EFFECTS OF SOLUBLE FRACTIONS OF USED LIGHT-WEIGHT
LIGNOSULFONATE TYPE MUD AND HEXAVALENT CHROMIUM ON THE COMPLETE
LARVAL DEVELOPMENT OF CRABS, RHITHROPANOPEUS HARRISII AND CALLINECTES SAPIDUS
by
Cazlyn G. Bookhout
*Robert Monroe
Richard Forward
John D. Costlow, Jr.
Duke University
Durham, NC 27706
and
*North Carolina State University
Raleigh, NC 27607
Grant No. CR807374
Project Officer
Charles McKenney, Jr.
Environmental Research Laboratory
U.S. Environmental Protection Agency
Gulf Breeze, FL 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 commercial products constitute endorsement or
recommendation for use.
11
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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 introduction
of these compounds into the environment be formulated on a sound scientific
basis. Accurate information describing dose-response relationships for
organisms and ecosystems under varying conditions is required. The
Environmental Research Laboratory, Gulf Breeze, contributes to this information
through research programs aimed at determining:
the effects of toxic organic pollutants on individual sc-ocies
and communities of organisms,
the effects of toxic organics on ecosystem processes and components, .
the significance of chemical carcinogens in the 'rstua-i n? and m'-n'ne
envi ronments.
This report describes the comparative toxicologies! errect: "r soluble
fractions of drilling fluids and a common component of drilling fluids,
chromium, on the complete larval development of two estuan'ne cr^h :n?:ies.
These data will be useful in assessing the possible effects o~ dulling fluids
and their components on the marine and estuarine environment and biota. The
study demonstrates a possible positive relationship between r?o!yc
hydrocarbons in the estuarine and coastal environment and cellular proli-
ferative diseases in bivalve molluscs.
Hen&v
F. Enos
Di rector
Envi ronnenta! "'esearch Laboratory
Gulf Breeze,
n i
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ABSTRACT
The mud aqueous fractions (MAP) and suspended particulate phase (SPP) of
lignosulf onate type mud were nontoxic to larvae during the complete larval
development of Rhithropanopeus harrisii. Five percent MAP and SPP were not
toxic to Callinectes sapidus . Survival of C_. sapidus larvae decreased as
concentrations of MAF and SPP increased from 5 to 50%. No larvae reached the
1st crab stage in 100% MAF and SPP. Statistical analyses of the data on
survival, mortality and behavior are presented.
Survival of R_. harrisii from hatching to 1st crab stage occurred in
Na2Cr04 concentrations from 1.1 to 29.1 ppm. Estimated LC50 for complete
zoeal development was 17.8 ppm Na2CrO^ and it was 13.7 ppm for development
to 1st crab stage. A concentration of 1.1 ppm Na2Cr04 was nontoxic, while
Na2Cr04 concentrations of 7.2 and 14.5 ppm were sublethal and
concentrations of 29.1 to 58.1 ppm were acutely toxic. Low concentrations of
Na2Cr04 caused an increase in swimming speed and high concentrations caused
a decline.
Survival of Callinectes sapidus occurred in Na2CrO^ concentrations
from 1.1 to 4.7 ppm. The LC50 for complete zoeal development was estimated to
be 2.9 ppm Na2Cr04 and the LC50 for development to 1st crab stage was
estimated to be 1.0 ppm Na2Cr04- Statistical analyses of the data on
survival, duration and mortality of .larvae are presented.
This report was submitted in fulfillment of Grant No. CR807374 by Duke
University under the sponsorship of the Environmental Protection Agency. This
report covers the period May 15, 1980 to May 14, 1982.
IV
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CONTENTS
Foreword iii
Abstract iv
Figures vi
Tables vii
Acknowledgments ix
1. Summary and Conclusions 1
2. Recommendations 4
3. General Materials and Methods 5
4. Lignosulfonate Type Mud 10
Introduction 10
Results 12
Discussion 28
5. Hexavalent Chromium 32
Introduction 32
Results 34
Discussion 54
Literature Cited 58
Glossary 62
v
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FIGURES
Number
1 Effect of percent of mud aqueous fraction (MAF) of used 18
light-weight lignosulf onate type mud on survival of
C_. sapidus larvae.
2 Effect of percent of suspended particulate phase (SPP) 19
of used light-weight lignosulf onate type mud on survival
of C_. sapidus larvae.
3 Effect of MAF of used light-weight lignosulf onate type mud 25
on mortality by stages of development of C_. sapidus
4 Effect of SPP of used light-weight lignosulf onate type mud 26
on mortality by stages of development of C_. sapidus
5 Effect of concentration of Na2CrC>4 in ppm on survival 37
of R_. harrisii larvae
6 Duration of zoeal development (DZ) and duration to 1st crab 39
(DC) in R_. harrisii vs concentration of Na2CrC>4 in ppm
Effect of Na2Cr04 in ppm on, rate of molting from hatch 40
to megalopa and hatch to 1st crab in R_. harrisii
Effect of Na2CrC>4 in ppm on mortality of R_. harrisii 45
larvae
9 Effect of concentration of Na2Cr04 in ppm on survival 48
of C. sapidus larvae
10 Duration of zoeal development (DZ) and duration to 1st crab 49
(DC) in C_. sapidus vs concentraion of Na2Cr04 in ppm
11 Effect of Na2Cr04 in ppm on rate of molting from 50
hatch to megalopa and hatch to 1st crab in £. sapidus
12 Effect of Na2Cr04 in ppm on mortality of £. sapidus 53
larvae
VI
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TABLES
Number Page
1 Summary of the analyses of light-weight lignosulfonate type 5
mud with ferrochrome added (No. 4 mud) from the Jay, Florida
Exxon Well by Dr. Robert Shokes
2 Percent survival and duration in days through zoeal and 14
megalopa development of three series (RhI-III) of II.
harrisii reared in seawater control and in A concentrations
of Mud Aqueous Fraction (MAF) and 4 concentrations of
Suspended Particulate Phase (SPP) of used lignosulfonate type
mud
3 Average percent survival and average duration in days of 15
zoeal and megalopa development of three series (RhI-III) of
R_. harrisii reared in seawater control, 4 concentrations of
MAF and 4 concentrations of SPP of used lignosulfonate tvpe
mud (No. 4 mud)
4 Percent mortality in developmental stages of R_. harrisii 16
reared in seawater control and different concentrations of
MAF and SPP of used lignosulfonate type mud
5 Average percent mortality in developmental stages of R. 17
harrisii reared in saltwater control and different
concentrations of MAF and SPP of used lignosulfonate type
mud
6 Percent survival and duration in days through zoeal and 21
megalopa development of three series of Callinectes sapidus
reared in seawater control and in different concentrations of
MAF and SPP in lignosulfonate type mud
7 Average percent survival and average duration in days of 22
zoeal and megalopa development of three series of C. sapidus
in seawater control and different concentrations of MAF and
SPP in lignosulfonate type mud
8 Percent mortality in developmental stages of three series of 23
C. sapidus reared in saltwater control and different
concentrations of MAF and SPP in lignosulfonate type mud
vn
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TABLES Continued
9 Average percent mortality in developmental stages of three 2^
series of C_- sapidus reared in saltwater control and
different concentrations of MAP and SPP in lignosulf onate
type mud
10 Swimming speed (mm/min) of control £. sapidus first zoea 27
and those treated with different concentrations of MAF and
SPP *
11 Percent survival and duration in days through zoeal and 35
megalopa development of R_. harrisii reared in seawater
control and in different concentrations of hexavalent
chromium
12 Average percent survival and average duration in days of 36
zoeal and megalopa development of R_. harrisii in seawater
control and different concentrations of hexavalent chromium
13 Percent mortality in developmental stages of R_. harrisii 41
reared in saltwater control and different concentrations
of hexavalent chromium
14 Average percent mortality in developmental stages of 43
II. harrisii reared in saltwater control and different
concentrations of hexavalent chromium
15 Swimming speed (mm/min) for different zoeal stages of 44
control R. harrisii larvae, and those treated with
16 Percent survival and duration in days through zoeal 46
and megalopa development of three series of Gallinectes
sapidus reared in seawater control and in different
concentrations of hexavalent chromium
17 Average percent survival and average duration in days 47
through zoeal and megalopa development of three series of
Callinectes sapidus reared in seawater control and in
different concentrations of hexavalent chromium
18 Percent mortality in developmental stages of three series 51
of £. sapidus reared in saltwater control and different
concentrations of hexavalent chromium
19 Average percent mortality in developmental stages of three 52
series of £. sapidus reared in saltwater control and
different concentrations of hexavalent chromium
Vlll
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ACKNOWLEDGEMENTS
We would like to thank Mr. Joseph Goy for preparing the figures; Dr. R.
Shokes of Science Applications Inc. for chemical analysis; Miss Anne DuCharme
for technical assistance in the summer of 1981 and Mr. Steven G. Morgan for
technical assistance for the total span of the project. The untiring efforts
of Mrs. Norma Jean Buck in typing the manuscript are greatly appreciated.
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SECTION 1
SUMMARY AND CONCLUSIONS
1. Survival of Rhithropanopeus harrisii from hatching to 1st crab stage
occurred in mud aqueous fraction (MAP) concentration from 5% (5,000 ppm)
to 100% (100,000 ppm) and in suspended particulate phase (SPP)
concentrations from 5% (5,000 ppm) to 100% (100,000 ppm). The percent
survival to megalopa and to 1st crab stage was 90% or over in seawater
control and in 5, 25, 50 and 100% MAF and SPP in three replicate series of
larvae tested.
2. Differential survival of Callinectes sapidus from hatching to 1st crab
stage occurred in MAF and SPP concentrations from 5 to 50%. No larvae
reached the 1st crab stage in either 100% MAF or 100% SPP- Five percent
MAF and 5% SPP were nontoxic to larvae tested. Statistical analysis
revealed for zoeal survival that there was approximately 4% decrease/10%
increase in MAF @ 50% concentration (CONG), and for survival to 1st crab
there was approximately 3% decrease/10% increase in MAF @ 50% CONC. For
zoeal development and development from hatching to 1st crab, there was
approximately 5% decrease in survival for a 10% increase in SPP near 50%
SPP CONC.
3. There was no significant difference in duration in zoeal development and
in hatch to 1st crab of C^ sapidus in seawater control and in
concentrations of MAF and SPP employed.
4. Mortality of larvae reared in 5 and 25% MAF was not significantly
different from larval mortality in seawater control in any of the nine
developmental stages of C_. sapidus, but mortality of larvae reared in 50
and 100% MAF was significantly different from the control in every
developmental stage. Even though larvae in zoeal stage I were most
sensitive, larvae in zoeal stage II were also very sensitive.
5. Mortality of larvae in 5% SPP was not significantly different from
mortality in the control in any of the nine developmental stages, but
mortality of larvae reared in 50 and 100% SPP was significantly different
from the control in every developmental stage of C. sapidus. In zoeal
stage I .25% SPP was significantly different from "the control at the 0.05
alpha level. As in the MAF experiment, although larvae in zoeal stage I
were most sensitive, larvae in zoeal stage II were also very sensitive.
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6. Blue crab larval behavior is affected by exposure to MAF and SPP with the
general effect being a decline in swimming speed. A significant reduction
was only observed in 100% MAF and 5, 25, 50 and 100% SPP-
7. Callinectes sapidus larvae could be in the vicinity of drilling operations
during development and might be found in the upper turbidity plume, but
the chances of many of the larvae remaining in the 3 m highly toxic zone,
or even in the 15 m intermediate toxic zone, around the discharge source
long enough to suffer mortality are very remote. If by chance a few 1st
or 2nd stage zoeae of C. sapidus in the process of molting happened to be
entrained within 15 m of discharge, they might be killed or receive
irreversible stress, for these zoeae are extremely sensitive. Larvae in
other stages could be affected, but not as quickly.
8. Survival of Rhithropanopeus harrisii from hatching to 1st crab stage
occurred in Na2Cr04 concentrations from 1.1 to 29.1 ppn. No larvae
reached the 1st crab stage in concentrations of 40.6, 46.4 and 58.1 ppm
Na2Cr04. A concentration of 1.1 ppm Na2Cr04 was non toxic to
larvae tested. The estimated LC50 for complete zoeal development was 17.8
ppm Na2CrG4 and for development to 1st crab was 13.7 ppm Nao
Statistical analysis of the data on R. harrisii duration revealed that
there was 0.120 + 0.021 days increase in duration of zoeal development
from hatching to megalopa for each ppm added Na2Cr04 , and that there
was 0.122 + 0.021 days increase in total duration time from hatching to
1st crab for each ppm added
10. There was differential mortality of R_. harrisii larvae from concentrations
of 1.1 to 58.1 ppm Na2Cr04- In 1.1 ppm Na2Cr04, there was no more
mortality than in seawater control. Na9Cr04 concentrations of 7.2 ppm
and 14.5 ppm were sublethal and those of 29 to 58 ppm were found to be
acutely toxic.
11. Rhithropanopeus larval swimming speed was affected by exposure to
Na2Cr04 . The general pattern was for the swimming rate to be elevated
with short-term exposure to concentrations from 1.1 to 29.1 ppm
Na2CrC>4 and with long-term exposure to low concentrations of 1.1 and
7.2 ppm Na2Cr04 . Swimming rates were only depressed in later stages
upon long term exposure to high Na2Cr04 concentrations of 14.5 and
29.1 ppm. Thus in general, low concentrations caused an increase in
swimming speed and high concentrations caused a decline.
12. Survival of Callinectes sapidus from hatching to 1st crab stage occurred
in Na9Cr04 concentrations from 1.1 to 4.7 ppm. There was better
survival in 1.1 ppm than in seawater control, but there was differential
survival from 1.1 to 7.2 ppm Na2Cr04. The LC50 for complete zoeal
development was estimated to be 2.9 ppm Na2Cr04 , and the LC50 for
development from hatching to 1st crab was estimated to be 1.0 ppm
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13. Statistical analysis of the data on £. sapidus duration revealed that
there was 1.65 + 0.29 days increase in duration of zoeal development from
hatching to megalopa for each ppm added Na2Cr04, and that there was
1.31 -h 0.29 days increase in total duration time from hatching to 1st
stage for each ppm added
14. There was significantly less mortality in 1-1 ppm Na2CrQ^ than in
seawater control. A Na2Cr04 concentration of 2.4 ppm was nontoxic.
There was differential mortality from 4.7 to 7.2 ppm Na2Cr04 . These
concentrations are considered acutely toxic since less than 10% of £•
sapidus larvae reached the 1st crab stage. Zoeae in zoeal stage III were
extremely sensitive to 7.2 ppm Na2Cr04, and zoeae in zoeal stages III,
IV and V were most sensitive to 4.7 ppm ^2^04. In seawater control,
1.1 and 2.4 ppm Na2Cr04, there was a highly significant increase in
mortality in the megalopa stage over the previous stage.
15. For most discharges, the background level for chromium has been reported
to be reached approximately 100 to 150 meters from the point of discharge.
Within this area, entrained crab larvae would undoubtedly absorb Cr
more readily than Cr , if both were present, and bioaccumulate
chromium. It is very questionable, however, whether crab larvae would
remain in the upper turbidity plume long enough to bioaccumulate enough
chromium to kill the larvae or to produce sublethal stress. Hence, it is
probable that chromium in drilling fluids is not likely to reduce the
population of crab larvae and other planktonic organisms in the area
around an oil well, except possibly in the immediate vicinity of the
discharge pipe.
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SECTION 2
RECOMMENDATIONS
1- Records of constituents of new drilling fluids should be kept on file with
a central agency together with the log of the time and the amount of
specific additives made at different depths. This information, should be
made available to investigators who will conduct acute and chronic
toxicity tests.
2. Approximately 90% of the main constituents of drilling fluids have been
reported to be nontoxic, but some of the remaining 10% additives are
toxic. Separate and joint toxicity studies should be made of the latter
to determine if the components are nontoxic, less than additive, additive,
or more than additive in toxicity, or antagonistic to one another
following the experimental design of Sprague and Logan (1979).
3. There is a need for more detailed chemical analysis of trace metals in the
suspended particulate fraction and the water-soluble fraction of drilling
fluids after discharge into the ocean. Particular attention should be
given to speciation of chromium if there is evidence that Cr ''" is
present.
4. Residue analyses of planktonic organisms for trace metals should be made
from the 150 meter zone around the discharge source and compared to
comparable samples taken from outside. If there is evidence of
bioaccumulation of trace metals by larvae or small crustaceans within the
vicinity of the upper turbidity plume, would the bioaccumulated metals be
passed through a food web to higher organisms?
+3
5. Chronic tests on the effects of Cr on the complete larval
development of Rhithropanopeus harrisii and Callinectes sapidus should be
made in order to make comparisons with the findings in the current
investigation on the effect of Cr on the development of the same
species of crabs.
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SECTION 3
GENERAL MATERIALS AND METHODS
Preliminary experiments were conducted to determine the range of concen-
trations of the Mud Aqueous Fraction (MAF) and the Suspended Particulate Phase
(SPP) of (No. 4 Mud) lignosulfonate type mud with ferrochrome lignosulfonate
added to use in definitive chronic toxicity studies on the development of
Rhithropanopeus harrisii (Gould) and Callinectes sapidus Rathbun. Preliminary
experiments were also performed to determine the concentrations of sodium
chromate (Na2Cr04) to use in definitive chronic studies on the development
of R. harrisii and C_. sapidus.
The lignosulfonate type mud which was tested was sent to us in 5.7 liter
polyethylene containers by the U.S. Environmental Protection Agency,
Environmental Research Laboratory (ERL), Gulf Breeze, Florida. Upon arrival at
the Duke University Marine Laboratory, Beaufort, N.C., the container was placed
in a cold room where the temperature was 4°C. The mud was originally collected
on July 22, 1980 just after it went through the shaker table and the cuttings
were removed. The mud was collected at a depth of 3,735.9 meters (12,257 feet)
with a density of 4.1 kg/3.785 liters (9.1 Ib/gal), a viscosity of 58 sec/qt
API and water content of 88.3%. Further analysis of the mud furnished by
ERL-Gulf Breeze is given in TABLE 1.
TABLE 1. SUMMARY OF THE ANALYSES OF LIGHT-WEIGHT LIGNOSULFONATE TYPE MUD WITH
FERROCHROME ADDED (NO. 4 MUD) FROM THE JAY, FLORIDA EXXON WELL BY DR. ROBERT
SHORES, SCIENCE APPLICATIONS (PERSONAL COMMUNICATION)
Metals
%
Al
Fe
Ba
8
3
4
.00
.19
.10
Pb
Cr
Zn
PPm
40
96
225
Total Resolved
Aliphatics (y g/1)
.2
.4
.0
17
,000
Total Resolved
Aromatics ( ug/1)
27,300
H20
%
88.
3
The Mud Aqueous Fraction (MAF) was prepared following the methods of Neff
e_t_ a^., 1980 by mixing one part used drilling mud with nine parts of
20°/oo seawate'r for experiments on R_. harrisii larvae and 30°/00
seawater for studies on £. sapidus larvae. The mixture was stirred thoroughly
with an electric mixer and then allowed to settle for 20 hours. The dark
colored aqueous layer was siphoned off for use in toxicity tests. For MAF
toxicity tests on R. harrisii and C_. sapidus larvae, 100% MAF was prepared by
mixing 120 ml of undiluted mud with 1080 ml of seawater. Fifty percent MAF
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was prepared by adding 150 ml of 100% MAP to 150 ml of seawater, 25% MAF was
made by adding 75 ml of 100% MAF to 225 ml of seawater, and 5% MAF was prepared
by adding 15 ml of 100% MAF to 285 ml of seawater.
Suspended Particulate Phase (SPP) was prepared following the methods of
Neff e_^ a^. (1980). One hundred percent SPP was made by air mixing with
compressed air one part undiluted mud with nine parts seawater (20°/0o
salinity for R. harrisii and 30° /„<> for £. sapidus) for one-half hour with
manual stirring every 10 minutes. After aeration the suspension was allowed to
settle for four hours before the supernatant was siphoned off for use in
toxicity tests. The concentrations of SPP used for R.. harrisii and £. sapidus
were the same as those described for MAF. Our aeration was through two
airstones connected by hoses to a compressed air line.
Source of Animals
Three ovigerous Rhithropanopeus harrisii furnished larvae for series
designated as Rhl, Rhll and RhIII. The mother crab, which furnished larvae for
RhI on September 30, 1980, was collected in the Neuse River near Havelock,
N.C., and the crab which provided larvae for Rhll on November 8, i960 was found
in the Newport River near Morehead City, N.C- It became ovigerous after it had
been held in a habitat aquarium in the laboratory. Ovigerous R.. harrisii which
furnished larvae for RhIII on November 14, 1980 was collected in the vicinity
of Fort Pierce, Florida, and was shipped air freight to Beaufort, N.C. There
were sufficient larvae from one ovigerous crab to do one toxicity test on MAF
and SPP fractions of No. 4 mud.
Adult R. harrisii which furnished larvae for series Rhl to RhVl used in
the sodium chromate experiments were collected near Morehead City and near
Havelock, N.C. while they were in the refractory period during the fall of 1980
and winter of 1981. They were placed in an artificial habitat in the
laboratory where the temperature of the water was 30°C and there was 14 h light
and 10 h darkness per day. When the crabs became ovigerous, they were isolated
in separate large finger bowls (19.4 cm diam) and maintained in a constant
temperature cabinet at 25°C under a light regime of 12 h light and 12 h
darkness until hatching occurred. The largest and most healthy hatches were
selected for the experiments. Larvae for Rhl and Rhll hatched on January 20,
1981. The dates of hatching were February 17, 1981 for RhIII, February 23,
1981, for RhIV, March 18, 1981, for RhV and March 19, 1981 for RhVI.
In experiments on No. 4 mud, three ovigerous Callinectes sapidus were used
to obtain larvae for series Csl, CsII and CsIII. Larvae of these series
hatched on July 4, July 19 and July 24, 1981 from three ovigerous crabs which
were collected in the Beaufort Inlet.
In experiments on Na2Cr04, three ovigerous Callinectes sapidus
furnished larvae for series Csl, CsII and CsIII. Larvae of series Csl and II
hatched on September 10 and CsIII hatched on September 11, 1981.
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Long-Terro Exposure of R. harrisii and C. sapidus Larvae to MAF and SPP
In each series of R. harrisii (RhI, Rhll and RhIII), there were sufficient
larvae for five bowls with 10 larvae in each bowl for seawater control and fiv and the bowls of larvae were
maintained in a constant temperature cabinet of 25°C and in a light regime of
12 h light and 12 h darkness. When larvae molted to megalopa, each megalopa
was placed in a separate compartment of a plastic box. Daily records were kept
for the number of live and dead larvae as well as the stage of each dead larva.
The time each larva molted to a megalopa and each megalopa molted to a first
crab stage were also recorded.
When £. sapidus larvae were exposed to MAF and SPP, the same procedures as
outlined above were used except the salinity of the media was 30°/00 and
larvae from the time of hatching until the first crab stage was reached were
fed Arbacia embryos and Branchionus plicatilis, rotifers, plus Artemia nauplii
after the second zoea had been reached.
Behavior of C. sapidus Larvae; Effects of Exposure to Drilling Mud MAF and SPP
Ovigerous C_. sapidus were collected locally, and the larvae hatched in
the laboratory. Larvae were reared in 30°/Oo filtered seawater at 25°C on
a I2:l2h:light dark cycle. The day after hatching, larvae were divided into
groups of about 150 and each group exposed to a different set of conditions:
30°/00 seawater alone, 5% MAF, 25% MAF, 50% MAF, 100% MAF, 5% SPP, 25%
SPP, 50% SPP, and 100% SPP.. The MAF and SPP solutions were made up as
previously described. The solutions were tested because they represent the
entire range of concentration used for the developmental study. Each day the
larvae were placed in new solutions and fed fertilized sea urchin eggs and
Artemia nauplii. Larvae swimming speed was measured after 48 hours exposure in
the middle of the light phase.
The behavior measured as indicative of stress was swimming speed. For
these measurements larvae were placed in a cuvette positioned on the stage of a
dissecting microscope, which was coupled to a closed circuit television system.
The microscope illumination light was filtered to the near infra-red region.
The larvae are insensitive to wavelengths in this region.
The general procedure was to place light-adapted larvae on the microscope
stage, extinguish the room lights and after one minute record larval movements
on video tape. In this way swimming was observed in darkness. The tapes were
later analyzed for random swimming speeds. Although speeds measured during
random swimming tend to underestimate the true rates (Forward, 1977), the
obtained values still serve as an indicator of changes in activity (Forward and
Costlow, 1976, 1978). Speeds for 20 larvae were measured for each hatch and
condition. Since three separate hatches were tested, the total sample size for
each condition was 60. Mean swimming speeds under the various conditions were
compared by the Student's T test.
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Long-Term Exposure of R. harrisii and C. sapidus Larvae to Sodium Chromate
Hexavalent chromium, Na2CrC>4, was purchased from Fisher Scientific
Company as Certified Anhydrous Sodium Chromate. A 58.09 ppt stock solution was
prepared by dissolving a known weight of Na2CrC>4 in glass distilled water
and different concentrations were made from this stock solution by serial
dilution. For experiments on the effect of Na2Cr04 on the development of
R_. harrisii, 1 ml of stock solution of 1.12 parts per thousand (ppt)
(°/oo) (ml/1), 7.17 °/0o, 14.52 °/00, 29.09 °/oo, 40.6
°/oo, and 58.09 °/00 were added to 999 ml of 20°/oo filtered
seawater to give final concentrations from 1.12 parts per million (ppm) (mg/1)
to 58.09 ppm. The concentrations of Na2Cr04 given in this manuscript are
those determined by the Hazleton Laboratories America, Inc.
For experiments on the effect of Na2Cr04 on the development of C_.
sapidus , 1 ml of Na2Cr04 stock solution of 1.1 °/oo, 2.4 °/0o,
4.7 °/oo, and 7.2 °/o0 were added to 999 ml of 30°/0o filtered
seawater to give final concentrations from 1.1 ppm to 7.2 ppm NaoCrC^.
The methods for rearing larvae in a check series, 10 larvae per finger
bowl (8.9 cm diam), was the same as previously described. Fresh solutions of
Na2Cr04 were prepared every other day, placed in 1000 ml flask and
dispensed by a 50 ml pipettor to finger bowls. Between daily changes of larvae
to clean bowls, bowls of larvae were maintained in a constant temperature
cabinet at 25°C and a light regime of 12 h light and 12 h darkness.
Behavior of R. harrisii larvae: Effects of Exposure to NaoCr04
Ovigerous specimens of R_. harrisii were collected from the Neuse River,
North Carolina. The eggs hatched during the night after collection and the
experiments begun the next morning. Larvae were reared in 20°/0o filtered
seawater, at 25°C on a 12:12 h light-dark cycle. Hatches were divided into
groups of about 75 larvae and each group chronically exposed to a different set
of conditions: 20°/0o seawater alone, 1.2 ppm, 7.2 ppm, 14.5 ppm and
29.1 ppm sodium chromate. The sodium chromate solutions were made up as
described previously. These concentrations were tested because they span the
region from no effect upon larval mortality to concentrations that are almost
totally lethal. Each day the larvae were placed in new solutions and fed
Artemia nauplii. Larval behavior was monitored for all 4 zoeal stages on
intermolt days. Furthermore, to avoid complications due to a possible
biological rhythm in activity, larval behavior was measured between 4 to 10 h
after the beginning of the light phase. The techniques for monitoring swimming
speed are identical to those used for C_. sapidus larvae.
Statistical analysis
The statistical methods used for analysis of larval development of R.
harisii and C_. sapidus subjected to treatment by MAF, SPP or Na2Cr04 are
outlined in detail in two articles on Kepone (Bookhout et_ al_. , 1979; Bookhout
et al. 1980). The mean swimming speeds under various conditions were compared
by" Student's T test.
-------
Briefly, the methods relied on the angular transformation of survival
and/or mortality percentages to stabilize the experimental error variance in
order to use standard analysis of variance and regression techniques for the
appropriate estimates and tests of significance. Durations of molting time
expressed in days were also analyzed as "rates" (i.e. reciprocal days) since
in some data sets the relationship between concentrations and rates was more
nearly linear than the relationship with time in days.
In presenting the results of the several analyses mean values of survival
and/or mortality were obtained from the retransformed mean values in the
angular scale. Regression coefficients reflecting decreases in survival
measured in the angular scale also were reexpressed as % decrease in surival/%
change in concentration (or per 10 ppb increase when appropriate). Since the
retransformation of the regression coefficients is non-linear (i.e. depends
upon the level of survival) we usually chose the 50% survival as the point at
which to reexpress as % decrease. However, in the data from the drilling muds
that survival percentage was not achieved even the controls so the point of
reexpression was chosen at 50% concentration (approximately 8-10% survival).
-------
SECTION 4
EFFECTS OF SOLUBLE FRACTIONS OF USED LIGHT-WEIGHT LIGNOSULFONATE TYPE MUD ON
THE COMPLETE LARVAL DEVELOPMENT OF CRABS, Rhithropanopeus harrisii AND
Callinectes sapidus
INTRODUCTION
With the increase in number of oil wells and new leases for oil
exploration along the Atlantic Coast, it is natural that the public would be
concerned about the effect of the discharge of drilling fluids on marine fauna.
Accordingly, this investigation will focus on the effect of a low-density
lignosulfonate drilling mud with ferrochrome added (No. 4 mud) on the complete
larval development of two crabs, a mud crab, Rhithropanopeus harrisii (Gould)
and the blue crab, Callinectes sapidus Rathbun. The Jay Exxon well drilling
fluid to be tested had a density of 9.1 Ib/gal and came from a land based well
in Florida. The samples of No. 4 mud were taken at a depth of 3735.9 m (12,257
feet) and were provided for this investigation by the United States
Environmental Protection Agency, Environmental Research Laboratory, Gulf
Breeze, Florida. Although the well was land based, it was believed that the
chemical components and physical characteristics of the drilling fluid were
similar to those in offshore wells.
Extensive tests on the effects of four to five used lignosulfonate
drilling fluids on warm- and cold-water organisms from the Gulf of Mexico have
been made by Neff et_ al^. (1980, 1981), Carr et_ al_. (1980), McCulloch et al.
(1980) and Gerber et_ al. (1980). They used mud supplied by the American
Petroleum Institute. For comparative purposes, therefore, there is pertinent
information concerning the relative toxicity of spud mud with a density of 9.2
Ib/gal, a low-density lignosulfonate (LWLS) drilling fluid with a density of
10.0 Ib/gal, and a high density lignosulfonate (HWLS) with a density of 17.4
Ib/gal.
Investigators have evaluated the toxicity of five components of whole mud
following the classification originally proposed by Neff et_ al. (1980). The
mud aqueous fraction (MAF) and suspended particulate phase (SPP) are two
fractions which have been most intensively investigated in toxicity tests and
are the two fractions tested in this investigation. The 100% MAF contains
water-soluble and fine particulate fractions of 100,000 ppm mud in water. SPP
resembles MAF but contains a higher concentration of particulates and a lower
concentration of volatiles (Neff e_t_ aj^. , 1980). These two fractions are found
in the upper plume of discharge and remain in the water column longer than
other fractions (Ayers et al., 1980) and, hence, may be the fractions which
might be expected to affect larvae of marine organisms.
10
-------
Research on the environmental effects of drilling fluids is difficult
because of the complexity of the fluids and the lack of homogeneity of samples
from the same depth. Ninety percent of the major drilling fluid components are
barite, primarily barium sulfate, clays such as sodium bentonite and calcium
bentonite, lignosulfonates and lignite (Perricone, 1980). The fluids, however,
may include additives, such as pH-control substances, bactericides, calcium
removers, corrosion inhibitors, deformers, emulsifers, filtrate, reducers,
flocculants, foaming agents, lubricants, shale-control inhibitors, thinners,
dispersants, viscosifiers, and weighting agents (Richards, 1979). Furthermore,
produced waters generally contain appreciable concentrations of inorganic salts
and trace minerals compared to normal seawater.
Although most of the drilling mud is recovered and recycled, tons may be
discharged as a turbity plume in the surface layers of water. Ray and Meek
(1980) reported that 2,854 barrels of mud and cuttings were discharged,
representing about 863,390 kg of solids, over an 85 day period on Tanner Bank,
California. Hence, 10,000 kg or 10 metric tons would be discharged per day,
95% of which would be normal cuttings and 4% drilling mud.
At the present time, the impact of drilling fluids is incompletely known.
Most investigations have been acute toxicity tests and they show that drilling
fluids have little or no effect on adult marine organisms, but they reveal that
larvae and juvenile invertebrates are sensitive to exposure to drilling fluids
(Neff et_ al_., 1980, 1981; Carr et_ al_. , 1980; Lees and Houghton, 1980; Gerber et_
al. , 1980; Carls and Rice, 1981).
Although most of the components of drilling fluids are apparently
nontoxic, some of the additives, such as the bactericide, pentachlorophenol
(Tagatz et al. , 1977), as well as paraformaldehyde, caprylalcohol and some
surfactants are especially toxic (Sprague and Logan, 1979). Trace metals found
within drilling fluids may also have detrimental effects (Liss et al. , 1980;
Hrudey and Eng, 1979).
Sprague and Logan (1979) investigated separate and joint toxicity to
rainbow trout of substances used in drilling fluids for oil exploration in the
MacKenzie delta. When seven most toxic components were added singly to a
simulated fluid about half of the combinations showed joint action. In several
preparations, however, antagonism apparently occurred. The research of Sprague
and Logan (1979) illustrates the chemical complexity of one drilling mud used
in the MacKenzie delta. The potential effects of chemicals, however, will
differ with each type of mud in well drilling operations, with cutting
composition related to type of substrate drilled, with well depth, temperature
generated, etc. (Richards, 1979).
As far as known, there have been no investigations on the effect of the
soluble fractions of whole mud on the complete larval development of crabs,
such as the suspended particulate phase (SPP) and mud aqueous fraction (MAF),
which would be found in the upper plume of discharges.
The objectives of the current investigation were, therefore, to determine
the range of concentrations of MAF and SPP of used lignosulfonate type mud with
11
-------
ferrochrome added (No. 4 mud) which would affect swimming behavior, survival
and duration of development of the mud crab, Rhithropanopeus harrisii, and the
blue crab, Callinectes sapidus, from the time of hatching until the 1st crab
stage is reached. Further objectives were to ascertain the concentrations of
MAP and SPP which were nontoxic, sublethal and acutely toxic, the sublethal
effects, and the most sensitive periods of development of the two species.
RESULTS
Survival and Duration of Rhithropanopeus harrisii Larvae
TABLE 2 gives the percent survival from hatching to megalopa and to 1st
crab stage and the mean duration in days of zoeal and megalopa development, as
well as the time from hatching to 1st crab stage of series Rh-I, Rh-II and
Rh-III reared in seawater control, four concentrations of MAF and four
concentrations of SPP- TABLE 3 gives average survival and duration data of the
three series reared from hatching to 1st crab stage. From the results
tabulated, it can be noted that the percent survival to megalopa and to 1st
crab stage is 90% or over in seawater control and in all concentrations of MAF
and SPP- There is no consistent reduction in survival in concentrations of MAF
or SPP compared to survival in seawater control. TABLE 3 also shows that the
average duration in days from hatching to 1st crab stage is fairly uniform in
seawater control and in all concentrations of MAF and SPP- Percent mortality
in developmental stages of each of three series of R. harrisii reared in
seawater control and different concentrations of MAF and SPP is listed in TABLE
4, whereas TABLE 5 gives the average percent mortality of the three series.
Survival of Callinectes sapidus Larvae
The percent survival from hatching to megalopa and to 1st crab for C.
sapidus is given for replicate series reared in different concentrations of MAF
and SPP in TABLE 6. The average percent survival of all series reared in
seawater control and four concentrations of MAF and SPP is listed in TABLE 7.
There was little difference between C_. sapidus survival to megalopa and to 1st
crab in 5% MAF and seawater control, but survival in 5% SPP was less than in
the control. There was differential survival, however, from 5% MAF and SPP to
100% MAF and SPP.
Statistical Analysis of C. sapidus Survival in MAF
Statistical analysis idicated that:
(i) Survival to megalopa (TRFSZ) and to first crab (TRFSC)
are both linearly related to % MAF (CONG) over the entire
range 0-100%.
(ii) The two lines (Figure 1) are nearly parallel but statistical
tests indicate a significant difference in the slopes
(b values).
12
-------
The summary equations are:
Zoeal : TRFSZ = 37.9 - 0.3585 * CONC
b = -0.3585 + 0.0203 Degrees/% change in MAP
or = -3.585 + 0.203 Degrees/10% change in MAF
or approximately a 4% decrease in survival of
zoea/10% increase in MAF (measured @ 50% CONC).
First Crab : TRFSC = 33.5 - 0.3303 * CONC.
b = -0.3303 + 0.0660 Degrees/% change in MAF
or = -3.303 + 0.660 Degrees/10% change in MAF
or approximately 3% decrease in survival to first
crab/10% increase in MAF (measured @ 50% CONC).
Statistical Analysis of C. sapidus in SPP
Statistical analysis indicated that:
(i) Survival to megalopa (TRFSZ) and to first crab (TRFSC) are both
linearly related to % SPP (CONC) in the range 0-50%. There was no
survival at the 100% CONC of SPP- Extrapolation estimates of total
zoeal mortality at 75% SPP and of total first crab mortality at 70%
SPP-
(ii) The two lines (Figure 2) are parallel since no significant
difference was found between the two slopes. The single slope was
estimated from the pooled data.
The summary equations are:
Zoeal : TRFSZ = 35.9 - 0.4722 * CONC.
First Crab : TRFSC = 33.1 - 0.4722 * CONC.
b = -0.4722 + 0.1142 Degrees/% change in SPP
or = -4.722 + 1.142 Degrees/10% change in SPP
or approximately 5% decrease in survival (to
either stage)/10% increase in SPP (measured @
50% CONC).
Duration of C. sapidus in Larval Development
TABLE 6 gives the percent duration in days through zoeal and megalopa
development as well as duration from hatch to 1st crab of each of three series
(CsI-III) of Callinectes sapidus reared in seawater control and different
concentrations of MAF and SPP. TABLE 7 lists the average duration in days of
zoeal and megalopa development and duration from hatch to crab of the three
13
-------
TABLE: 2. PERCENT SURVIVAL AND DURATION IN DAYS THROUGH ZOEAL AND MEGALOPA DEVELOPMENT OF THREE
SERIES (Rh I-III) OF Rhithropanopeus harrisii REARED IN SEAWATER CONTROL AND IN 4 CONCENTRATIONS OF
MUD AQUEOUS FRACTION (MAE) AND 4 CONCENTRATIONS OF SUSPENDED PARTICULAR PHASE 'SP0' OF USED
LIGNOSULFONATE TYPE MUD (NO. 4 MUD).
Culture Media
Salinity 200/00
Temp. 25°C
Seawater Control
5% MAF
25% MAF
50% MAF
100?; MAF
5% SPP
25% SPP
50% SPP
100% SPP
Initial No.
of larvae
per series
RhI-50
RhII-50
RhIII-50
RhI-50
RhII-50
RhIII-50
RhI-50
RhII-50
RhIII-50
RhI-50
RhII-50
RhIII-50
RhI-50
RhII-50
RhIII-50
RhI-50
RhII-50
RhIII-50
RhI-50
RhII-50
RhIII-50
RhI-50
RhII-50
RhIII-50
RhI-50
RhII-50
RhIII-50
% Survival to
Meqalopa
100
92
96
96
100
100
94
92
96
94
96
96
92
94
90.
96
90
98
94
96
96
96
96
90
94
96
96
Mean Duration of Deveiooment in davs
1st crab Zoea
98
90
94
94
98
100
92
92
96
92
96
96
92
90
90
96
90
96
94
96
96
94
96
90
94
96
96
12.5
10.9
12.2
12.5
10.5
11 . 1
12.4
10.6
12.0
12.4
10.7
12.1
12.7
10.9
12.3
12.4
11.2
11.8
13.1
11.1
11.8
12.7
11.2
11.9
13.0
11.5
11.9
Meaaiopa
11. 1
6.7
6.0
10.8
b.6
5.6
O c
6.0
5.6
6.7
6.0
5.3
8.5
5.5
5.3
1C."
6.3
6.0
Q.9
6.6
<>.6
9.5
6.0
5.9
8.8
6.2
5.6
Hatch to 1st Crab
23.6
17 .6
18.2
2^.3
17.1
io . 7
21 .9
] t, . 6
1~ .6
1 '
- J. . i
16.7
1 / . ^
21.2
16.4
17.6
23.3
17.5
i^.g
23.0
17.7
17.4
22.2
17.2
17.8
21.8
17.7
17.5
-------
TABLE 3. AVERAGE PERCENT SURVIVAL AND AVERAGE DURATION IN DAYS OF ZOEAL AND MEGALOPA DEVELOPMENT
OF THREE SERIES (Rh I-III) OF R. harrisii REARED IN SEAWATER CONTROL, 4 CONCENTRATIONS OF MAF AND
4 CONCENTRATIONS OF SPP
Culture Media
Salinity 20°/°°
Temp. 25°C
Seawater Control
5% MAF
25% MAF
50% MAF
100% MAF
5% SPP
25% SPP
50% SPP
100% SPP
OF USED LIGNOSULFONATE TYPE MUD (NO. 4 MUD).
Initial No.
of larvae % Survival to Mean Duration of Development in days
per series Megalopa 1st crab Zoea Megalopa Hatch to 1st Crab
RhI-50
RhII-50 96.0 94.0 11.9 7.9 19.8
RhIII-50
RhI-50
RhII-50 98.7 97.3 11.4 7.7 19.0
RhIII-50
RhI-50
RhII-r-0 94.0 93.3 11.7 7.0 18.7
RhIII-50
RhI-50
RhII-50 95,3 94.7 11.7 6.7 18.4
RhIII-50
RhI-50
RhII-50 92.0 90.7 11.9 6.4 18.4
RhIII-50
RhI-50
RhII-50 94.7 94.0 11.8 7.7 19.4
RhIII-50
RhI-50
RhII-50 95.3 95.3 12.0 7.4 19.4
RhIII-50
RhI-50
RhII-50 94.0 93.3 11.9 7.1 19.0
RhIII-50
RhI-50
RhII-50 95.3 95.3 12.1 6.9 19.0
RhIII-50
15
-------
CONTROL AND DIFFERENT CONCENTRATIONS OF MAF AND SPP bF USED LIGNOSUIFHNATL T V^E MUD
Media
Seawater Control
5% MAF
25% MAF
50% MAF
100% MAF
5% SPP
25% SPP
50% SPP
100% SPP
Series
RhI-50
RhII-50
RhIII-50
RhI-50
RhII-50
RhIII-50
RhI-50
RhII-50
RhIII-50
Rhl-50
RhII-50
RhIII-50
RhI-50
RhII-50
RhIII-50
RhI-50
RhII-50
RhIII-50
RhI-50
RhII-50
RhIII-50
RhI-50
RhII-50
RhIII-50
RhI-50
RhII-50
RhIII-50
1
0
2
0
2
0
0
6
4
2
4
2
0
6
0
2
4
6
0
6
0
2
4
4
4
4
2
0
Zoeal Stages
II III
0
4
2
2
0
0
0
2
0
2
2
0
0
2
0
0
2
0
0
2
0
0
0
0
2
0
0
0
2
0
0
0
0
0
2
2
0
0
0
0
0
0
0
2
0
0
2
0
0
0
n
0
2
0
IV
0
0
2
0
0
0
0
0
0
0
0
4
2
4
8
0
0
2
0
0
o
0
0
6
0
0
4
Megajooa
2
2
"3
0
i
L
;";
o
0
0
2
0
0
0
A
0
0
0
^>
0
n
0
•?
0
n
0
0
0
\n. « MUD).
Total
2
10
6
6
2
0
8
8
4
8
4
4
8
10
10
4
10
4
6
4
4
6
4
10
6
4
4
16
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TABLE 5. AVERAGE PERCENT MORTALITY IN DEVELOPMENTAL STAGES OF R[. harrisii (Rh I-III) REARED IN
SALTWATER CONTROL
Media
Seawater Control
5% MAF
25% MAF
50% MAF
100% MAF
5% SPP
25% SPP
50% SPP
100% SPP
AND DIFFERENT CONCENTRATIONS
Series 1
RhI-50
RhII-50 0.7
RhIII-50
RhI-50
RhII-50 0.7
RhIII-50
Rhl-50
RhII-50 4.0
RhIII-50
RhI-50
RhII-50 2.0
RhIII-50
RhI-50
RhII-50 2.7
RhIII-50
RhI-50
RhII-50 3.3
RhIII-50
RhI-50
RhII-50 2.7
RhIII-50
RhI-50
RhII-50 4.0
RhIII-50
RhI-50
RhII-50 2.0
RhIII-50
OF MAF AND SPP OF USED LIGNOSULFONATE TYPE MUD.
Zoeal Stages
II III IV Megalopa Total
2.0 0.7 0.7 2.0 6.0
0.7 0 0 1.3 2.7
0.7 1.3 0 0.7 6.7
1.3 0 1.3 0.7 5.3
0.7 0 4.7 1.3 9.3
0.7 0.7 0.7 0.7 6.0
0.7 0.7 0.7 0 4.7
0 0 2.0 0.7 6.7
0.7 0.7 1.3 0 4.7
17
-------
10
20
30 40 50
% CONCENTRATION OF MAP
70
EO
90
100
Figure 1. Effect of percent of mud aqueous fraction O1AF)
of used light weight lignosulfonate type mud on
survival of C_. sapidus larvae.
Hatch to megalopa x x
Hatch to 1st crab o o
18
-------
.40-^
o-
UJ
S
a:
O
SZ = 35.87- 0.4722 *conc
1st CRAB
SC = 33.08 - 0.4722 »conc
0+-
0
10
20
30 40 50 60
% CONCENTRATION OF SPP
70
80
90
100
Figure 2. Effect of percent of suspended particulate phase
(SPP) of used light weight lignosulfonate type
mud on survival of C_. sapidus larvae.
Hatch to megalopa x x
Hatch to 1st crab o o
19
-------
series of C_. sapidus reared in seawater control and in four concentrations of
MAF and SPP. There is no significant difference in duration in zoeal
development and in hatch to crab in seawater control and the concentrations of
MAF and SPP employed.
Mortality of C. sapidus by Larval Stage
Callinectes sapidus may pass through seven, occasionally eight, zoeal
stages before it molts into a megalopa, a ninth stage of development. In an
effort to determine if larvae in one or more of the nine developmental stages
of C_. sapidus were particularly sensitive to different concentrations of MAF
and SPP, a record of deaths by stages was made for larvae from each of three
crabs, CsI-III, which had been reared in seawater control and four
concentrations of MAF and SPP (TABLE 8). Average percent mortality of larvae
in developmental stages of C^. sapidus of all series reared in seawater control
and different concentrations of MAF and SPP is given in TABLE 9.
Statistical Analysis of C. sapidus Cumulative Mortality by Stages When Reared
in MAF
The results illustrated in Figure 3 show the effect of MAF concentration
on the mortality of larvae at each stage of development. The percent
mortalities on the graph were obtained from the means of the transformed
variable. Mortality of larvae in 5 and 25% MAF was not significantly different
from mortality in the control in any of the nine developmental stages, but
mortality of larvae reared in 50 and 100% MAF was significantly different from
the control in every developmental stage. Although larvae in zoeal stage I
were most sensitive, but larvae in zoeal stage II were also very sensitive, for
significant increases in mortality over the previous stage occurred in this
stage in all media (Figure 3).
Statistical Analysis of C. sapidus Cumulative Mortality by Stages When Reared
in SPP
Mortality of larvae in 5% SPP was not significantly different from
mortality in the control in any of the nine developmental stages, but mortality
of larvae reared in 50 and 100% SPP was significantly different from the
control in every developmental stage. In zoeal stage I 25% SPP was
significantly different from the control at the 0.05 level. As in the MAF
experiment, larvae in zoeal stage I were most sensitive (Figure 4), but larvae
in zoeal stage II were also very sensitive, for significant increases in
mortality over the previous stage occurred in zoeal stage II in all media
(Figure 4).
Larval Behavior: Swimming of C. sapidus Larvae Upon Exposure to Drilling Mud
MAF and SPP
Blue crab larval behavior is affected by exposure to MAF and SPP with the
general effect being a decline in swimming speed (Table 10). A significant
reduction in speed is only observed in the 100% solution of MAF. However, all
solutions of SPP cause a significant decline. The highest two concentrations
cause the greatest reduction.
20
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TABLE 6. PERCENT SURVIVAL AND DURATION IN DAYS THROUGH ZOEAL AND MEGALOPA DEVELOPMENT OF THREE
SERIES (Cs I-III) OF Callinectes sapidus REARED IN SEAWATER CONTROL AND IN DIFFERENT
CONCENTRATIONS OF
Culture Media
Salinity 300/°°
Temp. 25°C
Seawater Control
5% MAF
25% MAF
50% MAF
100% MAF
5% SPP
25% SPP
50% SPP
100% SPP
MAF AND SPP OF USED
Initial No.
of larvae
per series
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
LIGNOSULFONATE
% Survival
TYPE MUD
to
Megalopa 1st crab
70
30
14
66
36
10
48
24
10
4
18
8
2
0
0
46
32
18
14
26
8
2
4
6
0
0
0
50
22
14
54
32
6
42
20
8
4
14
8
0
0
0
40
26
16
10
20
8
2
4
6
0
0
0
Mean
Zoea
30.0
41.9
32.8
32.3
38.9
33.2
32.3
35.0
37.0
34.0
39.9
33.8
34.0
0
-
32.4
34.1
32.1
34.1
41.5
33.0
32.0
39.0
36.3
_
_
Duration of
Megalopa
7.5
8.0
7.8
8.2
7.2
6.5
7.7 ,
6.4
7.3
6.0
7.4
6.5
_
-
-
7.5
6.8
7.7
7.0
7.8
7.0
7.0
9.0
7.3
_
Development in days
Hatch to 1st Crab
37.2
49.7
40.7
40.7
45.1
39.8
39.7
44.5
44.5
40.0
47.4
40.3
_
-
-
39.5
44.3
40.0
41.2
49.9
40.0
39.0
48.0
43.7
_
21
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TABLE 7. AVERAGE PERCENT SURVIVAL AND AVERAGE DURATION IN DAYS OF ZOEAL AND »-tGALDPA DEVELOPMENT
OF THREE SERIES (Cs I-III) OF C. sapidus IN SEAWATER CONTROL AND DIFFERENT CONCENTRATIONS OF MAF
AND SPP OF USED L1GNOSULFONATE TYPE MUD.
Culture Media
Salinity 30°/°0
Temp. 25°C
Seawater Control
5% MAF
25% MAF
50% MAF
100% MAF
5% SPP
25% SPP
50% SPP
100% SPP
Initial No.
of larvae % Survival to Mean Duration or Development in davs
per series Megalopa 1st crab Zoea Meqaiopa Hatch to 1st Crab
Csl-50
CsII-50 37.3 28.0 34.9 7.8 42.5
CsIII-50
Csl-50
CsII-50 38.0 31.3 34.8 7.3 41.9
CsllI-50
Csl-50
CsII-50 27.3 23.3 34. R ".1 42.9
CsIII-50
Csl-50
CsII-50 10.0 8.7 35.9 6.6 42.6
CsIII-50
Csl-50
CsII-50 0.7
CsIII-50
Csl-50
CsII-50 31.3 26.7 32.4 7.3 41.3
CsIII-50
Csl-50
CsII-50 15.3 12.0 36.2 7.3 43.7
CsIII-50
Csl-50
CsII-50 4.0 4.0 35.8 '.8 43.6
CsIII-50
Csl-50
CsII-50 00--
CsIII-50
22
-------
TABLE 8. PERCENT MORTALITY IN DEVELOPMENTAL STAGES OF THREE SERIES (Cs I-III) OF £.
REARED IN SALTWATER CONTROL AND DIFFERENT CONCENTRATIONS OF MAF AND SPP OF USED LIGNOSULFONATE
TYPE MUD.
Media
Seawater Control
5% MAF
25% MAF
5 OX MAF
100% MAF
5% SPP
25% SPP
503 SPP
100% SPP
Series
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
I
16
28
32
28
18
54
42
36
32
74
58
74
98
90
96
30
34
62
60
46
52
94
74
60
100
88
92
II
8
28
42
4
24
32
8
26
4?
12
18
12
0
10
4
14
26
16
24
18
20
2
20
26
0
12
6
III
2
12
8
0
8
2
0
6
10
8
4
2
0
0
0
4
2
2
2
8
14
0
2
6
0
0
2
Zoeal
IV
0
2
4
0
8
0
2
2
6
0
2
0
0
0
0
2
0
0
0
2
2
2
0
0
0
0
0
Stages
V
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
VI
0
0
0
0
2
0
0
2
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
VII
2
0
0
0
2
2
0
2
0
0
0
2
0
0
0
0
2
0
0
0
0
0
0
2
0
0
0
VIII
2
0
0
2
0
0
0
2
0
2
0
2
0
0
0
4
4
0
0
0
4
0
0
0
0
0
0
Megalopa
20
8
0
12
4
4
6
4
2
0
4
0
2
0
0
6
6
2
4
6
0
0
0
0
0
0
0
Total
50
78
86
46
68
94
58
80
92
96
86
92
100
100
100
60
74
84
90
80
92
98
96
94
100
100
100
23
-------
TABLE 9. AVERAGE PERCENT MORTALITY IN DEVELOPMENTAL STAGES OF THREE SERIES (Cs I-1II; OF £.
sapidus REARED IN SALTWATER CONTROL AND DIFFERENT CONCENTRATIONS OF MAF AND SPP Hr USED
LIGNOSULFONATE TYPE MUD.
Zoeal Stages
Media Series I II III IV V VI VII VIII Megaiopa Total
Seawater Control CsI-50
CsII-50 25.3 26.0 6.7 2.0 0 0 0.7 0.7 9.3 71.3
CsIIl-50
5% MAF CsI-50
CsII-50 33.3 20.0 3.3 2.7 0.7 0.7 1.3 0.7 6.7 69.4
csiii-50
25% MAF CsI-50
CsII-50 36.7 25.3 5.3 3.3 0 0.7 0.7 0.7 4.0 76.7
CsIII-50
50% MAF Csl-50
CsII-50 68.7 14.0 4.7 0.7 0 0 0.7 1.3 1.3 91.3
CsIII-50
100% MAF CsI-50
CsIII-50 94.7 4.7 0 0 0000 0.7 100
CsIII-50
5% SPP CsI-50
CsII-50 42.0 18.7 2.7 0.7 0 0.7 0.7 2.7 4.7 72.7
CsIII-50
2555 SPP CsI-50
CsII-50 52.7 20.7 8.0 1.3 0 00 1.3 3.3 87.3
CsIII-50
50% SPP CsI-50
CsII-50 76.0 16.0 2.7 0.7 0 0 0.7 0 0 96
CsIII-50
100% SPP CsI-50
CsII-50 93.3 6.7 6.7 0 0000 0 100
CsIII-50
24
-------
100-
fe fe-
100%-
90-
80-
70-
60-
< 5O-
50%'
<
^
D
: 40-
-0%
20-
10-
HI
IV
STAGE
VI
VIII
Figure 3. Effect of MAF of used light-weight
lignosulfonate type mud on mortality by stages
of development 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
-------
100-
90-
80-
70-
•60-
ir
O
"50-
40-i
30-
20-
1O-
10O%
50%'
0%'
IV
STAGE
VI
VII
Figure 4. Effect of SPP of used light-weight lignosulfonate type
mud on mortality by stages of development of £. 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)
26
-------
TABLE 10. SWIMMING SPEED (mm/min) OF CONTROL _C. sapidus FIRST ZOEAE AND THOSE TREATED WITH
DIFFERENT CONCENTRATIONS OF MAF AND SPP. THE MEAN (M) AND STANDARD DEVIATION (SD) ARE SHOWN.
THE SAMPLE SIZE FOR EACH CONDITION IS 60. * INDICATES P < 0.02 STATISTICAL DIFFERENCE BETWEEN
CONTROL LARVAE AND EXPOSURE TO A PARTICULAR CONCENTRATION, WHILE ** IS P < 0.01 AND *** P <
0.001.
Condition M SD
control 100.2 28.9
5% MAF 94.6 33.2
25% MAF 94.9 36.7
50% MAF 89.6 39.5
100% MAF 60.7** 24.7
5% SPP 81.9** 35.0
25% SPP 86.0* 31.6
50% SPP 60.2*** 24.3
100% SPP 61.2*** 25.3
27
-------
DISCUSSION
Survival
Mud aqueous fractions (MAP) and suspended particulate phase (SPP) of used
low density lignosulfonate type drilling fluid were nontoxic to the development
of Rhithropanopeus harrisii from the time of hatching to the 1st crab stage.
TABLES 2 and 3 reveal that survival was 90% percent or greater in seawater
control and in concentrations of 5 to 100% MAF and SPP. In a similar
experiment using Callinectes sapidus, there was differential survival from
hatching to megalopa and to 1st crab stage in concentrations of 5 to 100" MAF
and SPP (TABLES 6 and 7; Figures 1 and 2). The sensitivity of R. harrisii and
C_. sapidus larvae to a pollutant, such as mirex, may be similar (Bookhout et
al., 1972; Bookhout and Costlow, 1975), or very different; sublethal
concentrations of Kepone to II. harrisii larvae ranged from 35 to 80 ppb,
whereas the sublethal concentrations to C. sapidus larvae ranged from 0.1 to
1.0 ppb (Bookhout et al. , 1980). We do not know why R_. harrisii are so such
more resistant to Kepone and MAF and SPP of low density lignosulfonate.
In chronic toxicity studies of the larval development of crabs, sublethal
concentrrtions of a pollutant are arbitrarily defined as those ir. which there
is a reduction in survial 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 sta?e (Epifanio,
1971; Bookhout and Costlow, 1975). Since survival to the 1st crab ^taee in 5%
MAF was somewhat better than in seawater control, 5% is considered nontoxic.
Twenty-five percent MAF, 5 and 25% SPP are sublethal, and concentrations of 50
and 100% MAF and SPP are acutely toxic.
Survival to 1st crab was better in MAF than in SPP- In 5% (5,000 ppm),
25% (25,000 ppm), 50% (50,000 ppm) and 100% (100,000 ppm) MAF survival was
31.3, 23.3, and 8.7% and 0 respectively, whereas in 5% (5,000 prm), 25"', (25,000
ppm), 50% (50,000) ppm and 100% (100,000 ppm) SPP, it was 26.7, 12.0, anc 4.0%
and 0 respectively (TABLES 1 and 2).
Behavior
Blue crab larval behavior was affected by exposure to MAF and SPP with the
general effect being a decline in swimming speed (TABLE 10). A significant
reduction in speed was only observed in 100% MAF, whereas all solutions of SPP
caused a significant decline with the highest two concentrations causing the
greatest reduction. Carls and Rice (1981), in a similar study of stage 1 crab
and shrimp zoeae exposed to fractions of used drilling fluids from Alaska,
reported that behavioral observations were a more sensitive indicator of mud
toxicity than mortality. The effective concentrations (EC50 at 144 h), as
measured by the cessation of swimming, could be determined at lower
concentrations than LC50 at 144 h. Thus, we are of the opinion that a
significant change in the rate of swimming indicates sublethal stress.
28
-------
Chronic vs Acute Toxicity Tests
As far as known, no chronic studies have been made on the effect of MAF
and SPP of whole drilling fluids on the complete larval development of any
marine organism. In this investigation, it was used because, in our opinion, a
test covering the entire larval development of crabs would give a better
evaluation of the possible toxicity of drilling fluids in the field than acute
toxicity study of 96 h. A chronic toxicity test would include all periods of
molting when crustacean larvae are known to be very sensitive to toxic
substances. Furthermore, if records are kept of mortality at each stage of
development, it is possible to determine which stage or stages in the larval
period are particularly sensitive (Figures 3 and 4). An acute toxicity test of
96 h would not have the advantages given for chronic tests and would not
include the particuarly sensitive 1st molt in zoeal development of a blue
crab (Bookhout and Costlow, 1975).
Numerous acute toxicity studies have been made on the effects of MAF and
SPP of whole drilling fluids on adult marine organisms and some have been done
on individual larval stages. In these tests, the median lethal concentration,
LC50, is defined as that concentration lethal to 50% of the test organism
within a specified test period, usually 96 h. In this study, the results of
acute toxicity studies (96-h LC50) could not be compared directly with the
results of the long term chronic studies because less than 50% of £. sapidus
survived in seawater control. Generalizations may be made, however, from both
types of studies.
There is sufficient evidence in the literature to conclude that adult
marine invertebrates are generally not affected by any type of drilling fluid
but juveniles and larvae are. In a comparative study of spud mud and three
types of lignosulfonate muds of different densities, Neff et al. (1980)
reported that used spud mud was nontoxic to all larval and adult organisms
tested. This mud is the type used near the surface and contains aqueous
solutions of bentonite clay and some barite which are considered nontoxic.
Aqueous extracts of the three other lignosulfonate drilling fluids are similar
in their acute toxicity to larvae and juvenile crustaceans and were generally
nontoxic to adult marine organisms. Ninety-six hour LC50 for 1st zoeae of
grass shrimp, Palaemonetes pugio ranged from 18 to 35% (18,000 to 35,000 ppm)
MAF of three types of lignosulfonate mud. The acute toxicity measured as 96 h
LC50 of SPP of used high density lignosulfonate of 1 day (1st zoeae), 5 days
(3rd zoeae) and 10 days (4th and 5th zoeae) was 11.8, 13.2 and 11.7%
respectively. Thus SPP of high-weight lignosulfonate was more toxic than MAF
of chrome lignosulfonate, mid-weight and high-weight lignosulfonate, as found
in this investigation when C^. sapidus larvae were exposed to similar fractions
of used light-weight lignosulfonate.
Carr et_ aJ^. (1980) found that the 96 h LC50 values for one-day old
oppossum shrimp, Mysidopsis almyra, exposed to MAF of used chrome
lignosulfonate drilling fluid which was static but replaced daily was 27%
(27,000 ppm), as contrasted to 42% (42,000 ppm) when the culture was static but
not changed in 96 hours. They believed the difference indicates that some
toxic volatile components were lost during static exposure. They also found
that as mysids aged from one to 14 days their tolerance to MAF of used chrome
29
-------
lignosulfonate increased. In this investigation, as shown in TABLES A and 5
and Figures 3 and 4, the most sensitive zoeae of C. sapidus, as revealed by
percent mortality, were in stages I and II. In the remainining zoeal stages,
there was less mortality indicating that larvae became more tolerant in later
stages, or that the more susceptible portion of the population was eliminated
first.
Gerber et_ £l. (1980) studied the effects of five types of used drilling
fluids on 13 species of marine animals from the coastal Gulf of Maine waters.
Except for spud mud, they were surprised'to find that there was little
difference in the toxicity of the other four muds since they contain different
amounts of toxic substances. As in other investigations they found adult
organisms exhibited little or no mortality to the highest concentrations of MAF
drilling fluids. By contrast the acute toxicity measured as 96 h LC50 MAF of
used light-density lignosulfonate drilling fluid of stage V zoeae of the
American lobster, Homarus americanus, was 5% (5,000 ppm). Zoeal stage I of the
northern shrimp, Pandalus borealis, exposed to used medium-density
lignosulfonate had a 96-h LC50 of MAF and filtered MAF of 17 and 19%
respectively, whereas in high-density lignosulfonate the 96-h LC50 of MAF and
filtered MAF was 65 and 55%, respectively.
Environmental Implications
To determine the possible hazards of MAF and SPP of lignosulfonate on test
larvae, such as those of Callinectes sapidus, it is necessary to know the
extent of dispersion of the upper of two turbidity plumes from the point of
discharge in relation to the rate and volume of discharge. MAF and SPP are
fractions of whole mud that are incorporated in the upper turbidity plume. To
correlate laboratory findings to field conditions, it is important to ascertain
the extent of dispersion of the highest concentrations around the point of
discharge and the gradient of reduction during dispersion peripherally until
the area of background level is reached.
Ray and Meek (1980) studied eight discharge plumes for 85 days from an
offshore exploratory well on Tanner Bank. Six resulted from mud discharges of
10 to 754 barrels per hour and the remaining two resulted froc cutting
discharges. There was an average initial dilution of 500-1000:1 of total
suspended solids from lightly treated seawater lignosulfonate mud plumes within
the first 3 m from the point of discharge. An additional 100:1 dilution
occurred within the next 100 m and at 100 m suspended solids were reduced to
background levels of 1.0 mg/1. Metal concentrations reached background levels
at 100 to 150 m from the point of discharge. Gerber et_ al_. (1980) estimated if
within one to three meters of the discharge source mud components were diluted
from 500:1 to 1000:1 under discharge conditions of 10 to 15 bbl/h, 100% MAF
(100,000 ppm) and 5% MAF (5,000 ppm) would persist within a couple of meters
from the discharge pipe. Occasionally high mud discharge rates might occur for
less than an hour. Ayers et_ al. (1980) reported that under high discharge
rates of 275 to 1000 bbl/h background levels of suspended solids in the upper
turbidity plume were reached 600 to 1500 m from the source. Under short
periods, 5% (5,000 ppm) MAF might represent conditions 20 to 35 m from the
source. Petrazzuolo (1981 draft unpublished) reported that for almost all of
30
-------
the species and fluids tested to date, acute lethal effects of drilling fluids
would not be expected further than 15 m from one discharge.
If drilling operations were taking place in the shallow shelf waters off
the southeast coast of United States or the coast of Gulf of Mexico, crab
larvae probably would be in the vicinity of operations from late spring to
early fall. Ovigerous blue crabs, Callinectes sapidus, are known to migrate
from estuaries to the mouths of rivers and beyond before they shed
approximately 2,500,000 zoeae per crab. Their larvae would become a part of
the plankton and would be distributed by currents of shallow shelf waters off
the southeast coast of the United States and Gulf of Mexico. Other crabs which
belong to the same subfamily (Portuninae) as the commercial blue crab, such as
other species of Callinectes, Portunus and Araeneus, also shed their larvae off
the southeast and Gulf states for this is where the adults live. Although
these larvae could be in the vicinity of drilling operations and might be found
in the upper turbidity plume, the chances of many of the larvae remaining in
the 3 m highly toxic zone or even in the 15 m intermediate toxic zone around
the discharge source long enough to suffer mortality are very remote. If by
chance a few 1st or 2nd stage zoeae of C_. sapidus in the process of molting
happened to be entrained in the near zone area of discharge, they might be
killed or receive an irreversible stress, for in this investigation it was
found that zoeae in stage I and II were very sensitive, especially at the time
of molting.
31
-------
SECTION 5
EFFECTS OF HEXAVALENT CHROMIUM ON THE COMPLETE LARVAL DEVELOPMENT OF CRABS,
Rhithropanopeus harrisii AND Callinectes sapidus
INTRODUCTION
One of the trace metals in drilling fluids which may have a detrimental
environmental effect is chromium. The toxicity of chromium to marine organisms
will vary with valence state, pH and oxidation states. Hexavalent chromium
(Cr &) is stable in seawater. It often appears as a soluble chromate or
dichromate, powerful oxidants which can readily penetrate biological membranes
and irritate cells (Mertz, 1969). Hexavalent chromium, as chromic oxide,
chromate or dichromate, reacts with organic matter in acidic solution, leading
to the trivalent form (Cr ). The trivalent form is associated chiefly
with particulate matter, such as clay, which suggests that organic particulate
matter may reduce and bind the hexavalent form in solution [National Academy of
Science (NAS), 1974; Curl e_t_ a_l_. , 1965]. Hexavalent chromium is much more
toxic to organisms than trivalent chromium, in part because hexavalent chromium
is water soluble and trivalent chromium has a very low solubility in seawater.
Chromium is contributed to drilling fluids chiefly by lignosulfonate which
is added in greater amounts as mud weight is increased (Hrudey and Eng, 1979).
Ferrochrome lignosulfonate, brand name "Q-Broxin," and chrome lignosulfonate
are common additives to drilling fluids which contribute to Cr enrichment.
Liss et al. (1980) reported that Q-Broxin included a metallic composition of 7%
Na, 3%~~Cr7 1% Fe and 0.3% Ca W/W. Initially, Q-Broxin contains hexavalent
chromate salts, but at temperatures between 120 to 175°C hexavalent chromium
is converted to the trivalent state. The thinning property of Q-Broxin can be
restored at temperatures between 120° and 175°C by adding sore C"*"0. Above
175°C no more C will restore lost thinning ability.
Chrome lignosulfonate, containing hexavalent salts, is added to drilling
fluids to improve their thermal stability and for corrosion protection. Carr
et al. (1980) reviewed pertinent literature in reference to chrome
TTgnosulfonate, including a Master's thesis by Knox (1978). According to Knox
(1978) the lignosulfonate is attached to clay by being adsorbed to metals
through phenolic oxygens, sulfonate groups and carboxilic acid groups. The
rate of adsorption and the conversion of Cr to Cr " is slow at room
temperature, but rapid at high temperature. Additional chromate salts
(Cr ) are often added to drilling fluids to further improve their thermal
stability and corrosion protection. After drilling fluids have been used for
an extended period of time, it is very probable that most of the chromium is
associated with the clay fraction and the chromium is trivalent.
32
-------
Knox (1978) has suggested that after drilling fluids are discharged into
the ocean, chromium and associated material are released slowly in soluble form
from clay particles into the water. 0ncJ freed from clay particles, Ca
through slow oxidation may revert to Cr as Cranston and Murray (1980)
discovered in their experiments. In other research, Cr oxidation rates
of 3% in 30 days occurred at 22 to 26°C, and at 35°C and 45°C the same extent
of oxidation occurred at 10 days and less than 3 days, respectively. Fukai and
Vas (1969) reported Cr oxidation rates of 7% Cr in one week.
Most investigators assume that all of the chromium in drilling fluids is
trivalent even though analyses were not made to determine the valence. Other
investigators, as Liss et_ a.U (1980), apparently are not certain that all of
the chromium in drilling fluids is trivalent. Personal communication with
several investigators concerning the presence of hexavalent chromium in
discharged drilling fluids brought forth comments such as: "doubtful", "under
certain conditions", "might vary from 5 to 20% depending upon input into
drilling fluid and time sample was taken."
In used chrome lignosulfonate drilling mud taken from an offshore well
after 20 days of drilling at a depth of 3,650 meters (12,000 feet), the whole
mud contained approximately 500 ppm total chromium on a dry weight basis (Neff
et al., 1981). The mud filtrate contained 27 ppm total chromium and the mud
aqueous fraction had less than 1 ppm total chromium. Carr et al. (1981)
reported that in a preliminary analysis more than 75% of the chromium was found
in a trivalent state.
We can conclude from the above discussion that the possibility exists that
under certain conditions both trivalent and hexavalent chromium may be
discharged. Most of the discharge would undoubtedly include trivalent chromium
and not be too bioavailable for planktonic organisms.
If hexavalent chromium were included in the discharge, it would be more
bioavailable than trivalent chromium and in the course of time would
bioaccumulate if assimilated. Mearns et al. (1976) and other investigators
have shown that hexavalent chromium is many times more toxic than trivalent
chromium.
This investigation was undertaken to determine the concentrations of
hexavalent chromium, Na2Cr04, which are nontoxic, sublethal and acutely
toxic to the complete larval development of the mud crab, Rhithropanopeus
harrisii and the blue crab, Callinectes sapidus.
33
-------
RESULTS
Survival of Rhithropanopeus harrisii Larvae
The percent survival of R.. harrisii from hatching to raegalepH. and to 1st
crab is given for each replicate series in TABLE 11. The average percent
survival of all R.. harrisii series reared in seawater control and four
concentrations of Na CrO, is given in TABLE 12. There are no significant
differences between survival to megalopa and to 1st crab stage reared in
seawater control and 1 ppm (TABLE 12). There was a decrease ir. survival with
an increase in concentration from 1 to 29 ppm Na^CrO..
Statistical Analysis of R. harrisii Larval Survival
Statistical analysis indicated that:
(i) survival to megalopa (TRFSZ) is linearly related to
concentration of Na CrO, (CONG) in the rar.go 0-40 ppm.
Survival to first crab (TRESC) is linearly related to
concentration of Na^CrO. (CONG) in the ranee '"i-2Ct r>Dm.
24 ' -
(ii) the two lines (Figure 5) are not parallel since tr.e two slopes
differ significantly.
The summary equations are:
Zoea: TRFSZ = 79.5 - 1.936 * CONG.
b = -1.936 ± 0.088 Degrees/ppm CONG,
or = -19.36 ± 0.88 Degrees/10 ppm CONG,
or approximately 34% decrease in survival of zoea/iO ppm
increase in Na^CrO, (measured @ 50% survival).
2 4 '
First Crab: TRFSC = 76.1 - 2.274 * CONG.
b = -2.274 ± 0.088 Degrees/ppm CONG,
or = -22.74 ± 0.88 Degrees/10 ppm CONG,
or approximately 40% decrease in survival to first
crab/10 ppm increase in Na^CrO (measured @ 50% survival).
Estimated LC50 values were obtained by setting each equation equal to
45 degrees (50% survival) and solving each equation for the value of
CONG.
Estimated LC50 values were:
Zoea: 17.8 ppm N
First Crab: 13.7 ppm N
34
-------
TABLE 11. PERCENT SURVIVAL AND DURATION IN DAYS THROUGH ZOEAL AND MEGALOPA DEVELOPMENT OF
Rhithropanopeus harrisii REARED IN SEAWATER CONTROL AND IN DIFFERENT CONCENTRATIONS OF HEXAVALENT
CHROMIUM, Na2CrOA.
Culture Media
Salinity 20 V"
Temp. 25°C
Seawater Control
1.12 ppm
NajCrO^
7.17 ppm
Na2Cr04
14.52 ppm
Na2Cr04
29.09 ppm
Na2Cr04
Initial No.
of larvae
per series
RhI-50
RhII-50
RhIII-50
RhIV -50
RhV-50
RhVI-50
RhI-50
RhII-50
RhIII-50
RhIV-50
RhV-50
RhVI-50
Rhl-50
RhII-50
RhIII-50
RhIV-50
RhV-50
RhVI-50
RhI-50
RhII-50
RhIII-50
RhIV-50
RhV-50
RhVI-50
RhI-50
RhII-50
RhIII-50
RhIV-50
RhV-50
RhVI-50
% Survival to
Megalopa
96
90
96
94
98
96
94
90
90
98
98
98
90
72
84
80
94
80
98
56
58
18
84
62
60
4
0
0
34
34
1st crab
96
88
92
94
96
96
94
90
86
96
98
98
90
38
62
56
92
74
82
18
48
8
76
52
14
0
0
0
6
22
Mean Duration of
Zoea
12.4
13.0
10.6
11.8
11.8
11.7
12.4
13.0
11.8
11.8
11.8
11.9
12.7
14.2
12.1
12.3
12.3
12.6
13.7
14.7
12.6
12.8
13.6
14.3
15.4
15.5
_
-
15.9
14,9
Megalopa
8.6
6.1
6.7
7.9
5.4
5.6
9.1
6.1
6.2
7.0
5.4
5.5
7.5
6.2
6.0
6.9
5.5
5.6
6.7
6.6
6.0
6.3
5.4
7.1
6.3
-
_
8.0
7.8
Develooment in days
Hatch to 1st Crab
21.0
19.2
17.3
21.7
17.2
17.3
21.5
19.1
18.0
19.4
17.2
17.4
20.2
20.3
18.1
18.9
18.0
18.4
20.5
20.9
18.5
18.9
19.1
21.0
21.4
_
_
_
23.0
22.8
40.60 ppm
46.40 ppm
58.09 ppm
RhIII-50— RhVI-50
RhI,II,V,VI-50
RhI,II,V,VI-50
35
-------
TABLE 12. AVERAGE PERCENT SURVIVAL AND AVERAGE DURATION IN DAYS OF ZOEAL .AND 'If.-LOPi [
OF R_. harrisii IN SEAWATER CONTROL AND DIFFERENT CONCENTRATIONS OF HEXAVALENT CHROMIUM,
Na2Cr04.
Culture Media
Salinity 20°/°°
Temp. 25°C
Seawater Control
1.12 ppm
Na2Cr04
7.17 ppm
Na2Cr04
14.52 ppm
Na2Cr04
29.09 ppm
Na?CrOA
Initial No.
of larvae °» Survival to Mean Duration of De\ eioorrent ;n davs
per series Meqalopa 1st crab Zoea Meaalooa Hatch to Crab
RhI-50
RhII-50
RhIII-50 95.0 93.7 11.9 6.7 19.0
RhIV-50
RhV-50
RhVI-50
RhI-50
RhII-50
RhIII-50 94.7 93.7 12.1 6.6 IS. 8
RhIV-50
RhV-50
RhVI-50
Rhl-50
RhII-50
RhIII-50 83.3 68.7 12.7 o.3 ^.0
RhIV-50
RhV-50
RhVI-50
RhI-50
RhII-50
RhIII-50 62.7 47.3 13.6 t.a i°.8
RhIV-50
RhV-50
RhVI-50
RhI-50
RhII-50
RhIII-50 22.0 7.0
RhIV-50
RhV-50
RhVI-50
36
-------
80-
70-
*«H
en
o
UJ
2
cc
04(H
30-
20-
10-
ZOEA
Y=79.5-1.936*conc
1st CRAB ^
Y= 76.1-2.274* cone
10
20 30 40 50
CONCENTRATION OF Na2Cr04lppml
60
Figure 5. Effect of concentration of hexavalent
Na2CrO^ in ppm on survival of R. harrisii.
Hatch to megalopa x x
Hatch to 1st crab o o
37
-------
Duration of R. harrisii Larval Development
Table 11 gives the mean duration in days of R.. harrisii zoeal and
megalopa development and the mean time in days from hatching tc the 1st crab
stage for each series reared in seawater control and in different
concentrations of Na2Cr02. TABLE 12 lists the mean duration of development in
days for _R. harrisii larvae reared in all series.
Statistical Analysis of R. harrisii Larval Duration
1. Significant linear regressions of both days to megalopa (DZ) and days
from hate
with no s
29.1 ppm.
from hatch to 1st crab (DC) upon Na-CrO, concentrations (CONC) were found
with no significant deviations from linearity in concentration of 0 to
DZ = 11.90 + 0.120 * CONC
DC = 18.52 + 0.122 * CONC
Where CONC is in ppm of Na0CrO,. These results are shown in Figure 6.
The regression coefficient may be interpreted as follows:
for DZ : 0.120 ± 0.021 days increase in duration of zoeal
development for each ppm added Na^CrO
for DC : 0.122 ± 0.021 days increase in total duration time to 1st
crab for each ppm added Na CrO,.
These increases in duration can be scaled up, for example to 10 ppm by
multiplication, i.e., DZ 1.20 ± 0.21 days for each 10 ppm Na_CrO added
and DC 1.22 ± 0.21 days increase for each 10 ppm added.
2. Nearly analogous results were obtained when RATE = 100/DAYS was used as
the dependent variable:
RZ = 8.38 - 0.066 * CONC
RC = 5.41 - 0.030 * CONC
These results are shown in Figure 7.
b(RZ) = -0.066 ± 0.007 (DAYS"1 * 100)/ppm CONC
b(RC) = -0.030 ± 0.007 (DAYS"1 * 100)/ppm CONC
Mortality of R. harrisii by Larval Stage
Rhithropanopeus harrisii passes through four zoeal stages and a megalopa
stage before molting into a. 1st crab stage. In an effort to determine if
larvae in one or more stages were particularly sensitive to different
concentrations of Na CrO,, a record of deaths by stage was made of each of the
replicate series of larvae (TABLE 13). Average percent mortality of larvae in
38
-------
24-r
22-
20-
18-
IstCRAB
= 18.5 + 0.122*conc
in
>
<
14
12
2OEA
02 = 11.9 + 0.120 *conc
10-
10 15 20 25
CONCENTRATION OF Na2C rO4 Ippcnl
30
35
Figure 6. Duration of zoeal development (DZ) and duration to
1st crab (DC) in R.. harrisii vs. concentration of
Na2CrO^ in pptn.
DZ: x- x Hatch to megalopa
DC: o o Hatch to 1st crab
39
-------
8H
ZOEA
RZ = 8.38—0.066 »-conc
O
o
sH
-1st CRAB
RC =5.11-0 030 *conc
3H
10
15
2O
25
CONCENTRATION OF N»2C rO4 Ippml
30
35
40
Figure 7. Effect of Na2CrO^ in ppm on rate of molting
from hatch to megalopa and hatch to 1st crab in
R. harrisii.
RZ : x x Hatch to megalopa
RC: o o Hatch to 1st crab
40
-------
AND DIFFERENT CONCENTRATIONS OF HEXAVALENT CHROMIUM, Na2CrO^.
Zoeal Stages
Media
Seawater Control
1.12 ppm
Na2Cr04
7.17 ppm
Na2Cr04
14.52 ppm
Na2Cr04
29.09 ppm
Na2Cr04
40.60 ppm
Na CrO
2 4
Series
RhI-50
RhII-50
RhIII-50
RhIV-50
RhV-50
RhVI-50
RhI-50
RhII-50
RhIII-50
RhIV-50
RhV-50
RhVI-50
RhI-50
RhII-50
RhIII-50
RhIV-50
RhV-50
RhVI-50
RhI-50
RhII-50
RhIII-50
RhIV-50
RhV-50
RhVI-50
RhI-50
RhII-50
RhIII-50
RhIV-50
RhV-50
RhVI-50
RhIII-50
RhIV-50
RhV-50
RhVI-50
I
2
4
2
2
2
2
4
6
8
0
2
2
8
18
10
4
0
2
2
10
10
2
0
2
6
24
10
4
2
0
16
2
16
10
I!
2
2
2
0
0
0
0
0
0
2
0
0
2
0
2
0
0
2
0
8
0
10
2
0
4
32
20
40
24
40
34
88
64
86
III
0
2
0
0
0
0
0
0
0
0
0
0
0
2
0
2
0
4
0
6
14
36
2
28
10
20
70
44
24
6
44
10
18
4
IV
0
2
0
4
0
2
2
4
2
0
0
0
0
8
4
14
6
12
0
20
18
34
12
8
20
20
12
16
20
6
_
2
-
Megalopa
0
2
4
0
2
0
0
0
4
2
0
0
0
34
22
24
2
6
16
38
10
10
8
10
46
4
_
_
28
12
—
_
_
Total
4
12
8
6
4
4
6
10
14
4
2
2
10
62
38
44
. 8
26
18
82
42
92
24
48
86
100
100
100
94
78
100
100
100
100
-continued-
41
-------
TABLE 13 Continued
Zoeal Stapes
Media Series I II III IV Meaaiooa Total
46.40 ppm
Na2CrO^
58.09 ppm
Na2CrOA
RhI-50
RhII-50
RhV-50
RhVl-50
RhI-50
RhII-50
RhV-50
RhVl-50
18
6
44
14
100
100
90
42
70 12
92 2 -
56 -
86
-
10
58
100
100
100
100
100
100
.00
.00
developmental stages of _R. harrisii in all series reared in saltwater control
and different concentrations of Na^CrO. is given in TABLE 1-. Frcr Ficure 3,
it can be seen that 1 ppm Na^CrO, is non-toxic, for there is no ircre mortality
in this concentration than in seawater control. There is differential
mortality, however, between larvae exposed to concentration? of 1 ?DT^ and
larvae exposed to 58 ppm Na0CrO . Concentrations of 7 ppm to 15 T^DI? Na^CrO,
are considered sublethal since more than 10 percent of R. harrisii larvae
reached the 1st crab stage. Concentrations of 29, 41, 46,"and 5? ?pir Xa.CrO
are acutely toxic to R.. harrisii larvae since less than 10 percent reached the
1st crab stage in 29 ppm and none reached the 1st crab stage in 41, 4r, £nd
58 ppm.
Statistical Analysis of R. harrisii Cumulative Mortality by Stares
The results illustrated in Figure 8 show the effect of Na.CrC,
concentrations on the mortality at each stage of development. ~As indicated
earlier (see Statistical Analysis in Materials and Methods) the analyses were
performed on the transformed mortality percentages. The means were adjusted
to account for the unequal number of replications at some concentrations of
Na^CrO, and the tests of significance completed, all in the transformed scale.
The means were retransfonned to percent mortality for presentation in
Figure 8.
Larval Behavior: Swimming Speed of R. harrisii Upon Exposure to Na,,CrO,
Generally, the swimming speed of seawater control treated _R. harrisii
larvae (TABLE 15) increases throughout development. Swimming speed is
affected by exposure to Na?CrO,. However, the direction of the speed change
(increase or decrease) and larval sensitivity to sodium chromate changes with
developmental stage. For stage I zoeae all test concentrations cause an
elevation in swimming speed. This elevation is observed in later stages at
42
-------
TABLE 14. AVERAGE PERCENT MORTALITY IN DEVELOPMENTAL STAGES OF JR. harrisii REARED IN SALTWATER
CONTROL AND DIFFERENT CONCENTRATIONS OF HEXAVALENT CHROMIUM, Na2Cr04.
Media
Series
Zoeal Stages
II III IV
Megalopa Total
Seawater
Control
1.12 ppm
7.17 ppm
14.52 ppm
29.09 ppm
40.60 ppm
46.40 ppm
Na2Cr04
58.09 ppm
Rhl-50 - RhVI-50 2.3 1.0 0.3 1.3
RhI-50 - RhVI-50 3.7 0.3 0 1.3
Rhl-50 RhVI-50 7.0 1.0 1.3 7.3
Rhl-50 RhVI-50 4.3 3.3 14.3 15.3
RhIII-50
RhIV-50
RhV-50
RhVI-50
Rhl-50
RhII-50
RhV-50
RhVI-50
Rhl-50
RhII-50
RhV-50
RhVI-50
11.0 68.0 19.0 2.0
20.5 76.0 3.5
83.0 17.0
1.3
6.3
1.0 6.3
14.7 31.3
15.3 51.0
Rhl-50 RhVI-50 7.7 26.7 29.0 14.7 15.0 93.0
100.0
100.0
100.0
the two lower concentrations. Depression in swimming rate is first observed
in stage II zoeae at the highest test concentration. Stages II and IV zoeae
show a depression in swimming rate at the two highest concentrations. Thus
the general pattern is for the swimming rate to be elevated by acute exposure
to all concentrations and chronic exposure to low sublethal concentrations
(1.2 and 7.1 ppm). Swimming rates are only depressed upon chronic exposure to
higher sublethal and lethal concentrations (14.5 and 29.1 ppm). These results
agree with the normal pattern for the effects of pollutants upon larval
swimming. The lower sublethal concentrations cause increases in swimming
speed and higher concentrations cause a decline (e.g. Lang et al. 1980).
43
-------
TABLE 15. SWIMMING SPEED (mm/min) FOR DIFFERENT ZOEAL STAGES OF CONTROL R_. Hql-r^. LARVAE AND
THOSE TREATED WITH Na2Cr04. THE MEAN (M; AND STANDARD DEVIATION fSO, ARE SHOWN. THE SAMPLE
SIZE FOR EACH CONDITION IS 60. * INDICATES P < O.Ob STATISTICAL DIFFERENCE BETWEEN CIN'TROL
LARVAE AND EXPOSURE TO A PARTICULAR CONCENTRATION WHILE ** IS P < 0.02 -:p »»» IS p •' O.OOi.
Zoeal Seawater Control 1.12 ppm
Stage M S.D. M S.D.
I 79.6 36.2 95.0* 36.2
II 112.2 45.6 105.2 40.8
III 117.0 47.2 138.8* 65.2
IV 126.4 56.0 125.6 53.0
7.17 ppm li.52 pom 29.0° ppm
M S.D. M S.D. M S.D
110.4* 43.0 113.8*** 46.3 136. a*** 52
130.4* 51.6 121.8 54. a 85.8*** ^9
122.4 65.2 9Q.o 52.6 Q6.4** 46
152.2* 72.4 104.8* 52.0 104.2* a7
•
.1
.5
.0
.0
Survival of C. sapidus Larvae
The percent survival of £. sapidus larvae from hatching to megalopa and
to 1st crab is given for each replicate in TABLE 16. The average percent
survival of all series reared in seawater control and four concentrations of
Na^CrO. is listed in TABLE 17. There are significant difference? between
survival of _C_. sapidus larvae to megalopa and to 1st crab stase reared in
seawater control and those larvae reared in Na CrO concentrations frcrr.
1.1 ppm to 4.7 ppm (TABLE 17). There was better survival of larvae in 1.1 ppm
Na CrO, than in the control and differential survival between 1.1 to 7.2 ppm
Statistical Analysis of C. sapidus Larval Survival
Statistical analysis indicated that:
(i) survival to megalopa (TRFSZ) and to first crab (TRFSC) are both
linearly related to concentration of Na^CrO, in ppin (CONC) in
the range 1.1 to 7.2 ppm.
(ii) the slopes of the two lines (Figure 9) differ significantly in
the statistical test.
The summary equations are:
Zoea: TRFSZ = 76.8 - 11.1 * CONC
b = -11.1 ± 0.83 Degrees/ppm CONC
or approximately 19% decrease in survival of zoea/ppm
increase in Na^rO^ @ 50% survival
44
-------
100-
9O-
80-
70-
-60H
K
cr
O
in
> 50H
I
3
U
« 40-
30-
20-
10-
29ppm
ISppm
III IV
STAGE
Figure 8. Effect of
R. harrisii.
in ppm on mortality of
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)
45
-------
TABLE 16. PERCENT SURVIVAL AND bURATION IN DAYS THROUGH ZOEAL AND MEGALOPA CE'. eLP°f'LNT OF THREE
SERIES (Cs I-III) OF Callinectes saoidus REARED IN SEAWATER CONTROL AND IN DIFFERENT
CONCENTRATIONS OF HEXAVALENT CHROMIUM, Na2Cr04
Culture Media
Salinity 30°/00
Temp. 25°C
Seawater Control
1 . 1 ppm
Na2Cr04
2.4 ppm
Na2Cr04
4.7 ppm
Na2Cr04
7.2 ppm
Na2Cr04
Initial No.
of larvae
per series
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-5U
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
% Survival to
Megalopa
64
58
62
38
78
76
74
72
50
24
18
-
_
-
-
1st crab
36
36
42
54
46
34
64
32
28
16
6
-
_
-
Mean Duration of Development in days
Zoea MeoaiOpa Hatch to 1st Crab
34.8 7.6 40.5
32.2 7.7 40.0
33.5 7. a 33.3
33.6 7.0 41.5
31.9 7.6 3°.6
37.9 6.9 44.8
36.0 7.3 ^3.1
34.1 8.1 41.6
38.2 7.4 44.9
41.8 8.3 47.3
39.0 7.7 43.3
-
_
-
-
First Crab: TRFSC = -52.5 - 7.5 * CONG
b = -7.5 ± 0.83 Degrees/ppm CONG
or approximately 13% decrease in survival to first
crab/ppm increase in Na CrO, @ 50% survival.
Estimated LC50 values were obtained by setting each equation equal to
45 degrees (50% survival) and solving each equation for the value of
CONC.
Estimated LC50 values were:
Zoea: 2.9 ppm Na
First Crab: 1.0 ppm Na
L. T
Duration of C. sapidus Larval Development
TABLE 16 gives the mean duration in days of zoeal and inegalopa
development for C. sapidus and the mean time in days from hatching to the 1st
crab stage for each series reared in seawater control and in different
46
-------
TABLE 17. AVERAGE PERCENT SURVIVAL AND AVERAGE DURATION IN DAYS THROUGH ZOEAL AND MEGALOPA
DEVELOPMENT OF THREE SERIES (Cs I-III) OF Callinectes sapidus REARED IN SEAWATER CONTROL AND IN
DIFFERENT CONCENTRATIONS
Culture Media
Salinity 30°/°°
Temp. 25°C
Seawater Control
1.1 ppm
Na2Cr04
2.4 ppm
4.7 ppm
7 . 2 ppm
OF HEXAVALENT CHROMIUM, Na2Cr04
Initial No. % Survival to Mean Duration of Development in days
of larvae Megalopa 1st crab Zoea Megalopa Hatch to 1st Crab
per series
CsI-50
CsII-50 61.3 38.0 33.5 7.6 39.6
CsIII-50
CsI-50
CsII-50 80.6 44.7 34.5 7.2 42.0
CsIII-50
CsI-50
CsII-50 65.3 41.3 36.1 7.6 43.2
CsIII-50
CsI-50
CsII-50 14.0 7.3 40.0 8.0 45.3
CsIII-50
CsI-50
CsII-50 -
CsIII-50
concentrations of Na0CrO,. TABLE 17 lists the mean duration of development in
days for C_. sapidus larvae reared in all series.
Statistical Analysis of C. sapidus Larval Duration
1. Significant linear regressions of Days to Megalopa (DZ) and Days to First
Crab (DC) upon concentration of Na CrO, in ppm (CONG) were found. No
significant deviations from linearity occurred in the range 0 to 4.7 ppm.
The most compact summary is in these equations:
DZ = 32.9 + 1.65 * CONG
DC = 40.0 + 1.31 * CONG
Where CONC is in ppm of Na CrO . These results are shown in Figure 10.
The regression coefficient may be interpreted as follows:
47
-------
2345
CONCENTRATION OF Na^CrO4 ippml
Figure 9- Effect of concentration of
of C. sapidus.
R. harrisii.
Hatch to megalopa x - x
Hatch to 1st crab o - o
in oom on survival
48
-------
3O
2345
CONCENTRATION OF Na2Cr04lppml
Figure 10. Duration of zoeal development (DZ) and duration to 1st
crab (DC) in C_. sapidus vs. concentration of NaoCrCv
in ppm.
DZ: x x Hatch to megalopa
DC: o o Hatch to 1st crab
49
-------
3.O
2.1
1 2 ~T~ 4 5
CONCENTRATION OF N*2C r04 Ippml
Figure 11. Effect of ^2^0^ in ppm on rate of molting from hatch
to megalopa and hatch to 1st crab in C_. sapidus.
RZ: x x Hatch to megalopa
RC: o o Hatch to 1st crab
50
-------
TABLE 18. PEPCENT MORTALITY IN DEVELOPMENTAL STAGES OF THREE SERIES (Cs I-III) OF £. sapidus
REARED IN SALTWATER CONTROL AND DIFFERENT CONCENTRATIONS OF HEXAVALENT CHROMIUM, Na2CrO^
Media
Seawater Control
1 . 1 ppm
Na?CrOi
2.4 ppm
Na2CrOA
4.7 ppm
Na2Cr04
7.2 ppm
Na2CrO^
Series
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
CsI-50
CsII-50
CsIII-50
I
2
8
4
4
6
4
4
6
14
18
10
2
14
12
0
II
16
10
6
0
6
2
8
2
6
4
8
6
6
18
2
III
4
14
20
0
6
2
4
14
18
12
16
30
54
60
98
Zoeal
IV
4
6
2
4
0
2
4
2
2
24
16
38
22
8
0
Stages
V
0
0
6
0
0
4
4
2
0
12
12
20
4
2
0
VI
4
2
0
4
0
4
2
0
4
6
12
4
0
0
0
VII
6
2
0
0
4
4
0
2
4
0
6
0
0
0
0
VIII
0
0
0
0
0
2
0
0
2
0
2
0
0
0
0
Meqalopa
28
22
20
34
32
42
10
40
22
8
12
0
0
0
0
Total
64
64
58
46
54
66
36
68
72
84
94
100
100
100
100
2.
for DZ: 1.65 ± 0.29 days increase in duration of zoeal development
for each ppm added Na.CrO,
for DC: 1.31 ± 0.29 days increase in total duration time to 1st
crab for each ppm added Na CrO,.
Nearly analagous results were obtained when RATE = 100/DAYS was used as
the dependent variable:
RZ = 3.03 - 0.122 * CONG
RC = 2.50 - 0.074 * CONG
These results are shown in Figure 11.
b(R2) = -0.122 ± 0.020 (DAYS""1 * 100)/ppm CONG
b(RC) = -0.074 ± 0.020 (DAYS"1 * 100)/ppm CONG
51
-------
TABLE 19. AVERAGE PERCENT MORTALITY IN DEVELOPMENTAL STAGES OF THREE SERIES ,' Ci i-III
sapidus REARED IN SALTWATER CONTROL AND DIFFERENT CONCENTRATIONS OF HEVAVALFV C^T^IU
.
Media
Series
Zoeal Stages
I II III IV V VI VII VII; Meoaiopa Total
Seawater Control CsI-50
CsII-50
CsIII-50
4.7 10.7 12.7 4.0 2.0 2.0
62.0
1.1 ppm
Na2CrO^
2 . 4 ppm
Na2Cr04
4.7 ppm
Na2Cr04
7.2 ppm
Na2Cr04
CsI-50
CsII-50 4.7 2.7 2.7 1.3 2.0 2.7 2.7 0.7
CsIII-50
CsI-50
CsII-50 8.0 5.3 12.0 2.7 2.0 2.0 2.0 0.7
CsIII-50
CsI-50
CsII-50 10.0 6.0 19.3 26.0 14.7 7.3 2.0 0.7
CsIII-50
CsI-50
CsII-50 8.6 8.7 70.7 10.0 2.0 0 00
CsIII-50
3&.C 55.3
24.0 58 . 7
o.7 92.7
0 100
Mortality of C. sapidus by Larval Stage
Callinectes sapidus passes through seven to eight zoeal staees and a
megalopa stage before molting into a 1st crab stage. In ar. cffcr; to
determine if larvae in one or more stages were particularly sensitive to
different concentrations of Na?CrO,, deaths were recorded by stage for each
replicate series of larvae (TAfiLE 18). Average percent mortalitv of larvae in
developmental stages of C_. sapidus in all series reared in saltwater control
and different concentrations of Na9CrOx is given in TABLE 19. From Figure 12,
it can be seen that there is significantly less mortality in l.i ppm Na^CrO.
than in seawater control. There is also less mortality in 2.4 ppm Na0CrO,,
but it is not significantly different from the control and hence it is
considered nontoxic. There is differential mortality from concentrations of
4.7 to 7.2 ppm Na0CrO, , and these concentrations are considered acutely toxic
since less than 10 percent of _£. sapidus larvae reached the 1st crab stage.
Statistical Analysis of C. sapidus Cumulative Mortality by Stages
The results illustrated in Figure 12 show the effect of Na^CrO,
concentrations on the mortality at each stage of £. sapidus larval
development. Zoeae in zoeal stage III were extremely sensitive to 7.2 ppm
-------
100-
9O-
80-
70-
60
550-
40-
30-
20-
10
-1.1 ppm
V
STAGE
VI
VII
VIII
Figure 12. Effect of Na2Cr04 in ppm on mortality 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)
53
-------
Na CrO^ (TABLE 19 and Figure 12). In 4.7 ppm, zoeae in zoeal stages III, IV
ana V were the most sensitive and mortality was significantly different from
the control in all developmental stages after zoeal stage III (Figure 12). In
the control, 1.1 and 2.4 ppm Na CrO, there was a very significant increase in
mortality in the megalopa stage compared to the previous stage (TABLE 19 and
Figure 12).
DISCUSSION
Survival
The range of concentrations of Na CrO, in which development of
Rhithropanopeus harrisii occurred from hatch to 1st crab was from 1.1 to
29.1 ppm Na^CrO, (TABLE 12), whereas in Callinectes sapidus it was from 1.1 to
4.7 ppm (TABLE 17). Na^CrO, concentrations of 1.1 ppm to R.. harrisii and 1.1
and 2.4 ppm to (3. sapidus were nontoxic. Actually survival of C. sapidus
larvae was significantly better in 1.1 ppm Na.CrO, and somewhat better in
2.4 ppm than in seawater control, possibly because these concentrations may
have killed bacteria or neutralized other toxic substances which were in
seawater. Concentrations of 7.2 and 14.5 ppm Na CrO. were sublethal
concentrations to R.. harrisii, because more than 10% reached the 1st crab
stage and there was a reduction in survival with an increase in concentration
compared to survival in seawater control (Epifanio, 1971; Bookhout and
Costlow, 1975). Sublethal concentrations for C_. sapidus would probably be
between 2.4 and 4.7 ppm Na^CrO, , but they were not employed in this
investigation. Acutely toxic concentrations of Na CrO, to R.. harrisii were
29.1, 40.6, 46.4 and 58.1 ppm, for only 7% of the larvae reached the 1st crab
stage in 29.1 ppm and no larvae reached the 1st crab stage in the other
concentrations of Na^CrO,. The acutely toxic concentrations to £. sapidus
were 4.7 ppm, in which 7.3% of the larvae became 1st crabs, and 7.2 ppm
Na?CrO,, in which no larvae reached the 1st crab stage.
The estimated 96-h LC50 for zoeal development from hatch to ir.egalopa was
17.8 ppm Na9CrO, for R.. harrisii and 2.9 ppm for C_. sapidus, and the estimated
96-h LC50 for development from hatch to 1st crab was 13.7 ppm Na^CrO for
R. harrisii and 1.0 ppm for C_. sapidus.
Comparative Toxicity
Most of the research on the effect of hexavalent chromium has been acute
toxicity studies on adult organisms, not on larvae. Eisler and Hennekey
(1977) using K CrO, reported the 7-day LC-100 for the sandworm, Nereis virens,
was 5 ppm; 20 ppm for the hermit crab, Pagurus longicarpus; 50 ppm for the
soft shell clam, Mya arenaria; 20 ppm for the starfish, Asterias forbesi;
20 ppm for the snail, Nassarius obsoletus; and 100 ppm for the mummichog
Fundulus heteroclitus. All of these species with the exception of Nereis
virens were less susceptible to hexavalent chromium than _C. sapidus during
larval development, but only the soft shell clam and the mummichog were less
susceptible to hexavalent chromium than R. harrisii during larval development.
54
-------
In making comparisons of the effects of Cr on other crustaceans,
differences in toxicity may depend upon the temperature and salinity of the
medium. Fales (1978) found that the susceptibility of the grass shrimp,
Palaemonetes pugio, to potassium chromate was greatest at 25°C and a
10°/0o salinity and least at 10°C and 20°/00 . In the first case the 48-h TL
(median tolerance limit) value was 21 ± 4 mg Cr/1 and in the second case the
TL value was 147 ± 16 mg Cr/1. Frank and Robertson (1979) also reported the
influence of salinity on the toxicity of Cr to juvenile blue crabs,
Callinectes sapidus. Using K^Cr.O , they found that the 96-h LC50 to juvenile
crabs was 34.2 ppm Cr in a salinity of l°/0o, whereas the 96-h LC50 to
juvenile blue crabs of the same size was 98 ppm Cr in 350/00 salinity.
From the above discussion, the implications are that when _R. harrisii and
C. sapidus are reared from hatching to 1st crab at a temperature of 25°C, Cr
would tend to adversely affect development. Since R.. harrisii and C_. sapidus
were reared in salinities of 20°/00 and 30°/00, respectively, however, these
high salinities might counter the adverse effects of high temperatures.
Sublethal Effects
The increase in duration with each increase in Na CrO, in zoeal
development from hatching to megalopa and in development from hatching to 1st
crab stage in R.. harrisii (TABLE 12 and Figure 6) and £. sapidus (TABLE 17 and
Figure 10) is considered a sublethal effect of hexavalent chromium. Swimming
behavior of R.. harrisii is also modified by Na.CrO,. Exposure to sublethal
concentrations of Na CrO, caused an elevation in swimming speed, while near
lethal concentrations produced a depression (TABLE 15).
Environmental Implications
Sodium chromate, Na^CrO,, is one of the potentially hazardous materials
being discharged into saline 'environments from metal processing facilities,
chemical industries and other sources. The total chromium in sodium chromate
is 32 percent by weight according to Dr. Tacy of Hazleton Laboratories. In
chronic bioassays on the effects of Na^CrO, on the complete larval development
of R.. harrisii and _C_. sapidus, it was observed that 1.1 ppm Na^CrO, with total
chromium of 0.36 ppm was nontoxic during the complete larval development of
R.. harrisii and CJ_. sapidus. This concentration was also nontoxic to the
fathead minnow, Pimephales promeles, in the first and second generation in
hard water (Pickering, 1980). Concentrations of 7.2 ppm Na?CrO, with total
chromium of 2.3 ppm and 14.5 ppm Na CrO with total chromium of^4.66 ppm were
sublethal to R.. harrisii, and it was estimated that concentrations between
2.4 ppm Na CrO, with total chromium of 0.77 ppm and 4.7 ppm Na CrO, with total
chromium of 1.5 ppm would be sublethal to C_. sapidus. These concentrations
would undoubtedly be absorbed by crab larvae, and they would bioaccumulate in
the tissues of the larvae in the course of time. Eventually the
bioaccumulated chromium would produce stress and more mortality than in the
control, especially in later zoeal stages as shown in Figure 8. Acutely toxic
concentrations in which less than 10% reach the 1st crab stage, ranged from
29.1 ppm Na^CrO^ with total chromium of 9.31 ppm to 58.1 ppm Na CrO with
total chromium of 18.65 ppm for R.. harrisii and 4.7 ppm Na CrO, with* total
chromium of 1.5 ppm and 7.2 ppm Na2Cr04 with 2.3 ppm total chromium for
55
-------
£. sapidus. In acutely toxic concentrations, there is marked mortality in the
first few zoeal stages as shown in Figures 8 and 12. The larval development
°f .R. harrisii might be considered one of the most resistant to Na^CrO. and
the larval development of _C_. sapidus among the most sensitive.
There is a question in the literature whether hexavalent chromium,
Na»CrO , which is incorporated into chrome lignosulfonate or ferrochrome
lignosulfonate before they are added to drilling fluids, has any detrimental
effect on the complete development of crabs or other planktonic organisms. If
hexavalent chromium plays an active part, it is more indirect and complex than
when Na.CrO^ is discharged from manufacturing plants into the marine
environment.
It is generally assumed that most of the chromium in the discharge of
drilling fluids is trivalent chromium (Cr ) which is not as readily
bioayailable or toxic to planktonic organisms compared to hexavalent chromium
(Cr ), which will pass through biological membranes readily (Hertz, 1969) and
is known to be toxic to marine organisms. If discharges of whole used
lignosulfonate or chrome lignosulfonate type muds were emitted at the same
time as additions of chrome lignosulfonate or ferrochrome ligncsulfonate were
being made, it is possible that both Cr and Cr would be present ir, the
discharge. Initially both chrome lignosulfonate and ferrochrome
lignosulfonate, "Q-Broxin," contain hexavalent chromate salts, but at
temperatures between 120 to 175°C hexavalent chromium is converted to the
trivalent state. The property of these two additives can be restored at
temperatures between 120 to 175°C by adding more hexavalent salts (Liss
et al_. , 1980).
Neff et al. (1981) found approximately 500 ppm total chromium in whole
used chromium'lignosulfonate drilling fluid and less than 1 ppm total chromium
in the filtered mud aqueous fraction (MAF) which is found in the upper
turbidity plume together with the suspended particulate phase (SPP). It is
these two mud fractions which were found to be toxic to R.. harrisii and
C. sapidus as described in Section 4 of this manuscript. Carr _et_ a_l. (1981)
evaluated the bioavailability of chromium from chrome lignosulfonate drilling
fluid to five species of marine invertebrates. The shrimp, Palaemoneres
pugio, exposed to MAF with a concentration of 0.25 ppm chromium for seven
days, accumulated 23.7 ppm, but released it within 96 hours of depuration to
control levels. Clams, Rangia cuneata, exposed to MAF for 16 aays,
accumulated up to 19 ppm chromium in their tissues. When the clams were
returned to clean water, they rapidly lost about half of tne chromium, but
retained 11 ppm even after 11 days. McCulloch ££ a.l_. (1980) confirmed the
findings of Carr £t_ al. (1981) in respect to the bioavailability of chromium
when Rangia was exposed to MAF. They also found that Rangia could accumulate
chromium from different concentrations of MAF and retain about half after
depuration. They also exposed oyster spat, Crassostrea gigas, to MAF and SPP,
and found that they accumulated more chromium from SPP than MAF. Thus
suggesting that they had a limited ability to accumulate particle-adsorbed
chromium, possibly by pinocytosis, whereas Rangia might absorb moderate
concentrations of chromium, possibly chiefly in the form of soluble chrome
lignosulfonate complex (Knox, 1978). Neff _et_ al. (1979), Carr _e^ al. (1981)
56
-------
and McCulloch et al. (1980) reported that a preliminary analysis of total
chromium in MAF revealed that more than 75% of it was in the trivalent state.
It seems reasonable to assume that if trivalent chromium in MAF can be
accumulated up to 19 ppm in the tissues of Rangia (Carr et al., 1981), and if
Na9Cr, were also present in the medium, even at a lower concentration than
trivalent chromium, it would be absorbed more readily and the total chromium
bioaccumulated would be greater. This condition could account for the
variations in bioaccumulation of chromium reported in the literature.
Although Palaemonetes accumulated 23.7 ppm chromium in seven days, it was
released within a 96-h depuration period to control levels. This may imply
that Palaemonetes merely adsorbed trivalent chromium and never absorbed it
into tissue, possibly because its cells could not absorb trivalent chromium,
nor were the cells able to take in chromium associated particles of clay by
pinocytosis. If this conclusion is valid, it is very possible that crab
larvae and Palaemonetes could only absorb hexavalent chromium.
It has recently been suggested that after drilling fluids are discharged
into the ocean, chromium and associated material are released slowly in
soluble form from clay particles into the water (Knox, 1978), Once freed from
clay particles, Cr through slow oxidation may revert to Cr as Fukai and
Vas (1969), Schroeder and Lee (1975), and Cranston and Murray (1980) reported.
This would take place over 7 to 30 days and involve from 3 to 7% of trivalent
chromium.
The decrease in concentration of suspended solids in the upper turbidity
plume from the point of discharge peripherally to> background levels has been
discussed in Section 4 of this report. For most discharges the background
concentration for chromium has been reported to be approximately 100 to 150
meters from the point of discharge. The distance will vary depending on the
amount and rate of discharge, as well as the currents (Ray and Meek, 1980).
Within this area entrained crab larvae might absorb and bioaccumulate Cr as
Cr , as occurs when Cr _ in human blood plasma enters the corpuscles and is
quickly converted to Cr (NAS, 1974). It is questionable whether crab larvae
would remain in the upper turbidity plume long enough to bioaccumulate enough
chromium to kill the larvae or to produce sublethal stress. This is
especially true since the initial response of the larvae upon exposure to Cr
concentrations is an increase in random swimming speed (TABLE 15) which
increases the probability of the larvae leaving the area. Hence, it is
probable that chromium in drilling fluids, whether Cr or Cr+ , is not likely
to reduce the population of crab larvae and other planktonic organisms in the
area around oil wells except possibly in the immediate vicinity of the
discharge pipes.
57
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LITERATURE CITED
Ayers, R.C., Jr., T.C. Sauer, Jr., D.O. Stuebner. 1980. An environmental
study to assess the effect of drilling fluids on water quality parameters
during high rate, high volume discharges to the ocean. In: Proceedings of
the Symposium: Research on Environmental Fate and Effects of Drilling
Fluids and Cuttings, Lake Buena Vista, FL. pp. 351-381.
Bookhout, C.G., A.J. Wilson, Jr., T.W. Duke and J.I. Lowe. 1972. Effects of
mirex on larval development of two crabs. Water Air Soil Pollut.
1:165-180.
Bookhout, C.G. and J.D. Costlow, Jr. 1975. Effects of cire:: on the larval
development of blue crab. Water Air Soil Pollut. 4:112-126.
Bookhout, C.G., J.D. Costlow, Jr. and R. Monroe. 1979. Kepone? effects of
larval development of Callinectes sapidus and Rhithropanopeus harrisii .
U.S. EPA, Environmental Research Laboratory, Gulf Breeze, FL . EPA Ecol.
Res. Ser. 600/3-79-104, 34 pp.
Bookhout, C.G., J.D. Costlow, Jr- and R. Monroe. 1980. Kepone effects on
larval development of mud-crab and blue-crab. Water Air Soil Pollut.
13:57-77.
Carls, M.G. and S.D. Rice. 1981. Toxicity of oil well drilling sjds to
Alaskan larval shrimp and crabs. Final report for Outer Continental Shelf
Energy Assessment Program. U.S. Department of the Interior, Bureau of
Land Management. 33 pp.
Carr, R.S., L.A. Reitsema and J.M. Neff. (1980). Influence of a used chrome
lignosulf onate drilling mud on the survival, respiration, feeding activity
and net growth efficiency of the opossum shrimp Mysidoosis almvra. In:
Proceedings of the Symposium: Research on Environmental fate and Effects
of Drilling Fluids and Cuttings, Lake Buena Vista, FL. pp. 944-963.
Carr, R.S., W.L. McCulloch and J.M. Neff. 1981. Bioavaibility of chromium
from a used chrome lignosulf onate drilling mud to five species of marine
invertebrates. Mar. Environ. Res. (In press)
Cranston, R.E. and J.W. Murray. 1980. Chromium species in the Columbian River
and estuary. Limnol. Oceanogr. 25(6) :1104-1112.
Curl, H.C., Jr., N. Cutshall and C. Osterberg. 1965. Uptake of chromium (III)
'by particles in seawater. Nature 205:275-276.
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2+ 6+
Eisler, R. and R.J. Hennekey. 1977- Acute toxicities of Cd , Cr ,
Hg , Ni and Zn to estuarine macrofauna. Arch.
Environ. Contain. Toxicol. 6:315-323.
Epifanio, C.E. 1971. Effects of dieldrin in seawater on the development of
two species of crab larvae, Leptodius floridanns and Panopeus herbstii.
Mar. Biol. 11(4):356-362.
Fales, R.R. 1978. Influence of temperature and salinity on the toxicity of
hexavalent chromium to the grass shrimp Palaemonetes p-ugio (Solthuis).
Bull. Environ. Contam. Toxicol., 20:447-450.
Forward, R.B., Jr. 1977. Occurrence of a shadow response among brachyuran
larvae. Mar. Biol. 39:311-341.
Forward, R.B., Jr. and J.D. Costlow, Jr. 1976. Crustacean larval behavior as
an indicator of sublethal effects of an insect juvenile hormona mimic.
Estuar. Process. 1:279-289.
Forward, R.B., Jr. and J.D. Costlow, Jr. 1978. Sublethal effects of insect
growth regulation upon crab larval behavior. Water Air Soil Pollut.
9:227-238.
Frank, P.M. and P.B. Robertson. 1979. The influence of salinity on toxicity
of cadmium and chromium to the blue crab, Callinectes sapidus. Bull.
Environ. Contam. Toxicol., 21:74-78.
Fukai, T. and D. Vas. 1969. Changes in the chemical forms of chromium in the
standing of seawater samples. J. Oceanogr. Soc. Jap. 25:47-49.
Gerber, R.P., E.S. Gilfillan, B.T. Page, D.S. Page and J.B. Hotham. i960.
Short and long term effects of used drilling fluids on marine organisms.
In: Proceedings of the Symposium: Research on Environmental Fate and
Effects of Drilling Fluids and Cuttings, Lake Buena Vista, FL. pp.
882-912.
Hrudey, S.E. and P. Eng. 1979. Sources and chracteristics of liquid process
wastes from Artie offshore hydrocarbon exploration. Arctic 39:3-21.
Knox, F. 1978. The behavior of ferrochrome lignosulfonate in natural waters.
Master's Thesis, Mass. Inst. of Technology. 62 pp.
Lang, W.H., R.B. Forward, Jr., D.C. Miller and M. Marcy. 1980. Acute
toxicology and sublethal behavioral effects of copper on barnacle nauplii
(Balanus improvisus). Mar. Biol. 58:139-145.
Lees, D.C. and J.P. Houghton. 1980. Effects of drilling fluids on benthic
communities at the Lower Cook Inlet. In: Proceedings of the Symposium:
Research On Environmental Fate and Effects of Drilling Fluids and
Cuttings, Lake Buena Vista, FL. pp. 309-350.
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Liss, R.G., F. Knox, D. Wayne and T.R. Gilbert. 1980. Availability of trace
elements in drilling fluids to the marine environment. In: Proceedings of
the Symposium; Research on Environmental Fate and Effects of Drilling
Fluids and Cuttings, Lake Buenea Vista, FL. pp. 691-722.
McCulloch, W.L., J.M. Neff and R.S. Carr. 1980. Bioavailability of selected
metals from used offshore drilling muds to the clam Rangia cuneata and the
oyster Crassostrea gigas. In: Proceedings to the Symposium: Research on
Environmental Fate and Effects of Drilling Fluids and Cuttings, Lake Buena
Vista, FL. pp. 964-983.
Mearns, A.J., P.S. Oshida, M.J. Sherwood, D.R. Young and D.J. Reish. 1976.
Chromium effects on costal organisms. J. Water Pollut. Cntr. Fed.
48:1929-1939.
Mertz, W. 1969. Chromium occurrence and function in biological systems.
Physiol. Rev. 49:163-235.
National Academy of Sciences. 1974. Medical and Biological Effects of
Environmental Pollutants: Chromium. Division of Medical Sciences,
National Research Council. Wash., D.C., 155 pp.
Neff, J.M., R.S. Carr and W.L. McCulloch. 1981. Acute toxicity of a used
chrome lignosulfonate drilling mud to several species of marine
invertebrates. Mar. Environ. Res. 4:251-266.
Neff, J.M., W.L. McCulloch, R.S. Carr and K.A. Retzer. 1980. Comparative
toxicity of four used offshore drilling muds to several snecies of marine
animals from the Gulf of Mexico. In: Proceedings of the Svcnosium:
Research on Environmental Fate and Effects of Drilling Fluids and
Cuttings, Lake Buena Vista, FL. pp. 866-881.
Perricone, C. 1980. Major drilling fluid additives - 1979. In: Proceedings
of the Symposium: Research on Environmental Fate and Effects of Drilling
Fluids and Cuttings, Lake Buena Vista, FL. pp. 15-29.
Petrazzuolo, G. 1981. Preliminary report: an environmental assessment of
drilling fluids and cuttings released onto the outer continental shelf.
Prepared by Industrial Permits Branch, Office of Water Enforcement and the
Ocean Programs Branch, Office of Water and Waste Management. Unpublished
draft.
Pickering, Q.H. 1980. Chronic toxicity of hexavalent chromium to the fathead
minnow (Pimephales promelas). Arch. Environm. Contam. Toxicol.
9:405-413.
Ray, J.P- and R.P. Meek. 1980. Water column characterization of drilling
fluids dispersion from an offshore exploratory well on Tanner Bank. In:
Proceedings of the Symposium: Research on Environmental Fate and Effects
of Drilling Fluids and Cuttings, Lake Buena Vista, FL. pp. 223-258.
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Richards, N.L. 1979. Effects of chemicals used in oil and gas well-drilling
operations in aquatic environments. ERL-Gulf Breeze, Contr. No. 392.
Schroeder, B.C. and G.F. Lee. 1975. Potential transformations of chromium in
natural waters. Water Air Soil Pollut. 4:355-365.
Sprague, J.B. and W.J. Logan. 1979. Separate and joint toxicity to rainbow
trout of substances used in drilling fluids for oil exploration. Environ.
Pollut. 19:269-281.
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pentachlorophenol on the development of estuarine communities. J.
Toxicol. Environ. Health 3:501-506.
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 pollutant 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
pollutant.
dosage—response relationship: the characterization of the change in response
(e.g. survival) with changing stimulus (e.g. concentrations of
pollutant). 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 morphology
with abdomen bent under cephalothorax, but is sexually irmature.
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+ 6 x + £ ,^and the relevant statistics
are estimates of the parameters, a, B, and a", the variance of the random
error, £.
general linear models technique: an attempt to characterize a given response
(e.g. survival) as a linear function of factors, experimentally 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.
62
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h: hour
LC50: lethal concentration; the concentration of toxicant in water estimated
to be lethal to 50 percent of test aimals 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.
g/g: micrograms per gram = parts per million.
g/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.
mud aqueous fraction (MAF): one part by volume of used drilling mud with nine
parts seawater of the appropriate salinity. The mixture is stirred
thoroughly with an electric mixer and then allowed to settle for 20 hours.
The dark colored aqueous layer is siphoned off for immediate use in
bioassays. The undiluted supernate is 100% MAF and contains the water
soluble and fine particulate fractions of 100,000 ppm mud in water (Neff
et_ al_. 1980). Other fractions are prepared by diluting 100% MAF with
seawater.
ppb: parts per billion.
ppm: parts per million.
°/0o: 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 'ppm,1 the units of
slope are DAYS/ppm.
sublethal concentrations: concentrations of pollutant 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 concentrations 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.
63
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suspended particulate phase (SPP): one part of volume of used drilling inud
with nine parts seawater of appropriate salinity. The mud-seawater slurry
is air mixed with filtered compressed air for 30 minutes, with manual
stirring every 10 minutes. After aeration the suspension is allowed to
settle before the supernate (100% SPP) is siphoned off for immediate use
in bioassays. The SPP resembles the MAF except that SPP contains a higher
concentration of particulates and a lower concentration of volatiles.
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.
transformed to angular scale: the transformation of data expressed in
'percent* to a new scale where the V 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 pollutant.
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).
64
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
EFFECTS OF SOLUBLE FRACTIONS OF DRILLING FLUID^
AND HEXAVALENT CHROMIUM ON THE DEVELOPMENT OF
THE CRABS, RHITHROPANOP EUS HARRISII AND CALLINEi
5. REPORT DATE
g PERFORMING ORGANIZATION CODE
CTES SAPIDUS
. AUTHOR(S)
C.G. Bookhout, Robert Monroe*, Richard Foward,
J - D . Costlow, Jr., *North Carolina State Univ
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Duke University Marine Laboratory
Beaufort, North Carolina 28516
10. PROGRAM ELEMENT NO.
11 CONTRACT/GRANT NO.
CR808374
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
13 TYPE OF REPORT AND PERIOD COVERED
Final Scientific
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
1.1
The mud aqueous fractions (MAF) and suspended particulate phase (SPP)
of lignosulfonate type mud were nontoxic to the complete larval development
of Rhithropanopeus harrisii. Five percent MAF and SPP were not toxic to
Callinectes sapidus. Differential survival of C_. sapidus larvae occurred
from 5 to 50% MAF and SPP. No larvae reached the 1st crab stage in 100% MAF
and SPP. Statistical analyses of the data on survival, mortality and
behavior are presented.
Survival of R_. harrisii from hatching to 1st crab stage occurred in
to 29.1 ppm Na?CKL. Estimated LCn^ for complete zoeal developme
was 17.8 ppm NaoCr(J4 and was 13.7 Tor development to 1st crab
stage. A concentration of 1.1 ppm was nontoxic, 7.2 and 14.5 He-
were sublethal and concentrations of 29.1 to 58.1 ppm were acutely toxic.
Low concentrations of Na^CrO* caused an increase in swimming speed and
high concentrations caused a decline.
Survival of Callinectes sapidus occurred in 1.1 to 4.7 Ma^CrO,,. The
for comolete zoeal development was estimated to be 2.9 pom and the
nt
LC
50
for development to 1st crab stage was estimated to be 1.0 ppm.
istical analyses of the data on survival, duration and mortality of
larvae are presented.
Stati
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
COSATi Field/Group
Drilling fluids
HexavaIent Chromi urn
B i o a s s a y
Crustacea
Crabs
Drilling fluid toxici
Na., C-| -0^ toxi ci ty
Blue crabs
Mud crabs
Larval development
ty
18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS fTlus Report!
U n c 1 a s s i f i e d
21 . NO. OF PAGES
20. SECURITY CLASS ( This p
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ORD CLEARANCE FORM
1 EPA Report No. 2. Series 3 uab
[P/I-/TT/S '.J>- 'W EPA/600-3 ERL,GE
Document Title (State in comments block exact title as it
FECTS OF SOLfl§t?7^fflgr12fe6rDfflTLTN"n"L'^D^
D HEXAVALENT CHROMIUM ON THE DEVELOPMENT OF '
E CRABS, RHITHROPANOPEU^ HWISII AND
LLINECTES SAPfDUS "
', Product (check one)
v
[? Project Report/Summary G Research Report
C Unpublished Report D Journal Article
heck one and specify product (instructions on back):
D Meeting/Publication C Application Guide
D Response Reoort
2. Is this a "major" publication? C Yes DjNo
Check why:
D Scientific or Technical Uncertainties
EH Policy Implications
D Exceeds Fund Limitations
D Periodical
t. Signature/Date
u. ,_~<~, 4- )vo ks_~^ v 3 , j s->_
j -j
/f 0 O- "^' d_ -./ -/-
X'1^ vj // , /^ ( -=., /^'/.-- -
/
/U^£?^ 3-• ,y.. — -i' i
(/
>• Comments
Original title in TIP: EFFECTS OF SOLUBLE
DEVELOPMENT OF CALLINECTES SAPIDUS AND RHT
Office Drar; No 4 Copvr;gnt Permission
Z Yes iA;!3cned) I N. A
0205
5 Author, Orcanization. arc; Accress
C.G. ;3ckhout, -,ODe--t '-o-^ce, Richard Foward,
and J.D. Cost low, Jr.
Duke University '!arins Laboratory
8 D'ol I
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