluegills and aquatic invertebrates. U. S. Bur. Sport Fish.
Wildl. Technical Paper No. 53. 18 pp.
Kuo, C-M., Z.H. Shehadeh and K. K. Milisen. 1973. A preliminary report on
the development, growth and survival of laboratory reared larvae of the
grey mullet, Mugil cephalus L. J. Fish Biol. 5:459-470.
Lauger, P., H. Martin and P. Muller. 1944. Uber Konstitution und toxische
Wirkung von naturlichen und neuen synthetischen insektentotenden Stoffen.
Helv. Chim. Acta. 27:892-928.
Lowe, J.I., P. R. Parrish, A.J. Wilson, Jr., P.D. Wilson and T. W. Duke.
1971. Effects of mirex on selected estuarine organisms. In: Proceedings
of the 35th N. Amer. Wild. Nat. Resources Conf., Portland, Oregon.
pp. 171-186.
Ludke, L., M. T. Finley and C. Lusk. 1971. Toxicity of mirex to crayfish,
Procambarus blandigingi. Bull. Environ. Contam. Toxicol. 6:89-96.
Menzie, C. M. 1969. Metabolism of pesticides. U.S. Bur. Sport Fish. Wildl.
Spec. Sci. Rep. -Wildl. 127. 487 pp.
Merna, J.W., M. E. Bender and H. R. Novy. 1972. The effects of methoxychlor
on fish. I. Acute toxicity and breakdown studies. Trans. Am. Fish. Soc.
101:298-301.
Metcalf, R. L., G. K. Sangha and I. P. Kapoor. 1971. Model ecosystems for
the evaluation of pesticide biodegradability and ecological magnification.
Environ. Sci. & Tech. 5:709-713.
Naqvi, S.M., A. Armando and A. A. de la Cruz. 1973. Mirex incorporation in
the environment: Toxicity in selected freshwater organisms. Bull. Environ.
Contam. Toxicol. 10(5):305-308.
Negherbon, W. C. 1959. Handbook ,of toxicology. Vol. m. Insecticides. Natl.
Acad. of Sci., Div. of Biology and Agriculture, Washington, D. C. 854 pp.
Reinbold, K.A., I. P. Kapoor, W.F. Childers, W.N. Bruce and R.L. Metcalf.
1971. Comparative uptake and biodegradability of DDT and methoxychlor
by aquatic organisms. HI. Hist. Serv. BuU. 30:405-417.
Sanders, H. O. 1970. Pesticide toxicity to tadpoles of the western chorus frog,
pseudacris triseriata and Fowler's toad, Bufo woodhousii fowlerii.
Copeia 2:246-301.
17
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Sprague, J. B. 1969. Measurement of pollutant toxicity to fish. I. Bioassay
methods for acute toxicity. J. Wat. Pollut. Res. 3:798-821.
Sylvester, J. R., C.E. Nash, and C. R. Emberson. 1974. Preliminary study
of temperature tolerance in juvenile Hawaiian mullet (Mugil cephalus).
Progr. Fish Cult. 36:99-100.
Thompson, J. F. 1971. Analysis of pesticide residues in human and environ-
mental samples. Perrine Primate Research Lab, U. S. Environmental
Protection Agency, Perrine, Florida. Section 1-5, 10.
Thompson, J.M. 1966. The grey mullets. Oceanogr. Mar. Biol. Ann. Rev.
4:301-335.
Van Valin, C.C., A.K. Andrews and L. L. Eller. 1968. Some effects of mirex
on two warm-water fishes. Trans. Amer. Fish. Soc. 97:185-196.
Wolfe, J. L. and B. R. Norment. 1973. Accumulation of mirex residues in
selected organisms after an aerial treatment, Mississippi 1971-72. Pestic.
Monit. J. 7:112-116.
18
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-660/3-75-015
2.
3. RECIPIENT'S ACCESSION* NO.
4. TITLE AND SUBTITLE
EFFECTS OF MIREX.AND METHOXYCHLOR ON STRIPED
MULLET, Mugil cephalus L.
5. REPORT DATE
Date of preparation: 4/3Q/74
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Jong H. Lee, Colin E. Nash and Joseph R. Sylvester
8. PERFORMING ORGANIZATION REPORT NO.
01-120
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Oceanic Foundation
Makapuu Point
Waimanalo, Hawaii 96795
10. PROGRAM ELEMENT NO.
1EA077
11. CONTRACT/GRANT NO.
R 802348
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency, Gulf Breeze
Environmental Research Laboratory, Sabine Island,
Gulf Breeze, Florida 32561
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
The effects of two chlorinated insecticides, mirex and methoxychlor, on striped
mullet, Mugil cephalus L., were studied. Test concentrations of both insecticides used were
0. 01, Q. 1, L 0 and 10.0 ppm in dynamic bioassay. Young juveniles were more susceptible to
mirex exposure than older juveniles or adults. No mortalities occurred in older juveniles and
adults exposed to mirex for 96 hours. For young juveniles, mortalities were highest in concen-
trations of 0.1 and 1. 0 ppm and were less in concentrations of 0. 01 and 10. 0 ppm. Significant
amounts of mirex residues were accumulated in the body tissues of the test fish; concentrations
increased with increased environmental concentrations. Methoxychlor was more toxic to mul-
let than mirex. Mortalities were greater than 90 percent over a 96-hour period for all life
stages studied at concentrations of 0.1, 1. 0 and 10.0 ppm. Mortality at a concentration of 0.01
was 5.1 percent or less for 96 hours. Relative to mirex, small amounts of methoxychlor resi-
dues accumulated in the tissues of the test fish. Results of the experiments on eggs and
larvae were inconclusive. Egg survival was slightly better in mirex than in methoxychlor over
a 96-hour period. Larval survival was generally better in mirex than methoxychlor.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Chlorinated hydrocarbon pesticides,
Mortality, Pesticide toxicity, Bioassay,
Pesticide residues, Water pollution effects,
Fishes
Mullets, Mugil species,
Fishkill, Mirex,
Methoxychlor, Egg and
Larval Survival
Biological and
Medical Sciences,
Toxicology/Fishej
18. DISTRIBUTION STATEMENT
Release unlimited
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
24
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
ft U.S. GOVERNMENT PRINTING OFFICE: 1975-696-269(121 REGION 10
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