EPA-600/3-76-007
March 1976
Ecological Research Series
EFFECTS OF MIREX, METHOXYCHLOR, AND
MALATHION OK DEVELOPMENT OF CRABS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
-------
RESEARCH REPORTING SERIES
Research reports of the Off ice of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal
species, and materials. Problems are assessed for their long- and short-term
influences. Investigations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work provides the technical
basis for setting standards to minimize undesirable changes in living organisms
in the aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
-------
EPA-600/3-76-007
March 1976
EFFECTS OF MIREX, METHOXYCHLOR, AND MALATHION
ON DEVELOPMENT OF CRABS
by
Cazlyn G. Bookhout and
John D. Costlow, Jr.
Duke University
Beaufort, North Carolina 28516
Grant No. R-801128-02-2
Project Officer
Jack Lowe
Gulf Breeze Environmental Research Laboratory
Gulf Breeze, Florida 32561
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
GULF BREEZE ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE, FLORIDA 32561
-------
DISCLAIMER
This report has been reviewed by the Gulf Breeze Environmental
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.
-ii-
-------
FOREWORD
Man and his environment must be protected from the
adverse effects of pesticides, radiation, noise, and other
forms of pollution, and the unwise management of solid
waste. Efforts to protect the environment require a focus
that recognizes the interplay between the components of
our physical environment--air, water, and land. The Environ-
mental Research Laboratory, Gulf Breeze contributes to this
multidisciplinary focus through a research program which
emphasizes the ecological effects on the marine environment
of pesticides and other organic and inorganic pollutants.
Specific research outputs include
• information essential for the EPA pesticide regis-
tration and control program
* information essential for the development of the
EPA Water Quality Criteria
*• toxic effects of pollutants, singly and in combina-
tion, on marine and estuarine organisms
* effects of pollutants, singly and in combination,
on the physiology and development of marine and
estuarine organisms
• assessment of the hazards of human exposure to
pollutants which reach man through bioconcentration
in marine and estuarine food chains
• impact on marine and estuarine ecosystems of off-
shore dumping of petrochemicals
This report describes the effects of three insecticides
on the development of crabs. The data will be useful in
establishing estuarine water quality criteria for mirex,
methoxychlor, and malathion.
Thomas W. Duke
Director
Environmental Research
Laboratory, Gulf Breeze
-iii-
-------
ABSTRACT
Laboratory experiments were conducted to determine the
effects of mirex, methoxychlor and malathion on the larval
development of Callinectes sapidus from the time of hatching
until the first crab stage is reached. For comparison,
similar investigations were made to ascertain the effects of
methoxychlor and malathion on larval development of Rhithro-
panopeus harrisii.
The effect of a range of concentrations of each insecticide
on survival of larvae of C. sapidus and R. harrisii was de-
termined, as well as concentrations whicE" were sublethal and
lethal. Zoeal and total development to the first crab stage
of R. harrisii and C. sapidus was prolonged in relation to
increased concentrations of methoxychlor and malathion.
Other sublethal effects of methoxychlor and malathion inclu-
ded abnormal development of the pleopods of male R. harrisii
and male C. sapidus early crab stages, and autotomy of the
legs of R. harrisix megalopa and early crab stages. The
developmental stages in which larvae are particularly sensi-
tive vary in the two species and with the three insecticides.
Mirex residues of C. sapidus larvae reared in different con-
centrations of mirex, and methoxychlor residues of R. harri-
sii and C_. sapidus larvae reared in concentrations of meth-
oxychlor were determined.
This report was submitted in fulfillment of Grant No. R-801-
128-02-2 by Duke University under the sponsorship of the
Environmental Protection Agency. Work was completed as of
October 15, 1975.
-iv-
-------
CONTENTS
Section Page
I General Introduction •*•
II Conclusions *
III Recommendations
IV General Materials and Methods 9
V Mirex 16
Introduction 16
Results 18
Discussion 24
VI Methoxychlor 30
Introduction 30
Results 32
Discussion 48
VII Malathion 53
Introduction 53
Results 55
Discussion 69
VIII General Discussion 73
IX References 78
X Glossary 84
-v-
-------
FIGURES
No.
1. Average percent survival of two replicate series 20
of Callinectes sapidus larvae, Cs II and Cs VI,
reared from hatching to megalopa (M) and to 1st
crab (C) in different concentrations of mirex.
2. Average percent survival of four replicate 35
series of Rhithropanopeus harrisii larvae, Rh '
I-IV, reared £rom hatching to megalopa (M) and
to 1st crab (C) in different concentrations of
methoxychlor.
3. Average percent survival of four replicate 42
series of Callinectes sapidus larvae, Cs I-IV,
reared from hatching to megalopa (M) and to
1st crab (C) in different concentrations of
methoxychlor.
4. Average percent survival of four replicate 57
series of Rhithrppanopeus harrisii, Rh I-IV,
reared from hatching to megalopa (M) and to
1st crab (C) in different concentrations of
malathion.
5. Average percent survival of four replicate 64
series of Callinectes sapidus, Cs I-IV, reared
from hatching to megalopa (M) and to 1st crab
(C) in different concentrations of malathion.
-vi-
-------
LIST OF TABLES
Title Page
EFFECT OF MIREX ON PERCENT SURVIVAL AND DURA- 19
TION IN DAYS THROUGH ZOEAL AND MEGALOPA DEVEL-
OPMENT OF Callinectes sapidus II AND VI
2. TWO-WAY ANOVA WITH REPLICATION; EFFECT OF MIREX , 21
ON PERCENT SURVIVAL THROUGH ZOEAL AND MEGALOPA
DEVELOPMENT OF C. sapidus II AND VI
3. MEAN PERCENT SURVIVAL TO MEGALOPA AND TO FIRST 22
CRAB IN C. sapidus II AND VI
4. PERCENT MORTALITY IN NINE DEVELOPMENTAL STAGES 23
OF C. sapidus II AND VI
5. RESIDUES OF DDE, DDD AND DDT IN FIVE MOTHER 24
CRABS
6. MIREX RESIDUES IN BLUE CRABS AND THEIR LARVAE 25
7. EFFECT OF METHOXYCHLOR ON PERCENT SURVIVAL AND 34
DURATION IN DAYS THROUGH ZOEAL AND MEGALOPA
DEVELOPMENT OF Rhithropanopeus harrisii I-IV
8. TWO-WAY ANOVA WITH REPLICATION; EFFECT OF 36
METHOXYCHLOR ON PERCENT SURVIVAL THROUGH
ZOEAL AND MEGALOPA DEVELOPMENT OF R. harrisii
I-IV ~
9. AVERAGE DURATION IN DAYS OF ZOEAL AND MEGALOPA 36
DEVELOPMENT OF R. harrisii I-IV
10. TWO-WAY ANOVA WITH REPLICATION; EFFECT OF 37
METHOXYCHLOR ON DURATION IN DAYS THROUGH
ZOEAL AND MEGALOPA DEVELOPMENT OF R. harrisii
I-IV ~
11. PERCENT MORTALITY IN FIVE DEVELOPMENTAL STAGES 38
OF R. harrisii I-IV
-vii-
-------
LIST OF TABLES (continued)
Table Title Page
12. TWO-WAY ANOVA WITH REPLICATION; EFFECT OF 39
METHOXYCHLOR ON MORTALITY IN FIVE DEVELOP-
MENTAL STAGES OF R. harrisii I-IV
13. METHOXYCHLOR RESIDUES IN R. harrisii LARVAE 40
14. EFFECT OF METHOXYCHLOR ON PERCENT SURVIVAL AND 41
DURATION IN DAYS THROUGH ZOEAL AND MEGALOPA
DEVELOPMENT OF Callinectes sapidus I-IV
15. TWO-WAY ANOVA WITH REPLICATION; EFFECT OF 43
METHOXYCHLOR ON PERCENT SURVIVAL THROUGH
ZOEAL AND MEGALOPA DEVELOPMENT OF C. sapidus
I-IV
16. AVERAGE DURATION IN DAYS OF ZOEAL AND MEGALOPA 44
DEVELOPMEPNT OF C. sapidus I-IV
17. TWO-WAY ANOVA WITH REPLICATION; EFFECT OF 44
METHOXYCHLOR ON DURATION OF ZOEAL AND MEGALOPA
DEVELOPMENT OF C. sapidus I-IV
18. PERCENT MORTALITY IN NINE DEVELOPMENTAL STAGES 46
OF C. sapidus I-IV
19. TWO-WAY ANOVA WITH REPLICATION; EFFECT OF 47
METHOXYCHLOR ON MORTALITY IN NINE DEVELOP-
MENTAL STAGES OF C. sapidus I-IV
20. METHOXYCHLOR RESIDUES IN C. sapidus LARVAE 48
21. EFFECT OF MALATHION ON PERCENT SURVIVAL AND 56
DURATION IN DAYS THROUGH ZOEAL AND MEGALOPA
DEVELOPMENT OF Rhithropanopeus harrisii I-IV
22. TWO-WAY ANOVA WITH REPLICATION; EFFECT OF 58
MALATHION ON SURVIVAL THROUGH ZOEAL AND
MEGALOPA DEVELOPMENT OF R. harrisii I-IV
23. AVERAGE DURATION IN DAYS OF ZOEAL AND MEGALOPA 58
DEVELOPMENT OF R. harrisii I-IV
-viii-
-------
LIST OF TABLES (continued)
Table Title Page
24. TWO-WAY ANOVA WITH REPLICATION; EFFECT OF 59
MALATHION ON DURATION IN DAYS THROUGH ZOEAL
AND MEGALOPA DEVELOPMENT OF R. harrisii I-IV
25. PERCENT MORTALITY IN FIVE DEVELOPMENTAL STAGES 60
OF R. harrisii I-IV
26. TWO-WAY ANOVA WITH REPLICATION; EFFECT OF MALA- 61
THION ON MORTALITY IN FIVE DEVELOPMENTAL STAGES
OF R. harrisii I-IV
27. RESIDUES IN FOUR MOTHER CRABS, Rh I-IV 62
28. EFFECT OF MALATHION ON PERCENT SURVIVAL AND 63
DURATION IN DAYS THROUGH ZOEAL AND MEGALOPA
DEVELOPMENT OF Callinectes sapidus I-IV
29. TWO-WAY ANOVA WITH REPLICATION; EFFECT OF MALA- 65
THION ON PERCENT SURVIVAL THROUGH ZOEAL AND
MEGALOPA DEVELOPMENT OF C. sapidus I-IV
30. AVERAGE DURATION IN DAYS OF ZOEAL AND MEGALOPA 66
DEVELOPMENT OF C. sapidus I-IV
31. TWO-WAY ANOVA WITH REPLICATION; EFFECT OF MALA- 66
THION ON DURATION IN DAYS THROUGH ZOEAL AND
MEGALOPA DEVELOPMENT OF C. sapidus I-IV
32. PERCENT MORTALITY IN NINE DEVELOPMENTAL STAGES 67
OF C. sapidus I-IV
33. TWO-WAY ANOVA WITH REPLICATION; EFFECT OF MALA- 68
THION ON MORTALITY IN NINE DEVELOPMENTAL STAGES
OF C. sapidus I-IV
-ix-
-------
ACKNOWLEDGMENTS
The help of the following persons has been invaluable and is
gratefully acknowledged. Dr. Marit Christiansen, University
of Oslo, Oslo, Norway, assisted in technical aspects of the
research and in discussing topics relevant to the project.
Dr. Genevieve Payen, University of Paris, Paris, France, did
the research on the effect of methoxychlor and malathion on
the external and internal sexual characters of Rhithropano-
peus harrisii and Callinectes sapidus. Dr. Robert Monroe,
Department of Experimental Statistics, North Carolina State
University, Raleigh, North Carolina, served as statistical
consultant.
The technical assistance of Mrs. Charles Johnson throughout
the project and the untiring efforts of Ms. Mamre Wilson in
typing the manuscript are greatly appreciated.
-x-
-------
SECTION I
GENERAL INTRODUCTION
Crabs are phylogenetically related to insects, and juvenile
crabs show extreme sensitivity to certain insecticides.
Crabs in larval stages might be expected to be more sensi-
tive than juveniles, and, therefore, more valuable in esta-
blishing safe levels of insecticides in estuaries. There
are relatively few papers in the literature, however, on the
effects of different concentrations of pesticides on the lar-
val development of crabs.
Buchanan et al. (1970) studied the effect of Sevin, a carba-
mate, on £Ee survival and development of larvae in the first
zoeal stage of the dungeness crab, Cancer magister. They
found that the change from sublethal to acute toxicity
occurred between 3.2 mg/1 (milligrams per liter - parts per
million) and 10.0 mg/1, and that duration of the first lar-
val stage increased with concentration. Larvae in first
zoeal stages were more sensitive to Sevin than juveniles,
and juveniles more sensitive than adults.
Epifanio (1971) made a thorough study of the effects of
various concentrations of dieldrin on the larval development
of two species of xanthid crabs, Leptodius floridanus and
Panopeus herbstii. He reported that concentrations of di-
eldrln in seawater of 1.0 ppb (parts per billion = vg/1
micrograms per liter) or higher are deleterious to xanthid
crab larvae in the laboratory. Early stage larvae were more
sensitive than larvae in later stages, and L. floridanus lar-
vae were more sensitive to dieldrin than P. herbstil larvae.
Leptodius floridanus larvae accumulated dieldrin 19.1 times
as fast from 0.5 ppb in seawater as from 213.0 ppb in food.
This, he believed, was because the pumping rate was much
higher than the feeding rate (Epifanio, 1973).
Courtenay and Roberts (1973) conducted laboratory studies to
determine lethal limits (96 hr - TL^Q, tolerance limit) for
toxaphene, temperature, salinity and their interaction
effects on developmental stages of four species of decapods.
-l-
-------
The bioassay organisms were the blue crab, Callinectes
dus; the pink shrimp, Penaeus duorarum; the drift line cra
Sesarma cinereum; and the mud-crab, REithropanopeus harrisii.
The decapod larvae became more tolerant to toxaphehe in
later developmental stages than earlier stages. Larvae of
C. sapidus, !3. cinereum and R. harrisii exposed to high con-
centrations of toxaphene showed contraction of the intestine
and progressive vacuolation and necrosis of the hepatopan-
creas.
From the literature cited, it appears that larvae are more
sensitive to insecticides than juveniles (Buchanan et al.,
1971) and larvae in early stages are more sensitiveThan
those in later stages (Epifanio, 1971; Courtenay and
Roberts, 1973). There are so few publications on species in
which the effects of insecticides on larvae and juveniles
are known, however, that it is premature to make generali-
zations.
Most of the data on the effects of pesticides on aquatic or-
ganisms concern short-term effects with lethal concentra-
tions. Long-term chronic exposure to low concentrations of
an insecticide would be closer to what larvae or juveniles
would receive in the field and the effects on organisms in
the laboratory and field might be similar. When the larvae
of the mud-crab, Rhithropanopeus harrisii, and the stone
crab, Menippe mercenaria, were reared from the time of
hatching to the first crab stage in a range of concentra-
tions of mirex, each species showed different sublethal
effects, but only slight differences in susceptibility to
the same concentration of mirex (Bookhout et al., 1972) .
There is no information in the literature,"However, on the
effect of mirex on the development of the commercial blue
crab, Callinectes sapidus, from the time of hatching to the
first crab stage, nor is there information on the effect of
methoxychlor or malathion on the complete larval develop-
ment of any crab.
In the current investigation, the objective will be to de-
termine the limits of concentrations of mirex, methoxychlor
and malathion within which the blue crab, Callinectes sapi-
dus can be reared from the time of hatching to the first
crab stage. For comparison, similar chronic tests will be
made to determine the concentrations of methoxychlor and
malathion in which the mud-crab, Rhithropanopeus harrisii.
will develop. From these studies7 it should be possible to
determine sublethal effects, the most sensitive larval
stages and the sublethal and acutely toxic concentrations
of the three common insecticides. From the data obtained
-2-
-------
in this investigation and from chronic tests on juvenile and
adult crabs by other investigators, it should be possible to
set more reliable standards of water quality for estuaries.
-3-
-------
SECTION II
CONCLUSIONS
1. Concentrations of mirex from 0.01 ppb to 10.0 ppb have
no effect on mortality of Callinectes sapidus larvae
for five days after hatching. _Thereafter, there is
differential survival in relation to concentration.
Concentrations of 0.01 ppb and 0.1 ppb mirex are suble-
thal and 1.0 ppb and 10.0 ppb acutely toxic to larvae.
2. There is no statistical difference in duration from
hatching to megalopa, or hatching to 1st crab stage, in
£. sapidus larvae reared in acetone control and in 0.01
ppb and U.1 ppb mirex.
3. Significantly greater mortality of larvae occurs in
zoeal stages III and VII and megalopa of C. sapidus in
0.1 ppb mirex, in zoeal stages II and III in 1.0 ppb
mirex, and in zoeal stages I and II in 10.0 ppb mirex
than in other stages.
4. Callinectes sapidus larvae reared in a range of concen-
trations of mirex show increased residues with concen-
tration, but biological magnification of mirex is
greatest in larvae reared in 0.01 ppb and decreases
with concentrations ranging from 0.01 ppb to 10.0 ppb.
5. The range of concentrations of methoxychlor in which
differential survival of Rhithropanopeus harrisii
occurs from the time of hatching to the first crab
stage is from 1.0 ppb to 7.0 ppb, whereas the range for
similar development of Callinectes sapidus is from 0.7
ppb to 1.9 ppb. Concentrations of 1.0 ppb, 2.5 ppb,
4.0 ppb and 5.5 ppb are sublethal and 7.0 ppb methoxy-
chlor is acutely toxic to R. harrisii larvae. Suble-
thal concentrations of metKoxychlor for C. sapidus
development are 0.7 ppb and 1.0 ppb, whereas 1.3 ppb,
1.6 ppb and 1.9 ppb are acutely toxic.
-4-
-------
6. The total time from hatching to 1st crab stage in R.
harrisii and C. sapidus is prolonged with each concen-
tration primarily due to effects of methoxychlor on
zoeal development. The reduction in molting rate is
considered a sublethal effect of methoxychlor on both
R. harrisii and C_. sapidus zoeae. During sexual mor-
phogenesis from megalopa to the 6th crab stage of R.
harrisii and C. sapidus, methoxychlor has no apparent
effect on the development of the gonads and their
ducts, but has sublethal effects on the male pleopods.
7. In 1.0 ppb, 2.5 ppb and 4.0 ppb methoxychlor, stage I
zoeae of R. harrisii are significantly sensitive as are
zoeae of stages I and II to 5.5 ppb and 7.0 ppb. In
the acutely toxic concentrations of 1.3 ppb, zoeae of
stages II and III of C_. sapidus are significantly sen-
sitive, as are zoeae of stage II to 1.6 ppb and zoeae
of stages I and II to 1.9 ppb.
8. Biological magnification of methoxychlor residues at 5
days and 10-15 days is much greater in C. sapidus than
in R. harrisii. There is no evidence from the limited
data that residues at any one concentration increases
with age.
9. The range of concentrations in which differential sur-
vival of Rhithropanppeus harrisii occurs from the time
of hatching to the 1st crab stage is 0.011 ppm to 0.02
ppm malathion. Two of the four concentrations are sub-
lethal, 0.011 ppm and 0.014 ppm, and two are acutely
toxic, 0.017 ppm and 0.02 ppm, to developmental stages
of R. harrisii. Larvae reared in 0.05 ppm malathion
do not survive beyond the 2nd zoeal stage. The range
for similar development of Callinectes sapidus is from
0.02 ppm to 0.11 ppm malathion.Concentrations of 0.02
ppm and 0.05 ppm are sublethal and 0.08 ppm and 0.11
ppm are acutely toxic.
10. The average duration of zoeal development and total
time from hatching to 1st crab stage is shortest in
acetone control and is lengthened with each increase
in concentration of malathion in R. harrisii from 0.011
ppm to 0.02 ppm and in C. sapidus from 0.02 ppm to 0.11
ppm. Other sublethal eFfects^include abnormal develop-
ment of the pleopods in the first three crab stages
of both species and autotomy of legs in the megalopa
and crab stages in R. harrisii.
-5-
-------
11. In the development of R. harrisii from hatching to the
1st crab stage, zoeae Tn stage II and megalopa are sig-
nificantly sensitive to 0.011 ppm malathion, as are
zoeae in stage II to 0.014 ppm, 0.017 ppm and 0.02 ppm
and zoeae in stage I to 0.05 ppm. In the larval devel-
opment of C. sapidus, zoeae in stages II and III are
significantly sensitive to 0.05 ppm malathion, as are
zoeae in stages I, II and III to 0.07 ppm and zoeae in
stage I to 0.11 ppm malathion.
-6-
-------
SECTION III
RECOMMENDATIONS
1. To establish meaningful water quality standards for
estuaries, the long-term effects of low concentrations
of insecticides on the complete larval development of a
number of marine organisms, including shrimp and crabs,
should be considered, as well as chronic effects of in-
secticides on juveniles and adults of the same species.
2. Estuaries should be monitored for background levels of
mirex and methoxychlor when the following conditions
apply: when mirex is sprayed or applied directly to
land areas to control fire ants and these areas are
near estuaries, and when estuaries can receive drainage
from areas such as fruit orchards, which are sprayed
repeatedly with methoxychlor. Marsh areas, which are
sprayed repeatedly to kill mosquito larvae, should be
monitored for background levels of malathion after each
spraying.
3. The current report outlined the effects of three insec-
ticides on the development of crabs when the larvae
were reared under optimum temperature and salinity con-
ditions. Further investigation should be made to de-
termine the effect of these insecticides on the com-
plete larval development of crabs when the larvae are
reared in a range of salinities and temperatures.
4. Methods should be developed to make accurate residue
analyses of chlorinated hydrocarbon insecticides from
samples of larvae weighing much less than 0.20 g wet
weight. In the current project, it was a time consum-
ing task to obtain 0.20 g of larvae in low concentra-
tions of an insecticide and impossible to obtain late
stages in high concentrations. Replicate analyses of
larvae for residues of methoxychlor should be made as
a supplement to those given in this report.
-7-
-------
5. Micro methods for measurement of the enzyme acetylcho-
linesterase should be developed, so this test could be
applied to crab larvae reared in different concentra-
tions of malathion.
-8-
-------
SECTION IV
GENERAL MATERIAL AND METHODS
Preliminary experiments were conducted to determine the range
of concentrations of mirex, methoxychlor and malathion to use
in the definitive chronic studies on the effect of mirex on
the development of Callinectes sapidus and methoxychlor and
malathion on the development of Rhithropanopeus harrisii and
Callinectes sapidus.
Pesticide analytical grade acetone was used as a carrier for
mirex, methoxychlor and malathion, because in preliminary
studies it did not affect larval development of crabs. There
was no significant difference in survival of larvae reared
in seawater and 1.0 ppt (parts per thousand) insecticide
grade acetone. Acetone control for experiments with each of
the three pesticides was prepared by adding 1 ml of full
strength acetone of insecticide grade to 999 ml of 20%o fil-
tered seawater for R. harrisii larvae or 30%„ filtered sea-
water for C_. sapidus larvae to give a final concentration of
1.0 ppt.
The source and purity of the three insecticides used by
Hazleton Laboratories America, Inc., to make stock solutions
for this project is given below. Mirex (dodecachloroocta-
hydro-l,3,4-metheno-14-cyclobuta[cd]mentalene) was obtained
from Allied Chemical Corporation and had a purity of 100%.
Methoxychlor (1,1,l-trichlor-2,2-bis p-methoxyphenyl ethane)
was secured from E. I. duPont de Nemours Company and had a
purity of 99%. Malathion (0,0-dimethyl phosphoro-dithionate
of diethy1 mercapto-succinate) was obtained from American
Cyanamid Company and had a purity of 96%.
All of the above were prepared in the following manner:
known weight was dissolved in pesticide analytical grade
acetone and various concentrations were made up from this
stock solution.
One ml of each stock solution in concentrations of 0.01 ppm,
0.1 ppm, 1.0 ppm and 10.0 ppm mirex was added to 999 ml of
-9-
-------
30%o filtered seawater daily to give the final concentrations
of 0.01 ppb to 10.0 ppb used to determine the effects of mi-
rex on the development of Callinectes sapidus.
For experiments on the effect of methoxychlor on Rhithropa-
nopeus harrisii development, one ml of stock solutions I.U
ppm, 2.5 ppm, 4.0 ppm, 5.5 ppm and 7.0 ppm was added to 999
ml of 20°/00 filtered seawater daily to give final concentra-
tions of 1.0 ppb to 7.0 ppb methoxychlor.
For experiments on the effects of methoxychlor on £. sapidus,
one ml of stock solutions of 0.7 ppm, 1.0 ppm, 1.3 ppm, 1.5
ppm and 1.9 ppm was added to 999 ml of 30%o filtered seawater
daily to give final concentrations of 0.7 ppb to 1.9 ppb.
For experiments on the effects of malathion on R. harrisii,
one ml of stock solutions of 0.011 ppt, 0.014 ppt, U.U1/
ppt, 0.02 ppt, and 0.05 ppt malathion was added to 999 ml of
20°/00 filtered seawater daily to give final concentrations of
0.011 ppm to 0.05 ppm malathion.
For experiments on the effects of malathion on C. sapidus,
one ml of stock solutions of 0.02 ppt, 0.05 ppt, 0.08 ppt,
and 0.11 ppt malathion was added to 999 ml of 30%0 filtered
seawater daily to give the final concentrations of 0.02 ppm
and 0.11 ppm.
Fresh stock solutions of malathion were shipped to Beaufort,
N. C., by Hazleton Laboratories before each experiment with
larvae of one of the species of crabs. The stock solutions
were stored in a coldroom at 5°C at the Duke University
Marine Laboratory to prevent deterioration during the course
of an experiment.
Source of mother crabs and hatching of eggs
Ovigerous Rhithropanopeus harrisii Gould, small mud-crabs
belonging to the family Xanthidae, were collected from the
west side of Newfound Harbor, a small estuary which is be-
tween the Indian River to the west and the Banana River to
the east in the vicinity of Cocoa Beach, Florida. They
were shipped to North Carolina from Florida by air freight
in January and February 1974 and 1975. Upon arrival in
Beaufort, N. C., each small mud-crab was placed in a large
glass finger bowl (19.4 cm diam.) containing filtered sea-
water with a salinity of 20700, the salinity to be used
during rearing of larvae for experiments with methoxychlor
-10-
-------
and malathion. The ovigerous crabs were maintained in a cul-
ture cabinet at 25°C and with a light regime of 12 hours
light and 12 hours darkness.
Ovigerous Callinectes sapidus Rathbun, commercial blue crabs
belonging to the family Portunidae, were collected from the
Beaufort Inlet, N.C. They furnished larvae for experiments
with mirex in 1973 and with methoxychlor and malathion in
1974. The crabs used carried black eggs which had eyes and
visible heartbeat. The method for developing the eggs was
the same as Costlow and Bookhout described in 1960. Pleo-
pods bearing eggs were removed, placed in large glass finger
bowls (19.4 cm diam.) of filtered seawater at 30%o, the
salinity to be used for rearing larvae. Setae bearing eggs
were removed from pleopods with fine scissors, and washed
3-5 times in filtered seawater.
The eggs were further dissociated with glass needles and
transferred to freshly filtered seawater. They were washed
in an additional bowl of filtered seawater and then placed
in plastic compartmented boxes, 32.5 cm x 22.7 cm, or 2000
ml flasks of filtered 30%o seawater. These containers were
then placed on an Eberbach variable speed shaker regulated
for 60 oscillations min~l. Black eggs with eyes and a visi-
ble heartbeat usually hatched within one or two days after
they were placed on the shaker.
Rearing of larvae in check series
The methods of rearing larvae of Rhithropanopeus harrisii
and Callinectes sapidus in acetone control and 4- to 5 con-
centrations ot the insecticides mirex, methoxychlor and mala-
thion were essentially the same for the two species. Larvae
of R. harrisii were reared in a salinity of 20%0 and at a
temperature ot 25°C, because this was the salinity and temp-
erature found to be optimum in earlier studies (Costlow,
Bookhout and Monroe, 1966). They were reared on Artemia
salina nauplii. Callinectes sapidus larvae were reared at
a salinity of 30%0 and a temperature of 25°C, the salinity
and temperature found to be optimum in rearing studies at
the Duke University Marine Laboratory from 1959 (Costlow
and Bookhout, 1959) to the present. Callinectes sapidus lar-
vae were fed Artemia salina nauplii and fertilized Arbacia
eggs.
As soon as the eggs from one mother crab hatched, 10 larvae
were placed in each of the finger bowls (8.9 cm in diameter)
to be used in the series. The series included a given num-
ber of bowls of control larvae in 1 ppt acetone in filtered
seawater, and the same number of bowls of larvae in 4 or 5
-11-
-------
concentrations of the insecticide. A drop of Artemia salina
nauplii, hatched from California eggs, and a drop or ferti-
lized Arbacia eggs were added to each bowl containing C.
sapidus larvae, but only Artemia salina nauplii were fed to
R. harrisii larvae. On the following day and each day
thereatter, the number of living larvae, and the number and
stages of dead larvae in each bowl were recorded. ^Hence,
this series is referred to as a check series. Living lar-
vae were transferred to clean bowls with fresh media, and
food was added daily. As each zoea molted to a megalopa, it
was transferred to a separate division of a compartmented
box containing 25 ml of the desired medium. The liquid con-
tents were removed from each compartment and fresh medium
and food were added daily until a megalopa molted to the 1st
crab stage. Larvae from each replicate mother crab were
handled in the same manner. Thus each mother crab furnished
enough larvae to be reared in acetone control and the con-
centrations of the insecticide for each replicate.
Rearing of larvae for residue analysis
Mass cultures of R. harrisii and C. sapidus were maintained
for residue analyses in large glass finger bowls (19.4 cm
diam.) containing the same media as the check series. Ini-
tially approximately 1000 freshly hatched larvae were placed
in a bowl containing 700 ml of one of the media. A medicine
dropper full of Artemia nauplii and the same volume of ferti-
lized Arbacia eggs were added to each bowl, if the larvae
were CT sapidus, but were fed Artemia nauplii only if the lar-
vae were R. harrisii. Living larvae were transferred to a
clean bowT and fed daily. To obtain 0.20 to 0.25 grams of
larvae (wet weight) for residue analyses, 10 large bowls of
larvae in acetone control and 10 to 16 bowls in each of the
concentrations of the insecticide were maintained. Mortali-
ty of larvae was much greater in mass cultures than in check
series, hence in the higher concentration of insecticides,
it was not possible to obtain enough larvae in late stages
to analyze. If larvae survived to the 1st crab stages in
check series, these were obtained for analysis.
Larvae collected for analysis were washed seven times in
filtered seawater, blotted on filter paper, weighed, wrapped
in aluminum foil and stored frozen. Wrapped samples in alu-
minum foil were shipped in dry ice to Hazleton Laboratories
for analysis.
-12-
-------
Residue analyses
Determination of chlorinated pesticide residues in mother
crabs, Rhithropanppeus harrisii and Callinectes sapidus, was
made by Hazleton Laboratories, Inc., in 19/3, and by the
same laboratory but known as Hazleton Laboratories America,
Inc., in 1974 and 1975. This company also made mirex deter-
minations of C. sapidus mother crabs and their larvae in
1973 and methoxychlor determinations in larvae of both spec-
ies in 1974 and 1975. Their methods are described below.
Weighed samples were ground with 15 g anhydrous sodium sul-
fate to absorb the moisture and transferred with petroleum
ether rinsings to a 500-ml round bottom flask. The sodium
sulfate and crabs were extracted three times with 10-ml pet-
roleum ether and filtered through 10 g sodium sulfate re-
tained on a fritted glass funnel into a 250-ml round bottom
flask. The extract was transferred with petroleum ether
rinsings into a 125-ml separatory funnel and extracted four
times with 25-ml acetonitrile saturated with petroleum ether.
Each 25-ml was transferred to a 500-ml separatory funnel
containing 300-ml of 2% sodium chloride and 100-ml petroleum
ether. After combining all extracts in the 500-ml separa-
tory funnel, it was shaken gently for 1 minute to transfer
the pesticides to the petroleum ether. The lower aqueous
layer was discarded and the petroleum ether washed twice
more with 50-ml 2% sodium chloride. After discarding the
washings, the lower petroleum ether layer was drawn off into
a glass stoppered bottle containing 10 g sodium sulfate.
The extract was transferred to an activated (5 hours at 130°
C) florisil (pesticide grade floradin) column (I.D. 22 mm)
containing 100-mm after packing and topped with about % inch
of anhydrous sodium sulfate. The petroleum ether extract was
passed through the column at a rate of 3 ml/minute. When
the last of the extract had sunk into the column the pesti-
cides (BHC, Lindane, Chlordane, Aldrin, Heptachlor, Hepta-
chlor Epoxide, DDE, ODD, DDT, Aroclor 1242, Aroclor 1254,
HCB, and Methoxychlor) were eluted with 110-ml of 6% ethyl
ether in petroleum ether at the same rate. After the last
of the 6% ethyl ether fraction had sunk into the sodium sul-
fate layer the receptacle was changed and the column was elu-
ted with 200-ml of 5070 ethyl ether in petroleum ether at
the same rate. This fraction elutes the remaining pesticides
(Endrin and Dieldrin). Both elutes were then concentrated
to 2-ml on a flash evaporator (water bath, 40°C). The con-
centrated extracts were transferred with rinsings to 15-ml
centrifuge tubes and concentrated further, if necessary,
using a stream of dry nitrogen and warm water bath.
-13-
-------
Gas Chromatography
The concentrated samples were analyzed by gas chromatography
using an electron capture detector. The gas chromatograph
was operated using the following parameters:
Apparatus: Micro-Tek, MT-220
f q
Detector Type: Electron Capture, Nio:),
RF Pulsed
Recorder Ranger: 1 mv Full Scale, Chart Speed
1/3 inch per minute
Column: 6' x %" O.D., Glass, 3% 0V
17 on 100/120 mesh, Gas
Chrom-Q
Temperatures:
Injection Port - 270°C
Detector - 280°C
Column Oven - 225"Isothermal
Gas Flow: Nitrogen, 5.5 on Flow Meter
and 40 psi at the Tank
Attenuation: 10 x 2
Dr. Richard Stanovick, Director of the Biochemistry Division,
Hazleton Laboratories America, Inc., stated that the expect-
ed recovery of residues in the analyses for mirex would be
at the level of 82%, for methoxychlor at the level of 90%
and for the analyses of pesticides in ovigerous crabs be-
tween 80-90%.
Studies related to sexual morphogenesis
To provide material to determine sexual variations due to
methoxychlor and malathion, Rhithropanopeus harrisii and
Callinectes sapidus were reared from the last zoeal stage to
the bth crab stage in acetone control and in the same con-
centrations of methoxychlor and malathion as described pre-
viously. For Dr. Genevieve Payen, University of Paris, to
conduct a study of sexual morphogenesis, we fixed in alco-
holic Bouin's (Dubosq-Brasil) 15 megalopa and up to 15 of
each of the 6 crab stages which had been reared in each of
-14-
-------
the concentrations of methoxychlor and malathion. The sex-
ual variants which received particular attention were the
pleopods, abdominal appendages, and the primary sexual char-
acters, such as the gonads, their ducts and the androgenic
gland.
The sequence of observations started with larvae which had
been reared in the highest concentration and continued with
larvae reared in lower concentrations of the insecticide un-
til no modification of pleopods could be detected. Such
larvae were found in the following concentrations: 7.0 ppb,
5.5 ppb and 4.0 ppb methoxychlor for R. harrisii; 1.9 ppb
and 1.6 ppb methoxychlor for C. sapidus;0.02 ppm and 0.017
ppm malathion for R. harrisii and 0.11 ppm and 0.08 ppm mala-
thion for £. sapidus.
Pleopods of control and contaminated crabs were dissected
either from the intact or isolated abdomen. The number and
character of setae and any morphological abnormality of the
pleopods were recorded. Whole mounts were stained with fast
gree or safran, and lactic acid was used to increase the
transparency of the tissues.
In order to determine the extent of development of the gonads,
its ducts and the androgenic glands, it was necessary to make
parasagittal and transverse sections. These were cut at 5y
and stained with picroindigo-carmin and hematoxylin. Serial
sections were examined to determine if the genital apparatus
was developing normally or abnormally compared to the con-
trols and to reference slides of each species made by Dr.
Payen before this project started.
-15-
-------
SECTION V
MIREX
INTRODUCTION
Mirex, a chlorinated hydrocarbon, is the active ingredient
in bait used to control the imported red ant, Solenopsis in-
vecta Buren, in the Southeastern United States, and soienop-
sis richteri in restricted areas in northeastern Mississippi
ana" northern Alabama (Alley, 1973). The bait, first used by
the U.S. Department of Agriculture (USDA) is designated as
4X bait. It consists of 84.7% corn-cob grits, 15% soybean
oil, and 0.3% mirex. It was applied at a rate of 1.4 kg/ha
(kilograms per hectare); (1.25 pounds per acre) (Coon and
Fleet, 1970). Within recent years 2X bait with 0.15% mirex,
and IX bait with 0.1% mirex has been used (Alley, 1973).
Lowe et al. (1971) found 4X mirex bait and/or technical mirex
to be toxic to decapod crustaceans in the laboratory and in
simulated field conditions. Juvenile brown shrimp, Penaeus
aztecus, grass shrimp, Palaemonetes pugio, juvenile blue
crabs, Callinectes sapidus, and fiddler crabs, Uca pug il at or,
were poisoned. ATthough adult and subadult blue crabs (76
to 127 mm) were not affected by ten times the USDA's suggest-
ed application rate of 1.4 kg/ha, McKenzie (1970), and Lowe
et al. (1971) found that juvenile blue crabs are more sensi-
tive to mirex bait, the smaller they are.
Crayfish, Procambarus blandingi, are sensitive to technical
mirex concentrations from U.I ppb to 5.0 ppb, to 4X bait,
and also to mirex which leaches out of 0.3% bait (Ludke e£
al., 1971). Redmann (1973) reported that grass shrimp,
Falaemonetes pugio, are poisoned by concentrations of 0.01
ppm to 1.0 ppm technical mirex, to 0.15% mirex bait, and to
mirex which leached out of the bait.
At a time when mirex was being aerially applied to coastal
areas from North Carolina to Florida, Mahood e£ al. (1970)
found mirex residues to be the second most common insecti-
cide in monthly samples of blue crabs. The percent of blue
-16-
-------
crabs with residues of mirex was directly proportional to
the amount of spraying.
Borthwick e£ al. (1973) monitored the movement and accumula-
tion of mirexTn selected estuaries from 1969-71, when mirex
was aerially applied on October, 1969, June and October,
1970, to coastal areas to eradicate fire ants near Charles-
ton, S.C. Mirex was not detected in any pretreatment samples
of crabs, shrimp, fish, water or sediment. After treatment,
the greatest number of organisms with mirex in estuaries
were taken at stations located closest to treated areas. Or-
ganisms downstream from the treated areas also accumulated
mirex, but in lesser amounts. Residues of mirex were more
often found in crabs than in shrimp or fish. The authors
were of the opinion that mirex moved-from treated land to
water after each treatment. They did not explain the mecha-
nisms involved, but they suspected surface runoff after heavy
rainfall was a likely method. Naqvi and de la Cruz (1973)
made a similar study of accumulation of mirex by freshwater
aquatic organisms near treated areas in Mississippi, and re-
ported widespread movement of mirex in the environment.
They were of the opinion that mirex residues in terrestrial
organisms were the result of animals consuming mirex bait,
whereas mirex residues in aquatic organisms were obtained
from leaching of the pesticide from bait.
Since it has been shown that juvenile blue crabs are more
sensitive to mirex, the smaller they are, it might be sus-
pected that larvae in developmental stages of Callinectes
sapidus might be even more sensitive than juveniles.In-
direct evidence for this suspicion was given by Buchanan et
al. (1970), for he determined that early larvae of the
Huhgeness crab, Cancer magister, were more sensitive to
Sevin, a carbamate pesticide,than juveniles, and juveniles
were more susceptible than adults. There was no direct evi-
dence to support or disprove this suspicion in the case of
blue crab larvae, however, until the portion of this report
dealing with mirex was published (Bookhout and Costlow,
1975). Only one other paper had appeared on the effect of
mirex on the complete development of crabs. It concerned
the chronic effects of mirex on the mud-crab, Rhithropano-
peus harrisii, and the stone crab, Menippe mercenana
(Bootchout et al., 1972). The objectives of the current
study were tolletermine the effects of technical mirex on
the complete larval development of Callinectes sapidus from
the time of hatching to the 1st crab stage.Specific answers
to the following questions were sought: (1) what concentra-
tions of mirex are sublethal and which are acutely toxic;
-17-
-------
(2) what are the effects of sublethal concentrations; (3)
are larvae in one or more developmental stages particularly
sensitive to different concentrations of mirex; (4) what
residues of mirex are present in larvae of different ages in
reference to concentration and length of exposure; and, (5)
what are the differences in the effects of mirex on the com-
plete development of C_. sapidus, R. harrisii and M. mercena-
ria?
This section is a final report of what has been done and
published (Bookhout and Costlow, 1975) on the effect of
mirex on the complete development of the blue crab, Calli-
nectes sapidus.
RESEARCH RESULTS
EFFECTS OF MIREX ON DEVELOPMENT OF Callinectes sapidus
Survival
Records of day to day percent survival of 200 Callinectes
sapidus larvae from mother crab, Cs II, indicate that there
was no appreciable difference in survival of larvae reared
in acetone control and in 0.01 ppb, 0.1 ppb, 1.0 ppb and
10.0 ppb mirex during the first five days. Thereafter,
there was a sharp reduction in survival of larvae reared in
10.0 ppb mirex until there were none living by day 13. Lar-
vae reared in 1.0 ppb mirex showed a sharp reduction in sur-
vival from 95% to 35% between the days of 8 and 15. From
day 15 to 58 there was a more gradual reduction until only
one (0.5%) 1st crab out of 200 of the original number of
larvae was left. Larvae reared in acetone control, 0.01 ppb
and 0.1 ppb mirex showed differential survival with the
highest percent surviving to megalopa and to 1st crab stage
in acetone control and the lowest in 0.1 ppb (Table 1).
One hundred larvae hatched from mother crab, Cs VI, were
also reared in acetone control and in the same concentra-
tions of mirex as larvae from Cs II. Again there was no
appreciable difference in survival of larvae reared in any
of the media for the first 5 days. Larvae reared in 10.0
ppb mirex only survived to 12 days and those maintained in
1.0 ppb mirex lived to 20 days. Percent survival to megalo-
pa and to 1st crab stage is given in Table 1.
The average percent survival of 300 larvae from Cs II and
Cs VI reared in five media is plotted in Figure 1.
-18-
-------
TABLE 1
EFFECT OF MIREX ON PERCENT SURVIVAL AND DURATION
IN DAYS THROUGH ZOEAL AND MEGALOPA
DEVELOPMENT OF Callinectes
sapidus II and VI
Culture Media Initial #
Salinity 300/o«, of Larvae
Temp. 25°C per Series
Acetone
Control
Mirex
0.01 ppb
Mirex
0.1 ppb
Mirex
1.0 ppb
Mirex
10.0 ppb
Acetone
Control
Mirex
0.01 ppb
Mirex
0.1 ppb
Mirex
1.0 ppb
Mirex
10.0 ppb
Cs II 200
Cs II 200
Cs II 200
Cs II 200
Cs II 200
Cs VI 100
Cs VI 100
Cs VI 100
Cs VI 100
Cs VI 100
Mean Duration of % Survival
Zoeal
Develop-
ment in
Days
32.28
32.90
31.06
42.50
37.75
38.07
38.49
Megalopa
Develop-
ment in
Days
8.55
8.76
8.83
7.00
(one)
.11.16
11.73
10.63
Hatching to to
1st Crab in Mega-
Days lopa
41.21 60.5
41.60 56.0
41.00 50.5
51.00 2.0
(one)
48.75 60
49.55 44
49.13 41
1st
Crab
52.0
41.5
30.5
0.5
55
40
30
-19-
-------
60
50
£ 40
> 30
o:
05
20
10
535%
C
60.3%
M
0 ACETONE
CONTROL
40B%
C
50.0%
M
45.8%
M
I.3%M
VZZZZZl -3% C
0.01 O.I 1.0
MIREX, ppb
10.0
Figure 1. Average percent survival of two replicate series
of Callinectes sapidus larvae, Cs II and Cs VI,
reared from hatching to megalopa (M) and to 1st
crab (C) in different concentrations of mirex.
-20-
-------
To determine if there were significant differences between
percent survival to megalopa and to 1st crab stages in ace-
tone control and 0.01 ppb and 0.1 ppb mirex, a two-way analy-
sis of variance (ANOVA) was made. There were replicate lar-
vae from crabs Cs II and Cs VI per combination of media and
stage (Table 1). The ANOVA (Table 2) shows that the percent
survival differs significantly (0.01 probability) between
megalopa and crab stage and among the three media.
TABLE 2
TWO-WAY ANOVA WITH REPLICATION; EFFECT OF MIREX ON
PERCENT SURVIVAL THROUGH ZOEAL AND MEGALOPA
DEVELOPMENT OF C. sapidus II and VI
Source of variation
Subgroups
A (Stages)
B (Media)
A x B (Interaction)
Within subgroups (error)
Total
DF
5
1
2
2
6
11
Sum of
Squares
1095.25
330.75
723.88
40.63
123.00
1218.25
Mean
Square
219.05
330.75
361.94
20.31
20.50
Fs
16.13 **
17.66 **
0.99 n.s.
Significant F ratios: F .01 [1,6] - 13.75, F .01 [2,6] = 10.93
Since the interaction between stage and media is clearly non-
significant (Table 2), the results may be summarized by the
stage difference and the media effect (Table 3). The survi-
val to megalopa is 10.5% ± 2.6% greater than that to crab
stage. The media effect may be expressed as a decrease of
9,4% ± 1.6% in survival for each 10-fold increase in mirex
concentration in the range studied.
-21-
-------
TABLE 3
MEAN PERCENT SURVIVAL TO MEGALOPA AND TO
FIRST CRAB IN C. sapidus II and VI
Media
Acetone control
0.01 ppb
0.1 ppb
Survival to
Megalopa
60.3
50.0
45.8
Survival to
1st Crab
53.5
40.8
30.3
Average
56.9
45.4
38.0
Average 52.0 41.5
Duration of Development
Larvae hatched from mother crab, Cs II, on May 1, 1973, and
reared in acetone control, 0.01 ppb and 0.1 ppb mirex showed
remarkable consistency in mean duration of zoeal development,
megalopa development and in total time from hatching to 1st
crab stage (Table 1). Larvae hatched from mother crab, Cs
VI, on June 18, 1973, and reared in the same media, took a
longer time to complete zoeal and megalopa development than
larvae from Cs II, but the time span of larvae reared in
each of the three media was similar (Table 1). Larvae of
blue crabs reared later in the breeding season showed a
longer period of development than those reported here. Thus
the increased time of development of larvae from Cs VI may
be related to time in the breeding season the larvae hatched,
rather than to the media in which they were reared. Larvae
from each crab which were reared in 1.0 ppb and 10.0 ppb
mirex were slower to molt than larvae reared from each crab
in 0.01 ppb and 1.0 ppb. This is considered a sublethal
effect of mirex. All of these larvae died with the except-
ion of 2% of larvae from Cs II which completed zoeal devel-
opment in 1.0 ppb mirex in 42.50 days compared to 31 to 32
days in other media (Table 1).
Mortality
A record of deaths in each of the eight zoeal stages and
megalopa was made for larvae reared from mother crabs, Cs
II and Cs VI (Table 4), in an effort to determine if larvae
-22-
-------
in one or more stages of development were particularly sensi-
tive to different concentrations of mirex.
A single classification ANOVA of the mortality data in Table
4, using replicates from larvae of mother crabs Cs II and Cs
VI, indicates that larvae in no one stage were significantly
sensitive statistically to 0.01 ppb. On the other hand,
larvae in zoeal stages III, VII and tnegalopa in 0.1 ppb mirex,
and larvae in zoeal stages II and III in 1.0 ppb. showed
significantly greater mortality than other stages. In 10.0
ppb mirex, all larvae died in zoeal stage I and II.
TABLE 4
PERCENT MORALITY IN NINE DEVELOPMENTAL STAGES
OF C. sapidus II and VI
Stage Control Mirex
Acetone
Cs II Cs VI
I
II
III
IV
V
VI
VII
VIII
Megalopa
Total
1.0
0.5
7.5
0.5
0.5
0.5
24.0
5.0
8.5
48.0
3
11
7
4
1
4
9
1
5
45
0.01 ppb
Cs II Cs VI
0.5
2.0
10.5
3.5
0.0
1.0
26.0
0.5
14.5
58.5
7
28
7
2
1
0
10
1
4
60
0.1 ppb
Cs II Cs VI
0.0
2.5
16.5
1.5
0.0
0.5
28.5
0.0
20.0
69.5
2
7
13
4
2
4
20
7
11
70
1.0
Cs II
6.0
34.0
24.5
10.0
2.0
1.0
13.0
3.5
1.5
99.5
ppb 10.0
Cs VI Cs II
9 53.5
78 46.5
13
- -
- -
- -
- -
-
-
100 100.0
ppb
Cs VI
95
5
-
-
-
-
-
-
-
100
Residue Analysis
Larvae of three mother crabs Cs II, Cs III and Cs V were
reared in mass cultures in acetone control, 0.01 ppb, 0.1
ppb, 1.0 ppb and 10.0'ppb mirex to obtain enough wet weight
of larvae (0.30 to 0.20 g) for residue analysis. These
three crabs plus mother crab Cs VI and Cs VII were analyzed
by the Hazleton Laboratories for residues of the following
pesticides: BHC, Lindane, Chlordane, Aldrin, Heptachlor,
Heptachlor Epoxide, DDE, ODD, DDT, Aroclor 1242, Aroclor
-23-
-------
1254, Endrin, Dieldrin and HCB. None of the pesticides were
found in detectable amounts, <0.01 ppm, with the exception
of DDE, ODD and DDT. The latter are given in Table 5.
TABLE 5
RESIDUES OF DDE, ODD AND DDT IN FIVE MOTHER CRABS
Sample DDE (ppm) DDD (ppm) DDT (ppm)
Cs II
Cs III
Cs V
Cs VI
Cs VII
0.03
0.03
0.03 ,
0.02
0.01
0.02
<0.01
0.04
<0.01
<0.01
0.07
0.06
0.03
0.01
0.01
Residue analyses in larvae were also made (Table 6) to ob-
tain information concerning the relationship between length
of time in each concentration and residues of mirex, and
between increase in residues and increase in concentration.
No detectable mirex, <5 ppb, was found in freshly hatched
larvae in seawater, or in 5 and 15 day larvae, megalopa, or
in 1st and 2nd crabs which had been reared in acetone con-
trol. Larvae reared in a range of concentrations of mirex
from 0.01 ppb to 10.0 ppb showed increased residues of mirex
with increase in concentration (Table 6).
According to the residue analyses, the biological magnifica-
tion of mirex was greatest in larvae reared in 0.01 ppb and
decreased with concentration thereafter.
Mother crabs whose larvae were used to obtain enough larvae
for residue analysis had no detectable mirex, <5 ppb, except
Cs II which had 10.0 ppb.
DISCUSSION
Effect of Mirex on Survival
Mirex, in a range from 0.01 ppb to 10.0 ppb, had no appre-
ciable effect on day to day survival of two replicate series
of blue crab larvae for five days after hatching. Delayed
mortality was also noted in the early stages of zoeal de-
velopment of Rhithropanopeus harrisii and Menippe mercenaria
-24-
-------
TABLE 6
MIREX RESIDUES IN BLUE CRABS AND THEIR LARVAE
Sample Identification and Treatment ppb Mirex
First day larvae - Seawater <5
5 day larvae - Acetone Control <5
15 day larvae - Acetone Control <5
Megalopa - Acetone Control <5
Crabs, 1st & 2nd - Acetone Control <5
5 day larvae - 0.01 ppb Mirex 11
15 day larvae - 0.01 ppb Mirex 30
Megalopa - 0.01 ppb Mirex 20
Crabs, 1st & 2nd - 0.01 ppb Mirex 9
5 day larvae - 0.10 ppb Mirex 33
15 day larvae - 0.10 ppb Mirex 70
Megalopa - 0.10 ppb Mirex 65
Crabs, 1st & 2nd - 0.10 ppb Mirex 77
5 day larvae - 1.0 ppb Mirex 301
8 day larvae - 1.0 ppb Mirex 406
5 day larvae - 10.0 ppb Mirex 1620
8 day larvae - 10.0 ppb Mirex 1370
Mother Crab Cs II - Seawater 10
Mother Crab Cs III - Seawater <5
Mother Crab Cs V - Seawater <5
reared in the same concentrations of mirex (Bookhout ejt al.,
1972). The only significant mortality of first stage zoeae
was observed in M. mercenaria reared in 10.0 ppb. Delayed
toxicity of mirex to juvenile blue crabs has also been re-
ported by MacKenzie (1970); by Lowe et al. (1971) to juve-
nile pink shrimp, Penaeus duorarum; By Eudke et al. (1971)
to two species of crayfish, Frocambarus bIandingi~and P.
hayi; and by Plapp (1973) to house flies, Musca domestica.
MacFarlane e£ al. (1975) noted that the characteristic
feature of mirex poisoning to the field cricket, Gryllus
pennsylvanicus, was the long latent period of 72 hours after
it was treated with a lethal dose of mirex. During this
-25-
-------
period the activity of the ventral nerve cord was the same
as untreated individuals and the behavior of treated crickets
was the same as untreated individuals. The long latent per-
iod before poisoning of the nerve cord may also account for
the delayed toxicity to adult and larval crustaceans. Inves-
tigators do not often define what they mean by sublethal and
lethal concentrations of an insecticide in chronic bioassay
studies of decapods. In a chronic bioassay investigation of
the effect of dieldrin on the development of two crabs, Epi-
fanio (1971) considered sublethal concentrations as those in
which there is differential survival with increased concen-
tration of the toxicant in relation to survival in the con-
trol medium and those in which more than 10% of the larvae
reach the 1st crab stage. Acutely toxic concentrations are
those in which 10% or less of the larvae reach the 1st crab
stage. These definitions, in our opinion, are valid when
survival to the 1st crab stage is high (above 30%) in the
control, but not when survival is low (below 30%).
After a five day period of delayed mortality in all experi-
mental concentrations of mirex, two concentrations were
shown to be sublethal and two acutely toxic to blue crab lar-
vae. Since 41.5% to 30% of two replicate series of larvae
of blue crabs reached the 1st crab stage when reared in 0.01
ppb and 0.1 ppb mirex, these concentrations are considered
to be sublethal. These concentrations of mirex are also sub-
lethal to Rhithropanopeus harrisii and Menippe mercenaria
(Bookhout et al., 1972) . Sixty-seven percent and 4yy0 of R.
harrisii larvae reached the 1st crab stage compared to 9%
and 5% of M. mercenaria larvae. In the control, survival to
the 1st crab stage in R. harrisii was 88% and 27% in M. mer-
cenaria . Concentrations of 1.0 ppb and 10.0 ppb proved to
be acutely toxic in the three species of crabs which have
been reared in mirex. In one series of blue crab larvae 0.5%
out of 200 larvae reached the crab stage in 1.0 ppb, but no
larvae from the second series survived beyond 20 days. In R.
harrisii, 7% reached the crab stage in 1.0 ppb, and no larvae
survived beyond the megalopa in 10.0 ppb mirex. In M. mer-
cenaria , no larvae developed beyond the megalopa stage in
1.0 ppb, or beyond the 3rd zoeal stage in 10.0 ppb mirex
(Bookhout et a^., 1972).
Juvenile blue crabs showed no symptoms of poisoning during a
96 h exposure to 0.1 ppm technical mirex in flowing seawater,
but died within 18 days in mirex-free seawater (Lowe e_t al.,
1970). Tagatz et al. (in press) found mirex in concentra-
tions of 0.04 ppb" to 0.12 ppb which had leached from mirex
bait to be lethal to juvenile blue crabs within 6 to 28 days
of exposure. Although these experiments are different from
-26-
-------
the chronic study of blue crab larvae, it appears that Calli-
nectes sapidus larvae are about as sensitive to mirex as small
juvenile blue crabs.
Larvae of C. sapidus, R. harrisii and M. mercenaria seem to
be as sensitive to mirex as juvenile craytish and juvenile
pink shrimp. Sixty-five percent and 71% of the juvenile cray-
fish, Procambarus blandingi and P_. hayi, died after being ex-
posed to U.I ppb and U.5 ppb mirex tor 48 h (Ludke et al.,
1971). Eleven percent of 36 juvenile pink shrimp, Fehaeus
duorarum, died during 3 weeks exposure to 0.1 ppb technical
mirex in flowing seawater and 25% died in mirex-free water
during a two-weeks post-treatment period (Lowe et_ al., 1971).
Adult grass shrimp, Palaemonetes pugio, seem to be interme-
diate in sensitivity to mirex between juvenile blue crabs and
blue crab larvae. After adult grass shrimp were exposed to
0.01 ppm mirex for 48 h, 40% died after 12 days. When they
were exposed to 0.1 ppm and 1.0 ppm for 48 h, 91% died in 0.1
ppm after 10 days, and 100% died in 1.0 ppb after 5 days
(Redmann, 1973).
Sublethal Effects
The duration of zoeal development and the time from hatching
to the 2nd crab stage of Rhithropanopeus harrisii increased
significantly with increase in concentration of mirex from
0.01 to 10.0 ppb (Bookhout et al., 1972). The reduction in
molting rate is considered a suETlethal effect of mirex. In
the two series of Callinectes sapidus larvae from mother
crabs, Cs II and Cs Vl,the duration from hatching to mega-
lopa and to 1st crab reared in acetone control and in 0.01
ppb and 0.1 ppb showed no statistical difference in duration.
In the acutely toxic concentration of 1.0 ppb mirex only 2%
of the larvae of Cs II reached the megalopa stage, but it
took them 11 days longer to do so than those reared in sub-
lethal concentrations of 0.01 ppb and 0.1 ppb mirex (Table 1).
Larvae in 1.0 ppb and 10.0 mirex showed a slower rate of
molting compared to larvae reared in acetone control and 0.01
ppb and 0.1 ppb mirex, hence this trend is considered a sub-
lethal effect of mirex.
In Menippe mercenaria, there were no significant differences
in duration o£ developmental stages or time to reach the
megalopa stage in relation to concentration of mirex, but
extra 6th zoeal stages increased from 65% to 66% to 90% in
concentrations of 0.01 ppb, 0.1 ppb and 1.0 ppb mirex, re-
spectively, compared to 2.5% in seawater control. This is
-27-
-------
another type of sublethal effect of mirex. The majority of
the 6th zoeal stages died before reaching the megalopa stage
(Bookhout et al., 1972). Blue crabs normally pass through
seven zoeaT~siEages, and occasionally eight zoeal stages, be-
fore molting into a megalopa stage (Costlow and Bookhout,
1959). Based on the tabulation of mortality of the 8th
zoeal stages in acetone control and in concentrations of
mirex from 0.01 ppb to 1.0 ppb (Table 4), there is no evi-
dence that extra 8th zoeal stages increased with higher con-
centrations of mirex. The majority of 8th zoeal stages of
C. sapidus, like extra 6th stages of M. mercenaria, usually
(Tied before molting into megalopa.
Residues
Table 6 shows that residues of mirex increased with concen-
tration from 0.01 ppb to 10.0 ppb. In the sublethal concen-
trations of 0.01 ppb and 0.1 ppb mirex, residues increased
in 5 to 15 days from 11 ppb to 30 ppb, and 33 ppb to 77 ppb,
respectively. Larvae reared in 0.01 ppb mirex had a residue
of 20 ppb in the megalopa and 9 ppb in the crab stage, but
larvae reared in 0.1 ppb had residues of 65 ppb in the mega-
lopa and 70 ppb in the crab stage, or about the same as the
residue in laryae at 15 days. Ludke et al. (1971) also
noted that residues in crayfish, whicE~TiacT been exposed to
two granules of mirex bait, increased initially, but remain-
ed fairly constant after the second day.
In the acutely toxic concentration of 1.0 ppb mirex, resi-
dues were 301 ppb at 5 days and 406 ppb at 8 days, whereas
in the lethal concentration of 10.0 ppb, residues were 1620
ppb at 5 days and 1370 ppb at 8 days. At 8 days, larvae in
these concentrations were dying in large culture bowls.
Hence, it was not possible to obtain enough larvae for resi-
due analysis at 15 days.
Biological magnification of mirex during larval development
of blue crabs was greatest in larvae reared in 0.01 ppb and
decreased with concentration from 0.1 ppb to 10.0 ppb mirex.
When Jernelov et al. (1972) examined the accumulation of
components 1-6 ofThe EDC-tar, a mixture of short chained
aliphatic hydrocarbons, in the shrimp, Leander adspersus,
and the mussel, Mytilus edulis, they too found that accumu-
lation was highest when EDC-concentration was lowest and
lowest when EDC-concentration was highest.
After 54 hours of exposure to 0.86 ppb mirex which had
leached from mirex bait, 33 of 35 juvenile crayfish died.
-28-
-------
These were divided into three samples for analysis. They
had body residues of 1.602, 1.409, and 1.339 ppm mirex (Lud-
ke et al., 1971). These residues are comparable to those
founcT in blue crab larvae at five and eight days which had
been reared in 10.0 ppb.
Juvenile shrimp, Penaeus duorarum, exposed to 0.1 ppb of
technical mirex for three weelcs and analyzed for residues
two weeks after being in mirex-free seawater contained 0.32
ppm (Lowe et al., 1971) which is comparable to residues de-
tected in ETue crab larvae reared in 1.0 ppb mirex at five
days.
Three juvenile blue crabs, which were fed a fish a day for
five days that contained 1.0 ppm mirex and uncontaminated
fish for three weeks thereafter, exhibited symptoms of mirex
poisoning or were dead (Lowe ejt al., 1971). These crabs had
residues of 0.13 ppm, 0.22 ppm ami 0.25 ppm mirex. These
values are slightly less, but comparable to residues of mirex
found in blue crab larvae reared in 1.0 ppb at five and eight
days.
Pooled samples of Crustacea, collected from lakes, ponds,
creeks and an estuary in areas which had varying degrees of
mirex treatment, had mirex residues which averaged 0.44 ppm
(Naqvi and de la Cruz, 1973). This value is very close to
the residue of mirex found in blue crab larvae reared in 1.0
ppb at eight days.
From the examples given, it appears that juvenile Prpcambarus
blandingi and juvenile Penaeus duorarum concentrate leached
or technical mirex, presumably through the gills, more effi-
ciently than blue crab larvae, since they reached approxi-
mately the same residue level when reared in 1/10 less con-
centrated medium of mirex. When mirex is obtained through
food, as in the cited case of juvenile blue crabs, accumula-
tion is slower than when mirex is obtained from technical
mirex. Epifanio (1973) reported that Leptodius floridanus
larvae accumulated dieldrin 19.1 times as fast from U.S ppb
in seawater as from 213 ppb in food. This, he believed, is
because the pumping rate is much higher than the feeding'
rate.
-29-
-------
SECTION VI
METHOXYCHLOR
INTRODUCTION
Methoxychlor is ore of the chlorinated hydrocarbon insecti-
cides which is used to replace DDT, now banned in the United
States. It is employed chiefly to control blackfly larvae
in streams, the smaller European elm bark beetle, and a var-
iety of pests on fruits, vegetables and forage (Burdick et
al., 1968; Wallner et a^., 1969; U.S. Department of Agricul-
ture, 1968). As a substitute chemical for DDT, methoxychlor
is nearly as toxic to the target organism, yet biodegradable.
Hence, it does not give rise to such long lasting residues
as DDT (Burdick et al., 1968; Metcalf ejt al., 1971).
Methoxychlor is stable to heat and resistant to oxidation
and relatively stable to ultraviolet radiation. Henderson
et al. (1959) concluded that pH and alkalinity has no major
eTfect on the toxicity of methoxychlor to fish, but found
that the insecticide was somewhat more toxic to fish in hard
water, 400 ppm, than in soft water, 20 ppm. Merna et al.
(1972) reported that hydrogen ion concentration in cTTstTlled
water within a range of pH 7 to 9 had no effect on the break-
down rate of methoxychlor.
Several investigators have been concerned with the breakdown
of methoxychlor and the accumulation of methoxychlor residues
in the environment. Burdick et al. (1968) exposed brook
trout to 5 pg/1 (micrograms per TTter-parts per billion)
methoxychlor for two day periods a week apart. At the end
of one week in clean water, the trout lost 41.3% of the resi-
due they gained during the two days of exposure. After three
months no residue was detected. Wallner e_t al. (1969) repor-
ted that after American elms were sprayed witE methoxychlor
from a helicopter, substantial residues were found in twig
crotches and soil beneath the trees for one year after spray-
ing. Water samples from a stream adjacent to the treated
trees showed most residues in the top inch, but the residues
were diluted by the current of the stream and were undetect-
ed after 24 hours. Samples taken later at two week intervals
-30-
-------
from spring to fall showed 1 part per trillion methoxychlor
in water and 1.0 ppm in silt.
Using freshwater mussels, Lampsilis siliquoidea and L. ven-
tricosa, as monitors, Bedford et al. (iye>8) ±ound that tne
mussels concentrated methoxychlor"from river water adjacent
to an area where there had been a spraying for Dutch elm
disease. The concentrations at two stations were 0.1190 ppm
and 0.0702 ppm after two weeks, increased to a high of 0.1310
ppm and 0.2221 ppm after six weeks and returned after ten
weeks to a level found after two weeks. These results indi-
cate that methoxychlor may remain in water for a longer time
than Wallner et_ al. (1969) reported.
The retention of methoxychlor in silt may be associated with
biomagnification of the insecticide by bacteria. Johnson
and Kennedy (1973) reported that Aerobacter aerogenes and
Baccillus subtilis accumulated l^C-labeled methoxychlor dir-
ectly from water. The uptake by both species was rapid; 80
to 907o of the 24-h residues occurred within 30 min. In
water ranging from 0.5 to 50 yg/liter, the residue magnifi-
cation factors from the water were between 1,400 to 4,300
fold. If bacteria can accumulate methoxychlor and concentra-
te it over one thousand times above ambient water levels,
they could be the means of transfer of the insecticide to
higher levels of the food chain for they serve as a nutrient
source for filter feeding organisms.
Although methoxychlor is considered a moderately hazardous
insecticide, it may be highly toxic to some aquatic organisms.
When juvenile and adult striped mullet, Mugil cephalus, were
exposed to a concentration of 0.1 mg/1, 95% of the juveniles
and 63% of the adults died within 48 hours. In 1.0 mg/1 all
juveniles were killed in 9 hours and adults in 15 hours (Lee
e_t al., 1975). Merna and Eisele (1973) reported that meth-
oxycHlor is highly toxic to freshwater organisms and contin-
uous exposure to 1.0 yg/1 methoxychlor may cause serious sub-
lethal effects. Methoxychlor in concentrations between 1.0
yg/1 to 0.125 yg/1 inhibited hatching of fathead minnow eggs
and 2 yg/1 prevented spawning. Concentrations of 50 yg/1
inhibited phytoplankton production. When yellow perch were
exposed to concentrations of methoxychlor from 0.625 to 6.00
yg/1 for eight and a half months, growth was retarded as
concentrations were increased. At 10.0 yg/1 perch died.
The 96 hour TLsq (tolerance limit for 50% survival) values
for the amphipod, Gammarus pseudolimaeus, was 0.61 yg/1 and
7.05 yg/1 for the craytish, Orconectes~v'irilis.
-31-
-------
There is a dearth of papers on the effect of methoxychlor on
estuarine and marine decapods. Eisler (1969) determined the
concentrations of seven organochloride and five organophos>-
phorus insecticides that kill 50% of the sand shrimp, Crangon
septemspinosa, the grass shrimp, Palaemonetes vulgaris, and
the hermit crab, Pagurus longicarpus, in 9b hours.Methoxy-
chlor was the third most toxic organochloride' insecticide to
Crangon septemspinosa, the fifth most toxic organochloride
to Falaemonetes vulgaris and the third most toxic organo-
chloride to Fag'urus longicarpus. With few minor exceptions
pesticide-induced mortality seemed to be related to tempera-
ture of the water. Mortality was least in the lowest test
temperature of 10° C and greatest in the highest test tempera-
ture of 30°C.
Although methoxychlor might be considered a moderately hazar-
dous organochloride insecticide, it may be highly toxic to
marine organisms, especially near areas whre repeated appli-
cations of the insecticide are used. The Florida estuary
from which we obtained ovigerous Rhithropanopeus harrisii in
January and February, 1975, for experiments on malathion
must have been such a place for four of the mother crabs had
residues of methoxychlor ranging from 0.57 ppm to 5.50 ppm.
As far as known, there have been on investigations on the
effect of methoxychlor on the complete larval development of
crabs. The objectives of our present study were to determine
the effects of methoxychlor on the complete larval develop-
ment of Rhithropanopeus harrisii and Callinectes sapidus from
the time of hatching until the 1st crab stage is reached.
Specifically answers to the following questions will be sought
(1) what concentrations are sublethal and which are acutely
toxic; (2) what are the sublethal effects of these concen-
trations; (3) what are the effects of methoxychlor on the
development of the external and internal sex characters; (4)
is there one or more larval stages particularly sensitive to
different concentrations of methoxychlor; (5) what residues
of methoxychlor are present in larvae of different ages in
reference to concentration and length of exposure; and (6)
what are the differences in the effects of methoxychlor on
the complete development of R. harrisii and C. sapidus?
RESEARCH RESULTS
A. EFFECTS OF METHOXYCHLOR ON DEVELOPMENT OF Rhithropano-
sus harrisii
Survival
-32-
-------
Rhithropanopeus harrisil larvae hatched from four mother
crabs, Rn I-IV, were reared in acetone control and in 1.0
ppb, 2.5 ppb, 4.0 ppb, 5.5 ppb, and 7.0 ppb methoxychlor.
The percent survival of each of the four series of larvae
which passed through zoeal and megalopa development is given
in Table 7, and the average percent survival of larvae of
four replicate series reared in six media is plotted in
Figure 2. Survival of larvae from four crabs reared in ace-
tone control through zoeal and megalopa development only
varied from 98% to 100% and 96% to 100%, respectively (Table
7). Such high survival indicates that each of the four ser-
ies of larvae used in the experiment were very healthy. In
five concentrations of methoxychlor from 1.0 ppb to 7.0 ppb,
there was differential survival (Figure 2). Survival through
zoeal and through megalopa development to the 1st crab stage
in 1.0 ppb methoxychlor was almost as high as in acetone con-
trol, whereas only 0.5% survived in 7.0 ppb, that is, only
one larva out of 200 larvae in the four replicate series
completed zoeal and megalopa development (Figure 2).
A two-way ANOVA with replication (Table 8) shows that there
is no statistical difference in survival between the number
of larvae which completed zoeal and megalopa development.
The differences in survival in acetone, 1.0 ppb, 2.5 ppb and
4.0 ppb methoxychlor are highly significant (P < 0.001).
Duration of Development
&
Table 7 gives the mean duration of zoeal and megalopa devel-
opment and the time from hatching to the 1st crab stage in
each of four replicate series of larvae reared in six media.
An average duration of zoeal and megalopa development of lar-
vae from all replicate series is listed in Table 9. It shows
that the duration of zoeal development is prolonged with each
increase in concentration of methoxychlor from 1.0 ppb to 5.0
ppb. This is considered a sublethal effect of methoxychlor.
The average duration of megalopa development in days, however,
is similar in all experimental media (Table 9). The total
times from hatch to 1st crab are prolonged with each increase
in concentration, and therefore are related primarily to the
effects of different concentrations of methoxychlor on zoeal
development.
A two-way ANOVA with replication indicates that variation of
duration of R. harrisii development is related to differences
in zoeal and megalopa development, the media or treatment used
and an interaction of the two, at the 0.001 level of probabi-
lity (Table 10).
-33-
-------
TABLE 7
EFFECT OF METHOXYCHLOR ON PERCENT SURVIVAL AND DURATION
IN DAYS THROUGH ZOEAL AND MEGALOPA DEVELOPMENT
OF Rhithropanopeus harrisli I-IV
Culture Media
Salinity 20%,
Temp. 25 °C
Acetone
Control
Methoyxchlor
1.0 ppb
Methoxychlor
2.5 ppb
Methoxychlor
4.0 ppb
Methoxychlor
5. 5 ppb
Methoxychlor
7.0 ppb
Initial #
of Larvae
per Series
Rh 1-50
Rh 11-50
Rh 111-50
Rh IV-50
Rh 1-50
Rh 11-50
Rh 111-50
Rh IV-50
Rh 1-50
Rh 11-50
Rh 111-50
Rh IV-50
Rh 1-50
Rh 11-50
Rh 111-50
Rh IV-50
Rh 1-50
Rh 11-50
Rh 111-50
Rh IV-50
Rh 1-50
Rh 11-50
Rh I I 1-50
Rh IV-50
Mean Duration of
Zoeal
Develop-
ment in
Days
10.86
11.04
9.98
10.74
12.06
11.69
11.20
11.31
15.03
12.53
12.36
12.51
17.13
14.42
14.00
14.63
_
15.14
15.14
15.64
_
15.00
-
_
Megalopa
Develop-
ment in
Days
4.63
6.04
4.80
5.06
4.49
6.00
5.14
5.57
4.47
5.48
4.91
5.17
4.75
5.18
5.09
5.28
_
5.00
5.31
5.09
_
5.00
-
_
Hatching to
1st Crab in
Days
15.48
17.08
14.78
15.83
16.55
17.69
16.35
16.89
19.50
17.93
17.28
18.67
21.88
19.61
19.06
19.91
_
20.14
20.31
20.73
_
20.00
—
_
% Survival
to
Mega-
lopa
98
100
98
100
94
96
100
96
76
94
90
78
16
66
66
64
__
28
28
22
_
2
_
„__
1st
Crab
96
100
98
96
94
96
98
92
76
84
86
72
16
66
66
64
_
28
26
22
_
2
_
_
-34-
-------
100
90
80
70
60
_, 50
> 40
ID
C/)
30
20
10
r\
••*
-
^
^ ,
—
975%
C
99%
M
y^^^^
95%
C
96.!
M
%
9,
795%
C
84.5%
M
53%
C
M
V «•
0.5 %M
0.5%C
f^^mm^—mm^
CONTROL
2.5
4.0
5.5
7.0
METHOXYCHLOR, ppb
Figure 2. Average percent survival of four replicate series
°f Rhithropanopeus harrisii larvae, Rh I-IV, rear-
ed trom hatcning to megalopa (M) and to 1st'crab
(C) in different concentrations of methoxychlor.
-35-
-------
TABLE 8
TWO-WAY ANOVA WITH REPLICATION; EFFECT OF METHOXYCHLOR
ON PERCENT SURVIVAL THROUGH ZOEAL AND MEGALOPA
DEVELOPMENT OF R. harrisii I-IV
Source of variation
Subgroups
A (Stages)
B Qfedia)
A x B (Interaction)
Within subgroups (error)
Total
DF
7
1
3
3
24
31
Sum of
Squares
10410.00
32.00
10350.87
27.13
4076.00
14486.00
Mean
Square Fs
32.00 0.1884 n.s.
3450.29 20.316 ***
9.04 0.053 n.s.
169.83
Significant F ratio: F.001 [3,24] = 7.55
TABLE 9
AVERAGE DURATION IN DAYS OF ZOEAL AND MEGALOPA
DEVELOPMENT OF R. harrisii I-IV
Medium
Duration of Zoeal
Development
Duration of Megalopa
Development
Time from Hatch
to First Crab
Acetone Control
10.66
5.14
15.8
Methoxychlor
1.0 ppb
2.5ppb
4.0 ppb
5.5 ppb
11.56
13.04
14.56
15.28
5.30
5.01
5.15
5.13
16.87
18.30
20.17
20.37
Mortality
Rhithropanppeus harrisii passes through four zoeal stages
and a megalopa, a ti±th stage of development. In an effort
-36-
-------
TABLE 10
TWO-WAY ANOVA WITH REPLICATION; EFFECT OF METHOXYCHLOR
ON DURATION IN DAYS THROUGH ZOEAL AND MEGALOPA
DEVELOPMENT OF R. harrisii I-IV
Source of Variation
Subgroups
A (Stages)
B (Media)
A x B (Interaction)
Within subgroups (error)
Total
DF
7
1
3
3
24
31
Sum of
Squares
490.28
445.73
20.76
23.79
14.57
504.85
Mean
Square
70.04
445.73
6.92
7.93
0.61
Fs
730.70 ***
11.34 ***
13.00 ***
Significant F ratios: F.001 [1,24] = 14.0, F.001 [3,24] =7.55
to determine if larvae in one or more of the five develop-
mental stages of R. harrisii were particularly sensitive to
different concentrations of methoxychlor, a record of deaths
by stage was made for larvae from four mother crabs, Rh I-
IV, which had been reared in acetone control and five concen-
trations of methoxychlor (Table 11).
A two-way ANOVA with replication (Table 12) shows that the
main effects of methoxychlor on the mortality of five devel-
opment stages of R. harrisii are related to differences in
stages, the media used and the interaction of the two (P <
0.001).
To determine which stage of development is siginificantly
sensitive to a particular concentration of methoxychlor, a
single classification ANOVA of mortality data was made for
each medium in Table 11, using replicates from larvae of
mother crabs, Rh I-IV. If the means of the series were sig-
nificantly different, the least significant difference (LSD)
was calculated. In acetone control, no single stage was
significantly different (t0 Ql) from anY other- Larvae in
zoeal stage I were significantly sensitive (tQ m) to 1.0
ppb, 2.5 ppb and 4.0 ppb methoxychlor, as were larvae in
zoeal stages I and II to 5.5 ppb and 7.0 ppb methoxychlor.
-37-
-------
TABLE 11
PERCENT MORTALITY IN FIVE DEVELOPMENTAL STAGES
OF R. harrisii I-IV
Stage
Acetone Control
Methoxychlor
1.0 ppb
Methoxychlor
2.5 ppb
Methoxychlor
4.0 ppb
Methoxychlor
5.5 ppb
Methoxychlor
7.0 ppb
Rh I
Rh 11
Rh III
Rh IV
Rh I
Rh II
Rh III
Rh IV
Rh I
Rh II
Rh III
Rh IV
Rh I
Rh II
Rh III
Rh IV
Rh I
Rh II
Rh III
Rh IV
Rh I
Rh II
Rh III
Rh IV
I
0
0
0
0
4
4
0
0
20
6
2
20
42
30
16
20
76
50
56
40
100
72
74
68
II
2
0
0
0
0
0
0
0
4
0
2
0
20
4
8
10
22
20
16
38
-
26
24 '
28
III
0
0
0
0
0
0
0
0
0
0
4
0
22
0
10
4
2
2
0
0
_
0
2
2
IV
0
0
2
0
2
0
0
2
0
0
2
2
0
0
0
0
_
0
0
0
_
0
-
2
Megalopa
2
0
0
4
0
0
2
2
0
10
4
6
0
0
0
0
_
0
2
0
_
0
_
_
Total
4
0
2
4
6
4
2
4
24
16
14
28
84
34
34
36
100
72
74
78
100
98
100
100
-38-
-------
TABLE 12
TWO-WAY ANOVA WITH REPLICATION; EFFECTS OF
METHOXYCHLOR ON MORTALITY IN FIVE
DEVELOPMENTAL STAGES OF
R. harrisii I-IV
Source of variation
Subgroups
A (Stages)
B (Media)
A x B (Interaction)
Within Subgroups (error)
Total
DF
29
4
5
20
90
119
Sun of
Squares
36611.87
14049.87
6650.67
15911.33
3286.00
39897.87
Mean
Square
1262.48
3512.47
1330.13
795.56
36.51
Fs
96.21
36.43
21.79
-t—f^r.
TvTCTC
•fafcfc
•JUJLJL*
TvTCTC
Significant F ratios:
F.001 [4,90] = 5.13,
F.001 [5,90] = 4.59,
F.001 [20,90] = 2.68
Residue Analysis
Larvae used in the experiments reported here were hatched
from four ovigerous Rhithropanopeus harrisii, Rh I-IV. They
were collected from the west side of Newfound Harbor, a small
estuary which is between the Indian River to the west and the
Banana River to the east in the vicinity of Cocoa Beach. Flo-
rida. To determine if the mother crabs came from a contami-
nated area in Florida, Hazleton Laboratories America, Inc.,
were engaged to analyze the crabs for chlorinated hydrocarbon
pesticide residues (BHC, Lindane, Chlordane, Aldrin, Hepta-
chlor Epoxide, Endrin, Dieldrin and HCB). None were found in
detectable amounts (< 0.01 ppm) except Aroclor 1242 and Aro-
clor 1254. In Rh I-IV, Aroclor 1242 residues were 0.04 ppm,
0.29 ppm, 0.07 ppm and 0.07 ppm. Aroclor 12454 residues in
Rh I-IV were 0.05 ppm, 0.018 ppm, 0.03 ppm and 0.04 ppm.
Analyses were also made to determine methoxychlor residues in
R. harrisii larvae. No detectable methoxychlor (< 0.1 ppm)
was found in newly hatched larvae, five and ten day larvae,
megalopa and first crab stages reared in seawater control, or
-39-
-------
in acetone control in five and ten day larvae, megalopa and
1st crab stages. The results of the determination of meth-
oxychlor residues in crab larvae reared in 1.0 ppb, 2.5 ppb,
4.0 ppb and 5.5 ppb are given in Table 13.
TABLE 13
METHOXYCHLOR RESIDUES IN R. harrisii LARVAE
Sample Identification and Treatment
Methoxychlor ppm
5 day larvae - 1.0 ppb Methoxychlor
10 day larvae - 1.0 ppb Methoxychlor
Megalopa - 1.0 ppb Methoxychlor
First Crab - 1.0 ppb Methoxychlor
5 day larvae - 2.5 ppb Methoxychlor
10 day larvae - 2.5 ppb Methoxychlor
Megalopa - 2.5 ppb Methoxychlor
First Crab - 2.5 ppb Methoxychlor
5 day larvae - 4.0 ppb Methoxychlor
10 day larvae - 4.0 ppb Methoxychlor
Megalopa -4.0 ppb Methoxychlor
First Crab - 4.0 ppb Methoxychlor
5 day larvae - 5.5 ppb Methoxychlor
First Crab - 5.5 ppb Methoxychlor
< 0.1
0.40
< 0.1
< 0.1
0.56
0.51
1.12
0.23
lost
0.59
0.55
0.76
1.28
0.55
B. EFFECTS OF METHOXYCHLOR ON DEVELOPMENT OF Callinectes
sapidus
Survival
Callinectes sapidus larvae hatched from four mother crabs
(Cs L-LV) were reared in acetone control and in 0.7 ppb, 1.0
ppb, 1.3 ppb, 1.6 ppb and 1.9 ppb methoxychlor from the time
of hatching through zoeal and megalopa development to the 1st
crab stage. The percent survival of each of the four series
of larvae which passed through zoeal and megalopa development
is given in Table 14. The average percent survival of larvae
of all replicate series reared in six media through zoeal and
megalopa development is plotted in Figure 3. There is differ-
ential survival through zoeal and megalopa development which
-40-
-------
TABLE 14
EFFECT OF METHOXYCHLOR ON PERCENT SURVIVAL AND DURATION
IN DAYS THROUGH ZOEAL AND MEGALOPA DEVELOPMENT
OF Callinectes sapidus I-IV
Culture Media
Salinity 307~
Temp. 25°C
Initial #
of
Larvae
per Series
Zoeal
Mean
Duration
of
% Survival
Megalopa Hatching to
Develop- Develop- 1st Crab in Mega-
ment in ment in Days lopa
Days
Acetone
Control
Methoxychlor
0.7 ppb
Methoxychlor
1.0 ppb
Methoxychlor
1.3 ppb .
Methoxychlor
1.6 ppb
Methoxychlor
1.9 ppb
Cs
Cs
Cs
Cs
Cs
Cs
Cs
Cs
Cs
Cs
Cs
Cs
Cs
Cs
Cs
Cs
Cs
Cs
Cs
Cs
Cs
Cs
Cs
Cs
1-50
11-50
111-50
IV-50
1-50
11-50
111-50
IV-50
1-50
11-50
111-50
IV-50
1-50
11-50
I I 1-50
IV-50
1-50
11-50
111-50
IV-50
1-50
11-50
111-50
IV-50
32.
28.
32.
35.
38.
32.
37.
37.
43.
36.
43.
40.
43.
41.
37.
46.
35.
*••
50.
«•
73
79
28
20
47
58
34
97
15
12
20
67
67
00
50
00
00
00
•
to
1st
Crab
Days
10.
8.
8.
9.
10.
8.
10.
10.
14.
8.
11.
12.
10.
9.
(two) 8.
10.
(two) 12.
(one) 13.
09
71
87
17
58
82
00
00
50
80
30
77
60
67
00 (one)
67
00 (one)
00 (one)
•
43
37
40
44
49
42
46
48
58
45
57
53
56
50
52
56
46
63
.61
.79
.70
.38
.42
.27
.36
.35
.10
.60
.60
.38
.60
.83
-
.00
.00
.00
-
—
.00
—
-
-
74
58
78
80
38
62
52
66
40
68
30
36
30
18
-
4
8
4
-
—
2
—
—
-
46
28
60
80
24
44
44
62
20
40
20
26
20
12
-
2
6
2
—
—
2
—
—
—
-41-
-------
CO
70
60
50
40
30
20
10
£35%
C
72.5%
M
43.5%
C
26.5%
C
435%
M
ACETONE 0 7
CONTROL W''
8.5%
C
13%
M
0.5% M
1.6
1.9
METHOXYCHLOR, ppb
Figure 3. Average percent survival of four replicate series
of Callinectes sapidus larvae, Cs I-IV, reared
from hatching to megalopa (M) and to 1st crab (C)
in different concentrations of methoxychlor.
-42-
-------
is related to increase in concentration of methoxychlor
(Figure 3).
A^two-way ANOVA with replication (Table 15) shows that the
difference between survival to the megalopa stage and to the
1st crab stage is statistically significant at the 0.05 level
of probability. The differences in survival related to media
(i.e., acetone control, 0.7 ppb and 1.0 ppb methoxychlor) are
significant at the 0.01 level. The interaction between stages
and media is not significant statistically.
TABLE 15
TWO-WAY ANOVA WITH REPLICATION; EFFECT OF METHOXYCHLOR
ON PERCENT SURVIVAL THROUGH ZOEAL AND MEGALOPA
DEVELOPMENT OF C. sapidus I-IV
Source of variation
Subgroups
A (Stages)
B (Media)
A x B (Interaction)
Within subgroups (error)
Total
DF
5
1
2
2
18
23
Sum of
Squares
4700.83
1441.50
3192.33
67.00
4125.00
8825.83
Mean
Square
940.17
1441.50
1596.17
33.50
229.17
Fs
6.29 *
6.97 **
0.146n.s.
Significant F ratios: F.05 [1,18] = 4.41,
F.01 [2,18] = 6.01
Duration of Development
The mean duration of zoeal and megalopa development and the
time from hatching to the 1st crab stage are given in Table
14 for larvae hatched from each mother crab, Cs I-IV, and
reared in six media. The average duration of zoeal and mega-
lopa development of all crabs, Cs I-IV, is shorter in ace-
tone control than in any of the concentrations of methoxy-
chlor (Table 16). In zoeal development and total time from
hatching to 1st crab, the duration increases with concentra-
tion from 0.7 ppb to 1.3 ppb methoxychlor.
-43-
-------
TABLE 16
AVERAGE DURATION IN DAYS OF ZOEAL AND MEGALOPA
DEVELOPMENT OF C. sapidus I-IV
Medium
Duration of Zoeal Duration of Megalopa Time from Hatch
Development Development to First Crab
Acetone Control
32.50
9.22
42.32
Methoxychlor
0.7 ppb
1.0 ppb
1.3 ppb
1.6 ppb
1.9 ppb
36.38
39.89
42.73
42.33
50.00 (one)
9.78
11.32
10.12
11.00
13.00 (one)
46.46
52.13
54.44
53.50
63.00 (one)
TABLE 17
TWO-WAY ANOVA WITH REPLICATION; EFFECT OF METHOXYCHLOR
ON DURATION OF ZOEAL AND MEGALOPA DEVELOPMENT
OF C. sapidus I-IV
Source of variation
Subgroups
A (Stages)
B (Media)
A x B (Interaction)
Within subgroups (error)
Total
DF
5
1
2
2
18
23
Sum of
Squares
4292.28
4131.49
125.20
35.59
104.75
4397.03
Mean
Square Fs
858.46
4131.49 709.88 ***
62.60 10.76
17.80 3.06 n.s.
5.82
Significant F ratios: F.001 [1,18] = 15.4,
F.001 [2,18] - 10.4
-44-
-------
It was only in acetone control and in 0.7 ppb and 1.0 ppb
that 20% or more of the larvae from each of the four mother
crabs survived to both the megalopa and 1st crab stage (Table
14) . A two-way ANOVA with replication of the data on dura-
tion of zoeal and megalopa development in these three media
indicates that variation of duration of C. sapidus develop-
ment is related to stage and to media at "the 0.001 level of
probability (Table 17). The interaction of stage and media
is not significant statistically at P = 0.05.
Sexual morphogenesis
The differentiation of the external and internal sexual cha-
racters of R. harrisii and C. sapidus does not appear to be
affected by dirterent concentrations of methoxychlor. Sex
ratio remains normal in all cases. Upon close examination
of the pleopods of crab stages I through III, however, one
to five plumose setae of the type characteristic of the mega-
lopa stage were found at the tip of each crab pleopod. Fur-
thermore, the pleopods of the male and female developed at a
different rate in the higher concentrations of methoxychlor
in both R. harrisii and C. sapidus than in acetone control.
Mortality
Callinectes sapidus may pass through seven, occasionally
eight,zoeai stages before it molts into a megalopa, a ninth
stage of development. In an effort to determine if there
were one or more of the nine developmental stages of C, sapi-
dus which were particularly sensitive to different concentra-
tions of methoxychlor, a record of deaths by stage was made
for larvae from each of the four mother crabs, Cs I-IV, which
had been reared in acetone control and five concentrations of
methoxychlor (Table 18).
A two-way ANOVA with replication (Table 19) shows that the
main effects of methoxychlor on the mortality of nine devel-
opmental stages of C. sapidus are related significantly to
differences in stages, the media used and interaction of the
two.
To determine which stage of development is significantly sen-
sitive to a particular concentration of methoxychlor, a single
classification ANOVA of mortality data was made for each me-
dium in Table 18, using replicates from larvae of mother crabs,
Cs I-IV. If the means of the series were significantly dif-
ferent, the least significant difference was calculated. In
-45-
-------
TABLE 18
PERCENT MORTALITY IN NINE DEVELOPMENTAL STAGES OF
C. sapidus I-IV
Stage
Acetone
Control
Methoxy-
chlor
0.7 ppb
Methoxy-
chlor
1.0 ppb
Methoxy-
chlor
1.3 ppb
Methoxy-
chlor
1.6 ppb
Methoxy-
chlor •
1.9 ppb
Cs I
Cs II
Cs III
Cs IV
Cs I
Cs II
Cs III
Cs IV
Cs I
Cs II
Cs III
Cs IV
Cs I
Cs II
Cs III
Cs IV
Cs I
Cs II
Cs III
Cs IV
Cs I
Cs II
Cs III
Cs IV
I
2
4
2
8
0
2
6
2
4
4
2
8
12
6
6
12
8
2
28
26
26
12
36
46
II
6
2
0
4
4
10
20
16
6
8
20
16
0
32
78
48
48
42
72
70
40
80
64
54
III
8
0
2
0
16
14
16
6
22
8
20
26
40
28
16
28
26
48
—
4
30
8
-
—
IV
0
4
4
2
4
2
0
6
0
2
8
8
6
10
-
4
4
4
—
—
0
—
—
—
V
0
0
0
2
0
2
2
0
0
0
4
2
0
0
—
2
2
0
—
-
0
—
—
—
VI
0
0
0
0
2
0
0
0
0
4
0
0
0
0
—
0
0
0
—
-
0
—
—
_
VII
10
20
8
2
36
4
4
2
26
2
14
4
12
4
—
2
4
2
—
-
2
_
—
_
VIII
0
12
6
2
0
4
0
2
2
4
2
0
0
2
—
0
0
0
—
-
0
—
—
—
Afega-
lopa
28
30
18
0
14
18
8
4
20
28
10
10
10
6
—
2
2
0
_
-
0
_
_
_
Total
54
72
40
20
76
56
56
38
80
60
80
74
80
88
100
98
94
98
100
100
98
100
100
100
-46-
-------
TABLE 19
TWO-WAY ANOVA WITH REPLICATION; EFFECTS OF
METHOXYCHLOR ON MORTALITY IN NINE
DEVELOPMENTAL STAGES OF
C. sapidus LARVAE
Source of variation
Subgroups
A (Stages)
B (Media)
A x B (Interaction)
Within subgroups (error)
Total
DF
53
8
5
40
162
215
Sum of
Squares
36493.87
12771.16
1119.20
22603.51
11603.00
48096.87
Maan
Square
688.56
1596.40
223.84
565.09
71.62
Fs
22.29 ***
3.13 **
7.89 ***
Significant F ratios: F.001 [8,162] = 3.46,
F.01 [5,162] = 3.13,
F.001 [40,162] = 2.03
acetone control a significantly greater number of deaths
(tn QI) occurred in the megalopa than in any other. No de-
velopmental stages were significantly different (tn Ql) ^ro
one another when the larvae were reared in 0.07 ppb'and 1.0
ppb methoxychlor. Larvae in zoeal stages II and III were
significantly sensitive statistically (P < 0.01) to 1.3 ppb
methoxychlor, as were larvae in zoeal stage II to 1.6 ppb
and zoeal stages I and II to 1.9 ppb methoxychlor.
Residue Analysis
Larvae used in the experiments reported here were hatched
from four ovigerous Callinectes sapidus. The crabs were
collected from the Beautort inlet, approximately two miles
from Duke University Marine Laboratory, Beaufort, North
Carolina. To determine if the mother crabs came from a con
taminated area, Hazleton Laboratories were asked to make .a
determination of chlorinated hydrocarbon pesticide residues
in mother crabs Cs I-IV and to determine methoxychlor resi-
dues in C. sapidus larvae.
-47-
-------
No detectable (< 0.01 ppm) chlorinated hydrocarbon pesticide
residues were found in crabs Cs I-IV of the following pesti-
cides: BHC, Lindane, Chlordane, Aldrin, Heptachlor, Hepta-
chlor Epoxide, DDE, ODD, DDT, Aroclor 1242, Aroclor 1254,
Endrin, Dieldrin and HCB. Furthermore, no detectable (< 0.1
ppm) methoxychlor was found in newly hatched C_. sapidus lar-
vae in seawater, megalopa and 1st crabs in seawater control,
or in acetone control in 5 and 15 day larvae, megalopa and
1st crab stages. The results of determination of methoxy-
chlor residues in crab larvae reared in 0.7 ppb, 1.0 ppb,
1.3 ppb, 1.6 ppb and 1.9 ppb methoxychlor are given in Table
20.
TABLE 20
METHOXYCHLOR RESIDUES IN C. sapidus LARVAE
Sample Identification and Treatment
ppm Methoxychlor
5 day larvae - 0.7 ppb Methoxychlor
15 day larvae - 0.7 ppb Methoxychlor
Megalopa - 0.7 ppb Methoxychlor
First Crab - 0.7 ppb Methoxychlor
5 day larvae - 1.0 ppb Methoxychlor
10 day larvae - 1.0 ppb Methoxychlor
First Crab - 1.0 ppb Methoxychlor
5 day larvae - 1.3 ppb Methoxychlor
13 day larvae - 1.3 ppb Methoxychlor
First Crab - 1.3 ppb Methoxychlor
4 day larvae - 1.6 ppb Methoxychlor
First Crab - 1.6 ppb Methoxychlor
5 day larvae - 1.9 ppb Methoxychlor
0.1
0.1
0.51
0.1
0.52
2.87
0.15
0.76
2.62
0.34
1.25
2.68
0.81
DISCUSSION
Survival in methoxychlor
The range of concentrations in which differential survival
of Rhithropanopeus harrisii occurred from the time of hatch-
-48-
-------
ing to the • 1st crab stage was from 1 . 0 ppb to 7 . 0 ppb methoxy-
chlor (Fig. 2), whereas the range for similar development of
Callinectes sapidus was from 0.7 ppb to 1.9 ppb (Fig. 3).
Since y57» of the R. harrisii larvae of the four series sur-
vived in 1.0 ppb, 79'.'Mo in 2.5 ppb, 53% in 4.0 ppb and 19% in
5.5 ppb methoxychlor , these ! concentrations are considered sub-
lethal . Only one larva survived to the megalopa and crab
stage in one of the four series in 7.0 ppb methoxychlor. There-
fore, this concentration is acutely toxic to R. harrisii lar-
yae. The sublethal concentrations of methoxychlor, tor' C. sap-
idus development were 0.7 ppb, in which there was 43.5% sur-
vival to the 1st crab stage, and 1.0 ppb, in which there was
26.5% survival. Survival to the 1st crab stage in 1.3 ppb,
1.6 ppb and 1.9 ppb was less than 10%, and hence, these con-
centrations are considered acutely toxic. Not only were R.
harrisii larvae much more resistant to methoxychlor than C.
sapidus larvae, but there was no significant difference in
survival to the megalopa and to the 1st crab in R. harrisii
development, but there was in C. sapidus . This means that
megalopa of R. harrisii were not sensitive to methoxychlor
but C. sapidus megalopa were (Figs. 2 and 3) .
The differences in sensitivity to methoxychlor between the
two species were marked, but the same species showed similar
degrees of sensitivity when they were reared in a range of
concentrations of mirex from 0.01 ppb to 10.0 ppb (Bookhout
Bookhout and Costlow, 1975).
Young Gammarus pseudolimnaeus are more sensitive to methoxy-
chlor tnan K. narrisii and c7 sapidus larvae. At two, four
and six weelcs the TLSQ in pg/1 was U.30, 0.29 and 0.24, res-
pectively. Chironomus tentans , however, when exposed to con-
centrations ot u.i ug/1 to 2.0 vg/1 methoxychlor for 28 days,
appear to be about as sensitive as C. sapidus larvae. Appro-
ximately 50% survived at 0.5 yg/1, T7% at i.o yg/1 and none
at 2.0 yg/1. At concentrations of 0.125 yg/1 and 0.25 yg/1
more C. tentans survived and pupated or emerged than in the
control media (Merna and Eisele, 1973).
Sublethal effects
The duration of zoeal development of R. harrisii was prolong-
ed with each concentration of methoxychlor trom 1.0 ppb to
5.0 ppb (Table 7), and there was a similar increase in dura-
tion of zoeal development of C. sapidus as concentrations of
methoxychlor were increased from 0.7 PPD to 1.3 ppb (Table
14). The average duration of megalopa development was simi-
-49-
-------
lar for both species in these concentrations, therefore, the
total time for hatching to 1st crab was prolonged with each
concentration primarily due to effects of methoxychlor on
zoeal development (Tables 7 and 14). The reduction in molt-
ing rate is considered a sublethal effect of methoxychlor on
both R. harrisii and C. sapidus zoeae. A similar increase
in duration ot zoeal development of R. harrisii with increase
of concentration of mirex occurred (Ebokhout et al., 1972),
but there was no significant prolongation of zoeaT develop-
ment of C. sapidus in sublethal concentrations of mirex, only
in acutely toxic concentrations (Bookhout and Costlow, 1975).
During sexual morphogenesis from the megalopa to the 6th crab
stage of R. harrisii and £. sapidus, methoxychlor has no
apparent effect on the genital apparatus, but has sublethal
effects on the pleopods. Since the sex ratio is normal in
crabs reared in different concentrations of methoxychlor,
this signifies that male or female sexual differentiation
occurs normally. The dimorphism of abdominal appendages
evolves according to the genetic sex. In males, only the
fonopods, the 1st and 2nd pleopods, differentiate, whereas in
emales, pleopods 2-5 develop.
Natatory plumose setae, characteristic of megalopa, persist in
crab stages I-III reared in different concentrations. This
indicates an abnormal metamorphosis, since these setae com-
pletely disappear at the molt between megalopa and 1st crab
stage in crabs reared in acetone control. Furthermore, male
differentiation of pleopods is slower in crab stages I-VI
reared in 4.0 ppb, 5.5 ppb and 7.0 ppb methoxychlor in the
case of R. harrisii and 1.6 ppb and 1.9 ppb in the case of C.
sapidus. The regression of the 3rd, 4th and 5th pair of ~
pleopods occurs more slowly in the crabs reared in the above
concentrations of methoxychlor than in the crabs reared in
acetone. This delay in regression of 3rd, 4th and 5th pleo-
pods and in loss of setae in treated crabs does not seem to
be a handicap for further development of crabs for in the
4th, 5th and 6th crab stages the pleopods of the male re-
sembled those in the control. In the female, the persistance
of the megalopa setae on the last three pairs of pleopods is
normal, they don't regress but develop.
Mortality
The main effects of methoxychlor on the mortality of larvae
in four zoeal stages and a megalopa stage of R. harrisii and
larvae in eight zoeal stages and a megalopa stage or u. sapi-
-50-
-------
dus are related significantly (Tables 12 and 19) to differ-
ences in stages, media used and the interaction of the two.
A single classification ANOVA of mortality data for each me-
dium in Table 11, and a calculation of the LSD of each, shows
that there isn't any single stage of R. harrisii which is
significantly different (to.Ol) from any other in acetone
control. In 1.0 ppb, 2.5 ppb and 4.0 ppb methoxychlor, how-
ever, stage I zoeae were significantly sensitive (to.Ol),
and in 5.5 ppb and 7.0 ppb methoxychlor, larvae in zoeal
stages I and II were sensitive statistically. Mirex, on the
other hand, in concentrations of 0.01 ppb, 0.1 ppb, 1.0 ppb
and 10.0 ppb seldom had any effect on larvae in zoeal stage
I of the same species (Bookhout et_ al., 1972).
Larvae in zoeal stage I of C, sapidus were not significantly
sensitive to any of the concentrations of methoxychlor used
except the highest, 1.9 ppm. In acetone control, a signi-
ficantly greater (tQ QI) number of deaths occurred in the
megalopa than in any'of the eight zoeal stages. No develop-
mental stages were significantly different (to.Ol) from any
other when reared in the sublethal concentrations of 0.07
ppb and 1.0 ppb methoxychlor. In the acutely toxic concen-
tration of 1.3 ppb, larvae in stages II and III were signi-
ficantly sensitive (tQ QI), as were larvae in zoeal stage II
to 1.6 ppb and larvae in zoeal stages I and II to 1.9 ppb.
When two series of C. sapidus larvae were reared in acetone
control and four concentrations of mirex, there were no stages
which were significantly sensitive in acetone control or to
0.01 ppb mirex. By contrast, larvae in zoeal stages III, VII
and megalopa in 0.1 ppb mirex and larvae in zoeal stages II
and III in 1.0 ppb mirex showed significantly greater morta-
lity than did larvae in other stages. In 10.0 ppb mirex, all
larvae died in zoeal stages I and II.
Residues
Biological magnification of methoxychlor at 5 days and 10-15
days is much greater in C. sapidus than R. harrisii. There
is no evidence from the limited data available tnat residues
at any one concentration increase with age of larvae (Tables
13 and 20). In R. harrisii, there is an increase of residue
with concentration at tive and ten days, but this trend is
not shown in megalopa and in 1st crab. In C. sapidus, there
is an increase in residue and biological magnification with
concentration in 5 day larvae and in 1st crabs which had been
reared in 1.0 ppb, 1.3 ppb and 1.6 ppb methoxychlor.
-51-
-------
Lee et al. (1975) reported that small amounts of methoxychlor,
0.1 yg/g in juveniles and 0.2 yg/g in adults, accumulated in
striped mullets which had been exposed to 0.01 mg/1 methoxy-
chlor for 96 hours. Juveniles or adults exposed to 0.1, 1.0,
and 10.0 mg/1 for 96 hours did not have significantly differ-
ent residues, although adults carried a heavier load of meth-
oxychlor than juveniles.
Kennedy et al. (1970) made residue analyses for methoxychlor
of whole bluegills from an untreated pond and two treated
ponds on day 1, 3, 7, 14, 28, 56 and 84. No residues were
detected in fish taken from untreated ponds. Fish from ponds
treated with 0.01 ppm methoxychlor had residues of 2.11 ppm
and 2.78 ppm on day one, peaked at 7 days with residues of
3.35 ppm and 4.00 ppm, and had no detectable residues at 56
or 84 days. Fish from ponds treated with 0.04 ppm methoxy-
chlor had residues of 9.80 ppm and 13.90 ppm at day one and
peaked at 3 days with 20.60 ppm and 21.10 ppm arid no resi-
dues were detected at 56 and 84 days.
After snails, mosquitoes and fish had been exposed to 1.6 ppb
methoxychlor for 28 days, residues of methoxychlor were 15.7
ppm in a snail, 0.48 ppm in mosquito, and 0.33 ppm in the
fish, Gambusia (Metcalf e_t ajL., 1971) .
If the biologial magnification of methoxychlor were calcula-
ted for examples cited, the snail exhibited the highest rate.
It apparently could not metabolize methoxychlor rapidly, and
as a result stored it at a substantial level (Metcalf et al.,
1971). The biological magnification of methoxychlor in de-
velopmental stages of C. sapidus was generally above that re-
ported in the literature for_fish with peaks reaching 2870
and 2015.6 times the medium in which they were reared. The
rate of biological magnification of methoxychlor of fish at
peak periods was somewhat above (Kennedy e_t al., 1970) that
found for R. harrisii larvae.
-52-
-------
SECTION VII
MALATHION
INTRODUCTION
Malathion, like many other organophosphate antichloinesterase
insecticides, is used widely to control crop pests, flies and
mosquitoes. The estimated production of malathion in the
United States was 24 million pounds in 1972 (EPA-540/1-75-
005) . The use of chlorinated hydrocarbon insecticides are
being questioned because of their persistence and the resi-
dues which are concentrated through biological accumulation.
The current trend, therefore, will probably be to replace
chlorinated hydrocarbons with less persistent organophos-
phates, including malathion, and carbamate pesticides (Cop-
page and Duke, 1971).
The toxicity of malathion to aquatic organisms apparently de-
pends upon the exposure time, temperature, pH of the water,
and the species. Malathion decomposes at high temperatures.
It is reported to be quite stable under neutral or acid pH
conditions. Susceptibility to hydrolysis increases, however,
with increasing alkalinity (Walker and Stojanovic, 1973). At
pH 5 to 7, no hydrolysis could be detected after 12 days; at
pH 9, 50% hydrolysis occurs in 12 hours, and at pH 12, hydro-
lysis takes place immediately (EPA-540/1-75-005). Malathion
is rapidly dissipated in non-sterile soils due to microflora
in the soil and chemical mechanisms (Walker and Stojanovic,
1973) . In the absence of residues of malathion in animals
from treated areas, measurements of the enzyme acetylchlin-
esterase (AChE) are probably the best general indicator of
organophosphate pesticide pollution (Coppage and Matthews,
1974). Inhibition of AChE is believed to be the cause of
death of higher vertebrates by blocking neurotransmission in
the respiratory center of the brain or neuromuscular junc-
tions of the respiratory apparatus. Inhibition of AChE is
also thought to be the mode of action of these pesticides on
arthropods (O'Brien, 1967).
Even though malathion is biodegradable, it may be highly tox-
ic to target and non-target organisms alike. As a non-per-
-53-
-------
sistent insecticide, repeated applications of malathion may
be necessary for control of pests, and cumulative reduction
of AChE may occur (Coppage and Matthews, 1974). Furthermore,
there is documented evidence from the literature that arthro-
pod pests develop resistance to malathion (Mengle and Lewal-
len, 1963; Busvine, 1959; Brown and Abedi, 1960; La Brecque
and Wilson, 1960; Mount, Seawright and Pierce, 1974). Hence,
increasing concentrations of malathion have to bemused to
control pests, and the danger to non-target organisms be-
comes greater.
Few laboratory and field studies have been conducted on the
effects of malathion on estuarine animals . Darsie and Cor-
riden (1959) reported that malathion formulated in fuel oil
and sprayed by airplane [560 g/ha (8 wt.oz/acre)] on tidal
marshes in Delaware was toxic within four hours to Fundulus
ocellaris held in tubs. Tagatz et al. (1974) studied tne
effects of thermal fog 420 g/ha TF wt.oz/acre) and ULV
aerosol spray 57 g/ha (0.64 fl.oz/acre) applications of
malathion 95 on salt marsh environments in northwestern Flo-
rida. After three treatments deaths due to malathion were
not observed to confined blue crabs, Callinectes sapidus;
grass shrimps, Palaemonetes vulgaris and F. pugio; pink shr-
imp, Penaeus duorarum; or sheepshead minnows, Cyprinodon
variegatus . Conte and Parker (1971), however, reported that
an aerial application of (256 g/ha) 3 fl. oz/acre to marsh
embayments in Texas killed 14 to 80% of commercial shrimp,
Penaeus aztecus and P. setiferus. They found residues of
malathion of 0.8 to 7.2 ppm in the water and in the tissues
of living shrimp residues from 0.28 to 2.67 ppm up to 48
hours after spraying.
To date, however, there have been no publications on the
effects of malathion on the larval development of crabs. If
aerial spraying of malathion can kill 14 to 80% of commercial
shrimp, similar treatment could have sublethal and/or lethal
effects on the larval development of crabs.
The objective of our study was to determine the effects of
malathionron the complete larval development of Rhithropano-
peus harrisii and Callinectes sapidus from the time of hat-
ching until the 1st crab stage is reached. Specifically
answers to the following questions will be sought: (1) what
concentrations of malathion are sublethal and which are acu-
tely toxic; (2) what are the sublethal effects of these con-
centrations; (3) what are the effects of malathion on the
development of the external and internal sex characters; (4)
is there one or more larval stages particularly sensitive to
different concentrations of malathion; and, (5) what are the
-54-
-------
differences in the effects of malathion on the complete de-
velopment of R. harrisii and C. sapidus?
RESEARCH RESULTS
A. EFFECTS OF MALATHION ON DEVELOPMENT OF Rhithropanopeus
harrisii
Survival
Rhithropanopeus harrisii larvae hatched from mother crabs,
Kn I-iv, were reared in acetone control and in 0.011 ppm,
0.014 ppm, 0.017 ppm, 0.02 ppm and 0.05 ppm malathion. The
percent survival of four series of larvae, which passed
through zoeal and megalopa development, is given in Table 21,
and the average percent survival of larvae of four replicate
series in five media is plotted in Figure 4. Since 97.5% of
the larvae survived zoeal development and reached the megalo-
pa stage in acetone control and 94.5% of the megalopa molted
to the 1st crab stage, it can be concluded that the larvae
used in the experiment were very healthy. There was a reduc-
tion in survival of larvae with each increase in concentra-
tion of malathion from 0.011 ppm to 0.02 ppm (Fig. 4). Lar-
vae reared in 0.05 ppm did not survive beyond the second
zoeal stage.
A two-way ANOVA with replication, Table 22, shows that the
differences between the number of larvae which completed
zoeal and megalopa development and differences in survival
in acetone control and in 0.011 ppm, 0.014 ppm, 0.017 ppm
and 0.02 ppm are both highly significant (0.001 probability).
Duration of Development
Table 21 gives the mean duration of zoeal and megalopa de-
velopment and the mean time from hatching to the 1st crab
stage in each of the four replicate series of larvae reared
in five media. The average duration of zoeal and megalopa
development of larvae from four replicate mother crabs is
tabulated in Table 23. The duration of zoeal development's
shortest in acetone control, and is lengthened with each in-
crease in concentration of malathion from 0.011 ppm to 0.02
ppm. The duration of the megalopa stage in the five media
does not follow this trend, but the total time from hatching
to the 1st crab stage does (Table 23).
-55-
-------
TABLE 21
EFFECT. OF MALATHION ON PERCENT SURVIVAL AND DURATION
IN DAYS THROUGH ZOEAL AND MEGALOPA DEVELOPMENT
OF Rhithropanopeus harrlsii I-IV
Culture Media
Salinity 20%,
Temp. 25 °C
Acetone
Control
Malathi-on
0.011 ppm
Malathion
0.014 ppm
Malathion
0.017 ppm
Malathion
0.02 ppm
Initial #
of Larvae
per Series
Rh 1-50
Rh 11-50
Rh 111-50
Rh IV-50
Rh 1-50
Rh 11-50
Rh 111-50
Rh IV-50
Rh 1-50
Rh 11-50
Rh 111-50
Rh IV-50
Rh 1-50
Rh II-50
Rh 111-50
Rh IV-50
Rh 1-50
Rh 11-50
Rh 111-50
Rh IV-50
Mean Duration of
Zoeal
Develop-
ment in
Days
10.92
11.38
10.73
11.02
11.46
11.26
11.37
11.24
11.47
10.95
11.52
11.63
12.21
10.95
12.57
12.56
12.67
12.00
12.00
Megalopa
Develop-
ment in
Days
4.96
5.28
5.47
6.00
4.79
5.20
5.00
5.21
5.00
5.13
4.88
5.36
5.13
5.13
6.00
4.60
5.50
—
-
Hatching to
1st Crab in
Days
15.88
16.66
16.20
16.63
16.30
16.43
16.18
16.35
15.96
16.50
16.11
16.86
18.00
16.50
17.00
17.40
17.00
—
—
"
% Survival
to
Mega-
lopa
100
100
98
92
88
84
82
84
68
44
44
38
38
30
14
18
6
6
2
0
1st
Crab
100
100
98
80
78
60
66
68
48
16
36
28
16
10
2
10
2
0
0
0
-56-
-------
100
80
60
|
1 40
CO
20
M
0 ACETONE
CONTROL
68%
C
8V%
32%
C
3.5% M
1.0% C
0.011
0.014 0.017 0.02
MALATHION, pprr\
Figure 4. Average percent survival of four replicate series
of Rhithropanopeus harrisii, Rh I-IV, reared from
hatching to megalopa (M) and to 1st crab (C) in
different concentrations of malathion.
-57-
-------
TABLE 22
TWO-WAY ANOVA WITH REPLICATION; EFFECT OF MALATHION
ON SURVIVAL THROUGH ZOEAL 'AND MEGALOPA
DEVELOPMENT OF R. harrisii I-IV
Source of variation
Subgroups
A (Stages)
B (Media)
A x B (Interaction)
Within subgroups (error)
Total
DF
9
1
4
4
30
39
'Sum of
Squares
51103.6
1166.4
49378.6
558.6
2090.0
53193.6
Mean
Square Fs
5678.2
1166.4 16.74 ***
12344.7 177.19 ***
139.7 2. 01 its.
69.67
Significant F ratios:
F.001 [1,30] = 13.3,
F.001 [4,30] = 6.12
TABLE 23
AVERAGE DURATION IN DAYS OF ZOEAL AND MEGALOPA
DEVELOPMENT OF R. harrisii I-IV
Duration of Zoeal
Medium Development
Duration of Megalopa
Development
Time from Hatch
to First Crab
Acetone Control
Malathion
0.011 ppm
0.014 ppm
0.017 ppm
0.02 ppm
11.01
11.33
11.39
12.07
12.22
5.43
5.05
5.09
5.22
5.50 (one)
16.19
16.32
16.36
-17.23
17.00 (one)
-58-
-------
A two-way ANOVA with replication indicates that variation of
duration of R. harrisii larvae reared in acetone control and
four concentrations ot malathion is related to stages and to
media at the 0.001 and 0.05 level of probability, respective-
ly (Table 24).
TABLE 24
TWO-WAY ANOVA WITH REPLICATION; EFFECT OF MALATHION ON
DURATION IN DAYS THROUGH ZOEAL AND MEGALOPA
DEVELOPMENT OF R. harrisii I-IV
Source of variation
Subgroups
A (Stages)
B (Media)
A x B (Interaction)
Within subgroups (error)
Total
DF
9
1
4
4
30
39
Sum of
Squares
494.94
430.79
60.75
4.40
140.15
636.09
Mean
Square
55.10
430.79
15.19
1.10
4.67
Fs
92.10 ***
3'.25 *
0.24n.s.
Significant F ratios: F.001 [1,30] = 13.3,
F.05 [4,30] = 2.69
Sexual Morphogenesis
Malathion in the higher concentrations had no effect on the
development of the genital apparatus of R. harrisii and C.
sapidus in crab stages I-VI. It had the sublethal effect in
the male of retarding the loss of pleopods 3-5 and the loss
of megalopa setae after metamorphosis, as was described pre-
viously for methoxychlor.
Effect of Malathion on Mortality
Rhithropanopeus harrisii passes through four zoeal stages
and a megalopa. In an effort to determine if larvae in one
or more stages of development were particularly sensitive to
different concentrations of malathion, a record of deaths by
stage was made for larvae reared from four mother crabs, Rh
-59-
-------
I-IV (Table 25).
TABLE 25
PERCENT MORTALITY IN FIVE DEVELOPMENTAL STAGES
OF R. harrisii I-IV
Stage
Acetone Control
Malathion
0.011 ppm
Malathion
0.014 ppm
Malathion
0.017 ppm
Malathion
0.02 ppm
Malathion
0.05 ppm
Rh I
Rh II
Rh III
Rh IV
Rh I
Rh II
Rh III
Rh IV
Rh I
Rh II
Rh III
Rh IV
Rh I
Rhll
Rh III
Rh IV
RhI
Rh II
Rh III
Rh IV
Rh I
Rh II
Rh III
Rh IV
I
0
0
0
2
2
6
0
2
2
6
0
0
0
8
0
2
2
0
14
0
80
30
98
80
II
0
0
0
0
8
6
12
14
22
18
14
44
58
32
50
74
82
60
70
92
20
70
2
20
III
0
0
0
0
0
4
2
0
4
4
12
8
0
12
14
0
8
26
12
8
—
-
-
™
IV
0
0
2
6
2
0
4
0
4
28
30
10
4
18
22
6
2
8
2
0
9m
-
-
™
Megalopa
0
0
0
12
10
24
16
16
20
28
8
10
22
20
12
8
2
6
2
0
*
-
-
—
Total
0
0
2
20
22
40
34
32
52
84
64
72
84
90
98
90
96
100
100
100
100
100
100
100
A two-way ANOVA with replication of the data in Table 25
shows that the main effects of malathion on mortality of
five larval stages of R. harrisii are related to stages, me-
dia and interaction of the two at the 0.001 level of proba-
bility (Table 26).
-60-
-------
TABLE 26
TWO-WAY ANOVA WITH REPLICATION; EFFECT OF MALATHION
ON THE MORTALITY IN; FIVE LARVAL STAGES
OF R. harrisii
Source of variation
Subgroups
A (Stages)
B (Media)
A x B (Interaction)
Within subgroups (error)
Total
DF
29
4
5
20
90
119
Sum of
Squares
46838.67
11819.00
6132.27
28887.40
9310.00
56148.67
Mean
Square
1615.13
2954.75
1226.45
1444.37
103.44
Fs
28.56 ***
11.86 ***
13.96 ***
Significant F ratios:
F.001 [4,90] = 5.13,
F.001 [5,90] = 4.59,
F.001 [20,90] - 2.68
To determine which stage of development is significantly sen-
sitive to a particular concentration of malathion, a single
classification ANOVA of the mortality data was made for each
medium in Table 25, using replicates from larvae of mother
crabs, Rh I-IV. If the means of the series were significant-
ly different, the least significant difference was calcula-
ted. In acetone control, no single stage was significantly
different in mortality from any other. Larvae in t zoeal stage
II and megalopa were significantly sensitive statistically
(tQ 01) to 0.011 ppm malathion, as were larvae in zoeal stage
II to 0.014 ppm, 0.017 ppm and 0.02 ppm, and larvae in zoeal
stage I to 0.05 ppm malathion.
Residue Analysis
Since Hazleton Laboratories in 1973 found no detectable mala-
thion in samples of blue crab larvae reared in concentrations
ranging from 0.1 ppb to 100.0 ppb, it was,not deemed advis-
able by Mr. Jack Lowe, the project director, to^have Rhithro-
panopeus larvae analyzed for residues of malathion. The four
mother crabs, Rh I-IV, which furnished the larvae for the in-
-61-
-------
vestigation in 1975 were analyzed, however, for pesticides by
Hazleton Laboratories in May, 1975. No Lindane, Chlordane,
Aldrin, Heptachlor, Heptachlor Epoxide, DDE or ODD were de-
tected. The results determined for these pesticides in the
6/94 ethyl ether/petroleum ether fraction could not be detec-
ted lower than 0.05 ppm due to the interference of Aroclor
1242 peaks. No Aroclor (<. 5 ppm), Mirex (<.2 ppm), Dieldrin
(<.01 ppm) or Endrin (<.01ppm) was found either. The pesti-
cides which were detected are given in Table 27.
TABLE 27
RESIDUES IN FOUR MOTHER CRABS, Rh I-IV
Pesticide in ppm Rh I Rh II Rh III Rh IV
DDT
Aroclor 1242
Methoxychlor
0.19
1.75
0.57
0.08
0.57
1.56
0.37
2.32
5.50
0.32
0.70
0.80
RESEARCH RESULTS
B. EFFECTS OF MALATHION ON DEVELOPMENT OF Callinectes sapi-
dus
Survival
Callinectes sapidus larvae hatched from mother crabs, Cs I-IV,
were reared in acetone control, and in 0.02 ppm, 0.05 ppm,
0.08 ppm and 0.11 ppm malathion. The percent survival of four
series of larvae which completed zoeal and megalopa develop-
ment is given in Table 28. The average percent survival of
larvae from four replicate series reared in five media is
plotted in Figure 5. Survival to megalopa and 1st crab stage
in all concentrations of malathion was less than in acetone
control. There was differential survival in concentrations
of malathion from 0.02 ppm to 0.08 ppm (Fig. 5). Survival in
0.11 ppm malathion was somewhat better than in 0.08 ppm but
lower than in 0.05 ppm. This apparent discrepancy will be
considered later in the discussion.
-62-
-------
TABLE 28
EFFECT OF MALATHION ON PERCENT SURVIVAL AND DURATION
IN DAYS THROUGH ZOEAL AND MEGALOPA
DEVELOPMENT OF Callinectes sapidus I-IV
f
Culture Media
Salinity 30%>
Temp. 25 C°
Acetone
Control
Malathion
0.02 ppm
Malathion
0.05 ppm
Malathion
0.08 ppm
Malathion
0.11 ppm
^ -^
Initial #
of Larvae
per Series
Cs 1-50
Cs 11-50
Cs 111-50
Cs IV-50
Cs 1-50
Cs 11-50
Cs 111-50
Cs IV-50
Cs 1-50
Cs 11-50
Cs 111-50
Cs IV-50
Cs 1-50
Cs 11-50
Cs 111-50
Cs IV-50
Cs 1-50
Cs 11-50
Cs 111-50
Cs IV-50
Mean Duration of
Zoeal
Develop-
ment in
Days
40.81
35.45
37.17
35.68
41.84
37.68
41.43
38.70
44.38
39.30
40.25
40.67
48.00
46.33
43.00
43.00
41.00
45.25
47.25
44.00
Megalopa
Develop-
ment in
Days
7.82
7.64
7.42
8.12
10.38
8.33
10.33
7.90
11.57
9.50
9.75
8.47
10.50
8.66
8.00
7.00
7.80
7.88
9.00
7.20
Hatching to
1st Crab in
Days
48.41
43.09
44.36
44.00
51.88
45.88
52.67
46.60
56.29
48.80
50.00
49.13
55.50
55.00
49.00
50.00
48.80
53.13
56.33
51.20
% Survival
Mega-
lopa
42
66
72
56
38
50
14
40
16
20
8
30
6
6
6
8
10
16
8
10
to
- 1st
Crab
34
66
66
52
32
48
12
40
14
20
8
30
4
6
4
8
10
<"• X^
16
10
-63-
-------
60
50
40
< 30
a:
:D
10
54.5K
C
59%
M
ACETONE
CONTROL
33%
C
35.5%
M
18%
C
18.5%
M
5.5%
C
M
•"""•
IQ.5%1
C
11%
M
0.02
0.05 0.08
O.I I
MALATHION, ppm
Figure 5. Average percent survival of four replicate series
of Callihectes sapidus, Cs I-IV, reared from
hatching to megalopa (M) and to 1st crab (C) in
different concentrations of malathion.
-64-
-------
A two-way ANOVA with replication (Table 29) shows that the
variation in media accounted for the differences in survival
(P < 0.001). The stage and interaction between stages and
media were not significant.
TABLE 29
TWO-WAY ANOVA WITH REPLICATION; EFFECT OF MALATHION ON
PERCENT SURVIVAL THROUGH ZOEAL AND MEGALOPA
DEVELOPMENT OF C. sapidus I-IV
Source of variation
Subgroups
A (Stages)
B (Media)
A x B (Interaction)
Within subgroups (error)
Total
DF
9
1
4
4
30
39
Sum of
Squares
13680.4
32.4
13624.4
23.6
3238.0
16914.4
Mean
Square
1520.04
32.40
3406.10
5.90
107.93
Fs
0.30 n.s.
31.56 ***
0.05 n.s.
Significant F ratio: F.001 [4,30] - 6.12
Duration of Development
Table 28 shows the variation in mean duration of zoeal and
megalopa development and the time from hatching to the 1st
crab stage in each of the four replicate series of larvae
reared in five media. The average duration of zoeal and meg-
alopa development of larvae from four replicate mother crabs
is tabulated in Table 30. The duration of zoeal development
is shortest in acetone control, and is lengthened with each
increase in concentration of malathion from 0.02 ppm to 0.08
ppm. The duration of the megalopa stage does not follow this
trend, but the total time from hatching to the 1st crab stage
does (Table 30).
A two-way ANOVA with replication (Table 31) reveals that var-
iation of duration of C. sapidus larvae reared in acetone
control and four concentrations of malathion is related to
stages, media and interaction of the two at the 0.001 level
of probability.
-65-
-------
TABLE 30
AVERAGE DURATION IN DAYS OF ZOEAL AND MEGALOPA
DEVELOPMENT OF C. sapidus I-IV
Medium
Duration of Zoeal
Development
Duration of Megalopa
Development
Time from Hatch
to First Crab
Acetone Control
Malathion
0.02 ppm
0.05 ppm
0.08 ppm
0.11 ppm
36.98
39.45
41.05
44.92
44.36
7.72
8.85
9.50
8.27
7.86
44.52
48.16
50.53
52.18
52.10
TABLE 31
TWO-WAY ANOVA WITH REPLICATION; EFFECT OF MALATHION ON
DURATION IN DAYS THROUGH ZOEAL AND MEGALOPA
DEVELOPMENT OF C. sapidus I-IV
Source of variation
Subgroups
A (Stages)
B (Media)
A x B (Interaction)
Within subgroups (error)
Total
Significant F ratios:
DF
9
1
4
4
30
39
F.001
F.001
Sum of
Squares
10999.68
10821.47
89.54
88.67
103.61
11103.29
[1,30] = 13
[4,30] - 6.
Mean
Square
1222.19
10821.47
22.39
22.17
3.45
.3,
12
Fs
3136.66 ***
6.49 ***
6.43 ***
-66-
-------
Effect of Malathidh on Mortality
Cairinectes sapidus usually passes through seven zoeal stages
and a megalopa stage before reaching a first crab stage. A
frequent variation from the norm in the life history of the
blue crab is the addition of an 8th zoeal stage before a molt
to a megalopa stage. To determine if larvae in one or more
of the nine stages of development of C. sapidus were particu-
larly sensitive to different concentrations ot malathion, a
record of deaths by stage was made for larvae reared from four
mother crabs, Cs I-IV (Table 32).
TABLE 32
PERCENT MORTALITY IN NINE DEVELOPMENTAL STAGES OF
C. sapidus I-IV
Stage
Acetone
Control
Malathion
0.02 ppm
IT IT
Malathion
0.05 ppm
Malathion
0.08 ppm
Malathion
0.11 ppm
Cs I
Cs II
Cs III
Cs IV
Cs I
Cs II
Cs III
Cs IV
Cs I
Cs II
Cs III
Cs IV
Cs I
Cs II
Cs III
Cs IV
Cs I
Cs II
Cs III
Cs IV
I
2
0
8
2
4
8
4
4
0
16
20
4
18
22
16
14
44
46
48
68
II
50
4
14
24
30
28
44
22
52
42
46
22
72
40
42
42
44
28
40
10
III
2
20
6
14
16
12
22
30
28
20
14
38
4
30
32
34
0
10
2
8
IV
0
8
0
0
4
2
4
2
2
2
. 4
6
0
0
4
0
0
0
0
4
V
0
0
0
0
6
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
VI
0
2
0
0
0
0
4
0
0
- 0
0
0
0
0
0
2
0
0
0
0
VII
4
0
0
4
2
0
6
0
0
0
4
0
0
0
0
0
0
0
2
0
VIII
0
0
0
0
0
0
2
2
2
0
4
0
0
0
0
0
2
0
0
0
Mega-
lopa
8
0
6
4
6
2
2
0
2
0
0
0
2
2
0
0
0
2
Total
66
34
34
48
68
52
88
60
86
80
92
70
96
96
92
90
84
94
f\/\
90
-67-
-------
A two-way ANOVA with replication of the data in Table 32 shows
that the main effects of malathion on mortality are associ-
ated significantly with stages, media and interaction of the
two (Table 33).
TABLE 33
TWO-WAY ANOVA WITH REPLICATION; EFFECT OF MALATHION
ON MORTALITY IN NINE DEVELOPMENTAL STAGES .
OF C. sapidus I-IV
Source of variation
Subgroups
A (Stages)
B (Media)
A x B (Interaction)
Within subgroups (error)
Total
DF
44
8
4
32
135
179
Sum of
Squares
32910.58
23580.58
698.36
8631.64
5711.00
38621.58
Mean
Square
2947.50
174.59
269.74
42.30
Fs
69.68 ***
4.12 **
6.38 ***
Significant F ratios: F.001 [8,135] = 3.51,
F.005 [4,135] = 3.89,
F.001 [32,135] = 2.19
To ascertain which stage of development is significantly sen-
sitive to a particular concentration of malathion a single
classification ANOVA of the mortality data was made for each
medium in Table 32, using replicates from larvae of mother
crabs, Cs I-IV. If the means of the series were significant-
ly different, the least significant difference was calculated.
Zoeal stage II was significantly different from the other
developmental stages in acetone control, hence a two-way ANOVA
of stages II and III was performed to compare the malathion
concentrations with an acetone control. From all calculations
no stages were significantly sensitive to 0.02 malathion. Lar-
vae in zoeal stages II and III were significantly sensitive
statistically (P < 0.05) to 0.05 ppm malathion, as were lar-
vae in zoeal stages I, II and III to 0.08 ppm and larvae in
stage I to 0.11 ppm malathion.
-68-
-------
DISCUSSION
Survival in Malathion
The range of concentrations in which differential survival of
Rhithropanopeus harrisii occurred from the time of hatching
to tne 1st crab stage was from 0.011 ppm to 0.02 ppm malathion
(Fig. 4), whereas the range for similar development of Calli-
nectes sapidus was from 0.02 ppm to 0.11 ppm malathion (Fig.
57"!D"f the four concentrations in which there was survival
to the 1st crab stage, in R. harrisii two were sublethal,
0.011 ppm and 0.014 ppm, and two were acutely toxic, 0.017
ppm and 0.02 ppm (Fig. 4). In 0.02 ppm malathion, there was
a small number of larvae which reached the megalopa stage in
three of the four series, but only two larvae reached the 1st
crab stage in one of the four series (Table 21). Larvae rear-
ed in 0.05 ppm malathion did not survive beyond the 2nd zoeal
stage. In C. sapidus there was survival to the 1st crab stage
in each concentration of each of the replicate series of lar-
vae. Survival of larvae to the 1st crab stage varied in the
replicate series reared in acetone control. Nevertheless,
if the number of larvae which survived to megalopa and to 1st
crab is noted for the same replicate, it can be seen that
there is a reduction in survival in concentrations from 0.02
ppm to 0.08 ppm. Survival in 0.11 ppm was slightly higher
than in 0.08 ppm. Concentrations of 0.02 ppm and 0.05 ppm
were sublethal, and 0.08 ppm and 0.11 ppm were acutely toxic.
A two-way ANOVA shows that the differences in survival of R.
harrisii are related to stages (P < 0.001) and media (P<0.05)
used (Table 22), but the differences in survival to the lst_
crab stage of C. sapidus are mainly associated with the vari-
ation in media (P < 0.001). Stage and interaction between
stage and media were not significant (Table 29).
When R. harrisii was exposed to different concentrations of
methoxychlor throughout larval development, there was no sta-
tistical difference between the number of larvae which com-
pleted zoeal and megalopa development, but the variations in
survival in different media were highly significant (Table 8),
whereas the variations in survival in C. sapidus development
were related to differences between survival to the megalopa
stage and to the 1st crab stage, as well as to media (Table
15) . This is just the opposite to the reaction of the lar-
vae of the two species to malathion.
The developmental stages of C. sapidus are much more resist-
ant to malathion than the larvae of R. harrisii. A concen-
tration of 0.02 ppm malathion was suFlethal to C. sapidus but
-69-
-------
acutely toxic to R. harrisii even though £. sapidus larvae
were exposed to daily changes of 0.02 ppm malathion from 46
to 52 days as compared to an exposure of 17 days in the case
of R. harrisii larvae. The sensitivity of R. harrisii and
C. sapidus larvae to methoxychlor was just the opposite that
observed to the sensitivity to malathion. Larvae of each
species were about equally sensitive to concentrations of
mirex in a range from 0.01 ppb to 10.0 ppb.
If residues of malathion of 0.8 to 3.2 ppm can remain in the
water after aerial application of 256 g/ha as Conte »and Par-
ker (1971) reported, these concentrations would be lethal to
R. harrisii and £. sapidus larvae.
Although it is known that hydrolysis increases as alkalinity
does (Walker and Stojanovic, 1973), the half-life of 0.5 ppm
malathion in water is reported to be about one month at pH 8
and 28°C (EPA-540/1-75-005). These are similar to the condi-
tions in which R. harrisii and C. sapidus larvae develop and
further supports the belief that sprays of malathion for mos-
quito control could be hazardous to crab larvae.
Sublethal effect
The average duration of zoeal development and total time from
hatching to 1st crab stage were shortest in acetone control
and were lengthened with each increase in concentration of
malathion in R. harrisii from 0.011 ppm to 0.02 ppm (Table
23) and in C. sapidus trom 0.02 ppm to 0.11 ppm (Table 30).
The variation in duration of larval development of R. harri-
sii is related statistically to stages (P < 0.001) and media
TF~< 0.05) (Table 24), whereas the variation in duration of
larval development of C. sapidus is related to stages, media
and the interaction of the two at the 0.001 level of probabi-
lity.
Sexual morphogenesis
No modification of the structure of the genital apparatus of
R. harrisii and C. sapidus was related to increase of concen-
trations of methoxychlor or malathion. The anlage of germi-
nal cells, protogonia, develop as in crabs reared in acetone
control. The genital ducts and androgenic gland differenti-
ate progressively in the males, as well as the spermatheca
and vagina in the females. The onset of oogenesis takes
place earlier than spermatogenesis, just as it does in the
controls. No particular effect of insecticides has been no-
-70-
-------
ticed during the process of gametogenesis in the male or fe-
male.
Though not associated with sexual morphogenesis it was noti-
ced that 0.017 ppm and 0.02 ppm malathion induces autotomy
of legs^at^the megalopa stage, as well as in crab stages, of
R. harrisii. This occurrence does not prevent the megalopa
from molting to the crab stage. It is known that autotomy
is usually associated with mechanical stimulation of legs of
crabs. In this case, a poisonous fluid, malathion, induces
autotomy as early as the megalopa stage.
Mortality
The main effects of malathion on the mortality of four zoeal
stages and a megalopa stage of R. harrisii and eight zoeal
stages and a megalopa stage of ET. sapidus "are related in a
highly significant way to stages, media and interaction of
the two (Tables 26 and 33).
A single classification ANOVA of mortality data from each me-
dium in Table 25 and a calculation of the least significant
difference, shows that there isn't any single stage of R.
harrisii which is significantly different (tQ.gi) in mortali-
ty from any other in acetone control. Larvae'in zoeal stage
II and megalopa were significantly sensitive (tQ.oi) to 0.011
§pm malathion, as were larvae in zoeal stage II to 0.014 ppm,
.017 ppm and 0.02 ppm, and larvae in zoeal stage I to 0.05
ppm.
In C. sapidus, no stages were significantly sensitive to 0.02
ppm malathion. Larvae in zoeal stages II and III were signi-
ficantly sensitive (P < 0.05) to 0.05 ppm malathion, as were
larvae in zoeal stages I, II and III to 0.08 ppm and larvae
in stage I to 0.11 ppm malathion.
An examination of the percent mortality of larvae in develop-
mental stages of C. sapidus in Table 32 shows that more lar-
vae in the first zoeal stage died in every replicate of 0.11
ppm malathion than in any replicate of the other concentra-
tions of malathion. It is believed, therefore, that the con-
centration of 0.11 ppm prepared by Hazleton Laboratories Amer-
ica, Inc., is higher than 0.08 ppm, even though the total mor-
tality to 1st crab was slightly higher in 0.08 ppm tiian 0.11
ppm malathion.
Many acute toxicity tests, but few chronic bioassays have
been made to determine the effect of malathion on aquatic or-
-71-
-------
ganisms. It is difficult, therefore, to compare our results
with those of other investigators. If the concentration of
malathion necessary to immobilize or kill 50% of the organ-
isms being tested for 24, 48 or 96 hours is lower than ob-
served in our chronic bioassays with R. harrisii and C. sapi-
dus larvae, it can be assumed that crab larvae are more re-
sTs~tant. The following common freshwater organisms fit into
this category. Daphnia magna. is immobilized in 50 hours by
0.9 yg/1 malathion wnen kept at 68°F, and Simocephalus serra-
latus is immobilized by 6.2 yg/1 in 48 hours at 70 "tf. Increase
in temperature from 50°F to 70°F resulted in an eight-fold
decrease in toxicity to the latter species (Sanders and Cope,
1966). The 96-hour TI^ for Gammarus lacustris was 0.002 ppm
(Gaufin et al., 1965) and the Z4-hour LCso tor larval Chiro-
nomus tentans was also 0.002 ppm (Karnak and Collins, 1974).
The 24-hour LCgo for larval mosquitoes, Culex pipiens quin-
quefasciatus ranged from 0.05 to 0.120 ppm in polluted water
and u. 1UU to 0.240 ppm in tap water (Lewallen and Wilder,
1963).
No studies have been made on the short or long-term effects
of malathion on larval decapods, but Eisler (1969") made acute
toxicity bioassays of three species of marine decapods. He
found that the 96-hour LCso values of malathion to be 33 yg/1
for the sand shrimp, Crangon septemspinosa, 82 yg/1 for grass
shrimp, Palaemonetes vulgaris, and 83 yg/1 for the hermit
crab, Fagurus longicarpus.
These concentrations are within the range of media used in C.
gapidus development but are higher than those employed in tEe
R1". harr'isii experiment.
Eisler (1969) was of the opinion that crustaceans are much more
sensitive to most organophosphorus insecticides than fish.
This may be true for many fish but not for the bluegill, Lep-
omis macrochiris. Concentrations of 66 and 28 yg/1 malathion
were lethal to all bluegills within 16 and 54 days, respect-
ively. Reproduction and early fry were unaffected by 7.4
yg/1 malathion which crippled adult fish after several months
of exposure. The safe concentration of malathion to bluegills
was experimentally determined to be 3.6 yg/1 and the unsafe
concentration to be 7.4 yg/1. The maximum acceptable concen-
tration is between the two (Eaton, 1970). It would appear,
therefore, that C_. sapidus larvae are about as sensitive to
malathion as bluegills, but R. harrisii larvae are more sen-
sitive than bluegills.
-72-
-------
SECTION VIII
GENERAL DISCUSSION
ECOLOGICAL IMPLICATIONS
Before considering the possible" effects of different concen-
trations of mirex, methoxychlor and malathion on the complete
development of R. harrisii and C. sapidus from hatching to
1st crab stage Tn tne field, it is important to know in what
range of salinities and temperatures normal development of
these bioassay crustaceans will take place. Costlow et al.
(1966) reported that R. harrisii can develop from hatcEing
to 1st crab stage in Ehe laboratory in salinities from 2.5%0
to 40%o, but that the best survival, 66% to 90%, will take
place in salinities from 15°/00 to 25°/00. In lower and higher
salinities, there is a reduction in survival. In salinities
below 15%0, there is higher survival in 30°C than in 20°C,
but in salinities from 15%o to 40%o survival is higher at
lower temperatures. In the Newport River Estuary, N.C., Pin-
schmidt (1963) collected R. harrisii larvae when water temp-
eratures were between 16°C" and 34°C, with the greatest abun-
dance at the higher temperatures. Stage I zoeae through
stage IV zoeae were present in the plankton in salinities be-
tween 0 and 33%o,with the range of greatest abundance between
5700 and 2i°/oo. The megalopa were collected at salinities be-
tween 7%0 and 19%<,.
Callinectes sapidus will develop in the laboratory from hatch-
ing to the 1st crab stage in salinities from 20%o to 35%„
and in temperatures from 20°C to 30°C. Zoeae of C. sapidus
were found in the plankton of the Newport River Estuary in
salinities from 18°/00 to 36700 and temperatures from 16°C to
31°C. Megalopa were collected in salinities from 19700 to
32%0 and in waters between 25°C and 30°C (Pinschmidt, 1963).
The megalopa in the laboratory, however, can withstand sali-
nities from 57 to 40700 and temperatures of 15°C to 30°C
(Costlow, 1967).
-73-
-------
01
oo
Mi rex
For mirex to affect blue crab larvae directly, fire ant bait
or leached mirex would have to be in water from 18 %o to 35°/(
salinity. Blue crab zoeae are so small that they could not
eat a flake of mirex bait. In our opinion, zoeae could be
affected by mirex which leaches out of the bait, provided
they settled on or near the bait between spurts of swimming.
Larvae of later zoeal stages are known to sink further down
in the water column than.do larvae of earlier stages. Ludke
e_t al. (1971) reported that 19 out of 20 crayfish died in
seven days from mirex which leached from 4X mirex bait. In
a similar experiment 33 of 35 crayfish died after 54 hours.
Water samples contained 0.86 ppb mirex and three pooled sam-
ples of crayfish had body residues of 1.602, 1.409 and 1.339
ppm mirex. In this investigation it was determined that
0.01 ppb and 0.1 ppb mirex were sublethal concentrations and
that 1.0 ppb was an acutely toxic concentration. It is prob-
able, therefore, that a concentration of 0.86 ppb or lower
could cause sublethal effects and mortality of blue crab lar-
vae in the field. Additional mirex could be obtained through
the food chain. Naqvi and de la Cruz (1973) reported the
presence of mirex in freshwater oganisms which did not receive
direct mirex treatment. This indicates widespread movement
of mirex in the environment and a spread of mirex through the
food chain. The resistance of mirex to degradation in the
environment *and its storage unaltered in animals favors bio-
accumulation (Metcalf et al^., 1973) .
Megalopa, in our opinion, are much more likely to be affected
directly and indirectly by mirex bait than are zoeae. It is
possible that they may nibble on mirex bait in the field, as
they do in the laboratory. They are also more apt to come in
contact with mirex which leaches from the bait, because they
often crawl or swim near the bottom. They are capable of
eating more and larger organisms than zoeae and thus obtain
more mirex through the food chain. Since megalopa are often
found in the lower part of the water column, they are in a
position to be carried landward in estuaries as Bousfield
(1955) reported for late-stage barnacles. Pinschmidt (1963)
and others have found blue crab megalopa throughout most of
the 17 mile Newport River Estuary, N. C. Being more widely
distributed in an estuary, they are more apt to be affected
by mirex than zoeae which are confined to waters of a sali-
nity of 20%0 and above.
Methoxychlor
As a substitute insecticide for DDT, methoxychlor is nearly
-74-
-------
as toxic to target organisms as DDT, yet it is biodegradable
5™C?£ 1t does not give rise to such long lasting residues as
DDT (Burdick et al., 1968; Metcalf et al 1971) Although
most of the literature on methoxychlor"effects concern fresh-
water organisms, Henderson et al. (1959) concluded that pH
and^alkalinity have no major erTect on toxicity of fish, and
so it is reasonable to assume that the insecticide is not any
less toxic to marine than freshwater organisms.
It is not known what concentrations of methoxychlor would have
sublethal and lethal effects on larval development of R. har-
risii and C. sapidus in the field. One can only make Ihe
following postuiations based on the current investigation and
on incomplete information in the literature.
It is probable that methoxychlor would have similar effects
on the complete development of R. harrisii and C. sapidus in
the field to those described in the laboratory Tf the salini-
ty and temperature of the water were favorable for complete
development. Continuous exposure to 1.0 ppb methoxychlor
would have little effect on the complete development of the
mud-crab, R. harrisii, from hatching to the 1st crab stage.
It is probable that continuous exposure to concentrations of
2.5 ppb to 5.5 ppb would reduce survival markedly and expo-
sure to 7.0 ppb would be lethal. C_. sapidus larvae, on the
other hand, would be much more sensitive to lower concentra-
tions of methoxychlor than R. harrisii. Concentrations of
0.7 ppb to 1.0 ppb would probably reduce survival of larvae
to the 1st crab stage and concentrations of 1.3 ppb to 1.9
ppb would be acutely toxic.
The effect of methoxychlor on R. harrisii and C. sapidus lar-
vae would also depend upon the rate of breakdown of the in-
secticide, its dilution in the estuary and bioaccumulation.
There is a difference of opinion as to how long methoxychlor
will remain in the water. Bedford e_t al. (1968), using fresh-
water mussels as monitors of river water, reported residues
from spraying for the Dutch elm disease after two weeks equal
to residues at ten weeks and peak concentrations at six weeks.
Kennedy et al. (1970) made residue analyses of bluegills in
ponds whichTTad been treated with 0.01 pptn methoxychlor. After
one day the fish had residues of 2.11 ppm and 2.78 ppm, after
seven days the residues peaked at 3.35 ppm and 4.0 ppm, and
after 56 to 84 days there were no detectable residues. Fish
from ponds treated with 0.04 ppm methoxychlor had residues
of 9.80 ppm and 13.90 ppm at day one, peaked with residues
of 20.60 ppm and 21.10 ppm at day three and had no residues
at 56 and 84 days.
-75-
-------
If methoxychlor can remain in river water for ten weeks, as
Bedford et aJL. (1968) reported, it could be carried by river
water from a recently sprayed area to an estuary. In the es-
tuary, it might have sublethal and/or highly toxic effects on
R. harrisii and C. sapidus larvae, especially if repeated
applications of the insecticide were used. The Florida estu-
ary from which we obtained ovigerous R. harrisii for mala-
thion experiments in January and February^19757 must have
been such an area, for these crabs had residues of methoxy-
chlor ranging from 0.57 ppm to 5.50 ppm. In this environ-
ment both species could obtain methoxychlor from water and
by way of the food chain. Larvae of C. sapidus would prob-
ably be affected before larvae of R. Eamsii, not only be-
cause they accumulate residues at a faster rate than R. har-
risii, but because they reach higher peaks.
Malathion
The toxicity of malathion to aquatic organisms apparently de-
pends upon the exposure time, temperature and pH of the water
and the species. Malathion decomposes at high temperatures
and is susceptible to hydrolysis as alkalinity increases. Its
residues are short-lived (Walker and Stojanovic, 1973; EPA-
540/1-75-005). Even though malathion is biodegradable, it
may be highly toxic to target and non-target organisms alike.
Since it is a non-persistent insecticide, repeated applica-
tions of malathion may be necessary for control of pests, and
cumulative reduction of acetylcholinesterase may occur (Cop-
page and Matthews, 1974). Furthermore there is documented
evidence from the literature that arthropod pests develop re-
sistance to malathion (Mengle and Lewallen, 1963; Busvine,
1959; Brown and Abedi, 1960; LaBrecque and Wilson, 1960; Mount;
Seawright and Pierce, 1974). Hence, increasing concentration?
of malathion have to be used to control pests and the danger
to non-target organisms becomes greater.
Few laboratory and field studies have been conducted on the
effects of malathion on estuarine animals, and, as far as
known, there have been on publications on the effects of mal-
athion on the larval development of crabs.
Tagatz e_t al. (1974) found that thermal fogging [420 g/ha (6
wt oz/acreJT and ULV aerosol spraying [57 g/ha (0.64 fl oz)]
left residues in marsh water as high as 5.2 ppb and 0.49 ppb,
respectively. Since R. harrisii development is affected by
concentrations from IT.0 ppb to 20.0 ppb malathion and C_.
sapidus larvae by concentrations from 20.0 ppb to 110.0 ppb,
tne residues Tagatz e_t al. (1974) found in the water would
probably not have any direct effect on either R. harrisii or
-76-
-------
C. sapidus larvae. The initial spraying and bioaccumulation
Had no etrect on confined blue crabs, grass shrimps, pink
shrimp or sheepshead minnow (Tagatz et al., 1974), but could
have affected crab larvae. Conte ancTTIrker (1971) reported
that aerial application of 256 g/ha (3 fl oz/acre) to marsh
embayments in Texas killed 14 to 80% of commercial shrimp,
Penaeus aztecus and P_. setiferous. They found residues of
malathion of 0.8 ppm to ^.Z^ppm in the water 48 hours after-
spraying. These concentrations of malathion would undoubte'd-
ly kill the larvae of R. harrisii and C. sapidus for they are
above the range of concentrations which" were tound to be acu-
tely toxic to each species.
-77-
-------
SECTION IX
REFERENCES
Alley, E. G. 1973. The use of mirex in control of the im-
ported fire ant. J. Environ. Quality. 2_(1):52-61.
Bedford, J. W., E. W. Roelofs, and M. J. Zabik. 1968. The
freshwater mussel as a biological monitor of pesticide
concentrations in a lotic environment. Limnol. Oceano-
gr. 13(1):118-126.
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. ^: 165-180.
Bookhout, C. G., and J. D. Costlow, Jr. 1975. Effects of
mirex on the larval development of blue crab. Water Air
Soil Pollut. 4:113-126.
Borthwick, P. W., T. W. Duke, A. J. Wilson, Jr., J. I. Lowe,
J. M. Patrick, Jr., and J. C. Oberheu. 1973. Accumula-
tion and movement of mirex in selected estuaries of South
Carolina, 1969-71. Pestic. Monit. J. 7^(1): 6-26.
Bousfield, E. L. 1955. Ecological control of the occurrence
of barnacles in the Miramichi Estuary. Bull. Nat. Mus.
Canada (Ottawa). 137. 69 p.
Brown, A. W. A., and Z. H. Abedi. 1960. Cross-resistance
characteristics of a malathion-tolerant strain developed
in Aedes aegypti. Mosquito News. 2_0(2) : 118-124.
Buchanan, D. V., R. E. Milleman, and N. E. Stewart. 1970.
Effects of the insecticide Sevin on various stages of
the dungeness crab, Cancer magister. J. Fish. Res. Bd.
Can. 2_7: 93-104.
Burdick, G. E., H. J. Dean, E. J. Harris, J. Skea, C. Frisa,
and C. Sweeney. 1968. Methoxychlor as a blackfly lar-
vicide, persistence of its residues in fish and its ef-
fect on stream arthropods. N.Y. Fish Game J. 15(2): 121-
142. —
-78-
-------
Busvine, J. R. 1959. Patterns of insecticide resistance to
organophosphorus compounds in strains of houseflies frcm
2*58-67 SOUrCeS' Eat«K>logia exp. appl. (Amsterdam).
Churchill, E. P. 1919. Life history of the blue crab. Bull
U. S. Bur. Fish. 3J5:91-128.
Conte, F. S., and J. C. Parker. 1971. Ecological aspects of
selected Crustacea of two marsh embayments of the Texas
coast. Texas A. and M. Univ., Sea Grant Publ. No TAMU-
SG-71-211. 184 p.
Coon, D. W., and R. R. Fleet. 1970. The ant war. Environ-
ment. 12_(10) : 28-38.
Coppage, D. L., and T. W. Duke. 1971. Effects of pesticides
in estuaries along the Gulf and Southeast Atlantic Coasts.
In: Proceeding of the 2nd Gulf Coast Conference on Mos-
quito Suppression and Wildlife Management, Schmidt, C.
H. (ed.). Washington, D. C. p. 24-31.
Coppage, D. L., and E. Matthews. 1974. Short-term effects
of organophosphate pesticides on cholinesterases of es-
tuarine fishes and pink shrimp. Bull. Environ. Contam.
Toxicol. 11(5):483-488.
Costlow, J. D. 1967. The effect of salinity and temperature
on survival and metamorphosis of megalops of the blue
crab Callinectes sapidus. Helgolander Wiss. Meeresun-
ters (Kiel).15:84-97.
Costlow, J. D. , Jr., and C. G. Bookhout. 1959. The larval
development of Callinectes sapidus Rathbun reared in
the laboratory. Biol. Bull. 116(7);373-396.
Costlow, J. D., Jr., and C. G. Bookhout. 1960. A method
for developing brachyuran eggs in vitro. Limnol. Ocean-
ogr. 5(2):212-215.
Costlow, J. D., Jr., C. G. Bookhout, and R. J. Monroe. 1966.
Studies on the larval development of the crab, Rhithro-
panopeus harrisii (Gould). I. The effect of salinity
and temperature on larval development. Physiol. Zool.
39(2):81-100.
Court enay, W. R., Jr., and M. H. Roberts, Jr. Environmental
effects on toxaphene toxicity to selected fishes and
crustaceans. Environmental Protectional Agency. Wasn-
-79-
-------
ington, D. C. EPA-R3-73-035. April 1973. 73 p.
Darsie, R. F., Jr., and F. E. Corriden. 1959. The toxicity
of malathion to killifish (Cyprinodontidae) in Delaware.
J. econ. Ent. 52(4):696-700.
Eaton, J. G. 1970. Chronic malathion toxicity to the blue-
gill (Lepomis macrochirus Rafinesque). Wat. Res. 4(10):
673-68ZT
Eisler, R. 1969. Acute toxicities of insecticides to marine
decapod crustaceans. Crustaceana. 16:302-310.
Epifanio, C. E. 1971. Effects of dieldrin in seawater on
the development of two species of crab larvae, Leptodius
flpridanus and Panopeus herbstii. Mar. Biol. 11(4):356-
76T.
Epifanio, C. E. 1973. Dieldrin uptake by larvae of the crab
Leptodius floridanus. Mar. Biol. 19:320-322.
Gaufin, A. R., L. D. Jensen, A. V. Nebeker, T. Nelson, and
R. W. Teel. 1965. The toxicity of ten organic insecti-
cides to various aquatic invertebrates. Water Sewage
Works. 112:276-279.
Henderson, C., Q. H. Pickering, and C. M. Tarzwell. 1959.
Relative toxicity of ten chlorinated hydrocarbon insec-
ticides to four species of fish. Trans. Amer. Fish.
Soc. 8^:23-32.
Initial scientific and minieconomic review of malathion.
Environmental Protection Agency. Washington, D. C.
EPA-540/1-75-005. March 1975. 14 p.
Jernelov, A., R. Rosenberg, and S. Jensen. 1972. Biological
effects and physical properties in the marine environ-
ment of aliphatic chlorinated by-products from vinyl
chloride production. Wat. Res. (London). 6_:1181-1191.
Johnson, B. T. , and J. 0. Kennedy. 1973. Biomagnification
of p,p'-DDT and methoxychlor by bacteria. Appl. Micro-
biol. 2^(1):66-71.
Karnak, R. E., and W. J. Collins. 1974. The susceptibility
to selected insecticides and acetylcholinesterase acti-
vity in a laboratory colony of midge larvae, Chironomus
tentans (Diptera: Chironomidae). Bull. Environ.contam.
toxicol. 12(1):62-69.
-80-
-------
Kennedy, H. D. , L. L. Eller, and D. F. Walsh. 1970. Chronic
effects of methoxychlor on bluegills and aquatic inver-
tebrates. U. S. Bur. Sport Fish. Wildl. Technical Paper
No. 53. 18 p.
LaBrecque, G. C., and H. G. Wilson. 1960. Effect of DDT re-
sistance on the development of malathion resistance in
house flies. J. econ. Ent. 53(2) -.320-321.
Lee, J. H., C. E. Nash, and J. R. Sylvester. Effects of mi-
rex and^methoxychlor on striped mullet, Mugil cephalus
L. Environmental Protection Agency. Corva His, Oregon .
EPA-660/3-75-015. May 1975. 18 p.
Lewallen, L. L. , and W. H. Wilder. 1963. Laboratory tests
of insecticides on mosquito larvae in polluted and tap
water. J. econ. Ent. 56_(6) : 834-835.
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 36th N.
Amer. Wild. Nat. Resources Conf., Portland, Oregon, p.
171-186.
Ludke, J. L., M. T. Finley, and C. Lusk. 1971. Toxicity of
mirex to crayfish, Procambarus blandingi . Bull. Envi-
ron. Con tarn. Toxicol. Ql):«y-95T
MacFarlane, J., T. Dirks, and S. Uk. 1975. Symptoms of mi-
rex, dieldrin, and DDT poisoning in the field cricket,
Gryllus pennsylvanicus Burmeister, and effect on acti-
vity of the central nerve cord. Pestic. Biochem. Phy-
siol. 5:57-64.
Mahood, R. K. , M. D. McKenzie, D. P. Middaugh, S. J. Bollar,
J. R. Davis, and D. Spitsbergen. 1970. A report on
the cooperative blue crab study - South Atlantic States.
Fla. Dept. of Nat. Resour., Div. Mar. Res., Contribu-
tion Series No. 139., Study in cooperation with the U.
S. Dept. of Interior, Bureau of Commercial Fisheries.
32 p.
McKenzie M. D. 1970. Fluctuations in abundance of the
blue crab and factors affecting mortalities. S. Carol.
Wildl. Res. Dept., Mar. Resour. Div., Tech. Rep. 45 p.
Mengle, D. C., and L. L. Lewallen. 1963 Metabolism of mal-
athion by a resistant and a susceptible strain of Culex
tarsalls : I. Degradation in vivo and identification
-81-
-------
of organic soluble metabolites. Mosquito News. 23(3):
226-233.
Merna, J. W., M. E. Bender, and J. R. Novy. 1972. The effects
of methoxychlor on fishes. I. Acute toxicity and break-
down studies. Trans. Amer. Fish. Soc. 101:298-301.
Merna, J. W., and P. J. Eisele. The effects of methoxychlor
on aquatic biota. Environmental Protection >Agency, Wash-
ington, D. C.~ EPA-R3-73-046. Sept. 1973. 59 p.
Metcalf, R,". L.f G. K. Sangha, and I. P. Kapoor. 1971. Model
ecosystem for the evaluation of pesticide biodegradabili-
ty and ecological magnification. Environ. Sci. Tech. 5_
(8):709-713.
Metcalf, R. L., I. P. Kappor, P. Lu, C. K. Schuth, and P.
Sherman. 1973. Model ecosystem studies of the environ-
mental fate of six organochlorine pesticides. Environ.
Health Perspectives. 4:35-44.
Mount, G. A., J. A. Seawright, and N. W. Pierce. 1974. Se-
lection response and cross-susceptibility of a malathion-
resistant strain of Aedes taeniorhynchus (Wiedemann) to
other adulticides. Mosquito News. 34T3T:276-277.
Naqvi, S. M., and A. A. de la Cruz. 1973. Mirex incorpora-
tion in the environment: Residues in nontarget organ-
isms - 1972. Pestic. Monit. J. 7^(2) : 104-111.
O'Brien, R. D. 1967. Insecticides. New York, Academic Press.
332 p.
Pinschmidt, W. C., Jr. 1963. Distribution of crab larvae in
relation to some environmental conditions in the Newport
River Estuary, North Carolina. Manuscr. Duke Univ. (un-
publ.). 112 p.
Plapp, F. W. 1973. Mirex: Toxicity, tolerance, and metabo-
lism in the house fly (Musea domestica L.). Environ.
Entomol. £: 1058-1061.
Redmann, G. 1973. Studies on the toxicity of mirex to the
estuarine grass shrimp, Palaemonetes pugio. Gulf Res.
Rep. 4(2):272-277. S—
Sanders, H. 0., and 0. B. Cope. 1966. Toxicities of several
pesticides to two species of Cladocerans. Trans. Am.
Fish. Soc. 95:165-169.
-82-
-------
Tagatz, M. E., P. W. Borthwick, G. H. Cook, and D. L. Coppage
1974. Effects of ground applications of malathion on
salt-marsh environments in northwestern Florida. Mos-
quito News. 34(3)-.309-315.
Tagatz, M. E., P. W. Borthwick, and J. Forester. 1975. Sea-
sonal effects of leached mirex on selected estuarine
animals. Arch. Environ. Contam. Toxicol. 3(3)(In press).
U. S. Department of Agriculture.. 1968. Suggested guide for
the use of insecticides to control insects affecting
crops, livestock, households:, stored products, f.drestsv
aird. forest products. Agriculture Research Service and
Forest Service-. Agriculture Handbook 331. 273 p.
Walker, ¥. W., and B. J. Stojanovic. 1973. Microbial ver-
sus chemical degradation of malathion in soil. J. En-
viron. Quality. 2(2):229-232.
Wallner, W. E., N. C. Leeling, and M. J. Zabik. 1969. The
fate of methoxychlor applied by helicopter for smaller
European elm bark beetle control. J. econ. Ent. 62(5):
1039-1042.
-83-
-------
SECTION X
GLOSSARY
acute toxicity tests: short-term exposure to concentrations
of toxicant which will be lethal to 50% of the larvae
in a short interval of time - 24 h, 48 h, or 96 h.
acutely toxic concentrations: concentrations of insecticide
in which 10% of the larvae or less survive to the 1st
crab stage.
ANOVA: analysis of variance.
chronic tests: long-term exposure to toxicant.
differential survival: reduction of survival with each in-
crease in insecticide.
external sex characters: secondary sex characters which dif-
fer in male and female crab stages, such as pleopods.
first crab stage: first stage after molt from megalopa; has
adult morphology with abdomen bent under cephatothorax,
but is sexually immature.
g/ha: grams per hectare.
h: hour.
internal sex characters: development of gonads and their
ducts.
juvenile: crab stages which have adult morphology but are
sexually immature.
megalopa: stage of development of a crab between last zoeal
stage and 1st crab stage; is dorso-ventrally depressed;
has all cephalothoracic and abdominal appendages present
and functional; and has extended abdomen.
-84-
-------
wg/g: micrpgrams per gram = parts per million.
yg/1: micrograms per liter = parts per billion.
mg/1: milligrams per liter = parts per million.
molt: the process of shedding the exoskeleton which is nec-
essary for growth during larval and juvenile development
in arthropods, including crustaceans.
non-target organism: organism which is not an intended tar-
get of an insecticide.
pleopods: paired abdominal appendages which are functional
in megalopa, juvenile and adult crab.
ppb: parts per billion.
ppm: parts per million.
PPt: %0 * parts per thousand.
setae: bristle-like extensions of the exoskeleton.
sublethal concentrations: concentrations of insecticide in
which more than 10% of the larvae survive to the 1st
crab stage.
sublethal effects: effects which occur in larvae reared in
sublethal concentrations, but not in acetone control,
and which become more pronounced as concentrations are
increased.
target organism: organism which is the intended target of
an insecticide.
TL50: tolerance limit; the concentration of toxicant in
water at which fifty percent of the animals are able to
survive for a specified period of exposure.
zoea(e): a planktotropic larval stage of a crab with a lat-
erally compressed cephalothorax and abdomen, and two
thoracic appendages (maxillipeds) for swimming.
zoeal development: refers to all zoeal stages from time of
hatching to megalopa stage (i.e., four zoeal stages in
R. harrisii and seven to eight zoeal stages in C. sap-
Tdus).
-85-
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-76-007
3. RECIPIENT'S ACCESSION'NO.
4. TITLE AND SUBTITLE
EFFECTS OF MIREX, METHOXYCHLOR, AND MALATHION ON
DEVELOPMENT OF CRABS
5. REPORT DATE
March 1976 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
Cazlyn G. Bookhout and John D. Costlow, Jr.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Duke University Marine Laboratory
Beaufort, North Carolina 28516
10. PROGRAM ELEMENT NO.
1EA077;ROAP 10AKC;Task 37
11. CONTRACT/GRANT NO.
R-801128-02-2
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency
Office of Research arid Development
Environmental Research Laboratory
Gulf Breeze, Florida 32561
13. TYPE OF REPORT AND PERIOD COVERED
10/72 to 4/75
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Laboratory experiments were conducted to determine the effects of mirex, methoxychlor
and malathion on the larval development of Callinectes sapidus from the time of
hatching until the first crab stage is reached. For comparison, similar investiga-
tions were made to ascertain the effects of methoxychlor and malathion on larval
development of Rhithropanopeus harrisii.
The effect of a range of concentrations of each insecticide on survival of larvae of
(}. sapidus and R.. harrisii was determined, as well as concentrations which were
sublethal and lethal. Zoeal and total development to the first crab stage of
A' harrisii and C^. sapidus was prolonged in relation to increased concentrations of
methoxychlor and malathion. Other sublethal effects of methoxychlor and malathion
included abnormal development of the pleopods of male II. harrisii and male C^. sapidus
early crab stages, and autotomy-of the legs of R.. harrisii megalopa and early crab
stages. The developmental stages in which larvae are particularly sensitive vary
in the two species and with the three insecticides. Mirex residues of JC. sapidus
larvae reared in different concentrations of mirex, and methoxychlor residues of
R. harrisii and (^. sapidus larvae reared in concentrations of methoxychlor were
determined.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Pesticides
Bioassay
Crustacea
Crabs
Malathion
Insecticide toxicity
Mirex
Methoxychlor
Blue crabs
Mud'crabs
Larval development
6F
13. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
96
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
-86-
•fr U.S. GOVERNMENT PRINTING OFFICE: 1976-657-695/5389 'Region Tfo7 5-11
------- |