United Statos
Environmental Protection
Agency
Environmental Research
Laboratory
Gulf Breeze FL 32561
EPA-600/3-79-104
September 1979
Research and Development
Kepone® Effects on
Development of
Callinectes sapid us
and Rhithropanopeus
harrisii
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U S Environmental
Protection Agency, have been grouped into nine series These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interlace in related fields
The nine series are
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2 Environmental Protection Technology
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4 Environmental Monitoring
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6 Scientific and Technical Assessment Reports (STAR)
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describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials Problems are assessed for their long- and short-term influ-
ences Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
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This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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KEPONE® EFFECTS ON DEVELOPMENT OF
CALLINECTES SAPIDUS AND RHITHROPANOPEUS HARISII
by
Cazlyn G. Bookhout and
John D. Costlow, Jr.
Duke University
Durham, N.C. 27706
and
Robert Monroe
North Carolina State University
Raleigh, N.C. 27607
Grant No. R-803838
Project Officer
Dana Beth Tyler-Schroeder
Environmental Research Laboratory
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FLORIDA 32561
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DISCLAIMER
This report has been reviewed by the Environmental Research
Laboratory, Gulf Breeze, 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 commerical products
constitute endorsement or recommendation for use.
-i i-
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FOREWORD
The protection of our estuarine and coastal areas from damage caused by
toxic organic pollutants requires that regulations restricting the introduc-
tion of these compounds into the environment be formulated on a sound scienti
fie basis. Accurate information describing dose-response relationships for
organisms and ecosystems under varying conditions is required. The EPA
Environmental Research Laboratory, Gulf Breeze, contributes to this informa-
tion through research programs aimed at determining:
"the effects of toxic organic pollutants on individual species and
communities of organisms
'the effects of toxic organics on ecosystem processes and components
"the significance of chemical carcinogens in the estuarine and marine
envi ronments
The investigation described in this report sought to determine the sub-
lethal and acutely toxic effects of the pesticide Kepone^' on the mud crab,
Rhithorpanopeus harrisii (Gould), and the blue crab, Callinectes sapidus
Rathbun, during larval development and early crab stages. These data should
contribute to our knowledge of Kepone^ concentrations affecting larval sur-
vival, length of larval development, molting, and ultimately the population
biology of the two species.
Thomas W. Duke
Director
Environmental Research Laboratory
Gulf Breeze, Florida
-m -
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ABSTRACT
Laboratory experiments were conducted to determine the effect of
Kepone on the development of Callinectes sapidus from the time of hatching
until the 1st crab stage. For comparison, similar investigations were
made to ascertain the effects of Kepone on larval development of
Rhithropanopeus harrisii.
Of the concentrations tested, 35, 50, 65,and 80 parts per billion (ppb)
Kepone were found to be sublethal and 95, 110,and 125 ppb Kepone were acutely
toxic to R.. harrisii larvae; whereas 0.1, 0.5,and 0.75 were sublethal and
1.0 ppb Kepone was acutely toxic to C_. sapidus larvae. The duration of zoeal
development and total time from hatching to 1st crab were in general
prolonged with concentration in f^. harrisii; whereas, in C_. sapidus, no
significant relationship could be detected between Kepone concentration and
duration of zoeal development, but there was a significant relationship
to 1st crab. The developmental stages in which the larvae are particularly
sensitive differ in the two species.'
This report was submitted in fulfillment of Grant No. R803838 by Duke
University under the sponsorship of the Environmental Protection Agency.
This report covers the period August 30, 1977, to May 31, 1979, and work
was completed as of July 30, 1979.
-IV-
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CONTENTS
Foreword iii
Abstract iv
Figures vi
Tables vii
Acknowledgments viii
1. Introduction 1
2. Summary and Conclusions 3
3. Recommendations 4
4. Materials and Methods 6
5. Types of Statistical Analyses 8
6. Results 11
7. Discussion 26
References 31
Glossary * 34
-v-
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FIGURES
Number Page
1 Effect of Kepone concentration on survival of R_. harrisii . . 14
2 Duration, zoeal development and hatch to crab in
R_. harrisii vs concentration of Kepone 16
3 Effect of Kepone on rate of molting from hatch to megalopa
and hatch to crab in R_. harrisii 18
4 Effect of Kepone on mortality by stages of development
of R^. harrisii 19
5 Effect of Kepone concentration on survival of C_. sapidus . . 21
6 Duration of hatch to 1st crab in C_. sapidus vs.
concentration of Kepone 23
7 Effect of Kepone on mortality by stages of C_. sapidus .... 25
-Vi-
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TABLES
Number Page
1 Effects of Kepone on percent survival and duration in days
through zoeal and megalopa development in Rhithropanopeus
harrisii IX, X, XIII, XIV and XVII 12
2 Average percent survival and average duration in days of zoeal
and megalopa development in R^. harrisii reared in acetone
control and different concentrations of Kepone 13
3 Percent mortality in developmental stages of R_. harrisii. ... 17
4 Effects of Kepone on percent survival and duration in days
through zoeal and megalopa development of Callinectes sapidus
XII, XIII and XIV 20
5 Average percent survival and average duration in days of zoeal
and megalopa development of C^. sapidus reared in acetone
control and different concentrations of Kepone 22
6 Percent mortality in developmental stages of C_. sapidus .... 24
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ACKNOWLEDGMENTS
The help of Dr. Adam Zsolnay, Chemist at Duke University Marine
Laboratory, is gratefully acknowledged.
The technical assistance of Mrs. Sally Herring and Mr. Joe Goy and
the untiring efforts of Mrs. Norma Jean Buck in typing the manuscript
are greatly appreciated.
-vm-
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SECTION 1
INTRODUCTION
Kepon
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(1977) studied the chronic effects of Kepone on embryo, fry, juvenile and
adult sheepshead minnows (Cyprinodon variegatus). The only life history
toxicity tests on a malacostracan crustacean have been made on mysids,
Mysidopsis bahia (Nimmo et_ aj_., 1977). In a 19-day bioassay survival was
reduced from 84.4% in 0.39 yg/1 to 0% in 8.7 yg/1 Kepone. Sublethal effects
included reduction in young, delay in formation of brood pouches, delay
in release of young and reduced growth.
As far as known, there have been no publications on the effects of
Kepone on the complete development of any crab. The objectives of the
current investigation, therefore, are to determine the limits of concen-
tration of Kepone within which the mud crab, Rhithropanopeus harrisii (Gould)
and the blue crab, Callinectes sapidus Rathbun, can be reared from the time
of hatching until the 1st crab is reached, and to ascertain the sublethal
and acutely toxic concentrations of Kepone. From these studies, it should
be possible to determine if there are sublethal effects, and if there are
one or more larval stages of development which are particularly sensitive.
A final objective is to determine if there is a statistically significant
effect of Kepone on survival to megalopa and to first crab, duration from
hatching to megalopa and to 1st crab, and cumulative mortality by stages of
development of R_. harrisii and C. sapidus.
-2-
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SECTION 2
SUMMARY AND CONCLUSIONS
1. Differential survival of Rhithropanopeus harrisii from hatching to 1st
crab stage occurred in a range of concentrations of Kepone from 35 to
125 ppb. Concentrations were sublethal from 35 to 80 ppb and acutely
toxic from 95 to 125 ppb. Statistical analysis indicated a 15?; decrease
in survival for each 10 ppb increase in Kepone in the range of 30 to 100
ppb Kepone.
2. „ Differential survival of Callinectes sapidus from hatching to 1st crab
stage occurred in a range of concentrations of Kepone from 0.1 to 1.0
ppb. Concentrations were sublethal from 0.1 to 0.75 ppb and were acutely
toxic at 1.0 ppb. Statistical analysis indicated a decrease of 2.2c,'o
in survival for each 0.1 ppb increase in Kepone in the range of 0.1 to
0.8 ppb Kepone.
3. In R^. harrisi i, the time from hatching to megalopa and hatching to 1st
crab is generally prolonged with each concentration of Kepone from 35 to
100 ppb, primarily due to prolongation of zoeal development. Statistical
analysis indicates that, for every 10 ppb Kepone added, duration from
hatching to 1st crab stage is increased by 0.391 j^ 0.043 days.
4.- In C_. sapidus, no significant relation could be detected between Kepone
concentration and duration of zoeal development; however, a significant
linear relationship was established between Kepone levels and duration
to 1st crab. For each increase of 0.1 ppb, the duration from hatching
to 1st crab is prolonged by 0.38 +_ 0.10 days.
5. The 1st and 2nd zoeal stages of R_. harrisii were the most sensitive
developmental stages to Kepone, and thereafter there was less mortality
among the remaining resistant larvae.
6. The 1st zoeal stage of C_. sapidus was not sensitive, statistically, to
any concentration of Kepone tested. In zoeal stages II, III, and IV,
there were significant increases in mortality over the previous stage
in all concentrations and in the control. There was a trend of
increasing mortality with concentration, but only in the highest
concentration (1.0 ppb) was the mortality significantly greater than
in the control.
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SECTION 3
RECOMMENDATIONS
1. It is important to determine the effects of Kepone on the ]arval
development of R_. harrisii in the middle estuary of the James and on
the development of C_. sapidus in the lower Chesapeake Bay, because
larvae are a natural component of the food chain in these waters and any
detrimental effect of Kepone on developmental stages will reduce the
adult population. Therefore, larvae should not be disregarded when
safe water standards for estuaries are set.
2. To determine the effect of Kepone on the development of crabs in the
field, there should be an annual program of monitoring sediment, water
and zooplankton for Kepone. Residue analyses should be made of adult
R_. harrisii and C_. sapidus, their eggs and larvae at periodic intervals
throughout the breeding season.
3. With reliable field data available, laboratory experiments should be
designed to simulate field conditions. The concentration of Kepone
in eggs of each species could be obtained from field studies, but the
additional uptake from seawater and from food, with concentrations
similar to those in the field, can be determined best through
laboratory studies. A correlation of Kepone residues with mortality
and exposure time should provide an indication of the effect of Kepone
on larval development of each species in the field.
4. The current report outlined the effects of Kepone on the development
of crabs when larvae were reared under optimum temperature and salinity
conditions. Further investigation should be made to determine the
effect of Kepone on the larval development of crabs when reared in a
range of salinities and temperatures. Since there is some evidence
that larval survival of C_. sapidus is better in the early part of the
breeding season and that total duration from hatching to 1st crab is
shorter than in the latter part of the breeding season, further
experiments should be done to determine if the larvae are more resistant
to Kepone in the early part of the breeding season than the later part.
5. Residue analyses of larvae reared in different concentrations of Kepone
for varying lengths of time were not a part of the current project,
but they would have been desirable. From past experience, we found it
to be a time-consuming task to obtain 0.20 g of larvae, wet weight,
in low concentrations of a chlorinated hydrocarbon insecticide and
impossible to obtain enough late-stage larvae in high concentrations
to make a residue anlaysis. Methods should be developed, therefore,
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to make accurate residue analyses of insecticides from samples of larvae
weighing less than 0.20 g wet weight. For a stable compound, such as
Kepone, it would seem useful to utilize radiolabeled material for such
small samples, especially in a static test.
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SECTION 4
MATERIALS AND METHODS
Preparation
Preliminary experiments were conducted to determine the range of concen-
trations to use in definitive chronic experiments on the effect of Kepone
on the development of the mud crab, Rhithropanopeus harrisii (Gould).and the
blue crab, Callinectes sapidus Rathbun.
Pesticide grade acetone was used as a carrier for Kepone. Acetone
control for experiments with Kepone was prepared by adding 1 ml of full
strength acetone to 999 ml of 20 /oo filtered seawater for R. harrisii larvae
-'n
, j orw /.
and 30 /oo filtered seawater for C_. sapidus larvae to give a final concen-
tration of 1.0 /oo (parts per thousand).
Keponen[decachlorooctahydro-l,3,4-metheno-2H-cylobuta[cd] pentalene
2-one) was purchased from Chemical Service, West Chester, PA, as 99% pure,
but analysis at Environmental Research Laboratory, Gulf Breeze, Florida,
indicated 88% purity after exposure to air (personal communication). Dr.
Adam Zsolnay, chemist at Duke University Marine Laboratory, prepared stock
solutions by dissolving a known weight of Kepone in pesticide analytical
grade acetone, and different concentrations were made up from this stock
solution.
For experiments on the effect of Kepone on the development of R_.
harrisij^ 1 ml of stock solutions of 35.0 parts per million (ppm) (mg/1),
50.0 ppm, 65.0 opm, 80.0 ppm, 95.0 ppm, 110.0 ppm and 125.0 ppm was added
to 999 ml of 20 /oo filtered seawater daily to give final concentrations of
35.0 parts per billion (ppb) (yg/1) to 125.0 ppb Kepone.
For experiments on the effect of Kepone on the development of C^. sapidus
one ml of stock solutions of 0.1 ppm, 0.5 ppm, 0.75 ppm and 1.0 ppm was
added to 999 ml of 30 /oo filtered seawater daily to give final concentra-
tions of 0.1 ppb to 1.0 ppb.
Source of mother crabs and hatching of eggs
Ovigerous R^. harrisii, small mud crabs belonging to the family
Xanthidae, were collected in the vicinity of Fort Pierce, Florida, in
February, April, and May 1978. They were shipped to Beaufort, North
Carolina, by air freight. Upon arrival at Duke University Marine Laboratory
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(DUML), Beaufort, North Carolina, each mud crab was placed in a large glass
finger bowl (19.4 cm d) containing 20 /oo filtered seawater, the salinity to
be used during the rearing experiments with Kepone. Thg ovigerous crabs
were maintained in a constant temperature cabinet at 25 C and with a light
regime of 12h light and 12h darkness until hatching of larvae occurred.
Ovigerous C^. sapidus, commerical blue crabs belonging to family
Portunidae, were collected off Beaufort Inlet, North Carolina, in July 1978.
The crabs used had black eggs with a visible heart beat. The method for
developing eggs has been described previously (Costlow and Bookhout, 1960;
Bookhout and Costlow, 1975).
Rearing of larvae
Five replicate series of R^. harrisii larvae and three of C^. sapidus
were reared in acetone control and different concentrations of Kepone. Each
series of larvae was hatched from a different mother crab. Series IX of
R_. harrisii was designated as Rh IX and the date of hatch was 2-19-78. The
dates of the other series are as follows: Rh X, 2-28-78; Rh XIII, 4-9-78;
Rh XIV, 4-13-78; and Rh XVII, 5-14-78. Series Cs XII and Cs XIII of £.
sapidus were hatched 7-26-78, and Cs XIV, 7-27-78. The methods for rearing
larvae of each species in a check series, 10 larvae per finger bowl (8.9 cm
diam), and an' insecticide, were described previously CBookhout e_t al_., 1976).
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SECTION 5
STATISTICAL ANALYSIS OF LARVAL DEVELOPMENT OF
R. HARRISII AND C. SAPIDUS SUBJECTED TO TREATMENT BY KEPONE
General
Three types of analysis were done on R_. harrisii and C_. sapidus data:
(i) Analysis of survival percentages using survival to megalopa and
to the first crab stage
(ii) Analysis of duration of molting for the times to megalopa and to
the first crab stage. Duration was measured in DAYS and analyzed as such.
In addition a new variable was constructed which has units of reciprocal days
and is therefore proportional to rate of molting. The exact transform was
RATE = TOO/DAYS where the constant 100 is a convenient scale factor. In many
biological systems where time is a relevant variable, the use of the reciprocal
transform often simplifies relationships to experimental factors,and when the
variability of the time measurement tends to increase with large values of
time, the reciprocal transform usually has nearly constant variance
(iii) Analysis of cumulative mortality by stage of development
All computations were done using the Statistical Analysis System (SAS)
(Barret_al_., 1976). PROC GLM (The General Linear Models Procedure) with
least squares means and standard error options was the primary procedure.
Analyses of survival percentages
The percent survival to raegalopa and to first crab was first transformed
to tbe angle Y, such that Sin Y = P (percent survival) or Y = arcsin /P~ =
sin" /P~. This transformation tends to stabilize the error variance across
the experiment and may help to linearize the response to the several Kepone
levels (Snedecor and Cochran, 1967).
The general linear model technique (Searle, 1971) was used to account
for the unequal numbers of replicates at each Kepone level. The transformed
% survival, Y, was regressed on the several levels of Kepone, the regression
coefficient and its standard error computed, and the deviations from linearity
tested for significant departure from a straight line relationship. The
implications of this approach is (i) that if a functional relationship can
be established between a response, say Y, and the concentration of Kepone,
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then the regression coefficient expressed in degrees/ppb Kepone is the most
efficient expression of that relationship, and (ii) if significant departures
from linearity are observed, then the simple model either must be restricted
to that range of concentrations in which the relationship is linear, or
discarded in favor of a more complicated model that contains terms to allow
for the nonlinearity of response. Linearity in the transformed survival
implies nonlinearity in the relationship between % survival and Kepone concen-
tration; but approximate expressions may be obtained for the rate of change of
% survival with respect to concentration by restricting the range of concen-
trations to one in which the derivatives are nearly constant. More formally
we have:
Observed, Y = a + bX, b measured in degrees/ppb.
wanted, dP/dX, where Y = arcsin /P,
and dP/dX = dY/dX ' dP/dY = 2b , °/o/ppb.
Thus, when the quantity /P(l - P) is approximately constant for a range of P
from 0.10 to 0.90, we can also express the results in the original, and more
familiar, scale of percent. The constant 57.3 converts angles in degrees
to angles in radians.
It is appropriate to mention at this point that a "significant"
regression coefficient is one which has been judged not zero and implies
by itself nothing about the biological importance or implications.
Analysis of duration/rate
The variables analyzed and their abbreviations are:
DZ = DAYS to megalopa
RZ = 100/DAYS, the variable proportional to molting rate measured in
reciprocal DAYS x 100.
DC = DAYS to first crab
RC = 100/DC analogous to RZ.
The general linear model technique was employed to estimate the regression
coefficients of each of the above variables on the Kepone concentration,
adjusting for unequal replication. The departure from linearity was tested
at the same time.
The regression coefficient has units of DAYS/ppb (or 100/DAYS/ppb) and
is said to measure a significant effect of Kepone if it is more than twice
Us estimated standard error. If, in addition, there is no significant
departure from linearity, it is then the single best expression of the
relationship between molting time (or rate) and Kepone concentration.
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As noted earlier, the rate variable is often linear with, say, concen-
tration if duration in DAYS is not. If best linearity is found in Rate (RZ),
then the equivalent value for duration would be obtained by
DAYS/ppb = b-) where
b = linear regression coefficient of
RZ on Concentration (ppb).
This value is clearly not now a constant but an approximate figure may be
obtained by using the average value of RZ for the several Kepone levels
involved.
Analysis of cumulative mortality
The percent cumulative mortality by stage of development was also
transformed to the angular scale for analysis. The analysis of these data,
however, had to account for the additional factors STAGE and the STAGE*
CONCENTRATION interaction following the technique for a split-plot analysis
of variance (Cochran and Cox, 1957). The introduction of another level of
classification required that the error structure be subdivided into two
components usually designated as the sub-plot error (E, ) and the whole-plot
error (Ea).
Comparisons of mean mortalities among STAGES at the same concentration
of Kepone were made using standard errors computed from E. only. Comparisons
of mean mortalities among concentration levels at the same STAGE or at
different STAGES were made using a weighted standard error involving both
estimates of error. Specifically the weighted mean square error,
w
where c = number of levels of concentration. Since Ea> Eb then also
All tests of differences between mean mortalities among STAGES and
CONCENTRATIONS were completed in the transformed scale but the results were
reported in the original percentage scale.
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SECTION 6
RESULTS
EFFECTS OF KEPONE ON DEVELOPMENT OF RHITHROPANOPEUS HARRISII
Survival
The percent survival to megalopa and to the first crab stage is given
for five series in TABLE 1. The average percent survival of all series reared
in various media is given in TABLE 2. Survival of larvae from hatching to
megalopa and to first crab stage in five replicate series reared in acetone
control only varied from 98 to 100% and 94 to 100%, respectively (TABLE 1).
There was differential survival in concentrations of Kepone from 35 ppb to
125 ppb (TABLE 2).
Statistical analysis of R. harrisii survival
The results shown in Figure 1 indicate the linear relationship between
the concentration of Kepone and survival to the megalopa, and to 1st crab
stages. Statistical analyses indicated that:
(i) the dose-response relationship is linear only in the range of
35-110 ppb Kepone. When the control and 125 ppb survivals are
included, the deviations from linearity become significant.
(ii) In the range of 35-100 ppb no significant differences in the dose-
response relationship exist between survival from hatching to
megalopa and survival from hatching to first crab either with
respect to the slope of the line or the level of survival. This
result suggests that survival of megalopa is not affected much by
the Kepone levels in this range. The results in Figure 1 are the
combined data.
b = -0.9854 +_ 0.0557 degrees decrease in survival
for the increase of 1 ppb Kepone.
The b-value here is not easily converted back to the original units
of percent survival, but would correspond approximately to a 15% decrease
in survival for each 10 ppb Kepone in the range of 30 to 100 ppb concen-
tration.
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TABLE 1. EFFECTS OF KEPONE ON PERCENT SURVIVAL AND DURATION IN DAYS THROUGH ZOEAL AND MEGALOPA DEVELOPMENT
IN Rhithropanopeus harrlsil IX, X, XIII, XIV AND XVII
rv>
i
Culture mecb'a
Salinity 20 /oo
Temp. 25°C
Acetone
Control
Kepone
35 ppb
Kepone
50 ppb
Kepone
65 ppb
Kepone
80 ppb
Kepone
95 ppb
Kepone
110 ppb
Kepone
125 ppb
Initial No.
of larvae per
per series
Rh IX-50
Rh X-50
Rh XIII-50
Rh XIV-50
Rh XVII-50
Rh XIII-5Q
Rh XIV-50
Rh XVII-50
Rh X-50
Rh XVII-50
Rh IX-50
Rh X-50
Rh XIII-50
Rh XIV-50
Rh XVII-50
Rh IX-50
Rh X-50
Rh XVII-50
Rh IX-50
Rh X-50
Rh XIII-50
Rh XIV-50
Rh XVII-50
Rh IX-50
Rh X-50
Rh XIII-50
Rh XIV-50
Rh IX-50
Rh X-50
Rh XIII-50
Rh XIV-50
% Survival
to
Megalopa
98
100
98
100
100
90
92
94
78
70
48
80
86
98
48
26
44
4
6
20
0
0
4
6
12
0
0
0
4
0
0
1st Crab
94
98
94
100
98
80
88
90
64
64
32
68
74
94
38
10
26
4
2
16
0
0
4
2
2
0
0
0
2
0
0
Mean
Zoea
11.20
9.88
10.78
10.10
11.02
12.31
12.04
13.04
11.64
13.91
12.63
11.93
13.33
11.63
15.00
14.77
12.59
15.50
14.00
13.10
-
_
17.50
14.33
15.50
-
-
_
14.50
_
-
duration
i
Megalopa
5.74
6.12
4.82
5.56
6.29
5.90
5.55
5.73
5.34
6.00
5.94
5.91
6.76
6.02
7.21
6.60
5.46
6.00
6.00
6.25
_
_
7.50
7.00
6.00
_
-
_
6.00
_
-
of development
n days
Hatch to 1st Crab
16.94
16.00
15.79
15.68
17.30
18.13
17.61
19.20
16.91
19.90
18.56
17.50
20.10
17.35
21.90
20.80
17.77
21.50
19.00
18.75
_
_
25.00
20.00
19.00
_
-
_
19.00
_
-
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TABLE 2. AVERAGE PERCENT SURVIVAL AND AVERAGE DURATION IN DAYS OF ZOEAL AND MEGALOPA DEVELOPMENT OF
R. harrisii REARED IN ACETONE CONTROL AND DIFFERENT CONCENTRATIONS OF KEPONE
OJ
I
Culture media
Salinity 20 /oo
Temp. 25°C
Acetone
Control
Kepone
35 ppb
Kepone
50 ppb
Kepone
65 ppb
Kepone
80 ppb
Kepone
95 ppb
Kepone
110 ppb
Kepone
125 ppb
Initial No. % Survival
of larvae to
per series Megalopa 1st Crab
Rh IX-50
Rh X-50
Rh XIII-50 99.2 96.8
Rh XIV-50
Rh XVII-50
Rh XIII-50
Rh XIV-50 92 86
Rh XVII-50
Rh X-50 74 64
Rh XVII-50
Rh IX-50
Rh X-50
Rh XIII-50 72 63.2
Rh XIV-50
Rh XVII-50
Rh IX-50
Rh X-50 26 13.3
Rh XVII-50
Rh IX-50
Rh X-50
Rh XIII-50 6 4.4
Rh XIV-50
Rh XVII-50
Rh IX-50
Rh X-50 4.5 1.0
Rh XIII-50
Rh XIV-50
Rh IX-50
Rh X-50 1 0.5
Rh XIII-50
Rh XIV-50
Mean duration of development
in da^s
Zoea Megalopa Hatch to 1st crab
10.59 5.70 16.34
12.59 5.72 18.33
12.72 5.67 18.41
12.69 6.32 18.75
13.51 5.80 18.90
13.87 6.45 19.91
15.11 6.50 19.50
-------
90
•75
CO
II
i60
>
oc
Q
LU
130
o
Li-
en
z
Y=108.35-0.9854 *Conc
35 50 65 80
KEPONE ppb
95
110
125
Figure 1. Effect of Kepone concentration on survival
of R. harrisii.
x x
o
Hatch to megalopa
Hatch to 1st crab o
Duration
TABLE 1 gives the mean duration in days of zoeal and megalopa
development and the mean time in days from hatching to the 1st crab stage
for each series reared in acetone control and in different concentrations
of Kepone. TABLE 2 lists the mean duration of development in days for all
larvae reared in all series. Each table shows that the duration of zoeal
development is in general prolonged with each concentration from 35 ppb
to 110 ppb Kepone. Since the mean duration of megalopa development was
somewhat similar in these concentrations, the 'total time from hatching to
the 1st crab stage was prolonged with each higher concentration primarily
due to the effect of Kepone on zoeal development.
-14-
-------
Statistical analysis of R. harrisii duration
1. Significant linear regressions of both DZ (days to megalopa) and DC
(days to 1st crab) upon Kepone levels were found with no significant
deviations from linearity. The most compact summary is in these
equations:
DZ = 10.5 + 0.0423 * CONC
DC = 16.5 + 0.0359 * CONC
where CONC is in ppb of Kepone. These results are shown in Figure 2.
The regression coefficients may be interpreted as follows:
for DZ: 0.0423 +_ 0.0051 days increase in duration of zoeal
development for each ppb added Kepone
for DC: 0.0359 +_ 0.0069 days increase in total duration time for
each ppb added Kepone.
These increases in duration can be scaled up, for example to 10 ppb,
by multiplication, i.e.,0.423 +_ 0.051 days increase for each 10 ppb
added Kepone.
The test of significance of the difference between the two slopes is also
not significant which suggests that a pooled estimate can be used to
characterize the concentration effect on duration. This estimate is
b = 0.0391 +_ 0.0043 DAYS/ppb Kepone
2. Nearly analogous results were obtained when RATE = 100/DAYS was used
as the dependent variable:
RZ - 9.31 - 0.0251 * CONC
RC = 6.01 - 0.0100 * CONC
These results are shown in Figure 3:
b(RZ) = -0.0251 +_ 0.0024 reciprocal days decrease
in RATE for each ppb increase in Kepone and
b(RC) = -0.0100 +_ 0.0015 reciprocal days decrease in RATE
for each ppb increase in Kepone.
Now these two coefficients do differ significantly from each other which
may suggest that the Kepone effect is greater on zoeal stage than on the
megalopa.
-15-
-------
2
-------
TABLE 3. PERCENT MORTALITY IN DEVELOPMENTAL STAGES OF R. harrisii
Media
Acetone
Control
Kepone
35 ppb
Kepone
50 ppb
Kepone
65 ppb
Kepone
80 ppb
Kepone
95 ppb
Kepone
110 ppb
Kepone
125 ppb
Series
Rh IX
Rh X
Rh XIII
Rh XIV
Rh XVII
Rh XIII
Rh XIV
Rh XVII
Rh X
Rh XVII
Rh IX
Rh X
Rh XIII
Rh XIV
Rh XVII
Rh IX
Rh X
Rh XVII
Rh IX
Rh X
Rh XIII
Rh XIV
Rh XVII
Rh IX
Rh X
Rh XIII
Rh XIV
Rh IX
Rh X
Rh XIII
Rh XIV
I
0
0
2
0
0
2
8
0
0
20
4
0
0
0
14
2
16
36
34
44
100
98
28
32
48
100
100
34
66
92
96
Zoeal
II
2
0
0
0
0
4
0
6
8
10
32
4
0
0
34
52
20
52
46
22
-
0
60
58
24
-
60
20
8
4
Stages
III
0
0
0
0
0
0
0
0
0
0
4
4
0
0
0
8
8
8
6
10
-
0
8
2
8
-
-
4
8
-
_
IV
0
0
0
0
0
4
0
0
14
0
12
12
14
2
4
12
12
0
8
4
-
2
0
2
8
-
-
2
2
-
_
Megalopa
4
2
4
0
2
10
4
4
14
6
16
12
12
4
10
16
18
0
4
4
-
-
0
4
10
-
-
0
2
-
-
Total
6
2
6
0
2
20
12
10
36
36
68
32
26
6
62
92
74
96
98
84
100
100
96
98
98
100
100
100
98
100
100
-17-
-------
RZ = 9.31- 0.0251 *Conc
RC=6.01-0.0100 *Conc
50 65 80
KEPONE ppb
125
Figure 3. Effect of Kepone on rate of molting from
hatch to megalopa and hatch to crab in
FL harrisii.
RZ: x x Hatch to megalopa
RC: o o Hatch to 1st crab
Statistical analysis of R. harrisii cumulative mortality by stages
The results given in Figure 4 show the effect of Kepone levels on the
mortality at each stage of development. The percent mortalities on the
graph were obtained from the means of the transformed variable adjusted for
the unequal number of replications for several treatments.
-18-
-------
10
IV
M
STAGE
Figure 4. Effect of Kepone on mortality by stages of
development of R_. ham'sii.
a. Significantly different from control (0.05)
Significantly different from control (0.01)
Significant increase over previous stage
(0.05)
Significant increase over previous stage
(0.01)
b.
*
**
EFFECTS OF KEPONE ON DEVELOPMENT OF CALLINECTES SAPIDUS
Survival
The percent survival to megalopa and to the 1st crab stage is given
for three series in TABLE 4. The average percent survival of all series
-19-
-------
TABLE 4. EFFECTS OF KEPONE ON PERCENT SURVIVAL AND DURATION IN DAYS THROUGH
ZOEAL AND MEGALOPA DEVELOPMENT OF Callinectes sapidus XII, XIII
and XIV
Culture media
Salinity 30 / oo
Temp. 25°C
Acetone
Control
Kepone
0.10 ppb
Kepone
0.50 ppb
Kepone
0.75 ppb
Kepone
1 .0 ppb
Initial No.
of larvae
per series
Cs XII-50
Cs XIII-50
Cs XIV-50
Cs XII-50
Cs XIII-50
Cs XIV-50
Cs XII-50
Cs XIII-50
Cs XIV-50
Cs XII-50
Cs XIII-50
Cs XIV-50
Cs XII-50
Cs XIII-50
Cs XIV-50
% Survival
to
Megalopa
30
32
24
34
30
4
38
10
10
38
10
6
14
0
0
1st Crab
20
26
22
32
28
4
28
10
8
24
8
6
14
0
0
Mean
Zoea
44.8
40.4
46.9
42.2
41.7
50.0
44.4
43.0
49.2
47.2
41.0
48.0
46.3
-
-
duration of
in days
Megalopa
9.6
9.7
11.1
9.9
9.4
11.0
9.9
8.8
13.5
9.8
10.0
13.7
10.4
-
-
development
Hatching to
1st Crab
54.3
50.4
57.0
52.3
51.1
60.1
53.4
51.8
59.8
54.8
51.5
61.7
57.1
_
-
of larvae reared in acetone control and in 0.1 ppb, 0.5 ppb, 0.75 ppb and
1.0 ppb is given in TABLE 5. There is differential survival with increase
in concentration to megalopa and to first crab.
Statistical analysis of C. sapidus survival
As in R_. harrisii, a significant linear decrease in survival with
Kepone concentration was detected for both zoeal and megalopa survival,
and there was no detectable difference between slopes nor between average
survival. The combined results are presented in Figure 5 which shows the
best prediction equation for the ranges of concentration given.
Again the regression coefficient
b = -18.5942 +_ 3.2397 degrees decrease for each ppb increase
i.e., -1.859 +0.324 degrees for each 0.1 ppb increase
-20-
-------
30-
C/5
>20
cc
c/)
Q15
UJ
CC
o
O-
£10
Y= 30,6339 -18.5942 *Conc .
0.1 0-2 0.3 0.4 0.5 0.6
KEPONE ppb
0.7
0-8
0.9
1.0
Figure 5. Effect of Kepone concentration on survival of C_
sapidus.
Hatch to megalopa x x
Hatch to 1st crab o o
cannot be easily interpreted in terms of percent survival. However, in the
range of 0.1 to 0.8 ppb of Kepone concentration,the decrease of survival
is approximately 2.2% for each 0.1 ppb increase in Kepone.
Duration
TABLE 4 gives the mean duration in days of zoeal and megalopa develop-
ment and the mean time in days from hatching to 1st crab stage for each
series reared in acetone control and in 0.1 ppb, 0.5 ppb, 0.75 ppb and 1.0
ppb Kepone. TABLE 5 lists the mean duration of development in days for
all larvae reared in all series.
-21-
-------
TABLE 5. AVERAGE PERCENT SURVIVAL AND AVERAGE DURATION IN DAYS OF ZOEAL AND
MEGALOPA DEVELOPMENT OF Callinectes sapidus REARED IN ACETONE
CONTROL AND DIFFERENT CONCENTRATIONS OF KEPONE.
Culture media, Initial No,
Salinity 30 /oo of larvae
% Survival Mean duration of development
to in days
Temp. 25UC
Acetone
Control
Kepone
0.10 ppb
Kepone
0.50 ppb
Kepone
0.75 ppb
Kepone
1 .0 ppb
per series Megalopa
Cs XII-50
Cs XIII-50 28.7
Cs XIV-50
Cs XII-50
Cs XIII-50 22.7
Cs XIV-50
Cs XII-50
Cs XIII-50 19.3
Cs XIV-50
Cs XII-50
Cs XIII-50 18
Cs XIV-50
Cs XII-50
Cs XIII-50 4.67
Cs XIV-50
1st Crab Zoea Megalopa Hatching to
1st Crab
22.7 43.7 10.1 54.7
21.3 42.47 9.75 52.25
15.3 44.97 10.26 54.13
12.7 46.11 10.42 55.16
4.67 46.3 10.43 57.14
Statistical analysis of C. sapidus duration/rate
1. No significant relationship could be detected between Kepone concentration
and duration of zoeal development (DZ) or rates (RZ).
2. A significant linear relationship was established, however, between
Kepone levels and duration to first crab (DC) (Figure 6).
DC:b = 3.7667 +0.9907 DAYS increase in time from hatch to 1st
crab for each ppb increase in Kepone, i.e., 0.38 +_ 0.10
increase for each increase of 0.1 ppb.
RC:b - -0.1219 +_ 0.0303 reciprocal DAYS decrease for each ppb
increase in Kepone, i.e., -0.012 +_ 0.003 reciprocal for
each increase of 0.1 ppb.
-22-
-------
58
57-
56-
co ,. ,
> 55
54
53
52
= 53.76 + 3.7667 -Cone
0.1 0.2 0.3 0.4 0.5 0.6
KEPONE ppb
0.7
0.8
0.9 1.0
Figure 6. Duration of hatch to 1st crab in C_. sapidus vs
concentration of Kepone.
DC: Hatch to 1st crab
Mortality
Callinectes sapidus may pass through seven, occasionally eight, zoeal
stages before it molts into a megalopa, a ninth stage of development. In
these experiments a zoea in an eighth zoeal stage was not observed. In an
effort to determine if larvae in one or more of eight developmental stages
of C_. sapidus were particularly sensitive to different concentrations of
Kepone, a record of deaths by stages was made for larvae from each of
three crabs, Cs XII - XIV, which had been reared in acetone control and four
concentrations of Kepone (TABLE 6).
-23-
-------
TABLE 6. PERCENT MORTALITY IN DEVELOPMENTAL STAGES OF C. sapidus
Media
Acetone
Control
Kepone
0.10 ppb
Kepone
0.50 ppb
Kepone
0.75 ppb
Kepone
1 .0 ppb
Series
XII
XIII
XIV
XII
XIII
XIV
XII
XIII
XIV
XII
XIII
XIV
XII
XIII
XIV
I
4
2
0
8
2
0
10
2
0
2
6
2
8
10
2
II
30
18
10
12
16
36
12
38
38
10
38
38
38
36
40
Zoeal
III
6
16
28
10
12
28
18
24
16
32
22
26
16
42
22
Stages
IV
14
14
24
22
10
20
14
14
26
6
14
20
14
8
34
V
6
6
6
2
8
12
8
6
8
10
8
6
6
4
2
VI
8
0
2
6
10
0
0
0
0
0
0
0
2
0
0
Megalopa
VII
2
12
6
6
12
0
0
6
2
2
2
2
2
0
0
10
6
2
2
2
0
10
0
2
14
2
0
0
0
0
Total
80
74
78
68
72
96
72
90
92
76
92
94
86
100
100
Statistical analysis of C. sapidus cumulative mortality by stages
The results in Figure 7 show significant increases in mortality
at zoeal stages 2, 3, and 4 in all concentrations including the control
-24-
-------
VII
M
Figure 7. Effect of Kepone on mortality by stages of C sapidus
a. Significantly different from control (0.05)
b. Significantly different from control (0.01)
*. Significant increase over previous stage (0.05)
**. Significant increase over previous stage (0.01)
-25-
-------
SECTION 7
DISCUSSION
Survival
In chronic bioassay studies of the larval development of crabs, sub-
lethal concentrations of a pollutant are arbitrarily defined as those in
which there is a reduction in survival with increased concentration of
the pollutant and in which at least 10% reach the 1st crab stage. Acutely
toxic concentrations are those in which less than 10% of the larvae reach the
1st crab stage (Epifanio,1971; Bookhout and Costlow, 1975). Since the
average survival of R_. harrisii from the time of hatching to 1st crab stage
was 86% in 35 ppb, 64% in 50 ppb, 63.2% in 65 ppb and 13.3% in 80 ppb Kepone
(TABLE 2), these concentrations are considered sublethal. Less than 10% of
R. harrisii larvae reached the 1st crab stage in 95, 110 and 125 ppb Kepone
^"TABLE 2); therefore, these concentrations are considered acutely toxic
to R_. harrisii larvae. The sublethal concentrations of Kepone to C_. sapidus
developmental stages were 0.10, 0.50, and 0.75 ppb Kepone and the acutely
toxic concentration was 1.0 ppb (TABLE 5).
The sensitivity of C_. sapidus larvae to Kepone and to mirex is similar,
as might be expected, since mirex is structurally similar to Kepone. Actual
concentrations tested revealed 0.1, 0.5 and 0.75 ppb Kepone and 0.01 and
0.1 ppb mirex to be sublethal concentrations and 1.0 ppb Kepone and 1.0 ppb
mirex to be acutely toxic concentrations (Bookhout and Costlow, 1975).
There was a marked difference, however, in the sensitivity of R_. harrisii
larvae to Kepone and to mirex. Concentrations tested revealed 35, 50, 65
and 80 ppb Kepone and 0.01 and 0.1 ppb mirex to be sublethal concen-
trations, and 95, 110 and 125 ppb Kepone and 1.0 and 10.0 ppb mirex to be
acutely toxic concentrations (Bookhout et a]_., 1972). We have no explanation
for R_. harrisii larvae being so much more resistant to Kepone than to mirex.
The range of concentrations of Kepone in which development of the
decapod, C_. sapidus, occurred (TABLE 5) was much more narrow than the range
reported for the survival of 48-h-old juvenile Mysidopsis bahia, a mysid,
exposed to different concentrations of Kepone for 19 days (Nimmo et al . ,1977).
Survival of mysids was 90.6% in 0 y g/1, 84.4% in 0.39 yg/1, 50% in 1.55 yg/1,
3.1% in 4.4 y g/1 and 0% in 8.7 yg/l. Thus M_. bahia was not as sensitive
to Kepone as C_. sapidus larvae, but much more sensitive than R_. harrisii
larvae. When adult sheephead minnows, Cyprinodon variegatus, were exposed
to Kepone, survival decreased in relation to increase in concentration and
duration of exposure. All fish died by day 15 when exposed to 7.8 and
-26-
-------
24 yg/l Kepone and 20% died in 0.8 yg/l. The effect of Kepone in water on
embryos, fry and juveniles exposed to 0.08, 0.18, 0.72, 6.6 and 32 yg/l
was less than that observed when adult fish were exposed to the same
concentrations. The 36-day LC50 to developing fish exposed to Kepone in
water was 6.7 yg/l (Hansen et_ aK, 1977). Thus the sensitivity of sheeps-
head minnows to Kepone in chronic bioassays is closer to that found for
C_. sapidus larvae than for R_. harrisii larvae.
Duration
The reduction in molting rate with increase in concentration of Kepone
in zoeal development of R. harrisii and in development from hatching to 1st
crab in R_. harrisii (Figure 3) and C_. sapidus is considered a sublethal effect
of Kepone (Figure 6). In these species, a similar reduction occurred with
each increase in concentration of methoxychlor (Bookhout e_t aj_., 1976,
malathion (Bookhout and Monroe, 1977) and mirex (Bookhout et_ al_., 1972);
however, in mirex the reduction occurred only when C_. sapidus was reared
in acutely toxic concentrations (Bookhout and Costlow, 1975). Costlow et al.
(1960, 1962, 1966) reported that high and low salinities reduce the molting
rate of Sesarma cinereum, Panopeus herbstii and Rhithropanopeus harrisii.
Thus it is possible that the reduction in molting rate is a response of larvae
to any physiological stress and not a direct action of Kepone on the crab's
neuro-endocrine system which controls molting.
Seasonal considerations
There is some evidence of seasonal variability in survival and duration
of C_. sapidus larvae from the time of hatching to the 1st crab stage. Based
on published (Bookhout and Costlow, 1975; Bookhout ejt aj_., 1976; Bookhout
and Monroe, 1977) and unpublished data, it can be hypothesized that C_. sapidus
larvae hatched in the early part of the breeding season, May to mid-June, at
Beaufort, North Carolina, have a higher survival and a shorter duration of
development than those reared in the latter part of the breeding season, mid-
July to September. According to this concept, a survival of 22.7% and a
duration of 54.7 days to 1st crab in acetone control is what might be
expected for hatches of C_. sapidus at the end of July. It is obvious that
there is need for further studies designed to develop a seasonal baseline and
also to determine whether there are seasonal biochemical changes in the egg
associated with an increase in water temperature.
Mortality
The effect of Kepone on mortality within the range of sensitivity for
R_. harrisii larvae is shown in Figure 4. The changes in larval mortality by
stages and concentrations are consistent with the postulation that as early-
stage larval mortality increases with increasing Kepone levels, the remaining
larvae at subsequent stages are those most resistant to the insecticide.
This process of eliminating larger and larger fractions of susceptible larvae
-27-
-------
continues at each increasing concentration of Kepone until a level is reached
at which all but the most resistant larvae succumb during the first and second
zoeal stages.
The contrast of Figure 4 with Figure 7 shows clearly that larvae of
R,. harrisii are susceptible to the higher concentrations of Kepone during
the first zoeal stage and C_. sapidus are not. The results in Figure 7 show
significant increases in mortality over the previous stage of zoeal stages
II, III and IV in the control and in all concentrations of Kepone. Only at
the 1.0 ppb level are mortalities significantly different from the control,
but the trend of increasing mortality with concentration is quite consistent
with the concentration effects seen in the analysis of survival to megalopa
and to 1st crab.
Ecological implications
The presence of Kepone in water, biota and sediments of the Appomatox
and James Rivers, Virginia, has been well-documented (Environmental Protection
Agency, 1975). It is the consensus that the James River will remain
contaminated for years. There may be other contaminated rivers and estuaries,
especially outside of the United States, where Kepone is used to control pests
that attack bananas and potatoes.
Up to this time there is no direct evidence of toxic effects of Kepone on
crab larvae in the James River, the Chesapeake or its tributaries; hence, the
potential impact of Kepone on R^. harrisii and £. sapidus larvae in the field
is inferred only from a few field studies and a number of laboratory investi-
gations on other estuarine organisms.
Rhithropanopeus harrisii larvae are common to abundant in the upper York
and Pamunkey Rivers where Sandifer (1973) collected their larvae in a
range of salinities from 0 to 21.03 /oo but primarily within a range of
0 to 10 /oo. They were found as early as May and as late as October, but were
most numerous from July to September. A similar distribution of R_. harrisii
larvae in relation to salinity and seasonal abundance in the York River might
be expected in the two-layered James River estuary. Since there is a net
horizontal flow down the James estuary in the upper layer and a net hori-
zontal flow up the estuary (Prichard, 1953), most R_. harrisii larvae should
be retained in the middle estuary where salinities are low.
Peak abundance of larvae is where salinities range from 0 to 5°/oo
in the York River (Sandifer, 1973); therefore, ovigerous R_. harrisii would be
expected to be in the same salinities in the James. Thus adults would be in
the middle estuary of the James and in adjacent creeks and embayments south
of Jamestown.
Since the middle estuary of the James River is known to be polluted with
Kepone (Huggett e_t ^1_., in press), it is reasonable to assume that adult
R_. harrisii and their eggs are polluted. Newly hatched larvae would have an
initial burden of Kepone from eggs, and during development larvae would
-28-
-------
acquire additional Kepone from water and food. In the middle estuary,
dissolved Kepone is estimated to be in parts per trillion (Water Control
Board, 1977; Batelle, 1978; Saleh and Lee, 1978),and phytoplankton, zooplankton
and detritus have Kepone residues of 1.30, 4.80 and 0.67 yg/g-dry weight,
respectively (Huggett et_ aj_., in press). Sediments associated with organic
matter are more heavily contaminated with Kepone in the middle estuary than
in the lower estuary (Huggett et al., in press).
Bioconcentration of Kepone from water during a developmental period of
16 to 19 days in R,. harrisii is not known, but Bahner et_ al_. (1977) reported
that it was up to 13,000 times the amount measured in exposure water when
mysids were exposed to 0.026 yg/1 for 14 days. Epifanio (1973) estimated that
if crab larvae were exposed to a field concentration of dieldrin of 2 parts
per trillion in seawater and 20 ppb of the pesticide in food, crab larvae
would take up the pesticide in 1.23 times as fast from water as from food.
Larvae of R_. harrisii would obtain an additional burden of Kepone by
ingesting zooplankton. In the middle estuary, zooplankton is estimated to
have a residue of 4.80 yg/g dry weight (Huggett et_ aj_., in press). Kepone
once bioconcentrated, is not readily lost, because depuration in crustaceans
is slow (Bahner et_ a]_., 1977).
The migration of ovigerous Callinectes sapidus from brackish waters to
the mouths of estuaries or beyond to spawn has been known for many years
(Churchill, 1921). Male blue crabs from the James River had an average
Kepone residue of 0.81 pg/g and females 0.19 ^g/g. The difference is
possibly related to the time spent in the river (Huggett e^t al_., in press).
It is very probable that the eggs of the blue crab are also contaminated with
Kepone and the body burden of Kepone is in freshly hatched zoeae. Van Engel
(1958) found the greatest number of zoeae in channels between Cape Charles
and Cape Henry with diminishing numbers up-bay and seaward. Sandifer (1973)
collected most zoeae in salinities of 20 to 30 /oo. In the lower Chesapeake
and its mouth, Kepone in water, biota and sediments is very much diluted
compared to the middle estuary. Larvae of C_. sapidus, however, are much more
sensitive than the larvae of R_. harrisii. Concentrations which were sublethal
were 0.1 ppb to 0.75 ppb, and 1.0 ppb was acutely toxic. Furthermore,
Provenzano et_ a_l_. (1978) reported that the female and eggs of grass shrimp,
Palaemonetes pugio, collected from the mouth of the James River had Kepone
residues of 0.63 and 0.7 ppm, respectively, and samples from Lafayette River
in the vicinity of Norfolk had residues of 0.04 and 0.4 ppm, respectively.
This is further evidence that the eggs of C_. sapidus would be contaminated
with Kepone, and from the time of hatching to the 1st crab stage C_. sapidus
zoeae would obtain additional Kepone from water and zooplankton. Megalopa
of C_. sapidus might be found up the James River where they would receive
the highest concentrations of Kepone of any developing stage. Williams
(1971) found that a significant number of C_. sapidus megalopa move toward
the upper reaches of North Carolina estuaries.
It is obvious that the relationship between Kepone residues and
mortality during development of JR. harrisii and C_. sapidus in the laboratory
needs to be determined. It seems reasonably certain that eggs and larvae
of R_. harrisii in the middle estuary of the James River and the eggs and
larvae of C_. sapidus in the vicinity of the mouth of the Chesapeake Bay
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are polluted with Kepone, but the concentrations are not known. If chemical
analysis could be made of eggs in an advanced stage of development, of zoeae,
and of megalopa, and compared to residue analyses of similar stages in the
laboratory, it should be possible to predict the potential effect of Kepone
on populations of the two species.
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REFERENCES
Bahner, L.H., A.J. Wilson, Jr., J.M. Sheppard, L.R. Goodman, G.E. Walsh, and
J.M. Patrick, Jr. 1977. Kepone bioconcentration, accumulation, loss,
and transfer through food chains. Chesapeake Sci. 18_ (3):299-308.
Barr, A.J., J.H. Goodnight, J.P. Sail, and J.T. Helwig. 1976. A users
guide to SAS. SAS Institute, Raleigh, N.C. 329 pp.
Battelle Memorial Institute. 1978. The feasibility of mitigating Kepone
contamination in the James River basin. In: Kepone mitigation feasi-
bility project. Appendix A: Final Report June 1978 for U.S. EPA
Criteria and Standards Division, Washington, DC. EPA-440-5/78/004A. 730 p.
Bookhout, C.G., A.J. Wilson, Jr., T.W. Duke, and J.I. Lowe. 1972. Effects
of mi rex on larval development of two crabs. Water Air Soil
Pollut. 1:165-180.
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.
Bookhout, C.G., J.D. Costlow, Jr., and R. Monroe. 1976. Effects of
methoxychlor on larval development of mud crab and blue crab. Water
Air Soil Pollut. 5^:349-365.
Bookhout, C.G., and Robert J. Monroe. 1977. Effects of malathion on
development of crabs. In: Physiological Responses of Marine Biota
to pollutants. Vernberg, F.J., A. Calabrese, F.P. Thurberg, and W.B.
Vernberg (Eds.). New York, Academic Press, p. 1-19.
Cochran, W.G., and G.M. Cox. 1957. Experimental Design, Chapter 7. John
Wiley and Sons, New York.
Churchill, E.P. 1921. Life history of the blue crab. U.S. Fish Wild!.
Serv. Fish. Bull. 36^:91-128.
Costlow, J.D., Jr., and C.G. Bookhout. 1960. A method for developing
brachyuran eggs in vitro. Limnol. Oceanogr. 5_(2) :212-215.
Costlow, J.D, Jr., C.G. Bookhout, and R. Monroe. 1960. The effect of
salinity and temperature on larval development of Sesarma cinereum
(Bosc) reared in the laboratory. Biol. Bull. 118:183-202.
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Costlow, J.D., Jr., C.G. Bookhout, and R. Monroe. 1962. Salinity-tempera-
ture effects on larval development of the crab, Panopeus herbstii
Milne-Edwards, reared in the laboratory. Physiol. Zool. 35:79-93.
Costlow, J.D., Jr., C.G. Bookhout, and R. Monroe. 1966. Studies on the
larval development of the crab, Rhithropaopeus harrisii (Gould). I.
The effect of salinity and temperature on larval development. Physiol.
Zool. 39^:81-100.
Environmental Protection Agency. 1975. Preliminary report on Kepone levels
found in environmental samples from the Hopewell, Va. area. EPA Health
Effects Research Laboratory, Research Triangle Park, M.C.,
unpublished, 33 pp.
Epifanio, C.E. 1971. Effects of dieldrin in seawater on the development
of two species of crab larvae, Leptodius floridanus and Panopeus
herbstii. Mar. Biol. Vl_ (4):356-362.
Epifanio, C.E. 1973. Dieldrin uptake by larvae of the crab Leptodius
floridanus. Mar. Biol. l_9:320-322.
Hansen, D.J., A.J. Wilson, D.R. Nimmo, S.C. Schimmel, L.H. Bahner, and R.
Huggett. 1976. Kepone: Hazard to aquatic organisms. Science. 193:528.
Hansen, D.J., L.R. Goodman, and A.J. Wilson, Jr. 1977. Kepone: Chronic
effects on embryo, fry, juvenile, and adult sheepshead minnows
(Cyprinodon variegatus). Chesapeake Sci. _1_8 (2):227-232.
Hansen, D.J., D.R. Nimmo, S.C. Schimmel, G.E. Walsh, and A.J. Wilson, Jr.
1977. Effects of Kepone on estuarine organisms. In: Recent Advances
in Fish Toxicology: A Symposium. U.S. EPA, Environmental Research
Laboratory, Corvallis, OR. EPA Ecol. Res. Ser. 600/3-77-085 p. 20-30.
Huggett, R.J., M. Nichols, and M.E. Bender. Kepone contamination of the James
River. American Chem. Society (In press).
Nimmo, D.R., L.H. Bahner, R.H. Rigby, J.M. Sheppard, and A.J. Wilson, Jr.
1977. Mysidopsis bahia: An estuarine species suitable for life-cycle
toxicity tests to determine the effects of a pollutant. In: Aquatic
Toxicology and Hazard Evaluation, ASTM STP 634, F.L. Mayer and J.L.
Hamelink, (Eds.). American Society for Testing and Materials, 109-116.
Prichard, D.W. 1972. Salinity distribution and circulation in the
Chesapeake Bay Estuarine System. J. Mar. Res. Vh 106-123.
Provenzano, Anthony, Jr., Katheleen B. Schmitz,and Mark A. Boston. 1978.
Survival, duration of larval stages, and size of postlarvae of grass
shrimp, Palaemonetes pugio. reared from Kepone^contaminated and
uncontaminated populations in Chesapeake Bay. Estuaries 1 (4):239-244.
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Saleh, F.Y., and G.F. Lee. 1978. Analytical methodology for Kepone in
water and sediment. Environ. Sci. Technol. ]_2_ (3):297-301.
Sandifer, Paul. 1973. Distribution and abundance of decapod crustacean
larvae in the York River estuary and adjacent lower Chesapeake Bay,
Virginia, 1968-1969. Chesapeake Sci. Jj4:235-257.
Schimmel, S.C., and A.J. Wilson, Jr. 1977. Acute toxicity of Kepone^ to
four estuarine animals. Chesapeake Sci. J_8 (2):223-227.
Searle, S.R. 1971. Linear Models. John Wiley and Sons, New York. 532 pp
Snedecor, G.W. and W.G. Cochran. 1967. Statistical Methods. Iowa State
University Press, Ames, Iowa. pp. 324-337.
Van Engel, W.A. 1958. The blue crab and its fishery in Chesapeake Bay.
Part I - Reproduction, early development, growth, and migration.
Commer. Fish. Rev. 20_ (6):6-17.
Water Control Board. 1977. Richmond, Virginia. Unpublished data.
Williams, A.B. 1971. A ten-year study of meroplankton in North Carolina
estuaries: annual occurrence of some brachyuran developmental stages.
Chesapeake Sci. J_2 (2):53-61.
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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 less
than 10% of the larvae survive to the 1st crab stage.
analysis of variance: a special application of the linear models technique
which can be used effectively when the experimental design is balanced
with respect to factors and replication.
chronic tests: long-term exposure to toxicant.
cummulative mortality: the total number of larval deaths incurred at any
given stage of development expressed as a percent of the initial
number of larvae.
differential survival: reduction of survival with each increase in
insecticide.
dosage-response relationship: the characterization of the change in
response (e.g. survival) with changing stimulus (e.g. concentrations
of insecticide). Typically such responses vary from 0% at some
threshold level of the stimulant to 100% at some uniformly lethal level
of the stimulant. An intermediate point is the ED50, the "effective
dose" at which 50% of the organisms react to the stimulant.
first crab stage: first stage after molt from megalopa; has adult morpho-
logy with abdomen bent under cephatothorax, but is sexually immature.
fitting a linear regression: another special application of the linear
models technique where the response variable is a simple linear
function of a single independent variable, y = a+e x + e, and the
relevant statistics are estimates of the parameters, a, g, and a2,
the variance of the random error, e.
general linear models technique: an attempt to characterize a given
response (e.g. survival) as a linear function of factors, experi-
mentally imposed and environmentally existent, and their interactions.
Statistical analysis of the resulting model quantitatively evaluates
the relative importance of the several factors and the experimental
errors.
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h: hour
LC50: lethal concentration; the concentration of toxicant in water estimated
to be lethal to 50 percent of test animals for a specified period of
exposure.
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.
yg/g: micrograms 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 necessary for growth
during larval and juvenile development in arthropods, including
crustaceans.
ppb: parts per billion.
ppm: parts per million.
°/oo = parts per thousand.
regression coefficient, in the linear regression model: the regression
coefficient of the independent variable and the slope of the
straight line relating y to x. If y is measured in 'DAYS' and x
in 'ppb,1 the units of slope are DAYS/ppb.
sublethal concentrations: concentrations of insecticide in which 10% or more
of the larvae survive to the 1st crab stage.
sublethal effects: effects in larvae reared in sublethal concentrations,
but not in acetone control; they become more pronounced as concen-
trations are increased.
sub-plot error: the component of experimental error that affects the
repeated measurements on-the same experimental unit, e.g. cumulative
mortality of an original unit of 100 larvae.
technique of split-plot analysis of variance: sometimes called a "repeated
measurement design" when successive measurements are taken on the same
experimental unit, e.g., survival at each stage of development. The
resulting analysis provides for two or more levels at which different
components of experimental error may affect the response.
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transformed to angular scale: the transformation of data expressed in
'percent1 to a new scale where the /percent is treated as the sine of
an angle. While 'percent' varies from 0 to 100 the corresponding
'angles' vary from 0 to 90 . The angular scale is more amenable
to statistical analysis because the sampling variance is approximately
constant whereas the variance in the percent scale is not.
weighted standard error: a standard error that combines the estimate of
error associated with experimental units treated alike (whole-plot
error) with the estimate of sub-plot error to provide an appropriate
basis for comparing sub-plot means at different levels of whole-plot
factors, e.g., to compare the mortality at a given zoeal stage at
several different concentrations of insecticide.
zoea(e): a planktotropic larval stage of a crab with a laterally 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^. sapidus).
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
2.
4. TITLE AND SUBTITLE
KEPONER EFFECTS ON DEVELOPMENT OF CALLINECTES SAPIDUS
AND RHITHROPANOPEUS HARRISII
7 AUTHOR(S)
C.G. Bookhout, J.D. Costlow, Jr. , R.J. Monroe*
*North Carolina State University
9. PERFORMING ORGANIZATION NAME AN
Duke University Marine Lab
Beaufort, N.C. 28516
D ADDRESS
Dratory
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
15. SUPPLEMENTARY NOTES
3. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
R803838
13. TYPE OF REPORT AND PERIOD COVERED
Final Scientific Report
14. SPONSORING AGENCY CODE
EPA/600/4
16. ABSTRACT
Laboratory experiments were conducted to determine the effect of
Kepone on the development of Callinectes sapidus from the time of hatching
until the 1st crab stage was reached. For comparison, similar investigations
were made to ascertain the effects of Kepone on larval development of
Rhithropanopeus harrisii.
Of the concentrations tested, 35, 50, 65 and 80 ppb Kepone were found
to be sublethal and 95, 110 and 125 ppb Kepone were acutely toxic to
R. harrisii larvae; whereas 0.1, 0.5 and 0.75 were sublethal and 1.0 ppb
Kepone were acutely toxic to C. sapidus larvae. The duration of zoeal
development and total time
prolonged with concentratii
significant relationship c<
duration of zoeal developm
to 1st crab. The developm
sensitive differ in the tw<
17.
a- DESCRIPTORS
Insecticides
Bioassay
Crustacea
Crabs
Kepone
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
from hatching to 1st crab was in general
Dn in R. harrisii; whereas, in C. sapidus, no
Duld be detected between Kepone concentration and
2nt, but there was a significant relationship
=ntal stages in which the larvae are particularly
D species.
KEY WORDS AND DOCUMENT ANALYSIS
b. IDENTIFIERS/OPEN ENDED TERMS C. COS ATI Field/Group
Insecticide toxicity
Kepone
Blue crabs
Mud crabs
Larval development
19. SECURITY CLASS (This Report) 21. NO. OF PAGES
UNCLASSIFIED 36
20. SECURITY CLASS (This page) 22. PRICE
UNCLASSIFIED
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
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