United States
 Environmental Protection
 Agency
 Environmental Research
 Laboratory
 Gulf Breeze PL 32561
 Research.and Development
 EPA-600/S3-82-018  June 1982
 Project Summary
 Effects of Soluble  Fractions of
 Drilling  Fluids and Hexavalent
 Chromium on the Development
 of the Crabs, Rhithropanopeus
 harr/si/and Callinectes sapidus
j*
      V

 C. G. Bookhout, R. Monroe, R. Forward, and J. D. Costlow, Jr.
  This study is part of a series of investi-
 gations  undertaken to determine the
 effect of the discharge of drilling fluids
 on marine fauna. The first division of this
 project was to determine the range of
 concentrations of the mud aqueous frac-
 tion (MAP) and the suspended particulate
 phase (SPP) of a low-density Kgnosul-
 f onate type mud with f errochrome added
 (No. 4 mud) which would affect swim-
 ming behavior, survival and duration of
 development of the mud crab, Rhithrop-
 anopeus harrisii, and the blue crab, CaH-
 nectes sapUus, from the time of hatching
 until the 1 st crab stage is reached. As far
 as is known, this is the first investigation
 on the effect of MAP and SPP, soluble
 fractions of whole mud, on the complete
 larval development of crabs. These frac-
 tions would be found in the upper plume
 of discharges of an oil well.
  The percent survival from hatching to
 megalopa and to 1st crab stage was
 90% or over in seawatar control and in
 concentrations from 5% (5,000 ppm) to
 100% (100,000 ppm) MAP and SPP in
 three replicate series of R. harrisii larvae
 tested.
  There was differential survival of Calli-
nectes sapidus from hatching to 1 st crab
stage in concentrations from 5% to 50%
MAP and SPP, but no larvae reached the
1 st crab stage in 100% MAP or SPP. Sta-
tistical analyses of the data on survival,
mortality and behavior are presented.
  A second division of the project was to
determine the  effect of  hexavalent
chromium, Na2CrO4, on the complete
larval development of R. harristt and C.
sapidus from the time of patching to 1 st
crab stage. The effects of different con-
centrations of Na2CrO4 on survival and
duration from hatch to megalopa and
hatch to 1 st crab stage are given, and
the most sensitive stages of larvalaVtvel-
opment were determined for each •JPcies.
Statistical analyses of the data fiH sur-
vival, duration, rate of molting, mortality
and behavior are presented.
  This Project Summary was developed
by EPA's Environmental Research Labo-
ratory, GuHBreexa, FL, to announce key
findings of the research project that is
f titty documented in a separate report of
the same title {sea Project Report ordering
information at back).

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                            Part I—Effects of Soluble Fractions of Used Light-Weight
                   Lignosulfonate Type Mud on the Complete Larval Development of Crabs,
                                 Rhithropanopeus harrisii and Callinectes sapidus
Introduction
  The Jay Exxon well drilling fluid 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  U.S.   Environmental  Protection
Agency, Environmental Research Labo-
ratory, Gulf Breeze,  Florida. Although
the well was land-based, it was believed
that the chemical components and phys-
ical characteristics of the drilling fluid
were similar to those of offshore wells.

  Investigators have evaluated the tox-
icity of five components of whole mud
following  the classification originally
proposed by Neff eta/. (1980). The mud
aqueous fraction (MAF) and suspended
paniculate phase (SPP) aretwofractions
which have been most intensively inves-
tigated in toxicity tests and are two frac-
tions 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 it contains a higher concentration of
particulates and a lower concentration
of volatiles. These  two  fractions are
found in the upper plume of discharge
and remain in the water column longer
than other fractions, and, hence, may be
the fractions which might be expected
to affect larvae of marine organisms  as
well as plankton.

  At present, the impact of drilling fluids
is incompletely known. Most investiga-
tions  have been  acute toxicity  tests.
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.  In this investigation,
chronic tests were employed because a
study covering the entire larval develop-
ment of crabs would give a better evalu-
ation of possible toxicity of drilling fluids
in the field than an acute toxicity study
of 96 h. A  chronic toxicity test would in-
clude all periods of molting when crusta-
cean larvae are known to be very sensitive
to toxic substances. Furthermore, it is
possible to determine which stage or
stages in the larval development are par-
ticularly sensitive if mortality is recorded
at each stage of development.
Results and Conclusions
  The percent survival to megalopa and
to 1st crab stage of Rhithropanopeus
harrisii was 90% or over in seawater
control and in all concentrations of MAF
and SPP. There was no consistent reduc-
tion in survival in concentrations of MAF
and SPP compared to  survival  in sea-
water control. Hence, 100% MAF and
100% SPP light-weight lignosulfonate
type mud is non-toxic to developing lar-
vae of R.  harrisii throughout complete
larval development.

   The average percent survival of three
 series of  Callinectes sapidus reared in
 seawater control and four concentrations
 of MAF and SPP is listed in Table 1. There
 was little difference between C. sapidus
 survival to megalopa and to 1 st crab in
 5% MAF and seawater control, but sur-
 vival in 5% SPP was less than in control.
 There was differential survival, however,
 from 5%  MAF and SPP to 100% MAF
 and SPP.

   Statistical analysis  revealed for C.
 sapidus zoeal survival to megalopa that
 there was approximately 4% decrease/
 10% increase in  MAF  @ 50% CONC.,
 and for survival to 1 st crab, there is ap-
 proximately 3% decrease/10% increase
 in MAF @ 50% CONC. For C. sapidus
 zoeal survival to megalopa and for survival
 to 1 st crab, there was approximately a
 5% decrease in survival for a  10% in-
 crease in SPP near 50% SPP CONC.
   From Table 1 it can be observed that
 there was no significant  difference in
 duration in zoeal development of C. sapi-
 dus and in hatch to 1 st crab in seawater
 control and in the concentrations of MAF
 and SPP employed.

  The results illustrated in Figure 1 show
the effect of MAF levels on the mortality of
larvae at each of the seven to eight zoeal
stages and a megalopa stage through
which C. sapidus passes. 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 sensi-
tive, larvae in zoeal stage II were also
very sensitive, for significant  increases
in mortality over the previous  stage oc-
curred in this stage in all media (Figure
1).

  Mortality of C.  sapidus 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 (Figure 2).
For zoeal stage  I, mortality in 25% SPP
was significantly different from the con-
trol at the  0.05 level. As in  the  MAF
experiment, zoeal stages I and II of C.
sapidus were the most  sensitive in all
media. In general, SPP was more toxic to
blue crab larvae than  MAF.
  Blue crab larval behavior is affected by
exposure to MAF and SPP with the gen-
eral effect being a decline in swimming
speed. A significant reduction in speed
was only observed in  100% MAF, but it
was found in  all percentages of SPP
tested.

  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 15m
intermediate toxic zone, around the dis-
charge source  long  enough  to  suffer
mortality are remote.  If by chance a few
1 st 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.

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Table 1. Average Percent Survival and Average Duration in Days of Zoeal and Megalopa Development of Three Series
(Cs l-lll) of C . sapidus in Seawater Control and Different Concentrations of MAP and SPP of Used Lignosulfonate Type
Mud
Culture Media
Salinity 30°/oo
Temp. 25 °C
Seawater Control
5% MAP
25% MAP
50% MAF
100% MAF
5% SPP
25% SPP
50% SPP
100% SPP
Initial No.
of Larvae % Survival to Mean Duration of Development in Days
per Series Megalopa 1st Crab Zoea Megalopa Hatch to 1st Crab
Csl-50
Csll-50 37.3 28.0 34.9 7.8 42.5
Cslll-50
Csl-50
Csll-50 38.0 31.3 34.8 7.3 41.9
Cslll-50
Csl-50
Csll-50 27.3 23.3 34.8 7.1 42.9
Cslll-50
Csl-50
Csll-50 10.0 8.7 35.9 6.6 42.6
Cslll-50
Csl-50
Csll-50 0.7 - - - -
Cslll-50
Csl-50
Csll-50 31.3 26.7 32.4 7.3 41.3
Cslll-50
Csl-50
Csll-50 15.3 12.0 36.2 7.3 43.7
Cslll-50
Csl-50
Csll-50 4.0 4.0 35.8 7.8 43.6
Cslll-50
Csl-50
Csll-50 00-- -
Cslll-50

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  100
   SO
   70
   60
   50
•
   40
   30
   20
   10
       700%-?
                                                              WO
                                                               90
                                                               80
                                                               70
                      K 60
                      fc

                     I
                     f 50

                     I
                     * 40
                                                               30
                                                               20
                                                               10
                                                                    100%
                                                                    50%
                                                                   25%
             /     //    ///    IV    V

                             Stage
VI   VII   VIII   M    Figun2
 Figure 1.     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. Sionificantly different from control (0.01)
               *. Significant increase over previous stage (0.05)
               **. Significant increase over previous stage (O.O1)
~/    ll   Hi    Iv    V    W    VIIVlJl   ~M

                 Stage

 Effect of SPP of used light- weight lignosulfonate
 type mud on mortality by stages of development
 ofC. sapidus.
  a.  Significantly different from control (O.O5)
  b.  Significantly different from control (0.01)
  *.  Significant increase over previous stage (0.05)
  **.Significant increase over previous stage (0.01)

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                       Part II—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 environ-
mental effect is chromium. The toxicity
of chromium to marine organisms varies
with valence state, pH and oxidation
states. Hexavalent chromium (Cr + 6) is
stable in seawater.  It often appears as a
soluble  chromate  or dichromate  and
both are powerful  oxidants which can
readily penetrate biological membranes
and irritate cells (Mertz, 1969). Hexava-
lent chromium, as chromic oxide, chro-
mate or dichromate, reacts with organic
matter in acid solution, leading to the tri-
valent form (Cr+3). The trivalent form is
associated chiefly with paniculate matter
such as clay, which suggests that organic
paniculate matter may reduce and bind
the hexavalent form in  solution. Hexa-
valent 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. Ferrochrome lignosulfonate,
brandname "Q-Broxin," and chrome lig-
nosulfonate are  common additives to
drilling fluids which contribute to  Cr
enrichment.  Initially, these  additives
contain hexavalent salts, but at tempera-
tures between 120 and  175 °C hexava-
lent chromium is converted  to the trivalent
state. The  properties of both of these
additives can be restored at temperatures
between 120 and  175°C  by adding
moreCr + 6.

  It has 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. Once freed from
clay particles, 3 to 7% Cr + 3 through
slow oxidation may revert to Cr + 6.
  From the above discussion, we con-
clude  that under certain conditions the
possibility exists that both trivalent and
hexavalent  chromium may  be in  the
vicinity of the discharge source.
  This investigation was undertaken to
determine the concentrations of hexa-
valent chromium, Na2CrO4, which are
nontoxic, sublethal and acutely toxic to
the complete larval development of the
mud crab, Rhithropanopeus harrisii, and
the blue crab, Callinectes sapidus.
Results and Conclusions

  In experiments on the effect of hexa-
valent chromium, Na2Cr04, on the de-
velopment of Rhithropanopeus harrisii, it
was observed that there were no signifi-
cant differences between survival from
hatching to  megalopa and hatching  to
1 st crab in  seawater control and 1.1
ppm Na2Cr04, but there was differential
survival from 1.1  to 29.1 ppmNa2Cr04
(Table 2). The estimated LC50 for zoeal
development from hatching to megalopa
was 17.8 ppmNa2Cr04, and from hatch-
ing to 1 st crab, the estimated LC50 was
13.7 ppm Na2CrO4.
  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 there was 0.122 ± 0.021 days  in-
crease in total duration time from hatching
to 1 st crab for each ppm added Na2Cr04.
  From Figure 3, it  can be seen that 1
ppm Na2Cr04 is nontoxic to /?.  harrisii
larvae, for there is no more mortality in
this concentration  than  in seawater
control. There is differential larval  mor-
tality from concentrations of 1.1 ppm to
58.1  ppm Na2CrO4. Concentrations  of
7.2 ppm and  14.5 ppm  Na2CrO4 are
considered sublethal,  since more  than
10 percent of R. harrisii larvae reached
the 1st crab stage. Concentrations  of
29, 41, 46 and  58 ppm Na2CrO4 are
acutely toxic to R. harrisii larvae, since
less than  10 percent reached the 1st
crab stage in 29 ppm Na2Cr04 and none
reached the 1 st crab stage in 41,46 and
58  ppm Na2Cr04.
  Rhithropanopeus larval swimming speed
was affected by exposure to Na2CrO4.
In general, low sublethal concentrations
caused an increase in swimming speed
and concentrations near those which are
acutely toxic caused a decline.
  In experiments on the effect of hexa-
valent  chromium, Na2Cr04, on the de-
velopment of Callinectes sapidus, it was
observed that survival from hatching to
1st crab occurred in 1.1 to 4.7  ppm
Na2Cr04 (Table  3). There was better
survival in 1.1 ppm Na2CrO4 than in sea-
water control, but there was differential
survival from 1.1 to  7.2 ppm Na2CrO4.
The LC50 for complete zoeal develop-
ment of C. sapidus was estimated to be
2.9 ppm Na2CrO4, and the LC50 for de-
velopment  from  hatching to  1st  crab
stage was estimated to  be 1.0  ppm
Na2Cr04.
  Statistical analysis of the data on C.
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 Na2CrO4,
and that there was 1.31 ±0.29 days in-
crease in total duration time from hatch-
ing to 1 st crab stage for each ppm added
Na2Cr04.

  From Figure 4, it  can  be seen  that
there is significantly  less mortality of C.
sapidus larvae in 1.1  ppm Na2Cr04 than
in seawater control. There is also less lar-
val mortality in 2.4 ppm Na2Cr04, but it
is not  significantly  different from the
control, and hence it is considered  non-
toxic. There is differential mortality of C.
sapidus larvae from  concentrations of
4.7 to 7.2 ppm Na2CrO4, and these con-
centrations are considered acutely toxic,
since less than 10 percent of C. sapidus
larvae reached the 1 st crab stage.  Blue
crab larvae in  zoeal stage III were ex-
tremely sensitive to  7.2 ppm Na2CrO4,
and larvae in zoeal stages III, IV and V
were most sensitive in 4.7 ppm Na2CrO4.

  For most discharges, the background
concentration for chromium has been
reported  to be reached  approximately
100 to 150 meters from the point of dis-
charge, depending on the amount and the
rate of discharge, as well as the currents.
Within this area, entrained crab larvae
would  undoubtedly  absorb Cr + 6 more
readily than Cr + 3, if  both were present,
and bioaccumulate chromium. It is ques-

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Table 2. Average Perct
Control and ir
Culture Media
Salinity 3O°/oo
Temp. 25 °C
Seawater Control





1. 12 ppm
Na2CrO4




7.17 ppm
Na2CrO4




14.52 ppm
Na2CrO4




29.09 ppm
Na2CrO4




int Survival and Average Duration in Days of Zoeal and Megalopa Development of R. harrisii in Seawatei
i Different Concentrations of Hexavalent Chromium, Na2Cr04
Initial No.
of Larvae % Survival to Mean Duration of Development in Days
per Series Megalopa 1st Crab
Rhl-50
Rhll-50
Rhlll-50 95.0 93.7
RhlV-5O
RhV-50
RhVI-50
Rhl-50
Rhll-50
Rhlll-50 94. 7 93. 7
RhlV-50
RhV-50
RhVI-50
Rhl-50
Rhll-50
Rhlll-50 83.3 68.7
RhlV-50
RhV-50
RhVI-5O
Rhl-50
Rhll-50
Rhlll-50 62.7 47.3
RhlV-50
RhV-50
RhVI-5O
Rhl-50
Rhll-50
Rhlll-50 22.0 7.0
RhlV-50
RhV-50
RhVI-50
Zoea Megalopa Hatch to Crab


11.9 6.7 19.0





12.1 6.6 18.8





12.7 6.3 19.0





13.6 6.4 19.8









tionable, 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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Table 3.     Average Percent Survival and Average Duration in Days Through Zoeal and Megalopa Development of Three Series
            (Cs l-lll) of Callinectes sapidus Reared in Seawater Control and in Different Concentrations of Hsxavalent Chromium,
            Na2Cr04
       Culture Media
      Salinity 3O%o
        Temp. 25 °C
Initial No.
of Larvae
per Series
      % Survival to
                  Mean Duration of Development in Days
Megalopa
1st Crab
Zoea
Megalopa
Hatch to 1st Crab
Seawater Control


1. 1 ppm
Na2Cr04

Csl-50
Csll-50
Cslll-50
Csl-50
Csll-50
Cslll-50

61.3


80.6


38.0


44.7


33.5


34.5


7.6


7.2


39.6


42.0

          2.4 ppm
          Na2CrO4

          4.7 ppm
          Na2Cr04

          7.2 ppm
          Na2Cr04
 Csl-50
 Csll-50
 Cslll-50
 Csl-50
 Csll-50
 Cslll-50
 Csl-50
 Csll-50
 Cslll-50
  65.3
   14.0
 41.3
   7.3
36.1
40.0
   7.6
   8.0
      43.2
      45.3
   100

    90


    80}
 ft  70
 o
 S
    60
 I  50
    30

    20


    10i
     0
                               1 ppm
                 100
         I     II    III   IV    M

                 Stage

Figure 3.  Effect of Na2CrOt in ppm on
           mortality of ft. harrisii larvae.
           a. Significantly different from
             control (0.051
           b. Significantly different from
             control (0.01)
           * Significant increase over
             previous stage (0.05)
          **. Significant increase over
             previous stage (0.01)
                      '   II  III  IV V  VI  VII VIIIM

                                Stage

               Figure 4.   Effect of Na2CrO4 in ppm
                          on mortality of C. sapidus
                          larvae.
                          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)

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       C. G. Bookhout, Robert Monroe, RichardFoward, andJ. D. Costlow. Jr.. are with
         Duke University Marine Laboratory. Beaufort. NC28516,
       Charles McKenney, Jr., is the EPA Project Officer (see below).
       The complete report, entitled "Effects of Soluble Fractions of Drilling Fluids and
         Hexavalent Chromium on the Development of the Crabs,  Rhithropanopeus
         harrissi and Callinectes sapidus," (Order No. PB 82-197 203; Cost: $9.00,
         subject to change) will be available only from:
              National Technical Information Service
              5285 Port Royal Road
              Springfield, VA 22161
              Telephone: 703-487-4650
       The EPA Project Officer can be contacted at:
              Environmental Research Laboratory
              U.S. Environmental Protection Agency
              Gulf Breeze, FL 32561
      U.S GOVERNMENT PRINTING OFFICE. 1982—559-092/3417
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Postage and
Fees Paid
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Agency
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Official Business
Penalty for Private Use $300
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