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.

-------
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

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     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

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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

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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

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   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

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.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

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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

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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

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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

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       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

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          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

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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

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                                  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

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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

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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

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                                   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

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     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

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                                    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

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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

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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

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                                 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

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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

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 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

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  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

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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

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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

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  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

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        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

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       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

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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

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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

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          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

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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

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     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

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£. 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

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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.


                                      58

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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.
                                      59

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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.
                                      60

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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.

Tagatz, M.E., J.M. Ivey, J.C. Moore and M. Tobia.   1977. Effects  of
     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
-------
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
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j -j
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>• 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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