PHYSIOLOGICAL EFFECTS OF DRILLING MUDS ON REEF CORALS
                Alina. Szmant-Froelich
              Department of Oceanography
               Florida State University
             Tallahassee, Florida  32306
                   CR-807345-01-0
                   Project Officer

                    Thomas W. Duke
          Environmental Research Laboratory
             Gulf Breeze, Florida  32561
          Environmental Research Laboratory
          Office of Research and Development
              U.S. Environmental Agency
             Gulf Breeze, Florida  32561

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                                 DISCLAIMER

     Although the research described  in this article  has  been  funded  wholly
or in part by the United States Enviromental Protection Agency under
Cooperative Agreement 807345 to Alina Szmant-Froelich, Florida State
University, Tallahassee, Florida,  it  has  not been  subjected  to the  Agency's
required peer and administrative review and, therefore, does not  necessarily
reflect the view of the Agency; no  official endorsement should be  inferred.

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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  EPA
Environmental Research Laboratory, Gulf Breeze, contributes to this  information
through research  programs aimed at determining:

       the effects of toxic  organic pollutants  on individual  species  and
       communities of organisms;

       the effects of toxic  organics on ecosystem processes and components;

       the significance of chemical carcinogens in  the estuarine  and  marine
       environments.

     This report  describes the toxicological  and  several physiological
responses of two  species  of  coral, Montastrea annularis and Acropora
cervicornis, after exposure  to fluids produced  by drilling operations  for  oil
exploration.  Although these fluids originated  from a land-based  operation and
were not to be disposed at sea, their characteristics closely resembled those
that are released in marine  waters.  The research data when coupled  with
information related to environmental levels  of  fluids used in offshore drilling
will contribute to a hazard  assessment  of the impact of drilling  fluids on the
marine environment.
                                           Henry  F.  Enos
                                           Director
                                           Environmental Research  Laboratory
                                           Gulf Breeze, Florida

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                                  ABSTRACT

        Discharge of drilling muds, a by-product  of oil  drilling,  could have
a detrimental effect on some marine organisms.  This research  was  designed  to
test the effects of exposure to drilling muds  on  coral  physiology.

        Coral from two species, Montastrea annularis and Acropora
cervicorni s, were exposed in the laboratory to  concentrations of  0,  1,  10
and 100 ppm drilling mud for two days to seven  weeks.   Several  physiological
functions of the coral animal (calcification rate,  respiration  rate)  and of
their zooxanthellae (photosynthesis rate, nutrient  uptake rate) were
monitored at regular intervals during the exposure  periods.   In addition,
biomass parameters (tissue nitrogen, zooxanthellae  cell  density,  chlorophyll
content) were measured at two-week intervals during the  long  exposure and  at
the end of each shorter exposure.

        Initial long-term exposures of pieces  of  _M. annularis to  a  series  of
drill muds (designated JX-2 through JX-7) collected from a  Jay  (Florida)
oil-field well  produced a significant reduction  in  calcification,
respiration, and N03-uptake rates during the fourth week of  exposure  to 100
ppm drill mud.   Photosynthesis and NH4-uptake  rates also decreased  during
the fifth week of exposure.  Normal feeding behavior was absent from  these
corals when tested during the sixth and seventh  weeks  of exposure.   Several
corals exposed to 100 ppm died during the fifth  and sixth weeks.

        Short-term (2 to 5 days) exposures of  pieces of  _M.  annularis  to 100
ppm JX-7 mud (the drill mud used during weeks  5  and 6,  which  had  a  much
higher Cr and hydrocarbon content than muds used  during  weeks 1 to  3) caused
large reductions in calcification, and, to a lesser degree,  in  respiration,
gross photosynthesis, and N03 uptake rates in  one of two experiments.  A_.
cervicornis showed a large reduction in calcification  after  12  hours  of
exposure to 100 ppm JX-7, and a decrease in N03-uptake  within 24  hours.  No
coral deaths occurred during these short tests.

        Implications of the results are discussed,  and  recommendations  are
given for future studies.

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                               CONTENTS







Foreword	iii



Abstract	   iv




Figures and Tables	   vi



Acknowledgments 	  vii





   1.  Introduction  	    1



   2.  Conclusions   	    4



   3.  Recommendations 	    6



   4.  Materials and Methods



            First Experimental Series - Stage I 	     7



            Second Experimental Series - Puerto Rico  	    12



            Data Analysis 	 .....    14



   5.  Results and Discussion



            Coral Survivorship	    15



            Physiological Rates - Stage I 	    15



            Physiological Rates - Puerto Rico 	    23



References	    29



Appendices



   A. Stage I experimental data summary	    32



   B. Summary of statistical analyses of Stage I data 	    35

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                          FIGURES

1  Diagram of exposure system 	  10
2  Daytime calcification rates, Stage I experiment  	  16
3  Respiration (A02) rates, Stage I experiment  	  17
4  Respiration (AC02) rates, Stage I experiment 	  17
5  Gross photosynthesis (A02) rates, Stage I experiment    .  .  19
6  Gross photosynthesis (AC02) rates, Stage I experiment   .  .  19
7  NH4+ Uptake rates, Stage I experiment  	  20
8  N03- Uptake rates, Stage I experiment  .	20
                           TABLES

1  Testing schedule, Stage I experiment 	  8
2  Drilling mud schedule for Stage I experiment 	  9
3  Feeding behavior of drill mud-exposed coral colonies ... 21
4  Summary of coral and algal biomass for Stage I
     experimental corals  ...... 	 22
5  Summary of results of five-day exposure of Montastrea
     annularis to JX-7 drilling mud (Puerto Rico -Test 1)  .  . 24
6  Summary of results of three-day exposure of Montastrea
     annularis to JX-7 drilling mud (Puerto Rico -Test 2)  .  . 26
7  Summary of results of two-day exposure of Acropora
     cervicornis to JX-7 drilling mud (Puerto Rico -Test 3)  . 27
8  Summary of coral and algal biomass of Puerto Rican
     experiments	28
                             VI

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                                ACKNOWLEDGEMENTS

        This study is the result of the dedicated  effort  of  many  students  and
technicians who spent many days and nights,  often  under  unpleasant  conditions,
in order to keep the experiments going twenty-four hours  a day.   Virginia
Johnson and Ted Hoehn did most of the alkalinity titrations  and  ran the  Auto
Analyzer; Dr. Roger Adam and Jeff Parker did the TC02  analyses,  built  our
experimental facilities and organized efforts the  first  summer;  James  Battey,
Jason Smith, and Esther Fleischmann, graduate students of Dr.  James W. Porter,
Univiversity of Georgia, did the oxygen analyses both  summers, and  constructed
the experimental equipment the second summer.  J.  Parker, T. Hoehn, J. Battey,
and E. Fleishmann did the chlorophyll analyses, and T. Hoehn also did  many of
the zooxanthellae counts and carbohydrate analyses.  Gregg Stanton, Les
Parker, Dr. James Porter, and J. Parker collected  the  corals used the  first
summer.  Many staff members and students at  the department of  Marine Sciences,
University of Puerto Rico, made our second summer  effort  successful  and
enjoyable; principal among them are Dr. Manuel  Hernandez-Avila,  the director,
who gave us his open cooperation, Dr. Tom Tosteson, Sofia Gil-Turnes,  and
Linda Riggs.

        We also thank EPA personnel Ted Gaetz and  Herb Fredickson for  their
patience and help with logistics during our  summer on  Stage  I.
                                      VI 1

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                                SECTION  1.

                               INTRODUCTION


        Drilling muds  are  a  necessary  by-product  of  oil-drilling.   The muds
serve to lubricate the drill-string,  remove  cuttings,  maintain  hydrostatic
pressure, prevent pipe corrosion,  and  seal the  bore  hole  in  porous
formations.  Drilling  muds are' a complex mixture  of  clay  minerals  or
polymers, barite, and  a series of  chemical additives which  vary to  suit the
drilling conditions.   Many of  these additives,  such  as ferrochrome
lignosulfonate,  fuel  oil,  and  some proprietary  chemical  additives  are
considered toxic and  hazardous to  living organisms  (Richards,  1981).

        Disposal of used drilling  muds recently has  become  an  environmental
concern.  A common procedure is to discharge the  muds  from  the  drilling rig
into surrounding waters.  An alternative used in  many  nearshore areas is to
remove the spent muds  by barge, either to deeper  waters,  or  to  chemical
waste burial  sites on  land.   The latter procedure is obviously  more
expensive and has only been  used in selected sites where  there  was  concern
that the toxic mud components  might enter the human  food  chain  or  damage
ecologically  sensitive marine  communities.

        Since used muds are  generally  dumped into the  immediate vicinity of
the drilling  rig, it  is important  to  identify marine communities or
organisms that might  be adversely  affected by exposure to drilling  muds.

        Drilling activities  on the outer continental shelf  of  the  Gulf of
Mexico are approaching the East and West Texas  Flower  Gardens—two  unique,
submerged coral  reefs  (Bright  and  Pequegnat, 1974).  These  reefs are the
only two extensive coral communities  in the  northern Gulf of Mexico, and
have formed on salt domes—formations  that often  contain  gas or oil.
There is concern about how the prolonged discharge  of  large  quantities of
drilling mud  on  or near the  Texas  Flower Gardens  will  affect the health and
viability of  these reef ecosystems (Science, 1979).  The  ecologically
dominant reef corals  are known to  be  sensitive  to high siltation (Dodge and
Vaisnys, 1977; Dodge  et_ ji]_., 1974; Loya, 1976), such as would  result from
the discharge of muds  onto the reef,  and also to  oil  pollution (Loya and
Rinkevich, 1980), which might  result  from an oil  additive or contaminant in
the drilling  muds or  from an accidental  oil  spill.   Since reef corals are
responsible for  reef  framework building, as  well  as  for much of the primary
production in the reef ecosystem,  their survival  is  essential  to the
integrity of  the reef  system as a  whole.  Previous  studies  have
concentrated  on  behavioral (polyp  expansion), growth  rate,  and lethal

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effects of short-term exposure of corals to drilling muds (Thompson,  1930;
Thompson _et_ a_l_., 1980).  Those studies showed that concentrations greater
than 100 ppm of drilling mud cause reduced polyp expansion in several
species, and concentrations greater than 1000 ppm cause death in several
species within 65 hours.  Other experiments, some of which were
non-quantitative and thus difficult to reproduce, indicated a decreased
growth rate after direct application of drilling mud slurries to the  coral
surface.

        Building on these results, the present study has focused on several
coral physiological and biochemical processes that might be affected  by
short- and long-term exposure to drilling muds.  Calcification and
respiration rates were chosen as indicators of animal  functions, and
nutrient uptake and photosynthesis rates as indicators of zooxanthellae
function.  (Zooxanthellae are the small, symbiotic algae that live within
most reef coral tissues.)  Animal and algal biomass were also measured as a
function of time to monitor for any deterioration of nutritional status
during the exposure period.  When coral polyps are fully retracted they
cannot feed, and the amount of light that reaches their zooxanthellae is
reduced.  Therefore, prolonged periods of polyp retraction could gradually
starve the corals.

        Montastrea annularis was chosen as the primary test species because
of its ecological importance in the Texas Flower Gardens (Tresslar, 1974)
and throughout the Caribbean (Goreau, 1959).  A second species, Acropora
cervicorni s, was used in later tests to compare our experimental
procedures and results with those of EPA-funded investigators studying this
species (E. Powell, Texas A & M).

        Initially, groups of corals were exposed in the laboratory to four
mud concentrations (0 ppm, 1 ppm, 10 ppm, and 100 ppm) for six weeks.  The
mud-exposed corals were fed during the experiments.  Two control groups
were used:  one control group was fed periodically throughout the
experimental  period; the second control group was not fed to simulate the
starvation effects expected in the exposed groups.  Previously listed
physiological  parameters were measured at biweekly intervals.  Respiration
and photosynthesis were measured both as changes in 03 and changes in C02
in the media;  calcification was measured as the decrease in
total-alkalinity (TA) of the media, and nutrient uptake was measured  as the
disappearance of N03" and NH4+ from the media.  All methods chosen were
non-destructive, which allowed us to test individual corals repeatedly
throughout the exposure period.  A second set of experiments measuring the
same physiological  parameters, focused on the short term (2 to 5 days)
effects of one of the more toxic muds used in the first experimental
series.

        The studies were conducted as a cooperative agreement between
Florida State  University and U.S. EPA Environmental Research Laboratory,
ERL, Gulf Breeze Florida, with the additional participation of Dr. James  W.
Porter and several  of his graduate students from the University of Georgia.
The first experiments were conducted during July and August, 1980, in a
laboratory provided by the EPA. on the U.S. Navy Stage I platform located

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12 miles offshore from Panama City, Florida.  The site was  selected because
its clear oceanic seawater was suitable for maintaining corals in a healthy
state, and because of its proximity to both to both Florida State
University and ERL, Gulf Breeze.  The second experiments were conducted a
year later at the marine laboratory of the Department of Marine Sciences,
University of Puerto Rico, La Parguera, P.R., which offered easy access to
freshly collected coral  specimens.

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                                  SECTION 2.

                                 CONCLUSIONS

        The results of the first experiment show that the reef coral
Montastrea annularis can be adversely affected by long-term (more than
three weeks) exposure to drilling muds.  Adverse effects ranged from an 84%
reduction in calcification rate and 40% reduction in coral  respiration rate
after a six-week exposure to 100 ppm drilling mud to lesser effects on
photosynthesis by the zooxanthellae of these same corals.  Several'of the
corals exposed to 100 ppm drilling mud died during the fifth and sixth
weeks, while none of the corals in the other treatments died.( In addition,
the corals exposed to 100 ppm drilling mud for six weeks lost  normal
feeding response and 20% of their zooxanthellae, while those in the other
treatments did not.

        Since different batches of drilling mud were used during the 6-week
experiment (collected from one oil well in Jay, Florida, during an ongoing
drilling operation and presented to the corals in the same  time-sequence as
collected), it was not clear whether the absence of any discernible
physiological effect during the first three weeks, and the  sudden decay in
the corals' physiological functions during the fourth week, were due to a
cumulative time effect or to a greater toxicity of the batches of drilling
mud used after the third week.  The muds used during the last  three weeks
of exposure (JX-5 and JX-7) contained higher concentrations of chromium and
hydrocarbons than mud used earlier in the experiment (JX-2  to  JX-4)
(Gilbert and Kakareka, New England Aquarium, unpublished).

        The second set of experiments, in which we exposed  specimens of M.
annularis and Acropora cervicornis to mud JX-7 for up to five  days, showed
that there is a considerable amount of variability in the response of
different coral colonies to drilling mud.  The first specimens of M_.
annularis exposed to 100 ppm of JX-7 suffered a 20% decrease in   ~~
calcification within 24 hours of exposure and a 40% decrease by the fifth
day of exposure, with smaller decreases in respiration, photosynthesis, and
nutrient uptake rates.  A second set of specimens of _M. annularis collected
from a single large colony (from the same reef where the first specimens
were collected), showed no adverse effects after three days of exposure to
100 ppm JX-7.  _A. cervicornis suffered a 50% decrease in calcification
within 12 hours~of exposure to 100 ppm of JX-7, and a 40% reduction in
N03~uptake within 36 hours.

        The conclusion from both sets of experiments is that short-term
exposures (less than two days) to concentrations of 100 ppm drilling mud
may cause a large decrease in calcification rate in some colonies of these
coral  species.  Longer exposures, however, especially when  more toxic

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drilling mud additives  are  used,  increase the  chance  that  sublethal  and
lethal  effects  will  occur.   Concentrations higher  than  100 ppm  will
probably have an  effect much sooner,  and  concentrations of 10 ppm or less
are unlikely to have an effect  in  exposures as long as  one to two months.
These results,  however, are only  indicative of what might  occur in a fully
developed oil field  where corals  may  be exposed for prolonged periods (six
months  to several  years) to intermittent  and variable doses of  drilling
mud.

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                                  SECTION 3.

                               RECOMMENDATIONS

         Initial studies were undertaken and designed with little
information on expected exposure concentrations and duration.  It appears
that a realistic exposure regime for corals on a reef adjacent (within 1
km) to a single drilling rig would be frequent (1 to 3 times per week)
exposures of short duration  (3 to 12 hours) to concentrations below 100 ppm
of whole muds over a period of three to six months.  Corals on a reef
situated amidst an oil field probably would be subjected to higher
concentrations for a longer duration of time.  Only corals situated within
about 100 meters of a  rig should encounter higher concentrations or
problems of burial beneath drilling mud.  Therefore, any future studies
should concentrate on  experiments designed to determine the effects of
repeated exposures and the factors that might affect recovery between
exposure episodes.

         A second recommendation is that the composition of the drill muds
to be used be determined before the tests are conducted, or that "typical"
muds for the drill site in question be used in the tests.  Tests with
individual additives would also be useful to identify the source of the
toxicity.  Critics of  drilling mud studies contend that the muds used to
expose the organisms was "atypical" or "not meant for discharge."  The Jay
muds used in our study were from a terrestrial, not an offshore well; but
we have  no information to indicate that the ingredients of these muds, save
one, were any different from those of muds used offshore.  Present U.S.
regulations prohibit the use of fuel oils as a lubricant in drilling muds
discharged offshore, and fuel oil was a component of the JX-5 and JX-7
muds.  However, in spite of the regulations, hydrocarbon residues
indicative of fuel oil have been detected in discharged offshore muds in
the Gulf of Mexico (Weichert _et_ _aj_., 1981).  While not added as a routine
ingredient, fuel oil  is used "aT the discretion of the drilling engineer on
an as-needed basis to  free stuck drill strings, even in offshore waters.
In addition, other countries where drilling is occurring near or on coral
reefs, such as the Philippines, Mexico and Trinidad, do not regulate the
composition of the drilling muds used as strictly as the United States.

        A final recommendation is that future studies should be concerned
with dispersal characteristics of different fractions of the mud.  Heavy
particulates will  settle quickly over a small downstream area where corals
may be both buried and poisoned.  Light particulates and dissolved
fractions will disperse over larger areas, but in lower concentrations, and
potential effects will  be limited to those associated with chemical
toxicity.  The solubility of many of the biologically active additives
gives reason to believe that much of the potential toxic activity will be
in the dissolved fraction.

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                                  SECTION 4.

                            MATERIALS AND METHODS

FIRST EXPERIMENTAL SERIES - STAGE I

Coral Collection and Maintenance

        At the outset EPA agreed to  provide  coral  specimens  from the Texas
Flower Gardens for our study, but there were  logistical  problems in
providing them and we were authorized to obtain  corals  from  the Florida
Keys.  There are no reasons to believe that  there are any basic
physiological differences between the two populations.

        Colonies of Montastrea annularis and  Madracis decactis  were
collected by scuba divers at depths  of 4 to  10 m from reefs  near Big Pine
Key, Florida.  Large heads of M_._ annularis were  broken  into  smaller  pieces
with a chisel and hammer.  The pieces of coral were  placed in  submerged
buckets and transferred to large coolers without exposure to air.  The
coolers were kept overnight in a running seawater holding tank  and
air-shipped the next morning to Panama City-,  Florida.  The pieces of coral
were put into individual plastic bags to minimize damage from  abrasion
during shipping.  The coolers were immediately transferred to  Stage  I by
boat, where the individual bags were suspended in large aquaria of running
seawater.  Stage I seawater was admitted to  the  bags slowly  over a 2 h
period to minimize shocking from change in temperature  and water
conditions.  The corals appeared to  be in good condition, and  most were
fully expanded within a few hours after transfer to  tanks.

        The Madracis colonies were used by EPA personnel for behavior
studies.

        The corals were maintained in five 202-L glass  aquaria  housed
on water tables in an air-conditioned Butler  building located  on the lower
level of the Stage I platform.  The  aquaria  were outfitted with plastic
"eggcrate" bottom racks to help support the  irregularly shaped  pieces in an
upright position.  Fresh unfiltered  seawater, pumped up from a  depth of
28 m, was drawn into the aquaria by  Little Giant water pumps at a rate of
5.2 L/min, resulting in a turnover time of 40 min.  Details  of  the seawater
system are illustrated in Figure 1.   A 12-h  light/dark cycle was provided
with banks of VHO cool-white fluorescent bulbs.   The average light level
was 100 yEin rrr2s-l.  The water tables housing the aquaria were surrounded
with dark, opaque shower curtains to shield  the  corals from the laboratory
lights at night.  The aquaria were cleaned as needed to remove  algae and
other fouling organisms.  During the exposure period, drill  mud that

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settled in the tanks was siphoned out.   The exposed  skeletons  of  the  coral
pieces were also scrubbed with soft bottle brushes to remove  algae.   Corals
in four of the aquaria (Tanks 1 to 4)  were fed periodically with  freshly
collected zooplankton or with brine shrimp nauplii.


Experimental Design

        Forty pieces of coral were selected and randomly assigned:   eight
pieces to each of five aquaria.  The aquaria were then randomly  assigned to
one of five treatments:  control unfed, control fed,  1 ppm drill  mud,  10
ppm drill mud, and 100 ppm drill mud.   Since only eight corals could  be
tested each day, a scheme was devised  to divide the  40 corals  among  the
five incubation days.  Four of the eight corals in each treatment (the
lowest numbered ones) were designated  the "A" subgroup, and the  remaining
four were designated the "B" subgroup.   The ten "A"  and "B" subgroups  were
assigned to the five incubation days accordingly:  1) there were  one  A
subgroup and one B subgroup per day;- 2) the A and B  subgroups  were  from
different treatments, and 3) there was  only one control subgroup  per  day.
The final incubation sequence is summarized in Table  1.


TABLE 1.  ALLOCATION OF TREATMENT SUBGROUPS TO 5-DAY  INCUBATION  SEQUENCE
                                   Week day
Sub Group

1 2
A Control fed 1 ppm

3
100 ppm

4
10 ppm

5
Control


unfed
 B         100 ppm      10 ppm     Control unfed  Control  fed      1 ppm


        Oxygen consumption and production rates (respiration and
photosynthesis) were measured once for the 24 corals in the 1,  10,  and 100
ppm treatments during the two days before exposure to mud.  Mud exposure
began on July 21, 1980, and continued until  September 3, 1980.   The 5-day
incubation sequence began on July 22, 1980 (Week 1) and was repeated
beginning on July 28, (Week 2), August 4  (Week 3), August  11 (Week  4),
August 18 (Week 5) and August 24 (Week 6).  The corals were exposed to the
various mud concentrations continuously except when removed from the
exposure tanks for tests.  Each coral was tested once per  week; each test
consisted of a light and a dark incubation.
        During Week 1, only A0£ was measured, with incubations lasting two
hours.  Four chambers with coral and one control chamber without coral
could be measured simultaneously; thus, each complete incubation series
consisted of two 2-h incubations in the light and two 2-h incubations in
the dark.  During subsequent weeks, 02 incubations were shortened to one

                                     8

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hour, since the A02 rates  were  found  to  be  constant  throughout  the  2-h
period.  The nutrient  uptake, calcification,  and  TCOg  measurements  began
the second week and were  done during  a  separate  3-h  incubation  of all eight
corals simultaneously, using  seawater supplemented with  NH4C1 and NaNOs to
elevate the initial nutrient  concentrations.   During Week 2,  the  initial
incubation water concentrations were  about  1  to  2 pM N03~ and NH4+; during
subsequent weeks, about 3 to  6  pM N03~  and  NH4+.

        At the end of  the six-week exposure,  the  40  experimental  corals
were sacrificed and their surface area  and  biomass measured (see  below).
In addition, four corals  were sacrificed before  the  exposure  to mud began,
and three corals from  each treatment  were sacrificed after two  and  four
weeks of mud exposure  to  detect any differences  in the biochemical
composition of the corals with  duration  of  exposure  to drill  muds.


Mud Delivery System
                                    •«»
        The mud delivery  system consisted of  two  separatory funnels (36.8 L
capacity) to hold diluted mud stock,  and two  multichannel peristaltic
pumps used to deliver  the mud at a constant rate  from the funnels to the
inflowing seawater lines  of the treatment aquaria (Figure 1).  The
separatory funnels were stirred continuously  to  keep the muds in
suspension.  Muds were collected from a  well  in  the  Jay  oil field,  Jay,
Florida, by EPA personnel.  Mud batches  were  changed in  our exposure system
to approximate the sequence and timing of collection of  these muds.  Table
2 summarizes the collection dates of the muds and their  use in  our
experiments.


TABLE 2.  DATES OF COLLECTION OF JAY DRILLING MUDS AND THEIR  USE  IN TEST
EXPOSURES.
       Date Mud Collected         7-9   7-11  7-22  7-29  8-4

       Designation of Mud Used    JX-2  JX-3  JX-4  JX-5  JX-7

       Date Exposure Began        7-21  7-27  8-3   8-10  8-24



Incubation Procedures

Oxygen Incubation -- Five 15-cm diameter Plexiglass chambers with 0-ring
sealed lids were used for respiration and photosynthesis measurements.  The
lids were fitted with openings to accommodate Orbisphere self-stirring BOD
probes.  The oxygen probes were calibrated daily against Winkler titrations
(Strickland and Parsons, 1972).  Four corals from each treatment were run
simultaneously, one chamber serving as a control.  All incubations were
conducted in filtered seawater (Honey-Comb Superfine, 1 ym filters).  The

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                                                        DETAIL OF DRY-WELL

                                                          PUMP  SYSTEM
                                               WATER LEVEL
           STAGE  I MUD  EXPOSURE   SYSTEM
              LOW
              MUD
            CONCENTRATION
              FUNNEL
            44 ml of mud/
            36.BL seowoter
  TANK I
  CONTROL FED
  ./»!» SI«w>Tt» ^  S.r L/.l. !C«W«Tt»
       ill        :
TANK 2
1 PPM MU
J L
ruur

D
                OUTGOING

                SEAWATER ——

                MUD MIXTURE
   HIGH
   MUD
CONCENTRATION
  FUNNEL
440ml of mud/
36.6L seowoter
                                                           LITTLE  GIANT
                                                         PICKUP FOR
                                                        RECIRCULATING
                                                         WATER  	
                                                                     [PLEXIGLASS DRY-WELL
                                                                     -AQUARIUM SIDE
     \\ ,^-MUO SLURRY

|J-	"^— FRESH INCOMING
 	—— SEAWATER
PI
43

TANK
IMP A
6ml/ mm
| Vtzzzz:
-nl
5 puyp
IOO PPM MUD
" "/_" " • " "°=^___ — . .x


Y
TANK 4 ru«r
IOPPM MUD
^
TANK 5 [fun?
CONTROL UNFED
Figure  1.   Diagram of the system  used  for exposing corals to a constant
            drilling mud concentration.
rack  of  five chambers was placed  in  a  Plexiglass trough, where a  continuous
flow  of  filtered seawater acted as  a constant temperature water bath
(±1°C).   Salinities, measured  periodically with a refractometer,  remained
constant  at  35 °/°°.  Light  incubations  were conducted under a bank of  two
G.E.  1500 watt cool-white fluorescent  bulbs, that yielded an average  light
intensity of 94 uEin rrr^s'l  inside  the incubation chambers.

Nutrients, C02 and Calcification  Incubation -- Glass chambers with stirring
bars  and  support racks were  placed  in  the same trough and light source  as
above.   A measured amount of NaN03  and NH4C1 stock was mixed into the
trough before the chambers were put  into place.  Initial water samples  for
pH, total  alkalinity (TA) and  for nutrient analysis were withdrawn from
each  chamber; pH was measured  immediately and the samples were capped to
prevent  evaporation before TA  analysis.   Sampling was repeated at the end
of the incubation period.

      N03~ determinations were made  by the standard Technicon Auto Analyzer
N03~  technique for seawater  and wastewater.  NH4+ determinations  were made
by an adaptation of the automated method of Berg et_ a_l_.  (1977), with  the
ethanol  eliminated from the  phenol  reagent.  pH determinations were made
with  an  Orion model 701 pH meter, using  a calomel combination electrode,
the slope of which was checked periodically with N.B.S.  buffers of known
pH.   TA  was  determined by potentiometric titration of duplicate 10-ml
aliquots  of  the seawater used  for the  pH determinations.  The endpoint  of
                                        10

-------
the titration was calculated by the Gran method (Stumm and Morgan,  1970).
The TA of the seawater sample was calculated as:

                       TA (ineq/1) = N -
                                      V2

where:  v is the volume of sample titrated in ml, _y_£ is the titration
equivalence point in ml, and _N is the normality of acid in meq/1.'


     The total inorganic C02 concentrations were determined by thermal
conductivity detection of C02 stripped from acidified seawater samples
after gas chromatographic separation.  A Shimadzu Model 3BT gas
chromatograph (GC) and a Shimadzu integrator-data processor were used for
the analyses.  The stripping was done in a Swinnerton-type stripping
chamber  (Swinnerton et_ a_l_.,  1962).  The gas stream passed through a 15 cm
Drierite column before entering the GC.  Gas separation was achieved with a
2m x 1/8" Porapak Q column at 30°C.  Two to five replicates were run for
each sample.  The mean coefficient of variation of the replicates was less
than 1%.  Initial tests with Na2C03 standards showed that the response of
the system was linear up to at least 3 mM NagCOs.  Unfortunately, standard
Na2C03 solutions were not run routinely during the experimental runs.  We
later discovered that the response of the machine to a given amount of TC02
was affected by the way in which the Drierite columns were packed.   We
could analyze the results of each run only by calibrating results against
the initial TC02 concentration calculated from the pH and alkalinity data
(see below).  We calculated calibration factors (f) for each test run, as:


              (TC02 cone, of "initial" samples calculated from pH and TA
        f =   (integrated peak area of TC02 in "initial" samples from GC

where "Initial" samples were the samples taken at the beginning of each
incubation set.  TC02 concentrations in the final samples were then
calculated as:
                   TC02(mM) = f x (peak area from GC)
Biomass Analysis
        The surface areas of the living portion of the 40 intact
experimental corals were determined by the aluminum foil method (Marsh,
1970).  These surface areas were used to normalize the metabolic rates
measured in the above incubations.

        All coral colonies processed for biomass analysis and biochemical
composition were first scrubbed to remove any mud or encrusting organisms
adhering to the dead portions of the skeletons.  A chisel and hammer were
used to break the colonies into smaller pieces to be used for the
individual  analyses.  Surface areas of these pieces were determined as
above.

                                    11

-------
        Pieces of each coral were placed into wide-mouth glass jars with
 100 ml of a chloroform:methanol:seawater  solution (1:2:0.5; Bligh and
 Dyer,  1959).  The caps of the jars were lined with aluminum foil  and the
 jars were pre-washed with Bligh-Dyer solution.  These samples were returned
 to Florida State University for lipid analysis by Dr. D.C. White who will
 report the results separately.

        The remainder of the tissue analyses were conducted on a smaller
 piece  of each coral.  Tissues were removed from the skeletons by first
 breaking up the tissue with a small wire brush powered by an electric hand
 drill, then blasting the remaining tissue off with a fine jet of filtered
 seawater from a Water-Pik (Johannes and Wiebe, 1970).  The tissue slurry
 was homogenized in a blender for 3 min and the volume of the homogenate
 recorded.  Duplicate 1-ml aliquots of the homogenate were analyzed for
 total-nitrogen by the persulfate oxidation method of D'Elia et aj_. (1977).
 Subsamples of homogenate were frozen in polyethylene bottlesTor later
 carbohydrate analysis (Dubois et al., 1956); 30-50 ml volumes of homogenate
 were filtered onto glass fiberTiTtfers for chlorophyll determinations
 following the procedures of Strickland and Parsons (1972).  Additional
 subsamples of homogenate were preserved with Lugol's iodine solution for
 zooxanthellae counts (Szmant-Froelich and Pilson, 1980).

 Other  Procedures
                                      V
 Histological Fixation--Smal1 pieces of coral from each treatment were fixed
 with a seawater-Zenkers fixative (Yevich and Barszcz, 1977) for 24 to 48 h.
 The fixed samples were washed in running seawater for another 24 to 48 h
 and then preserved in 70% ethanol.  All  further processing and examination
 were done by the EPA histopathology unit, at ERL, Narragansett under the
 direction of Mr. P. Yevich, who will report results separately.

 Staining with Alizarin-Red-S--Two days before mud exposures began, twelve
 large colonies were placed into two ten-gallon aquaria filled with an
 al izarin/seawater solution (15 mg alizarin/L of seawater).  The corals were
 left in the solution for 8 h before running seawater was restored to the
 aquaria.  A second treatment with alizarin was repeated the following day
 to ensure that a strong stain mark had been incorporated into the skeleton.
 Three of the stained colonies were placed into each of the four "fed"
 treatment tanks (control, 1 ppm, 10 ppm, and 100 ppm) on the first day the
 exposures to mud began.  They remained in these tanks until sacrificed on
 September 7, 1980 (except for two of the 100 ppm corals which died before
the end of the experiment).  Tissues were removed by first soaking the
 corals in buckets of fresh tapwater, and then squirting the corals with a
jet of water from a garden hose.  The skeletons were shipped for analysis
to Dr. R. Dodge (Nova University) (Dodge, Marine Biology, in press).


 SECOND EXPERIMENTAL SERIES - PUERTO RICO

Coral  Collection and Maintenance

        Specimens of Montastrea annularis were collected fom the reef Cabo

                                    12

-------
de la Raya at a depth of 2 to 5 m.  Corals for Test 1 were collected from
several adjacent colonies, but those for Test 2 were from a single
large colony.  The corals were kept in aquaria with running seawater for
48 to 72 hours until  used in the experiments.

        Specimens of Acropora cervicornis in Test 3 were collected from the
lagoon (2 to 3 m depth) of San~Cristobal reef the day before the experiment
began.  Whole colonies were returned to the laboratory, where individual
branches 6 to 9 cm long were clipped-off and placed upright into small
Plexiglass stands.  Thus, we could move the 'fingers' without having to
touch live coral tissue.
Experimental Design

        Two exposure series were conducted with M. annularis:  In Test 1,
corals were exposed to 0, 10 ppm, and 100 ppm drTlling mud (6 replicate
corals each) for five days; In Test .2, 9 replicate corals were exposed to 0
and 100 ppm drilling mud for three days.  From the day before mud exposure
was to begin (2 days for Test 2), the corals were incubated for two hours
in the daytime and for one hour at night.  Parameters measured during the
daytime incubations were 02 concentration, TA, N03~, and NH4+
concentrations (nutrients not measured during Test 2); only Og
concentrations were measured at night.

        The A. cervicornis experiments consisted of exposing coral fingers
(four replicates each) to 0, 10 ppm,  and 100 ppm drilling mud for 48 hours.
The corals were incubated as above beginning one day before exposure.
Concentrations of 02, TA, N03~, and NH4+ were measured as above.

        All corals were sacrificed at the end of the experiments to
determine their surface area, chlorophyll, zooxanthellae, and
tissue-nitrogen content by the methods described above.


Mud Delivery System

        The mud delivery system was a scaled-down version of the Stage I
system.  We used 80-L aquaria, and maintained a flow rate of 1 L/min.
The aquaria were kept on a shaded water table and received supplemental
light from cool-white fluorescent bulbs.  Light levels in the tanks were
about 100-150 yEin rrr^ s~l.  Dark curtains around the water table shielded
the corals from extraneous lights at  night.

        The mud tested was JX-7 collected the previous summer from the Jay
oil field and preserved by refrigeration.


Incubation Procedures

        The same glass chambers used  on Stage I were used for the M.
annularis incubations.  Chambers were filled with seawater, which TFad been

                                    13

-------
allowed to sit  in cubitainers for one hour to degas, and placed in a through
with a continuous flow of seawater to maintain constant temperature.
Salinities measured with a  ref ractometer, remained constant at 35 °/°°.
Light  incubations were conducted under a bank of 40-watt cool-white bulbs,
which yielded an average light  intensity of 450 yEin m-2s~1 inside the
incubation chambers.  Water samples for 02, TA, pH, and nutrient analyses
were taken from the cubitainers at the beginning of the incubation and from
each chamber at the end of  the  incubation.

        A_. cervicornis was  incubated in 500 ml cylindrical chambers
constructed trom 3-inch diameter Plexiglass tubing, using the same trough
and procedures  as above.


DATA ANALYSIS

        The changes in concentration of the incubation media were corrected
for water volume, incubation duration, and concentration changes in the
control chambers, then normalized to the living surface area of the coral to
give a rate per surface area of coral (nmol cirr2h-l) for each physiological
function.

Calcification rates (ACaCOs) from the Stage I experiments was calculated as:

                ACaC03 = 1/2 [ATA - ANH4+ + A(N03- + N02')]
to correct for any changes  in TA caused by the uptake of the added nutrients
(Brewer and Goldman,  1976;  Jacques and Pilson, 1980).  Calcification rates
from the Puerto Rican experiments were not corrected for nutrient uptake.
Total-C02 was calculated from the pH and alkalinity data, using the
relationships expressed in  Riley and Chester  (1971).  The change in TC02 due
to respiration and photosynthesis [AC02P/R] was calculated from the
equation:

               [AC02P/R] =  ATC02 - ACaC03

Derivations and discussions of the above equations and their application to
metabolic measurements on corals have been previously described (Smith and
Kinsey, 1978; Jacques and Pilson, 1980).

        One-way ANOVA using SPSS (Nie _et_ a_\_. , 1975) was used to analyze the
Stage I data.  We tested trends over time within each treatment group and
differences between treatments within each of the six weekly incubation
series.  The program also calculated t-tests between specified treatment
groups.  The 1 ppm coral rates were not significantly different from the
controls; therefore the 10  ppm and 100 ppm coral rates were tested against
the mean of the two control and 1 ppm groups.
                                    14

-------
                                  SECTION 5

                            RESULTS AND DISCUSSION

CORAL SURVIVORSHIP

        One of the ~90 pieces of j^. annularis  collected from the Florida
Keys for use in the Stage I  experiments died within  a  week of collection.
All the surviving corals appeared healthy at the  beginning of the exposure
experiment; polyps readily expanded both in  the aquaria and the
experimental chambers.

        Further coral  mortality occurred only  in  the 100 ppm treatment
tank.  One of the eight experimental  corals  lost  most  of its zooxanthellae
during week 5 and one-third  of its polyps after 34 days of exposure to  100
ppm mud.  A white flocculent film covered the  dead portion of the coral.
Two other colonies from the  100 ppm tank had partially bleached after 34
days of exposure and were dead by 43 days. Portions  of several  other coral
pieces from this tank were dead by the end of  the experimental  period.

        No deaths occurred among the corals  used  in  the short exposures to
JX-7 mud in Puerto Rican studies.


PHYSIOLOGICAL RATES - STAGE  I.

        The mean rates of calcification, respiration,  photosynthesis and
nutrient uptake for the 6-week exposure period can be  found in Appendix A
and are plotted in Figures 2 to 8.  Results  of feeding behavior studies
with exposed corals at the end of the exposure period  and of the coral  and
algal biomass determinations are given in Tables  3 and 4 respectively.

Calcification

        Daytime and nighttime calcification  rates showed the same trends
with time and treatment, but daytime rates were two to three times greater
than nighttime.  Only daytime rates are discussed here.

        There were no significant week-to-week differences in the
calcification rate for either control or 1 ppm treatments, but for both 10
ppm and 100 ppm treatments the rates decreased with time (Figure 2).
Between-treatment comparisons for each week (Appendix B) showed no
                                    15

-------
 statistically  significant differences between treatments until  the fourth
 week, when  100 ppm daytime calcification rates dropped to 16% of control and
 1  ppm coral  rates  (p  = 0.005).  During the sixth week, the 10 ppm corals
 calcified at 67% of the rate of the controls, but the difference was  not
 significant  (p = 0.084).




















CALCIFICATION RATES IN THE LIGHT
±95% CONFIDENCE LIMITS
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STATISTICAL
SIGNIFICANCE

+ P S.05
t P £.01
x ps. ooi

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                               3        4

                           WEEKS  EXPOSURE
Figure 2.  Daytime calcification rates of _M. Annularis measured  as  changes  in
           total alkalinity. n=8.  Legend applies to Figures  2 to 8.


Respi ration

        Respiration rates were measured in two ways:  as  decreases  in  02
concentration and as increases in C02 concentration.  The respiratory  quotient
(RQ = AC02/A02) reflects the degree of reduction of material  being
catabolized, as well as differences in analytical methodology.   The overall
C02 to 02 ratio for all the measurements was 0.85 (r2 = 0.95; n  = 39).  There
were no obvious changes in RQ over the six weeks, nor any significant
differences in RQ between treatments.
                                     16

-------
          MEAN RESPIRATION (AOz) RATES
           ±95% CONFIDENCE LIMITS
1400
1200
1
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                    1234

                         WEEKS EXPOSURE
Figure 3. Respiration  of _M.  annularis measured as changes  in  oxygen
          concentration.  n=8.   For symbols, see key to Figure 2.
      1400
      1200
      1000
    2
    o

    CM
    O
    O
800
      600
    o
    2
      400
      200
           MEAN RESPIRATION(ACOz) RATES
           ±95% CONFIDENCE  LIMITS
                           234

                          WEEKS  EXPOSURE
Figure 4. Respiration of _M.  annularis  measured as changes  in  ^6
          concentration. n=8~For  symbols, see key to Figure 2.
                                       17

-------
        Respiration  rates of all except the 100 ppm corals increased
gradually with time  (Figures 3 and 4).  The 100 ppm corals, whose
respiration  rate decreased over the six-week exposure period, had
significantly lower  respiration rates than the controls following the second
week of mud  exposure  (Figure 4); by the sixth week, their respiration rate
was reduced  to 60% of that of the controls (p < .001).


.Photosynthesis

        Gross photosynthesis was measured as both increase in 02 and
/Dn       in  C°2 concentrations.  The overall photosynthetic quotient
(PQ =  A02/AC02) was  0.98  (r2 =  .93; n = 39).  As with RQ's, there were no
trends over  time nor  differences between treatments.

        Photosynthetic  rates gradually increased with time for all
treatments except the 100 ppm treatment (Figures 5 and 6).  02 production by
the 100 ppm  corals decreased to 74% and 83% of the control rate during weeks
5 and  6 respectively, while C02 estimates decreased to 75% and 67%.  Tissue
analyses  of  corals sacrificed during the seventh week revealed that the
zooxanthellae content of  the 100 ppm corals was 20% lower than that of the
control corals (p =  .05)  (Table 4).  Therefore, most of the decrease in
photosynthesis rate  and a portion of the decrease in respiration rate of the
100 ppm corals during the last two weeks of exposure may have been  due to a
loss of zooxanthellae biomass.


Nutrient  Uptake

        The  control-unfed corals consistently took up more NH4+ than the
control-fed  corals,  and the differences were frequently statistically
significant.  However,  there was no consistent difference in N03~ uptake
between the  two control groups.  A possible explanation is that the
zooxanthellae of unfed  corals had less NH4+ available from coral metabolic
waste and, therefore, took up more NH4+ from the media.

        Nutrient uptake rates by zooxanthellae are known to follow
Michaelis-Menton kinetics (D'Elia, 1977; Muscatine and D'Elia, 1978).
Therefore, net nutrient uptake  in these type of experiments will depend on
the initial  nutrient  concentration of the incubation media.  Nitrogen uptake
rates were lowest for all treatments during week 2 (Figures 7 and 8) because
of the lower initial  nutrient concentrations and there were no significant
differences  in that week  between the control and the exposed corals.
Significant  differences between the 100 ppm corals and the controls were
first seen during the fourth week of exposure (Figures 7 and 8), and between
the 10 ppm corals and the controls, during the fifth week.  N03~ uptake
appeared to  be affected slightly more than NH4+ uptake.  By the sixth week,
N03~ uptake  by the 100  ppm corals had dropped to 42% of the control rate and
NH4+ uptake had dropped to 51% of the control rate.  Since zooxanthellae
densities decreased by only 20% (see above), there must have been a
decrease in  the capacity of the 100 ppm zooxanthellae to take up nutrients.
                                     18

-------
2000r GROSS PHOTOSYNTHESIS (AOz)

1800
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1400
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50


40


30


20


10
           MEAN AMMONIUM  UPTAKE RATES
           ±95% CONFIDENCE  LIMITS
                                  -M'
                                           WT
            01        23456

                           WEEKS EXPOSURE


Figure 7.  Ammonium uptake  by ^. annularis during both light  and  dark
           incubations.   n= 167  For symbols, see key to Figure  2.
           MEAN  NITRATE  UPTAKE  RATES

i
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Figure 8,
     Nitrate uptake by _M. annularis  during  both  light and dark
     incubations.  n= 16".  For symbols,  see key  to Figure 2.
                                    20

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Feeding Behavior

        During the fourth  and  fifth  week of  exposure  the  polyps  of the 100
ppm corals no longer expanded  during the incubations.  At the  end of the
6-week exposure period,  several  corals  from  each  treatment were  placed in
finger bowls containing  filtered seawater.   The two  100 ppm colonies
selected appeared the healthiest of  those surviving that  treatment.  Small
pieces of filter paper soaked  with Artemia nauplii homogenate  were
presented to five polyps per colony"! The individual  polyps were observed
for normal feeding behavior (Lenhoff, 1968;  Mariscal, 1971), the criteria
for which were swallowing  the  papers within  10 min and retaining them for
at least 5 min.  After initial  testing, all  the colonies  were  placed in an
aquarium with clean running seawater and retested twice daily  for six days
(Table 3).  The corals previously exposed to 100  ppm  did  not exhibit normal
feeding behavior even after almost a week of relief from  the exposure.  On
the sixth day of testing a few polyps from one of the 100 ppm  corals
appeared to be trying to capture the papers  but were  unable to swallow
them.  One of the three  10 ppm corals tested also exhibited depressed
feeding behavior.


TABLE 3.  FEEDING BEHAVIOR OF  M. ANNULARIS,  AFTER SIX-WEEK EXPOSURE TO
DRILL MUD, IN RESPONSE TO PIEClS" OF  FILTER PAPER  SOAKED IN BRINE SHRIMP
HOMOGENATE. t
                                   TREATMENT
 DAY/TEST No.  	  	
                    CONTROL       1 PPM      10 PPM        100 PPM
0 + + +
1/1 + + +
1/2 + + -
2/1 + + +
2/2 + ± -
3/1 + + +
3/2 	
4/1 	
4/2 	
5/1 + ± -
5/2 + + +
6/1 + - +
6/2 + ± -
+ + -
+ + + + - - -
+ + + + - - -
+ + + + - - -
± + - - -
+ + ± - -
+ + - - -
+ + - - -
- - _ _ _
+ _ _ _ _
+ + + - -
+ ± - + -
+ + - + -
t Each colony was scored:   (+)  if papers were captured and swallowed, (±) if
papers were captured but not swallowed and (-)  if there was no response.
Number of symbols represents the number of colonies tested.  Corals were
tested after six weeks of  exposure (day 0),  then allowed to recover in
clean seawater (days 1 to  6).

                                     21

-------
 IABLE 4.  SUMMARY OF CORAL AND ALGAL BIOMASS OF MONTASTREA ANNULARIS EXPOSED
 'U JAY DRILLING MUDS FOR UP TO SEVEN WEEKS, t
TREATMENT
Freshly Collected
n «« r- . .
TISSUE-N
yg-at. N
per cm*-
(n = 4)
58±10
ZOOX.
DENSITY
106 cells Chla

yg Ch 1 a
per ~
10" zoox.

Chla/
Chl£

  - — _'\f-"joui c 1(1 = Q )

 (2 weeks on Stage 1)     66±12    3.3±1.6    13.8±3.4    5.0±2.3    1.2±0.1


 Two Weeks Exposure (n = 3)
   /"^-.^.A.-!    _     »      /
^uniroi , unfed
Control , fed
1 ppm
10 ppm
100 ppm
Four Weeks Exposure (
Control , unfed
Control , fed
1 ppm
10 ppm
100 ppm
Seven Weeks Exposure
(experimental coral
Control , unfed
Control , fed
1 ppm
10 ppm
100 ppm
67±20
60± 3
60±35
7U24
78±21
n = 3)
100±17
92± 4
72±28
95±20
73±10
(n = 8)
s)
61±16
67±20
66±20
67±11
56±14
4.5±1
2.3±0
3.5±0
4.5±0
5.0±1

5.8±0
6.6±0
4.8±1
5.5±0
3.9±0


4.6±0
5.3±1
4.8±1
4.8±1
3.9±0
.0
.5
.7
.8
.5

.7
.6
.5
.8
_**
• »J


.7
.5
.2
01**
• £-
14
11
11
12
16

16
17
15
19
16


11
17
14
15
14
.6±6
.0±4
.7±4
.2±1
.0±7

.4±3
.5±3
.9±4
.0±3
.4±1


.6±2
.2±5
.0+2
.2±3
.6±3
.1
.0
.7
.3
.6

.2
.0
.6
.3
.3


.4
.6
.6
.1
.2
3
5
3
2
3

2
2
3
3
4


2
3
3
3
3
.2±0
.1±2
.3±0
.8±0
.1±0

.8+0
.7±0
.4±0
.5±0
.2±0


.5+0
.3±0
.1±0
.2±0
.7±0
.6
.9
.8
.7
.8

.2
.6
.6
.2
.5


.3
.7
.8
.6
.8
1.3±0
1.6±0
1.3±0
1.5±0
1.3±0

1.4±0
1.3±0
1.4±0
1.4±0
1.4±0


1.0±0
1.3±0
1.2±0
1.3±0
1.3±0
.1
.7
.1
.2
.2

.1
.1
.1
.2
.1


.1
.1
.1
.1
.2
 t  Mean ± std. dev. of eight pieces of coral.
 **
   Significantly different from control at p <.05
 Coral  and Algal  Biomass

         Table 4 summarizes the data on nitrogen and carbohydrate content  of
 coral  tissue, and on density and chlorophyll content of zooxanthellae.

         Nitrogen content is an indicator of the amount of coral  tissue
 protein,  and  thus a measure of coral biomass.  Earlier studies have shown
.that  coral  tissue N and biomass vary with the nutritional state  of  the
 animal  (Szmant-Froelich and Pilson, 1980).  We expected a lower  N content
 in  tissues  of unfed control corals and corals exposed to 100 ppm that
 exhibited reduced feeding behavior.  Although the mean tissue N  of  these

                                      22

-------
 two  groups was  slightly  lower than that of the  rest, the differences were
 not  statistically  significant.  There was also  no difference in the tissue
 carbohydrate  content.

         The zooxanthellae  density, but not the  chlorophyll content, of the
 100  ppm  corals  was  significantly  lower than that of the other groups of
 coral  (Table  4).   It  is  not  clear whether the 100 ppm corals expelled some
 of their original  symbionts  or whether the internal conditions of these
 corals were unfavorable  for  the continued growth and survival of the
 zooxanthellae.   It  is  clear, however, that the  zooxanthellae remaining in
 the  100  ppm corals  had a higher chlorophyll concentration per algal cell,
 presumably an adaptation to  the lower light level in the 100 ppm exposure
 tank.
 PHYSIOLOGICAL  RATES  -  PUERTO  RICO

         It  was  not clear  from the  Stage  1 experiments whether the
 detrimental  effects  on coral  calcification, respiration, nutrient uptake,
 feeding  behavior, and  zooxanthellae  content observed after the third week of
 exposure were  due to the  prolonged exposure to drilling mud, or to the use
 of more  toxic  drilling mud  during  the  last three weeks of exposure (see
 Table 2).  Drilling  muds  JX-5 and  JX-7 had much higher chromium and
 hydrocarbon content  than  some of the earlier muds  (Gilbert and Kakareka,
 unpublished).   Thus  we wanted to see whether detrimental effects could be
 induced  in  _M.  annularis by  short exposures to the  more toxic JX-7 mud.  Two
 tests were  conducted with ^.  annularis and a third test with Acropora
 cervicornis (shown to  be  extremely sensitive to Mobile Bay muds, E. Powell,
 personal  communication).  Results  of the physiological measurements are
 summarized  in  Tables 5 to 7 and the  biomass analyses in Table 8.
 Test 1
         Calcification  was  the most  sensitive physiological function to
 drilling mud  stress.   Within  12  hours, corals exposed to  100  ppm  drilling
 mud had depressed  calcification  rates  relative to the controls.   By^the
 fifth  day their  calcification rate  was only 22% of the control  rate,  and
 26% of their  own pre-exposure rate  (Table  5).  Corals exposed to  10 ppm
 drilling mud  also  exhibited a depressed  calcification rate beginning  the
 second day of exposure.  Results  of the  respiration measurements  were
 variable (Table  5), those  of the  control group being more variable than
 those  of the  exposed groups.  All three  groups had depressed  respiration
 rates  on day  5,  possibly indicating a  slowing of metabolism due to reduced
 nourishment under  laboratory conditions.   Although respiration  rates  of 10
 ppm and 100 ppm  corals were significantly  lower than those of controls on
 day 5, they were not significantly  lower than their pe-exposure rates.
 Tissue nitrogen  results  (Table 8) indicated no differences in coral biomass
_among  the three  groups that might account  for the differences in
 respi ration.
                                      23

-------
                               I 1C

TABLE  5.   PHYSIOLOGICAL RATES f(MEAN l STD. DEV.)  OF  MONTASTREA ANNULARIS
tXPn^sFn TH IV  ~i r\r\ T i i TH^V .*• ,r> /A-i-^-r- .. v   .               ' ' - - — • -       - - -•	
"rubtu 10 JX-7 DRILLING MUD (
-------
        The photosynthesis rates of the control  corals were also quite
variable:  their coefficient of variation was at least twice that of the
exposed groups.  Therefore, although a trend of decreasing photosynthesis
with time exists for the 100 ppm corals, it is not statistically
significant.  Inspection of the zooxanthellae density and chlorophyll  data
(Table 8) showed no differences in these parameters among the three groups.
Therefore differences in photosynthesis among the groups were due to
differences in physiological rate, not differences in algal biomass.

        The photosynthesis to respiration ratio (P/R, Table 5) is generally
viewed as an index of autotrophic potential.  P/R was generally less than
one indicating that photosynthesis could not meet the demands of
respiration.  Although the P/R of the control and 100 ppm corals tended to
decrease in time, that of the 10 ppm corals remained relatively constant.

        Nitrate and ammonium uptake rates (Table 5) were measured at
ambient concentrations (about lyM N03~ and 0.2 yM NH4+).  Little confidence
can be placed on NH4+ uptake rates measured at this low initial
concentration that approached the sensitivity of the analytical technique;
therefore, only the N03- data is discussed.  The control and 10 ppm corals
showed a definite trend of increasing N03~ uptake rate with time (p <0.01
and p <0.05, respectively) while the 100 ppm corals did not.  Therefore, by
day 5 the N03~ uptake rate of the 100 ppm corals was significantly lower
than that of the controls (p<0.02), but not significantly different from
their own pre-exposure rate.  It is possible that the increase in uptake
rate of the control and 10 ppm corals was a result of the adaptation of the
zooxanthellae to the reduced light levels of our experimental system,  which
were much lower than ambient light levels where the corals were collected.
The light levels in the 100 ppm exposure tank were even lower due to
turbidity from the suspended drilling mud; the zooxanthellae may not have
been able to adjust to it.

Test 2

        The purpose of Test 2 was to replicate the adverse effects of  100
ppm drilling mud observed in Test 1, then stop the stress, and observe the
time course of recovery.  All specimens were collected from a single large
colony chosen from a slightly greater depth and thus adapted to lower  light
levels.  Two treatments (100 ppm JX-7 and control) with nine replicates
were used.  Also, the physiological rates of interest were monitored for
two days prior to initiation of exposure for a more extensive baseline.  By
exposure day 3, no difference could be observed between the exposed and
control corals in any of the parameters measured (Table 6) and both groups
showed a significant decrease in photosynthesis.  Since this decrease
indicated a possible uncontrolled external source of stress, we terminated
the experiment.  The biomass analyses (Table 8)  showed the two groups  of
corals to be similar in tissue N and algal biomass.  Their algal biomass
was similar to that of corals used in Test 1 and in the Stage I test (Table
4), but their tissue N was about 20% lower than that of the Test 1 corals.
However, the M_. annularis from both Puerto Rican tests had 20-40% more
coral  tissue N than the ^. annularis collected from the Florida Keys for
the Stage I tests.

                                    25

-------
TABLE  6.  PHYSIOLOGICAL  RATES  (MEAN  ±  STD. DEV.)  EXPOSED MONTASTREA ANNULAR IS
TO  100 PPM  JX-7  DRILLING  MUD  (TEST  2).  t                	
   PARAMETER
                 TREATMENT
                              DAYS  PRE-EXPOSURE
                           DAYS  EXPOSURE
 Calcification
   (nmoi .cnr^.h-1)
                  Control
                  100  PPM

 Respiration  (R)
   (nmol  02.cm-2.h-l)
                  Control
                  100  PPM

 Gross  Photosynthesis  (P)
   (mnol  02.cm-2.h--L)
                  Control
                  100  PPM
P/R
                  Control
                  100  PPM
 597±209   72U140  728±217   478±112    576±255
 426±172   573±192  522±219   490±153    551±201
 791±309  1091±200
 713±372  1010±181
2037±436  2183±302
1844±621  2057±306
1.36±.27  1.0U.07
1.49±.53  1.03±.ll
1010±282
 999±224
929±359
852±237
2053±301  1335±616
2074±338  1239±611
1.17±.74  0.72±.23
1.07±.21  0.70±.22
t n =  9  for  each  treatment.
Test 3
        As was true  for  M.  annularis,  daytime calcification rates of
Acropora cervicornis  were  approximately  twice as  fast as nighttime rates
(Table 7")~  The calcification  process  of _A.  cervicornis also appears to be
the more sensitive to drilling mud.  Both  daytime and nighttime
calcification  rates of  the 100 ppm corals  decreased  by 40% during the first
day of exposure to drilling mud (Table 7).   By the second day of exposure,
calcification  rates had decreased by approximately 60%.  The only other
physiological  function  to  show a difference  was nitrate uptake.  Nitrate
uptake rates of the control and 10 ppm corals were higher than their
pre-exposure rates (p<0.01) but those  of the 100  ppm corals were not
significantly  different  from their pre-exposure rates.  The biomass
analyses (Table 8) show no differences in  animal  or  algal biomass among the
three groups.
                                      26

-------
TABLE 7.  PHYSIOLOGICAL RATES (MEAN ± STD. DEV.) OF ACROPORA CERVICORNIS
EXPOSED TO JX-7 drilling mud for 48 HOURS (TEST 3). t
                   PRE-EXPOSURE
PARAMETER
                      DAYS EXPOSURE
       TREATMENT   DAY
NIGHT
                                       Day
                    Night
Day
Night
Calcification
(nmol .cm~2.h~l)
Control
10 PPM
100 PPM

Respi ration
(nmol 02«cm~ »h
Control
10 PPM
100 PPM
Photosynthesis


575H43
5971 86
4951 48


~~)






3631 72
330H14
3031 57



4461 63
4801 49
4621 56



6091 74
548H08
3071 84*
*








382160
410190
180195*



504134
570117
546162



576H81
6871113
2271 45*
*








336149
357148
118155*
*


497137
565120
478172

(nmol 02.cm~2.h~1)
Control
10 PPM
100 PPM
P/R
Control
10 PPM
100 PPM
N03- Uptake
(nmol ,N03»cm- .
Control
10 PPM
100 PPM

NH4+ Uptake
(nmol .NH4.cm~2.
Control
10 PPM
100 PPM
t Controls, n =
* Statistically
* Statistically
8331171
886H05
7931 53

0.931.09
0.921.07
0.871.07

h~l)
2.6411.92
2.5011.91
2.131 .99


h~l)



4; 10 PPM,
different
different









3.591.63
3.381.23
3.361.88






n -• 3; 100
980+105
9691 94
8911 44

0.971.07
0.851.07
0.821.09


2.591.33
2.871.07
1.631.53*






PPM, n =
from control at p <0
from control at p <0









3.291.53
3.491.23
2.621.59



1.441.15
1.521.42
1.501.34
4. Mean
.05
.01
8851 98
84U319
8071 13

0.891.06
0.741.27
0.861.13


5.381.59
5.221.65
3.191.60*
*


1.551.29
1.34+.12
1.521.41
i std. dev.











4.651.60
4.501.75
2.971.61*
*


1.621.14
1.551.31
1.581.15



                                      27

-------
TABLE 8.  CORAL AND ALGAL BIOMASS (MEAN ± STD. OEV. OF CORAL SPECIMENS
EXPOSED TO ox-? DRILLING MUD. t
TREATMENT
Tissue N
yg-at-N
per cm2
Zooxanthellae
Density
106 cells/cm2
Chla
ug/crrf2
yg Chla
per ~
106cells
Chla
to
Chl£
 Test 1 (6)
   Control
   10 PPM
   100 PPM
 91+25
103+14
106±22
5.2±1.8     17.0±4.9     3.30±0.57
5.2±1.2     17.4±2.7     3.49±1.00
5.2±1.5     17.8±3.2(3)   3.41±1.28(3)
1.0±.3
 Test 2 (9)
   Control
   100 PPM
 80±11        4.9±0.5      16.0±3.2(8)   3.32±0.78(8)  1.7±.3
 76±12l/)      4.8±0.9      18.4±2.3(7)   3.90±0.44     1.4±.l
Test 3
Control (4)
10 PPM(3)
100 PPM(4)

24+ 3
25± 2
23± 4

1.8±0.9
2. 1±1.1
2.1±o!5

8.1±1.5
8.4±1.8
9.5±2.2

6.65+6.05
3.36±0.08
4.48±0.75

1.5±1.0
2.4±1.5
1.U0.2
t ( ) = number of replicates
  chl = chlorophyl1

  Test 1: Montastrea annularis exposed for 5 days,
  Test 2: M. annularis exposed for 3 days.
  Test 3: Acropora cervicornis exposed for 2 days,
                                      28

-------
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                                       31

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                                   APPENDIX A
 TABLE A-l.  MEAN (± 1 s.d.) RESPIRATION AND GROSS PHOTOSYNTHFST S RATTS
                                  ™'*    "u bKUi5 ™U I UMIN I HtM i> KAItb

TREATMENT
— • 	 — 	
RESPIRATION
Control :
Unfed
Control :
Fed
1 PPM:
10 PPM:
100 PPM:
-
0
~ . _
-
727
±127
806
±138
860
±139
	
I
•— - — 	 — .
795
±179
858
±124
796
±183
790
±129
800
±124
i v urv i LL 1 1
1 	 — -
nmol
2
	
921
±201
780
±119
854
±138
908
±169
753
±99
Nia nuu run
02 crn~2 h
WEEK
3
1240
±164
903t
±139
865
±137
902
±166
887
±169
. MA WttN
-1
4
1073
±187
1024
±205
972
±241
908
±130
843*
±151
.i UIN 51Mb

5
1074
±187
981
±167
1106
±252
967
±158
704**
±93
t i .

6
1183
±116
1002
±269
1115
±235
979
±190
693**
±72
GROSS PHOTOSYNTHESIS
Control :
Unfed
Control :
Fed
1 PPM:
10 PPM:
100 PPM:
-
-
1114
±229
1030
±245
1240
±194
1095
±221
1127
±193
1032
±229
1062
±290
1256
±232
1292
±327
1287
±194
1400
±258
1335
±364
1431
±296
1583
±279
1206t
±233
1236
±269
1303
±341
1481
±207
1486
±308
1506
±206
1331
±347
1350
±356
1278
±171
1476
±349
1365
±245
1498
±353
1414
±423
1066
±70
1562
±399
1433
±294
1497
±338
1347
±426
1241
±266
t Controls significantly  different  from  each  other  (p<0.05)

* Significantly different  from  controls  and  1  ppm corals at p<0.05.

** Significantly different  from controls  and  1  ppm  corals at p<0.001.
                                     32

-------
TABLE A-2.  MEAN (± 1 s.d.) RESPIRATION AND GROSS PHOTOSYNTHESIS RATES (AC02)
OF MONTASTREA ANNULARIS EXPOSED TO DRILLING MUD FOR SIX WEEKS ON STAGE I.





Al kal inity and pH
TREATMENT
RESPIRATION
Control :
Unfed
Control :
Fed
1 PPM:
10 PPM:
100 PPM:

2
859
±112
796
±139
703
±102
755
±104
822
±257

3
858
±134
846
±186
782
±205
745
±129
800
±124
WEEK
4
789
±143
733
±132
805
±198
755
±195
561*
±137
nmol
method
C02 cnr2
h-1



Gas Chromatography Method
WEEK
5
970
±174
933
±192
851
±239
726
±183
685*
±173
6
816
±144
782
±150
863
±262
775
±132
487**
±60
3
1003
±135
818
±270
861
±202
820
±92
704**
±93
4
801
±369
937
±335
786
±106
717
±76
742
±295
5
925
±200
994
±365
833
±298
608*
±153
675*
±86
6
931
±162
1021
±298
937
±225
782
±199
675*
±216
GROSS PHOTOSYNTHESIS
Control :
Unfed
Control :
Fed
1 PPM:
10 PPM:
100 PPM:
1138
±320
1429
±427
1058
±312
1083
±237
1522
±466
1319
±224
1419
±291
1196
±303
1306
±363
1176
±321
1249
±297
1301
±372
1300
±378
1208
±363
1033
±419
1595
±413
1534
±314
1499
±270
1414
±504
1153*
±286
1570
±415
1595
±280
1581
±465
1450
±478
1059**
±250
1588
±317
1710
±304
1205
±493
1307
±426
1285
±536
1351
±422
1533
±455
1190
±131
1129
±149
1974
±476
1672
±325
1807
±356
1537
±383
1558
±353
1185*
±388
1856
±478
1841
±502
1744
±453
1640
±495
1358**
±246
* Significantly different from controls at p<0.01
** Significantly different from controls at p<0.001.

                                      33

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TABLE A-3.  MEAN (±1 s.d.) NH4+ UPTAKE, N03-  UPTAKE  AND  CALCIFICATION RATES OF MONTASTREA ANNULAR IS
EXPOSED TO DRILLING MUD FOR SIX WEEKS ON  STAGE  I.  CU  =  CONTROL  UNFED; CF = CONTROL FED; L=LIGHT; 0=DARK.


                                                          nmol  cm~2  h~l
  TREATMENT
WEEK 2
WEEK 3
WEEK 4
WEEK 5
WEEK 6

NH4+ Uptake
CU:
CF:
1 PPM:
10 PPM:
100 PPM:
N03~ Uptake
CU:
CF:
1 PPM:
10 PPM:
11)0 PPM:
Calci f icat i
CU:
CF:
1 PPM:
10 PPM:
100 PPM:
L

13±6
7 + 2t
14+5
9±2
12±3

1316
5±2t
7±3
812
3±5
on
682±264
679±223
546±255
689±239
6381388
0

13±6
11±4
9±2
10± 3
13±5

6 + 2
6 + 2
612
7±3
612

2911166
335±141
3261157
254±165
211±260
L

33±13
23±llt
39± 9
35±14
23± 7

17t7
26 + 9
25±7
26±5
19±9

590±297
553±166
555±162
623±210
552±129
D

45±13
32±14
34±13
26± 8
36 + 8

29i7
27±9
23±9
25 ±8
26±3

387U66
280+197
182±106
331±183
315H13
L

42±16
32 + 13
44±25
38±12
30H2*

35±14
37t 8
26± 7
37± 9
18± 8**

538+225
624+154
542+266
574+160
**266t213
D

38+10
45+ 9
34± 9
38 + 11
32 + 15

33± 9
29± 5
29+ 6
37±12
21± 3

214+153
244+107
158+173
244+111
122+ 88
L

39±10
26 + 9t
26 + 9
20+10*
2118**

'28+11
30+ 6
30+ 9
20+ R*
18+ 4**

695+341
417+226
720+299
518+209
**189l 80
D

38+13
30+ 7
29+ 9
25±10
20+ 7

291 8
31±10
25 + 11
24± 8
2()i 5

214±154
229+131
204+124
177+156
45+ 32
L

48+15
32± 7
34± 9
31 + 11
D

41 + 12
37 + 16
4U13
32 + 10
26tlO** 14+10

41±12
34+ 6
39 + 12
31+ 8
19+ 6

745+247
6431277
7391209
4751283
1161 91

41+21
331 9
38H1
301 7
131 6

261+85
219+147
179H39
188+119
33t 31
 t Controls significantly different  from each  other  (p<0.05)
 * Significantly different from controls and  1 PPM at  p<0.01
 ** Significantly different from controls and  1  PPM  at p<0.001.

-------
                                  APPENDIX B

TABLE B-l.  SUMMARY OF RESULTS OF T-TESTS PERFORMED TO TEST WHETHER
DIFFERENCES BETWEEN TREATMENTS FOR EACH WEEK OF DRILLING MUD EXPOSURE DURING
STAGE I EXPERIMENT WERE STATISTICALLY DIFFERENT, t
                                             PARAMETER
TEST
WEEK
1 CU
C1
C1
2 CU
C1
C1
3 CU
C'
C1
4 CU
C1
C1
5 CU
C1
C1
6 CU
C1
C1
STATISTICAL
TEST
S
S

S
S

S
S

S
S

S
S

S
S
vs
vs
vs
vs
vs
vs
vs
vs
vs
vs
vs
vs
vs
vs
vs
vs
vs
vs
CF
10
100
CF
10
100
CF
10
100
CF
10
100
CF
10
100
CF
10
100
PPM
PPM

PPM
PPM

PPM
PPM

PPM
PPM

PPM
PPM

PPM
PPM
CALC
-
-
.99
.64
.98
.76
.64
.82
.39
.94
.005
.241
.233
.000
.44
.08
.000
R-02
.43
.65
.77
.12 "
.52
.06
.000*
.12
.07
,59
.08
.02
.31
.23
.000
.13
.16
.000
R-C02
-
-
.35
.50
.71
.94
.14
.000
.43
.80
.003
.70
.027
.007
.57
.50
.000
P-02
.76
.87
.09
.97
.95
.39
.02*
.76
.14
.84
.54
.07
.48
.84
.000
.48
.39
.06
P-C02
-
—
.18
.28
.04±
.46
.97
.31
.76
.68
.16
.74
.52
.007
.89
.50
.001
NH4+
-
-
.007*
.028
.53
.03*
.25
.13
.78
.77
.05
.009*
.004
.000
.19
.06
.000 ,
N03-


.007*
.85
.73
.36
.74
.29
.87
,07±
.000
.46
.002
.000
.11
.008
.000
t Numbers are the probabilities (P)  that the means are the same (P=1.00 when
two means are identical; P<0.05 for  differences to be significant).

Abbreviations:  CU = control  unfed;  CF = control  fed; C's = mean of CU, CF and
1 PPM; * = CU higher rates than CF;  ± = exposed higher rates than C's;
CALC = calcification in the light;  R-02 = oxygen  respiration; R-C02=C02
respiration;  P-02 = oxygen photosynthesis; P-C02  = C02 photosynthesis;
     = ammonium uptake; N03~  = nitrate uptake.


                                      35

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