United States
Environmental Protection
Agency
Environmental Research
Laboratory
Gulf Breeze FL 32561
Research and Development
EPA-600/S3-83-013 July 1983
Project Summary
Physiological Effects of Drilling
Muds on Reef Corals
Alina Szmant-Froelich
Coral from two species, Montastrea
annularis and Acropora cervicornis.
were exposed in the laboratory to
concentrations of 0,1,10 and 100 ppm
drilling mud for two to five days and for
seven weeks. The drilling muds
(designated JX-2 through JX-7) were
collected from a Jay (Florida) oil field,
and those fluids were not intended to be
disposed of on the Outer Continental
Shelf. Physiological functions of the
coral (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 lengthy
exposure and at the end of each shorter
exposure. Significant reductions in
calcification, respiration and NO3-
uptake rates of M. annularis were
observed during the fourth week of
exposure to 100 ppm drilling mud.
Photosynthesis and NH4 +uptake rates
also decreased during the fifth week of
exposure. Normal feeding was absent
from these corals when they were
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
day) exposure of M. annularis to 100
ppm JX-7 mud (the drilling mud used
during weeks 5 and 6, which had a
much higher chromium and hydrocar-
bon content than muds used during
weeks 1 to 3) caused great reductions in
calcification and lesser reductions in
respiration, gross photosynthesis, and
NO3- uptake rates in one of two
experiments. Acropora cervicornis
showed a great reduction in calcifica-
tion after 12 hours of exposure to
100 ppm JX-7 and a decrease in NO3-
uptake within 24 hours. No coral deaths
occurred during these short tests.
Implications of the results are discussed,
and future studies are recommended.
This Project Summary was developed
by EPA's Environmental Research Lab-
oratory, Gulf Breeze, FL, to announce
key findings of the research project that
is fully documented in a separate report
of the same title (see Project Report
ordering information at back).
Introduction
Drilling muds are necessary to oil-
drilling operations in that they lubricate
the drill string, remove cuttings, maintain
hydrostatic pressure, prevent pipe
corrosion, and seal the bore hole in
porous formations. They 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 are
considered toxic and hazardous to
organisms.
Disposal of used drilling muds recently
has become an environmental concern.
Since used drilling muds are generally
dumped into waters immediately
adjacent to the drilling rig, it is important
to identify local marine communities or
organisms that might be adversely
affected by exposure to them.
Drilling on the outer continental shelf
of the Gulf of Mexico is conducted near the
East and West Texas Flower Gardens-
two unique, submerged coral reefs.
Since reef corals are responsible for reef
framework building as well as for much of
the primary production in the reef eco-
system, their survival is essential to the
integrity of the reef system as a whole.
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The purpose of this study was to
examine several physiological and
biochemical processes in coral 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
algae that live symbiotically within most
reef coral tissue.) Animal and algal
biomass were also measured as a
function of time to monitor for any
deterioration in nutrition during the
exposure period. Montastrea annularis
was chosen as the primary test species
because of its ecological importance in
the Texas Flower Gardens and
throughout the Caribbean. A second
species, Acropora cervicornis, was used
in later tests to compare the experimental
procedures and results of this project
with those of other EPA-f unded studies of
this species.
During the first laboratory experiment,
groups of corals were exposed to four
drilling mud concentrations (0 ppm, 1
ppm, 10 ppm, and 100 ppm) for seven
weeks. The mud-exposed corals were fed
during the experiments. Two control
groups were used. One control group was
fed periodically throughout the
experiment; the second was not fed so as
to simulate the starvation effects
expected in the exposed groups. Previ-
ously listed physiological parameters
were measured biweekly. Respiration
and photosynthesis were measured as
changes both in 02 and 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 during exposure. A second set
of experiments measuring the same
physiological parameters focused on the
short-term (2 to 5 day) effects of one of
the more toxic muds used in the first
experimental series. The first experiment
was conducted during July and August,
1980, on the U.S. Navy Stage I platform,
located 12 miles offshore from Panama
City, Florida. 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.
Experimental Procedures
First Experimental Series: Stage I - Coral
specimens were obtained from the
Florida Keys. They were immediately
taken to Stage I by boat, where they were
transferred to large aquaria of running
sea water. The corals appeared to be in
good condition, and most were fully
expanded within a few hours after
transfer to tanks. Forty pieces of coral
were selected and randomly assigned to
the five treatments: control unfed, control
fed, 1 ppm drilling mud, 10 ppm drilling
mud, and 100 ppm drilling mud. Exposed
and control-fed corals were fed
periodically with freshly collected
zooplankton or with brine shrimp nauplii.
Oxygen consumption and production
rates (respiration and photosynthesis)
were measured once for the 24 corals in
the 1,10, andlOOppmtreatmentsduring
the two days before exposure to mud.
Mud exposure began on July 21, 1980,
and continued until September 3, 1980.
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 AO2 was
measured, with incubations lasting two
hours. During subsequent weeks, 02
incubations were shortened to one hour
and the nutrient uptake, calcification, and
TC02 were measured during a separate
3-hour incubation using seawater
supplemented with NH4CI and NaN03 to
elevate the initial nutrient concentra-
tions. During Week 2, the initial
incubation water concentrations were
about 1 to 2 //M N03- and NH4+; during
subsequent weeks, about 3 to 6 yuM
N03- and NH4+.
At the end of the six-week exposure,
the 40 experimental corals were
sacrificed and their surface area, tissue-N,
carbohydrate, zooxanthellae density, and
zooxanthellae chlorophyll content were
measured. 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 drilling muds.
The system for delivering the mud
consisted of two funnels to hold diluted
mud stock and two multichannel
peristaltic pumps to deliver the mud at a
constant rate to the inflowing seawater
lines of the treatment aquaria. Since
drilling mud composition varies with
depth of drilling, mud batches used to
expose the corals were changed to
approximate the sequence and timing of
collection of these muds so as to simulate
as much as possible the long-term
exposure that would have resulted from
discharge of these muds. Table 1
summarizes the collection dates of the
muds and their use in our experiments.
Second Experimental Series: Puerto Rico-
The mud tested was JX-7 collected the
previous summer from the Jay oil field
and preserved by refrigeration.
Specimens of M. annularis were
collected from the reef Cabo 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 they were used in the
experiments. In Test 1, corals were
exposed to 0, 10, and 100 ppm drilling
mud (six replicate corals each) for five
days; in Test 2, nine replicate corals were
exposed to 0 and 100 ppm drilling mud for
three days. From the day before mud
exposure was to begin (two 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 concentra-
tion, TA, N03-, and NH4+ concentrations
(nutrients not measured during Test 2);
only O2 concentrations were measured at
night.
Specimens of A. cervicornis for Test 3
were collected from the lagoon (2 to 3 m
depth) of San Cristobal reef the day before
the experiment began. Branch tips were
exposed (four replicates each) to 0 ppm,
10 ppm, and 100 ppm drilling mud for 48
hours. The corals were incubated as
above beginning one day before
exposure. Changes in concentration of
02, TA, NO3-, and NH4+ were measured
during both day and night incubations.
Table 1. Dates of Collection of Jay Drilling Muds and Their Use in Test Exposures*
Date Mud Collected
Designation of Mud Used
Date Exposure Began
7-9
JX-2
7-21
7-11
JX-3
7-27
7-22
JX-4
8-3
7-29
JX-5
8-10
8-4
JX-7
8-24
*These muds were collected from terrestrial wells near Jay, Florida, and were not intended for
disposal on the Outer Continental Shelf.
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All corals were sacrificed at the end of
the experiments to determine their
surface area, chlorophyll, zooxanthellae,
and tissue-nitrogen content.
Data Analysis
The changes in concentration of the
incubation media were corrected for
water volume, incubation duration, con-
centration 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 cnr2rr1)
for each physiological function.
Calcification rates (ACaC03) from the
Stage I experiments were calculated as:
ACaCOs = 1/2 [ATA - ANH4 + A(N03
+ N02)] to correct for any changes in TA
caused by the uptake of the added nutri-
ents. Total-CO2 (TCO2) was calculated
from the pH and alkalinity data. The
change in CO2 due to respiration and
photosynthesis [AC02 P/R] was calcula-
ted from the equation:
[AC02P/R] = ATC02 - ACaC03
One-way analysis of variance (ANOVA)
was used to analyze the Stage I data.
Trends over time were tested 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 differ-
ent from the controls; thus, the 10 ppm
and the 100 ppm coral rates were tested
against the mean of the two control and 1
ppm groups.
Results
Coral Survival -- Coral deaths occurred
only in the 100 ppm treatment group on
Stage I. 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. 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 the
Puerto Rican experiments.
Physiological Rates: Stage I - Drilling
mud had the greatest measured detri-
mental effect on calcification in coral
physiology. For the Stage I experiment, no
statistically significant differences
occurred between treatments until the
fourth week, when calcification rates of
the 100-ppm-exposed corals dropped to
16% of rates of control and 1 ppm
exposed coral (Figure 1). During the sixth
week, the coral exposed to 10 ppm
calcified at 67% of the rate of the controls,
but the difference was not significant (p =
0.084).
Respiration rates (measured both as
decreases in 02 concentration and as
increases in C02 concentration) of all
except the corals exposed to 100 ppm
mud increased gradually with time
(Figure 2). 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 exposure to
mud and by the sixth week, their
respiration rate was reduced to 60% of
that of the controls (p < .001).
Photosynthetic rates also increased
with time for all treatments except the
100 ppm treatment (Figure 3). O2
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
Calcification Rates in the Light
± 95% Confidence Limits
O
o
a
I
1000r
800
600
400
200
revealed that the zooxanthellae content
of the 100 ppm corals was 20% lower
than that of the control corals (p = .05).
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 rates by zooxanthellae
are known to follow Michaelis-Menton
kinetics and therefore depend on the
initial nutrient concentration of the
incubation media. Nitrogen uptake rates
were lowest for all treatments during
week 2 (Figure 4) because of the lower
initial nutrient concentrations, but 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 appeared during the fourth week
of exposure, and between the 10 ppm
corals and the controls, during the fifth
week. In both cases, NO3- uptake was
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
Key
Treatment
Control unfed
o Control fed
* 7 ppm
A 10 ppm.
100 ppm
Statistical
Significance
+ P<.05
* P<.001
CO
'I*
Figure 1.
123456
Weeks Exposure
Daytime calcification rates of M. annularis measured as changes in total
alkalinity, n = 8.
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Mean Respiration (b.0i) Rates
± 95% Confidence Limits
7400
7200
7000
800
6°°
SJ
^ 400
200
I
Key
Treatment
Control unfed
o Control fed
* 1 ppm
& 10 ppm
100 ppm
Statistical
Significance
tP<.01
P<.OO1
0 723456
Weeks Exposure
Figure 2. Respiration of M. annularis measured as changes in O2 concentration, n = 8.
to 51% of the control rate. Since
zooxanthellae densities decreased by
only 20%, there must have been a
decrease in the capacity of the 100 ppm
zooxanthellae to take up nutrients.
Feeding Behavior -- 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 to be the healthiest of
those surviving that treatment. Small
pieces of filter paper soaked with brine
shrimp nauplii homogenate were
presented to five polyps per colony. The
individual polyps were observed for
normal feeding behavior, the criteria for
which were swallowing the papers
within ten minutes and retaining them for
at least five minutes. After initial testing,
all the colonies were placed in an
aquarium with clean running seawater
and retested twice daily for six days. 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.
Coral and Algal Biomass -- 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 biomass varies with the
nutritional state of the animal. 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 two groups was slightly
lower than that of the rest, the
differences were not statistically signifi-
cant. 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. 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 con-
centration 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 I 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 1).
Drilling muds JX-5 and JX-7 had much
higher chromium and hydrocarbon
content than some of the earlier muds.
Thus we wanted to see whether
detrimental effects on corals could be
induced by short exposures to the more
toxic JX-7 mud. Two tests were
conducted with M. annularis and a third
test with A. cervicornis.
The first test with M. annularis (five day
exposure) again showed calcification to
be 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. Corals exposed to 10
ppm drilling mud also exhibited a
depressed calcification rate beginning
the second day of exposure.
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 own pre-exposure rates.
A trend of decreasing photosynthesis
with time was observed for the 100 ppm
corals, but it was not statistically signifi-
cant. Inspection of the zooxanthellae
density and chlorophyll data showed no
differences in these parameters among
the three groups that might account for
the differences in photosynthesis.
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
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2000
7800
7600
7400
7200
7000
800
600
Gross Photosynthesis (&0i) Rates
± 95% Confidence Limits
Key
Treatment
Control unfed
o Control fed
* 7 ppm
m 100 ppm
Statistical
Significance
+ />< .05
tA><.07
*/»<.007
2 3
Weeks Exposure
Figure 3. Photosynthesis by M. annularis measured as changes in02. n = 8.
controls (p<0.02), but not significantly
different from their own pre-exposure
rate.
The purpose of the second test with M.
annularis 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.
However, by exposure-day 3, no
difference could be observed between the
exposed and control corals in any of the
parameters measured and both groups
showed a significant decrease in
photosynthesis. Since this decrease
indicated a possible uncontrolled
external source of stress, we terminated
the experiment.
As was true for M. annularis, the
calcification process of A. cervicornis was
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. By the second day of exposure,
calcification rates had decreased by
pproximately 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-expo-
sure rates (p<0.01) but those of the 100
ppm corals were not significantly differ-
ent from their pre-exposure rates. The
biomass analyses showed no differences
in animal or algal biomass among the
three groups.
Conclusions
Based on the results of the first
experiment we can conclude that:
The reef coral Montastrea annularis
can be adversely affected by long-
term (more than three weeks)
exposure to 100 ppm drilling mud.
Adverse effects ranged from an 84%
reduction in calcification rate and
40% reduction in coral respiration
rate to lesser effects on photosyn-
thesis by the zooxanthellae of these
same corals.
Corals exposed to 100 ppm drilling
mud for six weeks lost normal
feeding response and 20% of their
zooxanthellae, and several of them
died durmg the fifth and sixth weeks.
Since different batches of drilling mud
were used during the 6-week
experiment, it was not clear whether the
absence of any discernible physiological
effect during the first three weeks was
due to a cumulative time effect or to a
greater toxicity of the batches of drilling
mud used after the third week. The
second set of experiments, in which we
exposed specimens of M. annularis and
A. 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; specimens of M. annularis
exposed to 100 ppm of JX-7 during
one test suffered a 20% decrease in
calcification within 24 hours of
exposure and a 40% decrease by the
fifth day of exposure, while those
used in a second test showed no
adverse effects after three days of
exposure.
Some coral species are more
sensitive to drilling mud than others:
A. cervicornis suffered a 50% de-
crease in calcification within 12
hours of exposure to 100 ppm of
JX-7, and a 40% reduction in NO3-
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 (or greater) may cause a
large decrease in calcification rate in
some colonies of these coral
species.
Longer exposures, especially when
more toxic drilling mud additives are
used, increase the chance that
sublethal and lethal effects will
occur.
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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o
i
I
Mean Nitrate Uptake Rates
gg ± 95% Confidence Limits
50
40
30
20
10
Key
Treatment
Control unfed
o Control fed
A 1 ppm
m 100 ppm
Statistical
Significance
+ P < -OS
*P<.001
0 1 23456
Weeks Exposure
Figure 4. Nitrate uptake by M. annularis during both light and dark incubations, n = 16.
Recommendations
Initial studies were undertaken and
designed with little information on
expected exposure concentrations and
duration. It appears that real exposures
will consist of frequent (several per week)
doses of drilling mud of varying
concentrations over prolonged periods
(three months to a year). Recent field
studies indicate that only corals situated
within about 100 meters of a rig should
encounter concentrations higher than
100 ppm drilling mud 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.
A final recommendation is that future
studies should be concerned with
dispersal characteristics of different
fractions of the mud. Whole muds were
used in the present experiments for lack
of better information on which fractions
were more likely to affect reef areas.
However, heavy particulates will settle
quickly over a small downstream area,
where corals may both be buried and
poisoned, while light particulates and
dissolved fractions will disperse over
larger areas, 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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Alina Szmant-Froelich is with Florida State University. Tallahassee, FL 32306.
Thomas W. Duke is the EPA Project Officer (see below).
The complete report, entitled "Physiological Effects of Drilling Muds on Reef
Corals," (Order No. PB 83-181 560; Cost: $8.50, 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
Sabine Island
Gulf Breeze, FL 32561
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Information
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